Advances in
INSECT PHYSIOLOGY
VOLUME 9
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Insect Physiology Edited b...
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Advances in
INSECT PHYSIOLOGY
VOLUME 9
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Advances in
Insect Physiology Edited by
J. E. TREHERNE, M. J. BERRIDGE and V. B. WIGGLESWORTH Department of Zoology, The University, Cambridge, England
VOLUME 9
1972
ACADEMIC PRESS London and New York
ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road, London NW1
United States Edition published by ACADEMIC PRESS INC. 1 11 Fifth Avenue New York, New York 10003
Copyright 0 1972 by ACADEMIC PRESS INC. (LONDON) LTD.
All Rights Reserved
No part of this book may be reproduced in any form by photostat, microfdm, or any other means, without written permission from the publishers Library of Congress Catalog Card Number: 63-14039 ISBN: 0-12-024209-5
PRINTED IN GREAT BRITAIN BY THE WHITEFRIARS PRESS LTD., LONDON AND TONBRIDGE
List of Contributors to Volume 9 B. BACCETTI, Institute of Zoology, University of Siena, Italy (p. 3 15) M. J. BERRIDGE, Agricultural Research Council Unit of Invertebrate Chemistry and Physiology, Department of Zoology of Cambridge, Downing Street, Cambridge, England (p. 1 ) R. G. BRIDGES, Agricultural Research Council Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge, England (p. 5 1 ) E. M. EISENSTEIN, Department of Biophysics, 128 Chemistry Building, Michigan State University, East Lansing, Michigan 48823, U.S.A. (p. 11 1 ) P. W. MILES, School of Natural Sciences, University of Zambia, P.O. Box 2379, Lusaka, Zambia (p. 183) Y. PICHON, Institut de Neurophysiologie, C.N.R.S., Gif-mr-Yvette 92, France (P. 257) W. T. PRINCE, Agricultural Research Council Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge, England (p. 1 ) J . E. TREHERNE, Agricultural Research Council Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge, England (p.257)
V
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Contents LIST OF CONTRIBUTORS TO VOLUME 9
. . . . . . . . . .
v
THE ROLE OF CYCLIC AMP AND CALCIUM IN HORMONE ACTION MICHAEL J . BERRIDGE AND WILLIAM T . PRINCE Introduction . . . . . . . . . . . . . . . . . . I1. The Structure and Function of Calliphora Salivary Glands . . . 111. The 5-HT-receptor Interaction . . . . . . . . . . . . IV . The Role of Cyclic AMP and Calcium as Intracellular Messengers . A. The Role of Cyclic AMP . . . . . . . . . . . . B. The Role of Calcium . . . . . . . . . . . . . . V. TheMode of Action of Cyclic AMP andcalcium . . . . . . A. The Effect of 5-HT and Cyclic AMP on Potential . . . . B. Evidence for the Independent Action of Cyclic AMP and . . . . . . . . . . . Calcium on Ion Transport C. The Time Course of the Cyclic AMP and Calcium Effects . VI . Control of Secretion-A Model of Hormone Action . . . . . VII . Comparison of 5-HT Action with that of Other Hormones . . . A. Aggregation of the Slime Mould, Dictyostelium discoideum . B. Synaptic Transmission-The Role of Cyclic AMP and Calcium inPre-andPost-synaptic Events . . . . . . . C. The Action of Epinephrine on the Heart . . . . . . . D. Excitation-Secretion Coupling . . . . . . . . . . E. Control of Metabolism . . . . . . . . . . . . . F. Transporting Epithelia-The Toad Bladder . . . . . . VIII . Conclusion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . Notes Added in Proof . . . . . . . . . . . . . . . . . . I.
1 2 5 12 12 19 21 23 26 28 31 32 33 34 36 36 37 39 41 42 49
CHOLINE METABOLISM IN INSECTS R . G. BRIDGES I. I1.
111.
IV .
Introduction . . . . . . . . . . . . . . . . . . 5 1 Choline Metabolism in Vertebrates . . . . . . . . . . 53 Nutritional Requirements of Insects for Choline . . . . . 55 A. Choline in Insect Development . . . . . . . . . 55 B. Substitutes for Choline in the Diet of Insects . . . . 59 Water-soluble Choline Metabolites . . . . . . . . . . 63 A. Acetylcholine . . . . . . . . . . . . . . . 63 B. Phosphorylcholine . . . . . . . . . . . . . . 66 vii
viii
CONTENTS
. . Cytidinediphosphorylcholine (CDP-choline) Glycerylphosphorylcholine . . . . . . . . V . Lipid-soluble Choline Metabolites . . . . . . . . A. Phosphatidylcholine . . . . . . . . . . B. Lysophosphatidylcholine . . . . . . . . . C. Sphingomyelin . . . . . . . . . . . . VI . Enzymes Involved in Choline Metabolism . . . . . A. Cholineacetylase and Acetylcholinesterase . . . B. Enzymic Synthesis of Lipids Containing Choline C. Hydrolysis of Phosphatidylcholine . . . . . D . Oxidation of Choline . . . . . . . . . . E. Synthesis of Choline . . . . . . . . . . VII . The Metabolic Role of Choline in Insects . . . . . Acknowledgements . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . C.
D.
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . .
69 70 71 71 82 83 84 84 85 87 88 89 91 100 100
LEARNING AND MEMORY IN ISOLATED INSECT GANGLIA E. M . EISENSTEIN I.
I1.
Introduction . . . . . . . . . . . . . . . . . A . The Concept of Learning . . . . . . . . . . . B. Towards a Reformulation of the Concept of Learning . C. The Use of “Model” Systems in Biology . . . . . . Behavioral Investigations . . . . . . . . . . . . . A. Intact and Headless Preparations . . . . . . . . B. Isolated Ganglion-P and R as Separate Preparations . .
.
C
IV .
V.
. .
.
112 113 115 117 118 118 128
. . . 132
Effects of Other CNS Lesions on P and R Behavior . . . Behavior of Ganglionless P and R Preparations . . . . . Discussion of P and R Leg Behavior in the Presence of Ganglionic Innervation of the Legs . . . . . . . . . G. Discussion of P and R Leg Behavior in the Absence of Ganglionic Innervation of the Legs (Ganglionless) . . . . Some Histological and Anatomical Findings Potentially Relevant to Understanding the Cellular Basis of Learning in the Cockroach . A. Introduction . . . . . . . . . . . . . . . . B. Mapping of the Cockroach Metathoracic Ganglion . . . C. Ganglion Transplantation Studies in the Cockroach . . . Electrophysiological Investigations of Learning . . . . . . A. Introduction . . . . . . . . . . . . . . . . B. Habituation Studies . . . . . . . . . . . . . C. Discussion of Habituation . . . . . . . . . . . D. Instrumental Learning Studies . . . . . . . . . . E . Classical Conditioning Studies . . . . . . . . . . F. Some Newer Electrophysiological and Analytical Approaches to Learning and Memory . . . . . . . . Molecular Approaches to Learning and Memory Storage . . . A . Introduction . . . . . . . . . . . . . . . . D. E. F.
111.
Isolated Ganglion-P and R in the Same Preparations
. . . .
136 137 140
146 149 149 149 150 150 150 150 156 157 162 164 167 167
ix
CONTENTS
B.
Assessing the Effects of Drugs on Learning and Memory in the Cockroach . . . . . . . . . . . . . . Some Speculations on the Future of the Chemical Approach C. VI . Summary . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
168 175 176 178 178
THE SALIVA OF HEMIPTERA PETER W . MILES I.
Introduction . . . . . . . . . . . . . . . . . . Methods of Investigation . . . . . . . . . . . . . . 111. Modes of Feeding . . . . . . . . . . . . . . . . IV. Stylet-Sheath Feeding . . . . . . . . . . . . . . . A. Sampling the Surface . . . . . . . . . . . . . Secretion of the Flange . . . . . . . . . . . . B. Formation of the Sheath . . . . . . . . . . . . C. D. Discharge of Watery Saliva . . . . . . . . . . . E. Ingestion . . . . . . . . . . . . . . . . . F. Withdrawal of the Stylets . . . . . . . . . . . . V. Lacerate-and-Flush Feeding . . . . . . . . . . . . . VI . Feeding by Carnivores . . . . . . . . . . . . . . . VII. Chemical Composition and Function of the Saliva . . . . . A . Sheath Material . . . . . . . . . . . . . . . Watery Saliva . . . . . . . . . . . . . . . . B. VIII . Phytopathogenicity . . . . . . . . . . . . . . . . IX. Salivary Glands and Ducts . . . . . . . . . . . . . A. Aphidoidea . . . . . . . . . . . . . . . . Jassomorpha . . . . . . . . . . . . . . . . B. C. Fulguromorpha . . . . . . . . . . . . . . . D. Other Auchenorrhyncha . . . . . . . . . . . . E. Heteroptera . . . . . . . . . . . . . . . . X . Origins of the Saliva . . . . . . . . . . . . . . . A . Functions of the Accessory Gland . . . . . . . . . B. Functions of the Principal Gland . . . . . . . . . C. Sources of Oxidases . . . . . . . . . . . . . D. Sources in the Homoptera . . . . . . . . . . . E. Salivary Carbohydrate and Lipid . . . . . . . . . XI . The Saliva as a Vehicle for Pathogens . . . . . . . . . . XI1. Evolution of Salivary Function in the Hemiptera: a Summary . . XI11. A Survey of Problems . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
I1.
183 185 191 194 194 195 196 197 200 201 202 203 205 205 208 217 225 226 229 232 233 234 236 236 237 238 239 240 241 244 247 250
THE INSECT BLOOD-BRAIN BARRIER
J . E. TREHERNE AND Y . PICHON
I. I1.
Introduction . . . . . . . . . Organization of Insect Nervous Tissues A. Extraneural Fat Body Deposits .
. . . . . . . . . . . . . . . . . . . . . . . . . . .
257 260 260
CONTENTS
X
B . The Neural Lamella . . . . . . . . . . . . . . C. The Perineurium . . . . . . . . . . . . . . . D. Glial Cells. Neurones and Extracellular Spaces . . . . . 111. The Ionic Composition of the Haemolymph and Nervous Tissues . IV . Electrical Aspects of Nervous Function . . . . . . . . . A. The Ionic Basis of Electrical Activity in Insect Nerve Cells . B. The Role of the Neural Fat Body Sheath . . . . . . . C. Neuronal Function in Intact Nervous System . . . . . D. Neuronal Function in Experimentally Treated Preparations . V . Exchanges of Radioactive Ions and Molecules . . . . . . . VI . Concluding Remarks . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . .
264 266 268 274 277 277 278 281 289 291 299 305 305 312
INSECT SPERM CELLS BACCIO BACCETTI I.
I1.
111.
Introduction . . . . . . . . . . . . . . . . . . 316 The Cell Surface . . . . . . . . . . . . . . . . . 317 The Acrosomal Complex . . . . . . . . . . . . . . 324 A. The Typical Triple-layered Insect Acrosomal Complex . . 324 B. The Bilayered Acrosomal Complex . . . . . . . . 326 C. The Acrosomal Complex with Only Two Outer Layers . 327 D. Monolayered Acrosomal Complex . . . . . . . . . 327 E. Total Absence of Acrosomal Complex . . . . . . . 328 The Nucleus . . . . . . . . . . . . . . . . . . 328 . . . . . . . . . . . . . . . 328 A. Nuclear Shape B . Submicroscopic Structure . . . . . . . . . . . 329 C. Chemical Characteristics . . . . . . . . . . . . 331 . . . . . . . . . . . . 331 D. Physical Characteristics The Centriolar Region . . . . . . . . . . . . . . . 332 A. The Centriole . . . . . . . . . . . . . . . . 332 B. The Centriole Adjunct . . . . . . . . . . . . . 333 C. The Initial Segment of the Axoneme . . . . . . . . 335 . . . . . . . . 336 The Axial Flagellar Filament or Axoneme A. The Microtubules . . . . . . . . . . . . . . 338 B. The Central Sheath . . . . . . . . . . . . . . 349 C. The Link-heads . . . . . . . . . . . . . . . 349 D. The Coarse Fibres . . . . . . . . . . . . . . 350 E. The Axonemal Matrix . . . . . . . . . . . . . 352 Mitochondria . . . . . . . . . . . . . . . . . . 354 A. Normal Mitochondria . . . . . . . . . . . . . 354 B . Mitochondria Transformed into Derivatives with a Crystalline Core . . . . . . . . . . . . . . . 354 C. Absence of Mitochondria . . . . . . . . . . . . 360 Accessory Ordered Flagellar Bodies . . . . . . . . . . 363 A. Structured Bodies Flanking Normal Mitochondria . . . . 363
.
IV.
V.
VI.
VII .
VIII .
xi
CONTENTS
B.
Structured Bodies Flanking the Mitochondrial Derivatives with a Crystalline Matrix . . . . . . . . . . . . C. Structured Bodies Replacing Mitochondria . . . . . . IX . Spermatozoa Possessing a Double Flagellar Apparatus or Being Devoid of it . . . . . . . . . . . . . . . . . . A. The Paired Spermatozoa . . . . . . . . . . . . B . Spermatozoa Possessing Two Axonemes . . . . . . . C. Non-flagellate Spermatozoa . . . . . . . . . . . X . Motility . . . . . . . . . . . . . . . . . . . A . Motile Mechanisms . . . . . . . . . . . . . . Metabolic Aspects of Motion . . . . . . . . . . B. The Problem of Sperm Capacitation . . . . . . . . C. XI . Spermatozoa Polymorphism and Genetics . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .
AUTHOR INDEX SUBJECT INDEX CUMULATIVE LIST OF AUTHORS CUMULATIVE LIST OF CHAPTER TITLES
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . . . . . . . . . . . .
365 365 367 367 369 370 374 374 380 381 382 384 384 399 413 435 437
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The Role of Cyclic AMP and Calcium in Hormone Action MICHAEL J. BERRIDGE AND WILLIAM T. PRINCE
A.R. C Unit of Invertebrate Chemistry and Physiology, Department of Zoology, Cambridge, England I. Introduction . . . . . . . . . . . . . . . . . . 11. The Structure and Function of Calliphora Salivary Glands . . .
The 5-HT-receptor Interaction . . . . . . . . . . . . The Role of Cyclic AMP and Calcium as Intracellular Messengers . A. The Role of Cyclic AMP . . . . . . . . . . . . B. The Role of Calcium . . . . . . . . . . . . . V. The Mode of Actionof Cyclic AMPand Calcium . . . . . . A. The Effect of 5-HT and Cyclic AMP on Potential . . . . B. Evidence for the Independent Action of Cyclic AMP and Calcium on Ion Transport . . . . . . . . . C. The Time Course of the Cyclic AMP and Calcium Effects . VI. Control of Secretion-A Model of Hormone Action . . . . . VII. Comparison of 5-HT Action with that of Other Hormones . . A. Aggregation of the Slime Mould, Dictyostelium discordeum . B. Synaptic Transmission-The Role of Cyclic AMP and Calcium in Pre- and Post-synaptic Events . . . . . . C. The Action of Epinephrine on the Heart . . . . . . D. Excitation-Secretion Coupling . . . . . . . . . . E. Control of Metabolism . . . . . . . . . . . . . F. Transporting Epithelia-The Toad Bladder . . . . . . VIII. Conclusion . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . Notes Added in Proof . . . . . . . . . . . . . . . . . . 111. IV.
.
.
1 2 5 12 12 19 21 23 26 28 31 32 33 34 36 36 37 39 41 42 49
I. INTRODUCTION
Coordination of cell activity in higher animals is achieved through various chemical messengers which are released by one cell population to influence another. Most hormones function in long range communication where the blood system carries the chemical messages to the target cells. However, communication can also occur over a short distance, such as in the synapse, where chemicals diffuse across the narrow synaptic gaps to transfer information between two AIP-2
1
2
M. J. BERRIDGE AND W. T. PRINCE
excitable cells. In both forms of communication the basic mechanism is the same-a simple chemical message is used to transfer information from one cell to another. This review attempts to summarize the progress which has been made in understanding how one particular cell system, the salivary gland of an insect, receives a chemical stimulus and translates the information into a change in activity. The salivary glands of the adult blowfly, Cullipkoru erytkrocephulu have unique structural and physiological properties which have enabled us to analyse the sequence of events which occur during cell activation by a specific hormone. Berridge and Pate1 (1 968) found that fluid secretion by isolated salivary glands was regulated by 5-hydroxytryptamine (5-HT). This simple biogenic amine may be an important secretogogue in insects because Whitehead (197 1) has evidence that 5-HT may be released from the nerves which innervate the salivary glands of the cockroach, Periplanetu americuna. In the cockroach, the increase in fluid secretion observed during nerve stimulation can be mimicked by externally applied 5-HT. Three major events have been recognized during stimulation of fluid secretion by 5-HT in the salivary glands of Cullipkoru (Berridge, 1970, 1972; Berridge and Prince, 1971, 1972a, b; Prince and Berridge, 1972; Prince et ul., 1972). Firstly, 5-HT recognizes and interacts with a specific cellular receptor. Secondly, the result of a successful 5-HT-receptor interaction is decoded into an increase in the concentration of two intracellular messengers, cyclic AMP and calcium. Thirdly, cyclic AMP and calcium are then responsible for stimulating the transport processes of the cell to produce an increase in fluid secretion. This work on Calliphoru salivary glands which has led to a simple hypothesis of the mode of action of 5-HT will form the first part of this review. In the second part, some of the general concepts which have emerged from these salivary gland studies will be discussed in relation to the mode of action of other hormones. 11. THE STRUCTURE AND FUNCTION OF CALLIPHORA
SALIVARY GLANDS
The salivary glands of the adult blowfly are paired tubules which extend down the length of the animal. That part of the gland which lies in the abdomen is uncoiled but after entering the thorax the gland forms a tangled mass which lies on either side of the
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
3
proventriculus. Each tube empties into a small elongated bulb which is connected anteriorly to a cuticle-lined duct. The two salivary ducts converge and unite in the thorax to form a common duct which conveys saliva to the mouthparts. The gland has three major cell types: secretory, reabsorptive and duct cells (Oschman and Berridge, 1970). The abdominal region of the gland and most of the convolutions in the thorax consist of a single cell type (Fig. 1A). These secretory cells are characterized by extensive canaliculi formed by elaborate infoldings of the apical plasma membrane (Oschman and Berridge, 1970). These canaliculi and the free apical surface are lined with sheet-like microvilli arranged in parallel arrays. The lateral plasma membrane is relatively straight and there are septate desmosomes in the apical region. The basal plasma membrane has infoldings which are closely associated with mitochondria. These basal infoldings often come very close (less than 1 p ) to the bottom of the canaliculi. Large mitochondria with perforated cristae are scattered throughout the cytoplasm. When stimulated with 5-HT these secretory cells produce an isosmotic fluid consisting primarily of potassium and chloride (Fig. 1) (Oschman and Berridge, 1970; Berridge and Prince, unpublished). This primary secretion then passes down through a short region of the gland which is lined with a simple squamous epithelium (reabsorptive cells). Reabsorptive cells (Fig. 1B) are much less elaborate in that there are no secretory canaliculi. The apical surface has a few short microvilli resembling those of the secretory cells. The short wide basal infoldings are often closely associated with mitochondria. Various experimental procedures have revealed that this region of the gland reabsorbs potassium thus diluting the saliva (Fig. 1). This mechanism of isosmotic fluid secretion followed by hyperosmotic reabsorption to produce a dilute fluid closely resembles the mechanism of saliva production in mammalian salivary glands (Mangos e t al., 1966; Young and Schogel, 1966). That part of the gland which lies in the abdomen and is responsible for secreting the primary saliva has been used to study the mode of action of 5-HT. Secretory activity can be monitored in vitro using a technique originally devised by Ramsay ( 1954) to study isolated Malpighian tubules. When set up in control saline salivary glands secrete very slowly (0.5-1 .O nl/min) but 1 min after addition of 1 x M 5-HT the rate suddenly accelerates to more than 40 nl/min (Berridge, 1970). This high rate of secretion is maintained
4 M. J. BERRIDGE AND W. T. PRINCE
Fig. 1. The salivary gland of the blowfly. Each gland is a tubular structure divided into a secretory (A) and a reabsorptive (B) region. Schematic diagrams of the cells found in these two regions are shown. In the secretory region (A) water and chloride follow the transport of potassium resulting in the formation of an isosmotic fluid. In the reabsorptive region (B) potassium and chloride are reabsorbed so that the secreted saliva is hypotonic. bm, basement membrane; bi, basal infold; c, canaliculus;
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
5
for as long as 5-HT is present but returns to the resting level 1-2 min after 5-HT is withdrawn. This very rapid response and recovery implies that the 5-HT-receptor interaction is readily reversible. Since these glands are composed of a single cell type whose secretory activity can be monitored continuously, they are particularly suited for analysing the sequence of events which occur during hormone action. The first of these events is the molecular interaction between the stimulating molecule, in this case 5-HT, and its receptor. 111. THE 5-HT-RECEPTOR INTERACTION
The molecular pharmacology of 5-HT has been approached by a study of the structure-activity relationships of a wide range of analogues of 5-HT (Berridge, 1972).
I
5-HT (I) consists of an indole ring nucleus with an ethylamine chain at the 3-position and a hydroxyl group at the 5-position. The indole ring system has a high electron density with the delocalizedn electrons smeared over the two rings to form a rigid planar structure. The positions of the hydroxyl group and the 0-carbon atom are fixed by their connection to this rigid ring system. The only parts of the molecule which have any degree of freedom are the a-carbon atom and the terminal nitrogen atom of the ethylamine side chain. The activity of such compounds is usually determined by the charge distribution over the molecule. The main centre of charge resides on the ethylamine nitrogen atom which is basic and accepts a proton at physiological hydrogen ion concentrations to become positively charged (I). The hydroxyl group at the 5-position has a partial negative charge resulting from its negative inductive effect on the 71 electrons of the indole ring. These two centres of charge will be separated from each other by the hydrophobic indole ring. The activity of molecules such as 5-HT depends on two important features: (i) a high affinity for the receptor which enables them to recognize and interact with the receptor at very low concentrations and
6
M. J. BERRIDGE A N D W. T. PRINCE
(ii) once attached to the receptor they must be capable of inducing an effect often referred to as intrinsic activity. Antagonists have a high affinity for the receptor but cannot induce an effect, i.e. they have no intrinsic activity. The first experiments on 5-HT pharmacology determined which part of the molecule was responsible for its intrinsic activity (i.e. the active site on the molecule) and which part confers upon it such a high degree of specificity. A positively charged ethylamine side-chain is an important component of a wide range of biologically active compounds including histamine, y-aminobutyric acid and dopamine and is probably the active site on all these molecules. When the two major parts of the 5-HT molecule, 5-hydroxyindole and ethylamine, were tested separately they failed to activate salivary glands. When these two major parts were presented together at very high concentrations ( l oe2 M ) they still failed to stimulate secretion. The action of 5-HT thus depends on the corporate effect of both these moieties. The importance of the ethylamine chain can be demonstrated by testing a range of homologues in which the length of the chain is increased by the addition of extra carbon atoms (Fig. 2). Increasing the chain length causes a progressive increase in activity so that amylamine, which has a 5-carbon chain, stimulates the glands maximally (i.e. it has the same intrinsic activity as 5-HT). However, very high concentrations were required to stimulate secretion indicating that these simple straight chain compounds have a very low affinity for the receptor. The groups used for increasing the chain length must be hydrophobic because there is no activity if the chain terminates with a hydrophilic group as in ethanolamine (11), ethylenediamine (111) or y-aminobutyric acid (GABA) (IV). COOHCHz CH2 CH2 NH; 'NH3 CHZ CH2 NH; HOCHz CHZ NH: (11) (111) The requirement for a hydrophobic group at one end of the ethylamine chain implies that interaction of the positively charged quartenary nitrogen atom with an anionic site on the receptor requires hydrophobic interaction with the opposite end of the molecule. A progressive increase in the length of the carbon chain permits this hydrophobic interaction to develop with a resulting increase in activity (Fig. 2). That high concentrations of these compounds are needed to produce activity suggests that they have low affinities for the receptor possibly because their interaction with the hydrophobic site is unspecific.
(rv)
7
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
The specificity of the hydrophobic site was investigated by testing compounds where ethylamine was attached to various ring structures (Berridge, 1972). Molecules containing a benzene ring (phenethylamine) or an imidazole ring (histamine) were more active than the straight chain homologues of ethylamine, but were still very much less active then tryptamine or 5-HT which have an indole ring (Fig. 2). Thus we can conclude that the specificity of 5-HT depends on a high degree of complementarity between the indole ring and a hydrophobic site on the receptor. 5-Hydroxytryptamine
c'
N*
Tryptamine 'N
Phenethylamine C/N+ C '",
+
Histamine 0N C C." \
+ +
uc "rc c-c-c-c-N
Log concentration
I
I
I
10-s
10-4
10-3
I
+
ethylamine
10-2
(M)
Fig. 2. The dose-response curves of isolated salivary glands to 5-HT and some 5-HT-like compounds. (From Berridge, 1972.)
Another problem to consider is the role of the hydroxyl group in the 5-HT-receptor interaction. Removal of the hydroxyl group causes a thousand-fold reduction in affinity but no change in intrinsic activity because tryptamine is capable of inducing a maximal response (Fig. 2). A similar reduction in activity is observed when dimethyl tryptamine (V) is compared with the hydroxylated derivative bufotenine (VI) (Berridge, 1972). The effect of the hydroxyl group was also assessed by comparing the activity of tryptophane (VII) and 5-hydroxytryptophane (VIII). The former is totally inactive whereas the latter is capable of stimulating secretion although both the affinity and intrinsic activity are reduced. The ability of the hydroxyl group to increase the activity of the three combinations tested implies that this group increases the affinity
8
M. J. BERRIDGE AND W. T. PRINCE
+p C-C-NH
+ /CH3
3
C-C-NH
m
Y COOH
COOH
possibly through hydrogen bonding. The position of the hydroxyl group is critical because its displacement to the 4- or 6-positions causes a thousand-fold decrease in activity similar to that observed in the absence of the hydroxyl group. These results imply that there is a hydrogen bonding site on the receptor which has a precise orientation with respect to the hydrophobic portion of the receptor which reacts with the indole ring. Having determined the contribution of the different regions of the molecule to its interaction with the receptor, it remains to establish the conformation of the ethylamine side chain as the molecule attaches itself to the receptor. As mentioned earlier, the a-carbon atom and the terminal nitrogen atom have considerable degrees of freedom and could assume various configurations as shown in Fig. 3. The &-carbon atom is free to rotate around the 0-carbon atom and can thus assume any position represented by the base of the cone I. Similarly, for most of the possible positions of the a-carbon atom, the nitrogen atom could assume a number of positions represented by the base of the second cone 11. A clue to the natural configuration of 5-HT can be obtained by testing certain molecules where this ethylamine side chain is fixed in a rigid configuration by being incorporated into additional ring systems. Preliminary unpublished experiments have shown that the lysergic acid derivative bromolysergic acid diethylamide (BOL) (IX) can stimulate secretion whereas various harmala alkaloids such as harmaline (X) are totally inactive. However, these harmala alkaloids have a high affinity for the receptor because they are potent competitors; this indicates that they have most of the attributes for a successful 5-HT-receptor interaction but once attached to the receptor they cannot induce an effect. This can be explained by a comparison of the position of the
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
9
HO
a
b
C
Fig. 3.
The orientation of the ethylamine sidechah of 5-HT. Rotation can occur around the p and a carbon atoms as indicated by the cones I and I1 respectively. a, b, and c show three possible conformations. Structure-activity relationship studies indicate that the active conformation is probably that in a.
nitrogen atom in the harmaline molecule with that in the active BOL molecule which suggests that the natural configuration of the ethylamine side chain is similar to that shown in Fig. 3a.
CH3
Ix
X
On the basis of these structure-activity relationships we can speculate about some of the properties of the 5-HT-receptor and the sequence of events which occur during a 5-HT-receptor interaction. The specificity of 5-HT resides in the 5-hydroxyindole ring system which suggests that the receptor must have a hydrophobic site which is precisely designed to accept the indole ring (Fig. 4).In relation to
10
M. J. BERRIDGE AND W. T. PRINCE
I
'
Electrostatic
\ forces
I I
I I
a
b
C
Fig. 4. The proposed 5-HT-receptor interaction. The receptor consists of three major regions-a hydrophobic site, an anionic site and a hydrogen bonding site (6' ). a. The anionic site attracts the positively-charged group of 5-HTby long range electrostatic forces. b. The final approach on to the receptor depends on hydrogen bonding and van der Waal's forces. c. The indole ring occupies the hydrophobic site and allows the po8itively-charged amino group to interact with the anionic site to produce a successful 5-HT-receptor interaction.
this hydrophobic site the position of the hydrogen bonding site is critical since displacement of the hydroxyl group from the 5- to the 4-or 6-position markedly reduces activity. Situated on the opposite side of the hydrophobic site there must be an anionic site which reacts with the positively charged nitrogen atom. The results with BOL and the harmala alkaloids suggest that this anionic site has a precise location relative to the other parts of the receptor as shown in Fig. 4. Thus, we suggest that the 5-HT molecule is firmly attached to the receptor by means of three different chemical bonds: (i) an electrostatic bond with the anionic site; (ii) hydrophobic bonding (e.g. van der Waal's forces) with the hydrophobic site; (iii) hydrogen bonding involving the 5-hydroxyl group.
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
11
It is of some interest to speculate on the way in which these various interactions develop as 5-HT approaches and occupies the receptor. There is every reason to suppose that the event is not a haphazard collision but entails an orderly approach sequence with some degree of mutual moulding as the agonist approaches the receptor. The initial attraction and guidance towards the receptor is probably carried out by long range electrostatic forces operating between the positively charged nitrogen atom and the anionic site on the receptor (Fig. 4). Such long range forces are non-specific and a wide range of positively charged molecules will be drawn towards the anionic site. However, interaction does not occur unless there is a high degree of complementarity between the molecule and the whole receptor; in order to get closer to the receptor the approaching molecule must satisfy the more stringent requirements of the hydrophobic and hydrogen bonding sites. lndeed the hydrophobic site may protect the anionic site from the many and varied positively charged ions and molecules which will be drawn towards the negative charge on the receptor. Only 5-HT and its closely related derivatives have chemical conformations satisfying the precise requirements for combination with the hydrophobic site and the hydrogen bonding site. The hydroxyl group on 5-HT may play an additional role by ensuring that the indole ring is correctly positioned over the hydrophobic receptor site during this final approach towards the receptor. As these short-range forces pull the molecule down onto the receptor the cationic head is brought close enough to the anionic site to form an effective electrostatic bond. Probably it is this neutralization of the anionic site that initiates the hormonal effect. These 5-HT structure-activity relationships in Culliphoru salivary glands resemble those found in the isolated rat stomach strip (Vane, 1959; Offermeier and Ariens, 1966) or the heart of a mollusc (Greenberg, 1960). This basic similarity suggests that 5-HT receptors from widely different tissues possess many common features. An interesting exception to this generalization is the action of 5-HT on the Malpighian tubules of Rhodnius. Under normal conditions fluid secretion by these Malpighian tubules is stimulated by a diuretic hormone released from the fused ganglionic mass in the mesothorax (Maddrell, 1963). The action of the diuretic hormone can be simulated by 5-HT (Maddrell et al., 1971). However, structureactivity studies revealed that the receptor was very different to the 5-HT receptors described above. In particular, the presence of a hydroxyl group at the 5-position is essential for the activity of 5-HT in Rhodnius. Removal of the hydroxyl group from a series of active
12
M. J . BERRIDGE AND W. T. PRINCE
5-HT analogues converts them into powerful antagonists. In the case of these Malpighian tubules, therefore, 5-HT may be acting on the receptor normally occupied by the diuretic hormone which is thought to be a polypeptide (Maddrell, personal communication). It has been suggested that the chemical conformation of 5-HT may closely resemble the configuration of the active site on the diuretic hormone (Maddrell et al., 197 1). IV. THE ROLE OF CYCLIC AMP AND CALCIUM AS INTRACELLULAR MESSENGERS
Once 5-HT has interacted with the receptor the resulting chemical signal input is translated into intracellular messengers which are then responsible for altering cell activity. Adenosine 3',5'-monophosphate (cyclic AMP) plays a central role in mediating the action of a wide range of hormones (Robison et al., 1968). However, Rasmussen (1970) has recently suggested that calcium may be an equally important intracellular mediator of hormone action. Both these messengers appear to mediate the action of 5-HT in salivary glands. A. THE ROLE OF CYCLIC AMP
The importance of cyclic AMP in hormone action was first recognized by Sutherland and Rall(l958) who showed that it played an essential role in the hyperglycemic actions of glucagon and epinephrine on the liver. Since then this compound has been implicated in cellular control mechanisms in a wide range of organisms from bacteria to mammals (Robison et al., 1968). The concentration of cyclic AMP in most cells is determined by the balance which exists between the synthetic enzyme, adenyl cyclase, and the degradative enzyme, phosphodiesterase (Fig. 5). The level of cyclic AMP is regulated by altering the activity of adenyl cyclase which represents the site of action of most hormones as well as certain neurotransmitters (Robison et al., 1967, 1968). One action of 5-HT on salivary glands is to stimulate adenyl cyclase which increases the intracellular level of this second messenger (Berridge, 1970; Prince et al., 1972). Direct measurement of the cyclic AMP concentration showed that the control level was 0.06-0.10 p mol/gland but 2 min after the addition of 5-HT there was a 3-4-fold increase in cyclic AMP level (Fig. 6 ) . After this initial rise the concentration of cyclic AMP declines slightly to an equilibrium level which then remains constant. In the absence of calcium, the cyclic
13
CYCLIC AMP AND CALCIUM IN HORMONE ACTION Basal plasma membrane
NH2
Theophylline
5'-AMP
ATP
Fig. 5. Regulation of the internal cyclic AMP level. 5-HT stimulates the enzyme adenyl cyclase which synthesizes cyclic AMP from ATP. Cyclic AMP is hydrolysed to 5'-AMP by phosphodiesterase. The methyl xanthine theophylline can increase the intracellular concentration of cyclic AMP by inhibiting (-) phosphodiesterase.
3
I t Control
0 1
/
I
I
5
10 15 Minutes
I
I
1
20
25
Fig. 6. The effect of lO-'M 5-HT on the concentration of cyclic 3';5'-AMP in the salivary of calcium. The hatched area gland in the absence (0- - - 0 ) and presence (0-0) represents the level of cyclic AMP in unstimulated glands. (From Prince et aZ., 1972.)
14
M. J. BERRIDGE AND W. T. PRINCE
AMP level does not increase so rapidly but it does reach and maintain a higher equilibrium level; the significance of this observation will be apparent later when the various feedback relationships between the two messengers are discussed (Section IV B). A similar effect of calcium has been observed when parathyroid hormone increases cyclic AMP levels in renal tubules (Nagata and Rasmussen, 1970). The change in intracellular cyclic AMP concentration during the action of 5-HT has also been studied by labelling the nucleotide pool with 3H-adenine. Subsequent treatment with 5-HT in the presence of the phosphodiesterase inhibitor theophylline causes a large increase in labelled cyclic AMP compared to the untreated controls. This experiment confirms that 5-HT acts to speed up the conversion of ATP to cyclic AMP as depicted in Fig. 5. The increase in cyclic AMP concentration observed on addition of 5-HT is an important link between receptor activation and the subsequent increase in fluid secretion. This direct role of cyclic AMP in cell activation is illustrated by its ability to simulate the action of 5-HT on fluid secretion (Fig. 7). The onset of the response to cyclic AMP is slightly slower than that for 5-HT. Apart from these minor temporal differences, there is a remarkable similarity in the action of these two agents. 40
-
35
-
30-
-25.E
E
-g o 1
i;
5-HT Cyclic AMP
I
.c
6$15-
I
Minutes
Fig. 7. The effect of l o - * M 5-HT, lO-’M cyclic AMP and lO-’M theophylline on the rate of fluid secretion of isolated salivary glands. All these compounds were applied at the arrow.
15
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
The very high concentrations of cyclic AMP required to activate secretion when applied exogenously (Figs 7 and 8) may be determined by several factors. Firstly, cell membranes may be relatively impermeable to these large nucleotides and the cell must be flooded with a very high concentration to enable enough cyclic AMP to enter the cell to raise the intracellular level sufficiently to activate secretion. Robison et al. (1965) have shown that cyclic AMP does not enter cardiac cells fast enough to achieve an effective concentration. Secondly, since phosphodiesterase is continually hydrolysing cyclic AMP, sufficient cyclic AMP must enter the cell to counteract this degradative process before stimulation of the effector system is apparent. This particular aspect can be demonstrated by studying the effect of a phosphodiesterase inhibitor on the dose-response curve for cyclic AMP (Fig. 8). After treatment with theophylline salivary glands become much more sensitive to exogenous cyclic AMP than glands without theophylline where phosphodiesterase is capable of degrading cyclic AMP maximally (Berridge, 1970). Thirdly, the natural hormone may induce an increase in other intermediates such as calcium (Section IV B) so that higher concentrations of cyclic AMP are required to overcome the lack of these other intermediates. Various cyclic AMP derivatives have been prepared in an attempt to overcome the problem of the slow rate of penetration of cyclic AMP into cells. The dibutyryl derivative of cyclic AMP (XI) introduced by Posternak et al. (1 962) has been widely employed but with very varied results. The action of the dibutyryl derivative is worth considering in some detail since it is likely to be used in many future experiments. Certain tissues such as vertebrate fat pads, salivary glands, adrenals, heart and thyroid, which are all insensitive to cyclic AMP, will respond to dibutyryl cyclic AMP (Bdolah and Schramm, 1965; Butcher et al., 1965; Imura et aZ., 1965; Pastan,
" i0-e
8
;0-7
Log concentration
1'0-6
A-5
4 0 3
I&
(MI
Fig. 8. The dose-response curves to 5-HT and cyclic AMP (c.AMP) in the presence (open circles) or absence (filled circles) of theophylline (T). (After Berridge, 1970.)
16
M. J. BERRIDGE AND W. T. PRINCE
b -0’
b C 0 CsH-,
I 0-
XI
1966; Babad et al., 1967; Skelton et al., 1970). When applied t o Calliphora salivary glands the dibutyryl derivative stimulated secretion but the nature of the response was very different to that recorded for cyclic AMP (Berridge, 1970). The presence of substituents at the N 6 - and 2’-positions not only delay the onset of the secretory response but they also greatly prolong the recovery time after removal of the dibutyryl analogue. Considerable delays have also been observed during the action of dibutyryl cyclic AMP on the parotid gland (Babad et al., 1967) isolated fat cells (Perry and Hales, 1970) and heart (Skelton et al., 1970). There is some indication that the potency of cyclic AMP depends on having a free hydroxyl group at the 2’-position on the ribose ring (Henion et al., 1967). Consequently, the slow response of salivary glands and other tissues to dibutyryl cyclic AMP may depend on the time required for intracellular esterases t o remove the butyryl substituent from the 2‘-position before this derivative can become active. The slow recovery after removing dibutyryl cyclic AMP from salivary glands (Berridge, 1970) may be explained by the observation that this compound is less susceptible than cyclic AMP t o degradation by phosphodiesterase (Posternak et al., 1962). All these factors must be taken into consideration when attempting t o interpret results obtained with these substituted derivatives. The specificity of cyclic AMP has been tested by comparing its response with a number of related cyclic nucleotides (Fig. 9). Cyclic nucleotides consist of three major regions: (i) a purine or pyrimidine base (R in Fig. 9); (ii) the sugar ribose which is linked to the base by a glycosidic bond; (iii) a phosphate group which forms a ring through its connection to the 3’- and 5’-carbon atoms of the ribose sugar (see Fig. 5).
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
17
The structure-activity studies reveal that the cyclic AMP receptor can accommodate certain modification of the base region (Fig. 9). Indeed the tubercidin and uridine cyclic phosphates were more active than cyclic AMP. Drummond and Powell (1 970) have also observed that cyclic tubercidin 3',5'-monophosphate is more active than cyclic AMP in stimulating phosphorylase b kinase from skeletal muscle. In salivary glands cyclic inosine monophosphate is less active than cyclic AMP and also shows a reduction in intrinsic activity in that it cannot induce a maximal effect (Fig. 9). Cyclic guanosine monophosphate is totally inactive even though this compound has been identified in various vertebrate tissues (Hardman et aL, 1971). All compounds tested which contained modifications of the ribose or phosphate rings were totally inactive. Some of the most interesting compounds tested were 5'-adenosine monophosphate (see Fig. 5), desoxyadenosine 3',5'-monophosphate (XII), adenine 9-/3-D-xylofuranosyl 3',5'-monophosphate (XIII) and adenosine 3',5'-cyclic phosphorothioate (XIV). In 5' AMP the phosphate ring has been opened (the 5'derivatives of various other cyclic nucleotides were also totally inactive, e.g. 5'UMP, 5'IMP, 5'GMP). In XI1 the 2'-hydroxyl group is missing. The 3'-hydroxyl group in XI11 is positioned above the plane of the ring which allows the formation of an unstrained phosphate R
10. Log concentration (M)
Fig. 9. The dose-response curves of salivmy glands produced by different cyclic 3'3'-nucleotide monophosphates. c.TMP-cyclic tubercidin monophosphate. c.UMP-cyclic uridine monophosphate. c.IMP-cyclic inosine monophosphate. c.GMP-cyclic guanosine monophosphate.The structure of the different bases (R) is shown.
18
M. J. BERRIDGE AND W. T. PRINCE
diester linkage in contrast to the bond in cyclic AMP which is very strained (Drummond and Powell, 1970). Activity is also lost if the double bond oxygen is replaced with the much bulkier sulphur atom (XIV). The latter compound is of particular interest because it is the only nucleotide tested which appeared to compete with cyclic AMP. There have been few reports of cyclic AMP competitors. Kulka Adenine
Adenjne
Adenine
and Sternlicht (1 968) have shown that adenosine 3'-monophosphate may inhibit the ability of cyclic AMP to stimulate enzyme secretion in mouse pancreas. Adenosine 3'-monophosphate had no effect on salivary glands. Further evidence to implicate cyclic AMP in the control of secretion has come from studies with the phosphodiesterase inhibitor theophylline (Berridge, 1970). By reducing the breakdown of cyclic AMP within the cell, theophylline can lead to higher than normal intracellular levels of cyclic AMP which in turn lead to several predictable consequences. Firstly, theophylline can stimulate secretion directly but the onset of the response takes much longer than that for either 5-HT or cyclic AMP (Fig. 7). Evidence will be presented later (page 27) t o show that the long delay represents the time required for the unstimulated adenyl cyclase to raise the internal cyclic AMP concentration high enough to stimulate secretion. The ability of theophylline to potentiate the action of cyclic AMP has already been described (Fig. 8 ) and this potentiation can extend to the action of the primary stimulus 5-HT. If the internal destruction of cyclic AMP is decreased, it becomes much easier for adenyl cyclase to raise the cyclic AMP concentration high enough to stimulate secretion. In other words, adenyl cyclase need not be activated maximally which in turn means fewer 5-HT-receptor interactions and therefore much lower concentrations of 5-HT are required. Figure 8 illustrates the marked potentiation which can be obtained with theophylline. The dose-response curve for 5-HT is shifted to the left. Thus a simple chemical signal, 5-HT, arriving at the surface is
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
19
translated into an intracellular secondary messenger in the form of cyclic AMP through the activation of the enzyme adenyl cyclase. B. THE ROLE OF CALCIUM
The actions of both 5-HT and cyclic AMP are critically dependent upon calcium. In the absence of calcium the onset of the secretory response of salivary glands to 5-HT and cyclic AMP is slower than normal (cf. Figs 7 and 10). This is particularly true for the action of cyclic AMP which showed a 2-min delay before the onset of secretion in a calcium-free medium. In both cases, a brief stimulation of secretion was followed by a decline to very low rates of secretion. In the case of the 5-HT-stimulated glands, this inability to secrete in a calcium-free environment is not caused by any impairment of adenyl cyclase activity since the cyclic AMP level under such conditions is higher than in the presence of calcium (Fig. 6). The limiting factor is calcium availability as shown by the dramatic recoveries evident when this divalent cation is returned to the bathing medium (Fig. 10). Flux studies with radiocalcium show that 5-HT, but not cyclic AMP, stimulates 45Ca influx into isolated salivary glands (Prince et a l , 1972). The efflux of 45Ca from prelabelled cells, however, was stimulated by both 5-HT and cyclic AMP. The ability of both agents to influence calcium efflux points to a complicated feedback
0
10
20
30
40
50
60
Minutes
Fig. 10. The effect of l o - * M 5-HT(0-0) and lo-’ M cyclic AMP (0- - - 0 ) applied at the first arrow,on the rate of fluid secretion in the absence of calcium. 2.0 mM calcium was added to the bathing medium at the second arrow. (After Prince et oZ., 1972.)
20
M . J. BERRIDGE AND W. T. PRINCE
relationship operating between these two second messengers. There is some evidence that cyclic AMP can affect intracellular calcium levels by influencing the intracellular pools of calcium contained in the mitochondria or sarcoplasmic reticulum (Rasmussen, 1970; Epstein et al.. 1971). The ability of 5-HT and exogenous cyclic AMP to increase calcium efflux from salivary glands could be explained if increasing the intracellular concentration of cyclic AMP leads t o greater release of calcium from some intracellular pool. This ability of one intracellular messenger to influence the concentration of the other messenger raises an important question concerning the role of various feedback relationships during hormone action. These feedback mechanisms are poorly understood but they are sufficiently important to warrant some discussion at this early stage because they may further our understanding of intracellular calcium homeostasis. The free calcium which enters the cytoplasm can originate from both the external bathing medium and from intracellular pools such as the mitochondria. One action of 5-HT is to increase calcium influx from the external medium and there is circumstantial evidence from the efflux studies that cyclic AMP exerts a positive feedback control on the release of calcium from some intracellular pool (Fig. 11). The slow onset of secretion Basal plasma membrane
Ca2
+
5-HT
/“O AMP
u lntracellular calcium pool
Fig. 11. The interrelationships of calcium and cyclic AMP in the salivary gland. The cytoplasmic calcium is derived from two sources: the external medium and an intracellular pool, probably mitochondria. Cyclic AMP may release calcium from this pool (+) and the calcium may inhibit adenyl cyclase (-) thus controlling cyclic AMP levels. The entry of calcium and the production of cyclic AMP are both stimulated by 5-HT.
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
21
induced by 5-HT and cyclic AMP in a calcium-free medium (Fig. 10) may depend on this proposed action of cyclic AMP on calcium release from the intracellular pool. As this calcium enters the cytoplasm, secretion is possible and accounts for the temporary burst of fluid secretion (Fig. 10). However, as this calcium is lost to the external calcium-free environment and is not replaced, secretion fails as the intracellular calcium reserves are depleted. Calcium, in turn, might be able to regulate cyclic AMP levels through a negative feedback control on adenyl cyclase activity as suggested by Chase et al. (1 969). This divalent cation inhibits adenyl cyclase activity in broken cell preparations of heart (Drummond and Duncan, 1970), kidney (Streeto, 1969), and foetal rat calvaria (Chase et al., 1969). The increased intracellular levels of cyclic AMP found in salivary glands incubated in a calcium-free medium (Fig. 6) is certainly consistent with such a feedback relationship. A similar mechanism could account for the higher than normal levels of cyclic AMP observed in calcium-free solutions during the action of epinephrine on the heart (Namm et al., 1968) and parathyroid hormone on the kidney (Nagata and Rasmussen, 1970). It is not claimed that the internal control mechanism in salivary glands can be described in terms as simple as the feedback relationships depicted in Fig. 1 1. There may be more feedback loops and more complex modes of interaction of the various processes which regulate the concentrations of cyclic AMP and calcium. The important point to establish at this stage is that such feedback relationships may exist and may play an important role in integrating hormone action within the cell. These various feedback mechanisms may be considered as sophisticated devices to ensure that the rapid changes in cell activity produced by hormonal stimuli are orderly and regulated. These devices may also prevent the second messenger concentrations from exceeding that required for a maximal activation of the cell. An important consequence of keeping such a check on the concentration of the second messengers is that it enables the cells t o recover rapidly on withdrawal of the hormonal stimulus. V. THE MODE OF ACTION OF CYCLIC AMP AND CALCIUM
The way in which the two intracellular messengers, cyclic AMP and calcium, react with the transport mechanisms responsible for
22
M. J . BERRIDGE AND W. T. PRINCE
secreting fluid has been investigated using electrophysiological techniques. The osmotic gradients necessary to promote a passive flow of water across salivary glands are generated by the transport of potassium and chloride (Oschman and Berridge, 1970; Prince and Berridge, 1972, unpublished observations). Ion transport across an epithelium usually generates an electrical potential which can be used to analyse the nature of the underlying ionic events. We have used this approach to try and uncover the site and mode of action of cyclic AMP and calcium. The sucrose-gap technique used by neurophysiologists has been adapted to study the electrical responses of isolated salivary glands (Berridge and Prince, 1971, 1972a, b; Prince and Berridge, 1972). The salivary gland (SG) is ligated at both ends with fine silk threads (SL) and transferred to a perspex chamber consisting of three parallel baths (Fig. 12). The gland which lies in a groove connecting all three baths is held in position by inserting the ligatures into Vaseline on the two outer walls. The closed end of the gland lies in the perfusion bath (PB) which is constantly perfused with saline (arrows). The open end of the gland lies in the saliva bath (SB) which also contains saline so that the lumen of the gland is in electrical contact with this bath. These two outer baths are also insulated from each other by liquid paraffin (LP) contained in the middle bath. The transepithelial PB
Fig. 12. Diagram of the perspex perfusion chamber used to record the electric potentials of isolated salivary glands. (From Berridge and Prince, 1972a.) See text for further details.
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
23
potential can be measured by connecting each outer bath to an electrometer through agar bridges (AB) and calomel electrodes. Intracellular potentials can be recorded by inserting a microelectrode into gland cells lying in the perfusion bath (Prince and Berridge, 1972). Agar blocks (A) support the gland during penetration with electrodes and also increase mixing in the perfusion bath. A. THE EFFECT OF 5-HT AND CYCLIC AMP ON POTENTIAL
The transepithelial potential of resting glands was approximately 4mV with the lumen positive to the bathing medium but a few seconds after treatment with 5-HT this potential changed to about 16 mV negative of the resting value and remained at this level as long as 5-HT was perfused over the gland (Fig. 13). After removal of 5-HT
"b
Fig. 13. The electrical response of an isolated salivary gland to 5-HT (lo-' M). The positions of the recording and reference electrodes for each trace are shown on the right; the hatched area represents the liquid paraffin gap which insulates the perfusion bath, containing the closed end of the gland, from the saliva bath. a. The transepithelial potential (VT ) recorded by means of calomel electrodes connected to the two outer baths. b. The potential across the basal surface (V,,), recorded by means of a microelectrode with the perfusion bath calomel electrode as a reference (note that the recording across this surface is greatly amplified). c. The potential across the apical surface (V,) recorded by using the calomel electrode in the saliva bath as a reference. The potential change across this surface is very similar to the change in transepithelial potential. (From Berridge and Prince, 1972b.)
24
M. J. BERRIDGE AND W. T. PRINCE
the potential returned to its resting level. The transepithelial potential is the sum of the potential changes occurring across the basal and apical surfaces of the salivary gland cells. Intracellular microelectrode recordings revealed that most of the transepithelial potential can be accounted for by a large depolarization of the apical plasma membrane (Fig. I3c). The small hyperpolarization (2-4 mV) across the basal plasma membranes developed much more slowly than that across the apical surface and may simply reflect changes in the concentration of intracellular ions which develop during the increased ion flux induced by 5-HT. The effect of cyclic AMP on potential (Fig. 14) was very different from that just described for 5-HT even though these two agents have such similar stimulatory effects on secretion (Fig. 7). Instead of going negative as with 5-HT, the transepithelial potential went even more positive after treatment of salivary glands with 1W 2M cyclic AMP (Fig. 14). Analysis of this response again revealed that the potential change occurs predominantly across the apical surface. Cyclic AMP caused the apical membrane to hyperpolarize. The small change which occurred across the basal plasma membrane was similar to that recorded on addition of 5-HT.
U
Cyclic AMP
lmin
Fig. 14. The electrical response of an isolated salivary gland to cyclic AMP (10- '11). The position of the recording and reference electrodes are shown on the right as described in Fig. 13. a. The transepithelial potential (V,) shows an increase in lumen positivity after treatment with cyclic AMP. b. There is a small hyperpolarization across the basal surface (V,). c.Most of the transepithelial potential can be accounted for by a large hyperpolarization of the apical surface (VJ. (From Berridge and Prince, 1972b.)
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
25
These potential responses to 5-HT and cyclic AMP can be summarized in the form of potential profiles shown in Fig. 15. 5-HT causes the transepithelial potential to go negative through a large depolarization of the apical plasma membrane (Fig. 15b). In contrast, cyclic AMP induces an increase in positivity by hyperpolarizing this apical surface (Fig. 15c). These observations apparently cast some doubt on the proposed role of cyclic AMP as a second messenger because if it was an intermediary it should exactly *30-
'20 +lomV
0-10
-
-20 -30 -40 -50 -60Resting
5-HT
Cyclic AMP
Fig. 15. A summary of the average potentials recorded across the basal (B) and apical (A) surfaces of the salivary gland cells in the resting state and during stimulation with either 5-HT or cyclic AMP. The opposite effects of 5-HT and cyclic AMP on the transepithelial potential (hatched area) are restricted to changes across the apical surface. 5-HTdepolarizes whereas cyclic AMP hyperpolarizes this membrane. (From Berridge and Prince, 1972b.)
duplicate the effects of the first messenger 5-HT on the transepithelial potential. However, this dilemma can be resolved if 5-HT has two actions only one of which is mediated by cyclic AMP. The electrical recordings indicate that cyclic AMP stimulates cation transport to account for the large increase in positivity seen when this compound acts in the absence of 5-HT. The increase in negativity observed with 5-HT suggests that there is stimulation of anion transport which is independent of cyclic AMP. Subsequent studies (e.g. Prince et al, 1972) which will be described later, have shown that this action of 5-HT on anion transport is mediated by calcium. Thus the two intracellular mediators of 5-HT action have very different effects on the ionic mechanisms responsible for secretion. The electrical measurements suggest that potassium transport is driven by an electrogenic pump located on the apical plasma membrane whereas chloride movement is mainly passive and is linked t o active cation transport. The potential generated across the gland depends both on the activity of the electrogenic pump and also on the ease with which
26
M. J. BERRIDGE AND W. T. PRINCE
chloride ions can enter the lumen to counteract this flow of cations (Prince and Berridge, 1972). Cyclic AMP appears to regulate the active cation pump whereas the intracellular calcium concentration determines either the permeability to anions or the degree of linkage of anions to cation transport. Evidence for this hypothesis has been obtained from a number of experiments designed to discriminate between the two different actions of 5-HT. The interpretation of such experiments must take into account the complex feedback relationships which may exist between cyclic AMP and calcium mentioned earlier (Fig. 11). Since the regulation of these two messengers is intimately related there are a limited number of procedures for obtaining an increase in the concentration of one without a simultaneous rise in the other. Most of the experiments are concerned with studying the effects of increased levels of cyclic AMP in the absence of calcium. B. EVIDENCE FOR THE INDEPENDENT ACTION OF CYCLIC AMP AND CALCIUM ON ION TRANSPORT
The simplest way of increasing the internal concentration of cyclic AMP without the mediation of 5-HT is to add this nucleotide to the bathing medium. The resulting increase in positive potential described earlier (Fig. 14) is consistent with the idea that cyclic AMP stimulates a cation pump and consequently increases the apical membrane potential. Since cyclic AMP mimics the action of 5-HT on secretion (Fig. 7), chloride movement must be high enough to support the high rate of cation transport necessary for a maximal rate of secretion during the action of exogenous cyclic AMP. It is conceivable that the ability of cyclic AMP to release calcium from an intracellular pool (as postulated in Fig. 1 1 ) keeps the internal calcium concentration just above the critical level for solute transport but not high enough to produce depolarization of the apical membrane. However, when salivary glands were treated with 5-HT or cyclic AMP in a calcium-free medium the internal calcium levels, and hence anion movement, were apparently limiting because the rate of secretion was very low (Fig. 10). In calcium-free media the internal cyclic AMP concentration is high, as measured directly in the presence of 5-HT (Fig. 6). In a calcium-free medium, 5-HT and cyclic AMP produce large positive potentials which are again consistent with an action of cyclic AMP on cation transport in the absence of the calcium-dependent increase in chloride movement
27
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
(Prince et al., 1972; Berridge and Prince, 1972b). When calcium is returned to the medium bathing a gland stimulated by 5-HT in a calcium-free medium, the transepithelial potential immediately returns to the negative value characteristic of the action of 5-HT in normal saline (Berridge and Prince, 1972b). Another way of studying the action of cyclic AMP in the absence of the 5-HT-induced increase in calcium influx is to use the phosphodiesterase inhibitor theophylline. As is discussed on p. 18, theophylline can stimulate secretion by inhibiting the intracellular breakdown of the cyclic AMP generated by the unstimulated adenyl cyclase. As one might expect, there is an increase in positive potential which has a very slow onset like that observed for the onset of secretion (Fig. 16) (Berridge and Prince, 1971). The slow time
t20L I
0
1
I
I
1
I
II)
I
I
I
I
1
20
I
I
I
I
timelmin
Fig. 16. The effect of l O - * M theophylline (horizontal bar) on rate of fluid secretion (nl/min) and the transepithelial potential (mV)of isolated salivary glands. (From Berridge and Prince, 1971.)
course for the onset of the secretory and potential response is probably indicative of the slow build up in the concentration of intracellular cyclic AMP which relies on the activity of the unstimulated adenyl cyclase. If this is the case, it should be possible to induce the action of theophylline much more rapidly by increasing the activity of adenyl cyclase. If at the beginning of the theophylline treatment, the turnover of adenyl cyclase is briefly enhanced by a 6 s treatment with 5-HT, the positive potential develops more rapidly (Berfidge and Prince, 1972a). This experiment clearly illustrates that the delayed secretory and potential responses
28
M . J. BERRIDGE AND W. T. PRINCE
observed during theophylline action cannot be accounted for by any delay in the development of phosphodiesterase inhibition. Indeed the inhibition develops very fast and the long delays depend solely on the time required for the unstimulated adenyl cyclase to raise the cyclic AMP concentration to the level necessary to stimulate the secretory and potential responses of the cell. It is also possible to distinguish between the different actions of cyclic AMP and calcium by preventing the expression of their effects by altering the ionic composition of the bathing medium. If salivary glands are stimulated with 5-HT in a saline where chloride has been
1
0
1
1
1
1
5
1
1
1
1
~
10
1
timelmin
Fig. 17. The transepithelial potential response of isolated salivary glands to lO-'M 5-HT (horizontal bar) when all the chloride in the saline was replaced with isethionate. (From Berridge and Prince, 197 1 .)
replaced with the large impermeant anion isethionate, the normal increase in negativity is not seen and the lumen becomes strongly positive (Fig. 17). Under these conditions 5-HT will function normally to increase the intracellular concentrations of both cyclic AMP and calcium. The former will stimulate cation transport but the action of calcium on anion movement is not expressed because isethionate cannot permeate the cell, the net result is a large increase in positivity (Berridge and Prince, 197 1, 1972a). C. THE TIME COURSE OF THE CYCLIC AMP A N D CALCIUM EFFECTS
A careful analysis of the electrical events which occur during stimulation with 5-HT in different conditions has revealed that the time course of the actions of the two messengers are very different. The increase in negativity, which is thought to reflect an increase in anion movement caused by a rise in the internal calcium concentration, develops very rapidly with a half time of about 5 s. The time course for the increase in the internal cyclic AMP
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
29
concentration is more difficult to determine but the isethionate experiments just described provide an indirect measure of the build up of cyclic AMP. The method depends on the assumption that during the action of 5-HT in an isethionate saline, the action of cyclic AMP can be unmasked since the movement of anions is prevented. In an isethionate saline, the increase in positivity has a half time of 30 s which is much longer than the increase in negativity induced by 5-HT in a normal chloride-containing saline (half time of 5 s). Thus, these measurements indicate that during the action of 5-HT the build up of calcium and cyclic AMP within the cell occurs at very different rates. The action of 5-HT on calcium influx leads to a very rapid increase in internal calcium concentration which in turn stimulates chloride movement with a rapid increase in negativity. The increase in cyclic AMP concentration, which depends on the action of 5-HT on adenyl cyclase, occurs much more slowly. The slow increase in cyclic AMP concentration may account for the delay in the onset of fluid secretion after administration of 5-HT. Although calcium will flood into the cell very rapidly and activate chloride movement, fluid secretion will not begin until active cation transport is stimulated by cyclic AMP. The half time for the onset of secretion is 35 s (Fig. 7) which is very close to the indirect estimate from potential measurements of 3 0 s for the build up of internal cyclic AMP concentration (Berridge and Prince, 1972a). The different time course for the actions of the two intracellular messengers provides another way of distinguishing their separate effects under normal conditions. If salivary glands are treated with 5-HT for very short periods, the normal increase in negativity is always followed by a marked increase in positivity. This “positive undershoot” gradually returns to the normal resting potential. The hypothetical curves in Fig. 18 attempt to describe this biphasic response in terms of the postulated differences in the time course for the actions of cyclic AMP and calcium. During a brief treatment with 5-HT, calcium will rapidly flood into the cell to cause an immediate increase in negativity through its action on chloride movement. At the same time 5-HT will also begin to activate adenyl cyclase to increase the intracellular concentration of cyclic AMP. At about the time the cyclic AMP level begins to rise, 5-HT begins to leave the bath causing an immediate decline in calcium influx and a subsequent return of the internal calcium concentration to unstimulated levels. The cyclic AMP system apparently does not respond so rapidly to the removal of 5-HT. The marked positive undershoot can
30
M. J. BERRIDGE AND W. T. PRINCE a
-20-
-10
-
0-
> E
+lo-
+20
-
t30
-
b
I
0
~
1
I
2
I
1
3 4 5 Minutes
'
6
'
7
'
'
8
Fig. 18. a. 'Ihe response of an isolated salivary gland to treatment with 1 x lO-'M 5-HT for 15 s (horizontal bar). b. Hypothetical curves illustrating the proposed changes in internal and cyclic AMP (---) concentrations during the response shown in a. The calcium (-) stippling represents the period when cyclic AMP acts in the absence of calcium which may account for the positive phase of the biphasic response in a.
be interpreted on the basis that cyclic AMP continues to activate cation transport for a short period after the action of 5-HT on anion movement has waned. The stippling represents the period when the internal cyclic AMP concentration is elevated in the absence of calcium (Fig. 18b). The steepness of the rising phase of this parameter may account for the sudden positive potential plunge observed during the biphasic response to short treatments of 5-HT (Fig. 18a). The recovery of the potential possibly indicates the destruction of cyclic AMP by phosphodiesterase. Some proof for the involvement of cyclic AMP in the development of the positive
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
31
undershoot has come from studies using theophylline which can stabilize the potential at the bottom of the positive undershoot and prevent the gradual recovery to an unstimulated potential (Berridge and Prince, 1972a). VI. CONTROL OF SECRETION-A MODEL OF HORMONE ACTION
Since thidiscovery of cyclic AMP by Sutherland and Rall(1958) this compound has been implicated in the control of a wide range of cellular processes. The original hypothesis, which has gained wide acceptance, that the actions of most hormones are mediated solely by cyclic AMP was clearly an oversimplification and Robison et al. (1968) have suggested that there may be other second messengers. Recently it has been postulated that calcium may have an equally important role in mediating hormone action (Rasmussen, 1970). This idea is particularly appealing because intracellular ions are known to play an important role in regulating many cellular functions (Bygrave, 1967). These studies on salivary glands suggest a model of hormone action which may be applicable to other hormones. The primary hormonal message arriving at the salivary gland cell is carried in the chemical configuration of the 5-HT molecule. This information is decoded into secondary messages in the form of cyclic AMP and calcium through a specific interaction with a cellular receptor (Fig. 19). The most important aspect of the 5-HT-receptor interaction appears to be the formation of an electrostatic bond between the positively charged quartenary nitrogen atom at the end of the ethylamine side chain and an anionic site on the receptor. The specificity of the response depends on precise hydrophobic and hydrogen bonding involving the indole ring and its hydroxyl group with corresponding sites on the receptor. The perturbation induced in the receptor as a result of this 5-HT interaction is then translated into an increase in the concentration of two intracellular messengers. The first event appears to be an increase in intracellular calcium concentration which may arise through a change in the calcium permeability of the basal plasma membrane. The simultaneous activation of the enzyme adenyl cyclase leads to a slightly slower increase in the internal cyclic AMP concentration. All further actions of 5-HT on cell activation are mediated by these two intracellular messengers. Cyclic AMP acts by stimulating cation transport whereas calcium functions by increasing the flow of anions across the apical plasma membrane. More information on the ionic basis of secretion
32
M . J. BERRIDGE AND W. T. PRINCE Apical membrane
Basal membrane
wir
I
I '
.- . -. ,
-.
Fig. 19. A model for hormone action. 5-HT acting on the receptor (stippling) at the basal membrane stimulates two processes: the entry of calcium and the production of cyclic AMP. The increase in the level of cyclic AMP stimulates the pumping of potassium across the apical membrane. Calcium controls the movement of chloride. Water follows the movement of potassium and chloride resulting in secretion. For clarity the details of cyclic AMP and calcium regulation (shown in Figs 5 and I 1 )are omitted.
is required before the actions of cyclic AMP and calcium can be described in greater detail. VII. COMPARISON OF 5-HT ACTION WITH THAT OF OTHER HORMONES
Animal hormones can be divided into two main groups. One group is concerned with regulating growth and development and is usually thought to act via the genome. Juvenile hormone and ecdysone are two such insect hormones which determine the long-term structure and function of cell populations. The mode of action of such hormones have been reviewed by Wigglesworth ( 1964, 1970) and Highnam and Hill (1969). It has been suggested that cyclic AMP may be involved with the action of ecdysterone (Leenders et al., 1970). The second group of hormones is more concerned with regulating the day-today activity of cells at each developmental stage during the life of the animal. For example, insect hormones have been discovered which regulate the transient colour changes observed in mantids (reviewed in Wigglesworth, 1970), the contraction of heart and visceral muscles (Davey, 1964), the conversion of glycogen to trehalose by fat body (Steele, 1963) and the activity of the excretory
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
33
system (reviewed by Highnam and Hill, 1969). The Malpighian tubules of Rhodnius are regulated by a diuretic hormone released from the fused ganglionic mass in the mesothorax (Maddrell, 1963). The action of this hormone may have many similarities to that of 5-HT on Culliphoru salivary glands. One action of the diuretic hormone is to increase the permeability of the Malpighian tubules to chloride ions and this accounts for a sudden increase in negativity which precedes the onset of secretion by several minutes (Maddrell, 1972). The subsequent activation of cation and anion pumps then induces a biphasic potential response associated with the beginning of fluid secretion. The latter response may depend on cyclic AMP which is thought to mediate the action of the diuretic hormone of Rhodnius (Maddrell et ul., 1971). Apart from these studies on the diuretic hormones of Rhodnius there is very little information on the mode of action of this second group of hormones in insects. The present study on the control of fluid secretion by salivary glands may provide a basic model for hormones concerned with regulating the short-term activity of insect cells. Due to our lack of knowledge about the mode of action of insect hormones, the general significance of this model for the control of secretion by salivary glands must be assessed by comparing the mode of action of 5-HT (summarized in Fig. 19) with that of hormones found in other animal groups. The following general features of the model outlined in Fig. 19 will be considered: (i) Interaction of the hormone with the receptor causes a change in membrane permeability to calcium, and possibly to other ions as well, in addition to adenyl cyclase activation. (ii) The increase in intracellular concentration of cyclic AMP and calcium mediate all further actions of the hormone. (iii) The intracellular concentrations of calcium and cyclic AMP are controlled by various internal feedback mechanisms. The following examples are mainly taken from vertebrate systems. In some examples the action of the hormone is not fully understood but they all display certain features seen in the action of 5-HT on salivary glands. In every case cyclic AMP and calcium are implicated in the control of cellular activity. A. AGGREGATION OF THE SLIME MOULD, DICTYOSTELIUM DISCOIDEUM
This slime mould can exist in two forms, a motile single-cell amoeba or aggregates of hundreds of individuals differentiated into a AIP-3
34
M. J. BERRIDGE AND W. T. PRINCE
spore. The ability of cyclic AMP to induce the motile individuals to aggregate (Konijn et al., 1967) requires the presence of calcium (Mason et al., 197 1). Chi and Francis (1 97 1) have shown that cyclic AMP stimulates calcium efflux from prelabelled amoebae suggesting a mobilization of intracellular stores as noted in salivary glands (p. 20). Thus an important relationship between cyclic AMP and calcium can be recognized even in this very primitive system. B. SYNAPTIC TRANSMISSION-THE ROLE OF CYCLIC AMP AND CALCIUM IN PRE- AND POST-SYNAPTIC EVENTS
Some of the most active preparations of adenyl cyclase have been obtained from nervous tissue (Sutherland et al., 1962; Weiss and Costa, 1968). Both electrical stimulation and treatment with catecholamines promotes an accumulation of cyclic AMP in brain slices (Kakiuchi and Rall, 1968a, b; Kakiuchi et al., 1969). There is no evidence that cyclic AMP plays a direct role in synaptic events but it may function in neural integration by modulating pre- and post-synaptic events. Although it is dangerous to generalize when so little is known, there are clear indications that cyclic AMP may facilitate pre-synaptic events but inhibit post-synaptic activity. Further, cyclic AMP also appears t o mediate most of the synaptic events which are controlled by catecholamines.
Pre-sy nap tic Activity The primary role of calcium in regulating the release of a wide range of cellular secretions is well established (Simpson, 1968; Smith, 1971). A role for cyclic AMP in the release process of cells including synaptic vesicles has also been recognized. The control of hormone and enzyme release will be described in greater detail later. Direct evidence for an involvement of cyclic AMP in pre-synaptic events has come from studies on neuromuscular transmission. Catecholamines can facilitate neuromuscular transmission by promoting the release of acetylcholine from motor neurones (KrnjeviC and Miledi, 1958; Jenkinson et al., 1968). This action of catecholamines can be simulated by both dibutyryl cyclic AMP and theophylline (Breckenridge et al., 1967; Goldberg and Singer, 1969) which can increase the frequency of the miniature end-plate potentials (m.e.p.p.’s). The exact mode of action of cyclic AMP is unclear, but Singer and Goldberg (1 970) suggest that it may function by altering calcium levels. Since external calcium concentration has
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
35
an important effect on the frequency of m.e.p.p.’s (Hubbard et al., 1968), cyclic AMP may modulate synaptic transmission by adjusting the intracellular level of calcium. Whether or not the present observations on neuromuscular transmission can be extended to the release of transmitters in the central nervous system is unknown. However, the observation that the very high activities of adenyl cyclase and phosphodiesterase are located in the synaptosomal fraction (DeRobertis et al., 1967) is very suggestive. Post-synaptic Activity The action of catecholamines in post-synaptic events in both the central and peripheral nervous system may also depend on cyclic AMP. Cyclic AMP mediates the inhibitory action of norepinephrine on the spontaneous discharge rate of cerebellar Purkinje cells (Siggins et al., 1969, 197 la). Intracellular recordings reveal that inhibition results from a large hyperpolarization achieved through an increase in membrane resistance (Siggins e t al., 197 1b). Dopamine has a similar inhibitory action in the peripheral nervous system (McAfee et al., 1971). Dopamine released from interneurones stimulates adenyl cyclase on the post-ganglionic neurone and the resulting increase in cyclic AMP leads to a slow wave of hyperpolarization. Cyclic AMP also plays an important role in the function of smooth muscle. As in other types of muscle, calcium is the agent responsible for the coupling between excitation and contraction. The contractile state of the muscle is thought to be related to the free calcium level in the cytoplasm; it is this level which is altered by excitatory and inhibitory transmitters. In most smooth muscles the action potentials induced by the excitatory transmitters are responsible for triggering contraction. During each action potential, calcium which is the charge carrier (Brading e t al., 1969; Bulbring and Tomita, 1970) enters the cytoplasm and induces a brief mechanical response (phasic response). There must be a rapid train of impulses in order to produce a full contracture. Cyclic AMP is apparently not involved in the development of such contractures but appears to be associated more with the relaxant effect of catecholamines. The possibility that cyclic AMP relaxes muscles by reducing the calcium level is suggested from studies on the uterus where the ability of calcium to induce contractions can be blocked by cyclic AMP (Mitznegg e t al., 1970). 0-Adrenergic agents induce an increase in the cyclic AMP content of smooth muscle from the intestine (Bueding et al., 1966) and uterus (Dobbs and Robison, 1968). This increase in cyclic AMP hyper-
36
M. J. BERRIDGE AND W.
T.
PRINCE
polarizes the membrane possibly through the activation of a sodium and/or calcium pump (Somlyo et al., 1970). Both cyclic AMP and calcium are thus intimately connected with the ability of catecholamines to modulate the activity of excitable cells. C. THE ACTION OF EPINEPHRINE ON THE HEART
Myocardial contractility is modulated by epinephrine which increases both the force of contraction (inotropic effect) and activates phosphorylase to accelerate glycogenolysis. After the administration of epinephrine there is a prompt rise in the cyclic AMP level of the heart which precedes the increase in phosphorylase and inotropism (Robison et aZ., 1965; Williamson, 1966) and leads to the suggestion that these last two effects are mediated by cyclic AMP. However, evidence is accumulating t o show that cyclic AMP may not play a direct role in regulating inotropism because Shanfeld et aZ. (1 969) observed an increase in inotropism without an increase in cyclic AMP. Langslet and @ye (1970) have also shown that cyclic AMP can increase phosphorylase without any increase in inotropism. These inconsistencies can be reconciled if calcium is included in the model linking the action of epinephrine with changes in heart function. As proposed by Meinertz and Scholz (1 969) and later by Rasmussen ( 1970), epinephrine may act by increasing calcium influx during the excitation process. The subsequent increase in calcium level may influence the force of contraction whereas cyclic AMP functions to stimulate metabolism. The activation process is complicated by the observation that this action of cyclic AMP on metabolism requires calcium for the conversion of phosphorylase B to phosphorylase A (Namm et aZ., 1968). Further complications may arise through the existence of feedback loops operating between cyclic AMP and calcium which closely resemble those described earlier for salivary glands (Section IV B). Calcium may inhibit adenyl cyclase (Namm et al., 1968) whereas cyclic AMP may regulate the ionized calcium level in the cytoplasm (Williamson, 1966) possibly by altering the permeability of the sarcoplasmic reticulum (Entman et al., 1969). D. EXCITATION-SECRETION COUPLING
Both cyclic AMP and calcium have been implicated in the release of a variety of cellular products such as adrenocorticotropic hormone
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
37
(Fleischer et al., 1969; Kraicer et al., 1969), luteinizing hormone (Samli and Geschwind, 1968; Ratner, 1970), thyroid stimulating hormone (Wilber et al., 1969; Vale et al., 1967) and amylase from the pancreas (Kulka and Sternlicht, 1968; Hokin, 1966). The way in which the various releasing factors influence the concentration of cyclic AMP and calcium and the subsequent action of these two agents in the secretory process is not clear. The control of secretion in salivary glands and the pancreas is particularly interesting since these systems are also capable of secreting large volumes of fluid. The two processes of enzyme and fluid secretion can be regulated independently of each other. For example, the submaxillary gland produces a copious watery secretion during parasympathetic (cholinergic) stimulation, while sympathetic (adrenergic) stimulation yields an enzyme-rich saliva (Bloom and Fawcett, 1968). Similarly, fluid transport by the pancreas is regulated by secretin whereas pancreozymin stimulates enzyme secretion. The way in which two or more distinct cellular activities are regulated independently of each other introduces an intriguing problem in cellular control. One possibility is that there may be a hierarchy of cellular activities which are sensitive to different concentrations of a single second messenger such as cyclic AMP. The capability of each hormone to stimulate adenyl cyclase could be different and will determine which cellular process will be stimulated. Another possibility is that each process is regulated by a different second messenger or a combination of second messengers. There is some circumstantial evidence to suggest that the latter control mechanism may operate in vertebrate salivary glands. Douglas and Poisner (1 963) showed that calcium is essential for fluid secretion during parasympathetic stimulation of parotid glands. Since there is little evidence that cholinergic agents can activate adenyl cyclase, it is conceivable that stimulation of fluid secretion is mediated by calcium but independently of cyclic AMP. However, cyclic AMP has been clearly implicated in the release of amylase by epinephrine in parotid glands (Bdolah and Schramm, 1965; Babad et al., 1967). In the case of these salivary glands, therefore, the two intracellular messengers may be independently regulated by separate primary hormones. E. CONTROL OF METABOLISM
Many behavioural patterns require instant bursts of energy and there are numerous hormonal and nervous control mechanisms which
38
M. J. BERRIDGE A N D W. T. PRINCE
assure instantaneous activation of various metabolic processes. Catecholamines are particularly important in regulating the fluctuating energy requirements of the body. Cyclic AMP can stimulate muscle and liver phosphorylase and can also activate specific lipases in adipose tissue leading to the formation of free fatty acids from triglycerides. In all the examples studied so far, however, the primary hormone can also induce changes in membrane permeability to ions in addition t o activating adenyl cyclase. Epinephrine acts on the liver t o increase the intracellular concentration of cyclic AMP which then leads to an increased release of glucose resulting from the activation of phosphorylase. The efflux of glucose precedes the increase in cyclic AMP levels (Sutherland and Robison, 1966). However, this efflux of glucose is, in turn, preceded by an efflux of potassium and calcium (Craig and Honig, 1963; Finder et al., 1964; Friedman and Park, 1968; Friedman and Rasmussen, 1970). The increased efflux of potassium and calcium probably accounts for the marked hyperpolarization of liver cells induced by catecholamines or glucagon (Haylett and Jenkinson, 1969; Friedman et al., 197 1). However, more information is required before the relationship of the ionic changes can be correlated with the hyperglycemic action of cyclic AMP. Cyclic AMP mediates the lipolytic action of various hormones by activating a specific lipase (Butcher et al., 1965; Steinberg, 1966). Apart from this metabolic effect induced by cyclic AMP, there is also evidence that lipolytic agents can induce changes in membrane potential and ion flux resembling those just described for the liver. Norepinephrine produces a marked depolarization and a rapid efflux of potassium whereas cyclic AMP and theophylline has no effect (Girardier et al., 1968; Horowitz et al., 1969; Krishna et al., 1970). Even though theophylline had no effect on the potential it was capable of stimulating heat production suggesting that the changes in membrane permeability preceded the activation of adenyl cyclase which is similar to the sequence of events observed in salivary glands (Section V C). A relationship between cyclic AMP and calcium is clearly evident in the action of parathyroid hormone (PTH) on the metabolism of kidney cells. PTH can activate renal adenyl cyclase (Chase and Aurbach, 1968) and calcium influx into kidney cells in tissue culture (Borle, 1968). As observed in salivary glands (Section IV A), the activation of adenyl cyclase by the primary hormone can occur independently of calcium (Nagata and Rasmussen, 1970). However,
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
39
calcium is clearly required for the ability of both PTH and dibutyryl cyclic AMP to stimulate renal gluconeogenesis suggesting that calcium is required for the expression of the cyclic AMP effect (Nagata and Rasmussen, 1970). F. TRANSPORTING EPITHELIA-THE TOAD BLADDER
Water and sodium transport by the bladder of the toad (Bufo marinus) are stimulated by the hormone vasotocin. The effects of vasotocin are mimicked by other octopeptide hormones such as vasopressin (ADH) and oxytocin. These hormones also effect the water permeability and sodium transport in other transporting epithelia such as frog skin and kidney tubules (Orloff and Handler, 1967). Calcium and cyclic AMP appear to be intimately involved in hormone action on all these tissues. Orloff and Handler (1 962) have shown that cyclic AMP and theophylline will mimic the effects of ADH on both the osmotic flow of water and sodium transport and have suggested that cyclic AMP is the intracellular mediator of the effects produced by ADH. Later they demonstrated that ADH and theophylline increase the intracellular concentration of cyclic AMP (Handler et al., 1965). Although cyclic AMP is the suggested intermediary in both actions of ADH the situation is complicated by the fact that the two effects of the hormone can be dissociated from each other yet cyclic AMP is postulated as mediating both actions. Different analogues of vasopressin have quantitatively different effects on water permeability and sodium transport (Bentley, 1967). This is suggestive of two different receptors and is seen in other amphibian epithelia (Elliot, 1967; Bourguet and Morel, 1967), In toad skin, arginine-vasotocin has a potency equal to argininevasopressin on water permeability yet is 200-400 times more potent than arginine-vasopressin on sodium transport. The effects on water and sodium movements can also be dissociated by using calcium. Increasing the serosal calcium concentration has no effect on sodium transport or stimulation by ADH, yet the effect of ADH on osmotic flow is inhibited (Petersen and Edelmen, 1964). This latter effect seems to be competitive at the receptor as it can be overcome by higher doses of ADH and as the response t o externally applied cyclic AMP is unaltered. Our understanding of toad bladder is further complicated by the presence of several cell types (Choi, 1963). The most predominant
40
M. J. BERRIDGE A N D W. T. PRINCE
cell type is the granular cell. The action of ADH on water movement appears to be at the mucosal surface of these cells as changes in the volume of these cells are seen during stimulation of osmotic water movement (Peachey and Rasmussen, 1961;Di Bona et al., 1969; Jard et al., 197 1). The situation regarding sodium transport is less clear. Sodium transport probably occurs through the mitochondria-rich cells and/or the granular cells. The latter cell type is the more plentiful but the mitochondria-rich cells appear to be more suited to active transport-they have many mitochondria and have microvilli thus resembling the parietal cells of the stomach (Choi, 1963). No changes are seen in either cell type when sodium transport is stimulated (Jard et al., 1971). Frazier suggests that the granular cells participate in ADH stimulated sodium transport whilst the mitochondria-rich cells are more suited to stimulation by aldosterone. If the granular cells are the sites of sodium transport then any theory has to explain the differences in the receptors responsible for the triggering of water and sodium movements in the same cell using the same common mediator, cyclic AMP. However, if the mitochondrialrich cells are the site of sodium transport (as suggested by Choi (1 963) and Matty and Guinness (1 964)) then separate cells would be responsible for the two effects of ADH and the explanation is somewhat easier. The receptors could have different pharmacological properties yet use similar intracellular mediators. Calcium plays an important role in the function of the toad bladder. It is important for the maintenance of the epithelial structure of the bladder and also for the action of ADH (Bentley, 1959, 1960). Removal of calcium causes the cells to dissociate from one another with a consequent reduction in transepithelial resistance and sodium transport (Hays et aL, 1965). Strontium, magnesium and barium will substitute for calcium in this respect but they will not substitute for calcium in stimulation by ADH. As well as being involved in the structural integrity of the bladder calcium may also be involved more directly in the action of the hormone. ADH will increase the efflux of 45Ca from preloaded tissue (Thorn and Schwartz, 1965) and will displace calcium from monomolecular films (Kafka and Pak, 1969). The implication of calcium involvement is also indicated from work with prostaglandins which are active in the toad bladder (Lipson and Sharp, 1971). These substances may well effect the distribution of calcium (Ramwell and Shaw, 1970). As adenyl cyclase prepared from toad bladder is inhibited by calcium (Hynie and Sharp, 1971) the same feedback mechanisms put forward
CYCLIC AMP AND CALCIUM IN HORMONE ACTION
41
in this review may function too in this tissue. Therefore both calcium and cyclic AMP are involved in bladder function but their respective roles have yet to be fully ascertained.
VII. CONCLUSION
Comparison of the mode of action of 5-HT in Calliphora salivary glands with that of many other hormones reveals numerous similarities. External chemical stimuli responsible for regulating cellular activity may thus share a common mode of action. The various differences which are apparent today may represent modification of this basic mechanism developed during the course of evolution to adapt cells systems t o new environments. The main feature of this control system concerns the transduction of hormonal information arriving at the cell into internal chemical signals which can be recognized by the various effector mechanisms responsible for carrying out the actions of the hormone. In the original cyclic AMP-hypothesis the hormonal signal input was transduced into cyclic AMP through an activation of the membrane-bound enzyme adenyl cyclase and cyclic AMP alone then mediated all further cellular events. The current hypothesis proposed by Rasmussen (1970) and verified in these salivary gland studies is that the conformational changes induced in the membrane by successful hormone-receptor interactions can be transduced into more than one secondary message. There is a general labilization of the plasma membrane resulting in changes in ion permeability together with activation of adenyl cyclase. The ensuing increase in cyclic AMP concentration together with the redistribution of ions, such as calcium, are then responsible for carrying out the hormonal effect. The intracellular concentration of cyclic AMP and calcium may also be regulated by various feedback loops. The ability of cyclic AMP to regulate the internal calcium concentration may be an important component in the control of both excitable and nonexcitable cells. In some cases, for instance the slime mould, liver and Calliphora salivary glands, cyclic AMP appears t o lead to a mobilization of internal calcium whereas in heart and smooth muscle it may have the opposite effect. This very close relationship which exists between these two internal second messengers in a wide range of control mechanisms warrants careful consideration in future studies on hormone action.
42
M. J. BERRIDGE A N D W. T. PRINCE
REFERENCES Babad, H., Ben-Zvi, R., Bdolah, A. and Schramm, M. (1967). The mechanism of enzyme secretion by the cell. 4. Effects of inducers, substrates and inhibitors on amylase secretion by rat parotid slices. Eur. J. Biochem. 1, 96-101. Bdolah, A. and Schramm, M. (1965). The function of 3‘,5‘ cyclic AMP in enzyme secretion. Biochem. biophys. Res. Commun. 18,452-454. Bentley, P. J . (1 959). The effects of ionic changes on water transfer across the isolated urinary bladder of the toad, Bufo marinus. J. Endocr. 18, 327-333. Bentley, P. J. (1960). The effects of vasopressin on the short-circuit current across the wall of the isolated toad bladder, Bufo marinus. J. Endocr. 21, 161-170. Bentley, P. J . (1 967). Natriferic and hydro-osmotic effects on the toad bladder of vasopressin analogues with selective antidiuretic activity. J. Endocr. 39, 299-304. Berridge, M. J. (1 970). The role of 5-hydroxytryptamine and cyclic AMP in the control of fluid secretion by isolated salivary glands. J. exp. Biol. 5 3 , 171-186. Berridge, M. J. (1972). The mode of action of 5-hydroxytryptamine. J. exp. Biol. (In Press.) Berridge, M. J. and Patel, N. G. (1 968). Insect salivary glands: stiyufation of fluid secretion by 5-hydroxytryptamine and adenosine 3 ,5 monophosphate. Science 162,462-463. Berridge, M. J. and Prince, W. T. (1971). The electrical response of isolated salivary glands during stimulation with 5-hydroxytryptamine and cyclic AMP. Phil. Trans. R . SOC.262, 11 1-120. Berridge, M. J. and Prince, W. T. ( 1 972a). Transepithelial potential changes during stimulation of isolated salivary glands with 5-hydroxytryptamine and cyclic AMP. J. exp. Biol. 56, 139-1 53. Berridge, M. J. and Prince,-W. T. (1972b). The role of cyclic AMP in the control of fluid secretion. In “Advances in Cyclic Nucleotide Research”, Vol. I. Raven Press, New York. Bloom, W. and Fawcett, D. W. (1968). “A Textbook of Histology”. Saunders and Company, Philadelphia. Borle, A. B. (1968). Effects of purified parathyroid hormone on the calcium metabolism of monkey kidney cells. Endocrinology 83, 13 16-1322. Bourguet, J. and Morel, F. (1967). Independance des variations de permeabilite a l’eau et au sodium produites par les hormones neurohypophysaires sur la vessie de grenouille. Biochim. Biophys. Acta. 135, 693-700. Brading, A. F., Bulbring, E. and Tomita, T. (1969). The effect of sodium and calcium on the action potential of the smooth muscle of the guinea-pig taenia coli. J. Physiol. 200, 637-654. Breckenridge, B. McL., Burn, J . H. and Matschinsky, F. M. (1967). Theophylline, epinephrine and neostigmine facilitation of neuromuscular transmission. Proc. natn. Acad. Sci. U.S.A. 57, 1893-1897.
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Bueding, E., Butcher, R. W., Hawkins, J., Timms, A. R. and,Sytherland, E. W. (1 966). Effect of epinephrine on cyclic adenosine 3 ,5 -phosphate and hexose phosphates in intestinal smooth muscle. Biochim. Biophys. Acta. 115, 173-178. Bulbring, E. and Tomita, T. (1 970). Calcium and the action potential in smooth muscle. In “Calcium and Cellular Function” (A. W. Cuthbert, ed.), pp. 249-260. Macmillan, London. Butchel;, R . W., Ho, R. J., Meng, H. C. and Sutherland, E. W. (1 965). Adenosine 3,,5,-phosphate in biological materials. 11. The measurement of adenosine 3 ,5 -monophosphate in tissues and the role of cyclic nucleotide in the lipolytic response of fat t o epinephrine. J. biol. Chem. 4 0 , 4 5 15-4523. Bygrave, F. L. (1 967). The ionic environment and metabolic control. Nature, Lond. 214,667-671. Chase, L. R. and Aurbach, G. D. (1968). Renal adenyl cyclase: anatomically separate sites for parathyroid hormone and vasopressin. Science 159, 545-547. Chase, L. R.. Fedak, S. A. and Aurbach. G. D. (1969). Activation of skeletal adenyl cyclasd by parathyroid hormone in vitro. Endocrinology 84, 76 1-768. Chi, Y. Y. and Francis, D. (1971). Cyclic AMP and calcium exchange in a cellular slime mold. J. Cell Physiol. 77, 169-173. Choi, J. K. (1963). The fine structure of the urinary bladder of the toad, Bufo marinus. J. Cell Biol. 16, 53-72. Craig, A. B. and Honig, C. R. (1963). Hepatic metabolic and vascular responses t o epinephrine: a unifying hypothesis. A m . J. Physiol. 205, 1132-1 138. Davey, K. G. (1 964). The control of visceral muscles in insects. In “Advances in Insect Physiology” (J. W. L. Beaument, J. E. Treherne and V. B. Wigglesworth, eds), Vol. 2, pp. 2 19-245. Academic Press, London and New York. DeRobertis, E., Arnaiz, G. R. D., Alberici, M., Butcher, R. W. and Sutherland, E. W. (1967). Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex. J. biol. Chem. 242, 3487-3493. DiBona, D. R., Curan, M. M. and Leaf, A. (1 969). The cellular specificity of the effect of vasopressin on toad urinary bladder. J. memb. Biol. 1, 79-9 1. Dobbs, J. W. and Robison, G. A. (1968). Functional biochemistry of beta receptors in the uterus. Fedn. Proc. A m , SOC. exp. Biol. 27, 352. Douglas, W. W. and Poisner, A. M. (1963). The influence of calcium on the secretory response of the submaxillary gland t o acetylcholine or to noradrenaline. J. Physiol. 165, 528-541. Drummond, G. I. and Duncan, L. (1970). Adenyl cyclase in cardiac tissue. J. biol. Chem. 245,976-983. Drummond, G . I. and Powell, C. A. (1 970). Analogues of adenosine 3’,5’-cyclic phosph?e as activators of phosphorylase b kinase and as a substrate for cyclic 3 ,5’-nucleotide phosphodiesterase. Molec. Pharmac. 6, 24-30. Elliot, A. B. (1 967). Similar effects of arginine-vasopressin and argininevasotocin on permeability of toad skin. Experientia 23, 220-221. Entman, M. L., Levey, G. S. and Epstein, S. E. (1969). Mechanism of action of epinephrine and glucagon on canine heart. $vidence for increase in sarcotubular calcium stores mediated by cyclic 3 ,5 -AMP. Circulation Res. 25,429-438. ’
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Perry, M. C. and Hales, C. N. (1970). Factors affecting the permeability of isolated fat-cells from the rat to [42K] potassium and [36C1] chloride ions. Biochem. J. 1 1 7 , 6 15-62 1. Petersen, M. J. and Edelman, I. S. (1964). Calcium inhibition of the action of vasopressin on the urinary bladder of the toad. J. clin. Invest. 43, 583-594. Postern,akl T., Sutherland, E. W. and Henion, W. F. (1 962). Derivatives of cyclic 3 ,5 -adenosine monophosphate. Biochim. biophys. Acta 65, 558-560. Prince, W. T. and Berridge, M. J. (1 972). The effects of 5-hydroxytryptamine and cyclic AMP on the potential profile across isolated salivary glands. J. exp. Biol. (In Press.) Prince, W. T., Berridge, M. J. and Rasmussen, H. (1 972). The role of calcium and cyclic AMP in the secretory response of the blowfly salivary gland to 5-hydroxytryptamine. Proc. natn. Acad. Sci. U.S.A. (In Press). Ramsay, J. A. (1954). Active transport of water by Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera, Phasmidae). J. exp. Biol. 31, 104-1 13. Ramwell, P. W. and Shaw, J . E. (1970). Biological significance of the prostaglandins. Recent Prog. Horm. Res. 26, 139-187. Rasmussen, H. ( 1 970). Cell communication, calcium ion and cyclic adenosine monophosphate. Science 170,404-412. Ratner, A. (1970). Stimulation of luteinizing hormone release i n vitro by dibutyryl-cyclic-AMP and theophylline. Life Sci. 9, 1221-1226. Robison, G. A., Butcher, R. W., 9ye, I., Morgan, M. E. a;d Sutherland, E. W. (1965). The effects of epinephrine on adenosine 3’,5 -phosphate levels in the isolated perfused rat heart. Molec. Pharmacol. 1, 168-177. Robison, G. A., Butcher, R. W. and Sutherland, E. W. (1 967). Adenyl cyclase as an adrenergic receptor. Ann. N. Y. Acad. Sci. 139, 703-723. Robison, G. A., Butcher, R. W. and Sutherland, E. W. (1968). Cyclic AMP, Ann. Rev. Biochem 37, 149-174. Samli, M. H. and Geschwind, I. I. (1968). Some effects of energy-transfer inhibitors and of Ca2+-free or K+-enhanced media on the release of luteinizing hormone (LH) from the rat pituitary gland in vitro. Endocrinology 82, 225-231. Shanfeld, J., Frazer, A. and Hess, M. E. (1969). Dissociation of the increased formation of cardiac adenosine 3’,5’-monophosphate from the positive inotropic effect of norepinephrine. J. Pharmac. exp. Ther. 169, 3 15-320. Siggins, G. R., Hoffer, B. J . and Bloom, F. E. (1969). Cyclic adenosine monophosphate: possible mediator for norepinephrine effects on cerebellar Purkinje cells. Science 165, 1018-1020. Siggins, G. R., Hoffer, B. H. and Bloom, F. E. (1971a). Studies on norepinephrine-containing afferents to Purkinje cells of rat cerebelly?. 111. Evidence for mediation of norepinephrine effects by cyclic 3 ,5 adenosine monophosphate. Brain Res. 2 5 , 535-553. Siggins, G. R., Oliver, A. P., Hoffer, B. J. and Bloom, F. E. (1971b). Cyclic adenosine monophosphate and norepinephrine: effects on transmembrane properties of cerebellar Purkinje cells. Science 17 1, 192-194. Simpson, L. L. ( 1 968). The role of calcium in neurohumoral and neurohormonal extrusion processes. J. Pharm Pharmac. 20, 889-9 10.
