FISH PHYSIOLOGY PHYSIOLOGY FISH VOLUME XI XI VOLUME The Physiology Physiology of of Developing Developing Fish Fish The...
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FISH PHYSIOLOGY PHYSIOLOGY FISH VOLUME XI XI VOLUME The Physiology Physiology of of Developing Developing Fish Fish The Part A A Part Eggs and and Larvae Larvae Eggs
CONTRIBUTORS CONTRIBUTORS D. D. F. F. ALDERDICE
THOMAS P. P. MOMMSEN J. PETER J. ROMBOUGH J. H. S. S. BLAXTER BLAXTER H. VON WESTERNHAGEN RANDAL K. BUDDINGTON PATRICKJ. THOMAS A. HEMING PATRICK J. WALSH KENJIRO KENJIRO YAMAGAMI
FISH PHYSIOLOGY PHYSIOLOGY FISH Edited by by Edited
W . SS.. H HO OA R W. DEPARTMENT OF OF ZOOLOGY ZOOLOGY DEPARTMENT UNIVERSITY OF OF BRITISH BRITISH COLUMBIA COLUMBIA UNIVERSITY VANCOUVER, BRITISH BRITISH COLUMBIA, COLUMBIA, CANADA CANADA VANCOUVER,
D.. J. J. RANDA AL LL L D DEPARTMENT OF ZOOLOGY ZOOLOGY DEPARTMENT OF BRITISH BRITISH COLUMBIA COLUMBIA UNIVERSITY OF VANCOUVER, BRITISH BRITISH COLUMBIA, COLUMBIA, CANADA VANCOUVER,
VOLUME XI XI VOLUME
The Physiology Physiology of of Developing Developing Fish Fish The Part A A Part Eggs and and Larvae Larvae
ACADEMIC ACADEMIC PRESS, PRESS, INC. INC.
Harcourt Harcourt Brace Brace Jovanovich, Jovanovich, Publishers Publishers
San San Diego Diego New New York York Berkeley Berkeley Boston Boston London London Sydney Sydney Tokyo Tokyo Toronto Toronto
COPYRIGHT CJ COPYRIGHT @ 1988 1988 BY BY ACADEMIC ACADEMICPRESS. PRESS. INC. INC. ALL ALL RIGHTS RIGHTS RESERVED. RESERVED. NO PART PART O F THIS PUBLICATION PUBLICATION MAY MAY BE BE REPRODUCED REPRODUCED OR OR NO OF TRANSMITTED IN IN ANY ANY FORM FORM OR BY ANY ANY MEANS. MEANS, ELECTRONIC ELECTRONIC OR BY OR MECHANICAL, INCLUDING INCLUDING PHOTOCOPY. PHOTOCOPY, RECORDING. RECORDING, OR OR MECHANICAL. OR ANY ANY INFORMATION INFORMATION STORAGE STORAGE AND AND RETRIEVAL RETRIEVAL SYSTEM. SYSTEM, WITHOUT WITHOUT PERMISSION IN IN WRITING WRITING FROM FROM TTHE H E PUBLISHER. PUBLISHER PERMISSION
ACADEMIC PRESS, ACADEMIC PRESS, INC. INC.
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United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road. 24-28 Oval Road. London London NW NWII 7DX 7DX
United Kin dom Edition ublished by ACADEME PRESS I&. (LONDON) LTD.
(Revised for (Revised for vol. vol. 11) 1 1)
Library Library of of Congress Congress Cataloging Cataloging in i n Publication Publication Data Data
Hoar, Hoar, William William Stewart, Date Date
Fish physiology. Fish Vols.8Vols. 8-
edited edited by by W. S. S. Hoar Hoar let [ e t al.J al.]
Includes Includes bibliographies bibliographies and and inpexes. indexes.
v. 1. 1. Excretion, ionic regulation, and Contents: v.
-
-
metabolism metabolism - v. v. 2. 2. The The endocrine endocrine system system -
-
v. 11. 11 - v.
developing fish. pt. A. A. Eggs Eggs and The physiology of developing larvae. juveniles larvae. pt. pt. B. B. Viviparity Viviparity and and posthatching juveniles (2 (2 v.) v.1
1 . Fishes-Physiology-Collected Fishes-Physiology-Collected works. works. 1.
I. I. Randall, Randall, D. D. J. J.
II. I I . Conte, Conte, Frank P., P., Date Date
III. I l l . Title. Title.
QL639.1 .H6 QL639.1.H6
597'.01 597l.01
76-84233 76-84233
ISBN 0-12-350433-3 0-12-350433-3 (v. 11, 11, pt. A) (alk. paper) ISBN (v. PRINTED STATES Of PRINTED IN IN THE THE UNITED UNITEDSTATES OF "MERle" AMERICA 88 89 90
91
987654321
CONTENTS CONTENTS
CONTRIBUTORS CONTRIBUTORS PREFACE PREFACE CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES CONTENTS
ix ix
xi xi xiii xiii
1. Pattern Pattern and and Variety Variety in in Development Development 1. ]. H. S. S. Blaxter J. I. I. II. 11. III. 111. IV. IV. V. VI. VI. VII. VII. VIII. VIII. IX. IX. X. X. XI. XI. XII. XII. XIII. XIII.
2. 2.
Introduction Introduction Rearing Rearing Techniques Techniques Progress and Diversity Diversity of Development Progress and of Development Terminology Terminology of of Early Early Life Life History History Stages Stages Egg Egg Size Size and and Egg Egg Quality Quality The The Effect Effect of of Starvation Starvation The The Effect Effect of of Captivity Captivity The The Effect Effect of of Fixation Fixation on on Shrinkage Shrinkage Rate Rate of of Development Development Organ Organ Systems Systems Structure Structure and and Function Function Critical Critical Periods Periods Conclusions Conclusions References References
1 2 4 15 15 17 23 27 30 31 31 33 33 41 41 44 44 47 47 48 48
Respiratory Respiratory Gas Gas Exchange, Exchange, Aerobic Aerobic Metabolism, Metabolism, and and Effects Effects of of Hypoxia Hypoxia during during Early Early Life Life
Peter PeterJ. J.Rombough Rornbough I.I. Introduction Introduction II. 11. Respiratory Respiratory Gas Gas Exchange Exchange III. 111. Aerobic Aerobic Metabolism Metabolism IV. IV. Effect Effectof of Hypoxia Hypoxia V. V. Conclusions Conclusions References References
59 59 60 60 82 82 123 123 143 143 144 144 vV
vi vi
CONTENTS CONTENTS
Osmotic and and Ionic Ionic Regulation Regulation in in Teleost Teleost Eggs 3. Osmotic 3. and Larvae and D. FF.. Alderdice Alderdice D. Introduction I. Introduction
II. Oogenesis 1 1. Oogenesis
III. Fertilization Fertilization 111. Development IV. Development V. Conclusions References References
163 167 176 183 236 242
of Pollutants on on Fish Eggs 4. Sublethal Effects of 4.
and Larvae H.. uon von Westernhagen Westernhagen H Introduction I. Introduction
II. Sublethal Sublethal Effects Effects during during Development Development 11. III. Sublethal Sublethal Effects Effects Displayed Displayed by by Larvae Larvae Hatched Hatched 111.
from Treated Treated Eggs Eggs from IV. Sublethal Sublethal Effects Effects on on Larvae Larvae Not Not Exposed Exposed as as Eggs Eggs IV. V. Discussion, Discussion, Problems, Problems, and and the the Future V. Future References References
253 258 296 3 15 319 330
55.. Vitellogenesis Vitellogenesis and and Oocyte Oocyte Assembly Assembly Thomas P. P . Mommsen and Patrick J. J. Walsh Walsh I. I. Introduction Introduction II. 11. Vitellogenesis Vitellogenesis III. 111. Oocyte Oocyte Assembly Assembly
IV. IV. Epilogue Epilogue References References
6. 6.
348 349 377 391 391 395
Yolk Absorption in Embryonic and Larval Fishes
Thomas A. A . Heming and Randal K. K . Buddington I. I. Introduction Introduction II. 11. Structural Structural Aspects Aspects of of Yolk Yolk Absorption Absorption III. 111. Yolk Yolk Composition Composition during during Development Development IV. of Yolk Yolk Absorption Absorption IV. Rate Rate of V. V. Efficiency Efficiency of of Yolk Yolk Utilization Utilization VI. VI. Nonyolk Nonyolk Nutrient Nutrient Sources Sources during during Early Early Development Development VII. of Embryos Embryos and and Larvae Larvae VII. Nutrition Nutrition of References References
408 408 410 410 414 414 424 424 430 430 434 434 437 437 438 438
CONTENTS CONTENTS
vii vii
7. Mechanisms of Hatching in Fish 7. Kenjiro Yamagami I. I. 11. II. 111. III. IV. IV. V.
Introduction-Early Studies Studies on Fish Hatching Introduction-Early Hatching-Gland Cells Cells Hatching-Gland Hatching Enzyme and Choriolysis Hatching Hatching in Fish Physiology of Hatching Epilogue-Problems to Be Solved in the Future Epilogue-Problems References References
447 447 449 449 459 459 480 480 489 489 490 490
AUTHORINDEX INDEX AUTHOR
501 501
SYSTEMATIC INDEX SYSTEMATIC INDEX
525 525
SUBJECTINDEX INDEX SUBJECT
537 537
This Page Intentionally Left Blank
CONTRIBUTORS CONTRIBUTORS
Numbers contributions begin. Numbers in parentheses indicate the pages on which the authors’ authors' contributions
D. F. ALDERDICE (163), Department of of Fisheries and Oceans, Fish FishF. ALDERDICE (163), Paci$c Biological Biological Station, Nanaimo, Brit Briteries Research Branch, Pacific V 9 R 5K6 ish Columbia, Canada V9R
J.
( l ) ,Dunstaf Dunstaffnage SS.. BLAXTER BLAXTER (1), fnage Marine Research Laboratory, Oban, Scotland Oban, Argyll PA34 4AD, Scotland
H. J. H.
RANDALK. BUDDINGTON BUDDINGTON (407), Department RANDAL (407),
of Physiology, Physiology, University of of California, Los Angeles, California 90024 of
THOMAS HEMING(407), (407), Pulmonary THOMAS A. HEMING
of Inter InterDivision, Department of of Texas Medical Branch, Galveston, Galveston, nal Medicine, University of Texas 77550-2780 77550-2780
THOMAS P. (347), THOMAS P. MOMMSEN MOMMSEN (347), Department
of of Zoology, University of of British Columbia, Columbia, Vancouver, Vancouver, British Columbia, Columbia, Canada V6T 2A9
PETER]. (59), PETERJ. ROMBOUGH ROMBOUGH (59), Zoology
Department, Brandon University, University, Brandon, Manitoba, Canada R7A 6A9
H . VON VON WESTERNHAGEN WESTERNHAGEN (253), H. (253),
(ZenBiologische Anstalt Helgoland (Zen trale), 52, Federal Republic of 0-2000 Hamburg 52, of Germany trale), D-2000
PATRICK ]. PATRICK J . WALSH WALSH(347), (347),
Rosenstiel School of of Marine and Atmo Atmoof Miami, Miami, Florida 33149 spheric Science, University of
KENJIRO YAMAGAMI YAMAGAMI(447), (447), Life KENJIRO
Science Institute, Sophia University, University, Chiyoda-ku, Chiyoda-ku, Tokyo 102, 102, Japan
ix
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PREFACE
Dramatic changes occur in the physiology of most animals during their their development. development. Among Among the the vertebrates, vertebrates, birds birds are are entirely entirely ovipa oviparous, live for variable periods in a cleidoic egg, and show fundamental hatchalterations in excretion, nutrition, and respiration at the time of hatch ing. In contrast, contrast, the eutherian mammals mammals are all viviparous, depending ing. on on the the maternal maternal circulation circulation and and aa specialized placenta placenta to to provide provide food, food, exchange gases, and exchange gases, and discharge discharge wastes. wastes. The The physiology physiology of of both both mother mother and is highly and fetus fetus is highly specialized specialized during during gestation gestation and and changes changes funda fundamentally time of birth. Fishes oviparous and mentally at at the the time of birth. Fishes exemplify exemplify both both the the oviparous and the the viviparous viviparous modes modes of of development, development, with with some examples examples that that are are intermediate two. In intermediate between between the the two. In these these two two volumes, volumes, we we present present reviews of many, but not all, aspects of development. The chapters in Part A relate to the physiology of eggs and larvae: different patterns of larval eflarval development development osmotic osmotic and and ionic ionic regulation, regulation, gas gas exchange, exchange, ef fects fects of of pollutants, pollutants, vitellogenesis, vitellogenesis, the absorption absorption of of yolk, yolk, and the the mechanisms hatching. Chapters Chapters in in Part Part B deal with with maternal-fetal maternal-fetal mechanisms of hatching. relations, meristic variation, smolting salmonids, the ontogeny of of be behavior, havior, and and the the development development of of sensory sensory systems. systems. The editors editors wish wish to to thank thank the the authors authors for their their cooperation cooperation and and dedication to this project and also to express express their deep appreciation to the many reviewers whose careful readings and constructive criti criticisms cisms have have greatly greatly improved improved the the final final presentations. presentations. W. W. S. S. HOAR
D. J. RANDALL D.
xi xi
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CONTENTS OF OTHER VOLUMES CONTENTS
Volume I The Compartments and and the of Electrolytes Electrolytes The Body Body Compartments the Distribution Distribution of
W. W. N. N . Holmes and Edward M. M . Donaldson
The Kidney Cleveland P. P . Hickman, Hickman, Jr., Jr., and BetYamin Benjamin F. F . Trump Salt Salt Secretion Secretion
Frank P. P . Conte The The Effects Effects of Salinity on the Eggs Eggs and Larvae Larvae of Teleosts
F. F . G. G . T. T . Holliday
Formation Formation of of Excretory Excretory Products Products
Roy P. Forster and Leon Goldstein Intermediary Intermediary Metabolism Metabolism in in Fishes Fishes
P. P . W. W. Hochachka Nutrition, Nutrition, Digestion, Digestion, and and Energy Energy Utilization Utilization
Arthur M. M. Phillips, Jr. Jr.
AUTHOR INDEX-SUBJECT AUTHOR INDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECTINDEX INDEX
Volume Volume II 11 The The Pituitary Pituitary Gland: Gland: Anatomy Anatomy and and Histophysiology Histophysiology ]. J. N. Ball and Bridget 1. 1. Baker
The The Neurohypophysis Neurohypophysis
A. A . M. M. Perks Prolactin Paralactin) and Prolactin (Fish (Fish Prolactin Prolactin or or Paralactin) and Growth Growth Hormone Hormone ]. J. N. N. Ball Ball Thyroid Function Function and Its Control in Fishes Fishes
Aubrey Gorbman Gorbmun xiii xiii
xiv xiv
CONTENTS CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES
Endocrine Pancreas The Endocrine August Epple The Adrenocortical Adrenocortical Steroids, Steroids, Adrenocorticotropin Adrenocorticotropin and and the the Corpuscles Corpuscles The of Stannius Stannius of I. ChesterJones, Chester Jones, D D.. K K.. 0. o. Clzan, Chan, 1. I. W W.. Henderson, aand]. N.. Ball Ball 1. nd]. N The Ultiinobranchial Ultimobranchial Glands Glands and and Calcium Calcium Regulation Regulation The D.. Harold Copp D Neurosecretory System Urophysis and Caudal Neurosecretory Howard A.. Bern Howard A AUTHOR INDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX INDEX AUTHOR
III Volume 1 11 Reproduction Reproduction William S. Williaiit S . Hoar
Hormones and Reproductive Reproductive Behavior in in Fishes Fishes N. N . R. R . Liley Sex Sex Differentiation Differentiation
Toki-o Toki-o Yamamoto Yainainoto Development: Development: Eggs Eggs and and Larvae Larvae ]. 1. H. H . S. S . Blaxter Fish Cell and Tissue Tissue Culture Culture
Ken Wolf Wolf and M. M . C. C . Quimby Quiinby Chromatophores and Pigments
Ryozo Fujii Bioluminescence Bioluminescence
]. 1.A. A. C. C . Nicol Poisons Poisons and and Venoms Venoms
Findlay Findla y E. E. Russell AUTHOR AUTHOR INDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX INDEX
Volume Volume IV IV Anatomy Anatomy and Physiology Physiology of of the the Central Central Nervous Nervous System System
Jerald Jerald J. 1.Bernstein Bernstein The The Pineal Pineal Organ Organ
James Jaines Clarke Clarke Fenwick Fenwick
CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES CONTENTS
Autonomic Nervous Nervous System Systems Autonoinic Graeme Campbell Campbell Craeine The Circulatory Circulatory System System The D. ]. Randall D. J . Randall Acid -Base Balance Balance Acid-Base C.. Albers Albers C Properties of of Fish Fish Heinoglobins Hemoglobins Properties Austen Riggs Riggs Austen Gas Exchange Exchange in in Fish Fish Gas D. ]. Randall D. J . Randall The Regulation Regulation of of Breathing Breathing The G. Shelton Shelton G. Air Breathing Breathing in in Fishes Fishes Air Kjell Johansen Kjell Johansen The Swiin Swim Bladder Bladder as as aa Hydrostatic Hydrostatic Organ Organ The Johan B. Steen Steen Johan Hydrostatic Pressure Pressure Hydrostatic Malcolm S. Gordon Gordon Malcolm S. Immunology of Fish Fish Immunology of
John Cushing John E. E. Cushing
AUTHOR AUTHOR INDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX INDEX
Volume Volume V V Vision: Vision: Visual Visual Pigments Pigments
F. F . W. W. Munz Munz Vision: Vision: Electrophysiology Electrophysiology of of the the Retina Retina
T. T . Tomita Tomita
Vision: of Visual Visual Behavior Behavior Vision: The The Experimental Experimental Analysis Analysis of
David David Ingle lngle Chemoreception Cheinoreception
Toshiaki]. Toshiaki J . Hara Hara Temperature Temperature Receptors Receptors
R. R . W. W . Murray Murray
Sound Sound Production Production and and Detection Detection
William Williain N. N. Tavolga Taoolga
xv xv
xvi xvi
CONTENTS CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES
The Labyrinth Labyrinth The O. Lowenstein Lowenstein 0, The Lateral Lateral Organ Organ Mechanoreceptors Mechanoreceptors The A e Flock l kke Flock The Mauthner Mauthner Cell Cell The ]. Diamond'. J. Diamond Electric Organs Organs Electric M. V. L. Bennett Bennett M . V . L. Electroreception Electroreception M.. V V.. L. Bennett Bennett M AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX AUTHOR
Volume VI VI Volume The Effect Effect of of Environmental Environmental Factors Factors on on the the Physiology Physiology of of Fish Fish The F. E.]. Fry F . E. J. F r y Biochemical Environment Biochemical Adaptation Adaptation to to the the Environment
P. P . W. W. Hochachka and G. G . N. N. Somero Freezing Resistance in Freezing Resistance in Fishes Fishes
Arthur L. L. DeVries Learning Learning and and Memory Memory
Henry Henry Gleitman Gleitrnun and Paul Rozin Rozin The The Ethological Ethological Analysis Analysis of of Fish Fish Behavior Behavior
Gerard Gerard P. P . Baerends Baerends Biological Biological Rhythms Rhythms
Horst Horst O. 0. Schwassmann Schwassmunn Orientation Orientation and and Fish Fish Migration Migration
Arthur Arthur D. D . Hasler Hasler Special SpecialTechniques Techniques D. D .].J. Randall Randall and and W. W . S. S . Hoar Hoar AUTHOR AUTHORINDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX INDEX
Volume Volume VII VII Form, Form, Function, Function, and and Locomotory Locornotory Habits Habits in in Fish Fish
C. C.C. C.Lindsey Lindsey
CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES CONTENTS
Swimming Capacity W.. H. H. Beamish Beamish FF.. W Hydrodynamics: Nonscombroid Nonscombroid Fish Fish Hydrodynamics: W.. Webb Paul W Hydromechanics, Morphology, Locomotion by Scombrid Fishes: Hydromechanics, and Behavior Behavior and John]. Magnuson John J . Magnuson Especially Skipjack Body Temperature Relations of Tunas, Especially William H H.. Neil1 Neill EE.. Don Stevens and William
Locomotor Muscle Quentin Bone The Respiratory and Circulatory Systems Systems during Exercise David R. Jones Jones and David J. J . Randall Metabolism Metabolism in in Fish during Exercise
William R. Driedzic and P. W W.. Hochachka William AUTHOR AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX INDEX
Volume VIII VIII Nutrition Nutrition
C. C . B. B. Cowey and]. and]. R. Sargent Feeding Feeding Strategy Strategy
Kim D. D. Hyatt The The Brain Brain and and Feeding Feeding Behavior Behavior
Richard E. Peter Digestion Digestion
Ragnar Fiinge Fange and David Grove Grove Metabolism Metabolism and and Energy Energy Conversion Conversion during during Early Early Development Development
Charles Charles Terner Terner Physiological Physiological Energetics Energetics
]. J . R. R . Brett Brett and and T. T . D. D. D. D. Groves Groves Cytogenetics Cytogenetics
]. J . R. R. Gold Gold Population Population Genetics Genetics
Fred W .Allendorf Allendorf and and Fred Fred M. M . Utter Utter Fred W.
xvii xvii
xviii xviii
CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES CONTENTS
Enhancement of of Growth Hormonal Enhancement M.. Donaldson, Ulf Ulf H H.. M M.. Fagerlund, David David A. Higgs, Edward M J. R. McBride and J.
Environmental Factors and Growth R.. Brett J}.. R Growth Rates and Models
W. E. Ricker W. AUTHOR INDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SUBJECT INDEX AUTHOR
Volume IXA Reproduction Reproduction in Cyclostome Fishes and Its Regulation
Gorbman Aubrey Gorbmun
Reproduction in Cartilaginous Fishes (Chondrichthyes) (Chondrichthyes) M.. Dodd }. J,M Reproduction The Brain and Neurohormones Neurohormones in Teleost Reproduction Richard EE.. Peter The Cellular Origin of of Pituitary Gonadotropins in Teleosts P. G. W. J. }. van Oordt and J. J. Peute G. W. Teleost Gonadotropins: Isolation, Biochemistry, and Function
T. Bun N Ng g David R. Idler ldler and T. The Functional Functional Morphology of of Teleost Gonads
Yoshitaka Nagahama Nagahntna The Gonadal Steroids
A. Fostier, B. B.Jalabert, Jalabert, R. Billard, B. B . Breton, and Y. Y. Zohar
Differentiation in Teleost Fishes Yolk Formation and Differentiation T. T . Bun Ng N g and David R. Idler ldler .'An An Introduction to Gonadotropin Receptor Studies in Fish
Glen Van Van Der Kraak AUTHOR INDEX-SUBJECT AUTHORINDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECTINDEX INDEX
Volume Volume IXB IXB Hormones, Pheromones, and Reproductive Behavior in Fish
N . R. R . Liley and N. N . E. E . Stacey N.
CONTENTS CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES
xix xix
Environinenta~Influences on Gonadal Activity in Fish Environmental T . J . Lam T.}. Hormonal Control of Oocyte Final Maturation in Fishes Maturation and Ovulation in
Frederick W. Goetz Frederick Sex Control and Sex Reversal in Fish under Natural Natural Conditions
S . T. T . H. H . Chan Chan and W. S. S. B. B . Yeung S. Hormonal Horinonal Sex Control and Its Application Application to Fish Culture
M . Donaldson George A. Hunter and Edward M. Fish Gamete Preservation and Spermatozoan Spermatozoan Physiology
Joachim Joachiin Stoss Stoss Induced Final Maturation, Ovulation, and Spermiation in Cultured Fish
Edward M. Donaldson and George A. Hunter Chromosome Set Manipulation Manipulation and Sex Control in Fish
Gary G a y H. H . Thorgaard AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECT INDEX-SYSTEMATIC INDEX-SUBJECTINDEX INDEX
Volume XA XA General Anatomy of of the Gills
George Hughes Gill Internal Morphology
Pierre Laurent Innervation and Pharmacology of the Gills
Stefan Nilsson Model Analysis of Gas Transfer in Fish Gills
Johannes Piiper and Peter Scheid Oxygen and Carbon Dioxide Transfer across Fish Gills
David Datiid Randall and Charles Ch.arle.9Daxboeck Daxboeck
Acid-Base Regulation in Fishes Acid -Base Regulation Norbert Heisler Physicochemical Physicochemical Parameters for Use Use in Fish Respiratory Respiratory Physiology
Robert G. G . Boutilier, Boutilier, Thomas A. A. Heming, and George K. K . lwama lwaina Robert AUTHOR AUTHORINDEX-SYSTEMATIC INDEX-SYSTEMATICINDEX-SUBJECT INDEX-SUBJECTINDEX INDEX
xx xx
CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES CONTENTS
Volume XB Volume
Permeation Water and Nonelectrolyte Permeation Isaia Jacques Zsaia Teleosts : The Roles of of Respiratory Branchial Ion Movements in Teleosts: and Chloride Cells and N N.. Mayer-Gostan P. Payan, JJ.. P. Girard, and ATPases Ion Transport and Gill ATPases Guy de Renzis and and Michel Bornancin
in Fish Gills Transepithelial Potentials in W.. T. T. W W.. Potts W of Chloride C hloride The Chloride Cell: The Active Transport of and the Paracellular Pathways J. A. A. Zadunaisky
Hormonal Control of o f Water Movement across the Gills J. J . C. Rankin and Liana Bolis Metabolism of o f the Fish Gill
Thomas P. Mommsen
The Roles ooff Gill Permeability and Transport Mechanisms in Euryhalinity David H. Etians Evans David H. The Pseudobranch: Pseudobranch: Morphology and Function
Pierre Laurent and Suzanne Dunel-Erb Perfusion Methods for the Study of of Gill Physiology
S. FF.. Perry, P. SS.. Davie, C C.. Daxboeck, A. A. G. G. Ellis, S. and D. G. G. Smith AUTHOR INDEX-SUBJECT INDEX AUTHORINDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX
Volume Volume XIB
The Maternal-Embryonic Maternal-Embryonic Relationship in Viviparous Fishes
Wourms, Bryon D. Grove, Grooe, and Julian Lombardi John P. Wourms, First Metamorphosis Metamorphosis
John H. H . Youson Factors Controlling Meristic Variation
C. C.C. C. Lindsey
CONTENTS CONTENTS OF OF OTHER OTHER VOLUMES VOLUMES
The Physiology of Smolting Smoking Salmonids
W. SS.. Hoar Ontogeny of Behavior and Concurrent Developmental Changes in Sensory Systems in Teleost Fishes
L. G. G. Noakes and ]ean-Guy Jean-Guy ]. J . Godin David L. AUTHORINDEX-SYSTEMATIC INDEX-SYSTEMATIC INDEX-SUBJECT INDEX INDEX AUTHOR INDEX-SUBJECT
xxi xxi
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1 PATTERN AND VARIETY IN DEVELOPMENT ]. J.
H. S. BLAXTER H.
Dunstaffnage Marine Marine Research Research Laboratory Laboratory Dunstaffnage Oban, Argyll Argyll PA34 PA34 4AD, 4AD, Scotland Scotland Oban, I. Introduction Introduction I. II. Rearing Rearing Techniques Techniques 11. III. Progress and Diversity of of Development 111.
IV. Terminology Terminology of of Early Early Life Life History History Stages Stages IV. V. Egg Egg Size and and Egg Quality A. Egg Egg Size A. Size B. Egg Quality Quality B. Egg The Effect Effect of of Starvation Starvation VI. The VII. The Effect VII. The Effect of of Captivity Captivity VIII. VIII. The The Effect Effect of of Fixation Fixation on on Shrinkage Shrinkage IX. Rate Rate of of Development Development X. Organ X. Organ Systems Systems A. A. Alimentary Alimentary System System B. B. Respiratory Respiratory System System C. C. Locomotor Locomotor System System D. D. Sense Sense Organs Organs Xl. Structure XI. Structure and and Function Function XII. Critical XII. Critical Periods Periods XIII. XIII. Conclusions Conclusions References References
I. I. INTRODUCTION INTRODUCTION
Present Present interest interest in in the the development development of of fish fish has has aa twofold twofold basis. basis. First, First, the the factors factors that that determine determine brood brood strength strength and and so so recruitment recruitment to to commercial commercial fisheries fisheries may may well well operate operate during during the the early early life life history history stages. stages. Second, Second, the the practice practice of of aquaculture aquaculture and and the the range range of of species species used used are are expanding expanding rapidly. rapidly. Improvements Improvements in in hatchery hatchery techniques techniques during two decades decades have have made made it it possible possible to to rear rear almost almost any any during the the last last two species, even the halibut Hippoglossus hippoglossus (V. 0iestad, species, even the halibut Hippoglossus hippoglossus (V. Oiestad, per personal sonal communication) communication) if if fertile fertile eggs eggs are are available. available. Manipulation Manipulation of of 1 FISH FISH PHYSIOLOGY. VOL. VOL. XIA XIA
PHYSIOLOGY,
1 CopyrightIt)0 1988 1988by by Academic Academic Press, Press, Inc. Inc. Copyright All All rights rights of of reproduction reproductionin in any any form form reserved. reserved.
2
J. S. BLAXTER J. H. S.
spawning time has also meant that a steady supply of of larvae-for larvae-for example, of turbot Scophthalmus Scophthalmus maximus and northern anchovy En Engraulis m ordax can be maintained throughout the year, even with mordar-can temperate species. species. Experimental material is thus readily available for the study of optimum rearing conditions, nutrition and growth, critical periods in development, toxicity testing, and the like, all germane to the assessments of the fishery biologist and the practices of the fish farmer. This material is also available to experimental embryologists farmer. and to physiologists interested in the ontogeny of organ systems, or to behaviorists interested in the ontogeny of behavior. Great insight into function is possible in larval stages that lack certain organs or have them only partially developed. In many species, it is the transparency of the larvae, their lack of hemoglobin, their simple intestinal tracts, undifferentiated skeleton, and incompletely developed nervous sys system and sense organs that can make them especially useful as experi experimental animals animals.. fish The progress of work oon n the developmental biology ooff fi s h since 1970s can be traced in a series of symposia and other meet meetthe early 1970s ings. International Early Life History Symposia were held in 1973, 1973, 1979, and 1984 1984 in Scotland, the United States, and Canada (Blaxter, (Blaxter, 1979, 1974; 1 ; Marliave, 1974; Lasker and Sherman, 198 1981; Marliave, 1985). 1985). A symposium on the "The “The Ontogeny and Systematics of Fishes," Fishes,” dedicated to the 1983 (Moser (Moser et memory of E. E. H. H. Ahlstrom, Ahlstrom, was held in California in 1983 al., 1984), he Ameri 1984),and larval fish conferences are held annually by tthe Ameri(e.g., Hubbs, 1986). 1986). can Fisheries Society (e.g., of “Fish Physiology,” a brief brief By way of introduction to this volume of "Fish Physiology," account will first be given of recent advances in techniques of rearing, variageneral life history stages, and terminology. It is intended that varia tion of experimental material and the sources of variation will be an paunderlying but continuing theme. Thus diversity of development, pa rental effects on the young, and the effects of captivity, starvation, and fixation will be discussed. FinaIly, Finally, the development of structure will be related to the development of function with particular reference to possible critical periods in ontogeny. ontogeny. -
11. II. REARING TECHNIQUES Over the past few years improvements in techniques for rearing fish marine fi sh have increased the number of species available for experiexperi ment and for aquaculture. These techniques are summarized by
11..
PATTERN PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT
3
Kinne ((1977), (1984). The greatest advance 1977), Blaxter ((1981), 1981), and Hunter (1984). of the rotifer has been the use of very small food items, especially of G ymnodinium Brachionus plicatilis, but also the naked dinoflagellate Gymnodinium splendens and other organisms such as Mytilus trochophores and sieved natural zooplankton, as a food source in the very young stages 34). Interest is now when the size size of the mouth is limiting ((see see page 34). “green-water” culture, where the larvae are increasing in the use of "green-water" of algae such as Chlorella, Chlorella, which maintained in fairly high densities of may damp out metabolite fluctuations, perhaps improve oxygenation, and provide a secondary food source for the larvae (Houde, 1977; 1977; Morita, 1985). 1985). In the future the use of compounded diets, especially in the form of microcapsules small enough to be eaten whole, may pro provide a further breakthrough. Appelbaum ((1985) 1985) reared Dover sole Solea solea entirely on compounded diets and cited other similar suc successful work on plaice Pleuronectes platessa, vendace Coregonus al alhula, labrax, turbot, catfish Clarias ga gabula, sea bass Dicentrarchus lahrax, riepinus, and the Atlantic silverside Menidia menidia. The best survival rate of sole on an artificial diet was obtained when live brine shrimp Anemia Artemia nauplii were provided provided for the first 10 10 days of of feeding. feeding. Artemia is certainly still the staple live food, food, both in experimental and Anemia (Sorgeloos, 1980). 1980). It has become increasingly ob obapplied fish culture (Sorgeloos, vious that Anemia Artemia from different sources can vary in quality-for quality-for example, in fatty acid "profi le"-and success and failure in the past example, “profile”-and unappreciated factor (van (van Ballaer et may have hinged on this hitherto unappreciated Kanazawa, 1985). 1985; Kanazawa, 1985). Dabrowski ((1984) 1 984) reviewed work on some al., 1985; of of the nutritional aspects of rearing fish larvae and the relevance of digestive processes. Other factors in rearing, such as optimum food density, stocking density, type of tank, light, and other environmental conditions, have now been established. Production of eggs out of season by the use of of artificial photoperiods, photoperiods, temperature, and hormone injections have also greatly improved the availability oflarvae (see Lam, 1982). of larvae year-round (see 1982). One of the most striking advances has been the improvement in survival and growth when larvae are reared in the absence of preda predators in large-scale facilities, or "mesocosms," “mesocosms,” in the form of of large on onshore tanks, large plastic-walled cylinders sited in sheltered coastal orjmpounded (Kvenseth and 0iestad, Oiestad, waters, or .impounded coastal bays or lagoons lagoons (Kvenseth 1984; 0iestad 1984; Oiestad et al., 1985; 1985; Gamble et al., 1985; 1985; Morita, 1985; 1985; Paulsen et al., al., 1985; ai., 1985). 1985; Sturmer Stunner et al., 1985). Atlantic cod Gadus morhua, turbot, and red drum Sciaenops ocellatus have been reared with unprece unprecedented success.
4
J. J. H. H. S. S. BLAXTER BLAXTER
PROGRESS AND D DIVERSITY III. PROGRESS IVERSITY OF DEVELOPMENT 111. Some Some comprehensive keys have recently appeared in the literature that give a good insight into the variety of eggs and larvae and their development. 1976) describes the eggs and planktonic stages development. Russell ((1976) (1983)the ichthyoplankton of the west westof British marine fishes, Fahay (1983) ern North Atlantic, and Auer ((1982) 1982) that of the Lake Michigan region of the Great Lakes Basin. Basin. The early life history stages of fish and their characteristics are discussed more generally by Blaxter (1969), (1969), Hem Hemcharacteristics pel ((1979), 1979), and Kendall et al. al. (1984). (1984). Great variety exists from species to species and, in particular, the size and extent of differentiation signifiwhen the young fish first becomes free-living is of considerable signifi cance for its chance of survival. final fish.such During the fi nal ovarian maturation of the eggs of marine fish. such MeZanoas the Atlantic cod, whiting Merlangius merlangus, haddock Melano grammus aeglefinus, aeglefinus, and plaice, there is a massive uptake of water and concomitant reduction in protein phosphate (Craik (Craik and Harvey, 1984a).This influx influx of water, such that the water content may reach as 1984a). high as 92% of the egg weight, weight, is an adaptation to pelagic life because the egg fluids are hypotonic and make the eggs buoyant. Freshwater fish with demersal eggs, like the rainbow trout Salmo gairdneri, po fish polauarcticus, and pike Esox lucius, do not show these wan Coregonus lavarcticus, changes. changes. initiaI buoyancy of pelagic fi fish eggs, like those of of the flounder sh eggs, The initial P. P . jlesus, depends on the salinity in which the female is kept before (Solemdal, 1973). 1973). Females from low salinities tend to pro prospawning (Solemdal, duce eggs that are neutrally buoyant at lower salinities, buoyancy, of osmocourse, not only being affected by water content but also by the osmo fluid. larity of the egg fluid. There is great variety in the reproductive styles of fish (Table I). I). In most species the eggs develop independently, but there are many (Breder and Rosen, 1966). In littoral species instances of parental care (Breder Rosen, 1966). gobies, this takes the form of guarding such as cottids, blennies, and gobies, the eggs, eggs, but nests may be built with one or other parent guarding and is found often ventilating the nest. Mouth brooding of eggs and larvae is in cichlids such as tilapia and in ariid catfish. catfish. Other species have evolved ovoviviparity or viviparity, the former where the eggs de develop within the female, female, the latter where nourishment is provided via “placental” structures (trophotaenia) (trophotaenia) within the female (see (see Wourms "placental" XIB). Recently Ridley (1978) (1978) and Blumer (1979) (1979) and Grove, volume XIB). significance. While summarized parental care and its evolutionary significance. (in 61 families), care by the care is more common by the male parent (in 61 families),
1. PATTERN AND VARIETY IN DEVELOPMENT 1. DEVELOPMENT
5
41 female occurs in 4 1 families. Care by the male is clearly linked to the prevalence of external fertilization in fish and generally to polygi\my polygamy and male territoriality. The morphological characteristics of of fish eggs are described by (1980), and Matarese and Russell ((1976), 1976), Ahlstrom and Moser (1980), Sandknop ((1984). 1984). Typically marine eggs are single, buoyant, and with of about 11mm (although the range is from 0.6 to 4.0 a modal diameter of mm) sh lay demersal eggs with a modal diameter mm).. Most freshwater fi fish somewhat greater than 11 mm. mm. The eggs may merely rest on the subTable Table II Classifi cation of Classification of Reproductive Styles· Styles" A.
Nonguarders N on guarders 1. Open 1. Open and and substratum substratum spawners spawners a. Pelagic spawners b. Rock and gravel spawners with pelagic larvae c. Rock and gravel spawners with benthic larvae c. d. Nonobligatory plant spawners d. e. e. Obligatory plant spawners f. Sand spawners g. g. Terrestrial spawners, in damp conditions 2. Brood hiders 2. a. b. c. d. d. e. e.
Beach spawners; above waterline at high tides Annual spawners; eggs estivate Rock and gravel gravel spawners spawners Cave Cave spawners spawners Spawners in live invertebrates
B. B. Guarders 1. Substratum spawners 1. a. a. Pelagic spawners; at surface of hypoxic waters Above-water spawners; male splashes clutch b. Above-water b. c. Rock spawners d. Plant spawners 2. Nest spawners 2. a. a. Froth Froth nesters nesters b. b. Miscellaneous substratum and materials nesters c. Rock and gravel nesters d. Glue-making Glue-making nesters e. e. Plant material nesters f. Sand nesters g. Hole nesters g. of host h. Anemone nesters; at base of
(continued) (continued)
S. BLAXTER J. H. S.
6 Table Table 11 (Continued) (Continued)
C. C. Bearers 1. 1. External bearers a. Transfer brooders; brooders; eggs carried before deposition b. Auxiliary brooders; adhesive eggs carried on skin under fins etc. c. Mouth brooders d. Gill-chamber Gill-chamber brooders e. Pouch brooders e. 2.
a
Internal bearers a. Facultative internal bearers; occasional internal fertilization of normally oviparous fish, fish, eggs rarely retained long b. Obligate lecithotrophic lecithotrophic live bearers; no maternal-embryonic maternal-embryonic nutrient transfer c. M atrotrophous oophages and adelphophages or a few eggs develop developMatrotrophous adelphophages;; one or ing at expense of other eggs or embryos d. Viviparous trophoderms; trophoderms; nutrition partially or entirely from female via "placental" “placental” structures structures
Adapted from Balon (1981a). (1981a).
stratum, or have some means of attachment such as adhesive threads or a supporting pedestal. In species such as salmonids salmonids the eggs are buried in the gravel, and the grunion Leuresthes tenuis lays its eggs intertidally in the sand. Other types of of demersal eggs are found in some littoral marine species and, in the more offshore Atlantic her herring, capelin Mallotus villosus and Pacific cod Gadus macrocephalus. While While teleosts teleosts usually usually have have round round eggs, eggs, most most engraulids engraulids have have eggs eggs that are ellipsoidal, thought to be an adaptation to reduce cannibalism by the filter-feeding parents after spawning. spawning. Other families like the gobies gobies have have slightly slightly flattened flattened eggs, and and demersal eggs eggs are are sometimes sometimes irregular in shape. Oviparous elasmobranchs have eggs of unusual shapes (the "mermaid's purse") with tendrils for attachment. Tendrils “mermaid’s purse”) are also found in the silverside Atherinopsis and the gar Relone, Belone, while the flying fish Oxyporhamphus has spines (Boehlert, (Boehlert, 1984). 1984). It is easy speto understand the adaptive value of tendrils in distantly related spe cies, but it is much more difficult to explain the ornamentations of of the chorion. Most teleosts have a smooth surface to the chorion, but the chorion. unrelated inshore dragonet Callionymus and bathypelagic gonostogonosto matid Maurolicus muelleri have chorions with hexagonal facets, and the fl atfish Pleuronichthys coenosus has a chorion with very many flatfish small facets. small The yolk is usually translucent, unpigmented, and homogeneous in texture, but may be segmented in primitive species like the pil-
11.. PATTERN AND VARIETY IIN N
DEVELOPMENT
7
chard Sardina pilchardus and sprat Sprattus sprattus. sprattus. In some soleids the segmentation is confined to the periphery of the yolk, and in other species like the jack mackerel Trachurus symmetricus segmentation appears progressively during early development. pe development. Most commonly, pelagic fi sh eggs have a single oil globule in the yolk. yolk. Of a total of 515 fish species checked by Ahlstrom and Moser ((1980), 1980), 60% 60% had one oil glob glob15% had multiple oil globules. The ule, 25% had no oil globule, and 15% oil globule, when single, usually lies at the vegetal pole in Marine fi sh larvae. It is generally thought that the oil globules are a spespe fish cialized form of nourishment and have a minimal effect on buoy buoyancy. Following activation or fertilization the egg absorbs water, the perivitelline space forms, forms, and the chorion hardens. The perivitelline space is usually narrow but is wide in some "primitive" “primitive” species such as the pilchard, in some eels, eels, and in unrelated species like the striped bass Morone labrax and long rough dab Hippoglossoides platessoides. Cleavage is meroblastic in hagfish, hagfish, elasmobranchs, elasmobranchs, and teleosts, al although in the lampreys it is holoblastic but with the formation of of micro- and macromeres. n Amia, gar macromeres. In primitive groups like the bowfi bowfin Lepisosteus, and sturgeon Acipenser, cleavage is is intermediate or semiholoblastic. semiholoblastic. The embryo develops as a blastodisc at the animal pole. The periphery of the blastodisc overgrows the yolk (epiboly), (epiboly), eventually enclosing it to form a gastrula but leaving an opening, the blastopore. The embryonic axis axis forms forms by a process of convergence and concentration in relation to the dorsal lip lip of the blastopore at the neurula stage but the quantity of uences the timing of such of yolk infl influences events. The head and eye eye cups are soon soon identifiable and the trunk lengthens and separates from the yolk sac. sac. The heart functions well before hatching, and in some demersal eggs a vitelline circulation can sac. Examples of the development of a marine be seen within the yolk sac. (dab)and a freshwater egg (rainbow (rainbow trout) are given in Figs. 11 and egg (dab) 2. 2. Before hatching, the embryo becomes very active and the chorion is ssoftened is oftened as a result of enzymes secreted by hatching glands. The larva depends very degree of differentiation of the newly hatched larva much on the species and egg size, size, and the incubation period depends factors and on temperature (see (see Fig. 18). 18). In many many marine on these factors species the mouth and jaws are are not formed, the eye eye is is not pelagic species pigmented, the yolk yolk sac sac is is huge, and a primordial finfold finfold runs around pigmented, position. Apart Apart from a few melanophores, the the trunk in the median position. larva is is very transparent. All newly hatched larvae larvae have free neuro neurolarva masts on the head and trunk, and otoliths are present in the otic capcapmasts
8
J. J. H. H. S. BLAXTER BLAXTER
Fig. 1. 1. Development of of the dab Limanda Limanda limanda, using Apstein’s Fig. Apstein's stages. stages. [From of Academic Press.) Press.] Russell (1976), with permission of
sule. sule. Other marine species hatch with the alimentary system nearly Some larvae are very advanced, functional and with pigmented eyes. Some and in loricariids the dorsal and caudal fin are partly developed at hatching (Fuiman, 1984); in flying fish, flexion of the notochord (Fuiman, 1984); (which (which precedes caudal fin formation) actually occurs before hatching. In the salmonids-for (Fig. 2)-although salmonids-for example, rainbow trout (Fig. 2)-although the yolk sac is still large, the larva (alevin) (alevin) is better developed and espe especially the vascular system and vitelline circulation are conspicuous with the blood containing hemoglobin. The young of cichlid and ariid also further developed and adapted to early life mouth brooders are also within the parental mouth. In ovoviviparous ovoviviparous and viviparous species
11.. PATTERN
AND VARIETY IN DEVELOPMENT
9
� :. ,
0
...,
'
-"
.
�
•
Fig. 2. Development of the rainbow trout Salmo Salmo gairdneri, gairdneri. (A) (A) 8-Blastomeres. (B) (B) one-third epiboly. (C) (C) 0-5 0-5 Somites, one-half one-half epiboly. (D) (D) Otic Early embryo apparent, one-third 10-20 somites, total somites placodes, three-fourths three-fourths epiboly. placodes, epiboly. (E) (E) Caudal bud with 10-20 somites 5158, heart beating. (F) (F) Posterior Posterior cardinal veins formed, choroid of of eye pigmented. (G) (G) Near hatching, pelvic fins fins develop. (H) (H) Hatched alevin, first anal and dorsal fin rays. (1.1) (1,J) Later alevin stages as yolk yolk is resorbed. Scale bars 2 mm long. [Redrawn from Vernier (1969).] (1969).]
(Amoroso, (Amoroso, 1960) 1960) the the young young may may hatch hatch effectively effectively as as postmetamorphic postmetamorphic juveniles. juveniles. mackerel, Examples of early life history stages, those of the jack mackerel, northern Figs. 3 and northern anchovy, anchovy, and and Pacific Pacific hake, hake, are are illustrated illustrated in in Figs. and 4. 4. The The changing changing shape shape of the the larvae larvae is is clearly clearly shown, shown, with with the the imporimpor-
J. H. S. BLAXTER J. H. S.
10 10
EGGS
YOLK SAC
PRE FLEXION
]
]
FLEXION
POST FLEXION
JUVENILE
Fig. 3. 3. Early Early life history history stages stages of the thejack mackerel mackerel Trachurus symmetricus. symmetricus. [From [From Fig. the original original drawings drawings of Ahlstrom Ahlstrom and and Ball Ball in Kendall Kendall et al. al. ((1984), with permission of 1984), with the American Society Society of Ichthyologists Ichthyologists and and Herpetologists.] Herpetologists.] the
11.. PATTERN AND
VARIETY IN DEVELOPMENT
1 1 11
F
Fig. 4. Northern anchovy Engraulis 4. Development of teleost larvae. larvae. (A-E) (A-E) Northern Engruulis mordax, mordux, 2.5,7.5,11.5,18.4,31.0 [Redrawn from Kramer and Ahlstrom ((1968).] (F-H) Pacifi Pacific 2.5, 7.5, 1 1.5, 18.4, 31.0 mm. mm. [Redrawn 1968).] (F-H) c 4.3, 7.7, 7.7, 111.0 1.0 mm. hake Merluccius productus, 4.3, mm. [Redrawn [Redrawn from Ahlstrom and Counts ((1955).] 1955).]
tance of exion of of fl flexion of the notochord notochord and development of of the caudal fin being emphasized, with implications for improved swimming. The duration of of the yolk-sac yolk-sac period depends on both species and temperature but als o on egg size (see also (see also p. p. 17). 17). The argentine Argentina silus and the halibut have egg diameters of of 3.0-3.5 3.0-3.5 mm. Unexpectedly, the newly hatched larvae are very undeveloped but the halibut takes 50 days to resorb its yolk (at 5.3°C) 5.3"C) and reaches a length of 1. 5 mm (Blaxter et ai., 983a) and the argentine reaches a of 111.5 al., 11983a) prodigious length of 17 mm on of 17 on its yolk supply (Russell, (Russell, 1976). 1976). During the yolk-sac period the mouth and gut and the eyes be period become functional to allow the larva to switch from endogenous to exo exogenous nutrition. The subsequent larval period ranges from a few days to some months (and even 2-3 2-3 years in eels), eels), depending on tempera-
12 12
H. S. S. BLAXTER BLAXTER J. H.
ture and species. species. During During this this time time the the larva is likely likely at at least least to to double double ture and larva is its length and to increase its weight by 10 to 100 times. Transient its length and to increase its weight by 10 to 100 times. Transient characters, such such as as spines, spines, may may appear appear (Fig. (Fig. 5), 5), which are presumably presumably characters, which are antipredator Other bizarre bizarre structures, structures, such as eyestalks, eyestalks, antipredator adaptations. adaptations. Other such as elongated elongated fin fin rays, rays, or or tentacles, tentacles, may may also also appear, appear, often often as as larval larval charac characters ters (Fig. (Fig. 5), 5), to to be be lost lost later later in in development. development. The The cobitid Misgurnus fossilis laments for time (Fuiman, fossilis even even has has external external gill gill fi filaments for aa time (Fuiman, 1984). 1984). The The importance importance of of allometric allometric growth growth during during larval larval development development has 1983) and and Fuiman Fuiman has been been emphasized emphasized by by Fuiman Fuiman ((1983) and Strauss Straws and ((1985). 1985). In some some species relative relative growth growth intensity intensity follows follows aa U-shaped U-shaped gradient gradient along along the the body body with with fastest fastest growth growth in in the the caudal caudal region, region,
f
Fig. 5. 5. Teleost Teleost larvae larvae showing showing spines spines and and other other processes. processes. (A) (A) Holocentrus vexilla uexillaFig. rius 5.0 5.0 mm. mm. (B) (B) Sebastes macdonaldi tnacdonaldi 9.0 9.0 mm. mm. (C) (C)Lophius piscatorius piscatorius 26 26 mm. mm. (D) (D) rius Acanthurid 77 mm. mm. (E) (E)Ranzania Ranzania laevis laeois 2.8 2.8mm. mm. (F) (F)Myctophum aurolaternatum 26 26 mm. Acanthurid (G)Campus Carupus aC1l8 ucus 3.8 3.8 mm. mm. (H) (H) Trachipterus Trachipterus sp. sp. 7.6 7.6 mm. (l) (I) Zu cristatu8 cristatus 6.5 6.5 mm. mm. [Re [Re(G) drawn drawn from from Moser Moser (1981).] (1981).]
11. . PATTERN PATTERN AND AND VARIETY VARIETY IN IN
DEVELOPMENT DEVELOPMENT
13 13
of the body. Growth is also linked to an increase in the propulsive area of of feeding and respiratory fast in the head region, where elaboration of functions may be taking place. IIn n sculpins, however, the caudal rere gion grows grows more slowly than the rest of of the body and growth is fastest in the head region. of adult characters (such (such as fi fin Progressive differentiation of n rays and skeleton) occurs. The larvae eventually pass through a process of metamorphosis to the juvenile stage. stage. This process may be rather be prolonged. Typically the blood becomes pig pigabrupt or it may he mented, scales and pigment appear on the body surface, the meristic characters such as fin rays are complete, and the body shape becomes like the adult. The juvenile appears as a small adult. In fl atfish, meta flatfish, metamorphosis is a remarkable process as the fish starts to change from the bilaterally symmetrical larva to an asymmetrical juvenile lying on on one (abocular or blind) side side (Fig. 6). Changes take place to the skull and (Fig. 6). sense organs and, in particular, particular, the eye of the abocular side migrates across the top of of the skull. By way of of summary, summary, Table II I1 lists some of of the characteristics of of a few species to emphasize the diversity that is is to be found. The young of many elasmobranchs, with a long incubation period, effectively hatch as juveniles, albeit with a yolk sac. sac. In species with parental care the early larvae may also be advanced or "precocial." “precocial.” This variety makes it difficult to categorize early life histories in a neat and con convincing way.
c
Fig. 6. platessa. (A) 6. Development Development and and metamorphosis metamorphosis in in the the plaice plaice Pleuronectes Pleuronectes platessa. (A) Yolk-sac 6.6 mm. 7.3 mm. (C-F) Stages morpho Yolk-sac larva, larva, 6.6 mm. (B) Larva Larva at at first first feeding, feeding, 7.3 mm. (C-F) Stages of of meta metamorphosis with scale bar 2 mm. mm. [Redrawn [Redrawn from from Ryland Ryland (1966).] (1966).1 sis with eye eye migration; migration; scale bar 2
Table II Early Life History Characteristics·
Species
Common name
Egg diameter (mm)
Hatching length (mm)
Hatch
First feed
Metamorphosis
Temperature range (0C)
3-4 6-7 3-4 2-3 2-3
1.4-2.9 2.5 0.8-1.5 0.6-0.9 0.4-0.5
1.9-3.6 4.0 1.3-2.0 1 .1-l.7 0.9- 1 . 1
4.2-5.0 10-12 1 1-13 5-6 1 1-15
4-12 7-11 9-15 13-18 13-15
5-8 6-7 1.7
1.0-3.0 2.0-3.0 0.15
2.0-4.5 9.0-10.0 0.7
12�24 ?15-16 ?
6-14 4-7 26
4 4-5 15-25 100 240-310
0.6-0.7 1.5-2.0 20-22 24-32 -104
1.7-2.6 2.0-2.4 26-28 28-36 -104
? Not clearcut Not clearcut 28-36 < 104
28 20-25 1-7 4-12 4-12
Gadus morhua Pleuronectes platessa Scomber scombrus Scophthalmus maximus Engraulis mordax
Cod Plaice Mackerel Turbot Northern anchovy
Clupea harengus Hippoglossus hippoglossus Acanthurus triostegus Oreochromis (=Tilapia) mossambicus Oryzias latipes Salmo salar Scyliorhinus caniculus Squalus acanthias
Herring Halibut Convict surgeonfish
1.1-1.9 1 .7-2.2 1.0-1.4 0.9-1.2 0.6-0.7 x 1.3-1.4b 0.9-1.7 3.0-3.2 0.7
Tilapia Medaka Salmon Spotted dogfish Spur dogfish
1.7-2.2 1.0-1.3 5-6 65 (long) 24-32
Weeks from fertilization to
Data from Blaxter (1969), Blaxter et al. (1983a), Howell (1979), J. R. Hunter and C. Kimbrell (personal communication), Iversen and Danielssen ( 1 984), Jones ( 1972), Kuhlmann et al. (1981), Rana (1985), and Russell ( 1976). b Eggs are ellipsoidal, minor and major axes given. •
11.. PATTERN PATTERN AND AND VARIETY VARIETY IIN N DEVELOPMENT DEVELOPMENT
15 15
IV. TERMINOLOGY TERMINOLOGY OF EARLY LIFE HISTORY STAGES A terminology is important both for understanding the literature and for brevity in describing development. A good terminology should so easily should be be as as simple simple as as possible possible (and (and so easily remembered) remembered) and and linked to both form and function. The production of of a generally ac accepted is aa current cepted terminology terminology is current issue issue in in ichthyology, ichthyology, and and some some of of the the varied attempts attempts to to produce produce standardization standardization are are shown in in Fig. 7. 7. The problems problems are are discussed discussed by Snyder Snyder and and Holt Holt (1983). (1983). The The diffi difficulties lie in producing a terminology that embraces all species and all patterns fish, and patterns of of development development in in fish, and it it almost becomes an an intellectual intellectual challenge challenge to to achieve this. this. Some Some workers favor favor aa large large number of of stages, stages, others very very few; few; one point point of view suggests suggests terminology terminology based based on size alone, on size alone, another another that that ecological ecological considerations considerations should should be para paramount. Some Some workers (e.g., (e.g., Balon, Balon, 1984; 1984; see see Fig. 22) 22) use the term "embryo" cover the “embryo” to to cover the period period from from fertilization fertilization to to first first feeding feeding and and consider consider hatching to to be be aa relatively relatively insignificant insignificant process. process. While While it it is is certainly certainly true true that that the the change change from from endogenous endogenous to to exogenous exogenous food food supply supply is is aa major major hurdle hurdle for for the the organism organism to to overcome, overcome, it it should should not not be be forgotten forgotten that that eggs eggs cannot cannot avoid avoid predators predators although although hatched hatched larvae larvae can. can. Many Many species species of of fish fish hatch hatch in in aa very very well developed developed state, state, espe especially cially where where ovoviviparity, ovoviviparity, viviparity, viviparity, or or other other parental parental care care is is in involved, volved, or or where where the the incubation incubation period period within within the the egg egg is is long; long; other other species development. It species hatch hatch in in aa much much earlier earlier state state of of development. It is is difficult difficult to to resolve such aa wide resolve aa nomenclature nomenclature to to cover cover such wide variation variation in in ontogeny. ontogeny. The The present present author author prefers prefers to to use use the the term term "embryo" “embryo” only only to to the the point of point of hatching, hatching, does does not not accept accept the the terms terms "prelarva" “prelarva” and and "post “postlarva," larva,” which which suggest suggest stages stages before before and and after after aa larval larval stage, stage, and and uses uses the the term term "larva" “larva” to to cover cover development development from from hatching hatching to to metamorpho metamorphosis and sis and the term term "juvenile" “juvenile” from from metamorphosis metamorphosis to to first first spawning. spawning. Terms such as "fingerling" “fingerling” or "young-of-the-year" “young-of-the-year” are unsatisfactory: the former former can can hardly hardly be applied applied to to very very short short fish fish or or the the latter latter to to species species with with aa short short generation generation time. time. A simplistic simplistic approach approach to to termi terminology may well well require require additional qualification to be given, given, such such as as “ "yolk-sac" yolk-sac” larva, larva, or or it it may may have have to to be be made made clear clear that that some some species species hatch in an an advanced state state of development. development. This is is in broad agree agreement 1984), who also ment with with Kendall Kendall et al. al. ((1984), also favor dividing the larval stage stage into "preflexion," “preflexion,” "flexion," “flexion,” and "postflexion" “postflexion” substages, substages, refer referring ring to to the the turning turning up up of of the the notochord notochord tip tip during during the the first first stages stages of of development of the caudal fin fin (Fig. (Fig. 3). 3). Since flexion flexion is is accompanied by n rays by rather rather rapid rapid development development of of other other characters characters such such as as the the fi fin rays and and change change of of body body shape, shape, as as well well as as aa dramatic dramatic improvement improvement in in
Full finray compleme-nt present,
END POINT POINT EVENTS EVENTS END TERMINOLOGY TERMINOLOGY
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Fig. 7. Terminology of life history stages. [From Kendall et et aZ. al. (1984), (1984), in which see original references; with permission permission of o f the of Ichthyologists and and Herpetologists.] Herpetologists.] American Society of American
11. . PATTERN AND
17 17
VARIETY IN DEVELOPMENT
of the terminol terminollocomotor ability, this seems a valuable elaboration of ogy. ogy. V. EGG SIZE AND EGG QUALITY QUALITY A. Egg Size The influence of initial egg size on subsequent survival and devel development has important ecological implications. implications. Although a large yolk sac may reduce locomotor performance, plentiful yolk is likely to ex extend the period for switching from endogenous to exogenous feeding. Any factor that might provide larger larvae is is likely to reduce the dangers from predation, since large larvae are likely to be able to escape faster and to be less susceptible generally to small predators. Furthermore, any factors factors that might cause early improvements in growth and size are likely to reduce the range of predators more rap rapidly. Interspecifically, egg size and fecundity tend to be inversely re related (see (see Table 11in Blaxter, 1969). 1969). Egg size also influences the rate of of development, those species with large eggs having a longer incuba incubation time. While the small eggs of many fecund marine species hatch within a few hours or days (see Fig. 18), 18),salmonids may take several weeks to hatch, and the very large eggs of elasmobranchs, several months. The ovoviviparous dogfish Squalus acanthias has a gestation period of about 2 years. d survival has The effect of intraspecific egg size on larval size an and been investigated. Egg size does not seem to affect incubation time (at (at any one temperature) either in Atlantic herring (Blaxter (Blaxter and Hempel, 1963), 1963), Atlantic salmon (Kazakov, (Kazakov, 1981), 1981), Arctic charr Salvelinus SalveZinus al aZpinus (Wallace 1984), tilapia Oreochromis ((= Tilapia) (Wallace and Aasjord, 1984), Tilapia) mossambicus (Rana, (Rana, 1985), 1985), or the the orangethroat darter Etheostoma spectabile (Marsh, 1986). However, egg size does influence larval (Marsh, 1986). size, with larger larvae, at first feeding, feeding, being produced from larger eggs. This was shown for Atlantic herring by Blaxter and Hempel ((1963), 1963), for Atlantic salmon by Kazakov ((1981) 1981) and Thorpe et al. al. (1984), (1984), for Chinook salmon Oncorhynchus tshawytscha by Fowler ((1972), 1972), for Arctic charr by Wallace and Aasjord (1984), (1984), for rainbow trout by Gall ((1974) 1974) and Springate and Bromage (1985), 1 985), (1985), for tilapia by Rana ((1985), for dace Leuciscus leuciscus by Mann and Mills ((1985), 1985), and for the orangethroat darter by Marsh (1986). (1986). Furthermore, the larvae from =
18 18
J. H. H. S. BLAXTER
Blaxter and Hempel, larger eggs live longer on their yolk reserves ((Blaxter 1963; 98 1 ; Mann 8B), 1963;Theilacker, Theilacker, 11981; Mann and and Mills, 1985; 1985; Marsh, Marsh, 1986; 1986; Fig. Fig. 8B), although the effect on ultimate survival may not always be established (Fig. 8A). (Fig. 8A). Wallace and Aasjord ((1984) 1984) found the effect of of egg size on the length of 140 days post of Arctic charr alevins was still clearly evident 140 post1985) also found that the greater size of hatching; Springate et al. a2. ((1985) rainbow trout fry from females fed on a high ration could be followed for 2-3 1979) showed that the length of 2-3 months (Fig. (Fig. 9). 9). Glebe et al. ((1979) fry of of Atlantic salmon from four New Brunswick rivers was still related to the original egg diameter some 8 8 months later (Fig. (Fig. 10). 10). K. J. Hana Rana (personal communication) (personal communication) reared tilapia and found that the weight of 20-, fish was significantly related to the original 20-, 40-, and 60-day-old 60-day-old fish egg size, although the degree of association decreased over the 2020-to 60-day period. Thorpe eett al. 1984) and Springate and Bromage ((1983, 1985), al. ((1984) however, working on Atlantic salmon and rainbow trout, respectively, concluded that the benefi ts of large egg size were soon lost during benefits subsequent growth. Intraspecifically, there are also many factors factors that control egg size. size. These are: 11.. Parental size. size. Larger females produce larger eggs in Atlantic salmon Salmo Salmo salar (see review by Thorpe et al., al., 1984). 1984). In larger) addition, females that spend longer at sea (and so are larger) also produce larger eggs ((Kazakov, Kazakov, 1981). 1981).In Atlantic herring, Hempel and Blaxter ((1967) 1967) found that the eggs of rst-time of fi first-time spawners were the smallest but that there was no influence influence of parental size on egg weight in repeat spawners. 2. Spawning group. There is an extensive literature showing how 2. salmonid eggs vary in size from different rivers (Bagenal, 1; (Bagenal, 197 1971; Thorpe et al., 1 979) mea al., 1984). 1984). For example, Glebe et al. ((1979) measured the calorific calorific value of Atlantic salmon eggs from four New Brunswick rivers and found a range from 1067-1576 J/egg. 3. 3. Season. The influence of spawning group is closely linked to different spawning seasons that may occur within a species. This is most dramatically shown in the herring (Fig. (Fig. 1l l1), ) , where different seasons may have egg dry weights varying by a factor of four, with the largest eggs being produced in the winter and spring and smallest in the summer and fall. fall. This general intra intraspecific trend toward smaller eggs as the spring season prog progresses was shown in a wide range of species by Bagenal ( 1971) Bagenal(l971) and for clupeoids by Blaxter and Hunter ((1982) 1982) (Figs. 1 and (Figs. 111 12). 12).
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Fig. 8, 8. (A) (A)Percent Percent survival survival of of Atlantic Atlantic salmon salmon parr parr Salrno Salmo saZar salar from from eggs eggs of of differ differFig, ent diameter. diameter. Each Each point point refers refers to to aa different different female. female.There There isis no no significant significant correlation correlation ent et al. al. (1979).] (1979).1(B) (B)The Thetime time to to50% 50% between egg egg size size and and survival. survival. [Redrawn [Redrawnfrom from Glebe Glebe et between survival from from hatching hatching of of unfed unfed tilapia tilapia Oreochrornis Oreochromis (Tilapia) (Tilapia)mossarnbicus mossambicus fry fry related related survival of the the eggs. eggs. Each Each point point refers refers to to aa different differentclutch clutch of of eggs. eggs. The The to the the mean mean dry dry weight weight of to (r = = 0.923, 0.923, d.f. d.f. == 23, 23, pp < < 0.01). 0.01). [Redrawn [Redrawn from from Rana Rana (1985).] (1985).] regression is is significant significant (r regression
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20
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Fig. n subsequent length ooff fry iin n four New Bruns Fig. 10. 10. The effect of egg diameter oon Brunswick (Eastern (Eastern Canada) Canada) Atlantic salmon Salrno Salmo salar S Q Z U ~ stocks. stocks. The four stocks stocks are shown by different symbols. symbols. Each point refers to a different female. female. [Redrawn [Redrawn from Glebe et et refers to al. (1979).] (1979).]
4. 4. Diet. The way in which the diet of the female affects fecundity and egg size needs clarification. clarification. Much may depend on the phase of the egg maturation cycle during which an experimen experimengiven. Diets may delay or accelerate spawning, so tal diet is given. allowing more or less time for material to be laid down in the comegg, or diets may affect the processes of atresia. The most com mon effect of starvation or overcrowding is to reduce fecun fecundity, and for good feeding or low stocking density, to increase fecundity (Wooton, (Wooton, 1979). 1979). Concomitant changes of of egg size often, but not always, occur; with poor feeding, egg size is sometimes increased (Wooton, (Wooton, 1979) 1979) and sometimes de deai., 1978f and creased, for example, in haddock (see (see Hislop et al., 1978jand ai., 1985). in rainbow trout (Springate et al., 1985). Despite the extensive experimental work on egg size, it is not clear the extent to which changes of fecundity or egg size have adaptive value in the wild and whether egg survival can be enhanced or not.
11..
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Fig. Fig. 12. 12. The seasonal seasonaldecrease decrease in mean mean egg volume volume of (1) (1)Pleuronectes Pleuronectes platessa, platessa, (2) (2) Trlgla Trigla gurnardus, gurnardus, (3) ( 3 )Melanogrammus Melanogrammus aeglejinus, aeglefinus, (4) (4)Gadus Gadus morhua, morhua, (5) (5)Solea Solea solea, solea, (6) (6) Merlangius Merlangius merlangus, merlangus, (7) (7) Sprattus Sprattus sprattus, sprattus, (8) (8) Platichthys Platichthys jlesus, Jesus, (9) (9)Rhinonemus Rhinonemus cimbrlus, cimbrius, and (10) (10)Limanda Limanda limanda. limanda. Square Square symbols symbols show dry weight of herring eggs. [From Bagenal (1971), with with permission permission of The Fisheries Society of the British Isles.] Bagenal (1971),
B. Egg Egg Quality Quality A key key to to early early success success in in rearing rearing is is the the production production of of eggs eggs of of good good quality. broodstock are are maintained maintained in in captivity, captivity, as as is is so often often the the quality. If broodstock case case in in aquaculture, aquaculture, it it is is desirable desirable to to strip strip the the fish fish soon soon after after ovulation, ovulation, unless the holding conditions will allow natural spawning. spawning. It is cer certainly tainly possible possible for for ovulated ovulated eggs eggs to to remain remain in in the the body body cavity cavity of of the the female female for for many many days, days, often often culminating culminating in in their their resorption resorption into into the the maternal 1984b,c) found maternal tissues. tissues. Craik Craik and and Harvey Harvey ((1984b,c) found that that the eggs eggs of of rainbow rainbow trout trout S. S. gairdneri held held for for 30 or or more more days days postovulation postovulation underwent underwent changes changes of of water water content content and and biochemical biochemical components components that that must have reflected a deterioration and decomposition of the yolk. yolk. They also also found that the percentage of eggs that that hatched hatched fell fell sharply sharply when when the the eggs eggs were were held held for for 18 18days days postovulation. postovulation. Springate Springate et al. aZ. (1984) (1984)found found maximum maximum egg egg and and fry fry survival survival if if rainbow rainbow trout trout were were
l. PATTERN 1. PATTERN AND
VARIETY IN DEVELOPMENT
23 23
4-6 days after ovulation. In contrast, Mollah and Tan ((1983) stripped 4-6 1983) found that the eggs of catfish Clarias macrocephalus only remained postovuIation at 26-31°C. viable for up to 10 10 h postovulation 26-31 ac. The need to fertilize particularly important in cases of hand eggs at an optimal time is particularly (Limanda stripping, or if spawning is induced by hormones. Flounder (Limanda yokahamae) yokahamae) eggs remain in good condition for 2-3 2-3 days after ovula ovula(Hirose tion, depending on the hormone used for inducing spawning (Hirose
et al., al., 1979). 1979). The postovulatory decline in egg viability seems much more sig significant than small differences in dry weight, protein, and lipid con content of eggs from different rainbow trout females, which may also be related to hatching success. Possibly a more important biochemical component of salmonid eggs is the carotenoid. 1985) reviewed carotenoid. Craik ((1985) the extensive Japanese and Russian literature on the role of of carot carotenoids in development. They may act as precursors of of vitamin A and play aa part in respiration; they are certainly a source source of pigment for the chromatophores. It is likely that they have to be above a critical con concentration to give high hatch rates. Variation in egg quality may arise in other ways. Billard et al. factors on ((1981) 1981) review the effect of stress and other environmental factors teleost reproduction. Adverse feeding, temperature, light, and water quality, as well as crowding, can cause low fertilization and hatching rates. In the red sea bream Chrysophrys major a low-protein, low-protein, phos phosphorus-deficient diet produced eggs with poor hatchability and de deformed hatched larvae (Watanabe (Watanabe et al., al., 1984). 1984). In other species, such as the rainbow trout, ayu Plecoglossus Plecoglossus altivelis, altiuelis, and carp Cyprinus carpio, deficiencies of fatty acids and vitamin E in the diet of the female may effect egg viability (T. (T. Watanabe, 1985) 1985).. Whipple et al. al. (198 1 ) found that striped bass migrating through polluted water (1981) showed poor condition of the parents, low fecundity, and low egg viability, as well as a high level of lesions and parasites. Poor quality can also be manifested in other forms. In Atlantic cod, poor-quality poor-quality eggs may show irregular shape, abnormal fertilization, soft chorions, or negative buoyancy (Kjf'Srsvik (Kjflrsvikand Lf'Snning, Lflnning, 1983). 1983). In a unique study of a wild stock, Kjf'Srsvik 1984) found that 6-60% Kjflrsvik et al. 640% al. ((1984) of cod eggs had abnormal mitoses or chromosome aberrations. VI. THE EFFECT OF STARVATION
During starvation a number of of morphological and chemical changes occur. There is a progressive collapse of the larval head and trunk in Atlantic herring and plaice (Ehrlich (Ehrlich et al., al., 1976) 1976) and in jack mackerel (Theilacker, (Theilacker, 1978), 1978), so that body weight or head height rela-
24
J. J. H. H. S . BLAXTER
tive to the length can be used as an index of starvation. The condition 3) has been used on sea-caught Atlantic her factor (dry (dry weightJlength weighVlength3) herring larvae (Blaxter, (Blaxter, 1971) 1971) to assess their nutritional condition. A U Ushaped relationship is is found between condition factor and length (see (see Fig. 17) 17) because the larval body is denser when yolk is present and later as ossifi cation occurs. Chenoweth ((1970) 1 970) and Ehrlich et al. ((1976) 1976) ossification therefore used relative condition factor (dry weightJlengthb) weighVlengthb) where b is the slope of the regression line relating weight to length (i.e., (i.e., weight oc a length b), b ) , which prevents the right-hand arm of of the U U appear appearing. Ehrlich 1 976) and 1 985) also Ehrlich et al. al. ((1976) and Y. Y. Watanabe Watanabe ((1985) also found found changes changes in in the height of the gut epithelia and shrinkage of the liver. O'Connell O’Connell ((1976) 1976) used 111 1 histological criteria to assess starvation in northern anchovy larvae and found the appearance of the pancreas, trunk mus muscle and liver cytoplasm gave the most reliable results. Theilacker ((1978) 1978) later used histological characteristics of the pancreas and gut to estimate the extent of of starvation of the larvae of jack mackerel. There were also changes in biochemical components in plaice and herring larvae during starvation (Ehrlich, (Ehrlich, 1974a,b). 1974a,b).Percentage of wa water increased and percentage of triglyceride, carbohydrate, carbohydrate, and carbon decreased. Percentage nitrogen decreased in plaice but not in herring. Recently B uckley ((1981) 1981 ) and Clemmesen ((1987) 1987) have shown that the Buckley RNA/DNA Hounder, her RNNDNA ratio drops dramatically in starving winter flounder, herring, and turbot larvae. The time over which starvation takes place depends on a number of factors. 1980) and McGurk ((1984) 1984) summa factors. Theilacker and Dorsey ((1980) summarized the time to reach the point-of-no-return (PNR; (PNR; Blaxter and Hem Hempel, 1963), 1963),or irreversible starvation, in some 25 marine species if they failed to feed once the endogenous yolk supply had been exhausted. There were very large differences, ranging from 3.5 3.5 days postfertiliza postfertilization in Anchoa mitchilli at 28°C 28°C to 36.5 36.5 days postfertilization for for Pa Pacific herring at 6°C. 6°C. Generally the time to the PNR for fi rst-feeding cific first-feeding larvae increases for species with long incubation periods and at low temperatures (Fig. 18). Once (Fig. 13) 13) and when the eggs are large (page (page 18). feeding is established the time to the PNR increases with age, as shown in Fig. 14, 14, presumably because of greater body reserves. of starvation dur durAlthough the morphological and chemical effects of ing development are now fairly well established, established, it is not certain how much these changes are reHected reflected in locomotor performance and be behavior. Blaxter and Ehrlich ((1974) 1974) found a decrease in sinking rate (or an increase in buoyancy) buoyancy) in plaice and Atlantic herring larvae as a result of of increasing hypotonicity of body Huids fluids and a decrease in body
40
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Fig. 3 . The time from fertilization sh Fig. 113. fertilization to irreversible irreversible starvation (PNR) (PNR) of of unfed fi fish larvae of25 species circles are species at different temperatures temperatures (open (open circles). circles). The black circles are larvae larvae of25 of Pacifi c herring Clupea pallasi. pallasi. [Redrawn [Redrawn from McGurk (1984).] (1984).] Pacific herring Clupea
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Fig. 14. The number of (PNR) of different aged plaice Fig. of days to irreversible starvation starvation (PNR) Pleuronectes platessa and Atlantic herring Clupea harengus harengus larvae. larvae. Temperatures Temperatures 7.57.511.5"C 11.5°C (Blaxter (Blaxter and Ehrlich, 1974). 1974).
H. S. S. BLAXTER J. BLAXTER J . H.
26
protein during during starvation (Fig. 15). 15). Activity was maintained maintained for for some some protein starvation (Fig. Activity was days decrease drastically drastically only only late late in in the of days and and tended tended to to decrease the period period of starvation, presumably presumably as as aa way maintain food-searching food-searching ability ability as as starvation, way to to maintain long as as possible, even if if it it was was energetically expensive. Huse Huse and and long possible, even energetically expensive. Skiftesvik 1985) found found that that starving starving turbot turbot larvae larvae swam swam faster faster for for Skiftesvik ((1985) shorter shorter periods periods and and searched searched for for food food less less efficiently efficiently than than feeding feeding larvae. larvae.
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Fig. 15. 15. Buoyancy Buoyancy forces forces due due to to chemical chemical components components in in Atlantic herring Clupea Clupea Fig. harengus harengus and and plaice plaice Pleuronectes Pleuronectes platessa larvae larvae at the the end of the the yolk-sac yolk-sac stage stage and at the point-of-no-return point-of-no-return (PNR). (PNR). All f,)Tces f<mesare are dynes/wet dynedwet weight (mg); (mg); arrows arrows show show direc directhe of forces. forces. Note that that the the larvae larvae become increasingly increasingly buoyant buoyant as as they they starve. starve. [From [From tion of tion (1974),with with permission of Springer Verlag.] Verlag.] Blaxter and and Ehrlich Ehrlich (1974), Blaxter
1. 1. PATTERN AND VARIETY IN DEVELOPMENT DEVELOPMENT
27
C APTIVITY VII. THE EFFECT OF CAPTIVITY
The effect effect of of captivity captivity on on growth growth and and morphology morphology was was summarized summarized The ( 1976). A common phenomenon is the “size "size hierarchy” hierarchy" by Blaxter (1976). (sometimes is, an increasing (sometimes called depensation of of growth)-that growth)-that is, range of of size as the larvae grow. Some examples are given in Fig. 16. 16. Whether Whether size size hierarchies hierarchies occur occur in in natural natural conditions conditions is is uncertain, uncertain, egg size phe since variations variations in in egg size and and spawning time time may may obscure obscure the phenomenon, and selective predation on small individuals may limit the of the size range. The increase in length range with age lower end of may be due to a natural variation early in development. For example, 10% variability in length of of 10-11 10- 1 1 mm early in development would a 10% of range in result in a range from 50 to 55 mm later. The increase of length is often well beyond beyond such such considerations, and it seems likely that size hierarchies are a tank phenomenon caused by competition for food, social dominance of of some individuals in crowded conditions, and lack of of predation on smaller individuals. Thus Eaton and Farley 40
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Fig. Fig. 16. 16. Size hierarchies. Increase of of length range with age in northern anchovy Engraulis mordax, mordar, Atlantic herring Clupea harengus, turbot turbot Scophthalmus maximus, and ayu Plecoglossus Plecoglossus altiuelis reared in the laboratory. [Redrawn from Blaxter (1981) altivelis reared ( 1981) and Tanaka (1973).] (1973).]
S. BLAXTER H. S. BLAXTER JJ.. H.
28
((1974) 1974) found a reduction in the size hierarchy of of the zebrafish Danio ((= = Brachydanio) rerio fed an adequate diet. After 32 days, larvae fed Brachydanio) on a low ration ranged from 5 to 12.5 mm (mean 9.5 mm), and after 39 12.5 (mean 9.5 mm), days another group fed on a high ration ranged from 7.5 mm days another group fed on a high ration ranged from 16.0 16.0 to to 117.5 mm (mean mm). (mean 16.9 16.9 mm). Rearing in captivity also tends to produce shorter, fatter fish, fish, with high (Fig. 17) 17) and high condition condition factors factors (Fig. and growth growth abnormalities abnormalities such such as as fore foreshortened snouts, neoplasms of the head, and failure of of eye migration and fin development in flatfish. 1980) found that the hearts of of flatfish. Arthur ((1980) northern anchovy larvae, as determined by length of of the ventricle,
0‘/o/0 FFAT AT
DRY W GH HTT DRY W EE II G
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Fig. 17. 17. A A general comparison of changes changes in in dry weight, fat, and condition factor of Fig.
“wild” fish fish as as they grow, grow, based mainly on Atlantic herring; see see text for reared and "wild" details. The change of condition factor (shown (shown bottom right) in the the sea results from from details. loss of yolk, causing a fall, fall, followed by progressive ossification, causing a rise. initial loss Condition factors of reared starved larvae tend tend to to be higher than "wild" “wild” larvae, showing Condition that starvation criteria criteria cannot cannot be obtained obtained satisfactorily from from captive larvae. larvae. that
1. 1. PATTERN AND VARIETY
IN DEVELOPMENT
29
were as much as 40% longer in laboratory-reared laboratory-reared compared with sea seacaught individuals. Excessive contact with the walls of the tank, other forms of stress, and social factors factors can be implicated in causing these forms abnormalities. of the volume of of the rearing container was investigated The effect of by Theilacker ((1980b) 1 980b) using jack mackerel larvae reared at the same density in 10-1 10-1 and 100-1 100-1circular tanks. The beneficial effect of the larger volume was apparent after only 4-5 4-5 days of of feeding, when the larvae were larger and in better better nutritional condition. The dramatic effect of using rearing facilities on the mesocosm scale was shown by Morita ((1985) 1985) and Paulsen et al. al. ((19851, 1 985), who reported excellent growth and survival of Pacific herring larvae and turbot larvae respec respec3 on-shore tanks. Sturmer et al. tively in 20-m al. ((1985) 1985) found similar 20-m3 benefi cial effects using red drum larvae in large on-shore beneficial on-shore tanks and Kvenseth and 0iestad 1 984) and 0iestad al. ((1985) 1985) using cod larvae giestad ((1984) Oiestad et al. 3• in a coastal impoundment of 60,000 60,000 m m3. High density of larvae may also create problems other than those associated with physical interactions. Crowded conditions may cause the production of inhibitory substances. Although originally demon demonstrated in amphibians, such substances may be implicated in popula populawhite cloud tion control of of the guppy Lebistes (Poecilia) (Poecilia) reticulatus, whitecloud mountain minnow Tanichthys albonubes, tiger barb Barbus tetra tetrazona, sp. (Rose, (Rose, 1960). 1960). Laale xona, rainbow trout, and ciscoes Leucichthys sp. and McCallion ((1968) 1 968) found that the development of zebrafish could be arrested before gastrulation by the use of of supernatant homogenates produced from other zebrafish embryos embryos.. Such effects are likely to be relevant in the wild only to species that live in crowded conditions or in stagnant water. In biochemical terms, hatchery fish fish often have a higher percentage fat content than their wild counterparts but lower percentage protein and ash (Table 11 in Blaxter, (Fig. 17). Blaxter, 1976) 1976) (Fig. 17). The biochemical and morphological changes occurring in hatchery fi sh can be refl ected in fish reflected their behavior and especially in their locomotor performance. Crowd Crowding induces stress and aggression that can be partly alleviated by adequate food, for example, in the medaka Oryzias latipes (Magnu (Magnuson, 1962) 1962) and Atlantic salmon (Symons, 1967) found (Symons, 1968). 1968). Barns Bams ((1967) that the fry fry of of wild migrant sockeye salmon Oncorhynchus nerka could stem a water current and avoid predation better than hatchery fry, fry, although mainly this was due to the fact that they were bigger. crowding, sensory deprivation may be As the inverse of stress and crowding, implicated as a feature of hatcheries or rearing tanks (Blaxter, (Blaxter, 1970). 1970). Developing fi sh may not be subjected to the normal interplay of fish of light and shade, nor to the very high light intensities appertaining in the
30
J. J. H. H. S. S. BLAXTER
wild. They may not be able to practice avoidance or other responses in the absence of a natural substratum, typical water currents, or preda predators. Such deprivation will be particularly harmful where learning is involved in the development of behavior patterns. The problems are well demonstrated by attempts to establish hatchery-reared plaice in the sea (Blaxter, (Blaxter, 1976). 1976). Survival was negligible, and it is is likely that inadequately developed predator-avoidance, burying behavior, and feeding mechanisms were to blame. A number of workers have assessed the effect of of blinding, or rear rearing in darkness, on the eye and optic tectum of fish larvae. PHugfelder Pflugfelder (1952) (1952) found that unilateral blinding of of newly hatched swordtails swordtails Xiphophorus and guppy Lebistes caused a reduction in the develop development of of the contralateral optic tectum, mainly by a decrease in volume of the ganglion cells. Experiments in darkness confirmed that the effect was caused by lack of of visual input rather than by degeneration products released from dying axons 1 970) axons of the optic tract. Blaxter ((1970) could find no retinal degeneration in dark-reared Atlantic herring lar larvae. Zeutzius et al. al. (1984) apia Sarothero (1984) found that dark-rearing of til tilapia Sarotherodon ((= = Tilapia) Tilapia) mossambicus did not affect the normal outgrowth of the nerve fibers of of the optic tectum into the retina. It did, however, reduce the optic layer and the differentiation of the synapses, where the number of synaptic vesicles increased. Dark-rearing can affect behavior. Blaxter ((1970) 1970) found that newly hatched Atlantic herring larvae were very inactive when returned to the light after rearing in the dark and subsequently showed a high mortality. Zeutzius and Rahmann (1984) (1984)found that dark-reared larvae apia failed to swim up after yolk resorption; visual acuity was of til tilapia impaired after 20-30 20-30 days in the dark, and after 50 days no optokinetic mininystagmus was present. Effects on body weight and length were mini mal, although the increase in body depth was substantial compared with control fish. Sensory deprivation may operate at aa more subtle level. For exam example, both Breder and Halpern (1946) 1960, 1961) (1946) and Shaw ((1960, 1961) had diffi diffiof zebrafish and Menidia. culty in rearing isolated individuals of Menidia. SurSur vival was poor, and in Menidia schooling was retarded when isolates were brought together.
VIII. SHRINKAGE VIII. THE EFFECT OF FIXATION ON SHRINKAGE The interpretation of developmental events related to size, and factor, will depend on whether estimates of growth rate and condition factor, ossification, or those with live or fixed material is used. Larvae lacking ossification,
1. PATTERN 1. PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT
3 311
form, will be especially prone to shrinkage, shrinkage, not only a long thin body form, fixatives, but also as a result of capture by plankton net if sam samfrom fixatives, pled from the wild. This problem has been addressed in the larvae of of (1963), in Atlantic Californian sardine Sardinops caerulea by Farris (1963), 1971), in Pacifi c herring by Schnack and Rosenthal herring by Blaxter ((1971), Pacific ((1977-1978) 1977-1978) and Hay ((1981) 1981) and northern anchovy, Pacific Pacific mackerel PaScomber japonicus, jack mackerel Trachurus symmetricus, and Pa cific barracuda Sphyraena argentea by Theilacker ((1980a) 1980a) and Theilacker and Dorsey ((1980), 1980), and in southern fl ounder Paralichthys flounder lethostigma 1 984). The degree of of shrinkage Zethostigma by Tucker and Chester ((1984). depends on the concentration and osmolality of the fixative, the pe period of fi xation, and the age of fixation, of the larvae. A shrinkage of of 5-10% 5-10% is normally experienced in long thin larvae, especially when young, but may be as little as 2% 2%in older stages. stages. Capture by net, either simulated (Blaxter, 11971; (Blaxter, 97 1 ; Theilacker, 1980a) 1980a) or after release of larvae in the path of a net (Hay, xation, may cause shrinkage as great (Hay, 1981), 1981),followed by fi fixation, as 20%, fixation is delayed. The effect is much less 20%, or even more if if fixation serious in older larvae with ossified skeletons. skeletons. Theilacker and Dorsey ((1980) 1980) also mention the loss of of dry weight following fixation. Formalin fixation caused a 30% loss in weight of fixation. Fonnalin of larval Pacifi c sardines and fi xation in ethyl alcohol a 30-80% loss in Pacific fixation 30-80% loss Pacifi c mackerel larvae. Pacific larvae. Methods of of preservation and curation are discussed by Lavenberg et al. ((1984), 1 984), who recommend final preservation in 70% ethanol to fixatives Probobviate problems of buffering acid fi xatives such as formalin. Prob lems of buffering are, however, trivial compared with the amount of shrinkage in formalin or alcohol. IX. RATE OF DEVELOPMENT IX. DEVELOPMENT
The rate of development is clearly under genetical control. Within a species it is most strongly influenced influenced by temperature, and this is exemplified by data on days to hatch at different temperatures in 13 13 species (Fig. 1 986) give further data on three (Fig. 18). 18).Herzig and Winkler ((1986) cyprinids. Blaxter ((1969) 1969) and Herzig and Winkler ((1986) 1986) discuss the mathematical relationship between temperature and incubation and the use of QlO Q l o and other temperature coefficients. In particular Q Q llOo is found to vary with range of temperature, and it may be that optimum temperatures for development, where hatching rates are highest, take place where the QlO 9 1 0 is between 2 and 3. Temperature can also influence size at hatching, efficiency of of yolk utilization, growth, feeding rate, time to metamorphose, behavior and
J. J. H. H. S. S. BLAXTER
32 32 200 200
2
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34 34
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TEMP. ( O C )
Fig. lB. 18. The time from fertilization to hatching for 111 of teleost related to Fig. 1 species of (1) Desert temperature. Data originally cited in Blaxter ((1969) 1969) except where stated. (1) pup fish Cyprinodon Cyprinodon macularius, macularius, (2) ( 2 )brook trout Salvelinus Salvelinus fontinalis, fontinalis, (3) ( 3 )rainbow trout pupfish Salmo gairdneri, galrdneri, (4) (4) smelt Osmerus eperlanus, (5) (5)Atlantic Atlantic herring Clupea Clupea harengus, (6) (6) plaice Pleuronectes platessa, platessa, (7) (7) Pacifi Pacific Gadus macrocephalus, (8) (8)rockling rockling Enche Enchec cod Gadus lyopus cimbrius, cimbrius, (9) (9) mackerel mackerel Scomber scombrus, (10) (10) grey mullet Mugil cephalus (Nash 1 1) striped bass Morone (Nash and Kuo, Kuo, 1975), 1975), and ((11) Morone saxatilis.
rates, and metabolic swimming speed, digestion and gut evacuation rates, demand. Temperature Temperature also has indirect indirect effects on larvae through the demand. viscosity, and phytoplankton blooms oxygen capacity of water, its viscosity, fish (Theilacker and Dorsey, 11980). 980). Much of the data on marine fi sh are summarized by these authors and in more general terms by Kinne Brett (1970). In particular, Brett (pp. (pp. 524-527) 524-527) gives a com com((1963) 1963) and B rett (1970). prehensive table of upper and lower lethal temperatures of embryonic fish. and postembryonic stages of marine and estuarine fish. is discussed by Garside The structural response to temperature is
11.. PATTERN PATTERN AND AND VARIETY VARIETY IN IN
DEVELOPMENT DEVELOPMENT
33
((1970) 1 970) with particular reference to the effect of of temperature on the interaction of differentiation and growth. Salmonids Salmon ids and clupeids inin cubated at high temperatures tend to weigh less at hatching. The best known effect is on meristic characters-counts characters-counts of serial structures scales, and gill rakers, which are labile such as vertebrae, fin rays, scales, (within limits) and susceptible to various environmental factors factors (Tan (Tining, 1952; 1952; Barlow, 1961; 1961; Blaxter, 1969; 1969; also see Lindsey, volume XIB). Until recently it has not been clear what adaptive advantage might exist for varying numbers of meristic characters. It has been shown that individuals of a particular species with more vertebrae are often longer, and it seems plausible that they might also be more flexible and able to swim faster. 1984) have recently faster. Swain and Lindsey ((1984) shown that there was selective predation for vertebral number in young sticklebacks sticklebacks Gasterosteus aculeatus preyed on by sunfish Le Lepomis gibbosus. The survival of8.2-mm-Iong 1.7 of 8.2-mm-long sticklebacks was 1.31.3-1.7 fish 31 times greater for fi s h with 3 1 vertebrae than with 32. This effect was 8.9-mm-long sticklebacks, nor was there any influence not found with 8.9-mm-Iong of temperature. Early development can be enhanced hormonally. Dales and Hoar of chum salmon 0. ((1954) 1954) treated the eggs of O. keta with thyroxine and thiourea, an antithyroid compound. Thyroxine accelerated growth of of the body wall and pectoral fi n s, increased guanine deposition, de fins, decreased pigmentation, caused exophthalmia, exophthalmia, but reduced the rate of increase of body length. Thiourea decreased guanine deposition and also decreased the rate of growth in length. length. More recently, it has been shown that immersion in thyroxine accelerates growth in larval til apia tilapia Sarotherodon ((= = Tilapia) Tilapia) mossambicus, carp Cyprinus carpio, and milkfish Chanos chanos chan os and enhances survival in tilapia and carp (Lam, 1980; (Lam, 1980; Lam and Sharma, 1985; 1985; Lam et al., al., 1985). 1985). Further infor information-for example, on the influence of hormones on smoltification mation-for salmonids-will be found in Hoar (volume XIB XIS). of salmonids-will ). X X.. ORGAN SYSTEMS
A. Alimentary System A. Many species hatch without a mouth, but this develops rapidly to allow for the transfer from endogenous yolk to exogenous food. food. Feed Feeding in many species is a predatory act requiring vision, and feeding does not occur in the dark, especially in the very young stages (Blax(Blax-
34
J. H. H. S. S. BLAXTER J. BLAXTER
ter, 1981, 1986; Hunter, 1980, 1981,1986; 1980, 1981). 1981). The size or gape of the mouth at first feeding, and therefore the size of food that can be taken, is crucial first for survival at the end of the yolk-sac yolk-sac stage (Fig. (Fig. 19A,B). 19A,B).Experiments show that the size of of food taken is is related to the gape of the jaw and that both increase with age of the larvae. Length and complexity of the alimentary tract increase as the lar larvae grow. 1984) describes three groups of fish larvae grow. Dabrowski ((1984) based on the morphology of the alimentary tract and gut enzymes. Most species have early larvae without a functional stomach or gastric beglands; salmonids, on the other hand, have a functional stomach be fore changing to external feeding. Tanaka ((1973) 1973) gives a full account of of the development of the alimentary tract in 21 21 Japanese marine and freshwater species. species. At first feeding there are no pharyngeal teeth and few, if mu if any, taste buds. The esophagus has longitudinal folds and mucous cells; the intestine and rectum are lined with columnar epitheepithe lium, and it is likely that most digestion occurs here. Cilia are present in the gut of early clupeoid and salangid larvae and may help to pass food along the gut (Iwai (Iwai and Rosenthal, 1981). 1981). The liver, gallbladder, and pancreas are also formed early. In many species, but not in salmosalmo nids like the rainbow trout, the stomach and pyloric caeca develop late in larval life as the pattern and quantity of feeding changes. These subsequent processes are well described by O'Connell 1981) in the O'Connell ((1981) northern anchovy and Govoni Govoni ((1980) 1980) in the spot Leiostomus xan xan-
thurus. thurus. The length of the gut influences the passage time for food. food. For example, in roach Rutilus rutilus larvae the food is is retained for only 2.5 2.5 h at 20°C, 20°C, whereas in the adult it is 6 h (Hofer, (Hofer, 1985). 1985). The time for digestion and resorption and for the recovery of digestive enzymes is therefore reduced in the larval stages. Dabrowski ((1984) 1984) reviewed work on the appearance of digestive enzymes during development. Clark et al. al. ((1985) 1985) found an increase in protease activity of Dover sole stage. Similarly, Similarly, Lauff and from the age of 24 days up to the adult stage. Hofer ((1984) 1984) found a progressive increase in activity and number of of proteolytic enzymes with age in whitefish Coregonus hybrids, in rain rainbow trout, and in roach. Higher proteolytic activity in the roach could be correlated with the lack of a stomach. stomach. In the rainbow trout the well welldeveloped Ideveloped digestive tract, which is differentiated into a stomach, py pyloric caeca, and a short intestine at first feeding, may compensate for a lower level of proteolytic activity. The relatively underdeveloped state of the alimentary system may explain why most species have carnivorous larvae, although later, when the gut lengthens, they may become herbivorous. It may also
PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT 11. . PATTERN
35
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Fig. of the mouth in the Fig. 119. 9 . (A) (A) The relationship between body length and width of larvae of Pacific mackerel Scomber Scomber of hake Merluccius merluccius, cod Gadus morhua, Pacific (1980).] (B) “Profile” "Profile" japonicus, and anchovy Engraulis ringens. [Redrawn from Hunter (1980).] of of the body length: vertical jaw gape relationship in the larvae of of 19 teleost teleost species. species. [Redrawn [Redrawn from Shirota (1970). (1970).]J
J. H. S. S. BLAXTER BLAXTER J. H.
36
explain why why artificial artificial food food is is less less satisfactory satisfactory for for young young larvae, larvae, since since explain live food food contains contains exogenous exogenous enzymes enzymes that that may may aid aid digestion digestion (Da(Da live browski and and Glogowski, Glogowski, 1977). 1977). browski B. Respiratory Respiratory System System B. By first first feeding feeding most most species species have have larvae larvae with gill slits slits and gill By with gill and gill arches (Tanaka, (Tanaka, 1973), 1973), but but gill gill filaments filaments develop develop later. later. The The cobitid cobitid arches Misgurnusfossilis fossilis has has external external gill gill filaments filaments for for aa time time (Fuiman, (Fuiman, 1984). 1984). Misgurnus Harder (1954) ( 1954) gave gave aa particularly particularly thorough thorough account account for for the the herring, herring, Harder where the the filaments filaments first first appear appear at at aa body body length length of of 20 mm mm several several where weeks after after hatching. hatching. De De Silva Silva (1974) ( 1974) measured measured the the gill gill area area of of both both weeks herring and plaice larvae during early development and related them herring and plaice larvae during early development and related them the surface surface area area of of the the body body (Fig. (Fig. 20). 20). In In the the early early stages stages it it is is clear clear to the to respiration is cutaneous. The larval heart is present even before that that respiration is cutaneous. The larval heart is present even before hatching and and pumps pumps aa colorless colorless body body fluid fluid around around an an as as yet yet unknown unknown hatching A
10
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Length l e n g t h (mm) (mm)
Fig. 20. 20. The effect of of size on on oxygen oxygen uptake uptake (ILl (p1 O�), O a ) ,Q0 QO,2 (ILl (pl Odmg Odmg dry dry weight/h), weighvh), Fig.
2/mg wet gill area area (mm (mm2/mg wet weight) weight) and and body body area area (mm2/mg (mm2/mgwet wet weight) weight) in in (A) (A) Atlantic Atlantic gill Clupea harengus harengus and and (B) (B) plaice plaice Pleuronectes Pleuronectes platessa platessa larvae. larvae. [Data [Data from from De De herring Clupea herring Silva and and TytIer Tytler (1973) (1973) and and De De Silva Silva (1974).] (1974).] Silva
1. 1.
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I N DEVELOPMENT PATTERN AND VARIETY IN
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system. Weihs ((1980b) vascular system. 1980b) found that yolk-sac larvae of northern oxygen saturation of the water anchovy were more active when the oxygen ambidropped below 60%, suggesting the requirement to renew the ambi limiting. ent water close to the body when oxygen is limiting. conclusions In relating size to respiration the following theoretical conclusions may be drawn: 2 a body length length2 Total body area for cutaneous respiration Q( 3 = body length length3 Body weight Q( a body lengthlength-'I Respiratory area per unit weight Q( 0 22 requirement requirement Q( body weightD'8 weighP8 or body length l e ~ ~ 2g'4 t(see (see h ~ 'Winberg, ~ O 1960) 1960) 002 or body a body weightweight-0'2 (QO2) Specific oxygen requirement (Q0 2) Q( OoB 1ength-O"j lengthThus the body surface area per unit weight declines as the body (QO,) delength-',I , whereas the oxygen requirement per unit weight (Q0 length2 ) de ooB. This would lead to a critical length-0,6. clines less rapidly, as body length-
38 38
J. H. H. S. S. BLAXTER BLAXTER J.
situation situation without without the the development development of of gills gills to to increase increase the the respiratory respiratory surface surface area area (Fig. (Fig. 20). 20). Although Although the the blood blood of of herring herring and and plaice plaice and and many many other other species species does does not not appear appear pink pink until until metamorphosis, metamorphosis, its its precursors, precursors, or or related related substances substances such such as as myoglobin, myoglobin, can can be identified identified histochemically histochemically soon soon after after hatching. hatching. The The circulating circulating body body fluids fluids were were reported reported to to be be acellu acellular lar until until about about 16 16 mm mm in in the the herring herring and and 10 10 mm mm length length in in the the plaice plaice (De 1 982) found 1974), but but Hickey Hickey ((1982) found unidentified unidentified cells cells 8-14 8-14 JLm pm (De Silva, Silva, 1974), in in diameter diameter in in newly newly hatched hatched larvae larvae of of both both species species and and an an efficient efficient wound-healing wound-healing mechanism mechanism in in herring, herring, plaice, plaice, and and Atlantic Atlantic salmon salmon lar larvae. vae. In system has In the the walleye walleye Stizostedion vitreum vitreum the the circulatory circulatory system has been 1 979). The The blood blood becomes becomes been described described by by McElman McElman and and Balon Balon ((1979). red red before before hatching, hatching, although although the the development development is is not not precocial. precocial. In In the the precocial hemoglobin may may be be precocial larvae larvae of of salmonids salmonids and and elasmobranchs, elasmobranchs,hemoglobin present, present, and and the the blood blood pink, pink, at at hatching. hatching. In In these these species species the the body body size so size is is large large enough enough for for cutaneous cutaneous respiration respiration to to be be inadequate inadequate and and so increase increase in in oxygen-carrying oxygen-carrying capacity capacity of of the the blood blood is is essential, essential, even even if if the the gills gills are are in in aa rudimentary rudimentary state. state.
C. Locomotor System Most species hatch with V-shaped myotomes acting against the notochord as a hydrostatic skeleton. Additional myotomes myotomes may be added posteriorly, but the final number is attained during the early larval period. Generally the myotomes, myotomes, which comprise white muscle, become progressively more complex in shape and interdigitate with adjacent myotomes. myotomes . The red muscle develops initially as a myotube, a superficial cylindrical sheath around the body, e.g., in northern anan chovy (O’Connell, (O'Connell, 1981), 1981), zebrafish (van Raamsdonk et al., 1982), 1982), herher ring (Batty, 1984), 1984), and red sea bream Pagrms Pagrus major (Matsuoka and Iwai, 1984). 1984). In Coregonus sp. (Forstner et al., 1983) 1983) the red muscle extends as a thin layer dorsally and ventrally from the lateral line. In all these species it later concentrates in a strip in the midlateral posiposi 21). tion on the flank (Fig. tion (Fig. 21). Larvae usually hatch with a primordial primordial median median finfold; the median fins often first appear appear as a discontinuity in the margin margin of of the finfold; a few fin rays then appear, which gradually increase in number number and size. In species with a homocercal tail the caudal fin develops after the tip of of the notochord notochord turns up (flexion, (flexion, see p. 15). 15). Lateral fins, used for stability, maneuvering, and sometimes for propulsion, develop differdiffer ently. Pectoral buds, fin-like structures that that lack lack rays, are often present present at hatching. Pelvic Pelvic buds buds and fin rays develop later.
ch
DEVELOPMENT 1. PATTERN 1. N DEVELOPMENT PATTERN AND AND VARIETY VARIETY IIN
L�,
Reynolds R eynolds number n u m b e r R= R= du
R R
RR
10 -- 200 200 10
<10 <10
cDN YR~
Co
39
IIC
R
V iscous forces Viscous forces m portant ii rnportant
�
~
Serpen t i ne Serpentine
� V-
0 body
sheet heel
RR
>20 >20 0 0
cD= Co = k k
Inertial I nertia l forces forces ii rn m portanf portant
Intermediate Intermediate z one zone
n develops, caudal develops c a udal ffiin
red muscle mu sc l e changes
>
� a
C a ra n g i f o r m Carangiform
’00 lateral lateral line
Fig. 21. Changes in hydrodynamic considerations considerations as fish larvae grow related to the Reynolds number (R), (R), where L is body length, U U is swimming speed, and V is the kinematic viscosity (see Webb, 1975). 1975). An inertial regime exists where R > > 200 and a viscous regime where R < 10, 10, with an intermediate zone between. The drag coefficient also changes. At the same time, the larvae change from a a serpentine to a carangicarangi (CD) (Gn) also form swimming mode as the tail flexion occurs and the red muscle develops from a myotube surrounding the whole body in a thin sheet (TS) (TS) to a strip situated along the midlateral position position on the flank. flank.
A number of workers (Webb, (Webb, 1975; 1975; Weihs, 1980a; 1980a; Batty, 1984; 1984; Webb Webb and and Weihs, Weihs, 1986; 1986; Blaxter, Blaxter, 1986) 1986) have have considered considered the the "locomo “locomotor regime" of of larvae of different size (Fig. (Fig. 21). 21). Where tor regime” larvae of Where the Reynolds number 10 (in very small larvae), viscous forces are parapara number (R) is below 10 effi mount and continuous high-speed swimming is energetically efficient. Where R is greater than 200, inertial forces are more important and beat-and-glide swimming is more efficient. Since R depends on as the body length and velocity, the hydrodynamic regime changes as larvae grow or alter their swimming speed. Often they may occupy an intermediate intermediate zone between between the the viscous viscous and and inertial inertial regime. During During later later development development there there are, are, in in many many species, increases increases in in the the surface surface area for propulsion as a result of of the appearance of of the the caudal caudal fin fin and and allometric allometric growth growth (Fuiman, (Fuiman, 1983). 1983). Linked to swimming is the buoyancy of of larvae. larvae. Although unim unimportant portant for for demersal demersal larvae, larvae, pelagic pelagic larvae larvae can can potentially potentially waste waste much much energy energy in maintaining their position in the water column. As larvae grow the skeleton they become grow and and the skeleton ossifies ossifies they become heavier heavier (Fig. (Fig. 17) 17) and and tend tend
40
BLAXTER H. SS.. BLAXTER JJ.. H.
more and more to negative buoyancy. Conversely, Conversely, if if they starve, they will tend to neutral buoyancy (Fig. (Fig. 15). 15).The larvae of many species fill the the swimbladder soon after hatching, or at the end of of the yolk resorp resorption, probably by swallowing air at the surface (Doroshev et al., al., 1981). 1981). This stage, sometimes called "swim-up," “swim-up,” can be critical for successful later development, e.g., e.g., in the turbot. The time of appearance of of the swimbladder varies widely between species. For example, the her herring fi lls its swimbladder at a length of about 30 mm, while the north fills northern anchovy inflates its swimbladder at about 10 10 mm (Hunter and Sanchez, 1976) and the menhaden Brevoortia tyrannus Sanchez, 1976) tyrunnus at 13 13 mm (Hoss 1982). In the physostomatous northern anchovy (Hoss and Blaxter, Blaxter, 1982). and menhaden there is is a diel die1 rhythm, the larvae filling their swimbladders by swallowing air at the surface at night (Hunter and Sanchez, 1976; 1976; Hoss and Phonlor, 1984). 1984). Sense Organs D. Sense 1. YE 1. THE THEE EYE In some species the eyes are free of of pigment at hatching and histo histological examination confirms confirms that they must be nonfunctional. By fi rst first feeding the eyes eyes are pigmented, and vision plays a major part in feed feeding. Of 10 1970), 10 teleost families examined by Blaxter and Staines ((1970), eight had larvae at first feeding with a pure-cone retina, and only an anguillid and a macrourid, caught in deep water, had a pure-rod ret retina. A pure-cone retina at first feeding was also found in the northern ina. anchovy by O'Connell 1981) and in the goldfish by Johns ((1982). 1982). In O’Connell ((1981) zebrafish, rods can be identified identified 9 days after hatching (Branchek and Bremiller, 1984), 1984), and at hatching in Pacific salmon, Oncorhynchus Pacific salmon, spp. (Ali, (Ali, 1959). spp. 1959).In the advanced young of the viviparous guppy Poeci Poecilia al. ((1983) 1 983) found a well-differentiated duplex Zia reticulata, Kunz et aZ. retina even before birth. Retinomotor movements of the masking pig pigment and the photoreceptors usually develop concomitantly with the rods (Ali, 1959; Blaxter and Staines, 1970; Kunz and Ennis, 1983; (Ali, 1959; Staines, 1970; 1983; Neave, 1984) 1984) so that the process of light-and-dark adaptation is linked to the establishment of a duplex retina. The development of of visual performance is described by Blaxter ((1986), 1986), improvements in acuity being the most noticeable feature. 2. MECHANORECEPTORS 2. MECHANORECEPTORS
Free neuromasts are present at hatching in all species examined (Iwai, 1967, 1967, 1980), (Iwai, 1980), usually on the head and sometimes on the trunk. of the development of of the Disler ((1971) 1971) gives a very detailed account of
11.. PATTERN PATTERN AND AND
VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT
41
free neuromasts and lateral line of of many freshwater species, including the sturgeon Acipenser Acipenser stellatus, stellatus, Pacific salmon O. 0 . keta, and some percids and cyprinids. Generally the initial number of of free neuro neuromasts is low but they proliferate and may become regularly arranged along the flank. flank. In marine species similar systems are found in gadids (Fridgeirsson, (Fridgeirsson, 1978), 1978), northern anchovy (O'Connell, (O’Connell, 1981), 1981), Atlantic herring (Blaxter et al., al., 1983b), 1983b), halibut (Blaxter (Blaxter et al., al., 1983a), 1983a), plaice and turbot (Neave, 1986), 1986), and spotted bass Micropterus punctatus (Kokkala (Kokkala and Hoyt, 1985). 1985). The lateral line canals almost invariably develop some time after hatching: at 17 17 mm in menhaden, 18-20 18-20 mm in northern anchovy, 242410 mm in the plaice, 26 mm in Atlantic herring, 8 mm in the turbot, 10 and 12 12 mm in spotted bass bass.. Thus young larvae have very incomplete mechanoreceptors mechanoreceptors.. The development of of the inner ear is not well known, except that larvae have one or more pairs of of otoliths at hatch hatching, which must give them a basic perception of of posture. 3. 3. CHEMORECEPTORS CHEMORECEPTORS Olfactory pits are described in the early larval stages of of Atlantic 1 978), tilapia by Iwai (1980), herring by Dempsey ((1978), (1980), northern anchovy by O'Connell 1981) walleye Stizostedion O’Connell ((1981) Stixostedion vitreum by Elston et al. al. ((1981), 1981), carp by Appelbaum ((1981), 1981), and striped bass Morone saxatilis by Bodammer ((1985). 1985). Kokkala and Hoyt ((1985) 1985) described taste buds in larval 1 980) found them between 11and 14 14 days larva1 spotted bass and Iwai ((1980) posthatching in tilapia, pond smelt Hypomesus transpacijicus, transpacijkus, gold goldfish, sea bass Lateolabrax japonicus, puffer Fugu niphobles, flatfish Kareius bicoloratus, and red sea bream Pagrus major. major. XI. STRUCTURE XI. STRUCTURE AND FUNCTION FUNCTION
Clearly most larvae, apart from some highly developed ovovivipa ovoviviparous or viviparous species, or species with very large eggs, go through struca massive increase in complexity while free-swimming. Since struc tures are often absent or incompletely developed, the associated be behaviors are also absent or poorly developed. When relating structure to function, mention should be made of the theory propounded by 1981b, 1984) Balon ((1981b, 1984) that ontogeny is saltatory, meaning that develop development proceeds by a series of rather rapid changes in both structure and function with relatively prolonged intervals in between, during which a more-or-Iess more-or-less steady state exists as the organism prepares itself itself for the next rapid change. change. Thus development does not proceed by a
42
J. S. BLAXTER J. H. H. S. BLAXTER
continuous continuous accumulation accumulation of of small small changes. changes. As As an an example, example, Balon Balon cites cites aa cyprinid, cyprinid, the the bream bream Abramis ballerus (Fig. (Fig. 22). 22). A A number number of of studies studies link link the the development development of of structure structure with with func function. best examples tion. One One of of the the best examples is is found found in in the the work work of of Hunter Hunter and and Coyne 1982) on Coyne ((1982) on northern northern anchovy anchovy (Fig. (Fig. 23), 23), where where age age and and length length are are related related to to the the development development of of the the sensory, sensory, respiratory, respiratory, digestive, digestive, and and locomotor systems and locomotor systems and associated associated behavior behavior and and ability ability to to withstand withstand starvation. 1985) gives starvation. Fukuhara Fukuhara ((1985) gives aa similarly similarly comprehensive comprehensive account account of sea bream of the the functional functional morphology morphology of of the the red red sea bream Pagrus major. major. Allen et al. al. ((1976) 1 976) related development of swallowing swallowing behav Allen related the the development behavior, avoidance avoidance responses, responses, and and shoaling shoaling specifically specifically to to the the develop development pro-otic bullae bullae and and swimbladder swimbladder of of Atlantic Atlantic herring herring larvae larvae ment of of the pro-otic (Fig. 1 985) (Fig. 24). 24). In aa somewhat somewhat similar similar fashion, fashion, Kawamura Kawamura and and Ishida Ishida ((1985) compared compared the development development of the the whole whole sensory sensory system system of the the floun flounder der Paralichthys olivaceus to to primary primary orientation, orientation, feeding, feeding, migration, migration, and and activity activity (Fig. (Fig. 25). 25). Considerable Considerable differences differences in in morphological morphological and and
Itep 4
slep 3
L A R VA E M8 R 'tO
$lep 2
,'.p 1 AGE
consecutive steps in the ontogeny of of the Danubian bream Fig. 22. A scheme of consecutive Abrarnis ballerus, demonstrating saltatory development. During each step various Abramis structures structures grow and differentiate at different rates but are completed and become func functional at the same time, at the end of of the step, thus enabling the larvae to make substan substanauthor’s terminology that the tial and rapid changes in behavior. Note according to the author's embryo changes to the larva at fi rst feeding, after step 3. ed and redrawn from 3. [Modifi [Modified first (1984).] Balon (1984).]
1. PATTERN 1. PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT
43 43
species; the behavioral events can be seen in these two unrelated species; herring, with an extended larval period metamorphosing into a pe peflatfish lagic schooling species, the flounder, a flatfi sh with a shorter larval period ending in settlement. (1980)gives a detailed but similar style of summary of of de deBalon (1980) Salvelinus. Despite their five velopmental events in fi ve species of charr Salvelinus. of close relationship, relationship, they they show show substantial substantial differences differences in in the the timing timing of the the appearance appearance of of both both morphological morphological and and behavioral behavioral features features such such as as fi ns, melanophores, fins, melanophores, branchial branchial respiration respiration and and swimbladder swimbladder filling. filling. The ontogeny of behavior, especially in salmonids and cichlids, is (1978) and Noakes and Godin (volume (volume XIB). XIB). discussed by Noakes (1978) of the early life These groups are of particular interest because much of history may be passed in gravel beds or under the care of a parent. Such a lifestyle may enhance the protection of im of the young but it imposes other problems, such as the avoidance of abrasion or cannibal-
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Fig Fig.. 23. 23. Developmental Developmental events events during during the the early early life life history history of of the the northern northernanchovy anchovy Engraulis Engruulis mordax. mordax. RBC, RBC, Red blood blood cells; cells; time time to to 50% 50%starvation starvation is is the the number number of days days after after which which 50% 50% of of unfed unfed larvae larvae died died (equivalent (equivalent to to point-of-no-return). point-of-no-return).For For further further discussion 1982).] discussion of of Reynolds Reynolds numbers numbers see see Fig. Fig. 21. 21. [From [From Hunter Hunter and and Coyne Coyne ((1982).1
• eo
J. J. H. H. SS.. BLAXTER
44
SENSE SENSE ORGANS ORGANS
BEHAVIOR BEHAVIOR Length Length
(mm) (mm)
-
Hatch--Hatch
Feeding starts starts
-
10
Eyes we w ellll pigmented pigmented Pure
cone r retina e t i n a present present 30-40 free uromasts 30-40 f r e e ne neuromasts Ol pits O lfactory factory p i t s present present
Respond to t o probes probes
Otic bulla Swallowing behavior behavior
Startle S t a r t l e response r e s p o n s e to to auditory a u d i t o r y stimuli stimuli
-
20
2o
30
-
30
Start Start o off metamorphosis metamorphosis Start S t a r t of of
shoaling shoaling
t -
i
-
-
-
develops
B Buullll a starts s t a r t s to t o fi f i ll l l with w i t h gas gas Lateral L a t e r a l line line
Rods Rods sstart tart
ccanals a n a l s sstart t a r t on head
to t o recruit recruit
Bulla B u l l a usually u s u a l l y gasg a s -fi f i llled led Swimbladder develops develops
Twin
cones c o n e s appear
Pure
cone
area d e v e l o p s area
develops
Retinomotor movements movements commence
40
-
Metamorphosis complete complete --
-
-
Head lateral lateral
line line
complete
Fig. 24. 24. The The development development of of parts parts of of the acoustic acoustic system system and and swimbladder swimbladder of of Atlan Atlantic al. ((1976).] 1976).] tic herring herring Clupea Clupea harengus harengus in in relation relation to to behavior. behavior. [Adapted [Adapted from Allen Allen et et al.
ism, which are quite different from free-living fish larvae. Both the behaviorist and the physiologist need to be aware of the ecology and reproductive habits of their experimental material before making evaluations of their data. XII. MI. CRITICAL PERIODS
The high fecundity of most fish and the low survival rate of of their offspring imply a high mortality from a number of possible causes: inherited defects, egg quality, starvation, disease, predation. Whether these sources of mortality occur continuously or sporadically or
1. 1.
45
PATTERN PATTERN AND AND VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT BEHAVIOR BEHAVIOR
SENSE ORGANS SENSE Time Time Hatch
First F i r s t horizontal horizontal orientation orientation Avoi d obstacles Avoid o b s t a c l e s by mechanoreception mechanoreception Positive P o s i t i v e phototaxis phototaxis First F i r s t feeding feeding
S e l e c t food food ii n sea sea Select
Migrate
coastward coastward
S e t t l e on on substratum substratum Settle
d)) ((d
''> i I
T
Body length length
2.5 2.5
112 2
115 5
16 16
f
Eyes poorly loped p o o r l y deve developed
O l f actory a c t o r y p ii tt ci c i lliated iated Olf P a i r of of Pair
f r e e neuromasts neuromasts on on head free
Otic O t i c hair h a i r ce c ellll ss c ii ll iiaated ted
0 . 38 0.38 33
(mm) (mm)
Two Two pairs pairs o off
free sts f r e e neuroma neuromasts
o n h e a d and t on head t rrunk unk
33.5 .5
w ellll pigmented pigmented Eyes we morphology of of eye Gross morphology
complete
7 .0 7.0
F i r s t taste t a s t e buds on g ii ll ll arches arches First
8 4 8 .. 4
i n oral oral T a s t e buds in Taste
cavity cavity
8.8 B .B
223 3
112.5 2.5
113.3 3.3
225 5
First F i r s t retinal r e t i n a l rods rods
F i r s t head lateral lateral First
l i n e canal canal line
Nares formed Nares
229 9
First F i r s t twin twin
cones cones
Taste buds on on lips lips T a s t e buds Pelagic P e l a g i c tt o benthic b e n t h i c ii n sea sea Positive P o s i t i v e phototaxis phototaxis di i ssaappe p p ears ars Nocturnally N o c t u r n a l l y active active
30 30
333 3
553 3
7 No chemi cal prey chemical p r e y de d etection t e c t i o n 667 Bury Bury entirely e n t i r e l y ii n sand sand
N e g a t i v e l y phototactic phototactic Negatively
7 1 71
1
Complete Complete head late l a t e ral ral
Complete Comp lete
t rrunk u n k lateral lateral t
line line
line line
Fig. 25. The development of of the sense sense organs organs and and behavior behavior in the the Hounder· flounderPamFig. Para oliuaceus. [Adapted [Adapted from from Kawamura and Ishida (1985).] (1985).] Kawamura and lichthys olivaceus.
whether there are particularly "critical “critical periods" periods” of high mortality is is often uncertain. The previous sections sections and and Figs. Figs. 22-25 22-25 illustrate illustrate well the the accretion of new structures and functions functions as as larvae grow grow until the (apart from from reproductive behavior) behavior) is is achieved at full adult repertoire (apart stage. At the present time time it is is not possible to the onset of the juvenile stage. conclude the extent to which developmerit 98 1b, development is is saltatory (Balon, (Balon, 11981b, 1984)or gradual. gradual. Such Such a conclusion requires a thorough study study of phys phys1984) iology and and behavior behavior as as well as as anatomy. anatomy. iology Because Because the larvae lack certain behavioral responses, and because
46
J. J. H. H. S. S. BLAXTER BLAXTER
they are going going through through aa massive massive morphogenesis, morphogenesis, it it seems seems almost almost they are inevitable that critical periods arise through which larvae have to pass inevitable that critical periods arise through which larvae have to pass to allow development to proceed. These critical periods are especially to allow development to proceed. These critical periods are especially related to feeding and predation but but also also to to respiration. respiration. Some Some potenpoten related to feeding and predation tially critical periods may be listed as follows : listed as follows: tially critical periods may 11.. Hatching. This depends hatching en Hatching. This depends on on the the production production of of hatching enzymes to zymes to break break down down the the tough tough chorion chorion that that protects protects the the em embryo from the of wave wave action action or or the pressures pressures and abra and abrabryo from the rigors rigors of sion within within or or on on aa spawning spawning substratum substratum (see (see Blaxter, Blaxter, 1969, 1969, for for sion discussion). aa discussion). 2 . First-feeding. Both high mortality at first-feeding first-feeding in rearing 2. of brood survival under natunatu experiments and and considerations of ral conditions conditions have have suggested suggested in in the the past past that that one one of of the the main main ral phases of larvae change from phases of high high mortality mortality occurs occurs when when the the larvae change from endogenous (sometimes called endogenous to to exogenous exogenous sources sources of of food food (sometimes called the this thesis thesis is the mixed mixed feeding feeding stage). stage). Obviously Obviously relevant relevant to to this is the (see Fig. 19) in relation relation to to the the size size of of prey prey the gape gape of of the the jaw jaw (see Fig. 19) available and quantity and quality of of the May (1974), ( 1974), available and the the quantity and quality the prey. prey. May Vladimirov (1975), ( 1975), and and Blaxter Blaxter (1984) (1984) discuss discuss this and Vladimirov this problem problem and conclude it is is likely likely that mortality often often takes takes place place rather rather conclude that that it that mortality steadily over over the the early early life life history. history. Mortality Mortality rates rates are are somesome steadily times as as high high in in the the egg egg stage stage of of pelagic marine fish fish as as in in the the times pelagic marine larval stage per day), predation can can day), suggesting suggesting that that predation Iarval stage (5-20% per predominate over starvation on some occasions. Furthermore, rearing rates rearing in in the the absence absence of of predators predators can can lead lead to to excellent excellent rates of the major major critical pe 3). First-feeding First-feeding as as the critical peof survival survival (see (see page page 3). riod in development, while sometimes being applicable, should treated with caution as as aa general general concept. concept. with caution should thus thus be treated Respiration. The The disadvantageous disadvantageous decline decline in in body body surface surface area area 3. Respiration. with size in relation to oxygen requirements has already been discussed (Fig. (Fig. 20). 20). There There is is some evidence of of aa high some evidence high phase phase of of discussed mortality associated associated with with early early development development of of the gill filafila mortality the gill ments, for for example, example, in in herring herring (De (De Silva, Silva, 1974). 1974). ments, S wim-up. The The first-filling of the swimbladder (and of the bulla 4. Swim-up. first-filling of the swimbladder (and of the bulla system buoyancy and system in in clupeoids) clupeoids) is is essential essential for for maintaining maintaining buoyancy and other functions such as hearing associated with a gas-filled swimbladder. species, such swimbladder. In In many many species, such as as sea sea bass bass and and sea bream bream (Chatain, 1986), 1986), the the swimbladder swimbladder appears appears soon soon after after hatching. hatching. (Chatain, In the physostomatous salmonids, salmonids, such as brown brown trout, trout, rainrain In the physostomatous such as bow trout, and whitefish whitefish C C.. clupea jormis, the the swimbladder swimbladder is is clupeuformis, bow trout, and by swallowing swallowing air air at the surface surface at the end yolk resorpresorpfilled by at the at the end of of yolk
11.. PATTERN PATTERN AND AND
VARIETY VARIETY IN IN DEVELOPMENT DEVELOPMENT
47
tion (Tait, 1960). If access to the surface is prevented, the (Tait, 1960). pneumatic duct remains open and the swim bladder can be swimbladder filled later. filled later. The The motivation motivation to to fill the the swimbladder swimbladder is is very very strong; Salvelinus (Cristivomer) (Cristivomer) namaycush strong; in in experiments experiments Saiveiinus fill the will swim up for at least 270 m without fatigue to fill swimbladder is empty. swimbladder if if it it is empty. In In physoclists physoclists failure failure to to fill the the swimbladder can lead to abnormal behavior and sometimes to death (e.g., al., (e.g., in mullet, sea bass, and turbot) (Doroshev et ai., 1981). 1981). In clupeoids (Blaxter (Blaxter and Batty, 1985) 1985) avoidance re responses sponses to to sound sound stimuli stimuli fail fail to to develop unless unless the the bulla bulla con contains tains gas. gas. 5. Metamorphosis. 5. Metamorphosis. In In those those species species where where aa marked marked metamor metamorphosis the larval larval stage, stage, aa number number of of major major phosis occurs at at the the end end of the changes changes occur. occur. Increasing Increasing conspicuousness, conspicuousness, as as the the transparent transparent larval is lost, it essential develop other lost, makes makes it essential for for fish fish to to develop other larval form form is protective schooling or or burying burying behavior, behavior, protective mechanisms, mechanisms, such such as as schooling in in order order to to avoid avoid predation. predation. Protective Protective coloration coloration mechanisms mechanisms also also develop develop as as the the scales, scales, pigment, pigment, and and new new chromatophores chromatophores appear. must be vulnerable vulnerable at at this this time, time, especially especially appear. Single Single fish fish must in pelagic habitat, habitat, and and survival survival in in aa schooling schooling species species may may in the pelagic well depend well depend on on early early successful successful aggregation aggregation with with conspecifics. conspecifics. At At or or near near metamorphosis metamorphosis many many marine marine species species move move inshore inshore to need to nursery grounds grounds and and flatfish flatfish need to settle settle on on an an appro approto seek nursery priate such as priate substratum substratum such as sand. sand. XIII. C ONCLU S IONS CONCLUSIONS
The aim of this chapter is to give the less initiated reader, espe especially cially potential potential experimentalists, experimentalists, an an account account of of the the variety variety of of material material now within the This variety now available available within the early early life life history history stages stages of of fish. fish. This variety has has its its attractions attractions and and its its disadvantages. disadvantages. Fish Fish eggs eggs and and larvae larvae present present aa wealth of new new and and interesting interesting problems problems related related to to development. development. In In wealth the the case case of of marine marine fish fish larvae, larvae, new new sources sources of of material, material, hitherto hitherto ne neglected glected because because of of difficulties difficulties in in rearing, rearing, are are now now available. available. The The re research aware of of the pace pace at at which which search worker worker needs, needs, however, however, to to be aware events events are are occurring. occurring. Body Body shape shape is is changing changing rapidly, rapidly, new new external external structures modified, and new internal or modified, and new internal organs organs are are structures are are being being added added or appearing For example, or being being elaborated. elaborated. For example, aa sensory sensory physiologist physiologist appearing or needs some insight needs to to have have some insight into into the the state state of of development development of of the the sense sense organ and its connections to the central nervous system, a student of osmoregulation needs to osmoregulation needs to know know the the state state of of development development of of the the kidney, kidney,
S. BLAXTER BLAXTER H. S. J, J . H.
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and aa nutritionist nutritionist requires requires some some knowledge knowledge of of the the repertoire repertoire and and and activity of the digestive enzymes . Major changes in organs can occur activity of the digestive enzymes. Major changes in organs can occur within the duration of of an experiment. Staging of of experimental material is therefore paramount. material is therefore paramount. Within the material can Within aa species, species, the the viability viability of ofthematerial can depend depend on on parental parental effects size and effects such such as as egg egg size and egg egg quality. quality. Successful Successful experimentation experimentation often requires some some expertise often requires expertise in in husbandry husbandry and and handling handling to to ensure is in in good ensure that that the the material material is good condition condition and and performing performing optimally. optimally. Small size may Small size may demand demand sensitive sensitive and and delicate delicate techniques. techniques. One One of of the the main main rewards rewards for for perseverence perseverence is is that that behavioral behavioral and and physiological physiological experiments experiments can can be be done done in in the the absence absence of of certain certain organs organs or or structures structures and and again again as as such such organs organs and and structures structures develop. develop. Invasive or or ablation ablation techniques techniques can can thus thus be be avoided avoided and and experiments experiments can on intact can be be done done on intact animals. animals.
ACKNOWLEDGMENTS I am am extremely grateful to Dr. L. A. Fuiman, at present present on a National Science Foundation Post Doctoral Fellowship at the Scottish Marine Biological Biological Association, Oban, Oban, for for reading reading and and commenting commenting on on this this chapter chapter in in draft draft form form and and for for making making many many constructive constructive and and useful useful comments comments to to improve improve it. it. The following have been most helpful in correspondence, correspondence, allowing me to use draw drawings, or Balon, Dr. Dr. B. D. D. Glebe, Glebe, Prof. Prof. R. ings, or in in other ways ways providing providing information: information: Prof. Prof. E. K. Balon, D. D. Hoyt, Dr. J. R. Hunter, Dr. A. A. W. Kendall, Jr., Jr., Dr. H. G. G. Moser, Moser, Dr. K. Rana, Prof. Prof. J. S. S. Ryland, Dr. Dr. D. E. E. Snyder, Snyder, Dr. Dr. J. E. E. Thorpe, Thorpe, and Dr. Dr. J.-M. Vernier. II am also grateful to Catriona Stewart, Stewart, who who drew several of the figures. figures.
