Advances in Marine Biology, Volume 16

Advances in

MARINE BIOLOGY VOLUME 16

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Advances in

MARINE BIOLOGY VOLUME 16 Edited by

SIR FREDERICK S. RUSSELL Plymouth, England

and

SIR MAURICE YONGE Edinburgh, Scotland

Academic Press London New York

San Francisco 1979

A Sulsidiay of Harcourt Brace Jovanovich, Publishers

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

Copyright

10003

0 1979 by Aca.demicPress Inc. (London) Ltd.

All rights reserved

NO 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: 0-12-02611&2

PRINTED IN QEUCAT BRITAIN BY TEE WHITEPRIARS PRESS LTD.

LONDON A N D TONBRIDQE

CONTRIBUTORS TO VOLUME 16 R. V. GOTTO,Department of Zoology, Queen’s University, Belfast, United Kingdum.

ROGERP. HARRIS,Plymouth Laboratory of the Marine Biological Association of the United Kingdom, Plymouth, England. G. Y. KENNEDY, The University of Shefield, England.

GUSTAV-ADOLF PAFFENHOFER, Skidaway Institute of Oceanogrqhy, Savannah, Georgia, U.S.A. A. J. UNDERWOOD, Department of Zoology, School of Biological Sciences, University of Sydney, N.S.W . 2006, Australia.

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CONTENTS

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111. Classification of Associated Forms and Other Systematic .. .. .. . . Considerations . . ..

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COXTRIBWTORS TO

VOLUME 16

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The Association of Copepods with Marine lnvertebrates

R. V. GOTTO

.. Previous Reviews . .

I. Introduction 11.

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IV. General Studies of Single Species

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IX. Effect on Host and Host Reaction

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VIII. Preferential Host Niche . .

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VI. Host Specificity

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VII. Attraction to Host

X. Morphological Variability at Infraspecific Level XI.

Sibling Speciation

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XII. Population Studies XIII. Larval Studies

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OONTENTS

XIV. Associated Harpacticoids and Calanoids A. Harpacticoids .. .. B. Calanoids . . .. .. ..

XV. Future Investigations XVI. References XVII. Addenda

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The Ecology of intertidal Gastropods A. J. UNDERWOOD I. Introduction

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11. Factors Affecting the Establishment of Patterns of Distribution .. .. .. .. .. 113 A. Large-scale Patterns .. .. .. .. 113 B. Local Patterns .. .. .. .. .. 120 C. Summary and Conclusions .. .. .. 136 1 .

111. Ma,intenanceof Patterns of Distribution by Behavioural .. .. .. .. .. Adaptations . . A. Patterns of Zonation .. .. .. .. B. Dispersion within Zones: Homing Behaviour . . C. Migrations and Aggregations . . .. .. D. Summary and Conclusions .. .. ..

IV. Maintenance of Patterns of Distribution by Physiological Stress . . .. .. .. .. .. .. A. Temperature and Desiccation . . .. .. B. Salinity and Osmoregulation . . . . .. C. Other Factors .. .. .. .. . . D. Summary and Conclusions .. .. . .

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V. Competition and the Distribution and Abundance of Populations .. .. .. .. .. .. 170 VI. Predation and the Distribution and Abundance of .. .. .. Populationa

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VII. Reproductit-e Biology and Geographical Distribution. . 183

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VIII. Influences of Gastropods on the Structure of Intertidal Communities . . .. .. .. .. .. A. The Effects of Grazers on Sessile Animals B. The Effects of Grazers on Algae . . .. .. .. C. The Effects of Predators on Sessile Animals D. Summary and Conclusions .. .. .. ~

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X. Acknowledgements XI. References

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Laboratory Culture of Marine Holozooplankton and its Contribution t o Studies of Marine Planktonic Food Webs

GUSTAV-ADOLF PAFFENROFER AND ROGER P. HARRIS

I. Introduction

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11. Cultivation Techniques A. Protozoa . B. Cnidaria .. C. Ctenophora D. Rotifera .. E. Chaetognatha F. Mollusca .. G. Amphipoda .. H. Mysidacea ., I. Euphausiacea J. Ostracoda . . K. Decapoda . . L. Copepoda . . 13. Cladocera . . N. Tunicata ..

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111. Contribution of Cultivation to the Study of Plankton Ecology .. .. . . .. .. .. A. Taxonomy and Morphology .. .. .. B. Experimental Studies Relating to Secondary .. .. .. Production .. .. ,. .. C. Simulation Studies . , .. ..

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Pigments of Marine Invertebrates

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X . Annelida. Echiuroidea. Sipunculoidea. Priapuloidea and .. .. .. .. .. 333 Phoronidea . .

X I . Arthropoda A . Crustacea B. Arachnida C . Myriapoda

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Polyzoa

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XIX. Comment

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XXI. References . . .. Taxonomic Index . . Subject Index . . .. Cumulative Index of Titles Cumulative Index of Authors

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Adv. mar. BioZ., Vol. 16 1979 pp. 1-109.

THE ASSOCIATION OF COPEPODS WITH MARINE INVERTEBRATES

R. V. GOTTO Department of ZooJogy, Queen's University, Belfast, United Kingdom

I. 11. 111. IV. V.

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VI. VII. VIII. IX. X. XI. XII. XIII. XIV.

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Introduotion .. .. .. .. PreviousReviews .. .. .. .. .. Classification of Associated Forms and Other Systematio Considerations .. General Studies of Single Speoies .. . .. .. .. .. Anatomical and Functional Aspects .. .. .. .. .. A. Integument .. .. .. .. .. . . B. Sensory Struotures .. .. .. . . .. .. , . C. Food and Feeding .. .. .. .. .. .. .. .. .. D. Struotural Studies of the Alimentary Canal .. E. Reproduction and Allied Topics . .. .. .. . .. .. Host Speoifioity . . .. .. .. .. Attrmtion to Host . . .. .. .. .. . . .. .. Preferentiml Host Niohe . .. . . .. .. . . .. Effeot on Host and Host Reaction .. .. .. .. * . Morphological Variability at Infraspeoifio Level . Sibling Speoiation . . .. . . .. .. .. .. . . Population Studies . .. .. . . .. .. .. .. .. Larval Studies .. .. .. . . .. .. .. Assooiated Harpacticoids and Calanoids .. .. A. Harpacticoids . . .. .. .. .. .. .. .. B. Calanoids .. .. .. .. .. Future Investigations. . .. .. Referenoes . .. .. .. Addenda , .. .. .. ..

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I. INTRODUOTION In contrast to the fish parasites, those copepods which habitually partner marine invertebrates have received scant attention until fairly recent times. This discriminatory treatment is hardly surprising, for while the fish associates are frequently conspicuous, often bizarre and, 1

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above all, linked with hosts of economic importance, the latter are mainly unobtrusive forms, their ecology clandestine and their economic significance limited. It might indeed be fair to say that, up to the last half century, the handful of such species then known were regarded almost as aberrations on the part of an otherwise well-ordered Naturea few curious types generated in odd moments of evolutionary whimsy. Although described and recorded as occasion offered by the carcinologists of the nineteenth century, sustained study of these copepods attracted comparatively few workers. Thorell, Hesse, Claus, Giesbrecht, Canu and the Sars may be numbered among those who contributed more significantly to our knowledge in this earlier epoch. They were followed by such researchers as de Zulueta, working on the lamippids associated with alcyonarians, and Chatton who, in collaboration with Br6ment and Harant, concentrated on ascidicolous species. It is, however, only within the past thirty years that the immense variety of such copepods has become apparent, and, in particular, the full extent of their host spectrum realized. It is now, indeed, difficult to name a marine phylum some a t least of whose members do not harbour these versatile and little-known associates. At this point, we should perhaps define two terms rather more closely. The word " associate " was suggested by Gooding (1957) to describe those copepods which habitually partner other organisms, but whose precise ecological relationship with the host may be currently obscure. This neatly avoids the use of such vague terms as " semiparasite " but does not prejudge future application of more rigidly defined categories when further information becomes available. On the host side, the term 'L invertebrate " is here taken to include not only the phyla universally recognized as such, but also certain of the " acraniate chordates "-in practice, ascidians, salps, enteropneusts and pterobranchs. Since the present paper is a review rather than a monograph, some constraints in treatment at once become operative. There would seem little point, for example, in repeating lengthy morphological descriptions (which in fact represent the bulk of recent work) when these are readily available elsewhere. Again, certain aspects of structure, function and indeed general biology have remained virtually unexplored and thus unreviewable. Finally, hard information on many taxa of associated copepods is so sparse that it is difficult to treat such scanty material in an organized manner under appropriate headings. It is for this latter reason that I have referred symbiotic harpacticoids and calanoids to a single section, rather than including them with the more extensively studied associated cyclopoids.

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11.PREVIOUS REVIEWS Only one, rather brief review of a wide ranging nature has been devoted largely to the copepod partners of marine invertebrates within recent years. Bocquet and Stock (1963) have discussed the interrelationships between the major groups of parasitic copepods, showing that the Copepoda purasitica of earlier workers certainly constitutes no monophyletic unit, but should probably be apportioned between the poecilostomatous and the siphonostomatous cyclopoids. I n like manner the old order, or sub-order, Notodelphyoida is a completely artificial assemblage, most of its members belonging by right to the gnathostomatous cyclopoids while others should more properly be referred to the poecilostomes. As regards the latter, Bocquet and Stock have reviewed the old argument as to the presence or absence of a mandible in these copepods. They point out that there is general agreement amongst recent workers that the following mouth-parts can be attributed to all cyclopoids: mandible, maxillule (= Grst maxilla), maxilla (= second maxilla) and maxilliped-although any or all of these may be subject to reductions or specializations of varying degree. The same paper incorporates a discussion on the origin of parasitism and it5 specificity in copepods and concludes with some brief observations on sexuality, ecology, behaviour, development and larval cycles. The only other papers of note in this context are those of Bouligand (1966a), who has contributed a useful review of copepods found in association with coelenterates, and Cheng (1967),who has dealt with the copepod parasites and commensals of commercially important marine molluscs. To some extent, this last paper updates and amplifies the earlier work of Monod and Dollfus (1932a, b, 1934) on the copepods associated with molluscs in general.

111. CLASSIF1:CATION OF ASSOCIATED FORMS AND OTHER SYSTEMATIC CONSIDERATIONS Some sort of taxonomic framework, however skeletal, must now be attempted. Recent discoveries of new families and genera, many of them clearly annectant, make this task somewhat easier than it would have been even a few years ago, but it nonetheless remains a daunting proposition. Let us take the broad view first. Although opinions still differ as to the circumscription of major divisions within the Copepoda, eight groupings are often cited as meriting recognition at ordinal or sub-

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ordinal level : the Caligoida, Lernaeoida, Calanoida, Harpacticoida, Monst rilloida , Not odelphyoida , Herpyllobioida and Cy clopoida. Of these, the first two, as fish parasites, are largely outside the scope of this paper.* Such harpacticoids and calanoids as are known to be associated forms will be dealt with, as already mentioned, in a separate section. The monstrilloids are associated with other animals only in their larval instars, and the notodelphyoids, by general agreement, retain no further claim to ordinal or sub-ordinal status. Despite recent

TABLEI.

SIPIiONOSTOME CYCLOPOIDS AND THEIR

HOSTS*

Hosta

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-Calvocheridae Stellicomitidae Nanaspididae Caricerillidae Micropontiidae Entomolepidae CYCLOPOIDA Artotrogidae SIPHONOSTOWAAsterocheridae

Echinoids Asteroids Holothurians Ophiuroids Irregular echinoids Sponges Nudibranchs (a few reports) Sponges, anthozoans, echinoderms, ascidians Dinopontiidae Sponges, actinians Megapontiidae (Found free) Dyspontiidae Sponges, scleractinians, ascidians Myzopontiidae (Found free or with algae) cBrychiopontiidae Abyssal holothurian

* For the recently erected family Namakosiramiidae, see Addenda (Ho and Perkins, 1977). excellent studies by Liitzen (1964b, 1966, 1968a) those highly modified associates of polychaets, the herpyllobioids, remain taxonomically enigmatic. This leaves us with the large order Cyclopoida, an assemblage containing a great many associated species. If pitfalls lurk even today in classifying the Copepoda as a whole, the detailed systematics of the cyclopoids constitute a veritable mine-

* It is true that a caligoid,Anchicaligua RautiZi (Willey), has been recorded from a nautiloid, but no description or figures of this species have been published (see Margolis et al., 1976).

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field for the unwary taxonomist. Kozloff (1974) in his key to marine invertebrates, cogently summarizes a current attitude to this problem: “The clmsification of symbiotic cyclopoids is so involved that no attempt will be made here to categorize the families or other taxa.” I n view of this confused and complex situation, the systematic tables presented here must be treated with considerable caution. I n them I TABLE111. POECILOSTOME CYOLOPOIDSAND

THEIR

HOSTS*

Hosts Ergasilidae Sabelliphilidae Lichomolgidae Pseudanthessiidae Rhynchomolgidae Urocopiidae Sapphirhinidae Clausidiidae Clausiidee CYCLOPOIDA < Anomoclausiidae POECILOSTOMA Catiniidae Eunicicolidae Nereicolidae Mytilicolidae Myicolidae Ventrjculinidae Xari6idae Vahiniidae Corallovexiidae Taeniacmthidae Gastrodelphyidae

Bivalves (but principally fish) Wide host spectrum Very wide host spectrum Wide host spectrum Scleractinians (Found free) Predatory on salps Sponge, octocoral, polychaets, bivalves, sipunculid, nudibranch, stomatopod, thalassinideans Polychaets, teredinid bivalves (Found free) Sipunculid Polychaets Polychaets Bivalves, gastropods Molluscs Sipunculids, gastropods Scleractinians Antipatharians Scleractinians Echinoids (but principally fish) Polychaets

* For the recently erected family Philoblennidae, see Addenda (Izawa, 1976). have attempted to summarize and, where possible, amalgamate those views which appear to me as rational interpretations of the presently available systematic data. It will be noted that in parts of these tables no formal categories are named as such--e.g. tribes, sub-families, etc.-since I believe our information is still too fragmentary for such definitive assignment. At present, probably only families, genera and species can be delimited with any degree of real confidence in their

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validity as natural units-and even then, anomalies are likely to remain. However, certain “ groupings ” of related forms can be discerned and are outlined below. Some disagreement in this area is clearly inevitable, especially as regards taxa of which I have no personal experience. I n justification, therefore, I can only plead the words of Lang (1948a): “. . . certainly much would be gained if everyone working on a group of copepods would venture outside his group and give his opinion as to which other group is most closely related to his own. It is evident that this may easily lead to mistakes and perhaps to an interpretation of the phenomena of convergence and parallelism as proofs of relationship. But the result will perhaps lead to a discussion, out of which the truth will later crystallize.” It may now be useful to give short summaries of the families named in Tables I, I1 and 111. These notes should not be regarded as complete diagnoses, still less as familial keys. However, in conjunction with Figures 1-41, they will provide some indication as to habitus, general structure and host spectrum. For the siphonostome families, I have relied mainly on the selected characters listed by Humes (1974b). (a) Siphonostomes Family Calvocheridae: Cephalosome bulbous. Only one postgenital segment. Mandible absent. Gall formers on echinoid spines (Fig. 1). Family Stellicomitidae: Body minute, swollen, highly modified, without external segmentation. On asteroids (Fig. 2). Family Nanaspididae : Body minute, prosome shield-shaped and flattened, urosome very small. Antenna without an exopod. Legs reduced, with legs 3 and 4 lacking an endopod. On holothurians, but with one endoparasitic genus, Allantogynw (Fig. 3). Family Cancerillidae: Body minute. Antennule with 5-9 segments in female. Legs reduced (in some, legs 3 and 4 absent). On ophiuroids (Fig. 4). Family Micropontiidae: Body minute. Antenna without an exopod. Legs 3 and 4 lacking an endopod. On irregular echinoids (Fig. 5 ) . Family Entomolepidae : Body shield-shaped and flattened. Mandible with a 2-segmented palp. I n sponges (Fig. 6). Family Artotrogidae: Body shield-shaped. Leg 4 absent. I n a few cases reported on nudibranchs (Fig. 7). Family Asterocheridae: Oral cone produced to form a siphon. Mandible with a palp. Leg 5 with a free segment. Associated with sponges, anthozoans, echinoderms and ascidians (Fig. 8). Family Dinopontiidae: Antenna with exopod absent or reduced to a

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small spine. Leg 5 with a free segment. With sponges and actiniana (Fig. 9). Family Megapontiidae: Antennule of female 11-segmented. Oral cone with trumpet-like opening. Leg 5 with a free segment. Found free a t great depths (Fig. 10). Family Dyspontiidae: Antennule with aesthete on last segment. Generally with a long, slender siphon. Leg 4 with endopod reduced or absent. Associated with sponges, scleractinians and ascidians (Fig. 11). Family Myzopontiidae: Antennule with aesthete on last segment. Leg 5 with a free segment. Found free or with algae (Fig. 12). Family Brychiopontiidae : Body shield-shaped. Antennule of female Wsegmented, with aesthete on segment 15. Antenna and maxilliped each bear terminally a broad lamelliform element. Associated with an abyssal holothurian (Fig. 13). (b) Gnathostomes Family Archinotodelphyidae : General form of cyclopinid type. First leg-bearing segment free. Antennule of male prehensile. Antenna of female with one apical claw, plus a number of setae. No brood-pouch, the eggs being carried in two dorsal sacs. I n ascidians (Fig. 14). (The assignment to this family of Nearchinotodelphys indicw Ummerkutty, found in a bivalve by Ummerkutty (1960) is somewhat dubious-see Monniot, 1968.) Family Notodelphyidae : Body form very varied. Dorsal broodpouch present. Terminal armature of antenna includes an articulated claw. I n ascidians and (a few species) in octocorals (Fig. 15). Family Buproridae :Cephalic region well defined, but body otherwise an ovate sac, due to great development of brood pouch. Urosome vestigial. Antenna 3-jointed. Maxilliped lamellate, with four spines. Pereiopods somewhat reduced, fifth leg a small lobe tipped with four setae. I n ascidians (Fig. 16). Family Ascidicolidae : Body slender, almost vermiform. Antenna prehensile. No brood-pouch, but exopodites of 5th legs act as oostegites, partially protecting the egg sacs. I n ascidians (Fig. 17). FIGURES 1-41. Representatives of associated oopepod families. All figures (redrawn from various authors) are of females, unless otherwise stated. Approximate lengths. where known, of the specimens figured m e given in mm after the name. 1. Calwocheres engeli (0.72), lateral. 2 . Stellicornea aupplicam (0*44), dorsal. 3. Nanaspia mixta (0.45), dorsal. 4. Cancerilla tubulata (0.80). dorsal. 6 . Mtcropntiw, glaber (0-46), doreal. 6 . Entomolepis adriae, male (0.7), dorsal. 7. Artotrogua orbicularia (2.0), ventral. 8. Aaterocheree vhlaceua (1*0),dorsal. 9. Dinopontiua acuticauda (1.3), dorsal.

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Family Botryllophilidae : Urosome well defined and distinct from metasome. Antenna not prehensile. 5th legs dorso-lateral, cylindrical or lamellate, supporting a globular egg mass. I n ascidians (Fig. 18). Family Enterocolidae :Body generally cruciform* or sausage-shaped. Mouth-parts showing various degrees of reduction. 5th legs dorsolateral, lamelliform, lappet-like or bluntly conical, exceptionally absent. No brood-pouch. I n ascidians, and possibly other invertebrates-see p. 21 (Fig. 19). Family Enteropsidae : Body eruciform, with indistinct segmentation. Antenna and mouth-parts show extensive spinulation. Leg 5 absent. I n ascidians (Fig. 20). (c) Poecilostomes Family Ergasilidae : Body generally cyclopiform. 5th thoracic somite much reduced, or fused with genital segment. Antenna with strong prehensile claw. Mandible with apical armature of several elements. Maxilliped absent in female. Legs 1-4 well developed. Leg 5 uniramous. Mainly parasites of fish, but one genus (Ostrincola) in mantle cavity of bivalves (Fig. 21). Family Sabelliphilidae : Body' generally cyclopiform. Legs 1-4 endopods 3-segmented in most genera. If leg 4 endopod 2-segmented, then legs 1-3 endopods also 2-segmented, the reduction of endopods occurring in an anterior to posterior series. Wide host spectrum (Fig. 22).

Family Lichomolgidae : Body generally cyclopiform. Leg 4 endopod 2-segmented, 1-segmented, reduced to a small knob, or absent. Legs 1-3 usually 3-segmented. Very wide host spectrum (Fig. 23). Family Pseudanthessiidae : Body often modified or transformed. Exopods of legs 1 and 2 in the female %segmented, in the male at least 2-segmented. Leg 4 endopod 1-segmented, reduced to a small knob, or absent. Leg 5 without a free segment. Wide host spectrum (Fig. 24). Family Rhynchomolgidae : Body transformed. Female with expanded cephalosome, but narrow metasome and urosome. Male elongate, almost vermiform. Rostrum a conspicuous, tumid snout-like lobe. In scleractinian corals (Fig. 25). Family Urocopiidae : Female only known. Body cyclopiform. 10. Megqmntiua pleuroapinoeua (6.6), lateral. 11. Dyapontiua atriatua (1.46), dorsal. 12. Myzopontiua auatralia (0.74), dorsal. 13. Brychiopontiue fdcatua (1.3), dorsal. 14. Pararchinotodelphya gurneyi (2.1), dorsal. 16. Notodelphye allmani (4.3), dorsal. 16. B u p m h e n i ( l e l ) , lateral. 17. Aacidiwla roaea (4.0), lateral. 18. Botr2/llophilua ruber (1.6). lateral. * caterpillar-like.

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THE ASSOCIATION OF UOPEPODS WITH MARINE INVERTEBRATES

13

Caudal rami large, lamellate. Leg 5 rudimentary, no free segment. Found free in plankton, 600-700 m depth (Fig. 26). Family Sapphirinidae : Often submerged in the family Oncaeidae. Body more or less flattened, due to lateral expansion of segments. Often iridescent. Corneal lenses generally present. Marked sexual dimorphism. Antennule short. Antenna prehensile. Maxilla and maxilliped claw-like. Usually free, pelagic, but known to be associated with salps in a predatory capacity (Fig. 27). Along with the Oncaeidae, the families Sabelliphilidae, Lichomolgidae, Pseudanthessiidae, Rhynchomolgidae and Urocopiidae are considered by Humes and Stock (1973) to constitute a superfamily, the Lichomolgoidea. Family Clausidiidae: Body cyclopiform to elongate. Terminal joint of antenna implanted excentrically. Mandible usually with accessory pieces. Paragnaths generally present. Some species with sucking discs on antenna or 1st leg. Wide host spectrum (Fig. 28). Family Clausiidae : Body cyclopiform to elongate or vermiform. Antenna 3- or 4-segmented, armed with setae and one or more subprehensile claws. Mandible reduced in size. Maxillule simple, lobate with setae. Two to five pairs of legs, variously reduced. With polychaets, teredinid bivalves, ophiuroids and holothurians (Fig. 29). Family Anomoclausiidae: Body elongate, eruciform-cylindrical. 7-segmented antennule. 4-segmented antenna. Mandible elongate, bearing distally three long, posteriorly-directed pointed processes. Rami of legs 1-4 with spines only. Leg 5 massive, uniramous, bimerous. Found free (Fig. 30). Family Catiniidae : Prosome dorso-ventrally flattened and wide, urosome narrow. Antenna 4-segmented, with a sucking disc on third segment. Mandible small and weakly cuticularized. Maxilliped prehensile, present only in male. On a sipunculid (Fig. 31). Family Eunicicolidae : Prosome broadly ovate. Large ventral sucking disc (absent in male) just anterior to mouth. Antenna 3-jointed, distal segment including two large setae, each terminating in a small sucking disc. Mandible an elongate blade carrying two scoop-like expansions. Three pairs of legs, last pair uniramous. On eunicid polychaets (Fig. 32). Family Nereicolidae : Segmentation of metasome generally indis19. Enterocola petiti (1.2), dorsal. 20. Enteropsis chattoni (4.0),lateral. 21. Ostrincola gracilis (Ial), dorsal. 22. Sabelliphilwc sarei (1*0),dorsal. 23. Lichonaolgue tridacnae (1.8), dorsal. 24. Pseudanthesaiwc madrasensis (0-7), dorsal. 26. Rhynchomolgm corallophilua (l*l), dorsal. 26. Urowpia aingularis (lag), dorsd. 27. Bapphirina angueta (3-0), dorsal.

I'

,\

30

29

2a

32

34

35

36

THE ASSOCIATION OF COPEPODS WITH

MARINE INVERTEBRATES

15

tinct or absent. Urosome segments fused into a single genito-anal complex or reduced to two segments only. Legs reduced or absent, fifth pair always absent. On polychaets (Fig. 33). Of the above-mentioned families, Gooding (1963, unpublished thesis) considered the following as being, at that time, impossible to separate adequately : Clausidiidae, Clausiidae, Catiniidae, Eunicicolidae, Nereicolidae and Synaptiphilidae-regarding the last-named as a separate entity, although synaptiphilids are believed by Bocquet and Stock (1957b)to fall within the limits of the Clausiidae. Gooding treated these " families " as an informal unit, the " nereicoliform group ". Family MytiIicolidae: Body vermiform, with fusion of the more posterior segments. Antenna 4-segmented, prehensile. No mandible. In female, no maxilliped. Legs of female short, broad, lamellate. I n bivalves and gastropods (Fig. 34). Family Myicolidae : Body cyclopoid to oblong. Antennule with 6-7 segments, some species with a conspicuous spine on basal segment. Antenna 3-segmented, often furnished with elaborately articulated claws. Female maxilliped knob-like, or terminating in a knob-like structure. Legs 1-4 biramous, trimerous. Leg 5 uniramous, bimerous. With bivalves, gastropods and tectibranchs (Fig. 35). Family Ventriculinidae : Body subcylindrical in female, more typically cyclopiform in male. Antennule 4-6 segmented. Antenna prehensile. Mandible and maxillule present, maxilla absent, maxilliped present or absent. Legs reduced. I n gastropods and sipunculids (Fig. 36). Family Xariflidae : Body elongate, rather slender, with weakly defined segmentation. With or without posteriorly directed processes arising from dorsum above the fifth legs. Antennule 3-6 segmented. Antenna 3-4 segmented. With or without a mandible. On or in scleractinian corals (Fig. 37). Family Vahiniidae : Body elongate, slender. Labrum and labium form a low cone. Mandible and maxillule minute, slender, styliform. Female maxilliped reduced to a small process, but 4-segmented in male. In antipatharian corals (Fig. 38). Family Corallovexiidae : Body vermiform, with, in female, a t least four pairs of fleshy lateral processes. Mandible absent in female, all mouth-parts except maxilliped absent in male. Thoracic appendages 28. llkrsiliodes cylindracea (2*8),dorsal. 29. Ctauaia uniseta (2.4), dorsal. 30. Anomoclauaia idrehzlsae (24),ventral. 31. Catinia plana (1*0),dorsal. 32. Eunicicola clausi (0.9), dorsal. 33.8elioidea bocqueti (1.6), ventral. 34. Mytilimb porreota (4.9). ventral. 35. Antheasius solidus (2-6), dorsal. 36. Endocheree obacurus (6.7),dorsal.

39

38 I

w

1

42

44

as 43

37. Xar@a naaldivenaia (1.4), dorsal. 38. Vahinius petax (0+3),ventral. 39. Corallooexia mediobrachium (3.4), dorsal. 40. Echinirus laxatus (2.1), dorsal. 41. @a.?trodelphys dale& (143, lateral. FIGURES 42-62. Representatives of families of disputable systematic position. 42. Larnippella faurei (1.2), ventral. 43. Spongiocnimn verrnifforrnis (l.6), ventral. 44. Nicothoe mtmi (1.4), dorsal. 46. Chonhsphaera canerorurn (0.4).

lateral.

THE ASSOCIATION OF COPEPODS WITH MARINE INVERTEBRATES

17

represented a t the most by two pairs of globular swellings. Caudal rami consist of fleshy, tapering lobes. Endoparasitic in stony corals (Fig. 39). Family Taeniacanthidae : Body usually flattened and rather elongate. Fifth legs generally large, prominent, tending t o project laterally. Antenna prehensile. On echinoids, but better known as fish parasites (Fig. 40). Family Gastrodelphyidae : Body segmentation largely obscured in female, but well defined in male. Most specieswith brood pouch formed by enlarged fourth metasomal segment. Urosome reduced in female. Very long caudal setae. Maxilliped absent in female. Fifth legs lacking in both sexes. On sabellid polychaets (Fig. 41). Systematically speaking, appropriate niches for the following families are still a matter for discussion, and their precise location within the broader taxonomy of associated copepods currently remains obscure. Family Spongiocnizontidae : Body horn-shaped, cylindrical or vermiform. Antennule biramous, strongly modified in male. No trace of mandible, maxillule or maxilla. Maxilliped present. I n sponges (Fig. 42).

Family Lamippidae : Body elongate, fusiform, or ovoid-globular, the shape frequently alterable by peristaltic contraction. Head appendages consist of antennule, antenna and (in some species) maxilliped. Mouth region complex and variable. Two pairs of legs. Furca variously developed. I n octocorals (Fig. 43). Family Phyllodicolidae : Body of adult female inflated, unsegmented, attached to host by chitinous ring from which arise two long rhizoids which penetrate the host coelome. Antennule, antenna and maxilliped present. Eggs attached individually by short peduncles to two common axial filaments. On phyllodocid polychaets (Fig. 44). Family Xenocoelomidae : Body of female unsegmented, sausageshaped or cucumber-shaped. No appendages of any sort. Genital openings withdrawn into an invagination. Cryptogonochorism (i.e. invasion of female by subsequently implanted degenerate dwarf males) probably occurs in all species. Internal parasites of terebellid polychaets (Fig. 45). Family Antheacheridae : Body of female clearly segmented, rather elongate, with two to several pairs of digitiform protuberances. Antennule, antenna and 2-3 mouth-parts present. Male considerably smaller than female. I n galls on mesenteries of actinians (Fig. 46). Family Splanchnotrophidae : Body of female indistinctly segmented, with or without several pairs of long, lateral processes. Mandible distinctive, armed with teeth and lacking a palp. Maxilliped generally

46. Splanchnotrophwr dellechiajei (2.0), ventral. 47. Echiurophilwr jizei (3.0), ventral. 48. Staurosma parasiticurn (5.0), dorsal. 49. dphanodomus terebellae (6.0), lateral. 60. Phyllodicola petiti (0.5), ventral. 51. Melinnacheres steenstmpi (1.8), ventral. 62. Herpyllobiua arcticua (1.9), oblique lateral. FIQUFCES 53-76. Anomalous genera. 63. Akessoaia occultu (l4),ventral. 64. Apodomyzon Zongicorne (0*7), lateral.

THE ASSOCIATION OF COPEPODS WITH MARINE INVERTEBRATES

19

absent. Males (where known) dwarfed. I n nudibranchs ;two candidates on gymnosome pterobranchs (Fig. 47). Family Echiurophilidae : Body of female elongate, with four pairs of long lateral processes. Antenna with distally-toothed setae. Mandible and maxillule reduced. Legs reduced. Eggs in uniserid strings. Male cyclopiform, with five pairs of legs. I n an echiuroid (Fig. 48). Family Nicothoidae : Thoracic segments of female either hypertrophied into large “ wings ” or swellings of various shape, or else enlarged to give either an ovoid or an almost spherical appearance. The only certain adult male known is cyclopiform. Mouth area tubular or suckerlike. Mainly on gills (one species on rostrum) of astacuran decapods, and (one species) in incubatory cavity of a bopyrid isopod (Fig. 49). Family Choniostomatidae :Females considerably swollen, sometimes almost spherical. Males dwarfed, squat. Mouth cone present, expanded into a disc. Mandible simple, blade-like, With malacostracan crustaceans and ostracods, generally amongst the host eggs (Fig. 50). Family Melinnacheridae : Body ovoid, externally unsegmented, attached to host by a short stalk terminating in a frontal bulla. Three or four pairs of rudimentary appendages. Dwarf males attach to ventral side of female. On polychaets (Fig:51). Family Herpyllobiidae : Body consisting of two portions, the ectosoma and endosoma, the latter located within the host, and connected to ectosoma by a short stalk. No appendages present. Males (where known) small, bottle-shaped, attached to female. Parasites of polynoid polychaets (Fig. 52). Some observations on the systematic resume given above will now be in order. As Lang (1948a)has pointed out, there is little difficulty in regarding the siphonostomes as a coherent natural group (Table I).However, they may, as he suggests, have some links with certain of the caligiformid copepods-in the sense used by Oakley (1930)-for example, the Dichelesthiidae. The less modified gnathostomes likewise present relatively few problems as regards their inter-familial and inter-generic affinities. It will be noted (Table 11)that I have in large measure, though with some modification, adopted Lang’s system of dividing the gnathostomes into the Cyclopoida gnathostoma cyclopinidiformes and the Cyclopoida gnathostoma notodelphyidiformes-“ tribes ”, according to Lang. I am aware that Dudley (1966) in her excellent study of development in symbiotic copepods, regards such a division as perhaps premature, but I believe that it has some merit even with our presently limited information. It is, of course, true that Lang outlined his scheme before the establishment (Lang, 1949) of his family Archinotodel-

20

R. V. GOTTO

phyidae, when he amended his tribal definition accordingly. This family bears so many resemblances to the free living Cyclopinidae as wellas to the associated Notodelphyidae, that its position as an annectant group can scarcely be doubted, making any dividing line between the two tribes extremely tenuous. That said, however, it may, for the moment, still be useful to emphasize the different modes of existence exemplified by them. I have also retained Lang’s division of the notodelphyidiform gnathostomes into two groups, the Notodelphyidimorpha and the Ascidicolidimorpha, though again with certain amendments. To the former, Lang assigned three families-Notodelphyidae, Doropygidae and Buproridae-all possessing a brood pouch and ventrally situated fifth legs. However, Illg (1958) rightly broadened the concept of the Notodelphyidae by incorporating the doropygids, originally promoted to sub-familial rank by Brady (1878), although treated as a family by Sars (1921). These latter, therefore, are no longer regarded as constituting a family unit. I n his Ascidicolidimorpha, Lang placed the Ascidicolidae and the Botryllophilidae. I n earlier days, the Ascidicolidae was often employed as a convenient repository for almost any copepod found within a seasquirt. Lang very properly restricted it to the type genus Ascidicola. One other, clearly related form (Styelicola) has since been added by Lutzen (196813). This latter genus is of considerable interest in showing that the dorsally sited lamellae which protect the ovisacs unquestionably represent exopodites of fifth legs, since (unlike the condition in Ascidicola) a small endopod is also present. I n part at least, this discovery resolves a long-standing dispute as to the nature of such protective lamellae or oostegites. Some workers, notably Chatton and Brkment (1915) and Chatton and Harant (1924), were inclined to regard them either as folds of the dorsal integument (pterostegites) or as compound structures comprising dorsal folds plus a dorsally displaced fifth pereiopod. Although pterostegites certainly occur on the metasoma1 segments in several associated copepods, I believe that in cases where partial or complete protection of this type is afforded to the eggs, the structures involved are almost invariably modified fifth pereiopods. The other family (Botryllophilidae) included by Lang in the Ascidicolidimorpha agrees with the Ascidicolidae in lacking a brood pouch and in the dorsal positioning of the fifth legs. I n Botryllophilus, the latter are more or less elongate, seta-bearing lappets, whilst in the other two genera referred to this family (Pteropygus and Schizoproctzcs) they form lamellate expansions. Where known, the extruded eggs are carried dorsally in a globular or ovate mass.

