Advances in Marine Biology (v. 5)

Advances in

MARINE BIOLOGY VOLUME 5

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Advances in

MARINE BIOLOGY VOLUME 5 Edited by

SIR FREDERICK S. RUSSELL Plymouth, England

Academic Press

London and New York

1967

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Copyright 0 1967 by Academic Press Inc. (London) Ltd.

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EDITOR’S NOTE I n Volume I it was stated that one of the objects of this series was to make available to readers information on aspects of marine biology the literature on which was scattered over a wide range of publications. While many such review articles encourage further research, some of them soon become out of date. Others of a more substantial nature form milestones in the literature of the subject. It is the intention that occasional single author volumes shall be published. These will bring together such an amount of material that the volume should become a work of reference for all future workers in the field covered. While such volumes may not be of general interest they will nevertheless form works of permanent value. Such a work is that by Professor Thomas C. Cheng. By collecting together all the accounts and illustrations of known parasites of commercial molluscs he has made it possible for present and future investigators to ensure not only that they have a complete bibliography but also that they can use the volume as a reference text when much of the literature quoted may not be available to them in a library. All interested in the culture of molluscs for food will be grateful to Professor Cheng for his labours. I am pleased to say that Sir Maurice Yonge is now joining me as co-editor for future volumes,

July 1967

F. 5. RUSSELL

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES WITH A REVIEW OF K N O W N PARASITES OF COM ME RCIALLY IMPORTANT S PEClES

THOMAS C. CHENG Department of Zoology, University of Hawaii, Honolulu, Hawaii, U.S.A.

This review is jointly dedicated to Professor Leslie A. Stauber and Professor Harold H. Haskin, both of Rutgers University, who not only have educated so many outstanding molluscan parasitologists, ecologists and physiologists, but also have themselves contributed extensively to our understanding of symbionts of marine molluscs and the internal defense mechanisms of molluscs.

ACKNOWLEDGEMENTS Numerous individuals have given direct or indirect assistance during the preparation of this review. Along scientific and technical lines, I wish to acknowledge Dr. Sidney J. Townsley, Dr. Lary V. Davis, Dr. Ernst S. Reese, Dr. Berry S. Muir, Dr. Peiter van Wee1 and Dr. Fred I. Kamemoto, all of the Department of Zoology, University of Hawaii, for providing technical information, reprints, and unpublished data. Thanks are also due to Dr. E. Alison Kay, Department of Science, University of Hawaii, for her assistance relative to the Mollusca ; Dr. Aage Mdler Christensen, Marinbiologiske Laboratorium, Helsingerr, Denmark, for information on crabs ; Dr. Demorest Davenport, University of California, Santa Barbara, for information pertaining to host-symbiont attractions ; Dr. Kenneth K. Chew, University of Washington, for information pertaining to parasites found in molluscs on the west coast of the United States; Mrs. M. B. Chitwood, U.S. Department of Agriculture, Beltsville, Maryland, for information pertaining to nematodes; Dr. Eugene C. Bovee, University of California, Los Angeles, for information pertaining to the amoebae; and Dr. Horton H. Hobbs, Jr., U.S. National Museum, for assistance relative to the taxonomy of certain animals mentioned herein. Relative to my researches quoted in this review, I wish to acknowledge several individuals who have served as research associates or assistants during these studies. My thanks go to Dr. Bob G. Sanders, California Institute of Technology, Dr. Randall W. Snyder, Jr., University of Virginia School of Medicine, Mr. Alan B. Blumenthal, California Institute of Technology, Mr. Arthur W. Rourke, University of Connecticut, Dr. Alan H. Anderson, University of Rhode Island, and Mr. Richard W. Burton, Rhode Island Department of Fish and Game. Financial support for my studies was provided by grants from the National Science Foundation, the National Institutes of Health, the Office of Research Administration of the University of Hawaii, and the American Cancer Society. I wish to acknowledge the fact that the preparation of this paper was initiated while I was on the staff of the Northeast Shellfish Research Center, U.S. Public Health Service, and I am grateful to the Director, Dr. Carl N. Shuster, Jr., who made certain facilities available to me. My thanks also go to the library staffs at the Marine Biological Laboratory, Woods Hole, Massachusetts, the University of Rhode ix