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Pseudobranchien von Fischen. Fischen. Wilhelm Wilhelm Raux' Rour’ Arch. Arch. Entwicklungsmech. Entwicklungsmech. Org. Org. 145, 145, 549-560. 549-560. Rana, 1985). Influence Rana, K K. J. J. ((1985). Influence of of egg egg size size on on the the growth, growth, onset onset of of feeding, feeding, point-of-no point-of-no1 19return, of unfed unfed Oreochromis Oreochromis mossambicus mossambicus fry. fry. Aquaculture Aquaculture 46, 46,119return, and and survival survival of 131. 131. Ridley, M 1978). Paternal care. M.. ((1978). care. Anim. Anim. Behav. Behav. 26, 26, 904-932. 904-932. Rose, 1960). A 1, Rose, S. S. M. M. ((1960). A feedback feedback mechanism mechanism of of growth growth control control in in tadpoles. tadpoles. Ecology Ecology 441, 188-199. 188-199. Russell, 1976). "Eggs Russell, F. F. S. S. ((1976). “Eggs and and Planktonic Planktonic Stages Stages of of Marine Marine Fishes." Fishes.” Academic Academic Press, Press, London. London. 1966). Observations Ryland, S. ((1966). Observations on on the the development development of of larvae larvae of of the the plaice plaice Ryland, J. J. S. Pleuronectes Cons., Cons. Cons. Int. Pleuronectes platessa platessa L. L. in in aquaria. aquaria. ]. J. Cons., Int. Explor. Erplor. Mer Mer 30, 30, 177-195. 177-195. Schnack, Schnack, D., D., and and Rosenthal, Rosenthal, H. H. (1977/1978). (1977/1978). Shrinkage Shrinkage of of Pacific Pacific herring herring larvae larvae due due to to Ber. Dtsch. 222formalin Dtsch. Wiss. Wiss.Komm. Komm. Meeresforsch. Meeresforsch. 26, 26,222formalin fixation fixation and and preservation. preservation. Ber. 226. 226. Shaw, E. ((1960). 1960). The Zool. 33, Shaw, E. The development development of of schooling behavior in in fishes. fishes. Physiol. Physiol. 2001. 33, 79-86. 79-86. Shaw, 1961). The fishes. II. 34, 263-272. Shaw, E. ((1961). The development of of schooling in in fishes. 11. Physiol. Physiol. Zool. Zool.34,263-272. Shirota, A. sh larvae (in A. (1970). (1970). Studies on the mouth size of fi fish (in Japanese). Japanese). Bull.]pn. Bull.Jpn.Soc. SOC. Sci. 353-368. Sci. Fish. Fish. 36, 36,353-368. Snyder, D. D. E., and Holt, J. J. G. ((1983). 1983). Terminology Workshop. (Mimeo) (Mimeo) Univ. Texas Mar. Sci. Sci. Inst. Port Aransas, Texas. Texas. Solemdal, P. ((1973). 1973). Transfer of of Baltic flatfish to a marine environment and the long term effects on reproduction. 15, 268-276. Oikos 15, 268-276. reproduction. Oikos Sorgeloos, P. (1980). (1980). The use of of brine shrimp Artemia Artemia in aquaculture. aquaculture. In In "The “The Brine Shrimp Artemia" Artemia” (G. (G. Persoone, P. Sorgeloos, O. 0. Roels, and E. Jaspers, eds.), eds.), Vol. 3, 3, pp. 25-46. 25-46. Universa Universa Press, Wetteren, Belgium. 1985). Effects of Springate, J. R. C., and Bromage, N. R R. ((1985). of egg size on early growth and survival in rainbow trout (Salmo (Salmo gairdneri Richardson). Aquaculture 47, 47, 163-172. 163-172. Springate, J. 1984). The J. R. C., Bromage, N. R, R., Elliott, J. J. A. K, K., and Hudson, D. L. ((1984). timing of ovulation and stripping and their effects on the rates of of fertilization and survival to eyeing, hatch and swim-up in the rainbow trout (Salmo R). (Salmo gairdneri R.). Aquaculture 313-322. Aquaculture 43, 43,313-322. Springate, J. R. C., Bromage, N. R, R., and Cumaranatunga, Cumaranatunga, P. R R. T. (1985). (1985). The effects of different fecundity and egg quality in the rainbow trout gairdneri). different ration on fecundity trout (Salmo gairdneri). G.. Bell, “Nutrition and Feeding in Fish" Fish” (C. B. Cowey, A. M. Mackie, and J. G In "Nutrition eds.), pp. 371-393. 371-393. Academic Press, London. eds.), Strauss, R. R E., and Fuiman, L. A. (1985). (1835). Quantitative Quantitative comparisons of of body form and Straws, Can.]. 2001.63, Zool. 63, (Teleostei: Cottidae). Cottidae). Can.]. allometry in larval and adult Pacific sculpins (Teleostei: 1582-1589. Stunner, L. N., Sturmer, N., McCarthy, C. E., and Rutledge, W. P. (1985). ( 1985). Hatchery production production of of red Larval Fish Con$ Conf Am. Fish. SOC., Soc., drum fingerlings in Texas. Abstr. 9th Annu. Larval 1985. 1985. Swain, D. P., and Lindsey, C. C. (1984). ( 1984). Selective predation for vertebral of vertebral number of Gasterosteus aculeatus. aculeatus. Can. Can. J].. Fish. Fish. Aquat. Aquat. Sci. Sci. 41, 41, 12311231young sticklebacks Gasterosteus 1233. 1233. Symons, P. E. E. K. K ((1968). 1968). Increase in aggression and in strength of of the social hierarchy among of food. J].. Fish. Fish. Res. Res. Board Board Can. Can. 25, 25, among juvenile Atlantic Atlantic salmon deprived of 2387-2401. 2387-240 1. Can . J].. Zool. Zool. 38, 38, Tait, J. S. S. (1960). (1960). The first filling ooff the swim bladder iinn salmonids. Can. 179-187. 179187. -
_
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Tanaka, M. (1973). (1973). Studies in the structure and function of the digestive system of of teleost larvae. D. Agric. Thesis, Kyoto University, Japan. Taning, Bioi. Rev. (1952). Experimental study of of meristic characters in fishes. Biol. Rev. T h i n g , A. V. (1952). Cambridge Philos. Soc. SOC. 27, 169-193. Theilacker, C. 1978). Effect G. H. ((1978). Effect of of starvation on the histological and morphological Fish. Bull. Bull. 76, 403characteristics Trachurus symmetricus symmetricus larvae. Fish. 76,403characteristics ofjack mackerel Trachurus 414. Theilacker, C. G. H H.. (1980a). (1980a). Changes in body measurements of of larval northern anchovy Engraulis Bull. 78, Engraulis mordax mordax and other fishes due to handling and preservation. Fish. Fish. Bull. 685-692. 685-692. Theilacker, C. H. (1980b). Theilacker, G. H. (1980b). Rearing container size affects morphology and nutritional condition of larval jack mackerel Trachurus Bull. 78, 789-791 . Trachurus symmetricus. symmetricus. Fish. Fish. Bull. 78,789-791. 1981). Effect o Theilacker, C. G. H H.. ((1981). off feeding history and egg size oonn the morphology of of Rapp. P.-V. Cons. Int. jack mackerel, mackerel, Trachurus Trachurus symmetricus. symmetricus. larvae. Rapp. P.-V.Reun. Reun. Cons. Znt. Explor. Explor. Mer 432-440. Mer 178, 178,432-440. Dorsey, K. (1980). Theilacker, C. (1980). Larval fish diversity, a summary of of laboratory G. H., and Dorsey, and field research. Intergov. (IOC) Workshop 105-142. Intergou. Oceanogr. Oceanogr. Comm. Comm. (ZOC) Workshop Rep. Rep. 28, 28,105-142. Thorpe, 1984). Development rate, fecundity and egg J. E., Miles, Miles, M M.. S., and Keay, Keay, D. D. S. S. ((1984). Thorpe, J. size in Atlantic salmon Salmo 289-305. Salmo salar salar L. Aquaculture Aquaculture 43, 43,289-305. Tucker, J. Chester, A. J. J, W., Jr., and Chester, J. (1984). (1984). Effects of salinity, formalin concentration concentration and buffer on quality of preservation of southern flounder (Paralichthys (Paralichthys lethos Zethostigma) Copeia, pp. 981-988. 981-988. tigma) larvae. Copeia, F., Leger, LBger, P., P., and Sorgeloos, P. (1985). (1985). Preliminary Amat, F., Hontoria, F., van Ballaer, E., Amat, results on the nutritional Artemia nauplii for nutritional evaluation of w3-HUFA-enriched o3-HUFA-enriched Artemia 49, 223-229. larvae of the the sea bass Dicentrarchus labrax. labrax. Aquaculture 49,223-229. van tin, C., and Pool, C. van Raamsdonk, W., W., van't van’t Veer, L., L., Veeken, K., Hey Heytin, C. W. W. (1982). (1982). Differentiation Brachydanio rerio, rerio, the the zebrafish. Differentiation of muscle fiber types types in the teleost Brachydanio Anat. 51-62. Anat. Embryol. Embryol. 164, 164,51-62. Vernier, J.-M. ((1969). chronologique du developpement embryonnaire de la Vernier, 1969). Table chronologique truite arc-en-ciel, Salmo Salmo gairdneri gairdneri Rich. 1836. 1836. Ann. Ann. Embryol. Embryol. Morphog. Morphog. 2, 495495520. 520. Vladimirov, V. I. 1975). Critical periods in the development of fishes (translated I. ((1975). (translated from Russian). ]. J . Ichthyol. Ichthyol. 15(6), 15(6), 851-868. 851-868. Wallace, J. C., C., and Aasjord, D. (1984). (1984).An investigation investigation of the the consequences of egg size Wallace, J. Arctic chaIT, cham, Salvelinus Saluelinus alpinus (L.). (L.).I. J. Fish. Fish. Bioi. Biol. 24, 24,427-435. for the culture of Arctic 427-435. T. ((1985). nutrition ffor develor further devel Watanabe, T. 1985). Importance of the study of broodstock nutrition opment of aquaculture. In "Nutrition “Nutrition and Feeding in Fish" Fish” (C. (C. B B.. Cowey, A. M M.. G. Bell, eds.), ppp. 394-414. Academic Press, London. London. p . 394-414. MacKie, and J. C. Watanabe, Y. 1985). Histological changes Y. ((1985). changes in the liver and intestine of freshwater goby larvae during short-term starvation. Bull. Bull. lpn. Jpn. Soc. SOC. Sci. Sci. Fish. Fish. 51, 707-709. 707-709. Watanabe, T., T., Kitajima, Kitajima, C., C., and Fujita, S. S. (1984). (1984). Effect of of nutritional nutritional T., Arakawa, T., quality on reproduction reproduction of of red red sea sea bream. bream. Bull. Bull. lpn. Jpn. Soc. S O C . Sci. Sci. quality of of broodstock broodstock diets diets on Fish. 50, 495-501 495-501. . Fish. 50, Webb, W. (1975). (1975). Hydrodynamics Hydrodynamics and and energetics energetics ooff fish fish propulsion. propulsion. Bull. Bull. Fish. Fish. Res. Res. Webb, PP.. W. Board Can. Can. 190, 190, 1-158. Webb, Webb, P. P. W., W., and and Weihs, Weihs, D. D. (1986). (1986). Functional Functional locomotor locomotor morphology morphology of of early early life life history 15-127. Trans. Am. Am. Fish. Fish. Soc. SOC. 115, 115, 1115-127. history stages. stages. Trans. Weihs, D. (1980a). (1980a). Energetic significance of �hanges changes in swimming swimming modes modes during Weihs, D. growth 597-604. Engraulis mordax. mordax. Fish. Fish. Bull. Bull. 77, 77,597-604. growth of larval anchovy Engraulis Weihs, 1980b). Respiration Weihs, D. D. ((1980b). Respiration and and depth depth control control as as possible possible reasons reasons for for swimming swimming of of northern anchovy Engraulis 17. Engraulis mordax mordax yolk-sac larvae. Fish. Fish. Bull. Bull. 78, 78, 109-1 109-117.
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Whipple, ]., J., Eldridge, M., M., Benville, P., Bowers, M., Jarvis, Jarvis, B., and Stapp, N. (1981). (1981). The condition and viability in striped bass effect of inherent parental factors on gamete condition Morone. Rapp. P.-V. Reun., 93-94. Reun., Cons. Cons. Int. Znt. Explor. Erplor. Mer 178, 178,93-94. Winberg, G. G. (1960). (1960). Rate of of metabolism metabolism and food requirements of of fishes. Fish. Fish. Res. Res.
Board Can. Transl. Board Can. Transl. 194. 194.
J. (1979). (1979). Energy costs of of egg production production and environmental determinants of of Wooton, R. ]. fecundity in teleost fish. Symp. Zool. Soc. Soc. London 44, 133-159. 133-159. Zeutzius, I., I., and Rahmann, H. ((1984). 1984). Infl uence of dark-rearing on the ontogenetic Influence (Cichlidae Teleostei). I. Effects of Sarotherodon mossambicus mossambicus (Cichlidae Teleostei). I. of development of Sarotherodon Biol. body weight, body growth pattern, swimming activity and visual acuity. Exp. Bioi. 43, 77-85. 43,77-85. Zeutzius, t, 1984). Influence of Zeutzius, I., Probst, W., and Rahmann, H. ((1984). of dark-rearing on the (Cichlidae, Teleostei). ontogenetic Sarotherodon mossambicus mossambicus (Cichlidae, Teleostei). II. 11. ontogenetic development of of Sarotherodon Effects on allometric allometric and growth relations and differentiation differentiation of of the optic tectum. Exp. Erp. Bioi. Biol. 43, 87-96. 87-96.
2 RESPIRATORY GAS EXCHANGE, AEROBIC METABOLISM, AND EFFECTS OF HYPOXIA DURING EARLY LIFE
ROMBOUGH PETER J].. ROMBOUGH Department Zoology Department Brandon Brandon University University Brandon, anitoba, Canada R7 A 6A9 Brandon, M Manitoba, R7A 6A9 11.. Introduction II. Exchange 11. Respiratory Respiratory Gas Gas Exchange A. A. The The Boundary Boundary Layer Layer B. The B. The Egg Egg Capsule Capsule C. C. The The Perivitelline Perivitelline Fluid Fluid D. Cutaneous Gas Exchange E. Respiratory Pigments F. F. Branchia Branchiall Gas Gas Exchange III. 111. Aerobic Aerobic Metabolism Metabolism A. A. Measurement Techniques B. B. Biotic Biotic Factors C. C. Abiotic Abiotic Factors Factors IV. IV. Effect Effect of of Hypoxia Hypoxia A. A. Environmental Environmental Hypoxia Hypoxia B. Physiological Physiological Hypoxia Hypoxia V. V. Conclusions References References
I. INTRODUCTION The The basic basic mechanisms mechanisms involved involved in in respiratory respiratory gas gas exchange exchange in in juvenile juvenile and and adult adult fish fish are are fairly fairly well well established established (see (see reviews reviews by by Jones Jones and and Randall, Randall, 1978; 1978; Randall, Randall, 1982; 1982; Randall Randall et al., al., 1982; 1982; Randall Randall and and Daxboeck, Daxboeck, 1984). 1984). The The study study of of oxygen oxygen metabolism metabolism in older older fish, fish, similarly, 1 ; Beamish, similarly, is is well well advanced advanced (reviewed (reviewed by by Fry, Fry, 1957, 1957, 197 1971; Beamish, 1978; 1978; Brett Brett and and Groves Groves,, 1979; 1979; Tytler Tytler and and Calow, Calow, 1985). 1985). In In contrast, contrast, relatively relatively little little is is known known of of respiratory respiratory gas gas exchange exchange and and energy energy usage usage during during early early life. life. This This arises arises not not so so much much from from aa lack lack of of effort effort on on the the FISH FISH PHYSIOLOGY. PHYSIOLOGY, VOL. VOL. XIA XIA
59 59
Copyright Copyright © 0 1988 1988 by by Academic Academic Press, Press, Inc. Inc. All orm reserved. All rights rights of of reproduction reproduction in in any any fform reserved.
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PETER JJ.. ROMBOUGH
part of of researchers-in researchers-in excess of 500 papers dealing with various as aspects of oxygen supply and demand during early life have been pub published in the last 20 years-but years-but rather from the lack of of a systematic approach approach to to the the problem. problem. The The aim aim of of this this review review is is to to collate collate the the large large amounts data that that are are currently currently available available and and to to fi fitt it it into into aa concep concepamounts of data tual framework that can be used as the basis for future investigations. investigations.
II. 11. RESPIRATORY GAS EXCHANGE
Analytical models provide a useful framework for the study of of re reexchange. The cascade model, in particular, has been spiratory gas exchange. used widely to describe various aspects of vertebrate respiratory func func(Dejours, 198 1981; 1982; Weibel, 1984; 1984; DiPrampero, 1985). 1985). tion (Dejours, 1 ; Piiper, 1982; In this model, respiratory gases are viewed as passing through a series specific of resistances, each of which is correlated with a specifi c process or structure. indi of the indistructure. The overall resistance of of the system is the sum of steady-state conditions, overall flow vidual resistances and, under steady-state through through the the system system is is equal equal to to the the flow flow through through each each of of the the elements. elements. The model is especially useful in helping to define the nature and magnitude of the various resistances in the respiratory pathway and the partial pressure gradients necessary to overcome them. Such analanal ysis is complicated for early life stages because of changes in respira respiratory rate and the nature and relative importance of of resistances during development. development. These problems, while formidable, formidable, are are not not insurmount insurmountable, of the cascade model able, as evidenced by the successful application of to the study of gas transport in mammalian (Dejours, (Dejours, 1981) 1981) and avian embryos (Dejours, (Dejours, 198 1981; 1984). Unfortunately, 1 ; Piiper and Scheid, 1984). insufficient information to apply the model rigor rigorthere is currently insufRcient ontogeny. However, ously to the study of of gas exchange during teleost ontogeny. enough enough is known known of of gas gas exchange exchange in in developing fish to to use use the the cascade cascade model in a more general way, that is, as a guide to help identify and major resistances resistances during development. development. describe the major In many ways gas exchange is very similar in embryos and larvae, particularly once organogenesis is complete. In both stages gas exex change is primarily cutaneous. Branchial exchange typically becomes dominant only near the end of the larval period. However, in spite of of sepathis similarity, it is often convenient to treat embryos and larvae sepa of the major impact the egg capsule (zona radiata) radiata) has rately because of on gas exchange during embryonic life. The egg capsule, in addition significant to acting as a signifi cant barrier in its own right, creates two other barriers, uid, that barriers, the the external external boundary boundary layer layer and and the the perivitelline perivitelline fl fluid, that together ). together have have an an equal equal or or greater greater impact (Fig. (Fig. 11).
2. 2. RESPIRATORY RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM
61 61
boundary l ayer
t:..I,....\-- pore canal
vitell ine membrane
perivite l l i ne space
Fig. Fig. 1. 1. Schematic Schematic diagram diagram showing showing the the major major resistances resistances to to respiratory respiratory gas gas ex exchange change for for fish fish embryos embryos (not in proportion). proportion). The heavy arrow indicates indicates the direction of of water water How. flow. The The actual actual shape shape of of the the trailing edge edge of of the the boundary boundary layer layer will will depend depend on on water water velocity velocity and and egg egg size. size.
A. A. The Boundary Layer The laminar boundary layer is is a semistagnant region of water adja adjacent to the egg urface where oxygen egg ssurface oxygen is depleted and metabolic wastes accumulate. The The boundary layer actually has no outer limit but for s ually defined as for practical purposes is uusually as the distance from from the egg surface where local local conditions are equivalent to 99% 99% of free-stream conditions conditions (Vogel, (Vogel, 1981). 1981). For the laminar flow flow regimes that eggs are typically exposed to (Reynolds (Reynolds numbers < < 75; 75; Johnson, 1980), 1980), the thickness of ize and in of the boundary layer is proportional to egg ssize in1965) indicated that versely proportional to water water velocity. velocity. Daykin ((1965) the the thicknes thicknesss of the solute boundary layer, layer, d" d,, is is less than the thick thickness of the velocity boundary layer and can be estimated from from the Sherwood Sherwood number (Sh) (Sh) and egg diameter (d) (d) using the equation
d,
=
d(Sh-')
(1) (1)
PETER PETER J. ROMBOUGH ROMBOUGH
62
The Sherwood number number is number dependent dependent on The Sherwood is aa dimensionless dimensionless number on two other other dimensionless dimensionless numbers, numbers, the the Reynolds number (Re) (Re) and and the the Schmidt number ((Sc). Sc). For spherical eggs, Sh can be estimated as
+
l3 · Re) lIZ Sc1 = 2.0 + 0.8(B 0.8(B.Re)1'2 Sclm Sh =
(2) (2)
dv 1 , Sc where l , and B where Re = = p, pdu-l, Sc = = VDOD-', B is is the the ratio ratio of of the the interstitial interstitial and and bulk bulk velocities velocities (Daykin, (Daykin, 1965; 1965; Johnson, Johnson, 1980). 1980). Here Here p, p is is bulk bulk water water velocity, velocity, d is is egg egg diameter, diameter, vu is is kinematic kinematic viscosity, viscosity, and and D D is is the the diffusion diffusion coefficient. coefficient. Interstitial Interstitial velocity velocity is is equal equal to to bulk bulk velocity velocity for for isolated eggs. eggs. According to Eq. (2), (2),the thickness of the oxygen bound boundary layer would be about 0.02 0.02 and 0.05 0.05 cm, respectively, for single eggs with diameters of 1 and 0.5 cm in a 100 of 0. 0.1 100cm h-1 h-l current. Increas Increasing current velocity to 1000 1000 cm h-1 h-' would reduce boundary layer thickness to approximately 0.0008 0.0008 and 0.02 cm, respectively. The higher metabolic (Winnicki, 1968; DiMichele and Powers, 1984a) (Winnicki, 1968; 1984a) and growth (Silver et al., 1963; Shumway et al., al., 1963; al., 1964) 1964) rates reported at higher water velocities are probably due to reductions in boundary boundarylayer thickness (Daykin, 1965; Wickett, 1975). 1975). Estimating interstitial (Daykin, 1965; velocities is a problem for eggs laid in masses or in substrate. If If a hexagonal array is assumed, the interstitial velocity in an egg mass averages about 9. 1 times the bulk velocity through the mass (Daykin, 9.1 (Daykin, 1965; Wickett, 1975). esti 1965; 1975). Interstitial velocities in substrate can be esti1 mated from porous bed theory as P,i = p,e, where P,i is interstitial pi = ~ E - I , pi velocity, p, p is bulk velocity, and eE is the empirically determined poros porosity (Johnson, (Johnson, 1980). 1980). The driving force required to overcome the resistance imposed by the boundary layer can be predicted by rearranging the Fick's equa equation for diffusion through a plane to yield -
C c11 -- C coo == YOzds(47TrZD)-1 b02dS(4~r2D)-l
(3) (3)
where C C11 is the free-stream oxygen concentration, C Coo is the oxygen concentration at the egg surface, YOz VO2 is the rate of oxygen consump consumpuivalent to the more tion, and rr is the egg radius. This equation is eq equivalent widely used mass transport equation (Daykin, 1975; (Daykin, 1965; 1965; Wickett, 1975; Johnson, 1980) 1980) in which the reciprocal of the mass transport constant, 1 , replaces dsD-l. kk-l, d,D-'. The value of k is estimated as k = = (Sh)Dd-1• (Sh)Dd-'. E quation (3) Equation (3) indicates that the driving force required to meet meta metabolic demands is directly proportional to the rate of oxygen consump consumption and thus increases more or less steadily throughout embryonic development. Daykin ((1965) 1965) estimated that a partial pressure gradient across across the boundary layer of about 52 mm Hg was required to meet the oxygen requirements of chum (Oncorhynchus 0.37 cm) (Oncorhynchus keta) keta) eggs (r (T = = 0.37 cm) near hatch at 10°C 10°C and a flow rate of of 85 cm h-1• h-l. Smaller pressure
2. 2.
RESPIRATORY AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC METABOLISM
63
differences are required for smaller eggs because of generally lower metabolic rates and thinner boundary layers. layers. For example, Wickett (1975) 0.05 cm) (1975) estimated that cod (Gadus (Gadus macrocephalus) eggs (r (T = = 0.05 cm) incubated at 5°C - 1 would require a pressure 5°C in a current of 170 170 cm h h-’ difference across the boundary layer of only about 17 17 mm Hg to fully satisfy satisfy their oxygen requirements. Until recently it was assumed that forced convection (i.e., bulk water flow) flow) was the major means of of supplying oxygen to eggs. It now appears that under certain circumstances natural (free) (free) convection may be important as well. Embryonic metabolism gives rise to solute concentration gradients across the boundary layer. O'Brien O’Brien et al. al. ((1978) 1978) have shown that the oxygen-depleted, carbon dioxide-rich wa water immediately adjacent to the egg is is denser than the well oxygen oxygenated, low C O2 water in the free stream. COZ stream. In still water, this sets up a toroidal flow as the denser solution adjacent to the egg sinks (Fig. (Fig. 2). 2). Water velocities in the toroid can be relatively high. For example, O'Brien al. ((1978) 1 978) observed an average velocity of 72 cm h-l h-1 in the O’Brien et al. toroid set up by eyed eggs (400 (400 degree-days, 100C) 10°C)of coho salmon (0. (0. kisutch). kisutch).At this velocity, natural convection would be about 150 times as eeffective ffective as simple diffusion in supplying oxygen under "“static” static" conditions. The effectiveness of of natural convection can be expected to increase as metabolic rate increases, due to greater depletion of of oxy oxygen and accumulation of carbon dioxide in the boundary layer. Thus, natural convection may act as a homeostatic mechanism helping to balance oxygen supply and demand. Whether natural convection plays a significant role in nature will depend on bulk water velocity and the orientation of the egg mass. Analysis vecAnalysis of mixed regimes is complex but, in general, natural con convec-
.... , .."--- ,,, /' J A' " I , "
Fig. 2. Stylized depiction of the toroidal toroidal fl flow around a respiring ow of water set up around respiring egg as a result of natural convection in the absence of bulk water movements. [From [From 1978).] O'Brien O’Brien et al. al. ((1978).]
64
PETER J. ROMBOUGH PETER J. ROMBOUGH
tion will play a greater role than forced convection at low bulk veloci velocities [see [see Vogel ((1981, 198 1, pp. 178-195) on how the ratio of of the Grashof number and the square of of the Reynolds number can be used to esti estimate the relative importance of ] . In nature, bulk veloci of the two forces forces]. velocities are often low. For example, the bulk flow in many spawning beds is less than the toroidal velocity set up by coho eggs in still water (O'Brien (O’Brien et al., 1978). 1978). Similarly, interstitial velocities in egg masses of species such as lumpfish (Cyclopterus (Cyclopterus lumpus) lumpus) and lingcod (Ophiodon (Ophiodon elongatus) elongatus) can be expected to be rather low. low. It has been suggested, but not demonstrated, that voids in such masses may pro provide avenues for natural convection to supply oxygenated water to the interior (O'Brien (O’Brien et al., 1978; 1978; Giorgi and Congleton, 1984). 1984). Natural convection will be most effective if eggs are oriented so that the heavier oxygen-depleted water can sink and its movement is is not op opal., 1978; 1978; Johnson, 1980). posed by forced convection (O'Brien (O’Brien et al., 1980).
B. The Egg Capsule The egg capsule traditionally has been viewed as the major barrier to diffusive gas exchange during embryonic life. This may seem obvi obvious, but the empirical evidence supporting this viewpoint is is actually rather scanty. Strongest support comes from observations that incipi incipient limiting oxygen tensions (Pc) (P,)drop significantly on removal of of the capsule (Hayes (Hayes et al., 1951; 1951; Rombough, 1986, 1986, 1987). 1987). If If metabolic rate and the drop in Pc P, are known, the diffusion coefficient of the capsule (Dc) Fick's equation for diffusion through a (DJ can be calculated using Fick’s plane [Eq. [Eq. (3)]. (3)]. Several investigators have done this (Hayes (Hayes et al., 1951; 1951; Alderdice et al., al., 1958; 1958; Daykin, 1965; 1965; Wickett, 1975), 1975), but the 1 , is that worked out by 2 Sx 10-5 cm cm2 s-l, value most often cited, 0.18 x Wickett (first (first presented presented in Daykin, 1965) 1965) using data provided by Hayes et al. ((1951) 1951) for Atlantic salmon (Salmo 1975) (Salmo salar). salar). Wickett ((1975) recognized that his estimate was based on rather sketchy data and indicated that standard diffusion tests should be conducted to check its validity. Unfortunately, this has not been done for teleosts, al al(1987) recently used a Krogh-type Krogh-type diffu diffuthough Diez and Davenport (1987) sion chamber to estimate the oxygen oxygen diffusion coefficient of sion of the egg case of the dogfish, Scyliorhinus canicula. canicula. Interestingly, the value 1 , is 2 Sthey arrived at, 0.285 x Wickett's value for X 10-5 cm cm2 s-l, is similar to Wickett’s Atlantic salmon. However, given the structural differences between the dogfish egg case and the salmon capsule, this cannot be taken as confirmation of Wickett's Wickett’s value.
2. 2.
RESPIRATORY GAS EXCHANGE, AEROBIC METABOLISM RESPIRATORY GAS EXCHANGE, AEROBIC METABOLISM
65
Wickett’s value for D, of water. Wickett Wickett's Dc is about one-tenth that of pene((1975) 1975) pointed out that the surface area of the radial pores that pene
trate the capsule is similarly about one-tenth the total surface area of of the capsule and speculated that this may indicate that diffusion takes place primarily through the pore canals rather than through the cap capsule matrix. If this is true, true, doubt is cast on on the the validity of the the practice sule matrix. (e.g., Daykin, 1965; 1965; Wickett, 1975; 1975; Kamler, Kamler, of some investigators (e.g., 1976; 1984) applying diffusion coefficients, 1976; DiMichele and Powers, 1984) calculated for one species, to another unrelated species. There is con considerable (Lfbnning, siderable variation variation among among teleosts teleosts in in capsule capsule structure structure (Lqhning, 1972; Stehr 1979; Groot 1985). Even 1972; Stehr and and Hawkes, 1979; Groot and and Alderdice, Alderdice, 1985). Even in in closely related salmonids, salmonids, pore pore area can vary anywhere between 7% and Groot and 30% of of the the total total surface surface area area ((Groot and Alderdice, Alderdice, 1985). 1985). and 30% Recent evidence suggests that the capsule may not be as great a barrier to diffusive gas exchange Bereexchange as was supposed previously. Bere zovsky 1979) used microelectrodes zovsky et aI. al. ((1979) microelectrodes to to measure measure the the dissolved dissolved oxygen uid, and capsule, perivitelline perivitelline fl fluid, and vitelline oxygen profile profile across across the capsule, membrane fossilis). membrane of of the the recently recently fertilized fertilized loach loach eggs eggs (Misgurnis fossilis). Surprisingly, they recorded very little drop in oxygen concentration across across the the capsule. capsule. In contrast, contrast, there there was was aa gradual gradual decline decline in in oxygen oxygen tension across the perivitelline fluid and a sharp drop across the vitel vitel(Fig. 3). 3). Sushko ((1982) 1982) reported a similar oxygen pro proline membrane (Fig. fi le for loach eggs incubated in helium file helium-oxygen nitrogen-oxygen -oxygen and nitrogen-oxygen gas Alderdice et aI. al. (1984) (1984) may may pro progas mixtures mixtures.. Observations Observations made made by Alderdice vide an explanation for why the capsule appears to offer relatively little resistance to gas exchange. exchange. Alderdice et al. al. (1984) (1984) observed that the hydrostatic pressure exerted on the capsule of steelhead (S. (S. gairdneri) gairdneri) was considerably less than the osmotic pressure of the perivitelline fluid and calculated an perivitelline fluid and calculated an effective effective filtration filtration pressure pressure of of about -62 -62 mm Hg driving water into the perivitelline space. space. They reasoned reasoned that that according according to to Starling's Starling’s hypothesis hypothesis this this should should lead lead to to the the movement movement of of water water into into the the perivitelline perivitelline space. space. Since Since the the egg egg is is in in volume volume equilibrium, equilibrium, this this must must be be balanced balanced by by an an equal equal outflow outflow by by fi ltration. Exactly is not not clear, clear, since since filtration. Exactly how how this this would would be be accomplished accomplished is the the capsule is not a linear structure like a capillary. Alderdice et al. (1984) (1984) suggested that the capsule may act like a balloon with micro microsieves accompanied sieves in in its its wall. wall. Increasing Increasing internal internal pressure pressure would would be accompanied by volume expansion. As the capsule expanded the pores would enen large and more water would fl ow out. Volume and tension increases flow thus (1984) proposed proposed that that this this thus would would be be self-limiting. self-limiting. Alderdice Alderdice et al. (1984) process process would would tend tend to to facilitate facilitate respiratory respiratory gas gas exchange exchange and and point point to to radiotracer and Rudy, (Potts and Rudy, 1969; 1969; Loeffler Loeffler and and Lovtrup, Lovtrup, 1970; 1970; radiotracer studies studies (Potts
PETER J. ROMBOUGH PETER J . ROMBOUGH
66
1 60
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I
140 1 1 20 E E ...
I I I I I I I I I I I I I I I I I bl I I I
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Z III c:I >)C o
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: 2ow
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Fig. Fig. 3. 3. Oxygen Oxygen profiles across across recently recently fertilized fertilized roach roach eggs measured using plati platinum microelectrodes. symbols, normal eggs; symbols, eggs in which rate eggs; closed symbols, microelectrodes. Open symbols, of oxygen oxygen uptake uptake was stimulated 3.3-fold 2,4-dinitrophenol;pvf, pvf, perivitel perivitel3.3-foldusing 10-4 M 2,4-dinitrophenol; layer. [After [After Berezovsky et al. al. (1979).] (1979).] fluid; line fl uid; bl, boundary layer.
Loeffler, LoeMler, 1971) 1971) indicating an exchange of water across the capsule equivalent to the volume of the perivitelline fluid every 1-4 1-4 min. A connective flux of this magnitude would add to the diffusive flux of of oxygen across the capsule but, perhaps more importantly, the currents generated would tend to prevent the establishment of of a large concenconcen tration gradient across the capsule. capsule. C. The Perivitelline Fluid As noted earlier, microelectrode studies (Berezowsky et al., al., 1979; 1979; Sushko, 1982) 1982) indicated that, at least for early embryos, much of the Sushko, acturesistance to gas exchange previously attributed to the capsule actu ally resides in the perivitelline fluid. The perivitelline fluid can be expected to have an oxygen diffusion coefficient similar to that for ~ 2 . 5x water ((=2.5 X 10-5 cm2 cm2 s-l) S - I ) (Dejours, (Dejours, 1981), 1981), but because diffusion of the perivitelline perivitelline fluid may distances are much larger the net impact of of the capsule. capsule. While capsule thicknesses in be greater than that of
2. 2.
RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM RESPIRATORY
10 1 0-
-!
gI
IJJ w
....J
7
....J
6
> > IJJ z IJJ
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x o
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5
fi1 4 � � :3 (j) B
l> _ 6°C 6'C 90C o 1 2°C • 15°C
9
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67
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o
P50=0.968+ 0 . 2 3 7 ( V O ~ )
2 4 6 8 1 0 12 1 4 1 6 8 10 12 14 16 METABOLIC p g02 0 2 individual-' META BOLIC RATE ((�g Individual- I h-'1 h-I ) I
Fig. 4. Relationships between critical dissolved oxygen levels (P,) (Pc) and. and routine "02 = metabolic rate and Pso concentration at which V02 = 0.5 rV02) rV02) and Pa values (oxygen concentration routine routine metabolic rate for steelhead embryos embryos incubated incubated at constant constant temperatures. temperatures. Equa Equaportions of of the curves. curves. [From [From Rombough Rombough (1987).] (1987).] tions are for the linear portions
salmonids range between /Lm (Groot Alderdice, 1985), 1985), salmonids range between 15 and and 70 pm (Groot and and Alderdice, the across the perivitelline perivitelline space in excess excess of of 500 /Lm. the distance distance across space can can be in pm. Absolute distances distances tend tend to smaller in in pelagic pelagic eggs, eggs, but but because because of of aa Absolute to be smaller thinner capsule capsule and larger amount amount of of perivitelline perivitelline thinner and comparatively comparatively larger fluid, the the relative relative impact perivitelline fluid on gas exchange fluid, impact of of the the perivitelline fluid on gas exchange would be even larger in salmonids. would be even larger than than in salmonids. The impact the perivitelline uptake can impact of of the perivitelline fluid fluid on on oxygen oxygen uptake can be seen seen in P, values values during during early early development development (Rom (Romin the the rapid rapid increase increase in in Pc bough, bough, 1987). 1987). Critical Critical oxygen oxygen tensions tensions for for steelhead steelhead increase increase very very rapidly rapidly in in relation relation to to metabolic metabolic rate rate until until about about the the time time embryos embryos began move and uid. Thereafter, values P, values began to to move and stir stir the the perivitelline perivitelline fl fluid. Thereafter, Pc increase more increase more slowly slowly and and in in direct direct proportion proportion to to metabolic metabolic rate rate (Fig. (Fig. 4). sense, stirring 4). Thus Thus in in aa teleological teleological sense, stirring of of the the perivitelline perivitelline appears appears to to be if metabolic met. RezniReznibe necessary necessary if metabolic oxygen oxygen demands demands are are to to be met.
68
PETER PETER J. J. ROMBOUGH ROMBOUGH
chenko et al. al. (1977) polarographic electrode chenko (1977) used used aa modified modified polarographic electrode to to model model oxygen uid. H Hee oxygen exchange exchange across across the the egg egg capsule capsule and and perivitelline perivitelline fl fluid. found found that that when when the the analog analog of of the the perivitelline perivitelline fluid fluid was was stirred, stirred, the while that PO22 at at the the body body (electrode) (electrode) surface surface increased increased while that under under the the the P0 capsule decreased. This This had capsule (membrane) (membrane) decreased. had the the effect effect of of increasing increasing the the steepness en steepness of of the the concentration concentration gradient gradient across across the the capsule capsule and and enhancing 1983) hancing net net oxygen oxygen transport. transport. Peterson Peterson and and Martin-Robichaud Martin-Robichaud ((1983) observed observed that that Atlantic Atlantic salmon salmon embryos embryos began began to to stir stir the the perivitelline perivitelline fluid fluid fairly fairly early early in in development. development. Trunk Trunk movements movements began began abruptly abruptly at at about 200 degree-days with an of degree-days of of development development with an initial initial frequency frequency of about about 60-120 - I . Dye Dye studies exures 60-120 flexures flexures h h-l. studies indicated indicated that that trunk trunk fl flexures about resulted movement along resulted in in rapid rapid water water movement along the the trunk trunk and and from from one one side side of uid to of the the perivitelline perivitelline fl fluid to the the other. other. These These movements movements were were appar apparently of nature, since ently of aa respiratory respiratory nature, since an an unexpected unexpected water water failure failure lead leading hypoxia resulted ing to to hypoxia resulted in in an an increase increase in in frequency frequency of of trunk trunk flexures flexures.. Trunk Trunk movements movements normally normally decline decline rather rather abruptly abruptly to to aa frequency frequency of of only every 2 2-4 350-400 degree-days degree-days in in Atlantic Atlantic salmon salmon (Peter (Peteronly 11 every -4 h by 350-400 son and 1983). However, However, by by this this time time the the embryo embryo son and Martin-Robichaud, Martin-Robichaud, 1983). had begun to ns rapidly 40-150 min-I. min-l. had begun to move move its its pectoral pectoral fi fins rapidly at at aa rate rate of of 40-150 This This rate rate was was maintained maintained until until hatch. hatch. Dye Dye studies studies indicated indicated that that these these movements generated a rapid water fl ow (=300 (=300 cm h-1) h-l) in the imme immeflow diate ns but ex flexdiate area area of of the the pectoral pectoral fi fins but were were not not as as effective effective as as trunk trunk fl ures in completely mixing the perivitelline fluid. fluid. Complete Complete mixing was exures. periodic trunk trunk fl flexures. was accomplished accomplished by periodic Recent Recent studies studies of of amphibians amphibians suggest suggest that that stirring stirring of of the the perivitel perivitelline line fluid fluid may may facilitate facilitate oxygen oxygen transport transport within within an an egg egg mass mass as as well well as as individual eggs. Burggren ((1985) within individual 1985) noted that oxygen partial pressures were higher and carbon dioxide partial pressures were lower in the interior of the egg mass of the frog Rana palustris than simple diffusion diffusion was was the only process process involved. involved. would be b e expected expected if simple perivitelline fl fluid. (1985) Frog embryos use cilia to stir the perivitelline uid. Burggren (1985) cilia could suggested that the currents generated by movement of of the cilia lead to oxygen being transported to the interior of the egg mass by capconvection as well as diffusion. Oxygen would diffuse across the cap sule sule at the surface of the egg closest to the outside of the egg mass. of oxygen-rich water would then be moved by ciliary action This mass of to the opposite side side of the egg where oxygen then would diffuse of the egg mass. An outward across the capsule toward the center of oxygen molecule thus could be passed from egg to egg in a manner water being passed along a bucket bucket brigade. somewhat analogous to water Carbon dioxide would pass in the opposite direction. This This appears to be a plausible mechanism for supplying oxygen to
2. 2.
RESPIRATORY EXCHANGE, AEROBIC AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, METABOLISM
69
the interior of teleost as well as amphibian egg masses. Many teleosts linglay large, compact masses of eggs. For example, the egg mass of ling 1984). At cod may be up to 5 liters in volume (Giorgi and Congleton, 1984). propresent, there is not enough information on oxygen and current pro files “bucket-brigade” fi les within such egg masses to adequately test the "bucket-brigade" prohypothesis. However, there is some circumstantial evidence that pro cesses in addition to bulk water flows flows may be involved. Giorgi and (1984) noted that while oxygen concentrations in the cen cenCongleton (1984) ter of a lingcod egg mass declined rather sharply following cessation of current flow, flow, levels did not decline to zero as expected but stabi stabiof 10%air saturation. saturation. Davenport ((1983) lized at about 10% 1983) similarly indicated lumpfish that oxygen levels in the egg mass of lumpfi sh declined more slowly intriguthan expected when aeration ceased. These observations are intrigu previing but say little about the mechanisms involved. As discussed previ ously, these observations can be explained equally as well by natural “bucket-brigade” hypothesis. They do, convection as by the "bucket-brigade" do, however, suggest that egg masses do not depend solely on forced convection to meet metabolic oxygen demands. (1973)noted that the deeper eggs in the egg mass of species Braum (1973) such as herring (Clupea (Clupea harengus) harengus) are threatened with asphyxia as a circulation. He suggested that the perivitelline result of poor water circulation. fluid fl uid could function as an oxygen reservoir to tide embryos over short of much significance. The periods of anoxia. anoxia. This is unlikely to be of fluid, 100%satu satuamount of oxygen in the perivitelline fl uid, assuming it is is 100% rated, rated, is only only sufficient sufficient to to meet meet the the oxygen oxygen requirements requirements of of advanced advanced embryos for 1-2 min. calculation assumes would not not min. This This calculation assumes that that oxygen oxygen would embryos for 1-2 diffuse course it would under under hy capsule-which of of course it would hydiffuse back back out out of of the capsule-which poxic conditions-and conditions-and that there is no convective exchange between fluid water-which is likely. the perivitelline fl uid and the surrounding water-which The perivitelline fluid provides the immediate environment for perivitelthe developing embryo, and it is the gas concentration in the perivitel line fluid-not of physiological signifi signififluid-not the surrounding water-that water-that is of cance. As predicted by the mass transport equation, equation, Eq. Eq. (3), (3), oxygen concentrations in the perivitelline fl uid decline progressively as de fluid development proceeds. Assuming relatively constant capsule conducconduc tance rising metabolic POZ, the the only only way way the the rising metabolic demands demands tance and and ambient ambient P02, associated associated with with tissue tissue growth growth can can be be met met is is by by an an increase increase in in the the driving capsule. This necessitates driving force force across across the the capsule. necessitates aa reduction reduction in in the the P02 of the PO2 of the perivitelline perivitelline fluid. fluid. Berezovsky Berezovsky et al. al. (1979) (1979) demonstrated demonstrated such aa drop fluid P02 PO2 when when the the metabolic metabolic rate rate of of loach loach such drop in in perivitelline perivitelline fluid of dinitrophenol (Fig. embryos was stimulated by low concentrations concentrations.of (Fig. 3). 3). A decline decline in in perivitelline perivitelline fluid fluid P02 PO2 is is also also implied implied by by the the gradual gradual
70
PETER J. ROMBOUGH PETER J. ROMBOUGH
increase in Pc P, that was seen during the course of steelhead develop development (Fig. (Fig. 4; Rombough, 1987). 1987). Recently, Diez and Davenport (1987) (1987) showed that the P02 PO2 of of the fluid in the egg case of of the dogfish declined as development proceeded. Finally, similar declines in P02 PO2 have been well documented for reptilian and avian eggs (Dejours, (Dejours, 1981). 1981). Bird eggs in particular have been studied extensively, and since many of the structures in bird and fish eggs can be considered analogous, the type of relationships seen in bird eggs probably apply to fish eggs as well. For example in the hen egg, oxygen levels in the air space, fluid, which is analogous to the perivitelline fl uid, decrease as metabolic rate increases (Wangensteen, 1972). 1972).This increases the diffusion gradi gradient across the shell, which like the teleost capsule is pierced by tiny pores, and automatically ensures a greater rate of diffusive flux. flux. It does so, however, at the expense of arterial P02 PO2 levels which gradually decline as development proceeds. Blood gas relationships have not been examined in fish embryos, but if the analogy with bird eggs holds, they probably follow a similar pattern. A decrease in perivitelline fluid P02 PO2 late in embryonic develop development appears to be the trigger that initiates hatching in at least some teleosts. If If advanced embryos are placed in hypoxic water, premature hatching occurs (Yamagami, (Yamagami, 1981 1981;; DiMichele and Powers, 1984a; 1984a; Ishida, 1985). 1985).Conversely, hatching can be delayed more or less indefi indefinitely under hyperoxic conditions (Taylor 1977; DiMichele and (Taylor et al., 1977; Taylor, 1980; 1980; Ishida, 1985). 1985). Low oxygen levels do not appear to act directly on the hatching glands. Studies involving various anesthetics suggest the response is mediated by the central nervous system (Ishida, (Ishida, 1985). 1985). The location of the oxygen sensor is not known. Hatching can be regarded as an adaptive response to physiological hypoxia. nes of the egg capsule reduces the hypoxia. Escape from the confi confines ambient oxygen level required to meet metabolic requirements by 30-50 30-50 mm Hg (Rombough, 1987). 1987). However, removal of the capsule does not alter the basic mechanisms involved in respiratory gas ex exchange.
D. Cutaneous Gas Exchange Respiratory gas exchange in fish, fish, and indeed in all vertebrates, is ininitially cutaneous. As development proceeds there is a gradual in crease in the relative importance of gills, although in many species the skin remains the major site of gas exchange throughout the embryonic periods.. Recent evidence indicates that even in adults the and larval periods skin may persist as an important site for respiratory gas exchange
2.
RESPIRATORY AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC METABOLISM
71 71
(Kirsch and Nonnotte, 1977; 1977; Lomholt and Johansen, 1979; 1979; Steffenson (Kirsch and Lomholt, 1985; 1985; Feder and Burggren, 1985). 1985). Studies of gas exchange during the early life stages of teleosts have tended to be descriptive. descriptive. As a result, most of what we know of respira respiraof tory mechanisms has been inferred from studies of the morphology of adaptawhat are assumed to be respiratory structures. Morphological adapta tions to facilitate gas exchange appear early in development. Boulekbache and Devillers ((1977) 1977) suggested that the function of the microvilli present on the outer surfaces of blastomeres of rainbow trout (S. (S. gairdneri) was to increase the surface area for respiratory gas exchange. In many species well-developed vascular networks form just under the skin during early organogenesis (Fig. (Fig. 5). 5). These capil capillary beds are often associated with specialized cutaneous structures, such as an enlarged yolk sac, expansive medial finfolds, finfolds, or enlarged pectoral fins fins,, that greatly increase the surface area available for gas exchange. Detailed descriptions of such specialized structures are provided by Taylor (1913), Sawaya (1942), 1942), Kry (1913), Sawaya (1942), Wu and Liu ((1942), Kryzanowsky ((1934), 1934), Smimov 1975), Lanzing Smirnov (1953), (1953), Soin (1966), (1966), Balon ((1975), (1976), McElman and Balon ((1979), (1981) al. (1976), 1979), Liem (198 1 ) and Hughes et al. ((1986), 1986), among others. The degree to which embryonic and larval larval respiratory structure are elaborated varies widely among species. species. Several authors have sugA
.'
B
f S I I u: e vs
,
s
I�
Fig. 5. 5. (A) Cutaneous gas exchange structures structures in in 5-day-old 5-day-old Tilapia mossambica. mossambica. (B) (B) Fig. Schematic Schematic diagram diagram showing showing blood blood flow flow through through caudal caudal and and rectal rectal vascular vascular systems: systems: cvs, cvs, caudal vascular system; system; h, system; vv, vitelline h, heart; heart; r, rectum; rectum; rvs, rvs, rectal vascular vascular system; vein; ym, yolk mass. mass. [[From From Lanzing ((1976).] 1976).]
72 72
PETER J. ROMBOUGH PETER J. ROMBOUGH
gested that this refl ects variations in oxygen levels in spawning habi reflects habitat and can be used as the basis for a functional classification system (Kryzanowsky, 1934; Soin, 1966; 1966; Balon, 1975). 1975).It is beyond beyond the scope (Kryzanowsky, 1934; of of the the current current discussion to to examine examine the the merits merits of of such such systems, but but attention attention will will be be drawn drawn to to one one characteristic characteristic that that these these authors authors have have considered particularly important, lays considered particularly important, that that is, is, whether whether the the species species lays pelagic or pelagic or demersal demersal eggs. eggs. In In the the marine marine and and temperate temperate freshwater freshwater environments, environments, pelagic embryos tend tend to to have have relatively poorly devel developed oped capillary capillary plexes plexes near near the the body body surface. surface. Respiratory Respiratory pigments pigments (hemoglobin, myoglobin, myoglobin, perhaps (hemoglobin, perhaps carotenoids) carotenoids) usually usually do do not not appear appear until gills may until late late in in development, development, and and gills may not not become become functional functional until until near (Balon, 1975). near the the end end of of the the larval larval period period (Balon, 1975). Pelagic Pelagic eggs eggs tend tend to to be small and are normally found in well-oxygenated waters. As a result, it has been suggested that oxygen is not normally a limiting factor and thus thus specialized specialized respiratory respiratory structures structures are are not not necessary necessary (Hempel, (Hempel, 1979). ex1979). In contrast, demersal eggs are usually larger and often are ex posed to to relatively relatively low low oxygen oxygen concentrations concentrations for for extended extended periods. periods. According According to to Balon Balon (1975), (1975), this this has has resulted resulted in in selection selection for for extensive extensive vascularization vascularization of of the the body body surface surface and and the the elaboration elaboration of of specialized specialized cutaneous cutaneous gas gas exchange exchange structures. structures. Such Such structures structures tend tend to to develop develop early persist throughout early and and often often persist throughout the the larval larval period. period. Respiratory Respiratory pig pigments ments appear appear early, early, and and gills gills become become functional functional soon soon after after hatch. hatch. It should be recognized, though, that as with most generalizations generalizations in zoology, zoology, there there are are exceptions. exceptions. For For example, example, the the Indian Indian air-breather air-breather pelagic eggs, Anabas testudineus lays lays small small pelagic eggs, but but the the body body surface surface is is well vascularized, pigments appear well vascularized, and and respiratory respiratory pigments appear early early in in develop development 1986). These These adaptations adaptations are are not not particularly particularly ment (Hughes (Hughes et al., 1986). surprising realizes that surprising when when one one realizes that the the eggs eggs are are laid laid in in the the very very oxygen oxygenpoor waters of tropical swamps, and that it is only in the surface layer that levels are develop that oxygen oxygen levels are high high enough enough to to sustain sustain embryonic embryonic development. The effective surface area, the length of the diffusive pathway, the magnitude of the partial pressure gradient between the water and blood, the amount of blood perfusing the structure, and the convective movement of water past the structure are among the most important factors On factors influencing influencing the the performance performance of of respiratory respiratory gas gas exchangers. exchangers. On emthe basis of these characteristics, cutaneous gas exchange in fish em bryos highly efficient. efficient. As As mentioned mentioned bryos and and larvae larvae would would appear appear to to be highly previously, previously, specialized specialized exchange exchange structures structures comprise comprise aa relatively relatively large fraction of total body surface area in many species. For example, the well-vascularized (C. well-vascularized medial and paired fins of larval herring (C. harengus) harengus) and and plaice plaice (Pleuronectes (Pleuronectes platessa) platessa) account account for for about about 40% of of
2. 2.
RESPIRATORY AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC METABOLISM
73 73
total surface area at hatch (DeSilva (DeSilva and Tytler, 1973). 1973). In addition, the surface/volume ratio of most larvae is large because of their small surface/volume absolute size. size. Total surface area for a 11.6 .6 mg carp (Cyprinus (Cyprinus carpio) carpio) 2 glarvae is is in the order of 12,000 12,000 mm mm2 g-l.l. In contrast, total surface area 2 g-l of 1400 mm of a 350 350 mg juvenile is only about 1400 mm2 g-' (Oikawa (Oikawa and Itazawa, 1985). 1985). Cutaneous diffusion distances have been estimated for only a few species, but the available evidence indicates that distances are only slightly greater than lamellar diffusion distances in juveniles and adults. The skin is only two cells thick over most of the body surface in young larvae (Lasker, (Lasker, 1962; 1962; Jones et al., 1966; 1966; Roberts et al., 1973). 1973). Lasker ((1962) 1962) reported that the skin thickness of of larval sardine (Sar (Sardinops caerislea) . 7 f.Lm caerislea) ranged from 11.7 pm on the finfold to 3.0 tLm pm on the al. ((1966) 1 966) estimated a minimum lateral portion of the trunk. Jones et ul. skin thickness in larval herring (C. (C. harengus) 2.3 f.Lm. harengus) of about 2.3 pm. The actual length of the diffusive pathway is is somewhat greater. Many, particularly particularly pelagic, species have a relatively thick fluid layer be between the dermis and epidennis epidermis:: 5.0 5.0 tLm pm in the case oflarval of larval plaice (P. (P. platessa; Roberts et al., 1973). 1973). In addition, distances associated with diffusion across capillary walls and through the plasma should be taken into account. Even when this is is done, distances remain rela relatively small. Webb and Brett ((1972a) 1972a) measured a mean distance of 4.7 4.7 f.Lm pm from the surface of the skin to the center of of blood capillaries in embryos of of two species species of of viviparous seaperch (Rhacohilus (Rhacohilus vacca and Embioteca Iateralis). lateralis). Liem (1981) (1981) estimated 8-15 8-15 f.Lm pm for the total thickness of the water-blood water-blood barrier in larval Monopterus. Monopterms. These dis distances are considerably less than the cutaneous diffusion distances of of larval amphibians. amphibians. Burggren and Mwalukoma (1983) (1983) estimated a blood-water 20-50 f.Lm blood-water barrier of of 20-50 pm for larval bullfrog ((R. R . catesbeiana). catesbeiana). In this species up to 60% of of total gas exchange takes place across the skin (Burggren and West, 1982). 1982). The skin of of larval teleosts is probably at least as effective as an organ of gas exchange, given the shorter diffusion distances and, in many cases, more elaborate vasculariza vascularization. Cutaneous gas exchange in other vertebrates frequently suffers from a relatively small partial pressure gradient across the skin as a result of central mixing of oxygenated and deoxygenated blood prior to transit to the skin (Burggren, 1984). This problem appears to be (Burggren, 1984). minimized in many teleosts. Cutaneous gas exchange structures-for structures-for example, the caudal and rectal vascular systems of larval tilapia (Fig. (Fig. 5) 5) or the vitelline circulation of of salmonids-typically salmonids-typically receive blood that has already passed through at least a portion of of the systemic
74
PETER ROMBOUGH PETER J. ROMBOUGH
the made, the been made, have been measurements have situ measurements in situ no in Although no circulation. Although circulation. poor comparatively poor be comparatively should be structures should exchange structures the exchange entering the blood entering blood in oxygen thus maximizing maximizing the partial pressure gradient between the water. the water. and the blood and blood te of tephysiology of cardiovascular physiology the cardiovascular of the known of is known little is Extremely little Extremely effec more effecbe more would be obviously would exchange obviously Cutaneous exchange embryos. Cutaneous leost embryos. leost ( 1 979) Balon (1979) and Balon McElman and regulated. McElrnan be regulated. could be flow could blood flow if blood tive if tive capillary cutaneous capillary through cutaneous passing through blood passing of blood amount of the amount that the noted that noted vi walleye (Stizostedion (Stizostedion uiof walleye development of the development during the varied during beds varied beds shunting for shunting mechanism for implied aa mechanism this implied that this suggested that treum) and and suggested treum) result as aa result recruitment as gas exchange. optimize gas to optimize as to so as blood so blood exchange. Vascular Vascular recruitment dur rate durheart rate increased heart by increased blood pressure of higher of higher systemic systemic blood pressure caused caused by there However, there proposed. However, was proposed. hypoxia was physiological hypoxia of physiological periods of ing periods ing pat flow patblood flow in blood changes in ontogenetic changes between ontogenetic differences between be differences may be may oxygen in oxygen alterations in to transient due to changes due compensatory changes terns terns and and compensatory transient alterations perfusing blood perfusing of blood amount of the amount of the Reflex control demand. Reflex supply or supply or demand. control of recruit capillary recruitoccur, capillary well occur, may well structures may exchange structures cutaneous gas cutaneous gas exchange amphibian in amphibian demonstrated in been demonstrated has been hypoxia has to hypoxia response to in response ment in ment be demonstrated has yet 1984), but larvae larvae (Burggren, (Burggren, 1984), but it it has yet to to be demonstrated in in teleost teleost embryos embryos or or larvae. larvae. E. Respiratory Pigments E.
apAs mentioned previously, the stage at which hemoglobin first ap pears is highly variable. In many demersal species, such as salmonids, large numbers of pigmented erythrocytes are evident well before hatch, (Bahatch. This is thought to be an adaptation to hypoxic conditions (Ba 1975). In contrast, hemoglobin may not appear in the circulation lon, 1975). of pelagic species, such as herring, until after metamorphosis (De Silva, 1974). 1974). Lack of of hemoglobin has been proposed as a mechanism Silva, Howto limit predation by making pelagic larvae less conspicuous. How well-oxyever, it simply may be that hemoglobin is not required in well-oxy genated waters. Holeton ((1971) 1971) reported that rainbow trout larvae showed little distress when their hemoglobin was poisoned by carbon monoxide. Similarly, Iuchi (1985) (1985) reported that rainbow trout larvae survived to to the fry f.y stage after having their erythrocytes erythrocytes destroyed by treatment with phenylhydrazine. Indeed, it may not even be neces necessary for small larvae to have a functioning circulatory system. Burg Burggren (1984) (1984) points out that the so-called "cardiac “cardiac lethal" lethal” larval mutant of the amphibian Ambystoma, in which the heart forms forms but fails fails to beat, is able to survive after hatching for many days in well-oxygen well-oxygenated water.
2.
RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM RESPIRATORY
75
hemoglo The evidence to date indicates that embryonic and larval hemogloof juveniles bins are structurally and functionally distinct from those of and adults. Iuchi and Yamagami (1969) (1969) reported a gradual change in the electrophoretic banding pattern for the hemoglobins of of rainbow trout during the period between hatch and gravel emergence. Similar shifts in electrophoretic banding patterns have been observed in Ho Homasu salmon (0. (0.rhodurus) rhodurus) and brook trout (Salvelinus (Salvelinus fontenalis; Iuchi et al., 1975) 1975) and in coho salmon (0. (0. kisutch; Giles and Vanstone, 1976). Distinct embryonic and adult hemoglobins also have been rere 1976). Terwil liger, ported for several viviparous species (Ingermann and Tenvilliger, 1981a,b, 1982,1984; 1982, 1984; Ingermann et al., al., 1984; 1984; Weber and Hartvig, 1984; 1984; 1981a,b, 1984). Hartvig and Weber, 1984). ( 1973b) compared the chemicaI chemical and physiological properties Iuchi (197313) of larval and adult hemoglobins of of rainbow trout. Both were tetratetra of meric, but larval hemoglobin displayed a higher oxygen affinity, affinity, less of of a Bohr effect, virtually no Root effect, and greater cooperativity at physiological pH than adult hemoglobin. Larval and adult hemoglo hemogloof 31 31 mm Hg and 57.5 mm Hg, respectively, at pH bins had PSO P50 values of 25°C. The Bohr effect (A (a log PSdpH) P5o/pH) was 0.023 for larval hemohemo 7.2 and 25°C. of larval globin but 0.64 for adult hemoglobin. The oxygen capacity of hemoglobin was virtually unaffected by pH, while a drop to pH 6.5 of adult hemoglobins to 50% of of that at pH reduced the oxygen capacity of 8.0. Slopes S lopes of of Hill plots were n = 2.33 and n = 1.62, 1 .62, respectively, at 8.0. pH 7.2 for larval and adult hemoglobins. The high oxygen affinity and pH independence of larval hemoglobins are clearly advantageous to embryos and larvae exposed to the oxygen-poor, oxygen-poor, low-pH and high highCO2 C02 environments of of the perivitelline fluid and interstices of of the redd. The shift in electrophoretic banding patterns suggests that the of embryonic and larval blood is due primarily greater oxygen affinity of of the hemoglobins rather than to intrinsic differences in the structure of of modulators of of hemoglobin (Hb) (Hb) affinaffin to changes in concentrations of ity. This was shown to be the case for the viviparous eelpout Zoarces 1984; Hartvig and Weber, 1984). 1984). WeWe viviparous (Weber and Hartvig, 1984; (1984) reported that fetal hemoglobin had a higher 0O22 ber and Hartvig (1984) affinity affinity (P50 (Pm values of9 of 9 mm Hg and 23 mm Hg, respectively, at pH 7.5 and lOOC), 10°C), reduced Bohr effect, and greater cooperativity than adult hemoglobin in nucleoside triphosphate-free preparations. Measure Measureof intraerythrocyte nucleoside triphosphate triphosphate (NTP) (NTP) concentraconcentra ment of tions revealed no significant difference in the NTP/Hb ratios of fetal and adult blood. Differences in modulator concentrations, though, do appear to be important in some species. Ingermann and Terwilliger =
=
76
PETER PETER JJ.. ROMBOUGH
((1981b, 1981b, 1982, 1982, 1984) 1984) reported that part of the reason for the higher O2 0 2 affinity of the hemoglobin of fetal seaperch E E.. lateralis was a lower NTP/Hb ratio. ATP was the most abundant modulator modulator (82% (82% total NTP), 18% of total NTP), but there was also a significant amount of GTP ((18% evidence suggests NTP) present within fetal erythrocytes. Indirect evidence cofactors may modulate the O2 0 2 affinity of larval hemoglobins in some oviparous species species as well. DiMichele and Powers (1982) (1982) attributed of different lactate dehydrogenase dehydrogenase differences in hatching times of (LDH) (LDH) genotypes of Fundulus heteroclitus to the ability to deliver oxygen to tissues. The LDH genotype (LDH BaBa) that hatched (LDH B"B") hatched earli earliest was also the genotype that had the highest concentration of ATP in their erythrocytes as adults, and presumably as embryos. Increased ATP concentrations would lower hemoglobin O 0 22 affinity and thus re reperivi duce O 0 22 delivery to tissues near hatch when P02 PO2 levels in the perivitelline fluid are low. This would trigger release of the hatching en enzyme and lead to early hatch. Iuchi and Yamamoto (1983) em (1983) demonstrated that the shift from embryonic-larval bryonic-larval hemoglobins to juvenile hemoglobins in rainbow trout was the result of erythrocyte replacement. Erythrocytes containing embryonic hemoglobins were formed in the extraembryonic blood intermediate cell mass beginning about one-third through islands and intermediate producembryonic development. development. These hemopoietic centers ceased produc tion shortly after hatch, and centers in the kidney and spleen began to produce morphologically and antigenically distinct erythrocytes con containing juvenile-type hemo juvenile-type hemoglobins. Similar shifts in the site of of hemopoiesis have been reported for Atlantic salmon (Vernidub, 1966) 1966) and anglefish Pterophyllum scalare (AI-Adhami (Al-Adhami and Kunz, 1976). 1976). Hemo Hemoglobin switching based on erythrocyte replacement replacement may turn out to be widespread in lower vertebrates. Kobel Kobe1 and Wolff (1983) (1983) recently re reported that the shift from embryonic embryonic to larval type hemoglobins in the amphibian Xenopus borealis also was associated with a shift in the site of erythropoiesis. It has been suggested that pigments other than hemoglobin may be involved in respiratory gas exchange. De Silva ((1974) 1974) noted that myoglobin was formed prior to hemoglobin in herring larvae and indi indicated that this may refl ect a respiratory role during early development. reflect This hypothesis has not been tested. tested. Other authors have proposed a respiratory role for the carotenoid pigments (Smirnov, 1953; 1953; Volodin, 1956; Balon, 1975, 1975, 1984; 1984; Mikulin and Soin, Czec1956; Soin, 1975; 1975; Soin, 1977; 1977; Czec zuga, 1979). 1979). This hypothesis is is based largely on circumstantial evi evidence, namely, that eggs of species containing high concentrations of carotenoids are often relatively large and, thus, are faced with rela-
2.
RESPIRATORY EXCHANGE, AEROBIC AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, METABOLISM
77
tively large diffusion distances gas exchange is com distances.. The problem of gas compounded for some of these species by the fact that they must develop in low oxygen in comparatively comparatively low oxygen environments. environments. Unfortunately, Unfortunately, there there is is little experimental evidence to indicate that carotenoids actually aid in oxygen transport under such conditions. In fact, con fact, there is now considerable evidence indicating indicating that that low carotenoid carotenoid levels in in eggs, eggs, nor normally rich cantly reduce mally rich in in carotenoids, carotenoids, do do not not signifi significantly reduce survival survival (Steven, (Steven, 1949; 1949; Craik, 1985; 1985; Craik and Harvey, 1986; 1986; Tveranger, 1986). 1986). Craik (1985) (1985) speculates that carotenoids may play some minor, as yet unde undefined, fined, role role in in oxygen oxygen transport transport but but that that present present evidence evidence does does not not warrant warrant acceptance acceptance of of carotenoid-based carotenoid-based respiration respiration as as an an established established fact, 1975, 1984) fact, as as Balon Balon ((1975, 1984) would would suggest. suggest. We We have have seen seen how how the the movement movement of of water water past past the the egg egg and and stirring stirring of embryonic gas of the the perivitelline perivitelline fluid fluid enhance enhance embryonic gas exchange. exchange. Adequate Adequate ventilation of body surfaces is similarly necessary for efficient gas exchange after hatch. Fish have adopted a number of tactics to ensure that this occurs. Some, such as nest-fanning, mouth-brooding, and occurs. Some, wriggler-hanging, involve the parents. Others involve behavioral and physiological adaptations on the part of the larvae. Hunter 1972) noted noted that (Engrulis mor morHunter ((1972) that newly newly hatched hatched anchovy anchovy (Engralis dax) dux) exhibited regular bouts of swimming that did not appear to be associated with feeding or predator avoidance. avoidance. He suggested that enswimming might have a respiratory significance, presumably by en hancing hancing ventilation ventilation of of the the body body surface. surface. This This hypothesis hypothesis was was exam examined in some detail Weihs (1980) pointed out Weihs (1980, (1980, 1981). 1981). Weihs (1980) pointed out ined in some detail by Weihs that that because of of their their small small size, size, anchovy anchovy larvae larvae existed existed in in aa viscous viscous environment Re). Consequently, environment (Le. (i.e.,, low low Reynolds Reynolds number, number, Re). Consequently, both both the the larva tend to transported together larva and and its its immediate immediate surroundings surroundings tend to be be transported together by oceanic would remain oceanic currents currents.. A nonswimming nonswimming larva larva thus thus would remain in in the the same oxygen. Weihs same mass of of water water and and gradually gradually deplete deplete the the available available oxygen. Weihs (1980) (1980) developed developed aa mathematical mathematical model model for for diffusive diffusive oxygen oxygen uptake uptake by the the larvae larvae and and estimated estimated that that oxygen oxygen would would become become limiting limiting for for aa stationary saturation stationary day-old day-old larva larva at at concentrations concentrations below below 63% 63% air air saturation (ASV). (ASV). He He then then tested tested this this prediction prediction by by observing observing larval larval swimming swimming behavior As predicted, predicted, both both the the fre frebehavior at at various various oxygen oxygen concentrations. concentrations. As quency quency and and duration duration of of swimming swimming significantly significantly increased increased at at oxygen oxygen levels (Fig. 6). 6). levels below below 60% 60% ASV (Fig. Weihs' Weihs’ (1980) (1980) study study indicates indicates that that the the limiting limiting step step in in cutaneous cutaneous gas least in gas exchange, exchange, at at least in anchovy anchovy larvae, larvae, is is the the convective convective flow flow of of water remembered that that an anwater past past the the exchange exchange surface. surface. It It should should be remembered chovy chovy larvae larvae live live in in aa comparatively comparatively oxygen-rich oxygen-rich environment. environment. It It would even more more important important for for larvae larvae inhabiting inhabiting would thus thus appear appear to to be even
78
PETER J. ROMBOUGH PETER J. ROMBOUGH
'" c
0.15
'E e 'j ..
; 0.10'
.�
'l;
�
1; � 0.05
II.
0:
20% 20%
60% 40% 60% 40% Percent Percent saturation soturotlon
80% 80%
100% loo% 1
Fig. Fig, 6. 6. Fraction Fraction of of time time spent actively actively swimming as as aa function function of ambient oxygen oxygen 1) northern anchovy concentration concentrationfor newly hatched (day (day 0) 0) and 24-h-old 24-h-old (day (day 1) anchovy larvae. larvae. [From [From Weihs (1980).] (1980).1
hypoxic environments to be able to generate large convective flows. This is accomplished in larvae of the Austrailian lungfish (Neocera (Neocerutodes forsteri) by means of cilia that direct a water current posteriorly todesforsteri) of many along the body surface (Whiting and Bone, 1980). 1980). Larvae of warm-water teleosts use their pectoral fins to create a similar water flow. Liem ((1981) 1981) reported that larvae of the air-breathing fish Monop flow. Monopterus albus use large, well-vascularized pectoral fins to direct a flow of of relatively oxygen-rich surface water backward along their body sur surface. face. The yolk sac and caudal region of Monopterus larvae are also extensively vascularized. vascularized. Microscopic observations indicated that sur surficial blood flow in these regions ran in the opposite direction to the flow of water generated by the pectoral fins. fins. When larvae were placed in a tube with water fl owing in the normally anterior to posterior flowing 41%.When the direction the oxygen extraction efficiency was about 41%. direction of water fl ow was reversed, extraction efficiency dropped to flow (Fig. 7). about 20% (Fig. 7). Liem ((1981) 1981) pointed out that on this basis the whole larva could be considered a functional analog of the gill iamel lamelfish. He noted that similar elaborate vascular networks and lae of adult fish. mobile pectoral fi ns were common in larvae of other species inhabit fins inhabiting hypoxic waters and speculated that such countercurrent flow mechanisms might be widespread. It should be noted, though, that pectoral fin movements are not always always associated with ventilating cutaneous gas exchange surfaces. Peterson (1975) (1975)found that, contrary to his expectations, the rapid pectoral fin movements of Atlantic salmon alevins did not direct water over the well-vascularized yolk sac. Instead, they appeared to be involved in drawing water over the sac. gills. gills.
2.
RESPIRATORY RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM
55
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79
10
20 30 20 30 Time Time (minutes)
(minutes)
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Fig. Fig. 7. (A) Larval Monopterus albus (4 days old) old) showing (1) (1) large vascular pectoral pectoral (2) well-developed network in the yolk sac, and (3) fins, (2) well-developed capillary network (3) well-vascularized well-vascularized generated by moveunpaired medial fin. (B) (B) Schematic representation of of water currents generated move ment of of pectoral pectoral fins. The stippled area shows the region from which water is drawn by water flowing the pectoral fins. (C) (C) The effectiveness of of oxygen extraction from water flowing in an anterior to posterior direction (countercurrent to blood flow) flow) or in a posterior to anterior direction (concurrent to blood flow). 130 ml I-’. I-I. (Reprinted with flow). The flow rate was 130 1981; copyright 0 © AAAS.) permission from Liem, 1981;
F. Branchial Gas Exchange The larval period is characterized by a shift in the site of respira respiratory gas exchange from the skin to the gills. Unfortunately, relatively little is is known of the physiology of this transition. It is known that species vary in the stage at which gills first appear and the speed with which they are elaborated. For example, gill arches begin to form in rainbow trout shortly after gastrulation and by hatch are complete with functional filaments and secondary lamellae (Morgan, (Morgan, 1974a,b). 1974a,b). Arctic char (Salvelinus alpinus) laments at hatch but alpinus) also possess fi filaments secondary lamellae do not begin to form until about 8 days posthatch (at 6.5°C; 6.5”C; McDonald and McMahon, 1977). 1977). In smallmouth bass (Mi (Micopterus dolomieu), dolomieu), filaments do not appear until about 7 days post posthatch (at (at 16°C; 16°C; Coughlan and Gloss, Gloss, 1984). 1984). Lamellae begin to form about 4 4 days days later, about the time the larvae larvae becomes free-swimming� free-swimming. Filaments and secondary lamellae first appear on gill arches of herring and plaice larvae about midway through the larval period, at body lengths of 10 10 and 8 mm, respectively (De (De Silva, Silva, 1974). 1974). Such description provides relatively little information about the relative importance of the gills in larval gas gas exchange. This question
PETER PETER J. J. ROMBOUGH ROMBOUGH
80
can be answered answered best best by by directly directly measuring measuring gas fluxes across across the the gills gills can gas fluxes and skin. This This has has been been done done for for amphibian amphibian larvae larvae (Burggren (Burggren and and skin. and West, 1982; Burggren, Burggren, 1984) 1984) but but not not for for teleost teleost larvae, larvae, even even though though West, 1982; the techniques techniques developed developed for for amphibians amphibians would would be relatively easy to to relatively easy the apply the absence parti apply to to some some of of the the larger larger fish fish larvae. larvae. In In the absence of of such such partitioning gills and and skin skin in in fish fish larvae larvae tioning studies, studies, the the relative relative importance importance of of gills must be inferred amount of of information information availavail inferred from from the the rather rather limited limited amount must able morphometrics of the two mentioned previ previable on on the the morphometrics of the two structures. structures. As mentioned ously, the the factors factors of of particular particular importance importance in in determining determining the the effieffi ously, ciency of of gas exchange structures structures are are total total surface surface area, area, diffusion diffusion ciency gas exchange distance, partial partial pressure pressure gradients, gradients, and and the convective supplies of distance, the convective supplies of blood and and water. water. blood (1974) reported reported that, at hatch, the gills of of both herring and De Silva (1974) plaice accounted accounted for for an an insignificant insignificant portion portion (<0.5%) « 0.5%) of of total total body body plaice surface area. The surface area of of the gills expanded rapidly during larval gill area were 3.36 3.36 and and 1.59, 1.59, larval development; development; gill area weight weight exponents exponents were respectively. sur respectively. However, However, even even at the the end end of of the the larval period, gill surface 10% for for herring herring and and face area was was only aa relatively relatively small fraction fraction ((-10% <5% for for plaice) plaice) of of the the total surface area over which which diffusive diffusive gas gas <5% total surface area over exchange of the gills of of exchange could take take place. Similarly, the surface area area of newly hatched larvae accounted accounted for for < 0.2% 0.2% of of the the total area total area newly hatched carp carp larvae available for for gas gas exchange exchange (gills, (gills, fins, fin s, body body surface) surface) (Oikawa (Oikawa and and ItaIta available 1985). Gills grew rapidly, with allometric mass exponents of of zawa, 1985). 7.066 between between 1.6 1.6 and and 3.0 mg mg and and 1.222 1.222 between between 3.0 3.0 and and 200 mg. mg. 7.066 of the the surface area of the gills gills However, even even for a 140 mg postlarva, the remained remained less less than than one-third one-third of of the the total total potential potential exchange exchange area. area. Thus, morphometric would suggest suggest that Thus, morphometric analysis analysis would that in in these these species species the the skin most important important organ organ for for gas gas exchange exchange throughout throughout skin remains remains the most larval development. Gills Gills appear appear to to be relatively relatively more more important important in in larval development. signifi salmonid larvae, but even here, cutaneous exchange remains signifi( 1977) rere cant until long after swim-up. McDonald and McMahon (1977) ported that that early early development development of of Arctic char gills gills was was characterized characterized ported Arctic char increase in in filament filament number number and and size size followed followed by an an inin by aa rapid rapid increase crease in in the the number number and and size size of of the secondary secondary lamellae. lamellae. At At 15 days days crease posthatch lamellae lamellae only only accounted for about about 10% 10% of of gill gill surface surface area, area, posthatch accounted for while by days posthatch posthatch (at (at 6.5°C) 6.5"C)they they comprised comprised 23% 23%of of total total gill gill while by 47 days (45% area. However, this was only about half half the lamellar contribution (45% area. total gill area) 1977) calcu area) in yearling char. McDonald and McMahon ((1977) calculated that on the the basis basis of of body body weight weight Arctic Arctic char char larvae larvae would would require require lated that on a lamellar surface area about four times that actually measured 47 days posthatch to meet metabolic oxygen demands if if the gills were the only posthatch site of of gas exchange. =
2. 2.
RESPIRATORY EXCHANGE, AEROBIC AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, METABOLISM
8 811
of the Only a few measurements have been made of the thickness of blood-water diffusion barrier in the gill of larval fi fish. blood-water s h. Morgan ((1974b) 1974b) blood-water pathway in rainbow reported that the thickness of the blood-water pm at hatch to 6.8 JLm pm 102 102 days trout decreased gradually from 111.1 1 . 1 JLm posthatch. This decrease was due primarily to a shift from aa multilayer respiratory epithelium at hatch to one that was only two cells thick. There was also a modest decrease in the thickness of the basement barrier is is membrane. A reduction in the thickness of the diffusion barrier lamelprobably typical of obligate water-breathing larvae. In contrast, lamel lar diffusion distances increase as development proceeds in bimodal breathers. In Anabas testudineus, diffusion distances increased from 1-2 JLm pm prior to air breathing to 4-8 4-8 JLm pm after the start of air breathing 1-2 1979). In Channa striatus, diffusion distances in in(Mishra and Singh, Singh, 1979). pm during the transition to air breathing creased similarly from 4 to 9 JLm (Prasad is prob (Prasad and Prasad, 1984). 1984).This increase in diffusion distances is probably an adaptation to prevent lamellar collapse while the gills are out of water. Development of branchial blood vessels has been described for (Solewski, 1949; 1949; Markiewicz, 1960; 1960; Morgan, 1974a,b; 1974a,b; many species (Solewski, 1979; Paine and Balon, 1984, 1984, among others). others). McElman and Balon, 1979; of the branchial circulation. Much less is known about the functioning of Morgan ((197413) 1974b) suggested that rainbow trout larvae might not be able to regulate blood How flow through specifi specificc secondary lamellae. This was based on histological observations that pillar cells did not appear to be contractile. ow to the gills presumably can be regulated contractile. Overall fl flow regulated by alterations in heart rate. Holeton ((1971) 1971) and McDonald and McMahon ((1977) 1977) noted that larvae responded to moderate hypoxia by increasing heart rate. Holeton ((1971) 1971) noted that ventilatory movements of of newly hatched rainbow trout were weak and poorly coordinated and suggested that the respiratory pump was not fully functional at hatch. Peterson similarly, observed that opercular movements were infrequent ((1975), 1975), similarly, and irregular in newly hatched Atlantic salmon. In contrast, Morgan ((1974b) 1974b) repOlted reported that the buccal and opercular pumps of rainbow trout were fully functional at hatch. The cause of of these apparently contra contradictory results may lie in the oxygen concentrations at which the ob observations were made. McDonald and McMahon ((1977) 1977) noted that the buccal and opercular movements of newly hatched Arctic char were infrequent and poorly coordinated in normoxic water, but if if the larvae were placed in hypoxic water, ventilatory movements become stronger and fully coordinated. McDonald and McMahon ((1977) 1977) pro proposed that this indicated that the larvae possessed the necessary neu-
82
PETER PETER ]. J. ROMBOUGH ROMBOUGH
ral and musculoskeletal mechanisms to irrigate their gills effectively but �did so unless oxygen was limiting. In well-oxygenated did not do so water, oxygen requirements could be satisfied by cutaneous exchange at less cost. cost. This raises the question of whether branchial gas exex change is cost-effective for very small larvae. Small larvae reside in a viscous environment (low (low Re). Re). As a result, fluid accelerated by the buccal and opercular pumps would rapidly come to halt between cy cycles. cles. Irrigating the gills using the buccal and opercular pumps thus can be expected to be relatively expensive and will be avoided by small larvae in favor of of less expensive cutaneous exchange under nor normoxie moxic conditions. A low Reynolds number environment may be the n movements to assist in reason Atlantic salmon larvae use pectoral fi fin n movements are rapid (= 100 moving water over the gills. Pectoral fi fin (-100 cycles min-I; min-’; Peterson, 1975) 1975) and can be expected to produce a more or less constant flow, flow, which at low Re may be more efficient than a pulsatile fl ow. flow. It has been suggested that larvae experience respiratory distress during the transition from cutaneous to branchial gas exchange. Iwai and Hughes (1977) (1977) reported that a preliminary study of of the morpho morphometrics of gill development in the black sea bream (Acanthopagrus schlegi) indicated a decline in the surface/volume surfaceholume ratio of of the gills 5-8 5-8 schlegi) days after hatch. They speculated that this would produce a situation unfavorable to gas exchange and might be the reason for high larval mortality during the so-called "critical “critical period." period.” McElman and Balon (1979) (1979)suggested that walleye larvae similarly may experience respira respiratory distress leading to mortality during the period when blood fl ow flow through the vitelline circulation is is in the process of being reduced but before the gills are fully functional. functional. Additional support for respiratory involvement in critical-period mortality in some species is provided by observations that critical oxygen levels rise (DeSilva (DeSilva and Tytler, 1973; Davenport, 1983) 1983) and resistance to hypoxia decreases (DeSilva 1973; and Tytler, 1973; 1973; Spoor, Spoor, 1977, 1977, 1984) 1984) midway through the larval pe period. These observations will be discussed later in more detail. III. 111. AEROBIC METABOLISM METABOLISM
A. Measurement Techniques
factors influence the metabolic rate of embryos and larvae. It Many factors factors when measuring meta metais virtually impossible to control all the factors bolic rates and, as a result, the measurement technique itself often significantly affects the rate that is recorded. Three general techsignifi cantly affects
2. 2.
RESPIRATORY RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM
83
nigues have been used to measure metabolic rates: indirect calorime calorimeof these tech techtry, bioenergetic analysis, and direct calorimetry. Each of briefly niques will be examined briefl y with regard to those aspects that are most pertinent to their use with fish embryos and larvae. NDIRECT C ALORIMETRY 11.. IINDIRECT CALORIMETRY
Indirect calorimetry is by far the most widely used technique. In virtually all cases the rate of energy production is is estimated by mea measuring the rate of oxygen consumption. It is possible theoretically to estimate energy production by measuring the rate of of carbon dioxide production, but because of practical difficulties this approach is rarely used. The few instances in which CO2 COZ production has been measured have been in conjunction with measurements of oxygen consumption. This allows calculation of respiratory quotients and provides some (see Devillers, 1965; insight into energy sources during development (see Devillers, 1965; Kamler, 1976; Gnaiger, 1983a). Kamler, 1976; 1983a). A variety of indirect methods have been used with fish embryos and larvae. The most widely used method seems to be the manometric technique using Warburg- or Gilson-type respirometers. The method is relatively easy to master and because of the standard design of the respirometers, results of different studies can be compared directly. However, the technique has a number of disadvantages, disadvantages, the most serious being the fact that respirometers are agitated. agitated. This disturbs the animals and may seriously affect the rate of oxygen consumption (Gnaiger, 983b). It is usually assumed that shaking elevates meta (Gnaiger, 11983b). metabolic rate (e.g., ldridge et ai., (e.g., E Eldridge al., 1977) 1977) but several investigators have speculated that in some species shaking may depress rather than enen hance larval metabolism (Hunter and Kimbrell, 1980; 1980; Theilacker and Dorsey, 1980; 1980; Solberg and Tilseth, 1984). 1984). The impact of shaking can be reduced by only agitating flasks flasks just prior to readings (e.g., (e.g., DeSilva et ai., 1986), but if this is done, continuous readings of al., 1986), of oxygen uptake cannot be made and much information is lost. lost. Another problem with the method is is the fairly long time required for equilibrium to occur, and as a result early responses may be unreliable. It is often suggested that because the system is closed, metabolic waste products accumu accumulate and may influence oxygen uptake. Given the relative insensitivity of of fish embryos and larvae to metabolic byproducts, this is probably not a major problem. In a few instances, Cartesian divers have been used to measure oxygen consumption. The technique is very sensitive, and accuracies in the order of of ± 10-5 ILl pl hh-' 1 have been reported (Davenport, (Davenport, 1976; 1976; Gnaiger, 1983b). 1983b). The problem is that divers are not readily available
PETER J. ROMBOUGH PETER J. ROMBOUGH
84
and require aa good and require good deal deal of of skill skill to to operate. operate. As aa result, result, their their applica application has been In addition, tion has been limited. limited. In addition, measurements measurements involving involving active active larvae small size larvae are are difficult difficult because because of of the the relatively relatively small size of of the the divers. divers. The The Winkler Winkler method method has has been been used used frequently frequently in in the the past past to to mea measure sure oxygen oxygen utilization. utilization. It It has has the the advantages advantages of of being being inexpensive, inexpensive, simple, and reasonably accurate. It allows for a variety of of respirometer designs designs and and can can be be applied applied to to either either flow-through flow-through or or closed closed systems. systems. The the technique technique is is that that it it does does not not permit permit The major major disadvantage disadvantage of the continuous monitoring of oxygen levels and in recent years has been largely superceeded by the use of polarographic oxygen sensors. sensors. The general use of of polarographic oxygen sensors to measure rates of oxygen environment has oxygen consumption consumption in in the the aquatic aquatic environment has been been reviewed reviewed thoroughly 1978; Forstner, 1983; Gnaiger, thoroughly (Hitchman, (Hitchman, 1978; Forstner, 1983; Gnaiger, 1983b). 1983b). Daven Davenport (1976) JLI (1976) reported that with care, accuracies in the order of ±0.02 k0.02 pl h-1 used in h-' are are possible. possible. Polarographic Polarographic sensors sensors can can be used in conjunction conjunction with with virtually virtually any any type type of of respirometer. respirometer. This This flexability flexability allows allows respi respirometers rometers to to be be designed designed to to suit suit the the particular particular circumstances. circumstances. For For ex example, ample, Dabrowski Dabrowski (1986) (1986)recently recently designed designed aa circular circular respirometer respirometer to to measure active metabolism in larvae. A banded paper drum moving around around the the respirometer respirometer induces induces activity, activity, which which is is monitored monitored visually. visually. Polarographic monitored contin continPolarographic electrodes electrodes allow allow oxygen oxygen uptake uptake to to be monitored uously and uously and correlated correlated with with activity. activity. Both Both closed closed and and flow-through flow-through sys systems embryos and and tems have have been been used used to to measure measure oxygen oxygen uptake uptake by by fish embryos larvae. Forstner (1983) points out, (1983) points out, each each .has .has advantages advantages.. Closed Closed larvae. As Forstner systems systems are are easier easier to to construct construct and and accuracies accuracies are are usually usually greater greater be because monitored (it (it is is assumed assumed cause only only oxygen oxygen concentration concentration has has to to be monitored that time is recorded in all cases). cases). In flow-through flow-through tests both oxygen concentrations concentrations and and flow flow must be monitored and accuracy may suffer. On the other hand, flow-through flow-through systems provide a more constant environment environment and and eliminate eliminate the the possibility possibility of of metabolic metabolic waste waste prod products In addition, ucts influencing influencing metabolic metabolic rate. rate. In addition, long-term long-term trends trends in in oxy oxygen uptake can be investigated. Gnaiger (1983b) (198313) points out that the choice design requires requires a choice of of respirometer respirometer design a compromise compromise between between factors factors such as as sensitivity, sensitivity, time time resolution, resolution, aa more more or or aa less less disturbed disturbed environ environment, ease of ment, and and ease of construction. construction. Forstner Forstner (1983) (1983) described described an an intermit intermittent-fl ow system fish eggs combines attrib tent-flow system suitable suitable for for fish eggs or or larvae larvae that that combines attributes utes of of both both flow-through flow-through and and closed systems. systems. Plastics Plastics are are frequently frequently used used in in the the construction construction of of respirometers. respirometers. Researchers Researchers should should be be aware aware that that plastics plastics vary vary considerably considerably in in their their permeability some, such silas tic [P cc cm cm as silastic [P = 60 60 X x 10-9 cc permeability to to gases, gases, and and some, such as 2 cm Hgt 2 cm Hg)l ] and acrylic [P l] , (s (s cm cm2 Hg)-lI [ P = 27 Xx 10-9 lop9cc cm (s(s cm cm2 Hg)-'], are are relatively relatively permeable permeable to to oxygen oxygen (see (see the the Cole-Parmer Cole-Parmer 1987-1988 1987-1988 catologue catologue for for aa listing listing of of N N2, 02, and CO C022 permeability permeability coefficients coefficients for for 2, O 2 , and =
=
2. 2.
RESPIRATORY AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC METABOLISM
85
significant the more common plastics). As a result, signifi cant amounts of oxygen if the walls of the respirometer respirometer may diffuse into or out of the system if are thin. thin. This is particularly a problem with plastic tubing because of the high surface-to-volume ratio. Plastics can be a problem even when chamber walls are relatively relatively thick, since they have a tendency to act as "sponges" “sponges” and thus dampen response times. Eriksen and Feldmeth (1967) permeability of plastics could be reduced bbyy (1967) indicated that the permeability coating them with water-soluble silicone (e.g., Siliclad, Clay-Adams Co.). Co.). The efficacy of of this procedure has not been documented, documented, but even if it does not significantly reduce gas permeability, it may still be formaworthwhile because of its effectiveness in inhibiting bubble forma tion. When all is said, probably the best procedure is to follow Forstner's (1983)recommendation recommendation and and use use glass glass or or stainless stainless steel steel in inForstner’s (1983) stead of plastic wherever possible. Control Control of of flow flow is is often often overlooked overlooked when when respirometers respirometers are are de deflows signed. As we have seen, low fl ows reduce oxygen uptake in eggs by effectively increasing the thickness of the boundary layer (Winnicki, 1968; Wickett, 1975; 1975; DiMichele and Powers, 1984a,b) 1984a,b).. In addition, 1968; adequate organadequate flow flow is is required required to to ensure ensure rapid rapid mixing mixing between between the organ isms and the oxygen sensor to avoid the problem of excessive lag. influences Flow also infl uences oxygen uptake by larvae, but the situation is more complex than is the case for eggs. Low fl ows can reduce uptake flows by by effectively effectively increasing increasing boundary layer thickness, thickness, while while high flows flows may elevate rates of oxygen consumption by increasing activity. In flows adjustflow-through respirometers, fl ows can be regulated by simply adjust ing the head of the water entering the system. In closed systems systems,, some type choice of of aa suitable suitable pump pump is is not not aa trivial trivial type of of pump pump is is required. required. The choice task. Centrifugal pumps often experience cavitation, which can lead to bubble formation. Reciprocating or peristaltic pumps cause cause pressure uence the changes in in the the system system that that can can infl influence the operation operation of of the the oxygen oxygen sensor. Low-speed gear pumps avoid these problems and would ap appear to be one of the most effective ways of regulating flows in closed closed1987). system respirometers (Rombough, 1987). (1983)addresses the problem of bacterial contamination Dalla Via (1983) of of respirometers respirometers as as aa source source of of error error when when measuring low rates rates of of H e points out that the oxygen uptake such as those typical of fish eggs. He use of antibiotics is not particularly particularly effective because of their potential appreciable toxicity to the test organism and the fact that many take an appreciable length of time to reduce bacterial populations. In some instances if if bacteria are eliminated, eliminated, fungal growth merely increases. Dalla Via ((1983) 1983) suggests that perhaps the most effective way to deal with the problem is to run blank controls and subtract bacterial uptake from that of the test animal. This procedure compensates for microbes
PETER PETER J. J. ROMBOUGH ROMBOUGH
86
growing growing on on the the respirometer respirometer and and in in the the water, water, but but it it does does not not take take into into account account epibiotic epibiotic contamination contamination of of the the test test organisms. organisms. Eggs Eggs in in particu particular shimizu et ai., lar can can harbor harbor large large numbers numbers of of bacteria bacteria (Yo (Yoshimizu al., 1980). 1980). Giorgi Giorgi and and Congleton Congleton (1984) (1984) tried tried to to account account for for oxygen oxygen consumed consumed by by microbes microbes growing growing on on lingcod lingcod eggs eggs by by excising excising the the capsule capsule and and mea measuring suring its its rate rate of of oxygen oxygen uptake uptake separately. separately. This This technique technique indicated indicated that that between between 10 10 and and 24% 24% of of total total oxygen oxygen consumption consumption was was due due to to epibiotic contamination. contamination. 2. ANALYSIS 2. BIOENERGETIC BIOENERGETIC ANALYSIS Rates Rates of oxygen consumption can be estimated from energetic of the relation relationanalysis of embryonic and larval growth on the basis of ship
A G+ +R R+ +U U A= =G
(4) (4)
assimilated, G is the energy equiva equivawhere A is the amount of energy assimilated, elaborated, R is the energy equivalent of the amount of of lent of tissue elaborated, oxygen consumed, and U is the amount of energy lost in excretory products. The rate of oxygen consumption is estimated by rearranging =A - (G V ) .Most investigators have used the equation to yield R = (G + U). (1966) and Laurence (1969). (1969). Caloric the dry-weight method of Toetz (1966) equivalents are given by Elliot and Davison ((1975), 1975), Brett and Groves (1979),and Gnaiger (1983a). (1983a).Balanced energy budgets are are rarely used (1979), as the sole means of estimating rates of oxygen consumption. Their main use has been to check the accuracy of metabolic rates measured (Gray, 1926; 1926; Smith, 1947; 1947; Laurence, 1969, 1969, 1973, 1973, by other methods (Gray, 1978; 1978; Johns and Howell, 1980; 1980; Gruber and Wieser, 1983; 1983; Houde and Schekter, Schekter, 1983; 1983; Rombough, 1987). 1987). Investigators cannot be sure that measured rates of oxygen oxygen consumption are representative of those in nature. Fry ((1957) 1 957) showed that simply placing fish in a respirometer significantly elevated their metabolic rate for a considerable length of time. However, if energy budgets can be shown to balance when Eq. (4), (4), the investi investimeasured rates of oxygen are substituted for R in Eq. repregator can be reasonably assured that measured rates are in fact repre laborasentative of those in the natural environment (or (or at least in the labora tory rearing system). Direct calorimetry has been used to estimate metabolic rates of fish embryos and larvae on only a few occasions (Smith, (Smith, 1947; 1947; fish 1979; Gnaiger et al., al., 198 1981). Gnaiger, 1979; 1 ). The technique measures total heat production, but this can be converted to oxygen consumption
+
2. 2.
RESPIRATORY GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM RESPIRATORY GAS METABOLISM
87
using oxycaloric equivalents and assuming aerobic metabolism. The technique has the advantage of of allowing the anaerobic contribution to if oxygen uptake is measured simul simultotal metabolism to be estimated if taneously (Gnaiger, ow (Gnaiger, 1983a). 1983a). Descriptions of of modern continuous-fl continuous-flow microcalorimeters are provided by Gnaiger (1983a), (1983a), Knudsen et al. al. (1983), (1983),and Pamatmat (1983). (1983).According to Gnaiger ((1983a), sensi1983a), the sensi of 2 IL pW, tivity of such calorimeters is in the order of W, equivalent to about 0.35 plO2 h-'1 assuming aerobic metabolism. This would appear to be 0.35 ILl O2 hadequate for measuring heat production of individual late embryos and larvae. Simultaneous direct and indirect calorimetry holds much promise for future investigations, since measurement of oxygen up upparticularly during take alone fails to reflect total metabolic activity, particularly periods of activity or hypoxia. Both these areas are of current interest. 3. DEVELOPMENT 3. OXYGEN UPTAKE DURING DURING EARLY EARLY DEVELOPMENT The early literature concerning the respiratory metabolism of fish embryos and larvae has been reviewed by Needham ((1931, 1931 , 1942), 1942), Smith ((1957, 1957, 1958), 1965), and Blaxter ((1969). 1969). More recent 1958), Devillers ((1965), but less comprehensive reviews are provided by Kamler (1976) (1976) and 1 979). According to Needham ((1931), 1931), the measurement of Hempel ((1979). respiratory rates during early development began in 1896 1896 with the publication of a monograph by Bataillon. Since then, hundreds of studies have been conducted, yet our understanding of many of the more basic aspects of aerobic metabolism in fish embryos and larvae is is still rudimentary. For instance, controversy persists as to the manner in which metabolic rates vary during development and the relation relationship between metabolic rate and body size. size. Activity is is known to have a profound effect on the rate of oxygen consumption, particularly in larvae, larvae, yet only recently have there been attempts to quantify the relationship. Fish vary considerably in their spawning habitats. Tem Temperature and dissolved oxygen are probably the two most important environmental variables, yet their effects on metabolism during early life have been documented in only a few species. A A host of of other factors, salinity, light level, factors, such as carbon dioxide concentration, pH, salinity, group size, and food intake, may also significantly affect metabolic limited. The aim rates, but our knowledge in these areas is is extremely limited. of this section of the review is is to summarize what is is known about the effects of of the more important biotic and abiotic factors factors on the rate of aerobic metabolism of fish embryos and larvae. Before beginning this examination, it would be useful to make a few general comments. comments, Literature reports of of oxygen uptake during
88
PETER J. J. ROMBOUGH ROMBOUGH PETER
early individual basis. This is is early life are are most most often often expressed expressed on on an an individual basis. This perfectly some purposes, general perfectly adequate adequate for for some purposes, such such as as illustrating illustrating general trends trends or or calculating calculating the the total total amount amount of of oxygen oxygen consumed consumed during during specific phases of specific phases of development. development. It It is is not not particularly particularly appropriate, appropriate, though, though, if if the the aim aim is is to to compare compare species, species, different different studies studies with with the the same study, same species, species, or or even even different different treatments treatments within within the the same same study, because mass has because of of the the overwhelming overwhelming effect effect body body mass has on on oxygen oxygen con consumption. size can effect of of body body size can be largely largely eliminated eliminated by by ex exsumption. The effect V02/M; also pressing also termed pressing oxygen oxygen uptake uptake on on aa mass-specific mass-specific basis basis ((VOz/M; termed metabolic relatively few metabolic intensity). intensity). Unfortunately, Unfortunately, relatively few investigators investigators have have done so. Even Even in cases in which metabolic metabolic rate in those those cases in which rate is is purported purported to to done so. be expressed expressed on on aa mass-specific mass-specific basis, basis, care care must be taken taken in in interpret interpreting results. mass (i.e., mass of ing results. Many Many investigators investigators have have used used total total mass (i.e,, mass of the the embryo yolk or some cases cases mass mass of embryo plus plus yolk or in in some of the the embryo, embryo, yolk, yolk, egg egg capsule, fluid) instead mass of capsule, and and perivitelline perivitelline fluid) instead of of just just the the mass of formed formed tissue tissue to to calculate calculate metabolic metabolic intensity. intensity. This This results results in in considerable considerable underestimation when underestimation of of metabolic metabolic intensities intensities early early in in development development when the nonrespiring yolk the ratio ratio of of nonrespiring yolk to to metabolically metabolically active active tissue tissue is is large. large. Care between values on taken to to distinguish distinguish between values calculated calculated on Care also also should should be taken the interpreting the the basis basis of of wet wet and and dry dry masses. masses. Another Another problem problem in in interpreting the literature is uncertainty Rates literature is uncertainty as as to to the the level level of of metabolism metabolism measured. measured. Rates of oxygen uptake uptake can during early early life, on 3- to to lO-fold 10-fold during life, depending depending on of oxygen can vary vary 3the given in the level level of of activity. activity. Activity Activity levels levels are are rarely rarely given in the the literature. literature.
B. Biotic Biotic Factors Factors 11. . STAGE STAGEOF OF DEVELOPMENT DEVELOPMENT
a. Metabolic Rate. Rate. The most a. most important important factor factor affecting affecting metabolic metabolic rate is the mass of actively metabolizing tissue, which under given conditions conditions is is aa direct direct reflection reflection of of the the stage stage of of development. Rates Rates of of V02) shortly after fertilization are low, in the oxygen consumption ((VOZ) 3.7 ng hh-Il (Gadus (Gadus morhua; morhua; Davenport et al., 1979) 1979) to 70 ng range of 3.7 h-'l (S. (S. gairdneri; 1979),depending on species and water hgairdneri; Czihak et al., 1979), temperature. These values increase on average about 20-fold as a result of tissue growth during the period from fertilization to hatch, spealthough the literature suggests a great deal of variation among spe cies. al. ((1982) cies. Kaushik et al. 1982) indicated that there was only about a 3-fold VO2 (Cyprinus increase in V 02 during embryonic development of carp (Cyprinus carpio), while Lukina (1973) (1973) reported a 50-fold increase for chum carpio),
2. 2.
RESPIRATORY GAS EXCHANGE, EXCHANGE, AEROBIC METABOLISM RESPIRATORY GAS AEROBIC METABOLISM
89
(0.ketal. keta). Relative increases in V VOZ 02 tend to be greater for salmon (0. (1984) indi inditemperatures. Dabrowski et al. (1984) larger eggs and at higher temperatures. cated that oxygen consumption at hatch can be predicted if initial egg mass is known using the equation VOZ V = 0.1334MO.fi634T0.7143 02 = 0. 1334M o.6634To.7 143 (5)
(5)
T is "C. in which V VO2 0 2 h-l, 02 is pl JLl O2 h-I, M M is mg dry mass egg-l, egg-I, and T °C. This equation was based on data for 111 1 species. species. It is open to question interestwhether this relationship can be applied generally, but it is interest VOz (i.e., ing that V 02 at hatch was not directly proportional to egg mass (i.e., < 1.0). 1.0).Dabrowski et al. (1984) (1984) attributed metabolic mass exponent, b < this to a higher proportion of metabolically inactive yolk in larger eggs. eggs. The rate of oxygen consumption continues to increase after hatch-for fed larvae in a roughly exponential fashion (Laurence, (Laurence, hatch-for 1973; Kamler, Kamler, 1976). of relative increases dur dur1973; 1976). There are few reports of ing the larval period, but they would appear to be of roughly the same order of magnitude as during the embryonic period. DeSilva and (1973) indicated that the rate of of oxygen uptake of plaice and Tytler (1973) herring at metamorphosis was about 12.5 12.5 and 35 times, respectively, the rate of hatch. In many studies the larvae are not fed. If this is the case, larval V VO2 %fold, although again there 02 typically increases about 3-fold, is considerable species variation, before endogenous food supplies is decline. For unfed larvae, a para parabecome limiting and metabolic rates decline. bolic model rather than an exponential model usually provides the VOz) VOz best fit (up (up to maximum V 02) for data relating V 02 and developmental 1 ; Rombough, 1951; Rombough, 1987). 1987). period (Hayes (Hayes et al., 195 The general trend of increasing metabolic rates is well estab established, but some controversy remains as to whether there are signifi signifi- cant deviations from this trend associated with specific developmental events. events. The The events events that that have have been been considered considered most most likely likely to to sharply sharply alter the rate at which oxygen is taken up are fertilization, gastrulation, the the formation formation of of the the embryonic embryonic circulation, circulation, hatching, hatching, and and the the transi transigas exchange. Early investigators investigators tion from cutaneous to branchial gas VO2 fersuggested that there was a sharp increase in V 02 associated with fer 1928; Needham, Needham, 1931). 1931). More recently studies studies indi indi(Boyd, 1928; tilization (Boyd, cate that this is not the case and that V 02 actually increases rather VO2 (Nakano, 1953; 1953; Hishida and Nakano, Nakano, 1954; 1954; gradually after fertilization (Nakano, Kamler, Kamler, 1972, 1972, 1976; 1976; Czihak et al., ul., 1979). 1979). Trifonova (1937) (1937) reported VO2 neufluctuations 02 during gastrulation and neu that there were large fl uctuations in V (Percafluviatus). fluviatus).The reliability of these obser obserrulation in the perch (Perea vations 1 ; Winnicki, e t al., 195 1951; Winnicki, 1968). 1968). HowHowvations was later disputed (Hayes (Hayes et
PETER J. J. ROMBOUGH PETER ROMBOUGH
90
ever, uctuations in ever, several several more more recent recent studies have have also also shown shown flfluctuations in " V 02 02 during during this this period, period, although although not not of of the the magnitude magnitude reported reported by by Tri Trifonova (Stelzer et al., 197 1 ; Kamler, fonova (Stelzer 1971; Kamler, 1972, 1972, 1976; 1976; Hamor Hamor and and Garside, Garside, 1976). These 1976). These studies studies have have also tended tended to to support support early early observations observations (Hyman, 1 ; Amberson and Armstrong, (Hyman, 192 1921; Armstrong, 1933) 1933) that " V 02 0 2 increases sharply sharply as as soon soon as as the the embryonic embryonic circulation circulation is is established. established. Many Many 02 shortly studies have indicated a leveling off off or even a decrease in " V02 before 1933; Hayes 1; before hatch hatch (Amberson (Amberson and and Armstrong, Armstrong, 1933; Hayes et al., 195 1951; 1 ; Braum, Hamdorf, 196 1 ; Stelzer et al., 197 1961; 1971; Braum, 1973; 1973; DiMichele and Powers, 1984a,b). 1984a,b). This is usually attributed to the capsule limiting the rate rate at at which oxygen can can be be delivered to to the the embryo. embryo. Conversely, Conversely, sharp increases in metabolic rate immediately after hatch (Eldridge et et al., 1977; 1977; Davenport Davenport and and L�nning, Lgnning, 1980; 1980; Cetta Cetta and and Capuzzo, Capuzzo, 1982; 1982; Davenport, Davenport, 1983; 1983; Solberg Solberg and and Tilseth, Tilseth, 1984) 1984) have have been been attributed attributed to to the no longer the fact fact that that the the capsule capsule.no longer restricts restricts oxygen oxygen supply supply or or activity. activity. �reases in " The magnitude of the in 02 associated with removal of the increases VOZ al. ((1977) 1977) reported a roughly capsule is highly variable. Eldridge et al. lO-fold c herring (Clupea pallasi). Daven 10-fold increase in Pacifi Pacific (Clupea harengus pallasi). Davenport and L�nning 1980) observed only about a 40-60% Lgnning ((1980) 40-60% increase in cod. cod. They suggested that part of the reason for for the relatively small increase was that the egg capsule of cod is comparatively thin (=7 /Lm) pm) and thus is less of a barrier to diffusion than the capsules of of other species. The capsule normally does not appear to restrict oxygen con species. consumption to all in some species. 1974) data indicate little species. Robertson's Robertson’s ((1974) change in " sh (Congiopodus leucopaecilus) on V 02 0 2 of southern pigfi pigfish (Congiopodus leucopaecilus) hatch. Kamler ((1976) 1976) similarly failed to observe an appreciable change in " al. ((1982) 1982) and V 02 0 2 on hatch in carp (Cyprinus (Cyprinus carpio). carpio). Kaushik et al. Dabrowski et al. (1984) (1984) actually reported a small decline in " V 02 0 2 im immediately after hatch in carp and whitefi sh (Coregonus whitefish (Coregonus lavaretus), lavaretus), respectively. During larval development, the transition from cutane cutaneous to branchial gas exchange has been linked to both an increase (Lukina, 1973) and a decrease (Kamler, 1972, 1976) (Lukina, 1973) (Kamler, 1972, 1976) in " V 002• 2.
b. h.
VOZ particuMetabolic Intensity. Attempts to link changes in " 02 to particu lar developmental events have, on the whole, proved inconclusive because of the overwhelming effect tissue mass has on oxygen con consumption. previously, changing tissue mass can be sumption. As pointed out previously, largely compensated for by expressing oxygen consumption on a mass-specifi c basis. The few studies in which this has been done mass-specific indicate a more or less consistent pattern during embryonic develop development. It appears that values increase following fertilization to reach a maxima sometime prior to blastopore closure (Trifonova, (Trifonova, 1937; 1937; Smith, Smith,
2. 2.
91
RESPIRATORY GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM RESPIRATORY GAS
1957, 1958) (see 8). Trifonova 1957, 1958) (see Fig. Fig, 8). Trifonova (1937) (1937) indicated indicated that that there there were were two one two peaks peaks during during early early development: development: one one during during cleavage cleavage and and one during epiboly. only aa single during epiboly. Most Most other other researchers researchers have have reported reported only single peak during during epiboly, epiboly, but but this this may be simply simply a reflection reflection of of insufficient insufficient peak measurements during early development. Metabolic intensity de declines minimum about clines following following blastopore blastopore closure, closure, to to reach reach aa minimum about mid midway (Hayes et al., 1 ; Smith, 1957, 1958; at., 195 1951; Smith, 1957, 1958; way through through organogenesis organogenesis (Hayes 1958; Gruber and Wieser, 1983). 1983). Thereafter there is a Alderdice et al., al., 1958; gradual increase to hatch. hatch. It It is is interesting that metabolic metabolic intensity intensity gradual increase to interesting that �s to increase near hatch while \1 continu 02 values are either stable or 60, continues declining. This would suggest that oxygen supplies are maintained to
���
600
500
400
'�
BlASTOPORE CLOSl>IE
:
5.0 ° C
10.0° C .... IO.2°C
I I
C>-<J
\
1 400
300
MTW
.,
I I I 1 I I
HATCH
.�
200
600
12.5 °C 12.5OC
7.3 °C 7.3oc I
MTW
,
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BlASTOPORE CLOSURE MTW
300
400
200
300
o ��__ 01 . o 0 40 I
.
.
.
.
-+�����--__���
� � � � � -� � � � -� � ,
40
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,
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,
160 Ibo
,
, 200
NUMBER POSTFERTILIZATION NVMBER OF OF DAYS DAYS POSTFERTILIZATITION
240
0
40
80
120
160
NUMBER OF POSTFERTILIZATION NUMBER OF DAYS DAYS POSTFERTILIZATITION
Fig. (Oncorhynchus tshawytscha) Fig. 8. 8. Metabolic Metabolic intensities intensities of chinook chinook (Oncorhynchus tshawytscha) embryos embryos and and larvae constant temperatures. larvae incubated at constant temperatures.
PETER PETER JJ.. ROMBOUGH ROMBOUGH
92
existing embryos compensate existing tissues tissues and and that that embryos compensate for for the fact fact that that the the capsule 1957, 1958) 1958) capsule limits limits oxygen oxygen uptake uptake by by reducing reducing growth. growth. Smith Smith ((1957, demonstrated demonstrated that that in in both both brown brown trout trout and and Atlantic Atlantic salmon salmon metabolic metabolic intensity intensity was was directly directly correlated correlated with with specific specific growth growth rate. rate. There sharp increase increase in in metabolic metabolic intensity intensity on on hatch hatchThere is is typically typically aa sharp this increase increase can ing. As case for V 02, 0 2 , this can be attributed attributed to to removal removal ing. As was was the the case for V of the the capsule capsule and and perivitelline perivitelline fluid fluid as as barriers barriers to to diffusion diffusion and and to to 1964; Davenport enhanced enhanced activity activity (Holliday (Holliday et al., 1964; Davenport and and L�nning, Lgnning, 1980; 1983; Davenport, Davenport, 1983). 1983). Patterns Patterns after after hatch hatch 1980; Gruber Gruber and and Wieser, Wieser, 1983; 021M (routine) are V02/M (routine) are variable. variable. Wieser Wieser and and Forstner Forstner (1986) (1986) reported reported that that V began decline exponentially species of of began to to decline exponentially almost almost immediately immediately in in three three species cyprinids cyprinids (Rutilis (Rutilis rutilis, Scardinius erythrophthalmus, Leuciscus decline in cephaIus). DeSilva et al. al. (1986) (1986) noted noted aa similar similar decline in the the nile nile cephalus). DeSilva tilapia (Oreochromis niloticus). niloticus).A A more more common common pattern pattern is is for for specific specific tilapia (Oreochromis oxygen oxygen consumption consumption to to continue continue to to increase increase until until about about midway midway through period of endogenous endogenous feeding. feeding. This This pattern pattern has has been been through the the period reported for aa number number of of sal�onids salmonids (Smith, (Smith, 1957, 1957, 1958; 1958; Gruber Gruber and and reported for Wieser, Wieser, 1983; 1983; Wieser Wieser and and Forstner, Forstner, 1986; 1986; Rombough, Rombough, 1987), 1987), sardine sardine (Lasker 1964), cod cod (Lasker and and Theilacker, Theilacker, 1962), 1962), herring herring (Holliday (Holliday et al., 1964), (Davenport 1983).In In (Davenport and and L�nning, LZnning, 1980), 1980), and and lumpfish lumpfish (Davenport, (Davenport, 1983). Oz/M declined most of studies the fed, so so that that V VOdM declined most of these these studies the larvae larvae were were not not fed, rapidly rapidly once once endogenous endogenous food food supplies supplies became became limiting. limiting. For For fed lar larvae, vae, specific specific oxygen oxygen consumption consumption may may continue continue to to rise rise until until metamor metamorphosis relatively stable 1983), remain remain relatively stable (Holeton, (Holeton, 1973), 1973), phosis (Forstner (Forstner et al., 1983), or or decline decline gradually gradually (DeSilva (DeSilva and and Tytler, Tytler, 1973), 1973), depending depending on on the the species. species.
2. MASS MASSRELATIONSHIPS RELATIONSHIPS Some investigators, rather than reporting mass-specifi mass-specificc rates of ox oxygen consumption, have chosen to express the relationship between V V 02 0 2 and body mass (M) ( M ) in terms of the allometric power functiOII functiorl
V V 02 0 2
b = aM aMb
(6) (6)
=
where a and b are constants. Such allometric relationships have long princiinterested physiologists, although the underlying biological princi ples remain rather rather obscure. Expressing oxygen consumption in this potentially important fluctuations in fashion has the disadvantage that potentially V V 02 0 2 tend to be obscured by the overall trend. Nonetheless, the rela relationship is widely used, particularly for larvae. [b,Eq. (6)] (6)] calculated for larvae are The metabolic mass exponents [b, often Winberg's (1956) often compared compared to to Winberg’s (1956) generalized generalized value value of b = 0.80. 0.80. =
2. 2.
RESPIRATORY EXCHANGE, AEROBIC AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, METABOLISM
93 93
However, However, Winberg's Winberg's value was based primarily on studies involving juvenile and adult fish, fish, and there is no reason to assume that the same exponent applies to exponent applies to earlier earlier life life stages stages.. Indeed, Indeed, ontogenic ontogenic variations variations in in the value of the metabolic mass exponent are widespread throughout the animal kingdom (Zeuthen, 1970; Adolph, 1983; 1950, 1970; 1983; Wieser, (Zeuthen, 1950, 1984). 1984). In virtually all cases, values decrease as development pro proceeds. ceeds. This generalization appears to hold for many fish species. Kamler (1976) (1976) recognized four periods during the ontogeny of carp on the basis of different metabolic mass exponents: embryos, prefeeding larvae, feeding larvae, and postlarvae. Values were not calculated for embryos and prefeeding larvae, but graphic presentation of the data indicates cantly greater .0. indicates that the the mass mass exponents exponents were were signifi significantly greater than than 11.0. The mass mass exponent exponent of of feeding feeding larvae larvae was was approximately approximately unity unity (b while that (b = = 0.97) 0.97) while that of of postlarvae postlarvae was was typical typical of of juvenile juvenile and and adult adult fi sh (b (b= = 0.80). 0.80). fish High High mass mass exponents exponents during during early early life life appear appear to to be widespread, widespread, 16 species for although not universal, among teleosts. Table I lists 16 which the larval mass exponent approached or exceeded unity. In addition, inferred for for larvae larvae of of addition, mass mass exponents exponents close close to to unity unity can can be inferred brown trout (Gray, 1926; Wood, 1932), 1932), Pacifi Pacificc sardine sardine (Sardinops (Gray, 1926; caerule; Lasker and Theilacker, 1962), 1962), cod (Davenport and L�nning, L@nning, caerule; 1980), Pacific mackerel (Scomber (Scomber japonicus; Hunter and Kimbrell, 1980), 1980), 1980), and and largemouth largemouth bass bass (Micropterus (Micropterus salmoides; Laurence, Laurence, 1969) 1969) from the way in which metabolic intensity varies during development or from graphs showing 02 and M. VOZ M . Many showing the relationship between \1' investigators, investigators, however, however, have have reported reported metabolic metabolic mass mass exponents exponents for for are not significantly different from those expected for juve juvelarvae that are I). Not all such values, though, can be taken (Table I). nile and adult fish (Table VOZ M dur duras accurately representing the relationship between V 02 and M differening much of early life. In most cases no attempt was made to differen tiate between data collected for prefeeding and feeding larvae. Cetta (1982) point out that this practice tends to mask any and Capuzzo (1982) stage-specific differences in the value of the mass exponent. It will actuprobably turn out that in many cases larval mass exponents are actu ally considerably higher prior to exogenous feeding feeding than literature ally suggest. For example, example, careful examination of data pre prereports would suggest. that V VOZ sented by Laurence ((1978) sented 1978) indicates that 02 tended to increase more rapidly with tissue mass for very young cod and and haddock haddock larvae than it larvae. In the one case case in which calculations were made did for older larvae. using only data data for small small larvae larvae (haddock, (haddock, 4°C), P C ) , it was found that the mass (b = = 1.267). 1.267). In In mass exponent exponent was was significantly significantly greater greater than than unity unity (b contrast, data for for all all larval larval stages stages were were combined, combined, mass mass contrast, when when the data
Table Table II Rate-Mass Relationships of of Fish Larvae" Larvae" Metabolic Rate-Mass V02 = aM*) aMb) Allometric Allometric relationship ((VO, =
Species Species
co .a;...
Temp. (0C) ("C)
-
Stage Stage
Size Size range
aa
V vo, 02 units
b
2 1.0 1.0 b� ILl pl h-1 h-'
Cyprinus carpio carpio Cyprinus
20
L
< 6674 mg 74m g
0.64
0.97
Cyprinus carpio carpio Cyprinus Cyprinus carpio carpio Cyprinus
20 20
F L L
Cyprinus carpioh Cyprinus carpiob Abramis Abramis brama brama Anabas testudiAnahas testudineus neus Channa punctatus punctatus Channa Coregonus Coregonus sp. sp. b Acipenser haeri baeri Acipenser
23 20 28
L L L
1.2-4.6 g 1.2-4.6 < ll g ' l lg g 2-70mg 2-70 mg 1.6-40 mg 1.6-40mg 5-50 5-50 mg
1.27 0.60 0.60 1.27 1.27 1.56 1.56 0.45 1.00 1.00
0.80 0.95 0.98 0.98 0.80 0.80 0.054 -0.054 0.93 0.95
28 10 10 20
Saloelinus alpinus alpinus Salvelinus Salmo gairdneri Salmo gairdneri
2 4
L L L F L L-F L-F
Salmo Salmo gairdneri gairdneri
12 12
L-F L-F
1-10 mg 1-10mg 6-65 mg 6-65mg 111-28 1-28 mg 111-390 1-390 mg 50-124mg 50-124 mg 80 mg-7 g 80mg-7g mg-7 g 80 mg-7 80 mg-7 g 80mg-7g
1.00 14.8 14.8 2.4 0.35 2.45 3.1 3.1 111.0 1.0 6.8 19.9 19.9
Clupea harengus harengus Clupea Clupea Clupea harengus harengus
8 8 8
0. 1-0.7 mg 0.1-0.7 dry dry
11.19 . 19
0.91 0.12 1.31 1.31 0.85 1.09 1.09 0.96 11.11 .11 0.93 11.14 . 14 11.1 .1 0.74
L L
M M units mg wet
Comments
Referencec Referencec
Slope signifi cantly different significantly for small and large larvae
11
ILl pl h-1 h-' pl h-1 h-' ILl
mg wet mg wet
mg g-'h-' g-1 h-1 ml hh-'1 h-' mg h-1
mg wet g wet g wet
V 02lM recalculated VO&f
h-' mg h-1 ILmol pmol h-1 h-I g-1 g-' h-' mg h-1
g wet mg wet g wet
mg hh-11 ILmol pmol h-1 h-'
g wet g wet
ILmol -1 pmol h h-1
g wet
p1l h-1 h-' IL ILl pl hh-'1
mg mg dry dry mg mg dry dry
Recalculated V VOdM, 02lM, to metamorphosis Prolarvae Feeding larvae Yolk-sac larvae V02 rrVO2 aV02 V02 rrVO2 V02 aVO, a Hatch to MLDW
Recalculated
avo,
11 2 3 3 3 4 5 5 6 7 8 9
10 10 111 1 111 1
12 12 13 13
13 13
L L
18 18
L L
88
L L
13 13
L
18 18
L
Pseudopleuroneetes Pseudopleuronectes amerieanus americanus
7 7
L L
Melanogrammus aeglefinus aeglefinus
4
L
7 7
L
9 9
L L
Anehoa mitehilli Anchoa mitchilli
26
L L
Aehirus lineatus Achirus lineatus
26
L L
HypophthalmiehHypophthalmichthys thys molitrix molitrix Sparus Sparus aurata aurata
20
L L
19 19
L L
24
L
20
L
Pleuroneetes Pleuronectes platessa platessa
CD tit
Stizostedion lucioperea perca
0.1-0.7 mg 0.1-0.7 dry dry 0.1-0.7 mg 0.1-0.7 dry dry 0.7- 1 .2 mg mg 0.7-1.2 dry dry 0.07-1.2 mg 0.07-1.2 dry dry 0.07-1.2 mg 0.07-1.2 dry dry 7-10 7-10 pg ILg protein protein (dry) (dry) 70-200 mg 70-200 dry dry 50-1000 mg 50-1000mg dry dry 50-1000 mg 50-1000mg dry dry 8.9-424 8.9-424 lL pgg dry dry 14.3-248 14.3-248 ILg pg dry dry 1-50 mg 1-50mg
3.63 3.63
1.33 1.33
3.33
0.87
1.21 1.21
0.78 0.78
2.20
0.96
3.29
0.74
0.006 0.006
28-1000 ILg 28-1OOOpg dry dry 28-1OOOpg 28-lOOO lLg dry dry
0.8-79 mg 0.8-79
ILl h-' h-1 pl
mg dry dry mg
13 13
1.03 1.03
p 1 h-' ILl h-1
pg protein ILg (dry) (dry)
14
0.0053
1.27 1 .27
p I h-' ILl h-1
pg dry ILg
Small larvae only
15 15
0.071 0.071
0.68
0.179
0.55 0.55
0.0077
0.98
ILl h-' h-1 pl
pg dry ILg
Feeding larvae
16 16
0.014
0.94
ILl pl h-1 h-'
pg dry ILg
Feeding larvae
16 16
0.33 0.33
LOS 1.05
ILl p1 h-1 h-'
mg wet
Recalculated Recalculated
17 17
7.09 7.09
1.00 1.00
ILg pg h-1 h-'
mg mg dry dry
18 18
6.47 6.47
11.03 .03
0.31 0.31
0.82
g wet
5
5 0.8 b :5 h-* ml h-1
(continued) (continued)
Table I (Continued) (Continued) V02 = aMb) aMb) Allometric relationship ((VO, =
Species
Perca jluviatilis Perea Rutilis rutilis Heteropneustes H eteropneustes fossilis fossilis Morone saxatalis saxatalis Morone calbasu Lebeo ealbasu Oreoehromis Oreochromis niloticus nilotieus Clupea Clupea harengus harengus Pleuroneetes Pleuronectes platessa platessa Pseudopleuroneetes Pseudopleuronectes americanus amerieanus Gadus Gadus morhua morhua
Arehosargus Archosargus rhomboidalis rhomboidalis
Temp. OC) (("C)
Stage
Size range
aa
b
V 30, 02 units
M units
20 20 20 28
L L L
1.5-32 mg 1.5--32 1.6-62 1.6-62 mg 2-20 2-20 mg
0.29 0.29 0.29 1.00 1.00
0.78 0.82 0.88 0.88
h-I1 ml hh-'- I ml h mg h h-'-1
18 18 28 30
E-L E-L L F
50-1500 50-1500 p. pg 100-300 100-300 mg 1-10 1-10 mg?
0.028 0.028 0.50 9.68 9.68
0.72 0.84 0.42 0.42
p l hh-'I p. -I mg h h-' pl.l hh-'I p
g dry p. dry Pg g wet mg mg dry dry
10 10 10 10
L L
0. 1-10 mg 0.1-10 0.5-10 0.5-10 mg
1.88 1.88 1.67 1.67
0.82 0.65
pIl hh-'1 p. p1l h h-'- I p.
mg dry mg mg dry dry
7
F
0.016
0.78 0.78
pll h h-'- I p.
44 7 10 10 26
L L L L
8-150 p. pg g 8-150 protein 50-1000 p. 50-1000 pg 50-1000 50-1000 p. pgg 50-1000 pg 50-1000 p. 18-66 18-66 p. pg
0.018 0.018 0.017 0.017 0.054 0.054 0.018 0.018
0.71 0.71 0.78 0.69 0.84 0.84
pll h h-'- 1 p.
g protein pg p. dry dry p.g dry dry
-I pll h h-' p.
p.g dry
Comments
Referencec
5 5 19 19
g wet g wet g wet
Recalculated
Recalculated Recalculated
20 2211 22 22 23 23 23 23 14 14
15 15 115 5 15 15 16 16
Abbreviations: L, larvae; F, fry; fry; MLDW, maximum larval dry weight. wejght. VO�M) and tissue mass. b Regression describes relationship between metabolic metabolic intensity ((VOdM) References: (1) (1) Kamler, 1976; 1972; (4) (4) Kaushik and Dabrowski, 1983; 1976; (2) (2)Winberg and Khartova, 1953 1953(cited in Kamler, 1976) 1976) (3) (3) Kamler, 1972; 1983; (5) (5) Kudrinskaya, 1969; (9) Khakimullin, 1985; 10) Holeton, 1973; 1973; (11) (11) 1969; (6) (6) Mishra and Singh, 1979; 1979; (7) (7) Singh et et al., 1982; 1982; (8) (8)Forstner et et al., al., 1983; 1983; (9) 1985; ((10) 1985; ((12) 1966; ((13) 1984; ((14) 1978; ((16) Wieser, 1985; 12) Blaxter and Hempel, 1966; 13) Almatar, 1984; 14) Cetta and Capuzzo, 1982; 1982; ((15) 15) Laurence, 1978; 16) Houde and Schekter, 1983; ((17) 17) Mukhamedova, 1977; 18) Quantz and Tandler, 1982; 1982; ((19) 19) Sheel and Singh, 1981 (20) Eldridge et 1982; (21) (21) Durve and Sharma, 1983; 1977; ((18) 1981;; (20) et al., 1982; 1977; (22) al., 1986; 1986; (23) 1977; (22) DeSilva et et al., (23) De Silva and Tytler, 1973. 1973. a
e
2. 2.
RESPIRATORY GAS EXCHANGE, AEROBIC METABOLISM RESPIRATORY GAS EXCHANGE. AEROBIC METABOLISM
97
exponents were found to be typical of of those seen in much older fish (b (b = = 0.55-0.78). 0.55-0.78). It It should should be recognized, recognized, however, however, that that mass mass exponents exponents are not always high, even for very young larvae. For example, a careful (1973) showed the metabolic mass expo expostudy by DeSilva and Tytler (1973) nents of both herring and plaice larvae to be considerably less than (0.82and 0.65, respectively). The reason why some species have unity (0.82 such relatively low mass exponents as larvae has not been established. Several Several reasons reasons have have been been advanced advanced to to explain explain high high metabolic metabolic (1976) suggested mass exponents during early development. Kamler (1976) that the transition from the high larval value to the lower juvenile value in carp was the result of of morphological changes affecting gas exchange. Pauly (1981). (1981).Pauly Pauly (1981) (1981) exchange. This This idea idea was was expanded expanded upon upon by Pauly proposed proposed that that the the underlying underlying factor factor limiting limiting metabolic metabolic rate rate and and hence hence growth was the the availability availability of of oxygen, oxygen, rather rather than than food food or or some some growth in in fish was intrinsic intrinsic control control mechanism. mechanism. He He reasoned reasoned that that if if oxygen oxygen was was not not limit limiting, ing, metabolic metabolic rate rate should should be be directly directly proportional proportional to to body body mass. mass. To To support support his his contention contention that that oxygen oxygen is is not not limiting limiting during during early early life, life, he noted noted that that gill surface surface area area of of larval larval herring herring and and plaice plaice increased at at aa much faster body mass mass exponents and much faster rate rate than than body mass (gill (gill area area mass exponents of of 3.36 and 1.59, respectively; De Silva, Silva, 1974). 1974). This was rather an unfortunate 1.59, choice larval metabolic metabolic choice of of species, species, since since in in both both herring herring and and plaice the larval mass exponent is significantly less than unity (0.82 (0.82 and 0.65, 0.65, respec respectively; 1973). This tively; De De Silva Silva and and Tytler, Tytler, 1973). This does does not not necessarily necessarily disprove disprove Pauly's Pauly’s basic basic hypothesis hypothesis (i.e., (i.e., that that the the ability ability to to supply oxygen oxygen to to tissues metabolic rate) tissues limits limits metabolic rate) but but indicates indicates that that more more sophisticated sophisticated analysis of total exchange capacity, perhaps similar to that done by Ultsch 1973) for is required required to adequately test for lungless lungless salamanders, salamanders, is to adequately test Ultsch ((1973) the hypothesis. As discussed earlier, cutaneous gas exchange is ex extremely must be included included in in any any analysis analysis of of tremely important important in in larvae larvae and and must diffusing diffusing capacity. capacity. In In both both herring herring and and plaice plaice cutaneous cutaneous surface surface area area expands body mass mass (mass 0.58 and and expands at at aa slower slower rate rate than than body (mass exponents exponents of of 0.58 0.50, capacity 0.50, respectively; respectively; De De Silva, Silva, 1974). 1974). Thus, Thus, the the total total diffuSing diffusing capacity of these larvae, larvae, in about the of these in fact, fact, may may be be expanding expanding at at about the same same rate rate as as metabolism. must be pointed pointed out out that that not not all all investigators investigators agree agree metabolism. It It must with this In particular, Itazawa ((1985) 1985) re rewith this hypothesis. hypothesis. In particular, Oikawa Oikawa and and Itazawa cently show no cently presented presented data data for for carp carp that that they they contend contend show no direct direct rela relaresting metabolism. tionship tionship between between respiratory respiratory surface surface area area and and resting metabolism. Forstner (1983) attributed attributed the the high high mass mass exponent exponent of of corego coregoForstner et al. (1983) muscle and nid to aa preponderence preponderence of of red red muscle and concommitant concommitant high high nid larvae larvae to activity mass activity of of oxidative oxidative enzymes. enzymes. The The decline decline in in the the value value of of the the mass exponent exponent following following metamorphosis metamorphosis was was attributed attributed to to aa major major reorganireorgani-
98
PETER J. ROMBOUGH PETER J. ROMBOUGH
zation of metabolism in which glycolytic activity becomes progresprogres sively more important (Forstner et al., 1983; 1983; Hinterleitner et al., 1987). 1987). There are, however, major differences among species in the developmental enzymes (Hin developmental trajectories trajectories of of oxidative oxidative and and glycolytic glycolytic enzymes (Hinterleitner et al., d.,1987), 1987), and it is difficult to believe that high oxidative enzyme enzyme activity activity per per se se is is sufficient to to explain explain high high mass mass exponents exponents early salmonids generally early in in life. life. For For example, example, salmonids generally display display high high mass mass ex exponents least the yolksac stage ponents up up to to at at least the end end of of the the yolksac stage (Table (Table I, Wieser Wieser and Forstner, 1986; 1986; Rombough, 1987), 1987), yet activity is borne almost entirely 1983), and and levels levels of of oxida oxidaentirely by by white white muscle muscle (Forstner (Forstner et al., 1983), tive tive enzymes are are very very low (Hinterleitner (Hinterleitner et al., 1987). 1987). Quantz and Tandler ((1982) 1982) attributed the high metabolic mass ex exponent of larval gil thead seabream (Sparus gilthead (Sparus aurata) aurata) to their high feed feeding rate. They speculated that since the larvae were feeding more or less continuously, they should have a high specific dynamic action (heat (heat increment). increment). Noting Noting that that juvenile juvenile and and adult adult fish had had aa mass expo exponent near unity for active metabolism (Brett and Groves, 1979), 1979), they suggested that it was not surprising that mass exponents for larvae were were of of aa similar similar magnitude. magnitude. The allometric equations relating \102 VO, and M M frequently have been used to compare metabolic levels of different species or ecological groupings. Konstantinov (1980) (1980) points out that such comparisons are only valid if weight exponents are equal. equal. This severely restricts the use of such equations. Metabolic weight exponents are seldom the same species, values for b vary same for different species. species. Even in the same during development uenced by such as during development and and are are infl influenced by factors factors such as tempera temperature (Laurence, (Laurence, 1978; 1978; Konstantinov, 1980; 1980; Almatar, Almatar, 1984) 1984) and activity (De Silva and Tytler, Tytler, 1973; 1973; Wieser, 1985). 1985).
