Advances in Marine Biology, Volume 11

Advances in

MARINE BIOLOGY VOLUME 11

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Advances irc

MARINE BIOLOGY VOLUME 11 Edited by

SIR FREDERICK S. RUSSELL Plymouth, England

and

SIR MAURICE YONGE Edinburgh, Scotland

Academic Press London and New York A Subsidiary of Harcoun Brace Jovanovich, Publishers

1973

ACADEMIC PRESS INC. (LONDON) LTD.

24-28

OVAL ROAD

LONDON NW1 7DX

U.S. Edition published by ACADEMIC PRESS INC.

111

FIFTH AVENUE

NEW YORK, NEW YORK

10003

Copyright 0 1973 by Academic Press Inc. (London) Ltd.

All rights reserved

N O PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM

THE PUBLISHERS

Library of Congress Catalog Card Number: 63-14040 ISBN : 0- 12-0261 11- 1

PRINTED IN GREAT BRITAIN BY THE WHITEFRIARS PRESS LTD. LONDON AND TONBRIDGE

CONTRIBUTORS TO VOLUME II E. J. DENTON, The Plymouth Laboratory of the Marine Biological Association of the United Kingdom, Plymouth, England.

J. B. GILPIN-BROWN, The Plymouth Laboratory of the Marine Biological Association of the United Kingdom, Plymouth, England. ELMER R. NOBLE, University of California, Santa Barbara, California, U.S.A. SHEINAM. MARSHALL, University Marine Station, Millport, Isle of Cumbrae, Scotland.

DENNIS L. TAYLOR, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Florida, U.S.A.

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CONTENTS

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I. Introduction: Multicellularity, Symbiosis and the Functional Unit . . .. .. .. .. ..

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CONTRIBUTORS TO VOLUME 11

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The Cellular Interactions of Algal-Invertebrate Symbiosis DENNISL. TAYLOR

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11. Hosts . . .. .. A. Phylogenetic Range . . B. Qualities of a Suitable Host

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C. Host-Symbiont Specificities D. Adaptations .. .. ..

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111. Symbionts . . .. .. A. Phylogenetic Range . . .. B. Qualities of a Suitable Symbiont

IV. Establishment of a Functional Symbiotic Unit A. Origins .. .. B. Transmission of Symbionts

V. Nutrition of the Functional Unit A. Sources .. B. Intercellular changes. . C. Growth ..

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VI. Conclusions VII. References

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Respiration and Feeding in Copepods

SHEINA M. MARSHALL I. Introduction

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11. Respiration .. .. Effect of Crowding . . .. A. B. Effect of Time after Capture .. C. Variation with Season .. D. Relation 60 Size . .. Effect of Light .. E. .. . F. Effect of Temperature . . .. G . Effect of Salinity . .. H. Effect of Pressure I. Effect of Oxygen Content .. . . .. J. Effect of Feeding 1 .

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IV. Conclusion

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Parasites and Fishes in a Deep-sea Environment

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I. Introduction 11. Methods

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.. .. IV. Fishes and Their Parasites . . A. Organization and Behaviour of Deep-water Fishes B. Parasites of Fishes-Introduction .. .. C. Inshore Fishes .. .. .. .. .. D. Selachians . . .. .. .. .. .. E. Midwater Fishes and Their Parasites-North .. .. .. .. .. .. Atlantic F. Midwater Fishes and Their Parasites-Eastern Pacific and Indian Ocean . . .. .. .. .. .. G. Fishes of the Family Macrouridae . .

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.. .. .. V. Discussion . . A. Food .. .. .. .. B. Life Cycles of Parasites .. .. C. Parasites as Biological Tags .. D. The Uniqueness of Deep-sea Parasitism VI . Conclusions and Summary VII. Acknowledgements VIII. References

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Floatation Mechanisms in Modern and Fossil Cephalopods

E. J. DENTON AND J. B. GILPIN-BROWN

I. Introduction

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11. Animals Without any Special Buoyancy Mechanism .. .. .. 111. Buoyancy Given by Fats . . IV. Buoyancy Given by Tissue Fluids

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VI. Buoyancy in Fossil Cephalopods A. The Fine Structure of the Siphuncle B. Posture .. .. .. .. C. Liquid in the Chambers of the Shell .. .. .. D. Strength of Shell VII. Conclusion

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IX. References

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AUTHOR INDEX

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TAXONOMIU INDEX. . SUBJECT INDEX

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CTJMTLATIVEINDEX OF AUTHORS

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Adv. mar. Biol., Vol. 11, 1973, pp. 1-66

THE CELLULAR INTERACTIONS OF ALGALINVERTEBRATE SYMBIOSIS* DENNIS L. TAYLOR Rosenatiel School of Marine and Atmospheric Science, University of Miami, Plorida, U.S.A. I. Introduction: Multicellularity, Symbiosis and the Functional Unit II. Hosts .. .. .. .. .. A. Phylogenetic Range . . .. .. .. . . . . B. Qualities of a suitable Host . . .. .. .. III. Symbionts . .. A. Phylogenetic Range .. .. B. Qualities of a Suitable Symbiont . .. .. N. Establishment of a Functional Symbiotic Unit A. Origins .. .. .. .. .. .. .. B. Transmission of Symbionts .. .. .. .. C. HostSymbiont Specificities. . .. .. D. Adaptations . . . . .. . . . . V. Nutrition of the Funotional Unit . .. A. Sources .. .. B. IntercellulaxExchanges .. . . . . . . C. Urowth . .. .. .. .. .. VI. Conclusions .. .. VII. References.

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" , why expend pain and labour on insignifkant creatures when so much remains to discover with respect to the higher animals, including man himself? " (Frederick Keeble, Plant Animala, 1910)

I. INTRODUCTION : MULTICELLULARITY, AND THE FITNCTIONAL UNIT SYMBIOSIS Although the autonomous activities of individual cells and noncellular organisms are fairly well understood, our knowledge of mechanisms co-ordinating inter-cellular function in metazoa is comparatively deficient. One of the most challenging biological problems of our time is the question of neoplastic growth and the way it is induced. As a possible consequence of biological organization, neoplasia is essentially a disease of multicellularity. Uncontrolled patterns of cellular growth may not be entirely due to a breakdown in cell function. Instead, they

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Contribution No. 1614. Rosenstiel School of Marine and Atmospherio Science. 1

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DENNIS L. TAYLOR

may be related to a failure of inter-cellular co-ordination, resulting in a breakdown in cell tolerances, and a progressive independence of specific metabolites excreted by neighbouring cells (Klein and Klein, 1967). Regulation of complex inter-cellular functions is of paramount importance to what we term " normal " growth of an organism (Foulds, 1969). The ability of cells to excrete metabolites into the surrounding medium is of particular significance, since it is believed to control the mechanisms of co-ordination in multicellular as well as multi-organismal systems. The influence of extracellular products on the behaviour of individual cells and organisms has been widely discussed (e.g. Barker, 1970), and is the subject of numerous reviews (Lucas, 1947, 1949, 1955, 1968 ; Provasoli, 1963 ; Whittaker, 1969 ; Whittaker and Feeny, 1971). The effect of an individual's external metabolites on organisms in the same medium can manifest itself in various ways, depending upon the degree of integration that is achieved in the system. This may include on the one hand, broadly defined " chemical symbiosis " (sensu Lucas, 1947) such as the dependence of marine phytoplankton on vitamin B,, which is produced in the environment (Daisley, 1957; Droop, 1957; Provasoli, 1958), and the fully integrated symbiotic associations of microalgae and invertebrates (as defined by DeBary, 1879) on the other. The latter represent the extreme degree of integration that can result from these broadly defined, non-predatory relationships involving excreted metabolites, and are of particular interest to the biologist investigating cellular interrelationships, since they present a suitable opportunity for studying the way in which co-ordination is established and maintained in multicellular systems. As a functional unit, their value resides in expanding the concept of organism to include heterogeneous systems which extend beyond the limitations of genetic uniformity (Gregory, 1951). Such associations are ideal material for the investigation of the way in which cells and tissues manage to live together. At their simplest they are readily studied as examples of a biological " field ", in which biological organization is first established by, and then becomes wholly dependent upon, the relatedness of its components (Waddington, 1956). The basic theme emerging from an investigation of algal-invertebrate symbiosis is the fundamental 'nature of cellular interaction. It is no coincidence that this is also the most distinctive characteristic of multicellularity itself, a reliance on interdependency of components rather than a fixed framework-a dependence upon ordered interactions (Weiss, 1962, 1963). Within this context, the ability of organisms and organismel assemblages to regulate must be a fundamental property. Examination of algal-invertebrate symbiosis is a legitimate form of

INTERACTIONS O F AJiGAL-INVERTEBRATE SYMBIOSIS

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enquiry into the nature of these interactions. The participation of phylogenetically distinct cell types in these associations provides a valuable experimental simplification which makes them a useful tool of enquiry. Associations between unicellular algae and invertebrates have captured the interest of biologists for an exceptionally long period of time. The history of opinion and theory relative to this area is both complex and controversial, but may be generally divided into a succession of attitudes which have culminated in the experimental analyses of the past decade. As might be expected, the status of the subject has been reviewed on frequent occasions. The recent increase of interest in algal-invertebrate symbiosis has produced both a flood of data and a proportional rise in expressions of the reviewer’s craft. The present effort is no exception. Hopefully, however, a viewpoint can be offered which will stimulate new interests in this comparatively old problem. In the following sections, the properties of the participants will be examined separately and then the resulting symbiosis will be reviewed in the context of cellular interactions that are common to all multicellular systems. The review of Droop (1963) is an excellent survey of the literature prior to 1962, and has provided much of the impetus for modern studies of algal symbiosis. The survey of carbon translocation in symbiotic associations given by Smith, D. et al. (1969) is extremely useful as a supplement. The recent reviews of Muscatine (1971, 1973) should also be consulted. For the purposes of this survey, emphasis will be placed on work published since 1960.

11. HOSTS There are extensive listings of invertebrate species in symbiosis with unicellular algae (Buchner, 1930, 1953 ; Droop, 1963 ; McLaughlin and Zahl, 1966). Recently, there has been an attempt to go beyond the non-specific designation “ zooxanthella ”, and assign particular hosts to individual algal symbionts of known taxonomic position (Taylor, 1972). This has strengthened our knowledge of algal symbionts (see below) ; but, like previous listings, it tells us practically nothing about the properties which make particular invertebrate species suitable as hosts. It seems likely that this suitability is dependent upon the character and degree of cellular organization in the host species themselves, and that this will be reflected in the phylogenetic distribution of species known to harbour symbionts. Pre-adaptation and post-adaptation of the host’s biology in the presence of algal cells

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are significant aspects of host suitability that may also be dependent on pre-existing cellular conditions. A. Phylogenetic range Marine algal symbionts have been recorded in the cells and tissues of invertebrate species belonging to several major phyla, and are also believed to occur in some chordates (Tunicata only) (Droop, 1963; McLaughlin and Zahl, 1966; Taylor, 1972). The reader should be warned, however, that many of the records that have become enshrined in the literature are the result of chance observations on casual associations, and general misinterpretation of animal cell structure and inclusions. For example, symbiosis among the tunicates recorded by Smith, H. G. (1935)has not been confirmed by this author (cf. Hastings, 1931), and the “algae ” reported in Beroe and several genera of Chaetopteridae (Berkeley, 1930a, b) have been identified as animal cell inclusions in electron micrographs of the animal tissues (Taylor, unpublished). Numerous other examples exist, and extensive reinvestigations of earlier reports will be required. This is especially true of reports involving the more unusual host types. Reliable reports suggest that symbiosis is exceptionally common among the Protozoa and Coelenterata (Lenhoff, 1968; Ball, 1969). Together, they may be regarded as a major host grouping characterized by highly successful and diversified associations. Symbiosis involving marine Porifera is poorly studied and somewhat atypical. Available literature suggests that only species of Cyanophyceae are involved (see below) (Feldman, 1933; Sara, and Liaci, 1964a, b ; S a d , 1965, 1966, 1971). Among the Platyhelminthes studied, only acoelus Turbellaria are known with certainty to be hosts for symbiotic algae (Ax and Apelt, 1965 ;Dorey, 1965; Sarfatti and Bedini, 1965;Provasoli et al., 1968; Apelt, 1969a, b ; Ax, 1970; Taylor, 1971b, 1972),although broader studies may reveal new associations. Symbiosis involving species of Mollusca is apparently restricted to a few highly specialized examples, most notably the Tridacnids (Yonge, 1936, 1953; Taylor, 1969a ; Fankboner, 1971) and the Sacoglossa (Taylor, 1970). Other phyla listed in Buchner (1930,1953),Droop (1963)and McLaughlin and Zahl (1966) include Ctenophora, Annelida, Polyzoa, Echinodermata and Chordata. Preliminary re-investigations of examples from each of these phyla have failed t o produce a valid instance of symbiosis with an alga (Taylor, unpublished). More intensive studies are indicated before they can be completely excluded from the ranks of potential hosts.

