Advances in Marine Biology, Volume 22

Advances in

MARINE BIOLOGY VOLUME 22

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Advances in

MARINE BIOLOGY

SAN Diso

VOLUME 22 Edited by

J. H. S. BLAXTER Dunstaflnage Marine Research Laboratory, Oban, Scotland

Thelate

SIR FREDERICK S. RUSSELL

Reading, England and

SIR MAURICE YONGE Edinburgh, Scotland

Academic Press ( A Subsidiary of Harcourt Brace Jouanouich) London Orlando San Diego New York

Toronto Montreal Sydney Tokyo

1985

COPYRIGHT 0 1985, BY ACADEMIC PRESS INC. (LONDON) LTD. ALL RIGHTS RESERVED. N O PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road

LONDON NW1 7DX

United States Edition published by ACADEMIC PRESS, INC. Orlando, Florida 32887

LIBRARYO F CONGRESS CATALOG CARD NUMBER: 63-14040 ISBN: 0-12-026122-7 PRINTED IN THE UNITED STATES OF AMERICA 8.5 M 87 88

9 8 7 . 5 5 4 3 2 1

CONTENTS ..

CONTRIBUTORS TO VOLUME22

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xi

Assessing the Effects of “Stress” on Reef Corals B. E. BROWNA N D L. I. Introduction

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s. HOWARD ..

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11. Natural Fluctuations and Man-made Influences

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A. Assessing changes on coral reefs . . .. .. .. B. lnterpreting temporal changes on coral reefs . . .. C. Effects and apparent lack of effects of pollution on coral .. .. .. .. .. reefs . . .. .. D. Predicting recovery of reefs .. .. .. ..

9 17

111. Experimental Studies on Effects of Pollutants on Corals . . .. .. .. .. .. A. Growth rate . . .. .. .. .. .. .. B. Metabolism . . .. C. Loss of zooxanthellae .. .. .. .. .. D. Behavioural responses .. .. .. .. .. E. Reproductive biology .. .. .. .. .. F. Histopathology . . .. .. .. .. .. G. Biochemical and cytochemical indexes . . .. ..

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IV. Discussion and Future Research Needs .. References .. .. . .

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Nutrition of Sea Anemones M. VAN-PRAGT I. Introduction

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11. Chemoreception and Feeding Behaviour A. Feeding behaviour . . .. .. V

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B. Nature of activators. . .. .. C. The conducting systems . . .. D. The control of feeding behaviour . .

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67 69 69

111. Absorption of Dissolved Organic Matter .. .. .. A. Localization of uptake .. .. .. .. .. B. Uptake systems for glucose and amino acids . . .. C. Ecological importance of dissolved compounds for the nutrition of sea anemones . . .. .. .. ..

70 70 71

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IV. Gathering and Digestion of Particulate Organic Matter . . A. Suspension-feeding structures . . .. . . .. B. Endodermal currents and the role of the trilobed portion .. .. .. .. .. of mesenteric filaments C. Phagocytic cells, cytological and enzymological aspects of intracellular digestion . . .. .. .. . . .. .. D. Importance of particulate organic matter

72 72

V. Predation and Digestion of Prey .. .. ,. A. Role of tentacles, acontia, and cnidae . . .. B. Extracellular digestion of prey, cytological and enzymological aspects .. . . .. .. .. .. C. Excretion .. .. .. . .

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81 81

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82 89

.. .. .. .. VI. Symbiosis . . .. .. .. A. Localization of algal symbionts . . .. .. .. B. Regulation of the concentration of algae in the tissues . C . Translocation of metabolites .. .. .. ..

89 89 90 91

VII. Sea Anemones as Prey and Remarks on the Diet .. .. .. Anemones. . .. .. A. Predators of sea anemones. . .. .. B. Diet of sea anemones .. .. .. .. .. References .. .. . .

of Sea

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74 76 79

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92 92 93 94

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I02 102

Effects of Environmental Stress on Marine Bivalve Molluscs H. B. AKBERALI A N D E. R. TRUEMAN

I. Introduction

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A. Definition of stress

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B. Occurrence of natural and man-made stresses . . C. Threshold levels of pollutant stress .. .. D. Development of experimental techniques ..

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I04 I04 105

11. Behavioural Responses to Stress .. .. .. .. A. Valve closure as a protective mechanism .. .. B. Relationship between heart rate, valve movement, and .. .. .. .. .. .. pumping activity C. Behavioural response to some pollutant stressors .. D. Relevance of valve closure in epifaunal and infaunal species .. .. .. .. .. .. .. E. Effect of subthreshold levels on behaviour .. .. F. Effect of temperature on heart rate .. .. . .

I08

111. Detection of Stress .. .. .. .. .. A. The significance of registering changes in the environment . . .. .. .. .. .. B. Sites of reception . . .. .. .. .. C. Detection and response to environmental changes

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121 I28 130 I32

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V. The Role of the Shell . . .. .. .. .. .. A. Physical protection and isolation from environmental stress . . .. .. .. .. .. . . . . B. Shell closure and calcium reabsorption . . .. .. C. Effect of prolonged stress on shell strength .. ..

VII. Conclusions References

111 117

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IV. Respiratory Physiology during Stress. . .. .. .. .. A. Valve closure and cessation of aerobic processes B. Relationship between heart rate, valve movements, POZ, and pC02 .. .. .. .. .. .. C. Anaerobic respiration during valve closure .. .. D. Valve activity and pH changes . . .. .. ~.

VI. Action of Heavy Metal Stressors .. .. .. A. Accumulation of heavy metals . . .. .. B. Effects of heavy metals on tissues .. .. C. Effects of heavy metals on released gametes and embryonic and juvenile stages .. .. .. D. Effects of heavy metals on cellular organelles . .

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146 146 146 I48 151

I55 155 156 161

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162 162 163

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168 175

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182 f83

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Vlll

Growth in Barnacles D. J. CRISPA N D E. BOURGET I. Evolution of Barnacles and Their Shells 11. Mechanisms of Growth . . .. .. A . Growth of individual shell plates . .

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203 203 204

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208 208 209 211

B . Primordial valves . . .. .. .. C. Orientation of barnacles at settlement and during .. .. .. .. .. growth. . ..

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IV. Factors Influencing Growth Rate .. A. Temperature . . .. .. .. .. .. .. B. Light .. .. C. Current, tidal level, and nutrition.. D. Surface contour .. .. .. E. Orientation to current .. .. F. Population density . . .. .. G . Competing organisms .. .. H. Parasites . . .. .. .. I. Reproduction. . .. .. ..

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V . Age and Growth-the

111. Modification of Shape

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A. Effects of crowding . . .. .. €3. Influence of substratum on shape . . C. Influence of salinity on shape ..

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211 215 216 216 217 217 217 219 219 219

Growth Curve..

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234 234 236 237 237

VI. Growth Rates of Various Species

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VII. Histology and Fine Structure of the Integument: Growth and Ecdysis .. .. . . .. . . . . VIII. Shell Structure in Relation to Function

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IX. Cyclical Factors in Growth A. Tidal influences .. B. Daily influences .. C. Other lunar influences D. Annual influences . .

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E. Other cyclic influences .. .. .. .. F. Frequency, scale, and precision of measurement .. .. . . .. .. .. References Taxonomic Index Subject Index

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238 238 239

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Cumulative Index of Titles

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Cumulative Index of Authors

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CONTRIBUTORS TO VOLUME 22 H . B. A K B E R A LDepartments I, of Zoology und Botany, University of Manchester, Manchester MI3 9PL, England. E . BOURGET,Dkpartement de Biologie, UniversitP Laval, QuPbec GI K 7P4, Canada. B. E . BROWN,Department of Zoology, University of Newcastle upon Tyne, Newcastle upon Tyne NEI 7RU, England.

D . J . CRISP,Natural Environment Research Council, Unit of Marine Invertebrate Biology, Marine Science Laboratories, Menai Bridge, Gwynedd LL59 5EH, United Kingdom. L. S . HOWARD, Department of Zoology, University of Newcastle upon Tyne, Newcastle upon Tyne NEI 7RU, England.

E. R . TRUEMAN, Department of Zoology, University of Manchester, Manchester MI3 9PL, England.

M . VAN-PRAET,Laborutoire de Biologie des Inverte'bre's Marins et Malacologie, MusPum National d'Histoire Naturelle, 75005 Paris, France.

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Assessing the Effects of “Stress” on Reef Corals B. E. Brown and L. S. Howard Department of Zoology, University of Newcastle upon Tyne Newcastle upon Tyne, England

I. Introduction . . . . . . . . . . . . . . . . . . 11. Natural Fluctuations and Man-made Influences . . . . . . . . A. Assessing changes on coral reefs . . . . . . . . . . B. Interpreting temporal changes on coral reefs . . . . . . C. Effects and apparent lack of effects of pollution on coral reefs . . D. Predicting recovery of reefs . . . . . . . . . . . . 111. Experimental Studies on Effects of Pollutants on Corals . . . . A. Growth rate . . . . . . . . . . . . . . . . B. Metabolism . . . . . . . . . . . . . . . . C. Loss of zooxanthellae . . . . . . . . . . . . . . D. Behavioural responses . . . . . . . . . . . . . . E. Reproductive biology . . . . . . . . . . . . . . F. Histopathology . . . . . . . . . . . . . . . . G. Biochemical and cytochemical indexes . . . . . . . . IV. Discussion and Future Research Needs . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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Introduction

Some years ago Johannes (1975) published the first major literature review on the effects of marine pollutants on coral reefs. At that time he 1 ADVANCES I N MARlNh BIOLOGY. VOL 22

Copyright

(0 19x7. by Academic Press I n i (London) Ltd All rights of reproduction in any form reserved ISBN 0-12-026122-7

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B . E. BROWN A N D L. S. HOWARD

highlighted the paucity of knowledge in many areas of the subjects. Although research efforts in the field have increased, particularly with respect to potential effects of pollution by oil (Loya and Rinkevich, 1980) and drilling muds (Dodge and Szmant-Froelich, 1984) there are still enormous gaps in our knowledge and serious contradictions in the existing literature. Much of this lack of information may be attributed to our limited understanding of the physiology of corals, although recent papers have contributed valuable data on growth (Highsmith, 1979), reproduction (Highsmith, 1982; Kojis and Quinn, 1981, 1982; van Moorsel, 1983), behavior patterns (Lasker, 1979), carbon turnover (Crossland ef ul., 1980b), calcification (Barnes and Crossland, 1978, 1982; Gladfelter 1982a), mucus production (Crossland et al., 1980a), and associated bacterial populations on living corals (Ducklow and Mitchell, 1979). The aims of the present article are to consider long-term ecological studies in the light of known effects of disturbances on coral reefs and to ask whether the effects of disturbances can be distinguished from longterm fluctuations on the reef and also where other difficulties lie in assessment of pollution in the field. In addition, in an attempt to improve understanding of the overall susceptibility of reef corals to marine pollution, an assessment is made of the responses of corals to stress and methods by which these responses have been monitored to date. The definition of “stress” has been much discussed in the literature (Grime, 1979; Pickering, 1981; Stebbing, 1981; Rosen, 1982), particularly with reference to problems involved in identifying and quantifying such a condition. Working with plants, Grime (1979) defined stress as the external constraints limiting dry matter production by all or part of the vegetation, while Rosen (l982), considering corals, described stressful conditions as those resulting in restricted growth and reproduction. Such specific definitions as these have not always been applied in many of the publications referred to in this article. For our purposes Rosen’s broader view of stress as a gradient between ideal conditions and the ultimate limits of survival will be adopted. As noted by Stebbing (1981), the term stress may be used as a cause or as an effect. In agreement with Stebbing and most other authors, we choose to view stress as an external force or stimulus. The article is divided into three sections, the first section dealing with observations in the field, the second with laboratory assessment of pollutant effects, and the third section incorporating a general discussion of the validity of generalizations made to date on the overall vulnerability of coral reefs to man-made disturbance.

EFFECTS O F STRESS O N REEF CORALS

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II. Natural Fluctuations and Man-made Influences A. Assessing Changes on Coral Reefs

Assessing change necessarily implies that reefs are monitored regularly by standard, repeatable methods. It is only in recent years, however, that quantitative methods have become routinely employed on permanent transects over time intervals. The first review of field methods applied on coral reefs was published by Stoddart in 1972. Subsequently other workers (Loya, 1972, 1978; Done, 1977; Bouchon, 1983; Dodge er al., 1982) have successfully tested various quantitative and semiquantitative methods on the reef. Strictly quantitative techniques vary from plotless (Loya, 1978) to quadrat methods (Bak and Engel, 1979), and more recently workers have compared both approaches at the same sites in an attempt to gauge their relative efficiencies (Dodge et al., 1982; Bouchon, 1983). Although there appear to be no major differences in the results obtained by either methods, there are variations in the quantity and type of information generated and the time required for use; the line transect or “intersected length” method generally makes the most efficient use of the time spent underwater. Measurements made on coral reefs using these techniques include coral cover, diversity, evenness (Loya, 1972, 1976a; Brown and Holley, 1982; Dodge et a f . , 1982; Bouchon, 1983), colony number and colony size (Loya, 1972; Fishelson, 1977), and more recently spatial complexity (Rogers er a f . , 1982, 1983) and spatial arrangement of both living and dead substratum components (Bak and Luckhurst, 1980). Although monitoring of coral cover and diversity may yield fundamental information about coral assemblages, few studies incorporate measurements of the cover and diversity of other components of the coral community, such as soft corals, zoanthids, algae, sponges, and ascidians. Recently, the importance of monitoring these groups has been highlighted by the publications of Benayahu and Loya (1977), Bak er af. (1981), and Tursch and Tursch (1982). Invasion and/or overgrowth of scleractinian corals by many species of soft corals (Nishihira, 1981; Tursch and Tursch, 1982) and ascidians (Bak er al., 1981; Sammarco er al., 1983; Sya’rani, 1983) has frequently been observed in both the Indo-Pacific and the Caribbean provinces. In studies where dominant components of the coral community have been considered (Rogers er a f . , 1982), the effect of disturbance on coral reef diversity may be complex. Diversity of scleractinian corals as a result of hurricane damage in St. Croix was shown to decrease in shallow reef

4

B . E. BROWN A N D L. S . HOWARD

zones, whereas diversity of the community as a whole actually increased because of colonization of new substrata by a wide variety of reef organisms, e.g., algae, sponges, tunicates, bryozoa, and hydroids. Clearly quantitative measurements on coral reefs affected by disturbance should include some account of all major components of the reef community. Such measurements are also improved by an appreciation of the structural complexity of the coral reef environment. Rogers et al. (1982) used a modified transect method, with a linked chain following the contours of the reef, to obtain an index of reef topography or structural complexity. Done (1981) has applied the use of stereophotography to permanent transects on the Great Barrier Reef. A stereo pair of photographs provides a great resolution of detail, a means of determining the three-dimensional coordinates of colonies and substratum, and a means of determining true dimensions and shapes of benthic organisms at any depth in the photograph. With automated stereoanalysis it should be possible to accurately map three-dimensional growth patterns of living corals and/or surface area of other substratum components. So complex are the interactions on coral reefs (Bak et al., 1982; Porter et al., 1982) that standard measurements of areal coverage, diversity, and abundance may not always be sensitive to changes in interactions which would be detected in a threedimensional approach to community analysis. Such an analysis should also consider the nonliving components of the substratum. Bak and Luckhurst (1980) have highlighted the importance of monitoring not only living cover but also nonliving substrata such as rock and sediments. Their study showed that alteration of spatial arrangement through dislodgement and collapse of substrata and changes in sediment flow were of paramount importance in describing the community, particularly in shallow-water (10- and 20-m) quadrats. As the authors note, a continuous change in the cover of nonliving components must have serious implications for the settlement and survival of juvenile benthos. One further factor should be considered when assessing changes on coral reefs, and that is the measurement of colony size. This parameter has been used by various workers (Loya, 1972, 1967a; Fishelson, 1977) involved in monitoring the effects of disturbance on coral reefs. It has recently been recognized (Hughes and Jackson, 1980) that partial colony mortality, colony fission, and colony fusion may affect any simple relationship between the size and age of reef corals. Following known corals in photographs for successive years demonstrated that, in foliose Caribbean corals, size and age are seldom related. Measurements such as those of Fishelson (1977) on age groups of faviids from polluted and nonpolluted sites, estimated from size dimensions, may require reinterpretation in the

EFFECTS OF STRESS ON REEF CORALS

5

light of these more recent studies. Loya (1976a), however, recognizes the regenerative ability of corals when interpreting the effects of low tides at Eilat, and stresses that in the few cases where corals did not fully regenerate the separate parts were considered as one individual.colony. His results suggested that whereas before the low tide coral colonies on the control reef fell into relatively large size categories, in 1973 after the catastrophic low tide, colonies fell into small size categories. Corals with marked regenerative ability included Cyphastrea microphthalma (Lamarck), Pauona decussata (Dana), Millepora dichotoma (Forskal), and Porites lutea (Milne-Edwards and Haime). Generally the recovery of the control reef was mainly due to recolonization by coral planulae rather than regeneration of survivors. Nevertheless, regeneration of corals after partial mortality is an important process on all reefs, and it may be very difficult to decide if a small coral has recently settled or whether it is actually part of a much larger colony which has suffered partial colony mortality or colony fission. Such difficulties may be compounded in polluted areas, particularly those suffering from a high sediment load (personal observation) (Fig. 1). Clearly this aspect requires further study on reefs affected by sedimentation where the growth form of massive species such as P. lutea and Goniastrea retiformis (Lamarck) appears nodular and where partial colony mortality is high. Many long-term monitoring programmes incorporating techniques described earlier have now been initiated on coral reefs in both the Caribbean and the Indo-Pacific, and much interesting information should gradually become available over the next decade-to quote Lewis (1976), considering long-term ecological surveillance on temperate rocky shores, “to record ‘change’ is no problem. There is much and it would be a remarkable investigation that showed none. The major need is to ensure that the change recorded is real and relevant.”

B. Interpreting Temporal Changes on Coral Reex7 Table I demonstrates major long-term changes observed as a result of mainly natural disturbances, while Table I1 records instances of manmade damage on coral reefs. It is clear from these tables that recent regular monitoring of fixed stations and transects in CuraGao (Bak and Luckhurst, 1980) and Eilat (Loya, 1976a) have produced interesting data on coral distributions and their spatial distributions with time. In addition, surveys before and after damaging natural events such as hurricanes (Stoddart, 1974; Shinn, 1976; Rogers et al., 1982), low temperatures (Shinn, 1976), and low tides (Loya, 1972) provide some insights into reef recovery and development.

TABLEI. LONG-TERM SURVEILLANCE OF NATURAL DISTURBANCES ON REEFS Time span

Environmental history

British Honduras

1964- 1966

Heron Island, Australia Key Largo, Florida

1963-1970

Humcane damage (1961) Hurricane damage (1966) Hurricane damage (1960)

Key Largo, Florida

1965-1967

St. Croix, U.S. Virgin Islands

1978-1979

Site

Gulf of Eilat

1950-1965

1970

Repeated hurricane damage (1965) Humcane damage ( 1979)

Catastrophic low tide

Major changes observed

Reference

Branching corals more susceptible than massive species

Stoddart (1974)

No marked change in coral abundance

Connell (1973)

Although colonies broken and much destruction within 1 year, difficult to recognize damage; by 1965 damage completely healed Damage not noticeable by 1967

Shinn (1976)

Effect of humcanes complex-may result in reduction in coral diversity but increase in community diversity due to provision of more light for slower growing corals and new substrate for algae and other invertebrates Change in the community structure with rare species affected

Rogers er a / . (1982)

Shinn (1976)

Loya (1972, 1976a)

Qatar, Persian Gulf

1965-1967

Low temperatures

Dry Tortugas, Florida St. Croix, U.S. Virgin Islands

1881-1976

Thermal shock (1976-1977) Bacterial infection

Carysfort Reef, Key Largo, Florida

MarchNo obvious natural November disturbances 1975 1973-1978 No obvious natural disturbances

CuraGao

John Brewer Reef, Australia Discovery Bay, Jamaica

1976-1979

1976-1980 1976-1980

No obvious natural disturbances Humcane Allen (1979)

Regeneration of Acroporu sp. after chill; 2 years later colonies 2-20 cm high Little change in area occupied by hermatypic corals; major changes were in coral species distributions Death of Acropora palmara as result of “white band disease” caused decrease in structural complexity of reef surface, decrease in living coral tissue and a reduction in CaC03 deposition on reef Estimates of net recruitment and mortality of reef corals suggested decline over 14-month study period Cover of living and nonliving components relatively constant throughout study; major differences lay in spatial arrangement of substrate components Net increase in colony abundance with a peak in recruitment in 1979 A trend of reduction in number of rarer coral species on the reef was reversed by Humcane Allen with storm-induced mortality being greatest in the most abundant species (Acroporu spp.)

Shinn (1976) Davis (1982) Gladfelter (1982b)

Dustan (1977)

Bak and Luckhurst (1980) Done (1981) Porter et a / . (1981)

1 FIG.

0.1 m

i

I . Portion of transect on intertidal reef affected by sedimentation where new recruits and colonies affected by partial mortality

and subsequent regeneration are difficult to distinguish (Brown, unpublished).

EFFECTS OF STRESS ON REEF CORALS

9

Studies of strictly long-term changes on coral reefs, however, are limited to those of Dahl and Lamberts (1978) and Davis (1982), who reassessed transects established 56 and 85 years before these recent surveys, respectively. In Dahl and Lamberts’ study in American Samoa, where results of dredging and cannery effluents were suspected of exerting influences on Arua Reef between 1917 and 1973, the total number of coral heads decreased by 28% during this 56-year period. Although the same genera were dominant in 1917 and 1973, the relative proportions of each differed in 1973 from that recorded in 1917. These authors conclude that the status of the reef in 1973 may reflect gradual recovery, or alternatively, a reef subject to intermittent “stress/sfresses.” Although little change (6%) in the overall area occupied by corals was noted in Dry Tortugas between 1881 and 1976 (Davis, 1982) and in Curaqao between 1973 and 1978 (Bak and Luckhurst, 1980), a major difference in the distribution and spatial arrangement of major coral species was observed in both studies during these time intervals. In the Dry Tortugas (Davis, 1982) in 1976 a lush Acropora ceruicornis (Lamarck) reef occupied what had been an octocoral-dominated region in 1881, while a considerable area of Acropora palmata (Lamarck) on the reef crest in 1881 was reduced to 600 m in 1976. During the relatively short time span of 5 years at Curaqao (Bak and Luckhurst, 1980) the combined effects of settlement, growth, dislodgement, and death of corals, coupled with variations in sedimentation, resulted in considerable temporal instability of both living and nonliving components. Generally shallow reefs show less short-term stability and lower predictability than deep reefs (Loya, 1976a; Bak and Luckhurst, 1980), the latter study demonstrating considerable change in spatial arrangement of substratum components and less stability at depths of 10-20 m when compared with deeper sites at 30-40 m. During the 5-year period of their study, Bak and Luckhurst comment on the constancy of coral cover in the reef habitats studied as compared with the shallow reef at Heron Island, Australia, where the area covered by living coral varied by a factor of 2-3 over 7 years of study (Connell, 1973). Similar variability was recorded by Glynn (1976) in Panama, where coral cover was observed to decrease by a factor of 2.5 in 15 months. C. Effects and Apparent Lack of Effects of Pollution on Coral Reefs Table I1 documents selected examples of recent studies on the effects of man-made disturbances on coral reefs. While studies on the effects of chronic oil and mineral pollution (Fishelson, 1973; Loya, 1975, 1976a), thermal pollution (Jokiel and Coles, 1974), sewage (Walker and Ormond,

TABLE11. EFFECTS,OR LACKOF EFFECTS,OBSERVED IN Pollutant

Site

Time scale

“Suspected” man-made influences (possibly dredging and cannery effluent)

Arua Reef, Samoa

191 7- 1973

Sedimentation from change in agricultural practice

Low Isles, Australia

1928-1978

Dredging and increased sedimenta. tion

Castle Harbour Bermuda

1974

Dredging

Piscadera Bay, CuraGao

1972

Dredging Kaolin clay spill

Diego Garcia Hawaii

Offshore oil drilling

N.W. Palawan Island, Philippines

1980

1981

THE

FIELDFROM “SUSPECTED”POLLUTION Species

Reference

Acroporu continued to be important throughout; Psummucoru reduced 213. Pocilloporu increased 5 x , Poritc7.s trndrcwsii reduced (three genera missing in 1973-Merrtlinrr. Goniopor~r , Cyplirist r-eu) Reef flat with few surviving corals but holothurians very abundant

Dahl and Lamberts (1978)

Diploria strigoscr more susceptible to sedimentation than Diplorio lubyrintirifortnis; most susceptible would appear to be Stepliancoeniu tnichilini Porites ustreoides (plating form) died as a result of inability to reject sediment; calcification rates of Modrueis mirubilis and Agoriciu oguricites decreased by 33% over 4-week period at least Coral diversity unaffected by dredging Corals survived discharge, although some Pocilloporrr meondrinu were temporarily bleached: coral cover in area dominated by P . meundrinu and Porites lobatu Massive species (e.g., P . Irrteu) appear to have survived in preference to branching species (Pocil/oporo, A croporu) which showed est imated 70-9096 reduction in nonstained area around wellheads

C. M. Yonge, personal communication Dodge and Vaisnys ( 1977)

Bak (1978)

Sheppard (1980) Dollar and Grigg (1981)

Hudson ct 01. (1982)

Experimental shading

San Cristobal Reef, S.W. Puerto Rico

Chronic oil pollution (and mineral dust)

Gulf of Eilat

1966-1972

Chronic oil pollution

Gulf of Eilat (Nature Reserve)

1966- I972

Thermal pollution

Kahe Point, Oahu, Hawaii

1971- 1972

Sewage pollution

Gulf of Aqaba

1979

A . ceruicornis most susceptible to partial shading; A . uguricires, Montustreu unnuluris, D. labyrinthiformis, Siderustreu sidereo, and Colpophylliu nutuns show bleaching and variable recovery; no visible response in Eitsmiliu fustigiutu, Montustreo cuuernosa,

or Mussu ungulosa Seriutoporu AcroPorU These genera were all reduced Stylophora Milleporu dichotomu: unchanged Nature reserve colonized after low tide by: P . luteu (16 colonies); M . dichotomu (8 CO~Onies); C . microphrhulmu (7 colonies); Fuvia favites ( 5 colonies); Acanthastreu echinata (2 colonies); Stylophoru pistillatu and C . microphtholmu suffered Order of resistance to high T" from field observations: strongly resistant-~epra.~rreu purpurea, Porites compressu, P . lobatu, Montiporu pufula, and Montiporu uerrucosu; least resistant-P. meandrinu Only surviving coral species was S . pistillutu at polluted site, while control site displayed Fuvia spp., Fuvites spp., Seriutoporu hystrix, Pocilloporu dunue, and S . pistillutu

I

Rogers (1979)

Fishelson (1973)

Loya ( 1976a)

Jokiel and Coles ( 1974)

Walker and Ormond (1982)

(continued)

TABLE11. (CONTINUED) Pollutant

Site

Time scale

Rotenone derivative (fish-collecting chemical)

E. Sambo Reef,

Recreational activities

Biscayne National Park, Florida

1977-1980

Heavy metal pollution and sedimentat ion

Intertidal reef Rats, Phuket, Thailand

1979

1973-1974

Florida

Species Octocorals apparently less susceptible than scleractinians; for the rotenone derivative, A . ceruicornis was more resistant than A. pulrnura, S.siderecc, D.strigo~ci.or Dichocoeniu stokesii; for quinaldine (generally less toxic to all scleractinia) A. qyiricites proved to be the most susceptible to the chemical Scleractinian damage greatest in branching species A . ceruicornis, A . pcclnzutrc, and P. porifes: Millepora highly susceptible; generally soft corals suffered more than scleractinians No apparent effect upon coral diversity and coral cover at site affected by heavy metal pollution, although increased incidence of partial mortality of faviids suspected at this site

Reference Jaap and Wheaton (1975)

Tilmant and Schmahl (1983)

Brown and Holley 1982; Brown (unpublished)

EFFECTS OF STRESS O N REEF CORALS

13

1982), fish-collecting chemicals (Jaap and Wheaton, 1975), dredging (Dodge and Vaisnys, 1977; Bak, 1978), and recreational activities (Tilmant and Schmahl, 1983) clearly show the impact of these disturbances on corals in the field, there are also a number of studies which demonstrate apparent lack of serious damage as a result of man-made interference. Such studies include the limited effects of a major spill of 2200 tons of kaolin clay on a reef in the Hawaiian Islands (Dollar and Grigg, 1981), of elevated metal levels from tin smelting and tin dredging activities on intertidal reef flats at Phuket, Thailand (Brown and Holley, 1982), of dredging activities in Diego Garcia Lagoon (Sheppard, 1980), and of drilling muds in the Palawan Islands, Philippines (Hudson et al., 1982). It may be argued that application of more detailed and longer term survey techniques may yet reveal subtle changes in the community structure at these sites. Nevertheless, no major deterioration in reef structure was evident in any of these examples. The possible reasons for this apparent lack of effect have been documented by the authors concerned. In the case of the kaolin spill, factors contributing to the lack of extensive damage were cited as the nontoxic nature of the kaolin, the small particle size of the clay, the presence of a wetting agent, and the rapid dispersal of the kaolin plume. In addition, rapid removal of sediments by coral cleansing aided the recovery process in the majority of coral colonies which were lobate and branching and hence less likely than platelike varieties to suffer heavy mortality (Dollar and Grigg, 1981). Apparent lack of effect of tin smelting and tin dredging processes at Phuket were ascribed to the possible reduced “biological availability” of toxic metals to corals, the general tolerance of intertidal reef species to stresses, and the possible acquisition of specific metal tolerance mechanisms by the corals themselves (Brown and Holley, 1982). At Diego Garcia dredging during the last decade was probably short term and limited in extent, any resulting damage being overcome by rapid recovery (Sheppard, 1980). Limited damage to branching corals only (an area 115 X 85 m2) was recorded in the vicinity of wellheads around Palawan Island, but the authors (Hudson et al., 1982) conclude that drilling mud probably constitutes a minor threat to coral growth under the conditions described in the study. In terms of overall tolerance of reef corals to disturbance in the field, the literature contains several references inferring the likely ability of intertidal and shallow-water corals to withstand physical stresses (Edmondson, 1928; Loya, 1972; Kojis and Quinn, 1981). Some authors also suggest that reef flat corals differ from deeper water species not only in

14

B. E. BROWN A N D L. S . HOWARD

increased physical tolerances, such as exposure to elevated temperatures (Jones and Randall, 1973), but also in reproductive strategies which they have evolved to reduce planktonic life to a minimum and to retain larvae on the reef (Stimson, 1978). Not surprisingly, then, it would appear that corals from shallow-water environments are more likely to be tolerant of environmental extremes, a finding reflected by Hudson (1981) on transplanting Montastrea annularis (Ellis and Solander) from a deep-water to a shallow-water location in the Florida Keys. The transfer resulted in severely reduced growth rates and mortality in one case. In contrast, transplantation of inshore M. annularis to offshore sites produced only slightly reduced growth rates compared with resident colonies. Should such increased environmental tolerances exist in shallow water corals, then there may be parallels with estuarine species in temperate ecosystems which have been described as “preadapted” to pollution stresses (Jones, 1975; Reeve et al., 1976). Indeed, Jokiel and Coles (1974) describe the Caribbean reef coral Siderastrea siderea (Ellis and Solander) as a “hardy estuarine coral” capable of establishing itself within a zone of maximum thermal effect around a thermal power plant in Florida during the winter and spring months of 1971. Although there may be some overall pattern in increased tolerances to stress shown by corals from different reef habitats and even varying geographical locations (Coles and Jokiel, 1977, having demonstrated lethal temperatures for Enewetak corals to be 2-5°C higher than for Hawaiian corals), Table I1 highlights the variability in response of different corals to the same stress at any one individual site. Considering, for example, temperature effects, Mayor (1914, 1918) was the first worker to note the ability of coral species to resist high temperatures in laboratory tests was inversely related to their metabolic rate. Since this date, Jokiel and Coles (1974) have camed out field observations in Hawaii, and their results confirm that this generalization also holds in the field-the most temperature-resistant coral being the large polyped species Leptasirea purpurea (Dana) with a low metabolic rate, and the most sensitive, Pocillopora meandrina (Dana) with a high metabolic rate. Recent observations by Neudecker (1981) in Guam showed Pocillopora damicornis (Linnaeus) to be more sensitive to thermal stress than Porites andrewsii (Vaughan), the most sensitive species being Acropora formosa (Dana). In the latter study, differences in temperature tolerance were considerable, colonies of P . andrewsii surviving up to 77 days in elevated (4-6°C above ambient) temperatures, whereas A . formosa generally died within 2 days. Whereas Pocillopora is cited in many studies as being relatively sensi-

EFFECTS OF STRESS ON REEF CORALS

I5

tive to stress in the form of increased temperatures, some authors (Dahl and Lamberts, 1978) describe P . dumicornis in American Samoa as “most tolerant of adverse conditions, being found near shore where there is silt and fluctuating water temperatures.” Since Mayor’s (1924) original transect at this site in 1917, P . dumicornis had increased fivefold, while P . andrewsii, once dominant in the midzone at Arua transect, was considerably reduced. Factors suspected of affecting the reef included dredging activities and discharge of cannery waste. Increased sedimentation resulting from drilling processes at Palawan Island, Philippines (Hudson et al., 1982), caused branching corals (including Pocillopora sp.) considerable mortality when compared with head corals such as P . futeu; Dollar and Grigg (1981) also describe short-term effects of a kaolin spill affecting P . meandrinu but cite no short-term damage in P . lobata as a result of sedimentation. Clearly, then, tolerance of Pocilloporu to either increased temperature or sedimentation, as described in Dahl and Lamberts’ study (1978), would appear to be at variance with observations of the above authors. It is, of course, acknowledged that there are probably few cases where a single factor is responsible for damaging effects observed in the field. Increased sedimentation, for example, will present at least three problems to exposed corals. These are decreased light values, increased energy-consuming processes such as sediment cleansing, and possibly reduction in planktonic food (Bak, 1978). One feature which does appear to be consistent throughout most studies involving sedimentation and/or shading is the particular susceptibility of branching corals to these stresses when compared with massive species. Considering shading alone, such observations would be in line with Porter’s (1976) conclusions that branching corals with small polyps may depend more upon light than upon planktonic capture and so are less able to withstand reduced light intensities than massive corals (Rogers, 1979), though evidence for such resource partitioning is now questioned (Rosen, 1982). With respect to sedimentation, branching corals are very effective in passive rejection of sediment because of their colony morphology (Hubbard and Pocock, 1972; Bak and Elgershuizen, 1976), and as Rogers (1979) showed, the branching Caribbean coral A . ceruicornis was unaffected by daily exposure to sediments. Plating colonies of Porites ustreoides (Lamark), however, which are reported as inefficient sediment rejectors (Bak, 1978), were unable to reject sediments resulting from dredging activities at Curasao and either wholly or partially died. Damage due to sedimentation summarized in Table I1 may therefore be variously interpreted. Where branching corals are observed to have suf-

16

B. E. BROWN A N D L . S . HOWARD

fered (Dollar and Grigg, 1981; Hudson et al., 1982) it may be speculated that light levels were sufficiently reduced to induce damage. Where plating varieties, e.g., P . astreoides, suffered more than branching species, e.g., Madracis mirabilis (Duchassaing and Michelotti) (Bak, 1978), it is likely that lack of sediment rejection capabilities proved to be more lethal than reduction in light intensity and subsequent zooxanthellae loss. As Jokiel and Coles (1977) point out, zooxanthellae loss may be temporary and reversible, bleached corals recovering within 2 months of return to normal conditions. In considering the response of a branching coral such as Pocillopora to temperature increase or sedimentation, it becomes apparent that generalizations must be applied with care when attempting to predict the response of an individual species to a pollutant or indeed the effect of a pollutant on a reef community. One further example will serve to demonstrate the need for more critical data on responses of corals to pollution. Stylophora pistillata (Esper) has been described as an opportunistic or “weedy” species, a typical colonizer of unpredictable environments (Loya, 1976a,b) and polluted habitats (Walker and Ormond, 1982). The latter study demonstrated that S. pistillata was the only surviving coral species on a reef flat in Aqaba affected by sewage and phosphate pollution. Fishelson (1973) also showed that Stylophora was apparently relatively resistant to oil pollution and phosphate dust at Eilat, the coral representing 47% of all branching species in 1966 and 73.7% of the sample in 1968. The combined effects of a low tide and chronic oil pollution, however, severely reduced recolonization by S. pistillata at the nature reserve at Eilat, the coral previously having been dominant at this site (Loya, 1975). In subsequent surveys, P . lutea proved to show more successful recolonization than Stylophora, with a relatively large number of small-sized colonies being recorded. P . lutea, however, could hardly be described as an opportunistic species; according to Highsmith (1982), the life history characteristics of P . lutea include a high growth rate, large adult size, a long life expectancy, but no apparent release of larvae. So, in contrast to Endean’s speculation that opportunistic species should be well represented among early colonizers of polluted habitats (Endean, 1973), in this instance-the only detailed study of its kind to date-such a hypothesis does not appear to hold. The reproductive biology of the adult and the settling behaviour of the larvae of Stylophora are detrimentally affected by exposure to oil (Rinkevich and Loya, 1977, 1979a), but no similar information is available for P . lutea, so it is impossible at this stage to say where the observed tolerance in the latter species lies.

EFFECTS OF STRESS ON REEF CORALS

17

D. Predicting Recouevy of Reefs Short-term phenomena on reefs are highlighted in Davis’ study (1982) on Dry Tortugas, with the destruction of 90% of an extensive A. ceruicornis stand in 1976-1977 as a result of lowered seawater temperatures in January, 1977. A . cervicornis, however, is known to show rapid recovery rates after partial destruction (Shinn, 1976). A growth rate of 10 cm/year, combined with a geometrical progression of branch formation, has been attributed to the short-term recovery (5 years) of A. ceruicornis reefs after storm damage in the Florida Keys (Shinn, 1976). Speedy partial recovery of Acropora sp. in Qatar, Persian Gulf, 2 years after death due to lowered seawater temperatures has also been reported by Shinn (1976). Highsmith (1982) concludes that S ~ recovery W occurs when disturbance is so severe that hardly any fragments of reef-building corals survive and when survival depends upon sexual reproduction, rapid recovery ensuing when asexual reproduction and regeneration are possible. Such conclusions are supported by the long-term recovery (10-20 years) of A. cervicornis in Belize after suffering high mortality as a result of Hurricane Hattie (Stoddart, 1974) and the relatively short-term recovery of A. cervicornis in studies of limited reef damage in Florida cited earlier (Shinn, 1976). In other instances, however, high rates of recruitment have been attributed to rapid recovery of reefs (Loya, 1975, 1976a), but care must be taken in noting the time scale of such “rapid” recovery. Initial recolonization of reef flats at Eilat between 1969-1973 was shown to be 23X greater on a control reef when compared to a reef affected by chronic oil pollution (Loya, 1976a), although by 1973 there was still a significantly lower coral cover on the control reef compared with the initial survey in 1969. It is interesting to note that the dominant corals on reef flats at Eilat, e.g., Stylophora and Cyphastrea, are species cited as showing life histories in which sexual reproduction is predominant over asexual reproduction, and therefore possibly a slower recovery when compared with corals reproducing primarily by asexual methods (Highsmith, 1982). Corals with high rates of recruitment have been observed to be among the most common species on submerged lava flows in Hawaii, where 20-50 years were required for recovery (Grigg and Maragos, 1974), and at Heron Island, Australia (Connell, 1973). Such a correlation was absent in CuraGao and Bonaire (Bak and Engel, 1979), where common species such as S . siderea, M . annularis, and Montastrea cauernosa (Linnaeus) had very few recruits. The authors suggest that such a finding may indicate a higher level of environmental disturbance on the examined shallow reefs in the Pacific when compared with those studied in the Caribbean.

18

B . E. BROWN A N D L . S . HOWARD

The importance of considering recruitment rates as only part of the overall life history strategy of a coral when attempting to explain distribution and abundance patterns has been shown in recent studies (Bak and Engel, 1979; Bak and Luckhurst, 1980). Two of the most common corals on the reef slopes of Curaqao are Agaricia agaricites (Linnaeus) and M . annularis. Agaricia displays a high recruitment rate, a low rate of survival, and a high mortality, whereas Montastrea combines a low recruitment rate, moderately good survival, and low mortality (Bak and Engel, 1979). Clearly, then, life history strategies of individual corals are all important in determining the rate of recovery of a reef after disturbance. Interpretation of data is further complicated by species exhibiting different life history features in different geographical locations. Highsmith (1982) describes P . darnicornis in Hawaii as a fugitive species, competitively subordinate, with a low growth rate, small adult size, and noted for production of planulae larvae. However, in Panama it is the major reef builder, competitively dominant, with a high growth rate and described as rarely showing recruitment of planulae. Contrasting characteristics are similarly shown in geographically isolated Porites haddoni (= P . lutea), which is reported as planulating from January to June at Low Isles, Australia, by Marshall and Stephenson (1933); no planulation, however, has been observed in P . futea at Enewetak (J. S. Stimson, personal communication, in Highsmith, 1982). As mentioned earlier, the depth of the reef may be important in assessing damage and also in predicting recovery. Hurricane damage has been reported to depths of 20 m (Highsmith et al., 1980; Luckhurst in Bak and Luckhurst, 1980), although damage is generally considered to be greatest in shallow waters (see Endean, 1971, 1973, for reviews). Shallow-water habitats, however, may not always show the greatest effects of hurricanes, as recent studies demonstrate. Rogers et al. (1982) describe the number of broken branches of A . palmata per metre as decreasing with depth (0.6-6.1 m) as a result of hurricane damage at St. Croix, U.S. Virgin Islands, but whereas shallow branches broke only at their distal ends, exposing relatively little surface area for healing, deeper branches broke at their bases and consequently exposed much greater surface areas for healing and recolonization. Generally the larger branches took longer to heal, the healing process being more effective in fractured small branches which predominated in shallow water. Theoretical aspects of recovery of coral reef communities devastated by catastrophic events have previously been reviewed by Endean (1973). At that time Endean speculated that in cases of extreme disturbance, opportunistic species with a high fecundity might be expected to be well

EFFECTS OF STRESS ON REEF CORALS

19

represented among the early colonizers; that substrata for colonization might be successfully invaded by benthic organisms other than corals such as algae and alcyonarians; that mortality of juvenile corals might be high; and that coral growth could be retarded, so increasing the time taken for the newly established coral to grow to maturity. Considering that Endean published his review 10 years ago, it is interesting to reflect how his speculations have been borne out by recent work. Earlier discussions in this article would suggest that while opportunistic species with a high fecundity might be found in polluted environments (Loya, 1976a; Walker and Ormond, 1982), they are not necessarily well represented among early colonizers in certain polluted conditions (see Section II,C of this article). Colonization of substrata by algae rather than corals has been observed in areas receiving sewage discharge such as Kaneohe Bay (Smith, 1977) and Aqaba (Walker and Ormond, 1982). Although algal growth was greatly stimulated in polluted areas at Aqaba, the authors maintained this factor was not the direct cause of coral death. They suggested that enhanced algal growth, stimulated by increased nutrient concentrations, may have acted as a sediment trap, thus exposing corals to a considerable sediment load. Other workers (Benayahu and Loya, 1977), looking at reef flats affected by periodic low tides at Eilat, have shown that resulting mass mortality of benthic communities opens up new spaces for settlement. Such unpredictable disturbances are believed to prevent potential dominant competitors from monopolizing the available space, the observed coexistence of stony corals, soft corals, and algae being due to different environmental tolerances and competitive abilities of each group. More recently, useful papers on space monopolization by some of the less well-known groups such as the soft corals have appeared in the literature (Samrnarco et af., 1983; Tursch and Tursch, 1982). Studies on the mortality ofjuvenile corals are still limited. The work of Connell(l973) and Bak and Engel (1979) suggests that on reefs unaffected by human disturbance, approximately 36 and 32% of the juvenile corals died or disappeared during the 1I-month and 6-month study periods, respectively. Bak and Engel cited causes of mortality as sedimentation and competition from coralline algae on shallow reefs and possibly random grazing and/or direct predation by parrot fishes on the reef slope. Another third of the juvenile population in this study were described as limited in growth by factors such as spatial competition, which was similar at all depths. Mortality of juvenile corals in polluted environments is unknown, apart from related work by Rinkevich and Loya (1977) on the effects of crude oil on planulae and juveniles of S. pistillata. Effects observed in the laboratory included a decrease in the viability and successful settlement

20

B . E. BROWN A N D L . S . HOWARD

of the planulae, which was manifest in the field by a limited recolonization by this coral at the polluted Nature Reserve at Eilat. It is clear that we are still lacking much fundamental information on aspects of recovery and recolonization of reefs (Pearson, 1981). From the limited knowledge we have gained during the last 10 years, it would seem that both generalizations and predictions are dangerous and that until more evidence is forthcoming we should consider each case individually. Recent work by Grigg (1983) suggests that disturbance is a primary mechanism governing diversity, community structure, and succession of coral reefs in Hawaii. Furthermore, Grigg depicts the effects of disturbance occurring at different stages of successional processes on coral reefs and concludes that in Hawaii, reef community structure is primarily a function of the interaction between disturbance and recovery time.

111.

Experimental Studies on Effects of Pollutants on Corals

The tolerance of scleractinian corals to factors such as increased temperature and sedimentation was first studied over 50 years ago by Mayor (1914, 1918), Edmondson (1928), and Marshall and Orr (1931). These early workers established the broad tolerances of a variety of coral species to physical disturbances which might be encountered in the field. Experiments were largely performed in the laboratory, where mortality was used as a measure of tolerance, though Mayor and others (Vaughan, 1915) saw the value of actually transplanting corals to a variety of environments and measuring growth rate and survival in siru as a reflection of environmental quality. Despite the short-term nature of laboratory experiments and their shortcomings, a variety of responses have been monitored, both in the laboratory and experimentally in the field, by exposure of corals to a wide selection of chemical and physical parameters (Tables 111-VIII). These responses are discussed below.

A. Growth Rate Growth rate of corals has been cited as one of the best quantitative measures of testing stress due to a disturbance since this parameter integrates a variety of physiological processes (Birkeland et al., 1976; Neudecker, 1983). It is also widely accepted, however, that coral growth rates may be inherently variable (Buddemeier and Kinzie, 1976; Barnes and Crossland, 1982) for a single species within reef zones (Gladfelter et al., 1978) and

EFFECTS OF STRESS ON REEF CORALS

21

even within individual colonies (Rogers, 1979; Brown et al., 1983). Gladfelter et al. (1978) have described some species as “conservative” in their growth whereas others are not. They cite M . annularis as showing relatively little response in growth rate to varying environmental conditions, while A . ceruicarnis shows marked variations under similar circumstances. Methods employed to measure growth rates of corals have been reviewed by Buddemeier and Kinzie (1976) and Gladfelter et al. (1978). Table I11 illustrates that the majority of studies, in which growth rate has been used as a parameter to measure the effect of disturbance, have involved either x-radiography (Dodge and Vaisnys, 1977; Hudson, 1981, 1983; Hudson and Robbin, 1980; Hudson et al., 1982), reference marking by alizarin red S stain or a fixed base line (Shinn, 1976; Rogers, 1979; Dodge, 1982; Bak and Criens, 1983; Neudecker, 1983), measurement of an increase in skeletal weight (Jokiel and Coles, 1977; Bak, 1978), or 4SCa deposition rate in the skeleton (Neff and Anderson, 1981). The use of x-radiography, in the above context, has been applied solely to massive corals such as P . lutea (Indo-Pacific) and M . annularis, Diploria strigosa (Dana), and Diploria labyrinthiformis (Linnaeus) (Caribbean). Significant suppression of coral growth as a result of disturbance has been observed using this method during short-term exposure of M . annularis to “extremely high” concentrations of drilling mud (Hudson and Robbin, 1980). Inhibition of coral growth was also obtained on transferring M . annularis from an offshore location to a more stressful inshore site (Hudson, 1983) and in D . strigosa and D . labyrinthiformis as a result of dredging in Bermuda (Dodge and Vaisnys, 1977). No suppression of growth was observed in M . annularis and P . lutea as a result of bombing activities at Vieques, Puerto Rico (Dodge, 1983) and drilling processes off N.W. Palawan, Philippines (Hudson et al., 1982), respectively. Results of the latter study, however, showed a 70-90% reduction in area coverage of branching coral species around the wellhead. Death of these low-profile corals was believed to be due to smothering by a prolonged and localized buildup of cuttings, surviving corals being primarily massive head corals in an elevated position above the bottom. Such differences between apparent tolerances of branching and massive species of coral emphasize the need for sensitive methods of assay. In long-term growth studies of M . annularis from the East Flower Gardens, Texas (Hudson et al., 1982), where exploratory drilling sites have been set up in recent years, and also at sites within the Key Largo coral reef marine sanctuary, Florida (Hudson, 1981), a decline in growth rates has been observed. However, in both studies the authors cannot directly attribute apparent growth suppression to any single environmen-

TABLE111. THEUSE OF GROWTHRATE TO ASSESSTHE EFFECTSOF DISTURBANCE ON CORALS IN A N D EXPERIMENTALLY I N THE FIELD Criterion: growth rate

As determined by x-ray analysis

Nature of study and location Analysis after field collection, N.W. Palawan, Philippines Analysis after application of drilling mud by divers to transplanted specimens, Florida Keys, USA, and after field collection, Texas Analysis after transplanting specimens, Florida Keys, USA Analysis after field collection, Castle Harbour, Bermuda Analysis after field collection, Vieques, Puerto Rico Analysis after field collection, Florida Keys, USA

Species

Disturbance

P. lutea

Drilling activities

M . annularis

Drilling muds

M . annularis

THE

LABORATORY

Results

Reference

Little apparent suppression of growth due to drilling Possible decrease in growth shown (though other factors may be responsible)

Hudson et a / . (1982)

Change of habitat from offshore to inshore site Dredging activities

Reduced growth rate and deposition of dense skeleton Decline in growth prior to death

Hudson (1983)

M . annularis

Military bombing activities

Apparent lack of effect

Dodge (1983)

M . annularis

Increased dredge and refill operations (?)

Decline in coral growth (1953-1968) at some midshore and inshore reef sites

Hudson (1981)

D . strigosa and D . labyrinrhifiormis

Hudson and Robbin (1980)

Dodge and Vaisnys (1977)

As determined

by alizarin S stain

Analysis after transplanting corals to site of thermal enrichment, Guam

P . andrewsii, P .

Experimental quadrats in the field, CuraCao

M . mirabilis, A . palmata, A . ceruicornis M . annularis

Laboratory experiment

damicornis, A . formosa

As determined by skeletal growth from a baseline

Analysis after shading and application of sediments to corals in experimental channels and on the reef, San Cristobal, Puerto Rico

A. ceruicornis

As determined by weight of skeletal growth deposited

Laboratory experiment using corals from a Hawaiian reef

P . damicornis, Montipora uerrucosa, Fungia scutaria

Thermal pollution 4-6°C above ambient

Fragmentation

1, 10, 100 ppm drilling mud doses for 6 weeks Experimental shading and sedimentation (receiving up to 800 mglcm2 and 200 mgicm2 once a day, once a week, and once a month during 40day period) Temperature increase

Coral growth impeded by higher temperature; suggests slower growing species more tolerant of high temperature than faster growing species M . mirabilis grew significantly more slowly after fragmentation Skeletal extension declined significantly in 100 pprn treatment

Neudecker (1983)

Shading significantly affected growth rate but no observed effect on growth rate as a result of exposure to sedimentation

Rogers (1979)

Exposure of corals to temperatures of 30°C reduced calcification

Jokiel and Coles (1977)

Bak and Criens (1981) Dodge (1983)

(continued)

TABLE111. (CONTINUED) Criterion: growth rate

As determined by "Ca incorporation

Nature of study and location

Species

Observations in the field, CuraGao

Selection of Caribbean corals

Dredging

Laboratory experiment

M . annularis

Drilling mud 1, 10, 100 ppm doses for 6 weeks

Laboratory experiment

Millepora sp., Madracis decactis, M . annularis, Oculina diffusu, Favia fragitrn

Water-soluble fractions of fuel oil and Louisiana crude oil

Disturbance

Results Exposure to increased turbidity and sedimentation caused a decrease in calcification rates of M . rnirabilis and A . agaricites Calcification rates decreased at 100 ppm dose after 4 weeks' exposure Variable results with no indications of a significant effect of hydrocarbons on 4SCaincorporation

Reference Bak (1978)

Szmant-Froelich et al. (1981)

Neff and Anderson (1983)

EFFECTS O F STRESS ON REEF CORALS

25

tal disturbance, although reduced growth rates in the Florida Keys coincide with a period of dredge and fill operations. Dodge and Lang (in Dodge and Szmant-Froelich, 1984) suggest that the decline in coral growth at the Flower Gardens may be due to water temperature fluctuations and increasing river discharge in the area. Another feature revealed by x-radiography is the presence of highdensity skeletal deposits or “stress” bands which have been observed in sections of M . annularis during periods of rapid chilling and mixing of shallow inshore waters (Hudson et a f . , 1976; Hudson, 1977, 1981) (Fig. 2). In addition, Highsmith (1979) has noted that, in M . annulavis from Belize, the high-density bands appear to be deposited for only short periods of

2.2 cm FIG.2. “Stress bands” revealed in sections of M . annularis exposed to periods of chilling and mixing of shallow inshore waters. B and C indicate the boring activities of sponges (Hudson, 1977).