ACTIVITY 3. ACTIVITY As for juveniles and adults (Brett, (Brett, 1970), 1970), activity is the single most important factor influencing the metabolic intensity of of larvae (Blaxter, (Blaxter, 1969). 1969).The relationship between activity and oxygen consumption has ' been inves tigated in some detail for juvenile and adult fi sh (Beamish, investigated fish (Beamish, 1964, 1964, 1978; 1978; Brett, 1964, 1964, 1970, 1970, 1972; 1972; Brett and Groves, 1979; 1979; Fry, 1971). 1971). Yet only recently has there been much interest in the earlier life stages, although there are compelling reasons to suspect that ener energetic relationships may be significantly different from those of of older fish. The body musculature and supporting metabolic machinery de fish. develop gradually during the embryonic and larval periods and in many species do not assume typical juvenile patterns until after metamor-
2. 2.
RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM RESPIRATORY
99
1982; Forstner et al., ai., 1983; 1983; Batty, 1984; 1984; Wieser et phosis (Johnston, 1982; ai., 1985). 1985). In addition, juveniles and larvae are exposed to different al., hydrodynamic regimes at routine swimming speeds. In larvae, routine activities occur in an intermediate hydrodynamic environment where both resistive and inertial forces are important (Webb (Webb and Weihs, 1986). 1986). As larvae grow or or during burst swimming they move into the adult hydrodynamic regime, where inertial forces dominate. In many yle species this transition is accompanied by a change in swimming st style from continuous movement of of body and tail to a beat and glide pattern (Hunter, (Hunter, 1972, 1972, 1981; 1981; Weihs, 1980; 1980; Batty, 1984). 1984). To further confound matters, there are species-specific differences in development of of mus muscle types and biochemical pathways (Wieser et al., 1985; 1985; Hinterleitner Hinterleitner et al., ai., 1987). 1987). The study of of the physiological energetics of of the early life stages has been hampered by the lack of clear defi nitions for the various definitions levels of metabolism associated with activity. In juvenile and adult fish, fish, energy expenditure is is normally described in terms of standard V0 2 ) and active (a V0 2), routine (r V02) metabolism (see (s (sVO~), (rVO2) (aVO2) (see Brett and 1979, for definitions). Groves, 1979, definitions). These terms have been applied to to the early stages, but it should be noted that conditions conditions are somewhat different during early life. In older fish fish standard metabolism refers to the postabsorptive state, a condition that is obviously not met during endogenous feeding. feeding. In addition, the standard metabolic rate of em embryos and larvae includes a sizeable sizeable growth component so that, unlike in older fish, fish, metabolic rates rates can be depressed considerably below the so-called standard level without affecting survival. survival. What most investi investigators gators consider standard metabolism during early early life is actually sim simply the metabolic metabolic rate under conditions of minimal neuromuscular activity. The vast majority of investigators have have attempted to measure the embryonic embryonic and larval larval equivalent of routine routine metabolism. This is a rather nebulous nebuIous term, term, since, since, as as Wieser (1985) (1985)points out, out, it can vary at least twofold in resporise response to a variety of intrinsic and extrinsic factors. factors. For embryos and larvae, routine metabolism probably can be defined best as the average rate of aerobic metabolism under normal rearing conditions. conditions. Active Active metabolism in older fish fish usually refers refers to sustained activity. activity. The The young of many species, species, however, however, are are not capable of sustained activity. activity. As a result, result, most estimates of active metabolism during early life are are based based on burst activity. activity. It is is a moot moot point point whether such results are comparable with values for juveniles and adults. adults. V02 Absolute aerobic aerobic scope, scope, defined as as the difference between between aaV02 Y02 , is and ssV02, is of particular interest to physiologists because it repre represents the amount of energy available to a fish fish to cover the the cost of
100 100
PETER PETER J. ROMBOUGH ROMBOUCH
various biological activities. Unfortunately, it has proved difficult to estimate absolute scope during early life, although a new type of of metabolic chamber recently described by Dabrowski ((1986) 1986) may make such determinations easier in the future. future. It has proved some somewhat easier to estimate selected portions of absolute scope, in particu particuV02 (termed V02 and lar between aaV02 lar the the difference difference between and rrV02 (termed the the relative relative scope; scope; V02 and Wieser, (termed the Wieser, 1985) 1985) and and the the difference difference between rrVO2 and ss V V 02 0 2 (termed the routine scope; scope; Beamish, Beamish, 1964). 1964). It It thus is possible possible to to obtain rough rough estimates of absolute scope indirectly by adding values reported for routine and relative scopes. scopes. Some Some investigators have used the ratios of metabolic rates at the various expressvarious activity levels, instead of their differences, as a way of express V02/s V02 are re V02/rV02, and V02/s V02, aaVOZ/rV02, ing ing scope. scope. The The ratios ratios aaV02hV02, and rrVOzlsV02 referred to, respectively, as absolute factorial scope, scope, relative factorial scope, scope, and routine factorial factorial scope. scope. This is convenient for comparative purposes but does not tell the whole tale, since it gives no indication of absolute absolute costs, which are of profound ecological importance. As will be discussed later, scopes and factorial factorial scopes can change in response to various intrinsic and extrinsic factors. factors. It should be recognized, how however, that because of the manner in which they are calculated, they V02 may not always change in the same direction. For instance, if aaVO2 V 0 2 decline in parallel, absolute scope will remain constant but and s V 02 absolute factorial scope will increase. V02-s V02) is routinely estimated for ju Absolute aerobic scope (a (aVOz-sVOZ) juvenile and adult fish by forcing them to swim against a current at progressively faster speeds. speeds. The oxygen uptake at maximum sustained V02, while extrapolation of swimming speed is taken to represent aaVO2, the power-performance power-performance curve back to zero swimming speed yields V02• Ivlev ((1960a) 1 960a) appears to have been the only investigator to have ssVO2. applied this technique to very young fish. fish. He reported that young Atlantic salmon (400 mass) were capable of sustaining a rate of (400 mg wet mass) oxygen uptake 22.5 22.5 times their standard rate. This value is sometimes cited as the degree of metabolic expansibility that can be expected for fish fish larvae (e.g., (e.g., Blaxter, Blaxter, 1969). 1969). However, more recent evidence sug suggests that this is a gross gross overestimation and that absolute factorial scopes actually range from about 2.5 to 10, 10, depending on species, fish size, and temperature (Table (Table II). 11). of the standard Dabrowski ((1986) 1 986) recently used a modification of power-performance power-performance procedure to estimate aerobic scope for salmon fry. The fish were induced to swim at progres progresalevins and coregonid fry. sively faster speeds by making use of their optomotor reaction to a background. Swimming speeds were monitored and correlmoving background.
2.
RESPIRATORY AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC METABOLISM
101 101
ated with rates of of oxygen uptake. For young Atlantic salmon, the maxi maximum V 02 recorded was about 3.3 3.3 times the value estimated for zero VOZ activity. scope of Coregonus schinizi fry was activity. The absolute factorial scope somewhat lower, about 2.5. A less common way of of estimating aerobic scope in juvenile and sh is to take the difference between the maximum and mini adult fi fish miniVOZ exmum V 02 recorded while fish are held in a respirometer for an ex tended period. The value obtained is is correctly termed the aerobic scope for spontaneous activity but, at least in some species, it appears scope (Ultsch et al., al., 1980). investigato approximate absolute scope (Ultsch 1980). Several investiga tors have applied this technique to fish larvae. Holliday et al. 1964) al. ((1964) indicated that there was approximately a 10-fold 10-fold difference between the minimum and maximum V 02 of herring larvae. For sardine larvae VOZ the maximum difference in V 02 recorded was about 3.5-fold (Lasker VOZ and Theilacker, 1962), 1962), while in winter founder (Pseudopleuronecter americanus) americanus) the maximum difference was only about 3.0-fold (Cetta and C apuzzo, 1982). Capuzzo, 1982). It is not clear why the apparent scope of herring should be so much greater than that of the other species, but it is interesting that larvae of Pacifi c herring (Clupea Pacific (Clupea pallasi) pallasi) also appear capable of increasing their metabolic rate about lO-fold 10-fold (Eldridge et al., 1977). 1977). In the case of of the Pacific herring, it was stress caused by the shaking of the respirometer, rather than spontaneous activity, that led to elevated metabolic rates. As mentioned previously, it has proved difficult to induce larvae to swim in a respirometer and, as a consequence, other techniques have V02 and aaVO2. V02. The most common had to be developed to estimate ssV02 V02 has been to anesthetize the larvae (Holli method for estimating ssV02 (Holliday et al., al., 1964; 1964; DeSilva and Tytler, Tytler, 1973; 1973; Davenport and L�nning, Lgnning, 1986). This technique indicates routine factorial 1980; 1980; DeSilva et al., 1986). V02/s V02) in the range of scopes (r (rVO2lsV02) of 1.4-3.3, 1.4-3.3, depending on species and stage of development (Table (Table II). 11). Another method has been to assume that the capsule severely restricts activity just before hatch (Daven (Davenport and Lgnning, L�nning, 1980; 1980; Davenport, 1983). 1983). Davenport and L�nning L9nning ((1980) 1980) demonstrated that, at least in in cod, the metabolic rate of of em embryos just before hatch was not signifi cantly different from that of significantly anaesthetized larvae shortly after hatch. Assuming that the metabolic rate of unanesthetized larvae shortly after hatch is is representative of V02, this method gives routine factorial scopes for cod (Davenport rrVOz, (Davenport and L�nning, 1980) and lumpfish (Davenport, 1983) of about 2.0. Lgnning, 1980) (Davenport, 1983) The evidence is is rather sketchy, sketchy, but it appears that the difference V02 (routine V02 and ssVOZ between rrVOz (routine scope) scope) tends to decrease as larvae mature (DeSilva (DeSilva and Tytler, 1973; 1973; Davenport and L�nning, L$nning, 1980; 1980; De-
Table II I1 Table Scope for Activity Scope ... 0 CI 0 �
te
Species
Clupea Clupea harengus harengus
Stage Stage
Temp. (“C) (0C)
Gadus Gadus morhua morhua Cyclopteros Cyclopterus lumpus lumpus Oreochromis Oreochromis niloticus niloticus Salmo salar salar
E L L L L L L L L L L
8 8 8 8 8 10 10 10 10 5 5 5 5 5 5 5 5 30 20
Salvelinus Salvelinus alpinus alpinus
L
4
Pleuronectes plattesa Pleuronectes plattesa Gadus Gadus morhua morhua
Scope
Technique
VOv's V02) Routine factorial factorial scope (r (rV0dsVOp) V 2.1 rrvodanesthetized Ov'anesthetized V 2.1 302 02 VOv'anesthetized V V02 1.4 02 rrV0daneseetized 2 .5 Posthatch V Ov'prehatch V 2.5 VOdprehatch V02 02 VOv'anesthetized V 1.9 VOp 1.9 rrVOdanesthetized 02 VOv'anesthetized V 1.6 rrVOdanesthetized VOp 02 VOv'anesthetized V 2.2 rrV0daneseetized 2.2 V02 02 VOdprehatch VO 2.0 Posthatch V Ov'prehatch V 02p V tN02 in dark , 11.6 .6 02 in ligh VOp ligh.hO2 02 .0 2.0 VOdprehatch VOz 2 Posthatch V Ov'prehatch V VOv'anesthetized V 3.5 rrvodanesthetized 3.5 VOp 02 1.5 Extrapolated from power-perf or1.5 power-performance curve Posthatch V Oprehatch V 2 .6 2.6 VOflrehatch V 02 Op
Reference
Holliday et al. (1964) et al. (1964) Holliday et al. ((1964) 1964) et al. ((1964) 1964) Holliday et (1973) De Silva and Tytler (1973) De Silva and Tytler ((1973) 1973) Davenport and Lonning (1980) (1980) Davenport and Lonning ((1980) 1980) Solberg and Tilseth (1984) (1984) Davenport ((1983) 1983) 1986) De Silva et et al. ((1986) Ivlev (1960a) (1960a) Gruber and Wieser ((1983) 1983)
V02!'rV02) (aVO$rVOz) Relative factorial scope (a
Alburnus alburnus Salmo Salmo gairdneri gairdneri
(3 species) species) Cyprinids (3
9.7" 9.P 2.7 1.9 1.9 1 .8 -1.8 2.4 2.4 2. 1 2.1
L L L L L
20 4 12 12 20 12 12 20
E L L
14 14 14 14 7
2.8 2.8 3.5 3.0
L L
8 12.5 12.5
10.4 10.4 10.0 10.0
E L
10.0 10.0 8
L L F
20 22 14
Power-perfonnance Power-performance relationship Forced bursts Forced bursts Forced bursts Forced bursts Forced bursts
Ivlev ((1960b) 1 960b) Wieser et al. ((1985) 1985) Wieser et al. (1985) (1985) Wieser et et al. (1985) (1985) Wieser and Forstner ((1986) 1986) Wieser and Forstner (1986) (1986)
Spontaneous factorial scope
Sardinops Sardinops caerulea caerulea
.... Q �
Pseudopleuronectes Pseudopleuronectes americanus americanus Cltlpea Clupea harengtls harengus harengus Clupea harengtls pallasi pallasi Salmo salar Salvelinus Salvelinus alpinus alpinus
Salmo salar Salmo salar Coregonus Coregonus sp. sp. a 0
Unrealistically high, see text.
Observed max. V VOdmin. 02!'min. 02!'min. Observed max. V VOdmin. Observed max. 02!'min. ma.V VOdmin.
V 02 VOZ V 02 VO Z V 02 VOZ
Lasker and Theilacker (1962) (1962) Lasker and Theilacker (1962) (1962) Cetta and Capuzzo Capuzzo ((1982) 1982)
Observed 02!'min. V 02 , Observed max. max. V VOdmin. VO2 Posthatch max. 02!'prehatch V 02 max. V VOdprehatch VOZ
.
Observed max. V 02!'min. V 02 VO2 VOdmin. Posthatch max. V VOdprehatch VOZ Posthatch 02!'prehatch V 02 V02!'s V02) Absolute factorial scope (a (aVOdsV0,) 3.0 5.0
22.5" 22.5" 3.3 3.3 2.5 2.5
Power-perfonnance Power-performance relationship Power-performance relationship relationship Power-perfonnance Power-performance relationship relationship Power-perfonnance
Holliday et 1964) et aZ. al. ((1964) aE. ((1977) 1977) Eldridge et al. 1951) et al. ((1951) Hayes et Gruber and Wieser Wieser (1983) (1983) 1960a). Ivlev ((1960a). Dabrowski ((1986) 1986) Dabrowski ((1986) Dabrowski 1986)
104 104
PETER PETER J. ROMBOUGH ROMBOUGH
Silva et al., 1986). 1986). For example, exampIe, the routine scope oflarval of larval herring and plaice at metamorphosis, measured as the difference between un unanaesthetized 02, was 25% of of that that at at anaesthetized and and anaesthetized anaesthetized V V02, was only only about about 25% hatch The decrease in routine 1973). The decrease in routine scope scope re rehatch (DeSilva (DeSilva and and TytIer, Tytler, 1973). V02. V02 than flected flected aa more more rapid rapid decline decline in in rrV02 than in in ssVOz. Wieser Wieser and and his his co-workers co-workers (Wieser, (Wieser, 1985; 1985; Wieser Wieser et al., 1985; 1985; Wieser Wieser and and Forstner, Forstner, 1986) 1986) have have attempted attempted to to obtain obtain estimates estimates of of ac active metabolism by forcing larvae (using electrical or mechanical stim stimulation) to swim at burst speeds speeds for short periods (30-60 (30-60 s). s). The main driving force behind such activity was shown to be anaerobic (Wieser et al., 1985), 1985), but it was felt that the maximum rate of oxygen uptake during activity activity or or the the first few few minutes minutes of of recovery recovery (the (the response response time of 02 was of the the system system was was not not fast fast enough enough to to say say precisely precisely when when V V02 V02. Using maximal) maximal) approached approached aaVO2. Using the the average average rate rate of of oxygen oxygen uptake uptake V02, this prior technique gave this technique gave estimates estimates of of 1.9-2.7 1.9-2.7 and and prior to to activity activity for rrVO2, V02/rV02) of young rainbow 2.4-2.9 for the relative factorial scope (a (aVO2lrVOz) trout (Wieser (Wieser et ai., (Wieser and Forstner, al., 1985) 1985) and larval cyprinids (Wieser V02 is V02, absolute 1986), 1986),respectively. respectively. Assuming Assuming that that aaV02 is about about twice twice ssV02, absolute factorial to 6. 6. factorial scopes scopes would appear to to range range from from about about 4 to In In juvenile juvenile and and adult adult fish, fish, aerobic aerobic scope scope tends tends to to increase increase as as the the 1973; Wieser, 1985). 1985). The pattern is is not as fish grows (Brett and Glass, 1973; obvious for younger fish. rainfish. The metabolic expansibility of young rain bow body mass, bow trout trout increased increased with with body mass, more more or or less less as as expected expected (Wieser, (Wieser, 1985; Wieser ai., 1985). 1985; Wieser et al., 1985). For For example example at at 4°C, 4"C,relative relative factorial factorial scope scope increased from about 2.7 for yolk-sac larvae (80-120 mg) mg) to to about about 5.2 5.2 increased from about 2.7 for yolk-sac larvae for fry weighing between 3 and 10 g (Wieser et al., 1985). 1985). On the other hand, in· i n cyprinids, relative factorial scope was independent of body mass between 1 and 400 mg (Wieser (Wieser and Forstner, 1986). 1986). Wieser and Forstner ((1986) 1986) suggested that this may reflect the need of very small larvae to avoid the constraints small size normally places on aerobic scope if if they are to escape predation. The influence of temperature on aerobic scope appears to vary depending on the species. As was the case involving the effect of body mass, young rainbow trout follow a pattern similar to that seen in 1985). Routine meta metajuveniles (Wieser, 1985; juveniles and adults (Wieser, 1985; Wieser et al., ai., 1985). bolic rate increases steadily with temperature 4°C and 20°C temperature between 4°C (Fig. 9). 9). Up to about 12°C, 12"C,active metabolism also increases with tem tem(Fig. perature, but at a faster rate than routine metabolism, so that relative 12"C, however, there is a descope increases. At temperatures above 12°C, de crease in the rate at which active metabolism expands so so that relative V02/rV02) remains remains constant scope (a scope (aVOzlrVO2) constant or or even even declines. declines. The The reasons reasons for for this decline decline have have not not been been demonstrated, demonstrated, but it it may may be that, that, as as in
2. 2.
RESPIRATORY RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM
105 105
• yolk yolk sac sac • 80-200 80-200 m9 me
32
32i
"i
�
mo--1000 mg mg • 200-1000 V 3-10 3-10 9 B ...
"iGl 24
1� 20
� t-
ift z III t2
u
... o • ...
t III :I
I I I
1 l'
.,.
12 8
.,
" " I " "
I
"
"
'
I, I
I
I
"
I
" "
� '"'" /' " ,/ � / ', " / i<,
f\
28
"
,-
"
,�
I
.,, '
�'
,
I
/
I,' I
I
'r
rt" '" " ........ "
4 2 0 4 20 112 2 4 T E M P E AATU A E - ° c TEMPERATUREOC
Fig.. 9. 9. Temperature Temperature dependence of rates rates of oxygen consumption of four size classes Fig rate; dashed lines, maximum maximum rate rainbow trout Salmo gairdneri. Solid lines, routine rate; of rainbow et al. ((1985).] 1985).] during or immediately following forced burst activity. [After [After Wieser et
adults adults (Fry, (Fry, 1971), 1971), oxygen oxygen limits limits active active metabolism metabolism at at high high ambient ambient temperatures. scope in temperatures. Relative Relative factorial factorial scope in cyprinids, unlike unlike in in rainbow rainbow trout, relatively independent independent of of temperature temperature between between trout, appears appears to to be relatively 12°C 1986). This 12°C and and 24°C 24°C (Wieser (Wieser and and Forstner, Forstner, 1986). This relationship, relationship, how however, should probably be checked since Wieser and Forstner (1986) (1986) used used data data pooled pooled from from three three species species (Rutilus rutilus, Leuciscus cepha cephalis, Scardinius erythrophthalmus), erythrophthalmus), and and the the apparent apparent temperature temperature in independence simply reflect dependence of of aerobic aerobic scope scope in in cyprinids cyprinids may may simply reflect compen compensating sating differences differences in in temperature temperature optima. optima. Y02 is It or less less fixed for for aa given given It is is usually assumed assumed that that aaV02 is more more or Y02 would temperature size of temperature and and size of fish. fish. Thus Thus factors factors that that increase increase rrV0, would be (1985) indicated that this expected to reduce relative scope. Wieser (1985) may not be the case. He noted that, within a given size class of of rain rainY02, Y02 also had a high a bow trout, those individuals with a high rrV0, avo,, Y02 • Wieser ((1985) Y02 had a low aaV02. while individuals with a low rrV02 1985) speculated ?) may speculated that that as as yet yet undefined undefined controlling controlling factors factors (hormones (hormones?) may influence influence individual individual metabolism metabolism in in the the same same way way temperature temperature does. does.
106
PETER PETER J. ROMBOUGH J . ROMBOUGH
Thus, an and aa an increase increase in in rV02 r\102 would would result result in in an an increase increase in in aVO2 a\102 and Thus, concomitant increase in relative scope. This hypothesis appears to be be concomitant increase in relative scope. This hypothesis appears to worth testing because if true, fish have a greater flexibility in their worth testing because if true, fish have a greater flexibility in their metabolic response response to stress than than previously previously assumed. assumed. metabolic to stress
ENDOGENOUS RHYTHMS 4. 4. ENDOGENOUS RHYTHMS
of oxygen consumpconsump It is recognized that diurnal variation in rates of tion can be a major source of of error in energetic analyses of of early 1983). Empirical estimates of of the magnimagni growth (Houde and Schekter, 1983). tude of of such variations during early life, though, are scanty. Ryzhkov (1965) reported oxygen uptake by eggs of of the Sevan trout (Salma isis (1965) trout (Salmo chan) peaked at about 0700 h and again at about 2100 2 100 h. Minimum and chan) \102 al. (1964) (1964) 3-fold. Holliday et al. maximum values for V 0 2 differed 2- to 3-fold. \102 unanes present data showing that the V 0 2 of of both anesthetized and unanesthetized herring larvae was lower during the “night” "night" than during the "day" “day” even though all measurements were taken under identical in indoor lighting conditions. This pattern was confirmed by De Silva and Tytler (1973). (1973). Oxygen uptake by both anesthetized and unanesunanes thetized herring larvae declined at "dusk" “dusk” and then gradually rose to typical daylight levels by "dawn." “dawn.” Again, all tests were conducted under indoor lighting. Minimun night-time values were about 50% of of typical daytime values. De Silva et al. al. (1986) ( 1986) recently reported diurnal \102 variation of the nile tilapia (Oreochronis (Oreachronis variation in in V 0 2 from larvae larvae and fry of 02 peaked twice in the course of a day. nilaticus). V02 day. nitoticus).Unlike in herring, \1 For newly hatched larvae, the peaks occurred just after sunset andjust and just before dawn. The \1 V 02 0 2 varied about 3-fold, 3-fold, with average nighttime rates being somewhat higher than average daytime rates. For older larvae (5 (5days) days) and fry (3 (3weeks), V\102 0 2 peaked at sunrise and then again at about noon. Rates varied 2- to 3-fold, 3-fold, with average daytime rates being somewhat higher than average nighttime rates. Geffen (1983) (1983) examined the effect of of various light-dark light-dark regimes on oxygen uptake \102 by Atlantic salmon embryos. During a 24-h period, V 0 2 rose to a single peak at about 1200 1200 h when embryos were held in constant darkness. Under a 12 12:: 12 12 light-dark light-dark cycle, V 02 0 2 peaked toward the cycle, \1 end of the dark period ((1000 1000 h) h) at about 0600 h. Under a 6 :: 6 light lightdark regime (two day), \1 (two cycles per day), V 02 0 2 peaked in the middle of the first dark period, again at about 0600 h. There was no peak during the second dark period. Under all light regimes peaks were rather sharp, with \1 V 02 0 2 remaining elevated for only 2-6 2-6 h. Peak \1 V 02 0 2 values were from 2 to 4 times the average nonpeak \1 V 02• 02.
2. 2.
RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM RESPIRATORY
107 107
5. GROUP FFECT GROUPE EFFECT
20-50% de deMetabolic rates of juvenile and adult fish can vary 20-50% pending on the number of of individuals in the group (Brett, (Brett, 1970; 1970; Ita Itazawa 1978; Kanda Kanda and and Itazawa, Itazawa, 1981). 1981). The The possibility possibility of of aa simi simizawa et al., 1978; lar lar "group “group effect" effect” during during the the early early life life stages stages has has received received considerable attention from Soviet researchers. Most of the earlier studies indicated that metabolic rates were significantly higher for (Ryzhkov, 1968; 1968; Grigor'yeva, Grigor’yeva, isolated individuals than for groups groups (Ryzhkov, 1967; Malyukina 1969; Kudrinskaya, 1969; Korwin 1967; Malyukina and and Konchin, Konchin, 1969; Kudrinskaya, 1969; Korwin Kos Kossakowski et al., al., 1981). 1981).This appeared to hold true for embryos as well as larvae and for schooling and nonschooling species. Reduced oxy oxygen gen uptake uptake was was attributed attributed to to reduced reduced motor motor activity, activity, chemicals chemicals re released into How water by by other other fish, fish, or or reduction reduction in in territoriality. territoriality. Howleased into the water ever, 1971, 1981, 1982) ever, Konchin Konchin ((1971,1981, 1982) has has recently recently reexamined reexamined the the "group “group effect" effect” in in several several schooling schooling and and nonschooling nonschooling species and and concluded concluded that earlier reports of reduced oxygen consumption by individuals in dropgroups were simply the result of oxygen levels in respirometers drop ping critical level level and and hence hence limiting limiting gas gas exchange. exchange. He He ping below below the critical found found that that if if fish fish to to respirometer respirometer volume volume ratios ratios were were kept kept constant constant there of eggs eggs and and there was was no no significant significant difference difference in in oxygen oxygen consumption consumption of larvae of the summer bakhtah (Salmo (Salmo ischan), ischan), roach (Rutilus (Rutilus rutilus), rutilus), or pike (Esox lucius) as or pike (Esox lucius) as groups groups or or as as individuals individuals.. The The rate rate of of oxygen oxygen uptake of isolated individuals was not affected by visual contact with larvae larvae of of that that particular particular species. species. Similarly, Similarly, "conditioned" “conditioned” water water (water (water in which larvae were previously held) held) did not affect oxygen uptake if it was aerated.
C. Abiotic Factors 11.. TEMPERATURE TEMPERATURE Next to has the Next to stage stage of of development development and and activity, activity, temperature temperature has the greatest greatest influence influence on on the the metabolic metabolic rates rates of of embryos embryos and and larvae. larvae. In In spite spite of of this, this, temperature temperature relationships relationships have have been been examined examined in in detail detail in only aa few some ways in only few species species.. In In some ways thermal thermal relationships relationships during during early early life those in For example, similar to to those in later later life. life. For example, when when the the life appear appear to to be similar logarithms the routine routine metabolic metabolic rates rates of of embryos embryos and and larvae larvae of of the the logarithms of the various Table III plotted against various species species listed listed in in Table I11 are are plotted against temperature, temperature, the the resulting curve assumes the convex shape shape typical of that for juveniles and 10). In In other other ways, ways, however, however, and adults adults (Krogh's (Krogh’s standard standard curve) curve) (Fig. (Fig. 10).
Table 1 III Table 11 Routine Routine Metabolic Intensities during during Early Early Life" Lifea
Species Species
Stage
Temp. (0C) ("C)
L, newly hatched hatched hatched L, newly hatched 19-50 dpf dpf E, 19-50 1 10 rng mg L, 110 half E, latter half 120-200 mg L, 120-200 120-200 mg L, 120-200 120-200 mg L, 120-200 16.4 mg E, 16.4 E-L, 23.2 mg E-L, E-L, 27.8 mg E-L, E-L, 24.9 mg E-L, E-L, 21.6 mg E-L, L, 70 mg mg L, 70 70 mg L, 70 70 mg L, 70 mg L, 70 L, 70 70 mg E-L E-L E-L E-L E-L E-L L, 45-70 45-70 dph L, newly hatched hatched E E
4.0 14 14 10.0 9.0 10.0 10.0 4.0 12.0 20.0 10.0 10.0 6.0 9.0 12.0 12.0 15.0 15.0 10.0 10.0 12.0 12.0 14.0 14.0 16.0 16.0 18.0 18.0 3.0 7.0 12.0 12.0 10.0 10.0 8.0 8.0 4.0 4.0 8.0 8.0
Metabolic Metabolic intensity intensity wt.)-l h-'1 h- 1] wet wt.)-' [lA-g(g wet [pg(g
Technique
Comments
Referenceb
Freshwater Freshwater salmonid
Salmo salar salar SaEmo
Salmo gairdneri gairdneri Salmo ' .... r
0 0 (Xl 0
Salmo Salmo trotta trutta
Salve linus alpinus Salvelinus alpinus
164 164 257 205 354 442 96 269 448 280 200 311 311 405 548 57 1 571 486 514 571 571 786 291 291 383 836 190 190 131 131
64 64
107 107
W W HG W DC POS POS POS W POS POS POS POS W W W W W M M M M M M POS POS POS
Mean, nn = = 11, 1 1, tissue wt. only At yolk absorption Average Tissue wt. only, start free-swimming
Mean fertilization to MLWW Mean Mean Mean
MLWW Mean, just before hatch to M LWW
5.1 DW, 5. 1 mg dry wt. wt. only near hatch DW, tissue wt. DW, tissue wt. only, near hatch
1 1 1 1 2 3 3
4 5 5 5 6 7 7 7 7 8 8 8 8 8 9 9 9 10 10 111 1 12 12 12 12
Oncorhynchus Oncorhy nchus gorbuscha gorbuscha Oncorhynchus Oncorhynchus keta keta Oncorhynchus Oncorhynchus tshawytscha tshawytscha
110 llO
W
At emergence
13 13
8 10 10
929 231 231
POS W
Mean
14 14 15 15
5
182 182
POS
Mean
16 16
248 303 303 339 366
POS POS POS POS
Mean Mean Mean Mean
16 16 16 16 16 16 16 16
Estimated from allometric equation Estimated from allometric equation Estimated from allometric equation Estimated from allometric equation Mean, stock differences differences
17 17 18 18 19 19 19 19 20 21 21 21 21 22 23 23 24
3.6
L, 245 mg L, MLWW E, 2.3-29 mg E, 2.3-29 E-L, 1-500 mg E-L, 1-500 wet E-L, E-L, 1-500 1-500 mg E-L, E-L, 1-500 1-500 mg E-L, E-L, 1-500 1-500 mg E-L, 1-500 mg E-L, 1-500
7.5 10.0 10.0 10.2 10.2 12.5 12.5
L, L, 100 100 mg L, 100 100 mg L, L, < < 1000 1000 mg L, L, < < 100 100 mg 22-24 dph L, 22-24 L, 5.5-15.8 5.5-15.8 mg L, 22.1-11.1 . 1- 1 1 . 1 L, 2.0 L, 2-70 2-70 mg L, 40-400 40-400 mg
20 20 10 10 20 20 22 28 28 23 23 23 23 12 12
796 782 350 829 829 639 727 1700 1700 857 857 1205 1205 208 208
M M M M ? ? M M M M M M M
L, 40-400 40-400 L, L, 40-400 40-400 L, 40-400 40-400 L L, L, 2.6-3.6 2.6-3.6
16 16 20 24 221 1 20
384 384 509 509 733 733 859 859 l1134 l34
POS POS POS W ??
nonsalmonid Freshwater, nonsalmonid
Cyprinus carpio Cyprinus carpio .... c. C> 0 co (D
Cyprinids (three (three species species combined) bined)
Rutilus Rutilus rutilus rutilus
mg mg mg mg
POS
Mean Mean
Mean Mean Mean Swimbladder Swimbladder filled
24 24 24 25 25 26
(continued) (continued)
Table Table III III (Continued) (Continued)
Species Species
Abramis Abramis bramis bramis S tizostedion tizostedion lucioperea lucioperca Perea Perca jluviatis jluviatis
.... F ... c Q 0
Esox Esor lucius lucius Mieropterus Micropterus dolomieui dolomieui Mieropterus Micropterus salmoides salmoides Morone Morone saxatilis saratilis Anguilla Anguilla rostrata rostratu Hyophthalmiehthys Hyophthalmichthys molitrix molitrir Channa Channa punetapunctutus tus H eteopneustes Heteopneustes fossilis fossilis Anabas Anabas testuditestudineus neus Etroplus Etroplus maeulamaculatus tus Lebeo h b e o ealbasu calbasu Coregonus sp. Coregonus sp. Coregonus Coregonus lavarelavaretus tus
Stage
2.62.9 L, 2.6-2.9 2.3-4.6 L, 2.3-4.6 L, 2.3-3.4 2.3-3.4 E, 5-1 5-111 dpf dpf L L, 1-12 1-12 dph
Temp. (0C) (“C)
Metabolic intensity [p.g(g [pg(g wet wet wt.)-l wt.)-’ h-1] h-’1
20 20
876 876 1214 1214
? ?
26 26
20
1486 1486 1905 1905 892 892 847 847
? W W POS
Start exogenous exogenous feeding Mean
26 27 25 28
W
Mean
29
22 22
21.5 20
Technique
Comments
Referenceb Referenceb
L, 18-148 18-148 g
19 19
796
L, 11.4-2.4 .4-2.4 mg L, 7 dpf dpf L, 300 300 mg L, L, 33 mg mg
20 18 18
23 23
20
1320 1320 1333 1333 737 706 706
DOS M POS W
4-9 dpf dpf Mean 4-9 DW, start feeding Elver Start Start exogenous exogenous feeding
30 31 31 32 33 33
L, L, 4.7 mg
28 28
1846 1846
POS
breathe1 Bimodal breather
34 34
L, 5.4 5.4 mg
28 28
1046 1046
POS POS
Bimodal breather
35 35
L, < < 18.5 18.5 mg
28 28
1255 1255
POS POS
Bimodal breather Bimodal
36 36
L, ??
27
573 573
M M
DW, prior to free-swimming
37
L, 100-260 100-260 mg L, 10-100 10-100 mg L, L, 88 mg mg
28 28 10 10 5
680 680 640 640 588 588
W POS W
38 38 24 39 39
Coregonus Coregonus peled peled Coregonus Coregonus lavarelavaretus tus Coregonus Coregonus schinzi schinzi Coregonus Coregonus sp. Coregonus Coregonus schinzi schinzi Coregonus Coregonus nasus nasus Acipenser Acipenser haeri baeri Oreochromis Oreochromis nilotis nilotis
L, mg L, 88 mg L, mg L, 8 8 mg L, 4.5 mg L, 4.5 mg L, 4.7 4.7 E, near hatch E,
10 10 15 15 5 10 10 15 15 12 12
650 875 875 667 778 1894 1894 331 331
W W W W W POS
L, 15 15 dph E, near hatch L, 15 15 mg
12.8 12.8 12.5 12.5 10 10 14.5 14.5 12 12 20 30
734 429 685 685 485 1057 1057 717 1213 1213
POS POS POS POS W ? M
DW DW At yolk absorption
DW
40 40 4 1 41 42 43 44 45
499 282 571 571 724 476 591 591 800 952 438 6343 6343
M M M M M M M M W M
DW 2-10 days post yolk absorption DW, 2-10 DW DW DW, tissue wt. wt. only wt. only DW, tissue wt. DW, tissue wt. only DW, tissue wt. only DW DW, near yolk absorption
46 47 47 47 48 48 48 48 49 50
L, ? L, 6.8 mg L, 20 mg L, feeding
39 39 39 39 39 40
DW
Marine c ... ... I... c
Clupea harengus harengus Clupea
L, yolk sac first-feeding L, fi rst-feeding
Clupea Clupea harengus harengus
L, newly hatched L, newly hatched L, newly hatched L, newly hatched L, sac larvae L, yolk sac
10 10 8 8 13 13 18 18 6 8 8 111 1 14 14 10 10 12.5 12.5
L, yolk absorbed L, yolk absorbed L, L, 55 55 /Lg Pg dry dry L, 11 dph
5 5 5 5
286 278 362 381 381
POS POS M POS
DW DW DW, at MTW DW
51 51 52 53 53 51 51
10 10 8
785 265
M M
DW DW
46 47
Clupea Clupea harengus harengus pallasi pallasi Gadus Cadus morhua morhua Pleuronectes Pleuronectes platessa platessa
L, 5 dph L, fi rst-feeding first-feeding
~~
(continued) (continued)
Table Table III I11 (Continued) (Continued) Temp. (0C) 1°C)
Metabolic intensity [ILg(g - 1] [pg(g wet wet wt.)-1 wt.)-' h h-'1
L, fi rst-feeding first-feeding L, fi rst-feeding first-feeding 70-180 hph L, 70-180
13 13 18 18 14 14
436 724 476
L 1.8dph L, 1.8 L, 4 dph
14 14 20 28
p g dry L, 50-150 50-ISO ILg L, 50-150 50-150 ILg p g dry L L
Stage
Species Species
Sardinops Sardinops eaerucaerulea lea sugar Sardinops sagax Cheilopogon Cheilopogon unicolor unieolor SSparus p Q W aurata QUrQtQ .... .... �
5
Engrualis sp. sp. Engrualis Seomber Scomber japonijaponieus cus
Comments
Referenceb Referenceb
M M D
DW DW DW, estimated
47 47 54 54
253 253 3293 3293 1215 1215
D POS POS
DW, inactive DW(?) DW(?) DW(?) DW(?)
55 55 56 56
119 9 24 18 18 18 18
1499 1499 1670 1670 857 857 11162 162
POS POS M ( ?) M(?) M(?) M(?)
DW DW DW DW
57 57 58 58 58 58
18 18 22 2
1162 1162 2171 2171 343 343
W W W
DW DW, estimated from graph DW, estimated from graph
59 59 60
5 8 8 6 22
495 743 560 1608 1608
W W W POS
DW DW
DW(?), end endogenous feeding DW(?),
60 60 61 61 56
L, feeding L, feeding
26 26
1352 1352 1886 1886
POS POS
DW DW
62 62
L, feeding L, 10.9 10.9 mg dry L, 11 II mg dry
28 16 16 19 19
2248 5371 5371 4838
POS M M M
DW 5% yolk remaining DW, 5% DW, 2.7% 2.7% yolk remaining
62 63 63 63 63
L, 3-5 3-5 dph L, 3-5 3-5 dph Pseudopleuroneetes Pseudopleuronectes L, 1000 ILg L, 1000
amerieanus americanus
Parophrys Parophrys retulus retulus Hirundichthys Hirundiehthys marginatus marginatus Anchoa mitchilli mitchilli Anehoa Arehosargus Archosargus rhomboidulis rhomboidalis Aehirus lineatus Achirus lineatus Tautoga Tautoga onitus onitus
Technique
L, L, E, L,
1000 1000 ILg pg 1000 ILg pg 1000 near hatch 1.4 1.4 mg
Lagodon rhomboides xanLeistomus xanthurus thums Congiopodus leucopaecilus leucopnecilus Breooortia tyrantyranBrevoortia nus
1 mg dry L, 111 L, 25 mg
22 15 15
7810 7810 500
M M
L, 42 mg
15 15
500
M
64 64
L, 18 dph L, 18
111.5 1.5
541 541
M
65
L, 47-55 47-55 mg
14 14
780
POS
66
47-55 mg L, 47-55 L, 47-55 47-55 mg
19 19 24
1000 1000 1555 1555
POS POS
66 66
254 mg 180-380 mg 180-380 46.3 46.3 mg
5 5 111 1 16 16
92 134 134 676
POS M M
389 mg 3.8 g 3.8
10 10 18 18
103 103 222
W W
DW, 10% 10% yolk remaining
63 64
Viviparous
oioiparus Zoarces viviparus
.... I.... c1 I:.)
Clinus superciliosus liosus Rhaeoehilus Rhacochilus raeca racca
w
10-27 days prepartum 10-27
At parturition
67 68 69 70 70
0 Abbreviations: Abbreviations: L, larva; larva; E, embryo; dpf, days postfertilization; dph, dph, days posthatch; hph, hours posthatch; W, Winkler; DC, direct calorimecalorime try; POS, polarographic oxygen sensor; M, manometric; D, diver; Hg, dropping mecury electrode; DW, original data expressed on dry weight M LWW, maximum larval wet weight; MTW, maximum tissue weight. basis, converted to wet weight assuming dry weight = 13.3% 13.3% wet weight; MLWW, basis, (1)Lindroth, 1942 1942 (cited in Hayes et al., 1951); 1951);(2) Komarova, 1970 (2) Hayes et al., 1951; 1951; (3) (3) Tamarin and Komarova, 1970 (4) (4) Smith, 1958; 1958; (5) (5) Wieser b References: (1) et al., 1985; 1987; (8) 1973; (9) 1932; (10) ( 10) Gray, 1926; 1926; et a/., 1985; (6) (6)Hamor, 1967 1967(cited in Hamor and Garside, 1975); 1975);(7) (7)Rombough, 1987; (8) Penaz and Prokes, 1973; (9)Wood, 1932; ((11) l l) Gnaiger, 1983b; 12) Gruber and Wieser, 1983; 13) Bailey et Smimov, 1982; 1982; ((15) 15) Alderdice et 16) P. J. 198313; ((12) 1983; ((13) et al., 1980; 1980; (14) (14) Storozhyk and Smirnov, et al., 1958; 1958; ((16) Rombough, 17) Kamler, 1976; 18) Winberg and Hartov, Kamler, 1972); 1972); (19) (19) Kamler, 1972; 1972; (20) et al., al., Rombough, unpublished unpublished data; ((17) 1976; ((18) Hartov, 1953 1953 (cited in Kamler, (20) Jitariu et 197 1 ; (21) (21) Korwin-Kossakowski 1981; (22) (24) Wieser and Forstner, 1986; 1986; (25) (25) 1971; Konvin-Kossakowski et et al., 1981; (22) Kamler et et al., 1974; 1974; (23) (23) Kaushik and Dabrowski, 1983; 1983; (24) Konchin, (27) Trifonova 1937 Spoor, 1984; 1984; (29) (30) Spoor, 1977; 1977; (31) (31) E ldridge et et al., ai., 1982; (32) Eldridge 1982; (32) Konchin, 1981 1981;; (26) (26)Kudrinskaya, Kudrinskaya, 1969 1969 (27) 1937 (28) (28) Spoor, (29)Laurence, 1969; 1969; (30) 1984 (33) (33) Mukhamedova, Mukhamedova, 1977; (34) Singh et al., 1982; 1982; (35) (35)Sheel and Singh, 1981; (36) (36) Mishra and Singh, 1979; Gallagher et al., 1984 1977; (34) Singh, 1981; 1979; (37) (37) Zoran and al., 1984; 1984; (41) 1983; (42) Kaushik, Ward, Ward, 1983; 1983; (38) (38) Durve and Sharma, Sharma, 1977; 1977; (39) (39) Prokes, 1973; 1973; (40) (40) Dabrowski et et al., (41) Forstner et et al., al., 1983; (42) Dabrowski and Kaushik, 1984; (43) Chemikova, Chernikova, 1964; (44) Khakimullin, Khakimullin, 1985; 1985; (45) (45) DeSilva et a/., (48) Holliday et 1964; (44) al., 1986; 1986; (46) (46) DeSilva and Tytler, 1973; 1973; (47) (47) Almatar, 1984; 1984; (48) 1984; (43) al., (50) Eldridge et al., 1977; (51) 1964; (49) (49) Marshall Marshall et al., 1937; 1937; (50) 1977; (51 ) Davenport and Lonning, 1980; 1980; (52) (52) Davenport et a/., al., 1979: 1979: (53) (53) Solberg and al., 1964; 1962; (55) 1962; (56) Klekowski et 1980; (57) Tandler, 1982; (58) Hunter, 1981; 1984; (54) Tilseth, 1984; (54)Lasker, 1962; (55)Lasker and Theilacker, 1962; (56)Klekowski et al., 1980; (57) Quantz and Tandler, 1982; (58) 1981; (59) 197 1 ; (62) (64) (59) Hunter and Kimbrell, 1980; 1980; (60) (60) Laurence, 1975; 1975; (61 (61)) Alderdice and Forrester, 1971; (62)Houde Houde and Schekter, 1983; 1983; (63) (63) Laurence, 1973; 1973; (64) Kjelson and Johnson, Johnson, 1976; 1976; (65) 1983; (68) (68) Korsgaard and Andersen, 1985; 1985; (69) (69) (65) Robertson, 1974; 1974; (66) (66) Hettler, 1976; 1976; (67) (67)Broberg and Kristofferson, Kristofferson, 1983; Veith, 1979; 1979; (70) (70) Webb and Brett, 1972b. 1972b. a
=
1114 14
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10. Relationship between routine metabolic intensities (log (log scale) scale) and temper temperFig. 10. 111. larvae. Data were taken from Table III. ature for fish embryos and larvae.
there are significant differences. In general, temperature changes have a more profound effect during early life. Literature values for the 9 10for metabolic rate in embryos 1.5 embryos and larvae range from about 1.5 010 1973) to 4.9 4.9 (Salvelinus (Salvelinus alpinus, Gru Gru(Coregonus lavaretus; Prokes, 1973) 1983), with an average value of about 3.0 (Table IV). ber and Wieser, 1983), 3.0 (Table IV). In juvenile and adult fish, Qlo 2.0 (Fry, 1971). fish, 0 values average about 2.0 (Fry, 1971). 0 1 The higher values during early life may reflect the fact that embryos and larvae tend to be more stenothermal than juveniles and adults. It is interesting that 0 9 11 0 0 values for the rate of development are often
2. 2.
RESPIRATORY RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM
1115 15
Table Table IV IV mbryos and Larvae Qlo Values for E Embryos 010
Species Species
Salrno Salmo gairdneri gairdneri Salrno Salmo gairdneri gairdneri
Salrno Salmo trutta trutta
Salmo trutta trutta Salrno Salmo salar salar Salvelinus Salvelinus alpinus alpinus Onchorynchus Onchorynchus tshaytshaywtscha wtscha Cyprinus Cyprinus carpio carpio Cyprinids
Coregonus Coregonus lavaretus lavaretus Coregonus Coregonus peled peled "Chalcalburnus “Chalcalburnus” "Taran “Taran” Oreochromis Oreochromis niloticus niloticus "
Clupea Clupea harengus harengus Clupea harengus Clupea harengus Pleuronectes Pleuronectes platessa platessa Pseudopleuronectes Pseudopleuronectes arnericanus ameri canus Brevoortia Brevoortia tyrannus tyrannus Tautoga Tautoga onitus onitus Scornber Scomber japonicus japonicus
Temp. range (OC) (“C)
OW 910
Reference
4-12 4-12 12-20 12-20 6-9 6-9 9-12 9-12
2.90 2.90 1.80 1.80 4.37 4.37 2.41 2.41
Wieser et et al. (1985) (1985) Wieser et et al. (1985) (1985) Rombough ((1987) 1987) Rombough (1987) (1987)
12-15 12-15 3-7 3-7 7-12 7-12 10-20 10-20 5-10 5- 10 4-8 4-8 5-7.5 5-7.5 7.5-10 7.5-10 10-12.5 10-12.5 10-20 10-20 12-20 12-20 5-1 5-155 5-15 5-15 19-23 19-23 15.5-19 15.5-19 25-30 25-30 30-35 30-35 6-8 6-8 8-1 8-111 111-14 1 - 14 8-13 8-13 13-18 13-18 8-13 8-13 13-18 13-18 2-5 2-5
2.74 2.74 1.98 1.98 4.76 4.76 1.87 1.87 3.67 3.67 4.9 4.9 3.45 3.45 2.23 2.23 2.12 2.12 2.37 2.37 3.09 3.09 1.49 1.49 2.84 2.84 3.00 3.00 4.68 4.68 2.42 2.42 2.09 2.09 2.93 2.93 2.75 2.75 1.79 1.79 1.9 1.9 3.0 3.0 1.8 1.8 6.4 6.4 3.40 3.40
Rombough ((1987) 1987) Wood (1932) (1932) Wood ((1932) 1932) (1973) Penaz and Prokes (1973) Hamor and Garside ((1977) 1977) (1983) Gruber and Wieser (1983) P. P. J. J. Rombough (unpublished) (unpublished) P. J. J. Rombough (unpublished) (unpublished) P. J. J. Rombough (unpublished) Kamler (1972) (1972) Wieser and Forstner ((1986) 1986) Prokes ((1973) 1973) Prokes ((1973) 1973) Karpenko and Proskurina ((1970) 1970) Karpenko and Proskurina Proskurina (1970) (1970) DeSilva et et al. (1986) (1986) Holliday et 1 964) et al. al. ((1964) Holliday et et al. al. (1964) (1964) Holliday et et al. (1964) (1964) Almatar (1984) (1984) Almatar (1984) (1984) Almatar (1984) (1984) Almatar ((1984) 1984) Laurence (1975) (1975)
5-8 5-8 14-19 14-19 19-24 19-24 16-22 16-22 18-22 18-22
3.86 3.86 11.64 .64 2.40 1.87 1.87 4.77 4.77
Laurence (1975) (1975) Hettler (1976) (1976) Hettler (1976) (1976) Laurence (1975) (1975) Hunter and Kimbrell (1980) (1980)
1 16 116
J. ROMBOUGH PETER PETER J. ROMBOUGH
similar to those for metabolism. Johns and Howell ((1980) 1980) suggested that this similarity may explain why growth efficiency remains rela relatively constant over a relatively broad temperature range in many species. species. The evidence is rather sketchy, sketchy, but it appears that at least some species of of fish may not be capable of of thermal acclimation during early life. Clements and Hoss ((1977) 1977) monitored rates of oxygen consump consumption of larval flounder (Paralichthys (Paralichthys dentatus and P. P . lethostigwa) transferred directly from environmental temperatures of 1O-12°C 10-12°C to constant temperatures of 10 and 15°C. oxygen uptake, mea of 10 15°C. Rates of oxygen measured daily for 4 days, did not vary following transfer. This would suggest that thermal acclimation occurs either very rapidly «(el 1 day) or not at all. The latter is more likely. Hinterleitner et al. ((1987) 1987) found no evidence of metabolic temperature compensation in larval roach or chub (Levciscus (Levciscus cephalis). cephalis). Preliminary analysis of of data indicates that steelhead embryos similarly do not show any signs of thermal acclima acclimation upon reciprocal transfer between 5 and 10°C (P. J. J. Rombough, 10°C (P. unpublished data). data). V 0 22 are sometimes difficult The effects of temperature change on V0 to interpret because of of changes in activity levels associated with the temperature change. change. For example, example, Gruber and Wieser (1983) (1983) attrib attribO uted the high Q (4.9) calculated for Arctic char larvae held at 4 and (310 (4.9) I 8°C 8°C to what they termed "warm “warm stimulation" stimulation” of activity at the higher temperature. Unfortunately, activity levels were not measured. Het Het(1976) did measure changes in activity levels of of larval menhaden tler (1976) (Breuoottia tyrannus) tyrunnus) transferred to differto.lt differ6 ,it temperatures. He found (Brevoortia that activity increased significantly as the temperature was raised but, interestingly, this was not reflected in a large increase in V V0 0 22 (Ql (QloO = = 2. 1). 2.1). 2. DISSOLVED OXYGEN DISSOLVED OXYGEN Dissolved oxygen concentrations obviously greatly influence metabolic rate. The relationship between V V0 0 22 and oxygen concentra concentration, however, is not simple. Most, Most, if if not all, fish can be classified as metabolic regulators on the basis of of their standard V V0 0 22 as juveniles and adults (Beamish, (Beamish, 1964; 1964; Fry, 1971 1971;; Ultsch et al., 1981). 1981). As men menV02 during the early life tioned previously, it is difficult to measure ssV02 V02 is used as the basis for classification stages, but if if rrV02 classification instead of of V02 , it appears that embryos and larvae, for the most part, also be SsV02, beconcenhave as metabolic regulators. This means that at high oxygen concen trations their metabolic rate is independent of the ambient oxygen is concentration, but if oxygen levels are gradually reduced, a point is
2. 2.
RESPIRATORY EXCHANGE, AEROBIC RESPIRATORY GAS GAS EXCHANGE, AEROBIC METABOLISM METABOLISM
117 117
eventually reached below which metabolic rate becomes dependent on the ambient oxygen concentration. This point, termed the critical oxygen tension (P,), (Pc), defines the oxygen oxygen concentration required to oxygen maintain a particular level of metabolism. It is important to recognize that Pc xed but varies in response to a variety of P, is not fi fixed of intrinsic and factors. In juvenile and adult fish the two most important extrinsic factors. factors influencing Pc P, are activity and temperature (Beamish, (Beamish, 1964; 1964; factors 1971; 1980). Combined high activity and temperature Fry, 197 1 ; Ott et al., 1980). can result in Pc P , values near 100% 100% air saturation (Brett, 1970). 1970). Activity (Broberg (Broberg and Kristofferson, Kristofferson, 1983) 1983) and temperature (Rombough 1986, 1986, 1987; Diez and Davenport, 1987) 1987) are also important factors influenc influenc1987; P, during early life. In addition, the stage of development (Lin (Lining Pc droth, 1942; 1 ; Rombough, 1986, 1987) and the water 1942; Hayes et al., 195 1951; 1986,1987) (Fry, 1971) in flow (Fry, 1971) have profound effects. Temperature and activity inoxygen demands. Stage of fluence fluence Pc P, by altering oxygen of development affects affects both oxygen demand and supply, supply, while water velocity primarily af afsupply. fects oxygen supply. V02) is directly dependent on Routine Pc P , (the Pc P, associated with rrV0,) the stage of development and temperature. Values increase more or less steadily throughout embryonic development (Lindroth, 1942; 1942; Hayes et al., 195 1 ; Davenport, 1983; 1951; 1983; Rombough, 1987) 1987) and at any given stage of development are greater at higher temperatures (Rom (Rombough, 1987) 1987) (Fig. (Fig. 111). 1). The effect of of temperature is an indirect one resulting from higher metabolic rates at higher temperatures, as indi indi02 all points fall on cated by the fact that when Pc P, is is plotted against \1 002 the same line regardless of incubation temperature (Rombough, (Rombough, 1987) 1987) (Fig. (Fig. 4). 4). At high temperatures, routine Pc P, for large eggs, such as those of salmonids, may approach 100% 100% air saturation near hatch. Such higher Pc P , values have led some investigators (e.g., (e.g., Davenport, 1983; 1983; Gruber and Wieser, 1983) 1983) to classify teleost embryos as oxyconfor oxyconformers. However, this apparent conformity is is simply a consequence of supply problems associated with the presence of the capsule and not intrinsic to the embryo itself. itself, Hatching or artificial artificial removal of the 1 ; Gnaiger, capsule results in an abrupt drop in Pc P, (Hayes (Hayes et al., 195 1951; 1983b; Gruber and Wieser, 1983; 1983; Rombough, 1987). 1983b; 1987). It must be noted that not all studies have shown fish embryos to be (1979) noted that \1 6 0 0 22 was metabolic regulators. Hamor and Garside (1979) 100% air saturation at all lower at 30% and 50% 50% air saturation than at 100% stages during the embryonic development of of Atlantic salmon. This is is difficult to explain since Pc P , values should have been well below 50% ASV during early development (Hayes (Hayes et al., 1951). 1951). It may be that metabolic response to chronic hypoxia is not the same as that to acute
1118 18
PETER PETER J. J. ROMBOUGH ROMBOUGH
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Fig Fig.. n. 11. Critical dissolved oxygen oxygen concentrations for for steelhead embryos embryos and larvae reared at constant temperatures of 6.0, 6.0, 9.1, 9.1, 12.0, 12.0, and 15.PC. 15.1"C. The horizontal arrow indicates 100% 100% air saturation. saturation. The point indicated by an asterisk represents the Pc P, for unhatched embryos h, hatch; 80% hatch; be, bc, blastopore closure; e, e, well eyed; h, embryos at about 80% mtw, mtw, maximum maximum tissue weight. [From Rombough (1987).] (1987).]
hypoxia. hypoxia. Other studies studies have indicated that embryos behave as neither 1983; Gruber and (Davenport, 1983; true conformers nor true regulators (Davenport, 1983). Such inconclusive results may arise, at least in part, Wieser, 1983). P, (and (and \1 V0 2). 02). from practical difficulties associated with determining Pc seen, \1 V 02 0 2 and hence Pc P, are dependent on many factors. factors. As we have seen, control. Simply Excitement and activity in particular are difficult to control. significantly elevate their metabolic placing fish in a respirometer can significantly rate (Fry, (Fry, 1957). 1957).Typically, Typically, \1 V 02 0 2 is is high when the fi fish is first first placed in rate sh is the respirometer and and declines as as it adapts adapts to the system. system. Associated the V 02 0 2 is a gradual decline in Pc. P,. Metabolic with this gradual decline in \1 \102, but if the respirometer is rates should should eventually stabilize at rrVO2, is rates P, as a closed system, system, oxygen oxygen levels may drop below routine Pc operated as can occur. occur. If this occurs, occurs, critical levels will not be apparent before this can as an oxyconformer. oxyconformer. Even if meta metaand the animal may be classified as P,, the oxygen levels drop drop below routine Pc, bolic rates stabilize before oxygen
do
2. 2.
RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM RESPIRATORY
119 119
ob transition from oxygen independence ttoo dependence may be ob( 1983) presents data illustrating this point. LumpLump scured. Davenport (1983) scured. V02 was fish eggs or larvae were placed in closed respirometers and VO2 V02 declined monitored until all oxygen was exhausted. Embryonic VOZ VO, rapidly at first but, after a while, V 02 tended to stabilize even though ambient oxygen levels continued to decline. Oxygen uptake during V02 . Eventually oxygen levels in this period probably approximated rrVOz. P,, and V VOZ the respirometer dropped below routine Pc, 02 again declined. The problem is that the period of of stable V 0 0 0 22 is not always easily P, is is rela relarecognized, particularly later in development when routine Pc tively high. Compensatory adjustments, such as changes in activity of gas exchange, of the embryo or level or the efficiency of exchange, on the part of larvae may also make it difficult difficult to discern routine Pc. P,. Additional problems in estimating Pc P, using closed respirometers arise from response lags associated with such systems and instability of polarographic sensors caused by pressure changes when the system is is closed. Both have the effect of appearing to to increase initial V V0 0 22 •. Rombough (1987) (1987) was able to minimize these problems, and that of initial excitement on being placed in the respirometer, by allowing the fish sufficient sufficient time to adapt to the system before closing the respirometer and, and, in the case of advanced embryos, embryos, initiating tests at moderate levels of hyperoxia (1 10-160% air saturation). (110-160% saturation). This ensured that apparent V 02 stablized well before oxygen levels in the respirom VOZ respirometer reached routine Pc, P,, allowing Pc P, to be easily estimated. estimated. The tech technique appears to have been effective since since energy budgets calculated on the basis of V 0 2 at the eestimated stimated routine Pc VOZ P, balanced [[mean(G [[meanfG + + R)]A -1 = R)]A-’ = 102%]. 102%]. Some Some of the problems associated with using "closed “closedsystem" system” respirometers to estimate estimate Pc P, can be avoided by using flow flowthrough systems, systems, but this is very very time-consuming, time-consuming, and in rapidly de developing species species routine Pc P, may have have changed before the tests are completed. completed. The metabolic response response of larvae to declining oxygen levels levels ap appears to be more variable variable than in embryos. As expected, activity is more of a problem, but even taking this into account there appear to be significant differences among among species. species. All All species species can can be expected to show 1987) show an abrupt decrease in routine routine Pc P , at at hatch. hatch. Rombough ((1987) noted noted that the routine Pc P, for steelhead larvae larvae continued to to decline after hatch up to to about midway midway through yolk absorption. absorption. The Pc P, then remained remained relatively relatively stable stable until until the end of yolk absorption, absorption, when ex experiments were terminated. terminated. Routine Pc P, was was directly directly dependent on temperature. Levels during the latter half of the period of endogenous endogenous feeding ranged ranged from from 2.3 2.3 mg I1-II at 6°C 6°C to to 4.8 4.8 mg I-I 1-’ at at 15°C. 15°C. Values
120
PETER ROMBOUGH PETER J. J . ROMBOUGH
were not not determined determined for for feeding feeding larvae, larvae, but but they they probably probably continued continued were Pc values during endogenous feeding to decline slowly since routine to decline slowly since routine P, values during endogenous feeding were somewhat higher than those reported for juvenile salmonids. De were somewhat higher than those reported for juvenile salmonids. De Silva and Tytler ( 1973) reported a different pattern for herring and Silva and Tytler (1973) reported a different pattern for herring and plaice larvae. In In both species Pc levels were were lower lower for for younger larvae P, levels younger larvae plaice larvae. both species than for older larvae. In fact, older larvae behaved as oxyconformers than for older larvae. In fact, older larvae behaved as oxyconformers up to to close close to to 100% 100% air air saturation. saturation. After After metamorphosis, metamorphosis, P, Pc levels levels up dropped and both species again behaved as metabolic regulators. dropped and both species again behaved as metabolic regulators. Davenport (1983) ( 1983) presented presented data data indicating indicating that Pc levels levels for for lumpfish lumpfish that P, Davenport 26 days of the larval period larvae similarly increased during the fi r st larvae similarly increased during the first days of the larval period (at 5°C). The different patterns reported for steelhead and for herring, different patterns reported for steelhead and for herring, (at 5°C). plaice, and and lumpfish lumpfish may may reflect reflect differences differences in in the the rate rate of of transition transition plaice, from cutaneous to branchial gas exchange. The gills of salmonids are from cutaneous to branchial gas exchange. The gills of salmonids are fairly well developed at hatch and probably assume an increasingly fairly well developed at hatch and probably assume an increasingly important important role role in in respiratory respiratory gas gas exchange exchange as as development development proceeds. proceeds. In contrast, the gills of larval herring and plaice In contrast, the gills of larval herring and plaice appear appear to to be be of of little little functional signifi c ance until close to metamorphosis. The skin skin re refunctional significance until close to metamorphosis. mains the the primary primary site gas exchange, exchange, and because of increasingly mains site of of gas and because of an an increasingly unfavorable surfaceholume surface/volume ratio ratio the the ability to extract extract sufficient sufficient oxyoxy unfavorable ability to gen from the environment may become limiting. The reduction in P, gen from the environment may become limiting. The reduction in Pc after after metamorphosis metamorphosis can can be attributed attributed to to rapid rapid elaboration elaboration of of the the gills gills and and the the appearance appearance of of hemoglobin hemoglobin in in the the blood. blood.
3. 3. SALINITY SALINITY The influence of salinity on the rate of oxygen consumption of species, but embryos and larvae has been investigated in only a few species, results negligible results to to date date suggest suggest that that net net ionoregulatory ionoregulatory costs costs are are negligible once acclimation has occurred. It is not clear whether this is because of salinities or because costs remain fairly constant over a broad range of increases in costs are paralleled by proportional decreases in other metabolic processes. Evidence that net ionoregulatory costs are small comes (1962)reported no comes from several sources. Lasker and Theilacker (1962) V0 2 of sardine embryos held in half-, half-, significant difference in the rrVO2 al. ((1964) similarly full-, and double-strength seawater. seawater. Holliday et al. full-, 1964) Similarly significant anesobserved no signifi cant difference in standard metabolic rates of anes embryos and newly hatched hatched larvae incubated at con conthetized herring embryos 5x0 and 50%0. 5 0 L . DeSilva et al. al. (1986) (1986) recently salinities between 5%0 stant salinities significant (anesreported no signifi cant difference in either routine or standard (anes thetized) metabolic intensities of larvae of the eurhyaline species thetized) salinities between 0 and 18%0. 18%0. Oreochromis niloticus reared at salinities changes in salinity do affect affect rates of oxygen oxygen consumption. consumption. Abrupt changes
2. 2.
RESPIRATORY EXCHANGE, AEROBIC AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, METABOLISM
121 121
Holliday et al. ((1964) 1 964) reported up to a lO-fold 10-fold increase in oxygen uptake by anesthetized herring embryos and larvae abruptly transfer transferred from 35%0 35%0to 5%0. 5%0.Rates gradually declined to the pretransfer level over a period of 6-8 6-8 h. Holliday et al. aZ. (1964) (1964) indicated that the period of elevated metabolism coincided with the length of of time required required to restore osmotic imbalance brought about by the abrupt change in salinity. The pattern of oxygen uptake in unanaesthetized larvae was 35% to both 15%0 15%0and 5%0 5% resulted in a quite different. Transfer from 35%0 decrease in metabolic rate followed by aa very gradual increase over the next 24 h to values typical of constant exposure to 35%0. 35%. Holliday et uZ. (1964) (1964) attributed the initial reduction in V VOz 02 to reduced activity ai. changes.. Almatar (1984) (1984) similarly reported a associated with buoyancy changes reduction in oxygen oxygen uptake rates of herring and plaice yolk-sac yolk-sac larvae (32%~~) (5 and 12.7%0). 12.7%). In transferred from seawater (32%0) to low salinities (5 feeding larvae, however, metabolic rates were elevated on transfer to 12.7%0 compared with control larvae (constant 32%0) and with larvae 12.7%~~ 32%~~) 5%0and 40%0. 40%0. Almatar ((1984) transferred to 5%0 1 984) attributed the apparent V0 0 22 to the fact that the larvae used in the 12.7%0 12.7% test were increase in " somewhat smaller than those used in the other tests, although why this should make a difference is not made clear. clear. 4. LIGHT 4. LIGHT Within Within limits, light light exerts exerts aa direct direct tonic tonic effect effect on on metabolic metabolic rate rate (MacCrimmon (MacCrimmon and and Kwain, Kwain, 1969; 1969; Hamor Hamor and and Garside, Garside, 1975; 1975; Konchin, Konchin, 1982a; 1984). Konchin (1982a) 1982a; Solberg and Tilseth, 1984). (1982a) indicated that the V 02 of V0, of roach roach (Rutilus (RutiZusrutilus) rutilus) larvae larvae was was 18-25% 18-25% higher higher in in the the "light" ‘‘light’’ than olberg and 1984) reported than in in the the dark. dark. SSolberg and Tilseth Tilseth ((1984) reported that that the the " V0 0 22 of of larval larval cod cod was was 25-70% 25-70% higher higher in in the the "light" “light” than than in in the the dark, dark, appar apparently because because of of greater greater activity. activity. Light Light intensities intensities were were not not specified specified in 1975) found in either either study. study. Hamor Hamor and and Garside Garside ((1975) found that that " V0 0 22 of of Atlantic Atlantic salmon salmon embryos embryos only increased increased in in response response to to increasing increasing light light inten inten0 2 began sities sities up up to to about about 200 200 lux; lux; at at 250 250 lux, lux, V VOz began to to decline. decline. 5. 5. OTHER OTHERFACTORS FACTORS Very little is known of how potentially important factors such as pH, carbon dioxide concentration, and ration level influence aerobic metabolism during the early life stages. Oxygen uptake by steelhead embryos was not significantly affected affected by low pH until levels dropped (Rombough, 1987). below pH pH 4.0 4.0 (Rombough, 1987). Alderdice Alderdice and and Wickett Wickett (1958) (1958) re reported ported that that the the metabolic metabolic rate rate of of chum chum salmon embryos embryos was was indepenindepen-
122 122
PETER PETER J. J. ROMBOUGH ROMBOUGH
dent ambient CO 125 mg dent of of ambient C022 levels levels below below 125 mg I-I 1-' (PC0 (PC02 not given). given). The 2 not relative embryos to relative insensitivity insensitivity of of embryos to pH pH and and carbon carbon dioxide dioxide is is not not sur surprising light of Bohr and prising in in light of the the weak weak Bohr and Root Root effects effects seen seen in in larval larval hemo hemoglobins globins (Iuchi, (Iuchi, 1973b). 1973b).As As mentioned mentioned previously, previously, Quantz Quantz and and Tandler Tandler (1982) (SDA) may (1982) suggested that that aa high high specific dynamic dynamic action action (SDA) may ac account for the high routine metabolic rate of of larval gilthead seabream. Kaushik DA of Kaushik and and Dabrowski Dabrowski (1983) (1983) attempted attempted to to measure measure the the SSDA young cant postprandial young carp carp but but were were not not able able to to detect detect aa signifi significant postprandial in increase crease in in \1 V0 0 22 until until the the carp carp had had attained attained aa mass mass greater greater than than 11 g. g. Dabrowski Dabrowski (1986) (1986) has has since since developed developed aa more more sensitive sensitive system system for for detecting changes 02 and c detecting changes in in \1 VO, and has has used used it it to to estimate estimate the the specifi specific dynamic action of Atlantic salmon alevins species, an increase alevins.. In this species, in metabolic rate equivalent to about 30% 30%of standard metabolism and peaking 2.5-3 h postfeeding postfeeding appears appears to to be be attributible attributible to to SDA. SDA. One One of of peaking 2.5-3 the reasons SDA has been difficult to detect is that it is partially compensated compensated for for by by aa decrease decrease in in active active metabolism. metabolism. The effects of water-borne pollutants on the early stages of fish are dealt with elsewhere in this volume (von (von Westernhagen; Chapter 4), 4), but it would be apropos to comment briefl y on the effect of briefly of such substances substances on on embryonic embryonic and and larval larval \1 V0 0 22.. A A wide wide variety variety of of sub substances, stances, including including pesticides pesticides (Kamler (Kamler et al., al., 1974; 1974; Klekowski Klekowski et al., al., 1977), 1977), metals metals (Storozhk (Storozhk and and Smirnov, Smirnov, 1982; 1982; Akberali Akberali and and Earnshaw, Earnshaw, 1984), al., 1979; 1984),and and hydrocarbons hydrocarbons (Eldridge (Eldridge et ai., al., 1977; 1977; Davenport Davenport et al., 1979; Hose and Puffer, 1984), 02 . If a 1984), have been tested for their effect on \1 V02. generalization is to be made, it is that results are highly variable and often difficult to interpret. A particular pollutant may have signifi cant significant effects on one stage but not on another (Davenport (Davenport et al., al., 1979). 1979).A high concentration of the toxicant may have no effect, while a much lower significant effect. For concentration of the toxicant will produce a signifi cant effect. (Leuresthes tenuis) tenuis) instance, oxygen uptake by embryonic grunion (Leuresthes was not affected by a high body burden of benzo[a]pyrene but was (Hose and significantly elevated in response to a low body burden (Hose 1984). Elevated \1 V0 0 22 at low exposure levels was attributed to Puffer, 1984). Puffer, (overcompensation to inhibitory challenge). challenge). Benzo[a]py Benzo[a]pyhormesis (overcompensation appears to be somewhat of an exception, and normally a rela relarene appears is required to produce a signifi signifi- tively high concentration of a pollutant is V0 0 22 ., Davenport et al. al. (1979) (1979)estimated that because of cant change in \1 \10 would be normal variability in \1 V0 0 22 a change change of about 40-50% rrVO2 normal 2 to be statistically signifi significant. this reason, \1 V0 0 22 would would not required to cant. For this indicator of sublethal toxicity toxicity for appear to be a particularly useful indicator most pollutants, especially since other more sensitive and easier to indicators are available available (e.g., (e.g., growth). growth). determine indicators
2. 2.
RESPIRATORY AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC METABOLISM
123 123
IV. EFFECT OF HYPOXIA
A. Environmental Hypoxia
Exposure to low levels of of dissolved oxygen during early life can elicit a wide variety of responses, some compensatory, others clearly particular response and its magnitude depends on pathological. The particular of hypoxia, and duration of of expo expospecies, stage of development, level of sure. factors, such as temperature and water flow, also may be sure. Other factors, of the important. The most obvious place to begin an examination of toleranceeffects of environmental hypoxia is to describe the zone of tolerance that is, to what levels oxygen can fall before mortality occurs.
1. 1. LETHAL LETHALLEVELS LEVELS Many studies have shown shown that fish fish are extremely sensitive to low levels of dissolved oxygen (see reviews by Doudoroff oxygen during early life (see and Shumway, Shumway, 1970; 1970; European Inland Fisheries Advisory Advisory Commis Commission, 1973; 1973; Davis, 1975; 1975; Alabaster and Lloyd, 1980; 1980; Chapman, Chapman, 1986). 1986). Incipient lethal levels, however, remain poorly defined. There are two two major reasons for this. The first arises from the failure of most investigators to follow standard bioassay procedures. Experimental investigators conditions are often not adequately controlled (e.g., (e.g., variable tempera temperature regimes). regimes). Sufficient numbers of test levels to obtain anything more than the roughest estimate of lethal levels are rarely used. Con Control mortality is often not taken into account, or even reported. Levels of significance are seldom given, and comparisons are frequently made on the basis of variable exposure periods. If past performance is any indicator, future investigators would do well to review basic toxi toxicological methods (e.g., (e.g., Shepard, Shepard, 1955; 1955; Sprague, Sprague, 1973) procological 1973) before pro ceeding. The The second major reason why incipient lethal levels remain defined poorly defi ned is inherent to the early stages themselves. Gottwald (1965) pointed out some problems:: the slow response of em em(1965) some of the problems bryos to hypoxia and the difficulty in assessing mortality during the stages.. The most significant difficulty, difficulty, though, though, is is that the organ organearly stages isms are are undergoing profound developmental developmental changes changes that that alter their isms as it is is being assessed. This makes it ex exsensitivity to hypoxia even as tremely difficult to determine precise response thresholds. is fairly well established that sensitivity to hypoxia tends to It is increase increase as as development proceeds proceeds.. Maximum Maximum sensitivity occurs occurs dur during the larval period, with the precise stage depending on the particu particuing species. Increasing sensitivity is is implied by mortality patterns durdurlar species.
124 124
PETER J. ROMBOUGH PETER J. ROMBOUGH
ing chronic exposures to low levels of dissolved oxygen. oxygen. For example, larval mortality is often recorded at oxygen levels that permitted sur survival to hatch (Brungs, 1 ; Eddy, 1972; al., 1973, 1974; (Brungs, 197 1971; 1972; Siefert et d., 1973, 1974; Dudley and Eipper, 1975). 1975). Mortality patterns are not always valid indicators of stage sensitivity. Rosenthal and Alderdice ((1976) 1976) noted that with many toxicants, injury may be sustained at an early stage but not manifested until later in development. development. In the case of hypoxia, however, studies of of acute toxicity also indicate increasing sensitivity as development proceeds. Alderdice et al. (1958) (1958) noted that the 7-day LC embryos (0. keta) increased from 0.4 0.4 mg 1LC50 (0.keta) 1-’1 shortly 50 for chum embryos after blastopore closure to 11.0-1.4 .0- 1.4 mg 11-’1 shortly before hatch (Table (Table values for V). Likewise, Gottwald ((1965) 1 965) reported reported that 3-day LC L C50~ O rainbow trout increased from <0.9 CO.9 mg 1I-’ 1 at blastopore closure to 0.90.92.7 2.7 mg 11-’1 near hatch. Studies that have compared sensitivities shortly before and after hatch indicate that the newly hatched larvae are sigsig nificantly more sensitive (Peterka 1976; Spoor, Spoor, 1977). (Peterka and Kent, 1976; 1977). This is is somewhat surprising given the fact that the embryos are effectively pres exposed to a much lower ambient concentration because of of the presence of the capsule. The particular stage at which larvae are most sensitive is highly variable. Bishai (1960) (1960)indicated that the sensitivity of young Atlantic salmon salmon and brook trout continued to increase up to at least 80 80 and 127 days posthatch (at 5°C), SOC), respectively. Tamarin and Komarova (1972) (1972) reported that the "threshold “threshold level" level” (asphyxiation level in a closed container) of of Atlantic salmon increased steadily to a maximum 42-60 42-60 days posthatch (at 8°C) 8°C) and then declined to reach typical juvenile and adult levels 240 days posthatch. De Silva and Tytler (1973) 12-h LC (1973) reported that 12-h LC50 in50 values for herring larvae in creased from 2.8 mg 1-’ I-I shortly after hatch to reach a peak of5. 1 mg Iof 5.1 1-’I after 5-6 1 mg 15-6 weeks of feeding before declining to 3. 3.1 1-’1 at metamor metamorphosiS. phosis. Smallmouth bass larvae (Micropterus (Micropterus dolomieui) dolornieui) were least resistant to severe hypoxia ((1.0 1.0 mg I-I) 1-l) at about the start of exogenous feeding (9 1984). In contrast, large (9 days posthatch at 20°C; 20°C; Spoor, Spoor, 1984). largemouth bass larvae (M. salmoides) were least resistant shortly after (M.salrnoides) hatch while still feeding endogenously (3 (3 days posthatch at 20°C; 20°C; Spoor, Spoor, 1977). 1977). Larval plaice were similarly most sensitive shortly after 1973). hatch (De (De Silva and Tytler, 1973). Changes in larval sensitivity have been linked to changes in the site and efficiency of respiratory gas exchange (DeSilva and Tytler, 1973; Spoor, 1977). 1973; Spoor, 1977). The argument is basically as follows. follows. The skin is is the major site of gas exchange during much of early life. life. Young larvae with relatively low metabolic rates and large surface/volume surfaceivolume ratios require a relatively small partial pressure gradient to meet oxygen oxygen
Table V V Table Lower Lethal Levels of of Dissolved Oxygen for Teleost Embryos and Larvaea Larvae"
Species
Temperature ("C) (0C)
Stage Stage tested
Duration of test of
LCm LC50 (mg - I) (mg I1-l)
No-effect level (mg (mg l-I) I-')
Comment
Reference
Chronic exposure, freshwater salmonids
Salmo Salmo gairdneri gairdneri Salmo Salmo gairdneri gairdneri Oncorhynchus tshawytscha tshaw ytscha Oncorhynchus Oncorhynchus kisutch Salmo salar Salmo salar Oncorhynchus kisutch kisutch Salvelinus Salvelinus fontinalis Salvelinus Salvelinus namaycush namaycush Salvelinus alpinus alpinus
10.4 10.4
E
f-h (-30 days) f-h (-30 days)
<2.8
3.0-4.5 3.0-4.5
al. ((1964) 1964) Shumway et al.
10.0 10.0
E
f-h f-h (-36 (-36 days) days)
1.6-2.5 1.6-2.5
1.6-2.5 1.6-2.5
Silver et et al. al. (1963) (1963)
10 10
E
f-h (-45 days) days) f-h (-45
1.6-2.5 1.6-2.5
1.6-2.5 1.6-2.5
E
f-h (-50 days) f-h (-50 days)
<2.5
2.8-4.1 2.8-4.1
1964) Shumway et al. ((1964)
5 5 10 10 8.5
E E E-L E-L
f-h (77 (77 days) f-h days) f-h (43 (43 days) f-h days) 1119 19 days
<3.7 <3.9 1.4-2.9 1.4-2.9
6.8-11.9 6.8-11.9 5.8-11.6 5.8-1 1.6 1.4-2.9 1.4-2.9
(1976) Hamor and Garside (1976)
8
E-L E-L
133 133 days
2.3-2.9 2.3-2.9
2.3-2.9 2.3-2.9
7
E-L E-L
131 days 131
2.4-4.3 2.4-4.3
4.3-6.0 4.3-6.0
10 10
E-L E-L
108 days 108
4 8
E-L E-L E-L E-L
93 days 77 days
5.65.610.5 10.5 <2.6 4.6 "'3.6 -3.6
5.6-10.5 5.6-10.5 <2.6 -3.6 3.6-5.9 3.6-5.9
12.3 12.3
E
f-h (-22 days) (-22 days) f-h
<3. <3.00
3.0-5.0 3.0-5.0
12.3 12.3
E
f-h ((-15 f-h - 15 days) days)
<3.0
<3. C3.0 0
9.5
(1974) Siefert and Spoor (1974)
(1974) Carlson and Siefert (1974) Near upper lethal temp. Gruber Gruher and Wieser (1983) (1983) Near upper lethal temp.
Chronic exposure, freshwater nonsalmonids
Stizostedion Stizostedion vitreum Catostomus commersoni commersoni
Walleye
1971a) Oseid and Smith ((1971a)
White sucker
1971b) Oseid and Smith ((1971h) ~
(continued) (continued)
Table V V (Continued) (Continued) ~~~~~
Species Species
Morone chyrops rops Fundulus Fundulus heteroclitus Morone Morone saxatisaxatilis lis Coregonus Coregonus artedii artedii .... c I!;I C)
$?
Micropterus Micropterus salmoides salmoides Cyprinus carpio carpio S tizostedion Stizostedion vitreum vitreum Catostomus Catostomus commersoni Porornis nigronigroPoxomis maculatus Prosopium williamsoni williamsoni Micropterus Micropterns dolomieui Morone Morone chryschrysops ODs
Temperature (0C) (“C)
~~
Duration of of test
Stage tested
LCm LCoo (mg l-I) I-’)
No-effect level (mg I-I) 1-I)
16 16
E
f-h (4 days) days) f-h
<1.8 < 1.8
<1.8
20
E
f-h days) f-h (20 (20 days)
2.4-4.5 2.4-4.5
4.5-7.5 4.5-7.5
20
E
f-h f-h (4 (4 days) days)
2
E
f-h ((166 f-h 166 days) days)
<1.0 < 1 .0
4 6 8 15 15
E E E E
f-h ((122 days) 122 days) f-h f-h days) f-h (84 (84 days) days) f-h (54days) f-h (54 f-h f-h
< 1.0 <1.0 1.0-2.0 1.0-2.0 2.0-4.0 2.0-4.0
20 25 25
E E E
f-h f-h f-h f-h f-h (70 (70 h) h) f-h
117 7
E-L
18 18
Comment
Reference Reference
Siefert S iefert et al. ((1974) 1974) Voyer and Hennekey ((1972) 1972) Turner and Farley ((1971) 1971)
>5. >5.0 0 1.0
Lake herring
Brooke and Colby (1980) (1980)
1.0-2.0 1.0-2.0 1.0-2.0 2.0-4.0 2.0-4.0 >2.4
Largemouth bass
Dudley and Eipper 1975) Eipper ((1975)
3.0-6.0 3.0-6.0
>2.4 >2.4 6.0-9.0 6.0-9.0
Carp carp
Kaur and Toor (1978) (1978)
20 days
3.4-4.8
3.4-4.8
Walleye Walleye
Siefert and Spoor ((1974) 1974)
E-L
22 days
1.2-2.5 1.2-2.5
1.2-2.5 1.2-2.5
White sucker
20
E-L
?
<2.7
<2.7
Black crappie
Siefert and Herman ((1977) 1977)
4 7 20
E-L E-L E-L E-L E-L E-L
193 193 days 158 158 days 14 14 days
3.3-4.6 3.3-4.6 3.1-6.0 3. 1-6.0 2.5-4.4 2.5-4.4
4.6-6.5 4.6-6.5 3.1-6.0 3.1-6.0 4.4-8.7 4.4-8.7
Mountain whitefish
Siefert 1 974) S iefert et et al. ((1974)
Smallmouth Small mouth bass
16 16
E-L E-L
111 1 days
<1.8 < 1.8
1.8-3.4 1.8-3.4
White bass
c1 ..... t-:) .l ....
Micropterus Micropteros salmoides Ictalurus Ictaluros puncta tus punctatus Esox lucius Pimephales promelas
20 23 23 25 29 15 15 24
E-L E-L E-L E-L E-L E-L E-L E-L E-L E-L L
20 days days days 20 days 19 19 days 19 days days 19 20 days 30 days
Oncorhynchus keta
10 10
E
Salmo gairdneri
1.5 111.5
E
Salmo solar salar Salmo
5
L
trutta Salmo trotta
5
L
6.5 11.5 I I.5 19.0 19.0 10 10
L L L E L L
7 days 7 days 7 days 7 7 days 3 3 days days 3 days 3 3 3 days 120 h 120 72 h 48 h 72 h 72 h 48 h 120 h 120 72 h 72 h 72 h 48 h ? ? ? 8h 8h 8h
Esor lucius Zucius Esox
3.1-4.5 3.1-4.5 1.7-4.2 1.7-4.2 4.2-5.0 4.2-5.0 3.8-4.6 3.8-4.6 2.6-4.9 2.6-4.9 4.02-5.01 4.02-5.01
Largemouth Largemouth bass
1 974) Carlson Carlson and Siefert ((1974)
sh Channel catfi catfish
al. (1974) Carlson et Carlson et 01. (1974)
Northern pike 1971) Brungs Brungs ((1971)
freshwater species Acute exposure, exposure, freshwater species
ta
Coregonus sp.
1.7-3.1 1.7-3.0 1.7-3.0 2.4-4.2 2.4-4.2 2.3-3.8 2.3-3.8 2.6-4. 2.6-4.99 4.02
0.4 0.6 0.6 0.6 0.6 1.0-1.4 1.0- 1.4 <0.9 <0.9 -0.9 -0.9 0.9-2. 0.9-2.77
3.3 3.3 3.7 5.5 -0.3 -0.3 1.6-3.5 1.6-3.5
0.9-1.7 0.9-1.7 1.7-2.7 1.7-2.7 2.7-4.3 2.7-4.3 0.4 0.7 0.7 2.0 3.1 3.1 2.8 0.4 0.7 2.0 2.4 2.3 6.0 6.9 7.5 7.5 <0.6 0.3-1.8 0.3-1.8 1.6-3.5 1.6-3.5
12 12 dpf 22 dpf dpf 32 dpf dpf 48 dpf dpf Blastopore Blastopore closure Start Start circulation circulation Near hatch Newly hatched 10 10 dph 40 dph 54 dph 80 dph 135 135 dph Newly hatched lO dph 10 54 dph 127 127 dph 180 dph 180
1958) Alderdice Alderdice et et al. al. ((1958)
Gottwald (1965) (1965)
Bishai (1960) (1960)
( 1965) Einsele (1965)
3 days prehatch larvae) (yolk-sac larvae) I dph (yolk-sac 1 Feeding larvae Feeding
( 1 976) Peterka and Kent (1976)
(continued) (continued)
V (Continued) (Continued) Table V
Species
Micropterus icropterus M
dolomieui dolomieui Lepomis Lepomis mamacrochirus
Temperature (“C) (0C)
21.5 2 1 .5 26.5
Stage Stage tested
Duration of of test
LC50 LCFn (mg (mg l-1) I-’)
E L E L
6h 6h 4h 4h 24 h 24 h 24 h 12 12 h 12 h 12 12 h 12 12 h 12 12 12 h 12 12 h 12 h 12 12 12 h 12 h 12
2.8 4.4 5.1 5.1 4.2 3.1 3.1 3.9 3.8 3.8 3.6 2.4
0.5
0.5-3.7 0.5-3.7
No-effect level (mg (mg l-1) 1-I) 0.5-1.8 0.5-1.8 0.5-2.2 0.5-2.2 <0 <0.5 .5 0.5-3.7 0.5-3.7
Comment
Reference
2 d prehatch Yolk-sac larvae
1-4 dph (yolk (yolk sac) sac) 1-4
Marine species
-
N w t:) (Xl 0
Clupea harengus
14 14
L
Clupea Clupea harengus harengus
8-13 8-13
L
Pleuronectes platessa
8-13 8-13
L
Cyclopterus C yclopterus lumpus Ophiodon elongatus elongatus Chasmodes bosquianus Gobiosoma Gobiosomu bosci
12.7 12.7
L
24 h
3.6
8.6 8.6
E
96 h
3.0
20.8
L
24 h
2.5
20.8
L
24 h
1.3 1.3
2.2-2.6 2.2-2.6 4.4 5.1 5. 1
4.6-7.7 4.6-7.7
Newly hatched 3 dph 3 4 dph Yolk-sac larvae 2-3 weeks feeding 2-3 5-6 5-6 weeks feeding 7-8 weeks feeding 7-8 Metamorphosis Yolk-sac larvae 2-3 weeks feeding 2-3 6-7 6-7 weeks feeding 3-4 weeks metamorpho3-4 sis 24 dph
(1960) Bishai (1960)
Lingcod
Giorgi and Congleton ((1984) 1984) Saksena and Joseph ((1972) 1 972)
Striped blenny, newly hatched Naked goby
DeSilva and Tytier Tytler (1973) (1973)
DeSilva and Tytler ((1973) 1973)
Bishai (1960) (1960)
Gobiesox strumosus Mugil cephalus
c
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20.5 20.5
L
24 h
0.7- 1.2 1.2 0.7-
20
E
48 h
4.5-5.0 4.5-5.0
13(?) 13(?)
L L L
48 h 96 h 24 h
4.8-5.4 4.8-5.4 6.4-7.9 6.4-7.9 3.5
Near hatch, striped mullet Newly hatched 4.8-5.4 4.8-5.4 Newly hatched 6.4-7.9 6.4-7.9 4.0 4.0 (Lew) (LC,~) First-feeding static test
L
24 h
3.2
3.5 (Lew) 3.5 (LCd
First-feeding static test
L
24 h
3.6 3.6
3.9 (Lew) 3.9 (LClO)
First-feeding static test
L L
24 h 24 h
<2.3 <2.3 3.6
4.2 (LelO)
First-feeding static test First-feeding static test
L
24 h
2.5 2.1 2. 1
2.9 (Lew) 2.7 (Lew)
L
24 h
6.9
1 .2 (LelO)
Skillet Skillet fish
4.5-5.0 4.5-5.0
1975) Sylvester et aZ. al. ((1975) Sylvester et
(1980) Brownell (1980)
First-feeding static test BowFirst-feeding flowFirst-feeding through test First-feeding flowthrough test ~~
a Abbreviations: E, embryos; L, larvae; dph, days posthatch; posthatch; f-h, f-h, fertilization to hatch.
a
130 130
PETER PETER J. J. ROMBOUGH ROMBOUGH
demands. As the larvae grows its metabolic rate increases, but expan expansion of the area for cutaneous gas exchange fails to keep pace. If If metabolic demands are to be met, the partial pressure gradient across the skin must increase (recall (recall V02=G02P02)' V02=G02P02). The result is an increase in Pc P, (standard) (standard) and a reduction in the ability of the larva to tolerate hypoxia. As development proceeds, the gills become progressively more important as a site of respiratory gas exchange. exchange. Gills are as assumed to be a more efficient organ of of gas exchange and, as a result, Pc P, (standard) (standard) values gradually decline. This is reHected reflected in a gradual de decrease in sensitivity to hypoxia. hypoxia. Species vary in the timing of the transition from cutaneous to branchial gas exchange, and thus it is not surprising that they vary in the particular stage at which they are most sensitive to hypoxia. 1973) indicated that the hypoxia. De Silva and Tytler ((1973) stage at which hemoglobin first appears also also may be important. Spoor (1984) (1984) linked increased resistance of smallmouth bass larvae near the start of exogenous feeding to inHation un inflation of the swimbladder. It is unlikely that this represents a true increase in tolerance to hypoxia but more likely reHects reflects the fact that the gas in the swimbladder can act as a temporary reservoir when oxygen oxygen is in short supply. It is generally assumed that the ability of fish to resist hypoxia decreases with increasing temperature (European Inland Fisheries Advisory Commission, Commission, 1973; 1973; Alabaster and Lloyd, 1980; 1980; Chapman, 1986), 1986), although Chapman (1986) (1986) points out that the data supporting this contention is rather spotty. There is some evidence indicating that, within limits, hypoxic sensitivity may actually vary relatively little with temperature. Davis (1975) (1975) found that incipient sublethal response thresholds of adult fish were insensitive to temperature 1 ). This still represents when expressed as a concentration (i.e., (i.e., mg 11-l). an increase in the driving force required to meet oxygen demands, since, at a given concentration, partial pressures are greater at higher temperatures (because 1978), (because of decreased capacitance). capacitance). Ultsch et ai. al. ((1978), on the other hand, reported that asphyxiation levels were actually lower at 20°C than at 10°C, lWC, even when expressed as partial pressures, in adults of six species of darters (Etheostoma). (Etheostorna). Few studies have specifically examined the effect of of temperature on the ability of embryos and larvae to tolerate a lack of of oxygen. oxygen. Temperature variations within a narrow range (3-5°C) appear to have little effect in many species (Siefert et ai., al., 1974; 1974; Carlson and Siefert, Siefert, 1974; 1974; Carlson et ai., al., 1974; 1974; Hamor and Garside, 1976). 1976).There are some exceptions though. Increasing the temperature from 7 to lOOC 10°C resulted in a significant increase in the sensitivity of young lake trout (Saive (Saluelinus (Carlson and Siefert, 1974). Zinus namaycush) narnaycush) (Carlson 1974). Similarly, Similarly, Arctic char
2.
RESPIRATORY EXCHANGE, AEROBIC AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, METABOLISM
131 131
embryos and larvae were less tolerant of of hypoxia at 88 than at 4°C 4°C (Gruber and Wieser, 1983). 1983). Both species are stenothermal, and in both studies the higher temperature was near the upper limit of the zone of tolerance. Lethal levels for embryonic (Brooke (Brooke and Colby, 1980) 1980) and larval (Einsele, 1965) coregonids increased with temperature, but (Einsele, 1965) again increases were greatest at the higher temperatures. These results suggest that lethal oxygen concentrations may be relatively independent of temperature within the normal temperature range of a particular species but that at high temperatures, there is a strong likestrong like lihood of an additive or synergistic interaction. High flow rates can be expected to increase the hypoxic tolerance of embryos by reducing the thickness of the boundary layer. The flow oxygen level has been interaction between fl ow rate and dissolved oxygen well documented using sublethal indicators such as growth (Silver (Silver et al., 11963; 1964).There is less information on its effect 963; Shumway et al., 1964). on lethality. Shumway et al. al. (1964) (1964) reported that mortality among coho and steelhead embryos exposed to low oxygen oxygen levels tended to flows. Miller ((1972) be greater at low flows. 1972) reported that mortality among "unfanned" “unfanned” largemouth bass embryos was greater than among fanned embryos. embryos. High embryonic mortality in nature is often associated with low flows flows (Wickett, 1954; Coble, 11961; 961; Phillips and Campbell, 11961; 961; (Wickett, 1954; McNeil, 1966; 1966; Taylor, 1971 1971;; Hempel and Hempel, 1971; 1971; Giorgi, 1981). cases, however, flow rate and oxygen concentration 1981). In most cases, varied simultaneously so it is difficult to separate effects due to low oxygen oxygen from those due due to low flow. flow. Salinity, Salinity, like temperature, appears to have little effect on hypoxic (1971) indi inditolerance within normal limits. Alderdice and Forrester (1971) cated that viable hatch of Pacific cod (Gadus (Gadus macrocephalus) was largely independent of oxygen, provided levels were above 2-3 2-3 mg I-I 1-’ within the optimal range of of temperatures (3-4.5°C) (3-4.5”C) and salinities (17(1723%0). 23%). Oxygen requirements tended to increase at higher salinities. Similarly, embryonic survival of of pilchard (Sardinops (Sardinops ocellata) ocellata) was largely independent of oxygen oxygen levels greater than 2. 2.11 mg I1-’I within the optimal range of temperature ((16-21°C) 16-21°C) and salinity (33-36%0) (33-36%0) (King, (King, 1977). 1977). There are significant differences in the abilities of different spe species to tolerate hypoxia. There does not appear to be any phylogenetic pattern, but rather the sensitivity of a particular species seems to re refl ect oxygen levels in its normal habitat. For example, flect example, Spoor (1984) (1984) linked differences in habitat selection by larvae of smallmouth bass and largemouth bass to differences in their sensitivity to hypoxia. hypoxia. (1986) pointed out that while salmonids salmonids are more sensitive Chapman (1986)
132 132
PETER PETER J. ROMBOUGH J . ROMBOUGH
to hypoxia than most nonsalmonid freshwater fish as juveniles, they are less sensitive than many other groups as embryos and larvae. ( 1975) concept of of These observations lend some support to Balon's (1975) embryonic reproductive guilds. Data in Table V indicate a mean embryoniclarval LC5 2.7 mg 11-' - 1 for salmonids at normal temperatures LCw0 of of 2.7 temperatures.. The nonsalmonid freshwater species fall into two groups: white sucker, black crappie, white bass, and largemouth bass, with a mean embry embryonic-larval - 1, and walleye, mountain whitefish, onic-larval LC5 LCa0 of of 2.3 2.3 mg 1l-l, embryonic-larval LC5o LC50 of of 3.8 smallmouth bass, and pike with a mean embryonic-larval 1- 1 . Marine species are generally less tolerant than freshwater mg 1-'. (1973) attributed species. Among marine species, De Silva and Tytler (1973) the greater resistance of of newly hatched hatched herring larvae compared with newly hatched plaice larvae to the fact that that the former hatch from lev demersal eggs and are thus more likely to encounter low oxygen levels. A glaring deficiency in the literature is is the absence of any data on the hypoxia tolerance of tropical freshwater species, some of of which spawn in virtually anoxic water. Durhorow and Avault (1985) Durborow (1985) reported significant differences among full-sib families of of channel catfish (Zctalurus (Ictalurus punctatus) punctatus) in possibility of of selecting larval resistance to hypoxia. This raises the possibility strains that are resistant to hypoxia for use in aquaculture. (1955) demonstrated that acclimation to low oxygen levels Shepard (1955) ofjuvenile brook trout to tolerate hypoxia. There increased the ability of resis are limited data data indicating that acclimation increases at at least the resisof larvae. McDonald (1977) reported that chronic tance of McDonald and McMahon (1977) exposure of of Arctic Arctic char char larvae larvae to to low oxygen oxygen (2.5 (2.5 mg 1-1) 1-I) resulted in a a 2.5-fold increase in median resistance time in 11.1 . 1 mg 11-' 1 compared . with norm oxic-reared larvae. normoxic-reared of hyperoxia have been reported to enhance sursur Moderate levels of of embryos (Gulidov, 1969, 1969, 1974; 1974; Gulidov and Popova, 1978) 1978) vival of and larvae (Sylvester et al., ai., 1975). 1975). At higher concentrations (>300% (>300% saturation), though, oxygen becomes toxic. Species vary in their air saturation), ability to tolerate hyperoxia. Gulidov (1969) (1969) reported significant mormor (Esox Zucius) lucius) eggs incubated in 36.4 36.4 mg 1-' 1-1 (336% (336% ASV). ASV). tality in pike (Esox 45.3 mg 1-I 1-1 (418% (418% ASV), ASV), apparently bebe No pike embryos hatched in 45.3 cause of of suppressed neuromuscular activity. Embryos of of verkhovka 1974) and roach (Rutilis (Rutilis rutilis) (Leucospuis delineatus) delineatus) (Gulidov, (Gulidov, 1974) rmtilis) (Gulidov and Popova, 1978), 1978), on the other hand, both hatched success success1-1. fully in about 40 mg 1-' . In L. delineatus, hatching was delayed at high oxygen concentrations. Newly hatched larvae tended to have more body segments than normal, and no red blood cells were present. The initial phase of of erythropoeisis was not inhibited, and red blood cells
2. 2.
RESPIRATORY RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM
133 133
of appeared in the circulation at about the normal time. The number of Aberythrocytes later declined, and red cells were absent at hatch. Ab sence of red blood cells did not adversely affect survival, which is not concentration. Gulidov surprising given the high ambient oxygen concentration. L. delineatus and roach to hyper hyper((1974) 1974) linked the high tolerance of L. oxia to the fact that their eggs are frequently laid on vegetation and may thus be exposed to high oxygen concentrations in their natural habitat.
2. RESPONSES 2. SUBLETHAL SUBLETHAL RESPONSES Hypoxia elicits a broad spectrum of sublethal responses in emem bryos and larvae. These include reduced rates of growth and develop development, morphological changes, behavioral alterations, and a wide vari variety of metabolic and physiological adjustments. adjustments. In general, sublethal response thresholds are even more poorly defi ned than lethal thresh defined thresholds. olds. The reasons for this are basically the same as those discussed previously: defi cient experimental design and changes in the intrinsic deficient sensitivity of the organism. The situation is actually somewhat worse than for studies of lethality. Sublethal response thresholds have tended to be higher than anticipated. Many investigators have chosen inappropriate experimental levels (i.e., (i.e., too low) low) and, as a result, have been able to defi ne response thresholds only very broadly (typically define (typically as lying somewhere between 30-50 100% air saturation), 30-50 and 100% saturation), if at all. The following discussion, therefore, will be restricted for the most part to a qualitative description of of the sublethal effects of hypoxia. hypoxia.
a. a. Development and Growth. Growth. Developmental velocity and early growth are highly sensitive to reductions in ambient oxygen levels. levels. In many species incipient limiting levels appear to be close to, or even in excess of, 100% 100% air saturation (Silver et al., al., 1963; 1963; Shumway et al., al., 1964; 1964; Eddy, 1972; 1972; Gulidov, 1974; 1974; Gulidov and Popova, 1978). 1978). Re Reduced growth and delays in development under moderate hypoxia should be regarded as compensatory responses whereby the animal adjusts metabolic demands to match available supply. Growth and development are normally closely linked, although there is some sug suggestion that developmental rates are less plastic than growth rates (Silver et al., al., 1963). 1963). Embryonic development is progressively progressively retarded by continuous exposure to low levels of dissolved oxygen (Garside, 1966; Win (Garside, 1959, 1959,1966; Winnicki, 1968). 1968). Delays are typically insignificant during early develop development at moderate levels (> 20-30% ASV) ASV) of of hypoxia. hypoxia. During later development, times to defined stages become progressively progressively more de-
134 134
PETER PETER J. J. ROMBOUGH ROMBOUGH
layed normoxia. As layed compared compared with with normoxia. As expected, expected, delays delays are are more more pro pronounced at lower oxygen levels. Most investigators have not monitored developmental velocities closely. Instead they have looked at the effect of of hypoxia on times to easily identifiable events such as hatch, emergence, or the onset of of feeding. The problem with this approach is that times to some of these events are not indicative of the overall effect of of hypoxia on develop developmental rate. For example, time to hatch is widely used as an indicator of sublethal hypoxic stress. stress. Low oxygen levels elicit two responses that have opposing effects on time to hatch. Hypoxia reduces the over overall rate of development and thus tends to delay hatching. However, once embryos have reached a certain stage, low oxygen levels initiate the release of of hatching enzyme (DiMichele (DiMichele and Taylor, Taylor, 1980; 1980; DiMi DiMichele and Powers, 1982; 1982; Yamagami et aZ., al., 1984) 1984) and thus tend to reduce time to hatch. Which effect dominates depends on the particu particular species. Continuous exposure of coho, coho, brook trout (Siefert (Siefert and Spoor, Spoor, 1974), 1974), walleye (Oseid (Oseid and Smith, Smith, 1971a), 1971a), mountain whitefish Siefert et aZ., al., 1974), 1974), mummichog (FunduZus (Fundulus heteroclitus) (Voyer (Voyer and Hennekey, 1972), Hennekey, 1972), lake trout (Garside, (Garside, 1959; 1959; Carlson and Siefert, Siefert, 1974), 1974),and lake herring (Brooke (Brooke and Colby, 1980) 1980)to to low oxygen oxygen levels resulted in significant delays in time to hatch. Continuous exposure of white white sucker (Oseid (Oseid and Smith, Smith, 1971b; 1971b; Siefert and Spoor, Spoor, 1974) 1974) and sockeye sockeye (Brannon, (Brannon, 1965) 1965)had no significant effect, while hypoxic incu incubation of smallmouth bass (Siefert aZ., 1974) (Siefert et al., 1974) and largemouth bass (Carlson (Carlson and Siefert, 1974) 1974) caused premature hatch. These results suggest that if embryonic development is is relatively rapid, as in smallmouth smalImouth and largemouth bass, the premature release of hatching enzyme outweighs any general delay in development. development. The effect of low oxygen on time to emergence is similarly complicated by the fact that moderate levels of hypoxia can act as a directive factor and induce substrate prematurely. As a result, hypoxia has the larvae to leave the substrate been reported variously to delay (Phillips aZ., 1966; (Phillips et al., 1966; Mason, Mason, 1969) 1969)or advance (Witzel 1 ; Bailey et aZ., (Witzel and MacCrimmon, 198 1981; al., 1980; 1980; Barns, Bams, 1983) 1983) emergence. Growth Growth has been the most widely used indicator of hypoxic stress during early life (Silver et aZ., al., 1963; 1963; Shumway Shumway et aZ., al., 1964; 1964; Oseid and during Smith, Smith, 1971a,b; 1971a,b; Carlson et aZ., al., 1974; 1974; Siefert et aZ., al., 1973, 1973, 1974; 1974; Siefert and Spoor, Spoor, 1974; 1974; Carlson and Siefert, Siefert, 1974; 1974;Hamor and Garside, Garside, 1977; 1977; and Wieser, 1983; 1983; Florez, Florez, 1972). 1972). As mentioned earlier, earlier, re reGruber and sponse thresholds are high. Embryos appear to be more sensitive than larvae, although this has not been well documented. documented. Embryonic Embryonic larvae, growth, at least, least, is more severely inhibited by low oxygen oxygen at higher
2. 2.
RESPIRATORY RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM
D ”0 10
.! -E. to Z
8
III •
6
g
I a
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135
..
wa1.r vvelocity wat.r elocity lcm (em h-’l h-1 ) BOO • 800 100
•
0
4 2
2 4 10 10 4 2 m g 1-1 OXYQEN LEVEL 1-11) OXYG EN L EV E L (Img
12 1’2
Fig. 12. 12. Mean dry weights of newly hatched coho salmon kisutch) Fig. salmon (Oncorhynchus kisutch) reared at various various constant constant oxygen oxygen concentrations concentrationsand and bulk water velocities. [After larvae reared et al. 01. (1964).] Shumway et Shumway
(Hamor and Garside, 1977; 1977; Eddy, 1972; 1972; Gruber and temperatures (Hamor P , values tend Wieser, 1983), 1983), as would be predicted from the fact that Pc temperature. Also, Also, as expected, low flow rates accen accento increase with temperature. limiting effect of low oxygen oxygen on embryonic growth (Silver et tuate the limiting al., 1963; 1963; Shumway et al., al., 1964; 1964; Hamor and Garside, 1977) 1977) (Fig. (Fig. 12). 12). al., flow appears to be more pronounced for larger eggs The effect of low flow than for smaller eggs (Silver (Silver et al., al., 1963; 1963; Shumway et al., al., 1964; 1964; Bran Branthan non, 1965) 1965).. Apparently the more favorable surface/volume ratios of non, eggs make variations in the thickness of the boundary layer less small eggs important. High flow rates during the larval period can have the oppo oppoimportant. ensite effect and reduce growth efficiency, apparently as a result of en hanced activity levels (Brannon, (Brannon, 1965). 1965). There is is some some question question as as to to the the significance significance of the reductions in growth noted at moderate levels levels of hypoxia. hypoxia. Eddy (1972) (1972) points out that although although low low oxygen oxygen levels result in smaller salmonid larvae larvae at that hatch and greatly extend the period of endogenous endogenous feeding, feeding, there is relatively little difference in the final final size achieved. achieved. Growth efficien efficiencies of Atlantic salmon (Hamor (Hamor and Garside, Garside, 1977) 1977) and and Arctic Arctic char cies (Gruber (Gruber and Wieser, Wieser, 1983) 1983) embryos were little affected by oxygen oxygen as low low as as 20-30% air saturation. saturation. Analysis Analysis of larval larval growth growth is levels as complicated. The The growth efficiency of Atlantic salmon (Hamor (Hamor more complicated. and Garside, 1977) 1977) and Arctic Arctic char char (Gruber (Gruber and Wieser, Wieser, 1983) 1983) larvae was was significantly reduced at 20-30% ASV ASV compared with at 100% 100%ASV (Fig. 13). 13). In both species, species, however, however, growth efficiency efficiency was was signifi signifi(Fig. cantly higher at 50% 50% ASV than than at normoxia. normoxia. This This was was attributed to to cantly
PETER PETER JJ.. ROMBOUGH ROMBOUGH
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Fig. Fig. 13. 13. Efficiency of early growth under conditions of of chronic hypoxia. (ASV, air saturation.) (A) of yolk (A) Gross conversion efficiencies (weight of tissue produced/weight of consumed during a given period) of embryos (e) (e) and larvae (L) (L)of Atlantic salmon (Salmo 1977).] (B) (B) Growth (Salmo salar) salar) reared reared at 55 and lODC. 10°C. [After Hamor and Garside ((1977).] efficiencies (energy content of of tissue elaborated/energy elaboratedlenergy expended on growth and me me(L) of tabolism during a given period) period) of embryos (e) (e) and larvae (L) of embryos of Arctic char (Salvelinus (Saluelinus 1983). ] alpinus) alpinus) reared at 44 and 8°C. 8°C. [after Gruber and Wieser ((1983).1
reduced locomotor activity under moderate hypoxia (Hamor (Hamor and Gar Garside, 1977; 1977; Gruber and Wieser, 1983). 1983).There is is another possibility that serves to illustrate the difficulties in comparing comparing early growth efficien efficiencies. cies. It arises from the fact that the larvae were not all at equivalent stages when the comparisons were made. For example, Hamor and
2. 2.
RESPIRATORY AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, EXCHANGE, AEROBIC METABOLISM
137 137
(1977) terminated their tests when between 80 and I111%(?) Garside (1977) I I %(?) of the yolk present at hatch was consumed. Yolk conversion efficiencies exhaustion. In steelhead, instan instandecline sharply as yolk reserves near exhaustion. 45% to 0% 0% between 80 taneous yolk conversion efficiencies efficiencies fall from 45% 100%yolk utilization (Rombough, (Rombough, 1987). and 100% 1987). It would require only a relatively minor difference in the stages that are compared to produce efficiency. an apparently significant difference in growth efficiency. Even if growth efficiency is signifi significantly cantly reduced at low oxygen levels levels,, the environmental significance is not always clear. It is gener generally assumed that smaller larvae are at a competitive disadvantage. Smaller absolute swimming Smaller larvae larvae display display slower slower absolute swimming speeds speeds and and hence hence themcan search a smaller volume for prey in a given time and may them selves be more susceptible to predation (Giorgi, (Giorgi, 1981). 1981). Smaller larvae selves are also less effective at competing for territories (Mason, 1969).How How(Mason, 1969). ever, territories may not always be fully occupied or food always limit limit(1969)observed that coho coho incubated under hypoxic condi condiing. Mason (1969) tions were smaller at emergence and could not compete successfully for territory with larger normoxic larvae. As a result, they were forced to migrate from the vicinity of the redd but subsequently did well providing 1983) reported reported providing the the stream stream was was not not heavily heavily populated. populated. Barns Bams ((1983) incubators were smaller and that chum larvae from Japanese-style incubators emerged earier than larvae incubated in upwelling gravel boxes, ap apparently because of hypoxic conditions in the Japanese-style incuba incubators. These alevins, tors. These alevins, though though smaller smaller at at emergence, emergence, subsequently subsequently grew grew faster than the alevins from upwelling boxes boxes because of earlier feed feeding. The hatchery situation may be unusual because of the abundance ing. of 1983) points of food, food, but, but, as as Barns Bams ((1983) points out, out, it it emphasizes emphasizes that that fitness fitness components defined carefully carefully and and must must be viewed viewed in in relation relation components must must be defined conditions. to the particular set of conditions.
b. b. Morphology. Teratogenic Teratogenic effects effects have have been been observed observed at at oxygen oxygen concentrations al., 1963; 1963; Shumway Shumway concentrations close close to to the the lethal lethal level level (Silver (Silver et al., al., 1964; 1964; Garside, 1959; 1959; Alderdice et al., 1958; 1958; Braum, Braum, 1973; 1973; et al., 1980). Deformities of the axial axial skeleton, skeleton, jaws, and Brooke and Colby, 1980). vitelline circulation appear most changes in the vitel vitelmost common. common. The changes line circulation appear to be adaptive. adaptive. Blood Blood vessels become more fi nely divided finely divided and and cover cover aa greater greater proportion proportion of of the the yolk. yolk. This This can can be expected to enhance respiratory gas gas exchange. Burggren and Mwalu Mwalukoma 1983) noted that that chronic exposure to low oxygen oxygen levels caused koma ((1983) a similar similar increase increase in in the density density of of capillaries capillaries supplying supplying the the skin skin of of larval larval amphibians. amphibians. This was associated with a reduction in the thick thickness ness of of the the blood-water blood-water diffusion diffusion barrier. barrier. Diffusion Diffusion distances distances have have
138 138
PETER PETER JJ.. ROMBOUGH ROMBOUGH
not not been reported reported for for fish embryos embryos or or larvae larvae chronically chronically exposed exposed to to low oxygen, but observations yolk sac hypoxic embryos low oxygen, but observations that that the the yolk sac of of hypoxic embryos ruptures (Garside, 1959; ruptures readily readily (Garside, 1959; Brooke Brooke and and Colby, 1980) 1980) suggest suggest that that this this might be be the the case. case. Chronic Chronic hypoxia also brings about about morphologi morphological cal adjustments adjustments in in the the gills that that can can be be expected expected to to enhance enhance gas gas ex exchange. change. McDonald McDonald and and McMahon McMahon (1977) (1977) observed observed lamellar lamellar hypertro hypertrophy in Arctic char larvae reared in 2.6 2.6 mg I1-’ - I (at (at 6,SOC). 6.5”C). Branchial development overall was inhibited as a consequence of a general inhibition of growth, so so that by 47 days posthatch hypoxic larvae had 22% fewer filaments and 40% 40% fewer lamellae than normoxic larvae. larvae. The surface area of individual lamellae lamellae,, however, was signifi cantly significantly larger in the hypoxic larvae to the extent that there was no signifi cant significant difference in total lamellar surface. The fact that the hypoxic larvae were more resistant to lethal oxygen concentrations led McDonald and McMahon ((1977) 1977) to suggest that other adjustments, such as re reduced blood-water blood-water distances, increases in the area of of lamellar blood spaces, and increased lamellar perfusion, were also involved. Pinder and Burggren (1983) (1983) suggested that such morphological adjustments leading to increased conductance of gas-exchange gas-exchange organs organs was typical of the early stages of of lower vertebrates. They contrasted this with the usual adult response to hypoxia of of increased blood-carrying capacity and increased hemoglobin oxygen oxygen affinity. affinity.
c. Behavior and Physiology. Larvae respond to acute hypoxia with c. an increase in random movements (Spoor, (Spoor, 1977, 1977, 1984). 1984). The level at which they respond appears to be directly related to the lethal level, suggesting they are responding to hypoxemia rather than ambient oxygen levels. Spoor ((1984) 1984) noted that the oxygen concentration at which smallmouth bass began to become agitated increased steadily 8-10 days after hatch. During this period the length of of during the first 8-10 time the larvae would resist acutely lethal oxygen concentrations also progressively. About 12 12 days posthatch the larvae became declined progressively. less responsive to low oxygen levels. This coincided with increased resistance. of fish embryos and larvae to acute The physiological responses of hypoxia are significantly different from those of juveniles and adults. adults. (Fisher, 1942; 1942; Hole HoleMost notable is the absence of reflex bradycardia (Fisher, 1971; McMahon, 1977). 1977). In this regard they are ton, 197 1 ; McDonald and McMahon, (West and Burggren, 1982; 1982; Feder, similar to amphibian larvae (West 1983a,b; Quinn and Burggren, 1983). 1983). 1983a,b; emPhysiological and behavioral responses designed to facilitate em bryonic and larval gas exchange were discussed in a previous section.
2. 2.
139 139
RESPIRATORY EXCHANGE, AEROBIC AEROBIC METABOLISM RESPIRATORY GAS GAS EXCHANGE, METABOLISM
Not Not unexpectedly, unexpectedly, such such responses responses tend tend to to intensify intensify during during hypoxia. hypoxia. Peterson and Martin-Robichaud (1983) (1983) noted that the frequency of of trunk Atlantic salmon embryos increased trunk movements movements of Atlantic salmon embryos increased during during hy hypoxia. 1971 ) noted that 1-8 days poxia. Holeton Holeton ((1971) that young rainbow rainbow trout trout larvae larvae ((1-8 old) responded acute exposure exposure to to moderate moderate levels levels of of hypoxia with with old) responded to acute increased pectoral fin movements, increases in rate and amplitude of of breathing, breathing, and and tachycardia. tachycardia. Only Only when when oxygen oxygen levels levels dropped dropped below about mm Hg Hg (at (at lO°C) l0OC) did did heart heart rate rate and and breathing breathing rate rate decline decline about 40 mm (Fig. 14). Heart Heart rate rate and and breathing breathing rate rate recovered recovered slowly slowly in in restoration restoration (Fig. 14). of normoxia, quick recovery normoxia, unlike unlike the the quick recovery seen seen in in adult adult fish. fish. Peterson Peterson (1975) (1975) reported reported that that the the ventilatory ventilatory rate rate also also increased increased in in Atlantic Atlantic salmon larvae Hg at (30-80 mm mm Hg at 9°C). 9OC). salmon larvae in in response response to to acute acute hypoxia hypoxia (30-80 Increased in Increased opercular opercular movements movements were were accompanied accompanied by increases increases in the n movements. pectoral-fin movements. Movements Movements of of pectoral pectoral fins fins the frequency frequency of pectoral-fi appear in appear to to help help draw draw water water across across the the gills, gills, since since ablation ablation of of the the fins fins in 1 00
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Fig. line) and Fig. 14. 14. Effect Effect of of acute acute hypoxia hypoxia on on heart heart rate rate (solid (solid line) and breathing breathing rate rate (dotted (dotted line) of line) of rainbow rainbow trout trout larvae larvae 8 8 days days after after hatch hatch in in lOoe 10°C water. water. Heavy Heavy line line indicates indicates P0 PO22 1971).] of Holeton ((1971).] of the water. water. [From Holeton
140 140
PETER J. ROMBOUGH
normoxia results in a significant increase in opercular rate. McDonald and McMahon ((1977) 1977) indicated that ventilatory and cardiovascular responses during chronic hypoxia were similar to those during acute hypoxia. Heart and circulatory rates of Arctic char larvae remained hypoxia. elevated in 33 mm Hg (at 6.5"C) 6.5°C) for at least 47 days after hatch. As in Atlantic salmon (Peterson, 1975), 1975), ventilatory movements of newly hatched Arctic char were infrequent and uncoordinated. In contrast, rhythmic and rapid (60 (60 min-I) min-') ventilatory movements were observed in hypoxic larvae within 11h of 1977) of hatch. McDonald and McMahon ((1977) speculated that cutaneous respiration could provide sufficient oxygen to meet the respiratory demands of of newly hatched larvae under norm normoxic conditions but not during hypoxia. hypoxia. The larvae compensate for a reduced driving force during hypoxia by branchial recruitment, effec effectively increasing the surface area for respiratory gas exchange (i.e., (i.e., increasing conductance). 1971) nor McDonald and conductance). Neither Holeton ((1971) McMahon ((1977) 1977) observed the reflex bradycardia bradycardia typical of of juvenile and adult fish. fish. Holeton (1971) (1971) speculated that the basic response to hypoxemia was increased breathing and heart rates and that the bradycardia observed in older fish was a superimposed mechanism designed to balance ventilation-perfusion ventilation-perfusion rates for efficient delivery of oxygen to tissues. McDonald and McMahon ((1977) 1977) argued that tachycardia was a more appropriate response during early life, since higher cardiac output would lead to opening of of vascular channels within the gills and effectively increase the surface area for gas ex exchange. change.
d. d . Metabolism. During severe hypoxia, hypoxia, physiological adjustments may not be sufficient to allow the organism to meet its energy require requirechannelments aerobically. Partial compensation can be achieved by channel ing energy through glycolytic pathways. Arctic char embryos are able V02) using anaerobic to supply energy at 111-23% 1 -23% the aerobic rate (r (rVOz) pathways (Gnaiger, (Gnaiger, 1979; 1979; Gnaiger et al., al., 1981). 1981). The ability to produce a relatively high proportion of normal energy nquirements r >quirementsanaerobi anaerobically probably accounts for the high anoxic resistance of of the embry embryonic stages, especially when it is remembered that a large proportion of normal metabolism goes toward growth rather than maintenance (Smith, 1957, 1958). Gnaiger et al. 1981) indicated that lactic acid was (Smith, 1957,1958). al. ((1981) the predominant end product in Arctic char embryos, although small amounts of succinic acid were also produced. There was a transient increase in alanine concentration but no change in volitile fatty acid concentration. concentration. It thus appears that the high anoxic tolerance is due to
2. 2. RESPIRATORY RESPIRATORY
GAS GAS EXCHANGE, EXCHANGE, AEROBIC AEROBIC METABOLISM METABOLISM
141 141
high tolerance of lactic acid, rather than activation of more efficient anaerobic pathways. Recovery from anoxia is slow. slow. Gnaiger ((1979) 1979) observed that it took up to 48 h after restoration of normoxia for the levmetabolic rate of Arctic char embryos to return to typical aerobic lev els. els. The metabolic responses of larvae to acute hypoxia are complex and apparently involve both activation of anaerobic pathways and utilizaphysiological or biochemical changes leading to more efficient utiliza aerotion of oxygen. Gnaiger ((1983a) 1983a) monitored changes in total and aero bic energy production of Arctic char larvae following abrupt transfer 100% to 48% 48%air saturation using simultaneous direct and indi indifrom 100% half the rect calorimetry. Heat production dropped rapidly to about half original rate but over the next 24 h gradually increased again to typical normoxic rates. During this period, period, total heat production measured directly was greater than that calculated on the basis of oxygen con consumption, indicating a significant anaerobic contribution (~20% mid(=20% mid way through recovery). Gnaiger ((1983a) 1 983a) termed this period the phase of anoxic compensation, although compensatory changes involving gradaerobic metabolism were also taking place as evidenced by the grad ual increase in the rate of oxygen h, the oxygen consumption. After about 24 h, rate of oxygen oxygen consumption stabilized at approximately normoxic lev levels but total heat production began to decline so els so that apparent aerobic energy production was greater than total energy production. Gnaiger ((1983a) 1983a) termed this the phase of conservative compensation and sug sugglyconeogenesis utilizing lactic acid gested that it reflected coupled glyconeogenesis accumulated during the period of anoxic compensation. compensation. Young salmo salmonids apparently either oxidize or reconvert most of the lactate accumu accumual., lated during periods of oxygen debt back to glycogen (Wieser (Wieser eett al., 1985). 1985).This may not be true in all species. species. Broberg and Kristoffersson Kristoffersson ((1983) 1983) reported that young of the viviparous species Zoarces vivipa uiuiparous excrete a large proportion of accumulated lactate.
B. Physiological Hypoxia Even oxic conditions, Even under under norm normoxic conditions, young young fish fish may may be be subjected subjected to to hypoxemia as a consequence of developmental events or activity. hypoxemia as a consequence of developmental events or activity. Sev Several eral investigators investigators have have attempted attempted to to link link physiological physiological hypoxia, hypoxia, as as evidenced by increased lactic acid concentrations, with specific evidenced by increased lactic acid concentrations, with specific de developmental velopmental events. events. It It appears appears that that in in general general there there is is aa rise rise in in lactic lactic acid levels during gastrulation, a drop during early organogenesis, acid levels during gastrulation, a drop during early organogenesis, and and
142 142
PETER J. ROMBOUGH
aa gradual gradual rise rise toward toward hatch hatch (Hishida (Hishida and and Nakano, Nakano, 1954; 1954; Kamler, Kamler, 1976; 1976; Boulekbache, Boulekbache, 1981). 1981). Kamler Kamler (1976) (1976) reported reported that that lactic lactic acid acid levels levels continued to rise in carp larvae until near the end of of endogenous feeding. The ontogeny of energy metabolism is beyond the scope of of of this review-recent review-recent reviews of of this area are provided by Terner (1979) (1979) and 1 98 1)-but it of and Boulekbache Boulekbache ((1981)-but it is is important important to to recognize recognize that that many many of the responses seen as result of responses seen as aa result of environmental environmental hypoxia hypoxia also also may may be elicited elicited during during normal normal development. development. Wieser et al. al. (1985) (1985) recently examined the metabolic responses of of young rainbow trout during physiological hypoxia induced by forced swimming. Anaerobic energy production (on swimming. (on a mass basis) was found to be independent of of body mass and temperature. temperature. It was pointed out previously that maximum aerobic energy production of young rain rainbow trout increases with body size and temperature, up to a maximum at about 12°C 12°C (Wieser 1985; Wieser, 1985). (Wieser et al., 1985; 1985).The net result is that anaerobic sources are proportionally more important during larval ac activity than in older fish. fish. In rainbow trout, anaerobic energy production during a l-min I-min burst of activity was 6-9 6-9 times the aerobic production in times the in yolk yolk sac sac larvae larvae but but only only 2-6 2-6 times the aerobic aerobic production production in in free freeswimming swimming larvae larvae and and fry. fry. Anaerobic Anaerobic energy energy production production was was derived derived entirely TP reserves entirely from from depletion depletion of of phosphocreatine phosphocreatine and and A ATP reserves during during the the first first 30 ss of of activity. activity. Glycolysis, Glycolysis, as as evidenced evidenced by by increased increased lactic lactic acid acid production, began to be important during the second 30 s of activity AMP and activity and and during during the the period period of of recovery. recovery. AMP and ADP ADP concentra concentrations did not increase in response to ATP depletion, indicating the probable presence of a very active AMP deaminase. During recovery, phosphocreatine stores were the first to be replenished (67% (67%control values within 5 min, 80% 10 min). 80%within 10 min). ATP levels remained low for the first 5 min of recovery but had regained 80% 80%of the control value after 10 min. Wieser et al. (1985) after 10 (1985) compared the energy debts acquired during burst swimming (60 s) with energy liberated during recovery. (60 s) recovery, For yolk-sac larvae the A TP required to replenish body energy stores ATP V02) supplied by balanced well with the amount of excess excess energy (> (> rrV0,) anaerobic anaerobic and and aerobic aerobic processes processes during during recovery. recovery. Larger Larger fish, fish, how however, consumed progressively progressively more energy during recovery than was required to repay the true oxygen debt. debt. This was attributed to poststim poststimulus excitement. Energy production during burst activity has not been if significant reported for other species, but it would not be surprising if differences are found given the variations in patterns of muscle devel development and partitioning of energy between oxidative and glycolytic pathways that are known to occur (Forstner et al., 1983; 1983; Wieser et al., 1985; 1985; Hintleitner et al., 1987). 1987).
2. RESPIRATORY GAS EXCHANGE, AEROBIC AEROBIC METABOLISM 2.
143 143
V. C ONCLUSIONS CONCLUSIONS
general, the ontogeny of respiration is In general, is not as well understood for teleosts as for higher vertebrate classes. However, there are signs that the situation is improving, thanks in large part to technological advances that have made it easier to work with such small and delicate organisms. Culture techniques have improved considerably during the past decade, allowing species that previously were difficult to obtain to be reared conveniently in the laboratory. Polarographic elec electrodes, including microelectrodes microelectrodes,, have become readily available. Ad Advances in electronics have permitted the construction of of extremely sensitive microcalorimeters, while methods for assaying metabolite concentrations in small samples have improved greatly. 1969). Since The last major review of this field was that of of Blaxter ((1969). then, a number of important advances have been made. It is now clearly estabished that the boundary layer represents a major barrier to diffusion (Daykin, 1965; 1965; Wickett, 1975; 1975; Vogel, 1981). 1981). A start has Bere been made in recording P02 PO2 profiles within the respiring egg ((Berezovsky et al., 1979; Sushko, 1982; 1982; Diez and Davenport, 1987). 1987). Studies al., 1979; by Burgreen and co-workers (e.g., (e.g., Burggren, 1985) 1985) with amphibians and Liem ((1981) 1 981) with fish larvae have brought about a greater appreci appreciation of the sophistication of cutaneous gas-exchange structures. ReRe searchers have begun to examine the transition from cutaneous to branchial gas exchange (e.g., organ, 1974a,b; (e.g., De Silva, Silva, 1974; 1974; M Morgan, 1974a,b; Mc McDonald and McMahon, 1977) 1977) and from water breathing to air breath breathing (Hughes (Hughes et al., 1986) 1986) and the possible ecological implications of of these transactions (Iwai and Hughes, 1977). 1985) and others 1977). Iuchi ((1985) have provided details concerning the ontogeny of respiratory pig pigments in fish. fish. Information on energy partitioning during development is now available for a number of 1957; Laurence, of species (e.g., (e.g., Smith, Smith, 1957; 1969, 1977, 1978; 1978; Houde and Schekter, 1983; 1969, 1975, 1975, 1977, 1983; Gruber and Wieser, 1983; 1983; Rombough, 1987). 1987). Researchers have begun to examine the energetics of larval activity in detail, in terms both of of functional morphology (Webb 1986) and of energy production (Webb and Weihs, 1986) (Wieser, 1985; 1985; Wieser and Forstner, 1986; (Wieser, 1985; Wieser et al., al., 1985; 1986; Hinter Hinterleitner et al., al., 1987). 1987). The physiological and morphological responses of the early stages to hypoxia have been shown to be quite different from those of adult fish ((Holeton, Holeton, 1973; 1973; McDonald and McMahon, 1977). 1977). The idea of of using critical dissolved oxygen levels to predict oxygen requirements and sublethal response thresholds has been advanced (Rombough, 1986, 1986, 1987). 1987). Much Much work, though, remains to be done. Most of what is is known
144 144
PETER J. ROMBOUGH
about respiration during early life is derived from studies of only a few species. Teleosts are a diverse group, and comparative studies of the physiological and anatomical adaptations that permit survival in hy hypoxic environments environments are a necessity. Other potentially productive ar areas of research include studies of how P02 PO2 profiles within the egg change during development, the problem of gas transfer in egg masses, and the functional aspects of of the transition from cutaneous to branchial gas exchange. exchange. So far studies of this transition have been restricted to the structural aspects. The nature of the relationship be beVOZ2 and tissue mass during early life remains controversial, tween \1'0 and further investigations in this area would seem appropriate. Impor Important aspects of aerobic metabolism in very young fish, fish, such as power powerperfonnance performance relationships and the nature of endogenous rhythms, have received little attention. It would appear to be especially impor important to be able to relate metabolic rates measured in the laboratory conwith those that occur in the field. More research needs to be con ducted on the physiological, morphological, and metabolic responses of embryos and larvae to chronic hypoxia and the ecological signifi significance of such adjustments. Finally, there is the problem of of factor interactions. The recent development of of a general multivariate dose doseresponse model (Schnute and Jensen, 1986; al., 1986) 1986; Jensen et d., 1986) now would appear to make this problem more tractable.