X

ACKNOWLEDGEMENTS

Island, Brown University, and the University of Hawaii for their patient assistance in searching out obscure pieces of literature. Also, my sincerest thanks to Mr. George P. Hoskin of my laboratory and Mrs. Barbara Downs and M i . Frank Vaughan, Jr., Staff Artists in this Department, for executing many of the illustrations included in this review, and to Miss Linda Tanaka, the Departmental Secretary, who was most helpful during the preparation of the manuscript. I should also like to acknowledge the co-operation and assistance rendered by Sir Frederick S. Russell, the Editor of this series, during the four years this contribution has been in preparation, and to Academic Press who provided invaluable editorial assistance. Finally, I wish to express my deepest gratitude to my graduate students, Messrs. Herbert W. F. Yee, Peter Castro, Erik Rifkin, Amar S. Thakur, George P. Hoskin, and Miss Donna M. Hindelang, with whom I have spent many enjoyable hours discussing topics relevant to various sections of this review, for untiring assistance in reading proofs and rechecking some of the literature.

THOMAS c. CRENU

ADDENDUM Just as this volume was being passed for press, my attention was drawn to several papers dealing with aspects of antagonism between two species of larval trematodes within the same mollusc. Interested readers are referred to the following publications: P. F. Basch and K. J. Lie, 2. ParasitKde 2'9, 252-259, 260-270 (1966); K. J. Lie, Nature, Lond. 211, 1213-1214 (1966), J . Parasit. 53, (October, 1967); K. J. Lie, P. F. Basch and T. Umathevy, Nature, Lond. 206, 422-423 (1965), J. Parasit. 52, 454-457 (1966); and K. J. Lie, P. F. Basch, and M. A. Hoffman, J . Parasit. (in press).

T. C. C.

CONTENTS

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EDITOR’SNOTE

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ACKNOWLEDGEMENTS .

I . Introduction

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2. Definitions of Types of Symbioses I. Symbiosis . . A. B. C. D.

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Parasitism. . Mutualism.. Commensalism Phoresis . .

11. Predation

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PAQE

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3. Significance of Understanding Sym bionts of Marine Molluscs

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I. Importance t o Shellfisheries 11. Importance t o Public Health 111. Importance to Biology

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4. An Analysis of the Factors Involved in Symbiosis

I. Host-Symbiont Contact .. .. .. A. Accidental Contact .. .. .. B. Contact Dependent upon Hosts’ Feeding Mechanisms C. Contact Influenced by Chemotaxis . . .. . . D. Contact Influenced by Other Natural Taxes . . .. E. Selectivity of Symbiont . . .. .. .. F. Influence of Nature of Substrate . ,.

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xi

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18 18 18 19 46 49

53

xii

CONTENTS

11. Establishment of the Symbiont .. .. A. Physiological Resistance .. .. B. Behavioral and Mechanical Resistance

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111. Escape of the Symbiont

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54 55 132

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5. Parasites of Commercially Important Marine Molluscs. The Phylum Protozoa

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I. Phylum Protozoa

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A. Subphylum Sarcomastigophorea Superclass Mastigophora ..

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B. Subphylum Sarcomastigophorea Superclass Sarcodina . ..

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C. Subphylum Sporozoa Class Telosporea

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D. Subphylum Sporozoa Class Haplosporea

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Class Microsporidea

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F. Subphylum Ciliophora Class Ciliatea .