26

B . E . BROWN A N D L. S. HOWARD

time while the low-density band is produced for a greater part of the year when compared with M . cavernosa and P . astreoides from the same locality. Highsmith attributes this difference to the contrasting distribution pattern of the corals ( M . cauernosa and P . astreoides being relatively restricted with respect to the broader tolerances of M. annularis), which may be reflected in the density banding pattern. It would seem, then, that massive corals living under similar environmental conditions are likely to reflect environmental variables to different degrees and that, while M . annularis is widely used in sclerochronological techniques because of the clarity of its banding pattern and wide distribution, other massive corals may be more sensitive indicators of changing environmental conditions. One alternative suggested by Hudson (1981) is S . sidereu which, although environmentally tolerant, does have a very close banding pattern enabling test cores to record longer time periods when compared with M . annularis. Whether S . siderea reflects lesser or greater sensitivity to environmental change than M . annularis remains to be seen. Reduction in growth rate of branching corals as a result of thermal discharge (Neudecker, 1983) and fragmentation (Bak and Criens, 1983) has been observed using alizarin staining. Neudecker concluded that slower growing coral species were more tolerant of high temperatures than faster growing species, the fastest growing coral in his study being A . formosa, extending at a rate of 4.9 k 0.3 mm, while the slower growing coral P . andrewsii grew at a rate of 4.2 2 0.2 mm over the same 63-day period. No significance values were attributed to this comparison, and since there were no measurements of the weight of calcium carbonate deposited or the density of the skeleton laid down, care must be taken in interpreting the data as suggesting slower growing corals are more tolerant of thermal enrichment than faster growing species. Measurements of skeletal growth (as weight of CaC03 deposited) in Hawaiian corals exposed to increased temperatures (4-5°C) did not indicate similar results (Jokiel and Coles, 1977). The order of increasing thermal tolerance was P . damicornis < Montipora verrucosa (Lamarck) and Fungia scutaria (Lamarck), while M . verrucosa calcified most rapidly and F . scutaria least rapidly of the three corals tested. Rogers (1979), in her estimation of the effects of sedimentation on growth rate in A . cervicornis (determined by measuring skeletal growth from a base line), stresses the importance of making adequate measurements of branch extension on a large number of branches from different colonies of the same species before reliable data can be obtained. Results of this study indicated that even daily sediment doses of 200 mg/cm2for 45 days did not affect growth rates of treated corals when compared with controls.

EFFECTS OF STRESS ON REEF CORALS

27

In contrast, Bak (1978) demonstrated an acute decrease in growth rate (measured as a 33% decrease in calcification) of M. mirabilis and A . agaricites as a result of increased sedimentation from dredging activites in CuraGao. Depressed calcification rates were noted for more than 1 month after reduction in light levels and suggest that the decrease in growth is not just the result of reduced light but also of metabolic shock that exceeded the period of environmental disturbance. The effects of water-soluble fractions of a fuel oil and Louisiana crude oil on the rate of calcium deposition (measured as 45Caincorporation into the skeleton) in Millepora sp., Madracis decactis (Lyman), M . annuluris, Oculina diffusa (Lamarck), and Favia fragum (Esper) were quite variable, with sample variability being greater in hydrocarbon-exposed animals than in controls (Neff and Anderson, 1981). Such variability was attributed to the individual variation between colonies or parts of colonies in their sensitivity to oil, an explanation also favoured by Birkeland et ul. (1976) using coral growth as a parameter in assessing the effects of bunker oils on corals. Despite the variability encountered in growth rate data, it would appear that this parameter has considerable value in many field observations, particularly since both branching and massive species can be transplanted into different reef sites. B. Metabolism

A criticism of the use of metabolism as an indicator of stress in short-term experiments carried out in temperate waters has been the environmentally unrealistic levels of pollutants required to produce any effect (R. C. Newell, personal communication). In the limited number of experiments carried out with tropical scleractinians (Table IV) efforts have been made in many cases to carry out experimental manipulations in the field (Rogers, 1979; Dallmeyer et al., 1982), and in all examples cited some impairment of an aspect of metabolism has been noted as a result of experimental disturbance (Rogers, 1979; Dallmeyer et al., 1982; Szmant-Froelich et al., 1983). It is difficult to say, however, how experimental conditions in each case correspond to those observed in the field. Exposure of M . annularis to gradually increasing suspended peat levels of 175, 350, and 525 mg/litre may reduce photosynthesis during the day and oxygen levels during the night (Dallmeyer et al., 1982), but no indication is given in this article of the levels of peat in natural waters at Negril, even in the brown plume reported in the field (Dallmeyer et al., 1982). A need for improved information on levels of pollutants in the field and experimental designs that more accurately approach water quality conditions in situ has been

TABLE1v. THE USE

OF

METABOLISM TO

Criterion Metabolism Primary productivity and respiration Respiration, gross photosynthesis. NO1 UPtake, NH, uptake Respiration and net photosynthetic production Photosynthesis and respiration

Nature of study and location

EFFECTSO F DISTURBANCE O N CORALS EXPERIMENTALLY I N THE FIELD

ASSESS T H E

AND

Species

Disturbance

IN THE

LABORATORY

Results

Reference

Experimental channels in the field, San Cristobal

Caribbean corals

Experimental shading

Primary productivity and respiration decreased as a result of shading

Rogers (1979)

Laboratory experiment in Row-through seawater system

M . unnuluris

Exposure to 1. 10, and 100 ppm drilling mud for 6 weeks

Respiration and photosynthesis, NO3 and NH4 uptake all decreased as a result of exposure to 100 ppm drilling mud

Szmant-Froelich crl. (1983)

I n situ measurement of

M . unnuluris

Exposure to suspended peat

Reduced net oxygen production as a result of exposure to suspended peat

Dallmeyer et 01. (1982)

P . darnicornis, M . uerrucosa, P . compressa, Fungin scutariu

Thermal increase

Coral metabolism closely adapted to ambient T o conditions; results suggest lethal temperatures for Enewetak specimens to be 2-5°C higher than for Hawaiian corals

Cotes and Jokiel (1977)

oxygen metabolism, Negril, Jamaica

Laboratory experiment, Hawaii and Enewetak

EFFECTS O F STRESS ON REEF CORALS

29

highlighted by Hudson et al. (1982) and Dodge and Szmant-Froelich (1984), respectively. In all examples cited, a key factor leading to reduced production as a result of shading (Rogers, 1979), exposure to drilling mud (SzmantFroelich et al., 1983), and exposure to suspended peat (Dallmeyer et al., 1982) was expulsion of zooxanthellae-a response which will be discussed in detail in Section II1,C. Short-term (1- to 2-h intervals) exposure of M. annularis to suspended peat concentrations of a maximum of 525 mg/litre resulted in a 50% fall in production and respiration rates when pre- and postexposure rates were compared over a 24-h period. Longer term exposure of M. annuluris to 100 mg/litre drilling mud in a flowthrough system for 4 weeks resulted in a 25% fall in respiration rate and a decline in gross photosynthesis of 75% when compared with controls after 5 weeks (Szmant-Froelich et al., 1983). Shading alone of a selection of Caribbean corals (including M. annularis) produced a fall in production of approximately 50% after cover of 4 weeks (Rogers, 1979). Recent work by Barnes (1983) may have some application to the study of stressed environments. Using a buoy equipped with pH and oxygen electrodes and a sensitive thermistor, he obtained measurements of changes in oxygen concentration, pH, and temperature of water across the reef flat, from which he deduced values for reef productivity and calcification. Barnes cites Kinsey (1979), who suggested that reef flats operate within narrow metabolic limits, any departures from these limits possibly reflecting perturbation. Once the respiratory and metabolic characteristics of reef communities are better understood, such a method as that described above may have a place in pollution studies. The technique has one distinct advantage over other “metabolism” studies in that it could be carried out in the field and potentially could give a direct measurement of the “health” of similar reef areas.

C. Loss oJ’ Zooxunthellur The loss of zooxanthellae from coral tissue has been described by several authors (Franzicket, 1970; Jokiel and Coles, 1974; Jaap and Wheaton, 1975; Neff and Anderson, 1981) as a useful indicator of stress. Discolouration of corals as a consequence of zooxanthellae release may result from natural factors such as elevated temperatures and low tides (Vaughan, 1916; Yonge and Nicholls, 1931; Jaap, 1979), decreased temperatures (Wells, personal communication, in Jaap, 1979), salinity changes due to storms (Goreau, 1964), and also laboratory-induced elevated temperatures, darkness, and starvation (Yonge and Nicholls, 1931). In addition, as illustrated in Table V, loss of zooxanthellae may result from man-made

TABLEv. Criterion Expulsion of zooxanthellae

LOSS OF

ZOOXANTHELLAE AS

Nature of study and location

A

RESULTOF MAN-MADE A N D NATURAL DISTURBANCES

Species

Disturbance

Results

Reference Coles (1975); Jokiel and Coles (1974); Jokiel and Coles (1977) Jaap and Wheaton (1975)

Field and laboratory observations. Hawaii

Hawaiian reef corals

Elevated temperatures of 2-4°C

Loss of zooxanthellar pig-

Application of pollutant by divers in the field at Western and Eastern Sambo. Florida

Caribbean reef corals

Exposure to quinaldine and rotenone derivatives

Field observations at CuraGao

Caribbean reef corals

Dredging activities

Field observations at Middle Sambo, Florida

Caribbean reef corals

Combined high temperatures and low midday tides

Experimental chambers in the field, San Cristobal, Puerto Rico

Caribbean reef corals

Shading for 4 weeks

A . ceruicmwis. A . pulmcttu. S . sidereu. D . strigosu. and Dichocoeniu stokesii showed bleaching as a result of application of chemicals P. astrvoidrs lost zooxanBak (1978) thellae and subsequently died Millrporcr complunutu Jaap ( 1979) displayed greatest discolouration; A. pulmutu, M . unnuloris, and Polvtlioo sp. were mildly discoloured A . c<jruic,ornis.A . t ~ g ~ r i c ' i - Rogers (1979) les, Millepora ulcicornis, M . unnuluris. D . lubyrinthiformis, S .

ment

A . ceruicornis

Sedimentation

Experimental chambers in the field. Carysfort Reef, Florida

Seven Caribbean reef species

Drilling mud

Field observations, Ha. waii

Hawaiian reef corals

Kaolin spill

Transplant of corals to varying depths, Discovery Bay. Jamaica Experimental studies in situ. Negril, Jamaica

M . einnirltrri~

Transplanting of coral to 10 and 30 m depth Addition of suspended peat to environmental chambers. during night and day Elevated temperatures 4-6°C above ambient

Transplant studies at sites of thermal enrichment, Guam

M . cinnirloris

sidereit. and C . n c i i o n s all show some zooxanthellae expulsion; large polyped species least affected Both controls and test colonies showed small areas of bleached tissue A . ceruicornis exposed to “mud 3” lost all zooxanthellae after 41 h: all tissue disintegrated within 52 h Expulsion of zooxanthellae in P. mcwndrino and Poc~illlporcievdomi Corals transplanted from 30 to 15 m showed loss of zooxanthellae Loss of zooxanthellae by corals exposed to peat

A . fornio.sii and P. dciniicornis showed loss of

zooxant hellae

Thompson (1980)

1’1

it/.

Dollar and Grigg (1981) Dustan ( 1979)

Dallmeyer et ril. (1982)

Neudecker (1983)

32

B. E. BROWN A N D L. S. HOWARD

disturbances such as thermal discharge (Coles, 1975; Jokiel and Coles, 1974, 1977; Neudecker, 1983), dredging (Bak, 1978), exposure to drilling mud (Thompson et al., 1980), kaolin (Dollar and Grigg, 1981), peat (Dallmeyer et al., 1982), quinaldine (Jaap and Wheaton, 1975), oil (Peters et al., 1981), shading (Rogers, 1979), and transplantations from deep- to shallow-water reefs (Lang, 1973; Dustan, 1979) and from offshore to inshore sites (Shinn, 1976). The loss of zooxanthellae in different coral species subject to similar stresses under the same environmental conditions is extremely variable; the response within an individual coral may even differ over the colony (Rogers, 1979; Neff and Anderson, 1981). Triggering mechanisms for zooxanthellae expulsion have been cited as reduced supplies of nutrients available to the algae from the stressed coral host (Yonge and Nicholls, 1931; Muscatine, 1971), decrease in space available to the algae caused by atrophied host tissues (Muscatine, 1971), and secretion of substances by the coral host which produce a hostile environment around the algae (Jaap, 1979). Such mechanisms do not seem a wholly adequate explanation of the phenomenon for at least two reasons. First, the response may take place very rapidly, loss of zooxanthellae being reported within hours by Jokiel and Coles (1974), Thompson et al. (1980), Dallmeyer et al. (1982), and Neudecker (1981), and within 5 min by Jaap and Wheaton (1975). Second, although space may decrease in atrophied host tissues (Muscatine, 1971), Peters et al. (1981) have shown that under such conditions many zooxanthellae actually degenerate in situ. Clearly this aspect requires further clarification; it may well be that the speed and intensity of polyp retraction following disturbance is sufficient to expel zooxanthellae via the coelenteron, differing responses observed in corals simply reflecting the intensity of the reaction. In two studies where other aspects of coral behaviour were noted, in addition to tissue colouration, bleaching generally followed a period of polyp retraction which continued for longer than 5 min posttreatment (Jaap and Wheaton, 1975) or for the whole 96-h period of the experiment (Thompson et al., 1980). The route taken during expulsion is via the absorptive zone of the mesenteries, just below the filaments (Fig. 3), although the actual stimulus that causes the algae to be conveyed is unknown. Some authors have described connections between zooxanthellae loss, polyp size, and other stressful environmental parameters (Rogers, 1979), but results obtained in other studies do not appear to show any consistent patterns (Goreau, 1964). In Goreau’s study (1964) of the effect of salinity changes, following Hurricane Flora, on 16 Caribbean coral species, M . annularis was observed to be the species most susceptible to zooxanthellae loss. In this study no correlation between tendency to lose zooxanthel-

EFFECTS OF STRESS ON REEF CORALS

33

FIG. 3. Transverse section through a portion of mesenterial filament of Goniu.s~rmsp. after exposure to elevated temperatures, showing zooxanthella in process of ejection. Abbreviations: az, absorptive zone; z, zooxanthella; ze, zooxanthella being ejected; m, mesogloea; gm, glandular margin (after Yonge and Nicholls, 1931).

lae and polyp size was observed. Other studies indicate rather variable results, with Millepora complanata (Lamarck) displaying a greater tendency to discolour over A . palmata and M . annularis as a result of ternperature stress at low tide; the latter two species, with small and mediumsized polyps, respectively, both showing medium discolouration (Jaap, 1979). P. astreoides displayed a marked loss of zooxanthellae as a result of dredging (Bak, 1978) when compared to other small polyped corals such as A . agaricites, and in other small polyped varieties A . formosa lost greater numbers of zooxanthellae than P. damicornis when exposed to a 5 4 ° C temperature increase (Neudecker, 1983). The two studies where a correlation between polyp size and zooxanthellae loss has been described are those of Jokiel and Coles (1974) and Rogers (1979). Jokiel and Coles (1974) noted that the larger polyped coral L . purpurea was more tolerant of thermal increases than the smaller polyped species P . rrteandrina and P. lobata, which lost their zooxanthellae more rapidly on response to temperature increases 4-5°C above ambient. Rogers (1979) described a fairly close connection between increased susceptibility to shading, as demonstrated by bleaching, with decreased polyp size, e.g., A. cervicornis, with small polyps being the first species to bleach, followed by M. annularis, D. labyrinthiformis, and D . strigosa,

34

B. E . B R O W N A N D L. S. H O W A R D

with medium-sized polyps and large polyped species such as Eusrnalia fastigiata (Pallas), Mussa angulosa (Pallas), and M . cauernosa appearing relatively unaffected by shading. The author acknowledges, however, that the latter species were growing at the edge of experimental channels where light may not have been totally excluded. The apparent lack of a relationship between polyp size and the tendency for zooxanthellae loss in some studies, and yet the existence of an apparent relationship between the two parameters in others, probably reflects the likely complexity of the response. Apart from the obvious variability in responses from different corals at a given depth, the same species collected from a variety of depths may also exhibit widely differing responses. M . annularis from different depths contains zooxanthellae which are photoadapted to specific habitats (Dustan, 1979). In transplant experiments, zooxanthellae adapted to high light intensities function poorly in deeper water habitats, while zooxanthellae adapted to low light intensities are actually damaged by high light intensities in shallow waters. At deep water sites, corals transplanted from shallower depths show some reduction in algal content; in contrast, corals transplanted from deep water to shallow water display considerable bleaching and high mortality (Dustan, 1982). Hence, the tendency to lose zooxanthellae in response to changing light intensities is very much dependent upon the original position of the coral within the reef environment. Considering, then, the limited literature to date, it would seem that loss of zooxanthellae in response to a particular stress does give some indication of the relative tolerance of a coral species to parameters such as temperature increase, salinity change, etc. There are problems, however, in interpreting such responses since they may be reversible (Jokiel and Coles, 1974, 1977; Jaap, 1979); they may be very localized, the undersurface of an affected coral displaying little apparent loss of zooxanthellae (Jokiel and Coles, 1977); or they may reflect not only one stress but a combination of two or more, such as combined temperature and strong light intensity (Jokiel and Coles, 1977). In addition, loss of zooxanthellae may occur unperceived by the observer but calculated through some other measurable parameter such as reduced calcification (Bak, 1978) or production (Dallmeyer et al., 1982). Quantification of the response is possible by extraction of photosynthetic pigment from coral tissues, and this has been successfully carried out by a number of workers (Jokiel and Coles, 1974; Dallmeyer et al., 1982) in response to thermal stress and addition of peat, respectively. The value of the response of loss of zooxanthellae by coral tissues as an indicator of stress could be considerably improved (1) by quantification of the magnitude of algal loss using pigment extraction techniques, (2) by better understanding of the mechanism

EFFECTS O F STRESS ON REEF CORALS

35

of expulsion of the algae, and (3) by relating this response to other physiological parameters. D. 1.

Behaviourul Responsvs

Feeding and mesenterial filament extrusion

Mesenterial filament extrusion by scleractinian corals has been widely reported by early workers (Duerden, 1902; Carpenter, 1910; Vaughan, 1912; Matthai, 1918; Yonge, 1930) in connection with feeding activities. These early studies revealed the ability of many scleractinians to engage in extracoelenteric digestion of food material by means of filaments extruded either through the mouth or through temporary openings on the colony surface (Fig. 4). Lewis and Price (1975) failed to observe corals feeding routinely by this method and commented that in many cases where extracoelenteric digestion was described in the literature this was connected with the presence of large particles of food on the oral disc. Nevertheless, numerous other workers have noted mesenterial filament extrusion as a result of exposure of scleractinians to feeding inducers such as the amino acids glutathione and proline (Brown and Phillips, unpublished; Mariscal and Lenhoff, 1968; Goreau et al., 1971), where tactile stimuli were not involved.

FIG.4. The extrusion of mesenterial filaments through the mouth and body wall of a coral polyp. Abbreviations: t , tentacle; m, mesentery; f, filament; c, calyx; s, skeleton (after Goreau et at., 1979).

36

B . E. BROWN A N D L . S . HOWARD

The extrusion of mesenterial filaments in interspecific aggression responses by corals has been reported both in the Caribbean (Lang, 1970, 1971; Bak et al., 1982) and in the Indo-Pacific (C. R. C. Sheppard, personal communication). Sheppard (1979) implies that the response is triggered as a result of chemoreception properties of the aggressor, a suggestion first put forward by Muscatine (1973) when considering the nutrition of corals. Filament extrusion has also been cited in recent years as a response of corals to adverse conditions (Table VI). Goreau et al. (1971) report severe starvation resulting in extrusion of mesenterial filaments by M . angulosa; Lewis (1971) describes the same response in Madracis asperula (Milne, Edwards, and Haime) when exposed to crude oil and oil dispersant; Bak and Elgershuizen (1976) recorded filament extrusion in M. decactis as the result of the presence of oil droplets in the gastrovascular cavity, while M . annularis was observed to extrude mesenterial filaments in response to exposure to drilling muds (Thompson et a/., 1980). Similar behaviour has also been recently reported by S. Wyers (personal communication) in D . strigosa exposed to fuel oil. All these observations apply to laboratory experiments, although J. H. Thompson (personal communication, in Dodge and Szmant-Froelich, (1984) observed mesenterial filament extrusion on four heads of M . annularis in the field after application of 5 ml drilling mud slurry to each. The same behaviour was observed on four heads of M . annularis treated with a similar amount of carbonate sand. The majority of examples of filament extrusion cited above have been the result of exposure of animals to oil. Both Loya and Rinkevich (1980) and Ormond and Caldwell (1982), reviewing the literature concerning anthozoan feeding behaviour and oil exposure, highlight the work of Blumer et al. (1971), which discussed the possible interference of crude oil products with chemoreception in marine invertebrates. The mechanism suggested involved the mimicking of natural stimuli by the oil product, which in turn elicited feeding behaviour. Tentacle responses and/or mouth-opening reactions also associated with feeding (Table VI) have been observed in anthozoans in response to crude oil pollution (Reimer, 1975a,b; Loya and Rinkevich, 1980; Ormond and Caldwell, 1982). Experiments with the temperate anthozoan Actinia equina (Linnaeus) suggest that crude oil or some component of crude oil does act as a feeding inducer, whereas the pure hydrocarbons tested did not. It was also shown that crude oil presented on filter paper to the anemones either interfered with or diluted the action of natural feeding inducers present in fish muscle extract (Ormond and Caldwell, 1982). Clearly the mechanism(s) involved in the appearance of mesenterial filaments and other feeding responses such as mouth opening as a result of exposure to pollutants are unclear. The use of filament extrusion as an

TABLEVI. THE USE OF MESENTERIAL FILAMENT EXTRUSION TO ASSESSTHE EFFECTSOF DISTURBANCE O N CORALSIN EXPERIMENTALLY I N THE FIELD Criterion Mesenterial filament extrusion

Nature of study and location

THE

Species

Disturbance

Results

Laboratory experiment, Barbados, West Indies

Porites porites, Madracis asperula, F. fragum, A . agaricites

Exposure to crude oil and oil spill dispersant

Laboratory experiment at Caribbean Marine Biological Institute, CuraGao

Nineteen Caribbean coral species

Exposure to crude oil and sediments

Experimental chambers sited on a sand flat at Carysfort Reef, Key Largo. Florida

Seven Caribbean coral species

Exposure to drilling mud

Field experiment, Key Largo, Florida

M . annularis

Exposure to drill mud slurry or carbonate sand

Flow-through laboratory experiment, Bermuda

D . strigosa

Exposure to crude oil

M . asperula exhibited greatest effects on addition of 50 ppm oil, involving an increase of extrusion of mesenterial filaments; all species were more affected by the dispersant than by the crude oil Extrusion of mesenterial filaments in response to introduction of oil drops into the gastrovascular cavity Some polyps of M . annularis extruded mesenterial filaments; other corals tested did not show this response Although sediments were cleared within 2 h, all corals showed mesenterial filament extrusion later Extrusion of mesenterial filaments

LABORATORY AND

Reference Lewis (1971)

Bak and Elgershuizen ( 1976)

Thompson et a / . (1980)

Thompson, in Dodge and Szmant-Froelich (1984) S. Wyers, personal communication

38

B . E . BROWN A N D L . S . HOWARD

indicator of stress, often cited by workers (Lewis, 1971; Bak and Elgershuizen, 1976; Thompson, et al., 1980), would appear to be complicated by the variability in response of individual corals (S. Wyers, personal communication), the often temporary nature of the response (Thompson et al., 1980), its regular appearance in certain coral species and not in others (Goreau et al., 1971), and a lack of understanding of natural stimuli which might elicit such behaviour. Other behavioural responses normally associated with feeding and also sediment shedding, such as coenosarc distension, have been observed on exposure of certain coral species to pollutants (Thompson et al., 1980; Dallmeyer et al., 1982). Bak and Elgershuizen (1979) describe the sequence of events which follows a sediment rain on an expanded coral surface as initial contraction of the polyps followed by a marked expansion of tissues including the coenosarc. Such behaviour was also observed in M . annularis at night after exposure to drilling mud, although other corals, e.g., Porites diuaricata (Lesueur), Porites frrrcata (Lamarck), P . astreoides, A . cervicornis, A . agaricites, and Dichocoeniu stokesii (Edwards and Haime), under similar conditions did not display coenosarc expansion (Thompson et al., 1980). Addition of peat to M . annularis (Dallmeyer et al., 1982) also resulted in conspicuous coenosarc distension at night. A reverse phenomenon, i.e., polyp retraction, has been used in some studies to assess the toxicity of pollutants (Jaap and Wheaton, 1975; Thompson et al., 1980). Contraction of polyps as a reaction to stress in the form of electrical stimulation was described by Horridge (1957), and to extreme water currents by Hubbard (1974), while Bak and Elgershuizen (1979) observed contraction as a reaction to contact with nonfood particles. Polyp retraction was a common reaction of all 12 Caribbean corals exposed to fish-collecting chemicals (Jaap and Wheaton, 1975), while in experiments with drilling muds five species ( M . annularis, A . agaricites, A . cervicornis, P . furcata, and P . astreoides) demonstrated significant polyp retraction to 100 ppm and higher concentrations of drilling mud. D . stokesii, however, showed no reaction to drilling muds at any of the tested concentrations, while P . diuaricata displayed significant retraction at 316 ppm. Exposure of corals to water-soluble fractions of fuel oil resulted in polyps of M . decactis and M . annularis remaining partially or totally retracted while polyps of control corals were fully expanded during all or most of the exposure period (Neff and Anderson, 1981). These authors describe retraction of polyps as a sign of the effects of severe stress in corals. While acknowledging that individual polyps can alternate between an expanded and a retracted state (Sweeney, 1976; Sebens and de Riemer,

EFFECTS OF STRESS ON REEF CORALS

39

1977), with some species remaining expanded for long periods of time while others engage in die1 cycles of expansion, Dodge and SzmantFroelich (in press) believe coral polyp retraction may be useful as a bioassay technique for pollutants. Lasker (1979, 1981), however, has shown that in at least one example ( M . cauernosa) expansion cycles may vary within a species. On the Caribbean coast of Panama, colonies of M . cauernosa can be divided into two morphs based on their activity cycles and polyp morphology (Lehman and Porter, 1973; Lasker, 1976, 1977, 1979), with polyps of the diurnal morph of M . cauernosa expanded both day and night, while those of the nocturnal morph expand only at night. Furthermore, in diurnal morph colonies which exhibited zooxanthellae loss and bleaching, only the “normal” part of the colony expanded during the day and night, while the “bleached” area expanded solely at night. The loss of daytime expansion appeared to be linked to the absence of zooxanthellae. Since zooxanthellae loss has been cited as a response of many scleractinians to exposure by pollutants (see Section lll,C), careful consideration should be given to behavioural responses involving polyp expansion and retraction which may be influenced by the presence or absence of these symbionts. 2. Mucus production Mucus production by corals has been described during the course of feeding (Lewis and Price, 1975; Lewis, 1977), sediment rejection (Yonge, 1930; Bak, 1978), and shading (Rogers, 1979) and also as a result of exposure to pollutants such as crude oil (Mitchell and Chet, 1975; Neff and Anderson, 1981), copper sulphate (Mitchell and Chet, 1973, fishcollecting chemicals (Jaap and Wheaton, 1973, drilling muds (Thompson et al., 1980), peat (Dallmeyer et al., 1982), and increased temperatures (Neudecker, 1983) (see Table V11). In histological studies an increase in the number and size of mucous secretory cells was observed as a result of exposure of Manicina areolata (Linnaeus) to chronic oil pollution (Peters, et al., 1981). Although excess mucus production as a result of man-made stress has often been cited in the literature, Bak and Elgershuizen (1976) showed that the response of 19 hermatypic Caribbean corals to oiled sediments was not an obvious increase in mucus secretion compared with secretion resulting from exposure to clean sediments. Mucus production may, in some cases, actually delay cleaning of the coral surface, particularly when very small particles (e.g., carborundum powder) become trapped in the mucus. In Bak and Elgershuizen’s study such a process caused death in P . astreoides and A . agaricites.

TABLEVII. THEUSE OF Mucus PRODUCTION TO ASSESSTHE EFFECTSOF DISTURBANCE ON CORALSIN EXPERIMENTALLY IN THE FIELD Criterion

Nature of study and location

Species

Disturbance

Results ~

Mucus production

Fish-collecting chemicals applied in the field

Caribbean reef corals

Exposure to quinaldine and a rotenone derivative

Laboratory experiment

Platygyra spp.

Laboratory experiment at Caribbean Marine Biological Institute, CuraCao

Nineteen Caribbean coral species

Exposure to crude oil, copper sulphate, potassium phosphate or dextrase Exposure to crude oil and sediments

Field experiment, San Cnstobal Reef, S.W. Puerto Rico

Ten Caribbean coral species

Shading

Experimental chambers sited on a sand flat at Carysfort Reef, Key Largo, Florida

Seven Caribbean coral species

Exposure to drilling mud

LABORATORY AND

THE

Reference ~

All corals secreted increased amounts of mucus, although no long-term damage was evident All corals stimulated to produce large quantities of mucus

Jaap and Wheaton (1975)

Certain species (e.g., A . palmata, A. cervicornis, P . porites, and P . astreoides) proved to be copious mucus secreters Some areas on collines of M. annularis secreting mucus after 8 weeks’ shading All corals exhibited an increased mucus production, mucus being produced either as a sheath or as strands

Bak and Elgerhuizen ( 1976)

Mitchell and Chet (1975)

Rogers (1979)

Thompson et al. ( 1980)

Exposure to surface oil slick of south Louisiana crude oil Exposure to elevated temperatures (4-6°C above ambient)

Flow-through experiment on corals from Carysfort Reef, Florida

M . annularis, A . cervicornis, A . palmata

Experimental transplantation in the field, Guam

P . andrewsii, P . damicornis, A . for mosa

Laboratory experiment

Manicina areolata

Exposure to 0.15 ppm oil hydrocarbons for 3 months

Laboratory experiment, Jamaica

M . annularis

Exposure to particulate peat

Mucus production particularly stimulated in M . annuluris

Neff and Anderson (1981)

A . formosa appeared

Neudecker (1983)

most sensitive to elevated temperatures and produced large quantities of mucus after a few hours of exposure; mucus production was also noted during alizarin staining in P . andrewsii, although these colonies were not used in subsequent experiments Histological studies revealed an increase in the number of mucussecreting cells in tissues of exposed corals Clumps of mucus produced on the surface of the coral trapped peat, but were subsequently removed. After 15 h the coral showed a normal appearance

Peters er al. (1981)

Dallmeyer et al. (1982)

42

B. E. BROWN AND L. S . HOWARD

Under normal circumstances mucus production may result in up to 40% net carbon fixation being lost from a coral such as Acropora acuminata (Verrill) (Crossland, Barnes and Borowitzka, 1980), so mucus production in corals under stress may constitute a considerable energy loss, as noted by other authors (Loya and Rinkevich, 1980). Workers also suggest that mucus may bind or absorb pollutants such as aromatic hydrocarbons (Neff and Anderson, 1981) or heavy metals (Howard and Brown, 1984) and so confer some protection to the underlying coral tissues either by physically protecting them or by acting as an avenue for hydrocarbon release from contaminated corals (Neff and Anderson, 1981). However, it is also well known that mucus strands and nets may be ingested by corals (Lewis and Price, 1975); should oil particles be trapped in the mucus, then these may also be ingested during the feeding process (Bak and Elgershuizen, 1976). An increase in mucus production by a coral under stress may also result in significant increases in the bacterial population of the mucus (Ducklow and Mitchell, 1979), and these effects will be discussed later in this article (see Section 111,F).The existing literature on effects of drilling muds and increased temperature exposure on selected corals suggests that Acropora spp. readily produce mucus when compared with other coral species exposed to the same stress. In experiments with drilling muds (Thompson et al., 1980), A . cervicornis produced mucus strands after 30 min exposure, whereas mucus production in other species (P. divaricata, P. furcata, P. astreoides, and M . annularis) was not observed until at least 24 h after mud application. (It should be noted that these responses were not shown by all colonies of the test species.) Exposure of A . formosa, P . andrewsii, and P . damicornis to 5-6°C temperature increase at the Cabros Power Plant, Guam, resulted in considerable mucus production in A. formosa after a few hours; all test colonies in one experiment were dead within 48 h of exposure, whereas P . damicornis died within 30 days and P. andrewsii survived the entire test period of 77 days. No note of excessive mucus production in Pocillopora or Porites was reported (Neudecker, 1983). If mucus production were to be used as a measure of stress, then some attempts would have to be made to assess the amount of mucus produced during exposure to a pollutant. Using techniques adopted by Crossland et al. (1980a), it would be possible to quantify the amount of mucus produced and also the rate of production in a stressed coral. Quantification of mucus production rates is limited to A . acuminata, where the rate of mucus output does not appear to vary diurnally. Extension of quantitative methods of mucus production to corals under stress would necessarily demand some base line information of the type already obtained for A . acuminata.

EFFECTS OF STRESS ON R E E F CORALS

3.

43

Sediment shedding

Sediment-shedding behaviour has been observed in a variety of corals by numerous authors (Hubbard and Pocock, 1972; Hubbard, 1974; Schumacher, 1979; Lasker, 1980; Fisk, 1983). Hubbard and Pocock described a ranking in the ability of Caribbean coral species to reject sediments which was related to the diameter of coral colonies and their abundance on the colony surface, but subsequent work by Bak and Elgershuizen (1976) failed to demonstrate such a relationship in Caribbean corals. Other workers, however, have used sediment-shedding behaviour to assess the effects of drilling mud (Thompson, no date, in Dodge and Szmant-Froelich, 1984; Thompson and Bright, 1980), oiled sediments (Bak and Elgershuizen, 1976), and heavy metals (Brown and Holley, unpublished) on reef corals (Table VIII). Results of experiments with drilling muds showed that while clearing rates varied from species to species, all three species tested ( D . strigosa, M. annufaris, and M . cauernosa) could clear barite, bentonite, and CaC03 but no species was able to remove the used drilling mud. The authors suggested that dissolved, toxic components of the mud were responsible for this dramatic difference in response. Similar results were obtained with M . annularis and P . astreoides exposed to drilling muds and carbonate sands. Montastrea failed to move the drilling mud and died within 15 h, while treated P. astreoides died within 10 days. Those specimens covered by carbonate sand recovered completely. In the 19 hermatypic coral species tested by Bak and Elgershuizen (1976), the efficiency of removal of oil sediment particles was the same and performed by an identical rejection mechanism as when they were covered with clean particles of the same size or quality. These authors highlighted the complexity of mechanisms involved in sediment rejection by corals, a feature noted recently by Fisk (1983) working with sediment shedding in fungiids. Bak and Elgershuizen state that the relationship between rejection efficiency and density of sediment particles and their size may involve a very rapid rejection, a rejection over time, or no rejection at all; e.g., M . cauernosa removes large oil-sand particles (3-mm diameter) by movements of the tentacles and polyp expansion, whereas smaller oil-sand particles (1-mm diameter) fall between the polyps and are rejected by ciliary activity; much smaller particles (0. I-mm diameter) are transported very rapidly by the cilia. Bak and Elgershuizen also noted that in some species rejection patterns varied considerably depending on the degree of expansion of the living tissue, a finding supported by Brown and Holley (unpublished) working with Fungia fungites (Linnaeus) exposed to metal-laden sediments.

TABLEVIII. THE USEOF SEDIMENT SHEDDING TO ASSESS THE EFFECTS OF DISTURBANCE ON CORALS I N THE LABORATORY AND EXPERIMENTALLY IN THE FIELD

Laboratory study

D . strigosa, M . annularis, M . cauernosa

Exposure to crude oil and sediments

Patterns of oil sediment rejection are identical to patterns of rejection of clean sediments; rates of sediment shedding are dependent on size and density of particles Cleaning rates varied from species to species; all corals could remove barite, bentonite, and calcium carbonate; no species was able to remove all the drilling mud

Laboratory experiment at Caribbean Marine Biological Institute

Exposure to drilling mud, barite, bentonite, and calcium carbonate

Sediment shed1 ding \

Nineteen Caribbean coral species

Results

Criterion

Laboratory experiment at Caribbean Marine Biological Institute

Disturbance

Laboratory study

Nature of study and location

Nineteen Caribbean coral species Species

Sediment shedding 1

Species

D . strigosa, M . annularis, M . cauernosa

\

Nature of study and location

Exposure to drilling mud, barite, bentonite, and calcium carbonate

Patterns of rejection to of clean rates of ding are size and particles Cleaning from all barite, calcium species remove mud

Criterion

Reference Bak and Elgershuizen (1976)

Thompson and Bright (1980)

Exposure to crude oil and sediments Disturbance

TABLEVIII. THE USEOF SEDIMENT SHEDDING TO ASSESS THE EFFECTS OF DISTURBANCE ON EXPERIMENTALLY IN THE FIELD

Laboratory study

Field experiment San Cristobal, Puerto Rico

M . annularis

M . annularis, D. srrigosa, Diploria cliuosa, A. palmata, A . cervicornis

Exposure to 10-ml aliquots of different (A and B) drilling muds and natural finegrained muds Exposure to calcareous sediments in different frequencies and doses

Corals were able to clear natural sediments and one type of drilling mud (A); only one specimen was able to clear the more concentrated drilling mud (B) A. palmata was the least tolerant of all species tested; although A . cervicornis and D . srrigosa were not significantly affected, single applications of 800 mglcm* to M . annularis and 200 mgl cm2 to A . palmata colonies caused death of underlying coral tissue

Thompson, in Dodge and SzmantFroelic h (1984)

Rogers (1984)

46

B. E. BROWN A N D L . S. HOWARD

Overall then, sediment-shedding efficiencies in corals exposed to stress may have some value as a bioassay, particularly when the environmental influence under consideration involves a combination of increased sedimentation and a pollutant. However, it must be borne in mind that patterns and efficiency of sediment rejection are often specific to particular coral species, that feeding status may affect ciliary rates (Holley, unpublished), and that observations in the laboratory must be extrapolated to the field with care since rates of sediment rejection in the natural environment may be accelerated by water movements (Bak and Elgershuizen, 1976; Dodge and Szmant-Froelich, 1984).

E. Reproductive Biology Information on sexual (Rinkevich and Loya, 1979b,c; Kojis and Quinn, 1981, 1982; van Moorsel, 1983) and asexual (see Highsmith, 1982, for review) reproduction in corals has increased considerably in recent years. Several authors (Loya and Rinkevich, 1980; Dodge and Szmant-Froelich, 1984) reviewing the effects of pollutants on corals, recognize the value of considering reproductive biology and larval recruitment as top priorities in any toxicity assessment. Not surprisingly in view of our recently acquired knowledge of reproductive strategies in corals, studies on the effects of pollutants on reproductive biology are limited, being restrkted to those of Rinkevich and Loya (1977, 1979a). Observations in thk field and laboratory suggested that populations of S . pistillata at a chronically oil-polluted reef at Eilat showed a smaller number of breeding colonies, a decrease in the average number of ovaria per polyp, a smaller number of planulae produced per coral head, and a lower settlement rate of planulae on artificial objects when compared with control colonies. The authors attributed the significant decrease in the number of ovaria in the oldest colonies from the oilpolluted site to the expulsion of premature and mature planulae, an observation recorded also by Cohen (1973) in the alcyonarian Hetoroxemia fuscescens (Hemprich and Ehrenberg) and Ormond and Caldwell (1982) in the anemone A . equina exposed to oil pollutants. In further laboratory experiments, Rinkevich and Loya (I979a) cut large and mature colonies of S . pistillata into two halves, exposing one half to pollutant and the other to a control solution. Such a procedure was devised to reduce expected variatjbn between colonies, and after 2 months a significant decrease in t& number of female gonads per polyp were recorded in 75% of the polluted halves as compared with control halves of the colonies in clean sea water. Similar detrimental effects of oil have been demonstrated by Peters et

EFFECTS OF STRESS ON REEF CORALS

47

al. (1981) in the Caribbean coral M . areolata and by Ormond and Caldwell (1982) in the temperate anemone A . equina. In these studies the anthozoans showed either degenerating ova and abnormal gonad development (Peters et al., 1981) or reduced size of ova (Ormond and Caldwell, 1982). The settlement of planulae on artificial substrata in chronic exposures to pollutants in the laboratory would appear to be an attractive bioassay, but care must be taken in selection of planulae for use in such experiments. Rinkevich and Loya (1979a) do not state how planulae were collected for settlement experiments, but experience has shown that planulae released by P . dumicornis in the laboratory display variable development, which may subsequently be reflected in their settlement behaviour during experiments (Brown and Holley, unpublished). In much earlier studies, Marshall and Stephenson (1933) speculated that coral collection in the field produced sufficient disturbance to trigger release of planulae in certain corals. Premature ejection of juveniles as a result of field collection of A . equina has also been described by Ormond and Caldwell (1982). It may be, then, that planulae which are to be used in settlement experiments in the laboratory should be collected in situ by methods adopted by Rinkevich and Loya (1977) when investigating the number of planulae expelled by individual colonies on the reef. In these experiments plankton nets (mesh size 125-pn diameter) were secured to cover individual coral colonies in the late afternoon, the nets being removed 2 h after sunset when the planulae were collected. Although the assessment of oil (Loya and Rinkevich, 1980) and heavy metal (Brown and Holley, unpublished) pollution using criteria based on sexual aspects of reproductive biology of corals may have yielded interesting results, the importance of asexual reproduction in reef coral life histories should not be neglected. Highsmith (1982) has already demonstrated that asexual reproduction by fragmentation is an important mode of reproduction in major reef-building corals. Separation of single colonies into independent units may also occur as a result of fission on partial mortality. Based on field data, Brown and Holley (1982, and unpublished) believe that the incidence of partial mortality in massive species may be increased on intertidal reefs affected by heavy metals and sedimentation in Thailand. Such observations could not be demonstrated easily in the laboratory, but nevertheless they do highlight the need to consider all life history characteristics when assessing the effects of pollutants, especially when many corals reproduce predominately by asexu$l methods (Highsmith, 1982). Coral fragments exposed to stress in thedorm of coral disease proved to be more vulnerable than undisturbed colonies in studies on survival after fragmentation in Curaqao (Bak and Criens, 1983). An unusual regeneration phenomenon, described as “polyp bail-out, ”

48

B. E. BROWN A N D L . S. HOWARD

has recently been demonstrated in Seriatopora hystrix (Dana) (Sammarco, 1982). This response, in which polyps detach from the skeleton and subsequently settle and calcify, has been described as an escape response to environmental stress. Polyp bail-out was induced in the laboratory by maintaining coral colonies in nonaerated, noncirculating sea water. However, the response was also noted in the field where no obvious adverse environmental conditions were apparent. Clearly, this mode of “reproduction” warrants further investigation in both the field and the laboratory. F. Histopathology

In a recent review on the pathology of reef corals (Antonius, 1983a) the author summarizes the four known coral diseases as bacterial infections, algal infections, white band disease, and shutdown reaction. According to Antonius, bacterial infections are often the result of corals protecting themselves against outside stresses by mucus secretion. Mitchell and Chett (1975) provide evidence of the involvement of predatory bacteria, Desulfouibrio, and Beggiatoa in the destruction of living tissue of Platygyra exposed to chemical pollutants. Excessive mucus production resulting from natural and man-made influences (e.g., increased sedimentation, toxic chemicals) may also enhance the numbers of the blue-green alga Oscillatoria submembranacea (Ardissone and Strafforella) thought to be responsible for black band disease. This disease may prove to be more damaging to some corals than to others, and Antonius (1983b) cites a tentative list of susceptibility for Caribbean corals, with D. strigosa and M . annularis as most prone to the disease and A . palmata, A . ceruicornis, and Acropora prolifera (Lamarck) as most resistant to black band infections. No pathogen is yet known to be responsible for white band disease (Antonius, 1983b), although work by E. C. Peters (personal communication, in Gladfelter, 1982b) suggests that bacteria may be responsible. The disease is currently widespread in the northeast Caribbean, Panama, and South Florida, and although Antonius cites A , pafmata as the most affected coral in certain parts of the Caribbean, Bak and Criens (1983) report that A . ceruicornis was more affected by the disease than A . palmata in Curasao. Shutdown reaction has been obydrved as the result of exposure of corals to continuous sedimentation or excessive temperature plus a slight additional stress (e.g., a scratch on the coral surface) which would normally not cause any effect in a healthy coral. The speed of this response, which involves tissue regression, may be spectacular (10 c d h ) , and it now appears that shutdown reactions are highly contagious (Antonius,

EFFECTS OF STRESS ON REEF CORALS

49

1983a). Antonius suggests that this response may be a purely physiological reaction, although the mechanism is unknown. Despite the apparent lack of understanding of causative agents involved in coral diseases, tumours which incorporate filamentous algae in Caribbean gorgonids have been cited as potential indicators of environmental stress (Morse rt a f . , 1977, 1981). According to these authors, tumour-like growths have previously been observed in scleractinia from the Pacific (Squires, 1965; Soule, 1965; Cheney, 1975). Although the etiology of these growths is unclear in most cases, Kaufman (1980) has described gall-like nodules in Acropora in response to predation by damselfish. The incidence of algal tumours in populations of the Caribbean gorgonid Gorgoniu uentulina (Linnaeus) is highly localized at Bonaire, and Morse et al. (1981) infer that the tumours may be produced in response to high levels of chronic or intermittent hydrocarbon pollution from nearby petroleum tanker lanes and loading depots. Similar tumours, however, have been described in the gorgonian coral Pseudoplexaura spp. in the Florida Keys (Goldman and Makemson, 1983), where the authors make no environmental implications in detailing the widespread occurrence of the condition. Tumours have been previously reported in the gonad tissues of clams collected from an oil-polluted area (Barry and Yevich, 1975), but there is no direct evidence that such pollutants result in malignant tumours. Similar histopathological studies in corals are restricted to observations made by Peters et al. (1981) on the effects of No. 2 fuel oil on M. arraluta after 3 months’ chronic exposure to 0. I ppm and 0.5 ppm concentrations. Cellular degeneration and atrophy of coral tissues were noted in both high and low concentrations of oil, a finding corroborated by S. Wyers (personal communication) working on the effects of oil on the Caribbean coral D . strigosa. Peters et a f . (1981) describe histopathological examinations as providing an early indication of tissue damage which might only be realized at a much later date in benthic ecological studies (i.e., mortality of adults and species recruitment). Despite this statement, it would seem that the general histology of coral tissues is poorly known, and the possible effects of laboratory entrainment and starvation (no mention of feeding of corals is given in the above experiment) on coral tissues remain to be documented. Effects observed by Peters et ul. may not have been the result of only one stress, b u t d m b i n a t i o n of pollutant and starvation. In addition, histopathological techniques generally involve the use of a limited number of specimens (three per assay). Considering the variability highlighted by other workers in measurement of physiological parameters between and even within different coral colonies, the potential pitfalls in histopathological examination of coral tissues are clear.

50

B. E. B R O W N A N D L. S. H O W A R D

G . Biochemical and Cytochemical Indexes Interest in biochemical indexes of effects of stress in reef corals has been shown by those workers studying the effects of drilling muds on Caribbean species (Szmant-Froelich et al., 1983; Dodge and Szmant-Froelich, 1984). Szmant-Froelich and Pilson (1980) had earlier shown that the ratio of lipid to protein reflected the nutritional status of an ahermatypic coral, Astrangia danae (Agassiz). Similar analyses were applied to M . annularis, and although there was no significant difference in nitrogen content (a measure of protein content) between control corals and corals exposed to 100 ppm drilling mud, changes were evident in the lipid content and composition of exposed corals (Dodge and Szmant-Froelich, 1984). These authors also describe several unpublished studies in which biochemical indexes have been used in assessing effects of drilling muds. These include a study by Powell on A . ceruicornis in which exposure to 100 and 500 ppm drilling mud for 24 h produced dramatic increases in the protein content and in the total ninhydrin-positive substances (mostly free amino acids) of coral tissues, and work by Prassad in which he found that treatment of M . decactis with drilling mud at 10 and 100 ppm concentrations actually inhibited protein synthesis. Clearly such measurements need to be interpreted in the light of further data on coral material. Nevertheless, the use of biochemical indexes represents an interesting development in the assessment of effects of stress in reef corals. Such indexes have been used with some success by temperate workers (Jeffries, 1972; Bayne et al., 1976; Moore, 1976; Moore and Stebbing, 1976). Measurements of the taurine/glycine ratio appear to offer some promise as an empirical indicator of stress, at least when the stressor is temperature. Another index of stress used by these workers has been the cytochemical demonstration of latency of lysosomal hydrolases in mussels exposed to temperature stress (Moore, 1976), in hydroids exposed to increased concentrations of Cu, Cd, and Hg (Moore and Stebbing, 1976), and in homogenates of the sterile septa of the anemone Cerianthus lloydii (Gosse) (Tiffon, 1971). The exposure of Mytilus edulis (Linnaeus) to temperatures of 25-28°C over a period of 4 days induced a significant decrease in the latency of lysosomal glucosaminidase. In experiments with (Hincks), cytochemical threshold the hydroid CampanulaQexuosa concentrations were comparable to known environmental levels and were about one order of magnitude lower than those obtained by measuring inhibitory effects in colony growth rates (Moore and Stebbing, 1976). As such, then, these techniques appear to offer a very sensitive way of assessing effects of stress in invertebrate tissues.

EFFECTS OF STRESS ON REEF CORALS

IV.