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Mechanisms of hatching in fish: Secretion of of hatching enzyme and Yamagami, K. ((1981). 1981). Mechanisms 459-471. enzymatic choriolysis. Amer. Zool. 21, 21,459-471. Yamagami, 1984). Retardation of Yamagami, K. K.,, Yamamoto, M., Iuchi, I., and Taguchi, Taguchi, S. ((1984). of matura maturachanges of tion associated associated and secretion associated ultrastructural ultrastructural changes of hatching gland in the medaka embryos incubated in air. Annot. ZooLJap. Zool. Jap. 56, 266-274. 56,266-274. Yoshimizu, M., Kimura, T., T., and Sakai, 1 980). Microflora Microllora of Sakai, M M.. ((1980). of the embryo and fry fry of Bull. Jpn. Soc. 967-975. salmonids. salmonids. Bull.]pn. SOC. Sci. Sci. Fish. 46, 46, 967-975. Zeuthen, E. E. ((1950). 1 950). Cartesian diver respirometer. Bioi. Bull. Bull. (Woods Hole, Mass.) respirometer. Biol. Mass.) 98, 98, 303-318. 303-318. Zeuthen, E. E. (1970). (1970).Rate of of living as related to body body size in organisms. Pol. Arch. Arch. 2 1-30. Hydrobiol. Hydrobiol. 17, 17,21-30. Zoran, M. M. J., and Ward, J. 1983). Parental egg care, behavior J. A. ((1983). behavior and farming activity for the orange chromide Etroplus maculatus. Environ. Bioi. Biol. Fishes 8,301-310. 8, 301-310.
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3 OSMOTIC AND IONIC REGULATION IN TELEOST EGGS AND LARVAE
D.. FF.. ALDERDICE D of Fisheries and Oceans Department of Fisheries Research Branch Pacific Biological Biological Station Nanaimo, British Columbia, Canada V9R 5K6 5K6 I. Introduction I. II. Oogenesis 11. A. Nonteleosts B. Teleosts B. III. 111. Fertilization IV. Development Development A. Properties of of the Plasma Membrane B. Influence of of the Perivitelline Fluid and Zona Radiata C. First Cell Division to Beginning of Epiboly C. D. D. Epiboly to Hatching E. Transition to the Adult M echanism for Regulation Mechanism V. Conclusions A. Oocytes Oocytes B. Fertilization C. First Cell Division to Beginning of of Epiboly D. D. Epiboly to Hatching E. Transition to the Adult Mechanism for Hatching References References
I. INTRODUCTION The The fishes fishes have have evolved evolved to to occupy occupy all all but but aa few few types types of of natural natural waters waters ranging ranging from from low low ionic ionic strength strength ("near-distilled") (“near-distilled”) fresh fresh waters waters to those with 1964; Parry, 1966; Grif to those with salinities salinities of of 80-142.4%o 80-142.4% (Kinne, (Kinne, 1964; Parry, 1966; Griffiths, 1974). fiths, 1974). Some Some are are restricted restricted to to narrow narrow ranges ranges of of salinity salinity (stenoha (stenohaline); line); others tolerate broad ranges (euryhaline). (euryhaline). Some Some spend a major part part of of their their pre-adult pre-adult lives lives in in fresh fresh water water and and migrate migrate to to spawn spawn in in the the 163 163 FISH PHYSIOLOGY. PHYSIOLOGY. VOL. VOL. XIA XIA
Copyright Copyright © B 1988 1988 by by Academic Academic Press. Press, Inc. Inc. All rights rights of reproduction reproduction in in any any fform reserved. All orm reserved.
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D. D. F. F. ALDERDICE ALDERDICE
sea begin life sea as sea (catadromous); (catadromous); others others begin life in in fresh fresh water, water, go go to to sea as juve juveniles, niles, grow grow and and mature, mature, and and return return to to spawn spawn in in fresh fresh water water (anadro (anadromous). mous). Some Some marine marine forms forms spawn spawn in in estuarine estuarine or or coastal coastal habitats habitats of of reduced salinities, or in oceanic oceanic salinities, or reduced salinity; salinity; others others spend spend their their entire entire life in in also is is much variin fresh fresh water. water. There There also much variation variation within within species. species. The vari ous ous developmental developmental states-the states-the fertilized fertilized egg, egg, larva, larva, juvenile-may juvenile-may have have particular particular salinity salinity optima, optima, including including those those catadromous catadromous and and anadromous anadromous forms forms where where relatively relatively rapid rapid changes changes occur occur in in salinity salinity tolerance juvenile metamorphosis tolerance with with juvenile metamorphosis or or prespawning prespawning migration. migration. Some uids Some fishes fishes are are osmoconformers osmoconformers (poikilosmotic) (poikilosmotic);; their body fl fluids tend tend to to follow follow and and conform conform with with changes changes in in osmotic osmotic properties properties of of the the external Teleost fishes (homo external medium. medium. Teleost fishes generally generally are are osmoregulators osmoregulators (homoiosmotic); iosmotic); their body body fluids remain remain relatively relatively constant constant with with altera alteration of the external medium. Many teleosts regulate in the central portion of their ranges of of tolerance and conform at the extremities. Hyperosmotic regulators (most freshwater teleosts) maintain body bodyfl uid concentration Con fluid concentration above above that that of of their their external external surroundings. surroundings. Conversely, hypoosmotic hypoosmotic regulators (most marine versely, regulators (most marine teleosts) teleosts) maintain maintain body body fl uid concentration medium. Osmoconfor fluid concentration below below that that of of the the external external medium. Osmoconformers wider varition mers tolerate tolerate wider varition in in internal internal concentration; concentration; osmoregulators osmoregulators tolerate wider range of external external concentration concentration (Prosser, (Prosser, 1973). 1973). tolerate aa wider range of Although Although the the terminology terminology associated associated with with osmosis osmosis is is thoroughly thoroughly discussed 1959a; Potts discussed in in most most fundamental fundamental texts texts (Dick, (Dick, 1959a; Potts and and Parry, Parry, 1965; Florey, 1966), 1964; 1964; Dainty, 1965; 1966), some basic terms are presented here. Osmoticity is a general term referring nonspecifi cally to the nonspecificalIy osmotic osmotic properties properties of of aa solution. solution. Osmotic Osmotic concentration concentration defines defines the the number of nonpermeating nonpermeating particles per unit volume. The difference between solutions separated separated by by aa between the the osmotic osmotic concentrations concentrations of of two solutions semipermeable membrane is referred to as an osmotic gradient. Water tends flow by membrane toward tends to to flow by osmosis osmosis across across aa membrane toward the the solution solution of of higher higher osmotic osmotic concentration concentration (osmoconcentration). (osmoconcentration). The The osmotic osmotic activ activity is defined molar concentration ity of of aa solution solution is defined as as the the molar concentration of of an an ideal ideal nonelectrolyte solution having the same osmotic effects. An osmole is noneIectrolyte solution having the same osmotic effects. An osmole is the amount of solute that exerts the same osmotic pressure as 1 mole of same osmotic pressure as 1mole of the amount of solute that exerts ideal same volume. volume. An An osmotic osmotic gradi gradiideal nonelectrolyte nonelectrolyte dissolved dissolved in in the same ent generates an osmotic pressure (7T) that ultimately is just sufficient sufficient ent generates an osmotic pressure (r)that ultimately is just to prevent further osmotic flow (flux) (flux) across a membrane. A 11 molar 22.4 (osmolar) solution of of nonelectrolyte has an osmotic pressure of of 22.4 (osmolar) atm atm (at DoC) OOC) and depresses the freezing point (d) (A) of the solution by mOsm/l) con conof 11 Osm/l ((1000 11.86"C. .86°C. A solution with an osmolarity of 1000 mOsm/l) tains 11 gram molecular weight (gmw) (gmw) of solute in 11 I1 of solution. A solution with an osmolality of 1000 mOsm/kg) contains a of 11 Osmikg Osmkg ((1000
3. 3.
OSMOTIC AND IONIC REGULATION REGULATION IN TELEOST EGGS OSMOTIC AND IONIC IN TELEOST EGGS AND AND LARVAE LARVAE
165 165
of solvent (water). (water). Isotonicity is a state of gmw of solute per kilogram of volume equilibrium in which a cell bathed in an aqueous solution neither shrinks nor swells. swells. The The primary primary basis basis for for regulative regulative capacity capacity can can be be considered considered as as the the semipermeable membrane of the individual cell and its tolerance and response to external and internal osmotic and ionic alteration. A sec second ond level level of of regulative regulative capacity capacity is is provided provided in in the the development development of of particular tissues and effector organs, whose cells have specific regu regulatory level, in third level, in the the intact intact animal, animal, involves involves neurose neuroselatory functions. functions. A third cretory cell, tissue, tissue, or cretory activity, activity, which which tends tends to to modulate modulate cell, or organ organ func functions governing regulation. Osmotic and ionic regulation during early development of teleosts then poses certain questions questions:: What are the mechanisms that may provide regulative capacity in the the egg, egg, embryo, embryo, and and larva? larva? What identified in in particular particular spe speWhat patterns patterns of of regulation regulation can can be identified cies? cies? When, When, or or at at what what developmental developmental states, states, do do these these regulatory regulatory mechmechanisms anisms become become functional? functional? The first first question question has has received received much much attention attention often often from from studies studies of of oocytes, fertilized eggs, eggs, juveniles, or adults of nonteleosts. A wealth of information has been gathered on structure and function of individual mechanisms, yet a full understanding of their integrated action in the intact intact animal animal seems seems near near but but remains remains elusive. elusive. With With respect respect to to the the second 1979, 1980) second question, question, Evans Evans ((1979, 1980) suggests suggests that that there there are are three three general s hes. One general patterns patterns of of regulation regulation among among the the fi fishes. One is is seen in in the the myxinid myxinid (hagfish) (hagfish)agnathids, agnathids, who who show show electrolyte electrolyte iso-osmolarity iso-osmolarity with with sea water. water. Renal ion regulation is important, and some some ion excretion may occur in the integumental slime. slime. The role of the branchial epithe epithe(1984a) offers offers intriguing lium is not fully understood, although Evans (1984a) Na+/H+ and CI-/HC0 Cl-/HC03systems, evidence that parallel Na+/H+ 3 - exchange systems, NaCl regulation in freshwater vertebrates, generally associated with NaCI are found in the branchial epithelium of these early marine verte verteelasmobranchs; the main effec effecbrates.. A second pattern involves the elasmobranchs; brates epithelium, the renal complex, complex, and the tor organs are the branchial epithelium, extrarenal rectal gland. gland. In the elasmobranchs the blood level level of NaCI NaCl extrarenal is is below below that that of of the the external external medium, medium, but but fluid fluid levels levels are are maintained maintained triisosmotic or somewhat hypertonic by the retention of urea and tri methylamine oxide. oxide. In a third pattern, pattern, found in the teleosts, the prin principal cipal effector effector organs organs are are the the branchial branchial epithelium, epithelium, the the renal renal complex, complex, the the gut, gut, and and (Marshall (Marshall and and Nishioka, Nishioka, 1980) 1980) the the integument. integument. Similari Similarities ties with with the the teleost teleost patterns patterns are are found found in in the the chondrosteans chondrosteans (stur(stur-
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D. F. F. ALDERDICE
geons), (gars, bowfin), petromy geons), holosteans holosteans (gars, bowfin), dipnoans (lungfish), (lungfish), and and petromyzonid (lamprey) (lamprey) agnathids. agnathids. These These patterns patterns refer refer entirely to to the the juvenile or adults stages, which brings the third question, basic to this chapter. What processes or tissues have a regulatory function in the egg and embryo, what are the effector mechanisms, and when do they become functional during development? In considering this question, attention will be directed to the te teleosts. leosts. Where relevant data are unavailable, insights will be sought among the nonteleosts. A second constraint arises from the fact that few studies have been conducted on the chronology of development of prejuvenile regulatory processes. Instead, a variety of of species and a limited number of development states have been used as a conse consequence either of their local availability, their usefulness in approach approaching a particular question posed, or because of the tradition of of particu particular laboratories. Indeed, initial attention has been centered, with good reason, on a search for understanding of regulatory mechanisms and on the development of improved technique for their examination, not on the order and timing of regulatory events during development. development. Studies of osmoosmo- and ionoregulation have arisen from the work in the last century ofVan't of Van’t Hoff, Arrhenius, and De Vries, Vries, who described relationships between the gas laws and osmotic pressure. In the inter interval to date, an enormous body of literature on cell, tissue, and whole wholeanimal regulation and regulatory mechanisms has been published, and the basis for enquiry has expanded into a number number of diverse but related fields of study. For these reasons much use has been made in this chapter of reviews on regulation that have appeared in the past few decades 1964) tends to integrate the decades.. That of Potts and Parry ((1964) earlier work. work. More specific considerations of regulation reguIation are found in Parry ((1966), 1966), Potts ((1968, 1968, 1976, 1 969), Hagiwara and 1976, 1977), 1977), Holliday ((1969), Jaffe ((1979), 1979), Folmar and Dickhoff ((1980), 1980), Heisler ((1980, 1980, 1982), 1982), Evans ((1980, 1980, 1984b), al. ((1982), 1 982), Eddy ((1982), 1982), Hoar and Randall 1984b), Evans et al. ((1984), 1984), and Metz and Monroy ((1985). 1985). An appreciation of the chronol chronology of development of the theory of particle transport is provided by Florey ((1966), 1 966), Katchalsky 1965), and Friedman ((1986). 1 986). Katchalsky and Curran ((1965), These were used to establish a perspective and to provide a frame frame“at what stage and by what ask, more specifically, "at work from which to ask, means is regulation achieved in the embryonic teleost?" teleost?” An early decision was made to provide an overview of the subject, rather than a review. The decision was based on the number of ex expanding and diverging areas of enquiry relevant to the subject, the enormous size of the literature, the rapidity with which enquiry is moving, and the variability apparent in the different animal groups
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LARVAE OSMOTIC AND IONIC IONIC REGULATION IN TELEOST EGGS AND LARVAE
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teleosts.. under investigation that could contribute to understanding in teleosts regulaMajor advances occurring in recent years in knowledge of regula tory processes are rooted in biochemistry, biophysics, and molecular probiology. Of numerous technological advances contributing to this pro gress, those of electron microscopy, use of isotopes, and most recently electrophysiology and cytochemistry provide outstanding examples. applicaAspects of the foregoing areas of inquiry will be drawn on, as applica ble, in the following sections on regulatory processes in various devel developmental states of fishes. fishes. Attention will focus primarily on biological influence mechanisms that could infl uence developmental events elicited through changes in membrane permeability at the cellular level. Where evidence for teleosts is sparse, comparisons will be attempted mamusing available information on invertebrates, amphibians, and mam mal s mals. a
II. 11. OOGENESIS
In the animal kingdom there are four general classes involving differing differing temporal temporal relationships relationships between between maturation maturation of of the the female female gamete Moreau et al., 1985; 1985). Hence, 1985; Masui, Masui, 1985). Hence, gamete and and fertilization fertilization ((Moreau defining there is a logical problem in defi ning the term oocyte. Of the four classes, classes, class I includes representatives from the nematodes, bivalve molluscs, and echiuroids, and insemination normally occurs at the (GVBD).In class end of prophase prior to germinal vesicle breakdown (GVBD). oo11, including representative annelids, molluscs, and ascidians, the oo II, cyte cyte normally normally is is inseminated inseminated at at metaphase metaphase I. I. In In class class III, 111, which which in inmetacludes Amphioxus and most vertebrates, insemination occurs at meta II following extrusion of the first polar body. In class IV, phase I1 including representative coelenterates and sea urchins, insemination occurs completion of second polar occurs at at completion of telophase telophase and and extrusion extrusion of of the the second polar body. classes is is arrested, arrested, respectively, respectively, at at body. In In effect, effect, meiosis meiosis in in the the four four classes the four stages indicated. Notwithstanding the fact that insemination normally stimu normally occurs occurs at at different different stages stages of of reductive reductive division, division, if if that that stimulus removes the the block block to to completion completion of of meiosis meiosis and and the the oocyte oocyte be becomes comes fertilizable, fertilizable, then then the the fertilizable fertilizable oocyte oocyte will will be be called called an an egg. egg. process by Activation is is the the process by which which an an arrested arrested oocyte oocyte (at (at prophase, prophase, metaphase II, or telophase) telophase) resumes resumes development development following following in inmetaphase II or or 11, semination, semination, or or some some other other physical physical or or chemical chemical stimulus. stimulus. Fertilization refers refers more more precisely precisely to to the the fusion fusion of of pronuclei pronuclei following following insemina insemination. tion. It appears that transmembrane shifts in ion concentration and hor-
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D. F. ALDERDICE ALDERDICE
of events leading to resumed mones act as triggers, stimulating a chain of of meiosis. Electrophysiological Electrophysiological oocyte development and completion of particularly useful in examining and cytological studies have been particularly these events. In these investigations, substantial changes may be seen in the resting and action potentials of of the oocyte plasma membrane. of influx or efflux of gener These signal sudden changes in rates of of ions, ions, generCa2 +, K+, K+, Na+, Na+, and C1-, CI-, resulting from the electrical ally involving Ca2+, concentra gradients generated in association with the established ion concentration gradients across the membrane. The sudden shifts in membrane of the potential are consistent with rapid changes in the permeability of oocyte plasma membrane to particular permeation particular ions. The ion permeation or pathways mechanisms or pathways through which these transmembrane fluxes occur in response to particular stimuli are described as as channels. MorMor phologically, these channels behave like protein protein structures with a 1984; Miller et ul., al., 1984). 1984). They may be central pore (Barry and Gage, 1984; "gated," gradi “gated,” opening and closing in response to transmembrane ion gradior by membrane depolarization (Heinz and Grassl, 1984; 1984; ents or Loewenstein, 1984). 1984). However, while electrically excitable channels, demonstrating action potentials, have been described in oocytes of of tunicates, annelids, molluscs, echinoderms, amphibians, and mammam mals, they have not been unequivocally demonstrated in teleost eggs. Documented evidence on the electrical excitability of of teleost oocytes and eggs is sparse; sparse ; the medaka egg (Oryzias (Oryzias Zatipes) latipes) is said to be 1979). electrically inexcitable (Hagiwara and Jaffe, 1979). A. Nonteleosts In starfish oocytes (Kanatani (Kanatani and Nagahama, Nagahama, 1980; 1980; Kanatani, Kanatani, 1985), 1985), development of the oocyte is is arrested in late prophase; then, just before spawning occurs, meiosis resumes, GVBD occurs, and devel development proceeds to metaphase II. 11. A first mediator initiating this re reof meiosis is the hormone gonad-stimulating substance sumption of (GSS), coelomic fluid. fluid. GSS acts on the ovary to produce (GSS), present in the coelomic a second mediator, maturation-induCing maturution-inducing substance (MIS), (MIS), identified as I-methyladenine I-MeAde). MIS MIS stimulates the production of a l-methyladenine ((l-MeAde). maturation-promoting maturution-promoting factor (MPF) (MPF) in the oocyte cytoplasm cytoplasm (Kana (Kanatani and Nagahama, Nagahama, 1980), 1980),and MPF production is is amplified by germi germinal vesicle material. cation produces GVBD, material. MPF amplifi amplification GVBD, oocyte mat maturation, and a fertilizable egg. MPF activity is is considered to be 2 + -dependent and Ca 2 + -sensitive. Absence of Ca 2 + delays GVBD Mg Mg2+-dependent Ca2+-sensitive. Ca2+ in MIS-induced oocyte maturation. A transient stimulation of an Na+ Na+
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-K+ electrogenic pump and increased passive Na+ Na+ permeability also -K+ 1981) in oocytes treated with MIS. It was concluded are noted (Dorke, (Don�e, 1981) 2 + ] . MIS [Ca2+]. that these events are regulated by a transient increase in [Ca 2 Ca2+. considerappears to act by releasing internally sequestered Ca + . In consider ing the resting potential of an oocyte, it must be remembered that modified ionic composition of the oocyte cytoplasm will be modifi ed by that of itself will be influenced by the compo compothe parental body fluid, which itself environment. The starfi starfish sition of the external environment. sh oocyte in seawater has -60 to -70 (Hagiwara and Jaffe, 1979). 1979). The a resting potential of -60 70 mV (Hagiwara K+; Na+ or C1perplasma membrane is predominately permeable to K+ ; Na+ CI- per meability is negligible. The resting potential is close to the Nernst K+ activity. In various species in the starfish estimate on the basis of K+ K+ permeability, or a decrease group there appears to be a decrease in K+ Na+,as maturation proceeds. In the with respect to other ions such as Na+, sea urchin egg the resting potential ranges between 60 and -60 -80 mV, 80 mY, Kf-dependent. Significantly, and it also is K+ -dependent. Signifi cantly, in their review Hagiwara memJaffe ((1979) and Jaffe 1 979) note that conductance of the oocyte plasma mem brane always decreases during oocyte maturation. In K+ ionophore ionophore valinomycin valinomycin was was found found to to In the the amphibians, amphibians, the the K+ K+, trigger GVBD in the Xenopus oocyte in the absence of external K+, which concommitantly resulted in a sharp reduction in intracellular [K+] GVBD is is accompa 1985). In In amphibian amphibian oocytes, oocytes, GVBD accompa[K+](Moreau (Moreau et al., 1985). (Lessman and Marshall, nied by marked membrane depolarization (Lessman 1984), reduction in number of ouabain-binding ouabain-binding sites, and markedly 1984), K+ and CIC1- ions (Weinstein et aI., al., reduced membrane conductance to K+ 1982). 1985) it 1982). In In other other studies studies reviewed reviewed by by Moreau Moreau et al. ((1985) it was was con coninflux in K+ deprivation, or reduction of active K+ K+ influx cluded that external K+ ouabain-treated oocytes, facilitated progesterone-induced maturation (Vitto al., (Vitto and and Wallace, Wallace, 1976; 1976; Wallace Wallace and and Steinhardt, Steinhardt, 1977; 1977; Kofoid Kofoid eett aI., 1979). 1979). In In addition, addition, amiloride, amiloride, which which blocks blocks Na+ Na+ channels channels in in the the oo oocyte reinitiation of meiosis cyte plasma plasma membrane, membrane, prevented prevented reinitiation meiosis in in arrested arrested oocytes. 1979), external oocytes. In In another another study study (Robinson, (Robinson, 1979), external current current was was found found to to enter the the oocyte oocyte at at the the animal animal pole pole and and exit exit at at the the vegetal vegetal pole. This This current, CI- ions, pole. current, associated associated primarily primarily with with movement movement of of C1ions, 2 + . Tracer appeared appeared to to be be regulated regulated by by Ca Ca2+. Tracer experiments experiments with with Xenopus oocytes (O'Connor et al., 1977) 1977) have shown a sustained increase in 2 + uptake, Ca maturaCa2+ uptake, which which decreased decreased before before GVBD GVBD was was blocked blocked by matura 2 + efflux tion-inhibiting There was rapid, consistent tion-inhibiting drugs. drugs. There was aa rapid, consistent Ca Ca2+ efflux and and aa parallel K+ K+ efflux; efflux; it was concluded that progesterone treatment of the 2 + ] . Amphibian oocyte oocyte resulted in an increase in internal [Ca [Ca2+]. membrane resting potentials, in appropriate Ringer's solution (Hagi (Hagi1979), are -50 -70 wara and Jaffe, 1979), 70 mV and the potential is 50 to -
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K+ -dependent, although Nernst estimate. Oocytes K+-dependent, although it it departs departs from from the Nernst estimate. Oocytes isolated increased negative potential, isolated from from follicular follicular tissue tissue showed showed an an increased negative potential, which some part which was was sensitive sensitive to to ouabain. ouabain. Hence, Hence, some part of of the the negative negative potential electrogenic, ac potential in in the the preparation preparation appeared appeared to to originate originate in in electrogenic, active tive ion ion transport. transport. Amphibian Amphibian oocytes oocytes treated treated with with progesterone progesterone showed decrease in 10 to showed aa decrease in membrane membrane potential potential to to aa final final value value of of -10 to -40 V, suggesting increase in -40 m mV, suggesting aa progressive progressive increase in ion ion permeability permeability of of the the plasma plasma membrane membrane coincident coincident with with progressive progressive oocyte oocyte maturation. maturation. Action Action potentials potentials also also provide provide information information on on the the presence presence of of ion ion channels. channels. In In echinoderms, echinoderms, two types types of of ion ion channel channel have have been been noted noted in in starfish starfish oocytes oocytes (Hagiwara (Hagiwara and and Jaffe, J&e, 1979). 1979). The The properties properties of of one one are are similar similar to to those those of of Ca2+ Ca2+channels. channels. The The second second type type differs differs from from the the first however, it first in in electrophysiological electrophysiological properties properties;; however, it appears appears to to be be aa special Ca2+ channel, special form form of of Ca2+ channel, the the current current being being carried carried by by Ca2+. Ca2+.A Ca2+ -dependent action probably analogous Ca2+-dependent action potential, potential, probably analogous to to the the special special Ca2+ is found sea urchin Ca2+ channel channel of of the the starfish, starfish, is found in in the the sea urchin egg. egg. With With reference K+ channels reference to to K+ K+ permeability, permeability, three three types types of of K+ channels have have been been identified in the oocytes of K+ channel, outward of starfish: starfish: the inward K+ K+ prop K+ channel, channel, and and the the fast-inactivating fast-inactivating K+ K+ channel. channel. The The electrical electrical properties of these erties these channels channels appear appear to to vary vary with with species species and and development development state. state. In In the first first two types types the the current current is is carried carried by by K+ K+ ions; ions; however, however, the respond the channels channels appear appear to to differ differ in in molecular molecular structure, structure, and and they they respond differently differently to to variations variations in in external external conditions. conditions. In In the the latter latter type, type, when when the the membrane membrane potential potential is is held held at at aa level level somewhat somewhat more more posi positive tive that that the the resting resting potential, potential, the the channel channel is is fully fully inactivated. inactivated.
B. Teleosts One is impressed by the paucity of of investigative data on teleost oocytes, particularly particularly on the processes that may parallel those in the invertebrates and amphibians. Among the chordates, Masui ((1985) 1 985) of meiotic arrest in oocytes (meta (metalists two fishes in which the stage of phase II) 11) is known: Catostomus (Lessman and Huver, AciHuver, 11981) 981) and Aci penser. 1 958) recorded penser. Yanagimachi Yanagimachi ((1958) recorded the same stage in meiotic arrest Pacific herring, Clupea pallasi. in the Pacific pallasi. The resting potential of the Oryzias 40 mV (Ringer's Oqzias latipes oocyte probably is in the range of -40 (Ringer’s solution) to -50 (0.1 Ringer’s) (Hagiwara (Hagiwara and Jaffe, Jaffe, 1979). 1979). Ion Ionsolution) 50 mV (0. 1 Ringer's) substitution experiments showed a membrane potential dependent on K+ and Na+, Na+, but independent of of Ca2+ Ca2+or CI-. C1-. Although Na+ Na+ channels, K+ several types of Ca2+ Ca2+ channel, and three types of K+ K+ channel have been identified, their role in ion transport across across the plasma mem-
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of teleost oocytes is relatively unknown. Hagiwara and Jaffe brane of ((1979) 1979) speculate that during oocyte and egg development various ion channels may be created, eliminated, or localized; some may be in inserted or removed while others may be present continually. Salvelinus fontinalis, Marshall et al. In a study of the brook trout Saivelinus ((1985) 1985) compared the electrophysiological characteristics of of oocytes, eggs. Control separated from follicular tissue, with those of ovulated eggs. genninal vesicle present were compared with those oocytes with the germinal 17a-20/3-dihydroxyprogesterone (DHP) to promote treated with 17 a-20,B-dihydroxyprogesterone (DHP) GVBD. Cortland's saline and examined in GVBD. The gametes were stored in Cortland’s various modifications of Ringer's Ringer’s solution, all having a constant osmo osmolality of 310-320 310-320 mOsm/kg. mOsm/kg. The electrical characteristics at the three stages of maturation are shown in Table I. I. Compared with oocytes in which the germinal vesicle was present, there was a significant reduc reduction in membrane potential in the ovulated eggs. On the same basis, membrane resistance was significantly higher in the oocyte after GVBD, ovulation. Ion conductance GVBD, and was greatly increased after ovulation. before and after GVBD GVBD was approximately constant, while K+ K+ and Na+ Na+ conductances decreased substantially with ovulation. There was no evidence of change in Clevidence C1- conductance. Treatment of control oocytes in low-Na+ high-K+ Ringer's low-Naf and high-K+ Ringer’s solutions resulted in decreased Rrn R, (membrane resistance) suggest (membrane resistance) and Vrn V, (membrane (membrane potential) values, suggesting that depolarization induces conductive pathways. Significantly, Significantly, one explanation for this effect would be a voltage-dependent voltage-dependent conduc conductance; electrode limitation prevented prevented confirmation confirmation of of this possibility. The study showed that GVBD GVBD and ovulation increased membrane TABLE TABLE II E lectrical Characteristics Electrical Characteristics (Mean (Mean :±: % SE) SE) of of Oocytes and Ovulated Ovulated Eggs of of Salvelinus Saluelinus Plasma M embrane Potential (V), fontinalis: Membrane (V), Membrane Membrane Resistance (R), ( R ) ,and fontinalis: Plasma of Change in Ion Concentration, Concentration, �C)a AC). (mV per Decade of Conductance (mV
V V Treatment Treatment
(mV) (mV)
Denuded oocytes GV presenti> presentb GV absent Ovulated Ovulated eggs
-46.3 .2 -46.3 ± 2 11.2 -41.5 ± f 3.0 3.0 -38.1 .8 -38.1 ± f 11.8
a From Marshall et al. al. ((1985). 1985). b GV, GV, Germinal Germinal vesicle. vesicle.
R 2) (kn (kfl cm cmz)
*
Conductance Conductance CIR C/R (mV per decade �C) AC) (mV K-
Na+ Na+
26.5 .7 29. 1 :±: 26.5 :±: 11.7 29.1 t 2.9 2.9 13.5 13.5 :±: f 5.7 5.7 40.5 1.2 12.4 40.5 :±: t 7.0 7.0 29.5 29.5 ± t 111.2 12.4 :±: t 4.5 4.5 1640 1640 ± f 150 150 7.1 7.1 ± & 1.8 1.8 0.5 0.5 ± t 0.2 0.2
CIc1-
t 0.2 0.2 2.7 ± 2.6 2.6 :±: f 0.7 0.7 2.3 2.3 ± t 0.4 0.4
172 172
D. D. F. F. ALDERDICE ALDERDICE
resistance, decreased membrane con resistance, decreased membrane current, current, and and that that Na+ Na+ and and K+ K+ conductances decreased with ductances and and membrane membrane depolarization depolarization decreased with ovulation. ovulation. Hence, as salmonid, the Hence, as aa representative representative salmonid, the mature mature egg egg of of the the brook brook trout trout should be relatively impermeable to K+ Na+ ions prior to fertiliza K+ and Na+ fertilization. tion. In general, the presence of the germinal vesicle in late prophase and rst metaphase and its its breakdown breakdown in in fi first metaphase appears appears to to trigger trigger aa chain chain of of maturation-inducing hormones, and and accompanied accompanied maturation-inducing events events mediated mediated by hormones, by by ion ion fluxes. fluxes. Most Most of of the the latter latter accompany accompany shifts shifts in in membrane membrane electri electrical cal activity activity and and signal signal changes changes in in plasma plasma membrane membrane permeability permeability to to particular particular ions. ions. Changes Changes in in membrane membrane potentials potentials may may not not necessarily necessarily be they may may be side effects effects be instigators instigators of of maturation-inducing maturation-inducing events events :: they incidental incidental to to the the process. process. Nevertheless, Nevertheless, membrane membrane potential potential and and per per2 +, K+, meability meability changes changes in in oocytes oocytes are are associated associated with with fluxes fluxes of of Ca Ca2+, K+, 2 + ions Na+, Cl-. In Na+, or or C1-. In amphibians amphibians Ca Ca2+ ions seem seem to to control control the the resting resting potential and ClK+ and Na+ Na+ permeabilities. C1- currents, as well as K+ 2 + re Moreau 1985) favor favor the the hypothesis hypothesis that that intracellular intracellular Ca Ca2+ reMoreau et al. ((1985) lease is lease is the the primary primary trigger trigger for for following following events. events. In In the the starfish starfish oocyte, oocyte, hormone -K+ electrogenic hormone application application induces induces Na+ Na+-K+ electrogenic pump pump activity, activity, 2 + release, which pump activation which itself itself results results from from Ca Ca2+ release, although although pump activation is is not not necessary necessary for for restarting restarting meiosis. meiosis. Many Many egg egg membranes membranes in in Ringer's Ringer’s solution have K+ -dependent resting potentials (approximately -70 -70 K+-dependent mY); mV); others have have smaller smaller potentials potentials and and relatively relatively nonspecific nonspecific ion permeability. course, also also reflect reflect conditions conditions permeability. Membrane Membrane potentials, potentials, of course, in being one in the the external external medium-the medium-the latter latter being one terminal terminal in in the the ion ion current-voltage relation for the starfish gradient. The steady-state current-voltage (Mediaster (Mediaster aequalis) aequalis) oocyte oocyte membrane membrane at at the the resting resting potential potential (0 (0 /LA pA 50 mV M membrane membrane current) current) (Hagiwara (Hagiwara and and Jaffe, Jaffe, 1979) 1979) was was -50 mV in in 25 m mM K+ M K+) K+) seawater. 73 mV K+ seawater seawater and and about about -73 mV in in normal normal (10 (10 m mM seawater. In In terms of of [H+], [H+],Peterson and Martin-Robichaud (1986) (1986) found the (well (well(Salmo salar) salar) in fresh water to have a eyed) eggs of Atlantic salmon (Salmo -65 mV -50 mV 3.5.The membrane potential of 65 m V near pH 6, 6, and V at pH 3.5. 50 m experimensmaller negative membrane potentials frequently found experimen 10 to -20 -20 m mV) artifacts, resulting from leaks around the tally ((-- 10 V) may be artifacts, (1979) sug sugimpaling microelectrodes. However, Hagiwara and Jaffe (1979) relagest these could be real in certain instances, resulting from the rela tively nonselective properties of a membrane in freshwater eggs in (1986) found that the natural state. Peterson and Martin-Robichaud (1986) “eggs” in fresh water the membrane potential for Atlantic salmon "eggs" -70 mV, which would argue against that suggestion. suggestion. 70 mY, would be near “gated”: that is, Ion channels in the plasma membrane may be "gated": they may open and close in response to certain stimuli. stimuli. Horn (1984) (1984)
3. 3.
LARVAE OSMOTIC AND IONIC IONIC REGULATION REGULATION IN TELEOST EGGS AND LARVAE
173 173
properties:: (1) lists such stimuli and related channel properties (1) transmembrane potential-channels may open more frequently or stay open longer at potential-channels (2)neurotransmitters and drugs may open or voltages;; (2) hyperpolarized voltages (3) after being activated, channels tend to desensitize close channels; (3) (4) open time may be influenced by the type and close spontaneously; (4) 5 ) gating speed is temperature-dependent, of permeant ion present; ((5) of involving conformational changes in membrane-bound proteins; and (6) single channels tend to gate very rapidly, the open-to-closed transi transi(6) <10 ps. tion being < 10 ILS. As alluded 1979) suggest alluded to to earlier, earlier, Hagiwara Hagiwara and and Jaffe Jaffe ((1979) suggest that that ion ion channels may be created or eliminated as required during develop development, ment, and and that that they they may may be be present present in in the the membrane membrane for for future future use use after 1983). This This reasoning reasoning may may be be ap apafter oocyte oocyte maturation maturation (Hagiwara, (Hagiwara, 1983). plied (On c salmon (Onplied to a current problem in the manipulation of Pacifi Pacific corhynchus). corhynchus). These anadromous species often are captured on their final approach to fresh water and held in saltwater pens, to be stripped final of when ripe, ripe, for Normally, the of gametes gametes when for hatchery hatchery culture culture of of the the eggs. eggs. Normally, the adults would enter adults would enter fresh fresh water water during during the the last last stages stages of of gamete gamete matu maturation ration.. Held Held in in sea sea water, water, the the adults adults may may suffer suffer substantial substantial mortality; mortality; the lower fertiliz the gametes gametes of of the the survivors survivors show show highly highly variable variable but but lower fertilizability. salmon (0. (0. keta), keta), about about to to enter enter fresh fresh ability. In In the the female female chum chum salmon water to spawn, the osmolality and "a+] [Na+] of the blood plasma and ovarian 340-350 mOsm/kg mOsm/kg (Na+ (Na+ 170170ovarian fluid fluid were were very very similar, similar, about 340-350 mM/1) (Hirano (Hirano et al., 1978; 1982; Stoss Stoss and Fagerlund, 180 mM/I) 1978; Lam et al., 1982; 180 1982). 1982). In In fresh fresh water, water, these values values declined declined to to 295-300 295-300 mOsm/kg mOsm/kg (Na+ 140-160 mMIl) mM/1).. With With chinook chinook salmon salmon (0. (0.tshawytscha) held held in in (Na+ 140-160 1981) found found that that blood blood plasma plasma of of the the females females seawater, Sower Sower ((1981) . seawater, 415-430 (Na+200 meq/l) meq/l) during oocyte reached maxima of 4 1 5-430 mOsm/kg (Na+ maturation, declining to about 390 mOsm/kg (Na+ (Na+ 175-180 175-180 meq/l) meq/l) at ovulation. ovulation. In fresh water, blood plasma osmolality and [Na+] "a+] declined to 305 mOsm/kg (Na+ (Na+ 136 136 meq/l) during oocyte maturation and were 320 mOsm/kg (Na+ (Na+ 144 144 meq/l) meq/l) at ovulation. ovulation. The comparison suggests that the female Pacific salmon, held in seawater at ovulation, probably 40-50 mOsm/kg fluid carries body-cavity eggs bathed in ovarian fl uid about 40-50 (Na+ 25-30 mM/1) mM/I) above natural 1982) made natural levels. levels. Lam Lam et al. ((1982) made recip recip(Na+25-30 rocal crosses between seawater- and freshwater-held males and fe females. Fertilization success was higher in crosses between fresh freshwater-held there was was aa 30- to to water-held fish; fish; in in crosses crosses of of seawater-held seawater-held fish, there 40-fold and embryonic 40-fold increase increase in in abnormal abnormal embryonic embryonic development, development, and embryonic survival lower. Sower 1981) concluded concluded that that salmon salmon held held in in sea seasurvival was was lower. Sower ((1981) water unable to medium while water were were unable to adapt adapt to to that that medium while undergoing undergoing matura maturation noted aa strong strong correlation tion and and ovulation. ovulation. She She noted correlation between between elevated elevated
174
D. F. ALDERDICE
[Na+] and osmolality and increased adult mortality. When ovulation “a+] al. (1982) (1982) concluded that was induced in seawater, Sower et al. that hormonal of osmoregulatory difficulties in the responses were reduced because of ( 1 984) sums up the problem, suggesting that adults. Wertheimer (1984) degenera salmon maturing in seawater seem to be in a race between degeneraosmoregulatory capacity with resultant loss of of reproductive reproductive tion of osmoregulatory of maturation of viable gametes competence or death, and completion of maturation of with resultant successful spawning. When the ovulated female is held in seawater, the eggs apparently are unable to cope with their their hy hyperosmotic ovarian fluid medium. Hence, the ovulated egg appears to depend on the adult for osmotic control of of its immediate environment, and could be limited in its capacity to regulate ion or water flows across the plasma membrane. The plasma membrane of of the mature salar) egg is highly permeable to water. On Atlantic salmon (Salmo (Salmo salar) the other hand, Marshall et al. 1985) noted signifi cant reductions in al. ((1985) significant Na+ Na+ and K+ K+ conductance in the plasma membrane of of the maturing brook trout (Salvelinus (Saluelinus fontinalis) fontinalis) egg compared to the oocyte after GVBD. GVBD. Therefore, lower fertilizability and increased abnormal em embryonic development in eggs of Pacific salmon (Oncorhynchus) (Oncorhynchus) fe females held in seawater could be a result of of increased water efflux and ion imbalance from dehydration. The data are limited, limited, but if if ion chan channels are present in the plasma membrane of the mature salmon egg, their role would seem to be one of passive resistance to ion transfer rather than regulation. Another problem receiving increasing attention is the influence of 2 +, Na+, low pH ("acid (“acid rain") rain”) on hydromineral ion requirements (Ca (Ca2+, Na+, K+) K+) during oocyte and postfertilization postfertilization egg development in salmonids salmonids and other freshwater teleosts. teleosts. In general, reproductive success success in spawning spawning salmonids salmonids is is lowered at pH levels below 5.5 5.5 (Tam (Tam and Pay Payson, son, 1986; 1986;Weiner et al., al., 1986), 1986), and plasma ion levels are are disturbed in adults 1-5.5 (Booth adults and juveniles below pH 5. 5.1-5.5 (Booth et al., al., 1982; 1982; Peterson and and Martin-Robichaud, 1986). 1986).Salmonid eggs incubating at low low pH are more more sensitive, showing a threshold for deleterious effects effects near pH 6.5-6.7 (Menendez, (Menendez, 1976; 1976; Craig and and Baksi, Baksi, 1977; 1977; Ruby et al., al., 1977; 1977; Rombough 1 969) showed Rombough and and Jensen, 1985). 1985). Rudy and and Potts Potts ((1969) showed that Atlantic Atlantic salmon (Salmo (Salmo salar) salar) embryos embryos require and take take up Na+ Na+ from from the external 1981 ) external medium during embryogenesis. embryogenesis. Brown and and Lynam Lynam ((1981) and Brown 1 982) found survival to hatch iin n brown trout (S. Brown ((1982) ( S . trutta) trutta)at 2 + levels. pH pH 4.5 4.5to be a function function of pH-dependent external Na+ Na+and Ca Ca2+ levels. Bradley and 1 985) related mortality in juvenile steelhead and Rourke Rourke ((1985) trout trout (S. ( S . gairdneri) gairdneri) to to low low levels levels of of environmental environmental Na+, Na+, postulating that that mortality mortality resulted resulted from from reduced reduced NH NH4+ excretion via via aa Na+/NH Na+/NH4+ 4 + excretion 4+
3. 3.
OSMOTIC AND IONIC REGULATION TELEOST EGGS OSMOTIC AND IONIC REGULATION IN IN TELEOST EGGS AND AND LARVAE LARVAE
175 175
exchange mechanism. A similar problem is is under study in chinook tshawytscha),where early alevin mortality appears to in in(0. tshawytscha), salmon (0. (Aldervolve genetic, osmoregulatory, and pathological components (Alder 1987). Susceptibility varies among spawning dice and Harding, 1987). 2 + ] in the incubation stocks;; in a susceptible stock, low [Na+] “a+] and [Ca [Ca2+] stocks water appear to result in osmotic stress in the alevins, and the debili debilitated alevins succumb to the effects of an unknown pathogen. Prelimi Preliminary recommendations in this instance are that eggs and alevins 2+ ] � 3 6.5, [Ca2+] s 88should be incubated under conditions in which pH � 6.5, [Ca 10 10 mg/l, and [Na+] “a+] � 3 11mg/l. The [K+] [K+]seems less critical. In summary, summary, although oocytes show substantial changes in electri electrical characteristics and specifi specificc ion permeabilities throughout oogene oogenesis, these changes appear to be interdependent processes associated sis, with development and maturation rather than with independent regu regulation. That is, is, adjustments in the oocyte and fertilizable egg appear to be passive, and dependent on the homeostatic mechanisms and regu regulatory capacity of the gravid adult. adult. Although such assumptions seem warranted, it is difficult difficult to extend the argument based on the current, limited knowledge of teleost oocytes. oocytes. It is is evident that much has been learned about ion transport and levels in oocytes of nonteleosts, yet the the more more modern modern investigative investigative techniques techniques appear appear to to have have been been ap applied plied only only infrequently infrequently to to teleosts. teleosts. Although Although there there are are likely likely to to be major major differences differences in in detail detail of of regulatory regulatory mechanisms mechanisms in in coelenterates, coelenterates, amphibians, compared to teleosts, the ground groundechinoderms, and amphibians, work thereby laid for substantial development of a similar knowl knowledge in teleosts appears to be in place. Some Some of the more obvious possibilities for further investigation of teleost oocytes oocytes would include:
meiosis, including The time course of oocyte development during meiosis, temperature (often (often not stated). stated). The stage of meiosis attained when the oocyte is is mature and ready for insemination. States of meiotic arrest and the cytochemical chain of events events be beGVBD and ending with maturation, maturation, including ginning with GVBD intracellular and extracellular hormonal and hydromineral involvements in the maturation process process.. Information oon n oocyte membrane permeability at intervals 2 +, K+, throughout oogenesis, oogenesis, at least to Ca Ca2+, K+, Na+, Na+, and CI-. Cl-. The The use use of a series of of representative, representative, generally available species species of differing plasticity: (a) (a) marine eurhyaline (e.g., (e.g., Clupea Clupea harengus, C. C. pallasi), pallasi), (b) (b) marine marine stenohaline stenohaline (Hippoglos(Hippoglos-
176 176
D. D. F. F. ALDERDICE ALDERDICE
sus stenolepis, H. H . hippoglossus), hippoglossus), (C) (c) freshwater freshwater (Cyprinus (Cyprinus carpio, Carrasius auratus), (d) anadromous anadromous (Salmo (Salmo auratus), (d) gairdneri), and (e) (e) catadromous catadromous (Anguilla (Anguilla rostrata, A. A . ja jagairdneri), and ponica). ponica). Other forms forms requiring consideration would in include clude Fundulus sp. sp. and and Oryzias Oryxias latipes. latipes. III. 111. FERTILIZATION FERTILIZATION
As As with with oocytes, oocytes, in in the the teleosts teleosts there there is is aa relative relative paucity paucity of of infor information mation on on the chronology chronology of of events events at at the the molecular molecular level level during during fertilization. fertilization. Much Much of of what what is is known known about about the the process process of of fertilization fertilization has has been been obtained obtained from from studies studies involving involving polychaetes, polychaetes, echinoderms, echinoderms, and and amphibians. amphibians. Our Our knowledge knowledge of of events events during during fertilization fertilization of of te teleosts, as with oocytes, is based primarily on the Japanese freshwater medaka rst used 1939) as medaka (Oryzias (Oryzias latipes), Zatipes), fi first used by by Yamamoto Yamamoto ((1939) as an an appro appropriate priate experimental experimental organism. organism. A number of of major major events events occurring occurring at at fertilization in the sea-urchin egg (Lytechinus (Lytechinus pictus) pictus) (Whitaker and Steinhardt, Steinhardt, 1985) 1985)may may serve serve as as aa guide guide to to the the teleosts teleosts (Table (Table II). 11). From From this this may be gained an appreciation of the number and complexity of events events triggered triggered in in the the arrested arrested oocyte oocyte by by contact contact with with the the sperm. sperm. The The list must be applied with caution to teleosts; between echinoderms echinoderms and and teleosts teleosts there there are are major major differences differences in in morphology, morphology, likely likely varia variations in timing of events, and possible differences in the events events them themselves. The teleost egg differs in one major respect from those of the polychaetes, polychaetes, echinoderms, echinoderms, and and amphibians, amphibians, in in having having aa micropyle micropyle in in the the outer egg membrane (zona (zona radiata). radiata). The micropyle, a pore whose inner diameter is about 1.5 1.5 /Lm pm (Ginzburg, (Ginzburg, 1968), 1968), allows sperm to enter and advance into the canal canal in le. The The first enter and advance into in single single fi file. first sperm sperm to to penetrate the plasma egg sets penetrate the the canal canal and and contact contact the plasma membrane membrane of of the the egg sets aa chain chain of of events in in motion that that effectively effectively prevents further further sperm sperm entry entry (polyspermy). From the point of sperm contact, the cortical alveoli (polyspermy). underlying the oocyte plasma membrane begin discharging their con contents 1975); the tents (Vacquier, (Vacquier, 1975); the reaction reaction proceeds proceeds in in the the medaka medaka egg egg in in association association with with aa pulse of of free free cytosolic cytosolic calcium calcium and and spreads spreads as as aa wave over the wave over the oocyte oocyte membrane membrane (Gilkey, (Gilkey, 1983; 1983; Gilkey Gilkey et al., al., 1978). 1978).At At the everted cortical alveolar material the micropyle micropyle the the everted cortical alveolar material pushes pushes into into the the 1969), prevent micropylar canal canaI to form a plug-like deposit (Nakano, (Nakano, 1969), preventing further sperm entry. entry. Immediately Immediately prior prior to to sperm sperm contact contact with with the the plasma plasma membrane, the resting potential of the membrane in freshwater teleosts is about
3. 3.
REGULATION IN TELEOST EGGS AND LARVAE OSMOTIC AND IONIC REGULATION
177
TABLE TABLE II I1
of Timing of Major Events (16-18°C) (16-18°C) Occurring at and Following Estimates of (Lytechinus pictus)o pictus)" Insemination of the Sea-Urchin Egg (Lytechinus Event
Time
Membrane potential Ca-Na Ca-Na action potential Na activation potential Increases in K conductance (remains (remains at higher levels) levels) Intracellular calcium release Cortical reaction Activation of NAD kinase Increases in reduced nicotinamide nucleotides (remains (remains at higher levels) levels) Acid efflux Increases in intracellular pH (remains (remains at higher levels) levels) Increased Increased oxygen oxygen consumption (initial (initial burst) Initiation of protein synthesis synthesis Activation of amino acid transport Initiation of DNA synthesis synthesis Mitosis First cleavage
Before 3 ss 3-120 s 3-120 500-3000 ss 500-3000 40-120 ss 40-120 40-100 40-100 ss 40-120 40120 s 40-900 ss 40-900
1-5 1-5 min 1-5 min 1-5 1-3 min After 5 min 15 min After 15 20-40 min 20-40 60-80 60-80 min 85-95 min 85-95
~~
a
From Whitaker and Steinhardt (1985). (1985).
-50 V, which, -50 to to -90 -90 m mV, which, in in eggs eggs fertilized fertilized externally, externally, will will vary vary in in relation relation to medium (Ito, to ion ion concentration concentration of of the the external external environmental environmental medium (Ito, 1963; 1963; Hagiwara Hagiwara and and Jaffe, Jaffe, 1979). 1979). In In most most materials materials examined, examined, this this potential potential results K+ results from from the the selective selective penneability permeability of of the the egg egg membrane membrane to to K+ ions ions and and aa higher higher [K+]i [K+Ii (inside) (inside) than than in in the the external external medium. medium. In In the sea-urchin egg, at the instant of sperm contact with the plasma membrane, evidence supports the occurrence occurrence of three consec consecutive electrical events: an initial depolarization, a Na+ -Ca2 + action Na+-Ca2+ potential, and an activation (fertilization) (fertilization)potential that exists until the egg repolarizes (Whitaker (Whitaker and Steinhardt, Steinhardt, 1985). 1985). The initial depolar depolar(-gated); it seems to trigger an appears to be sperm-dependent (-gated); ization appears 2 +, and suggests Na+ and Ca Ca2+, suggests action potential dependent on external Na+ calcium-carrying potential-gated membrane channel is is primar primarthat a calcium-carrying ily responsible. responsible. The The latter phase of the action potential overlaps overlaps the phase of the activation potential, and the latter results from initial phase potenNa+ conductance conductance by the membrane. The activation poten increased Na+ 2 + -dependent change in mem with a Ca Ca2+-dependent memtial may also be associated with tial brane penneability. permeability. Finally, repolarization at at the the end of the activation brane 2 +, and a Ca2+, appears to be the result of a resequestering of Ca potential appears
D. F. ALDERDICE
178
pH-dependent increase increase in in K+ K+ conductance conductance (Whitaker (Whitaker and and Steinhardt, Steinhardt, pH-dependent 1985). The total fertilization potential may then involve gated mem 1985). The total fertilization potential may then involve gated membrane channels channels and and Ca2+, Ca2 + , K+, Na+, Na+, and H+ ions. ions. and H+ brane As documented by by Hagiwara Hagiwara and Jaffe (1979) ( 1979) and Hagiwara (1983), ( 1 983), As documented and Jaffe and Hagiwara a very instructive examination of the activation potential and related a very instructive examination of the activation potential and related events is is provided provided by by Nuccitelli Nuccitelli (1980a,b) ( 1980a,b) for for the the medaka egg (Ory(Ory medaka egg events Figure lA is an example of the electrical activity of the zias Zatipes). latipes). Figure 1A is an example of the electrical activity of the average plasma membrane during activation, for which the following plasma membrane during activation, for which the following were obtained obtained (20-22”C, (20-22°C, in in 10% 10% Yamamoto values were Yamamoto Ringer’s). Ringer's). 1. Membrane potential. 1. - 39 f (a) ± 9 mV (Yama(Yama (a) Resting potential, prior to fertilization: -39 - 47 +. moto Ringer’s); Ringer's); -47 ± 10 10 mV (10% ( 10% Yamamoto Ringer’s) Ringer's) mot0 (b) At activation: small depolarization of mY; duration, of 4 4 ± 3 mV; (b) ± 10 10 s. s. 20 r - 66 (c) 1 ? ± 12 12 mV (e.g., to -66 (c) Hyperpolarization: Hyperpolarization: amplitude, amplitude, 331 mY) mV)
*
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Fertilization
potential. Fig. 1. Medaka egg. (A) Electrical events associated with the fertilization potential. (B, C) Dependence of potential on [K+], of plasma membrane potential [K+)o and [Na+lo, [Na+)o, respectively. respectively. (Al) (V)shows (a) (a) resting potential, (b) depolarization at (b) small depolarization (AI) Membrane potential (V) hyperpolarization phase, (d) recovery phase, and (e) fertilization, (c) (c) hyperpolarization (d) fast recovery (e) slower recovrecov 10%Yamamoto's Yamamoto’s Ringer's Ringer’s solution). (A2) (A2)Changes in membrane resistance resistance ery phase (in 10% (Yamamoto Ringer's). Ringer’s). (3, C) Ion dependence of the membrane (B, C) (R) over the same period (Yamamoto C )in [Na+]o, “a+],,, in (1) (1)unfertilized unfertilized egg, (2) (2) at of change (B) potential per decade of (B) in [K+]o, ((C) (10% Yamamoto Ringer's), Ringer’s), and (3) (3)fertilized egg about B 8 min hyperpolarization peak (10% hyperpolarization K+ and Na+ Na+ dependence is shown by the unfertilized unfertilized egg; conductance at later. Some K+ hyperpolarization peak peak is strongly K+-dependent; K+-dependent; in the fertilized egg, K+ K+ depen depenthe hyperpolarization Na+ increases slightly. [Modifi [Modified Nuccitelli dence falls to near zero while that for Na+ ed from Nuccitelli ((1980a).] I980a).]
3. 3.
OSMOTIC AND IONIC REGULATION IN TELEOST EGGS AND LARVAE
.
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179
180 180
ALDERDICE D. F. ALDERDICE
(d) phase of (d) Fast Fast phase of recovery recovery from from hyperpolarization; hyperpolarization; duration, duration, 155 155 ± f 18 18 s. s. (e) (e) Slower Slower phase phase of of recovery, recovery, reaching reaching aa steady, steady, postactivation postactivation resting potential of 19 ± 15 mV min after resting potential of -19 -C 15 mV by by 9.4 ± +. 11 min after activation. activation. 2. 2. Membrane Membrane resistance. resistance. A A lO-fold 10-fold decrease decrease in in resistance resistance from from 40 40 to MOa occurred occurred over min following to 33 M over the the first first 2 min following activation. activation. Thereafter, 8 min, min, Thereafter, there there was was aa slow slow recovery recovery over over the the next next 8 resistance resistance ultimately ultimately reaching reaching aa value value 30% 30% greater greater than than the the preactivation value. preactivation value. From electrical events, the time of activation (Fig. 1A) was deter deter(Fig. lA) mined to be about 5 s after a sperm entered the micropyle. Nuccitelli ((19804 1980a) also examined the ionic basis for changes in membrane poten potential. The resting potential showed a limited dependence on external 2+ K+ independence of external Ca K+ and Na+ Na+ ions (Figs. (Figs. IB, lB, lC), lC), but independence Ca2+ and CI-. C1-. The plasma membrane of the unfertilized egg depolarized 99 mV/decade increase in [K+]o [K+], (outside) (outside) and 6 mY/decade mV/decade increase in mY/decade [Na+]o. 1 963) obtained a value of 16 “a+],. Ito ((1963) 16 mY/decade mV/decade increase in [K+]o [K+], in mature oocytes (eggs) Oryzias. Nuccitelli concluded that the (eggs) of of Oryzias. small leaks at the membrane membranesmall ion dependencies were due in part to leaks K+ and Na+ Na+ membrane per perelectrode seal and in part to a significant K+ studies indicated that the small initial meability. Other, preliminary, studies 2 + and Na+ Ca2+ Na+ entry as well as K+ K+ depolarization pulse is carried by Ca efflux. The strong K+ K+ dependence (28 (28 mY/decade mV/decade [K+]o [K+], (Fig. (Fig. IB) 1B) and efflux. hyNa+ response (6 (6 mY/decade mV/decade [Na+]o “a+], (Fig. (Fig. 1C) much lower Na+ lC) during hy perpolarization suggest that the increase in conductance during this K+ membrane permeability. About 88 phase is due mainly to increased K+ K+ permeability fell to near zero min after peak hyperpolarization, K+ mV/decade [Na+]o). “a+],). Na+ permeability increased slightly ((11 while Na+ 1 1 mY/decade flux resultThe hyperpolarization and increased K+ fl ux are suggested as result ing K+ channels channels due due ing from two possible mechanisms: ((1) 1 ) an opening of K+ 2 +]j and (2) to a rise in [Ca (2) the addition of K+ K+ channels, present in the [Ca2+]i vesicuvesicular membranes, that fuse with the plasma membrane by vesicu lar exocytosis. K+ permeability increase exocytosis. The transient nature of the K+ 2 +]j after [Ca2+Ii may be associated with the concomitant decrease in free [Ca passage of the calcium wave over the cytoplasm at activation, and a K+ channels channels in the mem memcorresponding closing of newly inserted K+ brane through endocytosis endocytosis of the vesicular membrane material brane (Gilkey, 11981, 1983; Kobayashi, Kobayashi, 1985). 1985). (Gilkey, 98 1 , 1983; events that coincide with with or follow follow the activation Of these several events potential immediately, an important one is is the very large increase in
3.
OSMOTIC AND IONIC REGULATION REGULATION IN IN TELEOST EGGS AND LARVAE LARVAE OSMOTIC
181
of free calcium. the internal concentration of calcium. In the sea-urchin egg this increase likely approaches the 300-fold 300-fold increase seen in the medaka (Jaffe, 1985). egg (Jaffe, 1985). Second is the eversion of the cortical alveoli, which moves as a wave over the egg surface from the point of of sperm contact. With exocytosis, the cortical alveoli deliver their contents into the presumptive perivitelline space immediately above the plasma memmem brane. There, the alveoli contents imbibe water from the external medium, raising the fertilization membrane (echinoderms) (echinoderms) or zona radiata (teleosts) radiata (teleosts) away from the plasma membrane of of the egg proper as the perivitelline fluid forms 1983) injected buffered calcium forms.. Gilkey ((1983) eggs. He found the level of free calcium into unfertilized medaka eggs. required to elicit a transient increase in cytoplasmic free calcium to be 0.51 and 55.1 (7.0-7.5). The between 0.51 . 1 mM, depending on internal pH (7.0-7.5). desequestering of free internal calcium appears to be an autocatalytic 2 + release. This threshold, required to elicit the 2 + -stimulated Ca Ca2+-stimulated Ca2+ Ca transient calcium wave, is well below the 30 mM of of free calcium reached at the peak of the transient. He concluded that the calcium wave is necessary and sufficient to activate the medaka egg. In the fertilization, the internal calcium content begins sea-urchin egg, after fertilization, to fall; cytosol and into the external fall; it appears to be pumped out of the cytosol 1976). In some (protomedium (Azarnia (Azarnia and Chambers, 1976). some invertebrates (proto somes) egg activation requires calcium, calcium, but it appears to enter the somes) cytosol from the external medium through voltage-gated channels in response al., 1979). 1979). response to to aa shift shift in in membrane membrane potential potential (Jaffe (Jaffe et al., Internal le in egg activation-or in suppression of Internal pH pH may may play play aa rrqle in egg activation-or in suppression of q activation. M edaka eggs with an apparent internal pH of 77.1 . 1 (Jaffe, (Jaffe, Medaka 1985) 1985) show a slowing of the calcium wave at pH 6.9 6.9 and its accelera acceleration at pH 7.3. 7.3. Reduction of internal pH from 7.0 7.0 to 6.5 6.5 in the medaka egg increases threefold the calcium threshold required for initiation of the calcium surge. The ovarian eggs eggs of frogs, frogs, taken directly from the surge. The oviduct, oviduct, were were shown to be unfertilizable because of their high C0 C022 content. Hence, low low ovarian pH may act as as a brake to activation prior is an apparent apparent doubling of to egg deposition. In sea-urchin eggs there is ions shortly after activation; activation; this alkalinization of the cytosol [OH-] ions -H+ ion appears, in in part, part, to to be the result of an electrically electrically neutral Na+ Na+-H+ appears, transfer. trans fer What can can be inferred from the the available available data regarding ionoiono- and osmoregulation in the teleost oocyte and in the egg at fertilization? osmoregulation Few Few data data are are available available on on molecular molecular processes, processes, and and the the general general nature nature of of possible possible ionic ionic involvements involvements must must be be cautiously cautiously inferred inferred from from other other animal animal groups. groups. Basically, Basically, various various types types of of ion-transporting ion-transporting channels channels appear appear in in the the plasma plasma membrane membrane of of the the developing developing oocyte. oocyte. At At various various
.
182 182
D. D. F. F. ALDERDICE ALDERDICE
times these may become functional, cease times these may become functional, may may proliferate, proliferate, or or may may cease functioning; functioning; they they may may be be electrically electrically neutral, neutral, electrogenic, electrogenic, or or voltage voltagegated. blocked by pH levels gated. Some Some may may be be blocked by particular particular pH levels in in the the surround surrounding of the the plasma membrane membrane is is depen depening medium. The The resting potential potential of dent dent on on its its selective selective ion ion permeability permeability and and the the voltage voltage gradient gradient set set up up by by differences differences in in ion ion concentrations concentrations on on either either side of of the the membrane. membrane. Generally becomes more Generally it it becomes more negative negative as as ion ion concentration concentration decreases decreases in in the medium. Hence, the external external (freshwater) (freshwater) medium. Hence, the the resting resting potential potential may may change (such as change in in relation relation to to external external stimuli stimuli (such as sperm sperm contact) contact) or or inter internal ionic activity nal stimuli stimuli (Ca2+ (Ca2+release), release), and and to to the the level level of of ionic activity in in the the external medium. As ions move, so so too will water move across the membrane in relation to membrane permeability, and to differences in in molar molar concentrations concentrations of of solute solute particles particles on on the the two two sides sides of of the the membrane. membrane. Relatively speaking, speaking, the oocyte prior to release is subject to the protective regulative mechanisms of of the parent. The ovulated egg responds responds to to changes in in the the ovarian ovarian fluid fluid (Sower (Sower et al., 1982), 1982),the the ovar ovarian fluid osmotically is very similar to the blood plasma (Hirano aZ., (Hirano et al., 1978), 1978), and the blood plasma is in balance physiologically with the osmotic activity of the external environment (Sower (Sower and Schreck, 1982). However, when shed externally at spawning, mature eggs will 1982). be be subject subject to to major major changes changes in in osmotic osmotic and and ionic ionic composition composition of the the external medium; in general they will be hypotonic to seawater and hypertonic to fresh water. It would seem that the first major regulatory challenge will occur at spawning, adding to the large number of mo molecular activities set in motion by activation and fertilization. fertilization. In the newly fertilized egg the only structure available with regulatory ca capacity would be the plasma membrane. It seems reasonable to assume that the membrane should be well prepared, in terms of of permeation, ion-channel function, and electrical activity, to cope with the changes occurring when the eggs are shed and fertilized over a range and variety of external conditions, conditions, whose extent would be related to the normal normal habitat habitat of of the the species. species. In In terms terms of of tolerance, no no doubt doubt these these ranges will have limits related both to permeability characteristics of of the plasma membrane and to the tolerance of the initial cell and its successors in the newly fertilized egg. In summary, it appears that an understanding of regulative charac characteristics of teleost eggs at fertilization will be centered on the plasma membrane and will require, initially, the development of basic, de descriptive data on the electrical and ion permeation characteristics of of the teleost egg. Further, examples need to be developed for stenoplas stenoplastic and euryplastic species from the marine and estuarine environ environments, as well as from fresh water; patterns that may exist would be
3. 3.
REGULATION IN IN TELEOST AND LARVAE OSMOTIC AND IONIC REGULATION TELEOST EGGS AND
183 183
difficult to identify from the limited information currently available. available. One use of One may may also also plead plead for for aa greater greater use of normal normal external external media media during during such such studies studies to to make make the the physiological physiological results results obtained obtained ecologically ecologically more meaningful. Areas of interest in specific studies would include: Resting ion permeation Resting potentials, potentials, ion permeation characteristics, characteristics, and and electrical electrical activity activity in mature, unfertilized unfertilized eggs. eggs. The The activation activation potential, potential, and and membrane membrane resistance resistance and and conduc conduc2 +, K+, tance Na+, and l - in K+, Na+, and C C1in the the external external tance with with respect respect to to Ca Ca2+, medium. medium. Action they occur. occur. Action potentials, potentials, if if they Ion-channel function in preactivation to postfertilization postfertilization stages. stages. Internal Internal ion ion events events during during activation activation and and fertilization. fertilization. 2 + , K+, Influence low-ionic-strength fresh fresh water water (e.g., (e.g., low Ca Ca2+, K+, Influence of low-ionie-strength Na+) Na+) on activation. The timing of major events during activation and fertilization in relation relation to to temperature temperature and and species. species. IV. DEVELOPMENT
Of the three levels Of the three levels of of potential potential regulatory regulatory capacity capacity mentioned mentioned ear earlier-cellular, lier-cellular, tissue, tissue, and and neurosecretory neurosecretory involvement-it involvement-it is is assumed assumed the oocyte oocyte and and mature mature eggs eggs are are restricted restricted to to the the first first of of these, these, continu continuing, ing, in in the the fertilized fertilized egg, egg, for for aa period period extending extending into into very very early early cell cell division. division. It It is is assumed assumed also also that that the the second second level, level, proliferation proliferation of of tis tissues sues with with regulatory regulatory capacity, capacity, may may begin begin with with blastodermal blastodermal over overgrowth earlier, with yolk, and and possibly possibly earlier, with an an initial initial level level of of regula regulagrowth of of the yolk, tory plug closure tory capacity capacity achieved achieved by by yolk yolk plug closure (Holliday (Holliday and and Jones, Jones, 1965). 1965). It It would would seem seem that that neurosecretory neurosecretory involvement involvement would would of of ne necessity development of specialized tissues, yolk plug plug cessity await await the the development of specialized tissues, after after yolk closure, process of continuing continuing elaboration elaboration through through the the larval larval and and closure, in in aa process later viewpoint the later stages. stages. From From this this viewpoint the components components of of the the fertilized fertilized egg egg will be examined examined that that may may contribute contribute to to regulation. regulation. In In order order of of devel develwill opment these would included the plasma membrane, the perivitelline fl uid and fluid and zona zona radiata, radiata, tissues tissues of of the the blastoderm, blastoderm, the the embryonal embryonal epi epidermis, cells, and, dermis, chloride chloride cells, and, briefly, briefly, aa consideration consideration of of the the transition transition from from the the embryonic embryonic regulative regulative mechanisms mechanisms to to those those of of the the juvenile, including gills, gut, Possible responses responses to evolving, including the the gills, gut, and and kidney. kidney. Possible to evolving. regulatory regulatory capacity capacity could could involve involve changes changes in in egg egg volume, volume, water water con content, levels of tent, levels of tissue tissue osmolarity, osmolarity, internal internal ion ion concentration, concentration, hydro hydrostatic These will will be static pressure, pressure, and and buoyancy buoyancy and and specific specific gravity. gravity. These be touched briefly, but emphasis is is placed placed on on regulation, regulation, regulative regulative touched on on briefly, but emphasis processes, where and when these might occur. processes, and and where and when these might occur.
184
F. ALDERDICE ALDERDICE D. F.
Properties of of the the Plasma Plasma Membrane Membrane A. Properties of a lipid The plasma membrane of a cell generally has the form of bilayer matrix matrix in in which which islands islands of of protein protein are (Korenbrot, bilayer are interspersed interspersed (Korenbrot, 1977). The lipid matrix phos 1977). The lipid matrix comprises comprises some some 5-10 5-10 lipids, including phosphatidyl phatidyl derivatives, derivatives, sphingomyelin, sphingomyelin, cholesterol, cholesterol, neutral neutral lipids, lipids, and and glycolipids. protein derive their characteristics glycolipids. The islands islands of of protein derive some some of of their characteristics from the the composition composition of of the the lipid lipid matrix. They may extend across across the from matrix. They may extend the entire of the membrane and provide access to reactants at its entire thickness of oc two surfaces. Most water transport across a membrane appears appears to occur by aa solubility-diffusion solubility-diffusion process process through through the hydrophobic bilayer cur mem lipid matrix. On the other hand, hand, ions appear to move across the memof the islands brane, by active or passive transport, by way way of the protein islandsreferred to as channels. As most cells do not require high generally referred water permeability, permeability, plasma membrane channels devoted to water movement would seem redundant. Nevertheless, water molecules may may penetrate penetrate aa membrane via via channels channels and and do do so in relation to to chan channel diameter. diameter. In In such such instances, instances, flows flows through through channels channels may may involve involve nel water-water, water-ion, ion-ion interactions (Levitt, (Levitt, 1984). 1984). water-water, water-ion, or ion-ion Three of a membrane include its wawa Three important functional aspects of ter of ions, and its ter permeability, transfer of its electrical properties. Various techniques have been used, refined and redefined for measuring the of particles across a membrane. There is a potential movement of potential for confusion because in the the because of of the the various various terminologies terminologies and and measures measures in confusion literature, as pointed out by Potts and Parry (1964). ( 1 964). The flow of of soZute solute z/s), across across aa membrane membrane may may be be measured as as aa diffusion diffusion constant (cm (cm2/s), or or as as aa permeability permeability constant (cm/s). (cm/s). Permeability Permeability of of aa membrane membrane to to aa solvent (water) (water) is measured in terms of of permeability coefficients. coefficients. ( 1961) and As demonstrated by Kedem and and Katchalsky (1961) and Katchalsky ( 1 965), the of particles across a membrane is and Curran Curran (1965), the movement of of dependent on the flows and forces operating in the system. A set of phenomenological equations, relating these flows and forces, may be written as
h = Lll Xl + LIZ Xz + . 12 = LZI Xl + LZ2 X2 + .
= Jn Jn =
L,1 Lnl x1 Xl
+ L,z LnnX, X2 + . -. -. + LnnXn Ln2 xz *
of the XXkk forces; that is, where the JJii flows are linear functions of
3. 3.
OSMOTIC AND IONIC REGULATION IN TELEOST EGGS EGGS AND LARVAE
i J ii = =
n n
2: C LL iikXk kXk k= I
k= l
185 185
2, . .. .,, nn)) (i = = 1, 2, .
.
X ii forces will be linear functions of of the theJk Hence, the X h flows, n
X i = 2: R ik h k= l
(i = 1 , 2,
.
.
.
, n)
The L L iikk = = U;lXk)xi ( J i / X k ) X , coefficients coefficients are flows characflows per unit force and are charac Rik = (X/h)); (Xi/Jk)licoefficients terized as conductances or mobilities. The R ik = of force per unit flow and represent resistances or have dimensions of J ii or Xj X i equations may be converted frictions. In practice, either set ofi into the other by standard matrix algebra. algebra. Furthermore, the matrices are symmetric, so that Lik = Lki
(i ( i =1= # k) k)
Hence, in ow, two-force Hence, in aa two-fl two-flow, two-force system system (one (one solute, solute, one one solvent), solvent), there there are three coefficients; in a three by three system, there are six coeffi coefficients. In general,
ILI 2:: 2 0 0,, LiiLa 2 L i and ILl is advantageous to trans In considering membrane properties, it is transform the original (thermodynamic) (thermodynamic) forces into simpler quantities. For example, it follows, in a binary system, that
L Lii ii
2 2::
0, 0,
Jv = = Lp L p ilP AP + + LPD LPDil1T AT iv iD ]D = = LDP LDPilP AP + + LD LDil1T AT J v is the volume flow of solvent (e.g. (e.g. water); iD J D is the velocity of where iv AP is the hydro hydrosolute relative to solvent, similar to a diffusional flow; ilP static pressure difference AT is is the the osmotic osmotic static pressure difference across across the the membrane; membrane; il1T pressure membrane; Lp L p is is the the mechanical mechanical filtra filtrapressure difference difference across across the the membrane; tion coefficient, to ilP; L is the diffusional mobility coeffi AP; L D is diffusional mobility coeffition coefficient, relating relatingfv to D iv cient, J D to to il1T; AT; and and LP L pD D is is the the coefficient coefficient of of osmotic osmotic flow, flow, cient, relating relating iD which equals equals LDP, L D p , the the ultrafiltration ultrafiltration coefficient. coefficient. Several Several examples examples may may show value of show the the value of these these multiflow-multiforce multiflow-multiforce relationships. relationships.
1. If 1. If il1T AT = = 0: 0: iv Jv = = Lp L p ilP, AP,
D = LDP i JD L DilP AP ~ =
Thus, with will produce Thus, with il1T AT = 00,, hydrostatic hydrostatic pressure pressure will produce both both aa volume volume flow ultrafiltration. flow and and aa diffusional diffusional flow, the the latter latter by ultrafiltration. =
If ilP AP = = 0: 2. If J v = L p D AT,
D= i ]D = LD LDil1T AT
186
D. F. ALDERDICE
Hence, with AP tlP = = 0, the osmotic pressure gradient will produce an flow. There also is a relation between osmotic flow and a diffusional flow. osmotic flow flow and and ultrafiltration, ultrafiltration, as as osmotic
(h) tl1T -
IfJv = 0 0:: 3. If lv =
!J.p=o
�
Lpv = Lvp
(tlP),v-o
=
=
(tlP k) -
�1T=O
-
LpD - Lp tl1T
That [(tlP),v=o = equ_ilibrium [(AP)Jv=o = AT] a1T] occurs only if if -Lpg That is, true osmotic equilibrium = - LPD = = LD LD in in an an ideal ideal Semipermeable semipermeable membrane membrane where where the the solute solute flow flow Lp = is zero. zero. is of the solute: solute : 4. Where the membrane permits passage of LpD -LpD
_
Lp LP
< <11
The ratio ratio v-L is called the the reflection coefficient u. (T. When When (T (T = = 1, 1, all all LpDILp The p ~ / L is p called reflection coefficient of the the solute solute is is "reflected" membrane (e.g., (e.g., ideal the membrane ideal semiperme semipermeof “reflected” by the < 1, 1 , part of of the mem (T < the solute permeates the memable membrane); when (T brane; when when u (T = = 0, the the membrane membrane is is nonselective nonselective and solute and and brane; and solute solvent move move with with equal equal velocities. velocities. solvent flow is 5. By rearrangement and in terms of of (T, u,the volume flow
-
= Lp(AP LPD AT tl1T = Lp(tlP tlP + + Lpg Jv = = Lp Lp AP lv
(T (T
tl1T) AT)
6. In a binary system, the total solute flow is 6. (T) Jv G s (1 Js = Cs - U) + w0 tl1T AT lv + ls = (1 where (7s of solute in the membrane where membrane and G s is the mean concentration of w = Gs
[LpLv - L�D] Lp
Then, whenJv when lv = = 0, Then, s w = l
tl1T
w is the coefficient of of solute permeability at zero volume The term w flow. For an ideal semipermeable membrane, as before, -LPD = Lp = - LPD = LD,, Lv" and w w = = 0. O. For a nonselective membrane, where the solute difdif fuses freely, LpD = = 0 and w w = = CSLD. G sLv. =
3. 3.
REGULATION IN TELEOST EGGS OSMOTIC AND IONIC REGULATION EGGS AND LARVAE
187 187
coefficients Lp, Lp, u, u,and w w is generally accepted as being The set of coefficients the most convenient for the description of membrane properties. For n solutes, solutes, the equation for volume flow frequently seen is a system of n
iV= L ~ ( A P- k= 1 uiA
~ J
For a binary system of water and one solute, the equations for volume (Jv) ( j s ) simplify to flow U flow v) and solute flow Us)
Jv Jv = = Lp(ap Lp(AP -u CT a7T) AT) Js )J v + Js = = 6s(1 Cs(1 -u U)JV + Ww a7T AT or their equivalents or their equivalents
Jv Jv = = Lp L p (.6.P (AP - uRT uRT .6.Cs) ACs) Js Js = = 6s(1 Cs(1 - u)Jv u)Jv + + wRT .6.Cs ACs where where 7T T ==RRTCs T C S (atm) (atm) R= = 0.082 11 atm atm °C-I "C-I molemole-ll R and where T is the absolute temperature (K), .6.C ACSs is the molal concen concentration AT7T = = RT RT .6.Cs. ACs. tration difference difference across across the the membrane, membrane, and and .6. Two Two widely widely used used permeation permeation coefficients coefficients may may now now be introduced. introduced. P= . These often often are are estimated estimated on on the the assumption assumption that that a AP = 0 and and u cr = = 11. Hence 1 ) the Hence we we have have ((1) the filtration filtration permeability permeability coefficient coefficient Lp
Pf = V- w and and (2) (2) the the diffusion diffusion permeability permeability coefficient coefficient
Pd
-
[ Js ]
w .6.7T -
1v=0
Pjf measures the net flow, flows The term P flow, the difference between two flows Pd measures one of opposite directions across across a membrane; Pd moving in opposite flow flows separately, and is used to estimate the unidirectional fl these fl ows separately, ow studies. of tritiated water in isotope studies. When values of the two permeation coefficients coefficients are obtained for Pff > > PP dd .. Earlier it was same membrane system, system, it often occurs occurs that P the same this inequality resulted from the fact that diffusion was assumed that this flow, and that isotopic methods accelerated in the direction of the net flow, against the net fl flow. Poiseuille's law always measure diffusion against ow. Using Poiseuille's
188
D. D. F. F. ALDERDICE ALDERDICE
of volume volume flow, flow, the difference difference between between the the two two measures measures provided provided aa of means of of calculating calculating the width width of of pores pores in in aa membrane membrane through through which which means such flows flows could could occur. occur. The validity validity of of this this inequality, inequality, however, however, has has such been argued argued for for some some time time (Hansson (Hansson Mild and and Lgvtrup, Lf/lvtrup, 1985). 1985). AlAl been though the earlier earlier assumptions assumptions now now generally generally are considered unrealunreal though the are considered istic, comparison comparison of of the two two coefficients coefficients remains remains valid. valid. Hence, Hence; where where istic,
Pf
=
LP
Pd
b.P AP
= w,
W
then then
(!)
Pf l:P = Pd Vw
=
w
=
=
0, 0,
and
CT = = 1 1 u
!::L Vww
If one one wishes wishes to to examine examine the the influence influence of of the the variables variables on on the the ratio, ratio, If CT to to vary, vary, where where allowing b.P AP and and v allowing Lp Pf Vw - (b.P _
_
-
Jv CT
b.7T)VW
then then b.7T JJvv AT b.7T) +J Vw Pd (Vw b.P lvCTGs) (Js - lv eT (VW AP VW AT) US J VC CsS + V~CS) pd
f P =!1
-
Jv JV
VdAP AT)^ Vw(b.P - v CT b.7T)W
Friedman 1986) provides an excellent survey of these and associated Friedman ((1986) transrelationships, and their utilization in problems of biological trans port. Water Water permeability permeability coefficients coefficients measure measure the the volume volume of of water water pass passing across a unit area of membrane per unit time. Although Pr P f and Pd usually usually are are reported reported in in centimeters centimeters per per second, second, this this actually actually repre repre3 cmP2 sents cm-2 Salso may sents aa contraction contraction from from cm cm3 s - lI.. The latter latter (( x X 104) lo4)also may be 3 pm+? reported /Lm-2 Ss-I, - I , equivalent - I . Other also have reported as as /Lm pm3 equivalent to to /Lm pm Ss-l. Other units units also have been used (Potts 1964). Where Where possible, possible, the the original original units units been used (Potts and and Parry, Parry, 1964). 3 pm-2 J.tm-2 Sused converted to to /Lm pm3 s-l1 used in in articles articles reviewed reviewed will be converted I ). (J.tm (pm ss-l). conA long series of most informative investigations has been con ducted ducted by by Lf/lvtrup, Lgvtrup, Hansson Hansson Mild, Mild, and and their their associates associates that that deserves deserves particular particular mention; mention; an an excellent excellent summary summary of of these these studies studies is is found found in Lf/lvtrup 1981). Using an electromagnetic diver Lgvtrup and Hansson Mild ((1981). balance, balance recorded recorded aa composite composite response response balance, they they found found that that the balance involving involving both both cytoplasmic cytoplasmic diffusion diffusion and and plasma plasma membrane membrane permeperme-
3. 3.
OSMOTIC AND IONIC REGULATION IN IN TELEOST EGGS AND LARVAE IONIC REGULATION
189 189
ation. That is, the measure of membrane permeability for molecules passing into a cell was influenced by the rate of diffusion of those cytoplasm. They molecules moving away from the membrane in the cytoplasm. of isotopic water diffusion selected an approach providing an estimate of 2 ss-') - l ) in the cytoplasm (D,cm cm2 cytoplasm';l ; this allowed an independent independent estimate (D, of the exchange permeation coefficient ((E, E , cm S-I) s-l) for the plasma of conmembrane. Further, they found that the amphibian oocyte, in con trast to the mature egg, has no measurable barrier to water perme permeation; that there is a relation between cortical tension, tightness of the plasma membrane, and tonicity of the external medium (low (low tonicity increases cortical tension and tightness of the membrane); membrane); that diffu diffusion in the cytoplasm is a complex function of temperature; and that and cell density is a function of water content. Finally, they derived Pf Pfand Pd ( =E, = E , cm S-I) s-l) coefficients for the plasma membrane, and questioned the popular interpretation that Pf Pf > > PPdd is related to the presence of of membrane channels. Finkelstein ((1984) 1984) has examined the water permeability coeffi coefficients obtained for various bilayer membranes, and fi nds they range finds 2 /-Lm X 10lo-'1 to 11 XX 10 lo2 pm S-I. s-l. For plasma membranes examined, the from 2 X range extends from 0.96 0.96 x . 14 X 1.14 X 10- 2 /-Lm p n S-I s-l ~ for midgastrula X 10- 2 to 1 2 to 2 X 2 /-Lm eggs of Fundulus sp. sp. at 23°C (Dunham et al., 1970) 1970)2 x 10 lo2 pm S-I s-l for erythrocytes; most values are found to be around 2 X x 10-1 lo-' /-Lm pm S-I. s-l. Dunham et al. (1970) (1970)concluded from the low Pd values they obtained for for the the eggs eggs that that water water transport transport across across the the egg egg membrane membrane was was very very slow, suggesting that no special mechanisms are necessary for volume regulation of the Fundulus egg. egg. The unfertilized oocyte of of the plaice (Pleuronectes platessa) shed into seawater (Potts (Potts and Eddy, 1973) 1973) had 8.6 X x 1010-22 /-Lm pm S-I, s-1, reducing to 1.7 1.7 X x 10- 3 /-Lm pm Ss-1 an initial Pd of 8.6 - I after 11 day. 1969) estimated Pd for the ovarian egg of day. Potts and Rudy ((1969) of the Atlantic salmon (Salmo salar), prior to shedding, to be 6 Xx 10-2 (Salmo salar), pm S-I, s-l, falling to <4 Xx 10- 3 /-Lm pm s-I s-l after water hardening. For fertil fertil/-Lm ized eggs of the same species (gastrula (gastrula to myomere stage) stage) previously stored in Ringer's solution and examined in that medium at 5.5°C, 5.5"C, Loeffler and LfiSvtrup LZvtrup (1970) (1970) obtained a permeability coefficient (E) ( E ) of 1.0 x - I. Loeffler ((1971) 1971) also noted (his 1.0 x 10- 2 /-Lm pm Ss-l. (his Fig. 3) 3 ) that ovarian eggs of the zebrafish (Brachydanio (Brachydanio rerio) had a permeability ((E) E ) near 6.4 x 10-1 lo-' /-Lm p m S-I. s-l. Prescott and Zeuthen ((1953), 1953), using mature un6.4 X I For the derivation of vtrup and Hansson Hansson Mild ((1981); 1981); for E, of D, D,see Lfll Lzvtrup E , see L�vtrup Lzvtrup (1963). (1963). 2 There is a typographical typographical error in Finkelstein (1984); (1984); the original original record from I pm Ss-l), 1970) places Finkelstein's fi gure at 11 x 10-6 10-6 cm S - 1 (1 (1 x et aZ. al. ((1970) figure Dunham et - ) , not x 10- 2 JLm 11 x 10-16 cm ssl S-1 as shown in Finkelstein ((1984). 1 984).
190 190
D. D. F. F. ALDERDICE ALDERDICE
fertilized fertilized eggs eggs of of the the same same species species in in Ringer's Ringer’s solution solution (estimated (estimated temperature temperature 20-22°C; 20-22°C; Dick, Dick, 1959b), 1959b), obtained obtained an an average average Pf value value of of 4.5 4.5 xX 10lo-’1 J.Lm pm S-I. s-l. Prescott Prescott (1955) (1955)obtained obtained aa Pd P d of of 16.8 16.8 xX 10-1 10-l J.Lm pm S-1 s-l for for unactivated, unactivated, unfertilized unfertilized mature mature eggs eggs of of the the chinook chinook salmon salmon (On (Oncorhynchus tshawytscha) tshawytscha) in in Ringer's Ringer’s solution solution at at 15°C. 15°C. Although Although the the range range indicated indicated for for these these permeation permeation estimates estimates of of teleost teleost plasma plasma membranes membranes is is not not extensive, extensive, there there is is evidence evidence that that membrane membrane perme permeability 1) state ability varies varies in in relation relation to to ((1) state of of embryonic embryonic development, development, (2) (2) composition composition of of the the external external medium, medium, (3) (3)temperature, temperature, (4) (4)surface-to surface-tovolume presence or volume ratio, ratio, and and (5) (5) presence or absence absence of of membrane membrane channels. channels. These These will will be be considered considered in in turn. turn. TATE OF E MBRYONIC D EVELOPMENT 1. SSTATE 1. EMBRYONIC DEVELOPMENT
An examination of permeation characteristics at various various stages of embryonic development (Table (1) mature oocytes (Table III) 111) suggests that (1) (ovarian speaking, have a highly permeable plasma (ovarian eggs), eggs), relatively speaking, membrane, (2) (2) at fertilization there may be an increase in membrane permeabilpermeability for a short period, (3) (3) following fertilization, permeabil ity decreases rapidly to a minimum, and (4) (4)subsequently there may be a minor increase in permeability. Potts and Eddy (1973) (1973) compared 3 values of of of the rate constant (K) (K)3for tritiated water exchange in eggs of the plaice (Pleuronectes (Pleuronectes platessa) (Table (Table III). 111). Their data show that mature eggs shed into isotonic saline were highly permeable initially, retained higher permeability longer, but eyentually permeability de declined to levels similar to those for eggs shed into seawater. seawater. In seawa seawater, the reduction in permeability to a minimum occurred about 11day after fertilization. fertilization. Both plaice and Dover sole (Solea (Solea vulgaris) uulgaris) were shown to have K K values of of 0.03-0.07 0.03-0.07 later in development, a small 1 day following fertilization. increase from the minimum achieved 1 Potts and Rudy (1969) (1969) noted a similar decline in K K values (Table 111) III) for Salmo salar, equivalent to a reduction in Pf from 6 x x 10-2 pm J.Lm s-l s-1 in freshly stripped eggs to <4 xx 1 0- 3 J.Lm water-hardened eggs. pm sS-1 - l in water-hardened They found that the permeability permeability of of unfertilized eggs declined almost de immediately following their placement in water; it continued to decline for 7-8 7-8 h to a minimum, remaining at or slightly above the following 50 days. The resting value of of the unfertilunfertil minimum in the folIowing = 0.4 h-l) h-1) again appears to rise somewhat after its placeplace ized egg ((K K= ment in water. Loeffler (1971) (1971) carried out a similar study with fertilfertil ized and unfertilized unfertilized eggs of of the pike (Esox lucius) lucius) (Table 111) III) stored E Bll
3K = = (IIT) ( liT) In[(A In[(A - B$(A B1)/(A - €is)], B2)], where A A is the activity in the external medium and s pecific activities at two times, time T and B B2z are specific T apart, apart.
TABLE TABLE 1II I11 Changes in Water Permeation Rates of of Oocytes, Oocytes, and Fertilized Eggs of of Three Teleosts at Various Stages of of Development
salarb Salmo salarb
Pleuronectes Pleuronectes platessaa platema“ Time from fertilizationd fertilizationd
K (hI) (h-’)
-
11.0 .0
h 224H
0.5 0.5
-
0.05 0.05
Il h 2h 24 h 2!
0.2 0.2 0.1 0.1 0.05 0.05 0.02-0.04 0.02-0.04 0.02 0.03-0.07 0.03-0.07
>10 h >10 h Id 14 14 d
Remarks Remarks
Time from fertilizationd fertilizationd
K K (h-I (h-l))
E
Remarks Remarks
Initial observation in isotonic saline Unfertilized in isotonic saline
Oocytes Oocytes
00
-0.4 -0.4 >0.5 >0.5
"Resting “Resting value" value” At fertilization
H 14 h
-0. 10 -0.10
Final observation in isotonic saline Fert., in SWe SW Fert., Fert., in in SW SW Fert., in SW Fert., Fert., in in SW Fert., in SW Fert., in SW
A few hours 40d 40d
0.02-0.03 0.02-0.03 0.05 0.05
50 50 d
0.023 0.023
In river water, water hardened In river water In river water, " eyed" “eyed” In river water, unfertilized unfertilized
a Potts and Eddy ((1973), 1973), marine. (1969), freshwater. b Potts and Rudy (1969), Loeffier 1971), freshwater. LoefAer ((1971), d Time from fertilization in hours (h) (h) and days (d). (d). SW, seawater. e Fert., Fertilized; SW,
c
Esox luciusc luciusc Stage Unfertilized Unhardened Unhardened
(/Lm (pm S-I) s-’)
Remarks Remarks
1.7 x 10-1 lo-’ 1.7 1.9 x 10-1 lo-’ 1.9
100% Ringer's Ringer’s In 100% 100% Ringer's Ringer’s In 100%
Hardened
3.8 100% Ringer's Ringer’s 3.8 x 10-2 In 100%
Fert., early cleavage Blastoderm Beginning of of epiboly Advanced myomere myomere
1.8 x 10-2 lo-% In 7.5% Ringer's Ringer’s 1.8 2.2 X 10-2 In 7.5% Ringer's Ringer’s 2.2 x 1.9 1.9 x 10-2
In 7.5% 7.5% Ringer's Ringer’s
2.2 x 2.2 X 10-2
In 7.5% 7.5% Ringer's Ringer’s
192 192
D. F. ALDERDICE
in Ringer's Ringer's and and examined examined at at 9.O"C. 9.0°C. From From aa maximum maximum of of 1.7 1.7 X x lo-' 10-1 pm p.m in S - 1 , in in the the unfertilized unfertilized oocyte, (=Pf) rose rose slightly, slightly, then fell in in the the s-l, oocyte, E (=Pf) then fell fertilized egg to a minimum of of 1.8-2.2 1 .8-2.2 X x 10- 2 pm p.m s-l S- 1 from early No subsequent rise in Pf is noted in these data cleavage onward. No during 1980a) found in the during later later development. development. However, However, Guggino Guggino ((1980a) found in the embryos embryos of of Fundulus heteroclitus following following fertilization fertilization (25°C) (25°C)that that P P dd 1 1 2 p.m 3 p.m increased - at S - at 7 days increased from from 44 x x 10lop3 pm Ss-l at 44 days days to to 11 x x 10low2 pm s-l days and 1.6 10 days. Dunham et al. (1970) ( 1970) obtained PPdd 1.6 x x 10- 2 p.m pm Ss -- l1 at 10 estimates for midgastrula eggs of of Fundulus [presumed heteroclitus by Guggino Guggino (1980a)] (1980a)l at at 23°C. 23°C. For For the the pregastrula pregastrula eggs, eggs, an an earlier earlier devel developmental stage than Guggino's 4-day embryos, Pd P d was 9.6 x X 10- 3 to X 10- 3 11.14 . 14 X X 10- 2 p.m pm Ss -- lI.. The two two lowest values (4 (4 x X 10- 3 , 9.6 X I - ) were the p.m pm Ss-l) the lowest lowest values values for for teleosts teleosts found found in this review. review. Other studies based on differing methodologies methodologies show that water uptake by the egg or embryo may vary throughout development. Zotin (1965) ve phases of water uptake by embryos of the (1965) distinguished fifive sturgeon (Acipenser gilldenstiidti huso), and guldenstadti colchiens, beluga (Huso (Huso huso), sevruga (Acipenser stellatus). stellatus). LJiSvtrup Lgjvtrup (1960) (1960) demonstrated complex changes changes in in rate rate constants constants (k),4 (k),4 diffusion diffusion permeabilities permeabilities and and water water vol volumes in eggs of two amphibians (Siredon (Siredon mexicanum, Rana platyr platyrrhina) development. Harvey and Chamberlain (1982) (1982) showed rhina) during development. that the eggs of the zebrafish (Brachydanio (Brachydanio rerio) rerio) undergo complex density changes throughout development, apparently refl ecting reflecting changes in water content. Other simple to complex changes in density throughout development have been noted in marine pelagic eggs by Alderdice and Forrester (1968, (1968, 1971, 1971, 1974) 1974) (Parophrys (Parophrys vetulus, vetulus, Eop Eopsetta jordani, Hippoglossoides elassodon), Forrester and Alderdice jordani, elassodon), ((1973) 1973) (Hippoglossus (Hippoglossus stenolepis), stenolepis),and Alderdice et al. al. (1987) (1987)(Anoplo (AnoploThese changes appear to have survival values in rela poma fimbria). appear relapomafimbria). tion to depth distribution and lateral transport of eggs eggs and larvae in the natural environment. In summary, permeability of the plasma membrane of the mature teleost egg to water appears to be relatively high. In mature amphib amphibian eggs it appears to be so high that the plasma membrane provides the movement of water; water; that that re reno measurable diffusion barrier to the exchange experiments is is now concluded by LJiSvtrup Lgjvtrup corded in isotope exchange diffusion in the cyto cytoand Hansson Mild ((1981) 1981) to be attributable to diffusion plasm. When the egg is is shed, shed, the permeability of the egg membrane plasm. D/2.303, where where Q = = 61d, 6/d,dd is the the diameter diameter of the the egg, egg,and and D is the diffusion diffusion 4 k = Q D/2.303, permeability constant constant (cm (cm Ss-1). permeability I). =
3. 3.
OSMOTIC AND IONIC REGULATION REGULATION IN IN TELEOST EGGS AND LARVAE LARVAE
193 193
appears to increase briefl y, then drop rapidly to very low values, to briefly, “tightness” of of the increase again, slightly, in later development. The "tightness" membrane that develops a few hours after fertilization would seem an appropriate appropriate form of "passive" “passive” regulation in an embryonic stage obvi obviously lacking regulatory tissue and apparently depending wholly on the plasma membrane to maintain its integrity. There are no other obvious means by which the egg proper could be protected from the mahypotonic and hypertonic media into which the freshwater and ma rine teleost eggs are shed. One may speculate that the initial period of of high water permeabil permeability would allow osmotic water fl ows to occur, assisting in the achieve flows achievement of a steady state of the internal environment of of the newly fertil fertilfluxes ized egg relative to the ion fl uxes that may occur during the activation availpotential (Nuccitelli, (Nuccitelli, 1980a). 1980a). Little regulative control would be avail able to the egg following fertilization if water permeation of the mem membrane brane were were minimized minimized but but substantial substantial ion ion flows flows were were to to continue. continue. It It then assumed that that when when water water permeation permeation is is attenuated, attenuated, ion ion then. could could be assumed flows flows across across the the membrane membrane must must also also reach reach low low values. values. This This appears appears to (Rudy and in the the Atlantic Atlantic salmon salmon (Salmo (Salmo salar) salar) egg egg (Rudy and Potts, Potts, to be so in 1969); 4Na uptake 1969);prior prior to to the the "eyed" “eyed” stage, stage, 224Na uptake was was confined confined to to the the zona zona radiata uid. The radiata and and perivitelline fl fluid. The latter latter also also was was found found to to concentrate concentrate cations in dilute cations when when immersed immersed in dilute external external solutions, solutions, which which would would tend tend to to reduce reduce the the concentration concentration gradient gradient across across the the plasma plasma mem membrane, yolk. of cations cations from from the the yolk. brane, and and therefore therefore the the potential potential rate rate of of loss of Potts 1973) noted noted aa similar similar pattern pattern in in early early eggs eggs of of Potts and and Eddy ((1973) Pleuronectes platessa; it Na by the it appears appears that that some some uptake uptake of of 22 22Na the yolk yolk may rst five may have have occurred occurred in in the the fi first five hours hours after after fertilization, fertilization, but but not not thereafter. thereafter. Zotin 1 965) found ve periods influx in Zotin ((1965) found fi five periods of of differing differing rates rates of of water water influx in the the decapsulated decapsulated eggs eggs of of sturgeon, sturgeon, beluga, beluga, and and sevruga. sevruga. The The first, first, between rapid wa wabetween fertilization fertilization and and gastrulation, gastrulation, was was characterized characterized by rapid ter In the ter uptake. uptake. In the second second period, period, from from gastrulation gastrulation to to yolk yolk plug plug clo closure, sure, water water uptake uptake virtually virtually ceased. ceased. In In the the third third period, period, from from yolk yolk plug plug closure closure to to the the appearance appearance of of the the heart heart anlage, anlage, water water uptake uptake was was rapid. rapid. In pulsation, some some water lost. In fth In the the fourth fourth stage, stage, to to heart heart pulsation, water was was lost. In the the fi fifth period, period, to to hatching, hatching, no water water uptake uptake occurred. occurred. One One may speculate speculate that that the with volume the first first period period coincides coincides approximately approximately with volume stabilization stabilization fol following lowing fertilization fertilization and and attenuation attenuation of of membrane membrane permeability. permeability. Ex Exchange change may may then then cease cease or or almost almost cease cease until until development development of of the the blas blastoderm toderm and and its its overgrowth overgrowth of of the the yolk yolk at at epiboly, epiboly, tissue tissue suspected suspected to to have 1966). have osmo-ionoregulatory osmo-ionoregulatory function function (Jones (Jones et al., 1966).
194 194
D. D. F. ALDERDICE ALDERDICE
XTERNAL M 2. E EDIUM 2. EXTERNAL MEDIUM
Composition of the external medium also influences the permea permeability of the plasma membrane. Potts and Rudy (1969) (1969) stripped mature eggs of Salmo salar into various media (3.5”C) (3.5°C) and compared their permeation characteristics. characteristics. Those in isotonic saline had a rate con constant (K) 0.4 h-1, (K)of 0.4 h-’, falling to 0.2-0.3 hh-’1 over 2-3 2-3 h. In isotonic l ), but these fell rap glucose initial K K values were higher (0.9-1 (0.9-1.0.0 hh-l), rapl idly to a rather constant level of 0.05-0. 1 hwithin about !1 h. In river of 0.05-0.1 h-’ water (0.45 (0.45 mM 1 1 mM - 1 de mM Ca, 0. 0.11 mM Mg/I), Mg/l), initial K K values near 0.5 hh-’ de2 + ions delayed clined to about 0. 1 h-1 0.1 h-’ within H li h. The presence of Ca Ca2+ onset of the initial stage of high permeability for a short time; high Na+ Na+ ion concentration delayed the onset indefinitely. Membrane per permeability was highest in solutions solutions containing 0.5-10 0.5-10 mM mM Call C d l and o 0lD 1971) found that the exchange exchange coefficient (E 10mM mM Nail. Ndl. Loeffler ((1971) (E = = PI) Pf) for water in the pike (Esox lucius) egg was greater in 100% (Esox lucius) 100% Ringer's Ringer’s 1 ) than in 7.5% .0 X solution (4 (4 xx 10 - 2 to 11.0 x 10-1 lo-’ /Lm pm ss-l) 7.5% Ringer's Ringer’s (1.8 (1.8 xX 2 /Lm S -1), although the comparison is confounded by 10 - 2 to 2.2 x 1 O2.2 x pm s-l), is differences in developmental stages. Berntsson et al. ( 1964) compared stages. al. (1964) the permeability of the plasma membrane of eggs of the amphibian Siredon mexicanum mexicanum after storage for various periods in 100% 100% or 7.5% 7.5% Ringer's solution (Table IV). Although the data are minimal, it can be Ringer’s (Table IV). seen that eggs stored in 100% 100% Ringer's Ringer’s (isotonic) (isotonic) retain a higher mem membrane permeability and probably for a longer period than when stored in 7.5% Ringer's. Ringer’s. Similar results were obtained by Haglund and L�vtrup (1966) oocytes and fertilized eggs at a later stage of develLZvtrup (1966) for oocytes TABLE TABLE IV
Permeability Coefficients Coefficients (IJ.m (pm S-I SKI) Plasma Membrane of Penneability ) for the Plasma Siredon mexicanum mexicanum Stored for Two Eggs of the Amphibian Siredon Eggs day, <1 day) in 100% 100% (Isotonic) (Isotonic) or 7.5% 7.5% Periods (>1 day, <1 day) (Hypotonic) Ringer's Ringer’s Solution Solution and Examined in 100% 100% or 7.5% 7.5% (Hypotonic) Ringer’s Ringer's Stored in
Tested in Tested (% (% Ringer's) Ringer’s) 100 100 7.5 7.5
100% 100% Ringer's Ringer’s
> day day >
<1 day day <1
(3) 11.16 . 16 (3)
0.84 (1) (1) 0.84
-
-
7.5% Ringer's Ringer’s 7.5% >11 day >
<11 day day <
-
0.52 (1) (1) 0.52 0.71 (3) (3) 0.71
(2) 0.49 (2)
the table table are are averages averages for for n= n=1-3 trials. Data from from Figures in the 1-3 trials. al. (1964). (1964). Berntsson et al. a
3. 3.
OSMOTIC AND IONIC IONIC REGULATION REGULATION IN TELEOST EGGS AND LARVAE LARVAE
195 195
opment in the same species species.. Variations Variations in the original trial values for - I) E for for eggs stored or examined in 7.5% 7.5% Ringer's Ringer’s (0.51-0.94 /Lm pm Ss-l) suggest that the three values on the right side of the table (7.5% (7.5%Ring Ringer's) er’s) are not significantly significantly different. Berntsson et al. al. (1964) (1964) also mea measured cortical stiffness in the eggs of Siredon. They attributed the decrease in permeability in hypotonic solution to an increase in stiff stiff(force required to deform the cell) of the egg cortex and vitelline vitelline55 ness (force membrane, and an increase in hydrostatic pressure within the egg that would occur in hypotonic media. One would conclude that the plasma membrane is tighter, and less permeant, in hypotonic media. Hansson Lflvtrup ((1974a) P dd in mature oocytes oocytes of the Mild and L�vtrup 1974a) determined P amphibian Rana temporaria, temporaria, stored in Ringer's Ringer’s solution, and exam examined in several dilutions of the medium. The P Pd d values were 1.57, 1.57, - l for oocytes in 100, 0.75 /Lm pm Ss-l 100,25, 7.5%Ringer's, Ringer’s, respec respec25, and 7.5% 11.07, .07, and 0.75 tively, over test periods of 2.8-4.6 h. The P Pd d estimates also were ob ob8-9 h for oocytes in the same three solu solutained continuously over 8-9 tions. In 100% .5 ± I /Lm 100%Ringer's, Ringer’s, P Pd d remained constant at 11.5 ? O. 0.1 pm Ss-’.I . In the dilutions of Ringer's, Ringer’s, P Pd d declined markedly with time more or less proportionate to the degree of of dilution; dilution; in 7.5% 7.5%Ringer's, Ringer’s, P P dd declined from 11.4 .4 to O.4 0.4 /Lm pm Ss-l1 in 8 h. These authors also computed the tension in the vitelline membrane as a function of oocyte radius and internal hydrostatic pressure. They reasoned that tonicity per se would have no direct influence on permeability of the plasma membrane and that tension in the vitelline membrane is indirect, resulting from the influ influence of tonicity on imbibition of water, change in egg radius, and internal pressure, all of which influence tension. Their results (Fig. (Fig. 2) 2) support this contention. They reasoned further that increased vitel vitelproportionline membrane tension is transmitted either directly or proportion ately to the plasma membrane, and they concluded that permeability of the plasma membrane (in the amphibian oocyte) oocyte) varies with the tension in the vitelline membrane surrounding it. It would seem rea reasonable to conclude that the same relation should apply to the plasma membrane and zona radiata of teleost oocytes and eggs. eggs. The internal pressure in an egg may vary considerably with species, state of devel develconditions, including salinity. Alderdice Alderdice et e t al. opment, and external conditions, ((1984) 1 984) measured hydrostatic pressure in the eggs of five salmonids salmonids throughout development (range (range when water-hardened, 30-90 30-90 mm Hg) 1 954) Hg) and compared the results with those of Kao and Chambers ((1954) for Fundulus (marine 150 mm Hg). (maiine species) species) eggs ((150 Hg). In addition, D. D. F. Alderdice and J. O. 0. T. T. Jensen (unpublished (unpublished data) data) obtained an internal 5
The outer egg membrane membrane in amphibians, amphibians, equivalent to the zona radiata radiata of teleosts.
196 196
D. F. ALDERDICE ALDERDICE
1.6
-
u V CIJ III " \
%
E
11.2 .2
.3-
5
Y
0. '0
nu
8
I
.@.
• 01 •d •
o*lt
o o�
Percentage 0 o
I),
""a A an
,0 . . . . .
•
-
�
0.8
.
I),
Ringers Ringers
1100 00
Sol ufion Solution o O
I), A
50
25 7.5 7.5
[J 0
I), A
0 [J
0 0 o 1), An 0 0
A I),
I), A I), A
I), A I), A
0.4
0 0
110 0
20 20
30 30
4 0 40
60 60
d ynes/em )1 TENSION TENSION ((dyneslcm
penneability coefficient Fig. 2. 2. Relation between the water permeability coefficient Pd P d and the tension of of the vitelline membrane in eggs of of the amphibian Rana Rana temporaria temporaria held in 7.5, 25, 50, and 100% 100% Ringer's solution. solution. Note the inverse relationship; relationship: as tension increases, Pd Pd decreases. [Modified 1974a).] [Modified from Hansson Mild and L�vtrup Lprvtrup ((1974a)J
pressure of 162 (8.5"C, 17%0), 17%), 162 mm Hg in 72-h-old Clupea pallasi eggs (8Se, first with internal pressures increasing in the fi rst 200 h of incubation from 103 to 188 188 mm Hg. These data show that the tension on the zona 103 x 10 lo33 and 11.1 x 104 lo4dyne cm-I, cm-l, approxi approxiradiata may vary between 5.8 5.8 X .1 x argumately a twofold change in tension. Therefore, following the argu Lzvtrup (l974a), (1974a),plasma membrane perme permement of Hansson Mild and Lpivtrup ability could vary considerably and inversely as a function of hydrostatic pressure. Although plasma membrane permeability reduces to and remains near a minimum during incubation, water exchange across across the mem membrane does not cease. cease. Loeffler and Lpivtrup L@vtrup(1970) (1970) calculated influx rates for developing eggs of Salrno Salma salar to be about 1I300th 1/300th of the egg day. Over the incubation period (-50 (-50 days), days), the total net volume per day. infl ux would be about one-sixth of influx of the egg volume. The salmon egg appears to come into an osmotic steady state with its external environ environment by a process whereby the concentration difference between the cytoplasm and the external medium is reduced by the sequestering of cations by the perivitelline fluid, and assisted by the increased hydro hydrostatic pressure resulting from water imbibition, sustained by the ten tension of the zona radiata. In addition, the increased tension in the zona radiata may be directly or proportionally transmitted to the plasma
3. 3.
OSMOTIC AND IONIC REGULATION TELEOST EGGS OSMOTIC AND IONIC REGULATION IN IN TELEOST EGGS AND AND LARVAE LARVAE
197 197
1974a) as a further means of of membrane (Hansson Mild and LZvtrup, L�vtrup, 1974a) interlimiting water permeation in the plasma membrane. This latter, inter esting observation should be examined further. coefficients of the plasma mem memIn summary, summary, water permeation coefficients relatively large in mature oocytes. They remain high at brane are relatively fertilization, and under normal circumstances, permeability declines fertilization, precipitously thereafter. This period of high permeability may allow the egg to attain a new set of steady-state steady-state conditions in relation to the fluxes that may occur across the plasma membrane as a various ion fluxes consequence consequence of of electrical electrical activity activity at at activation. activation. The The absolute absolute value of the the permeability permeability coefficient at at activation activation is is affected affected by the the nature nature of of the medium into which the egg is shed. In isotonic media, membrane permeability tends to remain higher longer and decrease more slowly. In hypotonic media, membrane permeability decreases at a rate re related lated to to the the level of of tonicity tonicity of of the the medium. medium. In In fresh fresh water, water, the the rates rates of of 2+ Ca2+ change are altered by ions in the medium; significant effects of Ca and Na+ Na+ ions have been demonstrated. However, the permeability coefficient does not vary directly with tonicity of of the medium. Rather, there appears to be a direct relation between P P dd and tension on the plasma membrane arising through intermediate steps involving imbi imbibition of water, change in egg size, and internal egg pressure. Hence, apas the hydrostatic pressure gradient increases, wall tension rises, ap pears transmitted to the the plasma plasma membrane, membrane, and and P P dd falls. falls. These These pears to to be transmitted differdifferences in permeability must be recognized when eggs of differ ent species or size are compared, compared, or when differences in tonicity of the permeexternal medium are involved. It appears that differences in perme ation coefficients may be small in isotonic media and large in hypo hypotonic media. However, However, ecologically tonic media. ecologically meaningful meaningful estimates estimates of of permea permeability coefficients require the use of media in which the egg normally is is found, found, aa point point frequently frequently recognized recognized but but one one that that has has seldom been been addressed permeation theory theory addressed in in the the long, long, complex complex development development of permeation and hyand methods methods of of measurement. measurement. Even Even then, then, minor minor differences differences in in hy dromineral content of fresh waters may significantly affect the course of change of permeation characteristics characteristics of the plasma mem memand rate of brane. 3. EMPERATURE 3. T TEMPERATURE Temperature is known to to influence influence membrane membrane permeability. permeability. Temperature also also is L$vtrup (1966) (1966) obtained exchange permeability coeffi coeffiHaglund and L�vtrup (E = =P P d) d ) for unfertilized eggs of the amphibian Siredon mex mexcients (E icanum in in Ringer's Ringer’s solution solution after after pre-adaptation pre-adaptation in in Ringer's Ringer’s at at each each
198 198
D. F. F. ALDERDICE
test temperature (4-28°C). values. increased from 0.1 - 1 at (4-28OC). The valuesqincreased 0.1 J1.m pm Ss-l 4°C, .S J1.m - 1 at 4”C, more or less linearly to �0.6 -0.6 J1.m pm S-1 s-l at 20°C, 2O”C, and to 11.5 pm Ss-l 28°C. Using two amphibians with different natural temperature 28°C. ranges, Rana temporaria (1O-2S°C) (10-25%) and R. pipiens (6-17°C), (6-17“C), Hansson Mild et al. 1974a) and Hansson Mild and L�vtrup 1974b) noted a al. ((1974a) Lplvtrup ((1974b) general trend toward increased plasma membrane permeability at higher temperatures. The Pf Pf values for mature oocytes ("body-cavity (“body-cavity eggs”) varied between 2.4 ± f 0.6 pm ss - l1 in 7.S, 7.5,25, 50% Ringer's Ringer’s at eggs") 0.6 J1.m 2S, and SO% 22-23”C, and 11.8 f0 0.1 pm Ss-l 7.5% Ringer's Ringer’s at 19°C. 19°C. An Arrhenius 22-23°C, .8 ± . 1 J1.m - 1 in 7.S% R.. temporaria oocytes ("ovarian plot of Pf Pi for R (“ovarian eggs") eggs”) in SO% 50% Ringer's Ringer’s (Hansson Mild et al., al., 1974a) 1974a) is complex: there is a local maximum at 16”C, a minimum at 19”C, 16°C, 19°C, and a trend to higher permeabilities at (Fig. 3A). 3A). In comparison, Fig. 3B higher temperatures (Fig. 3B shows a similar Arrhenius plot for Pd P d (Hansson (Hansson Mild and L�vtrup, Lplvtrup, 1974b) 1974b) for mature oocytes ("body-cavity oocytes (“body-cavity eggs") eggs”) for both ranid species at isotonic condi condi(100% Ringer's). Ringer’s). Not only do Pd P d values vary with temperature, tions (100% they also vary with tonicity of the medium as discussed in the previous section. It is worthy of note, as pointed out by Hansson Mild and L�vtrup Lplvtrup (1974a), (1974a), that the effect of of higher higher temperature on P P can be countered by an increase in plasma membrane tension, as occurs when the egg is exposed to a more hypotonic medium. medium. In summary, summary, osmotic permeability of the plasma membrane in increases at higher temperatures. The nature of this relation may also change with alteration of the tonicity of the external medium. Changes in permeability of ect complex tem of the membrane also refl reflect temperature-induced changes in rates of diffusion of water in the cyto cytoplasm, and in the external medium, on each side of the membrane (Hansson also recall argu (Hansson Mild and L�vtrup, Lplvtrup, 1974b); 1974b); these authors also arguments supporting the contention that structural changes changes in the proper properties of water may occur with temperature change. Phase transitions frequently are found in the 13-16°C 13-16°C range, suggesting an association with the discontinuities found for cytoplasmic diffusion and plasma membrane permeation at 16°C. 1974b) 16°C. Hansson Mild and L�vtrup Lplvtrup ((1974b) also note the well-known fact that the degree of of unsaturation of body lipids tends to be correlated with the general environmental tempera temperature range for a given species (Baranska There (Barariska and Wlodawer, 1969). 1969). Therefore, fore, the temperature-associated changes in the plasma membrane of the two ranid species examined, at least in part, may reflect a broad thermal phase transition in the structure or composition of membrane lipids.
TEMPERATURE ( O C ) 26
30
70
18
22
14
10
-
60:l\
A
R. temporaria
50 ... CII III "
E l:I.
40 -
30
-
20 3.30
3.34 3.34
3.38 3.38
3.42
3.46
3.50
lII/ T ((lo3 10 K K ))
3.54 3.54
3
TEMPERATURE TEMPERATURE (OC ("C)1 26
00
t
40
20
22 2 2
10 10
14 14
8 118
6
R R.. temporaria
B 8
10 8
� III
E 2"
6 4
\
2 2-
II 0.8 0.8 0.6 0.6
0.4 3.36 3.36
3.40 3.44
3-48 3.48
3.52
3
lV I T ((101 Fig. Fig. 33..
3.56 3.60
K)~ ~ 1 0
Arrhenius Arrhenius plots plots ooff permeability permeability coefficients coefficients ooff the the plasma plasma membrane membrane iinn two ranid species. (A) (A) Prestimates Rana temporaria PFestimates for ovarian eggs of of Rana temporaria in 50% 50%Ringer's. Ringer's. (B) Comparison of of Pd Pd estimates for body-cavity eggs (mature (mature oocytes) of of R. temporaria temporaria and R. R. pipiens pipiens in 100% 100% Ringer's. Ringer's. Note that the permeability permeability of of the oocytes is an order of of magnitude higher than that of the mature eggs. [Modified from Hansson Mild et et al. al. (1974a) and Hansson Mild and LJ<'.iVtru L@vtrup (1974b)J (1974a) p (1974b).J
200 200
ALDERDICE D. F. ALDERDICE
VOLUME 4. 4. EGG EGGVOLUME
One might assume that osmoregulation would pose a greater prob problem in smaller eggs, which have aa higher surface-to-volume surface-to-volume ratio than larger eggs. That is, given equal rates of water permeation across the plasma membrane, smaller eggs with their smaller volume would tend to come into a steady state with the external medium more rap rapidly than larger eggs. Dick (1959b) (195913)examined the question earlier; he found, initially, an inverse correlation between cell size and permea permeability coefficient in a variety of cell types and sizes. sizes. This could be expressed as a direct correlation between the permeability coefficient and the surface/volume (SlY) spheres) for groups of cells of (S/V) ratio (for (for spheres) of similar phylogenetic or histological type. When the same parameters were examined for all cells without grouping, the increased scatter in the pattern was suggestive of several confounded relationships. The confounding was removed when the diffusion coefficients coefficients for water movement in the cytoplasm were examined; examined; an inverse relation was found with SlY, S/V, the lower S/V ratio of the larger eggs being associated with higher measured values for diffusion of water in the cytoplasm. cytoplasm. Dick (1959b) -Pf (195913) suggested an attractive interpretation of the S/V SIV-Pj correlation: the apparent decrease in permeability in the plasma membrane of larger cells (with (with smaller SlY S/V ratios) ratios) is due to the greater length of time water takes to diffuse through the larger volume of is, measures of membrane permeability (P) (P) internal cytoplasm. That is, and cytoplasmic diffusion (D) (D) were confounded and interactive, each influencing the measured value of the other. The development of this problem and its resolution is well described by L�vtrup LZvtrup (1963), (1963), Hans Hans1972), Hansson Mild et ai. son Mild ((1971, 1971, 1972), al. (1974b), (1974b),and L�vtrup L g ~ t r u pand perHansson Mild ((1981). 1981). More recent determinations of membrane per cytomeability take into account the rate of diffusion of water in the cyto (01) and in the external medium (D (D2) (Hansson Mild and plasm (D1) 2) (Hansson LZvtrup, 1974b). 197413). L�vtrup, Lzvtrup (1963) (1963) confirmed Dick's argument, and Hansson Mild and L�vtrup LZvtrup (1985) (1985) describe how permeability of a cell membrane (P ( p d) d ) is a L�vtrup cell size. size. For function of both the rate of cytoplasmic diffusion and cell (r)of 100 100 fJ-m pm and Pd Pd = = 101, 101, example, for a spherical egg with radius (r) l loo,or 1010-11 fJ-m pm Ss-l, (01)must be below about - , cytoplasmic diffusion (D1) 10°, - l , respectively, before D 2 Ss-l, 103.5,102.5, 102.5,or 101.5 fJ-m pm2 D11 has a significant 103.5, effect on water exchange. exchange. In terms of cell size size and P p dd value of 101 10' fJ-m pm effect 100,10, ss-l, -1, cytoplasmic diffusion cannot be neglected in eggs of r= 100, 10, or l pm when D1 D1 values are below about 103.5, 103.5,102.5, 102.5,or 101.5 101.5 fJ-m pm22 Ss-1, 11 fJ-m - , respectively. respectively.
3. 3.
OSMOTIC AND IONIC REGULATION IN TELEOST TELEOST EGGS AND LARVAE
201 201
In summary, the direct correlation between the permeability coeffi coeffiS/V ratio discussed in the earlier cient of a cell membrane and its SlY literature is now considered spurious. There is an inverse curvilinear relation between Pd P d and Dl D1 such that Pd P d tends to be overestimated as a result of cytoplasmic diffusion at lower values of D uence of D1. influence 1 • The infl Dl D1 on Pd P d is greater greater in large cells (with (with a smaller SlY S/V ratio) ratio) when the tighter, At a given cell size, D membrane is tighter. Dl1 must decrease in order not to inflate the real value of Pd. P d . A further correction to earlier esti estimates mates takes into account the the presence presence of of stirred stirred or or unstirred unstirred layers layers external to the membrane (Dz). (D2). Hence, earlier estimates of perme of the permeability coefficient P were biased by the effect of Dl D1 and D2 Dz on perme permeation ation influenced by cell size. Yet, the bias may lie in how one inter interprets If one wishes to estimate the ultimate capacity prets the information. If for transfer of of water through a plasma membrane, then the peripheral effects effects of of cytoplasmic cytoplasmic diffusion, diffusion, and and of of unstirred unstirred layers layers external external to to the b e removed. On the other hand, if one wishes to membrane, must be know know how how aa given given membrane membrane capacity capacity is is utilized under under natural natural condi conditions, then the “biased” "biased" estimate, at least relative to D , probably D1, is 1 more meaningful. As an example of the differences involved, Prescott and Zeuthen (1953) P d of 0.75 (1953) obtained an uncorrected uncorrected estimate for Pd }Lm - 1 for mature oocytes (body-cavity eggs) of Rana temporaria in pm Ss-1 Ringer's l.6 }Lm - I ; when Ringer’s (22°C). (ZZOC). Corrected Corrected for for D D1, this rises rises to to 1.6 pm Ss-l; when cor cor1 , this rected Dl - 1 (Hansson 1 and and D2 D2 its its value is 2.6 2.6 }Lm p m Ss-l (Hansson Mild Mild and and rected for for D L�vtrup, reexamination and and adjustment adjustment of of earlier earlier uncor uncorLgvtrup, 1974b). 197413). A reexamination rected rected estimates, estimates, where where possible, possible, would would seem highly highly appropriate. appropriate. "bias" remains. of interpretation interpretation of of “bias” remains. The cor corHowever, the the question question of rected measure of ultimate capacity of of the rected value of P may provide a measure membrane; Db may may be more more mean meanmembrane; the the value, value, at at least least uncorrected uncorrected for for D1, ingful ingful in in terms terms of of how how aa given given capacity capacity is is utilized utilized by by the the living living cell. cell. 5. PflPd ATIOS Pf/Pd R RATIOS It It is recognized that water molecules may cross plasma mem membranes in two ways: through the matrix of the membrane by a solubilsolubil ity-diffusion ity-diffusion process, process, or or by by passage passage through through transmembrane transmembrane chan channels 1 984) gives examples of special materials that nels.. Finkelstein FinkeIstein ((1984) "sieve" having PflPd ratios of 5. Their P f / P d ratios of 3 to to 5. “sieve” nonelectrolyte nonelectrolyte molecules, molecules, having characteristics characteristics indicate indicate that that they they would would pass pass water water molecules molecules through through channels in file array. is discussed array. The question question is discussed by by Friedman Friedman channels in single single file ((1986). 1986). When solute molecules pass through a pore in single file, the factors factors that determine permeation permeation rate differ from those governing diffusion higher solute-solute larger pore, pore, and and the the probability probability of of higher solute-solute diffusion in in a larger
202 202
D. F. F. ALDERDICE ALDERDICE
interactions is greater. In the previously described relation, P PfIPd = IPd = VnW, where water molecules pass through a pore in single file and n Lpl Lp/V,,w, molecules fill fill the pore, Lea ((1963) 1963) shows shows that osmosis osmosis is n + + 11times more "efficient" “efficient” than tracer diffusion; that is, is, P Pff -- n + l1 _ = n + Pd pd
Hence the number of molecules needed to fill the pore is of of less consequence. consequence. If P Pff lP / P dd is large, transport takes place through large pores. In smaller pores the ratio will be closer to unity. When pores are very small or absent and solvent cannot pass through, water will cross cross the membrane by diffusion only, so that P Pfj = Pd. P d . Of importance 1, and a pore transport mechanism is implied. is the case when PrJP P f / P dd > > 1, implied, Some Some examples are examined from from this viewpoint. Prescott and Zeuthen ((1953) 1953) compared P Pff and Pd p d values for oocytes of the zebrafish (Brachydanio (Brachydanio reno) rerio) (a) (a) teased from the follicular membranes, and (b) (b) as eggs normally shed, without manipulation. In the former, PrJPd = 29.310. 68 (/-Lm - I ) gives a ratio of 43; in the latter, PrJ Pf/Pd=29.3/0.68 (pm Ss-I) Pf/ I Pd .3. At first glance it appears that Pd = = 0.45/0.36 (/-Lm (pm Ss-’),), aa ratio of 11.3. water permeation of the membrane of the teased (ovarian) (ovarian) oocytes (PrJ (Pf/ P Pd d = = 43) 43) would involve transmembrane channels, which are absent in the mature (body 1). Dick (1959b) (body cavity) cavity) egg egg (P ( pff IP / Pdd = 1). (195913)and others have questioned the validity of of this interpretation, suggesting that it re reflects flects injury to the plasma membrane resulting from from dissection of the oocytes from the ovarian tissue. Potts and Rudy (1969) (1969) calculated the oocytes fluxes across the plasma pIasma membrane in filtration and diffusional water fluxes the egg of Atlantic salmon (Salmo (SaZmo salar). salar). The ratio of these inflows inflows was about 4. The authors noted that correction for unstirred layers was not made, nor apparently was a correction made for diffusion in the cytoplasm. cytoplasm. The authors concluded that the plasma membrane was an differapparent barrier to diffusion. If the order of magnitude of the differ ence between corrected and uncorrected values of Of P P dd should be simi similar to that for Rana temporaria (Hansson (Hansson Mild and Lf1vtrup, L#vtrup, 1974b), 1974b), discussed in the previous section (2.6/0.75=3.5), (2.6/0.75=3.5), then the ratio found by Potts and Rudy (1969) (1969) for SS.. salar saZar would reduce from near 4 to about 11.1; . 1 ; on this basis one might conclude that channels were not involved in transport of water across the plasma membrane of the S. S. salar egg. egg. Hansson Mild et aI. al. (1974a) (1974a) also compared P PIf and Pd p d esti estiR. temporaria. temporaria. They obtained mates, using oocytes of the amphibian R. I (/-Lm S ) (Pm s-l) =
=
3. 3.
OSMOTIC AND AND IONIC IONIC REGULATION REGULATION IN TELEOST EGGS AND AND LARVAE LARVAE
P PfI - 2.4 k 0.6 0.6 2.4 ± = .83 =0 0.83 Pd
and
=
*
2.9 2.9 ± 0.5
k 0.3 0.3 11.8 .8 ± . 06 = = 11.06 1.7 ± ? 0.2 0.2 1.7
203 203
at 22°C
at
19°C
In this particular instance experimental error is taken into consider consideration, and there seems to be no justification in assuming PI P f> > P Pd; d; in PfIPd = 11.. each case PIPd In summary, n the literature where PIPd summary, there are few instances iin PfIPd comparison. It is possible that some ratios are capable of statistical comparison. eggs eggs may may possess possess transmembrane transmembrane channels channels;; further further data data on on teleosts teleosts (1984) con conFinkelstein (1984) would help clarify the question. Moreover, Finkelstein cludes that there are too few channels in most plasma membranes to provide a significant path for water transport, and that the major route across most plasma membranes must be through their lipid bilayers. permeabilHe also points out that most cells do not require high water permeabil find ities, and that one therefore would not expect to fi nd channels devoted to water transport in such membranes. It is of of interest to note that teleost egg plasma membranes are highly permeable to water when formaimbibition of water and processes related to perivitelline fluid forma hytion occur naturally, shutting down thereafter when the internal hy drostatic pressure gradient is higher and the water permeability of the plasma membrane is presumed to be at a minimum. Although there is impreslittle further evidence evidence to add regarding teleosts, the current impres sion sion that that there there are are no no special special mechanisms mechanisms devoted devoted to to transmembrane transmembrane Finkelstein’s ((1984) water movement does not appear to contradict Finkelstein's 1984) concl usions. conclusions. =
B. Influence Zona Radiata B. Influence of of the the Perivitelline Perivitelline Fluid Fluid and and Zona Radiata 1. F ORMATION 1. FORMATION The outermost eggs has outermost nongelatinous nongelatinous membrane membrane in in teleost teleost eggs has been been called called variously variously the the zona zona radiata, radiata, chorion, chorion, or or capsule. capsule. The The egg egg proper, proper, bounded is bathed bathed by the perivitelline perivitelline fluid fluid bounded by by its its plasma plasma membrane, membrane, is after after activation activation and and imbibition, imbibition, and and both both are are enclosed enclosed by by the the outer outermost membrane. Arguments regarding most membrane. Arguments regarding the the naming naming of of the the outermost outermost membrane are based primarily on its origin (Anderson, 1967). Here Here membrane are based primarily on its origin (Anderson, 1967). the outermost coat is called the zona radiata. The term "vitelline term “vitelline the outermost coat is called the zona radiata.
204 204
D. F. F. ALDERDICE ALDERDICE
membrane" membrane” also also is is used used in in the the nonteleost nonteleost literature literature for for the the outermost outermost egg membrane. To avoid confusion, the lipid membrane egg membrane. To avoid confusion, the lipid membrane here here has has been called the plasma membrane. The structure of the zona been called the plasma membrane. The structure of the zona radiata radiata varies varies substantially, substantially, particularly particularly between between marine marine and and freshwater freshwater te teleosts. Among the anadromous salmonids (Oncorhynchus, Salmo), the the leosts. Among the anadromous salmonids (Oncorhynchus, Salmo), zona main layers, layers, the the internus internus and and zona radiata radiata appears appears to to consist consist of two main externus; in some species the internus may be further externus; in some species the internus may be further subdivided subdivided (Groot 1985). These ex(Groot and and Alderdice, Alderdice, 1985). These authors authors believe believe the the very very thin thin ex (0. 1-0.3 JLm) primarily is responsible for the permeation ternus ternus (0.1-0.3 pm) primarily is responsible for the permeation char characteristics zona radiata, (30-60 acteristics of of the the zona radiata, the the internus internus and and the the subinternus subinternus (30-60 JLm), providing structural integrity to the zona radiata. pm), providing structural integrity to the zona radiata. Many Many marine marine teleost eggs smaller and zona teleost eggs are are smaller and possess possess aa proportionately proportionately thinner thinner zona radiata (Lanning, 1972; Stehr and Hawkes, 1979; Grierson and radiata (Lonning, 1972; Stehr and Hawkes, 1979; Grierson and Neville, 198 1 ). Neville, 1981). At in At fertilization, fertilization, and and as as aa consequence consequence of of the the changes changes occurring occurring in the the plasma plasma membrane membrane resting resting potential potential and and correlated correlated cytoplasmic cytoplasmic calcium calcium surge, surge, cortical cortical alveolar alveolar exocytosis exocytosis occurs, occurs, the the contents contents of of the the alveoli alveoli underlying underlying the the plasma plasma membrane membrane being being everted everted into into the the pre presumptive sumptive perivitelline perivitelline space space between between the the plasma plasma membrane membrane and and zona zona radiata. radiata. There There the the alveolar alveolar contents contents cause cause imbibition imbibition of of water water across across the the zona zona radiata radiata from from the the external external medium medium into into the the presumptive presumptive perivitelline space. The plasma mem perivitelline space. The zona zona radiata radiata lifts lifts away away from from the the plasma membrane brane by by displacement, displacement, and and some some shrinkage shrinkage occurs occurs of of the the egg egg proper. proper. The action of of The initially initially flaccid flaccid egg egg swells swells and and becomes becomes turgid turgid under under the action the colloid osmotic fluid. Swell the colloid osmotic pressure pressure of of the the forming forming perivitelline perivitelline fluid. Swelling ing continues continues as as water water and and ions ions enter enter the the forming forming perivitelline perivitelline fluid, fluid, until until aa steady steady state state ensues ensues between between the the increasing increasing tension tension of of the the zona zona radiata hydrostatic pressure pressure resulting osradiata and and the the increasing increasing hydrostatic resulting from from the the os motic pressure pressure difference difference between perivitelline fluid fluid and ex motic between the the perivitelline and the the exal. (1984) ( 1984) measured hydrostatic pressures ternal medium. Alderdice et aE. (Oncorhynchus, Salmo), Salmo), which varied with five in eggs of of fi ve salmonids (Oncorhynchus, species of development. In of SS.. gairdneri an effective species and stage stage of In eggs of filtration pressure (Starling, (Starling, 1895; Florey, 1966) 1966) of of 62 mm mm Hg Hg was was filtration pressure 1895; Florey, calculated, which would would result an efflux efflux of of water water and and ions ions from from the the calculated, which result in in an perivitelline fluid to the external medium against the established osos of water and motic gradient. The The expected rapid turnover of and ions asso associated ciated with this this effective filtration pressure is supported by evidence from a number of of sources (Potts (Potts and Rudy, 1969; 1969; Rudy and Potts, 1969; Loeffler and Lflvtrup, LjZlvtrup, 1970; Loeffler, 1971; 1971; Eddy, 1974). 1974). The 1969; 1970; Loeffler, tension of of the zona radiata, resulting from the internal hydrostatic of the perivitelline perivitelline fluid, pressure created by the osmotic properties of produces aa cushioned cushioned environment environment for for the embryo against against external external produces the embryo
3.