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B. Qualities of a suitable host 1. Pre-aduptations

With the exception of molluscan hosts (recognized here as a special case), the species participating in symbiotic associations are characterized by a comparatively low level of intercellular co-ordination. This facet of host biology has not been studied experimentally. It could, however, be of significance in the establishment and maintenance of a symbiosis, since the introduction of alien cells into the animal’s tissues would appear to do little to disrupt their underlying organization. A related, and extremely important aspect of this same problem is the ability of a host’s cells and tissues to recognize “ self ”. Among higher animals this is a very well developed biological property, which poses major difficulties in the field of tissue transplantation (e.g., Billingham and Silvers, 1961). Among symbiotic hosts it may be poorly developed or even non-existant. I n the absence of immunological barriers, cohabitation with algal cells would pose very few cellular problems for the host. The failure of a host to digest or otherwise destroy invading algal symbionts is dependent on properties present in both the animal and the alga. The vast majority of symbiotic hosts are carnivores, and it seems likely that they are incapable of digesting their algae (cf. Kawaguti, 1965; Fankboner, 1971) (see below, p. 33). This fact has been adequately pointed out by Droop (1963) and Yonge (1944,196s). When considered in the context of processes of intracellular digestion employed by the majority of hosts, a partial explanation for the success of intracellular symbionts is apparent. Among herbivorous hosts, the establishment of a symbiosis must necessarily follow a different set of rules, and rely heavily upon the resistance of the alga and the weakening or susceptibility of the host (Yonge, 1944; Droop, 1963). The most familiar examples include the Tridacnids where the possibility of the host digesting its algae still exists (Fankboner, 1971 ; Goreau et al., 1966, 1973), and the Sacoglossa where chloroplast symbiosis has evolved as an alternative to whole algal associations (Taylor, 1970). In both instances molluscan hosts are involved and, as noted previously, these are not typical of the overwhelming majority of associations found in the marine environment. Symbiosis between marine sponges and blue-green algae represents another example of a herbivore-algal association that is atypical of most marine examples. Among freshwater hosts, associations involving herbivores are more common (Droop, 1963; McLaughlin and Zahl, 1966).

DENNIS L. TAYLOR

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111. SYMBIONTS Available information on the biology of symbiotic microalgae is poor compared with that on the various host species. Traditionally, the principal symbionts of marine hosts have been referred to as “ Zooxanthellae ”, in recognition of their yellow-brown colour. Green and blue-green symbionts are referred to as “ Zoochlorellae ” and “ Cyanellae ” respectively. Droop (1963) has convincingly pointed out the inadequacy of these epithets. Their continued use in contemporary studies a decade later still effectively denies the properties of individual symbiont species, thereby ignoring most of the “ algal ” aspects of the symbiosis. Because they are the primary producers in symbiotic associations, it is essential to have an understanding of their distributions as well as their individual biochemical, physiological, structural and taxonomic properties. This information can give insight into the quality of their input, and contribute to a fuller appreciation of their role in the total metabolism of the host (e.g., Goreau et al., 1971). A. Phylogenetic range The current state of taxonomic knowledge relative to marine algal symbionts has been summarized in a recent review (Taylor, 1972). A complete listing of specific algal symbionts and their hosts is given there. At least three algal classes have been positively identified in successful associations with invertebrate hosts. These include the Cyanophyceae, Dinophyceae and Chlorophyceae (Geitler, 1959 ; Norris, 1967; Taylor, 1972). At least two others, the Cryptophyceae and Bacillariophyceae, have been tentatively involved in associations with invertebrate hosts (Lee and Zucker, 1969 ; Ax and Apelt, 1965 ; Apelt, 1969a). These are excluded from the present discussion, because a positive relationship with a cryptomonad or diatom species has not been determined experimentally. This proof must rest heavily on the re-infection of alga-free hosts using cultured algal symbionts, i.e. satisfaction of Koch’s postulates (Taylor, 1972). Until that is done, the validity of reports involving Cryptophyceae and Bacillariophyceae must remain in doubt (cf. Fritsch, 1935, 1952 ; Caullery, 1952). 1.

Cyanophyceae

Blue-green algae are frequently encountered as symbionts within the cells of marine planktonic diatoms. They have been generally ignored by most workers and consequently reports of their existence are sparse. Lebour (1930) illustrates an association between a bluegreen alga and the diatom Coscinodiscus concinnus Wm. Smith. This has been tentatively identified as a species of Anabaena (Taylor,

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unpublished). Desikachary (1959) describes the diatom Rhizosolenia sp. in symbiosis with Richelia intracellularis Schmidt. Other casual reports involving marine species lack data on host and symbiont identities and are of little value. The function of these associations is obscure, although the possibility of nitrogen fixation by the symbiont could be an important factor. Marine sponges are the most common hosts of blue-green algal symbionts (Desikachary, 1959 ; Geitler, 1959; cf. Lewin, 1966). Several Cyanophyceae have been described by Feldmann (1933) in associations found commonly in the Mediterranean, and their general biology and relationship with the host has been described in studies by Sara (1948, 1964a, b, 1965, 1966, 1969, 1971), SarB and Liaci (1964a, b) and Vacelet (1971) (see below). Common symbiont species are known to include Aphanocapsa raspaigellae (Hauck) FrBmy, and A. feldmanni FrBmy. Most of these occur frequently in a freeliving state. 2. Dinophyceae

Dinoflagellates are perhaps the most ubiquitous of all marine algal symbionts. Recent studies (summarized by Taylor, 1972) suggest that Cymnodinium microadriaticum (Freudenthal) ( = Symbiodinium microadriaticum Freudenthal) is the most common species occurring among benthic dwelling hosts (Taylor, 1969c, 1971c, 1972). Similar investigations of pelagic hosts, most notably Radiolaria, Foraminifera and Chondrophora, and benthic dwelling acoelus Turbellaria have demonstrated the existence of a second free-living genus with symbiotic members. Amphidinium chattonii (Hovasse) D. Taylor has been described in associations with the chondrophores Velella velella (L.) and Porpita porpita (L.) (Taylor, 1969c, 1971c, 1972), Amphidinium klebsii exists in a remarkable morphologic state within the tissues of the acoel Amphiscolops langerhansi (Taylor, 1971b) and several unidentified Amphidinium species have been found in associations with Radiolaria and Foraminifera (Taylor, 1972). With the single exception of Amphidinium klebsii Kof. et Swezy, dinoflagellate symbioiits have not been collected as free-living stages in the environment adjacent to their hosts. Studies on their lifehistories in culture suggest, however, that such stages do occur, and they may play a significant role in transmission of the symbiosis (see below). Clear understanding of symbiont life-histories is important if host distributions and symbiont identities are to be accurately determined. Extended studies of Gymnodinium microadriaticum both in situ and A.M.B.-11

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in culture suggest that this alga has a complex life-history involving a series of free-living and encysted stages. Sexual reproduction does occur to a limited extent in culture, and some evidence of early stages of meiosis have been found in situ. The total picture differs somewhat

FIG.1. Life history of aymnodinium microadriaticum i n vivo and in vitro. Modification of the scheme proposed by Freudenthal (1962), based on optical and electron microscope studies. (1) Immature cyst, (2) mature cyst, (3) unequal division of cyst (Taylor, 1969e), physiologically younger daughter cell reverts to immature cyst, (4)physiologically older daughter cell degenerates and is excreted (Taylor, IQGQe), ( 5 ) zoosporangium, (6) motile zoospore, (7)stage in meiotic division?, (8) developing gametes, (9) mature gametes released. Chloroplast (cp), calcium oxalate (c), accumulation body (a), pyrenoid (p), nucleus (n), cyst wall (w).

from that originally described by Freudenthal (1962), although the basic elements are similar (Fig. 1). Studies on a variety of hosts harbouring this species, indicate that different stages may be characteristically associated with a particular host. In the absence of information on the alga's life history these are frequently described as " new species. ))

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3. Chlorophyceae

Although they are the rule among freshwater hosts, green algal symbionts are exceptionally rare in the marine environment. This, as pointed out by Droop (1963), is most probably a reflection of the fact that the Chlorophyta are more widely distributed in freshwater habitats, and the Chromophyta tend t o be more common in the sea. The acoelus Turbellaria apparently harbour the only known symbiotic marine Chlorophyceae (Parke and Manton, 1967 ; Taylor, 1972). This symbiont has been identified as Platymonas convolutae Parke et Manton, a member of the Prasinophyceae (Parke and Manton, 1967). It has been collected from a t least four species of Convolutidae (Taylor, 1972), and is known t o occur commonly in a free-living state. As a group, species of Prasinophyceae, most notably those belonging t o the genera Platymonas, Prasinocladus and Tetraselmis, are compatible with a symbiotic condition although they do not occur naturally in associations with invertebrates (Provasoli et al., 1968). It seems reasonable t o assume that there are some morphologic or metabolic characteristics of this group which may account for their success with marine hosts.

B. Qualities of a suitable symbiont 1. Pre-adaptation

As a rule, symbiotic algae appear to differ very little from their free-living relations, a fact which complicates any assessment of their success as symbionts. Except for Platymonas convolutae, which achieves an exceptional structural and metabolic integration with its hosts, and Amphidinium klebsii which lacks apparent structural adaptations (Taylor, 1971b), most symbiotic algae exhibit a tendency towards a coccoid habit. All of these forms are, however, expressions of a high degree of morphological plasticity that is common to most free-living and symbiotic unicellular algae. Their existence in a given host should not be regarded as species specific (see Karakashian, S. J., 19701, and care should be exercised in assessing their significance. Among Cyanophyceae symbionts generally belong t o the Chroococcales although Anabaena (Nostocales) is an exception. Unlike their freshwater counterparts (Hall and Claus, 1963, 1966 ; Echlin, 1967), symbiotic marine Cyanophyceae have retained a thickened cell wall similar to that of free-living species (Sarii, 1971; Vacelet, 1971). Dinoflagellate symbionts tend t o assume a condition that is best described as encystment, a habit that is widespread in the class and

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could account for their successes as symbionts. Their life histories have parallels with those of free-living species (Freudenthal, 1962 ; Droop, 1963; Wall and Dale, 1967 ; Wall et al., 1967 ; Bibby and Dodge, 1972), and their encysted condition is therefore best regarded as a fortuitous pre-adaptation. The function of a thickened cyst wall has received little experimental study. Droop (1963) and Taylor (1968) postulate that in an intracellular situation its advantages may be protective, serving to isolate the alga from the environment of the host cell thereby preventing digestion or outright poisoning. Studies of carbon translocation in symbiosis (Smith, D. et al., 1969; Taylor, 1969d; Trench, 1971a, b, c) suggest to the contrary that permeability barriers do not exist in situ or in in vitro experiments on freshly isolated symbionts. Selective permeability towards translocated algal photosynthate and essential host-supplied nutrients is a distinct possibility. The problem of regulation and complex systems of host-symbiont interactions related to these questions will be discussed below. Because the nutritional and biochemical properties of micro-algae in general are poorly understood, it is difficult to comment upon possible pre-adaptations to symbiosis in these areas. It seems likely, however, that the success of dinoflagellates in particular may rest heavily on some pre-existing metabolic characteristics which would serve to establish a nutritional compatability with the host. Similar properties may also explain the singular success of the Prasinophyceae in both natural and artificially established associations. Many algal species are not wholly autotrophic ; but are capable of either heterotrophic or photo-heterotrophic modes of nutrition, i.e., growth dependent upon or stimulated by the presence of organic substrates, the latter being light dependent. This property is most common among species isolated from nutrient-rich environments, a situation obviously analogous with the host cell. Successful growth of symbionts in the host milieu could be dependent upon or stimulated by key host-supplied compounds (see below, p. 32; Hutner et al., 1972). Similarly, excretion of polyols is a common property of unicellular algae (Hellebust, 1965), that has come to prominence in symbiotic associations. Among the associations reviewed by Smith, D. et al. (1969), excreted polyols are significant as the principal compounds translocated from symbiont to host (see below, p. 36). OF A FUNCTIONAL SYMBIOTIC UNIT IV. ESTABLISHMENT Basic cellular properties of permanent symbiotic associations may be examined in terms of their origins, mechanisms of inter- and intra-

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specific transmission of the established symbiosis and the accommodations required for perpetuation in the environment (i.e., the degree of integration). The latter also includes dependencies arising from the elimination of duplicate functions as the association evolves. A. Origins The origins of algal-invertebrate symbiosis are obscure. Most theories are based on inherent properties of the organisms which have already been noted here, i.e., the animal habit of phagotrophy, the inability of carnivorous hosts to digest plant material and the alga’s resist’ance to digestion (Droop, 1963 ; McLaughlin and Zahl, 1966). The role of the host is generally regarded as active, and that of the alga passive. There are apparently no records of an actively invasive algal symbiont, although pathological situations have been recorded, e.g. in Hydra spp. (Goetsch, 1924) and the Giant Scallop, Placopecten magellanicus (Gmelin) (Stevenson, 1972). Experimental proof of the theory is lacking. Nevertheless, there have been promising studies on the origins and hereditary aspects of symbiosis among freshwater organisms (Karakashian, M. W. and Karakashian, S. J., 1964; Karakashian, S. J. and Karakashian, M. W., 1965; Hirshon, 1969). The mystique of marine biology has unfortunately led to the widely held belief that marine examples of algal-invertebrate symbiosis are difficult ” experimental material. On the contrary, laboratory cultivation of Convoluta roscoffensis (Graff ), G . psammophida BeM., Amphiscolops langerhansi and Aiptasia sp. has opened the way for serious experimental study in this area (Provasoli et al., 1968; Dr. L. Provasoli, personal communication ; Taylor, 1971b). Axenic cultivation of C. roscoflensis at the Haskins Laboratories (Dr. L. Provasoli, personal communication) is a satisfying realization of one of Droop’s (1963) major projections. Preliminary studies on initiation and development of symbiosis in laboratory cultures of C. roscoffensis (Oschman, 1966) have prepared the way for a full-scale study of symbiotic origins in marine associations. Hopefully, comparative work with coelenterate hosts will follow. Examination of major forms of marine symbiosis (dinoflagellatecoelenterate) suggests that it is a comparatively old phenomenon in geological time. The probable age of associations based on the spectacularly successful hermatypic corals may date from the middle Triassic (Wells, 1956; cf. Woodhead and Weber, 1970). This could well be representative of coelenterate symbiosis as a whole. It is difficult to determine whether such a symbiosis arose once or several times during the evolution of coelenterates. Undoubtedly, the biology ((