21

THE ASSOCIATION O F COPEPODS WITH MARINE INVERTEBRATES

I n Table 11,it will be seen that I have included two other families in the ascidicolidimorph group-the Enterocolidae and the Enteropsidae, both highly modified associates of ascidians. Under the influence of Sars (1921) and later, Lang (1948a) the enterocolids have usually been assigned to the poecilostomatous rather than the gnathostomatous cyclopoids. Since then, Dudley (1966) has carried out a very detailed examination of the naupliar and copepodid stages of two enterocolids, an enteropsid and a botryllophilid, comparing these with one another and with one of the several notodelphyids similarly investigated by her. After discussing the rather weak nature of Lang's arguments regarding enterocolid affinities, Dudley concludes that if ontogeny indeed furnishes valid phylogenetic clues, the enterocolids and enteropsids should properly be placed in the gnathostomatous division, not too far removed from the Botryllophilidae. Her analysis and interpretation of larval similarities is certainly convincing, whilst her thesis regarding a probable relationship between enterocolids and botryllophilids is, of course, supported by some adult features as well-notably the structure of the antenna and the form and dorsal position of the fifth legs. Naupliar studies show Enteropsis to be a slightly more isolated genus, though it still exhibits gnathostome affinities. Since enteropsids lack the fifth pereiopods, we must therefore modify Lang's definition of the Ascidicolidimorpha from " no brood pouch ; fifth pair of legs placed dorsally " to " no brood pouch ; fifth pair of legs, when present, placed dorsally." The Enterocolidae perhaps merit our further attention for an altogether different reason. The better known forms are currently apportioned between two sub-families, the Enterocolinae and the Haplostominae (Chatton and Harant, 1924). Most are gut parasites of ascidians, inhabiting the stomach or intestine, and it may be that all ascidicolous enterocolids started their evolutionary careers from a base in the alimentary canal (Gotto, 1970b). However, in describing several new species referable t o this family, Chatton and Harant (Zoc. cit.) suggested that a number of ambiguous and little known genera might ultimately be recruited to it. These were Bactropus and Entobius from polychaet hosts, Ventriculina from a sipunculid, Enterognathus from a crinoid and Zanclopus from a pterobranch. Although all of these require further investigation, we may say a t present that the first two seemto belong, or be close to, the Clausiidae, whilst Ventriculina,currently placed in a small, inadequately known family, may have affinities with the dichelesthiids and eudactylinids (Bocquet and Stock, 1956). Enterognathus and Zanclopus, however, remain as reasonable candidates for inclusion in the Enterocolidae. If we compare Enterognathus with Enterocola, similarities are at once A.M.B.-16

2

22

R. V. OOTTO

apparent in the structure of the pereiopods, especially as regards the modified fifth legs. Resemblances between the head appendages of Enterognathus and enterocolids generally are much more difficult to find. It should be remembered, however, that wide variation in cephalic structure, both as regards detailed architecture and even presence or absence of mouth parts, already exists within the Enterocolidae. Zanclopus, in so far as it is known from the brief description and sketchy figures of Calman (1908) reveals marked similarities to both of the above-mentioned copepods. I n particular, the exopods of legs two and four seem almost identical to those of Enterognathus lateripes Stock (1966a). Moreover, both types resemble the majority of enterocolids in being gut-dwellers. To this putatively related assemblage, I should like here to propose another possible candidate-Gomphopodrion byssoicum Humes (1974b) from the abyssal holothurian Oneirophanta mutabilis Theel. It is almost certainly an internal parasite, though its exact site within the host could not be determined. Humes diagnosed his copepod as a poecilostome but, in the absence of a male specimen,was reluctantto assign it to a definite family. I n fact, G. byssoicum seems to exhibit an intriguing mixture of features “ borrowed ”, as it were, from the “ enterocolid ” genera previously considered. The antenna and mandible, for example, are strongly reminiscent of those belonging to Enterognathus lateripes. Even more marked is the resemblance between the maxilliped of Gomphpodarion and the posterior mouth part of E. lateripes. Although Stock (1966a) was unwilling to commit himself as to the homology of this appendage, on balance it seems probable that it is indeed a maxilliped. The anterior pereiopods, too, can readily be homologised with those of enterocolids, and the fifth leg is likewise highly comparable. Now if we are correct in claiming membership of the Enterocolidae for these copepods, an interesting aspect of this family’s host spectrum at once becomes apparent. The hosts involved are tunicates, echinoderms and pterobranchs-the very groups believed, on good circumstantial evidence, to be somehow implicated in the genesis of the phylum Chordata. It may therefore be that the hypothesis of a close phylogenetic relationship between these host animals in the remote past is marginally strengthened by the occurrence in their respective guts of highly modified copepods belonging to a single, relatively small family. A further point which emerges is, of course, the consequent antiquity of this family. It is at least conceivable that the enterocolids were already specialized parasites well before the chordates came into existence. The great assemblage of poecilostomatous cyclopoids, which we must

THE ASSOCIATION OF COPBPODS WITH MARINE INVERTEBRATES

23

now consider, presents us with no shortage of systematic problems (Table 111).I n these copepods, appendages of all sorts appear and disappear like rabbits in a magician's hat. Part of the trouble in delimiting the group resides in its very definition and the subsequent interpretation of this by different authorities. To a large extent, its circumscription has relied on negative, or supposedly negative, features-always dubious criteria where forms moulded by the exigencies of long-continued associative existence are involved. A few phrases from the poecilostome diagnosis given by Gurney (1933) will illustrate this point : " Antennules of male not prehensile . . . upper lip not transformed into a tube . . . maxilla not prehensile . . ." However, the allegedly diagnostic feature which has caused most confusion is undoubtedly the supposed absence of a mandible, already mentioned (p. 3). The chief proponent of this idea was G. 0. Sara, though he, in fact, was merely following the founder of the poecilostome concept, namely Thorell(l859). Paradoxically, it is indeed a tribute to the reputation and influence of the great Norwegian carcinologist that this unfortunate misconception has permeated so much of the literature for so long-and this despite quite early-expressed objections. We now know, of course, that a mandible is generally present in these copepods, though' its structure and relative development may vary considerably. But some of the other criteria are also a little doubtful with respect to their phylogenetic value. Thus the presence of non-prehensile antennules in male poecilostomes is in fact a feature shared by a fair number of male gnathostomes-though geniculate male antennules are usually considered as diagnostic of the Gnathostoma! On the whole, the nature of the maxillipeds would Beem to offer a reasonably reliable character. I n most gnathostomes they are nonprehensile and substantially similar in both sexes ; in poecilostomes, they are prehensile in the male, but structurally reduced and sometimes absent in the female. It should be remembered, however, that in a number of poecilostomes the male is still unknown, so the exclusivity of this feature remains unproven. Clearly, a thorough reappraisal of the poecilostomatous cyclopoids is required, but this must await further detailed studies of many genera and families. A welcome trend towards research in this direction is now evident, but for the moment we must content ourselves with outlining such ideas as currently exist regarding family relationships within this group. The Ergasilidae, although mainly gill parasites of fish, include at least one genus (Ostrincola)which occurs in the mantle cavity of marine bivalves. This family was suspected by Gurney (1933) to occupy a very basal position in the general roster of parasitic copepods. Bocquet and

24

R. V. QOTTO

Stock (1963) consider it as holding a transitional niche between such strongly modified forms as the chondracanthids on the one hand and the barely altered lichomolgids on the other, with the families Mytilicolidae and Myicolidae as annectant groups. The Sapphirinidae are very closely allied to the Oncaeidae, and indeed are often submerged in the latter family. The oncaeids are reckoned (Humes and Stock, 1973) to belong to the superfamily Lichomolgoidea and, within this complex, to be near the Sabelliphilidae and Lichomolgidae. Gooding (1963, unpublished thesis) considers them to represent a separate lineage from his " nereicoliform group '' (see p. 15), though likewise derivable from gnathostomatous cyclopoids. The superfamily Lichomolgoidea is a new entity, arising from revision of the Lichomolgidae, a family which, over the years, had become something of a melting-pot. The massive and superbly executed work of Humes and Stock (1973) is here of inestimable value, dealing with 76 genera and 324 species, which between them encompass an enormously wide spectrum of hosts. The Sabelliphilidae are regarded as the most primitive group, from which the Lichomolgidae s.str. may have arisen. The latter, in turn, seem to be ancestral to the Pseudanthessiidae, and from these the Rhynchomolgidae have apparently evolved. The relationship of the little known Urocopiidae is still doubtful, but origin from an early pseudanthessiid stem is possible. The Gastrodelphyidae, a small family of strongly modified copepods associated with sabellid worms, shows an interesting parallel with the notodelphyids in the transformation of the fourth metasomal segment into a capacious brood pouch. So far, only one species (Gastrodelphys fernaldi Dudley) is known to possess external ovisacs. Dudley (1964) has commented on the resemblances between these copepods and the Sabelliphilidae, in particular the genus Sabelliphilus, which also partner sabellid polychaets. The similarities are certainly very striking and amply justify Dudley's conclusion that this family may have diverged from lichomolgoids via a form such as Sabelliphilus. Gooding (1963) had already remarked on certain resemblances between the Gastrodelphyidae and his " nereicoliform group " of poecilostome families, with which a rather more distant relationship might perhaps be postulated. There is general agreement that the families Clausidiidae and Clausiidae, despite remarkable plasticity in body-plan and a wide host spectrum, are closely allied (Wilson and Illg, 1955; Bocquet and Stock, 1957a ;Gotto, 1964). Pronounced similarities are apparent, especially in the structure of the antenna and the mandible. The Clausidiidae, in fact, can be regarded as a central family, " primitive " in the aense that its genetic potentialities have been expressed in genera some of which

THE ASSOCIATION

OF COPEPODS WITH MARINE INVERTEBRATES

25

are relatively unmodified, others profoundly transformed (Bocquet and Stock, loc. cit.). From such a basic but euryplastic assemblage, the Clausiidae can be envisaged as arising by a process whose main theme is simplification, while more specialized descendent families would be represented by the Eunicicolidae and the Catiniidae. Some useful revisionary notes on the genera of the Clausidiidae have been supplied by Vervoort and Ramirez (1966). Closely aligned to this complex is the presently monotypic Anomoclausiidae. When I erected this family (Gotto, 1964)it seemed undesirable to extend yet further the already very broad definition of the Clausiidae, within which Anomoclausia could otherwise have been accommodated. However, any future appraisal of the entire complex will almost certainly submerge the Anomoclausiidae in a wider group concept. Such an enlargement may well incorporate also the curious cyclopoids found by Southward (1964) in serpulid tubes, namely Rhabdopus and Serpulidicolu (Bresciani, 1964). The Nereicolidae is another family with pronounced clausiid affinities. All are associated with polychaets and have been keyed recently to generic level by Stock (1968a) who includes the genera Nereicola, Vectoriella, Pherma, Anomopsyllus, Sigecheres, Selius and Selioides. The poorly-known family Ventriculinidae is claimed by Bocquet and Stock (1956) to have close ties with the fish parasites belonging to the Dichelesthiidae and the Eudactylinidae. They base this opinion largely on their study of Endocheres obscurus Bocquet & Stock, a rare associate of the prosobranch gastropod Calliostoma zizyphinum (L.) on the Channel coast of France. The other two genera referable to this family, Ventriculina and Heliogabulus, both associates of sipunculids, require further investigation. The Taeniacanthidae are, of course, well documented as cyclopoids associated with fish. I n recent years, however, three new genera (Echinosocius, Echinirus and Cluvisodalis) have been described from sea-urchins (Humes and Cressey, 1961 ; Gooding, 1965; Humes, 1970). It is a somewhat startling fact that these echinophilous species differ remarkably little from their piscicolous relatives. The final three families probably eligible for inclusion in the Poecilostoma are all found in scleractinian corals. The Xarifiidae are elongate copepods showing a greater or lesser degree of segmentation and with relatively well developed head appendages. Some are capable of crawling over the surface of the coral, but they seem to live mainly within the polyps, on which they feed. Humes (1960a)has suggested a relationship with the Lamippidae, a highly enigmatic group associated with soft

26

R. V. QOTTO

corals. Lamippids, however, lack evident segmentation, while their mouth parts (and indeed most appendages) are so reduced and obscure that this takes us little further in determining.a possible rapprochement with the xari€iids. The Vahiniidae are represented by a single species, Vahinizcs petax Humes from the antipatharian Stichopathes echinulata Brook. Humes (1967a) places it near the Lamippidae and the Xarifiidae, but remarks that its mouth parts " appear to be fundamentally different from those of either . . . and indeed from those of any poecilostome known to me." The Vahiniidae would therefore seem to occupy a somewhat isolated position. The Corallovexiidae, large endoparasites of stony corals, find a taxonomic niche amongst the poecilostomes mainly on the grounds that the general organization of their cephalic appendages is neither of gnathostome nor of siphonostome type (Stock, 1975). If anything, these appendages bear most resemblance to the mytilicolid condition, but in certain other structural features the family is reminiscent of the Antheacheridae, an anomalous group parasitizing sea anemones. The families which we must now briefly consider comprise units of associated forms often so bizarre that it is at present very difficult to slot them in with currently recognized higher taxa. The Lamippidae (Fig. 42) have already been mentioned as falling into this category, despite the excellent anatomical studies of Bouligand (1960a, b, 1961, 1965, 1966a, b, and Bouligand and Delamare Deboutteville, 1959). Even more difficult to interpret systematically is the sponge-dwelling family Spongiocnizontidae (Stock and Kleeton, 1964). These copepods show little or no segmentation and lack all mouth parts except for a powerfully developed maxilliped (Fig. 43). Their antennules are almost unique* amongst copepods in being biramous-a feature so striking that to accord spongiocnizontids familial status appears fully justified, though simultaneously making accurate determination of their appropriate taxonomic position virtually impossible. The Choniostomatidae and the Nicothoidae-both parasites of other crustaceans-can be considered together (Figs 44, 45). Since their discovery many years ago these copepods have enjoyed little peace at the hands of systematists. Hansen (1897) believed there were some similarities between the choniostomatids and the lernaeopodids. LeighSharpe (1926)referred Nicothoe to the siphonostome family Ascomyzontidae, but also remarked on certain resemblances to the caligoids. Gurney (1929)was the first to suggest that the affinities of this genus lay * The male of Paranicothoe cladoceru Carton. an associate of a bopyrid isopod, also possesses a biramous antennule (Carton, 1970).

THE ASSOCLATION OF COPEPODS WITH MARINEINVERTEBRATES

27

rather with the choniostomatids-a view amply confirmed by the comparative studies of Lemercier (1965) and further supported by Kabata (1967) and Carton (1970). The latter also implies (though without specific detail) a possible connection with Eunicicola. Kabata (loc.cit.) noted that both families bear many resemblances to the rather loose group of copepods referred to by Heegaard (1947) as Pectinata ”. As Lang (1948a) observed, Heegaard’s Pectinata ” coincides with Oakley’s (1930) group Caligiformes, encompassing the families Caligidae, Dichelesthiidae, Lernaeidae, Lernaeopodidae, Choniostomatidae and Herpyllobiidae. Kabata stresses the basic similarity of mandibular structure in the (‘Pectinata ”, the nicothoids and the choniostomatids. Just how much significance we should attach to this is doubtful, since the mouth part in question consists solely of a simple blade with cutting edges. If we accept that there is a limited number of ways in which copepods can attack host tissue and that mandibles are likely to be employed as tissue-cutting tools by many parasitic species, a blade such as that described above might well evolve irrespective of the animal’s phylogenetic past. I n short, no more than convergent adaptation need be involved. However, the other common.feature invoked by Kabata, namely the type of development, is probably of fundamental significance. A chalimus or pupal stage, attached to the host by a frontal filament, occurs in many choniostomatids as well as in the other families attributed to the Pectinata ”. Unfortunately, development in the nicothoids cannot yet be compared with these, since, despite exhaustive researches by Mason (1959), the life cycle in this family is still not fully known. We may say, therefore, that while the Nicothoidae and Choniostomatidae are unquestionably related, with the former representing a more “ primitive ” condition, their precise location within the Copepoda as a whole must remain for the moment sub judice. The small family Splanchnotrophidae are highly specialized parasites of molluscs, for the most part living in the body cavity of nudibranchs. According to Laubier (1964), the classically known forms should be restricted to the single genus Splanchnotrophus (Fig. 46). They are generally reckoned to have close ties with the Chondracanthidae, copepods associated primarily with fish. This latter family, in turn, should probably be considered as forming but one unit of a larger grouping, the Chondracanthoidea, within which the splanchnotrophids might retain familial rank, Stock (1971 ; 1973) has recently described two monotypic genera (MicraZtectoand Nannallecto) of very small copepods parasitic on gymnosome pterobranchs, which he considers as probably referable to the Splanchnotrophidae. They are the only copepods so far known from ((

((

28

R. V. GOTTO

gymnosomes, and it is possibly significant that these hosts are not too distant systematically from nudibranchs-the usual partners of splanchnotrophids. To the chondracanthoidean complex may likewise belong the Echiurophilidae, so far monotypically represented by Ec?t,iurophilus Jizei Delamare Deboutteville & Nunes-Ruivo (Fig. 47) an intestinal parasite of an echiurid (Delamare Deboutteville and Nunes-Ruivo, 1955). However, according to Gooding (1963, unpublished thesis) this form requires further investigation before its relationships can be firmly established. The Antheacheridae includes two genera, Antheacheres and Xtaurosoma (Fig. 48), which form galls in sea anemones, and possibly a third (Gmtroecus) which might, however, ultimately prove identical with Staurosoma (Stock, 1975). The family is perhaps better known as the Staurosomidae-but this name, as Vader (1970) has pointed out, is probably a junior synonym of Antheacheridae M. Sars (1870). These poorly known forms are again strongly suspected to have chondracanthoidean affinities (Caullery and Mesnil, 1902 ; Okada, 1927 ; Laubier and Schmidt, 1971),though Bouligand (1966a) has postulated, rather surprisingly, possible kinship with the Xarifiidae. I n passing, it should be mentioned that relationship between fish parasites of the family Chondracanthidae and poecilostome associates of invertebrates is no new idea (Bocquet and Stock, 1963). It dates back, in fact, to Vogt (1878) before being reinforced by Oakley (1930). The Xenocoelomidae might well be regarded as an end point in parasitically transformed copepods. Two genera, Xenocoeloma and Aphanodomus (Fig. 49) are included, both of which are internal parasites of terebellid polychaets. No trace of segmentation or appendages can be discerned, and the ovisacs issue from an unpaired opening. Much argument has centred on the nature of their sexuality-a point which will be discussed in more detail later. Although Jespersen (1939)placed Aphanodomus in the family Herpyllobiidae, there seems to be no doubt that Bresciani and Lutzen (1966) are correct in creating a separate family to receive these curious genera. The Phyllodicolidae is another recently erected family (Delamare DebouttevilIe and Laubier, 1960). The name originally proposed (Phyllocolidae)was amended by the same authors the following year. I n their account of the then only known form, Phyllodicola petiti Delainare & Laubier (Fig. 50) a parasite of phyllodocid polychaets, the French authors discussed possible affinities, but were able only to stress the distinctiveness of this species. The same is true of the much fuller description given by Laubier (1961). In a short paper, Gotto (1961~)made the

THE ASSOCIATION OF COPEPODS WITH MARINE INVERTEBRATES

29

tentative suggestion that P. petiti might be derived neotenously from monstrillid or monstrillid-like ancestors-an idea partly based on the occurrence of nutritive filaments in both larval monstrillids and adult Phyllodicola. However, Laubier (1966), while not denying the possible evolutionary significance of neotenic processes, considers the monstrillids as structurally too dissimilar to be thought of as ancestral forms. Liitzen (1964b) has transferred the genus Cyclorhiza (Heegaard, 1942), also a parasite of phyllodocids, from the Herpyllobiidae to the Phyllodicolidae-a much more convincing taxonomic niche. It would, however, be interesting to know just how closely the long ovisacs of Cyclorhiza resemble the very peculiar egg strings of Phyllodicolapetiti (see Laubier, 1966). The Melinnacheridne owe their status as a familial entity to Lutzen (1964a) who originally proposed the name Saccopsidae. Further discoveries, however, and restudy of the genus Melinnacheres (Fig. 51) have shown this latter to be congeneric with the later-described Saccopsis, which therefore becomes a junior synonym (Bresciani and Lutzen, 1975). The three known species are externally located parasites of terebellid polychaets, and although their very simple habitus would suggest profound internal modification, this is not altogether the case. Bresciani and Lutzen, in fact, were able to verify " the presence of nearly all the organ systems which could only be expected in a far less deformed parasitic copepod." Although sometimes considered to be nereicolids, they have more frequently been ascribed to the Herpyllobiidae (Hansen, 1892; Haddon, 1912). This view is not, however, supported by the recent detailed studies mentioned above. I n particular, the structure of the dwarfed males differs in the two groups, as does the degree to which the female's body is implanted in the host. The development and differentiation of the alimentary system is also dissimilar.'Lutzen (1966) believes that an ancestral type resembling the melinnacherids may have provided an evolutionary starting point for the herpyllobiids. At present, however, we must regard the affinities of these copepods as uncertain. Our last family, the Herpyllobiidae, comprises forms so aberrant that several authors have accepted a category-the Herpyllobioida, above familial level for their reception. The seven genera formerly attributed to this group have now been reduced to three-Herpyllobius (Fig. 52), Eurysilenium and, more doubtfully, Phallusiella (Liitzen, 1964b). All are associates of polynoid worms, and consist essentially of an ectosomal body portion situated outside the host, and an endosomal portion located within. Appendages are completely absent. The males, where known, are dwarfed, bottle-shaped forms attached to the female,

30

R. V. QOTTO

through whose skin they transmit sperms via filamentous tubules. Possible affinities of this extraordinary group have already been touched on when dealing with certain of the families mentioned above. It must be admitted, however, that apart from the suggestion that these copepods may have arisen from melinnacherid-like predecessors, their precise relationships are still quite obscure. There remain a number of anomalous genera deserving some attention. These comprise associated copepods which, for one reason or another, have so far defied attempts to place them in the context of an established family. The subjoined list (in alphabetical order) is not exhaustive. I have, for example, omitted (a) forms which have been described so briefly, vaguely, or otherwise inadequately, as to be virtually unrecognizable even if re-discovered, (b) forms now recognized as synonymous with established genera, and (c) forms not seen since their original description, but which are currently assignable with B fair degree of certainty to known family units. Practically all those listed, however, require a more searching investigation than has hitherto proved possible. Akessonia. One species, A. occulta Bresciani & Lutzen 1962, in the coelomic cavity of the sipunculid Goljngia minuta (Keferstein), west coast of Sweden (Fig. 53). Length of female lefimrn, of male 0.5 mm. Female body cylindrical, oblong, with a number of blunt diverticula. Presence of antennules and antennae problematical. Mandibles of conical type, and one other large post-oral appendage, probably a maxilliped, present. Posterior end of body pointed. Egg strings very long, coiled, eggs arranged uniserially. Male with small naked furca, and one pair of two-jointed head appendages, ending in strongly chitinized hooks.

Apodomyxon. Two species, Apodomyzon brevicorne Stock and A . longicorne Stock (1970a) both in the sponge Haliclona indistincta (Bowerbank), Roscoff, France (Fig. 54). Length of female 0.84 mm, of male 1.0 mm. Body small, gherkin-shaped, unsegmented. Four minute terminal setae probably represent caudal rami. Antennules well segmented and possessing in the male, a sub-chelate structure. Antenna 4-segmented. Mandible a simple stylet. Siphon large, pear-shaped. Legs absent. Considered by Stock to be the most profoundly modified siphonostome genus known at present. I n certain respects, resembles some members of the Artotrogidae sensu Eiselt (1962). Arthrochordeumium. Two species, A . appendiculosum Mortensen & Stephensen 1918, from the ophiuroid Astrocharis gracilis, western Pacific, and A . asteromorphae Stephensen 1933, from the ophiuroid

THE ASSOOUTION

OF COPEPODS WITH

M ~ R I N E INVERTEBRATES

31

Asteromorpha koehleri (DGderlein),western Pacific (Fig. 5 5 ) . Both found in swellings at the base of the host’s arm. Female body, 1-1-26 mm long, with more or less distinct segmentation (especially anteriorly) and with 3 pairs of short lateral processes. Antennule 1-segmented,bipartite at tip. Antenna (or ? maxilla) %segmented. Masses of very small eggs wrapped irregularly around body. Male ( A . appendiculosum) 1.7 mm long, body sausage-shaped or saccate, curved, lacking distinct segmentation. Antennule I-segmented, with bipartite tip. ( ‘2 ) Maxilla 3-segmented, with terminal claw. Axinophilus. One species, A . thyasirue Bresciani & Ockelmann 1966, attached to the anterior adductor muscle of the bivalves Thyasira Jtezuoaa (Montagu) and T.sarsi (Philippi), North Sea, Norwegian Sea, Baltic Sea (Fig. 56). Female body about 2 mm long, divided into distinct cephalic, metasomal and abdominal portions. Segmentation almost obsolete. Two lateral, distally tapering horns arise from oral area, and are embedded in the host muscle. Metasome with a pair of large lateral wing-like expansions, containing the ovaries. Abdomen with well-developed genital portion, followed by a long, fusiform, tapered part. No caudal rami. Antennules and antennae minute, poorly segmented. No other appendages present. Double ovisacs on each side, resembling a pair of bent sausages. Male unknown. A very similar, possibly identical copepod has been found in Thyusiru gouldi (Philippi) from west Greenland and Spitzbergen. Briarella. Three species (which may or may not be valid) in various nudibranchs from the western Pacific and Red Sea. General resemblance to Echiurophilus, but with shorter lateral processes and only two pairs of legs (Fig. 57). Placed by Delamare Deboutteville and Nunes-Ruivo (1955)in the Splanchnotrophidae, but not considered a member of this family by Laubier (1966). Chordeumium. One species, Chordeumium obesum (Jungersen) (Jungersen, 1912). I n galls on the ophiuroid Asteronyx Zoveni Muller & Troschel, Skagerrak. Female body 4.0-5.3 mm long, sausage-shaped, with distinct cephalon, four thoracic segments and unsegmented “ postabdomen ” (Fig. 58). Antennule 1-segmented. Antenna 1-segmented, papilliform. Maxilla 3-segmented, with hook. No mandibles or maxillules. Legs uniramous, 1-segmented. Male (2.0 mm long) slender, sub-cylindrical, curved. Codoba. One species, C . discoveryi Heegaard 1951, from a gall in the ophiuroid Ophiura meridionalis (Lyman), South Georgia (Fig. 59). Female only known. Within the gall, the copepod is enclosed by a mem-

55

THE ASSOCIATION O F COPEPODS WITH MARINE INVERTEBRATES

33

brane, which also contains loose eggs. Length approximately 2 mm. Cephalic and first thoracic segments fused, otherwise clearly demarcated into five thoracic and three abdominal segments. The first three thoracic segments bear small lateral extensions. Caudal rami naked. Antennule long, slender, many-jointed, highly setose. Antenna and mandible apparently missing. Mouth cone present. ( 1 ) Maxillule with basal joint and two branches, each tipped with a seta. ( 1 ) Maxilla trimerous, terminating in a sickle-shaped claw. Maxilliped also trimerous, bearing terminally a short conical claw. Four pairs of reduced, biramous pereiopods are illustrated, but not further described. Heegaard’s account would lead one to suppose that Codoba is a strongly modified siphonostome. The presence of a membrane enclosing both the copepod and her eggs is somewhat reminiscent of the condition found in the nanaspid Allantogynus-likewise an internal parasite of echinoderms. Conchocheres. One species, C. malleolatus G. 0. Sars 1918, from the pallial cavity of the septibranch bivalve Neaera (now Cuspidaria) obesa LovBn, west coast of Norway (Fig. 60). Female body slender, 3.3 mm long. Cephalic region with blunt lateral protuberance on each side. Antennule 7-segmented. Antenna with powerful claw. Legs 1-4 well developed, leg 5 uniramous. Caudal rami slender, with setae. Egg sacs curved, sausage-shaped. Male 1.6 mm long, cyclopiform, lacking cephalic protuberances. Considered by Sars to be a clausiid, but this view is not altogether shared by Wilson and Illg (1955) who reserve judgment until more detailed studies can be undertaken. Cucumaricola. One species, C. notabilis Paterson 1958, in amorphous cysts from the coelom of the holothurian Cucumariafrauenfeldi Ludwig, South Africa (Fig. 61). Mature females 20-40 mm in length, with elongate, curved cylindrical body, indistinctly segmented. Antennules, antennae and maxillipeds present, the latter as bulbous protuberances. Three pairs of peculiarly lobed legs, the second and third pairs massively developed. Caudal rami large, fleshy, digitiform. Small ovisacs, containing numerous eggs, are deposited within the cyst. Male up to 5 mm long, with a cylindrical body consisting of a small cephalothorax and six well-defined trunk segments. Three pairs of relatively short legs. Caudal rami fleshy, up to two-thirds the length of the body. Paterson suggested chondracanthid affinities for this genus. Bouligand and 66. Arthrochordeumium mteromorphae ( l . l ) ,ventral. 66. Axinophilus thyakrae (2.0), dorsal. 67. Briarella risbeci (13.0), ventral. 68. Ghordeumiuna obesum (4.0), lateral. 69. Codoba diecoveryi (2.0), ventral. 60. Conchocheres malleolatu8 (3*3),dorsal. 61. Cucumaricola notabdie (40.0), lateral. 62. Dichelina eeticauda (2.1), ventral. 63. FlabelZicola neapolitana (W), diagrammatic reconstruction.

34

R. V. QOTTO

Delamare Deboutteville (1959) believe it sufficiently distinctive to warrant separate familial status.

Dichelina. Two species, D. phormosomae Stephensen 1933, and D. seticauda Stock (1968b) both from deep-water echinids, the former certainly inhabiting the intestine ; Indonesian waters. Female of D. phormosomae 5-7 mm long, of D. seticauda 2.2 mm (Fig. 62). Cephalosome clearly demarcated from unsegmented remainder, caudal area rounded with two setae on each side representing rudimentary caudal rami. Antennule 6-segmented. Antenna 4-segmented. Mandibles and maxillules finger-shaped. Maxilla with pointed claw. Maxilliped 3segmented, with many spinules. First leg 2-segmented, second reduced to a single seta, others absent. Male (D . phormosomae) smaller than female, with prehensile antennule. D. seticauda bears some rather haunting resemblances to the small siphonostomatous nanaspids associated with holothurians, although it is an appreciably larger copepod. As well as a general similarity of habitus, the form and proportions of the antennules, antennae, maxillae and maxillipeds are reminiscent of this family. Furthermore, we know at least one nanaspid genus (Allantogynus) which, like Dichelina, is endoparasitic. It should, however, be stressed that such resemblances as may exist might well be due to mere convergence. Ftabelticola. One species, F . neapolitana Gravier (1918a), from the polychaet Flabelligera diplochaitos (Otto), Gulf of Naples (Fig. 63). Female body an unsegmented, sausage-shaped sac, 3-5-4.0 mm long, lacking appendages, situated inside the host and connected by a short neck to a very small vesicular portion 0.3 mm long, which protrudes through the body wall of the host at sexual maturity, and from which the egg-sacs emerge. No male known. Gravier regarded this copepod as being related to the herpyllobiids. Flabel1iphilu.s. One species, F . inersus Bresciani & Lutzen 1962, from the polychaet Flabelligera afJinis Sars, west coast of Sweden (Fig. 64). Male only described. Body 1 mm long, somewhat cylindrical and slender, tapering distally and with simple caudal rami. Antennules and antennae 4-jointed. Mandibles highly chitinized, with an apical toothed projection and a smaller projection, also with teeth. Maxilliped 4jointed with long terminal claw. Two pairs of rather reduced legs. Bresciani (1 964) considers Flabelliphilus a possible candidate for the Clausiidae, while Stock (1968a) lists it among the genera inquirenda appended to the Nereicolidae.

THE-ASSOCIATION OF COPEPODS WITH MARINE INVERTEBRATES

35

Gonophysema. Probably one species, G . gullmarensis Bresciani & Lutzen 1960, attached to the peribranchial wall of the ascidians Ascidiella aspersa (MiiUer), A . scabra (Muller) and Distomus variolosus Gaertner, southern Scandinavia and western Mediterranean (Fig. 65). Possibly another species from the interstitial ascidian Heterostigma reptans C1. & F. Monniot, western European coastline. Female body up to 7 mm long by 8 mm broad, posteriorly subconical and anteriorly produced into blunt branched diverticula. Antennae vestigial, no other appendages. Ovisacs issue from an apical slit. Very reduced dwarf males lie in a deep ectoderm-lined invagination of the female’s body. Mediterranean specimens from D . variolosus attain sexual maturity a t a much smaller size (Bresciani et aZ., 1970). Ive. One species, I . balanoglossi Mayer 1879, from coelom of the enteropneust Glossobalanus minutus (Kowalevsky),Mediterranean. Female, up to 12 mm long, elongate, somewhat truncate anteriorly, tapering posteriorly to a small furca (Fig. 66). Segmentation very indistinct. Four pairs of small blunt lobes are spaced along body length. Reduced antennules, antennae, mandible and two pairs of legs are described. Eggs in strings. Male up to 5 mm long, vermiform, with reduced number of lobes. Thought by Mayer to have lernaeoid affiities, but suggested by Kesteven (1913) to be a chondracanthid. Lernaeosaccus. One species, L. ophiacanthae Heegaard 195 1, endoparasitic in the ophiuroid Ophiacantha disjuncta (Koehler), Palmer Archipelago, Antarctica (Fig. 67). Female only known. Length 1-5 mm. Entire body bag-like, unsegmented. Anterior end bearing two short lateral processes and a short mouth cone enclosing mandibles. Antennules, antennae and maxillules apparently lacking. Two pairs of mouth parts (2 maxillae and maxillipeds) posterior to cone, the former pair two-jointed, terminating in a small claw, the latter stout, almost globular. Pereiopods absent. Two very large egg sacs, enclosing numerous eggs. Heegaard believed this copepod to be closely related to the Lernaeopodidae. MesoglicoZa. One species, M . delagei Quidor 1906, lying in sub-ectodermal galls on the column of the anemone Corynactis viridis Allman, Atlantic coast of France and western Mediterranean (Fig. 68). Body 6-7 mm long, cylindro-conical, no sexual dimorphism. Antennule, antenna, buccal siphon and three pairs of mouth parts present. Bouligand (1966a) considers Mesoglicola to bear a strong resemblance to the lamippids.

66

64

V

69

6a 67

70

71

72

64. Flabelliphilus inersus, male (1.0), ventral. 65. Bonophysema gullmarensis (2.01, dorsal. 66. Ive balanoglossi (12.0), dorsal. 67. Lernaeosaccus ophiacanthae (1.5), lateral. 68. Mesoglicola delagei (7.0), ventro-lateral. 69. Ophioicodes asymmetrica (2.0),ventral reconstruction. 70. Ophioika tenuibrachia (3.0), dorso-lateral. 7 1 . Ophioithys amphiurae, dorsal. 72. Parachordeumium tetraceroa (0*5), ventral.

THE ASSOCIATION O F COPEPODS WITH MARINE INVERTEBRATES

37

Ophioicodes. One species, 0. asymmetrica (Pyefinch 1940, under Ophioika asymmetrica), from Ophiacantha imago Lyman (probably in genital bursa), southern Indian Ocean and Ross Sea. Female body about 2 mm long, more or less amorphous, with three pairs of large serpentine processes arising ventro-laterally and an unpaired median process near the mouth (Fig. 69). Other processes arise asymmetrically from a dorso-lateral position. I n ovigerous females additional processes occur, and an abdominal region can be distinguished. A median groove is also present, in which lies the male. The female is firmly embedded in host tissue, causing a slight swelling. Egg masses are retained in a cyst of host tissue origin. Male about 1 mm long, simpler, with two lengthy processes. (See Heegaard, 1951.) Ophioika. Three species, 0. ophiacanthae Stephensen 1933, 0. appendiculata Stephensen 1935, and 0. tenuibrachia Heegaard 1951 (Fig. 70), endoparasitic in ophiuroids, all in the genital bursae ; Southern Hemisphere. Female body (about 1.5 mm long in 0. ophiacanthae, and 3 mm in 0. tenuibrachia) a lobate sac bearing a number of serpentine processes of varying length. Four other long, curved processes (‘1 modified thoracic limbs) coalesce with a very thin body wall to form a brood sac, containing four ovate egg masses. Within this sac is a short segmented urosome, terminating in small caudal rami. Two pairs of blunt anterior horns probably represent antennules and antennae. Males dwarfed, sausage-shaped, one or more largely inserted in the female’s body. Heegaard suggests a possible relationship with fish copepods of the family Philichthyidae. Ophioithys. One species, 0. amphiurae (HBrouard 1906, under Philichthys amphiurae), parasitic in the genital bursae of the ophiuroid Amphipholis squamata Della Chiaje at Roscoff, France (Fig. 71). Another possible record from the same host at Rhode Island, U.S.A. (see Fewkes, 1888). Female body globular anteriorly, cylindro-conical posteriorly. Antennules small, conical. ( ?) Antennae large, bifid, unsegmented. Two pairs of tubercle-like mouth parts are described. A large lateral, bifurcated projection occurs on each side of the swollen body and a single pair of filiform processes on the more slender ‘‘ abdomen ”. Male dwarfed (0.5 mm long), nearly triangular in form, with, anteriorly, a pair of small hooks which attach it to the female, and a pair of filiform processes. Apparently destroys the reproductive capability of the infected host bursa. Thought by HBrouard to be referable to the fish-parasitizing genus Philichthys, but transferred by Heegaard (1951) to the new genus Ophioithys, which he believes is close to Ophioicodes. If a connection with the philichthyids is indeed tenable,

38

R. V. QOTTO

relationship with a chondracanthoidean complex is again implied (Delamare Deboutteville and Nunes-Ruivo, 1955).