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E. Subphylum Cnidospora

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6. Parasites of Commercially Important Marine Molluscs. The Phyla Porifera, Cnidaria and Platyhelminthes

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198

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111. Phylum Platyhelminthes A. Class Turbellaria . B. Class Trematoda . C. Class Cestoidea

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199 199 202 254

I. Phylum Porifera 11. Phylum Cnideria

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xiii

CONTENTS

7. Parasites of Commercially Important Marine Molluscs. The Phyla Nemertinea, Aschelminthes and Annelida I. Phylum Nemertinea 11. Phylum Aschelminthes Class Nematoda

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9. Parasites of Commercially Important Marine Molluscs. The Class Crustacea

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8. Parasites of Commercially Important Marine Molluscs. The Phylum Mollusca

262

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111. Phylum Annelida

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I. Class Crustacea . .. A. Subclass Copepoda B. Subclass Malacostraca

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263 273

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286 286 315

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Appendix. A List of Commercially Important Marine Molluscs .. .. .. .. 336 and their Known Parasites

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References.

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AUTHOR INDEX

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TAXONOMIC INDEX ..

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SUBJEUT INDEX . .

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401 415

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CHAPTER 1

INTRODUCTION Although the role of molluscs as hosts of zooparasites has been known for over two centuries, ever since Swammerdam found the redial stage of trematodes in a snail in 1737, it has only been in relatively recent years that host-parasite relationships between molluscs and their parasites have been studied in any detail, and even now a great deal remains to be elucidated. Understandably it has been the freshwater gastropods that have been most extensively studied, since it is among these that are found those species which serve as intermediate hosts of parasites of medical and veterinary importance. For example, the relationships between Lymnaea spp. and Fasciola hepatica and that between Australorbis gbbratus and Schistosoma mansoni are among the most extensively investigated. During the last two decades, however, interest has developed in the symbionts of marine molluscs. This interest has developed along two distinct, although artificial, lines. The interest in parasites has stemmed from the realization that parasitism of marine molluscs, especially the economically important species, has far-reaching economic implications. The interest in other types of associations involving marine molluscs, although long in existence, has been accelerated as the result of the surge of interest in marine biology and biological oceanography all over the world. I n the case of economically and potentially important parasites, it has been primarily the parasites of pelecypods, such as edible oysters, clams and mussels, that have received concentrated attention. Even in investigations concerned with fundamental problems manifested by marine symbioses, of which molluscs represent one partner, it has been almost exclusively the estuarine species that have been studied. Little is yet known about the symbionts of and their relationships with deep-sea molluscs. Although the latter part of this review is concerned with the parasites of commercially important marine molluscs, it should be emphasized that the biology of zoosymbionts of marine molluscs, irrespective of their economic or medical importance, has a great deal to contribute to our understanding of the nature of symbiotic relationships and deserves increased attention. All too often in the past, and still true to some extent, parasitologists have concerned themselves primarily with medically and economically important parasites, thus 1 A.Dd.B.-5

2

’