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of potential hosts and their compatibility with a pioneering alga is as significant for success as the alga’s availability and symbiotic fitness. Host suitability alone could account for the absence of algal symbionts among ahermatypic corals. This assumes that hermatypic and ahermatypic species had already diverged prior t o the emergence of algal symbiosis as a potent evolutionary factor, and that the successful association in hermatypic corals only accentuated differences already in existence. The pandemic nature of Gymnodinium microadriaticum (Taylor, 1972), suggests that the spread of associations based on this alga was both rapid and extensive, encompassing all of the world’s oceans. I n this circumstance, it is likely that ahermatypic species were exposed t o possible “ infection ” a t the same time as their hermatypic relations yet they failed t o acquire the symbiosis. B. Transmission of symbionts Transmission of symbionts has as its goals the perpetuation and spread of the association within the same host species, and extended to other real or potential hosts of different species. It may be an evolved active process, a passive result of the host’s reproductive biology or a fortuitous accident of nature. 1. Intra-speci$c

(a) Non-cellular. For many hosts, perpetuation of t’he symbiosis is depcndmt upon re-infection of offspring at each successive generation. These include a number of casual or seasonally based symbioses, as well as examples of totally integrated and highly dependent symbioses such as Convoluta, and rely heavily 011 symbiont motility and availability. Many Protozoa transmit their symbionts in this fashion, although their principal means is via asexual reproduction. Noctiluca is a typical example of a seasonal or regionally based host (Sweeney, 197 1). The ectocominensal ciliate Urceolaria patella usually acquires its symbionts through phagocytosis, and later transmits them during reproduction (Taylor, 1972, unpublished). The unique symbiosis of Mesodinium rubrum (Lohmann) (Taylor et al., 1969, 1971) also appears to depend on host phagocytosis for its perpetuation. Further examples may be found in the review of Ball (1969). Among Porifera, where symbiosis tends t o be a more casual or precarious situation (for the alga), transmission relies heavily or almost exclusively, on phagocytosis (Sara, 1964a, b ; Sara and Liaci, 1964a, b). Coelenterates, on the other hand, usually do not rely on this mechanism. Kinsey (1970) (see also Gohar, 1940, 1948; Theodor, 1969) reports

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13

that the planulae of the gorgonids Briareum asbestinum (Pallas) and Muriceopsis Jlavida (Lamarck) lack symbionts, and later acquire them through feeding on free-living stages of Gymnodinium microadriaticum. More precise experiments are required before this interesting possibility is confirmed (see below, p. 14). I n spite of the fact that G. microadriaticum produces motile stages in culture (Freudenthal, 1962 ; Taylor, 1972), and encysted stages are commonly excreted by coelenterate hosts (Taylor, 1969e), it has not been collected free-living in the environment during extensive searches by this author (cf. Kawaguti, 1944). I n this context it is of interest that other algal symbionts commonly occur outside their hosts (e'.g.,Parke and Manton, 1967; Taylor, 1971b). The age (in an evolutionary sense), and consequent degree of host-symbiont integration could be the controlling factor in these instances (see below, p. 16). Until the presence of G. microadriaticum is established in the environment with certainty, one must assume that the alga is not readily available for infection via host phagocytosis. The alternative, of secondary acquisition of symbionts through ingestion of Protozoa and plankton infected with the alga, could be an important vehicle of transmission among coelenterate hosts. This approach has proved to be highly effective in laboratory re-infections of the anemone Aiptasia in alga-free cultures, and stands in sharp contrast to the failure experienced when the same hosts were simply exposed to suspensions of symbionts in culture. Transmission in associations involving acoelus Turbellaria is based exclusively on re-infection of alga-free larvae following hatching of the egg (Ax and Apelt, 1965; Provasoli et al., 1968 ; Apelt, 1969a, b ; Ax, 1970; Taylor, 1971b). At first sight, it is surprising that such a casual method of transmission has persisted, particularly when the survival of a species like Convoluta roscoffensis is a t stake. This apparent randomness has been partially overcome through the evolution of precise chemical and behavioural clues. Platymonas convolutae is attracted to the egg cases laid by Convoluta roscoffensis and C. psammophila, and tends to remain attached for a considerable period after hatching. Since newly hatched larvae remain near their discarded egg cases, and feed voraceously during the first 3-7 days, re-infection is all but guaranteed for the species (Keeble, 1910). Synchronization of host reproduction with lunar cycles provides further assistance by ensuring favourable tidal conditions (Gamble and Keeble, 1904 ; Keeble and Gamble, 1907 ; Keeble, 1910), and the psammophilic habit of P. convolutae (Parke and Manton, 1967) contributes favourably to symbiont availability in the vicinity of the animals habitat. The same principles apply to the less well integrated symbioses of Convoluta

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convoluta (Keeble, 1908, 1910; Ax and Apelt, 1965; Apelt, 1969a), and Amphiscolops langerhansi (Taylor, 197 lb). Inadequate knowledge of the life histories of the tridacnids makes analysis of symbiont transmission impossible. Presumably, it is cellular, i.e. eggs carry the symbiont. However, an uninfected veliger stage is a distinct possibility, and new studies are required before proper assessments can be made. I n the case of associations between chloroplasts and species of Sacoglossa, each new generation is reinfected through feeding on the host plant (Taylor, 1967, 1968b, 1970; Trench, 1969; Trench et al., 1969; Greene, 1970 a,b,c). (b) Cellular. Sexually reproducing organisms typically transmit symbionts either directly via the egg or indirectly prior to the release of offspring (e.g., as in coral planulae, Marshall, 1932). Studies of coelenterate reproductive biology provide the best examples of sexual transmission of algal symbionts between host gznerations. Indeed, it appears as if the coelenterates are the only major group that has utilized this option. Possibly, studies of tridacnid reproduction and development will alter this view. Direct entry of zooxanthellae into the eggs of Millepora has been recorded by Mangan (1909) (see also literature cited by Droop, 1963). Similar studies on hermatypic corals (Atoda, 1951), the hydroids Myrionema amboinense (Fraser, 1931) and Aglaophenia pluma (L.) (Faure, 1960) and the chondrophore Velella velella (Kuskop, 1921 ; Brinkmann, 1964) serve to confirm these observations. Promising work on the scyphozoan Stephanoscyphus (Werner, 1970) should eventually lead t o a detailed understanding of reproduction and symbiont transmission in this genus. I n all of these instances, early stages of egg development are alga-free ; however, a t some time prior t o fertilization, symbionts pass from the surrounding gonadal tissues and enter the egg (e.g. in Millepora). Precise details of the transmission are not available. Some doubt exists as to whether sexual transmission of symbionts occurs in the octocorals. Studies of xeniids suggest the absence of algae in early planular stages (Gohar, 1940, 1948). Similarly, work on Eunicella, Briarium and Nuriceopsis (Theodor, 1969 ;Kinsey, 1970) failed t o reveal the presence of symbionts in newly released planulae, Planulae maintained in pasteurized, sterile seawater did not develop symbionts, while those kept in running seawater tanks eventually became infected (Kinsey, 1970). Despite these observations, there are no apparent reasons why octocorals should not follow the typical coelenterate patterns. Clearly, more critical studies are required. Close attention must be paid to essential symbiont growth factors that cannot be supplied by the host. Pasteurized

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seawater that has been heated t o boiling may be deficient in key nutrients or cofactors and this would inhibit the development of an " embryo " symbiont population carried by the planula. Asexual reproduction is the rule for the vast majority of symbiotic hosts. As a simple cellular mechanism, it provides an effective means of partitioning symbionts among successive generations, and ensures the perpetuation of the association in a way that is unequalled by other methods. Symbiont transmission of this nature recalls the concept of " plasmids " (Lederberg, 1952 ; Karakashian, S. J . and Siegel, 196.5)) and has prompted numerous theoretical reviews on the symbiotic origins of csllular organelles (Sagan, 1967 ; Taylor, 1970 ; Stainer, 1970). This matter will be discussed further when the question of host-symbiont adaptation is considered below. Early literature on the asexual transmission of symbionts is contained in Droop (1963) and McLaughlin and Zahl (1966). The process is a familiar one. Recent studies of particular interest include work on symbiont effects on strobilation in the scyphozoan coelenterate Cassiopea andromeda Esch. (Ludwig, 1969) and a study of the role of architomy in the acoel Amphiscolops Eangerhansi (Hanson, 1960 ; Taylor, 1971b). A useful discussion of architomy is given by Marcus and Macnae (1 954). 2. Inter-specijic

Transmission of symbionts between hosts belonging t o different species depends heavily on the production and availability of motile or free-living stages in the symbiont's life history. To date, interspecific transmission of symbionts by this mechanism has not been examined. The apparent absence of free-living G'ymnodinium microadriaticum in the marine environment suggests that such a mechanism has little current value, although it may have been historically important in the initial spread of symbiosis based on this ubiquitous alga. Ingestion of food organisms harbouring symbiotic algae could be a more likely means of inter-specific transmission. The successful introduction of algal symbionts into cultured Aiptasia sp. using intermediary hosts or temporary carriers (i,e., Artemia or liver injected with suspensions of cultured algae) (Taylor, unpublished), suggests that this may have been an important mechanism for the introduction and spread of symbiosis among coelenterate hosts. Predation on infected Protozoa, planulae or other zooplankton is an obvious source of potentially compatable symbionts. Recent observations on interspecific aggression among corals (Lang, 1970, 1973) provides still another vehicle for symbiont transfer between hosts. Such predation

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between species may have contributed to the rapid spread of symbiotic algae among hermatypic corals. Undoubtedly, predatory acquisition of symbiotic algae is an effective and powerful mechanism for the perpetuation and transmission of established symbioses in large communities and organismic assemblages (e.g. coral reef communities),

C. Host-symbiont specijicities Precise taxonomic recognition of individual genera and species of symbiotic algae has provided the necessary tools for a serious investigation of the preferences which hosts and algae exhibit in their union with a suitable partner (Parke and Manton, 1967 ; Kevin et al., 1969 ; Taylor, 1969a, b, c, 1971b, c, 1972; SarB, 1971; Vacelet, 1971). Experimental studies in this area are still in their infancy, and centre on associations found exclusively in acoel turbellarians. Similar principles should apply to symbioses involving protozoan, sponge and coelenterate hosts, although these have not been examined in this context. Host-symbiont preferences have been studied extensively in Convoluta roscoffensis (Provasoli et al., 1968), and to a lesser degree in C. psammophila (L. Provasoli, personal communication) and Amphiscolops langerhansi (Taylor, 1971b). All of these hosts are currently maintained in laboratory culture, and their symbionts and potential symbionts (both symbiotic and free-living) are similarly available. Several genera and species of free-living Prasinophyceae occur in the intertidal habitat of Convoluta roscoffensis (Parke and Manton, 1967). During the early larval stages all of these can be ingested, and at some point a selective process takes place which ensures that the correct symbiont (Platymonas convolutae) is established before the host stops feeding and becomes totally dependent on its algae for survival. The mechanics of this have been examined in the laboratory in studies by Provasoli et al. (1968). They report that successful symbioses may be established with most of the species of Prasinophyceae available in the host’s native environment (e.g., Platymonas convolutae, Platymonas sp. (Plymouth 315), Prasinocladus marinus (Cienk.) Waern, Tetraselmis verrucosa Butch, etc.). Using growth, time to maturity and reproductive rate (i.e. eggs laid) as criteria, a spectrum of symbiotic successes were demonstrated. Associations based on Prasinocladus marinus proved to be equal to and frequently better than natural controls, those based on Tetraselmis verrucosa were generally poorer, and those involving Platymonas sp. (Plymouth 315) were extremely poor and frequently unsuccessful. Subsequent studies of photosynthesis in these same associations show parallel results (Nozawa et al., 1972). Preferences for a particular symbiont species were investigated in a series of

FIG.2 . Ultrastructure of Platymonas convolutae in symbiosis with Convoluta roscoffensis, illustrating the plasticity and structural conformation of the natural symbiont. Nucleus (N), pyrenoid (P), chloroplast (CP). Unpublished micrograph, courtesy I. Manton. x 6900.

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DENNIS L. TAYLOR

FIQ.3. Ultrastructure of Prasinocladus marinus in symbiosis with Convoluta roscoffensis, showing the absence of cellular plasticity and conformation with the host. From Provasoli et al. (1968). x 9000.