51

Discussion and Future Research Needs

Statements such as those of Johannes (1972) and Rogers et af. (1982) that coral reefs are adapted to natural catastrophes but susceptible to unnatural stresses, and that of Loya (1976a) who concluded that human-perturbed reefs may not return to their former configurations, while naturally denuded areas of reef may recover, given time, are obviously very broad generalizations. In the light of recent data, these conclusions are not necessarily supported, for lack of recovery as a result of natural damage has been noted in several cases (see Endean, 1973, for review), and both chronic and acute man-made pollution have been shown to have limited impact in a significant number of examples (see Section 11,C of this article). Furthermore, stresses (e.g., salinity and temperature) in the tropics have been cited as greater than those in temperate waters (Moore, 1972). Moore concluded that tolerances shown by tropical animals would tend to decrease under stress, and that as a result the effect of any additional stress would be exaggerated. This, in turn, led Johannes (1975) to consider that tropical marine communities would appear to be less tolerant to pollution than their temperate counterparts, which has further contributed to the view that c o r d reefs are fragile communities, highly sensitive to a wide range of man-induced pollutants (a view discussed by Dollar and Grigg, 1981). Perhaps we should question the basic premise in the above statement. Considering temperature stress, then, tropical and temperate organisms have similar metabolic rates (measured as O2 consumption) when determined at their respective habitat temperatures (Vernberg, 1962; Vernberg and Vernberg, 1972), although tropical animals may have a limited ability to alter their metabolic response to different temperatures (Vernberg, 1981) when compared with homeostatic mechanisms shown by temperate organisms. Certainly data on one metabolic parameterprimary production-show no clear pattern when compared between tropical and temperate systems, higher values being obtained in tropical benthic communities but lower values in tropical phytoplankton (Johannes, 1975). It is our view, in accor ance with that of Dollar and Grigg (1981), that be as fragile as previous generalizations would reef ecosystems may lead us to believe. We believe that greater attention should be paid in the tropics to interpretation of individual examples of natural and man-made disturbances. Given the variability in responses of corals both in the field and in the laboratory to stress, as highlighted in this article, how should we approach each problem? Here a generalized approach to the assessment of natural and man-made disturbances may have some value in

,;d

52

B . E . BROWN A N D L . S . HOWARD

providing comparative data on tolerances both within and between different coral species and also between different provinces. Conclusions from the foregoing article may be summarized as follows: 1 . Of primary importance is the assessment of the disturbance (manmade or natural) in the field. A lesson learned from temperate studies is that relatively little can be gained from isolated laboratory experiments executed without any field context. Obviously, methods employed to characterize man-made pollution in the field will depend on the form of pollution. It is important that the pollutant should be both physically and chemically characterized in the environment (i.e., waters, sediments, biota, etc.) as completely as possible. Such analyses would ideally be carried out over time, particularly in a region which displayed a marked seasonal regime, i.e., reversing monsoon, where heavy rains and increased sediment loads may alter the biological availability of the pollutant at different times of the year. 2. Measurement of the response of the reef community in the field should involve estimates of abundance and diversity of all dominant members of the benthic community, as described in earlier sections of this article. Estimates of diversity, however, should be interpreted with care, since they may result in misleading conclusions (Hedgepeth, 1973; Johannes, 1975). High diversities may be variously interpreted (Rogers et al., 1982; Brown and Holley, 1982). The latter study showed a high diversity of scleractinians at a polluted site which could be directly attributable to a high diversity of faviids when compared with a control reef. Branching corals, however, were restricted in both number of species and abundance at the polluted location, a feature masked by the high diversity value obtained overall. Estimates of colony size of scleractinians, recruitment, mortality of juveniles, and spatial distribution of living and dead cover in permanent quadrats over time enable man-made disturbances to be evaluated with respect to natural fluctuations at both polluted and unpolluted sites (Bak and Engel, 1979; Bak and Luckhurst, 1980; Bak and Criens, 1983). Such observations constitute a vitally important part of any monitoring program. 3. Experimentahfanipulationsin the field offer considerable scope in assessing the effects of both man-made (Hudson, 1981; Neudecker, 1983) and natural (Bak and Criens, 1983) disturbances. Where appropriate, sediment-shifting capabilities of scleractinians measured in the field (Rogers, 1979, 1984) provide comparative data on a variety of species in different habitats. In addition, contaminated sediments may be used to assess the

EFFECTS OF STRESS ON REEF CORALS

53

relative tolerances of species from polluted and unpolluted sites under natural conditions. Growth rate measurements also offer a useful and sensitive technique for assessing stress in the field, especially when both branching and massive species are analyzed at selected sites. Unfortunately, very few studies to date have incorporated measurements of both types of coral, so we have no comparative data on tolerances of these species to stress. Techniques such as x-radiography and alizarin staining lend themselves well to transplantation of corals to different habitats, which again enables intra- and interspecific differences between sites to be established. Assessment of the reproductive potential of corals transplanted to different environments may also highlight the effects of stress at polluted sites. Measurement of the number of planulae produced per colony and the fecundity of individual species will ultimately reflect the structure of the whole community. Such techniques (Rinkevich and Loya, 1977) have already clearly demonstrated that pollutants such as oil, previously thought to be relatively harmless to adult corals (for review see Johannes, 1975), are in fact capable of exerting a significant effect on reproductive processes. Asexual reproduction, recognized as an important process on coral reefs (Highsmith, 1982; Tunnicliffe, 1983), may also be assessed in unpolluted and polluted sites by experimental manipulation (Bak and Criens, 1983). Using alizarin red S staining, skeletal extension, weight of calcium carbonate deposited and calcification rates, and growth of fragments may be compared with intact colony measurements in different habitats. 4. Experimental biossays in the laboratory should be closely related to field measurements and if possible be used to extend or confirm observations made in the field. There is little convincing evidence in the literature reviewed so w o f a sensitive laboratory bioassay to any pollutant tested; neither is there any indication of how many of the parameters measured relate to the ultimate survival of the coral. Work in progress in Bermuda (S. Wyers, personal communication) attempts to combine a series of laboratory assays on the effects of oil on scleractinians with a closely monitored recovery period in the field. Overall, however, results of laboratory exposure of corals to pollutants tend to be very variable, particularly with respect to behavioural responses, and it may well be argued that a new approach is urgently required here. Such an approach has already been adopted with temperate marine organisms in the use of a “scope for growth” model which measures the response of individuals to environmental stress and pollution (Bayne and Widdows, 1978). These authors argue that it is unlikely that analyses of physiological parameters such as behaviour, growth, and reproduction will provide information on

54

B . E. BROWN A N D L. S. HOWARD

specific environmental stresses since changes in the biochemical targets of particular pollutants will be integrated into more generalized physiological changes as part of a response syndrome. The equation for the scope for growth is as follows:

P

=

A

-

(R

+ U)

where P = production or scope for growth A = product of consumption and efficiency of absorption of energy from food R = respiratory heat loss U = energy lost as excreta Using the mussel M. edulis from natural environments, the authors were able to show a reduced scope for growth in animals from polluted habitats. Such an equation may not be entirely applicable to corals, for it appears that less than 1% of assimilated input appears as growth (P. Spencer Davies, personal communication). In addition, it would be extremely difficult to measure the energy intake from carnivorous sources in many corals. However, energy budgets have been constructed for several species, including Pocillopora eydouxi (Milne, Edwards, and Haime) (P. Spencer Davies, personal communication). A study of the overall energy budget of the coral, together with measurements of growth rate, may therefore be more appropriate. Combining this approach in in situ studies with biochemical and cytochemical assays of coral tissues from field and laboratory exposures would permit a reasonable interpretation of the condition of corals in the environment. The disadvantageLo3 these methods are clear-all require sophisticated equipment and techniques which are not always readily available at remote tropical laboratories. Although it is possible that any meaningful laboratory measurement of effects of stress in reef corals will involve a much more subtle approach than has been applied to date, the value of critical methods of evaluation of pollution in the field, such as those described here, should not be underestimated. On the basis of these field methods alone, considerable advances in our understanding of the tolerances of reef corals to effects of stress-both man-made and naturalcould be made during the next decade. Acknowledgments We would like to acknowledge authors who allowed us to see copies of papers in press and also the staff of the library at the Marine Biological Association, Plymouth, especially David Moulder, for their cooperation.

EFFECTS OF STRESS ON REEF CORALS

55

We should also like to acknowledge the helpful comments of Sir Maurice Yonge, Dr. Peter Spencer Davies, Dr. Tony Stebbing, and Mr. Martin Le Tissier on the manuscript draft, and the useful discussion with Dr. B. Widdows and Dr. Moore of the Institute of Marine Environmental Research, Plymouth.

References Antonius, A. (1983a). Coral reef pathology: A review. Proceedings of the 4th International Coral Reef Symposium, Manila 1981 2, 3-6. Antonius, A. (1983b). The “band” diseases in coral reefs. Proceedings of the 4th International Coral Reef Symposium, Manila 1981 2, 7-14. Bak, R. P. M. (1978). Lethal and sublethal effects of dredging on reef corals. Marine Pollution Bulletin 9, 14-16. Bak, R. P. M., and Criens, S. R. (1983). Survival after fragmentation of colonies of Madracis mirabilis, Acropora palmata, and A . cervicornis (Scleractinia) and the subsequent impact of a coral disease. Proceedings of the 4th International Coral Reef Symposium, Manila, 1981 2, 221-227. Bak, R. P. M., and Elgershuizen, J. H. B. W. (1976). Patterns of oil-sediment rejection in corals. Marine Biology 37, 105-1 13. Bak, R. P. M., and Engel, M. S. (1979). Distribution, abundance, and survival ofjuvenile hermatypic corals (Scleractinia) and the importance of life history strategies in the parent coral community. Marine Biology 54, 341-352. Bak, R. P. M., and Luckhurst, B. E. (1980). Constancy and change in coral reef habitats along depth gradients at CuraGao. Oceologia (Berlin) 47, 145-155. Bak, R. P. M., Sybesma, J., and van Duyl, F. C. (1981). The ecology of the tropical compound ascidian Tridemnum solidum. 11. Abundance, Growth and Survival. Marine Ecology Progress Series 6 , 43-52. Bak, R. P. M., Termaat, R. M., and Dekker, R. (1982). Complexity of coral interactions: i n f l d c e of time, location of interaction, and epifauna. Marine Biology 69, 215-222. Barnes, D. J. (1983). Profiling coral reef productivity and calcification using pH and oxygen electrodes. Journal of Experimental Marine Ecology 66, 149-161. Barnes, D. J., and Crossland, C. J. (1978). Diurnal productivity and apparent I4C calcification in the staghorn coral Acropora acuminata. Comparative Biochemistry and Physiology 59A, 133-138. Barnes, D. J., and Crossland, C. J. (1982). Variability in the calcification rate of Acropora acuminata measured with radioisotopes. Coral Reefs 1, 53-57. Barry, M., and Yevich, P. P. (1975). The ecological, chemical and histopathological evaluation of an oilspill site. 111. Histopathological studies. Marine Pollution Bulletin 6 , 171-173. Bayne, B. L., and Widdows, J. (1978). The physiological ecology of two populations of Mytilus edulis L.Oceologia (Berlin) 37, 137-162. Bayne, B. L., Livingstone, D. R., Moore, M. N., and Widdows, J. (1976). A cytochemical and a biochemical index of stress in Mytilus edulis L. Marine Pollution Bulletin 7, 221224. Benayahu, Y., and Loya, Y. (1977). Space partitioning by stony corals and benthic algae on the coral reefs of the northern Gulf of Eilat (Red Sea). Helgolander wissenschafiliche Meeresuntersuchungen 30, 362-282. Birkeland, C., Reimer, A. A., and Young, J. R. (1976). Survey of marine communities in Panama and experiments with oil. Environmental Protection Agency Ecological Research Series.

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Nutrition of Sea Anemones

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M Van-Praet Laboratoire de Biologie des Inverte'bre's Marins et Malacologie Muse'um National d'Histoire Naturelle. Paris. France

I . Introduction . . . . . . . . . . . . . . . . . . . . I1. Chemoreception and Feeding Behaviour . . . . . . . . . . . . A . Feeding behaviour . . . . . . . . . . . . . . . .

B . Nature of activators . . . . . . . . . . . . . . . . C . The conducting systems . . . . . . . . . . . . . D . The control of feeding behaviour . . . . . . . . . . . . I11. Absorption of Dissolved Organic Matter . . . . . . . . . . . . A . Localization of uptake . . . . . . . . . . . . . . . . B . Uptake systems for glucose and amino acids . . . . . . . . C . Ecological importance of dissolved compounds for the nutrition of sea anemones . . . . . . . . . . . . . . . . . . IV . Gathering and Digestion of Particulate Organic Matter . . . . . . . A . Suspension-feeding structures . . . . . . . . . . . . B . Endodermal currents and the role of the trilobed portion of mesenteric filaments . . . . . . . . . . . . . . . . . . . . C . Phagocytic cells, cytological and enzymological aspects of intracellular digestion . . . . . . . . . . . . . . . . . . . . D . Importance of particulate organic matter . . . . . . . . . . V . Predation and Digestion of Prey . . . . . . . . . . . . . . A . Role of tentacles, acontia, and cnidae . . . . . . . . . . B . Extracellular digestion of prey, cytological and enzymological aspects . C . Excretion . . . . . . . . . . . . . . . . . . VI . Symbiosis . . . . . . . . . . . . . . . . . . . . A . Localization of algal symbionts . . . . . . . . . . . . B . Regulation of the concentration of algae in the tissues . . . . . . C. Translocation of metabolites . . . . . . . . . . . . . . VII . Sea Anemones as Prey and Remarks on the Diet of Sea .4nemones . . . . A . Predators of sea anemones . . . . . . . . . . . . . . B . Diet of sea anemones . . . . . . . . . . . . . . . . References . . . . . . . . . . .. . . . . . . . .

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65 ADVANCES IN MARINE BIOL.OGY, VOL . 22

Copyright 0 1985. by Academic Press Inc . (London) Ltd . All rights of reproduction in any form reserved . ISBN 0- 12-026122-7

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1.

Introduction

In the nineteenth century, the progress of histological techniques permitted zoologists such as the Hertwig brothers (1879), Metschnikoff (1880), Brandt (1881), and Faurot (1895) to extend knowledge of the biology of sea anemones. But paradoxically, the discovery of phagocytosis by Metschnikoff (1880, 1882) reduced the importance of other studies on digestion. Accurate information about digestion of prey was not established until the studies of Krijgsman and Talbot (1953) and Nicol (1959). More generally, the absence of cytological studies on sea anemones during the first 60 years of the twentieth century led biologists to interpret their observations by reference to those of the nineteenth century or to those known in the most studied cnidarian, Hydra. Knowledge about nutrition of sea anemones is, therefore, restricted to one particular aspect, namely, the chemoreception of prey and its related behaviour (Lenhoff et al., 1976), and the symbiosis with zooxanthellae (Taylor, 1973; Muscatine, 1974). Our present knowledge of digestion and absorption of particulate organic matter and dissolved organic compounds has recently benefited from the progress of electron microscopy, enzymology, and the use of labelled radioactive compounds. These advances and new trends will be discussed in order to determine the importance of prey, particulate organic matter, dissolved compounds, and symbionts in the diet of sea anemones.

II. Chemoreception and Feeding Behaviour A.

Feeding Behaviour

Pollock (1883) was the first to describe how, in sea anemones, the presence of nearby food evoked the induction of a particular behaviour. All common species studied by Pollock open their discs and wave their tentacles after the introduction of pieces of animal prey. Parker (1905) observed, during this behaviour in Metridium senile (L.), that ciliary currents reversed in the throat and on the lips. In the normal beat, the actual stroke and the propagation waves are directed outward. Shortly after the introduction of pieces of crab, both reverse (Holley and Shelton, 1984). Yonge (1968, 1973) observed identical structure and behaviour in corals. Recent studies on the nature of activators and the transmission of stimuli permit the differentiation of two distinct phases: the prefeeding response, which correspond to Pollock’s observations, and the feeding response,

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which ensures the ingestion of prey. The prefeeding response, with the expansion of the oral disc, the movements of its tentacles, and the extension and swaying of the column, increases the chance of catching nearby food. The feeding response, after contact of prey or particulate animal matter with the tentacles or the oral disc, includes the discharge of nematocysts and ingestive movements. These movements include ciliary currents, movements of the tentacles, the oral disc and the throat, to varying extents depending on the species. These two responses are mediated by two distinct systems: an ectoderma1 conducting system (SS1) for the prefeeding response and an endoderma1 slow conducting system (SS2) for the feeding response (McFarlane, 1970, 1975). Both these conducting systems seem to be interconnected with the nerve nets of the deep part of the tissue layers, but may be nonnervous. McFarlane and Jackson (1976) suggest that SSI corresponds to a cell-to-cell conduction in the supporting layer of the ectoderm, including the secretory mucous cells, and that the SS2 is a low-resistance pathway between endodermal cells linked to ectodermal chemosensory cells. The induction of these two distinct responses requires different activators at least in certain species of sea anemones (Lindstedt, 1971b; Williams, 1972; Nagai and Nagai, 1973). The induction of the feeding response is normally elicited by a combination of chemical and mechanical stimuli (Pantin and Pantin, 1943), but starvation may lower the threshold of the response such that nematocyst discharges and ingestive movements are elicited by mechanical stimulations alone. Conversely, feeding with Artemia has been correlated with the inhibition of nematocyst discharges (Sandberg et al., 1971) and of ingestive movements of the oral disc (Logan, 1979).

B. Nature of Activators Carbohydrates and lipids do not induce feeding behaviour, and Pantin and Pantin (1943) demonstrated that the active substances of natural food are closely associated with proteins. They found that dissolved polypeptides and amino acids induce prefeeding behaviour. In Hydrozoa the only known activator molecules of the prefeeding and feeding responses are a tripeptide [reduced glutathione (GSH)] and an amino acid (proline). In sea anemones, GSH, a few amino acids, and vitamins induce or facilitate this behaviour (Table I). Lindstedt et al. (1968) demonstrated that lo-’ mol/l of valine induces the extension of the oral disc in Boloceroides and allows the ingestion by

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TABLEI. CHEMICAL ACTIVATORS OF FEEDING BEHAVIOUR I N SEAANEMONES Anemonia sulcata (Pennant) Actinia equina (L.) Boloceroides sp. Haliplanella luciae (Verrill) Anthopleura elegantissima (Brandt) Diadumene luciae (Haliplanella luciae)

Anthopleura midorii (Parulekar)

Culliacfispolypus (Forskll)

A few amino acids and glutathione (Pantin and Pantin, 1943) Glutamate (Steiner, 1957) Valine (Lindstedt et al., 1968) Leucine (Lindstedt, 1971a) Asparagine (prefeeding response), GSH (feeding response) (Lindstedt, 1971b) Proline, glutamic acid (prefeeding response); GSH, six amino acids, and vitamin B (feeding response) (Williams, 1972) Alanine, histidine, glycine (prefeeding response); proline (?), cysteine, and GSH (feeding response) (Nagai and Nagai, 1973) GSH, proline, and nine amino acids slightly (Reimer, 1973)

these sea anemones of inorganic particles which are usually refused. Lindstedt (1971b) demonstrated that in Anthopleura elegantissima (Brandt), asparagine induces prefeeding behaviour and that GSH controls feeding behaviour. In 81% of his experiments, Lindstedt obtained both phases of behaviour by placing a paper soaked with asparagine on the tentacles of sea anemones in aquaria containing mol/l of GSH. The comparative study of chemical activators in cnidarians and the results of experiments with structural analogues of GSH led Lenhoff et al. (1976) to propose a process of evolution of chemoreceptors in Cnidaria. According to these authors, primitive receptors could respond to many amino acids and to GSH as it is known in Chrysaora. They could then have evolved to become specific receptors of one amino acid and GSH, and then into two distinct receptors, one to GSH and another to proline. The chemosensitive sites could evolve from those associated with the induction of pinocytosis. This last hypothesis of Lenhoff et al. (1976) is based on the observations of Slautterback (1969), who found that certain amino acids stimulate the formation of microvilli and apical vesicles of pinocytosis in the digestive cells of Hydra. However, these cells can be considered as effectors, like the muscular cells of the tentacles and the oral discs, and not as the chemoreceptive cells themselves. There is a lack of cytological description of these chemoreceptors and the conducting systems of stimuli; electrophysiological studies permit us to locate them, but not precisely.

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C . The Conducting Systems

The chemoreceptors of the prefeeding behaviour are located in the ectoderm (McFarlane, 1970). By placing chemical microdoses of activators (with tubular electrodes of l-mm tip diameter) Lawn (1976) demonstrated that these prefeeding chemoreceptors are dispersed throughout the column ectoderm but are particularly abundant at its base. According to this author, the ectoderm of the pedal disc, the oral disc, the tentacles, and the throat is devoid of these chemoreceptors, even if these tissues conduct SSl pulses, but they may contain mechanoreceptors. On the other hand, the chemoreceptors for feeding behaviour are situated in the ectoderm of the tentacles and the oral disc. Both these slow conduction systems act by inhibition of different muscles. The SS1 causes the opening of the oral disc by relaxation of its radial muscles. The SS2 causes inhibition of inherent contractions of circular and parietal muscles of the column endoderm. Its continuous stimulation evokes mouth opening and pharynx protrusion (McFarlane, 1975; Shelton and McFarlane, 1976). McFarlane and Shelton (1975) have suggested that the nematocyst discharge threshold may also be influenced by the SS 1. McFarlane and Jackson (1976) consider the possibility of four interacting systems in sea anemones; two are nerve-net, but SSl and SS2 could be nonnervous and may involve cell-to-cell conduction. The septate junctions which link all epithelial cells in all sea anemones studied (Van-Praet, 1982b) or gap junctions are known for their properties of high permeability and could be implicated in the process of slow conduction of pulses (Green, 1984).

D. The Control of Feeding Behaviour The detection of nearby prey by sea anemones is due to the emission by this prey of small dissolved molecules: amino acids, tripeptides (GSH), and probably vitamins (Table I). Their ectodermal chemoreception induces a stereotyped behaviour mediated by slow ectodermal conducting systems: opening of the oral disc and movements of the tentacles and the column, which increase the food capture range of the tentacles. The contact of solid food with the tentacles induces nematocyst discharges. Ingestion is controlled by chemical and mechanical stimuli originating from immobilized prey. These phenomena, due to the chemoreception of food, also induce behaviour propitious for the gathering of particles (opening of the oral disc, reversal of ciliary currents of the throat) and the absorption of the dissolved organic matter is particularly effective in the ectoderm of the tentacles.

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111.

Absorption of Dissolved Organic Matter A. Localization of Uptake

The absorption by sea anemones of amino acids dissolved in sea water is principally carried out at the level of the ectoderm of the tentacles and the column (Chia, 1972; Schlichter, 1975; Fig. 1). The opening of the oral disc and the enlargement of the ectodermal body surface by the exposure of the numerous tentacles are anatomical and behavioural factors which increase the capacity for absorption. The endoderm is capable of effectively absorbing glucose (Chia, 1972) and, contrary to Schlichter’s opinion, amino acids, but this occurs in particular zones, the mesenteric fila-

FIG.1. Autoradiography of a section of Actinia equina which was incubated 2 h in I4Clabelled amino acids. Most developed grains are over the ectoderm (EC), which is responsible for absorption of molecules dissolved in the sea water. Abbreviations: E, filaments; EN, endoderm; C, coelenteron; EC, ectoderm. FIG. 2. Autoradiography of a section of a dissected mesenterial filament which was incubated 2 h in 14C-labelledamino acids such as in Fig. 1. Developed grains are concentrated over the filaments (ELThe absorption of dissolved molecules by the filaments may be important during extracellular digestion of prey. Abbreviations: P, zone of phagocytosis; E, filaments.

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ments (Van-Praet, 1978, 1980; Fig. 2). This endodermal absorption may be particularly important during extracellular digestion of prey in intimate contact with the convoluted portions of the mesenteric filaments. On the other hand, the digestive cells of the mesenteric filaments, which ensure phagocytosis, absorb very few of the dissolved molecules. B.

Uptake Systems for Glucose and Amino Acids

The absorption of dissolved organic molecules is energy-dependent, and reciprocal inhibitions between different amino acids exist. Schlichter (1978a) used this phenomenon of competitive inhibition to determine the number and the specificity of the uptake systems for amino acids in Anemonia sulcata (Pennant). With this method, Schlichter identified the existence of at least three systems. The first is highly specific for acidic amino acids. It is not inhibited by either basic or neutral amino acids. The second ensures the absorption of basic amino acids, but it is less specific than the first and it is influenced by neutral amino acids. The third system, for neutral amino acids, is defined by Schlichter as a single broad specific system or several systems with overlapping specificity. Bennet and Stroud [ 1981) have histochemically located the enzyme glutamyl transpeptidase in the ectodenn of the column and the tentacles. They state that this enzyme may be involved in the uptake of amino acids and small peptides from sea water. It is still not possible, however, to relate the occurrence of this enzyme to the three systems described by Schlichter (1978a,b) or to known sodium-coupled transport systems. C. Ecological Importance of Dissolved Compounds for the Nutrition of Sea Anemones

The uptake systems appear effective and permit sea anemones to absorb and accumulate in their tissues molecules dissolved in the sea. From experiments with [3H]glucose, Schlichter (1975) concluded that the energy profit provided may supply 50% of that needed for basal metabolism in the seashore actinian A . sulcata, and a maximal incorporation after 6 h of incubation. The use of tritiated molecules to study quantitative metabolism introduces artifacts due to the spontaneous exchange of the tritium with the hydrogen of sea water during incubation and the process of absorption. Metabolic studies require amino acids labelled with carbon14. The results of these experiments indicate a slightly longer period than the 6 h mentioned by Schlichter as maximal incorporation of amino acids such as proline and leucine in macromolecules is observed in 9-10 h (Gosline, 1971; Van-Praet, 1982b).

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The dissolved molecules and their absorption must be considered from another point of view. Murdock (1971) showed that extracellular digestion of prey produced amino acids, and Chia (1972) demonstrated the capacity of the embryos of Actinia equina (L.) to absorb dissolved molecules. This capacity permits the embryos of incubator species to benefit from the amino acids and carbohydrates produced in the coelenteron by the parent during extracellular digestion of prey. This fact is important if one considers the large number of species which incubate their embryos in the coelenteron. Numerous common European species such as A . equina, Cereus pedunculatus (Pennant), Sagartia troglodytes (Price), and Bunodactis uerrucosa (Pennant) are incubator species. Sea anemones seem to be well adapted for the absorption of dissolved organic matter. The importance of their surface epidermis is increased by their numerous tentacles and the presence of microvilli on the apical face of most cells. These anatomical adaptations are complemented by the possession of effective carrier uptake systems. Schlichter (1978a) demonstrated the nonsaturation of these systems with higher concentration than normal. He considered that sea anemones may quickly absorb a high level of dissolved compounds if these conditions briefly appear seasonally. For Schlichter the energy gain resulting from the absorption of dissolved compounds is particularly important for animals with low metabolic rates, such as sea anemones, and even though their energy content becomes negligible, the absorption of a few molecules such as vitamins could be vital.

IV. Gathering and Digestion of Particulate Organic Matter A.

Suspension-Feeding Structures

All sea anemone species are able to collect macromolecules and particles with the ciliary current of the pharynx. Many species have developed complementary adaptations, such as ectodermal ciliary currents on their tentacles, oral discs, and columns, as well as appropriate positioning in water currents. Parker (1905) demonstrated the influence of nearby food on the direction of the ciliary currents in the pharynx of M. senile. Carlgren (1905) studied the extension of the cilia on the epidermis and the currents that they induce in some species. He correlated these observations with the diet of the species studied. In the genera Protanthea and Gonactinia the

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entire ectoderm, from the column to the tentacles and the oral disc, is ciliated. The currents extend along the column to the oral disc and to the top of the tentacles. In the presence of food the tentacles bend toward the throat, where particles are also carried along by the centripetal currents of the oral disc (Fig. 3A). This ciliary disposition may be restricted to strictly microphagous sea anemones specialized in the gathering of particulate organic matter in sandy or muddy environments. In the genera Halcampa, Metridium, and Sagartia, cilia are less developed on the column and do not induce currents on its surface. On the epidermis of the tentacles, the currents are similar to those described previously, but those on the oral disc are centrifugal (Fig. 3B). On the throat the direction of current depends on the microanatomical zone and on the physiological state of the individual. In the siphonoglyphs, the currents are permanently directed toward the coelenteron. On the other zones of the throat they are directed outward, but they are reversed in the presence of food, or if the individual is starved. This d i a r y disposition may be correlated with microphagous feeding, such as in S . troglodytes, in which the coelenteron contains diatoms, green algae, copepods, poly-

A

1;

FIG. 3. (A) Currents on the epidermis of microphagous sea anemones (Proranrhea, Gonactinia) (after Carlgren, 1905); (B) currents in the genera Halcampa, Metridium, and Sagartia. On the syphonoglyphs (S), the currents are permanently directed toward the coelenteron (C). On the other zones of the throat (T), they are directed outward, but they are reversed in the presence of food (after Carlgren, 1905).

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chaetes, and detritus (Riemann-Ziirneck, 1969). In the genera Tealia, Bolocera, and Actinostola, the column, the tentacles and the oral disc seem devoid of oriented ciliary currents. As in both preceding groups, the currents are permanently directed toward the coelenteron in the siphonoglyphs, but are reversed in the other parts of the throat. This disposition with restricted ectodermal currents may be characterisitic of macrophagous species. The gathering of particles is also favoured by adaptations like the development of the oral disc in the large deep-sea actinians, such as Actinoscyphia (Aldred et al., 1979) and Phelliactis (personal observations in the Bay of Biscay from the submersible “Cyana”). Phelliactis robusta (Carlgren), living at the surface of muddy sediments, appear as large orientable particle traps, with their two hypertrophied contractible lobes of the oral disc. In the closely related order of Ceriantharia, Tiffon and Daireaux (1974) described a ferritin endocytosis by the ectoderm of the labial tentacles in Cerianthus lloydi (Gosse). In sea anemones, absorption of dissolved molecules by the ectoderm is effective, but endocytosis of particles seems negligible. The collected particles are ingested in the coelenteron, where phagocytosis occurs.

B. Endodermal Currents and the Role of the Trilobed Portion of Mesenteric Filaments The endocytosis of macromolecules and particles of a few micrometres is restricted to certain tissues of the coelenteron, characterized by phagocytic cells. These tissues correspond to the zone of phagocytosis defined by Metschnikoff (1880) in his study on the absorption of carmine powder, which contributed to the establishment of the concept of phagocytosis. However, zones other than in the mesenterial filaments absorb particles, namely the endoderm of the tentacles and of the column between the insertions of the mesenteries and the upper trilobed part of the filaments (Fig. 16, section V,B). The cnidoglandular tracts of filaments and their ciliated tracts ensure coelenteric currents which have mainly been studied by RiemannZiirneck (1969) in S. troglodytes. She observed that after contact with feeding suspensions the cilia of the cnidoglandular tracts of the trilobed filaments, which ensure outward currents, come to a standstill. She presumed that this phenomenon favoured the phagocytosis of particles by the mesenterial filaments. In fact, experiments with 14C-labelledCyanophyceae revealed a high endocytosis by the intermediate tract of the

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upper trilobed part of the filaments superior to those in the mesenterial filaments (Van-Praet, 1980, 1982b). These intermediate tracts lie transverse to the currents induced by the ciliated tracts. They are situated between them and the cnidoglandular tracts and are deep depressions mainly composed of phagocytic cells (Figs. 4 and 5). The trilobed portion of the filaments may have two main functions. First, it traps the particles which are too small (a few micrometres or less) to be caught and immobilized in the mesenterial filaments. Second, it contributes to the ejection of the indigestible elements (faecal pellets coated with mucus) and the pseudofaecal pellets of zooxanthellae (in the

FIG.4. (A) Edge of a mesentery; (B) diagrammatic section in the upper “trilobed” part of a mesentery. The cnidoglandular tract (CGT) is mainly composed of mucous cells (MI, nematocysts, and a few zymogen cells (Z). Abbreviations and symbols: CT, ciliated tract; IT, intermediate tracts, mainly composed of phagocytic cells; RT, reticular tracts, with vacuolar cells and cells with concretions (b);-, currents gathering the particles toward the intermediate tracts.

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FIG. 5. Section of Actinia equina stained with paradimethyl aminobenzaldehyde (pDMAB). The zymogen cells stained with pDMAB are located at the level of the filament sectioned in its lower portion (E), but are absent at the level of the filament sectioned in its upper portion ( C G , cnidoglandular tract). Zones of phagocytosis of mesenteries (P) and of the intermediate tracts (I) appear in black in this sea anemone placed in china ink 24 h before fixation. Other abbreviations: C, ciliated tract; R , reticular tract.

symbiotic species; section VI) carried to the throat by the outward ciliary currents of the cnidoglandular tracts.

C. Phagocytic Cells, Cytological and Enzymological Aspects of Intracellular Digestion The digestion of macromolecules and particles (of 10 pm or less) is carried out in the phagocytic cells without prior extracellular digestion. These phagocytic cells (Figs. 6-9), also called digestive cells, possess one cilium and several apical microvilli. They have many pinocytic vesicles with an inner layer similar to those of the coated vesicles described by Slautterback (1967) in the digestive cells of Hydra. Digestion occurs in vacuoles of a few micrometres diameter. Using absorption experiments with lit-

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FIG.6 . Electron micrograph of phagocytic cells in rnesenterial filaments of Edwardsia callimorpha. Abbreviations: C, cilium; G, Golgi apparatus; MV, microvilli.

mus powder, Chapeaux (1893) revealed the acid pH of the small vesicles. According to recent finds, acid phosphatase activities have been located by cytochemistry in these phagosomes surrounded by primary lysosomes and an active Golgi apparatus (Van-Praet, 1976). These cytoenzymological observations in sea anemones are similar to those in other Anthozoa, such as Zoantharia (Trench, 1974) and Ceriantharia (Tiffon and Hugon, 1977), where acid phosphatases were also revealed. These observations confirm that their localization in mucous cells by Yamao and Makino (1954) is a mistaken interpretation due to the very positive reaction of the phagocytic cells in the mesenterial filaments. The absence of enzymological investigation on purified lysosomal fractions does not permit us to correlate the hydrolase activities revealed in the ground mesenterial filaments of sea anemones (Van-Praet, 1982a) and Ceriantharia (Tiffon, 1972). However, it is likely that a large proportion of the enzymes, such as acid phosphatases, glucuronidases, and amylases, originates from the numerous phagocytic cells. These phagocytic cells also ensure the storage

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FIG. 7. Electron micrograph of a phagocytic cell of the deep sea anemone Phelliactis rubusta. Abbreviations and symbols: C, cilium; G, Golgi apparatus; MV, microvilli; + , coated vesicles.

for many weeks of indigestible endocytosed particles (Mouchet, 1930) in large postphagosomes of a few micrometres, and the accumulation of glycogenolipidic reserves at their base. These reserves are composed of wax droplets surrounded by glycogen. Their metabolism into triglycerides (Hill-Manning and Blanquet, 1979) seems to induce enzymatic synthesis of esterase-lipases detectable in the reticulum around the droplets (VanPraet, 1982b) during starvation. Other cellular types are capable of endocytosis. It is mainly the function of vacuolar cells and of cells with concretions which absorb macromolecules (Van-Praet, 1978). These cells are located in the reticular tract of the trilobed filaments (Fig. 4) and the inner layer of the throat. These cells may be two stages of the same cell type evolving from the vacuolar state to the “concretion” stage (Van-Praet, 1982b; Figs. 10, 11). In experiments with ferritin, the process of absorption by coated vesicles is similar to that in phagocytic cells. The endocytosed macromolecules are accumulated in postphagosomes of a few micrometres, but never appear in the

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FIG.8. Electron micrograph of phagocytic cells ofActiniu equinu which was incubated 1 h with femtin. Dark apical vesicles of pinocytosis (P) meet in phagosomes of a few micrometres. Abbreviations: G , Golgi apparatus; MV, microvilli; SJ, septate junctions; P, pinocytosis.

large vacuoles. Their function is unclear, but they may contribute to the excretory processes (see section V,C). D. Importance of Particulate Organic Matter The early observations on the ciliary currents, as well as recent findings of microphytoplankton and bacteria in phagocytic cells by electron microscopy (Van-Praet, 1980, 1982b; Fig. 9) and the studies on phagocytosis, allow us to consider that particulate organic matter (microzooplankton, phytoplankton, detritus, etc.) constitutes a source of food for sea anemones. It is possible to postulate that it may be the main source of food for the entirely ectodermal ciliated species studied by Carlgren (Section IV,A) and the deep-sea trap species. We have investigated its importance in a seashore anemone considered as macrophagous, A . equina. Experiments with Cyanobacteria permit us to estimate their ability to collect,

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FIG.9. Aspect of the inclusions of phagocytic cells of Actinia equina fixed on the sea shore. Bacteria (B) and fragments of prey (muscle, M) are observable in phagosomes. They are digested in a few hours.

ingest, and endocytose these particles (Van-Praet, 1980). Many studies on amylase and laminarinase of crustaceans show that the levels of these enzymes are characteristic of the trophic situation of the population, and especially of the richness of carbohydrates and/or microalgae in their diet. These enzymes are also known in sea anemones (Krijgsman and Talbot, 1953; Sova et al., 1970). We have thus looked for the existence of seasonal variations in the amylase levels and the amylase/chymotrypsin ratio (NC)and their eventual contribution to the diet of A . equina. The results show the increase of amylase levels in June as well as the A/C ratio (VanPraet, 1982a). The values seem to diminish in summer and reach their lowest level in winter (Fig. 12). These variations parallel those of microalgae in the sea and confirm our hypothesis of the role of the microalgae as well as bacteria and plant fragments (terrigenous, macroalgae) in the diet of A . equina. This species, as well as numerous others, should no longer be considered as a strictly carnivorous sea anemone, but future studies may determine the real importance of particulate organic matter, and of its origin from plants, in their diet.

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FIG.10. Thin section in a reticular tract of a filament of Acrinia equina. Concretions (*) are observable around the vacuole (V) of a vacuolar cell. Vacuolar cells and cells with concretions may be two stages of the same cell type. Abbreviations: P, vesicles of pinocytosis; MV, microvilli; V, vacuole.

V.

Predation and Digestion of Prey

A. Role of Tentacles, Acontia, and Cnidae Chemoreception of nearby food induces prefeeding behaviour, which increases the rate of capture (section 11). Prey immobilized by the Cnidae of the tentacles are carried to the throat by movements of tentacles and often by contractions of the oral disc. Ciliary currents, protrusion, and peristaltic movements of the throat contribute to the ingestion of prey in the coelenteron. The cnidae which ensure the capture of prey are mainly spirocysts and a few nematocysts. The function of the numerous spirocysts of sea anemone tentacles seems essentially to be an adhesive one. Food stimuli are effective in causing a massive discharge of spirocysts (Mariscal, 1974), which hold prey while the nematocysts inject their toxins. The acontia and the particular tentacles rich in nematocysts, “catch-

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FIG.1 1 . Cell with concretions in a reticular tract of a filament ofActiniu equina. Abbreviations: MV, microvilli; m, mitochondria.

tentacles,” and acrorhagi have a defensive function; they are not predatory. On the other hand, in the coelenteron the prey is immobilized, killed, and enclosed by mesenterial filaments whose cnidoglandular tracts contain nematocysts but no spirocysts. The penetrating filaments of these nematocysts (principally basitrichs, holotrichs, and microbasic mastigophores) inject toxin which is composed of several active compounds. Two major polypeptide compounds, one cytolytic and the other acting on the sodium channel of cell membranes, are known. Prey in contact with the cnidoglandular tracts are subject to the action of the secretory cells, which ensure their extracellular digestion.

B. Extracellular Digestion of Prey, Cytological and Enzymological Aspects

Fredericq (1878) and Krukenberg (1880) revealed protease activity in extracts of tissues of sea anemones, but they could not detect activity in the coelenteric fluid. Metschnikoff (1880) formulated the concept of phagocytosis and described this function in sea anemones. He considered that

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83

.... ...

.. ..

...

.,.

80

..

.. ..

70 60. 5040.

30. 20. 10. I

J ' F ' M ' A ' M h n e ' J ' A ' S ' O '

FIG. 12. Seasonal variations of the amylasekhymotrypsin ratio (AK) in extracts of Acrinia equina from the Brest Channel. This ratio is considered characteristic of the richness of microalgae and plant detritus in their diet (Van-Praet, 1982a,b). Symbols: - - - , mean value; . . . . , standard deviation.

digestion is exclusively intracellular in lower invertebrates. In spite of the observations of secretory cells in filaments by Hollard (1851) and Hertwig and Hertwig (1879), and the experiments of Chapeaux (1893), who revealed a fragmentation of prey in the coelenteron, Metschnikoff (1880, 1882) and then Mesnil(l901) maintained the hypothesis of a solely intracellular digestion. This mistaken hypothesis was corroborated by the impossibility of revealing any enzymological activities in the coelenteric fluid until the study of Krijgsman and Talbot (1953). These authors detected protease, lipase, and amylolytic activities in the tissues of Pseudactinia flagellifera (Hertwig) and were the first to describe slight protease and very weak amylolytic activities in the coelenteric fluid during digestion. They suggested the hypothesis that these weak enzymatic activities ensure digestion because they are not diluted in the fluid, but are stored in the mucous film covering the prey during digestion. Nicol(l959) considered that this nondilution of enzymes results from the disposition of mesenterial filaments around the food, which is enveloped in a saclike mass of tissues which adhere closely to the surface of prey. Thus this author confirmed the hypothesis of extracellular predigestion in contact with mesenteries, which was first formulated by Krukenberg as early as 1880! Jeuniaux (1962) revealed chitinolytic activities (chitinase and chitobiase) in the endodermal extracts of Adamsia palliata (Bohadsch), A . sulcata, Anthopleura ballii (Cocks), and Edwardsia dlimorpha (Gosse).

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He confirmed that digestion of chitin occurs in the coelenteron in close contact with filaments. Elyakova (1972) also described strong chitinase activity in extracts of M . senile. Gibson and Dixon (1969) purified three serine proteases from extracts of M . senile. One of them possesses a chymotrypsin-like zymogen activation mechanism, a specificity for substrates and reactions with inhibitors which strongly suggests that this enzyme is closely related to the mammalian chymotrypsin. At least two proteases, trypsin-like and chymotrypsin-like, are found in zymogen form in extracts of mesenterial filaments of all seashore and deep-sea species of tribes Athenaria and Thenaria which we have studied (Van-Praet, 1981, 1982a,b), A . equina, E. callimorpha, P . robusta, Paracalliactis stephensoni (Carlgren), and a sea anemone from hydrothermal vents (unpublished). The study of Michaelis constants at different temperatures demonstrates that enzyme-substrate affinity decreases rapidly in measurements carried out well outside physiological temperatures. The hypothesis of a regulation correlated with the existence of different isoenzymes has thus been formulated (Van-Praet, 1982a). Quantitative measurements of protease activity and histochemical studies allow us to differentiate two parts in the filaments and to define the zymogen secretory cells. The histochemical reaction with paradimethyl aminobenzaldehyde (pDMAB) specific to tryptophan is considered indicative of the zymogen cells (Gabe, 1968). In the upper trilobed part, the cnidoglandular tract of the filaments has a few pDMAB-positive secretory cells and the extracts possess weak proteolytic activity. On the other hand, in the lower convoluted part of the filaments, at the level of mesenterial filaments the cnidoglandular tracts show the highest pDMAB-positive cell concentration and the extracts present considerable trypsin and chymotrypsin activities (Van-Praet, 1978; 1982a,b; Fig. 5 ) . The secretion of pDMAB-positive cells also appears as basic proteins included in granules of 1 pm. In electron microscopy these granules, surrounded by a membrane, appear electron dense and homogeneous (Figs. 13, 14). The ,Golgi apparatus is not prominent in these cells, which contain an extensive rough endoplasmic reticulum (Van-Praet, 1978). These ultrastructural characteristics are similar to those of zymogen cells described by Rose and Burnett (1968) in the gastrodermis of Hydra. They resemble exocrine mammalian pancreatic cells. Vader and Lonning (1975) indicate that granules are variable in diameter, up to 3-4 pm, and that endoplasmic reticulum is more or less extensive in the type 111 cells (= zymogen cells) of Bolocera tuediae (Johnston). These observations were possible because the sea anemones were starved for a long period. In the same conditions we regularly observed an accumulation of granules, which invaded the entire cytoplasm, and a correlated decrease of the reticulum.

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FIG. 13. Secretory cells in a cnidoglandular tract of a filament: zymogen cells (Z) and nematocysts (N). Abbreviation: er, endoplasmic reticulum.

On the other hand in fed individuals, or in individuals fixed immediately on the sea shore or after trawling, it is possible to observe a majority of zymogen cells with an abundant reticulum, and at the bottom of the cnidoglandular tract, near the mesoglea, some young zymogen cells with an active Golgi apparatus (Van-Praet, 1982b; Fig. 15). The results of protease measurements and of cytological observations definitely confirm that the lower part of filaments, at the level of mesenterial filaments, constitutes the main zone of extracellular digestion of prey from some 10 pm to a few cm in the bigger macrophagous species. The presence of a few pDMAB-positive secretory cells in the ectoderm of tentacles and the detection of trypsin and chymotrypsin activity in the extracts of dissected tentacles do not allow us to consider as likely an “external” digestion of large prey. The products of these cells may, with mucous secretions of other cells, contribute to bacterial protection and ectodermal cleaning (Van-Praet, 1982a). In the cnidoglandular tracts, secretory cells other than zymogen cells can be identified by histochemistry as well as electron microscopy. In their study on the mesenteries of B . tuediue, Vader and Loning (1975)

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FIG.14. Secretory cells in a cnidoglandulartract of a filament of Actiniu equinar spumous mucous cell (M) and zymogen cells (Z). Zymogen cells contain an extensive rough endoplasmic reticulum (er).

described two types of mucous cells, a spumous type and a granular mucous type which exist in all the sea anemones studied (Van-Praet, 1978, 1981, 1982b). The spumous type possesses an acid poiysaccharide secretion practically devoid of protein (Van-Praet, 1978). Its acidity is probably due to carboxylic groups detected by histochemistry (Van-Praet, 1978, 1982b)and to phosphonic compounds detected by biochemistry (Bunde et al., 1978; Dearlove et al., 1979). In electron microscopy the secretion is a dense filamentous material packed into granules of a few micrometres (Fig. 14). The bigger granules show aggregation and fusion of their membrane. The granular mucous type has smaller granules of 0.4-0.8 rum. The granules contain a fine granular compound which seems acid from histochemical tests (Van-Praet, 1982b). Bouillon (1966) presumed that granular mucous cells (= “cellules phkruleuses hypostomiales”), in the Hydrozoa contribute to extracellular digestion. Krijgsman and Talbot (1953) described very weak amylolytic activity in the coelenteric fluid, but the possibility of weak contamination by frag-

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coelenteron

A

FIG.I S . (A) Edge of a mesentery; (B) section of the convoluted portion of a mesenterial filament. The cnidoglandular tract of the filament (enteroid) is mainly zymogen cells (Z), nematocysts, and a few mucous cells (M). Abbreviations: aa+ , zone of absorption of small molecules such as amino acids; P, phagocytic cells; p+ , zone of endocytosis of particles, preferentially in the dip between the enteroid and the zone of phagocytosis.

ments of the mesenteries cannot be excluded. The highest amylase levels have been detected in the extracts of mesenterial filaments, but levels are slightly different from those from other dissected tissues (Van-Praet, 1982a). In our hypothesis this enzyme acts essentially during digestion of plant detritus and microalgae and may be in the phagocytic cells of the mesenterial filaments and numerous other zones of the endoderm (Section IV,D). The fragmentation of prey ends within a few hours and produces macromolecules and particles which are caught by the phagocytic cells of the mesenterial filaments enclosing the food (Fig. 16). Murdock (1971) indicated that this extracellular digestion produces amino acids. The absorption of small molecules, such as amino acids, is mainly carried out by the cells of the filaments in contact with prey (Figs. 2, 15, and 16). 'The cnidoglandular tracts appear as effective as the ectoderm in absorbing small molecules (Van-Praet, 1978, 1982b). An emulsification of lipids has been demonstrated (Chapeaux, 1893;

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FIG.16. Diagrammatic representation of the zone of absorption and digestion in Actinia equina (after Van-Praet, 1980). Abbreviations and symbols: . , zones of absorption of small molecules [ectoderm (EC) and filaments (E)]; H , zones of absorption of macromolecules (intermediate and reticular tracts and zone of phagocytosis of mesenteries); 0 , zones of absorption of particles of a few micrometres [zone of phagocytosis of mesenteries (d), intermediate tracts ( S ) ] ;CT, ciliated tracts; M, mesentery; EN, endoderm; PREY, zone of extracellular digestion of prey in contact with mesenterial filaments; t , zone of storage of lipids.

Krijgsman and Talbot, 1953), but digestion in the coelenteron of high esters has never been proved. The absorption of lipids seems to occur by pinocytosis (Van-Praet, 1982b). Digestion of the products of prey fragmentation (lipids, macromolecules, fragments of a few micrometres) ends in vacuoles of a few micrometres in the phagocytic cells, as described in section IV,C, for the particles. Numerous coated vesicles may be seen in individuals fed with Arternia or fragments of mussel. They seem to meet and to form vesicles of 1 pm or less. One or two days later, large heterogeneous phagosomes of a diameter of a few micrometres and associated acid phosphatase activities persist. After a few days of starvation, the number of these phagosomes

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of more than 1 p m decreases, but a few heterogeneous postphagosomes, sometimes with crystalline inclusions, are observable. C. Excretion This function, in cnidarians, is not well known. Some indigestible compounds seem to be stored, many in the phagocytic cells. The processes of catabolism remain unknown. Mouchet (1929) described the disappearance of the concretions of reticular tracts by the use of solvents such as ammonia and piperazine hydrate. This author concluded that xanthine was present. Raman spectrophotometry microanalysis of sections embedded in epon, after Carnoy fixation, showed a proteinaceous complex at the level of concretions, but did not permit identification of any major compound (Van-Praet, 1982b). The nature of these crystalline concretions (Van-Praet, 1977; Fig. 11) and the function of these cells (able to pinocytose, Van-Praet, 1982b) cannot be definitely established, but they appear to be the terminal state of the vacuolar cells (Van-Praet, 1982b). A molecule, homarine, has been isolated in extracts of many marine invertebrates. It is particularly abundant in sea anemones (Mathias ef al., 1960) and could intervene in excretion and osmoregulation (Beers, 1967; Gaill, 1981). Lewis and Smith (1971) and Cates and McLaughlin (1976) described excretion of ammonia by sea anemones. According to these authors, the symbiotic zooxanthellae remove a large part of ammonia and carbon dioxide excreted by the host sea anemone.

VI.

Symbiosis

Symbiosis has given rise to many studies and reviews (Taylor, 1973; Muscatine, 1974), and we consider here only recent results related to nutrition of sea anemones.

A. Localization of Algal Symbionts Brandt (1881) created the genus name Zooxanthella. In marine cnidarians, he was the first to determine that these organisms were microalgae and not parasites. Recent studies of their motile phase and the observations by electron microscopy indicate the similarity of structure of all the Dinophyceae of sea anemones, but the authors are still discussing the validity of the term “Zooxanthelfa.” Freudenthal (1962) proposed the term Symbiodinium, Taylor (1973, 1974) the term Gymnodinium, but

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Loeblich and Sherley (1979) reintroduced Zooxanthella. The presence of other microalgae was described by Muscatine (1971) in Anthopleura xanthogrammica (Brandt) and A . elegantissima (Brandt). These Chlorophyceae (= zoochlorellae) have been mainly investigated by O’Brien (1978, 1980). Zooxanthellae, like zoochlorellae, are always located in endodermal cells. They are abundant in the endoderm of tentacles, the oral disc, and the intermediate tracts of the filaments. During gametogenesis they accumulate in the mesenteries around the gametes. In a few genera they are located in specialized organs, such as pseudotentacles in Lebrunia, column vesicles in Bunodeopsis, or ruff on the upper part of the column in Phyllactis. Their accumulation in the tentacles facilitates the exposure of these algae to light. Sea anemones with specialized organs present a specialized behaviour. During daylight, organs with zooxanthellae are expanded, but the tentacles are contracted. On the other hand, the tentacles are expanded at night when prey is available (Gladfelter, 1975; Steele and Goreau, 1977; Sebens and Reimer, 1977). Phototaxis of symbiotic species may be related to local variations of the oxygen level in the tissues and to the metabolism of zooxanthellae. The cycle of expansion in the light and contraction in the dark, however, persists in Anthopleura which have lost their symbionts, so other factors may exist (Pearse, 1974). B. Regulation of the Concentration of Algae in the Tissues The efficiency of symbiosis seems a function of the type of symbionts (zooxanthellae or zoochlorellae) and of their number. Unlike zooxanthellae, zoochlorellae translocate only a small part of the products of their photosynthesis (O’Brien, 1980). Two processes maintain the quantity and balance of algae in the tissues: first, the growth rate by binary fission of the algae; and second, the expulsion rate of these symbionts by the host. Expulsion of algae may be continuous or sequential. It increases during starvation or after environmental stress (increase of temperature, darkness, variation of salinity). Steele (1976) considered that the density of symbionts in host tissues is directly related to the intensity of light and that the rate of expulsion is controlled by the presence or absence of altered metabolites. These metabolites are produced by zooxanthellae when they degenerate or when their number increases. The degeneration of algae is itself dependent on the environmental stress mentioned. In this scheme, Steele explains the egestion of zooxanthellae during starvation by the relative increase of algae, which accompanies shrinkage of host tissues, so leading to rapid passing of the critical level of metabolites within these tissues. The presence of altered metabolites initiates the

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extrusion of zooxanthellae by the sea anemone, and this continues until the situation has reverted to normal; then it stops. This scheme seems realistic for many species. In Phyllactis jlosculifera (Lesueur), Steele and Goreau (1977) have described a more complete regulation, and a real “farming” of zooxanthellae by their host. As in all sea anemones, living zooxanthellae are never digested, but Phyllactis can secrete a proteinaceous substance which has a destructive effect on symbionts. Regulation of the secretion of this protein permits this actinian to use zooxanthellae as a source of food according to its needs. In general, the nutritional significance of symbiosis is not considered to be the direct utilization of symbionts by the sea anemone but as the translocation of metabolites produced during photosynthesis.