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Williamson, J. R. ( 1 966). Kinetic studies of epinephrine effects in the perfused rat heart. Pharmac. Rev. 18,205-210. Young, J. A. and Schogel, E. (1 966). Micropuncture investigation of sodium and potassium excretion in rat submaxillary saliva. Pflugers Arch. 291, 85-98.
NOTES ADDED IN PROOF The following subjects have come to our attention since this review was submitted for publication. (1) T H E M O D E O F A C T I O N O F A D R E N O C O R T I C O T R O P H I C H O R M O N E (ACTH)
The ability of ACTH to stimulate adrenal steroid synthesis is mediated by cyclic AMP. This effect of cyclic AMP depends on calcium which appears to have a direct effect on the transfer of amino acids from aminoacyl transfer RNA to protein (Farese, 1971). Farese (1971) suggests that these observations support Rasmussen’s (1971) hypothesis that both cyclic AMP and calcium regulate intracellular events. (2) CYCLIC A M P IN I N S E C T S (a) The glycogen level of cockroach nerve cords is depleted by octopamine which acts by stimulating phosphorylase activity. This action of octopamine can be mimicked by cyclic AMP (Robertson and Steele, 1972). (b) In many vertebrate tissues examined so far, cyclic AMP appears to act by stimulating a specific protein kinase. Kuo et al. (1971) have studied the distribution and properties of protein kinases extracted from a range of tissues from different species. This study indicates that cyclic GMP may also be an important intracellular mediator of hormone action in insects.
REFERENCES Farese, R. V. (1971). Calcium as a mediator of adrenocorticotrophic hormone action in adrenal protein synthesis. Science 173,447-450. Kuo, J. F., Wyatt, G. R. and Greengard, P. (1971). Cyclic nucleotide-dependent protein kinases. IX. Partial purification and some properties of guanosine 3’,S1-monophosphate-dependent and adenosine 3 ,5 -monophosphatedependent protein kinases from various tissues and species of arthropoda. J. biol. Chem. 246, 7159-7167. Robertson, H. A. and Steele, J. E. (1972). Activation of insect nerve cord phosphorylase by octopamine and adenosine 3’,5’-monophosphate. J. Neurochem. (In Press).
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Choline Metabolism in Insects R . G . BRIDGES A . R . C Unit o f Invertebrate Chemistry and Physiology. Department of Zoology. Cambridge. England I. I1.
Introduction . . . . . . . . . . . . . . Choline Metabolism in Vertebrates . . . . . . . 111. Nutritional Requirements of Insects for Choline . . A. Choline in Insect Development . . . . . . B . Substitutes for Choline in the Diet of Insects . . IV . Water-soluble Choline Metabolites . . . . . . . A. Acetylcholine . . . . . . . . . . . . B . Phosphorylcholine . . . . . . . . . . . C. Cytidinediphosphorylcholine (CDP-choline) . . D. Glycerylphosphorylcholine . . . . . . . . V. Lipid-soluble Choline Metabolites . . . . . . . A. Phosphatidylcholine . . . . . . . . . . B . Lysophosphatidylcholine . . . . . . . . C. Sphingomyelin . . . . . . . . . . . . VI . Enzymes Involved in Choline Metabolism . . . . . A. Cholineacetylase and Acetylcholinesterase . . . B . Enzymic Synthesis of Lipids Containing Choline . C. Hydrolysis of Phosphatidylcholine . . . . . D. Oxidation of Choline . . . . . . . . . . E . Synthesis of Choline . . . . . . . . . . VII . The Metabolic Role of Choline in Insects . . . . . Acknowledgements . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
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I . INTRODUCTION
The importance of choline in the metabolic processes of vertebrates has been established for many years . Nutritional studies with animals fed on low protein and high fat regimes have demonstrated that large amounts of choline are required in the diet. if normal growth is to be maintained and fatty infiltration of the liver prevented . This nutritional requirement is not. however. an 51
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absolute one and is greatly influenced by the protein content of the diet because of the interrelationship of choline, methionine and serine. Choline can be synthesized by mammals provided an adequate source of labile me thyl-groups is available. Animals possessing choline oxidase can, in turn, oxidise it to betaine which can furnish methyl-groups for the synthesis of methionine. Most symptoms of choline-deficiency can be alleviated provided adequate amounts of methionine or betaine, plus homocysteine, are available to the animal. Because of its involvement in methyl-group transfer many metabolic pathways are related to choline metabolism and this makes the results obtained from nutritional studies difficult to interpret. Choline is found in almost all living cells, predominantly or exclusively in a combined form, largely in the phospholipid fraction. Quantitatively the role of choline as a constituent of the phospholipids of the lecithin and sphingosine type overshadows its other possible functions. It is well documented that in this form it is an important component of cell membranes and that cholinecontaining lipids are the major phospholipid types in vertebrates. Qualitatively , however, choline esters play a fundamental role in neurophysiology. In vertebrates, acetylcholine plays a vital role in preganglionic transmission in the automatic nervous system, at nerve endings of the parasympathetic system, at skeletal neuromuscular junctions and in the brain. In insects, these various aspects of choline metabolism have been looked at in differing detail. The cholinergic system has received a great deal of attention because of its importance in relation to the mode of action of organophosphorus and carbamate insecticides. The composition of insect phospholipids has been analysed and results from a large number of species have accumulated over the past few years. The present review will consider only the choline-containing lipids, but in almost all cases a fairly complete analysis of the different phospholipid types has been carried out and this may be obtained by consulting the appropriate reference. A number of studies have been made following the change of phospholipid pattern during development but little has been achieved in understanding the metabolic role of the phospholipid. The need for choline in the growth of insects has been examined by dietary means in a large number of species but the replacement of this need by other related compounds has received less attention. The discovery that carnitine is an essential dietary component for tenebrionid beetles led to an examination of its ability to substitute for choline in the diet of
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other insects. This in turn revealed that other choline-like compounds could spare the insect’s dietary need for choline and that these were incorporated into phospholipids in a similar manner to choline itself. These findings raised several important questions. How essential was the normally occurring phosphatidylcholine to the functioning of the insect? Could neurotransmitter substances other than acetylcholine be successfully used by the insect? What was the relationship between the choline involved in lipid metabolism and in acetylcholine synthesis? One of the objects of this review is to see if these questions can be answered. In doing so much of the scattered information available on choline metabolism has been gathered together. After summarizing the evidence for the insects’ need for choline and the ability of other compounds to satisfy this need, the review will deal with the occurrence of the different choline metabolites and the enzymes involved in their metabolism so that the significance of their roles may be assessed. Initially, however, an outline of the different metabolic pathways involving choline which have been demonstrated in vertebrates will be given. This will illustrate their interrela tionship and provide a basis for comparison with those found in insects. 11. CHOLINE METABOLISM IN VERTEBRATES
Figure 1 illustrates the main metabolic pathways in which choline plays a key role in vertebrates and how these pathways are Acet ylchdine
f 3 . GIycer ylphosphory ICh
y lC h
y
a
t
T
i
Betaine aldehyde
d y lCh Betaine
Sphingmyelin
PhasphatidylCh Methionine
N-Dimethylglycine
S-Adenosylmethionine
-
Phosphatidylethanolarnme
Phosphatidylserme
Fig. 1. Metabolism of choline in vertebrates.
54
R. G. BRIDGES
interlinked. Four main pathways are involved. First, the choline to acetylcholine cycle which makes up the cholinergic system involving acetyl CoA, cholineacetylase and cholinesterase (Hebb and Morris 1969). Secondly, the synthesis and catabolism of phosphatidylcholine. This involves a complex of enzymes. Choline is phosphorylated with adenosine triphosphate and cholinekinase (Wittenberg and Kornberg, 1953) and the phosphorylcholine reacts with cytidine triphosphate and choline phosphate cytidyltransferase yielding cytidinediphosphorylcholine (Kennedy and Weiss, 1956). The latter acts as the precursor for both phosphatidylcholine and sphingomy elin by transferring its phosphorylcholine moiety to a diglyceride involving 1,Zdiglyceride cholinephosphotransferase (Kennedy and Weiss, 1956) or to a ceramide involving ceramide cholinephosphotransferase (Sribney and Kennedy, 1958). Hydrolysis of phosphatidylcholine back to choline is a three step process. One of the esterified fatty acids is first removed by the action of a phospholipase A (Rimon and Shapiro, 1959) followed by the removal of the second by phospholipase B (Dawson, 1956a) and the glycerylphosphorylcholine produced finally hydrolysed to choline and cu-glycerophosphate by a glycerophosphorylcholinediesterase (Dawson, 1956b). Sphingomyelin is apparently hydrolysed back to a ceramide and phosphorylcholine (Kanfer et al., 1966). Thirdly, choline can be oxidised to betaine via betaine aldehyde involving choline dehydrogenase (Redina and Singer, 1959) and betaine aldehyde dehydrogenase (Rothschild and Barron, 1954). One of the methyl-groups of the betaine is transferred to homocysteine to give methionine by betaine homocysteine methyltransferase (Klee et aL, 1961) and the dimethylglycine further demethylated to glycine (Handler et al., 1941). Glycine and serine are inter-convertible by a serine transhydroxymethylase (Blakley , 1960) and serine can exchange with the ethanolamine-moiety of phosphatidylethanolamine to give phosphatidylserine which can be decarboxylated back to phosphatidylethanomine (Borkenhagen et al., 196 1 ). Fourthly, choline can be synthsized from ethanolamine by stepwise methylation (Bremer and Greenberg, 1961). This only occurs when it is attached to the phosphate-moiety of the phospholipid molecule. This methylation is performed by S-adenosylmethionine which is synthesized from adenosine triphosphate and methionine with the enzyme methionine adenosyltransferase (Cantoni, 1951). It should be noted that the sequence of reactions from serine to phosphatidylcholine does not lead to a net increase in phospholipid but merely an alteration in the
CHOLINE METABOLISM IN INSECTS
55
base moiety. However, ethanolamine is released on serine exchange with phosphatidylethanolamine and this is available for incorporation de novo into phospholipid after phosphorylation and formation of cytidinediphosphorylethanolamine in a series of reactions analogous t o the synthesis of phosphatidylcholine. Thus many of the compounds involved in these sequences are interrelated although some such as methionine, glycine or serine are participants in reactions other than those directly concerned with choline metab olism. 111. THE NUTRITIONAL REQUIREMENTS OF INSECTS FOR CHOLINE A. CHOLINE IN INSECT DEVELOPMENT
Insects which have been shown t o need the addition of choline to a basal diet if maximum growth of their juvenile stage is t o be achieved, are listed in Table I. In many of the early experiments casein has been used as an amino acid source. The presence of trace amounts of choline in this casein cannot be ruled out, so that the omission of choline may merely have reduced that available to the insect to some undetermined amount. Again many of the early experiments were not carried out under asceptic conditions so that micro-organisms and symbionts may have supplied some of the insects’ choline. This was well demonstrated with Lasioderma serricorne and Sitodrepa panicea which were shown to have a very definite requirement for choline in the larval diet for normal growth when freed of symbionts (Pant and Fraenkel, 1954). In spite of these provisos, the list of insects showing a requirement for choline is impressive. An attempt has been made to summarize the effects of choline deficiency on growth in Table I. The value of this may be suspect mainly because of the unknown amounts of choline present in some diets. For example, more recent experiments with Tenebrio molitor (Bieber and Monroe, 1969) have indicated that the effect of choline deficiency is much more marked in this insect than had previously been reported (Fraenkel et al., 1950). However, the effect on growth seems particularly critical in members of the Orthoptera and Lepidoptera where omission of choline from the diet results in practically no growth of the juvenile stage. Reported effects with the Coleoptera and Diptera are more variable. Many of the Coleoptera fail to grow on diets lacking choline but others merely exhibit slower
Table I Choline requirement for growth and development of juvenile stages of insects Order Coleoptera
Species Attagenus spp. Cardophilus hemipterus Dermestes vulpinus Lasioderma serricorne Palorus ratzeburgi Ptinus tectus Stegobium paniceum Tenebrio molitor Tribolium confusum
Diptera
Trogoderma granarium Aedes aegypti
Calliphora ery throcephala Cochliomyia hominivorax Drosophila melanogaster
Effect of omission of choline from diet No larval growth No larval growth Slow growth, some pupae obtained No growth in absence of symbionts No larval growth Growth retarded. Few adults obtained Growth retarded in absence of symbionts Some retardation of growth. More than 95% mortality of larvae Retardation of growth
Reference Moore (1946) Stride (1953) Fraenkel(l95 1a) Blewett and Fraenkel (1 944) Pant and Fraenkel(l954) Cooper and Fraenkel(l952) Fraenkel and Blewett (1 943) Blewett and Fraenkel (1944) {Pant and Fraenkel(l954) Fraenkel et al. (1 950) Bieber and Monroe (1 969)
Lemonde and Bernard (1 955) Fraenkel and Blewett (1943) Pant (1 956) No development Larvae reached 3rd. instar. Lea and DeLong ( 1956) Poor larval growth. Some adults. Singh and Brown (1957) Some larvae reached Akov (1962a) 3rd or 4th instar. Slow growth Sedee (1953, 1958) No larval growth Gingrich (1 964) No larval growth. No Hinton et a/. (1951) pupae. Retarded growth Reached 3rd instar. No pupae
Drosophiln subobscura
Drosophila ambigua Drosophila obscura Drosop hila funebris Drosophila immigrans Hylemya antiqua Musca domestica
Phormia regina
Pseudosarcop haga af f inis Hemiptera
Myzus persicae
Lepidoptera
Agro tis orthogonia Anthonomus grandis Argyrotaenia velutinana
Larvae reached 3rd instar. Few pupae. No affect on larval growth Mortality at pupation. Retarded larval growth. Few pupae. Near normal larval growth. Choline required for optimal growth. Poor larval growth. Some larvae reached 3rd instar. Retarded growth. Adults obtained Very retarded larval growth. No pupae
Tineola bisselliella Acheta domesticus Blattella germanica
100% mortality before 3rd instar Very retarded growth Retarded growth. Some pupae and adults Retarded growth. No pupae Very retarded larval growth. N o pupae Larval growth markedly retarded. Retarded growth Very retarded growth Very retarded growth
Locusta migratoria Schistocerca gregaria
Very retarded growth Very retarded growth
Bombyx mori Ephestia elutella Ephestia kuehniella Heliothis zea Pectinoph or gossypiella
1
Pyrausta nu bialis Orthoptera
Royes and Robertson (1 964)
Choline required for normal growth of larvae but less than for D. melanogaster Friend and Patton (1 956) House and Barlow (1 95 8) Bridges et al. (1965) Brust and Fraenkel ( I 955) McGinnis et al. (1956) Hodgson et al, ( 1 956, 1960) House (1954) Dadd e t al. ( 1 967) Kastings and McGinnis (1 967) Vanderzant (1 963) Rock (1969) Horie and Ito (1 965) Fraenkel and Blewett (1946b) Vanderzant (1968) Vanderzant and Reiser (1956) Beck et al. (1949) Fraenkel and Blewett (1 946a) Ritchot and McFarlane (1961) Noland and Baumann (1949) Noland et al. (1949) Dadd (1961) Dadd (1961)
58
R. G. BRIDGES
growth. With the Diptera the reported effects range from no larval growth for Cochliomyia hominivorax to near normal growth of larvae for Musca domestica and Phoimia regina. The report by Singh and Brown (1 957) that adults of Aedes aegypti were obtained, was due almost certainly to the presence of lecithin which was included in the diet. It seems likely that some Diptera require relatively small amounts of choline when compared with other orders and that the variations in the observations are due to trace contamination of choline in the other compounds of the diet. For example, the addition of as little as 0.25 pmole of choline/g of diet is sufficient to enable housefly larvae t o develop to give adults (Bridges and Ricketts, 1968) whereas at least 5 pmole/g of diet is required for the normal development of Bombyx mori (Horie and Ito, 1965). The need for choline by Hemiptera is not clearly established. Choline deprived diets affected the growth of Myzus persicae but adults were obtained, although in reduced numbers. The black citrus aphid, Toxoptera aurantii, could be reared for at least two generations on an amino acid diet lacking all B vitamins (Tahori and Hazan, 1970). However, the authors suggested that this was due to the presence of the necessary symbionts in the aphid. The need for a dietary supply of choline by the adult insect has been studied in only a few cases. Some insects do not feed during their adult life and others such as the housefly will live on a diet of glucose and water but will not lay eggs. Rasso and Fraenkel (1 954) showed that the addition of choline to the diet of adult female blowflies, Phormia regina, somewhat accelerated egg production. Similarly Vanderzant and Richardson ( 1964) concluded that the adult Anthonomus grandis required choline in the diet for continued laying of viable eggs, although choline had been supplied in the larval diet. The utilization of dietary supplied choline by the adult Drosophila melanogaster has been quantified by Geer et al. (1 970). Choline is required for both egg and sperm production by this insect. If it is not supplied to the adult female some eggs will be laid at the expense of the insect’s reserves of choline. Thus at least in the few orders which have received most attention, choline is required by the insect for normal growth. Differences recorded in the effects of deficiency may be due t o variation in the needs of the insect combined with the presence of trace amounts of choline contaminants in the diet. From the limited amount of information available, it seems likely that once the adult stage is reached, the insects main need for choline is for reproduction.
CHOLINE METABOLISM IN INSECTS
59
B. SUBSTITUTES FOR CHOLINE IN THE DIET OF INSECTS
Table I1 summarizes information available on the ability of various compounds related to choline to act as a substitute for an established need for choline in the diet of different insects. All diets contained methionine either as the free amino acid or as a constituent of the casein used as an amino acid source and any relationship between the choline and methionine content of the diet has not been studied in detail. Apart from work with Blattella germanica, betaine has not been found to be a substitute for choline in the diets of the insects examined. In the case of the cockroach, the sparing action of betaine may have been due to the presence of internal symbionts. Betaine aldehyde has been shown to be an effective dietary substituent for choline when fed at high concentrations to Drosophila melanogaster. Addition of ethanolamine has failed to stimulate the growth of any of the insects tested. N-Monomethylaminoethanol has some slight stimulatory effect on growth with the exception of Musca domestica where larval growth was found to be more retarded. N-Dimethylaminoethanol stimulated growth of larvae and pupation occurred in Phormia regina, Drosophila melanogaster and Musca domestica. No adults were obtained however. The same compound also stimulated growth of Tribolium confusum, Aedes aegypti and Blattella germanica, although in the latter case it was fed along with methionine. This stimulation by dimethylaminoethanol has led some authors to suggest that synthesis of choline from this precursor may be occurring. However, it has been shown (see Section VA) that this compound can be incorporated into the phospholipids of Phormia regina and Musca domestica and this may well be connected with its growth stimulating property in these and other insects. Considerable attention has been paid to carnitine as a possible substituent for choline in insect diets, although there is no evidence that it will act as a choline substitute in mammals (Fitz and DuPont, 1957). Carnitine has been established as a dietary essential for the normal development of Tribolium confusum (French and Fraenkel, 1954), Tribolium castaneum (Magis, 1954), Tenebrio molitor and Palorus ratzeburgi (Fraenkel, 195 1b; Cooper and Fraenkel, 1952). However, there is no evidence that it would replace the choline requirements of Palorus ratzeburgi, Tenebrio molitor, Blattella germanica, Bombyx mori, Anthonomus grandis, Aedes aegypti or Cochliomylia hominiuorax. It would, however, act as a partial replacement for choline in the diets of larvae of three Diptera,
Table I1 The effectiveness of various compounds as substitutes for choline in the diet of insects. Betaine
N-Dimethylaminoethanol
Lasioderma serricorne
-
-
Palorus ratzeburgi
-
N-Monomethylaminoethanol -
Ethanolamine -
Carnitine
Referencc
No substitute
Fraenkel et al. (1955)
Requires carnitine Fraenkel et al. as vitamin but will (1955) not substitute for choline Requires carnitine Bieber and as vitamin but will Monroe (1 969) not substitute for choline
Tenebrio molitor
Tribolium No substitute confusum Aedes aegypti
No substitute
Cochliomyia
N o substitute
hornin ovorax
Slight stimulation of growth Slightly enhanced growth, not stimulated with additional methionine
Very slight stimulation of growth
-
No substitute
-
N o substitute
Requires carnitine Lemonde and Bernard (1 955) as vitamin No substitute
Akov (1 962b)
N o substitute
Gingrich (1 964)
Drosophila
melanogaster
No substitute.
Increase in
S o m e increase
Betaine aldehyde effective at high concentrationsa
larval growth. No adults."
in larval growth. Not so effective as dimethylaminoethanola
Musca No substitute dom estica
Increases larval growth. Pupae obtained No adults
Retards larval growth
Anthonomus No substitute grandis Bombyx mori
-
Blattella Adequate germanica substituteC
Phormia regina
No substitute
No substitutea Effective
substitute. in smaller
-
Increase in Some increase in larval growth larval growth. Pupae obtained. No adults
(1965) Geer e t al. (1968) bFraenkel et al. (1955)
No substitute
Effective Bridges e t al. substitute. Adults (1965) obtained.
No substitute
No substitute
Vanderzant (1 963)
N o substitute
Horie and Ito (1965)
-
N o substitute but with methionine some enhancement of growthC
"Geer and Vovis
No substituteC N o substituted
No substitute
CNoland and Baumann (1 949) Fraenkel e t al. (1955)
Effective Hodgson et al. substitute. Adults (1956, 1960, obtained but not 1969) considered normal
62
R. G. BRIDGES
Phormia regina, Musca domestica and Drosophila melanogaster. With these insects larval growth was normal when carnitine was supplied in the diet in place of choline and pupation occurred. Adult flies were obtained, although in the case of Phormia regina none were considered t o be normal. The finding that carnitine was decarboxylated to 0-methylcholine which was incorporated into the phospholipids of Phormia regina larvae (Bieber et al., 1963) led to an intensive investigation into the ability of various compounds related to choline to act as dietary substitutes in the three Diptera. The results are summarized in Table 111. In general a common pattern emerges for all three insects. With Phormia regina, adults were obtained when /3 -meth ylcholine, a-methylcholine , dimethylethylcholine and dimethyl-isopropylcholine were included in the larval diet in place of choline. 0-Methylcholine and dimethylethylcholine were the most successful choline substitutes for the housefly but a-methylcholine failed t o support growth t o adult stage. 0-Methylcholine and dimethylethylcholine supported growth of Drosophila melanogaster to the adult stage. Most other choline analogues increased larval growth and enabled the larvae to pupate but very few adults were obtained. It would seem that the 1-hydroxy-2-quaternary ammonium grouping was essential for the compound to be an adequate substitute for choline. A single methyl-group substituent on the 1- or 2-position was acceptable but further substituents in the 2-position made the compounds less effective. Alkyl-substituents of the quaternary nitrogen groups were permissible if one methyl was replaced by an ethyl-group or an isopropyl-group (Phormia regina) but compounds with alkyl-groups of increasing chain length were progressively less effective although the diethyl-compound stimulated larval growth in the case of Phormia regina and yielded a few adults in the case of Drosophila melanogaster. Thus only very close analogues of choline are capable of acting as dietary substituents which enable the insect to develop to the adult stage. It is of interest to note that adults of Drosophila melanogaster (Geer et al., 1968) from larvae fed on a medium containing carnitine in place of choline and themselves fed on a similar medium laid only few eggs, none of which hatched; so that although certain choline analogues enable adult development, it is likely that choline will be required if they are to produce viable eggs. So far studies with choline analogues have been carried out almost entirely with the three related species of Diptera. The failure of carnitine to act as a replacement for choline on other insects does not imply that they are incapable of utilizing choline analogues, rather that they are unable
CHOLINE METABOLISM IN INSECTS
63
to decarboxylate carnitine t o 0-methylcholine. Bieber and Monroe (1969) have shown in fact that Tenbrio molitor larvae were unable to decarboxylate carnitine but that 0-methylcholine could act as a substitute for their choline requirement, enabling near normal growth. From the results obtained from all these nutritional studies it seems that choline, or some close analogue, is a dietary necessity for the development of insects. This would suggest that, if insects can synthesise choline, they are unable to synthesise adequate amounts to meet their requirements. Apart from the results with Blattella germanica, the failure of betaine, ethanolamine or N-methylated ethanolamines to substitute for choline suggests that transfer of me thyl-groups is lacking which implies a complete inability to synthesise choline. However, the interpretation of results from these nutritional experiments is difficult and only from a detailed investigation of the biochemistry of each insect can a definite conclusion be drawn. IV. WATER-SOLUBLE CHOLINE METABOLITES A. ACETYLCHOLINE
0
+ II (CH3)3-N-CH2 CH2 0-C-CH3 The presence of acetylcholine in insects is now well established and has been the subject of several recent reviews (Colhoun, 1963; Treherne, 1966; Boistel, 1968; Smallman and Mansingh, 1969). Acetylcholine is present in high concentrations in insects when compared with most other invertebrates and vertebrates. It is now generally accepted that it is an excitory synaptic transmitter in insect nerve (Kerkut et al., 1969) but not at the neuromuscular junction. Colhoun (1963) and Smallman and Mansingh (1969) have summarized data showing the distribution of acetylcholine in various insect tissues and at different stages of development. In Periplaneta americana, acetylcholine is confined to nervous tissue and recent unpublished work by the author has also indicated that it is largely associated with the fused ganglia of the larva of the housefly. High concentrations of acetylcholine have been reported in the eggs of insects. The most complete study has been that of Mehrotra (1960) on Musca domestica and Oncopeltus fasciatus. A large increase in the acetylcholine content of the eggs occurred just prior
Table 111 Utilization of choline analogues in place of choline by Drosophila melanogaster, Musca domestica a n d Phormia regina
D. melanogaster
M. domestica
P. regina
A (CH3)3fi-R
R=
OH
1
-CH, CHCH,
p-methylcholine
Normal L growth, P and some A 0btainedC.f
Normal L growth, P and some A ob t h e & * k
Normal L growth, P but no A obtainede
Normal L growth, P and some A obtainedg
Some enhancement of L growth. Few P and no A obtaineda
Normal L growth, some P but no A obtainede
Some enhancement of L growth. No P obtainedg
-
Normal L growth, P but no A obtainede
Enhances larval growth. P and some A obtaineda
CH, OH 1 1 -CH-CH, (YH3)2yH -C
-
CH,
a,&-dimethylcholine
C,H, OH
I
1
CH,
a-e thylcholine
-CH, CH, CH, OH
Homocholine
-C
-
Enhancement of L growth. P and some A obtaineda
R
I B (CH,),-N+-CH,CH20H
R= N-DimethylaminoEthan01
Enhancement of L growth. Some
P and few
Normal L growth, P but
Enhancement of L growth,
no A obtainedc.d
P but no A obtained8.i
C*H, -
Dimethylethylcholine
C,H, (CH,), -CHC,H, -
c5 HI,
-
Enhancement of L growth.
Normal L growth,P and
P and A obtaineda
A obtainede
Dimethyl-n-propylcholine Dimethyl-isopropyl- . choline Dimethyl-n-bu tylcholine Dimethyl-n-amylcholine
-
-
Enhanced L growth, P but very few A obtainede -
Enhanced L growth, P but very few A obtainede Some enhancement of L growth, few P and no A obtainede
Normal L growth. P and
some A obtained89h.i Enhancement of L growth, some P, no A obtainedg.ki Normal L growth, P and A obtainedg9h.i Enhanced L growth, some P but no A obtainedg.h,i -
C Miscellaneous N-Monomethylaminoethanol CH,N-CH,CH,OH 7-Butyrobetaine (CH,), - N'- CH, CH, CH, COOH Diethy lmethylcholine (CH&
Enhancement of L growth. Some P no A obtained4 b Enhancement of L growth. P and some A obtaineda
Decrease in L growth. No P 0btainedc.d -
-
Enhancement of L growth. No P obtained8 Enhancement of L growth. Some P and A obtainedgsk Enhancement of L growth. Some P but no A
I
(C,H5)l -N'CH,CH,OH Sulphocholine (CH,), - S+ CH, CH,OH
Enhancement of L growth. Some P and A obtained0
Triethylcholine (C,Hs)3- N+-CH, CH, OH
Enhancement of L growth. Few P and no A obtaineda
and Vovis (1965) b Geer et al. (1968) C Bridges et al. (1965) Bridges and Ricketts (1967) Bridges and Ricketts (1970) fMoulton et al. (1970)
a Geer
L=Larval
g Hodgson et al. (1969) h Mehendale et al. (1970) i
Hodgson and Dauterman (1964)
i Bieber and Newburgh (1963) k Bieber et al. (1963
P = Pupae
A = Adults
ob tainedg. i Enhancement of L growth. Some P but no A ob tainedg -
66
R. G. BRIDGES
to hatching, rising to 3 pmole/g in eggs of Musca domestica and 1 pmole/g in eggs of Oncopeltus fasciatus. This is very much higher than values obtained for 6-day-old larvae of Musca domestica which were between 0.01 8-0.024 pmole/g (Bridges and Ricketts, 1968, 1970). It is difficult to explain these high levels in terms of function and the source of the large amount of choline necessary for its synthesis is not at all clear. From the data collected by Smallman and Mansingh (1969) little change in the acetylcholine content is observed during the late larval and pupal stages of Antheraea polyphemus, Antheraea pernyi, Malacosoma americanum, Galleria mellonella, Ostrinia nubilalis and Hyalophora cecropia. During development of the adult, a rise occurs which appears to be associated with the differentiation and elaboration of the imaginal nervous system. Colhoun (1963) has noted that there are some events, remote from the insect nervous system, that involve acetylcholine. For example a high concentration has been reported in royal jelly of Apis mellifera and also in the venom of Vespa crabro. It is also present in the male and female reproductive organs of certain Lepidoptera. A recent paper by Grzelak et al. (1 970) suggests that changes observed in the acetylcholine content of the abdomen of Celerio euphorbiae during the first eight days after pupation do not seem to involve the central nervous system. Thus although insects have a basic need for choline to provide acetylcholine for the normal functioning of the nervous system some may have an additional need to supply the acetylcholine required for more specialized purposes. B. PHOSPHORYLCHOLINE
+
0 II
(CH3)3N CHZCHZ-0-P-OH
I OH Phosphorylcholine has been found in high concentrations in the haemolymph of certain Lepidoptera. Table IV summarizes the information available giving the concentration of phosphorylcholine in the lymph or tissues and its percentage of the acid-soluble phosphorus. The presence of undetermined amounts of phosphorylcholine has been demonstrated in adult Celerio euphorbiue (Chojnacki, 196 l ) , adult Arctia caia (Chojnacki and Korzybski,
67
CHOLINE METABOLISM IN INSECTS
Table IV Phosphorylcholine content in insects
pmole/mlOr g wet wt. Musca Whole L (6 day-old) dornesticaalb Whole A Antheraea Haemolymph P, 5 months at 6zC polyphemusC P, 9 months at 6 C P, 11 months at 6°C Bombyx morF Haemolymph L, midstar V L, instar V pooled P, early Galleria Haemolymph L, Days fasting 0 mellonellad Days fasting 5 Days fasting 19 Days fasting 37 Days fasting 42 Galleria Intestine L mellonellae Fat body L Remaining tissue L Whole L Hyalophora Haemolymph L, instar V cecropiaC P, 25OC A? 2 days old A? 13 days old Hyalophora Fat body P, diapausing cecropiaf P, 2 days after start of adult development Protoparce Haemolymph L, fully grown sex tac qb Bridges and Ricketts (1968,1970)
dLenartowicz and Niermierko (1964) fGarey and Wyatt (1963) L = Larva
P = Pupa
% of acid soluble phosphorus
0.19-0.45 0.84 10.7 11.1 15.6 6.7 5.7 18.0 21.2 24.6 35.4 28.6 43.9 6.2 4.1 5.9 11.7 2.3 11.5 8.7 20.7 0.4
48 33 47 13 15 62 29 36 48 35 57 14 20 17 35 8 31 24 64 18
1.7 w.7
25 3-3
Wyatt e f al. (1963) Lenartowicz et a1. (1964) A = Adult
1962), larvae of Heliothis zea (Willis and Hodgson, 1970), in haemolymph of Acan tholyda nemorulis larvae (Zielinska and Dominas, 1967), larvae of Hylobis pales (Willis and Hodgson, 1970), adult Periplaneta umericana (Kumar, 1968) and in larvae of Phormia regina (Willis and Hodgson, 1970; Mehendale et al., 1970). Phosphorylated choline-analogues have been demonstrated in Phorrnia regina larvae when fed on diets containing dimethylethyl-
68
R. G. BRIDGES
choline, dimethyl-n-propylcholine, dimethyl-isopropylcholine and dimethyl-n-butylcholine (Mehendale et al., 1970). Phosphorylcholine was found in extracts of Musca dornestica larvae and phosphoryl-0methylcholine when 0-methylcholine, carnitine or y-butyrobetaine were fed (Moulton et al., 1970; Bieber et al., 1969). The concentration of phosphoryl-p-methylcholine in these larvae was estimated to be between 0.3 and 0.5 pmole/g wet weight. Estimates of the amount of phosphorylcholine have been made in larva and adults of Musca domestica (Bridges and Ricketts, 1968, 1970) and it has been shown to depend on the amount of choline available to the larvae in its diet. The figures given in Table IV are for housefly larvae fed on high choline diets (16 pmole choline/g casein). When fed on diets containing 1 pmole and 0.04 pmole choline/g casein the levels of phosphorylcholine in the larvae fell to 0.02 and 0.001 pmole/g wet weight respectively. Phosphorylcholine was not reported as a component in the phosphorus-containing compounds present in the acid-soluble fraction of thoraces of Musca dornestica (Winteringham et al., 1955). However, using their figure of 10 pg acid-soluble phosphorus/7 mg thoracic tissue, the amount of phosphorylcholine present in the adult fly (Table IV) would be only 2% of the total phosphorus-containing compounds and could well have remained undetected. The low proportion of phosphorylcholine in this insect is very different from the figures obtained for the Lepidoptera (apart from Protoparce sexta), where even in tissue extracts as distinct from haemolymph, about 20% of the acid-soluble phosphorus was phosphorylcholine. The high levels of phosphorylcholine in the haemolymph of the Lepidoptera listed in Table IV were found at all stages of development there being some suggestion of an increase from larva to adult. It was particularly high in larvae of Galleria mellonella and it increased during fasting. Lenartowicz and Niemierko (1964) suggested that this was due to the breakdown of phosphatidylcholine. This implies the action of a phospholipase C or some factor affecting the reincorporation of phosphorylcholine into phospholipid such as a lower availability of &glyceride. No role has been suggested for the high concentrations of phosphorylcholine in the haemolymph of these insects. Whether they are related to the amount of choline in the diet is not known with certainty but the high level found in fasting Galleria mellonella larvae suggests that they may be independent. Whatever the role of this compound is in these insects, choline from the diet will be required for its synthesis. In summary, the presence of phosphorylcholine has been
69
CHOLINE METABOLISM IN INSECTS
demonstrated in a number of different orders of insects; the Coleoptera, Diptera, Hymenoptera, Lepidoptera and Orthoptera. The amounts present show some dependence on the stage of development and, in the case of the housefly, on the amount of choline available to the insect in its diet. Tissue levels in most cases are comparable with those reported for vertebrates (Ansell and Hawthorne, 1964) but the concentration in the haemolymph of the Lepidoptera studied (apart from Protoparce sexta) is very much higher. C. CYTIDINEDIPHOSPHORYLCHOLINE (CDP-CHOLINE)
R B
CH2-0-P-0-P-0-CH2CH2 I
OH
4.
N(CH3)3
I
OH
Very little information is available on the occurrence of this choline metabolite in insects. It is the intermediate between phosphorylcholine and the lipids phosphatidylcholine and sphingomyelin. In vertebrates the reported levels of CDP-choline do not exceed 0.1 pmole/g (Ansell and Hawthorne, 1964). Results from feeding isotopically labelled choline have shown the presence of CDP-choline in larvae of Phormia regina (Mehendale et al., 1970) and in larvae and adults of Musca domestica (Bridges and Ricketts, 1968, 1970). In the case of the housefly, the level of CDP-choline depended on the level of choline in the larval diet and was 0.006 pmole/g wet weight of tissue (dietary concentration of choline 16 pmole/g casein) 0.002 pmole/g ( 1 pmole/g casein) and 0.0003 pmole/g (0.04 pmole/g casein). Adult flies from larvae fed on diets containing 16 pmole choline/g casein contained 0.009 pmole CDP-choline/g of fly. The formation of CDP-choline has been demonstrated in vitro in fat body preparations from larvae of Phormia regina (Shelley and Hodgson, 1970). Willis and Hodgson (1970) stated that CDP-choline could be detected in extracts from larvae of Heliothis zea and Hylobius pales as well as Phormia regina. Thus, where it has been looked for in insects, CDP-choline has been found. In the housefly larva, the concentration of CDP-choline is dependent on the amount of choline in the diet and appears to be considerably lower than that found in vertebrates.
70
R. G. BRIDGES
D. GLYCERYLPHOSPHORYLCHOLINE
CH2OH
I
CH OH
I
OH Glycerylphosphorylcholine can be produced by the action of phospholipases on phosphatidylcholine. It has been found in fairly high concentrations in the fat body of the pupae of Hyalophora cecropia (Garey and Wyatt, 1963). Diapausing pupae, one month after pupation stored at 25-30" contained 0.1 1 pmole/g (5% of the acid-soluble phosphorus); diapausing pupae, debrained two months after pupation and stored at 25" for three months and then at 6" for three months contained 4.65 pmole/g (40% of the acid-soluble phosphorus) and pupae at second day of adult development after storage for seven months at 6°C and two days at 25°C contained 0.68 pmole/g (1 0% of the acid-soluble phosphorous). Wyatt et al. (1 963) failed to detect glycerylphosphorylcholine in the haemolymph of Hyalophora cecropia but they suggested that this might be due to its hydrolysis to a-glycerophosphate and choline during storage of the lymph prior to analysis. Kumar ( 1968) found injected methyl-14C-choline appearing in glycerylphosphorylcholine in Periplaneta americana. Mehendale et al. ( 1970) found radioactive glycerylphosphorylcholine in extracts from Phormiu regina larvae fed on diets containing methy1-l4C-choline. Unpublished results by Bridges have demonstrated very small concentrations of g1ycerylphosphorylcholine in housefly larvae of the order of 0.01 pmole/g wet weight. Glycerylphosphoryl0-methylcholine has been detected in housefly larvae fed on diets containing 0-methylcholine, carnitine or y-butyrobetaine (Bieber et al., 1969). The presence of large amounts of glycerylphosphorylcholine in fat body of Hyalophora cecropia during pupation may be due to breakdown of phospholipid during this stage of development. However, the amount which accumulates is too great to be entirely derived from pre-existing phospholipid and suggests a steady
CHOLINE METABOLISM IN INSECTS
71
turnover of phospholipid leading to this product. The presence of this choline metabolite has been detected also in Orthoptera and Diptera but apparently in much smaller amounts. V. LIPID-SOLUBLE CHOLINE-METABOLITES A. PHOSPHATIDYLCHOLINE
A
CH, -0-C-R
I
‘ 1
CH-0-
-R”
OH R‘ and R” = long chain alkyl groups Table V lists the percentage of the phospholipid present as phosphatidylcholine in lipid extracts from different orders of insects at different stages of development. It is an extension of the major survey carried out by Fast (1966) and adds further confirmation to his conclusion that, as in vertebrates, phosphatidylcholine is the predominant phospholipid type in Hymenoptera, Lepidoptera and Orthoptera. The Coleoptera, Hemiptera and the one species of Thysanura examined contain a rather lower percentage of their phospholipids as phosphatidylcholine. The Diptera, apart from a few exceptions (Ceratis capita eggs and larvae, Culex pipiens fatigans eggs, Glossina morsitans adults, Phytophaga rigidae larvae and Rhabdophaga swainei larvae) all contain a much lower level of phosphatidylcholine than other insects and vertebrates, varying between 14 to 29% of the total phospholipid. The figure of 22% reported for larvae of Trogoderma granarium is the only other exception to these generalizations. Tables VI and VII summarize the
4 N
Table V Lipids containing choline in whole insects Results are based on phosphorus estimation and are expressed as a percentage of the total lipid phosphorus
Coleoptera Altica am biens alni Chrysomela crotchi Pissodes strobi Tenebrio molitor Tribolium confusum Trogoderma granarium Diptera Aedes aegypti Calliphora ery throcephala Ceratis capita
Stage
Phosphatidylcholine
L L P L L L P A L A
34.3 39.8 36.0 39.3 43 42-53 39-44 35-36 22.2 37.7
L L E
14.4 24 31.8 34.3 26.0 17.5 26.1 33 26 23 20 27.5 27.4 14.9 Present
? ? 0.1 1.9 1.o 1.1 1.8 ? ? ? ? 1.8 0.4 1.5
17.5
?
L Chironomus spp. Culex pipiens fatigans
Dacus oleae
P A A
E L P A L P A
Drosophila melanogaster iigy&1s.itB~ikTsa
A L
Lysophospha- Sphingomyelin tidylcholine ? ? ? ? ?
trace 1-7 7
? ? ? ?
Reference
Fast (1 966) Fast ( 1966) Fast (1966) Fast (1966) Kamienski et al. (1 965)
6.8
9 Beaudoin et al. (1 968) 9-10
1.3a Absent Absent >
Fast and Brown (1 962) Bridges (unpublished work)
} Castillbn et al. (1971)
Absent Absent Absent Absent Absent >
Absent Absent Absent) Absent Absent Absent ? Absent
W
E
U
Rao and Argarwal(1969)
?
P
}
I
Fast (1 966) Kalra et al. (1969)
Castillbn et 41. (1 971) Wren and Mitchell (1959) Fast (1966)
8
v1
Glossina morsitans Hylemia antiqua Lucilia cuprina Musca domestica Nephrotoma sodalis Phormia regina Phytophaga rigidae Rhabdophaga swainei Strauzia longipennis Hemipt era Anuraphis bakeri Aphrophora parallela Ericerus pela Oncopeltus fasciatus Prociphilus tesselatus Schizolachnus pini-radiatae Hymenoptera A can tholyda nemoralis Arge pectoralis Monoctenus juniperinus Neodiprion sertifer Pikonema alaskensis Lepidoptera Archips cerasivoranus Arctia caia Bom byx mori Datana integerrima Erannis tiliaria
A
A P&A L A A L E L A L L L
Au N.A L E,N,A A All All
17.4 26.0 16.0 13.9 17.2 26.0 19 21 19 37.5 48.1 29.0
Absent ? ? ? ? ? ? ? ? ?
27.2 46.0 30 Major phospholipid 31 29.7 33.7
L L L L L
64.5 41.6 52.1 49.2 47.8
L A
45.9 47
P L L
62.5 37.6 45.4
Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent
? ? ?
35
-
? ?
? ? ?
Cmelik et aL (1 969)
Fast (1 966) D'Costa and Birt (1966) Bridges and Price (1 970a) Fast (1 966) Crone and Bridges (1963) Fast (1966) Bieber et al. (1 96 1) Fast (1 966) Fast (1 966) Fast (1 966)
?
?
7
7
Fast (1 966) Fast (1966) Hashimoto and Mukai (1 967) Kinsella (1 966a) Yurkiewicz and Whelchel(l969) Fast (1 966) Fast (1 966)
? ? ? ?
? ? ? ?
Zielinska and Domina (1 967) Fast (1 966) Fast ( 1966) Fast (1 966) Fast (1966)
? ?
1W
trace Present
Present
1
u 7.8
? ? ?
?
16" ? ?
Fast (1 966) Chojnacki and Korzybski (1 962) Sridhara and Bhat (1 965) Fast (1 966) Fast (1966)
w
4 P
-
Table V-cont.
Galleria mellonella Heliothis zea Hyphantria cunea Paleacrita vernata Plodia in terpu n c tella Protoparce sexta Orthoptera Acheta domesticus Blattella germanica Diapheromera femorata Gryllus bimaculatus Locusta migratoria Periplaneta americana
L
?
60
A 39 L & P Major component
Not detected Wlodawer and Wihiewska (1 965) 5 Yurkiewicz (1 968) Present Lambremont and Graves (1 969) ? Fast (1 966) 7 Fast (1966) Yurkiewicz (1 967) 8 Not detected Hodgson ( 1 965)
?
L L E
40 48.7 38-45
? ?
u
7 p
A
ca 40
?
W
E L
58 40 45 53 57.4 54 58.6-70.5 5 7-6 5 57.7 60 44
? ? ? ? ? ? ? ? Some detected ? ?
A A A A E
E N A A
11
}
? ?
Present 3.5-4.W 5.2-6.5 6.1 5.9 ?
}
Lipsitz and McFarlane (1 970) Crone and Bridges (1 963) Fast (1 966) Fast (1 967) Allais et al. (1 964) Kinsella (1 966b) Crone and Bridges (1 963)
Thysanura Lepisma saccharina a Sphingomyelinnot fully characterized.
4.0
36.3 E = Egg;
L = Larva;
10.4 N = Nymph;
Kinsella (1 969) P = Pupa;
A = Adult.
E U Q g
Table VI Lipids containing choline in different insect tissues Results are based on phosphorus estimations and are expressed as a percentage of the total lipid phosphorus Phosphatidylcholine
Lysophosphatidylcholine
Sphingomyelin Absent Absent Absent Absent
Reference 0
Musca domestica L Brain + imaginal discs Cuticle Muscle + tracheae Gut + salivary glands + Malpighian tubules Fat body Haemolymph A Head Thorax Abdomen Sarcophaga bullata L Fat body Apis mellifera A Brain Galleria mellonella L Haemolymph
17.7 20.6 19.1 19.5
? ? ? ?
19.2 11.3 19.5 13.5 19.0 21 12 57
? ? ? ? ? 13 ? ?
Hyalophora cecropia PA Muscle A Muscle Acheta domesticus L Haemolymph Schistocerca gregaria A Haemolymph
46 43
X 0
'I
c Bridges and Price ( 1970a)
d z m 4
>40
Absent Absent Absent Absent Absent Absent 9 Not detected
I
Crone ( 1 964)
h-
All detected but not quantified Detected Detected PA = Pharate adult;
}
sE z
2 Allen and Newburgh (1 965) Patterson et al. (1945) Wlodawer and Wiiniewska ( 1 96s)
? ?
L = Larva;
k
. - - - - I
Thomas and Gilbert (1 967)
.
Wang and Patton (1 969) Mehrotra et al. (1 966)
A = Adult.
a
c4l
m
0
2
Table VII Lipids containing choline in various subcellular fractions from insect tissue
4 o\
Results are based on phosphorus estimations and are expressed as a percentage of the total lipid phosphorus Phosphatidylcholine Lucilia cuprina Flight muscle sarcosomes PA 20 A at emergence 27 A at 5 day 21 Musca domestica Gut 600g Fraction L, 6 day 7.1 10,OOOg Fraction 14.4 100,OOOg Fraction 20.5 Muscle 600g Fraction L, 6 day 13.8 10,OOOg Fraction 11.6 100,OOOg Fraction <19.1 14.2 Fat body 600g Fraction L, 6 day 10,OOOg Fraction 18.5
P=Pupa;
Lysophospha- Sphingomyelin tidylcholine ? ? ?
Reference
I
Not detected Not detected D’Costa and Birt (1 966) Not detected
4.4 2.2 Absent Absent Absent Absent Absent Absent Absent ? ?
6.0 7.1 3.0 0
Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent
?
3.6
?
?
?
?
PA = Pharate adult;
1
p Bridges and Price (1 970a)
I Crone (1 964) Chan (1 970) Khan and Hodgson (1 967a)
Thomas and Gilbert (1 967) Chan and Lester (1 970)
A = Adult.
n m
zU 0 E
CHOLINE METABOLISM IN INSECTS
77
distribution of phosphatidylcholine in the phospholipids extracted from various types of insect tissue and also subcellular fractions made from different tissues. The low percentage of phosphatidylcholine present in extracts of whole larvae of Musca domestica was also seen throughout the different tissues isolated from the larvae. The larval haemolymph contained an even smaller percentage. Similarly, heads and abdomina from Musca domestica adults contained only a slightly greater percentage of their phospholipids as phosphatidylcholine than did the whole insect. The rather smaller percentage found in the thoraces would seem to be correlated with that found in the thoracic flight muscle sarcosomes (Table VII). Comparison of all three tables shows that in only one case, the brain of Apis mellifera, does the percentage of phosphatidylcholine present in the phospholipids of the different tissues or subcellular fractions differ from that expected assuming that the different phospholipids are evenly distributed throughout the whole insect. The figure for the bee brain may be suspect as it was obtained before the more refined techniques for phospholipid separation were available. Although not shown in Tables V-VII, Diptera which contained this unusually low percentage of phosphatidylcholine, also contained a correspondingly higher proportion of phosphatidylethanolamine which was the major phospholipid in these insects. The combined phosphatidylcholine plus-ethanolamine content was between 70-80% of the total phospholipid. Clearly therefore, some phosphatidylethanolamine must be utilized by Diptera to perform similar functions to those for which much of the phosphatidylcholine is used in other insects and vertebrates. Fast (1966) linked this observation with the occurrence of large amounts of esterified palmitoleic acid in Diptera and suggested that this modified the physical and physiological effects of the phosphatidylethanolamine to that of phosphatidylcholine containing the more usual fatty acids. However, the fatty acid composition of Musca domestica can be altered considerably by dietary means (Bridges, 197 1) and palmitoleic acid has been reported in other orders of insects, being particularly high in some Lepidopteran larvae which overwinter (Bracken and Harris, 1969). Table VIII gives some estimated values for phosphatidylcholine on a comparable tissue concentration basis. The figures have been obtained by making certain assumptions, i.e. that 1 pmole of phospholipid contains only one patom of phosphorus and that the phosphorus content is 4% of the phospholipid content. The Diptera
78
R. G. BRIDGES
Table VIII Phosphatidylcholine in insects Nmolelg wet wt. Coleoptera Tenebrio molitor Tribolium confusum Trogoderma granarium Diptera Aedes aegypti Culex pipiens fatigans
Musca domestica Phormia regina Hemiptera Oncopeltus fasciatus Lepidoptera Bombyx mori Galleria mellonella Plodia interpunctella Orthoptera Acheta domestica
E = Egg;
L = Larva;
L L P A L A L E L P A E L A E L A
5
:}
Bieber and Monroe (1 969) Beaudoin e t al. (1 968)
8 7
Rao and Argarwal(1969)
'I
Fast and Brown (1 962)
5
Bridges (unpublished work) Bridges and Ricketts (1 968) Crone and Bridges (1 963)
1
43
3 4
Kalra e t al. (1969)
Bieber e t al. (1 96 1)
A
5
Yurkiewicz and Whelchel(l969)
P
10
L E
15 8
Ito e t al. (1958); Sridhara and Bhat (1 965) Wlodawer and Wihiewski (1 965) Yurkiewicz (1 967)
E L A
';}
Lipsitz and McFarlane (1970)
10
P = Pupa;
A = Adult.
contain, as one would expect if the total phospholipid content is similar for all insects, less phosphatidylcholine than other orders. Where observations have been made on changes of phospholipid composition during development, little differences are apparent in either the percentage of phospholipid present as phosphatidylcholine or in its amount. In general the egg is richer in the choline-lipid than other stages but this largely reflects the larger concentration of total phospholipid.
CHOLINE METABOLISM IN INSECTS
79
Larvae of Musca domesticu can grow on diets to which no choline has been added. The phosphatidylcholine content of such larvae was 0.9% of the total phospholipid present (Bridges and Price, 1970b) with a corresponding increase in phosphatidylethanolamine. Thus in this extreme case much of the phosphatidylcholine which would have been present, if the larva had been fed adequate amounts of choline, had been replaced by phosphatidylethanolamine. The addition of only small amounts of choline to the larval diet enabled the normal development of the insect through to the adult. Larvae containing less than one quarter of the maximum amount of phosphatidylcholine that could be achieved by feeding them diets containing greater concentrations of choline, went on to pupate and gave normal adults (Bridges and Ricketts, 1968). The level of phosphatidylcholine increased in larvae with increasing amounts of choline in their diet up to 4pmole choline/g of casein. Further increase in the concentration of choline in the diet produced no further increase in phosphatidylcholine. While the phosphatidylcholine content was below the maximum attainable, 97-98% of the choline taken up by the larvae was present as phosphatidylcholine (Bridges and Ricketts, 1968, 1970). In larvae fed on diets containing no added choline, the phosphatidylcholine was not evenly distributed among the different tissues (Bridges and Price, I970b). There was a considerable concentration of the small amount of choline present in such insects into the nervous system. The phosphatidylcholine content of whole larvae and non-nervous tissues was 1% of the total phospholipid present in the corresponding tissue whereas that of the nervous tissue was 5% of the phospholipid content. Some 8% of the total phosphatidylcholine content of such larvae was recovered from the nervous tissue whereas only 0.6% of the total protein was present. Clearly, under conditions of choline deficiency some mechanism exists which allows choline to be taken up preferentially by the nervous system. Thus in the housefly full development of the insect from larvae to adult occurred even when most of the tissues contained relatively small amounts of phosphatidylcholine. The satisfactory development of this insect would seem to be linked with the accumulation of phosphatidylcholine in the nervous system. Whether this higher level of phosphatidylcholine is necessary for the normal functioning of the nervous system or whether it is a consequence of the more rapid uptake of choline by the nerve to provide for synthesis of acetylcholine will be discussed later (Section VII).
80
R. G. BRIDGES
The incorporation of analogues of choline into the phospholipids of insects in place of phosphatidylcholine has been mentioned earlier. Both Phormia regina and Musca dornestica have been studied and a wide range of hydroxy-ammonium compounds have been found to be utilized for phospholipid synthesis when they are fed to the growing larvae. The ability of some of these analogues to stimulate the growth of larvae grown on choline-deficient diets has been dealt with (Section 111, B) and some attempt has been made to correlate this ability with the effectiveness of the compound as a phospholipid precursor. Hodgson et al. (1969) have shown that all the compounds which stimulate the growth of Phormia regina larvae, in the absence of a dietary source of choline, were incorporated into the larval phospholipids as the corresponding phosphatidyl-derivatives. Mehendale et al. (1970) have linked the amount of 14C-activity present in the phospholipids of Phormia regina larvae fed on diets containing dimethylethyl-, dimethyl-n -propy1-, dimethy1-isopropy1-, dimethyl-n-butyl- and 0-methyl-cholines or carnitine labelled with 14C in the N-methyl groups, with the extent to which they supported growth and development. Earlier work with this insect had shown that phosphatidyl-0-methylcholinewas present in the larvae when fed on either 0-methylcholine, carnitine or y-butyrobetaine (Bieber et al. 1963; Agarwal and Houx, 1964). The combined phosphatidylcholine plus phosphatidyl-0-methylcholinewas between 2 1-27% of the total phospholipid in these insects while the phosphatidylcholine content alone was between 1-576 of the total phospholipid. Bieber and Newburgh ( 1963) reported that both N-dimethylaminoethanol and N-dimethylamino-isopropanol were also incorporated into Phormia regina larvae in the absence of choline. The grown larvae contained about 30% of their phospholipids as phosphatidyldimethylaminoethanol and 18% as phosphatidyldimethylaminoisopropanol. The percentage of phosphatidylcholine decreased from 20% (when choline was fed) to 2.3% when the dimethylaminoalcohols were fed. A similar situation was found to exist with Musca dornestica larvae. P-Methylcholine and carnitine when fed to larvae in place of choline resulted in 11-14% of the total phospholipids of the six-day-old larva being recovered as phosphatidyl-0-methylcholine and 2-3% as phosphatidylcholine (Bridges et al., 1965). N-Dimethylaminoethanol and also N-monomethylaminoethanol were extensively incorporated into phospholipid by the larvae when included in their diet in both the absence and presence of added choline. When
CHOLINE METABOLISM IN INSECTS
81
choline was included along with the aminoalcohols, the proportion of the phospholipid in the larvae that was the phosphatidyl-methylated-aminoalcohol decreased as the phosphatidylcholine increased. The incorporation of a large range of choline-analogues into the phospholipids of the larval stage of this insect has also been studied (Bridges and Ricketts, 1970). By using analogues in which one of the quaternary me thyl-groups of the choline-moiety was replaced by a series of alkyl-groups of increasing chain length and feeding them at equimolar concentrations it was shown that the longer the alkyl-substituent the lower was the percentage of the corresponding phosphatidyl-derivative in the six-day-old larva. When dimethylethylcholine was fed, 17% of the phospholipid was phosphatidyldimethylethylcholine when dimethyl-n-amylcholine was fed only 6% of the phospholipid was phosphatidyldimethyl-n-amylcholine.Adult flies were obtained when either 0-methylcholine or dimethylethylcholine was included in the larval diet in place of choline and the adults also contained the phosphatidyl-choline-analogue in place of much of what would have been phosphatidylcholine, if choline had been fed to the larvae. When any of the choline-analogues were fed with equimolar amounts of choline competition between the two for incorporation into phospholipid was demonstrated, the closer the structure of the analogue to choline itself the more effectively did it compete for incorporation. Similar observations were made by Mehendale et al. (1970) with larvae of Phormia regina. It is likely that similar incorporation into phospholipid occurs in the case of Drosophila melanogaster but no detailed examination of the lipid-fraction has been made (Geer et al., 1968). The incorporation of &me thylcholine into the phospholipids of Tenebrio molitor has been reported (Bieber and Monroe, 1969) indicating that the ability to utilise choline analogues is not confined to the Diptera. Very little effort has been made to determine the choline plasmalogen content of insects. Choline plasmalogen differs from phosphatidylcholine in that one of the esterified long chain fatty acids is replaced by an ether linked, a,0 unsaturated long chain hydrocarbon. In some cases positive tests for the presence of plasmalogens in separated lipid fractions have been obtained, e.g. in Phormia regina (Bieber et al., 196l), in Tenebrio molitor (Kamienski et al., 1965) and in the haemolymph of Acheta domesticus (Wang and Patton, 1969). However, no positive identification of choline plasmalogen has been made. Crone and Bridges (1 963) found only 1.3% of the phospholipids from adult Musca domestica behaved as an
82
R. G. BRIDGES
uncharacterized plasmalogen fraction. Plasmalogens could not be detected in Protoparce sexta (Hodgson, 1965) or Culex pipiens fatigans (Kalra et al., 1969). In summary, phosphatidylcholine has been found to be a constituent of the phospholipid fraction in all insects examined. It is the major phospholipid type in Hymenoptera, Lepidoptera and Orthoptera. In most Diptera it is quantitatively less important than phosphatidylethanolamine. In Coleoptera and Hemioptera both these lipids are in approximately similar amounts. Most insects will require much of their available choline to meet the requirements for the synthesis of phosphatidylcholine although in the case of Galleria mellonellu a similar amount will be required for phosphorylcholine synthesis (cf.Table IV and VIII). In the case of the housefly, development through to the adult stage can occur before the maximum levels of phosphatidylcholine are attained in most tissues although higher levels are found in the nervous tissue due to more rapid uptake of choline. It is not known how typical this is of other insects. Choline-analogues are incorporated into phospholipids in place of choline. This has been demonstrated most extensively in two species of Diptera. B. LYSOPHOSPHATIDYLCHOLINE
0
II CH2 -0-C-R
I
CHOH
I
0
I1
CH2 -0-P-OCH2
-+ CH2 N(CH3 )3
OH Lysophosphatidylcholine is formed enzymically by the action of phospholipase A on phosphatidylcholine with the hydrolysis of one of the fatty acid residues. It has been found in very small amounts in most vertebrate tissues and can cause serious damage to the cellular and intra-cellular frameworks at certain concentrations. Its presence may be in part an artefact due to hydrolysis of phosphatidylcholine during the preparation and extraction of the material. Information on its occurrence in insect lipids is fragmentary and has been
83
CHOLINE METABOLISM IN INSECTS
summarized in TablesV, VI and VIII. A detailed study of the changes occurring in the phospholipids during metamorphosis of Tribolium confusum showed a big increase in the lysophosphatidylcholine at the beginning of the pupal period followed by a fall (Beaudoin et al., 1968). This might be associated with the general histolysis of larval tissue that occurs at this time although equally large amounts were reported in the adult. It is interesting to note that no significant amounts of lysophosphatidylcholine were detected in extracts of larvae of Musca domestica but appreciable amounts were found in the subcellular fractions prepared from gut tissue suggesting the presence of an active phospholipase A in this tissue (Bridges and Price, 1970a). It was also detected in subcellular fractions of flight muscle from Musca dornestica (Khan and Hodgson, 1967a) where again its presence may well have been due to hydrolysis during the fractionation procedure. It seems clear that lysophosphatidylcholine can only be present in very small amounts in insects otherwise more reports of its occurrence would be available. C. SPHINGOMYELIN
0 CH3(CH2),2CH=CH-CH-CH-CH2
I
I
OH NH
I
II
0-P-0
CH2CH2 h(CH,
)3
OH
T=O R
R = long chain alkylgroup. Sphingomyelin is a fatty acid amide of sphingosylphosphorylcholine. It has been reported to be present in a number of insects (summarized in Tables IV, V and VI). In many cases, it has not been completely characterized but evidence is good for its presence in some Lepidoptera, Coleoptera and Orthoptera. Its absence has been clearly established for a number of Diptera. Only in the case of Aedes aegypti has there been any report of its presence in this order of insect and here it was not characterized, so that it may well have
84
R. G. BRIDGES
been confused with lysophosphatidylcholine. Evidence for the ethanolamine-analogue of sphingomyelin in the lipids of Diptera is considerable, e.g. Musca dornestica (Crone and Bridges, 1963; Khan and Hodgson, 1967a), Phorrnia regina (Bieber et aZ., 1963) and Calliphora erythrocephala (Dawson and Kemp, 1968). Here again one sees the curious inversion of ethanolamine and choline in the lipids of this order of insects. Where sphingomyelin has been found it has been shown to be a relatively minor component of the phospholipid fraction in almost all cases being less than 10% of the total phospholipids. VI. ENZYMES INVOLVED IN CHOLINE METABOLISM A. CHOLINEACETYLASE AND ACETYLCHOLINESTERASE
N o attempt will be made to list the occurrence of these two enzymes in insects as this subject has been covered in numerous reviews. The recent review of Smallman and Mansingh (1969) summarizes data for their presence and the relationship between them and the developing nervous system. Appearance of the two enzymes in the egg is associated with neuroblast development and in the developing adult with the differentiation and elaboration of the nervous system. Of interest in the context of the present review is the study of Mehrotra and Dauterman (1963) on the specificity of cholineacetylase in preparations from Musca dornestica. They examined a number of N-alkylcholine compounds and found that with increasing chain length of one of the quaternary alkyl-groups of the choline moiety, progressively slower rates of acetylation were obtained. The dimethyl-isopropylcholine was however not acetylated. Some differences between the specificity of the insect acetylase and rat brain acetylase were noted. The latter was only able to utilize dimethyl-derivatives of choline whereas the insect preparation could acetylate both diethyl- and triethylderivatives. The cholinesterase from housefly heads was found to have a considerably broader specificity than the acetylase and hydrolysis of a range of N-dimethyl and Ndiethyl derivatives of choline was observed (Dauterman and Mehrotra, 1963). Thus the possibility exists, at least in the housefly, that N-dime thylethylcholine and possibly Ndimethyl-n-propylcholine can be acetylated and the acetylderivatives hydrolysed by the cholineacetylase and cholinesterase system. Such reactions might become important when choline analogues are available to the insect especially under conditions of choline-deficiency.
CHOLINE METABOLISM IN INSECTS
85
B. ENZYMIC SYNTHESIS OF LIPIDS CONTAINING CHOLINE
The incorporation, in uiuo, of radioactive choline into the phosphatidylcholine of a number of insects has been demonstrated. The insects include Musca domestica (Bridges and Ricketts, 1968, 1970), Phormia regina (Mehendale et al., 1970; Willis and Hodgson, 1970), Periplaneta americana (Kumar, 1968), Heliothis zea and Hylobius pales (Willis and Hodgson, 1970). In all these cases, phosphorylcholine was shown to contain radioactive choline, as was CDP-choline with the exception of the experiments with Periplaneta americana where only phosphorylcholine was reported to be labelled. As both these compounds are intermediates between choline and phosphatidylcholine (Fig. 1) these observations provide some evidence that the cytidine pathway is operative in these insects. Evidence for this pathway in other insects has been provided by workers using 32P-labelled precursors. Garey and Wyatt (1963) studied the incorporation of radioactive ortho-phosphate into the phosphorus containing compounds of Hyalophora cecropia and concluded that phosphorylcholine was a precursor of phosphatidylcholine. The rate of incorporation of 32P-ortho-phosphate into phosphorylcholine and phosphatidylcholine indicated that the former was the precursor of the latter in adult Celerio euphorbiae (Chojnacki, 1961 ) and adult Arctia caia (Chojnacki and Korzybski, 1962). Incorporation of 32 P-labelled phosphorylcholine into phosphatidylcholine was observed in homogenates of larvae, pupae and adults of Celerio euphorbiae (Chojnacki and Piechowska, 1961). A similar incorporation of phosphorylcholine into lipids of fat body preparations of Sarcophaga bullata was reported by Crone et al. (1 966). Chojnacki and Korzybski (1 963) demonstrated the incorporation of 32P-labelled cytidinediphosphorylcholine into phosphatidylcholine in homogenates of the brain and fat body of Locusta migratoria and Bombyx mori. They showed that deoxycytidinediphosphorylcholine was a less effective precursor in these preparations and that 6-N-monomethylcytidinediphosphorylcholine and adenosinediphosphorylcholine were both ineffective. An interesting experiment by Bieber ( 1968) demonstrated that the phosphonate of choline could be incorporated into the lipids of larvae of Musca domestica to give a phosphonolipid. This indicated that such compounds were precursors of “lecithins”. More detailed studies have been made on the biosynthesis of phosphatidylcholine in uitro using preparations of fat body from
86
R. 0.BRIDGES
larvae of Phormia regina (Habibulla and Newburgh, 1969; Shelley and Hodgson, 1970). Using choline phosphorylcholine and CDPcholine labelled with carbon-14 in the methyl- or 1,2-positions of the choline-moiety the latter authors showed that CDP-choline was the immediate precursor of phosphatidylcholine and that the pathway proceeded in the following sequence: choline + phosphorylcholines + CDP-cholines+ phosphatidylcholines The choline kinase and phosphorylcholinecytidine transferase were located in the supernatant whereas the phosphorylcholinediglyceridetransferase was present in both the mitochondria1 and microsomal fractions. The choline kinases from homogenates of gut tissue from Periplaneta americana (Kumar and Hodgson, 1970) and fat body from larvae of Phormiu regina (Shelley and Hodgson, 1971) have been partially purified and their properties studied. Thus there is good evidence for the synthesis of phosphatidylcholine from choline by the triple enzyme system of the cytidine pathway in some insects of the Coleoptera, Diptera, Lepidoptera and Orthoptera. The possibility of its synthesis by stepwise methylation of phosphatidylethanolamine will be considered later when the evidence for choline synthesis is discussed (Section VI, E). A third possible route is by the exchange of free choline with the base of another phospholipid such as ethanolamine or serine. This was considered by Crone (1 967) and some indirect evidence was obtained for such an exchange using fat body preparations of Musca domestica but it seems unlikely that such a pathway would have any great significance in vivo. The pathway for the incorporation of analogues of choline into the phospholipids of Phormia regina and Musca domestica appears to be analogous with that for choline. Mehendale et al. (1 970) showed the presence of the phosphates of dimethylethylcholine, dimethyl-npropylcholine, dimethyl-isopropylcholine and dimethyl-n-butylcholine in larvae of Phormia regina when fed on diets containing these analogues. A small fraction was tentatively identified as the CDP-derivative of dimethylethylcholine in larvae fed with this choline analogue but no indication of the presence of CDP-derivatives of the other analogues was obtained. The authors suggested that this was because a degree of specificity was manifest at the level of formation of the CDP-derivatives and that once formed they were readily incorporated into phospholipid. In the housefly, evidence for
CHOLINE METABOLISM IN INSECTS
87
the involvement of the cytidine pathway in the formation of phosphatidyl-2-aminobu tan- 1-01 and phosphatidy 1-N-monomethylaminoethanol has been given (Bridges and Ricketts, 1968; Bridges and Holden, 1969). An increase in the composite phosphoryl-base fraction isolated from larvae of Musca domestica fed on diets containing dimethylethylcholine and trimethyl(3-hydroxypropy1)ammonium acetate was obtained (Bridges and Ricketts, 1970). Phosphoryl-/3-methylcholine was found in housefly larvae fed on /3-methylcholine, carnitine or y-butyrobetaine (Bieber e t al., 1969; Moulton e t al., 1970). The forma tion of phosphatidylcarnitine has been suggested but the evidence available is not very conclusive. Mehendale et al. (1 966) showed the formation of a lipid, identified as phosphatidylcarnitine when larvae of Phormia regina were fed on diets containing carnitine and trime thyl-(3-hydroxypropyl)ammonium acetate. The latter compound had been found t o reduce the effectiveness of carnitine as a replacement for choline in the diet. No evidence for the formation of a similar lipid was found in larvae fed on diets containing carnitine alone. A small lipid-fraction, tentatively identified as phosphatidylcarnitine was found when fat body preparations from Phormia regina were incubated with carnitine (Mezei and Newburgh, 1967). However, phosphokinase preparations from Phormia regina (Habibulla and Newburgh, 1969; Shelley and Hodgson, 1971) and Periplaneta americana (Kumar and Hodgson, 1970) would not phosphorylate carnitine. The presence of a carnitine decarboxylase was demonstrated in the gut but not the fat body of Phormia regina (Habibulla and Newburgh, 1969). Bieber e t al. (1969) failed t o detect phosphatidylcarnitine in larvae of Musca domestica fed on diets containing carnitine but did not rule out the possibility that it might occur in trace amounts. It would seem most likely, however, that carnitine is decarboxylated t o /3-methylcholine in the gut before absorption and incorporation into phospholipid. C. HYDROLYSIS OF PHOSPHATIDYLCHOLINE
The hydrolysis of the two fatty acid ester groups of phosphatidylcholine is brought about in a stepwise manner by the action of phospholipases A and B (Fig. 1). The presence of a phospholipase A, stimulated by calcium ions, was shown to be present in homogenates of fat body from larvae of Musca domestica (Crone, 1967). A similar enzyme was also demonstrated in subcellular fractions prepared from
88
R. G. BRIDGES
homogenates of adult Musca domesticu and in preparations from larvae and adults of Phormia regina (Khan and Hodgson, 1967). The presence of a phospholipase B hydrolysing the lysophosphatidylcholine to water-soluble choline-metabolites was also demonstrated in these preparations. Both enzymes had similar properties to those of comparable enzymes studied from vertebrate sources. PhosphoIipasesA and B have been reported in homogenates of larvae of Culex pipiens fatigans (Rao and Subrahmanyan, 1969). From results obtained in a study on the incorporation of 32P-labelled orthophosphate into the phosphorus containing compounds of Hyalophoru cecropia, Garey and Wyatt (1 963) concluded that glycerylphosphorylcholine was a degradation product of phosphatidylcholine. This implies that the two phospholipases occur in this insect. A similar result was obtained by Kumar (1968) in studies with radioactive choline using Periplaneta americuna. The conversion of phosphatidylP-methylcholine to glycerylphosphoryl-0-methylcholine which can then be hydrolysed further to a-glycerophosphate and 0-methylcholine has been demonstrated in larvae of Musca domestica (Bieber et al. ( 1969). From the scanty evidence available, it is probable that phosphatidylcholine is metabolized in insects by enzymes similar to those in vertebrates and that the products resulting from this hydrolysis are fatty acids, a-glycerophosphate and choline. The failure to find appreciable amounts of lysophosphatidylcholine (Section I , B) is probably due to the presence of an efficient phospholipase B which prevents the harmful effects that would result from an accumulation of this intermediate in the tissues. D. OXIDATION OF CHOLINE
If enzymes are present in insects which oxidize choline to betaine and then N-demethylate the betaine to glycine, it would be expected that when choline labelled with carbon-14 in either the methyl-group or the 1,2 position is introduced into the insect the radioactivity would become incorporated into a large number of metabolites. In fact, where such experiments have been carried out, the activity has been recovered only in metabolites which are derivatives of choline. When radioactive choline was fed to larvae of Musca domestica only labelled choline, acetylcholine, phosphorylcholine and CDP-choline were recovered in the acid-soluble extract and phosphatidylcholine in the lipid extract (Bridges and Ricketts, 1968, 1970). Similarly
CHOLINE METABOLISM IN INSECTS
89
Mehendale et al. (1970) and Willis and Hodgson (1970) found radioactive choline incorporated only into phosphorylcholine, CDP-choline and phosphatidylcholine in larvae of Phormia regina. Some glycerylphosphorylcholine was detected by the former authors. Willis and Hodgson (1970) obtained similar results with Heliothis zea and Hylobius pales. Beaudoin and Lemonde (1970) found that radioactive choline was incorporated only into phosphatidylcholine, sphingomyelin and lysophosphatidylcholine by Tribolium confusum. No significant amounts of radioactivity were present in any of the other lipids. When methyl-14C-betainewas fed to Phormia regina, Heliothis zea and Hylobius pales all the radioactivity was recovered in the water-soluble extracts from these insects and this was shown to be unchanged betaine (Willis and Hodgson, 1970). In a survey of occurrence of betain-homocysteinemethyl-transferase in a large number of organisms, Ericson (1960) failed to demonstrate its presence in whole homogenates of Formica rufa, Apis mellifera and Drosophila melanogaster. Thus, the enzyme systems, present in vertebrates, for the oxidation of choline and N-demethylation of betaine have not been demonstrated in insects. This is confirmation of results from dietary experiments which show the failure of betaine to substitute for most insects’ requirements of choline (Section 111, B). The successful substitution of choline by betaine or betaine nitrile (Noland and Baumann, 1949; Gordon, 1959) in the diet of Blattella germanica is apparently an exception to the above generalization. This result suggests that oxidation of choline may be occurring in this insect and that betaine has its effect by sparing the oxidation of what choline is available to the insect in its diet, but further work with radioactive materials is necessary to establish this point. No evidence for the oxidative metabolism of a number of choline-analogues by Phormia regina has been obtained (Mehendale et al., 1970). E. SYNTHESIS OF CHOLINE
The synthesis of choline by the stepwise methylation of ethanolamine with S-adenosylmethionine has been demonstrated in vertebrates but only after the ethanolamine has been incorporated into phospholipid (Bremer and Greenberg, 1961). It is therefore a synthesis of phosphatidylcholine from phosphatidylethanolamine and free choline only becomes available to the animal from this process after hydrolysis of the phospholipid. Experiments on the
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R. G. BRIDGES
metabolism of L-serine labelled in the 1,2 position with carbon-1 4 by larvae of Musca domestica gave no evidence for the incorporation of the label into the choline-moiety of the phospholipid although labelling of ethanolamine occurred (Bridges and Ricketts, 1965). Also although both N-monomethyl- and N-dimethyl- aminoethanol were extensively incorporated into the phospholipids of the housefly fed on choline deficient diets no increase in the phosphatidylcholine was observed. As the phosphatidyl-derivatives of both these compounds are expected intermediates between phosphatidylethanolamine and phosphatidylcholine it was concluded that synthesis of choline by the methylation pathway did not occur in Musca domestica. Additional evidence has been obtained by Bieber (1968) who was unable to show any methylation of the phosphonolipid synthesized by larvae of Musca domestica when fed on diets containing dimethylaminoethylphosphonic acid. More direct evidence was obtained by Moulton et al. (1 970) who were unable to show any radioactivity in choline present in housefly larvae fed on diets containing methyl- 14C-methionine. Similar conclusions have been reached by other workers studying Phormia regina. Bieber and Newburgh (1 963) failed to show any increase in the phosphatidylcholine in larvae fed on choline-deficient diets containing N-dimethylaminoethanol although considerable amounts of the corresponding phospholipid were present. Failure to detect any radioactive choline in larvae of Phormia regina, Heliothis zea and Hylobius pales when they were fed on diets containing methyl14C-methionine was reported by Willis and Hodgson (1 970). These authors, whose investigation was designed to look at transmethylation reactions in insects, concluded that there was no evidence for the transfer of the methyl-groups of methionine, betaine or choline in the three insects. Beaudoin and Lemonde (1 970) failed to show any labelling of choline when 14C-labelled ethanolamine or serine were fed to Tribolium confusum. They concluded that synthesis of choline by stepwise methylation did not occur in this insect. Thus the absence of choline synthesis has been clearly established for insects drawn from three different orders, namely the Coleoptera, Diptera and Lepidoptera. How typical this is for other insects in these or other orders is difficult to assess, although it may well be so in view of the wide ranging necessity for choline in insect nutrition. However, Zieliriska and Dominas (1 967) have given some evidence for the synthesis of choline from 14C-labelled formate which was injected into larvae of Acantholyda nernoralis, an
CHOLINE METABOLISM IN INSECTS
91
Hymenopteran. They suggested that the methyl-group was transferred into phospholipid via the methyl-group of methionine which in turn was derived from the hydroxymethyl-group of serine. Unless these observations were due to the presence of symbionts, a generalization that all insects are unable to synthesize choline cannot be made. The inability of both Musca domestica and Phormia regina to synthesize choline means that the source of the small amounts of choline found in these insects grown on diets to which no choline has been added (Section V, A) requires some explanation. The amount carried over from the egg is unlikely to be sufficient to account for the choline found in the larva. The phosphatidylcholine content of six-day-old larvae of Musca domestica fed on diets to which no choline was added was 0.001 5 pmole/larva (Bridges and Ricketts, 1970). This is some six times the amount of phosphatidylcholine in the egg (Bridges, unpublished work). Most of the choline present in the larvae must have been derived from the diet. When casein was replaced by an amino acid mixture, similar levels of phosphatidylcholine were obtained in the larvae (Bridges, et aZ., 1965). It is not clear whether the experiments with Phormia regina were carried out on diet based on casein or an amino acid mixture, however, these insects also contained some phosphatidylcholine (Bieber et al., 1961). The source of the choline contamination may be not only from the casein but also from some other component of the diet. It seems likely that if this contamination could be eliminated growth of the larvae would be much more severely restricted. VII. THE METABOLIC ROLE OF CHOLINE IN INSECTS
The previous sections have established that choline is required by insects for two major metabolic pathways, i.e. the synthesis of acetylcholine and the synthesis of lipids containing choline. The failure to show any oxidation of choline in insects means that the choline available will be utilized entirely for these two pathways. The evidence indicates that most insects are unable to synthesize choline so that the choline required must be supplied from the diet. The minimum amounts of choline required by the insect will therefore be related to the minimum amounts of acetylcholine and phospholipid choline necessary for the functioning of the metabolic systems in which they are involved. However, as choline is the common precursor of both metabolites this minimum requirement may only
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be related to the requirement for one; the formation of the other merely reducing the amount of choline available for the synthesis of the essential component. Quantitatively the major choline metabolites in most insects are phosphatidylcholine and sphingomyelin although phosphorylcholine is almost equally important in some Lepidoptera. The requirement for choline from the diet is likely to be particularly great for insects containing high levels of these compounds such as the Orthoptera, Lepidoptera and possibly the Hymenoptera and to be less for the Diptera whose lipid-choline requirements are less. This seems to be borne out by the results obtained from nutritional studies (Section 111, A). The situation may be further complicated in insects which pass through a pupal stage. Here adequate amounts of choline may have to be attained during the larval period to meet the requirements of the adult. If these are met then it would seem that the adult insect has no further need for choline, apart for that required in egg production and for certain secretions such as the royal jelly of Apis mellifera and the venom of Vespu crabro. This requirement for egg production has been definitely established for Drosophila melunogaster (Geer et al., 1 970). Non-reproducing insects will however maintain their levels of choline for long periods on choline-free diets and this indicates that the adult insect has a very efficient mechanism for retaining choline. The amount of choline taken up by the larval stage will depend on the choline content of the diet on which the larvae feed. This has been clearly shown for larva of Musca domesticu (Bridges and Ricketts, 1968, 1970). The requirement for choline to synthesize lipid-choline in this insect is satisfied when the choline in the larval diet reaches a particular level. Further increase in the dietary supply of choline beyond this level does not increase the lipid-choline (Section V, A) but does increase the amounts of TCA-soluble metabolites, largely free choline and phosphorylcholine. The acetylcholine in these larvae increases with increasing choline in the diet, the proportion of the total choline in these larvae recovered as acetylcholine remaining reasonably constant (Table IX). However, when one considers the acetylcholine in relation to the non-lipid choline recovered from such insects it is apparent that it represents a much larger proportion when the choline available to the insect is low (Table IX). The relationship between the choline available in the diet, the amount of choline taken up from it and its distribution between the various choline-metabolites is therefore not a simple
Table IX Distribution of choline and acetylcholine in 6-day-old larvae of Musca dornestica fed on diets containing various concentrations of choline Choline Concentration of choline in diet pmolelg casein
0.04a 1.ob
1 6.2a 16.@
pmolelg wet wt. of larva
0.002 0.009 0.44 0.54
% of all choline % of TCA-soluble choline derivatives in derivatives in larva larva
0.8 0.7 12.7 13.9
40 24 66 41
Bridges and Ricketts (1968)
pmole/g wet wt. of larva
0.0015 0.0044 0.024 0.018
-
3 \
% of all choline % Of TCA-soluble choline derivatives in derivatives in larva larva 0.6
30
0.3 0.7 0.5
12
Bridges and Ricketts (1970)
t: z m
Acetvlcholine - /
/
P0 e m
kt3
z
2
3.6
z
1.4
cn
m
0 +I
94
R. G. BRIDGES
one. As acetylcholine is confined to the nervous system and as it has been shown that an accumulation of choline occurs in the nervous system of the housefly when choline levels in the diet are low (Bridges and Price, 1970b) it is necessary to consider the metabolism of choline in nerve as distinct from that in non-nervous tissue. In non-nervous tissue of most insects, the only need for choline would seem to be for synthesis of lipid-choline. The need for this does not seem to be critical, at least, as far as Musca dornestica is concerned. Larvae grown on diets containing no added choline have only some 6% of the phosphatidylcholine that the larvae are capable of synthesizing when sufficient choline is present in their diet (Bridges and Price, 1970b). This is true for all separate tissues examined, apart from nervous tissue. Although such larvae will not pupate and give adults, addition of relatively small amounts of choline to the diet will enable them to do so. Larvae capable of developing normally can have as little as 20% of the phosphatidylcholine that they are capable of synthesizing when sufficient choline is available to them (Bridges and Ricketts, 1968). The reduction of the phosphatidylcholine in such larvae and larval tissue is accompanied by an increase in phosphatidylethanolamine and this is apparently acting as a completely adequate replacement. It can be concluded that, at least in the case of Musca dornestica, the need for choline to synthesize phosphatidylcholine in non-nervous tissue is not a critical one and the insect can develop normally with very low levels of this phospholipid in these tissues. The accumulation of choline in the nervous system of housefly larvae fed on diets deficient in choline suggests that some carrier-mediated transport system similar t o that observed in vertebrate tissue also occurs in insects. The concentration of choline into mouse cerebral cortex slices has been demonstrated by Schuberth et al. (1 966). This depended on the molarity of choline in the incubation medium and the concentrative ability fell as the molarity increased. A preparation of synaptosomes (pinched-off nerve endings) has also been studied and a carrier-mediated transport system for the uptake of both choline and acetylcholine demonstrated (Marchbanks, 1969). It was concluded that choline was the natural substrate for this carrier system. Similar observations have been made with isolated giant axons of Loligo (Hodgkin and Martin, 1965). Here again the concentrative ability fell off as the external concentration of choline increased and under comparable conditions choline was taken up eight times as fast as acetylcholine. The
CHOLINE METABOLISM IN INSECTS
95
presence of such a system in the nervous tissue of the insect could thus account for the results obtained with the housefly. However, the choline must first reach the extracellular space surrounding the neuron from the haemolymph and to do this, it must first cross the so-called “ion-impermeable barrier”. Until recently, the neural lamella surrounding the insect nervous system was considered to act as an ion barrier. However, Treherne and Smith (1 965a) have shown that acetylcholine can be readily taken up by non-eserinized preparations of the abdominal nerve chord of Periplaneta americana and the acetyl-moiety rapidly hydrolysed (Treherne and Smith, 1965b). A slower rate of uptake was observed by these authors when hydrolysis was prevented using a medium containing eserine. Essentially the same results were obtained by Eldefrawi and O’Brien (1 967) and these authors extended their observations to include the uptake of a series of quaternary ammonium compounds. They concluded that there was some restriction t o the penetration of ionized molecules when compared with unionized ones but that the difference was relatively small and depended on the polarity and size of the molecule, and the extent of its metabolism after uptake. More recent work by Treherne et al. (1970) and Pichon and Treherne (1970) has indicated that an extremely complex situation exists and that, because the type of treatment a preparation receives has a profound effect on its accessibility to small ions, the earlier work must be interpreted with caution. These authors have produced evidence for the existence of a restriction to ion movements associated with tight junctions at the inner margin of the intercellular perineurial cleft. However, a finite permeability for choline ions through this peripheral barrier has been demonstrated although it is approximately one-half that for sodium and one-tenth that for potassium (Pichon et al., 197 1). An alternative or additional route by which choline could reach the extra-neuronal fluid could be by diffusion within the glial system, this involving the penetration of at least two glial membranes (see p. 257). What part such barriers may play in the control of access of choline to the neurons during the growth and development of the nervous system, which is the time when the concentration of choline is occurring in the cholinedeficient insect, is not known. Also the form in which the choline could cross such barriers is uncertain. Recent studies by Ansell and Spanner (1 97 1) on the uptake, in vivo, of choline into rat brain have suggested that it is transported to the brain by the blood in a lipid-bound form. Phosphatidylcholine is isoelectric over a wide pH
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R. G. BRIDGES
range and having crossed the ion barrier would have to be metabolized to free choline prior to uptake by the carrier-mediated transport system referred to earlier. Both these features would facilitate its uptake into the nervous system. Once inside the cell, the choline will be available for both acetylcholine and phosphatidylcholine synthesis and the distribution between these two products will be a function of both the amounts and activities of the two enzyme systems involved in their synthesis and breakdown. The higher percentage of phosphatidylcholine in the phospholipids of the nervous tissue from larvae of Musca domestica fed on diets deficient in choline compared with that in other tissues may thus be merely a consequence of the carrier-mediated uptake of choline by the nervous system. As phosphatidylethanolamine seems to be an adequate replacement for phosphatidylcholine in nonnervous tissue where it would normally be functioning as a membrane component the same situation could exist in nervous tissue and the essential need for choline would be for acetylcholine synthesis. Whether or not this is so, phosphatidylcholine is the major choline-metabolite in the nervous system of larvae of Musca domestica (Bridges, unpublished work) and accounts for some 90% of the choline present. Acetylcholinesterase is associated with the synaptic membrane in the central nervous system of Periplaneta americana (Smith and Treherne, 1965) and from studies with vertebrate preparations it is likely to be on the outside of the membrane in the synaptic cleft (Whittaker, 1969). Choline-acetylase appears to be a cytoplasmic enzyme and much of the acetylcholine will be expected to be contained in synaptic vesicles (Whittaker, 1969). All the enzymes for the synthesis and hydrolysis of phosphatidylcholine have been demonstrated in mammalian brain (Ansell and Hawthorne, 1964) but apart from the incorporation of CDP-choline into phosphatidylcholine by brain preparations of Locusta migratoria and Bombyx mori (Chojnacki and Korzybski, 1963) nothing is known about their presence in the insect nervous system. However, if their distribution in this tissue is similar to that found in fat body of Phormia regina (Shelley and Hodgson, 1970) then the situation that may exist near a synaptic ending can be summarized in Fig. 2. The level of choline in the cytoplasm will be a function of the relative rates of turnover of membrane-bound lipid-choline and of acetylcholine. Turnover of the latter will involve release of vesicular acetylcholine into the synaptic cleft, hydrolysis there and the reabsorption of the choline back into
97
CHOLINE METABOLISM IN INSECTS Synaptic cleft
S
I
T
CDPCh
A\\\\
GPCh
?
\\\\\\ ‘I.\\\\\\\\\ I
L
+*r ‘i 1
Fig. 2. Diagrammatic representation of reactions occurring at a synapse. ACh = acetylcholine; @ = vesicular acetylcholine; Ch = choline; PCh = phosphorylcholine; CDPCh = cytidinediphosphorylcholine;GPCh = a-glycerylphosphorylcholine.
the cell. This reabsorption process is likely to be the same as that discussed earlier in connection with the uptake of choline from the extracellular space and must be very efficient if the nerve is not to lose choline back into the haemolymph. The importance of the recycling of the choline produced by cholinesterase action is further emphasized when the rapid speed of turnover of acetylcholine is considered. This has been estimated as 6.1 x 10 - 5 pmole/min/head of adult Musca domestica (Winteringham, 1966) which is equivalent to 0.09pmole, more than the lipid-choline content of the entire housefly (0.06 pmole), turning over in one day. The ability of certain analogues of choline to allow normal growth and development of Musca domestica, Phormia regina and Drosophila melanogaster (Table 111) when included in the larval diet in place of choline may also be connected with the amount of the very small quantity of choline available to these insects that is taken up by the nervous system. When P-methylcholine is fed to housefly larvae, 10%of all the lipid-choline present in the larvae is found to be in the nervous system and, after pupation of such larvae, 42% of all the lipid-choline present in the adult fly is found in the head and thoracic ganglion (Bridges, unpublished work). The choline-analogue appears to exert its sparing action by competing with the small amount of choline available from the diet and taken over from the egg for incorporation into phospholipids of non-nervous tissue. This allows rather more of the choline to be taken up by the nervous system, sufficient to supply the insect’s needs for acetylcholine synthesis. The acetylcholine content in the heads of such flies was reduced to only 30% of its control value whereas the phosphatidylAIP-5
98
R. G. BRIDGES
choline content of the fly was reduced to 6% of the control value (Bridges et a/., 1965). An alternative explanation for the replacement of the insect’s needs for choline by other choline-like compounds could be that the analogues are themselves acetylated and used as neurotransmitter substances. This may occur in the case of the analogue dimethylethylcholine. Again one must consider the results obtained with the housefly (Bridges and Ricketts, 1970) which showed that when this analogue was supplied to the larvae in their diet much smaller amounts of choline were found in the larvae than when it was not supplied. The dimethylethylcholine appears t o compete with the small amounts of choline in the larval diet for uptake from the gut. Acetylcholine levels in these analogue-fed insects and the phosphatidylcholine levels in the nervous system are much lower than in larvae fed on diets containing no dimethylethylcholine. The former go on to pupate and give adults, whereas the latter do not. Adults from larvae fed on dimethylethylcholine contain only one tenth of the acetylcholine present in normal adults. Mehrotra and Dauterman (1 963) have shown that dimethylethylcholine is a good substrate for housefly cholineacetylase, so that it is possible that acetyldimethylethylcholine is being synthesized and that this is acting as a neurotransmitter in place of acetylcholine in this situation. It is interesting to note these workers were unable to demonstrate acety lation of dimethy 1-isopropylcholine. This analogue has been shown to be a good substitute for choline in the diets of Phormia regina (Hodgson et al., 1969). It is likely therefore that the sparing action of this compound is similar to that proposed for p-me thylcholine in Musca domesticu. The ability of choline-analogues to become incorporated into insect phospholipids in place of choline has been studied only in three species of Diptera and one of Coleoptera (Section V,A). However it is likely that many other insects will possess this ability as the enzyme systems involved in phosphatidylcholine synthesis seem to be similar to those in vertebrates, where a comparable lack of specificity seems to exist (for a summary of references see Bridges and Ricketts, 1967; Hodgson et aL, 1969). Because other orders of insects require larger amounts of choline for optimal growth than do the Diptera, the effectiveness of choline-analogues to act as choline substitutes would be expected to be less than for Diptera. It seems likely however that if choline was completely absent from any insect diet no analogues would have a great growth promoting effect except perhaps dimethylethylcholine.