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experiments involving competition between algae. When mixed cultures of natural and unnatural symbionts were presented to a single host, only associations with the natural symbiont were found. Similarly, when hosts infected previously with unnatural symbionts were presented with cultures of P. convolutae, the established symbiosis was rejected in favour of a new one based on the natural symbiont. Other studies involving varying combinations of the different Prasinophyceae noted, establish a definite " pecking order '' for the various natural and unnatural symbionts. These observations have been confirmed in a second acoel, Amphiscolops langerhansi, whose potential symbionts are drawn exclusively from the Dinophyceae (Taylor, 197 1b). The principal criteria for host-symbiont preferences of this nature would appear to be based on structural, physiological and biochemical compatibilities. Structural compatibility is particularly apparent in Convoluta roscoffensis. Natural syrpbionta produce elahorate interdigitating processes to forin a mu& more intimate cellular contact w&h their host than do unnatural symbionts (Oschman and Grey, 1965; Parke and Manton, 1967 ; Provasoli et al., 1968) (Figs 2 and 3). Replacement of Prasinocladzcs marinus by Platymonas convolutae in competitive experiments may be partially explained on this basis (Provasoli et al., 1968; Nozawa et al., 1952). The fact that hosts infected with Prasinoclaadus marinus actually grow and reproduce at faster rates than controls with natural symbionts (Provasoli et al., 1968), suggests that structural compatibility is an important factor for stability and ultimate symbiotic success in Convoluta roscoffensis. The same may be true for Convoluta convoluta (Ax and Apelt, 1965). In Amphiscolops langerhansi, intimacy of contact and structural compatability are apparently not obvious factors in the selection of symbionts (Taylor, 1971b) (Fig. 4). Physiological and biochemical factors effecting symbiont selection are insufficiently studied to permit thoughtful comment. Examination of photosynthetic O2 production in Convoluta roscoffensis fails to elaborate any sound principles based on this criterion, although it does point out some of the inherent problems of photosynthetic studies of symbiotic associations (see below, p. 30; Nozawa et al., 1972). One hopes that an elaboration of symbiont photosynthetic pathways and intermediary metabolism may provide some useful guiding principles. At the same time, the role of subtle and poorly studied nutritional factors (vitamins, cofactors, tracemetals, etc.) should not be overlooked in the quest to follow carbon. They may be the most significant factors in the final analysis. Parallel studies of host nutrition and excreted

FIG.4 . Ultrastructure of Arnphidinium klebsii in symbiosis with Amphiscolops langerCell is not structurally altered by the symbiotic condition. Epicone (E), ha&. Hypone (H). From Taylor (1971b). x 8500.

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metabolites will also be required. Hopefully these investigations will expand the foundation of data already available for the major symbiont phyla (Florkin and Scheer, 1967, 1968). Photosynthetic carbon fixation by algal symbionts is an ideal means of introducing label into animal metabolic pathways. Its full potential in studies of animal nutrition has not been realized.

D. Adaptations Once established, symbiotic associations tend to undergo integrative processes (post-adaptations), which eventually bring about increased behavioural, structural and functional (i.e., biochemical-physiological) compatabilities. These adaptations are in effect, a manifestation of the tendency for natural, multi-organismal systems to stabilize, and increase their efficiency within a given frame of time and space (Iberall, 1972). Within highly evolved associations, the price of increased stability and eEciency is increased dependence on the survival of the functional unit (i.e., the symbiosis). 1. Behavioural adaptations

The response of intact symbioses to the presence and direction of light is universal, regardless of the host’s dependence on the photosynthetic carbon fixation of its algal symbionts. Without exception, the known behavioural adaptations of host species can be traced to this single source, although it may be mediated through a response to an internal oxygen gradient (Stanier and Cohen-Bazire, 1957 ; Droop, 1963). Loss of the symbiont almost always results in loss of the behavioural response (McLaughlin and Zahl, 1969). Among freshwater associations, the role of light and its effects on host-symbiont interaction has received review in the work of Lytle et al. (1971). Marine associations are, by comparison, poorly studied in this respect. The work of Gohar (1963) presents a contemporary view of the problem. Behavioural adaptations mediated by light or an accompanying photosynthetic oxygen gradient may be divided roughly into two categories, (a) directive responses of individual hosts and (b) habitat selection related t o light requirements.

(a) Directive responses. Algal symbionts in culture are known to exhibit strong phototactic responses. These may be reflected in the patterns of growth observed with encysted symbionts, and are clearly dependent upon the strong phototaxis of motile stages. Cultures of cu~ marine Protozoa infected with ~ m n o a ~ n mi ~u c~r o a d r ~ a t ~exhibit

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DENNIS L. TAYLOR

similar rasponses to light. This is particularly true of the ciliates Urceolaria patella and Paraeuplotes tortugensis which rapidly collect on the lighted sides of culture dishes and glass slides. The anemone, Anemonia sulcata (Pennant), has been widely studied as an example of algal-coelenterate symbiosis (e.g. Taylor, 1968a). Its response to light is typical of anemones in general and consists of a direct posturing of the animal’s body and tentacles in the direction of, and parallel with ambient light. The same phenomenon has been observed widely in tropical and temperate species (McLaughlin and Zahl, 1959 ; Yonge, 1963). I n contrast, corals are generally believed to exhibit a negative phototaxis with respect to posture. It is widely (though incorrectly) held that polyps expand only a t night. Use of S.C.U.B.A. for reef exploration in recent years has clearly shown that this is definitely not the case. The primary stimulus for polyp expansion is the presence of food, however, not light (Goreau et al., 1971 ; Muscatine, 1973). Overall growth form and orientation of entire colonies is, nevertheless, directly attributable to light (Yonge, 1963; Goreau, 1963; Barnes, 1970, 1973). Studies of other coelenterate groups show varied responses. For example, the octocorals appear to be more like anemones in their response to directional illumination (Gohar, 1948 ; Kinsey, 1970). Light oriented behaviour of the acoels Convoluta roscoffensis and Convoluta convoluta is complex, involving diurnal and tidal variations in response to shifting environmental conditions. The principal aspects of this behaviour are summarized by Keeble (1910), who illustrates the survival value of simple light-dependent responses. Details of lightoriented ecological preferences and responses of individuals may also be found in studies by Guerin (1960) and Fraenkel (1961). I n nature, animals optimize available illumination in preferentially selected habitats, through patterns of body posture and the construction of mucus lined “ reflectors ” in the sandy substrate. Similar observations are recorded for molluscan-chloroplast symbiosis (Fraenkel, 1927 ; Kawaguti, 1 9 4 l a ; Kawaguti and Yamasu, 1965; Kawaguti et al., 1965). The sea slug, Elysia viridis (Montagu), orients to a directional light source through the use of its photoreceptors (Fraenkel, 1927). This may be the rule for Sacoglossa. The role of oxygen gradients has not been examined, although ‘‘ organelle ” symbiosis of this type could rely heavily on this mechanism. Species of Tridacnidae also exhibit positive responses to illumination (Yonge, 1936, 1953 ; Kawaguti, 1966). In the genus Tridacna the alga-rich siphonal tissues are expanded out over the partially opened valves. The single species of Hippopus differs slightly in that expansion of siphonal tissues is not as great. The valves are opened to a greater extent, however, and the same

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purpose is accomplished. The Pacific heart shell Corculum cardissa (Linnk) exposes its algal inhabited tissues through the translucent shell (Kawaguti, 1968). ( b ) Habitat selection. For sedentary hosts harbouring algal symbionts, habitat selection is governed principally by phototactic behaviour of free-swimming larval stages. This aspect of host biology has been well studied in the hermatypic corals (see reviews of Droop, 1963; Yonge, 1963 ; McLaughlin and Zahl, 1966). The general principles defined there may be expected to hold true for other sedentary hosts such as the Tridacnidae. Particular note should be made of important studies on coral planulae and settlement behaviour by Marshall (1932), Kawaguti (1941b) and Atoda (1951). Recent work on laboratory rearing of coral planulae and newly settled polyps (Reed, 1971), and skeleto-genesis of newly settled polyps (Vandermeulen and Watabe, 1973) provide a useful basis for further experimental studies in this area. Once settlement has taken place, the quality, quantity and direction of available light exerts profound influence over the rate of growth and general form of the developing hermatypic coral (Goreau and Goreau, 1959; Yonge, 1963; Goreau, 1963; Barnes, 1970, 1973). This factor alone can account for the variability of colony morphology observed with increasing depth, e.g. in some species hemispherical growth forms predominate in shallow water. These progress towards flat plates with increasing depth (Goreau, 1963 ; Barnes, 1973), and light also limits the ultimate depth penetration of hermatypic corals in general. Alga-free ahermatypic corals are not restricted in this same fashion (Droop, 1963 ; Yonge, 1963 ; Stoddart and Yonge, 1971). Mobile hosts are capable of shifting positions to optimize available light intensities. This type of behaviour is seen commonly in anemones such as Anemonia sulcata (Taylor, unpublished) and Condylactis sp. (McLaughlin and Zahl, 1959) and is also known in the acoel Convoluta roscoflensis (Guerin, 1960 ; Fraenkel, 1961). It is complementary to, and part of the general posturing of host species discussed above. 2. ~ t r u c t u ~ a da a~~ t a t i o n ~

Both hosts and symbionts exhibit observable structural modifications resulting from the symbiosis which reinforce its integrative cellular aspects. Extreme situations typified by the alga Platymonm convolutae or the Tridacnidae are well studied, and provide instructive examples of how structural changes can serve the cellular or organismic ends of a symbiosis. Oschman (1966) has studied the ultrastructure of cellular accommodation in P. convolutae during the period when it becomes

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DENNIS L. TAYLOR

established in the tissues of Convoluta roscoffensis. Supplementary observations may also be found in the work of Parke and Manton (1967). The ultimate success of a symbiosis in C. roscoffensis depends to a large extent upon the degree of intimate cell contact that is achieved between host and alga (Fig. 2, p. 19). Platymonas convolutae satisfies this requirement during the period of infection, by undergoing a transformation from a motile, quadriflagellate unicell possessing an eyespot and cell wall, to become a naked protoplast without eyespot or flagella. In this state it is capable of achieving the required degree of cell to cell contact with the host and, in the case of natural symbionts, complex interdigitating cell processes can be formed (Fig. 2, p. 19) (Oschman, 1966). It is of interest to note that artificial symbionts employed in competitive studies also undergo this same structural modification (Provasoli et al., 1968). In contrast, Amphidinium klebsii is completely lacking in any structural modification when symbiotic with Amphiscolops Zangerhansi (Taylor, 1971b) (Fig. 4, p. 19) ; and as noted previously, the encysted state of Gymnodinium microadriaticum does not appear to be the result of symbiotic interaction (Fig. 5 ) . Symbiosis among acoels such as Convoluta roscoffensis is unique in that the symbionts are situated intercellularly, not intracellularly as with other invertebrate hosts (e.g., Coelenterates, Fig. 5; cf. Kawaguti, 1964). The outstanding success of the symbiosis in C. roscoffensis may have resulted from the achievement of close cellular contact mediated by the establishment of symbionts in protoplast form. Similar forms of cell wall reduction have been reported for the symbionts of Convoluta psammophila (Sarfatti and Bedini, 1965), Cyanophora paradoxa Korschikoff (Hall and Claus, 1963), Glaucocystis nostochinearum Itzigsohn (Hall and Claus, 1967 ; Echlin, 1967), Paramecium bursaria Focke (Karakashian, s.J., 1970), Hydra viridis (Park et al., 1967) and the diatom genus Rhopalodia (Drum and Pankratz, 1965).

Within the range of marine hosts, the most outstanding examples of structural modification resulting from associations with algae are found among the Tridacnidae. Yonge (1936,1953) provides a complete description of structural adaptations (anatomical alterations, light filtering pigments, light concentrating organs), based on the host’s need to “ house ” its symbionts (see also Kawaguti, 1966; Taylor, 1969a; Fankboner, 1971 ; Goreau et al., 1973). Comparison of tridacnid organization with that of their Cardium-like relations clearly indicates just how extensive these modifications have been (Yonge, 1936, 1953). In the genus Tridacna it is most apparent, being somewhat less evident in Hippopus, and still less evident in Corculum (Kawaguti,

FIQ.5. Ultrastructure of Gymnodiniurn microadriaticum in symbiosis with Montastrea annularis. Accumulation body (A). Unpublished micrograph, D. L. Taylor. x 9000.

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DENNIS L. TAYLOR

1968). I n all species, the primary function of these structural modifications is t o facilitate optimum exposure of the algae to ambient illumination. Pigmentation of mantle tissues may serve a dual function, acting to reduce excessive light intensity and to alter light quality. The influence of light quality on the character of symbiont photosynthate will be discussed below (also p. 31). Hermatypic corals provide another striking example of structural modification in response to a symbiosis. There is little doubt that their successful evolution, like that of the Tridacnidae, has been largely dependent upon associations with algae (Yonge, 1960, 1963, 1968). Nevertheless, structural elaboration of reef-building corals can be regarded as a “ secondary attribute ”, arising from possible increases in metabolic efficiency and high rates of calcification imposed on the animal by the presence of the alga. It does not obviously function in the service of the symbiosis, as does the migration and elaboration of siphonal tissues in the Tridacnidae. The concept that structural modification of hermatypic corals results from an increase in efficiency of excretory pathways, provided by the symbionts (the “renal function ” of Geddes, 1882), has a long history. It has recently been discussed at length by Woodhead and Weber (1970). Such a mechanism may have some value. However, removal of wastes from the simple cellular system of coelenterates (even when organized in complex colonial forms) in an aquatic environment seems a minor problem (see Muscatine, 1973). More significant contributing factors would appear to be the provision of a stable supplementary source of nutrients in a form that is rapidly assimilated, and enhancement of basal rates of calcification. Together, these would contribute substantially towards the structural complexity seen in modern reef building corals, and serve to explain their comparatively rapid evolution. Further access to pertinent literature may be found in recent reviews (Goreau, 1963 ; Yonge, 1968 ; Woodhead and Weber, 1970; Muscatine, 1971, 1973; Barnes, 1973). Pigmentation of host species is widespread among coelenterates and the Tridacnidae (noted above). Where light intensity is excessive, animal pigments serve to protect the algal symbionts and optimize conditions for photosynthesis. They may also affect light quality (spectral composition), and in this way influence the character of symbiont photosynthate (see below, p. 31). It is of interest that these pigments generally serve to eliminate shorter wavelengths, and transmit blue light. The effect which the presence or absence of pigmentation has on the distribution with depth of a species is well known. Pigmented varieties of Anemonia sulcata are found high on the shore, in shallow rock pools exposed to full sunlight. Unpigmented forms