C . Translocation of Metabolites Any sea anemone species may be considered as autotrophic. The experiments of Muscatine and Hand (1958), Trench (1971), and Lewis and Smith (1971) have demonstrated the translocation of products of photosynthesis from zooxanthellae to their hosts. These studies demonstrated that zooxanthellae incorporate C 0 2 and release as much as 50% of this carbon, mainly as glycerol and a small amount as glucose and alanine. Trench (197 1) found that 75% of these translocated compounds were incorporated by the host into its lipids and 25% into its proteins. These authors described how the homogenated host tissues stimulate the release of soluble molecules from free zooxanthellae. They deduced a regulation of the phenomenonon of translocation by the host, and Muscatine (1974) considered zooxanthellae as ‘‘efficient primary producers. Recent studies indicate some further factors which must be assessed. The temperature of the environment has a great influence on carbon fixation by zooxanthellae and on carbon budget partitioning in symbiosis. In Aiptasia pallida, carbon fixation appears very low at less than lTC, and translocated molecules seem to be incorporated less in lipid and protein molecules of the sea anemone (Clark and Jensen, 1982). In sea anemones such as Aiptasia, which possess zooxanthellae and/or zoochlorellae, 0’Brien (1980) demonstrated that with zoochlorellae translocation is very low, less than 3% from the fixed carbon. The nutritional benefit appears generally in experiments under conditions of starvation (Taylor, 1969). In symbiotic A . sulcata fed daily with Euphausia, the wet weight increases at the same rate in light or in darkness (Janssen and Moller, 1981). It seems obvious that this symbiosis may be considered as a nutritional benefit during the seasonal periods when food decreases. The quantitative evaluation of this advantage under normal feeding conditions will become ”

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possible when the reversed translocation has been studied more thoroughly. The utilization by zooxanthellae of catabolic compounds of the sea anemone host has been demonstrated by Lewis and Smith (1971) and Cates and McLaughlin (1976). However, the reversed translocation seems to concern more compounds than carbon dioxide, ammonia, and nitrites. By feeding Aiptasia with 35S-labelledfood, Cook (1971) demonstrated the reversed translocation of compounds in a few hours and that 15-30% of the radioactivity was retained in zooxanthellae for at least 3-5 days.

VII.

Sea Anemones as Prey and Remarks on the Diet of Sea Anemones A. Predators of Sea Anemones

Many species of sea anemones live in commensalism with crustaceans (amphipods, decapods) or fish (Amphiprion). In a few cases, this association with amphipods may, in fact, be considered as parasitism. A few Caprellidae and Gammaridae find protection between the tentacles of sea anemones and catch their food in the sea, but a few Stenothoidae collect a part of the food caught by their actinian host. In the genera Onisismus, Aristias, and Allogausia, the amphipods live in the coelenteron of their host and collect a part of the ingested food. Onisismus normani (Sars) is associated with B. tuediae, Actinostola callosa (Verrill), and Hormathia spp.; 20 amphipods can live in the coelenteron of one Bolocera. The young of 3 mm infest their host, live in it for 18 months, and leave only at the oviger state (Vader, 1970). Amphipods that feed on the tissues of their host may exist in the genus Acidostoma (Lyssianassidae). In spite of their nematocysts, sea anemones constitute a source of food for many animals. The gastropods Aeolidae which stock nematocysts of their prey are their best known predators. Each species of aeolid seems to show an alimentary preference; thus Aeolidia papillosa (L.) feeds frequently on A . elegantissima, less on Anthopleura artemisia (Pickering), and never on Corynactis (Waters, 1973). Harris (1971) considered that fluctuations in the numbers of A . papillosa influence the distribution patterns of M. senile. Other gastropods, such as Trochidae and above all Epitoniidae, frequently feed on sea anemones. Pycnogonids, asterids, fish, and worms such as Hermodice are known to feed on sea anemones (Ottaway, 1977). Hermodice may affect the local

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distribution of Stoichactis (Lizama and Blanquet, 1975). Predation by fish is effective; Webb (1973) found that Anthopleura spp. constitute 5% of the gut content of the flounder Rhombosolea, but in all the reports of predation by fish it seems that sea anemones may have been ingested accidentally along with commensal crustaceans and molluscs.

B. Diet of Sea Anemones In these sedentary coelenterates, prey is composed of small motile animals (zooplanktonic larvae, isopods, amphipods, polychaetes). On the sea shore some bigger sessile prey species dislodged by wave action, or occasionally by foraging predators, complete the diet of sea anemones. In the anemone species commensal with crustaceans, such as A . palliata and Calliactis parasitica (Couch), or with fish (Amphiprion), such as the Stoichactis and Stilodactys, pieces of prey caught by the commensal species are collected by the anemones. Generally the prey size is small considering the diameter of the sea anemone, and many species may be considered as microphagous (Section IV). In the nine common species of Caribbean reef sea anemones, seven are planktivores, and the two species Condylactis gigantea (Weinland) and Stoichactis helianthus (Ellis), which can eat macroscopic prey such as gastropods and echinoids, are probably dependent on heavy wave action on the reef to supply prey (Sebens, 1976). For common seashore species such as A . elegantissima and M . senile, the prey size ratio to the diameter of sea anemones is 0.08-0.09. In A . xanthogrammica, the value of this ratio attains 0.4, but this high value is due to the ingestion of mussels and barnacles dislodged by wave action and by asteroid foraging (Dayton, 1973; Sebens, 1981). The two most common species on the European coast, A . sulcata and A . equina, are able to immobilize large prey, but the observation of coelenteric contents demonstrates the scarcity of large crustaceans and molluscs (Moller, 1978; Van-Praet, 1982b). The most common prey are small crustaceans (amphipods, isopods), POlychaetes, and gastropods. Fairly often, fragments of appendages of Carcinus maenas (L.), insects, and detritus of macroalgae can be observed. As mentioned earlier, dissolved molecules and particulate organic matter are absorbed and used by sea anemones. The diet of these sessile cnidarians is less rigid than that of mobile animals. The classification into mainly microphagous, but with a few macrophagous species, does really reveal the extent of opportunism in the diet. Future studies may better determine the respective part constituted by prey, particulate organic matter, and dissolved molecules in their diet and its seasonal variation.

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Acknowledgments The author wishes to thank Professor C. Levi (Laboratoire de Biologie des Inverttbrts Marins et Malacologie, Musturn, Paris), Dr. J. F. Samain (Centre Octanologique de Bretagne, Brest), and Dr. C. Milet (Laboratoire de Physiologie, Museum, Paris) for their valuable suggestions and for providing laboratory facilities.

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Krijgsman, B. J., and Talbot, F. H. (1953). Experiments in digestion in sea anemone. Archives Internationales de Physiologie 61, 277-291. Krukenberg, C. (1880). Ueber den Verdauungsmodus der Actinien. Vergleichend-Physiologische Studien 1, 33-56. Lawn, I. D. (1976). Chemoreception and conduction systems in sea anemones. I n “Coelenterate Biology and Behavior” (G. 0. Mackie, ed.), pp. 581-590. Plenum, New York. Lenhoff, H. M., Heagy, W., and Danner, J. (1976). A view of the evolution of chemoreceptors based on research with Cnidarians. In “Coelenterate Biology and Ecology” (G. 0. Mackie, ed.), pp. 571-580. Plenum, New York. Lewis, D. H., and Smith, D. C. (1971). The autotrotophic nutrition of marine coelenterates with special reference to hermatypic corals. I. Movement of photosynthetic products between the symbionts. Proceedings of the Royal Society of London 178, 111-119.

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Effects of Environmental Stress on Marine Bivalve Molluscs HeBeAkberali Departments of Zoology and Botany, University of Manchester, England

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E. R. Trueman Department of Zoology, University of Manchester, England

I. Introduction

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A. Definition of stress .. . . . . . . . . .. . . B. Occurrence of natural and man-made stresses .. .. .. C. Threshold levels of pollutant stress . . .. .. .. .. D. Development of experimental techniques . . . . .. . . 11. Behavioural Responses to Stress . . .. .. .. .. .. A. Valve closure as a protective mechanism . . .. .. .. B. Relationship between heart rate, valve movement, and pumping ., .. .. .. .. .. .. .. activity . . C. Behavioural response to some pollutant stressors .. .. D. Relevance of valve closure in epifaunal and infaunal species . . E. Effect of subthreshold levels on behaviour .. .. .. F. Effect of temperature on heart rate . . .. .. .. .. 111. Detection of Stress . . . . . . . . .. . . . . . . A. The significance of registering changes in the environment .. B. Sites of reception .. .. .. . . . . . . .. C. Detection and response to environmental changes .. ..

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.. Respiratory Physiology during Stress . . . . . . . . . . .. A. Valve closure and cessation of aerobic processes. . . . . . B. Relationship between heart rate, valve movements, PO>. and pC02 . . C. Anaerobic respiration during valve closure.. . . . . .. .. .. D. Valve activity and pH changes . . . . . . . . . . V. The Role of the Shell . . . . . . . . . . . . . . .. .. A. Physical protection and isolation from environmental stress . . .. B. Shell closure and calcium reabsorption . . . . . . . . .. C. Effect of prolonged stress on shell strength . . . . . . VI. Action of Heavy Metal Stressors . . . . . . . . . . .. .. A. Accumulation of heavy metals . . . . . . . . .. .. .. B. Effects of heavy metals on tissues . . . . . . . . . . C . Effects of heavy metals on released gametes and embryonic and juvenile stages . . . . . . . . . . . . . . . . . . . . D. Effects of heavy metals on cellular organelles . . . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . IV.

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I. Introduction A. Definition of Stress Concern about changes in the environment and their effect on animals has recently evoked extensive research on Bivalvia based on behavioural, physiological, and biochemical responses at organismal, tissue, or cellular levels. Bayne (1975) offered the following working definition of stress as applied to marine bivalve molluscs: “Stress is a measurable alteration of a physiological (behavioural, biochemical, or cytological) steady-state which is induced by an environmental change, and which renders the individuals (or the population) more vulnerable to further environmental change.” Most adult bivalves are relatively immobile, and any change in the environment of transient, recurrent, or permanent occurrence has to be accommodated by the animal. In the aquatic environment, many low levels of natural or man-made changes are within their normal adaptive range and are tolerated without serious consequences. More extreme changes may be temporarily resisted but can ultimately result in the death of organisms. Between these limits of tolerance and resistance to environmental stressors, a region of sublethal response occurs whereby the capability for survival may be reduced as a consequence of the stressor. The species used in this article are listed in Table I with their authorities.

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TABLEI. THE LIST OF SPECIES WITH THEIRAUTHORITIES USED I N THISARTICLE Species

Authority

Anadara s e n i h Anodonta anatina Anodonta cygnea Arctica islandica Cardium (Cerastoderma) edule Cardium (Cerastoderma) gluucum Chlamys opercularis Choromytilus meridionalis Crassostrea commercialis Crassostrea gigas Crassostrea margaritacea Crassostrea uirginica Donax denticulatus Donax juliane = Donax trunculus Donax serra Donax trunculus Helix uspersa Isognomon alatus Ligumia subrostrata Lima scabra Loligo pealii Lolliquneula breuis Macoma batthica Mercenaria (Venus) mercenaria Modiolus demissus Modiolus modiolus Mya arenaria Mytilus californianus Mytilus edulis Mytilus edulis planulatus Mytilus galloprovincialis Mytilus uiridis Neotrigonia margaritacea Ostrea edulis Pecten irradians Pecten maximus Perna perna Pimpehales promelas Pleurobema coccineum Scrobicularia plana Spisula solidissima Tellina fabula Unio tumidus Venerupis decussuta Venus striatula

Linnaeus Linnaeus Linnaeus Linnaeus Linnaeus Poiret Linnaeus Krauss Iredale and Roughley Thunberg Lamarck Grnelin Linnt Linnaeus Roding Linnaeus Linnaeus Gmelin Say Born Lesueur Blainville Linnaeus Linnaeus Dillwyn Linnaeus Linnaeus Conrad Linnaeus Lamarck Lamarck LinnC Lamarck Linnaeus Lamarck Linnaeus Linnaeus Rafinesque Conrad da Costa Dillwyn Gmelin Philipsson Linnaeus da Costa

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B.

Occurrence of Nuturul and Man-made Stresses

Bivalve molluscs are found in aquatic habitats ranging from oceanic waters to fresh water. The open marine environment is relatively stable with respect to its physicochemical composition, but in coastal and estuarine habitats significant fluctuations may occur in salinity, temperature, oxygen levels, and turbidity. Bivalves may also be subjected to further stresses arising from man’s activities, such as off-shore drilling, dredging, or the release of pollutants.

C. Threshold Levels of Pollutant Stress Bivalve molluscs may be either suspension or deposit feeders, and these feeding strategies pose different problems with respect to threshold levels of environmental stress. Suspension-feeding bivalves, e.g., Mytilus edulis, Cerastoderma edule, or Anodonta cygnea, filter suspended particles from the water current entering the mantle cavity, whereas deposit-feeding bivalves, e.g., Scrobiculuria plana, feed on the sediment surface with long extensible siphons but are also capable of suspension feeding (Hughes, 1969; Earll, 1975b). The principal source of pollutant stress in suspension feeders is from pollutants in solution and associated with suspended particles, whereas in deposit-feeding animals the most important source will be the sediment. In Scrobicularia, which is both a deposit and suspension feeder, the uptake of pollutants will be related to both modes of feeding. Different feeding strategies may determine the stressors likely to affect different bivalves; for example, several heavy metals, such as zinc, manganese, cadmium, or selenium, are largely taken up with particulate matter either in suspension or from the sediment, whereas the more acutely toxic metals, such as mercury, copper, and silver, are absorbed most rapidly from solution (Bryan, 1976, 1979). Evidence of significant differential absorption of metals from solution by various tissues has been reported; for example, copper is absorbed from solution about three times more rapidly by the gills and mantle/siphons than by the digestive gland and other tissues (Bryan and Uysal, 1978). The uptake of heavy metals bound to suspended food or inorganic particulate matter is less harmful than direct availability from solution since bound heavy metals have first to be isolated by digestive processes. They may thus be dealt with by metal scavenging systems, such as metallothionein proteins, which have been shown to exist in bivalve molluscs (see Section VI,A). It is also possible that the low toxicity threshold level for mercury and copper is partly related to their direct uptake from the solution. Metals such as zinc and cadmium have a higher toxicity threshold level (Bryan, 1976, 1979)

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because they are less readily taken up in the free form from the solution. The sensitivity of bivalve molluscs to zinc in, for example, Mya arenaria (Eider, 1977), Mytilus viridis (D’Silva and Kureishy, 1978), Mytilus edulis planulatus and Neotrigonia margaritacea (Ahsanullah, 1976), and S. plana (Akberali et al., 1981), in terms of both the threshold for any behavioral response and acute toxicity is one order of magnitude lower than the corresponding values for copper in S . plana (Akberali and Black, 1980), M. edulis (Davenport and Manley, 1978), and other bivalve molluscs (D’Silva and Kureishy, 1978; Manley and Davenport, 1979). Differences in the threshold levels of response to salinity decline have also been reported in terms of siphonal closure and shell valve adduction, which occur at about 25 and 20%0,respectively, in M . edulis (Davenport, 1979) and S. plana (Akberali and Davenport, 1981).

D. Development of Experimental Techniques The development of electronic and other analytical techniques has led to significant advances in our knowledge of bivalve behaviour. These techniques allow continuous monitoring of heart activity and valve movements both in the laboratory and under field conditions (Hoggarth and Trueman, 1967; Trueman, 1967; Vero and Salanki, 1969; Trueman et al., 1973; Coleman, 1974; Earl1 and Evans, 1974; Brand, 1976) and have stimulated interest in the physiological and biochemical adaptations associated with behavioural changes. Electronic techniques are a considerable advance over previous studies, which involved cutting holes in the shell to observe the heart or the use of a cumbersome system of threads and levers to record valve movements (Koch, 1917; Brown, 1954; Barnes, 1955; Schleiper, 1955; Segal, 1956; Pickens, 1965). To minimize disturbance to the animals, attempts have been made to observe heart activity without cutting the shell, e.g., in A . cygnea (Koch, 1917) and in M. edulis (Schleiper, 1957), observation of heart activity being made through the almost transparent shell of young individuals. Hers (1943) etched away the outer shell layers of older Anodonta with acid and viewed the heart through the thin, transparent, inner nacreous sheet. Although these modifications of the “shell hole” technique might reduce interference with the animal, the use of direct observation is time consuming and impractical in the field. Monitoring of heart activity using the impedance technique developed by Trueman (1967) requires the positioning of electrodes through the shell on either side of the heart. Once the electrodes are properly positioned, the animal need not be disturbed further and recordings can be made remotely, hence facilitating laboratory and field studies. Recordings of

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hydrostatic pressure in the pericardium and electrocardiographs have confirmed that the heart rate is satisfactorily recorded by the impedance technique (Helm and Trueman, 1967; Brand, 1976). The impedance technique has been used, both in the laboratory and in the field, to record heart and valve activity of bivalve molluscs and other marine invertebrates in relation to a variety of environmental changes such as tidal exposure (Trueman, 1967; Helm and Trueman, 1967; Coleman and Trueman, 1971; Coleman, 1972, 1974; Earll, 1975a), feeding (Thompson and Bayne, 1972; Widdows, 1973), temperature fluctuations (Segal, 1962; Trueman and Lowe, 1971; Lowe, 1974; Coleman, 1973, 1974; Parker, 1978; De Fur and Mangum, 1979; Dietz and Tomkins, 1980; Stone, 1980; Davenport and Carrion-Cotrina, 1981), environmental oxygen levels (Bayne, 1971; Lowe and Trueman, 1972; Badman, 1974; Brand and Roberts, 1973), salinity (Davenport, 1976, 1979; Akberali, 1978; Akberali and Davenport, 1981), pollution studies (Davenport, 1977; Manley and Davenport, 1979; Akberali and Black, 1980; Akberali et al., 1981, 1982b; Manley, 1983), and mantle cavity ventilation (Walne, 1972; Akberali and Trueman, 1979; Davenport, 1979). The relative size of the animals has also been shown to affect heart rate; in general, the heart beat in small specimens is faster than that of larger individuals in similar conditions (Pickens, 1965; Earll, 1975a). Monitoring of the water-pumping activity of bivalves has also been improved with the development of thermistor flow meters (Heusner and Enright, 1966; Brand and Taylor, 1974; Earll, 1975a,b; Brand, 1976; Foster-Smith, 1976; Akberali, 1978; Parker, 1978; Stone, 1980), although these have yet to be adapted for field recordings. This technique was, however, a great improvement on previous attempts at measuring the water flow through the mantle cavity involving the direct determination of water volumes by the insertion of tubes into the mantle cavity (Galtsoff, 1926, 1928) or the attachment of “rubber aprons” (Loosanoff and Engle, 1947) which considerably disturbed the animal being monitored. An alternative approach is the use of indirect particle filtration techniques which have posed a number of problems in the interpretation of the results, for these depend on several assumptions, any of which may not be met during the course of an experiment (Coughlan, 1969). In Pecten irradians after initial clearance on the gills, phytoplankton such as Nitzschia and Chlarnydornonas may be returned to the suspension (Chipman and Hopkins, 1954). Further doubt on the accuracy of plankton filtration studies has been cast by Hildreth (1980), who observed that considerable amounts of particulate material were produced from the faeces of M . edulis, which may be added to the suspension, resulting in an underestimate of filtration rate on the order of 35%. Furthermore, the flow rate of water across the

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gills can vary enormously, depending upon experimental procedure (Jergensen, 1966). A laser diffraction technique has recently been employed to measure the shell growth of Mytilus when subjected to fluctuating salinity regimes (Gruffydd et al., 1984) or the presence of copper or zinc in the sea water (Manley et al., 1984). This technique appears to be particularly suitable for future laboratory studies of the effect of stressors on shell growth. The use of electronic techniques has given further insight as to what constitutes “activity” in bivalve molluscs. The isolated observation of valve movements or indirect filtration methods led to a widely held interpretation of bivalve behaviour as a “steady state” of continuous feeding and activity (Loosanoff, 1939; Jgrgensen, 1966; Purchon, 1968). Recently, with the development of electronic techniques, this view has been questioned, for many examples exist in the literature which indicate differing levels of pumping, feeding, valve movements, and heart rate when bivalves are in the “active” state (Earll, 1975a,b). The results of recent studies (Purchon, 1971; Morton, 1973) make a “steady state,” or continuously active, concept difficult to apply, especially in an environmental context, for example, littoral bivalves are subject to tidal rhythms. Irregular discontinuity in pumping rate has also been observed in the sublittoral bivalves Zsognomon alutus (Trueman and Lowe, 1971) and Arctica islandicu (Brand and Taylor, 1974). Morton (1973) has put forward the view that in the long term feeding is discontinuous, exhibiting a distinct progression of events often closely synchronized with environmental rhythms. The concept of activity put forward by Loosanoff (1939), Jergensen (1966), and Purchon (1968) conceals the possibility of different levels of activity in “active” animals. The description of “active,” “routine,” and “standard” levels of metabolism in M . edulis, as defined by Bayne et al. (1973), would all fall into the category of animals described as “active” using Loosanoff s criteria (Earll, 1975a). Many other examples exist of bivalves which show differing levels of pumping, feeding, valve movements, heart rate, and shell deposition when they are in an “active” state (Earll,\,l975a; Coleman, 1974; Morton, 1969, 1970, 1971, 1973; SalAnki, 1966,a,b; Stone, 1980). In M . edulis, Bayne et ul. (1973) have suggested that, in the laboratory, once a period of behavioural adaptation has elapsed the main activities are those concerned with the maintenance of pumping for both feeding and respiratory purposes. Earll (1975a,b) defines “activity” as the maintenance of the pumping current for feeding, respiration, and excretion and “inactivity” as the cessation of pumping. However, most bivalves can show short-term discontinuous pumping patterns while still active (Earll, 1975a,b; Brand, 1976; Akberali, 1978; Parker, 1978; Stone, 1980), and accordingly Earll has defined inactivity as

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a period of prolonged cessation of pumping (exceeding an arbitrary period of 1 h) to differentiate between a short cessation of pumping during activity . The effects of environmental variables on bivalve molluscs have been studied at the organismal, tissue, and cellular level. In the past, the main approach adopted has been the “steady state” or “direct transfer” experiments where either a particular response or a multitide of responses of the animal is monitored in relation to experimental variation of a single environmental variable, e.g., salinity, temperature, or pollutant, while maintaining other factors as constant as possible. Although such studies provide a less complicated basis for understanding interactions with a particular environmental variable, doubts have been recently expressed as to whether animals in their natural habitat will ever encounter changes in a single environmental variable. For example, salinity variation in the natural habitat may be expected to alter other related factors such as temperature or oxygen solubility. Further doubts have been raised as to whether changes in the media reach a steady state in the natural habitat, especially with respect to intertidal bivalves (Davenport, 1982). This has led to the design of multivariate dynamic experiments in an attempt to simulate naturally variable conditions. Regimes such as salinity-temperature, salinity-pollutant, temperature-pollutant, and temperature-oxygen have been attempted. It is not intended to review the numerous papers on this subject here, but the following should be consulted: Stickle and Ahokas (1975), Davenport et al. (1975), Shumway (1977), Widdows (1978), Cawthorne (1979), and Manley (1980), together with a review written by Davenport (1982) on the subject of environmental simulation experiments on marine and estuarine animals.

II. Behavioural Responses to Stress A. Valve Closure as a Protective Mechanism Bivalve molluscs can isolate their tissues from the external environment by closing their valves, and some have been shown to minimize desiccation by valve adduction during periods of emersion at low tides. In M . edulis (Helm and Trueman, 1967; Coleman and Trueman, 1971), Cardium glaucum, and C. edule (Trueman, 1967; Boyden, 1972a) bradycardia occurs in response to littoral exposure when the valves become closed; activity is depressed, resulting in the animal respiring at lowered levels. In I . alatus (Trueman and Lowe, 1971) and Modiolus demissus (Lent, 1969), controlled valve opening exposes a film of water between the mantle

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

109

margins when exposed to air. This may serve both for oxygen exchange and as a source of moisture for evaporative cooling (Lent, 1968). When the level of water in an experimental tank was lowered beneath S . plana, bradycardia occurred and the valves closed (Earll, 1975b). Similarly, when Scrobicularia were transferred from normal sea water to a lowered salinity, the valves tended to remain closed; the’greater the drop in salinity, the greater the tendency to remain closed (Freeman and Rigler, 1956; Akberali, 1978). The activity of both intertidal and subtidal species of Modiolus, as measured by valve movements, was also reduced when the animals were exposed to either a high or a low salinity. The valve movements appeared to return to normal after a period of time the duration of which was dependent on the magnitude of the salinity change (Pierce, 1971). Valve closure in response to the presence of heavy metal pollutants has also been reported in a number of bivalve molluscs (Davenport, 1977; Manley and Davenport, 1979; Akberali and Black, 1980; Akberali et al., 1981). It has also been shown that low temperatures induce “hibernation” in oysters (Galtsoff, 1928). At low temperatures Mercenaria (Venus)mercenaria may remain completely closed for a period of several days; one clam was closed for a period of 18 days (Loosanoff, 1939). Valve closure response in M . edulis has been shown to operate at about -1.5”C when subjected to simulated subarctic conditions, which ensures that the tissues of mussels are exposed to little or no osmotic stress (Davenport and Carrion-Cotrina, 198 1). Furthermore, Loosanoff (1942) has also reported that M . edulis remained open more than 75% of the total time at temperatures as low as -1.O”C. The freshwater clam Pleurobema coccineum showed a continuous level of activity under well-aerated conditions, whereas in the absence of oxygen a remarkable activity shift occurred, consisting of prolonged shell closure interrupted by periods of intense valve activity (Badman, 1974). Bivalves respond to the presence of predators either by taking avoiding action or by closing the shell as a protective measure. The swimming of scallops by the rapid flapping of the valves in response to starfish is well known (Brand and Roberts, 1973), and the leaping movements of two species of the Asaphidae by flexure of the foot have been observed by Ansell (1967). Simple experiments (A. Jenkins and A. R. Brand, personal communication) during investigations of the feeding behaviour of the starfish Astropecten, using Venus striatula and Tellina fabula, indicate that the former may survive ingestion by the starfish for periods of longer than 1 week with its valves closed, but that Tellina appears to be unable to close its valves for more than brief periods and is fairly rapidly consumed after ingestion. Astropecten is only able to consume T . fabula when placed in aquaria without sand, for a shallow layer of sand allows this

110

H . B. AKBERALI A N D E. R . TRUEMAN

species to escape. This suggests two alternative strategies by which bivalves may respond to predator stress, either by rapid locomotion and escape or by the closure of thickened valves with the ability to remain quiescent utilizing anaerobic respiration (Section IV,C). Further experiments with a range of sessile and active infaunal bivalves are required to elucidate this (Trueman, 1983b). Valve closure of bivalves prevents drastic changes in osmotic concentration of their body fluids when exposed to short-term fluctuations in salinities (Shumway, 1977). With long-term exposure to salinity change, acclimatization of the internal body fluids does occur (Akberali, 1980a), suggesting that the valve closure mechanism allows the animal a period of grace and thus prevents osmotic shock. In M. edulis, Davenport (1979) showed that the valve closure response to short-term gradual decline in salinity is partially dependent upon rate of change of salinity. This led him to the discovery that the isolation of the mantle cavity fluid is not simply produced by valve closure, but comprises a three-part sequence. The first part of this sequence is the closure of the exhalant siphon, which effectively ceases the irrigation of the mantle cavity. Further decline in external salinity results in the closure of the inhalant siphon, which is then followed by valve closure. A similar sequence has also been reported in S . plana (Akberali and Davenport, 1981). Although “shell closing” in bivalve molluscs may help the organism to withstand transient adverse changes in the environment, it cannot contribute to its long-term survival in situations where a change in the environment may be of a permanent or recurrent nature. This is because during any period of valve closure the animal incurs penalties related to feeding, reproduction, or exchanges of gases and metabolites. The term “valve closure” does not necessarily imply that there is no contact between the tissues and the media. In the strictest sense, valve closure means the complete sealing of the ventral margins of the shell. Studies have shown that during valve closure some exchange or contact with the environment is often maintained. In M. edulis, for example, during exposure at low tides, there is a reduction in valve gape and apposition of the mantle margins but usually a small gape remains (Coleman and Trueman, 1971). Low salinity stress produces a similar feature in Donax denticulatus (Trueman, 1983a), and Perna perna has been observed to respond to copper ions by incomplete valve closure where the periostracal margins of the two mantle lobes are brought into contact so as to seal the shell from the exterior (Hodgson, unpublished). Furthermore, bivalves such as Mytilus californianus, Cardium edule, M . edulis, and S . plana, with the valves forcibly clamped together show a greater drop in the oxygen tension or increase in calcium concentration of the

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

111

mantle cavity than unclamped clams (Moon and Pritchard, 1970; Boyden, 1972a; Coleman, 1972; Akberali et al., 1977; Akberali and Trueman, 1979). When a bivalve is exposed to salinity or pollutant stress, the maintenance of some slight tissue contact with the media which may be intermittent is advantageous, for it allows the organism to monitor the situation and become active once the prevailing conditions become favorable (see Section 11,B). The animal must rely on the registering of chemical changes by a slight tissue contact with the media. This is in contrast to bivalves when exposed at low tide, for these may register vibrations due to water movement or temperature changes at reimmersion. B. Relationship between Heart Rate, Valve Movement, and Pumping Activity The heart rate of bivalves is a useful physiological rate function since it may easily be monitored together with valve movements and pumping activity (Trueman et al., 1973). Amplitude of heart beat is not, however, easy to monitor and interpret successfully using impedance techniques. A relationship between heart rate and valve movements was demonstrated many years ago by Koch (1917), who showed in Anodonta that the heart rate is reduced during periods of valve closure. More recently this phenomenon has been reported in a number of other bivalve molluscs (Bayne, 1971; Coleman and Trueman, 1971; Coleman, 1974; Earll, 1975a,b; Brand, 1976; Akberali, 1978; Dietz and Tomkins, 1980; Akberali et al., 1981). In bivalve molluscs high levels of heart rate are associated with periods of activity as indicated by valve gape, high rates of respiration, and filtering/pumping activity, whereas periods of inactivity are associated with a low heart rate (Fig. 1). Such marked changes in heart rate accompany different activity levels in bivalves, such as those which occur when pumping ceases and the valves close. Studies of some species, e.g., M . edulis, Anodonta anatina, M . arenaria, and Ostrea edulis, have demonstrated that when kept in constant conditions, bivalves show a particular activity level with relatively little change in the heart rate. The heart rate of these bivalves during activity is largely independent of gross changes in pumping activity. Depending on the species, heart rate might be expected to vary by 2, 3, or 4 beatslmin over a period of hours (Coleman, 1974; Earll, 197%). By far the most marked changes of heart rate occur when animals become inactive, for in these situations pumping ceases and in some species the valves are often completely closed (Fig. 1). This inactive period is associated with a marked slowing of the heart or bradycardia. Bradycardia is a term usually applied to describe major changes of heart rate in bivalves, such as be-

1 I2

H . B . AKBERALI AND E. R. TRUEMAN

L

c L

m

a

I

15 r

-I

lot

2

/---

A

-

\ Hours

FIG. 1. Analysis of events during experiments on the effect of exposure on the heart rate and valve gape of M. edulis (A), and the effect of exposure on the heart rate of Modiolus modiolus (B). The rate is measured as beatshinute, and gape in degrees. The period of exposure is indicated by the horizontal line above each (from Coleman and Trueman, 1971).

tween different activity levels, rather than to small fluctuations in rate during a particular activity level in the sense used by Boyden (1972a). Following periods of inactivity, pumping recommences and the heart rate increases markedly, often to a level greatly exceeding the heart rate of continual activity. This is commonly referred to as an “overshoot” effect. Within an hour of the onset of activity, the heart rate usually falls to a rate which is similar to that shown throughout the rest of the period of activity (Trueman, 1967; Helm and Trueman, 1967; Coleman and Trueman, 1971; Brand and Roberts, 1973; Coleman, 1974; Earll, 1975b; Brand, 1976; Taylor, 1976a,b; Akberali, 1978; Parker, 1978). Pumping, associated as it is in bivalves with both feeding and respiration, has been considered of primary importance in studies of activity. While recognizing the importance of pumping, it is often more convenient to obtain an indication of pumping activity by monitoring valve movements. Since pumping activity usually occurs when the valves are gaping, it is possible to obtain some indication of pumping activity from valve movements, but in some species these may be at a superficial level and lead to wrong conclusions. For example, in M . edulis, although the valves may be gaping and at the same time be exhibiting a high heart rate, the mussel need not necessarily be actively pumping (Bayne et al., 1973).

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EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

Davenport (1979, 1982) showed the closure of the exhalant siphon, thus preventing pumping, was the crucial event which largely isolated the mantle cavity of the mussel from falling external salinities; shell valve closure occurred at rather lower salinities to produce virtually complete isolation. Akberali and Davenport (1981) have shown that closure of the exhalant siphon is even more important to Scrobicularia, in which absolute valve closure is impossible, and in which valve adduction is delayed until low external salinities occur. This is reasonable for an infaunal estuarine species since the interstitial salinities of estuarine muds tend to be high and relatively stable (Kinne, 1971). Scrobicularia would hardly be exposed to the rigours of the salinity fluctuations in the estuarine water column except in respect of water flowing through the mantle cavity. In some other species of bivalves, variations of heart rate during activity in response to large changes in pumping level seem either disproportionately small, e.g., 0. edulis (Walne, 1972), or nonexistent as in M. edulis (Thompson and Bayne, 1972). A “regular” heart rate is maintained in M. arenaria during activity while its pumping is essentially a discontinuous process, being interrupted for varying periods by closure of the exhalant siphonal aperture (Earll, 1975a) (Fig. 2). In Scrobicularia plana, pumping during activity is also a discontinuous process but the heart rate is sensitive to changes in pumping level (Earll, 1975a,b; Akberali, 1978). During activity, S . plana shows a high heart rate and trace amplitude so

1 min I

I

I

I

I

I

h

I

I

I

I

I

I

,

I

1

1

I

FIG,2. M . arenaria: simultaneous records of pumping activity (exhalant current speed H, high and 0, zero pumping level), impedance heart trace and midventral valve movement (dc impedance). Abbreviations: imp, impedance; 0, open; C, closed. Discontinuous pumping is not related to the heart record. A burst of water from the exhalant siphon (V)coincides with the elimination of faeces (from Earll, 1975a).

FIG.3. S. plana: simultaneous records of pumping level, heart activity, and shell movement during short-term variation of heart activity; open (0)and closed (C) (from Earll, 1975a).

I15

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

long as pumping is maintained (Fig. 3). When pumping is interrupted during activity, however, a gradual fall occurs in the heart rate and the amplitude of the heart trace falls rapidly to a low level (Fig. 3). Earll (1975a,b) has broadly defined the short-term variation in the heart pattern of Scrobicularia plana as a repeating cycle of heart activity beginning with a transition to a high rate. This is then followed by a slowing down in a variable manner until a period of lowered heart rate is reached, which precedes the transition to the high heart rate once again (Fig. 4). In Scrobicularia, the heart rate and heart amplitude coincide significantly with changes in pumping level, suggesting that heart function is more sensitive to the cessation of pumping. This close relationship of heart rate and

I

10

5

I

Time fh)

0

20

c

5

I

30

60

T i me (min)

FIG. 4. Heart activity patterns of M. arenaria (A) and S. plana (B) when constantly submerged at 10°C: heart rate in Mya during behavioural inactivity shown by the continuous line (-); in Scrobicularia during activity, a cycle of heart rate variation as shown by the broken line (----) between transition, T, from low to high rates (from Earll, 1975b).

I16

H . B. AKBERALI AND E. R. TRUEMAN

pumping during activity, described by Earll (1975b), and confirmed by Akberali (1978), is uncommon in other bivalves which have been studied (Coleman, 1974; Earll, 1975b). Another example occurs in Donax serra, buried normally in sand, where valve adductions may be observed when the exhalant current stops flowing and the heart beat immediately ceases (Trueman, unpublished). Replacement of stressful change in environment, e.g., polluted, salinity, aerial exposure, with clean sea water is associated with a rapid resumption of activity (Fig. 5). An initial series of valve adductions, which may be associated both with testing behaviour and hyperventilation of the mantle cavity, is usually evident within 5-10 min, which is then followed by the progressive opening of the valves and extension of siphons. Accompanying this is a marked increase in heart rate, often beyond normal activity rates (Figs. 7, 10). This “overshoot” response is more marked in clams subjected to severe environmental changes and is comparable to the “overshoot” response observed in a number of studies with bivalve molluscs (see Section 11,B). The greater the change in the environment from normal conditions, the longer are the inactivity periods resulting in the cessation of pumping activity, isolation of the clam from the media, and pronounced bradycardia. The longer the inactive period the greater the “overshoot” response to compensate for the physiological needs in terms of respiration, feeding, and excretion.

MINUTES

FIG.5. Examples of recordings of heart beat and valve movements of S. p l u m at 10°C in control (A) and animal exposed to 10 ppm added zinc (B) for 6 h when test solutions were replaced by fresh sea water; c (J) and e (4) indicate removal of test solutions and d (1) subsequent replacement with fresh sea water (op, open; cl, closed). (From Akberali et al., 1981).

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

117

C. Behavioural Response to Some Pollutant Stressors

Since the heart rate in S . plana is so closely related to pumping, the heart rate may be utilized as a means of monitoring interactions with environmental stress (Akberali and Black, 1980). Whenever a high heart rate is recorded from this clam, pumping is indicated, thus implying interaction with the particular environment, and conversely. An example of this is found in the response of Scrubicularia to sea water with added copper to final concentrations of 0.01-0.5 ppm. The immediate response is valve closure, but the timing and duration of this varies with the copper concentration (Fig. 6) and is associated with an

B

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FIG.6. Heart rate (a) and valve movements (b) of individual Scrobiculuriu during the first hour when subjected to various copper concentrations. A, 0.5; B, 0.1; C, 0.05; and D, 0.01 ppm copper concentrations in sea water (S, 31%0). Arrows indicate addition of copper solution. Heart rate (HR) sampled by counting heart beats per minute for each minute. Valves open (o),closed (c) (from Akberali and Black, 1980).

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H. B. AKBERALI A N D E. R. TRUEMAN

immediate drop in the heart rate indicating a cessation in pumping activity (Akberali and Black, 1980). In 0.01 and 0.05 ppm, heart rate subsequently increased, while in contrast, the heart rate in 0.1 and 0.5 ppm kept falling during the first hour of recordings. Over the 6-h exposure period to 0.5 ppm, the heart rate was maintained at about 6-8 beatdmin and the valves remained closed (Fig. 7). After 2-3 h in lower concentrations, there was an increase in heart rate and intense valve activity was observed, indicating interaction with the polluted sea water (Figs. 7 and 8). Akberali er ul. (1981) have also used heart rate and valve movements to examine the behavioural response of S. p l u m to various zinc concentrations. At concentrations of 0.1-0.5 ppm, the clams did not behave differently from the controls. At higher concentrations, however, the siphons were withdrawn and valves remained closed, with a marked lowering of A

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2

3

4

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6

7

8

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FIG.7. Heart rate (HR) of Scrobicularia subjected to various copper concentrations over a 6-h exposure period. A, 0.5; B, 0.1; C, 0.05; and D, 0.01 ppm copper concentrations in sea water (S, 31%0). Addition of copper solution indicated by J, replacement with normal sea water by t. HR of each Scrobiculariu over the 6-h exposure period sampled by averaging 5min periods. Horizontal bars (-) refer to increased activity in heart rate and valve movements during the 6-h exposure period. Each point is a mean of four animals recorded in each concentration. Vertical bars (I) represent the range of individual variation (from Akberali and Black, 1980).

EFFECTS O F STRESS ON MARINE BIVALVE MOLLUSCS

I19

A

B

C

D U

I

5 min

FIG. 8. Examples of recordings of heart activity (HA) and valve movements (VM) of Scrobicularia 2-3 h after subjection to various copper concentrations and corresponding to the state of increased activity referred to in Fig. 7 by horizontal bars (-). A, 0.5; B , 0.1; C, 0.05; and D, 0.01 ppm. Copper concentrations in sea water (S, 31%0). Valves open (o), closed (c) (from Akberali and Black, 1980).

heart rate (Fig. 9). Concentrations of 1-5 ppm zinc resulted in a partial resumption of activities in some clams, while animals exposed to 10 ppm zinc maintained valve closure over the experimental period, accompanied by pronounced bradycardia (Fig. 10). At lower zinc concentrations, the frequency and duration of interaction with the environment became greater over a 6-h period, as reflected by increase in heart rate. Similar behavioural responses have been reported in S. plana exposed to abrupt salinity changes or the first hydrolytic product (1-naphthol) of the insecticide Sevin (Akberali, 1978; Akberali et al., 1982b). Lethal toxicity tests based on a fixed exposure period, e.g., 24-h LCso,

H. B. AKBERALI A N D E. R. TRUEMAN

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FIG.9. Examples of recordings of heart beat and valve movements of S. plana at 10°C on addition of various concentrations of zinc as described in the original paper. AJ, addition of stock zinc solution; BJ, attainment of final zinc concentrations. Valves open (op), closed (cl) (from Akberali et al., 1981).

48-h LCso,or 96-h LC50,may lead to erroneous interpretation of results in terms of toxicity threshold. In some bivalves, e.g., S . plana, it has been shown that the clam can withstand valve closure for periods of 5-7 days (Akberali et al., 1977; Akberali, 1978) while effectively isolating its tissues from the stress environment. In such animals, care should be taken in interpreting toxicity results based on fixed exposure periods. In these bivalves, median lethal time exposures would be more meaningful than fixed exposure periods. For example, when M. edulis (Davenport, 1977) and S. plana (Akberali and Black, 1980) were subjected to short-term (6h) 0.5 ppm copper concentration they suffered no mortality (Table 11) since they closed their valves immediately. Similar observations have

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

121

4’

a l ~ , ~ H o u r s 0 1 2 3 4 5 6 FIG.10. Effects of various levels of added zinc to normal sea water on the heart rate of S. plana at 10°C. Arrow (4)at 0 h indicates application of test concentration of zinc, while f at 6 h indicates replacement with fresh sea water. Each point is a mean of 4-8 animals. Vertical 0.5 ppm zinc ( N = 4); A-A, 1 ppm zinc ( N = bars represent the standard error. W-H, 7); A--A, 5 ppm zinc ( N = 5 ) ; 0-0, 10 ppm zinc ( N = 8). 50 ml normal sea water (1) applied to controls (0-0, N = 5). Treatment for controls after 6 h as in test solutions (from Akberali et al., 1981). I~~~~~~~~~

been made in Scrobicularia subjected to short-term (6-h) 5-10 ppm zinc (Akberali er al., 1981) or 1-naphthol (Akberali et al., 1982b). A longer exposure period, however, resulted in an increase in mortality, with a median lethal time of 2-7 days (Table 11). Akberali and Black (1980) and Akberali et al. (198 1) have suggested that there is a limit to the duration of anaerobic respiration, since the clam must eventually eliminate its accumulated wastes. When this occurs, the tissues then come into contact with the media, poisoning takes place, and death results.

D. Relevance of Valve Closure in Epifaunal and Infaunal Species Not all bivalve molluscs are capable of complete valve closure; e.g., infaunal species such as S. plana and M . arenaria show a reduction in heart rate indicative of a period of reduced activity which can frequently be related to the closure and the partial or complete retraction of the siphons into the burrow (Coleman, 1974; Earll, 1975a,b; Akberali, 1978; Akberali and Davenport, 1981). In littorally adapted epifaunal M . edulis, water is retained in the mantle cavity during exposure at low tide by the apposition of the mantle margins coupled with reduction of valve gape, but usually an opening remains (Coleman and Trueman, 1971). The retention of mantle cavity water reduces the danger of desiccation from aerial exposure or from osmotic shock during a decline in external salinity.

TABLE11. ADULTBIVALVE MORTALITYIN RELATIONTO THE PERIOD OF EXPOSURE TO POLLUTANT Species

Pollutant stressor

Concentration

STRESS'

Duration of exposure

Observed response

5 mg/l 39 Fg/l 35 P d l

48 h 96 h 168 h

LCJO LCSO LCSO

Eisler (1977)

Reference

M . arenaria

Copper

M . arenaria

Zinc

52 mg/l 5.2 mg/l 1.6 mgA

48 h 96 h 168 h

LCso LCSO LCSO

Eisler (1977)

M . edulis

Copper

300 pg/l

Continuous exposure for 7 days

LCSO

Scott and Major (1972); Martin e f al. (1975)

M . edulis

Copper

500 c~gfl

6h Continuous exposure Continuous exposure

No mortality MLT 4-5 days MLT 2 days

250 pg/l 500 pg/l

Davenport (1977)

M. edulis

Zinc

S. plana

Copper

S. plana

Zinc

> 5 mgfl

0.1-20 mg/l 10 mgA

20 mg/l

S.plana

1-Naphthol

1-10 mg/l

5 mgA 10 mg/l

Martin et al. (1975)

Continuous exposure for 7 days

6h Continuous exposure

No mortality MLT 5-7 days

Akberali and Black (1980)

6h Continuous exposure Continuous exposure

No mortality MLT 6 days MLT 5 days

Akberali et al. (1981)

6h Continuous exposure Continuous exposure

No mortality MLT 15 days MLT 9 days

Akberali et al. (1982b)

Abbreviations: MLT, median lethal time for 50% mortality; LCs0, lethal concentration for 50% mortality.

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H . B. AKBERALI A N D E. R. TRUEMAN

In the M . edulis, a 50% fall in heart rate occurs within 20 min of being exposed at low tides (Coleman and Trueman, 1971; Coleman, 1972, 1973). The behaviour of M . edulis is similar to M. californianus (Moon and Pritchard, 1970; Bayne et al., 1976b) in that the valves are gradually closed at the onset of aerial exposure to retain water in the mantle cavity. Oxygen from the air, however, diffuses into the mantle cavity, leading to a higher oxygen tension in the mantle cavity than if the valves had been tightly closed (Moon and Pritchard, 1970). M . edulis respires during exposure at a level which approaches the lowest levels animals show when immersed (Widdows et al., 1979). Oxygen uptake, however, is erratic, and it appears to depend on an occasional opening of the valve (Coleman, 1973; Bayne and Livingstone, 1977). In the epifaunal M . demissus (Kuenzler, 1961; Lent, 1968), some water is expelled from the mantle cavity at the onset of exposure and the valve gape is reduced to about half that found when the animals are pumping actively. This enables air to enter the mantle cavity, so allowing aerobic respiration (Table 111). In M. edulis and Mytilus galloprovincialis, an aerial rate of between 4 and 17% of the rate of oxygen consumption in water occurs, and in C . edule and M . demissus between 28 and 78% has been reported (Widdows et al., 1979), the species differences being related to the degree of shell gape during exposure (Table 111). In C . edule, as in M . demissus (Kuenzler, 1961; Lent, 1968), some water is also expelled from the mantle cavity at the onset of exposure (Boyden, 1972a,b), and the heart rate of Cardium initially rises on exposure and then falls, but is maintained at a relatively high level throughout exposure (Trueman, 1967; Boyden, 1972a,b). I . alatus, a tropical epifaunal species, maintains a small valve gape during exposure, and it has been suggested that this species also respires aerobically (Trueman and Lowe, 1971). Another behavioural pattern is adopted by infaunal species such as C . glaucum, S. plana, and M . arenaria (Boyden, 1972a; Earll, 1975a,b). In these species, for example, little or no valve movement occurs during exposure, a pattern in contrast with M . demissus, C . edule, and M . edulis, which show a reduced but regular sequence of valve activity and the ability to utilize atmospheric oxygen during exposure at low tides (Table 111). For example, C . glaucum (Boyden, 1972a) seals its valves tightly, whereas M . arenaria (Dicks, 1972) and S. plana (Earll, 1975b) close the siphonal apertures and withdraw the siphons into the burrow. Neither S. plana, C. glaucum, nor M . arenaria has been reported to utilize atmospheric oxygen during exposure at low tide, and oxygen comsumption in these species is very low during exposure and difficult to demonstrate (Collip, 1920, 1921; van Dam, 1935; Boyden, 1972a; Dicks, 1972). Furthermore, in these infaunal species a complete cessation of heart rate and

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

125

valve movement occurs during exposure, and it is possible that they are entirely dependent upon anaerobic respiration during exposure at low tides (Table 111). Sublittoral species and those which live at low intertidal levels show erratic behaviour during experimentally simulated tidal exposure. This in part accounts for the greater variability of the heart and valve movement records found in these species during aerial exposure at low tides, e.g., M . modiolus (Coleman and Trueman, 1971; Coleman, 1972) and Pecten maximus (Brand and Roberts, 1973). Modiolus and Pecren appear unable to regulate either valve movements or the retention of water in the mantle cavity during aerial exposure. The behavioural differences and the subsequent biochemical and physiological adaptations between littoral epifaunal and infaunal bivalves pose a few interesting questions. Although the respiratory system of bivalves is characteristic of aquatic life, several epifaunal bivalve species, such as M . edulis, M . demissus, M . californianus, M . galloprovincialis, and also C. edule, are capable of utilizing atmospheric oxygen and may, during aerial exposure, obtain part of their energy requirement by aerobic respiration (Moon and Pritchard, 1970; Boyden, 1972a,b; Coleman, 1973; Bayne el al., 1976a,b; Bayne and Livingstone, 1977; Widdows et al., 1979). In infaunal species such as C. glaucum, S. plana, and M . arenaria, the opportunity for aerial respiration is reduced by the restricted atmospheric contact and the potentially anoxic conditions prevailing in the interstitial mud (Brafield, 1964). In these species there will thus be a far greater need for anaerobic respiration to sustain basal metabolism, either during exposure at low tides which results in the cessation of pumping activity or when exposed to other environmental stress conditions, such as saiinity or pollutants. These may induce valve closure and anaerobiosis in all bivalves, except those such as M . modiolus which appears unable to maintain valve closure. Moreover, among species shown to utilize atmospheric oxygen during exposure at low tides, differences in biochemical and physiological adaptations do exist in response to exposure at low tides. For example, M . edulis responds to aerial exposure by maintaining a tighter control over its valve gape than C . edule and M . demissus (Kuenzler, 1961; Coleman and Trueman, 1971; Boyden, 1972a,b; Coleman, 1973; Widdows et al., 19791, and this is reflected in the increase in aerial rate of oxygen uptake in Cardium and Modiolus compared with Mytilus (Table 111). This is also associated with greater need for utilizing anaerobic pathways in M . edulis, with subsequent greater accumulation of end products in the tissues than in C. edule (Widdows et al., 1979). The accessibility of the tissues of Cardium and Myrilus to atmospheric I4CO2, and hence to atmospheric oxygen during air exposure, has been investi-

TAESLE 111. COMPARISON OF BEHAVIORAL RESPONSES A N D ABILITYTO UTILIZE ATMOSPHERIC OXYGEN DURING EXPOSURE AT Low TIDEIN SOMEBIVALVE MOLLUSCS~ Species

Zone

Habitat

Valve activity

Oxygen utilization

Aquatic rate

Anaerobiosis demonstrated

Reference

M . edulis

Littoral

Epifaunal

Closure with a small gape

+

4-6%

+

Coleman and Trueman (1971); Coleman (1974); Bayne and Livingstone (1977); Widdows et al. (1979)

M . californianus

Littoral

Epifaunal

Closure with a small gape

+

74%

+

Moon and Pritchard (1970); Bayne et al. (1976b)

M . galloprovincialis

Littoral

Epifaunal

Closure with a small gape

+

11-17%

+

Widdows et al. (1979)

M. demissus

Littoral

Epifaunal

Closure with a controlled wide gape

+

56-65%

+

Kuenzler (1961); Lent (1968, 1969); Widdows et al. (1979)

I. alatus

Littoral

Epifaunal

Closure with a controlled wide gape

M . modiolus

Sublittoral

Epifaunal

Unable to maintain valve closure

C . edule

Littoral

Surface infaunal

Wide shell gape

C. glaucum

Littoral

Surface infaunal

Closure

M . arenaria

Littoral

Deep infaunal

S. plana

Littoral

Deep infaunal

Symbols:

+, shown to occur; -,

t

+?

Trueman and Lowe (1971)

Coleman and Trueman (1971); Coleman (1976)

+

+

Boyden (1972a,b); Widdows et al. (1979)

+

Boyden (1972a,b)

Closure

+?

Collip (1920, 1921); Dam (1935); Dicks (1972); Earll (1975a)

Closure

+?

Earll (1975a,b); Akberali er al. (1977)

-

shown not to occur; +?, not shown, but likely.

28-78%

128

H . B . AKBERALI A N D E. R. TRUEMAN

gated by Ahmad and Chaplin (1977). They showed that C. edule is more efficient than M . edulis at incorporating 14C02into its tissues, which is in part a reflection on the wider shell gape of Cardium during exposure to low tides. Furthermore, in M . edulis a greater proportion of the total radioactivity was recovered from anaerobic end products such as succinate than in C . edule (Ahmad and Chaplin, 1977). These authors concluded that Mytilus uses anaerobiosis to a greater extent than Cardium. At present, it is not known whether such differences exist between epifaunal and infaunal species in their capacity for anaerobic respiration.