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Once the insect has acquired sufficient choline in its nervous system t o satisfy its minimal needs for acetylcholine synthesis in the developed adult, normal development will proceed. The need for this minimal amount will only become apparent when the adult is about to emerge from the pupa. This would seem t o be linked with the fact that a much wider range of analogues will bring about development to the pupal stage than those which allow full development. However even those compounds which allow adults to develop do not allow them to lay viable eggs until choline is supplied to the adult. This was clearly shown by Geer et ul. (1968) in the case of Drosophilu melunoguster with adults obtained from larvae fed on diets containing carnitine in place of choline. It can be concluded that if the insect is to complete its life cycle no substitute is possible for choline. Finally, it would seem appropriate to consider the action of anticholinesterases in relation t o this discussion. Many organophosphorus insecticides bring about an increase in acetylcholine in insects and this is undoubtedly connected with the inhibition of cholinesterase (Smallman and Fischer, 1958). These authors studied the increase of acetylcholine in the heads and whole insects of Muscu domestica poisoned with parathion, malathion, TEPP (tetraethylpyrophosphate) and DFP (di-isopropylphosphofluoridate). The first three compounds led t o a steady increase in the acetylcholine content of both heads and whole flies followed by a fall in the case of the insects treated with TEPP and malathion due t o a gradual reactivation of the cholinesterase. The acetylcholine increased to 5.7 times its normal level in flies treated with parathion. Only slight increases were observed with DFP but this was accompanied by a redistribution of the acetylcholine away from the head to the abdomen (Lewis and Fowler, 1956). Winteringham (1 966) observed a reduction in the rate of turnover of 14C-acetate into acetylcholine in houseflies in the presence of anticholinesterases. He found this surprising in view of the earlier results indicating an increase in total acetylcholine level under these conditions. He postulated therefore that in the synaptic region acetylcholine synthesis and enzymic hydrolysis are tightly coupled so that synthesis proceeds only as one or both of the products of cholinesterase activity become available. He suggested that the rate of synthesis may be effectively controlled by local choline availability. If one views these findings together with the situation pictured in Fig. 2 concerning the metabolism of choline in the region of the synapse, it seems likely that, in the presence of
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an anticholinesterase, acetylcholine will accumulate in the synaptic cleft. Even if the acetylcholine is retaken up by the neuron using the carrier-mediated system which would normally take up choline it will not be involved in turnover with acetylCoA and would therefore not have become labelled in the experiments of Winteringham. The source of free choline for further acetylation could be either from outside the nervous system of from the phosphatidylcholine within the cell. The former seems unlikely if the carrier system is already saturated with acetylcholine and the slow build up of acetylcholine on prolonged poisoning could be due to the gradual release of choline from the phosphatidylcholine with its subsequent acetylation. If this is so then what Winteringham was measuring was the rate of de novo synthesis of acetylcholine in the poisoned insect. With its build up in the synaptic cleft, acetylcholine may then diffuse out of the synaptic region and out of the nervous system itself and become translocated to the abdominal regions. DFP does not fit in with this pattern but a paper by Nelson and Barnum (1960) has shown an effect of this compound on the synthesis of phosphatidylcholine in mouse brain. If this compound could effectively block the choline available to the system from hydrolysis of phosphatidylcholine as well as inhibiting acetylcholine hydrolysis then only a slight increase in the amount of acetylcholine would be expected. This would be due to the acetylation of the free choline in the cell. This not only explains the lack of increase in acetylcholine in DFP-poisoned insects but also the very low rates of “turnover” found in such insects by Winteringham (1966) when compared with those in TEPP and malathion-poisoned flies. ACKNOWLEDGEMENTS
The author wishes to acknowledge the helpful discussions with Dr.
J. E. Treherne.
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Magis, N. (1954). Nutrition comparke des tribolium. 111. Action vitaminique de la DL-carnitine et la DL-carnitine chez Tribolium castaneum Herbst (Insecte Colbopthre). Archs. int. Physiol. 62, 505-51 1. Marchbanks, R. M. (1 969). Biochemical organisation of cholinergic nerve terminals in the cerebral cortex. In “Cellular Dynamics of the Neuron” (S. H. Barondes, ed.). pp. 115-135. Academic Press, New York and London. McGinnis, A. J., Newburgh, R. W. and Cheldelin, V. H. (1956). Nutritional studies on the blowfly, Phormia regina (Meig.). J. Nutr. 58,309-323. Mehendale, H. M., Dauterman, W. C . and Hodgson, E. (1966). Phosphatidylcarnitine: a possible intermediate in the biosynthesis of phosphatidylP-methylcholine in Phormia regina (Meigen). Nature, Lond. 21 1, 759-76 1. Mehendale, H. M., Dauterman, W. C. and Hodgson, E. (1 970). The incorporation of choline analogues into the phospholipids of Phormia regina. Int. J. Biochem 1,429-437. Mehrotra, K. N. (1 960). Development of the cholinergic system in insect eggs. J. Insect Physiol. 5, 129-142. Mehrotra, K. N. and Dauterman, W. C . (1963). The N-alkyl specificity of cholineacetylase from the housefly, Musca domestica L. and the two spotted spider mite Tetranychus telarius L. J. Insect Physiol. 9, 293-298. Mehrotra, K. N., Sethi, G. R. and Bhamburkar, M. W. (1966). Phospholipids in the-haemolymph of the desert locust, Schistocerca gregaria, Forkal. Znd. J. Ent. 28,468-476. Mezei, C. and Newburgh, R. W. (1967). The formation of lipid-bound carnitine derivatives in the fat body of Phormia regina, J. Insect Physiol. 13, 1489-1499. Moore, W . (1946). Nutrition of Attagenus (?) sp. I1 (Coleoptera: Dermestidae). Ann. ent. SOC.A m . 39,513-521. Moulton, B., Rottman, F., Kumar, S. S. and Bieber, L. L. (1 970). Utilization of methionine, choline and 0-methylcholine by Musca domestica. Biochim. biophys. Acta 210, 181-185. Nelson, W. L. and Barnum, C. P. (1960). The effect of diisopropylphosphofluoridate (DFP) on mouse brain phosphorus metabolism. J. Neurochem. 6,43-50. Noland, J . L. and Baumann, C. A. (1949). Requirement of the german cockroach for choline and related compounds. Proc. Soc. exp. Biol. Med. 70, 198-201. Noland, J. L., Lilly, J. H. and Baumann, C. A. (1949). Vitamin requirements of the cockroach Blatella germanica (L). Ann. ent. SOC.A m . 42, 154-164. Pant, N. C . (1 956). Nutritional studies on Trogoderma granarium Everts. Basic food and vitamin requirements. Zndian J. Ent. 18,259-266. Pant, N. C . and Fraenkel, G. (1954). Studies on symbiotic yeasts of two insect species, Lasioderma serricorne F. and Stegobium paniceum L. Biol. Bull. mar. biol. Lab., Woods Hole 107,420-432. Patterson, E. K., Dumm, M. E. and Richards, A. G. (1 945). Lipids in the central nervous system of the honey bee. Archs. Biochem. 7,201-21 0. Pichon, Y. and Treherne, J. E. (1 970). Extraneuronal potentials and potassium depolarization in cockroach giant axons. J. exp. Biol. 53,485-493. ~
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Pichon, Y.,Moreton, R. B. and Treherne, J. E. (1971). A quantitative study of the ionic basis of extraneuronal potential changes in the central nervous system of the cockroach (Periplaneta americana L.) J. exp. Biol. 54, 75 7-777. Rao, K. 0. P. and Agarwal, H. C. (1969). Lipids of the larvae and adults of Trogoderma grananum (Coleoptera). Comp. Biochem. Physiol. 30, 161-167. Rao, R. H. and Subrahmanyam, D. (1 969). Studies on the phospholipase A in larvae of Culex pipiens fatigans. J. Insect Physiol. 15, 149-159. Rasso, S. C. and Fraenkel, G. (1954). The food requirements of the adult female blowfly Phormia regina (Meigen) in relation to ovarian development. Ann. ent. SOC.A m . 47,636-645. Redina, G . and Singer, T. P. (1 959). Studies on choline-dehydrogenase. I. Extraction in soluble form, assay and some properties of the enzyme. J. biol. Chem. 234, 1605-16 10. Rimon, A. and Shapiro, B. (1959). Properties and specificity of pancreatic phospholipase A. Biochem J. 71, 620-623. Ritchot, C. and McFarlane, J. E. (1961). VitaminB requirements of the housecricket. Can. J. 2001.39, 11-15. Rock, G. C. (1969). Sterol and water-soluble vitamin requirements for larvae of the red banded leafroller, Argyrotaenia velutinana. Ann. ent. SOC.A m . 62, 61 1-613. Rothschild, H. A. and Barron, E. S. G. (1954). The oxidation of betaine aldehyde by betaine aldehyde dehydrogenase. J. biol. Chem. 209, 5 11-523. Royes, W . V. and Robertson, R. W. (1964). The nutritional requirements and growth relations in different species of Drosophila. J. exp. 2001. 156, 105-135. Sang, J. H. (1 956). The quantitative nutritional requirements of Drosophila melanogaster. J. exp. Biol. 33,45-72. Schuberth, H. J., Sundwall, A., Sorbo, B. and Lindwell, J. 0. (1966). Uptake of choline by mouse brain slices. J. Neurochem. 13, 347-352. Sedee, P. D. J. W. (1953). Qualitative vitamin requirements for growth of larvae of Calliphora erythrocephala (Meig.). Experimentia 9, 142-143. Sedee. P. D. J. W. (1958). Qualitative B vitamin requirements of the larvae of a blowfly, Calliphora erythrocephala (Meig.). Physiol. 2001. 31,3 10-316. Shelley, R. M. and Hodgson, E. (1 970). Biosynthesis of phosphatidylcholine in the fat body of Phormia regina larvae. J. Insect Physiol. 16, 131-139. Shelley, R. M. and Hodgson, E. (1971). Choline kinase from the fat body of Phormia regina larvae. J. Insect Physiol, 17,545-558. Singh, K. R. P. and Brown, A. W. A. (1 957). Nutritional requirements of Aedes aegypti L.J. Insect Physiol. 1, 199-220. Smallman, B. N. and Fischer, R. W. (1958). Effect of anticholinesterases on acetylcholine levels in insects. Can. J. Biochem. Physiol. 36, 575-586. Smallman, B. N. and Mansingh, A. (1969). The cholinergic system in insect development. A . Rev. Ent. 14, 387408. Smith, D. S. and Treherne, J. E. (1 965). The electron microscopic localization of cholinesterase activity in the central nervous system of an insect Periplaneta americana L. J. Cell Biol. 26,445-465.
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Wlodawer, P. and Wihieska, A. (1965). Lipids in the haemolymph of wax moth larvae during starvation. J. Insect Physiol. 11, 11-20. Wren, J. J. and Mitchell, H. K. (1 959). Extraction methods and an investigation of Drosophila lipids. J. biol. Chem. 234,2823-2828. Wyatt, G. R., Kropf, R. B. and Carey, F. G. (1963). The chemistry of insect haemolymph. IV. Acid soluble phosphates. J. Insect Physiol. 9 , 137-152. Yurkiewicz, W. (1 967). Phospholipid metabolism in the embryo of the Indian meal moth, Plodia interpunctella. Proc. Pa. Acad. Sci. 41, 27-29. Yurkiewicz, W. (1968). Glass fibre silica gel1 sheets for insect phospholipid fractionation. Ann. ent. Soc. A m . 61, 1026-1027. Yurkiewicz, W. J. and Whelchel, K. E. (1969). Lipids of melanic mutant of the large milkweed bug, Oncopeltus fasciatus. Comp. Biochem. Physiol. 29, 879-883. Zielihska, Z. M. and Dominas, H. (1967). The origin of phospholipid ethanolamine and choline in a saw fly, Acantholyda nemoralis. J. Insect Physiol. 13, 1769-1 779.
Learning and Memory in Isolated Insect Ganglia E. M. EISENSTEIN Department of Biophysics. Michigan State University East Lansing, Michigan, U.S.A. Introduction . . . . . . . . . . . . . . . . , . A. The Concept of Learning . . . . . . . . . . . B. Towards a Reformulation of the Concept of Learning. , . C. The Use of “Model” Systems in Biology . . . . . . 11. Behavioral Investigations . . . . . . . . . . . . . . A. Intact and Headless Preparations. . . . . . . . . B. Isolated Ganglion-P and R as Separate Preparations . . . C. Isolated Ganglion-P and R in the Same Preparation . , . D. Effects of Other CNS Lesions on P and R Behavior . , . E. Behavior of Ganglionless P and R Preparations . . . . . F. Discussion of P and R Leg Behavior in the Presence of Ganglionic Innervation of the Legs. . . . . . . . . . . G. Discussion of P and R Leg Behavior in the Absence o# Ganglionic Innervation of the Legs (Ganglionless) . . . . . . 111. Some Histological and Anatomical Findings Potentially Relevant to Understanding the Cellular Basis of Learning in the Cockroach . . A. Introduction . . . . . . . . . . . . . . . . B. Mapping of the Cockroach Metathoracic Ganglion . . . . C. Ganglion Transplantation Studies in the Cockroach . . . IV. Electrophysiological Investigations of Learning . . . . . . . A. Introduction . . . . . . . . . . . . . . . , . . . . . . . . B. Habituation Studies . . . . C. Discussion of Habituation . . , . . . . . . . . D. Instrumental Learning Studies . . . . . . . . , . . . . . . . . E. Classical Conditioning Studies F. Some Newer Electrophysiologi a1 nd Analytical Approaches to Learning and Memory . . . , . . . . . . . . V. Molecular Approaches to Learning and Memory Storage . . . . A. Introduction . . . . . . . . . . . . . . . . B. Assessing the Effects of Drugs on Learning and Memory in the Cockroach . . . . . . , . . . . . . . . C. Some Speculations on the Future of the Chemical Approach VI. Summary . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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I. INTRODUCTION
The concept of learning is generally defined in terms of certain arbitrary procedures (such as the methods used in instrumental and Pavlovian conditioning) and the results obtained with them. It is assumed by many that specific structural and/or biochemical processes encode what shows up behaviorally under certain conditions as learning. It is pragmatically necessary at present, when virtually nothing about these encoding mechanisms is known, to define learning in terms of the procedures which presumably are responsible for the encoding process. It may be advantageous in thinking about learning, however, to distinguish between its definition (primarily in terms of such procedures and their results) and its actual underlying basis. Such a distinction emphasizes the need to be flexible in our consideration of what phenomena to include for consideration at present under the term “learning”. Eventually when the biological bases of the various classes of behavioral phenomena are determined, it may well be that certain classes of behavior arbitrarily distinguished at present on procedural grounds (e.g. instrumental and Pavlovian conditioning) may be merged on the basis of identical underlying processes while certain behaviors viewed at present by some as identical or closely related (e.g. habituation as a form of learning) may turn out to have an entirely different underlying basis. It would seem desirable, therefore, to have a working formulation of learning which, with minimal restrictive characteristics, included the bulk of phenomena traditionally related to learning, but which also provided a guide for research into areas previously not considered at all, or only considered tangentially to the learning process; but which may shed important light on fundamental mechanisms underlying much of the learning process. The purposes of the present paper are: (1) to discuss and restate a formulation of learning which was previously published and which I think meets the above requirements; (2) to review a variety of behavioral, physiological, histological and chemical studies on insect systems which bear directly on the question of site and changes associated with learning and memory storage and (3) to raise certain questions about the nature and site of the
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learning process and to propose certain experimental approaches to the problem. The basic organization of the paper will be in sections, each dealing with a major approach to learning and memory (viz. behavioral, histological, electrophysiological and chemical). A. THE CONCEPT OF LEARNING
Most commonly, learning is said to occur in a system when it can be shown that an association between a stimulus and a response is developed de novo or that a previous association has been strengthened. The procedure giving rise to this association is termed a learning procedure. The development of an association is commonly measured in terms of some behavioral change, such as response frequency, latency, or magnitude. Two broad classes of procedures give rise to the development of an association between a stimulus and a response. These are the classical conditioning and instrumental learning procedures. 1. Classical conditioning
This procedure is also known as respondent, Pavlovian, type I or type S conditioning. A stimulus that invariably elicits a particular reflex (such as food in the mouth reflexly producing salivation) is presented repeatedly, together with a stimulus that initially does not elicit the reflex or does so only weakly (such as a bell or a light). After several presentations of the two stimuli, the original ineffective or conditioned stimulus (CS, bell or light) now elicits a response similar to that originally produced by the unconditioned stimulus (UCS,food). This strengthening of the relationship between the conditioned stimulus and the response is defined as learning. An adequate control for changes resulting from the sheer number of stimulus presentations often is achieved by comparing behavior resulting from a forward conditioning procedure with that resulting from a backward conditioning procedure. In the former, the onset of the CS precedes the onset of the UCS.In the latter, the onset of the CS alwaysfollows the onset of the UCS.Generally, the response level is much higher in the former.
2. Instrumental learning This learning procedure is often referred to as operant, type 11, typeR, trial-and-error and simple problem solving. The animal is
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placed in a situation in which the experimenter has designated that a given response will be followed by a stimulus selected to alter the probability that the response will recur in that situation. The change in probability is termed learning. The stimulus event that follows the response and is responsible for the change is termed the reinforcer. (The reinforcer with a Pavlovian procedure is the UCS, because it is the stimulus event that alters the occurrence probability of a particular response to the CS.) The reinforcer may be either positive or negative, depending upon whether the behavioral change is in the direction of attaining the reinforcer more rapidly (positive) or of avoiding it (negative). Suppose the rat is placed on the white side of a two-chamber compartment and is shocked until it jumps over a hurdle to the black side. Following several such trials, the rat, when placed on the white side, will jump over to the black side more rapidly than it did initially. To be able to call this change-or any part of it-learning, it is generally considered necessary to demonstrate that the response of hurdle-jumping is caused by the presence of the white-shock-jump pattern in the apparatus and not just shock, independent of this temporal order. Thus, if one group were shocked only on the white side of the hurdle box and another group received the same number of shocks either on the black side or outside the training situation, we would expect that the group receiving shocks on the white side, if later placed on that side without shock, would make more crossings to the black side than the other groups if, indeed, there were any association between shock onset on the white side and the hurdle-crossing response. The difference between such groups indexes the occurrence of learning. In both of these examples the organism must demonstrate the ability to generate a difference in output or response to a given stimulus input (CS in the Pavlovian experiment and the white side of the hurdle box in the instrumental experiment) as a function of the previous sequence of stimuli and responses to which it has been exposed, before the phenomenon of learning is generally acknowledged to have occurred. With a Pavlovian procedure, the response to the CS when a forward conditioning sequence has been used, must be different from that to the same CS when a backward conditioning procedure has been employed. The probability of hurdle jumping from the white side also must be different in animals when shock has followed the placement of the animal on the white side, compared to animals in which shock has occurred independently of whether the
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animals were on the white or black side. These relationships are schematized in Fig. 1 and form the basis of the reformulation of learning discussed below. B. TOWARD A REFORMULATION OF THE CONCEPT OF LEARNING
I am basing this definition of learning on an abstraction of factors generally considered important in experiments accepted as demonstrated learning and including both the learning procedures just described. Formal statement. A system is said to demonstrate learning when its output (response) to a given test input (stimulus) is a function of the total previous input-output pattern of which the test input was a part. That is, a system can be said to have learned if its output to a given test input is a function of the specific input-output pattern to which it has been exposed. (Whether any given example of learning involves the encoding of a pattern of inputs and outputs (stimuli and responses) or just the stimulus pattern alone, is not known. The Group
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Fig. 1. Scheme showing that with both classical and instrumental training procedures, the essential variable manipulated in experimental and control groups to demonstrate learning is the same, i.e. input-output sequence. (If responses are the same during the test period, no learning; If different, learning is said to occur.) S is the stimulus to which the system learns to respond, i.e. appropriate cues in a maze or hurdle box or bell or light with a Pavlovian procedure. R is the response designated as correct, i.e., turning right or left in a maze, jumping a hurdle, or salivating to a bell or light. Srf is the “reinforcer” responsible for strengthening the association-commonly food, water or electric shock Often with an instrumental procedure as in maze learning, variation in temporal pattern of stimuli and responses between experimental and control groups is achieved by following a right turn with reinforcement in one group and a left turn with reinforcement in the other group (Right turn + reinforcement. left turn + no reinforcement, leads to a highter probability of right turns than in the group in which the sequence has been left turn -t reinforcement right turn no reinforcement.) Ability of animals to discriminate such sequences allows conclusion that learning has occurred. F r o m Eisenstein, 1967a.) --+
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present formulation is stated in operational terms denoting both the stimulus and the response elements whose temporal sequence is actually varied with learning procedures and is not a theoretical commitment to the actual elements being associated.) The encoding of some temporal sequence of events seems to me to be one of the most fundamental aspects of learning. An input-output pattern difference is interpreted as any distinguishable temporal feature between two sets of stimulus and response elements. Commonly, this will be a difference in the temporal order of the stimulus and response events. It may also include other temporal variations, such as spacing of the events. The pattern difference may also show up over several trials rather than on any single given trial, as when a partial versus a 100%reinforcement schedule is employed. It is critical, in demonstrating that differences in output to a test stimulus are determined by the previous pattern, to show that the output varies although the same total amount of stimulus input in the same time period occurred for both patterns. “System” is being used in its broadest sense to include any arbitrary integrating unit from a single cell to a whole organism or any subdivision thereof. Such a formulation considers changes as learning when the inputs may be direct electrical stimulation of a peripheral sensory nerve and the output is measured by discharges or some other appropriate response-for example, from a motor nerve. This concept of learning has two key points: (1) traditional learning procedures are seen as varying inputoutput sequence, and the encoding of sequence is the basis of learning; (2) there is no control group in the sense of a group showing non-learned changes, because to show a system’s ability to encode sequence one must vary the temporal pattern in at least two groups to demonstrate it is the responsible factor. The “control group” receives the second pattern and its difference from the first group in response output shows that the system under consideration can undergo learning. Such a formulation of learning considers experimental and control groups of both instrumental and Pavlovian procedures in terms of the one variable that distinguishes them most consistently. That variable is the temporal pattern of some input-output function. Such a formulation of learning, it is hoped, will allow investigations of systems previously used only transiently in learning
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studies. That is because the terminology frequently employed in discussing learning includes concepts such as motivation, incentive, reward, approach, avoidance, in a word, adaptive behavior. Such terminology has meaning in relation to intact, evolutionally advanced systems, but little or none for isolated sub-systems, such as a ganglion in higher or lower organisms. Often it is even difficult to employ such words when discussing the behavior of intact lower invertebrates. Thus, systems most likely to make important contributions to the understanding of fundamental mechanisms of pattern storage are often not acknowledged as showing the phenomena of learning, primarily because the above terminology does not always easily apply (Eisenstein, 1966, 1967a).
C. THE USE OF “MODEL” SYSTEMS IN BIOLOGY
The purpose in utilizing any biological model is to simplify the variables involved in the analysis. Fundamental to the use of a model system is the notion that the variables and their interaction in the model also apply to the intact and more complex biological system which is being modelled. Understanding the relevant neural pathways and their interaction in learning and memory storage in an intact mammalian brain which may contain more than 1 0 ’ O neurones with some neurones making as many as l o 5 synapses appears to many as extremely difficult (if at all possible). If one assumes that many of the neural variables important in learning and memory storage in such a complex system have evolved, then it seems reasonable to look for less complex interactions of similar (or identical variables) in less evolved forms. Hence the interest in an arthropod nervous system, such as a cockroach, which has on the order of lo5 neurones in its nervous system. Furthermore, the nervous system of the cockroach consists of ganglia, each of which may contain between lo2 to lo3 neurones. By pruning the system down further surgically it is possible to isolate one ganglion for study. (Each ganglion can be considered as a mini-brain.) Such pruned down systems are considered in this review as models for investigating relevant neural factors underlying learning and memory. It is assumed that information gained by these studies will apply both to the intact cockroach nervous system as well as to the nervous systems of more complex and evolved organisms (Miller, 1970).
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11. BEHAVIORAL INVESTIGATIONS A. INTACT AND HEADLESS PREPARATIONS
Measuring learning during a testing period In two important papers in 1962, Horridge demonstrated the feasibility of investigating learning and memory processes in isolated portions of the CNS of insects. He described a procedure for studying the learning of leg position in headless locusts and cockroaches (Horridge, 1962a, b). A
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Fig. 2. The arrangement of the connections to P and R animals from the stimulator as originally used by Horridge. “A: In the initial training the two animals are arranged in series and both animals receive shocks when P lowers its leg below the critical level. B: For retest animals are connected so that either receives a shock when it lowers its leg below the critical level. Any of the six legs may be employed and the animal may be trained on one leg but tested afterward on a different leg.” (From Horridge, 1965.)
In initial training procedures, corresponding legs of two animals were connected in a series circuit using 25-micron silver wire. One of the animals, called the positional or P-animal, could complete the circuit by lowering its leg lead into a saline bath. When this occurred, both animals received shocks until the P-animal lifted its leg out of the saline. The second animal, called the random or R-animal, could therefore receive shocks when its leg was in a variety of different positions, while the P-animal was only shocked when its leg was extended. Following a 45 min training period, the circuit was
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altered so that each animal received shock independently when its leg lead was extended into its own saline bath. If any association between leg extension and shock occurred during the initial training period, then during the test period the P-member of the pair would be expected to take fewer shocks than the former R-member. This is because during the training period leg extension was always associated with shock in the P-animals and only occasionally in the R-animals. Horridge indeed found that the P-animals initiated significantly fewer shocks than the former R-animals during the testing period. He interpreted this as a demonstration of learning by the insect’s ventral nerve cord. Apparently, the brain was not necessary for this kind of learning. Disterhoft et al. (1968) have been able to demonstrate that if-intact roaches are given 45 min of shock avoidance training using the left mesothoracic leg, a P-R difference (with P taking fewer shocks than R) can be observed from 15 min after training up to at least 24 h later. In addition, they report a slight tendency (not statistically significant) for R to be better than P 48 h after training. The latter needs to be confirmed. The shape of the curves of the 24 h group, particularly the initial worsening of the R-group at the start of testing, before improving, is remarkably similar to those curves obtained by Eisenstein and Cohen (1964, 1965) (Fig. 8) using the left prothoracic leg of an isolated ganglion preparation tested within 15 min after training. The results of Disterhoft et al. (1968) tend to support a hypothesis first put forward by Eisenstein and Cohen (1 965) that both P and R animals learn during training. This hypothesis suggested that P learns that leg extension initiates shock and flexion avoids it, while R learns that any leg position adopted during training may lead to shock onset or offset (depending upon whether P extends its leg to initiate shock or not while R is in any given position). The initial worsening of the R curve during testing before improving to the level of the P curve (see Fig. 8) suggested to us that R may first have to extinguish competing responses learned during training before it can learn as P that leg extension initiates and flexion avoids shock. In a recent more direct test of this hypothesis Disterhoft et al. (1971) report that animals shocked initially with the leg only in the extended position learned better at a later time to lift the leg to avoid shock than did those animals given the same amount of initial shock but with the leg in the flexed position.
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2. Measuring learning during a training period
Another measure of learning involves comparing changes in P and R behavior during training when only P controls the shock." Figure 3A shows such data for a group of headless roaches over a forty-five minute training period (Eisenstein, 1968a). As can be seen, there was a divergence of the two curves during training with P-animals holding their legs flexed more often and therefore receiving fewer shocks, while R-animals were holding their legs progressively more often extended during this period. Such divergence would be expected if the P member were learning to lift its leg to avoid shock and the R member, after flexing its leg to the initial shocks, stopped flexing because it had no control over the shock. A difference between P and R leg positions is often manifest to some extent by the end of the first minute of training, even though the leg leads of P and R are in the saline the same amount and the leg positions are similar at the start of training. Figure 3B shows the leg positions of two preparations wired similarly to R in 3A so neither is shocked. Contact of the saline by the leg lead only records leg position. Without shock to the leg there is no maintained improvement over the first 10 min nor divergence of the two curves over time. Over a 45 min period however there is a downward drift of both curves showing a gradual increase in maintained flexion of the legs as a function of time. It is too early to determine which behavioral criteria during training are the most accurate indices of the underlying molecular associative changes responsible for the behavioral difference seen between P and former R members during testing. Once this is known, however, a test period will not be necessary. The difference in behavior between P and former R members during testing is presently the best index that learning occurs during training. However, a test period, in which both P and R animals are now retrained as P animals, has the disadvantage of confounding any molecular differences between two such groups produced during training. Such considerations would be important for any investigations of the macromolecular bases of P and R learning (Eisenstein, 1968b). * When P extended its tarsal lead into saline, a 3 ms shock was delivered to both legs and an event recorder counted how many pulses were delivered as an index of how long the P lead was in. The shock was terminated when P lifted its leg free of the saline. Contact with the saline by the tarsal lead of the R leg initiated pulses at 4 CPS which were recorded through a separate circuit as an index of how long the R leg was extended but these pulses did not shock either leg.
0
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Fig. 3A. Divergence of P and R curves during training. Mean number of pulses initiated by the P and R tarsal leads of two animals making contact with the surface of a dish of saline. The decrease in the number of shock pulses initiated by the P group from the 1st to the 10th minute is significant at the P = 0.006 level (N = 8, one tailed Wilcoxon matched pairs test). The first 10 min of these curves have been published (Eisenstein, 1968a).
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Fig. 3B. The legs of two animals are wired similarly to that of the R leg in Fig. 3A so that the leads on the legs do not shock either leg but only record that contact with the saline has been made. P and R designations only refer to the fact that the same recording circuitry as was used in Fig. 3 A is preserved here (N = 10 pair).
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Pritchatt (1968) has reported a study on intact roaches in which he records metathoracic leg behavior of P and R animals to electric shock during a 78 min training period. His results on training the roach to flex its leg to avoid shock confirm our own results with this procedure in demonstrating an increasing divergence of P and R curves during training (Eisenstein, 1968a). There are indications from his data also that R is in fact learning something: (1) the P and R curves diverge significantly; (2) R animals which have been yoked to P for the first 30 min failed to perform as P over the next 48 min when they were no longer yoked but controlled their own shocks as P. Pritchatt interprets these results as we have (Eisenstein and Cohen, 1965; Pritchatt, 1968), viz. that R is learning competing responses when yoked to P which have to be extinguished before it can relearn to perform as P. Of interest also, are Pritchatt’s resulting demonstrating that as the shock intensity approaches 60 microamperes, the P-R training difference diminishes. Perhaps of greatest interest are his results demonstrating that the intact roach could be taught to extend as well as flex the leg to avoid shock. This finding further substantiates that we are dealing with a learning phenomenon and not just reflex sensitization (i.e., leg flexion) to shock. (Howson (personal communication)), using P-R differences during a test period, reports that preliminary results in his laboratory confirm Pritchatt’s results on the ability of the cockroach to learn to extend its leg to avoid shock). These findings correlate well with Hoyle’s results in the locust (Hoyle, 1965) (see section on electrophysiology) that the frequency of spontaneous discharge to the coxal adductor muscle of the leg could be either increased or decreased by making electric shock to the tibia contingent upon spontaneous decreases or increases in discharge frequency respectively.
3. Activity and position response measures of learning The behavioral response measure employed in Fig. 3 (pulses initiated by P and R tarsal leads) is essentially a measure of the amount of time that the leg spends in either an extended position, in which case it initiates pulse, or in a flexed position, in which case it does not initiate pulses. However, in addition to recording leg position in this manner, it is also possible to record leg activity by noting how frequently a given leg makes and breaks contact with the
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Fig. 4. Relationship of measures of leg position and. leg activity during the initial five minutes of training and testing in headless cockroaches (N = 14 pair). The upper part of the figure indicates the mean number of pulses initiated by P and R tarsal leads and the lower part of the figure indicates the mean number of times the leg extends and flexes, making and breaking contact with the saline respectively (Eisenstein, 1970a).
saline per minute. If such an activity measure is recorded, the results shown in Fig. 4 are frequently obtained (Eisenstein, 1970a). Several interesting results emerge: (1) P stays considerably more flexed during training and testing than R, as shown by the measure of leg position (Fig. 4 upper). (2) Often during training, however, particularly for the f i s t few minutes, P is more active than R. That is, P makes and breaks contact with the saline more frequently than R does (Fig. 4 lower). (3) During testing the reverse appears to be true, namely that R is more active than P. (4) The latter result often is achieved, as seen from the figure, by P becoming less active in going from training to testing while R often becomes more active in going from training to testing. (In several replications, points 3 and 4 are more consistent than point 2. Often in training there is little difference in P and R activity after the first few minutes with R on occasion being more active than P.) These results suggest that R has not been injured or damaged by its experience during the training period. At the start of testing R may be exploring by repeated flexions and extensions the relationship of
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leg position to shock occurrence as P did at the start of training. (See Section II,B for a further discussion of R learning). It is important to note that although the two response measures are related they are not the same. During training P is relatively more flexed than R (Fig. 4 upper) but P is more active than R (Fig. 4 lower). During testing P is again relatively more flexed than R (upper) but is somewhat less active than R (lower).
4. “Transfer o f training” between ganglia Horridge also demonstrated in the cockroach, P. Americana, that if the prothoracic legs of P and R animals were trained, a significant P-R difference could be obtained from the metathoracic legs during testing (Horridge, 1962a). He interpreted this as evidence of transfer of learned information from one segment of the cockroach to another. This apparent ability of the cockroach to demonstrate “transfer” of learned information from one ganglion to another in the same animal is of particular interest with respect to the question of how learned information in a central nervous system might be encoded. Recently Harris (1 97 l), has repeated and extended these observations in the cockroach, Leucophaea. He trained the right prothoracic legs and tested right mesothoracic legs of P and R preparations. By using lesions of the connectives between the proand meso-ganglia, either before or after training, as well as using a reversible cold block to the interganglionic connectives during or after training, he has demonstrated the “on-line” nature of the transfer. His results show that “information is stored in the mesothoracic ganglion at about the same time it is stored in the prothoracic ganglion and that once it is stored in the prothoracic ganglion it cannot be transferred to another ganglion” (Harris, 1971). Furthermore he reports transfer occurs equally well if only one connective is left intact. Apparently it makes no difference whether the intact connective is on the same or opposite side as the leg being trained. The first five to ten minutes of training seem to be the critical ones since no transfer occurs if a cold block is applied to the connectives during this time. (It is an open and interesting question whether transfer would occur if the cold block were applied five to ten minutes after training started). One of the questions to consider in both the Horridge and Harris results on transfer of training is to what degree has transfer of learning from the pro- to the meso- and meta-ganglia been
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demonstrated and to what degree has learning occurred simultaneously in these ganglia? There are at least three possibilities as to what “transfers” down the connectives from the pro- to the meso- and meta-ganglia: (1) a corollary discharge of the primary sensory input to the prothoracic ganglion is transferred posteriorly via the interganglionic connectives. If, for example, leg flexion or extension was simultaneously initiated in the three thoracic segments (even though it was not recorded) and if shock to the prothoracic leg was represented by interneurone discharge in the meso- and meta-ganglion, then the possibility of simultaneous learning, that is, the associating of a particular “central state” of the ganglion with shock input exists. (2) Transfer of “raw” sensory and motor data from the prothoracic to posterior ganglia (i.e. what response the prothoracic ganglion made and what sensory input followed but the posterior ganglia must associate them). (3) Transfer of a pre-formed input-output association, i.e. a true transfer of learned information from the prothoracic to posterior ganglia. Some of the information transferred from the prothoracic to more posterior ganglia may be in a humoral form, rather than in terms of nerve spikes and transferred from one ganglion to another by axoplasmic flow. This possibility cannot be excluded. If axoplasmic flow is involved then the time it takes for various substances to be transported by such flow from one ganglion to the next should be correlated with the time it takes for the transfer phenomenon to occur. If such a correlation is found then attempts to chemically inactivate or block the transport of certain substances (e.g. proteins) down the connectives and its effect on the transfer must be considered. The distinct advantage this system has for investigating how a central nervous system (CNS) might encode information in terms of nerve impulses for transfer from one part of the CNS to another is that the regions between which the information is transferred (ganglia) are specifiable, as well as the pathways (connectives) over which the transfer occurs. Since Harris (1 97 1 ) has shown no transfer when the connectives are cut or blocked before training, it is reasonable to conclude that all transfer is via these nerves. Further, until evidence to the contrary is obtained it also is reasonable to assume that an important mode of transfer is via nerve impulses.
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If there is a nerve impulse code for learned information which “transfers” the learning from one ganglion to the next then this system may provide a means of examining the nature of the correspondence between nerve impulse changes and the verbalization of what is learned, e.g. “flex the leg to avoid shock”, “extend the leg to avoid shock” or “shock occurs irrespective of leg position”. Such analyses of P and R preparations may prove useful in providing more general information as t o how nervous systems encode behavior. 5. Retention of shock avoidance learning
Two questions of interest when learning is discussed in phylogenetically simpler systems which have been simplified further by surgical means are:
(1) how long is such learning retained? and (2) how does the retention curve compare to that seen in intact (usually more phylogenetically advanced) systems? Recently we undertook such an investigation using the original Horridge procedure with headless cockroaches. They were trained for 45 min and various sub-groups tested for retention at 10 min, 30 min, 1 h, 2 h and 12 h after training. Our measures of retention were P-R differences-the larger the P-R difference the greater the retention was presumed to be. Figure 5 shows our results employing two different response measures with the 24 h value of Disterhoft et al. (1 968) on the intact cockroach added and a comparison made to the results obtained by Kamin (1957) for shock avoidance retention in the intact white rat. The points in the upper two curves represent the difference over the first ten minutes of testing between P and R. Two response measures were used-the number of shocks taken per minute and the number of times per minute the preparation made and broke contact with the saline. With both measures the response in testing right after training is for R to take more shocks than P and to be more active. Hence, the difference (R minus P) for both measures at this time is positive. For both response measures the R minus P difference diminishes, reaching its lowest value at 1 h and the positive difference increases after this. The reliability of the shape of these curves is strengthened by the following additional results: (1) Aranda and Luco ( 1969) report that P-animals retrained up to 30 min after training show a significant saving-i.e. take fewer
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Fig. 5. A comparison of the retention curves of shock avoidance learning in the headless cockroach, P. arnericana (Eisenstein, 1970b) with that of the intact white rat (Kamin, 1957). The 24 h value for the intact cockroach is from Disterhoft et al. (1968). Response measures are discussed in the text.
shocks to retrain than they did originally, whereas at one hour there is no significant savings. This supports the data shown in Fig. 5 (upper curves) at 1 h. (2) Disterhoft et ul. (1 968) studying shock avoidance learning in intact roaches report a statistically significant difference between P and R 24 h after training (see upper curve in Fig. 5). (3) Harris (personal communication) has seen P-R differences as long as 72 h after training in headless cockroach preparations. (4) Howson (personal communication) reports retention as
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measured by P-R differences for at least 8 h in an isolated me tathoracic ganglion preparation. (5) Perhaps one of the most interesting aspects of these results is how closely they follow the time course of shock avoidance retention shown by Kamin (1957) for the intact rat (see lower curve in Fig. 5 ) . Such retention for avoidance learning does not follow the classic Ebbinghaus curve where retention falls off gradually as a function of time since learning. That Aranda and Luco (1969) did not find retention beyond 1 h need not be at odds with the results of Disterhoft et al. at 24 h or our own at 2 and 12 h. There were several noteworthy experimental differences in these studies. Both Disterhoft et al. and our results are based on 45 min of training and P-R differences; whereas Aranda and Luco used only 10 min of training, P savings from training to test and did not sample between 1 and 24h. In addition they only studied four animals at the 24 h delay (see section on electrophysiology for a further discussion and figures of Aranda and Luco’s neural correlates of this behavior). Halstead and Rucker (1968) recently have postulated a 3-phase model of memory in which they predict the “Kamin effect”, that is, the retention decrement after one hour, followed by a return of retention as shown in Fig. 5. Whether the similarity of the time course of the “Kamin effect” in the intact rat and ventral nerve cord of a cockroach are fortuitous or do in fact reflect fundamental similarities in the molecular processes underlying memory in the two systems remains to be elucidated. B. ISOLATED GANGLION-P AND R AS SEPARATE PREPARATIONS
M. J. Cohen and I were interested in whether the shock avoidance learning described by Horridge could occur in a single thoracic ganglion of a cockroach when the ganglion had been isolated from the rest of the central nervous system but the peripheral sensory and motor innervation to the legs were left intact (Fig. 6) (Eisenstein and Cohen, 1965). Although shock was delivered across the femoral-tibial joint, the predominant avoidance response was flexion at the coxal-trochanter joint (Fig. 7). The R-animals received the same shock current and temporal pattern as the P-animals because of the series connection between them during training. During testing (Fig. 8) the P-animals held their legs out of the saline for longer periods than the former R-group, and therefore took fewer shocks. The difference between the two groups in number of shocks taken
Fig. 6. Ventral view of the isolated prothoracic ganglion as used in both P and R animals. The head has been removed and the posterior connectives between the pro- and mesothoracic ganglia have been cut. At anterior connectives; G: ganglion; P: posterior connectives; T: tracheole; C: coxa. (From Eisenstein and Cohen, 1965 .)
Fig. 7. Anterior view of the isolated prothoracic ganglion preparation attached by its dorsal surface to a glass rod. The petroleum jelly sealing the cut made by removing the head is seen just above the coxa. A: Position of the left prothoracic leg before training. B: Position of the leg in the same preparation at the end of training. The leg is lifted by flexion at the coxal-trochanter joint. C: coxa; CT: coxal-trochanter joint; F: femur, T: tibia; G: glass rod. Grid squares 1.5 x I .5 mm. (From Eisenstein and Cohen, 1965.) AIP-6
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P
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Minutes From Stort of Troining
Minutes From Stort of Testing
Fig. 8. The left curve illustrates the progressive decrease in the medium number of shocks taken per minute by the P animals during training. 'Ihe right curves show the median number of shocks taken by P and former R animals during testing when both are retrained with a P procedure. (Shock: 3 ms pulse at 4 CPS) (Redrawn from Eisenstein and Cohen, 1965).
over the first 28 min of testing is significant. If the improvements seen during training represented only a general shock effect, then no difference between the two groups would be expected during testing, as both groups received an identical amount of current, number and pattern of shocks during training. That the P-group took significantly fewer shocks during testing than the former R-group, suggests that during training the P-group established a specific association, i.e. it learned to lift the leg to avoid shock. The predominant trend of the R-group during the first 18 min of testing was toward an increase in the number of shocks received per minute. This cannot be a deterioration or fatigue effect, as the P-group received the same current, number and time course of shocks during training and yet showed rapid and maintained improvement during testing. We think that the upward trend of the R-group curve and the difference between the two groups are the result of one experimental variable-the relationship of leg position to shock occurrence during training. The P-group relationship was always constant; leg extension initiated shock, flexion avoided it. The
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R-group had no control over the shock during training. Sometimes shock onset occurred when the leg was flexed and sometimes when it was extended. Shock offset might also occur in any leg position. The R-group was then given the same task as the P-group during testing, that is, to avoid shock by lifting the leg. It first may have had to extinguish any previous chance association between leg lift and shock onset that had occurred during training. Such a competing response tendency would have to be extinguished before avoidance learning by leg-lifting could occur. That the R-group performance gets worse early in the testing period and then improves supports the idea of an initial period during which competing responses are extinguished, followed by relearning to perform as P. After 28 m of testing, the R-group closely approaches the performance level of the P-group. One interpretation of these phenomena is that both P and R groups learn during training. What is learned, as shown by their different behaviors during testing, is dependent on the input-output (stimulusresponse) relation to which P and R were exposed during training. The avoidance behavior seen in P and R animals showed considerable variability. The predominant avoidance response was a flexion of the coxal-trochanter joint as seen in Fig. 7. This was frequently accompanied by extension of the femoral-tibia1 joint resulting in elevation of the tibia and tarsus as seen in Fig. 9; flexion of this latter joint only occurred rarely. Occasionally the response involved complex coxal movements in which the leg was extended laterally or anteriorly. This form of avoidance response generally would be used when flexion of the coxal-trochanter joint together with tarsal and tibial elevation did not raise the leg enough to avoid shock. On several occasions the saline level was deliberately raised gradually after the animal had previously learned to avoid shock by coxal-trochanter flexion. As the saline level rose, the leg showed an increasing coxal-trochanter flexion. Each new avoidance level often was achieved after fewer shocks than the previous level. As the maximum possible coxal-trochanter flexion was approached, tibial extension would often occur (cf. Figs 7 and 9). It was only after these responses failed to avoid shock that a variety of other complex leg movements primarily involving the coxa would occur. Those movements leading to shock avoidance were maintained the longest. Occasionally it appeared that these “new” responses achieved maintained avoidance with fewer shocks than during the original learning, suggesting a transfer of training to neurons involving new
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muscle groups. However further substantiation of this point is necessary. Hence, the behavior was by no means stereotyped. The isolated ganglion was capable of generating a variety of leg movements when necessary. Proprioceptive feedback appears involved in the maintenance of the avoidance behavior. Often the flexed leg would slowly descend toward the saline and then rapidly be raised before any shock was received. This behavior would occur repeatedly in the same preparation. It suggests that the proprioceptive input evoked when the leg drops toward the saline acts as a conditioned stimulus which triggers the conditioned reflex of leg raising before the leg descends far enough to be shocked. This behavior has been seen in several laboratories (Eisenstein and Cohen, 1965; Horridge, 1962a; Prichatt, 1968). (Compare these results to those obtained by Hoyle (1965) on the locust in which proprioception was not involved in the learning.) Pringle (1939) has examined the innervation of the metathoracic leg of the cockroach, Periplaneta americana. On the basis of his work, it appears likely that Nerves 3b and 6 probably are the main motor output for the predominant avoidance responses. Nerve 3b provides motor output to both the flexor trochanteris and extensor tibiae muscles, while Nerve 6 innervates the flexor trochanteris. The major sensory input is most likely through Nerve 5 as it is the main sensory nerve of the leg (Guthrie, 1962; Pipa and Cook, 1959). The woik of Aranda and Luco (1 969) on learning in the isolated me tathoracic ganglion of the roach, Blatta orientalis, further confirms the original work of Horridge (1962a) on learning in headless preparations and of Eisenstein and Cohen (1965) on learning in the isolated prothoracic ganglion of the roach, Periplaneta americana. Howson (personal communication) also has demonstrated learning in both the isolated meta and mesothoracic ganglia of the cockroach, P. Americana. C. ISOLATED GANGLION-P AND R IN THE SAME PREPARATION
For quantitative studies of such learning it is desirable to use P and R preparations with minimal genetic variability and to obtain behavioral information from both during training, rather than during a test period when the learned difference in behavior has been abolished, especially if one wishes to examine the histological correlates of the behavior. We have investigated the use of a single preparation in which one
2U
Fig. 9. Anterior view of the preparation attached by its dorsal surface to a glass rod. The petroleum jelly sealing the cut made by removing the head is seen just above the coxa. A: Anterior extension of the prothoracic leg produced in a trained preparation by raising the saline level so that maximal coxal-trochanter flexion is not sufficient to avoid shock. Note attachment of 25 fi silver wire leads to the leg. B: Pronounced tibial extension laterally in conjunction with maximal coxal-trochanter flexion in a trained preparation. CT: Coxal-trochanter joint. FT: Femoral-tibia1joint. (From Eisenstein and Cohen, 1965.)
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leg is wired as the P leg, controlling the onset of the shock as before, and the contralateral leg is wired as the R leg. It is possible to record how often during the training period the P leg lowers its tarsal lead into the saline, shocking both P and R legs, and how often during the same period the R leg lowers its tarsal lead into the saline; lowering of the R lead produces no shock for either leg but only gives a record of R leg position. (Cohen et al., 1968; Eisenstein and Krasilovsky, 1967). Our work on this preparation has gone through several stages of development. In our first published report of such a preparation (Eisenstein and Krasilovsky, 1967) we used 50 volt monophasic pulses. The results showed maximal P-R differences (P leg in contact with saline less than the R leg) within the first minute of training and no gradual improvement in P. In fact, there was a general tendency for both legs to spend more time in the saline as training progressed-a kind of “fatigue” effect noticed also by Pritchatt (1968) who used considerably greater duration of shock than either Horridge or we used. In the next development we found that if the shock voltage was reduced to 10-15 volt pulses, the results as seen in Fig. 10 were obtained (Cohen et al., 1968). Thus, the “fatigue” we noticed with 50 volt pulses of 3 ms each could be eliminated if lower shock intensities were used.* Here we see a gradual improvement in P. The R behavior initially improves as P (probably a combination of rapid and similar learning in P and R and initial reflex sensitization to shock) and then the R leg stays extended progressively more than P, as would be expected as R has no control over shock occurrence. Also, R’s behavior shows considerably more variability from moment to moment than does P’s behavior. This again would be expected if P were learning that a particular leg position (flexion) led to shock avoidance whereas no such preferred position exists for R. Over time there is a gradual
* Our own work has shown that in a single isolated prothoracic ganglion where one leg is P and its contralateral mate is R, if a lower shock current is used (10-20 pa, 3 ms pulses) there is a gradual divergence of P and R curves with the overall P-R difference becoming greater than when a larger current is used (approximately 50-100 pa). (Compare Fig. 10 in this paper with Fig. 2 of Eisenstein and Krasilovsky (1967)). Pritchatt (1968) reports that as the shock current is increased from 20-60 pa, the P-R difference linearly decreases and there is a marked “fatigue” in the ability of the P and R legs to remain withdrawn from the saline over the training period. This suggests that relatively short (e g. in the range of 3 ms) pulses of 10-20 pa intensity yields gradually developing and maximal P-R differences with minimal evidence of leg fatigue. (Although shock is a factor in producing the fatigue observed by Pritchatt, he also observed some in his non-shock group, whereas we did not (see Fig 3B)J
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250r
Time (Min)
Fig. 10. The median number of pulses per minute initiated by tarsal leads attached to the
“P” and “R” legs of the same animal. The inset shows the predominant positions of the “P” and “R” legs during the experiment. The shock was a 3 ms pulse at 4 cps. The difference between “P” and “R” curves over the 30 min of training is significant using a one-tailed . Wilcoxon matched pairs test, p= 0.025, N = 6 . (From Cohen e t al., 1968.)
divergence of the P and R curves. These P-R differences were independent of shock polarity as essentially the same results were obtained if polarity were reversed. More recently we have used biphasic pulses, and modifications of the wiring such that the pulse travels up one leg and then down the other, reversing direction as the polarity changed. We have also systematically varied right and left prothoracic legs as P and R. The results obtained essentially confirmed those shown in Fig. 10. Horridge (1 965) also has reported results on a single intact locust in which he shocked the left metathoracic leg when it extended (P leg) and the contralateral leg was shocked independent of its leg position (R leg). After he turned the shock off there was a period in which the P leg remained above the level at which it was shocked while the R leg dropped below this level in its activity. There is similarity in the divergence and other characteristics of P and R curves during initial training (when R does not control the shock) whether a single isolated ganglion is used with one leg as P and its contralateral mate as R (Fig. 10) or whether two separate
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headless preparations are used with the leg of one being the P leg and that of the other preparation an R leg (Fig. 3A). It is not possible at present to state to what extent the P-R difference, when a single preparation is used, is due to independent underlying processes as in the work with separate “P” and “R” ganglia, and to what extent composite processes (e.g. crossed extension reflexes) may be involved. In the event that a crossed extension reflex is involved, the fact that the divergence occurs consistently with the “P” leg becoming progressively more flexed (shock avoidance) rather than with the “R” leg more flexed (no avoidance) qualifies such a composite response as learned. Such a developing difference in P and R behavior suggests the formation of an association between leg position and shock occurrence. D. EFFECTS O F OTHER CNS LESIONS ON P AND R BEHAVIOR
I . Inter-ganglionic lesions The ability of the roach to demonstrate learning was studied as a function of lesions made in various parts of the CNS. Preliminary results indicate that P-R differences during a test period are greatest when the prothoracic ganglion is isolated from the head (headless) or from the posterior ganglia (head attached but posterior connectives of the prothoracic ganglion are severed); surprisingly the P-R difference appeared markedly reduced relative to the above two groups if both anterior and posterior influences on the prothoracic ganglion are left to operate simultaneously (intact animals). If the brain is removed and only the subesophageal and prothoracic ganglia are joined, the P-R difference is abolished due to flexion of P and R legs to shock (Eisenstein, 1967b).
2. Intra4split) ganglionic lesions Initial attempts several years ago to split the ganglion longitudinally suggested that the attached prothoracic leg can still undergo complex coordinated movements when stimulated either by stroking tibia1 spines or by electric shock. A long lasting response to shock, quite similar to that shown in Fig. 7B, was seen in a half-ganglion P preparation as long as 7 h after the operation. Without a matched R member, however, it could not be said whether the half-ganglion could learn. These experiments were pursued to determine how small an amount of CNS tissue was necessary to support avoidance learning (Eisenstein and Cohen, 1965.)
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In more recent experiments of this kind an isolated prothoracic ganglion was split longitudinally into two symmetrical halves and one half was attached to a “P” leg and the other to an “R” leg. There is no clear divergence of “P” and “R” curves during training as seen in intact prothoracic ganglion systems (cf. Fig. 10). In addition, the leg responses to shock are not as good as in the intact ganglion, suggesting, perhaps, that in the process of splitting the ganglion there was damage to the motor output. However, if in addition to splitting the ganglion the head or posterior ganglia are left attached, then the leg responses are normal and there is excellent divergence of the P and R curves during training. This would suggest that splitting per se does not impair the motor output, but that more CNS tissue than is present in a half-ganglion is essential for normal coordinated behavior and P-R divergence during training. No consistent P-R difference was observed during testing in intact or split prothoracic ganglia preparations where one leg is P and its contralateral mate is R. With the exception of the isolated split-ganglion preparation (which was poorly coordinated and showed no separation during training), all other split preparations had some connection between the two halves during training (through either anterior or posterior ganglia left attached to the split prothoracic ganglia). Therefore it is not unreasonable that while P and R learned their appropriate behaviors during training, they also had access to information about the behavior of the other leg, thereby minimizing the necessity of R .to relearn during testing to perform as a P leg and hence, the minimization or abolition of the P-R difference during testing. Further research is still required on: (1) the reasons for the lack of a consistent P-R difference during testing when P and R are in the same preparation; (2) the role of communication between the two halves during training and testing and (3) the ability of half an isolated ganglion to support a developing P-R difference and hence, show learning. E. BEHAVIOR OF GANGLIONLESS P AND R PREPARATIONS
In order to determine if the difference in the number of shocks taken during testing by the P and R groups with an isolated ganglion was due to a ganglionic process or to direct electrical stimulation of
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nerve and muscle in the leg, the prothoracic ganglion was removed in six pairs of animals (N = 12). Such a preparation provides a baseline upon which to evaluate CNS contributions to P and R behavior seen in preparations with a ganglion. These “ganglionless” preparations were subjected to the same training and testing procedures as described for the prothoracic “isolates” (Eisenstein and Cohen, 1965). Figure 11A illustrates the predominant resting leg position found
Fig. 1 1. Anterior view of the ganglionless preparation attached by its dorsal surface to a glass rod. A Position of the left prothoracic leg before training. B: Position of the leg in the same preparation at the end of training. The.leg is lifted solely by posteromedial flexion at the femoral-tibialjoint. FT:femoral-tibial joint. (From Eisenstein and Cohen, 1965.)
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in preparations in which the prothoracic ganglion has been removed. Figure 1 1B shows the way in which the same leg responds to electric shock. The response involves a slow postero-medial movement of the tibia until the leg lead is brought out of contact with the saline. The tibia then slowly drifts back until a shock is received at which time the cycle repeats itself. The response was very slow and stereotyped, showing neither the speed nor the variety of movements of the prothoracic “isolate” preparations. It always consisted of flexion at the femoral-tibia1 joint. The lack of movement at the coxaltrochanter joint in the ganglionless preparation might possibly result from injury to the neuromuscular apparatus of the joint while removing the ganglion. This possibility was ruled out, however, by the fact that flexion or extension could be produced at this joint by crushing the cuticle over the flexor trochanteiis or extensor trochanteris muscles respectively. The peripheral neuromusculature of the leg responds directly to electric shock between the femur and tarsus with a characteristic stereotyped response. When the ganglion is present this peripheral effect is generally over-ridden by the centrally evoked ganglionic responses (cf. Figs 7 and 9). The ganghonless preparations were subjected to the training and testing procedures described above for the prothoracic “isolates” and the results plotted in Fig. 12 (cf. Fig. 8). The left curve of Fig. 12
TRAINING
TESTING P x-x R X---X
Minutes From Start of Training
Minutes From Start of Testing
Fig. 12. The left curve illustrates the decrease in the median number of shocks taken per minute by the P ganglionless animals during training. The right curves show the median number of shocks taken by the ganglionlessP and former R groups during testing. Note lack of difference between P and R groups during testing. (Redrawn from Eisenstein and Cohen, 1965.)
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shows the decrease in the median number of shocks taken per minute by the P-animals during the first 10 min of training. The difference in number of shocks taken in the first and tenth minutes is significant (p = 0.053, N = 5, Wilcoxon matched pairs test). During testing the P and R groups take about the same number of shocks as shown by the close apposition of the curves on the right side of Fig. 12. The ganglionless P group takes progressively fewer shocks over training, thereby demonstrating some type of generalized prolonged excitability change in the leg neuromusculature evoked by electric shock. The stereotyped nature of the response and the lack of a difference between the P and R groups during testing suggests an entirely different process underlying this behavior from that underlying the behavior of the prothoracic “isolates”. The specific association established between the reinforcing shock stimulus and a particular leg response distinguishes the behavior shown in Fig. 8 from the longlasting changes shown in Fig. 12 that may be evoked by direct stimulation of the leg neuromusculature. F. DISCUSSION OF P AND R LEG BEHAVIOR IN THE PRESENCE OF GANGLIONIC INNERVATION OF THE LEGS
The curves in Figs 3A, 8 and 10 show several features in common with respect to P and R behavior. They are: (1) a rapid and maintained improvement in P; (2) a P-R difference within the first minute; (3) a marked improvement in R over the first two minutes of both training and testing; (4) following initial improvement in R, there is a gradual divergence of the R from the P curve; (5) more variability in R than in P behavior; (6) the P-R divergence is maintained over the duration of the training period (Figs 3A and 10) whereas R eventually approaches the P level during testing (Fig. 8). Many of these common features emerge whether one utilizes a single preparation for P and R, uses two separate P and R preparations, studies an isolated prothoracic ganglion preparation, examines an entire ventral nerve cord preparation or measures P and R behavior during training or testing. Each of these behavioral characteristics may represent a different underlying molecular process. Which, and how much, of any of these behavioral characteristics represent underlying learning and memory processes await the identification of the molecular bases of these
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phenomena. However, it may be useful for further experimental work to hypothesize the possible significance these behaviors have for learning and memory in the cockroach system. The improvement in both P and R in the first two minutes of training may represent similar learning in both, since both legs begin from the same starting position and it is likely in the first few shocks to find P and R legs in similar positions. (Some of the improvement at the start in P and R legs may also represent reflex sensitization to shock producing leg flexion,) As discrepancies in leg positions for P and R develop, there will be corresponding discrepancies in the relationship of leg position and shock occurrence for them. The difference between P and R seen in the first minute suggests that, while both P and R may be learning the same thing at the start, (e.g., leg extension initiates shock and flexion avoids it) P nevertheless forms such an association more rapidly and strongly (presumably because it controls the shock onset and offset). The eventual divergence of R from P may represent two different processes occurring in the R preparation-an initial extinction in R of the P contingency (i.e. leg extension intiates shock and flexion avoids it) which it learned in the first few minutes of training when both legs were in similar positions, followed by the development of a unique R learning, i.e. the contingency that shock onset or offset may occur independently of whether the leg is flexed or extended. How much of this latter learning in R occurs will probably depend on how many times P initiates shock after the curves begin t o diverge in training. If P initiates very few shocks, then the dominant component in the diverging R curve will be the extinction of its original P learning. If P initiates many shocks following divergence, then an increasing percentage of the R curve will probably represent a unique R learning component. Both the extinction component and the unique learning component could be expected to produce a diverging R curve, since in both cases R has no control over the shock and might well adopt the leg position requiring minimal energy expenditure (viz. a more extended position). Also R might well show more variability than P over time in where it kept its leg since it can be shocked or not shocked in any position. During testing it may be possible to sort out the relative contributions that extinction and learning played during training in the diverging R curve. If R only extinguished its P Iearning during training, then during testing when R is treated as a P leg, it should describe a similar time course of improvement as P did at the start of training. (Perhaps this is what the locust curve in the original
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Horridge (1962a) paper on this subject shows.) If R also learned during training that shock may occur independent of its leg position, then during testing R might be expected to show competing response tendencies and so behave differently than P did in training or testing, as manifested by a different time course of behavioral change. The initial worsening of the R-animals at the start of testing followed by improvement (see Fig. 8 and its ensuing discussion) may represent the extinction of just such a competing response tendency. It may well represent extinction of the unique R learning which occurred during training and which now competes with P learning, followed by R relearning to behave as a P-animal. The shape of the R curve during testing appears to represent a reliable phenomenon and is not unique to the isolated ganglion. (I would predict that the more shocks R received during training while it was diverging from P, the greater the period of competing response behavior during testing.) Evidence for these assumptions about unique R learning during training and the need for its extinction during testing before it can learn to behave as a P-animal, comes from several lines, some of which were previously mentioned:
(1 ) Disterhoft et al. (197 1) report that in intact roaches if a leg of one preparation is stimulated in a flexed position and that of another in an extended position, and then at a later time both are trained to flex the leg to avoid shock, the one previously shocked in a flexed position will find this more difficult to do; (2) Pritchatt (1968) reports that following a 30 min period of R learning in intact roaches, preparations now given a P contingency (i.e. lift the leg to avoid shock) do not learn to perform as a P animal within the next 48 min and in fact at the end of 48 min they are more extended than a non-shock control group; (3) Horridge (1962a) also showed that often the R leg improved with difficulty, if at all, during the testing period (this is a common fmding (Disterhoft et al, 1968; Eisenstein and Cohen, 1965; Horridge, 1962a;Pritchatt, 1968), in several laboratories); (4) Hoyle (1965) (see section on electrophysiology) reported that if shock occurred independently of the response, then later it is extremely difficult to build a specific contingency (that is a particular response and shock relationship); (5) the fact that R gets worse (i.e., takes more shocks) early in testing before improving to the level of P suggests the interaction of competing response tendencies (cf. Fig. 8 and its discussion); (6) Howson (personal communication) reports that M. E. P.
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Seligman of Cornell University has “examined avoidance conditioning in dogs in which one group was given random shock before conditioning. The conclusion of these studies was that dogs subjected to a period of shock unrelated to the animals’ behavior could not learn to avoid shock in later attempts at conditioning”. This finding appears related to the difficulty in retraining R roaches during testing to behave as P animals; (7) if one measures activity of P and R animals i.e., the number of times each leg makes and breaks contact with the saline per minute (cf. Fig. 4) P is usually more active than R at the start of training, while R is the more active in testing. This certainly demonstrates R is responsive. It also suggests that one of the processes occurring in R during testing is that it is now going through a learning process similar to P at the start of training. Some P-R differences in training and testing, it may be argued, might be due to differences in the voltage drop and/or current density in P and R legs as a result of different leg positions when the shock occurs. Thus if leg resistance varied with leg position, and voltage drop across the leg were important in determining sensory input, then P-R differences might be obtained because of this. (No consistent gross differences in leg resistance were observed between P and R preparations at the end of avoidance training or after testing.) Likewise, current density across the sensory nerves may vary with leg position and so influence the sensory input in P and R legs to the same current. Thus, P-R differences might reflect differences in sensory input to P and R legs, rather than a difference in P and R learning that different leg positions lead to shock onset and offset. In addition the shock, it might be argued, produces different effects at the neuromuscular junctions as a function of leg position when the shock occurs and so effect the ability of P and R legs to flex equally well. The question here then is to what extent P-R differences reflect differences in the ganglion of the association of leg position and shock occurrence in P and R animals and to what extent they reflect differences in peripheral sensory input and/or neuromuscular changes. The factors arguing for a strong central associative interpretation of P-R differences are the following:
(1) R describes a different shaped curve than P in both training and testing. If R were only receiving more or less sensory input than P then the R curve should be shaped essentially the same as the P
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curve, only showing a greater or lesser P effect. (cf. Figs 3A, 8 and 10; also see original Horridge paper, 1962a). (2) Shocking across several different leg joints (femoral-tarsal, femoral-tibial, upper tibia and tarsus and the lower tibial-tarsal joint) produces similar P-R differences in training and testing. This would tend to argue against an interpretation based only on current density differences and/or voltage drop differences in P and R due to leg position. It seems unlikely that such uniform results would be obtained when shock occurs across so many different parts of the leg if that were the case. It also does not seem reasonable that for all these joints P would always get more or less input than R. Furthermore, the primary joint involved in shock avoidance (the coxal-trochanter joint) does not receive shock across it at all. (3) P-R differences can be shown up to 24 h after training. At this time (Disterhoft et al., 1968), in intact preparations, R describes a curve similiar to that of the isolated ganglion shown in Fig. 8. Furthermore, these P-R differences disappear within 20-30 min of testing during which both P and R are retrained as P-animals. Such long retention as measured by differences in P and R behavior at the start of testing which disappears within 20-30 min of retraining argues for different associations being formed in P and R ganglia during training. The shape of retention curve itself (cf. Fig. 5 ) also argues for different ganglionic associations in P and R preparations and against interpretations of P and R behavior in terms of neuromuscular effects and differences in amount of shock input during training. (4) Both Horridge ( 1962a) and Harris (1 97 1) have shown P-R differences in meso and meta P and R legs in testing when prothoracic legs were trained. Their results thus tend to minimize the importance of peripheral shock input per se in accounting for the P-R differences seen in testing. ( 5 ) Pritchatt (1968) can train a P leg to avoid shock by flexion or extension relative to an R leg. This argues against current and/or voltage differences in the leg due to leg position when shock occurs in accounting for the P-R differences seen. (See Hoyle (1965), Fig. 16, for an electrophysiological counterpart to this latter study.) (6) The results presented for the ganglionless preparation certainly argue for the role of the ganglion in generating different leg positions which lead, presumably, to different associations in P and R ganglia of leg position and shock occurrence. The above results are meant to indicate that peripheral changes per
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se, that is, differences in sensory neuron discharge in P-R legs as a
result of long-lasting sensory threshold changes, or differences in voltage drop and/or current density in P and R legs and neuromuscular changes, as a result of shock occurring in different leg positions are not sufficient to explain the P-R differences obtained either in training or in testing. Rather, it is suggested that these results are consistent with the interpretation that P and R ganglia are learning different things about the relationship of leg position to shock onset and offset. This is not to suggest that peripheral factors such as those mentioned do not in fact play a role in the behaviors seen. One of the major limitations of behavioral investigations of learning of this kind is that it is not possible to know what sensory inputs (both due to shock as well as proprioceptive feedback) actually occur in different leg positions. It is with this limitation in mind that electrophysiological investigations of such preparations in which sensory inputs and motor outputs of the ganglia are directly monitored become essential. As noted, Horridge (1962a), Pritchatt (1968) and our own laboratory (Eisenstein and Cohen, 1964, 1965) have seen what would appear to be proprioceptive feedback in the maintenance of roach avoidance behavior. Murphy (1967) studied P savings from training to testing when hairplate cells were removed in the roach. This only partially interferes with proprioceptive feedback as there is still some input left. He found some interference with the savings of P from training to testing when such cells were removed. It is not clear as to the exact role of proprioception in this behavior, whether it is with respect to learning, retention or in the motor act of lifting the leg itself. Overall the results of P and R behavior suggest to me that there are two types of learning going on during training: P learning which involves the association of a particular leg directional movement (extension) with shock onset and another specific directional movement (flexion) with shock offset and R learning which can involve the association of either directional movement with shock onset or offset depending on what R s leg is doing when P activates or turns off the shock by leg extension and flexion respectively. The great uniformity and reproducibility of results from several laboratories in this area is encouraging. This occurs in spite of the great diversity in training and testing procedures, species of cockroach used, ganglion used, training time, shock duration and intensity, whether the leg makes direct contact with saline or AIP-7
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through a wire, whether learning is measured as divergence in training, P-R difference in testing or P savings from training to testing and whether the response is leg flexion or extension. This indicates the phenomena seen are not unique or peculiar to a methodology and thus most likely reflect something fundamental about the learning and retention abilities of the systems under study. G. DISCUSSION OF P AND R LEG BEHAVIOR IN THE ABSENCE OF GANGLIONIC INNERVATION OF THE LEGS (GANGLIONLESS)
The results with the ganglionless preparations (Figs 11, 12) pose some interesting problems of interpretation of data. Our interpretation of these results is that those animals with ganglionic innervation of P and R legs which showed a P-R difference in their behaviour during testing demonstrated learning and those without such ganglionic innervation which showed no difference in their P-R behavior during testing demonstrated long-lasting changes that may be evoked by direct stimulation of the leg neuromusculature (compare Figs 7 and 8 with Figs 11 and 12). There are, of course, other possible interpretations of the meaning of P and R results in animals with and without ganglionic innervation of their legs. These interpretations depend to a large extent upon whether one chooses to focus on the similarities or on the differences in the curves obtained from those with and without a ganglion. Thompson (1967), for example, suggests as one possibility that both P and R ganglionless animals learn. He feels on the basis of the curve shown in Fig. 12, that they in fact learn the shock avoidance response as well as the P ganglionic preparations (cf. Fig. 8). It should be noted, however, that a similarity in the decrease in the number of shocks taken per minute is not really the whole measure of the change that one can observe in the behavior of these animals. The character of the response is quite different in preparations with a ganglion compared to those without. The latter, for example, show extremely stereotyped response patterns. If one uses a weak definition of learning, that is, improvement per se as measured by a decrease in shocks taken per minute, then one could argue that ganglionless animals learn as well as those P animals with a ganglion. However, a stronger definition requires that a temporal relationship be shown between stimulus (shock) and response as varied in our experimental set-up by having P and R preparations and measured by
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a P-R difference. If such a measure is used, then there is no learning in the ganglionless P and R preparations of the kind seen in P and R preparations with a ganglion. However, we do not wish to exclude the real possibility of peripheral storage of learning in the arthropods. In fact, it was partly to test such a possibility that a ganglionless experiment was even performed. It is well known that arthropods (particularly in the skeletal muscle systems of crustaceans) show multiple innervation of single muscle fibers by a single neuron, polyneuronal (i.e. fast and slow innervation) as well as inhibitory axon innervation of the same muscle fiber. In addition, the graded electrical and mechanical responses in such muscles provide a potentially rich region for integration in the periphery, in this sense, not unlike CNS neuropil. Pattern-sensitive neuromuscular synapses are also known; in these, the magnitude of muscle contraction may be greater to shocks initiated in doublets in the motor nerve, than t o equally spaced shocks of the same frequency (Wiersma and Adams, 1950). This is evidence of pattern recognition at a neuromuscular level. The possibility that higher integrative processes akin to learning occur in the neuromuscular region of arthropods (particularly crustaceans) should not, therefore, be overlooked. Thompson (1967) also raises the possibility that “the ganglion functions to prevent learning” because the R group with the ganglion did not learn to withdraw the leg to shock during training (measured by their lack of improvement over the first 18 min of testing (cf. Fig. 8)) as did P animals with a ganglion and as did the P and R ganglionless preparations (cf. Fig. 12). This interpretation is based on Thompson’s suggestion that improvement in the P leg may be due to habituation of the leg searching movements (with the result that P extends its leg progressively less over time) and that the R training prevents this habituation of the leg searching response in R preparations. A comparison of Figs 3A and 3B, however, shows that both the decrement in the number of shocks taken by P and the divergence of the R curve are facilitated by the shock. Still another interpretation, contradictory to that above, of the R group behavior in those animals with the ganglion is that only the K group learns. Since P and R ganglionless preparations behave the same way as P preparations with the ganglion, then one might argue that the behavior of the “ganglionless” systems represents the ground state, so to speak, that is the unlearned state. Any divergence from this state such as shown by R animals with a ganglion represents learning.
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An important point to emphasize again is the marked difference in behavior between animals with and without a ganglion. The former are active, variable and plastic in their behavior; the latter are inactive and highly stereotyped in their behavior using as an avoidance response flexion at the joint they were shocked across (femoral-tibia1 joint). Such flexion was not seen in preparations with a ganglion as a means of shock avoidance except when they were deteriorating. It could be that the stereotyped behavior seen in those preparations without a ganglion prevents the development of a P-R difference, that is the ganglion provides response variability so that shocks could in fact occur in P and R legs when they were in different positions. One approach to testing this possibility might be to train P and R animals with a ganglion, then remove the ganglion and test to see if a P-R difference shows up. If it does, this would argue for at least some peripheral storage of learned information in preparations when the CNS innervation to the legs was intact. There would, of course, be many advantages in looking at the molecular basis of learning and memory storage in such a neuromuscular system if learning could occur there. It is a considerably simpler system histologically than the isolated ganglion preparation. It is easier to locate pre- and post-synaptic elements in the tissue since the pre-synaptic elements would be peripheral nerve and the post-synaptic element would be muscle. If peripheral neuromuscular learning could occur, then such a system would provide a relatively simple and accessible system for electrophysiological and morphological investigations on the cellular basis of such integrative phenomena. It is a fact that a P-R difference is manifest during testing in P and R preparations with ganglionic innervation of their legs and that no such difference exists during testing in ganglionless P and R preparations. Further, the nature, the plasticity and the variability of leg movements in the former are quite different from the latter. It is possible to conclude from these results that a P-R difference represents a state of learning. Whether only P or only R or, in fact, both learn cannot categorically be answered at present.’ Whether some learning occurs in ganglionless preparations and is stored peripherally likewise cannot be answered at present. Such questions as these will depend for their answers on empirical studies of the molecular bases of P and R behavioral changes in preparations with and without a ganglion.
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111. SOME HISTOLOGICAL AND ANATOMICAL FINDINGS POTENTIALLY RELEVANT TO UNDERSTANDING THE CELLULAR BASIS OF LEARNING IN THE COCKROACH A. INTRODUCTION
Because of the complex organization of neuropil where presumably all synapses within the ganglion occur, it would seem extremely difficult to pinpoint sites relevant to the occurrence of any particular innate behavior, let alone identify the occurrence of new functional sites associated with learning and memory. Since, however, an isolated ganglion is capable of supporting learning (Eisenstein and Cohen, 1965) and since it consists of a small volume of tissue (probably less than 1 cubic millimeter) and a relatively small number of cells (on the order of 1O3 or less) it would seem desirable to examine such tissue microscopically for possible sites and changes forming the bases of such learning (Fig. 6).* B. MAPPING OF THE COCKROACH METATHORACIC GANGLION
The feasibility of determining the pathways, synapses and cytochemical changes associated with (and possibly causally related to) such discrete behavior as shock avoidance learning in the roach is considerably enhanced by the detailed histochemical studies of the metathoracic ganglion by Cohen and Jacklet (1 967). They have mapped out the major bilateral distribution of motor cell bodies and
* Some very preliminary exploration of electron microscope sections of prothoracic ganglia of cockroaches (P.americana) showed dense particles of approximately 250-280 A in diameter within certain axons and pre-synaptic boutons within the neuropile. (Our original report indicated the vesicles were about 150 A in diameter; however further work showed they were larger.) These occur in addition to the profile of neurotubules, neurosectetory droplets, and synaptic vesicles usually found in these locations (Johnson and Eisenstein, 1966). The most striking feature of these particles was their discrete distribution. In a given section the axons and synapses in which they are seen make up only a few per cent of the total. The few axons and synapses in which they are found however contained them in large numbers. Typically they occurred in clusters of about 5-10 scattered along the length of an axon or among secretory vesicles. They are electron dense in unstained sections and their density is enhanced by uranyl ions. Their nature is presently unknown. The presence of aggregates of these particles in just a fex axons and synapses raises the possibility, however, that they may be related t o specific neural functions. There is the possibility that they may occur more in stimulated than in unstimulated ganglia. If this proves to be the case then, in principle, particles such as these might provide useful “morphological markers” for active sites within the neuropile associated with a given behavior.
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the peripheral nerves through which they send their axons. This makes it possible to identify a specific motor cell and to ask whether it is involved in a particular behavior and, if so, what cellular changes are associated with the behavior. The marked bilateral symmetry on a cellular level makes it possible to use one cell as a genetic control for changes produced in a contralateral mate. Perhaps the ability of an isolated ganglion to show learning when one leg serves as P and its contralateral mate as R (see Fig. 10) will be useful for such cellular studies on the bases of learning. C. GANGLION TRANSPLANTATION STUDIES IN THE COCKROACH
Jacklet and Cohen (1967), also found that if a metathoracic ganglion of one roach were transplanted into the coxa of another roach, it would innervate within 30 days only those muscles whose normal innervation was previously cut. Thus, the “transplanted thoracic ganglion offers the possibility of examining the establishment of connections between motor neurons and their muscles under relatively controlled circumstances’’ (Cohen et al., 1 968). If the connections on the sensory side of the system could be as well specified as on the motor side, then it may be possible as Cohen et al. (1968) noted to “construct an excitable system in a stepwise manner and correlate each step with discrete behavioral capabilities.” IV. ELECTROPHYSIOLOGICAL INVESTIGATIONS OF LEARNING A. INTRODUCTION
In the context of specifying the molecular mechanisms of learning and memory storage, I view electrophysiological measurements as a bridge between behavior and biochemistry. They tell us where in the system activity relevant to the behavior is occurring so that more fundamental molecular studies can be done. B. HABITUATION STUDIES
The term habituation is used to describe the progressive decrease in magnitude and/or probability of a response resulting from its repeated elicitation by an intermittent stimulus. Thus, if a loud bell is sounded for the first time, an animal may show a startle response, indicated by a host of autonomic and skeletal reactions. Following
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several presentations of the bell, there is progressively less response and in some cases the response may diminish to zero. That this decrease is not caused by fatigue of the effector apparatus often can be demonstrated through reinstatement of the original response by changing the stimulus. By using an intermittent stimulus, receptor adaptation often can be ruled out as well and so the response decrement is assumed to be due t o processes within the CNS. Kandel and Spencer ( 1968) have listed a number of criteria which define habituation. Briefly, they are: ( 1) response decrement with repeated intermittent stimuli,
(2) greater and more rapid decrement with higher stimulation frequencies, (3) greater decrement with weaker stimuli, (4) delay of recovery if stimulation continues while the response is abolished, (5) generalization of response may occur to other stimuli, (6) spontaneous recovery of response if stimulation is stopped, (7) rate of habituation increases after several periods of habituation training and spontaneous recovery, (8) a strong extra stimulus may produce restoration of the habituated response (dishabituation) and (9) the effectiveness of a dishabituating stimulus diminishes after several presentations of it. Electrophysiological studies of habituation often use progressive decrement in number of spikes elicited to a repeated stimulus as a response measure. Horridge (Horridge et al., 1965; Horridge, 1965), for example, reports that the number of spikes elicited in a higher order neuron in the optic lobe of the locust to a spot of light diminishes rapidly by the third stimulus presentation, and the response returns if the light stimulus is moved to a new area of the eye (Fig. 13). Other characteristics of habituated responses were observed in various units of the locust optic lobe. For example, Horridge reports that habituation of a unit through its ipsilateral input leads to diminished output of the unit when its contralateral input is stimulated (contralateral transfer of habituation). Further, a unit previously habituated to light can be dishabituated to light (i.e., the response to light recovers) by presenting a sound stimulus. Baxter (1957) studied habituation of the running response in cockroaches to puffs of air delivered to the anal cerci. Hairs on the
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Fig. 13. "Typical responses of a novelty unit of the locust optic lobe to a repeated visual stimulus which is changed to a new location after the third presentation. The stimulus was a flash from a small green light source as shown by the thickening in the trace, at a repetition interval of 5 s. There is rapid habituation as shown by the f i s t three traces, whereupon at A the light was moved 10" in the visual field without change in the repetition rate or other features. A response almost identical to that f i s t found now reappears". (From Horridge, 1965.)
cerci are sensitive to air movement and the cockroach exhibits rapid locomotion to such stimulation. He restrained adult specimens of Periplaneta arnericana and delivered puffs of air well above threshold for the startle response and observed a decline in motor activity visually. If one-second puffs of air are delivered at the rate of six per minute, the roach soon ceases to give the characteristic avoiding motor response and exhibits either quiescence or greatly diminished activity. Recording electrical activity of the giant fibers making contact with the primary cercal nerves in the sixth abdominal ganglion, Baxter found no diminution in the electrical activity of these giant fibers to puffs of air, even though there is a decrease in the motor activity of the animal. If it is the giant fiber discharges to more anterior portions of the nerve cord that are responsible for the motor activity to the puffs of air, then these results would suggest that the decrease in motor activity was due to some type of synaptic change anterior to the cercal-giant fiber synapses in the sixth abdominal ganglion (very likely in the metathoracic ganglion). He also notes preliminary work that indicates habituation may take much longer to occur if the higher brain centers are removed by decapitation. Recovery of the habituated response occurs with 5 to 15 min of rest.
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Pumphrey and Rawdon-Smith (1 936, 1937) have studied synaptic transmission in the last abdominal ganglion of the cockroach, Periplaneta americana, using acoustic stimuli. They described the cercus as a primitive hearing organ. Recording from the primary cercal nerve and stimulating the cercus with tone stimuli, they note that the primary cercal nerve discharges are synchronous with tone stimuli from 50-400 CPS. The response is only partially synchronized for stimulus frequencies between 400 and 800 CPS; above 800 CPS the response is asynchronous, but they were still able to get a response up to approximately 3000CPS. They note with some surprise that so primitive a hearing organ as the cockroach cercus can “reproduce so many of the physiological phenomena associated with the vastly more complex mammalian cochlea” (Pumphrey and Rawdon-Smith, 1936; see also Wosniak e t al., 1967). Figure 14 shows the anatomy of the sixth abdominal ganglion in which the primary cercal nerve enters. As can be seen from this throuah fibers
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Fig. 14. Sensory input, synapses with giant fibers and motor output pathways of the 6th abdominal ganglion of the cockroach, P. americana. (From Pumphrey and Rawdon-Smith, 1937.)
figure, the “through fibers” mostly run on the same side of the cord as the cercus from which they are derived. These are fibers that originate in the cercus and continue right on through the sixth abdominal ganglion to higher parts of the cord. “Giant fibers” respond in both connectives from a given cercal input-but there is a lesser response on the side contralateral to that from which the cercal input came. Figure 15 shows the arrangement of stimulating and recording electrodes that Pumphrey and Rawdon-Smith (1 937) used in studying habituation in the last abdominal ganglion of the
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Fig. IS. “Sketch of the posterior part of the abdominal nerve cord of the cockroach (Q) with the principal tracheae and the positions of stimulating and recording electrodes shown. The external genitalia have been removed. R = recording electrodes. S,, S, = alternative positions of electrodes for stimulating the afferent cercal nerve. (a) cercal nerve (sensory); (b) cercal nerve (motor); (c) nerves to genitalia (cut). IV, V, VI = last three abdominal ganglia.” (From Pumphrey and Rawdon-Smith, 1937.)
cockroach. They noted that when the pre-ganglionic fibers are subjected to repeated supramaximal electrical stimulation at regular intervals, the post-ganglionic giant fiber response in the last abdominal ganglion is approximately the same, provided that the stimulating frequency is low. The authors noted, however, that with sub-maximal stimulation of the cercal nerve there was a decrease in the giant fiber output. Such a response decrease occurs even though it appears that the pre-ganglionic response is the same throughout the stimulating period. They attributed this result to a change in the synapses of the last abdominal ganglion which they designated as “adaptation”. The post-ganglionic response may be brought back in one of three ways:
(1) “by increasing the number of peripheral fibers stimulated, i.e. raising the stimulus intensity;
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(2) by increasing the stimulus frequency, without modification of its intensity; or (3) by the interpolation of an extra stimulus into the series” (Pumphrey and Rawdon-Smith, 1937). The return of an adapted (habituated) response, particularly by the third way, is known as dishabituation. It can be brought about by an interpolated stimulus that is less intense than the stimuli of the series in which it is inserted. The dishabituated response generally occurs to the stimulus in the regular series immediately following the interpolated one. The stimulus frequency used by these authors was generally in the range of about 25 pulses per second. It is the third way of bringing about the return of the response that is of such great interest when considering how nervous systems encode temporal sequences of events. In our own laboratory (Eisenstein, unpublished), we have studied response decrement in the anterior connectives of the isolated last abdominal ganglion of the cockroach, Periplunetu americuna, using electric stimuli delivered through bipolar electrodes inserted in a single cercus and recording nerve impulses with separate suction electrodes placed over the cut ends of the left and right anterior connectives. The motor outputs from the last ganglion to the cerci are cut and only the sensory inputs from the cerci are left. Our stimuli consisted of 1% ms pulses of submaximal strength delivered at the rate of 1 per 2% s and in which the polarity of the stimulus was reversed on alternate presentations; this latter was to minimize electrode polarization. There was a marked decrease in response output over the first few trials and generally an asymptote was reached within about 10 presentations. Both Wozniak et ul. (1 967) and Pumphrey and Rawdon-Smith (1937) have shown that even at frequencies of cercal stimulation as high as 10 CPS there is no reduction in the cercal sensory input to the last abdominal ganglion. Thus the reduction we saw in output of the last abdominal connectives most likely represents a ganglionic process rather than a decrease in pre-ganglionic input. Although a given cercal input produced spikes in both connectives, the discharges in the connective ipsilateral to the cercal input was greater (see Fig. 14). Some preliminary observations also suggest that habituation to one cercal input may be dishabituated by stimulation of the contralateral cercal input and that interpolating extra ipsilateral stimuli may, as h m p h r e y and Rawdon-Smith observed (1 937), also produce dishabituation.
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Hughes (1 965) has studied habituation in the isolated abdominal nerve cord of the cockroach, Periplaneta americana, using a puff of air to an anal cercus as a stimulus. He noted that only if stimuli were repeated at intervals greater than five minutes was there no reduction in the response over time. He concluded that at least part of the change seen during habituation takes place at synapses of the last abdominal ganglion. C. DISCUSSION OF HABITUATION
One of the major problems in any discussion of habituation is the fact that response diminution can occur as a result of a number of conditions and it may occur by several different mechanisms. Horn (1 967), for example, in discussing neuronal mechanisms of habituation, supposes that the gradual waning of the response of neurones to a repeated stimulus is caused by a “self-generated depression of sensitivity at one or more places in the group”. He uses the term “self-generated depression of sensitivity” as a generic term to include “such phenomena as after-hyperpolarization, conduction block due to accumulation of potassium ions in the extracellular clefts surrounding an active fiber, synaptic depression which probably represents an imbalance between the mobilization and utilization of transmitter, in certain circumstances recurrent inhibition in prolonged primary afferent depolarization as found in the spinal cord and thought to be partly responsible for presynaptic inhibition”. This, of course, allows for a wide range of possible mechanisms to account for any waning of a response. In addition, a response may diminish over time as the result of deterioration of the system, accumulation of waste metabolites etc. Thus, it is not a simple matter to decide how to label any given response decrease over time and to determine which, either singly or in combination, of the above mechanisms accounts for the waning response. Most of the criteria previously given to define habituation (Kandel and Spencer, 1968) would not necessarily exclude interpretations of a response decrease which may be due to changes at the primary receptor area itself or at the effector. While it is true that any response decrease that met all the criteria proposed (Kandel and Spencer, 1968) would unlikely be due solely to either primary receptor or effector changes, nevertheless, in many habituation studies only a few of these criteria are ever demonstrated, thus leaving open whether the decrease is in fact in the central processing
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area (probably central synaptic) of the nervous system or if its explanation lies more peripherally. One of the better criteria that one might employ to demonstrate convincingly that any response decrease was not due solely to receptor or effector changes, but in fact involves central processing of input would be to demonstrate dishabituation by the interpolation of an extra weaker stimulus out of the normal temporal order of the other stimuli (see Pumphrey and Rawdon-Smith, 1937). Horn ( 1967) has postulated a number of possible neural circuits to account for some of the phenomena of habituation, such as response decrement, recovery, dishabituation and stimulus generalization as well as time dependent effects. He cautions that several mechanisms may be used in the central nervous system to bring about the phenomena that have been termed habituation. (See the review by Kandel and Spencer (1968) for a further discussion of possible mechanisms such as IPSP recruitment and EPSP diminution, as possible bases for short-term and long-term .habituation.) Examination of the studies reported on habituation reveals that at present it is not at all obvious what similarities exist among the various studies as to either the sites at which the changes responsible for the response decrease occurred or the nature of the mechanisms responsible for decrement. D. INSTRUMENTAL LEARNING STUDIES
Studies of instrumental and Pavlovian learning in simpler systems offer the promise of understanding the cellular bases of these kinds of learning in higher systems. Some of the most intriguing studies of electrophysiological correlates of instrumental learning in simpler systems have been done in insects. Hoyle ( 1 9 6 9 , for example, has studied electrophysiological characteristics of learning in the locust. Recording from single muscle fibers in the anterior coxal-adductor muscle of the metathoracic leg of a locust, Hoyle found that there are spontaneous EPSP discharges at the rate of about 10 per s. He found that he could modify this rate by applying shocks to the tibia of the ipsilateral metathoracic leg. He was able to demonstrate that if following a spontaneous decrease in discharge rate of single fibers of the coxal-adductor muscle he shocked the tibia, he could over a period of time, by selectively timing the shocks, raise the average discharge frequency to the coxal-adductor muscle by as much as 300%. On the
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other hand, if he selectively timed the shocks to the tibia to follow increases in coxal-adductor discharge frequency, he could over a period of time cause a decrease in the discharge rate. Perhaps most significantly in demonstrating that this alteration in frequency was due to the special contingency between the response and the tibia1 shock and not just to amount of shock per se was the demonstration that if shocks were applied to the tibia independently of the discharge frequency in coxal-adductor muscles, there was in fact no progressive change in either direction in the average frequency of the coxal-adductor muscle discharge. Furthermore, following such “random” stimulation it was quite difficult to build a specific contingency into the system. These phenomena are shown in Fig. 16. Hoyle was able to show also that such changes did not depend on proprioceptive feedback as he could still get them even when proprioceptive feedback was removed. Thus, it would appear that instrumental learning in the true sense could occur in such a simplified system. It is most interesting that the observations Hoyle made on the effects of “random” stimulation on such systems were quite similar to those observed originally by Horridge and then by us on the difficulty of a “randomly” stimulated animal (R-animal) to later learn to perform as a P-animal. Such a preparation has advantages over the behavioral preparations in that: (1 ) feedback from the response can be controlled, monitored and/or eliminated; (2) the effector response itself can be eliminated and recordings made directly from the motor nerve and (3) other sensory input into the ganglia responsible for this response modification can be controlled and monitored directly so that the information that the system is processing can be known. Such a system invites close scrutiny for its potential use in understanding macromolecular changes that may underlie such learning. Luco and Aranda (Aranda and Luco, 1969; Luco and Aranda, 1964a, b, 1966) in a series of intriguing papers dealing with different kinds of instrumental learning (postural learning as well as avoidance learning) have shown both changes in synaptic latency in the metathoracic ganglion of the cockroach, Bluttu orientalis, as well as in the spontaneous and evoked discharges of the fifth leg nerve of this ganglion following a learning procedure.
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Fig. 16A. “Learning”. “Results of an experiment in which a shock was given to the right metathoracic anterior coxal adductor muscle of the locust S. greguria, each time its mean frequency over a 10 s period fell below a prescribed arbitrary level (“demand” level), indicated by the broken line. Moments of stimulation are indicated by arrows. The dots show the mean, frequency over 10 s periods whilst the bars show the mean frequency over 100 s periods to show the general trend more clearly. After a time the demand level was raised. Note that after a few stimuli have been received the mean frequency rises.” (From Hoyle, 1965.) V
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Fig. 16B. “Stimuli not correlated with output. Stimulus times (indicated by A) were taken from a successful experiment of the kind given in Fig. 16A. They were thus not correlated with output for the test animal and no maintained increase in frequency resulted. Subsequent stimuli selectively timed to follow rises in mean frequency (indicated by V) failed to depress the background and stimuli timed to follow falls in mean frequency (A) caused only a small rise in the background frequency.” (From Hoyle, 1965.)