Parachordeumium. One species, P. tetraceros Le Calvee 1938, from the coelom of the ophiuroid Amphipholis squarnata, Villefranche-sur-Mer, France (Fig. 72). Female only known. Body almost square, very small (0.5 mm diameter) with no distinct segmentation, but bilaterally symmetrical. At anterior end, two pairs of large, blunt horns, possibly for absorption of nutrients, are lodged in the host's stomach. Posterodorsally a median ridge or keel separates two lateral folds containing eggs. Ventro-medially a pair of stout, curved appendages act as pincers to hold the copepod in position close to the pore of the gall. Posterior to these are a pair of flat, slightly curved and tapered processes. Two pairs of ventrally situated tubercles complete the tally of possible appendages. Alimentary canal absent. Le Calvez considered this copepod to have affinities with Arthrochordeumium. Pionodesmotes. One species, P. phormosomae Bonnier 1898, in galls on the inner test wall of the echinoid Hygrosoma petersi (A. Agaeziz) (Fig. 73). Body of female almost spherical, about 2.7 mm long. Segmentation still apparent in thoracic region. Seven-segmented antennules, 4-segmented antennae, rudimentary mandibles, a pair of maxillae and a pair of maxillipeds are described. Caudal rami reduced. Long, curved egg strings. Male smaller, up to 1.9 mm, less modified. The galls measure 7-11 mm in diameter and communicate with the exterior by a small hole, 1.6 mm in diameter. Bonnier drew attention to resemblances with such forms as the choniostomatids, the nereicolids and the herpyllobiids, but felt it was sufficiently distinct to warrant separate familial status. Wilson (1932) included it in his group Lernaeopodoida. Sponginticola. One species, S. uncifer Topsent 1928, from the sponges Cliona celata Grant, lMycale macitenta (Bowerbank) and Stylopus coriaceus Fristedt, western coasts of Europe, Irish Sea and Mediterranean (Fig. 74). More fully described by S i l h (1963) under the name

Clionophilus vermicularis and commented upon by Stock and Kleeton (1964). Body vermiform, unsegmented, shape variable due to elasticity of cuticle. Female 1 mm long, male 0.8 mm. The sole oral appendages are 3-jointed maxillipeds terminating in a hook. There are two pairs of caudal claws at the posterior extremity. Sil6n regards this copepod as probably related to the lamippids.

Teredoika. One species, T . serpentina Stock (1959a),from the stomach of the bivalve Teredo utricutw Gmelin, Gulf of Naples (Fig. 75). Female

THE ASSOCUTION OF COPEPODS WITH MARINE INVERTEBRATES

39

73 74

~

75

76

73. Pionodestnotes phormosomae (2-7), ventral. 74. Sponginticola uncifer (l-O), ventral. 76. Feredoika serpentina (2*6),dorsal. 78. Ubiua hi% (6.41, lateral.

body 2.5 mm long, with a central, vermiform part and four lateral serpentine processes. No cephalic appendages or legs. Two ovisacs, four-lobed in outline, containing multiserially arranged eggs. Male unknown (see also Stock, 1961).

Ubius. One species, U . hilli Kesteven 1913, from enlarged edges of the genital wings of the enteropneust Balanoglossus australis (Hill), New South Wales (Fig. 76). Female 6.4 mm long, with cylindrical unsegmented body, abruptly truncated anteriorly, posterior eighth tapering to a point. Antennules l-jointed, triangular in shape. Antenna apparently 3-jointed, the second and third joints constituting a chela. Mandibles 2-jointed, the second segment small, flattened and triangular, with a notched apex. Fourth and fifth appendages (? pereiopods) biramous, with unimerous endopodite deeply notched terminalIy, and unimerous exopodite of similar appearance, but chelate rather than notched. All appendages extremely small. Anus absent. Ovary paired.

40

R. V. QOTTO

Male 2.6 mm long, similar in general shape, but posterior extremity bifid. Kesteven believed that Ubius should be classified with the Chondracanthidae.

IV. GENERAL STUDIES OF SINGLE SPECIES Although short notes of general biological interest are scattered through many of the mainly systematic accounts cited above, more extensive studies devoted to single associated species are comparatively few in number. However, some ecological aspects of Ascidicola rosea Thorell, a copepod living in a variety of ascidian hosts, have been investigated by Gotto (1957). By studying its association with the very transparent Corella parallelogramma (Miiller),it was possible to observe this commensal’s feeding behaviour and general pattern of activity. Ascidicola rosea feeds on particles which it removes from the food-string as the latter is passing through the host’s oesophagus. Since the string is in constant, slow, downward movement, the copepod adjusts its position periodically by upward climbing. When active feeding is not taking place it remains quiescent in the oesophageal funnel. Sometimes an inverted position is adopted ;this is generally associated with meagre development of the food-string. Certain peculiar structural featuresnotably the spinous pad on the penultimate segment and the very long, finely serrated endopodal setae-are shown to be adaptive, assisting the copepod to maintain its hold. Feeding and orientation of Sabelliphilus elongatus M. Sars on its fanworm host Sabella pavonina Savigny have also been investigated by Gotto (1960). It is believed that this copepod’s characteristic alignment on the worm’s branchial filament (with the head towards the filament’s base) might be achieved by its sensitivity to the unidirectional beat of cilia on a small rejection tract which runs along the lateral surface of the filament. Although S a ~ e l ~ i ~elongatus h i l ~ is found on the food-gathering organ of its filter feeding host, there is strong evidence to suggest that its life style is parasitic rather than commensalistic-an interpretation reinforced by the observations of Carton (1967) on the closely related 8.sarsi Claparbde, which lives on the body of an allied fan-worm, Spirographis spallanzani Viviani. Another annelidicolous species, Eunicicola insolens (T. & A. Scott), studied by Gotto (1963), occurs on the polychaet Eunice harassii Audouin & Milne-Edwards. This copepod uses its large ventral sucker as an adhesion mechanism when clinging to the smooth body surface of its host. When it moves on to the finely pectinate gills, however, minute antenna1 suckers come into play. These tiny discs, mounted on

THE ASSOCIATION OF COPEPODS WITH

MARINE INVERTEBRATES

41

the last antenna1 segment, possess a remarkably wide range of movement. Some ecological notes on Octopicola superbus Humes have been supplied by Delamare Deboutteville et al. (1957). This cyclopoid remains in the pallial cavity of Octopus vulgaris Lamarck by day, but becomes more active after dark, moving out along the arms of its host. The biology of the mussel-infesting Mytilicola intestinalis Steuer has claimed the attention of several workers-an interest not altogether surprising when we remember that this is one of the few associated species of major commercial significance. Hockley (1951) observed that large female parasites (up to 8 mm in length) prefer the recurrent intestine or rectum of the mussel, occupying most of the lumen. They appear to be strongly thigmotactic, loss of contact with the gut wall evoking vigorous peristaltic contractions. Adult females are orientated with the head towards the oncoming food, whereas the smaller males move about more freely. Grainger (1951) found the heaviest infestations in that part of the recurrent intestine which is embedded in the digestive gland. He also noted a tendency for Mytilicola to move down the gut of mussels kept in poorly aerated water. I n the context of this last observation, K8 (1961) investigated the survival of three cyclopoids (Ostrincola koe Tanaka, Conchyliurus quintus Tanaka and Modiolicola bijidus Tanaka) in the clam host Tapesjaponica Deshayes exposed to air for varying periods. Even after six days exposure, a few copepods were still alive. Mason (1959) has contributed much useful information on Nicothoe astaci Audouin & Milne-Edwards, the gill-infesting parasite of lobsters. He points out that when the lobster moults, the copepods are shed along with the discarded shell and die, reinfestation by settlement of last stage copepodids only occurring in significant numbers shortly after the moult, before the lobster’s gills have hardened. Excellent comprehensive studies on the ecology of two siphonostomes, Scottomyzon gibberum (T. & A. Scott) on Asterias rubens L. and Asterocheres lilljeborgi (Boeck) on Henricia sanguinolenta (Muller),have been carried out)by Rbttger (1969) and Rbttger et al. (1972). Preferred host site, food and feeding method, growth and egg number are among the topics covered. Another associate of sea-stars, the curiously transformed lichomolgid Botulosoma endoarrhenum Carton, has been recently investigated (Carton, 1974). Females live in an integumental cavity of the host (Othilia purpurea Gray) whilst the males move freely in the general coelomic space. On the basis of histological studies, Carton believes that the invasive stage gains access to the host via the thin tissue of the

42

R. V. OOTTO

papulae which project through perforations of the asteroid’s thick body wall. Changeux (1961) has provided a very detailed account of AEZantogynus delamarei Changeux, now recognized as a nanaspid, from the body cavity of two holothurians, Holothuria stellati Marenzeller and H . tubulosa Gmelin. Many aspects of ecological interest are appended to the descriptive part of this paper. The ecology of Paranthessius anemmiae Claus, a sabelliphilid inhabiting the body surface of the snake-locks anemone Anemonia sulcata (Pennant) has been well covered by Briggs (1976). As well as a two year population study, Briggs carried out some interesting immunity and host-transfer experiments, and also investigated the ectoderm of Anemonia in the context of its significance as a habitat for Paranthessius.

V. ANATOMICAL AND FUNCTIONAL ASPECTS A, Integument Few papers deal with the integument of copepods in general and fewer still with that of associated species. A detailed description, however, is given by Briggs (1974, unpublished thesis) for Paranthessius anemoniae. At EM level, division of the cuticle into endocuticle, exocuticle and epicuticle is apparent, with the exocuticle subdivided into an inner electron-dense zone and a thinner, outer layer which presents a honeycombed appearance. The epicuticle seems to be of a membranous nature, and is covered by a surface coat or “ fuzz ”. A hypodermis of flattened cells underlies the cuticular elements. Unicellular hypodermal glands, opening to the surface by pores, are probably responsible for secreting the “ fuzz ” which clings to the epicuticle, and which is thought to consist mainly of mucus. Briggs believes that substances may be secreted by these glands conferring immunity to the toxins elaborated by the host anemone. Bouligand (1966b) has studied the integument of a number of copepods, including the associated PennaEulicola pteroidis (Della Valle), Ascidicola rosea, Notopterophorus elongatus Buchholz and four lamippid species. I n barely modified associates, he finds the structure to be essentially similar to that of free living copepods, and is much the same as that described above for Paranthessius. I n the strongly transformed lamippids, however, the cuticle is characterized by the predominance of zones suggesting greater flexibility. Linaresia mammillifera (De Zulueta), a lamippid lacking a clearly differentiated gut, possesses minute villosities on the surface of the epicuticle. Sections of this copepod in situ reveal that these tiny projections are in intimate contact with

THE ASSOUIATION OF COPEPODS WITH MARINE INVERTEBRATES

43

the cells of the octocorallian host Paramuricea chmaeleon (Von Koch), and the inference may be drawn that absorption of nutrients can occur at the host-parasite interface. Microvilli-like projections arising from the epicuticle have also been observed in two other internal parasites, Gmqhysema and Anthemheres (Bresciani and Lutzen, 1972). It may be noted here that some associates possess a remarkably thin and soft integument. This is the case, for example, in the annelidicolous Acaenomolgw protulae Stock (Stock, 1969b; Gotto, 196lb). The latter author has suggested that such a delicate cuticle might possibly facilitate respiratory exchange when the host worm (Protula intestinum (Lamarck))withdraws into its tightly sealed calcareous tube. At such time, the copepod would be enveloped by the closely packed mucusladen filaments of the pseudobranchial fan-a situation in which the efficient use of available oxygen might be of critical importance. One further cuticular attribute of some associated species may be mentioned, namely a capacity for extra-ecdysial growth. This has been noted in Nereicola ovatus Keferstein, by Laubier (1966) and analysed in Nimthoe astaci by Bocquet et al. (1968). After the copepodid of N . astaci has moulted for the last time, the second, third and fourth thoracic segments undergo lateral hypertrophy, forming large " wings " which enclose elemenh of the reproductive and alimentary systems. These wings continue to grow in spectacular fashion relative to the remaining, unaltered part of the body, but without any further moult taking place. Bocquet and his colleagues consider that the wing-forming metameres, under genetic control, alone possess epithelial cells capable of prolonged mitotic activity and able to secrete a cuticle which permits continued growth-certainly a novel development in the general context of arthropodan growth patterns. It seems likely that a similar facility could account for the often monatrous deformation seen in certain other parasitic copepods.

B . Sensory structures There are not many detailed modern accounts of sensory receptors in associated copepods. A notable exception, however, is the work of Dudley (1969)on the fine structure and development of the nauplius eye in the ascidian-dwelling notodelphyid Doropygus sectusus Illg. Using electron microscopy, Dudley found the eye of adult and copepodid stages to consist of forty cells-two central primary pigment cells, eight accessory pigmented glial cells, six tapetal cells and twenty-four photosensory retinular cells. The retinular cells have a microvillous borderthe rhabdomere-nearest the tapetum. The general arrangement of the microvilli is such that they would be perpendicular to incoming light.

44

R. V. GOTTO

Innervation is purely efferent. During the h s t four naupliar stages, neither rhabdomeres nor axons are apparent, but these begin to form during the fifth and final naupliar instar. All the components of the adult eye are present in at least rudimentary form in the free-living first copepodid, although microvilli are still differentiating as is the pigment in the accessory pigmented cells. The second copepodid has an eye smaller than that of the adult, but otherwise very similar. It is at this (host-infective) stage that a marked change from positive to negative phototaxis takes place, the copepodid apparently sinking down to enter its benthic host. Absence of eyes has been reported in the ascidicolid Styelicola bahusia Lutzen. Since the styelid hosts have opaque, leathery tests and occur in depths exceeding 100 m, the lack of visual organs is presumably related to the permanent darkness in which this commensal must live (Liitzen, 1968b). The occurrence of cuticular hair-like structures, of probable sensory function, has been noted from time to time. Kabata (1966) describes such “ hairs ”, with a length of 10 p, arising from the cephalothoracic rim of Nicothoe analata Kabata, and seemingly associatedwith fine ducts traversing the cuticle. Briggs (1974, unpublished thesis) has also observed cuticular r‘ hairs ” on the dorsal surface of Paranthessius anemoniae. They are about 4 p long, are spaced some 20-30 p apart, and are situated in cuticular cups. Such “ hairs ” have an electron-dense core which divides into two flanges near the base. These flanges overlie an ovoid, electron-dense body from which a cytoplasmic area passes down through the cuticle towards the hypodermis. Briggs compares them to the hair-peg organs of the lobster (Laverack, 1962)and believes that they are concerned with rheoreception, pointing out that Paranthessius is markedly sensitive to local water currents when installed on its anemone host. There can be no doubt that chemosensitivity in general is of great significance in the life of associated copepods (see, e.g. Gotto, 1962; Carton, 1968a). It may indeed prove of paramount importance in establishing contact with a host, but almost nothing is known about the receptors involved. .However, a sense organ which may well function in this way has been recently described by Dudley (1972)in the nauplii and copepodids of the ascidicolous Doropygus seclusus. This receptor, located bilaterally in the anterodorsal head region, is composed of dendrites of extra-optic protocerebral origin which have ciliary protrusions with basal bodies, no rootlets, and a basal infrastructure of the 9 0 type. The cilia do not branch and their distal terminations contain only one to four microtubules. I n nauplii and free-livingcopepodids,

+

THE ASSOCIATIOIG' O F COPEPODS WITH MAFLINE INVERTEBRATES

45

a large epidermal supporting cell encapsulates the end of one dendrite and its cilia in a sac. Other dendrites and their cilia pass through the supporting cell. Terminally, these latter cilia escape to form a whorled fascicle which contacts the anterolateral cephalic cuticle-an area, in nauplii at all events, of apparent specialization, as evidenced by the presence of minute striations. This end organ reaches its greatest development in the second copepodidinstar-the stagewhichinvades the host ascidian. All the subsequent symbiotic sta.ges of the copepod have a proportionately smaller organ of the saccular type only, apparently lacking the organ consisting of whorls of ciliary ends. Dudley suggests that the organ which disappears in the symbiotic stages is used by second copepodids in host recognition. Gotto (1959, 1962) has drawn attention to the frequent utilization by copepods of hosts such as bivalves, ascidians and other invertebrates which constantly expel a water current, and believes that these exhalant streams, presumably loaded with metabolites and other clues as to their origin, may provide a chemical " homing beam for infective larval stages. Possibly it is to just such a current that this enigmatic end organ of D. seclusus is receptive. I n the same paper, Dudley mentions the exaggerated development of the antennular aesthetascs in the second copepodid. Crustacean aesthetascs in general have, of course, long been regarded as chemosensory structures. However, in so far as associated copepods are concerned, no detailed study of them seems to have been published. ')

C. Food and feeding Speculation in the literature regarding the nature of food taken and the mechanics of obtaining it, is more frequently encountered than hard factual data. Nevertheless, some observations have been made, and the more significant of these are listed below. It will perhaps be useful to attempt a rough classification of feeding methods in the light of our present fragmentary information. It is entirely possible that the categories suggested may ultimately prove inappropriate, and that modifications will have to be made as data accumulates. At best, therefore, the following should be considered merely as guide lines for future investigation. 1. Debris feeders

Under this heading we can probably place such forms as Lichomolgides cuanensis Gotto which is found only in the large cloaca1 cavities of its compound ascidian host Trididemnum tenerum (Verrill) (Gotto, 195413). In this situation, the copepod would be bathed in a constant

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R. V. GOTTO

exhalant stream rich in faecal material, mucus strands and other small organic particles. Very likely the same is true of Zygomolgus didemni (Gotto) occurring in colonies of the sea-squirt Didemnum maculosum (Milne-Edwards) (Gotto, 1956). The ovoid shape of the notodelphyid Ooneides ameba Chatton & BrBment, with greatly reduced appendages and genera1 immobility, suggests similarly passive feeding habits in the cloaca1 cavities of didemnids. The clausidiid genus Hemicyclops is regarded by Gooding (1963, unpublished thesis) as perhaps representative of the most primitive poecilostomes, and the feeding habits of these copepods are therefore of some interest. MacGinitie (1935) believes that H . thysanotus Wilson, from the gill chamber and body surface of callianassid shrimps, may remove debris from the host’s eggs and, if so, it may also help to keep the gill chamber clean. On the other hand, the droplets of red material appearing among the organs of the prosome in this species may be products of carotenoid metabolism originating from the host itself, and thus imply a food source other than incidental debris. Eunicicola insolens, an associate of eunicid polychaets, may also come into this general category. Certainly the tiny fringed scoops which adosn the mandibles, together with the rapid fore-and-aft twitching movement of these mouth parts (Gotto, 1963) could sweep minute particles of organic material towards the oral aperture. 2.

‘‘ Larder ” feeders

These could be considered as commensals or mess-mates in the original strict sense of the term. However, as commensalism has acquired so many other connotations over the years, it seems necessary to emphasize the purely trophic aspect implied by the term “ larder ”. Various degrees of larder feeding can of course be discerned ; one might say that some copepods have penetrated further into the host’s larder than others. Most of the Notodelphys species living in the ascidian pharynx are almost certainly feeders of this type, removing particles from the mucus sheets which line the pharyngeal wall. The ascidicolids Styelicola and Ascidicola are a little more specialized. Less mobile than the notodelphyids, they are virtually restricted to the food string as it passes through the oesophagus of the ascidian and thus obtain food in more concentrated form (Lutzen, 1968b ; Gotto, 1957). Pachypygus gibber (Thorell), a bulky and sluggish notodelphyid, also prefers the oesophageal region, and likewise appears to rely on the host food string (Gotto, 1955). Some, though not all, of the cyclopoids associated with the pseudo-

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branchial fan of sabellid and serpulid polychaets, are probably larder feeders. Thus Acaenomolgus protulae, from the serpulid Protula tubularia (Montagu), has been observed clinging to the frontal surface of the branchial filaments (Gotto, 196lb). I n this position it is, of course, astride a food-collecting tract. The same may hold for Acaenomolgus serpulae (Stock), from Serpula vermicula~isL., though in this case we lack detailed information as to the precise position of the copepod vis-&-visthe host’s filament. Dudley (1964)believes that a similar mode of food gathering is practised by most of the gastrodelphyids which live on the branchial fans of sabellids. Deeper penetration of the host alimentary system is achieved by a number of associated species. One could probably regard such gutdwellers as parasites in the generally accepted sense. However, Hockley (1951) found that the intestinal wall of mussels infected by Mytilicola intestinalis was not directly attacked, and it appears that this large copepod subsists solely on the food present in the host’s gut. Hockley also describes the feeding process : the maxillules contribute to this only slightly, food being pushed into the mouth by action of the maxillae. I n ascidian hosts, several species of Enterocola likewise inhabit the stomach and presumably live in the same way as Mytilicola (Brbment, 1911). I n Enteropsia sphinx (Aurivillius),however, an associate of the colonial tunicate Diazona violacea Savigny, only the immature stages occur in the stomach ; mature females migrate through the long, slender oesophagus to reach the more capacious pharynx (Gotto, 1961a). A much transformed clausiid genus, Entobius, has been found in the midgut of terebellids (Dogiel, 1908; Gotto, 1966) and the peculiar little Zanclopus in the stomach of pterobranchs (Calman, 1908). Almost 7 000 specimens of the small siphonostome Collocherides astroboae Stock have been recorded from the stomach of a single basket-star, Astroboa nuda (Lyman),by Humes (1973). It is probably safe to assume that all these, along with Enterognathus from the intestine of crinoids, utilize the food already ingested by the host. 3. Mucus feeders

A considerable number of marine invertebrates secrete large quantities of mucus, and since this substance contains many molecules of potential food value, it almost certainly constitutes a major nutrient source for a variety of associated copepods. Although few direct observations have been made, we may cite the recent work of Yoshikoshi and K6 (1974) on the sabelliphilid Modiolicola bijdus, the claudidiid Conchyliurus quintus and the ergasilid Ostrincola koe. All three inhabit the mantle cavity of the clam Tapes philippinarum (Adams & Reeve).

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Histochemical tests on the midgut contents of these species reveal the presence of protein containing sulphated muco-substances markedly similar to the secretions produced by the mucus glands of the host gill. Only a negligible amount of tissue or cellular debris of gill origin can be detected. It may therefore be concluded that mucus secreted by the clam gill is the principal food of these copepods. Briggs (1977a) in a careful analysis of feeding in the actinian-infesting Paranthessius anemoniae, likewise regards mucus as the chief food taken. However, copepods transferred from the usual host (Anemonia sulcata) to red specimens of Actinia equina L. take up the colouration of this alternative host within 48-72 hours, suggesting that host pigments -mainly xanthophyll esters-are also being absorbed. Lipid present in the columnar cells of the commensal’s gut shows marked depletion in starved copepods, and survival in the absence of a host for periods of up to ten days testifies to the importance of such stored material in the trophic economy of associated cyclopoids. Briggs has furthermore shown that the column ectoderm of Anemonia possesses microvillus-like extensions. If, as seems likely, these structures are concerned with nutrient absorption from the surrounding water, an additional source of food might be available to a copepod capable of “ grazing ” on such a surface. Some preliminary experiments on the clausiid Synaptiphilus tridens (T. & A. Scott), an eoto-associate of the burrowing holothurian Leptosynapta inhaerens (Muller),again indicate that mucus is utilized as food, though brownish-red pigment granules (presumably of host origin) are often present in the copepod’s gut. That pigment ingestion is not, however, vital to Synaptiphilus is suggested by lengthy survival on hosts which have become clepigmented under laboratory conditions (Gotto, unpublished). S. tridens shows a marked preference for the anterior third of the leptosynaptid’s body, and some admittedly rather crude staining experiments reveal that the mucus glands located in this region elaborate a somewhat different secretion to that produced by other areas of the host integument. I n passing, it may be noted that mucus-eating habits are no monopoly of associated species. Richman et al. (1975) have found that the free-living reef copepod Acartia negligem Dana actively feeds on mucus particles produced by reef corals, assimilating up to 50% of the organic matter present. Coral mucus has been shown to contain energy-rich wax esters similar to the wax found in many pelagic copepods (Benson and Lee, 1975). It may well be that the availability of these substances could, in part at least, account for the large and varied assemblage of copepods now known to be coral associates.

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4. Integument feeders

Copepods which utilize elements of the host integument may represent a step from mucus feeding towards more committed parasitism. Sabelliphilus elongatus erodes the epithelial tissue of its host’s branchial filaments, and its gut contents appear as a rust-brown mass, matching almost perfectly the carotenoid pigments of its partner, Sabella pavonina (Gotto, 1960). There is indeed some evidence that this copepod shows a preference for locating itself on the narrow, heavily pigmented bands which occur a t regular intervals along the filament. Globules of fat or oil, possibly associated with the carotenoids, can also be detected in the alimentary canal. It is interesting to note, however, that Carton (1967) considers adult Sabelliphilus sarsi, living on the body of the sabellid Spirographis spallanzani, to feed on secretions rather than host tissue, since the epithelium in this case suffers only minimal damage. Rottger (1969) and Rottger et al. (1972) have demonstrated that two siphonostomes of asteroids-Scottomyzon gibberurn on Asterias rubens and Asterocheres lilljeborgi on Henricia sanguinolenta-digest the host skin extra-intestinally before sucking the food into the pharynx. No doubt many other siphonostomes feed in a similar manner. 5. General tissue feeders

It must be frankly admitted that this heading covers a field of which our ignorance is almost total. Sil6n (1963) studied Rponginticola uncifer Topsent (under the name Clionophilusvermicularis SilBn), an anomalous form living in the canal system of various sponges. S i l h believes that this copepod uses its appendages to tear small fragments from the canal wall, which are then ingested by the suctorial mouth. Gerlach, quoted by Humes (1960a) has observed living specimens of the coral-inhabiting genus XariJia on its host Pocillopora. Although the copepods generally crawl about on the surface of the coral, they may at times enter the polyps, where they seem to tear up the tissue. 6. Blood feeders

Host blood is an obviously rich source of nutriment for those associated copepods sufficiently specialized to obtain it. Nicothoe astaci, with its suctorial mouth and piercing mandibles, is thus admirably equipped to penetrate the soft gill tissue of newly moulted lobsters, and tap the host circulation (Mason, 1958, 1959). Up to 1 700 individuals have been recorded from a single lobster, but such massive infestations are exceptional. Bresciani and Lutzen (1974) have carried out a fine study of the

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xenocoelomid Aphunodomus terebellae (Levinsen), establishing that it too is a blood feeder. An internal associate of the polychaet Thelepus cincinnatus (Fabricius), it adheres to a blood vessel of the intestinal wall. Initial access to the host’s vascular system may possibly be achieved through penetration of the thin walled gills by a small infective stage. The female of the nereicolid Selioides bocqueti Carton, although occurring externally on the polynoid worm Scalisetosus assimilis McIntosh, penetrates the host integument by means of the mandibles and affixes itself to the dorsal blood vessel. Its hold is maintained by the maxillae and maxillipeds (Carton and Lecher, 1963). The nutritional habits of the dwarf male, attached to his female partner, remain problematical, although food material of some sort has been observed in the gut. Another external parasite of polychaets is Melinnacheres steenstrupi (Bresciani & Lutzen) from the terebellid Terebellides stroemi M. Sars. I n this case, attachment to the worm’s gill is effected by a frontal bulla on which opens the anterior part of the alimentary canal, and host blood is pumped into the gut by muscular action (Bresciani and Lutzen, 1961a). The related Melinnacheres ergasiloides M. Sars, however, from the ampharetid polychaet Melinna cristata (M. Sars), shows certain significant differences in alimentary arrangements, which will be commented on in the next section. Kystodelphys drachi Monniot, a species in which only the male is known, is a notodelphyid found in spherical cysts in the branchial circulatory sinuses of the solitary ascidian Microcosmus savignyi Monniot. The cysts, apparently of host origin, are three-layered structures) the middle layer consisting of lymphocytes derived from the host’s blood. These lymphocytes squeeze through the inner cyst wall and thus enter the lumen, where they are eaten by the copepod (Monniot, 1963). Finally, blood is probably utilized as food by the vermiform enterocolid Mychophilus roseus Hesse, frequently encountered in the circulatory canal systems of the compound ascidians Botryllus schlosseri (Pallas) and Botrylloides leachi (Savigny) (Gotto, 1954a). 7. Feeders on other body fluids

Lutzen (1966) has investigated the structure and function of the gut in the herpyllobiids, a group which parasitize polynoid worms, and which have proved difficult to interpret systematically. This is largely because the body consists of two distinct portions, the ectosoma and endosoma, the former external and the latter buried in the host tissue ;

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the two parts are connected by a narrow stalk. A cuticular canal puts the endosomally situated intestine in communication with the host’s coelomic cavity, and in this way the copepod can feed on the body fluids. After digestion, food passes into a mesenchymatous canal system (somewhat modified in Eurysilenium) which transports it to the ectosoma. Lutzen believes that body movements of the host may play an important part in renewing the food content of this curious alimentary system, though he regards diffusion as the chief means of nutrient transport. Melinnacheres ergasiloides, although closely related to the bloodfeeding M . steenstrupi (see above), exhibits one major difference in the make up of its alimentary system-unlike its congener, it possesses an undoubted anus. Bresciani and Lutzen (1975) suggest that differences in utilization of food, or a different food source, could account for this dissimilarity, and speculate on the possibility that M . ergasiloides obtains food from the body cavity rather than from the blood. Food uptake in Gonophysema gullmarensis Bresciani & Liitzen is also dealt with by Bresciani and Lutzen (1960). This peculiar form attaches to the peribranchial wall of simple ascidians and lacks all trace of an alimentary canal. The chitinous covering of the copepod, however, is extremely thin (only 5-10 p ) and it seems likely that the tissue fluid or lymph of the host is taken up by absorption through this layer. The numerous blunt processes which develop over the parasite’s body as it grows would, of course, greatly increase the absorptive area available for food uptake of this type. The large Antheucheres duebeni M. Sam, living in galls on the mesenteries of its anemone host Bolocera tuediae (Johnston), also lacks an alimentary canal. The closed galls contain a clear fluid in which the copepods lie, sometimes as many as twelve in a single gall. Nourishment must again be derived by diffusion through an extremely thin body wall (Vader, 1970). Paterson (1958) offers a similar explanation regarding food uptake by adults of the enigmatic Cucumaricola notabilis, a galldweller in holothurians. Other associates can probably be added to this list of forms which have apparently abandoned the more traditional method of food intake in favour of nutrient absorption through areas of the body surface. Phyllodicola petiti, anchored to the body of a phyllodocid worm, sends two long unbranched rhizoids into the host’s coelomic cavity, which insinuate themselves by contractile movements among the parapodial muscles. Since, in the adult copepod, the mouth has closed over and the alimentary canal regressed, these rhizoids must surely function in an absorptive capacity, taking up nutrients from the coelomic fluid by

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diffusion or osmosis (Laubier, 1961). Similarly, the aberrant lamippid Linaresia mummillifera is thought by Bouligand (1960a)to absorb food material from its alcyonarian host via large capitate projections of the body wall. The cellular structure of these projections indicate that they are sites of considerable metabolic activity, and can presumably compensate for the absence of an alimentary canal. In the same context, we may also recall the lengthy horns of monstrillid larvae, protelean endoparasites of polychaets and prosobranchs. These horns have long been regarded as filling an absorptive role during this phase of the monstrillid life-cycle (Malaquin, 1901). 8. Egg feeders

That host eggs, with their inbuilt reserves of food, should prove attractive targets for certain specialized copepods, is not altogether surprising. Bouligand (1960a) has recorded that the lamippid Enalcyoniurn rubieundum Olsson eats the eggs of its alcyonarian host as well as endodermal debris. However, the most highly adapted egg-eaters are probably to be found amongst the Choniostomatidae, although Hansen (1897) considered members of this family to be blood feeders. This is probably true of those which inhabit the branchial chambers of their crustacean hosts, but the species which live among the host eggs are well equipped for predation on the latter-as has been demonstrated for species of Sphaeronella and Choniosphaera by Connolly (1929),Bloch and Gallien (1933) and Lemercier (1963). Anyone viewing a crab egg mass thus parasitized by Choniosphaera maenadis (Bloch & Gallien), cannot fail to be impressed by the remarkable mimicry between the little globular females and the host eggs among which they live-size, colour and shape match almost perfectly (Gotto, 1970a). More research, however, is required on the feeding habits of choniostomatids in general, since some observations indicate that the presence of adult females actually inhibits host egg production (Bowman and Kornicker, 1967 ; Hamond, 1973). 9. Feeders on other host-elaborated material I n t.his category we can consider such species as Myxomolgus myxieolae (Bocquet & Stock) and M . proximus Humes & Stock, associates of the sabellid genus Myxicola. The host worm secretes a mucous tube in successive layers, and it is between these layers that the copepods are found. According to Bocquet and Stock (1958a)the mucoproteins of the tube probably constitute the most significant part of their diet. Scolecodes huntsmani (Henderson), a very large, vermiform notodel-

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phyid, is found in cysts of host origin in the subendostylar blood vessel of simple ascidians. I n the cyst lumen, a spherical cell containing lipid globules and glycogen has been described by Dudley (1968). These cells arise directly from cyst cells, and have been also observed in the gut of the female Scolecodes, which thus appears to utilize them as food. Another elongate notodelphyid from simple ascidians is Ophioseides joubini Chatton, some ecological aspects of which have been studied by Bresciani and Lutzen (1962). 0.joubini tunnels between the inner layers of the ascidian’s tunic and its epidermis, the galleries so formed being easily followed due t o the deposition of coloured excretory material within them. Chatton (1909) believed that host blood was ingested along with the yellow semi-fluid material resulting from tunicin breakdown. Illg and Dudley (1965) also imply that blood may be utilized by such tunnelers in the host tunic. The siphonostome Allantogynus delamarei is found in the body cavity of sea-cucumbers belonging to the genus Holothuria. Changeux (1961) has observed that this species feeds on matter scraped from the surface of the coelomic epithelium-amoebocytes, epithelial cells, and perhaps other incidental debris.

D. Structural studies of the alimentary canal Comparatively little modern work has been carried out on this aspect. However, Bresciani and Lutzen (1961a, 1975) have given good accounts of the melinnacherid gut, and Lutzen (1966) has similarly investigated that of the herpyllobiids. Carton and Lecher (1963) have included a description of the alimentary canal of the nereicolid Selioides bocqueti in a general anatomical study of this species. Heptner (1968) has contributed a functional and morphological account of the sucking mouth apparatus of the siphonostome Megapontius pbeurospinosus Heptner, and Lemercier (1963) has done much the same for two choniostomatids and Nicothoe astaci. Investigations of the gut which include histological detail are those of Bouligand (1960a, b) for the lamippids, Changeux (1961) for the nanaspid Allantogynus delarnarei, and Briggs (1977a) for Paranthessius anemoniae. Of particular interest in the latter study is the occurrence of characteristic amoeboid cells in the mid-gut wall of P. anemoniae. These each contain a large electron-dense membrane-bound vacuole, and seem to be sloughed off periodically. Changeux (loc. cit.) noted cells of rather similar type in Allantogynus, and considered them essentially excretory in nature. Briggs suggests that the vacuole acts either to concentrate food material prior to becoming loaded with indigestible remnants and subA.M.B.-16

3

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R. V. GOTTO

sequently shed, or else that it is concerned with the selective engulfment and elimination of toxic wastes.