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27

(brown in colour due to the algal symbionts) occur further down in shaded situations (Taylor, unpublished). Kawaguti ( 1 937) has made similar observations on the effect of light on the colour and form of reef-building corals. Pigmented varieties of Indo-Pacific corals appear to be more abundant than those found in Atlantic reefs. This may account for the success which the former have had in shallow situations of high light intensity. Conversely, absence of pigmentation could favour distributions to greater depths as seen in Atlantic reefs (Goreau and Wells, 1967). 3. Functional adaptations

The biochemical, physiological and genetic adaptations of hosts and symbionts have not been studied in proportion to their significance as indicators of cellular integration. Full understanding of these facets of a symbiosis is relevant to the theoretical concept of the plasmid (Lederberg, 1952), and provides an understanding of the evolutionary potential inherent in the intimate cellular contacts of phylogenetically distinct individuals. Recent interest in the evolutionary aspects of symbiosis has given rise to several reviews (e.g., Sagan, 1967 ; Allsopp, 1969; Stanier, 1970; Taylor, 1970), and at least one important book which explores the subject in depth (Margulis, 1970). Among marine symbioses, functional adaptations are usually manifested through nutritional or nutritionally related dependencies. There are apparently no known genetically integrated equivalents of Cyanophora paradoxa (Korschikov, 1924 ; Hall and Claus, 1963) or Paramecium bursaria (Siegel, 1960; Karakashian, S. J., 1963; Karakashian, M. W. and Karakashian, S. J., 1964), although undoubtedly these could exist. Nutritional interdependencies arise through complete elimination of duplicate metabolic pathways, or the sharing of complementary portions of the same pathway. Such adaptations find expression in a range of symbioses progressing from obligate to facultative dependence on the survival of the functional unit. I n general, hosts sacrifice nutritional independence in favour of the symbiosis, a possible consequence of inherent reliance on autotrophic organisms for nutritional input. Symbiotic marine algae can be isolated and grown in standard media used for free-living species (Taylor, 1972), and appear to have no obligate dependence on an association with a host. Even species such as Gymnodinium microadriaticum, which have long histories as symbionts, can be grown autotrophically in mineral media supplemented with vitamins B,, and thiamine (standard growth requirements of free-living species). I n contrast, some algal species symbiotic with freshwater hosts are known t o have undefined nutritional and genetic

28

DENNIS L. TAYLOR

dependencies, and have not been cultivated separately (e.g. Chlorella sp. symbiotic with Hydra viridis and Cyanocyta korschikofiana symbiotic with Cyanophora paradoza). Convoluta roscoffensis has an obligate dependence on its symbiosis with Platymonas convolutae, that is based on a loss of the animal’s capacity for phagotrophy and apparent reliance on the alga for essential sources of nutrients and energy (Geddes, 1878, 1880 ; Gamble and Keeble, 1904 ; Keeble and Gamble, 1907 ; Keeble, 1910 ; Parke and Manton, 1967; Provasoli et al., 1968; Provasoli et al., 1969; Taylor, 197la). The possibility that dissolved organic compounds, trace minerals and vitamins present in the surrounding milieu, may supplement the host’s nutritional requirements, cannot be entirely excluded a t this time. Extended studies of the symbiosis maintained in sequential laboratory cultures suggest that this is not an important source ; since the intact association can be grown autotrophically in a defined mineral media without organic supplements (Provasoli et al.) 1968). This view is further reinforced by the successful cultivation of Convoluta axenically (L. Provasoli, personal communication). The biochemical pathways underlying the host’s extreme nutritional adaptation t o the symbiosis have not been studied t o date. Recent work on the nutrition and intermediary metabolism of Platymonas convolutae in culture (Gooday, 1970; Taylor, 1971a), and of the intact association (Taylor, 1971a), provide some preliminary information on the kinds of materials that the symbiont is supplying the host (e.g. alanine, glucose, fructose, mannitol and lactic acid). This is, however, a long way from understanding the full nature of the alga’s nutritional input, its regulation and the way the host’s metabolism is dependent upon it. Comparative studies of other acoelus hosts exhibiting a full spectruni of nutritional integration may provide some useful insights. Recent observations suggest that fecundity may be a good index of successful nutritional balance between endogenous (symbiont derived) and exogenous food sources. Convoluta psammophila, also symbiotic with Platymonas convolutae (M. Parke, personal communication), is believed t o feed during its life in nature ; but will survive sequential laboratory culture in mineral media without exogenous food (Provasoli et al., 1969). Growth and reproduction of the animal are not optimum under these conditions, and successive generations progressively lose fecundity until reproduction ceases. Feeding hosts with suitably sized ciliates and copepods collected from natural populations will alleviate this reproductive. loss (Taylor, unpublished), suggesting that Platymonas convolutae is unable t o supply trace amounts of a key nutrient required by the host. This problem is unknown with Convoluta roscoffensis,

INTERACTIONS O F ALGAL-INVERTEBRATE SYMBIOSIS

29

Amphiscolops langerhansi feeds voraciously throughout its life (Hyman, 1937, 1939 ; Taylor, 1971b). Its symbiosis with Amphidinium klebsii is closely analogous to the associations found among coelenterates (e.g. corals). The alga’s contribution is part of a multi-faceted nutritional input that includes both endogenous and exogenous sources (soluble and particulate organic compounds, vitamins, trace elements, zooplankton, etc.). A recent study of this species in culture has shown that hosts reared to adult size without their symbionts, or with certain artificial symbionts, fail t o achieve sexual maturity despite the fact that they have substantial supplies of exogenous food (Taylor, 1971b). Apparently a key growth factor for Amphiscolops can only be supplied by the alga. Despite the apparent lack of structural adaptation (see above), and the broadly based nutritional sources of this animal, functional accommodations have evolved which make the symbiosis obligate for the survival of the species. Similar, subtle interdependencies may eventually be elucidated in other types of associations, where their analysis might serve t o broaden our understanding of the functional adaptations that evolve from the cellular associations of algae and invertebrates. Examples of a single metabolic pathway shared by a host and symbiont are not common. Frequently, these involve the production of biochemically exotic compounds, and are best known from studies of terpenoid biosynthesis among species of gorgonians (Weinheimer et al., 1967; Weinheimer et al., 1968). Papastephanou (1972) has demonstrated the role of symbiotic algae in the synthesis of crassin acetate, and it seems likely that this will prove to be a normal condition. Similar shared pathways may also function in the biosynthesis of prostaglandins produced by species such as the commercially important Plexaura homomalla (Esper), although their existence has not been demonstrated.

V. NUTRITIONOP THE FUNCTIONAL UNIT Inclusion of primary producers and consumers in the same functional unit enhances metabolic efficiences in symbiotic associations, and makes the study of their nutrition and maintenance of special interest. At the cellular level, fundamental aspects of intercellular exchange (e.g. control of the quantity and quality of excreted metabolites, regulation of rates of translocation, etc.), cellular tolerances and the regulation of cell growth can be examined. Serious investigations of these problems have concentrated primarily on the question of carbon translocation in in vitro systems (e.g., Smith, D. et al., 1969). These give a necessarily abstract, but useful view of potentially

30

DENNIS L. TAYLOE

important nutrients and the mechanisms which control their quantity, quality and movement. Efforts in this area have produced at least two important reviews which explore the available data and significant problems (Smith, D., et al., 1969; Muscatine, 1973). In order to avoid unnecessary duplications, these works will be cited freely below. It is the purpose of this section to explore the nutritional aspects of symbiosis, examining potential sources, internal exchanges, avenues of utilization and resulting growth, including its regulation.

A. Xources Emphasis on the significance of individual nutrient and energy sources is a traditional focus for extreme and controversial views. This is particularly true of studies dealing with algal-coelenterate associations, notably the reef-building corals (see Droop, 1963; Yonge, 1963, 1968 ; McLaughlin and Zahl, 1966 ; Muscatine, 1973), and relates to the relative dependence or independence which hosts exhibit towards the metabolites excreted by their algae. This relationship will vary somewhat with each association. Most evidence suggests, however, that the nutritional foundations of any symbiosis are many. Each individual source must be assessed in terms of the total input of all available sources, a view recently supported by Goreau et al., (1971). The ability of hosts and symbionts to utilize various nutritional inputs is also an important and related consideration (Sorokin, 1972). 1. Algal

( a ) Photosynthesis. Studies on the growth of symbionts in culture show that photosynthesis can provide the total nutritional and energy requirements of the alga (Craigie et al., 1966; McLaughlin and Zahl, 1966; Gooday, 1970; Taylor, 1971a). Symbiotic algae are similar t o their free-living relations in this respect. Photosynthesis is also a major source of nutrients for the host species. The path of carbon in photosynthesis is generally assumed to follow that proposed by Bassham and Calvin (1957) (Bassham, 1962), although there is no experimental evidence to support this, and comparative data on free-living marine spscies are lacking. The possibility that the recently proposed HatchSlack pathway (Hatch and Slack, 1966; Dagley and Nicholson, 1970) might function in symbiotic, as well as free-living micro-algae, makes investigation of this point an important priority. Physiological aspects of symbiont photosynthesis in vivo and in vitro have been examined in detail employing polarographic and chemical methods (Burkholder and Burkholder, 1960; Kanwisher and Wainwright, 1967 ; Roffman, 1968 ; Halldal, 1968 ; Franzisket, 1969a ;

INTERACTIONS OF ALOAL-INVERTEBRATE SYMBIOSIS

31

Nozawa et al., 1972). Despite wide differences in hosts and symbionts employed in these studies most observations are remarkably uniform. Saturating light intensities are reached around 3000 foot candles, and the symbiotic unit reaches a compensation point at or about 300-500 foot candles. Such profound similarities bring the methods into question. It seems legitimate to ask whether they are real or merely artifacts brought about by the techniques themselves when combined with the complexities of respiration and photosynthesis in an intact symbiosis. Internal compensations and physiological mechanisms which balance algal photosynthesis against algal photorespiration and animal respiration would go undetected by these methods. Photosynthetic products differ little from those encountered among free-living species, although quantities and qualities do vary in symbionts freshly isolated from their hosts (Trench, 1971~).This is probably due to host factors which control the quality and quantity of translocation in the symbiosis. Cultured symbionts do not exhibit these same traits (Gooday, 1970; Taylor, 1972). Trench (1971 a,b,c) has studied photosynthetic products produced in algal-coelenterate associations involving Gymnodinium microadriaticum. Principal products believed to be important to the symbiosis include glycerol, glucose, alanine, lipids, organic acids and organic phosphates. Similar studies of Platymonas convolutae reveal mannitol, fructose, glucose, lactic acid, amino acids, lipids and organic acids as the principal excreted products (Craigie et al., 1966; Gooday, 1970; Taylor, 1971a, unpublished). Some significance has been attached to the production and excretion of polyols by symbiotic algae (Smith, D. et al., 1969), although their production is common among free-living species (Hellebust, 1965). Lewis and Smith (1971) have noted the importance of alanine translocation in algal-coelenterate associations as a possible mechanism of nitrogen conservation. Successful nutritional balance depends substantially on the quality of the photosynthate produced. Studies of free-living micro-algae demonstrate the effects that variations in spectral composition may have on the major distribution of compounds synthesized by natural and cultivated populations (Hauschild et al., 1962a, b ; Hess and Tolbert, 1967 ; Wallen and Geen, 1971 a,b,c). As spectral composition is shifted in favour of shorter wavelengths, there is an enhancement of protein synthesis relative to carbohydrates. Ethanol soluble fractions of cells grown in green light or bluealight contain higher proportions of incorporated 14C in alanine, serine, aspartic, glutamic, fumaric and malic acids (Wallen and Geen, 1971a). Blue light also enhances basal rates of photosynthesis and could favour the Hatch-Slack (Hatch and

32

DENNIS L. TAYLOR

Slack, 1966) pathway of CO, fixation (Wallen and Geen, 1971a). I n contrast, white light favours carbohydrate production. Most natural symbiotic associations tend to favour conditions where shorter wavelengths of light prevail. Shallow water intertidal species of Convolutidae are exceptions. I n situations of full exposure, this may be accomplished through the filtering properties of host accessory pigments such as those found among coelenterates and tridacnids (see above, p. 26). Species living a t greater depths would normally be exposed to blue light, as a result of the natural absorptive properties of water. Assessments of nutritional relationships in these circumstances must consider spectral quality as an important factor in the determination of nutrient quality. ( b ) Heterotrophy and photo-assimilation. Growth of free-living algal species is commonly stimulated by the presence of organic substrates (Hutner et al., 1972). Frequently, this is dependent upon the presence TABLEI A . klebsii P. convolutae B. microadriaticum Acetate Lactate Succinate Pyruvate Glycerol Glucose

+ + + + + +

+ +-

+ + + + +-

P. marinus

+ +-

-

T . verrucosa -

-

-

of light, i.e., photoassimilation, photoheterotrophy. Symbiotic species are generally believed to be strict autotrophs (Droop, 1963 ; McLaughlin and Zahl, 1966), an unusual view if one considers that heterotrophy is specially characteristic of species living in nutrient-rich environments. Recent studies of symbionts in culture employing the radioisotope technique of Parsons and Strickland (1962) show that a wide range of compounds can be assimilated by algal symbionts (Table I) (D. L. Taylor, unpublished). Studies of their utilization by the algae are currently in progress. The intermediary metabolism of Amphidinium klebsii has been partially characterized by analysis of key TCA-cycle enzymes. Activities recorded are characteristic of species normally classed as strictly autotrophic (B. C. Chalker, unpublished). Nevertheless, this species can assimilate the organic substrates noted in Table I. Uptake is an inducible phenomenon. Heterotrophic modes of nutrition may serve several functions in the symbiosis. For example, they may serve to enhance overall rates of calcification by corals. Recent studies of calcification in 'coccolithophorids (Isenberg et al., 1965), show that