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Specifically, they have shown that following new postural learning there is an increased synaptic efficacy as measured by decreased synaptic delay from the time a pulse is delivered to the subesophageal connective until it is measured in the fifth leg nerve (Luco and Aranda, 1964a).* (The contribution of leg amputation to the synaptic changes observed requires investigation.) They also studied shock avoidance learning using a Horridge procedure with separate P and R animals. They showed that if following such learning the thoracic ganglia are removed and recordings made from the fifth leg nerve to P and R legs, there is a much higher rate of spontaneous discharge in the P fifth nerve than in the R for at least a half hour after training, and furthermore, there is no difference in the spontaneous discharges of the fifth leg nerve on the contralateral side of P and R (Aranda and Luco, 1969) (Fig. 17 upper). In addition, they find the threshold for eliciting the discharge in this nerve from stimulating an anterior connective is much lower in the P fifth nerve than in either the contralateral or the R fifth nerve (Fig. 17 lower). It is of interest that both the difference in spontaneous and evoked electrical activity in P and R nerves disappear within about 30 min. Their behavioral work on retraining the P leg showed a saving up to 30 min. The apparent independence of the two sides of the ganglion (as measured by electrical activity) in the P preparation emphasizes again the probable value of a single ganglion for molecular and histological studies of learning in which one side receives input from the P leg and the mirror image side from the R leg. As previously mentioned, our behavioral studies of such a preparation show that P and R legs in the same preparation develop different behaviors during training depending on the relationship of leg position to shock occurrence (cf. Fig. 10). It would appear from these studies of Luco and Aranda that there may be specific synaptic changes in the metathoracic ganglion (related to diminished threshold and latencies) that are closely associated with, if not at the basis of, the instrumental learning seen in their studies. Such ganglionic systems invite close analysis, particularly of a histochemical and electron microscopic nature for changes that may be at the basis of the behavioral changes. Perhaps if
* Normally a cockroach balances himself on his meso and meta legs while using a foreleg to manipulate the antennae through the maxillae for cleaning. If both forelegs are removed he must often learn a new posture to balance himself with one meso and two meta legs and learn to use one meso leg to assist in cleaning the antennae.
Fig. 17. Upper. “Spontaneous activity in the metathoracic ganglion of a learned P preparation, recorded from the fifth nerve o f A: the untrained and B: the trained sides. 2, 5 and 10 sweep speeds in mseclcm. Calibrations: 1 om and 500 pV.” (From Aranda and Luco. 1969.)
Fig. 17. Lower. “Evoked responses in the metathoracic ganglion of a learned P preparation. Stimulation applied to the thoracic connectives. Recording from the fifth nerve of A: the untrained and B: the trained side. Number and calibrations as in Figure 17 Upper.” (From Aranda and Luco, 1969.) AIP-8
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differences of a macromolecular nature were seen in the metathoracic ganglia of P and R animals after avoidance learning, it might be possible to specifically inhibit the formation of such changes or to obliterate these changes by various drugs and observe whether by doing so new learning was prevented from occurring or previously established learning was eliminated respectively. It is of interest that both Luco and Horridge have observed synaptic changes in the metathoracic ganglion associated with training. Luco found a diminished synaptic delay, whereas Horridge found an increase in synaptic delay (although Horridge was careful to point out that he was not sure whether this delay had any significance at all for the avoidance learning he was studying). Kandel and Spencer’s review (1968) gives a more complete discussion of possible synaptic changes associated with learning. A most intriguing approach to electrophysiological investigations of instrumental learning is that provided by the studies of Kandel (1967) on modification of the parabolic burster cell rhythm of Aplysia. He has been able to show that an elementary monosynaptic IPSP on this cell produced by stimulation of an identified interneuron and made to occur either just before burst onset or during the interburst interval led to increases or decreases, respectively, in the rate of bursting. Occasionally such modifications in bursting rhythm lasted up to 30 min (Fig. 18). It is of particular interest that the alteration in bursting rhythm occurred gradually rather than immediately with alteration in contingency and furthermore that the effects lasted for a while after the contingency ceased. It would be particularly interesting to know whether or not (apropos our previous discussions of P and R learning) a randomly presented reinforcing stimulus in the interburst interval would make it more difficult at a later time to produce a specific contingency between burst onset and a reinforcing stimulus. It has often been suggested that central inhibition may play an important role in learning (particularly in mammals). The use of an identifiable inhibitory neuron input as a reinforcing stimulus deserves further work in this context. E. CLASSICAL CONDITIONING STUDIES
The use of a classical conditioning procedure offers several advantages for studying the molecular bases of learning and memory
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Fig. 18. “Analogs of Type I1 conditioning in Aplysia. Contingency-specific effects in identifiable cell (L3). The reinforcing stimulus is indicated by arrows. With two exceptions, a 3 and 2.5 minute interruption before lines 6 and 10 respectively, these are continuous data with sections at the ends of some lines omitted in order to line up the first burst in each line. This permits comparison of burst onset intervals before, during, and after contingent stimulation. The time from the first spike of the first burst in line 1 to the vertical line indicates the control burst onset interval. A: Line 1 is the last part of the control period. ‘Ihe same reinforcing stimulus (a weak 6 s train of 1 s duration) is first applied at the beginning of the burst (lines 2, 3, and 4), and later during the silent period (lines 7 and 8). Note ,opposite effects produced by the two types of contingencies.” (From Kandel, 1967.)
storage. Unlike instrumental learning, it involves the use of specifiable and therefore monitorable inputs, one of which initiates by reflex the response to be studied. Because of the latter it is possible to know in advance at least a portion of the pathway that will be involved in learning. It would seem in many respects to be a
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simpler and more “mechanical type” of learning than instrumental learning. Surprisingly little work has been done on insects using classical conditioning procedures. Several studies on possible Pavlovian conditioning in the isolated abdominal ganglion of the marine mollusc Aplysiu have been done, but at this time it is not clear as to just how these studies are to be interpreted (Kandel, 1967; Von Baumgarten and Djahanparwar, 1967). Horridge et ul. (1 965), making use of the interneurons in the optic lobe of the locust that were responsive to more than one sensory modality (notably auditory and visual), attempted unsuccessfully to pair such inputs and to see if an originally ineffective input could be made to produce a discharge in such interneurons when paired with an effective input. They were unsuccessful even though they used many temporal orders and several input combinations. The last abdominal ganglion of the cockroach would appear to be a system worthy of careful investigation for possible Pavlovian learning. It is a large ganglion and easily accessible. Its major anatomical pathways and much about its pharmacology are known (Boistel, 1968; Pumphrey and Rawdon-Smith, 1937). In addition, it has bilaterally symmetrical inputs through the cercal nerves that are primarily mechano-receptive. By use of puffs of air a relatively pure mechano-sensory input can be obtained and monitored from the cercal nerves. Such a system would seem to be unusually well-suited for investigations of electron-microscopic and histo-chemical changes that may underlie both habituation and Pavlovian conditioning. F. SOME NEWER ELECTROPHYSIOLOGICAL AND ANALYTICAL APPROACHES TO LEARNING AND MEMORY
One of the major reasons for going to phylogenetically simpler systems, which have been further simplified surgically, for investigations of the macromolecular bases of learning and memory storage is the reduction in complexity of the interaction of the elements, particularly feedback loops. It would be desirable to know and to separate out, for example, the relative contributions that primary sensory input, motor output, efferent gating and afferent feedback played in determining the final learning achieved (Fig. 19). Figure 19 attempts to schematize ways of experimentally separating out such contributions to learning and memory storage. If one were to record from the motor nerve directly, for example, and sever this distally from the effector (b), it should be possible to
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Fig. 19. Conceptual preparations for studying learning phenomena. a: “Usual” learning system of intact organism; it may also serve as a model for part of an organism where sensory and motor units are intact. (REC. = Receptor; INT. = integrator (ganglion or brain); AFF. = afferent; EFF. = efferent. EFF. = effector). b: Bypassing of effector and removal of afferent feedback (proprioceptive) from system. c : By passing receptor (and perhaps even primary afferent neurone) and removal of efferent gating or control of primary input to system. d: Bypassing receptor and effector thus eliminating from system studied any efferent gating of sensory input or proprioceptive feedback from effector response. (Eisenstein, unpublished.)
eliminate from our learning situation the role of afferent feedback on our system. Correspondingly, if one stimulates primary sensory nerve input directly, bypassing the primary receptor (c), it should be possible in principle to separate out the effects of efferent gating on the system. By stimulating primary sensory input directly and recording the motor nerve output (d), it should be possible to remove both sensory feedback and efferent gating. Such approaches as shown in Fig. 19 should make it possible to ascertain for any given system at any phylogenetic level what relative contributions these factors make to learning and memory storage. In principle they allow us to ask as we ascend the phylogenetic scale whether or not and in what ways, phenomena such as feedback and gating are involved in lqarning and memory storage. One might expect, for example, that at lqwer phylogenetic levels there is relatively little difference between a ‘Pven system when we stimulate the primary sensory nerve directly aqd record from the motor nerve (d) compared to the same system when we stimulate through its receptor and record its effector ou‘tput (a) (assuming that there is relatively little if any contribution from feedback and gating processes on learning in this system). However, as we ascend the phylogenetic scale we may well expect to
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see a marked difference in certain systems when we have eliminated its receptor and its effector responses from our consideration. Another advantage of such an approach as shown in Fig. 19 is that it allows us to do comparative studies of the relative integrative capacities with respect to learning and memory storage of different nervous systems at different phylogenetic levels. In addition it becomes possible to do comparative studies of the relative integrative abilities of parts of the nervous system, one with respect to another, within the same animal. One of the problems in comparative studies of learning and memory storage has been in assessing the relative contributions to any given results, of differences in the apparatus used as well as in sensory and effector capabilities of different animals. So, for example, it has been difficult to meaningfully compare the learning of a maze by an octopus with bar pressing learning in a rat-where both effector and sensory systems are so different and the manipulations which they perform are so different. How does one assess or determine the specific contribution, to any obtained results, which is due to integrative capabilities of the nervous system per se from these other considerations? Using such a schematic approach as I have described above, one can, for example, monitor nervous output from a motor nerve and put into the system direct electrical stimulation of sensory nerves and monitor what gets in. It now becomes possible to compare nervous systems directly. All inputs and outputs are measured in the same units, namely, some parameter of nerve discharges. It is now possible to directly relate input and output. It becomes possible to ask, for example, how a thoracic ganglion of a cockroach and a crayfish compare in their ability to demonstrate Pavlovian or instrumental learning. One can put in known sensory inputs, monitor motor outputs and ask about rates of learning, retention, stimulus patterns (both spatial and temporal) that are discriminible, generalization; in fact, all the questions that are normally asked about learning, memory and perception in any system-except that now we have the ability to know exactly what is coming into the system, what is going out and t o talk about them in the same units. It then also becomes possible to correlate meaningfully the neuroanatomy of these systems with their learning and memory abilities. With such simplified and pruned down systems it becomes possible to answer the question as to how any biological system can encode in
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some three-dimensional structural way (as measured by histochemical, electron microscopic and other molecular techniques) various temporal sequences of events that form the basis of instrumental and Pavlovian learning procedures. Once such mechanisms in any simple system have been uncovered, it then becomes an empirical question as to what relationship, if any, the changes found in such a system have with normal learning in that system in the intact animal and what relationship, if any, such changes have to the bases of learning in other more complicated systems. If the bases of such temporal encoding can be established in any system, then we have a lever with which t o look for specific changes in that same system in the intact animal and in other animals and to find, as we well may, that in some cases the results obtained from the pruned down system fit what happens in the intact animal and in other cases it does not. V. MOLECULAR APPROACHES TO LEARNING AND MEMORY STORAGE A. INTRODUCTION
The approaches that may be utilized for getting at the chemical bases of learning and memory storage are varied. They include such techniques as: (1 ) injecting pharmacological agents of various kinds in animals and observing their influences on learning and memory storage; (2) extraction of brain tissue after learning and comparing its various chemical contents to that existing in such a brain before learning; (3) extracting brain components from trained animals and injecting them in various places in untrained animals and observing effects on learning in the recipient animals; (4) injecting radio-labelled isotopes in untrained animals and observing uptake in various parts of the brain after learning and (5) using autoradiographic techniques in conjunction with electron microscopy to correlate macromolecular changes with various anatomical sites within and between nerve cells before and after a learning procedure. Perhaps the most commonly used approaches at this time involve the
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use of radioisotopes and the use of various drugs (principally antibiotics whose action on specific metabolic sites within the system are known). The methodological problems in assigning “causal roles” to any molecular changes accompanying behavioral changes are formidable. For example, in observing chemical changes in a system (such as increased protein formation or increased RNA turnover) after a learning experiment, one has the problem of separating out changes due to activity of the organism per se, sensory feedback into the system as a result of effector activity, the influence of efferent gating on the input, from those chemical changes which in fact are encoding or storing the learned behavior being investigated (Eisenstein, 1968b) Dingman and Sporn (1 964) have, among others, suggested certain criteria by which a given molecular change could be established as being the basis of one class of behavior, i.e. learning and memory storage. They “suggest that the following criteria must be satisfied in order to demonstrate that a given molecule, set of molecules, structure, or set of structures is indeed a permanent memory trace: (i) It must undergo a change of state in response to the experience to be remembered. (ii) The altered state must persist as long as the memory can be demonstrated. (iii) Specific destruction of the altered state must result in permanent loss of the memory ” Point (iii) has been satisfied often using ablation techniques. Point (ii) has not been shown in any system and demonstration of point (i) is complicated by the considerations discussed above. B. ASSESSING THE EFFECTS OF DRUGS ON LEARNING AND MEMORY IN THE COCKROACH
While there has been a great deal of work on the pharmacology of the central nervous system of the insect and particularly on the last abdominal ganglion of the cockroach, there has been very little work on the pharmacology of learning and memory storage in insects. In view of the unexpected recent results demonstrating similarity in the shapes of the retention curves of shock avoidance learning in the headless cockroach and the white rat (Fig. 5 ) it would seem reasonable as a first approximation to suggest that the processes underlying retention in two such widely separated species may be similar. In view of the great amount of pharmacological work that has been done on the learning process in mammals it would seem
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reasonable to pursue similar work on a relatively simpler system such as the ventral nerve cord of the cockroach, where the chances of specifying mechanisms are greater. We had, in fact, several years ago examined the effects of sodium pentobarbitol and strychnine sulphate on shock avoidance behavior in an isolated prothoracic insect ganglion, since these substances had been shown to have markedly opposite effects on learning and retention in the mammal (McGaugh and Petrinovich, 1965; Petrinovich and Eisenstein, 1965). Results were quite preliminary. They suggested that the leg withdrawal responses to shock in an isolated ganglion preparation were differentially effected by these drugs (Eisenstein, 1965). There are methodological pitfalls in using behavior to assess the influence of pharmacological agents on learning because it is often extremely difficult to sort out the effects of these agents on behavior that are due to threshold changes in sensory and motor neurones, neuromuscular and muscular effects from effects on the learning process. Because of this I undertook a methodological analysis to consider the conditions under which a change in behavior may be considered to represent learning in a system and particularly on what interpretation is to be placed on behavioral changes produced by pharmacological agents (Eisenstein, 1968b). Although the analysis mainly concerned assessing shock avoidance learning in the isolated ganglion of the cockroach, it would also apply to other systems. In addition, the analyses also considered specific predictions which could be made about behavioral changes to be expected in a system such as an isolated cockroach ganglion, if, in fact, various pharmacological agents do influence learning and memory storage. Figure 20 is hypothetical and attempts t o show in somewhat idealized fashion several points about my thinking regarding P and R learning under the influence of pharmacological agents. Let us examine the saline control curves initially. The first point is that both P and R groups learn during training. P learns that leg extension initiates shock and flexion avoids it. R learns it may be shocked in any number of different leg positions. What position it is shocked in during training, of course, is determined by P. These different kinds of P and R learning manifest themselves during training by P keeping its leg progressively more flexed and by R, perhaps after flexing to the initial shocks, keeping its leg extended for longer periods as it has no control over the shock during training. During a test period P avoids the shock more rapidly than the former R leg and R takes
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Minutes From Start of Training
Minutes From Start of Testing
Fig. 20. Hypothetical curves predicting the P and R changes to be seen during training and testing in preparations receiving injections of saline (saline control SC), substances which facilitate (F) and substances which inhibit (I) learning See text for discussion of these curves. (From Eisenstein, I968b.)
progressively more shocks over the first few minutes of testing before it too approaches the P level. The second point is that if both P and R learn during training then any drug which facilitated this learning should cause a larger P-R difference during training. Correspondingly, any drug which interfered with this learning should cause a smaller P-R difference. The third point is that any drug which facilitated learning during training should cause a larger P-R difference during testing by causing the drug P (i.e. the P group receiving a drug injection) to improve more rapidly than a saline control P (a P group receiving a saline injection). This would be expected since the former learned better during training that leg flexion avoids shocks. On the other hand, the drug R should more slowly approach the drug P than should a saline R approach the saline P level since there was facilitation of drug R’s learning that any number of leg positions it adopted during training (when P made contact) could lead to shock. Therefore, it would be more difficult for this R-animal to extinguish competing responses and relearn to perform as a P-animal during testing. The last point is that any drug which interfered with P and R learning during training should produce P and R curves during a test
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period that do not differ much from each other. Ideally, of course, one would like to use drugs and concentrations which were effective during training but which did not interfere with the ability to perform during testing. However, it should be noted that one of the advantages of using yoked P and R preparations is that nonassociative factors which affect motor performance should operate the same way on P and R. Thus, factors which make the legs more or less active should, in principle, affect the level but not the difference between P and R members during the test period. Hence, if the difference between P and R members is greater during a test period in a drug group relative to the difference in a saline control, because the drug P takes fewer shocks than the saline P and the drug R takes more shocks than the saline R (as hypothesized in Fig. 20), then clearly the effect of the drug is not on performance alone. The logic behind these hypothetical curves is that any drug which acts during training to produce a larger (or smaller) difference between P and R members during testing relative to a saline control, especially when this difference is due to the curves of P and R members moving in opposite directions relative to their saline control counterparts, must be effecting some aspect of the associative process regardless of any other effect it may be having on non-associative processes. Thus, in Fig. 20, both P and R get the drug and it is the difference between their behaviors relative to saline P and R controls that makes it possible to pull out associative from non-associative effects of a drug. Again I would like to emphasize that obtaining differences such as those shown in Fig. 20 makes it possible, on a behavioral level, to infer associative effects on a more molecular (neural and biochemical) level. Furthermore, making use of yoked P and R preparations makes it easier for biochemical analyses to separate possible non-associative activity effects (shock, movement, hormones etc.) from associative ones. (As a first approximation one might assume that biochemical changes common to P and R represent that treatment which was common to both whereas biochemical changes which were different in P and R would be related to differences in P and R behaviors. See Roberts (1 966) for a discussion of the problem of biochemically separating changes due to activity per se from associative changes.) Lack of a P-R difference with a drug condition relative to a saline control condition does not allow one to state the drug had no effect on an associative process. Like most pegative results, the factors responsible for the lack of difference are unknown. However, once the molecular substrates of an associative P-R difference are
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established, one may use these as markers to see what other behavioral changes (e.g. no P-R difference) also may occur with some of, or variants of, the molecular changes underlying the associative P-R behavioral difference. Figure 21 shows another possible procedure for assessing the effects of drugs on learning, by reversing which leg is P and which is R during training and then continuing the training. The logic underlying what is to be expected follows that discussed for Fig. 20. Recently Brown and Noble ( 1 967, 1968) attempted to study the effects of cycloheximide on learning in the isolated prothoracic ganglion of the cockroach. They had two groups of Panimals, one
TRAINING *-------9 cQ------
.'
Minutes From Start of Troining
P-R REVERSAL TRAINING
-
P R I er *--SC H k---N F 0.---o
Minutes From Start of Reversal Training
Fig. 21. Hypothetical curves predicting the P-R differences to be obtained during training and reversal in animals injected with saline (saline control SC), as well as substances facilitating (F) and inhibiting (I) learning. Reversing which leg is P (controlling the shock) and which R and then continuing the training is another possible way to separate out associative from non-associative changes in behavior. In both Figs 20 and 21, a former R leg is being asked to perform as a P leg, and whether a test procedure (Fig. 20) or a reversal procedure (Fig. 21) is used to measure R's ability, one would predict that the better the learning of R during the original training (i.e. that shock can occur in any leg position) the harder it should be for R to relearn to perform as P. Likewise the better P learned during training the harder it should be for it to relearn to perform as an R leg with reversal training. Arrows on the abscissa hypothesize the relative points in time at which the different injection groups wiU fust successfully show reversal of former P and R leg positions. (From Eisenstein, 1968b.)
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which had their prothoracic ganglia bathed in cycloheximide and another which had their ganglia bathed in saline. They then trained both groups to lift their legs to avoid shock and found, in fact, that the saline animals improved and reached a lower level of taking shocks much sooner than was reached by the cycloheximide groups. The slopes of improvement they show for both the cycloheximide and saline groups over the first five minutes of training are virtually the same, only the levels at which they start and finish differ (Brown and Noble, 1967, their Fig. 1). These results demonstrate that ganglia perfused with cycloheximide control a leg which is considerably more responsive to shock than is a leg attached to a ganglion perfused with saline. Brown and Noble (1 967) did not use P and R animals in training nor did they run a test. Rather as previously noted they used two P groups, one given cycloheximide and one given saline. Both were independently trained, using the following as response measures: (1) the amount of activity (i.e., the frequency with which the leg was extended and flexed, making and breaking the shock circuit) per minute (Fig. 1 of Brown and Noble, 1967) and (2) the number of shock pulses taken each time the leg was extended, making contact, before the leg was lifted, turning the shock off (Fig. 2 of Brown and Noble, 1967). Suppose the effect of cycloheximide was to increase the spontaneous activity level of both flexor and extensor peripheral motor neurons or to lower their threshold for discharge. The improvement would thus begin from a higher baseline, i.e., a more active leg in the cycloheximide group, relative to the saline group. Would not the differences they show in their Fig. 1 and 2 be seen? This hypothetical explanation assumes the effects seen may be due to peripheral motor neuron changes alone. The point here is not whether cycloheximide does or does not effect learning in this system nor whether the above peripheral neuron explanation is or is not correct, but that without some means of observing, for example, an emerging difference during training or a test period between two groups matched in all respects except for the relationship of leg position to shock during training, it is difficult to conclude that a behavioral change per se is indicative of learning in a system or, more to the point, that a difference between the behavior of their two groups is due t o learning. Recently, a confirmation of this hypothesis, i.e. that cycloheximide may produce its effect primarily
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by altering leg activity per se, which I suggested in 1968 (Eisenstein, 1968b) has been reported (Glassmann et al., 1970). More recently Brown and Noble (1968) examined the possible effect of cycloheximide on the inhibition of protein synthesis in the isolated cockroach ganglion and state that “as protein synthesis declines, so does learning”. However, because the possible effects of cycloheximide on free amino acid pool size were not evaluated (e.g. by use of pulse labelling, double labelling or turnover rate experiments) it is not possible to be certain at this time that the cycloheximide did in fact exert its effect on inhibition of protein synthesis in the cockroach ganglion. As Howson (personal communication) has suggested, an apparent decrease in protein synthesis could be explained by a decreased entry of the label into the free amino acid pool. In addition, the arguments and criticisms raised above for their first paper hold for this one as well. As in their previous work (Brown and Noble, 1967) the possibility is real that cycloheximide might be affecting sensory thresholds as well as the peripheral motor apparatus directly rather than CNS integration vis a vis learning. For example, Fig. 1 in their most recent paper (Brown and Noble, 1968) shows an approximately linear relationship between dosage of cycloheximide and length of time it takes before the leg is kept flexed for a long enough period t o meet their criterion of “learning”. The slopes of improvement over time that they show for the various dose groups (their Fig. 4) are similar (as might be expected if all dose groups were learning at the same rate that leg extension initiated shock and flexion avoided it). The main difference among the groups is in the degree of activity they show. As in their previous paper, they record two separate response measures-the number of shocks taken each time the leg extends before the leg withdraws and the number of times per minute the leg makes and breaks contact with the saline. An increase in either or both of these measures means more shocks to the leg and therefore a longer time before the leg meets their criterion of learning, viz. keeping the leg flexed. If thresholds are changed which either lead to increased leg activity or affect its ability to flex, it will take longer to reach criterion. The more active a leg is then, the longer it will take for it to reach the arbitrarily defined level of inactivity (i.e. keeping the leg flexed) which the authors define as learning. Changes in leg activity and/or responsiveness over time do not seem sufficient criteria for inferring an association (i.e. learning) in a
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system. There may well be differences in the starting base line (i.e. the amount of activity and/or number of shocks taken as a function of threshold and effector changes) which would determine how long it took those starting at a higher base line to reach a criterion. Thus, those dosage groups starting at a higher base line may actually have to learn more to reach the arbitrary criterion of maintained leg flexion used by these authors as a measure of learning. One criterion which I think meets the requirement for concluding that changes seen in P and R behavior are due to the relationship of leg position to shock occurrence and therefore represent associative changes, is that during training the difference between .P and R increases (Fig. 3A). Almost any developing difference found between P and R during training could be used for a criterion if the only variable known to differentiate P and R were the relationship of the response (leg position) to the shock stimulus. It is not yet possible to determine which behavioral criterion during training is the most accurate index of the underlying molecular associative changes responsible for the behavioral difference seen between P and former R members during testing. At the present time all that Brown and Noble have demonstrated is that cycloheximide treated ganglia generate considerably more activity in a shocked leg than do saline treated ganglia, but they have raised the interesting and exciting possibility, that cycloheximide also may effect the associative process. The latter possibility, I think, remains yet t o be proven. While one cannot be too rigid in what will or will not be accepted as operational behavioral criteria for the demonstration of learning, it does seem that one is on safer ground in this area at present if one uses the fundamental criterion met most often in studies where learning is generally agreed to occur, which is, in the context of the present discussion, that the development of a behavioral difference between P and R be shown to be due to the nature o f t h e temporal relationship between the response output (i.e. leg position) and the stimulus input (probably both proprioceptive and cutaneous input due to electric shock) (Eisenstein, 1967a, 1968b). C. SOME SPECULATIONS ON THE FUTURE O F THE CHEMICAL APPROACH
Structural changes on a molecular level probably will play a critical role in our understanding of the biological bases of learning and memory storage. Behavioral and physiological investigations are
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necessary to establish the boundary conditions, so to speak, of the problem. A real understanding of how the system performs learning and stores memory, it seems to me, requires understanding at a molecular and perhaps even submolecular level. * Developments in the use of quantitative cytochemical techniques in studies of single cells along with recent developments in the use of ESR (Hubbell and McConnell, 1968) and fluorescent dye techniques (Stryer, 1968; Tasaki et al., 1968) for studying conformational changes in membranes will probably prove extremely valuable tools in eventually understanding the cellular and molecular bases of learning and memory storage. Little can be said at this time about the molecular substrates of learning and memory storage. In view of the broad generality of biochemical pathways among the various animal species, it seems reasonable to suppose that chemical mechanisms isolated at one phylogenetic level for one kind of learning may be similar in many respects over a broad phylogenetic range. Perhaps one of the greatest barriers to an understanding of the molecular basis of learning and memory storage is that associated with finding an optimal system for investigating the problem. Most investigations have involved mammals and lower invertebrate systems which are enormously complex. It may well be that learning and memory storage are, like irritability, a fundamental characteristic of protoplasm and have only become specialized in nervous tissue when it evolved. If this is so, then the protozoa may be worthy of very careful consideration in the investigation of the chemical bases of learning and memory storage. They have no nervous system and therefore only changes associated with learning and memory storage would occur in one cell (Gelber, 1965).
VI. SUMMARY
The traditional concept of learning has been redefined in terms more amenable to molecular investigations in simpler systems. A survey was undertaken of behavioral, histological, electrophysiological and molecular studies of learning and memory storage in isolated insect ganglia, principally the cockroach. The preparations
* Recently Kerkut er al. (1970) showed that after training P animals had incorporated much more uridine into their metathoracic ganglion RNA than had R animals.
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studied ranged from intact central nervous systems to an isolated half-ganglion. In addition, a number of hypotheses about learning in such systems was discussed and specific experimental approaches suggested. Methodological problems in establishing the molecular bases of learning and memory were discussed and solutions suggested. A conceptual model for studying the comparative learning and retention abilities of different ganglia within a given nervous system and among nervous systems at different phylogenetic levels was proposed. For the behavioral studies of learning both activity and position response measures were employed. Learning was measured by these response changes during the training session as well as during the testing session. Retention of such learning can be shown 24 h later and may last considerably longer. Central nervous systems pruned down to an isolated ganglion showed clear-cut learning. It is dubious whether less than a ganglion is capable of supporting learning as measured by a developing P-R difference during training or the presence of such a difference during testing. Animals in which the ganglionic innervation of the legs has been removed are capable of highly stereotyped, relatively long term behavioral changes that are distinctly different from those seen in animals with ganglionic innervation of the legs. Recent histological studies have been concerned with mapping the distribution of motorneurons in the metathoracic ganglion of the cockroach and have shown marked bilateral symmetry on a cellular level. A single isolated ganglion can support shock avoidance learning when one leg serves as P controlling the onset and offset of shock and the contralateral leg serves as R. Thus there is the distinct possibility of examining bilaterally matched cells within a ganglion for determining at least part of the cellular bases of such learning and memory storage. Electrophysiological studies of habituation, instrumental learning and classical conditioning in isolated systems were discussed. It was suggested that isolated insect ganglia could be most useful in elaborating the molecular bases of such phenomena. Although there has been relatively little work on the chemistry and pharmacology of learning and memory storage in isolated insect ganglia, a methodological analysis of the problem involved in such studies was presented which should make such studies more meaningful in the future. Because of their discrete input-output channels, relatively few
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neurons, marked bilateral symmetry and ability to demonstrate learning and relatively long term memory storage, isolated insect ganglia would appear to be desirable miniature model systems for investigating the cellular bases of learning and memory storage. ACKNOWLEDGMENTS
The research work reported from my laboratory has been supported in its various stages by U.S. Public Health Service Grants NB 05827, MH 11012-01, NB 08369 and NSF GB 23371 t o the author. I would like to thank the following individuals for their critical and constructive comments during the preparation of this review: Drs. Elloise Kuntz, William Kilmer, Donald Maynard, Kenneth Friedman, William Coming, Peter Miller, John I. Johnson, M. Herz, Barnett Rosenberg, Jon Harris, Horton A. Johnson and Mr. David Howson. My graduate students, Mr. Thomas C. Hamilton and Mr. Ira Skurnick, provided many excellent criticisms and suggestions. In addition I would like to thank my former technician, Mrs. Lillian Harry, who performed many of the experiments discussed from my laboratory. I am grateful to Dr. W. Coming, Dr. J. T. Harris and Mr. David Howson for making available to me their unpublished data.
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Hams, J. T. (1971). Aspects of Learning and Memory in the cockroach. (A doctoral dissertation to be presented in partial fulfillment for the Ph.D. degree at Tufts University.) Harris, J. T. (Personal communication). Horn, G. (1967). Neuronal mechanisms of habituation. Nature, Lond. 215, (5 102), 707-7 1 1. Horridge, G . A. (1 962a). Learning of leg position by the central nerve cord in headless insects. Proc. R . SOC. 157, 33-52. Horridge, G. A. (1962b). Learning of leg position by headless insects. Nature, Lond. 193, 697-698. Horridge, G. A. (1965). The electrophysiological approach t o learning in isolatable ganglia. In “Learning and Associated Phenomena in Invertebrates” (W. H. Thorpe and D. Davenport, eds). Animal Beh. Supplement 1. Horridge, G. A., Scholes, J. H., Shaw, S. and Tunstall, J. (1965). Extracellular recordings from single neurons in the optic lobe and brain of the locust. In “The Physiology of the Insect Central Nervous System” (J. E. Treherne and J. W. L. Beament, eds.). Academic Press, London and New York. Howson, D. (Personal communication, Dept. Neurobiology, Cornell University, Ithaca, New York). Hoyle, G. (1965). Neurophysiological studies of “learning” in headless insects. In “The Physiology of the Insect Central Nervous System” (J. E. Treherne and J. W. L. Beament, eds.). Academic Press, London and New York. Hubbell, W. L. and McConnell, H. M. (1968). Spin-label studies of the excitable membranes of nerve and muscle. Roc. natn. Acad. Sci. U.S.A. 61, 12-16. Hughes, G. M. (1 965). Neuronal pathways in the insect central nervous system. In “The Physiology of the Insect Central Nervous System”. (J. E. Treherne and J. W. L. Beament, eds.). Academic Press, London and yew York. Jacklet, J. W. and Cohen, M. J. (1967). Synaptic connections between a transplanted insect ganglion and muscles of the host. Science, 156, 1638-1640. Johnson, H. and Eisenstein, E. M. (1966). Dense particles in axons and presynaptic boutons of the prothoracic ganglion of the cockroach. Am. Zool. 6, (4), (Abstract). Kamin, L. J. (1957). The retention of an incompletely learned avoidance response. J. Comp. Physiol. 50,457-460. Kandel, E. R. (1967). Cellular studies of learning. In “The Neurosciences: A Study Program” (G. C. Quarton, T. Melnechuk and F. 0. Schmitt, eds.). Rockefeller University Press, New York. Kandel, E. R. and Spencer, W. A. (1968). Cellular neurophysiological approaches in the study of learning. Physiol. Rev. 48,65-134. Kerkut, G. A., Oliver, G., Rick, J. T., Walker, R. J. (1970). Biochemical changes during learning in an insect ganglion. Nature, Lond. 227,722-723. Luco, J. V. and Aranda, L. C. (1964a). An electrical correlate t o the process of learning. Acta physiol. Lat. Amer. 14, (3), 274-288. Luco, J. V. and Aranda, L. C. (1964b). An electrical correlate to the process of learning. Experiments in Blatta orientalis. Nature, Lond. 201, 1330-1331. Luco, J. V. and Aranda, L. C. (1966). Reversibility of an electrical correlate t o the process of learning. Nature, Lond. 209,205-206.
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McGaugh, J. L. and Petrinovich, L. F. (1 965). Effects of drugs on learning and memory. In “International Review of Neurobiology” (C. C. Pfeiffer and J. R. Smythies, eds.). Vol. 8, pp. 139-191. Academic Press, New York and London. Miller, P. L. (1 970). Studies of learning in the insect central nervous system. In “Short Term Changes in Neural Activity and Behavior” (G. Horn and R. Hind, eds.), pp. 475-498. Cambridge University Press. Murphy, R. K. (1967). Proprioception and learning of leg position in cockroaches. (A paper in partial fulfillment of the requirements of the degree of Master of Arts at the University of Minnesota). Petrinovich, L. F. and Eisenstein, E. M. (1965). Drug facilitation of learning-problems of strain and species. Paper read at A.A.A.S., Berkeley, California. Pipa, R. L. and Cook, E. F. (-1959). Studies on the hexapod nervous system. 1. The peripheral distribution of the thoracic nerves of the adult cockroach, Periplaneta americana. Ann. Ent. SOC.A m . 52,695-710. Pringle, J. W. S. (1939). The motor mechanism of the insect leg. J. exp. Biol. 16, 220-231. Pritchatt, D. (1968). Avoidance of electric shock by the cockroach Periplaneta americana. Animal Beh. 16, 178-185. Pumphrey, R. J. and Rawdon-Smith, A. F. (1936). Synchronized action potentials in the cercal nerve of the cockroach (Periplaneta americana) in response to auditory stimuli. J. Physiol. Lond. 87,4p-5p. Pumphrey, R. J. and Rawdon-Smith, A. F. (1937). Synaptic transmission of nervous impulses through the last abdominal ganglion of the cockroach. Proc. R . SOC.122, 106-1 18. Roberts, E. (1 966). Models for correlative thinking about brain, behavior, and biochemistry. Brain Res. 2, 109-144. Stryer, L. (1968). Fluorescence spectroscopy of proteins. Science 162, (3853), 526-5 3 3. Tasaki, I., Watanabe, A., Sandlin, R. and Carnay, L. (1968). Changes in fluorescence, turbidity, and birefringence associated with nerve excitation. Proc. natn. Acad. Sci. U.S.A. 61, (3), 883-888. Thompson. R. F. (1 967). “Foundations of Physiological Psychology”, Harper and Row, New York. Von Baumgarten, R. and Djahanparwar, B. (1967). Time course of repetitive heterosynaptic Facilitation in Aplysia californica. Brain Res. 4, 295-297. Wiersma, C. A. G. and Adams, R. T. (1950). The influence of nerve impulse sequence on the contractions of different crustacean muscles. Physiol. comp. Oecol. 3,20-33. Wozniak, A., Alvarez, R., Wilson, E. and Ausit, E. G. (1967). Cercal potentials in the Periplaneta americana. Acta physiol. Lat. Amer. 17, 102-1 11.
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The Saliva of Hemiptera PETER W . MILES School of Natural Sciences. University of Zambia Introduction . . . . . . . . . . . . . . . . Methods of Investigation . . . . . . . . . . . . Modes of Feeding . . . . . . . . . . . . . . Stylet-Sheath Feeding . . . . . . . . . . . . . A. Sampling the Surface . . . . . . . . . . . B. Secretion of the Flange . . . . . . . . . . . C. Formation of the Sheath . . . . . . . . . . D. Discharge of Watery Saliva . . . . . . . . . . . E. Ingestion . . . . . . . . . . . . . . . F . Withdrawal of the Stylets . . . . . . . . . . V . Lacerate-and-Flush Feeding . . . . . . . . . . . VI . Feeding by Carnivores . . . . . . . . . . . . . VII . Chemical Composition and Function of the Saliva . . . . A. Sheath Material . . . . . . . . . . . . . B. Watery Saliva . . . . . . . . . . . . . . VIII . Phytopathogenicity . . . . . . . . . . . . . . IX . Salivary Glands and Ducts . . . . . . . . . . . . A . Aphidoidea . . . . . . . . . . . . . . B . Jassomorpha . . . . . . . . . . . . . . C. Fulguromorpha . . . . . . . . . . . . . D. Other Auchenorrhyncha . . . . . . . . . . E. Heteroptera . . . . . . . . . . . . . . X. Origins of the Saliva . . . . . . . . . . . . . . A. Functions of the Accessory Gland . . . . . . . B . Functions of the Principal Gland . . . . . . . C. Sources of Oxidases . . . . . . . . . . . . D. Sources in the Homoptera . . . . . . . . . . E . Salivary Carbohydrate and Lipid . . . . . . . . XI . The Saliva as a Vehicle for Pathogens . . . . . . . . XI1. Evolution of Salivary Function in the Hemiptera: a Summary XI11. A Survey of Problems . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . I. I1. 111. IV .
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I . INTRODUCTION
Salivary function is especially interesting in Hemiptera because of the effects the saliva has on the living and surviving organisms on 183
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which many of these insects feed. Some of the exclusively phytophagous suborder Homoptera cause “phytotoxaemias” including galls in their food plants; some Homoptera and a few phytophagous Heteroptera transmit diseases to plants in their saliva and some of the blood-sucking Heteroptera similarly transmit diseases to their hosts. Even when Hemiptera transmit diseases apparently by contamination of their mouthparts, they do not function as mere “flying pins”; there is evidence that the saliva can modify the effectiveness of transmission. The saliva of Hemiptera is by no means a simple secretion: in addition to the usual salivary functions of moistening food and mixing it with hydrolytic enzymes before ingestion, the saliva of phytophagous species plays an important physiochemical role during the mechanical penetration of plant tissues by the piercing and sucking mouthparts and, in accomplishing this task, the saliva may vary in its chemical composition and physical consistency from one moment to the next. Moreover, deposits of solidifying components of the saliva of many species persist in the food plants, modifying the long term effects of feeding by the insects. At the same time, other salivary components dissolve solid materials during gustatory exploration of substrates and it seems highly probable that the saliva thus acts as a carrier of gustatory stimuli to sensilla remote from the “functional mouth” at the tip of the stylet-like mouthparts. Finally, there is evidence that the saliva may subserve an excretory function in some species, varying in composition in relation to the compositim of the haemolymph-indeed it would appear to provide the only means of excretion in those few, highly specialized Homoptera that lack an anus. In relation to the complex composition and varied functions of the saliva, the salivary glands of Hemiptera are themselves complex and in the suborder Homoptera may present a seemingly bewildering array of cell types that appear to secrete different products and to undergo individual cycles of secretory activity. Identification of the products of individual types of cells with externally recognizable salivary functions is difficult in the Homoptera, in which the main secretory parts of the salivary apparatus are without a distinct lumen; but in the Heteroptera the salivary glands take the form of sac-like lobes, and accumulations of the salivary secretions can be identified and even collected from the salivary glands of the larger species. The history of investigations of salivary functions in Hemiptera is largely one of independent studies: on the one hand of economically
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significant Homoptera, in which research on the saliva is beset with problems connected with the small size of the insect; and on the other of the larger Heteroptera which are easier to deal with but have evoked less interest because of their lesser economic importance. Only recently has a measure of similarity in salivary functions in the two taxa been recognized and explored. It is the intention of this article to bring together the various types of investigation on salivary functions in the Homoptera and Heteroptera, and to suggest profitable lines of future investigation based on analogous functions in different taxonomic groups. 11. METHODS OF INVESTIGATION
The effects of the feeding of phytophagous Hemiptera were first investigated by histological study of plants on which these insects had fed. Histochemical analyses were made of the tissues surrounding feeding punctures and physiological inferences drawn as long ago as Busgen (1 89 1); although the usefulness of such interpretations was limited by the uncertainty of whether observable effects were caused by the insects directly or were due to reactions on the part of the plant (Petri, 1909). A refinement in histological studies was the examination of tissues on which the insects had fed for a known length of time and in which the insects’ stylets remained in situ. This was achieved by rapid killing of the insects with drops of very hot wax (Smith, 1920), electric shocks or by snipping or shaving off the stylet bundle while the insect was still feeding (Mittler, 1957). Major advances in the determination of the nature of the salivary secretions of Hemiptera and of their possible roles during feeding became possible when it was found that the insects could be induced to feed on transparent media such as agar gels or solutions enclosed by membranes. McLean and Kinsey (1 967) brilliantly exploited the ability of aphids to probe through parafilm by passing a small electric current through the aphid and substrate and correlating changes in the current with observable feeding activities involving salivation and ingestion. By this means, McLean and Kinsey (1 968) were then able to determine the sequence of salivation and ingestion in opaque, natural substrates. Refinements and details of the method have been published by McLean and Weigt (1 968): electrical contact is made with the aphid by cementing a gold wire of 15.0 p thickness to the dorsum with a quick drying silver conducting paint. An example of the circuitry is outlined in Fig. 1. Patterns of change in the
. . Fig. 1. Block and schematic diagram of the various devices and components used in a system to record pea aphid salivation and ingestion. (From McLean and We&, 1968.) Rl-IOK; R2-150-500K; R3-99M; R4-47K; R5-16; R6-1M; R7-1M; R8-470K; R9-8; R10-1M; Rll-16; D1 to D4-1N1614; M-high impedance A-C voltmeter; T-Triad F-16X filament transformer; S1A,B-DPDT toggle switch; S2A,B-DPDT toggle switch.
Fig. 2. Strip chart recordings showing sequences of feeding activity by the pea aphid. To be read from right to left: base line at the bottom of chart Omv. S-salivation waveform associated with penetration of tissues by the stylets and the secretion of a stylet sheath; X-waveform associated with penetration of phloem sieve tube; t-probably a period of brief ingestion between unblocking of food canal by discharge of saliva; Y-waveform of unknown significance; I-prolonged ingestion. (From McLean and Kinsey, 1967.)
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conductance of the system can be recorded on a cathode ray oscilloscope, through a loud-speaker or on a chart recorder or any combination; although the chart recorder is probably the most useful as a means of recording data permanently (Fig. 2). A similar system has been used by Smith and Friend (1971) to record the feeding of the blood-sucking bug Rhodnius prolixus. These authors inserted a platinum electrode through the cuticle and fixed it in place with beeswax and colophony (2: 1). The insects were permitted to feed through a rubber membrane on artificial diets containing 0.15 M NaCl with or without M ATP sodium salt. Flows in the liquid were observed by adding washed frog erythrocytes. The authors used DC current rather than AC and added the refinement of television monitoring of the activity of the mouthparts (Fig. 3). Saliva has been obtained for chemical analysis by a number of means. Salivary deposits have been collected from solid surfaces or recovered from inert gels or membranes (Edwards, 1961; Miles, 1959b, 1965). Saliva has also been collected directly from the mouthparts (Anders, 196 1 ; Miles, 1967b; Strong, 1970), sometimes after the insects have been stimulated to salivate by placing them in a humid environment or by injecting them with solutions containing 10% pilocarpine (Miles and Slowiak, 1970). Saliva has also been collected by allowing the insects to probe inert absorbent materials. Feir and Beck (1961) collected the saliva of the milkweed bug (Lygaeidae) by allowing the insect to probe through the testa of a milkweed seed into powdered cellulose. Schaller ( 1968a) collected
0
P 47, recwder
Oscilloscope
recorder
Monitor
Fig. 3. System for simultaneously recording and displaying optical and electrical data from a feeding insect. (From Smith and Friend, 1971).
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the saliva of several hundred aphids at a time by placing them in successive batches on the undersurface of a piece of damp filter paper, illuminated from above with yellow-green light. The aphids were first deprived of food and water so that they no longer produced honeydew as a contaminant. The saliva was eventually eluted from the paper for analysis. Adams and McAllan (1958) analysed saliva for one particular enzyme by enclosing the insects directly over a spot of the substrate on a strip of filter paper. A membrane was interposed between the insects and substrate and the exploratory probes of the insects through e.g. parafilm caused their saliva to be directly applied to the substrate. Subsequent chromatography of the spot then revealed whether any enzymic hydrolysis of the substrate had occurred. In the larger Heteroptera particularly, recourse has been made to analysis of enzymic activity of the whole glands or of the contents of particular lobes (Miles, 1967a; Hori, 1968b). Chromatographic separation of components of the secretions of the glands can be conveniently carried out by using the whole glands themselves as the “spot” at the origin of the chromatogram (Miles, 1967b). Radioisotopes have been used both as a means of determining the extent of salivary deposition in substrates (Fig. 4)and as a method of tracing metabolites into the saliva and thus identifying salivary components. Kloft et al. (1968) reviewed the methods used for labelling the saliva of aphids by incorporating radioisotopes, 32P in particular, into host plants or “sachets” of artificial diet enclosed in parafilm. Onion leaves which had their cut stems placed in a solution containing one or two mCi/ml Na2 32P04became radioactive within three or more hours and aphids or Thysanoptera placed on the labelled leaves acquired sufficient radioactivity over 24 h to make their saliva traceable by autoradiography when the insects were transferred to non-radioactive hosts (Kloft and Ehrhardt 1959a, b; Kloft, 1960). Hennig (1 963) similarly placed the cut stalks of Vicia faba leaves in solution containing 16pCi/ml for 5 to 20 h to label the leaves in preparation for transfer of the label to Aphis fabae Scop. Injection of from 0.5 to 10 pCi of radioisotope-labelled metabolites directly into the haemolymph of Heteroptera has been used to trace the transport of materials from the haemolymph into the saliva and to demonstrate metabolic pathways leading to the occurrence of individual compounds or classes of compounds in the saliva (Miles, 1967b, 1969a). It became apparent that any compound injected into the haemolymph was likely to appear in the saliva
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Fig. 4. Autoradiograph: Distriibytion of saliva labelled with '*P 'discharged by Myzus ascolonicus in leaf of violet. (From Kloft, 1960).
within a few minutes unless it was first rapidly metabolized in the haemolymph; but by injecting a labelled metabolite already known to occur in the haemolymph, it was possible to demonstrate the normal presence in the saliva of those compounds of which the labelled metabolite was a precursor.
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111. MODES OF FEEDING
I. Scratch-and-suck Feeding The Hemiptera, according to Goodchild (1 966), probably arose from insects that scratched the surfaces of growing plants, sucking out the cell contents (Fig. 5). Such an architypal scratch-and-suck mode of feeding is practised by the Thysanoptera, and probably also by the Tingidae within the Heteroptera. Little is known about the feeding of the tingids, however, except that these small insects feed on cells immediately below the epidermis, for the most part causing insignificant lesions. They are specialized Hemiptera nevertheless, and if their mode of feeding should indeed prove primitive, it is likely that it arose as a simplification of a more complex mode such as the stylet-sheath feeding described below. Even during the scratch-and-suck feeding of Thysanoptera, radioisotopic labelling of the saliva indicates that some of it is deposited in the cells on which the insects have fed (Kloft and Ehrhardt, 1959b) and species of both Thysanoptera and Tingidae are known to cause galls. Discharge of saliva into food sources is characteristic of the Hemiptera and it is convenient to distinguish between at least four major modes of feeding among the Hemiptera based partly on food source and partly on the function of the saliva.
2. Stylet-sheath feeding All the Homoptera and the Heter0ptera:Pentatomorpha are able to discharge at least two different kinds of saliva; one that solidifies rapidly on ejection and another that always remains watery and that leaves only a trace of solid matter evaporation. During the normal course of feeding on growing plant tissues, the solidifying kind of saliva is ejected wherever the stylets penetrate and is moulded by the action of the stylets to form a tubular lining to the path taken by them, thus forming the so-called “stylet-sheath”. The watery saliva is also secreted before and during penetration of the substrate, as well as on withdrawal of the stylets and the functions of these secretions will be discussed in detail in Section VII.
3. Lacerate-and-gush feeding Some members of the Pentatomorpha, particularly the Lygaeidae and Pyrrhocoridae, feed on seeds. When they do so they secrete only a surface “flange” of sheath material, that is not continued as a stylet-sheath into the seed. Instead, the stylets and watery saliva are
Reduviidae Cimicidae etc. predatory forms
Hydrocorisae \
\
subaquatic / Corixidae algophagous
Miriabe Tingidae return to plant-feeding carnivorous Geocorinae Asopinae Coreoidea \ / \ Amphibicorisae Cimicomorpha Pen tatornoidea - -- surface Saldidae skating litoral Pen tatornorpha habit stylet-sheath feeding on sap, shbre lacerate-and-flush on seeds or prey litter \? / to secrete a stylzsheath ability abhity to secrete a cornplite stylet sheath lost: mostly carnivorous retained: phytophagous
\
I
/
I
/
Dipso corimorpha
Jassomorpha Cicadomorpha
/ Geocorisae
\? litter-inhabiting OmniVOrOUS forms, lacerate-and-flush feeding evolved
other Sternorrhyncha /
Malpighian tubules reduced ;
Fulguromorpha
/
I
\
posterior junctidn antehor junition of mid and hind gut of mid and hind gut stblet-sheath fehing Coleorrhyncha evolved; mesophyllfeeding, true Hemiptera
>
\ tubules lost
\
A phidoidea
I
scratch-and-suck pre-Hemipterans -THYSANOPTERA Fig. 5 . Evohtionary interrelationshipsbetween divisions of Hernipternbased on physiology of feeding. OVIodifi from Goodchild. 1966.)
Y
v, h)
THE SALIVA OF HEMIPTERA
193
used to lacerate and macerate a pocket of cells or the entire contents of the seed according to its size. The contents are finally flushed out with a copious flow of dilute saliva. Elsewhere among the Heteroptera, phytophagous forms are found within the Cimicomorpha: namely the Tingidae and many species of Miridae. These insects produce no stylet-sheath, yet they feed on the roots and shoots of growing plants. The tingids are mostly very small and probably feed on individual cells or small numbers of them near the epidermis, but the phytophagous mirids apparently use a lacerate-and-flush method of feeding on pockets of cells in a manner very much like that of the Pentatomorpha when they feed on seeds. Thus the derivation of lacerate-and-flush feeding on plant tissues by the Cimicomorpha is perhaps to be seen as a necessary consequence of their loss of ability to secrete a stylet-sheath.
4. Predation Many Heteroptera feed on small prey and they employ what is in effect lacerate-and-flush feeding. Indeed, some of the mirids among the Cimicomorpha are carnivorous as larvae and phytophagous as adults. In the Pentatomorpha too, cannibalism is practised and undoubtedly the seed-feeding forms employ much the same technique whether feeding on seeds or individuals of their own kind. The main difference between phytophagous lacerate-and-flush feeding and predation proper lies in the composition of the saliva; for the saliva of the predators causes rapid immobilization of their prey, while the phytophagous bugs can practise cannibalism only on incapacitated individuals or on eggs. Further, the true predators are not known to produce a stylet-sheath. 5. Blood-sucking Blood-sucking Hemiptera are found within the Cimicomorpha only. Despite the observation that the phytophagous Cimicomorpha secrete no sheath material, in at least one of the blood-sucking Reduvioidea, Rhodnius prolixus Stbl, traces of a solidifying secretion are deposited at the point on the surface of the skin about to be penetrated (Friend and Smith, 197 1). No stylet-sheath is formed, however. The mandibles force their way a short distance into the skin and the maxillae alone penetrate deeper, thrusting first one way and then another until a blood vessel is penetrated (Lavoipierre et al., 1959; Smith and Friend, 1971). Little or no histolysis of the tissues of the host occurs. Indeed, the success of feeding depends on avoidance of sensory reaction on the part of the host. Alp-9
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IV. STYLET-SHEATH FEEDING A . SAMPLING THE SURFACE
All phytophagous Hemiptera, Homoptera as well as Heteroptera, often touch a surface several times with the labium before settling down to feed. It has been suggested (Miles, 1959b) that during this initial period, gustatory stimuli are picked up by watery saliva that is ejected and almost immediately sucked back into contact with the gustatory receptors that form the epipharyngeal organ (Wensler and Filshie, 1969). Exploration of surfaces with the mouthparts by aphids may or may not be accompanied by actual penetration. Hennig ( 1963) observed that when the black bean aphid, Aphis fabae Scop. touched leaf surfaces several times with the labium, each touch could last between 5 and 60 s and that the epidermis was sometimes penetrated, sometimes not. Often sheath material was deposited to form an external flange (Fig. 6) and when the stylets penetrated the surface, more sheath material was sometimes secreted and formed a short canal through the surface. At other times, however, deposits of sheath material were almost imperceptible or non-existent.
I
200p d
Fig. 6. Flanges of sheath material. a: aphid probes into a leaf surface; a short sheath accompanies the probe into a mid-lamella. (After Hennig, 1963). b: aphid penetrating parafilm membrane and building a sheath in a solution-showing formation of the flange within the tip of the labium and the bead-like appearance of the sheath. (After Miles, 1968b). c: formation of flange by Rhodnius on rubber membrane. (After Friend and Smith, 1971). d: flange formed on testa of milkweed seed by Oncopeltus. (After Miles, 1967a). e: formation of flange external to labium by Dysdercus feeding on cotton seed. (After Saxena, 1963).
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Such observations make it important to distinguish between an actual penetration of a surface-a true probe-and a mere touching of the surface-a dab. Hennig ( 1963) was unable to obtain any evidence that during these dabs and probes, any radioactivity was taken up from plants labelled with 32P; on the other hand, his results indicate only that aphids had not taken up quantities of radioactivity large enough to be measured; it is also possible that the sucking back of watery saliva brought into very brief contact with the surface of a leaf plant labelled with 3? would not pick up the label at all. Miles (1 959a) observed that aphids with their stylets exposed and in contact with glass ejected and sucked back minute quantities of watery saliva quite distinct from the solidifying sheath material; while McLean and Kinsey ( 1964), during the electronic recording of feeding by aphids, also obtained evidence that some fluid was ejected and sucked back on impenetrable surfaces. Heteroptera will spend a considerable time dabbing surfaces that contain soluble food materials, while at the same time ejecting. and sucking back watery saliva (Miles, 1959b; Saxena, 1963; Bongers, 1969). B. SECRETION OF THE FLANGE
When Homoptera and Heteroptera : Pentatomorpha feed on plant tissues, typically a small amount of sheath material is first secreted on the surface and the stylets then push through the solidifying material and begin to penetrate the substrate. This external sheath material has been termed a “flange” (Nault and Gyrisco, 1966). Exceptions have been noted. Saxena (1963) stated that the pyrrhocorid Dysdercus koenigii ( F . ) produced a flange on the hard testa of a cotton seed or on the intact cuticle of stems and leaves, but no sheath material was secreted at the surface of slices of cottonseed kernel. Hori (1968a) claimed that when the pentatomid Eurydema rugosa Motschulsky fed on cabbage leaves, it did not always secrete a flange or any other sheath material when the stylets subsequently remained solely within the mesophyll; but the insect did produce both flange and sheath when the destination of the stylets was a phloem vessel. Hennig (1 963) indicated that short probes by Aphis fubae Scop. into epidermal cells were not necessarily accompanied by a flange or sheath. Saxena’s observation on Dysdercus can be interpreted as indicating that this insect secretes a sheath only when its stylets encounter a hard substrate during attempts at penetration. But there is no simple
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explanation for the observations of Hori and Hennig. They would seem to imply that, at the moment of penetration, the subsequent pattern of feeding is already determined-i.e. if no flange has been secreted, subsequent feeding will be on mesophyll cells (and not on vascular tissue) or only a short “exploratory” probe will be made (and the stylets will not penetrate further). Presumably such predetermination of stylet activity would have t o be due to the physiological state of the individual. One possibility is that subsequent feeding behaviour depends on the ability of the insect to produce sheath material when it first attempts penetration of the substrate. Nevertheless, McLean and Kinsey (1 969) found no loss of ability of an aphid to secrete sheath material with starvation and further investigation of this problem is clearly required. Despite the few reports to the contrary noted above, the usual course of events during feeding by Homoptera and Heteroptera: Pentatomorpha is the secretion of a drop of solidifying saliva on the surface at the point where penetration of the stylets will begin; and this now seems to be true even of a blood-sucking reduviid Rhodnius prolixus Stll (Friend and Smith, 1971) that until recently was thought unable to produce any secretion similar to the stylet sheath of the phytophagous bugs. The initial secretion of sheath material on to a surface results in a flange of more or less circular cross section surrounding the point of penetration. The flange extends either around the labium or into the labial groove (Fig. 6). C. FORMATION OF THE SHEATH
A flange is not necessarily continued as a stylet-sheath into the substrate below. When the seed-feeding lygaeids and pyrrhocorids penetrate a hard surface, the flange is continued only a short way beyond the surface (Miles, 1959a, 1967a; Saxena, 1963). When Aphis fubae probes the body of an epidermal cell, further sheath material is unlikely to be secreted; whereas if the stylets have struck an intercellular groove, secretion of a stylet sheath is more likely, whether or not the penetration continues further into the plant (Hennig, 1963). Mostly, however, when Homoptera and Heter0ptera:Pentatomorpha feed on the roots or shoots of plants, it is found that a flange of sheath material is secreted on the surface and is
THE SALIVA OF HEMIPTERA
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continuous with a stylet-sheath laid down wherever the tips of the stylets penetrate (Fig. 7). Precise details of the way in which a stylet-sheath is formed have been furnished by McLean and Kinsey (1965, 1968). The stylets characteristically move into tissues by a series of backward and forward movements. When the stylets are at the end of a backward sequence, a drop of sheath material is secreted; watery saliva is ejected into this, making it balloon out; the stylets then push forward through the material and pierce the end of it (McLean and Kinsey, 1968). At this stage, the substrate may be sampled by sucking. The stylets then move back again and sheath material is again secreted. In this way, each new drop of sheath material is secreted in contact with the last piece and becomes part of a coherent sheath. In a soft, uniform medium, the stylet-sheaths of aphids thus have a typically beaded structure (Fig. 6); but in materials of varied structure and texture, the sheath may flow into the space surrounding the stylets or be squeezed into a space so narrow that it almost disappears. When stylet-sheaths pass through solid materials (e.g. when an aphid’s stylets pass through unstretched parafilm) it would seem that the insect is sensitive to the backpressure caused by the medium and that less material is secreted. Even so, it is under these circumstances that some aphids begin to suck back some of the sheath material before it has completely solidified and it then remains as an indigestible deposit in the mid-gut. (Moericke and Mittler, 1965). Possibly the thinness of the sheath when it is secreted within narrow confines in relatively rigid structures accounts for some confusion on whether stylet sheaths are continuous within plant tissues or not (Miles, 1968b). D. DISCHARGE OF WATERY SALIVA
Some watery saliva is ejected by aphids as part of the process of formation of the sheath (McLean and Kinsey, 1965); and as the stylets push through each new drop of sheath material, this watery saliva is released. When the secretions of aphids have been labelled with 3?, diffusible components of the saliva can be traced for some distance beyond the stylet-sheath in, for instance, leaves (Fig. 4).In aphids and some other Hemiptera the diffusing secretion contains enzymes such as pectinase and cellulase (see Section VI1,B) that presumably aid penetration of the plant tissues. The phytophagous Hemiptera continually sample any substrate
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PETER W. MILES
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.-c
Fig. 7. The feeding of Aulacaspis tetalensis (Zhnt.), Coccidoidea, in sugar-cane stems. a-c: stylets and stylet-sheaths in cortical cells: . Williams, 1970.) c: pierced cell that has collapsed; d: barbed tip of stylet (scale = 5 0 ~ )(From
\o
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W. MILES
through which the stylets are passing by sucking back small amounts of liquid. Aphids periodically suck whatever fluids are present when the stylets have just emerged from the last drop of sheath material secreted; this has been indicated by the electronic methods of McLean and Kinsey (1967), the uptake of radioactivity from very heavily labelled plants (Garrett, personal communication) and from the sucking back of sheath material that has been unable to solidify when aphids probe unstretched parafilm (Moericke and Mittler, 1965). All Hemiptera would seem to react to an inability to suck fluids when they attempt to do so by alternately discharging small quantities of watery saliva and sucking back (Miles, 1959b; Saxena, 1963; McLean and Kinsey, 1967). In this way insoluble materials are brought into solution (Saxena, 1963) or viscous solutions diluted (Miles, 1959b). E. INGESTION
Once the stylets have penetrated a source of ingestible food (e.g. a phloem seive tube), no more saliva is ejected (Kloft, 1960) unless the food canal should subsequently become blocked. Rapid movements of the tips of the stylets then occur, and if this does not dislodge the
b
Dendrites
C
Fig. 8. Stylets of Myzus persicae. a: tip of left maxilla. b: cross section of maxillae. c: cross section of whole bundle. fc-food canal; md-mandible; mx-maxillae; sc-salivary canal. (After Forbes, 1969).
THE SALIVA OF HEMIPTERA
20 1
particle, a small amount of sheath material is secreted (McLean and Kinsey, 1965). This last technique is effective because the functional opening of the salivary canal lies a short distance behind the functional opening of the food canal (Fig. 8). McLean and Kinsey (1967) ascribe a characteristic sequence of change of electrical resistance between aphid and substrate (the “X-waveform” Fig. 2) to the accumulation of callose slime around the tips of the stylets when they have been inserted into phloem sieve tubes and the clearing of the slime by short bursts of salivation. Those insects that feed on mesophyll tissue by the stylet-sheath method eject watery saliva continually (Kloft, 1960). Presumably they eject saliva into each cell on which they feed and suck out the contents before the secretion of the sheath is continued and the stylets move on to another cell. F. WITHDRAWAL OF THE STYLETS
The watery saliva has immediate effects on plant cells into which it diffuses, causing increased respiration and streaming of the protoplasm (Kloft, 1960); effects very likely caused primarily by an increase in the permeability of the cell membranes. Hemiptera may withdraw their stylets through the stylet-sheath completely, or they may draw back the tips of the stylets for a short distance before the stylet bundle is thrust through the side of the sheath to produce a branch in the sheath. Whenever the stylets retreat, even during the final withdrawal of the stylets, the surrounding cells again show transient increases in respiration, as though once more affected by the emission of saliva (Kloft, 1960). It has in fact been shown by Kinsey and McLean (1967) that when aphids withdraw their stylets voluntarily from plants, the end of the stylet sheath is sealed and the central canal filled up with secretion. It seems likely that any effects on the plant tissues that occur during withdrawal of the stylets are due to the saliva emitted at the moment withdrawal begins, as it is improbable that mechanical stimulation of the plants’ cells during withdrawal of the stylets would be significant and Kinsey and McLean (1967) have demonstrated that once the end of the sheath is sealed by the insect, dyes will not diffuse from the central canal to the exterior of the sheath. They conclude therefore that all ingestion by aphids is through the open end of the sheath and not from materials diffusing through its walls; at the same time it follows that saliva would be unlikely to diffuse outwards through the walls of the sheath.
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These observations are a clear indication of the sealing properties of the sheath. Since the central canal of the sheath is moulded by the stylets themselves, the insect may be forced to fill the canal of the sheath with saliva during withdrawal of the stylets by the suction that would occur otherwise. What kind of saliva remains in the central canal of the sheath has not been determined with certainty. The photographic evidence produced by Kinsey and McLean (1 967) would seem to indicate that it is sheath material itself; although observations on lygaeids (Miles, 1959b) were that the central canal of the sheath is filled with watery saliva on voluntary withdrawal of the stylets. V. LACERATE-AND-FLUSH FEEDING
Lygaeids, pyrrhocorids and some pentatomids will feed either on seeds or on the growing tissues of plants. When feeding on phloem sap, the lygaeid Oncopeltus fusciutus (Dall.) secretes a complete stylet-sheath, but when feeding on seeds, it secretes a flange of sheath material which is continued as an abbreviated tubular structure within the seed (Miles, 1967a; Bongers, 1969), be it a milkweed seed on which the insect normally feeds or a peanut (Miles, 1959b). Saxena (1963) points out that Dysdercus secretes such a flange only when the surface covering the food material is non-porous and insoluble: the cut surface of a seed kernel does not induce the secretion of sheath material. Once the stylets of a seed-feeder are within the kernel, the feeding activity is very different from typical stylet-sheath feeding. The stylets are pushed first one way and then another for periods of up to two hours when Oncopeltus feeds on milkweed-seeds or Dysdercus on cotton seeds (Saxena, 1963). During this period, the whole contents of a small seed (Miles, 1969b) or a pocket of cells in a larger seed (Miles, 1959b; Saxena, 1963) will be rendered fluid and removed. How this is achieved may differ, however, with different species. Oncopeltus when feeding on a milkweed seed, reduces the solid contents to a thin juice which can be squirted out of the seed, the liquid being derived from the watery saliva. The insect finally sucks out this juice, leaving the testa practically empty. Bongers (1 969) calculated that an individual Oncopeltus injected some 1.14 mg of salivary solids into a milkweed seed during the process of digesting and flushing out its contents. How great a volume of saliva this
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represented was difficult to estimate, since Bongers found almost no solids at all in the watery saliva he was able to collect. This apparent anomaly is probably explicable in terms of the ability of Hemiptera to vary the dilution of the watery saliva they secrete (see Section VI1,B) Dysdercus appears to suck back its watery saliva immediately after it is discharged and thus significant quantities of watery saliva do not accumulate within the cotton seed kernels on which it feeds. External digestion of, for instance, starch grains too large to be sucked into the salivary food canal of the stylets can easily occur when Oncopeltus is feeding, but Saxena (1 963) concluded that this was impossible when Dysdercus fed and that disintegration of larger particles was due mainly to mechanical action aided, at the most, by physical solution in the watery saliva. Nevertheless, all seed feeders seem to require relatively large amounts of water to drink if they continue to feed exclusively on dry seeds and liquefaction of the contents of seeds, whether by physical solution and disruption or by enzymic action, requires the insect to secrete large amounts of watery saliva during feeding (Saxena, 1963; Bongers, 1969). The mirids and tingids of the Cimicomorpha secrete no sheath material in the sense of producing a coherent tubular structure; although early descriptions of feeding by mirids included accounts of “granular”, deeply staining material within histological sections through feeding punctures (Smith, 1926). In the light of recent reports of a secretion akin to sheath material being secreted by even blood-sucking reduviids (Friend and Smith, 197 l ) , the categorical statement that sheath material is not secreted by the Cimicomorpha (Miles, 1968b) must be revised. Mirids lacerate and flush out pockets of adjacent cells when they feed. They do not seem to be adapted to feed on dry seeds, and feed mostly on growing tissues and fruits. The general process of feeding is similar to that of lygaeids and pyrrhocorids, however, as indicated by the water-soaked patches of tissue that they produce, and the silvery appearance of the air pockets left behind when they have finished. VI. FEEDING BY CARNIVORES
Lacerate-and-flush feeding is clearly akin to feeding by predators; some species of mirids are carnivorous, as are some pentatomids and lygaeids and even the phytophagous Pentatomorpha are capable of
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PETER W. MILES
cannibalism and suck eggs. It is interesting to note that when Oncopeltus feeds on the eggs or other individuals of its own species, it first secretes a flange of sheath material on the surface; thus seeds and prey are treated alike. The seed feeders, however, require an immobile food source and will attack only disabled individuals, while the true carnivores must rapidly immobilize their prey. The difference in the feeding process between the two forms thus resides in the chemical content of the saliva. Predatory (as opposed to blood-sucking) reduviids inflict painful bites on man. Some species spit their saliva for distances up to 30 cm as a defensive mechanism. As Edwards (1 96 1) showed, this spitting is without effect on the normal invertebrate prey of the insect, for the saliva is harmless when applied topically to arthropods or even when ingested by them. The spitting is apparently “aimed” at the sensitive mucous membranes of the eyes, nose and mouth of potential vertebrate enemies. Once the saliva is injected into an arthropod, however, it is highly toxic. The reduviid Platymerus rhadamanthus Gaerst immobilizes a large Periplanetu americana within two t o five s by injecting about 10 mg of saliva (Edwards, 1961). The saliva of predators in the Hydrocorisae and in other Geocorisae similarly rapidly immobilizes the prey. The offensive function of spitting by Platymerus probably explains the concentrated nature of this secretion, which Edwards used as a source for analysis of the saliva. In sharp contrast, the saliva ejected by the phytophagous forms outside their food is very dilute indeed (Strong and Kruitwagen, 1968; Bongers, 1969). Edwards (1961) compared the saliva of Platymerus to snake venom: both contain hyaluronidase, proteinase and phospholipase, although the activity of the salivary phospholipase of the insect was somewhat weaker than the phospholipase of snake venom. The hyaluronidase acted as a spreading agent, reducing the viscosity of some body fluids and attacking the intercellular matrix, thus speeding tissue lysis by the other enzymes. There was particularly rapid disruption of neural function, mainly due to lysis of the outer phospholipid membranes of nerves. The insect did not, however, contain ATP-ase or serotin, which are commonly present in snake venom, but which are presumably specifically adapted to vertebrate prey. By comparison, the saliva of the insects that suck vertebrate blood seems to contain no venom-indeed the habit depends for its effectiveness on the absence of immediate reaction on the part of the host (Table I). Little is known of the composition of the saliva of the blood-suckers, however.
205
THE SALIVA OF HEMIPTERA
Table I Toxicity of Hemiptera salivary gland homogenates as indicated b y effect o n heart beat of Periplanetaa Donor species
Dilution factorb
Action
I. Predators Naucoris cimicoides Platymeris rhadamanthus R h in coris carm elita Reduvius personatus
40
Immediate cessation
40
Immediate cessation
11. Phytophagous bugs Pentatoma rufipes
1
Oncopeltus fasciatus
1 ,
Slight increase in rate: cessation after some minutes Slow decrease in amplitude
111. Blood-suckers
Rhodnius prolixus Triatoma protracta
i
10
No action
Data from Edwards (196 1). The unit is a pair of glands homogenized in 0.05 ml saline; larger numbers indicate relative increases in dilution. (I
VII. CHEMICAL COMPOSITION AND FUNCTION OF THE SALIVA A. SHEATH MATERIAL
The Homoptera and Heter0ptera:Pentatomorpha produce an oral secretion that originates from the salivary glands and that solidifies very rapidly after secretion. Such recent studies as have been done on this material, whether from aphids, pentatomids or lygaeids, indicate that it is mainly protein, but contains about 10% phospholipid and probably some conjugated carbohydrate (Miles, 1964b, 1967b). It is mainly hydrogen bonded, but is stabilized by disulphide linkages (Miles, 1967a, 1969a). Once formed it is impermeable (Kinsey and McLean, 1967). It is also inert, for when aphids ingest their own sheath material, they are unable to digest it (Moericke and Mittler, 1965, 1966). When first secreted, the precursors of the sheath material are admixed with soluble amino acids and these diffuse from
206
PETER W. MILES
the sheath during the second or less while it is solidifying (Miles, 1964b). Oxidizing enzymes are secreted with the sheath material, which in some species actually appears to be tanned by these enzymes (Miles, 1964b). The sheath can be formed in the absence of oxidizing enzymes however (Miles, 1967a), and these enzymes are probably also secreted by Hemiptera that secrete no sheath (Miles, 1964a). The precursors of the sheath material are secreted by specialized lobes of the salivary glands and are discharged more or less independently of the other products of the glands. The secretions of all parts of the glands are nevertheless freely miscible with one another and the sheath material shows no signs of beginning to solidify if for instance the contents of the glands are mixed together in vitro, as long as oxygen is rigorously excluded (Miles, 1959b, 1967a). Within the glands, the precursors of the sheath material are maintained in solution by reducing conditions, probably due to the presence of free sulphydryl groups (Miles, 1967a). The precursors are intimately mixed with amino acids that are presumed to prevent the formation of hydrogen bonds by maintaining a high dielectric constant (Miles, 1964b). Any other secretions of the salivary glands that are likely to be mixed with the sheath precursors before they are ejected are similarly reducing and contain free amino acids, although the analysis of freely soluble metabolites differs in different parts of the gland (Miles, 1967a, b). Once the precursors are secreted, diffusion of amino acids out of the mixture and the presence of free oxygen rapidly bring about solidification by hydrogen bonding and disulphide bonding respectively. The oxidizing enzymes present in the saliva undoubtedly assist in the oxidative part of the process of solidification, although they do not appear to be essential (Miles, 1967a). The work of Friend and Smith (1 97 1) indicates that the secretion of sheath material by Homoptera and Pentatomorpha is matched in Rhodnius prolixus, a blood-sucking bug within the Cimicomorpha which secretes a flange at the surfaces it penetrates; but nothing is yet known of its composition. The function of the stylet-sheath remains problematical, despite discussion by several authors (Mittler, 1954; Saxena, 1963; Miles, 1968b; Bongers, 1969). Mittler’s suggestion that it serves t o seal the stylets of Homoptera into cells (e.g. phloem sieve tubes), the contents of which are under turgor pressure, still seems sound when
THE SALIVA O F HEMIPTERA
207
applied to sap-sucking aphids, particularly since recent studies have confirmed that these insects require a diet under pressure for their optimum growth (Wearing, 1968). The claim by Hori (1 968a) that the pentatomid Eurydema secretes a sheath when feeding on phloem sap, but not when feeding on mesophyll cells, would seem to be evidence that in the Pentatomorpha also, the sheath is an adaptation to feeding on pressure systems. But the foregoing does not explain why no sheath is produced by Dysdercus when it feeds on phloem sap (Saxena, 1963) or why Homoptera that feed only on mesophyll tissue do produce a stylet-sheat h. Saxena suggested that the flange secreted on to surfaces by Dysdercus enabled the insect to attach the labium firmly to the substrate without the danger of the mouthparts slipping sideways while the stylets were forced into the substrate. This explanation is supported by the cement-like nature of the sheath material and its ability to adhere to waxy surfaces of plants by virtue of its phospholipid content (Miles, 1968b), but such an explanation ignores the complete stylet-sheath that most phytophagous bugs secrete within plant tissues. Miles (1968b) contrasted the relatively small lesions made by stylet-sheath feeders of the Pentatomorpha with the large lesions produced by lacerate-and-flush feeders of comparable size in the Miridae. He suggested that if the stylet-sheath is seen as maximizing the efficiency of feeding on individual cells by preventing losses into intercellular spaces, then, in contrast, lacerate-and-flush feeders probably need to scavenge the contents of ruptured cells with large quantities of dilute saliva. Possibly the functional significance of the stylet-sheath can properly be appreciated only in an evolutionary context. Goodchild (1966) discussed the evolution of the physiology of the digestive system of Hemiptera in relation to sources of food. Although he did not refer to the secretion of sheath material, his ideas are relevant to it: as already noted in Section 11, he would consider the primitive mode of feeding in the Hemiptera to be stylet-sheath feeding (that arose from scratch-and-suck feeding, presumably when the insects had developed the ability to secrete a solidifying saliva). Secondarily, the Heteroptera became omnivorous. Even among the Homoptera, the secretion of a stylet-sheath may not occur automatically whenever the stylets are used (Hennig, 1963) and presumably some of the ancestral Heteroptera lost the ability to secrete a stylet-sheath
208
PETER W. MILES
almost entirely when they became predators, using the stylets and saliva as a lacerate-and-flush system. Goodchild assumed the mirids to be evolved from such predatory ancestors. If so, they retained the lacerate-and-flush system perforce because unable to secrete a stylet-sheath; indeed Goodchild specifically compares the feeding of the bryocorine mirids with that of the predatory Heteroptera. At the same time, it would appear that predators such as the reduviids include species that retain some of the ancestral ability to secrete sheath material, as the recent discovery of the salivary flange secreted by Rhodnius prolixus has demonstrated (Friend and Smith, 197 1 ). Within this evolutionary interpretation, the Heter0ptera:Pentatomorpha must be seen as Heteroptera that have retained the stylet-sheath feeding habit, but that nevertheless display some of the possible intermediate kinds of feeding between stylet-sheath and lacerate-and-flush. Thus the seed-feeders use both and also perhaps so do the pentatomids such as Eurydema which according t o Hori (1968a), uses the stylet-sheath method when it sucks phloem sap and the equivalent of a lacerate-and-flush technique when feeding on mesophyll. According to Saxena’s descriptions, Dysdercus has almost lost the use of a stylet-sheath, but retains the flange alone-perhaps because of its value in steadying the stylets during the initial penetration of a surface which, if a cotton seed, may be particularly hard to penetrate. An evolutionary interpretation in this way allows for the retention of ancestral habits and for changes in function that would be difficult to envisage in a strictly functional analysis. B. WATERY SALIVA
Some of the known components of the non-gelling secretions (watery saliva) of the salivary glands are summarized in Table 11. The watery saliva is a vehicle for hydrolysing (“digestive”) enzymes. Many of the Heteroptera can be induced t o secrete a watery liquid from the free tips of the stylets while these are outside food substrates; e.g. by manipulation of the labium, especially after subjecting the insects to a humid atmosphere or after injecting them with pilocarpine (Miles and Slowiak, 1970) or by amputation of the antennae (Clarke and Wilde, 1970a). Nevertheless, there is doubt whether this experimentally induced secretion properly represents the watery saliva secreted within substrates. Strong and Kruitwagen
Table I1
-$
Some recent studies of the soluble contents of the salivary secretions of Hemiptera
I
0
Compounds
Insects studied
Features noted
References
1. Enzymes Amylase
Oncopeltus fasciatus (Dall) (Lygaeidae) Heteroptera (various families) Lygus disponsi Linnavouri (Miridae)
Cellulase Oligosaccharases
Pectinpolyglacturonase
Empoasca fabae (Harris) (Jassoidea) Aphididae Oncopeltus fasciatus Dolycoris bascarum L. (Pentatomidae) Lygus disponsi Empoasca fabae Aphididae Auchenorrhyncha Heteroptera Lygus hesperus Knight (Miridae)
Occurrence
Feir and Beck (1 96 1)
Origin in glands Activation by C1and NO3 Characteristics
Miles (1 967a) Hori (1969)
Variability in quantity Role in digestion Occurrence
Hori (1 970b) Hori (1 970c) Berlin and Hibbs (1 963)
Occurrence Heat stability Occurrence . Occurrence
Adams and Drew (1963a) Adams and Drew (1963b) Feir and Beck (1 96 1 ) Nuorteva and Laurema ( 1 96 1b)
Absence Occurrence Variable occurrence
Hori (1 970c) Berlin and Hibbs (1 963) Adams and McAllan (1958)
Absence Occurrence in Miridae only Role as spreader in phytotoxicity
Laurema and Nuorteva ( 1 96 1) Laurema and Nuorteva (1961)
Hori (1970a, d)
Strong (1 970)
N
0 \D
Table 11-cont.
Compounds
Insects studied
Features noted
References
1. Enzymes Hyaluronidase Proteinase
Platymeris rhadamanthus Gaerst. (Reduviidae) Platymeris rhadamanthus Oncopeltus fasciatus (Lygaeidae) Empoasca fabae (Jassoidea) Dolycoris baccarum L. (Pentatomidae) Lygus disponsi (Miridae) Aphididae
EsteraseILipase
Oncopeltus fasciatus (Lygaeidae)
Phospholipase
Platymeris rhadamanthus (Reduviidae) Pyrrhocoris apterus L. (Pyrrhocoridae) Pyrrhocoris apterus L. (Pyrrhocoridae) Hemiptera
Acid phosphatase Phosphorylase Phenolase/ peroxidase
Pentatomorpha (Pentatomidae and Lygaeidae)
Role as spreader in toxicity to prey Role in toxicity Occurrence
Edwards (1 96 1)
Occurrence
Berlin and Hibbs (1963)
Variability in quantity Variability in quantity
Nuorteva and Laurema (196 1b)
General properties Occurrence Occurrence
Hori (1970d) Schaller (1 968a) Feir and Beck (1 96 1)
Origins in glands Role as neurotoxin
Miles (1967a) Edwards (1 96 1)
Occurrence
Kloft (1 960)
Occurrence
Kloft (1960)
Presence in glands of phytophagous and predatory species
Miles (1 964a)
Occurrence
Edwards (1 96 1) Feir and Beck (1961)
Hori (1970b)
Miles (1964a) and Miles and Slowiak (1 970)
Oncopeltus fasciatus (Lygaeidae) Aphididae
Origin in glands
Miles (1 967a)
Occurrence
General
Discussion of role
Miles (1965) and Miles and Slowiak (1970) Miles (1 969b)
2. Metabolites Amino acids
Phenylalanine/ Tyrosine/DOPA Cysteine and derivatives
Aphididae
Occurrence and phytotoxicity
Kloft (1960); Anders (1961) and Schaller (1 968b)
Occurrence and transfer to saliva Role in experimental induction of galls
Miles (1964a, 1967a, b)
Pentatomorpha (Pentatomidae and Lygaeidae) Pentatomorpha (Pentatomidae and Lygaeidae) Aphids
Occurrence
Schaller (1968a)
Eumecopus punctiventris Stil (Pentat omidae)
Occurrence and m etab o h m
Miles ( 1 969a)
Miles (1964b, 1968a)
212
PETER W. MILES
(1968) have shown that the secretion discharged from the free tips of stylets of Lygus hesperus Knight outside plant tissue lacks detectable quantities of the enzymes the insects discharge within feeding punctures. The lygaeid Oncopeltus is known to secrete esterase into substrates (Feir and Beck, 1961) and the enzyme can be found in its salivary glands-yet Miles (1967a) was unable t o detect it in saliva collected directly from the tips of the stylets. It would thus seem that the watery saliva of phytophagous Hemiptera is of varying composition. When secreted within plants it contains hydrolysing enzymes, but it is subject t o varying degrees of dilution and when discharged outside plants may contain little but water. Studies based on the watery saliva collected from the free tips of the mouthparts are thus of limited use in determining the composition of the saliva; yet it is indeed difficult to collect the watery saliva secreted within plants. Saxena (1 963) managed to collect drops of saliva discharged by Dysdercus while it was feeding on thin slices of cotton seed kernel. The best attempts so far t o analyse watery saliva as actually discharged into plants have been made by allowing aphids t o probe into damp filter paper (Schaller, 1963); by caging Homoptera over filter paper covered with plastic membrane or parafilm (Adams and McAllan, 1958) or by allowing larger insects to probe through the surface of a normal food material into a pocket of powdered cellulose (Feir and Beck, 1961). The results of such experiments are still open to some doubt. One of the functions of the very dilute saliva discharged by Hemiptera is probably the bringing back of soluble materials to the gustatory sensilla of the epipharyngeal organ; thus in the absence of the gustatory stimuli normally associated with food-for instance in filter paper or powdered cellulose-the watery saliva discharged might still lack the normal concentration of salivary enzymes secreted into food. The closest approach to determining salivary enzymes under the natural conditions of their secretion is probably that of Adams and McAllan (1 958) who have caged insects over filter paper impregnated with the pure substrates for the enzyme. By determining the breakdown or otherwise of these substances chromatographically, they were the first t o demonstrate the secretion of pectin polygalacturonase by Hemiptera. Other studies of this enzyme are summarized in Table 111. Salivary polygalacturonases are assumed to aid the disruption of the midlamellae of the cell walls of plants rather
THE SALIVA OF HEMIPTERA
213
Table 111 Occurrence of pectinase in the saliva of Hemiptera ~
1. Present Aphididaea Aphis abbreviata Patch Dactynotus sp. Macrosiphoniella millefolii (DeGeer) Myzus persicae (Sulz) Rhopalosiphum rhois (Monell) Aphis fabae Scop. Aphis sedi Kltb. Aphis spireacola Patch Eriosoma americanum (Riley) Eriosoma lanigerum (Hausm.) Phorodon humili (Schrank) Aulacorthum solani Cinara spp. Periphyllus negundinis (Thomas) Pterocomma spp. Schiz olach n is pin i-radiata (Davidson) Thomasiniellula populicola (Thomas) Ceruraphis viburnicola (Gill)
In both apterae and alatae In both apterae and alatae In both apterae and alatae In both apterae and alatae In both apterae and alatae In apterae, not always in alatae In apterae, not always in alatae In apterae, not always in alatae In apterae, not always in alatae In apterae, not always in alatae In apterae, not always in alatae Known from apterae only Known from apterae only Known from apterae only Known from apterae only Known from apterae only Known from apterae only Known only from immature alatae
Auchenorrhyncha (Cicadellidae)" Dalbulus maidis (Del and Wohl)
Demonstrated in adults
Heteroptera (Miridae)b ~~~
~
Adelphocoris seticornis (F) Poeciloscytus unifasciatus (F) Miris dolabratus (L) Stenodema calcaratum (Fall.) Capsus ater (L) Lygus pratensis (L) Lygus hesperus KnightC
~
~~
In males and females In males and females In males and females In males and females Demonstrated in female only Demonstrated in female only In saliva discharged in plants, but not in saliva discharged outside plants
214
PETER W. MILES
Table 111-cont. Heteroptera (Lygaeidae)' Demonstrated in adults
Liocoris lineolaris (Beauv.)
2. Absent Aphididae:' Aphis cardui L. Ceruraphis eriophori (Wlk.) Melaphis rhois (Fitch) Myzus cerasi (F) Prociphilus tessellata (Fitch)
Psyllidae:' Unidentified spp. Auchenorrhyncha:b Various spp. in Fulguroidea (5), Cercopoidea (2) and Jassoidea (10). Heteroptera:b ~
Various spp. in Lygaeidae ( l ) , Coreidae (2) and Pentatomidae (4) a
In saliva secreted into filter paper (Adams and McAllan, 1958). In salivary glands (Laurema and Nuorteva, 1961). (Strong, 1970).
than to function primarily in the digestion of a major nutrient (Adams and McAllan, 1958; Strong, 1970). Many workers have contented themselves with analysis of whole gland homogenates or (from the larger Heteroptera) the secretion expressed from the sac-like lobes of the principal gland. The main generalizations that have emerged from such studies can be summarized as follows:
(1) The saliva of phytophagous Hemiptera most often contains amylase. Other carbohydrases are sometimes present, but Hori ( 1970c) states that Lygus disponsi Linnavouri secretes a salivary amylase and no enzymes that hydrolyse oligosaccharides; all further digestion of carbohydrates occurring in the midgut. (2) Those insects that suck phloem sap as their principal food
THE SALIVA OF HEMIPTERA
215
are said not t o have hydrolytic enzymes other than carbohydrases in their saliva, although among these enzymes may be counted the pectin polygalacturonases secreted mainly by aphids and mirids (Adams and McAllan, 1958; Laurema and Nuorteva, 1961). (3) Those insects that feed on mesophyll tissues or seeds however, are likely to have both proteinases and esterases (or lipase) in their saliva (Nuorteva, 1958; Feir and Beck, 196 I ). Other salivary enzymes have also been found. Kloft (1960) showed that the mesophyll-feeder Pyrrhocoris upterus L. secreted acid phosphatase and a phorphorylase among the other enzymes in its saliva (whereas these enzymes were not secreted by sap-sucking aphids). Miles (1968b) reported that the saliva of all the Hemiptera excepting those that suck vertebrate blood secrete a salivary polyphenol oxidase (PPO). The latter generalization is perhaps too sweeping, however, for the Jopeicidae in the Cimicomorpha are predatory bugs that apparently have no .detectable oxidase in the glands (Davis and Usinger, 1970). In addition t o PPO, Miles and Slowiak ( 1970) report that the insects also secrete peroxidase and despite the possible confusion of the two enzymes (van Loon, 1971), the discovery of insects that secrete the one without the other would seem to corroborate the finding. The function of the oxidases is discussed in the concluding section; so far no function for them has been convincingly demonstrated. The enzymic content of the saliva of any particular species cannot be assumed t o be constant. There is considerable evidence for the variation in content of proteinase especially. Nuorteva ( 1 956b) found that in the mirid Miris dolubrutus L. salivary proteinases occurred in the juveniles and in only female adults and Nuorteva and Laurema ( 196 1b) found that the amount of proteinase in the salivary glands of the pentatomid Dolycoris buccururn (L) increased when the amount of protein in the diet increased. Similarly Hori (1970b) found that not only did both amylase and proteinase content of the salivary glands of the mirid Lygus disponsi vary with the physiological state of the insect, but that the proteinase content was variable even between the two glands of a single individual and that there was no correlation between the amount of proteinase in the glands and the less variable amount of amylase present. Similarly, Adams and McAllan (1 958) found that whether detectable pectinase occurred in the saliva of aphids depended on the species of plant on which they were feeding, or whether they were apterous or alate; sexual differences were also noted in the latter (Table 111).
216
PETER W. MILES
The saliva of Hemiptera is noted for its content of amino acids and, as indicated in SectionVI1,A the presence of these may be essential in those species that secrete a stylet-sheath. In the grape phylloxera, Viteus vitifolii Fitch, Anders (1 96 1) and Schaller (1 960) have shown that especially large quantities of amino acids are secreted, no doubt because this insect is without an anus and must excrete via the salivary glands. A distinctive feature of the saliva of Viteus is that it contains an unusually high content of the basic amino acids asparagine and glutamine, which presumably represent the end points of nitrogenous excretion in the insect. In other species of aphid, however, there are only trace quantities of the dibasic amino acids, although aspartic acid and glutamic acid may be present in significant amounts (Table IV). Lately, the work of Miles (1967b) with radioisotopes has shown that any soluble metabolite introduced into the haemolymph will appear also in the watery saliva. Miles worked mainly with the watery saliva discharged from the free tips of the mouthparts and this presumably represented direct excretion of water from the haemolymph along with traces of any metabolites present in it. Nevertheless, analysis of the contents of the whole glands (Miles, 1967b, 1969a) suggests that some metabolites are selectively sequestered from the haemolymph by the salivary glands. Thus a large part of labelled cysteine injected into a pentatomid subsequently appeared in the salivary glands and secretions; Hagley (1 969) noted a disproportionately large amount of phenylalanine in the salivary glands of the cercopid Aeneolamia compared with the composition of the haemolymph. It is therefore safe to conclude that all the soluble metabolites that occur in the haemolymph, be they salts, amino acids, sugars or the precursors of lipids (e.g. glycerol), will occur in the salivary glands and will be secreted (or excreted) in the very dilute secretion that comes mainly from the accessory gland (see below); but some metabolites are specifically sequestered in the principal glands. Of particular interest in relation to phytopathogenicity is the possible occurrence of the plant hormone p-indolyl acetic acid (IAA) in the saliva. Duspiva (1954) and Miles (1968a) claimed to have demonstrated the in vitro production of IAA from tryptophan by homogenates of the salivary glands of an aphid and heteropteron respectively. Nuorteva ( 1962) claimed evidence for the transfer of IAA and gibberellic acid from diet to saliva of the auchenorrhychan Calligyponu pellucidu. On the other hand, IAA is a common
THE SALIVA OF HEMIPTERA
217
endpoint of indole metabolism in animals, and the recent work of Schaller (1 965, 1968a) has demonstrated conclusively that the saliva of some aphids contains IAA: whether in sufficient quantity to account for their phytopathogenicity will be discussed in the next section. A possible means of demonstrating the origin of a salivary secretion is provided if the pH of the secretion can be determined. Thus the pH of the sheath material is slightly on the acid side of neutral (Saxena, 1963; Miles, 1965, 1968b); and the pH of the watery saliva is alkaline, with pH values of up to 9 (Miles, 1965, 1968a). On the other hand, the contents of the principal salivary gland never have as high a pH as this, and thus the more strongly alkaline the watery saliva, the more it is likely to be the secretion of the accessory gland (see Section X) and the less it comes from the principal gland and thus contains the digestive enzymes and other compounds secreted by that gland. VIII. PHYTOPATHOGENICITY
Large numbers of some species of aphids or leafhoppers can abound on plants and apart from some stunting, or contamination of the foliage with honeydew, little effect on the growth pattern of the plant can be observed. Yet individual mirids of some species produce large lesions or such violent necrosis at the points where they feed that the fruit will absciss prematurely or grow with characteristic deformations (Carter, 1962). Although lacerate-and-flush feeding necessarily results in necrosis, the lesions produced by cocoa capsids on young stems may become surrounded by a zone of increased growth, such that in time the lesion becomes occluded (Carter, 1962). Stylet-sheath feeding is usually accompanied by less immediate damage, and is more likely to cause longer-term disturbances in growth. Colonies of the woolly aphis, Erisoma lunigerum (Hausm.) produce intumescences on the part of the stem or root on which they feed. The grape phylloxera, Viteus vitifolii Fitch causes similar swellings on roots; by depressing growth at the point where it feeds and stimulating it further away, this insect causes characteristic pocket galls on the vine leaves (Fig. 9). Similarly, many (but not all) sedentary psyllids and coccids produce galls. Among the scratch-and-suck feeders also, some tingids (and Thysanoptera) cause pocket galls in which they live colonially.