2

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

paralleling the role of the medical bacteriologist of the previous two decades. Like many modern-day bacteriologists, or microbiologists as they prefer to be referred to today, increasingly more parasitologists now realize that the subject of their speciality-parasitism-is a widely distributed phenomenon, a natural way of life among at least half of the known species of animals, and need not always be associated with diseases of man and domesticated animals. Furthermore, as is now widely realized, if science is to enjoy a more complete picture of symbiosis, including parasitism, it is insufficient that studies be confined to the symbiont alone. Symbiosis has no meaning if the host organism is artificially removed from the scene. The unique contribution a symbiologist or parasitologist by his special training can make is to uncover and explain the facts, mechanisms and eventually the principles underlying the host-symbiont relationship. Lest I be accused of belittling the practical scientist and technologist and claiming unwarranted glory for the generalist, I hasten to state that applied and basic research do not differ, especially in this era, in their tools or even in the ends that are sought in many instances, but sometimes differences do exist in the basic philosophy of the investigator. Good science, as has been reiterated many times, commences with the wise choice of a model from which observational and experimental data can be extracted to support or reject a hypothesis. I n studying symbiosis, this rule is no exception. If the investigator should find what he considers an ideal working model in some medically or economically important host-parasite association, he should not be prejudiced against the experimental animals. On the other hand, experimental animals should not be rejected merely because they are of no immediate practical importance. It is from the viewpoint expressed in the previous two paragraphs that I embarked on this review of our present knowledge concerning symbionts and host-symbiont relationships involving marine molluscs. Research concerned with this category of associations actually comprises a hybrid as far as disciplinary boundaries are concerned, but then so many aspects of modern biology are hybrids in this sense. Investigation into this area involves competence in invertebrate zoology, behavior biology, parasitology, coupled with an understanding of marine biology, especially molluscan ecology and physiology, and utilizes techniques hitherto considered to be the tools of the taxonomist. morphologist, immunologist, physiologist and biochemist. Furthermore, this area of symbiology is somewhat unique since the ambient environment is marine, and hence represents a portion of the discipline concerned with the overall biotic activity in the sea.

1. INTRODUCTION

3

It appears appropriate a t this point to quote from Laing (1937) who has stated : The objects sought by any animal are different at successive periods of existence-food at one time, shelter, a mate, or a medium for oviposition in others. Which of these objects should be sought at any particular time depends, clearly, upon the internal conditions of the animal, for example, its state of hunger or the ripeness of its germ cells. In addition, the immediate behavior of the animal is directly affected by external factors. Thus, in dealing with marine symbioses, certain factors contributed by the marine environment obviously must differ from factors contributed by a terrestrial or freshwater environment. It is these different " external factors ", along with obvious differences that exist between distinct taxonomic categories of hosts and symbionts, which render marine symbiology unique. As this review serves t o point out, a t this time very little is yet known about " external factors " that influence symbioses in the marine environment, although such factors undoubtedly exist.

CHAPTER 2

DEFINITIONS OF TYPES OF SYMBIOSES I n studying heterospecific associations among organisms, a number of terms have been coined to describe types of relationships. These, however, like so many biological terms, are essentially operational words that are defineable only within broad limits. They are nevertheless useful in that they permit the filing of data into convenient, although in some instances poorly defined and overlapping, compartments. Specifically, the terms symbiosis, parasitism, commensalism, mutualism, inquilinism, phoresis and even predation are often found in the literature pertaining to heterospecific associations. In recent years, various authors (including Lapage, 1958 ; Baer, 1952; Caullery, 1952; Cameron, 1956; Dales, 1957, 1966; Hopkins, 1957b; Yonge, 1957; Dogiel, 1962; Olsen, 1962; Noble and Noble, 1961; Smyth, 1962 ; Sprent, 1963 ; Lincicome, 1963; Cheng, 1964b ; Geiman, 1964; Henry, 1966; Croll, 1966) have presented and discussed definitions for terms that describe heterospecific associations, and, as to be expected, differences, depending upon the immediate interests of the author, exist, sometimes only slightly. I n order that the meanings implied by me in the subsequent pages are clear, those terms that I consider useful are defined below.

I. SYMBIOSIS The term symbiosis, as originally coined by De Bary (1879) to mean no more than " living together ", is being retained in its original sense, although some authors have used symbiosis as a synonym of mutualism. Thus symbiosis is the broad, all-encompassingterm used to describe all types of heterospecific associations, excluding predation, during which there exists physical contact or intimate proximity between the two members. There are no implications of benefit acquirement or giving, nutritional dependency, or infliction or receipt of harm. Thus symbiosis is a broad ecological term under which can be categorized parasitism, commensalism, mutualism and phoresis. AS explained later, I have chosen to consider inquilinism to be no different from commensalism. It is with this or a very'similar definition in mind that Read (1958a) has suggested " a science of symbiosis " and Noble 4