INTERACTIONS O F ALGAL-INVERTEBRATE SYMBIOSIS

33

heterotrophy has profound effects on calcification rates. Similar phenomena may exist with symbiosis involving reef-building corals. Other important nutrients, vitamins and trace metals may enter the symbiont via osmosis, diffusion or active transport. The requirement for vitamin B,, is almost universal among unicellular algae (Droop, 1961, 1969; Hutner et al., 1972). Symbiotic species are apparently no exception (McLaughlin and Zahl, 1966). Such requirements should be readily satisfied from the surrounding milieu. Experimental evidence suggests that nitrates and phosphates may be acquired either in the same fashion or as part of conservative mechanisms operating in the symbiosis (Gooday, 1970; Lewis and Smith, 1971; Simkiss, 1964a, b ; Pomeroy and Kuenzler, 1969). For some associations, removal of nitrates and phosphates may be an essential function of the alga that serves the need for removal of host wastes. Simkiss (1964a, b) cites removal of phosphates as a key factor in successful calcification in reef corals, and Gooday (1970) has noted the significance or uric acid utilization by Platymonas convolutae. ( c ) Digestion of the alga. Digestion of symbionts by hosts has frequently been postulated on the basis of algal fragments within animal cells (for historical literature, see Droop, 1963). Kawaguti (1965) illustrates degenerate symbionts within cells of the coral Oulastrea. Fankboner (1971) shows similar stages in the amebocytes of the digestive gland in tridacnids. Degenerate symbionts have been observed in studies of regulatory mechanisms in the symbiosis of Anemonia sulcata (Taylor, 1969e). These are interpreted as part of the normal regulation of symbiont numbers in this association, and host digestion is not involved. Digestive processes are dynamic cellular functions that cannot be adequately demonstrated in static cytological and cytochemical studies. Conclusive evidence favouring host digestion of symbionts should demonstrate the hydrolysis of algal substrates and their assimilation by the host (Muscatine, 1973). The near classic account of symbiont digestion in the symbiosis of Convoluta roscoffensis (Keeble, 1910 ; Droop, 1963) may appeal to one’s romantic sense, but conclusive proof is lacking and present evidence (obtained from work with laboratory cultures) shows that such an interpretation is quite incorrect. Digestion of the alga in symbiotic associations remains as a potential, but generally unproved nutrient source. 2. Animal (a)Phagotrophy. Ingestion of particulate food by the majority of symbiotic hosts is a significant nutrient source for the functional unit (cf. Johannes et al., 1970). Known exceptions include Convoluta

34

DENNIS L. TAYLOR

roscoffensis (Keeble, 1910) and possibly some xeniids and zoanthids (Gohar, 1940, 1948; von Holt and von Holt, 1968a, b ; Goreau et al., 1971) (cf. Muscatine, 1973). The role of phagotrophy in the nutrition and metabolism of Protozoa has been recently reviewed (Hutner et al., 1972). I n symbiotic systems its function is the same. Providing a variety of metabolites essential to host nutrition, and through digestion by the animal, a source of excreted metabolites which may support symbiont heterotrophy. Similar patterns may be expected to exist in other host phyla. The somewhat precarious symbiosis of Porifera are probably sustained to a large extent by host filter-feeding. Some insight into aspects of their nutrition and metabolism may be gained from the review of Rasmont (1968). Phagotrophy by coelenterates in general and the corals in particular, has been extensively reviewed (Lenhoff, 1968 ; Muscatine, 1973). Nutrition of free-living acoels is poorly understood (Jennings, 1968), but species such as Gonvoluta convoluta and Amphiscolops langerhansi are known to feed extensively. Host phagotrophy is essential for reproduction in Amphiscolops (Taylor, 1971b) (see above, p. 29). Traditional arguments have centred on the relative significance which host phagotrophy and symbiont photosynthesis have to the nutritional success of coelenterate symbiosis. I n the extreme, this may be interpreted as autotrophy v. heterotrophy as a nutritional basis for these associations. A view that ignores the significance of multiple nutritional inputs. Studies of reef corals in Hawaii have been interpreted as indications that these associations are wholly autotrophic (Franzisket, 1969b, 1970). Comparable work on Bermudan reefs also de-emphasizes the role of host feeding (Johannes et al., 1970), allowing only that it may provide an essential source of limiting nutrients such as phosphorus. Opposing views, summarized by Yonge (1963, 1968), de-emphasize the role of symbiotic photosynthesis. Adoption of the concept of corals as organisms with a multi-faceted nutritional basis goes some way towards reconciling these opposed and unproductive arguments (Goreau et al., 1971 ; Sorokin, 1972). Incorrect interpretation of corals as filter feeders, rather than specialized carnivores (Muscatine, 1973) can lead to unfortunate experimental approaches. Johannes et a,?., (1970) have made efforts to sample zooplankton and calculate its availability to corals as a source of food. Accurate sampling is difficult and the methods used fail to account for " patchiness " in zooplankton distributions over the reef (Emery, 1968). As carnivores, corals might be expected to feed in large amounts a t irregular intervals, a habit that is compatible with zooplankton " patchiness ". Studies of anemones in the laboratory support the view that host phagotrophy

INTERACTIONS OF ALGAL-INVERTEBRATE SYMBIOSIS

35

is indispensable for total growth of the association (Muscatine, 1961 ; Taylor, 1969e). The concept that each individual association is different and not comparable to others is too beguiling. Some basic underlying principles must exist which will allow a generalized assessment of nutritional resources. ( b ) Osmotrophy. A wide range of dissolved organic compounds is available in the aquatic environment (Parsons and Seki, 1970). Comparatively little is known about their utilization, but it is generally agreed that uptake of these substances can be demonstrated repeatedly. Many compounds, notably carbohydrates, amino acids and vitamins would serve as significant resources for a symbiotic host and its algae. Research has centred particularly on coelenterates and has been reviewed by Muscatine (1973). Goreau (1956) first noted specific adaptations for absorptive feeding in corals (see also Goreau and Philpott, 1956; Goreau et al., 1971). Subsequent studies by Stephens (1960a, b, 1962) and North and Stephens (1971), clearly demonstrate uptake and some possible controlling factors (see also Lewis and Smith, 1971). Unpublished studies suggest that acoelus Turbellaria behave in a similar fashion. Larval Convoluta roscoflensis and Amphiscolops langerhansi without symbionts can incorporate a variety of organic acids, amino acids and carbohydrates from solution. I4C-lactate is rapidly assimilated and incorporated into cellular constituents. Approximately 30% of the total is respired by both hosts in microrespirometric studies. Patterns of uptake of I4C-mannitol and 14Cfructose differ somewhat. In Convoluta, a period of induction is required for both compounds before uptake begins. If animals are pre-induced with cold mannitol, uptake of fructose is immediate upon presentation. Induction of mannitol (cytochrome) dehydrogenase has been demonstrated in both Convoluta and Amphiscolops using the methods of Edson and Shaw (1966). Translocated algal photosynthate also represents an available source of dissolved organic material for host osmotrophy. Other compounds required in trace amounts may also enter the host in this fashion.

B. Intercellular exchanges A significant portion of nutrients and metabolites entering a functional symbiosis are translocated or recycled internally. These mechanisms provide a basis for the progressive integration of hostsymbiont metabolic pathways, and serve to conserve energy and resources.

36

DENNIS L. TAYLOR

1. Nutrients

(a) Soluble metabolites. Translocation of nutrients from the alga to host in marine symbioses has been almost exclusively studied in coelenterates (see reviews of Smith, D. et al., 1969; Muscatine, 1971, 1973). Authoritative reviews on the subject make a truly detailed discussion of this aspect of exchange redundant. The reader is referred t o those works cited above. Key compounds which could satisfy host requirements have been noted (see above, p. 31). These all represent soluble materials which can enter specific host pathways and satisfy requirements for energy and biosynthetic building blocks (e.g., carbohydrates, amino acids, organic acids, organic phosphates, etc.). They are in one sense, a reservoir of reduced organic carbon and essential nutrients. Other compounds conserved by the alga and translocated to the host include nitrates and phosphates. Demonstration of alanine movement in substrate inhibition experiments (Lewis and Smith, 1971) provides positive proof for the translocation of nitrogen from symbiont to host. Subsequent incorporation into protein has been postulated (Muscatine and Cernichiari, 1969). Conceivably, the organic phosphates detected by Trench (1971b) could function to conserve and recycle phosphorous in a similar fashion. Host to algal translooation is a major resource for mechanisms of algal heterotrophy and photoassimilation (see above, p. 32). Excreted nitrates and phosphates would provide a reliable supply of these limiting factors in algal growth. Nutritional studies of Gymnodinium microadriaticum in culture (McLaughlin and Zahl, 1959 ; McLaughlin et al., 1964) show that this alga can utilize a broad spectrum of host excretory products. Urea, uric acid, guanine, adenine and several amino acids can all serve as a single nitrogen source for growth. Similarly glycero-phosphoric, cytidylic, adenylic and guanylic acids may serve as suitable sources of phosphorus. Gooday (1970) has made similar observations in studies of Platymonas convolutae. Uptake of amino acids by free-living Platymonas in culture is increased in nitrogen depleted cells (North and Stephens, 1971). Analogous situations may exist in symbiotic associations. Investigations of acetate utilization by Platymonas convolutae and Amphidinium klebsii show that this substrate is metabolized by the alga, and respired CO, is incorporated photosynthetically (Taylor, unpublished). Such " anaerobic " photosynthesis (Pringsheim and Wiessner, 1960 ; Droop, 1961, 1963) may serve important functions in acoel symbiosis where available CO, can be limiting. ( b ) Particulate metabolites Translocation of particulate material

INTERAOTIONS OF ALQAL-INVERTEBRATE SYMBIOSIS

37

has not received adequate proof in the few associations where it has been mentioned, and it is impossible to give a reasonable assessment of its significance. Kawaguti (1965) interprets dissociation of symbiont thecal elements shown in electron micrographs of reef corals as a possible path of particulate nutrients. This view is not supported by studies of symbionts from Anemonia sulcata (Taylor, 1968a, 1969e). It is conceivable that the membrane fragments seen by Kawaguti are artifacts of preparation. Oschman and Grey (1965) also describe movements of particulate material between Convoluta roscoffensis and Platymonas convolutae. Their conclusions are based on similarities between stellate lipid bodies found in host and symbiont cells. Conclusive biochemical proof is lacking. I n culture, Gymnodinium microadriaticum produces an array of insoluble particulate compounds, mostly mucopolysaccharides (McLaughlin et al., 1963). Speculations suggest that these may be of use to the host, but actual translocation in vivo is not known. ( c ) Regulation of translocation. Biochemical or physical mechanisms for the regulation of the quality and quantity of translocation exist in several associations. Excretion of photosynthate by symbionts isolated in vitro from corals and Tridacna is markedly increased by exposure to host tissue homogenates (Muscatine, 1967). The phenomenon is widespread in associations involving Gymnodinium microadriaticum (Taylor, 1969d; Muscatine, 1971 ; Trench, 1971b) ; and is known to be a naturally occurring stimulatory mechanism (Trench, 1971~).Muscatine et al., (1972) describe optimum conditions for the action of host homogenates. The precise identity of the chemical factors involved remains elusive. However, it is known to be heat labile, associated with the soluble fraction of disrupted host cells and inactivated by proteases (Muscatine, 1967 ; Muscatine et al., 1972 ; Taylor, unpublished). Successful action of host homogenates is dependent on symbiont receptivity, an attribute that declines with time. Excretion is stimulated only in freshly isolated symbionts in in vitro systems (Trench, 1971~).It is not effected in cells taken from axenic cultures (Taylor, 1972). Excretion of photosynthate by Platymonas convolutae in associations with Convoluta roscoffensis is controlled by the maintenance of low intercellular p H in the symbiotic unit (Taylor, 1971a). The same mechanism has been described in Hydra (Cernichiari et al., 1969). Studies of Platymonas in axenic culture show that low p H operates to alter the quality of excreted photosynthate and increase the rate of excretion. Comparative work with Qymnodinium microadriaticum was negative (Taylor, 1971a).