Table IV Analyses of aphid saliva for some metabolites Viteus Eriosoma vitifolii lanigerum
Serine Alanine Aspartic acid Glutamic acid Glycine “Underasparagine” Leucine Valine Asparagine Glutamine Arginine Lysine Histidine Cysteine/Cystine Tyrosine PAlanine Threonine a-Aminobutyric acid Methionine
++ ++ ++ + + + + f
+++
++ + +
Aphis pomi
+
+++
+
f
+++
i
-? -
f f
+
+
Sappaphis Myzus Hyalopterus Crypto- Aphis myz.us smnbuci mali cerasi pruni rib is Amino acids
Myzus ascalonicus
Megoura viciae
f f f f
?
++ f f f -
i i
-
-
f f
*3M %
Phenolics Naringenin Phloridzin? Quercitrin Quercetin Chlorogenic acid? Rutin? Hyperin? Robinetinaglycone? Protocatechuic acid? Ferrulic acid? pCumaric acid?
+ +
?
+ +
f
?
+
+ ++ + + f +
f -
f f
+ f +
+
-
+
f 4
-
?
?
-
-
? ?
f
Indole acetic acid
*
*
+
Proteinase
+
?
-
?
-
-
-
+
-
-
-
-
Results for some unidentified amino acids and phenolics not included: Number of ++ indicates strength of reaction, * indicates trace only, and ? an uncertain result. Results for Viteus vitifolii represent the pooled results for seven biotypes of the species. (From Schiiller, 1968a).
E
Bt: <
E
s
M
p
>
220
PETER W. MILES
a. Dreyfusia spp. Increase in cell protein but not cell growth (from Kloft, 1960).
b. Eriosma lanigerum Simple surface gall.
a
d. Wteus vitifolii At root tip of vine causes swelling surrounding feeding puncture (from Anders,1961).
e. Viteus vitifoii on foliage causes occluded gall and with daughters o calonial gall forms (adapted from AndersJ961).
c. Cocoa capsids Cause occluded necrosis on young shoots by stimulating peripheral growth.
a
Fig. 9. Formation of galls: a: “physiological gall”. b: surface gall; c: occluded necrosis; d: root gall; e: pocket gall on leaf.
Such localized effects of feeding must be differentiated from systemic stunting or deformation that are caused by viruses or by mycoplasmas (Raine and Forbes, 1969; Whitcomb and Davis, 1970). Localized effects, whether or not they take the form of necrotic lesions or galls, can usually be shown to be independent of any disease organism and to be due to the toxic effects of the saliva discharged by the insects into the plants during feeding. The identity of the salivary toxins responsible has elicited much interest. In recent work on the causal agents of galls, a major controversy has been whether phytopathogenicity resides in free amino acids present in the saliva of plant bugs (Anders, 1961 ; Kloft, 1960) or in the IAA that is either secreted in the saliva (Schaller, 1968a) or may be formed from tryptophan by oxidative deamination brought about by salivary oxidases (Miles, 1968a, b). Recently an explanation of the formation of lesions by mirids has been advanced as a separate hypothesis; namely that pectin polygalacturonase in the saliva causes a rapid spread of other salivary enzymes into the tissues of plants during the lacerate-and-flush type of feeding of these insects (Strong, 1970). Kloft (1 960) showed that the amino acids secreted by aphids in their saliva caused increases in the permeability of plant cells and
THE SALIVA O F HEMIPTERA
22 1
probably thereby increased respiration, transpiration and protoplasmic streaming. Insects that suck phloem sap release their saliva only while penetrating to the phloem or at the moment of withdrawal of the stylets and the pathological effects of an insect such as the onion aphid, Myzus ascalonicus Donc. are thus not pronounced. In contrast, the Dreyfusi spp. (Adelgidae) that multiply on silver fir, feed on the parenchyma and secrete saliva continually; the net result of their feeding is similarly progressive, resulting in the accumulation of proteins in the bark just under the points of feeding of the insects. Such an accumulation of nutrient materials without any obvious hypertrophy of the plant tissues Kloft called a “physiological gall” and ascribed it principally to a stimulation of protein synthesis brought about by the amino acids discharged by the insect in its saliva. Anders in a series of papers ( 1960a, by 196 1) described his analysis of the saliva of larvae of phylloxera that were producing galls on vines. As has been discussed in Section VI1,B the unusually high concentrations of amino acids that this species has in its saliva have been considered excretory in nature. Anders claimed t o have identified three amino acids in particularly high concentrations namely lysine, histidine and tryptophan, with significant quantities also of glutamic acid and valine. When germinated in solutions of these amino acids (but not others), the roots of grape seedlings produced subapical swellings that Anders compared to the subapical galls produced by phylloxera feeding just behind the root cap (Fig. 9d). On the other hand, similar results have been described when vine roots are grown in potassium phosphate solution (Miles, 1968a) and perhaps solutions of other compounds that affect the permeability of the cells in the meristematic subapical region of the rootlet would cause similar growth disturbances. Careful analysis of the saliva of phylloxera and several other species of aphids with varying degrees of phytopathogenicity led Schaller ( 1 968a) to conclude that in fact the amino acids named by Anders were not typical of the saliva of phylloxera, in which he found the principal amino acids to be glutamine, serine, alanine and aspartic acid. The saliva analysed by Schaller was collected from dampened filter paper and there is thus the problem of whether the saliva discharged in what were presumably exploratory probes was of the normal strength and composition secreted by the insects when they feed on plants.
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Nevertheless, that amino acids were found at all gives a good indication of those that feature most prominently in the salivary secretions of the various species. Schaller also found IAA and several phenolic compounds in the saliva of aphids. The average quantities of compounds collected from an individual aphid during the 6 to 8 h when it was permitted to probe the filter paper were up to a maximum of 2 x 10-Fg each of the major amino acids present in the saliva. Eriosomu Zunigerum secreted a similar quantity of IAA in its saliva and somewhat less IAA was retrieved from the saliva of Viteus vitifolii and of Myzus cerusi (Table IV). Schaller concluded that although the analysis of the saliva of aphids showed each to have a specific composition qualitatively and quantitatively, certain generalizations emerged. The greater the ability to cause hypertropy of tissues in their host plants, the greater the overall concentration of amino acids, the greater the concentration of IAA and the lower the concentration of phenolics. He concluded that IAA must work together with free amino acids to produce galls while the phenolic compounds, by producing quinones that reacted with amino acids and destroyed IAA, reduced the likelihood of gall formation. In a series of brilliant experiments, Schaller (1968b) tested his conclusions by introducing solutions that simulated the saliva of aphids into the petioles of developing vine leaves. To do this he filled hollow glass microneedles with the solution and allowed the needles to remain permanently in place while the leaf grew (Fig. 10). He found he could imitate the galls of phylloxera with remarkable felicity and the nearer the solution approached the actual composition of phylloxera saliva with respect to amino acids and IAA, the more “perfect” the gall produced. Strong ( 1970), meanwhile, has investigated the destructive lesions produced by Lygus hesperus Knight. He concluded that the saliva contained no IAA and lacked any physiological system by which IAA could be synthesized by the saliva released within plants. He ascribed the large lesions produced by individual bugs to the salivary pectin polygalacturonase which, by greatly increasing the effective spread of the digestive enzymes in the saliva during the lacerate-andflush feeding practised by this and other mirids, caused a much larger lesion than would be produced by a sap-sucking insect of comparable size. Presumably the effects of the feeding of the mirids that attack cocoa must involve more than the destructive processes in the feeding of Lygus, since Strong’s explanation does not dispose of the
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Fig. 10. Formation of “histoid” gall on petiole of vine leaf by a mixture of amino acids and IAA simulating the saliva of the grape phylloxera, V . vitifolii. (After Schiller, 1968b).
observation that hypertrophy of cells can occur at the edge of the lesions produced by the “cocoa capsids” (Carter, 1962). In fact, Scott (1 970) showed that the feeding both of L. hesperus and of L. elisus Van Duzee on either carrot seeds or beans, while depressing initial growth of the seedlings after germination, actually increased their eventual growth compared with plants grown from seeds on which the insects had not fed. A third approach to the phytotoxicity of the saliva of Hemiptera is the proposal that the salivary phenols and phenolases of Hemiptera directly interfere with the growth-controlling mechanisms of meristematic cells. As Gordon and Paleg (1 96 1) demonstrated in vitro, quinones produced by a system such as dihydroxy phenylalanine (DOPA) plus polyphenol oxidase (PPO) will transform tryptophan by oxidative deamination to IAA. On this basis, Miles (1969b) proposed that an increase in IAA content of cells surrounding the feeding punctures of Hemiptera would be likely. The PPO would seem to be provided in the saliva of all the phytophagous forms; phenylalanine is transformed in the salivary glands to DOPA and the tryptophan, although present in small amounts only, would
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nevertheless occur in the haemolymph and therefore saliva as the free metabolite. Miles thus considered all phytophagous Hemiptera as possessing the potential for forming galls on their food plants and he considered they would do so provided the precursors of the IAA-producing reaction were present in sufficient concentration and the insect injected enough of its saliva in a place where the plant’s cells were still sufficiently meristematic. As a demonstration of this hypothesis, he made a non-cecidogenic lygaeid temporarily produce galls on cotyledons of a sunflower seedling by implanting into the bug flakes of agar containing phenylalanine and tryptophan (Miles, 1968a). Scott (1970) similarly suggested that the potential of the saliva to form IAA was a likely explanation for the enhanced growth of plants grown from seeds that had been attacked by Lygus spp. A number of problems arise from the hypothesis that salivary phenols and phenolases cause increases in IAA concentrations in the tissues of plants. Phenolases have also been implicated in the breakdown of IAA (Pilet et al., 1970); Schaller (1 968a) correlated an increase in salivary phenolics with decreases in both IAA-content and phytopathogenicity of the saliva. Moreover, as Schaller ( 1968b) has pointed out, the total amounts of known metabolites injected by the insects into plant tissues are small compared with the amounts already present within the tissues themselves. Indeed the amino acids in the saliva of a sap-sucking aphid represent merely a small proportion of the amino acids obtained by the insect from the plant’s own phloem sieve tubes and the galls induced by phylloxera are much richer in IAA than the saliva injected into them by the insects. The question thus arises whether the observations so far made on possible gall-inducing substances have in fact been observations of secondary effects: the amino acids and IAA in the saliva of cecidogenic species being derived from the metabolites already present in rich supply in the galls, rather than the galls being due to the substances introduced in the saliva of the aphids. Nuorteva’s (1962) work on the transfer of dietary amino acids t o the saliva of Calligypona and the evidence that even enzymes are readily transported unaltered from haemolymph to the saliva of plant bugs (Miles and Slowiak, 1970) would add weight to such an interpretation. Schaller’s successful demonstration that the injection of amino acids and IAA does cause galls must tip the balance in favour of the explanation that cecidogenesis indeed lies in the presence of these
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compounds in the saliva of the insects, even though the apparent anomaly that they are already present in higher quantities in the plant remains. A definitive explanation of cecidogenesis is likely to be concerned with the precise location and movement of physiologically active substances, rather than overall concentrations. A sap-sucking insect in effect removes amino acids from sieve tubes and subsequently reinjects them elsewhere in the plant. The hypertrophy that occurs at the edges of the lesions of cocoa-capsids must be due either to a gradient in concentration of substances that cause necrosis at higher concentration and increased growth at lower or to differential rates of diffusion of necrosis-causing metabolites and growth-promoting ones. And it is possible to reconcile the in vitro production of IAA by a phenol-phenolase system, with demonstrations that the same system causes the destruction of IAA, by assuming that the end point of the reaction is dependent on a precise relation between the concentrations of the reactants. IX. SALIVARY GLANDS AND DUCTS
The salivary glands of Hemiptera are labial glands, lying mainly in the anterior region of the thorax, alongside and partly above the gut. Typically, there is on each side a principal gland and an accessory gland. The duct of the accessory gland unites with the duct of the principal gland near where the latter duct leaves the principal gland (Fig. 1 l), and the principal ducts unite to form a common salivary duct that discharges into the salivary pump. From the pump there is a short meatus leading to the salivary canal, the posterior of the canals formed by the opposition of the maxillae (Fig. 8). There is usually a high degree of uniformity of structure in the glands of members of the same family and a degree of likeness is also apparent within higher taxa. At the same time, considerable differences exist between the glands of such taxa as the Sternorrhyncha and Auchenorrhyncha in the Homoptera and between the superfamilies of the Auchenorrhyncha. In contrast, the glands of all the Heteroptera show a remarkable similarity of anatomical pattern, seemingly derived from the relatively simple pattern within the Coleorrhyncha, a small unspecialized group once regarded as heteropteran, but now usually placed in a separate division of the Homoptera.
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Fig. 1 1. Salivary glands of Hemiodoecus veitchi Hawker (Co1eorrhyncha:Peloridiidae). (From Prendergast, 1962). A nerve (not shown) also innervates the principal gland.
A. APHIDOIDEA
The salivary glands, although apparently simple, have been shown by Weidemann (1968, 1970) to be composed of a number of different kinds of cells (Fig. 12) that show very different cycles of activity and produce visibly different kinds of secretion (Moericke and Wohlfarth-Bottermann, 1960a). At least nine histological types of cell occur in the principal gland apart from the canal cells. Weidemann arranged them into four functional groups, at least three of which produce components of the saliva. Using the electron microscope, Moericke and Wohlfarth-Bottermann ( 1960b) have shown that the secretory products accumulate in vesicles that are extruded from the cell border, and the membrane surrounding the vesicle then breaks down. The principal salivary glands of Aphididae exhibit a partial bilateral division into two symmetrical halves. Down each half runs a stout walled central canal, formed intracellularly by the “canal cells” through which it runs. In the principal glands there is a region of at least seven kinds of “Main Cells”, and at its proximal end, the region of main cells fits into a region of “Cover Cells” rather as an acorn fits into its cup. Both main and cover cells are secretory and small canalulae ramify among the cover cells from the central canals.
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gland
b
Fig. 12. Salivary glands ofMyzus persicae. a: principal gland; left side showing cell types and numbers (on each side), right side showing nuclei; canal cells and their nuclei not shown; cross-hatching indicates histochemical grouping: A and F are rich in cysteine; B, C, and I in hyaluronic acid; D and E in carbohydrate. (After Weidemann; 1968, 1970). b: principal and accessory gland showing ducts. c: schematic diagram of the structure of the salivary ducts, showing the canal wall surrounded by intercellular spaces and the interdigitating processes of the canal cells which form the corona sinuosa (Faltenkranz). (Redrawn from Moericke and Wohlfarth-Bottermann, 1960b).
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According to Moericke and Wohlfarth-Bottermann ( 1 960b) the cells of the principal gland discharge vesicles containing their secretory products into the intercellular spaces that surround and indent the canal cells. The surface area of the cells of the central canal is increased by numerous radiating folds, forming the corona sinuosa (Moericke and Wohlfarth-Bottermann, 1 960b) and this is particularly well developed in the region of the cover cells. The secretion of the main cells appears to be relatively electron transparent, while in the region of the cover cells the contents of the canal become electron dense. It is not certain, however, whether this can be ascribed solely to the secretory products of the cover cells or to changes occurring to the products within the canal, possibly mediated by activity of the canal cells themselves. The accessory gland of Aphids is usually a small structure with a few cells producing a very electron transparent secretion. Its canal is continuous with numerous intracellular canalulae that ramify into the secretory cells-an arrangement consistent with the rapid discharge of a dilute secretion. In the phylloxera, Viteus vitifolii, as Rilling (1 966) has shown, the accessory gland is enormously increased in size and is much larger than the principal gland (Fig. 13). The cells of the accessory gland form a syncytium and appear to be greatly enlarged canal cells. Surrounding the intracellular accessory canal is a system of radial, laminar vacuoles and surrounding these again vesicular vacuoles that occupy most of the bulk of the cells. Rilling relates this development of the accessory gland to the gall-forming propensities of the insects, but it is more probable that the hypertrophy of the accessory gland is related primarily to its assumption of the main excretory role in the insects, which not only lack Malpighian tubes, in common with the other Aphidoidea, but also have no anus and must therefore excrete, if at all, through their salivary glands. Viteus show other interesting phenomena. The partial division of the principal glands of aphids into bilaterally symmetrical halves has apparently become complete in Viteus for the principal gland is now completely bilobed, although each lobe has a histologically similar structure. Each is composed of four “giant cells” and two “eosinophilous cells”, and a further group of cells that Rilling homologizes with cover cells, although they are arranged centrally in a “hilus”. The giant cells, the eosinophilous cells, and the cover cells all show secretory activity. In addition there are the presumably
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Fig. 13. Salivary glands of Viteus vitifolii (Phylloxeridae), semidiagrammatic. a: bilobed principal gland and multilobular (semisyncytial) accessory gland. b: one lobe of the principal gland, showing the four “giant” cells, the branched, double nuclei and the hilus cells. c: cross section through one lobe of the principal gland, principal duct and part of the accessory gland. I to 1V-giant cells; e-one of the two eosinophilous cells; hilus cells in c cross hatched. (Adapted from Rilling, 1966).
non-secreting canal cells. The secretory cells are typically binucleate, and the nuclei of the giant cells are greatly enlarged and branched structures. €3.
JASSOMORPHA
In the superfamilies Jassoidea and Cercopoidea, the various kinds of secretory cells of the principal salivary glands are grouped
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separately into distinct lobes or regions, while the accessory gland takes on a more or less elongated, vesicular appearance. In Empoasca fabae (Hausm.), the four separate lobes of the principal gland remain a solid mass of cells (Fig. 14a), although between the cells at the centre of the lobe there is an intercellular space, sometimes extending as indentations (“intracellular canniculae”) into the
Fig. 14. Salivary glands of Jassoidea, semidiagrammatic. a: Empoasca fabae (Harris) (Typhlocybidae). (After Berlin and Hibbs, 1963). b: Erythroneura limbatu Matsumura (Cicadellidae). (After Sogawa, 1965). c: Nephotetrix cincticeps Uhler (Deltocephalidae). (After Sogawa, 1965).
secretory cells (Fig. 15). This space communicates at the origin of the lobe with a chitin-lined duct. The accessory gland is typically elongated with at least one portion having a distinct lumen and looking similar in structure to a Malpighian tube. The glandular cells of the salivary apparatus are typically binucleate and the nuclei of the large secretory cells may be ramified (Berlin and Hibbs, 1963). In other Jassoidea, the cells in each lobe tend to remain separated
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Fig. 15. Lumen of glands. a: Xenophyes cuscus Bergroth (Coleorrhyncha), with canniculae only. (After Prendergast, 1962). b: Empwscu f a h e (Harris) (Jassoidea), with confluent intracellular vesicles. (After Berlin and Hibbs, 1963). c: Notonectu glaucu L. (Hydrocorisae), with true but poorly developed lumen approximating the condition in Empouscu. (After Baptist, 1941).
from one another (Fig. 14b) and in the principal glands of some families, Sogawa (1965) described up to seven kinds of secretory cells, arranged not into distinct lobes, but as rosettes of cells (Fig. 14c). He nevertheless distinguished two groups of these cells: the principal salivary duct branches just as it enters the gland into two “ducteoles”; about the end of the one (“posterior”) branch, four kinds of cells form a concentric rosette, while two other kinds of cells are arranged in radial layers along the “anterior” branch. The anterior ducteole remains short in some species, but becomes more elongate in others and may end in a terminal appendage formed from a cluster of small cells of yet another type. The accessory gland of the Jassoidea is typically elongated and may be differentiated into a tubular distal (“tail”) region and a more
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bulbous proximal (“head” or “cephalic”) region in which the lumen becomes less evident. Sogawa believes the bulbous region t o be secretory or resorptive, possibly increasing the analogy between the accessory gland and an excretory organ. Very little information is available about the glands of the Cercopoidea. Nuorteva ( 1956a) provided illustrations of some glands (Fig. 16), indicating that there occur in them compact groups of cells
Fig. 16. Salivary glands of Cercopidae. a: Aphrophora alni (Fall.) b: Philaenus spumarius (L.) (After Nuorteva, 1956a).
corresponding to Sogawa’s “anterior” lobe, and a “crown” of sometimes tubular acini corresponding to the rosettes of the posterior lobe of some Jassoidea. C . FULGUROMORPHA
The principal gland in the Delphacidae is composed of a large number of multicellular acini, each with its separate ducteole (Fig. 17). It differs from the gland of Empoasca (Fig. 14) chiefly in the larger number of acini and in the absence of a tubular accessory gland. Sogawa (1 965) describes nine kinds of acini, one of which he designates an accessory gland, although it is reniform and not always well separated from the other acini. Balasubramanian and Davies (1968) also distinguish nine commonly occurring acini in the family Delphacidae and a tenth type occurring in some species. Miles ( 1 964a) similarly described ten lobes in the glands of a flatid. Neither Balasubramanian and Davies nor Miles distinguished an accessory gland. Sogawa lists the numbers of cells in each type of acinus as
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d
I
loop
Fig. 17. Salivary gland of Laodelphax striatellus (Fu1guromorpha:Delphacidae). a to h-distinguishable acini of the principal gland; Ag-accessory gland. (After Sogawa, 1965).
varying between two and eight, while Balasubramanian and Davis list two kinds of acinus as unicellular. The latter authors point out that all cells are binucleate; a feature they appear to share with the Jassomorpha. The ducts in the Fulguromorpha are similar to those of the Cicadellidae in the Jassomorpha: intracellular spaces in the cells of the acini finally join up with ducteoles that have their own characteristic small cells with compact ovoid nuclei. The ducteoles join up with one another and eventually a common salivary duct is formed on each side of the body; these join to form a very short median duct just before this enters the salivary syringe. D.OTHERAUCHENORRHYNCHA
Very little work has been done on the other groups within the Homoptera. The Cicadomorpha are described by Nuorteva ( 1956a) as having bilobed principal glands and a tubular accessory gland, but this account was based on early studies and, as Balasubramanian and Davies (1 968) point out, many past generalizations may need careful revision as more information on the comparative morphology of the salivary glands of Homoptera begins to accumulate. The salivary glands of the small family Peloridiidae, now placed in the taxon Coleorrhyncha separate from the other Hornoptera, are of especial interest. These glands bear a superficial resemblance to the glands of many of the relatively unspeciaIized, carnivorous
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Heteroptera. Balasubramanian and Davies object that this similarity is more apparent than real, quoting as the major difference between the glands of the heteroptera and Homoptera the presence or absence of a lumen. The glands of the Peloridiidae have no distinct lumen and therefore are typically Homopteran , according to these authors. But the distinction is not necessarily valid, for the salivary glands of many of the Heteroptera Cryptocerata, as Baptist (1941) has shown, have no more lumen than those of the Cicadellidae (Fig. 15). E. HETEROPTERA
Most of the Heteroptera have principal salivary glands that are clearly differentiated into an anterior and a posterior lobe, each of which has a distinct lumen. In most, also, the accessory gland is vesicular. In the Cimicomorpha the accessory gland is a thin-walled bladder, whereas in the Pentatomorpha, the terminal vesicle is completely lost, but the cells at the distal end of the elongated accessory duct take on a swollen, glandular appearance. The nerve supply in the lygaeid Oncopeltus (Bronskill et al., 1958; Miles, 1960b) is two-fold, one nerve supplying the anterior lobe and the other, which travels alongside the principal salivary duct, supplying the posterior lobe and accessory gland and duct (Fig. 18). Although Baptist ( 1 941) illustrated only a single nerve supplying the gland of any one species of heteropteran, that nerve might variously appear to be the homologue of either of those described in Oncopeltus. It is therefore possible that the double nerve supply is typical throughout. According to Miles, this arrangement provides for the discharges either of the anterior and related lobes of the principal gland or of the posterior lobe and accessory gland, thus accounting for the independent discharge of the two kinds of saliva secreted by the Pentatomorpha. In the Cimicomorpha, blood-sucking forms tend to lose the division between the anterior and posterior lobes of the principal gland (Rhodnius) or lose the anterior lobe entirely as in Cimex. Whereas, in the Pentatomorpha, only the Pentatomidae retain the simple bilobed condition; in all other families, the glands are multilobed. Baptist (1941) claimed that such proliferation of lobes was due to subdivisions of the posterior lobe, but studies of innervation and of physico-chemical composition of the secretions could indicate that, on the contrary, the subdivisions are of the
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Fig. 18. Salivary glands of Heteroptera. a: a predator, Notonecta glauca L. (Hydrocorisae) b: a blood-sucker, Rhodnius prolixus Stal (Reduviidae). c: a blood-sucker, Cimex lectularius L. (Cimicidae). d: a phytophagous lacerateand-flush feeder, Lygus pratensis L. (Miridae). e: a stylet-sheath feeder, Pentatoma rufipes L. (Pentatomidae). f a stylet-sheath feeder, Oncopeltus fusciatus (Dall.) (Lygaeidae). Ag-accessory gland; anterior-anterior lobe; lateral-lateral lobe; n, ,na -nerves; posterior-posterior lobe. (a to e from Baptist, 1941; f from Miles, 1960b). All drawings approximately to scale.
anterior lobe and are concerned with the secretion of sheath material (Miles, 1967a). If, as Goodchild (1 966) has suggested, all Heteroptera are to be derived from phytophagous Hemiptera through omnivorous forms in which the number of lobes was reduced or primitively small as in the aquatic Heteroptera, then the return to a phytophagous condition in the Pentatomorpha has apparently required a more complex division of labour in the salivary secretions and a convergent elaboration of the salivary glands in the Homoptera on the one hand and Heteroptera : Pentatomorpha on the other.
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X. ORIGINS OF THE SALIVA
Tracing the origins of the salivary components within the salivary glands is easiest in the Heteroptera because the great majority have glands that are morphologically simple and each lobe of the gland has its own lumen in which the secretory products of the lobe collect. Little is known of the contents of the salivary glands of Heteroptera other than the Geocorisae, however. Work has been done mainly on the glands of Pentatomorpha; but amongst these, only the Pentatomidae retain a simple two-lobed principal gland and in the other families the problem of identifying the origins of any specific salivary secretion has proved more difficult because of secondary multiplication of the lobes.
A. FUNCTION OF THE ACCESSORY GLAND As a result of the work of Goodchild (1966) it can be accepted that, throughout the Hemiptera, the function of the accessory gland is the production of the copious flows of very dilute secretion that the insects employ when they “taste” surfaces with which the stylets come into contact, as a means of flushing out food sources or as a means of excreting excess water. In all Hemiptera, with only the possible exception of the Fulguromorpha, that part of the salivary apparatus identifiable as the accessory gland is either vesicular or tubular. When the accessory gland is a compact organ, it often has greatly enlarged intracellular vesicles or extensive canniculae; i.e. ways of increasing the surface area of the functional ducts. In the Fulguromorpha, Sogawa ( 1965) identified the lobe in which the duct had a distinctive “corona sinuosa” as the accessory gland. Where it has been specifically identified, the secretion of the accessory gland is alkaline (Miles, 1968b) and presumably the accessory gland thus functions as the first stage of a typical diuretic organ, producing a dilute ultrafiltrate of the circulatory fluid by the active secretion of cations. In a complete excretory organ, there would follow a part that served to resorb useful metabolites such as amino acids and sugars, while exchanging the cations for hydroxonium ions, thus “acidifying” the original secretion. Clearly, however, in the Heteroptera at least, such a resorptive region of the accessory gland does not occur (or is at best only partially effective).
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B. FUNCTIONS OF THE PRINCIPAL GLAND
In the two-lobed gland of pentatomids, the contents of the anterior lobe rapidly gel on exposure to gaseous or dissolved air or to hypotonic liquids, whereas the contents of the posterior lobe do not. The anterior lobe has thus been identified as the origin of the sheath material. The contents of the anterior lobe gel particularly rapidly in water, in which the free amino acids that are presumed to keep the solution of sheath precursors (Section VI1,A) stable, can rapidly diffuse from the secretion (Miles, 1964b); whereas under paraffin oil, the contents remain fluid. Nevertheless, paraffin oil readily dissolves oxygen and a skin of solidified material forms at the interface of the secretion and the oil. The cells of the anterior lobe in pentatomids do not show uniform chemical activity, however. The cells that contribute most of the sulphydryl groups of the sheath precursors are located at the anterior end of the lobe. In lygaeids, both anterior and lateral lobe of the three-lobed principal gland produce sheath precursors, but the secretion of the lateral lobe contains less sulphydryl groups than does that of the anterior lobe. Thus Miles (1 967a) concluded that the two lobes (anterior and lateral) of lygaeids represent a secondarily bifid anterior lobe (of pentatomids) with a division of function between the two; the lateral lobe providing a protein that readily forms hydrogen bonds and that provides the bulk of the protein (or lipoprotein) of the sheath, while the anterior lobe provides the sulphydryl groups that give the sheath chemical stability and initiate particularly rapid gelling in oxidizing conditions. In the Pyrrhocoridae, the principal gland is four-lobed and the contents of all the lobes except the posterior can form irreversible gels; thus Miles (1960b, 1967a) considered that in the Pentatomorpha as a whole, any more than two distinct lobes (not counting digitations) in the principal gland represented subdivisions of the anterior lobe. This point of view is at variance with that of Baptist (1941) who believed any subdivision to belong morphologically to the posterior lobe. But so little is known of the functions of the various lobes in the multilobed glands of some families of Pentatomorpha, that any conclusion must be considered conjectural at the present time. The posterior lobe of the salivary glands of Heter0ptera:Pentatomorpha secretes salivary enzymes. Miles (1 967a, 1968b) stated that the digestive enzymes occur only in the posterior lobe of the lygaeid
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Oncopeltus, but other authors claim the occurrence of such enzymes in other lobes of the salivary glands of Heteroptera (Baptist, 1941; Bronskill et al., 1958; Salkeld, 1960). In Heteroptera that secrete no sheath, the division of function between anterior and posterior lobes is almost unknown. Edwards (1961) found that in the carnivorous bug Plutymerus, extracts of both anterior and posterior lobes were proteolytic and zootoxic, whereas the contents of the accessory gland were neither. Hori (1968b) stated that the main source of salivary amylase in the mirid Lygus disponsi was the posterior lobe and that only traces of activity occurred in the anterior lobe and accessory gland; but it has also been pointed out (Miles, 1960b) that very careful separation of the lobes and removal of contaminants from the haemolymph is necessary to pinpoint the origin especially of amylase in view of its rapid activity and the sensitivity of tests for it. In the blood-sucking bugs, the distinction between the lobes in the principal gland tends to disappear and Cimex lacks an anterior lobe entirely. This secondary reduction of structure is presumably ascribable to a reduction in the number of functions performed by the saliva and comparative investigation of salivary function in the Cimicomorpha, phytophagous, predatory and blood-sucking, should be most rewarding, especially in the light of the discovery by Friend and Smith (197 1) of the secretion of traces of a sheath-like material by Rhodnius. C. SOURCES OF OXIDASES
In most Hemiptera, some part of the salivary glands secrete polyphenol oxidase (Miles, 1965) and perhaps peroxidase as well (Miles and Slowiak, 1970). In the Homoptera it is cells of the principal glands and in Heteroptera it is the cells of the accessory gland or its ducts and often those of the principal salivary duct also that secrete polyphenol oxidase. Within the phytophagous Hemiptera, these secretions could be expected to aid the oxidative process accompanying sheath formation; but experiments in which the oxidase of the accessory gland was effectively isolated from the feeding mechanism failed to prevent the normal formation of a stylet sheath within plant tissues (Miles, 1967a). At the same time, carnivorous bugs which are presumed to secrete no stylet-sheath also secrete a polyphenol oxidase in the accessory glands or ducts (Miles, 1964a).
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It is noteworthy that the cells reacting with DOPA in the Heteroptera are those surrounding the ducts, all of which have a chitinous intima: the oxidase could thus be part of the normal cuticle-producing mechanism of epidermal cells, except in those insects in which there is definite evidence of secretion of the enzyme in the saliva itself. Locke ( 1969) has recently shown that peroxidase too is involved in the oxidative processes carried out by the epidermal cells when they produce cuticle, and peroxidase is the latest enzyme to join the list of those secreted in the saliva of Hemiptera (Miles and Slowiak, 1970). One puzzling feature of the secretion of salivary oxidases by Hemiptera is that the enzymes are secreted in high concentration along with the sheath of aphids (Miles, 1965) and Pentatomorpha, and they can be found in high concentration in secretions remaining within the lumen of the sheath (Miles, 1967a); yet in the Pentatomorpha these enzymes are secreted by the accessory gland and this produces so dilute a secretion that .it is often impossible to detect any solutes in it. Possibly it is always secreted by the cells of some parts of the accessory gland or duct (Miles, 1964a) and is subject to varying degrees of dilution by water discharged by other parts. D. SOURCES IN THE HOMOPTERA
In the Homoptera, discovery of the origin of salivary components is considerably more difficult than in the Heteroptera, and recourse has mainly been had to histochemistry. It is not always easy to differentiate between the secretory products and the synthetic processes that give rise to them, however, and although prosecretory granules are separately identifiable by electron microscopy in the cells of the salivary glands of Homoptera (Moericke and WohlfarthBottermann, 1960a) the composition of the secretion cannot be determined by this means. Comparison between the work of different authors on the histochemistry of the salivary glands of Homoptera is also complicated by their use of different dyes and reagents. Another difficulty, pointed out by Weidemann (1970) is that in Myzus persicae the cells do not react consistently with reagents-a likely result in view of the cycles of activity which the cells undergo (Moericke and Wohlfarth-Bottermann, 1960a). Miles (1964a) published a comparison of the cells that produce
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PETER W. MILES
PPO in the salivary glands of Homoptera; Sogawa ( 1 965, 1967) made a histochemical comparison of the glands of delphacids (Fulguromorpha) and deltophacids (Jassomorpha) and Weidemann (1 970) made a histochemical study of the glands of the aphid Myzus persicae. Since the sheath material secreted by Hornoptera, like that secreted by Heteroptera, most probably contains sulphydryl groups (Miles, 1965), attempts have been made to trace the origins of the sheath material by looking for a major source of the sulphydryl compounds, for example cysteine/cystine in the glands. Results have tended to depend very much on the particular reagent chosen, however. Perhaps the most specific test for sulphydryl groups is the nitroprusside reaction, but it is insensitive and unsuitable for small sections; a highly sensitive method, said to be specific, is with DDD (dihydroxy dinaphthyldisulphide), but this test gave no clearly positive results with the salivary glands of leaf hoppers (Sogawa, 1967). Other methods, found more likely to react positively, were of less certain chemical protocol-e.g. the alkaline tetrazolium reaction (Sogawa, 1967) and performic acid-alcian blue (Weidemann, 1970). Finally, quite apart from the occurrence nt sulphydryl groups in the sheath, any other secretion with which the sheath precursors are likely to come into contact within the conducting system of the glands are also likely to contain reducing groups (Miles, 1967a). Other properties of sheath material that may be of use in identifying its origins in the glands of Homoptera are its content of phospholipid and its periodic acid-Schiff (PAS) reaction. Using tests for lipids and the PAS-reaction, Sogawa homologized some lobes of the salivary glands of the jassomorph N. cincticeps and the fulguromorph L. striatellus (Figs 14, 17). These cells gave somewhat different combinations of reactions, but Sogawa concluded that two or three sets of cells were responsible for the secretion of the sheath precursors out of the six kinds of cells in the principal glands of N. cincticeps and out of eight kinds in the glands of L. striatellus. The strongest homologies according to Sogawa are between the IV cells of N. cinticeps and the A cells of L. striatellus (main secretion of protein); the I11 cells and the H and G cells (main secretion of lipids) and the V cells and the Ag cells (secretion of conjugated carbohydrate). E. SALIVARY CARBOHYDRATE AND LIPID
One histochemical peculiarity of sheath material and of some cells in the salivary glands may be of use in future studies of its glandular
THE SALIVA OF HEMIPTERA
24 1
origins. The PAS-reaction is usually given by carbohydrates, but also by some lipids: these sources can be differentiated by either treating with an acetylating agent, which blocks the groups in carbohydrates that are PAS-positive or by extraction with solvents that remove the source of the reaction when it is due to lipids. One or both of these pre-treatments should prevent the PAS-reaction, thus indicating the identity of the PAS-positive source. Sheath material (Miles, 1960a) and certain cells of the Homoptera (Sogawa, 1967) and Heteroptera (Miles, 1960b) remain PAS-positive despite prior blocking of carbohydrates or extraction of lipids. Sogawa (1 967) suggested this was due to the presence of both sources of reactivity in a conjugated form. His hypothesis received some support from experiments with injected radioactive glucose and glycerol (Miles, 1967b), both of which are incorporated into the sheath precursors of the pentatomid Eumecopus, the glycerol presumably being metabolized into the 10% or so lipid moiety. According to Hori (1968b), the mirid Lygus also has a strongly PAS-reacting secretion in the anterior lobe (compared with the posterior) of the principal gland. This insect produces no sheath material and the anterior lobe secretes relatively little protein. In the Pentatomorpha, by comparison, the anterior lobe, which does secrete sheath precursors, produces a secretion that is strongly PAS-positive and also contains a large amount of protein. These results, taken with the finding that the blood-sucking reduviid, Rhodnius produces a salivary material akin to sheath material, may mean that in all Heteroptera the anterior lobe retains an ability to secrete protein conjugated with carbohydrate and lipid, although in the forms that secrete no sheath the amount of protein secreted is reduced. Even in the Pentatomorpha, there is apparently a tendency for the most anterior part of the anterior lobe to lose its ability to secrete a large bulk of protein (Miles, 1967b), this function being taken over by another part of the glands or perhaps by the hind region of the anterior lobe, which becomes separated off as a distinct lateral lobe. XI. THE SALIVA AS A VEHICLE FOR PATHOGENS
The pentatomid Acrosternum hilare (Say) acquires and transmits the fungus Nematospora coryli Peglion when it feeds on lima or soy beans or the berries of dogwood, Cornus drummondi Meyer; and Dysdercus transmits N. gossypii to cotton bolls similarly (Clarke and Wilde, 1970a, b). The spores contaminate the stylets and salivary syringe and infectivity is lost during moulting. Clarke and Wilde AIP-
11
242
PETER W. MILES
(1970a) were unable to recover the fungus from watery saliva secreted outside plants (the insect was induced to salivate by amputation of its antennae). Even so, the authors did not discard salivary transmission as the most likely and they had not entertained the possibility that this secretion might have been atypical of those normally secreted within plant tissues (see Section VI1,B)-perhaps a more viscous secretion, such as the sheath material, is normally required to dislodge the contaminating spores. By far the most significant instances of transmission of plant pathogens by Hemiptera are the virus diseases carried by Homoptera. The “non-persistent” viruses are transmitted immediately after they have been acquired by the insects and the latter very rapidly lose their infectivity; the “persistent” viruses are transmitted only after a period of hours to weeks, the latent period, and usually the insect then continues to be infectious until it dies. The non-persistent viruses are not ingested before transmission and they are somehow contaminants of the stylets or food canal of the feeding mechanism; whereas the persistent viruses are ingested, transferred first to the haemolymph and thence to the saliva. The non-persistent viruses are typically transmitted by aphids and are acquired from and transmitted to the subepidermal tissues of plants. The persistent viruses are typically transmitted by the jassomorph Homoptera and are acquired from and transmitted to the vascular tissues of the plant. A number of authors have suggested that the saliva is by no means an inert carrier of the viral particles and may either inactivate them or modify the plant cells in such a way that they become more or less susceptible (Sylvester, 1962; Miles, 1968b). Of most recent interest in the field of salivary transmission of plant diseases is the finding of mycoplasma-like bodies (Fig. 19) in the salivary syringe and ducts of the jassomorph leafhopper Mucrosteles fascifrons (Raine and Forbes, 197 1) and in its ejected saliva (Raine and Forbes, 1969), and the evidence that such bodies are normally associated with individuals carrying the Aster Yellows disease of China Aster and annual Chrysanthemum (Davis and Whitcomb, 1970). Moreover, both the disease and the organisms within the insect are alike attacked by certain antibiotics, according to Whitcomb and Davis (1970), who nevertheless warn that some non-infective individuals also carried the mycoplasma-like bodies and that an absolute link of organism with disease had thus not been established beyond all possible doubt.
THE SALIVA O F HEMIPTERA
243 m
$
c E
P 3
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PETER W. MILES
Observations on identifiable infective organisms in the salivary glands are of especial interest in the light of the report by Ossiannilsson ( 196 1) and Moericke and Wohlfarth-Bottermann (1960a, b) that the wall of the gut and the basement membrane of the salivary glands respectively have no pores in them large enough to allow viral particles (e.g. 32 x 300 mp in size) to move through. Moericke (1961) found large viral bodies up to 50 x 400 mp in the cells of the salivary glands of Myzus persicue infected with potato leaf-roll disease; he reasoned that the infective unit must be very much smaller and was probably molecular nucleic acid. The mycoplasma-like bodies described by Raine and Forbes in the salivary glands and ducts of Mucrosteles were up to 400 mp across. Even larger particles, up to 800 mp across, have been described in the cells of the glands themselves by Hirumi and Maramarosch (1 969); Raine and Forbes (1 97 1) concluded that these must produce small intermediate bodies that leave the cells and develop into the organisms found in the saliva. On the other hand, Moericke and Wolfarth-Bottermann ( 1960a) describe the discharge of secretory granules up to 260 mp across from the cells of the salivary glands of Myzus persicae by a membrane flow mechanism-i.e. a prosecretory granule bulges out from the secretory cell into a canal cell, a part of the cell membrane surrounding the granule detaches itself from the parent membrane and the granule is eventually liberated when the membrane dissolves. Just how large a particle can pass all the way through the glands is now known, however. Slama (personal communication) found that the concentration of enzymes in the saliva of the phyrrocorid Pyrrhocon's apterus was much the same as their concentration in the haemolymph and the possibility must therefore be considered that the enzymes merely found their way into the saliva by transport from the haemolymph. In the only known check of this possibility (Miles and Slowiak, 1970), peroxidase, with a molecular weight of 40,000, was shown to pass into the saliva of a pentatomid after injection into the haemolymph, but this is a relatively small molecule for a protein. XII. EVOLUTION OF SALIVARY FUNCTION IN THE HEMIPTERA: A SUMMARY
The salivary glands of Hemiptera can be thought of as having two kinds of function:
THE SALIVA O F HEMIPTERA
245
(1) those of excretory organs due to their origin as the segmental nephridia of the labial segment and (2) those of epidermal cells due to their ectodermal origin. The excretory function resides mainly in the accessory gland and this is seen very clearly indeed in the Phylloxeridae. Whereas, in the related aphids, the main excretion occurs into the midgut and the accessory gland is small, in Viteus there is no anus and the accessory gland is enormously increased in size. Characteristically, the cells concerned with salivary excretion contain either large intracellular vacuoles or an elaborate system of intracellular canals as in the Aphidoidea or much of the accessory gland takes on the characteristics of a Malpighian tube, as in the Jassoidea and the He terop tera. Epidermal cells, as Locke (1 970) has shown, can carry out a series of functions simultaneously : they secrete the carbohydrate and protein of the cuticle, pass on the lipid elements that are eventually incorporated in the exocuticle and epicuticle, secrete the polyphenol oxidase and peroxidase involved in the oxidative bonding that stabilizes the cuticle, at the same time they secrete the hydrolysing enzymes that digest the untanned parts of the old cuticle and they assimilate the products. The cells of the principal salivary gland and ducts of the salivary glands of Hemiptera similarly secrete the precursors of the sheath material, as well as oxidizing enzymes and hydrolysing enzymes, although there would seem to be a considerable separation of specialized functions between the various parts of the glands. It is noteworthy that the ducts of the salivary glands have chitinous linings. Miles (1 960a) drew attention t o the histochemical and histological similarity of newly secreted sheath material and newly secreted cockroach cuticle. It was found later that gelling of the sheath material involved the formation of disulphide linkages, at a time when it was widely reported that the cuticle of insects contained no sulphydryl amino acids. Recently, however, this generalization has been shown to be incorrect (Hackmann and Goldberg, 197 1) and perhaps in the cuticle, as in sheath material, the oxidative bonding of sulphydryl groups plays a part in the gelling and stabilization of the precursor proteins. If all the Hemiptera are to be derived from forms similar to the Peloridiidae (Coleorrhyncha), then the salivary glands of the Heteroptera are the more primitive in shape and form within the Hemiptera as a whole. In both Homoptera and Heteroptera the
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PETER W. MILES
accessory gland has retained a diuretic function, although there is doubt whether a true accessory gland persists in the Fulguromorpha (Balasubramanian and Davies, 1968). In the Heteroptera, the accessory gland assumes a definitive form as a chitin-lined duct surrounded towards its distal end by more or less glandular cells. In all but the Pentatomorpha, the accessory gland has a thin walled vesicle at its distal extremity. This vesicle, far from being a reservoir for secretory products (Baptist, 1941), appears to be a bladder that collects a dilute ultrafiltrate of the haemolymph. It is probably an adaptation to predatory and other lacerate-and-flush feeding, in which a copious flow of water is finally required to flush out the food source. In the Pentatomorpha, although lacerate-and-flush can be practised by the seed-feeders, the absence of a vesicle on the accessory gland is possibly associated with a reduced need for large quantities of watery secretion by insects adapted to the more conservative stylet-sheath feeding. Within all the Hemiptera, there is a clear division of labour between the different lobes or types of lobe of the principal gland. Even in the Peloridiidae, the poorly developed principal gland is composed of an anterior and posterior lobe that have different staining reactions (Prendergast, 1962). In the Heteroptera, the posterior lobe is probably the main source of the hydrolysing enzymes of the saliva. All the secretions of the principal gland are relatively concentrated and it is likely that the posterior lobe in particular produces a secretion that in normal function requires dilution by the secretion of the accessory gland. Although the anterior lobe in the Heter0ptera:Pentatomorpha produces components of the sheath material, its primary function throughout the Heteroptera may be the production of mucoid substances and conjugated lipids that both lubricate the stylets and are sufficiently viscous to dislodge particles at their tips; for, as McLean and Kinsey (1965) have shown, this is one of the functions of the sheath material of aphids. At the very beginning of the evolution of the Hemiptera, some species must have begun to utilize the solidification of this viscous secretion, brought about perhaps by the release into the saliva of oxidases secreted by the ectodermal cells of the ducts, perhaps by the increased content of sulphydryl groups within the protein moiety of the secretion, perhaps by a combination of both. However evolved, this “sheath material” may originally have been used to fix the stylets at one point on a hard or slippery surface
THE SALIVA OF HEMIPTERA
247
during its initial penetration (Saxena, 1963): but for all those species that fed on phloem sap, the evolution of stylet-sheath feeding has clearly become the most unique and characteristic of their salivary functions. It also seems likely that evolution of the production of sheath material has tended to result in a further division of labour and proliferation of morphological divisions in those parts of the glands responsible for its secretion; a convergent tendency in both Homo p tera and Heteroptera :Penta tomorpha despite their p hy letic separation. XIII. A SURVEY OF PROBLEMS
Of the problems concerned with salivary function in the Hemiptera that remain t o be solved, perhaps one of the most intriguing is the function of the salivary oxidases. Little is known about their secretion by Hemiptera other than that they can be readily demonstrated in the stylet-sheaths of aphids and Pentatomorpha in concentrations that must be physiologically active. Miles (1969b) has attempted to rationalize the occurrence of oxidase in the salivary glands and saliva of Hemiptera by suggesting that the enzymes when first secreted may have subserved a detoxifying function. This suggestion was based on the facts that:
(1) many of the toxic and repellant substances produced by insects are based on phenolic compounds (their production no doubt also being derived from the abilities of ectodermal cells to secrete substances that tan the cuticle) and (2) the reaction of plants t o wounding also involves the secretion of toxic phenols. Thus for insects that feed either on plants or other arthropods, the possession of an antidote against toxic phenols and quinones, by oxidizing them all the way to insoluble polymers, would be an advantage. It would seem t o be possible t o test this hypothesis by experimental inactivation of the salivary phenolase system, either by surgery on the salivary gland (Miles, 1967a) or by the use of reducing compounds in artificial diets. It could then be determined whether the insects’ sensitivity t o toxins was increased. A related problem is the occurrence and function of salivary peroxidases and their interaction with the phenol/phenolase system. A further line of investigation of a possible detoxicant function of
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PETER W. MILES
the saliva of phytophagous Hemiptera is indicated by a recent investigation of the degree of resistance of various tissues from apple trees to attack by the woolly aphis, Eriosoma Zanigerum (Sen Gupta and Miles, unpublished). These insects will begin to colonize a variety of seedlings germinated on wet filter paper, but survive on only apple as the seedlings grow. In the field and greenhouse, a relation existed between how readily the insect attacked different varieties of apple-or different parts of any one tree-and the chemical composition of the tissues. High amino acid content predisposed tissues to attack, but phenolics appeared t o protect them; thus the ratio of phenolics t o amino acids provided a rough means of prediction of the resistance of any particular tissue t o attack (Table V). Of further interest was the discovery that the galls produced by the insects were more readily colonizable than surrounding tissues. A number of contingent questions are raised by these observations and as yet remain unanswered. Is host specificity due to the presence of specific phagostimulants (e.g. in apple and not in other plants) or absence of repellents (in young as opposed to older seedlings)? Is Table V The susceptibility of tissues of the apple to attack by E. lanigerum in relation to their chemical composition Susceptibility ratin&
a-amino Nb
phenolics (8)"
@/N
14.5f0.04 lO.lf0.04 8.5f0.12
115 105 59
2 1 .OfO.1 1 16.3f0.11 13.2f0.33
43 20 12
Shoot
+
tip base gall
++ +++
0.126f0.002 0.096f0.002 0.143fO.007 Root
+++ ++++
8mm 4mm gall
*U+
0.493f0.02 0.806f0.02 1.069f0.05
0 Based on measurement of rate of development of colonies, expressed semiquantitatively because of incomplete equivalence of, for instance, physical conditions. b mg N/g wet weight: colorimetric determination with ninhydrin against a D-alanine standard, standard deviation. mg/g wet weight: colorimetric determination with Folin and Denis reagent against a catechin standard, standard deviation.
*
*
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249
ability to feed on apple due to the possession by the insect of a specific detoxicant for repellent (e.g. phenolic) substances present in apple? Is the reduced toxicity of galls a direct result of salivary action or an indirect result of the stimulation of cells to grow at a rate greater than they can accumulate phenolics? The problem of cecidogenesis itself is still far from closed. Despite the elegant way in which Schaller (1 968b) has shown that the amino acids and IAA injected by some aphids cause galls in plant tissues-how the IAA comes to be present is an interesting problem in insect physiology, while the roles of the salivary IAA and the specific amino acids said to determine gall morphogenesis represent an even more challenging problem in plant physiology. The possible functions of the stylet-sheath and the salivary oxidases in moderating the effect of feeding on the plants also requires investigation. It has been suggested that the sheath, besides increasing the efficiency of feeding by the phytophagous Hemiptera, may also reduce those interactions between insect and plant that cause necrosis in the latter (Miles, 1969b) with an obvious advantage to both. According to Hille Ris Lambers (personal communication), however, the aphid Aulucorthum soluni is a stylet-sheath feeder that causes necrosis as a result of a single feed and this possible exception should be especially worthy of investigation. The problem of the role of the phenolic compounds as distinct from the phenolases in the saliva of phytophagous insects also require elucidation. Schaller ( 1 968a) has demonstrated that phenolic compounds occur in the saliva of aphids and, at the same time, has correlated their presence with a lack of pathogenicity of the insect. Although some of these phenolics could well have been transferred directly from the diet, Miles (1964a) demonstrated that one of the phenolics present in the saliva of the phytophagous Heteroptera is DOPA and that it is the end product of a specific metabolic pathway in the salivary glands. Thus any investigation of the salivary oxidases should take into account the complete phenol/phenolase system and its eventual products. The functions of the salivary glands and saliva of Heteroptera other than the Pentatomorpha are still almost unknown. The division of labour between the various lobes of their salivary glands remains undetermined; the way in which Rhodnius produces its flange of sheath material and in what way the loss of the anterior lobe has affected the salivary physiology of the Cimicids remain further intriguing problems.
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W. MILES
How viral or mycoplasmal particles find their way seemingly from the gut to the saliva of Homoptera and details of the transmission of rickettsia1 diseases by reduviids are further aspects of the salivary physiology of Hemiptera that remain undetermined. Indeed, the degree to which salivary composition is normally due to transport of whole molecules including proteins from the haemolymph to the saliva is largely unknown. Finally, the control of salivary discharge and compositionwhether entirely by neural pathways or partly subject to hormonal influences-remains apparently unexplored. Yet this knowledge will be necessary to integrate what is known of salivary function into the broader, but still unique, subject of the physiology of feeding of these highly specialized and economically important insects.
REFERENCES Adams, J. B. and Drew, M. E. (1 963a). A cellulose-hydrolysing factor in aphids. Can. J. Zool. 41, 1205-1212. Adams, J . B. and Drew, M. E. (1 963b). The effects of heating on the hydrolytic activity of aphid extracts on soluble cellulose substrates. Can. J. 2001.41. Adams, J. B. and McAllan, J. W. (1958). Pectinase in certain insects. Can. J. ZOO^, 36, 305-308. Anders, F. (1960a). Untersuchungen uber das cecidogene Prinzip der Reblaus ( Viteus vitifolii Shimer) I. Untersuchungen an der Reblausgalle. Biol. Zbl. 79,47-58. Anders, F. ( 1960b). Untersuchungen iiber das cecidogene Prinzip der Reblaus ( Viteus vitifolii Shimer) 11. Biologische Untersuchungen uber das galleninduzierende Sekret der Reblaus. Biol. Zbl. 79,679-700. Anders, F. (1961). Untersuchungen Uber das cecidogene Prinzip der Reblaus ( Viteus vitifolii Shimer) 111. Biochemische Untersuchungen iiber das galleninduzierende Agenus. Biol. Zbl. 80, 199-233. Balasubramanian, A. and Davies, R. G. (1 968). The histology of the labial glands of some Delphacidae (Hemiptera:Homoptera). Trans. R . ent. SOC.Lond. 120, 239-251. Baptist, B. A. (1 941). The morphology and physiology of the salivary glands of Hemiptera-Heteroptera. Quart. J. micr. Sci. 82, 9 1-139. Berlin, L. C. and Hibbs, E. J. (1963). Digestive system morphology and salivary enzymes of the potato leafhopper, Empousca fabae (Harris). Iowa Acud. Sci. 7 0 , 527-540. Bongers, J. ( 1969). Saugverhalten und Nahrungsaufnahme von Oncopeltus fusciatus Dallas (Heteroptera, Lygaeidae). Oecologia, Berl. 3 , 374-389. Bronskill, J. F., Salkeld, E. H. and Friend, W. G . (1 958). Anatomy, histology, and secretions of salivary glands of the large milkweed bug Oncopeltus fasciatus (Dallas) (Hemiptera:Lygaeidae). Can. J. Zool. 36,961-968. BUsgen, M. (1891). Der Honigtau Biologische Studien an Pflanzen und Pflanzenlausen. Z . Naturwiss. 2 5 , 340-428. Carter, W. (1962). “Insects in Relation to Plant Disease”. Interscience
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Publications, New York. Clarke, R. G. and Wilde, G. E. (1 970a). Association of the green stink bug and the Yeast-Spot Disease organism of soybeans. I. Length of retention, effect of moulting, isolation from faeces and saliva. J. econ. Ent. 63,200-204. Clarke, R. G. and Wilde, G. E. (1 970b). Association of the green stink bug and the Yeast-Spot Disease organism of soybeans. 11. Frequency of transmission to soybeans, transmission from insect to insect, isolation from field population. J. econ. Ent. 63, 355-357. Davis, N. T. and Usinger, R. L. (1970). The biology and relationships of the Joppeicidae (Heteroptera). Ann. en?. SOC.A m . 63, 577-587. Davis, R. E. and Whitcomb, R. F. (1970). Evidence on possible mycoplasma etiology of Aster Yellows Disease. I. Suppression of symptom development in plants by antibiotics. Infect. Immun. 2, 201-208. Duspiva, F. (1 954). Weitere Untersuchungen uber stoffwechsel-physiologische Beziehungen zwischen Rhynchoten und ihren Wirtspflanzen. Mitt. biol. BundAnst. Ld-u.-Forstw. 80, 155-162. Edwards, J. S. (1961). The action and composition of the saliva of an assassin bug Platymeris rhadamanthus Gaerst. (Hemiptera, Reduviidae). J. exp. Biol. 38,61-77. Feir, D. and Beck, S. D. (1961). Salivary secretions of Oncopeltus fasciatus (Hemiptera:Lygaeidae). Ann ent. SOC.A m . 54,316. Forbes, A. R. (1969). The stylets of the green peach aphid Myzus persicae (Hom0ptera:Aphididae). Can. En?. 101, 31-41. Friend, W. G. and Smith, J. J. B. (1971). Feeding in Rhodniusprolixus: mouthpart activity and salivation, and their correlation with changes of electrical resistance. J. Insect Physiol. 17, 233-243. Goodchild, A. J. P. (1 966). Evolution of the alimentary canal in the Hemiptera. Biol. Rev. 41, 97-140. Gordon, S. A. and Paleg, L. G. (1961). Formation of auxin from tryptophan through action of polyphenols. Plant Physiol. 36,838-845. Hackman, R. H. and Goldberg, M. (1971). Studies on the hardening and darkening of insect cuticles. J. Insect Physiol. 17,335-347. Hagley, E. A. C. (1969). Free amino acids and sugars of the adult sugar cane froghopper (Aeneolamia varia saccharinn) (Homoptera) (Homoptera: Cercopidae) in relation to those of its host. Entomologia exp. appl. 12, 23 5-239. Hennig, E. (1 963). Zum Probieren oder sogenannten Probesaugen der schwarzen Bohnenlaus (Aphis fabae Scop). Entomologia exp. appl. 6,326-336. Hirumi, H. and Maramorosch, K. (1 969). Mycoplasma-like bodies in the salivary glands of insect vectors carrying the aster yellows agent. J. Virol. 3,82-84. Hori, K. (1968a). Feeding behaviour of the cabbage bug, Eurydema rugosa Motschulsky (Hemiptera:Pentatomidae)on cruciferous plants. Jap. J. appl. Ent. Zool. 3, 26-36. Hori, K. (1 968b). Histological and histochemical observations on the salivary gland of Lygus disponsi Linnavouri (Hemiptera, Miridae). Res. Bull. Obihiro Zootech. Univ. 5, 735-744. Hori, K. (1969). Some properties of salivary amylases of Adelphocoris suturalis (Miridae), Dolycoris baccarum (Pentatomidae), and several other heteropteran species. Entomologia exp. appl. 12,454-466. Hori, K. (1970a). Some properties of amylase in the salivary gland of Lygus disponsi (Hemiptera). J. Insect Physiol. 16, 373-386.
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Hori, K. (1970b). Some variations in the activities of salivary amylase and protease of Lygus disponsi Linnavouri (Hemiptera:Miridae). Jap. J. appl. Ent. Zool. 5, 51-61. Hori, K. ( 1 9 7 0 ~ ) Carbohydrases . in the gut and salivary gland, and the nature of the amylase in the gut homogenate of Lygus disponsi Linnavouri (Hemiptera:Miridae). Jap. J. appl. Ent. Zool. 5, 13-22. Hori, K. (1970d). Some properties of proteases in the gut and in the salivary gland of Lygus disponsi Linnavouri (Hemiptera:Miridae). Res. Bull. Obihiro Zootech. Univ. 6 , 3 18-324. Kinsey, M. G. and McLean, D. L. (1 967). Additional evidence that aphids ingest through an open stylet sheath. Ann. ent. SOC.A m . 60, 1263-1265. Kloft, W. (1 960). Wechselwirkungen zwischen pflanzensaugenden Insekten und den von ihnen besogenen Pflanzengeweben. Z . angew. Ent. 45, 337-381 and 46,42-70. Kloft, W. and Ehrhardt, P. (1959a). Untersuchungen uber saugtatigkeit und schadwirkung der Sitkafichtenlaus Liosomaphis abietina (Walk.) (Neomyzaphis abietina Walk.). Phytopath. Z. 35,401-410. Kloft, W. and Ehrhardt, P. (1959b). Zur frage der Speichelinjektion beim Saugakt von Thrips tabaci Lind. (Thysanoptera, Terebrantia). Naturwiss. 20, 586-587. Kloft, W., Ehrhardt, P. and Kunkel, H. (1968). Radioisotopes in the investigation of interrelationships between aphids and host-plants. In “Isotopes and Radiation in Entomology”, pp. 23-30. IAEA. Vienna. Laurema, S. and Nuorteva, P. (1961). On the occurrence of pectin polygalacturonase in the salivary glands of Heteroptera and Homoptera Auchenorrhyncha. Ann. Ent. Fenn. 27, 89-93. Lavoipierre, M. M. J., Dickerson, G. and Gordon, R. M. (1959). Studies on the methods of feeding blood-sucking arthropods I. The manner in which triatomine bugs obtain their blood-meal as observed in the tissues of the living rodent, with some remarks on the effects of the bite on human volunteers. Ann. trop. Med. Parasit. 5 3 , 235-250. Locke, M. (1 969). The localization of a peroxidase associated with hard cuticle formation in an insect, Calpodes ethlius Stoll, Lepidoptera, Hesperiidae. Tissue Cell 1, 555-574. Locke, M. (1 970). The control of activities in insect epidermal cells. Mem. SOC. Endocrin. 18,285-299. McLean, D. L. and Kinsey, M. G. (1964). A technique for electronically recording aphid feeding and salivation. Nature, Lond. 202, 1358-1 359. McLean, D. L. and Kinsey, M. G. (1 965). Identification of electrically recorded curve patterns associated with aphid salivation and ingestion. Nature, Lond. 205, 1130-1131. McLean, D. L. and Kinsey, M. G. (1967). Probing behavior of the pea aphid, Acyrthosiphon pisum I. Definitive correlation of electronically recorded waveforms with aphid probing activities. Ann. ent. SOC.A m . 60,400-406. McLean, D. L. and Kinsey, M. G. (1968). Probing behavior of the pea aphid, Acyrthosiphon pisum 11. Comparisons of salivation and ingestion in host and non-host plant leaves. Ann. ent. SOC.A m . 61, 730-739. McLean, D. L. and Kinsey, M. G. (1969). Probing behavior of the pea aphid, Acyrthosiphon pisum IV. Effects of starvation on certain probing activities. Ann. ent. SOC.A m . 6 2 , 987-994.
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McLean, D. L. and Weigt, W. A. (1968). An electronic measuring system to record aphid salivation and ingestion.Ann. ent. SOC.A m . 61, 180-185. Miles, P. W. (1 959a). The secretion of two types of saliva by an aphid. Nature, Lond. 183,756. Miles, P. W. (1959b). The salivary secretions of a plant-sucking bug, Oncopeltus fasciatus (Dall.) (Heteroptera: Lygaeidae) I. The types of secretion and their roles during feeding. J. Insect Physiol. 3, 243-255. Miles, P. W. (1960a). The salivary secretions of a plant-sucking bug, Oncopeltus fasciatus (Dall.) (Heteroptera : Lygaeidae) 11. Physical and chemical properties. J. Insect Phys. 4 209-2 19. Miles, P. W. (1 960b). The salivary secretions of a plant-sucking bug, Oncopeltus fasciatus (Dall.) (Heter0ptera:Lygaeidae) 111. Origins in the salivary glands. J. Insect Physiol. 4,271-282. Miles, P. W. (1 964a). Studies on the salivary physiology of plant-bugs: oxidase activity in the salivary apparatus and saliva. J. Insect Physiol. 10, 121-129. Miles, P. W. (1964b). Studies on the salivary physiology of plant-bugs: the chemistry of formation of the sheath material. J. Insect Physiol. 10, 147-160. Miles, P. W. (1 965). Studies on the salivary physiology of plant-bugs: the salivary secretions of aphids. J. Insect Physiol. 11, 126 1-1268. Miles, P. W. (1967a). The physiological division of labour in the salivary glands of Oncopeltus fasciatus (Dall.) (Heteroptera: Lygaeidae). Aust. J. biol. Sci. 20, 785-797. Miles, P. W. (1 967b). Studies on the salivary physiology of plant-bugs: transport from haemolymph to saliva. J. Insect Physiol. 18, 1787-1801. Miles, P. W. (1968a). Studies on the salivary physiology of plant-bugs: experimental induction of galls. J. Insect Physiol. 14, 97-1 06. Miles, P. W. (196813). Insect secretions in plants. Ann. Rev. Phytopathol. 6, 137-164. Miles, P. W. (1969a). Incorporation and metabolism of cysteine in the haemolymph and saliva of a plant bug. Aust. J. biol. Sci. 22, 1271-1276. Miles, P. W. (1 969b). Interaction of plant phenols and salivary phenolases in the relationship between plants and Hemiptera. Entomologia exp. appl. 12, 1364744. Miles, P. W. and Slowiak, D. (1970). Transport of whole protein molecules from blood to saliva of a plant-bug. Experientia 26, 6 1 1. Mittler, T. E. (1954). “The Feeding and Nutrition of Aphids”. Doctoral Thesis, University of Cambridge, England. Mittler, T. E. (1957). Studies on the feeding and nutrition of Tuberolachnus salignus (Gmelin) (Homoptera, Aphididae) I. The uptake of phloem sap. J. exp. Biol. 3, 334-341. Moericke, V. (196 1). Virusartige Korper in Speicheldrusen von Kartoffelblattrollinfektiosen Myzus persicae (Sulz.) Z. PflKrankh. PflPath. PflSchtz 68, 581-587. Moericke, V. and Mittler, T. E. (1965). Schlucken Blattlause ihren eigenen Speichel? Z. PflKrankh. PflPath. PflSchutz 72, 5 13-515. Moericke, V. and Mittler, T. E. (1966). Oesophageal and stomach inclusions of aphids feeding on various cruciferae. Entomologia exp. appl. 9 , 287-297. Moericke, V. and Wohlfarth-Bottermann, K. E. (1 960a). Zur funktionellen Morphologie der Speicheldrusen von Homopteren I. Die Hauptzellen der
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Hauptdruse von Myzus persicae (Sulz.), Aphididae. Z . Zellforsch. 5 1, 157-184. Moericke, V. and Wohlfarth-Bottermann, K. E. (1 960b). Zur funktionellen Morphologie der Speicheldrusen von Homopteran IV. Die Ausfuhrgange der Speicheldriisen von Myzus persicae (Sulz.), Aphididae. Z . Zellforsch. 53, 25-49. Nault, L. R. and Gyrisco, G. G. (1966). Relation of the feeding process of the pea aphid t o the inoculation of pea enation mosaic virus. Ann. ent. Soc. A m . 59, 1185-1 197. Nuorteva, P. (1 956a). Notes on the anatomy of the salivary glands and on the occurrence of proteases in these organs in some leafhoppers (Hom., Auchenorrhyncha). Ann. Ent. Fenn. 22,103-108. Nuorteva, P. (1956b). Developmental changes in the occurrence of the salivary proteases in Miris dolabratus L. (Hem., Miridae). Ann. Ent. Fenn. 22, 117-1 19. Nuorteva, P. (1958). Die Rolle der Speichelsekrete im Wechselverhaltnis zwischen Tier und Nahrungspflanze bei Homopteran und Heteropteran. Entomologia exp. appl. 1,41-49. Nuorteva, P. (1962). Studies on the causes of the phytopathogenicity of Calligypona pellucida (F.) (Hom., Araeopidae). Ann. Zool. SOC. Vanamo 23, 1-58. Nuorteva, P. and Laurema, S. (1 96 1a). The effect of diet on the amino acids and salivary glands of Heteroptera. Ann. Ent. Fenn. 27, 57-65. Nuorteva, P. and Laurema, S. (1961b). Observations on the activity of salivary proteases and amylases in Dolycoris baccarum (L.) (Het., Pentatomidae). A n n . Ent. Fenn. 27, 93-97. Ossiannilsson, F. (1961). Negative evidence against passage of whole virus particles through gut wall in aphid vectors. Nature, Lond. 190, 1222. Petri, L. (1 909). Uber die Wurzelfaule Phylloxierter Weinstocke. 2. PflKrankh. PflPath. Pflschtz. 19, 18-48. Pilet, P. E., Lavanchy, P. and Sevhonkian, S. (1970). Interactions between peroxidases, polyphenoloxidases and auxin oxidases. Physiologia P1. 23, 8 00-804. Prendergast, J. G. (1962). The internal anatomy of the Peloridiidae (Homoptera:Coleorrhyncha). Trans. R. ent. SOC.Lond. 114, 49-65. Raine, J. and Forbes, A. R. (1 969). Mycoplasma-like bodies in the saliva of the leafhopper Macrosteles fascifrons (Stil) (Homoptera:Cicadellidae). Can. J. Microbiol. 15, 1105-1 107. Raine, J. and Forbes, A. R. (1971). The salivary syringe of the leafhopper Macrosteles fascifrons (Homoptera:Cicadellidae) and the occurrence of mycoplasma-like organisms in its ducts. Can. Ent. 103, 110-116. Rilling, G. (1 966). Die Speicheldrusen der Reblaus (Dactylosphaera vitifolii Shimer). Vitis 6 , 136-150. Salkeld, E. H. (1960). A histochemical study of the secretions in the principal salivary glands of the large milkweed bug, Oncopeltus fasciatus (Dallas). Can. J. 2001. 38,449-454. Saxena, K. N. (1963). Mode of ingestion in a heteropterous insect Dysdercus koenigii (F.) (Pyrrhocoridae). J. Znsect Physiol. 9,47-7 1. Schaller, G. ( 1960). Untersuchungen uber den Aminosauregehalt des Speicheldriisensekreter der Reblaus (Viteus [Phylloxera] vitifolii Shimer), Homoptera. Entomologia exp. appl. 3, 128-136.
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Schaller, G, (1 965). Untersuchungen uber den /3-Indolylessigsaure-Gehalt des Speichels von Aphidenarten mit unterschiedlicher Phytopathogenitat. Zool. Jb. Physiol. 71, 385-392. Schaller, G. (1 968a). Biochemische Analyse des Aphidenspeichels und seine Bedeutung fur die Gallenbildung. Zool Jb. Physiol. 74, 54-87. Schaller, G. (1968b). Untersuchungen zur Erzeugung kunstlicher Pflanzengallen. Marcellia 35, 131-153. Scott, D. R. (1970). Feeding of Lygus bugs (Hemiptera:Miridae) on developing carrot and bean seeds increased growth and yields of plants grown from that seed. Ann. ent. SOC.A m . 63, 1604-1608. Smith, K. M. (1920). Investigation of the nature and cause of the damage to plant tissue resulting from the feeding of capsid bugs. A n n appl. Biol. 7, 40-55. Smith, K. M. (1926). A comparative study of the feeding methods of certain Hemiptera and of the resulting effects upon plant tissue, with special reference to the potato plant. Ann. appl. Biol. 13, 109-139. Smith, J. J. B. and Friend, W. G . (1971). The application of split-screen television recording and electrical resistance measurement to the study of feeding in a blood-sucking insect (Rhodnius prolixus). Can. Ent. 103, 167-172. Sogawa, K. (1965). Studies on the salivary glands of rice plant leafhoppers I. Morphology and histology. Jap. J. appl. Ent. 2001.9,275-290. . Sogawa, K. (1967). Studies on the salivary glands of rice plant leafhoppers 11. Origins of the structural precursors of the sheath material. Jap. J. appl. Ent. Zool. 2, 195-202. Strong, F. E. (1970). Physiology of injury caused by Lygus hesperus. J. econ. Ent. 63,808-814. Strong, F. E. and Kruitwagen, E. C. (1968). Polygalacturonase in the salivary apparatus of Lygus hesperus (Hemiptera). J. Insect Physiol. 14, 11 13-1 119. Sylvester, E. S. (1962). Mechanisms of plant virus transmission by aphids. Zn “Biological Transmission of Disease Agents” (K. Maramorosch, ed.), pp. 1 1-3 1. Academic Press, New Y ork and London. van Loon, L. C . (1971). Tobacco polyphenoloxidases: a specific staining method indicating non-identity with peroxidases. Phytochemistry 10, 503-507. Wearing, C. H. (1968). Responses of aphids to pressure applied to liquid diet behind parafilm membrane-longetivity and larviposition of Myzus persicae (Sulz.) and Brevicoryne brassicae (L.) (Hom0ptera:Aphididae) feeding on sucrose and sinigrin solutions. N.Z. J. Sci. 11, 105-121. Weidemann, H. L. (1 968). Zur Morphologie der Hauptspeicheldruse von Myzus persicae. Entomologia exp. appl. 11, 450-454. Weidemann, H. L. (1 970). Histochemische Differenzierung verschiedener Zellen in der Hauptspeicheldriise von Myzus persicae. Entomologia exp. appl. 13, 153-161. Wensler, R. J. and Filshie, B. K. (1 969). Gustatory sense organs in the food canal of aphids. J. Morph. 129,473-492. Whitcomb, R. F. and Davis, R. E. (1970). Evidence on possible mycoplasma etiology of Aster Yellows Disease 11. Suppression of Aster yellows in insect vectors. Infect. Zmmun. 2,209-215. Williams, J . R. (1970). Studies on the biology, ecology and economic importance of the sugar-cane insect, Aulacaspis tegalensis (Zhnt.) (Diaspididae), in Mauritius. Bull. ent. Res. 60, 61-95.
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The Insect Blood-Brain Barrier J. E. TREHERNE AND Y . PICHON" A.R.C. Unit of Invertebrate Chemistry and Physiology Department of Zoology, University of Cambridge, England Introduction . . . . . . . . . . . . . . . . . . Organization of Insect Nervous Tissues . . . . . . . . . A. Extraneural Fat Body Deposits . . . . . . . . . . B. The Neural Lamella . . . . . . . . . . . . . . C. The Perineurium . . . . . . . . . . . . . . . D. Glial Cells, Neurones and Extracellular Spaces . . . . . 111. The Ionic Composition of the Haemolymph and Nervous Tissues . IV. Electrical Aspects of Nervous Function. . . . . . . . . . A. The Ionic Basis of Electrical Activity in Insect Nerve Cells . B. The Role of t h e Neural F a t Body Sheath . . . . . . . C. Neuronal Function in Intact Nervous Systems . . . . . D. Neuronal Function in Experimentally Treated Preparations . V. Exchangesof RadioactiveIonsand Molecules . . . . . . . VI. Concluding Remarks . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . . I. 11.
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I. INTRODUCTION
The concept of the vertebrate blood-brain barrier has been extant since the original observation by Paul Ehrlich, in 1885, that certain aniline dyes stained all the tissues of the body except those of the central nervous system. During the subsequent years this concept has been employed to explain the exclusion of a wide variety of ions and molecules from the central nervous system. The available evidence indicates, however, that this concept is not generally applicable to invertebrate animals. In most of the higher invertebrates which have been studied there appears to be no exclusion of small water-soluble substances from the central nervous system equivalent to that attributed to the blood-brain barrier of vertebrates (c.f. Treherne and * Senior Research Fellow of King's College, Cambridge. Present Address: Institut de Neurophysiologie, C.N.R.S., Gif-sur-Yvette (92), France. 251
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Moreton, 1970; Treherne, 1970a). This is well exemplified by the results of investigations on an annelid (Nicholls and Kuffler, 1964), a freshwater lamellibranch (Treherne et al., 1969; Lane and Treherne, 1972) and a marine crustacean (Abbott, 1969, 1970). Insect species, however, seem to constitute a ,notable exception to the above generalization for it has been repeatedly shown that changes in the chemical composition of the medium, bathing intact central and peripheral nervous systems are not reflected in equivalent rapid changes in neuronal function. This observation was first made by Hoyle (1952, 1953) when he demonstrated the delayed potassium depolarization of locust peripheral nerve and was confirmed for the central nervous system by Twarog and Roeder (1956) in their study of the cockroach abdominal nerve cord. These authors attributed this effect to the presence of a peripheral diffusion barrier which they identified with the connective tissue sheath that envelops the insect nervous system. Toxicological data (O’Brien, 1957; O’Brien and Fisher, 1958) and the relative insensitivity of insect ganglia (Roeder, 1948b) to extraneously applied acetylcholine, were also attributed to such a peripheral neural diffusion barrier (Twarog and Roeder, 1957, Yamasaki and Narahashi, 1960). The postulation of a peripheral diffusion barrier surrounding the central nervous system of insects although providing an explanation for the delayed effects of inorganic ions and pharmacologically-active compounds in intact (as compared with desheathed) preparations also presented some conceptual difficulties. In the absence of an intraneural circulating system it was not apparent, for example, how the supply of nutrient materials to the underlying nervous tissues could be achieved in the presence of such a peripheral diffusion barrier. A radioisotope study showed, however, that the uptake of trehalose and glucose molecules took place rapidly from the haemolymph in the abdominal nerve cord of Periplanetu americana (Treherne, 1960, 1961a). Rather unexpectedly, in the light of the various electrophysiological studies, the use of radioisotopes also revealed the existence of relatively rapid exchanges of monovalent and divalent cations between the blood and bathing medium and the central nervous tissues of this insect (Treherne, 1961b, c, d, e, f, 1962a, c). On the basis of the above results it was concluded that a dynamic steady state rather than a static impermeability must exist across the peripheral nerve sheath of P. americana (Treherne, 1961a, f). It was further speculated that the effects of the desheathing procedure on
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the electrical responses of connectives and ganglia produced, for example, by sodiumdeficient or high-potassium solutions might result from effects other than the removal of a peripheral diffusion barrier (Treherne, 1962a, b). In particular, it was suggested that enhanced rates of potassium depolarization obtained in desheathed preparations might result, in part at least, from changes to the extraneuronal environment resulting from the disruption of a postulated Donnan equilibrium between the extracellular fluid and the haemolymph (Treheme, 1962b, c; Pichon and Boistel, 1967). Much of the subsequent work confirmed and amplified both the original electrophysiological and the radioisotopic investigations (c.f. Treherne and Moreton, 1970), leaving what was essentially a paradoxical situation. Relatively recently, however, a number of studies, largely carried out in this laboratory, have gone some way towards the resolution of this paradox. It is the purpose of this review to consider the results of these recent investigations in relation to earlier work on the nature of the processes involved in the exchanges of ions and molecules between the haemolymph, or bathing medium, and the central nervous tissues. The implications of our conception of the insect blood-brain barrier are of importance for a variety of reasons. Our understanding of the mechanism of insecticidal action and, possibly of resistance, require a knowledge of the processes involved in the access of poison molecules to synaptic and axonal surfaces. The interpretation of neuronal function in intact central nervous tissues also requires a knowledge of the chemical composition of the fluid immediately bathing the neuronal surfaces, for such function is profoundly affected by the composition of the extraneuronal environment. This problem is particularly acute in some insect species which possess haemolymph of specialized ion concentrations, for example, with extremely low concentrations of sodium ions and surprisingly high levels of potassium and magnesium (c.f. Shaw and Stobbart, 1963; Treherne, 1966a; Usherwood, 1969). The axons of such largely phytophagous insects appear, however, to require relatively high concentrations of sodium ions for excitation and conduction (Treherne and Maddrell, 1967b; Weidler and Diecke, 1969; Treherne, 1971 ; Pichon et al., 1972). This “conventional” extraneuronal environment must be maintained by specialized physiological mechanisms within the central nervous tissues. As the maintenance of the appropriate concentration of sodium ions in the extraneuronal fluid ultimately depends upon exchanges with these
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cations in the haemolymph it is clear that these phytophagous insects present the possibility of some intriguing specializations in bloodbrain barrier function. As will become apparent in the succeeding pages the relatively simple organization of insect central nervous tissues offers some unique advantages for the study of blood-brain barrier phenomena as compared with vertebrate central nervous systems. In particular the use of the electrical response of nerve cells to act as indicators of the chemical composition of the extraneuronal fluid has enabled quantitative analyses to be made of the nature of the exchanges taking place between the haemolymph and the extraneuronal fluid. 11. THE ORGANIZATION OF INSECT NERVOUS TISSUES
The interpretation of physiological data on the exchanges of ions and molecules requires some consideration of the histology and ultra-structure of the insect nervous system. As some fairly detailed accounts are available (c.f. Smith and Treherne, 1963; Smith, 1967, 1968) consideration here will be limited to a brief account of the relevant physiological data presented in succeeding sections of this review. A. EXTRANEURAL FAT BODY DEPOSITS
In some insects the surfaces of the central nervous system are overlaid by scattered deposits of fat body cells. In the cockroach, P. urnericunu, these deposits are largely confined to small areas on the ganglionic surfaces (Smith and Treherne, 1963). In the latter species the fat body cells are closely applied to the underlying neural lamella, leaving little discernible intervening space. In other insect species, notably the stick insect, Curuusius morosus (Maddrell and Treherne, 1966), and the locust, Schistocerca greguriu (Boulton and Rowell, 1968), the fat body deposits have become modified to form a continuous sheath covering the central nervous system (Fig. 1). The cytological organization of these cells in C. rnorosus appears to be essentially similar to those in fat body deposits in other parts of the body (Lane and Treherne, 1971). In the latter species and S. greguriu this cellular sheath delimits an additional fluid compartment, the extraneural space, at the surface of the central nervous system. In conventionally-fixed material from C. rnorosus the extraneural space appears to be relatively large
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Fig. 1. Micrograph showing, in transverse section, the neural fat body sheath surrounding a connective (between pro- and meso-thoracic ganglia) in the nerve cord of Curausius morosus. The cylinder of fat body cells cfbc) is continuous, delimiting an underlying fluid compartment, the extraneural space (ens). The neural lamella, or perilemma ( p l ) , and the underlying cellular perineurium ( p n ) are seen as concentric sheaths surrounding the connective which contains the axons (ax). Several tracheal branches (fr) are seen in this section. (From Treherne and Maddrell, 1967a.)
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(Fig. 1) and has been estimated to represent approximately 7.4% of the combined volume of the fat body sheath, extraneural space and central nervous tissues (Weidler and Diecke, 1970a). Recent experiments using peroxidase (MW 40,000) as an extracellular marker suggests, however, that this relatively large extraneural space may be artefactual (Lane and Treherne, 197 1). It has been found, for example, that the fat body sheath was closely applied to the underlying neural lamella over fairly extensive areas. In these areas the restricted extraneural space (c.f. approximately 0.11 in width) was completely filled with the reaction product resulting from the presence of peroxidase molecules. In areas showing relatively capacious extraneural spaces, on the other hand, the distribution of the peroxidase was limited to thin deposits at the neural lamella and on the inner surface of the fat body sheath, the intervening space being devoid of reaction product. These observations have been tentatively explained in terms of appreciable cellular shrinkage, following peroxidase perfusion, during fixation (Lane and Treheme, 197 1). In C morosus the neural fat body sheath is absent from the peripheral nervous system (Lane and Treherne, 197 1). In this species the peripheral nerves are surrounded only by a discontinuous connective tissue layer. It has also been shown that the extraneural space is accessible to Indian Ink particles, to molecules as large as those of peroxidase (MW 40,000) and, apparently, to haemocytes (Lane and Treherne, 1971). The penetration of the peroxidase molecules to the surface of the nervous system clearly occurred via intercellular channels in the fat body sheath (Fig. 2). Quantitative analysis indicates that leakage via these intercellular spaces would preclude the possibility of any significant involvement of the fat body cells in extraneuronal sodium regulation as proposed by Weidler and Diecke (1969). Fig. 2a. Electron micrograph showing penetration of horseradish peroxidase into the intercellular spaces of the neural fat body sheath of Carausius morosus. The peroxidase was injected into the haemolymph 12 h prior to fixation. The reaction product is localized in the intercellular spaces formed by adjacent fat body cell membranes (S) as well as in the occasional pinocytotic vesicle or lysosome (arrows) within the fat body cytoplasm. Stained with lead citrate x 16,590. Fig. 2b. Section through the base of a neural fat-body cell in C. morosus showing its associated connective tissue (C) lying adjacent to the neural lamella (NL). The reaction product, corresponding to the distribution of peroxidase molecules, is shown in the connective tissue, the extraneural space (S) and in the neural lamella. Peroxidase was also taken up by pinocytotic vesicles in the fat body cells (arrows) and had penetrated the the perineurium (P) of the connective. Unstained x 23,600. (From Lane and Treherne, 1971.)
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B. THE NEURAL LAMELLA
Both the peripheral and central nervous systems are enclosed within this superficial connective tissue layer which, in the central nervous system at least, is closely associated with a specialized underlying cellular layer, the perineurium (Fig. 3). In most insects the neural lamella appears to consist of two structural elements. In an insect such as P. americana the outer zone consists of amorphous material with only faint indications of further organization; the inner one containing more obvious striated fibres (Hess, 1958; Ashhurst and Chapman, 196 1 ; Smith and Treherne, 1963). The fairly intensive investigation of recent years (using a variety of histological and biophysical techniques) indicates that the outer zone is mucopolysaccharide in nature, the inner one consisting of collagen fibrils embedded in a neutral mucopolysaccharide matrix (see Ashhurst (1968) for references). The present consideration of the neural lamella is relevant for three fairly obvious reasons. First, it is frequently assumed, for example by Ashhurst (1 968) and Ashhurst and Costin (1 97 l), that the permeability properties of the neural lamella may influence the access of ions and molecules into the underlying nervous tissues. Secondly, the mechanical properties of this layer are likely to be of importance in counteracting hydrostatic pressure resulting from Donnan forces operating in the underlying cellular and extracellular compartments. Thirdly, the presence of anion groups associated with the collagenous components of the nerve sheath are likely to be of importance as cation reservoirs. This factor is particularly relevant in the case of some lepidopteran nervous systems (Treherne and Moreton, 1970), for in Galleria mellonella (Ashhurst and Richards, 1964) and Manduca sexta (Pichon e t al., 1971b) the neural lamella Fig. 3a. The peripheral margin of an interganglionic connective of Carausius morosus. This transverse section shows the superficial fibrous neural lamella (ns). The tortuous path of an intercellular cleft (ic) formed by the lateral walls of the perineurial cells, can be followed from its opening beneath the fibrous sheath (arrow at upper right of picture) to the channel separating the perineurium from the underlying glial cells (arrow at left). The lateral walls are held together near the bases of the cells by septate desmosomes (sd) and “tight” junctions (tj). Microtubules (mt) are shown in the type I1 cells (11) and the darkly staining granules (g) together with the cluster of possible Golgi vesicles (ves) in the type I cell (I). The underlying extracellular system contains deposits of electrondense material (asterisks). Other visible structures include axons (ax) and glial, or Schwann cells (sc) x 34,000. Fig. 3b. High magnification electron micrograph showing the lateral walls of the perineurium held together at the inner margins of the perineurial clefts by septate desmosomes and “tight” junctions. x 81,000. (From Maddrell and Treherne, 1967.)
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and the associated dorsal connective tissue mass exceeds the volume of the underlying nervous tissues. As will be apparent from electrophysiological and radioisotopic data presented in succeeding sections of this article the first of the above factors is unlikely to be of significance in relation t o the blood-brain barrier phenomena for small water-soluble ions and molecules. The available structural evidence also appears to militate against the hypothesis of an appreciable restriction t o diffusion offered by the neural lamella. This is most convincingly shown in the penetration, observed at the ultrastructural level, of the large peroxidase molecules (MW 40,000) into the neural lamella of P. americana (Lane and Treherne, 1969, 1970), C. morosus (Lane and Treherne, 1971) and M . sexta (Lane, 1972) and in the apparent relatively free permeability of this layer t o charged water-soluble dye molecules seen in histological investigations (Wigglesworth, 1960; Eldefrawi et al., 1968). C. THE PERINEURIUM
The cellular layer of the nerve sheath, underlying the neural lamella, has been described in a number of histological and ultrastructural investigations (c.f. Smith and Treherne, 1963; Smith 1968; Treherne and Moreton, 1970). Maddrell and Treherne (1 967) recognized the following salient ultrastructural features in the perineuria of P. americana and C. morosus: (i) an extensive system of long, tortuous channels between the lateral walls of the perineurial cells, apparently opening at the outer margin immediately adjacent to the neural lamella; (ii) the presence of septate desmosomes and/or “tight” junctions between the lateral cell walls towards the inner margin of the perineurium; (iii) an increased surface area at the inner margin of the perineurial cells produced by the presence of long, inwardlyprojecting flanges and (iv) the presence of an electron-dense coat immediately beneath the plasma membrane of type I1 perineurial cells at the points of contact with the underlying extracellular system and at considerable areas of the outer surface adjacent to the neural lamella (Fig. 3). In a lepidopteran, M. sexta, the perineurium approximates to the
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above organization with the exception that the intercellular perineurial channels show considerable expansion at their outer margins underlying the neural lamella (Pichon et al., 1971b). These relatively large spaces contain material similar to that of the neural lamella (i.e. collagen-like fibrils embedded in an amorphous matrix). The perineurial surfaces in this species are attached to the neural lamella and to the connective tissue material in the enlarged extracellular spaces by hemi-desmosomes and to the underlying glial cells by desmosomes. The striking similarity of the perineurial organization with fluidsecreting epithelia (c.f. Diamond and Tormey, 1966; Berridge and Gupta, 1967) was noted by Maddrell and Treherne (1967). The use of peroxidase as an extracellular marker in an electron microscopic study has revealed that these molecules penetrate into the intercellular perineurial channels (Fig. 4) (Lane and Treheme,
Fig. 4. Electron micrograph showing penetration of peroxidase into the perineurial clefts of an abdominal connective of Periplanefa americana. The reaction product for this large exagenous protein is shown in the neural lamella (NL) and lying between the perineurial cells (arrows). x 61,560. (From Lane and Treherne, 1970.)
268
J. E. TREHERNE AND Y . PICHON
1969, 1970, 1971; Lane, 1972). An apparent restriction to the movement of peroxidase molecules was observed at the inner margin of these channels corresponding to the intercellular occlusions described by Maddrell and Treherne (1 967) (Fig. 3). Histological and ultrastructural examination has shown that the standard desheathing procedure employed by electrophysiologists involves substantial damage to the perineurium (Fig. 5 ) . The rupture of at least the outer perineurial membrane during the removal of the neural lamella has been described by Twarog and Roeder ( 1956) and Treherne et al. (1 970) in I? americana and by Treherne (1 97 1) in C morosus. The extent of this perineurial damage is illustrated by the extensive in tracellular invasion of perineurial and glial cytoplasm by peroxidase molecules in desheathed connectives of P. americana (Lane and Treherne, 1969, 1970) (Fig. 6). These ultrastructural observations also provide an explanation for the massive increase in the measured inulin space in desheathed preparations, for it follows that this molecule must also have penetrated extensively into the glial cytoplasm (Treherne, 1962a) despite the essentially normal ultrastructural appearance of the glial and neuronal elements seen in conventionally-fixed material (Fig. 5 ) . D. GLIAL CELLS, NEURONES AND EXTRACELLULAR SPACES
Wigglesworth ( 1960) has classified the sub-perineurial glial system, in the ganglion of P. americana, into two loosely-divided regions. These consist of a peripheral layer of cells, which invest the neuronal cell bodies, and deeper elements which form a complex system of processes in the neuropile. These two latter layers are separated by extensive extracellular spaces, the “glial lacunar system”. The organization of the glial elements in the interganglionic connectives correspond to the above neuropilar organization. Glial-axon relationships are encountered in insect central nervous systems in which a single axon is enveloped by a single glial process or in which a number of axons are surrounded by a single glial process or processes Fig. 5. Both electron micrographs show transverse sections of connectives from the abdominal nerve cord of Periplaneta americana. In both cases the neural lamella (N)and perineurium (P) are shown ensheathing the underlying axons (A), glial elements (G) and a mesaxon (MI. (a) is from an intact connective, while (b) shows the effects of the conventional desheathing procedure employed by neurophysiologists. It will be seen that at the point of removal of the neural lamella there is substantial damage to the perineurium. The underlying glia and axons, however, appear essentially similar to those observed in intact connectives. x 10,000. (From Treherne e t aL, 1970.)
270
J. E. TREHERNE A N D Y. PICHON
Fig. 6a. Unstained section from the neuropile in a desheathed preparation of the terminal abdominal ganglion of Periplaneta americana incubated for 30 min in horseradish peroxidase. The distribution of the electrondense reaction product indicates that the peroxidase had penetrated deeply into the ganglion, being situated in the glial elements (G) which ensheath the neurones (N). x 17,000. Fig. 6b. Section, between adjacent glial cell processes in a desheathed ganglion which had been incubated in horseradish peroxidase for 1 h prior to fixation. Note the “tight” junctions between the glial cells. x 69,500. (From Lane and Treherne, 1970.)
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(Fig. 7) (c.f. Smith and Treherne, 1963; Treherne and Maddrell, 1967b; Pichon e t al., 197 1b). In conventional electron micrographs of cockroach connectives the above glial-axon relationships delimit an extracellular system which consists of a complex three-dimensional network of narrow
Fig. 7. Diagrammatic representation of the relationship between axons and the ensheathing gIiaI cells in the central nervous system of P. urnericunu. A larger axon (ax) is invested in a concentric glial or Schwann cell sheath (sc). The mesaxon invagination (mx), which defines a channel 100-150 A in width, follows an approximately spiral course and contains frequent dilatations (d). The dilatations and the &ger extracellular spaces (ECS) contain an electron-dense material (*), most probably acid mucopolysaccharide.The smaller axons may be associated with separate mesaxon invaginations, as between the long and short arrows at ax, (From Smith and Treherne, 1963.)