2. DEFINITIONS OF TYPES OF SYMBIOSES

5

and Noble (1961) have expressed the view that because of the modern trends in parasitology, parasitologists might be designated as “ symbiontologists ”. I, however, prefer the term symbiology (Cheng, 196410) over “ symbiontology ”, since the latter implies that the investigator and his discipline are only concerned with one member of the association, the symbiont, and not with the host. As stated earlier, if the study of relationships between heterospecific organisms is to be comprehensive and unique, both the host and the symbiont, plus the nature of the relationship, must be taken into consideration. As stated, several subcategories of symbioses have long been in use in the biological literature. These are redefined below, and are drawn from what are considered to be the most appropriate aspects of previous definitions, although the basic interpretations are those of Smyth (1962).

A. Parasitism Parasitism describes a heterospecific relationship, be it permanent or temporary, during which there exists metabolic dependence of the parasite, the smaller of the two species, on its host. This metabolic dependency may be in the form of nutritional materials, digestive enzymes, developmental stimuli, or control of maturation. With the acceptance of this definition, Smyth (1962) has pointed out that it is now possible “ t o draw up a list of parasitic species which show an increasing degree of metabolic dependence on their hosts ”. Along this hypothetical scale (Fig. l), one can assign free-living organisms to one METABOLICDEPENDENCE Free-living

1 0%

1 Totally parasitic 100%

FIG.1. Diagram showing the relative concept of parasitism based on the degree of metabolic dependence. (After Smyth, 1962.)

terminal and complete dependence, or total parasitism, to the other. It should be pointed out, however, that if the parasite is metabolically dependent on the host even for a single factor, which cannot be obtained from the microenvironment, the relationship becomes an obligatory one if the parasite is to survive and perpetuate its species. Thus “ total dependence ” describes the number of metabolic factors on which the parasite is dependent and not necessarily the extreme at one end of a gradient of relationships.

6

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

B. Mutualism Mutualism describes an intimate relationship during which both the mutualist and the host are metabolically dependent on each other. An often cited example of mutualism is that relationship between the cnidarian Chlorohydra viridissima and the green alga Zoochlorella, which lives in the cytoplasm of the nutritive-muscular cells of the cnidarian’s gastrodermis. The alga produces oxygen which Chlorohydra utilizes, and Zoochlorella makes use of the nitrogenous waste products of Chlorohydra for its synthetic processes. Thus there exists interdependency between the mutualist and host. I n addition to this aspect of metabolic dependency, others exist (Muscatine and Lenhoff, 1965a,b ; Muscatine, 1965). What appears to be a concise distinction between parasitism and mutualism has been partially shattered by Smyth’s (1962) viewpoint that mutualism is actually a specialized form of parasitism during which some metabolic by-products of the parasite are of value to the host. Nevertheless, the mutual dependency is a real, recognizable and obligatory one during mutualistic, but not during parasitic, relationships.

C. Commensalism Commensalism describes that type of more or less intimate relationship during which the commensal generally derives physical shelter from the host, is nourished on foods that are associated but not a part of the host, and is not metabolically dependent on the host. Literally, commensalism means “ eating at the same table ”. It is thus a loose type of relationship and is not an obligatory one. I n accepting this definition, there is no longer a need to have a special category of inquilinism as defined by Caullery (1952). One of the best known examples of commensalism is found within the realms of marine biology. This is the association between certain species of hermit crabs and sea anemones. The anemone lives on the shell sheltering the hermit crab, At this location, it benefits directly in that it has access to the food caught and scattered by the crab. I n return, the crab benefits from the presence of the anemone which aids in warding off predators. Yet each animal can live without the other. Not all anemone-hermit crab associations are commensalistic. For example, Faurot (1910) has shown that the relationship between the hermit crab Eupagurus prideauxi and the anemone Adamsia palliata is an obligatory one, since neither of the partners will survive alone. Here the relationship appears to have developed into a mutualistic one.