OSMOTIC AND IONIC IONIC REGULATION REGULATION IN IN TELEOST TELEOST EGGS AND LARVAE LARVAE OSMOTIC
205
deformation deformation and, as indicated earlier, a decrease in the permeability permeability of of the plasma membrane. INCLUSIONS PERIVITELLINE FLUID FLUID INCLUSIONS 2. PERIVITELLINE
( 1974) found the perivitelline fluid of of the Atlantic salmon ((S. Eddy (1974) S. salar) egg to consist of of 58% water, 25% protein, 12% 12% lipid, and 1.7% 1 .7% salar) if the colloidal osmotic pressure were carbohydrate. He suggested that if of the organic due to a single molecular species, the molecular weight of fraction of of the perivitelline fluid would be about 300,000. 300,000. Schuel ( 1985) noted ever (1985) noted that sea-urchin cortical granules prior prior to alveolar evermembrane-bound calcium, P-1,3-glucanase, ,8-1,3-glucanase, protease, sion contain membrane-bound con peroxidase, acid mucopolysaccharides, structural proteins that contribute to the hardening of of the fertilization envelope, and hyalin. DurDur tribute to ing and following imbibition, the zona radiata is impermeable, or almost so, to higher-molecular-weight substances such as dextran (Eddy, 1974). 1974). Hence, the mucopolysaccharides of of the perivitelline (Eddy, fluid, assumed to be largely responsible fluid's colloid osmotic responsible for the fluid’s pressure, are confined to the perivitelline fluid by the zona radiata. At macromolecular inin normal internal and external pH conditions, the macromolecular clusions likely will be above their isoelectric point and will be pre preten dominately acidic and negatively charged. Hence there will be a tendency for the perivitelline fluid to accumulate cations in excess of of in the external medium. Because the perivitel their concentrations in perivitelline fluid contains diffusible as well as nondiffusible ions, a Donnan type equilibrium will be established resulting in an electrical poten potential difference, the perivitelline fluid in freshwater eggs being nega negative relative to the external medium. In most marine teleost eggs the perivitelline potential is likely to be neutral to positive (Peterson and Martin-Robichaud, 11986). 986). Rudy and Potts (1969) Na+ a (1969) obtained for Na+ perivitelline fluid-to-external medium ratio of 11:: 11 for 150 150 mM NaClIl NaCM 0.1 NaCl/l the ratio was about 10 in the external medium. At 0. 1 mM NaClIl 10 :: 11.. In the more dilute condition an appreciable number of the available cations is is assumed to associate with the negative charges on the perivitelline fl uid colloids, thereby reducing the ionic excess excess (Peter fluid (Peter2 +, and H+ 2 son, 1 984). Under normal circumstances Na+, K+, Ca son, 1984). Na+, K+, Ca2+, Mg2+, H+ +, Mg probably are involved in establishment of these equilibria. The perivitelline fluid also also appears to change in composition as embryonic development proceeds. In newly fertilized eggs of of Clupea CZupea pallasi, p a l h i , the perivitelline fluid is is watery in appearance; as develop development proceeds it becomes increasingly viscous, suggesting that water content is decreasing or that larger molecule metabolites metabolites are are accumuaccumu-
206
D. F. ALDERDICE
lating. 1971) listed lating. Yarzhombek Yarzhombek and and Maslennikova Maslennikova ((1971) listed 18 nitrogenous nitrogenous metabolites S. trutta, metabolites that that leached leached from from the the perivitelline perivitelline fluid fluid of of S. trutta, S. S. gairdneri, Acipenser nudiventris, nudiuentris, and and Huso huso, ooff which which urea, urea, am ammonia, and ornithine were the major components. Among the remain remainder were 14 14 amino acids. These authors also reported previously hav having found creatine and creatinine in the perivitelline fl uid of fluid of eggs of far eastern salmon, presumably Oncorhynchus. OTENTIAL ERIVITELLINE P 3. P 3. PERIVITELLINE POTENTIAL
Peterson ((1984) 1 984) examined the perivitelline potential (PVP) (PVP) of the Atlantic salmon (S. ( S . salar) sa2ar) egg using microelectrodes filled with 4 M NaCl in 1% agar. The PVP is the voltage gradient between the perivi periviNaCI 1% agar. telline fl uid and the external medium, expressed by the Nernst equa fluid equation as a function of the cation concentration difference across the zona radiata. Varying [H+], [ H+], Peterson obtained for fertilized, water waterhardened eggs prior to the "eyed" “eyed” stage a value very close to Nernst - 56.5 mY/decade equation prediction ((-56.5 mV/decade change in [H+]). [H+]).Reversal of of potential occurred at pH=4.0, and maximum and minimum potentials of 80 and + 50 mV were obtained at [H+] 5.3 and -80 +50 [H+]equivalent to pH 5.3 pH 2.0, 2.0, respectively. In deionized water the change in potential for [H+], ] , and [Na+] 56, 40, [H+], [K+ [K+], “a+] was 56, 40, and 30 mV per decade change in concentration, respectively. Peterson suggests that these differences in response may reflect differences in mobility of the ions in aqueous solution, solution, resulting in differing permeabilities through the zona ra radiata. For Ca2+ Ca2+and Mg2+ Mg2+the change in potential followed a Nernst slope of 26-28 V decade - 1 ; a high selectivity coefficient suggested 26-28 m mV decade-’; that Ca2+ Ca2+ was being adsorbed at binding sites in the zona radiata. Departures from Nernst slopes were also noted for Na+ Ca2+ in Na+ and Ca2+ more acid media, indicative of cation interference where the measur measuring electrode is differentially more sensitive to H+, H+,relative to Na+ Na+ and Ca2+, water (pH (pH 6.8, Ca2+,at more acid pH conditions. In normal tap tapwater 6.8, Na+ Na+ 0. 1-0.2 mM, Ca2+ 44 mY. 0.1-0.2 Ca2+50 I'M, pM, Mg2+ Mg2+24 I'M) pM) the mean PVP was -44 mV. - 44 mY) The difference between the low value obtained in tapwater ((-44 mV) - 80 m and the maximum obtained in deionized water at pH 5.8 V) 5.8 ((-80 mV) could largely be explained by the residual levels of of [Na+] “a+] and [Ca2+] [Ca2+] occurring in the tapwater. The data indicate the presence of a net negative charge of the perivitelline colloids at normal (near 7.0) 7.0) pH, and a tendency for the perivitelline fl uid to concentrate cations. This fluid net negative charge would reduce to zero at a pH of 4.0. At pH 2.0 2.0 the of 4.0. system would be saturated with H+ 4.0 there could be H+ ions; below pH 4.0 a tendency for the perivitelline fluid to concentrate anions. For devel-
3. 3.
AND IONIC IONIC REGULATION REGULATION IN IN TELEOST TELEOST EGGS EGGS AND AND LARVAE LARVAE OSMOTIC AND
207
oping salmonid eggs, pH levels of of 6.0-6.5 or lower are of of increasing environmental concern with the growing acidification of of natural wawa ( 1985) found water uptake and resistance ters. Rombough and Jensen (1985) to deformation in eggs of gairdneri to be signiikancly of S. S . gairdneri significancly reduced at pH 5.5 5.5 and 6.0 6.0 respectively, although the trends in each case, after 24 h of of exposure to various [H+], [H+l,appear to begin at a threshold near pH 6.5. 6.5. Hence, in fresh waters inhabited by salmonids, the PVP likely could range between 45 and -80 -45 -80 mY, mV, depending on the ionic strength of of an ion species present present in the water involved, with Na+ Na+ and 2 + being the most important ions. Peterson and Martin-Robichaud Ca Ca2+ (1986) (1986)also examined PVPs in eggs of of Gadus morhua, the white sucker Catostomus commersoni, S. salar, commersani, and Atlantic salmon S. saEar, using micro microelectrodes filled with 3 M M KCl. The authors note that the smaller PVPs obtained with KCI electrodes likely are a function of of the greater simi similarity in mobilities of K+ K+ and CIC1- ions, compared with Na+ Na+ and CI C1ions. With KCI KC1 electrodes they obtained, respectively, for H+, H+,K+, K+,Na+ Na+ and Ca2+, 27, 19, Ca2+,perivitelline potentials of 40, 40,27, 19, and 15 15 mV per decade change in ion concentration for S. S. salar. The PVP values for Catosto Catostomus and Salmo 15 mV decade-I) Salmo were very similar ((15 decade-’) for [Cd2+] [Cd2+]be be5 M, tween 10-3 and 1050 and 10 mV lop5 M , zero slopes occurring near -50 -10 ( 10-6 and 1 0-2 M Cd2+), Cd2+),respectively. The PVP for Gadus became more positive as the external medium (30%0 (30%0sea water) water) was diluted; the response was attributed to diffusion of salts across the zona ra raPVPs were approximately 0 to +40 mV at [Na+] diata. For the cod, PVPs “a+] of 11 to 10-4 M, M , respectively. Plasma membrane potentials also were mea measured for eggs eggs of S. salar. salar. At "norma}" “normal” conditions of high ambient pH and and low ambient ion levels the yolk was was 25-30 25-30 mV negative to the perivitelline fluid. The plasma membrane potential so so measured was less responsive to change ] ((10 10 mV decade-I) change in [H+ [H+] decade-l) compared with the PVP (40 (40 mV decade-I); decade-’); the two measures would appear (Peterson (Peterson and Martin-Robichaud, Martin-Robichaud, 1986, 1986, their Fig. Fig. 5) 5) to converge at a potential near 70 mV at a pH near neutrality (pH -70 (pH -6.7). At pH 3.5 3.5 the plasma membrane potential was 60 m V relative to the perivitelline fluid and -60 mV 40 mV relative to the external medium of deionized water. Hence, -40 the uid and the potential difference difference between between the the perivitelline flfluid and external medium at 20 mY, at pH 3.5 3.5 should be about ++20 mV, a value obtained by Peterson and Martin-Robichaud (their (their Fig. Fig. 5) 5) and one one close to the Nernst estimate estimate (Peterson, (Peterson, 1984, 1984, his Fig. Fig. 1). 1).These These results suggest that that in the developing salmonid egg the plasma membrane has a low low per permeability and the plasma membrane uid potential is membrane-perivitelline fluid is -perivitelline fl much less less responsive to ambient pH compared with the potential between the perivitelline fluid and the external external medium. medium. The perivi-
208
D. D. F. F. ALDERDICE ALDERDICE
telline fluid fluid should have a substantial capacity to accumulate K+ K+ and Na+ Na+ ions in natural fresh waters. As the salmonid embryo may begin to Na+ from the perivitelline fl uid at the "eyed" (Rudy and fluid “eyed” stage (Rudy take up Na+ Potts, 1969), 1969), this accumulatory capacity could be of distinct advantage to eyed salmonid eggs and the resulting larvae, particularly in waters 2 +, Na+, of very low hydromineral content involving Ca Na+, and K+ Ca2+, K+ in particular, (Alderdice (Alderdice and Harding, 1987). 1987). Peterson and Martin-Robi Martin-Robichaud ((1986) 1986) question whether the PVP of the cod egg is the result of a Donnan mechanism or of an ion selectivity of the zona radiata. The substantial movement ofK+, of K+, Na+, Na+,and CIC1- ions across the zona radiata of G. 15 and 35%0 G. morhua eggs held in 15%0 15% salinity and transferred to 15 35x0 (Kandler ( E n d l e r and Tan, 1965) 1965) would seem to negate the latter suggestion. suggestion. From the results of Peterson ((1984) 1984) and Peterson and Martin-Robi Martin-Robichaud ((1986), 1986), several general comments may be made. ((1) 1 ) Over a pH range of 4.0-6.7 4.0-6.7 the plasma membrane of the freshwater teleost egg maintains a substantial negative potential relative to the external me medium. It appears to be a tight membrane with a low permeability to H+, (Table II) (Rudy and Potts, 1969). H+,as well as to water (Table 11) and Na+ Na+ (Rudy 1969). (2) (2) meThe perivitelline fluid is very responsive to H+ H+ in the external me dium; it appears to act as a buffer, minimizing changes in plasma [H+]in the membrane potential with reference to large variations in [H+] external medium. (3) (3)The ability of the perivitelline fluid to accumu accumulate hydromineral ions is maximized, relative to PVP, at neutral pH in strength, and minimized in acid medium. fresh waters of low ionic strength, advantageous to the developing salmonid em emThe former could be advantageous bryo, which may incubate in natural waters neutral to slightly acid and oflow of low ionic activity. Under these conditions the sequestering of phys physiologically important ions by the perivitelline fluid may be essential Confor continued embryonic development beyond the eyed stage. Con (“acid versely, the acidification of low-ionic-strength fresh waters ("acid rain”) may deny the accumulation of hydromineral ions at a level rain") sufficient for post-eyed-stage post-eyed-stage development. development. (4) (4) Relative to freshwater sufficient maincubation, where the PVP is substantially negative, that of the ma rine egg perivitelline fluid may be near zero or positive in estuarine waters. Hence, the marine egg perivitelline fluid may sequester an anwaters. C1-. (5) interpreions, such as CI-. (5) There remains some uncertainty in the interpre 2 + ions and their necessary 2 +, and Mg K+, Na+, Na+, Ca Ca2+, Mg2+ tation of the role of K+, levels during freshwater incubation. Brown and Lynam ((1981) 1981) show that S. S . trutta eggs incubating at low pH (4.5) (4.5) were most successful in waters containing calcium at 10 10 mg/l, independent of [Na+] “a+] (0, (0, 1, 10 1, 10 mg/l). After hatching, however, survival of alevins was high in water mg/l). mg/l). Eyed eggs of of Oncorhynchus Oncorhynchus tshacontaining sodium ((1 1 or 10 10 mg/l).
3. 3.
OSMOTIC AND IONIC REGULATION REGULATION IN TELEOST EGGS LARVAE EGGS AND LARVAE
209 209
(Swinehart and Cheney, 1984) 1984) wytscha exposed to acid pH conditions (Swinehart 2 + and Mg 2 + at pH 5.0, Ca2+ Mg2+ showed losses of Ca 5.0, exceeding those losses that water. Leaching of of primary amines at pH 5.0 5.0 did occurred in distilled water. water; some losses of amines could be not exceed the rate in distilled water; expected under normal conditions (Yarzhombek (Yarzhombek and Maslennikova, 1971). The fact that Swinehart and Cheney (1984) (1984) noted divalent ion 1971). losses within minutes of exposure to acid water would suggest the source of this loss to be the perivitelline fluid or desorption from zone radiata binding sites. sites. Bradley and Rourke ((1985) 1985) found a correlation Na+ plasma levels, and mortality, between elevated NHl NHt and reduced Na+ (Satmo gairdneri) gairdrzeri) cultured in low-ionic low-ionicin juvenile steelhead trout (Salmo “a+] strength fresh water. They suggested that low environmental [Na+] NH4f excretion occurring normally by an NHtl NH$ likely was inhibiting NHt Na+ exchange mechanism. Before the gills are functional, Na+ exchange functional, the block blockage of transfer of NHt +/Na+ or NHtINa+ NHd to the exterior via H H+/Na+ NHd/Na+ ion (Heisler, 1982) 1982)presumably could lead to acido acidoexchange mechanisms (Heisler, toxicity, and loss of regulatory capacity. capacity. Alderdice and sis, ammonia toxicity, 2 + and Na+ Na+ to hatchery water Harding (1987) (1987) found the addition of Ca Ca2+ of low ionic activity resulted in a signifi cant reduction in mortality of significant chinook salmon (0. (0. tshawytscha) alevins alevins.. In this instance, however, the problem is complicated by genetic and pathological components (Alderdice (Alderdice and Harding, 1987). 1987). The point to be made is that pH and ionic activity of fresh waters in which teleost eggs incubate may have a much more important bearing is currently realized. In this respect, more on incubation success than is work on the minimum requirements of hydromineral ions ions during in incubation in fresh water would seem highly appropriate. appropriate. The zero to positive perivitelline potentials obtained for eggs of marine species by Peterson (1984) 1986) indi (1984) and Peterson and Martin-Robichaud ((1986) indicate that major differences exist in the electrochemistry of the various compartments, compared to fresh water eggs. eggs. Yet, concentration dif differences between the embryo and the external medium in cod eggs (Gadus (Gadus morhua) rnorhua) obtained by Leivestad (1971) (1971) suggest a plasma mem membrane potential near -61 mV, not unlike that found in in freshwater 61 mY, eggs. eggs. In summary, a number of questions emerge whose further study could lead to an improved understanding of the role of the zona ra rauid on the function of the plasma membrane diata and perivitelline fl fluid division: during early cell division: macHow does the zona radiata inhibit the transzonal passage of mac romolecules, including those of the perivitelline fl uid? The fluid?
210 210
D. F. ALDERDICE
microvillar canals appear to be too large to inhibit such movements. Is the thin externus layer responsible for the semipermeable characteristics characteristics of the zona radiata? The relation between plasma membrane tension and permeabil permeability as influenced by internal hydrostatic pressure, first noted by Hansson Mild and Lfivtrup Lgvtrup (1974a), (1974a), is is unex unexplored. Low plasma membrane permeability during early cell division appears to be the major source of ionic and osmotic control. Factors that influence hydrostatic pres pressure, such as activity of the external medium, could lower hydrostatic pressure and increase plasma membrane per permeability. 2 +, and Mg 2 + on the What is the influence of H+, H+, K+, K+, Na+ Na+,, Ca Ca2+, Mg2+ electrical gradients between the yolk and perivitelline fluid, the yolk and the external medium, and the perivitel perivitelline fluid and external medium in freshwater eggs eggs?? What ions are involved in the foregoing relationships in marine eggs eggs?? It appears that the perivitelline fluid buffers [H+] [H+]in the external medium, thereby preventing large variations in the plasma membrane potential of eggs in fresh water. The perivitelline fluid accumulates accumulates cations in fresh waters of low ionic ionic strength, and salmonid embryos embryos begin taking up Na+ Na+ beginning at the eyed stage. stage. Is the sequestering of of cations essential to the later development of the embryo? If so, so, what are the characteristics of a water supply that would make these minimum requirements available? available? In other words, what are the hydromineral requirements in the ex external medium that are necessary for continued embryonic development? ? development? How are these influenced by [H+] [H+]?
C.. First Cell Division to Beginning of Epiboly C To recapitulate, during during development in the the ovary ovary the the oocyte oocyte ap apTo pears to be relatively permeable to water up to the time of activation. the transfer of nutrients and ions ions across across During that formative period the occurs mainly through the contacting the oocyte membrane probably occurs oocyte and follicular cell cell microvilli. Hence ionic ionic and osmotic osmotic control oocyte system. At ovula ovulalikely would be a function of the parental regulatory system. egg is is removed from intimate contact with the follicu follicution the mature egg cells, and becomes becomes free in the ovary ovary or body cavity cavity of the adult. lar cells,
3. 3.
OSMOTIC OSMOTIC AND IONIC REGULATION IN TELEOST TELEOST EGGS AND LARVAE
2 11 211
There the ovarian fl uid, under control ooff the adult regulatory system, fluid, maintains the egg in a medium very similar to that of the adult blood mem(Lam et al., al., 1982). 1982). Changes in ion permeability of the plasma mem brane prior to gamete maturation do occur, but they appear to be short-term short-term changes changes associated associated with with transitory transitory events events during during meiosis. meiosis. During this period the oocyte appears to be relatively permeable to K+ +, but K+ and and Na Na+, but independent independent of of Ca2+ Ca2+and and CIC1- in in the the external external medium. medium. At At activation, activation, and and concurrent concurrent with with increase increase in in free free [Ca2+]j [Ca2+Iiassociated associated with K+ permeability of the plasma with the the calcium wave, wave, is is an an increase in K+ membrane, both events being transitory. During imbibition plasma membrane membrane water water permeability permeability reaches reaches aa maximum, maximum, then then begins begins to to shut shut down, down, reaching reaching aa state state of of very very low low permeability permeability aa few few hours hours following following activation. activation. The The presence presence of of ion ion channels channels and and their their function function in appear to to be be in the the plasma membrane membrane remains remains enigmatic. enigmatic. K+ channels appear ubiquitous ubiquitous in in their their phylogenetic phylogenetic distribution, distribution, which which includes includes teleosts. teleosts. To provide a basis for further examination of potential regulatory capacity during development, three examples are illustrated in Fig. 4 of yolk osmoconcentration during incubation of of the plaice (Pleuronectes platessa) platessa) (Holliday 1967), Atlantic herring (Holliday and Jones, Jones, 1967), (Clupea (Clupea harengus) harengus) (Holliday (Holliday and and Jones, Jones, 1965), 1965),and and Pacific Pacific herring herring (C. (C. pallasi) (Alderdice (Alderdice et al., al., 1979). 1979). The plaice plaice eggs eggs (Fig. (Fig. 4A), 4A), fertilized fertilized 3.5, 50%0), show and and incubated incubated in in four four salinities (5, (5, 17.5, 17.5,3.5,50%0), show aa remarkable remarkable consistency yolk osmotic osmotic concentration 12 h onward. onward. consistency in in yolk concentration from from about about 12 For the first 132 132 h yolk osmotic concentration is highly uniform; from day 7 to day 13 13 it tends to rise, particularly particularly at the extreme salinities (5, (5, 50%0), 17). The au (day 1313-17). au50%0),yet recovery appears to occur thereafter (day thors thors suggest that that regulation regulation occurs occurs within within relatively relatively narrow narrow limits from fertilization fertilization to to hatching. hatching. Early Early mortality mortality occurred occurred in in salinities salinities lower 17.5%and and was was highest highest during during gastrulation gastrulation and and before before yolk yolk lower than than 17.5%0 plug plug closure. closure. Following Following hatching, hatching, yolk-sac yolk-sac larvae larvae tolerated tolerated salinities salinities ranging from 5 to 65%0 65% for 24 h, 10 to 60%0 60%0for 48 h, and 15 to 60%0 60% for 1 week. limits of sa week. Between Between yolk-sac yolk-sac absorption absorption and and metamorphosis, metamorphosis, limits of salinity 1 - 5 0 s for for 24 h and and 2.5-45%0 2.5-45760 for for linity tolerance tolerance shifted shifted downward downward to to 1-50%0 11 week week of this example example is of exposure. exposure. Of Of major major interest interest in in this is the the constancy constancy of It appears 12 h onward. onward. It appears from from Holli Holliof yolk yolk osmotic osmotic concentration concentration from from 12 day (1967) that that aa temperature temperature of of 7°C could could be applied applied to to day and and Jones Jones (1967) these 12 h probably these incubations: incubations: therefore therefore egg development development at at 12 probably would would not 1890). The The not have have proceeded proceeded beyond beyond the the early early blastula blastula (Fullarton, (Fullarton, 1890). osmotically yolk of osmotically equivalent equivalent salinity salinity of of the the yolk of the the unfertilized unfertilized oocyte oocyte was 10.5% and and of of the the activated activated egg egg at at fertilization fertilization about about 13%0. 13%0. was about about 10.5%0 From From examination examination of of rate rate constants constants for for water water permeation permeation across across the the plasma P . platessa (Table (Table II) 11) there there is is no no indication indication of of aa plasma membrane membrane in in P.
D. D. F. F. ALDERDICE ALDERDICE
212 212
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Fig. 4. 4. Changes Changes in in osmoticity osmoticity of of the the yolk yolk (A, (A, B, B, AGC; A'C; C, C, mOsm) mOsm) in in embryos embryos of of three three Fig. (A)Pleuronectes Pleuronectes plattessa plattessa eggs eggs fertilized fertilized and and teleosts throughout throughout the the incubation incubationperiod. period. (A) teleosts
5, 17.5, 17.5, 35, 35, and and 50%0. 50%~. (B) Clupea Clupea harengus harengus eggs eggs incubated in in each each of of four four salinities: salinities: 5, incubated (B) 35%and and tran�f transferred to 5, 5, 17.5, 17.5,35, 35,and and 50%0, 50%, 30 30 min min after after fertilization. fertilization. (C) (C) fertilized in in 35%0 fertilized erred to Clupea pallasi pallasi eggs eggs fertilized fertilized in in 20%0 20% and and transferred transferred to to 5, 5, 20, 20, and and 35%0, 35%, 10 10 min min after after Clupea
(A) between between 66 and and 12°C; 12°C;(Bl (B)between between 88 and and !l°C; 11°C;(C) (C)10 10 :t:2 fertilization. Temperatures Temperatures:: (A) fertilization. 0.1"C. Symbols: CRE, GRE, germ g e m ring ring at at equator; equator; YPC, YPC, yolk yolk plug plug closure; closure; E, E, eyed eyed stage; stage; H, H, O. IOC. Symbols: hatching. [Redrawn [Redrawn ffrom (A) Holliday Holliday and and Jones Jones ((1967); (B)Holliday Holliday and and Jones Jones (1965); (1965); hatching. rom (A) 1967); (B) (C)Alderdice Alderdice et et ai. al. ((1979).] (C) 1979).]
3. 3.
OSMOTIC TELEOST EGGS OSMOTIC AND AND IONIC IONIC REGULATION REGULATION IN IN TELEOST EGGS AND AND LARVAE LARVAE 213
700 600
100
c f------ 2 0 1.2
GRE
YPC
100
E
,
400 500 200 300 TIME FROM FERTILIZATION ( hr) Fig.
4.
H
5 5, 220 0, ,35 35 600
!i
700
3
(Continued)
more rapid than usual shutdown in membrane permeability. From the results of Guggino (1980b) (1980b) for Fundulus Fundulus and Peterson and Martin MartinG. morhua one might expect a yolk-to-perivitel yolk-to-perivitelRobichaud ((1986) 1986) for G. line fluid potential of +40 to + 50 mV in P. +50 P . platessa, and an increase in [Na+] "a+] and a decrease in [K+] [K+] over the first few days of development. Such evidence would indicate rather constant osmotic concentration, similar to that in P. P . platessa (Fig. (Fig. 4A). 4A). Although the argument is spec speculative, it appears that P. P . platessa maintains osmotic equilibrium fluxes and low water permeation rates, at through compensating ion fluxes least in the period between fertilization and the early blastula stage. stage. In comparison, (C. harengus) comparison, the Atlantic herring (C. harengus) egg (Holliday 1965) shows develand Jones, 1965) shows major differences in osmolarity during devel patopment (Fig. (Fig. 4b). 4b). Major differences in the osmotic concentration pat tern in this species involve the rapid change in yolk osmoconcentra osmoconcentration in the 112 2 h following fertilization, particularly at higher salinities, of maximum values between 12 12 and 96 h, and as well as the reaching of the subsequent recovery to a rather narrow, apparently regulated level at an osmotic concentration equivalent to 17.5%0 17.5%:0salinity. In this species there appears to be little or no regulation of the yolk in the 1212h period following fertilization. fertilization. The equivalent salinity of the unfertil unfertilized egg was 12.5%0. 12 and 96 h the yolk 12.5%. In the interval between 12
214
D. D. F. F. ALDERDICE ALDERDICE
becomes approximately isosmotic in relation to the external medium, except for the suggestion of some regulation occurring in the higher salinities (35, (35, 50%0). 5%). At the apparent incubation temperature of 7°C, TC, this period would include development of of the blastula and gastrula, epiboly, and growth to the stage of yolk plug nearing closure at about 84 h. Between 84 and 96 h (yolk (yolk plug closure, closure, 90 h), h), corrective trends begin and are well established by 120 120 h, at which time the embryo is well defined. In the initial 12-h 12-h period there must be a major ion infl ux influx into or water efflux from the yolk, followed between 84 and 96 h by the beginning of regulative function. function. Following hatching, herring lar larvae survived salinities of .4-60 . 1%0 for 24 h, and 2.5-52.5%0 of 11.4-60.1% 2.5-52.5s for 11 sa, one would conclude that the week. Compared with P. P . plates platessa, C. harengus egg must be considerably more plasma membrane of the C. permeable to influx of ions up to the development of of the blastula. It seems appropriate to suspect the development of some regulative ca capacity pacity in the cells of the blastoderm, particularly at higher salinities salinities.. Holliday and Jones ((1965) 1965) suggested that this capacity could relate to the intucking of blastula cells with the commencement of epiboly. From Guggino ((1980b) 1 980b) it would appear that regulative cells may be found in the cell sheet established with germ ring overgrowth of the yolk, yolk, as differentiated chloride cells are to be found in the resulting embryo. Depeche DBp6che ((1973) epithelium of the yolk sac of the later embryo. 1 973) found chloride cells in the superficial ectoderm of the blastodermal sheet during epiboly in Poecilia reticulata. The work of Holliday and Jones ((1965) 1 965) supports the contention that these cells become func functional prior to, and become an effective regulatory mechanism follow following, yolk plug closure. 1966) examined the epidermis of closure. Jones et al. al. ((1966) of C. C. harengus larvae 12-24 12-24 h after hatching in a search for cells with function. They examined the outer mem mempotential ion regulatory function. branes and buccal membranes of the head, the lateral and ventral yolk sac, and the tail, in larvae incubated and hatched in salinities of 5, 5, sac, 35, and 50%0. 50% In spite of their extensive examination of fine 117.5, 7.5, 35, epidermis, no special cells were found. Guggino structure of the epidermis, ((1980b) 1980b) found chloride cells in close association with the vitelline F . heteroclitus. He first first recorded their presence in blood vessels in F. days after fertilization (25°C), (25”C),a stage stage about 1-2 1-2 days after yolk larvae 44days functional plug closure and shortly before the pronephros becomes functional (Armstrong and Child, Child, 1965). 1965). The removal of excess excess ions ions from the (Armstrong yolk-sac circulation circulation would seem seem to be a natural and effec effecestablished yolk-sac Is there such such an association between tive approach to ion regulation. regulation. Is tive cells and subepithelial subepithelial Circulatory circulatory vessels, such as as those on chloride cells the yolk sac? sac? Might not these be present in herring embryos as as well as as
3. 3.
OSMOTIC REGULATION IN TELEOST TELEOST EGGS AND LARVAE OSMOTIC AND IONIC IONIC REGULATION
215
marine teleost embryos in general general?? However, if the chloride cells do appear after yolk plug closure, they cannot be the source of regulation seen in Atlantic herring embryos in the period between 12 12 and 84 h postfertilization (Fig. (Fig. 4b). 4b). One seems forced to reexamine the function of the blastoderm, which initially forms a two-layered cell sheet en enveloping the yolk during epiboly. Pacific pallasi) (Fig. (Fig. The third species compared is the Pacifi c herring (C. (C. pallasi) 4C). Alderdice et al. al. ((1979) 1979) fertilized Pacific herring eggs in sea water 4C). (20%0, 5°C) 20, and (20%0,V C ) and and incubated incubated groups of of these these eggs eggs at at 5, 5,20, and 35%0, 35%, the the transfer occurring 10 10 min after fertilization. Compared with Atlantic herring (Fig. (Fig. 4b), 4b), a greater similarity in response might have been anticipated for two forms whose similarities and taxonomic relations (species al. (1979) (species or subspecies) are still being debated. Alderdice et al. (1979) conclude salini conclude that Atlantic herring eggs are more tolerant of of higher salinities; ties; maximum maximum hatch hatch of of Atlantic Atlantic herring herring eggs eggs occurs occurs near near 25%0, 25%0,while while that in Pacifi c herring occurs near 17%0. Pacific 17% Under these circumstances yolk osmotic should be minimal throughout develop yolk osmotic changes changes should be near near minimal throughout development in both species at an external salinity near 20%0 (Fig. 4b, 17.5%0; 20% (Fig. 17.5%0; Fig. 4C, 20%0), 20%0),and they are. At 55 and 35%0, 35%0,respectively, initial under undershoot and overshoot of yolk osmolality occur more rapidly and reach a higher final level of osmotic concentration in 35%0 35% in the Atlantic herring. In both species stabilization of yolk osmoconcentration oc occurs curs at at or or shortly shortly after after yolk plug closure. closure. The The major major difference difference in response to external salinity occurs at this time. In the Atlantic herring there is a return from undershoots and overshoots toward a medium osmotic c herring osmotic value value near near the the equivalent equivalent of of 17.5%0; 17.5%; in in the the Pacifi Pacific herring egg egg in the same period (yolk (yolk plug closure to hatching) hatching) there is a slow increase in yolk osmolality and the egg appears to achieve a steady state at the equivalent external salinity of 12%0. 12%. An An inference inference to to be be drawn from the comparison is that the Atlantic herring egg plasma blastomembrane is more permeable and that the cells of the early blasto derm derm are are more more tolerant tolerant of of higher higher salinity salinity than than in in the the Pacific Pacific herring herring egg. egg. From the comparison of the eggs of the three species, the follow following inferences are drawn, which agree with Holliday and Jones ((1967) 1967) that that survival survival is is based based on on aa combination combination of tissue tissue tolerance tolerance and and regula regulation. tion.
Plaice. When plaice eggs Plaice. When plaice eggs are are transferred transferred between between salinities, salinities, mea measures of osmoconcentration of the yolk prior sures of osmoconcentration of the yolk prior to to gastrulation gastrulation ((Holliday Holliday and egg, blastodermal blastodermal cap, and Jones, Jones, 1967), 1967), and and of of egg, cap, and blastula cell size (Holliday, 1965), suggest and blastula cell size (Holliday, 1965), suggest the the plasma plasma
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membrane is relatively impermeable to water and ions from from fertilization fertilization to to yolk yolk plug plug closure. closure. The The individual individual blas blastula cells therefore may be less tolerant of internal ion con concentration centration changes, changes, particularly particularly at at low salinities. salinities. Atlantic herring. In similar transfers and from measures of yolk osmoconcentration (Holliday and Jones, 1965) 1965) and egg size in various salinities (Holliday (Holliday and Blaxter, Blaxter, 1960), 1960),the Atlan Atlantic tic herring herring egg appears appears to to be be more more poikilosmotic, poikilosmotic, the the plasma membrane being more permeable to water and ions ions.. The cells of the blastula therefore are presumed to be more tolerant of internal ion concentration changes. Pacific c herring appear to be interme PaciJc herring. Eggs of the Pacifi Pacific intermediate in response to salinity change, being less poikilos poikilosmotic than the Atlantic herring and less homoiosmotic than the the plaice. plaice. However, However, the the individual individual blastula cells cells are are as ascon sumed to be only slightly less tolerant of internal ion con(Aldercentration changes than those of the Atlantic herring (Alder 1979). dice et al., 1979). From the assumptions made in the foregoing comparison, the fol following argument will be examined. The first passive stage of "regula “regulation" tion” following fertilization may more properly be considered one of of resistive maintenance maintenance of the integrity of of the egg proper, achieved through water and ion permeation permeation characteristics of the plasma mem membrane. The second stage, and the first involving embryonic “tissue,” "tissue," is assumed to commence with development of the blastoderm, a tissue whose transitory regulatory function would be an assumption of of the role of the diminishing area of the plasma membrane, and provision of greater regulative capacity. The third stage, beginning with gastrula gastrulation, would seem to be an increasing restriction of water and ion transfer across the developing ectodermal layer of the blastoderm as it spreads to cover the yolk sac and pericardial region of of the embryo, ending with the appearance of of chloride cells near or following yolk plug closure. The fact that chloride cells have been found in some embryos at epiboly (Poecilia), (Poecilia),in later embryos (Fundulus), (Fundulus),and not at all in others ((C. C . harengus) harengus) suggests suggests natural natural flexibility in in the the regulatory regulatory process. process. If the three stages of embryonic regulatory development-involving development-involving successively the plasma membrane, the blastoderm, blastodem, and chloride cells-provide cells-provide aa regulative regulative function function of of greater greater and and growing growing effective effectiveness, it seems reasonable to assume that the basic ground ness, it seems reasonable to assume that basic ground plan plan would would not necessarily be followed by all teleost embryos in an identical
3. IONIC REGULATION 3. OSMOTIC OSMOTIC AND AND IONIC REGULATION
IN TELEOST EGGS IN TELEOST EGGS AND AND LARVAE LARVAE
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manner. manner. If, as as suggested, suggested, the the plaice plaice blastula blastula cells cells were were relatively relatively in intolerant tolerant of of ion concentration concentration extremes, extremes, the the egg egg might might compensate compensate by by providing providing greater greater conb'ol control over over water water and and ion ion fluxes fluxes across across the the limiting limiting cell membrane. Conversely, if herring blastula cells are relatively more ionic change more tolerant tolerant of of internal internal ionic change in in concentration, concentration, there there would would be less less need need to to maintain maintain aa homoiosmotic homoiosmotic environment environment around around the the cells, cells, as as suggested suggested by the the larger larger variations variations in in yolk yolk osmolarity osmolarity in in C. C. harengus and to a lesser extent in C C.. pallasi. The same argument would support the would support the apparent apparent lack lack of of chloride chloride cells cells in in the the yolk yolk sac sac and and C. harengus; it also would would suggest those cells pericardial it also suggest those cells pericardial epidermis epidermis of of C. should should be be present present in in the the plaice plaice embryo. embryo. Guggino's ((1980b) 1 980b) fi ndings in it seems seems highly highly Based on on Guggino’s findings in Fundulus, it appropriate should be found close associa chloride cells cells should found in in close associaappropriate that that the chloride tion vessels of of the the yolk yolk sac. sac. Their Their juxtaposition juxtaposition would would tion with with the the blood vessels support support removal removal of of Na+ Na+ and and CIC1- from from the the circulating circulating plasma. plasma. Follow Following nal stage ing this this pattern, pattern, the the fourth fourth and and fi final stage of of regulatory regulatory development, development, probably would see probably following following hatching, hatching, would see development development of of chloride chloride cells cells in rst appearance of in the the branchial branchial epithelium, epithelium, the the fi first appearance of of this this component component of the ultimate regulatory array (gills, (gills, gut, kidney) of of the adult teleost. Much Much of of the the foregoing foregoing is is circumstantial, circumstantial, based based on on incomplete incomplete evi evidence, (1)what what are are the the dence, yet yet it it serves serves to to pose the the following following questions: questions: (1) electrical electrical characteristics characteristics of of the the limiting limiting membrane membrane (the (the earlier earlier plasma plasma membrane) membrane) of of the the blastula blastula cells cells and and how how do do they they vary vary near near the the bound boundaries of (2) do the undifferentiated blas blasof the egg's egg’s salinity tolerance; (2) tula cells have c (3) what what specifi specific tula cells have an an ionoiono- or or osmoregulatory osmoregulatory function; function; (3) ion ion channels channels and and cell-to-cell cell-to-cell channels channels may may occur occur in in the the blastoderm; blastoderm; (4) cells first (4) when when do do the the chloride chloride cells first appear appear in in embryonic embryonic development; development; (5) ( 5 ) do do the the yolk yolk sac sac and and pericardial pericardial epithelia epithelia and and their their chloride chloride cells cells form (6) are are chloride chloride form the the primary primary embryonic embryonic ion ion transport transport system; system; (6) cells absent or individual cells cells absent or less less developed developed in in forms forms where where the the individual cells have (7)do do forms forms with with aa low low have aa high high tolerance tolerance to to salinity salinity change; change; and and (7) tolerance tolerance to to salinity salinity change change compensate compensate by by having having aa tighter tighter limiting limiting membrane? questions are membrane? These These questions are addressed addressed in in the the following following sections. sections.
1. PLASMA (LIMITING) MEMBRANE 1. PLASMA (LIMITING) MEMBRANE Activation egg increases Activation of of the teleost teleost egg increases internal internal free free calcium, calcium, possi possibly modifi ed by modified by membrane membrane potential-dependent potential-dependent changes changes in in Ca2+ Ca2+in infl ux (Nuccitelli, A temporary temporary incorporation incorporation of of K channels channels into into flux (Nuccitelli, 1980a). 1980a). A the occurs through the plasma plasma membrane membrane also also occurs through condensation condensation of of cortical cortical alveolar vesicular membrane (Hagiwara and Jaffe, 1979; 1979; Gilkey, 1981). 1981). In In addition, addition, transmembrane transmembrane movement movement of of Ca2+, Ca2+,K+, K+, and and Na+ Na+
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may occur in relation to changes in the membrane potential. The K+ K+ channels added to the membrane in Oryzias latipes eggs appear dur during hyperpolarization, reaching a maximum within about 11 min fol following sperm contact. The fast and slow recovery phases following hyperpolarization are coincident with removal of the K+ K+ channels, and hence K+ K+ permeability of the membrane returns to an undetectable Nuc level about 88 min after the conductance peak (Nuccitelli, (Nuccitelli, 1980a). 1980a). Nuc1 980b) also noted that the duration of citelli ((1980b) of the phase of increased K+ K+ conductance was dependent on voltage; the greater the level of of polar polarization in voltage-clamped eggs, the more rapidly K+ K+ conductance diminished. Conversely, eggs clamped at positive voltage levels re retained a higher conductance, suggesting that K+ K+ channels remained open. In addition, Na+ open. Na+ permeability of the membrane increased slightly as K+ K+ permeability fell during posthyperpolarization recov recovery. ery. Nuccitelli ((1980a,b) 1980a,b) indicated that increased ion fl uxes could occur fluxes between the yolk and cytoplasm and the external medium during activation. Yet these adjustments may be minimal in view of the tran transient nature of the electrical events. events. Further adjustments of of a longer but still transient nature must occur in the period of higher and attenu attenuating water permeability at and immediately after ((12-24 1 2-24 h) h) activa activation. Although there is evidence of increased Na+ Na+ and K+ K+ exchange from the eyed egg stage until hatching (Rudy (Rudy and Potts, 1969; 1969; Shen and Leatherland, 1978a), 1978a),there is little on which to judge the chronol chronology of of possible changes in limiting membrane permeability or ion channel function between the recovery phase after hyperpolarization and yolk plug closure. Apparently there is no evidence of po of action potentials during this period (Hagiwara and Jaffe, 1979). 1979). Since the rest resting potential of the limiting membrane relative to the external me medium is a function of of the concentration gradient, the lower the ionic activity of a freshwater medium, the more negative the resting poten potenconcentratial should be with respect to external hydromineral ion concentra tion. In S. S . salar this negative potential appears to reach an asymptote at a pH of 6.7-7.0 6.7-7.0 (Peterson (Peterson and Martin-Robichaud, 1986). 1986). Is it possi possible, at such levels (-50 80 mY), (-50 to -80 mV), that channels in the limiting membrane cells might be activated to open in response to physiologiphysiologi cal requirements for Ca2+, K+, or Na+ ? On the other hand, the period Ca2+,K+, Na+? from shortly after fertilization until the end of the blastula stage may be one of very limited regulatory capability, where a tight limiting membrane would provide a major defence of internal stability. stability. In acidified fresh water, or fresh water with a low hydromineral content-and content-and low buffering capacity-egg capacity-egg mortality can be rapid and
3. 3.
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substantial in the early egg stage. Over the first 26 days of develop development, mortality of s. S. trutta eggs at 8°C 4.5 was 90-100% in 8°C and pH 4.5 media containing CaCl 10 mg/ CaCl2z at 0 or 11 mg/l, and 10-33% mortality at 10 1, Na+ was added or not (0, (0, 1, 1, 10 10 mg/l of NaCl) (Brown 1, whether Na+ (Brown and 1981). A control with 10 of NaCl and CaCl CaC12, Lynam, 1981). 10 and 18 18 mg/l of z, 12%mortality. respectively, resulted in 12% mortality. Although NaCl NaCI addition had little influence on early egg survival, later survival of alevins was maximal at 10 10 mg CaCl CaCl2z and l-lO 1-10 mg NaC1/1. NaClII. These results suggest maximal z + in the incubation water is of that some minimum level of Ca Ca2+ of prime importance, while Na+ Na+ requirements probably are met from internal stores or from perivitelline fluid accumulation up to the eyed stage. stage. Peterson's 1984) study of S. salar, relative Peterson’s ((1984) of perivitelline potentials in S. to the external medium, indicates that lowering external pH and the attendant reduction in potential across the zona radiata can result in loss of cations from the perivitelline fluid. Yet under these conditions the yolk-to-external medium potential appears to remain substantially negative (Peterson and Martin-Robichaud, 1986). Hence, the limiting Martin-Robichaud, 1986). membrane appears to remain tight, even when the perivitelline fluid is less effective in acid media as an "ion “ion trap" trap” (Eddy (Eddy and Talbot, 1985) 1985) is in binding Na+ Na+ and other ions as as they diffuse from the embryo. Eddy and Talbot ((1985) 1 985) also found the Na+ Na+ level in the yolk of newly stripped eggs of S. S. salar to be higher than in the eyed stage, indicating (influx versus efflux) a low net loss (influx efflux) during early development. development. This loss was very small and unaffected by the presence of + ; however, of H H+; similar conditions at and after the eyed stage resulted in marked loss of Na+ Na+ from the embryo. In summary, the period between activation and the end of the blastula stage appears to be transitional in the teleost egg. egg. The oocyte of the adult through the prior to spawning is under regulatory control of be ovarian fluid; beginning with gastrulation, the embryonic tissue begins to assume this control (Fig. (Fig. 4). 4). For a short period during and z + , K+, following activation there could be movement of Ca Ca2+, K+, and Na+ Na+ ions across the plasma membrane concurrent with a high initial water permeability attenuating within 12-24 12-24 h. From that time until the end of the blastula stage it appears that the egg probably depends, for its internal regulation, on three interrelated factors: 1 ) control of factors: ((1) of water (2) tightness of permeation at a low rate, (2) of the plasma (limiting) (limiting) mem membrane to ion flows, flows, and (3) (3)compensatory control of these water and ion permeation rates relative to the innate tolerance of the blastula cells to z + seems osmotic and ionic variation. During this period external Ca Ca2+ essential for normal development, while internal stores of Na+ Na+ seem sufficient for development to the eyed stage. stage. The role of of K+ K+ during
220 220
D. F. F. ALDERDICE
this this period period is is less less clear. clear. An An examination examination of of the the electrical electrical properties properties of of the the plasma plasma (limiting) (limiting) membrane membrane during during the the blastula blastula stage stage would would be be instructive, instructive, particularly particularly regarding regarding the the presence presence and and function function of of spe specific mineral ions cific ion ion channels channels and and the the influence influence of of hydro hydromineral ions in in the the exter external nal medium. medium. Since Since ion ion concentration concentration gradients gradients may may differ differ between between the the yolk yolk and and perivitelline perivitelline fluid, fluid, and and between between the the perivitelline perivitelline fluid fluid and uenced and the the external external medium, medium, and and the the associated associated potentials potentials are are infl influenced by by pH, pH, aa comparison comparison of of intact intact and and dechorionated dechorionated eggs eggs would would also also be be useful. useful. 2. B LASTODERM 2. BLASTODERM Limited evidence suggests the blastula stage is one in which there is a major limitation in exchange of water and ions between the egg and the external environment, a holding action awaiting the develop development of new tissue with regulatory capacity. There is is growing evi evidence (Bennett et al., al., 1981 1981;; Caveney, 1985) 1985)that the individual blasto blastomeres begin very early to acquire properties properties associated with homeostasis, growth control, pattern pattern formation, formation, and tissue differentia differentiation through electrical coupling of adjacent cells. Electrical coupling assumes the presence of special pathways for current flow where the resistance between adjacent cells is known to be low. The sharing of electrical or chemical information by an array of cells is suggestive of of the function of a primitive tissue. In initial cell division in an egg this connection may occur over cytoplasmic bridges, where cell division is incomplete. In most instances, however, primary control of of intercellu intercellular communication appears to be associated with "gap “gap junctions" junctions” (Revel al., 1985) (Revel et al., 1985)between adjacent cells. Gap junctions form cell-to cell-tocell channels through adjacent plasma membranes. They are permeapermea ble to inorganic ions and small organic molecules with a diameter up 1.2 nm (molecular weight about 450-1500) to 1.2 450-1500) (Bennett et al., al., 1981). 198 1) . ( 1 984) estimates these channel diameters, using fluoresfluores Loewenstein (1984) cent labeled molecular probes, to be 16-20 16-20 A A (1.6-2.0 (1.6-2.0 nm). The chanchan nel behaves as though it has a fixed or induced charge, and it selecselec tively discriminates against negatively charged molecules. These gap 1980) or cell-to-cell channels junctions (Unwin and Zampighi, 1980) (Loewenstein, (Loewenstein, 1984) 1984) have been convincingly demonstrated by dye tracer studies, electron microscopy, and X-ray diffraction techniques (Fig. ( Fig. 5). 5). Gap junctions are found in most metazoan tissues, at least from annelids to mammals (Bennett et al., al., 1981). 1981). Four-cell mouse embryos are coupled in pairs by cytoplasmic bridges; genbridges ; coupling is gen eral and gap junctions are present present in the eight-cell stage (Dhcibella (Ducibella et
3.
OSMOTIC OSMOTIC AND AND IONIC IONIC REGULATION .REGULATION IN TELEOST TELEOST EGGS AND LARVAE
221
of connexons, units of closed (right). Fig. 5. Model of of gap junction, open (left) (left) and closed lattice and presumed constructed constructed of of protein oligomers, the Organized as a hexagonal lattice hi long, 25 hi unit is about 75 A A in diameter, and bridges a gap of of 30-40 hi A of of extracellular extracellular juxtapositioned cells. The central opening space between the plasma plasma membranes of of juxtapositioned 20 hi the radial displace displacewidens to a diameter of about 20 A at the cytoplasmic face. Closed, the threeA. The unit acts as a three ment of each subunit (cytoplasmic side) side) would be about 6 hi. closed. [Reprinted by dimensional iris diaphragm with two apparent states, open and closed. (1980),copyright copyright 1980, Jourpermission from Nature, Unwin and Zampighi (1980), 1980, Macmillan Jour nals Limited.]
ai., 975; Lo and Cilula, al., 11975; Gilula, 1979). 1979). Early Fundulus blastomeres re-form junctions within minutes of cell separation and reaggregation (Ne'eman (Ne’eman et al., 1980). 1980). Hagiwara (1983) (1983) noted that in the tunicate Halocynthia HaZocynthia roretzi roretxi all blastomeres are electrically coupled by the 1616to 64-cell stage. In the limpet (Patella), (Patella),gap junctions appear at the two-cell stage but junctional communication may not occur until the 32-cell stage (Caveney, (Caveney, 1985). 1985). Blastoderm cells of Fundulus show a rapid distribution of injected fluorescent dye, indicative of cell-to-cell communication (Bennett et al., 1978). 1978). Increase of [H+]i [H+Iireduces the conductance of treated cells, as does an increase in either internal or external [Ca2+] ; the sensitivity of gap junctions to Ca2+, [Ca2+]; Ca2+,however, is much lower than that to H + .. Blastomeres may be uncoupled by appli H+ application of polarizing current and channels may close with membrane Gap junctions are stable in the open state (Loewen (Loewendepolarization. Cap stein, 1984), 1984), whereas open time in other types of channel is is of short duration. Dye molecules that cross cross high conductance junctions do do not cross cross when the junctions are are held at low conductance by transjunc transjunctional voltage. In a comparison with amphibian eggs, eggs, Bennett et al. al.
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D. F. ALDERDICE
((1981) 1981) also note that there is very little junctional voltage dependence in teleost eggs. Cell-to-cell recognition and adhesion prior to junction formation appear to require the presence of of Ca2+ Ca2+or Mg2+ Mg2+and a glyco glycoprotein on the membrane surface. The junctional permeability of of vari various mammalian cell types in culture increases when [cyclic AMP] is is elevated internally. internally, This increase, which depends on protein synthe synthesis, is correlated with the number of of gap junctions present (Loewen (Loewenstein, 1984). 1984). At later stages of development, patterns of of junctional communica communication appear in which "communication “communication compartments" compartments” form (Caveney, (Caveney, 1985). 1985).Within such compartments there is low resistance to intercellu intercellular communication, while at the periphery of a compartment, com communication with an adjoining compartment is severely restricted. The gap junctions of these border cells have a reduced junctional permea permeability; they effectively block cell-to-cell movement of tracer dyes, al al(Caveney, 1985). 1985).The outer mem memthough ionic coupling is unaffected (Caveney, branes of border cells that form enveloping layers may be of very high resistance, and be joined at their apical surfaces by "tight “tight junctions" junctions” 'eman et al., (Ne (Ne’eman al., 1980), 1980),which prevent ion flow as well as water perme permeation (Bennett et d., al., 1981). 1981). Loss of cell coupling through gap junc junctions also appears to occur during final stages of of cell differentiation that lead to normal physiological function in mature tissues (Caveney, 1985). 1985). The function of specific ion channels and gap junctions in the blastomeres of of teleosts is not yet well understood. Gap junctions in compothe blastomeres and blastoderm may be only one of various compo nents contributing to the later development of homeostasis. There of ion entry into the yolk of of the appears to be little or no limitation of Atlantic herring (C. (C. harengus) harengus) egg during initial cell division and at least to the end of the bIastula blastula stage (Fig. (Fig. 4b); 4b); evidence suggests gap junctions could be functional during this period. Based on available evidence, the following inferences are drawn regarding regulatory developments from first cell division to the end stage. Presumably gap junctions between blastomeres of the blastula stage. may appear as early as the third cell division, division, and the junctions appear to provide for the establishment of ionic and osmotic stabilization within the compartments of connected cells. The outer enveloping border cells form tight junctions, which would limit ion and water transport between the cell compartment interior and the perivitelline fluid. Perhaps the differences in particle transport across across the apical fluid. compartments of the three species cell surfaces of the developing compartments
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illustrated in Fig. 4 is related to species variation in the function of of the of tight junctions between enveloping layer cells and the permeability of their apical membranes on the one hand, and tolerance of of the cell cytoplasm to salinity (osmotic and ionic) variation on the other. As suggested by Holliday and Jones ((19651, 1 965), the in-tucking of blasblas of gastrulation produces a cell sheet of of two tula cells at the beginning of layers, the inner layer changing polarity in the process. The cells at of the blastoderm uncouple selectively from the yolk in the margin of Fundulus (Caveney, 1985) in the late blastula, just prior to gastrula (Caveney, 1985) gastrulaof gap junction channels and tion. The uncoupling involves closure of would appear to create two communication domains, the blastoderm and the syncitial periblast. Recent evidence (Balinsky, 1975) suggests (Balinsky, 1975) that the "in-tucked" “in-tucked” cells of of the endomesoderm actually are formed by a rearrangement of of deep-lying cells within the blastoderm. Hence, with gastrulation, gastrulation, compartmentalization could result in the establish establishment of three major communication domains-the domains-the outer "firmly “firmly ad adherent" herent” covering layer of the blastoderm (Balinsky, (Balinsky, 1975), 1975), the deeper blastoderm, and the intermediate endomesoderm. Evidence indicates that the outer blastodermal layer would be relatively impermeant. impermeant. The periblast would be involved with yolk metabolism. Hence, any regulatory activity would seem of necessity to be restricted to the deeper blastoderm or the endomesoderm. Positionally, the endome endomesoderm would seem to be unfavorably located if if it were to have a regulatory role. In F. F . heteroclitus the cells of the enveloping layer have typical gap junctions in their lateral lateral and basal membranes. The deeper blastodermal cells cells also also have gap junctions, both in the blastula and gastrula, but in contrast to the enveloping layer, there are no tight junctions (Ne'eman (Ne’eman et al., 1980). 1980). In In the amphibian Xenopus, Xenopus, the blas blastomeres during early cell division show electrogenic sodium pumping (Turin, (Turin, 1984); 1984);there is little evidence evidence of active ion transport in early teleost embryos embryos prior to gastrulation. gastrulation. In summary, summary, it seems that embryonic regulation per se does not begin in the blastula, although compartmentalization compartmentalization probably signals signals the beginning of tissue formation. formation. As As suggested earlier, unmodulated “ "regulation" regulation” may occur from from the eight-cell eight-cell stage to the end of the blastula stage through limitations limitations on particle transfer provided by by the high resistance and tight junctions of the cells of the enveloping layer of the outer blastoderm. The The establishment of osmotic regulation be beginning after gastrulation, gastrulation, and in place place by yolk plug closure (Figs. (Figs. 4b, 4b, 4c), 4c),indicates indicates that that embryonic embryonic regulation begins with with epiboly, the the stage at which chloride cells have first first been observed (Depeche, (DBpkche, 1973). 1973).
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D. D. Epiboly to Hatching From From the the large large number number of of investigations investigations conducted conducted on on aspects aspects of of ionoiono- and and osmoregulation osmoregulation in in juvenile juvenile and and adult adult teleosts teleosts (Evans, (Evans, 1979, 1979, 1980), 1980), it it is is well well recognized recognized that that the the renal renal complex, complex, the the gut, gut, and and the the branchial branchial epithelium epithelium are are primary primary sites sites of of regulation. regulation. The The evolving evolving integument integument would would be be included included among among these these primary primary sites sites of of regula regulation tion (Marshall (Marshall and and Nishioka, Nishioka, 1980; 1980; Marshall, Marshall, 1985). 1985). All All of of these these sys systems tems are are as as yet yet undeveloped undeveloped at at gastrulation. gastrulation. From From the the rather rather imper impermeant meant limiting limiting membrane membrane of of the the blastula blastula and and gastrula gastrula (Bennett (Bennett et al., 1981) 1981) arises arises the the next next stage stage in in the the development development of of regulatory regulatory capac capacity-the ity-the appearance appearance of of chloride chloride cells cells in in the the integument, integument, likely likely cen centered in the yolk-sac yolk-sac epithelium, and becoming functional at the earli earliest est in in the the period period from from beginning beginning of of epiboly epiboly until until shortly shortly after after yolk yolk plug closure.
1. THE CHLORIDE CELL 1. T HLORIDE CELL HE C Shelbourne ((1957) 1 957) was the first to show that the integument of the platessa) was the probable site of teleost larva (plaice, (plaice, Pleuronectes platessa) iono- and osmoregulation in teleosts. He found activity in the whole ionoof chloride cells, but particularly in the integintegument, suggestive of integ ument of of the yolk sac. sac. Since then chloride cells have been found in number of the integument of of a number of embyronic and larval forms, summasumma rized by Hwang and Hirano (1985) ( 1985) to include (a) (a) saltwater speciesspecies P . platessa), niphobzes), red seabream (Pargues plaice ((P. platessa), puffer (Fugu (Fugu niphobles), (Pargues major), major), northern anchovy (Engraulis (Engraulis mordax), mordax), sardine (Sardinops (Kareius bicoloratus); bicoloratus); (b) (b) freshwater caerulea) [[So sajax], and flounder (Kareius caermlea) S . sajux], species-carp (C (Cyprinus carpio); (c) (c) anadromous or estuarine formsforms species-carp yprinus carpio); gairdneri), killfish (Fundulus (Fundulus heteroclitus), heteroclitus), rainbow trout trout (Salmo gairdneri), reticulata), and ayu (Plecoglossus altivelis). altivelis). Chloride molly (Poecilia reticulata), of the cells were not found in the embryonic or larval integument of harengus), nor in the anadromous coho Atlantic herring (Clupea (Clupea harengus), salmon (Oncorhynchus kisutch). kisutch). In many of of these studies embryos were examined at intervals and it is not possible to determine the developmental stage at which chloride cells first appeared. Nor is the incubation temperature always given, from which one might estimate the developmental stage of of interest. In such descriptive studies, incuincu bation bation temperatures and descriptions of of embryonic stages allow concon firmation or comparison and should always be given. The function of of “chloride-secreting "chloride-secreting cells,” cells," first proposed by Keys
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OSMOTIC TELEOST EGGS EGGS AND OSMOTIC AND AND IONIC REGULATION REGULATION IN IN TELEOST AND LARVAE LARVAE
225
and Willmer ((1932), 1932), has until recently been the subject of of continuing (1980) showed that physiologi physiologicontroversy. Marshall and Nishioka (1980) (Gillichthys mirabilis) mirubilis) skin cally important chloride current in teleost (Gillichthys epithelium, as varies directly with the density of chloride cells in the epithelium, detected by fl uorescence microscopy. 1982), fluorescence microscopy. Foskett and Scheffey ((1982), using a technique that localized conductance and chloride current with with reference reference to to individual cells, cells, now now have have shown shown rather rather conclu conclu(Oreochromis mossambicus) mossambicus) [Sarotherodon [Sarotherodon mos mossively in the tilapia (Oreochromis sambicus] sambicus] [Tilapia [Tilapia mossambica] mossambica] that that the the so-called so-called chloride chloride or or mito mitochondria-rich cells indeed are the site of salt transport. In the teleost species studied, there appears to be considerable variation in the location and time of appearance of embryonic and larval integu larval chloride cells. cells. They They have have been found found primarily primarily in the the integusac, on the ment covering the epicardial region of the anterior yolk sac, sac, and ( 1981) notes the yolk sac, and in in the the tail tail region of of the the trunk. trunk. O'Connell O’Connell(l981) the close close association association of of the the yolk-sac yolk-sac blood blood vessels vessels with with the the yolk yolk syncytium syncytium and the yolk-sac yolk-sac ectoderm in the anterior portion of sac. of the yolk sac. In summary, summary, chloride-cell chloride-cell density density varies varies between between integumental integumental regions and between regions and between species. species. The The cells cells may may develop develop or or degenerate degenerate at at various ages, or following transfer between saltwater and fresh water. They may appear during epiboly, at yolk plug closure, at the eyed stage, or shortly before hatching, and they may disappear in the postpost yolk-sac stage, presumably with concurrent development of branchial and As yet yet the the data data are are too too frag fragand other other adult-type adult-type regulatory regulatory tissues. tissues. As mentary to allow close definition of the chronology of of chloride-cell chloride-cell development tissue. The development in in extra-branchial extra-branchial tissue. The interesting interesting point point is is the the fact cells provide fact that that these these cells provide continuity, continuity, with with increasing increasing complexity, complexity, between between the the cellular cellular property property of of very very limited limited transmembrane transmembrane particle particle exchange of the the exchange in in the the blastomeres blastomeres and and blastula, blastula, and and the the final final function function of tissues constituting the regulatory apparatus of the juvenile and adult. 2. SSTRUCTURE TRUCTURE AND F UNCTION IN ALT W ATER AND RESH 2. FUNCTION IN S SALT WATER AND F FRESH W ATER WATER Zadunaisky 1 984) recently Zadunaisky ((1984) recently reviewed reviewed the the structure structure and and function function of of the in the the chloride chloride cells cells in the teleost teleost branchial branchial epithelium. epithelium. The The morphol morphology cells (D6p&che, (Depeche, 1973; 1973; Guggino, ogy of of extrabranchial extrabranchial chloride chloride cells Guggino, 1980b) 1980b) is less well documented. On the other hand, Hwang and Hirano ((1985) 1 985) conclude cell types conclude that that the the same same general general cell types occur occur in in both both integumental integumental and morphology of of the the and branchial branchial tissues. tissues. On On that that basis basis the the general general morphology chloride cell cell may 1979) illustrated from from the the studies studies of of Sardet Sardet et al. ((1979) chloride may be illustrated
226 226
D. F. F. ALDERDICE ALDERDICE D.
and 1980) (Fig. and Sardet Sardet ((1980) (Fig. 6) 6 ) on on mullet mullet (Mugil capito), capito), eel eel (Anguilla (Anguilla anguilla), anguilla), trout trout (Salmo (Salmo gairdneri), gairdneri), killfish, killfish, (F. ( F . heteroclitus), heteroclitus), and and the the molly [Lebistes reticulatus] reticulatus]. molly Peocilia reticulata [Lebistes IIn n the integument, integument, the chloride chloride cell underlies and protrudes through through an an outer outer enveloping enveloping layer layer of of epidermal epidermal cells. cells. Discontinuity Discontinuity in in the the surface surface epithelial epithelial cells cells surrounding surrounding aa chloride chloride cell cell forms forms an an orifice orifice or or "pore" “pore” over over the the chloride chloride cell cell apical apical surface. surface. The The apical apical surface surface
.
Fig. 6. 6. Schematic drawing of of chloride cells of of the branchial epithelium in fresh water water (FW) and saltwater (SW). (SW). With some minor minor differences differences the sketch is representarepresenta tive of chloride cells in the embryonic integument. C, Chloride cell; A, adjacent, accesacces sory, or small chloride cell; R, epithelial cell (in the integument); TVS, tubulo-vesicutubulo-vesicu lar system; M, mitochondrion; mitochondrion; BLM, basolateral basolateral membrane membrane (serosal side); side); AP, apical pit of of the chloride cell (mucosal side); (1) (1) tight tightjunction junction between between chloride cell and adjacent junction between epithelia1 epithelial cell; (2) leaky junction between chloride cell and adjacent or smaller chloride cell. The interdigitations between between these cells provide provide channels for the the TVS to commucommu nicate nicate via the extracellular extracellular spaces (ECS) with with the the apical pit. pit. It is also assumed that that vesicles of of the TVS may communicate directly directly with with the apical apical membrane in both both fresh water of Cell BiolBiol water and and saltwater (3). (3). [Reproduced (slightly modified) from the the Journal of ogy, ogy, Sardet et al. (1979), ( 1979), by by copyright copyright permission permission of of the the Rockefeller Rockefeller University University Press.] Press.]
3. 3. OSMOTIC AND IONIC REGULATION
IN TELEOST EGGS EGGS AND IN TELEOST AND LARVAE LARVAE
227 227
forms a pit or crypt, most obvious in seawater-adapted cells of older fonns individuals; there may be little or no concavity to the crypt in fresh freshindividuals; water-adapted cells or in seawater-adapted larvae. The body of the cell is rich in mitochondria and tubules of the tubulovesicular system (Fig. 6). 6). The latter is an extension of the bastolateral border (Sardet (Sardet et (Fig. al., at., 11979) 979) and extends throughout the body of the cell as far as the region below the apical pit, which is relatively free of mitochondria ouabainand tubules. The tubulovesicular system contains many ouabain binding sites. sites. Sardet et al. ((1979) 1979) speculated that small projections on the tubule walls may be Na+,K+-dependent Na+,K+-dependentATPase pumping units, Na+-coupling ouabainwith the N a+ -coupling site on the cytoplasmic side and the ouabain K+-coupling sensitive K+ -coupling site toward the lumen of the tubule. Recent (Karnaky, 1986). 1986).The tubu tubuautoradiographic studies support this view (Karnaky, lar system appears to terminate in small vesicles below the apical crypt; these vesicles could be secretory in function. Sardet et at. al. (1979)also suggested that the vesicles could be a means of transport of (1979) particles between the tubular reticuli and the apical membrane, acros acrosss the "vesicular-tubular" “vesicular-tubular” space. Larger molecules do not pene penetrate the space; space; the function of the vesicles and the vesicular-tubular vesicular-tubular space remains somewhat uncertain, although there is little doubt that chloride efflux efflux occurs across the apical membrane in seawater seawateradapted fishes (Degnan, (Degnan, 1986; 1986; Karnaky, Karnaky, 1986). 1986). In both freshwater- and seawater-adapted individuals the epithe epithelial cells of the enveloping layer fonn form tight junctions between them themselves selves,, and with adjacent chloride cells cells at the apical surface. surface. The tight junctions appear to fonn uxes, limiting or form a barrier to ion or water fl fluxes, flows between paracellular pathways (Fig. preventing flows (Fig. 6, 6, EeS) ECS) and the mucosal side side of the epithelium. The junctions are deep (0.2-0.4 f.Lm), pm), consist of five to nine anastomosing strands, and are presumed to improvide the high electrical resistance characteristic of other tight, im penneant permeant epithelia. In freshwater-adapted freshwater-adapted teleosts the apical pits generally are associ associated with single chloride cells. In seawater adaptation the accessory cells adjacent to the chloride cells differentiate to form "“small small chlo chlocells”; these are sheet-like sheet-like in cross cross section. Such accessory cells cells ride cells"; have not been reported in freshwater forms. forms. The pluricellular pluricellular chlo chloform interdigitations in the apical region, and ride cell complexes fonn (paracellular) channels (Fischbarg et at., al., 1977; extracellular (paracellular) 1977; Degnan and Zadunaisky, 1980; 1980; Lewis et at., al., 1984) 1984)fonn form between the adjoining chloride cells, pathways not present in freshwater-adapted cells. In terminate seawater-adapted cells the extracellular channels appear to tenninate at leaky junctions in the apical crypt (Fig. (Fig. 6). 6).
228
D. F. ALDERDICE D. F. ALDERDICE
In seawater-adapted seawater-adapted cells cells there there are are large large internal internal and and external external fluxes 1986) suggests the following model for the fluxes of NaCl. NaC1. Karnaky ((1986) a+ ,K+ chloride cell. The primary driving force force is the tubular system N Na+,K+Na+ gradient ATPase (primary (primary active transport), which creates a large Na+ with high concentrations in the tubular system lumen and low con concentrations in the cell cytoplasm. Na+ Na+ entering the cell is recycled for K+ K+ via the Na+,K+-ATPase. Na+,K+-ATPase.This gradient drives a (secondary (secondary active transport) NaCI carrier, also located in the tubular membrane; CI transport) NaCl C1enters the cell via the carrier and diffuses to the mucosal side through the apical membrane into the apical crypt. The blood (serosal is (serosal side) side) is electropositive relative to sea water, and Na+ Na+ ions concentrating in the tubular reticulum would diffuse down the concentration gradient to the paracellular pathways, to exit at the mucosal side through the leaky junctions between the chloride cell and accessory cell interdigi interdigitations into the apical crypt. Foskett et al. al. ((1982) 1982) note that chloride secretion by the chloride cell can be modified by two different hormohormo nally controlled pathways: (1) (1)slow changes in the number and differ differentiation of the cells, as described by Hwang and Hirano (1985), (1985), and (2) (2) rapid changes by direct effects on the cell transport mechanisms. 1984), Numerous examples of the latter are given by Zadunaisky ((1984), Foskett et al. 1982), and Degnan (1986), al. ((1982), (1986), including epinephrine, so somatostatin, cortisol, prolactin, urotensin I and 11, II, and phosphodies phosphodiesterase inhibitors. In freshwater and euryhaline species the situation remains un unclear. However, Karnaky ((1986) 1986) made an interesting observation in F. F. heteroclitus, adapted for several weeks to 1% 1% seawater. seawater. The chloride cells remained in pluricellular complexes and shared apical crypts. He noted a new type of deep, tight junction between the cells. In such epithelia, placed in normal Ringer's Ringer’s solution, these deep tight junc junctions disassembled within 30-50 30-50 min to the shallow junctions charac characterizing chloride cells from seawater-adapted fish. Laurent and Dunel 1980) provided some provocative information Dune1 ((1980) on chloride cell function in the gills of of teleosts (Anguilla (Anguilla anguilla, S. S. gairdneri, Mugil cephalus, cephalus, F. F . heteroclitus) heteroclitus) in fresh water. They noted that in gill development there are two major tissues: primary and secondary epithelium. The former covers the primary lamellae and the interlamellar region, and the latter the free part of the secondary lamellae. lamellae. Chloride cells are associated largely with the primary epi epithelium. In S. S. gairdneri larvae, at hatching, the embryonic primary lamellae are sheathed in pavement cells, and interspersed among them are fully developed chloride cells cells.. Chloride cells are not incor incorporated into the epithelium of the secondary lamellae. When transfer-
3. 3.
OSMOTIC AND IONIC IONIC REGULATION REGULATION IN IN TELEOST TELEOST EGGS AND LARVAE LARVAE OSMOTIC AND EGGS AND
229 229
red to seawater, the fish may show an accelerated development of of “ fast renewal cells” in seawater acclimation, and these cells originate "fast cells" waepithelium. Fish transferred to deionized wa from the inner primary epithelium. ter show a proliferation of chloride cells in the secondary epithelium (S. gairdneri, A. 10% seawater, many of (S. A. anguilla). anguilla). On their return to 10% (S. gairdneri) degeneration. Trout (S. the chloride cells showed signs signs of degeneration. proliferamoved to deionized water show an intensive ion loss and a prolifera (“freshtion of chloride cells, apparently in the secondary lamellae ("fresh type”).. Eels transferred to seawater show a proliferation of chlo chlowater type") ride cells in the primary epithelium, but not in the secondary epithelium. Transfer of fishes to deionized water stimulates ion flux 2 + removal. Therefore the acute development of secondary la Ca2+ laby Ca mellae chloride cells could provide a compensatory mechanism for increasing net ion uptake in fresh water. It would be of interest to know whether external acid pH conditions (acid (acid rain) rain) might also pro provoke the acute development of secondary lamellar chloride cells. In that regard, Perry and Wood ((1985) 1985) showed that calcium uptake in S. S. 2 + 36.07 gairdneri in freshwater (Ca (Ca2+ 36.07 mg!l, mg/l, Na+ Na+ 46.75 46.75 mg/I) mg/l) occurred at equal equal rates rates through through the the gills gills and general general body body surface. surface. Exposure Exposure of of 2 + fl 2 + ((1 the fish to low Ca 1 mg/l) ux, which was correl Ca2+ mg/l) increased the Ca Ca2+ flux, correl2 + ] re ated proliferation of of lamellar chloride chloride cells. cells. Plasma [Ca [Ca2+] reated with with a proliferation 2 mained constant regardless of the external [Ca + ] . However, Na+ Na+ and [Ca2+]. C1- initially were reduced internally, but recovered in 7 days. Hence, CINaC1, it while chloride cells in saltwater-adapted fish serve to excrete NaCl, cells may would seem that in freshwater-adapted fish similar chloride cells Na+ and Cl-. C1-. The differences between promote the net influx influx of Na+ seawater and freshwater adaptation, in relation to chloride-cell func function, may require a close examination of the origin and location of branchial (and (and extrabranchial) extrabranchial) chloride cells and their relation to par particular compartments of the circulatory system, as hypothesized by Laurent and Dunel 1980). Dune1 ((1980). Hwang and Hirano ((1985) 1985) compared survival with morphology of chloride cells in the skin and branchial epithelium of the freshwater ayu (Plecoglossus altivelis), altiuelis), carp carp (Cyprinus (Cyprinus carpio), carpio),and and marine marine floun flounayu (Plecoglossus der (Kareius bicoloratus) in in seawater seawater (30%0), (30%0),fresh fresh water, water, and and in in trans transder (Kareius fers between the two media. In transfers of larval ayu to seawater or larval flounder to fresh water, these authors found that interdigitations and leaky junctions may form or be lost rapidly (3 h). Transfer of ( 3 h). seawater-adapted individuals to fresh water generally, but not always, resulted in the degeneration of chloride cells or a reversion to the structure within a few days. days. No interdigitations typical freshwater cell structure or leaky junctions were found in the chloride cells of larval ayu incu-
230
F. ALDERDICE ALDERDICE D. F.
seawater at at intervals intervals up up in fresh fresh water. water. Ayu6 Ayu6 eggs, eggs, transferred transferred to to seawater bated bated in 120 h following fertilization, showed no survival to hatching (17( 17to 120 20°C). When transferred at or after 144 144 h of of incubation, survival to 20°C). 2 1-28%. Apparently these embryos hatching rose to about about 21-28%. embryos formed interdigitations and leaky junctions within 24 h after transfer as 144-h 144-h embryos. embryos. Freshwater-incubated ayu, challenged at hatching, ap appeared peared to to begin begin developing developing interdigitations interdigitations and and leaky leaky junctions within 3 h; after 24 h these chloride cells showed the morphological characteristics of fish. Juvenile (60-day) of seawater-adapted fish. (60-day)ayu in fresh water showed no interdigitations or leaky junctions; transferred to seawater (30%0), modification of of (30%),they died within 66 h and showed no modification the epithelium. epithelium. However, transfer to salinities below 20%0 20%0 resulted in limited formation of of interdigitations and leaky junctions. Larval floun flounder incubated in seawater possessed interdigitations and leaky junc junctions between integumental chloride cells. Newly hatched fl ounder flounder larvae transferred to fresh water or mild seawater «(<10%0) 10%0) were unable to survive; survive; chloride cells showed either abnormalities or no morpho morphological change, while the integument was severely disrupted. disrupted. Juve Juvenile (60-day) ounder showed typical interdigitations and leaky junc (60-day) fl flounder junctions in seawater. Transferred to lower salinities or fresh water the juveniles survived and the interdigitations and leaky junctions began to degenerate within 3 h; after 3 days, chloride-cell morphology was typical of freshwater cells cells.. Finally, juvenile carp showed no interdigi interdigitations or leaky junctions in fresh water. When juveniles were trans transferred to dilute saltwater these structures did not form, form, there was extensive damage to the skin and branchial epithelium, and the juve juveniles did not survive beyond 12 12 h in 15%0. 15%. In the three species, species, sur survival was correlated with the elaboration or degeneration of chloride cells cells following transfer between external media. In this regard, the juvenile carp tolerated only fresh water. Larval or embryonic embryonic ayu gen generated chloride cells, but that adaptive ability seems to be lost in the juvenile. Larval fl ounder could not tolerate fresh water but the juve flounder juveniles were relatively euryhaline. euryhaline. The results of Hwang and Hirano ((1985) 1985) parallel those of Sardet et al. 1979) and Sardet (1980). al. ((1979) (1980).Interdig Interdigitations and leaky junctions occurred occurred in those species species normally found in seawater, seawater, and not in those restricted to to fresh water. In species Kashiwagi eett al. al. (1986) (1986) have have reported reported on the relation relation between temperature temperature and and 6 Kashiwagi 50% hatch hatch in in the ayu ayu egg. egg. The Schnute Schnute ((1981) general growth growth time to to 50% development time 1981) general model provides provides the follOWing following relation relation between between their their data data (their Table Table 5) 5) and and tempera temperamodel of 11-26°C 11-26°C:: tures of tures 1) 1 4556. Y (days) (days) = = [9.2092 r9.2092 - [[6.3693(1-e-0."24sXvc 1~37281114556. 6.3693(I - e - O.I248X·C 1.3728 + +
3. 3.
OSMOTIC AND IONIC REGULATION REGULATION IN TELEOST LARVAE TELEOST EGGS AND LARVAE
231 231
showing evidence of eurhalinity these structures either formed or degenerated after transfer. Although differences in adaptability varied with species, age, and embryonic salinity experience (ayu), (ayu), there seems little doubt that chloride cells, either integumental or bran branchial, were involved with regulation and survival. The observations of Hwang and Hirano (1985) (1985) regarding morpho morphological changes in the chloride cells, after adaptation of of the organism to saltwater or fresh water, are convincing. However, the assumption that morphologically shallow (leaky, (leaky, one-to-four-junctional strand) strand) and deep (tight, junctions are and deep (tight, Hve-to-nine-junctional five-to-nine-junctional strand) strand) junctions are fully fully cor correlated functionally with high and low permeabilities may be too sim simplistic. plistic. Where Where there there are are leaky leaky junctions junctions between between the the cells, cells, paracellu paracellular cellular conductances lar conductances conductances are are greater greater than than trans transcellular conductances.. With With tight tight junctions, junctions, the the paracellular paracellular pathway pathway is is more more resistive. resistive. In spite spite of of atthe distinctive differences between the two types of junction, at tempts tempts to to correlate correlate junctional junctional resistance resistance with with the the number number of of junctional junctional strands (Friedman, 1986) . 1986). strands remain remain arguable arguable (Friedman, Depeche D6peche (1973) (1973) found found the the yolk yolk sac sac and and pericardial pericardial epithelium epithelium were homeostasis in were involved involved in in embryonic embryonic homeostasis in the the live-bearer live-bearer molly, molly, Poecilia 1001 PoeciZia reticulata. Chloride Chloride cells cells occurred occurred at at aa density density of of 8080-100/ 2 on the yolk sac, and 60-80/mm mm mm2 60-80/mm22 on the pericardial epithelium in 3.5-mm 3.5-mm embryos. embryos. Embryos Embryos exhibited exhibited osmoregulative osmoregulative capacity capacity after after the the seventeenth seventeenth day day of of development. development. Guggino Guggino (1980b), (1980b), who who found found chloride cells in close association yolk chloride cells in close association with with the the vitelline vitelline vessels vessels in in the the yolk sac epithelium in F.. heteroclitus heteruclitus embryos, embryos, found found no no sac and and pericardial pericardial epithelium in F chloride cells in the branchial epithelium before hatching. In F F.. he heteroclitus (7-, lO-day) termlitus dechorionated (7-, 10-day) embryos and in hatchlings, [Na+]j [Na+Iitended tended to to increase increase while while [K+l [K+Iiand and [CI-]j [Cl-Ii tended tended to to decrease decrease toward toward hatching. hatching. In intact intact eggs eggs the the [Na+ “a+] ] and and [K+ [K+l] of of the the perivitelline perivitelline fluid fluid were were similar similar to to that that of of the the seawater seawater medium; medium; that that in in the the pericar pericardial the dial cavity cavity resembled resembled the the seawater-acclimated seawater-acclimated blood blood plasma plasma of the adult. adult. From From transmembrane transmembrane potentials potentials and and ion ion replacement replacement trials, trials, Guggino K+ and and Na+, Na+, Guggino concluded concluded that that the the embryos embryos were were permeable permeable to to K+ the former more so than the latter, and that the epithelium was cation cationselective. The trans epithelial potential could not be explained by sim transepithelial simple diffusion, as expressed by the Goldman equation, suggesting Na+ efflux is diffusional, C1strongly that while Na+ Cl- is moved out of the emembryo by electrogenic pumping. Guggino concluded that the em bryo CI- excretion bryo maintains maintains osmotic osmotic and and ionic ionic balance balance by by Na+ Na+ and and C1excretion across yolk sac sac and and embryonic embryonic cavity, cavity, the the site site of of across the the epithelium epithelium of of the yolk ion transport moving to the gills later, after hatching. Guggino l 980a) made Guggino ((1980a) made aa further further observation observation of of major major interest, interest, rere-
232
D. F. F. ALDERDICE
garding water water balance balance in in the marine marine embryo. embryo. If If the the osmolality osmolality of of an an garding embryonic tissue tissue fluid, fluid, for for example, example, is is 350 mOsm/kg mOsm/kg and and that that of of the the embryonic perivitelline fluid fluid is is 600 600 mOsm/kg mOsm/kg (e.g., (e.g., Fig. Fig. 4C), 4C), then then the the tissue tissue fluid fluid perivitelline is hypotonic hypotonic relative relative to to the the perivitelline perivitelline fluid fluid and and the the gradient gradient of of 250 250 is mOsm/kg between between them them will will tend tend to to remove remove water from the the tissues. tissues. If If mOsm/kg water from the embryonic embryonic epithelium epithelium is is not not completely completely impermeant, impermeant, which which is is aa the reasonable generalization, generalization, there there will will be aa tendency tendency for for the the embryo embryo to to reasonable lose water. water. Guggino Guggino estimates estimates these these losses losses in in F F.. heteroclitus heteroclitus at at 2.6 2.6 and and lose 12% per per day day for for 4.74.7- and and 10-day-old lO-day-old embryos, embryos, respectively. respectively. Juvenile Juvenile 12% and adult adult teleosts teleosts tend to balance such water water losses losses by by drinking drinking or or and tend to balance such presumably by using using the fluid intake intake provided provided by by their their food, food, removing removing presumably by the fluid the ions so so gained gained and eliminating them them through through the gills, gills, kidney, kidney, and and and eliminating the ions region of of the 10-day F F.. heteroclitus embryo (ZSOC), (25°C), gut. IIn n the head region Guggino found lateroventral lateroventral pores, pores, anterior anterior to the pectoral pectoral fins. fins. These These Guggino found to the were open to to the perivitelline fluid fluid and and connected bran were open the perivitelline connected via via paired paired branchial chambers chambers and and embryonic embryonic gill gill arches arches with with an an open open pharynx, pharynx, chial which extended forward forward to to aa position position posterior posterior to to the eyes. These These which extended the eyes. structures, in in rudimentary rudimentary form, in the 4-day embryo (as 4-day embryo (asstructures, form, are are present present in sumed pharynx anteriorly anteriorly sumed 1-2 1-2 days days after after yolk yolk plug plug closure), closure), and and the the pharynx and undeveloped. In In the lO-day embryo embryo the the and the the gut gut posteriorly posteriorly are are undeveloped. the 10-day mouth is still nonfunctional; the gut is formed, but it is a blind sac, closed is aa channel available channel available closed at at the the cloaca. cloaca. Hence, Hence, in in the the embryo embryo there there is for fluid to to enter the pharynx pharynx and and gut. gut. There There water for the the perivitelline perivitelline fluid enter the water presumably absorbed, from from which ions would would be be returned via the the presumably is is absorbed, which ions returned via integumental chloride cells the perivitelline fluid. One surely integumental chloride cells to to the perivitelline fluid. One must must surely question question whether whether aa similar similar water-conservation water-conservation mechanism mechanism may may be found in larvae of of other marine teleosts. Development of salinity tolerance in eggs of the freshwater ayu (Hwang and Hirano, 1985) 1985)provides a possible interpretation for simi simi(Clupea lar observations in embryonic development of Pacific herring (Clupea pallasi). Duenas Duefias ((1981) herring eggs at two tempera temperapallasi). 1981) incubated Pacific herring (13, 221, 29%0),and cultured the (6, 12°C) 12°C) and three salinities (13, tures (6, 1 , 29%0), resulting larvae at three temperatures (6, (6, 9, 12°C). 12OC). Estimates of salin salinity Dso, %0) ity tolerance (72-h (72-h E EDSO, %o) were obtained for samples of newly hatched 18 incubation-culture incubation-culture conditions. conditions. Incu Incuhatched larvae larvae for for each each of of the the 18 bation salinity (IS), (IS), incubation temperature (IT), (IT), and the IS x x IT interaction had a significant effect on salinity tolerance of the larvae. Larvae from eggs incubated at 13,21, 13, 21, and 29%0 29% had median tolerance 48.2%0,respectively. Those Those from the 6 and estimates of 441.7,43.6, 1 .7, 43.6, and 48.2%0, 12°C incubations had median tolerance values of 48.5 and 40.4%0, 40.4%0, 12°C respectively. With reference to the interaction term, larvae were more tolerant tolerant of of higher higher salinities salinities when when incubated incubated as as eggs eggs at at the the lower lower temtem-
3. OSMOTIC OSMOTIC
AND IONIC REGULATION TELEOST EGGS AND IONIC REGULATION IN IN TELEOST EGGS AND AND LARVAE LARVAE
233 233
perature. (6,9,12”C) perature. Culture temperature (6, 9, 12°C) had a small small but insignificant effect on salinity tolerance. These data are examined in greater detail by Alderdice and Hourston ((1985). 1985). Duefias ((1981), In relation to the results of Duenas 1981), Hwang and Hirano re((1985) 1985) show that the extent of chloride-cell development can be re lated to the extent of salinity change involved in a challenge; that is, of chloride cells is not an "all-or-nothing" “all-or-nothing” response. the development of This would suggest that the greater larval salinity tolerance associated differentiwith higher incubation salinities could result from a greater differenti tissue-such as an increased density of chloride ation of regulative tissue-such cells. occur, are yolk-sac integument? cells. If If such such cells cells occur, are they they in in the the yolk-sac integument? In the the (C. harengus), harengus), functional functional gill filaments filaments do do not occur occur Atlantic herring herring (C. some weeks after hatching (Blaxter, (Blaxter, 1969). until some 1969). One might assume, from the regulatory capabilities of the embryos of both Atlantic and Pacific Pacific herring, that integumental chloride cells would be found in both forms. forms. They were not found in the Atlantic herring larva (Jones (Jones et 1966), yet a reexamination of their results (e.g., (e.g., their plate 1A2) 1A2) al., 1966), suggests these cells may have been present but unrecognized. The inference is that chloride cells may be found in the yolk-sac integu integuspecies, a suggestion in need of further exam examment of embryos of both species, ination. As an alternative explanation, ination. explanation, a greater basic cell tolerance to salinity might account for the greater tolerance of Duenas' Dueiias’ ((1981) Pa1981) Pa cific cific herring larvae hatched from higher incubation salinities, al although this would seem less probable. In summary, summary, there is considerable evidence that true osmoregula osmoregulation in teleost embryos begins following gastrulation and is functional apby yolk plug closure. Although the timing is variable, regulation ap pears pears to to be be correlated with with the the development development of of chloride chloride cells cells in the the integument of the yolk sac, sac, particularly over the pericardial region, and on the trunk. In freshwater forms forms the chloride cells are sparse and are joined to surrounding epithelial cells by impermeant tight junc juncforms there is an elaboration of tions at their apical margins. In marine forms chloride cells, whose chloride cells cells and and accessory accessory chloride chloride cells, whose interdigitations interdigitations pro prosysvide paracellular channels connected with the tubular vesicular sys tem of the chloride cell interior and with the apical crypt via leaky junctions at the mucosal ends of the paracellular channels. It appears Na+ may be concentrated electrogenically in the tubular vesicular that Na+ system, an extension of the basolateral membrane, where it diffuses system, down the concentration gradient, via the paracellular channels, to the C1- ions appear to diffuse passively across the cytoplasm apical crypt. CIto the apical crypt, although Guggino's 1980b) results indicate Cl Guggino’s ((1980b) C1forms, tight juncefflux may involve active transport. As in freshwater forms,
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D. F. F. ALDERDICE ALDERDICE D.
tions occur occur between between the the chloride chloride cells cells and and adjoining adjoining epithelial epithelial cells. cells. tions Transfer of of eggs eggs or or larvae larvae between between seawater seawater and and fresh fresh water water can can propro Transfer duce rapid rapid changes changes in in chloride-cell chloride-cell morphology; morphology; elaboration elaboration or or dede duce generation of of chloride chloride cells cells may may begin begin in in as as little little as as 3 h, h, and and be be generation complete 1-3 1-3 days after transfer. transfer. Some Some early early larvae larvae respond in this this complete days after respond in manner to to transfer, transfer, while while later later juveniles juveniles cannot. cannot. Alternatively, Alternatively, in in manner some species early larvae larvae do not respond while later juveniles do. Others show no morphological change in chloride cells, and cannot (seawater) media tend tolerate transfer. Marine embryos in hypertonic hypertonic (seawater) to to lose tissue water water at at aa slow rate; rate; juveniles and adults balance water loss embryo appears accomplish loss by by "drinking." “drinking.” The The Fundulus embryo appears to to accomplish “ "drinking" drinking” by by aa mechanism mechanism involving involving external external pores pores communicating communicating with with the the pharynx. pharynx. Perivitelline Perivitelline fluid apparently apparently is taken taken into the the phar pharynx, is absorbed, absorbed, and and the excess ions ynx, water water is the excess ions are are returned returned to to the the perivi perivitelline system, balancing telline fluid. fluid. Such aa system, balancing water water loss, loss, could could occur occur in in other other marine embryos. embryos. Evidence also indicates that Clupea pallasi embryos are are more more salt-tolerant salt-tolerant when when incubated incubated in in higher higher salinities, salinities, suggesting suggesting that that the the extent extent of of regulative regulative tissue tissue development development may may correlate correlate with with the salinity of the external medium. Although chloride cells were not found found earlier earlier in in C. C . harengus, harengus, growing growing evidence evidence suggests suggests both both species species be examined for the presence of chloride cells. cells.
E. Transition to the Adult Mechanism for Regulation E. is a tendency Marine teleosts function in a medium where there is loss of water water and diffusional gain of Na+ Na+ and CI-. C1-. In fresh for osmotic loss occurs:: there is is an an osmotic gain of water and and a water the reverse occurs loss of Na+ Na+ and Cl-. C1-. The preceding section section indicated indicated that diffusional loss many marine teleosts may may develop extrabranchial extrabranchial chloride cells cells in many embryonic (epiboly (epiboly and and later) later) or postembryonic stages, stages, with these cells functioning functioning to eliminate excess excess ions ions and stabilize stabilize extracellular cells fluid osmoconcentration. osmoconcentration. In In the the postembryonic postembryonic stage, stage, regulation shifts shifts fluid adult-type tissues (kidney, (kidney, gills, gills, and gut), gut), and water water uptake to to offset offset to adult-type osmotic loss loss in in sea sea water water is is considered considered to to occur occur largely largely by by drinking. drinking. osmotic “Drinking” to to offset offset osmotic osmotic water water loss loss appears appears to to have have been been antici antici"Drinking" pated by by the the apparent apparent pharyngeal pharyngeal and and gut gut absorption absorption of of water water from from the the pated perivitelline fluid fluid as as documented documented by by Guggino Guggino ((1980a) in F. F . heterocli heterocliperivitelline 1980a) in tus and and F. F. bermudae bermudae embryos. embryos. However, However, in in marine marine teleosts, teleosts, absorbed absorbed tus water carries carries with with it it ions ions that, that, if if they they remained, remained,, would would defeat defeat the the water ' purpose of of "drinking." “drinking.” In In adult-type adult-type regulation regulation in in marine marine teleosts teleosts purpose most divalent divalent ions ions remain remain in in the the gut gut and and are are excreted excreted via via the the anus; anus; most
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some are absorbed through the epithelium of the gut and esophagus and are excreted by the kidney. Monovalent ions are absorbed by the gut and excreted either by the kidney or by the branchial epithelium. In fresh water, osmotic influx of water is balanced by renal production of copious volumes of hypoosmotic urine. Na+ Na+ and CIC1- ions also are exresorbed from the urine by the kidney, while divalent ions are ex furcreted. In those freshwater teleosts possessing a urinary bladder, fur Na+ and CIC1- are actively resorbed through an Na+,K+ Na+,K+-activated ther Na+ -activated ATPase mediating an Na+-K+ Na+ -K+ exchange system resulting in sodium uptake (Evans, (Evans, 1980). 1980).Absorption of Na+ Na+ and CIC1- by the renal system is not total; if the kidney were the only strucutre involved, there would be a net loss of NaCI. NaCl. This potential net loss by the kidney Na+ and C1appears to be offset through active uptake of Na+ CI- by the branchial epithelium. epithelium. Various models of the chloride cell in marine teleosts have been proposed, based on growing detailed evidence of their salt-secreting function (Sardet (Sardet et al., 1979, 1979, Zadunaisky, 1984; 1984; Karnaky, 1980) and Aronson ((1985) 1985) have proposed Karnaky, 1986). 1986). Heisler ((1980) models for salt balance and pH control in marine and freshwater te teleosts. To these systems may be added the probability that the resisresis leosts. tance of individual cells to salinity change may vary between species, as suggested by Weisbart ((1968) 1968) for the five species of of North American Pacific Pacific salmon (Oncorhynchus). (Oncorhynchus). On the other hand, Shen and “mitochondria-rich” chloride chlorideLeatherland ((1978b) 1978b) found very few "mitochondria-rich" type cells in the yolksac yolksac epithelium of the salmonid S. S. gairdneri. They also found a small small number of chloride-type cells at the base of the gill filaments in the branchial epithelium 11 week before hatching. They concluded that there did not appear to be a significant number of chloride-type cells and that their role in regulation during the early alevin period might be a limited one. The chronology of chloride-cell appearance, density, location, and transfer of regulative function from the integumental system to the postembryonic renal-branchial-gut renal-branchial-gut system is is not well documented. From the scattered evidence available, it appears that the timing of appearance and properties of integumental chloride cells and the transfer of regulatory function to the adult-type regulatory system in the larvae or juvenile may be highly variable. Chronology may vary in relation to the ecology of the species-whether species-whether it is is obligatory marine and stenohaline, obligatory fresh water, euryhaline with episodes of of marine, estuarine, or freshwater experience in its life history, and catadromous. anadromous or catadromous. In summary, summary, the timing of the transition from integumental regula regulation to the adult type is is not well documented, but it would seem to be
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restricted mainly just prior prior to until devel restricted mainly to to the the period period from from just to hatching hatching until development opment of of the the branchial branchial epithelium epithelium occurs occurs some some days days after after hatching. hatching. As As the adult-type adult-type of of regulative regulative system system involves involves the the renal renal complex complex and and gut, gut, as as well as as the the branchial branchial epithelium, epithelium, the the inference inference is is compelling compelling that that all all of of these these structures structures should should be be examined examined concurrently concurrently to to obtain obtain aa full appreciation question of whether appreciation of of regulative regulative function. function. The The question whether and and how how chloride chloride cells cells function function in in freshwater freshwater teleosts, teleosts, such such as as the salmo salmonids, remains obscure. To be noted is the fact that in fresh waters of acid acid pH pH or or very very low low ion ion content, content, sequestering sequestering of of Na+ Na+ by by the the embryo embryo 2+ from ofNa+ from the the eyed eyed stage stage onward onward may may be be subnormal, subnormal, levels levels of Na+ and and Ca Ca2+ in the resulting hatched alevins may be low, low, and mortality may occur in these fish prior to their reaching the first feeding stage when exoge exogenous sources minerals would become available in the food. sources of hydro hydrominerals food. If chloride cells are present under chloride cells are present under such such circumstances, circumstances, it it would would seem seem reasonable reasonable that that they they would would be be involved, involved, at at least least in in the the uptake uptake of of Na+ Na+ ions from the medium.
V. CONCLUSIONS V.
Three questions were posed earlier regarding regulatory mecha mechanisms, patterns of regulatory activity, and the developmental states at which regulatory processes become functional. functional. A wealth of informa information is accumulating that addresses these questions in particular stages and species of invertebrates, invertebrates, lower chordates, and vertebrates. However, its consolidation and integration into cause-effect cause-effect associa associations and developmental chronologies has yet to be achieved, particu particularly in teleosts. Major phylogenetic differences in cell function and regulatory behavior in different groups of organisms are such that a sequence of events determined for an echinoderm or amphibian may not apply among teleosts. Even among the teleosts a surprising num number of gaps occur in our understanding of of general as well as specific aspects of regulatory development. A. Oocytes The oocyte appears to depend largely on its adult female carrier for osmoosmo- and ionoregulation. Teleosts, being homoiosmotic regulators, maintain a gradient between the external medium and the oocyte via the circulatory system, system, which serves to regulate the tissues including
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237
the follicular cells of intimate cellular of the the ovary. Following ovulation, intimate contact is replaced by the ovarian fluid, which also follows the gradigradi ent. To what extent might the oocyte take part in these regulatory regulatory processes? processes ? The oocyte plasma membrane would seem to be the only structure that could partake in regulatory activities. Yet changes that do occur in the properties of of the membrane appear to be no more than transient adjustments associated with episodes of of development within the bounds of of a regulated regulated ovarian or coelomic environment. A number of between GVBD of changes in the properties of of the membrane occur between and ovulation. The membrane potential of of the teleost oocyte is dependepen dent largely on K+ and Na+ Na+ concentrations, and developmental epiepi sodes may involve the freeing of of bound bound Ca2+ Ca2 + and its movement across the membrane. The ovulated oocyte likely will show a lower memmem potential, increased electrical resistance, a decrease in K+ and brane potential, Na+ Na+ conductance, and high water permeability compared with the immature oocyte. oocyte.
B. Fertilization Fertilization E gg extrusion present the the first major regulatory regulatory Egg extrusion and and fertilization fertilization present first major challenge On leaving leaving the the protective protective internal internal challenge to to the the teleost teleost gamete. gamete. On environment of body cavity, cavity, the the fertilized fertilized egg egg is is immediimmedi environment of the the adult adult body ately hypotonic (freshwater) (freshwater) exterexter ately bathed bathed in in aa hypertonic hypertonic (marine) (marine) or or hypotonic nal of the extruded egg, egg, just nal medium. The resting resting potential potential of just prior to activation, 50 to 90 mV the -50 to -90 mV in in fresh fresh water, water, the activation, will will probably probably be be around around potential potential being being the the result result of of aa limited, limited, selective selective permeability permeability to to K+ K+ 2 + or and and Na+, Na+, but but independent independent of of Ca Ca2+ or CI-. C1-. The The membrane membrane potential potential in in general general is is aa function function of of sperm sperm contact, contact, internal internal factors factors such such as as calcium calcium release release at at activation, activation, and and the the ions ions present present and and their their concentrations concentrations in in the sperm contact instant of of sperm contact with with the the plasma plasma the external external medium. medium. At At the instant membrane, set in membrane, aa number number of of molecular molecular and and electrical electrical events events are are set in motion. As shown shown in in the the medaka medaka egg, egg, the the activation activation potential potential consists consists motion. As consecutively consecutively of of an an initial initial small small depolarization, depolarization, aa major major hyperpolariza hyperpolarization, slow recovery, phases of of fast fast and and slow recovery, the the whole whole process process tion, followed followed by phases lasting 10 min. min. During During this this period, period, membrane membrane resistance resistance shows shows lasting about about 10 aa marked rst few marked decrease decrease for for the the fi first few minutes, minutes, followed followed by by recovery recovery to to aa final final resistance resistance value value somewhat somewhat greater greater than than the the initial initial level. level. The The initial by Na+ Na+ and and initial small small depolarization depolarization apparently apparently may may be be carried carried by 2 + influx Ca K+ efflux. efflux. In In the the following following hyperpolarization hyperpolarization phase, phase, Ca2+ influx and and K+ cortical cortical aleveolar aleveolar exocytosis exocytosis occurs occurs with with aa concurrent concurrent large large increase increase in in cytosolic cytosolic free free calcium; calcium; this this occurs occurs as as aa wave, wave, spreading spreading from from the the point point
238 238
D. D. F. F. ALDERDICE ALDERDICE
of encompass the of sperm sperm contact contact to to encompass the egg egg and and terminate terminate at at the the vegetal vegetal pole. membrane shows in pole. During During hyperpolarization hyperpolarization the the plasma plasma membrane shows an an increased conductance due mainly to increased K+ K+ permeability. This may result from the opening of K+ K+ channels in the membrane, or from the transient addition of vesicular membrane, and its K+ K+ channels, to the plasma membrane. After peak hyperpolarization the vesicular membrane material appears to be removed from the plasma mem membrane: K+ K + permeability of the membrane falls rapidly, while Na+ Na+ per permeability increases slightly. After the main Ca2+ Ca2+surge, internal Ca2+ Ca2+ is pumped out into the external medium, the net internal loss being about one-third of the initial free calcium concentration. The Ca2+ Ca2+ wave also is p H-dependent; acid pH slows the Ca2+ pH-dependent; Ca2+wave and in increases the [Ca2+] [Ca2+]required for initiation of of the Ca surge. surge. What do these changing rates of permeation and ion concentration mean to the activated egg? egg? Rates of water permeation are very high at activation, reducing to a minimum within approximately 24 h or less. Water fluxes fluxes are certain to occur in response to changes in ion concen concentrations in the cytoplasm, presumably involving Na+, Na+, K+, K+, and Ca2+. Ca2+. Hence, for a limited period following activation the plasma membrane may be selectively permeable and the concentration gradient be between the cytoplasm and the new external medium may be of particu particular importance in the establishment of a new steady state between the yolk and the external medium. medium. Before turning to the first stage of cell division several several factors factors may Before be mentioned that influence following events. events. They comprise condi conditions influenced by properties properties of the plasma membrane and perivitel perivitelline fluid. fluid. The available evidence rather definitely shows shows that plasma membrane permeability, high at activation, decreases rapidly within so, to reach a minimum within within 24 h. h. This tightening of the first hour or so, plasma membrane appears appears to restrict transmembrane flows of both the plasma water and ions. The reduction in membrane permeability, starting water is correlated with increasing tension in the mem memwith activation, is from the increase in hydrostatic (internal) (internal) pressure brane, resulting from occurring as as a consequence of water imbibition during during formation of occurring the perivitelline fluid. fluid. Restricted permeability of the the membrane membrane ini inithe tially, and of its derivative limiting membrane in later later multicellular tially, stages, is is retained retained at at least to the the eyed eyed embryo embryo stage. stage. Following that, that, stages, small increase increase in permeability of the embryo embryo to both there may be a small ions. water and external ions. to be lowest in media of very low Membrane permeability tends to ionic activity. activity. Many studies studies of permeability have been conducted usionic
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Ringer’s solution or dilutions of Ringer's, Ringer’s, with the ing eggs bathed in Ringer's permeation characteristics tend to reflect the level of ionic result that penneation activity of the external medium used. Under such conditions standard measures may be obtained, but it is then difficult to appreciate the significance of penneation permeation characteristics characteristics in natural media. ecological significance potentials.. The same argument applies in the estimation of membrane potentials Hydromineral ion concentrations iin n freshwater media also influence permeability; Permeability; Ca2+ Ca2+and and Na+ Na+ appear appear important important in in this this regard regard for for contin continMg2+, Ca2+,Na+, ued successful embryonic development. While Ca2+, Na+, K+, K+, M g2+, and H H++ in the external medium may be of critical importance during surprising that development, it it is is surprising that few few hydromineral hydromineral ion ion criteria criteria are are particularly available for incubation of teleost eggs in fresh water, particularly where Na+/H+ Na+/H+and and Na+/NH Na+/NH4+ may be be of of critical critical concern in in 4 + exchange may low ionic strength natural waters with a pH of lower. The of 6.5-6.6 6.5-6.6 or lower. impermeant colloids of the perivitelline fluid are negatively charged in freshwater eggs, and the excess negative charge tends to sequester cations from the external medium. In fresh water, the perivitelline (PVP, between the perivitelline fluid and the external me mepotential (PVP, dium) appears explainable in terms of [Na+] “a+] and [Ca2+] [Ca2+]in the me medium) -40 -80 mV; in marine eggs dium. In fresh water the PVP may be 40 to 80 mY; mV, and there is a substan substanthe PVP varies between about 0 and +40 mY, C1- across across the zona radiata. Na+, K+, tial movement of Na+, K+, and CIpermeability is complex. It is The influence of temperature on penneability of water argued that that the the properties properties of water near near interfaces interfaces are are different different from from those of bulk water, and that phase transitions may be expected to occur as temperature changes (Hansson Mild and L¢vtrup, L@vtrup,1974b). 197413). These transitions frequently occur at 13-16°C. They appear to infl u influence the rate of diffusion of water in the cytoplasm, tension in the plasma membrane, plasma membrane, measures measures of of osmotic osmotic water water penneability permeability of of the the membrane, permeabilmembrane, and internal egg pressure. In ranid species species the penneabil counterity coefficient increases with higher temperature; this can be counter acted tonicity of of the the external external medium-presumably medium-presumably acted by reducing the tonicity through tension resulting through increased increased membrane membrane tension resulting from from increased increased hydro hydrostatic pressure occurring because of further water uptake in a medium beof lower ionic activity. Differences in temperature dependence be tween tween species species also also have have long long been been assumed assumed related related to to the the fatty fatty acid acid composition composition of of membrane membrane lipids. lipids. Hence Hence differences differences in in the the tempera temperature distribution of penneability permeability coefficients may also reflect differ differences in thennal thermal phase transitions associated with variations in mem membrane lipid composition between species. In In the the earlier earlier literature literature much much discussion discussion centered centered on on an an observed observed
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D. F. F. ALDERDICE
inverse relation relation between between the the size size of of aa cell cell and and its its permeability permeability coefficoeffi inverse cient, equivalent equivalent to to aa direct direct relation relation between between the the surface-to-volume surface-to-volume cient, (SIV) of ofaa cell cell and and its its membrane membrane permeability. permeability. That That is, is, larger larger eggs eggs ratio (SIV) ratio with aa smaller smaller S/V ratio ratio appeared appeared less less permeable permeable than than smaller smaller eggs eggs with S/V ratio. ratio. Dick Dick (1959b) ( 1959b) showed showed that that there is aa significant significant with with aa greater greater SIV there is S/V ratio and the the coefficient coefficient for for diffusion diffusion in in inverse relation relation between between SIV ratio and inverse the cytoplasm. He He reasoned reasoned that that the the relative relative effect effect of of plasma plasma memmem the cytoplasm. brane resistance resistance in in reducing reducing the the rate rate of of diffusion, diffusion, in in comparison comparison with with brane the effect effect of of resistance resistance of of the cytoplasm, cytoplasm, would would be be greater greater in in smaller smaller the cells (with (with aa greater greater S/V ratio) ratio) than in larger larger cells cells (with (with aa smaller smaller SIV S/V cells than in ratio). Hansson Hansson Mild Mild and and L@vtrup L�vtrup (1985) ( 1985) appear appear to to have have aa fi nal explaexpla ratio). final nation. Plasma Plasma membrane membrane permeation, permeation, cytoplasmic cytoplasmic diffusion diffusion and and cell cell nation. size are interrelated. The The value of Pd as aa function function of of D Dl (diffusion in in P d as 1 (diffusion size are interrelated. value of the cytoplasm) varies varies with cell size size when when the the effect effect of cytoplasmic the cytoplasm) with cell of cytoplasmic diffusion is taken diffusion is taken into into account. account. Another Another factor possibly involved in this relationship is hydrostatic pressure. Although Although exceptions exceptions occur, occur, smaller eggs tend tend to to have higher pressure. smaller eggs have higher hydrostatic pressures. pressures. Hence Hence smaller smaller eggs eggs (greater (greater S/V ratio), with with aa SIV ratio), hydrostatic greater tension in in the plasma membrane, membrane, may may have lower permeabilpermeabil greater tension the plasma have lower ity coefficients. Interestingly, the unbiased estimate of P P should should propro ity the unbiased estimate of of the the plasma plasma membrane. membrane. vide of the vide aa measure measure of the permeation permeation capacity of However, the manner in in which which that capacity is is utilized, in in an an ecologiecologi However, the manner that capacity cal sense, sense, may may be best best provided provided by the biased estimate, estimate, at at least least uncoruncor cal the biased rected for Dl. D1. rected for
C. First Cell Division to Beginning of of Epiboly C. activaThe first stage of "“regulation” regulation" in the teleost egg, following activa of resistive maintenance of of the integrity of of the tion, appears appears to be one of memegg proper, achieved through the presence of a tight plasma mem fluxes. variabrane and limited transmembrane water and ion fl u xes. Natural varia tions in this pattern may involve differences in membrane tightness, of the dividing cells of the together with differences in the tolerance of blastula to salinity change. change. Hence, it is suggested that a species with a extremes may compensate by shielding the low tolerance to salinity extremes conversely, a species with a blastomeres with aa tight membrane; conversely, transbroad cellular salinity tolerance may demonstrate less limited trans befluxes. The fact that compartmentalization compartmentalization of the cells be membrane fluxes. gins rather early in cell division suggests that such differentiation may gins signal the initial stages in development of real regulatory capacity,
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particularly where the enveloping layer cells show high membrane resistance and tight intercellular junctions. The fact that cells within fluxes compartments share communication and ion fl uxes via gap junctions of protected prototissues among suggests the emerging development of of osmoosmo- and ionoregulation. whose new functions may be those of
D. Epiboly to Hatching of extrabranchial chloride cells appears to signal The development of the beginning of of modulated and selective ion regulation in the teleost embryo. Prior to epiboly there was a suggestion of some regulatory capacity. However, this capacity seems to result from low transmem transmemof enveloping cell layers, and brane fluxes based on tight membranes of chronolas such appears to be neither modulated nor selective. The chronol ogy of development and first appearance of chloride cells is not yet defined. These well defined. These cells cells are recorded in the yolk-sac integument as early as during epiboly, epiboly, although in various species they may develop from that time until shortly before hatching. The transport of of Na+ Na+ and CI- by chloride cells has now been clearly demonstrated. The mor morC1phology of the chloride and epithelial cells and their tight and leaky junctions leads to various models of ion transport in a saltwater me medium. Although the picture is less clear in fresh water, it would seem that the simplest, most efficient process for ionoregulation would be that involving modification of one tissue to serve as an intake device in in fresh fresh water water or or an an output output device device in in saltwater. saltwater. Based on on available available data, one would expect to see considerable variation in the time of appearance, appearance, cell cell density, density, and and location location of of extrabranchial extrabranchial chloride chloride cells. cells. One embryos of marine, marine, estuarine, estuarine, and and One might might expect expect to to find find them them in in embryos catadromous absent in in strictly strictly freshwater freshwater spe specatadromous species species.. They They may may be absent cies, cies, and and they they might might be be induced induced to to form form in in some some freshwater freshwater forms, particularly particularly those those with with anadromous anadromous life life histories. histories. The The quantitative quantitative development development of of chloride chloride cells cells in in relation relation to to the the level of of salinity salinity expe experienced rienced during during incubation incubation is is not not as as yet yet fully fully substantiated, substantiated, but but is is sup supported by the fact that higher incubation salinity has been shown to ported 1980a) ob salinity tolerance tolerance in in resulting resulting larvae. larvae. Guggino's Guggino’s ((1980a) obincrease salinity servation of "pharangeal another avenue “pharangeal pores" pores” in Fundulus opens up another of of enquiry enquiry into into embryonic embryonic osmoregulation, osmoregulation, particularly particularly regarding regarding wa water ter uptake uptake by by hypoosmotic hypoosmotic embryos embryos in in seawater. seawater. In In general, general, true true regu regulation appears appears to begin in the embryo with the development of extra extrabranchial branchial chloride chloride cells, cells, and and the the apparent apparent capacity capacity to to provide provide water water
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D. F. F. ALDERDICE ALDERDICE D.
balance by the pharangeal pharangeal complex. complex. These These inferences, inferences, however, however, rere balance quire considerable considerable experimental experimental confirmation confirmation in in view view of of the the limited limited quire number of of species species examined examined and and the the variation variation in in the the results results obtained. obtained. number E. Transition Transition to to the the Adult Adult Mechanism Mechanism for for Regulation Regulation In major aspects aspects of of In teleosts, teleosts, as as well as as in in other other organisms, organisms, two major regulation teleosts regulation are are certain certain to to be be water water and and salt salt balance. balance. Marine Marine teleosts tend lose water tend to to lose water osmotically osmotically and and gain gain salts salts diffusionally. diffusionally. Freshwater Freshwater teleosts tend same processes. teleosts tend to to gain gain water water and and lose lose salts salts by by the the same processes. In In juvenile juvenile and and older older stages stages of of marine marine teleosts, teleosts, water water loss loss is is made made up up by by "drinking," “drinking,” the the ions ions absorbed absorbed in in the the process process being being excreted excreted by the renal renal complex complex and and branchial branchial epithelium epithelium (monovalent (monovalent cations) cations) and and gut gut (mainly divalent cations). cations). In juvenile juvenile and and older freshwater freshwater teleosts teleosts aa copious volume of hypoosmotic urine is is produced from which Na+ Na+ and C1Cl- are resorbed resorbed by the renal complex and, where present, by the urinary bladder. Some Na+ Na+ and ClC1- may be taken up from the external medium medium by by the the branchial branchial epithelium. epithelium. One might anticipate that the same regulatory strategies would apply apply in in the the late late embryo, embryo, although although the the tactics tactics employed employed may may vary vary with with developmental deveIopmenta1 stage stage and and species. species. The The chronology chronology of of such such develop development ment and and the timing timing of of the the transition transition between between embryonic embryonic extrabranch extrabranchial chloride-cell function and juvenile branchial function largely re remain to be documented. Perhaps Perhaps our fascination with chloride-cell function has diverted attention from the renal complex and gut, which may be more involved in embryonic regulation than has been appreci appreciated. Hence the search search for for further further understanding understanding of of patterns patterns of of regula regulation tion in in teleost teleost embryos embryos seems seems to to suggest, suggest, both both for for marine marine and and fresh freshwater forms, forms, that the the chronology of tissue development be examined more more closely closely and and concurrently concurrently in in the the gut gut and and pharynx, pharynx, the the renal renal com complex, plex, and and the the extrabranchial extrabranchial and and branchial branchial apparatus apparatus of of the the embryo embryo before, during, and after its transition to to the first first juvenile form. form.
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4 SUBLETHAL EFFECTS EFFECTS OF POLLUTANTS POLLUTANTS SUBLETHAL LARVAE ON FISH EGGS AND LARV AE H.VON VON WESTERNHAGEN H. Biologische Anstalt Helgoland (Zentrale) (Zentrale) 52, Federal Republic of of Germany D-2000 Hamburg 52,
I. Introduction 11. Sublethal Effects during Development II. Ovarian Eggs and Egg Deposition A. Ovarian Fertilization, Water Uptake, and Water Hardening B. Fertilization, C. Early Development C. D. Advanced D. Advanced and Late Development E. Effects Other than Morphological Aberrations E. F. F. Incubation Time and the Process of of Hatching G. Hatchability G. Hatchability and Viable Hatch 111. Sublethal Effects Displayed by Larvae Hatched from Treated Eggs III. A. Larval Length B. Yolk-Sac Size and Yolk Metabolism C. Morphological Aberrations: Eye Deformities, Skeletal C. Abnormalities D. Minor Morphological Aberrations D. E.. Metabolic Alterations E F. Behavioral Abnormalities Abnormalities IV. IV. Sublethal Effects on Larvae Not Exposed as Eggs V. Discussion, Problems, and the Future References References
I. INTRODUCTION The developing fish fish embryo or larva is generally considered the most sensitive stage in the life cycle of of a teleost, being particularly sensitive to all kinds of low-level environmental changes to which it might be exposed. Available data suggest that certain stages in the life 253 253 PHYSIOLOGY, VOL. XIA XIA FISH PHYSIOLOGY,
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cycle of marine and freshwater fishes are more susceptible to environenviron mental and pollutional stress than others (von Westernhagen, 1968, 1968, 1970; 1970; Rosenthal and Alderdice, 1976). 1976). Differences in susceptibility are particularly known to exist between the early developmental stages, that is, embryos embryos and larvae. The younger embryonic stages (before (before gastrulation) gastrulation) are more vulnerable than those that have com completed gastrulation. This has been shown by Kiihnhold 1972) for the reaction of cod Kuhnhold ((1972) (Gadus (Gadus morhua), morhua),herring (Clupea (Clupea harengus), harengus), and plaice (Pleuronectes (Pleuronectes platessa) platessa) embryos when exposed to crude oil extracts and could be confirmed for cod by Davenport et al. 1 979), Black Sea Hounder aZ. ((1979), flounder (Platichthysjlesus (Platichthysflesus luscus) Zuscus) by Mazmanidi and Bazhasvili (1975), (1975),north north(Esox lucius) lucius) by Hakkila and Niemi ((1973), 1973), and several other ern pike (Esox marine species (Wilson, (Wilson, 1972). 1972). The decrease of sensitivity of cod C. G. morhua embryos to methyl methylnaphthalene during development is similar. similar. Newly fertilized eggs are more sensitive than after completion of gastrulation (Stene and Lanning, 1984). develop Lonning, 1984). The effects of heavy metals on embryonic development are likewise particularly pronounced during early embryonic stages as documented experimentally by P. Weis and Weis ((1977) 1977) and Sharp and Neff 1980, 1982) 1982) for the effects Neff ((1980, effects of of methylmercury on em em(FunduZus heteroclitus) and Akiyama ((1970) bryos of the killifish (Fundulus 1970) and Dial (1978) (1978) on Oryzias Iatipes. latipes. Another toxicant, such as the lamprey larvicide TFM, was also found to exert its most detrimental effects on rainbow trout embryos immediately following fertilization (Niblett and McKeown, McKeown, 1980). 1980). Even though the relatively high susceptibility of of the early embry embryonic stages has been well documented, the yolk-sac yolk-sac or alevin stage is known to be the most sensitive stage in the teleost life cycle. Linden 10 for the higher sensitivity of of Baltic herring ((1974) 1974) reports a factor of 10 ((C. C .harengus membras) membras) larvae toward water-soluble crude oil compo components, while the larvae of northern pike (E. ( E . lucius) are 100 100 times more sensitive to oil than the embryos (Hakkila and Niemi, 1973). 1973).The same can be observed in the alevins of pink salmon (Oncorhynchus gorbus gorbus(Rice et al., 1975). cha) 1975). cha) (Rice Likewise, heavy-metal toxicity to early life stages of of fish expresses itself itself most sensitively in larvae and alevins, with significant inhibition of growth in Atlantic salmon (Salmo (0.47 (Salmo salar) salar) upon exposure to low (0.47 pg Cd/I) Cd/l) levels of cadmium, while a significant reduction in viable ILg pg Cd/l (Rombough and hatch is noticed only between 300 and 800 ILg 1982). Similar effects of copper on the larvae of of eight species Garside, 1982). of freshwater fi sh are known from McKim et al. ((1978). 1978). Low concentraoffreshwater fish
4. 4.
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255
methylmercuric (0.075 /Lgll), pg/l), cadmium (0.55 (0.55 ILg/I), pgll), and lead tions of methyl mercuric (0.075 (0.7 pg/l) ions appear to cause little "biochemical “biochemical stress" stress” on brook (0.7 ILgll) (Salvelinus fontinalis) fontinalis) embryos, but cause definite changes in trout (SaIvelinus alevins. The higher susceptibility of larvae compared to embryos has alevins. (Paflitsbeen also demonstrated for organophosphate insecticides (Pafl its 1979) as well as chlorinated hydrocarbons [Schimmel [Schimmel et aI. al. chek, 1979) Aroclor 1254; 1254; Dethlefsen ((1977), DDE].. ((1974), 1974), ArocIor 1977), DDT, DDE] fluctuating factors Thus, within a variety ooff fl uctuating natural environmental factors such as temperature, salinity, oxygen activoxygen content, etc., human-made activ ities have added a series of new parameters that may exert consider considerable impact on the developing fish. fish. It is during early life that fish seem to be particularly sensitive to substances, many of which are com commonly called pollutants; during early life the yolk-sac yolk-sac stage may be considered the most sensitive one, followed by the embryonic stage of gastrulation. gastrulation. prior to completion of The effects caused during these and other stages of development by pollutants may be very subtle and go unrecognized at the individ individual level. Yet these effects may proceed until they develop into very conspicious lethal effects. Ultimately their significance may be recogrecog nized at the population level. The urgent need for the determination of of legally applicable mea measures of pollutants and their effects on the aquatic biota has recently sures triggered a multitude of investigations on the effects of hitherto unrec unrecognized pollutants and the determination of their LC50 LCm (concentration at which 50% 50% of the experimental animals died within a given time span, usually 48 h) MATC (maximal (maximal acceptable toxicant con h) or their MATC concentration). The application of of the LCso Lc50 has found widespread entry into the methods for determination of water quality by means of of test organisms (Anonymous, (Anonymous, 1972a,b). 1972a,b). In combination with application factors, the LCso Spra LC50 is used to determine specifi specificc pollutant toxicity ((Sprague, 1969; 1969; Anonymous, 1976). 1976). Among the aquatic organisms, fish (Stephan (Stephan and Mount, 1973) 1973) as well as their eggs and larvae have at atbetracted considerable interest for toxicity testing, particularly so be cause an early life stage test with fish eggs can be considered equiva equivalent to life-cycle tests, as as was shown by McKim ((1985) 1985) when comparing 72 life-cycle tests in four freshwater fish with early life lifecycle tests of these species. Relatively few works have been devoted to the description of of sub sublethal effects; with the exception of the paper by Rosenthal and Alder Alderdice ((1976) 1 976) on sublethal effects of of environmental stressors, relatively little synthesis of data appears to have occurred with regard to suble sublethal effects of sh eggs and larvae. One of of pollutants on fi fish of the reasons for
256
H. VON VON WESTERNHAGEN
the the lack lack of of work work on on sublethal sublethal effects effects may may be the the need need of of decision decision makers makers for for clear-cut clear-cut answers answers regarding regarding damage damage to to aa test test organism. organism. Since LCSO Since death death of of the the individual individual is is easily easily recognized recognized in in most most cases, cases, LC 50 investigations investigations are are preferred preferred for for reasons reasons of of simplicity simplicity of interpretation interpretation (see requires more 1985).A sublethal sublethal response response requires more (see Rand Rand and and Petrocelli, Petrocelli, 1985). subtlety well understood. subtlety in in its its interpretation interpretation if if it it is is to to be be well understood. The easily defined. The term term "sublethal" “sublethal” is is not not easily defined. According According to to Rosenthal Rosenthal and 1976), sublethal defined as sublethal effects effects may may be defined as "those “those re reand Alderdice Alderdice ((1976), sponses sponses to to environmental environmental changes-histological, changes-histological7 morphological, morphological, physiological, or physiological, or ethological-that ethological-that may may be be induced induced in in one one stage stage of of development expressed at at aa later later stage stage of of organization organization or or devel develdevelopment but but be expressed opment in terms of reduced survival potential." potential.” This implies that a "sublethal" “sublethal” effect on an embryo may give rise to a lethally damaged larva. Thus the eggs applies applies to to larva. Thus the term term "sublethal" “sublethal” for for developing developing fish eggs the stages of the different different stages of development development in in the the early early life life history, history, which which may may begin begin as as early early as as in in the the ovarian, ovarian, unfertilized unfertilized egg; egg; even even impair impairment ment of of fertilization fertilization would would then then be be considered considered as as aa sublethal sublethal effect effect on on an an egg. egg. When When trying trying to to define define "sublethal" “sublethal” in in terms terms of of effects effects on on groups groups of of fish fish eggs eggs and and larvae larvae rather rather than than on on individuals, individuals, the the connotation connotation may may change exchange significantly. significantly. An An individual's individual’s sublethal sublethal response response always always ex cludes immediate cludes immediate death, death, though though its its life life expectancy expectancy may may be shortened shortened and and it it might might die die earlier earlier than than usual, usual, while while the the sublethal sublethal response response of, of, say, say, a batch of eggs toward environmental factors factors accepts the death of aa few few individuals individuals as as normal normal (such (such as as found found in in the the controls controls of of most most incubation eggs); thus thus even even an an increasing increasing death death incubation experiments experiments with with fish eggs); rate given experiment considered "sublethal" “sublethal” as as rate in in any any given experiment may may still still be considered long group concerned is not not lethally long as as the the group concerned is lethally damaged. damaged. That That is, is, before before we is so high high that that no no new new recruitment recruitment can can we observe observe aa mortality mortality rate rate that that is be assured, we may still call an observed effect "sublethal." “sublethal.” Thus Thus the the term term "sublethal" “sublethal” in in the the context context of of this this chapter chapter will will de describe a rather subjective situation, which is strongly dependent on the the biological biological end end point point considered considered and, and, as as indicated indicated above, above, allows allows aa stochastic approach (which stochastic approach (which applies applies for for dealing dealing with with sublethal sublethal effects effects on level) as on the the population population level) as well as as aa more more deterministic deterministic interpretation, interpretation, which referred to to when when discussing discussing responses responses on on the the individ individwhich may may be referred ual level. I shall thus discuss sublethal responses elicited in one onto ontogenetic stage (the egg), and its (the earliest of which may be the ovarian egg), Significance significance for for that that ontogenetic ontogenetic stage stage or or ensuing ensuing stages stages of of develop development ment in in terms terms of of consequences consequences to to the the afflicted afflicted individual individual or or group group (reduced (reduced survival survival percentage). percentage).
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EFFECTS FISH EGGS EGGS AND EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH AND LARVAE LARVAE
257
today’s knowledge of sublethal effects of pollutants As one looks looks at today's “not on fish eggs and larvae, larvae, it would be preferable to use the term "not lethal” rather than "sublethal," “sublethal,” since most of of the described acutely lethal" phenomena are defined for an arbitrarily chosen biological end point. Many will be lethal for the individual at a later stage of development. significance The signifi cance of the effects for the survival of the population will be discussed later. In this review, due to lack of information, impact on the genetic information of the population via selection pressure efmust be totally neglected. The only available data of mutagenic ef fish experifects of aquatic pollutants on the reproduction of fi sh are the experi exposments conducted by Prein et al. ((1978) 1978) and Alink et al. ((1980), 1980), expos ing the eastern mud minnow Umbra pygmaea to Rhine water. The chromamutation rate in the testes of exposed fish (measured as sister chroma b y the factor 2 and 3, respectively, when exchange) increased by tid exchange) days.. The water contained, among exposing the specimens for 3 or 111 1 days 16 phenolic compounds, 28 aromatic hydrocarbons, other substances, 16 and 16 16 aromatic bases, of which the fraction of of aromatic compounds was was found found to to have have aa mutagenic mutagenic potential potential (Prein (Prein et al., al., 1978). 1978). The The authors speculated that the common known mutagenic compounds responsible for increased mutation rate [i.e., [i.e., some chlorophenols, chlorobenzenes, nitrobenzene, bis(chloroisopropyl)ether] bis(chloroisopropyl)ether] that were detected fraction of of the the total total muta mutadetected in in the the water water represented represented only only a fraction genic load of the Rhine water, since many pqllutants pollutants that may be bio biologically logically active active are are present present in in concentrations concentrations below below detection detection levels. levels. This review is limited to sublethal effects defined by Rosenthal (1976) as histological, morphological, physiological, and Alderdice (1976) and and ethological. ethological. From From the the available available literature, literature, it it appears appears that that within within the framework of the review there are four main types of pollutants that stages of sh that are are likely likely to to produce produce sublethal sublethal effects effects in in the the early early stages of fi fish either either experimentally experimentally or or in in the the field. field. These These are are heavy heavy metals, metals, petro petroleum leum hydrocarbons, hydrocarbons, chlorinated chlorinated hydrocarbons, hydrocarbons, and and acidifying acidifying sub substances (pH). This This short short list is is not not quite quite consistent consistent in in itself itself (for (for aa more more stances (pH). complete 1975; McKim, McKim, 1985; 1985; complete enumeration, enumeration, see see McKim McKim et al., 1975; Nimmo, 1985; 1985; Russo, 1985), 1985), since with the fi first Nimmo, rst three pollutant types we while the we are are referring referring to to substances substances while the fourth fourth in in the the literature literature is is referred referred to to rather rather as as aa chemical chemical state-the state-the hydrogen hydrogen ion ion concentration concentration (pH) water-and not the (pH) in in the the water-and the substances substances causing causing aa possible possible shift shift in in the +/OH - equilibrium. H+/OHequilibrium. Thus this chapter chapter will be be devoted devoted to to the the the H above mentioned "substances" “substances” and their effects on gonadal tissue and ovarian eggs, eggs and sperm, embryos, and larvae, up to metamor metamorphosis.
258
H. VON WESTERNHAGEN
II. SUBLETHAL EFFECTS DURING DEVELOPMENT 11.
of pollutants on early developmental stages may Sublethal effects of of the parent be caused in two different ways. The first is by exposure of fish and and ensuing ensuing reduction reduction in in eggs eggs deposited. This This may may be be the the case case when when fish are are chronically chronically exposed to to low low levels of of metals metals or or pesti pesticides, and cuts down egg production by up to 80%; 80%; short-term expo exposure to cyclic hydrocarbons has similar effects. The second is by expoexpo of the extruded egg. Then exposure of of the unfertilized egg may sure of of the fertilization process and the hardening of of lead to an impairment of of the fertilized egg causes disturbances of of the egg shell. Exposure of early cleavage patterns or morphological aberrations, particularly in aberra axis formation and head and eye development. Morphological aberrapollutant-specific and may be caused also by natural tions are not pollutant-specific stressors. Other, nonmorphological, aberrations from the normal pat pattern of (metals, of development include depression of of embryo activity (metals, hydrocarbons), hydrocarbons), inhibition of of hatching enzyme (low pH), and alteration in incubation time (metals, (metals, hydrocarbons). hydrocarbons). All of of the pollutants dealt "viable hatch”-that hatch"-that is, the with reduce hatchability. The parameter “viable production of of viable larvae-is larvae-is a more sensitive indicator for sublesuble production thal effects than hatchability.