E. Reproduction and allied topics 1. Sexual dimorphism

This shows a wide range of expression in associated copepods, varying from slight to extreme. I n some notodelphyids, it becomes apparent at the fourth or fifth copepodid stage (Dudley, 1966). Apossible case of much earlier dimorphic expression has been recorded by Bresciani and Lutzen (1 962) who found nauplii of the monstrillid Thespesiopsyllus paradoxus (Sars) among the stomach folds of ophiuroids. Some of these nauplii were green whilst other, rather smaller individuals, were pink in colour. The former ultimately developed into female copepods, and it is assumed that the latter would have metamorphosed into males. In general, the sexual differences observable in free-living copepods occur also in associated species-body size, shape of the genital segment and structure of certain head appendages, notably the maxillipeds. I n the cyclopoids dimorphism is also frequently apparent in the antennules and antennae, and in the structure of legs 1 and 5 (Humes and Stock, 1973). Occasionally other features provide additional clues. Very strong sexual dimorphism, for example, has been noted by Stock (1966b)in the siphonostome Collocheres breei Stock, in which the caudal rami of the female are long and slender, but are much shorter and broader in the male. Again, in Acant7~omolgusvarirostratus Humes & Ho, a lichomolgid associated with alcyonarians, the rostrum of the female is rounded whilst that of the male is angled. For some species, too, an antenna1 sucker is the prerogative of the male, as in Aspidomolgus stoichactinus Humes from the anemone Stoichactis. I n a number of siphonostomes, and almost invariably in poecilostomes, the maxilliped shows very clear sexual dimorphism. An interesting exception, however, is provided by Stellicola femineus Humes & Ho, a lichomolgid from asteroids, in which this appendage is very similar in the two sexes. I n modified or transformed species, notable alterations of body shape are frequently confined. to females. Males tend to greater conservatism, retaining a more " primitive " (or at least a less specialized) facies. They are usually more mobile than their consorts and smaller in size. Exceptionally the male may be larger than the female, as in IndomoZgus brevisetosus Humes & Ho, Rhynchomolgus corablophilus Humes & Ho, Temnomolgus eurynotus Humes & Ho-all from coelenterate hostsand the sponge-inhabiting Apodomyzon brevicorne. The trend towards reduced size in males reaches its penultimate

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development with those families in which the males can properly be described as dwarfs-the Melinnacheridae, Choniostomatidae, Splanchnotrophidae and Herpyllobiidae. Such males are often more or less permanently attached to the female’s body. The climax of male reduction is reached in the phenomenon of cryptogonochorism, which occurs in Gonophysema gullmarensis and (in an even more advanced state) the xenocoelomid Aphanodomus terebellae. I n these species, the female reproductive system is invaded by a male stage which ultimately transforms itself into little more than testicular tissue (Bresciani and Liitzen, 1961b, 1966, 1974). An intriguing side issue is the discovery by Dudley (1966) of male dimorphism in the notodelphyid Doropygus seclusus. Here structurally identical males in the fifth copepodid stage moult either into anamorphic or metamorphic adults. These differ in a number of small morphological details and are, moreover,behaviourally distinguishable,the former being active “ walkers ’’ and the latter efficient swimmers. So far, ecdysis to the metamorphic form has been observed mainly in vitro, which may suggest that some factor from the ascidian host is necessary to evoke a moult into the anamorphic form. Dudley has put forward the interesting idea that species of the genus Agnathaner-so far known only from males-might in reality represent metamorphic males of some well known female notodelphyids. Finally, there are certain genera (Botryllophilus, Mytilicolu and Trochicola) in which the females of different species are practically indistinguishable, whereas the corresponding males are widely dissimilar (Stock, 1970b). 2. Mating

Stock (1962) has described mating posture in the lichomolgid Pennatulicola pterophilus (Stock), a sea-pen associate. Here, the male grasps a female copepodid with his maxillipeds and holds her against his ventral surface. Stock believes that actual fecundation probably does not take place before the female attains the fifth copepodid instar. Giesbrecht (1882) observed copulation in the notodelphyids Notopterophorus pupilio Hesse and N . elatus Giesbrecht. Before the female’s final moult, the male was attached to the dorsal surface. Just prior to her ecdysis, however, he released his hold, reattaching subsequently to the ventral surface by hooking his antennae to the bases of her fourth legs. Spermatophores were then extruded which became fixed to the vulva of the female. Thorell(1859, 1860)noticed a male Notopterophorus auritus Thorell remain attached to a female fifth copepodid for three days.

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Stock et al. (1963) have recorded mating in Pseudanthessius tortuosus Stock, Humes & Gooding, a cyclopoid from amphinomid polychaets. Third to fifth stage female copepodids are grasped around the fifth pedigerous segment by the maxillipeds of the male, which also seems to employ the antennae t o hold the female in place. The same authors (loc. cit.) also describe amplexus in the curiously transformed pseudanthessiid Meomicola aniplectans Stock, Humes & Gooding, an associate of echinoids, and have commented on the various morphological adaptations facilitating the embrace, which is both stable and lengthy. Despite the safeguards of sexual dimorphism and often prolonged copulatory behaviour, occasional mistakes are made by presumably over-ardent males. A male Synaptiphilus tridens has been found with spermatophores attached t o his urosome (Gotto, unpublished), and similar accidents ” have been recorded by other authors. (I

3. Eggs and egg laying The shape, size and number of eggs produced by associated copepods vary within wide limits. Generally, ova are more or less spherical, but when extruded in a uniserial string tend to be rather more discoidal, as in the ascidicolous enterocolid Haplostoma banyulensis (Brbment). Exceptionally, they are laid singly or in small groups within the host, as in Mychophilus roseus from botryllid ascidians (Lang, 1948a ; Gotto, 1954a) and in certain lamippids (Bresciani and Liitzen, 1962). I n some other ascidicoles, such as Enterocolides ecaudatus Chatton & Harant, egg masses are deposited a t the surface of the compound ascidian host (Chatton and Harant, 1924) although mature females are normally found in the depths of the colony. A deliberate egg-laying migration is thus implied. A similar movement by gravid females would seem t o be undertaken by Ophioseides joubini,which tunnels between the tunic and epidermis of styelid and pyurid ascidians. Breseiani and Liitzen (1962) have observed that one of the tunnels invariably leads t o the rim of a siphon-the only place where the biotope of the copepod has a penetrable interface with the outside world-and in this passage females can be seen with the posterior end pointing to the siphonal rim. Ascidicola rosea, usually inhabiting the oesophagus of simple sea-squirts, migrates into the host’s stomach t o deposit the egg masses. I n this case, however, the ripe eggs are carried with the host’s faeces to the anus, where hatching and expulsion of nauplii takes place (Gotto, 1957). A curious state of affairs obtains in Alhntogynus delanzarei, from the body cavity of holothurians. Here the entire female becomes enveloped in a relatively immense (‘ovigerous sac ” which, in the later stages of its development, contains not only eggs but faecal material as well. Just

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how this sac is secreted remains uncertain (Changeux, 1961). Another interesting case is provided by Mesoglicola delagei from the actinian Corynmtis viridis. Here a male and female occur together within a closed gall, in which can also be found two or three pairs of sacs, not attached to the female’s body, containing eggs in different stages of development (Laubier, 1966). I n the choniostornatid Sphaeronellopsis nzonothrix Bowman & Kornicker, parasitizing the ostracod Parasterope pollez Kornicker, Bowman and Kornicker (1967) have shown that the eggs are laid in groups of about 1 5 , each group being enclosed by a membrane. All the eggs in one such sac are at the same developmental stage, but those in different ovisacs may be in different stages. Each cluster measures approximately 0.20-0-30 mm-about the same as an individual ostracod egg. “ This similarity in size clearly seems t o be a case of egg mimicry having adaptive value for the Sphaeronellopsis. The third thoracic legs of myodocopid ostracods are veryflexible, adapted for removing foreign particles from the interior of the valves and from the eggs . . . Individual copepod eggs presumably would be removed from the brood chamber by the cleaning leg, but the copepod avoids this hazard by laying its eggs in groups within sacs, each sac mimicking one of the ostracod eggs in size and shape. Instead of being removed as a foreign particle, the Sphaeronellopsis ovisac is retained within the brood chamber and cleaned by the host with the same solicitude given to its own eggs. To a male ostracod, however, a n ostracod egg or a Sphaeronellopsis ovisac . . . is a foreign particle and therefore is removed by the cleaning leg. It is significant that in the few instances in which a female Sphaeronellopsis was found in a male ostracod, no ovisacs were present.” These authors also draw attention to the many choniostomatid species which inhabit the marsupia of amphipods, isopods, cumaceans and mysidaceanshosts which aerate and keep their eggs clean by circulating a water current through the pouch. Although individual copepod eggs would be in danger of being flushed from the brood chamber, ovisacs similar in size to the host’s eggs are too large to encounter this risk. Once again, therefore, advantage t o the copepod would seem to accrue from this particular method of egg deposition. I n the Notodelphyidae, Buproridae and most of the Gastrodelphyidae, the eggs are laid into a brood pouch. I n notodelphyids, this incubating cavity is essentially a product of one or more of the thoracic segments. Its development in this family has been studied by Chatton and Brement (1915) and by Dudley (1966). The pouch opens by a posteriorly situated aperture, and may account for a considerable proportion of the body volume of mature females. I n gastrodelphyids it

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is elaborated from the fourth metasomal segment, and may likewise be very capacious. I n buprorids, it occupies dorsally almost the entire length of the female’s rather squat body. I n most associated copepods, however, the extruded ova lie in paired sacs or strings, one on each side, and attached to the genital segment as in many free-living forms. The botryllophilids are exceptional in producing a single globular egg mass, neatly balanced between the dorsally positioned fifth legs. The great majority of associates produce oval, rounded or sausage-shaped sacs in which further development of the eggs proceeds. Occasionally, these sacs assume a more bizarre shape. Thus in the sabelliphilid Scambicornus lobulatus Humes, from a holothurian host, they are peculiarly lobed. Humes (1967b) has suggested that perhaps the eggs of S. lobulatus are extruded in spurts, so that the cement substance, when hardening, is responsible for this particular shape of sac. I n this connection, he quotes Heegaard (1959), who believes that egg string shape in species of Caligus may be determined by the movements of the female and by water currents related to the movements of the fish host. Pronounced lobulation of the sac is again apparent in Teredoika serpentina, an anomalous endoparasite of shipworms (Stock, 1959a). We must admit that our general information on this topic is woefully inadequate. The number of eggs produced by associated copepods has been the subject of some speculation (Gotto, 1962 ; Humes, 1967b ; Riittger, 1969; Rottger et al., 1972). I n general, we may say that the fewer the number in any one clutch, the larger are the individual eggs. It hasbeen suggested (Gotto, loc. cit.) that a high egg number might be related to : (i) the host being sparsely distributed, somewhat inaccessible, or not obviously attractive from a distance ; (ii) the host being highly mobile ; or (iii) the environment of the host being inimical to successful infestation (e.g. swift currents, wavebeaten shores, or exposure during low tides). All such factors could be regarded as providing stiff challenges to the successful finding of new hosts by each generation of infective stages, thus making a large number of larvae a sine qua non of survival. On the other hand, the production of few eggs may imply : (i) that the infective larvae possess strongly developed powers of host perception ; (ii) that the host is abundant, readily accessible, or chemically attractive from some distance ; (iii) that the degree of host specificity is low, so that any of several species may serve as hosts ; (iv) that environmental conditions are such as to encourage a high percentage of infection (e.g. quiet, sheltered waters) ; or (v) that autoinfestation takes place. Clearly, more work is needed to test the validity of these assumptions-but a welcome start in this direction is already apparent. Carton (1968~) for

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example, studying Cancerilla tubulata Dalyell on its ophiuroid host, believes that several of the above-mentioned factors may contribute significantly to the low egg number characteristic of this siphonostome. I n the context of egg number and egg size, it is of interest to note that in Octopicola superbus the sub-species antillensis from the West Indies carries only about half as many eggs as the European sub-species superbw, but individual eggs are rather larger (Stock et al., 1963). Within recent years, some information has become available regarding the number of clutches produced in the course of a season by associated species, but it will be appropriate t o consider this later under the heading of population studies. 4. Cryptogonochorism

This condition, recently discovered in certain copepods associated with marine invertebrates, is of sufficient significance to merit some discussion. Sexuality in most associated copepods is quite unambiguous, the reproductive apparatus of those investigated being, on the whole, closely similar to that of free-living copepads, though details of genital architecture may, of course, differ slightly from species to species. Accounts of the reproductive system have been furnished by Illg (1958) for notodelphyids ; by Mason (1959) for Nicothoe astaci ; by Bouligand (1960a) for lamippids ; by Changeux (1961) for Allantogynus delamarei ; by Bresciani and Liitzen (1960, 196lb) for Gonophysema gullmarensis ; by the same authors (1961a) for Melinnacheres steenstrupi ; by Carton and Lecher (1963) for Selioides bocqueti; by Lutzen (1966) for the herpyllobiids; and by Bresciani and Lutzen (1966, 1972, 1974) for the xenocoelomids. For many years, Xenocoeloma alleni (Brumpt), a strongly modified parasite of terebellid polychaets, was regarded as a self-fertilizing hermaphrodite (Caullery and Mesnil, 1919). A similar hermaphroditic condition was inferred by Gravier (1918b) for Plabellicola neapolitana, since males were never found in a very large material of this copepod. Certain other annelidicolous species, likewise characterized by an apparent absence of males, were also presumed to fall into this category. The researches, however, of Bresciani and Liitzen (loc. cit) on the ascidicolous Gonophysema gullmarensis and the polychaet-infesting Aphanodomus terebellae have now established that, in these forms a t least, hermaphroditism is apparent rather than real-what we are witnessing should properly be described as cryptogonochorism. A summary of the Gonophysema life cycle may best illustrate this phenomenon.

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I n G . gullmarensis, a typical nauplius undergoes a single moult to produce a cyclopiform copepodid stage. This has two pairs of swimming limbs, strongly prehensile antennae, and antennules provided with large aesthetascs. Both of these pelagic instars are of short duration, sometimes as little as 7-8 hours in all. The older copepodids display characteristic searching behaviour, apparently testing the substrate with their aesthetascs. If provided with a piece of ascidian tissue, they will attach to it and initiate the final ecdysis. This last stage larva, the onychopodid, is a simple elongate sac, retaining antennae as the sole appendages. It burrows into the host's mesenchymatous tissue and wanders slowly along the blood sinuses of the ascidian, before settling just below the epithelium of the peribranchial cavity, with its dorsal side orientated against the host epithelium. It then rounds off into an ovate mass and ramifications of the body develop in a regular manner. This branching proceeds until the definitive shape of the adult female is attained. Within the onychopodid's body, the only organs discernible are those concerned with reproduction-an ovary, a cement gland with a receptaculum seminis opening into its base, a chitin-lined atrium and an associated atrial gland. The aperture of the atrium is the only opening to the environment. Thus equipped, a newly settled young female is investigated by later-arriving onychopodids. One to several (usually three or four) of these now invade her atrium and make their way-apparently with some difficulty-to its inner depths, ultimately reaching the " testicular vesicle " and " testicular organ ". This region once attained, the invading larvae turn through 180" to lie with the cephalic portion towards the atrium. Very vigorous reduction of the onychopodid's body now takes place, until a male gonad remains as virtually the sole organ. Sperms produced by this gonad are evacuated through the cephalic region, ultimately coming to lie in the receptaculum seminis, and strategically positioned to fertilize the eggs. No histological difference is demonstrable between onychopodids which develop in male or female directions. Since, however, young females are very attractive to later-arriving larvae, it seems clear that subsequent development into males is somehow controlled by a female which has been thus successfully invaded. An analogous state of affairs may perhaps be postulated for the monstrillids, since Malaquin (1901) adduces evidence that in these copepods sex appears to be determined by the numerical ratio between hosts and parasites : if there is more than one parasite in each host, they will become males, whereas an isolated individual will develop in the female direction. Presumably, those onychopodids of Gonophysema which fail to find, or penetrate, an

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established female, will themselves develop into females in the tissue of the ascidian host. The case of Aphanodomus terebellae would seem to be basically similar, though details of the structure and development of the reproductive organs are somewhat different. The dissimilarities have led Bresciani and Lutzen to conclude that A . terebellae has advanced even further than Gonophysema along this bizarre reproductive road. I n particular, the ultimate reduction of the invading male is more complete, though its origin from a pelagic copepodid is verified by the discovery of moulted copepodid skins (exuviae) in the atrium of the female. Since there is now little doubt that Aphanodomus is closely related to Xenocoeloma, it is reasonable t o suppose that reproductive processes in the latter follow a similar pattern. Some of the recent findings by Bocquet et al. (1970) on X . alleni would seem to support this hypothesis, though others are less easy to interpret. For the moment, however, it may tentatively be concluded that the Copepoda represent an entirely gonochoristic group. VI. HOSTSPECIFICITY Few hard and fast rules can be applied to the incidence of host specificity. There are, of course, some species with a staggering degree of versatility in regard to host selection-Doridicola agilis Leydig, for example, has been recorded from nearly thirty species of nudibranchs, a tectibranch, a polychaet and, possibly, a cephalopod. Equally, many species appear immutably linked to a single host. However, earlier judgments regarding invariable partnership a t higher group level must be modified as informationaccumulates, and two recent instances of just such drastic reappraisal may be cited. The taeniacanthids, long known as exclusively fish parasites, have lately been shown to include genera which patronize echinoids. Even more startling is the case of the Notodelphyidae. For more than a century, ascidians were the only known hosts-an association so invariable that it could virtually be reckoned as a diagnostic feature. However, Stock and Humes (1970) have now discovered four species inhabiting an octocoral, and these show remarkably few morphological changes from closely related ascidicolous forms. Occasionally examples come to light which indicate that host availability may be an important factor in determining incidence of infection. Certain sheltered inlets of Strangford Lough, Northern Ireland, support a richly diversified ascidian fauna which provides habitats for a number of associated copepods. The little transparent enterogone Clavelina lepadiformis (Muller) is commonly found here, but is seldom infected.

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Some 190 km further north, in the equally enclosed bay of Sheephaven, ascidians are not only markedly depleted but are generally somewhat stunted, with the notable exception of Clavekina which occurs in abundant and thriving colonies. A high percentage of the zooids are here infected by at least three species of ascidicolous copepods (Gotto, 1961a). Such cases would seem to suggest an unexpected capacity for adaptability in commensals when normally preferred hosts may, for some reason, be absent. This said, however, it should be borne in mind that integration with atypical hosts must inevitably involve subtle refinements of physiological adjustment, so that such copepod populations might in reality represent distinct biological races. Humes and Stock (1973), in their monumental revision of the Lichomolgidae, have briefly analysed host specificity in these cyclopoids. Thus, in the sabelliphilids, half of the known genera are associated with holothurians, the remaining half being distributed amongst the Antipatharia, Actiniaria, Polychaeta, Bivalvia and Ascidiacea. I n the Lichomolgidae s. str., 42 genera occur with definite host groups which encompass much of the invertebrate spectrum, while nine have been found with more than one host group or have, so far, only been discovered in the free-living state. Four of the five known genera of the pseudanthessiids are restricted to single host groups, while Pseudanthessius itself patronizes no fewer than seven. Some of the data supplied by Humes and Stock merit further consideration. Not infrequently we encounter a genus, the constituent species of which occur in what seem, at first sight, to be widely disparate host m>ilieux. Species of the lichomolgid Macrochiron, for example, a m associated mainly with hydroids or with algae, but M . echinicolum Humes & Stock is found on various sea-urchins, and M . sargassi Sars with certain compound ascidians. Similarly, some species of Metaxymolgus are partners of coelenterates, others of opisthobranchs, and one ( M . claudus Humes & Stock) of an ophiuroid. Finally, Kelleria species have been discovered free living, on a crinoid, and in inter-tidal burrows. It may be suggested that these instances of closely allied forms partnering very different hosts could be explained in one of two ways. Either their trophic tastes and requirements are extraordinarily catholic, or else (and this appears to me the more likely explanation) they may in fact be highly stenotrophic at the generic level. If, as seems probable, the food of these species consists largely of host secretionsincluding mucoproteins and mucopolysaccharides-a situation could obtain in which availability of a precise nutrient is crucial for normal feeding, reproduction and, indeed, survival. Such an exact requirement might be met by the entirely fortuitous production of an

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identical food element by utterly diverse hosts. Specificity, in short, may in these cases be for specific mucoproteins or mucopolysaccharides rather than for the total entity represented by a host animal. Perhaps an analogue may be found here in the behaviour of certain polynoid worms commensalistic with other polychaets. I n some areas, Polynoe scolopendrina Savigny, ordinarily associated with the terebellid Polym nia nebulosa (Montagu), appears to have little interest in related terebellids, but responds very strongly to a member of a completely different family, the eunicid Lysidice ninetta Audouin & Milne-Edwards. It must surely be concluded that the attraction exerted by the latter is due to some degree of fortuitous chemical resemblance to the polynoid’s usual partner (Davenport, 1953). The notodelphyid genus Notopterophorus gives us another glimpse of seemingly erratic host preference. Six species of these large ascidicoles are currently recognized-N. elongatus, N . elatus, N. papilio, N . micropterus Sars, N. auritus and N . dimitus Illg & Dudley (Illg and Dudley, 1961). If older records could be relied an, the genus occurs in quite an array of ascidian hosts ; but this is almost certainly erroneous and stems from the confusing synonymies which have accreted over the years for ascidians and copepods alike. More recent information strongly suggests that in fact only two, rather distantly related, host families are implicated-the Ascidiidae and the Cionidae. With the exception of N. dimitus, which infects Ciona intestinalis L., the genus is restricted to the ascidiids Phallusia and Ascidia, whilst the closely allied Ascidiella is ignored as a host. We may, then, reasonably enquire what feature (if any) is common to the two chosen genera and to Ciona, but is lacking in Ascidiella. A possible answer resides in the detailed architecture of the pharyngeal wall. I n Ascidia, Phallusia and Ciona, secondary papillae project from the wall into the lumen of the branchial sac. These short, finger-like processes are absent in Ascidiella. Such elaboration of the pharynx is, according to Berrill(1950), a mere consequence of increased body size in the genera mentioned. The presence of papillae, however, imposes a slight difference on the topography assumed by the mucus sheet secreted by the endostyle in Phallusia, Ascidia and Ciona. As Millar ( 1 953) has shown in Ciona, the water current passing through the pharynx presses this sheet with its adherent food particles closely against the wall. Instead of presenting a completely flat surface, however, it is raised into small, blunt cones wherever it overrides an underlying papilla. I n short, the surface of the food-trapping sheet is regularly rugose rather than smooth. We may safely assume that the same condition obtains in the similarly endowed Phallusia and Ascidia. Although seemingly trivial, this feature may acquire significance

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vis-b-vis the biomechanics of feeding in species of Notopterophorus and therefore, in more general terms, of host specificity within this genus. I n these copepods, the head region is characteristically and permanently bent almost at right angles to the rest of the body. Since there is no compensating flexure of the mouth parts, the extraction of food particles from a completely flat sheet might well present difficulties, due to this structurally imposed angle of attack. The mouth parts, however, would seem to be ideally aligned for gathering food at those sites where the mucus rides up and over the papillary cones. One can visualize an evolutionary situation arising in which only a rugose food sheet could be effectively '' grazed )'by a copepod with the cephalosomic peculiarities of Notopterophorus. Once established in the few large ascidian species which fulfil this requirement, further commitment to such hosts would be enhanced by normal selective processes-the acquisition, for example, of host-specific chemotactic responses by infective larval stages. It must, of course, be emphasized that the above hypothesis is purely speculative, relying as it does on inference rather than detailed observation, which in vivo might well prove difficult. In Mediterranean notodelphyids, Illg and Dudley (1961) could detect some degree of correlated phylogenetic differentiation in copepods and their ascidian partners. Thus, at ordinal level, there are indications that two distinct but closely related groups of Notodelphys species are associated respectively with phlebobranchiate enterogones and stolidobranchiate pleurogones. As far as familial preferences are concerned, species of Periproctia favour hosts belonging to the Didemnidae, although one species has been recorded from a botryllid. On the generic plane, Gunenotophorus globularis Buchholz is restricted in the Mediterranean to species of Polycarpa, though in other areas its host spectrum is wider. Laubier and Lafargue (1974) have drawn attention to association patterns in some specialized notodelphyids and didemnid ascidians. They point out that two evolutionary lines are evident in the host family-one exemplified by Lissoclinum and Diplosoma, the other by Trididemnum and Didemnum. The genus Polysyncraton occupies an intermediate position, P . canetensis Brement greatly resembling Lissoclinum and P. bilobatum Lafargue being closely similar to Didemnum. Certain notodelphyid species are shared by P. bilobatum and Didemnum species, whilst P . canetensis shelters copepods occurring also with the Lissoclinum-L)iplosoma group. Brementia balneolensis Chatton & Brkment spans this host-spectrum, being found in both of the Polysyncraton species and in Didemnum commune (Della Valle). Such coincident trends of evolution, however, are not apparent in the

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gastrodelphyid copepods and their polychaet hosts (Dudley, 1964). For example, the two most closely related species, Sabellacheres gracilis M. Sars and S. illgi Dudley live on hosts belonging to different subfamilies, while the most distantly related forms, S. gracilis and Gastrodelphys myxicolae List both live on the cosmopolitan host Myxicola infundibulum (RBnier), though in geographically distinct areas. Although in most species the mature females seem to be associated with particular sabellids at specific level, the copepodids, adult males and immature females of S. illgi wander among worms of different genera and species which occur in the same area. We may mention here the by now familiar phenomenon of different copepod species occurring on or in the same individual host. A few examples will suffice. Humes and Stock (1965) report three species of the myicolid genus Anthessius from a single specimen of the clam Tridmna squamosa Lamarck. Cohabitation of the same inter-tidal burrow by three species of the clausidiid Hemicyclops has been noted by Humes (1965), and K8 et al. (1962) likewise record three cyclopoid species belonging to different genera as living together in the bivalve Tapes japonica. In such cases of cohabitation, we must suppose either that different microniches are occupied, or different food sources utilized. As regards the latter, it may well be that an ability to deal with food particles of a particular size is sufficient in itself to permit coexistence. A useful analysis of cohabitation by different copepod species of the ascidian Microcosmus has been provided by Monniot (1961). Although his findings are concerned with the aspect presently under discussion, they may more appropriately be considered under the subsequent heading of preferential host-niche. The seeming anomaly of a highly specialized parasite possessing an extensive host roster is exemplified by the remarkable notodelphyid Scolecodes huntsmani. This large vermiform copepod occupies a cyst of host origin in the Aubendostylar blood vessel of simple ascidians. The cyst reveals a high degree of organization (Dudley, 1968) and the hostparasite relationship as a whole would certainly suggest extraordinarily detailed integration of the two organisms (Illg, 1970). However, no less than five host species have now been recorded, assignable to four genera and two families! As Illg rightly remarks, this must represent an unexpectedly high degree of opportunistic adaptability. Pinally, host specificity may also, of course, be expressed in subspecific differentiation. A well documented example is that of the clausidiid Conchyliurus cardii Gooding (Bocquet and Stock, 1958~).The sub-species C. c. cardii Gooding is found inthe pallial cavity of Cardium

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echinatum L. and Meretrix chione L., whilst a second, clearly defined form (C. c. tapetis Bocquet & Stock) occurs in three species of Tapes. The divergence of Conchyliurus solenis Bocquet & Stock has reached specific level, and this copepod is restricted to Solen marginatus Montagu. In their paper on this genus, Bocquet and Stock include an interesting discussion on speciation and sub-speciation among associated copepods in general.

VII. ATTRACTION TO HOST Some twenty years ago, pioneer studies by Davenport and his coworkers on the polychaet commensals of echinoderms laid a secure foundation for further research into this facet of the biology of interspecific associations. Unfortunately, however, the small size of copepods as experimental animals and, above all, the difficulties attendant on rearing the larval instars up to the infective (and therefore host-sensitive) stage, have proved formidable impediments to work on this problem. Tribute is therefore due to Carton (1966, 1967, 1968a, b, c, d) who has provided a fascinating analysis of host attraction for two species of associated copepods. The first of these, Sabelliphilus sarsi, is restricted in nature to the large tube-dwelling sabellid Spirographis spallanzani. It can, however, n i on be induced to settle on the brevispira variety of S . ~ p a l ~ a n z a and the generically distinct Sabella pavonina. Using an ingenious choice apparatus, Carton has shown that the copepod is sensitive to water which has bathed the host, and is thus attracted from a distance by secretions of host origin. The secretions of the natural host are most effective in this respect, followed by those of the variety brevispira. No attraction from a distance can be demonstrated to Sabella pavonina. Biochemical studies, involving disc electrophoresis on polyacrylamide gel, reveal seven protein fractions (five of them mucoproteins) common to the two attractive hosts, but missing in S. pavonina. It seems reasonabIe to suppose that it is these common biochemical factors which furnish the clue as to host proximity. Carton has similarly investigated the siphonostome Cancerilla tubulata. Its normal host is the ophiuroid Amphipholis squamata, and the " artificial " host employed was the related Acrocnida brachiata Montagu. Once again, the host utilized in nature is preferred. I n this instance, however, successful fixation on the ophiuroid by the infective copepodid is greatly influenced by, firstly, the physiological state of the host, and secondly, by the age of the searching larva. Thus, to be maximally attractive, Amphipholis must be gravid, whilst the fifth day of the

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copepodid‘s life represents the optimal period for host contact to be established. An interesting suggestion put forward by Bocquet and Stock (1963) is of relevance in this context. These authors believe that host specificity in associated copepods may be acquired through a conditioning process operative during development up to the liberation of naupliar or postnaupliar stages. Once some “ host ” offers sufficiently favourable feeding conditions to induce a female copepod to remain on it, her eggs will be laid and will develop in that “ host’s ” biochemical ambience. Conditioned thus early, the resulting larvae will be attuned to seek out this particular partner when they themselves approach maturity. Selective processes may then be relied upon to cement and refine the relationship. Certain aspects of Carton’s work on the Cancerilla-Amphipholis association reinforce this theory. For example, copepodids of C. tubulata obtained from females reared on the atypical host Acrocnida show a reduced degree of success in affixing themselves to the natural host. Presumably the embryos developing in the ovigerous sacs have been influenced, perhaps both directly and indirectly, by the biochemical products of an atypical host, thus impairing their ability to recognize and settle on the normal partner. An investigation by Briggs (unpublished thesis) to detect a substance possibly released by Sabella pavonina which would attract its usual associate Sabelliphilus elongatus proved negative. A similar attempt to demonstrate “ host-factor ” in the Paranthessius-Anemonia association (Briggs, 1976) was likewise abortive. It should not, however, be concluded from tests on adult copepods only that such factors are nonexistent in these species. It seems much more probable that in many cases it is only the infective copepodid stage which is capable of receiving, interpreting and responding to chemical cues emanating from the host. It should also be noted that the presence of an entire host animal is not invariably necessary to trigger an orientation response in its associated copepod. Delamare Deboutteville et al. (1957) have observed that Octopus egg masses, even when virtually empty, are attractive to the cyclopoid Octopicola superbus. I n conclusion, we may therefore say with some confidence that, although more experimental evidence is desirable, attraction from a distance would seem to depend on a chemotactic awareness of host proximity. Contact once established, other considerations, such as recognition of the appropriate host ‘L terrain ”, no doubt become operative.

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VIII. PREFERENTIAL HOSTNICHE The successful finding of the preferred partner is not necessarily the end of the trail for a searching larva. Some copepods are highly selective as regards the precise niche finally occupied on or in the host’s body. Especially in ascidicolous forms, discrimination of this type can be understood in terms of specialized exploitation of the food supply. For example, Monniot (1961) has charted the preferred niches of eight “ guest ” species within simple ascidians of the genus Microcosmus. Two associates (a Lichomolgus species and the nemertine worm Tetrastemma JEavidum Ehrenberg) inhabit the cloaca1 cavity ; two copepods (Enteropsis chattoni Monniot and Ascidicola rosea) prefer the oesophageal region ; three others (two Notodelphys species and Doropygus pulex Thorell) live in the branchial sac, whilst Ophioseides joubiizi burrows in the host tunic. Within the pharynx, however, D . pulex tends to remain in the vicinity of the dorsal lamina, but Notodelphys is generally found close to the endostyle. Interestingly, the presence of adult doropygids appears to inhibit successful cohabitation by the nemertine, but Notodelphys and Doropygus would seem to reinforce each other’s infestive capability. The advantages accruing to commensals and parasites capable of exploiting different microniches within the host can be readily understood. Less easy to interpret, however, are certain other cases of rigidly restricted site-preferences. Xabelliphilus elongatus, for example, occurs more frequently on those filaments of its host’s branchial fan which are overlapped by other filaments, even though the former may represent less than 20% of the total number of filaments available. However, since these overlapped plumes are the ones least likely to brush against the edge of the tube when the worm withdraws, it is possible that copepods situated on them stand a better chance of avoiding dislodgement-hence producing the uneven distributional pattern so often seen (Gotto, 1960, 196lb). Go~ophysemagullmarensis exhibits an equally intriguing pattern in its sea-squirt host Ascidiella aspersa. Although the copepods may be found anywhere in the exterior wall of the peribranchial cavity, nearly two-thirds favour the left wall, and moreover settle in areas lying furthest away from the atrial siphon. Their final orientation is such that the egg strings, when produced, protrude into the atrial cavity, thus ensuring adequate aeration as long as the host is filtering. According to Lutzen (1964b) the herpyllobiids are distinctly conservative as regards site preference. Two species constantly attach to the prostomium of the polynoid host, four choose elytrophoral or

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pseudoelytrophoral sites, two more are found interparapodially, one occurs on the dorsum and one on the ventral part of the body. But even finer discrimination is apparent within these preferred situations. Thus Herpyllobius arcticus Steenstrup & Lutken attaches to elytrophores only on the front portion of the worm, H . haddoni Lutzen only between parapodia on the hind part, while Eurysilenium oblongum Hansen and E . truncatum M. Sars occupy their preferred sites almost exclusively in the central portion of the host’s body. Another parasite of polychmts, Aphanodomus terebellae, although wholly internal, is similarly restricted (Bresciani and Lutzen, 1974). Despite the fact that the host, Thelepus cincinnatus, may consist of more than 80 segments, 95% of the copepods are attached to intestinal blood vessels between segments 18-37-the region of the anterior intestine. Bresciani and Lutzen incline to the belief that this pattern may reflect a route taken through the host’s vascular system by infective stages which perhaps gain initial access to the blood vessels via the thin walled gills. The notodelphyid Scolecodes huntsmani is likewise highly selective, settling only in the subendostylar blood vessel of its ascidian host. As one of the largest vessels in the ascidian body, this is probably the only vascular channel capable of housing this very large vermiform copepod which may reach a length of 14 mm. Despite its size, however, multiple infections are frequently encountered. A final example of curiously narrow site preference is provided by Melinnacheres ergasiloides, which attaches only to the parapodia of the last few thoracic segments of its ampharetid partner-a situation which Bresciani and Lutzen (1975) find impossible to explain. I n the same paper, these authors touch on the unexpectedly high frequency of multiple infections in annelidicolous copepods. Certainly the data presented lends support to the idea that multiple infection can hardly be a random process. After considering various possibilities, Bresciani and Lutzen point out that in many polychaet-infesting copepods the females are accompanied by dwarf males. Now, if sex is not genetically fixed in the larvae, some of the latter, when attracted by females, would develop into males. Guided by the same stimulus, however, others would settle on the host instead and develop into additional females, thus producing a multiple infection. Although no corroboration is as yet available, general considerations of evolutionary advantage would tend to support such a theory. A different type of sex-related distribution within the host may be exemplified by Trochicola entericus Dollfus. I n this mytilicolid, females can be found in the intestine of the sea-snail Gibbula varia (L.), while males frequent the pallial cavity (Stock, 1960).