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DENNIS L. TAYLOR

2. Gases

( a ) Oxygen. Under optimum conditions, symbiotic algae tend to produce an excess of oxygen beyond that consumed in photorespiration and host respiration (Burkholder and Burkholder, 1960 ; Kanwisher and Wainwright, 1967 ; Roffman, 1968 ; Franzisket, 1969a ; Nozawa et al., 1972). The physiological importance of this is questionable, since few associations are found in situations where oxygen might be a limiting factor. As noted above (p. 30), measurements of 0, production with polarographic or chemical methods cannot take into account variables dependent upon host rates of respiration, algal photorespiration and the biochemical-physiological interactions of hosts and symbionts. The possibility that net photosynthetic 0, production of the association is influenced by internal compensations makes valid assessments of photosynthesis in these terms difficult. Conversion of net 0, values to grammes of carbon fixed/m2 suggests that net productivity is greater than that encountered with free-living phytoplankton (Kanwisher and Wainwright, 1967). Such estimates are only approximate, however, and it is unwise to extrapolate these data in order to judge productivity in complex symbiosis-based ecosystems such as coral reefs. ( b ) Carbon dioxide. All algal-invertebrate symbioses will take up CO, during photosynthesis. The difficulties in assessing the significance of CO, exchange between hosts and symbionts have been adequately pointed out (Droop, 1963). Most environments seem t o favour an abundant supply, and consequent rapid exchange with the hostsymbiont cellular complex, making CO, limitation remote. High symbiont densities could mediate against this, and cause severe local deficiencies (Droop, 1963 ; McLaughlin and Zahl, 1966). There is little experimental assessment of the fate of host respired CO,. Pearse (1970) has shown that it can become incorporated in the skeletal bicarbonate and matrix of corals. Preferences for host derived CO, are not known. It would be of value to know the proportional contributions of externally and internally derived CO,. Among hermatypic corals, removal of CO, by the algae is an important factor in skeletogenesis (Goreau and Goreau, 1959, 1960a, 1960b ; Yonge, 1963). The subject has received considerable attention because of its broad biogeochemical importance. Reviews by Yonge (1963, 1968) and Muscatine (1972) should be consulted. Recent studies clearly demonstrate that calcification is light dependent and that this dependency rests heavily on photosynthetic CO, removal (Pearse and Muscatine, 1971 ; Vandermuelen et al., 1972). Consideration should

39

INTERACTIONS OF ALGAL-INVERTEBRATE SYMBIOSIS

also be given to the role of algal heterotrophy in calcification (see above, p. 32). Reduced photosynthetic rates, due to reductions in available light, can have a profound effect on skeletal growth form (Barnes, 1973). Conversely, saturating light intensities, and resulting high rates of algal photorespiration can suppress rates of calcification in the Atlantic coral Montastrea annuluris (Ellis and Solander) studied in situ (Barnes and Taylor, 1973). C. Growth 1. Regulation

There is a natural balance between symbiont numbers and available host tissue (Muscatine, 1961 ; Droop, 1963; Taylor, 1969e). Regulation of this balance is determined primarily by nutrient levels (dependent upon the host’s metabolic rate) and available space (dependent upon the host’s potential for growth) (Taylor, 1969e). It is achieved either through suppression of the alga’s growth rate or by the physical expulsion of symbionts by the host. Experimental studies of symbiont growth in newly infected larval Convoluta roseoflensis illustrate the suppression of algal growth

2o01

TIME (DAYS) FIG. 6. Growth curve of Platymonas convolutae following introduction into larval Conuoluta roscoffensis. Taylor (unpublished). 11.I.R.-11

3

40

DENNIS L. TAYLOR

rates within the limits set by the host’s growth potential (Fig. 6). Growth curves obtained by cell counts from individual larvae show typical sigmoid patterns, an early lag phase followed by logarithmic growth lasting 2 to 3 days then near stationary growth. Limitations of the method make it impossible to follow algal numbers throughout the animal’s growth t o adult size (30 to 35 days), but it may be assumed that near stationary growth persists. The mechanism effecting growth suppression is believed to be mediated through a low intercellular pH ( 5 . 5 ) , resulting from algal synthesis of dimethyl-/3-propiothetin and its subsequent breakdown in situ t o yield dimethyl sulfide (released) and acrylic acid (accumulated) (Taylor, 1971a). The same mechanism regulates algal growth in the symbiosis of Arnphiscolops langerhansi. I n these associations, patterns of symbiont growth in situ bring to mind laboratory systems of continuous culture. Chemostat studies of algal symbionts may prove to be a useful experimental approach for examining pathways of algal biosynthesis and excretion, providing it can be shown that the symbiotic growth condition of the alga is perpetual stationary phase. Logarithmic growth of symbionts in host tissues is not known. Their physical expulsion by the host could favour this condition! establishing a situation roughly analagous t o t h a t found in turbidostats. However, the nutrient demands of logarithmically growing symbionts could deprive the host of essential growth requirements and mediate against such a system of “population control ”. Regulation of algal numbers in Anernonia sulcata depends on host recognition of aged or degenerate symbionts and their expulsion by normal excretory processes (Taylor, 1969e). Among coelenterates this is the preferred regulatory mechanism. Stages in symbiont degeneration, leading to eventual excretion have been studied with the electron microscope (Figs. 7 and 8). Unequal division of symbionts yields cells which are physiologically older (Taylor, 1968a). These carry the bulk of accumulated symbiont wastes and are recognized by host cells which transport them to the mesenteries where they are excreted. Possible toxicity may facilitate recognition by the host (Lucas, 1947). The process is believed t o be continuous. I n circumstances where the Fm. 7. A. Zooxanthellae from a mesentery of Anemonia sulcata that has been fed in the light. Note the degenerate appearance of these cells as compared with B. Micrograph x 2000. B. Zooxanthellae in a tentacle from a host that has been fed in the light. Micrograph x 2000. C. Zooxanthellae in a mesentery from a host that has been starved in the light. Micrograph x 2200. D. Zooxanthellae in a tentacle from a host that has been starved in the light. Compare with B and C. Micrograph x 2200. From Taylor (1969e).

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host is severely stressed (e.g., by starvation, elevated temperatures, low salinity, darkness), depression of host metabolism accelerates the excretory process, and the symbiont is " pruned '' back t o more compatible numbers (e.g. Yonge and Nicholls, 1931 ; Goreau, 1964). Extremely stressed conditions may introduce severe oscillations into this regulatory mechanism, and survival of the association may be imperiled. This may explain the observation that corals transplanted from deep to shallow water do not survive (Lang, 1970). VI. CONCLUSIONS Considering the very great variety of symbiotic associations involving marine algae and invertebrates, it should be possible to study all of the as yet unanswered problems which have been enumerated here. Judicious selection of material can narrow the numbers of different associations that are needed, making it possible to study all of the aspects of these in detail, and to define unifying principles. I n order t o obtain a logical framework for the assessment of cellular relationships in algal-invertebrate symbiosis, it is essential t o learn everything possible about each association that is selected for study. A great number of techniques should be applied to a few kinds of associations rather than the converse. The latter has been applied to the study of nutrient movement. We now know much about carbon translocation in many associations, but cannot integrate this into an understanding of the relationships in any single symbiosis because supporting data are lacking. For the purpose of studying the intercellular relationships of algalinvertebrate symbiosis, hosts can be selected on the basis of the following criteria : (1) host species should exhibit the full range of symbiotic integration, extending from obligate dependence upon the alga (i.e. host never feeds) to facultative dependence (i.e. host sometimes feeds) ; (2) closely allied host species should be capable of being infected with a variety of different algal symbionts in order t o facilitate comparative studies on nutrient pathways (ideally these should include algae from several different genera and classes); (3) hosts should be amenable to axenic cultivation in the laboratory ; and (4) re-infection FIG.8. A. Zooxanthellae in a mesentery of Anemonia sulcata that has been fed in the dark. Micrograph x 2000. B. Zooxanthellae in a tentacle from a host that has been fed in the dark. Micrograph x 2000. C. Zooxanthellae in a mesentery from a host that has been starved in the dark. Note the degenerate state of the host cells. Micrograph x 3000. D. Zooxanthellae in a tentacle from a host that has been starved in the dark. Compare with C. Micrograph x 3000. From Taylor (19690).

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of hosts should be able t o be controlled by the investigator. Among freshwater symbioses, Hydra goes a long way towards satisfying these requirements. Marine acoels belonging to the family Convolutidae are nearly ideal as experimental subjects. Extensive knowledge of their natiiral and experimental symbionts makes these associations extremely attractive. Similarly, expanded knowledge of coelenterates and their algae may eventually make them equally useful. The importance of algal-coelenterate associations in the primary organization of coral reef ecosystems makes their detailed study a priority. Artificial symbiosis between vertebrate cells and Chlorella has been established in the laboratory (Buchsbaum and Buchsbaum, 1934 ; Buchsbaum, 1937). More recently, the technique has been applied in studies using isolated chloroplasts (Nass, 1969). These artificial systems provide controlled experimental conditions where the mutual conforniance of animal and algal cells may be studied, and the basic biological criteria of a " good " host and a " good " symbiont may be assessed. Further investigations, possibly employing invertebrate tissue culture are extremely desirable, since they provide an immensely practical means of investigating the problems of symbiosis discussed here. This work was supported by grants from the National Science Foundation (GB 19790) and the Browne Fund of the Royal Society.

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McLaughlin, J. J. A., ZaN, P. A., Nowak, A. and Marchisotto, J. (1963). Some constituents of zooxanthellae grown in axenic culture. Proc. I Int. Congr. Protozool., Prague, pp. 204-205. McLaughlin, J. J. A., Zahl, P. A. and Nowak, A. (1964). I n vitro analysis of nutritional requirements and population dynamics of some free-living phytoplanktons and symbiotic algae (zooxanthellae). Proc. X Int. Bot. Congr., Edinburgh, p. 242. Mangan, J. (1909). The entry of zooxanthellae into the ovum of Millepora, and some particulars concerning the medusae. Q . Jl Microsc. Sci., 53, 697-709. Marcus, E. and MacNae, W. (1954). Architomy in a species of Convoluta. Natwre, Lond. 173, 130. Margulis, L. (1970). “ Origin of Eucaryotic Cells.” Yale University Press, New Haven. Marshall, S. M. (1932). Notes on oxygen production in coral planulae. Xcient. Rep. Gt Barrier Reef Exped. 1, 253-258. Muscatine, L. (1961). Symbiosis in marine and freshwater coelenterates. In “ Biology of Hydra ” (H. M. Lenhoff and W. F. Loomis, eds.) pp. 255-268. University of Miami Press, Miami. Muscatine, L. (1967). Glycerol excretion by symbiotic algae from corals and Tridacna and its control by the host. Science, N.Y. 156, 576-519. Muscatine, L. (1971). Endosymbiosis of algae and coelenterates. In “ Experimental Coelenterate Biology ” (H. M. Lenhoff, L. Muscatine and L. V. Davis, eds.), pp. 255-268. University of Hawaii Press, Honolulu. Muscatine, L. (1972). Influence of zooxanthellae on productivity and calcification in reef corals : critique and perspectives. I n “ Symbiosis in the Sea ” (W. B. Vernberg and F. J. Vernberg, eds.) In press. University of South Carolina Press, Columbia. Muscatine, L. (1973). Nutrition of Corals. In “ Biology of Coral Reefs ” (R. Endean, ed.) Vol. 2, pp. 77-115. Academic Press, New York. Muscatine, L. and Cernichiari, E. (1969). Assimilation of photosynthetic products of zooxanthellae by a reef coral. Biol. Bull. mar. biol. Lab.,Woods Hole, 137, 506-523. Muscatine, L., Pool, R. R. and Cernichiari, E. (1972). Some factors influencing selective release of soluble organic material by zooxanthellae from reef corals. Mar. Biol. 13, 298-308. Nass, M. M. K. (1969). Uptake of isolated chloroplasts by mammalian cells. Science, N . Y . 165, 1128-1131. Norris, R. E. (1967). Algal consortisms in marine plankton. In “ Proceedings of Seminar on Sea, Salt and Plants ” (V. Krishnamurthy, ed.) pp. 178-189. North, B. B. and Stephens, G. C. (1971). Uptake and assimilation of amino acids by P l a t y m o w . 11. Increased uptake in nitrogen deficient cells. Biol. Bull. mar. biol. Lab., Woods Hole, 140, 242-254. Nozawa, K. Taylor, D. L. and Provasoli, L. (1972). Respiration and photosynthesis in Convoluta roscoffensis Graff, infected with various symbionts. Biol. Bull. mar. biol. Lab., Woods Hole, 143, 420-430. Oschman, J. L. (1966). Development of the symbiosis of Convoluta roscoffensis Graff and Platymonas sp. J . Phycol. 2, 105-111. Oschman, J. L. and Grey, P. (1965). A study of the fine structure of Convoluta roscoffensie and its endosymbiotic algae. T r a m . Am. microsc. SOC.84, 368375.

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Weiss, P. (1963). The cell as unit. J . theoret. Biol. 5, 389-397. Wells, J. W. (1956). Scleractinia. I n “ Treatise of Paleontology ” (R. Moore, ed.) Part F, pp. 328-443. Geol. Soc. of America, University of Kansas Press. Werner, B. (1970). Contribution to the evolution in the genus Stephanoscyphus (Scyphozoa: Coronatae) and ecology and regeneration qualities of Stephanoscyphus racemosw Komai. Publ. Set0 Mar. Biol. Lab. 18, 1-20. Whittaker, R. H. (1969). ‘‘ Communities and Ecosystems.” MacMillan, New York. Whittaker, R. H. and Feeny, P. P. (1971). Allelochemics: Chemical interactions between species. Science, N . Y . 171, 757-770. Woodhead, P. M. J. and Weber, J. N. (1970). The evolution of reef-building corals and the significance of their association with xooxanthellae. Symposium on Biogeochemistry, Tokyo, Japan. In press. Yonge, C. M. (1936). Mode of life, feeding, digestion and symbiosis with zooxanthellae in the Tridacnidae. Scient. Rep. Gt Barrier Reef Exped. 1,283-321. Yonge, C. M. (1944). Experimental analysis of the association between invertebrates and unicellular algae. Biol. Rev. 18, 68-80. Yonge, C. M. (1953). Mantle chambers and water circulation in the Tridacnidae (Mollusca). Proc. zool. SOC.,Lond. 123, 551-561. Yonge, C. M. (1960). Ecology and physiology of reef building corals. In. “ Perspectives in Marine Biology ” (A. A. Buzzati-Traverso, ed.) pp. 117-135. University of California Press, Berkeley. Yonge, C. M. (1963). The biology of coral reefs. I n “Advances in Marine Biology ” (F. S. Russell, ed.) Vol. 1, pp. 209-260. Academic Press, London. Yonge, C. M. (1968). Living corals. Proc. R . SOC.B 169, 329-344. Yonge, C. M. and Nicholls, A. G. (1931). Studies on the physiology of corals. V. The effect of starvation in light and darkness on the relationship between corals and zooxanthellae. Scient. Rep. Gt. Barrier Reef. Exped. 1, 177-211.