E. EfJPct of Subthreshold Levels on Behaviour Marine bivalve molluscs are frequently exposed to a range of sublethal levels of environmental stresses such as salinity, temperature, oxygen, and pollutants. The adverse effects of environmental stress on aquatic organisms including bivalve molluscs have been generally identified with their acute and lethal impact. Mortality is an end point that can be readily recognized and quantified; hence the standard assay for acute toxicity testing of pollutants in aquatic organisms measures the particular stress condition or concentration that causes 50% mortality over a standard period of time (LCso). It is evident that death is a very crude index of stress in the environment, and that sublethal effects can be induced at much lower levels than the LC5,,. While not directly resulting in death, sublethal effects can affect survival through effects on behaviour, growth, physiology, and reproduction (Bayne et al., 1978, 1979, 1981; Viarengo et al., 1980b; Lowe et al., 1982; Calabrese et al., 1984). The ultimate test of significance of a sublethal effect of environmental stress is whether it has an impact on the propagation of a species and on its population (Waldichuk, 1979; Bayne et al., 1979, 1981). However, as Perkins (1979) has pointed out, the demonstration of a sublethal effect is often of limited use because the ecological significance of a change in the measured parameter is usually not established. The lower the level at which the effect is demonstrated, the more difficult it is to translate it into a meaningful ecological observation. Behavioural modification is one of the most sensitive indicators of environmental stress and may directly affect survival (Eisler, 1979). Available literature on bivalve behavioural response to stress is limited, but studies (Perkin, 1979; Eisler, 1979; Olla et al., 1983) carried out in the recent past indicate that sublethal effects on bivalve behaviour may give some insight into the observed physiological, biochemical, and reproductive responses.

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129

A behavioural avoidance mechanism to adverse environmental conditions has been shown to occur commonly in bivalves, and existing evidence indicates that below the sublethal threshold level the animal is capable of interacting with the environment. For example, it has been shown that siphonal and valve closure in M . edulis and S . plana is triggered at salinities of 25 and 20%0,respectively (Davenport, 1979; Akberali and Davenport, 1981). This implies that a drop from the normal salinity (32%0)to the respective salinities in these two species can be described as sublethal. Similarly, Akberali and Black (1980) and Akberali et al. (1981) have shown that S . plana interacts with 0.01-0.05 ppm copper and 0.10.5 ppm zinc in sea water. It is only when the organisms are subjected to a more extreme salinity or pollutant level that the behavioural avoidance mechanism is mediated. The present evidence indicates that during sublethal exposure, bivalve molluscs interact with stressors which may have long-term effects on metabolic processes. For example, sublethal stresses have been demonstrated to affect the behavioural and metabolic processes of bivalves in various ways. Both depth and rate of burrowing ofjuvenile hard clam M . mercenaria was affected by oil-contaminated sediments (Olla et al., 1983). These authors suggest that such effects indicated avoidance behaviour rather than oil-induced debilitation and may increase the vulnerability of this species to predation. McGreer (1979) studied the burrowing behaviour of the estuarine clam Macoma balthica in response to sublethal levels of mercury and cadmium. A correlation was found between higher concentration levels and decreased burrowing speed which was attributed to a behavioural avoidance mechanism. Similar effects on burrowing behaviour in the clam Protothaca (Phelps et d . , 1983) and in Venerupis decussata (Stephenson and Taylor, 1975) have been reported for copper in the sediment, the burrowing time being increased logarithmically with greater sediment copper concentrations. It has also been reported that sublethal levels of heavy metals decrease filtration rates in bivalve molluscs (Watling, 1981). In M . edulis, a 50% reduction in filtration rate was found at concentrations of only 0.04 ppm mercury, 0.15 ppm copper, and 1.6 ppm zinc (Abel, 1976). The rate of oxygen consumption of excised gill tissue of Crassostrea virginica showed a significant increase when continuously exposed to sublethal levels of 50 and 100 ppb copper (Engel and Fowler, 1979). The most obvious difference occurred after 14 days' exposure to 100 ppb copper, by which time the tissue concentration of copper had reached 0.8 pg/mg (dry wt). Robinson et al. (1984) have shown that in the surf clam Spisula solidissima, turbidity levels >100 mg/l of attapulgite clay resulted in a significant increase of pseudofaecal production and a

130

H . B. AKBERALI A N D E. R. TRUEMAN

decrease in the amount of algal food actually ingested. They have concluded that anthropogenic turbidity-producing discharges at low levels can possibly cause adverse effects on the energetics of surf clam populations. It has also been reported that continuous exposure to sublethal levels of copper and zinc suppresses gametogenesis in adult M . edulis, with copper being more toxic (Maung-Myint and Tyler, 1982).

F. Effect of Temperature on Heart Rate

It is apparent in many investigations (Pickens, 1965; Trueman and Lowe, 1971; Lowe and Trueman, 1972; Coleman, 1972, 1974; Davenport and Carrion-Cotrina, 1981) that heart rate is markedly affected by temperature. Increase in temperature leads to a rise in heart rate and a decrease in temperature results in a fall in heart rate in an intact M . edulis (Fig. 11). There is an almost linear relationship with a heart rate of 30 beats/min at 10°C decreasing to 3 beats/min at - 13°C (Davenport and Carrion-Cotrina, 1981). In the bivalve S. solidissima, an increase in temperature is associated with an increase in heart rate and vice versa (De Fur and Mangum, 1979). Similarly, in the freshwater Ligumia subrostrata the heart rate has been shown to be an exponential function of temperature (Dietz and Tomkins, 1980). In the bivalves M . arenaria and Crassostrea gigas, rapid temperature change also brings about an immediate response

I

0

n J '20

*10

0

-5

Temperature VC)

FIG. 11. M. edulis: effect of temperature on heart rate. Norwegian mussels (O), Welsh mussels (0) (from Davenport and Carrion-Cotrina, 1981).

EFFECTS O F STRESS ON MARINE BIVALVE MOLLUSCS

131

in heart rate (Lowe, 1974). In both these species, heart rate is dependent on the temperature of the bathing fluid, but during sudden changes of temperature there is significant relationship between heart rate and mantle cavity temperature, and it has been suggested by Lowe (1974) that thermoreceptors, possibly in the mantle tissue, play an important role with respect to the immediate response to temperature change. A number of examples of perfect, partial, and nonexistent acclimation of seasonal variation in heart rate have been described in the mussels M. californianus and M . edulis by Pickens (1965). He suggested that these results might be accounted for by considering the effects of condition, in particular the reproductive state and food availability on a seasonal basis. Widdows (1973), in a closely controlled experiment, confirmed many of the results obtained by Pickens (1965). Using M. edulis he studied the effects of acclimation on heart rate, oxygen consumption, and pumping rates, and the effect of starvation. M. edulis acclimated at a set temperature was transferred to higher or lower temperatures and showed examples of both partial and complete acclimation in terms of oxygen consumption and pumping rate. Bayne et al. (1973) have also reported that the acclimation of these functions varies consistently on a seasonal basis. Heart rate, however, shows no acclimation in the long term and remains dependent upon ambient water temperature. Heart rate has been used as a measure of temperature acclimation in a number of poikilothermic animals such as M. edulis and M . californianus (Pickens, 1965; Widdows, 1973). Perfect acclimation of heart rate to temperature alone was not evident in either of these species and consequently seasonal changes in heart rate were attributed to other factors. Widdows (1973) found that starvation of M. edufis produced a 35% reduction in heart rate over 9 days, emphasizing the point that in the field, heart rate may depend not only on the ambient water temperature but also on the nutritive and reproductive state of animals. In addition, temperature has been shown to affect burrowing behaviour in the tropical surf clam D . denticulatus (Trueman, 1971, 1983a; Ansell and Trueman, 1973) and in D . serru (McLachlan and Young, 1982). Environmental variables, especially temperature, do affect patterns of activity on a seasonal basis, and some species, such as M . mercenaria, show a reduction in the duration of active periods (Loosanoff, 1939) and shell deposition (Panella and McClintock, 1968; Jones et al., 1983) with seasonal fall in temperature. A similar reduction in activity was found in A. cygnea (Salanki et al., 1974; Parker, 1978), although short-term fluctuations in temperature, which were equivalent to natural daily variation, appeared to have a minimal effect on periodic activity.

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H . B . AKBERALI A N D E. R. TRUEMAN

111. A.

Detection of Stress

The Signijicance of Registering Changes in the Environment

Many bivalve molluscs, when exposed to lethal levels of environmental stress, rely on behavioural mechanisms which enable them to avoid contact with such conditions (see Section 11, Behavioral Responses to Stress). Mobile species have the ability to move away from regions of potentially harmful stress conditions, whereas sedentary species possess behavioural mechanisms such as burrowing into the substratum, retracting into existing burrows, or closing of valves. In employing such mechanisms for osmotic control in response to salinity fluctuations and in avoiding harmful effects of pollutants, bivalve molluscs must therefore be capable of detecting changes in the environment and implementing the appropriate response. The immediate detection of environmental changes is essential for the success of any protective response. This applies at both the commencement and the termination of stress so as to allow feeding to be resumed immediately on the removal of the stress. Detection of changes in the environment by bivalves is particularly important with respect to distinguishing between sublethal and lethal levels and eliciting appropriate behavioural responses to lethal levels. Occurrence of environmental stress is often intermittent, being affected by the duration of tidal cycles, currents, and variable freshwater runoff. These factors are particularly relevant in intertidal and estuarine conditions. Reference should be made to the detailed review by Davenport (1982) on environmental simulation experiments. Bivalve molluscs such as S . plana, M . edulis, C. edule, and 0. edulis have been shown to accumulate heavy metals in their tissues far in excess of the environmental levels (Phillips, 1977; Bryan, 1979; Bryan and Gibbs, 1983; Viarengo et al., 1981; Calabrese et al., 1984). Tissue concentrations in S . plana at Restrongnet Creek (Cornwall) show a wide variation in metal ion concentration. The highest concentrations of 7270, 101, and 25 pg/g (dry wt) for zinc, copper, and manganese, respectively, occurred in the digestive gland, whereas highest iron concentration of 2051 pg/g (dry wt) was found in the mantle and siphons (Bryan and Gibbs, 1983). Such differences in tissue concentration in relation to various heavy metals and also between different localities have been reported in numerous studies involving bivalve molluscs. Akberali and Black (1980) and Akberali et al. (1981) have shown that S . plana avoids levels of 100-500 pgfl (0.1-0.5 ppm) and > 5000 pg/l (> 5 ppm) of copper and zinc concentration, respectively, by prolonged valve

EFFECTS O F STRESS ON MARINE BlVALVE MOLLUSCS

133

closure and thus protects the tissues from the presence of these heavy metals. However, at concentrations below these levels the clams continue to interact with the pollutant after an initial valve closure response. This response to lower concentrations in S. plana, which is probably similar to other bivalves, implies that bivalves may discriminate between toxic and nontoxic effects of heavy metals. It is important to emphasize that the high tissue levels of heavy metals, for example copper and zinc, in Scrobicularia could only have accumulated from heavy metals associated with particulate food or in solution in the ambient medium at subthreshold levels. The temporary incidence of lethal levels could be avoided by valve closure and would not lead to accumulation in the tissues. It has been rightly pointed out, in Scrobicularia for example, that bivalves would avoid the worst of conditions by deposit feeding when sediment metal contamination is low and suspension feeding when dissolved metal level is low (Bryan and Gibbs, 1983). It has also been shown that osmoconforming bivalves such as M . edulis and S. plana minimize osmotic stress when exposed to low external salinities of short-term duration by isolating their tissues and body fluids from the water to a considerable extent (Shumway, 1977; Davenport, 1979; Akberali and Davenport, 1981). In M . edulis this is achieved by siphon and shell valve closure at a salinity of about 25%0, while S . plana closes and retracts the siphons at about 20%0 (Fig. 12). This implies that the behavioural avoidance mechanism was triggered at a lower external salinity level, the difference being 7 and 12%0in M . edulis and S. plana, respectively, indicating that a fluctuation in the media of this magnitude

Minutes

FIG.12. Shell valve recordings from anterior (a) and posterior (b) part of normal Scrobicularia subjected to a decline and rise in salinity of the external medium (graph below). Op, open, C1, closed (from Akberali and Davenport, 1981).

I34

H . 0. AKBERALI A N D E. R. TRUEMAN

can be tolerated without serious consequences. In both M. edulis and S . plana, closure of the exhalant siphon, which prevents pumping, was the crucial event in largely isolating the mantle cavity from falling external salinities, while shell valve closure occurred at rather lower salinities, so producing almost complete isolation (Davenport, 1979; Akberali and Davenport, 1981). Even when apparently isolated, bivalves can detect favorable changes, since they respond within a short period of time, resulting in valve opening and commencement of pumping (Figs. 5 and 12). The foregoing account suggests that bivalves may distinguish between lethal and sublethal levels of pollutant or salinity stress. During periods of valve closure the animal incurs penalties, and unnecessary valve closure at sublethal levels of pollutant or salinity would be of little survival value. B. Sites of Reception In the class Bivalvia, envelopment of the body by paired valves has resulted in the reduction of the head and the role of sensory perception has been taken over by the mantle margin and siphons, which are the main sites of contact with the external environment (Dakin, 1910; Bullock and Horridge, 1965). The structure of the mantle of a number of bivalves has been described and the sensory structures developed on the middle mantle fold discussed (Yonge, 1949, 1957; Kawaguti and Ikemoto, 1962; Gilmour, 1963; Beedham and Owen, 1965; Barber et al., 1967; Land, 1968; Petit et al., 1978). Tactile sensitivity in the mantle, which is probably a feature of all bivalve mantles, has been demonstrated in M. arenaria (Pumphrey, 1938), S . solidissima (Wilson and Nystrom, 1968), Lima scubra (Stephens, 1978a), Chlamys opercularis (Stephens and Boyle, 1978), and S . plana (Hodgson, 1982; Black, 1983). In the mantle of bivalves P . maximus (Thomas and Gruyffyd, 1971) and L . scabru (Stephens, 1978a) chemical sensitivity has also been reported. The application of extract from a predatory starfish onto the mantle in two species of Asaphidae (Ansell, 1967) triggered a violent escape response and caused leaping movements. The formation of siphons in bivalve molluscs involves partial or complete fusion of one or all of the mantle folds (Yonge, 1948). In some bivalves, such as C. edule, siphonal tentacles bearing eyes have been observed (Barber and Wright, 1969). Furthermore, tactile and chemical sensitivity in response to stimuli has been reported in bivalve siphons, e.g., touching the siphons of Ensis (Trueman, 1966a), Spisula (Mellon, 1965; Prior, 1972), and Scrobiculuriu (Hodgson, 1982; Black, 1983). The isolated and in situ siphon in S . plana responds to salinity decline and

EFFECTS OF STRESS O N MARINE BIVALVE MOLLUSCS

135

presence of pollutants (Akberali, 1981; Akberali et al., 1981, 1982a,b; Akberali and Davenport, 1982). Photosensitivity of the siphon has also been reported, exposure to high light intensities causing withdrawal (Light, 1930; Kennedy, 1960). In M. edulis it has been shown that salinity detection is carried out peripherally by salt-sensitive receptors on the tentaculate portion of the inhalant siphon (Davenport, 1981). In S. plana the sensory structures are more deeply situated, and it is thought that salinity or zinc detection is mediated by the central nervous system rather than by any peripheral neural network within the siphon (Akberali et al., 1981; Akberali and Davenport, 1981,1982). In none of these studies, however, have the sense organs been located or described structurally, and the areas of sensitivity are based on experimental studies related to observations, ablation, or isolation of likely sensory sites, and the recording of behavioural response to a particular environmental stress. In a number of bivalves, different types of ciliary tufts occur on the mantle and siphons (Moir, 1977; Owen and McCrae, 1979; Frenkiel and Moueza, 1980; Hodgson et al., 1982; Black, 1983); they show many characteristics of sensory receptors as described by Laverack (1968). It is difficult to assign functions for the receptors from their morphological features alone, but it is possible to assign some function by comparison with receptors whose functions have been identified, or by correlating receptor density in an area showing sensitivity to a particular stimulus (Zylstra, 1972). It has been shown in S. plana that the siphons have a higher density of ciliary tufts than the mantle, and this may explain a greater response of the inhalant siphon to chemical stimulation than the mantle (Black, 1983). It is likely that, in the mantle and siphons of bivalves, at least some of the different types of ciliary tufts are sensory and are the mechano- and chemoreceptors. The possibility of the cruciform muscle complex in S . plana functioning as a chemoreceptor has been suggested by Odiete (1978), who recorded a burst of electrical activity from this structure when the clam was exposed to “foul water.” There is some evidence that the cruciform muscle complex in Scrobiculuria is involved in the siphonal withdrawal response to changing salinity (Akberali and Davenport, 1982) and zinc ions (Akberali el al., 1981). Ablation of the cruciform muscle complex in Scrobicularia when exposed to changing salinity or zinc ions resulted in the siphonal withdrawal response being delayed or weakened (Figs. 17, 21, and 22). The withdrawal response was not, however, totally abolished, suggesting that receptors in other areas, such as mantle and siphons, were still functioning. In other Tellinacean bivalves, such as Donax trunculus (Moueza

136

H . B . AKBERALI A N D E. R . TRUEMAN

and Frenkiel, 1974; Frenkiel, 1980), it has been reported that the cruciform muscle complex functions not as a chemoreceptor but as a vibration receptor (Pichon et al., 1980). Studies using electrophysiological techniques have shown tactile and chemical sensitivity in the mantle edge of the bivalves L . scabra and Aequipecten (Stephens, 1978a,b). Afferent impulse activity in nerves innervating peripheral sensory regions of S. plana in response to tactile and chemical stimulation has also been demonstrated (Hodgson, 1982; Black, 1983). The visceral ganglion in Scrobicularia is involved in controlling adductor muscle rhythms (Odiete, 1976a,b; Black, 1983) and also in the withdrawal of the in situ siphon in response to decline in salinity (Fig. 17). Activity generated in the posterior adductor muscle nerve in response to tactile or chemical stimulation of peripheral regions may lead to valve adduction in the intact clam. Furthermore, Black (1983) has shown that, in Scrobicularia, the mantle and siphons possess chemoreceptors that respond to the presence of zinc. In mantle nerves, the electrical response to zinc or to tactile stimulation was less than that of the siphons, and she suggested that this may be either related to the mantle nerves being of smaller diameter than the siphonal nerves, to the number of chemoreceptors present in each site, or to their firing threshold. Black (1983) also made the important observation that the threshold for response in the lower mantle nerve was 2-3 ppm zinc, whereas for the inhalant siphon nerve it was less than 1 ppm. This finding is in close agreement with previously reported studies on the behavioural and inhalant siphon responses of S . plana to zinc, where 1 ppm zinc was the lowest concentration tested to cause a temporary reduction in heart rate, siphon contraction, and valve closure (Akberali et al., 1981). The thresholds for the exhalant siphon and mantle were higher than the inhalant siphon, which may reflect the lesser importance of these sites for chemoreception in the natural habitat (Black, 1983). Valve closure provides a useful behavioural avoidance mechanism during exposure to adverse environments (see Section II,A), but the site of perception has been less thoroughly investigated. In a few bivalve molluscs, including S . pluna, ciliated tufts on the mantle and siphons resemble sensory receptors and are probably involved as mechano- or chemoreceptors. Their stimulation may then elicit siphonal withdrawal and valve closure as a general stress avoidance response.

C. Detection and Response to Environmental Changes There have been few studies on mechanical and chemical detection in bivalve molluscs. From the literature, it is apparent that among marine

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

137

invertebrates there is no common mechanism for registering environmental changes. Studies on certain mobile species such as gastropod molluscs (Blandford and Little, 1983), crustaceans (Gross, 1957; Lagerspetz and Mattila, 1961; McLusky, 1970; Thomas e? al., 198l), and annelids (Janson, 1962) indicate that, when subjected to a choice of different salinities, these organisms are capable of detecting and discriminating salinity levels. In sedentary species such as bivalve molluscs, the underlying basis of salinity detection has been investigated by exposing animals to artificial sea waters of differing ionic and osmotic composition and observing the effect on behaviour (Davenport, 1979, 1981 ; Akberali and Davenport, 1981, 1982). In both mobile and sedentary species, salinity detection depends on the concentration of particular ions (Barnes, 1939, 1940; Davenport, 1981; Akberali and Davenport, 1982; Black, 1983), the osmotic pressure of the medium (Davenport, 1972; Bettison and Davenport, 1976; Blandford and Little, 1983), or to the combination of both (Barnes and Barnes, 1958). S. plana is an osmoconforming bivalve in which valve closure is mediated by detection of change in osmotic pressure, and not by measurement of any ionic constituent (Freeman and Rigler, 1956). Akberali and Davenport (1982) showed that siphon withdrawal is triggered by a change in groups of ions such as sodium, magnesium, calcium, and chloride (Fig. 13) rather than by changes in the gross osmotic pressure (Figs. 14 and 15)

-

i

MINUTES

FIG.13. The effects of exposure to NaC1, MgCI2, and CaClz followed by a salinity regime (graph below) on (a) an in siru Scrobicutnria siphon preparation in which the downward deflection of the trace represents isotonic siphon contraction [note the effect of a mechanical stimulus (s) applied by pinching with forceps] and on (b) intact mussels in which the horizontal bar represents activity: open sections, gaping; closed (black) portions, closed valves. The “ionic test medium” regime description represents an alteration from 0% (when animals were supplied solely with sea water of salinity 32%0)to 100% (when animals were supplied with pure “ionic test medium”) (from Akberali and Davenport, 1982).

138

H . B . AKBERALI AND E. R . TRUEMAN

-

-a

b

6

lb

1’5

2b

25 3b MINUTES

35

do

45

50 i

FIG.14. The effects of exposure to two test media, the first containing NaCI, MgC12,and CaCI2, the second containing NaCl alone: response of (a) an in situ Scrobiculan‘a siphon preparation and (b) of intact mussels: details as in Fig. 13, except that the “NaCI only” regime description represents an alteration from 0% (when animals were supplied solely with “ionic test medium” regime) to 100% (when animals were supplied with pure “NaCl only”) (from Akberali and Davenport, 1982).

or in the concentration of a single ion (Fig. 14). Similarly, in M . edulis, both siphon withdrawal and valve closure (Akberali and Davenport, 1982) and the opening reaction (Davenport, 1981) are mediated by a group of ions such as sodium, magnesium, and chloride rather than by changes in the osmotic pressure or in the concentration of a single ion (Figs. 13-15). Siphon withdrawal and valve closure in S . plana and valve closure in M . edulis are different with respect to the presence of one ion. In order to prevent siphon withdrawal and valve closure in Scrobiculariu, the presence of sodium, magnesium, calcium, and chloride is necessary, whereas in M . edulis valve closure can be prevented by the presence of only sodium, magnesium, and chloride (Figs. 14 and 15). The reasons underlying these differences in calcium ion specificity are not clear. They may be due to differences in the type of habitat occupied or to a variety of ionic and osmotic mechanisms of salinity detection of animals within the same class (Bettison and Davenport 1976). There is as yet no sign of coherent evolutionary or physiological pattern. This calcium dependency requirement for the closing response in S . plana with respect to salinity changes, and its absence from M . edulis, may reflect an additional physiological requirement in the former species. Both species use behavioural mechanisms to avoid salinity variations (Davenport, 1979; Akberali and Davenport, 1981). S. plana, however, has two long, mobile, and highly extensible siphons, whereas in the epifaunal M . edulis the siphons are of negligible length. In Scrobiculariu siphonal contractions are dependent on the presence of calcium ions in the external

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

139

4 7 0 m M NaCl a

h

NaCI.CaCI9

CholineCI.CaCl2. MgC12

h

b

010

d

MINUTES

FIG. 15. The effects of various test media on in situ Scrobicukuriu siphon preparations (a) and intact mussels (b); details as in Fig. 13. The graph at the bottom indicates percentage concentration (CONC.) change for ail media (from Akberali and Davenport, 1982).

medium (see Section VI,B), and this may reflect the requirement of calcium ions for detection of falling external salinities. The opening response of S. p l u m to rising salinities has been shown to be dependent on sodium, magnesium, and chloride ions, but the presence of calcium is not required (Black, 1983). In M. edulis, both the closing response to falling salinities and the opening response to rising salinities rely on the presence of the identical ions (Davenport, 1981; Akberali and Davenport, 1982). In M. edulis the salinity-sensitive receptors lie on the tentaculate portion of the inhalant siphon (Davenport, 1981). Therefore, when the mussels are open

140

H . B . AKBERALI A N D E. R. TRUEMAN

and pumping, the water entering the mantle cavity will be continuously monitored near the margins of the valves so that any behavioural reaction to adverse media will result in negligible exchange between the mantle fluid and the external medium. It has been reported that the opening response to rising salinities is mediated by changes in electrochemical gradients due to the diffusion of salts to the tentaculate portion of the inhalant siphon and not to any other portion of the mantle edge or to any more deeply located structures (Davenport, 1981). Such a diffusion of salts to the salinity-sensitive region can easily be achieved in an epifaunal species even when the valves are closed, since valve closure does not always provide complete isolation. The existence of fine passageways between the mantle margins may allow salt diffusion. An epifaunal bivalve mollusc can thus continuously monitor the environment. In the infaunal S. plana when open and pumping, as in M . edulis, the water entering the clam will be registered by the salinity-sensitive region and accordingly trigger the behavioral avoidance mechanism when conditions become unfavourable. Once siphons are retracted and valves closed, an infaunal species cannot rely on salt diffusion gradients from interstitial water. These would not give a realistic assessment of conditions in the water column, since interstitial salinities of estuarine muds tend to be high and relatively stable (Kinne, 1971). In the natural habitat during exposure at low tide Scrobicularia does not withdraw its siphons into the shell, nor are the valves completely closed; the siphons are just withdrawn to the entrance of the burrow and the clam stops pumping (Earll, 1975a,b). It is possible that this behaviour allows periodic sampling by opening the siphonal apertures and drawing in small amounts of the external medium. In doing so the clam is at risk by the introduction of the stressor into the mantle cavity, and whether such a strategy is actually adopted by infaunal bivalves is not yet clear. Another alternative would be to locate sensory structures at the siphonal tips and along the entire length of the siphon so that loss of the tips by predation (Edwards and Steele, 1968) would not deprive the organism of its sensory structures. The isolated inhalant siphon of S . plana (Fig. 16) does not respond to dilute sea water, indicating that salinity detection either does not occur in the siphon or is mediated by the central nervous system (Fig. 17) rather than by any peripheral neural network. The dependency of the closing and opening response of M . edulis to sodium, magnesium, and chloride ions (Davenport, 1981; Akberali and Davenport, 1982) is hardly surprising since these ions make up the bulk of the salt content of sea water. It is possible that adequate concentrations of sodium and magnesium for ATPase activity, probably involved in the neural control of the gaping response, are also important. In S. plana;

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

141

ISOLATED SIPHON

OJ

0

I

Ib

do

5; $0 215 MINUTES FIG.16. Effects of salinity decline (graph below) on an isolated and an in situ pieparation of Scrobiculuria: the downward deflection of the traces represents isotonic siphon contraction; mechanical stimulus (s) applied by pinching with forceps (from Akberali and Davenport, 1982). 5

calcium is an added requirement for the closing response (Akberali and Davenport, 1982), but it is not required for the opening response (Black, 1983). The reason for this difference may be related to the dependence of the siphonal contraction on this ion (Akberali et ul., 1982a). Future work is required on other infaunal and epifaunal bivalve molluscs to elucidate this interesting phenomenon and to indicate whether calcium ions are an added physiological requirement in infaunal species for the contraction of the long extensible siphons. It must be understood throughout that the metal ions referred to in the text are those forms of inorganic metallic salts tested in sea water without taking into account the form of metal ions in solution. Behavioural avoidance mechanism in response to the presence of copper and zinc have been reported in both S. p l u m (Akberali and Black, 1980; Akberali ef a/., 1981) and M . edulis (Manley and Davenport, 1979; Davenport, 1977). These studies have also shown how quickly the animal responds to the removal of the pollutant, by the valves opening and pumping commencing (Fig. 5). The first visible response to changes in salinity or presence of pollutant in S. plana and M . edulis is the closure of the siphonal aperture, followed