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IX. EFFECT ON HOST AND HOSTREACTION Thus far, we have considered copepods in the light of their capacity to infest other marine invertebrates and the stratagems of behaviour and structure which enable them to live as commensals or parasites. We must now enquire how the host fares when its defences have been breached by these versatile invaders. I n many cases, so far as we can tell, the effects are minimal. This would seem especially true of actively filter-feeding hosts, such as ascidians, endowed with a capacious food-gathering apparatus. At all events massive infestations by ascidicolous copepods are on record with no mention of host malfunction or lack of condition. Up to 123 individuals of the quite large notodelphyid Doropyglgzcsflexus Gotto have been found in the pharynx of a single, apparently healthy Pyura praeputialis (Heller) (Gotto, 1976). An interesting case of a combined onslaught on the resources on an individual Ascidia mentula Miiller has also been reported by Gotto (1959). Here seven " guests " (including three notodelphyids) representing six species, five orders, three classes and two phyla had been accommodated without noticeable ill-effects. Such examples presumably indicate a remarkably wide safety margin inherent in the ascidian method of feeding. A similar superfluity may also be characteristic of the feeding of basket-stars, if one can judge from the nearly 7 000 specimens of Collocherides astroboae found in the stomach of a single Astroboa nuda by Humes (1973). Many of the copepods associated with coelenterates, especially corals, likewise occur in considerable numbers on apparently healthy hosts. Since many corals produce abundant mucus, it may well be that ample food is available to support these high infestations. However, the curiously transformed Corallonoxia longicauda Stock is an internal parasite which possibly ingests host tissue. Stock (1975) reports that in heavily parasitized colonies of Meandrina meandrites (L.),an estimated 25% of the tissue weight is accounted for by these large parasitic copepods, and he believes that infestations of this magnitude must play a significant role in the carbon flux. It is not, of course, surprising that relatively large host animals can support an enhanced number of very small ecto-associates, but some of the figures available are nonetheless impressive. Thus a total of 750 specimens of the little siphonostome Nanaspis tonsa Humes & Cressey have been recovered from only three sea-cucumbers (Humes and Cressey, 1959). Not all invasions, however, are so innocuous. Southward (1964) noticed that the entire abdomen of the serpulid Omphalopoma stellata Southward was flattened when its tight-fitting calcareous tube was

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shared with the copepod Serpulidicola omphalopomae Southward. Again, while small individuals of Aphanodomus terebellae are easily accommodated within their polychaet host, space becomes limited as they grow. The intestine of the worm can be displaced, and the oblique and mesenteric muscles affected to the point of noticeable reduction or even total disappearance (Bresciani and Lutzen, 1974). I n mussels infected by Mytilicola intestinalis, Caspers (1939) observed a reduced rate of water filtration, but failed to find any direct evidence of otherwise harmful effects, and Hockley (1951) was likewise unable to detect tissue damage to the host’s intestinal region. On the other hand, Couteaux-Bargeton (1953) reported local lesions of the digestive epithelium associated with an excess of alkaline phosphatase activity, and there have been other accounts of heavy Mytilicola infestations incurring marked loss of condition followed by the death of the host mussel (Korringa, 1951, 1953 ; Cole and Savage, 1951). Metaplastic changes in the gut of the Pacific oyster Crassostrea gigas (Thunberg) when infested by Mytilicola orientalis Mori have been noted by Sparks (1962) and rather similar effects have been seen in Crassostrea glomerata (Gould) when parasitized by the myicolid Pseudomyicola spinosus (Raffaele & Monticelli) (Dinamani and Gordon, 1974). Perhaps the most interesting results are those involving disturbance or even destruction of the host’s reproductive capability. Over 60 years ago, Brhment (191 1) noted considerable degeneration of testicular follicles in zooids of the didemnid Diplosoma listerianum (MilneEdwards) when occupied by mature females of Enterocola pterophora Chatton & BrBment. Akesson (1958) observed that sterility in the sipunculoid CTolJingia minuta followed infection by the coelome-inhabiting copepod Akessonia occulta. Bocquet et al. (1968) believe that the terebellid Polycirrus caliendrum Claparhde produces fewer eggs when infected by Xenocoeloma alleni. The choniostomatid associates of ostracods certainly appear to inhibit egg laying (Bowman and Kornicker, 1967) and the same seems to be true of Sphaeronella paradoxa Hansen and S. leuckarti Salensky in their respective amphipod hosts, Bathyporeia, sarsi Watkin and Corophium volutator (Pallas) (Hamond, 1973). Such inhibitory effects are in a sense paradoxical, in view of the fact that other choniostomatids (e.g. Choniosphaera maenadis) actually rely on host eggs as a food source. Detailed documentation of host reaction is relatively sparse. At histological level, Carton (1967) has studied the response of sabellid worms to infestation by copepods of the genus Sabelliphilus. On Spirographis spall~nzani,its natural partner, 8.sarsi has little effect on the body wall. The epithelium is minimally disrupted, but partial lysis of

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the basal lamina and the underlying muscle layers is apparent in those areas gripped by the powerful antennae. Within a month of settlement, the main tissue response has been conversion of the normally thin boundary layer between the circular and longitudinal muscles into a compact network a t the site of attachment. The “reinforcement zone” thus created presumably contains and localizes further cellular damage. Settlement on the closely related but atypical host S. spallanzani variety brewispira induces a more profound response. The epithelium, basal lamina and circular muscles completely disappear at the fixation point, resulting in an insecure anchorage for the antenna1 claws. Initially, this appears to be a purely mechanical consequence of the parasite’s presence, but more prolonged fixation evokes a series of hosttissue reactions. Gradually the copepod sinks into the now lysed tissue, whilst cellular proliferation takes place around it. Simultaneously it is progressively engulfed by an exudate of blood and coelomic cells. After a month, less than half of the copepods which originally settled have survived this insidious entombment. On the more distantly allied Subella pavonina, 8. sarsi provokes a response of even more dramatic intensity. The tissues beneath the parasite disappear rapidly and completely, leaving the copepod plunged in a gaping wound which extends to the coelomic cavity, the lining of which starts to thicken. The dorsal blood sinus may be pierced and even, on occasion, the host’s alimentary canal. Once again, the invader is bathed in a blood and cellular exudate. After a month, only about one-fifth of the copepods remain on the host. According to Carton, three types of host defence reaction are apparent in this case :lysis of existing tissue, proliferation and enlargement of blood vessels near the wound, and the appearance of undifferentiated cell masses close to the point of fixation. The intensity of these reactions is particularly interesting, since 8. pavonina is regularly parasitized without obvious ill effects by the very closely related Sabelliphilus elongatus. The latter, however, is confined to the branchial fan rather than to the body of the sabellid-the chosen domain of S . sarsi. Although slight erosion of the branchial filaments may be observed at the site of attachment, no other damage can be discerned (Gotto, 1960).

Tissue reaction in the sponge Xuberites domuncula (Olivi) against a copepod probably referable to the genus Sponginticola has been described by Tuzet and Paris (1964). Although the naupliar stage evokes no response, once transformation to the adult has taken place, a marked reaction is evident. The copepod becomes enveloped in a sheath of

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amoebocytes characterized by large nuclei and granular cytoplasm, which arrange themselves in more or less concentric rows around the invader and effectively isolate it. Tuzet and Paris think it probable that some at least of these amoebocytes arise from dedifferentiation of certain other cell types-pinacocytes, collenocytes, etc. As previously mentioned, Monniot (1963) gives an account of the three-layered cysts of the notodelphyid Kystodelphys drachi, situated on the branchial sinuses of the ascidian Microcosmus savignyi. These spherical cysts, 0-3-0.8 mm in diameter, are composed of host-derived cells, and presumably represent a well-defined tissue reaction to the presence of the copepod. Almost certainly the cyst or galls which enclose Staurosoma parasiticum Will, Mesoglicola delagei and Antheacheres duebeni within their respective actinian hosts can likewise be attributed to a specialized titlsue response on the part of the coelenterate. A truly astonishing host reaction to the notodelphyid Scolecodes huntsmani has been revealed by Dudley (1968). This large vermiform copepod is found, as already stated, in cysts within the big subendostylar blood vessel of various simple ascidians. Having gained access to the host in some as yet undetermined way, the second copepodid moults to a third copepodid stage.which is enveloped in a small bubblelike cyst. Subsequent development, involving two further larval instars, leads either to adult males or adult females. Living adult males have never been found encysted, though it is clear that they must gain access to a female’s cyst for mating. The cyst, which may eventually reach 19 mm in length, is composed of cells derived from the host, and is anchored by connective tissue within the blood vessel. As growth proceeds, a funnel, profusely lined with cilia, develops at one end of the cyst. This funnel emerges from the wall of the blood vessel and opens into the host’s atrial cavity. The cells comprising the cyst are of columnar epithelial type, and Dudley believes that they may originate from free, totipotent lymphocytes. A t least part of the funnel complex, however, can be attributed to modification of cells forming the blood vessel wall. Bearing in mind the radical transformation involved in the production of ciliated elements from such unlikely precursors, the subtle complexity of this host-parasite interplay is a t once apparent. I n effect, the ascidian has been induced to provide an elaborate exit for its partner’s nauplii, and possibly a route of access for the male. Finally, some of the cyst cells become free within the lumen and are ingested by the copepod, as previously described in the section on feeding. Since even heavily infected hosts appear perfectly healthy, we may conclude that balanced, adaptive specialization could scarcely go further (see also Illg, 1970).

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X. MORPHOLOGICAL VARIABILITY AT INFRASPECIFIC LEVEL From time to time, authors have commented on slight structural differences which close scrutiny sometimes reveals in sufficiently large collections of an associated species. Such variations are too small to warrant recognition at specific level, and the erection of sub-species is a dubious procedure unless more is known of the total geographic range than is usually the case. The differences observed would frequently seem to be linked with occurrence on, or in, different hosts-but this is by no means invariable. Certain species of the ascidicolous genus Botryllophilus, for example, show remarkable asymmetric variation in pereiopod structure, even though the individuals concerned may come from the same host colony (Lang, 1948a; Stock, 1970b). The cause of such pronounced asymmetry-unique in Stock’s wide experience-is uncertain, though Lang is inclined to attribute it to a genetically controlled reduction which has not yet attained stability. Both Gotto (1961a) and Costanzo (1968) have recorded LichomoZgus canui Sars from the ascidian Clavelina lepadiformis, the former from the fully marine waters of Northern Ireland and the latter from the brackish Lago di Faro in Sicily. The Sicilian specimens are significantly smaller than those from Ireland, an effect which Costanzo regards as a possible consequence of reduced salinity. I n some species, small but constant differences, apparently hostcorrelated, are not hard to find. Mychophilus roseus, from botryllids, shows variation in the body outline of mature females,which enables one to distinguish specimens found in Botryllus schlosseri from those found in Botrylloides leachi. Individuals from B. schlosseri are of more or less uniform girth, whilst those from B. leachi are slightly inflated over much of the posterior third of the sausage-shaped body (Gotto, 1954a). Clear differences in the shape of both the body and the egg sac have been noted by Kabata (1967) in Nicothoe brucei Kabata from the prawns Nephrops sagamiensis Parisi and N . andamanicus Wood-Mason. Such host-linked variability is, of course, well known in a number of copepods associated with fish (Delamare Deboutteville and Nuncs, 1951;Cressey and Collette, 1971). Among notodelphyids, a distinctive form (forma spinulosa) almost equally referable to Notodelphys allmani Thorell or N . rufescens Thorell, is found in Ciona intestinah. This type is characterized mainly by the presence of many more spinules on the basal segment of the fifth leg than occur in the more typical form of either species found in ascidiid hosts (Bocquet and Stock, 1960a). I n a rather similar way, the lichomolgid Odontomolgus mundulus Humes exhibits minor differences according

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to the coral host infected (Humes, 1974a). Individuals from Alveopora catalai Wells possess a caudal ramus which is longer and more slender than that of specimens from A. mortenseni Crossland, and very slight differences in antennular proportions are also discernible. A host-correlated size difference has been detected in Pseudanthessius liber (Brady), an ecto-associate of sea-urchins. Both males and females from Echinus esculentus L. collected in depths of 40-60 m are significantly larger than those found on Psammechinw miliaris (Gmelin) and Paracentrotus lividm (Lamarck) from the intertidal zone of the French Channel coast (Bocquet et al., 1963c). An even more striking instance is furnished by Gonophysema. On Scandinavian coasts, G . gullmarensis has been found only in Ascidiella aspersa and A . scabra, whilst in the Mediterranean, a morphologically identical but much smaller form occurs in an altogether different ascidian, Distomw variolosus. This latter host is, in turn, appreciably smaller than either of the Ascidiella species infected in more northern waters, which may be of some relevance. Bresciani et al. (1970) have left the precise specific identity of these Mediterranean specimens open, but are reluctant to erect a new species of Gonophysema in view of the incontestably detailed resemblance to G . gullmarensis in virtually everything except size. Species which have a very extensive geographic range and/or a large roster of acceptable hosts seem particularly prone to morphological variation at infraspecific level. The myicolid Pseudomyicola spinosus has been recorded from 39 species of bivalves, and its range includes both sides of the Atlantic, the Mediterranean, the Black Sea and the Indian Ocean. Humes (1968)finds marked variation in body length and width, proportions of the caudal rami and degree of spinulation on the anal segment, but regards P. spinosus as a single, albeit variable, species. Perhaps the same may be said of the equally wide ranging notodelphyid Doropygm pulex. This ascidicole has a very extensive host-spectrum and exhibits a bewildering variety of subtle structural differences which are at present almost impossible to interpret (Illg and Dudley, 1961, 1965; Stock, 1967b; Gotto, 1975). I n such cases, it is difficult to determine which factor is of paramount significance in promoting variability-host-influence or mere geographic distance. XI. SIBLING SPECIATION Within recent years, some interesting examples of sibling speciation in associated copepods have come to light. These seem to follow the same pattern of evolutionary development putatively established for other animal groups. Summarizing in general terms, we may say that a

76

R. V. GOTTO

copepod (or its ancestor) forms a liaison with a particular host (or host ancestor) which occupies a continuous geographic range. A geophysical event now supervenes to interrupt this range, thus splitting the population. On either side of the barrier genetic differences will inevitably accumulate and express themselves morphologically, physiologically or ecologically. If the barrier is now removed, differentiation may have proceeded far enough to prohibit mating between the reunited populations, resulting in the occurrence of obviously related but distinct species which may occupy the same area, or even be found on the same individual host. The two closely allied species of Sabelliphilus, elongatus and sarsi, would seem to provide us with a classic example (Bocquet, 1953; Bocquet and Stock, 1963, 1964). S. elongatus is found only on the branchial filaments of Sabellapavonina and (where the host is available) Spirographis spallanzani. It extends over a great part of the western European coastline and into the Mediterranean. On balance, Bocquet and Stock (1964) believe that it is closest to the ancestral Sabelliphilus. S. sarsi, in the pre-adult stages of the female, is also found on the branchial filaments of its host, in this case exclusively Spirographis spallanzani, which does not, a t the present time, extend farther north in Europe than about 49"N latitude. After fecundation, however, females of S. sarsi move from the filaments on to the body of the worm where they remain to become ovigerous. Although the available data is susceptible to various interpretations, the following may be a reasonable theoretical account of the evolutionary path taken by this genus. Let us suppose that S. elongatus-or an elongatus-like ancestor-originally parasitized the pseudobranchial fan of sabellids over a wide, continuous range. Some geophysical event now supervened which effectively split the population into two. Such an event could well have been accompanied by climatic changes hostile to the northward extension or maintenance of Spirographis spallanzani. In the boreal area, Sabelliphilus elongatus became progressively committed as a fan parasite of Sabellapavonina, with no suitable, alternative host to infest. The Sabelliphilus constituting the now isolated " southern " population accumulated sufficient genetic changes to become more specialized parasites of Spirogrcvphis, including in their repertoire of altered behaviour an adaptive migration to the host's body. They could thus capitalize on the more capacious tube of this sabellid to ensure greater shelter and protection for the ovigerous females. In time, the modifications involved would have become recognizable a t specific level-Sabelliphilus sarsi, in short, would have come into being. Some subsequent geophysical happening must at this stage

THE ASSOCIATION OF COPEPODS WITH MARINE INVERTEBRATES

77

be postulated to have removed the barrier, allowing the now distinct species to establish contact. 8. elongatus had remained sufficiently adaptable to colonize the fan, but not the body, ofSpirogruphis, as well as continuing to infest the filaments of Subella. As a mated female, however, S. sarsi had become irrevocably committed to the body region of Spirographis. I n southern parts of the range at the present day, both copepods may be found on this latter host, but as adult females, are rigidly confined to their preferred niches. Somewhat similar case histories, involving the concurrent infestation by sibling species of the same host, have been postulated for Anthessius species in tridacnid clams and for Pseudanthessius species on crinoids (Stock, 1967a). A rather less complex evolutionary story may be suggested for the siphonostonies Asterocheres violaceus (Claus) and A . minutus (Claus) from regular echinoids on the western European coastline (Bocquet et ul., 1963b). These two copepods are structurally almost identical, but A . violaceus is significantly larger and enjoys a wide range on the Atlantic sea-board, in marked contrast to A. minutus which is restricted to the Mediterranean. I n the latter area, it has been recorded from three species of sea-urchin, though its preferred partner is almost certainly Paracentrotus lividus. Asterocheres violaceus has a more extensive host, roster of eight echinoid species. The two copepods overlap in the Mediterranean and may be found together on the same sea-urchin without displaying any territorial preference. Bocquet and co-workers consider A. minutus to be derived from the larger species and believe that the present situation can be interpreted as a consequence of allopatric speciation. I n this case, therefore, a single ancestral species of Asterocheres is envisaged, which parasitized sea-urchins over a wide geographical range, but became divided into " western " (Atlantic) and " eastern " (Mediterranean) components by a land barrier. The Atlantic population remained relatively unchanged, thanks to a stable oceanic environment. The Mediterranean group, however, trapped in a relatively small sea subject to considerable fluctuations throughout its history, accumulated sufficient mutations to transform it into the species minutus. By the time the Straits of Gibraltar had opened to reestablish communication, specific separation was complete. The new sea link allowed the euryplastic A. violaceus to recolonize the Mediterranean, but did not permit range-extension westward by A . minutus, a species by now stenoplastically adapted to the conditions peculiar to an inland sea (Bocquet. and Stock, 1963). Paired species of lichomolgids associated with the same coelenterate host have also been recorded in the genera Paramolgus, Plesiomolgus and Metaxymolgus (Humes, 1976). It may be presumed that their

78

R. V. OOTTO

evolution has followed a similar course to that suggested for Asterocheres. Though geographic isolation may have contributed to speciation within the genus Synaptiphilus, the destinies of these associates of apodous holothurians have probably been influenced to a greater degree by the evolutionary fates of their respective hosts. Bocquet and Stock (1957b)point out that Synaptiphilus tridens occurs on both Leptosynapta inhaerens and L. cruenta Cherbonnier which are very closely related. A quite distinct, less spinous species, Synaptiphilus luteus Canu & Cubnot, is found on Leptosynapta galliennei (Herapath) which, in turn, differs significantly from its congeners inhaerens and cruenta. Finally, Synaptiphilus cantacuzenei Bocquet & Stock has diverged markedly from the other two species and is restricted t o a generically distinct host, Labidoplax digitata (Montagu).* Bocquet and Stock believe that an ancestor common to both Leptosynapta and Labidoplax was originally parasitized by an ancestral species of Synaptiphilus. When the hosts underwent generic separation, the copepods synchronously differentiated into S. cantacuzenei on the one hand, and a species characterized by extensive body spinulation on the other. This htter, in parallel with. speciation of the leptosynaptine hosts, became stabilized as S. luteus on Leptosynapta galliennei and, in an exaggeratedly spinous form, as Synaptiphilus tridens on Leptosynapta inhaerens and L. cruenta. This very plausible reconstruction, ingeniously linking the palaeontological age of the hosts with sibling speciation of their copepod associates, should perhaps be slightlymodifiedand extended in the light of two more recent discoveries. Firstly, Guille and Laubier (1965) have described a sub-species of f3ynaptiphilus cantacuzenei on Labidoplax digitata in the western Mediterranean. They have named this form Synaptiphilus cantacuzenei miztus Guille & Laubier and it is clearly distinct from the typical (Atlantic) sub-species, now known as S. cantacuzenei cantacuzenei Bocquet & Stock. Secondly, in Strangford Lough, Northern Ireland, we have discovered that S. tridens infests not only inter-tidal Leptosynapta inhaerens but the sub-tidal Labidoplax media, a little known holothurian inhabiting black, glutinous mud (Gotto, unpublished; and see Gotto and Gotto, 1972). With such additional facts in mind, we may now envisage the

* It is true that Bare1 and Kramers (1970) have recorded S. Zutew from L. inhaerem and L. bergetwis (Oestergren), as well as from L. gdliemnei, in the Plymouth area. There is nothing, however, to indicate in their collecting methods that the hosts were kept separate, m c l since species of Synaptiphilus will readily attach temporarily to atypical hosts, this record must remain doubtful.

THE ASSOCIATION OF COPEPODS WrrR MARINE INVERTEBRATES

79

evolutionary history of these cyclopoids in a little more detail. To begin with, we might perhaps regard Synaptiphilus tridens as closest to the ancestral Synaptiphilus stock. On this view, its well-developed caudal rami, the possession of four setae (the full eomplement) on the distal joint of the P1 exopod, and the presence of three prongs on the antennular base, would be " primitive " features. Its very extensive body spinulation might also be considered an ancestral character, though possibly of adaptive advantage for adhesion to the host from an early stage. With reference to this point, it may be remarked that here we differ somewhat from the view of Guille and Laubier (loc. cit.) who seem to imply that hyperspinulation is secondarily adaptive in these copepods. S . tridens, then (or its immediate predecessor) may be visualized as becoming a host-labile ectoparasite infesting various species belonging to at least two synaptid genera, Labidoplax and Leptosynupta. This early absence of rigid host specificity has continued to the present day, with Leptosynapta inhaerens and L. cruenta, as well as Labidoplax media Oestergren constituting the currentIy known host roster. S ~ n a p t i p h i ~ u s luteus can be seen as a closely related offshoot from this ancestral stock, evolving in the direction of less pronounced spinulation, but retaining the three antennular hooks and alimb chaetotaxy similar to that of S . tridens. It has acquired a narrow degree of host specificity, infesting only Leptosynapta galliennei and ignoring other species of Leptosynapta and Labidoplax. Synaptiphilus cuntacuzenei, in its typical sub-specific form, appears to represent a more radical departure from the ancestral tridens type. The antennular hooks are reduced to two, the terminal podomere of the P1 exopod has lost a seta, the fourth thoracic segment has altered in shape, and general spinulation is but feebly developed. Moreover the ornamentation of the female maxilliped is markedly different from that of its congeners. This species may also have diverged early to form a strictly specific and continuing association with Labidoplax digitata. The sub-speciesSynaptiphilus c. mixtus, known so far only from the western Mediterranean, seems to represent a race derived in comparative isolation, as already suggested by Guille and Laubier. We might postulate genetic drift to have assumed a significant role in its inception. JQhatever the truth of this supposition, there have evidently been mutations towards a reacquisition of extensive spinulation and a return to the tridens-luteus type of female maxilliped. It would seem unlikely that the ornamentation of this appendage is an adaptive character, since that of the typical sub-species is completelydifferent,even though the latter successfully infests the very same host on the Atlantic seaboard.

80

R. V. OOTTO

That the evolutionary situation in this genus is one of continuing fluidity, may be attested by a further observation. On the north-eastern coast of Ireland we have some evidence, as yet incomplete, that not all populations of Leptosyna 200 days

d r h i a nauplii

EUPHAUMACEA Euphausia pacafrca

months

Brtmia nauplii

144-16.4

1 litre

1 year

h n y ; Thal; Dun;

6.5-16.0

1 litre

A r h i u nauplii Artmnia nauplii mixed diatom? i Artemio nauphi

8.3-124 5-15

2 litre 750 ml

Jerde and h k e r (1966) Fowler et d.(1971)

COSC.

12-15

1 litre

Paranjape (1967)

14.8-16.4

1 litre

83-12.2 8342.2 8.3-12.2 6.15

2 litre

h k e r and Theilacker (1965) Jerde and h k e r (1966) Jerde and h k e r (1966) J a d e and Laaka (1966) Fowler et d.(1971)

E. pacific0

44 daYB 7 months

40 days E. e z i m

months

E. m.ma E. rGcum

19 dam 7 days

B. giaaoidu E. kmhni

8

am

-

-

Platymonas 8ukoniqormia;

P l a t k sp. ThaIu&Ara k u l a ; drtsnsia nauplii drternia nauplii

d r h i a nauplii A r h h nauplii Arlstnia nauplii

maintenance

mixed

months

Phytophnktoq; d r h i a naupln A r h i a nauplii

egg-late juvenile Laud; Cocw; Cyclo; Coscinmfwcud grani ; A r h M nauplii mixed maintenance

11.8 11.8 14-20

8.5 litre

8.5 litre 500 ml

2 litre 2 litre 750 ml

14.8-16.4

1 litre

13

76-100 ml

Laaker and Theilacker (1965) ctopalakrishnan (1973)

5-15

750 ml

Fowler et d.(1971)

Nydiphamd muchi

N.couohi

11 montha 49 days

20 days Mq7an@phama Mzwpica several montha

N. rimpk

M.noroegica

60 days

M.7lowe&a

Thyuanoc8a radchii

15 days >30 days

T. lonsipcs

30 days

T.apinifera

30 days

T. apinifera Teesarabrachh ocddu8

20 day6 15 days

Thy8a-a

acpuilalsr

OSTRACODA Conelroaeiaepinirostris C. apinirculris Gtqantqpris m&ri Cypridma WstaMa DECAPODA Sergeeta l u m

TUNICATA Thdia &m@3&%

Artemia muplii Phaeo mixed phyto&mkton ; Artsmio nauplii Artemia nauplii mixed phytoplankton; Artemia nauplii Phaeo; Telroedmis s p i A r h h nauplii

.

-

ThoEclsmOsira rotula; Artemh nauplii COSC ; P ~ y H m a 8p s ; Talasuiauira rot&; Artemh nauplii cosc Pbtylnonassp * T&&ra eula;' A h h nauplir Artenria nauplii cosc ; Platynoluursp ; ThoEclsmoairardda; Artemh nauplii A r h h nauplii

.

Chaetooeros ocra&sporum; ,, Artmia naupbr

0. diOic0

FdiUaria w k Oikopleura dzOic0

0. diOim

>1 generation

Fowler el d.(1971)

10-20

50-80 ml

Le Roux (1973)

83-12.2 5-15

2 litrea 750 ml

Jerde and Lasker (19813) Fowler st al. (1971)

10-20

5&80 ml

Le Rnux (1974)

5-20

2.5 litre 1 litre

Baker (1963) Paranjape (1967)

11-15

1 litre

Paranjape (1967)

11-15

1 litre

Paranjape (1967)

8.3-124 11-15

2 litre 1 litre

Jerde and Laaker (1988) Paranjape (1967)

83-12.2

2 litre

J a d e and Lasker (1968)

--

0-20 0-20

2.5 Utre 2.5 litre

Lochhead (1988) Angel (1970) Baker (1963) Baker (1963j

18-25

50-1 000 ml

Omori (1971)

-

maintained maintained maintained multiple eeneratiom 20 days

750 ml

-

13 days 7 days

hatchingpostlarval stage V

615

cost ; Platylnonas sp ; 11-15

few days few days

multiple generations 2 generations 19 generatiom >1 gcneration

T.dsmonath

mixed phytoplanktoq;

Pavolva (1967) Pavolva (1987) Pavlova (1967) Inone and Aoki (1971)

25

natural seawater

14

natural seawater 180. Mono. Cyclo I30 Mono CyClO; natural seawater

13 13 7-18

-

-

Abbreviations for food organism : coOco. Coeodiihus h u w W C . coscinodisnrs Ovcln. CurlotsuR ~

I U I

-

14-22

4 litre 4

litre

4 litre 4 litre

Braconnot (1963) Heron (197%)

r.p.m. :rotator r.p.m. :rotator r.p.m. :rotator

Pa5enhllfer (1978~) Pa5enhllfer (1973) Pa5enhllfer (1976~) Femur (1978)

218

OUSTAV-ADOLF PAFFENHOFER

AND ROQER P. HARRIS

212 pm for animals from surface and below surface plankton samples. The oral diameter was a more constant feature of Iorica structure, showing little change (90-92 pm). Similar abnormal development in cultured Tintinnopsis beroidea is figured by Gold (1970) who also reported a decline in tintinnid viability in culture which he suggested was due to the failure to conjugate. I n a further study by the same author a vigorous strain of T. beroidea was established in vitro by mixing several isolates (Gold, 1971). It was hoped that different mating types would be introduced thus increasing the longevity of the Fluorescent lighting

I

1

Stirrer

* Fresh medium resevoir

FIa. 1. Schematic diagram of a system for the continuous-flow culture of the tintinnid, Tintinnopsi8 beroidea. Arrows indicate the direction of flow. (Redrawn from Gold, 1973.)

culture by facilitating conjugation. It appears that previous problems of tintinnid senescence were overcome in this way as the active culture was maintained for 1.5 years. Mass cultures were grown in 2.5 litre vessels a t 10°C feeding on the microflagellates used previously (Gold, 1968). Too high a food concentration was found to be inhibitory, and too low led to rapid starvation. Tintinnid densities of 1 0 0 0 cells ml-l were routinely obtained, doubling time ranging from 2.5-6 days. Though characteristic conjugation was not observed Gold presented evidence that the culture might retain its vigour, concluding that a useful species of tintinnid was now available for physiological studies.

LABORATORY CULTURE OF MARIXE HOLOZOOPLANRTON

219

In a further development of culture methods Gold (1973) maintained T . beroidea in continuous culture in 5 litre flasks (Fig. 1) at 9.O"C for 5 months using intermittent agitation and the same food organisms as in earlier studies. The three species studied by Gold (Favella campanula, Tintinnopsis beroidea and T . tubulosa) are all coastal forms. Another tintinnid, Stenosemella cf. nivalis, was cultured at 18" to 20°C in screw-cap tubes or 250 ml Erlenmyer flasks feeding on Cryptomonas sp., Isochrysis galbana and Monochrysis lutheri (Beers et al., 1970). Maximum ciliate densities approached 50 animals ml -l. It was found that high food concentrations lead to a reduced ciliate density, an observation similar to that of Gold (1971). Working in the same laboratory as Beers et al., Heinbokel (1978a) isolated five species of tintinnid from the Pacific Ocean off Southern California, which were subsequently cultured at 18°C. The species investigated were, Amphorella quadrilineata (Claperhde and Lachmann), Tintinnopsis cf. beroidea Stein, Tintinnopsis cf. acuminata Daday, Eutintinnus pectinis (Kofoid and Campbell) and Helicostomella. subulata (Ehrenberg). The food used consisted of the flagellates Isochrysis galbana, Monochrysis lutheri and Dunaliella tertiolecta Butch. Feeding experiments were conducted at initial concentrations ranging from 25 to 400 pg algal carbon litre-l. Ingestion rates of E . pectinis and H . subulata initially increased with increasing food concentration, but between 60 and 100 pg C litre-1 there was no further increase in ingestion with increasing food concentration. Ingestion rates of T . acuminata, however, continued to increase over the whole range of experimental food concentration used. Feeding on natural particulate material, tintinnids ingested particles up to 43% of their lorica diameter. The maximum diameter of particle that was ingested increased with increasing oxal diameter. Grazing rates decreased with increasing food concentration. They were a function of the oral diameter and ranged from 1-9 microlitre swept clear animal-1 h-I. (Heinbokel, 1978b). In contrast to the mostly neritic tintinnids that have proved amenable to laboratory culture Hamilton and Preslan (1969) succeeded in cultivating a species of pelagic hymenostome ciliate of the genus Uronema. The original isolate of Uronema sp. was made from an open ocean water sample taken aseptically from a depth of 385 m approximately 150 miles off the coast of Baja California. Cultural characteristics were investigated initially in batch culture overranges of temperature and salinity of 10" to 32°C and 17" %, to 43" X0. The optimum temperature for growth was about 25"C, contrasting with the work of Gold (1968) with Tintinnopsis where the importance of low

220

QUSTAV-ADOLF PAFFENHOFER

AND ROGER P. HARRIS

temperature (< 10°C) was emphasized. Marine strains of Chromobacterium, Pseudomonas, Vibrio,Micrococcus and Serratia were consumed by Uronema. I n the batch cultures there were large changes in cell volume in cultures of various ages. For example, during the lag phase there was a twelve-fold increase (550 pm3 to 7 000 pm3). Further aspects of trophodynamics of Uronema sp. feeding on the marine bacterium Serratia marinorubra under steady state conditions were studied in continuous culture by Hamilton and Preslan (1970). Protozoan cell volumes varied under steady state conditions in apparent response to cell numbers. It was found that the maximum growth rate, representing a doubling time of 4.6 h, was attained at a concentration of 1.5 x 106 bacteria ml -l (= 225 pg C litre -l). From their investigations Hamilton and Preslan concluded that this protozoan cannot feed effectively on free bacteria in the sea which they suggested occur at much lower concentrations than those employed in their continuous culture studies. However, they point out that it is well adapted to utilizing localized high concentrations of bacteria in association with particulate material. Thus, from an ecological standpoint, an interesting feature of their work is that it quantifies trophic relation ships of an open ocean organism, which preys on bacteria, which may in turn be preyed on by larger forms. Hamilton and Preslan cited observations by E. R. Brooks showing that the copepods Calanus helgolandicus Claus and Labidocera trispinosa Esterly will eat this ciliate. The ability to maintain planktonic protozoa in continuous culture (Gold, 1973; Hamilton and Preslan, 1970) makes the future development of quantitative models of protozoan trophodynamics a realistic objective. Such studies have already been made for a hymenostome ciliate Uronema marinum Dujardin (Ashby, 1976 ; Parker, 1976). However this species was non-planktonic being isolated from sediment. Techniques for culturing benthic forms of Uronema have also been described by Hanna and Lilly (1974) and Soldo and Merlin (1972) and such methods may also, with adaptation, be appropriate to planktonic forms.

B. Cnidaria The phylum Cnidaria is almost exclusively represented by species which at least during part of their life cycle are not planktonic, consequently most publications on maintenance, rearing or culture of Cnidaria concern species which are attached to surfaces during part of their life cycle, thus not being holoplanktonic. For example, the maintenance of medusae of the scyphozoan Cyanea eapillata (L.) and

LABORATORY CULTURE O F MARINE ROLOZOOPLANKTON

221

Chrysaora quinquecirrhu in 80 litre aquaria has been described by Ward (1974). C. Ctenophora Species of the phylum Ctenophora are holoplanktonic, excluding the order Platyctenea. Although they occur in large numbers in neritic waters and have been the objects of numerous studies, it was not until recently that a satisfactory technique was developed that enabled Air

Communicating tube

.................................. .............................

\\A\\\

\\\A\''

!I

ii Outlet

i i !! i :i :

:I

j.. ..g ... ... \!

Centre column

r

D \

U

10

Q

0

5.0

7

\

1.0 0.05

1,

0.5 I

I

I

I

5

10

15

20

I

25

30

35

40

I

45

50

55

Days after hatching

FIG. 14. Ingestion rates of Rhincalanus nasutus reared on Thalassiosira fluviatilis at two different temperatures. 0 and upper line represent data for 15'C (y= 0 . 0 4 6 ~0.74); X and lower line represent data for 10°C (y = 0 . 0 3 7 ~- 0.074). The right ordinate indicates the equivalent atering rate at a food concentration of 150 pg C/litre. (After Mullin and Brooks, 1970a.)

2. Respiration

I n comparison with detailed studies of feeding rates of holozooplankton in laboratory cultures relatively little work has been done on respiration despite its importance as a measure of metabolic rate. One reason for this may be that ingestion rates can be measured using small numbers of animals in relatively uncrowded conditions either by microscope counts or, more recently, by using electronic particle counters to measure changes in cell concentrations. I n contrast the majority of respiratory measurements require a reduction in oxygen concentration which can only be brought about by using large numbers of animals or relatively confined conditions. Clutter and Theilacker (1971) in their study of the energy budget of the pelagic mysid Metamysidopsis, which involved rearing animals in the laboratory, measured weight specific respiration rate, but it is not clear whether the animals used were actually derived from the laboratory cultures. Similarly Lasker (1966) measured the respiration of Euphausia pacisca using " young and old " animals. Conover and Lalli (1974) reported on respiration of Clione Zimacina, Hirota (1972) for Pleurobrachia bachei and Dagg (1976)on the respiration of Calliopius. Mullin and Brooks (1970a) report the only results on the respiration of laboratory reared copepods, measuring respiration rates for CIV/V

TABLEVIII. STUDIES ON GENERATION T I ~ OFS HOLOZOOPLANKTON IN LABORATORY CULTURES spceies

PROTOZOA Tintinwpm8 bermdm

T . cf. amnainata Eutintinnus pectinis l€elieostolneuo rubulata U r m a sp. CTENOPHORA Pleurobrachia bachei

Mnemiqpsis mccradyi

Foad organime

Temperature "C

10 18 18 18 20

15 21-31

Is0 * Rhodo. Platy. small phdtosynth&ic flaghate Is0 Mono. Dun. Is0 f Mono Dun. 180. Mono ' Dun. SeAatia m&inmubra

.