A. Ovarian Eggs and Egg Deposition Exposure of of zinc, of mature fish to low (micrograms (micrograms per liter) liter) levels of cadmium, copper, mercury, hydrocarbons, or pesticides may lead to an 80% reduction of (4.5-6.0) exerts similar of eggs produced. produced. Low pH (4.5-6.0) effects on some freshwater species. The requisite for successful reproduction is the production of of species. This enough eggs by the parental generation to preserve the species. first and basic requirement requirement may be considerably impeded by the action of of the above-mentioned pollutants. Thus, copper administered at concentrations of pgll to aquaria inhabited by fathead minof 18-32 18-32 ILgll min promelas) totally prevents egg deposition in this nows (Pimephales promelas) 1968; Mount and Stephan, 1969). species (Mount, (Mount, 1968; 1969). Although the com complete suppression of of ovarian egg development may be a rare case, a reduction in number of of eggs produced upon exposure to pollutants is ( 1969) for the relatively common and has been reported by Brungs (1969) of low levels of of zinc (0.18 (0. 18 mg/l). The number of of eggs produced effects of by female Pimephales promelas was only 17% 17% of of the eggs produced in the controls. In addition to zinc, cadmium and copper at low concen-
4. 4.
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259
3.7-31 p.g/l; pg/l; Cd, Cd, 0.6-60 0.6-60 p.gll) pgll) caused progressively de detrations (Cu, (Cu, 3.7-31 creasing spawning activity and egg numbers spawned per female (Eaton, 1973). 1973).A reduction in the number of eggs spawned (up (up to 21%) (Eaton, 21 %) 0.13 0.2 mg/l is known to occur in the after exposure to zinc at 0. 13 and 0.2 (Bengtsson, 1974); 1974); the number of eggs minnow Phoxinus phoxinus (Bengtsson, rerio, when spawned is also reduced in the zebrafish Brachydanio rerio, Znll for a 9-day period during the time of of gamete exposed to 5 mg Zn/l al., 1977). 1977). Effective zinc and cadmium conmaturation ((Speranza Speranza et al., con centrations appear to be fairly low, low, as documented above and also by al. ((1978) flagfish floridae, 1978) in experiments with Hagfi sh Jordanella floridae, Spehar et al. (ppb) range (micrograms (micrograms per liter). liter). This is even in the parts per billion (ppb) true also in the live-bearing guppy Poecilia reticulata, where the suffers a reduction down to 50% 50%upon exposure to clutch size of young suffers (0.36-17 pg/l) (Uviovo (Uviovo and Beatty, Beatty, 1979). 1979).Aside from low levels of zinc (0.3617 p.g/l) exthe above-mentioned metals, phenylmercuric acetate is known to ex B. ert detrimental effects on the number of eggs spawned by zebrafish B. rerio, at concentrations ooff 11 p.g/l pgll or less (Kihlstrom (Kihlstrom et al., at., 1971). 1971). How However, between this concentration and the controls, the authors ob obbeneficial (“hormesis”) of 0.2 pII of phenylmercuric served a benefi cial effect ("hormesis") 0.2 p./l freacetate on egg production, a phenomenon that one encounters fre quently in pollution research. Besides metals, other substances may reduce the number of viable ovarian eggs. eggs. Among the hydrocarbons, benzene, a toxic component of petroleum, is known to be very active. When female Pacifi c herring C. Pacific C. (ppb) levels of benzene for 48 h just prior to pallasi are exposed to low (ppb) significant respawning, a signifi cant reduction in survival of ovarian eggs is re corded in the range of 10-25% (Struhsaker, (Struhsaker, 1977). 1977). Effects on the number of eggs produced or spawned are also known known for for pesticides pesticides and and chlorinated chlorinated hydrocarbons hydrocarbons in in general. general. Again, Again, minnows minnows Pimephales promelas, Phoxinus phoxinus, and and Cyprinodon C yprinodon variegatus have been been used for for the the oral oral or or external external administration administration of of uariegatus have carbasubstances. In experiments with Pimephales promelas and the carba mate insecticide carbaryl lasting for 9 months, 0.68 0.68 p.g/l pgll of the insecti insecticide reduced the number of eggs produced per female significantly (Carlson, (Carlson, 1971). 1971). Also, diazinon, an organophosphate insecticide, em employed in continuous continuous flow-through tests with sheepshead (Cyprino (Cyprinodon variegatus) up to 55% 55% in the uariegatus) minnows caused a reduction of up number of eggs produced per female at concentrations higher than 0.47 0.47 p.g/l pg/l in seawater. Similar effects are known to be caused by PCBs (polychlorobiphenyl) when applied orally (Phoxinus (Phoxinus phoxinus; phoxinus; Bengtsson, solution (Pimephales (Pimephales promelas; Nebeker et al., Bengtsson, 1980) 1980) or or in in solution al., 1974) at low ((1.8 pgll) doses. 1974) 1.8 p.g/l)
260
VON WESTERNHAGEN WESTERNHAGEN H. VON
Impairment of of egg egg production production in in some some of of the the above-mentioned above-mentioned Impairment species is is also also known known to to be be caused caused by by depressed depressed pH pH of of the the holding holding species promeIas) (Mount, 1973) 1973) and Hagfish water. Thus fathead minnow ((P. P . promelas) and flagfish (J.I.jloridae) fiondae) when kept in water 4.5 and 6.0 ( water at pH levels between 4.5 produce produce significantly fewer eggs than in an an environment with higher pH 6.8) (Craig and Baksi, 1977). 1 977). In fact, fact, not only is the of pH (pH 6.8) and Baksi, the number of of fully mature (stage (stage 6) oocytes eggs laid reduced, but the production of of being fertilized is reduced to 20.7, 20.7, 15.8, 15.8, or 8.2% 8.2% at pH 6, capable of al., 1977). 1977). At the lowest pH, mature 5.5, or 4.5, respectively (Ruby et al., oocytes soon undergo resorption and become atretic, while there is a of early oocyte stages in the ovary. In brook trout preponderance of (Saivelinus fontinah), jontinalis), low pH delays ovulation considerably (Tam (Saluelinus 1986). Beamish et al. (1975) ( 1975) suggested that due to altered and Payson, 1986). matu pH, normal calcium metabolism required for successful ovarian matu( Urist and Schjeide, 1961) 1961) is altered, resulting in abnormally ration (Urist low calcium concentration in female serum, which in turn affects the female reproductive physiology. B. Fertilization, Water Uptake, and Water Hardening While heavy metals have only minor effects on the process of fer of fertilization, low pH through reduced sperm activity may lower fertiliza fertilization rate in freshwater fish considerably. Petroleum hydrocarbons disdis turb formation of of a fertilized egg, probably via effects on cleavage membrane formation, while chlorinated hydrocarbons inside the egg are responsible for a considerable reduction of fertilization rate due to cytogenetic damage. Impairment of osmotic activity of perivitelline colloids reduces water uptake in eggs reared in water of of low pH or high metal levels. An important step toward successful reproduction in fish is the of the extruded egg and the water-hardening process, two fertilization of two events that may very well be reversed in sequence depending on the fish species involved. Thus, in the pelagic eggs of of many marine fish, the process of of water hardening and water uptake may preceed the actual event of fertilization (Kjarsvik (Kjorsvik and Lanning, Lonning, 1983; 1983; Lanning Lonning et al., aE., 1984); 1984); fertilization may not be necessary for water hardening (Potts (Potts and Eddy, 1973), 1973), or fertilization is is required for the hardening process as in most demersal eggs, such as in herring and salmonids. There are few reports of of heavy metals exerting detrimental effects (C. harengus), harengus), Ojaveer et al. (1980) on the fertilization rate. In herring (C. al. (1980) observed reduced fertilization when this process was conducted in
4. 4.
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261 261
of 0.01 0.01 copper contaminated water at an ambient copper concentration of mg/l. Yet Blaxter ((1977) mg/I. 1977) was able to fertilize herring eggs at copper of 22%. These concentrations of 0.9 mg/l, obtaining a fertilization rate of Alderresults fall in line with information provided by Rosenthal and Alder dice (1976), (1976), who reported reported that that fertilization fertilization of of Pacific Pacific herring herring eggs is virtually unaffected by exposure of up to 10 10 mg/l cadmium/l prior to fertilization. fertilization. IIn n contrast to the action of metals metals,, the pH of the incubation and fertilization media seems to have a delicate bearing on fertilization rate as shown by Petit et al. al. (1973) (1973)through work with rainbow trout (Salmo gairdneri). gairdneri). At optimum sperm concentration, fertilization rate (Salmo drops from about 90% at pH 9.5 50% at pH 7.3, 7.3, although it is 9.5 to only 50% not not clear clear whether whether this this is is an an effect effect on on the the eggs eggs or or the the sperm sperm or or both. both. 1958) confirm confirm Earlier experiments with sperm of trout (Inaba (Inaba et al., 1958) that sperm mobility increases with rising pH; thus the reduced fertil fertilization rate described above may be an effect of the lowered sperm activity. activity. Reduced Reduced sperm sperm activity activity with with resulting resulting lowered lowered fertilization fertilization success success occurs occurs also also after after treatment treatment of of herring herring sperm sperm with with oil oil disper dispersants (Wilson, (S. gairdneri) gairdneri) (Wilson, 1976) 1976) as well as after the exposure of trout (S. sperm 1 mg/l (McIntyre, >1 (McIntyre, 1973). 1973). sperm to methylmercury at concentrations > While While the the latter latter author author used used seawater seawater of of higher higher salinity salinity for for the the incuba incubation 1980) were were tion of of herring herring eggs, eggs, the the experiments experiments of of Ojaveer Ojaveer et al. ((1980) conducted in seawater seawater of of only only 5.6-5.8%0 5.6-5.8%0 salinity. salinity. In In these these experi expericonducted in ments, ments, not only copper but also cadmium at concentrations higher than 0.005 mg/l mg/l affected affected fertilization fertilization negatively, negatively, ultimately ultimately yielding yielding than 0.005 only 1.0 mg only 62% 62% fertilized fertilized eggs eggs at at 1.0 mg copper/I, copper/l, and and 60% 60% at at 0.5 0.5 mg mg cad cadmium/I. mium/l. Due to the fact that the two experimental series were con conducted ducted in in different different salinities, salinities, they they are are difficult difficult to to compare compare in in terms terms of of metal efmetal effects effects on on fertilization, fertilization, since since we we know know that that particularly particularly the the ef fects of cadmium are salinity-infl salinity-influenced (von Westernhagen et al., fects uenced (von 1974). The origin of this sublethal response is not clear. clear. It might be 1974). caused by the interference of cadmium with the jelly coat of the egg, thus altering the site site of the penetration of the sperm into the egg, the micropyle. micropyle. A direct direct influence influence on on the the formation formation of of the the zygote zygote can can probably probably also also be excluded excluded as as the the mode mode of of action action of of pollutants pollutants such such as as aromatic aromatic hy hydrocarbons drocarbons (xylene), (xylene), which which prevent prevent the the formation formation of of aa fertilized fertilized egg egg and 10mg/l mg/l in in cod cod and early early cleavage cleavage stages stages at at concentrations concentrations higher higher than than 10 eggs 1982). The The action action of of aromatic aromatic hydrocarbons hydrocarbons such such eggs (Kj6rsvik (Kjorsvik et al., 1982). as as para-xylene para-xylene on on fertilization fertilization and and early early cleavage cleavage and and the generation generation of of the the characteristic characteristic small small cells cells as as described described by by Lanning Lonning (1977) (1977) in in the the reaction reaction of of plaice plaice (Pleuronectes (Pleuronectes platessa) eggs eggs to to xylene xylene and and benzene benzene
262
H. VON WESTERNHAGEN
are probably derived from their properties of causing membrane dam damage and increased membrane permeability (Roubal (Roubal and Collier, 1975; 1975; Morrow et al., 975). Mechanisms located in the cell surface are im al., 11975). important for the fonnation formation of the cleavage membrane, as shown by Rappaport (1977) (1977) in an investigation of of cleavage in eggs from different invertebrates invertebrates.. As Roubal and Collier (1975) (1975) pointed out, aromatic hy hydrocarbons attack the outer surface of of membranes and may thus influ influence the mechanism of of cleavage, as apparent in the photographic evidence of Kjarsvik 1982) in fertilized plaice (P. (P. platessa) eggs. Kjorsvik et al. al. ((1982) eggs. Similar effects are caused by the carcinogen benzo[a]pyrene (BAP) (BAP) in flatfish fladish embryos (sand sole, Psettichthys melanostichus; flathead sole, Hippoglossoides elassodon) al., 1982). 1982). elassodon) (Hose (Hose et al., hyStrong depression of fertilization is also caused by chlorinated hy drocarbons incorporated into the egg from parental sources such as DDT and dieldrin in eggs of of winterflounder (Pseudopleuronectes americanus (Smith 1973) or polychlorobiphenyl (PCB) (Smith and Cole, 1973) (PCB) (34 (34 ppm) ppm) in Atlantic salmon (S. ( S . salar) salar) (Jensen et al., al., 1971). 1971). In these cases, residues (4.6 ppm DDT; 1.2 1.2 ppm dieldrin) inside the egg give rise to considerable reduction of 12%, thus of fertilization rate down to 40% and 12%, suggesting direct cytogenetic effects. High DDT residues in the same range may be responsible for the failure of reproduction in seatrout Cynoscion nebulosus (Butler (Butler et al., al., 1972). 1972). Alderdice et al. 1979a) have shown that metals such as cadmium al. ((1979a) delay the water hardening and the process of water uptake in Pacific herring eggs, while at the same time the primary bursting pressures of exposed eggs are reduced from between 700 and 1300 1300 g to 200 and 350 g at cadmium concentrations near 11mg/I. mg/l. Brungs (1969) (1969) observed a similar effect in zinc-treated eggs of the fathead minnow, Pi Piggs in zinc concentrations higher than 0.18 mg/l mephales promelas. E Eggs remain in a fl accid condition resulting frequently in the rupturing of flaccid of the egg capsules during handling. Since the hardening of the egg chorion requires calcium (Kusa, (Kusa, 1949; 1949; Lanning Lonning et al., al., 1984) 1984) and the presence of an enzyme (Zotin, (Zotin, 1958), 1958), this process might very well be influenced by cadmium, a metal that is chemically closely related to calcium; a strong calcium/cadmium calciumlcadmium interaction could be assumed, since cadmium competes with calcium for binding sites in the egg (Maljkovic and Branica, 197 1971; capsule (Maljkovic 1 ; von Westernhagen et al., 1975), 1975), thus interfering with the hardening process. The bound cadmium might alter the physical properties of the capsule and its jelIy jelly coat, (Alderdice et al., al., 1979a) 1979a) and penneability permeability to salt reducing its strength (Alderdice and water. Although the cadmium effect on capsule strength may be explained, effects of of other metals and substances are subject of of prob problematic interpretation.
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263 263
Reduced water uptake by salmonid eggs is also known to be caused by low pH. Thus Peterson and Martin-Robichaud ((1982) 1982) and Eddy and Talbot ((1983) 1983) as well as Rombough and Jensen ((1985) 1985) report inhibited water uptake of newly fertilized eggs of Atlantic salmon and 5.5and lower. Together with the reduced rainbow trout in water of pH 5.5 water uptake goes a decrease in the ability to resist deformation when consubjected to mechanical loads. loads. Rombough and Jensen ((1985) 1985) con exocycluded that the low pH probably interferes with cortical vesicle exocy affects the osmotic activity of perivitelline colloids, for in intosis and affects (Rudy and stance through denaturation of the proteinaceous colloid (Rudy 1969), so active. The same mech mechPotts, 1969), so that it is no longer osmotically active. anism might apply in the action of heavy metals.
C. E Early C. arly Development Blockage Blockage of phosphorylation of ADP caused mainly by aromatic hydrocarbons and naphthalenes may lead to visible effects on early cleavage patterns. Initial irregular cleavages can be related to cytoge cytogenetic damage and can be traced through blastodisc and early gastrula formation by the appearance of opaque cell patches indicating irregu irregular cell cell sizes. sizes. In the meroblastic fi sh egg, the early cleavages on the surface of fish the yolk separate the clearly visible developing blastodisc from the yolk mass. Hence any deviation from the "typical" “typical” cleavage pattern is is easily recognized and has frequently been used to describe sublethal effects on early embryogenesis. In particular, effects effects of temperature and salinity (Lieder, (Lieder, 1964; 1964; Alderdice and Forrester, 1968; 1968; von Wes Wes1968, 1970, 1970, 1974) 1974) have attracted the attention of scientists ternhagen, 1968, (see (see Chapter 3, 3, this volume). volume). In general, sublethal effects effects of pollutants are seldom visible at the very early cleavage stages. stages. Experimentally only substances such as the aromatic compounds compounds benzene and xylene provided irregular cleavages cleavages in the two- to eight-cell stages of plaice platessa) (Lonning, (Lonning, 1977) 1977) and cod (G. morhua) eggs (Pleuronectes platessa) (G. morhua) (Kjorsvik (Kjorsvik et al., al., 1982). 1982). The appearance appearance of the early cleavage stages upon treatment with aromatic hydrocarbons clearly demonstrates the impairment of cell division. division. Similar disruptive early cleavage patterns in cod eggs have been reported by Dethlefsen ((1977) 1977) on treatment of incubated eggs with DDT and DDE. We may find other substances that have the same effects on fi sh eggs, fish eggs, since during our current re research (unpublished data) Baltic we have frequently found data) in the Baltic abnormal early cell stages in pelagic eggs (cod, (cod, plaice, flounder, flounder, sprat) sprat) (Fig. (Fig. 1). 1).The same observations have been made for cod eggs by Kjors-
264
H. VON WESTERNHAGEN H.
Fig. 1. Aberrant Fig. 1. Aberrant early early cleavages cleavages and and gastrula gastrula (arrows) (arrows) in in (a, (a, b, c) c) cod cod (Gadus (Gadus morhua) morhua) eggs, eggs, (d) (d)sprat sprat (Clupea (Clupea sprattus) sprattus) eggs, eggs, and and (e, (e, f) f) plaice plaice (Pleuronectes (Pleuronectes platessa) platessa) eggs, eggs, caught caught with with aa plankton plankton net net in in the the Baltic Baltic in in 1983. 1983. Horizontal Horizontal bars bars 200 200 /-Lm. pm.
vik al. ((1984) 1984) in by Dethlefsen 1985) vik et al. in Norwegian Norwegian waters waters and and by Dethlefsen et al. ((1985) for the eggs of fl ounder Platichthys fiesus, flounder ftesus, dab Limanda limanda, and whiting in the the German German Bight. Bight. whiting Merlangius merlangus in Sublethal effects on the developing embryo are more pronounced in blastodisc stage epiboly and in the the blastodisc stage and and during during beginning beginning epiboly and may may readily readily (1906) describes effects of of Liel LiCl be caused experimentally. Stockard (1906) solutions F . heteroclitus, provoking provoking unusual unusual enlargement enlargement of of the the solutions on on F. segmentation cavity under the blastodisc. Moreover, it is at the blasto blastodisc sh (B. ( B . rerio), rerio),produce produce proto protodisc stage stage that that zinc-treated zinc-treated eggs eggs of of zebrafi zebrafish plasmic protrusions projecting abnormally from the sides sides of the em embryo 1977). When When exposed to to naphthalenes, naphthalenes, bryo (Speranza (Speranza et al., 1977). methylnaphthalenes, methylnaphthalenes, and and aromatic aromatic hydrocarbons, hydrocarbons, eggs eggs of of several several ma marine species display retarded early cell division and differentiation into irregular blastodiscs with opaque patches, indicating different cell sizes 1984; Falk-Petersen et ai., al., 1982), 1982),prob probsizes (Stene (Stene and Lonning, 1984; brief treatment dur durably originating from the initial cleavages. Only brief ing early cleavage frequently permits the embryo to develop normally apparup to midgastrulation before abnormal development becomes appar ent. blastoent. Typical Typical effects effects during during epiboly epiboly are are irregular irregular margins margins of of the blasto cells giving rise to malformed gastrulae, the derm with different size size cells embryo being less distinct and often surrounded by irregular cells (Falk-Petersen 1982). These defects become particularly evi(Falk-Petersen et al., 1982).
4. 4.
EFFECTS OF POLLUTANTS ON FISH EGGS AND LARVAE
265
dent in experiments with aromatic hydrocarbons and DDT and DDE, (G. morhua) morhua) eggs and Smith as reported by Dethlefsen ((1977) 1977) for cod (G. and Cole ((1973) 1 973) for the eggs of winter Hounder flounder (Pseudopleuronectes (Pseudopleuronectes However, embryos with similar aberrations are found in americanus). americanus). the field field (Fig. (Fig. 1). 1). Investigations by Longwell and Hughes (1980) (1980) pro provide some evidence evidence for statistically significant associations between of the health of of cytological, cytogenetic, and embryological diagnoses of pelagic pelagic mackerel mackerel eggs eggs and and heavy heavy metal metal and and toxic toxic hydrocarbon hydrocarbon levels levels of some surface waters. Embryos from these areas (New York Bight) show increased incidence of chromosome and mitotic abnormalities, which to the the observed observed developmental developmental aberrations. aberrations, Par Parwhich probably probably led to ticularly high incidences (>50%) of chromosome bridging and translo translocations cations are are encountered. encountered. It It may may be be difficult difficult to to explain explain the the cause cause and and significance of these aberrations of of early development. development. There could be several reasons, but most of them are probably biochemical in origin, inhibiting metabolic processes responsible for differentiation and 1976) suggest suggest aa blockage blockage and maintenance. maintenance. Rosenthal Rosenthal and and Alderdice Alderdice ((1976) of phosphorylation of adenosine diphosphate (ADP), (ADP),thus inhibiting the formation of adenosine triphosphate (ATP), (ATP), which is a prerequi prerequisite for a multitude of metabolic processes necessary for differentia differentiation. tion. If the energy budget of the embryo is severely reduced either by direct an overload overload in in direct blockage blockage of of the the above above mentioned mentioned pathway pathway or by an metabolic metabolic work work required required for for detoxification detoxification of of hydrocarbons hydrocarbons through through enzymatic enzymatic degradation, degradation, no no coordinated coordinated differentiation differentiation takes takes place place and and development is retarded or arrested. Similar effects can be provoked by exposure exposure of of herring herring embryos embryos (C. (C.harengus) harengus) to to dinitrophenol dinitrophenol (DNP) (DNP) (Stelzer (Stelzer et al., al., 1971), 1971), aa decoupler decoupler of of oxidative oxidative phosphorylation. phosphorylation. Other Other causes the causes for for aberrations aberrations from from the the normal normal cleavage cleavage patterns patterns may may be the substances affecting cell cleavage directly, as does colchi colchiaction of substances cine through inactivation of the chromosome transport mechanism of hydrocarspindle apparatus apparatus.. Similar effects are exerted by cyclic hydrocar the spindle 1977), and the effects resemble those caused by DNP bons (Lanning, (Lonning, 1977), (Watennann, 1940). 1940).Yet short-term effects, when sublethal, might not (Watermann, necessarily be persistent. Particularly during the very early cleavage stages, irregularities caused by different stressors might be adjusted in stages, the course of development. development. If, If, for instance, irregular cell cell patterns undergo further cleavages, the previously noticed asymmetrical pat patterns terns may may disappear disappear in in the the morula morula stage stage and and macroscopically macroscopically no no traces traces (H. von Westernhagen et al., of the initial aberration can be noticed (H. unpublished). The only only remnants of unequal initial cleavages may may be unpublished). chromosome aberrations. Thus, Thus, particularly in early developmental developmental stages in situ, situ, investigations demonstrate relatively high rates of chrochrostages
266
H. VON WESTERNHAGEN H.
mosomal aberrations (anaphase (anaphase aberrations) aberrations) in pelagic fish eggs (Longwell 1980; Kjorsvik et al., 1984; (Longwell and Hughes, 1980; 1984; Dethlefsen et al., 1985). 1985). The significance of these anaphase aberrations for later em embryos is not clear, but Kjorsvik and co-workers suggest that this is is a sign of “bad "bad quality” quality" eggs, yielding low rates of of advanced embryos. embryos. D. Advanced and Late Development A multitude of observations describes the morphological reactions of advanced fish embryos to various pollutants. The most conspicious damages at this stage are abnormal development of the spinal column, abnormal head and eye development, and irregular proliferations from the main body over the yolk surface. surface. Neither of of the mentioned pollutants provokes typical, single-pollutant-specific reactions in the embryo. Morphological aberrations are not particularly pollutant-spe pollutant-specific cific and may be caused also by natural stressors. deThere exists relatively little experimental work on abnormal de velopment in fish embryos before neurulation and formation of the head and optic cups. cups. Most of the effects described are of of advanced and late development, although in nature the situation is different in the 10sense that the rate of malformed early embryos in the sea is 4- to 10(Kjorsvik et al., 1984; 1984; Dethelfsen et al., al., fold that of the late stages (Kjorsvik 1985).One of the reaons why most authors concentrate on later devel devel1985). opmental stages is probably because early aberrations are very incon inconspicious. Besides, in one group, the salmonids, the early stages are extremely delicate to handle and most investigations study the salmo salmonid only beginning at the eyed stage. In this context the term ad advanced development applies to embryos at or beyond stage II, 11, as described by von Westernhagen ((1970) 1970) for cod, flounder, flounder, and plaice, e y e formation, visible tail bud, that is, with the beginning of head and eye and yolk two-thirds surrounded by blastoderm. irreguThe most conspicious damages at and before this stage are irregu lar margins of the periblast on the yolk displaying a serrated appear appearance and emigrating groups of opaque cells in the blastoderm blastodenn layer or in the space between blastoderm and periblast. We have found these (Fig. lc), lc), but the period sorts of defects in live eggs taken in the Baltic (Fig. of epiboly prior to organ formation is rarely described in literature. literature. of Probably before this stage of organ differentiation a lot of early dam damif no more stress is age can be repaired and the embryo may recover if applied. At the same time, this period (gastrulation before closure of applied. of blastopore) blastopore) is considered especially sensitive, and stressed embryos
4.
EFFECTS OF POLLUTANTS ON FISH EGGS EGGS AND LARVAE
267
development. However, at die rather than compensate with aberrant development. the time of organ formation, effects become particularly pronounced in head and notochord, as described for effects of copper and zinc in (G. morhua) morhua) embryos (Swedmark (Swedmark and Granmo, 198 1981) cod (G. 1 ) and lead at B . rerio) rerio) 0.05 and 0.07 mg/l in dechorionated embryos of zebrafish ((B. (Ozoh, 1980). 1980). (Ozoh, These malformations are very similar to those caused by treatment of fish with petroleum hydrocarbons or derivates. Thus, offish Thus, Linden ((1974, 1974, 1976, 1978) reported on herring eggs, which, when treated with crude 1976,1978) oil and/or oil dispersants from 3. 1 to 59 mg/l water-soluble fraction 3.1 (WSF) of crude and number 11 fuel oil, (WSF) oil, display abnormal spinal columns, abnormal heads, and lack of of spinal column. column. In comparable 0.1 to 11.0 mgA, Lonexperiments with Ekofisk oil at concentrations of 0.1 .0 mg/l, Lan 1977) described the same effects on plaice (Pleuroneces ning ((1977) (Pleuroneces pla platessa) embryos. tessa) An embryo embryo damaged in such a manner is is called a typical "oil “oil larva," larva,” with poorly differentiated head, protruding eye lenses, and a bent notochord. Inhibited pigmentation also can be taken as a suble sublestresss (Kjarsvik (Kjorsvik et al., 1982). thal effect and reaction to oil stres 1982). Embryos with these malformations are frequently found after oil spills in the vicinity of oil slicks, slicks, an indication of the fast action of of the WSF of crude oil on fish embryos (Longwell, (Longwell, 1977). 1977). Figure 2 shows the differ different gross morphological abnormalities of cod eggs caused by treat treatment with Iranian crude oil. Available literature on teratogenic effects of of chlorinated hydrocar hydrocarbons on advanced and late fish embryos can be summarized as fol follows. lows. Cod embryos (G. (G. morhua) morhua) exposed to DDT concentrations of mg/l and more react with irregular proliferations at the yolk 0.025 mgtl zigzag-growing spinal surface, and the embryo develops a bent or zigzag-growing column (Dethlefsen, (Dethlefsen, 1977). 1977). Sheepshead minnow (Cyprinodon (Cyprinodon va variegatus) and killifish (F. ( F . heteroclitus) eggs, when subjected to DDT and malathion (organophosphate (organophosphate insecticide), insecticide), carbaryl, or parathion at 10 mg/l, 10 mg/l, display developmental arrest prior to the initiation of heart beat. Blood pigmentation does not occur. occur. Cyprinodon C yprinodon variegatus de develops a malformed spine (Weis (Weis and Weis, 1974, 1974, 1976). 1976). Experiments conducted by Kaur and Toor ((1977) 1977) with carp eggs (Cyprinus (Cyprinus carpio) carpio) and the insecticides diazinon, fenitrotion, carbaryl, malathion, and phosphamidon produce similar effects. Upon exposure to concentra concentrations around 0.008 0.008 (diazinon), (diazinon), 0.25 0.25 (fenitrothion), (fenitrothion), 1.0 1.0 (carbaryl) (carbaryl),, 2.5 (malathion), 12 (phosphamidon) (malathion), and 1112 (phosphamidon) mg/l the embryos show stunted growth, curving of the tail, deformed head regions, enlargement of the pericardial sac, sac, circulatory failure, deformed vertebral column, and
H. VON WESTERNHAGEN
268 268
1 . 5 mm
Fig. 2. Abnormalities in embryonic development of of cod (Gadus (Gadus morhua) rnorhua) eggs un unFig. der the influence influence of (WSF) of Iranian crude oil: (a) (a) early of the water-soluble water-soluble fraction (WSF) (d) twinning, (e) (e)axis deformation, and cleavage, (b) (b) gastrula, (c) (c) embryo without head, (d) (f) (f) microphthalmia. microphthalmia. [From Kuhnhold ((1974).] 1974).]
poorly poorly developed developed eye eye pigment pigment and and chromatophores. chromatophores. Virtually Virtually all all of of these sublethal effects can also be observed when fish embryos embryos de dethese sublethal effects can also be observed when Wesvelop under naturally stressed conditions, as described by von Wes of temperature and salin salinternhagen ((1970) 1970) for the effects of extremes of ity ity on on plaice plaice (P. (P. platessa) embryos embryos (Fig. (Fig. 3). 3). Grossly deformed embryos may be caused Grossly deformed embryos may caused by by still still other other factors, factors, for for (pH 4.5) of of the incubating water in fathead minnow instance, low pH (pH (Pimephales (Pimephales promelas) promelas) (Mount, 1973) 1973) and Atlantic salmon (S. (S. salar). salar). Yet the typical injuries to salmon embryos by low pH, such as altera alterations in vascular structures, cellular dysplasia, necrosis, and sloughing 1980),are similar to those (Daye and Garside, 1980), of superficial ectoderm (Daye caused by heavy metals, detergents, halogenated organic compounds, Thus, it is apparent that it might be and some petroleum fractions. Thus, difficult if not impossible to identify any particular substances respon responsible one sible for for one one or or several several sublethal sublethal morphological morphological effects. effects. On On the one hand, it is difficult to distinguish clearly between morphological, morphological, physiological, or behavioral abnormalities, since one may result from
4.
EFFECTS OF POLLUTANTS ON FISH FISH EGGS EGGS AND LARVAE
269
Fig. 3. 3. Plaice Plaice (Pleuronectes (Pleuronectes platessa) platessa) eggs eggs and and larvae larvae incubated incubated under under extreme extreme temperature aberrant development: development: (a) (a) temperature and and salinity salinity conditions. conditions. Arrows Arrows indicate indicate zones zones of aberrant salinity; (b) aberrant O T , 25%0 25% salinity; (b)gastrula gastrula with with irregular irregular cell proliferation, proliferation, aberrant early early cleavage, cleavage, ooe, lOoe, l O T , 25%0 25% salinity; salinity; (c) (c) embryo embryo not not able able to to close close blastopore, blastopore, lOoe, 10°C, 20%0 20% salinity; salinity; (d) (d) distorted n development, distorted notochord, notochord, failure failure in in pectoral pectoral fi fin development, 2°e, 2"C,33%0 33% salinity; salinity; (e, (e, f) f ) crip cripm. pled 15% salinity. salinity. Horizontal Horizontal bars bars 200 200 p.. pm. pled larvae, larvae, lOoe, IOT, 15%0
the other, as they are frequently related. n anomalies related. For example, example, fi fin may ed (reduced) may be be related related to to modifi modified (reduced) dermal dermal respiration respiration (Rosenthal (Rosenthal and and Alderdice, Alderdice, 1976). 1976). On On the the other other hand, hand, the the detrimental detrimental action action of aa metal · on embryogenesis may be indirect. For example, cadmium in high high concentrations concentrations may may alter alter the the properties properties of of the the egg egg membrane membrane and and its its "jelly '?jelly coat," as as known from from herring herring eggs eggs (Alderdice (Alderdice et al., 1979c) 1979c)or eggs eggs of garpike (von (von Westernhagen et al., 1975), 1975), ultimately impeding oxygen exchange. Thus, Thus, observed malformations malformations in these experiments experiments may may be attributed attributed simply simply to to lack lack of of oxygen, oxygen, as as described described by by Braum Braum for herring eggs eggs incubated experimentally under low low oxygen oxygen ((1973) 1973) for tension, rather than to direct effects of the the metal. metal. In In fact, fact, some some of of the the anomalies anomalies resemble resemble monstrosities monstrosities produced produced during during incubation incubation at at low low oxygen oxygen levels levels as as described described by by Alderdice Alderdice et aI. 1 958) for 1973) for al. ((1958) for salmon salmon (0. (0.keta) keta) and and Braum Braum ((1973) for herring herring eggs eggs (Clupea (Clupea harengus). harengus). The The general general retardation retardation or or arrest arrest of of development development is is also also aa phenomenon phenomenon occurring occurring at at low low oxygen oxygen levels levels (see (see also also Ham Hamdorf, dorf, 1961). 1961).Therefore, Therefore, in in general, general, one one may may say say that that the the major major morphomorpho-
270
H. VON VON WESTERNHAGEN
logical logical aberrations aberrations such such as as notochord notochord distortions distortions and and head head and and eye eye malformations in late embryos are malformations occurring occurring in late embryos are not not particularly particularly pollu pollutant-specific tant-specific but but are are the the expression expression of of an an embryo embryo in in aa stressed stressed condi condition. tion. E E.. Effects Other than Morphological Aberrations As nonmorphological aberrations, effects on egg shell (chorion), (chorion), embryo activity, and the hatching enzyme are prominent. Deviating from the nonspecific cause/effect relationship for gross morphological deformities and pollutants, reduction of chorion strength is mainly caused by heavy metals and at times by low pH. Low pH typically also depresses activity of the hatching enzyme, which results in low or retarded hatch. Petroleum hydrocarbons are extremely effective de depressors of embryo activity measured as heart beat, pectoral fin move movement, or body activity. Total embryo activity, though, is likewise re reduced by other pollutants. Aside from the occurrence of gross gross malformations, several func functions of the chorion and the embryo are drastically impaired during and after exposure to these pollutants. As already mentioned, cad cadmium severely impairs hardening of the egg membrane after fertiliza fertilization. Cadmium-exposed herring eggs never reach the maximum hard hardness attained by untreated eggs, and the egg capsules remain flaccid throughout embryogenesis (Rosenthal 1974; von WesWes (Rosenthal and Sperling, 1974; temhagen et al., 1974; 1974; Alderdice et al., 1979a). 1979a). In conjunction with this effect, herring eggs upon treatment with cadmium display smaller volumes than individuals incubated under uncontaminated condi conditions (Alderdice al., 1979a,b), (Alderdice et al., 1979a,b), leaving a smaller perivitelline perivitelline space. Also, zinc at a concentration of 6.0 mg/l causes the softening of of egg membranes in C. C. harengus eggs (Somasundaram et al., 1984b) 198413) and brook trout (Salvelinus (Holcombe et ai., 1979). In fathead (Salvelinusfontinalis) fontinalis) (Holcombe al., 1979). minnows (Pimephales (Pimephales promelas), promelas),at a concentration of 295 or 145 145 1Lg/1 pgIl touchand above, Zn reduces chorion strength so that eggs burst upon touch ing. Bursting pressure is only 15% 15% of the normal value at zinc concen concentrations of 1360 1360 ILg/I pgIl (Benoit and Holcombe, Holcombe, 1978). 1978). Chorion strength is also negatively influenced by low pH, as shown by the investigations of Mount ((1973) 1973) with fathead minnow eggs (P. 5.9. The same effects of ( P . promelas) exposed to pH lower than 5.9. low pH on capsule strength are known for eggs of of rainbow trout (S. (S. gairdneri) 1981) report gairdneri) (Kugel, (Kugel, 1984), 1984), although Haya and Waiwood ((1981) hardening of of Atlatnic salmon eggs in water of pH 4.5 due to a change
4. 4.
EFFECTS OF POLLUTANTS EGGS AND EFFECTS OF POLLUTANTS ON ON FISH FISH EGGS AND LARVAE LARVAE
271 271
in the physical structure of the outer mucopolysaccharide layer of the chorion. chorion. The significance of the softening of the egg shell to substrate spawning fish such as salmonids eggs buried salmonids is is evident since the soft eggs fact, in the gravel, when subjected to movement, may easily break. In fact, the eggs of the fathead minnow at low pH become so flaccid that the cleaning action of the male on the egg clutches breaks the egg shells (Mount, (Mount, 1973) 1973) and kills the embryos. embryos. Similar detrimental effects on eggs of the substrate spawning Pacific herring may be expected when high metal concentrations cause softening of the egg membranes. membranes. When the embryo starts to develop a heartbeat, this parameter has frequently been used as a measure of pollutant effects. effects. Typically, heartbeat frequency increases with age, age, and although subject to con considerable variation caused by by disturbances this increase is is consistant with ongoing development (Rosenthal, (Rosenthal, 1967). 1967). Metals such as cadmium reduce embryonic heart rate considera considerably. Thus in garpike Belone belone embryos, reduction in heart rate can be caused by incubation in water containing 1.0 1.0 mg/l or more of the metal, depending on the salinity of of the incubating medium (Fig. (Fig. 4). 4). This is true also for the heartbeat in the Japanese medaka Oryzias latipes when the embryos are reared in 60 p.g p g methylmercury/I, methylmercury/l, where heartbeat is reduced from 80-90 80-90 to 50 beats per minute (Dial, (Dial, 1978). 1978). Zinc at a concentration of of 2 mg/l cause a transient 2 :: 1 heart block (two (two beats of the atrium for every beat of of the ventricle) after 3-4 3-4 days of of exposure in fathead minnow P. P . promelas (Pickering and Vigor, Vigor, 1965). 1965). Linden (1974, 1976, 1978) and Kiihnhold (1974,1976,1978) Kuhnhold (1978) (1978) demonstrated that petroleum hydrocarbons are extremely effective in the impairment of the normal functioning of the heart. Crude oil, especially in conjunc conjunction with oil dispersants, reduces heart-beat of herring embryos by 50%. 50%.Also, killifish F. F . heteroclitus embryos show sublethal responses to the water-soluble fraction of crude oil, which reduces heartbeat and overall embryo activity (Sharp al., 1979). (Sharp et al., 1979). Other hydrocarbons, such as benzene, applied at concentrations of 177 and 45 mg/l to incubating jars with Pacifi c herring C. pallasi and anchovy Engraulis mordax also Pacific reduce heart-beat of late embryos (Struhsaker et al., al., 1974) 1974) or induce irregularities, as does toluene in the embryo of the Japanese medaka 0. (Stoss and Haines, 1979). 1979). O. latipes (Stoss Changes in embryonic heart rate have also been caused experi experimentally by other pollutants, including DNP (Rosenthal (Rosenthal and Stelzer, 1970: C. harengus), 1970: C. harengus), sulfuric acid from titanium dioxide production (Kinne (Kinne and Rosenthal, 1967: 1967: C. harengus), harengus), or the organophosphate insecticide malathion (Weis (Weis and Weis, 1976: 1976: Cyprinodon variegatus). variegatus). At a stage of of development where the regular heartbeat is well
272 272
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is developed far enough to enable the established, the embryonic axis is movements . These movements, embryo to make the first wriggling movements. accomplished by slight, repetitive body flexure, flexure, are presumed to circir accomplished perivitelline fluid, fluid, thus improving the increasing oxygen culate the perivitelline of the late embryo. In some species that release very advanced needs of larvae from their eggs (i.e., (Le., B B.. belone, 0. O. latipes, F F.. heteroclitus, salmonids), the undulating movements of of the body axis are supported salmonids), flapping of of the pectoral fins and the opercula. opercula. As is the case for by flapping heartbeat, embryonic movements may be severely impaired either directly through through the stressors or indirectly whenever egg volumes are reduced imposing mechanical blockage to movements. The influence of cadmium, for instance, on embryonic movements in herring eggs of becomes apparent when the embryo has grown to encircle the yolk. "wriggling" then is is replaced by “trembling,” "trembling," a high-frehigh-fre The normal “wriggling” is maintained maintained even in more advanced embryos, quency shivering that is "somersaults" within the which are normally performing rotations or “somersaults” of activities are influenced influenced by egg shell. As Fig. 5 shows, both types of (von Westernhagen et al., al., 1974). 1 974). cadmium in the incubating water (von 13 mg/l exert strong paralyzing efef Copper concentrations around 0. 0.13 be fects on herring embryos, which, with progressing development, become more and more immobilized (von Westernhagen et d al.,. , 1979). 1979). Other sublethal effects of of cadmium on embryo activity are displayed by the reduced pectoral fin movements of of garpike ((B. B . belone) embryos al., 1975) concentra (von Westernhagen Westernhagen et al., 1975) when exposed to cadmium concentramg/1. Stoss and Haines (1979) ( 1979) as well as Leung tions higher than 1.0 mg/l. and Bulkley ((1979) 1979) report influence of toluene on the opercular move move0. latipes, which ment of the late embryo of the Japanese medaka O. becomes erratic, irregular, and shallow at concentrations of of 80-100 80-100 pgIl (WSF). (WSF). A general decrease of embryonic activity is known for JLg/1 salar) embryos when reared at low pH of 4.0-4.5 4.0-4.5 compared salmon (S. salar) to controls in pH 6.7 (Peterson (Peterson and Martin-Robichaud, 1983). 1983).A possi possible explanation for the reduced activity in cadmium-treated embryos Pacific herring may be the effects of cadmium on enzyme activity. In Pacific eggs, Mounib et al. al. ((1976) 1976) found that exposure to 10 10 mg cadmium/l cadmiumll decreased activity of four important carbon dioxide-fixing dioxide-fixing enzymes: enzymes: propionyl coenzyme A (CoA) (CoA) carboxylase, nicotinamide adenine di dinucleotide (NAD) (NAD) and NADP NADP malic enzymes, and phosphoenolpy phosphoenolpyruvate (PEP) eggs, PEP (PEP) carboxykinase. In control eggs, PEP carboxykinase activ activity ity increases by two two orders orders of magnitude in the period from the early blastodisc just prior to hatching. The increase indicates the impor importance tance of the enzyme metabolism. metabolism. In contrast, there is considerably less less increase increase in in PEP PEP carboxykinase carboxykinase activity activity in in cadmium-exposed cadmium-exposed eggs eggs
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,
,\
,\
,\
\ " \.
0 01
0.5
to
50
erent cad Fig. Fig. 5. 5. Activity Activity of herring herring (Clupea (Clupea harengus) harengus)embryos embryos influenced by diff different cadmium mium concentrations concentrations in the incubating incubating water. water. [From [From von Westernhagen Westernhagen et et ai. al. (1974).] (1974).]
up to the beginning of embryo activity, while in the controls activity further. Final activity of of the enzyme is more than 25% less increases further. of the controls. Relative activity of of propionyl-CoA carboxyl carboxylthan that of ase in the cadmium exposed eggs is about 20% 20% of the control level closure 32% prior to hatching. Relative prior to blastopore clos ure and only 32% of NAD and NADP malic enzymes enzymes remains stable up to eye activity of reduced by about 20% 20%just prior to hatching, when pigmentation but is reduced the embryo has completed differentiation and growth is dominant. In of the important role played played by carbon dioxide-fixing dioxide-fixing enzymes view of effect of cadmium in depreSSing depressing en enin biosynthetic processes, the eff ect of developmental stages may result in lethargic zyme activity during the developmental larvae. embryos and small and inactive larvae.
4. 4.
EFFECTS EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS EGGS AND AND LARVAE LARVAE
275 275
As a consequence of the lowered activity of the embryo, embryo, the hatch hatching process may be severely impaired-on impaired-on the one hand, because the hatching enzyme is is not distributed throughout the perivitelline fluid and on the other hand, after the digestion of the inner layer of the zona radiata by the hatching enzyme, the emerging larva cannot break the nondigested outer part of the egg shell (Hagenmaier, (Hagenmaier, 1974a), 1974a),and thus remains longer in the egg casing or never hatches. Even though the proteolytic hatching enzyme may be produced and distributed sufficiently, heavy metals in the external and internal medium may not allow the enzyme to display its proteolytic functions fully. Hagenmaier ((1974b) 1974b) reports that manganese, zinc, mercury, or copper inhibits the proteolytic functions of of the enzyme. From experi experiments with Atlantic salmon (S. salar), brown trout ((S. ( S . salar), S . trutta) trutta),, and rainbow trout (S. 1967) thinks that zinc affects ( S . gairdneri), gairdneri), Grande ((1967) affects the enzymatic processes that soften the egg capsule. capsule. Also, Also, the pH of of the incubating medium has aa strong strong bearing on the functioning of of the hatching enzyme, which has a maximum activity at pH 8.5 8.5 in eggs of of the rainbow trout (Hagenmaier, 1974c) and from 7.5 to 8.0 in the (Hagenmaier, 1974c) 7.5 to 8.0 Pacific salmon (0. (0. keta) 1969). Thus, in experiments with keta) (Bell (Bell et al., 1969). perch (Perca fluviatilis) eggs, Runn et al. 1977) found strong impair (Perca$uuiatilis) aE. ((1977) impairment of of the activity of the hatching enzyme at low pH resulting in reduced hatch. Similarly, at pH 4.5, 4.5, chorionase activity of of Atlantic salmon eggs reached only 49% of its activity at pH 6.5 (Haya (Haya and Waiwood, 1981). Waiwood, 1981). F. ncubation Time and the Process of Hatching F. IIncubation Alterations in incubation time may be caused by premature or delayed hatching. Metals either shorten shorten or or lengthen lengthen this this period, period, delayed hatching. Metals may may either and no unequivocal prediction is possible; petroleum hydrocarbons as prediction petroleum WSF usually retard hatching and development. The effect of of chlorinchlorin ated hydrocarbons on hatching hatching have not been looked into closely. While low pH retards development in general, the major effects of oflow low of chorionase (hatching enzyme) 5.5, pH are on inactivation of enzyme) below pH 5.5, or prevents it entirely. which delays hatching or Onset of of hatching in teleost eggs begins with the secretion of of pro proteolytic hatching enzyme from the hatching glands around the head region of of the enzyme is of the embryo/larva. The secretion and activity of F.. heteroclitus heteroclitus eggs (Kaighn, relatively rapid, as seen in studies with F (Kaighn, 1964). Chorion treated with chorionase dissolves within 5-10 5-10 min. 1964). Thus 1970), Thus,, with the strong movements of of the late embryo (Poy, (Poy, 1970), hatching in F. F . heteroclitus should be completed within a few minutes,
276
H. VON VON \\"ESTERNHAGEN U’ESTERNHAGEN
while it may take several hours in salmonids with a thicker egg shell and lower incubation temperatures (Hayes, al., 1969). 1969). (Hayes, 1942; 1942; Bell et al., Incubation time, the period between fertilization and 50% 50% hatch, is mainly dependent on temperature, higher ranges rirnges accelerating devel development. Low oxygen concentrations, when maintained throughout development, lengthen the total incubation period (Hamdorf, 1 ; S. development, (Hamdorf, 196 1961; S.
gairdneri). gairdneri). Changes in time to hatch are common in fish embryos subjected to sublethal effects of pollutants. Many xenobiotic substances shorten the incubation period or cause premature hatch. Others, however, lengthen the development period or delay hatching. Due to the differ different effects of metals on the late embryo, incubation may be either shortened or lengthened. In most cases that have come to the atten attention of the reviewer, incubation time is shortened and larvae hatch prematurely. Rainbow trout (S. (S. gairdneri) gairdneri) has frequently been used for these essays. essays. Thus, Shabalina (1964) (1964) notes that cobaltous chloride in concentrations of up to 5 mg/l shortens the incubation period; hatching larvae are viable. Also, Also, nickel and copper when applied in concentrations concentrations of 11 mg/l accelerate development of of rainbow trout eggs by about 45% 45%with copper and by 20% 20%with nickel (Shaw (Shaw and Brown, 1971). 1971).The same is true for vanadium (44 (44mg/l) (Giles (Giles and Klaverkamp, Klaverkamp, 1982). 1982). Brook trout (Salvelinus (Salvelinus fontinalis) fontinalis) eggs incubated at 32.5 32.5 p.. pgg copperll copper/l hatch prematurely (McKim (McKim and Benoit, 1971) 1971) as do herring at even lower concentrations (>0. 1 mg/l) (>0.1 mg/l) (Ojaveer et al., 1980). 1980). Other metals such as cadmium and zinc have the same effects on fish em embryos as shown by Vladimirov (1969) (1969) for carp (Cyprinus carpio) carpio) and by Rosenthal and Sperling ((1974), 1974), von Westernhagen et al. al. ((1974), 1974), and Somasundaram et al. 1984a) for herring (Clupea al. ((1984a) (Clupea harengus). harengus). However, the reverse, a prolonged incubation period, is is also known to be caused by metals such as zinc, cadmium, copper, and mercury (Grande, (Grande, 1967; 1967; Servizi and Martens, 1978; 1978; Swedmark and Granmo, 198 1 ; Weis, 1984; 1981; 1984; Somasundaram et al., al., 1984a,b). 1984a,b).In the case concenof zinc, concentrations below 2.0 2.0 mg/l accelerate while higher concen trations retard development of of herring eggs. eggs. The above does not imply that embryos hatching early developed faster or that the late-hatching ones displayed delayed embryogene embryogenesis. In fact, fact, changes in differentiation pattern, known to occur in trout sis. (S. eggs, developing at low partial O ( S . gairdneri) eggs, 0 2 pressure (Hamdorf, (Hamdorf, 2 1961), (in the 1961), are not reported. Rather, the immature embryo hatches (in case of "acceleration") “acceleration”) or an over-mature larva hatches with a small yolk reserve and advanced differentiation (a functional mouth in the case of cod G. G. morhua; morhua; Swedmark and Granmo, 1981). 1981).
4. 4.
EFFECTS OF POLLUTANTS ON FISH EGGS AND LARVAE EFFECTS
277 277
ObThere may be no general explanation for this phenomenon. Ob effects depend on the application of the metal, its concentra concentraserved effects exposure, etc. Thus the rea reation, stage of development, duration of exposure, sons for early or late hatching are frequently found in the history of the egg. On the one hand, egg. hand, an early hatch might be caused by a beneficial low levels of, of, say, zinc, on the embryo (Somasundaram et al., effect of Iow 1984a; C. C . harengus), harengus), with truly accelerated development in the sense 1984a; “sufficient challenge" challenge” concept forwarded by Smyth (1967), (1967), or of the "sufficient 0.133 by the highly detrimental effects of copper at concentrations of 0. 133 mg/l, which immoblize herring embryos totally, so that the hatching (no distribution of glands produce a punctiform hole in the chorion (no enzyme), causing premature liberation of the embryo (von (von hatching enzyme), 1979) (Fig. (Fig. 6). 6). On the other hand, late hatch may Westernhagen et al., 1979) concenbe caused by a retarded development such as caused by high concen (>2 mg/I), mg/l), with resulting inability of the embryo to trations of zinc (>2 1981), or because of mal malbreak the chorion (Swedmark and Granmo, 1981), functioning of the hatching enzyme proper as suggested by Servizi (1978) for the delayed hatching of sockeye (Oncorhy (Oncorhyand Martens (1978) (0.gorbuscha) gorbuscha) exposed to copper con connerka) and pink salmon (0. nchus nerka) pgl1.. A similar explanation may apply for the centrations higher than 6 p,g/l other substances dealt with in this context and the sublethal effects of hydrocarbons, which may likewise lengthen the time from petroleum hydrocarbons, fertilization to hatching or may shorten it. In contrast to the common effects of heavy metals on incubation, most authors report a delayed effect of petroleum hydrocarbons hydrocarbons on hatching when applied as the water-soluble fraction (WSF). (WSF). At low concentrations ((12.5%) 12.5%) of the WSF of crude oil, development was F . grandis (Ernst (Ernst et al., 1977); the accelerated, with early hatch in F. al., 1977); early hatch of Japanese medaka Oryzias Iatipes latipes is likewise considered premature hatching resulting resulting from stimulation of the the hatching mech mecha premature anism by oil components (Leung (Leung and Bulkley, Bulkley, 1979). 1979). However, most authors report delayed hatch of larvae after treatment of developing (1978) eggs and embryos with petroleum hydrocarbons. Thus, Linden (1978) C. harengus mem memreported delayed hatching in Baltic herring larvae C. WSF of light fuel oil; oil; this is also also true when bras exposed to 54 mg/l WSF (24, 48, 96 h) h) to 40 40-45 concenherring are pulse-exposed (24, -45 mg/l initial concen trations of benzene (Struhsaker et al., 1974). 1974). Delay in hatching also occurs in eggs of winter flounder Pseudopleuronectes americanus if 100 p.g/l pgll WSF of number 2 fuel oil the parents have been exposed to 100 (Kiihnhold et al., 1978), 1978),and and in F. F . heterocli heterocliduring gonad maturation (Kiihnhold 25% WSF of this oil (Sharp (Sharp et al., 1979). 1979). Other reports tus exposed to 25% Kuhnhold of petroleum hydrocarbons delaying hatching are given by Kiihnhold
278
H. VON VON WESTERNHAGEN WESTERNHAGEN
a
Fig. 6. 6. Clupea harengus. harengus.(a) (a) Empty egg chorions with punctiform hole after incuba incubation -(133 (b) prematurely 133 p.g pg Cull); CuA); (b) prematurely hatching hatching crippled crippled tion in in copper-contaminated copper-contaminated water water .( herring mm. herring larva larva after after incubation incubation in in copper-contaminated copper-contaminatedwater. water. Horizontal Horizontal bar bar 11 mm.
4. 4.
EFFECTS EFFECTS OF POLLUTANTS POLLUTANTS ON ON FISH EGGS EGGS AND AND LARVAE LARVAE
279
((1974) 1974) (G. (G. morhua), morhua), Stoss Stoss and Haines (1979) (1979) (0. (0.latipes, Zatipes, toluene) and Carls and Rice (1984) (1984) (Theragra (Theragra chalcogramma). chalcogrumma). Further, the polycy polycyclic aromatic carcinogen, benzo[a]pyrene, is known to retard develop development and hatching in rainbow trout (S. ( S . gairdneri) (Kocan and Landolt, 1984; Hannah et al., 1982). 1982). The effect of high concentrations of petro 1984; petroleum hydrocarbons on fish embryos may sometimes be a narcotizing one that reduces metabolism, thus slowing down development (Struhsaker et al., 1974; 1974; Carls and Rice, 1984) 1984) as well as exerting narcotic effects on the late ready-to-hatch embryo. There is little information on the disturbance of pro of the hatching process by chlorinated hydrocarbons (mainly (mainly pesticides and PCB). PCB). No direct impairment of hatching has been reported. Reports of variations of time to hatch refer to abnormally shaped embryos that hatch late due to physical failure to break the egg shell (Dethlefsen, (Dethlefsen, 1977; 1977; G. morhua, morhua, DDT) DDT) or to premature hatch in coho salmon Oncorhynchus kisutsch eggs, eggs, treated with Arocolor 1254 1254 (PCB) (PCB) at 4.4 and 15.0 15.0 f'g/l pg/l (Halter and Johnson, 1974), 1974), where early hatching may be caused by an alteration of the chorion due to PCB treatment. Reduction in hatching time also occurs in eggs of minnow Phoxinus phoxinus when the pa parental fish have been administered PCB PCB orally and the eggs contained high amounts ((1.5-170 1.5-170 mg/kg mgkg fresh weight) of Clophen 50. 50. Hatching time in eggs containing > >15 mgkg fresh weight PCB is significantly 15 mg/kg reduced compared to controls (Bengtsson, (Bengtsson, 1980). 1980). In contrast to the effects of pesticides on the hatching process and hatching time, which are diffuse and probably relate to the general disturbed condition of the embryo, effects of pH on the hatching pro process are much better understood. Report of sublethal effects of of pH on fish eggs and larvae are mainly restricted on the effects of low pH related to acidification of of lakes in North America and northern northern Eu Europe. The effect of low pH on development and hatching is is very con consistent in all but one of the available reports. Only Trojnar ((1977b) 1 977b) in his experiments with brook trout (Salvelinus fontinalis) eggs reported (Sa2veZinusfontinaZis) faster development and hatching at pH 4.65 than at pH 8.07, 8.07, where hatching took place 12 12 days later at the higher pH. Early hatching larvae at pH 4.65 4.65 did not appear to be premature, but were fully developed. This report is contrary to all other information on suble sublethal effects of low pH on early developmental stages of fish, fish, where there is general agreement that low pH prolongs the period from fer fertilization to hatching. Thus, Peterson et al. ((1980a,b) 1980a,b) show that the eggs of Atlantic salmon, S . salar, salary exposed to water of pH 4.0-5.5, 4.0-5.5, following eye pigmentation, showed delayed hatch. The same effects are reported by Swarts et al. al. (1978) fontinalis) eggs (1978) for brook trout (S. (S.fontina1i.s)
280 280
H. VON H. VON WESTERNHAGEN WESTERNHAGEN
incubated at pH 4.75. The delay in hatching may be considerable, reaching 14 14 days for eyed eggs of rainbow trout ((S. S . gairdneri) gairdneri) sub subjected to pH 4.0-4.5 4.0-4.5 when compared to controls incubated in pH 7.87.8(Kugel, 1984). 1984). In yellow perch (Perea (Percajluviatilis), fluuiatilis), incubation time 8.0 (Kiigel, increased by 29% at pH 4.0 compared to pH 6.4 6.4 (Rask, (Rask, 1983). 1983). The prolongation of the period from fertilization to hatching at low pH occurs also in eggs of of zebrafish (B. ( B . reTio) rerio) (Johansson (Johansson et al., al., 1973) 1973) and fathead minnow (Pimephales (Pimephales promelas) (Mount, (Mount, 1973). 1973).Likewise, low pH retards development of Pacific herring (C. (C. pallasi) pallasi) embryos, as already observed by Kelley ((1946), 1 946), while high pH (pH (pH 10) 10) accelerates B.. rerio). development slightly (Johansson (Johansson et al., 1973; 1973; B rerio). The factors factors involved may be either a general retardation of development, or a delay in the process of hatching, or both. Evidence for retarded devel development is 1946), but in most cases the prolonged is provided by Kelley ((1946), incubation period is due to an impairment of the hatching process due to inhibition of enzyme (chorionase) (chorionase) activity at lower pH. Due to the permeability of the chorion for hydrogen ions (Peterson (Peterson et al., al., 1980a), 1980a), the perivitelline fluid rapidly adjusts to the pH of the incubating me medium, and the pH may be too low for maximum enzyme activity. Thus at pH 5.2, chorionase activity of rainbow trout SS.. gairdneri embryos, is reduced to 10% 10% of the optimal rate at a pH of of 8.5 8.5 (Hagenmaier, 1974a), 1974a), and the process of hatching takes several days rather than hours. In salmon SS.. salar, reduction of chorionase activity at low pH is less drastic, but still, at pH 4.5 only 49% of of the activity at pH 6.5 is ob observed (Haya (Haya and Waiwood, 1981). 1981). Alevins of of Atlantic salmon (S. ( S . sa salar) lar) delayed in hatching are thus larger than when hatched at the normal pH (Peterson et al., 1980a). 1980a). Other indications of of incomplete lysis of the chorion at low pH are tail-hatched larvae with the chorion around the yolk or partially hatched larvae with the head still inside the egg shell (Johansson 1 ; Kiigel, (Johansson et al., 1977; 1977; Brown and Lyman, Lyman, 198 1981; Kugel, 1984). 1984).
G . Hatchability and Viable Hatch G. effects of pollutants One of the main interests scientists had in the effects on fish eggs and larvae was the reduction in hatching success. Effects of pollutants on hatchability and viable hatch are dependent on the stage of development, and the species and type of pollutant. Exposure before closure of blastopore causes more severe reduction in hatching “Viable hatch" hatch” is a success than when advanced stages are exposed. "Viable effects than "hatchability." “hatchability.” more sensitive indicator of pollutant effects
4. 4.
EFFECTS EGGS AND EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS AND LARVAE LARVAE
281 281
SSince ince the total of all previously mentioned effects on eggs and embryos is expressed in the number of larvae emerging, it appears useful to present data on reduction of hatchability and viable hatch (percent) in a table (Table (Table I). (percent) I). This should enable the readers to deter determine readily the effects of pollutants on emergence of larvae. For the user's user’s convenience, zero hatch is included. Data in Table I show that hatchability as a parameter to assess sublethal effects is is limited in its use by the differences in toxicity of of the various pollutants. Thus, in saltwater species, cadmium exerts detrimental effects only at high (environmentally nonrelevant) nonrelevant) concentrations in the range of 100010002000 ILg/1 1975; Voyer et al., 1979) 1979) or pg/l (von (von Westernhagen et al., 1974, 1974, 1975; more (Rosenthal (Rosenthal and Sperling, 1974; 1974; von Westernhagen and Dethlef Dethlefsen, 1975). 1975). In freshwater species reactions toward cadmium are more sensitive (Pickering (Pickering and Gast, Gast, 1972; 1972; Spehar, Spehar, 1976; 1976; Rombough and Garside, 1982). 1982). The situation is similar regarding the effects of zinc. Copper, in tum, turn, even in saltwater, saltwater, causes substantial effects on hatch hatchability and viable hatch at concentrations between 30 and 90 ILg/l pg/l (Blaxter, (Blaxter, 1977; 1977; Servizi and Martens, 1978; 1978; Ojaveer et al., 1980; 1980; Cosson and Martin, 1981). 1981). These concentrations are frequently found in sur surface microlayers of polluted areas (Hardy et al., d., 1985), 1985), which have been found to cant reduction in hatchability of herring (C. to cause signifi significant (C. harengus) harengus) eggs (von Westernhagen et al., 1987). 1987). Sublethal effects on hatching caused by chlorinated hydrocarbons are dependent on the toxicity of of the pollutant but also on the mode of of application. Via a biomagnifi cation effect, DDT reduces hatching in biomagnification eggs from female fathead minnow (Pimephales promelas), promelas),kept in wa wap g DDT/I DDT/l until spawning, from 89% to 74%, 74%, and the PCB ter with 2 ILg Aroclor 1254 1254 prevents hatching entirely in eggs from female fathead minnows treated with 4.6 4.6 ILg/1 pg/l (Nebeker et al., 1974). 1974). When Aroclor 1254 1254 is is applied to Cypronodon variegatus eggs at 10 10 ILg/I, pg/l, hatchability is is only 57% (Schimmel et al., 1974). 1974). In general it is evident that hatchability of eggs increases when later developmental stages are exposed to pollutants. In early-stage exposures, effects on percent hatching are more severe. This is in line with Stockard's 192 1) ideas concerning effects of development arrest Stockard’s ((1921) in fish embryos through abiotic substances. Thus, in F. F . heteroclitus, egg development may be stopped safely shortly after gastrulation is completed. Critical stages are those before closure of completed. of the blastopore, during which marked inequalities of of cellular proliferations are taking place. Since body-axis body-axis formation takes place fairly early in develop development (beginning with neurulation and continuing throughout onto ontogenesis), genesis), impairment of this process has its ultimate bearing on hatch-
Table Table II Sublethal Effects of Hydrocarbons, Chlorinated Hydrocarbons, pH) of Pollutants (Heavy Metals, Petroleum Hydrocarbons, pH) on Hatchability and Viable Hatch of of Fish Eggs· Eggs"
Species
Pollutant
of Day of exposure
Concentration ((PdU lLgll)
11 11 11 11 11 11 11
C 100 100 500
Hatchability (W (%)
Viable hatch ((%) %)
"C °C
Salinity (%0) (W
Reference
Heavy metals
Belone belone Belone belone
IoQ N go 8 IoQ N
Cd Cd
Brachydanio Brachydunio reTio rerio
Cu+Pb Cu+Pb
Brachydanio Brachydunio reTio rerio
Cu cu Pb Zn Zn
Brachydanio Brachydanio reTio rerio
H g Hg
Cichlasoma Cichlasoma nigronigrofasciatum fascia tum
Pb Pb
11 11 11 11 P P P
p P
P P P
pc P' pc P" pc P"
pc F pc P" pc p"
1000 lo00
2000 5000 C C 36 72 72 72 C 5000 C C 11 0.2 C 50 100 100 300 400 500 600 600
73 73 62 68 73 73 2 1 21 0 86 86 38 38 56 47 42
100b lOOb
15 15
41 4 1
29 30 95 45 30 17 17 27 21 21 44
von von Westemhagen Westernhagen et al. al. ((1975) 1975)
100 100 90 65 0 26
63 63 11 44 44
25
2 7 27
25
Ozoh (1979a) (1979a)
Speranza et et al. al. ((1977) 1977) Kihlstrom eett al. ((1971) 1971)
Ozoh ((1979b) 1979b)
Clupea harengus
cCuu
Clupea harengus
Cu cu
Cd N Q!l w
Clupea harengus
Cd
Clupea harengus
Cd
Dicentrarchus labrar labrax
cCu u
4 4 4 4 4 4 11 11 11 11 11 11 11 11 11 11 11 11 11 11 1 11 11 11 11 11 11 11 11 11 11 11 11 11
C 30 90 90 300 900 C 30 90 300 C 55 10 10 100 100 C 3 5 50 500 500 C 100 100 500 1000 1000 5000 5000 C 100 100 1000 1000 5000 5000 10000 lo000 C C 55 10 10 50 50 100 100
45 45 44 44 0 0 0 0 0 0 25 22 12 12 0 0 96 96 93 93 98 98 98 98 97 97 91 91
89 89 95 95 84 75 14 14 100 100 74 79 79 18 18 11
9-10 9- 10
90 81 81 71 71 71 71 86 81 81 82 82 66 13 13 94 95 95 93 62 62 0 0 87 87 83 83 16 16 0 0 0 0
29-32 29-32
Blaxter ((1977) 1977)
Ojaveer Ojaveer et al
(1980) (1980)
10 10
16 16
von von Westemhagen Westemhagen
et 1974) et al. ((1974)
10 10
16 16
Rosenthal Rosenthal and Sperling Sperling (1974) (1974)
Casson Cosson and Martin
(( 198 198 1) 1)
I (continued) (continued)
Table II (Continued) (Continued) Table
Species Species
Pollutant Pollutant
Fundulus heteroclitus
Hg Hg
Jordanellafloridae jloridae Jordanella
Cd, Zn
Menidia menidia
Cd
saratilis Morone saxatilis
cu Cu
Oncorhynchus Oncorhynchus nerka
cu Cu
N Q!) lI>-
Day of exposure exposure
11 11 11 11 11 11 11 11 11 11 11 I1 11 11 5 5 5 5 2 2 2 2 2 2 2 22 2
Concentration Concentration
(p.g1I)
C
44 10 10 20 30 40 40 60 80
C, c, Cc
17,28 17, 28 4.1, 47 4.1, 8.1, 75 139 16, 139 31,267 31, 267 C
1170 70 390 750 C
10 10 100 100 500 2800 5000 18 18 37 78
HatchHatchability ability %) ((%)
Viable hatch hatch (%o) ) (%
"C °C
25 25
89 81 81 81 81 73 69 41 41 6 0 66,70 66, 70 66, 76 66,76 73, 72 66,59 66, 59 68, 73 68,73 0, 0, 0 0 90 100 100 94 74 100 100 45 27 45 0 0
Salinity (%0) (%)
20 20
Sharp Sharp and and Neff Neff
(1980) (1980)
Spehar (1976) Spehar (1976)
25
15-19 15-19
Reference Reference
20
al. (1979) (1979) Voyer et al.
O'Rear ((1972) 1972)
98 98 96 96 50 50
6-9 6-9
Servizi and Martens (1978) (1978)
Cd
Oncorhynchus Oncorhynchus nerka nerku
Hg
Oncorhynchus Oncorhy nchus tshawytscha tshaw ytscha
Cu cu
Oryzias latipes Oryzias latipes
Hg
Pimephales promelas melas
Cd
Platichthys flesus Platichthys flesus
Cd
I>:) 00 �
2 2 2 2 22 2 22 2 22 2 11 11 11 11 11 11 11 11 11 11 11 11 11 11
11 11 11 11 11 11 11
174 174
0 0 96 96 97 97 96 95 95 95 95 93 93 95 95 64 64 0 0
C
0.4 0.4 1.5 1.5 5.7 C 1.0 1.o 2.5 4.3 4.3 9.3 9.3 C 2211 40 40 80 80 C 10 10 15 15 20 30 C 7.8 7.8 14 14 27 57 57 C
100 100 500 500 1000 1000 2000 2000 3000 3000 5000 5000
80 76 82 82 78 78 47 47 58 58 21 21 00 00 95 95 97 97 95 95 94 94 78 78 87 83 83 84 84 81 81 80 80 67 67 22
6-9 6-9
6-9 6-9
Servizi and Martens ((1978) 1978)
1314 13-14
Hazel and Meith
(1970) (1970)
26
?
25 25
Heisinger and Green (1975) (1975)
Pickering and Gast
(1972) (1972)
5 5
32
Westernhagen von Westemhagen and Dethlefsen
(1975) (1975)
(continued) (continued)
Table II (Continued) (Continued) Table
Species
Pollutant
Pseudopleuronectes Pseudopleuronectes americanus americanus
Cd, Cd, Ag Ag
Pseudopleuronectes Pseudopleuronectes americanus americanus
Cd
� N 0 Q!) �
m
Salmo gairdneri
Cr
Day of exposure
Concentration (/Lg!l) (Pd)
11 11 11 11 11 11 22 2 2 22 2 2 2 11 11
C, C c
11
Salmo Salmo salar
Cd
SalveZinus fontiSaloelinus fontinalis naZis
Zn
11 11 11 11 11 11 11 11 11 11 11
Hatchability
(96) (%)
c,
100, 100, 18 18 99 550, 550,99 1000,180 1000, 180 1000 lo00 1000, 18 1000,18 C 100 100 320 1000 lo00 11150 150 1550 1550 2100 2100 C 20 200 2000 2000 C 2.8 111 1 29 90 270 270 870 343 343 724 709
93 80 80 87 90 100 100 98 88 98 98 100 100 84 84
93 97 95 95
Viable hatch (96) (%)
100 100 100 100 100 100 90 36 55 55 90 74 74 881 1 19 19 84 84 78 49
76 82 70 80 80 76 47 0 0
°C “C
Salinity (6) %,;)
Reference
99
2211
Voyer eett al. 1982) al. ((1982)
10 10
20 20
Voyer et et al. (1977) (1977)
5 12 12
Van der Putte et et 1982), expo al. exp. al. ((1982), at pH 6.5 6.5
5
Rombough and (1982). Garside (1982).
9
Holcombe et et al. ~ l
((1979) 1979)
.
Salvelinus fontifontinalis naZis
Salvelinus Salve linus fontifontinalis
Pb
P
mHg
t.:l CD -t
Salvelinus fontifontiSalvelinus nalis nalis
1 1 1 1 1 1 1 1 1 P
ceu u
P P P P P P P P P P 1 P, 1 P, P, 1 1 P, P, 1 1 P, P, 1 1 P, P, 1 1 P, P, P, 11
1382 1382 1353 1353 2017 2099 2099 4336 4336 4363 4363 e C 3 ,4 3,4 58 58 119 119 235 235 474 e C 0.03 0.09 0.09 0.3 0.3 0.9 0.9 2.9 2.9 e C 3.4 5.7 9.5 17.4 17.4 32.5 32.5
90 90 79 67 67 73 73 3 3 1.5 1.5 96-100 96-100 57-95 57-95 79 79 85-86 85-86 60-73 60-73 28 28 97 87 87 99 99 98 0,84 0 ,m 0 81 81 99 99 85 85 98 98 95 95 26
9 9
et al. al. Holcombe et ( 1 976) (1976)
McKim et et al. al.
(1976) (1976)
5-14 514
McKim and Benoit
( 1971) (1971)
Petroleum hydrocarbons
Clupea harengus
Oil
(WSF)
1 1 11 1 1 1 1
1 1 1
e C 600 600 1900 1900 5400 5400 17500 17500 36000 36000
32 32 50 50 26 26 28 28 24 24 44
14 14
6
Vuorinen and and AxeII (1980) h e l l (1980)
~~
(continued) (continued)
Table II (Continued) (Continued) Table
Species
� K2 co co
g
Pollutant Pollutant
Clupea harengus harengus Clupea membras membras
Oil (WSF) (WW
Clupea harengus harengus Clupea pallasi Clupea harengus harengus Clupea pallasi pallasi
Benzene Benzene
Fundulus heteroheteroFundulus clitus clitus
Fundulus Fundulus heteroheteroclitus clitus
Oil (WSF) (WW
Oil (WSF) (WSF)
Oil Oil (WSF) WSF)
Day of exposure
Concentration Concentration (PdU (,ug/l)
Hatchability (%) (%) 100 100
11 11 11 11 3 3 3 P P 5-6 5-6
C 50 500 5000 50 500 500 C C 800 C
4 0 99 9 1 91 0 93 67 53
5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 11 11 11 11 11
680 680 680 680 C 10% 10% WSF 20% 20%WSF 25%WSF 25% 25% WSF
44 32 27 0 90 90 72 7 85
11
25% 25% WSF
40
11 11 11 11
C 25% 25%WSF 50% 50%WSF 100% WSF 100%
100 100 100 100 60 0
60 60
Viable hatch (%)
Salinity
(W (%0)
"C °C
Reference
9-14 9-14
6
Linden Linden (1978)
111-12 1- 12
22
Struhsaker (1977) Struhsaker
20
Smith and Cameron ((1979) 1979) 8h h only only exposed exposed 24 h only exposed 8h h only exposed exposed 6 days days exposed exposed Sharp 1979) Sharp et et al. al. ((1979)
8-9 8-9
22
21 2 1
20
4 days only exexposed posed 8 days only exexposed posed Anderson et et al. al. ((1977) 1977)
Cyprinodon vavaCyprinodon riega tus riegatus Gadus morhua morhua Gadus
ta t-:) 0 co (D <.C>
Oil (WSF)
Mallotus villosus
Oil (WSF)
Platichthysflesus Platichthys flesus luscus
Oil (WSF)
l1
C C
11 11 11 11 11 11 11 55 55 55 55 14-20 14-20 14-20 14-20 14-20 14-20 14-20 14-20 14-20 14-20
25% WSF WSF 25% 50% WSF WSF 50% 100% WSF WSF 100% C C 100 100 1000 1000 10000 10000
d d
25 50 100 100 200 400 11700 700 2500 C C 10 10 100 100 10 10 100 100
d d
d d
d d
d d d d
d d
d d
Pseudop.- uronectes Pseudopleuronectes americanus
Oil (WSF)
P p P p P p
11 11
C C
100 100 1000 1000 10000 10000
C C 10 10 25 50 100 100 C C
100 100 88 88 62 62 00 20 20 14 14 4 00 53 53 24 24 19 19 17 17 100 100 100 100 88 88 75 75 68 68 90 90 89 89 89 89 86 86 88 88 89 90 90
58 58 55 55 44 44 24 24 15 15 99 00 00 63 63 65 65 53 53 60 60 42 42
221 1
20 20
5-6 5-6
29-34 29-34
Kiihnhold 1974) Kuhnhold ((1974)
6-7 6-7
34-35 34-35
Johannessen Johannessen
9-12 9- 12
((1976) 1976)
Mazmanidi Mazmanidi and Bazhasvili Bazhasvili
((1975) 1975)
1-10 1-10
31 31
Kiihnhold Kiihnhold et L al. al.
((1978) 1 978)
(continued) (continued)
Table II (Continued) (Continued) Table
Species
Pollutant
Day of exposure exposure
Concentration Concentration P d )) ((1Lg!l
HatchHatchability (%)
Viable hatch (YO) (%)
“C °C
Salinity ((%) %0)
Reference .Reference
Chlorinated hydrocarbons Chlorinated
Cyprinus Cypriflus carpio carpi0
lQ co 0
Cyprinodon vavaCyprinodon riegatus riegatus
Simazine
Gramaxone Gramaxone
11 11 11 11 11 11
Taficide
11
Aroclor hoclor 1254 1254
11
11 11 11 11
Cyprinodon vauaCyprinodon riega tus riegatus
Aroclor 1254 1254
11 11 P P P P P p P
20,000 30,000 40,000 60,000 80,000 80,000 40,000 60,000 80,000 80,000 90,000 25,000 30,000 40,000 50,000 50,000 100,000 100,000
79 68 50 9 0 86 63 9 0 92 68
C C 0.1 0.1 0.32 1.0 3.2 3.2 10.0 10.0 C 0.1 0.1 0.32 0.32 l1.0 .o 3.2 10 10
79 69 73 82 75 57 93 88 88 80 98 85 72
and Yadav Yadav Kapur and ((1982) 1982)
16 16
16 16
16 16
34 34
111 1 0 29
16-32 16-32
Schimmel et et al. ((1974) 1974)
30
10-27 10-27
al. Hansen et al. (1974) (1974)
Gadus morhua
%
I!O 10 c ,...
DDT DDT
phorinus Phoxinus phoxinus
Clophen 50 A 50
Pimphales proPimephales melas
Aroclor 1254 1254
Pimphales proPimephaies melas Oncorhynchus kisutch
Aroclor 1254 1254
I1 11 11 I1 11 P P
pe P" pe P' p e P"
P P p P
P P P P P P f f fJ fJ f f
80 80
C C
60 60
150 ISO
60 60 40 30 30 49 39 39 43 43 8 8 74 55 55 63 63 79 79 0 0 0 89 89
300 300 700 700 800 800 C 1.6 1.6 15 15 170 170 C 0.23 0.23 0.52 0.52 1.8 1.8 4.6 15 IS C 0.5 0.5 2.0 0 4.4 7.8 15.4 15.4 26.0 56.4 56.4
84 84 74 96 96 88 88 79
8
12-16 12-16
35 35
Dethlefsen (1977) (1977)
Bengtsson 1980) Bengtsson ((1980)
al. Nebeker et al. (1974) (1974)
Jarvinen et al. al. ((1977) 1977)
12-14 12-14
Halter and Johnson ((1974) 1974)
17-19 17-19
Trojnar ((1977a) 1977a)
47 63 63 pH
Catostomus commersoni
8.1 B.l
5.8 5.8 5.4 5.0 5.0 4.5 4.5 4.2
1 1 11 11 I1 I1
56 56 61 61
60 60
55 16 16 0
(continued) (continued)
Table II (Continued) (Continued) Table
Species Species
Cyprinodon nevanevaCyprinodon densis densis
Jordanella Jordanella jloridae floridae
1:0 cc 1:0
to
Perea jluviatilis jluviatilis Perca
3
Pimephales pmPimephales promelas rnelas Rutilus rutilus Rutilus rutilus
Perea Perca jluviatilis jluviatilis
Pollutant
8.3 7.0 6.5 6.0 6.0 5.5 5.5 6.8 6.0 5.0 5.0 4.5 7.3 5.5 5.0 4.5 5.5 5.0 4.5 7.5 6.6 6.6 5.9 5.9 5.2 5.2 7.7 6.1 6.1 5.6 5.2 4.7 8.0 5.6 5.1 5.1
Day of exposure exposure
1 1 1 11 11 11 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 14 14 14 14 14 P, P, 11 P, P, 11 P, p, 11 P, p, 11 11 2 2 2 2 11 2 2 2 2
Concentration Concentration (JLg/l) (CLgIl)
Hatchability (%) (%)
Viable hatch hatch (%0) (%)
°C "C
Salinity (%) (%)
51 51 32 9 3
0 44 57 51 51 17 17 0 100 96 70 8 98 44 51 51 78 80 42 0 89 82 40 30 6 53 53 12 12 6
Reference
Lee and Gerking ( 1980) (1980)
26
Craig and Baksi ((1977) 1977)
14
Runn et et al. (1977) ( 1977)
20-25 20-25
16 16
16 16
Mount (1973) (1973)
Johansson and Johansson Milbrink 1976) Milbrink ((1976)
Perca flucjiatilis jluviatilis Perca Salmo salar salar Salmo
salarg Salma salarg Salmo
Salmo salar Salmo salar � M to w
E Salmo Salmo tmttai trutta'
Salvelinus fontiSaloelinus fontinalis nalis
4.6 4.0 4.0 6.4 4.0 3.5 6.8 5.0 4.5 4.2 4.0
3.7 4.9 4.8 4.7 4.55 4.55 6.0-6.8 6.0-6.8 5.5 5.0 4.5 4.5 4.0 8.0 5.5 5.5 4.7 4.2 4.2 8.3 8.3 4.75 4.75 4.4 8.3 8.3 4.75 4.4 8.3 8.3 4.75 4.75 4.4 4.4
22 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 h h
h h h
11 11 11
11
35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35
2 0 90 41 0 96 91 92 94 89 0 70 60 50 40 100 100 100 100 97 -55 -55 0 0 98 98 90 70 90 91 91 2 1 21 100 100 64 12 12 80 80 75 75 50 50
15 15
Rask (1983) (1983)
4
Daye and Garside
(1979) (1979)
-10 -10
Lacroix (1985) ( 1985)
8
Peterson et al. al.
(1980a) (1980a)
4-6 4-6
Johansson et a1 al. ((1977) 1977)
10 10
Swarts et al. Swarts (3 differdiffer((1978) 1978) (3 strains) ent strains)
(continued) (continued)
Table II (Continued) (Continued) Table
Species
Salvelinus Salvelinus fonti fontinalis nalis
fontiSalvelinus fonti nalis nalis
Pollutant
of Day of exposure
Concentration ( P d u) (JLg/l
7.0 6.6 6.1 6.1 5.6 5.6 5.1 5.1 7.0 7.0 6.6 6.6 6.1 6.1 5.6 5.6 5.1 5.1 4.5 C 6.5 6.5 6.0 5.5 5.0 4.5
P P P P P 11 11 1 11 11 1 1 11 1 11 11 11
82 74 59
Hatchability (%) (%)
Viable hatch (L) (%0) 9
°C "C
Salinity (70) (%)
Reference
(1976) Menendez (1976)
54 54
26 74 65
54 54
47 44 44
0 99 95 98 82 65 65 48
95' 95j 911 91i 96i 9f3 701 70i 58i 581
Kwain and Rose (1985) (1985)
OJ 0'
percent of of successfully inseminated eggs; P, parental exposure prior to spawning; C, control. Hatchability and viable hatch expressed as percent of total hatch. Percent of As ppm per 100 100 g body weight (injected). (injected). d Exposure from gastrulation. e Orally dosed, concentration as mg kg-' kg-1 wet weight. f f Two weeks before hatching. g g Data extrapolated from graph. Fxposure at eyed stage. h Exposure j .A va strain. Avl j Survival to swim-up. •
b
c
4. 4.
EFFECTS EGGS AND EFFECTS OF O F POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS AND LARVAE LARVAE
295
ability of the embryo. In the majority of the cases, whenever hatching affected, embryos experimentally liberated from the chorion remain is affected, developin a curled position unable to swim, swim, indicating failures in develop prement that prevented hatching. This is not the case in embryos pre vented from hatching in waters of low pH. Within a time limit, em embryos not hatching at low pH can be induced to hatch almost immediately after transfer to high pH (see (see also Rask, 1983). 1983). This indi indihatching-via inhibition of the cho chocates that only the process of hatching-via rionase-is affected, while the development of the embryo has pro prorionase-is normally-an indication also for a true effect of low pH and ceeded normally-an not the secondary effect of high metal concentrations in the water due to low pH, as frequently suggested. As a consequence, though, when eggs are left throughout development in low pH, hatching success may be drastically reduced, as shown for Atlantic salmon (S. (S. salar) salar) (Daye and Garside, 1977, Lacroix, 1985), (Daye 1977, 1979; 1979; Lacroix, 1985), other salmonids salmonids (Jo (Jo1977), and various other species [Le., hansson et al., 1977), [i.e., white sucker (Trojnar, (Trojnar, 1977a), 1977a), walleye (Hulsman (Hulsman et al., 1983), 1983), desert pupfish (Lee 1980), roach and yellow perch (Milbrink (Milbrink and Johansson, and Gerking, 1980), 1975); 1975); Johansson and Milbrink, 1976; 1976; Runn et al., 1977; 1977; Rask, 1983)] 1983)l.. polluEven though hatchability iiss usually considered a measure ooff pollu tant effects on ontogenesis, it should be remembered that this ob obscures the fact that within these data substantial numbers of nonviable larvae may be included. Thus reference to the rate of "viable “viable hatch" hatch” as a means of assessing sublethal effects of pollutants would be prefer preferred, since since only the normal and viable larvae are of concern for recruit recruitment. It should be added that species such as gar pike, Relone Belone belone, Dicentrarchus (Paflitschek, 1979) 1979)even as larvae Dicentrurchus labrax, or the tilapias (Paflitschek, are so so vigorous that they live with major damage to their skeletal system. Yet when assessing effects on the frail clupeoid and “viable hatch" hatch” is is the more sensitive parameter, pleuronectid larvae, larvae, "viable (Rosenthal and Sperling, 1974; 1974; Maz Mazas can be seen from Table I (Rosenthal Bazhashvili, 1975; 1975; Voyer et al., 1977; Ojaveer et al., 1980). 1980). manidi and Bazhashvili, al., 1977; simultaWhenever values for viable hatch and hatchability are given simulta neously, viable hatch is substantially lower. lower. In many experiments, even the value for viable hatch overestimates the real fi gure for viable figure of effects may not be de delarvae, since, since, as already noted, a number of tected with the naked eye, eye, but require histological examination-for examination-for example, example, the effects of zinc on brain and muscle tissues of herring (C. (C. harengus) harengus) (Somasundaram (Somasundaram et al., 1984a,b; 1984a,b; Somasundaram, 1985). 1985). Other metabolic effects might appear after the young fish has passed the larval period. For instance, hatchability of rainbow trout (S. (S. gairdneri) gairdneri) is not affected by cromium at concentrations concentrations at 0.2 0.2 mg/l
296
H. VON VON WESTERNHAGEN WESTERNHAGEN
(van alevins is (van der der Putte Putte et al., 1982), 1982), yet yet the the survival survival of of alevins is affected affected after after 32 weeks, pH of weeks, depending depending on on the the pH of the the rearing rearing water. water. The inability inability for for complete and caused by cadmium cadmium treatment complete and early early calcification calcification caused treatment is is an another shows only only later alevin's life salmon (S. (S. other effect effect that that shows later in in the the alevin’s life in in salmon salar) salar) (Rombough (Rombough and and Garside, Garside, 1984). 1984). Similar Similar hidden hidden effects effects that that become become apparent apparent only only after after histological histological examination (Ca examination are are known known from from fish larvae larvae treated treated with with crude crude oil oil (Cameron Smith, 1980; 1980; Hawkes meron and and Smith, Hawkes and and Stehr, Stehr, 1982), 1982),and and it it is is likely that that aa large large number number of of hitherto hitherto undetected undetected effects effects of of pollutants pollutants have have consid considerable erable bearing bearing on on the the percentage percentage of of viable viable larvae. larvae. III. SUBLETHAL ISPLAYED BY LARVAE 111. SUBLETHAL EFFECTS D DISPLAYED HATCHED FROM FROM TREATED EGGS
The larval larval stage stage of a fish, fish, although very different from the egg in outer outer appearance, appearance, is is not not totally totally different different in in its its physiological physiological state. state. Any Any impairment of functions functions or organs afflicted in the embryo is carried over to the free-living larva. larva. Hatching is is a rather arbitrarily deter determined since it mined point, point, since it may may occur occur at at aa variety variety of of ontogenetic ontogenetic stages. stages. Many Many larvae, larvae, after after emerging emerging from from the the egg egg shell, shell, are are still still incapable incapable of of feeding, due to the fact that the mouth is not yet functioning (clupeids, (clupeids, gadids, gadids, pleuronectids, pleuronectids, cyprinids, cyprinids, and and others), others), or or because because they they are are not not (salmonids) or at atcapable of swimming and remain on the bottom (salmonids) tached in the tached to to plants plants in in the the water water (pike), (pike). Species Species such such as as found found in the family family Belonidae, Belonidae, Cyprinodontidae, Cyprinodontidae, or or members members of of the the mouth-breed mouth-breeding may rely rely for for aa longer longer ing cichlidae cichlidae are are able able to to feed feed upon upon hatching. hatching. All may or or shorter shorter period period on on yolk yolk reserves reserves for for metabolism. metabolism. However, However, for for the the sake of convenience, the newly hatched larvae are treated together in sake section. this section.
A. Larval Length A. larvae from from eggs eggs incubated Reduction of length in newly hatched larvae (notably heavy metals, petroleum under the influence of pollutants (notably commonly observed hydrocarbons) is a commonly hydrocarbons, and chlorinated hydrocarbons) feature. Reduced length of newly hatched larvae larvae is is frequently correl correlfeature. Reated with larger yolk-sac yolk-sac sizes, sizes, suggesting suggesting impaired development. development. Re duced length in itself is is not considered to lower larval larval fitness. fitness. One characteristic characteristic of of several several abiotic abiotic factors factors influencing fi fish sh larvae incubation is is the altered altered size size and shape shape of yolk sac sac and the length of
4. 4.
EFFECTS OF POLLUTANTS ON FISH EGGS EGGS AND AND LARVAE LARVAE
297 297
the newly hatched larvae. Basicly, yolk-sac size and shape as well as larval length change in relation to various abiotic factors, such as incu incubation salinity and temperature or oxygen. Examples for a decrease in S . gairdneri) gairdneri) at reduced oxy oxylength of newly hatched rainbow trout ((S. gen tension are given by Hamdorf 1961). Salinity and temperature Hamdorf ((1961). effects on length of newly hatched larvae are known to occur in the veincubation of several fish fish species such as English sole Parophrys ve 1968), herring C. C. harengus and C. tulus (Alderdice and Forrester, 1968), C. pallasi (von (von Westernhagen Westernhagen et al., 1974; 1974; Alderdice Alderdice and and Velsen, Velsen, 1971), 1971), and B. belone (Fonds (Fonds et aI., al., 1974). 1974). Frequently Frequently they they occur occur in in and garpike garpike B. conjunction conjunction with with aa prolonged prolonged (larger (larger larvae) larvae) or or shortened shortened (short (short lar larvae) vae) incubation incubation period period until until hatching. hatching. Variations Variations in in larval larval size size are are known known from from rearing rearing experiments experiments with with herring C.. pallasi) ( C . harengus, C pallasi) eggs eggs in in cadmium, cadmium, zinc, zinc, and and copper. copper. herring (C. In all effective treatments, larvae hatch early and total lengths are smaller smaller than than in in controls. controls. Effective Effective concentrations concentrations are are given given by by Rosen Rosenthal 1974) to 1.0 mg/l mg/l for for cadmium cadmium and and 0.1 0.1 mg/l mg/l in in aa thal and and Sperling Sperling ((1974) to be 1.0 pulse pulse exposure exposure of of copper copper (Rice (Rice and and Harrison, Harrison, 1978), 1978), while while Ojaveer Ojaveer et 1 980) report that cadmium concentrations as low as 3.0 al. 3.0 ILg/I pg/l reduce al. ((1980) larval larval length. length. Zinc Zinc increases increases larval larval total total length length in in concentrations concentrations up u p to to 2.0 2.0 mg/l; mg/l; starting starting at at 6.0 6.0 mg/I, mg/l, the the length length of larvae larvae hatched hatched from from zinc zinc incubated incubated eggs eggs decreases decreases (Somasundaram (Somasundaram et al., al., 1984b). 1984b). Yet Y e t under under chronic (Poecilia reticu reticuchronic exposure exposure to to sublethal sublethal levels levels of of zinc, zinc, guppies guppies (Poecilia .7 ILg lata) 0.88 and 11.7 p g zinc/l zincll (Uviovo (Uviovo Zata)are very sensitive to levels of only 0.88 and and Beatty, Beatty, 1979). 1979). Offspring Offspring produced produced under under these these conditions conditions are are smaller than controls smaller than controls and and have have not not absorbed absorbed the the yolk yolk completely, completely, indicating that zinc reduces energy utilization. The authors suggest that that zinc zinc has has an an "uncoupling" “uncoupling” effect effect in in the the mitochondria, mitochondria, similar similar to to the effect that Hiltibran ((1971) 1971) has demonstrated in the mitochondria of the bluegill Lepomus macrochirus liver. liver. Effects of cadmium on herring larvae are depicted in Table II, 11, show showlength of newly hatched herring decreasing length with increasing cadmium at different salinities salinities.. ing decreasing Eaton (1974) L. macrochirus (1974) also found that incubation ooff bluegill L. shorteggs in cadmium concentrations higher than 0.08 0.08 mg/l leads to a short ening of the total length of the hatching larvae. larvae. ening Reduced length of newly hatched larvae is frequently correlated Reduced as noted for yolk sacs sacs of herring larvae with larger yolk-sac sizes, as incubated at different different cadmium and salinity conditions. Besides the infl uence of salinity salinity on on yolk-sac yolk-sac size size (May, (May, 1974a; 1974a; Alderdice Alderdice and and influence Velsen, ndings indicate Velsen, 1971), 1971), these these fi findings indicate low low yolk yolk utilization utilization under under cad cadS . salar). salar). Reduced (Rombough and Garside, 1982; 1982; S. mium exposure (Rombough length of newly newly hatched larvae larvae is also also caused by the exposure of the
Table II I1 Table Clupea Clupea harengus horengus Larvae: Total Length, Diameter of of Eye, and Otic Capsule at Hatching" ~~~~~~~
~~
~
~
~
S (%0) (%)
Cd. conc.
n n
5 5 5 5 5
Control 0.1 ppm 0.1 0.5 0.5 ppm 1.0 ppm 5.0 ppm
80 91 9 1
16 16 16 16 16 16 16 16 16 16
Control 0.1 0.1 ppm 0.5 ppm 1.0 ppm 5.0 ppm
100 100 62 93 105 105
25 25 25 25 25 25
25
Control 0.1 0.1 ppm 0.5 ppm 1.0 ppm 1.0 5.0 ppm
32 32 32 32 32
Control 0 . 1 ppm 0.1 0.5 ppm 1.0 ppm 1.0 5.0 ppm
50 50
63 72 91 9 1 50 54 61 61 74
x r
ss
Eye diameter (mm) (mm)
Otic capsule (mm) (mm)
Total length (mm) (mm)
Experimental design design
si Sf
n
n
x f
SS
si. Sf
si. Sf
125 125 137 137 39
0.002 0.003 0.002 0.002 0.003
76 4 1 41 43 129 129 100 100
0.287±0.0l4 0.28720.014 0.284±0.017 0.28420.017 0.285-tO.009 0.285±0.009 0.283±0.014 0.283+0.014 0.258 2 0.020 0.258±0.020
0.002 0.003 0.001 0.001 0.001 0.001 0.002
0.311 1120.015 0.3 ± 0.015 0.312C0.021 0.312±0.021 0.315±0.017 0.315-tO.017 0.31120.034 0.31 1 ±0.034 0.24420.062 0.244±0.062
0.002 0.003 0.002 0.004 0.006
63 46 91 9 1 82
0.288 0.288 0.27720.018 0.277±0.018 0.289-tO.010 0.289±0.010 0.293±0.0 1l 0.293+0.011 0.263±0.024 0.26320.024
0.002 0.001 0.001 0.001 0.003
0.279±0.027 0.27920.027 0.286-tO.020 0.286±0.020 0.285±0.027 0.28520.027 0.28820.022 0.288±0.022 0.178±0.035 0.17820.035
0.004 0.003 0.003 0.003 0.002 0.006
0.274?0.008 0.274±0.008 0.26520.020 0.265±0.020 0.268 -t0.0 14 0.268±0.014 0.272+0.018 0.272±0.018 0.234-tO.019 0.234±0.019
0.002 0.001 0.001 0.002 0.002 0.002 0.003
0.002 0.002 0.002 0.002 0.007
0.06 0.08 0.05 0.04
100 100 65 93 129 129 141 141
0.3 1420.023 0.314±0.023 0.311 120.025 0.3 1±0.025 0.310±0.021 0.31020.021 0.301?0.027 0.30l±0.027 0.244±0.033 0.244 L0.033
7.9820.17 7.98±0. 17 7.90-tO.46 7.90±0.46 7.8820.41 7.88±0.41 7.48±0.60 7.4820.60 Not measurable
0.02 0.06 0.05 0.06
49 62 71 7 1 9 1 91 102 102
7.0550.28 7.05±0.28 7.0020.34 7.00±0.34 6.94±0.42 6.9420.42 6.8220.41 6.82±0.41 Not measurable
0.04 0.05 0.05 0.05
53 56 63 73 35
7.7820.58 7.78±0.58 7.8820.62 7.88±0.62 7.7020.51 7.70±0.51 7.13-tO.38 7. 13±0.38 Not measurable
S S
0.002 0.001 0.001 0.001 0.002 0.003 0.003
0.32720.020 0.327±0.020 0.3 16?0.024 0.316±0.024 0.313±0.026 0.31320.026 0.31420.025 0.314±0.025 0.2 12-t0.039 0.212±0.039
0.04 0.06
x f
0.288 20.0 18 0.288±0.018 0.280±0.01 l 0.28020.011 0.251 ±0.0l6 095120.016 0.25220.018 0.252±0.018 0.22020.016 0.220±0.016
80 89 171 171 179 179 33
8.2720.34 8.27±0.34 7.77?0.54 7.77±0.54 Not measurable Not measurable Not measurable
n n 55
64 64
25
50 50
38 74 45
a n, n, Number of of larvae measured; x, f , mean; s, s, standard deviation, s., si, error of of the mean. Larvae derived from incubation incubation trials in 32%0 32% salinity originated 1974). Westernhagen et et al. al. ((1974). originated from a second female. After von Westemhagen a
4. FISH 4. EFFECTS EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH
EGGS AND EGGS AND LARVAE LARVAE
299 299
eggs to petroleum hydrocarbons, usually applied as the water-soluble (WSF) of crude oil or its derivates derivates.. Thus Pacific herring (C. (C. fraction (WSF) pallasi) eggs exposed in a 48-h 48-h pulse exposure of of 12. 12.1 pallasi) 1 mg benzene/l 10.3 yield larvae with a mean standard length of 9.2 mm, compared to 10.3 controls.. The same is true for anchovy (E ( E.. mordax) mordax) eggs ex exmm in controls 40-55 ppm (Struhsaker et al., 1974). 1974). Also, Also, Baltic herring (C. (C. posed to 40-55 rnembras) eggs exposed to 5.4-5.8 5.4-5.8 mg/l total oil hydrocar hydrocarharengus membras) (Linden, bons yield significantly shorter hatching larvae than controls (Linden, 1978). The same effects are described by Carls and Rice ((1984) 1978). 1984) after of the walleye pollock T. T . chalcogramma chalcogrumma and embryos exposing eggs of of oil (Leung (Leung and O. latipes to the WSF of of the Japanese medaka 0. Bulkley, 1979). 1979). In the walleye pollock, reduction in length amounts to 0.5 mm at a total length of of only 4.5 mm. In the killifish F. F . heteroclitus, exposure of eggs to the WSF of number 2 fuel oil leads to a shortening of hatching larvae with increasing strength of of the applied WSF (Sharp (Sharp et al., 1979; 1979; Linden et al., 1980). 1980). In this species the latter authors note a simultaneous decrease in the number of vertebrae. vertebrae. Reduced lengths of newly hatched larvae are also known to occur after treatment of cod (G. (G. marhua) morhua) eggs with DDT and DDE. Within the range of DDT applied (0, 0.0095, 0.0413, 0.09, 0. 15, 0.39, 0.69 mg/l), (0,0.0095,0.0413,0.09,0.15,0.39,0.69 mg/l), emerging larvae are progressively smaller with increasing insecticide concentrations; the mean total length is only 4 mm at the highest DDT concentration, while control larvae measure 4.75 mm (Dethlefsen, (Dethlefsen, 1977). 1977).A reduction concentrain length of yolk-sac fry of pike Esox lucius incubated in concentra tions as low as 0.1 0.1 ng 2,3,7,8-tetratchlorodibenzo-p-dioxin (TCDD)/l (TCDD)/I is also known through the experiments of Helder ((1980). 1980). The significance of of the hatching size for the fitness of of fish larvae is not clear, although it is generally accepted that it is of of disadvantage for the larvae to hatch small. This assumption depends on an "uneasy “uneasy feeling" feeling” rather than on facts. facts. Swimming velocity, for instance, does not seem to be altered significantly in different-size different-size herring larvae 1.0 mm total length (von (von Westernhagen and Rosenthal, within 7.0 to 111.0 1979), 1979), and thus prey catching behavior will not be impeded. Of course, smaller larvae have aa smaller range of of food availability, since organs such as eyes and otic capsules (von Westernhagen et al., 1974) 1974) as well as the head and jaw apparatus are smaller, thus limiting the choice of food particles. However, the limitation to smaller food parti particles does not reduce survival if enough food is available, as shown by my unpublished lennius pavao unpublished data on larvae of B Blennius pauo. It seems reasonable to argue that it is not the absolute size of of a larva but its size in relation to its ontogenetic stage of development and the remaining yolk volume that is is important for survival. If If the
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H. VON VON WESTERNHAGEN
embryo encounters encounters unfavorable conditions at the end of its intracho intrachorion development and it "decides" “decides” to hatch prematurely, as reported reported for the effects of several abiotic factors including low oxygen, the prematurely liberated larva, unless otherwise damaged, is generally only shorter than normal, but is holding a larger yolk sac. sac. If If the caus causative agent of premature hatch does not have any lasting effects, de development will proceed normally. B Yolk-Sac Size and Yolk Metabolism B.. Yolk-Sac A large or deformed yolk sac is taken as an indicator for metabolic or osmotic disturbances that may be caused by mitochondrial mal malfunction, induced by b y heavy metals or petroleum hydrocarbons. It is not always true that a large yolk sac at hatching is is due to premature hatching. There are indications that a large yolk sac occurs because of metabolic or osmotic disturbances in the embryo/larva embryoAarva that prevent proper use of the energy stored in the yolk. Lanning 1977), Lonning ((1977), from observations of cod, plaice, and fl ounder eggs (G. flounder (G. morhua, Pleuronectes platessa, Platichthys Jesus) fiesus) exposed to Ekofisk oil, thinks that the use of the energy-rich substances in the yolk becomes delayed by an inhibition of the mitochondrial system. For instance, newly hatched larvae from Pacifi C. pallasi eggs exposed to Pacificc herring C. Prudhoe Bay crude oil for 4-144 h and then returned to uncontami uncontaminated seawater show no gross abnormalities micros abnormalities.. Yet transmission microscopy of exposed organisms reveals inter- and intracellular spaces in brain and muscle tissue that are not found in controls (Cameron and 1980). Many mitochondria ((13%) exSmith, 1980). 13%) in the body muscle of ex posed animals are swollen, some with deteriorating cristae. Changes in mitochondrial functions would affect the total respiration and me metabolism of the larvae and thus explain the previously previously mentioned gen general suppression of of embryo activity and metabolism after prolonged exposure to petroleum 1980) inferred petroleum hydrocarbons. Linden et al. ((1980) that at low hydrocarbon hydrocarbon levels, when the homeostatic mechanisms are ect increased costs of homeo not overwhelmed, overwhelmed, respiration rates refl reflect homeostasis. When the stress is more severe, but still sublethal, the response would be mediated by lack of metabolic integration because of of poorly functioning homeostatic mechanisms. They believe that exposure to oil predominately predominately impedes mobilization of of nutrient uptake from the yolk through the breakdown of mitochondria in in the cells, leading to glycogen and lipid depletion such as demonstrated by Sabo and SteSte geman ((1977). 1977). This would ultimately lead to reduced tissue growth, as
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found in embryonic embryonic herring C. C. pallasi exposed to sublethal sublethal concentra concentra1977). Yolk utilization is likewise (Eldrige et al., 1977). tions of benzene (Eldrige pentachlorophenate (Na (Na PCP), reduced by exposure to sodium pentachlorophenate PCP), as shown by Chapman and Shumway ((1978), for example, in the alevins 1978), of steelhead trout S. S. gairdneri. The bioenergetic data obtained in their consistent with the concept that PCP disrupts energy metab metabstudy are consistent deleterious to the early larva, since energy olism. This is particularly deleterious requirements increase rapidly by approximately tenfold shortly after 1977; C Clupea pallasi). Struhsaker et al. (Eldridge et al., 1977; hatching (Eldridge lupea pallasi). of yolk at high concentrations of of ((1974) 1974) relate the impaired utilization of of the animal; this aspect will benzene to an increasing narcotization of be treated later in a different context. Inhibited yolk utilization is also E . lucius larvae incubated at known under conditions of low pH. Pike E. p H 4.2 are smaller than controls but have larger yolk sacs, which pH 14 days, indicating poor utilization of yolk reserves. Yolk persist for 14 frequently appears appears coagulated, ultimately leading to death of the af affrequently 1975). Retarded yolk absorption fected fry (Johansson and Kihlstrom, 1975). at pH 55 is also known in brook trout (S. fontinalis) alevins (Menendez, (S.fontinaZis) (Menendez, 1976). 1976). outer appearance appearance of the yolk shows signs of abnor abnorFrequently, the outer appearance of an empty space between yolk mality or one notices the appearance flesus (Mazmanidi and Bazhashvili, 1975; 1975; Platichthys ./lesus sac and yolk (Mazmanidi luscus) or anteriorly adjacent to the pericardium as demonstrated by luscus) (1978)for newly newly hatched herring herring (C. ( C . harengus membras) membras) lar larLinden (1978) Linden These features are similar to those observed by von Westernha Westernhavae. These fish gen ((1970) 1970) and Alderdice and Velsen ((1971) 1971) when marine fi sh larvae outside their optimum temperature temperature and salinity re reare incubated outside indicating additional stress stress on the larvae, larvae, resulting resulting in gimes, thus indicating functions.. failure of osmoregulatory functions Cadmium and zinc are also known to interfere with osmoregula osmoregula(C. pallasi) pallasi) eggs (Alderdice et al., 1979c). 1 9 7 9 ~ )Cad Cad. Pacific tion in Pacifi c herring (C. reduces osmolality of perivitelline fl fluid, probmium exposure of eggs reduces uid, prob ably due to the marked tendency of cadmium to form complexes (Remy, (Remy, 1956), 1956), particularly with iodide, bromide, and chloride ions. ions. The effect of the the formation of these complexes is is to to marshal other ions The into complex formation [e.g. [e.g.,, Cd(I3h]' Cd(I&], reducing the number of active solution, thus reducing reducing osmotic pressure. Rosenthal and particles in solution, Sperling (1974) (1974) report a disproportionate shortening of the yolk sacs sacs of cadmium C. harengus larvae larvae at at cadmium cadmium levels levels of5.0 of 5.0 cadmium exposed exposed herring herring C. and 10.0 10.0 mg/l, mg/l, which may be caused by reduced perivitelline fluid turgor. turgor. al. effects on fish larvae. larvae. Somasundaram et al. Zinc has similar effects
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H. H. VON VON WESTERNHAGEN WESTERNHAGEN
((1984d), 1984d), working working with with C. C. harengus, harengus, speculate, speculate, on on the the basis basis of of their their histological histological investigations, investigations, that that the the swelling swelling of of mitochondria mitochondria and and sar sarcoplasmic coplasmic reticulum reticulum caused caused by by zinc zinc treatment treatment suggests suggests the the creation creation of of an an osmotic osmotic imbalance imbalance through through zinc. zinc. In In mammals mammals zinc zinc causes causes swelling swelling of of mitochondria mitochondria and and appears appears to to alter alter potassium potassium permeability, permeability, uncou uncouples ples oxydative oxydative phosphorylation, phosphorylation, and and inhibits inhibits the the electron electron transport transport chain chain (Cash (Cash et aZ., al., 1968; 1968; Kleiner, Kleiner, 1974; 1974; Bettger Bettger and and O'Dell, O’Dell, 1981}. 1981). Uncoupling ultimately lead Uncoupling of of oxydative oxydative phosphorylation phosphorylation will will ultimately lead to to an an energy deficit, even though the in energy deficit, even though the embryo embryo may may compensate compensate with with increased decomposition creased decomposition of of carbohydrates carbohydrates (Stelzer (Stelzer et aZ., al., 1971); 1971); this this would would have have its its bearing bearing on on the the osmoregulatory osmoregulatory capacities capacities of of the the em embryo. bryo. C. Morphological Aberrations C. Aberrations:: Eye Deformities, Skeletal Abnormalities Gross malformations such as eye Gross eye deformation and reduction, as well as skeletal deformities, are caused by all types of pollutants and are not pollutant-specific. Typical anomalies, which may also be caused by extreme temperatures and salinities, are spirality and cur curvature of the notochord and abnormal development of of the jaw. The severity of the effects can be generally related to the doses applied and diminishes with exposure during later stages of development. Since cadmium interferes with calcium metabolism, it is suspected to impair the calcification process directly. Petroleum hydrocarbons probably act as general stressors and do not have a specifi c effect on specific any any enzyme enzyme or or physiological physiological process. process. Besides aberrations of of length and yolk usage, newly hatched larlar vae display a vast array of gross deformities, such as lack of of organs, of gross extremities, etc. and/or abnormal behavior due to the action of pollu of pollutheir embryonic stage. behavtants during their stage. These malformations and behav ioral aberrations may play major or minor roles in their survival. In general, aberrations that stem from tissue injury or enzyme inhibition follows : various types of of during earlier stages can be categorized as follows: eye deformation or reduction, jaw anomalies, malformations of of the vertebral column, minor morphological aberrations (i.e., fin defects, otic capsule defects, change in color pattern), impairment of of swimswim of these ming and prey catching behavior, and reduced growth. Some of have obvious effects on survival. The bearing of of others may be insiginsig nificant or difficult to recognize. Eye deformations are common in fish larvae exposed to sublethal
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of stressors such as heavy metals. Microphthalmia (Ojaveer et levels of 1980) in Baltic herring incubated in copper and cadmium solusolu al., 1980) al., tions of of BO.01 >0.01 and 0.05 0.05 mg/l have been noted, as well as cyclopia in morhua) reared in sublethal (0.01-0.5 mg Cu/l; Cull; 0.5-10.0 0.5-10.0 mg cod (G. morhua) Zn/l) concentrations of of copper and zinc (Swedmark and Granmo, 1981). de198 1 ) . These gross abnormalities are similar to those already de scribed for the effects of of mercury on killifish ((F. F . heteroclitus) em embryos, by P. Weis and Weis (1977). (1977). The spectrum of of eye and head F . heteroclitus by exposure to inorganic mercury defects produced in F. reflects interference with inductive processes at a relatively early stage. stage. The severity of of the response diminishes with exposure during later stages of 0. S. S. Weis and Weis, 1977). of development (J. 1977). Fundulus of malforma malformaheteroclitus appears to have a propensity for this type of tion; further, Stockard ((1907) 1907) produced cyclopia in this species by treatment with magnesium chloride. chloride. It is is interesting to note that optic abnormalities are not produced in F. F . heteroclitus by exposure to to in insecticides (Weis (Weis and Weis, Weis, 1974). 1974). Other subtle deviations from the normal are displayed by herring (C. harengus) (C. harengus) embryos exposed to cadmium (Rosenthal (Rosenthal and Sperling, al., 1974). 1974). Exposure to cadmium concen 1974; 1974; von Westernhagen et al., concentrations higher than 1.0 1.0 mg/l leads to a reduction in eye diameter (Table 1984a) showed a significant (Table II). 11). Also, Somasundaram Somasundaram et al. al. ((1984a) reduction in eye size size at a concentration of 6 and 12 12 mg/l of zinc in herring (C. (C. harengus), harengus), even considering reduced length at hatching. The same is true for the the reduced otic capsule capsule diameter. diameter. Gross eye eye deformations deformations are are one of the typical effects occurring occurring after sublethal exposure exposure to metals and other teratogenic compounds compounds such such as ben benzo[a]pyrene (BAP). (BAP).When exposing rainbow trout (S. ( S . gairdneri) gairdneri) to the mutagen BAP, BAP, Kocan and Landolt (1984) (1984) and Hannah et al. al. (1982) (1982) always nd gross always fi find gross physical defects in the ocular and cephalic region of larvae similar to those resulting from exposure exposure to heavy metals metals.. Skele Skeletal tal and cephalic abnormalities of newly hatched fish, fish, encountered most frequently upon exposure to BAP, BAP, are believed to be caused by the mutagenic action of the BAP. BAP. Implied possible mutagenic action of copper and cadmium is is not supported by experimental evidence, and thus the the question remains open. open. Another sublethal stressor affecting the the eyes eyes of young fish fish is is low pH. 1980), when sectioning alevins pH. Daye and Garside ((1980), alevins from from exposed eggs eggs of Atlantic salmon (S. ( S . salar), salar), found that incubation at at pH 4.0 4.0 yields alevins alevins with with eye lens lens fibers fibers less less differentiated than that of con controls. trols. The The lenses also also suffer severe sloughing of epithelium, epithelium, a common common pathologic pathologic change change due due to to acid acid environment. environment. Anatomically, Anatomically, the the prime prime
304 30 4
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sites cial tissues. sites of of injury injury are are the the superfi superficial tissues. Internal Internal structures structures are are af affected fected secondarily, secondarily, both both in in time time and and degree. degree. A A wide wide array array of of skeletal skeletal malformations malformations (Le., (i.e., jaw, jaw, head, head, pelvic pelvic and and pectoral pectoral girdle, girdle, vertebral, vertebral, and and opercular opercular anomalies) anomalies) occurs occurs commonly commonly in in freshwater freshwater and and marine marine fish fish species species (Wunder, (Wunder, 1971 1971;; Kroger Kroger and and Guthrie, 1973; 1973; Dethlefsen, Dethlefsen, 1980, 1980, 1984). 1984). Accordingly, one would also expect expect these these anomalies anomalies to to occur occur in in fish fish larvae, larvae, and and this this is is the the case case under laboratory conditions. For example, anomalous formation of the jaw, mentioned during earlier investigations, is caused by extreme temperatures and salinities in several fish species [von [von Westernhagen 1974), Platichthys fiesus, jlesus, B ((1970, 1970, 1974), B.. belone; belone; Alderdice and Velsen C. pallasi] pallasi].. These anomalies are also caused by sublethal sublethal ef ef((1971), 1971), C. cranio-facial fects of metal pollutants and are likely to be expressed as cranio-facial as a reaction toward mer merand mandibular malformations; particularly as (Weis, 1984 1984;; F. F. heteroclitus) heteroctitus) and zinc (Somasundaram et al., cury (Weis, 1984a; C. C . harengus). harengus). Different types of of malformations of the head 1984a; (C. harengus) harengus) larvae (Fig. 7). Due to the region are found in herring (C. (Fig. 7). incomplete development of the feeding apparatus of many fish larvae (mouth opening still closed), closed), symptoms of jaw at the time of hatching (mouth immediately detectable, in particular when defects are not always immediately hatched larvae are not given additional time to de deexperimentally hatched 7 shows sev sevvelop their mouth apparatus before assessment. Figure 7 eral types of jaw deformations in herring. These differ depending on development. Jaw deformations are also known to occur the stage of development. spontaneously” in hatchery enterprises in North America. The open open"spontaneously" of salmonids is of of particular particular concern in hatchery-reared jaw syndrome of al., 1973). salmon (Crouch et al., 1973). Jaw deformities may also result from the treatment of treatment of eggs with crude oil (Tilseth et al., al., 1984; 1984; Solberg et al., 1984). At concentrations of of about 150-1245 1984). 150- 1245 kg/l J,tg/l (WSF), (WSF), cod (G. (G. of the upper jaw, which may morhua) larvae suffer from deformation of have a later bearing on feeding. Also, short-term exposure of of newly 24-96 h at concentrations spawned Pacific Pacific herring (C. (C. pallasi) pallasi) eggs for 24-96 of 4800-45,000 p g benzene/l causes severe anomalies in the head of 4800-45,000 J,tg region of of hatching larvae, including including jaw deformations (Struhsaker et al., 1974). of the head region of 1974). Similar pictures of of Baltic herring larvae after treatment with the WSF of crude oil (up g hydrocarafter treatment with of (up to 59,000 p J,tg hydrocar bondl) are given by Linden (1978). Also, when exposing 6-day-old bons/I) ( 1 978). Pacific herring embryos to the WSF of Prudhoe Bay oil at concentraof concentra tions of g total hydrocarbons/l for only 48 h, of around 1000 1000 p J,tg h ' advanced larvae display a high incidence of of gross morphological abnormalities, such as improperly formed mouth, misfit of of the lower jaw into the upper, missing of of the premaxillary bone, and failure of of the jaw to fully “
4. 4. EFFECTS
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Fig. Fig. 7. 7. Head of of herring (Clupea (Clupea harengus) harengus) larva showing different types of jaw malformations after incubation in Zn-contaminated 2, 55 Zn-contaminated water. Upper row, normal 11,, 2, days (arrow); third row, secondary days old; second row, rudimentary lower and/or upper jaw (arrow); pugheadedness, branchial arches (arrows); (arrows); fourth row, row, pugheadedness, cross bite, bite, and and protrusion protrusion of branchial normal lateral and dorsal view, and same view of of larva with exophthalmus. [From Somasundaram et al. ((1984a).] 1 984a).]
differentiate (Smith (Smith and Cameron, 1979). 1979). Another abnormality no noticed i ticed only only under the the electron electron microscope microscope was was the the absence of of branch branchiostegal membranes, a phenomenon observed also by von Westernha Westernhagen al. ((1987) 1987) after gen et al. after treatment treatment of of herring herring embryos embryos with with surface surface microlayer hexane extracts. extracts. Abnormal development development of the jaw is seen in larvae exposed as eggs to pesticides like the moluscicides Bayluscid and Lebaycid (Paflits (Paflitschek, 1979; Tilapia leucosticta, leucosticta, Heterotilapia multispinosa) chek, 1979; multispinosa) or or as as aa result result of of high PCB PCB content content (2.8 (2.8 JLg/g pg/g wet wet wt) wt) in in eggs eggs of of rainbow trout trout (S. (S. gairdneri) (Hogan (Hogan and and Brauhn, Brauhn, 1975). 1975). In In the the natural natural environment environment these anomalies are these jaw jaw anomalies are likely likely to to interfere interfere severely severely with with feeding feeding and and
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H. VON WESTERNHAGEN VON WESTERNHAGEN
thus reduce survival. In the most severe cases, starvation will follow the inability to feed. In my observations, I frequently noticed that swimming is greatly impaired in gaping-mouth larvae of of marine fish, fish, probably probably due to increased water resistance. Mouth and jaw anomalies constitute a relatively small part of the gross abnormalities occurring after treatment of eggs with pollutants. The bulk of the symptoms observed are related to axis formation. Injury to the vertebral column or its anlage in response to pollutants is commonly seen by most investigators working with fish eggs and lar larvae. The range of damage is extensive: from very slight flexures to bends or spiral distortions, shortening of the body axis, axis, or reductions of the brain. Within the range of possible damage, none of the sub substances seem to cause substance-specific damage, which can undoubt undoubtedly be attributed to a particular pollutant. Responses seem to be general and ubiquitous without regard to the stressor. Exposure time and substance concentration influence the severity of the symptoms. The physical appearance of affected larvae resembles that of individ individuals incubated under natural stress of of extremes of temperature and 1970) and described by sev salinity, as shown by von Westernhagen ((1970) several other authors. Damage of the vertebral column expressed as curvature of the larval body axis axis is caused by all metals currently termed "heavy “heavy metals" metals” when present in the incubating medium. The most common metal pollutants are cadmium, copper, mercury, lead, and zinc, em emof a few micro microployed singly or in combinations at concentrations of grams per liter in the case of the acutely toxic metals such as mercury, and up to several thousand micrograms micrograms per liter with metals such as lead or zinc. facThe toxic levels of the different metals differ and depend on fac tors such as susceptibility of fish species, temperature and salinity, or chemical chemical speciation of the metal. Thus cadmium causes vertebral damage in developing fish eggs at concentrations between 80 I-tg/l pg/l 1974; Lepomis macrochirus) and 300 I-tg/l pgll (Rombough (Rombough and (Eaton, 1974; 1982; S. S. salar) salar) in fresh or brackish water (Voyer (Voyer et ai., al., 1977; 1977; Garside, 1982; americanus), but at higher concentrations of be bePseudopleuronectes americanus), 1000 and 2000 I-tg/l pgIl in seawater (von Westernhagen et aZ., 1974, al., 1974, tween 1000 belone). Since cadmium interferes with calcium 1975; C. 1975; C. harengus, B B.. belone). metabolism-cadmium replacing calcium-the calcium-the effect of cadmium on metabolism-cadmium investivertebrae formation might be a direct one, as suggested by the investi gations of Rombough and Garside (1984) (1984) observing the impairment of of the calcification process in Atlantic salmon alevins. Earlier effects of the metal, prior to ossification, ossification, must be considered general effects on
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dismetabolism. Copper is also known to be effective in producing dis torted larvae at fairly low concentrations. At 30 IL p copper/I, copper/l, 30% 30% of of the hatching herring larvae are deformed (Blaxter, (Blaxter, 1977). 1977). At the same level zebrafish (B. ( B . rerio) rerio) develops scoliosis (Ozoh, (Ozoh, 1979a), 1979a), and for bass) this level is is only slightly higher ((100 Dicentrarchus labrax (sea bass) 100 pg/l) (Cos (Cosson 1981). The initial concentrations for mer merILg/I) son and Martin, 1981). cury causing sublethal effects on axis formation are even lower. J. J. S S.. Weis and Weis ((1977), 1977), in experiments with the killifish (F. ( F . heterocli heteroclitus), prove that concentrations of only 10 pg Hg/I Hg/l cause lordosis and tus), 10 ILg larvae. Although affected larvae are still scoliosis in newly hatched larvae. able to swim, movements are impaired. Experiments of Sharp and Neff 1 980, 1982) 1977) with F. Neff ((1980, 1982) and P. Weis and Weis ((1977) F . heteroclitus confirmed the low effective concentrations for mercury that cause sim similar effects to those described above. Still lower detrimental concen concentrations of 1978) with of mercury were reported by Servizi and Martens ((1978) sockeye (0. (0. gorbuscha) (0.nerka) nerka) and pink salmon (0. gorbuscha) eggs. Apparently, mercury concentrations above 2.5 ILg/1 pgIl increase vertebral deformities; at 4.3 ILg p g mercury/I, mercury/l, 46% of the alevins are crippled with impaired swimming. swimming. Exposure of brook trout (Salvelinus (Salvelinusfontinalis) over three genera generations shows that lead is also an effective teratogen at low concentra concentrations. Alevins of the third generation from eggs exposed to 1119 19 ILg pg lead/l display 2 1 % scoliosis, scoliosis, compared to only 2% 21% 2% in the controls al., 1976). 1976). In short exposures during embryonic devel devel(Holcombe et al., opment, lead causes sublethal effects on axis formation, but concen concentrations must be around 1000 1977; F. 1000ILg/1 pg/l (J.S. (J.S. Weis and Weis, 1977; F . hetero heteroclitus). clitus). Small aberrations from normal axis formation caused by zinc are first detectable at the micrograms per liter level. Slight bends of of the tail tip of herring larvae at hatching can be observed after incuba incubation at 50 ILg/1 pg/l (Somasundaram (Somasundaram et al., 1984a). 1984a).Animals with this type of deformity still swim like normal larvae. With increasing zinc concen concentrations, damage to the vertebral column becomes more severe. Fur Further information on damage of cod (G. (G. morhua) and herring (C. (C. harengus) harengus) larvae hatched from zinc-exposed eggs are given in reports by Swedmark and Granmo ((1981) 1981) and Ojaveer et al. ((1980); 1 980); effective concentrations for herring are in the same range, although higher for cod. A series of photographs of cod. of various degrees of of spinal malforma malformations caused by metal is given in Fig. 8 from our own experiments with herring and fl ounder. In the herring larva (Figs. flounder. (Figs. 8b,8c), 8b78c),the dam damage done to the vertebrae is is visible. Disturbed axis formation is also common in larvae incubated in hydrocarbons-although not a typical phenomsolutions of petroleum hydrocarbons-although
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e Fig. 8. Newly hatched larvae of herring (Clupea (Clupea harengus) harengus) and flounder (Platichy (Platichythysflesus) thysjesus) incubated in water contaminated with 55 mg cadmium/I. cadmium/l. (a) (a)Crippled herring larva with exophthalmus; (b, c) exophthalmus; (b, c) herring larvae with damaged or almost disintegrated notochord (arrows); (arrows); (d-f) (d-f) bent and severely crippled flounder larvae; y, yolk. Horizontal bars indicate pm. indicate 200 /Lm.
enon. According to the Sharp et al. 1979) interpretation of their exper al. ((1979) experF . heteroclitus) heteroclitus) embryos: embryos: imental results with oil-exposed killifish ((F. Hydrocarbon pollutants act iin n fish embryos embryos as general stressors and do not have a hydrocarbon pollu polluspecific effect on any single enzyme or physiological process. Thus hydrocarbon mortants may shunt limited metabolic energy away from critical differentiation and mor phogenetic processes to maintenance functions.
The depressant or retarding effects of hydrocarbons during early de development may be reflected in the effects effects on gross morphology of emergent fry, where the degree of the effects depends on the strength of the hydrocarbon applied. A variety of of species has been used in exposure studies with hydrocarbons (mostly (mostly WSF of crude oil), oil), one of the most common being herring (C. (C. harengus). harengus). Effects of of 680 ILg/l pgll WSF for 48 h on developing herring (C. (C. harengus pallasi) eggs leads abnormalito a significantly higher incidence of gross morphological abnormali (Smith and Cameron, Cameron, 1979). 1979). Most of the abnor abnorties than in controls (Smith malities are bent vertebral columns leading to larvae with L, S, or helical configurations. Affected larvae are usually unable to swim in aa straight line or not able to swim at all. The same aberrations have been provoked by Linden ((1976, 1 976, 1978) 1978) using somewhat higher concentra-
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EFFECTS EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS EGGS AND AND LARVAE LARVAE
30 309 9
bentions, 3100 to 111,900 1,900 pgll, JLg/I, and by Struhsaker et al. al. ((1974) 1974) using ben zene. The eggs of several other species have been subjected to the same or similar treatment using crude oil or other petroleum hydrocar same hydrocarbons. Thus Thus Mironov (1969) (1969) used anchovy Engraulis encrasicolus pon ponticus; Hakkila and Niemi (1973) (1973) used northern pike pike Esox lucius; Kiihnhold 1 974) used cod C. Kuhnhold ((1974) G. morhua; Mazmanidi and Bazhashvili ((1975) 1975) used flounder Platichthys flesus luscus; Stoss and Haines (1979) (1979) Japanese medaka Oryzias latipes; and Kiihnhold et al. (1978) (1978) used the winter fl ounder Pseudopleuronectes americanus. americanus. All authors flounder report deformation of axis axis to a greater or lesser extent. Effective con concentrations of of the WSF of petroleum hydrocarbons are in the range of 100 100 JLg/1 pgll (Mazmanidi and Bazhashvili, 1975) 1975) to 4000 JLg/1 pg/l (Stoss and Haines, 1979). 1979). When combining oil oil with oil-spill dispersants (Wilson, (Wilson, 1972; 1972; Linden, 1974, 1974, 1976), 1976), deleterious effects on axis formation are usually aggravated, reaching levels known from the teratogenic ef effects of benzo[a]pyrene 1983) on notochord benzo[a]pyrene (24 (24 JLg/I; pg/l; Winkler et al., al., 1983) abnormalities in the grunion Leuresthes tenuis. tenuis. Body flexure occurs also also in newly hatched larvae from eggs con containing chlorinated chlorinated hydrocarbons such as DDT [Dacre [Dacre and Scott ((1971); 1971); S. 1973), Pseudopleuronectes S. gairdneri; gairdneri; Smith and Cole ((1973), americanus] americanus] in in the range of 2.4-4.6 2.4-4.6 mg/kg mglkg wet weight. Hogan and 60-70% deformed rain rainBrauhn ((1975) 1975) assume that the occurrence of 60-70% bow trout in aa trout hatchery was caused by the high PCB (Aroclor (Aroclor 1242) 1242) content (2.7 (2.7 JLg/g) pg/g) in the eggs. eggs. When exposing eggs to these incubating water at concentrations of about 100 100 JLg pg substances in incubating DDTIl Dethlefsen, 1977; DDTl1 ((Dethlefsen, 1977; C. G. morhua) morhua) or only 13 13 JLg p g PCB/I PCBll (Mauk et al., 1978; Salvelinus fontinalis), fontinalis), a considerable percentage of the al., 1978; hatching larvae displays curvature of the body in different degrees. degrees. The severity of these effects increases with the concentration of of the organochlorine employed. From the data on viable hatch of cod under influence 10 JLg pg DDT/l DDTll or higher already the infl uence of DDT it appears that 10 reduces viable hatch. Similar effects can be caused by other pesti pestireduces cides, such as malathion, but at considerably higher concentrations of 10,000 JLg/l pgll for sheepshead sheepshead minnow (Cyprinodon (Cyprinodon variegatus) variegatus) eggs 10,000 (Weis and Weis, 1976) 1976) or carp carp (C. (C. carpio) carpio) eggs treated with various (Weis Yadav, 1982). 1982). herbicides (Kapur and Yadav, crippled and and distorted larvae is is also ob obOccasional hatching of crippled served when eggs eggs are incubated incubated at at low pH. Thus at pH p H 4.0 4.0 to 4.5, served perch Perca fluviatilis hatching is delayed and the few few (3%) (3%) larvae hatched show vertebral deformations (Runn (Runn et al., al., 1977). 1977). This effect is is known to occur at pH 5.0 5.0 in white sucker Catostomus commersoni
310 310
H. VON VON WESTERNHAGEN WESTERNHAGEN
(Trojnar, (Clupea pallasi) eggs (Trojnar, 1977a). 1977a). In Pacific Pacific herring (Clupea eggs subjected subjected to to (pH 6.7), low pH during development development (pH 6.7), hatching hatching is is reduced reduced to to almost almost zero although a few bent individuals emerge (Kelley, (Kelley, 1946). 1946). Runn et 1977) judged these deformities to be secondary and not caused by al. al. ((1977) a disturbance of the early organogenesis but developing during the prolonged nonhatch period at low pH-possibly pH-possibly aggrevated by the smaller inner volume of the egg and the reduction of of the diffusion of metabolites uid. The metabolites through through the the perivitelline perivitelline fl fluid. The same same phenomenon phenomenon is is known for cod (G. (G. morhua) morhua) embryos that fail to hatch. When the fully developed larva (with jaws and no more yolk) yolk) is liberated by dissec dissection, it remains curled in an embryonic posture (von (von Westemhagen, Westernhagen, 1970), indicating that the growth of the larva inside the egg was re 1970), responsible for the malformation. D. Minor Morphological Aberrations
Aside from the above-mentioned obvious gross gross malformations, an array of minor deformities and deficiencies are known to be caused by pollutants. These cannot be dealt with in detail, detail, although they may represent the real sublethal effects at the individual level. In other words, effects of the pollutants may be expressed as minor changes not large enough to cause immediate or ultimate death, but large enough to reduce overall fitness. Typical effects may follow different kinds of treatment. Thus, fi n erosion or sloughing of epithelial tissue fin occurs after exposing embryos to cadmium, copper, zinc, or lead (von Westernhagen et al., 1975; 1975; Ozoh, 1979a; 1979a; Somasundaram, 1985), 1985), as Westemhagen well as after incubation in water contaminated with petroleum hydro hydro(Kuhnhold, 1972; 1979; carbons (Kiihnhold, 1972; Linden, 1975; 1975; Smith and Cameron, 1979; Vuorinen and Axell, 1980) 1980) or rearing in organochlorines or or organophosphates (Pafl itschek, 1979; (Paflitschek, 1979; Helder, 1980). 1980). Other symptoms are impaired blood circulation or blockage of blood vessels leading to thrombosis, as seen in ?n larvae hatching from eggs incubated in cad cadmium solutions (Pickering and Cast, Gast, 1972; 1972; Eaton, 1974; 1974; Beattie and 1978), toluene (Stoss (Stoss and Haines, 1979), 1979), or tetrachlorodi tetrachlorodiPascoe, 1978), benzo-p-dioxin (TCDD) 1980). The same effects are known (TCDD) (Helder, (Helder, 1980). (Winkler to be caused in fish larvae by the carcinogen benzo[a]pyrene (Winkler et al., 1983). 1983). Very common is the poor development of pigmentation 1980) or exposure to petroleum hydrocar caused by cadmium (Ozoh, (Ozoh, 1980) hydrocarbons (Mazmanidi (Mazmanidi and Bazhashvili, 1975; 1975; Anderson et al., 1977; 1977; John John1979; Falk-Petersen Falk-Petersen et al., 1985), 1985),malathion (Weis (Weis and Weis, son et al., 1979; 1976), 1976), or low pH (Johansson (Johansson and Kihlstrom, 1975; 1975; Nelson, 1982). 1982).