Adv. mar. Biol., Vol. 11, 1973, pp. 57-120

RESPIRATION AND FEEDING IN COPEPODS SHEINAM. MARSHALL Institute of Marine Resources, University of California, and

University Marine Station, Millport, Isle of Cumbrae, Xcotland I. Introduction . . .. .. .. 11. Respiration .. .. .. .. A. Effect of Crowding .. .. B. Effect of Time after Capture . . .. C. Variation with Season . . D. Relation to Size. . .. . . E. Effect of Light . . .. .. .. F. Effect of Temperature . . .. G . Effect of Salinity .. .. H. Effect of Pressure .. .. I. Effect of Oxygen Content .. J. Effect of Feeding .. 111. Feeding .. .. .. .. .. .. A. Feeding Mechanisms . . B. Food .. .. .. .. C. Experimental Feeding . . .. .. IV. Conclusion . . .. .. .. V. Acknowledgements .. .. .. .. VI. References . . . . .. ..

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57 69 60 60 61 62 62 66 66 67 67 67 71 I1 82 95 110 110 111

I. INTRODUCTION Copepods are perhaps the most numerous animals in the world (Fig. 1). They form the bulk of most zooplankton hauls, they inhabit the vast expanse of the oceans and may be abundant to a depth of several hundred metres, so it is not surprising that they outnumber all other kinds of animal, even the insects, which may have more species but fewer individuals. Copepods are small, rarely exceeding 10 mm in length and usually much smaller ; many measure less than 1 mm. They are found in both fresh and salt water, near the coasts and in the open ocean, floating near the surface or crawling in the seashore sand. They are important in the sea because they are the main convertors of the phytoplankton into food suitable for higher organisms. For this reason a knowledge of their feeding habits and the amount of food they require is essential for an understanding of the processes of production in the sea. 57

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FIQ.1. Living copepods; different stages of Calanua helgolandicus. Photo: D. P. Wilson.

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I n recent years the breeding and rearing of marine pelagic copepods in the laboratory has led to greater possibilities for the accurate measurement of food ingested throughout the life cycle. Methods of measuring both feeding and respiration rates have also been improved and diversified. Nevertheless, the species of copepods used remain much the same. The large and easily obtainable genus Calanus heads the list among marine forms, Diaptomus and Cyclops among freshwater forms. I n the following pages the name Calanus (C.Jinrnarchicus (Gunnerus), C. helgolandicus (Claus), C . paci$cus Brodsky", C. hyperboreus ( K r ~ y e r) )will occur over and over again, whereas observations on other genera are scattered and sporadic. It is not safe to conclude, however, that what is true of one species will necessarily be true of another, even closely related, species ; the behaviour of copepods differs from one species to another, even from one individual to another. There remains a great deal to be done before we have a body of information about, for instance, the feeding and metabolism of predatory copepods comparable t o that which we have now for a few CaEanus species. 11. RESPIRATION Putter (1925) made some measurements on the respiration of copepods in bulk but, apart from a single experiment on Calanus hyperboreus (Ostenfeld, 1913), work on an individual species, Calanus Jinmarchicus, did not begin until the 1930s (Marshall et al., 1935; Clarke and Bonnet, 1939) ; it has now been extended to many different species of varying size from both salt and fresh water. The methods most often used have been estimations of oxygen consumption by either the Winkler method, the manometric respirometer or modifications of these. The polarographic oxygen electrode (Kanwisher, 1959; Teal and Halcrow, 1962; Nival et al., 1971) has more recently come into use. To obtain a measurable result in a short time (3-6 h) it is necessary to have a large number of copepods in a small bottle (Winkler) or, what in terms of the environment may come to the same thing, give each copepod only a small volume of water (respirometry). When a small number of animals are used and the time is prolonged, antibiotics must be added to prevent bacterial respiration interfering with the results. Penicillin cannot be used with the Winkler method since it reacts with the iodine in the final stages of the estimation, but strepto-

* Following Fleminger, the form off the Californian coast is recognized as the species, C. pncijiczcs.

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mycin and chloromycetin have often been used in combination. The last, however, is injurious to some copepods (Berner, 1962) and was found to decrease feeding in Calanus (Marshall and Orr, 1961). Bernard (1963a) found that penicillin and streptomycin were injurious t o copepods (she used them in high concentrations) but that sulfamethopyrazine was harmless. Among the factors influencing respiration which have been considered are crowding, time after capture, season, size, light, temperature, salinity, pressure and the oxygen content of the water.

A. Effect of crowding The effect of crowding has been considered by several workers with varying results. Some (Marshall and Orr, 1958 ; Comita and Comita, 1964; Conover and Corner, 1964) found that it made no appreciable difference; Satomi and Pomeroy (1965) found that it did. Zeiss (1963) made the most detailed experiments on the subject. To reduce the effect of any increased metabolites in a crowded culture he enclosed several copepods in short tubes, closed at each end with bolting silk, and suspended these in the experimental bottles. Using these the volume per Calanus finmarchicus seemed to make no significant difference to its oxygen consumption, although with Daphnia magna Straus there was a decided increase of oxygen uptake in this type of experiment. Increasing the number of Calanus in the experimental bottle did, however, decrease the oxygen consumption. This might be caused by the increased concentration of metabolites and Zeiss thought that the effect might vary between different types of crustaceans, relating it to their concentration in natural waters.

B. Effect of time after capture

It has often been observed that oxygen uptake is higher during the first hours after capture than subsequently (Marshall et al., 1935; Berner, 1962; Zeiss, 1963; Bishop, 1968) and to avoid this period experiments are often made on animals which have been kept 24 h or so in the laboratory. It is not certain whether the excitement of capture and handling raises oxygen uptake above normal, or whether under laboratory conditions there is a decline from normal values ; t h e first of these alternatives is usually assumed. S. K. Katona (personal communication) has stated that male Eurytemora aginis Poppe do not behave normally until one or two days after being isolated from a laboratory population into a separate vial.

61

RESPIRATION AND FEEDING I N COPEPODS

C. Variation with season There is a marked seasonal variation in oxygen consumption (Fig. 2). From a low value in winter months there is a sharp rise (per individual) in spring (Marshall and Orr, 1958; Conover, 1959; Haq, 1967 ; Gaudy, 1968). Several factors may be responsible for this. I n spring most copepods are at their maximum size and have a plentiful 3c

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FIG.2. Seasonal changes in oxygen consumption in various copepods. (a) Length of metasome in mm of ripe female CalanusJinmurchicusin 1957. (b) Oxygen consumption of ripe female C. Pnmurchicus in 1956 and 1957. (c,) oxygen consumption of PseudocaZunua elongatus (open circles), Temora Zongicornis (closed circles) and Acartia cZuusi (triangles) in 1956; (cii)Centropuges hamatus (open circles) Metridia Zucens (closed circles) and Oithona similis (closed triangles) all in 1956; 0. sirnilis (open triangles) in 1965.

62

SHEINA M. MARSHALL

food supply ; they are also reproducing actively. Temperature is rising although the maxima of temperature and respiration do not coincide, the first continuing t o rise after the second has begun to decline. Oxygen consumption rises even when calculated per unit weight (Anraku, 1964a) so that it is not caused only by increased size (Fig. 2). It is less in pre-adult stages, less in males than in females and in immature than in ripe females. The proportion of actively respiring tissue in ripe females must, because of the mass of large eggs in the oviducts, be higher than in males or Stage V which contain relatively more fat, and this may be one cause. Ripe females in summer, however, do not consume so much oxygen as ripe females in spring. Vollenweider and Ravera (1958) observed that egg-carrying Cyclops strenuzls Fischer females used more oxygen than non-egg-carryin g, but Coull and Vernberg (1970) found consumption lower in gravid than in non-gravid females of Longipedia helgolandica (Klie) ; they attributed this t o lessened activity. After the spring rise, consumption in ripe female Cabnus declines gradually t o minimal winter values.

D. Relation to size As one might expect the larger copepods use more oxygen but when uptake is expressed per unit of dry body weight the small forms are usually found to be more metabolically active. The same tendency is seen when the developmental stages of a single species are compared. Coull and Vernberg (1970), however, found that in benthic harpacticids, the activity of the animal mattered more than its size. Males and females have often been used together but, as mentioned above, their oxygen consumption is not always the same. The whole developmental range has been covered only in Calanus finmarchicus (Marshall and Orr, 1958), Acartia clausi Giesbrecht and A . latisetosa Kritcz (Petipa, 1966). The results are shown in Table I and Fig. 3, p. 69.

E. Effect of light Full sunlight is lethal t o Calanus as to many other marine animals (Huntsman, 1925). The effect of keeping C. finmarchicus at constant temperature in bright sunlight or even in shade out of doors on a bright day is to raise oxygen consumption considerably as well as to damage the animals. When the copepods are suspended in glass bottles in the sea (Marshall et al., 1935) the effect is not measurable below 2.5 m. Most respiration experiments are carried out in shade indoors or in darkness to avoid this effect, but Bishop (1968) states that sunlight had no effect on the oxygen consumption of some freshwater copepods (Diaptomus and Cyclops spp.).

r oYOWNC x STAGESor Acartia cla-i TABLE I. OXYGENC o m s ~ ~ ~ a IN

Species Acartia clausi, large

A. clausi, small A. clausi, young

Calanus Pnrnarchicus

Number used

Stage

P

+

2

? 6 ? d

212 45 28 95 63

Dry wt Range Mean 4.3-10.2 4.3- 5.9 2.6- 5.4 1.P 1.9 1 . P 1.6

AND

CaZanrur~Zmnaarchiclls

DuraOxygen consumption tion expt. pllcopepodlday pllmg dry wtlday (h) Range Mean Range Mean

Tt:p'

8.8 5.0 4.6 1.7 1.5

15-26 Most 24 7-24 23-25 8-24 7-24 2&26 16-24 24 24-26

0.90.41.20.30.7-

3.4 2.7 4.6 2.0 1.4

1.61 1.28 2.87 0.91 0.98

12689211-1 153-1 483-

525 595 295 770 950

13

1.8

24-26

24

2.02

1050

and V) C I V and V c I11 c I1 C I and I1 N V and V I

16 16 44 24 25 24

1.0 1.5 0.6 0.3 0.6 0.1

25 2P26 25-26 24 25 24

15 24 15-24 24 17 24

0.34 1.51 0.67 0.24 0.27 0.10

336 1008 1108 800 446 1056

(176)

10

c.48

6.0-13.9

7.6

(43)

(242)

10

c.48

10.3-17.8

15.1

(62)

(203)

10

c.48

6.8-12.4

10.4

(27)

(240)

10 10 10 10 10

c.48 19-48 19-48 19-48 19-48

247 1.40.90.50.3-

5.5 3.1 1.4 0.9 0.6

(23)

2 4 28

10 10 10 10

19-48 19-48 19-48 19-48

0.7- 0.8 0.2- 1.9 0.2- 0.8 0.19

874 720

10 10

19-48 19-48

? ?

c3

cv c IV c I11 c I1 C I N VI NV N IV N I11 N I1 and I1 and I11 N 1-11

386 (June-Mar.) 191 (Apr. May) 213 519 73 70 36 4 4

0.3- 1.0

9.3 4.0 1.9 1.3 0.9

0.07-0.09 0.05

Source

\

214 \ 267 753 632 676

CV

c I V ( + I11

Location

Black Sea

Petipa (1966)

Firth of Clyde

Marshall and Orr (1958)

0.19 0.08 0.05

Dry weights of Acartia calculated, according t o Petipa, as 16% wet weight. Calanus dry weights in brackets averaged from 112 samples taken throughout the years 1933 and 1961-64. The averages of the two sets of samples agreed well.

TABLE11. OXYGENCONSUMPTIONOF Calanus spp., FEMALE

r

-

-

320

May

4-6

8-24

(15.2)

47.4

Gulf of Maine

-

8-48

24

9.1 2.9

42.4 24.5

Conover and Corner, 1968 Conover, 1960 Anraku, 1964a

19 5

-

213 129

Aug. Aug., Dec.

7.5 8

5

-

152

May, June

8

24

7.2

50.0

386

2.37

176* June-Mar.

10

48

7.6

43.2*

Gulf of Maine Buzzards Bay and Cape Cod Bay Anraku, 1964a Buzzards Bay and Cape Cod Bay Marshall and Orr, Firth of Clyde

I91

2.79

242*

Apr., May

10

48

15.1

B2.4*

Firth of Clyde

5

-

141

Aug., Dec.

15

24

5.9

41.7

5

-

136

May, June

15

24

9.0

62.3

35

2.45

-

Aug., Sept.

3

19.2

-

240

-

-

Aug.

corr. to 17 20

4

18.8

-

Anraku, 19648 Buzzards Bay and Cape Cod Bay Buzzards Bay Anraku, 1964a and Cape Cod Bay Firth of Clyde Raymont and Gauld, 1951 Firth of Clyde Marshall et aZ.,

1958 (7.jinmarchicus

Marshall and Orr, 1958

i

1935

ca

x E w P

5 !d

b

E

133

0ct.-Dec.

8

24

(6.8)

51.5

English Channel Cowey and Corner,

158

Mar.-Sept.

8

24

(11.9)

75.5

English Channel Cowey and Corner,

-

0ct.-Apr.

10

48

9.2

-

Jan.-Feb.

15

4-8

8.7

1963 1963

Firth of Clyde

Marshall and Orr,

108.5

Villefranche

Nival et al., in press Midlin and Brooks, 1970 Mullin and ’ Brooks, 1970

1958 80

C . hyperboreus

C . gracilis

I:

{ -

(204)

-

10

22-29

9.0

(44.1)

La Jolla

(204)

-

15

22-29

11.7

(52.5)

La Jolla

-

-

8-24

13.5 27.5 28.0

-

2 332

Dec. Apr. Apr.

11.2

3 650

Aug.

8-48

25.0

5.8

-

June

6 8

14.4

-

3-7 3-7 P 6