Definition

Ueneratwn time

doubling time

2.5-6.0 days

doubling time doubling time doubling time doubling time

12 h 12 h 24 h 4.6 h

-

source

Gold (1971)

Heinbokel (1978a) Heinbokel(1978a) Heinbokel (1978a) Food concentration Hamilton and Prmlan (1970)

Labidocera and Galanus nauplii' Adult Acartia ;Artemia nauplii ' fipepods

egg-egg

60 days

-

Hirota (1972)

egg-sgg

minimum 13 days

-

Baker and Reeve (1974)

2.7-0.8 days

ROTIFERA Brachionus plicatilis

24

Mono ;Nannoehlm's sp ;Exuviella sp; Dunaliella sp.

doubling time

CHAETOGNATHA Sagitta hispida S.hispida

22-24 17-31

Microzooplankton Microzooplankton

hatching - maturity 33 days hatching maturity 19-45 days

AMPHIPODA CaUiquius laemusculus Hyp~rochenaedusarum

5-15 10

COSC calanoid copepods Herring larvae

hatching-maturity hatching-sexual maturity

29-50 days 69 days

MPSIDACEA Metamysidqpsis elongata

lG-14

Artemia nauplii

hatching-hatching

63 days

COPEP0.DA EuterpPW acutifrm.3

16-23

Phaeo.

hatching-maturity

10-25

PhaW.

egg-egg

10-25 days (J); 1.338 days ( 9 ) 5-40 days

Calanus helgolandicue

12

36 days

10-15 12-17

Thal :Cyclo :Dit ;Co8cinOd~BC9.48 wailesii Thal. Tbal; Gymno

egg-adult

C. hdgolandieus C. hdgdandieus

hatching-adult hatching-CIV

2 2 4 4 days 14-30 days

C. hdg0landiou.s

15

1.354 days

12

Laud; Skele; Gymno; Chaetoceros eurviaetus Thal ' Cyclo ;Dit ;Coreinodienur a w a d i ; ~ r t m i nauplii

hatching-adult

R h i d a n u s nasutus

egg-adult

2 8 4 9 days

E . acutifrmzs

Enwironnznztd variables

.

-

Food species and concentration

-

Theilacker and McMaster (1971)

Temperature

Reeve (1970a) Reeve and Walter (1972)

Temperature

Dagg (1976) Westernhagen (1976)

-

Clutter and Theilacker (1971)

-

Bernard (1963)

Temperature; sexual dimorphism Haq (1972) Yulliu and Brooks (1867)

-

Nullin and Brooks (1D70a) Temperature Temperature. food Nullin and Brooks (1970b) concentration' Food species and Paffenhofer (1970) concentration &fullinand Brooks (1867)

R.nagulus

10-15

Thal; Dit

hatching-adult

22-53 days

P8eudocalanus minutus Pseudodanus dongatus

11.9 12.5

180.

Thalassiosira rot&

hatching-adult hatching-hatching

P. dongatus C t m d a n u s vanw Temara longicornis

15 18 12.5

180 ; Skele ; Platymunas

egg-egg

T . lGn&O??&iS Euwtmora herdmani E. herdmani E. allinis Cenlropages typicus c. typkU8 0. haW24ZtU.S Oladioferensimparipes

20 10-15 2-23.5 2-23.5 20 18 20 15-25

Tetraselmisswcica Thalassiosira sp. Cyclo; Skele; Platymums sp 180; Cyclo; Skele; Platymonas sp Tetraselmia a&a 13 species of phytoplankton Tetraselmis euecia Dun; Cyclo; Phaeo.

hatching-maturity

35.5 days 2&32 davs (x = 28.5) 37 days 35 days 24-33 days (X = 28.2) 21 days 16-28 davs 19-73 days 10-105 days 25 days GO days 22 days 114-29.7 days

Labidocera triapinosa

Laud ; Gymno. Acartia and Calanus nauplii Proro ; Gony ; Gymnodinium 20 nelsoni: Artemia nauplii 15.5-25.5 Natural seawater 17 Is0 ; Rhodomonas sp : small diatom Tetraselmis sueeiea 20

hatching-maturity

36 days

NI-adult

18-25 days

egg-egg

7-13 days 25 days

Ponlella nteadi Acartia toma A. t m a A. A. A. A.

dausi clawri daun

sp. 13 species of phytoplankton Thalassiosim rotula

180; 180;

15

daugi

18 10-20

13 species of phytoplankton 180; Dun; Peri.

A. elatmi

15-20

180 ; Mono

A. grant

17-21

180; DiaCronsma vlkianum (?)

TUNICATA Thaldo democratice

-

hatching-hatching

hatching-maturity

-

-

-

20

hatching-adult

-

Oikopkwra dioico

0. dioica

13 7-18

0.dioica FriliUaria borealis

14-22 13-16

26 days 2-14 days

180. Mono ' Cyclo 180 Mono f Cyclo ; Natural

seawater

-

Natural seawater

Abbreviations for food organisms : Cosc. Coscinodiscpu,angsti Cyclo. Cydotella mna Dit. Ditylum brightwelli Dun. Dunaliella tertioleeta Gony. Qonyaulaz polyedra Gymno. lrymnodinium splendens

hatching-spawning hatchingapawning

hatching-spawning

9.5 days &24 days 3-12 days 7-9 days

180. Isochrysia galbam Laud. Lauderia borealis

Mono. Monochrysis lutheri Peri. Peridineum trochoideum Phaeo. Phaeodaetylum lricornutum

Mullin and Brooks (1970a) . .

-

Corkett (1970) Food concentration Harris and Paffenhofer(1976a)

-

hatona and Yoodie (1969) Kassogne (1970) Food concentration Harris and PaKenhOfer(l976a) Temperature Temperature Temperature

Person Le Ruyet (3975) McLaren (1976) Katona 11971))

-

-

Temperature

Temperature -

-

-

hatching+gg laying

-

Food species; temperature

Rippingale and Hodgkin (1974) Barnetc(1974) Gibson and Grice (1976) Heinle (1966) Zillioux and Wilson (1966) Person Le Rnyet (1975) Corkett (19G8) Nassogne (1970) Landry (1976)

Temperature; seasonal acclimation Temperature; Light Iwasaki et d.(1977) intensity Vilela (1972) Temperature; Food Heron (1972a) concentration Food concentration Paffenhofer (1973) Temperature Paffenhofer (19760) Temperature

-

Fenaux (1976) Paffenhofer (1976~)

Platy. Platynoma tetrathele Proro. Proroeentrum micans Rhodo. Rhodomonas lens Skele. Slcsletoma C08tUlUm Thal. Thalassiosirafluviatilis

E 0

268

OUSTAV-ADOLF PAFFENHOFER

AND ROGER P. HARRIS

and adult female Calanus and Rhincalanus raised on Ditylum and Thalassiosira, and comparing them with those of wild animals. The results of the latter comparison suggested that respiration per unit body weight of the laboratory reared animals was comparable t o that of animals from the wild. Measurements of excretion, for example of nitrogen and phosphorus, require the same large numbers of animals and degree of crowding as oxygen measurements. Probably for this reason excretion measurements are completely absent from culturing studies. Dagg (1976) reported on ammonia excretion by Calliopius, but relied on animals collected from Puget Sound. The use of laboratory cultures of copepods would seem t o have potential in providing animals of known age for excretion studies of the type performed by Corner, Cowey and Marshall (1967) who used Calanus from nauplius I t o adult ; the later naupliar and all copepodite stages being picked from plankton samples. 3. Growth and generation time

I n studies where holoplanktonic organisms have been maintained over more than one generation in the laboratory, the generation time is

\ 6 20

-

I

c c

m

-

r

-

0

i; 5-

I

I

I

1

1

5

10

15

20

Temperature 1°C)

FIG. 15. Generation time of Oikopleuru dioica reared in natural sea-water at different (After Paffenhofer, temperatures. log y = 1.8173 - 0 . 0 6 1 8 ~ ; r = -0.9939. 19760.)

usually reported though the actual definition of the interval designated as the ((generation time" is often not specified. I n addition t o observations on generation time incidental t o studies of culturing methodology a number of workers have used laboratory cultures to

LABORATORY CULTURE OF MARINE HOLOZOOPLANKTON

269

specifically measure generation time and development rates under varying environmental conditions (Table VIII). Information on generation times is a particularly useful product of laboratory cultures as estimates from wild populations may be inaccurate, depending as they do on cohort analysis or size frequency distributions together with the difficulty of sampling the same population of animals on repeated occasions. I n addition in areas, for example the tropics, where there is continuous reproduction with overlapping generations, laboratory estimation is often the only way of obtaining good estimates of generation time. The generation time of Oikopleura dioica at 13°C feeding on Isochrysis galbana, Monochrysis lutheri and Thulassiosira pseudonana ranged from 8 to 12 days (2 = 9.5 days) at food concentrations between 25 and 80 pg C.litre-I (Paffenhofer, 1973). The same author measured the generation time of Oikopleura dioica feeding on natural particulate matter, passed through 180 pm mesh, which between 7" and 18°C is a function of temperature being 5.5 days at 18" and 24 days at 7 O C (Paffenhofer 1976c) (Fig. 15). Fritillaria borealis has nearly the same generation time being 9 days at 13°C. Daily exponential growth rates ( k ) ranged from 0 . 5 7 to 1.09 at 13°C; this maximum value meaning that the weight of the appendicularian may multiply by a factor of 2.6 during 24 h. Feeding on natural particles, values of k increae from 0.23 ( T o ) , 0.47 (12") to 0.93 (17°C). I n similar laboratory experiments the generation times of Oikopleura dioica ranged from 3 to 5 days at 22°C and 10 to 12 days a t 14OC (Fenaux, 1976), his results on generation times at different temperatures essentially confirming those of Paffenhofer (1973) for animals raised on natural food. Reeve and Walter (1972) measured the time from hatching to maturity for Xagitta hispida and the generation time of Pleurobrachia pileus at 15°C was estimated to be 35 to 50 days (Greve, 1970). Apart from these estimates, incidental to general culturing investigations there have been a number of more specific investigations of generation time in laboratory cultures. Landry (1976) used cultures of Acartia ciausi to investigate the relationship between temperature and development of life stages for animals reared with excess food, confirming and complementing the initial findings of Corkett and McLaren ( 1970) for Pseudocalanus minutus, Eurytemora hirundoides and Temora longicornis. Data on development time from nauplius I to median CIV for Calanus helgolandicus are provided by Mullin and Brooks (1970a). Development a t two temperatures at a range of food concentrations decreased especially below 200 pg C slitre-l. Rearing Temora and Pseudocalanus at food concentration ranging from

270

QUSTAV-ADOLF PAFFENHOFER

AND ROQER P. HARRIS

25-200 pg C-litre'-l at one temperature (12°C) in contrast showed little apparent effect of food concentration on development, with the exception of the low food concentration of 25pg C .litre-l (Harris and Paffenhofer, 1976a; Paffenhofer and Harris, 1976). I n contrast Paffenhofer (1970) found that development times of Calanus helgolandicus were clearly affected by both food concentration and food species when feeding on unialgal diets (see Fig. 16). Heinle (1966) estimated development times in his work on production of Acartia tonsa a t a range of temperatures, rearing animals in estuarine water containing the natural phytoplankton assemblage. Detailed information on generation times of other estuarine copepods (Eurytemora afinis and E . herdmani) is given by Katona (1970) who investigated the relationship with temperature over a wide range (2-23.5"C). Also working with E . herdmani McLaren (1976) used cultures to provide the f i s t analysis in a marine copepod of inheritance of demographic and production parameters. One of those studied was the age of maturity of female offspring, which was shown to be strongly heritable among female offspring, a t 15°C. Growth rates of laboratory populations of planktonic Protozoa have been estimated by Heinbokel (1978a). Growth rates of Eutintinnus pectinis were described using a Michalis-Menten type equation. Growth rates of E. pectinis, Helicostomella subulata. and Tintinnopsis, cf. acuminata, increased with increasing food concentration staying even after reaching a maximum, except for T . acuminata the rate of which decreased at higher concentrations. The maximum growth rates yielded doubling times of about 12 h for E . pectinis and T . acuminata and 24 h for H . subulata. Preliminary experiments with the scope of determining the effects and interactions of four food species showed that Isochrysis galbana significantly enhanced the growth of E . pectinis and T . acuminata. I n comparison the following doubling times were obtained for the ciliates : Uronema 4.6 h at 25°C (Hamilton and Preslan, 1969))Xtenosemella 2 to 4 days at 20°C (Beers et al. 1970), Tintinnopsis beroidea 2.5 to 6 days at 10" and 15OC (Gold, 1971) and 12 to 24 h for unnamed tintinnids near 20°C (Heinbokel, 1975). The individual growth rates of asexual (blastozooid) and sexual (oozooid) stages of the tunicate T h l i a democratica were determined in the laboratory on animals collected from the sea (Heron, 1972a), being as high as a 14% increase in length-h-l. Medium growth rates, ranging from 1 to 5% length increase-h-l, could be sustained for up to 61 h. High growth rates were sustained for only 5 h, then dropped sharply. Heron suggested that this drop could be due to a marked reduction in phytoplankton concentration after 5 h preventing any

271

LABORATORY CULTURE O F MARINE HOLOZOOPLANKTON

‘‘ fast ” feeding thereafter. No information on handling, experimental temperature, container type and size is given in this study. The fact that constant growth rates could be maintained for only 2.5 days leads to the assumption that experimental conditions were in some way inadequate. The ability to rear holozooplankton animals in laboratory cultures facilitates the precise measurement of growth rates under controlled conditions. As with feeding and development time, the two major environmental variables that have been investigated are the effects of

I 5 0 100

200

800

400

Food concentration (pqC/litre

’

Fro. 16. Period from hatching to adulthood as a function of food concentration and food species for laboratory reared Calanus helgolandicua. Skeletonema coatatum ; 0 Chuetoceros curvisetua ; Lauderia borealis ; A Gymnodinium splendens. (Re-drawnfrom Paffenhofer, 1970.)

temperature and food concentration (Table IX). Growth has been measured in a number of ways. Some authors, for example Mullin and Brooks (1970a) and Paffenhofer (1976a) have removed animals a t intervals from cultures for carbon, nitrogen and dry weight measurements. But as such a sacrifice of animals from a small experimental population is often unacceptable, in many studies linear dimensions of animals temporarily removed from cultures have been measured. These are then used in conjunction with relationships between length and weight to estimate biomass increase. For larger planktonic crustacea such as mysids and euphausiids shed moults may be recovered from cultures for measurement of linear dimensions (Clutter and Theilacker, 1971; Lasker, 1966).

TABLEIX. STWDIES ON specie9

Temperdrre OC

CTENOPHORA Pkurobrachia p i k u s P. p ' k U 8 P. bachei

THE

GROWTHOF HOLOZOOPLANKTON MAINTAINEDIN

Food organism8

Units of lneasrreinsnt

P. bachei

15-20

copepods Copepods Labidocera and Calanus nauplii ; Adult Acnrtia; Artemia nauplii Copepod nauplii and adults

Bdinopsis infundibulum Beroe graeilis Mnemiopsis m w a d y i

16 15-18 21-31

Copepods Pleurobrachia pilers Natural zooplankton

body diameter body diameter Dry weight ; body diameter body diameter; organic carbon length length ash-free dry weight

CHAETOGINAT HA Sagitta hispida S.h i q i d a S.hispida S. hispida

22-24 12-33 17-33.5 21-31

Microzooplankton Artemia nauplii Microzooplankton Natural zooplankton

length length length ash-free dry weight

MOLLUBCA Clione limaclna C. limacina

12-14 15

Spiratella retroversa and S. Wicina dry weight Spiratella retroversa and S.helicina calories

AMPHIPODA C d i o p i u s lacviusculus

8-15

Cosc ; calauoid copepods

carbon and nitrogen

15 6-20 15

0.044.47

-

-

LABORATORY

Environmntal uatieblee

-

sour038

Temperature

Greve (1970) Greve (1972) Hirota (1972)

Body size

Hirota (1974)

-

Temperature

-

Temperature Temperature Temperature

Greve (1970) Greve (1870) Reeve and Baker (1975) Reeve .. 11970al Reeve (19703 Reeve and Walter (1972) Reeve and Baker (1975)

~

F

s

U

r +d tp

$

2

8: r M

0

10

Herring larvae

MYSIDACEA Metamysidopsis elongata

body length; eye diameter

10-14

Artemia nauplii

body length

EUPHAUSIACEA Euphausia pacifica Nemutoseelis diflcilis

65-16.0 13

Artemia uauplii Laud; Cocco; Cyclo; Coscimdiscus p a n i ; Artemia nauplii Phaeo ; mixed phytoplankton; Artemia nauplii Phaeo ; Tetrasdmis ;Artemia nauplii

Hyperoche medusarum

Coejioknt of daily exponential growth (k)

THE

Nyctiphams eouchi

10-20

Heganyctiphmm noruegica

10-20

0.001-0.143 0.21-050

-

-

Body size

-

Conover and Lalli (1972) Conover and Lalli (1974)

Temperature; body Dagg (1976) size Westernhagen (1976)

-

Clutter and Theilacker (1971)

dry weight length

-

Lasker (1866)

length

-

Le Roux (1973)

length

-

I& Roux (1974)

2!U 0 0 0 M Ea +d

*

X m

DECAPODA Sergestes l w n a

IS-25

Chaetoceros ceratosporum; Artemia nauplii

length

COPEPODA Calanun helgolandicus

15

Laud; Gony; Gymno; Proro

body weight

0.04-0.41

C. helgolandicus

10-15

Thal

body carbon

0.08-030

Rhinc&nus nasutus R. nasutus

10-15

Thal; Dit

body carbon

@OW64

PwAmJanus elongatus

12.5

Thalassiosira rotula

ash-free dry weight

0.01-0%3

Temro longieornia

13.5

Thalaasiosira rotula

ash-free dry weight

003-0.54

Labidocera trispimsa

15

Laud; Gymno; Acartia and Calanus nauplii

body length (mm)

TUNICATA Thalia democratica Oikopkura dioica

13

Is0 ;Mono ; Cyclo

length ash-free dry weight

-

0.57-1.09

-

Omori (1971)

Developmental Paffenhofer (1976a) stage. food species * food doncentration’ Mdlin and Brooks (1970s) Temperature; developmental stage Mullin and Brooks (1967) Temperature ; food Mullin and Brooks (1970a) species ;developmental stage Paffenhofer and Harris (1976) Developmental stage ; food concentration Harris and Paffenhofer Developmental staee : food (197th) coGeintration Barnett (1974)

Size, food concentration

Heron (1972a) Paffenhtifer (1976~)

Values for coefficient of exponential growth (k) represent range of values given. Qony. Gonyadax polyedra Abbreviations for food organisms: Cocco. Coccdithus huzleyi Gymno. Oymnodinium spkndem Cosc. Coscinodiscus angsti Cyclo. Cyclotella nana Iso. Isoehoyeis galbana Laud. Lauderia borealis Dit. Ditylum brightzoelli

Mono. Monochrysis lutheri Phaeo. Phaeadactylum tricornutum Proro. Proroceutrum micam Thal. Thalaasiotira fEuViatili8

274

GUSTAV-ADOLF PAFFENHOFER

AND ROGER P. HARRIS

Among studies of relationships between temperature and growth Reeve and co-workers (Reeve, 1970b; Baker and Reeve, 1974; Reeve and Baker, 1975; Reeve and Walter, 1972) provide extensive information for two planktonic carnivores from the subtropics. Growth of Sagitta hispida was initially measured in small animals collected from the sea and subsequently reared in the laboratory (Reeve, 1970), but further work by Reeve and Walter (1972) involved animals hatched

t

/

I . . . . . . . . . . 10

20

30

40

Days from hatching

50 Days from hatching

FIG. 17. Growth in the laboratory of the chaetognath, Sagitta hispida, and the ctenophore, MnWniOp8i8 mccradyi, at three different temperatures. (After Reeve and Baker, 1975.)

and reared in the laboratory. The period from hatching to 50% maturity of Sagitta hispida decreased with increasing temperature ranging from 45 days a t 17°C to 19 days at 31°C (Reeve and Walter, 1972). A similar approach was also used by Reeve and Baker (1975) in studies of both X. hispida and also the ctenophore Mnemiopsis mccradyi (Fig. 17) to obtain growth rates of both species a t three different temperatures as part of a study of production of these two planktonic carnivores. Also investigating ctenophores in laboratory culture

LABOltATORY CULTURE O F MARINE ISOLOZOOPLANICTON

275

Greve (1972) measured growth rates of Pleurobrachia pileus in relation to temperature. Greve's individuals seemed to have the fastest growth from days 20 to 45 after hatching (3 to 10 mm body diameter). The growth of P. bachei followed a sigmoidal curve, growth being slow during the first 40 days from 0.1 to 2 mm diameter (daily exponential growth rate k of 0.12 to 0.17)) a maximum from 2 to 7 mm (k = 0.21 to 0.47)) decreasing from 7 to 13 mm (k = 0.17 to 0.04) and levelled off above 13 mm (Hirota, 1972, 1974). Dagg (1976) used direct measurement of carbon and nitrogen to estimate growth in the amphipod Calliopius laeviusculus a t 8, 12 and 15°C the daily growth rate increasing with temperature (Fig. 18). Differentiation of the growth equations then enabled the relationship between growth rate and body weight to be investigated a t the three temperatures. Also working in terms of carbon and nitrogen Mullin and Brooks (1970a) investigated temperature effects on growth from egg t o adult in Rhincalanus and Calanus fed on two species of diatoms. Paffenhofer (1976a) working with Calanus helgolandicus investigated growth from egg to adult of animals maintained on different unialgal diets. Growth a t different environmental food concentrations was studied in Femora; and Pseudocalanus (Harris and Paffenhofer, 1976a ; Paffenhofer and Harris, 1976) temperature being maintained constant. From this work it may be concluded that in these small copepods, food concentration is less of an influence than temperature on growth rate. Changes in length of the lobate ctenophore Mnemiopsis were measured by Baker and Reeve (1974) for animals presented with a variety of food types. Daily growth rates were determined by rearing populations at 21"' 26" and 31°C offering naturally occurring zooplankton (no densities given), mainly being Acartia tonsa and Paracalanus parvus. Growth rates decreased with increasing size from 0.50 to 0.07 (21"), 0.78 to 0.07 (26*)and 0.65 to 0.07 (31°C). Growth only occurred when animals fed on natural zooplankton; phytoplankton and detritus apparently being inadequate diets. The effect of size of prey offered to the pteropod Clione limacina on growth rate was measured by Conover and Lalli (1972). Experimentally reared Clione feeding at 12°C to 14°C on Xpiratella from plankton tows showed varied growth responses. Repeatedly after initial rapid growth, feeding and with it growth declined resulting several times in weight losses which could be subsequently followed by intensive feeding (Conover and Lalli, 1972). Exponential growth rates were as high as 0.143, often between 0.02 and 0.04. There was a significant correlation between feeding rate and growth rate. The authors believe that the size of the available prey and not its abundance

Age ( days)

lor

15OC

1 1 1 1 1 1 1 120 40 00 Age (days )

40

00

Age (days)

FIQ.18. Growth of laboratory reared individuals of the amphipod Calliopius laeviusculua at three temperatures.

carbon ; 0 nitrogen. (After Dagg, 1976.)

LABORATORY CULTURE O F MARINE HOLOZOOPLANKTON

277

determine growth and ultimate size of the predator. I n additional experiments with Clione limacina, feeding on Spiratella retroversa and S. helicina at 15"C, Conover and Lalli (1974) determined instantaneous growth rates ranging from 0-155 to 0.419 based on weight, and from 0.208 to 0.504 based on calories. These growth rates, for animals weighing initially 0.31 to 4.19 mg (dry weight), are relatively high when compared with earlier findings (Conover and Lalli, 1972), being in the same range as those for actively growing nauplii and copepodites of Calanus helgolandicus (Paffenhofer, 1976a). 4. Growth eficiency

The ability to measure simultaneously parameters such as ingestion, respiration and growth in laboratory cultures has been of considerable importance in the construction of metabolic budgets for a number of groups of zooplankton, for example euphausiids (Lasker, 1966) and mysids (Clutter and Theilacker, 1971). Perhaps the most comprehensive of such studies is that of Dagg (1976)for the carnivorous amphipod Calliopius in which the effect of temperature and body size on all budget parameters was investigated in terms of carbon and nitrogen. This enabled the balance of each budget to be checked-unlike most other studies where one parameter (usually the most difficult to measure experimentally) has been determined by difference. Neither temperature nor body size affected the percentage of ingested matter assimilated by C. laeviusculus (Dagg, 1976). Thus, the mean assimilation efficiency was 90.4% (carbon) and 88.4% (nitrogen). Gross and net growth efficiency increased with increasing size from instar 1to 6, remained rather even over the 2 following instars and decreased from then on to instar 12 at all 3 experimental temperatures (So, 12" and 15°C). Minima and maxima of gross growth efficiency range from 25% (instar 1) to 45% (instar 8) to 1.0% (instar 12) at 8°C; from 34 to 46 to 1.1% at 12°C and from 25 to 48 to 1.8% at 15°C. E . pacifica at 10°C assimilated on an average 84% of the ingested food (Lasker, 1966). The percentage of carbon incorporated generally decreased with increasing animal size ranging from 30 to 6%. The average gross growth efficiency of animals from 1.3 to 8.9 mg dry weight was 25.6%. Other investigations have concentrated on utilizing laboratory determined growth or reproductive rates, together with ingestion rates, to determine growth eeciency, and with some exceptions for example, the pteropod, Clione limacina (Conover and Lalli, 1974) and Sagitta hispida (Reeve, 1970b)have involved herbivorous copepods (Table X). Reeve determined short-term gross growth efficiency in terms of dry A.M.B.-16

10

TABLEx. GROWTHEFFICLFNCIES DERIVEDFROM LABORATORY REARING EXPERIMENTS speeies

Temperature "C

Food organisms

Growth interval

Units of rnemuremnt

Gross growth

Emney (XI)

CTENOPHORA Pleurobrachia bachei

15

54-100 days from hatching

CHAETOGNATHA Sagitta hispida

Labidocera and Calanus nauplii ; Adult Acartia; nauplii

16-26

Artemia nauplii

immature stages dry weight; nitrogen

34.5% (mean)

MOLLUSCA Clime limacinu

15

Spiratella retrmersa and S. helicina

4-7 daya growth calories

49-78

AMPHIPODA Calliopius laeviusculus

8-15

Cosc ; calanoid copepods

pre-adult phase

carbon

MYSI DACEA Metam ysidopeis elonguta

14-20

Brtemia nauplii

hatchjug-adult

calories

EUPHAUSIACEA EupJmusia pacificu

66-16

Artemia nauplii

pre-adult growth carbon

COPEPODA Cala?azas helgolandicus

15

carbon

19-29s

-

organic carbon

20-29.9 % 0.744%

Thal

carbon

34-35%

Thal ;Gymno

NI-CIV

ratio of dry weight (CIV) to cumulative ingestion (pgC)

6. helgoZandicua

10-15

C. helgolandieus

12-17

-

-

Rhincdanus nasutus

10-15

Thal :Dit.

NI-Adult

carbon

30-45%

Pseudocalanulr dongatus T m o r a longicomis

12.5

Thalassiosira rotda

ash-free dry weight

12.5

!l'hhalassiosira rotula

Eurytemora afinis

20

Is0 ; Thalussiosira pseudonana; Chlamy.

hatching-50% adult hatching-50% adult egg-production

13.5-17.6 % (mean values) 17.3-26+3% (meanvalues) 8%-17.2%

Abbreviations for food organisms : Chlamy. C?damydomoluur reinhardti Cosc. Cosnnodascus angsti Dit. Ditylum brightwelli

ash-free dry weight carbon

Gony. (;lonyaulaz polyedra Gymno. Gymnodinium splenden.3 Isochrysis galbana

180.

-

%

organic carbon

Laud; Gymno ; Gony; Proro Laud

(Kl)

-

NII-Adult

15

eificiennd

< 60%

Adult weight increase NI-Adult

C. helgolandicus

Net growth

Ennvironmtal variables

Source

-

Hirota (1972)

TempeTature ; body size

Reeve (1970b)

-

43-4% (mean) Temperature

Conover and Lalli (1974) Dagg (1976)

32%

-

Clutter and Theilacker (1972)

6-30%

-

Lasker (1966)

-

-

-

-

-

Food concentration

Paffenhbfer (1976a)

Temperature

Mullin and Brooks (1970a) Mullin and Brooks (1970b)

-

Temperature: Food species and concentration Temperature Food concentration Food concentration Food concentration

Paffenhiifer (1976b)

Mullin and Brooks (1970a) Harris and Paffenhbfer(1976b) Harris and Paffenhiifer (1976b) Heinle et el. (1977)

Laud. Lauderia boredis Proro. Prorocentrum micans Thal. Thalassiosira jluviatilis

LABORATORY CULTURE OF MARINE HOLOZOOPLANKTON

279

weight and nitrogen a t three different temperatures. Gross growth efficiency including egg production determined in terms of nitrogen for X. hispida feeding on nauplii of Artemia salina did not change with increasing body weight ranging from 19 to 54%. Efficiency decreased with increasing temperature from 51% a t 16°C to 25% a t 26°C.

Cumulative ingestion (pq ash- free dry weight )

FIQ. 19. Relationships between the logarithm of gross growth efficiency (K,) and cumulative amount of food ingested (ration)for Ternoya Zongicornis and Pseudocalanus elongatwr. O - - - O ,N I - CI; @-,. CI - CIII; A---A, CII to 60% adult; A-A, complete development, hatching to adult. (After Harris and Paffenhofer, 1976b.)

Conover and Lalli (1974) used the K-line concept of Paloheimo and Dickie (1966) to investigate relations between first order growth efficiency, k,, and ration ingested, and body size. A similar approach was used by Harris and Paffenhbfer (1976b) to analyse relationships between k, and cumulative ration ingested by Temora and Pseudocalanus reared at different food concentrations (Fig. 19). Mullin and

280

GUSTAV-ADOLF PAFFENHOFER

AND ROGER P. HARRIS

Brooks (1970b) similarly observed an inverse relationship between a measure of growth efficiency and the total amount of food ingested during development by Calanus helgolandicus in laboratory cultures. The same authors (1970a)measured gross growth efficiency for Calanus and Rhinealanus raised at two different temperatures, concluding that growth efficiency was not affected by temperature. The efficiencies determined in this study (35-40%) were generally higher than those reported by Paffenhofer (1976a) using slightly different culturing techniques (Table X). I n this study growth efficiency during juvenile life was estimated in three different ways, as calories, organic carbon, and dry organic substance. Paffenhofer (1976b), in his study of continuous and nocturnal feeding by Calanus, determined gross growth efficiencies for laboratory reared animals and compared them with animals from a Deep Tank facility and from the Pacific Ocean, there being no great difference between those of the wild and cultured animals. At most of the initial experimental concentrations from 25 to 470 pg C-litre- gross growth efficiencies for the tintinnids Tintinnopsis acuminata and Eutintinnus pectinis were calculated to exceed 50% (Heinbokel, 1978a). If tintinnids indeed serve as a link between nanoplankton and large particle feeders this high efficiency would mean that the nanoplankton energy would be made available to higher trophic levels at a relatively low loss of energy. Clione limacina feeding on Xpiratella retroversa assimilated an average 94.7% of the ingested carbon and 99.0% of the ingested nitrogen (Conover and Lalli, 1974). Gross growth efficiencies based on measured weight ranged from 48 to 76%, based on calorific measurements from 49 to 85%. These values are the highest reported gross growth efficiencies excluding those for developing herring eggs (Paffenhoferand Rosenthal, 1968). It appears that the digestive system of C. limacina is extremely well developed for not only complete digestion but also assimilation of the animal food. This high efficiency is further explained by the specialized food habit of the predator. One may note that assimilation and gross growth efficiency seem to be higher for animal than for plant food (Corner et al., 1976). Conover and Lalli (1974) observed a decrease in gross growth efficiency with increasing predator size and increasing ration. 5. Reproduction

Rates of reproduction and total fecundity of planktonic invertebrates are difficult to estimate with accuracy in the field. An approach has been to capture animals from the wild population, maintain them in the laboratory, and estimate reproductive rates on this basis. This,

TABLEXI. FECUNDITY OF HOLOZOOPLANKTON MAINTAINEDFOR EXTENDED PERIODS OF TIME IN THE LABORATORY species CTENOPHORA Pleurobrachia bachei