2. DEFINITIONS O F TYPES O F SYMBIOSES

7

D. Phoresis Phoresis is most akin to commensalism but does not involve “ eating at the same table ”. This type of relationship is again a loose and nonobligatory one during which one organism, the host, merely provides shelter, support, or transport for the other. Metabolic dependency is not involved. According to this definition, those animals that are commonly referred to as being epizootic or epizoic can be considered as being engaged in phoretic associations with their hosts. Again, in marine biology an example of such a relationship can be cited in the case of fishes of the genus Fierasfer which live within the respiratory tracts of holothurians. Fierasfer is a relatively helpless fish that is readily preyed upon by others. Living in association with the holothurian, which is undisturbed by its presence, Fierasfer is provided with shelter and is transported from place to place. Another example of a phoretic relationship, but one which does not involve transport, is that between the hydroid Clytia balceri and certain intertidal molluscs, such as Donax gouldi and Tivela stultorum in Southern California. The hydroid is attached to the exposed surfaces of the host’s shell and no metabolic dependency occurs. The hydroid presumably does benefit, since it is prevented from being washed away with the tide. Since one of the major criteria employed to distinguish between types of symbioses, specifically to differentiate mutualism and parasitism from commensalism and phoresis, is metabolic dependency, it follows that definite assignments of associations can only be conclusively brought about through physiological and biochemical analyses. Such have been performed on relatively few species although increasingly more information of this nature is being contributed by modern symbiologists. Regrettably, in the case of the symbionts of marine molluscs not much is known other than indirect and inferential evidences. Herein lies a virtually untouched area of research for the imaginative mind. Having given definitions for ytegories of symbioses, it appears appropriate at this point to re-emphasize that overlaps do occur between the types of symbioses described. This is especially true between parasitism and mutualism, which share the feature of the occurrence of metabolic dependency, and between commensalism and phoresis, which do not involve metabolic dependency. The interrelationship between all four categories of symbioses is depicted in Fig. 2. From this diagram, it may be inferred that the greatest amount of overlapping can be expected to occur between commensalism and phoresis at one end, and between parasitism and mutualism at the

8

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

other; however, there may also be a slight overlapping between commensalism and parasitism. Cheng (1964b) has discussed the possible origins of symbiotic relationships. It has been concluded that although one type of association may evolve into another, and the occurrence of overlaps suggest this, such need not always be the case. I n certain instances, the types of relationships may also have evolved independently, not involving a transitional stage which could be interpreted as being one of the other established types of symbiosis. NON-OBLICATORY

NON-OBLIOATORY IFACULTATIVE)

IFADULTATWE)

SHARINO OF FOOD

MUTUAL DEPENDENCY

NUMO~IQATORY

ONE-SIDED DEPENDENCY I FACULTATlVE PARASTTISMI

FIQ.2. Diagram illustrating categories of symbioses and overlappings.

Notice that there are greater overlaps between mutualism and parasitism, and between commensalism and phoresis.

11. PREDATION A predatory relationship can be defined as one during which one member, the predator, as a rule, rapidly kills and devours the other, the prey. Furthermore, the two members need not be heterospecific, although they often are.* Another distinction between predator-prey and symbiotic relationships is that, as a rule, a prey reacts towards a predator, commonly attempting to escape from it. Such reactions have been studied in a number of marine invertebrates (Bullock, 1953; Feder, 1963 ; Margolin, 1964a,b ; Gonor, 1965; and others). However, in the case of sedentary prey escape reactions are generally wanting, and hence the use of this criterion in differentiating between symbiotic and predatory relationships is not always effective. The problem is compounded when one considers relationships during which the rapidity of the kill, which is a relative matter, is prolonged. Thus, when relation-

* A predatory relationship involving organisms of the same species is generally recognized as cannibalism.