I42

H . B. AKBERALI A N D E. R. TRUEMAN VISCERAL GANGLFIO ~-

~~~

~

.

'a CRUCIFORM MUSCLE

FOOT

?(I) El6 3

2

]

-

v

0 0

5

I

I

I

1

@

10

15

20

25

30

MINUTES

FIG.17. The effects of ablation of the visceral ganglion, cruciform muscle complex, foot (including pedal ganglion), or gills upon the response of in situ Scrobiculuviu siphon preparations to a fall in salinity; for other details see Fig. 16 (from Akberali and Davenport, 1982).

by siphonal withdrawal and valve closure. This led Akberali (1981) to examine the direct effects of copper on the isolated inhalant siphon of S. plana to investigate the possibility that the behavioural response observed in the intact clam may be due to the detection of copper by the siphons. The isolated siphon reacts to the presence of copper with a series of spontaneous contractions (Fig. 18). The copper concentrations were close to those found to trigger the behavioural avoidance response in the intact clam. Similarly, Akberali et ul. (1981) showed that the intact clam avoids lethal zinc concentrations by behavioural avoidance mechanisms and that zinc has no effect on the isolated siphon (Fig. 19). In an in situ siphon

143

EFFECTS OF STRESS O N MARINE BIVALVE MOLLUSCS

0

10

20

30

40

50

60

Time ( m i d

FIG.18. S. plana: Effects of addition of copper on an isolated inhalant siphon preparation. Upward deflection of the trace indicates isotonic siphonal contraction. The following experimental protocol was carried out in sequence: appropriate volume of stock copper solution (10 ppm copper nitrate) in normal sea water was added to give 0.25 ppm at (a) and 0.5 ppm at (b); removal of sea water containing 0.5 ppm copper (c), followed by washes (w) and replacement with normal sea water (d). Copper was added again to give a final concentration of 0.25 ppm (a’) and 0.5 ppm (b’) (from Akberali, 1981).

preparation where the clam is intact except for removal of one shell valve, the siphon reacts in a manner similar to that in the intact clam (Fig. 20). This led to the suggestion that zinc is registered by sensory structures on the mantle and ablation of the sensory cruciform muscle complex partially abolishes the in situ siphonal withdrawal response (Akberali et al., 1981) (Fig. 21). The possibility that the cruciform muscle complex in Scrobicularia has a chemoreceptive function has been suggested by Graham (1934), Yonge (1949), Moueza and Frenkiel (1974), and Odiete (1978); it appears to have a multiple function, facilitating siphonal movement vibration reception and acting as a chemoreceptor. However, as shown by Akberali et al. (1981) and Akberali and Davenport (1981, 1982), it is evident that ablation of the cruciform muscle complex affects the behavioural avoidance mechanisms to some extent but does not completely abolish the response to salinity decline (Figs. 17 and 22) or to the presence of zinc (Fig. 21). This suggests the possibility that the cruciform muscle complex together with other parts of the mantle lobes or siphons or ganglia may be involved.

t-httrmii i i i i i : : , , : <
0

10

20

t i i i i i j t

30

i i i i i i i : i i : i : ,

40

, i : i

i

50

i i i : : i : : i i bl’t-ti,

60

4-

70

f t 1

I

80

Minutes

FIG.19. Effects of various zinc and copper concentrations on an isolated inhalant siphon preparation of S. plann. Upward deflection of the trace indicates isotonic contraction of the inhalant siphon. The following experimental protocol was carried out in sequence: application of zinc at the final concentrations indicated (4)and removal of sea water containing 10 ppm zinc (C), followed by washes (w) and replacement with normal sea water (D). Prior to copper applications (J), the siphonal response was tested by a mechanical stimulus (s) (from Akberali et a / . , 1981).

1

+ +

I44

H. B. AKBERALI AND E. R. TRUEMAN

0

10

20

30 Minutes

50

40

FIG.20. Effects of various zinc concentrations on an in situ inhalant siphon preparation of

S.plana. Upward deflection of the trace indicates isotonic contraction of the siphon. The following experimental protocol was camed out in sequence: application of zinc at the final concentrations indicated (J) and removal of sea water containing 10 ppm zinc (C), followed by washes (w) and replacement with normal sea water (D), then mechanical stimulus (s) (from Akberali et al., 1981).

S. plana and M. edulis clearly avoid lethal levels of copper and zinc solutions by employing behavioural avoidance mechanisms, and are capable of discriminating sublethal levels. This response in Scrobicularia has also been demonstrated recently using other metal ions (Black, 1983). Black showed that silver and mercury cause siphon withdrawal and complete valve closure, whereas partial valve closure was observed in the presence of nickel and cobalt. The behaviour was least affected when Scrobiculuria were exposed to manganese, cadmium, or chromium (tri- or hexavalent forms). Observed differences in the behavioural responses of

* 0

20

10 Minutes

30

Frc. 21. Effects of various zinc concentrations on in situ inhalant siphon preparations of

S.plana. Arrows (1) mark the application of zinc stock solution to give the indicated concentrations. X, cutting of the cruciform muscle complex before addition of 10 ppm zinc; (A and B) Two examples of the recordings observed; s, mechanical stimulation (from Akberali et a / . , 1981).

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

145

I

CI

OJ I

0

20

I

I

40

I

I

I

I

60 80 Minutes

I

I

100

I

I

I

120

FIG.22. Shell valve recordings from anterior (a) and posterior (b) part of a specimen of S. plunu, with its cruciform muscle complex destroyed by making a median longitudinal incision through the muscle and then subjected to a decline and rise in salinity of the external medium (graph below). Op, Open; C1, closed (from Akberali and Davenport, 1981).

Scrobicularia can also be related to the toxicity of each metal ion over longer exposure periods. Black (1983) found that those metals which evoked closure responses were also acutely toxic to S . plana during prolonged exposures (median lethal time, ca. 1-7 days), whereas metal ions that did not cause complete valve closure had low toxic effects over a similar period of time. The isolated siphonal preparation of S. pfana was also used to test the effects of copper, zinc, cadmium, nickel, manganese, chromium (tri- or hexavalent), silver, and mercury (Black, 1983). Only copper was effective in causing spontaneous contractions of the isolated siphon, which is dependent on the presence of calcium ions in the external medium (Akberali et al., 1982a). The possible mode of action of copper is discussed later (Section V1,D). At present, it is not clear why copper, of all the metal ions studied so far, has this ability to induce contractions in the isolated inhalant siphon. Recent studies, using isolated myogenic heart of the land snail Helix aspersa, have shown that low copper concentrations induced continuous tonic contraction, whereas zinc does not have this effect (Akberali, unpublished). It is not known whether the direct effect of copper on the isolated siphon forms the basis of siphon withdrawal in the avoidance response of the intact animal or whether it acts in the same manner as other metal ions, through sensory reception sites. The evi-

146

H . B. AKBERALI AND E. R . TRUEMAN

dence so far indicates that the bivalve Scrobicularia is able to detect the presence of metal ions and discriminate potentially lethal levels.

IV. A.

Respiratory Physiology during Stress Valve Closure and Cessation of Aerobic Processes

Periods of valve closure are commonly associated with behavioural inactivity and cessation of pumping activity. The most obvious effects of this are limitation of time available for feeding and consequent reduced growth potential, cessation of aerobic respiration, and accumulation of excretory products. Despite these penalties, the behavioural avoidance strategy of bivalve molluscs is of great survival value. There is, however, a limit to the time an animal can remain isolated from the environment, depending on its overall physiological and biochemical condition and the adaptive mechanisms to sustain the basal metabolic processes. Such mechanisms will include the utilization of anaerobic respiration to sustain basal metabolism, availability of stored food reserves, and the ability to tolerate and accommodate levels of excretory products.

B. Relationship between Heart Rate, Valve Movements, P O ? ,and PC02 Numerous studies (Trueman, 1967; Coleman, 1974; Earll, 1975b; Brand, 1976; Akberali er al., 1981) have shown that the effect of valve closure on heart rate in bivalve molluscs is bradycardia, which may be associated with the depletion of oxygen tension in the mantle cavity. Oxygen depletion in the mantle cavity of M. californianus is rapid when the valves are clamped shut, falling from 115 mmHg to 20 mmHg O2 in 20 min or less (Moon and Pritchard, 1970). Similar rapid depletion of the oxygen in the mantle cavity of A . islandica (Brand and Taylor, 1974),S . plana (Akberali and Trueman, 1979), M. edulis (Davenport, 1979), and A. cygnea (Parker, 1978) has been reported. It is possible, as Bayne (1971) suggests, that the rapid fall of oxygen tension in the mantle cavity could account for the rapid bradycardia shown by M. edulis. Earll (1975a,b) considered why S . plana, of all the bivalve species investigated, alone shows a marked temporal variation in heart rate which is dependent upon pumping (Fig. 3). A number of factors may be responsible. The heart rate is reduced when the clam is subjected to reduced oxygen tensions. A high weight-specific oxygen consumption (Hughes, 1970) compared with a small mantle cavity volume would ensure an even

EFFECTS OF STRESS O N MARINE BIVALVE MOLLUSCS

147

faster depletion of mantle cavity oxygen tension than occurs in species such as A. islandica (Brand and Taylor, 1974) or the large M. arenaria which requires an average of 30 min for the heart rate to fall after the cessation of pumping (Earll, 1975a). Akberali and Trueman (1979) have examined the pattern of short-term variation in heart rate in S. plana in relation to p 0 2 and p C 0 2 changes in the mantle cavity. Samples of water were withdrawn from the mantle cavity at known heart rates (Fig. 23) and indicated that a high heart rate (16-18 beatshin, indicative of pumping) was correlated with a p 0 2value of between 140 and 160 mmHg. Decrease in heart rate to 10-13 beatshin was associated with a p 0 2 level of 80-1 10 mmHg. During activity, the p C 0 , rarely exceeded 2 mmHg, but on cessation of pumping never fell below this value. The factors causing suppression of heartbeat during inactivity are not clear. For example, Ptcsi and Salanki (1974), Brand (1976), and Parker

K

I

I" E E

O ' '

2

'

4 ' 6 .

e

' l o ' l z ' i 4

Individual Animal No.

FIG.23. Mantle cavity p O z ,pCOz, and heart rate (HR) of individual S. p l u m during active periods (0)and ventilatory pauses (0)when constantly immersed in sea water. Each point for the 14 animals during active periods is the mean of 6 determinations and each point for the 5 animals during ventilatory pauses is the mean of 4 determinations. Bars indicate SE; points for the same animal are joined by broken lines (from Akberali and Trueman, 1979).

148

H . B . AKBERALI A N D E. R. TRUEMAN

(1978) showed that bradycardia in A . cygnea and A . anatina is not merely an affect of shell closure. Schleiper (1957) suggested that bradycardia in M . edulis may be effected by a build-up of carbon dioxide, whereas Bayne (1971) postulated that depletion of oxygen within the mantle cavity of M . edulis was responsible. The factors and mechanisms of suppression of the heart during valve closure are thus largely uncertain, and further investigations are needed.

C. Anaerobic Respiration during Valve Closure It is well known that bivalves are able to withstand periods of shell closure and resultant lack of oxygen (Dugal, 1939; Trueman, 1967; Helm and Trueman, 1967; Crenshaw and Neff, 1969; Moon and Pritchard, 1970; Coleman and Trueman, 1971; Crenshaw, 1972; Akberali and Trueman, 1979; Davenport, 1979,1982; Widdows et al., 1979). The changeover from aerobic to anaerobic respiration in bivalves normally occurs when the oxygen tension of the mantle cavity falls to low levels, after the bivalve has closed its valves in response to environmental stress. Early work on this subject, reviewed by von Brand (1946), recorded a variety of species able to survive long periods in the absence of oxygen. Most of the earlier work reported for anaerobic metabolism in bivalves is based on the observation that, on return to aerobic conditions, there is a rapid rise in the rate of oxygen consumption to levels above normal (Schlieper, 1957; Moon and Pritchard, 1970; Coleman, 1973, 1974). Other authors have reported a similar “overshoot” in heart rate (Trueman, 1967; Helm and Trueman, 1967; Coleman, 1974; Earll, 1975b; Brand, 1976; Akberali, 1978; Parker, 1978; Stone, 1980). Shell closure in A . islandica is followed by an exponential decrease in the oxygen tension of the mantle cavity water and is accompanied by an initial increase in heart rate, which is then followed at lower oxygen tensions by bradycardia (Taylor, 1976a,b). On return to aerobic conditions, both the heart rate and oxygen consumption are increased to a high level but then decline gradually to normal levels. When the freshwater bivalve P . coccineum is kept out of water for 5 days, the gill tissue exhibits a well-developed oxygen debt (Badman and Chin, 1973). Crassostrea (Ishida, 1935) and M . arenaria (Collip, 1921; van Dam, 1935) exhibit a higher rate of oxygen consumption after anaerobiosis than normal. These observations have frequently been interpreted as representing an increase in circulatory and respiratory demands, associated with the repayment of an “oxygen debt” incurred during the period of anaerobiosis. In recent years, research carried out by a number of workers has led to a greater appreciation of the biochemical pathways operating in bivalve

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

I49

molluscs during anaerobiosis, and has shown that these pathways differ in some ways from the classical ones described in vertebrates (Stokes and Awapara, 1968; Hochachka and Mustafa, 1972; De Zwaan and Zandee, 1972). It is now evident that many invertebrates are true facultative anaerobes, capable of surviving indefinitely in the absence of oxygen and capable of active oxidative metabolism in its presence. In these organisms, as in primeval ones that possibly arose under reducing conditions in the absence of molecular oxygen (Wald, 1964), organic substrates, instead of oxygen, are the acceptors of electrons and protons. In intertidal bivalve molluscs, Hammen (1969) has shown that under aerobic conditions the catabolism of glycogen leads to pyruvate, which is then fully oxidized to carbon dioxide and water by the Krebs cycle reactions, a metabolic adaptation similar to that occurring in vertebrate muscle. Under anoxic conditions the breakdown of glycogen (or glucose) to the level of phosphoenolpyruvate (PEP) is also similar to the process in vertebrates, but the breakdown products are different. Whereas vertebrates convert PEP to pyruvate and accumulate lactate, the main end products of anaerobic glucose catabolism in intertidal bivalve molluscs are succinate and alanine (Stokes and Awapara, 1968; Chen and Awapara, 1969; Hammen, 1969; De Zwaan and Zandee, 1972; De Zwaan and Marrewijk, 1973). Metabolic pathways for the conversion of glucose into succinate and alanine have been discussed by numerous authors (Stokes and Awapara, 1968; Chen and Awapara, 1969; Hochachka and Mustafa, 1972; Hochachka et al., 1973; De Zwaan et al., 1973, 1975; De Zwaan and Wijsman, 1976; De Zwaan, 1977; Newell, 1979). Anaerobic conditions are common for intertidal bivalves, and it has become clear that their ability to survive these stress conditions is connected to a remarkably high tissue glycogen content (De Zwaan and Zandee, 1972; Hochachka and Mustafa, 1972) together with certain adaptations of their intermediary metabolism (De Zwaan, 1977; De Zwaan et al., 1973; De Zwaan and Wijsman, 1976; Newell, 1979). For example, the glycogen and glucose concentrations of the clam P. coccineurn under anaerobic conditions decrease rapidly from 24 to 48 h, but level off after 48 h. Clams in low, but not zero, oxygen concentration appear to be unaffected (Badman and Chin, 1973). The ability to respire anaerobically also varies in bivalve tissues, depending on the easy accessibility of oxygen from the environment or circulatory system. For example, deeply located tissues have a greater tendency to respire anaerobically than superficially located tissues as a result of relative oxygen availability. It has been suggested that in bivalve molluscs, some tissues may be adapted to function anaerobically while others which are near to the sites of gas exchange may be primarily aerobic (Booth and Mangum, 1978). This may be of particular importance in epifaunal species

I50

H. B. AKBERALI AND E. R. TRUEMAN

such as M . edulis, M . californianus, M . demissus, I . alatus, and C . edule during exposure at low tides when a small gape is maintained in their mantle margins during valve closure (Table 111). These bivalve molluscs have been shown to utilize atmospheric oxygen which diffuses into the mantle cavity fluid (Lent, 1968, 1969; Moon and Pritchard, 1970; Coleman and Trueman, 1971; Trueman and Lowe, 1971; Boyden, 1972a,b; Bayne et al., 1976a,b; Widdows et al., 1979). During valve closure, the oxygen obtained this way may sustain a slight level of aerobic respiration in superficially located tissues, e.g., mantle margins, gills, but it would be insufficient to meet the animal’s overall requirements. M . californianus is able to meet only part of its metabolic requirements by aerial oxygen consumption during the intertidal period, and approximately 25% of the minimal metabolic requirements are met in this animal by anaerobic pathways (Bayne ef al., 1976b). Also relevant here are the observations made on other molluscan species. For example, cephalopod mantle, which is rich in mitochondria, has been shown to carry out aerobic respiration by superficial contact with aerated water both externally and in the mantle cavity; deeply situated muscle is, however, anaerobic (Bone et al., 1981). In the squids Loligo pealii and Lolliquneula brevis the cardiac myofibre differs fundamentally from that of lamellibranchs in terms of mitochondrial and tubule density (Dykens and Mangum, 1979). The authors have concluded that these properties are correlated with a very high rate of oxidative metabolism in the intact animal, with little capacity for anaerobiosis. In the gastropod Bulliu, the foot, 1 mm thick, is entirely aerobic, whereas the deeply situated muscles, including the columellar muscle, may be anaerobic (Brown, 1982). In the large and actively burrowing bivalve D . serru, blood supply to the foot is probably cut off for 2-3 min while digging, and the respiratory demand of the pedal tissues is maximal (Trueman, unpublished). These observations suggest that anaerobic pathways play a significant role in molluscan respiration. In contrast to conditions in the sublittoral zone, intertidal organisms are normally subjected to marked changes in oxygen availability. In coarse sediments, for example, interstitial oxygen concentrations tend to be high, but as the deposits become finer, the redox-potential discontinuity layer may approach the surface so that burrowing organisms are effectively surrounded by nearly anoxic conditions (Brafield, 1964). Therefore, infaunal bivalve species, for example, S . plana and M . arenaria, retain a connection to the oxygenated water at the surface of the sediment by means of siphons. There may be no oxygenated surface water available to draw over the gills at low tide, and future studies should consider differences between the behaviour of epifaunal and infaunal species in relation to their capacity for anaerobic respiration.

EFFECTS OF STRESS O N MARINE BIVALVE MOLLUSCS

D.

15 I

Valve Activity and p H Changes

When the clam M . mereenaria was kept out of water for a period of days, dissolution of the inner layer of the shell occurred (Dugal, 1939). This was thought to buffer acid metabolites, principally lactic acid, but subsequent work has shown them to be largely succinic acid (Stokes and Awapara, 1968; De Zwaan et al., 1975). In Mercenaria measurements of calcium and succinic acid in the tissues and fluids indicate that succinic acid produced by the anaerobic metabolism is neutralized by the dissolution of previously deposited shell (Crenshaw and Neff, 1969; Crenshaw, 1972). The conversion of neutral substances into organic acids may cause a drop in the pH unless an animal has strong buffering systems at its disposal. In the absence of oxygen there is a rapid decrease in the glycogen content of the tissues and an increase in acid and carbon dioxide content. Evidence that the calcareous shell is involved in buffering acids has been found in various other bivalves when the animals are exposed to air or during the closure of the valves under normal or stress conditions (Collip, 1920, 1921; Dugal, 1939; Crenshaw and Neff, 1969; Crenshaw, 1972; Akberali et al., 1977, 1983; Parker, 1978; Akberali, 1980b; Akberali and Black, 1980). Crenshaw (1972) and Wijsman (1975) noticed that bivalves kept in aerated sea water closed their shells from time to time, with a concomitant drop in pH of the mantle cavity (Table IV). Measurements of oxygen tension in the extrapallial fluid of Mercenaria (Crenshaw and Neff, 1969; Crenshaw, 1972) revealed that the clam became completely anaerobic within 25 rnin of shell closure. Wijsman (1975) showed that the pH drop in aerially exposed M . edulis was even greater than in mussels kept in oxygen-free sea water (Table IV). The last group rarely kept their valves closed for longer than 5 h. When the valves of S. plana are closed (due to an osmotic shock), a significant increase in mantle cavity fluid calcium concentration occurs whereas clams in oxygen-free normal sea water keep their valves open with no noticeable rise in mantle cavity calcium concentrations (Fig. 30). It has been suggested that continuous water flow through the mantle cavity in the latter situation may explain the absence of a noticeable rise in calcium concentration, for under these conditions no anaerobic metabolites will accumulate, and in consequence little, if any, buffering will be required. In aerially exposed M . edulis, the lowest pH value recorded was 6.5, while in water-immersed “anaerobic” mussels it was 7.2 (Wijsman, 1975). A rapid rise in pH was noticed after opening the shell, suggesting that during exposure an increase in acidity is less harmful to Mytilus than the danger of desiccation. A drop in pH of the mantle cavity fluid also occurs in the bivalve S . plana due to prolonged valve closure in normal sea water (Fig. 24) or

152

H . B . AKBERALI AND E . R . TRUEMAN

TABLEIV. SPECIES SHOWING A CHANGE I N pH OF THE MANTLECAVITY OR EXTRAPALLIAL FLUIDDURING NATURALOR ENFORCEDPERIODOF VALVECLOSURE^ Period of valve closure

Species

Condition

M . mercenaria

Aerial exposure Natural quiescence Natural quiescence

M . edulis

S.plana

A . cygnea

pH change

2 weeks

7.48

lh

7.41 -+ 7.25 (EP)

4h

7.59

-+

-+

7.25 (MC)

6.91 (EP)

Reference Dugal ( I 939) Crenshaw (1972) Crenshaw and Neff (1 969)

Aerial exposure Oxygen-free sea water

30 h

7.5 -+ 6.5 (EP)

Wijsman (1975)

3h

7.5 -+ 7.2 (EP)

Wijsman (1975)

Natural quiescence Aerial exposure Salinity change

8h

7.8 + 7.2 (MC)

Original data

6h

7.8 --$ 7.65 (MC)

Original data

24 h

7.8 + 7.0 (MC)

Original data

24 h

7.8

Natural quiescence

-+

7.45 (MC)

Parker (1978)

Abbreviations: MC, mantle cavity fluid; EP, extrapallial fluid.

0

2

4

6

8

1 0 1 2

Hours

FIG. 24. S. plana: pH of mantle cavity water, heart rate (HR), and valve movements during quiescence (- - -) and activity when constantly submerged in normal sea water (S, 31%0) at 10°C. Onset of activity, T; open, 0; closed, C. Continuous pH and HR recordings are represented as a mean of alternate 5-min intervals (original data).

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

153

salinity stress (Fig. 25). When prolonged valve closure occurs during inactivity, the heart rate is low and the pH gradually drops (Fig. 24). At the termination of quiescence, the heart rate first increases, then the valves open before pumping activity recommences. Only when pumping recommences does the pH of the mantle cavity water gradually return to normal. Immersion in 20% sea water (Fig. 25) results in the siphons being withdrawn, the valves being tightly closed, and the heart rate falling within 2 h. The pH of the mantle cavity shows a gradual stepwise drop, with intermittent rise and fall, but the cause of this remains obscure (Figs. 25 and 26A). The valves are still closed after 24 h in 20% sea water, and the pH falls to 7.00, while the heart rate remains at 6-8 beatdmin (Fig. 25). On replacing the low-salinity water with normal sea water, the heart rate and pH return to the previous recorded levels within If h of the valves opening and recommencement of ciliary pumping. A common feature of recovery from quiescence in most intertidal bivalves (Coleman, 1974), including S. p l u m (Earll, 1975b), is the repeated sharp adduction and relaxation of the valves (see Fig. 5). This has been explained as effecting repayment of an oxygen debt or the “flushing out” of excretory products (Brand and Roberts, 1973). Resumption of activity in Scrobiculuriu, either in normal sea water or after salinity or pollutant stress, is also characterized by rapid valve adductions. There is a corresponding drop in the p H of the mantle cavity water a short time after each of these adductions (e.g., see Fig. 26B) followed by a slow rise as water circulates through the open mantle cavity. This suggests release of an acidic substance at each adduction which slowly diffuses out during the intervals between adductions. It is possible that acid metabolites, accumulated

FIG. 25. S . plana: pH of mantle cavity, heart rate (HR), and valve movements when transferred directly to S, 6.2%0sea water (V)from normal sea water (S, 31%0) at 10°C. At the end of 24 h, 20% medium replaced with normal sea water (A).Open, 0; closed, C. pH and HR sampled as in Fig. 24 (original data).

I54

H . B . AKBERALI AND E. R . TRUEMAN

:;--A

I,

78f 70

-

~-

0

C

0

FIG.26. S. piuna, examples of recordings of mantle cavity pH and valve movements: A, pH fluctuations seen in the mantle cavity water during a salinity stress (S, 6.2%0);B, rapid valve movements observed shortly after transference to normal sea water (S, 31%0) from a 24-h salinity stress; C, immersion in normal sea water (J) after a 6-h exposure period (original data, except for Fig. 26B, from Trueman and Akberali, 1981).

during anaerobiosis, are released from the tissues during valve adductions. Adduction of the valves causes the generation of simultaneous pressure pulses in both the mantle cavity and the tissues, resulting in rapid outflow of water from the mantle cavity (Trueman, 1966b). The pressure lasts longer in the tissues (1-2 s), however, than in the mantle and could well bring about the rapid flushing of metabolites from the renal tissues while normal ciliary pumping between adductions would remove these from the mantle cavity. During normal pumping activity, or when the pH drop in the mantle cavity is less, the pH is hardly affected by valve adductions (Fig. 26C); the feature shown in Fig. 26B is only observed after anaerobiosis and prolonged adduction of valves. Similar stepwise changes of p 0 2 and $ 0 2 levels in the mantle cavity of S . plana during recovery from anaerobiosis support this suggestion, for these changes imply a somewhat intermittent recovery compatible with hyperventilation of the mantle cavity by adduction of valves (Fig. 27). During 6-h emersion, S. plana showed a rather variable heart rate (12-22 beatdmin). In contrast to a drop in pH of the mantle cavity under normal conditions and salinity exposure (Figs. 24 and 2 3 , the pH in aerially exposed S. plana remained stable at 7.65 (Fig. 26C). Valve closure in bivalves such as S. plana, M. edulis, and M. mercenariu during short-term stress conditions or natural quiescence thus results in cessation of pumping, bradycardia, and the introduction of largely anaerobic respiration. The resulting accumulation of acid metabolites leads to the pH of the mantle cavity falling (Table IV). Resumption of

EFFECTS O F STRESS O N MARINE BIVALVE MOLLUSCS

0

40

80

I55

120

Time (rnin)

FIG.27. p o l , pCOz, and heart rate (HR) of S. plana transferred from normal sea water ( S , 31x0)to sea water (S, 6.2%0) for 24 h when they were replaced in normal sea water at 0 min. Each point for pOz and p C 0 2 is mean of seven animals, which were then discarded. Bars represent SE. Heart rate presented for one typical animal (from Akberali and Trueman, 1979).

activity is characterized by a rapid series of valve adductions whose probable function, at least in S . plana and possibly in other bivalves, is to hyperventilate the mantle cavity and probably to accelerate the removal of metabolites.

V.

The Role of the Shell

A. Physical Protection and Isolation from Environmental Stress Since most bivalve molluscs lead a relatively sedentary mode of life, it is essential that they have means of protection from predators and the prevailing physical and chemical environmental variables. This protection is provided by the shell. It is well known that molluscs, including bivalve

156

H . B. AKBERALI AND E. R. TRUEMAN

species, inhabiting exposed environments have stronger and thicker shells to protect their tissues from abrasive physical forces such as wave action than those inhabiting sheltered environments. Epifaunal species such as mussels and oysters have strong shells in order to protect their soft parts from predation and are also fixed to the substratum to prevent dislodgement. Infaunal species have different risks from predators, but may have thin slim shells to aid burrowing into the substratum. Apart from the use of the shell for protection from predation and physical factors, the shell can also provide valuable protection for the tissues from other environmental stress, e.g., salinity, pollutants, and aerial exposure. A sedentary mode of life restricts bivalves to a particular habitat where environmental stress cannot be avoided by migration, and under these circumstances the behavioural avoidance mechanism is of importance.

B. Shell Closure and Calcium Reabsorption It has often been suggested that bivalve molluscs are able to withstand long periods of valve closure through a buffering of accumulated acid metabolites during anaerobiosis by dissolution of previously deposited shell material (Table V). This was first proposed by Collip (1920, 1921) for M . arenaria and has recently been reiterated by a number of other researchers working on A . cygnea (Dottenveich and Elssner, 1935; Parker, 1978), M . mercenaria (Dhgal, 1939; Crenshaw and Neff, 1969; Crenshaw, 1972), M . edulis, C. edule, Donax juliane and M . arenaria (Alyakrynskaya, 1972), Unio tumidus (Stone, 1980), and S . plana (Akberali et al., 1977, 1983; Akberali, 1980b). The osmoconforming bivalve S . plana, when subjected to a long-term low-salinity stress, shows a gradual decline in the internal sodium, magnesium, chloride, and potassium concentrations becoming isosmotic with the external medium (Fig. 28). However, the calcium concentration in the body fluids examined shows an initial increase, reaching a maximum of 30 mM (about 3 times that in normal sea water) within 5-7 days, when it drops toward that of the external medium, stabilizing at a new low level at about 18 days (Fig. 29). To determine whether the increase of calcium over the first 7 days is related to anaerobic metabolism, clams were placed in oxygen-free sea water, where they exhibit only a slight rise in calcium levels (Fig. 30). It is only when the valves are closed by immersion in 20% sea water and the mantle cavity is not ventilated that calcium levels increase markedly. Forcible closure of the valves results in a rapid increase of calcium level over 2 days to about the same level as that reached by clams in 20% aerated sea water in 7 days (Fig. 29). A continuous flow of oxygen-free 100% sea water through the mantle cavity may explain the

TABLEV. ORGANISMS WHICHHAVEBEENSHOWNTO MOBILIZECALCIUM FROM

Species

Condition

Period of valve closure

THE

SHELLDURING PERIODSOF VALVECLOSURE^

Increase in calcium concentration 8.9 + 60 mM (MC)

Reference Collip (1920)

M . arenaria

Aerial exposure

96 h

M . mercenaria

Aerial exposure Natural quiescence Natural quiescence

13 days lh 4h

11.8 + 75.6 mM (MC) 21.1 + 24.6 rnEq/l (EP) 8.8 + 11 .O mM (EP)

Dugal(l939) Crenshaw (1972) Crenshaw and Neff (1969)

S. plana

Salinity change 0.5 ppm copper

5-7 days 6h

11.7 + 30.0 mM (MC) 8.7 --$ 11.0 mM (MC)

Akberali et al. (1977) Akberali and Black (1980)

U . tumidus

Natural quiescence

30 h

3.1 -+ 5.5 mM (MC)

Stone (1980)

A. cygnea

Natural quiescence

48 h

1.8+ 11.2mM(MC)

Parker (1978)

a

Abbreviations: MC, mantle cavity fluid; EP, extrapallial fluid.

I58

H . B . AKBERALI AND E. R. TRUEMAN

0

4

x

12

I6

20

24

2x

Time (days)

FIG.28. Na+ concentrations in S. pfana transferred directly from 30%0 sea water (day 0) to 6%0sea water in well-aerated conditions. (B-B), mantle cavity fluid; (8-0). blood from the ventricle; ( b a ) ,medium. Each point is the mean of four determinations made on samples pooled from 12-14 individual S. plana selected randomly (from Akberali et al., 1977; reprinted by permission from Nature (London) 266, No. 5605, 852; copyright 0 1977, Macmillan Journals Limited).

absence of a significant rise in calcium ions (Fig. 30A and B), for in this condition no anaerobic metabolites would accumulate and no buffering would be required. An increase in calcium concentration of the mantle cavity water has been shown in a number of bivalves during periods of valve closure arising from various environmental stressors or natural quiescence (Table V). The increase in body fluid calcium concentration suggests mobilization from the shell. The calcium ions are derived from the interior of the valves by dissolution during stress, as shown by the use of 45Cain S. plana (Akberali, 1980b) and M . mercenaria (Crenshaw and Neff, 1969). When Scrobicularia with 45Caincorporated in its shell is subjected to a longterm salinity stress, 50% of the incorporated 45Cais lost within the first

!h T 20

-

No

0

10

4

E

12

16

20

24

28

Time (days)

FIG.29. Calcium concentrations of S. plana transferred directly from 100% sea water (S, 30%0) at day 0 to sea water (S, 6%0).Mantle cavity fluid (B-B); blood from the ventricle, (8-8); medium ( b A ) . Each point is the mean of four determinations made on samples pooled from 12- 14 individual Scrobiculnria selected randomly (from Akberali et a / . , 1977; reprinted by permission from Nature (London) 266, No. 5605, 852; copyright 0 1977, Macmillan Journals Limited).

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

0

'

8

'

"

'

4

'

8

~

'

'

12

'

~'

16

20

I59

24

Time (days)

FIG.30. Calcium concentrations of intact Scrobicuhia placed in oxygen-free normal (S, 30%0)sea water at A. After 6 days, the clams were additionally subjected to salinity stress, by immersion in oxygen-free, dilute (S, 6%0) sea water at B and C. After 7 days the valves of 20 animals were forcibly closed (D). Mantle cavity fluid, (W-W); blood from the ventricle (0-0); medium ( b d )(from Akberali et a / . , 1977; reprinted from Nature (London) 266, No. 5605, 853; Copyright 0 1977, Macmillan Journals Limited).

24 h (Fig. 31). In contrast, clams left in normal sea water as a control show a fairly stable level of incorporated calcium in the shell. The rapid initial drop was also observed in clams exposed to a short-term (6-h) salinity stress where a rapid drop in 45Cawas caused in the 2 h after the onset of salinity stress (Fig. 32) and approached, after 24 h, levels similar to those of the previous experiment (Fig. 31). This suggests that the freshly deposited calcium is more labile than the remainder of the valve and is lost initially. This seems a relevant physiological adaptation; for example,

*'$ 9

\

lo: 8

X

s

f*- t*-*\*.

4-

2-

'

I

2

3

4

5

Time (days)

6

k7 \ 1 4

FIG.31. S. pluna with their valves covered with amyl acetate on the outside were left for 72 h in 45Ca-labelledsea water (5 pCi/l) for incorporation of labelled calcium in the valves on the inside, followed by placing those clams in unlabelled sea water for 48 h. The clams were then either subjected to a salinity stress at day 0 for 14 days (-) or placed in normal sea water (S, 31%0)as a control (----); values are presented as means of total 45Cacounts per minute (cpm) per valve (n = 8); bars represent SE (from Akberali, 1980b).

I60

H. B. AKBERALI A N D E. R. TRUEMAN 1

Time ( h )

FIG.32. Loss of incorporated 45Cacounts per minute (cpm) per valve in Scrobicularia subjected to sea water (S, 6.2%0) for 6 h (-); clams in unlabelled normal sea water (S, 31%0) as control (----); treatment of animals and experimental procedure as in Fig. 31 (n = 8); bars represent SE (from Akberali, 1980b).

during exposure at low tides or under short-term stress conditions reflecting tidal duration when the valves are closed, the calcium most recently secreted is most likely to be utilized. The disappearance of the freshly incorporated 45Cahas also been shown using autoradiographic technique in S. p l u m by Akberali (1980b). This provides visual evidence of the disappearance of calcium from the valves during anaerobiosis under stress conditions. Further evidence of the dissolution of the inner shell surface has also been shown using scanning electron microscopy (Akberali et al., 1983). Under long-term, laboratory-induced salinity stress, the shell surface is markedly eroded in comparison with the control shells over the same period. This has also been investigated in the natural habitat by examining the effects of tidal immersion and exposure. There is no visible sign of erosion, and the inner shell surface is smooth at the end of the period of tidal immersion. In contrast, the shells that were collected toward the end of the exposure period at low tide show areas of dissolution in the form of eroded localized pits like those of long-stressed shells, though the pits are not as extensive (Akberali et ul., 1983). The foregoing account shows the use of the shell in counteracting the harmful effects of both short-term and long-term environmental stressors. Although the shell may be weakened, this weakening is presumably a price worth paying for the survival of the organism during a short-term, unfavourable stress. A number of bivalve species utilize shell calcium in order to buffer acid end products of anaerobic respiration (Table V). Although both epifaunal and infaunal species mobilize shell calcium during anaerobiosis, it is likely that this phenomenon is more marked in

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

161

infaunal than epifaunal species. For example, the ability to utilize atmospheric oxygen during aerial exposure has been reported in a number of bivalves (Table 111), and evidence suggests that these species are not entirely dependent on anaerobic metabolism during aerial exposure. In contrast, such an option is not available to infaunal bivalve species, and this will result in a subsequent increase in demand for calcium to buffer the end product of anaerobiosis. Furthermore, in the infaunal bivalve S. plana valve closure may persist for 5-7 days during exposure to various environmental stressors such as copper and zinc, after which the necessity to open their valves results in pollutant poisoning and possibly death (Akberali and Black, 1980; Akberali et al., 1981). In the epifaunal bivalve M. edulis, continuous long-term exposure to 0.5 ppm copper results in an increase in mortality, with a median lethal time (MLT) of 2 days, whereas in Scrobicularia continuous long-term exposure to either copper or zinc results in a MLT between 5 and 7 days (Akberali and Black, 1980; Akberali et al., 1981). It is likely that this difference in MLT (Table 11) is due to the ability to survive varying periods of valve closure in these two species. The infaunal Scrobiculuria is probably better adapted for the prolonged use of behavioural avoidance mechanisms, and hence anoxic conditions, than the epifaunal M. edulis. Further work on habitat differences is necessary in order to enhance the understanding of the responses of bivalve molluscs to environmental stress.

C. Effect of Prolonged Stress on Shell Strength Mobilization of calcium from the shell in order to buffer the end products of anaerobic respiration is a relevant physiological adaptation to environmental stress which allows the animal to protect its tissues by valve closure while sustaining basal metabolism. As mentioned in the previous section, during short-term environmental stress freshly deposited calcium is more labile than the remainder of the valve and is certainly utilized during the initial stress period. Under long-term or repeated short-term stress conditions, however, there will be a greater demand for calcium due to the more prolonged periods of valve closure (Akberali et al., 1977, 1981; Akberali, 1978; Akberali and Black, 1980). It is likely that the more tightly bound previously deposited calcium will then be mobilized, which could lead to an overall decrease in shell mass. The effects of increasing duration of continuous salinity stress on shell mass and shell strength in Scrobicularia have been examined and indicate that calcium in the mantle cavity fluid is directly related (or proportional) to a reduction in shell mass (Akberali et al., 1983). Such a reduction in shell mass will also include the loss of organic and other inorganic constituents. The shell

162

H . B. AKBERALI A N D E. R. TRUEMAN

strength is reduced approximately in proportion to calcium dissolution from the valves and shows a general decrease with increased duration of the applied stress.

VI.

Action of Heavy Metal Stressors A. Accumulation of Heavy Metals

The capacity of bivalve molluscs to accumulate potentially toxic heavy metals in their tissues, far in excess of environmental levels, is well known and has become the focus of an increasing number of studies (Manley and George, 1977; Phillips, 1976, 1977; Bryan and Gibbs, 1983). Most of the evidence for absorption of metals and their radionucleotides from solution seems to involve passive diffusion of the metal, probably as uncharged soluble complexes, down gradients created by adsorption at the surface and binding by constituents of the surface cells, body fluids, and internal organs (Bryan, 1979; Carpene and George, 1981; Simkiss, 1983). However, this does not exclude, e.g., in higher organisms, the possibility of some movement of heavy metals by carrier systems used for calcium or magnesium transport (Bryan, 1976; Davies, 1978). The most important source of heavy metal bioaccumulation in bivalve molluscs is from suspended particles, in the case of suspension feeders, and from sediments in deposit feeders (Bryan, 1976, 1979). Tissue distribution of heavy metals as a result of bioaccumulation is typically uneven, and in some cases shows a high degree of organ specificity (Bryan, 1973; Bryan and Gibbs, 1983; George and Pirie, 1980; Viarengo et al., 1980a,b, 1981; Carmichael, 1980; Calabrese et al., 1984). The ability of bivalve molluscs to concentrate heavy metals at very high levels in the different tissues and yet to survive and apparently to reproduce normally indicates that they have evolved control or tolerance mechanisms at the cellular levels. Various systems for losing or immobilizing metals have been recognized in bivalve molluscs, apart from the possibility of diffusion. These include the immobilization of heavy metals in membrane-bound vesicles prior to their excretion from the kidney (Bryan 1973; George and Pirie, 1980; Carmichael, 1980; Viarengo et al., 1981; Simkiss et al., 1982; Calebrese et al., 1984) and by binding to wandering leucocytes, polysaccharides, amino acids, and proteins, e.g., metallothioneins (George et al., 1979; Bryan, 1979; Roesijadi, 1980; Viarengo et al., 1980a,b). A comprehensive review by Cunningham (1979) on the factors affecting accumulation, distribution, and loss of metals from the tissues due to intrinsic and extrinsic factors should also be consulted.

EFFECTS O F STRESS ON MARINE BIVALVE MOLLUSCS

I63

It has been suggested that the usefulness of bivalves such as M . edulis (Davenport, 1977) and S. plana (Akberali and Black, 1980; Akberali et al., 1981) as biological monitoring agents may be limited, since the bivalves may fail to register the transient or recurrent short-term presence of high pollutant levels as a result of their behavioural avoidance mechanisms (Figs. 6 and 9). It should be pointed out, however, that Scrobicularia shows behavioural avoidance of high concentrations of soluble zinc (5- 10 ppm) and copper (0.1-0.5 ppm) which will make it ineffective at the natural levels of between 1 and 52 pgll (ppb) zinc and 0.2-1.7 pg/l (ppb) copper currently recorded in coastal and estuarine water (Phillips, 1977; Coombs, 1980). In some polluted estuaries, copper and zinc levels in estuarine water may rise to only 0.1 and 2 ppm, respectively (Bryan and Gibbs, 1983). Even at such concentrations, bivalves such as Scrobicularia and Mytilus will remain active and interact with the medium, so that any determination of copper and zinc contained within the tissues will reflect the availability of metals in the environment. It should be stressed that quoted environmental levels of copper and zinc do not necessarily correspond with the concentration at the time of arrival of the pollutant, when the concentration may be far greater. During such an instance, if the environmental concentration exceeds the sublethal tolerance levels of the animal, it will result in the triggering of the behavioural avoidance mechanism.

B. Effects of Heavy Metals on Tissues Bivalves can protect and isolate their tissues from adverse lethal effects of heavy metal pollutants by closure of the shell. The first visible response of S. plana to adverse environmental stress, e.g., copper and zinc, is to retract the siphons rapidly into the shell. Accordingly, an isolated inhalant siphonal tissue preparation was developed to examine the direct effects of heavy metals and pesticides (Akberali, 1981; Akberali et al., 1981, 1982a,b). Scrobicularia possesses extensible inhalant and exhalant siphons through which the clam makes contact with the overlying sea water. The inhalant siphon of Scrobicularia comprises longitudinal, circular, and radial muscles with six longitudinal nerves (Yonge, 1949: Chapman and Newell, 1956; Hodgson and Trueman, 1981). The posterior pallial nerves pass from the visceral ganglion to the siphonal nerves and innervate the muscle fibres. Longitudinal muscles are the major muscular component, and their stimulation causes the siphons to retract rapidly into the shell. In the isolated siphonal preparation, the nerve tracts are detached from central ganglia so that responses do not occur by means of the normal motor nervous system. Addition of copper at a final concentration

164

H . B . AKBERALI A N D E. R . TRUEMAN

of 0.25 or 0.5 ppm causes the isolated inhalant siphon to go into a series of contractions (Fig. 18). Removal of copper from sea water terminates siphonal contractions, indicating that copper has a direct effect. Earlier studies (Akberali and Black, 1980) on the effects of copper on the behaviour of intact Scrubicularia revealed the complete withdrawal of siphons, followed by valve closure during a 6-h exposure period to concentrations of copper from 0.1 to 0.5 ppm (Fig. 6). The effect of copper on the isolated inhalant siphon of Scrobiculuria is comparable to its action on the behaviour of the intact clam and so the sensitivity of the clam to dilute copper solutions may be explained in terms of a direct effect of copper on the siphons. Additionally, it has been shown that the copper-induced contractions of the isolated siphon are dependent on the presence of calcium in sea water (Fig. 33). Such contractions may arise via the displacement of free calcium from intracellular reservoirs, resulting in a stimulation of the nerve/muscle system (see’ Section V1,D). In contrast, addition of 1-10 ppm zinc has no direct effect on isolated siphons (Fig. 19), whereas the same concentrations of zinc, when added to an in situ siphon preparation, caused marked contractions of the inhalant siphon (Fig. 20). It is well known that heavy metals can inhibit the activity of many enzymes (Dixon and Webb, $967)and affect the function of several ceHular constituents such as membranes (Rothstein, 1959), lysosomes (Moore, 1977), and mitochondria (Corner and Sparrow, 1956; Kleiner, 1974; Zaba and Harris, 1978; Akberali and Earnshaw, 1982a,c; Akberali et ul., 1984). With respect to tissues of marine organisms, and bivalves in particular (Table VI), it has been reported that heavy metals exert inhibitory effects on physiological processes, e.g., ciliary activity of the gills, oxygen consumption (Brown and Newell, 1972; Manley, 1983; Calabrese et al., 1984; Martin et al., 1984), heart rate (Scott and Major, 1972), byssus synthesis (Martin et al., 1975; Davenport, 1977), changes in ATP content, uptake of amino acids and protein synthesis following sublethal ASW __L- h h h

At

Bt

-----t

0

CtwtD -

20

40

-

+

60

at -

-

-

-

80

+

-

-

100

II

€7

bt -

-

i

* --t-*--l P

120

140

MINUTES

FIG.33. S. plana: effects of copper on an isolated inhalant siphon preparation in artificial sea water (ASW) (see original paper for formula). Upward deflection of the recording trace indicates isotonic contraction of the inhalant siphon. The following experimental protocol was carried out in sequence: application of copper at final concentrations of 0.25 ppm (A) and 0.5 ppm (B); removal of ASW containing 0.5 ppm copper (C), followed by washes (w) and replacement of Ca-free ASW (D); further application of copper at final concentrations of 0.25 ppm (a) and 0.5 ppm (b); and addition of CaCI2 to the organ bath to give a final concentration of 10 mM (E) (from Akberali et a / . , 1982a).

TABLEVI. SUMMARY OF SOMEOBSERVED RESPONSES TO HEAVYMETALSTRESSORS I N RELATION TO ALTERATION IN BEHAVIOURAL/BlOCHEMICAL/PHYSIOLOGICALPROCESSES IN MARINEBIVALVEMOLLUSCS

Species

Stage of organization where stressor is applied

Heavy metal stressor

Observed response to stressor

-

Reference

M . edulis, M . modiolus, C . gigas, Anadara senilis, M . demissus

Adult

Copper

Valve closure depending on concentration

Davenport (1977); Manley and Davenport (1979); Manley (1983)

S . plana

Adult

Copper, zinc, 1naphthol

Valve closure depending on concentration

Akberali and Black (1980); Akberali et al. (1981, 1982b)

M . edulis

Adult

Copper, zinc, mercury

Inhibition of byssal thread production

Martin et al. (1975); Davenport (1977)

M . edulis

Adult

Copper, zinc

Inhibition of respiration

Scott and Major (1972); Brown and Newell (1972); Manley (1983)

M . edulis, C . gigas, C . margaritacea, P . perna, C . meridionalis

Adult

Copper, zinc, cadmium, lead

Inhibition of filtration rate

Abel (1976); Watling (1981)

M . galloprovincialis

Adult

Copper

Inhibition of protein synthesis, ATP content, and uptake of amino acids by various tissues

Viarengo et al. (1980b)

TABLEV1 (CONTINUED)

Species

M . edulis

Stage of organization where stressor is applied Adult

Heavy metal stressor Copper

Zinc

Cadmium

Observed response to stressor

Reference

Most toxic-suppressed growth of young oocytes and vitellogenesis in large oocytes Less toxic-inhibited oocyte development and severe lysis of gametes Least toxic-suppressed gametogenesis only in the initial stages

Maung-Myint and Tyler (1982)

C. virginica

Adult

Copper

Increase in oxygen consumption by excised gill tissue; ultrastructural changes in terms of mitochondria1 swelling and damage in the ciliated epithelial cells

Engel and Fowler (1979)

M . californianus

Excised gill tissue

Copper, mercury, iron

Inhibition of glycine influx

Swinehart and Crowe (1980)

S. p l u m

Isolated siphonal tissue

Copper, 1-naphthol

Spontaneous contractions of muscle

Akberali (1981); Akberali et a/. (1982a,b)

C . virginica

48-h straight-hinge larval stage

Copper

Inhibition of growth

Calabrese et al. (1977)

M . mercennria

48-h straight-hinge larval stage

Copper, zinc

Inhibition of growth

Calabrese et a / . (1977)

C . gigas

Larval stage

Zinc

Inhibition of growth

Brereton et nl. (1973)

M . edulis

Unfertilized eggs

Copper Zinc

Stimulation of egg respiration Inhibition of egg respiration

Akberali et al. (1984)

M . edulis

Sperm

Copper Zinc

Inhibition of sperm respiration Inhibition of sperm respiration and motility

Akberali et al. (1985)

M . edulis

Digestive gland and mantle mitochondria

Copper

Biphasic effect-stimulation followed by inhibition of mitochondrial respiration Inhibition of mitochondrial respiration

Akberali et a/. (1984)

Copper

Inhibition of mitochondrial calcium transport

Akberali and Earnshaw (1982~)

Zinc

Inhibition of mitochondrial calcium transport

Zinc

M . edulis

Digestive gland and mantle mitochondria

Akberali and Earnshaw (1982a)

I68

H. B. AKBERALI A N D E. R. TRUEMAN

exposures to copper (Viarengo et al., 1980b), and mitochondria1 respiration and calcium transport (Akberali and Earnshaw, 1982a,c; Akberali et al., 1984). It has also been shown that concentrations of 0.05 ppm copper or 0.2 ppm zinc applied continuously to adult M . edulis severely inhibit gametogenesis by suppressing both the growth of young oocytes and vitellogenesis in larger oocytes (Maung-Myint and Tyler, 1982). When the oyster C. virginica was continuously exposed to 50 and 100 ppb copper, significant increases in the rate of oxygen consumption of the excised gill tissue after 14 days of continuous exposure to 100 ppb copper were reported (Engel and Fowler, 1979). It has also been shown that copper, cadmium, lead, and zinc cause reduction of the filtering rates in the bivalve molluscs C. gigas, Crassostrea margaritacea, P . perna, and Choromytilus meridionalis, with copper being most toxic to both oysters and mussels (Watling, 1981). Further evidence on the effect of heavy metals on filtration rates has also been reported for M . edulis (Abel, 1976). Mercury, copper, and zinc reduced filtration rate by 50% at concentrations of 0.04, 0.15, and 1.6 ppm, respectively. In the gills of M . californianus and Anodonta, mercury has a dramatic effect on amino acid efflux, whereas copper, mercury, and iron inhibit the influx of glycine into Mytilus gills (Swinehart and Crowe, 1980). C. Effects of Heavy Metals on Released Gametes and Embryonic and Juvenile Stages Although adult bivalves may be capable of tolerating high body concentration of heavy metals, little attention has been directed toward the effects these metal concentrations may have on gametogenesis, fertilization, and embryology. Laboratory techniques are now available for the rearing of early developmental stages of a variety of marine fishes and invertebrates so that it should be feasible to direct both field and laboratory work to emphasize the impact of heavy metal stress on various stages in the life cycle. The gametogenic cycle in bivalves, e.g., M . edulis, can be affected by variation in natural environmental parameters, such as temperature and food abundance (Seed, 1976; Bayne et al., 1978; Lowe et al., 1982) but is also subjected to stress imposed by heavy metal input. The application of environmental stress to adult marine bivalve molluscs can result in a reduction in reproductive capacity. For example, prolonged exposure of M. edulis to high temperatures and low ration results in stress to the organism owing to an increase in metabolic rate (Gabbot and Bayne, 1973). Although subsequent oocyte development, vitellogenesis, and fertilization appear to proceed normally, the larvae have a lower growth rate

EFFECTS OF STRESS O N MARINE BIVALVE MOLLUSCS

I69

than larvae from “unstressed” adults. Moreover, the “stressed” adults produce fewer and smaller eggs with less lipid and protein reserves (Bayne et al., 1978) and a larger proportion of abnormal trochophores resulting in a small proportion of normal prodissoconch I larvae (Bayne, 1972; Bayne et al., 1975). It has recently been shown that continuous exposure to sublethal levels of copper and zinc suppresses gametogenesis in M . edulis, with copper being more toxic (Maung-Myint and- Tyler, 1982). Little information is available in the literature concerning the mechanism of the action of heavy metals either with respect to the inhibition of gametogenesis or in their sublethal effects on the released gametes of bivalve molluscs and on the overall pattern of development. Due to a high surface area : volume ratio and in the absence of behavioural avoidance mechanisms, newly released gametes may well be more susceptible than adults to the direct input of heavy metal into the aquatic environment. The presence of heavy metals in the environment has been shown to inhibit the gametogenic cycle, embryonic development, and larval growth. For example, Wisely and Blick (1967) exposed M . edulis and C . commercialis larvae with developing dissoconchs to copper and mercury. Fifty percent of the mussel larvae died in 2 h at concentrations of 32.3 ppm copper and 13 ppm mercury, and oyster larvae at 180.5 ppm mercury. They concluded that this comparatively high resistance was a result of the ability of these organisms to withdraw their bodies into their shells, thereby reducing the penetration of the toxic material into the tissues. The lethal concentration values (LCso) obtained would thus be influenced by the length of time that a particular test species could remain closed (Table VII). In the presence of zinc concentrations of 125-500 ppb, growth and development of larvae of the Pacific oyster C . gigas were markedly slower (Brereton et al., 1973). This indicates that exposure of larvae to sublethal concentrations of heavy metals can have subtle but equally deleterious consequences on development as exposure to lethal concentration levels. Calabrese et al. (1973) have reported an LCs0 value for the development of embryos of C . virginica to straight-hinge larval stage of approximately 0.103 ppm copper, 0.0056 ppm mercury, and 0.31 ppm zinc, an indication that the embryos of bivalve molluscs may be more sensitive than the larvae to heavy metal pollution, while the adult animals are fairly resistant (Table VII). Furthermore, a comparison of studies on C . virginica and M . mercenaria larvae (Calabrese et al., 1977) during the development of embryos to the straight-hinge larval stage (Calabrese et al., 1973; Calabrese and Nelson, 1974) indicates that a slightly higher concentration of mercury, silver, and copper was required for 50% mortality of larvae than for the development of embryos. The early develop-

TABLEVII. COMPARISON OF TOXIC EFFECTSOF STRESSORS I N RELATIONTO VARIOUSLIFE STAGES^ Species

Stage of organization

Stressor

Concentration

Duration of exposure

M. mercenaria

Embryos and following development to straight-hinge larvae

Mercury Silver Zinc Nickel Lead

4.8 pg/l 21 pg/l 166 pg/l 3 10 pg/l 780 pg/l

42-48 42-48 42-48 42-48 42-48

M . edulis

Embryos and following development to straight-hinge larvae

Copper Mercury Silver Zinc Lead Cadmium

5.8 pg/l 5.8 pg/l 14 pg/l 175 pg/l 476 pg/l 1200 pg/l

48 h 48 h 48 h 48 h 48 h 48 h

h h h h h

M . edulis

Unfertilized egg First polar body Two-cell stage 64 cells or greater Ciliated blastula Trochophore Early veliger

Sevin Sevin Sevin Sevin Sevin Sevin Sevin

20 mgil 5.3 mg/l 7 mg/l 8.3 mg/l 16 mg/l 19 mg/l 24 mg/l

I h Ih l h Ih Ih l h Ih

M . edulis

Larvae

Sevin

2.3 mg/l

2h

M . edulis

Adult

Copper

250 pgll 500 pg/l

Continuous exposure Continuous exposure

Observed response

Reference

Lc50 Lc50 LC50 LC50 LCSO

Calabrese and Nelson (1974)

ECso ECso

Martin et al. (1981)

EC50 EC50

EC50 EC50 Armstrong and Millemann (1974)

EC50

Stewart et al. ( 1967)

MLT 4-5 days MLT 2 days

Davenport (1977)

M . edulis planulatus

Larvae with developing dissoconch

Mercury Copper

13 mg/l 32.2 mg/l

2h 2h

C . virginica

Embryos and following development to straight-hinge larvae

Mercury Silver Copper Zinc

5.6 pg/l 5.8 pgll 103 pg/l 310 pg/l

42-48 42-48 42-48 42-48

C . gigas

Embryos and following development to straight-hinge larvae

Copper Mercury Silver Zinc Cadmium Lead

5.3 pgll 6.7 pgll 22 pg/l 119 pg/l 611 pg/l 758 pg/l

48 h 48 h 48 h 48 h 48 h 48 h

EC50 ECso EC50

180.5 mg/l

2h

LCSO

Wisely and Blick ( 1967)

1 mgll 100 mg/l

96 h 96 h

LCJO LCJO

Walne (1964); Portman (1970)

Lc50 LCSO

Eisler (1977) Akberali and Black (1980) Akberali et a / . (1981)

h h h h

LCSO LCSO

Wisely and Blick (1967)

LCSO Lc50 LCSO LCSO

Calabrese et al. (1 973)

EGO

Martin et a / . (1981)

EGO EGO

C . commercialis

Larvae with developing dissoconch

Mercury

0 . edulis

Larvae Adult

Zinc Zinc

M . arenaria

Adult

Copper Zinc

5 mg/l 52 mg/l

48 h 48 h

S . plana

Adult

Copper

500 pg/l

Continuous exposure

MLT 5-7 days

Zinc

10 mg/l

Continuous exposure

MLT 6 days

1-Naphthol

20 mg/l 10 mg/l

Continuous exposure Continuous exposure

MLT 5 days MLT 9 days

Akberali et a / . (1982b)

Abbreviations: MLT, median lethal time for 50% mortality; LCso, lethal concentration for 50% mortality; ECso, concentrations that caused anomalous development of 50% test animals.

I72

H . B . AKBERALI A N D E. R. TRUEMAN

mental stages in M . edulis was most sensitive to the insecticide Sevin during the first hour after fertilization. The toxicity of Sevin decreased with increase in age of the larvae (Armstrong and Milleman, 1974). For example, the l-h EC50values for the first polar body stage and for the veliger were 5.3 and 24.0 mg/l, respectively (Table VII). The mechanisms involved in the disruption by heavy metal of the gametogenic cycle (Maung-Myint and Tyler, 1982) and embryonic development (Calabrese and Nelson, 1974) are unknown, but it is possible that these heavy metals inhibit metabolic processes (Table VI). Zinc is known to inhibit the respiration of mitochondria isolated from the mantle or digestive gland of M . edulis (Akberali and Earnshaw, 1982a), fish liver (Zaba and Harris, 1978), and rat liver (Kleiner and von Jagow, 1972; Kleiner, 1974; Akberali and Earnshaw, 1982a). In contrast, copper initially stimulates and then inhibits respiration of mitochondria isolated from fish liver (Zaba and Harris, 1978), rat liver (Zaba and Harris, 1976), and mantle tissue of M . edulis (Akberali et al., 1984). Recent studies have examined the action of copper and zinc on both the sperm and the unfertilized eggs of M . edulis, with emphasis placed on their effects on respiration (Akberali et al., 1984) and sperm motility, and on uptake and intracellular localization of heavy metals (Akberali, unpublished). This approach establishes the mode of action of copper and zinc on the released gametes at a cellular level, in order to avoid complexities involved when working at organismal and tissue levels. Such an approach would serve as a convenient starting point in seeking to understand the observed heavy metal toxicity associated with the gametogenic cycle and development. Many metal accumulation studies with adult bivalve molluscs have been undertaken with little thought as to their reproductive condition at the onset of the exposure period or the effect that spawning during the exposure period may have on metal elimination via the released gametes and on gamete viability itself. For example, in M . edulis, the toxicity of copper varies during the reproductive period (Delhaye and Cornet, 1975). In the months preceeding the spawning (January-February) the TL, is 9-10 days for a l-ppm copper concentration; this decreases to 6 days and 2-3 days during the spawning period (March) and postspawning period (April-May), respectively. It was concluded that this increase in sensitivity during the spawning season, which is characterized by a period of high respiration (de Vooys, 1976), was more a result of increased metabolic rate, resulting in a faster absorption of copper, than an increase in sensitivity. The decrease in T L , to 2-3 days observed during postspawning period may well be due to the metabolic condition of the adults. It is well known that during the period following spawning the metabolic reserves of the adults are at a minimum (Bayne, 1976); and

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

I73