Pleurobrachia pileus P . pileus

Temperature "C

10

16-26

AMPHIPODA CaUiopius laeviusculus 8-15

Hyperoche medusarum 10 MYSIDACEA Metamysidopsis dongata

10-14

COPEPODA Euterpina acutijrons E. acutifrons E . acutifrons

16-23 10-25 18

~~~

Units of measurement

Rate of productia

Total fecundity

Environmental variablas

w

0 Source

m b. e

0 15

Mmmiopsis mccradyi 21-31 CHAETOGNATHA Sagitta hispida

Food organisms

Labidocera and Calanus eggs p -124h nauplii ;Adult Acartia; Artemia nauplii

100-1000

-

Copepods Copepods

eggs y -124 h -I

Artemia nauplii

eggs p -I24 h

-'

Cosc ; calanoid copepods egg losa (pg C? -'24h-') Herring larvae

-

Artemia nauplii

-

-

1100-14100 eggs 0 -l

2 generations

3 903-7 075

body size

eggs 0

746 (average)

5 690-12 423 eggs p -l

10347.6 (means)

6-420 eggs p -1

38-75

-

-

50-90 eggs p -1

-

16-24 eggs p -1

Hirota (1972)

z

0

-

Greve (1970) Greve (1972) Baker and Reeve (1974) Reeve (1970b)

Temperature; body sire

-

nagg (1976) Westernhagen (1976)

-

Clutter and Theilacker (1972)

c F

! 0

w

E2 M

F

~~

Cdanus helqolondicw 15 Jthincalanus naarlrcs 12

Phaeo Phaeo Phaeo; Proro ; P1alyrnom.s sueciea; Ghaetmros danicus; Gymnodinium sp. Laud; Gymno Thal ; Cyclo ; Dit ; Coscinodiscus waiksii ; Artemia nauplii

-

-

9-68 eggs p -1 13-98 eggs p -l 14.6280.6 eggs .. 0 -l

-

1 704-2 080 eggs p -I 66 eggs p -1 (mean)

Food species and concentration

-

Bernard (1963) Haq (1972) Nassogne (1970)

PaffenhOfer (1970) Mullin and Brookg(1967)

0 2

TABLEXI-contd. Species

Temperature "C

Food organisms

Units of

Rats of production

measurement

Total feeunday

Environmental variables

sources

COPEPODA-contd. Pseudocalanus minutus P. elongatus P. d o n g a m

6-7

Is0 ;

Is0 ; 6-7 1.3-16.2 Is0 ;

Temora longicomis T.longicomis

Thalassiosira rotula ; Peri 4.1-15.4 Is0 12.5 ThahSiOsira rot ula

E u r y h r a afinis

20

P. dongatus

12.5

Gladioferena imparipes 15-25 L a M m r a lnkpinosa Acartia tonna Acartia dausi T UNICATA Oikqpleura dioica

0.dioica

Is0 . Thalassiosira ~s~;cdmucna; Chlamg Dun; Cyclo; Phaeo

Laud; Gymno; Amrtia and Calanus nanplii 3.9-20.2 Cryptomom8 baltiea Is0 ; Mono 15-20

egg sacs p -'24h

-

-l

ems P, -'24h I

-

-

--I

nauplii p -'24h

-l

eggs 9 -'24h nauplii 0 -'24h-'

egg masses 9 -124h -I

0.7 3.4 (mean) 3.1-4.0 (means) 4.7-17. 43-242

2-411 eggs 0 -l 84-871 nauplii p

-

38-125 eggs 0 -l

13

180; Mono; Cyclo

Is0 ; Mono ; Cyclo

2-136 nanplii

Abbreviations for food organisms : CNamy. Chlamydomonas reinhardti Cosc. Co6cinodiSCUS angsti Cyclo. Cyelotdla nana Dit. Dityluwa brightwdli Dun. Dunallida tertidwta

-l -l

1.6-18.4 3.1-24.7

-l

4-769 eggs p -l 44-896 eggs 0 -l 116-361 eggs p - 1

-

Food concentration ; Corkett and McLaren (1969)

v size

Starvation Temperature

Corkett and McLaren (1969) Corkett and Zillioux (1975)

Food species

Paffenh6fer and Harris (1976)

Temperature Food concentration

Corkett and Zillioux (1975) Harris and Paffenhilfer (1976s) Food concentration Heinle et al. (1977) Temperatwe

360 eggs egg 0 -'24h eggs p -'24h

p -I

0.3-0.7

15

7-18

8-11 egg sacs p -1 106 eggs p -l 3-88 eggs 0 -l

.-~-~-,

(mean)

51-877 eggs 0 -l ( X = 250)

Gymno. @mnodinium splendens Iso. ISOchrysiS qalbana Laud. Lauderia borealis Mono. MonochrySiS l u t L r i

-

Rippingale and Hodgkin (1974) Barnett (1974)

Temperature Corkett and Zillioux (1975) Food concentration; Iwasaki et al. (1977) light ; temperature

Food species and concentration

Paffenhilfer (1973) Paffenhilfer (1976~)

Peri. Peridinium trochoideum Phaeo. Phaeodaclylum tricarnecttm Proro. PrormnCrum micans Thal. Thalassiomra puviatilis

LABORATORY CULTURE OF MARINE HOLOZOOPLANKTON

283

however, presupposes that the particular organism can be maintained for an adequate duration in the laboratory, an ability which is often only developed as the result of establishing multiple generation cultures. In addition estimates based on wild caught animals suffer from two disadvantages. Firstly, the conditions of capture may affect the parameters measured, for example many species release large numbers of eggs on first being brought into the laboratory and this may result from the trauma of capture. Secondly, with the exception of cases such as spermatophore-bearing females of some calanoid copepods, such as Calanus helgolandicus, which may be assumed to be a t the start of egg production on capture, it is difficult to derive estimates of total fecundity for animals captured a t an unknown age. For these reasons development of laboratory culturing techniques has resulted in great advances in our knowledge of quantitative aspects of zooplankton reproduction (Table XI). Many observations on egg-production and fecundity in laboratory culture are incidental to attempts t o achieve multiple generation cultures. However, specific studies of egg production have been made, and as with growth and feeding studies the main interest has centred on the effects of temperature and food concentration on reproduction. General information on reproduction of ctenophores is given by Baker and Reeve (1974), Greve (1972), and Hirota (1972) and generally emphasizes their high fecundity. At 15°C two Pleurobrachia pileus, each being kept in 400 ml, produced 4 000 (over 7 days) and 7 000 (over 12 days) eggs, respectively, resulting in rates of 571 and 583 eggs. day-1 (Greve, 1972). After these intensive periods egg production comes to a sudden halt within one day. The animal’s body diameter decreased while reproducing. Beroe gracilis had a maximum reproductive rate of 600 eggs-day-1. Hirota (1972) noted that errors in the estimation of reproductive rates of Pleurobrachia bachei may be possible due to the feeding of the omnivorous prey Acartia tonsa on the ctenophore’s eggs and larvae. Reproduction starts at a mean body length of 8 mm (range 6 to 10 mm), between 45 to 70 days after hatching (Fig. 20). Egg production rate initially was near 100 eggs*day-l. animal-l attaining a maximum of close to 1000 eggs-day-l (Hirota, 1972). The first generation produced an average 2 800 eggs-animal-l, the second generation 14 100; the latter representing a rate of 350 eggs-day-1 * animal-1 over the period of 40 days. Self-fertilization was observed. Hirota (1972) offers no explanation for the difference in reproductive performance between the two generations. Baker and Reeve (1974) found that the maximum egg production by Mnemiopsis rnccradyi was proportional to body size. All lab-reared specimens (6)

284

OUSTAV-ADOLF PAFFENHOFER

AND ROQER

P. HARRIS

started reproduction at 13 days after hatching, the experiments being stopped 23 days after hatching when egg production rate was still high. Over 7 days of intensive reproduction the average was 8 210 eggs animal-I equivalent to a reproductive rate of 1 170 eggs’animal-l day-1. Field-collected specimens produced overnight 199 to 9 990 eggs. animal-1; lab-reared animals did not attain these maximum rates. Both Hirota and Baker and Reeve reported self-fertilization in individually reared ctenophores, the latter authors suggesting that the ability to self-fertilize coupled with high fecundity are most important

20

40

60

80

I00

Days from hatching

FIG.20. Reproductive rate of the ctenophore, Pleurobrachia bachei, as a function of age under laboratory conditions. Symbols represent data from three individual ctenophores. (After Hirota, 1972.)

in their ability to build up populations rapidly during favourable conditions of food supply. Hyperoche medusarum females reared in the laboratory at 10°C carried between 48 and 94 eggs in their marsupium (Westernhagen, 1976). The number of eggs produced by Parathemisto gaudichuudi depend on the size of the female (Sheader, 1977): 10 eggsmfemale-1 of 3 to 3-5 mm and 200 eggs-female-1 of 16 to 18 mm length. Eggs are not produced in the absence of males. The juveniles remain in the marsupium through 3 instars. OnIy stage 3 which is released from the marsupium can feed. A number of authors have compared laboratory fecundities with those of animals obtained from wild populations. For example, Bernard

LABORATORY CULTURE OF MARINE HOLOZOOPLANKTON

285

(1963) found that fecundity of laboratory reared Euterpina measured both in terms of the number of egg sacs and number of eggs per sac waa much lower when compared with wild animals. Of calanoid copepods, Rhincalanus is another species for which fecundity of laboratory reared populations has to date proved to be lower than that of locally obtained spermatophore-bearing wild animals (Mullin and Brooks, 1967). Mullin and Brooks investigated the effects of culture volume, stirring, use of antibiotics and an enrichment (Lewis, 1967) but none of these variables improved fecundity. Calanus helgolandicus cultured by Paffenhofer (1970) in contrast showed fecundities close to those of the spermatophore bearing females from the ocean. The average number of fertilized eggs laid by cultured females was 1991 as opposed to 2 267 for wild animals, and the hatching percentage was in fact higher in the laboratory fertilized animals (see Table XI). Another example of culture methods resulting in comparable fecundities to wild animals is that of Metamysidopsis (Clutter and Theilacker, 1971) where laboratory fecundities were in fact higher than those of animals from the field. Another group for which general data on fecundity are available are Appendicularia. Paffenhofer (1973, 1976c) reported a mean number of spawned eggs for females maintained on both mixtures of algae, and in natural sea water. His estimates of fecundity were comparable t o those obtained by Wyatt (1971) from the southern North Sea. A number of studies have investigated the effect of temperature on egg production using laboratory reared animals. Dagg (1976) working with the amphipod Calliopius defined relationships between daily reproductive loss measured in terms of carbon and nitrogen and the female body weight for different temperatures. From this work he concluded that, body size has a major effect on the amount of material put into eggs, temperature was considered to be a relatively insignificant factor. C. laeviusculus produces a brood pouch every 26 days a t 8", every 16 days at 12" and every 12 days a t 15°C (Dagg, 1976). Reproductive rate increased with increasing temperature and female size ; but production of offspring as a percentage of ingestion decreases with increasing temperature from l l . l y o to 9.2% a t 1 2 5 0 pg C body weight, and decreases with increasing body weight, as shown a t 8°C from l l . l y o (1 250 pg C) t o 6.5% ( 3 750 pg C female weight). I n an investigation of quantitative aspects of growth and reproduction of Sagitta hispida Reeve (1970b) reported on the effect of temperature on egg production, S. hispida produced a maximum of 420 eggs. animal-l over 20 days feeding on nauplii of Artemia salina. Total egg production changed with increasing temperature from 61 animal-1 a t 15" to 105 a t 26", dropping t o about 85 a t 32°C. The breeding in the laboratory

286

OUSTAV-ADOLF PAFFENHOFER

AND ROGER P. HARRIS

of Euterpina was investigated by Haq (1972) with special reference t o the role of the dimorphic males. Studies of egg production indicated that 20°C is the optimum temperature for egg production in this species. Perhaps the most intensive studies of temperature effects on reproduction using laboratory maintained animals are those of McLaren and co-workers for copepods. Much of this work concerns temperature effects on egg development, but two studies (Corkett and McLaren, 1969 ; Corkett and Zillioux, 1975) deal specifically with egg production. I n studying egg production in Pseudocalanus Corkett and McLaren in fact used wild animals selected from the plankton and maintained a t excess food concentrations using the methods of Corkett and Urry (1968). Animals were maintained in small volume containers and the possible deleterious effects of this have already been alluded to. Corkett and Zillioux (1975) studied temperature effects on egg-laying of Acartia tonsa, Temora longicornis and Pseudocalanus elongatus in the laboratory. However, as Temora and Pseudocalanus were taken from the sea and Acartia were removed from stock cultures of mixed age it is difficult to interpret the data on fecundity as the age of the females is unknown. Corkett and Zillioux suggested that egg production rate in Pseudocalanus, which carries eggs in a sac, may be lower in comparison with species which lay single eggs as a new sac cannot be laid until the old one has hatched. Studies of temperature effects on reproduction have often been performed under conditions of excess food (e.g. Dagg, 1976 ; Corkett and Zillioux, 1975), the implication being that effects of food concentration on egg production will be standardized in this way. However, there have been rather few specific studies of the relation between food concentration and egg production, and all have been for copepods. Nassogne (1970) using young adult female Euterpina investigated egg production and adult life span using five different algal diets and a mixture of all five foods. The best results were obtained with the mixture (Table 111),the total number of eggs per adult being 280.0 & 17.2, much higher than the total fecundity reported by Bernard (1963) for the same species. Heinle et al. (1977), as a preliminary to their study of detrital diets as food for Eurytemora afinis, investigated egg production at three algal food levels. The maximum number of eggs per brood a t the highest food level was significantly greater than the medium and low levels. As the animals were isolated when first seen mating from populations reared from hatching t o adulthood it is certain that the females used were a t the start of their adult egg production and that the estimates of fecundity obtained apply to the whole of the adult life-

LABORATORY CULTURE OF MARINE HOLOZOOPLANKTON

287

span in culture. Similar estimates for Temora and Pseudocalanus (Harris and Paffenhofer, 1976a; Paffenhofer and Harris, 1976) were made for defined temperatures and food conditions. Heinle (1970) reported on the effects of different rates of harvesting on egg production in laboratory cultures of Acartia tonsa and Eurytemora a@&. Information on effects of food concentration and temperature on egg production by Acartia ctausi is given by Iwasaki et al. (1977). 6. Other rate processes

I n addition to enabling measurements to be made of the major parameters discussed above, the use of laboratory cultures has facilitated a number of studies of more specialized aspects of zooplankton behaviour which are relevant to secondary production. For example, Paffenhofer (1973) made observations on the frequency of housebuilding by Oikopleura. The frequency of copulation has been measured in Euterpina by Haq (1972) and Wilson and Parrish (1974) investigated re-mating in Acartia. BB and Anderson (1976) studied aspects of gametogenesis in laboratory cultures of Foraminifera. The rate of moulting and the loss of material as moults has been estimated in copepods by Mullin and Brooks (1967), mysids (Clutter and Theilacker, 1971), and euphausiids (Fowler, et al., 1971 ; Jerde and Lasker, 1966; Lasker, 1966; Le ROUX,1973, 1974; Paranjape 1967). 7. Pollution studies

An ability to maintain zooplankton with low mortality over multiple generations under defined laboratory conditions has considerable potential for evaluating the effect of pollutants on planktonic organisms. Toget’herwith large scale ecosystem experiments (e.g. Reeve, Gamble and Walter, 1977) it enables comparisons to be made between populations subjected to low-level inputs of pollutants and those of untreated animals. For example, Berdugo, Harris and O’Hara (1977) and Ott, Harris and O’Hara (1978) studied effects of low-levels of a variety of petroleum hydrocarbons on reproduction of Eurytemora in laboratory cultures, and Harris, Berdugo, Corner, Kilvington and O’Hara (1977) investigated retention of ldc-naphthalene or its metabolites during growth of the same species from hatching to adulthood. Paffenhofer and Knowles (1978) studied the effects of cadmium on laboratory cultures of Pseudodiaptomus coronatus, and the effect of “red mud” from an aluminium production plant on growth and survival of the juvenile stages of Calanus helgolandicus was studied by Paffenhofer (1972). Studies of pollutants in relation to plankton are

288

OUSTAV-ADOLF PAFFENHOFER

AND ROQER P. HARRIS

covered in detail in the recent reviews by Corner (1978) and Davies (1978).

C. Simulation studies An awareness of the deficiencies of small-scale laboratory systems for studying many aspects of zooplankton behaviour and ecology has led a number of investigators to a recognition of the fact that to closely approach natural behaviour of zooplankton organisms relatively large volumes are required enabling a more realistic simulation of the animal's natural environment. Such large enclosures have low surface to volume ratios meaning that contact with the walls is considerably reduced, and in addition may provide at least limited scope for vertical movement which is a dominant feature of the behaviour of many of these organisms. Larger volumes may be necessary to permit uninhibited natural behaviour such as escape moves by large copepods or prey searching by predatory forms. I n addition such volumes may enable additional trophic levels to be maintained at approaching steady state conditions in multi-species food chain investigations. Studies using large volume enclosures may be considered as complementary to many of the small-scale attempts t o culture zooplankton in the laboratory. I n reviewing such developments emphasis will be placed on relevant studies in this context, a consideration of the value of such enclosures in more complex ecosystem studies is beyond the scope of the present treatment. The earliest of' such studies on marine zooplankton behaviour was carried out by Pettersson, Gross and Koczy (1939a, b) in their plankton tower at Goteborg, a structure of 12 m in height and 2 m diameter equipped with a cooling device. The tower was initially filled with filtered sea water to which natural plankton samples were subsequently added. Attempts were made to work with a stratified water column in this system, a la,yer of low salinity nutrient enriched water being introduced above high salinity water. I n this tower a population of copepods was maintained at a uniform density for three weeks at 7°C ; nauplii and females with egg sacs were observed in this culture. Raymont and Miller (1962) grew copepods over 2.5 months in 20 m3 of sea water in concrete tanks 5.6 m in diameter filled to a depth of 1.3 m, in a study of production. Nutrient additions were made to stimulate phytoplankton growth. The copepods introduced into the enclosures included the cyclopoids Oithona similis Claus and 0. brevicornis Giesbrecht, and the calanoids Temora Zongicornis, Centropages hamatus and C . typicus, Tortanus discaudatus (Thompson and Scott), Eurytemora herdrnani and E . hirundoides, Paracalanus crassirostris

289

LABORATORY CULTURE OF MARINE HOLOZOOPLANKTON

and dcartia tonsa. The dominant species P. crassirostris, E. hirundoides and A . tonsa all bred in the culture, Acartia probably producing 3 broods. The other species did not survive so well and as the temperature increased the phytoplankton and copepod species composition changed, the latter being dominated in the second half of the experiment by Acartia, with only brief reproductive bursts by Paracalanus and Oithona. The dominance of Acartia may be attributed to the high temperatures (up to 25.4OC) and to the fact that being omnivorous it may have preyed on the offspring of Oithona and Paracalanus.

-.

-.eggs

1-3

'1-3

adults

Extinction

Doys from stort of systemoiic harvest

FIG.21. Yield (copepods per litre) resulting from systematic harvesting of a laboratory population of Acartia tomsa. Harvest intervals were varied between 3.0 and 4.0 days. (After Heinle, 1970.)

Though not involving large-scale cultures the work of Heinle (1970) on population dynamics of exploited cultures of Acartia tonsa and Eurytemora afinis is one of the first examples of an attempt to

maintain a steady state zooplankton population under laboratory conditions. Heinle studied the effects of different harvesting regimes, on cultures lasting in excess of 250 days (Fig. 21), investigating parameters such as birth rate, eggs per female, sex ratio and yield per harvest. Person Le Ruyet (1975) similarly investigated the long-term

290

GUSTAV-ADOLF PAFFENHOFER

AND ROQER P. HARRIS

dynamics of populations of Acartia clausi in 20 litre containers maintained for 7 months. These two studies illustrate the potential of using laboratory cultures for studies of production and population dynamics of copepods. Other simulation studies using large enclosures on land include a series of investigations using a Deep Tank at Scripps Institution of Oceanography. This system, similar to the Goteborg plankton tower in dimensions ( 3 m diameter and 10 m deep) was first used to study phytoplankton growth over periods of about 2 weeks (Strickland, Holm-Hansen, Eppley and Linn, 1969). This tank was subsequently used in a number of studies of large scale zooplankton cultures. Mullin and Paffenhofer (1971, 1972) conducted two experiments in this 70 m3 facility on the growth of a population of Calanus helgolandicus. This species shows pronounced vertical migratory behaviour in nature, and as has been indicated in previous studies requires a volume of at least 20 litres for successful fertilization of females in the laboratory. The initial naupliar population in the 1971 experiment was derived from keeping 1 200 fertilized females in bags of 390 pm mesh for 12 days in the tank. The diatoms Skeletonema costatum and Lauderia borealis were added regularly from batch cultures. As no cooling was used the temperature increased from 13" to 19" over 84 days. The first two consecutive copepod generations indicated a generation time of about 20 days at approximately 15°C. This agrees with the shortest egg-adult intervals at this temperature observed by Paffenhofer (1970) and Mullin and Brooks (1 970a) at roughly comparable food concentrations. This suggests that these small-scalelaboratory experiments give a valid estimate of the rates of growth which animals exhibit under less confined conditions. I n the second experiment Mullin and Paffenhofer ( 1 972) the rate of food supply was measured and controlled. Acartia tonsa and Paracalanus parvus were added together with Calanus helgolandicus to find out whether one species would replace the other. Phytoplankton was added every few hours ; temperature increased from 13" to 19°C and then was maintained at 18°C. Initially Acartia tonsa grew well ; yet recruitment of copepodite stages was later limited resulting in the disappearance of A . tonsa after 8 weeks. Draining one third of the tank and refilling it resulted in strong reproduction of Calanus and Paracalanus suggesting that a decreasing population may resume reproduction as a result of a partial change of sea water. Generation times, initially at 14"C, were 21 days for Calanus and 18 to 21 days for Paracalanus. Later in the 24 week experiment, at 18"C, generation times were 28 (Calanus)and 24 days (Paracalanus). It is not apparent why the generation times were longer at the higher temperature. The average concentration of particulate organic carbon

LABORATORY CULTURE OF MARINE HOLOZOOPLANKTON

291

was 147 pg C. litre-l. The harvestable yield of copepod carbon as a percentage of the phytoplankton carbon added to tank was 6%, a value which approximates the food chain efficiency under steady state conditions. I n a further development of these two experiments, Mullin and Evans (1974) measured under quasi steady state conditions the transfer efficiency of organic carbon in B 3-member food chain at environmental concentrations consisting of Xkeletonema costatum,

10

-

ro 8

E 5

E Y)

6

z

0

0

e S

4

%

m

V

2

0

20

40

60

100

80

120

140

160

180

Days

FIG.22. Biomass of zooplankton components integrated over the water column of e deep tank during a study of planktonic food chain efficiency. Copepods; 0 ctenophores. The dotted line under " copepods " indicates the biomasses of Acartia and Paracalanua during transition from dominance of the former to the latter species. (After Mullin and Evans, 1974.)

Acartia tonsa plus Paracalanus parvus and Pleurobrachia bachei, in the same 70 m3 deep tank at temperatures between 14" and 16°C. The experiment consisted of 3 phases (Fig. 22). Phase I (fist 50 days) was largely dominated by A . tonsa being characterized by a food chain efficiency of 12.5%. During Phase I1 A. tonsa vanished and P. parvus took over (day 50 to 90) with a food chain efficiency of 10~4')'~(yield of herbivores related to phytoplankton supplied). At the beginning of Phase I11 (day 90 to 165) the predatory ctenophore Pleurobrachia bachei was introduced resulting in a food chain efficiency of 2.6% (yield of ctenophores as a percentage of the phytoplankton supplied).

292

GUSTAV-ADOLF PAFFENHOFER

AND ROGER P. HARRIS

The calculated increase of copepod food chain efficiency during Phase

I11 to 25% is partly explained by the reduction of dead or dying copepods due to predation thus reducing non-predator mortality ; and also by reducing the average age of the Paracalanus population which increases the eEciency as CV and adults have a relatively low gross growth efficiency. Mullin and Evans mentioned that interpretation of some of their results was made difficult by the lack of a control population, and concluded by suggesting the interest of further investigations of relationships between food supply, schedule and intensity of harvest, and standing stock (cf. the work of Heinle, 1970). Hirota (1972) also used the same facility to compare laboratory growth rates of Pleurobrachia bachei with those under the almost natural conditions in the deep tank. The growth rate of P. bachei in 1 to 3 litre beakers at 15°C resulted in a 2.5 mm increase in body diameter during 9 days ; in the deep tank, at food concentrations 10% of that in the beakers, it took 8 days for the same size increase. The reasons for the relatively fast growth in the deep tank despite the lower food concentration may include the fact that P . bachei in the deep tank can move almost unrestricted being able to extend its 2 tentacles fully (40 cm length or more), its behaviour being unaffected by walls. However, it was noted in a separate experiment that P . bachei and Calanus concentrated in the upper meters of the tank resulting in high prey densities which, being 10% of the laboratory’s when evenly distributed in the tank, could have been close to laboratory densities (Hirota, 1972) thus resulting in the same growth rate. These deep tank studies were the first controlled experiments allowing the observation of the development of distinct cohorts at known natural food densities over extended periods of time, resulting in generation times which can be considered realistic in relation to the neritic oceanic environment. The increased sophistication of this approach enabled the first marine planktonic phytoplankton-herbivore-carnivore food chain to be studied under controlled conditions providing significant information on the effect of predation on transfer efficiency at a lower level. A modern deep tank, the Aquatron facility at Dalhousie University, was used by Conover and Paranjape (1977) in studies of a variety of aspects of zooplankton behaviour. I n this 10 m deep, 3-66m diameter tower, zooplankton community structure remained intact for more than three months, with a reproducing population of Xagitta elegans being established. Using viewing ports in the side of the tower observations were made of swimming behaviour of Sagitta and Spiratella retroversa under unconfined conditions. Conover and Paranjape concluded that the ability to observe behaviour of zooplankton was the major advan-

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tage of such a system, especially in cold or turbid waters where in situ observations by divers are not possible. I n contrast to these large enclosures on land, considerable recent interest has developed in enclosing plankton populations in the sea itself to study directly aspects of ecosystem function. This approach to large scale plankton studies has been facilitated by the development of plastics combining high strength and transparency. The first use of plastic enclosures was in studies of in situ primary production (Antia, McAllister, Parsons, Stephens and Strickland, 1963 ;McAllister,Parsons, Stephens and Strickland, 1961 ; Strickland and Terhune, 1961). Subsequent developments have included large scale investigations of zooplankton populations. Much of the impetus for these studies has been provided by the need to study possible effects of pollutants such as metals and hydrocarbons on plankton populations and processes. Two major experimental programmes of this type, one in Europe (Gamble, Davies and Steele, 1977), and one in North America (Menzel and Case, 1977) have provided information on zooplankton ecology under unpolluted as well as polluted conditions. The Controlled Ecosystem Pollution Experiment (CEPEX) was designed to study long-term effects of pollutants on plankton populations, their structure and interactions, in an environment closely simulating the natural system (Grice, Reeve, Koeller and Menzel, 1977; Menzel and Case, 1977 ; Takahashi, Thomas, Seibert, Beers, Koeller and Parsons, 1975). In the original conception of this experimental programme several needs were stressed (Menzel and Case, 1977) : 1. A t least two trophic levels should be represented. 2. Zooplankton populations must be maintained through at least one

generation. 3. Several enclosures are simultaneously required for experimental

manipulations, replication and controls. 4. Removal of organisms should not exceed 1% of the zooplankton

population per day. 5. The water mass enclosed should initially contain all organisms

associated with it. I n all experiments carried out in Saanich Inlet, B.C., Canada, plastic bags of up to 30 m depth and 10 m diameter were filled with sea water and its plankton populations and each bag was raised from greater depths of the inlet to the surface where they were fastened to floats. The effects of copper concentrations ranging from 5 to 50 pg .litre-l on herbivorous zooplankton abundance and species composition were partly obscured by predation of carnivores (Gibson and Grice, 1977b).

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As ctenophores and medusae were more numerous in controls than in copper treated enclosures adverse effects of copper are thought to have occurred. It is not certain whether the copper effects were direct or indirect (through lower trophic levels). Pseudocalanus, Calanus, Euphausia and Pleurobrachia grown in 5 or 10 pg copper -litre-l had lower feeding rates than the same forms from non-contaminated enclosures. Egg and faecal pellet production seemed to be reduced too (Reeve et al., 1977) . The most sensitive short-term indicator of a sublethal effect of metals (1 to 10 pg of mercury or copper-litre-l) on planktonic marine copepods seems to be egg production (Reeve et al., 1977). Densities of copepod nauplii in these enclosures were similar in controls and at 5 pg copperelitre-1 but lower at 10 and 50 pg-litre-l (Beers, Stewart and Hosking, 1977). I n addition to studies of the effects of pollutants the CEPEX enclosures have considerable potential for investigating zooplankton populations under unpolluted conditions. For example observations made by Grice et al. (1977) in a control enclosure provided detailed information on Pseudocalanus population dynamics (CI to adult) over a period of 71 days. Similarly Reeve and Walter (1976) were able to study, on a large scale, the growth, production and predation potential of ctenophore populations. Growth of Pleurobrachia in the enclosures is at least as fast as in the laboratory in the presence of abundant food and at the same temperature. About 4% of the primary production was converted into ctenophore biomass, in comparison with the figure of 2.6% for the same species in a Deep Tank (Mullin and Evans, 1974). In an experimental study associated with the CEPEX programme plastic enclosures (17 m deep, 3 m diameter) were established in Loch Thurnaig, Scotland. The objective of this study was to investigate temporal changes in zooplankton populations in relation to differences in nutrient and population levels (Davies, Gamble and Steele, 1975; Gamble et al., 1977). I n bags with relatively high regular nutrient additions herbivore populations were maintained at a level close to that in the sea (outside the bags). Oithona similis dominated the zooplankton biomass, changes in abundance of Oithona being related to the abundance of the predator Bolinopsis infundibulum. The density of Evadne nordmanni was related to high concentrations of dinoflagellates (Peridinium depressum). Comparing data on egg production and feeding rates suggests that the copepod population was negatively affected by copper. The preceding bag experiment (Davies et al., 1975) yielded an interesting result which can be extrapolated to the sea. With an increase in the zooplankton population (copepods) the percentage of faecal material in the detritus settling on the bottom of the bag

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decreased from 85 to about 50% of the total detritus. It is assumed that the copepod population increase leads not only to an increase in faecal pellet densityelitre-l but also to a far larger amount of the water column being swept clear per day resulting in a lower percentage of pellets reaching the bottom. I n general the large enclosure studies mentioned have the advantage of simulating more closely conditions in the sea, but are limited in comparison with small-scale laboratory cultures by the possibility of manipulation and replication. Another major disadvantage is the cost of such large enclosures. Studies of the responses of zooplankton to artificial perturbations by pollutants have been affected by variability induced by parameters such as change of temperature, season, food concentration, and chemical complexing capacity of the sea water (Reeve et al., 1977). I n the future one may expect increased use of medium scale enclosures in conjunction with smaller scale controlled laboratory cultures for investigations of zooplankton populations and simple food chains. Such systems may either be on land such as, for example, the 757 litre polythene cylinders used by Ulanowicz, Flemer, Heinle and Mobley (1975), or submerged plastic enclosures such as the systems used by Brockmann, Eberlein, Junge, Trageser and Trahms (1974) and Kuiper (1977).

IV. CONCLUSIONS After a consideration of the research conducted so far on the cultivation of marine holozooplankton we would like to conclude by discussing, in relation to these past studies, possible future areas of cultivation research which, by being closely related to environmental conditions and processes, may be of considerable significance in furthering our understanding of food-web relationships in pelagic ecosystems. It is apparent that some groups of organisms, which are numerically important in the world’s oceans, have been the subject of relatively few cultivation studies ; many important holozooplankton groups remain virtually impossible to maintain in good condition in the laboratory. As has already been noted most experimental zooplankton research has focused on calanoid copepods which are considered to be the most numerous invertebrate metazoans in the oceans. The emphasis on copepods may have resulted in the neglect of other phyla and orders. Cyclopoid copepods, for example, of the genera Oithona, Oncaea and Corycaeus are often iiumerically important in both neritic and open ocean waters ; yet they have received little attention experimentally since Murphy’s studies in 1923. Planktonic chaetognaths, which may

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be considered as second in abundance in the ocean, have similarly been little studied. Our knowledge of these important animals is almost entirely due to the pioneering work of Reeve and co-workers (e.g. Reeve, 1970a, b ; Reeve and Walter, 1972) and is largely based on the one coastal species Xagitta hispida. Reeve (1970b) noted that X . hispida was virtually unique among the phylum in that it could be maintained for more than 24 hours in the laboratory, and he emphasized the virtual lack of physiological information on planktonic chaetognaths as a whole. Experimental studies of Appendicularia, thought to be third in overall abundance, so far include only Oikopleura dioica and Fritillaria borealis (Paffenhofer, 1973, 1976~). Thaliacea occur in many oceanic regions, at times in concentrations of up to 1 500 per cubic metre (Atkinson, Paffenhofer and Dunstan, 1978), but experimental studies on this group are only just beginning, having been only partly successful due to inadequate methods. Ostracoda, Cladocera, Radiolaria, Foraminifera and naked ciliates are other holoplanktonic groups which have been largely neglected. Even among calanoid copepods our knowledge of oceanic and deep water forms is fragmentary, the neritic species being most intensively studied. This latter generalization applies to all holozooplankton and though many of the coastal species studied are undoubtedly important in productive areas on the continental shelf, there is clearly scope for studies of open ocean representatives of all groups. The preponderance of cultivation studies on copepods may be due to the fact that this group as a whole seems to be relatively amenable to laboratory culture. Conversely the lack of information for other groups may be explained by the lack of adequate culture techniques. I n reviewing experiments on cultivating holozooplanktonthe importance of paying close attention to the organism in its natural environment as a prerequisite for successful culture recurs constantly. Lack of attention to this aspect together with difficulties in initially capturing undamaged animals is probably the major reason why many holozooplankton groups have proved so difficult to study in the laboratory. Considering successful studies of, for example, delicate animals such as ctenophores we may note that Greve (1970) provided a relatively natural physical environment for Pleurobrachia pileus combined with satisfactory food conditions in his planktonkreisel. Similarly Hirota (1972)in the Scripps deep tankofferednear natural physical conditionsfor Pleurobrachia bachei. Previously ctenophores had been considered to be difficultto work with experimentally. By attempting to simulate natural conditions there is no reason why survival of a zooplankton species should not be almost 100% in the laboratory. For example Paffenhofer

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( 1976a) by offering Calanus helgolandicus dinoflagellates which are

abundant in southern California neritic waters during summer (Reid, Fuglister and Jordan, 1970) obtained up to 100% survival from hatching to adulthood. I n this case the food organisms rather than the physical environment seemed to be the limiting factor. The same seemed to be true for the culture of hyperiid amphipods (e.g. Westernhagen, 1976). I n general sufficient attention to the choice of an appropriate food, and the provision of this food in good physiological condition, may be identified as one of the major reasons for the recent successes in zooplankton cultivation. I n considering groups of zooplankton that have not yet been successfully cultured, one may suggest that a careful evaluation of the species natural environment will in turn lead to the development of appropriate culture methods. For example, any study on the cultivation of pelagic Thaliacea through the complete life cycle would be difficult in 1 to 2 litre containers as the animals would be unable to develop asexually their long chains of beyond 2 m length as observed in the sea (D. R. Deibel, personal communication). Similarly, for actively moving animals such as pontellid copepods a profound knowledge of the animals behaviour in the wild may lead to its succesful culture. Pontellids may require certain light conditions or sufficiently large containers to permit uninhibited prey searching behaviour. Once the environmental requirements of an animal are known, it is possible to proceed with the design of a laboratory system permitting experimentation under nearly natural conditions. As has been already considered much of the recent interest in zooplankton cultivation has derived from attempts to make quantitative models of planktonic food chains, and planktonic ecosystems as a whole. Such an approach involves considering all possible pathways of qualitative interaction in the field, and this should provide much of the information on environmental conditions on which to base culture experiments. However, such an approach has so far been made in only a few studies. A notable example is the study by Petipa, Pavlova and Mironov (1970) combining a series of laboratory and field investigations in describing interactions in a Black Sea food web. Other studies, though not so comprehensive in their inclusion of all trophic levels, may be identified as being strongly orientated to the environment. For example, Hirota’s studies (1972, 1974) in the laboratory, Deep Tank, and the field of ctenophore predator-prey relationships, Barnett’s approach (1974) of offering a natural food spectrum of potential food to Labidocera trispinosa, and the experiments of Mullin and Evans (1 974) are significant steps towards the experimental investiga-