272
J. E. TREHERNE A N D Y. PICHON
intercellular channels with occasional larger spaces. Of particular interest is the relation of the glial processes with the giant axons of P americana which have been intensively studied during recent years. The glial cell associated with each giant axon is spirally arranged (as illustrated by ax in Fig. 7). The extra-axonal space, formed by the closely applied innermost glial membrane, is restricted being only approximately 200 A in width. The extra-axonal fluid appears to communicate with the mesaxon channel which spirals six or seven times around each giant axon (see also Fig. 7). Measurements from electron micrographs show that the length of a mesaxon channel averages 770p (Treherne et al., 1970). “Tight” junctions are frequently observed between adjacent glial membranes in the mesaxon. These appear to represent localized regions of Cell contact and d o not apparently occlude the intercellular channel linking the extra-axonal fluid with the outer end of the mesaxon channel (Treherne et al., 1970). Intercellular communication be tween the open end of the spiral mesaxon channel and the perineurium is achieved via the complex and ramifying system of, in general, narrow channels illustrated in Fig. 7 . The diffusion pathway from the haemolymph to the surface of a giant axon thus consists of four major portions: first, the intercellular perineurial channels; secondly, the complex network of channels formed by the glial membranes and peripheral axons; thirdly, the long spiral mesaxon channel and, finally, the extra-neural space delimited by the innermost glial membrane and the axolemma itself (Fig. 8). Quantitative prediction of the time course for the unrestricted diffusion of an inorganic cation, such as potassium, along this pathway has been attempted (Treherne et al., 1970). As will be apparent from the predicted time course (Fig. 9) for the change in extra-axonal potassium concentration, following elevation of the external potassium concentration the greater part of the diffusion pathway is accounted for by the extended mesaxon channel, which with a length of 7701.1 corresponds to a half-time of between 124.8 s (Curve A) and 154.9 s (Curve B). An additional 20p pathway (roughly equivalent to a peripheral intercellular pathway from the perineurium to the opening of the mesaxon channel) has a relatively small effect, the calculated half-time including this additional component being 158.1 s (Curve C). The relevance of these theoretical values to experimental observations on the central nervous system of P. americana will be considered in a subsequent section (p. 290).
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Fig. 8. Schematic representation of the organization of the tissues in the penultimate abdominal connective of Periplunetu umericuna, showing the appearance in histological and ultrastructural detail. Also included is the edge of a giant axon with a portion of the spirallyor neural lamella, which is directly bathed with the insect’s blood, and the underlying cellular perineurium. Smaller axons, with ensheathing dial processes, are shown in ultrastructuraldetail. Also included is the edge of a giant axon with a portion of the spirallyfolded mesaxon. The extended mesaxon channel is confluent with the complex network of interconnecting extracellular spaces and channels which lie between it and the perineurium.
A structural feature of the glial system which is likely to be of functional importance is the apparent linkage of the cytoplasm of glial processes by numerous “tight” junctions between adjacent glial membranes. These specialized membrane contacts have been described in the central nervous systems of P. umericunu (Lane and Treherne, 1969, 1970), Locustu migrutoriu (Schiirmann and Wechsler, 1969) and M. sextu (Pichon et ul., 1972). It has been suggested (Lane and Treherne, 1969, 1970) that these “tight” junctions may represent the low-resistance contact between glial cells demonstrated electrophysiologically in the leech (Kuffler and Potter, 1964). The possibility also exists that the extensive penetration of the glial cytoplasm by molecules as large as those of peroxidase (MW 40,000) from locally desheathed areas of cockroach ganglia (Fig. 5 ) AIP-12
274
4
J . E. TREHERNE AND Y. PICHON
Curve A to.5 = 125 sec Curve B to.5 = 155 sec Curve C to.5 = 158 sec
--
Time (t sac)
Fig. 9. The predicted time course for the change in extra-axonal potassium concentrations (Ca), following elevation of the external potassium concentration of the bathing medium (at time t = 0) from zero to C, in the cockroach nerve cord. The three theoretical curves illustrate the cumulative effects of three sections of the diffusion pathway as follows: (A) mesaxon channel, of 770p,alone, (B) mesaxon channel with terminating reservoir formed by the extra-axonal space and (C) with an additional 20p intercellular pathway (represented by the extracellular channels formed by the closely apposed glial and peripheral axon membranes) added to (B). (From Treherne et QL, 1970).
may be mediated by this intracellular pathway (Lane and Treherne, 1969, 1970; Treherne, 1970b).* An additional structural feature which may be of importance is the presence of extracellular amorphous or granular material of appreciable electron density. In the cockroach this material appears generally to pervade the extracellular system, being found in the large dial lacunar spaces as well as in the relatively restricted mesaxon dilatations (Smith and Treherne, 1963). Histochemical evidence indicates that this extracellular material is probably acid mucopolysaccharide (Ashhurst, 1961). In M. sexta, on the other hand, the large extracellular spaces do not contain equivalent deposits of electron opaque material (Pichon et al., 1972). 111. THE IONIC COMPOSITION OF THE HAEMOLYMPH
AND NERVOUS TISSUES
The ionic composition of insect haemolymph shows considerable variation at higher taxonomic levels (c.f. Wyatt, 1961; Shaw and * Although it should be borne in mind that the extreme complexity of the glial system makes it difficult to exclude the possibility that the sequential appearance of peroxidase in adjacent glial processes may have largely resulted from diffusion within the cytoplasm of a single glial cell.
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Stobbart, 1963; Sutcliffe, 1963; Usherwood, 1969). In general the sodium/potassium ratio tends to be lower than in the body fluids of other arthropods and in certain orders (e.g. Coleoptera, Lepidoptera and Phasmida) the absolute level of sodium in the haemolymph may be exceedingly low, approaching only a few millimoles/litre in some cases. The potassium concentration of some species, on the other hand, can be relatively high and can be associated with bizarre concentrations of divalent cations. This is well exemplified by a coleopteran, Timarchia tenebriosa, in which haemolymph sodium concentration has been reported to be as low as 1.6 mM/l (Table I and Duchlteau et al., 1953). Considerable individual variation is encountered in the ionic composition of haemolymph, depending upon the nutritional state of the insects (c.f. Hoyle, 1954; Pichon and Boistel, 1963a, b). In addition variations in concentrations of both monovalent (Na' and K') and a divalent cation (Ca2') has been reported in the haemolymph taken from different parts of the body of P. americana (Pichon, 1970a). This effect does not appear to result from variations in haemocyte density and must, therefore, be attributed to changes in the cation concentration of the plasma. It should be borne in mind, however, that evidence is available of a sequestration of a significant proportion of the haemolymph potassium in the haemocytes of P. americana (Brady, 1967a, b). An estimate of the intracellular concentration of sodium and potassium ions of cockroach giant axons has been made using voltage-clamp data (Pichon, unpublished). This yielded values of axoplasmic concentrations of 24.4 mM / 1 Na' and 180.0 mM / 1 K'. Thus despite marked differences in the absolute concentrations of these cations in the body fluids, as compared with cephalopods or the frog, the gradients for both of these cations across the giant axon membrane of the cockroach approximate to those for the node of Ranvier and the cephalopod giant axon. The other available estimates of the ionic composition of insect nervous tissues were obtained by direct chemical determination of whole nerve cords and thus only provide gross values of the concentrations of ions distributed between both intracellular and extracellular tissue compartments. These are summarized in Table I, together with the ionic composition of the haemolymph of species which have been studied in relation to neuronal function. It should be noted that in the cases of C. morosus and Romalea microptera the sodium concentration of the central nervous tissues are approximately equivalent to those of P. americana despite the relatively low
Table I The ionic composition (mM/kg) of the haemolymph and central nervous tissues (where available) of insect species mentioned in the text Species
Tissue
Na'
Dictyoptera Periplaneta americana (Adult)
Haemolymph Nerve cord Nervecord
156.5 60.9 75.6
7.7 107.4 132.1
-
Cheleutoptera Carausius morosus (Adult)
Haemolymph Nervecord and fat body sheath
15.0 63.8
18.0 313.4
53.0 22.1
7.5 30.2
101.0 -
-
Haemolymph
60.0
12.0
24.8
17.2
97.6
Haemolymph Nerve cord
56.5 69.5
17.9 89.0
-
-
Haemolymph
25.4
25.1
-
-
Haemolymph
13.9
38.7
50.3
Haemolymph
1.6
46.9
158.0
Orthoptera Locusts migratoria (Adult) Romalea microptera (Adult) Lepidoptera Manduca sexta (Adult) Bombyx mori (Larva) Coleoptera Timarchia tenebriosa (Larva)
K'
Mg2'
5.3 2.9
Caz+
C1-
4.2
144.4
P043-
HC03- SO,"-
-
-
-
-
-
-
-
-
-
-
16.0
5.0
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
18.1
21.0
-
-
4.9
82.0
-
-
-
-
-
~
References Asperen and Esch (1956) Tobias (1948a) Treherne (1 9 6 1b) Wood (1957) Treherne (1965b)
Duchhteau et al. (1953) Tobias (1948b) Tobias (1 948b) Pichon et al. (1972) Buck (1 953)
Duchhteau e t al. (1953)
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concentration of this cation in the haemolymph of the former species. IV. ELECTRICAL ASPECTS OF NERVOUS FUNCTION A. THE IONIC BASIS OF ELECTRICAL ACTIVITY IN INSECT NERVE CELLS
The axons of the relatively few insect species which have been studied appear to be “conventional” in that the ionic basis of excitation and conduction seems t o be essentially similar to that proposed for the classical cephalopod giant axon preparation (c.f. Hodgkin, 1964). Experiments with intracellular electrodes in desheathed preparations indicate that both the giant axons of €! americana (Boistel and Caraboeuf, 1954: Yamasaki and Narahashi, 1958, 1959) and the neurones of C. morosus (Treherne and Maddrell, 1967a) are profoundly affected by the potassium concentration of the bathing medium, suggesting that this cation plays a dominant role in determining the resting potential. However, both the resting axon and neurone membranes do not, apparently, function as ideal potassium electrodes. Not only did the measured resting potentials show some departure from the calculated potassium equilibrium potentials (c.f. Treherne, 1966a) but also showed only a 42.0mV slope in P. americana (Yamasaki and Narahashi, 1959) and a 37.0 mV one in C. morosus (Treherne and Maddrell, 1967b) for decade change in external potassium concentration instead of the 58 mV slope predicted by the Nernst equation. Sucrose-gap measurements have also shown that decade change in external potassium concentration yields slopes of approximately 42 mV in desheathed connectives of M . sextu (Pichon e t al., 1972) and P. americana (Schofield and Treherne, unpublished). As suggested by Narahashi (1963) the departure from the theoretical relation predicted for an ideal potassium electrode implies that the conductance of other ions are involved to a certain extent in determining the resting potential. It is of some interest, therefore, to record an apparent involvement of sodium ions in determining the resting potentials of desheathed connectives of M. sextu measured using the sucrose-gap method, a 16 mV slope being obtained for decade change in external sodium concentration (Pichon et al., 1972). The possibility of some electrogenic component, as shown for example, in Aplysia neurones (c.f. Carpenter and Alving, 1968) also cannot be eliminated at this stage.
278
J . E. TREHERNE AND Y . PICHON
All the available evidence indicates that in cockroach giant axons the action potential is largely mediated by sodium ions. Thus, the amplitude of the action potentials measured in desheathed connectives is closely related to the sodium concentration in the bathing medium (Boistel and Caraboeuf, 1958; Yamasaki and Narahashi, 1959). Voltage-clamp studies on isolated giant axons have also confirmed that the inward current responsible for the rising phase of the action potential is largely carried by sodium ions (Pichon, 1968). Furthermore, the specific action of tetrodotoxin and TEA confirm the essential similarity of the cockroach with that of the squid giant axon (Pichon, 1969a, by 1970b). Of particular interest in the present context are the observations which indicate that the action potentials of insects, possessing specialized haemolymph of low sodium concentrations, are apparently nevertheless mediated by sodium ions. In the case of both C. rnorosus (Treherne and Maddrell, 1967b; Weidler and Diecke, 1969; Treherne, 1971) and M. sextu (Pichon et al., 1972) there is a rapid decline of the action potentials recorded in desheathed preparations maintained in media of sodium concentrations equivalent to that of the haemolymph or in sodium-free solutions. Furthermore, in both species action potentials were blocked in desheathed preparations in the presence of dilute tetrodotoxin (Treherne and Maddrell, 1967b; Weidler and Diecke, 1969; Pichon et ul., 1972) and, in the case of C. morosus, procaine (Weidler and Diecke, 1969). The lack of effect of manganous ions in desheathed preparations has also been advanced as evidence of sodium-mediated action potentials in C. rnorosus (Weidler and Diecke, 1969), since this substance has been shown to suppress the increase in membrane conductance to calcium during propagation of authentic calciummediated action potentials in other excitable tissues (c.f. Hagiwara and Nakajima, 1966). B. THE ROLE OF THE NEURAL FAT BODY SHEATH
The suggestion has been made, (Weidler and Diecke, 1969, 1970a, b) that the neural fat body sheath (Fig. 1) which envelops the central nervous system of C rnorosus (Maddrell and Treherne, 1966) functions in the regulation of the extraneuronal sodium concentration. The former authors envisage that the fat body cells of this extraneural structure transport sodium ions so as to maintain a high sodium concentration (ca. 150-180 mM/ 1) in the fluid bathing
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the surface of the central nervous system relative to the low concentration (15.0 mM/1) of this cation in the haemolymph. Weidler and Diecke specifically exclude the involvement of glial elements in extraneuronal sodium regulation. It will be apparent from the preceding sections that some form of extraneuronal sodium regulation must be postulated to account for the ability of the sodiumdependent axons of C. morosus (p. 277) to function in haemolymph of such low concentration (p. 276). The electrophysiological evidence on which Weidler and Diecke base their hypothesis is largely derived from so-called “viability studies”. These involved the measurement of the rates of deterioration of axonal function in the presence and absence of the neural fat body sheath in isolated connectives bathed with a physiological solution containing 18.0 mM/ 1 Na’. The more rapid deterioration obtained in the absence of the neural fat body sheathing was attributed by Weidler and Diecke to the removal of a sodiumtransporting mechanism associated with the. fat body cells. The available evidence, both structural and physiological, would seem to militate against the above hypothesis. As has already been mentioned (p. 266) the neural fat body sheath is a relatively leaky structure, allowing intracellular penetration of peroxidase molecules into the underlying extraneural space (Fig. 2) (Lane and Treherne, 1971). Now a general feature of ion transporting epithelia is the presence of lateral occlusions which, by restricting passive intercellular ion movements, reduce such short-circuiting effects on the actively transported ions (c.f. Keynes, 1969). The absence of such restrictions to intercellular diffusion in the neural fat body sheaths has been calculated to involve the postulation of inappropriately high rates of sodium transport even in comparison with specialized ion transporting epithelia let alone with such relatively unspecialized cells as those of the insect fat body (Lane and Treherne, 1971). A recent electrophysiological study has also controverted the hypothesis that the neural fat body sheath functions as a significant organ in extraneuronal sodium regulation. In this study locally desheathed areas of connectives were used as indicators of the nature of the ionic environment underlying the neural fat body sheath in C rnorosus (Treherne, 1972). It was found that the axonal response to solutions of equivalent sodium concentration as the haemolymph (1 5 mM / 1) was essentially similar to that recorded in desheathed connectives in the absence of the fat body sheath (Fig. 10). The rapid decline in the amplitude of the recorded action ,potentials
280
J. E. TREHERNE AND Y. PICHON 180 mM/l Na+
15 mM/l N a +
180 mM/l Na’
20
10
30
Time (min)
Fig. 10. The effects of normal (15.0 mW1) and elevated (180.0 mM/1) sodium concentration on the amplitude of action potentials recorded in a locally desheathed connective contained within an intact length of neural fat body sheath. (From Treherne, 1972.)
observed on changing from elevated sodium concentration (1 80 mM / I ) to 15 mM / 1 thus confirms both the leaky nature of the neural fat body sheath and the inability of the fat body cells to maintain a high sodium environment at the outer surface of the central nervous system in C. morosus. The postulated ion regulatory role of the neural fat body sheath in any case represents an unnecessary complication for this structure is absent from the peripheral nerves in C rnorosus (Lane and Treherne, 1971) and is also not present in a phytophagous species such as M. sextu, which also has a low-sodium haemolymph and a requirement for a relatively high extra-axonal sodium level to maintain axonal function (Pichon e t al., 197 1b). The lack of correlation between the presence of a neural fat body sheath and the possession of low-sodium haemolymph can be further illustrated by the cockroach, Bluberus gigantea. which has a neural fat body that envelops both the central nervous system and the cercal nerves, but has “conventional” haemolymph with a relatively high sodium concentration (Pichon, unpublished observations). Boulton and Rowel1
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28 1
(1968) have also emphasized the lack of correlation between the structure of the extraneural sheath and any obvious features of the ionic composition of the haemolymph for some Orthopteran and Dictyopteran species. As emphasized by Lane and Treherne (1971) it is very easy to interpret the electrophysiological observations of Weidler and Diecke (1969) without the necessity of postulating the neural fat body sheath as the primary site of extraneuronal sodium regulation in C morosus, Thus, for example, the reduced “viability times” observed in the absence of the neural fat body sheath could most reasonably be attributed to the effects of tracheal damage caused by its removal. Alternatively, it is possible to explain the effects of the removal of the neural fat body sheath in terms of the disruption of perineurial intercellular occlusions which apparently restrict the access of inorganic ions to the underlying extracellular system (Treherne et al., 1970) (see p. 285) and which, in the cockroach at least, appear to be extremely sensitive to experimental treatment (Pichon and Treherne, 1970) such as any slight stretching during the removal of the fat body sheath. In the apparent absence of a significant role of the neural fat body sheath it seems reasonable to attribute a primarily trophic role to this structure (Lane and Treherne, 1971) such as has earlier been suggested for the scattered fat body deposits associated with the nerve cord of P. americana (Smith and Treherne, 1963). In the light of the above considerations it is clear that in C morosus, the regulation of the extraneuronal sodium concentration must be mediated within the central nervous system of C morosus, as was originally proposed (Treherne and Maddrell, 1967b; Treherne, 1967). C. NEURONAL FUNCTION IN INTACT NERVOUS SYSTEMS
The axonal response to alterations in the chemical composition of the bathing medium in intact preparations shows profound differences when compared with desheathed preparations or isolated axons. This effect is manifested in the delayed potassium depolarization observed in insect peripheral nerve (Hoyle, 1952, 1953), in central nervous connectives (Twarog and Roeder, 1956; Treherne, 1965a; Pichon and Boistel, 1967; Weidler and Diecke, 1969; Treherne et al., 1970; Pichon and Treherne, 1971) and ganglia (Roeder, 1948a, b; Twarog and Roeder, 1956; Treherne, 1962b;
282
J . E. TREHERNE AND Y . PICHON
Pichon and Boistel, 1965). An equivalent delay has also been observed in the decline in the amplitude of action potentials in intact preparations bathed in sodium-deficient saline (Twarog and Roeder, 1956; Treherne, 1965a; Treherne and Maddrell, 1967b; Weidler and Diecke, 1969; Treherne et al., 1970; Pichon et al., 1972) and in the effects of externally applied acetylcholine (Roeder, 1948b; Twarog and Roeder, 1956,1957). 0 sec
20 sec
I min
10 min
-
I.O msec
Fig. 11. Intracellularly recorded action potentials (from giant axons in an intact, unstretched cockroach abdominal connective) following exposure to a bathing solution in which sodium was replaced by potassium ions. (From Treherne et aZ., 1970.)
More recent experiments have revealed the existence of an apparent rapid depolarization, recorded intracellularly within giant axons, in intact cockroach connectives following elevation of the external potassium concentration (Treherne et al., 1970; Pichon and Treherne, 1970, 197 1). This apparent depolarization was not associated with any equivalent reduction in amplitude of the action potentials (Fig. 1 1). Essentially similar electrical responses were obtained when the tip of the microelectrode was withdrawn into an apparently extracellular position (Fig. 12). It is clear from the above observations, that the rapid potential changes associated with the elevation of the external potassium level could not have resulted from a significant reduction in the resting potential of the giant axons. It also follows that this effect must have resulted from the interpolation of an additional potential between some portion of the extracellular system and the bathing medium. The presence of such an extraneuronal potential, together with the relatively slow depolarization of the giant axons in response to an increased external potassium concentration, indicates that a considerable degree of restriction must occur in the penetration of these cations to the axon surfaces in intact preparations. The extent of this restriction can be illustrated by reference to the half-time of approximately 158 s (Treherne et al., 1970) calculated for the free diffusion of potassium ions along the tortuous and extended
INSECT BLOOD-BRAIN BARRIER
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a
2 1 4 . n K+ ~
-10 -20
-
-30
-
n 214.0 mM K+
-40 -
>^
5
-50
-
-60-
5 min
Fig. 12. Continuous recordings of the potential changes obtained during exposure of intact, unstretched cockroach connectives to a high-potassium/sodium-free solution. (a) Intracellularly recorded potential from a giant axon: (b) potential changes recorded with the tip of the microelectrode in an extracellular position. (From Treherne et aZ., 1970.)
extracellular diffusion pathway to the giant axon surfaces (see p. 272). Extraneuronal potential changes were also observed in response to the substitution of other inorganic and organic cations for the normal (2 14.0 mM / 1) sodium content of cockroach physiological saline (Pichon et al., 1971). Substitution of tris or choline ions was shown, for example, to result in potential changes of reversed polarity to those observed with potassium ions (Fig. 13). The relative potential changes resulting from the presence of these cations in the bathing medium is summarized in Fig. 14. It will be seen that it is possible to arrange the monovalent cations that were tested in the following sequence of effectiveness:
K' > Rb' > Cs' > TEA' > Na' > Li' > Choline' > Tris'
284
J. E. TREHERNE AND Y. PICHON
+40 -
+50
7
E +30-
1 (Y M
; +20c1
c
Y
+lon
0-10
-
-20 Minutes
Fig. 13. The effects of the substitute of tris and choline ions for those of sodium on the extraneuronal potentials measured using the “sucrosegap” technique. The potential changes produced by a high-potassium solution (214 mM/1) are included for comparison. (From Pichon etal., 1971.)
Fig. 14. The relative potential changes produced by substitution of various monovalent cations for those of sodium in the bathing medium. The potential changes are expressed as a fraction of the positivation produced by potassium ions. The vertical lines indicate the extent of twice the standard error of the mean. (From Pichon etal., 1971.)
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285
This order corresponds to sequence IV in the classification devised by Diamond and Wright (1 969) to describe the relative potencies of alkali cations in biological systems, that is:
K'
> Rb' > Cs' > Na' > Li'
This sequence also approximates to that deduced from the measurement of the relative potential changes produced at the inner surface of the frog skin (Lindley and Hoshiko, 1964), differing only in the greater potency of Li' relative to Na'. The similarity between the results obtained with the cockroach and the potential changes produced by substitution of other cations for those of sodium in the anuran skin provides a clue as to the nature of the insect extraneuronal potentials. In the frog skin changes in the ionic concentration are thought to affect only one of two membrane surfaces, due to the restriction to diffusion offered by lateral intercellular occlusions (cf. Ussing, 1965; Keynes, 1969). Lateral occlusion has also been observed'in the form of "tight" junctions at the inner margin of the intercellular perineurial channels in both C. morosus, P. americana and M . sexta (Maddrell and Treherne, 1967; Lane and Treherne, 1970; Pichon e t al., 1971b). A restriction to the inward diffusion of peroxidase molecules has also been observed towards the inner margins of the perineurial channels (see p. 268). It can thus be readily envisaged that in the insect perineurium, as in the frog skin, the effects of any rapid change in the ionic composition of the bathing medium would be largely confined to only one of two membrane surfaces. In the case of high external concentrations of potassium for example, there would thus be a more pronounced depolarization of the outwardly-directed perineurial membranes, the equivalent depolarization of the inwardly-directed one being reduced by the presence of the occluded regions at the inner ends of the perineurial clefts. As pointed out by Pichon et al. (1971) such a system would produce rapid potential changes, which can be measured using microelectrodes located in the giant axons in the absence of a significant depolarization of the axonal membranes. The above system can also be employed to explain the extraneuronal potential changes obtained on substitution of other cations for those of sodium in the bathing medium (Pichon e t al., 1971). The negative potential change observed in the presence of tris and choline (Fig. 13) can, for example, be cxplained in terms of an efflux of intracellular sodium ions through the outer perineurial membrane in the absence of this cation in the bathing medium. This
286
J. E. TREHERNE A N D Y. PICHON
postulated hyperpolarization would thus be essentially similar to that produced in gastropod neurones by substitution of organic cations for those of sodium in the bathing medium (Moreton, 1968a, b; Sattelle, 1971). It has, in addition, been pointed out (Pichon et al., 1971) that the extraneuronal potential changes induced in the presence of high external concentrations of rubidium and lithium ions (Fig. 14) also parallel the effects produced by these cations on excitable cells (c.f. Sjodin, 1959). The relatively small effects produced by TEA' and lithium ions can be related to their similarity to those of sodium. Thus it has been observed that both TEA' (Binstock and Lecar, 1969; Pichon, unpublished) and Li' (c.f. Chandler and Meves, 1965) can partly substitute for sodium ions in nerve membranes. Despite the qualitative similarities observed between the insect perineurial system and the frog skin preparation which have been emphasized earlier there are some quantitative differences between the insect and the anuran systems (Pichon et al., 1971). Alteration of the potassium concentration at the inner surface of the frog skin .has been shown, for example, to result in the 58 mV slope for decade concentration change predicted by the conventional Nernst relation (Koefoed-Johnsen and Ussing, 1958). The departure of the cockroach extraneuronal potentials from that predicted for an ideal potassium electrode is illustrated by the 17 mV slope obtained for decade change in potassium concentration of the bathing medium
+ 30
c
I
, I
/
I I I 1 I l l I J 6 810 20 40 6080100 200 400 Potassium concentration of bathing solution (mM/I)
I
2
'
4
Fig. 15. The relation between extraneuronal potential changes and the potassium concentration of the bathing medium. The broken line illustrates the 5 8 m V slope for decade concentration change predicted by the Nernst equation. (From Pichon e l oL, 1971.)
287
INSECT BLOOD-BRAIN BARRIER
(Fig. 15). The considerable departure of the extraneuronal potentials from the values predicted by the Nernst equation can be accounted for by the assumption that the perineurial “tight” junctions exhibit a finite ion permeability (Pichon et al., 1971). On the basis of a theoretical model system consisting of a perineurial diffusion barrier in series with an extended narrow channel (represented by the restricted intercellular spaces joining the perineurium to the extraaxonal space) it was concluded that the relative ion permeabilities of the diffusion barrier would be: P,: P,: PCholine=1.0: 0.19: 0.11 This theoretical analysis, using the constant-field theory to estimate ion fluxes at the diffusion barrier, showed that the behaviour of the potassium concentration at the inner surface of the barrier approximates to that required t o explain the observed potential changes (Fig. 16). IOOr,.I
I
I
I
I
I
I
I
I
1
.
1
I
I
I
I 200
90
180
80
160
70
140
. 60
120
50
100
40
80
30
60
20
40
10
20
-
*
.
0 t
(min)
Fig. 16. The movements of ions in a model system based on that of a peripheral diffusion barrier in series with a long narrow channel at the inner end of which is the giant axon (Fig. 8). The barrier is presumed to represent either the cell membranes of the perineurial cells, the “tight” junctions at the inner ends of perineurial clefts or a combination of the two. The external concentration is changed instantaneously at time t = 0. (a) and (b): Potassium concentrations at peripheral and axonal ends respectively of the extracellular system, following an increase of the external concentration from 3.1 to 100 mM/1. (c): Corresponding behaviour of the potential difference across the peripheral barrier. (d): Sodium concentration at the axonal end of the system, following a decrease of the external concentration from 200 mM/l to zero, sodium ions being replaced by those of choline. The left-hand scale of ordinates in mM/1 refers to curves (a) and (b); the scale of mV to curve (c) and the right-hand scale of mM/1 to curve (d). The arrows indicate half-times for potassium uptake and sodium efflux. (From Pichon et al., 1971.)
288
J. E. TREHERNE AND Y. PICHON
Very occasionally (in two experiments out of several hundred) larger extraneural potential than those illustrated in Figs 12 and 13 are encountered (Schofield and Treherne, unpublished; Tucker and Pichon, 1972). These maximal values yield slopes of approximately 58 mV for decade change of external potassium concentration. In these rare preparations it would appear that the leak component into the subperineurial extracellular system would be minimal and that the outwardly-facing perineurial membrane approximates to a potassium electrode. It is not clear, however, whether this represents a specialized physiological condition or whether the more commonly encountered extraneuronal potential changes are correlated with preparations in which intercellular leaks have been induced by the experimental procedure. More recent experiments on the central nervous system of M. sextu have revealed that an essential similarity exists between the physiological organization of the central nervous system of this lepidopteran and that outlined above for P. americana (Pichon e t ul., 1972). There appears to be a restricted access of inorganic ions to the axon surfaces in intact connectives which, as in P. americanu, are associated with the development of appreciable extraneuronal potential changes following elevation of the external concentration of potassium ions. The restricted access of inorganic ions to the extra-axonal fluid makes it easier to envisage physiological mechanisms by which a different ionic environment is maintained at the axon surfaces in comparison to the blood or bathing medium of the sodium ion in phytophagous insects, such as C morosus and M. sextu, in which the extra-axonal sodium concentration may be an order greater than that of the blood or bathing medium (c.f. Treherne and Maddrell, 1967b; Pichon et al., 1972). Differences in the ionic concentration of the extra-axonal fluid and the blood or bathing medium can also be inferred from observations on cockroach giant axons (Pichon and Boistel, 1967). This is manifested in the lower resting potentials (58.1 k 5.4 mV) observed in intact as compared with desheathed connectives (67.4 f 6.2 mV) and in the bigger action potentials (103.0 5.9 mV) observed in the former as compared with the latter preparations (85.9 k 4.6 mV). Significantly higher resting potentials were also observed in C morosus in connectives from which the neural fat body sheath was removed (Treherne and Maddrell, 1967a). In view of the apparent sensitivity of connectives to experimental handling the possibility cannot be eliminated that in the latter
*
INSECT BLOOD-BRAIN BARRIER
289
species this effect may have resulted from perineurial damage during the removal of the fat body sheath, thus implying that this phenomenon is similar to that in P. americana. Pichon and Boistel (1967) suggested that the “sheath” or “extracellularyy potentials might be an artefact determined by variations in the microelectrode tip potential between different ionic solutions. However, it is unlikely that the difference in the amplitude of the action potentials measured between desheathed and intact preparations would be affected in this way. The possibility remains that the greater amplitude of the action potentials in the intact preparations results from extra-axonal sodium concentrations which exceed those in the bathing medium. Treherne and Moreton (1970) have pointed out that (taking account of the average positive extracellular potential of 6.7 mV measured by Pichon and Boistel) there would be a difference of 19.7 mV between the overshoot measured in giant axons in desheathed and intact preparations. As the active membrane potential of cockroach giant axons shows a 48 mV increase for decade increase in external sodium concentration (Yamasaki and Narahashi, 1959) it can be calculated that concentration of sodium ions at the axon surfaces would exceed that of the bathing medium (210.2 mM/1) by a factor of 2.3. This would thus imply that the extra-axonal sodium concentration would be in the region of 483.0 mM/l in intact preparations. The possibility of secondary effects on the giant axons, produced by the desheathing procedure, would make it necessary to view this conclusion of Treherne and Moreton with some degree of caution. However, the results of Pichon and Boistel (1967) certainly suggest the possibility that the extra-axonal fluid may possess a sodium concentration which is very much higher than that of the haemolymph or bathing medium. The possible significance of these conclusion will be discussed later in this review (p. 304). D. NEURONAL FUNCTION IN EXPERIMENTALLY TREATED PREPARATIONS
In experiments in which intact cockroach connectives were maintained under tension or were dried and briefly exposed to air only relatively small extraneuronal potentials were observed in the presence of high external potassium concentrations (Treherne et aL, 1970; Pichon and Treherne, 1970). In these preparations the rate of potassium depolarization of the giant axons also occurred more rapidly: the half-time for the accumulation of potassium ions in the
290
J. E. TREHERNE AND Y. PICHON
extra-axonal fluid being estimated to average 173.0 k 22.6 s. The latter values approximate to the theoretical figure of 158.1 s calculated for the free diffusion of potassium ions from the perineurium through the extended intercellular pathway represented by the mesaxon and the overlying intercellular clefts (see p. 272). On the basis of the similarity of the theoretical and experimentally determined values, it was concluded that there was little appreciable restriction to the movement of potassium ions from the bathing medium to the giant axon surfaces in stretched preparations (Treherne et al., 1970). It was further concluded that the absence of appreciable extraneuronal potentials, correlated with relatively unrestricted extracellular diffusion, resulted from the disruption of the peripheral diffusion barrier, tentatively identified with the perineurial “tight” junctions. The apparent increased access of potassium ions to the s u b perineurial extracellular system in the experimentally treated preparations are of interest in relation to the relatively rapid exchanges of monovalent cations observed in earlier radioisotope experiments using isolated cockroach ganglia and connectives (c.f. Treherne, 1962a). The electrophysiological observations outlined above clearly suggest the possibility that these rapidly-exchanging ion fractions could have resulted from perineurial changes produced by the procedure involved in the radioisotope experiments (Pichon and Treherne, 1970). This factor will be considered in a subsequent section dealing with the evidence obtained from more recent radioisotope experiments on cockroach central nervous connectives (p. 296). As has already been mentioned the application of more drastic surgery to insect central nervous systems leads to more rapid axonal responses following alteration of the cation concentrations of the bathing media (p. 258). In desheathed cockroach connectives, for example, it was found that the depolarization, resulting from the elevation of the external potassium concentration to 60 or 100 mM/1, corresponded to a half-time of around 24.0 s for the movement of those cations between the bathing medium and the extra-axonal fluid (Treherne et al., 1970). The experimentally determined value is considerably smaller than the theoretical one of f0.5 = 158.1 s calculated for the unrestricted intercellular diffusion of potassium ions from the bathing medium to the larger axon surfaces. This relatively rapid movement of potassium ions clearly suggests that some additional diffusion pathway had been created by the desheathing procedure.
INSECT BLOOD-BRAIN BARRIER
29 1
Electron microscopic examination of desheathed cockroach connectives failed to reveal any significant changes in ultra-structural appearances of the axonal, glial or extracellular elements as compared with intact preparations (Treherne et al., 1970). The standard desheathing procedure was, however, found to involve considerable disruption of the outwardly-directed perineurial membranes (Fig. 5). As has already been mentioned (p. 268) this perineurial damage renders extensive areas of the cytoplasm of the underlying glial elements accessible to the relatively large molecules of peroxidase (Fig. 6) (Lane and Treherne, 1969, 1970). On the basis of this ultrastructural evidence it was suggested by Treherne et al. (1 970) that the additional diffusion to the axon surfaces produced by the desheathing procedure, occurred via the glial cytoplasm. Of particular significance in this respect are the local “tight” junctions between adjacent mesaxon membranes (Fig. 6b), for it has been suggested that these provide low-resistance junctions so as to short-circuit the extended mesaxon channel (Lane and Treherne, 1970; Treherne et al., 1970). According to the above analysis the potassium in the bathing medium diffuses via an intracellular glial pathway. Potassium ions diffusing by such a route would thus only have to traverse a single high-resistance cell membrane, that is the glial membrane immediately adjacent to the axon surface. The diffusion of potassium ions to the extra-axonal fluid through the above system has been examined quantitatively by Treherne et al. (1970). In this analysis an unrestricted access of cations to the glial fold was assumed. With this system it was calculated that the access of ions through the glial cytoplasm would be sufficiently rapid to account for the observed rates of depolarization, even if the permeability of the inner glial membrane were as low as cm s - I . A value considerably lower than, for example, that of the squid axon membrane, which is 1.8 x cm s-l (Hodgkin and Katz, 1949). It would appear, therefore, that the diffusion of potassium ions from a relatively high concentration in the glial cytoplasm could account for .the observed rates of accumulation of these cations in the extra-axonal fluid in desheathed preparations. V. EXCHANGES OF RADIOACTIVE IONS AND MOLECULES
Early investigations using radioactive ions revealed the existence of relatively rapid exchanges of sodium and potassium ions between the haemolymph or bathing medium and the central nervous tissues of P.
292
J. E. TREHERNE AND Y. PICHON
americana in experiments in which the radioisotopes were injected into the haemolymph (Treherne, 1961b). Experiments performed using isolated ganglia or connectives further indicated that the efflux of 24Na or '*Na appeared to be a complex process and to show considerable departure from the simple exponential relationship which would be expected from the presence of a single rate-limiting peripheral diffusion barrier associated with the nerve sheath (Treherne, 1961c, d, e, f, 1962a). The effluxes of monovalent and divalent cations of chloride ions and of tritiated water were found, in fact, to approximate to a two-stage process. In the case of sodium ions, for example, the efflux curve could be resolved into two components with mean half-times of 28.4 and 277.0 s respectively (Fig. 17). The escape of the initial, rapidly-exchanging, fraction was found to be unaffected by the presence of ouabain or metabolic inhibitors in the bathing medium; l00.000~
I
I
I
I
I
1 -
t
-
1
0 ' O .
\0
--
-
-
1000
0
I
100
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300
400
500
600
INSECT BLOOD-BRAIN BARRIER
293
the subsequent, more slowly-exchanging fraction, was on the other hand, found to be significantly reduced by lo-% ouabain, KCN or DNP (Treherne, 196 1f, 1966b). On the basis of the above results it was postulated that the second, ouabain-sensitive component must represent an intracellular sodium fraction and that the initial rapidly-exchanging one contained the extracellularly-located ions (Treheme, 1962a). Essentially similar interpretations have also subsequently been applied to the equivalent process occurring in both crustacean (Abbott, 1969, 1970) and mammalian (Ames and Nesbett, 1966) central nervous tissues. The rapidly-exchanging sodium fraction demonstrated in the radioisotope experiments was found to correspond to 32.6% of the exchangeable sodium in the nerve cord of P. americana (Treherne, 1962a). The total concentration of the rapidly exchanging sodium (44.9 k 2.3 mM/kg) and potassium 2.7 1 k 0.15 mM/kg) fractions was considerably higher than that for chloride ions (16.9 -I 1.5 mM /kg). On the basis of these results it was tentatively suggested that the distribution of the rapidly-exchanging monovalent cations and anions approximated to some sort of Donnan equilibrium resulting, in part at least, from the presence of anion groups associated with extracellular acid mucopolysaccharides (p. 274) (Ashhurst, 1961). Subsequent experiments using radioactive sodium and potassium ions have also revealed the existence of relatively rapid exchanges between the blood, or bathing medium, in C. rnorosus (Treherne, 1965b; Weidler and Diecke, 1970a, b). Equivalent exchanges of fatty acid ions and quaternary alkylammonium cations have, in addition, been observed in the central nervous system of P. americana (Eldefrawi and O'Brien, 1966, 1967). The majority of the above studies were carried out using isolated preparations which, as has been subsequently shown (Pichon and Treheme, 1970), could be reasonably expected to have been affected by the experimental procedures involved in these earlier radioisotope experiments. In particular, any appreciable longitudinal extension of the connectives or relatively gentle drying with filter-paper might be expected to increase the access of ions into the underlying extracellular fluid. To test the above possibility experiments have been recently carried out in this laboratory in which the form of '%a efflux has been related to the magnitude of potassium induced extraneuronal potentials (p. 282) in isolated cockroach nerve-cords (Tucker and Pichon, 1972a, b). In these experiments the electrical and isotopic
294
J. E. TREHERNE AND Y. PICHON
measurements were performed on approximately 2 mm lengths of the penultimate abdominal connectives of P. americana used in the preceding electrophysiological experiments (p. 283). These recent experiments confirm that even in the presence of large potassium-induced extraneuronal potentials there is a relatively rapid exchange of 22Na between the nerve cord a n c t h e bathing medium as demonstrated earlier by Treherne (1961b, c, d, e, f, 1962a). They also show, as suggested earlier by Pichon and Treherne (1970), that there is a clear relation with the state of the preparation: the higher the extraneuronal potential the slower the exchange of radio sodium. Since the extraneuronal potential has been supposed to originate at the level of perineurium (p. 285) it might be expected a priori that the main restriction to ion movement would be situated at the same level. In this case it could be reasonably considered to be effectively a single-compartment system, the efflux curve approximating to a simple first-order diffusion process defined by a curve whose slope would be related directly to the permeability of the peripheral barrier. As will be seen from Fig. 18 the form of the total efflux of "Na from the nerve departs markedly from the theoretically predicted values. This time course for the initial escape of "Na from connectives showed considerable variation between different preparations. The initial efflux curve could nevertheless be clearly related to the
0
4
12
8 time
16
20
(min)
Fig. 18. Theoretical curve (closed circles) for diffusion from a single compartment (calculated from Hill's (1928) equations for diffusion of a substance from a cylinder) and a "Na efflux curve obtained for cockroach connectives (open circles). (From Tucker and Pichon, 1972b.)
295
INSECT BLOOD-BRAIN BARRIER 100
80 62 mV
-g (u
r
35 mV
50
C C .GI
‘E ._ m
E, m z
23 mV
N N
20 1L mV
1(
1
1
1
1
40
20 time
1
1
60
(sec)
Fig. 19. Relationship between the rate of sodium efflux from isolated cockroach abdominal connectives, and the magnitude of the extraneuronal potentials developed when the connectives were exposed to a high-potassium solution. (From Tucker and Pichon, 1972b.)
magnitude of the recorded extraneuronal potential (Fig. 19). It has further been shown that the efflux curve can be graphically resolved into at least three arbitrary components: a slow one ( t o . 5 ~16.0 f 1.4min), an intermediate one = 102.2 f 17.7 s) and a fast one ( t o 4 = 29.7 f 10.1 s). The fast and intermediate components were essentially similar to those described earlier (p. 292), whereas the slow component is probably identical to the very slow one described by Treherne (1 96 1d, e) for 24Namovements (Pichon and Tucker, 1971; Tucker and Pichon, 1972a, b). The time courses of the efflux of these three ion fractions are illustrated in Fig. 20. Of these three components only the fast one showed a highly significant relation (P < 0.001) with the extent of potassiuminduced extraneuronal potentials (Fig. 21), the intermediate one showing a much less close correlation (0.2 > P > 0.1) and the slow fraction showing no significant correlation (P 0.8).
296
J. E. TREHERNE AND Y. PICHON
0
5
10
time
15
(min)
Fig. 20. Semi-logarithmic plot of "Na efflux from an intact, isolated cockroach connective illustrating analysis of the efflux curve into three components: slow component (linear portion of open circle curve), intermediate component (straight portion of squares curve) and fast component (triangles). From Tucker and Pichon, 1972b.)
The highly significant correlation between the fast efflux and the size of the extraneuronal potentials agrees with the quantitative analyses of the ionic basis of this potential (p. 287). In particular, these experiments support the postulated role of the intercellular perineurial channels in short-circuiting the trans-perineurial potentials observed in P. americuna (Pichon et al., 1971) and possibly also in M. sextu (Pichon et al., 1972). In desheathed cockroach connectives 22Na efflux of all three exchange components was increased as compared with intact preparations (Tucker and Pichon, 1972b). As will be seen from Table I1 the effects were most marked in the case of the rapidly-exchanging 22Na fraction. As has been emphasized (p. 268) the perineurial damage associated with the desheathing procedure renders extensive volumes of the glial cytoplasm accessible to peroxidase molecules and also causes a dramatic increase in the measured inulin space (Treherne, 1962a). These results have also been correlated with the
297
INSECT BLOOD-BRAIN BARRIER FAST
COMPONENT
I 1
0
I
I 2 time
3
(min)
Fig. 21. Relationship between the rate of sodium efflux in the fast component (Fig. 20) and the magnitude of the potassiuminduced extraneuronal potentials in intact cockroach connectives. The line drawn through the points is the calculated regression line. (From Tucker and Pichon, 1972b)
extremely rapid ion movements measured in electrophysiological experiments in desheathed preparations which have been postulated to result from an increased accessibility of glial cytoplasm to inorganic ions contained in the bathing medium (p. 268). It, therefore, seems reasonable to assume that the substantial decrease in the measured half-time of the rapidly-exchanging 22Na fraction results, in part at least, from the rapid escape of an appreciable intracellular ion fraction. The alkylammonium cations which penetrate into the tissues of the intact abdominal nerve cord of P. arnericana can be resolved, in Table I1 The effect of desheathing on 22Na efflux from nervous connectives in Periplaneta americana * Preparation
Fast component 10.5 (sec.)
Intermediate component ro.5 (sec.)
Slow component to.5 (min.)
Sheathed
29.7-110.1
102.2k17.7
16.0f1.4
P Desheathed
2.0k0.82
* From Tucker and Pichon (1972b)
< 0.001
33.0f2.72
P
< 0.05 P < 0.02 8.5f0.47
298
J. E. TREHERNE AND Y. PICHON
radiotracer experiments, into fast and slowly-exchanging fractions (Eldefrawi and O’Brien, 1967). It was suggested that these represent the distribution of cations between extracellular and intracellular phases or conceivably, between “free” and “bound” extracellular cations. The data presented by Eldefrawi and O’Brien show that increasing lipid solubility tended to increase the penetration into the central nervous tissues and that increasing ionic size decreased the rate of influx. Charged molecules appear to penetrate more slowly than uncharged ones of equivalent dimensions, the difference being, however, relatively small (of the order of a 5 to 15-fold change). It is very difficult, in our present state of knowledge, to relate the degree of accessibility of ions of differing physico-chemical properties to the extent of their penetration within the different structural compartments of the central nervous system and, in particular, to their accessibility to the neuronal elements. It is hoped that future research may elucidate the role of factors such as lipid solubility in the penetration of substances to the various neuronal surfaces of the insect central nervous system. Our knowledge of the uptake of nutrient substances is limited to a single early investigation on the uptake of radioactive glucose and trehalose molecules by the cockroach abdominal nerve cord (Treherne, 1960, 1961a). As in the case of cations a relatively rapid uptake of 14C-labelledglucose and trehalose was demonstrated, being equivalent to an influx of 1.09 mM glucose/l nerve cord water. The possible significance of these results can be best appreciated in relation to the contemporary histochemical study of Wigglesworth (1 960) on the uptake of carbohydrate by the central nervous system of P.americana (p. 305). In spite of the apparent insensitivity of insect ganglia to externally applied acetylcholine (p. 258) relatively rapid exchanges of l4C-labe1led acetylcholine have been demonstrated t o occur between the bathing medium and the intact central nervous tissues of P. americana (Treheme and Smith, 1965a; Eldefrawi and O’Brien, 1967). Despite the rapid influx of labelled acetylcholine molecules measured in these investigations the calculated extracellular acetylcholine concentration was found to be 8.1 x M in preparations bathed with 1O-* M acetylcholine (Treherne and Smith, 1965b). In these exReriments the products of the hydrolysis of acetyL3Hcholine accumulated largely as acetate and glutamate with the nerve cord tissues. It appeared, from these results, that an appreciable hydrolysis of acetylcholine occurred towards the periphery of the
INSECT BLOOD-BRAIN BARRIER
299
nerve cord. Thus, for example, when whole nerve cords were bathed in a solution of radioactive 10-2 M acetylcholine the concentration of this substance within the nervous tissues showed only a slow approach to the level obtained after a one second exposure in the presence of eserine. The amount of radioactivity taken up during this brief exposure of labelled acetylcholine solution was found to be roughly equivalent to that contained in the nerve sheath. It was postulated, therefore, that a considerable hydrolysis of the applied acetylcholine must have occurred at the periphery of the ganglion, possibly within the nerve sheath itself (Treherne and Smith, I965b). The peripheral sites of cholinesterase activity, which were shown to be localized on the glial membranes bordering peripheral extracellular channels (Smith and Treherne, 1965; Treherne and Smith, 1965b), would be strategically placed to effect hydrolysis of acetylcholine penetrating in to the perineurium and neural lamella. It would appear, therefore, that the access of acetylcholine molecules to the synaptic surfaces may be limited not only by diffusion barriers, but also by the presence of a “metabolic barrier” situated at the periphery of the ganglion. VI. CONCLUDING REMARKS
The overall picture which emerges from the measured electrophysiological responses of nerve cells is of a restricted access of ions and molecules to the extraneuronal fluid. In this sense it can be clearly stated that insects possess a well developed blood-brain barrier system; our present concept being that the main restriction to intercellular diffusion of water-soluble substances to the neuronal surfaces from the blood, results from the presence of intercellular occlusions at the inner ends of the perineurial clefts. It is also clear from radioactive studies that, despite this peripheral restriction to intercellular ion movements, relatively rapid steady-state exchanges nevertheless occur between the blood and the central nervous system. This is well exemplified by the rapidly-exchanging 22Na fraction (which, according to Tucker and Pichon (1972b) amounts to 45% of the exchangeable sodium) observed under conditions of maximal peripheral restriction to intercellular ion movements postulated on the basis of the observed extraneuronal potential changes in the cockroach nerve end. The steady-state exchanges of radioactive ions and molecules observed under these conditions strongly suggest the possibility that
300
J . E. TREHERNE AND Y. PICHON
rapid movements occur via an intracellular pathway within the nervous system. The most obvious candidate for this would seem to be the glial cytoplasm. This concept would accord with our present knowledge of the ultrastructural organization of insect central nervous tissues. In particular the apparent existence of low-resistance pathways linking the perineurial and underlying glial cytoplasm would ensure the rapid movement of substances within the glial compartment. The uptake of substances through the outwardly facing perineurial surface would be facilitated by the large effective membrane area resulting from the extensive infoldings. Phenomena such as exchange-diffusion mechanisms, for sodium at least, and active transport would also explain the rapid and extensive steady-state exchanges observed in intact central nervous preparations. It should, in addition, be borne in mind when considering the electrophysiological results in relation t o those obtained using radioisotopes that in the former case we are studying the effects of net ion movements resulting from substantial changes in concentration in the bathing medium. In these conditions electro-chemical gradients are created which may contribute to the modification of permeability characteristics of the system as compared with those pertaining in the steady state conditions. These factors are represented diagrammatically in Fig. 22. The evidence presented above, showing a significant restriction to diffusion of substances between the blood and the fluid bathing the neuronal surfaces in insects, contrasts markedly with the situation in the only other arthropod species which has been investigated: the shore crab, Curcinus maenas (Abbott, 1969, 1970). In this crustacean there appears to be no equivalent restriction to ionic and molecular exchanges with the blood which in this species is channelled into the brain by a well-developed capillary blood supply (see also Sandeman, 1967). Furthermore, as was emphasized in a recent review (Treherne and Moreton, 1970), there is no substantial evidence for the existence of noticeable restriction in access to the neuronal surfaces in the other higher invertebrates which have been studied. The closest parallel to the blood brain barrier of insect species appears to be that encountered in higher vertebrate animals. In mammalian brain, as in insect central nervous tissues, the restricted access to the fluid surrounding neurones appears to result from the presence of intercellular occlusions interposed between the blood and the extraneuronal fluid. These occlusions are in the form of “tight” junctions between the lateral membranes of the capillary
4 I
+t
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..... ................................................... (7)
...I......
/
Fig. 22. Simplified and speculative diagram of the exchange properties of the nervous tissue in Penplaneta americanu L. The large circles 1, 2 and 3 represent respectively the presumed extracellular, glial and axonal compartments. The continuous lines indicate the extracellular pathway, the interrupted lines an intracellular pathway. The resistors represent the proposed main sites of restriction. R, = axonal membrane resistance; R3+: inner glial mesaxon membrane resistance; R+,: restriction in the mesaxon channels; R2-l: glial membrane resistance; R2-; outer perineurial membrane resistance; Rl-o: tight junctions. Rl-o and R2-o are not readily distinguished by physiological experiments and are likely to be coupled (as indicated by dotted line). The arrows indicate the possible pathways used by aa Na ions to leave the nervous tissue in radio-isotope experiments. In fully intact preparations, the effective value of Rl-o is large compared to R,-o (because of the large membrane wea of the perineurial cells). The overall resistance of the outer barrier (R) approaches Rz-o and the permeability of the system to different molecules is a function of the properties of the outer perineurial membrane. In stretched preparations Rl-o is reduced and possibly Rz-o . The value of R is lower than in intact conditions: the nerve sheath is less effective in regulating the movements of ions and molecules from and into the nervous system. In very stretched or desheathed preparations, R approaches or equals zero and the restriction to the movements of ions and molecules from and into the axons is a function of the values of R,, R3-, and R3-2 . In non-steady state conditions, electrochemical gradients are created which affect the movements of ions through the system. For example, in high external K+,a potential is created between the outer and the inner perineurial membranes (the extraneuronal potential) which opposes inward movements of K + ions; this effect is more or less short circuited by R,-,, in mechanically treated preparations. In Na+ free solutions, R2-,, which is large in resting conditions (due to the properties of the outer perineurial membrane) becomes even larger due to the building-up of a potential across the perineurium (possibly associated with the disappearance of exchangediffusion movements of sodium); this restriction is also more or less short-circuited by Rle0 in mechanically treated preparations. (Modified from Tucker and Pichon, 1972b.)
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endothelium (in mouse, hamster and Necturus) or the perivascular glial end-feet (in elasmobranchs) which, as in their insect counterparts, restrict the entry of peroxidase (Brightman and Reese, 1970). Another striking similarity between the vertebrate and the insect situations are the presence of appreciable potential changes which have been recorded between the cerebrospinal fluid and the blood following alterations in the potassium concentration of the blood (c.f. Cameron and Kleeman, 1970). It would seem reasonable to conclude that these potential changes might have a functionally similar basis to those proposed (p. 285) for the insect central nervous system. A diagrammatic representation of our current conception of the blood-brain barrier in insects related to those for vertebrates, a crustacean and an annelid is given in Fig. 23. A distinctive feature of the physiological organization of insect central nervous tissues is their ability to maintain ionic concentrations in the extra-axonal fluid which differ markedly from those of the haemolymph. This is seen most dramatically in phytophagous species, such as C. morosus and M. sexta, in which the sodium concentration in the fluid bathing the axon surfaces may be approximately an order greater than that of the haemolymph. This phenomenon may not, however, be confined to phytophagous insects, for as has been pointed out, some electrophysiological evidence can be interpreted on the assumption that the extra-axonal sodium concentration of P. americana (which has a relatively high haemolymph sodium level) exceeds that of the haemolymph by a factor of approximately 2.3. The precise nature of physiological mechanisms involved in extra-axonal sodium regulation still remains to be elucidated. It is likely that relatively high extracellular sodium concentrations could be maintained by the anion groups associated with the acid mucopolysaccharide (c.f. Treherne, 1962a). However, it seems clear that the thermodynamic activity of sodium ions in such a fixed-anion system would only be equivalent to that in the haemolymph or bathing medium and could not, therefore, contribute to an elevation of the effective sodium concentration of the extra-axonal fluid (Treherne and Maddrell, 1967b; Treherne, 1967). It seems reasonable to suppose, however, that any sodium ions associated with extracellular fixed-anion groups would tend to act as a cation reservoir which might function in the short-term maintenance of the ‘extra-axonal sodium level. It could thus be envisaged that any fluctuations in the sodium concentrations in the vicinity of the
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Leech blood
nl
blood
blood
.
Insects
Teleost
. . ..
, ........, ...... . . . ... ,:.:.:.:.'.'.'.'.'*.'.'... . . . . ....:.:. ::::.:.: .:.:.:.: :.:.:.:. .'.'.:.:
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Fig. 23. Diagrammatic representation illustrating some current concepts of the structural basis of the insect and vertebrate blood-brain barriers, in comparison with the situation in an annelid and a crustacean species. The interpretation of the situation in vertebrate animals is based on data summarized by Brightman and Reese (1970), that for Curcinus maenus from Abbott (1969, 1970) and for Hirudo medicinalis from Coggeshall and Fawcett (1964), Nicholls and Kuffler (1964) and Kuffler and Potter (1964). The representation of the situation in the insect species is based on the various data presented in this article, especially that of Lane andTreherne (1969,1970,1971) and Lane (1972.)
a: axon; bm: basement membrane; e: endothelium; fbs: fat body sheath (present in some species); g: glia; nl: neural lamella; p: perineurium.
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axons, resulting from their electrical activity, would be compensated for by a release of cations associated with the fixed-anion system. The only other hypothetical physiological mechanism which has been advanced attempts to relate extra-axonal sodium regulation to the activity of glial elements (Treherne, 1967; Treherne and Maddrell, 1967b; Pichon and Boistel, 1967; Pichon 1969a). It has further been tentatively suggested that the positive extracellular potentials demonstrated by Pichon and Boistel (1967) might be a manifestation of a glial-mediated electrogenic sodium pump which could be involved in the maintenance of the high concentration of sodium ions in the fluid bathing the axon surfaces relative to that of the haemolymph (Treherne and Moreton, 1970). This hypothetical mechanism would also accord with the concept, advanced above, of an intracellular low-resistance pathway linking the perineurium with the innermost glial membrane adjacent to the axon surfaces in P. americana. Such an intracellular route would form an obvious pathway for the movement of sodium ions to the glial cytoplasm immediately adjacent to the extra-axonal fluid. It should be borne in mind considering the validity of the provisional hypothesis, outlined above, that the positive extracellular potential could arise from mechanisms other than glial electrogenic sodium pumps. It is conceivable, for example, that the positive extracellular potential could result from the juxtaposition of the positively-charged outer neuronal surfaces with the extracellular negatively-charged acid mucopolysaccharide in a manner analogous to that postulated for vertebrate malignant trophoblastic cells (Hause et al., 1970). However, the provisional hypothesis advanced above represents a system which should be susceptible to experimental analysis and, for this reason, may be of assistance in future research in this area of neurophysiology. Whatever the nature of the extra-axonal sodium regulating system in insects it seems obvious that the restriction to intercellular diffusion offered by the perineurial “tight” junctions would greatly facilitate the functioning of the system by reducing the escape of sodium ions from the extracellular system. The perineurial restriction to the intercellular diffusion of watersoluble ions from the haemolymph or bathing medium appears to exist, as was described above, in parallel with relatively large rapidly-exchanging ion fractions in the central nervous system of P. americana. These rapidly-exchanging ion and molecular components, demonstrated in radioisotope experiments, have been tentatively
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identified as being of glial origin. The existence of such rapidlyexchanging glial fractions suggest a possible mechanism by which nutrient substances could be transported from the haemolymph to the nerve cells in the presence of the peripheral intercellular diffusion barrier. The rate of uptake of nutrient molecules from the haemolymph is presumably enhanced by the relatively large outwardly-directed perineurial surface resulting from the extended and tortuous perineurial clefts: the rapid transport through the glial cytoplasm being facilitated by the low resistance junctions between adjacent glial cells. The existence of such a glial transport system was foreshadowed by the histochemical observations of Wigglesworth ( 1960) who demonstrated a sequential deposition of glycogen, through the nervous systems of starved cockroaches following injections of glucose into the blood. The sequence was terminated by the transfer of material from the glial cytoplasm to neuronal cell body via the trophospongium of Holmgren. It is possible, therefore, to visualize a dual system of exchanges in the insect central nervous system : a restricted access of water-soluble ions and molecules into the extracellular system, which is paralleled by relatively rapid exchanges with the glial cytoplasm. As should be apparent from the above account this arrangement facilitates both the regulation of the ionic environment of the neurones and, despite the peripheral restrictions to intercellular diffusion, allows rapid and effective exchanges of nutrient substances to occur between the haemolymph and the neurones.
ACKNOWLEDGEMENTS
We are indebted to Dr. N. J. Lane and Miss Y. Carter for their help in preparing some of the electron micrographs and to Mr. J. Rodford for supplying some of the illustrations included in this article.
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Treherne, J. E. (1966b). The effect of ouabain on the efflux of sodium ions in the nerve cord of two insect species (Periplaneta americana and Carausius morosus). J. exp. Biol. 44,355-362. Treherne, J. E. (1 967). Axonal function and ionic regulation in insect central nervous tissues. In “Insect and Physiology” (J. W. L. Beament and J. E. Treherne, eds.). Oliver and Boyd, Edinburgh and London. Treherne, J. E. (1 970a). Exchanges of ions and molecules in the central nervous system of invertebrate animals. In “Proceedings of the Wates Foundation Symposium on the Blood-Brain Barrier.” Truex Press, Oxford. Treherne, J. E. ( 1970b). Ultrastructure and physiological function. In “Insect Ultrastructure” (A. C. Neville, ed.). Blackwell, Oxford and Edinburgh. Treherne, J. E. (1 972). A study of the function of the neural fat-body sheath in the stick insect (Carausius morosus). J. exp. Biol. 5 6 , 129-137. Treherne, J. E. and Maddrell, S. H. P. (1967a). Membrane potentials in the central nervous system of a phytophagous insect (Carausius morosus). J. exp. Biol. 4 6 , 4 13-42 1. Treherne, J . E. and Maddrell, S. H. P. (1967b). Axonal function and ionic regulation in the central nervous system of a phytophagous insect (Carausius morosus). J. exp. Biol. 47,235-247. Treherne, J. E. and Moreton, R. B. (1 970). The. environment and function of invertebrate nerve cells. Int. Rev. Cytol. 28,45-88. Treherne, J. E. and Smith, D. S. (1 965a). The penetration of acetylcholine into the central nervous tissues of an insect (Periplaneta americana) J. exp. Biol. 43, 13-21. Treherne, J. E. and Smith, D. S. (1 965b). The metabolism of acetylcholine in the intact central nervous system of an insect (Periplaneta americana L.) J. exp. Biol. 43,441-454. Treherne, J. E., Mellon, D. and Carlson, A. D. (1 969). The ionic basis of axonal conduction in the central nervous system of Anodonta cygnea (Mollusca: Eulamellibranchia) J. exp. Biol. 50, 71 1-722. Treherne, J. E., Lane, N. J., Moreton, R. B. and Pichon, Y. (1970). A quantitative study of potassium movements in the central nervous system of Periplaneta americana. J. exp. Biol. 5 3 , 109-136. Tucker, L. E. and Pichon, Y . (1972a). Electrical and radioisotope study of an insect “blood-brain barrier”. Nature, Lond. (In press). Tucker, L. E. and Pichon, Y . (1 972b). Sodium efflux from the central nervous connectives of the cockroach. J. exp. Biol. (In press). Twarog, B. M. and Roeder, K. D. (1956). Properties of the connective tissue sheath of the cockroach abdominal nerve cord. Biol. Bull., Woods Hole, 1 11,278-286. Twarog, B. M. and Roeder, R. D. (1 957). Pharmacological observations on the desheathed last abdominal ganglion of the cockroach. Ann. ent. SOC.A m . 50, 231-237. Usherwood, P. N. R. (1 969). Electrochemistry of insect muscle. In “Advances in Insect Physiology” Vol. 6, 205-278. (J. W.L. Beament, J. E. Treherne, and V. B. Wigglesworth, eds.). Academic Press, New York and London. Ussing, H. H. (1965). Transport of electrolytes and water across epithelia. Harvey Lect. 5 9 , 1-30.
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Weidler, D. J. and Diecke, F. P. J. (1969). The role of cations in conduction in the central nervous system of the herbivorous insect Carausius morosus. 2. vergl. Physiol. 64, 372-399. Weidler, D. J. and Diecke, F. P. J . (1 970a). The regulation of sodium iojs in the central nervous system of a herbivorous insect, Carausius morosus. Z. vergl. Physiol. 6 7 , 160-1 78. Weidler, D. J. and Diecke, F. P. J. (1 970b). Hemolymph ionic concentrations and sodium ion regulation by the neural sheath in two stick insect species (Diapheromena femorata and Carausius morosus) 2. vergl. Physiol. 69, 3 11-325. Wigglesworth, V. B. (1960). The nutrition of the central nervous system of the cockroach, Periplaneta americana. The mobilization of reserves. J. exp. Biol. 37, 500-5 12. Wood, D. W. (1957). The effects of ions upon neuromuscular transmission in a herbivorous insect. J. Physiol. Lond. 138, 119-139. Wyatt, G. R. (1961). The biochemistry of insect haemolymph. Ann. Rev. Ent. 6, 75-102. Yamasaki, T. and Narahashi, T. (1 958). Effects of potassium and sodium ions on the resting and action potentials of the giant axon of the cockroach. Nature, Lond. 182,805. Yamasaki, T. and Narahashi, T. (1959). The effects of potassium and sodium ions on the resting and action potentials of the cockroach giant axon. J. ins. Physiol. 3, 146-158. Yamasaki, T. and Narahashi, T. (1960). Synaptic transmission in the last abdominal ganglion of the cockroach. J. ins. Physiol. 4, 1-13.
NOTE ADDED IN PROOF Since this article was submitted for publication the following relevant observations have been reported. A recent study, employing en bloc uranyl acetate staining (Karnovsky, 1967), has revealed that the perineurial intercellular occlusions in P. americana consist of septate desmosomes, gap junctions and tight junctions (zonula occludens) (Lane and Treherne, 1972). Microperoxidase (h4W 1900) showed extensive penetration of intercellular clefts and limited penetration of septate desmosomes. There was no evidence of penetration into the underlying extracellular spaces. Colloidal lanthanum was seen t o penetrate both septate desmosomes and gap junctions. It is concluded, therefore, that its relatively limited penetration to the underlying extracellular system probably results from its restricted access through the tight junctions (see p. 268). An account has been published on the efflux of 2%a from nerve cords of Carausius morosm and Periplaneta americana (Weidler e t al., 1971). Isolated nerve cords were used in these experiments and were “loaded” in radioactive solutions for periods of up to 14 h. The authors extracted no less than six components from their desaturation curves which were obtained using efflux periods of 26 h. With shorter periods, of about lOmin, only two efflux
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components were recognized. These results, which the authors claim to be consistent with the previous electrophysiological findings (see p. 28 l), appear to have little physiological significance. For example, the long duration of these experiments (up to 40 h) was quite inappropriate, especially as it is known that isolated nerve cords show significant changes in Na' and K + concentrations after only 1.5 h in normal physiological solution (Tucker, unpublished observations). Furthermore, no statistical analyses were employed in this investigation, neither was any attempt made either to correlate these components with any functional properties of the system or to test the excitability of the isolated nerve cords during the long experimental periods employed. The hypothesis that a significant proportion of the observed radioactive flux in short duration experiments originates in the fat body is also unacceptable for similar efflux components have been observed from isolated connectives of Periplaneta (see p. 294) which were free of fat body deposits (Tucker and Pichon, 1972b).
REFERENCES Karnovsky, M. J. (1 967). The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 3 5 , 21 3-236. Lane, N. J. and Treherne, J. E. (1972). Studies on perineurial junctional complexes and the sites of uptake of microperoxidase and lanthanum by the cockroach central nervous system. Tissue and Cell (In press). Weidler, D. J., Myers, G . C., Gardner, P. J., Bennet, A. L. and Earle, A. M. (1971). Defects in the experimental design of radioisotope studies on the insect nerve cord. 2.vergl. Physiol. 7 5 , 352-366.
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Insect Sperm Cells BACCIO BACCETTI Institute of Zoology. University of Siena. Italy
I. I1. 111.
IV .
V. VI .
VII .
VIII .
Introduction . . . . . . . . . . . . . . . . . The Cell Surface . . . . . . . . . . . . . . . . The Acrosomal Complex . . . . . . . . . . . . . A. The Typical Triple-layered Insect Acrosomal Complex . B. The Bilayered Acrosomal Complex. . . . . . . . . C. The Acrosomal Complex with Only Two Outer Layers . D. Monolayered Acrosomal Complex . . . . . . . . E. Total Absence of Acrosomal Complex . . . . . . . The Nucleus . . . . . . . . . . . . . . . . . A . Nuclear Shape . . . . . . . . . . . . . . . B. Submicroscopic Structure . . . . . . . . . . . C . Chemical Characteristics . . . . . . . . . . . D. Physical Characteristics . . . . . . . . . . . . The Centriolar Region . . . . . . . . . . . . . . A . The Centriole . . . . . . . . . . . . . . . B. The Centriole Adjunct . . . . . . . . . . . . C. The Initial Segment of the Axoneme . . . . . . . . The Axial Flagellar Filament or Axoneme . . . . . . . A. The Microtubules . . . . . . . . . . . . . B. The Central Sheath . . . . . . . . . . . . . C. The Link-heads . . . . . . . . . . . . . . D. The Coarse Fibres . . . . . . . . . . . . . E . The Axonemal Matrix . . . . . . . . . . . . Mitochondria . . . . . . . . . . . . . . . . . A. Normal Mitochondria . . . . . . . . . . . . B. Mitochondria Transformed into Derivatives with a Crystalline core . . . . . . . . . . . . . . . . . C. Absence of Mitochondria . . . . . . . . . . . Accessory Ordered Flagellar Bodies . . . . . . . . . . A . Structured Bodies Flanking Normal Mitochondria . . . B. Structured Bodies Flanking the Mitochondria1 Derivatives with a Crystalline Matrix . . . . . . . . . . . C. Structured Bodies Replacing Mitochondria . . . . . 315
316 317 324 324 326 327 321 328 328 328 329 331 331 332 332 333 335 336 338 349 349 350 352 354 354 354 360 363 363 365 365
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Spermatozoa Possessing a Double Flagellar Apparatus or Being Devoid of it. . . . . . . . . . . . . . . . . . A. The Paired Spermatozoa . . . . . . . . . . . B. SpermatozoaPossessing Two Axonemes . . . . . . C. Non-flagellate Spermatozoa . . . . . . . . . . X. Motility. . . . . . . . . . . . . . . . . . . A. Motile Mechanisms . . . . . . . . . . . . . B. Metabolic Aspects of Motion . . . . . . . . . . C. The Problem of Sperm Capacitation . . . . . . . XI. SpermatozoaPolymorphism and Genetics . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . IX.
367 367 369 370 374 374 380 38 1 382 384 384
I. INTRODUCTION
Among the animal phyla that have adapted themselves permanently to land life, the spermatozoon had to undergo thorough changes in its structure, metabolism and motility as a consequence of the onset of internal fertilization. The aquatic environment having been abandoned, spermatozoa were required to swim in a completely different medium, which was produced usually by the two partners at fertilization. Annelida, Mollusca, Arthropoda and the vertebrates have separately, but sometimes with amazing analogies, devised new forms of male gametes. In some arthropod groups still breeding in water, a “primitive” sperm model is retained which might be referred to as “aquatic” in type, e.g. in the Merostomata (AndrC, 1965; Baccetti, 1970; Shoger and Brown, 1970) and in the crustaceans Mystacocarida (Brown and Metz, 1967), Cirripedia and Branchiura (Brown, 1966, 1970). The outstanding features of this “aquatic” sperm type can be found in the most ancient representatives of almost all phyla, including Amphioxus among the Chordata (Baccetti et al., 1972f). These “primitive” features are represented by a roundish head, a great number of normal mitochondria rich in cristae and a relatively short flagellum with a plain axial filament simply organized according to the 9 + 2 pattern. This model was inherited by all arthropod classes, including those which are now definitely terrestrial. If allowance is made for some slight variations, this model is retained by primitive Arachnida, the scorpions (AndrC, 1963); among primitive Myriapoda, by the Pauropoda, Symphyla (Rosati et al., 1970) and Chilopoda (Horstmann, 1968); as well as by the Collembola and Diplura among primitive insects (Dallai, 1967, 1970; Baccetti et al., 1972d).
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When we follow the pathway of their evolution as revealed by extant forms, it may be observed that the Arachnida, Myriapoda and Insecta have subsequently refined their spermatozoa. Most Arachnida and Myriapoda produce “encysted” sperms (Baccetti, 1970) which are subsequently released in the female genital tract. All the spiders exhibit a peculiar 9 + 3 flagellum (Baccetti et al., 1970d; Reger, 1970) which is apparently of little functional significance because the encysted sperm is transported passively. As a consequence, the most evolved of the two lines, Opilionids (Reger, 1969) and Acarina (Reger, 1963; Breucker and Horstmann, 1968) on one side, and high Diplopoda (Reger and Cooper, 1968; Horstmann and Breucker, 1969a, b; Reger, 1971) on the other, have non-flagellate spermatozoa. Conversely, the spermatozoon of insects has evolved in the direction of increased motility: its shape, structure, metabolism and locomotory capabilities are tremendously differentiated. In many instances they attain an extreme specialization which is related to: (1 ) the mode of sperm transmission (internal fertilization always being the case); (2) the duration of its survival in the male genital tract or within spermatophores and spermathecas; (3) the type of fluid they have to swim in; and (4) the complexity of the egg envelopes they must penetrate. Typical features of the insect spermatozoon (Fig. 4) are the following: a generally very slim shape with an extremely elongated head; a bi- or three-layered acrosomal complex; an exceedingly long tail whose axial filament is flanked by accessory structures usually derived from mitochondria1 transformations. Along these lines evolutionary changes have resulted in a highly diversified and, in some respects, puzzling picture. Since some insight into the structure and function of spermatozoa has been gained during the past few years, it seems worth while to determine how far our knowledge on this subject has progressed. 11. THE CELL SURFACE
.
Mature insect spermatozoa are surrounded by the typical triple-layered membrane which appears markedly asymmetrical in electron microscopical sections (Fig. 9) due to the fact that with all fixation and staining procedures its outer electron opaque layer consistently is as thick as the intermediate light (30 A ) and opaque
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inner (20 A) layers put together. According to Baccetti et al. (1 97 la) both opaque layers show acid phosphatase activity (Fig. 1C). They appear smooth after freeze-etching whereas the intermediate transparent layer contains a number of scattered globules (Fig. IA). The outer layer reacts positively to all the methods for glycoproteins, which suggests that this membrane resembles a common plasma membrane with a strongly thickened glycocalyx. This model in which, moving from the inside to the outside, the layers are consecutively some 20, 30 and 60 A in thickness and the glycocalyx appears structureless, is shared by the vast majority of insect spermatozoa. It was referred to as the “fruit-fly type membrane” by Baccetti et al. (1 97 1a). In addition, three progressively more elaborate arrangements are known. The first one (Fig. 1) was described in Aphaniptera and given the name of “flea type membrane” again by Baccetti et al. (1971a). In this instance, the outer sperm wall is about 200 A thick, since a 130 A thick glycocalyx is attached to the normal plasma membrane which averages 70 A . The glycocalyx has a peculiar structure. It is arranged around the spermatozoon in transverse striae, with a periodicity of some 140 A , overlying the outer surface of the plasma membrane (Fig. IB). A more complex arrangement (Fig. 2) was found among locusts (“locust type membrane”) by Baccetti et al. (1971a). Here, as was described in earlier observations (Roth, 1957; Kessel, 1967), the sperm wall is as thick as 400 A, due to a glycocalyx exceeding 300 A in thickness (Fig. 2A) and comparable to the fuzzy coat of many protozoa. This coat is made up of rodlets 300 A long and 70 A wide inserted almost perpendicularly into the sperm surface. They are arranged in tetrads in which every rodlet is 2 0 A apart from its neighbour (Fig. 2B). Each tetrad is about 70 A away from the others. Both the striated envelope of the Aphaniptera spermatozoon and the fuzzy one in locusts are derived from an abundant amorphous glycoprotein material coating the spermatid. This material becomes organized over the plasma membrane during sperm maturation. More elaborate and less obvious is the origin of the most complex type of sheath so far known, namely that of Lepidoptera, which was described in butterflies by AndrC (1 959, 1962) and Phillips (1 970b), and in moths by Riemann (1970). This type, however, has been recently confined to the eupyrene sperms (Phillips, 197 1). This sheath (Fig. 3A) is made up of numerous appendages, called lacinate
Fig. 1. The cell periphery of the spermatozoon of Ctenocephalus canis. (From Baccetti et aZ., 1971a.) (A) Frozen-etched preparation, showing the striation of the outer surface (s) and the granulated (g) space of the cell membrane. ~60,000.(B) Longitudinal section of the plasma membrane (p) and of the array of fibres striating the cell coat (s). ~150,000.(C) Acid phosphatase on plasma membrane (p) and on the outer cell coats (s). ~80,000.
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Fig. 2. The cell periphery in the spermatozoon of Aiolopus strepens. (From Baccetti et al., 1971a.) (A) Cross section of mature sperms, showing the brush-structured outer coat (c) surrounding the plasma membrane (p). ~120,000.(B) Negative staining of the fragmented cell membrane, showing the frontal view of the rodlets arranged in tetrads (s). ~360,000.
Fig. 3. The lacinate coat of the Lepidopteran spermatozoon. (From Phillips, 1971.) (A) Transverse section of eupyrene testicular spermatozoa showing the radiate outer appendages. x 120,000. (B) Transverse section of eupyrene spermatozoa after entering the female. The radial appendages are substituted by two layers of extracellular material (arrows). x 106,000.
Fig. 3 AIP-14
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appendages by Andre (1959, 1962), radially arranged all around the spermatozoon. Each appendage consists of many juxtaposed laminae 90 A apart from one another. One of these appendages is lattice-like instead of laminar and lies at the level of doublet 1 of the flagellum. This crown of appendages seems to be derived from a peculiar amorphous small body referred to as “light band” by Yasuzumi and Oura (1964). In the spermatids, this body arises externally and differentiates into the radially arranged laminar structures. The glycocalyx is not the only structure to grow and develop during the last phases of spermiogenesis. There are also striking changes in the plasma membrane associated with the change in cell shape which occurs when the roundish spermatid turns into an impressively elongated spermatozoon. In the mature spermatid, progressively more complex membrane systems develop. These are flat cisternae which envelope the main organelles, notably the axial filament and mitochondria, demarcating the form of the future sperm and excluding wide regions of the spermatid cytoplasm which will later be shed. These membranes were regarded as endoplasmic reticulum by Yasuzumi et al. (1958) as well as by Ito (1 960). It was pointed out by Baccetti et al. (1 972b) that they are derived from the Golgi complex, and that during sperm maturation they usually fuse with the limiting membrane. In some places they simply adhere to the inner surface of the plasma membrane, doubling its thickness. Elsewhere a total fusion takes place, eventually resulting in a simple plasma membrane; again at other sites the original plasma membrane is eliminated and replaced by a newly formed membrane of Golgi origin. The morphological significance of these membranes, which clearly originate from the Golgi complex, is still debated. In spermatocytes, Sakai and Shigenaga (1 967) favour the view that the tubular endoplasmic reticulum originates from the Golgi. It may perhaps be preferable (Baccetti et al., 1972b) to call them Golgiderived membranes, mindful of the fact that many of them are going to build the definitive limiting membrane. Alteration of the membranes does not stop at the end of spermatogenesis, but undergoes further modifications during sperm transfer from the male to the female. The elaborate lacinate appendages of the eupyrene spermatozoa in the Lepidoptera undergo breakdown in the male genital duct. The material they are composed of is rearranged into concentric bands encircling the formerly naked apyrene sperms (Phillips, 1971). Once they have reached the female (Fig. 3B), both eupyrene and apyrene sperms possess identical
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Fig. 4. Schematic drawing of conventional models of a mature insect spermatozoon. The acrosome region is the three-layered one of Aiolopus (Orthoptera), the centriole region the radiated one of Bacillus (Phasmoidea), the tail the most complete known, of Tenebrio (Coleoptera): A, acrosome; AB, accessory bodies; AC, centriole adjunct; AT, accessory tubules; CC, central cylinder; CF, coarse fibres; CS, central sheath; CT, central tubules; D, doublets; DD, dissociated doublets; IC, inner cone; EL, extraacrosomal layer; GM, Golgi derived membranes; LH, link-heads; MD, mitochondria1 derivatives; N, nucleus: OC, outer cylinder; PM, plasma membrane; RL, radial links; RLA, radial laminae.
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envelopes. In moths, these envelopes differentiate into two parts having different paracrystalline structures (Riemann and Thorson, 1971). On the contrary, the “fuzzy coat” of the locust sperm is completely digested in the female genital tract (Renieri and Vegni, 1972). 111. THE ACROSOMAL COMPLEX
A multi-layered acrosomal complex is present among the most primitive insects such as the Diplura, Collembola and Thysanura (Dallai, 1970, 1972; Baccetti et al., 1972d). There are but small increases in complexity of this basic arrangement up to the most evolved Pterygota, like the Coleoptera (Baccetti et al., 1972a) and Hymenoptera (Hoage and Kessel, 1968). For these latter examples, therefore, it is possible to propose a general model of acrosomal structure for the whole class. The various modifications exhibited by single orders and species can be derived from this model and always consist of various structural simplifications. In extreme cases, this organelle is completely absent. The insect acrosome, as is the rule for all animals, is typically derived from the Golgi complex (the earliest evidence in this regard was provided by Beams et al. (1 956), Clayton et al. (1 958), Ito (1960), Gatenby and Tahmisian (1 959) and others). A. THE TYPICAL TRIPLE-LAYERED INSECT ACROSOMAL COMPLEX
This arrangement, with few variations, is present in almost all the Pterygota. It has been described in great detail for Orthoptera and Blattoidea (Kaye, 1962; Eddleman et al., 1970; Baccetti et al., 1971c), in Mecoptera (Baccetti et al., 1969c), in Aphaniptera (Baccetti, 1968), in the coleopteran Tenebrio (Baccetti et al., 1972a) and in the honey-bee (Hoage and Kessel, 1968). Generally, three juxtaposed layers (sometimes described as concentric) are encountered on the inside of the plasma membrane (Fig. 5A). The outermost “extraacrosomal layer” is an aggregation of granular cytoplasmic material which is concentrated between the plasma membrane and the acrosome proper during spermiogenesis. It occurs in a€l the instances mentioned here, but is particularly abundant in the more primitive orthopterans (Kaye, 1962; Shay and Biesele, 1968; Baccetti et al., 1971c). Its significance is utterly obscure. Beneath it the acrosome proper is found. This layer originates from the proacrosomal granule of the spermatid which is synthesized by
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Fig. 5. Three different arrangements of the acrosome complex in insects. (A) The three-layered model of Tenebrio: a, acrosome; el, extraacrosomal layer; ic, inner cone; pm, plasma membrane; n, nucleus. ~60,000. (From Baccetti el al., 1972a.) (B) The bilayered model of Campodea: a, acrosome; ic, inner cone. ~48,000.(From Baccetti and Dallai, 1972.) (C) The monolayered model of Chloeon: a, acrosome; n, nucleus. ~60,000 (From Baccetti et aZ., 1969b.)
the Golgi apparatus. Synthesis is most frequently associated with the concave side of its cisternae (Gatenby and Tahmisian, 1959; Kaye, 1962; Phillips, 1966a). In a single case it was seen to arise from the convex side (Phillips, 1970b). Histochemical investigations (Zylberberg, 1969; Baccetti et al., 1971c) have shown that the acrosome consists of a rather stable glycoprotein which is resistant to extraction. By negative staining the acrosome reveals a 95 vertical period and a 25 A horizontal period (Warner, 1971). The acrosome is
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entirely surrounded by its own triple-layered membrane, which previously encircled the proacrosomal granules derived from the Golgi (Kaye, 1962), and contains acid phosphatase (Baccetti et al., 1972b). Within the acrosome proper the third layer is encountered. This may be rodlike or conical, in the latter case being called “inner cone”. This is a compact structure, which is formed in the spermatid in an interspace between the acrosomal granule and the nuclear membrane, independently of the acrosome proper or of the Golgi region (Kaye, 1962). The characteristic interstitial membrane, which in the spermatid forms between the nuclear membrane and the proacrosomal granule (Kaye, 1962), is seen to break down at the site where this inner rodlet arises. Generally, no trace of the interstitial membrane is left in the mature sperm. The inner rodlet is endowed with histochemical properties diverging considerably from those of the acrosome; it consists of a protein core surrounded by a glycoprotein halo (Baccetti et al., 197 1 c). Information as to its origin and significance is entirely lacking. According to its position and ontogenesis, it might be regarded as comparable to the vertebrate perforatorium, which also contains carbohydrates (Sandoz, 1970), but its chemical nature is unknown. So far, the functional interpretation of the different components of the insect acrosomal complex is difficult to determine since even the mammalian one is as yet poorly understood. In mammals, according to Austin (1948) and Wada et al. (19S6), the acrosome contains hyaluronidase, while the perforatorium is believed to contain lysine (see Dan, 1967). Among the insects, the acrosomal hyaluronidase is completely absent (Baccetti et al., 197 1c). However it is not known whether lysines are similarly concentrated in the acrosome. This triple-layered acrosomal arrangement is generally conical in shape. However, in the tettigonioid Orthoptera it is arrow-like, and its stratification is also detectable inside the arrow limbs (Phillips, 1970b; Baccetti et al., 1 9 7 1 ~ ) In . the spermatozoon it generally lies apically to the nucleus. B. THE BILAYERED ACROSOMAL COMPLEX
This arrangement is very similar to the triple-layered acrosomal complex described above, the only difference is the absence of the outer extraacrosomal layer (Fig. SB). This arrangement is typical of the Apterygota. The extraacrosomal layer is consistently lacking in
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Collembola (Dallai, 1970), Protura, Diplura (Baccetti et al., 1972d) and Thysanura (Dallai, 1972). In this respect the bilayered arrangement might be regarded as more primitive than the triple-layered one, rather than the reverse. But, pending clarification of the nature and function of the extraacrosomal layer, nothing can be said about it at present.
C. THE ACROSOMAL COMPLEX WITH ONLY TWO OUTER LAYERS
This is a modification of the triple-layer arrangement; it consists of the acrosome proper and the extraacrosomal layer. Examples of this arrangement are found in phasmoidea (Baccetti et al., 1972b) and among a number of Homoptera (Folliot and Maillet, 1970). When the acrosome of Hemiptera has reached a considerable length, it becomes enriched by inner and outer microtubules which maintain its rigidity (Payne, 1966; Tandler and Moriber, 1966; Folliot and Maillet, 1970) and are still present in the mature spermatozoon. Since the inner rodlet is found in the more primitive arthropods and in the apterygotes as well, this arrangement might be interpreted as an involution of the triple-layered one. In Bacillus, glucose-6phosphatase is detectable in a restricted basal zone (Baccetti et al., 1972b) which might indicate a specialized area, although any speculation in this regard is far from easy.
D. MONOLAYERED ACROSOMAL COMPLEX
This structure consists of the acrosome alone (Fig. SC), enveloped by the acrosomal membrane and the plasma membrane. It has been reported in many groups: it occurs regularly in Ephemeroptera (Baccetti et al., 1969b), in Plecoptera (Baccetti et al., 1970e), in many Heteroptera (Barker and Riess, 1966; Herold and Munz, 1967; Mazzini, 1970), in Corrodentia , Mallophaga and Thysanoptera (Baccetti et al., 1969d), in those Trychoptera which possess the acrosome (Phillips, 1970b), in Lepidoptera (Phillips, 1971), in Diptera (Bairati and Perotti, 1970; Perotti, 1969; Tates, 1971, in Drosophila; Phillips, 1966b in Sciara) and in the Psychodidae (Baccetti et al., 1972c). This type of acrosome is always of very small size, and is often displaced to a lateral position with respect to the nucleus.
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E. TOTAL ABSENCE OF THE ACROSOMAL COMPLEX
This is occasionally found in some orders in which certain species possess an acrosome. Some instances are found in some of the more highly evolved species of the Isoptera (Baccetti et al., 1972e); in Coccoidea among Rhynchota, (Robison, 1966); in some species among Trychoptera (Baccetti et al., 1970e; Phillips, 1970b), in Oryctes among Coleoptera (Furieri, 1963a). In a whole order, Neuroptera (Baccetti et al., 1969c), the acrosome seems to be consistently lacking. This condition seems also due to involution. All the information summarized above seems to suggest that the triple-layered arrangement, typical of the pterygotes, was derived from a primitive bilayered acrosome model (rodlet and acrosome sensu stricto) as still retained by Apterygota. In some of these forms the acrosome may lose its rodlet and become bilayered (but the two outer layers are retained, hence not corresponding to the two found in Apterygota), monolayered or disappear altogether. The acrosomal layer itself is the only one present whatever the arrangement. IV. THE NUCLEUS
In a recent review on the sperm nucleus, Chevailler (1 970) points out that it is usually given less attention than the other sperm organelles, possibly due to its structural homogeneity and the uniformity of its transformations during spermiogenesis in various species. However, the survey carried out by this author brings into focus many important features, which will be discussed in the following section. A. NUCLEAR SHAPE
Among the insects, the sperm nucleus is as a rule fairly elongated, spindle-shaped anteriorly and truncated posteriorly. It is considerably compact, occasionally helicoidally arranged (Fig. 6b), sometimes even coiling around the flagellum as in the Mecoptera or Thysanura (Baccetti et al., 1969c, d). In Dahlbominus (Hymenoptera) some sperms have the helix twisted to the right, others to the left (Wilkes and Lee, 1965). Only in the spermatozoa provided with two axial filaments does the nucleus display a short flat shape as found in Anoplura and Mallophaga (Baccetti et al., 1969d). Aberrant
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329
non-flagellate sperms found in the most highly evolved Isoptera have a spheroidal or flattened nucleus (Baccetti e t al., 1972e). B. SUBMICROSCOPIC STRUCTURE
As a rule, in the mature insect sperm the nucleus appears compact and homogeneous in electron micrographs. This structure is attained during spermiogenesis. The chromatin in the young spermatid thick (Yasuzumi and Ishida, 1957; contains fibrils some 30-40 Gibbons and Bradfield, 1957; Dass and Ris, 1958; Gall and Bjork, 1958; Nebel, 1957), which are rapidly converted into thicker fibrils (1 00-200 arranged longitudinally (Werner, 1966; Chevaillier, 1970). These fibres then fuse into laminae which sooner or later coalesce. In the Apterygota the laminar pattern is also retained by the nearly mature spermatozoon (Bawa, 1964; Dallai, 1967) and the nucleus becomes compact just before its expulsion. In some instances, the nucleus instead of being homogeneous, exhibits a honey-comb texture (Yasuzumi and Ishida, 1957). The nuclear material does not undergo uniform morphological changes during spermiogenesis. Sometimes the central zone is the first to condense, at other times it is the periphery (Chevaillier, 1970). Among Psyllidae (Le Menn, 1966) only half the nucleus is occupied by chromatin, even in the mature sperm. During spermiogenesis the nuclear envelope is surrounded by a layer of microtubules which function in the compression and elongation of the nucleus, and which will disappear in the mature sperm (see, in particular, Kessel (1966, 1967)). DNA is seen to aggregate preferentially at the microtubule level (Baccetti e t al., 1972b) as is the case in most animals (Ferraguti and Lanzavecchia, 1971). Wide portions of the nuclear membrane are seen to form blebs which are pinched off into vesicles and dispersed into the cytoplasm (Kessel, 1970). Pores progressively decrease in number until they are no longer evident in the mature sperm. In some instances, however, the nucleus seems to take part in the elaboration of substances which are retained in the sperm cytoplasm. it was suggested by Werner (1 966) that these nuclear derivatives may give rise to the centriole adjunct and similar structures. However, this finding calls for further verification. Stable microtubules may also be endonuclear and protrude outwards in evaginations of the nuclear and plasma membrane. This was pointed out in the Neuroptera (Baccetti et al., 196%). This condition is quite common in the
a
a)
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B. BACCETTI
Fig. 6 . Two different forms of insect sperm nuclei. (A) The conical model of Bacillus, with an inner cavity containing polyribosome-like granules. ~ 9 0 , 0 0 0(From Baccetti et al, 1972b.) (B) The coiled arrangement found inMachi1is.x 90,000. (From Dallai, 1972.)
star-shaped nucleus of certain crustaceans (Decapoda), in which motile arms are found (Pochon-Masson, 1965, 1968a, b; Yasuzumi and Lee, 1966; Eliakova and Goriachkina, 1966;Anderson and Ellis, 1967),but is exceptional among insects. The scale insects, whose sperm is highly aberrant (see below), also seem to belong to this category.