hence the presence of copper or other heavy metals may well provide an added burden on the adult metabolic state. It is also reported by Cunningham and Trip (1975) that when C. uirginica were continuously exposed to sublethal levels of mercury, a 60% decline in the accumulated mercury levels in the tissues was observed during the spawning season. These authors have suggested that if mercury was accumulated in the gonadal tissue then an appreciable loss may occur during spawning, with the implication that the metal-gamete interaction during the spawning season may result in a decrease in reproductive potential, Indeed, Maung-Myint and Tyler (1982) have reported that continuous exposures to both copper and zinc suppress gametogenesis in adult M . edulis, with copper being more toxic (Table VI). Furthermore, transfer of heavy metals, e.g., cadmium or copper, from adult female oysters to their eggs has also been reported (Greig et al., 1975). Direct addition of 0.1-0.5 mM (6.3-31.5 ppm) copper to unfertilized eggs of M . edulis resulted in an immediate stimulation of respiration, but a similar direct addition of zinc (6.5-32.5 ppm) had no such effect (Fig. 34). By contrast, a similar direct addition of copper to sperm has no effect, but the respiration is inhibited by zinc (Fig. 34). Additional experiments, in which the gametes were preincubated with the heavy metal at 0°C for 20 min before measurement of respiration, showed that egg respiration was inhibited by zinc and sperm respiration by copper (Akberali et al., 1985). Presumably the time-dependent increase in the cytoplasmic concentration effects of copper and zinc is variable and appears to be relevant in its mode of action on sperm and egg respiration. Furthermore, the effects of direct application (Fig. 35) or preincubation (Akberali et al., 1985) with

9

cf

-

2 min

FIG.34. Oxygen electrode traces showing the effect of direct additions (4) of copper or zinc on egg and sperm respiration in filtered sea water at 10°C. Heavy metal ions were present at a final concentration of 0.5 mM. Numerals refer to respiration as nanomoles oxygen/minute/milligram protein (from Akberali ef a / ., 1984).

174

H . B . AKBERALI A N D E. R. TRUEMAN

0 '

I

I

r

I

1

2

3

4

cu2+ ( m M )

FIG.35. Egg respiration as afunction of direct additions of various copper concentrations. The experiment was carried out as in Fig. 34 (from Akberali et a / . , 1984).

copper shows that a concentration of ca. 0.5 mM copper causes a maximal stimulation in Mytilus of egg respiration, and that higher copper concentrations result in a progressively smaller stimulation of respiration. The uptake of copper and zinc by sperm and eggs of Mytilus, expressed on the basis of cell volume in order to take account of markedly different volumes, indicates that the uptake of both metal ions by the gametes increases with increasing metal concentration. The metal ion uptake in the sperm is approximately three times that in the eggs, with more zinc than copper uptake in both the egg and sperm (Akberali ct ul., 1985). Akberali et al. (1984) have further reported that respiration in unfertilized M. edulis eggs is only partially released, since the addition of uncoupler carbonyl cyanide rn-chlorophenylhydrazone (CCCP) causes a several-fold increase in egg respiration. Presumably the low rate of basal respiration in the unfertilized egg ensures that the substrate reserves are not depleted prior to fertilization. These authors have suggested that respiration in the unfertilized egg is inhibited by a high ATP/ADP ratio in the cytosol. Respiration can, therefore, be stimulated by either the addition of H+-translocating uncoupler (CCCP) or by copper which may act by stimulating mitochondria1 K + influx (see Section V1,D). It is tempting to suggest that the observed effects of copper and zinc in suppressing gameto-

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

175

genesis in adult M . edrnlis (Maung-Myint and Tyler, 1982) may be partly caused by their disruptive effects on normal cellular respiration of gametes (Table VI). Adult bivalve tissues, including gonads, accumulate high levels of heavy metals (Cunningham and Trip, 1975; Bryan and Gibbs, 1983). It is likely that low levels of heavy metal incorporated during gamete development from adult tissue (Greig et al., 1975) could well affect subsequent gamete viability and postfertilization development. Under natural conditions, the long-term effects of heavy metals on gametogenesis may well be of more importance to a species than, for example, their action in reducing the performance of gametes and larvae. The presence of a heavy metal scavenging system in M . edulis or other bivalve gametes has not yet been established. Recently, zinc-binding protein has been demonstrated in the unfertilized egg of the sea urchin Anthocidaris (Ohtake et al., 1983). It has been shown that zinc concentrations which were lethal to “naive” fathead minnow (Pimpehales promelas) cell cultures were not lethal to cells which were slowly adapted to higher levels of zinc (Adragna and Privitera, 1978). This suggests that the cells were able to tolerate a gradual increase in the metal load. Such an exciting approach, which has been adopted for vertebrate cell cultures, could well yield interesting results in heavy metal stressor studies of bivalves. D. Effects of Heavy Metals on Cellular Organelles The ability of bivalve molluscs to concentrate heavy metals in different tissues (Bryan and Gibbs, 1983) and apparently to survive and reproduce indicates that they have evolved control or tolerance mechanisms at the cellular level. The accumulation of heavy metals in various tissues in the estuarine bivalve molluscs, e.g., S . plana and M . edulis, has been shown to vary in different localities depending on the environmental concentration (Phillips, 1977; Bryan and Gibbs, 1983). Although studies on uptake and respiratory effects of heavy metals on cellular organelles, e.g., fish and rat liver mitochondria (Kleiner and von Jagow, 1972; Kleiner, 1974; Zaba and Harris, 1976, 1978) have been reported, there is little published work with cellular organelles from bivalve tissues. Emphasis should be placed especially with reference to tolerance and sensitivity, which may exist at the organelle levels in various tissues in relation to the observed differences in the amount of heavy metal accumulated by the tissues. In fish and mammals, the liver is known to be of considerable importance in the storage, uptake, and detoxification of heavy metals by metallothionein synthesis (O’Dell and Campbell, 1971 ;Zaba and Harris, 1976, 1978). Furthermore, studies on mammalian liver mitochondria indicate that these

I76

H . B . AKBERALI A N D E. R. TRUEMAN

organelles are capable of active uptake of divalent cations (Vainio el al., 1970; Bygrave, 1978). In M . edulis, digestive gland and mantle mitochondria have been shown to possess a calcium transporter comparable to vertebrate mitochondria (Akberali and Earnshaw, 1982b). The addition of calcium to M . edulis mitochondria results in a transient stimulation of respiration which is less pronounced than in rat liver mitochondria. This stimulatory response by mussel mitochondria to calcium ion addition is strongly indicative of an energy-linked uptake of calcium which is similar to that reported for vertebrate preparations (Lehninger, 1970; Bygrave, 1978; Zaba and Harris, 1978). Mitochondria are known to act as regulators of intracellular calcium levels (Bygrave, 1977; Carafoli and Crompton, 1978a; Nicholls and Crompton, 1980), and may play a role in both nerve conduction (Alnaes and Rahamimoff, 1975; Carafoli and Crompton, 1978b) and muscle contraction (Huddart and Price, 1976; Carafoli and Crompton, 1978a). Since bivalve molluscs accumulate excessive levels of heavy metals in their tissues, it is likely that mitochondria1 calcium transport may at some stage be affected prior to immob,ilizationor excretion of heavy metals. Akberali and Earnshaw (1982~)examined the effects of copper and zinc on the respiration of digestive gland mitochondria in M . edufis and their calcium transport in a sucrose reaction medium. Copper has little effect on the initial rate of basal respiration, but dramatically inhibits the initial rate of calcium transport, with 50% inhibition occurring at 50 p M copper (Fig. 36A). Calcium transport is also inhibited by copper in rat liver mitochondria (Zaba and Harris, 1976), probably in a fashion similar to lead, which at low concentrations is a competitive inhibitor of calcium transport and at high concentrations inhibits the production or use of respiratory energy (Parr and Harris, 1976). On the other hand, zinc produces a similar degree of inhibition in the initial rates of both basal respiration and calcium transport, with 50% inhibition occurring at approximately 60-80 p M zinc (Fig. 36B). Akberali and Earnshaw (1982~)suggested that the inhibition of calcium transport in M . edulis mitochondria caused by zinc is largely a respiratory inhibition. The effects of copper and zinc have also been examined on M . edulis digestive gland mitochondria which have been preloaded with calcium (Akberali and Earnshaw, 1982~).The addition of 100-200 p M copper results in an immediate efflux of calcium, whereas the accumulated mitochondrial calcium was stable on addition of 100 p M zinc (Fig. 37). A delayed and much slower rate of calcium efflux occurred at 200 p M zinc. On the basis of these findings, Akberali and Earnshaw (19824 put forward a hypothesis for the copper-induced contraction observed in the isolated bivalve mollusc siphon (Akberali, 1981; Akberali et al., 1982a). They

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS

I\

-

1

I77

0

-*-•

-

0 100

200

300

400

cuso4 OJM)

0 ' 100

ZOO

300

400

Z ~ S O( p~ M )

FIG.36. M. edulis digestive gland mitochondria: The effect of copper (A) and zinc (B) on the initial rate of basal respiration in the absence of calcium (0)and on the initial rate of calcium transport (0)in the sucrose reaction medium. Control rates (100%) for respiration were 12.3 ng atoms oxygen/min/mg mitochondria1 protein (A) and 12.7 ng atoms oxygen/ min/mg mitochondria1 protein (B). Control rates (100%) for calcium transport were 8.0 ng ions calcium/lO s/mg mitochondria1 protein (A) and 7.3 ng ions calcium/lO s/mg mitochondrial protein (B) (from Akberali and Earnshaw, 1982~).

I78

H . B. AKBERALI A N D E. R. TRUEMAN

10

I

C ._ m

-2 8 a

7

'n +

3 Z

.-

6

m

C

I

+ L 0

5

m

4

I

s

.-

U -

3

2

B

A 0 1

2

3

4

5

6

7

1

2

3

4

5

6

Time (rnln)

FIG.37. The effect of copper (A) and zinc (B) on accumulated mitochondria1 calcium-45: Mitochondria were isolated from M . edulis digestive gland. Mitochondria1 calcium transport was determined at 5°C using calcium-45. Copper was added at 2 min (J) at concentrations of 100 p M (0)and 200 pM (W), and ( 0 )represents calcium transport in the control (from Akberali and Earnshaw, 1982~).

suggested that, in the presence of copper, release of calcium ions takes place from mitochondria or possibly another cellular site, and that this increase in free intracellular calcium then induces excitation-contraction coupling in muscle cells. Mitochondria are thought to play a role in calcium movements during excitation-contraction coupling in muscle cells; this may be particularly important in molluscan smooth muscle in the absence of an organized sarcoplasmic reticulum (Huddart et al., 1977). Akberali and Earnshaw (1982~)have alternatively suggested that the induced calcium efflux by copper from mitochondria and possibly smooth endoplasmic reticulum in the presynaptic nerve terminal may result in an increase in intracellular calcium activity, which possibly triggers transmitter release (Katz and Miledi, 1965, 1967; Shapiro et al., 1980). It has been shown that fish liver mitochondria are capable of binding certain cations to a considerable degree (Zaba and Harris, 1978); thus calcium, copper, and manganese are taken up much more rapidly than

7

EFFECTS O F STRESS O N MARINE BIVALVE MOLLUSCS

I 79

zinc. Furthermore, copper possesses a biphasic reaction on state 4 oxidation of mitochondria isolated from rat liver (Zaba and Harris, 1976), fish liver (Zaba and Harris, 1978), and the mantle tissue of M. edulis (Akberali et al., 1984). Zaba and Harris (1976, 1978) have explained the biphasic mode of action of copper on vertebrate mitochondrial respiration. They have shown that the initial stimulation of mitochondrial respiration is a result of enhanced K + influx, which is accompanied by mitochondrial swelling. The ensuing progressive inhibition of mitochondrial respiration appears to be linked to K+ loss, which seems to take place at higher copper concentrations, where copper may have a general inhibitory effect upon respiratory enzymes. It seems reasonable to assume that the biphasic mode of action of copper on M. edulis mitochondrial respiration in KCI reaction medium (Fig. 38) is comparable, particularly as the addition of copper to M. edulis mitochondria in a reaction medium containing sucrose does not produce a respiratory stimulation (Fig. 36A). Furthermore, it is also reasonable to suppose that in the presence of copper, the stimulation of M. edulis unfertilized egg respiration (Figs. 34 and 35) and the increase in oxygen consumption rates of excised oyster (C. virginica) gill tissue (Engel and Fowler, 1979) is due to a similar uncoupling of mitochondrial respiration by copper, as previously described (Akberali er al., 1984). In fact, Engel and Fowler (1979) have demonstrated ultrastruc-

FIG.38. Oxygen electrode trace showing the action of copper on the respiration of mitochondria isolated from M. edulis mantle tissue. The substrate addition (J S) was made 1 min after addition of the mitochondria to the potassium chloride reaction medium and consisted of 6 mM succinate plus 6 mM glutamate. Copper (4)was added at a final concentration of 0.4 mM. Numerals refer to respiration as nanomoles oxygen/minute/milligram mitochondrial protein (from Akberali et al., 1984).

180

H . B . AKBERALI AND E. R . TRUEMAN

tural changes in the ciliated epithelial cells of gill tissue in relation to cellular swelling and mitochondrial damage. These authors have suggested that the observed increase in gill-tissue respiration may be related to increased cellular or mitochondrial membrane permeability. Akberali et al. (1984) have further reported a lack of inhibition of respiration in the eggs of M . edulis in the presence of copper as would be expected from the biphasic mode of action on mitochondrial respiration (Fig. 38). This could possibly be caused by low intracellular copper concentrations, since it is noticeable that the degree of stimulation from direct application (Fig. 35) or pretreatment (Akberali et al., 1985) with copper concentrations greater than 0.5 mM is reduced. This may be an important consideration in the case of the egg where the vitelline membrane, which surrounds the plasma membrane, could be responsible for binding a large proportion of heavy metal taken up from the medium, thus resulting in low intracellular metal concentrations. On the other hand, Akberali et al. (1984) reported that direct addition of copper has no effect on the respiration of the sperm (Fig. 34) of M . edulis, whereas pretreatment of sperm with copper inhibits respiration (Akberali et al., 1985). This different effect of copper on sperm and egg respiration results from greater copper uptake by the sperm (Akberali et d.,1985), which inhibits mitochondrial respiration; it may also be the result of different physiological states of the mitochondria in the two gametes. The latter authors have suggested that sperm mitochondria are presumably carrying out state 3 oxidation in which the rate of respiration is not limited by ADP, and therefore possess little potential for copper-induced uncoupling. Egg mitochondria, however, show a considerable potential for uncoupling by a H+-translocating uncoupler (CCCP), or by copper, and it has been suggested that their respiration is inhibited by a high cytoplasmic ATPIADP ratio. Unlike copper, zinc inhibits respiration of mitochondria isolated from the mantle or digestive gland of M . edulis (Akberali and Earnshaw, 1982a), fish liver (Zaba and Harris, 1978), and rat liver (Kleiner and von Jagow, 1972; Kleiner, 1974; Akberali and Earnshaw, 1982a). Approximate zinc concentrations giving a 50% inhibition of respiration are 70, 135, and 280 p M for rat liver, mantle, and digestive gland mitochondria, respectively (Akberali and Earnshaw, 1982a). The authors have suggested that the increased resistance of digestive gland and mantle mitochondria to zinc in M . edulis could be advantageous in an organism that accumulates excessive amounts of zinc (Fig. 39). The greater resistance of the digestive gland as compared to the mantle mitochondria could be an adaptation in a tissue that has been shown to accumulate greater zinc concentrations (Pentreath, 1973; George and Pirie, 1980; Bryan and Gibbs, 1983).

181

EFFECTS OF STRESS O N MARINE BIVALVE MOLLUSCS

)

100

1

200

,

300

1-

400

1000

Zinc added ( V M )

FIG.39. The effect of zinc on the oxidation of succinate plus glutamate by M. edulis digestive gland, M. edulis mantle, and rat liver mitochondria. The control rates (100%) were 10.22 -+ 1.40 (SE) nmol oxygenfmidmg mitochondrial protein for M. edulis digestive gland (-.-); I1 5 0 0.40 for M. edulis mantle (- - - -), and 53.31 ? 4.88 for rat liver (-). The points represent means of five to seven separate mitochondrial isolations from different groups of animals. Vertical bars represent SE (from Akberali and Earnshaw, 1982a).

*

In fish, Zaba and Harris (1978) have shown that zinc is taken up by the mitochondria to a very limited extent, for example, raising the external zinc concentration from 10 to 60 ng ions/mg protein resulted in an increase in uptake from 0.4 to only 1.2 ng ions/mg protein. They found, however, that zinc was the most potent inhibitor of mitochondrial respiration, 75% inhibition occurring at a mitochondrial uptake level of only 2 ng ions/mg protein. They also reported that for mitochondria to accumulate this amount of zinc, the external concentration must be raised to 65 p M . This concentration is lower than the 50% inhibition concentration of 135 and 280 p M for M . edulis mantle and digestive gland mitochondria, respectively. It seems that mitochondria isolated from bivalve tissues are more resistant than vertebrate mitochondria. Future work involving bivalve mollusc populations with differing tissue zinc and other metal con-

182

H . B . AKBERALI A N D E . R. TRUEMAN

centrations is certainly required in order to elucidate this difference in resistance between tissues and species at cellular organelle level. The acute effects of heavy metals in a number of bivalve molluscs have been extensively investigated, and it is now perhaps the right time for sublethal effects of heavy metals to be examined in detail at tissue and cellular level. The work reviewed here indicates that sublethal effects can have far more serious long-term consequences on various processes which can ultimately affect the survival and propagation of species. Studies on the effects of heavy metals on genetic material of cells are still unclear. There is some evidence for metal resistance (Bryan, 1976; Adragna and Privitera, 1978) whereby tolerance can be increased to the toxic effects of heavy metals by previous exposures to sublethal concentrations, but these studies are few and limited to fish, brown fouling algae, and seaweeds.

VII.

Conclusions

The development of electronic and other analytical techniques has led to significant advances in the understanding of the behavioural adaptations in bivalve molluscs to adverse environmental changes. It is evident that bivalves can distinguish between favourable and unfavourable environmental conditions and make an appropriate behavioural response either to minimize the harmful effects of unfavourable conditions or to exploit favourable conditions. Many bivalves studied so far utilize valve closure as a protective mechanism to counteract transient or recurrent short-term adverse environmental changes, and they are capable of sensing immediate changes in environmental conditions. Sensory receptors are present on the marginal lobes of the mantle and the siphons, and changes in the medium are detected even when the valves are apparently closed. During periods of valve closure, bivalve molluscs are capable of sustaining basal metabolism using anaerobic respiration. The mobilization of bound calcium from the shell is also of great significance to an organism in order to buffer the end products of anaerobic respiration and hence minimize the toxic effects of metabolites. Although valve closure allows the organism to overcome short-term adverse changes in environmental conditions, it is of no survival value to long-term or persisting environmental changes.

Acknowledgments Our gratitude is expressed to the Natural Environment Research Council (1978-1982), the Leverhulme Trust (1983-1985), and The Nuffield Foundation (April-June, 1983) for support

EFFECTS O F STRESS O N MARINE BIVALVE MOLLUSCS

I83

and to Professors E . G. Cutter and D. M. Guthrie for the generous hospitality of the Botany and Zoology Departments, University of Manchester. Sincere gratitude is expressed by H.B.A. to Dr. M. J. Earnshaw, a friend and colleague, for his constant encouragement, advice, and moral support. We would like to thank Mrs. S. E. Hardman for patiently typing the manuscript.

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Growth in Barnacles D. J. Crisp Natural Environment Research Council, Unit of Marine Invertebrate Biology, Marine Science Laboratories, Menai Bridge, Gwynedd, United Kingdom

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E. Bourget Dkpartement de Biologie, Universitk L a u d , QuPbec, Canada

I. Evolution of Barnacles and Their Shells .. .. ,, Mechanisms of Growth . . .. .. .. .. ,. A. Growth of individual shell plates . . .. .. .. B. Primordial valves .. .. .. .. .. .. C. Orientation of barnacles at settlement and during growth .. .. .. .. .. 111. Modification of Shape A. Effects of crowding . . .. .. .. .. .. .. B. Influence of substratum on shape . . C. Influence of salinity on shape .. .. .. .. IV. Factors Influencing Growth Rate. . .. .. .. .. A. Temperature . . .. .. .. .. . . . . B. Light . . .. .. .. .. .. .. .. C. Current, tidal level, and nutrition . . .. .. .. .. D. Surface contour .. .. .. .. .. .. .. E. Orientation to current .. .. .. .. F. Population density . . .. .. .. G. Competing organisms. . .. . . .. . . H. Parasites .. .. . . . . . . . . . . I. Reproduction . . .. .. .. .. .. .. V. Age and Growth-the Growth Curve . . .. . . . . 11.

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Growth Rates of Various Species . . . . . . . . . . Histology and Fine Structure of the Integument: Growth and Ecdysis Shell Structure in Relation to Function . . . . . . . . . . Cyclical Factors in Growth.. . . . . . . . . . . . . A. Tidal influences.. . . . . . . . . . . . . . . B. Daily influences.. . . . . . . . . . . . . . . C. Other lunar influences . . . . . . . . . . . . . . D. Annual influences . . . . . . . . . . . . . . E. Other cyclic influences . . . . . . . . . . . . F. Frequency, scale, and precision of measurement . . . . . . References . . . . . . . . . . . . . . . . . .

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22 1 222 227 234 234 236 237 237 238 238 239

Evolution of Barnacles and Their Shells

All three orders of cirripedes, the parasitic Rhizocephala, the burrowing Acrothoracica, and the sessile Thoracica, have a nauplius stage with frontal horns and a n antennulary fixation mechanism, but only the Thoracica have an external armament of calcareous plates. The earliest known cirripede, Cyprilepas, was a stalked, unarmoured epizoite of eurypterids dating from the upper Silurian (Wills, 1963), clearly a forerunner of the suborder of stalked barnacles, the Lepadidae (Fig. 1). The development of the calcareous armature of the capitulum, protecting the body, mouthparts and cirri, was probably necessary for the early Lepadidae to have become independent of the protection offered by a n epizoic life, for modern epizoic barnacles often remain poorly armoured in comparison with intertidal forms. During the evolution of the Lepadidae, or stalked barnacles, there was first an increased calcification of plates surrounding the aperture-the paired scuta and terga-then the addition of an unpaired carinal plate (Fig. lA), and finally a surrounding defence of numerous smaller plates at the top of the stalk, one of which, the rostral, was to become of greatest size and importance (Fig. 1B and C). Whereas the floating and often epizoic lepadomorphs such as Lepas, Conchoderma, Octolasmis, etc., are still relatively thin shelled, with naked stalks, benthic scalpellids (Fig. 1B and C) and the intertidal Pollicipes have become well armed against predators by this further array of plates reinforced by chitinous scales over a short leathery stalk. The Verrucidae and Balanidae, or sessile barnacles, have further shortened the stalk to a mere cemented disc and have reduced in number but increased in size the wall plates (parietes) surrounding the tergum and scutum. The Balanomorpha, which are the most advanced thoracicans, dating from the upper Cretaceous, have thus evolved through a basic pattern of two paired opercular plates (scutum and tergum) and eight wall plates, two unpaired (carina and rostrum) and six paired (Fig. 1D). These are

GROWTH IN BARNACLES

20 1

A 5- plated lepadomorphs

hypothetical balanomorph

scutum

tergum

FIG. I . Shell plates of lepadomorph (A-C) and hypothetical eight-plated balanomorph (DF). Abbreviations: s, scutum; t, tergum; c , carina; cap, capitulum; pe, peduncle or stalk, not visible in adult balanomorphs; ps, peduncle scales; r, rostrum; 1, rl, ul, cl, sc, other small shell plates developed around the base of the capitulum in benthic forms (the rostrum, r, becomes dominant in balanids); E, F, the inner surface of the opercular valves of balanomorphs; ar, adductor ridge; ad, insertion of adductor muscles; a, ala; r, radius at upper edges of each of the parietes, p, or shell plates r, rl, cl and c; cd, Id, rd, insertion of depressor muscles; oc, occludent margin of scuturn; am, articular margins where terga and scutum fit closely interlocking at the articular furrow, af, and articular groove, arg; bm, basal margins joined to the opercular membrane; cm,carinal margin of tergum; ax, apices, that of the tergum often produced into a beak; sp, spur of tergum (after Foster, 1978).

fused in various ways to produce the families as classified by Darwin (1854) and little modified since. Some species have also calcified the base, but whether membranous or calcified, the base must be perforated by the ducts of the cement glands in order to provide it with the adhesive mechanism by which it remains fixed to the substratum as it continues to grow outwards (Crisp, 1973). The cement, in Balanus at least, is a viscous material which relaxes under pressure and so allows barnacles to slide along the substratum under the force provided by surrounding growing individuals (Crisp, 1960a). It has nothing like the strength sometimes attributed to it (see Yule and Walker, 1984). Cyprids of the balanomorphs show clear evidence of recapitulation. Immediately after settlement they retain a broad, flexible peduncle which can be pulled out when exposed to strong currents (Crisp and Stubbings, 1957), but within a few days the animal is drawn down onto the substratum, to which its basal region adheres (Yule and Walker, 1984). Immediately after metamorphosis, the scuta and terga of the young barnacle are greatly exaggerated in size, as in the Lepadidae, with the wall plates

202

D. J. CRISP AND E. BOURGET

relatively small. However, during early growth the shape rapidly changes to that of the typical balanomorph (Bourget and Crisp, 1975a). Balanomorphs have evolved features which have rendered them highly successful in occupying the intertidal zone. These modifications not only seal them from desiccation but also protect them from predators. As we have seen, the vulnerable stalk has disappeared, the wall plates have become interlocked so that they invest the soft parts completely, and the opercular plates which seal the orifice, instead of being exposed to attack, are sunk below the crown of the barnacle. These plates usually fit rigidly against the parietes and are held closed by powerful muscles. The opening between the pairs of opercular plates is clamped by the adductor muscle of the scutum (Fig. l), and the occludent margin of the plates is covered by leathery strips or “flaps” so that, when the operculum is closed, the mantle cavity is completely sealed. When required, however, it can be ventilated through a tiny hole or micropyle left between the flaps (Barnes and Barnes, 1957). The opercular plates must clearly be mobile to allow the cirri to emerge and so are suspended by a tough flexible membrane attached to an overhang which projects downwards from the upper end of the paries. This additional calcareous ring was termed by Darwin “the sheath.” Thus, as the barnacle grows and the crown apex extends upward, the downward growth of the sheath keeps the opercular aperture well protected below (Fig. 2).

ou TER

SURFACE OPERCUL.UM

HYPODERMIS (inner surface)

F I X AT I O N F I BR’ES

BASAL MEMBRANE

FIG.2. Section through wall plate (paries) of a balanornorph to show disposition of sheath and operculurn.

G R O W T H I N BARNACLES

203

II. Mechanisms of Growth A.

Growth of Individual Shell Plates

When Darwin wrote his monograph, the mechanism by which a sessile barnacle grew was thought puzzling, as it still is by the uninitiated, who believe the wall plates to be sealed on to the substratum. Darwin beautifully unravelled the mystery (Darwin, 1854, pp. 33-61), showing that the individual shell plates can grow at their edges where they overlap each other, yet maintain complete protection of the underlying parts. The overlapping shell margin he called the radius and the underlapping of the adjacent plate the ala (Fig. 1). Clearly each must be separated from the other by thin slips of living tissue in order to allow growth to continue. The wall plates grow at their lower edges and the base at its periphery, so these also must be separated by two layers of hypodermis, which add to the paries above and to the base below. We shall term this important region the basal suture, following Darwin's apt nomenclature. How then, since there is no solid seal at the suture between paries and base, is the shell held in place? The answer must lie in the numerous fixation fibres (see Crisp, 1965), demonstrated by Gutmann (1960) in Balanus balanoides (L.)' and present, as far as we know, in other balanomorphs (Fig. 2). These are long, fine tendons attached to the inner surface of each paries and expanded into a very short piece of striated muscle, joined again to the basis by a much shorter tendon. Thus the barnacle shell is not itself cemented to the substratum, but, like the shell of a limpet, is held by tendon and muscle to that part of the animal which does adhere to the substratum-the cemented base of the barnacle-which can be compared with the foot of the limpet held by mucus (Crisp, 1973; Grenon and Walker, 1981). No one has yet demonstrated the role of the muscles of the fixation fibers, but we believe that they allow the deposition of thin laminae of shell when they relax and cause it, while still malleable, to conform to the substratum when they contract. There can be no doubt that barnacles are able to chisel the shell into the substratum. Fouling species thus strip off paint films, causing corrosion (Woods Hole Oceanographic Institution, 1952) while epizoic species such as Chelonobia resrudinaria and Coronula diadema drive their shells into the substance of their turtle and I In this article we are retaining the scientific names used in the original publications, so as to avoid confusion, except where an actual mistake in identity was made. Newman and Ross (1976) have raised the former subgenus Semibalanus to generic rank, and refer to B . balanoides as Semibalanus balanoides. Southward (1976) has divided Chrhamalus stellarus (Poli) into true C. stellatus and another species called Chthamalus montagui; it is likely that most experimental work on C. stellatus, referred to here, was actually carried out with C . montagui.

204

D. J . CRISP AND E. BOURGET

cetacean hosts. In many barnacles with calcareous bases, the junction between wall plate and base is reinforced by a closely interlocking series of holes and projections, as described on p. 227. Exceptionally, in some species the various shell plates have lost their capacity for independent growth and have become effectively fused. Some Chthamalus species, especially those belonging to the hembeli group (Euraphia), show this tendency. Older specimens of Chthamalus hembeli and Chthamalus intertextus have a fused or nearly fused tergum and scutum, while the basal margin of the parietes becomes tucked in below the base, forming a calcareous ledge which prevents further growth in diameter. The compartments of old specimens of Chthamalus stellatus, and probably of many other species of this genus, become so strongly bound that they can no longer enlarge. Similarly, in some genera, such as Acasta and Pyrgoma, the parietes become fused so that the shell can increase in diameter only at the base, causing the aperture to remain small. Increase in girth, with commensurate increase in orifice diameter, can then be achieved only by abrasion or disintegration of the crown. Darwin noted that in two species, Tetraclita purpurascens (Wood) and Balanus perforatus (Bruguike), in which the aperture was usually enlarged by erosion, specimens in which the shell remained intact had indeed well-developed radii, and so had succeeded in enlarging the circumference and orifice. As he writes, “It appeared, but of course erroneously, as if the lateral growth of the compartments had been subjected to the will of the animal” (Darwin, 1854, p. 56).

B. Primordial Valves In the primitive thoracican Lepas, the cyprid, or settling stage, already contains the primordia of the five calcareous valves, scuta, terga, and carina, though before its attachment to a substratum they are without trace of calcareous matter. They can be seen through the carapace as a single layer of cells which, according to Darwin, are hexagonal in outline, 1/6000-2/6000 in. (4-8 pm) across. Almost immediately after settlement calcification begins, and the characteristic form of the five most primitive shell plates can clearly be seen under light-field, dark-field, or phasecontrast illumination if the tissues of the cyprid are cleared away with a strong solution of sodium hydroxide (Figs. 3 and 4A-C). Under high magnification with polarizing filters, the hexagonal patterns mentioned by Darwin can be seen to be composed of interlocking garlands of tiny calcite crystals with variously orientated crystal axes (Fig. 3D), alternately sparkling as the plane of polarisation is changed. A primordial shell lies at the umbones of each of the five major plates of lepads, and can be distinctly

FIG.3. Primordial valves of Lepas cyprid, under dark-field illumination, showing primordial scuta (s), terga (t) and canna (c); cyprid 1.5 m m long.

GROWTH I N BARNACLES

207

identified in the adult shell by their peculiar patterning. The mature shell is laid down around the primordial shell with marks that indicate the position of the growing edge at each progressive growth episode (Fig. 4D). It is interesting that, in Lepadidae with numerous plates, only the most primitive five have primordial elements. Darwin claimed to have seen traces of these primordial valves in Chthamalus, a primitive balanomorph, but not in the more advanced Balanidae. However, a distinct element at the apex of the paries (Bourget and Crisp, 1975c), primordial in the sense of being laid down at or very soon after metamorphosis, can be identified in balanids. Contrary to the views of Kuhn and Fuchs (1954), there are no calcareous deposits in the unmetamorphosed larval stages. Yule et al. (1982), by analytical methods, found that, as in the lepad cyprid, calcite is laid down only after metamorphosis in Balanus. The white crystalline deposits thought by Kuhn and Fuchs to be calcium carbonate were described by Walley (1969) and are probably organic, like those in the limbs of the adult barnacle, in which there are white deposits of guanine (Waite and Walker, 1984).

C. Orientation of Barnacles at Settlement and during Growth At settlement the last act of the still mobile cyprid is to orientate itself to three environmental influences-the contour of the surface, the direction of light, and the current. These influences form a hierarchy to which the cyprid reacts in that order (see Crisp, 1975). The strongest influence is the direction of surface grooves or furrows (Crisp and Barnes, 1954). The cyprid is capable of aligning to the long axis of a cylindrical cavity whose radius of curvature is many times the length of the cyprid itself, though narrow grooves into which it can fit snugly are preferred. These situations clearly offer protection during the period immediately after metamorphosis. Secondly, the cyprid responds to face the direction of light (Barnes et al., 1951). Forbes et al. (1971) examined the shading response to light coming from various directions and found that the rostra1 area of the adult barnacle, beneath which lie the paired eyes, was the most sensitive. When it orientates t o light at settlement, the cyprid places this sensitive comFIG.4. (A) Detail of primordial scutum of cyprid under normal illumination; f, food body and associated pigment; (B) detail under phase contrast; (C) detail under polarised light; (D) fully calcified primordial valve forming the umbone of the scutum of a juvenile Lepas. Note the similar hexagonal pattern within the primordial valve and the growth banding in the growing shell. Valve length ca. 0.5 mm [cf. (A)].

208

D . J . CRISP AND E . BOURGET

partment away from the light. However, it is from this direction that predatory fish learn to attack, so that the orientation adopted is likely to be of survival value. Thirdly, the response to current is so weak that it is difficult to demonstrate in the laboratory unless the first two influences are eliminated by experimenting on smooth surfaces and in darkness (Crisp and Stubbings, 1957). However, the reaction may nevertheless be important in barnacles that settle on swimming animals such as portunid crabs (Forbes er a[., 1971), turtles, and whales (Crisp and Stubbings, 1957). The orientation displayed to current, which causes the posterior end of the cyprid to point upstream, is that which allows the cirral flet, after metamorphosis, to fish into the current with least distortion of the animal’s posture. If, after settlement, the orientation is otherwise, it can readjust during growth and optimise its orientation. This process of “torsion” was shown by Crisp (1953) to result from constant exposure to unidirectional or prevalent currents. In barnacles with radial canals in the calcareous base, for example, Balanus improvisus (Darwin) or Balanus amphitrite (Darwin), the evidence of torsion remains behind as a spiral twist in the canals. However, B. balanoides, which has a membranous base, also reorientates in the expected sense (Crisp, 1953; contra Moore, 1933). It is not known how the prevailing current causes the torsion, whether the animal senses the current and can change the direction of growth accordingly, or whether the orientation is a passive result of mechanical forces. The current, which causes the extended cirri to twist, may thereby apply a torque to the base.

111.

Modification of Shape

The morphology of a barnacle has great plasticity, so that its form may be much modified during growth without greatly hindering its vital activities. Although there have been attempts to relate shape to environment (Abel, 1926; Neu, 1935), only three main factors have been well documented as causing modification in shape: first, the forces produced by the growth of surrounding individuals; secondly, the shape of the surface on which the barnacle is growing; and thirdly, salinity. A. Effects of Crowding An isolated barnacle grown on a smooth flat plate might be expected to extend symmetrically about its point of attachment. In an experiment to test this, Bourget and Crisp (1975a) found that although there was com-

GROWTH IN BARNACLES

209

plete symmetry in lateral extension, extension along the rostral exceeded that along the carinal direction in Elminius modestus (Darwin), while in Balanus balanoides there was considerable variation in eccentricity along the rostrocarinal axis. We suspect that, since in most species the rostral compartment makes the more acute angle to the substratum, the rostrum should grow faster, as in Elminius. It is well known that when barnacles grow in close proximity, their shells become elongated and the base narrow (Trusheim, 1932; Schafer, 1938, 1948). Whether the species possesses a membraneous or a calcareous base, the shell-secreting tissue of the basal suture in such elongate specimens must become distorted; the parietes, instead of progressively thickening towards the base, are there reduced to thin slips of shell with greatly reduced adhesion. In contrast, growth of the opercular plates, the sheath and the summits of the radii and alae continues apace. As a result, the upper parts of the individuals expand and fan out, producing hummocks which are strengthened on the outside by the interlocking of the compartments (Gutmann, 1960) but are fragile within. Eventually, the hummock disrupts (Barnes and Powell, 1950). When individuals grow in this hummock form, the opercular plates are often a better guide to size and age than the shell as a whole. Nevertheless, if barnacles grow on a smooth surface with sufficient space outside the group in which to expand, even though the individuals come to touch one another, they will continue to adhere. The outer ones will slide along the surface centrifugally, and the whole group will continue to grow with little elongation upwards (Crisp, 1960a). Clearly barnacles compete avidly for whatever space is free, and only when there is no more space in which to spread are they distorted into the columnar form. Some species, such as Chthamulus depressus (Poli) and E. modestus, have a naturally flattened shape with a low angle of contact with the substratum; these can form only slightly elongated columns when crowded. Others, such as Balanus humeri (Ascanius) or Chamaesipho columna (Spengler), readily and naturally develop tall individuals. B. Influence of Substratum on Shape Gregg (1948) noticed how surface irregularities left their impression on the walls of barnacles and of other organisms that grew in close conformity to the surface. Gutmann (1960) described how isolated individuals of Balanus balanoides were modified, those settling in hollows becoming flattened, while those growing near an edge had to extend the shell plates in order to allow them to arch over it. Small individuals lying in grooves would become elongated in the direction of the groove. He interpreted

210

D. J. CRISP A N D E . BOURGET

these modifications in terms of the necessity for the shell to remain in contact with the substratum. Crisp and Patel (1967) saw the analogy between the shape of a barnacle on a surface and that of a droplet of water. Thus a droplet in a groove becomes similarly orientated along the length of the groove. This is not due merely to its remaining in contact with the surface, but rather because it maintains a constant angle of contact at all points at the periphery, as a result of which its shape is modified. Crisp and Patel calculated, on this basis, how the shape of a barnacle would be modified when grown on surfaces of positive (convex) and negative (concave) curvature. Their observations agreed well with mathematical prediction, both for B . bulunoides with its conical form, and for E . modestus with its more complex, flatter shape. Fig. 5A and B illustrates the variation in shapes observed

CONCAVE

PLANE

CONVEX

FIG.5. Variation in shape with surface contour. Left to right: concave, plane, and convex substratum. (A) E. modesfus in side view, all of equal age; (B) E. modestus in plan view, all of equal age; (C) shell plates of B . balanoides, all of equal age (from Crisp and Patel, 1967, reproduced by courtesy of Marine Biological Association of India).

GROWTH IN BARNACLES

21 I

when the latter species is grown on concave and convex surfaces. Crisp and Patel also noted that, when barnacles grow on concave surfaces, the weight of dry tissue relative to the total, including shell, was larger than when grown on convex surfaces, probably because of the greater capacity of the hollowed-out base, while the opercular valves and parieties were reduced. In absolute size, however, the individuals on convex surfaces grew more rapidly, probably because they had the advantage, due to their exposed position, of greater access to water flow. In both Balanus and Elminius the shorter and narrower parietes formed on the concave surfaces were distinctly more crenulated than those on convex surfaces (Fig. 5B, C). There is probably a morphogenetic norm for the surface area/ volume ratio, so that the reduction in area caused by the concave shape of the substratum is compensated by folding and crenulation of the paries at the basal margin. Another example of morphometric compensation was seen in the size of the lateral compartments. Thus, when there was a large left compartment it would usually be compensated for by a small right one and vice versa (Crisp and Patel, 1967). C. Influence of Salinity on Shape Barnes and Barnes (1962a) noted that in the region of the Danish Belts, where B. balanoides reached its salinity limit, individuals have fragile shells and become unusually flat, the measure of height/basal diameter falling to 0.2 in Jutland, compared with values of 0.4-0.5 on the North Sea coasts. The euryhaline barnacle B. irnprouisus, however, showed no comparable change in shape (Barnes and Barnes, 1961)nor, in our experience, does the cosmopolitan species B. amphitrite.

IV.

Factors Influencing Growth Rate

Since the Lepadidae are oceanic and only occasionally found washed up alive, growth studies depend on serendipity. Most of our information on barnacle growth, in consequence, relates to the Balanomorpha, and in particular to B . balanoides. This species has a highly synchronized settlement in spring, so that a single cohort can be followed readily. All individuals of a year class have been exposed to broadly similar seasonal conditions of water temperature, salinity and suspended nutrients. The effects of local factors, such as wave action and water flow, can be isolated quite easily. Table I summarizes growth data for B. balanoides, and Table I1 includes data for other species.

TABLEI. B. balanoides GROWTHDATA

Location Spitzbergen Herdla, Norway Millport, Scotland Port Erin, Isle of Man Liverpool Menai Straits St. Malo St. Andrews, New Brunswick Woods Hole, Rocky Beach Woods Hole, Buzzards Bay

Conditions Intertidal Midlittoral Raft (cleaned) Midlittoral Piles, midlittoral Raft (cleaned) Midlittoral, piles Several levels and exposures Midlittoral Raft (submerged and cleaned)

Approximate annual monthly temperature range (sea and air average, "C) 4.2- 13.7 6-13.9 6.8-13.4 6.3-14.3 5.0-16.1 6.1-15.4 5.5-15.5 7.0-17.1 18--2 24- 4-2 22-4-4

pnlday 25 26 120- I30 120-160 23

Mean rostrocarinal diameter at end of first season (mm)

Reference Feyling-Hanssen (1953) Runnstrom (1925) Barnes and Powell (1953)

158 63 33

7-9 13-14 17.5 5.3 5.9 12-17 5-7 2.5-4.0

Moore (1934) Corlett (1948) D.J. Crisp (unpublished) D.J. Crisp (unpublished) Hatton (1938)

36 44 75

Ca. 9 Ca. 10

Bousfield (1954) Barnes and Barnes (1959b) Barnes and Barnes (1959b)

TABLE11. GROWTHRATESI N VARIOUSSPECIES~

Species

C . stellatus St. Malo HW exposed MTL exposed Brixham HWS HWN MTL LWN Millport Continuous immersion (raft) Continuous immersion after keeping at HW E . modestus Menai Straits (raft-continuous immersion) Millport, raft Millport, high intertidal Stranraer, low intertidal Balanus glandula (Darwin) Pacific Coast, intertidal

Linear rate + half maximum size

Daily growth in linear phase (pm)

Approximate maximum size (mm)

10 I5 21.4 37.5 50 55

12 12 12 12 12 12

1.67 2.5 3.57 6.25 8.33 9.71

12-14

12

2.0-2.33

Barnes (1956)

50

12

8.33

Barnes (1956)

138 113 25 42

17 17 17 17

16.2 13.3 2.9 5.0

D.J. Crisp (unpublished) Barnes and Barnes (1962b) Barnes and Barnes (1962b) Barnes and Barnes (1962b)

51

25

4.1

Barnes and Barnes (1956)

Reference Hatton (1938)

D.J. Crisp (unpublished)

(continued)

TABLE11. GROWTHRATESIN VARIOUSSPECIES~ (CONTINUED) Daily growth in linear phase (wn)

Approximate maximum size (mm)

Linear rate + half maximum size

245

25

19.6

Balanus perforatus Brixham intertidal Raft

14 122

35 35

0.8 6.97

Emily Clay (unpublished) Emily Clay (unpublished)

Balanus crenatus (Bruguiere) Millport, raft Menai Straits, raft

220 284

25 25

17.6 22.12

Barnes and Powell (1953) D.J. Crisp (unpublished)

50

8

12.5

Barnes (1958)

130 36 70

43 43 43

6.05 1.7 3.26

Barnes and Barnes (1954) Barnes and Barnes (1954) Crisp (1954) as Balanus porcatus (Da Costa)

31*

2.12

Moore (1935)

Species

B . amphitrite Shoreham Harbour, Portslade Power Station, panels on wall of cooling pond

Verruca stroemia (Miiller) Millport, raft

Balanus balanus (L.) Millport, raft Two fast-growing individuals Average first year Beaumaris Bay, sublittoral Balanus humeri Isle of Man a

33h

For B . balanoides, see Table 1. Derived from volume measurements and Bertalanffy equation

Reference Crisp (unpublished)

GROWTH I N BARNACLES

215

Although it is the soft tissues that are responsible for growth, the shell is so much easier to measure and weigh that the greater part of our knowledge relates to length or weight of the shell. It is assumed that a relationship exists between shell and body weight, although seasonal influences must be large for a species such as B . balanoides, which sheds its gametes in autumn and then stops feeding and moulting for some time (Crisp and Patel, 1960, 1969; Ritz and Crisp, 1970). For this species Barnes and Barnes (1959a,b) have provided a large amount of data on seasonal changes in wet weight and nitrogen content of the “body” (prosoma and thorax) in relation to shell size. It should be noted that these weights are difficult to relate to those of other investigators where tissue weight includes the depressor muscles and other parts adhering to the shell and basis, as well as prosoma and thorax. Thus Barnes and Barnes show a wet weight of tissue comprising 7-10% of the total of shell and tissue, whereas Crisp and Patel (1967) and Fradette and Bourget (1980) found the dry weight of all tissues to be as high as 7-10% of the total weight in B . balanoides and as high as 15% in Elminius, which has a thinner shell. Other variations in tissuehhell weight ratios may well be found, in addition to seasonal changes. For example, barnacles growing at high tide levels, under nutritional stress, would be expected to have a relatively low ratio of tissue weight to shell weight. Conversely, the ratio should increase in low salinity habitats where the shell is obviously less well calcified. However, we are not aware of any researches expressly directed to these aspects of growth of barnacles. A.

Temperature

It is usually assumed that temperature will enhance growth rates, since the cirral beat, which represents the potential rate of food gathering, has been shown by several authors to be strongly temperature-dependent (e.g., Southward, 1955, 1957). Thus it is scarcely valid to attempt to isolate the effects of temperature from those of food supply. Seasonal variations in growth rates involve a complex set of factors, including the physiological state of the animal, as well as environmental temperature, nutrient levels and racial differences, none of which can readily be disentangled, as can be seen from the study by Barnes and Barnes (1959a,b). Temperature may well have differential effects on assimilative and respiratory metabolism, growth and gametogenesis being determined as the net gain to the animal of these processes. Wu and Levings (1978, 1979) compiled an energy budget for individual and population growth, respectively, over the annual cycle of Baianus glandula.

216

D . J . CRISP AND E. BOURGET

B. Light Neither we nor Barnes (1953), in a careful study where other factors were eliminated, could find any evidence of an influence of light on growth, despite the claims of Klugh and Newcombe (1935) (see also p. 236). C. Current, Tidal Level, and Nutrition Sessile organisms such as barnacles generally reach their greatest size when growing in estuaries, in tidal rapids, or on headlands exposed to swell, since they can receive greater quantities of suspended food under such conditions. Hatton (1938) demonstrated this effect by transplanting barnacles from still to flowing water, while Hatton and Fischer-Piette (1932) and Moore (1934) found faster growth at wave-exposed localities. By growing B. balanoides on panels in the same current flow but with some individuals protected by baffles, Crisp (1960b) showed that those directly exposed to current not only grew larger, but their rate of growth steadily rose to reach double that of those behind the baffles. The faster growth rate was attributed to the extending of the cirri beyond the boundary layer into the freely flowing current. Currents not only transport more food to the barnacle, but also stimulate cirral activity. Cirral activity is also stimulated by the presence of food particles and soluble organics in the water (Crisp and Southward, 1961; Crisp, 1967; Allison and Dorsett, 1977). The higher the tidal level occupied, the more is the food supply restricted by periods of emersion, as shown by Barnes and Powell (1953) (see also Table IV). If their data are reinterpreted as growth rate per daily submergence time, there is only a small influence of tidal level, those at high water growing slightly faster. Crisp (1960b) explained this effect by suggesting that those at high tidal levels would benefit from greater wave exposure. However, Southward (1955) showed that the feeding mode of cirral activity (fast beat) was more prevalent in high water animals, while Barnes (1956) found that Chthamalus previously kept at high levels grew faster (Table 11). Ritz and Crisp (1970) demonstrated that high water B. balunoides fed more avidly than those accustomed to live at lower tidal levels. Thus, higher ingestion and growth rate per unit of feeding time may be explained as an adaptation to nutritional stress-subjectively, to hunger-in those individuals which have shorter periods of immersion. In Balanus bulunoides the influence of current, wave exposure, and tidal level appears greatly to outweigh latitudinal temperature influences, as can be seen from Table I.

GROWTH IN BARNACLES

217

D. Surface Contour Hatton (1938) noted that barnacles settled in hollows grew more slowly than those on an exposed surface. Cyprids of B. balanoides, settled in pits only slightly larger than themselves, not only showed slower metamorphosis (Gamble, private communication) but also slower growth during the first 15 days. After this time, having outgrown the dimensions of the pit, they fed, respired, and grew normally.

E. Orientation to Current A heavy board suspended from an anchored chain on swivels, such that it always swung with the tide, provided natural conditions of growth with unidirectional current flow. Spat of B. balanoides that had settled within a few days of each other were arranged at various orientations to the current flow and measured at the end of the season's growth. Those with the rostra1 end towards the current source could collect plankton simply by extending the cirral net (see Crisp and Southward, 1961), but those with the lateral plates or the carina towards the current source had to twist the cirral net through 90 or 180" in order to set it across the current. A small but significant advantage in growth of approximately 10% by weight or 3% by linear dimension accrued to the former (Crisp, 1960b).

F. Population Density Since barnacles settled together on a surface remove particulate material from the same water mass, they compete for the same food resource. When sufficiently close, they may also interfere with each other's cirral activity. This latter possibility was investigated by comparing with single individuals, pairs of barnacle spat settled adjacent to each other so that they might interfere. Pairs and singletons with otherwise ample space from which to draw food grew at equal rates; hence their mutual interference was negligible (Crisp, 1960b). However, growth rates of populations of B. balanoides set up at increasing densities starting from 0.25/cm2 began to compete for space as soon as they touched. Beyond that point, growth in diameter almost ceased, but individual dry weight and volume continued to increase by growth in height. Above l/cm2, however, the growth rate by weight gradually fell with increasing density of settlement. In a further examination of intraspecific competition, Crisp (1964) compared dry weights of B. balanoides at the end of the season at various population densities and in three different situations:

218

D. J . CRISP A N D E. BOURGET

1. The most favourable: on a raft in the Menai Strait 2. On an intertidal support and 3 . In an enclosed box with limited water flow and planktonic food supply When expressed as a fraction of the weight of individuals grown in isolation, growth at given population densities was identical in all situations, though the absolute growth in situations (2) and ( 3 ) was only about onethird and one-tenth, respectively, of the raft individuals' (Fig. 6). Moreover, the total biomass per unit area was limited by insufficient settlement density only below five individuals/cm2. Above this critical settlement density, competition was such that any increase in food assimilation by the extra individuals was subtracted from that obtained by the others. The average weight to which the group occupying unit area would grow by the end of the season was therefore independent of the number of individuals settled per unit surface area. Since these barnacle populations came to monopolise the surface area at densities >5/cm2, the rate of food capture was maximal and strictly depended only on the rate at which the moving water could transport particles into the waving sheet of cirri below. The hydrodynamics of this system deserve further study. To carry particles toward the surface where the extended cirri capture them, the flow must be nonlaminar with velocity components normal to the surface. Moreover, this regime of turbulent flow must be uniform both up- and downstream of an array of barnacles, since there was little difference in growth a W

a 6

BOO

100

t -

z 3

a W

a 5c

s' w

3

m

ul

b-

> a <, .5,.0

I

o,.o

I5.,0

0

I N I T I A L S E T T L E M E N T D E N S I T Y [ern-'] FIG.6. The relationship between total oven-dry biomass at the end of the first growing season and initial settlement density, in each of three environments (from Crisp, 1964, by courtesy of Blackwell Scientific Publications, Oxford).

GROWTH IN BARNACLES

219

rate between the leading and trailing edges of the population (Crisp, 1960b).

G . Competing Organisms Many sessile barnacles form part of the primary fouling community settling on otherwise bare surfaces, usually after the primary film has been established. They grow best on wave-exposed shores where limpets control algal growth, or in dark, plant-free situations. Barnes (1955) documents reduced growth of submerged B . balanoides and Balanus crenatus (Bruguikre) when filamentous algae are present, since they reduce water flow and possibly interfere with cirral activity. Enteromorpha intestinalis is frequently abundant in eutrophic estuaries, especially in the cooler months, and similarly reduces the growth of E. modestus. The presence of foliaceous animal fouling, such as hydroids, bryozoans, etc., must similarly reduce growth, while secondary fouling by space monopolisers such as mussels, anemones, compound ascidians and sponges will at first depress barnacle growth and ultimately smother the settlements altogether (Scheer, 1945).

H. Parasites The cryptoniscid isopod Hemioniscus balani lives usually singly in the mantle cavity of a number of species (see Crisp and Fischer-Piette, 1959), where it destroys the ovary and reduces somatic growth (Crisp, 1960b). Barnacles are also host to a number of rhizocephalan parasites, but their effects on growth have not been researched. Examples are Chthamalophilus delagei, which is exceptional in being ectoparasitic (BocquetVCdrine, 1957), and Boschmaella, often present as a multiple parasite, which attaches to the internal mantle lining of balanids such as B. improuisus (Bocquet-VCdrine, 1969). Intertidal barnacles are often heavily infected with the metacerceria of trematodes; these cysts distort the body but, being passive, are unlikely to influence its growth seriously.

1. Reproduction Energy expended in reproduction cannot be available for growth, and vice versa. Crisp and Pate1 (1961) prevented breeding in E . modestus, an obligate cross-fertilizing hermaphrodite, by isolation. They compared growth of isolated individuals with paired individuals well spaced apart to reduce mechanical or competitive interaction, but which were nevertheless able to breed. The two sets grew at equal rates during the period

220

D. J . CRISP AND E. BOURGET

before the majority acquired mature ova (less than 6 mm; 0-52 days), but after breeding had begun (more than 5 mm; 38-157 days) growth rates diverged significantly, the breeding individuals growing more slowly. Lest this result should have arisen from competition between pairs, in a second experiment Elminius was paired either with its own species or with a faster growing species with which it could not breed. After 6 months the tissue weight of the breeding specimens had been reduced to about half that of virgin individuals. The loss of tissue was shown to correspond reasonably closely with that of the broods of nauplius larvae that had been liberated. The effects of reproduction in reducing growth rate have not been tested on barnacles with a single annual brood such as B . balanoides, where, in Europe, feeding and growth normally ceases in winter. Such an investigation would be of special interest since Barnes (1 962) attributed anecdysis and other aspects of reduced activity characterising the winter condition of B . balanoides (Crisp and Patel, 1960) to low environmental food levels and postbreeding “debilitation” rather than to a physiological change under hormonal control (Crisp and Patel, 1969). If laboratorymaintained animals are supplied with food during the winter, then the period of anecdysis is reduced and growth continues, although other factors regulating the seasonal cycle are present (Barnes et af., 1963). Barnes (1962) and Barnes et al. (1963) also detected an arrest of growth not entirely dependent on feeding, coinciding with the time when the ovary was initiated.

V.

Age and Growth-the

Growth Curve

As in most invertebrates, the rate of growth of juvenile barnacles at first accelerates and then, with increasing age and maturity, slows down. Thus, whatever the initial growth rate, a given species tends towards the same ultimate size if allowed long enough to reach it. Some authors have quoted “specific growth rates” or “relative growth rate,” (dx/dt)/x or d logs/dt. Since, except for exponentially growing cultures, this quantity declines even more rapidly with age than absolute growth rate, the concept has little to recommend it. In seeking a more useful function by which to describe the change in growth with age, it must be borne in mind that the curve representing absolute growth will depend critically on the units employed, whether linear, superficial or volumetric. Crisp (1960b) suggested that the linear increase in diameter was the most appropriate, since not only was it easily measured, but it also remained relatively constant over much of the animals’ early growth.

GROWTH I N BARNACLES

22 1

For that part of the growth curve beyond maturity in which growth rates are falling, the Bertalanffy equation is probably better. Crisp (1960b) suggested that, since for an isometrically growing barnacle the area of cirral net should increase as the square of a linear dimension L, so also would food uptake if the net captured suspended particles by extending passively in a steady current. He assumed that the rate of volume increase would therefore rise as Lz. This would, in turn, imply a constant increase in the linear dimensions of the animal, as is in fact observed in the rate of increase of basal diameter over a considerable part of the growth curve. Dr. R. N. Hughes has drawn to our notice that assimilated energy must be partitioned between increase in biomass and respiration. Fortunately, this correction does not influence the conclusion. The equations for the energy budget should be prefaced in terms of biomass (B) proportional to length3 (L3). B

KpL3

=

(1)

Production ( P ) is increase in biomass with time: P

=

dB1dt

=

3KpL2dL/dt

(2)

Respiration ( R ) is approximately proportional to L2 (the well-known “area law”):

R

KRL~

=

(3)

Assimilation ( A ) (Crisp, 1960b) is assumed proportional to food uptake and to cirral net area: A