4. 4.
EFFECTS EFFECTS OF OF POLLUTANTS POLLUTANTS ON FISH FISH EGGS EGGS AND AND LARVAE LARVAE
3 11 311
E. E. Metabolic Metabolic Alterations Alterations One metals and and oil oil is is an an altera alteraOne major major subcellular subcellular effect effect caused caused by metals tion leads to tion of of the the internal internal structure structure of of mitochondria, mitochondria, which which leads to an an im impairment of the inhi pairment of the intracellular intracellular energy energy transfer transfer system. system. Blockage Blockage or or inhibition bition of of this this system system may may be be the the cause cause for for inadequate inadequate use of of yolk reserves and reserves and retarded retarded development. development. More More subtle subtle impact impact of of pollutants pollutants (stressors) (stressors) may may easily easily escape escape at attention is still or resist resist interpretation. interpretation. Recent Recent studies studies show show that that there there is still aa tention or large large number number of of phenomena phenomena that that are are now being being looked looked at at more more closely closely but but are are not not yet yet fully fully understood. understood. This This is is particularly particularly true true for for events events at at the subcellular levels, which are known to be affected and in turn to affect affect the whole whole organism. organism. Thus Thus herring herring embryos, embryos, incubated incubated in in zinc zincpolluted signs of polluted water, water, show, show, in in addition addition to to signs of epithelial epithelial necrosis, necrosis, changes changes in in mitochondria mitochondria structure, structure, absence absence of of the the Golgi Golgi apparatus, apparatus, and and reduction reduction in in smooth smooth endoplasmic endoplasmic reticulum reticulum (Somasundaram, (Somasundaram, 1985). internal active 1985). Because Because of of reduced reduced mitochondria mitochondria internal active surface, surface, cell cell metabolism might be impaired and total energy budget of of the animal affected al., 1984c); 1984~); zinc is is known known to to interfere interfere with with affected (Somasundaram (Somasundaram et al., oxygen uptake of 1971) and might cause un unof mitochondria (Hiltibran, (Hiltibran, 1971) coupling of oxidative phosphorylation and inhibition of the electron transport chain (Kleiner, 1974; Bettger and O'Dell, O’Dell, 1981), 1981), a theory (Kleiner, 1974; 1979). Uviovo and and Beatty Beatty ((1979). also forwarded forwarded by Uviovo Similar Similar effects effects on on mitochondria mitochondria are are caused caused by by xylene xylene on on the the earli earliest cleavage 1986) and (G. morhua) morhua) eggs eggs (Kjorsvik, (Kjorsvik, 1986) and the the est cleavage stages stages of of cod cod (G. oil on WSF of crude crude oil on herring herring larvae larvae hatching hatching from from oil-treated oil-treated eggs eggs (Cameron and Smith, 1980). Enzyme activity 1980). Enzyme activity in in brook brook trout trout S. fonti fonti(Cameron and Smith, naZis, as as shown by by Christensen Christensen (1975), (1975), is is also also greatly greatly affected affected by naZis, metals such as metals such as cadmium, cadmium, mercury, mercury, and and lead. lead. Activity Activity of of glutamic glutamicoxaloacetic (ALP), acetyl acetyloxaloacetic transaminase transaminase (GOT), (GOT), alkaline phosphatase phosphatase (ALP), choline esterase choline esterase (ACH), (ACH), and and adenosine adenosine triphosphate triphosphate (ATP) (ATP) have have been been either either significantly significantly decreased decreased in in late late embryos embryos or or increased increased in in alevins. alevins. Probably Probably several several malformations malformations and and developmental developmental aberrations aberrations are are ultimately ultimately caused caused by by aa blockage blockage of of the the energy-transfer energy-transfer system, system, leading leading to an arrest of respiration and differentiation, or to dedifferentiation. Inhibition Inhibition of of acetylcholine acetylcholine esterase esterase in in neuromuscular neuromuscular and and brain brain tis tissue, (S. sue, for for instance, instance, as as demonstrated demonstrated to to occur occur in in rainbow rainbow trout trout (S. gairdneri) gairdneri) exposed exposed to to organophosphate organophosphate pesticides pesticides (Matton (Matton and and Lat Lat1969) or in Cyprinodon Cyprinodon variegatus treated with malathion (Weis (Weis tam, 1969) and Weis, 1976), 1976), will will severely severely impair impair locomotion locomotion and/or and/or cause cause death death and Weis, of asphyxia. of the the organism organism by asphyxia. Blockage inhibition of systems or Blockage or or inhibition of intracellular intracellular energy-transfer energy-transfer systems or
312 312
H. H. VON VON WESTERNHAGEN WESTERNHAGEN
shunting energy from differentiation metabolism to detoxification pro processes may also be the cause for the commonly observed retardation in growth and the inability of of the yolk sac larvae to use yolk reserves adequately. It has already been noted that herring (C. (C. harengus) harengus) lar larvae hatching in cadmium-contaminated water had larger yolk sacs than those from controls (von Westernhagen 1974). Obviously, Westernhagen et al., 1974). resorption of yolk under the influence of of metal stress is impaired. This utilization continues in the larval stage. Thus impairment of yolk utilization brook trout S. S. fontinalis alevins incubated in copper-contaminated copper-contaminated water (32.5 (32.5 JLg/I) pgll) take 4 weeks longer to complete yolk resorption and remain smaller than controls (McKim and Benoit, 1971). 1971). Impairment of yolk utilization on incubation in cadmium-contaminated water also occurs in Atlantic salmon alevins when reared through yolk absorp absorption (Rombough and Garside, 1982). 1982). At 9.6°C 9.6"C concentrations of of 0.47 JLg p g Cd/I Cd/l impaired yolk utilization to the extent that final weight of of alevins was significantly reduced when compared to controls. The same is is known for salmon fry reared at 40-55 40-55 JLg p g copper/l copped1 (Hazel and Meith, 1970; 1970; Servizi and Martens, 1978), 1978), and for the young of the zebrafish B. B.rerio under the influence of lead (Ozoh, (Ozoh, 1979a). 1979a). The last lasting effect of cadmium on yolk utilization is shown in rearing experi experiments with Atlantic salmon (S. ( S . salar). salar).When reared in concentrations of 2 JLg/l, pgll, the fish display reduced growth, growth, which continues even after initiation of feeding (Peterson et al., 1983). 1983). Impaired yolk utilization has also been reported as an effect of petroleum hydrocarbons on embryos of winter flounder Pseudo Pseudo1978) and the killifish F. pleuronectes americanus (Kiihnhold et al., 1978) F. heteroclitus (Sharp (Sharp et al., al., 1979). 1979). The effect of these pollutants is not entirely a direct one; these substances express their activity through storage in the lipid reserves of the yolk and are later mobilized during This system is is particularly active with lipophilic sub subyolk absorption. This stances such as the chlorinated hydrocarbons. Thus larvae of fathead 15JLg pg PCB/l minnows Pimephales promelas hatched after exposure to 15 controls (Nebeker et al., are severely retarded in growth compared to controls 1974), 1974), and this was also reported by Halter and Johnson ((1974) 1974) work work0. nerka eggs and ArocIor Aroclor 1254. 1254. Hogan and ing with Pacific salmon O. S. Brauhn ((1975) 1 975) observed that hatchery-reared rainbow trout S. gairdneri fry display a high percentage ooff deformed animals with a yolk utilization. On variety of skeletal abnormalities and impaired yolk analysis, the eggs eggs of parental fish fish showed 2.7 JLg p g PCB/g egg chemical analysis, 0.09 JLg pg DDT/g. Also, Also, S. S . salar incubated in DDT show wet weight and 0.09 alevin development, particularly in their their behavior (Dill (Dill and retarded alevin Saunders, 1974). 1974). Another substance, substance, sodium pentachlorophenate Saunders,
4. 4.
EFFECTS EGGS AND EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS AND LARVAE LARVAE
313 313
(Chapman and Shumway, 1978), 1978), when applied at 40 JLg/I, pgll, decreased (Chapman S. steelhead trout S. yolk utilization, growth, and development in steelhead
gairdneri. The significance of impaired yolk utilization is obvious. With a
given yolk reserve, larvae must develop to a certain ontogenetic stage span. If this is not attained, the larva is likely to find in a given time span. itself in an environment for which it is not yet prepared (swimming itself speed, orientation); in the case of salmonids, larvae may emerge and speed, fall prey to larger predators. In the limited environment of a body of fresh water, the proper timing might be crucial for survival. In the sea this factor might not be of paramount importance, but its bearing should not be underestimated. underestimated. F. F. Behavioral Behavioral Abnormalities Abnormalities E ggs incubated Eggs incubated under under the the influence influence of of metals metals or or petroleum petroleum hydro hydrocarbons carbons may may release release larvae larvae with with reduced reduced activity. activity. While While effects effects of of metals metals are are long-lasting, long-lasting, petroleum petroleum hydrocarbons act act twofold; twofold; tran tranhistopathsiently, with subsequent recovery, and permanently, when histopath ological damage took place. Also, the high chlorinated hydrocarbon contents yolk may responsible for for reduced reduced activity. activity. Low Lowcontents of of larval Iawal yolk may be responsible ered is an an indication indication of of reduced reduced fitness. fitness. ered larval larval activity activity is Several Several authors authors assume assume that that an an additional additional reason reason for for slow slow yolk yolk utilization utilization is is the the reduced reduced activity activity of of larvae. larvae. Frequently, Frequently, larvae larvae hatch hatching eggs lie lie motionless motionless on on the the bottom bottom of the the experimen experimening from from exposed exposed eggs tal sluggish movements tal containers containers or or perform perform only only sluggish movements not not equal equal to to nor normal immobile often mal swimming swimming activity. activity. Hatched Hatched larvae larvae that that remain remain immobile often come from eggs incubated in high concentrations of copper, zinc, or (Eaton, 1974; 1974; Swedmark and Granmo, 1981); 1981); the reason for cadmium (Eaton, a2. (1982) (1982) describe their immobilization is not quite clear. Voyer et al. winter flounder larvae Pseudopleuronectes Pseudopleuronectes americanus incubated in cadmium concentrations of up to 100 100 JLg/I pg/l that show reduced swim swim(10% salinity). salinity). This response sug sugming activity only in low salinity (10%0 susgests a potential long-term effect on larval feeding, growth, and sus gests ceptibility to predation. The reasons reasons for for the the reduced reduced activity activity of of metal-treated metal-treated larvae larvae are are definitely hydrodefi nitely different from those that occur after treatment with hydro carbons. The The effects of hydrocarbons on embryo activity activity can be b e ob obalso in newly hatched larvae. Crude oil fractions (WSF) (WSF) are served also activity. Hatched embryos and larvae suffer very effective in reducing activity. narcotic effects when swimming in water admixed with petroleum
314
H. VON VON WESTERNHAGEN WESTERNHAGEN
hydrocarbons (Sharp (Sharp et al., 1979). 1979). From several experiments by Kiihnhold 1 969, 1972; Kuhnhold ((1969, 1972; Kiihnhold Kuhnhold et al., 1978), 1978), we know that larvae may become completely stunned when swimming into clouds of of pe petroleum hydrocarbons or oil dispersants (Wilson, (Wilson, 1974) 1974) and sink to the processs is bottom. When they reach lower WSF concentrations the proces reversed (Hakkila and Niemi, 1973). 1973). Sometimes Sometimes larvae are not fully immbolized but show only reduced swimming activity (Mazmanidi (Mazmanidi and Bazhasvili, 1975). 1975). Partly anesthetized larvae, although still swim swimming, may lose equilibrium (Stene (Stene and Lanning, Lonning, 1984) 1984) and be unable to catch prey. When kept for prolonged periods at high concentrations of the WSF of petroleum hydrocarbons, growth is negatively affected due to nonfeeding [Struhsaker et al. al. ((1974), 1974), 4000 ILg/I pgll WSF benzene; Solberg et al., (1984), ] . The narcotic effects (1984),30-200 30-200 ILg/I pgll WSF crude oil oil]. of crude-oil extracts (8000 WSF) have a stronger impact on (8000 ILg/l pg/l WSF) starved larvae than on individuals with fully functional yolk sacs (Davenport et al., 1979). 1979). Effects are twofold, depending on WSF concentration and dura duration of exposure, and may be transient with subsequent recovery or long-lasting if there is is histopathological damage to the retina or fore forebrain, as shown for larvae of of the surf surf smelt Hypomesus pretiosus by Hawkes and Stehr ((1982). 1982). A larva with a damaged brain or eye is unable to survive. ed by other organic survive. Acute effects may be intensifi intensified al. (1984) compounds accumulated from the water. Solbakken et al. (1984) (G. morhua) morhua) eggs and larvae exposed to several naph naphfound that cod (G. PCB for 24 h accumu thalenes, phenanthrene, benzo[a]pyrene, and PCB accumulated these lipophilic xenobiotics in the yolk and stored them until the yolk was used, thus causing a delayed effect on later development, resulting in reduced activity, morphological aberrations, and the like. Extensive changes in in behavioral behavioral patterns, such as delay in the occur occurrence of certain swim positions in alevins from DDT-treated Atlantic salmon (Dill (Dill and Saunders, 1974), 1974), or reduced swimming activity, may be observed in fish larvae that contain chlorinated hydrocarbons. Ex Experiments with the cyprinodont Adinia xenica confirm that eggs from DDT- and mirex-contaminated loss in mirex-contaminated parents yield larvae that show loss equilibrium and effects of narcotization (Koenig, 1977). Since the yolk (Koenig, 1977). (Atchison, 1976; 1976; Guiney et is the main storage site for DDT and PCBs (Atchison, al., 1980),the larva, while consuming yolk, takes up more and more of al., 1980), the stored chlorinated hydrocarbons, producing the damaging effects. The consequences of the behavioral abnormalities abnormalities depend on If swimming and prey catching behavior their severity and duration. If anticare impaired, increasing risk of starvation or predation would be antic ipated, leading to reduced survival (see (see Rosenthal and Alderdice,
4. 4.
EFFECTS EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS AND AND LARVAE LARVAE
315
1976). 1976).Larvae with considerable amounts of of chlorinated hydrocarbons in their yolk (see (see Hogan and Brauhn, 1975) 1975) are usually not viable and are characterized by a high incidence of malformations, failure to com completely absorb their yolk sac, or death during the larval stage (Macek, (Macek, 1968). al. ((1974) 1 974) observed reduced survival of 1968). Schimmel et al. of sheeps sheepshead minnow Pimephales >0.32 p,g Pirnephales promelas fry after incubation in >0.32 pg Aroclor 1254/1. 125411. Reduced survival is common in larvae hatched from eggs treated with PCB or DDT during incubation (Hansen et al., al., 1974; 1974; Freeman and Idler, 1975; 1975; Dethlefsen, 1977). 1977). Proof Proof of of the detri detrimental effects of DDT in yolk does not stem only from laboratory experiments but is also available in fi eld data. 1971) field data. Dacre and Scott ((1971) and Hogan and Brauhn ((1975) 1975) report substantial loss of of trout fry due to hydro high DDT in larval tissues. Effects of high levels of of chlorinated hydrocarbons in eggs on percent viable hatch are also known from the investigations of von Westernhagen et al. 1981) with Baltic flounder al. ((1981) flews, Hansen et al. Platichthys flesus, al. ((1985) 1985) with herring C. harengus, Westin et al. 1985) with striped bass Morone saxatilis and Cameron al. ((1985) et al. al. ((1986) 1 986) with whiting, Merlangius merlangus. Effective chlorin chlorinated hydrocarbons are PCB at a gonad concentration of 120-180 120-180 p,g/kg pglkg wet weight and DDE at 18 18 p,g/kg. pglkg. IV. SUBLETHAL SUBLETHAL EFFECTS ON LARVAE NOT EXPOSED AS EGGS
Outstanding sublethal effects on larvae are impairment of of yolk utilization and ensuing depression in growth, which may be caused either by low levels of metals, petroleum hydrocarbons, or chlorinated hydrocarbons, or by low pH. In addition, petroleum hydrocarbons are particularly effective in reducing larval activity. In the previous section I have discussed deleterious effects of pollutants on larvae resulting from exposed eggs. All of the effects lower the individual's individual’s survival chances even if if the larva is released into an uncontaminated uncontaminated environment after hatching. However, pollutants may also exert their influence on normal lar larvae with (sublethal) (sublethal) reactions that result in reduced survival of of the young fish. fish. Larvae hatched in uncontamined water may come under the influence of of contaminants during larval drift by encountering an oil spill or plumes of of heavily polluted river water. Such experimen experimentally mimicked posthatching encounters with pollutant stressors will now be addressed. Larvae and alevins are generally considered more susceptible to
316
H. H. VON VON WESTERNHAGEN WESTERNHAGEN
abiotic factors factors than is the egg (Hiikkilii (Hakkila and Niemi, 1973; 1973; Linden, 1974; 1974; Rice et al., 1975). 1975). Effective concentrations concentrations for for the production of of suble sublethal effects should be low. For example, sublethal effects exhibited by larval salmon exposed to heavy metals are skeletal deformities after exposure to low mercury (9.3 (9.3 �g/l; pg/l; Servizi and Martens, Martens, 1978) 1978) or low cadmium (>0.78 (B0.78 �g/l; pgll; Rombough and Garside, 1984) 1984) concentrations. concentrations. Other responses may be inhibition of of enzymatic processes by cad cadmium, mium, lead, and mercury exposure, as seen in brook trout, S. S. fonti fontinalis; 1976; McKim et al., 1976); nalis; alevins (Holcombe et al., 1976; 1976); this may lead to reduced survival due to uncoupling or inhibition of of unknown metabolic functions [Benoit [Benoit ( 1975), 1975), Lepomis macrochirus; macrochirus; Spehar ((1976),JordanellafEoridae], 1976),jordanellajloridae] . Thus, one sublethal reaction of of larval spot Leiostomus xanthurus to cadmium is is a lowering of of its thermal maxi maxi1975). Most of of the information on sublethal mum (Middaugh et al., 1975). effects effects of metals is related to metal-induced depression of growth due to insufficient yolk utilization or decreased larval activity and reduced feeding. Blaxter ((1977), 1 977), using larval herring and plaice, found that in feeding plaice larvae there is a marked reduction in growth in length and development at 90 90 �g pg copper/l or above, above, while in herring, doses of 300 300 �g/l pg/l tend to inhibit activity during their dark phase of migration. The growth inhibiting effects of copper on alevins are documented also for king salmon O. 0. tshawytscha (21 (21 �g/I; pg/l; Hazel and Meith, 1970) 1970) as well as brook trout (McKim (McKim and Benoit, 1971) 1971)starting at 3.4 �g/I. pg/l. In the latter species the influence of copper on metabolism retards yolk absorption for about 4 weeks. Lowered rate of yolk utilization is is also considered to be the reason for the slow growth of alevins of Atlantic salmon salmon kept in water with 2 �g pg cadmium/I. cadmium/l. The growth-depressing effect of cadmium continues after the initiation of feeding (Peterson (Peterson et al., 1983). 1983). The possible role of metal-damaged mitochondria in the (Somasunimpairment of energy transfer and metabolism of protein (Somasun 1984b,c) has already been mentioned and might be re redaram et al., 1984b,c) sponsible for the inability of metal exposed larvae to make adequate effects on metabolism, the latter use of their yolk reserves. Besides the effects C. harengus lar larauthors report considerable brain damage in herring C. from zinc exposure; exposure; this will have a bearing on swim swimvae resulting from prey catching behavior. ming activity and prey effects of low low levels levels of heavy metals on fish fish larvae In general, the effects effects described for petroleum hydrocar hydrocarare less spectacular than effects bons. Due to the increasing numbers of oil spills, spills, many reports have bons. effects of petroleum hydrocarbons on recently been prepared on the effects fish larvae to oil oil has aroused particular marine life; the reaction of fish fish larvae exposed to petroleum interest. Generally, the reaction of fish
4.
O F POLLUTANTS ON FISH EGGS AND LARVAE EFFECTS OF
31 317 7
hydrocarbons resembles that of larvae hatched from eggs incubated in oil-polluted waters. Petroleum hydrocarbons and dispersants clearly yolkdecrease larval activity, as noted by a reduction of heartbeat of yolk 1973).At 18 18 mgn mgil of of sac larvae of pike Esox lucius (Hakkila and Niemi, 1973). (Neste A), A), a reduction in heartbeat from 90 beats min-I min-’ an emulsifier (Neste min-l is observed after 2 days, followed by a period of of to 30 beats min-I swimming activity and narcosis. Also, under the influence of of reduced swimming C. harengus and plaice an oil dispersant, larvae of herring C. Pleuronectes platessa show narcosis within 20 min of exposure to 8 abovemg/l; affected larvae may recover if exposure time is within the above mentioned range (Wilson, (Wilson, 1972, 1974). Linden ((1975) 1972, 1974). 1 975) found narcotic ml l-I) l-l) using her hereffects caused by oil/dispersant mixtures (0.01/0.005 mI ring larvae; Rosenthal and Gunkel ((1967) 1967) noted similar effects. These (>0.5 mg/l) induce impaired swimming and prey catching mixtures (>0.5 hydrobehavior, as do dispersants (Wilson, (Wilson, 1972) 1972) or other petroleum hydro carbons alone (Kuhnhold, (Kiihnhold, 1969; Lanning, 1984). 1969; Stene and Lonning, 1984). Concen Concentrations of oil/dispersants of 100/50 n pri 100/50 p,lpl-ll already destroy larval fi fin primordia (Linden, (Linden, 1975) 1975)or other tissue (Kiihnhold, (Kuhnhold, 1972), 1972), which might later kill the individual. Thus, Thus, one of the major effects of oil and dispersants is reducing larval activity. This may influence larval survival directly, since it has been demonstrated demonstrated experimentally that anesthetized anesthetized larvae are more susceptible to predation than others (see susceptible (see Rosenthal and Alderdice, 1976). uence of a narcotic such 1976). One reason may be that under the infl influence larva’s "critical “critical distance," distance,” that is, the as the WSF of crude oil, the larva's greatest distance between a larva and aa small object (hypothetical predator) to induce a flight reaction, is shortened (Johnson et al., 1979), and flight reactions may then be initiated too late to be success success1979), anesthetized larvae is their re reful. Yet the more obvious effect on anesthetized duced prey catching ability due to slower movements and uncoordin uncoordinated swimming swimming and prey catching maneuvers (Rosenthal, 1969). 1969). determines the volume of a water body a larva is Swimming capacity determines able to search for food per unit time (Rosenthal and Hempel, 1970); 1970); thus, reduced swimming swimming speed decreases the number of of encounters encounters detrimental effects of petro petrowith food particles. Starvation enhances detrimental leum hydrocarbons (Davenport (Davenport et al., 1979). 1979). While 35-day-old starved cod G. C. morhua m o r h a larvae exposed to 88 mg/l WSF (crude (crude oil) get narco narcotized, larvae with functional yolk sacs remain unaffected. An additional factor contributing to the deleterious effects of of petro petroleum hydrocarbons is the apparent reduction in larval growth in oil oilpolluted waters. Since petroleum petroleum hydrocarbons are readily accumu accumulated (Roubal et al., 1977), 1977), even fairly small quantities of of oil (0.075
318
H. VON VON WESTERNHAGEN
mIll) O. gorbuscha ml/l) in the water inhibit growth in Pacific Pacific salmon 0. alevins exposed for 10 10 days (Rice (Rice et ai., al., 1975). 1975). Length and weight were equally affected. 1980) speculated that the affected. Vuorinen and Axell Axel1 ((1980) poor growth of pike E. 1 mg! E . lucius larvae in oil-contamined water (>0. (>0.1 mg/ 1) 1) may be related to gill damage, which decreases oxygen supply and food utilization. A concentration-dependent concentration-dependent reduction in growth is known for cod C. G. morhua larvae continuously exposed to 50-200 50-200 JLg/l pgll of a WSF of crude oil. Besides a direct impairment of of yolk utilization in the presence of oil (Lanning, (Lonning, 1977) 1977) or benzene (Struhsaker (Struhsaker et al., al., 1974), ect alterations in the 1974), the suppressed larval growth may refl reflect metabolic rate with energy diverted from assimilation to detoxifica detoxification (Eldridge (Eldridge et ai., al., 1977). 1977). Poor yolk utilization in larvae and alevins exposed to chlorinated hydrocarbons, in particular PCBs, is the major feature observed with these pollutants. O. kisutch exposed to poIlutants. Thus, alevins of of coho salmon 0. 15 JLg 15 pg Aroclor 1254/1 1254/1 react with reduced growth and poor yolk absorp absorption (Halter and Johnson, 1974), min 1974), as do the young of of the fathead minnow P. P . promelas (Nebeker et al., al., 1974). 1974). The inhibition of the mito mitochondrial NADH oxidase system by PCB PCB as shown by Pardini ((1971) 1971) may very well be responsible for this phenomenon. Inhibited yolk consumption is also present in fry exposed after hatching to low pH. In pike E. E . lucius, pH<4.2 inhibits yolk absorption considerably (Jo (Johansson and Kihlstram, 1975), and this is true also for larvae of the Kihlstrom, 1975), white sucker Catostomus commersoni (Trojnar, (Trojnar, 1977a). 1977a). Likewise, growth of fry is impaired and survival significantly reduced in the flagfish }. jlo ridae at pH 4.5 4.5 to 5.5 5.5 (Craig and Baksi, 1977); flagfishJ.floridae 1977);brook trout Saivelinus Salvelinus fontinalis alevins react in a similar way (Kwain (Kwain and Rose, 1985). 21 days in brook trout 1985).Time for yolk absorption is increased by 21 pH <5.0 (Menendez, 1976), alevins at pHC5.0 1976), and larvae usually remain Swarts et al., 978). In fry of rainbow trout Salmo gairdneri, smaller ((Swarts al., 11978). low pH affects the whole metabolism, as shown by the decreased cardiac rate, decreased rate of ossification, ossification, slower growth, less pig pigmentation, and increased mortality rate (Nelson, (Nelson, 1982). 1982). This section demonstrates the outstanding effects of all types of of pollutants on yolk utilization. The proper and complete utilization of the yolk reserves is is prerequisite for survival and successful develop development. Larvae that are not able to use these reserves for differentiation to the feeding stage will perish, particularly since the period between yolk utilization and first feeding is critical in many species and re requires precise “timing,” "timing," as pOinted 1 974b) in the "critical pointed out by May ((197413) “critical period concept." concept.”
4. EFFECTS OF POLLUTANTS 4.
ON FISH EGGS AND LARVAE
319
V. DISCUSSION, PROBLEMS AND THE FUTURE
Present knowledge of sublethal responses of of fish eggs and larvae to different types of pollutants is summarized in Table III. gen 111. The general plan of Table III 1976) 111is adopted from Rosenthal and Alderdice ((1976) and may be used for a quick overview of of the mode of of action of of pollu pollutants. Major malformations during advanced development, such as bent body axis, axis, may be caused by varying groups of of pollutants, and it is interesting to note the absence of specific effects caused by one single factor. factor. This is surprising considering the enlarged scope for differentiated reactions in advanced stages of of development, particu particularly since the embryonic stages from gastrulation to organogenesis have been reported to be the stages wherein most teratogenic effects occur (Wilson, (Wilson, 1973). 1973). Yet after completion of gastrulation the fre frequency of teratogenic effects declines, because organogenesis is eses sentially complete ((Stoss Stoss and Haines, 1979). em 1979). Morphologically the embryo stabilizes itself during the later stages, and the seeming seeming lack of of diversity in morphological abnormalities may be considered an arti artifact, fact, due to the inability of techniques to register effects before they express themselves as gross malformations. Enzymatic and hormonal monitoring, which has received little attention in early life-stage test testing (McKim, 1985) would provide a measure of (McKim, 1985) of the disruption of of nor normal enzyme and/or hormone development during organogenesis in the growing embryo. Thus, gross malformations malformations may be considered belated reactions to stress, stress, just as elevated body temperature in mam mammals is an indication of of infection. infection. It does not tell us whether whether the cause is pneumonia, measles, an infected tooth, or appendicitis. appendicitis. This also explains why in Table III 111 we find a preponderance of of morphological abnormalities as indicators for sublethal responses. At present we are still far from the desirable goal of establishing clear cause-effect cause-effect rela relationships between pollution and cellular or physiological processes. Although research has been particularly particularly active, resulting in a large body of fundamental information, when compared to the experimental evidence available 10 10 years ago (Rosenthal (Rosenthal and Alderdice, 1976) 1976) we note a lack of of new approaches. The majority of of the contributions still focus on a simple listing of not acutely lethal responses of of eggs, em embryos, or larvae to what seems to be an irrelevant enumeration of of more or less toxic substances, substances, often employed in outrageously high concen concentrations. Few of these contributions increase our knowledge of the nature of sublethal effects, effects, uncovering the mechanistic cause-effect cause-effect relationship between stressor and recipient. Neither are we today in a
Table Table III 111 Summary of of Observed Responses to Heavy Metals, Petroleum Hydrocarbons, Hydrocarbons, Effectsn Chlorinated Hydrocarbons, and pH Considered as Sublethal Effectsa Stage of of organization organization where pollutant stress is imposed or recognized Ovarian eggs and egg deposi deposition Fertilization, Fertilization, water uptake, and water hardening W 0 N
Observed response response Reduced egg number, Chromosome damage Chromosome Rate of of fertilization Changes in properties of of egg membranes Water uptake during and after fertilization
Impaired or delayed water hardening Changes in egg volume
Pollutant
M, PH, ClH, CIH, ppH H PH
M, PH, ClH, CIH, pH CIH M, ClH M, pH
M H M,, p PH M M
Embryonic development (early and advanced)
Biochemical effects Biochemical ef fects Changes in ATP levels
M M
Observed or suspected of response consequences of
recruitment Reduced recruitment Embryonic malformations Reduced recruitment recruitment Reduced capsule strength Changes in osmoregulatory capacity of pelagic Changes in buoyancy of eggs (changes (changes in transport, transport, distribution and location in water column) column) Reduced capsule strength Lowered resistance to deforma deformation activReduced space for embryo activ ity ity Energy deficit Retarded development Necrotic tissue Dedifferentiation Dedifferen tiation Organ malformation
activ Changes in enzyme activity ity
Physiological effects effects Physiological Respiration effects effects Respiration
Co:> � ....
Embryonic heart rate Morphological ffects Morphological eeffects Unusual Unusual shape shape of of blastoblastodisc Deformation toof bIas blastoDeformation of meres meres Irregular cleavage of blastomeres blastomeres Amorphous Amorphous embryonic embryonic tissue nite embryo tissue (no (no defi definite embryo formed) formed) Necrosis Necrosis Pigment Pigment anomalies anomalies Yolk-sac Yolk-sac blood blood circulation circulation not not well well developed developed Organ Organ malformations malformations Bent Bent body body axis axis No No blood blood pigmentation pigmentation Elongated Elongated heart heart tube tube Eye Eye malformations malformations (see (see also also yolk yolk sac sac larvae) larvae)
M , PH pH M,
Interference with general meme tabolism tabolism and biosynthetic processes processes Retarded Retarded development Reduced yolk/energy yolklenergy conversion conversion Smaller larval size at hatching Reduced hatching success Retarded Retarded hatching
PH M, PH
Changes growth Changes in in embryonic growth rates Retarded development
M
(?) Embryonic malformations (?)
CIH ClH
(?) Embryonic malformations (?)
PH, ClH
Embryonic malformations (?)
M, PH, PH, ClH, pH M,
No viable hatch
M, PH, PH, ClH, ClH, pH M, M,, PH PH M M, pH pH M,
Embryonic malformations malformations Embryonic to predation (?) (?) SSusceptible usceptible to Impaired yolk yolk utilization utilization (?) (?) Impaired
M, PH, PH, CJH, ClH, pH pH M, M,, PH PH M M M M, PH PH M,
Respiration (?) (?) Respiration Impaired swimming, swimming, feeding feeding Impaired Impaired respiration respiration Impaired Impaired blood blood circulation circulation (?) (?) Impaired Impaired vision, vision, prey prey hunting, hunting, Impaired phototaxis phototaxis
(continued) (continued)
Table Table ill 111 (Continued) (Continued) Stage Stage of of organization pollutant stress where pollutant is imposed or recognized
Incubation time and the pro process of of hatching
Larvae (hatched from exposed eggs) eggs)
Observed response
Pollutant Pollntant
Observed or suspected consequences of of response
Malfonned Malformed otoliths and/or otic capsules Behavioral effects effects Behavioral Embryonic activity activity re reduced
M M
Impaired equilibrium, swim swimming, prey hunting
M M,, PH, PH, pH PH
Pectoral fi n and opercula fin movements reduced
M M,, PH
Reduced mixing of of perivitelline fluid affecting respiration distribution of of hatching enen zyme zyme Retarded development Abnonnal Abnormal hatching process Retarded hatching As above (embryonic activity) activity)
Altered hatching parameters Prolonged hatching process
PH, PH, pH PH
Increased or decreased incubation time Reduced viable hatch
M, PH, ClH, pH
Smaller Smaller larval size at hatching
M, PH, ClH CIH
Changes in subcellular morphology
M
M, M , PH, ClH, pH
preIncreased susceptibility to pre dation Desynchronizing Desynchronizing of of food avail availability at time of of fi rst feeding first potential at Reduced survival potential population level Reduced biomass, increased increased suspectibility to predation reduced cruising speed Impaired metabolic functions
Biochemical eeffects ffects Changes in enzyme activ-
M
Impaired metabolic functions
ity ity Swimming Swimming behavior behavior Impairment of of swimming Inability to swim
M, PH, CIH
Impaired ability to maintain position in water column Inability to capture food Reduced escape reaction Increased susceptibility to predation
Equilibrium anomalies of avoidance reaction Loss of
"" to ""
Altered yolk utilization utilization Altered Reduced conversion efficiency Changed rate of of yolk utilization Malformations M a l f o m tions fins Serrated fi ns
M, PH M, PH
M, PH, CIH, ClH, pH M, PH, pH M, PH, PH
M
of Reduced larval size at time of fi rst food intake first Shift in timing to first first food intake
Eye defects (anophthalcyclopia) mia, cyclopia)
M, PH, pH
Mouth, lower jaw, branchial apparatus Vertebral column
M, PH, CIH ClH
Sloughing of of epidermal tissue Faulty pigmentation
M, PH, CIH, pH
Altered dermal respiration, swimming ability, susceptibility to disease Reduced visual perception, phototaxis, prey hunting ability ity Impaired respiration, respiration, feeding success Impaired swimming ability, escape reaction, prey hunting Susceptibility to infections
M, PH, CIH, ClH, pH
Susceptibility to predation
M, PH, ClH, CIH, pH
(continued) (continued)
Table 1 III (Continued) Table 11 (Continued) of organization Stage of where pollutant stress recognized is imposed or recognized unex Larvae (hatched from unexposed eggs)
Pollutant Pollutant
Observed response response
Malformations Malformations Skeletal deformations Skin lesions Morphological effects effects Morphological Brain damage Biochemical effects effects Biochemical Metabolic depression Retarded ossification ossification Retarded Reduced conversion of yolk and efficiency of reduced growth Behavioral Behavioral effects effects Changed migratory be behavior Change in critical critical dis distance Decreased activity, re reduced swimming capaccapac ity ity
Observed Observed or suspected consequences of of response
M, PH PH
Impaired swimming ability, prey prey catching
M
Impaired swimming, feeding
M
Energy deficit, deficit, impaired food conversion, conversion, increased thermal thermal sensitivity Reduced swimming speed Loss of of competitive fitness
pH PH M, PH, ClH, pH
M
PH
Food deprivation, starvation, suspectibility to increased suspectibility predation, reduced water volume searched for food
PH
These are conse are shown in relation relation to ontogenic ontogenic stages of of development in which they they are imposed imposed or observed and to known known or possible consequences at later ClH, chlorinated hydrocarbons, pesticides. Adapted from of development; M, metal; PH, petroleum petroleum hydrocarbon; CIH, chlorinated hydrocarbons, later stages of 1976). Rosenthal and Alderdice ((1976). a
4. 4.
EFFECTS EGGS AND EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS AND LARVAE LARVAE
325
better position to recognize the consequences of sublethal responses, even though there have been substantial contributions in histology, morphology, physiology, and ethology, as called for by Rosenthal and Alerdice ((1976), 1 976), nor are we able to bridge the gap between inferences based on observed individual responses and the implications they generate regarding the cellular level at which they were elicited on the the one one hand hand and and their their effect effect on on survival survival potential potential of of the the population population on the other hand; our ultimate concern must be for the long-term (Bayne, 1985). 1985). effects of contamination on the ecosystem (Bayne, Although biochemistry, molecular molecular mecha mechaAlthough in in the the particular particular field of biochemistry, nisms nisms of of the the effects effects of of trace trace metals metals on on enzymatic-related enzymatic-related metal metal toxicity toxicity have (see Viarengo, Viarengo, 1985), 1985), the connection between between have been been identified (see the pollutant impact and the complete reaction of the organism is still uncertain uncertain for for most of of the the xenobiotic xenobiotic substances, substances, preventing preventing aa clear clear view of the more subtle events inside the cell. responsiveWith the differentiation of the embryo, its scope for responsive ness becomes progressively enlarged from cells cells to tissue to organs etc., etc., while the the complexity complexity of of physiological physiological and and enzymatic enzymatic reactions reactions and metabolic feedbacks and metabolic feedbacks is is diversified. diversified. In In view view of of the the logarithmically logarithmically growing growing numbers numbers of of conceivable conceivable responses to to stressors, we we are are left left with rst of with an an unsatisfying unsatisfying incapability incapability of of explaining explaining even even the the fi first of the the most obvious deviations from normal development-that development-that is, the oc occurrence and significance of aberrant early cell divisions. divisions. Do they have a lasting effect on differentiation or does the growing embryo compensate and repair the early disorder? disorder? We do not yet have answers exfor those basic questions and, with progressing development, the ex difficult. This has panding variety of responses makes matters more difficult. field (see also McKim, McKim, 1985). 1985). been recognized by most workers in this fi eld (see arises: should we In seeking a way out of this dilemma, the question arises: be studying the basic processes that direct ontogeny and differentia differentiaaption or should we, for the time being, accept a less sophisticated ap proach, which may be simpler, simpler, easier to comprehend, comprehend, and more work workmisinterpretation? Frequently the nature able, and accept the risk of misinterpretation? able, of the investigation and the experimental approach do not allow a detailed explanation, since only gross gross morphological abnormalities are described while subtle responses at the cellular or subcellular levels are not considered. Thus, by far, far, more publications provide only a superficial evaluation of the causes of observed effects and refer only vaguely to such topics as metabolic inhibitors, for example. example. Much has been said in this review about energy pathways, their inhibition, inhibition, and blockage leading to malformations, yet with much too little known about little is is known about how how subsublittle experimental experimental background. background. Too little
326 326
H. VON VON WESTERNHAGEN
stances stances or their metabolites interfere with energy transfer through oxidative phosphorylation, the citric acid cycle, or protein metabo metabolism. No information is available as to why certain pathways are not passable for the embryo or the larva after the application of of certain stressors. stressors. Observations Observations of of reduced reduced heartbeat, heartbeat, dedifferentiation dedifferentiation of of tis tissues, sues, or or incomplete incomplete yolk yolk absorption absorption suggest suggest impaired impaired energy energy metabo metabolism. Although this may be correct and the reason for almost identical effects from different types of pollutants, this answer does not tell us anything about the mechanisms involved or the true nature of the processes impaired by lack of cellular energy. Experiments designed to study metabolic processes at the subcel subcellular level are rare and the situation is seldom so simple that we can understand the action of a pollutant in terms of a cause-effect cause-effect rela relationship, as appears to be the case in the chorionase inhibition at low pH. There are several possible ways for heavy metals to interfere with metabolic processes, although their significance for individual onto ontogenetic processes can only be postulated. Thus, zinc interferes with lysosomal lysosomal membrane stability, while mercury and cadmium are able to disrupt ionic balance and interfere with osmoregulation. Other metals such as copper have been shown to be very effective inhibitors envi of behavioral patterns at pollutant levels present in the today's today’s environment. At concentrations of a few micrograms per liter in the water they impair feeding in yolk-sac yolk-sac larvae, thus reducing survival. Water-soluble fractions of of petroleum hydrocarbons are effective narcotics, anesthetizing fish embryos and larvae at concentrations found after accidental oil spills. However, in the environment effec effective concentrations are not usually maintained maintained over long periods, since oil-degrading bacteria quickly reduce toxicity; the probability of petroleum hydrocarbons significantly affecting fish larvae is minimal. Experimentally chlorinated hydrocarbons also reduce percent via viable hatch at low concentrations in the lipids of parental gonads. Our investigations with marine fish and those of others with salmonids have demonstrated drastic reduction of viable hatch due to chlorin chlorinated hydrocarbons accumulated in gonads from the natural environ environment. It appears from this review that most sublethal effects are bio biochemical in origin and that they are expressed as histological, morpho morphological, logical, physiological, or behavioral respones. In conjunction with a better understanding of physiological and biochemical events at the subcellular level goes the necessity to concentrate more on cytopatho cytopathological and ultrastructural effects on cells, tissues, and organs. From recent investigations we learn that even without an obvious external
4. 4.
EFFECTS EGGS AND EFFECTS OF OF POLLUTANTS POLLUTANTS ON O N FISH FISH EGGS AND LARVAE LARVAE
327 327
damage, pollutants such as metals (zinc) (zinc) and petroleum hydrocarbons may severely influence cellular and tissue organization, disrupting cell structure in brain and eyes or causing damage to ultrastructural cell bodies such as mitochondria or endoplasmic reticulum; possible effects on orientation or locomotion give clues to the mechanism of of the pollutants. As yet there are too few investigations of of detailed scru scrutiny in this field. The significance of of the gross malformations is generally better un understood than the alterations at the subcellular level. This is particu particularly true for the individual and represents a substantial part of of Table III, 111, where the majority of of the observable responses to pollutants ulti ultimately cause the death of of the individual. There are few responses, such as changes in color pattern or slight body flexure due to metal, that may not be crucial for survival or may be repaired repaired as development proceeds. Particularly hardy species species such as cyprinodontids, belonids, or some salmonids when reared in captivity may even survive major damages to fi n s, jaw, or vertebrae. In the field, however, the percent fins, percentage survivors with gross gross morphological abnormalities is negligible, since fish larvae are relatively fragile and natural mortality is is high even in healthy individuals individuals.. At this point aan n important question arises. arises. What iiss the significance to natural populations of the pollution-induced sublethal responses? The only one of the four factors factors reviewed in this chapter that at present shows shows clear effects on fish populations is is the low pH of fresh freshwater lakes and rivers in the northern hemisphere. From the available data fish eggs to low pH (pH (pH 4.0-5.0), data on the susceptibility of fish 4.0-5.0), it is evident that the prevailing pH of many lakes and rivers in northern Europe and America (Beamish, 1976; Jensen and Snekvik, 1972; (Beamish, 1974, 1974,1976; 1972; Schofield, Schofield, 1976; 1976; Wright and Snekvik, 1978; 1978; Harvey, 1980; 1980; Rosseland et al., ai., 1980; ai., 1980; 1980; Overrein et al., al., 1980; 1980; Sevaldrud et al., 1980; Haines, 198 1 ; Gunn, 1982) 1981; 1982) is already too low to guarantee normal ontogenetic processes in fish eggs. In these lakes, although adult populations of fish eggs. fish may still survive for a few years, years, the impairment of the hatching enzyme enzyme through through acid waters waters is is too great to allow more than a small small percent of larval eld and larval hatch hatch (Beamish (Beamish and Harvey, Harvey, 1972). 1972). From fi field experiments, Muniz and Leivestad ((1980) that the 1980) conclude that laboratory experiments, most common mechanism of population extinction in acidified lakes is postembryonic mortality and subsequent lack of recruitment. As we know (Harvey (Harvey and Lee, 1982), 1982), other lakes in particularly sensitive sensitive areas areas such such as as the La Cloche Cloche area in Canada or southern Norway are already seriously depleted of the original fish populations or are de-
328 328
H. H. VON VON WESTERNHAGEN WESTERNHAGEN
void of 1974; Sevaldrud et of fish altogether (Beamish, (Beamish, 1974; et ai., al., 1980; 1980; Gunn, 1982). 1 978) showed that no short-term adaptation 1982). Since Swarts et al. al. ((1978) of eggs or alevins to low pH can be expected, it seems probable that more and more lakes with low buffering capacity will be deprived of their fish populations. The effect of of low pH is usually exacerbated by elevated metal contents. Several of of these metals, such as copper (McKim 1 ; Horning and Neiheisel, 1979) (McKim and Benoit, 197 1971; 1979) or mercury (Huckabee (Huckabee and Griffith, Griffith, 1974), 1974), occur at concentrations (3.0-18.0 (3.0-18.0 p,g/l) pgll) toxic to embryos and larvae of freshwater fish (Haines, (Haines, 1981). 1981). The same may be true for aluminum (Grahn, 1980), although in the case of (Grahn, 1980), of aluminum we are dealing probably with asphyxia due to hydroxide preci pitation. precipitation. This is a social rather than a biological problem (see also Haines, 198 1 ; LaZerte and Dillon, 1984). 1981; 1984). As long as society and industry are not prepared to take measures against the increasing acidification of lakes, promoting a drastic reduction of sulfuric (S0 (SOz) (NO,.) 2 ) and nitric (NOx) exhaust-gas exhaust-gas emission, lake acidification will progress rapidly; for many fish populations the sublethal effects of decreased hatching will turn into a lethal effect when no viable hatch is possible. Aside from an ecological catastrophe, some areas must face substantial economic losses due to decreasing revenues from recreational fishing (see (see Tuomi, 1981). 1981). For the marine environment, pH has no bearing, since the effective low pH is far below the level that can be observed even at extreme conditions in the sea (Kelley, (Kelley, 1946; 1946; von Westernhagen and Dethlefsen, 1983). 1983). Petroleum hydrocarbons are likely to produce short-term effects on fish populations. These substances manifest their effects in two ways 1 ) as acute effects resulting from oil spills, blow-outs, or the ways:: ((1) like, these effects may be locally confi ned and, even if confined if severe (Longwell, (Longwell, 1978), 1978), will not have far reaching consequences because only a limited number of of eggs or larvae is affected in time and space; (2) (2) long-term sublethal effects may be expected for coastal and estua estuarine substrate spawning populations, since petroleum hydrocarbons and their metabolites concentrate in and on the substrate (sand) (sand)that is is used by demersal spawners (Chapman et al., 1982; Landolt and Ko al., 1982; Kocan, 1984). 1984). Some substances or their metabolites are highly mutagenic (Le., (i-e.,benzo[a]pyrene) benzo[a]pyrene) and in the long run produce chronic effects by increasing genetic load in a population (Hose (Hose et al., al., 1982; 1982; Kocan and Landolt, 1984). 1984). This applies particularly for harbors and heavily in industrialized areas of both fresh and saltwater bodies where small in indigenous fish populations exist. Although the mechanisms of the pro process are comprehensible, nothing is known about its dynamics and the
4. 4.
EFFECTS OF OF POLLUTANTS POLLUTANTS ON ON FISH FISH EGGS EGGS AND AND LARVAE LARVAE EFFECTS
329 329
scale involved. It may be that a fish fish population employing employing the time scale “low expenditure per progeny" progeny” in its its reproductive strat stratprinciple of "low certain-relatively high-percentage high-percentage of nonviable egy requires a certain-relatively eggs and larvae larvae before recruitment recruitment becomes detectibly impaired re reeggs sulting in collapse collapse of the the population. population. Fluctuations Fluctuations within natural pop popsulting fish are are known to surpass surpass the the factor factor 4. 4. Thus in Arctic Arctic cod ulations of fish the level of recruitment was the same same in 1953, 1953,1955, 1960 as as that the 1955, and 1960 even though the size size of the spawning stock stock of the years in 1944-1946, even 1953, 1955, 1955, and 1960 1960 was was only only 25% 25% of that that in 1944-1946. 1944-1946. If, If, on the 1953, hand, the population concerned is more of the "high “high expendi expendiother hand, progeny” type, as is the case in salmonids with with relatively few ture per progeny" eggs per female, then a 20% 20% reduction in reproductive success may eggs have a bearing on the population. This has been shown in a seatrout (Cynoscion nebulosus) nebulosus) population, which declined drastically due to (Cynoscion feimpaired reproductive success because of high DDT residues in fe gonads (Butler (Butler et al., aZ., 1972). 1972). Also, Willford et al. al. ((1981) conmale gonads 1981) con from the reproductive failure of lake trout Salvelinus namay nama ycluded from cush in Lake Michigan that increased mortality of fry due to high DDE and PCB PCB levels impedes restoration of the lake trout population DOE self-sustainability. to self-sustainability. Although the concentrations of these substances in the water are (Brugmann and Luckas, 1978) 1978) not high enough to cause acute effects (Briigmann (DDT, PCB; and are constantly declining (DDT, PCB; Olsson and Reutergardh, 1986), 1986), we believe that chlorinated hydrocarbons are causative agents such a process. The involved mechanism acts by biomagnifi biomagnifitriggering such cation:: the accumulated substances become effective on the embryo cation eggs. Investi Investior larva through the parental gonads and the yolk of the eggs. al. ((1981), gations of von Westernhagen et ai. 1981), Hansen et al. ((1985), 1 985), Westin et ai. 1 985), and Cameron et al. 1 986), conducted with several fish al. ((1985), al. ((1986), species, have shown that chlorinated hydrocarbons [PCB, [PCB, DDT, DDE, chlordane] in gonads reduce viable DOE, hexachlorobiphenyl (HCB), (HCB), chlordane] 2% and less in flounder Platichthys PlatichthysfEesus, hatch down to 2% fiesus, herring C. M . saxatilis, and whiting (Merlangius (Merlangius harengus, striped bass M. merlangus). of the popula populamedungus).However, the overall reproductive capacity of endangered-the mean viable hatch of floun flountion does not seem to be endangered-the of von Westernhagen et al. der as determined from data of al. ((1981) 1981) is still 55%. Similarly high values are found for herring (59%) (59%) (Han (Hanaround 55%. al., 1985). 1985). Yet results from the North Sea indicate that viable sen et ai., of whiting Merlangius merlangus (Cameron (Cameron et al., 1986) experihatch of 1986) experi mentally incubated, is usually low ((11%). 1 1%). These data fall in line with the high percentage of of aberrations found in whiting eggs in the North Sea and low hatching success of “wild” "wild" eggs (Dethlefsen et aZ., al., 1985). 1985).
330
H. VON VON WESTERNHAGEN H.
present there appears to be no direct impact on Thus, although at present of Hogan and Brauhn recruitment in these species, the investigations of (1975) and Butler et aZ. al. (1972) (1972) show that other species have already (1975) of chlorinated hydrocarbons displaying yielded to high gonad burden of failures failures in in recruitment. recruitment. A A great great variety variety of of heavy heavy metals metals has has been been investigated, investigated, but but very very few few relevant concentrations concentrations have have been been employed. employed. In most cases inordi inordinately nately high high metal metal levels have have been tested. tested. Even Even the the lower lower concentra concentrations, such as the low levels of copper that affect feeding of of herring and plaice larvae (Blaxter, (Blaxter, 1977), 1977), are still high when compared to actual concentrations in river water or seawater (Anonymous, 1983; (Anonymous, 1983; Kremling 1978). Only Kremling and and Petersen, Petersen, 1978; 1978; Duinker Duinker and and Nolting, Nolting, 1978). rarely rarely do do we we find find concentrations concentrations in in nature nature that that can can be be related related to infor information provided in this review and that might cause sublethal effects on eggs and larvae (Smith (Smith et aZ., al., 1981). 1981). Thus, for the time being we must conclude that heavy metals in the sea or natural fresh waters (except for mine tailings, etc.) etc.) do not influence recruitment. Yet there are indications that concentrations in nature are only one order of of magnitude or less away from metal levels known to cause sublethal effects, and sediment concentrations of metals if if mobilized are are in most cases high enough to unleash a series of of sublethal and lethal effects ((Forstner Forstner and MiilIer, Muller, 1973; 1973; Hershelman et aZ., 1981). 1981). In brief, the detrimental effects of of low pH on fish reproduction in fresh water are clearly discemable, discernable, while those of of petroleum hydro hydrocarbons and chlorinated hydrocarbons are about to show in fresh wa water and in the sea. For the time being, metals at concentrations concentrations exist existing ing in in the the field field do do not not yet yet exert exert sublethal subletha1 effects effects on on fish fish eggs eggs and and larvae that have a bearing on recruitment.
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4. 4.
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((1979). to zinc on reproduc reproduction in the guppy (Poecilia (Poecilia reticulata). reticulata). Bull. Bull. Environ. Environ. Contam. Contam. Toxicol. Toricol. 23, 650650657. 657. van der Putte, J. ]. 1982). Effects of hexavalent Putte, I., I., Galieen, W., W., and Strik, Strik, J. J. T. T. W. W. A. A. ((1982). hexavalent chromium in rainbow trout (Salmo (Salmo gairdneri) gairdneri) after prolonged exposure at two dif different pH levels. Ecotqxicol. Environ. Enuiron. Saf. Saf. 6, 6, 246-257. 246-257. levels. EcotO,xicol. Viarengo, A. ((1985). 1985). Biochemical effects of trace 153-158. trace metals. Mar. Mar. Pollut. Pollut. Bull. Bull. 16, 16,153-158. Vladimirov, V. 1969). Dependence of the embryonic V. J. J. ((1969). embryonic development and viability of the Vopr. Ikhtiol. Ikhtiol. 9, 687-696. 687-696. the carp on the trace element zinc. Vopr. von Westernhagen, H. ((1968). 1968). Versuche zur Erbriitung Westernhagen, H. Erbriitung der Eier des Schellfisches (Me (Melanogrammus L.) unter Linter kombinierten SalzgehaltsSalzgehalts- und Temperaturbe Temperaturbelanogrammus aeglefinus aeglejinus L.) dingungen. Ber. Ber. Dtsch. Dtsch. Wiss. Wiss. Komm. Komm. Meeresforsch. Meeresforsch. 19, 19, 270-287. 270-287. von Westernhagen, H. (1970). Westernhagen, H. (1970). Erbriitung Erbrutung der Eier vom Dorsch (Gadus (Gadus morhua), morhua), Flun Flunder (Pleuronectes (Pleuronectesflesus) Jesus) und Scholle (Pleuronectes (Pleuronectes platessa) platessa) unter Linter kombinierten TemperaturSalzgehaltsbedingungen. Helgol. 21, 21Helgol. Wiss. Wiss. Meeresunters. Meeresunters. 21, 21Temperatur- und Salzgehaltsbedingungen. 102. 102. von Westernhagen, 1974). Incubation of garpike Westernhagen, H. H. ((1974). garpike eggs (Belone (Belone belone belone Linne) Linne) under controlled 625f . Mar. Mar. BioI. B i d . Assoc. Assoc. U.K. U.K. 54, 54,625controlled temperature temperature and salinity conditions. ]. 634. 634. Westernhagen, H. H. and Dethlefsen, V. V. (1975). (1975). Combined effects effects of cadmium and von Westernhagen, J . Mar. Mar. Biol. Assoc. U.K. U.K. 55, survival of fl flounder eggs. ]. salinity on development and survival ounder eggs. Bioi. Assoc. 945-957. 945-957. Westernhagen, H., and Dethlefsen, Dethlefsen, V. V. (1983). (1983). North Sea Sea oxygen deficiency 1982 1982 von Westernhagen, 264-266. and its effects on the bottom fauna. fauna. Ambio Ambio 12, 12,264-266. Westernhagen, H., H., and Rosenthal, Rosenthal, H. H. (1979). (1979). Laboratory Laboratory and in situ studies studies on von Westernhagen, larval lupea harengus larval development and swimming swimming performance of Pacific herring C Clupea harengus pallasi. pallasi. Helgol. Helgol. Wiss. Wiss. Meeresunters. Meeresunters. 32, 32, 539-549. Westernhagen, H., H., Rosenthal, Rosenthal, H., H., and Sperling, Sperling, K.-R. (1974). (1974). Combined effects of von Westernhagen, development and and survival of herring eggs. eggs. Helgol. Helgol. Wiss. Wiss. cadmium and salinity on development Meeresunters. 4 16-433. 26,416-433. Meeresunters. 26, von Westernhagen, Westernhagen, H., H., Dethlefsen, Dethlefsen, V., V., and Rosenthal, H. H. (1975). (1975).Combined effects of and salinity on development and and survival of garpike eggs. Helgol. Helgol. Wiss. Wiss. cadmium and Meeresunters. 27, 268-282. Meeresunters. 27,
4. 4.
EFFECTS OF POLLUTANTS EGGS AND EFFECTS OF POLLUTANTS ON ON FISH FISH EGGS AND LARVAE LARVAE
345
Dethlefsen, V., and Rosenthal H. (1979). (1979). Combined effects of von Westernhagen, H., Dethlefsen, Wiss. cadmium, copper and lead on developing developing herring eggs and larvae. Helgol. Wiss.
32,257-278. Meeresunters. 32, Meeresunters. 257-278.
Rosenthal, H., Emst, W., Harms, U., and Han Hanvon Westernhagen, H., Rosenthal, H., Dethlefsen, V., V., Ernst, Bioaccumulating substances substances and reproductive success in Baltic sen, sen, P.-D. ((1981). 1981). Bioaccumulating Jesus. Aquat. Aquat. Toxicol. Toxicol. 1, 1,85-99. 85-99. flounder Platichthys flesus. Kremlvon Westernhagen, H., Kocan, R., Landolt, M., Furstenberg, G., C., Janssen, D., and Kreml ing, K. ((1987). 1987). Toxicity of sea surface microlayer: Effects on herring and turbot Mar. Environ. Enuiron. Res. Res. (in press). embryos. Mar. (1977). Viability of of Voyer, R. A., Wentworth, C. E., Berry, E., and Hennekey, R. J. (1977). comembryos of the winter flounder, Pseudopleuronectes americanus, exposed to com embryos Mar. Biol. (Berlin)44, binations of cadmium and salinity at selected temperatures. Mar. Bioi. (Berlin)
1117-124. 17-124.
J. F., F., and Kraus, Kraus, R. A. ((1979). Voyer, R. A., Heltsche, J. 1979). Hatching success and larval (Linnaeus), exposed to cadmium mortality in an estuarine teleost, Menidia menidia (Linnaeus), Enuiron. Contam. Contam. Toxicol. Toxicol. 23, fluctuating Bull. Environ. in constant and fl uctuating salinity regimes. Bull. 475-481. 475-481. F., and Hoffman, C. G. L. (1982). (1982). Viability of of Voyer, R. A., Cardin, J. A., Heltsche, J. F., Pseudopleuronectes americanus americanus exposed to mix mixembryos of embryos of the winter flounder Pseudopleuronectes tures of cadmium and silver in combination combination with selected fixed salinities Aquat. Aquat. 223-233. Toxicol. 2, 223-233. Taxicol.
M.-B. (1980). (1980). Effects of of the water soluble Vuorinen, P., and Axell, AxeIl, M.-B. soluble fraction of crude fry. Int. Counc. Counc. Explor. Sea, Counc. Counc. Meet. E:30, oil on herring eggs and pike fry.
11-10. - 10.
Watermann, A. J. (1940). (1940).Effects of of colchicine colchicine on the development of of the fish embryo,
Oryzias 29-34. Oryzias latipes. latipes. Bioi. Biol. Bull. Bull. (Woods (Woods Hole, Hole, Mass.) Mass.) 78, 78,29-34.
Weis, J. S., S., and Weis, P. 1977). Effects of P. ((1977). of heavy metals on development of of the killifish, heteroclitus. ]. J . Fish Bioi. Biol. 111,49-54. Fundulus heteroclitus. 1 , 49-54. Weis, P. (1984). (1984). Metallothionein Metallothionein and mercury tolerance in the killifish, Fundulus Fundulus he he-
teroclitus. Enuiron. Res. teroclitus. Mar. Mar. Environ. Res. 14, 14, 153-166.
Weis, P., and Weis, J. S. 1974). Cardiac malformations and other effects due to in S. ((1974). insecticides in embryos of Fundulus heteroclitus. heteroclitus. Teratology Teratology 10, 10, of the killifish, Fundulus
263-268. 263-268. S. (1976). Weis, P., aud and Weis, J. S. (1976). Abnormal locomotion associated with skeletal malfor malforvariegatus, exposed to malathion. mation in the sheepshead minnow Cyprinodon Cyprinodon uariegatus, Environ. Res. Res. 12, Enuiron. 12, 196-200. 196-200. 1 977). Methylmercury teratogenesis Weis, P., and Weis, J. S. ((1977). teratogenesis in the killifish, Fundulus Fundzllus heteroclitus. 317-326. heteroclitus. Teratology Teratology 16, 16,317-326. Westin, D. T., Olney, C. D. T., C. E., and Rogers, B. A. (1985). (1985). Effects of parental parental and dietary organochlorines Trans. Am. Am. organochlorines on survival and body burdens of striped bass larvae. Trans. Fish. 125-136. Soc. 114, 114, 125-136. Fish. Soc. Willford, W. A., Bergstedt, R. A., Berlin, W. H., Foster, N. R., Hesselberg, R. J., Mac, M. 1981). Introduction and J., Passino, D. R. M., Reinert, R. E., and Rottiers, D. V. ((1981). summary-Chlorinated summary-Chlorinated hydrocarbons as as a factor in the reproduction reproduction and survival of of lake trout (Salvelinus (Saluelinus namaycush) namaycush) in Lake Michigan. Tech. Tech. Pap. Pap. U. U.S. S . Fish Fish Wildl. Wildl. Ser. Ser. 105, 105, 1-4. 1-4. Wilson, Wilson, J. J. C. G. (1973). (1973). "Environment “Environment and and Birth Birth Defects." Defects.” Academic Academic Press, Press, New New York. York. Wilson, K. W. ((1972). 1972). Toxicity of of oil-spill dispersants to embryos and larvae of of some marine fish. In (M. Ruivo, ed.), In "Marine “Marine Pollution and Sea Life" Life” (M. ed.), pp. 318-322. 318-322. Fishing News (Books), (Books), London.
346 346
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Wilson, K. W. (1974). (1974). The ability of of herring and plaice larvae to avoid concentrations concentrations of of “The Early Life History of of Fish" Fish” 0. (J. H. S. 589oil dispersants. In "The S. Blaxter, ed.), pp. 589602. Springer-Verlag, Berlin and New York. York. Wilson, K. W. ((1976). 1976). Effects of oil dispersants on the developing embryos embryos of of marine 259-268. fi s h. Mar. Biol. (Berlin) (Berlin) 36, 36,259-268. fish. Winkler, D. L., L., Duncan, K. L., Hose, J. E., and Puffer, H. W. (1983). (1983). Effects of of ben benzo(a)pyrene zo(a)pyrene on the early development of of California grunion, Leuresthes tenuis (Pisces, Atherinidae). 473-481. Atherinidae). Fish. Bull. 81, 81,473-481. R., and Snekvik, E. (1978). (1978). Acid precipitation: precipitation: Chemistry and fish populations populations Wright, R., in 700 lakes in southernmost Norway. Verh.-Int. Verh.-lnt. Ver. Ver. Theor. Theor.Angew. Angew. Limnol. 20, 20, 765-775. 765-775. Wunder, W. ((1971). 1971). Missbildungen beim Kabeljau (Gadus (Gadus morhua) verursacht verursacht durch Helgol. Wiss. Wiss. Meeresunters. 20, 20,201-212. 201-212. Wirbelverkiirzung. Helgol. Zotin, A. J. J. (1958). (1958). The mechanism of of hardening of of the salmonid egg membrane after Exp. Morphol. 6, 549-568. fertilization or spontaneous spontaneous activation. ]. J . Erp. 549-568.
5 VITELLOGENESIS AND OOCYTE ASSEMBLY THOMAS PP.. M MOMMSEN THOMAS OMMSEN Department of Zoology Department of University of of British Columbia Vancouver, British Columbia, Canada V6T 2A9
PATRICK PATRICK ]. J . WALSH WALSH Rosenstiel Rosenstiel School School of of Marine and Atmospheric Science University of of Miami Miami, Florida 33149 Miami, 33149
1. I. Introduction
II. Vitellogenesis 11. A. A. Hormonal Induction B. Target Cells Cells B. Estrogen Receptors in Target C. C. Plasma Binding Proteins D. D. Hepatic Events E. E. Vitellogenin F. Posttranslational Modifications Modifications F. Posttranslational G. Actions of Estradiol G. Other Actions H. H. Male Male Fish I. I. Elasmobranch Fishes III. 111. Oocyte Oocyte Assembly Assembly A. A. Transport of Vitellogenin B. B. Uptake of Vitellogenin Vitellogenin C. C. Phosvitin and and Lipovitellin Lipovitellin D. D. Oocyte Oocyte Lipids Lipids E. E. Carotenoids Carotenoids F. F. Glycoproteins Glycoproteins G. G. Vitamin-Binding Vitamin-Binding Proteins Proteins H. H. Hormones Hormones I. I. Yolk-DNA Yolk-DNA J. J. Metabolism Metabolism IV. IV. Epilogue Epilogue References References
347 347 FISH FISH PHYSIOLOGY. PHYSIOLOGY. VOL. VOL. XIA XIA
Copyright tC 0 1988 1988 by by Academic Academic Press. Press, Inc. Inc. Copyright All rights rights of ofreproduction reproductionin in any any form form reserved. reserved. Al!
348
THOMAS MOMMSEN AND THOMAS P. MOMMSEN AND PATRICK PATRICK JJ.. WALSH
I. INTRODUCTION At their life At certain certain stages stages of of their life histories, histories, the the females females of of egg-laying egg-laying fishes, maturavertebrates, including including most species of fi s hes, enter a phase of matura tion of their oocytes in preparation for ovulation and spawning. Under the the mutifaceted mutifaceted influence influence of of hormonal hormonal centers centers such such as as the the hypothala hypothalamus and the pituitary gland (reviewed by Peter, 1983), 1983), the growing follicles synthesize and excrete into the systemic circulation circdation steriod hormones hormones that that govern govern aa variety variety of of different different metabolic metabolic processes. processes. One of the primary target organs for these steriods, particularly 17P-estra17f3-estra diol, is the liver. This organ, which possesses highly specific binding proteins for 117P-estradio1, 7f3-estradiol, in turn responds to such hormonal stimulus with the synthesis and export of vitellogenin. First named by Pan et 1969), vitellogenin constitutes the carrier molecule for various al. ((1969), al. classes of compounds accumulated by the developing oocyte. While the backbone of the vitellogenin molecule is a protein chain of sub sub250,000-600,000), it also carries copi copistantial size (molecular weight 250,000-600,000), ous amounts of lipid material, carbohydrate components, phosphate groups, and mineral salts. Following highly selective uptake into the vitellogenin is broken up and accumu accumuoocyte, the transport molecule vitellogenin lated as egg-specifi c yolk constituents, such as phosvitin and lipovitel egg-specific lipovitellin. In In addition addition to to these these well-known well-known egg egg components, components, growing growing fish fish suboocytes accumulate a variety of other compounds, sometimes in sub proper develop developstantial amounts, which play an integral part in the proper ment of the embryonic and larval fish. fish. Some compounds serve as a reservoir reservoir for for energy-demanding energy-demanding processes. processes. In some cases, cases, however, however, the these compounds compounds is is not not yet yet known known or or the physiological physiological importance importance of these evidence. Substances that can only be inferred from circumstantial evidence. fall into these categories include glycogen, carotenoids, lectins, sialoglycoproteins, wax esters, and sterol esters. The early developmental stages of many species species of fish entail long periods, sometimes weeks, of starvation prior to their first exogenous vitellogenin and the feeding. Therefore, the maternal production of vitellogenin assemdeposition of adequate supplies of yolk, yolk, as well as the proper assem bly of the oocyte, are essential to subsequent embryonic and larval survival. This chapter covers several aspects of vitellogenesis survival. This vitellogenesis and vitellogenin synthesis oocyte assembly, such as hormonal induction of vitellogenin in vitellogenin and its later fate in the devel develin the liver, the transport of vitellogenin oping oocyte, and the origin and nature of other egg components. priWhile these various topics for fishes have been addressed in the pri decades, this chapter will mary literature only during the last two decades,
5. 5.
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349 349
focus attention on recent developments and touch on comparative aspects that deserve future attention. Since this chapter was comcom pleted, a review appeared in press covering aspects of vitellogenesis (Waland oocyte developments, albeit from a widely differing angle (Wal 1985). lace, 1985). At this point it should be emphasized that research focusing on piscine systems field systems has been a relatively recent addition to a fi eld that has anilong been established for amphibians and birds as experimental ani mals. specific. mals. Readers interested in more comparative facets or specifi c molecmolec of those areas ular principles are therefore referred to recent reviews of (Soreq, 1985; 1985; Browder, 1985). (Soreq, Browder, 1985).
II. VITELLOGENESIS 11,
vitelA general framework for the hormonal control of exogenous vitel logenesis was first proposed by Bailey (1957), (1957),and over the years has been tested experimentally and modified accordingly. Environmental cues such as photoperiod, temperature, feeding, and social factors factors all regulate the production of gonadotropic hormones (GtH) (GtH) in the pitui pituitary (Peter, (Peter, 1983; 1983; Liley and Stacey, Stacey, 1983). 1983). In females, the ovaries ,a-estra respond to increased levels of GtH by enhancing estrogen (17 (17p-estradiol and possibly estrone) estrone) production and release into the blood bloodstream (Fostier (Fostier et al., 1983). 1983). Estrogens are transported to the possible specificc blood proteins or attached to the ubiq ubiqtarget tissues bound to specifi bIood albumins. faciliuitous blood albumins. They are thought to enter the tissues by facili tated diffusion and in the livers specifically induce the synthesis of of vitellogenin. Much of the experimental evidence in support of this recently (Ng (Ng and Idler, 1983). 1983). These stud studscheme has been reviewed recently effort, fall into ies, as well as a considerable amount of continued effort, several categories: categories: 1. In vitro studies make it possible to identify and characterize 1. highly specific estrogen binding proteins in fish hepatic tissue (Le Menn et al., 1980; 1980; Turner, Turner, et al., 198 1981; 1985; (Le 1 ; Lazier et al., 1985; Maitre et al., 1985b). 1985b). 2. The "natural" “natural” dynamics of hormone titers 'and ?and vitellogenesis 2. have been studied and indicate that estradiol levels and vitel vitellogenesis are positively correlated and increase in parallel (de (de Vlaming et al., 1984; 1984; Veda Ueda et al., 1984; 1984; van Bohemen and 1981), goLambert, 198 1), following photoperiod-related increases in go nadotropins (Bromage (Bromage et al., 1982; 1982; Breton et al., 1983). 1983).
350 350
THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH
3. The effects of experimental treatment of fish with various horhor 3. mones, hormonal metabolites, and analogs have been well disdocumented. Note that in these and many of the studies dis cussed later (Section (Section II,H), II,H), the observed effects are inducible in male and/or immature fish. fish. Injection with pituitary extracts or certain purification fractions fractions [concanavalin A (Con (Con A) A)Sepharose/carbohydrate-rich Sepharose/carbohydrate-rich fraction] fraction] induces estrogen syn synthesis and subsequent vitellogenesis (Idler and Campbell, 1980). 1980). Injection with estrogen alone (preferentially estradiol) or pharmacological doses of androgen induces the appearance ofvitellogenin 1 ; Korsgaard and of vitellogenin in the plasma (Plack (Plack et al., 197 1971; Petersen, 1979; 1979; Korsgaard et al., al., 1983; 1983; Hori et al., 1979; 1979; van Bohemen et al., 1982a,b) 1982a,b)as well as follicular fluid (Korsgaard, (Korsgaard, 1983). 1983). 4. In vitro studies of ovaries have confirmed the cells of the follic follicular epithelium as responsible for the production of estrogen (van 1 ; Sundararaj (van Bohemen and Lambert, 198 1981; Sundararaj et al., al., 1982b; 198213; Nagahama, 1983). 1983).However, certain parts of the fish brain have also been shown to be capable of synthesizing estrogen (van (van 1981; 1981), whereas Bohemen and Lambert, 198 1 ; Callard et al., 1981), the quantitative contribution of brain-derived estrogens to cir circulating estrogens and hence vitellogenesis has not been re resolved. solved. The major components of fish oocytes are derived from the blood bloodborne high-molecular-weight compound vitellogenin, which is syn synthesized in the liver of oviparous vertebrates vertebrates.. This supply of of oocyte components-especially components-especially yolk-from yolk-from extraovarian sources has been termed exogenous vitellogenesis. The classification of vitellogenin as a phospholipoglycoprotein already indicates the crucial functional groups that are carried on the protein backbone of of the molecule, addinamely, lipids, some carbohydrates, and phosphate groups. In addi tion, vitellogenin also has strong ion-binding properties and thus may serve as a major supply of minerals to the oocyte. oocyte. A. Hormonal Induction
of the normal events accompanying early vitello vitelloIn the course of oocyte are stim stimgenesis, the follicle cells surrounding the developing oocyte ulated to synthesize estradiol. Androgens constitute the prevailing precursors of estradiol, and a slow increase in estradiol during the annual cycle indicates early exogenous vitellogenesis. After the hor-
55..
VITELLOGENESIS VJTELLOGENESIS AND AND OOCYTE OOCYTE ASSEMBLY ASSEMBLY
351 35 1
mone mone has has entered entered the target target organs, organs, in in this this case case the the liver, liver, it it binds binds to to highly c estrogen highly specifi specific estrogen receptors. receptors. In In aa subsequent subsequent step, step, the the receptor/ receptor/ chromatin. estradiol complex will bind to high affinity sites on the chromatin. While While the the actual actual biochemistry biochemistry of of the the hormone-receptor hormone-receptor complex complex and and the mechanism of its binding to nuclear structures has not yet been al., 1984), 1984), it it has has been well docu docuentirely elucidated elucidated (Miesfeld (Miesfeld et ai., hormone-receptor complex with mented that the interaction of the hormone-receptor the DNA leads to modulation of the expression of c genes. In of specifi specific the case of estradiol administration to immature female or male fish, fish, specific directed toward toward the the vitellogenin vitellogenin gene, gene, which which is is specific activation activation is directed of the actual binding site located somewhat downstream on the DNA of receptorhormone complex. Thus, the estrogen receptor can be for the receptorihormone regarded as a gene regulatory protein. A diagrammatic representation of of the the feedback feedback system system between the the ovary ovary and and the the liver liver involving involving estradiol estradiol and and vitellogenin vitellogenin is is presented presented in in Fig. Fig. 11.. OOC Y T E
OOCYTE
B B LLO OO OD D
H HE EP P AATTO OC CY YT TE E
Fig. 1. 1. Simplified feedback system between ovary and liver during exogenous vitel vitelFig. the influence influence of pituitary pituitary hormones, hormones, the follicle folIicle cells cells release release estrogen logenesis. Under the logenesis. into the bloodstream. The estrogen estrogen enters enters the target target cells cells (hepatocyte) (hepatocyte) by facilitated into the events depicted in Figs. 22 and 3, 3, vitellogenin is is synthesized on the diffusion. After the Golgi apparatus, and ex exendoplasmatic reticulum, modified, packed into into the Golgi rough endoplasmatic vitellogenin binds to specific specific receptor pro prointo the bloodstream. Blood-borne vitellogenin creted into the oocyte oocyte membrane, is is taken taken up by micropinocytosis, and is moved in mi miteins on the teins yolk, vitellogenin is is broken up into into final deposition in the yolk, crovesicular bodies. Before fi nal deposition Iipovitellin and phosvitin components. components. Estrogen Estrogen is not involved in the uptake and lipovitellin processing the growing growing oocyte. oocyte. processing of vitellogenin by the
352 352
THOMAS P. MOMMSEN MOMMSEN AND AND PATRICK J. WALSH J . WALSH
of androgens into estrogens is is catalyzed by the The conversion of enzyme aromatase and occurs not only in the ovary but also in the of all vertebrates; vertebrates ; aromatase brain of aromatase activity activity is exceptionally high in al., 1981; 198 1 ; Lambert and van Oordt, 1982). 1982). However, teleosts (Callard et al., released the estrogen thus produced in the fish brain apparently is not released into the circulation, circulation, unless chemically chemically altered, altered, which results in its of the ultimate removal removal from the circulation by gill tissue. Exertion Exertion of of this hormone multifaceted biological effects of hormone will consequently be (Callard, 1982) 1982) and therefore cannot restricted to the local brain level (Callard, of exogenous vitellogenesis. be implicated in the initiation of B. Estrogen E strogen Receptors in Target Cells The prevailing model for the cellular distribution and action of of steriod hormone receptors has been the so-called "two-step "two-step model," model," which was first proposed by Gorski, Jensen, and co-workers co-workers (Gorski et HEPATOCYTE
/
I
7'
-
i i I
E E
//
\
E Estrogen
Cy1Q801
Fig. 2. Two-step Two-step model of estrogen-receptor estrogen-receptor mechanism. mechanism. Estrogen binds to cytoso cytosoFig. lic 5-5 receptor proteins in the target cell (liver). (liver). During translocation from the cytosol lie to the nuclear matrix, the estrogen-occupied estrogen-occupied receptor receptor (estrogen-receptor (estrogen-receptor complex) complex) changes from 5 SS to 4 SS and acquires acquires high affinity affinity for the chromatin. The estrogen estrogenchanges receptor complex binds to specifi specific other things (cf. (cf. Table receptor c sites on the DNA and among other II) 11) leads to activation of the the gene coding for viteIlogenin. vitellogenin.
5. 5.
VITELLOGENESIS VITELLOGENESIS AND AND OOCYTE OOCYTE ASSEMBLY ASSEMBLY
35 3 353
al., al., 1968; 1968; Jensen et al., al., 1968). 1968).According to this model, which is sum summarized 2, estradiol-or estradiol-or any any other other steriod steriod hormone hormone for for that that marized in in Fig. Fig. 2, matter-enters matter-enters the target cell by diffusion and binds to a specific cytosolic cytosolic form form of of the the receptor receptor protein. protein. Subsequently, Subsequently, the the estrogen estrogenreceptor bind receptor complex undergoes aa transformation transformation from from aa non-DNA binding ing form form to to aa species species that that does does and and is is translocated translocated to to the the nucleus. nucleus. Receptor Receptor transformation transformation is is also also reflected reflected in in aa decrease decrease in in receptor receptor size size from S. The to about about 4 4 S. The model model from aa sedimentation sedimentation coefficient coefficient of of around around 5 S to implies present with with or or implies that that the the cytosolic cytosolic form form of of the the receptor receptor can can be present without without bound bound steriod, steriod, while while the the nuclear nuclear form form only only exists exists in in the the hor hormone-occupied state. Ever Ever since since the the inception inception of of the the two-step two-step model, model, experimental experimental evi evidence been accumulating, dence has has been accumulating, which which is is difficult difficult to to reconcile reconcile with with the the model. Recently, model. Recently, aa different different model model of of steriod steriod receptor receptor localization localization has has been proposed, proposed, disbanding disbanding the the existence of of the the cytosolic cytosolic receptor. receptor. Using Using powerful powerful immunocytochemical immunocytochemical techniques, techniques, the the proponents proponents of of 3) localized localized all receptor molecules, molecules, unoc unocthis "nuclear “nuclear model" model” (Fig. (Fig. 3) cupied occupied, in Greene, 1984; cupied or or occupied, in the the nuclear nuclear region region (King (King and and Greene, 1984; HEPATOCYTE
E
E str o g e n
Fig. 3. 3. Nuclear Nuclear model model of estrogen-receptor estrogen-receptor mechanism. mechanism. Estrogen Estrogen enters enters the target target cell and binds c nuclear cell and binds to to highly highly specifi specific nuclear receptor receptor proteins. proteins. Estrogen-receptor Estrogen-receptor complex complex displays c sites displays high high affinity affinity for for chromatin, chromatin, binds binds to to specifi specific sites on on the the DNA, and, and, among among other vitellogenin gene. (cf. Table Table II), 11), leads leads to to activation activation of the the vitellogenin gene. other things things (cf.
354
P. MOMMSEN AND PATRICK J. J. WALSH THOMAS P.
1984). Since these authors found the cytosol to be Welshons et al., 1984). of appreciable amounts of of steriod binding sites, they came to devoid of experi the conclusion that the larger, cytosolic receptor may be an experiof receptor/ mental artifact. In this model, no nuclear translocation of affin hormone complex occurs and the receptor attains its increase in affinity ity for nuclear structures by binding hormone directly within the nuclear compartment. Highlighting the merits of of the comparative ap approach, the work of of Callard and Mak (1985) (1985) on the estrogen receptors of the testes in an elasmobranch fish (Squalus (Squalus acanthias) acanthias) support this of novel working model; both occupied and unoccupied receptors are exclusively associated with the nuclear compartment. re In recent years, evidence has accumulated that that many steriod reof proteins that ceptors fall into an interesting group of that are regulated by reversible covalent covalent modifications modifications through through phosphorylatioddephosphosphorylation/dephos reversible phorylation. phorylation. Experimental Experimental data suggest suggest that phosphorylation phosphorylation is is aa pre prerequisite for the hormone binding activity of of the receptor protein and that dephosphorylation leads to an inactivation of the receptor mole molecule. cule. While most proteins, including the chicken progesterone recep recepal., 1982) 1982) and the glucocorticoid receptor of of fibrofibro tor (Dougherty et al., 1983), are phosphorylated at serine residues blasts (Housley and Pratt, 1983), within pro within the polypeptide chain, the calf calf uterine estrogen receptor protein is phosphorylated on tyrosine (Migliaccio et al., 1984), 1984), a relarela tively unusual unusual phosphorylation phosphorylation site site for for covalent covalent modification (Krebs, tively modification (Krebs, 1985). 1985). of the present controversy over the actual cellular localiza In light of localization of of unoccupied estrogen receptors, where the evidence points to the cytosolic receptor receptor as a possible experimental artifact (King, (King, 1984), 1984), it is somewhat confusing to reconcile the new interpretation with the of the calf estrogen receptor is brought about fact that phosphorylation of specific, calcium- and calmodulin-dependent cytosolic receptor by a specific, kinase . Not surprisingly, however, the dephosphorylation-dependent dephosphorylation-dependent kinase. Of the receptor inactivation of receptor is caused by a phosphatase (Migliaccio et ai., 1984) 1984) with exclusive localization within the nuclear compartment. aZ., of the existing models has not been tested for While the validity of pis cine systems, the presence of of highly specific eses hepatic tissue in piscine trogen receptors has been verifi ed for a number of verified of different species of of fish, fish, namely, namely, Gobius niger (Le (Le Menn Menn et al., 1980), 1980), Pacific Pacific hagfish hagfish (Eptatretus stouti; Turner al., 1981), Turner et al., 1981), Atlantic Atlantic salmon salmon (Salmo (Salmo saIar; salar; 1985; Mommsen and Lazier, 1986), 1986), sea-raven (Hemitrip(Hemitrip Lazier et al., 1985; terus americanus), americanus), winter flounder (Pseudopleuronectes americanus), americanus), terns octodecimspinosus; Mann et al., 1988), 1988), rainrain sculpin (Myoxocephalus octodecimspinosus; (S. gairdneri; Maitre et al., 1985b; 1985b; Mann et al., 1988) 1988) and bow trout (S. 1986). most recently in the brown trout (S. trutta; Pottinger, 1986).
5. 5.
VITELLOGENESIS VITELLOGENESIS AND AND OOCYTE OOCYTE ASSEMBLY ASSEMBLY
355
The livers of nonvitellogenic or male Atlantic salmon (S. (S. salar) salar) contain specific high-affinity estrogen binding proteins in the cytoso cytosolie high lic fraction, while the liver nuclei reveal low concentrations of of highaffinity affinity estradiol-binding components. Injection of pharmacological doses of estradiol in the fish fish leads to a transient depletion of the cytosol binder and the appearance of of exceptionally high concentra concentrations of estradiol binding sites in nuclear salt extracts (Fig. (Fig. 4). 4).In naive fish, sites in the cytosol was 640 fmollg fish, the concentration of binding sites 150 frnol/g finol/g liver. liver, while their concentration in liver nuclei was 150 After the administration of a single dose of estradiol, the concentration of nuclear binding ssites ites increases almost 80-fold 1 pmol/g 80-fold to above 111 liver (Lazier et al., al., 1985). 1985). The induction is apparently highly specifi specificc for estradiol and, after a single injection of estradiol into an experi experimental fish, fish, reaches a maximum of receptor response after some 24 h. Such response can just as easily be elicited in cultured hepatocytes, using physiological, rather than the more usual pharmacological, doses of hormone (Fig. (Fig. 4A). 4A). The binding characteristics are identical for the in vivo or the in vitro response (Fig. (Fig. 4B). 4B). Put into the proper perspective of the cellular environment, environment, the maximum concentration of accumulated estrogen receptor is dwarfed in comparison with the products of some commonly expressed genes. metabolism, for instance, occur in Most enzymes involved in liver cell metabolism, 10 to 100 100 nmol per gram ofliver, of liver, that is, concentrations ranging from 10 is, at least two orders of magnitude higher than the maximum amount of estrogen receptor. receptor. One gram of liver contains about 0.9 ILg pg estrogen receptor, assuming a molecular mass of of about 80 80 kDa, compared with almost 500 500 ILg p g pyruvate kinase alone. alone. A similar increase in the total level of cellular estrogen-binding sites, where the increase in nuclear binding sites more than offsets offsets the decrease decrease in in cytosolic cytosolic sites, is is observed observed when when cultured cultured salmon salmon liver liver cells are exposed to physiological doses of estradiol (Mommsen (Mommsen and Lazier, 1986; 4). The time course and the peak concentration 1986; cf. Fig. 4). reached reached differ differ somewhat somewhat between between the the in in vivo vivo and and the the in in vitro vitro systems, systems, possibly due to the amounts of estradiol administered and enhanced metabolism in the isolated liver cells. cells. While this particular subject was fish, it was not the the focus focus of any any of of the the studies studies on on fish, it is interesting to to note note that that the metabolism metabolism of of the the steriod hormone itself itself and and not not so much much the the kinetics of the hormone-receptor uences the effectiveness hormone-receptor decay infl influences of of the the hormone hormone administration. administration. In In tissue tissue cultures cultures of of Xenopus hepato hepatocytes, cytes, for for instance, instance, estradiol estradiol turnover is rather rather rapid, especially especially in in cells is in cells derived derived from from male male toads. toads. Here, Here, the the half-life half-life of of the the molecule molecule is in the range of (Tenniswood et al., al., 1983) 1983) at at 20°C. 20°C. It It is is the range of only only 40 min (Tenniswood obvious that if similar conditions are found in isolated fish cells, very
356
THOMAS MOMMSEN AND THOMAS P. P. MOMMSEN AND PATRICK PATRICK JJ.. WALSH WALSH
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Fig. Fig. 44.. (A) Temporal changes in hepatic estrogen receptor after estrogen administra administra0 , e) O )or isolated isolated hepatocytes in culture ((*) of * ) were treated with a single dose of tion. Fish ((0, estradiol [5 [5mg (kg (kg live weight)-I weight)-l and I1 IJ-M gM for cultured cells, respectively]. respectively]. Estrogen (0,**)) and cytosolic binding sites were determined after the indicated time in nuclear (e, (0)fractions. Modified from Lazier et al. ((1985) (1986). (0) 1985) and Mommsen and Lazier (1986). temporarily depressed, Numbers of assayable binding sites in the cytosolic fraction are temporarily while numbers of of nuclear binding sites continue to increase until the end of of the experi experiof response is reached about mental period ((120 120 h). h). In cultured liver cells, a maximum of 24 h after the addition of of estradiol. Vitellogenin can first be detected by immunoprecipi immunoprecipitation (B) Estrogen-binding characteristics of 24 h. (B) of tation in the hepatocyte medium after 24 salmon liver nuclei. Cultured hepatocytes were treated with estradiol as indicated in (A). (A).At 12 12 h after hormone exposure, cells were harvested and nuclear nuclear extracts were analyzed for receptor activity and characteristics (Scatchard analysis). The dissociation I-I T. T. constant (�) 3.4 nM 1-I (&) for estradiol is computed by linear regression regression and found to be 3.4 P. Mommsen and C. B. B. Lazier (unpublished). (unpublished). The dissociation constant for highly 5.4and specificc estrogen binders from liver nuclei in estrogen primed fish is between 5.4 specifi al., 1985). 5.9 5.9 nM (Lazier et et al., 1985).
5.
357
VITELLOGENESIS VITELLOGENESIS AND OOCYTE ASSEMBLY
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little little exposure exposure is is needed to to initiate initiate the the transcription transcription of of estradiol-de estradiol-dependent genes, genes, in estrogen-receptor gene, gene, thus setting pendent in this this case case the the estrogen-receptor thus setting up an interesting positive feedback system. Compared vitellogenic vertebrates, vertebrates, the teleost liver liver apap Compared with with other other vitellogenic the teleost pears to to be the the richest richest source source of of highly highly specific specific estrogen estrogen receptors, receptors, pears making the fish fish liver liver an an ideal ideal model model system system to to study study the of making the induction induction of receptor and and analyze in detail the mechanism mechanism of of hormone-receptor hormone-receptor receptor analyze in detail the and receptor-chromatin receptor-chromatin interactions interactions in in lower lower vertebrates. vertebrates . The The values values and listed in in Table Table I compare compare the the magnitude magnitude of of the receptor induction induction in in the receptor listed S. salar with other oviparous vertebrates utilized in the analysis of S. of the biochemistry biochemistry of of the the estrogen estrogen receptor. receptor. Recently, Recently, it it was was found found that that the the salmon is not unique in this respect, nor is such largesse of of response restricted to to salmonid salmonid fish. fish. Liver nuclei isolated isolated from the sea-raven sea-raven restricted Liver nuclei americanus), the the longhorn longhorn sculpin sculpin ((M. octodecimspinosus), winter winter ((H. H . americanus), M . octodecimspinosus), flounder ((P. americanus), and the rainbow trout (S. gairdneri) all accuaccu P . americanus), flounder mulate similarly similarly high high concentrations concentrations of of estrogen estrogen binding binding proteins proteins as as mulate
358 358
THOMAS THOMAS P. MOMMSEN MOMMSEN AND AND PATRICK PATRICK JJ.. WALSH WALSH
Table Table I Magnitude Magnitude of of Estrogen-Receptor Estrogen-Receptor Response Response in in Vitellogenic Vitellogenic Vertebratesa VertebratesD Naive animals animals Salmo saiar salar Saimo laevis Xenopus iaevis Gallus domesticus domesticus Gallus
0.15 0.15 0.2 0.2 0.1 0.1
After After induction induction with with estradiol estradiol
> > 12 12
2.5 2.5 0.4 0.4
a0 Values Values are are given given as as 10-1 10-122 moles moles of of nuclear nuclear binding binding sites sites for for estradiol estradiol per per gram gram of of liver. liver. Concentration Concentration of of cytosolic cytosolic binding sites 0.6 x 10-12 binding sites in in the the untreated untreated salmon salmon was was about about 0.6 mol courses of see mol (g (g liver)-I. liver)-'. For For time time courses of receptor receptor abundance abundance see Fig. maximum number number of nuclear binding binding sites sites in Fig. 4. 4. The The maximum of nuclear in vivo vivo and computes to nuclear estrogen and in in vitro vitro computes to around around 25,000 25,000 nuclear estrogen re receptor molecules liver cell. Hayward et ceptor molecules per per liver cell. Sources: Sources: Hayward et ai. al. ((1980), 1980), Lazier 1975), and Lazier ((1975), and Lazier Lazier et et ai. al. (1985). (1985).
the Atlantic salmon (Mann et al., al., 1988). 1988). In a goby (Gobius (Gobius niger), niger), an elasmobranch (Potamotrygon, (Potamotrygon, ssp.), ssp.),and a hagfish (Eptatretus (Eptatretus stouti), stouti), on on the the other other hand, hand, numbers numbers of of estrogen-binding estrogen-binding sites sites in in the the liver liver are are (Le Menn more than an order of magnitude lower than in the salmon (Le et al., 1980; 1980; Callard and Mak, 1985; 1985; Turner et al., al., 1981). 1981). The The magnitude magnitude of of the the receptor receptor response, response, which which resembles resembles that that in in some mammalian systems (Walter et al., al., 1985), 1985), and the reported sta stability of the nuclear binding protein in many teleost fishes fishes (Lazier et al., 1985) 1985) should should make make it it possible possible to to attain attain receptor receptor preparations preparations of highest purity. To date, this goal has been hampered by the poor response in all other oviparous vertebrates (see (see Table I). I). The anticiantici pated availability of of receptor preparation of of extreme purity may help to shed light on the ongoing controversy over the cellular distribution of hepatic receptor (King, (King, 1984; of 1984; Szego and Pietras, 1985) 1985) and the comcom of occupied estrogen receptors in the cell nucleus (Shapiro, (Shapiro, plex fate of 1982). 1982). Furthermore, comparisons with estrogen receptors from mammam malian tissues will furnish insights into the evolutionary trends of of the receptor gene (Greene (Greene et al., 1986). 1986). While the actual source of of these comparatively large amounts of of receptor in the piscine the gene-regulatory estrogen receptor piscine system has not been elucidated yet, some indirect evidence in other vitellogenic verver tebrates indicates that de novo synthesis of of receptor protein is inin 1975; Perlman et al., 1984). Again, the highly sensitive volved (Lazier, (Lazier, 1975; al., 1984). piscine system seems ideally suited to supply mechanistic insight into
5. 5.
VITELLOGENESIS AND AND OOCYTE ASSEMBLY
359
the source of "induced" “induced” receptor molecules by molecular biology techniques, ssimilar imilar to the ones used to assess the transcriptional activ activity mRNA longevity albumin gene (Wolffe et ity and and mRNA longevity of of the the albumin gene in in Xenopus (Wolffe al., 1985), 1985), for instance. instance. al., In receptor proteins proteins resemble the the In all all parameters parameters analyzed, analyzed, the the fish receptor receptors from other vertebrates vertebrates.. The salmon receptors are character characterprogesterized by a high specificity for extradiol and they do not bind progester one, one, hydrocortisone, hydrocortisone, or or dihydrotestosterone. dihydrotestosterone. In In agreement agreement with with stud studies of receptors from many other vertebrate sources, the fish receptors display high high affinity affinity for for the the nonsteroidal nonsteroidal estrogen estrogen diethylstilbestrol diethylstilbestrol as as 4-hydroxytamoxifen. The es eswell as for the nonsteroidal antiestrogen 4-hydroxytamoxifen. E . stouti, on the of the hagfish E. trogen binding proteins in the liver of other hand, display global verte other hand, display unique unique features, features, different different from from the the global vertebrate brate picture. picture. This species species possesses possesses nuclear nuclear estrogen estrogen receptors with with a lower affinity for extradiol than other vertebrate counterparts (disso (disso&=38 3-6 nM in the salmon; salmon; see Fig. ciation constant Ki = 38 nM versus &= Ki= 3-6 4B). However, the hagfish system is unusual in that estrone or estriol 4B). effidisplaced estradiol from the nuclear binding components as effi ciently ciently as as estradiol estradiol or or diethylstilbestrol diethylstilbestrol (Turner (Turner et al., aE., 1981). 1981). In other other vertebrates, vertebrates, binding binding affinities affinities for for estriol estriol or or estrone estrone are are usually usually more more than an order of magnitude lower than for estradiol. estradiol. In the rainbow of vitellogenin trout, estrone administration leads to the induction of synthesis in the liver and its release into the bloodstream, but estrone displays 12% of the 5%to to 12% the potency potency of of estradiol estradiol (van (van Bohemen et displays only only 5% al., 1982a,b). 1982a,b). It seems that one of the functions of estrone in vivo may al., be to prime hepatic tissue for subsequent exposure to estradiol and the hepatocytes to to es esthus to to potentiate potentiate the the vitellogenic vitellogenic response response of the tradiol. The interesting characteristics of the hagfish receptor, to tocharacteristics of gether with the positioning of the cyclostomes within the vertebrate line, of steroid steroid sex sex line, might might in future future shed shed some some light light on on the the evolution evolution of hormones, their interactions, and receptor specificity. specificity. In In the the annual annual cycle cycle of of the the rainbow rainbow trout, trout, the the liver liver is is exposed exposed to to of estradiol and estrone. differing concentrations and ratios of estrone. While blood concentrations of both estrogens increase during early vitello vitellogenesis, the first phase of vitellogenesis is dominated by estrone, which increases by a factor of 10 10 altogether (van (van Bohemen and Lam Lambert, 981). During bert, 11981). During the the later later stages stages of of exogenous exogenous vitellogenesis, vitellogenesis, estra estradiol reaches blood concentrations of60 of 60 ng/ml, reflecting an increase of 60-fold. It would be interesting to analyze whether similar changes 60-fold. are specific estrogen abundance or or preference preference of of specific estrogen re reare reflected reflected in in the abundance ceptors in the nuclei of the liver, liver, the main target organ for ovarian estrogens.
360
THOMAS THOMAS P. P. MOMMSEN MOMMSEN AND AND PATRICK PATRICK JJ.. WALSH WALSH
In In addition addition to to the the estrogens, estrogens, androgens androgens are are able able to to elicit elicit aa vitello vitellogenic genic response response in in teleost teleost fish, fish, albeit albeit only only when when administered administered in in phar pharmacological macological doses, doses, (Le (Le Menn, Menn, 1979; 1979; Hori Hori et al., 1979). 1979).Interestingly, Interestingly, at at least in C. G. niger this response appears to be mediated by androgen binding binding to to the the estrogen estrogen receptor receptor rather rather than than through through the the nuclear nuclear an androgen drogen receptor itself itself (Le (Le Menn Menn et al., 1980). 1980). Similarly, Similarly, high high doses doses of of androgen androgen fed fed to to juvenile juvenile salmon salmon may may lead lead to to aa pronounced pronounced feminiza feminization tion of of some some fish fish (Solar (Solar et al., 1984), 19841, although although the the molecular molecular mecha mechanisms for these phenomena remain to be analyzed. The The sedimentation sedimentation coefficient coefficient of of the the salmon salmon receptor receptor protein protein of of 3.6 3.6 SS indicates indicates that that it it may may be be aa little little smaller smaller than than the the nuclear nuclear estrogen estrogen receptors receptors of of birds birds or or mammals, mammals, but but it it falls falls into into the the same same range range as as the the receptor (Lazier, 1978; receptor isolated isolated from from Xenopus laevis (Lazier, 1978; Wright Wright et al., al., 1983). 1983).
C. Plasma Binding Proteins C. The steroid hormones produced and released by the ovarian cells are transported to their target tissue in the systemic circulation. Al Although probably not a target issue in itself, fish plasma displays a certain degree of steroid-binding steroid-binding capacity. For a variety of of fishes as well as for other vertebrate groups, such "sex-steroid “sex-steroid binding pro proteins" teins” have been characterized numerous times (Wingfield, (Wingfield, 1980). 1980). Their specificities and properties clearly distinguish plasma binders from the cellular steroid receptors, while their exact physiological function, over and above the suggested role in steroid transport, is still under debate. Since steroid hormones exert their biological functions only in the free and not in the bound form, such plasma steroidsteroid binding proteins may serve to buffer free steroid concentrations in of high steroid turnover, thus obviating time consuming de conditions of novo synthesis. compo In the plasma of of SS.. salar, two differing estradiol binding components are abundant, one with high affinity and one with low affinity for al., 1985). 1985). In contrast to the highly estradiol-speestradiol-spe estradiol (Lazier et aZ., cific nuclear of the nuclear receptors inducible in the salmon liver, neither neither of plasma-binding components are competed for by the nonsteroidal eses diethyl s tilbestrol. Furthermore, again differing from the situatrogen diethylstilbestrol. situa 4-hydroxytamoxifen does not comcom tion in the liver, the antiestrogen 4-hydroxytamoxifen pete with estradiol for binding to the high-affinity (&=13 (KI= 13 nM) estrogen binder in plasma. Experiments also indicate that the androandro gen dihydrotestosterone as well as progesterone and estrone reveal
5. 5.
VITELLOGENESIS VITELLOGENESIS AND AND OOCYTE OOCYTE ASSEMBLY ASSEMBLY
361
affinity for the plasma binder and are likely to compete considerable affinity 1985). with extradiol in vivo as they do in vitro (Lazier et al., 1985). D. Hepatic Hepatic Events Events With a time delay of a few hours following the binding of the estrogenlreceptor complex to the nuclear DNA, DNA, a variety of changes in estrogen/receptor liver cells are initiated that are consistent with a substantial increase in the capacity for protein synthesis and export-plasma export-plasma concentra concentration tion of vitellogenin vitellogenin may may reach reach 50 50 mg/ml mg/ml (Ng (Ng and and Idler, Idler, 1983). 1983). Indeed, Indeed, naturally sh reveal fish reveal much much higher higher rates rates of of hepatic hepatic protein protein naturally vitellogenic vitellogenic fi synthesis than nonvitellogenic fi sh (e.g., fish (e.g., Haschemeyer and Mathews, 1983; 1983;Yu et al., 1980; 1980; Emmersen and Korsgaard, 1983), 1983),a phenomenon that can be provoked by estrogen administration in vivo as well as in vitro. vitro. Several Several ultrastructural differences are observed between liver cells from immature and vitellogenic fish. (Epifish. In the red grouper (Epi nephelus akaara), akaara), vitellogenic livers are characterized by expanded nuclear envelope envelope cisternae, swollen swollen mitochondria, and much en enhanced rough endoplasmic reticulum, Golgi apparatus apparatus and secretory vesicles (Ng sh treated with estra (Ng et al., al., 1984). 1984). Hepatocytes of naive fi fish estradiol imilar, but diol showed showed ssimilar, but not not entirely entirely identical identical ultrastructural ultrastructural changes changes (Ng (Ng et al., al., 1984). 1984). Several Several studies studies indicate indicate an an increase increase in in hepatosomatic hepatosomatic index (van Bohemen et al., al., 1982a,b; 1982a,b; Dasmahapatra et al., 1981) 1981)and, at least in the red grouper, this appears to be due to a rise in cell lipid and water content rather than proliferation of cell numbers (Ng al., (Ng et al., 1984). 1984). In the Atlantic salmon, as in the flounder, flounder, estradiol administra administration leads to increases in liver protein, total RNA, and total nuclear count (Korsgaard 1976). Since at (Korsgaard et al., al., 1986; 1986; Korsgaard and Petersen, 1976). the same time the liver volume and weight increase, calculated on a is augmented unit weight basis, only the amount of cellular RNA is signifi cantly. It can be concluded that in these two species of fish, significantly. fish, apdiffering from the grouper, hyperplasia rather than hypertrophy ap pears to be responsible for the enhanced liver weight (Korsgaard (Korsgaard et al., 1986). 1986).The more than 30% 30% increase in cellular RNA content (on (on a al., basis) is is yet another indication of the increased biosyn biosynunit weight basis) thetic activity of the liver (Korsgaard al., 1986), 1986),where where the de novo (Korsgaard et al., some of synthesis of messenger RNA for vitellogenin may account for some the observed increase in total RNA. RNA. A larger proportion of the newly synthesized RNA in hepatic tissue the exposure to estradiol is is due to apparent increases in the following the amounts amounts of ribosomal RNA, RNA, which can be explained by the massive
362
THOMAS P. MOMMSEN MOMMSEN AND PATRICK J. WALSH
increases in rough endoplasmatic reticulum observable in liver micro micrographs of fish and other oviparous vertebrates (Bast (Bast et al., al., 1977; 1977; Selman and Wallace, 1983a). 1983a). Since estrogen administration is respon responsible for the proliferation of of cell structures, such as endoplasmic retic reticulum (ER), (ER), Golgi vesicles (and turnover), turnover), and mitochondria, genes coding for any of these structures must have been activated or estro estrogen administration must have at least led to increased translational activity involving existing mRNAs. mRNAs. Obviously estradiol is able to or orchestrate cell metabolism and biosynthetic activities at a number of different levels. An ancillary question concerns the actual localization of hepatocy hepatocytes active in the synthesis and export of vitellogenin. Contrary to the identical, it widespread belief belief that all hepatocytes are metabolically identical, has been shown rather conclusively that rat hepatocytes in the perive perivenous and periportal regions of the liver possess differiong metabolic functions, with anabolic pathways such as gluconeogenesis, fat syn synthesis, and proteins synthesis being favored in the better oxygenated periportal cells (Jungermann (Jungennann and Katz, Katz, 1982). 1982). It would be interesting to know whether a similar hepatic zonation exists in the lower verte vertebrates in general and extends to estrogen receptors and the vitello vitellogenic response. E strogen treatment of fish also appears to result in a general gear Estrogen gearing up of of metabolism to provide the large amounts of of energy and reducing power (NADPH) (NADPH) necessary for protein and lipid synthesis (Ng 1984) fur (Ng et al., 1984; 1984; Petersen and Korsgaard, Korsgaard, 1977). 1977). Ng et al. al. ((1984) further report significant and substantial increases in transaminases and enzymes of the Krebs cycle and glycolysis. glycolysis. On the other hand, natu naturally vitellogenic vitello genic female sockeye salmon (Oncorhynchus (Oncorhynchus nerka) nerka) on their spawning migration do not increase any specific metabolic ma machinery in liver (on (on a weight basis, Mommsen et al., ul., 1980), 1980),apart from the general augmentation due to an-probably an-probably estradiol-dependent estradiol-dependentincrease in liver weight (Idler and Clemens, 1959). 1959). In vivo treatment of male flounder (Platichthys flesus) results in an (Platichthysjesus) increase of protein synthetic activity when assessed in an in vitro system (Korsgaard (Korsgaard et al., 1983). 1983).That such stimulation may be a direct effect of estrogen on liver cells was recently demonstrated in hepato hepatocytes isolated from juvenile coho salmon (0. (0.kisutch; Bhattacharya et al., al., 1985). 1985). Hepatocytes treated with 17f3-estradiol 17p-estradiol and exposed to 4C]serine or [['*C]glycine either [ l14C]serine 14C]glycine exhibit an increase in radioactivity precipitable by trichloroacetic acid (TCA), (TCA), a decrease in TCA-soluble radioactivity, and enhanced release of TCA-precipitable radioactivity into the medium compared with untreated untreated controls (Bhattacharya et
5. 5.
VITELLOGENESIS AND OOCYTE ASSEMBLY
363 363
Table Table II I1 Cellular Events Associated with the Estrogen-Dependent Induction Vitellogenesis in Teleost Hepatic Tissue of Vitellogenesis Transient decrease in cytosolic estrogen receptor protein Induction of nuclear estrogen receptor protein Increase in hepatosomatic hepatosomatic index due to hyperplasia or hypertrophy Golgi apparatus Proliferation of Colgi Increase in cisternae of the nuclear envelope Synthesis Synthesis of ribosomes Polysome assembly assembly Increase in rough rough endoplasmatic endoplasmatic reticulum reticulum of mitochondria mitochondria Swelling of Appearance species of mRNA (vitellogenin) Appearance of of a new species Increase in protein synthetic activity Synthesis Synthesis of vitellogenin Increase in cellular RNA (?)n Increase in lipid metabolism (?)" Increase lipoproteins (VLDL) (VLDL) Augmented output of very low density lipoproteins Decrease in glycogen content per cell Increase in metabolic enzymes enzymes Increase Higher amount of hepatic DNA Increase Increase in in hepatic hepatic water water content content "0 For the the fishes, fishes, to to date date only only circumstantial circumstantial evidence evidence suggests suggests this this particular particular alteration. alteration, See See text text for for relevant relevant references. references.
al., 1985). 1985). Originally Originally it it was was observed observed that that liver liver slices slices from from cod cod (G. (G. 1 4C]leucine into morhua) rnorhua)treated treated with with estradiol estradiol incorporated incorporated labeled labeled [[‘4C]leucine into "egg evidence confi rmed 1971).All this this evidence confirmed “egg proteins" proteins” (Plack (Plack and and Fraser, Fraser, 1971).
that teleost fishes, just as that in in the the teleost fishes, just as in in other other oviparous oviparous vertebrates, vertebrates, estra estradiol rapid and hepatic synthesis and specific specific hepatic synthesis of of the the egg-yolk egg-yolk diol leads leads to to the rapid precursor vitellogenin. The most apparent ultrastructural and bio bioexperience during exogenous vichemical changes that hepatocytes experience exogenous vi 11. tellogenesis are summarized in Table II.
E.. Vitellogenin E 10years vitellogenin molecules from a number of differ differIn the last lO biochemicent fishes have been isolated and partially characterized biochemic ally. Interestingly, Interestingly, the fishes fishes display a much higher variability in the ally. phosphorylsuch as as molecular weight, weight, degree of phosphoryl different parameters, such ation, degree of lipidation, or subunit subunit composition than their amphib amphibation, 111).As the example of the tetrameric counterparts (Table (Table III). ian or avian counterparts shows (Hara (Hara et al., al., 1980), 1980),not all fish vitellogenin in the Japanese eel shows are dimers-for dimers-for instance, and in the case of the brown vitellogenins are
364 364
THOMAS THOMAS P. MOMMSEN MOMMSEN AND AND PATRICK J. WALSH
Table Table III 111 Molecular Weights of of Native Vitellogenin and Subunits from Fish Other Than Rainbow Trout Native molecular mass (kD4 (kDa)
Subunit molecular mass (kDa) (kD4
Carassius Carassius auratus auratus (goldfi sh) (goldfish)
326 380
140-156 140-156 140-147 140-147
Gadus Gadus morhua morhua (cod) (cod) Anguilla japonica (Japanese eel) eel) Fundulus heteroclitus (killifish) (killifish) Ameiurus Ameiurus nebulosus nebulosus (brown bullhead) Platichthys Plotichthys flesus Jesus (flounder) (flounder) Oncorhynchus Oncorhynchus kisutch kisutch (coho (coho salmon) salmon) Salmo Salmo salar salar (Atlantic salmon) (Atlantic salmon) Salrno Salmo trutta trutta (brown trout) Heteropneustes Heteropneustes fossilis fossilis (catfish) (catfish)
400
-
350
85 85
-
200
-
145 145
550
-
Gel fi ltration filtration
390” 390c
-
Gel filtration
495 and 520 495and520
-
Gel fi ltration filtration
440
-
Gel filtration
550
-
Gel fi ltration filtration
Species
Method" Methodu
Reference
Native/SDS-PAGE 1979) Native/SDS-PAGE Hori et et al. al. ((1979) Native/SDS-PAGE Native/SDS-PAGE de Vlaming - et al. (1980) (1980) Plack et Gel filtration et al. (1971) (1971)
Gel filtration and SDS-PAGE SDS-PAGE SDS-PAGE SDS-PAGE b
Hara et al. ((1980) 1980) et al. Selman and Wallace ((1983a) 1983a) Roach and Davies (1980) (1980) Korsgaard and Petersen ((1976) 1976) Markert and Vanstone ((1971) 1971) (1985) So et al. al. (1985) Norberg and Haux ((1985) 1985) Nath and Sundararaj (198 1) (1981)
SDS-PAGE: : sodium dodecyl sulfate polyacrylamide polyacrylamide gel electrophoresis. " SDS-PAGE prevailing mRNA induced by estradiol treatment treatment of of bullhead b Translation product of the prevailing catfish. catfish. c Lipovitellin from coho salmon eggs. eggs. C
bullhead, is substantially bullhead, the the messenger messenger RNA RNA for for vigellogenin vigellogenin is substantially smaller smaller than that for any other vertebrate (Table III). 111). Even within the same species, S . gairdneri), gairdneri),aa large large variation variation in species, in in this this case case the the rainbow rainbow trout trout ((8. the the apparent apparent molecular molecular weight weight of of the the vitellogenin vitellogenin is is noticed, noticed, which which may be due to different methodologies used, different degrees of pro proteolytic breakdown, or teolytic breakdown, or dephosphorylation dephosphorylation occurring occurring during during the the isola isolation IV). Some Some degree degree of of heterogeneity heterogeneity in in vitellogenin may may be tion (Table (Table IV). due due to to the the fact fact that that in in the the fishes, fishes, as as in in other other vertebrates vertebrates,, vitellogenin vitellogenin is is not of slightly slightly differ differnot encoded encoded by by aa single single gene, gene, but but rather rather by by aa family family of ent ent genes. genes. As long as the translation of of one isolated vitellogenin messenger (mRNA) in this salmonid fish leads to different molecular-weight RNA (mRNA) estimates estimates for for the the vitellogenin monomer, monomer, more more attention attention will have have to to
5. VITELLOGENESIS AND 5.
365 365
OOCYTE ASSEMBLY
Table IV IV Molecular Weight of Native Vitellogenin and Subunits from gairdneri S. gairdneri Rainbow Trout S. Native molecular mass (kDa) Wa)
342 342 440 440 440 440 470 470
500 500 500 500 535 600
Subunit molecular molecular mass (kDa) (kDa)
170" 170" 200 200 250 250 16-103" 16103c 220
Method
Reference
Ultracentrifugation Ultracentrifugation Gel filtration Gel filtration Gradient PAGE Gradient PAGE poly(A)+ SSDS-PAGE DS-PAGE of poly(A)+ translation product filtration/SDS-PAGE Gel filtration/S DS-PAGE Not stated SDS and gradient PAGE SDS Gel fi ltration/SDS-PAGE filtration/SDS-PAGE
Campbell and Idler (1980)
(1985) Norberg and Haux (1985) Campbell and Idler ((1980) 1980) 1983) Chen ((1983) Valotaire et et al. (1984) (1984)
1981 ) Sumpter ((1981) (1984)b Y. Valotaire (1984)b Maitre et al. (1985a) (1985a) Hara and Hirai (1978)
modifications, polypeptide alone = = 160 160 kDa. "a Polypeptide plus posttranslational modifi cations, polypeptide al. (1984). (1984). b Unpublished observation, cited in Valotaire et al. prec Eleven polypeptides that represent breakdown products from handling or pre cursors.
be devoted to multiple vitellogenin genes, to strain differences within one species, or to possible partial degradation of of this large molecule or its mRNA-which mRNA-which do not critically affect the immunological reactiv reactivity-before ity-before a definitive answer with respect to molecular weight and phosphorylation sites can be given. Estradiol treatment leads to the appearance of a specific high-mo high-molecular-weight species of of messenger RNA (6300 (6300 or 7200 nucleotides) in the rainbow trout ((Chen, Chen, 1983; 1983; Valotaire et al., aI., 1984; 1984; see Table IV). Using cytoplasmic polyadenylated RNA isolated from thes thesee estradiol estradiolexposed trout in a cell-free translation system, Chen (1983) (1983)was able to synthesize a 160,000-Da 160,000-Da polypeptide poIypeptide that was chemically, immuno immunologically, and electrophoretically identical to the authentic vitello vitellogenin monomer. Similar results were obtained for the same species by Valotaire and co-workers ((1984), 1984), although their larger mRNA (7200 (7200 nucleotides) upon translation yielded a considerably larger (200,000 (200,000 Da) polypeptide, which was immunoprecipitable with antibodies Da) against trout serum vitellogenin. The same authors also synthesized DNA complimentary to the estrogen-stimulated mRNA and back-hy back-hybridized with liver RNA to determine the increase in RNA due to estrogen treatment; the treatment increased it by 9%.
366
THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH
F. F. Posttranslational Modifications Modifications The biochemical information concerning vitellogenin clearly indiindi cates that a great deal of posttranslational modification must occur in the liver cell to reach the finished product seen in the serum. serum. First the protein backbone of the vitellogenin is membrane is synthesized on membranebound ribosomes, a feature that it shares with other proteins destined to be secreted from the hepatocyte (Lewis al., 1976). 1976). In subsequent (Lewis et al., steps, the molecule must be lipidated, glycosylated, and phosphoryla phosphorylated. It has been suggested that all these processes occur on the mem membranes of the endoplasmatic reticulum and that they are already initi initiated while the polypeptide chain is being translated (Tata (Tata and Smith, 1979), although this view has been debated by Gottlieb and Wallace 1979), (1982). (1982). Finally, existing "pro" “pro” sequences or signal peptides have to be removed before vitellogenin is packaged into Golgi vesicles and se secreted into the bloodstream. While some information exists concerning the nature and extent of modifications on the vitellogenin molecule, rather limited information is is available for fish with respect to the mechanism, sequential events, or locale of these transformations. Therefore, the following discussion has to be confined to a description of of nonprotein components found on the circulating vitellogenin molecule. Just as do the vitellogenins from other oviparous vertebrates, fish vitellogenins carry a certain number of of phosphate groups, some of of it as protein phosphorus, in a region that in the mature oocytes becomes deposited as phosvitin. Generally, the molecule is phosphorylated on serine moieties, and since the degree of of phosphorylation of delipida delipidated piscine vitellogenins ranges around 0.6-0.7% 0.6-0.7% (by weight) weight) (Le., (i.e., only about 50% of the protein phosphate content in other vertebrates), vertebrates), the serine content must be comparatively lower. lower. Experimentally, this alkaline-labile protein phosphorus, which is specific to naturally or induced vitellogenic animals, has been utilized utilized repeatedly for the determination of the degree of the vitellogenic response in fish. fish. Vitel Vitellogenic female fish contain between 20 and 100 100 JLg pg of of protein phos phosphorus per milliliter of plasma, while untreated males contain less than 5JLg/mi 5pg/ml (Craik and Harvey, 1984). 1984). In spite of the large amounts of protein phosphate moved through the plasma compartment during vitellogenesis, in the unfertilized egg inorganic phosphate, and phos phospholipid, and not protein-bound phosphate, make up the bulk of of the 3 IP]phosphate (Grasdalen nuclear magnetic resonance (NMR) (NMR) visible [[31P]phosphate and JJgrgensen, flSrgensen, 1985). 1985). This observation indicates that additional ma maternal sources must supply phosphate and phospholipids to the 00oo-
5. 5. VITELLOGENESIS VITELLOGENESIS AND AND
OOCYTE OOCYTE ASSEMBLY ASSEMBLY
367
vitellogenin-derived cyte. Also substantial dephosphorylation of vitellogenin-derived phosphoproteins during transmit through the oocyte or following dep depof osition in the yolk may explain the low protein phosphate content of compo(Craik, 1982). mature oocytes (Craik, 1982). The highly charged phosphate compo nent also gives the vitellogenin molecule its high ion-binding capac capacity. Teleost vitellogenins are known to bind ions such as calcium, magnesium, or iron efficiently (Hara, (Hara, 1976; 1976; Hara and Hirai, 1978; 1978; Hara et al., 1980) thus may 1980) and and thus may designate designate an an important important vehicle vehicle for for mineral mineral supply In fact, supply to to the the growing growing oocyte. oocyte. In fact, the the competition competition of of vitellogenin vitellogenin with chelating substances has been used successfully to isolate fish vitellogenins from other plasma proteins (Ng (Ng and Idler, 1983). vitellogenins 1983). In In contrast contrast to to the the phosphate phosphate content content of of fish fish vitellogenins, vitellogenins, which which is is lower lower than than that that of of other other oviparous oviparous vertebrates, vertebrates, the the amounts amounts of of lipid lipid material material carried carried on on the the vitellogenin vitellogenin molecule molecule are are generally generally about about groups. The lipid content of twice as high as for other vertebrate groups. vitellogenin ranges around 20% fishes as different in 20% by weight in fishes lifestyle (21%; Hori et al., lifestyle and feeding preferences as the goldfish (21%; 1979), 1979), rainbow trout (21%; (21%;Wiegand and Idler, 1982; 1982; Fremont Frbmont et al., 1984; 19%; Norberg 1984; 18%, 18%, Norberg Norberg and and Haux, Haux, 1985), 1985), sea-trout sea-trout ((19%; Norberg and and Haux, 1985), 1985), or the elasmobranch dogfish ((18%; 18%; Craik, 1978a). 1978a). The bulk of this lipid material, moiety of bulk of this lipid material, which which later later forms forms the the lipovitellin lipovitellin moiety of the yolk, yolk, can can be classified classified as as polar polar lipid lipid (Hori (Hori et al., 1979). 1979). In In rainbow rainbow trout 82%of of the the trout vitellogenin, vitellogenin, for for instance, instance, polar polar lipids lipids make make up up some some 82% total oocyte, how 1982). Generally Generally the the mature mature oocyte, howtotal (Wiegand (Wiegand and and Idler, Idler, 1982). ever, ever, contains contains much much larger larger percentages percentages of of triglyceride, triglyceride, and and it it is is there therefore fore reasonable to to assume assume that that sources sources other other than than vitellogenin vitellogenin must must supply teryl supply the the oocyte oocyte with with nonpolar nonpolar lipids, lipids, such such as as triglycerides, triglycerides, ssteryl esters, is interesting interesting to esters, sterols, sterols, and and wax wax esters. esters. In In this this context context it it is to note note that that dietary manipulation of free fatty acids in trout is reflected in altered lipids, but altered fatty fatty acid acid composition composition of of serum serum lipids, but not not of of the the lipopro lipoproteins, which are most important during vitellogenesis (Fremont et al., 1984). 1984). While fish vitellogenin vitellogenin is known to contain carbohydrate groups, little of the the little concrete concrete information information on on the the amount, amount, nature, nature, and and linkages linkages of carbohydrates is available. However, it is known that for many pro proteins, of the the teins, successful successful glycosylation glycosylation is is aa prerequisite prerequisite for for excretion excretion of export protein. In other cases, such as the chicken ovalbumin, ovalbumin, which usually usually occurs occurs in in glycosylated glycosylated form, form, no no glycosylation glycosylation is is required required for for excretion. excretion. Experiments Experiments utilizing utilizing tunicamycin, tunicamycin, aa specific specific inhibitor inhibitor of of N-glycosylation, side N-glycosylation, revealed revealed that that the the absence absence of of the the oligosaccharide oligosaccharide side chains chains from from the the ovalbumin ovalbumin molecule had had no no effect effect on on its its secretion secretion (Colman 1981). For comparative comparative purposes purposes and and from from an an evoluevolu(Colman et al., 1981).
368 368
THOMAS P. MOMMSEN AND J. WALSH AND PATRICK J. WALSH
tionary perspective, it would be a rewarding task to determine the group of glycoproteins to which the fish vitellogenins belong and whether successful glycosylation is a prerequisite for excretion from Obviously, there is a large information gap between the hepatocyte. Obviously, the process of glycosylation of the vitellogenin molecule in the liver and the presence of large amounts of of sialoglycoproteins in fish eggs (Inoue (Inoue and Iwasaki, Iwasaki, 1980a,b). 1980a,b). Vitellogenin could be detected in the blood but not in the livers of of estradiol-treated rainbow trout, a result that was first interpreted to indicate that vitellogenin is rapidly secreted following synthesis (van (van Bohemen et al., 1982b). al. (1983), 1982b). However, Nunomora et al. (1983), using the peroxidase-anti peroxidase complex method (immunologically spe peroxidase-antiperoxidase specifi c for vitellogenin), were able to localize Significant cific significant amounts of vitellogenin in livers of of estradiol treated rainbow trout (Salmo (Salmo gairdneri), (0. keta), keta), or charr (Salvelinus leucomaenis). leucomaenis). gairdneri), chum salmon (0. Similarly, Similarly, So and co-workers (1985) (1985) detected cross-reactivity of anti antibodies against salmon (Salmo (Salmo salar) salar) vitellogenin with liver extracts of vitellogenic fish. fish. These results can be reconciled by the fact that van Bohemen et al. al. (1982b) (1982b) assayed for vitellogenin by molecular weight determination on sodium dodecyl sulfate (SDS) (SDS) polyacrylamide gels and thus screened for mature vitellogenin rather than immunoreactive components (see (see below). below). availMore information regarding posttranslational modification is avail able from other vertebrate systems. Recently, Recently, rooster hepatocytes were used to determine the probable sequence of events in hepatic vitellogenesis (Wang (Wang and Williams, 1982). 1982). Precursors (pVTG (pVTG I and pVTG II) 11)for each of of the two types of avian vitellogenin (VTG (VTG I and 11)were found in hepatocytes of of roosters treated with estrogen VTG II) 3H ]serine and pulse-chase experiments. The by pulse-labeling with [[3H]serine molecular weights of the precursors were lower than those of the SDS gel electrophoresis. How Howmature vitellogenins as determined by SDS of these polypeptides by immunological meth methever, further analysis of ods, peptide mapping, and molecular-weight determinations by gel chromatography revealed that the precursors are similar to mature vitellogenin in size and degree of glycosylation, but are not phos phosphorylated. Wang and Williams (1982) (1982) could also show that highly phosphorylated proteins, such as mature avian vitellogenin, will yield erroneously high molecular weights on SDS SDS gels. The very small quantities of phosphorylated vitellogenin inside of these hepatocytes (1982) to suggest that phosphorylation is is rap rapled Wang and Williams Williams (1982) idly followed by secretion. Their determination of vitellogenin molec molec(Wang ular weight by gel chromatography caused the same authors (Wang
5. 5.
VITELLOGENESIS AND AND OOCYTE OOCYTE ASSEMBLY ASSEMBLY VITELLOGENESIS
369
and Williams, 1982) “accepted” molecular weight for the 1982) to revise the "accepted" avian vitellogenin monomer from above 235,000 to 180,000. 180,000. In light of of the obvious controversies about the actual molecular weights of of pispis cine vitellogenins, even within the same species (see Table 111), III), this of multifaceted multifaceted approach represents a fertile area for research on type of fish. In this field, the isolated hepatocyte systems vitellogenesis in fish. would appear to be an excellent, as yet underutilized, experimental tool (Moon (Moon et al., aZ., 1985). 1985).Recently, several laboratories have been able to prove that fish hepatocytes in suspension or in primary culture are highly 1985; Mommsen Mommsen highly responsive responsive to to estrogen estrogen (Bhattacharya (Bhattacharya et al., 1985; 1986), and that hepatocytes isolated from primed fish will and Lazier, 1986), synthesize and excrete large amounts of of vitellogenin in vitro (Hasche (Haschemeyer and Mathews, 1983). 1983). From From the the reviewed reviewed studies studies on on fishes fishes and and other other egg-laying egg-laying verte vertebrates, brates, aa preliminary preliminary picture picture of of the the sequence sequence of of events events implicated implicated in in exogenous synthesized (Table (Table V). V). Unfortu Unfortuexogenous vitellogenesis vitellogenesis can can be synthesized nately, many parts fishes, many parts of of the the scheme scheme require require aa nately, especially especially for for the fishes, major major concerted concerted research research effort effort from from biochemists, biochemists, physiologists, physiologists, and and molecular replace speculation speculation and and add add information information molecular biologists biologists alike to replace on on actual actual mechanisms. mechanisms. The The major major task task would would be be to to successfully successfully utilize utilize the piscine system the vast vast potential potential of of piscine system to to elucidate elucidate and and understand understand the the estrogen with the estrogen receptor receptor mechanism, mechanism, its its interaction interaction with the nuclear nuclear DNA, DNA, and and not not least least the the subsequent subsequent gene gene activation. activation. Further Further challenging challenging topics topics include include the the diverse diverse posttranslational posttranslational modifications modifications of of the the vitel vitellogenin logenin molecule molecule occurring occurring in in the the liver liver cell. cell. Furthermore, Furthermore, the the particu particular lar intracellular intracellular structures structures where where the the individual individual steps steps occur occur have have to to date date eluded eluded identification. identification. Recent of similar similar vitellogenin vitellogenin molecules molecules of of Recent work work on on the the number of Xenopus has has revealed revealed that that the the situation situation is is not not quite quite as as clear-cut clear-cut or or simple simple as as it it first first appeared. appeared. Rather Rather than than being being just just one one protein, protein, coded coded for mRNA, aa whole whole family family of of vitellogenin vitellogenin genes genes is is in in for by by one one type type of mRNA, existence aZ., 1981), 1981), all all of of which which give give rise rise to to slightly slightly differ differexistence (Wahli (Wahli et al., ent ent vitellogenin vitellogenin molecules. molecules. These These in in turn turn supply supply the the growing growing oocyte oocyte with with the the different different building building blocks blocks for for at at least least five five different different types types of of yolk and 2, 2, phosvitin, phosvitin, and and phos phosyolk polypeptides, polypeptides, namely namely lipovitellins lipovitellins 11and vettes 198 1)], which and 22 [nomenclature [nomenclature of of Wiley Wiley and and Wallace Wallace ((198l)l, which in in vettes 11and themselves and 22 can can themselves are are somewhat somewhat heterogeneous. heterogeneous. Lipovitellins Lipovitellins 11 and each each be be resolved resolved into into three three differing differing polypeptide polypeptide components, components, while while dephosphorylated two polypeptide polypeptide bands bands of of different different dephosphorylated phosvitin phosvitin yields yields two molecular SDS electrophoresis. electrophoresis. Phosvettes Phosvettes are are relatively relatively molecular weights weights on on SDS small small phosphorylated components components with single single polypeptide chains. From on the the vitellogenin vitellogenin of of Xenopus, Wiley Wiley and and Wallace Wallace From their their study study on
370
THOMAS P. P. MOMMSEN MOMMSEN AND AND PATRICK PATRICK J. WALSH THOMAS J . WALSH
Table V V Table of Events during Hepatic Synthesis Synthesis of of Vitellogenin" Vitellogenina Suggested Sequence of Nuclear compartment compartment Nuclear of vitellogenin viteIlogenin gene through through binding binding of of receptor-homone receptor-honnone complex complex to to Activation of regions of of the the nuclear nuclear DNA specific regions Transcription and presence presence of of primary primary transcript transcript in the nuclear compartment compartment Transcription Processing of of primary primary transcript transcript Processing Translocation to cytoplasm cytoplasm Translocation reticulum Rough endoplasmatic reticulum Polysome assembly Translation of of vitellogenin viteIlogenin mRNA Translation Processing of of previtellogenin previteIlogenin subunits Processing Phosphorylation at serine residuesb residuesb Phosphorylation Lipidationc Lipidation' reticulum Translocation to smooth endoplasmatic reticulum Smooth endoplasmatic reticulum phosphorylation at serine residuesb residuesb Further phosphorylation Translocation Translocation to Golgi apparatus Golgi apparatus Glycosylation Glycos ylation Mannose N-Acetylglucosamine N-Acetylneuraminic acid, etc. Lipidationc LipidationC Removal of existing signal peptides Dimerization Phosphorylation Phosphorylation at serine residuesd Excretion into systemic circulation circulation Adapted from Tata and Smith (1979), (1979), Wang and Williams (1982), (1982), and Gottlieb and Wallace Wallace (1982). (1982). b b In Xenopus, Xenopus, phosphorylation phosphorylation occurs occurs in the rough rough endoplasmatic endoplasmatic reticulum reticulum and the the smooth smooth endoplasmatic endoplasmatic reticulum only. The exact cellular site covalent attachment of lipid to vitellogenin is still site of the non noncovalent under under debate. debate. d In the the chicken, chicken, vitellogenin is phosphorylated phosphorylated during its time in the the Golgi appa apparatus, followed by rapid excretion from the hepatocyte. •
C
((1981) 1981) came to the conclusion that the whole gamut of yolk yolk proteins is is also that the phos phosderived from multiple vitellogenin molecules and also vettes are are alternate cleavage products products from from homologous homologous regions of vettes different parent vitellogenins copies of vitellogenins.. Using complimentary DNA copies mRNA, Wahli Wahli et al. al. ((1981) that vitello vitelloXenopus vitellogenin mRNA, 1 98 1 ) deduced that genin is is encoded in a family family of at least four expressed genes. genes. A similar similar situation situation appears appears to to exist in the the chicken, chicken, where where three different genes genes of of the the vitellogenin vitellogenin family family are are expressed, expressed, producing different three polypeptide polypeptide chains chains with with molecular molecular weights weights ranging ranging from from three
5. 5.