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33 1
In the lower Arthropods, and in general among sea-dwelling invertebrates, the existence of a canal running within the nucleus, housing processes that are connected with acrosomal structures, is of common occurrence. As mentioned earlier, the sperm nucleus in insects is usually compact. However, in Bacillus, Baccetti and his collaborators (1 972b) describe a cribriform structure (Fig. 6A) full of polyribosomes positioned in an extranuclear space in the centre of the nucleus. C. CHEMICAL CHARACTERISTICS
It is well known that in the mature spermatozoon the nucleus consists essentially of the haploid DNA content. Atypical lines may lack DNA, as in apyrene lepidopteran sperms (Meves, 1903). In some instances, e.g. some carabids, polyploid sperms have been described (Bouix, 1963). Conversely, no RNA is present. In two insects, Philaenus and the house cricket, extrusion of ribonucleoprotein granules through the nuclear pores during the spermatid stage has been documented (Maillet and Gouranton, 1965; Kaye and McMaster Kaye, 1966). Along with DNA, proteins are contained in the sperm nucleus. These are mainly basic and consist of basic amino acids which replace the histones occurring in the nucleus of normal somatic cells and spermatogonias. Protamines, essentially consisting of arginine, lysine and histidine, chiefly studied in fish, are the most frequent. Other insects (Orthoptera and Drosophila) have, on the contrary, a category of histones particularly rich in arginine as a basic nuclear protein, which are synthesized in the cytoplasm during spermiogenesis (Das et al., 1964), using messenger RNA produced prior to meiosis (Bloch and Brack, 1964; Claypool and Bloch, 1967). Protamines and arginine-rich histones are essential for the compact nucleus of the mature spermatozoon. It was reported by Shoup (1967) that a particular Drosophila mutant being unable to achieve the transition from histones to arginine-rich histones possesses sterile sperms, with non-condensed nuclei. D. PHYSICAL CHARACTERISTICS
The important problem concerning the organization of material within the sperm nucleus, is still unresolved. Since X-ray observations in insects are not available, all information has been obtained from polarized light, fluorescence and electron microscopical investiga-
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tions. In other groups of animals we know that the nucleoprotein chains in the sperm nucleus are variably arranged, that is, ranging from an almost parallel array to haphazardly distributed chains. Working on the same material in insects, where the sperm nucleus is always exceedingly long and narrow, InouC and Sat0 (1 962, 1966) and MacInnes and Uretz (1968) came to opposite conclusions concerning Orthopteran spermatozoa. According t o the former workers, deoxyribonucleoproteins are organized into two superhelices of 8000 each, resulting from the coiling of a 1500 helix According to which consists in its turn of slender bundles (200 the latter workers, however, no superhelices exist and the DNA lies in arrays parallel t o the major axis of the sperm. The second interpretation has been substantially confirmed by Zirwer et al. (1970). Chevaillier (1 970), in an effort to resolve the controversy, proposes a scheme in which the chromosomes are oriented end-to-end along the length of the sperm nucleus. In this connection, Chevaillier mentions that Hughes-Schrader ( 1946) has reported a constant arrangement of the haploid chromosome set in Coccidae and quotes Taylor’s (1 964) work on Orthoptera, which by step-wise labelling with thymidine has revealed different levels of DNA in the sperm nucleus corresponding to a linear arrangement of chromosomes in the head.
a
a).
a
V. THE CENTRIOLAR REGION A. THE CENTRIOLE
In insect spermatids the normal orthogonal orientation of the two centrioles has been observed occasionally (Breland et al., 1966). More often either a single centriole is found (Friedlander and Wahrman, 1966), or two, as in biflagellate sperms (Fig. 7). However, the fine structure of this organelle has been established by only a few workers (Hoage and Kessel, 1968; Anderson and AndrC, 1968; Phillips, 1970b). They have the classical type of nine helicoidally arranged triplets (AndrC and Bernhard, 1964; Fawcett, 1966), comparable to those recently described by Ross (1968). Nevertheless, a more obscure problem today is the persistence of a centriole in the mature insect spermatozoon. As a matter of fact, no electron micrographs so far published show a classical centriole in a fully mature sperm and the presence of this organelle as late as the completion of spermiogenesis was denied by Phillips (1970b) in a
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Fig. 7. The centrioles (c) of the biflagellate sperm of Haemtopinus suis Both are surrounded by the adjunct centriole (ca). ~ 6 0 , 0 0 0(From . Baccetti el aZ., 1970a.)
recent discussion of this problem. At the same time, a centriole-like organelle was accurately described by Perotti ( 1970) in Drosophila sperm. However, in this case all the C tubules of the triplets are continuous with the accessory flagellar fibres (which, as will be reported presently, have a different origin) and the central pair of tubules is also present. These profiles could more easily be interpreted as the tip of the normal axial filament, where the accessory fibres may simulate triplets since they lie close to the peripheral doublets. At present, therefore, it may be assumed that a centriole does not occur in the mature insect sperm. B. THE CENTRIOLE ADJUNCT
This structure is a compact, basophilic sleeve, already known from light microscopy, characteristic of the spermatozoon in almost all insects. Called by various terms (“centriole adjunct”, which was
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coined by Gatenby and Tahmisian in 1959, is used most often), this organelle has been described in a variety of species in almost every order (see Cantacuzene’s survey (1 970)). It is generally of granular appearance (Fig. 7), with granules ranging between 160 and 320 a;it is fibrous in a few cases, e.g. in the house cricket and in Sciara (Kaye and McMaster-Kaye, 1966; Phillips, 1966a). Generally, it is more abundant in young spermatids (over 1 p 2 across) than in older ones where it becomes more compact (Breland et al., 1966). It is even more reduced in spermatozoa, where in some instances it may not be found (Phillips, 1970b). The origin of the centriole adjunct is still unknown. Werner (1965) favours the view of a nuclear origin starting from some material derived from a particular porous caudal portion of the nuclear membrane. Cantacuzhe (1970) showed that its synthesis was induced by the spermatid centriole. The aberrant multiplication of the centrioles is paralleled by a corresponding multiplication of the centriolar adjunct. Transitory contacts with Golgi cisternae and the endoplasmic reticulum are not sufficient to secure a supply of material by these organelles. Thus, the origin of the centriole adjunct is still uncertain. It seems to be responsible for the disappearance of the centriole around which it forms. Only recently has the nature of the centriolar adjunct been clarified by histochemical methods (Baccetti et al., 1969c; Yasuzumi et al., 1970; Gassner, 1970) and autoradiography (Baccetti et aZ., 1970a). There is little doubt that it consists of a ribonucleoprotein. This demonstration enabled Yasuzumi and his collaborators to propose a possible relationship between centriole adjunct and the chromatoid body of the spermatid. The ribonucleoprotein nature of the chromatoid body has long been claimed in many animals (Sud, 1961a, b; Daoust and Clermont, 1955) as well as in insects (Tandler and Moriber, 1965). But Eddy’s (1 970) recent demonstration that the chromatoid body is devoid of ribonucleoproteins, and that it arises not from the nucleus but rather from the interstices between the mitochondria (Fawcett et al., 1970) nullifies the main support to the derivation: nucleus-chromatoid body-centriole adjunct. The function of the centriole adjunct is not clear. Beginning with Gatenby and Tahmisian (1959) and Gatenby (1 96 l), most workers have always favoured a mechanical function for this compact collar, in the sense of it fastening the sperm head and tail together. This view is shared by Breland et al. (1966) and Fawcett and Phillips (1 969). Only after the more recent chemical analyses have other
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335
hypotheses been advanced, regarding it as a deposit of basic histones released by the nucleus during its enrichment with arginine-rich histones (Cantacuzkne, 1970) or as a concentration point of substances to be utilized in the assembly of the flagellar filaments (Yasuzumi et al., 1970). Since the initial segment of the axial filament is housed inside the centriole adjunct which apparently functions to initiate the movement of the flagellum (Baccetti et al., 1972b), a mechanical action of the centriole adjunct appears more and more probable. C. THE INITIAL SEGMENT OF THE AXONEME
Granted the absence of the centriole in insect spermatozoa, it follows that the first tubular units one encounters directly behind the sperm head are the fibres of the axial flagellar complex. In some instances, the accessory tubules are the first t o appear, in others the doublets; in all cases, the two central units arise shortly thereafter. This initial segment of the axoneme fits into ‘a corresponding nuclear indentation, or inside the sleeve formed by the condensed centriole adjunct. Its structure is complicated by the fact that it contains some units which are no longer found in the following tracts. The most widespread arrangement (Fig. 8C) consists of two short concentrically orientated cylinders connected by nine longitudinal radial laminae dividing the interspace into sectors (Fig. 4). Each doublet and each accessory tubule are contained in one sector, lying against a
Fig. 8. The “initiation motor” region of insect sperms. (A) and (B) Two different levels inDrosophiZa, showing cross links (arrows)between the opposite doublets. x 120,000. (From Perotti, 1970.) (C) Cross section in Euccillus, showing the outer ( 0 ) and the inner (i) cylinder and the radial laminae (I). ~90, 000.(From Baccetti et aZ., 1972b.)
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B. BACCETTI
lamina. The two central units are contained within the central cylinder. Tail sections in different functional moments show that laminae and cylinders lie at variable angles and the laminae themselves differ in their extension. These laminae are strongly reactive for ATPase activity. Baccetti and his collaborators ( 1972b) have recently proposed that this complex may function as a device to trigger the rotation of the inner cylinder within the outer one, thereby dragging the heads of the axoneme tubules into this motion, so as to initiate a wave which then propagates along the fibres (Section X A). An arrangement of this type is evident morphologically in Phasmoidea, Orthoptera, Dermaptera and Neuroptera. In the spermatozoa of Drosophilu, the two cylinders are not present in the neck region (Perotti, 1970), but cross links are evident between the opposed triplets (doublets plus accessory fibres) each embedded in a dense sheath (Fig. 8A, B). In the coleopteran Tenebrio, both the inner cylinder and the radial or cross laminae (or strands) are missing, while a thick outer cylinder is found, which connects the heads of the outer fibre crown one after the other. In point of fact, the wave pattern in the three arrangements is considerably different (see Section X A). The organization of the axoneme is undoubtedly very complex and is relevant to the general problem of flagellar motion. In a schematic diagram of a normal cilium at different levels, Gibbons and Grimstone (1 960) and Holwill(l966) illustrate a structure composed of two cylinders and radial laminae in proximity to the head, followed by another consisting of a single outer cylinder and one more with cross strands. As a working hypothesis, it can be assumed that from the primitive multiphase mechanism occurring in cilia one stage or another was preferentially developed by spermatozoa, resulting in the formation of different kinds of waves. VI. THE AXIAL FLAGELLAR FILAMENT OR AXONEME
By slightly modifying Warner’s definition (1970), the axoneme (Fig. 9) may be regarded as the whole of the flagella complex limited by the outermost crown of microtubules. Hence, the classical Fig. 9(A) Cross section of sperm tails of Ceratitis capitata (Diptera): a,axoneme; m, mitochondrial derivatives; p, plasma membrane. ~120,000.(B) Cross section of the
sperm tail of Tenebrio molitor (Coleoptera): ab, accessory bodies; at, accessory tubules; cf, coarse fibres; ct, central tubules; d, doublets; Ih,link heads; md, mitochondrial derivatives. ~240,000.(From Baccetti et aZ., 1972a.)
Fig. 9
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microtubules, arranged according to the fundamental 9 + 2 or 9 + 9 + 2 patterns, along with the elements connecting them with one another contribute to its formation, while both mitochondria and membranes are excluded. A. THE MICROTUBULES
In the vast majority of insects, the axoneme consists of two central tubules, nine doublets and nine accessory tubules (Figs 4,9B, 10A, D, E). This scheme was first brought to light by Rothschild (1955) in the honey-bee and by Yasuzumi (1956) in Drosophila. It was then established that most insect orders belong to this category. The only exceptions are the Protura (Fig. 12A), which have a bizarre axoneme with 12 (Acerentulus) or 14 (Acerentomon) doublets and nothing else (Baccetti et al., 1972d), and the other primitive Apterygota (Collembola and Japigidae), in which the accessory tubules are missing and the basic 9 + 2 pattern, typical of classical primitive sperms, is retained (Krzystofowicz and Byczkowska-Smyk, 1966; Dallai, 1967, 1970; Baccetti and Dallai, 1972). Accessory tubules are also lacking (Fig. 10B) in the two kindred orders Mecoptera (Baccetti et al., 1969c) and Aphaniptera (Baccetti, 1968; Phillips, 1969), as well as in two species of Trichoptera (Phillips, 1970b) and in Thysanoptera (Baccetti et al., 1969d). Other arrangements have occasionally been reported, e.g. the Ephemeroptera (Fig. 1OC) lack both central tubules, thus being 9 + 9 + 0 (Baccetti et al., 1969b; Phillips, 1969); the same is true of a Psocid, which however has a solid central fibre (Phillips, 1969). Culicid Diptera, on the contrary, have only one central tubule and are thus 9 + 9 + 1 (Breland et al., 1966). A non-identified mycetophilid dipteran has three central tubules, hence is 9 + 9 + 3 (Phillips, 1970b). The two trichopteran species mentioned above have as many as seven central tubules being therefore 9 + 7 (Phillips, 1969). The dipteran Sciara coprophila (Fig. 11) lacks central tubules, having instead 70 doublets and 70 spirally arranged accessory tubules in its huge flagellum (Makielski, 1966; Phillips, 1966b). In general, therefore, there are three kinds of tubules in the axoneme of insect spermatozoa: accessory, doublets and central tubules. These are most often present in the 9 + 9 + 2 pattern. Differences among the various tubule categories have already emerged from structural studies (Andre, 1961; Baccetti and Bairati, 1964) and were conclusively demonstrated by Behnke and Forer
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Fig. 10. Cross section of five different sperm tails: ab, accessory bodies; at, accessory tubules; ct, central tubules; d, doublets; md, mitochondria1 derivatives. (A) Neuronia 9 + 9 + 2 (Trichoptera). ~130,000. (From Phillips, 1970.) (B) Panorpa annexa, 9 + 2 (Mecoptera) ~ 9 0 , 0 0 0 .(From Baccetti et al., 1969c.) (C) Chloeon dipteron, 9 + 9 + 0 (Ephemeroptera). ~120,000.(From Baccetti et al., 1969b.) (D)Noronectuglauca, 9 + 9 + 2 (Rhynchota). ~90,000. (E) Chrysopa curnea 9 + 9 + 2 (Neuroptera). ~90,000.(From Baccetti e t al., 1969c.)
Fig. 11. Cross sections of the sperm tail of Sciuru coprophila. ~45,000.(From Phillips, 1966b.)
( 1967) by means of differential enzymatic extractions upon ultrathin
sections. The following knowledge is now available about this subject.
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34 1
1. Central Tubules a. Tubules. The diameter of central tubules from different axonemes
varies from 200 to 300 A. Their wall is about 70-80 A and its globular structure was first demonstrated by Bairati and Baccetti (1965). After dissociation it resolves into subunits appearing as fibrils made up of an alignment of globules or microcylinders, some 40 A thick. As a rule, the wall of each tubule consists of about 10 subunits. In various mammals, AndrC and ThiCry (1963) and Pease (1963) counted 10 of them, but among insects their number seems a little higher, i.e. 13 (Fig. 9B), according to the measurements of Phillips (1966), which were carried out on many species from different orders. There are 10-12 according to Danilova (1 969) in Bombyx mori. It is important to recall that 12-13 subunits are found in cytoplasmic microtubules and in those of the flagellum of protozoa (Ledbetter and Porter, 1964; Gall, 1965, 1966; Behnke and Zelander, 1967; Ringo, 1967; Fuge, 1968). In insects, no detailed chemical investigation has been performed'on the tubule wall. It is,
however, known to be proteinaceous (Behnke and Forer, 1967); possibly the tubulin of Mohri (1968). The latter is an actin-like protein with a 6 s sedimentation constant and molecular weight of about 120,000 changing to about 60,000 after denaturation. It contains a guanine nucleotide and binding sites for colchicine (Shelanski and Taylor, 1968). The above protein was studied in echinoderm sperm (Plowman and Nelson, 1962; Nelson, 1966; Shelanski and Taylor, 1968) as well as in the doublets of Tetrahymena cilia (Stevens et al., 1967; Renaud et aZ., 1968). The biochemical unit (mol. wt = 120,000) is likely to correspond to two of the 40 a microcylinders described by morphologists (Shelanski and Taylor, 1968). According to Fine (1971) it seems to be composed of two different subunits, with mol. wt. = 56,000 and 54,500 respectively, with different amino acid composition (Bryan and Wilson, 1971). These subunits would then be able to aggregate into longitudinal fibrils or to follow a helical course. This explains the different appearance shown by the same microtubules when examined longitudinally after dissociation in negative stain (Danilova, 19691, while in cross section they always look the same (Thomas, 1970; Henley, 1970). The central tubules may be hollow (Fig. 10B, E), as in Mecoptera, Plecoptera and Neuroptera (Baccetti et aZ., 1969c, 1970e) or may contain a globular central core (Fig. 9), of the same size as the wall subunits, as is the case among Diptera (Bairati and Baccetti, 1965; Phillips, 1966b; Perotti, 1969; Warner, 1970). In some cases, they may be filled with microcylinders, some
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Fig. 12(A) Cross section of the 14 + 0 axoneme of Acerentomon majus (Protura). ~80,000. (From Baccetti et al., 1972d.) (B) Cross section of the 9 + 9 + 2 sperms of Campodeu (Diplura) showing the disordered migration of accessory tubules near the mitochondrion (arrows). ~48,000.(From Baccetti and Dallai, 1972.) (C)A more caudal section of the 9 + 9 + 2 sperm of Campodea (Diplura) showing the ordered disposition of the nine accessory tubules (arrows) near the mitochondrion. ~75,000.(From Baccetti and Dallai, 1972.) (D) The tip of the sperm tail in Telamona spec. (Rhynchota). The doublets appear dissociated. ~62,000.(From Phillips, 1970a.)
40 A in diameter, about eight per tubule, as in Gryllus (Kaye, 1964, 1970). Very often (in Trichoptera, Lepidoptera, Phasmoidea), however, the two tubules (Fig. 14A, B, D) become filled not only by protein, but also by a glycogen-like polysaccharide (Baccetti et a l , 1969a, 1970e, 1972b). b. The projections. Short side-projections (Kessel, 1967) are seen to jut out from the tubule wall, 150-170 A apart from one another and helically coiled (Fig. 13B, C) around the tubules (Perotti, 1969). In the cilia and flagella of Protozoa these projections exist in two series in tubule I and in one series in tubule I1 (Hopkins, 1970), or are present only in tubule I (Chasey, 1969). They seem to be separated from the tubule wall and vertically interconnected by filaments (Chasey, 1969).
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According to Warner (1970) they might be the binding site of the tubule wall to the “central sheath” which is also helically coiled around both structures with a similar pitch of 160 A . The central tubules do not generally undergo transformations towards the tail end, where they usually terminate after the accessory tubules and shortly before the doublets. In a few instances, e.g. Drosophila and the Trichoptera (Perotti, 1969; Baccetti et al., 1970e) they end after the doublets. Rarely, as in Tenebrio, they are the first to disappear (Baccetti et al., 1972a). In Gryllotalpa they are filled with a glycoprotein which becomes tanned, thereby making the distal half of the tail rigid and stiff (Baccetti et al., 197 1b). 2. Doublet tubules
a. The tubules. These are the only structures which are consistently present in all types of flagellate spermatozoa. Doublets are, as a rule, nine in number, lying at some distance from one another. They can be oriented and numbered from 1 to 9 .according to the general pattern of cilia and flagella (Afzelius, 1959; Gibbons and Grimstone, 1960): so that doublet 1 lies on the median plane normal to the line linking the two central tubules. Doublet 2 follows in the arm direction and so on. Only Protura exhibit 12 or 14 doublets (Baccetti et al., 1972d); in Sciara there are 70 (Phillips, 1966a: Fig. 11). In cross section both tubules in each doublet (Fig. 9B) are found to consist of a B tubule (200-230 A ) slightly larger than the A tubule (200 A). From tubule A two arms are seen to extend. The outer one is about 35 mp long and directed towards the flagellum centre, the inner one is only 20 mp long. These measurements have been checked by Allen (1968) in Tetrahymena cilia and seem to be valid for the insect flagellum (Warner, 1970). The arm pairs, up to 100 in thickness, are found every 200-220 A along the tubule length (Phillips, 1970b). The doublet wall and the arms are not made up of the same material. Both are proteinaceous (Fig. 14C) but, like the central tubules, the doublet consists essentially of tubulin (although this evidence concerns protozoan cilia and sperms from Echinodermata as emerges from studies carried out primarily by Renaud et al. in 1968 and by Shelanski and Taylor in the same year). The arms, however, consist of dynein, a 14s protein unit with a molecular weight of 600,000, arranged in linear polymers endowed with ATPase activity (Fig. 14E). These observations are based on studies of Tetrahymena and the sea urchin by Gibbons (1963) and Gibbons and Rowe (1 965). It is most unlikely that the sperm doublets differ
a
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Fig. 13
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from this structure. Unlike the tubule walls, the arms of the A tubules are rich in ATPase activity (Baccetti et al., 1972b). Both consist of globular units of comparable size with those of tubulin. However, they differ from one another in features other than their B is not a real size. Firstly, as shown by Phillips ( 1 9 6 6 ~ )tubule ~ tubule, but a groove adhering to tubule A. Nevertheless, the subunit number seems to be consistently 13 and it may be supposed that B opens against A. Furthermore, a chemical difference between the two tubules has emerged from enzymatic extractions of various types as reported by Behnke and Forer (1967), though in both cases they are proteins (Baccetti et al., 1970e). As a rule, tubule A appears “solid” and tubule B “hollow”. When, as in Gryllus, both are filled up with globular units, these are in greater number in A than in B, though the latter is narrower (Kaye, 1970). In Bacillus spermatids tubule B has glucose-6-phosphatase activity (Baccetti et al., 1972b). A new technique of heat fractionation devised by Stephens (1970) separated the doublets of sea urchin spermatozoa into two units. He was thereby able to isolate A and B tubules which had a similar guanine nucleotide content, but different levels of amino acids, in particular cystein. This explains why only tubule A can bind to dynein and raises some questions about other microtubule categories. The contractility problem also remains open since, again in the sea urchin, tubulin does not possess antigenic properties like those of actin (Stephens, 1970). Recently, Behnke et al. (1971) were able to demonstrate in Nephrotoma sperm tails, bundles of 15-20 actin filaments independent of the axoneme tubules. This actin may be involved in sperm motility, but its exact localization in the sperm is still unknown. The doublets are usually the longest sperm tubules and are the last to terminate at the tail tip. Often they dissociate into their two units (Phillips, 1 9 6 6 ~ ) In . the Homoptera the tail splits into four strands (Fig. 12D), each containing some doublets, which eventually dissociate (Folliot, 1970). In Gryllotalpa the doublets are invaded in Fig. 13. Fine structural pattern of the insect axoneme. (A) Radial links (RL) and peripheral doublet (D) of Drosophila, negatively stained. The doublet (D) appears not dissociated. ~ 140,000.(From Bairati and Perotti, 1970.) (B) Central tubule of Drosophila, negatively stained. Projections spaced at 170 A are evident. ~200,000.(From Bairati and Perotti, 1970.) (C) Platinum shadowed central tubule of Aiolopus. A helicoidal surface pattern is evident. ~ 48, 000.(D) Acid phosphatase in Ceratitis (Diptera) spermatozoa. The activity is evident on the plasma membrane and in the matrix surrounding the tubules. ~120,000.(E) Glycogen on the link-heads in cumpodea (Diplura). Thi&y’s method. ~75,000.(From Baccetti and Dallai, 1972.)
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B. BACCETTI
Fig. 14
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the distal half by a glycoprotein material which undergoes tanning, thus stiffening the entire tail segment (Baccetti et al., 1971b). In a few cases (e.g. in Trichoptera and Drosophila), the tubules are the first to end among the axonemal tubules (Baccetti et al., 1970e; Perotti, 1969). b. The radial links. Besides arms, the doublets also possess other projections which are directed centripetally (Fig. 4). Cross-sectional profiles suggested that they were radial laminae, which was, in fact, the earliest interpretation. They are radial links consisting of straight rays some 325 A long and 70-125 A thick (Behnke and Forer, 1967; Perotti, 1969; Warner, 1970) more or less orthogonally oriented to the fibre and alternatively spaced at 320 A and 560 A . When viewed longitudinally, each closely adhering pair is separated by a wide interval from the other. On the two opposite sides of the flagellum, the radial links are staggered 200 A from each other in a helical coil (Warner, 1970). An exactly comparable situation was reported in Chlamydomonas (Hopkins, 1970), hence it may possibly be of general occurrence.
3. Accessory Tubules These are characteristic, apart from a few exceptions, of the most evolved insects, from Campodeidae Diplura upwards. Their structure and size are closely reminiscent of those of the two central tubules (Fig. 9A, B). However, in some species they are thinner, while in other species (Trichoptera, Lepidoptera) they are thicker (over 300 A in diameter). In cross-section they show 13 globular units in their wall as in the two central tubules (Phillips, 1 9 6 6 ~ )Where . their diameter is wider, these units are in greater numbers; for instance in a Fig. 14. Histochemistry of the tail of insect spermatozoa. (A) Glycogen (arrows) in the central and accessory tubules of Bacillus rossius sperm. ThiCry’s method. ~75,000.(From Bigliardi et Q L , 1970.) (B) Glycogen (arrows) in central and accessory tubule ofhlystacides azurea (Trichoptera) sperm. Thikry’s method. ~90,000.(From Baccetti et al., 1970e.) (C) Pepsin treated sperm tail of Nemoura cinerea (Plecoptera). Doublets and mitochondria1 derivatives (arrows) appear significantly extracted. ~30,000. (From Baccetti et at., 1970e.) (D) Amylase treated sperm tail of Ceratitis capitata (Diptera). Only the accessory tubules (From Bigliardi et al., 1970:) and the two central ones (arrows) appear extracted. ~60,000. (E) ATPase in the sperm tail of Bacillus rossius (Phasmoidea). The reaction (arrows) 1s positive on the coarse fibres, on the arms of the doublets, on the central sheath and on the accessory bodies. ~60,000.(From Bigliardi e t al., 1970.) (F)UTPase in the same material. The reaction appears similar to that of ATPase. ~60,000.(From Bigliardi et al., 1970.) (G) UTPase in the sperm tail of Ceratitis capitata (Diptera). The reaction is positive only in axonema1 (coarse fibres, arms, central sheath) structures. ~60,000.(From Bigliardi et a/., 1970.)
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Coleopteran, as many as 15 or 16 were counted by Shay et al. (1 969). During spermiogenesis, these tubules arise from laminar outgrowths of the B tubules of the doublets (Cameron, 1965). These projections (Fig. 25A, B) first extend outwards in the form of a groove, then bend and once the tubular shape is achieved they separate from the doublets. Later, the material filling them makes its appearance. Therefore, their major chemical component should resemble that of the B tubules, i.e. tubulin B, but no information about this is available. When viewed longitudinally, projections are also discernible which may possibly connect adjacent accessory tubules. The content of the accessory tubules often resembles that of the central ones. In some species which have hollow accessory tubules, the central ones are hollow as well, e.g. in Plecoptera (Baccetti et al., 1970e). In other species (e.g. among the Diptera mentioned earlier) a globular osmiophilic core is found in both categories (Fig. 9). In rare cases, the central tubules are hollow (Fig. lOE), while an osmiophilic core is found in the accessory ones (Neuroptera: Baccetti et al., 1969c). The invasion by microcylinders, typical of Orthoptera (Kaye, 1964, 1970), is more massive in the accessory tubules; up to 15 units can be found in each tubule. A similar event was also reported in Coleoptera (Cameron, 1965; Baccetti, et al., 1972a) where there are fewer, namely, 6-7 units. The most spectacular behaviour is provided by the accessory tubules containing the glycogen-like polysaccharide (Phasmoidea, Trichoptera, Lepidoptera) as reported by Baccetti et al. (1969a, 1970e, 1972b). This polysaccharide appears as a compact mass which fills up the huge tubules previously described as over 300 A in diameter (AndrB, 1961; Yasuzumi and Oura, 1964). In the spermatid stage, during which the accessory tubules are being filled, nine Golgi vesicles (Fig. 25A, D) surround the axoneme, each concentrating around one tubule. Transfer of material, however, could not be established (Baccetti et al., 1972b). In the flagellum of some species, these accessory tubules arise more apically than the other axonemal units (Phillips, 1 9 6 6 ~ ) Since . they are secreted by the B tubules, they are pushed forward after separating from the latter. As a rule, these accessory tubules terminate towards the tail tip before the doublets and the central units. In a few cases, however, such as Trichoptera or Tenebrio (Baccetti et al., 1970e, 1972a), they are the last ones to end.
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In Diplura Campodeidae a 9 + 9 + 2 arrangement is differentiated early in the spermatid. The accessory tubules soon migrate around it and are arranged in two crescents (Fig. 12B, C), one consisting of five and the other of four units aligned against doublets 1, 7 and 8 on one side only of the axoneme. They are separated by the mitochondrion which is juxtaposed to doublet 9 (Baccetti and Dallai, 1972). Similar behaviour is shown in a thysanuran (Machilidae) in which the two crescent-shaped accessory bodies which flank the axoneme displace the accessory tubules into two series, one consisting of four and the other of five elements (Dallai, 1972). The classical 9 + 9 + 2 arrangement is first found in the highest Thysanura, namely, the Lepismatidae (Bawa, 1964; Werner, 1964), and is retained thereafter by all the Pterygota. B. THE CENTRAL SHEATH
This is a structure which surrounds the two central tubules (Fig. 4). In cross-sectional view it shows a circular profile about 700 A in diameter and ranges in thickness from 150 A , in the sector facing doublet 1, t o 90 4 in the contralateral one facing doublets 5 and 6 (Warner, 1970). In longitudinal sections it appears as a helically coiled band, with a pitch of 120 A (Andre, 1961) or of 160 A (Warner, 1970). This pitch, equal to the period of the projections of the central tubules, suggests that the central sheath is somehow anchored to it, even though the latter does not seem to run, as suggested by longitudinal sections, in exact correspondence to the lateral edges of the central tubules. The central sheath is ill-defined from a chemical standpoint because it is an extremely labile structure difficult to isolate or examine by negative staining. How it comes to be connected to the heads of the radial links remains obscure. It certainly contains (Fig. 14E, F, G) ATPase and UTPase (Baccetti et al., 1972b) and is present when, as in Ephemeroptera (Fig. 1OC), the central tubules are completely absent (Baccetti et al., 1969b). C . THE LINK-HEADS
The presence of nine fibrous units between the central tubules and the doublets was demonstrated in the Flagellates by Gibbons and Grimstone (1 960), who called them secondary fibres. Afzelius (1959), in the sea urchin sperm, interpreted them as juxtaposed
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radial links. But their nature remains obscure. Other elements in favour of the existence of longitudinal filamentous connections between the thickenings of the links lying in the same row, have been provided by Andre (1 96 1) in insect spermatozoa, and by Birge and Doolin (1 969) in vertebrate cilia. Almost all other workers have disregarded this problem. More recently, the presence of secondary longitudinal fibres has been disproved both in the flagellate flagellum (Hopkins, 1970) and insect sperms (Perotti, 1969; Bairati and Perotti, 1970; Warner, 1970) as well as in mammalian and amphibian sperms (Fawcett, 1970). All the data available in the literature related to the secondary fibrils in spermatozoa must be ascribed to the link-heads. The link-heads were first described by Andr6 (1961). In cross section, it was shown by Bairati and Baccetti (1965) that they are bipartite; this finding has been consistently confirmed by careful measurements done recently by Warner (1 970): in cross section they measure 200 A by 280 A . This value (Fig. 9A, B) is valid for the Diptera, a Dipluran Cizmpodea, Coleoptera and Orthoptera (Baccetti and Dallai, 1972; Baccetti et al., 1972a). However, a second arrangement exists (Fig. 24A) in Phasmoidea (Baccetti et al., 1972b) and in another Dipluran, Japyx (Baccetti and Dallai, 1972) in which the bipartite structure is less evident and the link-head is smaller in diameter, i.e. about 150 A by 180 A . They are chemically and structurally rather labile, hence poorly understood. Baccetti and Dallai (1 972) showed histochemically the presence of glycogen (Fig. 13E) at the same sites as both the large and small forms of link-heads in Diplura, while such glycogen deposits are not found in all other insects (see Anderson and Personne, 1970). A similar distribution of glycogen was described in some Gastropoda (Anderson and Personne, 1970). D. THE COARSE FIBRES
In the typical insect spermatozoon, an important material exists between the accessory tubules, which is rarely studied because it is difficult to fix and preserve. Baccetti ( 1963) and Baccetti and Bairati (1964) were able to identify four components in what were called “outer fibres” (Andr6, 1961) in Drosophila and Dacus, one of which obviously corresponds to the accessory tubule. Later, Bairati and Baccetti (1965) after identifying the accessory tubules, regarded the interposed material as amorphous, but viewed the presence of the interposed “X granule” as a unit in itself. Generally, all the material
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35 1
lying between the accessory tubules was considered as amorphous by later workers and neglected by them. However, Daems et al. (1 963), as well as Bigliardi et al. (1970), found ATPase and also UTPase localized in it (Fig. 14E, F, G). In Sarcophaga, Warner (1970) has recently identified two coarse fibres lying between the accessory tubules, thus confirming an interpretation similar to that of Baccetti and Bairati (1964). On the basis of histochemical data partly obtained from Dipteran spermatozoa, both coarse fibres (Fig. 14E, F, G) had ATPase and UTPase activity. However, they are certainly not the same in all the insects. On the basis of the literature on this subject it emerges that in Diptera (Fig. 9A) the coarse fibres are generally as Warner (1970) described them and the same is true for Coleoptera (Baccetti et al., 1972a: Fig. 9B) and Phasmoidea (Baccetti et al., 1972b) (Fig. 24A) and even in Thysanura Lepismatidae (Bawa, 1964). In other cases (Fig. lOE), however, they are much attenuated (Neuroptera, Lepidoptera, Plecoptera) and occasionally (Ephemeroptera: Baccetti et ul., 1969b) entirely absent (Fig. 1OC). A peculiar case is found in Diplura Campodeidae in which
Fig. 15. Biflagellate spermatozoa of Rhynchotoidea. (From Baccetti et aZ., 1969d.) (A) The sperm tail ofMenopongallime (Mallophaga).Two axonemes (a) and two mitochondrial (m) derivatives are evident. ~ 6 0 , 0 0 0 .(B) The sperm tail of Chryptothrips latus (Thysanoptera). The axoneme is composed by 18 doublets and four single tubules.
~135.000.
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the accessory tubules are all assembled against doublets 1, 7 and 8 without intervening material. External to doublets 2, 3, 4, 5 and 6, however, there are a series of associated osmiophilic structures which look like coarse fibres and are not separated by tubules. In the case of Bacillus spermatozoa, various nucleoside-phosphate phosphatases (GTPase, ADPase, CTPase) were identified biochemically in the same fraction (Bigliardi et al., 1970). It may be speculated that these enzymes are also concentrated in the same zone together with ATP- and UTPase, thus being involved in flagellar motility . E. THE AXONEMAL MATRIX
This is represented by all the space lying between the structures listed above and separated from the compartment in which the mitochondria1 derivatives are located, by means of membrane residues originating from the Golgi. This space often appears empty in electron micrographs. However, it contains a number of enzymatic activities detectable histochemically or by means of biochemical assays of the supernatant fraction. Histochemically, the most prominent enzyme (Fig. 13D) in this space is acid phosphatase (Bigliardi e t al., 1970; Baccetti et al., 1970b). Lactic dehydrogenase and phosphorylase are also abundant. They may also be detected on the plasma membrane, though only on the inner cytoplasmic membranes (partly plasma membrane, partly Golgi-derived ones) surrounding the axoneme. These enzymes are concerned with carbohydrate metabolism. Biochemical assays carried out on lactic dehydrogenase show that it is essentially present in the supernatant fraction from an Orthopteran, Aiolopus (Baccetti et al., 1970c) while it is strongly associated with membranes in a Phasmoid, Bacillus rossius, where it is quantitatively more important (Baccetti et al., 1972b). The different localization in the two foregoing instances may indicate a different physiological role, bearing in mind that Aiolopus sperm is capable of aerobic metabolism, while that of Bacillus is devoid of mitochondria, hence of cytochrome oxidase. Other enzymes involved in carbohydrate metabolism, i.e. phosphofructokinase, fructose-diphosphate aldolase and glyceraldehyde-3phosphate-dehydrogenase were identified in Bacillus rossius spermatozoa (Baccetti et al., 1972b). So far, their localization is unknown. This enzyme assembly, along with the presence of a polysaccharide in the accessory tubules, points to the existence of a glycolytic
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Fig. 16. Cross sections of several spermatozoa of the Coccid insect Parlatoria aleae: c, chromatin; m, microtubules; n, nucleus. ~70,000. (From Robison, 1970.)
pathway in the axonemal compartment. As a rule, carbohydrate utilization goes t o completion in the reactions catalyzed by mitochondria1 structures. In the absence of the latter (see next Section) it may be deduced that lactic acid is the end metabolite (Baccetti et al., 1972b). AIP--15
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VII. MITOCHONDRIA
As mentioned in the introduction, one of the essential features of the insect sperm is the peculiar evolution of the mitochondria, which are transformed into elongated structures flanking the axoneme. Their organization, however, is not the same in all insects, neither is it simple nor fully understood. A few very primitive insects exist in which the mature sperm has normal mitochondria which are fused into elongated masses. In others, some impressive mitochondrial transformations have occurred, while others lack mitochondria altogether . A. NORMAL MITOCHONDRIA
We may consider as normal mitochondria those with a moderately opaque matrix free of crystalline materials containing abundant well-developed inner cristae with clear-cut cytochrome oxidase activity. This mitochondrial type (Fig. 21 E) is found only among the most primitive insect groups, namely, in all of the Collembola (Dallai, 1967, 1970) and Diplura (Baccetti and Dallai, 1972), in the primitive Machilidae among Thysanura (Dallai, 1972) and in the most primitive Pterygota (Fig. 1OC), the Ephemeroptera (Baccetti et aL, 1969b). In all these cases the mitochondria, though retaining their normal aspect, undergo complex rearrangements during the spermatid phase. At this stage, mitochondria are numerous, small in size and have a tendency to fuse into a few units, undergoing the same rearrangements that were studied in greater detail in the more evolved mitochondria. These transformations lead to three elongated mitochondria in the sperm tail of Collembola, t o two in that of Diplura and Machilidae and to one in the Ephemeroptera sperm tail. These mitochondria retain both normal structure and function throughout the life of the sperm. In some, among the most involuted spermatozoa (Fig. 27) (e.g. those from the higher Termites, Reticulitermes), the mitochondria are also of the conventional model, namely small in size, numerous and provided with cristae (Baccetti eta]., 1972e). B. MITOCHONDRIA TRANSFORMED INTO DERIVATIVES WITH A CRYSTALLINE CORE
This, the best known mitochondrial category, was first studied by light microscopy (Bowen, 1920). The fine structural details have
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been fully described by Andre (1959, 1962) in Lepidoptera, by Pratt (1968) in a Hemipteran as well as by occasional reports from several other authors. All this data was coordinated by Phillips (1970b) who provided a scheme which can be applied to almost all insects. During the last spermatogonial generations and spermatocyte I prophase, the small spherical mitochondria increase considerably in number, until they fuse into long chains, oriented by the poles, forming a “palisade around the spindle” (Andre, 1962). Thus, meiosis leads to an exact distribution of mitochondria between the two daughter cells, until, during the second telophase, the mitochondria pack together into a skein (Retzius’ Nebenkern, 1904). Then there begins in each spermatid, the slow and conspicuous metamorphosis (Fig. 17) of the mitochondria into the Nebenkern through stages which And& called “twinned”, “chromophobic envelope”, “onion” and “loaf”. During these rearrangements two extremely long, labyrinthically interwoven identical chondrioconts are formed (Pratt, 1968) which eventually separate and come to lie on either side of the incipient axoneme. Together with the latter, the mitochondria develop within a narrow evagination of the plasma membrane against which the nucleus is first located. A cytoplasmic space gradually (Figs 18, 19, and 20) forms between the nucleus and the periaxonemal membrane as the latter is dragged distalwards away from the nucleus by the ring centriole (Fawcett and Phillips, 1970). The two Nebenkern derivatives insert themselves within this space (Phillips, 1970b), progressively elongating and flanking the axoneme. They taper until they become equal in size to the axoneme itself. In the Pterygota there are usually two mitochondrial derivatives (Figs4, 9, and 10) which either remain alike or one of them overtakes the other in length or in width (see Phillips (1970b) for a detailed case history). Occasionally, one of them progressively diminishes until it vanishes, thus the mature sperm possesses but one mitochondrial derivative (Fig. 10A, B). This occurs in the Mecoptera (Baccetti et aZ., 1969c) and Trichoptera (Baccetti et aZ., 1970e; Phillips, 1970b). Likewise in Thysanoptera (Baccetti et al., 1969d) (Fig. 15B) and in Diptera (e.g. Psychodidae, Baccetti et al., 1972c) (Fig. 26) only one mitochondrial derivative was found. In these cases, however, the spermatid mitochondria fuse directly into a single mass rather than two initial ones. When the two mitochondrial derivatives have flanked the axoneme for almost its entire length (instances of their equalling the axoneme in length are extremely rare) (Folliot, 1970), two important changes
Fig. 1 7
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take place. First of all (Fig. 22C), the cristae become aligned, regularly spaced at right-angles to one side of the major axis of the derivative, looking like a series of short lamellae. Then within the mitochondrial matrix some masses of material contained in regular granules, with a paracrystalline structure (Fig. 21 D), make their appearance. These were studied first by Andr6 (1 962). Subsequently they were resolved by Meyer ( 1964) and by Phillips (1 966a) using negative stains. This paracrystalline material invades the space left free of cristae in all the insects so far studied. It consists (Fig. 14C) of a protein (Baccetti et al., 1969c, 1970e) whose three-dimensional organization was suspected of varying according t o species (Meyer, 1964). It contained various types of longitudinal and transverse periodicities (the most common being 450 a) and different unit arrangements, which generally are hexagonally spaced. Two diverse crystalline organizations within the same mitochondrion have been found. The example quoted by Phillips (1970b) is not relevant, however, since the mitochondria described ‘are in reality an ordered extramitochondrial material (Mazzini, 1970). Intramitochondrial crystals probably have a pattern which is very common to many, if not all, insect orders. In Fig. 22(A, B), the same structure is demonstrated in a Dipteran and in a Coleopteran sperm. This material does not arise spontaneously within the mitochondria (Baccetti et ul., 1972a). During the last phases of spermiogenesis it was noticed by Baccetti and his collaborators (1 972a) that the Golgi cisternae come into contact with the mitochondrial wall and deposition of the early proteinaceous aggregates takes place at this site (Fig. 21A, B, C). This material is of unknown significance. In several species, repeated histochemical investigations (Bigliardi et ul., 1970; Baccetti et al., 1970b, 1972a) have shown that cytochrome oxidase is confined to the short cristae (Fig. 22C) and that the pro teinaceous matrix contains none of the enzymes concerned with respiration, glycolysis or the hydrolysis of triphosphate nucleosides. In all the spermatozoa provided with mitochondria, therefore, there are two pathways for energy production in the flagellum.
Fig. 17. Various stages of mitochondrial condensation and Nebenkern formation in the spermatid of Murgantia hisrrionicu (Rhynchota). (From Pratt, 1970.) (A) and (B). Early cluster or “twinned” Nebenkern. ~18,000.(C) Later cluster or “twinned” Nebenkern. ~16,000.(D) “Small sheet” or “chromophobic envelope” Nebenkern. x 17,000. (E) “Four layered” or “onion” Nebenkern. x 16,000. (F) “Two layered” or “loaf” Nebenkern. x 17,000.
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Fig. 18. Young spermatid of Tenebrio (Coleoptera). The ring centriole (rc) is still very close to the centriole (c) whereas the Nebenkern (n) are far away (cf., Fig. 19). ~30,000. (From Baccetti et aL, 1972a.)
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Fig. 19. Later spermatid of Euchistus (Rhynchota). The ring centriole (rc) is some distance from the centriole (c) and the two Nebenkerns (nb) are nearer than in Fig. 18. ~ 1 3 , 0 0 0(From . Phillips, 1970b.)
Normal respiration in the mitochondria1 compartment is by means of the Krebs cycle and anaerobic glycolysis which occurs in the axonemal compartment at the expense of the polysaccharide contained within the accessory tubules.
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Fig. 20. A still later stage spermatid of Euchistus (Rhynchota). The Nebenkern (ntd surrounds the axoneme between the ring centriole (rc) and centriole (c) ~13,000. (From Phillips, 1970b.) C. ABSENCE OF MITOCHONDRIA
Only one insect group provided with a typical flagellum is devoid of mitochondria. It is the order Phasmoidea (Fig. 24A), in which the genera Bacillus (Baccetti et al., 1972b) and Clitumnus have been studied. The young spermatid has a moderate complement of
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36 1
Fig. 21. Mitochondria in insect sperms. In (A), (B) and (C) a system of narrow membranes (m) originating from Golgi vesicles (g) penetrates between the axoneme (a) and mitochondrial derivatives (md) of Tenebrio spermatozoon, fusing with its limiting membranes (arrows). The inner mitochondrial crystallization (c) occurs near the region of first contact with the Golgi membranes. ~90,000.(From Baccetti et al., 1972a.) (D) Frozen-etched preparation of Tenebrio spermatozoon. The crystalline material filling the mitochondrial derivatives is evident (arrows). ~60,000. (From Baccetti et aL, 1972a.) (E) A normal mitochondrion (m) in a mature insect sperm (Campodea). ~48,000.(From Baccetti and Dallai, 1972.)
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Fig. 22. Crystallized mitochondrial derivatives in insect sperms. (A) Crystallized material of Drosophila (Diptera) after phosphotungstic acid negative staining. ~260,000.(From Bairati and Perotti, 1970.) (B) Crystallized material of Tenebrio (Coleoptera) after uranyl acetate negative staining. ~306,000.(From Baccetti et aL, 1972a.) (C) CytochromeC oxidase in the peripheral region (containing cristae) of Tenebrio mitochondrial derivatives (arrows). The crystalliie region (c) is negative. ~60,000. (From Baccetti etaZ., 1972a.)
mitochondria (which show no tendency to evolve into a Nebenkern), which possess inner cristae rich in cytochrome oxidase (Fig. 24B). At this stage, cell respiration is conducted normally. Subsequently, mitochondria undergo degeneration and are definitely absent in the spermatozoon. Biochemical assays showed that both cytochrome oxidase and succinic dehydrogenase activities were extremely low (Bigliardi et al., 1970) and were probably the result of contamination by spermatids. Oxygen consumption in sperm preparations, as
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determined in the presence of cytochrome c and ascorbate, is also absent (Baccetti e t al., 1972b). As reported in the preceding section, metabolism in the axonemal compartment is definitely reduced to glycolysis taking place under strictly anaerobic conditions. Both mitochondria or any form of mitochondrial derivative are absent from the category of non-flagellate sperms (Termites of the genus Kalotermes and coccids). In both cases (Figs 27 and 28), the spermatozoon is supplied with a rich microtubule complement (see below). In the sperm of coccids, which are the best studied, ATPase is present (Moses, 1966). Therefore, an active energy source, though different from oxidative phosphorylation, may be postulated (Ross and Robinson, 1969). In all these cases, the solution of the energetic problem may be analogous to that found in Phasmoidea spermatozoa. However, this is still an open question.
VIII. ACCESSORY ORDERED FLAGELLAR BODIES
One of the essential features of the sperm tail in insects is the fact that, besides the mitochondrial derivatives, the axoneme is flanked by one or more elongated accessory structures of paracrystalline texture. These structures are reminiscent of the compact protein core which often invades the mitochondrial matrix in Pterygotes. However, some do not seem to be related to them, either chemically or functionally. In addition, several categories may be identified.
A. STRUCTURED BODIES FLANKING NORMAL MITOCHONDRIA
This condition occurs only in the most primitive insects, in which mitochondria do not undergo their characteristic metamorphosis. In some Thysanura (Dallai, 1972) two long crystalline rodlets were described (Fig. 23B), which flank the flagellum throughout its length. They have a crescent shape when viewed in cross section. In longitudinal sections they display a paracrystalline texture with a periodicity of 200 A . In Ephemeroptera two elongated crystalline bodies (Fig. lOC) are also seen to flank the single mitochondrion. They have an average periodicity of 160 A . They seem to arise in close contact with the Golgi cisternae (Baccetti et aZ., 1969b). Their function and chemical composition is unknown.
3 64
B. BACCETTI
Fig. 23. Patterns of accessory flagellar bodies in insect sperms. (A) Tenebrio molitor (Coleoptera). ATPase present around the two bodies (b) and absent from mitochondrial derivatives (m) ~120,000.(From Baccetti et al., 1972a.) (B) Muchilis distincta (Thysmura). The accessory bodies (b) are much larger than mitochondria (m). ~ 6 0 , 0 0 0(From . DdS, 1972.) (C) Cixius netvows (Rhynchota). The accessory bodies (b) are as developed as the mitochondrial derivatives (m) ~35,000.(From Folliot and Maillet, 1970.) (D) Megouru viciue (Rhynchota). Spermatid: The accessory bodies (b) originate in contact with Golg membranes (g). ~ 6 0 , 0 0 0(From . Mazzini, 1970.)
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€3. STRUCTURED BODIES FLANKING THE MITOCHONDRIAL
DERIVATIVES WITH A CRYSTALLINE MATRIX
I. Connected with the Centriole Adjunct These formations occur in greater number and are better known than the preceding ones. In the spermatid, they make their appearance first as caudal expansions of the centriole adjunct. As they become progressively enlarged and surrounded by microtubules, they flank the axoneme and mitochondrial derivatives and end up embedded in the flagellum. A single one is found in Plecoptera (Baccetti et al., 1970e) and in Cicindela (Werner, 1965). Two symmetrical ones are present in Tenebrio (Baccetti et al., 1972a) and in Aphaniptera (Baccetti, 1968). They are primarily of a protein nature, with a carbohydrate core, demonstrated histochemically in Tenebrio. Again in Tenebrio, these structured bodies (Fig. 23A) are seen to be rich in ATPase and UTPase at least in a cortical halo consisting of globular units of 80 A . They show a compact organization in their central zone where the polysaccharide is located. The function of these formations still has to be assessed; however, the occurrence of ATPase seems to suggest some role in flagellar motility. Their origin is also unknown. The Golgi-derived cisternae never come into contact with them (Baccetti et al., 1972a). In Cicindela, Werner (1965) has documented the origin of the centriolar adjunct and its derivative from a caudal zone of the nucleus, where the nuclear membrane is highly porous. 2. Products of the Golgi Complex This section deals with the origin of the two accessory formations found in Homoptera , which are symmetrically positioned, like the mitochondrial derivatives, on either side of the axoneme. They often exhibit bizarre profiles (Fig. 23C) and a crystalline texture. They were studied in Delphacids by Herold and Munz (1967), in Cicadellidae by Phillips ( 1970b), in several Auchenorhyncha by Folliot and Maillet (1970) and in Aphids by Mazzini (1970). Their chemical characteristics and function are obscure. Only in Aphids has their origin from the Golgi-derived cisternae (Fig. 23D) been ascertained (Mazzini, 1970). C. STRUCTURED BODIES REPLACING MITOCHONDRIA
This is an extreme event typical of Phasmoidea (Bigliardi et al., 1970; Baccetti et al., 1972b). In this order, mitochondria disappear
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Fig. 24(A) The sperm tail of Bacillus rossius: ab, accessory bodies; at, accessory tubules; cf, coarse fibres; ct, central tubules; d, doublets. Mitochondria are lacking. x 120,000, (From Baccetti e t al., 1972b.) (B) Cytochrome-C-oxidasein a normal mitochondrion (m)of the spermatid of Bacillus. Seligman et al. 's method. ~ 6 0 , 0 0 0(From . Baccetti et al., 1972b.)
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during spermiogenesis and are replaced by two large bodies (Fig. 24A). These consist of alignments of proteinaceous material arranged in periodically juxtaposed small laminae and longitudinal filainents. In all sections, along almost the entire length, they appear J-shaped. They arise from the Golgi at the spermatid stage (Fig. 25B, C), are made up of proteins and are extremely rich in ATPase and UTPase. Their role is important as regards the type of locomotion, which in this case is peculiar as it follows almost circular trajectories. Although they do not appear to be structured in alternative fashions according to their thickness and do not change organization when the spermatozoon is naturally or experimentally relaxed, it was supposed that they function in contraction in view of their enzymatic equipment (Baccetti et al., 1972b). IX. SPERMATOZOA POSSESSING A DOUBLE FLAGELLAR APPARATUS OR BEING DEVOID OF IT
In relation to the rule of 9 which seems. to dominate doublet arrangement, mention has already been made of the huge flagellum of Sciara coprophila studied by Phillips (1966a) and by Makielski (1966). This has 70-90 doublets (Fig. l l ) , each flanked by an accessory tubule, which are derived from a large centriole spirally coiled around a single mitochondria1 derivative. This case seems to depend on the particular type of spermatogenesis (Metz, 1938) which leads ultimately to a single spermatid with a chromosome complement, two Xs and one or two heterochromatic chromosomes, corresponding t o the germinal tissue. In other sperm categories, described earlier, either the absence or an excess number of both central and accessory tubules can be found. A different case is represented by germ cells which either have two normal flagellar devices or where the flagellum is completely absent. A. THE PAIRED SPERMATOZOA
This is a peculiar situation found occasionally in the animal kingdom. It is generally known in Thysanwa Lepismatidae (Bawa, 1964; Werner, 1964) as well as in the coleopteran Dytiscus marginalis (Ballowitz, 1895), the gastropod Turritella terebru (Retzius, 1906; Idelman, 1960) and in American marsupials (Biggers and Creed, 1962; Phillips, 1970c, d). In Thysanura Lepismatidae, membrane fusion unites sperms two by two at the level of the nucleus.
Fig. 25
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Movement is possible only under this condition and isolated sperms are non-motile. In these insects, unlike Dytiscus, the sperm pair remains united in the female genital tract. What happens when the sperm penetrates the egg is obscure (Bawa, 1964). The type of fusion of the membranes at the nuclear level also requires further investigations. Phillips ( 1 970c, d) has observed in Opossum that at the moment of sperm pairing a gap junction arises between the two sperm membranes. B. SPERMATOZOA POSSESSING TWO AXONEMES
This is a characteristic feature of the whole group of orders known as Rhynchotoids (Fig. 15A, B); namely, the Psocoptera, Mallophaga, Anoplura, Thysanoptera and Rhynchota. In all instances, the doubling of this structure concerns only the axonemes and the centriole adjuncts, with the exception of. the mitochondria1 derivatives which are two in number (almost in .all cases) or even a single one (in Thysanoptera). Psocoptera generally have a single axoneme (Phillips, 1969); however, in one species, Atropos puZsatorium (Baccetti et al., 1969d), a high percentage of biflagellate sperms are found among the normal ones. It should be kept in mind, with regard to this, that among insects the accidental appearance of sperms with two axonemes is relatively frequent. This may be experimentally enhanced by mercaptoethanol treatment (Friedlander and Wahrman, 1965). It was reported by Baccetti et al. (1 969d) that, among Mallophaga, two equal axonemes consistently originate from two closely apposed centrioles lying beneath a rather enlarged nucleus. An analogous condition is found in Anoplura, among which Pediculus (Ito, 1966) and Haemutopinus (Baccetti et al., 1970a) have been studied. Among Thysanoptera (Fig. 15B) two axonemes are still encountered (Baccetti et al., 1969d); here, however, the accessory tubules, radial links and central sheath are wholly absent and the four central tubules, together with the 18 doublets, haphazardly form into a single bundle. It is curious that in such an arrangement Fig. 25. The origin of accessory tubules and accessory bodies in the spermatozoon of Bacillus rossius. (From Baccetti ef nl., 1972b.) (A) The tubules B of the doublets form a long outer arm, (a), and are surrounded by a system of Golgi (g) cisternae symmetrically disposed. ~ 6 0 , 0 0 0 .(B) The outer arms of the B tubules produce the accessory tubules (arrows). A Colgi (g) membrane is folded forming the precursor of an accessory body (b). ~180,000. (C) A later spermatid, with complete accessory tubules (at) and the rudiment of an accessory body (b) arising from a Colgi vesicle (g). ~90,000.(D)Migration of Cold vesicles (g) towards the axoneme. ~ 6 0 , 0 0 0 .
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the axoneme still retains its motility (unpublished observations on Thrips). Among Rhynchota the condition with two axonemes has the tendency to disappear. The heteropteran, Pyrrhocoris (Furieri, 1963b) has two axonemes which lie side by side, one longer than the other-a situation not found in other species. Other Heteroptera, e.g. Gelastocoris (Payne, 1966) and Nepa (Werner, 1966) have a single axoneme. The same is true of the most evolved Rhynchotoids, i.e. the Hornoptera, whose highest forms, the coccids, have non-flagellate spermatozoa. The presence of two axonemes, therefore, does not seem to be a haphazard event; it is a condition which distinguishes the primitive Rhynchotoid orders but disappears in the vast majority of the species in the most evolved orders. The type of motility acquired is rather peculiar and disorderly (see below). It is difficult to foresee what its selective advantage can be. C. NON-FIaAGELLATE SPERMATOZOA
This seemingly paradoxical condition appears, as far as I know, only in four insect groups (in one of which in two different forms). In three cases it appears to be associated with non-motility (the sperm being passively displaced), in the other two cases there are alternative organelles concerned with movement. Among the latter, the most widely known example is that of coccids mentioned above. In these Rhynchotes the syncytial condition which is shared by all insects during a given period of spermiogenesis (64 spermatids in Drosophila; 256 in Dacus-according to Baccetti and Bairati (1964); 128 in Atropos-according to Baccetti (1967)-and in Trychopteraaccording to Phillips (1970b); always increasing by the 2nd power) is retained by the adult sperms. In the light microscope Nur (1962) observed sperm bundles consisting of 16 elements in Pseudococcus obscurus, 32 in Eriococcus and Parlatoria, 64 in Puto. Moses and Coleman (1964) found in Steatococcus tuberculatus 32 spermatozoa in every bundle, each of them exhibiting a set of parallel microtubules surrounding the nucleus two and a half times. Robison (1966, 1970) and Ross and Robison (1969) have studied Parlatoria oleae, Pseudococcus obscurus, Matsucoccus bisetosus, Stomacoccus plantani, Kermes sp. and Put0 albicans. In the last species, spermatozoa generally exhibit a “cork-screw” shape. A motile apparatus, consisting of an alignment of 20-250 microtubules, spirals around a central chromatin axis (Fig. 16). Nuclear membrane,
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acrosome, mitochondria, centrioles and axoneme are all absent. The microtubules contain ATPase (Moses, 1966) and their arrangement always follows asymmetrical patterns. According to Robison (1 970) these features suggest that the microtubule bundle is associated with sperm motility. Ross (1 97 1) points out that the cork-screw shape is attained by virtue of a second microtubule system helically coiled around the spermatid, thereby moulding its characteristic outline. On the contrary, in one case in the Termites, replacement devices do not exist, hence the absence of a flagellum is accompanied by non-motility. In another instance replacement structures are available. The loss of flagella is progressively acquired in the course of evolution of the different families, the most primitive of which seem to possess normal flagellate spermatozoa. Some examples were succinctly described at the light microscopical level by Springhetti (1963). In the more primitive Mastotermitidae and Termopsidae, the spermatozoon is elongated and bears a flagellum; it is likely to be of the orthopteroid type encountered also in Embioptera (Baccetti et ul., 1972e). But in Kalotermitidae and Rhinotermitidae, spermatozoa appear to lack the flagellum. Baccetti et al. (1972e) have described this spermatozoon in a specimen from the first family, Kalotermes flavicollis and in one belonging to the second family, Reticulitermes Zucifugus. In the latter (Fig. 27) the spermatozoon is a severely involuted cell. It has a compact almost perfectly spherical shape. The acrosome is absent. At the posterior pole, a small cytoplasmic rim accommodates a few mitochondria of a conventional pattern, namely spheroidal in shape and small in size. Also present are two extremely short parallel cylinders looking like centrioles, but which are actually two truncated axoneme apices lacking the two central tubules and only provided with nine doublets at their periphery. These two flagellar anlagen do not protrude from the roundish sperm body which is made up solely of a head and unable to move. In Kulotermes (Fig. 28) a much more bizzarre shape is encountered. During spermiogenesis, the nucleus of each spermatid is condensed and flattened into an ovoid shape by a set of microtubules which surround it. The plasma membrane is separated from the nuclear membrane by a most tenuous interspace in which the microtubules are seen to run in a single array even in the mature sperm. The plasma membrane is displaced at four angles into four long arms which are also completely occupied by microtubules. Thus, apart from the acrosome, this sperm is devoid of flagellar anlagen and mitochondria. This situation closely resembles that
Fig. 26. Cross sections of mature sperms of Thelmatoscopus albipunctatus: a, acrosome; m, mitochondria1derivative; n, nucleus. The axoneme is lacking. ~120,000.(From Baccetti e t al., 1972c.)
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Fig. 21. The spermatozoon ofReticulitermes lucifugus.~ 6 0 , 0 0 0 .
Fig. 28. The spermatozoon of Kalotermes jlavicollis. ~ 3 9 , 5 0 0 .
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found in coccids. How this spermatozoon moves, if it does, is unknown. However, the long flattened arms crammed with microtubules suggest a pattern of undulating membranes. In the Diptera Psychodidae, Baccetti et al. ( 1 9 7 2 ~have ) examined several species, in which a common behaviour was pointed out. Their spermatozoon (Fig. 26) looks like a rather stiff long needle, with a multilayered cortical zone. This is actually the result of an association of the plasma membrane with the secretory products occurring in the testis lumen, which surround each germ cell. Inside this theca there is a nucleus which is highly elongated, compact and wholly or partly enveloped by a profuse material which is derived from the Golgi and may be viewed as an acrosome. Embedded in the acrosome is a small mitochondria1 derivative which has few cristae. These spermatozoa, which distinguish the whole Psychodidae family, are also completely immotile. In the genus of Protura, Ementornon, the spermatozoon has the form of a disk when contained in the testes. The circular nucleus is perispherical and flattened mitochondria lie in the central area. After ejaculation, the cell assumes the form of a cup. X. MOTILITY A. MOTILE MECHANISMS
The problem of insect sperm motility is centred around an understanding of ciliary motion of which it is a variation. Indeed, there are a series of variations of increasing complexity which parallel an increase in structural complexity of both supporting and contractile tail structures. Most attempts to explain ciliary or flagellar motion have so far focused on the flagellum of flagellate Protozoa or on the sperm tail of the sea urchin, both of which have many similarities. They both have a plain axoneme of the 9 + 2 pattern. Some experiments have been performed on the bull spermatozoon, whose axoneme is more complex ( 9 + 9 + 2) but whose general pattern agrees with the preceding model, being a sperm with a large head and a short tail. From the overall information thus far collected (Holwill, 1966; Brokaw et al., 1970; Brokaw, 1971) two conclusions are evident: (1) In Protozoa and in sea urchin sperm, motion consists of consecutive waves propagated along the tail thus resulting in a planar beat. Bull spermatozoa, however, show a helicoidal motion.
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(2) In spermatozoa these waves originate from a point situated in the centriole zone (Bradfield, 1955). This was demonstrated by amputations performed by Bishop and Hoffmann-Berling (1959), Goldstein and Brokaw (1968) and Goldstein (1 969) with a laser microbeam, and by Van Herpen and Rikmenspoel's (1969) indirect estimates concerning the size of this control centre on the basis of its susceptibility to X-rays and protons. In Flagellata, on the contrary, the amputated portion of the flagellum can beat and a different origin of waves must be supposed (Goldstein et al., 1970). At present, any explanation as to the role played by the single fibrillar components in motility and the mechanisms involved in wave triggering and propagation, are a matter of speculation. The presence of directly contractile structures has been debated. Gray (1 955) and Machin (1 958) considered that they were not associated with any precise flagellar component, whereas others have identified them with the two central tubules (InouC, 1959) or with the nine doublets which are the only universally occurring structures also having ATPase in their arms (Bradfield, 1955; Silvester and Holwill, 1965). Satir (1961) advanced a hypothesis based on the twocomponent mechanism of muscle contraction. He suggested that a myosin-like component might exist in the tubules and an actin-like one in the matrix, which for the first time is thus believed to be involved in flagellar motility. Along this line, again under the influence of muscular contraction, Satir (1968) and Brokaw (1968, 1971) put forward a new view on a sliding model, excluding the matrix and proposing (Satir) a sliding of each B subfibre with respect to the others. This fact cannot be demonstrated in sperm tails, where the orientation of the bent tip is difficult to determine. A separation between A and B subfibres has occasionally been described at the tip of the tail in Insect spermatozoa, but this does not demonstrate any form of sliding. Another hypothesis has been put forward by Schreiner ( 197 1) on purely mathematical grounds. He excludes contraction along any type of tubule and postulates the existence of welding points between the matrix and motile tubules along the flagellum, which would lead to the displacement of the straight segments during wave propagation. The active part of the movement would therefore be the recovery stroke. Nothing is known about this in the insect sperm flagellum where the recent demonstration of actin filaments, apparently independent of the microtubules (Behnke et al., 1971), suggests the presence of a muscle-like
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Fig. 29. Sequences of frames from high-speed cinefilms showing flagellar beat of different insect sperms. (A) Ctenocephulus mnis: 9 + 2 model. Total time: 0.21 s. x380. (B) Gryllotulpu gryllotulpu: 9 + 9 + 2 model with a rigid caudal tip (t). Total time: 0.43 s. x560. (C) Huematopinus suis: 9 + 9 + 2, the axoneme twinned in each sperm. Total time: 0.21 s. x790.
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mechanism. In connection with the helical shape of the waves, the structures which exhibit a helical arrangement around the sperm main axis may be thought to play an important role in the progression of the contractile events. These structures are the central sheath and the radial links. It is interesting to recall that the doublets run parallel to the sperm main axis. Motion pictures of sperms from different species show at least three forms of movement which seem to correspond to a structural hierarchy. The spermatozoa (9 + 2 axonemal pattern) of some species show a simple, almost planar wave of low frequency with a wave length as long as, or longer than, that of the flagellum. Tails are generally inextensible and a single wave type arising in the centriolar zone is propagated along them. The example examined by us is the spermatozoon of Aphaniptera (Fig. 29A). This has the simplest type of motility which closely resembles that of flagellates, marine sperms and cilia in general. At a second level of complexity there is a helical wave (owing t o two harmonic movements of equal amplitude). This model (Fig. 29B) is typical of all the species possessing the 9 + 9 + 2 formula, with accessory tubules and ATPase-rich accessory fibres. This type of movement and structure occurs in many insect and mammalian sperms. At the third level of complexity there is a combination of the two types of undulation. This is the form of movement which occurs in spermatozoa having a 9 + 9 + 2 tail plus accessory bodies endowed with ATPase activity. In some cases these bodies enable the sperm to course a doubly helical trajectory which remains straight (Tenebrio: Fig. 30A), in other cases their shape compels them to follow always doubly helicated trajectories in a helical course (Bacillus: Fig. 30B). A detailed study on sperm motility (Baccetti et al., 1972b) deals with this last form of movement which incorporates the preceding ones. The relevant deductions are as follows. Initiation of motility takes place in the centriolar region and has been identified in the axoneme apex which is supplied with laminae showing ATPase activity. This motor unit is called initiation motor (it must be remembered that the mature insect sperms lack a centriole). In this region it is postulated that consecutive small range revolving movements occur around the central axis, leading to related displacements between the bundle of tubule apices and the centriole adjunct. This torsional impulse propagates passively along the tail, through the assembly of tubules by virtue of their inextensibility. The internal viscosity of this system involves some energy expendi-
Fig. 30
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twe during the transmission of the movement. For this reason the first deformation stops almost at once and is of limited mechanical extension. This deformation, however, is enough to trigger a contraction in all the axonemal structures possessing ATPase activity (central sheath, arms, coarse fibres), possibly reacting with the actin units and thus propagating the contractile wave along the flagellum; this wave propagation is an active one. Tubules function only to propagate the mechanical deformation to consecutive levels. This wave is not reflected, but it dies away near the end of the flagellum. Upon it, at a following moment, a second wave of greater amplitude is superimposed. It arises when the oscillation frequency of the initiation motor nears the natural oscillation frequency of the structures which surround the axoneme. The resulting resonance enhances the oscillation amplitude of the outer structures until a mechanical deformation is also produced outside the axoneme, such as to evoke the active reactions of the extraaxanemal bodies which also are endowed with ATPase activity. The greater structural consistency of these organelles results in wave propagation towards the caudal end, as if it was an elastic wave which is reflected by the tail tip. This reflection occurs in a reverse direction, since it may be tentatively postulated that the surrounding medium acts as a boundary for the tail. The helicoidal trajectory is determined by the non-rectilinear shape and non-linear reactivity of the structure of the sperm tail. When the above observations are extended to more simpler cases, it may be deduced that where extraaxonemal bodies are not available a single cylindrical-helicoidal wave type arises (simple 9 + 9 + 2 insects). When the ATPase-containing coarse fibres lying between the accessory tubules are also absent, the quasi-planar wave type is evoked. This wave form has a much lower frequency, but a greater length being as long as the sperm tail and it is characteristic of the 9 + 2 model (first type of movement, as for instance among Aphaniptera). Other motility patterns, related to a particular flagellar architecture have emerged. As already mentioned, Gryllotalpa (Baccetti et al., 1971b) has a tail divided into two segments. The first has a Fig. 30. Sequences of frames from high-speed cinefilms of flagellar beat of different insect sperms. (A) Tenebrio molitor: 9 + 9 + 2 model with little accessory bodies: the trajectory is double helicated, but linear. h, Head. Total time: 0.17 s. x460. (B) BuciZlus rossius: 9 + 9 + 2 model with enormousaccessory bodies. The trajectory is double helicated and circular. h, Head. Total time: 0.09 s. x430.
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conventional 9 + 9 + 2 pattern, without accessory tubules and moves according to the general insect model. The second, with sclerified tubules, is completely rigid (Fig. 29B). The biflagellate Rhynchotoid sperm also moves in an irregular fashion. For instance, in Anoplura a rather ill-coordinated movement is found (Fig. 29C), although it belongs to the category of a single cylindrical helicoidal wave, short and frequent (general type), as suggested by the absence of accessory bodies. Movement of the complex syncytium of sperms in Coccids is most peculiar (Robison, 1970) and can possibly be regarded as helicoidal, like the motion of flagella (on which, however, the various wave types have not been studied). Since the accessory bodies are lacking, it should be ascribed to the two simpler categories, possibly to the second one. B. METABOLIC ASPECTS OF MOTION
In the preceding sections it was seen that in insect spermatozoa an important energy source is due to intense anaerobic glycolysis occurring in the axonemal complex. Normal respiration effected in the mitochondria1 compartment, when present, may be added to it. In all cases the stored polysaccharide is glycogen, which usually accumulates in the accessory tubules at the spermatid stage. The mature spermatozoon can thus call upon an endogenous store of carbohydrate. However, since a high carbohydrate level exists in both the seminal fluids and the spermatheca secretions, these may be thought of as being somehow a part of the material metabolized by the spermatozoon, despite the fact that the uptake of carbohydrate, or even indications of any type of pinocytosis, have never been demonstrated. Indeed, in many cases, sperm locomotion was observed even without carbohydrate administration (Hughes and Davey, 1969). In normal respiring sperms, oxygen is an element which regulates their motion. It may be experimentally demonstrated not only that these sperms are immotile under anaerobic conditions, but also that they migrate towards oxygen gradients. It was suggested that the tracheae-rich female conceptacula can thereby direct the movement of spermatozoa (Rao and Davis, 1960). Spermatozoa devoid of mitochondria are insensitive to oxygen (Baccetti e t al., 1972b). Hydrogen concentration also exerts an important effect on sperm motility: an optimum between pH 7.0 and 7.4 was demonstrated in Cimex by Rao and Davis (1 969). Richards (1963a) was the first to show that in Periplaneta sperm
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motility is a function of temperature and attains a maximum at 39". Davey (1958) has made similar observations. In Bacillus sperm (Baccetti et al., 1972b) motility was enhanced up to 50"C, but then it decreased though still remaining high up to 60°C. At this elevated temperature, however, few sperms were seen to survive. Temperature does not seem to appreciably influence wave length, but it does enhance both wave frequency and velocity. Motility is obviously affected by the osmotic pressure of the medium. In Periplaneta, Davey (1 958) reported a maximum activity with a tonicity exceeding 400 mosmoles. Later, lower tonicities were tested (Hughes and Davey, 1969). Generally, in flagella, altered conditions of viscosity affect the frequency, but not the form of beat (Brokaw, 1963, 1966; Holwill, 1965). Nevertheless, the high viscosity seems to reduce to two dimensions the normal threedimensional motion of bull spermatozoa (Rothschild, 1961). Studies on this problem in insect sperms are in progress. . C. THE PROBLEM OF SPERM CAPACITATION
This subject has been little investigated in insects. As observed by Hughes and Davey (1969) the general concept we can reach at present is that in some species fully mature sperms are transmitted to the female, while in other species their maturation is achieved in the female organism, thereby leading to a form of capacitation. In Periplanetu, for example, the extraacrosomal space is reduced in the spermatheca (Hughes and Davey, 1969). Likewise, in Sciuru a portion of the Nebenkern is eliminated (Makielski, 1966). In coccid insects the spermatozoa originally assembled under syncytial conditions become free individuals (Robbins, 1965). As stated earlier (Section 11), the most outstanding transformations occurring in the spermatheca concern the digestion and remoulding of the glycoprotein materials overlying the plasma membrane (Payne, 1934, in a hemipteran; Riemann, 1970; Riemann and Thorson, 1971; Phillips, 1971, in many Lepidoptera; Renieri and Vegni, 1972, in Iocusts). A form of capacitation may perhaps be recognized in these aspects, which in the moths Trichoplusiu and Anugustu culminates in the formation of an extracellular rod-like crystalline structure running parallel to the sperm, embedded in two thick crystallized sheaths. All this complex comes into being exclusively within the female genital tract and it originates from the material which formed the laminated appendages and the satellite body (Riemann, 1970; Riemann and
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Thorson, 197 1). The fine structure of the penetrating spermatozoon still requires more detailed study. XI. SPERMATOZOA POLYMORPHISM AND GENETICS
After the earliest work by von Siebold (1836), who discovered an atypical spermatogenesis associated with a typical one in a Mollusc, a number of investigations have been focused on the typical two-fold spermatogenesis found in many invertebrates; in particular in Lepidoptera where it was brought to light by Meves (1903). Fain-Maurel ( 1966) and Zylberberg ( 1969) have summarized its most relevant aspects. Among the various insect orders, when an atypical sperm line exists, it is generally a diploid or polyploid hyperpyrene line (Richards, 1963b; Bouix, 1963) because of variations in the ratio of DNA to nuclear proteins (Ansley, 1958). On the contrary, in Lepidoptera either an apyrene line is found when the nuclei are entirely eliminated during spermiogenesis, or an oligopyrene line when some nuclear residues persist (Zylberberg, 1969). Hyperpyrene spermatozoa have a normal length, but much thicker heads and tails as compared to normal sperms. They have been little studied as yet; the apyrene ones in Lepidoptera have been the object of recent research on the parts of Zylberberg and of Phillips (197 1). In these latter cells the nucleus does not undergo its typical condensation during the spermatid stage, as is true of eupyrene sperms; it remains spherical, glides caudal along the flagellum and then is eliminated. The acrosome does not form and the centriole comes to lie in the anterior cell portion. The corpuscle, interpreted by Zylberberg (1969) as a centriole, is conversely interpreted as a cap of undefined material by Phillips (1971). Both workers have found defective mitochondria1 derivatives in the apyrene line. In addition, Phillips (1 97 1) finds hollow instead of solid accessory tubules and, as already stressed, an absence of lacinate appendages around the plasma membrane. The presence or absence of the nucleus is not therefore related to the absence of any other organelle (excluding the acrosome, which forms on the nucleus itself), but rather is seen to affect normal organization in the spermatocyte stage. In fact, the cytoplasm has already received all its necessary information from the nucleus, and the spermatid nucleus is dormant (Olivieri and Olivieri, 1965). Many important papers on this aspect have appeared describing the long series of investigations on Drosophila mutants lacking the Y chromosome (Meyer, 1964, 1968; Kiefer, 1966;
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Anderson, 1967). Drosophila heydei does not progress beyond the spermatocyte I stage (Hess and Meyer, 1963); in Drosophila melanogaster, on the contrary, spermiogenesis begins without going to completion. All the spermatozoa of these X-0 males possess: ( 1) ill-organized mitochondria1 derivatives, sometimes supernumerary ones, with an anomalous crystalline core; (2) distorted or broken axonemes, which may lack some of its units; (3) bundles each having an overall smaller number of sperms which have shorter tails because they are immature. On the other hand, in the XYY males, spermatozoa are twice as long as normal ones (Meyer, 1968). Hence, in Drosophila. the Y chromosome seems to be endowed with a given number of fertility factors essential for the development of normal functioning spermatozoa. These factors must come into play during spermatocyte I stage since in normal X males spermatozoa are also normal. In this phase, similar loops to those of the lampbrush chromosomes are seen to form. In special mutants in which the Y chromosome is shorter (Bairati and Baccetti, 1966) and lacks one or more loops, sperms do not undergo maturation, thus resembling those from X-0 males. These individuals are sterile (Hess and Meyer, 1968). A particular mutant with a short Y (KL- 1-), studied by Kiefer (1969), has appgrently normal and motile spermatozoa and is capable of fertilizing the female. However, these spermatozoa degenerate within the female. They probably bear biochemical deficiencies which cannot be detected at the morphological level. In summary, the hypothesis is forwarded that the Y chromosome does not code for the proteins required in the formation of the sperm organelles, but governs the coordination of the various synthetic and morphogenetic steps (Meyer, 1970). Similar malformations have been induced by heat (Anderson, 1967) and X-rays (Hess, 1965). Altering the haemolymph composition by adding K + and Mg2+ (Meyer, 1969) also induces similar effects. All these altered features may in fact be viewed as phenocopies of Y deficiencies. What happens in the normally X-0 species (Orthoptera, Protenor) under the same experimental conditions has not been studied. Neither is it known whether a dimorphism between X-sperms and 0-sperms is detectable in those species in which a large X occurs, while their autosomes are minute. Almost all the genetics of spermatozoa still need studying.
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ACKNOWLEDGEMENTS
The original research reported in this article was supported by C.N.R. I am indebted to Drs. A. Bairati, jr., R. Dallai, R. Folliot, P. L. Maillet, M. Mazzini, E. Perotti, D. M. Phillips, S . Pratt and W. G. Robison for permission to use figures. REFERENCES Afzelius, B. (1959). Electron microscopy of the sperm tail. Results obtained with a new fixative. J. biophys. biochem. Cytol. 5,269-278. Allen, R. D. (1 968). A reinvestigation of cross-sections of cilia. J. Cell Biol. 37, 825-83 1. Anderson, W. A. (1 967). Cytodifferentiation of spermatozoa in Drosophila melanogaster. The effect of elevated temperature on spermiogenesis. Molec gen. Genet. 99, 257-273. Anderson, W. A. and AndrB, J . (1968). The extraction of some cell components with pronase and pepsin from thin sections of tissue embedded in an epon-araldite mixture. J. Microsc. 7,343-354. Anderson, W. A. and Ellis, R. A. (1967). Cytodifferentiation of the crayfish spermatozoon: acrosome formation, transformation of mitochondria and development of rnicrotubules. 2.Zellforsch. mikrosk. Anat. 77,80-94. Anderson, W. A. and Personne, E. (1 970). The localization of glycogen in the spermatozoa of various invertebrate and vertebrate species. J. Cell Biol. 44, 29-5 1. AndrB, J. (1 959). Etude au microscope electronique de l’evolution du chondriome pendant le spermatogknkse du Papillon du Chou Pieris brassicae. Annls Sci. nat. Zool. 12 Ser., 283-308. AndrC. J. (1961). Sur quelques ddtails nouvellement connus de l’ultrastructure des organites vibratiles. J . Ultrastruct. Res. 5 , 86-1 08. AndrB, J. (1962). Contribution 2 la connaissance du chondriome: Btude de ses modifications ultrastructurales pendant la spermatogBnkse. J. Ultrastruct. Res. Suppl. 3, 185 pp. AndrC, J. (1963). Some aspects of specialization in sperm. In “General Physiology of Cell Specialization” (G. Mazia and A. Tyler, eds), pp. 9 1-1 15. McGraw-Hill, New York. Andrt, J. (1965). A propos d’une leCon sur la Limule. Annls Sci. Clermon t-Ferrand 26, 2 7-3 8. Andrb, J. and Bernhard, W. (1 964). The centriole and the centriolar region. In “XIth International Congress Cell Biology”. Providence, R.I.9. AndrB, J. and ThiBry, J.-P. (1963). Mise en Bvidence d’une sous-structure fibrillaire dans les filaments axonkmatiques des flagelles. J. Microsc. 2, 7 1-80. Ansley, H. R. (1958). Histones of mitosis and meiosis in Loxa flavicollis (Hemiptera). J. biophys. biochem. Cytol., 4, 59-62. Austin, C. R. (1948). Function of hyaluronidase in fertilization. Nature, Lond. 162. 63-64.
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Zirwer, D., Buder, E., Schiilike, W. and Wetzel, R. (1970). Lineardichroitische untersuchungen zur DNS-Organisation in Spermienkoffen von Locusta migratoria, L. J. Cell Biol. 4 1 , 4 3 1-434. Zylberberg, L. (1969). Contribution 21 l'btude de la double spermatogenbe chez un Lbpidoptkre (Pieris brassicae L. Pieridae). Annls Sci. nut. 2001.Ser. 12, 1 1 , 569-626.