=

KAL2

(4)

By the first law of thermodynamics, dBldt

=

P

=

A-R

Substituting for P , R , and A from Eqs. (2), (3) and (4): dLldt

(K*-KR)/3KP = a constant

(5) Barnacles would offer a suitable subject on which to test these relations.

VI.

=

Growth Rates of Various Species

The best records of growth rate have been made on sessile barnacles responsible for fouling (e.g., Barnes and Powell, 1953), or by repeated measurements in autecological investigations (e.g., Moore, 1934). Other, less reliable, data have been obtained from measurements of the maxi-

222

D . J . CRISP A N D E . BOURGET

mum dimensions of individual barnacles found on ships’ hulls after the completion of a voyage or on buoys left at sea for a known period. Table I, for a single species, B . balanoides, illustrates how much variation exists. Measurements apply only to the location and to the season quoted, although perhaps there is an optimal rate, characteristic of the species, which is approached when ideal conditions of temperature, current and food availability obtain. Table I1 supplements Table I, giving recorded growth rates of certain other species. In comparing species, Barnes (1958) sought to minimise the effects of differences in size by measuring relative growth rate at half maximum size. A simpler comparison, giving similar results, is shown in Table I1 by dividing the rate of linear growth by onehalf the maximal size. It can be seen that: 1. Growth rates are low in large species 2. Species growing on rafts, in flowing channels, or in the shallow sublittoral give the highest rates, those at high tide levels the lowest, and 3. Chthamalus, often a high shore barnacle, grows relatively slowly.

The few available observations on growth rates of lepadomorphs indicate fast growth despite the alleged paucity of plankton at the surface of oceanic waters. Dalley and Crisp (1981) fitted observed growth of Conchoderma aurita and Conchoderma uirgatum to the Bertalanffy equation, obtaining initial growth rates of the capitulum of 0.83 and 1.17 mm/d, respectively. Tropical and subtropical sessile barnacles grow, mature and breed faster than cool temperate species (e.g., Paul, 1942; Daniel, 1954).

VII.

Histology and Fine Structure of the Integument: Growth and Ecdysis

In cirripedes, most of the hypodermis has retained the ability to produce a flexible and frequently replaced uncalcified skeleton. However, the hypodermis underlying the shell plates can lay down an organic matrix containing calcium carbonate in the form of calcite crystals, while the outer surface of the shell is covered by a cuticle. The animal, like all other anthropods, is totally surrounded by a cuticle and epicuticle. However, unlike other crustaceans where the chitin-protein integument may be impregnated by calcium carbonate, in barnacles there is a complete separation between the outer cuticle and the accreted layers of calcite. Whereas in most crustaceans with calcified exoskeletons the mineral portion, before ecdysis, is either resorbed, often into a gastrolith, or shed with the old integument, in cirripedes the shell is a permanent structure

GROWTH IN BARNACLES

223

continuously added to during each period of intermoult. “A cirripede,” wrote Darwin of the Thoracica, “cannot like a crab crawl into some crevice and remain protected till its shell becomes hardened; hence, probably, it is that the shell is never wholly moulted.” The hypothesis put forward by Darwin (1854), that the shell was deposited by a hypodermis lying below previously formed layers of shell, has been widely accepted. Only Gruvel (1893) and Davadie (1963) thought that the epidermis lay immediately below the cuticular elements on the outer surface of the shell and that the shell was formed by addition of material from outside the preexisting shell. Newman et al. (1967) effectively disposed of this error. As noted by Darwin, there is evidence within the shell of frequently deposited laminae (1854, p. 57), which we now know as being formed at each tidal immersion (Bourget and Crisp, 1975b; Crisp and Richardson, 1975), as well as evidence on the surface of the shell of less frequent episodes of ecdysis. It must be emphasised at this point that we have insufficient information regarding the composition and origin of the shelled integument of barnacles to be able to homologise with certainty the various layers with the more usual structures in arthropods (Neville, 1975). The outermost horny layer, which is often lost, was called by Darwin the “epidermis”; it probably corresponds to epicuticle plus cuticle in whole or in part but is, in any case, excessively thin. The organic components of the shelly layers secreted by the underlying hypodermis may also represent part of the endocuticle. The amount of material is sparse (Barnes et al., 1976), yet often highly complex (see below). In Chthamalus, Klepal and Barnes (1975a,b) consider that this material includes persistent epicuticle left at each moult. It probably combines an arthropodin-like protein (Crisp and Meadows, 1962, 1963) with chitin in various proportions (Barnes et al., 1976). However, it is best not to assume any certain homology with classical cuticle at this stage, and in referring to the outer horny layer as “cuticle” we imply none. Darwin (1854, p. 59) describes how, as a result of shell expansion in three regions of sutures-between the opercular valves and the sheath, between the lateral edges of the parietes, and between the base and the shell wall-the adherent cuticle splits “at each period of exuviation.” This membrane “which when well preserved invests the walls of the shell is made up . . . of successive adherent slips which originally covered the lines of the sutures.” He also states that “little bristles . . . which arise from the slip of membranes left adherent to the opercular valves, sheath and walls stand in rows; a row corresponding to each period of exuviation . . . Darwin had clearly seen and appreciated the significance of the relationship between shell growth and moulting long before others had ”

224

D. J. CRISP AND E. BOURGET

redescribed it (von Bahls, 1903; Costlow, 1956; Costlow and Bookhout, 1953, 1956; Bocquet-VBdrine, 1963, 1964, 1965, 1966a,b; Bourget and Crisp, 1975c; Klepal and Barnes, 1975a). Darwin was not equipped to investigate the processes at work in these sutures at the cellular level. Histological studies carried out by Bocquet-VCdrine (1963, 1964, 1965, 1966a) on various operculates have shown that the activity of the hypodermis wedged in the basal suture is discontinuous, the events being synchronised with the animals’ moulting cycle. Klepal and Barnes (1975a) repeated these observations on C . depressus without demurring from Bocquet-VCdrine’s conclusions. Bubel (1975) made further advances by describing at the fine structure level the opercular hinge and the basal suture. According to Bubel and Bocquet-Vedrine, during each intermoult period the cells located here lay down first new epicuticle, then endocuticle, both within the previously formed cuticular membrane. The new layers are folded and contrast sharply with the older stretched layers beneath (Fig. 7). In order to lay down the increased length of membrane which will allow for subsequent growth, the surfaces of the secreting cells are greatly folded. Electron micrographs of the hypodermis underlying the opercular hinge of E . modestus show this folding clearly

0

FIG.7. Distribution of continuous shell growth during the intermoult period: Mean, with standard error bars, of cumulative percentage growth during the intermoult period plotted against the percentage of time elapsed during the intermoult period (from Bourget and Crisp, 1975c, by courtesy of the Marine Biological Association).

GROWTH IN BARNACLES

225

(Bubel, 1975). According to Bocquet-VCdrine, secretory activity starts at the extreme corner of the basal suture where the hypodermis abuts the rim of the calcareous shell and proceeds centripetally along the basal layer of hypodermis. The process begins at stage C of the ecdysis cycle (Drach, 1939), as judged by the condition of moulting of the limbs and body. The formation of the epicuticle is followed by secretion of cuticle at moult stage D2. At this point the limbs and body moult, but clearly nothing can be shed from beneath the persistent wall plates. During the premoult stages A and B, Bocquet-VCdrine (1965) states that the hypodermal cells deposit calcium on the older portion of the shell, thereby extending the length of the parietes and the circumference of the shell, so breaking the old cuticle and unfolding the new. Unfortunately, Bocquet-Vedrine does not record the time intervals of the various moult stages. Bourget and Crisp (1975b,c) in B. balanoides and Crisp and Richardson (1975) in E. modestus, one of the species studied by Bocquet-VCdrine (1963, demonstrated that discrete layers of calcium carbonate are formed whenever the barnacle is immersed. This appears to be so whatever the interval between immersions, so that deposition must be virtually continuous as long as sea water is supplied to the mantle cavity. Bourget and Crisp (1975b) found by direct observation of young immersed barnacles that there was little variation in the rate of shell growth throughout the moulting cycle, other than a slight increase during the first half and a small reduction towards the latter end of the intermoult period (Fig. 7). It follows that, if Bocquet-VCdrine is correct in assuming that epicuticle, cuticle and calcareous shell are laid down consecutively, the length of stage C to D2 for cuticle formation must be very short indeed compared with the premoult and intermoult stages of the cycle, when the shell is being produced. Bourget and Crisp (1975b) proposed a slightly different mechanism to account for the two distinct processes: cuticle formation and shell deposition. They agree that the epicuticle and cuticle must be secreted first, and are gradually stretched at the basal perimeter of the shell as shell is being laid down. Calcite is secreted periodically during tidal immersions by the hypodermis beneath the parietes, mainly towards the outer edges. These authors suggested that haemolymph pressure controlled by the muscles of the prosoma, together with adjustments made by the small muscular elements of the fixation fibres that join the shell plates to the base, periodically force the still plastic shell matrix down into the substratum, the pressure causing the new cuticle to form an annular bulge at the edge of the shell (Fig. 8). In B. balanoides these bulges are figured by Bourget and Crisp (1975~) as smaller ridges which do not coincide with tidal growth increments nor

226

D. J. CRISP AND E. BOURGET

D' Ce

FIG. 8. Growing edge of a barnacle: Abbreviations: A, A', calcium carbonate-secreting area of hypodermis (maximum secretion); B, B', calcium carbonate-secreting area of hypodermis (minimum secretion); C, C', basal cuticle-secreting area of hypodermis (premoult only), not exuviated; Ce, cement; Cuo, Ep,, remains of cuticle and epicuticle formed during a previous intermoult; C u , , E p , , old cuticle and epicuticle which will become broken by stretching; Cu2, Ep,, new folded cuticle and epicuticle; D, D', nonsecreting area of hypodermis; E, E', mantle lining hypodermis secreting a normal thin cuticle exuviated at each ecdysis; h, hair of hirsute ridge; G , , G 2 ,tidal growth increments separated by growth lines; r,, large moulting ridge or hirsute ridge; r , , rz. rl. ridges formed during growth; F, fixation fibre; M, muscle of fixation fibre. (Modified from Bourget and Crisp, 1975c.)

with the larger ridges and hairs (hirsute ridges) associated with moulting (Fig. 8). Klepal and Barnes (1975b) illustrated similar configurations in C . depressus. Eventually, as Darwin stated, shell growth so stretches the old underlying cuticle that it breaks, but the new folded cuticle continues to accommodate the enlarging shell until the next moult occurs. As the parietes grow from below, the new cuticle appears to move up the external surface of the shell, as on a conveyor belt, carrying the torn annular remnant representing the epidermis formed at the previous moult, together with the hirsute ridge, to occupy a position higher on each paries. These hirsute ridges can be seen at intervals up the shell, defining the occasion of a previous moult (Fig. 8). An important difference between the typical arthropod and the barnacle results from the separation of the cuticle from the largely calcite shell, each apparently being laid down by different parts of the hypodermis. In normal arthropod cuticle, the hypodermal cells elongate to a columnar form with a wavy surface to accommodate further growth at the onset of ecdysis. They lay down first an epicuticle; then, after secretion of moulting fluid and resorption of the old integument, the exo- and endocuticle are formed beneath the epicuticle by the same cells (Wigglesworth, 1962).

GROWTH IN BARNACLES

227

It is implied by Bocquet-VCdrine (1964, 1965, 1966a) and Bubel (1975), that barnacles differ fundamentally in having specialised hypodermal cells for each function. Elongated cuticle-secreting cells are found in the basal suture, and variously modified hypodermal cells underlie the basis, the opercular membrane, and the shell plates. However, we know nothing of the origin of these modifications. Do the cuticle-secreting cells remain permanently lodged within the suture? If so, before the onset of each ecdysis, the increased areas of hypodermis must have been made good by a multiplication of basis-secreting and shell-secreting cells on either side of the cuticle-secreting cells. Alternatively, do all the cells slide along the basement membrane, occupying successively basis-secreting, epidermissecreting and calcite-secreting positions and modifying their structure and function accordingly?

VIII.

Shell Structure in Relation t o Function

The thin laminae deposited along the inner surfaces of the shell of many species may be seen in radial sections (Fig. 9). These are the growth increments of Bourget and Crisp (1975b,c) which form at each tidal immersion. Darwin (1854) had observed these layers: “if now a section of one of the shelly zones of growth be carefully examined, it can in some cases be distinctly seen to be formed of successive, excessively fine laminae . . . ” They were noted also by Gutmann (1960) and Bassindale (1964), both of whom confused them with ecdysal marks on the outside of the shell and so wrongly associated them with ecdysis. The thickness of these laminae indicate where maximum deposition takes place; the regions of growth in balanids comprise the lower outer edge of the basis and lower parts of the paries, meeting at the basal suture, the downward protruding region of the sheath, the margins of the opercular plates, and the radii and alae (Darwin, 1854). These areas of enhanced shell deposition were also evident from autoradiograms obtained by Bourget and Crisp (1975b) after immersing living animals in 45Ca-treated sea water (Fig. lo). Younger individuals show a more general and intense pattern of deposition, but in older individuals deposition is more clearly concentrated in the regions identified above. In most balanids the sheath is a separate entity, but in the chthamalids a paries is a single entity with very thin zones of deposition laid down parallel to the inside of the shell (Fig. 8A). The chthamalid shell, therefore, increases in total thickness towards the apex. Intermediate forms, such as Elminius spp., show reduced growth rates between two regions of deposition, one at the basal suture

D. J . CRISP A N D E. BOURGET

228 cc -

osh +! osu

gs

gs

B

A

C

D

FIG.9. Diagram of radial sections of shell plates from different species illustrating banding and layering of shell: (A) one-layered type of shell (Chthamalus sp.); (B) two-layered type of shell (Balanus sp.); (C) change in shell banding of a very young barnacle showing the developing discontinuity of the bands and enlargement of the more basal increments; (D) section of Tetraclita squarnosa stalactifera (Lamarck), showing sublayered inner layer, Abbreviations: bs, base of sheath; cc, cuticular covering; d, discontinuity; gb, growth bands; gs, growing surface; il, inner layer; is, inner surface; is-I, inner sub-layer; mb, membranous base; 01, outer layer; osh, organic sheet; 0s-I, outer sub-layer; osu, outer surface; ps, primordial shell.

and the other at the base of a pseudosheath, the paries being a single element without discontinuity (Fig. 9C). Patterns of deposition, however, become more complicated when a thickened calcareous base interlocks with the lower end of the paries at the basal suture. Both may have evolved a complex system of canals in which secondary calcite is sometimes laid down. In such species the basal and parietal hypodermis must meet on each side of the interlocking parts and lay down calcite crystals perpendicular to the plane of suture. If the base and paries are rudely separated these interlocks are often broken, suggesting that in some instances, where one member forms a neck into which the opposing member fits, dissolution of shell may have to accompany growth. Such complex interlocks greatly strengthen the shell (Murdock and Currey, 1978), especially if the joints merit the accolade of being “workmanlike. If, at any point, there is a fold in the opposed sheets of hypodermal cells, the axes of the crystals formed by them will not lie parallel to each other but meet at a disjunction which is visible in the section of shell as a ”

GROWTH IN BARNACLES

229

FIG. 10. Autoradiograms of young (A) and older (B) specimens of B . bdanoides after immersion in sea water containing 45Ca. Abbreviations: bm, basal margin; op, opercular plate; os, outer surface of shell; sh, sheath; sp, shell plate (from Bourget and Crisp, 1975b, by courtesy of the Marine Biological Association).

discontinuity. The canals, separated by septa with their secondary teeth, are all formed by an infolding of hypodermis at the basal suture. The lines of teeth become gradually embedded in secondary deposition as they are moved up the shell by its growth from the base. Hence, higher up in the paries the inner wall becomes smoothed by secondary growth as the canals become embedded within it together with septa, teeth, and their lines of crystal discontinuity. These patterns, which can be seen in Fig. 1 1 A, were termed interlaminate figures by De Allessandri (1895) and were later studied exhaustively in many species by Cornwall (1956, 1958, 1959, 1960, 1962), Davadie (1963), Newman et al. (1967), and Bourget (1977). Some species with canals in the parietes (e.g., Tetraclita, B . perforatus, B . amphitrite) may also have complicated structures associated with sec-

230

D. J . CRISP AND E . BOURGET

FIG. 11. (A) Tranverse section near the apex of a shell plate of B . perforatus seen in polarised light (with crossed polars), showing filled canals and ovoid interlaminate figures (above); (B) fractured shell plate showing the ropelike organic fibers forming the tangential organic sheets of C. sfellatus (scale = 50 pm);(C) transverse bridges of organic material joining the ropelike fibers of the tangential organic sheets of C. stellatus (scale = 1 p n ) ; (D) decalcified shell plate of T . squamosa stalactifera showing the concentric organic sheets present in the longitudinal canals (scale = 200 pm).

ondary deposition within the canals, so that often the upper portion of the canal forms a column of infilled shell material (Fig. 11A and D). Elminius simplex and Elminius plicatus (Epopella group-Foster, 1978) have calcareous pillars depending from the inner side of the paries in radial series. Their origin lies just below the first formed outermost layers of shell, and they become embedded in the later formed inner layers through which they pass. The vertical bundles of shell crystals constituting a pillar are sharply defined from the ordinary laminae and must therefore be formed by specialised cells of the hypodermis.

GROWTH IN BARNACLES

23 1

Within the calcareous laminae of most barnacles may also be found various arrangements of organic material in the form of plane sheets, sheets enveloping crystals, or, as in many chthamalids, large or small threads or twisted ropelike bundles of fibres (Fig. 11B and C). Bourget (1977) examined the layering and orientation of the crystals, classifying them into the different crystalline types. He depicted the arrangement of the organic sheets within the shell matrix and was able to relate shell microstructure to shell function and cirripede classification. The microstructural features of the parietes are summarised for various taxa in Table 111. The table shows how these features are adapted to the mechanical function of the shell, for example, to resist the impact of floating objects, abrasion by particulate matter, attack by predators, competition by adjacent barnacles, crushing by grazers, or invasion by boring algae. The importance of biological factors was discussed by Palmer (1982). He provides evidence that easier access by predatory gastropods near the region of apposition of the parietes and at the margins of the opercular plates may account for the reduction in the number of plates and the development of thick external ribbing of the shell in some balanomorphs during evolution. Bourget (1977) suggested that the thin and highly orientated layer of crystals at the outer surface of the alae and radii might serve to give additional strength in the regions most exposed to attack by borers and to wear by abrasive particles. These suggestions call for experimental verification. Shell characteristics likely to increase the strength and resistance of the shell are 1 . Increased thickness to give added strength 2. Organic layers surrounding the crystallites to increase flexibility and strength by arresting the propagation of cracks (see Currey, 1964; Taylor and Layman, 1972; Murdock and Currey, 1978) 3. Laminated structures as in chthamalids, similarly increasing toughness and flexibility 4. Longitudinal canals which lighten the shell and, if surrounded by annuli of orientated fibrillar crystals or filled with columns of secondary calcite invested in organic matrix (Fig. llD), give even greater strength than a solid block Currey (1964) and Wainwright et al. (1976) show how hollow canals in bone structure can interrupt the spread of cracks; a similar function could be argued for the parietal canals in barnacle shell. The rather crude tests made by Barnes et al. (1970) and Murdock and Currey (1978) indicate that strength per unit weight of shell is relatively greater in the species with canals than in those with solid shells.

TABLE111. SUMMARY OF OBSERVATIONS ON ~~~~~~

~

Number of species examined

Number of layers

Verruca

1

1

Chthamalus

6

1

Balanus

9

2

Taxon

THE

SHELL MICROSTRUCTURE OF SOME BARNACLESO

~

Structure of the parietes

Potential advantages for resisting damage

Orientated microcrystals; plates heavily corrugated The parietes are stratified by alternate regions of thick organic sheets and fibrils and irregular prisms; Chthamalus rhizophorae ( C . hembeli group) differ from those of the C . stellatus group in having a thin stratum of convergent fibrils on the outer surface of the shell, and a wedge-shaped region of elongated transparent prisms delimiting the pseudosheath from the outer region of the shell plate Mainly composed of small disorientated crystals (granular structure and/or orientated microcrystals and prisms arranged in and around the interlaminate figures and sheath); the structure of the outer layer reflects the complexity of the shells; B . amphitrite and B . perforatus have filled canals

Corrugations may increase shell strength Many Chthamalus species do not have thick shells but live in high-water, wave-beaten areas; the laminated structure may allow relative movement between layers and hold the shell intact after minor breakage

Discontinuities around and in interlaminate figures, hollow or filled canals could serve to stop the propagation of cracks; crystallites of the longitudinal septa separating the canals are well orientated; the zone of tangential orientation around each canal probably compensates for the loss of solid material inside it; canals secondarily filled with radially orientated crystals and organic sheets within a tangentially orientated cylinder are expected to be highly resistant; these columns of shell material are usually the last to disintegrate

Acasta

1

2

Pyrgoma

2

2

Elminius

1

2

4

Pseudosheaths

Tetraclita

2

2

Chelonobia

2

2

Bourget, 1977.

Mainly granular, but also orientated prisms Mainly granular, but also orientated prisms as well as orientated microcrystals; the shell is made of one circular piece Outer layer mainly granular but inner layer (sheath) with large prisms Granular structure constitutes the basic structure of the parietes. Depending ridges of E . simplex and E . plicatus consist of crystallites arranged in long bundles, each bundle encased in an organic sheath; the bundles are secondarily associated in prisms invested in organic sheets; the shell contains a considerable amount of organic material Outer layer with small irregular disorientated crystals (granular structure), but longitudinal septa and inner laminae consist of orientated crystals packed into lamellar sheets; canals are secondarily filled with layers of large lamellar crystals tangentially arranged to the surface of the canals and separated by concentric organic sheets; some Terraclita have sublayered sheaths Pillars of convergent crystallites (lamellae and fibrils) surrounded by small crystallites disorientated in C. testudinaria but convergent in Chelonobia patula; the sheath extends downwards to the base

E. simplex has organic ribbons extending through the shell; E . plicatus, E. kingii have thick organic sheets as well as pillars of oriented crystals; this organic material might hold the shell intact after minor breakage, allowing relative movement of the shell laminae; specimens of E . plicatus with exposed organic layers on the outer surface are not uncommon and suggest a possible barrier against disintegration Columns of shell material tangentially orientated to the sides of the canals presumably create a ply structure highly resistant to forces in all directions; these columns of radially orientated prisms are clearly visible as ridges on heavily eroded shells, indicating their resistance to erosion: boreholes are also less common in the orientated structures of the columns than in the matrix surrounding them

234

D. J . CRISP AND E. BOURGET

According to Bourget (1977), species with obviously strong, thick shells (e.g., Balanus balanus (L.), Balanus psittacus, Balanus amaryllis, and B . perforatus) are not necessarily those found at high water in wavebeaten habitats where physical damage is most likely to occur. Indeed, of the two genera found in such regions, Chthamalus and Tetraclita, only the latter has a strong and thick shell. However, Tetraclita spp. are found somewhat below the levels of Chthamalids in the tropics and are probably more likely to be attacked by large boring gastropods or by fish well adapted to chipping the coral. The chthamalids are often too high upshore to be vulnerable to marine predators. Another obviously weak-shelled form, E. modestus, is highly prolific (Crisp and Davies, 1955) and may thus compensate for losses due to mechanical damage, but Verucca stroemia would appear very vulnerable unless it has other means of defence. On balance, it seems probable that strong shells have been evolved as a defence against predators rather than against the elements. More refined measurements of barnacle shell strength and hardness in relation to its physical structure would repay further effort.

IX.

Cyclical Factors in Growth

Shell growth in barnacles is usually measured as an increment over a given period of time, resulting from a series of influences, some endogenous, some exogenous. The factors that influence growth may act over a short or long term; they may operate only once or recur periodically. These will be considered and their magnitude assessed. Growth has usually been measured in various species at defined sites or geographical areas or the effect of individual factors examined under given conditions. However, growth is a dynamic process, and if we could evaluate all the periodic and aperiodic influences, then a more comprehensive picture could be put forward. Cyclical factors likely to influence growth at various periodicities are summarised in Fig. 12 for the wellstudied species B . balanoides. The figure clearly shows that a given factor may influence growth at several periodicities, of which the chief are tidal and annual. A.

Tidal Influences

When shell growth at the edge of the barnacle B . balanoides, was measured very precisely (k l pm) at hourly intervals under a microscope, a stepwise deposition was found to take place (Fig. 13). The animals grew

235

GROWTH IN BARNACLES

Q

6h

FXOGENOUS FACTORS illumination tidal regime temperature

j<

1 2 h j i 2 4 h \ < lwki< 2 w k i 4 lmoj< 4mo/< l y r

j

*-

food

I

moulting

-

reproduction

FIG. 12. Relative importance of cyclical factors likely to influence growth at various periodicities.

when in sea water but ceased soon after they became emersed. The periodicity of deposition was later traced within the shell structure as growth increments separated by darker bands in both B . balanoides (Bourget and Crisp, 197%) and E. modestus (Crisp and Richardson, 1975). By allowing these two species to grow for short periods in calcium-rich sea water, thereby forming thick increments as time marks, these workers were able to show that under tidal conditions each increment corresponded with a Wet

f 6

I

I

I

I

12

18

24

30

Hours

FIG. 13. Hourly changes in the position of the growing edge of B . balanoides settled on a coverglass and viewed from below with a high-power oil-immersion objective (from Bourget and Crisp, 197513, by courtesy of the Marine Biological Association).

236

D. J . CRISP AND E. BOURGET

TABLEIV. EFFECTOF TIDALLEVELON GROWTHRATEA N D GROWTHINCREMENT WIDTHI N B . balanoides AT MENAI BRIDGE,NORTHWALES Condition

Number of individuals

Mean growth rate (mm/day)

5

0.0602 0.0400 0.0243

~

Raft LWN HWN

~

3 3

Mean increment width ( p m ) ~

31.24 22.46 13.33

semidiurnal period of tidal immersion. The width of the increments was directly correlated with the duration of immersion. Moreover, when shells from different tidal conditions were compared, those immersed continuously formed diffuse bands of circa-tidal frequency, whereas those immersed intertidally all gave distinct bands of precisely tidal frequency. The banding was much more distinct in animals from higher tidal levels, despite the fact that the increments were narrower (Table IV). Sometimes, in barnacles living at or above the level of high water of neap tides, fewer than the theoretical number of bands were laid down because they were not always immersed. Some individuals at this level were marked and followed during 23 successive tidal periods. These were found to have certain bands missing and replaced by a darker “stress” band, notably when an unduly long period of emersion was associated with long insolation and high air temperature. Thus, shell deposition may temporarily cease during periods of heat stress (Bourget and Crisp, 1975~).Reduced growth at high tide level is evident from Tables I and 11, pp. 212, 214. B. Daily Influences We have already noted the suggestion by Klugh and Newcombe (1935) that direct sunlight reduced growth in B . balanoides. Barnes (1953) and Crisp and Pate1 (1960) showed that light did not influence growth, and any reduced growth observed in illuminated conditions probably resulted from interference by algae. Costlow and Bookhout (1956) measured growth every 24 h in “B. amphitrite niveus” [B. amphitrite amphitrite, actually (Ed.)] kept in the laboratory. They obtained faster growth in darkness. This question was further examined by Bourget and Crisp (1975b) by measuring very precisely the basal diameter of a group of newly metamorphosed B . balanoides kept under a 12-h light/l2-h dark regime. They were unable to show any significant effect of illumination on

GROWTH IN BARNACLES

23 7

growth in the laboratory. Possibly Costlow and Bookhout’s results may have resulted from a nightly carbon dioxide accumulation in the algal culture, which could stimulate cirral activity (Southward and Crisp, 1965). On the other hand, Trump and Bourget (1980), in reexamining the results of Bourget and Crisp (1975b), found slightly greater increments during daytime than at night. They suggested that in uncontrolled field situations sea surface and air temperatures would probably have been higher during the day and so increased the rate of feeding and growth. No influence on the regular production of tidal bands in the laboratory could be detected when the light regime was modified. Animals kept either under continuous illumination or in alternating 12-h lighti12-h dark periods, as well as animals kept since metamorphosis in a light-tight box in the field, continued to produce the usual two equal increments per day. Thus, any effect that light might have over a 24-h periodicity must be very small. However, where the tidal regime has a strong diurnal component, it might impose a daily (23.8 h) rhythm of growth, as was found by Evans (1972) in the cockle Clinocardiurn nuttalli growing on the Pacific Coast of the USA.

C . Other Lunar Influences Since the thickness of the tidal growth increment is correlated with the duration of tidal immersion, it follows that differences in the semidiurnal growth increment should be observed between the spring and neap periods of the lunar cycle (14 days), as Bourget and Crisp (197%) have shown. Similarly, the smaller differences in the new and full moon cycle (28 days) and the semiannual solar-lunar effect must also be present, although probably too small to be detected.

D. Annual injhences The tidal cycles of growth, other than semidiurnal, might not be easily separated from the seasonal cycle of growth which, for example in B . balanoides, is annual and characterised by a maximum in the spring, a reduction during summer, possibly a rise in autumn, and a virtual cessation during winter (Hatton and Fischer-Piette, 1932; Moore, 1934; Hatton, 1938; Barnes and Barnes, 1959a,b; Barnes, 1961, 1962; Ritz and Crisp, 1970). The annual cycle of growth is clearly evident in shell sections from the much thinner bands laid down during winter. In some species the annual cycle is also evident from discontinuities on the outer form of the shell (e.g., B. porcatus = B. balanus, Crisp, 1954; B . balanoides, Bourget, 1980).

238

D . J . CRISP AND E . BOURGET

E. Other Cyclic Influences The moulting cycle, at least in B . balanoides, will impose a further, but somewhat irregular, periodicity, since growth is maximal in the immediate postmoult period. The moulting frequency is itself increased by greater feeding and growth of the animal (Crisp and Patel, 1960), although the animal continues to moult even when not growing (Crisp and Patel, 1958), possibly to remove small organisms that settle and grow on the cuticle. Breeding, which competes with growth for resources (Crisp and Patel, 1961), is likely to have quite different effects on high-latitude species which breed once a year than on those which liberate broods continuously over the breeding season or those from low latitudes which breed continuously.

F. Frequency, Scale, and Precision of Measurement From Fig. 12, which illustrates the periodic factors influencing barnacle growth, it can be seen how some factors, notably immersion and emersion, are effective at several periodicities. The most important factors influence growth over 12-h tidal and yearly cycles. Most growth studies have been carried out using weekly or monthly measurements, and at precisions of approximately 1 mm. These time scales are too short to register annual effects and too long to register tidal changes. The precision of most measurements would also preclude any possibility of observing increases at tidal intervals. Since each successive growth band width is a short-term and accurate method of recording growth rate, Trump and Bourget (1980) suggested the use of these patterns to separate the influence of the various periodic and aperiodic effects by time-series analysis. Sufficiently long-term records were not available, but the technique was applied to an artifically created series of band widths and immersion periods, so that after filtering out the shorter term periodicities, it should be possible to isolate and describe long-term variations in growth rates, such as the annual cycle, the moulting cycle or the reproductive cycle, and to isolate and describe residual aperiodic events, such as unique periods of high or low temperature, spills of pollutants, or eutrophic algal blooms. By the development of such methods, the information locked within the shells of barnacles and other organisms such as corals, bivalves, gastropods and chitons, not forgetting fossil shells, could be of great practical and academic importance.

GROWTH IN BARNACLES

239

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24 1

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243

des c6tes rocheuses par les cirripkdes. Bulletin de l’lnstitut Oce‘anographique, Monaco No. 592. Klepal, W., and Barnes, H. (1975a). A histological and scanning electron microscope study of the formation of the wall plates In Chthamalus depressus (Poli). Journal of Experimental Marine Biology and Ecology 20, 183-198. Klepal, W., and Barnes, H. (L975b). The structure of the wall plate in Chthamalus depressus (Poli). Journal of Experimental Marine Biology and Ecology 20, 265-285. Klugh, A. B . , and Newcombe, C. L. (1935). Light as a controlling factor in the growth of Balanus balanoides. Canadian Journal of Research 13, 39-44. Kuhn, O., and Fuchs, H. (1954). Uber die Verbeitung der Kalkschalenbildung bei Balanidenlarven. Naturwissenschaffen 41, 286-287. Moore, H. B. (1933). Change of orientation of a barnacle after metamorphosis. Nature (London) 132, 969-970. Moore, H . B. (1934). The biology of Balanus balanoides. I. Growth rate and its relation to size, season and tidal level. Journal of the Marine Biological Association of the United Kingdom 19,851-868. Moore, H . B. (1935). The growth rate of Balanus humeri (Ascanius). Journal of the Marine Biological Association of the United Kingdom 20, 57-63. Murdock, G. R., and Currey, J. D. (1978). Strength and design of shells of the two ecologically distinct barnacles Balanus balanus and Semibalanus balanoides (Cirripedia). Biological Bulletin, Marine Biological Laboratory, Woods Hole 155, 169-192. Neu, W. (1935). Mitteilung einiger Beobachtungen zur Formbildung von Balanus balanoides L . und dessen Ansiedlung. Zoologischer Anzeiger 110, 169-179. Neville, A. C. (1975). “Biology of the Arthropod Cuticle.” Springer-Verlag, Berlin and New York. Newman, W. A,, and Ross, A. R. (1975). Revision of the balanomorph barnacles; including a catalog of the species. Memoirs Sun Diego Society of Natural History N o . 9, 108 pp. Newman, W. A., Zullo, V. A., and Wainwright, S . A. (1967). A critique on recent concepts of growth in Balanomorpha (Cirripedia, Thoracica). Crustaceana 12, 167-178. Palmer, A. R. (1982). Predation and parallel evolution: Recurrent parietal plate reduction in balanomorph barnacles. Palaeobiology 8, 3 1-44. Paul, M. D. (1942). Studies on the growth and breeding of certain sedentary organisms in the Madras Harbour. Proceedings of the Indian Academy of Sciences 15B, 1-42. Ritz, D. A., and Crisp, D. J. (1970). Seasonal changes in feeding rate in Balanus balanoides. Journal of the Marine Biological Association of the United Kingdom 50, 223-240. Runnstrom, S. (1925). Zur Biologie und Entwicklung von Balanus balanoides (Lime). Bergens Museums .&bog No. 5. Schafer, W. (1935). Bewuchs-Verteilung von Seepocken (Balaniden) im Gezeiten-Gurtel. Natur und Volk 68, 564-569. Schafer, W. (1948). Wuchformen von Seepocken (Balanus balanoides). Natur und Volk 78, 74-78. Scheer, B. T. (1945). The development of marine fouling communities. Biological Bulletin, Marine Biological Laboratory, Woods Hole 89, 103-121. Southward, A. J. (1955). On the behaviour of barnacles. I. The relation of cirral and other activities to temperature. Journal of rhe Marine Biological Association of the United Kingdom 34, 403-422. Southward, A. J. (1957). On the behaviour of barnacles. 111. Further observations on the influence of temperature and age on cirral activity. Journal of the Marine Biological Association of the United Kingdom 36, 323-334.

244

D. J . CRISP AND E . BOURGET

Southward, A. J. (1976). On the taxonomic status and distribution of Chthamalus stellatus (Cirripedia) in the northeast Atlantic region: With a key to the common intertidal barnacles of Britain. Journal of the Marine Biological Association of the United Kingdom 56, 1007- 1028. Southward, A. J., and Crisp, D. J. (1965). Activity rhythms in barnacles in relation to respiration and feeding. Journal of the Marine Biological Association of the United Kingdom 45, 161-185. Taylor, J. D., and Layman, M. (1972). The mechanical properties of bivalve (Mollusca) shell structures. Palaeontology 15, 73-87. Trump, C. L . , and Bourget, E . (1980). Study of barnacle shell growth band patterns using time-series analysis. In “Skeletal Growth of Aquatic Organisms” (D. C. Rhoads and R. A. Lutz, eds.), pp. 687-697. Plenum, New York. Trusheirn, F. (1932). Palaeontologisch bemerkenswertes aus der Okologie rezenter Nordsee-Balaniden. Senckenbergiana 14, 70-87. Von Bahls, H. (1903). Uber Struktur und Wachstum der Schale von Balanus improvisus. Diss Griefswald. Wainwright, S . , Biggs, W. D., Currey, J. D., and Gosline, J. M. (1976). “Mechanical Design in Organisms.” Arnold, London. Waite, M. E., and Walker, G . (1984). Guanine in Balanus balanoides (L.) and Balanus crenatus Bruguitre. Journal of Experimental Marine Biology and Ecology 77, 11-21. Walley, L. J. (1969). Studies on the larval structure and metamorphosis of Balanus balanoides (L.). Philosophical Transactions of the Royal Society, London, Series B . 256,237280, Wigglesworth, V. B. (1962). “The Principles of Insect Physiology,” 2nd Ed. Methuen, London. Wills, L. J. (1963). Cyprilepas holmi Wills 1962, a pedunculate cirripede from the Upper Silurian of Oesel, Esthonia. Palaeontology 6, 161-165. Woods Hole Oceanographic Institution (1952). “Marine Fouling and its Prevention.” Annapolis, Maryland. Wu, R. S. S . , and Levings, C. D. (1978). An energy budget for individual barnacles (Balanus glandula). Marine Biology 45, 225-235. Wu, R. R. S., and Levings, C. D. (1979). Energy flow and population dynamics of the barnacle Balanus glandula. Marine Biology 54, 83-89. Yule, A. B., and Walker, G . (1984). The adhesion of the barnacle Br‘anus balanoides to slate surfaces. Journal of the Marine Biological Association of the United Kingdom 64, 147- 156. Yule, A. B., Crisp, D. J . , and Cotton, I. H. (1982). The action of acetazolamide on calcification in juvenile Balanus balanoides. Marine Biology Letters 3, 273-288.

Taxonomic Index

A

Astropecten, 109 Astrungin dunne, 50

Acunthirstreu echinutu, I I Accrstu. 204, 233 A cidostomu, 92 Acroporu, 7 , 10. I I , 17, 42, 49 ucuminatu, 42 ceruicornis, 9, I I , 12, 15, 17, 21, 23. 26, 30, 31, 33, 38, 40-42, 45, 48, 50 formosa, 14, 23, 26, 31, 33, 41, 42 p~llmutu.7, 9, 12, 18, 23, 30, 33, 40, 41, 45, 48 prol$era, 48 Acrothorucicri, 200 Acliniu equinu, 36, 46, 47, 68, 70, 72, 76, 79-84, 86, 88, 93 sulcliru. 93 Actinostola, 74 cullosa, 92 Adumsiu pulliuta, 83 suIccrrc1, 83 Aelolidiu pupillosu, 92 Aequipecten, 136 Agariciu agurieiies, 10-12, 18, 21, 30, 33, 31-39 Aiptusiu, 92 pnllidu, 91 sulcutu, 91 Anudura senilis, 103, 165 Anemonim sulcatu, 68, 7 I Anodontu. I 1 1 unatina, 103, I l l , 148 cygnea, 103-105, 131, 146, 148, 152 156, 157 Anthopleuru, 90, 91 urtemisiu, 92 hullii, 83 rleguntissimu, 68, 90, 92, 93 midorii, 68 pulliutu, 93 xunthogrammicu, 90, 93 Arctica islandica, 103, 107, 146-148 Artemia, 67, 88 245

Btilmus, 201. 228, 232 umuryllis, 234 urnphitrite. 208, 214, 229. 232 urnphitrite umphitrite. 236 hulunoides (Semihulunirs hulanoides), 203, 208, 209-211, 215-217, 209, 220, 222, 225, 229, 234-238 hrilunns f B . porcwius), 214, 234, 237 crenatus, 214, 219 glcrndulu, 213, 215 . humeri, 214 improvisus, 208. 219 prrforutus, 204, 214, 229, 230, 232, 234 p.sittucus, 234 Billin, I 50 Boloceru. 74, 92 tuediue, 84, 85, 92 Boloceroides, 61, 68 Boschmuellu, 2 19 Bunoductis verrucosu, 72 Bunodeopsis, 90

c Culliuctis parusiticu, 93 polypits, 68 Campunuluriu Jexuosu, 50 Curcinus muenus, 93 Cardium (Cerastroderma) edule, 103, 104, 108, 110, 124, 125, 127, 128, 132, 134, 150, 156 glaucum, 103, 108, 124, 125, 127 Cereus pedunculotus, 12 Cerianthus Iloydii, 50, 74 Chumaesipho columna, 209 Chelonobia, 233 patula, 233 testudinariu, 203, 233

246

TAXONOMIC I N D E X

Chlomys opcwrrlrrris, 103, 134 Choromylihs meridiondis, 103, 165, 168 Ciifiicinrulopliilirsdelogei, 2 19 Chthumciii~s,207, 216, 223, 228, 232, 234 depressirs, 209, 224 hemheli. 204, 232 intertcxtrts, 204 stelkutii~,204, 2 13, 230, 232 Colpophyllcr nutuns, I I , 30 Conchodertnri, 200 Condylactis giganfeu, 93 Coronirlu diudemu, 203 Corynactis. 92 Crusstuireu, I 48 eomtnerciulis. 103, 169, 171 gigrrs. 103, 130, 165, 168, 169, 171 tnurgriritrrc~eu,103, 165, 168 virginica, 103, 129, 166-169, 171 Cyphasrreu, 10, 17 tnicrophthulmu, 5 , I I Cypri/epus, 200

Diciiocoeniu stokesii, 12, 30, 38 Diplora clivosn. 45 lubyrinthiformis, 10, I I , 21, 23, 30, 33 strigosu, 10, 12, 21, 22, 30. 33, 37, 43-45, 48, 49 Donax denticulurus, 103, I 10, I3 I jirliune (D. fruncirlus). 103, 156 serra, 103, 116, 131, 150 trunculus, 103, 135

E Edwurdsia callimorphu. 77. 83, 84 Elminius, 227, 233 kingii. 233 modestus, 209-211, 213, 215, 219, 220, 224, 225, 234, 235 plicutus, 230, 233 simplex, 230, 233 Enteromorpho intestinalis, 21 9 Euphuusiu, 91 Erisrnulia fustigiata, 11, 34

F Friviu. I I ,fiugimm, 24, 27, 37 Fuvites, I 1 Fungiri ,firngitrs. 43 scxtarici. 23, 26, 28

G

H Hulcumpci, 73 Hulipliinellu (Dicrduinene)Irrci(ie368 Helix uspersu, 103, 145 Hemioniscits bnluni. 2 19 Hermodice, 92 Heteroxetnia .fitscescens. 46 Hormantiu, 92 Hydru, 66, 68

I Isognomon altrfus, 103, 107, 108, 124. 127, 150

L Lebrrmiu. 90 Lepus, 200, 204, 205, 207 Leptostrea piirpurea, 11, 14, 33 Ligumia subrostrutu, 103, 130 Lima scabra, 103, 134, 136 Loligo peulii, 103, 150 Lo//igitneu/ubrevis, 103, 150

Macotnu halthic~rr.103, 129 Mudrucis usperulci, 36, 37 decac~is,24, 27. 36, 38 mirahilis. 10, 16, 23, 27 Manicina urcolutu. 39, 41, 47, 49

247

TAXONOMlC INDEX

Mercmuriu (Venics) mi>rcenuriu, 103, 109. 131. 151, 152, 154, 156-158, 167, 169, I70 Merrrlinci. 10 Metridium. 73 senile. 66, 72. 84, 92 Milleporu. 12, 24, 27 crlcicorrris, 30 c~implrincita,30, 33 dichotomu. 5, 1 I Modiolr4s, 109 demissirs, 103, 108, 124-126, 150, 165 modioliis, 103, 112, 125, 127, I65 Monustrcw cinnt~lciris, I I , 14, 18, 21-30, 32-34, 36-38. 41-45, 48 cuverno.su. 11, 17, 26, 34, 39, 41, 43, 44 Montiporci putiilu, 1 I verrucosu, 11, 23, 26, 28 Mirssti ungidosu. I I , 34, 36 Myu urenuriu, 103, 105, I l l , 113, 11.5, 121. 122, 124, 125, 127, 130, 134, 150, 156, 157, 171 Mytilirs col~&rniuniis, 103, 110, 124-126, 131, 146, 150, 166, 168 PdUliS, 50, 54, 103-113, 120-126, 128-135, 138-141, 144, 146, 148, 150-152, 154, 1.56, 161, 163, 165-170, 172- 18 I edltfis p/rnu/rctus. 103. 105. 171 gcilloprouincirrli.~, 103. 124-126, 165 viridis. 103, 105

N

Ptrvonu clecrr.sstrttr. 5 Pcc.tc,n irrudiuns. 103, 106 mciximirs. 103, 125. 134 P w z u p u m t i , 103. 110, 165, 168 Phc>lliccc~ti*v, 74 rohustu, 74, 78, 84 Phvlluc~ti.~, 90 poscr/l~fc,rci,9 1 Pimpehu1e.s promelus. 103, 175 Pluiygyru, 40, 48 Plctrrobemu cocrincwm. 103, 109, 148 Pocilloporu, 10, I I , 14-16, 42 domicornis. 14, 15, 18, 23, 26, 28, 31, 33, 41. 42, 47 dunue, I I eydorcxi. 3 1. 54 nieundrinu. 10, I I , 14, 15, 31. 33 Pollicipes, 200 Poriies. 42 andrewsii, 10, 14, 15, 23, 26, 41 42 ustreoidc.s, 10, IS, 16, 26, 30, 33, 38-40, 42, 43 c'ompressu. I I , 28 diuuricutu, 38, 42 ficrcrrto, 38, 42 Iobufu, 10, I I , 33 lritea (hriddoni),5, I I , 15, 16, 18, 21, 22 porites, 12, 37, 40 Protcinrheci, 73 Psammucoru. 10 Pseirductinim flugell$kr~, 83 Pseudoplexuriru, 49 Pyrgomu, 204, 233

Neotrigonici murgtirituceu, 103, 105 Rhizocephulu, 200 Ortolusmis, 200 Ociclina d$ficsu, 24, 27 Oni.ssimics, 92 normani, 92 O.scillatoriu suhmemhrunuceu, 48 Osfreu edulis, 103, I l l , 132, 171

P Paiythoa, 30 Puruculliurtis sfephensoni. 84

S Sugurtiu, 73 troglodyte.^, 72, 73, 74 Scmhicirluriu plunu, 103-105, 109, 110, 113-121, 123, 125, 127, 129, 132-147, 150-161, 163-167, 171, 175 Seriutoporu, 1 I hysirix, I I , 48 Siderustrea sidereu, I I , 12, 14, 17, 26, 30 Spisirla solidissirnu. 103, 129, 130, 134

248

TAXONOMIC INDEX

Stoichuc~ti.~. Y2 heliunthrrs Y 3 Stylophoru. I I , 11 pistilloru, I I , 16, 19, 46

U

.

T Teulia, 14 Tellinci.fiihulo, 103, 109 Tefruclita, 229, 233, 234 purpuruscens, 204 stutcictiferu, 228 Thorucicu. 200

Unio turnidus, 103, 156, 157

Venerupis decussutu. 103, 129 Venus striutulu, 103, 109 Verrucu, 232 strormiu, 214, 234

Z Zooxunthellu, 98, YO

Subject Index A

measurements, precision, 238 orientation during, 207-208 primordial valves, 204-207 rates in various species, 221-222 seasonal changes, 21 1-212, 237 shell plates, 203-204 tissue/shell weight ratio, 215 integument composition and origin, 223-225 cuticle formation, 225-227 shell deposition, 225-228 shape, effects of crowding, 208-209 salinity, 21 1 substratum, 209-21 I shell defence against predators, 234 deposition, 225-228 evolution, 200-202 structure, 228-233 microstructure in various species, 23 1-233 Bivalve molluscs, stress detection and responses to heavy and toxic metals, 132-133, 143-146 salinity, 133-134, 137-143. 145 calcium ion dependence, 138-139, 141 heart rate, 111-116 copper effect, 117-1 I9 temperat ure effect, 130- 132 zinc effect, 118-121 heavy metal effects, 104-105, 129, 132- 133, 143- 146 accumulation in tissues, 162-163 behaviour and physiology changes, 163-168 gametogenesis inhibition, 166, 168, 169, 173 growth inhibition, 167 mitochondria1 respiration and, 176-181 calcium role, 176-179

Algae barnacle growth and, 219 coral infection, 48 digestion by sea anemones, 79-80 substrate colonization, 19 Alizarin red S stain coral growth, 21, 23 Amino acids in sea anemones feeding activation, 67-69 metabolism, 71-72 uptake, 70-72, 87 Amylase in sea anemones, 80, 83, 86-87 Asterids sea anemone predators, 92

Bacteria coral infection, 7, 48 digestion by sea anemones, 79-80 Barnacles growth curve, equations, 220-221 daily changes, 213, 236-237 effects of breeding, 238 competing organisms, 219 food, 216, 235 light, 216, 235 lunar cycles, 237 moulting, 235, 238 orientation to current, 217 parasites, 219 population density, 217-219 reproduction, 219-220, 235 surface contour, 217 temperature, 212, 215, 235 tidal levels, 212-213, 216, 234-236 249

250

SUBJECT INDEX

unfertilized eggs and, 173-175 methods for monitoring of “activity ’ ’, 107- 108 heart activity, 105-106 shell growth, 107 valve movements, 106-107 water pumping, 106-107 mortality copper-induced, 122-123T zinc-induced, 122-123T nature of stress, 104-105 pollutants, threshold, 104-105 pumping activity, 112-1 16 salinity effect. 113, I I6 respiration anaerobic, 146-150 glycogen increase, 149 metabolism, 148- 150 heart rate and, 146-148, 155 pH decrease, 151-155 valve movements and, 146-148 sensory receptors in inhalant siphon, 134-136 in mantle, 134-136 shell calcium reabsorption from, 156-162 physical protection, 155-156 strength reduction, 161-162 sublethal stress levels, 128-130 avoidance behaviour and, 129-130 valve movements, 108-1 12, I16 effects of copper, 117-1 19 salinity, 110, 113 temperature, 109 zinc, 118-121 in epifaunal and infaunal species, 121, 124- 128 oxygen consumption and, 125-128

C Calcium, bivalve molluscs concentration under stress, 151 mitochondria1 respiration and, 176-179 reabsorption from shell, 156-162 in response to salinity, 138, 139, 141 Caribbean corals, stress histopathology, 48-49

loss of zooxanthellae, 30-3 IT metabolism, 28 mucus production, 40T sediment shedding, 44T C hemoreceptors prefeeding, sea anemones, 69 Conducting system sea anemones, 69 Copper, bivalve molluscs accumulation in tissues, 163 behavioural responses to, 117-1 19, 164- 168 effects on gametogenesis, 166, 168, 169, 172, 173 isolated siphons, 163-164, 167 mitochondrial respiration, 176-180 physiology, 164-168 unfertilized eggs, 173-175 mortality, 122-123T sublethal levels, 129-130 Coral reefs changes monitoring by coral colony size measurements, 4-5 nonliving substrate assay, 4 quantitative technique plotless, 13 quadrant, 13 stereophotography, 4 future research, 51-54 natural disturbances, 5-9 in various geographical areas, 6-7T pollution effects, 9-16 juvenile coral mortality, 19 responses to various pollutants, 10-12T tolerance in shallow water, 14 recovery rate, 16-20 temperature effects, 11. 14-16, 26 Corals, stress behaviour, 35-46 feeding and mesenterial filament extrusion, 35-39 mucus production, 39-42 polyp retraction, 38 sediment shedding, 43-46 biochemical indexes lipid/protein ratio, 50 lysosomal glucosaminidase, SO lysosomal hydrolases. 50 definition, 9

25 1

SUBJECT INDEX

growth rate, 20-27 alizarin red S stain, 21, 23 calcification, 24, 27 disturbance effects, 22-24T fixed base line, 21, 23 X-radiography, 21, 22. 25 histopathology, 48-SO algal infection, 48 bacterial infection, 48 shutdown, 48 tumours, 49 white band disease, 48 loss of zooxanthellae, 29-35, 39 metabolism, 27-29 photosynthesis, 28-29 respiration, 28, 29 reproduction, 46-48 laboratory studies, 47-48 oil pollution and, 46 polyp bail-out, 47-48 Crustaceans small, in sea anemone diet, 93 Current barnacle growth and, 216-217

E Eggs, bivalve molluscs heavy metal effects, 173-175 Endocytosis in sea anemones, 78-79 Evolution barnacles and their shells, 200-202

F Feeding corals under stress, 35-38 sea anemone behaviour, 66-69, 81 Fish sea anemone predator, 92-93 Fixed base line coral growth, 21, 23 Food, barnacle growth and, 216, 235

G Gametogenesis, bivalve molluscs heavy metal effects, 166, 168, 169, 173

Gastropods sea anemone predators, 92 small, in sea anemone diet, 93 Glucosaminidases in corals under stress, SO Glycogen in bivalve molluscs under stress, 146- 1-50

H Hawaian corals, stress loss of zooxanthellae, 30-31T Heart rate, bivalve molluscs effects of copper, 117-1 19 temperature, 130-132 valve closure, 146-148, 155 zinc, 117-1 19 methods, 105-106 pumping activity and, I 1 1-116 valve movements and, I 1 1-1 16 Heavy metals, bivalve molluscs accumulation in tissues, 162-163 detection and response to, 143-145 effects on behaviour and physiology, 163-168 gametogenesis, 166, 168, 169, 172, 173 growth, 167 life stages, 168-175 mitochondria1 respiration, 176- 181 unfertilized eggs, 173-175 toxicity, 104-105, 129, 132-133 zinc, see Zinc Histopat hology corals under stress, 48-50 Hurricane coral reefs and, 6 Hydrolases corals under stress, SO

L Laminarinase in sea anemones, 80 Light barnacle growth and, 216, 235 Lipid/protein ratio corals under stress, 50

252

SUBJECT INDEX

Lunar cycles barnacle growth and, 237

Mesenterial filaments, corals extrusion, feeding and, 35-39 Metabolism corals, stress effects, 27-29 Mitochondria, bivalve molluscs respiration, heavy metal effects, 176-181 cation transport and, 176-179 in eggs and sperm, 180 Mucus, corals production, stress effects, 39-42

Protease in sea anemones, 82-85 Pycnogonids sea anemone predators, 92

R Reproduction barnacle growth and, 219-220, 235 corals under stress, 46-48 Respiration bivalve molluscs anaerobic, 146-155 heavy metal effects, 164-168 in gametes, 173-175 corals, stress effects, 28, 29

0 Oil pollution bivalve molluscs and, 129 coral reefs and, 1 1 , 16 coral stress mesentenal filament extrusion, 36-37 reproduction and, 46 Oxygen, atmospheric consumption in bivalve molluscs, 125-128

P PH in bivalve molluscs under stress, 151-155 Phagoc ytosis in sea anemones, 74, 76-79, 88 Phosphate coral reef response to, 16 Photosynthesis in corals, stress effects, 28-29 in sea anemones, 91-92 Pollution effects on bivalve molluscs, see Bivalve molluscs, stress on corals, see Corals, stress Polychaetes in sea anemone diet, 93 Polysaccharide acid, secretion by sea anemones, 86

S Salinity barnacle shape and, 21 I bivalve mollusc responses calcium reabsorption from shell, 156, 1 58- I 60 detection and changes, 137-142 protective, 133-134 pumping activity, 113, I16 at sublethal level, 129 valve movements, 110, I13 Sea anemones amino acid metabolism, 71-72 amino acid uptake localization, 70-71, 87 systems of, 71-72 diet. 93 digestion acontia, 81 of bacteria, 79-80 cnidae, 81 cnidoglandular tracts, 82, 87 endocytosis, 78-79 endodermal current, 74-75 excretion, 89 extracellular, 82-89 acid polysaccharide secretion, 86 amylase, 83, 86-87 chymotrypsin, 83, 84

253

SUBJECT INDEX

protease, 82-85 of microalgae, 79-80 phagocytosis, 74, 76-79, 88 tentacles. 81 pharynx, ciliary current, 72-74 feeding behaviour activation by amino acids, 67-68 chemoreceptors, 69 conduction system, 69 control of, 69 feeding response, 66-67 prefeeding response, 66-69, 81 as prey, 92-93 symbiosis photosynthetic metabolites, 91-92 zoochlorellae, 90-91 zooxanthellae, 89-92 Sedimentation coral reef response to, 8, 10, 15-16 Sewage coral reef response to, 1 I , 16, 19 Stereophotography coral reef assay, 4 Stress, environmental corals, see Corals, stress marine bivalve molluscs, ,see Bivalve molluscs, stress Substratum barnacle shape and, 209-21 I Symbiosis microalgae in sea anemones content. 90-91 localization, 89-90 photosynthetic products, 91-92

T Temperature barnacle growth and, 212. 215, 235 bivalve mollusc heart rate and, 130-132 coral reefs and, I I , 14-16, 26 Tidal levels barnacle growth and, 212-213, 216, 234-236 Toxic metals bivalve mollusc stress and, 104, 129-130, 132-133 copper, see Copper

Tumours corals under stress, 49

w White band disease corals under stress, 48

X X-radiography coral growth, 21, 22, 25

Z Zinc, bivalve molluscs accumulation in tissues, 163 behavioural responses to, 117-1 19, 165- 168 detection and responses to, 143-145 effects on gametogenesis, 166, 168, 169, 172, 173 mitochondria1 respiration. 176- 177, 180- I8 I physiology, 165- I68 unfertilized eggs, 173-175 mortality, 122-123T sublethal levels, 130 Zoochlorellae in sea anemones content regulation. 90 localization, 90 photosynthetic products, 91 Zooxanthellae in corals loss as stress indicator, 29-35, 39 polyp size and. 32-34 by various species, 30-31T. 32 in sea anemones content regulation, 90-91 localization, 89-90 photosynthetic products, 91-92

This Page Intentionally Left Blank

Cumulative Index of Titles

Alimentary canal and digestion in teleosts, 13, 109 Antarctic benthos, 10, 1 Artificial propagation of marine fish, 2, 1 Aspects of stress in the tropical marine environment, 10, 217 Aspects of the biology of seaweeds of economic importance, 3, 105 Assessing the effects of “stress” on reef corals, 22, 1 Association of copepods with marine invertebrates, 16, I Behaviour and physiology of herring and other clupeids, 1, 262 Biological response in the sea to climatic changes, 14, 1 Biology of ascidians, 9, 1 Biology of clupeoid fishes, 20, 1 Biology of coral reefs, 1, 209 Biology of euphausiids, 7, 1; 18, 373 Biology of mysids, 18, 1 Biology of pelagic shrimps in the ocean, U ,233 Biology of Phoronida, 19, 1 Biology of Pseudomonus, 15, 1 Biology of wood-boring teredinid molluscs, 9, 336 Blood groups of marine animals, 2, 85 Breeding of the North Atlantic freshwater eels, 1, 137 Circadian periodicities in natural populations of marine phytoplankton, 12, 326 Competition between fisheries and seabird communities, 20, 225 Coral communities and their modification relative to past and present prospective Central American seaways, 19, 91 Diseases of marine fishes, 4, 1 Ecology and taxonomy of Hulimedu: primary producer of coral reefs, 17, 1 Ecology of intertidal gastropods, 16, 11 1 Effects of environmental stress on marine bivalve molluscs, 22, 101 Effects of heated effluents upon marine and estuarine organisms, 3, 63 Environmental simulation experiments upon marine and estuarine animals, 19, 133 Estuarine fish farming, 8, I19 Fish nutrition, 10, 383 Flotation mechanisms in modern and fossil cephalopods, 11, 197 General account of the fauna and flora of mangrove swamps and forests in the Indo-West Pacific region, 6, 74 255

256

CUMULATIVE INDEX OF TITLES

Growth in barnacles, 22, 199 Gustatory system in fish, 13, 53 Habitat selection by aquatic invertebrates, 10, 271 History of migratory salmon acclimatization experiments in parts of the Southern Hemisphere and the possible effects of oceanic currents and gyres upon their outcome, 17, 397 Influence of temperature on the maintenance of metabolic energy balance in marine invertebrates, 17, 329 Interactions of algal-invertebrate symbiosis, 11, 1 Laboratory culture of marine holozooplankton and its contribution to studies of marine planktonic food webs, 16, 21 I Learning by marine invertebrates, 3, 1 Management of fishery resources, 6, 1 Marine biology and human affairs, 15, 233 Marine molluscs as hosts for symbioses, 5, I Marine toxins and venomous and poisonous marine animals, 3, 256 Marine toxins and venomous and poisonous marine plants and animals, 21, 59 Methods of sampling the benthos, 2, 171 Nutrition of sea anemones, 22, 65 Nutritional ecology of ctenophores, 15, 249 Parasites and fishes in a deep-sea environment, 11, 121 Particulate and organic matter in sea water, 8, 1 Petroleum hydrocarbons and related compounds, 15, 289 Photosensitivity of echinoids, 13, 1 Physiological mechanisms in the migration of marine and amphihaline fish, 13, 248 Physiology and ecology of marine bryozoans, 14, 285 Physiology of ascidians, 12, 2 Pigments of marine invertebrates, 16, 309 Plankton as a factor in the nitrogen and phosphorus cycles in the sea, 9, 102 Pollution studies with marine plankton, Part 1 : Petroleum hydrocarbons and related compounds, 15, 289 Pollution studies with marine plankton, Part 2: Heavy metals, 15, 38 I Population biology of blue whiting in the North Atlantic, 19, 257 Present status of some aspects of marine microb~ology,2, 133 Problems of oil pollution of the sea, 8, 215 Rearing of bivalve mollusks, 1, 1 Recent advances in research on the marine alga Acetabularia, 14, 123 Recent developments in the Japanese oyster culture industry, 21, I Respiration and feeding in copepods, 11, 57 Review of the systematics and ecology of oceanic squids, 4, 93 Scallop industry in Japan, 20, 309 Scatological studies of the Bivalvia (Mollusca), 8, 307 Some aspects of the biology of the chaetognaths, 6, 271

CUMULATIVE INDEX OF TITLES

257

Some aspects of neoplasia in marine animals, 12, 151 Some aspects of photoreception and vision in fishes, 1, 171 Speciation in living oysters, 13, 357 Study in erratic distribution: the occurrence of the medusa Gonionemus in relation to the distribution of oysters, 14, 251 Taurine in marine invertebrates, 9, 205 Upwelling and production of fish, 9, 255

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Cumulative Index of Authors

Allen, J. A., 9, 205 Ahmed, M., 13, 357 Arakawa, K. Y., 8, 307 Bailey, R. S., 19, 257 Balakrishnan Nair, N., 9, 336 Blaxter, J. H. S., 1, 262, 20, I Boney, A. D., 3, 105 Bonotto, S . , 14, 123 Branch, G. M., 17, 329 Bruun, A. F., 1, 137 Campbell, J. I., 10, 271 Carroz, J. E., 6, 1 Cheng, T. C., 5, 1 Clarke, M. R., 4, 93 Corkett, C. J., 15, 1 Corner, E. D. S., 9, 102; 15, 289 Cowey, C. B., 10, 383 Cushing, D. H., 9, 255; 14, 1 Cushing, J. E., 2, 85 Davenport, J., 19, 133 Davies, A. G., 9, 102; 15, 381 Davis, H. C., 1, 1 Dell, R. K., 10, 1 Denton, E. J., 11, 197 Dickson, R. R., 14, 1 Edwards, C., 14, 251 Emig, C. C., 19, 1 Evans, H. E., 13, 53 Fisher, L. R., 7, I Fontaine, M., 13, 248 Furness, R. W., 20, 225 Garrett, M. P., 9, 205 Ghirardelli, E., 6, 271 Gilpin-Brown, J. B., 11, 197 Glynn, P. W., 19, 91 Goodbody, I., l2, 2 Gotto, R. V., 16, I Gulland, J. A., 6, 1 Harris, R. P., 16, 211 Hickling, C. F., 8, 119 Hillis-Colinvaux, L., 17, 1 Holliday, F. G. T., 1, 262 Hunter, J. R., 20, 1

Kapoor, B. G., 13, 53, 109 Kennedy, G . Y., 16, 309 Loosanoff, V . L., 1, 1 Lurquin, P . , 14, 123 McLaren, I. A., 15, 1 Macnae, W., 6, 74 Marshall, S. M., 11, 57 Mauchline, J . , 7, 1; 18, 1 Mawdesley-Thomas, L. E., 12, 151 Mazza, A . , 14, 123 Meadows, P. S., 10, 271 Millar, R. H., 9, 1 Millott, N., 13, 1 Moore, H. B., 10, 217 Naylor, E., 3, 63 Nelson-Smith, A., 8, 215 Newell, R. C., 17, 329 Nicol, J. A. C., 1, 171 Noble, E. R., 11, 121 Omori, M., U, 233 Paffenhofer, G-A., 16, 211 Pevzner, R. A., 13, 53 Reeve, M. R., 15, 249 Riley, G. A., 8, I Russell, F. E., 3, 256; 21, 60 Russell, F. S., 15, 233 Ryland, J. S., 14, 285 Saraswathy, M., 9, 336 Sargent, J. R., 10, 383 Scholes, R. B., 2, 133 Shelbourne, J. E., 2, 1 Shewan, J . M., 2, 133 Sindermann, C. J., 4, 1 Smit, H., 13, 109 Sournia, A., U, 236 Stewart, L., 17, 397 Taylor, D. L., 11, 1 Underwood, A. J., 16, 111 Ventilla, R. F., 20, 309; 21, 2 Verighina, 1. A., 13, 109 Walters, M. A., 15, 249 Wells, M. J., 3, 1 Yonge, C. M., 1, 209 259

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