371 371
VITELLOGENESIS AND OOCYTE ASSEMBLY
Table VI Estradiol-Dependent Lower Vertebrates· Vertebratesa Selected E stradiol-Dependent Genes in Lower Proteins Proteins
Organ
Organism(s) Organism(s)
Effect
Ovalbumin Lysozyme Conalbumin Ovomucoid Avidin
Oviduct
Birds
Induced Induced Induced Induced Induced
Vitellogenin Vitellogenin Albumin
Liver
Oviparous vertebrates Xenopus, Oncorhy Oncorhynchus ?) nchus nerka nerka ((?) (?) Chicken, Chicken, teleosts (?) Birds
Induced
ApoB, Apoll ApoII (VLDL) (VLDL) Vitamin-binding Vitamin-binding proteins Biotin Thiamin Cobalamin Ribofl avin Riboflavin Transferrin Transferrin receptor Estrogen receptor
Teleosts, chicken
Depressed Induced Induced Induced Induced Induced Induced Induced
For structural structural and further biochemical biochemical changes initiated in the hepatocyte under the influence of estradiol, II. References: Muniyappa estradiol, see Table 11. Muniyappa and Adiga (1980), (1980), Leger et 1981), Lazier et ai. (1985), et ai. al. ((1981), et al. (1985), White (1985), (1985), Wolffe et et ai. al. (1985). (1985). •
170,000 190,000. Moreover, 170,000 to to 190,000. Moreover, the the three three subunits subunits possess possess different different degrees degrees of of phosphorylation, phosphorylation, and and subsequently subsequently make make up up the the native native vitellogenin By analogy, vitellogenin dimer dimer (Wang (Wang et al., 1983). 1983). By analogy, aa similar similar multi multigene gene family family can can be expected expected to to code code for for vitellogenin vitellogenin in in the the fishes. fishes. The The observed observed multitude multitude of differing differing egg egg phosphoproteins phosphoproteins further further suggests suggests widespread widespread heterogeneity heterogeneity within within the the vitellogenin vitellogenin molecule. molecule. C.. Other Actions of of Estradiol G
The specific specific action of estradiol estradiol at nuclear level level in in hepatic hepatic tistis The action of at the the nuclear sue is is by by no no means means restricted restricted to to the the activation activation of of the vitellogenin gene, gene, sue the vitellogenin although, at least least in in the fishes, vitellogenin single most most although, at the fishes, vitellogenin constitutes constitutes the the single important de novo of protein. In the chicken, which which has has important de novo synthesis synthesis of protein. In the chicken, attracted most attention this respect, number of other genes genes are are attracted most attention in in this respect, aa number of other activated concomitantly, apoVLDLII-the activated concomitantly, including including those those coding coding for for apoVLDLII-the major lipoproteins) in in laying laying chickens chickens (Deel(Deel major VLDL (very-low-density (very-low-density lipoproteins) ey, 1985)-as well well as as various proteins. A A ey, et al., 1985)-as various vitamin-binding vitamin-binding proteins. preliminary (i.e., constantly constantly growing) growing) list list of genes that that are are directly directly preliminary (i,e., of genes affected by estradiol in various various vertebrates vertebrates is is given given in in Table Table VI. VI . In In aa affected by estradiol in
372 372
THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH
mechanistically unknown fashion, estradiol also induces the multi multitude of ultrastructural changes occuring in the liver of all vertebrates actively undergoing vitellogenesis (cf. (cf. Table II). 11). As As pointed out above, the teleost fishes add another estrogen-induced gene to this growing list, namely, the gene for the nuclear estrogen receptor pro protein. Estradiol also exerts negative effects effects on the synthesis of other ex export proteins, among them the ubiquitous serum albumins. In many vertebrates, estradiol administration leads to a pronounced reduction in the concentration of albumin circulating in blood, an effect that is especially especially apparent apparent in in chronically chronically estradiol-exposed estradiol-exposed male male Xenopus. Xenopus. Experiments conducted by Tata and co-workers co-workers (Wolffe (Wolffe et al., 1985) 1985) led to the conclusion that in this amphibian, two levels of estrogen action action on on albumin synthesis synthesis can can clearly clearly be be distinguished. distinguished. First, First, estra estradiol diol administration administration leads leads to to aa deinduction deinduction of of transcription transcription of of the the two two genes kDa) and more abundant albumin. genes coding coding for for the the larger larger (74 (74 kDa) albumin. Second, Second, it also causes a substantial destabilization of of the messenger RNA for albumin, which is reflected in a decrease in the actual half halflife of the messenger RNA by two-thirds (Wolffe (Wolffe et al., 1985). 1985). The same deinduction of albumin synthesis in the presence of of estradiol can also be observed in vitro using isolated hepatocytes (Wangh, (Wangh, 1982). 1982). In fishes, a similar reduction in the amount of of circulating albumin is apparent in naturally vitello genic sockeye salmon (0. (0. nerka) vitellogenic nerka) during their spawning migration (T. S. MookeIjea, C. French, (T. Mommsen, S. Mookerjea, and C. results). Conversely, in the rooster, estradiol withdrawal unpublished results). results in the destabilization of vitellogenin and apoVLDLII mRNAs, while the stability of of the serum albumin mRNA is not affected (Wisko (Wiskocil et al., 1980). 1980). In addition to inducing the de novo synthesis of of vitellogenin mRNA, estradiol has been shown to accelerate the rate of transcription of of other genes, while not necessarily necessariIy altering the amounts of mRNAs coding for different genes. of mRNAs genes. In all lower vertebrates, estradiol exerts a pronounced lipogenic action on peripheral tissues, while in Xenopus it also enhances the activities of enzymes involved in the hepatic synthesis of lipids (Phil (Phillips and Shapiro, Shapiro, 1981). 1981). It can be speculated that the de novo synthe synthesized lipid will be partly destined for the lipidation of vitellogenin and partly for inclusion in the increased output of VLDL by the liver. To date, only one study has addressed this topic in fishes: female capelin (Mallotus (Mallotus villosus) uillosus) displayed considerably higher total activi activities of of fatty acid-catabolizing acid-catabolizing enzymes than did their male counter counterparts (Henderson et al., 1984). 1984). However, as long as only one part of
5.
VITELLOGENESIS AND QOCYTE OOCYTE ASSEMBLY ASSEMBLY
3 73 373
fatty acid metabolism (either anabolic or catabolic direction) is anaana lyzed, no conclusive statement can be made about the lipid turnover turnover in the respective respective tissues. Ultimately, the ratio of of fluxes in the two net flux, and. and hence determine net directions will influence the actual net import or export. The increased potential in vitellogenic grouper to sup generate cytosolic NADPH furnishes circumstantial evidence supporting of increased hepatic fat synthesis during this period porting the notion of al., 1984). 1984). In light of of the general observation of of increased lipid (Ng et aZ., content in the blood of of vitellogenic fishes (Plack and Pritchard, 1968; 1968; 1 976; Sand et al., 1980), 1980), it can be speculated Petersen and Korsgaard, 1976; that while Iipid lipid turnover is stepped up, net flux is increased in the direction of lipid export from hepatic tissue. Strong lipogenic action of oflipid of Heteropneustes fossifossi estradiol has been reported for SS.. gairdneri and Heteropneustes lis (Haux and Norberg, 1985; 1985; Dasmahapatra and Medda, 1982). 1982). MicroMicro Zis graphs of of vitellogenic livers of heteroclitus contain less of Fundulus heteroclitus lipid depositions than livers from male fish (Selman and Wallace, 1983b), while the livers of cryso 1983b), of two other teleosts (Notemigonus crysoZeucas leu cas and Brachydanio rerio) redo) increased the amounts of of lipid under the influence of of estradiol in a dose-dependent fashion (de (de Vlaming et al., 1977; 1977; Peute et al., 1978). aI., 1978). In the blenny (Zoarces (Zoarces viviparus), uiuiparus),lipid is accumulated in the liver before vitellogenesis is hormonally induced, and subsequently, the vitello lipid is mobilized and can be found in the bloodstream during vitelloof pregnancy-a pregnancy-a genesis. Also, estradiol treatment during the course of nonvitellogenic period in the blenny-leads blenny-Ieads to a a dose-dependent ac accumulation in vitellogenin and a concomitant increase of lipids in the 979). blood (Korsgaard (Korsgaard and Petersen, 11979). In conclusion, two different strategies can be envisaged with re respect to lipid mobilization and estradiol action in different species of fish. The simpler situation exists in fishes that accumulate lipids fish. within the liver, such as the cod or the blenny. Here, estradiol is likely into first cause a mobilization of intrahepatic lipid stores and later in crease the output of VLDL from the liver. In fishes that use extrahe extrahe(Lepto patic sites for lipid deposition, such as salmonids or a sculpin (Leptococcus armatus; armatus; de Vlaming et aI., al., 1984), 1984), estradiol first induces the mobilization of extrahepatic extrahepatic lipids, and perhaps subsequently paces their uptake into the liver leading to increased hepatic output of VLDLs. The treatment of goldfish with salmon gonadotropin leads to an augmentation of plasma triglycerides and cholesterol (Wiegand (Wiegand and 1980)in goldfish with undeveloped ovaries, a phenomenon that Peter, 1980) is most likely mediated by gonadotropin-dependent gonadotropin-dependent estradiol producis
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THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH
tion by the ovary. ovary. In animals undergoing the final stages of of ovarian development, the same treatment decreases plasma lipid concentra concentration, which is possibly due to a gonadotropin-enhanced (progester (progesterone-dependent?) lipid uptake into the ovary. one-dependent?) ovary. Varied results are reported for the changes changes in intracellular glyco glycogen content following estradiol treatment, although the generally ob observed trend seems to support the notion that hepatic glycogen is is decreased in vitellogenic females. However, variable results for the contents of hepatic glycogen can be expected, expected, since of of all storage materials they are the most likely to be dependent on the preexperi preexperimental state of the experimental organism with respect to variables such as diet, photoperiod, and temperature. Vitellogenic females of the killifish F. F . heteroclitus or estrogen estrogeninjected males contained less glycogen in their livers than uninjected uninjected male fish (Selman and Wallace, 1983a). 1983a).A similar picture can be found in many other teleost fishes [H. [ H . fossilis, Dasmahapatra and Medda ((1982); 1982); Z. viviparus, 1979); S. uiuiparus, Korsgaard and Petersen ((1979); S . gairdneri, Haux and Norberg ((1985); 1985); Anguilla anguilla, Olivereau and Olivereau ((1979)], 1979)], where estradiol-primed vitellogenic fish generally contain less glycogen in their livers than do vehicle-injected controls. In the grouper, in contrast, induction of exogenous vitellogenesis leads to a marked increase in hepatic glycogen (Ng (Ng et at., al., 1984). 1984). Sockeye salmon (0 (0nerka) build up maximum liver glycogen levels at the end of the spawning migration, when exogenous vitellogenesis is approaching completion, and the fish subsequently call upon liver glycogen to fuel the exhausting spawning process (French (French et at., al., 1983). 1983). An integral part of of estradiol action is the observed hypercalcemia in vitellogenic fish, fish, which can largely be ascribed to the calcium calciumbinding properties of phosphorylated, and hence highly charged, components of the native vitellogenin molecule. Furthermore, this hypercalcemia has been employed to confirm the vitellogenic state of of experimental animals. animals. Fish scales have been singled out as the sug suggested source of the bound calcium (Mugiya and Watabe, 1977), 1977), while, for once, estradiol does not seem to be implicated in the uptake of environmental calcium, neither through the gills nor through the intestine (Mugiya (Mugiya and IchU, Ichii, 1981). 1981). As in the case of carotenoid bind binding, the actual site for the attachment of calcium to the vitellogenin molecule has not been identified, although liver seems the most likely If in the future it can be confirmed that other metals, such candidate. If as copper or cadmium, travel from their hepatic deposition site to the ovary bound to vitellogenin (Shackley et al., at., 1981), 1981), it will be appreci appreciated how easily heavy metals will be able to impair the fi ne-tuned ion fine-tuned balance of the growing oocyte. oocyte.
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It is interesting to note that that cortisol, a steroid hormone, which is known to exert direct metabolic effects by way of of enzyme induction and permissive effects on peptide hormones such as glucagon, also possesses a pronounced enhancing influence on the estrogen-induced H.. fossilis (Sundararaj (Sundararaj et al., synthesis of vitellogenin in the catfish H 1982a). This situation is somewhat reminiscent reminiscent of of the estrone-depen estrone-depen1982a). of vitellogenesis through estradiol. Glucocorticoid ad addent priming of ministration to cultured hepatocytes curtails the vitellogenic response to estradiol, while at the same time enhancing the production of of al albumin, a protein whose synthesis may be suppressed in the presence sugof estrogen (cf. (cf. Table VI). of VI). Furthermore, recent experiments also sug gest an important role for thyroxine, which is tightly bound by isolated fish liver nuclei (Bres and Eales, 1986), 1986), as an accelerating factor in exogenous vitellogenesis in the guppy (Poecilia reticulata; reticulata; Lam and Loy, Loy, 1985). 1985). Evidently, a number of other hormones interact in an as yet undetermined manner with estradiol during exogenous vitello vitellogenesis (cf., (cf., Leatherland, 1985); 1985); these interactions should provide a endocrinologists and mo momultitude of challenging topics of study for endocrinologists biologists. lecular biologists. H. H. Male Male Fish Fish It vitellogenesis that that the the It is is an an interesting interesting facet facet of of the the induction induction of vitellogenesis estrogenic elicited in in males males of of oviparous oviparous verte verteestrogenic response response can can also also be elicited brates 1979; Korsgaard Korsgaard et al., 1983; 1983; brates,, including including fish fish (Emmersen (Emmersen et al., 1979; Maitre Maitre et al., al., 1985a). 1985a). It It clearly clearly indicates indicates that that the the administration administration of of estradiol estradiol can can activate activate normally normally silent silent genes. genes. The The complete complete absence absence of of products products of of these these unexpressed unexpressed genes genes has has made made male male animals animals aa prime prime model model for for the the analysis analysis of of gene gene regulation regulation and and activation. activation. Basically, Basically, the the male “reprogrammed” to to synthesize synthesize and and export export large large male liver liver can can be "reprogrammed" amounts amounts of of vitellogenin vitellogenin and and other other proteins, proteins, aa process process that that appears appears to to occur occur without without involving involving DNA DNA replication. replication. Since Since an an appropriate appropriate depo deposition vitellogenin in in the sition site site is is lacking lacking in in the the male, male, the the fate fate of of the vitellogenin bloodstream bloodstream differs differs:: it it builds builds up up to to rather rather high high concentrations concentrations and and eventually eventually is is taken taken up up by by the the liver liver and and degraded degraded along along with with other other blood blood proteins. proteins. The The actual actual process process of of vitellogenesis vitellogenesis is is accompanied accompanied by by identical identical patterns patterns of of hepatocyte hepatocyte differentiation differentiation in in both both sexes, sexes, including including the the pro proliferation RNA liferation in in Golgi Golgi vesicles, vesicles, rough rough endoplasmatic endoplasmatic reticulum, reticulum, and and RNA mentioned mentioned (cf. (cf. Table Table II). 11).In In the the male male Atlantic Atlantic salmon salmon (S. ( S . salar), salar), the the estrogenic estrogenic response response also also includes includes an an increase increase in in the the amount amount of of assaya assayable ble nuclear nuclear estrogen estrogen receptor receptor to to levels levels characteristic characteristic of of induced induced fefe-
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THOMAS THOMAS P. P. MOMMSEN MOMMSEN AND AND PATRICK PATRICK JJ.. WALSH WALSH
male fish, fish, which is probably due to de novo synthesis of of the receptor al., 1985). 1985). The identical situation in hepatocytes protein (Lazier et al., from male Xenopus has made it possible to unequivocally identify tranreceptor synthesis as the rate-limiting step in vitellogenin gene tran scription (Perlman (Perlman et ai., al., 1984). 1984). In male fish, fish, vitellogenin synthesis synthesis cannot be stimulated by the administration of pituitary extracts, indicating two specifi c properties specific of the vitellogenic response in fishes: ( 1 ) with regard to exogenous fishes: (1) exogenous horvitellogenesis, the liver is not a direct target organ for pituitary hor mones, and (2) (2) in males, vitellogenesis is specifically dependent on estrogen administration, because of the inability of the gonad to pro produce estrogen (Idler and Campbell, Campbell, 1980). 1980). 1. I. Elasmobranch Fishes
In general, vitellogenesis and its hormonal control in the elasmo elasmobranch fishes have received much less attention than in teleost fishes. The few studies on elasmobranchs suggest that, even in species from temperate zones, vitellogenesis and oviposition appear to occur throughout the year, with a maximum during winter (Sumpter and Dodd, 1979). 1979). As a consequence, vitellogenin is detectable in dogfish 1978b) and skate (Raja (Raja erinacea; erinacea; T. T. P. (Scyliorhinus canicula; Craik, 1978b) Mommsen, unpublished) blood throughout the year, albeit in a low concentration compared with vitellogenic teleosts. The biochemical properties of the elasmobranch vitellogenins and their relationship to vitellogenins from other vertebrates remain to be analyzed. analyzed. Injection of estradiol results in a much smaller vitellogenic re re1978a). While the synthesis of vitello sponse than in teleosts (Craik, (Craik, 1978a). vitellogenin in the female dogfi sh is a slow process compared with teleosts, dogfish its uptake into the ovary is fine-tuned to the rate of its synthesis. synthesis. This results in an unusually long half-life for vitellogenin (9 (9 days; days; Craik, sh plasma, and a similar result can be expected for 197813) in dogfi dogfish 1978b) other elasmobranchs that are vitellogenic throughout the year. The systems where such such long half-lives half-lives for vitellogenin repre repreonly other systems sent the rule rather than the exception are the estrogen-primed males of other vertebrates that possess no tissue that would recognize vitel vitellogenin for uptake. In male, estrogen-injected Xenopus, for instance, vitellogenin is removed from the bloodstream at a rate of less than 11% % per day-which day-which resembles plasma protein turnover-compared turnover-compared to more than 12% 12% per day in the vitello genic female (Wallace vitellogenic (Wallace and Jared, 1968). 1968).
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111. III. OOCYTE ASSEMBLY ASSEMBLY A. Transport of Vitellogenin maAfter the Golgi vesicles of the hepatocyte have unloaded the ma ture vitellogenin into the plasma, the circulatory system delivers it to the ovary. ovary. It appears that vitellogenin is dissolved freely in the plasma, plasma, since since no no special special carrier carrier molecule molecule for for it it could could be be identified identified in in teleosts different situation situation is is found found in in the the blood blood of of teleosts or or amphibians. amphibians. A different birds. birds. In In these these vertebrates, vertebrates, vitellogenin vitellogenin is is carried carried from from its its hepatic hepatic site site of of synthesis to the gonad as part of of the high-density lipoproteins (HDL), (HDL), which are regularly synthesized and excreted by the avian liver.
B. Uptake of Vitellogenin In the females of oviparous vertebrates, with the possible excep excepfishes, tion of elasmobranch fi s hes, circulating vitellogenin is rapidly and specifi cally cleared from specifically from the the bloodstream bloodstream by by the the growing growing oocyte. oocyte. In In vitellogenic Xenopus, some 12% 12% of the vitellogenin circulating in the 1968). In blood is taken up by the gonad per day (Wallace (Wallace and Jared, 1968). specific the absence of a specifi c target tissue in estrogen-primed males, the vitellogenin continues to exist in the circulatory system until it is fi nally removed by the liver and degraded along with other plasma finally proteins. The mechanism of vitellogenin recognition and the selectivity of of its uptake into the oocyte remain open questions, especially for the fishes. fi shes. Here only a single study on the rainbow trout has critically 1979), with looked into into these mechanisms mechanisms (Campbell (Campbell and and Jalabert, Jalabert, 1979), with conclusions that do not support the picture that has emerged from a multitude of studies on Xenopus and the chicken. animals, it appears that vitellogenin In the latter two experimental animals, is bound on the oocyte membrane by specifi c, high-molecular-weight specific, (molecular weight -500,000), receptors (molecular -500,000), which are taken up into the oocyte independent of vitellogenin binding. binding. The oocyte and and turn turn over over independent of vitellogenin The recep recepnonspecificc binding, are saturable, and appar appartor proteins display low nonspecifi ently specifi cally recognize vitelently specifically recognize and and bind bind the the phosvitin phosvitin region region of of the vitel logenin molecule; logenin molecule; again, again, phosphorylation phosphorylation is is crucial crucial to to the the process process of of receptor 1; 1981; receptor recognition recognition and and vitellogenin vitellogenin uptake uptake (Opresko (Opresko et al., 198 Yusko eett al., 1981). 1981). Other studies, in addition, have implicated the
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THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH
importance importance of of N-glycosylation N-glycosylation of of the the vitellogenin vitellogenin molecule molecule on on its its up uptake Similar receptors take by by the the oocyte oocyte (Lane (Lane et al., aZ., 1983). 1983). Similar receptors are are presumed presumed to to exist exist for for VLDL in in avian avian oocytes, oocytes, and and it it has has been been pointed pointed out out else elsewhere where that that the the vitamin-binding vitamin-binding proteins proteins are are only only recognized recognized and and taken taken up up into into the the oocyte oocyte if if adequately adequately phosphorylated phosphorylated (Miller (Miller et al., al., 1982). 1982).After After its its binding binding to to the the oocyte oocyte surface surface receptor, receptor, the the vitellogenin vitellogenin molecule, possibly in conjunction with its receptor, is is taken up into the oocyte by micropinocytosis. In the fishes, vitellogenin contains only about about half the the protein protein phosphorus phosphorus of of other other vertebrates vertebrates,, and and the phosvitins comprise a more heterogeneous group altogether. There Therefore, fore, a critical analysis of the involvement of of phosphate groups in these lower vertebrates may lead to interesting insights into receptor recognition and receptor mechanism in general. In subsequent steps, the vitellogenin is is directed toward different yolk sites sites within the oocyte, depending on the stage during vitello vitellogenesis. While the receptor molecule appears to be recycled, the vi vitellogenin molecule is cleaved proteolytically into the main yolk com components in the course of its translocation from the oocyte surface to the yolk deposition sites. From an enzymatic point of of view, the system responsible for the cleavage of of the vitellogenin molecule is poorly characterized, but the lysosomal system seems to be implicated (see (see below). below). Finally, the components such as the lipovitellins, phosvitins, and phosvettes are deposited within membrane-bound spherical yolk bodies, in many marine teleosts constituting fluid yolk globules rather than the well-known insoluble platelets. Such yolk bodies form the so-called "extravesicular “extravesicular yolk," yolk,” which may fuse at some point during oocyte development (Wallace Selman, 1981). (Wallace and Selman, 1981). The "intravesicular “intravesicular yolks" yolks” that have been described for growing teleost oocytes are supposedly precursors of the cortical alveoli, which shed their endogenously synthesized "yolk" “yolk” at fertilization (te 1977; Wallace and Selman, Heesen, 1977; Selman, 1981). 1981). The discussion in this chapter will be restricted to the egg components derived from exoge exogeautonous vitellogenesis and will therefore not be concerned with the auto synthetic intravesicular yolk as defined above. Considering the rapidity and specificity of of vitellogenin deposition in teleost oocytes in the course of of exogenous vitellogenesis, Campbell and Jalabert (1979) (1979) obtained surprising results results:: developing trout 00oocytes in vitro did not take up vitellogenin selectively over serum al albumin and at a rate that amounted to less than 10% 10% of that observed in (Campbell and Xenopus under comparable experimental conditions (Campbell 1979). Obviously, more research is needed before any gen genJalabert, 1979). eral statements about diversity or conservation in the mechanism of
5. 5.
VITELLOGENESIS VITELLOGENESIS AND AND OOCYTE OOCYTE ASSEMBLY ASSEMBLY
37 379 9
of the vertebrate line can be vitellogenin uptake in the evolution of made. Interestingly, Xenopus oocytes oocytes are selective for vitellogenin vitelloover albumin or ferritin, while the vertebrate source of the vitello genin-which teleost-possessed little influence on the genin-which included a teleost-possessed rate of uptake (Wallace et aZ., 1980). Similarly, al., 1980). Similarly, microinjected vitello vitellogenin mRNAs from different species gave rise to mature vitellogenins and led to subsequent export from the Xenopus oocyte. Amphibian vitellogenin was later taken back up and deposited in the yolk plate platelets. Locust vitellogenin, on the other hand, was synthesized and exex (Lane et aZ., ported but not sequestered from the medium (Lane al., 1983). 1983). With respect to the hormonal regulation of vitellogenin uptake by the oocyte, aa rather scant body of information is available, apart from the fact that estrogen does not seem to be involved. involved. Instead, uptake may be dependent on the presence of progesterone, with its exact mode of action on the surface of of the oocyte and not on the transcrip transcriptional level still being under debate. This steroid may possess some general maturation function or act specifically to induce micropinocy micropinocytosis in vitellogenin (Tata (Tata and Smith, Smith, 1979). 1979). Studies on Xenopus have indicated that once the oocytes have entered into the vitellogenic state, the rate of vitellogenin sequestering is regulated by by the follicle cells and not by the oocyte itself (Wallace, (Wallace, 1983). 1983). There is an ongoing debate on the number of gonadotropins present in fishes, but inde independent of the outcome of this perceived perceived controversy, two statements can be made with respect to exogenous vitellogenesis. One pituitary hormone, rich in carbohydrate, leads estrogen production in the fe female gonad and thus initiates the events outlined in Fig. 11.. Another pituitary hormone, which is characteristically low in carbohydrate content, specifically enhances the uptake of of vitellogenin from the bloodstream into the growing oocyte while at the same time being devoid of vitellogenic action per se (Burzawa-Gerard, (Burzawa-Gerard, 1982). 1982). Only if the oocyte has taken up the vitellogenin by micropinocyto micropinocytosis will the molecule be processed correctly, cleaved at predeter predeteryolk. On the mined sites, and directed toward specific sites in the yolk. other hand, if if microinjected into the oocyte, the vitellogenin molecule is is rapidly degraded in its entirety and degradation products never reach the yolk platelet (in Xenopus; Wallace and Hollinger, 1979). 1979). These findings reconcile the observations made when messenger RNA for vitellogenin vitellogenin is microinjected into growing oocytes during translational or modification studies. In this case, after the mRNA has been translated and the molecule has undergone the required post posttranslational translational modifications, the mature vitellogenin is excreted from the oocyte and subsequently sequestered from the medium by micro-
380 380
THOMAS THOMAS P. P. MOMMSEN MOMMSEN AND AND PATRICK PATRICK JJ.. WALSH WALSH
pinocytosis and only then directed toward the yolk, where it is is stored as phosvitin and lipovitellin (Lane ai., 1983). (Lane et al., 1983). Campbell and Idler ((1976) 1976) found that some degree of dephosphorylation of cine vitello of pis piscine vitellogenin may occur during incorporation into ovarian yolk. yolk. At the later stage of meiotic maturation, many fi sh eggs take up fish up substantial amounts of water, and this hydration may be accompanied by a marked drop in protein phosphorus assayable in the oocyte (Craik, (Craik, 1982). 1982). During the previtellogenic part of of oocyte development in the trout, microvesicular bodies (MVB) (MVB) accumulate and later occupy the larger part of the cell. These bodies contain acid hydrolase activity and can be classified as a lysosomal-like compartment. compartment. In the course of of exogenous vitellogenesis, large yolk vesicles form, form, which contain yolk as well as the remnants of of the microvesicular bodies. At the comple completion of vitellogenesis, the microvesicular bodies have disappeared (Busson-Mabillot, (Busson-Mabillot, 1984) 1984) and as a general observation, observation, acid phospha phosphatases are absent from fully developed oocytes (Korfsmeier, (Korfsmeier, 1980), 1980), while cathepsin and a-glucosidase activities are present in unfertil unfertilized eggs (Vernier Sire, 1977). (Vernier and Sire, 1977). Although lysosomal lysosomal activities oc occur associated with yolk platelets in most lower vertebrates, this rule is not without exception. exception. For example, example, the oocytes oocytes of two species of (herring and plaice) plaice) are alto altomarine fishes with polylecithal egg cells (herring gether devoid of acid hydrolases (Korfsmeier, (Korfsmeier, 1980). 1980). The exact role of the lysosomes lysosomes in the proteolytic cleavage of vitel vitellogenin that has been sequestered from the bloodstream by micro micropinocytosis remains an enigma to date. date. From the informatiCill information that has been gathered from other vertebrate systems, systems, it is is not obvious what type of enzymes are responsible for the breakdown of vitellogenin originate. The observation of efficient, nonspecifi nonspecificc and where they originate. breakdown and subsequent removal of microinjected vitellogenin in the Xenopus oocyte suggests that micropinocytosed vitellogenin is not available for full lysosomal lysosomal attack and may be only exposed to en enavailable zymes that will specifi specifically zymes cally cleave it into lipovitellins, phosvitins, and phosvettes.. Obviously, the vitellogenin molecule itself is not resistant phosvettes attack. On the other hand, it seems seems that to other types of proteolytic attack. the microvesicular bodies bodies transporting vitellogenin and its its products to yolk are somewhat related to the lysosomal lysosomal system, system, since they the yolk display some some enzyme enzyme activities activities with characteristic characteristic acidic max maxclearly display ima. The microvesicular bodies, bodies, however, however, do not display the full com comima. lysosomal enzymes, which would most likely lead to the plement of lysosomal its receptor. receptor. degradation of vitellogenin and its
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381 38 1
C. C. Phosvitin and Lipovitellin of the vitellogenin mole moleThe general picture of cleavage products of noncule in piscine systems is not quite as clear-cut as in the other non mammalian vertebrates. Although it has long been known that fish eggs contain lipovitellins and phosvitins with by and large similar properties to those from other vertebrates, a much more pronounced apparent. Such variability is interspecific variation on the theme is apparent. of different components as well as some somereflected in high numbers of times unusual chromatographic behavior. The most extreme example to date is found in the eggs of an Antarctic fish (Chaenocephalus (Chaenocephalus aceratus) that possesses no less than nine different phosphorylated proteins 1984). Fish Fish lipovitellins lipovitellins are are proteins (Shigeura (Shigeura and and Haschemeyer, Haschemeyer, 1984). much more those from much more heterogeneous heterogeneous than than those from other other vertebrate vertebrate eggs, eggs, are are smaller, smaller, contain contain higher concentrations concentrations of of lipids, lipids, and and possess possess little little or or no protein phosphorus. Numerous low-molecular-weight low-molecular-weight phosvitins have been found in teleost eggs, characterized by widely varying, albeit generally low, low, amounts of alkali-labile protein phosphorus (Mano and Yoshida, 1969; 1969; Markert and Vanstone, 197 1 ; Inoue et al., 1971; (Mano 1971; 1971; de de Vlaming Vlaming et al., 1980; 1980; Craik, Craik, 1982). 1982). In In the the killifish killifish Fundulus the native native vitellogenin vitellogenin molecule molecule (200 (200 kDa; cf. cf. Table Table III) 111) heteroclitus, the cannot efficannot be be localized localized within within the the oocyte, oocyte, indicative indicative of of aa rapid rapid and and effi cient degradation into smaller components (Wallace (Wallace and Selman, 1985) 122, lO3, 45, 26, and kDa) 1985).. In In fact, fact, five five major major protein protein bands bands ((122, 103,45,26, and 20 kDa) of which are allegedly derived are abundant in growing oocytes, all of from from proteolytic proteolytic breakdown breakdown of of vitellogenin. vitellogenin. During During final final maturation, maturation, this 122- and and 45-kDa 45-kDa proteins proteins are are this pattern pattern is further further changed changed in in that that the the 122degraded It has number oflower-molecular-weight of lower-molecular-weight proteins. proteins. It has degraded to to yield yield aa number been implied that new proteins been implied that these these new proteins are are involved involved in in the the hydration hydration process during final 1985), but but their their process during final maturation maturation (Wallace (Wallace and and Selman, Selman, 1985), physiological the physiological function(s) function(s) and and their their relationship relationship to to phosvitin phosvitin or the nature of the proteolytic machinery responsible await identification. identification. D. Oocyte Lipids In the course of exogenous vitellogenesis, teleost oocytes accumu accumulate amounts of lipids in lipids delivered late large large amounts of lipids in addition addition to to the the polar polar lipids delivered as as part of the vitellogenin molecule. In spawned eggs, which contain between 8 and and 32% 32% lipid lipid (based (based on on dry dry weight), weight), several several classes classes of of between lipids are represented, where the emphasis varies strongly between different species of different species of fish. fish. Depending Depending on on the the preferred preferred type type of of lipid lipid
382 382
THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH WALSH
accumulated in the eggs, three strategies can be distinguished: distinguished: the first group, which includes rainbow trout, sole (SoZea (Solea vuZgaris), vulgaris), and a whitefish (Coregonus aZbula), atbula), is characterized by equally high levels of polar lipids and triglycerides (Kaitaranta 1 ; Devau (Kaitarantaand Ackman, Ackman, 198 1981; Devauchelle et aZ., al., 1982). 1982). Baltic herring, roach, and turbot (ScophthaZmus (Scophthalmus maximus) belong'to a second group, which accumulates mainly polar lipids (75-90%) 1 ; Devauchelle et aZ., (75-90%) (Kaitaranta (Kaitaranta and Ackman, 198 1981; al., 1982). 1982).A third group, encompassing a wide variety of species such as the gourami (Trichogaster (Trichogaster cosby; cosby; Sand et aZ" al., 1971), 1971), sea bass (Dicen (Dicen1982), striped bass (Morone trarchus Zabrax; al., 1982), (Morone sax saxlabrax; Devauchelle et aZ., atilis; Eldridge et aZ., jluviatilis), burbot (Lota al., 1983), 1983), perch (Perea (Perca fluviatilis), (Lota Zota; 1), and many others, accumulates lota; Kaitaranta and Ackman, 198 1981), large amounts (>80%) (>80%) of wax and steryl esters in the so-called egg oil globules. globules. In fact, all fish eggs harboring oil globules, which are dis distinct from the yolk or yolk platelets, have been shown to contain substantial amounts of wax and sterol esters (Kaitaranta (Kaitaranta and Ackman, 1981). consists almost 1981). In species such as M. M . saxatilis, the oil globule consists entirely of steryl esters and wax esters (90%) (90%)as well as some some triglycer triglycerides ((lo%), 10%), whereas the small the yolk lipids is small bulk of ofthe is dominated by phospholipids (79%, (79%, Eldridge et aZ., al., 1983). 1983).With wax esters generally belonging in the domain of the marine environment, environment, the above list shows that the occurrence of these compounds in fish eggs is by no means restricted to marine species. species. The physiological advantages of of accumulating large amounts of wax esters in eggs eggs (71% (71% of the total caloric value of the egg in M. M . saxatiZis; saxatilis; Eldridge et aZ., al., 1983) 1983)have not been analyzed yet, although it can be hypothesized that in addition to serving as an energy supply, supply, they will play an important role in buoy buoyancy control for the embryo and developing developing larva. exists in our knowledge with respect to Unfortunately, a large gap exists the maternal source of these wax and steryl esters. It appears that the glulipid components of vitellogenins from species accumulating oil glu bules in their eggs have not been given any attention to date. Vitello VitellobuIes fishes is known to transport some 20% (by (by weight) weight) of genin from other fishes some 20% (Wiegand, 1982; 1982; lipid, the bulk of which consists of phospholipids (Wiegand, Haux, 1985). 1985). If this is is verified verified for vitellogenins of fishes fishes Norberg and Haux, eggs, vitellogenin can be ruled ruled out that synthesize oil globules in their eggs, as the transport form form for their unique unique lipid complement. complement. Alternatively, as wax esters esters may be synthesized endogenously in the the oocyte from the wax acids delivered as part of lipoproteins or bound to serum al alfatty acids T . cosby, cosby, where wax esters esters constitute the bumins. In the gourami T. lipids of the egg, egg, the ovarian fatty acyl acyl alcohols alcohols can be synthemajor lipids
5. 5.
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sized de de novo novo from from dietary dietary acetate acetate or or longer longer dietary dietary carbon carbon chains, chains, but but 1971). the site of biosynthesis could not be identified (Sand et al., 1971). Since Wiegand and Idler ((1982) 1982) reported that the ovary of the rainbow trout possesses the metabolic machinery to reduce exogenously ad administered fatty acids to the corresponding alcohols, the endogenous excluded. Another interesting facet synthesis of wax esters cannot be excluded. of fish fish accumulating wax esters in their eggs is the fact that none of the adults of these species use wax esters as their lipid storage forms, forms, but generally rely on triglycerides instead. Comparable Comparable to to the the situation situation in in avian avian systems, systems, some some experimental experimental evidence evidence for for rainbow trout trout suggests suggests that that circulating circulating lipoproteins lipoproteins may may enter and serve the major major source source of of polyunsaturated polyunsaturated free free enter the the ovary ovary and serve as the fatty fatty acids, acids, the the bulk bulk of of which which is is transported transported in in lipoproteins lipoproteins and and not not on on the (Fremont et al., al., 1984). 1984). The experimentally experimentally the vitellogenin vitellogenin molecule molecule (Fremont induced fishes is induced or or naturally naturally occurring occurring vitellogenesis vitellogenesis in in fishes is accompanied accompanied by large increases in liver biosynthesis and export of of VLDL (cf. (cf. Ta Tables II and and VI). bles I1 VI). Just Just as as in in the the hepatic hepatic synthesis synthesis of of vitellogenin, vitellogenin, the the synthesis initiated in synthesis of of lipoproteins lipoproteins may may be be initiated in vivo vivo through through the the influ influence ence of of circulating circulating estradiol. estradiol. In In the the annual annual cycle cycle of of fishes, fishes, increases increases in in blood blood VLDL VLDL are are positively positively correlated correlated with with vitellogenesis. vitellogenesis. Comparative data on avian vitellogenesis and VLDL metabolism indicate that the ovary is capable of of the uptake of of lipoproteins directly from independent of of the the from the the bloodstream bloodstream and and that that this this process may may be independent sequestration vitellogenin through through micropinocytosis. micropinocytosis. While this sequestration of vitellogenin lipoproteins, it it has has general scheme scheme may not not be be applicable applicable to to all serum lipoproteins, been proven to hold for VLDL in the hen (Holdsworth at., 1974), 1974), (Holdsworth et al., where the basal lamina appears to be permeable to circulating VLDL al., 1979). 1979). (Evans et at., (Evans While Wiegand and Idler ((1982) 1 982) determined some capacity for endogenous in the endogenous triglyceride triglyceride biosynthesis biosynthesis from from acetate acetate in in the the ovary ovary in the rainbow trout, Leger and 1981) on of Leger and co-workers co-workers ((1981) on the the same same rainbow trout, the the results results of species suggest serum lipoproteins such as VLDL or LDL as the more likely sources for the triglycerides accumulated in the egg. Lipid of phospholipids, phospholipids, is is first first Lipid material, material, composed composed to to aa large large extent extent of accumulated accumulated in in the the perinuclear perinuclear cytoplasm cytoplasm of of the the oocyte. oocyte. However, However, these bodies, the these early early lipid lipid bodies, the source source of of which which still still awaits awaits identification, identification, do yolk since known to do not not constitute constitute true true yolk since they they are are known to disappear disappear before before or or during during exogenous exogenous vitellogenesis. vitellogenesis. The The study study of of Wiegand Wiegand and and Idler Idler ((1982), 1 982), which which showed showed for for an an in in vitro vitro system system that that labeled labeled acetate acetate was was incorporated incorporated into into ovarian ovarian polar polar lipids, lipids, remained remained inconclusive inconclusive with with regard cell fraction lipid was regard to to the the cell fraction with with which which polar polar lipid was associated. associated.
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E. Carotenoids Carotenoids Fish Fish eggs eggs are are known known to to contain contain other other secondary secondary products products such such as as carotenoids, carotenoids, which which sometimes sometimes contribute contribute to to the the colorful colorful appearance appearance of of the (0.keta), keta),almost almost 1% 1%of of the the fresh fresh weight weight the eggs. eggs. In In the the chum chum salmon salmon (0. of of the the spawned spawned egg egg consists of of carotenoids, carotenoids, mainly mainly astaxanthin astaxanthin (Kita (Kitahara, 1984). In this many other the egg egg carot 1984). In this species, species, as as in in many other salmonids, salmonids, the carothara, enoids enoids are are presumably presumably derived derived from from previous previous depositions depositions in in the the mus muscle 1970). In In view view of of the the absence absence of of aa specific specific transporting transporting cle (Crozier, (Crozier, 1970). vehicle, carotenoids are hypothesized that that the the carotenoids are passively passively vehicle, it it can can be hypothesized transported transported out out of of the the tissue tissue together together with with storage storage lipids lipids according according to to their In the the course the their lipid lipid solubility. solubility. In course of of the the spawning spawning migration, migration, the lipid lipid deposits deposits within within the salmon's salmon’s body are are mobilized mobilized in their their en entirety, tirety, partly partly for for energy energy production production during during migration migration and and in in the the female female also Depending on also as as part part of of the the estrogenic estrogenic response. response. Depending on the the composition composition of the individual lipids that the carotenoids are associated with, differ differing envisaged for for their their transport transport into into the the gonad gonad of of the the ing routes can can be envisaged vitellogenic female: female: the carotenoids may form part of the vitellogenin molecule itself or, alternatively, they may travel with the abundant lipoproteins, Rolipoproteins, especially especially VLDL, in in salmon salmon blood (cf., (cf., Skinner and and Ro gie, Sire and puri gie, 1978; 1978; Sire and Vernier, Vernier, 1983). 1983). The light light pink pink hue hue of of highly highly purified sockeye salmon salmon vitellogenin suggests that least some some of fied sockeye vitellogenin suggests that at at least of the the carotenoids travel to the ovary bound to the lipid component of vitel vitellogenin (T. (T. P. Mommsen and C. C. J. French, unpublished). In fact, it has been reported that a crustacean lipovitellin moiety possesses a cova covaaZ., 1983). 1983). Obviously, Obviously, lent binding binding site for for carotenoids carotenoids (Zagalsky (Zagalsky et ai., more cations of more research research on on posttranslational posttranslational modifi modifications of the the vitellogenin vitellogenin polypeptide polypeptide and and on on possible possible association association of of carotenoids carotenoids with with other other li lipophilic components of the fish blood is required before even a pre preliminary picture will emerge. emerge. Another Another interesting facet of the carotenoid deposition deposition in in the the oocyte oocyte is is the the fact that not all of the carotenoids are are localized localized in in the the yolk, yolk, but but some 20% 20%is associated with other structures in in the the oocyte oocyte (Kitahara, (Kitahara, some 1984) 1984) leaving the question of the physiological function function of of such such aa heterologous heterologous group as carotenoids in embryo embryo nutrition nutrition and and survival survival wide wide open. open. F. F. Glycoproteins In addition to their ubiquitous glycogen stores, fish eggs are protein-bound carbohydrate moieties, but their ex exknown to contain protein-bound act localization and their biochemical nature have only been given
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attention. Even more surprisingly, surprisingly, the physiological rather cursory attention. function of these carbohydrate-containing proteins, which have lately been been charaterized as sialoglycoproteins, is is entirely unknown, despite abunthe fact that these compounds may surpass the egg phosvitins in abun (Inoue and Iwasaki, 1980a,b). 1980a,b). dance by almost an order of magnitude (Inoue fish Sialoglycoproteins are apparently rather common constituents of fi sh eggs, eggs, since they have been described and partially characterized for a (Ctupea pallasii), pallasii), Alaskan number of species, namely Pacific Pacific herring (Clupea poIlack (Theragra (Theragru chalcogramma), chulcogramma), Japanese common charr (Salve (Sahepollack (Salmo gairdneri), gairdneri), and three spe speleucophaenus), rainbow trout (Salmo linus leucophaenus), 0. masou, and O. 0 . nerka) nerka) cies of Pacific salmon (Oncorhynchus keta, O. (Inoue and Iwasaki 1978, 1978, 1980a,b; 1980a,b; Iwasaki and Inoue, 1985). 1985). (Inoue sialoglycoproteins are associated with the soluble fraction of The sialoglycoproteins the egg, namely, the cortical vesicles, and thus do not form part of the purified demembrane superstructure of the egg. egg. The purifi ed molecules are de void of phosphorus and derive their acidity from the abundant sialic, acids. In fact, fact, these three strongly acidic com comglutamic, and aspartic acids. 20% (by weight) weight) of the sialoglycoproteins ponents make up more than 20% (Inoue and Iwasaki, 1980a). 1980a). In this species, the in the herring egg (Inoue molecular weights of the three main sialoglycoproteins range from 40-50% of 8800 to 13,000. 13,000. Their protein backbone, comprising some 40-50% the molecules, is unique in its amino acid composition and displays contrast, is very little variability. The carbohydrate composition, in contrast, variable, especially in the content of N-acetylglucosamine, constitut constituting 6.2% of the total weight of the sialoglycopro sialoglycoproing between 12.5 12.5 and 6.2% N-acetylteins. Other abundant carbohydrates, in addition to N-acetyl neuraminic acid (sialic (sialic acid) acid) and N-acetylglucosamine, are neurammIC N-acetylgalactosamine, fucose, galactose, and mannose. co-workers (Iwasaki (Iwasaki and Inoue, Inoue, More recent work by Inoue and co-workers 1985) on polysialoglycoproteins isolated from un un1985; Inoue et al., 1985) 1985; summafertilized egg from different species species of salmonid fishes fishes can be summa rized as follows: follows: l1.. The The glycoproteins glycoproteins are are characterized characterized by by
high high molecular molecular kDa. 150 to 300 kDa. weights, ranging from 150 2. 2. More More than than 50% 50% of of their their weight weight is is comprised comprised of sialic sialic acid, acid, and and total carbohydrate content may reach 85%. 85%. 3. 3. They They contain contain poly(oligo)sialyl poly(o1igo)sialyl groups groups linked linked to to O-glycosidic O-glycosidic carbohydrate carbohydrate units. units. 4. The polypeptide polypeptide backbone backbone is is made made up up of of seven seven acidic acidic or or neu neu4. tral tral amino amino acids acids only, only, namely, namely, alanine, alanine, aspartatic aspartatic acid, acid, glu glutamic acid, glycine, proline, threonine, and serine. serine.
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THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH
5. 5. Amino Amino acids acids are are arranged arranged in in two kinds kinds of of repeated repeated polypeptide polypeptide 13 amino sequences ((13 sequences amino acid acid residues). residues). 6. 6. All of of the the threonine threonine and and half half of of the the serine serine residues residues are are glycosy glycosylated. lated.
It It is interesting to to note note that that the the accumulated accumulated polysialoglycopro polysialoglycoproteins teins in in the the rainbow-trout rainbow-trout egg egg undergo undergo aa drastic drastic reduction reduction in in size upon upon fertilization, fertilization, at at which which time time they they decrease decrease from from 260 to to 9 kDa kDa (Inoue (Inoue et ai., 1985). 1985). A similar al., similar reduction reduction in in size size can can be be expected expected for for the the large large glycoproteins prevalent in is glycoproteins prevalent in other other salmonid salmonid fishes, fishes, and and the the situation situation is somewhat reminiscent of the further breakup of vitellogenin break breakdown products into even smaller units during final oocyte maturation in in F. heteroclitus (Wallace (Wallace and and Selman, Selman, 1985). 1985). In both both cases, cases, the the dras drastic decreases in size are due to highly specific proteolytic attack, and the carbohydrate moieties on the polysialoglycoproteins remain unal unaltered. In the case of of the glycoproteins, the drastic reduction in size occurs occurs simultaneously simultaneously with with cortical cortical vesicle vesicle breakdown breakdown and and exocytosis exocytosis (Inoue (Inoue et ai., al., 1985). 1985). Unfortunately, Unfortunately, despite despite the the fact fact that that the the sialoglycoproteins sialoglycoproteins com compounds are are prevalent prevalent in in fish eggs eggs and and that that the the timing of of their their break breakdown implies some involvement upon fertilization (block to poly polyspermy?), spermy?), no no data data exist exist on on such important important aspects aspects as as their their physiological physiological function, function, their their source, transport transport form form or or mechanism, mechanism, and and timing of uptake into the developing developing oocyte. oocyte. If If these multitudes of carbohydrates carbohydrates are synthesized in the maternal liver as part of of the posttranslational cation of posttranslational modifi modification of the the vitellogenin vitellogenin molecule, molecule, the the codes codes for for the the small, small, but but unique, unique, polypeptide polypeptide chains chains should be be identifiable identifiable with with relative relative ease ease within within the the recently recently purified purified vitellogenin vitellogenin messenger messenger RNA (Chen, (Chen, 1983; 1983; Valotaire Valotaire et ai., al., 1984). 1984). Biochemical and histochemical studies have identified lectins as an integral part of the soluble fraction of fish oocytes (Nosek et al., 1983). 1983). However, However, just just as as in in the the case case of of the the sialoglycoproteins sialoglycoproteins found found in in mature eggs, sources or physiological function is a matter of of specula speculation (Nosek (Nosek et ai., al., 1983). 1983). G. Vitamin-Binding Proteins As mentioned before for the chicken, estradiol induces the hepatic synthesis of a number of vitamin-binding proteins destined for uptake into the growing oocyte (Table (Table IV). IV). One of these vitamin-binding proteins is the well-characterized riboflavin-binding protein, which is glycosylated as well as phosphorylated and is responsible for the
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carbohytransport of riboflavin to the oocyte. While in many instances carbohy drate side chains are important in the recognition of glycoproteins by their target cells, it was recently demonstrated that, in the case of the glycosylariboflavin-binding protein, correct phosphorylation and not glycosyla tion is crucial for the uptake of the molecule into the oocyte, as was shown for the uptake of experimentally administered phosvitin (Miller et al., al., 1982). 1982). For the fishes, however, information on occur occur(Miller rence of such vitamin-binding proteins is limited to the observation (0. that the specific riboflavin-binding protein is absent from salmon (0. nerka) oocytes. Riboflavin, Riboflavin, on the other hand, occurs in salmon 00oonerka) (H. B. cytes in similar concentrations as in the chicken egg (H. B. White and M. of its source and its M. A. Letavic, unpublished), leaving the question of possible transport open to speculation. Vitamin-binding proteins can be expected to play an integral role in the survival of embryos or larvae, supplying them with vitamins at poscritical stages of their development. Such proteins may further pos unsess antimicrobial action by rendering vitamins stored in the egg un available to infesting bacteria. H. H. Hormones While it has been known for some time that fish larvae respond to exogenously administered hormones hormones,, the physiological relevance of such observations remained unclear, especially at a time when the intraembryonic existence and availability of such hormones had not been established. In the context of the hormonal status of fish oocytes, an avian concept may deserve attention by researchers interested in embryonic fish metabolism and morphogenesis. In addition to known nutrients and secondary compounds, the chicken egg contains signifi significant amounts of thyroxin, and the embryonic chick liver already dis displays highly specific specific steriod receptor activities activities for hormones such as 17,8-estradiol ) )(Bella 17p-estradiol (Lazier, (Lazier, 1978) 1978) and 3,5,3'-triiodothyronine 3,5,3’-triiodothyronine (T (T33 (Bellabarba and Lehoux, 11981). 981). Recent analyses by 1987) and Kobuke 1987) by Brown et al. al. ((1987) Kobuke et al. al. ((1987) unequivocally unequivocally established the presence of substantial amounts of thy thyroxin and T T33 in unfertilized ova and embryos embryos of salmonids salmonids (Oncorhy (Oncorhynchus sp.) s p . ) and striped bass bass (M. ( M . saxatilis). saxatilis). These hormones hormones,, which are localized preferentially in the embryonic yolk, yolk, are apparently of ma maternal origin. The The suggested route of transfer from from the maternal circu circulatory latory system into the growing oocyte is is through vitellogenin, since since this compound compound displays appreciable binding capacity for thyroid horhor-
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THOMAS THOMAS P. P. MOMMSEN MOMMSEN AND AND PATRICK PATRICK JJ.. WALSH WALSH
(0. ki mones in in the the plasma collected collected from from vitellogenic vitellogenic coho coho salmon (0. kiC. V. Sullivan, unpublished sutch; A. A. Hara, W. W. Dickhoff, and C. results). results). The absolute amount of hormone transferred into the ova, (in the range of 5 ng/oocyte, Brown et al., 1987; however, is small (in 1987; Kobuke Kobuke et al., 1987), 1987),and it is therefore unlikely that such transfer will be reflected in concentration changes of hormone in the maternal circulatory system. While the presence of a hormone does not necessarily imply its physical availability or functionality, 1987) functionality, the data of Brown et al. ((1987) and Kobuke et al. (1987) (1987) already indicate that yolk thyroxin and T T3 3 undergo turnover during early development. development. Thus both hormones can be assumed to be available to the embryo and to infl uence physiologi influence physiological functions. Considering how many other lipophilic compounds from the maternal system reach the growing oocyte, the deposition of of thyroxin is not surprising, and the same principle is likely to be appli applicable to other steriod hormones. hormones. However, as the present discussion reveals (cf., (cf., III,F, G, and I), I), similar arguments can also also be made for the potential transfer of peptide hormones (insulin, (insulin, glucagon, etc.) etc.) from the maternal system into the oocyte. oocyte. Two important conclusions conclusions can can be drawn from these novel findings findings:: 1. 1.The fact that thyroxin and T3 T3 are present in the growing oocyte oocyte and undergo undergo changes changes during oocyte development long before a functional hypothalamo-adenohy hypothalamo-adenohypophysial-thyroid axis axis is established, established, implies that these hormones hormonesyet unidentified-exert unidentified-exert physiological roles during early larval mor morphogenesis and 2. If, If, as it seems seems possible, hormone stores supplied by the maternal system are a common feature in fi sh eggs, fish eggs, an entirely new window on the endocrinology and physiology of developing fish has been opened. opened.
Yolk-DNA I. Yolk-DNA exclusive, route for the uptake of vitellogenin The main, if not exclusive, oocyte is micropinocytosis (Brummett (Brummett from the blood into the growing oocyte and Dumont, 1977). 1977). As pointed out elsewhere in this review, the and fate of the vitellogenin molecule inside inside of the oocyte oocyte is is subsequent fate yolk. cleavage into different components which are later stored in the yolk. However, it can can be hypothesized that the the uptake of the large molecule However, may not entirely exclude exclude smaller, smaller, vitellogenin by micropinocytosis may blood-borne molecules, such as as sugars, sugars, lipids, lipids, plasma proteins, or blood-borne even DNA. DNA. To To exemplify this phenomenon, phenomenon, a short short comparative comparative ex exeven cursion to amphibian systems systems is required, since to date no data on cursion similar phenomena phenomena have have been analyzed for fishes. fishes. similar
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of the oocyte chromatin, the yolk platelets In addition to the DNA of of the amphibian Xenopus laevis Iaevis contain yolk-bound DNA. DNA. This yolkyolk of DNA is found to be double-stranded, and characterized by a high molecular weight (Hanocq et al., al., 1972), 1972), but its actual concentration is small at about 20 ng per oocyte, compared with some 280 jLg pg vitello vitellogenin derived protein contained in the fully grown oocyte. When experimentally exposed to Xenopus, bovine, or bacterial DNA, iso isolated vitellogenic oocytes of of Xenopus sequestered it from the incuba incubation medium, and the DNA was later found to be associated with the yolk platelets (Opresko (Opresko et al., 1979). 1979). However, there was no discrimi discrimination in uptake rates for DNA from the different sources, and further furthermore yolk-DNA yolk-DNA was determined to undergo relatively rapid turn turnover. This circumstantial evidence suggests that in this particular amphibian system, the DNA associated with the yolk is not involved in information transfer during the embryonic development. On the other hand, the indisciminate uptake of DNA from the maternal bloodstream, which has been shown to contain small small (25 (25 jLg pg ml-1) ml-l) amounts of of DNA (Opresko (Opresko et al., 1979), 1979),presents a good example of of an adventitious uptake of maternal blood components into the growing oocyte, which is is solely a byproduct of the mode of vitellogenin uptake by micropinocytosis. It may also also help to explain the otherwise surpris surprising presence of other components of maternal plasma or their deriva derivatives in growing oocytes. J. M etabolism Metabolism In addition to the uptake and and processing of vitellogenin and other blood-borne proteins such as as VLDL, the growing fish fish oocyte synthe synthesizes sizes and and accumulates accumulates aa number number of of high-molecular-weight high-molecular-weight compo components. First, First, the oocyte displays displays aa whole complement of RNA (some (some 10 lo44 more than in somatic cells), cells), mainly rRNA (95%), (95%),mRNA (2-3%), (values (values for Xenopus) Xenopus) and tRNA, tRNA, including an oocyte-specific oocyte-specific 5-S 5-S RNA (Denis (Denis and Ie le Maire, 1983), 1983),which are likely to be of importance in early embryonic development. development. Second, Second, the oocyte oocyte can can perform pro protein tein biosynthesis as as well well as as aa multitude of of posttranslational modifica modifications, specifi cally glycosylation, specifically glycosylation, phosphorylation, and lipidation. In the the course course of of their their development, development, Xenopus oocytes oocytes increase increase their their bio biosynthetic synthetic activity by more than 100-fold, 100-fold,from from 4.3 4.3ng protein per day in stage stage 11 oocytes oocytes to over 0.5 0.5 jLg p g per day day in stage 66 oocytes oocytes (Taylor (Taylor and Smith, Smith, 1985). 1985). Each of the mentioned activities activities requires specific sub subsets sets of enzymes. enzymes. This high biosynthetic potential made made the Xenopus oocyte oocyte the system system of choice choice to study translation and posttranslational posttranslational
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THOMAS THOMAS P. MOMMSEN AND PATRICK JJ.. WALSH
modification of microinjected messenger RNAs from a variety of of ani animal sources, including insect mRNAs (Lane 1983; Soreq, Soreq, 1985). ul., 1983; 1985). (Lane et al., As the the example example with with microinjected microinjected vitellogenin vitellogenin shows shows (see (see above), above), the oocyte is also capable of totally degrading "foreign" “foreign” proteins. On account of these results and some histochemical studies, the oocyte en usually gets credited with a limited complement of lysosomal enzymes that are supposedly also involved in the breakdown of of vitello vitellogenin into phosvitin, etc. Furthermore, adventitiously sequestered yolk-DNA yolk-DNA has been shown to undergo turnover (Opresko (Opresko et al., 1979), 1979), again a metabolic activity that demands a specific set of en enzymes. zymes. Other metabolic activities of the growing teleost oocyte apparently include the synthesis of urea, which is absent in most adult teleosts, and results in oocyte urea concentrations surpassing those of the ma maternal system by two- to fivefold (Depeche (DBpGche et al., ul., 1979). 1979). All of these actions, as well as acid-base acid-base regulation and the active vesicle transport through the cell, require energy in the form of ATP. ATP. The ATP must somehow be generated inside of the oocyte, oocyte, since it is unlikely that it is furnished by the follicle cells cells.. Unfortunately, the questions concerning the energy supply and preferred substrates for the growing oocyte have yet to be investigated, particualarly for the fishes. fishes. This is a deplorable situation, especially since the answers to these questions may have particular relevance to the early survival of of the fish embryos and larvae. larvae. Even before exogenous vitellogenesis has been initiated, the 00oocytes of Misgurnus fossilis augment their contents of of metabolic en enzymes, specifically those involved in glycolysis, glycolysis, the pentose shunt, and gluconeogenesis. During the entire course of oocyte develop development, glucose sequestered from the maternal circulation serves as an important energy source and also supplies the building blocks for accumulating glycogen. In fact, the activity of one of the key enzymes in this pathway, glycogen synthetase, increases 100-fold 100-fold during vitel vitellogenesis (Yurowitzky 1975). Following maturation, the (Yurowitzky and Milman, 1975). Misgurnus oocyte completely loses hexokinase activity and with it the ability to use exogenously administered glucose. glucose. At the same time, the switch from exogenous to endogenous energy use, at least as far as carbohydrate metabolism is concerned, is reflected in alterations of the enzymes regulating glycogen synthesis and its degradation. The moment that hexokinase is lost from the oocyte, glycogen synthetase activity decreases by half, while glycogen phosphorylase activity in increases by an order of magnitude (Yurowitzky (Yurowitzky and Milman, 1972). 1972). It can can be be concluded concluded from from the the presence presence and and high high activities activities of of enzymes enzymes
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mainvolved in glucose catabolism that during oocyte development ma ternal glucose may constitute one of the major energy sources for the different ATP-demanding reactions. It would also be interesting to confirm whether vitellogenesis might be correlated with increased glucose output from the maternal liver, as the decrease in hepatic glycogen exact extent extent of of its its importance importance is is not not clear clear yet, yet, glycogen suggests. suggests. The exact mainly because data for enzymes involved in other pathways, such as fatty acid utilization, are lacking. It should be recalled that during exogenous vitellogenesis, the availability of of lipid material through increased VLDL output by the liver is stepped up, as is lipid turnover in general. Once the oocyte has been matabolically "cut “cut off' off’ from the hexmaternal continuum of energy supplies, as the disappearance of hex metabolically distinct, okinase from the oocyte suggests, suggests, it exists as a metabolically and closed, unit, which from this point on has to rely on accumulated substances glycogen will will serve substances for for survival. survival. It It can can be speculated speculated that that glycogen serve of the relative ease as the first supplier of metabolic energy, because of with which it can be mobilized. Considering their overall bulk and their caloric contents, yolk lipids will be of overwhelming importance during ensueing parts of of embryonic and larval development, while the accumulated amounts of protein and amino acids are most likely to be funneled into anabolic and not ATP-delivering pathways. IV. EPILOGUE
The processes of hepatic vitellogenin synthesis and yolk-compo yolk-component deposition in fishes represent nent deposition in the the oocyte oocyte in in the the fishes represent aa wide-open wide-open field for fields. From for researchers researchers in in aa variety variety of of fields. From comparative comparative and and evolution evolutionary pis cine systems ary viewpoints, viewpoints, studies studies on on piscine systems are are likely likely to to supply supply valu valuable able insights insights into into hepatic hepatic steriod steriod receptor receptor mechanisms, mechanisms, estrogen estrogen in interactions genes, and mechanisms of multitude of genes, and mechanisms of teractions with with a multitude posttranslational posttranslational modifications, modifications, as as well well as as into into the the nature nature of of hormone hormone interactions level. On interactions on on the the receptor receptor and and gene gene level. On the the level level of of the the oocyte, oocyte, central topics will include the vitellogenin receptor mechanism, the regulation regulation and and control control of of the the enzymatic enzymatic machinery machinery involved involved in in the limited breakdown of vitellogenin, and the sources-maternal sources-maternal or in internal-of sialoglyco ternal-of such differing compounds as wax esters, lectins, sialoglycoproteins, to proteins, or or hormones, hormones, or or vitamins. vitamins. In In each each case, case, the the fishes seem to present the experimenter with a variety of species ideally suited for ease with with which which massive massive the individual individual topic, topic, not not least least because because of the ease vitellogenesis 7,8-estradiol. vitellogenesis can can be induced induced by by the the administration administration of of 117P-estradiol. The with respect respect The apparent apparent variability variability among among different different species of of fish with
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to each each theme theme will will help help to to prevent prevent the the emergence emergence of of generalizing generalizing to statements from from the the study study of ofjust just one one species. species. This This approach approach is is unforunfor statements tunately prevalent prevalent in in the the literature literature on on other other vertebrates, vertebrates, where where one one tunately toad (Xenopus (Xenopus laevis) laevis) represents represents all all amphibians, amphibians, or or the the chicken chicken porpor toad trays the birds. birds. trays the Finally, it is imperative imperative to described biochemical biochemical events events into into Finally, it is to put put described context of of the the life life history history of of fishes. fishes. In In contrast contrast to to most most other other eggegg the context laying vertebrates, vertebrates, the the fishes are are known known to to invest invest large large amounts amounts of of laying their body body reserves reserves into into the the production production of of gonadal gonadal products. products. The The most most their extreme examples examples of of the striking metabolic metabolic effort effort exerted exerted by fishes fishes extreme the striking during the the time leading up up to to the the spawning spawning period period are are some some anadroanadro during time leading Pacific salmons (Oncorhynchus spp.) or the mous fishes such as the Pacific American shad shad ((Alosa (Idler and and Clemens, Clemens, 1959; 1959; Glebe Glebe A h a sapidissima) (Idler American and Leggett, Leggett, 1981). 1981). and It can be appreciated appreciated that that only only an an unperturbed unperturbed sequence sequence of the It can of the outlined events in the maternal system will lead to mature oocytes development. The with an optimized starting point for embryonic development. fine-tuning of of the the orchestrated orchestrated maternal events leading leading to to mature mature oo00fine-tuning maternal events cytes makes makes it necessary to to consider consider potential potential points points of of environmental environmental cytes it necessary interference. While potentially potentially interfering interfering infl uences range range from from interference. While influences acid-base disturbances disturbances (Tam (Tam et al., al., 1987) 1987) and and thermal thermal pollution pollution to acid-base anything that will invoke stress reactions reactions in in fish, fish, the the following following will will anything that will invoke stress focus with potentially potentially detrimental effects on the focus on two specific cases with survival of of the the young young of of the the ensuing ensuing generation, generation, namely, namely, lipophilic lipophilic survival toxicants and and heavy heavy metals. metals. toxicants Although Although carotenoids carotenoids are are possibly possibly rather rather ancillary ancillary compounds compounds in the the egg egg per per se, se, the the case case of of the the accumulated accumulated carotenoids carotenoids shall shall serve serve to emphasize emphasize the the point point of of the the potential potential importance importance that that the the maternal maternal history events may egg components. history and and events may bear bear to to the the formation formation of of egg components. Ca Carotenoids fish in usually deposited in their their food food and and usually deposited due due rotenoids are are taken taken up up by fish to to their their chemical chemical properties properties together together with with functional functional lipids-in lipids-in the the case As part part of of case of of salmonid salmonid fishes, fishes, usually usually in in the the white white muscle muscle tissue. tissue. As the the general general lipogenic lipogenic action action of of estradiol estradiol and and thus thus during during the the course course of of vitellogenesis, vitellogenesis, extrahepatic extrahepatic lipid lipid stores stores are are mobilized mobilized and and transported transported to to the the liver; liver; due due to to their their hydrophobicity, hydrophobicity, carotenoids carotenoids are are translocated translocated to liver together to the the liver together with with mobilized mobilized lipids. lipids. At At this this point point it it should should be recalled recalled that that during during exogenous exogenous vitellogenesis, vitellogenesis, hepatic hepatic tissue tissue consti constitutes central organ organ with with respect respect to to lipid lipid metabolism, metabolism, in in that that it it takes takes tutes the central up up triglycerides triglycerides and and phospholipids phospholipids to to utilize utilize them them for for different different meta meta(1) fatty acids serve as major oxidative substrates to fuel bolic tasks tasks:: (1) metabolic (2) as as part part of of the the posttranslational posttranslational modifications modifications metabolic processes; processes; (2) performed lipids are the liver, liver, lipids are attached attached to to that that particular particular part part of of the the performed by the
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forms the lipovitellin of the oocyte; vitellogenin molecule that later forms (3)the rate of hepatic lipoprotein synthesis and export is sharply and (3) increased during vitellogenesis. Carotenoids may associate passively of the lipid utilized in these processes, or it may actively be with any of bound to a covalent binding site on the vitellogenin molecule. The fi rst route will lead to carotenoid deposition in the liver. The second first option will result in carotenoid-colored vitellogenin, as the example of of the pink hue of sockeye-salmon vitellogenin shows. shows. The third alter alternative will also deliver carotenoids from the liver to the gonad, which during vitellogenesis displays possibly the highest rates of uptake for lipoproteins, especially VLDL, from the bloodstream. All lipophilic substances accumulated in the maternal system are likely to behave like the carotenoids. carotenoids. It is is known that chlorinated hydrocarbons and many other lipophi lipophilic pesticides are transported in the bloodstream by lipoproteins lipoproteinsDDT, for instance, has been found associated with serum lipoproteins (Salmo gairdneri; Plack et e t al., 1979). simiin exposed rainbow trout (Salrno 1979). A simi lar behavior can be anticipated for other lipophilic environmental toxicants, such as aliphatic or polycyclic hydrocarbons and many of of toxicants, their derivatives derivatives.. As a consequence, it is is reasonable to assume that sh such lipophilic compounds that have found their way into adult fi fish will eventually be translocated-just translocated-just as the carotenoids are-to are-to the exogenous vitellogenesis. Considering the facts facts that un ungonad during exogenous der the influence of estradiol, hepatic lipoprotein synthesis is in in(cf. Table II) 11) and that vitellogenin itself contains a highly creased (cf. lipophilic region, it does not come as a surprise that DDT and other fish (Plack et al., hydrophobic pesticides are accumulated in fi sh eggs (Plack 1979). Subsequently they will severely impair egg survival and hatch hatch1979). ability (Johnson and Pecor, 1969). 1969). The massive oil globules, com composed of wax esters and steryl esters, prevalent in the eggs of a large fish, number of fi s h, designate a potentially detrimental sink for pesticides, petrochemicals, or other lipophilic environmental toxicants. toxicants. Further Furthermore, at the level of the gonad, exposure of vitellogenic fish sublefish to suble thal concentration of pesticides led to a signifi cant decrease in the significant 32P]phosphate by the growing oocytes, uptake of [[32P]phosphate oocytes, thus probably com compromising their normal composition ((Singh Singh and Singh, Singh, 1981). 1981). hydrocarbons, it To compound the problems posed by halogenated hydrocarbons, has been reported that such compounds not only bind to the vitello vitellogenin molecule, but also decrease the estradiol-dependent vitello vitellogenic response in the rainbow-trout liver (Chen (Chen and Sonstegard, Sonstegard, 1984). 1984).Inducers of the hepatic mixed-function oxidase oxidase system, system, such as beta-naphthofl avone, exert an inhibitory influence on the production beta-naphthoflavone,
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THOMAS P. P. MOMMSEN AND PATRICK PATRICK JJ.. WALSH
of (Chen and Sonstegard, of vitellogenin mRNA in the rainbow trout (Chen Sonstegard, 1984). It should be recalled that during exogenous vitellogenesis, the 1984). matabolic demand put on the liver is enormous (cf. (cf. Table II). 11).Conse Consequently, it can be expected that any additional additiona1 metabolic require requirements placed on the liver, such as the synthesis of of elements involved in detoxification, are likely to reduce the effort expended on vitello vitellogenesis and thus may imbalance the maturing of of the oocytes. oocytes. A further example for the costly metabolic expenditure incurred is the occur occurrence of a novel vitellogenin-like protein in the blood of of pesticide pesticideexposed fish (Denison et al., 1981). 1981). Also on the level of the liver, vitellogenesis may be impaired or its timing imbalanced by the known estrogenic action of some insecticides insecticides.. Examples in mammals and birds show that the chlorinated insecticide chlordecone interacts directly and rather persistently with the uterine estrogen receptor (Hammond et al., 1979). 1979).As As pointed out, mammalian and piscine estro estrogen receptors reveal numerous similarities, similarities, making the exertion of biological effects highly likely in fish systems. A similar line of reasoning applies to the exposure of of fish to envi environmental heavy metals. In Blennius pholis, cadmium and copper are known to accumulate in hepatic tissue, and in the course of the final oocyte oocyte maturation and massive yolk deposition, these heavy metals are transferred from the liver to the gonad and accumulate in the egg (Shackley et al., 1981). (Shackley 1981). Whereas this designates one passive way for the female fish to decrease its own hepatic concentration of these trace metals, it may develop into an important, potentially lethal, strategy for the oocyte. It is not too far-fetched to suggest that in situations where the environmental load of these or other heavy metals to the adult is increased from trace amounts to sublethal levels, transfer to the gonad in the course of oocyte maturation may result in the accu accuHow of mulation of highly toxic levels in the oocyte. oocyte. While such flow of poten potential toxicants may presently not affect marine fish, it is already fright frightfully relevant for freshwater and brackish-water fishes in many parts of the world. The vitellogenin molecule itself may be implicated in the transport of hepatic heavy metals to the gonad due to its protein phos phosphorus-dependent phorus-dependent charge and ion-binding capacity (Hara et al., 1980; 1980; Hara and Hirai, 1978; 1978; Lange, 1981). 1981). An additional problem may be introduced through the potential competition of hepatically accumu accumulated heavy metals for those metal ions that are transported to the gonad during undisturbed vitellogenesis, namely magnesium, cal calcium, and iron. Although adult fish are able to bind and detoxify heavy metals quite efficiently through the specific hepatic synthesis of metallothio-
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(Roch and McCarter, 1984), 1984), the process does not rid the parent nein (Roch body of the heavy metal load rapidly and thus sets the stage for the oocyte. Also, since metallothionein is inpotential poisoning of the oocyte. in duced duced in in the the liver, liver, its its synthesis synthesis effectively effectively competes competes with with vitellogenin vitellogenin (cf. aZ., 1984) 1984) and and therefore therefore can can be be expected expected to to impair impair the the (cf. Seguin Seguin et al., balanced flow How of vitellogenin to the gonad. of Xenopus and some other amphibian eggs is a The green tinge of reflection of the maternal biliverdin deposited adventitiously. colorful reHection It also presents an additional example of how the maternal system may may dispose of of an an excretory excretory product product via via the the eggs. eggs. However, However, as as the the above compounds deposited deposited in in the the maternal maternal above examples examples show, show, not not all compounds liver and and eventually eventually accumulating in in the the eggs eggs are are as as inocuous inocuous as as bili biliverdin in Xenopus.
ACKNOWLEDGMENTS We would like to thank Dr. Catherine B. Lazier (Dalhousie (Dalhousie University) and Dr. Harold B. White III I11 (University of Delaware) Delaware) for helpful discussions. We are grateful to Dr. Bodil Korsgaard (Odense University) for critically reading the manuscript manuscript,:
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Sumpter, J. P., and Dodd, J. M. ((1979). 1979). The annual reproductive cycle cycle of of the female sh, Scyliorhinus Scyliorhinus canicula canicula L., and its endocrine control. }. J , Fish Fish lesser spotted dogfi dogfish, 687-695. Bioi. 15, Biol. 15,687-695. Sundararaj, B. I., Nath, P., Synthesis of P., and Burzawa-Gerard, Burzawa-Gerard, E. (1982a). (1982a). Synthesis of vitellogenin Sundararaj, B. and its uptake by the ovary in the catfish, (Bloch) in re Heteropneustes fossilis (Bloch) recatfish, Heteropneustes sponse to carp gonadotropin and its subunits. subunits. Gen. 93-98. Gen. Camp. Comp. Endocrinol. Endocrinol. 46, 46,93-98. Sundararaj, S. V., and Lamba, V. J. (1982b). Goswami, S. (1982b). Role of of testosterone, estra estraSundararaj, B. I., Goswami, Heteropneustes fossilis diol-17/3, and cortisol catfish, Heteropneustes diol-1713, cortisol during during vitellogenesis in the catfish, (Bloch). Compo Endocrinol. 390-397. (Bloch). Gen. Gen. Comp. Endocrinol. 48, 48,390-397. Szego, J. (1985). (1985). Subcellular distribution of of oestrogen receptors. Szego, C. C. M., and Pietras, R. J. Nature (London) 88-89. (London)317, 317,88-89. Tam, W. Payson, P. D., Whitney, D. K., and Yu, C. K.-C. K.4. W. H., Birkett, L., Makaran, Makaran, R., Payson, (1987). cation of carbohydrate metabolism and liver vitellogenic function in (1987).Modifi Modification brook trout (Salvelinus ]. Fish. Aquat. (Salvelinusfontinalis) by exposure to low pH. Can. Can.]. Aquat. Sci. 44,630-635. 44, 630-635. Tata, J. Smith, D. F. ((1979). 1979). Vitellogenesis: J. R., and Smith, Vitellogenesis: A versatile model for hormonal 47-95. regulation of Recent Prog. Prog. Harm. Horm. Res. Res. 35, 3547-95. of gene expression. expression. Recent Taylor, durin� (1985).Quantitative changes in protein synthesis during Taylor, M. A., and Smith, L. D. (1985). Bioi. 110,230-237. 110, 230-237. Xenopus laevis. laevis. Dev. Dev. Biol. oogenesis in Xenopus te Heesen, D. D. (1977). (1977). Immunologische Untersuchungen an exoexo- und endogenen Dot Dotterproteinen von Brachydanio Brachydanio rerio rerio (Teleostei, Cyprinidae) Cyprinidae) und verwandten venvandten Ar Arten. Zool. Jahrb., Abt Anat. Anat. Ontog. 566-582. 2001.Jahrb., Ontog. Tiere, Tiere, 97, 97,566-582. M.. P. R., R., Searle, P. F., Wolffe, Wolffe, A. P., and Tata, J. R. (1983). (1983).Rapid estrogen Tenniswood, M metabolism and vitellogenin gene expression in Xenopus hepatocyte cultures. Mol. Cell 329-345. Cell Endocrinol. Endocrinol. 30, 30,329-345. Dickhoff, W. W., Gorbman, A. ((1981). Turner, R. T., Dickhoff, W., and Gorbman, 1981). Estrogen binding to hepatic stouti. Gen. Camp. Endocrinol. pacific hagfish hagfish Eptatretus stouti. Gen. Comp. Endocn'nol. 45, 26-29. 26-29. nuclei of of pacific 1984). Changes in serum Ueda, O., Hara, A., Yamauchi, Yamauchi, K., and Nagahama, Nagahama, Y. Y.((1984). Ueda, H., Hiroi, 0., concentrations of of steroid hormones, thyroxine, thyroxine, and vitellogenin during spawning migration of keta. Gen. Gen. Comp. Comp. Endocrinol. 53, of the chum salmon, salmon, Oncorhynchus keta. 203-211.1 . 203-21 Valotaire, Y.,Tenniswood, Tenniswood, M M.,. , L Lee Cuellec, Guellec, C., and Tata, J. J. R R.. (1984). (1984).The preparation Valotaire, Y., and characterization of (Salmo of vitellogenin vitelIogenin messenger RNA from rainbow trout (Salmo gairdneri). 217, 73-77. gairdneri). Biochem. Biochem. }. J . 217,73-77. van Bohemen, C. G., C., and Lambert, G. D. (1981). (1981). Estrogen synthesis in relation Lambert, J. C. to estrone, estradiol and vitellogenin plasma levels during during the reproductive cycle Camp. Endocrinol. 45, 105of Gen. Comp. 105of the female rainbow trout, Salmo gairdneri. Gen. 114. 114. van Bohemen, C. C., G. D., Coos, Coos, H. J. T., and van Oordt, P. G. G. W. J. G., Lambert, J. C. (1982a). (1982a).Estrone and estradiol participation during exogenous exogenous vitellogenesis in the Compo Endocrinol. female rainbow trout, Salmo gairdneri. gairdneri. Gen. Gen. Comp. Endocrinol. 46, 81-92. 81-92. van Bohemen, C. C. C., G., Lambert, J. J. C. G. D., D., and van Oordt, P. G. G. W. J. (1982b). (1982b).Vitellogenin ViteIIogenin induction by estradiol in estrone-primed rainbow trout, Salmo Salmo gairdneri. gairdneri. Gen. Gen. 136-139. Camp. Comp. Endocrinol. 46, 46,136-139. J.-M., and Sire, Sire, M.-F. ((1977). activit6 hydrolasique Vernier, J.-M., 1977). Plaquettes vitellines et activite acide au cours du developpement dheloppement embryonnaire de Ie le truite arc-en-ciel. arc-en-ciel. Etude ultrastructurale et biochimique. Biol. Bioi. Cell. Cell. 29, 99-112. 99-112. Wahli, Ryffel, C. G. U., and Weber, Weber, R. (1981). (1981). Vitellogenesis and the Wahli, W., Dawid, II.. B., Ryffel, 298-304. vitellogenin gene family. family. Science 212, 298-304.
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Wallace, R. A. (1983). (1983). Interactions between between somatic cells and the growing oocyte of of WaUace, Xenopus cLaren Xenopus laevis. laeuis. In In "Current “Current Problems in Germ Cell Differentiation" Differentiation” (A. (A. M McLaren eds.), pp. 285-306. and C. L. Wylie, eds.), 285-306. Cambridge Univ. Press, London and New York. York. Vitellogenesis and oocyte growth in non-mammalian vertebrates. Wallace, R. A. ((1985). 1 985). Vitellogenesis In In "Developmental “Developmental Biology" Biology” (L. (L. Browder, ed.), ed.), Vol. Vol. 1, pp. 127-177. Pergamon, Pergamon, New York. York. Wallace, R. A., and Hollinger, T. G. (1979). T. G. (1979). Turnover of of endogenous, microinjected, and oocytes. Exp. 19, 277-287. sequestered protein in Xenopus Xenopus oocytes. E x p . Cell Cell Res. Res. 1119,277-287. Wallace, R. A., A., and Jared, D. W. (1968). (1968). Studies on on amphibian yolk. VII. Serum-phos Serum-phosphoprotein synthesis by vitellogenic Xenovitellogenic females and estrogen-treated estrogen-treated males of Xeno pus J. Biochem. Biochem. 46, 953-959. 953-959. pus laevis. laeuis. Can. Can.J. Wallace, R. A., and Selman, K. ((1981). 1981). Cellular and dynamic aspects of of oocyte growth in 325-343. teleosts. Am. Am. Zool. Zool. 21, 21,325-343. Wallace, R. A., A., and Selman, K. (1985). (1985). Major changes during vitellogenesis vitellogenesis and matura maturation of Fundulus Dev. Bioi. 10, 492-498. Fundulus oocytes. Deu. Biol. 1110,492-498. Wallace, R. A., A., Deufel, R. A., and Misulovin, Z. (1980). (1980).Protein incorporation by isolated amphibian oocytes. VI. Comparison of autologous and xenogeneic vitellogenins. vitellogenins. Comp. Biochem. Physiol. B 65B, 151151-155. Compo 155. Walter, P., Green, S., Greene, G., Krust, A., A., Bornert, J.-M., Jeltsch, J.-M., Staub, Staub, A., A,, Jensen, E., Scrace, G., Waterfield, M., M., and Chambon, P. (1985). (1985). Cloning of the Natl. Acad. Acad. Sci. Sci. V.SA. Proc. Natl. U S A . 82, 82, 7889-7893. 7889-7893. human estrogen receptor cDNA. Proc. Wang, S.-Y., S.-Y., and Williams, D. L. (1982). (1982). Biosynthesis of of the vitellogenins. vitellogenins. Identification Identification and characterization characterization of of nonphosphorylated precursors to avian vitellogenin I and Bioi. Chem. vitellogenin II. 11. J. J. Biol. Chem. 257, 3837-3846. 3837-3846. Wang, S.-Y., D. E., D. L. ((1983). 1983). Purification of S.-Y., Smith, Smith, D. E., and Williams, D. of avian vitellogenin III: II. Biochemistry 6206-6212. 111: comparisons with vitellogenins I and 11. Biochemistry 22, 22,6206-6212. Wangh, L. J. ((1982). 1 982). Glucocorticoids act together with estrogens estrogens and thyroid hormones in regulating the synthesis and secretion of Xenopus Xenopus vitellogenin, vitellogenin, serum albumin and fibrinogen. Dev. 294-298. D e n Bioi. Biol. 89, 89,294-298. Welshons, W. V., V., Lieberman, M. E Liebennan, M. E.,. , and Gorski, J. (1984). (1984).Nuclear Nuclear localization of of unoc unoc747-749. Nature (London) (London)307, 307,747-749. cupied oestrogen receptors. Nature White, H. H. B. ((1985). 1985). Biotin-binding oocytes. Ann. Biotin-binding proteins and biotin transport transport to oocytes. Ann. N. N.Y. Y. Acad. Sci. Sci. 447, 202-21 1. 447,202-211. Acad. Wiegand, M. D. ((1982). 1 982). Vitellogenesis Vitellogenesis in fishes. In In "Reproductive “Reproductive Physiology of Fish" Fish” (C. J. J. Richter and H. H. J. J. T. Goos, compilers), 146. Pudoc, Wageningen. compilers), pp. 136136-146. Wageningen. (1982).Synthesis of lipids by the rainbow trout (Salmo (Salmo Wiegand, M. D., and Idler, D. D. R. (1982). J. Zool. 60, 2683-2693. gairdneri) Can.J. 60,2683-2693. gafrdneri) ovary in vitro. Can. 1980). Effects of the salmon gonadotropin (SG-GlOO) Wiegand, M M.. D., and Peter, R. E. ((1980). (SG-G100) on plasma lipids in the goldfish, Carassius 967-972. Carussius auratus. aurutus. Can. Can.J. J. Zool. 58, 58,967-972. Wiley, H. S., ultiple vitello S . , and Wallace, R. A. (1981). (1981). The structure of of vitellogenin. M Multiple vitellogenins in Xenopus Xenopus laevis laeuis give rise to multiple forms forms of of the yolk proteins. J. J . Bioi. Biol. 8626-8634. Chem. 256,8626-8634. Chem. 256, Wingfield, J. J. C. ((1980). 1980). Sex-steroid binding proteins in vertebrate blood. In In Hormones Hormones:: Adaptation and Evolution" (S. Ishii, T. Hirono, and M. Wada, eds.), eds.), pp. 135-144. 135-144. Evolution” (S. Jpn. Soc. Press, Tokyo. Jpn. Sci. SOC. Wiskocil, R., Bensky, P., Dower, W., W., Goldberger, F., Gordon, J. I., and Deeley, R. G. Goldberger, R. F., (1980). (1980).Coordinate regulation regulation of of two two estrogen-dependent genes in avian liver. liver. Proc. Proc. Natl. Acad. Sci. A. 77, 4474-4478. Sci. U.S USA. 77,4474-4478. Natl. Acad.
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Wolffe, A. P., Glover, J. F., F., Martin, S. S. C., C., Tenniswood, Tenniswood, M. P. R., Williams, J. L., and Tata, J. J. R. ((1985). 1985). Deinduction of transcription transcription of of Xenopus Xenopus 74-kDa albumin genes ]' and destabilization ofmRNA mRNA by estrogen in in vivo uiuo and in hepatocyte cultures. Eur. Eur.J. destabilization of Biochem. 489-496. Biochem. 146, 146,489-496. Wright, C. V. E., E., Wright, S. S. C., C., and Knowland, J. (1983). (1983).Partial purification purification of of estradiol receptor from Xenopus Xenopus laevis laeuis liver and levels of of receptor in relation to estradiol 973-977. concentration. EMBO], EMBO], 2, 2,973-977. 1980). Sexual patterns of Yu, J. Y.-L., Dickhoff, W. W., and Gorbman, Gorbman, A. ((1980). of protein metab metabolism in liver and plasma of of hagfish, Eptatretus stouti, with special reference to Compo Biochem. Biochem. Physiol. 1 1- 1 17. vitellogenesis. Physiol. B B 65B, 65B, 1111-117. vitellogenesis. Comp. Yurowitzky, Y. G., and Milman, L. S. ((1972). 1972). Changes in enzyme activity of of glycogen fassilis L. and hexose metabolism Misqurnusfossilis metabolism during oocyte maturation in a teleost, Misqurnus Wilhelm 171, 48-54. Wilhelm Roux' Roux’ Arch. Entwicklungsmech. Org. Org. 171, 48-54. Yurowitzky, Y. G., and Milman, L. S. S. ((1975). 1975). Changes Changes in activity of of enzymes enzymes of of glycogen metabolism in loach oocytes and embryos. Biochemistry (Engl. 821(Engl. Transl.) Transl.) 40, 40,821825. 825. Yusko, S., 1981). Receptor-mediated Yusko, S., Roth, T. F., and Smith, T. T. ((1981). Receptor-mediated vitellogenin vitellogenin binding to 43-50. Biochem. ]. J . 200, 200,43-50. chicken oocyte. Biachem. Zagalsky, P. P. F., F., Gilchrist, Gilchrist, B. M., M., Clark, R. J. J. H., and Fairclough, D. D. P. P. (1983). (1983). The canthaxanthin-lipovitellin canthaxanthin-lipovitellinof Branchipus Branchipus stagnalis stagnalis (L). (L). (Crustacea: (Crustacea: Anostraca) Anostraca):: A A resonance resonance Raman and circular dichroism study. study. Compo Comp. Biochem. Biochem. Physial. Physiol. B 73B, 73B, 163-167. 163- 167.
6 YOLK ABSORPTION ABSORPTION IN EMBRYONIC EMBRYONIC YOLK LARVAL FISHES AND LARVAL
A . HEMING HEMING THOMAS A Pulmonary Division Division Pulmonary Department of of Internal Internal Medicine Medicine Department University of of Texas Texas Medical M edical Branch Branch University Galveston, Texas Texas 77550-2780 77550-2780 Galveston,
BUDDINGTON RANDAL KK.. BUDDINGTON Department of of Physiology Physiology Department University of of California California University Los Angeles, Angeles, California California 90024 90024 Los
I. Introduction I. Introduction II. 11. Structural Structural Aspects Aspects of of Yolk Yolk Absorption Absorption A. A. Yolk Yolk Morphology Morphology B. B. Meroblastic Meroblastic Fishes Fishes C. C . Holoblastic Holoblastic Fishes Fishes Ill. 111. Yolk Yolk Composition Composition during during Development Development A. A. Dry Dry Matter Matter and and Water Water Content Content B. B. Protein Protein C. C . Lipid Lipid D. D. Carbohydrates Carbohydrates E. E. Caloric Caloric Content Content IV. IV. Rate Rate of of Yolk Yolk Absorption Absorption V. V. Efficiency Efficiency of of Yolk Yolk Utilization Utilization A. A. Biotic Biotic Factors Factors B. B. Abiotic Abiotic Factors Factors VI. VI. Nonyolk Nonyolk Nutrient Nutrient Sources Sources during during Early Early Development Development A. A. Piitter's Putter’s Theory Theory B. embranes and B. Egg Egg M Membranes and Perivitelline Perivitelline Fluid Fluid C. C. Viviparity Viviparity D. D. Mixed Mixed Feeding Feeding VII. VII. Nutrition Nutrition of of Embryos Embryos and and Larvae Larvae References References 407 407 FISH FISHPHYSIOLOGY, PHYSIOLOGY, VOL. VOL.XIA XIA
Copyright©0 1988 1988by byAcademic AcademicPress, Press, Inc. Inc. Copyright All rights rightsof ofreproduction reproduction in inany anyform formreserved. reserved. All
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THOMAS A. A. HEMING HEMING AND AND RANDAL RANDAL K. K. BUDDINGTON BUDDING TON THOMAS
I. INTRODUCTION INTRODUCTION I.
of our knowledge regarding yolk absorption is A major portion of based on on species species possessing possessing large, large, demersal demersal eggs, eggs, which which are are adapted adapted based pre for colder waters and long incubation periods. This is evident in previous reviews reviews of of yolk yolk utilization by Hayes Hayes (1949), ( 1949), Smith Smith (1957, ( 1957, 1958), 1958), vious utilization by ( 1967), Blaxter (1969), ( 1969), Terner (1979), (1979), and Boulekbache Williams (1967), ( 1981). Development Development of of culture culture techniques techniques for for other other species species and and inin (1981). of addiaddi creasing ecological concerns, however, have elicited research of particularly marine fishes. This information has been tional groups, particularly incorporated and contrasted in in the present review. review. incorporated and contrasted the present A fish egg can be considered a semiclosed system. Once the egg membrane(s) has been hardened mem membrane(s) hardened by exposure to water, the membrane(s) permits permits gas gas exchange but is is relatively impervious to to most most brane(s) exchange but relatively impervious of fish embryos are dependent solutes. As a consequence, the majority of on endogenous yolk reserves to supply the substrates for energy pro pro(see Section duction and growth. Viviparous fishes are are an an exception (see VI,C). Both the rate of of yolk utiliutili VI,C). of yolk absorption and the efficiency of zation of early zation are are important determinants of early development, growth, and ultimately dependent on the availability survival. Larval Larval survival is ultimately of food food in in sufficient sufficient quantity and of of adequate quality after after yolk yolk rere of quantity and adequate quality pres serves are exhausted. It follows that there are strong selective presof yolk absorption, development sures synchronizing completion of of the capability of of feeding, and the availability of of suitable food of (Barns, 1969; 1969; Rosenthal and Alderdice, 1976). 1976). As well, since large (Barns, size size confers confers certain certain advantages advantages on on larvae, larvae, there there are are strong selective selective pressures pressures to to maximize the the efficiency with with which which yolk yolk is is converted converted into into tissues. tissues. Larger Larger larvae larvae of of aa given given species species can can be be expected to to be stronger swimmers (Hunter, 1972), 1972), less affected by competition (Hulata (Hulata et al., al., 1976), 1976), more more resistant resistant to to starvation starvation (Blaxter (Blaxter and and Hempel, Hempel, 1963), 1963), less susceptible to predation (Ware, (Ware, 1975), 1975), able to com commence mence feeding feeding earlier earlier (Wallace (Wallace and and Aasjord, Aasjord, 1984a), 1984a),and and able able to to have have increased success at first feeding (Braum, (Braum, 1967; 1967; Ellertsen et al., al., 1980). 1980). The The rate rate and and efficiency efficiency of of yolk yolk absorption absorption are are influenced influenced by by a number number of of environmental environmental factors, factors, including including temperature, temperature, light, light, oxy oxygen gen concentration, concentration, and and salinity. salinity. Fish Fish eggs eggs are are not not motile, motile, however, however, and and thus thus developing developing embryos embryos are are unable unable to to actively actively exploit exploit the the most most favorable favorable environments environments available, available, at at least least until until after after hatching. hatching. Only Only species species that that utilize utilize reproductive reproductive strategies strategies such such as as viviparity viviparity or or mouth mouth brooding may, through parental behaviors, be able to manipulate egg may,
6. 6.
YOLK ABSORPTION IN IN EMBRYONIC EMBRYONIC AND AND LARVAL FISHES YOLK LARVAL FISHES
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incubation conditions. It is selectively advantageous, therefore, for a species to produce eggs that can develop successfully within a range of “expected” incubation conditions. The scope of “expected” of "expected" of these "expected" conditions will depend on those conditions experienced during evo evoof the species. For some fishes, the fl fluctuation environmenlution of uctuation in environmen tal parameters may be relatively slight (e.g., abyssal marine habitats), while for others it may be large (e.g., some temperature freshwater habitats). habitats). From an applied standpoint, there has been an interest in of environmental factors factors on yolk absorp absorpdetermining the influence of tion-particularly the effects of temperature, since it is generally the tion-particularly most variable parameter and the most easily controlled in culture settings. settings, A review review of of present present knowledge knowledge regarding regarding yolk yolk absorption absorption in in fish is is hindered somewhat by the use of of many different and often impre imprestaging. For the pur purcisely defined terminologies for developmental staging. pose of our review, we have adopted the generalized terms defined below. below. 1. Embryo-the 1. Embryo-the developing developing fish prior prior to to hatch. hatch. 2. sh after 2. Eleutheroembryo-the Eleutheroembryo-the developing developing fi fish after hatch hatch until until the the fish, parturi parturior, in the case case of viviparous fish, initiation of feeding or, tion 1975). For For our our purposes, purposes, feeding feeding refers refers to to the the in intion (Balon, (Balon, 1975). gestion gestion of of exogenous exogenous matter matter into into the the stomach stomach or or the the capability capability to so, rather rather than than behavioral behavioral responses responses to to potential potential food food to do do so, items. comitems. Defined Defined in in this this way, way, feeding feeding is is independent independent of com plete independent of of the the capabil capabilplete yolk yolk absorption absorption and and may may be independent ity ity to to digest digest and and utilize utilize ingested ingested material. material. 3. 3. Larva-the Larva-the developing developing fish fish after after initiation initiation of of feeding feeding or or partu parturition juvenile, characterized characterized by by aa full full complement complement of of rition until until a juvenile, minute minute adult adult features, features, is is attained. attained. 4. 4. Yolk-the Yolk-the nutritional nutritional reserves reserves provided provided in in the ovum, ovum, includ including ing those those associated associated with with the the yolk yolk platelets platelets and and oil oil globules. globules. A number number of of authors authors categorize categorize the the yolk yolk sac sac contents contents into into "yolk" “yolk” and basis of of visual visual appearance. appearance. For For simplicity, simplicity, we we and "oil" “oil” on on the basis have have regarded regarded these these categories categories as as equivalent equivalent to to yolk yolk platelets platelets and and oil oil globules, globules, respectively. respectively. 5. 5. Tissues-the Tissues-the body body of of the the developing developing fish fish including including the the yolk yolk sac sac but but without without the the yolk. yolk. Throughout Throughout this this review, review, we we use use the the common common and and scientific scientific names names of of fishes 1980). by Robins Robins et ai. al. ((1980). fishes listed listed by
410
THOMAS A. HEMING AND RANDAL K. BUDDINGTON
II. STRUCTURAL 11. STRUCTURAL ASPECTS OF YOLK YOLK ABSORPTION
A. Yolk Morphology The structural components of fish yolk include yolk platelets and oil globules. The majority of yolk platelets are round or oval in shape, flattened in one plane, and 4-15 4-15 1Lm pm in length. Larger platelets appear to be characteristic of species possessing larger eggs (Grodzinski, (Grodzinski, 1973). 1973).Platelet size also varies within each egg, with the deeper, more centrally located platelets tending to be larger and more homogenous than the superficial peripheral ones (Vernier (Vernier and Sire, 1977; 1977; Hamlett and Wourms, 1984). 1984). Each platelet consists of an outer sheath and a central core (Fig. (Fig. 1). 1). The sheath forms forms a semipermeable bilayer around the core (Grodzinski, (Grodzinski, 1973) 1973)and contains mucopolysaccharides (Ohno al., 1964). (Ohno et al., 1964). The core is is composed of lipovitellin and phos phosvitin, or analogous lipoproteins and phosphoproteins (Fujii, (Fujii, 1960; 1960; Wallace et al., al., 1966; 1966; Jared and Wallace, 1968). 1968). These core proteins may or may not be arranged in 1 , 1982; in a crystalline lattice (Lange, (Lange, 198 1981,1982;
'-l----- sm
���\--- a l
�m1.mlt=fC �
cc
}
8
Fig. 1. structure of ofAmia Fig. 1. Model of a yolk platelet based mainly mainly on the structure Amia platelets. The (A) outer sheath; sheath; (B) (B) main body; body; sm, superficial membrane; aI, al, interior: (A) cuts reveal its interior: sm, superfi cial membrane; amorphous superficial superficial layer; layer; fc, fibrillar fibrillar cortex; cortex; cc, crystalline crystalline core. [From [From Grodzinski Grodzihski amorphous (1973).] (1973).]
6.
FISHES YOLK ABSORPTION ABSORPTION IN EMBRYONIC EMBRYONIC AND AND LARVAL LARVAL FISHES YOLK
411 411
Lange et et al., al., 1982). Moreover, M oreover, the crystalline structure may be lost as 1970). the ova mature (Balinsky, 1970). Oil globules are located among the yolk platelets. Globule number vary greatly among species, from innumerable small globules and size vary in the micrometer micrometer diameter range to singular large globules in the millimeter millimeter diameter range. The globules contain primarily triglycertriglycer ides, although proteins (Grodzinski, 1973), 1973), wax esters (Vetter et al., al., 1983), and carotenoid pigments (Nakagawa and Tsuchiya, 1971) 1983), 1971) are also present in some species. M eroblastic Fishes B. Meroblastic of most fishes fishes (elasmobranchs and Meroblastic cleavage, typical of teleosts), results in the formation of of an extraembryonic yolk sac. A characteristic feature of of this extraembryonic sac is the yolk syncytium, a specialized tissue responsible for absorption of yolk. The presumppresump tive yolk syncytium, the periblast, is recognizable in the fertilized 1982). As cleavage pro teleost egg at the one-cell stage (Yamamoto, (Yamamoto, 1982). proceeds, numerous free nuclei appear in the periblast, thus transforming transforming the layer into a true syncytium. meso In teleost eggs, the yolk syncytium together together with overlaying mesoEndo derm and ectoderm spreads to enclose the entire yolk mass. Endoderm does not follow the movement of of the teleost blastodisc rim and, consequently, Ab consequently, the yolk is not enclosed by an endodermal layer. Absorption of yolk nutrients in teleosts, therefore, occurs without any involvement of endodermal cells or the gut (Bachop (Bachop and Schwartz, 1974). 1974). A system of blood vessels, the vitelline circulation, develops within the walls of the yolk sac. In some areas, the endothelial wall of vitelline capillaries is incomplete incomplete and embryonic blood is in direct contact with the syncytium (Shimizu and Yamada, 1980) 1980).. Absorption of yolk involves endocytosis by the syncytium, intrasyncytial diges digestion and synthesis, and finally the release of yolk metabolites to the vitelline circulation circulation.. When yolk reserves are exhausted, the syncy syncytium is resorbed; it does not take part in formation of the permanent fish fish body (Yamada, (Yamada, 1959; 1959; Yamamoto, 1982). 1982). Two regions of the yolk syncytium can be distinguished distinguished on the basis of their fine structure (Shimizu and Yamada, 1980). 1980). One region, characterized by smooth endoplasmic endoplasmic reticulum, numerous mitochon mitochondria, and glycogen granules, is proposed to be responsible for carbo carbohydrate and/or lipid metabolism. This This region extends throughout the syncytium. syncytium. The The second second region region is is characterized characterized by by rough rough endoplasmic endoplasmic
412
BUDDINGTON THOMAS A. HEMING AND RANDAL K. BUDDING TON
reticulum reticulum and and Golgi Golgi complexes, complexes, and and extends extends in in portions portions across across the the syncytium region is is thought syncytium forming forming aa stratified stratified structure. structure. This This latter latter region thought to involved in proteinaceous sub to be be involved in the the synthesis synthesis and and transport transport of of proteinaceous subsoluble. protein must be dephosphorylated to become soluble. stances. Yolk protein Amirante 1972) suggested suggested that that fish yolk yolk proteins proteins are are solubilized solubilized by by Amirante ((1972) the Syncytial Golgi Golgi the action action of of calcium calcium and and phosphoprotein phosphoprotein phophatase. phophatase. Syncytial complexes complexes probably probably supply supply acid acid hydrolases hydrolases for for the the degradation degradation of of yolk Sire, 1977; 1977; Hamlett, Hamlett, et al., 1987). 1987). yolk platelets platelets (Vernier (Vernier and and Sire, In In addition addition to to the the syncytial syncytial layer, layer, the the yolk yolk itself itself contains contains enzymes enzymes (Hamor 1973) that that probably probably facilitate facilitate the the breakdown breakdown of of (Hamor and and GarSide, Garside, 1973) yolk (1977) described described yolk into into its its constituent constituent nutrients. nutrients. Vernier Vernier and and Sire Sire (1977) two types types of of yolk yolk platelets platelets with with different different enzyme enzyme contents. contents. One One form, form, the the embryonic embryonic platelet platelet type, type, has has an an enzyme enzyme load load that that allows allows nutrients nutrients to released prior second or of the the syncytium. syncytium. The The second or to be be released prior to to establishment establishment of
Ectoderm
Capill ories
Endoderm
Yol Syncyllum
Fig. 2. 2. Idealized diagram diagram of the cellular organization organization in a preimplantation preimplantation shark Fig. yolk sac. sac. The teleost yolk sac is similar in structure structure except that it it lacks endoderm. endodem. [From [From Woums (1984).] (1984).1 Hamlett and Wourms
6. 6.
YOLK ABSORPTION ABSORPTION IN IN EMBRYONIC EMBRYONIC AND AND LARVAL FISHES YOLK LARVAL FISHES
413
usual platelet type lacks this enzyme load and is digested by syncytial enzymes. While the extraembryonic yolk sac with its yolk syncytium is the sole site of of yolk absorption in teleosts, this is not the case in chonchon ratfish). In holocephali holocephalidrichthyean fishes (sharks, skates, rays and ratfish). of the yolk mass is enclosed by ans, for instance, only a small portion of the 1906). The The remainder remainder breaks breaks up up into into aa viscous viscous the yolk yolk sac sac (Dean, (Dean, 1906). fluid, fl uid, which is first absorbed via the external gills of the embryo and later ingested through the mouth. This ingestion of yolk nutrients is comparable, in a general sense, to that exhibited by oophagous sharks, (Fuwhose viviparous embryos ingest ova present in the same uteri (Fu 1981; 1983). jita, 198 1 ; Gilmore et al., 1983). In I n elasmobranchs, elasmobranchs, the the formation formation of of an an archenteron archenteron at at the posterior posterior edge blastodisc during during gastrulation gastrulation results results in in aa yolk yolk sac sac that that edge of of the blastodisc possesses 2). This endodermal layer medi medipossesses an an endodermal endodermal layer layer (Fig. (Fig. 2). ates transfer of yolk yolk metabolites metabolites from from the the syncytium syncytium to to the the vitel vitelates the transfer 1987). Moreover, the elasmobranch line circulation (Hamlett et al., 1987). yolk sac sac is continuous with the alimentary tract via a yolk stalk, and thus majority of of yolk yolk is is digested digested within within the the intestine. intestine. Yolk plate platethus the majority lets moved by yolk sac lets are are moved by ciliary ciliary action action from from the the yolk sac through through the yolk yolk stalk (TeWinkel, Winkel, 1943; 1943; Baranes Baranes and and Wen Wenstalk and and into into the the spiral spiral intestine intestine (Te dling, 1981). 1981). An An internal internal yolk yolk storage storage organ organ may may or or may may not not be dling, present. Enzymatic activity in the gut is established reilltively relgtively early in development, development, when when the the embryo embryo is is approximately approximately one-quarter one-quarter its its size size at 943). (Te Winkel, Winkel, 11943). at parturition parturition (Te C. C. Holoblastic Holoblastic Fishes Fishes A A few few fish fish species species develop develop holoblastically holoblastically (e.g., (e.g., lampreys lampreys and and chondrosteans). endodermal and and lateral lateral plates plates chondrosteans). In In these these species, species, the endodermal fuse ventral line fuse along along the the mid midventral line forming forming an an intraembryonic intraembryonic yolk yolk sac. sac. As As aa result, result, all all three three germ germ layers layers enclose enclose the the yolk yolk mass mass (Ballard (Ballard and and Ginzburg, 1980). The The resultant resultant intraembryonic intraembryonic yolk yolk sac sac directly directly par parGinzburg, 1980). ticipates ticipates in in formation formation of of the the alimentary alimentary canal. canal. During During posthatch posthatch devel development yolk sac sac is is separated separated into into two two major major opment of of chondrosteans, chondrosteans, the yolk regions, regions, each each of of which which develops develops separate separate blood blood drainages drainages (Ballard (Ballard and and Needham, 1964).The The distal distal region region comprises comprises the the intestine intestine and and spiral spiral Needham, 1964). valve, corresponding blood blood supply supply proceeds proceeds to to the the liver. liver. Yolk Yolk valve, and and the corresponding within is the the first first to to be utilized. utilized. The The second second region region in inwithin this this region region is cludes cludes the the stomach stomach and and esophagus esophagus and and develops develops aa blood blood supply supply that that proceeds sinus venosus. venosus. This This region region is is the the last last portion portion proceeds directly directly to to the sinus
4 14 414
THOMAS THOMAS A. A. HEMING HEMING AND AND RANDAL RANDAL K. K. BUDDINGTON BUDDINGTON
of of the the alimentary alimentary canal canal to to differentiate, differentiate, and and yolk yolk is is retained retained there there longer (Buddington and Christofferson, 1985). longer (Buddington and Christofferson, 1985). Although Although hydrolytic hydrolytic enzymes enzymes are are present present within within the the developing developing alimentary canal, their activities are low (Korzhuev and alimentary canal, their activities are low (Korzhuev and Sharkova, Sharkova, 1967; 1967; Buddington Buddington and and Doroshov, Doroshov, 1986). 1986). The The existence existence of of yolk yolk mate material yolk sac rial within within endodermal endodermaI cells cells lining lining the the yolk sac implies implies that that endocyto endocytosis and sis and intracellular intracellular digestion digestion may may be be the the primary primary mechanisms mechanisms by which which yolk yolk nutrients nutrients are are made made available available (Krayushkina, (Krayushkina, 1957; 1957; Bud Buddington dington and and Christofferson, Christofferson, 1985). 1985). Thus, Thus, the the intraembryonic intraembryonic yolk yolk sac sac of yolk sac of holoblastic holoblastic fishes fishes and and the the extraembryonic extraembryonic yolk sac of of meroblastic meroblastic fishes exhibit exhibit similar fishes similar mechanisms mechanisms for for mobilization mobilization of of yolk yolk nutrients. nutrients.
III. YOLK COMPOSITION DURING DEVELOPMENT 111.
Selective sh exploiting wide diverdiver Selective pressures pressures have have resulted resulted in in fi fish exploiting aa wide sity of reproductive strategies. As a consequence, egg size and fecun sity of reproductive strategies. a consequence, egg size and fecundity fishes from 0.7 mm dity vary vary among among species, species, in in oviparous oviparous fishes from about about 0.7 mm egg egg diameter (e.g., convict surgeon fish Acanthurus triostegus) to diameter (e.g., convict surgeon fish triostegus) to greater greater than 10 mm diameter (e.g., chinook salmon than 10 mm diameter (e.g., chinook salmon Oncorhynchus tsha tshawytscha), wytscha), with spawns varying from less than 100 100 eggs per female (e.g., (e.g., mouth-brooding mouth-brooding cichlid cichlid Labeotropheus fuelleborni) fuelleborni) to to more more than Viviparous fi sh tend than 9,000,000 9,000,000 (e.g., (e.g., Atlantic Atlantic cod cod Gadus morhua). morhua).Viviparous fish tend to produce fewer but proportionally proportionally larger eggs. These differences in egg egg size size and and number number imply imply maternal maternal investment investment per per egg egg differs differs widely widely among among species. species. The The deposition deposition of of nutrients nutrients into into the the egg egg during during oogenesis been reviewed Mommsen and oogenesis has has been reviewed in in this this volume volume by by Mommsen and Walsh Walsh (this volume, Chapter 5). 5). (this The nutrient composition of of fish eggs is species-specific (Table I). Within a given species, as well, welI, egg quality varies as a function of of maternal age, weight, and diet (Kamler, (Kamler, 1976; 1976; Kuznetsov and KhaliKhali tov, 1979). 1979). Despite these differences, the dynamics of of yolk absorption are similar among groups. Following fertilization, the developing emem bryo begins to utilize yolk nutrients. This is accompanied by increas increasing consumption of of oxygen, particularly particularly after the blastula stage is reached. As development proceeds, the absolute and relative compocompo 1972, 1974, 1976). sition of of the yolk changes (Nakagawa and Tsuchiya, 1972,1974,1976). Various approaches (proximal ra (proximal analysis, respiratory quotients, radiolabeled substrates) have been used to investigate the sequence Genwith which yolk nutrients are catabolized for energy production. Gen erally, erally, carbohydrate, lipid, and protein are consumed prior to hatch-
Table Table II Chemical Composition Composition of Fish Eggs Eggs Chemical Percentage Percentage of of dry dry weight weight
Dry weight
Species Species
mg
% %
Protein
Lipid
Carbohydrate
Ash
Acipenser transmontanus (white sturgeon) sturgeon) (white Coregonus albula (vendace) (vendace) Coregonus lavaretus (whitefish) (whitefish) Cyprinus carpio
6.25" 6.25"
23.8 23.8
67 67
30 30
-
33
16.2'ib 16.27b
-
64.4
25.8
-
8.5 8.5
Dabrowski Dabrowski and and Luczynski Luczynski (1984) (1984)
15.6b 15.6b
-
60.3
27.7
-
9.8 9.8
Dabrowski 1984) Dabrowski and and Luczynski Luczynski ((1984)
-
30.4" 30.4" 10.2b 1O.2b
64.3 64.3 58.3-59.2 58.3-59.2
5.9 5.4-29.3 5.4-29.3
3.7 3.7 1.5-6.2 1.5-6.2
6.3 6.3
0.283" 0.283" 0.29Bb 0.298b 0.232" 0.232a
22.1 22.1 10.4 10.4 46.3
66.4 66.4 56.7
-
-
20.5 16.8 16.8 52.0
-
2. 2.11 8.4 3.0 3.0
M oroz and 1976) Moroz and Luzhin Luzhin ((1976) M oroz and 1976); Moroz and Luzhin Luzhin ((1976); Kamler 1976) Kamler ((1976) Lapin and Lapin and Matsuk Matsuk (1979) (1979) atsuk (1979) Lapin Lapin and and M Matsuk (1979) 1981a) Eldridge Eldridge et et al. al. ((1981a)
0.051AF" 0.051AFa 0.049AFb 0.049AFb
-
79.3 77.4
15.4 15.4 19.4
5.3 5.3 3.2
7.2 7.2
42.1" 42.1a
-
41.3 33.Bb 33.8b
56.2 59.8-71.3 59.8-71.3
11.4 1 1.4
0.6
3.8-3.9 3.8-3.9
49.7b 49.7b
36.0
52.2
36.1
1.0
2.8
-
29.3"
71.6
13.0 13.0
<1 <1
77
Lasker (1962) ( 1962) Lasker
-
7.0b 7.0b
28.1
33.7
0.4
-
Vetter et al. al. (1983) (1983) Vetter et
(carp) (carp)
Eleginus navaga nauaga Eleginus (navaga) (navaga) Morone saxatilis saratilis (striped bass) Pseudopleuronectes Pseudopleuronectes arnericanus americanus (winter flounder) flounder) Salmo gairdneri gairdneri (rainbow trout) Salmo salar (Atlantic salmon) Sardinops Sardinops caerulea caerolea (Pacific (Pacific sardine) Sciaenops Sciaenops ocellata (red drum) " Unfertilized. b Fertilized; AF, ash-free.
-
Sources Sources Wang 1987) Wang et et al. al. ((1987)
Cetta and Capuzzo 1982) Cetta and Capuzzo ((1982) Cetta and and Capuzzo (1982) Cetta Capuzzo (1982)
et al. al. (1977) ( 1977) Zeitoun et Zeitoun Smith ( 1957); Smith (1957); Satia et et al. al. (1974) (1974) Satia Hamor and Garside (1977a) ( 1977a) Hamor and Garside
416
THOMAS A. HEMING AND RANDAL K. K. BUDDINGTON
while lipid lipid and and protein protein catabolism catabolism predominate predominate after after hatching. hatching. ing, while However, the precise sequence of nutrient consumption varies both However, the precise sequence of nutrient consumption varies both qualitatively and quantitatively. This is not surprising, since it is un qualitatively and quantitatively. This is not surprising, since it is unlikely that any one energetic scheme is adequate to describe the se likely that any one energetic scheme is adequate to describe the sequential utilization of energy substrates in all fishes, in other than quential utilization of energy substrates in all fishes, in other than general terms. terms. More More likely, likely, the the precise precise scheme scheme varies varies among among fishes fishes in in general relation to absolute egg composition, which in turn has probably relation to absolute egg composition, which in turn has probably evolved within within the the “expected” "expected" constraints constraints of of aa given given rearing habitat evolved rearing habitat and/or reproductive strategy. andor reproductive strategy. Dry Matter Matter and and Water Content A. Dry Water Content absolute amount of dry dry matter matter in in aa fish egg egg exhibits exhibits both both interinter The absolute amount of and intraspecific intraspecific variation. variation. The The percentage percentage dry dry weight weight per per egg, egg, and and and hence egg egg water water content, content, is is also also variable (Table I). Following Following fertilizafertiliza hence variable (Table tion, egg egg water water content content comes comes under under osmoregulatory osmoregulatory control. control. The The spespe tion¶ cific gravity of of fish fish eggs is inversely related to water content. content. Thus, Thus, the the eggs is inversely related to water cific gravity buoyant nature nature of of pelagic pelagic eggs eggs is is associated associated with with aa higher higher water water concon buoyant tent relative relative to to that of dense, dense, less less hydrated hydrated demersal demersal eggs. eggs. The The spespe tent that of cific gravity of of eggs eleutheroembryos can decrease with time eggs and and eleutheroembryos can decrease with time cific gravity and Ehrlich Ehrlich [e.g., Atlantic herring Clupea harengus harengus, Blaxter Blaxter and [e.g., Atlantic herring ((1974); 1974); Atlantic salmon Salmo salar, Peterson and Metcalfe Metcalfe (1977)l ( 1977)] or or Atlantic salmon Peterson and increase with time [e.g., plaice plaice Pleuronectes platessa, Blaxter Blaxter and and increase with time [e.g., Ehrlich ( 1 974); northern San northern anchovy Engraulis mordax, Hunter and SanEhrlich (1974); chez 1976)] . These These changes chez ((1976)l. changes are are aa function function of of osmoregulatory osmoregulatory adjust adjustments yolk and ments and and changes changes in in the the quantity quantity and and composition composition of of yolk and tis tissues. sues. The percent water content of yolk and tissue cannot be assumed to development. The percent water content remain constant throughout development. yolk, for example, example, has been reported to decrease during of salmonid yolk, 1942; Harvey, 1966), (Hayes and Armstrong, 1942; development (Hayes 1966), to increase 1984), or to remain unchanged (Gray, (Gray, 1926; 1926; Pe Pe(Escaffre and Bergot, Bergot, 1984), (Escaffre 1977). Similarly, the percent water content of terson and Metcalfe, 1977). salmonid tissues has been found to increase during development 1966; Escaffre and Bergot, Bergot, 1984) 1984) or to remain unchanged (Harvey, 1966; (Harvey, 1977). The documented changes are large in (Peterson and Metcalfe, 1977). (Peterson 20%increase in the relative water content of rainbow some cases cases [e.g., [e.g., 20% some ( S . gairdneri) gairdneri) yolk from hatch to complete yolk absorption; absorption; (Es (Estrout (S. caffre and Bergot, 1984)] 1984)l.. caffre changes in the weight of embryonic Figure 3 illustrates typical changes (yolk and tissues) tissues) with time. Generally, Generally, as high-density, components (yolk
6.
417
YOLK YOLK ABSORPTION ABSORPTION IN IN EMBRYONIC EMBRYONIC AND LARVAL LARVAL FISHES FISHES 700 700
1
I
/ m aww
600 600
-1
500
6 0 a. 4 0 0 E ..s
Y
� E m .2' .-
*/
I
egg capsule
I
!
i
I
� 300 30 0
w ater water
I
I I
I I
I I
200
100 100
yolk
0 0 0 0
60 60 Days
1I20 20
1I80 80
postfer t i l izat ion postfertilization
Fig. 3. Typical changes in the relative composition of fish during during the yolk absorp absorpFig. are from chinook salmon salmon (Oncorhynchus (Oncorhynchus tshawytscha) tshawytscha) at 8°C. 8°C. The tion period. period. Data are broken line represents 50% 50%hatching. hatching. Variability Variabilityabout some means is is indicated by 95% 95% broken pvf, perivitelline fluid; fluid; maww, maximum maximum eleutheroembryo wet confidence limits; limits; pvf, mhv, maximum tissue weight. [From [From Heming ((1982).] weight; mtw, 1982).]
low-moisture low-moisture yolk yolk is is converted converted into into low-density, low-density, high-moisture high-moisture tis tissues, sues, there there is is aa decrease decrease in in yolk yolk dry dry weight, weight, an an increase increase in in tissue tissue dry dry weight, weight, and and an an increase increase in in bulk bulk water water (yolk (yolk plus plus tissues). tissues). In In some some marine marine eleutheroembryos, eleutheroembryos, however, however, yolk yolk absorption absorption is is accompanied accompanied by bulk water Blaxter by aa decrease decrease in in bulk water content content [e.g. [e.g.,, plaice plaice P. platessa, Blaxter and (1974)], suggesting suggesting that that the the yolk yolk of of these these species species has has aa and Ehrlich Ehrlich (1974)J, higher higher moisture moisture content content than than the the developing developing tissues. tissues. Due Due to to the the catabolism catabolism of of some some yolk yolk materials materials for for energy energy produc production, of yolk yolk to to tissues tissues is is less less than than 100% 100% efficient efficient (see (see tion, the the conversion conversion of Section V). As As embryonic embryonic tissues tissues and and their their associated associated maintenance maintenance Section V). costs costs grow, grow, the the amount amount of of catabolic catabolic loss loss increases. increases. This This results results in in an an increasing 3).Tissue Tissue increasing loss loss of of bulk bulk dry dry matter matter (yolk (yolk plus plus tissues) tissues) (Fig. (Fig. 3).
418
THOMAS THOMAS A. HEMING AND RANDAL RANDAL K. K. BUDDINGTON
dry weight continues to increase, however, as yolk is absorbed and converted into tissues. As yolk reserves near exhaustion, the meta metabolic demands of maintenance and activity exceed the supply of yolk nutrients, and tissues begin to be catabolized for energy production. The resultant reduction in body weight causes a maximum tissue weight to be reached before yolk absorption is completed. Wallace and Aasjord (1984b) alpinus) at 3°e (1984b) found that Arctic char (Salvelinus (Saluelinus alpinus) 3°C reached maximum tissue weight at complete yolk absorption, whereas at 12°e 12°C maximum tissue weight was reached with 1.5 1.5 mg of of dry yolk remaining ((12% 12% of yolk reserves at hatching). The timing of hatching). of maximum tissue weight, and hence the end of growth utilizing yolk alone, would appear to be influenced by temperature, occurring earlier in develop development at higher temperatures. temperatures. This temperature dependency may re refl ect changes in maintenance costs (see (see Section V) or shifts in the flect relative rates of protein and lipid mobilization from yolk (see (see Section IV). The temperature effect may also explain why a maximum tissue IV). weight has not been evident prior to complete yolk absorption in some studies. The bulk wet weight of of the embryoleleutheroembryo embryo/eleutheroembryo increases during development despite the concurrent loss in bulk dry weight, because of of uptake of water. Gray (1928) (1928) modeled these early weight changes and predicted that, dependent on metabolic costs, costs, the bulk wet weight would reach a maximum before the tissue growth cycle was completed. completed. Thus, toward the end of the endogenous nutrition period, eleutheroembryo wet weight can be expected to decrease de despite a continued increase in tissue weight; the resultant maximum eleutheroembryo wet weight will be reached before the maximum (Fig. 3). 3). Maximum eleutheroembryo wet weight is tissue weight (Fig. reached earlier in development when relative metabolic costs are ele elevated, such as at higher rearing temperatures (Heming, (Heming, 1982) 1982) or in smaller eggs of a given species (Smith, (Smith, 1958; 1958; Escaffre and Bergot, 1984; 1984; Rombough, 1985). 1985).
v)
B. Protein B. Protein is the most abundant dry constitutent of sh eggs of many fi fish (Table I). Assuming the data in Table I1 (Table II reflect general trends, the majority of egg protein resides in the yolk; yolk; the remainder is associated primarily with the perivitelline fluid. fluid. Yolk protein serves two primary functions:: it provides amino acids for tissue growth and supplies en enfunctions ergy via catabolic processes. As a result, there is a continual loss of of
6. 6. YOLK YOLK ABSORPTION ABSORPTION
419 419
IN IN EMBRYONIC EMBRYONIC AND AND LARVAL LARVAL FISHES FISHES
Table II I1 Table Constituents of Freshly Fertilized and and Water-Hardened Water-Hardened Relative Distribution of the Constituents (Salmo salar)a salary Eggs of Atlantic Atlantic Salmon Salmon (Salmo ~~
Total Total
Percentage of total total weight Percentage
Constituent Constituent
weight weight (mg) (mg)
Egg membranes membranes
Perivitelline fluid fluid
Yolk Yolk
Water Dry Dry matter matter Protein Lipid Carbohydrate Carbohydrate Ash Ash
88.3 88.3 49.7 49.7 26.0 26.0 18.0 18.0 0.5 0.5 1.4 1.4
5.2 5.2 0.8 0.8 0.3 0.3 0. 1 0.1 48.0 48.0 7. 1 7.1
23.3 23.3 16.9 16.9 22.7 22.7 7.7 7.7 46.0 7. 1 7.1
71.8 71.8 82. 1 82.1 76.9 76.9 92.2 92.2 2.0 2.0 85.7 85.7
a
From 1977a). From Hamor Hamor and and Garside Garside ((1977a).
mass as it is is transferred to the developing tis tisprotein from the yolk mass sues. sues. In addition, addition, the catabolism of protein for energy production (tissue plus yolk). yolk). The results in a decline of bulk protein quantities (tissue significant exemplified importance of yolk protein is exemplifi ed by the signifi cant positive (Satia et correlation between egg protein content and early survival (Satia 1974). al., 1974). During early development, when the embryo is small and total is low, low, little if any protein is is utilized for energy metabolic activity is production; bulk protein quantities remain relatively constant. As growth proceeds and the metabolic rate increases, a larger portion of quanyolk protein is shunted into energy production and bulk protein quan tities decline. This is especially evident after hatch, in accordance with the higher levels of activity and energy demand. This trend has 1977; Dabrowski and been reported for salmonids (Zeitoun (Zeitoun et al., 1977; (Kamler, 1976), 1976), and gadids (Lapin (Lapin and Luczynski, 11984), 984), cyprinids (Kamler, 1979; Buckley, 198 1981), conMatsuk, 1979; 1 ), among others. Studies of oxygen con aE., 1982; (Kaushik et al., sumption and nitrogen metabolism (Kaushik 1982; Cetta and 1982; Dabrowski et al., al., 1984) 1984) also support an increased use Capuzzo, 1982; Capuzzo, difficult, however, of protein for energy production after hatching. It is difficult, to directly determine protein catabolism before hatching because of the low permeability of egg membranes to nitrogenous metabolites 1971; Stokes, 1974). 1974). (Yarzhombek and Maslennikova, 197 (Yarzhombek 1 ; Rice and Stokes, abunThere are three major classes of yolk proteins. In order of abun lipoproteins, glycoproteins, and phosphoproteins dance, these are lipoproteins, Nakagawa, 1970). develop(Nakagawa and Tsuchiya, 1969; (Nakagawa 1969; Nakagawa, 1970). During develop ment, there appears to be little change in the relative proportion of
420
THOMAS THOMAS A. A. HEMING HEMING AND AND RANDAL RANDAL K. K. BUDDINGTON BUDDINGTON
these yolk proteins these yolk proteins (Nakagawa (Nakagawa and and Tsuchiya, Tsuchiya, 1972). 1972). This This suggests suggests the the three utilized at However, the three protein protein types types are are utilized at the the same same relative relative rate. rate. However, the physical and chemical properties physical and chemical properties of of yolk yolk lipoprotein lipoprotein change change during during development molecular weight weight in 1974); molecular indevelopment (Nakagawa (Nakagawa and and Tsuchiya, Tsuchiya, 1974); creases, lipid groups specific amino acids creases, the the prosthetic prosthetic lipid groups decrease, decrease, and and specific amino acids are embryos develop yolk protein protein are released. released. Fish Fish embryos develop at at the the expense expense of of yolk degradation 1). 1961). degradation and and the the liberation liberation of of amino amino acids acids (Monroy (Monroy et al., 196 Changes Changes in in amino amino acid acid concentrations concentrations during during development development appear appear to to parallel (Zeitoun et parallel changes changes in in the the concentrations concentrations of of DNA DNA and and RNA (Zeitoun al., 1977). 1977). This This suggests suggests that that the the changes changes are are associated associated with with periods periods of tissue growth. of varying varying tissue growth. It is interesting It is interesting to to speculate speculate about about possible possible preferential preferential retention retention of of specific, specific, particularly particularly essential, essential, amino amino acids. acids. Preferential Preferential retention retention might expected to amino might be be expected to ensure ensure that that sufficient sufficient amounts amounts of of essential essential amino acids acids are are available available for for protein protein synthesis. synthesis. Presently, Presently, we we do do not not have have an an adequate understanding of specific amino adequate understanding of how how specific amino acids acids might might be be re retained. is possible possible that that processes processes associated associated with with their their catabolism catabolism tained. It It is are are suppressed suppressed or or not not developed developed until until later later in in development, development, thereby thereby preventing preventing early early losses. losses. C. C. Lipid
Following protein, lipids are the next most abundant dry constitu constituent of most fish eggs (Tables (Tables I and II). interspe 11).There is considerable interspecific however; Balon Balon ((1977) 1977) lists lists lipid lipid quantities quantities ranging ranging cific variation, variation, however; from 0. 1% of egg weight in the plaice P. 0.1% P . platessa to 45% in the mouth mouthbrooding cichlid Labeotropheus. Labeotropheus. This variation has been considered an 1977) or or to to an adaptation adaptation to to different different reproductive reproductive strategies strategies (Balon, (Balon, 1977) the the duration duration of of the the endogenous endogenous nutrition nutrition period period (Kaitaranta (Kaitaranta and and Ack Ackman, oil globules 1981). Lipid Lipid content content and and the presence presence of large large oil globules are are man, 1981). poorly correlated wi