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Author Index Numbers with an asterisk refer to pages on which references are listed at the end of the paper
B
A Abbott, N. J., 258, 293, 300, 303, 305 * Adams, J. B., 189, 209, 212, 214, 215, 250* Adams, R. T., 147, 181* Afzelius, B., 343, 349, 384* Agarwal, H. C., 72,78,80, loo*, 108* Akov, S., 56,60, 100* Alberici, M., 35,43* Allais, J. P., 74, 101* Allen, R. D., 343,384* Allen, R. R., 75, 101* Alvarez, R., 153, 155, 181* Alving, B. O., 277,306* Ames, A., 293, 306* Anders, F., 188, 211, 216, 220, 221, 250* Anderson, E., 324,386* Anderson, W. A., 330, 332, 350, 383, 384* And& J., 316, 318, 322, 332, 338, 341,348,349,350,355,357,384* Ansell,G. B.,69, 95, 96, 101* Ansley, H. R., 382,384* Aranda, L. C., 126, 128, 132, 158, 160, 161, 178*, 180* Argarwal, H. C., 72, 78, 80, loo*, 108* Ariens, E. J., 11,46* Arnaiz, G. R. D., 35,43* Ashhurst, D. E., 264, 274, 293, 306* Asperen, K. Van., 276,306* Aurbach, G. D., 21,38,43* Ausit, E. G., 153, 155, 181* Austin, C. R., 326, 384*
Babad, H., 16, 37,42* Baccetti, B., 316, 317, 318, 319,320, 322, 324, 325, 326,327,328,329, 330, 331, 333, 334, 335,336,338, 339, 341, 242, 243,345,347,348, 349, 350, 351, 352,353,354,355, 357, 358, 360, 361, 362,363,364, 365, 366, 367, 369,370,371,372, 374, 377, 379, 380, 381, 383, 385*, 386*, 394* Bairati, A., Jr., 327, 338, 341, 345, 350, 351, 362, 370, 383, 385*, 386* Balasubramanian, A., 232, 233, 234, 246,250* Ballowitz, E., 367,386* Baptist, B. A., 231, 234, 235, 237, 238,246,250* Barker, K. R., 327,386* Barkley, D. S., 34,45* Barlow, J. S., 57, 105* Barnum, C. P.,100,107* Barron, E. S. G., 54, 108* Baumann,C. A.,57,61,89, 107* Bawa, S. R., 329, 349, 351,367,369, 386 Baxter, C., 151, 178* Bdolah, A., 15, 16,37,42* Beams, H. W., 324,386* Beaudoin, A. R., 72, 78, 83, 89, 90, 101 * Beck, S. D., 57, 101*, 188, 209, 210, 212,215,251* Behnke, O., 338, 341, 345, 347, 375, 386*
399
400
AUTHOR INDEX
Bentley, P. J., 39,40,42* Ben-Zvi, R., 16, 37,42* Berendes, H. D., 32,45* Bergerard, J., 74, 101* Berlin, L. C., 209, 210, 230, 231, 250* Bernard, R., 56,60, 106* Bernard, W., 332, 384* Bernheim, M. L. C., 54, 105* Bernini, F., 328, 329, 354, 371, 386* Berridge, M. J., 2, 3, 5, 7, 12, 13, 15, 16, 18, 19, 22, 23, 24, 25, 26, 27, 28, 29, 31, 42*, 46*, 47*, 267, 306* Bhamburkar, M. W.,75, 107* Bhat, J. V., 73,78, 109* Bieber, L. L., 55, 56, 60, 62, 65, 68, 70, 73, 78, 80, 81, 84, 85, 87, 88, 90,91, 101*, 107* Biesele, J. J., 324, 332, 334, 338, 348, 387*, 388*, 395* Biggers, J. D., 367, 386* Bigliardi, E., 318, 319, 320,333,334, 342, 347, 348, 351,352,357, 362, 365,369,385*, 386* Binstock, L., 286,306* Birge, W. J., 350, 386* Birt, L. M., 73, 76, 103* Bisho, D. W.,375, 386* Bjork, L. B., 329,389* Blakley, R. L.,54, 101* Blewett, M., 55, 56, 57, 101*, 104* Bloch, D. P., 33 1,386*, 388* Bloom, F. E., 35,47* Bloom, W.,37,42* Boistel, J., 63, 101*, 164, 178*, 259, 275, 277, 278, 281, 282,288, 289, 304,306*, 308*. 309* Bongers, J., i95, 2b2, 203, 204, 206, 250* Bonner, J. T., 34,45* Borkenhagen, L. F., 54, 101* Borle, A. B., 38,42* Bouix, G., 331,381,387* Boulton, P. S., 260,280, 306* Bourguet, J., 39,40,42*, 45* Bowen, R. H., 354,387* Boyme, T., 38,44* Brack, S. D., 331, 386*
Bracken, C. K., 77, 101* Bradfield, J. R. C., 329, 374, 375, 387*, 389* Brading, A. F., 35,42* Brady, J., 275,306* Brady, R. O., 54, 106* Branson, C. M., 37,45* Braun, G., 3,46* Breckenridge, B. McL., 34,42* Breland, 0. P., 324, 332, 334, 338, 348,387*, 388*, 395* Bremer, J., 54, 89, 102* Breucker, H., 317, 385*, 390* Bridges, P. M., 68, 109" Bridges, R. G., 57, 58, 61, 65, 66,61, 68, 69, 73, 74, 75, 76, 77, 78, 79, 80, 81, 83, 84, 85, 87, 88, 90, 91, 92, 93, 94, 97, 98, 102*, 103* Brighman, M. W.,302,303,306* Brodie, B. B., 38,45* Brokaw, C. J., 374, 375, 381, 387*, 390* Bronskill, J. F., 234, 238, 250* Brookes, V. J., 72, 73, 78, 81, 91, 101*, 106* Brown, A. W. A., 56, 58,72,78, 104*, 108*
Brown, B. M., 172, 173, 174, 175, 178* Brown, G. G., 316,387*, 395* B r u t , M., 57, 102* Bryan, J., 341, 387* Buck, J. B., 276,306* Buder, E., 332, 397* Bueding, E., 35,43* Biilbnng, E., 35,42*, 43* Burgos, R., 37,48* Bum, J. H., 34,42* Burrini, A. G., 316, 322, 324, 325, 326, 327, 329, 330,331, 333,334, 335, 336, 342, 343,345,341,348, 349, 350, 351, 352,353,355,357, 358, 360, 361, 362,363,364,365, 366, 361, 369, 372,374,317,379, 380,381,385*, 386* Biisgen, M., 185,250* Butcher, R. W.,12, 15,31, 35,36,37, 38,39,43*, 44*, 47* Byczkowska-Smyk, W.,338,391*
40 1
AUTHOR INDEX
Bygrave, F. L., 3 1,43*
C Cameron, I. R., 302, 306* Cameron, M. L., 348, 387* Cantacuzsne, A. M., 334, 335, 387* Cantoni, G. L., 54, 102*, 106* Caraboeuf, E., 277, 278, 306 Carasso, N., 40, 45* Carlson, A. D., 259,3 11 * Carnay, L., 176, 181* Carpenter, D. O., 277, 306* Carter, W., 217, 223, 250* Castillbn, M. P., 72, 102* Catalh, R. E., 72, 102* Chan, S. K., 76, 102* Chandler, W. K., 286, 306* Chapman, J. A., 264, 306* Chase, L. R., 21, 38,43* Chasey, D., 342, 387* Cheldelin, V. H., 57, 61, 62, 65, 73, 78, 80, 81, 84, 91, 101*, 105*, 107* Chevailler, P., 328, 329, 332, 387* chi, Y. Y., 34,43* Choi. J. K.., 39.40.43* , Chojnacki, T., 66; 73, 85, 96, 102*, 103* Clarke, R. G., 208, 241, 251* Clausen, T., 38,44, Claypool, C. J., 331, 388* Clayton, B. P., 324, 388* Clermont, Y., 334, 388* Cmelik, S. H. W., 72, 103* Coggeshall, R. E., 303, 306* Cohen,M. J., 119, 122, 128, 129, 130, 132, 133, 134, 135, 136, 138, 139, 142, 149, 150, 178*, 179*, 180* Coleman, J. R., 370, 392* Coles, M., 55, 56, 104* Colhoun, E. H., 63, 66, 103* Collier, J. R., 326, 396* Conrad, R. G., 37,45* Cook, E. F., 132, 181* Cooper, D. P., 317,394* Cooper, M. I., 56, 59, 103*
Cordle, M., 174, 179* Corning, W. C., 119, 126, 127, 128, 142,144,179* Costa, E., 34,48* Costin, N. M., 264,306* Cox, J. T., 57, 61, 65, 80, 91, 98, 102* Craig, A. B., 38,43* Creed, R. F. S., 367, 386* Crone, H. D., 73, 74, 75, 76, 78, 81, 84, 85, 86, 87, 103* Curan, M. M., 40,43*
D Dadd, R. H., 57, 103* Daems, W. Th.,351,388* Dallai, R., 316, 317, 322, 324, 325, 326, 327, 328, 329, 330, 331, 333, 334, 335, 336, 338,339,341, 342, 343, 345, 347, 348,349,350,351, 352, 353, 354, 355,357,358,360 361, 362, 363, 364, 365,366, 367, 369, 371, 372, 374, 377,379, 380, 381,385*, 386*, 388*, 394* Dan, J. C., 326,388*, 396* Danilova, L. V., 341, 388* Daoust, R., 334,388* Das, C. C., 331, 388* Dass, C. M., 329,388* Dauterman, W. C., 61, 65, 67, 68,69, 70, 80, 81, 84, 85, 86, 87, 89, 98, 103*, 105*, 107* Davey, K. G., 32, 43*, 380, 381, 388*, 390* Davies, R. G., 232, 233, 234, 246, 250* Davis, N. T., 215, 242, 251*, 380, 393* Davis, R. E., 220,251*, 255* Dawson, R. M. C., 54,84, 103* D’Costa, M. A., 73,76, 103* DeLong, D. M., 56, 106* Dempsey, P., 38,45* De Robertis, E., 35, 43* Deutsch, K., 324,388* Devine, R. L., 324,386*
402
AUTHOR INDEX
Diamond, J. M., 267,285,307* Dibella, F., 34, 46* Di Bona, D. R., 40,43* Dickerson, G., 193,252* Diecke, F. P. J., 259, 262, 278, 279, 281,282,293,312* Dingman, W.,168, 179* Disterhoft, J. F., 119, 126, 127, 128, 142, 144, 179* Djahanparwar, B., 164, 181 * Dobbs, J. W., 35,43* Dobson, W.J., 348,395* Dominas, H., 73,90, 110* Donald, R. A., 37,44* Doolin, P. F., 350,386* Douglas, W.W.,37,43* Drew, M. E., 209,250* Drummond, G. I., 17, 18,21,43* Duchlteau, G., 275,276,307* Dumm, M. E., 75, 107* Duncan, L., 2 1 , 4 3* Dupont, P., 59, 104* Duspiva, F., 21 6, 25 1*
Epstein, S . E., 16, 20, 36, 43*, 44*, 48* Ericson, L. E., 89, 104* Erk, F. C., 56, 104* Esch, I. Van., 276, 306* Etienne, J., 74, 101*
F
Fain-Maurel, M. A., 382,388* Farese, R. V.,49,49* Fast, P. G., 71, 72, 73, 74, 77, 78, 104* Favard, P.,40,45* Fawcett, D. W., 37, 42*, 303, 306*, 332,334,350,355,388*, 389* Fedak, S. A., 21,43* Feir, D., 188, 209, 210, 212, 215, 251* Ferraguti, M., 329,389* Fielding, L., 54, 101* Filshie, B. K., 194,255* Finder, A. G., 38,44* Fine, R. E., 341,389* E Fischer, R. W.,99, 108* Fisher, R. W.,259,308* Eddy, E. M., 334,388*, 389* Fitz, I. B., 59, 104* Eddleman, C. D., 324,388* Fleischer, N., 37,44* Edelman, I. S., 39,47* Florkin, M., 275,276, 307* Edwards, J. S., 188, 204, 205, 210, Folliot, R., 327, 345, 355, 364, 365, 238,25 1* 389* Forbes, A. R., 200, 220, 242, 243, Ehrhardt, P., 189, 191,252* Ehrlich, P., 257, 307* 244,25 1*, 254* Eisenstein, E. M., 115, 117, 119, 120, Forer, A., 338, 341, 345, 347, 375, 386* 121, 122, 123, 128, 129, 130, 132, 133, 134, 135, 136, 138, 139, 142, Fowler, K. S., 99, 106* 145, 149, 150, 155,165,168,169, Fraenkel, G., 55, 56, 57, 58, 59, 60, 170, 172, 174, 175, 178*, 179*, 61, 101*, 102*, 103*, 104*, 107*, 180*, 181* 108* Eldefrawi, M. E., 95, 103*, 266, 293, Francis, D., 34,43* 298,307* Fratello, B., 316, 324, 327, 338, 342, Eliakova, G. V., 330,388* 343,386* Elliot, A. B., 39,43* Frazer, A., 36,47* Ellis, J., 56, 105* Frazier, H. S., 40,44* Ellis, R. A., 330,384* French, E. W., 59, 104* Emmersen, J., 345, 375, 386* Friedlander, M., 332, 369, 389* Entman, M. L., 36,43* Friedman, N., 38,44*
403
AUTHOR INDEX
Friedman, S., 60,61, 104* Friend, W. G., 57, 104*, 188, 193, 196, 203, 206, 208,234,238,238, 250*, 251*, 255* Fuge, H., 341,389* Fujimura, W., 322, 396* Furieri, P., 328, 370, 389*
Greengard, P., 35,46*, 49,49* Grimstone, A. V., 336,343,349,389* Grzelak, K., 66, 105* Guillemin, R., 37, 48* Guinness, F. E., 40,46* Gupta, B. L., 267, 306* Guthrie, D. M., 132, 179* Gyrisco, G. G., 195,254*
G Gall, J. G., 329, 341, 389* Gardiner, B. 0. C., 11, 12, 33, 46* Carey, F. G., 67, 70, 85, 88, 104*, 110* Gassner 111, G., 332, 334, 338, 387*, 389* Gatenby, J. B., 324,325, 334, 389* Gay, H., 33 1,388* Geer, B. W.,56, 58,61, 62, 65,81,92, 99, 104* Gelber, B., 176, 179* Geschwind, I. I., 3 7 , 4 7 * Gibbons, I. R., 329, 336, 341, 343, 349,389*, 390*, 394*, 395* Gilbert, L. I., 75, 76, 109* Gingrich, R. E., 56,60, 104* Girardier, L., 38,44* Giusti, F., 324, 325, 327, 328, 329, 336, 338, 339, 343,348,349, 350, 351, 354, 357, 358,361,362,363, 364,365,371,385*, 386* Glassman, E., 174, 179* Goldberg, A. L., 34,44*, 48* Goldberg, M., 245, 25 1* Goldstein, S. F., 374, 375, 390* Goodchild, A. J. P., 191, 192, 207, 208,235,236,25 1 * Gordon, H. I., 89, 105* Gordon, R. M., 193,252* Gordon, S. A., 223, 25 1 * Goriachkina, V.,330, 388* Gosbee, J. L., 37,45* Gouranton, J., 33 1,392* Graves, J. B., 75, 106* Gray, J., 375, 390* Greenberg, D. M., 54,89, 102* Greenberg, M. J., 11,44*
H Habibulla, A. M., 86, 87, 105* Hackman, R. H., 245,25 1* Haeusler, G., 36,48* Haggerty, R., 119, 142, 179* Hagiwara, S . , 278, 307* Hagley, E. A. C., 216, 251* Hales, C. N.,'16,47* Halstead, W.C., 128, 179* Hamann, K. F., 3 , 4 6 * Handler, J. S., 39, 44* Handler, P., 54, 105* Hardman, J. G., 17,44* Harris, J. T., 124, 125, 127, 144, 180* Harris, P., 77, 101* Hashimoto, A., 73, 105* Hause, L. L., 304, 307* Hawkins, J., 35,43* Hawthorne, J. N., 69, 96, 101* Haylett, D. G., 38, 44* Hays, R. M., 40,44* Hazan, A., 58, 109* Hebb, C., 54, 105* Heim, F., 35,46* Hellyer, G. C., 68, 109* Henderson, A., 174, 179* Henion, W. F., 15, 16,44*, 47* Henley, C., 390* Hennig; E.., 189, 194, 195, 196, 207, 251* Herold, F., 327, 365, 390* Hess, A., 264,307* Hess, M. E., 36,47* Hess, O., 383,390* Hibbs, E. J., 209,210,230, 231, 250* Highnam, K. C., 3 2 , 3 3 , 4 4 * Hill, A. V.,294,307*
404
AUTHOR INDEX
Hill, L., 32, 33,44* Hinton,T., 56, 60, 61, 104*, 105* Hirumi, H., 244,25 1* Ho, R. J., 1 5 , 3 8 , 4 3 * Hoage, T. R., 324, 332, 390* Hodgkin, A. L., 94, 105*, 277, 291, 307* Hodgson, E., 57, 61, 65, 67, 68, 69, 70, 73, 74, 76, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 96, 98, 101*, 105*, 106*, 107*, 108*, 109* Hoffer, B. J., 35,47* Hoffman-Berling, H., 375,386* Hokin, L. E., 37,44* Holden, J. S., 87, 102* Holwill, M. E. J., 336, 374, 375, 381, 390*, 395* Honig, C. R., 38,43* Hopkins, J. M., 342, 347, 350, 390* Hori, K., 189, 195, 196, 207, 209, 210,215,238,241,251*, 252* Horie,Y.,57,58,61,78, 105* Horn, G., 156, 157, 180* Horowitz, B. A., 38,44* Horowitz, J. M., Jr., 38, 44* Horridge, G. A., 118, 124, 132, 134, 135, 142, 144, 145, 151, 152, 164, 180* Horstmann, E., 316, 317, 387*, 390* Hoshiko, T., 308* House, H. L., 57, 105* Houx, N. W. H., 80,100* Howson, D., 122, 127, 132, 142, 180* Hoyle, G., 122, 132, 144, 157, 158, 159, 180*, 250,275,281,307* Hubbard, J. I., 35,45* Hubbell, W.L., 176, 180* Hughes, G. M., 155, 180* Hughes, M., 380,381,390* Hughes-Schrader, S., 332,390* Hurrell, D. P., 72, 103* Hynie, S., 40,45*
I Idelman, S., 367,390* Imura, H., 15,45*
InouB, S., 332,375,391 * Ishida, H., 322, 329, 396* Ito, S., 322, 324, 369,391 * Ito, T., 57, 58, 61, 78, 105*
J Jacklet, J. W., 134, 135, 149, 150, 178*, 180* Jard, S., 40,45 * Jenkinson, D. H., 34, 38,44*, 45* Johnson, H., 149, 180* Jones, S. F., 35,45* Jordon-Luke, B. M., 324,388*
K Kafka, M. C., 40,45* Kakiuchi, S., 34,45* Kalra R. L., 7 2 , 7 8 , 8 2 , 105* Kamienski, F. X., 7 2 , 8 1, 106* Kamin, L. J., 127, 128, 180* Kandel, E. R., 151, 156, 157, 162, 163,164, 180* Kanfer, J. N., 54, 106* Kastings, R., 57, 106* Katz, B., 291,307* Kaufman, B. P., 33 1,388* Kaye, J. S., 324, 325, 326, 331, 334, 342,345,348,391* Kemp, P., 84, 103* Kennedy, E. P., 54, 101*, 106*, 109' Kerkut,G. A.,63, 106*, 176, 180* Kessel, R. G., 318, 324, 329, 332, 342,390*, 391* Keynes, R. D., 269,285,307* Khan, M. A. Q., 6 1 , 6 5 , 7 6 , 8 0 , 8 3 , 8 4 , 88,98, 105*, 106* Kiefer, B. I., 382, 383, 391* Kinsella, J. E., 73, 74, 106* Kinsey, M. G., 185, 187, 195, 196, 197,200,201,202,205,246,252* Klee, W.A., 54, 106* Kleeman, C. R., 302,306 Klein, J. R., 54, 105*
405
AUTHOR INDEX
Kloft, W., 189, 190, 191, 200, 201, 210,211,215,220,252* Koefoed-Johnsen, V., 286, 307* Konijn, T. M., 34,45* Kornberg, A., 54, 109* Korzybski, T., 66, 73, 85,96, 102* Kraicer, J., 37,45* Krasilovsky, G. H., 134, 179* Krieger, D. L., 57, 103* Krishna, G., 38,45 * Krnjevik, K., 34,45* Kropf, R. B., 67,70, 110* Kruitwagon, E. C., 204,208,255* Krzysztofowicz, A., 338, 391 * Kuffler, S. W., 258, 273, 303, 307*, 308 * Kulka, R. G., 18,37,45* Kumar, S. S., 65, 67, 68, 70, 85, 86, 87,88,90,101*, 106*, 107* Kunkel, H., 189,252* Kuo, J. F., 49,49*
Leenders, H. J., 32,45* Le Menn, R., 329,391 * Lemonde, A., 56, 60, 72, 78, 83, 89, 90, 101*, 106* Lenartowicz, E., 67,68, 106* Leste, R. L., 76, 102* Levey, G. S., 16, 20, 36, 43*, 44*, 48 * Lewis, S. E., 99, 106* Lilly, J. H., 57, 101* Lindley, B. D., 308* Lindwell, J. O., 94, 108* Lipsitz, E. Y., 74, 78, 106* Lipson, L.C., 40,45* Locke, M., 239,245,252* Luco, J. V., 126, 128, 132, 158, 160, 161,178*, 180* Lunat, M., 72, 103*
M L Lambremont, E. N., 74, 106* Landau, E. M., 35,45* Lane, N. J., 95, 109*, 258, 259, 260, 262, 264, 266, 267, 268, 270, 271, 272, 273, 274, 276, 277,278, 279, 280, 281, 282, 283,285,288,289, 290, 291, 296, 303, 307*, 308*, 309*, 311* Langslet, A., 36, 45* Lanzavecchia, C., 329,389* Lassota, Z., 66, 105* Laszlo, S., 60,61, 104* Laurema, S., 209, 210, 214, 215, 252*, 254* Lavanchy, P., 193,224,254* Lavoipierre, M. M. J., 252* Lea, A. O., 56, 106* Leaf, A., 40, 43* Lecar, L., 286, 306* Leclercq, J., 275, 276,307* Ledbetter, M. C., 341, 391 * Lee, K. J., 330, 396* Lee, P. E., 328, 396*
Machin, K. E., 375,391* MacInnes, J. W.,332, 391* Maddrell, S. H. P., 11, 12, 33, 46*, 259, 260, 261, 264,266,267,268, 271, 277, 278, 281,282,285,288, 303,304,308*, 31 1* Magis, N., 59, 107* Maillet, P. L., 327, 331, 364, 365, 389*, 394* Makielski, S. K., 338, 367, 381, 392* Malamed, S., 40,44* Maltbie, M., 2 1, 36,46* Mangos, J. A., 3,46* Mansingh, A., 63,66,84, 108* Maramorosch, K., 244,25 1 * Marchbanks, R. M., 94, 107* Martin, K., 94, 105* Mason, J. W.,34,46 Matano, Y., 334,396* Matschinsky, F. M., 34,42* Matsukura, S., 15,45* Matsuyama, H., 15,45* Matingley, R. F., 304, 307* Matty, A. S., 40,46* Mayer, S. E., 21,36,46*
406
AUTHOR INDEX
Mazzini, M., 324, 325, 327, 336, 343, 348, 350, 351, 357, 358,361,362, 364,365,385*, 392* McAfee, D. A., 35,46* McAllan, J. W., 189, 209, 212, 214, 215,250* McConnell, H. M., 176, 180* McFarlane, J. E., 57, 74, 78, 106*, 108* McGaugh, J. L., 169, 180* McGinnis, A. J., 57, 106*, 107* McIlwain, H., 34,45* McLean, D. L., 185, 186, 187, 195, 196, 197, 200, 201, 202, 205, 246, 252*, 253* McMaster-Kaye, R., 331, 334, 391 * Mehendale, H. M., 61, 65, 67, 68, 69, 70, 80, 81, 85, 86, 87, 89, 98, 105*, 107* Mehrotra, K. N., 63, 75,84, 98, 103*, 107* Meinertz, T., 36, 46* Mellon, D., 259, 31 1* Meng, H. C., 15,38,43* Menon, T., 34,48* Metz, C. B., 316,387* Metz, C. W., 367, 392* Meves, F., 33 1, 382, 392* Meves, K., 286, 306* Meyer, G. F., 357, 382, 383, 390*, 392* Meythaler, B., 35,46* Mezei, C., 85, 87, 103*, 107* Miledi, R., 34,45* Miles, P. W., 188, 189, 194, 195, 196, 197, 200, 202, 203,205,206,207, 208, 209, 210, 211,212, 215,216, 217, 220, 221, 223,224,232,234, 236, 237, 238, 239,240, 241,244, 245,247,249,253* Miller,P. L., 117, 181* Miller, R. L.,374, 387* Milligan, J. V., 37,45* Mitchell, H. K., 72, 110* Mittler, T. E., 57, 103*, 185, 197, 200,205,206,253* Mitznegg, P., 35,46* Miyake, T., 15,45 * Moericke, V., 197, 200, 205, 226, 227,228,239,244,253*
Mohri, H., 341,392" Monroe, R. E., 55, 56, 6 0 , 6 2 , 7 8 , 8 1 , 101* Moon, H. M., 174, 179* Moore, W.,56, 107* Morel, F., 39,42* Moreton, R. B., 95, 108*, 109*, 258, 259, 264, 266, 268,272,274,281, 282, 283, 284, 285,286,287, 289, 290, 291, 296, 300, 304, 308*, 309*,311* Morgan, M. E., 15, 36,47* Moriber, L. G., 327,334,395* Morris, D., 54, 105* Moses, M. J., 363, 370, 371, 392* Moskowit, J., 38,45* Moulton, B., 65, 68,87, 90, 107* Mukai, K., 73, 105* Municio, A. M., 72, 102* Munz, K., 327,365,390* Murphy, R. K., 145, 181* N Nagata, N., 14,21,38,39,46* Nakajima, S., 278, 307* Namm, D. H., 21,36,46* Narahashi, T., 258, 277, 278, 289, 308*, 312* Nault, L. R., 195,254* Nebel, B. R., 329,392* Nelson, L., 341,392*, 393* Nelson, W.L.,100, 107* Nesbett, F. B., 293, 306* Newburgh, R. W., 57, 61, 62, 65, 72, 73, 75, 78, 80, 81, 84, 85, 86, 87, 90, 91, 101*, 103*, 105*, 106*, 107* Nicholls, J. G., 258,303,308* Niemierko, S., 67, 68, 106* Noble,E.P., 172, 173, 174, 175, 178* Noland, J. L., 57, 60, 61, 89, 104*, 107* Noyes, D. T., 56, 105* Nuorteva, P., 209, 210,214,215,216, 224,232,233,252*, 254* Nur, U., 370,392* Nurnberger, J., 1 19, 126, 127, 128, 144, 179*
AUTHOR INDEX
0
407
Petri, L., 185, 254* Petrinovich, L. F., 169, 180*, 181* O'Brien, R. D., 95, 103*, 258, 266, Phillips, D. M., 318, 320, 322, 325, 293,298,307*, 308* 326, 327, 328, 332,334,338, 339, Offermeier, J., 11, 46* 340, 341, 342, 343,345,347,348, Olander, R. M., 58,92, 104* 355, 357, 358, 360,365,367,369, Oliver, A. P., 35,47* 370,381,382,389*, 383* Oliver, G., 176, 180* Piantelli, F., 322, 326, 327, 329,330, Olivieri, A., 382, 392* 331, 335, 336, 342, 345, 348, 349, Olivieri, G., 382, 392* 350, 351, 352, 353,360, 363, 365, Orloff, J., 39,44*, 46* 366, 367, 369, 377,379,380,381, Oschman, J. L., 3, 22,46* 385* Ossiannilsson, F., 244, 254* Pichon, Y., 95, 107*, 108*, 109*, Oura, C., 322, 348, 396* 259, 264, 267, 268,271,272,273, (Bye, I., 15, 36, 45*, 47* 274, 275, 276, 277,278,280,281, 282, 283, 284, 285,286,287,288, 290, 291, 293, 294,295,296,297, 299, 301, 304, 308*, 309*, 31 1* P Piechowska, M. J., 85, 103* Pilcher, D. E. M., 11, 12,33,46* Pak, C. Y.C., 40,45* Pilet, P. E., 224, 254* Paleg, L. G., 223,25 1* Pipa, R. L., 132, 181* Pallini, V., 322, 326, 327, 329, 330, Pitman, R. M., 63, 106* 331, 335, 336, 342, 345, 347, 348, Plowman, K. M., 341,393* 349, 350, 351, 352,353,357,360, Pochon-Masson, J., 330,331,393* 362, 363, 365, 366,367,369,377, Poisner, A. M., 37,43* 379,380,381,385* Polonovski, J., 74, 101* Pant, N. C., 55, 56, 107* Porter, K. R., 341, 391* Park, C. R., 38,44* Posternak, T., 15, 16,44*, 47" Pastan, I., 15,46* Potter, D. D., 273, 303, 307* Patel, N. G., 2,42* Powell, C. A., 17, 18,43* Patterson, E. K., 75, 107* Pratt, S. A., 355,357, 393* Pattillo, R. A., 304,307* Prendergast, J. G., 226, 231, 246, Patton, R. L., 57, 75, 81, 104*, 109* 254* Payne, F., 327,370,381,392* Price, G. M., 73, 75, 76, 79, 83, 94, Peachey, L. D., 40,46* 102* Peake, G. T., 37,48* Prince, W. T., 2, 3, 12, 13, 19,22,23, Pease, D. C., 341,392* 2 4 , 2 5 , 2 6 , 2 7 , 2 8 , 2 9 , 3 1 , 4 2 * ,47* Periti, P., 322, 326, 327, 329, 330, Pringle, J. W. S., 132,181* 331, 335, 336, 342,345,348,349, Pritchatt, D., 122, 132, 134, 142, 144, 350, 351, 352, 353,360,363,365, 145, 181* 366; 367; 369; 377; 379; 380,381, Pumphrey, R. J., 153, 154, 155, 157, 385* 164,181* Perotti, M. E., 327, 333, 335, 336, 341, 342, 343, 345,347,350,362, 386*, 392*, 393* R Perry, M. C., 16,47* Persijn, J. P., 351,388* Raine, J., 220,242,243,244,254* Personne, E., 350,384* Rall,T. W., 12, 31,34,45*, 48* Petersen, M. J., 39,47* Ramsay, J. A., 3,47*
408
AUTHOR INDEX
Ramwell, P. W., 40,47* Rad, H. V., 380,393* Rao, K. 0. P.,72,78, 108* Rao, R. H., 88, 108* Rasmussen, H., 2, 12, 13, 14, 19, 20, 21, 25, 27, 31, 34, 36, 38, 39, 40, 41,44*, 46*, 47* Rasso, S. C., 58, 108* Ratner, A., 37,47* Rawdon-Smith, A. F., 153, 154, 155, 157,164, 181* Redina, G., 54, 108* Reger, J. F., 317,393* 394* Reese, T. S., 302, 303,306* Reiser, R., 57, 109* Renaud, F. L., 341, 343, 394*, 395* Renieri, T., 324, 325, 336, 343, 348, 350, 351, 357, 358,361,362,364, 365,381,385*, 394* Retzius, M. G., 355, 367, 394* Richards, A. G., 75, 107*, 264,306*, 380,382,394* Richards, H. H., 54, 106* Richardson, C. D., 58, 109* Rick, J. T., 176, 180* Ricketts, J., 57, 58,61, 65, 66, 67,68, 69, 78, 79, 80, 81, 85, 87, 88, 90, 9 1 , 9 2 , 9 3 , 9 4 , 9 8 , 102* Riemann, J. G., 318, 324, 381, 394* Riess, R. W.,321, 332,334,338, 387* Rikmenspoel, R., 375,396* Rilling, G., 228,229,254* Rimon, A., 54, 108* Ringo, D. L., 341, 394* Ris, H., 329, 388* Ritchot, C., 57, 108* Robbins, W. G., 38 1, 394* Roberts, E., 171, 181 * Robertson, H. A., 49,49* Robertson, R. W.,58, 108* Robison, G . A . , 12, 15, 17, 31,35,36, 38,43*, 44*,47*, 48* Robison, W. G., Jr., 328, 353, 363, 370,371,380,394* Rock, G. C., 57, 108* Roeder, K. D., 258, 268, 282, 309*, 311* Rosati, F., 316, 317, 318, 319, 320, 322, 324, 325, 326,327,328,329,
Rosati, F.-cont. 330, 331, 333, 334, 335,336, 338, 339, 341, 342, 343,345,347, 348, 349, 350, 351, 352, 353,354, 355, 357, 358, 360, 361, 362, 363,364, 365, 366, 367, 369, 371, 377, 379, 380,381,385*, 386*, 394* Ross, A., 332, 394* Ross, J., 332,363, 370, 371, 394* Roth, L. E., 318, 394* Rothschild, Lord, 338, 381, 394*, 395 * Rothschild, H. A., 54, 108* Rottman, F., 65, 68, 87, 90, 107* Rowe, A. J., 341,343,390*, 394* Rowell, C. H. F., 260,280, 306* Royes, W. V., 57, 108* Rucker, W. B., 128, 179* Rudzisz, B., 67, 106*
S Sakai, A., 322,395* Salkeld, E. H., 234,238,250*, 254* Salpeter, M. M., 266, 307* Samli,,M. H., 37,47* Sances, A,, 304,307* Sandeman, D. C., 300,309* Sandlin, R., 176, 181* Sandoz, D., 326,395* Sang, J. H., 56, 104*, 108* Satir, P.,375,395* Sato, H., 332,391 * Sattelle, D. B., 259, 264, 267, 271, 273, 274, 276, 277,278,280,285, 286,288,296,309* Saxena, K. N., 194, 195, 196, 200, 202, 203, 206, 207,208,212,217, 247,254* Schalike, W.,332, 397* Schaller, G., 188, 210, 211, 212, 216, 217, 219, 220, 221,222,224,249, 255* Schoemaker, W.C., 38,44* Schogel, E., 3 , 4 9 * Scholes, J. H., 151, 164, 186* Scholz, H., 36,46*
AUTHOR INDEX
409
Smith, A. D., 34,48* Schorderet, M., 35,46* Schramm, M., 15, 16,37,42* Smith, D. S., 95, 96, 108*, 109*, 260, Schreiner, K., 375, 395* 264, 266, 271, 274,281,298, 299, 309*, 310*, 311* Schuberth, H. J., 94, 108* Schiirmann, F. W., 273,309* Smith, E., 61,65, 80,98, 105* Schwartz, I. L., 40,48* Smith, J. J. B., 188, 193, 196, 203, Scott, D. R., 223, 224,255* 206,208,238,251*, 255* Sedee, P. D. J. W., 56, 108" Smith, K. M., 185,203,255* Sellers, L. G., 68,70,87,88, 101* Smith, R. E., 38,44* Selmi, G., 322, 324' 325, 326, 327, Sogawa, K., 230, 231, 232, 233, 236, 240,241,255* 328, 329, 330, 331,335,336,342, 343, 345, 347, 348,349,350,351, Somlyo, A.P., 36, 38,44*,48* 352, 353, 354, 357,358, 360, 361, Somlyd, A. V., 36,38,44*, 48* 362, 363, 364, 365,366,367,369, Sorbo, B., 94, 108* 371, 377, 379, 380, 381, 385*, Spanner, S., 95, 101* Spencer, W. A., 151, 156, 157, 162, 386* 180* Sethi G. R., 75, 107* Sporn, M. B., 168, 179* Setsuda, T., 15,45* Springhetti, A:, 371,395* Sevhonkian, S., 224,254* Squarez, A., 72, 102* Seydoux, J., 38,44* Sribney, M., 54,109* Shanfeld, J., 36,47* Sridhara, S., 73,78, 109* Shapiro, B., 54, 108* Stamenovik, B. A., 34,45* Shapiro, D., 54, 106* Stauffer, J. F., 57, 101* Sharp, G. W. G., 40,45* Steele, J. E., 32,48*, 49,49* Sharp, P. L., 58,92, 104* Steinberg, D., 38,48* Shaw, J., 259,271,309* Stephens, R. E., 343,395* Shaw, J. E., 40,47* Sternlicht, E., 18, 37,45* Shaw, S., 151, 164, 180* Stevens, R. E., 341,395* Shay, J. W., 324,348,395* Stobbart, R. H.,259,274,309* Sheladci, M. L., 341,343,395* Streeto, J. M., 21,48* Shelley, R. M., 69,86,87,96, 108* Stride, G. O., 56, 109* Shigamatzu, H., 78, 105* Strong, F. E., 188, 204, 208, 209, Shigenaga, M., 322, 395* Shoger, R. L., 316,395* 214,220,222,255* Shoup, J. R., 331,395* Stryer, L., 176, 181* Siebold, C. T. von, 382, 395* Subrahmanyam, D., 88, 108* Siggins, G. R., 35,47* Sud, B. N., 334,395* Silvester, N. R., 375, 395*, 390* Sugioka, T., 334,335,396* Simmons, E. E., 348,395* Sundwall, A., 94, 108* Simpson, L. L., 34,47* Sutcliffe, D. W., 274, 3 1O* Singer, B., 40,44* Sutherland, E. W.,12, 15, 16, 17, 31, Singer, J. J., 34, 44*, 48* 34, 35, 36, 38, 39, 43*, 44*, 47*, Singer, T. P., 54, 108* 48 * Singh, K. R.P., 56,58, 108* Sylvester, F. S., 242, 255* Sjodin, R. A., 286,309* Skelton, C. L., 16,20,44*, 48 * T Slowiak, D., 188, 208, 215, 224,238, 239,244,253* Tahmisian, T. N., 324, 325, 334, Smallman, B. N., 63, 66,84,99, 108* 386*, 389* AIP--17
410
AUTHOR INDEX
Tahori, A. S., 58, 109* Tandler, B., 327,334, 395* Tasaki, I., 176, 181* Tates, A. D., 327, 351,388*,396* Taylor, E. W., 341, 343, 395* Taylor, J. H., 332, 396* Thiiry, J.-P., 341,384* Thomas, K. K., 75,76, 109* Thomas, M. B., 396* Thompson, R. F., 146, 147, 181* Thorn, N. A., 40,48* Thorson, B. J., 324,381,382, 394* Timms, A. R., 35,43* Tobias, J. M., 276, 310* Tomita, T., 35,42*, 43* Toppozada, A., 266,307* Tormey, J. M., 267,307* Treherne, J. E., 63, 95, 96, 107*, 108*, 109*, 257, 258, 259, 260, 261, 262, 264, 266,267,268,270, 271, 272, 273, 274,275,276,217, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,289,290,291, 292, 293, 294, 296,298,299,300, 302, 303, 304, 308*, 309*, 310*, 311* Tsubo, I., 334,335,396* Tucker, L. E., 288, 293, 294, 295, 296,297,299,301,309*, 3 11 * Tunstall, J., 151, 164, 180* Twarog, B. M., 258, 268, 281, 282, 311*
U Uretz, R. B., 332, 391 * Usherwood, P. N. R., 259, 274, 31 1* Usinger, R. L., 215,251* Ussing, H. H., 285,286,307*, 31 1* Utiger, R. D., 37,48*
V Vale, W., 37, 48* van de Meene, J. G. C., 34,45*
Vanderzant, E. S., 57, 58,61, 109* Vane, J. R., 11,48* Van Herpen, G., 375,396* van Loon, L. C., 215,255* Vegni, M., 324,381,394* Venkitasubramian, T. A., 72, 78, 82, 105* Villeneuve, J.-L.,72, 78, 83, 101* Von Baumgarten, R., 164, 181* Vovis, G. F., 56, 61, 62, 65, 81, 99, 104*
W Wada, S. K., 326,396* Wahrman, J., 332,369,389* Walker, R. J., 63, 106*, 176, 180* Wang, C. M., 75,81, 109* Warner, F. D., 325, 336, 341, 343, 347,349,350,351,396* Watanabe, A., 176, 181* Wattal, B. L., 72,78,82, 105* Wearing, C. H., 207,255* Wechsler, W., 273, 309* Weidemann, H. L., 226, 239, 240, 255 * Weidler, D. J., 259, 262, 278, 279, 281,282,293,312* Weigt, W. A., 185, 186, 253* Weiss, B., 34, 48* Weiss, S. W., 54, 106* Wensler, R. J., 194, 255* Werner, G., 329, 334, 349, 365, 367, 370,396* Wetzel, R., 332, 397* Whelchell, K. E., 73, 78, 1l o * Whitaker, B. D. L., 34,45* Whitcomb, R. F., 220, 242, 251*, 255* Whitehead, A. T., 2,48* Whittaker, V. P., 96, 109* Wiersma, C. A. G., 147, 181* Wigglesworth, V. B., 32, 48*, 266, 268,298,305,3 12* Wilber, J. F., 37,48* Wilde, G. E., 208,241,25 1* Wilkes, A., 328,396*
41 1
AUTHOR INDEX
Williams, J. R., 199, 255* Williamson, J. R., 36,49* Willis, N. P., 67, 69, 85, 89, 90, 109* Wilson, E., 153, 155, 174, 181* Wilson, J. E., 179* Wilson, L., 341, 387* Winteringham, F. P. W., 68, 97, 99, 100, 109* WiSnieska, A., 74, 75, 78, 110* Wittenberg, J., 54, 109* Wlodawer, P., 74,75,78, 110* Wohlfarth-Bottermann, K. E., 226, 227,228,239,244,253*, 254* Wood, D. W., 276,312* Wozniak, A., 153, 155, 181* Wren, J. J., 72, 110* Wright, E. M., 285,307* Wroniszewska, A., 66, 105* Wullems, G. J., 32,45 * Wyatt, G. R., 49, 49*, 67, 70, 85,88, 104*, 1 lo*, 274,312*
Y Yamasaki, T., 258, 277, 278, 289, 312* Yasuzumi, F., 334, 335, 396* Yasuzumi, G., 322, 329, 330, 334, 335, 338, 348, 396* Young, J. A., 3 Young, 0. M., 54,106* Yund, M. A., 56, 61, 62, 65, 81, 99, 104* Yurkiewicz, W. J., 73,74, 78, 110*
Z Zelander, T., 341, 386* ZielMska, 2,M.,73,90, 110* Zirwer, D., 332, 397* Zylberberg, L., 325, 382, 397*
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Subject Index A Abdominal ganglion, and learning, 153-5 Abdominal nerve cord, ion uptake, 95 Abdominal tissues, choline, 75 Acantholyda nemoralis, choline metabolism, 67, 73, 90 Accessory salivary gland, 236, 246 Acerentomon, sperm axoneme, 338 A . majus, 342 Acerentulus, sperm axoneme, 338 Acetylcholine and choline metabolism, vertebrates, 53-5 and choline synthesis, 91-100 and catecholamines, 34 extraneous application, 258, 282 metabolism, 63-6 Acetylcholinesterase, in choline metabolism, 84, 96 Acheta domesticus, choline metabolism in development, 57 lipids containing choline, 74, 75 phosphatidylcholine, 7 8 , 8 1 Acid phosphatase saliva, 210,215 sperm axoneme, 352 Acrosomal complex, sperm, 324-28, 382 Acrosternum hilare, and fungus, 241 Actin, sperm flagellum, 345, 375, 379 Action potentials and extra-axonal ions, 282-3,288-9 and Na+, 278 Active transport, in CNS, 300 Adelgidae, and phytopathogenicity, 22 1
Adelphocoris seticornis, salivary pectinase, 2 13 S-adenosylmethionine, in choline metabolism, 53-5 Adenyl cyclase, and cyclic AMP, 12, 18-19, 21, 29, 31-8,40,41 ADPase, sperm, 352 p -adrenergetic agents, and cyclic AMP, 35 Adrenocortieotropic hormone, and cyclic AMP, 36 Aedes aegypti, choline metabolism in development, 5 6 , 5 8 lipids containing choline, 1 2 phosphatidylcholine, 78 sphingomyelin, 8 3 substitutes, 59, 60 Aeneolamia, composition of saliva, 216 Afferent feedback, and learning, 164-6 Aggregation, slime mould, and cyclic AMP, 33-4 Agrotis orthogonia, choline in development, 57 Aiolopus strepens, sperm axoneme, 344,352 cell surface, 3 19,,323 Alanine, Hemiptera saliva, 218, 221 Aldosterone, mitochondria-rich cells, 40 Alkaline tetrazolium reaction, sulphydryl groups, 240 Altica ambiens alni, choline, 72 Amino acids, Hemiptera saliva origins of saliva, 236, 237 and phytopathogenicity, 21 8, 220-5 in sheath material, 206 in watery saliva, 2 16
413
414
SUBJECT INDEX
a-aminobutyric acid, aphid saliva, 2 18 Animals other than insects-cont. 7-aminobutyric acid, and salivary Dictyostelium discordeum, and gland stimulation, 6 cyclic AMP, 33-4 Amphibicorisae, feeding, 192 Diplopoda, sperm, 3 17 Amylamine, salivary gland stimulation, 6 Dog, learning, 143 Amylase Echinodermata, sperm, 341,343 and cyclic AMP, 37 Elasmobranchs, blood-brain saliva, 209, 214, 215 barrier, 302, 303 Fish, sperm 33 1 Anagasta, sperm capacitation, 38 1 Flagellates, flagellum, 349, 350 Anal cerci, and habituation, 15 1-2, Frog 156 body fluid composition, 275 Animals other than insects potential changes, 285,286 Acarina, sperm, 3 17 water permeability, 39 Amphibia Gastropoda blood-brain barrier,303 glycogen, 350 epithelia, permeability, 39 hyperpolarisation, 286 sperm, 349 paired sperm, 367 Amphioxus, sperm, 3 16 Hamster, blood-brain barrier, 302 Annelida Hirudo medicinalis, blood-brain blood-brain barrier, 258, 302, 303 barrier, 303 sperm, 3 16 Lamellibranch, blood-brain barrier, A plysia 303 learning, 162, 163, 164 Leech neurones, 277 blood-brain barrier, 303 Arachnida, sperm, 3 16, 3 17 glial cells, 273 Arthropods Loligo, choline, 94 and Reduviid saliva, 204 Mammal sperm, 316,327,331 blood-brain barrier, 300, 302, Branchiura, sperm, 3 16 3 03 Bufo marinus, cyclic AMP, 39-41 choline metabolism, 52, 59, 96 Bull, sperm, 374, 381 cyclic AMP, 12 Carcinas maenas, blood-brain learning and drugs, 169 barrier, 300, 303 saliva production, 3 Cephalopods sperm, 326,341,350,382 body fluid composition, 275 Marsupials, paired sperm, 367 excitation and conduction, 277. Merostomata, sperm, 31 6 Chilopoda, sperm, 3 16 Mollusca Chlamydomonas, flagellum, 347 heart, 5-HT, 11 Chordata, sperm, 3 16 sperm, 316 Cirrepedia, sperm, 3 16 Mouse Crab, shore, blood-brain barrier, blood-brain barrier, 302 3 00 choline metabolism, 94 Crustacea cyclic AMP, 18 blood-brain barrier, 258, 300, Myriapoda, sperm, 3 16, 3 17 302,303 Mystacocarida, sperm, 3 16 muscle fibre innervation, 147 Necturus, blood-brain barrier, 302 sperm, 316 Opilionids, sperm, 3 17 Decapoda, sperm, 330 Opossum, paired sperm, 369
SUBJECT INDEX
Animals other than insects-conl Pauropoda, sperm, 3 16 Protozoa flagellum, 341, 342, 343, 374, 375 learning, 176 Rat choline uptake, 95 learning, 114, 115, 126, 127-8, 168 stomach, 5-HT,11 Scorpion, sperm, 3 16 Sea urchin, sperm, 343, 345, 349, 3 74 Slime mould aggregation, and cyclic AMP, 33-4,41 Snake venom, 209 Squid, giant axon, 278, 291 Symphyla, sperm, 3 16 Toad, bladder, and cyclic AMP, 39-41 Teleost, blood-brain barrier, 303 Tetrahymena, cilia, 341, 343 Turritella terebra, paired sperm, 3 67 Vertebrates blood-brain barrier, 25 1,302,303 choline metabolism, 52, 53-5, 69, 71,82,89, 96 cilia, 350 cyclic AMP, 15 malignant trophoblastic cells, 304 and reduviid saliva, 204 salivary glands, 37 sperm, 316 Anoplura, sperm, 328, 369, 380 Antheraea pernyi, acetylcholine, 66 Antheraea polyphemus acetylcholine, 66 phosphorylcholine, 67 Anthonomus grandis, choline metabolism, 5 7 , 5 8 , 5 9 , 61 Anticholinesterases, 99-1 00 Anuraphis bakeri, choline metabolism, 73 Aphaniptera, sperm acrosomal complex, 324 axoneme, 338
415
Aphaniptera, sperm-cont. cell surface, 3 18 motility, 377,379 Aphididae, saliva composition, 209, 210,211 pectinase, 2 13, 2 14 Aphidoidea, saliva, 226-9,245 Aphids saliva composition, 211, 212,215, 216 excretion, 245 metabolites, 218-9,220, 221 methods, 185-7, 189 necrosis, 249 oxidases, 239,247 phenolic compounds, 249 phytopathogenicity, 2 17-25 stylet-sheath feeding, 194-202, 205,246 virus transmission, 242 sperm, 365 Aphid, black bean, feeding, 194 Aphis abbreviata, pectinase, saliva, 21 3 Aphis cardui, pectinase, saliva, 2 14 Aphis fabae, saliva feeding, 194, 195, 196 methods, 189 pectinase, 213 Aphis pomi, metabolites, saliva, 2 18-9 Aphis sambuci, metabolites, saliva, 2 18-9 Aphis sedi, pectinase, saliva, 213 Aphis spireacola, pectinase, saliva, 2 13 Aphis, woolly, saliva detoxicant function, 248 phytopathogenicity, 2 17 Aphoidea, feeding, 192 Aphrophora alni, salivary glands, 232 Aphrophora parallela, choline metabolism, 73 Apis mellifica, choline metabolism acetylcholine, 66 lipids containing choline, 75, 77 oxidation, 89 requirements, 92 Apterygota, sperm, 326, 327, 328, 329,338 Archips cerasivoranus, lipids containing choline, 73
416
SUBJECT INDEX
Arctia caia, choline metabolism, 66, 73,85 Arge pectoralis, lipids containing choline, 73 Arginine saliva, aphids, 2 18 sperm, 33 1,335 Argyrotaenia velutinana, choline in development, 57 Asopinae, feeding, 192 Asparagine, saliva, 2 16, 2 18 Aspartic acid, saliva, 216, 218, 221 Attagenus spp., choline in development, 56 ATP, conversion to cyclic AMP, 14 ATPase, snake venom, 204 sperm, 336, 343, 345, 346, 349, 363-7 371,375,377, 379 Atropos pulsatorium, sperm, 369, 370 Auchenorrhy ncha saliva composition, 209, 216 glands, 225, 233-4 pectinase, 213, 214 sperm, 365 Aulacaspis tetalensis, feeding, 198-9 Aulacorthum solani, saliva, 213, 249 Axoneme, sperm, 336-53, 374-80 axonemal matrix, 352-3 central sheath, 349 coarse fibres, 350-2 links heads, 349-50 microtubules, 338-49 and motility, 374-80
Betaine, and choline metabolism, 52, 53-5,59,63,89 Blaberus gigantea, neural fat body sheath, 280 Blatta orientalis, learning, isolated ganglion, 132, 158 Blattella germanica, choline metabolism, 57, 59, 61, 74, 89 Blattoidea, sperm, 324 Blood-brain barrier, 257-3 12 electrical aspects, nerves, 277-9 1 ionic basis, 277-8 neural fat body sheath, 278-81 neuronal function, 28 1-9 1 ionic composition, haemolymph and nervous tissues, 274-7 nervous tissue, organisation, 260-74 extraneuronal fat body deposits, 200-3 glial cells, neurones and extracellular spaces, 268-74 neural lamella, 264-6 perineurium, 266-8 radioactive ions and molecules, exchanges, 291-9 Bloodsuckers, saliva, 203, 204, 205, 206,215,238 BOL (Bromolysergic acid diethylamide), and salivary glands, 8-10 Bombyx mori choline metabolism in development, 5 7, 5 8 lipids, 73 phosphatidylcholine, 78, 85, 96 phosphorylcholine, 67 substitutes, 59, 61 haemolymph, 276 sperm, 34 1 Brain catecholamines and cyclic AMP, 34 choline, 75,77 see also Blood-brain barrier Bufotenine, and salivary gland stimulation, 7 Butterfly, sperm, 3 18
B B vitamins in development, 58 Bacillus rossius, sperm absence of mitochondria, 360 accessory flagellar bodies, 366,368 acrosomal complex, 327 axoneme, 345,346,352 cell surface, 323 centriole region, 335 motility, 377,368, 381 C nucleus, 330,331 Bacteria, and cyclic, AMP, 12 Caesium ions, and potential changes, Barium. and stimulation bv ADH. 40
SUBJECT INDEX
Calcium haemolymph, 275 -mediated action potentials, 278 see also Cyclic AMP Calligypona pellucida, saliva, 2 16, 224 Calliphora ery th rocep hala choline metabolism, 56, 72, 84 salivary glands, cyclic AMP, 1-49, 1-49 see Cyclic AMP Campodea, sperm, 325,342,344, 350, 361 Campodeidae, sperm, 347,349,351 Capsus ater, pectinase, saliva, 2 13 Carabids, sperm, 331 Carasius morosus extra-axonal sodium regulation, 3 02 extra neuronal potentials, 285, 288 fat body deposits, 260-3 ionic basis, electrical activity, 277, 278 ionic composition, nervous tissues, 275,276 neural fat body sheath, 278-8 1 neural lamella, 265-6 perineurium, 266,268 Carbohydrates saliva, 240-1 sperm, 352-3. 380 Cardiac cells, and cyclic AMP, 15 Cardophilus hemipterus, choline, 56 Carnitine, and choline metabolism, 52, 59-62, 70, 80,87, 99 Carnivores, saliva, 203-5,238 Catecholamines, and cyclic AMP, 34, 35-6,38 Cation exchange, blood and CNS, 258 CDP-choline (cytidinediphosphorylcholine), and choline metabolism, 53-5, 69, 86, 96, 97 Cecidogenesis, and saliva, 224-5, 249 Celerio euphorbiae, choline metabolism, 6 6 , 8 5 Cell membrane and choline, 52 and watery saliva, 201 Cell surface, sperm, 3 17-24 Cellulase, saliva, 197, 209 Centriolar region, sperm centriole, 332-3, 382
417
Centriolar region, sperm --con t. centriole adjunct, 329, 333-5, 365 initial segment, axoneme, 335-6 Ceramide, and choline metabolism, 5 3-5 Ceratatis capita choline metabolism, 7 1, 72 sperm, 337,344,346 Cerci, anal, and habituation, 15 1-2, 153-5 Cercopoidea, saliva, 214, 229, 232 Cercopidae, saliva, 2 16 Ceruraphis eriophori, saliva, 214 Ceruraphis viburnicola, saliva, 2 13 Chironomus spp., choline metabolism, 72 ChlGon dipteron, sperm, 325, 339 Chloride and diuretic hormone, 33 salivary glands, 3, 22, 24-5, 28,29, 31 Chlorogenic acid, aphid saliva, 219 Choline metabolism, 5 1-109 enzymes, 84-91 cholineacetylase and acetylcholinesterase, 84 choline, oxidation, 88-9 choline, synthesis, 89-91 phosphatidylcholine, hydrolysis, 8 7-8 phosphatidylcholine, synthesis, 85-7 lipid-soluble metabolites, 7 1-84 lysophosphatidylcholine, 82-3 phosphatidylcholine, 7 1-82 sphingomyelin, 83-4 metabolic role of choline, 91100 nutritional requirements, 5 5-63 in development, 55-8 substitutes, 59-63 vertebrates, 52, 53-5 water-soluble choline metabolites, 63-7 1 acetylcholine, 63-6 CDP-choline, 69 glycerylphosphorylcholine, 70-1 phosphorylcholine, 66-9 Choline ions, and potential changes, 283-7
418
SUBJECT INDEX
Chromatoid body, and centriole adjunct, 334 Chromosomes, sperm, 332 Chryptothnps latus, sperm axoneme, 351 Chrysomela crotchi, choline, 72 Chrysopa carnea, sperm axoneme, 339 Cicadellidae saliva, 213,230,233 sperm, 365 Cicadomorpha, saliva, 192, 233 Cicindela, sperm, 3 65 Cimex lectularius, salivary glands, 234, 235,238 Cimicidae feeding, 192 salivary glands, 235, 249 Cimicomorpha composition of saliva, 2 15 composition of sheath material, 206 feeding, 192, 193,203 salivary glands, 234, 238 Cinara, spp., pectinase, saliva, 213 Cixius nervosus, sperm, 364 Classical conditioning, 1 13, 162-4 Clitumnus, sperm, 360 Coccidae phytopathogenicity, 2 17 sperm, 332, 353, 363, 370, 374, 380,381 Coccoidea, sperm, 328 Cochliomyia hominivorax, choline, 56, 5 8 ,5 9 ,6 0 Cockroach abdominal nerve cord, 258 choline substitutes, 59 fat body deposits, 260 learning, isolated ganglia see Learning Cocoa capsid, phytopathogenicity, 217,220,223,225 Coleop tera choline metabolism analogues, 98 in development, 5 5-6 enzymes, 8 6 , 90 lipid-soluble metabolites, 71, 72, 7 8 ,8 2 ,83
Coleoptera-cont. choline metabolism-cont. phosphor ylch oline , 6 9 haemolymph, ionic composition, 275 sperm accessory flagellar bodies, 364 acrosomal complex, 324,328 axoneme, 337,348,350, 351 cell surface, 323 centriolar region, 336 mitochondria, 357,358,362 paired sperm, 367 Coleorrhy ncha feeding, 192 salivary glands, 225, 226, 231,233, 245 Collembola, sperm, 316, 324, 327, 338,354 Colour changes, mantids, 32 Conditioning, classical, 113, 162-4 Coreidae, pectinase, saliva, 2 14 Coreoidea, feeding, 192 Corixidae, feeding, 192 Cornus drummondi, (dogwood), and Acrosternum, 241 Corrodentia, sperm, 327 Cricket, house, sperm, 33 1,334 Cryptocerata, salivary glands, 234 Cryptomyzus nbis, saliva, 218-9 Ctenocephalus canis. sperm, 318, 376 CTPase, sperm, 352 Culex pipiens fatigans, choline metabolism, 71,72, 78, 82, 88 Culicid Diptera, sperm, 338 p-Cumaric acid, aphid saliva, 2 19 Cuticle choline, 75 formation, 239, 245 Cyclic AMP and Calcium, and hormone action, 1-49 Calliphora salivary glands, 2-5 5-HT, comparison 32-41 control of metabolism, 37-9 epinephrine and heart, 36 excitation-secretion coupling, 36-7 pre- and post-synaptic transmission, 34-6
SUBJECT INDEX
Cyclic AMP and Calcium-cont. SHT, comparison-cont. slime mould aggregation, 33-4 transporting epithelia, 39-41 5-HT-receptor interaction, 5-1 2 Intracellular messengers, 12-2 1 calcium, 19-2 1 cyclic AMP, 12-19 mode of action, 21-32 ion transport, 26-8 potential effect, 23-6 time course, 28-3 1 model of hormone action, 31-2 Cyclic nucleotides, and specificity of cyclic AMP, 16-18 Cycloheximide, and learning, 172-5 Cysteine, saliva, 21 1, 216,218 Cytidine pathway, 85 Cytochrome-c-oxidase, sperm, 35 7, 362,363,366
419
Diapheromera femorata, choline, 74 Dibutyryl derivative, cyclic AMP, 15-16, 34,39 Dictyoptera, ionic composition, haemolymph, 281 Diglyceride, and choline metabolism, 53-5 Dihydroxy phenylalanine, saliva, 223, 239,249 N-Dimethylaminoethanol, and choline metabolism, 59 Dimethylethylcholine, as choline substitute, 62 N-Dimethylglycine, and choline metabolism, 53-5 Dimethyliso-propylchohe, as choline substitute, 62 Dimethyltryptamine, and salivary gland,.7 Diplura, sperm, 316, 324, 327, 342, 344,347,349,350,354 Dipsocorinomorpha, feeding, 192 Diptera choline metabolism D analogues, 98 enzymes involved, 86 Dacus, sperm, 350,370 lipid soluble metabolites, 7 1, Dacus oleae, choline, 72 72-3,77, 78,82,83,84 Dactynotus, sp., saliva, 2 13 requirements, 55-8, 62, 92 Dahlbominus, sperm, 328 Dalbulus maidis, saliva, 2 13 synthesis, 90 Datana integerrima, choline, 73 water soluble metabolites, 69, DDD (dihydroxydinaphthyldi70 sperm sulphide), test for sulphydryl acrosomal complex, 327 groups, 240 axoneme, 337, 339, 341, 344, Delphacidae salivary glands, 232,233 346,348,350,351 mitochondria, 355,357,362 sperm, 365 Deltocephalidae, salivary glands, 230 non-flagellate sperm, 374 Deoxyribonucleoproteins, sperm Diuretic hormone nucleus, 332 and 5-HT, 11-12 Dermaptera, sperm, 336 Rhodnius, 33 Dermestes vulpinus, choline in DNA, sperm, 329,33 1,332,382 development, 56 Dolycoris baccarum, composition of Desheathing, and axonal response, saliva, 209,210, 215 279,286-7,288,289 Donnan equilibrium, and peripheral Detoxicant function, saliva, 246-7 diffusion barrier, 259,264 Development, choline in, 55-8 DOPA, saliva, 223,239,249 DFP (di-iso-propylphosphofluoridate) Dopamine, and cyclic AMP, 6,35 and acetylcholine, 99, 100 Dreyfusia spp., and galls, 220, 221
420
SUBJECT INDEX
Drosop h ila m elanogaster choline metabolism metabolic role, 92, 97, 99 oxidation, 89 lipid-soluble metabolites, 72, 8 1 requirements, 56, 58, 59,61, 62 substitutes, 64-5 sperm nucleus, 33 acrosomal complex, 327 axoneme, 338, 343, 344, 349, 350 centriole region, 333, 335, 336 mitochondria, 362 polymorphism, 382-3 spermatids, 370 Drosophila spp. choline in development, 57 sperm polymorphism, 383 Drugs, and learning, cockroach, 168-75 Dysdercus feeding, 194, 202-3, 207, 208 and fungus, 241 salivary composition, 2 12 Dysdercus koenigii, secretion of flange, 195 Dytiscus marginalis, paired sperm, 367,369
E Ecdysone, mode of action, 32 Ecdysterone, and cyclic AMP, 32 Efferent gating, and learning, 164-6 Egg, choline metabolism, 63-4, 72-4, 91, 92, 99 Electrical aspects, nervous function, 277-91 ionic basis, 277-8 neural fat body sheath, 278-8 1 neuronal function in experimental preparations, 28 1-9 intact systems, 281-9 Electron spin resonance, and membranes, 176 Embioptera, non-flagellate sperm, 373
Empoasca fabae saliva, 209, 210, 230, 231,232 Enzymes choline metabolism, 84-9 1 saliva, 37, 197, 203, 204, 206, 209-17, 237-9, 245 sperm axoneme, 352 Eosentomon, non-flagellate sperm, 3 74 Ephemeroptera, sperm, 327,338, 339, 354,363 Epinephrine, and cyclic AMP, 12, 21, 36, 37, 38 Epipharyngeal organ, and saliva, 2 12 EPSP, and learning, 157 Esterases, in saliva, 210, 212, 215 Ethanolamine as choline substitute, 59, 63 and salivary gland, 6 Ethylamine chain, and 5-HT-receptor interaction, 6-9 Ethylenediamine, and salivary gland, 6 Erannis tiliaria, choline, 73 Ericerus pela, choline, 73 Eriococcus, non-flagellate sperm, 370 Erisoma americanum, saliva, 213 Erisoma lanigerum, saliva, 213, 217, 218-9,220,221,248 Erythroneura limbata, salivary glands, 230 Euchistus, sperm, 359, 360 Eumecopus punciventris, saliva, 21 1, 24 1 Eurydema rugosa, stylet-sheath feeding, 195,207,208 Euphestia elutella, choline in development, 57 Euphestia kuehniella, choline in development, 57 Euxesta notata, choline, 72 Evolution of salivary function, Hemiptera, 207, 244-7 Excretion, salivary glands, 184,245
F Fat body choline, 75, 76 glycogen conversion, 32
SUBJECT INDEX
42 1
Ganglia- con t. isolated, learning, 11 1-81, see Learning Gelastocoris sp., sperm axonemes, 370 Genetics, sperm, 382-3 Geocorinae, feeding, 192 Geocorisae, feeding, 192,204 Giant axons cation gradients, 275 excitation and conduction, 277, 278 extraneuronal potentials, 282, 285, 288, 289-90 and glial cells, 272 Gibberellic acid, in saliva, 2 16 Glands and ducts, salivary, Hemiptera, 225-35 Aphidoidea, 226-9 Fulguromorpha, 232-3 Heteroptera, 234-5 Jassomorpha, 229-32 other Auchenorrhyncha, 233-4 Glial cells choline uptake, 95 organisation, 268-74 and Sodium regulation, 279, 300, 304-5 Glossina morsitans, choline, 71, 73 Glucagon, and cyclic AMP, 12, 38 Gluconeogenesis, renal, and cyclic AMP, 39 Glucose, and cyclic AMP, 38 Glutamic acid, Hemipteran saliva, 216, 218,221 Glutarnine, Hemipteran saliva, 216, 218,221, Glyceraldehyde-3-phosphate dehydrogenase, sperm axoneme, 352 Glycerylphosphorylcholine, metabolism, 53-5, 70-1 Glycine G aphid saliva, 2 18 Galleria mellonella and choline metabolism, 53-5 choline metabolism, 66, 67, 68, 74, Glycogen 75, 78,82 deposition, nervous system, 305 neural lamella, 264 sperm axoneme, 346,348,350 Galls, and Hemipteran saliva, 184, to trehalose, hormones, 32 191,211,217, 220-5 Glycogenolysis, and epinephrine, 36 Ganglia Glycolysis, anaerobic, sperm, 359, K' depolarisation, 28 1 363,380
Fat cells, and dibutyryl cyclic AMP, 16 Fat body sheath, neural, role, 278-81 Fat body deposits, extraneural, 260-3 Fatty acids, free, and cyclic AMP, 38 Feedback cyclic AMP and Calcium, 19-21,36, 40,41 and learning, 132, 164-6 Ferrulic acid, aphid saliva, 2 19 Flagellar apparatus, sperm, 367-74 non-flagellate sperm, 370-4 paired sperm, 367-9 two axonemes, 369-70 Flagellar bodies, accessory ordered, 363-7 Flagellar filament, axial, sperm, 336-53 Flagellar motion, sperm, 335, 336, 374-82 Flea, sperm, 318 Flight muscle, choline metabolism, 76, 77,83 Fluid secretion epithelia compared with penneurium, 267 regulation, 3 7 Fluorescent dye techniques, and membranes, 176 Formica rufa, oxidation of choline, 89 Fulguroidea, pectinase, saliva, 2 14 Fulguromorpha, saliva, 192, 232-3, 236,240,246 Fungal disease, and saliva, 241-2 Fructose-diphosphate aldolase, sperm axoneme, 352 Fruit-fly, sperm, 3 18
422
SUBJECT INDEX
Glycolytic pathway, sperm axoneme, 352 Glycoproteins, sperm, 381 Gold complex, sperm, 322, 324, 325, 365 Granular cells, and Sodium transport, 40 Grape phylloxera, saliva, 2 1 6 , 2 17 Gryllotalpa gryllotalpa, sperm, 343, 345,376,379 Gryllus, sperm axoneme, 342, 345 Gryllus bimaculatus, choline, 1 4 GTPase, sperm, 352 Gustatory sensilla, and saliva, 2 12 Gut, choline, 7 5 ,76
H Habituation, electrophysiology, 150-7 Haematopinus suis, sperm, 333, 369, 376 Haemocytes, and extraneural space, 262 Haemolymph choline, 75 ionic composition, 274-7 Harmala alkaloids, and salivary gland stimulation, 8-9 Harmaline, and salivary gland stimulation, 8 Heart, and cyclic AMP, 16, 21,32, 41 Heliothis zea, choline metabolism in development, 57 enzymes, 8 5 ,8 9, 90 lipids, 74 water-soluble metabolites, 67, 69 Hemiodoecus veitchi, salivary glands, 226 Hemiptera choline metabolism, 56, 58,71,73, 78,82 saliva see Saliva sperm, 327,355,381 Heteroptera saliva composition, 205, 207, 208, 209,213,214,216
Heteroptera-con t. saliva-cont. evolution, 245-1 glands and ducts, 184, 225, 234-5 feeding, 191, 192, 193-6 methods, 189 origin, 236,241 sperm, 327,370 Histamine, and salivary gland, 6 , 7 Histidine saliva, 218,221 sperm, 33 1 Histones, sperm, 331, 335 Homocysteine, and choline metabolism, 52, 53-5 Homoptera saliva composition, 205,206,207,212 evolution, 245-7 feeding, 191, 192, 194-6 glands and ducts, 184-5,225,234 origins, 236, 237-8 sperm, 327,345,365,370 Honey bee, sperm, 324,338 Hormone action, role of cyclic AMP and Calcium 1-49, see Cyclic AMP Hormone, plant, in saliva, 216 Housefly, choline metabolism, 62, 63, 69, 70, 82 5-HT Calliphora salivary glands, 2-5 compared with other hormones, 32-41 control of metabolism, 37-9 epinephrine and heart, 36 excitation-secretion coupling, 3 6-7 pre- and post-synaptic transmission, 34-6 slime mould aggregation, 33-4 transporting epithelia, 39-41 intracellula messengers, 12-2 1 calcium, 19-2 1 cyclic AMP, 12-19 mode of action, cyclic AMP and Calcium, 21-32 effect on potential, 23-6
423
SUBJECT INDEX
5-HT-con t. Inotropism, and cyclic AMP, 36 mode of action-cont. Insecticides and blood-brain barrier, 259 ion transport, 26-8 and choline metabolism, 52, 99, time course, 28-31 100 model of hormone action, 3 1-2 Instrumental learning, 113-5, 157-62, receptor interaction, 5-12 164 Hyalophora cecropia, choline metaIntestine, and cyclic AMP, 35 bolism Intracellular messengers enzymes, 85, 88 calcium, 19-2 1 lipid-soluble metabolites, 75, 76 cyclic AMP, 12-19 water-soluble metabolites, 66, 67, Inulin penetration, nervous system, 70 268 Hyalopterus pruni, metabolites, saliva, Ion transport 2 18-9 abdominal nerve cord, 95 Hyaluronidase, saliva, 204,210 and cyclic AMP, 38, 4 1 Hydrocorisae, saliva, 204, 192, 23 1, and diuretic hormone, 33 235 intact and headless preparations, Hydrolysis of phosphatidylcholine, 118-28 87-8 5-hydroxytryptamine, see 5-HT isolated ganglion, 128-136 5-hydroxytryptophane, and salivary other CNS lesions, 136-7 dand stimulation. 7 and electrical activity, nerve cells, Hylemya antiqua, cholhe metabolism, 277-8 57.73 salivary gland, 22-5,26-8, 3 1 Hyalobius pales, choline metabolism IPSP’s, and learning, 157, 162 enzymes, 8 5 , 8 9 , 9 0 Isethionate, and 5-HT, salivary glands, Homoptera 67, 69 28,29 Hymen opt era Isoptera, sperm, 328,329 choline metabolism lipid-soluble metabolites, 71, 73, 82 phosphoryl choline, 69 J requirements, 92 synthesis, 91 327,345,365,370 Japyx, sperm axoneme, 350 Hyperin, aphid saliva, 2 19 Japigidae, sperm axoneme, 338 Hyphantria cunea, lipids containing Jassoidae, saliva, 209, 210 choline, 74 Jassoidea, saliva, 214, 229-30, 245 Jassomorpha, saliva, 192,229-32, 233, 242 Jopeicidae, saliva, 2 15 I Juvenile hormone, mode of action, 32
Imaginal discs, choline, 75 Indole acetic acid, saliva, and phytopathogenicity, 219, 220, 222, 223-5,249 p-indolyl acetic acid (IAA), in saliva, 2 16-7 Inhibition, central, and learning, 162
K Kalotermes, sperm, 363 K . flavicollis, 371, 373
424
SUBJECT INDEX
Kalotermitidae, non-flagellate sperm, 37 1 Kermes, sp., non-flagellate sperm, 370 Kidney, adenyl cyclase, 21, 38 Krebs cycle, sperm, mitochondria, 359 L Lacerateand-flush feeding, Hemiptera, 191-3, 202-3, 207, 208, ,217, 220,222,246 Lactic acid, sperm axoneme, 353 Lactic dehydrogenase, sperm axoneme, 352 Laodelphax striatellus, salivary glands, 233 Larva, choline requirements, 92 Lasioderma serricorne, choline requirements, 5 5 ,5 6, 60 Leaf hoppers, saliva, 217, 240 Learning and memory, isolated ganglia, 11 1-8 1 behavioural investigations, 1 18-48 ganglionless P and R preparations, 137-40 intact and headless preparations, 118-28 isolated ganglion, 128-136 other CNS lesions, 136-7 P and R behaviour, ganglionic innervation of legs, 140-6 P and R behaviour, ganglionless legs, 146-8 concept of learning, 113-5 electrophysiological studies, 150-67 classical conditioning, 162-4 habituation, 150-7 instrumental learning, 157-62 newer approaches, 164-7 histological and anatomical, cockroach, 149-50 ganglion transplantation, 150 metathoracic ganglion, mapping, 149-50 “model” systems, use, 1 17 molecular approaches, 167-76 drugs, cockroach, 168-75 speculations, 175-6 reformation of concept, 1 15-7
Lepidoptera blood-brain barrier haemolymph, ionic composition, 275 neural lamella, 264 choline metabolism in development, 55-6 enzymes, 8 6 , 9 0 lipid-soluble metabolites, 7 1,73, 78,83 metabolic role, 92 wat er-soh ble metabolites, 6 6, 68, 69, 77 sperm cells acrosomal complex, 327 axoneme, 342,347,348,35 1 capacitation, 38 1 cell surface, 318, 320,322 mitochondria, 355 nucleus, 331 spermatogenesis, 382 Lepisma saccharina, lipids containing choline, 74 Lepismatidae, sperm cells, 349, 35 1, 367,369 Leucine, aphid saliva, 21 8 Liocoris lineolaris, pectinase, saliva, 214 Lipases and cyclic AMP, 38 saliva, 2 15 Lipids containing choline, 72-6 lipid-soluble choline metabolites, 7 1-84 lysophosphatidylcholine, 82-3 phosphatidylcholine, 7 1-82 sphingomyelin, 83-4 in saliva, 240-1 Lithium ions, and potential changes, 283-6 Liver, and cyclic AMP, 12, 38, 41 Locust classical conditioning, 164 fat body deposits, 260 learning eye, 151, 152 leg position, 118-9, 122, 141, 157-9
SUBJECT INDEX
Locust-cont. sperm cells capacitation, 381 cell surface, 3 18, 324 Locusta migratoria blood-brain barrier glial system, 273 haemolymph, 276 choline metabolism in development, 57 lipids containing choline, 74 phosphatidylcholine, 85, 96 Lucilia cuprina, lipids containing choline, 73, 76 Luteinking hormone, and cyclic AMP, 37 Lygaidae, saliva composition, 205, 209, 210, 211, 214 feeding, 191,196, 202,203 galls, 224 glands, 235, 237 methods, 188 Lygaeoidea, feeding, 192 Lygus spp., saliva, 224,241 L . disponsi, 209, 210, 214, 215, 238 L. elisus, 223 L. hesperus, 209, 212, 213, 222-3 L. pratensis, 213, 235 Lysine Hemiptera saliva, 2 18,22 1 sperm cells, 326,331 Lysophosphatidylcholine, metabolism, 53-5,72-6,82-3,84
M Machilidae, sperm cells, 349,354 Machilis, sperm cells, 330 M. distincta, 364 Macrosiphoniella millefolii, pectinase, saliva, 2 13 Macrosteles fascifrons, salivary transmission of disease, 242-3,244 Magnesium and ADH, 4 0 and blood-brain barrier, 259
425
Magnesium-con t. and sperm malformation, 383 Malacosoma americanum, acetylcholine, 66 Malathion, and acetylcholine, 99, 100 Malpighian tubules choline, 75 diuretic hormone. 33 Mallophaga, sperm cells, 327, 329, 351.369 Manduca sex ta blood-brain barrier electrical aspects, 278,280, 285, 288.302 haemolymph, ionic composition, 276 nervous tissue, organisation, 264,266-7,273 lipids containing choline, 76 Manganous ions, and action potential, 278 Mapping, metathoracic ganglion, 149-50 Mastotermitidae, non-flagellate sperm, 371 Matsucoccus bisetosus, non-flagellate sperm, 370 Mecoptera, sperm cells acrosomal complex, 324 axoneme, 338,339,341 mitochondria, 355 nucleus, 328 Megoura viciae accessory flagellar bodies, sperm, 364 metabolites, saliva, 21 8-9 Melaphis rhois, pectinase, saliva, 214 Membranes conformational changes, and learning, 176 sperm cells, 322 Membrane permeability, and cyclic AMP, 38 Membrane potential, and Na+ concentration, 289 Memory and learning, isolated ganglia, 1 11-8 1, see Learning Menopon gallinae, sperm axoneme, 35 1
426
SUBJECT INDEX
Mercaptoethanol, induction of biflagellate sperm, 369 Mesothoracic, ganglion, and learning, 124-5 Mesothoracic leg, and learning, 1 19, 144 Messengers, intracellular calcium, 19-21 cyclic AMP, 12-19 Metabolism, control, and cyclic AMP, 37-9 Metathoracic ganglion and learning, 124-5, 132, 150, 158-62, 177 mapping, 149-50 Metathoracic leg, and learning, 122, 124, 132, 135,144,157 Methionine aphid saliva, 2 18 choline metabolism, 52,53-5,59 N-methylated ethanolamines, as choline substitutes, 63 LY and p-methylcholines, as choline substitutes, 62 Milkweed bug, saliva, 188 Miniature end-plate potentials, and cyclic AMP, 34-5 Miridae, saliva composition, 209, 210,213, 215 feeding, 192, 193,203,207,208 glands, 235 and phytopathogenicity, 2 17, 220, 222 Miris dolabratus, composition of saliva, 213,215 Mitochondria -rich cells, and Na* transport, 4 0 sperm cells absence, 360-3 accessory flagellar bodies, 363-7 axoneme, 353,363 derivatives, 354-60 normal mitochondria, 354 polymorphism, 382 Model of hormone action, and cyclic AMP, 3 1-2 Molecular approaches, learning, 167-76 drugs, cockroach, 168-75 speculations, 175-6
Monoctenus juniperinus, lipids containing choline, 73 N-Monomethylaminoethanol, and choline metabolism, 59 Monomolecular films, calcium, displacement by ADH, 40 Moths, sperm cells, 318, 324,381 Motility, spermatozoa, 345, 352, 367, 370,371 capacitation, 38 1-2 mechanisms, 374-80 metabolism, 380-1 Motor output, and learning, 164-6 Motor neurones, release of ACh, 34 Murgantia histrionica, sperm mitochondria, 356 Musca domestica, choline metabolism enzymes involved, 84, 85, 86-8,90, 91 lipid-soluble metabolites, 73, 75, 76,77, 78, 79,80,82, 83 metabolic role, 92, 93-5, 96,97,98, 99 nutritional requirements, 57, 58, 59,61 water-soluble metabolites, 63, 64-5, 66,67,69 Muscle choline, 75,76 heart and visceral, regulation, 32 smooth, and cyclic AMP, 35, 41 Mycetophilid Diptera, sperm axoneme, 338 Mycoplasmal particles, Hemipteran saliva, 242-3,250 Myosin, in flagellum, 375 Mystacides azurea, sperm axoneme, 347 Myzus ascolonicus, saliva, 190, 218-9 Myzus cerasi, Myzus cerasi, saliva, 214, 218-9, 221 Myzus persicae choline in development, 57,58 saliva, 200,213,227,239,240,244
N Naringerin, aphid saliva, 219 Naucoris cimicoides, saliva, 205
427
SUBJECT INDEX
Nematospora coryli, and Acrosternum, 24 1 Nematospora jossypii, and Dysdercus, 24 1 Nemoura cinerea, sperm axoneme, 346 Neodiprion sertifer, lipids containing choline, 73 Nepa, sperm, 370 Nephotettix cinticeps, saliva, 230, 240 Nephrotoma, sperm axoneme, 345 Nephrotoma sodalis, lipids containing choline, 73 Neural fat body sheath, role, 278-81 Neural lamella as ion barrier, 95 organisation, 264-6,273 Neuronia. sperm axoneme, 339 Neurones and glial cells, organisation, 268-74 neuronal function experimental preparations, 289-9 1 intact nervous systems, 28 1-9 Neuroptera, sperm cells, 328, 329, 336,339,341,348,35 1 Neurotoxin, phospholipase as, 210 Neurotransmitter substances, and choline, 53,98 Noradrenaline, and cyclic AMP, 35 Norepinephrine, and K+ efflux, 38 N o tonec ta glau ca salivary glands, 23 1,235 sperm axoneme, 339 Nucleotides, cyclic, and specificity of cyclic AMP, 16-18 Nucleus, sperm, 328-32 chemical characteristics, 33 1 physical characteristics, 33 1-2 shape, 328-29 submicroscopic structure, 329-3 1
0 Oligosaccharases, saliva, 209 Oncopeltus fasciatus choline metabolism, 63, 66, 73, 78 saliva composition, 209, 2 10, 2 1 1, 2 12
Oncopeltus fasciatus-cont. saliva-con t. enzymes, 238 feeding, 194, 202-3,204,205 glands, 234,235 Operant learning, 113-5, 157-62, 164 Orthop tera choline metabolism enzymes, 86 lipid-soluble metabolites, 7 1, 74, 78, 82, 83 requirements, 55-6, 92 water-soluble metabolies, 69, 70 haemolymph, ionic composition, 28 1 sperm cells acrosomal complex, 324, 326 axoneme, 348,350,352 cell surface, 323 centriolar region, 336 genetics, 383 nucleus, 33 1, 332 Oryctes, sperm, 328 Osmotic pressure, and sperm motility, 381 Ostrinia nubilalis, acetylcholine, 66 Oxidases, Hemipteran saliva, 220, 238-9,246,247 Oxidation of choline, 88-9 Oxygen, and sperm, 380 Oxytocin, and cyclic AMP, 39
P Paleacrita vernata, lipids containing choline, 74 Palmitoleic acid, and choline metabolism, 77 Palorus ratzeburg’, choline requirements, 56,59,60 Pancreas, regulation by secretin, 37 Pancreozymin, stimulation of pancreas Panorpa annexa, sperm axoneme, 339 Parafilms, use in Hemipteran feeding, 185, 189,194, 197,200,212 Parasympathetic stimulation, salivary glands, 37 Parathion, and acetylcholine, 99
428
SUBJECT INDEX
Parathyroid hormone, and cyclic AMP, 14,21, 38 Parlatoria oleae, sperm, 353,370 Parotid glands and calcium 37 and dibutyryl cyclic AMP, 16 Pathogens, and saliva, 241-4 Pavlovian conditioning, 1 13, 162-4 Pectinase, in saliva, 197, 2 13 Pectinophor gossypiella, choline requirements, 57 Pectinpolygalacturonase, in saliva, 209,212,215,220,222 Pediculus, sperm, 369 Peloridiidae, salivary glands, 233, 245, 246 Pentatoma rufipes, saliva, 205, 235 Pentatomidae, saliva, composition, 205, 208, 209, 210, 21 1,214,215,216 feeding, 202, 203 glands, 234,235,237 Pentatomoidea, feeding, 192 Pentatomorpha, saliva, composition, 205, 206, 207, 208, 210,211 feeding, 191, 192, 193, 195, 196, 203 glands, 234,235,246 oxidases, 239, 247 PAS reaction, 241 Performic acid-alcian blue, for sulphydryl groups, 240 Perineurium, and blood-brain barrier, 264-5, 266-8, 273, 285, 290, 29 1, 300-5 Perineurial cleft, “tight junctions”, 95 Periodic acid-Schiff (PAS) reaction, Hemipteran saliva, 240, 241 Periphyllus negundinis, pectinase, saliva, 2 13 Periplaneta arnericana blood-brain barrier cation exchange, blood and CNS, 25 8 extra-axonal sodium regulation, 302,304 extraneuronal potentials, 282-3, 285.286.288.289.290 , _ , ,
Periplaneta americana-cont. blood-brain barrier-con t. fat body deposits, 260, 281 glial system, 268-74 glycogen deposition, nervous system, 305 ionic basis, electrical activity, 277,278 ionic composition, haemalymph, 275 ionic composition, nervous tissues, 275, 276 nervous tissues, exchange properties, 301 neural lamella, 264,266 perineurium, 266-9 trehalose and glucose uptake, 258 choline metabolism acetylcholine, 63, 95 acetylcholinesterase, 96 glycerylphosphorylcholine, 70 lipids containing choline, 74 phosphatidylcholine, 85-8 phosphorylcholine, 67 5-HT, 2 and Hemipteran saliva, 204, 205 learning and memory, isolated ganglia, 111-8 1 see Learning sperm, motility, 380, 38 1 Peroxidase penetration, nervous system, 262, 266, 267, 273, 279, 285, 302 in saliva, 215, 238-9, 244,245,247 PH of saliva, 217 and sperm motility, 380 Phagostimulants, and host specificity, 248 Phasmida, ionic composition, haemolymph, 275 Phasmoidea, sperm cells absence of mitochondria, 360, 363 accessory flagellar bodies, 365 acrosomal complex, 327 axoneme, 342, 348, 350, 351, 352 cell surface, 323 centriolar region, 336 Phenols, Hemipteran saliva, 223, 224
SUBJECT INDEX
Phenol/phenolase system, Hemipteran saliva, 247, 249 Phenolases, Hemipteran saliva, 223, 224 Phenolase/peroxidase system, Hemipteran saliva, 2 10 Phenolic compounds, Hemipteran saliva, 219, 221, 247, 248,249 Phenylalanine, Hemipteran saliva, 2 16, 223,224 Phenylalanine/Tyrosine/DOPA, , Hemipteran saliva, 2 11 Phenethylamine, salivary gland stimulation, 7 Philaenus, sperm, nucleus, 331 Philaenus spumarius, salivary glands, 232 Phloridzin, aphid saliva, 219 Phormia regina, choline metabolism enzymes, 85-91 lipid-soluble metabolites, 73, 78, 80,81,84, metabolic role, 96, 97,98 nutritional requirements, 57, 58, 59,61,62 water-soluble metabolites, 64-5, 67, 69,70 Phorodon humili, pectinase, saliva, 213 Phosphatidylcarnitine, metabolism, 87 Phosphatidylcholine, and choline metabolism, 7 1-82 enzymic synthesis, 85-7 hydrolysis, 87-8 and metabolic role of choline, 92, 94, 95, 96,98, 100 in vertebrates, 5 3-5 Phosphatidyl DMAE, and choline metabolism, 53-5 P h o s p ha t id y l e t h a n olamine, and choline metabolism, 53-5,77, 79, 96 Phosphatidyl-MMAE, and choline metabolism, 53-5 Phosphatidylserine, and choline metabolism, 53-5 Phosphodiesterase, and cyclic AMP, 12-18,27, 35 Phosphofructokinase, sperm axoneme, 352
429
Phospholipase, saliva, 204, 210 Phospholipids and choline metabolism, 52-3 in saliva, 240 Phosphorylase and cyclic AMP, 38 and epinephrine, 36 in saliva, 210,215 sperm axoneme, 352 Phosphorylase b kinase, and cyclic AMP, 17 Phosphorylcholine, and choline metabolism, 53-5, 66-9, 92, 97 Phylloxera, galls, 22 1, 222, 224 Phylloxeridae, salivary glands, 228-9, 245 Phytopathogenicity, and Hemipteran saliva, 2 17-25 Phytophaga rigidae, lipids containing choline, 71,73 Phytophagous insects blood-brain barrier, 280, 288, 302 saliva, 184, 192, 193, 196, 197, 203, 204, 207, 210, 212, 224 Pikonema alaskensis, lipids containing choline, 73 Pilocarpine, and salivation, 188 Pissodes strobi, lipids containing choline, 72 Plasma membrane, labilisation, and cyclic AMP, 4 1 Platymerus rhadarnanthus, saliva, 204, 205,210,238 Plecoptera, sperm, 327,341,365 Plodia interpunctella, choline metabolism, 74,78 Poeciloscytus unifasciatus, pectinase, saliva, 2 13 Polymorphism, sperm, 382-3 Polyphenol oxidase (PPO), Hemipteran saliva, 215, 223, 238, 240, 245 Polysaccharide, sperm, 348, 352, 359, 365,380 Potassium and blood-brain barrier, 259, 272, 274 and cyclic AMP, 38 and extraneuronal potentials, 281-91
430
SUBJECT INDEX
Potassium-con t. haemolymph, 275 and osmotic gradients, salivary glands, 22,24 and resting potential, 277 in saliva, 3 and sperm malformation, 383 uptake, abdominal nerve cord, 95 Potentials extraneuronal, 282-9 membrane, and cyclic AMP, 38 miniature end-plate, and cyclic AMP, 34-5 salivary glands, effect of 5-HT and Cyclic AMP, 23-6,27 Predatory insects, saliva, 193, 203-5, 208,210,215 Procaine, and action potential, 278 Prociphilus tessellatus lipids containing choline, 73 pectinase, saliva, 214 Prostaglandins, and Calsium, 40 Protamines, sperm nucleus, 333 Protein synthesis increased after learning, 168 inhibition by cycloheximide, 174 Proteinases, in Hemipteran saliva, 204, 210,215,219 Protenor, sperm genetics, 383 Prothoracic ganglion, and learning, 124-5, 129, 132, 136, 137, 138-40, 149, 169, 172-3 Prothoracic leg, and learning, 124, 125, 129, 135, 138-40, 144 Protocatechuic acid, in aphid saliva, 219 Protoparce sex fa. choline metabolism, 67,68,69,74, 82 Protura, sperm acrosomal complex, 327 axoneme, 338,342,343 non-flagellate sperm, 374 Pseudococcus obscurus, non-flagellate sperm, 370 Pseudosarcophaga affinis, choline in development, 57 Psocidae, sperm axoneme, 338 Psocoptera, sperm, two axonemes, 369 Psychodidae, sperm, 327, 355, 374
Psyllidae saliva, 2 14, 2 17 sperm nucleus, 33 1 Pterocomma spp., pectinase saliva, 213 Pterygota, sperm acrosomal complex, 324 axoneme, 349 mitochondria, 354,355,363 Ptinus tectus, choline in development, 56 Purkinje cells, cerebellar, and cyclic AMP, 35 Puto, non-flagellate sperm, 370 P. albicans, 370 Pyrausta nubalis, choline in development, 57 Pyrrhocoridae, saliva composition, 21 0 feeding, 191, 196,202,203 principal gland, 237 Pyrrhocoris, sperm, two axonemes, 370 Pyrrhocoris apterus, saliva, 210, 215, 244
Q Quaternary ammonium compounds, uptake by abdominal nerve cord, 95 Quercitin, aphid saliva, 2 19 Quercitrin, aphid saliva, 219 Quinones, Hemipteran saliva, 222, 223,247
R Radioisotopes, and salivation, 189, 190 Receptor-5-HT interaction, 5-1 2 Reduviidae, saliva composition, 2 10 feeding, 192,203,204,208 glands, 235
SUBJECT INDEX
431
Reduviidae, saliva-cont. Rutin, aphid saliva, 219 and rickettsia1 diseases, 250 Reduvioidea, feeding, 193 Reduvius personatus, saliva, 205 Renal tubules, and cyclic AMP, 14 S Reproductive organs, acetylcholine content, 66 Saldidae, feeding, 192 Resting potential, and extra-axonal Saliva, Hemiptera, 183-255 ions, 277, 288 composition and function, 205-1 7 Reticulitermes, sperm, 354 sheath material, 205-8 R. lucifugus, 371 373 watery saliva, 208-1 7 Rhabdophaga swainei, lipids conevolution, 244-7 taining choline, 7 1, 73 feeding by carnivores, 203-5 Rhopalosiphum rhois, pectinase, lacerate-and-flush feeding, 202-3 saliva, 2 13 methods, 185-90 Rhincoris carmelita, saliva, 205 modes of feeding, 190-3 Rhinotermitidae, non-flagellate sperm, origins, 236-41 371 accessory gland, 236 Rhodnius prolixus principal gland, 237-8 diuretic hormone, 33 salivary carbohydrate and lipid, 5-HT and Malphighian tubules, 240-1 11-12 sources of oxidases, 238-9 saliva sources in Homoptera, 239-40 composition, 205,206, 208 phytopathogenicity, 2 17-25 feeding, 188, 193, 192, 196 salivary glands and ducts, 225-35 glands, 234,235 Aphidoidea, 226-9 origins, 238,241 Fulguromorpha, 232-3 Rhynchota, sperm Heteroptera, 234-5 accessory flagellar bodies, 364 Jassomorpha, 229-32 axoneme, 339,342 other Auchenorrhyncha, 2 3 3 4 cell surface, 328 stylet-sheath feeding, 194-202 mitochondria, 356,359,360 as vehicle for pathogens, 241-4 non-flagellate sperm, 370 Salivary glands, cyclic AMP and two axonemes, 369,370 Calcium, 1-49, see Cyclic AMP Rhynchotoidea, sperm, 35 1, 269-70, Saldidae, feeding, 192 380 Sappaphis, mali, metabolites, saliva, Ribonucleoproteins, in sperm centriole 218-9 adjunct, 329 Rickettsial diseases, Sarcophaga, sperm axoneme, 353 transmission, 250 Sarcophaga bullata, choline metaRNA bolism, 75,85 increased, after learning, 168 Sarcoplasmic reticulum, permeability, sperm nucleus, 33 1 and cyclic AMP, 36 Robinetinaglycone, aphid saliva, 2 19 Sarcosine, and choline metabolism, Romalea microptera, ionic composi53-5 tion, nervous tissues, 275,276 Sarcosomes, flight muscle, choline Royal jelly, acetylcholine content, 66, metabolism, 76 92 Scale insects, sperm nucleus, 330 Rubidium ions, and potential changes, Schistocerca gregaria 283-6 choline metabolism, 57, 75
432
SUBJECT INDEX
Schistocerca gregaria-cont. fat body deposits, 260 learning, leg position, 157-9 Schizolachnis pini-radiata lipids containing choline, 73 pectinase, saliva, 2 13 Sciara coprophila, sperm acrosomal complex, 327 axoneme, 338,340,343 capacitation, 38 1 centriole, 334 flagellum, 367 Scratch-and-suck feeding, 19 1, 2 17 Secretin, and fluid transport, pancreas, 37 Sensilla, gustatory, and saliva, 2 12 Sensory input, and learning, 164-6 Serine and choline metabolism, 5 2 , 5 3 4 Hemipteran saliva, 2 18,22 1 Serotin, snake venom, 204 Shock-avoidance learning, cockroach, 169-7 1 Simple problem solving, 1 13-5, 157-62, 164 Sitodrepa panicea, choline in develop ment, 5 5 Smooth muscle, and cyclic AMP, 35, 41 Sodium and cyclic AMP, 36,39-40 and fat body cells, 262 in haemolymph, 275 and neural fat-body sheath, 278-8 1 and neuronal function, 282-9, 299, 300, 302-4 and peripheral diffusion barrier, 259 and resting potential, 277 uptake, abdominal nerve cord, 95 Sodium pentobarbitol, and learning, 169 Sperm cells, 3 15-97 accessory ordered flagellar bodies, 363-7 acrosomal complex, 324-28 axoneme, 336-53 central sheath, 349 coarse fibres, 350-2 links heads, 349-50
Sperm cells-cont. axoneme-con t. matrix, 352-3 microtubdes, 338-49 cell surface, 3 17-24 centriolar region, 332-6 double flagellar apparatus, 367-74 mitochondria, 354-63 motility, 374-82 nucleus, 328-32 polymorphism and genetics, 382-3 Sphingomyelin, and choline metabolism, 53-5, 72-6, 83-4,92 Steatococcus tuberculatus, non-flagellate sperm, 370 Stegobium panicea, choline in development, 56 Stenodema calcaratum, salivary pectinase, 2 13 Sternorrhyncha, saliva, 192, 225 Stick insect, fat body deposits, 260 Stomacoccus plantani, non-flagellate sperm, 370 Strauzia longipennis, lipids containing choline, 73 Strontium and stimulation by ADH, 40 Structure-activity relationships, 5-HT, 5-12 Strychnine sulphate, and learning, 169 Stylet-sheath feeding, Hemiptera discharge of saliva, 197-200 formation of sheath, 196-7 ingestion, 200-1 and phytopathogenicity, 21 7 sampling the surface, 194-5 secretion of flange, 195-6 sheath material, composition, 205-8 withdrawal of stylets, 201-2 Subcellular fractions, lipids containing choline, 76 Suboesophageal ganglion, and learning, 136 Succinic dehydrogenase, sperm mitochondria, 364 Sucrose-gap technique, 22, 277, 284 Sulphydryl groups, Hemipteran saliva, 231,240,245,246 Symbionts, and choline metabolism, 58, 59
SUBJECT INDEX
Sympathetic stimulation, salivary glands, 37 Synapse, reactions at, 96-7 Synaptic changes, during learning, 162 Synaptic transmission, and cyclic AMP and Calcium post-synaptic, 35-6 pre-synaptic, 34-5
T TEA, and potentials, 278, 283, 284, 286 Telamona, sperm axoneme, 342 Temperature, and sperm motility, 381 Tenebrio molitor choline metabolism lipids containing choline, 72 nutritional requirements, 55, 56, 59, 60, 63 phosphatidylcholine, 78, 8 1 sperm accessory flagellar bodies, 364, 365 acrosomal complex, 324, 325 axoneme, 337,343,348 cell surface, 323 centriolar region, 336 mitochondria, 358,361,362 motility, 377, 378 Tenebrionid beetles, carnitine, 52 TEPP (tetraethylpyrophosphate) and acetylcholine, 99, 100 Termites, sperm, 354, 37 1 Termopsidae, sperm, 37 1 Tetrodotoxin, and action potential, 278 Tettigonioid Orthoptera, sperm, 326 Thelmatoscopus albipunctatus, nonflagellate sperm, 372 Theophylline, and cyclic AMP, 14, 15, 18,27, 31, 34,38, 39 Thomasiniellula populicola, salivary pectinase, 2 13 Thorax tissues, choline, 75 Threonine, aphid saliva, 218 Thrips, sperm, 370
433
Thyroid stimulating hormone, and cyclic AMP, 37 Thysan optera saliva, 189, 191, 192,217 sperm, 327,338,351,355,369 Thysanura lipids containing choline, 7 1,74 sperm accessory flagellar bodies, 363, 364 acrosomal complex, 324, 327 axoneme, 349,351 mitochondria, 354 nucleus, 328 paired sperm, 367-9 “Tight” junctions, perineurial, 95, 285, 287, 290, 291, 300, 302, 304 . Timarchia tenebriosa, ionic composition of haemolymph, 275,276 Tineola bisselliella, choline in development, 57 Tingidae, feeding, 191, 192, 193, 203, and phytopathogenicity, 21 7 Toxoptera aurantii, B vitamins in development, 58 Transplantation of ganglia, cockroach, 150 Transporting epithelia, hormone regulation, 39-41 Trehalose from glycogen, hormones, 32 Trial-and-error learning, 113-5, 157-62, 164 Triatoma protracta, saliva, 205 Tribolium castaneum, choline substitutes, 59 Tribolium confusum, choline metabolism enzymes, 8 9 , 9 0 lipid-soluble metabolites, 72, 78,83 nutritional requirements, 56,59, 60 Trichoplusia, sperm capacitation, 38 1 Trichoptera, sperm acrosomal complex, 327, 328 axoneme, 338, 339, 342, 343, 346, 347,348 mitochondria, 355 spermatids, 370
434
SUBJECT INDEX
Triglycerides, and cyclic AMP, 38 Tris ions, and potential changes, 283, 284,285 Trogoderma granarium, choline metabolism, 5 6 , 7 1 , 7 2 , 7 8 Tryptamine, salivary gland stimulation, 7 Tryptophane Hemipteran saliva, 221, 223,224 salivary gland stimulation, 7 Tubulin, sperm, 341,343,349 Type R learning, 113-5, 157-62, 164 Type I1 learning, 1 13-5, 157-62, 164 Typhlocybidae, salivary glands, 230 Tyrosine, aphid saliva, 218
Vasopressin (ADH), and cyclic AMP, 39-40 Vasotocin, and cyclic AMP, 39 Vespa crabro, venom, acetylcholine, 66,92 Vicia faba, saliva, 189 Virus transmission, and saliva, 242, 244,250 Visceral muscles, hormone regulation, 32 Viscosity, and sperm motility, 38 1 Viteus vitifolii, saliva composition, 2 16 glands, 228-9 metabolites, 2 18-9 origin, 245 and phytopathogenicity, 21 7 , 220, 22 1
U “Under-asparagine”, aphid saliva, 2 18 Uterus, and cyclic AMP, 35 UTPase, sperm axoneme, 346, 349, 351,352,365,367
V Valine, Hemipteran saliva, 218,221
W Water transport, toad bladder, 39-40 Watery saliva, composition and function, 208-17
X Xenophyes cascus, salivary glands, 231
Cumulative List of Authors Numbers in bold face indicate the volume number o f the series Aidley, D. J., 4, 1 Andersen, Sven Olav, 2, 1 Asahina, E., 6, 1 Ashburner, Michael, 7, 1 Baccetti, Baccio, 9, 3 15 Beament, J. W. L., 2, 67 Berridge, Michael J., 9, 1 Boistel, J., 5, 1 Bridges, R. G., 9, 5 1 Burkhardt, Dietrich, 2, 131 Bursell, E., 4, 33 Burtt, E. T., 3, 1 Carlson, A. D., 6, 5 1 Catton, W.T., 3, 1 Chen, P. S., 3, 53 Colhoun, E. H., 1, 1 Cottrell, C. B., 2, 175 Dadd, R. H., 1 , 4 7 Dagan, D., 8, 96 Davey, K. G., 2,219 Edwards, John S., 6,97 Eisenstein, E. M., 9, 11 1 Fraser Rowell, C. H., 8, 146 Gilbert, Lawrence, I., 4, 69 Goodman, Lesley, 7 , 9 7 Harmsen, Rudolf, 6,139 Harvey, W. R., 3, 133 Haskell, J. A., 3, 133
Hinton, H. E., 5,65 Hoyle, Graham, 7, 349 Kilby, B. A., 1, 11 1 Lawrence, Peter A., 7, 197 Lees, A. D., 3, 207 Maddrell, S. H. P., 8, 200 Miles, P. W., 9, 183 Miller, P. L;, 3, 279 Narahashi, Toshio, 1, 175; 8, 1 Neville, A. C., 4, 213 Parnas, I., 8, 96 Pichon, Y.,9, 257 Prince, William T., 9, 1 Pringle, J. W. S., 5, 163 Rudall, K. M., 1,257 Sacktor, Bertram, 7,268 Shaw, J., 1,315 Smith, D. S., 1,401 Stobbart, R. H., 1, 315 Treherne, J. E., 1,401; 9, 257 Usherwood, P. N. R., 6,205 Waldbauer, G. P., 5,229 Weis-Fogh, Torkel, 2, 1 Wigglesworth, V. B., 2, 247 Wilson, Donald M., 5,289 Wyatt, G. R., 4, 287 Ziegler, Irmgard, 6,139
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Cumulative List of Chapter Titles Numbers in bold face indicate the volume number of the series Active Transport and Passive Movement of Water in Insects, 2,67 Amino Acid and Protein Metabolism in Insect Development, 3, 53 Biochemistry of Sugars and Polysaccharides in Insects, 4, 287 Biochemistry of the Insect Fat Body, 1, 11 1 Biology of Pteridines in Insects, 6,139 Cellular Mechanisms Underlying Behaviour-Neuroethology, 7,349 Chitin Orientation in Cuticle and its Control, 4, 213 Chitin/Protein Complexes of Insect Cuticles, 1, 257 Choline Metabolism in Insects, 9, 5 1 Colour Discrimination in Insects, 2, 131 Comparative Physiology of the Flight Motor, 5, 163 Consumption and Utilization of Food by Insects, 5, 229 Control of Polymorphism in Aphids, 3, 207 Control of Visceral Muscles in Insects, 2, 2 19 Effects of Insecticides on Excitable Tissues, 8, 1 Electrochemistry of Insect Muscle, 6,205 Excitation of Insect Skeletal Muscles, 4, 1 Excretion of Nitrogen in Insects, 4, 33 Feeding Behaviour and Nutrition in Grasshoppers and Locusts, 1 , 4 7 Frost resistance in Insects, 6,1 Function and Structure of Polytene Chromosomes During Insect Development, 7, 1 Functional Aspects of the Organization of the Insect Nervous System, 1,401 Functional Organizations of Giant Axons in the Central Nervous Systems of Insects: New Aspects, 8, 96 Hormonal Regulation of Growth and Reproduction in Insects, 2, 247 Image Formation and Sensory Transmission in the Compound Eye, 3, 1 Insect Blood-Brain Barrier, 9, 257 Insect Ecdysis with Particular Emphasis on Cuticular Hardening and Darkening, 2, 175 Insect Sperm Cells, 9 , 315 Learning and Memory in Isolated Insect Ganglia, 9 , 11 1 Lipid Metabolism and Function in Insects, 4, 69 Mechanisms of Insect Excretory Systems, 8, 200 Metabolic Control Mechanisms in Insects, 3, 133 Nervous Control of Insect Flight and Related Behaviour, 5,289 Neural Control of Firefly Luminescence, 6,5 1 Osmotic and Ionic Regulation in Insects, 1, 3 15 '
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CUMULATIVE LIST OF CHAPTER TITLES
Physiological Significance of Acetylcholine in Insects and Observations upon other Pharmacologically Active Substances, 1, 1 Polarity and Patterns in the Postembryonic Development of Insects, 7 , 197 Postembryonic Development and Regeneration of the Insect Nervous System, 6, 97 Properties of Insect Axons, 1, 175 Regulation of Breathing in Insects, 3, 279 Regulation of Intermediary Metabolism, with Special Reference to the Control Mechanisms in Insect Muscle, 7 , 2 6 8 Resilin. A Rubberlike Protein in Arthropod Cuticle, 2, 1 Role of Cyclic AMP and Calcium in Hormone Action, 9, 1 Saliva of Hemiptera, 9 , 183 Spiracular Gills, 5, 65 Structure and Function of the Insect Dorsal Ocellus, 7 , 9 7 Synaptic Transmission and Related Phenomena in Insects, 5, 1 Variable Coloration of the Acridoid Grasshoppers, 8, 146