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tion of zooplankton rate processes a t near natural conditions. Mullin and Brooks (1976) provide a good example of the potential for understanding plankton ecology by a combination of careful field observations and laboratory experimentation. Future experiments apart from concentrating on the neglected groups of zooplankton discussed above may involve development of a number of approaches. I n small scale systems, further studies of steady state populations of the type carried out by Heinle (1970) may be envisaged. Large scale ecosystem enclosures may be used t o study populations of zooplankton under unconfined conditions as was done by Reeve and Walter (1976). Increasing emphasis can be expected on multispecies cultures in which simple food chains or even more complex food webs are cultivated under controlled conditions. Automated procedures are required, and continuous culture techniques for groups other than protozoa may be developed enabling a number of species to be added simultaneously t o a “food web container” a t various rates simulating environmental conditions (cf. Lampert, 1976). I n large scale enclosures on land future developments may involve increased control over the physical environment within the enclosure ; for example, by being able to create and disrupt stratification such as thermoclines and pycnoclines. I n addition, the design of systems enabling migratory species t o undergo diurnal vertical movements under controlled conditions is a major priority. Further more advanced stages of cultivation research may be envisaged as involving experimental field studies. These may be required when the water masses needed are too large t o be confined on land, as may be the case in experiments on Thaliacea. Such studies would either involve use of confined environments of several thousand cubic metres, allowing not only vertical but also horizontal movement of zooplankton, or continuous observation by automated devices within a water mass. After an initial phase of observation in which the physical, chemical and biological characteristics are described an experimentally reared population of feeders might be introduced t o determine their behaviour as a population in relation t o the environment. I n summary we suggest that small-scale laboratory cultivation experiments on zooplankton populations and conventional field studies will have to become much more closely integrated if we wish t o obtain a comprehensive understanding of biological processes in the ocean.

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V. ACKNOWLEDGEMENTS Part of this research was supported by the National Science Foundation (Grant OCE 76-01142).

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Pettersson, H., Gross, F. and Koczy, F. F. (1939b). Large-scale plankton cultures. Goteborgs K . vetenskap- vitterhetssamhdles handlingar sjatte foljden, 5, Ser B, 1-25. Provasoli, L., Shiriashi, K . and Lance, J. R. (1959). Nutritional idiosyncracies of Artemia and Tigriopus in monoxenic culture. Annals of the New York Academy of Sciences, 77, 250-261. Raymont, J. E. G. and Miller, R. S. (1962). Production of marine zooplankton with fertilization in an enclosed body of sea water. Internationale Revue der gesamten Hydrobiologie und Hydrographie,47, 169-209. Reeve, M. R. (1970a). Complete cycle of development of a pelagic chaetognath in culture. Nature, London, 227, 381. Reeve, M. R. (1970b). The biology of Chaetognatha I. Quantitative aspects of growth and egg production in Sagitta hispida. I n " Marine Food Chains " (J.H. Steele, Ed.) pp. 168-189. Oliver and Boyd, Edinburgh. Reeve, M. R . and Baker, L. D. (1975). Production of two planktonic carnivores (Chaetognath and ctenophore) in south Florida inshore waters. Fishery Bulletin of National Oceanic and Atmospheric Administration (U.S.) Washington, D.C., 73, 238-248. Reeve, M. R. and Walter, M. A. (1972). Conditions of culture, food-size selection, and the effects of temperature and salinity on growth rate and generation time in Sagitta hispoida Conant. Journal of Experimental Marine Biology and Ecology, 9, 191-200. Reeve, M. R. and Walter, M. A. (1976). A large-scale experiment on the growth and predation potential of ctenophore populations. I n " Coelenterate ecology and behaviour " (G. 0. Mackie, Ed.) pp. 187-199. Plenum Press, New York. Reeve, M. R., Gamble, J. C. and Walter, M. A. (1977). Experimental observations on the effects of copper on copepods and other zooplankton: controlled ecosystem pollution experiment. Bulletin of Marine Science, 27, 92-104. Reid, F. M. H., Fuglister, E. and Jordan, J. B. (1970). The ecology of the plankton off La Jolla, California, in the period April through September, 1967, V. Phytoplankton taxonomy and standing crop. Bulletin of the Scripps I n stitution of Oceanography, 17, 51-66. Rippingale, R. J. and Hodgkin, E. P. (1974). Population growth of a copepod Gladioferens imparipes Thompson. Australian Journal of Marine and Freshwater Research, 25, 351-360. Roman, M. R . (1977). Feeding of the copepod Acartia tonsa on the diatom Nitzchia closterium and brown algae (Fucus vesiculosus) detritus. Marine Biology, 42, 149-155. Sheader, M. (1977). Breeding and marsupial development in laboratorymaintained Parathemisto gaudichaudi (Amphipoda). Journal of the Marine Biological Association of the U.K., 57, 943-954. Sheader, M. and Evans, F. (1974). The taxonomic relationship of Parathemisto gaudichaudi (Guerin) and P. gracilipes (Norman), with a key to the genus Parathemisto. Journal of the Marine Biological Association of the U.K., 54, 915-924. Sheader, M. and Evans, F. (1975). Feeding and gut structure of Parathemisto gaudichaudi (Guerin) (Amphipoda, Hyperiida). Journal of the Marine Biological Association of the U.K., 55, 641-656. Soldo, A. T. and Merlin, E. (1972). The cultivation of symbiont-free marine ciliates in axenic medium. Journal of Protozoology, 19, 519-524.

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Steele,D. H. and Steele, V. J. (1973). Some aspects of tho biology of Calliopius laeviusculus (Krnyer) (Crustacea, Amphipoda) in the northwestern Atlantic. Canadian Journal of Zoology, 51, 723-728. Strickland, J. D. H. and Terhune, L. D. B. (1961). The study of in situ marine photosynthesis using a large plastic bag. Limnology and Oceanography, 6, 93-96. Strickland, J. D. H., Holm-Hansen, O., Eppley, R . W. and Linn, R . J. (1969). The use of a deep tank in plankton ecology. I. Studies of the growth and composition of phytoplankton crops a t low nutrient levels. Limnology and Oceanography, 14, 23-34. Takahashi, M., Thomas, W. H., Seibert, D. L. R.,Beers, J. R., Koeller, P. and Parsons, T. R. (1975). The replication of biological events in enclosed water columns. Archiv f u r Hydrobiologie, 76, 5-23. Takano, H. (1971). Notes on the raising of an estuarine copepod Gladioferens imparipes Thompson. Bulletin of the Tokai Regional Fisheries Research Laboratory, 64,81-88. Theilacker, G. H. and McMaster, M. F. (1971). Mass culture of the rotifer Brachionus plicatilis and its evaluation as a food for larval anchovies. Marine Biology, 10, 183-188. Ulanowicz, R. E., Flemer, D. A., Heinle, D. R. and Mobley, C. D. (1975). The a posteriori aspects of estuarine modeling. I n " Estuarine research " Vol. 1 (L. E. Cronin, ed.) pp. 602-616. Academic Press, New York, London. Vilela, M. H. (1972). The developmental stages of the marine calanoid copepod Acartia grani Sars bred in the laboratory. Notas Estudos do Institutodo Biologk Maritima (Lisbon),40, 1-14. Ward, W. W. (1974). Aquarium systems for the maintenance of ctenophores and jellyfish and for the hatching and harvesting of brine shrimp (Artemia salina) larvae. Chesapeake Science, 15, 116-1 18. Westernhagen, H. von (1976). Some aspects of the biology of the hyperiid amphipod Hyperoche medusarum. Helgolander wissenschaftliche Meeresuntersuchungen, 28, 43--50. Westernhagen, H. von and Rosenthal, H. (1976). Predator-prey relationship between Pacific herring Clupea harengus pallasii larvae and a predatory hyperiid amphipod (Hyperoche medusarum). Fishery Bulletin of National Oceanic and Atmospheric Administration ( U.S.) Washington, D.C., 74, 669-674. Wilson, D. F. and Parrish, K. K. (1974). Remating in a planktonic marine calanoid copepod. Marine Biology, 9, 202-204. Wyatt, T. (1971). Production dynamics of Oikopleura dioica in the southern North Sea, and the role of fish larvae which prey on them. Thalassia jugoalavica, 7, 435-444. Zillioux, E. J. (1969). A continuous recirculating culture system for planktonic copepods. Marine Biology, 4, 215-218. Zillioux, E. J. and Lackie, N. F. (1970). Advances in the continuous culture of planktonic copepods. Helgolander wissemchaftliche Meeresuntersuchungen, 20, 325-332. Zillioux, E. J. and Wilson, D. F. (1986). Culture of a planktonic calanoid copepod through multiple generations. Science, N . Y . , 151, 996-998.

Adv. Mar. Biol. Vol. 16, 1979, pp. 309-381

PIGMENTS OF MARINE INVERTEBRATES G. Y. KENNEDY The University of Shefield, Shefield, England

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" 0 Sea! Old Sea! who yet knows half Of thy wonders or thy pride? " Gosse: Aquarium 226-227.

I. INTRODUCTION Marine plants and animals are often very brilliantly coloured, especially those from tropical waters. Even in temperate climes many animals of the sea-shore, when viewed in quantity as in a rock pool association, present a fine sight, but in warmer waters, corals and their attendant fauna and flora provide a pageant of great beauty. Marine organisms also display many examples of pattern, an aspect discussed 309 A.P.B.-16

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The phylogenetic tree FIG.1. The phylogenetio tree (from Scheuer, 1973).

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in most fascinating style by the late T. A. Stephenson (1944) in his beautiful little book “Sea Shore Life and Pattern.” I n his stimulating article on Marine Natural Products, Thomson (1978) writes : “ There is no clear explanation for chemists’ neglect of marine products, although several contributory factors come to mind. Chief among these is the relative difficulty of collecting material which may be compounded by the problem of identification. While collecting intertidal species is easy enough, in deeper waters diving, trawling or dredging is necessary, and in some parts of the world the local marine fauna and flora have not been studied, and there is no taxonomic literature available. Sometimes there is no local expertise to hand where it might reasonably have been expected: e.g. there is no algologist in Aberdeen and only one in the whole of South Africa. Hence it is not unusual t o find in the current literature interesting compounds reported from unidentified sources.” Later on : “ Numerous marine animals are brightly coloured but little is known about the pigments.” It is true that at times the study of natural pigments has been desultory and empirical but, as we hope to show in this chapter, a good deal is known about many of them, and we owe our knowledge of many quinone pigments to Professor Thomson himself, and his school in Aberdeen. Marion Newbigin (1898) gave three reasons why the biologist should be interested in the colours of organisms : 1. Conspicuousness of colour phenomena in an objective survey of animals and plants ; 2. Relation of these colours to current theories of evolution ; 3. Their importance in comparative physiology.

The colours of living things are the visual result of three different processes : 1. Chemical:the metabolic formation of natural pigments, or the storage of ingested pigments, both consisting of coloured molecules which reflect and transmit parts of visible light : i.e. chemical pigments. 2. Physical: colourless structures which include laminations, striations, ridges, air bubbles, crystals, particles etc. which split light into its constituent colours by reflection, scattering and interference : i.e. structural colours.

3. A combination of 1 and 2. In this review, we shall consider only the chemical pigments of marine invertebrates by a discussion of their occurrence, phylum by

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phylum, with some mention of their metabolism and speculation on their functions. The order of classification followed is that of the Plymouth Marine Fauna List of the Marine Biological Association of the United Kingdom, 1957. There is a most useful table (Table 4.1) in the book by Needham (1974) embodying “ The major taxa of animals in relation to their chromatology.” “ Their colours and their forms were then t o me an appetite . . . . . . . Wordsworth: ‘‘ Lines composed a few mil@ from Tintern Abbey ”. 1,

11. PROTOZOA Very little has been done on the pigments of marine Protozoa, possibly because in many instances it is very difficult to obtain quantities sufficient to make a thorough chemical examination. The amoeba Janickina (Paramoeba)pigmentifera is parasitic in the coelom of the chaetognaths Sagitta and Spadella, and contains pigments which have not yet been identified (Hyman, 1959). Several blue-green, brown, yellow and purple pigments are found in some heterotrich ciliates ; some of these are fluorescent and photodynamic. Carotenoids have also been reported. Stentor coeruleus has been extensively studied by Tartar (1961)in a comprehensive monograph which describes some marine species : 8.multiformis is reported from salt or brackish water, and is bluegreen, with pigment stripes ; S.pygmaeus with a chitinoid case is found attached to some gammarids in the depths of the Sea of Baikal. This has a dark pigment; S. auriculata Kent and S. auriculatus Kahl were shown by Faur6Fremiet (1936a) to belong to the genus Condylostoma; S. acrobaticus said to be found on a branch of Fucus-reported unpigmented. Some chemistry has been done on the pigments of Stentor and its species and relatives. The blue-green pigment of S. coeruleus, ‘‘ stentorin”, is probably also found in S. multiformis, S. amethystinus and S. introversus. Stentorin is very similar to hypericin (the pigment from some species of Hypericum, notably St John’s Wort, H . perforatum) in fluorescence and U.V.visible absorption spectra. The pigment has the structure of a tetra-cr-hydroxynaphthodianthrone. Another pigment, “ stentorol”, was extracted from Stentor niger by Lankester (1873), and this yellow pigment was studied by Barbier, FaurB-Fremiet and Lederer (1956)who

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isolated it as a black powder giving a red solution in chloroform and red fluorescence in U.V. light. They suggested that it was a polyhydroxyquinone. Zoopurpurin extracted from Blepharisma undulans by Arcichovskij (1 905) and examined fairly recently by Sevenants (1 965) is a mixture of two compounds both very similar to stentorin and hypericin, and has been the subject of further study by Giese and Grainger (1970). It may be seen from the discussion of these pigments that investigation of the ma,rine species of Stentor and its relatives might be well worth while. The function of these pigments is still unkown. They may render the animals sensitive to light or, since they are toxic to other Protozoa, they may have some protective value. It is interesting that if Stentor engulfs another Stentor, the pigment of the victim is not assimilated but is ejected as a green excretion vacuole. I n the Folliculinidae, which often attach themselves to mollusc shells and tunicates, Faur6-Fremiet (1936b) found green, blue and reddish-violet pigments which may be polycyclic quinones. I n the blue Folliculina ampulla, closely related to the stentors, he found many blue granules close to the macronucleus. Another heterotrich Fabera salina, found in salt works in France and saline pools in Russia, Rumania and California, contains a dark pigment which, when extracted, is purple-red with a fine red fluorescence in U.V.(Fontaine, 1934). The yellow-green solution in pyridine gives absorption bands at 612 and 566 nm, and it has been suggested that this is also a polycyclic quinone. Although the hypotrich ciliate Holosticha rubra (previously known as Kernopsis rubra) found in aquarium tanks at the Plymouth Laboratory obviously invites investigation, nothing is known about its pigment ; there is also a yellowish variety H . rubra var. Jlava. Some Protozoa require pterins, principally biopterin and neopterin, as co-enzymes for some redox systems (Kidder, 1967) and others need pteroylglutamic acid (Kaufman, 1967). The hermit crab Eupagurus prideauxii is parasitized by ciliates Polyspira spp. and Gymnodioides spp., which take up blue carotenoproteins from the host. The carotenoid is split off from the protein by digestion in the food vacuole and is attached to another protein, producing a new carotenoprotein which imparts a violet-red colour, or even blue or green, to the ciliate. This pigment is passed on to the daughter cells in fission. I n similar vein, the copepod Idyafurcata contains a blue carotenoprotein in epidermis and retina, and a deep orange carotenolipoprotein in the blood and eggs. The parasitic ciliate Spirophrya takes up these pigments and reconstitutes them after digestion, to become pigmented itself.

314

Q.

Y. EBNNEDY

There is an unidentified ethanol-soluble red pigment in cytoplasmic granules in the foraminiferan Myxotheca arenilega-probably a carotenoid derived from the crustaceans on which it feeds. Newbigin (1898) drew attention to this pigment, and also to some species of the rhizopod Globigerina, which were described by Agassiz (1888) in his account of the cruise of the Blake, as " floating scarlet masses on the surface of the sea." Radiolarians often have a pigmented body, the phaeodium, which suggests a brown colour. It is well known that the protozoans Noctiluca miliaris and Pyrocystis noctiluca have great powers of phosphorescence, produced from minute granules of the system : Luciferin

-

+ oxygen luciferase oxyluciferin + water + light

Less well known is the pink colour of Noctiluca which " may be thrown upon the shore in such numbers as to form a coloured layer along the beach, the shore water resembling thick tomato soup " (Russell and Yonge, 1975). The identity of this pigment is unkown, but it is likely to be a form of oxyluciferin. The luciferins of known structure are heterocyclic chain-linked molecules whose colours, in their oxidized forms, may range from yellow through red to purple. The protozoan Opalina ranarum, parasitic in the intestine of frogs, does not fall within the marine category, but is mentioned because of its unique green pigmentation by biliverdin from the bile of the host. Another protozoan, Nassula, has a blue pigment which is probably derived from the Oscillatoria of the food. Nusslin (1884) described a beautiful violet pigment in Zoonomyxa violacea from the Herrenwieser Lake. The protoplasm is filled with many small violet vacuoles which impart a violet colour to the whole animal. Amphizonella violacea contains a granular pigment which is similar in many ways to that of Zoonomyxa.

111.PORIFERA The sponges provide many fine examples of vivid pigmentation as well as the more sombre browns, black and grey with off-white. The lipid-soluble nature of some of the yellow and red pigments of sponges was described by Krukenberg in 1880-1882 (see Krukenberg, 1882). MacMunn (1883, 1890) examined Halichondria albescens, Halma bucklanai and Leuconia gossei and found " lipochromes '' giving one strong absorption band in his simple spectroscope. The pigments producing the most vivid coloration of sponges are predominantly carotenoids

PIGMENTS OF MARINE INVERTEBRATES

316

(Figs. 2, 3, 4), with a preponderance of carotenes over xanthophylls, but there are instances of the occurrence of other types of pigment. Lonnberg (1931, 1932, 1933) examined many sponges (among other marine animals) for carotenoids, but his studies were not sufficiently

FIG.2. Structures of some cmotenoids.

complete to enable the pigments in his extracts to be characterized; this is a great misfortune, considering the amount of work done. None of the absorption spectra given by Lonnberg are near enough to the accepted maxima of authentic carotenoids to identify his pigments. However, because of the striking coloration of many sponges, their

316

0.Y. KENNEDY

FIG.3. Structures of carotenoids.

availability in quantity and the continuing active interest in carotenoids coupled with well-developed analytical methods, much of the work of Lonnberg has been repeated and extended. Astacene (Fig. 3) was isolated and crystallized from the red " cockscomb "Axinella crista-gulli by Karrer and Solmssen (1935) (but see later discussion on astacene). Lederer (1938), working with Suberites domuncula and Ficulina $GUS, reported that all the carotenoids in his extracts were epiphasic, even after saponification, and maintained that the pigments present were torulene (as in the red yeast Torula rubra), lycopene (Fig. 2) (as in the epidermis of the fruits of the tomato) and CH, \'

v

CH

CH,

/

CH,

CH,

CHa

CH,

CH,

I /\ I /"\ I I CH, C ~ C I I - C H ~ C - C H C H - C H ~ C - C H C H - C f i C H ~ C ~ C H - C H C H - C ~ C H - C 1 I ~ C CH, I HOCH

11C.CH,

/

I1,C.k

CH,

CH,

I

BOCH

AHOF

\/

Zeaxanthin

1I

H&.d

C.Ct1, Xant hophyll

FIG,4. Structures of carotenoids.

H !(OE

PIGMENTS OF MARINE INVERTEBRATES

317

/3- and y-carotenes (Fig. 2) with a small fraction of xanthophyll (Fig. 4) in Ficulina only. A propos this report of torulene by Lederer, Fox, Updegraff and Novelli (1944) often encountered this carotenoid in deep marine muds, and they suggested that the sponges which Lederer had examined may have ingested numbers of red Torula species known to occur in the sea (ZoBell, 1946.) If this is true, it seems odd that torulene has not been found in other detritus feeders, although of course there are isolated cases of specific retention of carotenoids and other pigments by marine animals. Drumm and O’Connor (1940) and Drumm, O’Connor and Renouf (1945) isolated and crystallized echinenone (4-keto-fi-carotene) from Hymeniacidon perleve ( = Hymeniacidon sanguinea) and also detected a-carotene in traces. Lederer (1938) also reported a brown-orange carotenoprotein in Ficulina Jicus. There are many papers describing carotenoids of sponges, and reference should be made to the books of Karrer and Jucker (1950), Goodwin (1952)) Fox (1953, 1976) and the short review by Goodwin ( 1 96th).

a-,

CH3 FIU.5. Struoture of renieratene.

I n recent times, the work of Yamaguchi (1957)who worked with the sponge Reniera japonica brought to light two new carotenoid hydrocarbons, together with ,%carotene. These were named renieratene, iso-renieratene and renierapurpurin. The two first-named are unique carotenoids in that they have aromatic rings (Fig. 5). Their absorption spectra are very near to those of y-carotene and /I-carotene,respectively. The sponge is able to aromatize the cyclic end-groups. Leprotene, present in some mycobacteria (Goodwin and Jamikorn, 1956) has been shown by Liaaen-Jensen and Weedon (1964) to be identical with isorenieratene. Yamaguchi (1957) also found a pigment spectroscopically close to torulene in R. japonica, but it differs from torulene in having a keto-group. Goodwin (1968a) raises the question of the reported occurrence of torulene and y-carotene in sponges and wonders whether this will be supported by further work.

318

0. Y. KENNEDY

Smith (1968) working with two red siliceous sponges Cyamon neon and Trikentrion helium, common on the sea floor off La Jolla, California, extracted a mixture of carotenoids containing unusual proportions of hydrocarbons from both. These included ,%carotene, 3, 4-dehydro8-carotene and some resembling 8-iso-renieratene. There was also a carotenoid tentatively identified as monohydroxy-fl-iso-renierateneand some 40% of the total carotenoid was a partially esterified dihydroxycarotene not previously encountered, suggested by Smith to be a dihydroxy-bis-dehydro-8-carotene. Tyrosinase has been found in the tissue fluids of the black Suberitee domuncub, the orange Tethya aurantia and Cydomium gigas by Cotte (1903), so that some of the dark colours and greys and blacks of sponges may be due to melanins. Some of the Aplysinidae contain pigments of the type described by Krukenberg (1882) as ‘‘ uranidines ”. These are yellow pigments, soluble in water and organic solvents with a green fluorescence (cf. Holothuria). The yellow pigment of A p l y s i m aerophoba blackens in situ after death, or when extracted ; it is blackened by boiling, alkaline pH or when shaken with air or oxygen. Acid pH prevents this change to some extent. The black material becomes insoluble and is precipitated or flocculated ; it is probably melanin. I have noticed that when the encrusting sponges Halichodria, Hymeniacidon and Hicrociona are collected,the torn edges readily darken during the period from the shore to the laboratory, and any material left overnight out of water is very dark the next day. Dark pigments also occur in Chondrosia. Ray Lankester sent specimens of the Australian sponge Suberites wilsoni to MacMunn in 1890. Lankester had already named the striking purple pigment “ spongioporphyrin ”, and MacMunn found that it had a two-banded absorption spectrum with peaks at 571 and 627 nm. However, he could not obtain a porphyrin after treating the pigment with concentrated sulphuric acid, and concluded that the name spongioporphyrin, while descriptively apt, was misleading. The pigment is still unidentified, but considering the absorption spectrum, colour and behaviour in various solvents with acid and alkali, one might speculate that it could be a trihydroxyanthraquinone. MacMunn (1890) also examined another purple sponge, the hexacinellid Polypogon gigas, and found that the pigment was quite different from that of Suberites iuilsoni and very unstable. Kennedy and Vevers (1954) reported a small amount of a red-fluorescent pigment in Tethya aurantia. This had an acid number of 5 (non-esterified)so that it could have been free protoporphyrin but further investigation was prevented by scarcity of material. There were no chlorophyll derivatives.

PIQMENTS OF MARIXE INVERTEBRATES

319

MacMunn (1890) extracted red-fluorescent “ chlorophyll-like ” pigments from a number of sponges, including Hymeniacidon sanguinea, Grantia seriata and Hircinia variabilis. These had a phaeophytin-like absorption spectrum and almost certainly were derived from the chlorophyll of algal symbionts or epiphytes. The green colour of the fresh-water sponge Spongilb viridis is due to symbiotic algae but that of the marine Halichondria panicea is not. This sponge contains two pigments-a yellow carotenoid resembling p-carotenone, and a blue one soluble in water which is pH and redox sensitive. This pigment is also soluble in ethanol but not in ether or acetone and is destroyed by boiling (Abeloos and Abeloos, 1932). It is interesting that the blue pigment is accumulated in the liver of the nudibranch Archidoris pseudoargus ( = Doris tuberculata) which feeds on the sponge. This may be a biliprotein like phycocyanin. Hircinia variabilis and some of the sponges which Krukenberg examined (cited by Newbigin, 1898) were said by him to contain a red pigment “ similar to the pigment of red algae and which is readily decolourized by reducing agents.” This could be a biliprotein like phy coerythrin . IV.COELENTERATA The Coelenterata have some of the most beautiful and spectacular coloration, with the molluscs and echinoderms as runners-up. Members of the phylum, which includes the anemones, jellyfishes and corals, are found in greatest abundance in warm seas but, even in temperate climates, the colours to be seen in shallow water or in rock pools are striking. There have been several reviews of coelenterate pigments, many of them extensive, the most recent being Fox (1974, 1976), Goodwin (1968b), and Fox and Pantin (1944), but the older accounts of Newbigin (1898) and of Verne (1926, 1930) still make fascinating reading and often provide useful and interesting facts, some of them long forgotten, from a time more tranquil and leisured than ours today. The reviews also provide many references to the original work so that the account to be given here will be confined to the most interesting work and the more recent discoveries. There is good evidence that carotenoids are distributed through every branch of the coelenterates, as may be seen by referring to the useful table (Table 28) by Goodwin (1952). The red pigment “ zoonerythrin ” first described by Bogdanow (1858) and later renamed tetronerythrin was found by Merejkowski (1881,1883) in Actinia ~ e ~ e ~ b ( =~A . ~ eguina), a ~Aiptasia ~ ~ spp., e ~

u

~

320

0. Y. KENNEDY

Cereactis spp and some hydrozoans. M. and R. Abeloos-Parize (1926) working with the red, brown and green forms of A. equina found an orange carotenoid in the red and brown variants and another red one in the red form. Further work by Lederer (1933) and Fabre and Lederer (1934) led to the isolation of a-and /3- carotenes, a xanthophyll and a red esterified carotenoid, actinioerythrin as carotenoprotein. A beautiful blue acidic carotenoid, violerythrin, was isolated by Heilbron, Jackson and Jones (1935) from hydrolysis of actinioerythrin. Actinioerythrin is now known (Weedon, 1971) to be a mixture of fatty-acid esters of the parent carotenoid actinioerythrol, which has 2-nor end groups. Actinioerythrol has not been reported in nature, and neither has violerythrin, the exact structure of which is still unknown, but which certainly has cyclopentenedione end-groups. The green variety of Actinia equina yielded a xanthophyll ester probably of taraxanthin or dinaxanthin, the green colour being due to the conjugation of the carotenoid with protein (see Cheesman et al., 1967).

Sulcatoxanthin, isolated by Heilbron et al. (1935) from Anemonia sulcata, has been identified with peridinin, the pigment of the alga Peridinium, but this may not be so (Weedon, 1971). MacMunn (1890) investigated the bright red pigment from the red form of Actinoloba dianthus ( = Metridium senile) and much later, Heilbron et al. (1935) found this to be an ester of low melting-point which upon hydrolysis with sodium hydroxide gave a red sodium salt. Fox and Pantin (1941) reported the pigment as distinct from astacene and named it " metridene " or more correctly, metridioxanthin. It is interesting that within the colour varieties of M . senile, the white ones contain some astaxanthin esters with free astaxanthin ; the brown have the least carotenoid, with esters of astaxanthin or metridioxanthin, carotenes and xanthophyll esters plus melanin ;the yellow, orange and red forms have large amounts of carotenoid with metridioxanthin or astaxanthin esters and some free pigments as well. This suggests that even very white animals (in any phylum) are worth investigating for pigments. Fox et al. (1967) examined M . senile jimbriatum from the Pacific Coast and found rich quantities of red carotenoids in the eggs of the white genotype which rendered very little pigment while unripe. The carotenoid was reported as mainly astaxanthin accompanied by some unfamiliar ketones and " zeaxanthin-like " esters. The same was true of the ovarian tissue of the red and brown variants. MacMunn (1885a) extracted a purple-brown pigment from Actinia mesembryanthemum ( = A . equina) with glycerine and named it actiniohaematin, a haemoprotein. Reduction produced a haemochromogen

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321

spectrum, concentrated sulphuric acid formed haematoporphyrin. The same pigment was detected in Tealia felina and the white form of Metridium senile. Roche (1936) found actiniohaematin to be a mixture of cytochromes a,, b, and c with b, predominating. The whole spectrum of reduced cytochrome with b very intense may be seen in the musculature of Hormathia coronata and Cereus pedunculatus. Band d of the cytochrome absorption spectrum can be seen in muscles of Actinia equina, Anemonia sulcata and Adamsia palliata. MacMunn also found biliverdin in the green base of Actinia equina. The first reported observation of an invertebrate porphyrin was that of Moseley (1877)-the same year as the discovery of haematoporphyrin by Hoppe-Seyler. Moseley extracted a red pigment which he named “ polyperythrin ” from the anemones Discosoma and Actinia, the scyphozoans Cassiopeia, Rhizostoma and Cyanea capillata; he also obtained it from the corals Ceratotrochus diadema, Flabellurn variabile, Fungia symmetrica and Xtephanophyllia formosissima. MacMunn (1886) examined polyperythrin and considered it to be identical with haematoporphyrin. This work has never been repeated, so far as I know, certainly because of the dificulty in obtaining material, but quoting MacMunn’s original paper : “ As the colouring matters in Uraster [ = Asterias], Limax, Arion and Lumbricus described above are identical with haematoporphyrin, and as polyperythrin is identical with them, polyperythrin must also be identical with haematoporphyrin ”

and later : I n the eggshell of the Cochin-China hen, the bands almost exactly coincide with those of polyperythrin ”. “

By the same reasoning, we now know that Asterias porphyrin is protoporphyrin (Kennedy and Vevers, 1953a and b), Lumbricus porphyrin is protoporphyrin (Hausmann, 1916 ; DhBrB, 1932)and the main porphyrin of avian eggshells is protoporphyrin (Kennedy and Vevers 1973, 1976), axiomatically polyperythrin must be protoporphyrin too. It is therefore almost certain that the coelenterates which Moseley and MacMunn examined contained protoporphyrin (Fig. 6). However, it is now known that Arion and Limax contain uroporphyrin I (Kennedy, 1959), so that there must have been some spectroscopic error here. Herring (1972) found free protoporphyrin in the bathypelagic scyphozoans Atolb wyvillei and Periphylla periphylla, but not in either of the shallow-living medusae Pelagia and Aurelia. He suggested that

322

Q.

Y. KENNEDY

the distribution of the pigment pointed to its possible function as a light absorber for the bioluminescence of prey in the gut, prevention of reflection of ambient day-light or bioluminescence from some of the tissues of the animals. The porphyrin fluorescence in vivo is quenched, yet it cannot be extracted as a protein complex by using buffers : it may be a complex with polysaccharide. Bonnett, Head and Herring (personal communication) have confirmed that the pigment is protoporphyrin present in the free state in crystalline cell-inclusions. The purple-brown colour i n many deep-sea medusae may be due partially or even entirely to porphyrin.

CH?

CH2

LOOH

COOH

I

FIQ.6. Protoporphyrin IX.

Some medusae are conspicuous by the presence of striking blue pigments in the integument, and these are usually caroteno proteins. Merejkowski (1883) described such a blue pigment in the oceanic siphonophores Velella spirans ( ‘ I by-the-wind-sailor ”) and Porpita, and called the pigment “ velelline ”. He converted the blue pigment to “ zoonerythrin ” by adding alcohol. S. C. Crane (cited by Fox, 1976) demonstrated that the blue chromoprotein of Velella lata has astaxanthin as chromophore, and Fox and Haxo (1959)confirmed this, suggesting also that the coloration protects the symbiotic zooanthellae against excessive sunlight. Herring (1971a) found that the blue carotenoproteins from Velella and Porpita show reversibility in colour changes due to rising temperature or depression of salinity, when normal conditions return. Herring (1971b) reported a biliprotein in the venomous jellyfish Physalia physalia, the Portuguese Man-0’-War. He found the chromo-

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phore to be a bilatriene, similar to but yet different from biliverdin. He suggested also that the lavender colour of the float, the pink crest and the green or purple of the gonodendra and gastrozoids might be due to other biliproteins. The reddish, purple and brown stripes of the large venomous jellyfish Pelagia colorata Russell (as P.noctiluca) were found to be due to melanins by Fox and Millott (1954). The violet chromoproteins described by them are not carotenoproteins. The sea-fan Eugorgia ampla is found at depths of up to 50 metres off the Baja California Coast. It is yellow-orange and has a pale yellow carotenoid with the cumbersome name of eugorgiaenoic acid firmly bound t o the calcareous microspicules which are embedded in the soft parts. There are other yellow carotenoids and long-chain fatty acids bound to calcium carbonate. The eugorgiaenoic acid is a non-fluorescent, non-aromatic unstable polyene resembling dihydrobixin (Fox, 1976). Goldman (1953) cultured isolated perisarc-enclosed segments of the hydroid Tubularia, without any source of exogenous pigment and found that the segments reconstituted with normal red pigmentation. The pigment is reported as astaxanthin (Goodwin, 1952) so that is the fist authenticated report of any animal being able to synthesize carotenoid de novo. Astaxanthin was the only carotenoid extracted from the vermilion skeleton of the hydrocoral Distichopora violacea from Eniwetok Island in the Marshall group. From the purple aragonitic skeleton of the hydrocoral Allopora californica from depths of 50 metres near Catalina Island off Southern California came the same pigment (Fox and Wilkie, 1970). Other species of Distichopora, D. coccinea and D . nitida also had astaxanthin as the only carotenoid but the coenenchyme of Allopora californicayielded astaxanthin ester, free phoenicoxanthin, free astaxanthin, a free polyhydroxy-/3-carotene and an unfamiliar hydroxydiketonic carotenoid. Fox (1972) investigated three species of Stylaster and reported that 8.roseus had astaxanthin as the only carotenoid in the purple skeleton, while the pink and orange skeleton of S. elegans and that of S. sanguineum revealed the enolically acidogenic astaxanthin and low concentrations of a neutral dihydroxyxanthophyll which Fox considered to be '' bonded through formation of their respective calcium acid carbonate ester." Not all coloured coral skeltons contain carotenoids. The brilliant blue Heliopora coerulea, the alcyonarian coral of Australian and WestIndo-Pacific waters has in its calcareous skeleton a bilichrome which was named helioporobilin by Tixier (1945). Riidiger et al. (1968)

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G . Y. KENNEDY

isolated the bilin, identified it as biliverdin IXa and suggested that helioporobilin was a mixture of biliverdin and the partially oxidized pigment. The free carboxylic radicals probably render the pigment sufficiently acid to form a calcium salt with the bicarbonate and carbonate of the skeleton. The deep red skeletons of the Organ-pipe Coral Tubipora musica and the Precious Coral Corallium rubrum are not coloured by carotenoid. Ranson and Durivault (1937) found that these corals contain iron, confirmed in some experiments in the laboratory of the writer, which further suggest that the red pigmentation is due to ferric hydroxide bound in the form of a complex salt with aluminium and calcium to a mucopolysaccharide. This work is still under way. Alcyonium palmatum also contains iron in the spicules (Durivault, 1937). Read andco-workers (1968)examined some coelenterates in blue light, and reported that the madreporarian corals Montastrea cavernosa and Mussa angulosa were red-fluorescent. They were red or blue-grey a t a depth of 40 metres but brown at the surface :the zooantharianCorynactis californicus was also red-fluorescent. The pigments were not identified but, recalling the work of MacMunn, it is tempting to expect them to be porphyrins. The zooantharian may also be fluorescent because of symbiotic algae although if the chlorophyll (or derivatives) is linked to a protein, fluorescence would be quenched. The pigment actiniochrome, occurring in the violet tentacle tips and stomodaeum of some anemones, e.g. Tealia felina, Anthopleura ballii and Anemonia sulcata, is still of unkown composition. It was first described by Moseley (1873) who found it to be a red pigment with an absorption band in the yellow-green. MacMunn (1885a) confirmed this and obtained the pigment from Anemonia sulcata. Fox and Vevers (1960) give the absorption band as 572 nm, and that of the green pigment which colours the tentacles of Anemonia sulcata as 512 mn. Both pigments are destroyed by boiling water (H. M. Fox, unpublished), the green one turning grey and losing its fluorescence, suggesting that the pigments may be linked to protein or, more likely, to a polysaccharide. Actiniochrome may be extracted from fresh tissue by glycerol as a red solution and this is changed to violet by alkali and darkened by ammonium sulphide, possibly on account of the presence of iron. That is all that is known about it so far. Christomanos (1953) described a purple pigment from the acontia of Adamsia rondeleti, which had a blue fluorescence in u.v., was sensitive to pH and gave an absorption spectrum of 555, 465, 450 and 435 nm. He took matters no farther. A violet pigment in the anemone Xagartia parasitica ( = Calliactis

PIGMENTS O F MAXINE INVERTEBRATES

325

eSfoeta) was described by Abeloos and Teissier (1926) and then isolated in crystalline form by Lederer et al. (1940), who named the pigment calliactin, reporting its close chemical relationship to the bile pigments. Calliactin can be oxidized and reduced and is yellow with acid and blue, violet and red with increasing pH. Riidiger (1970) maintains that this is not a pyrrol pigment. The beautiful anthozoan Cerianthus membranaceus from the Mediterranean may be violet, purple or even red. Krukenberg (1882) extracted a violet pigment from it, which he called “ purpuridin ” ; there was no definite absorption spectrum, and the colour was not discharged or changed by boiling. There seems to be a small amount of this pigment in C. lloydii. The chemical nature of this substance is unknown. Bullock ( 1 970), working with sea-pens, reported free protoporphyrin in the soft parts of Pennatula borealis and Bolticina jinmarchia. The concentration was particularly high in the tentacles and was such that the animal could be photosensitive. Pennatula aculeata had only minute amounts of porphyrin, too small to characterize. Bullock suggested that the pigment must have a function of some selective value to the animals : they live a t moderate depths and it is possible that the photosensitivity may be an advantage.

V.CTEHOPHORA There do not seem to be any reports of pigments in this phylum of beautiful, transparent and infinitely fragile animals. Fox and Vevers (1960) mention the fine colours produced by diffraction in ctenophores seen in the aquarium of the Stazione Zoologica at Naples. The diffraction grating is supplied by the moving ciliary combs. These combs, which may be reddish in Pleurobrachia pileus ( = Cydippe pileus), also give out waves of a greenish luminescence, which flashes for a few seconds, is extinguished and then returns. This is probably based on the luciferinluciferase system. The food of ctenophores consists of plankton, which may be worms, crustaceans, larvae, little fish, etc.-all pigmented-and in the words of Dr Strethill Wright, a naturalist of the last century, observing Cydippe pomiformia: “ The bright colouring of the prey so swallowed contrasts most conspicuously with the crystalline transparency of the body in which they are enclosed.” The ctenophores do not seem to store pigment from their food so must get rid of it by ejecting it or by metabolizing it into colourless compounds. VI. PLATYHELMINTHES