9

2. DEFINITIONS OF TYPES O P SYMBIOSES

ships between organisms in the marine environment, or in any environment for that matter, are examined, the line of demarcation between parasitism and predation sometimes becomes extremely difficult to recognize. This is especially true if the often used criterion of “ inflicting injury to host ” is employed in defining parasitism, since this would indicate that both predation and parasitism are types of interactions that result in negative effects on the survival of one of the populations. Although this is true with certain parasites, it is by no means the rule as it is with predators. With the acceptance of the definition of parasitism given earlier, the distinction between parasitism and predation becomes more recognizable although the problem is by no means completely resolved. For example, in cases where one member of the association feeds on the tissues of the other and yet does not rapidly destroy the latter, should this be considered as parasitism or predation? It would appear that the solution to this dilemma lies in the qualifying phrase obligatory metabolic dependence ” that is used to define parasitism. If the aggressive member of the association is obligatorily and specifically dependent upon the tissues of the passive member, such a relationship may be categorized as parasitism. However, if the whole or parts of other organisms, within a broad spectrum, may be substituted for the passive member, the association may be considered as a predator-prey relationship. Since this review is only concerned with symbiotic associations, predators of marine molluscs are not considered although a few invertebrates whose role as either parasite or predator remains uncertain are mentioned briefly. (I

UHAPTER 3

SIGNIFICANCE OF UNDERSTANDING SY MBlONTS OF MARINE MOLLUSCS The original intent of this review was to summarize what is known about the parasites of commercially important marine molluscs. However, as the search of the literature progressed, it became increasingly evident that very little is known about the more subtle, yet important, aspects of parasitism among these associations. As would be expected, many organisms have been reported t o parasitize commercial molluscs and there have been some not too discrete statements as to the pathogenicity and lethality of certain of these parasites. There is also some information pertaining to geographic distribution. Unfortunately, these pieces of information hardly sufficed in assembling a continuous and natural description of parasitism. For this reason, the topic was broadened to include information that exemplifies the mechanisms involved in all types of symbiotic associations and on all types of molluscs, but without losing sight that the marine species are of primary concern in this review. Despite this change in plans, an annotated list of known parasites of economically important marine molluscs is given in Chapters 5-9. Many individuals have urged that this be done because up until this time no such list exists ; this has resulted in hardships for certain marine biologists, shellfishermen and fisheries biologists who do not have access to the large variety of journals in which such information is distributed. The significance of understanding symbioses of which marine molluscs, especially the commercial species, serve as hosts can be explained from three major viewpoints: (a) that of the fisheries biologists and shellfishermen ; (b) that of the public health officer, and (c) the more fundamental one of the biologist.

I. IMPORTANCE TO SHELLFISHERIES There is no need to belabor the importance of symbionts, especially parasites, to the fisheries biologist and shellfishermen. Several mass mortalities of shellfish, particularly of oysters, throughout the world during the last five decades have aroused the concern of the industry. Oyster mortalities since the 1910s throughout the world have been la

3. UNDERSTANDING SYMBIONTS OF MARINE MOLLUSCS

11

reviewed in detail by Korringa (1952) and hence need not be given in detail here. I n brief, the heaviest blow dealt to the industry in Europe occurred during 1920 and 1921, when large numbers of dead oysters appeared with relative suddenness in France, England, Germany, Denmark and the Netherlands. Many natural oyster beds were wiped out and have yet to recover. The cause has never been satisfactorily determined. I n 1919, another serious mortality of Ostrea edulis occurred in the Mar Piccolo near Taranto, Italy (Cerruti, 1941s). In the New World, Needler (1941) and Needler and Logie (1947) have reported the devastating mortality of the American oyster, Crassostrea virginica, which was initiated during 1914 in the waters of Prince Edward Island, Canada. The cause of this vast mortality remains uncertain, but the designation " Malpeque disease " has been coined for identification. This designation was borrowed from Malpeque Bay, Prince Edward Island, where the f i s t deaths were noticed. During subsequent years, the mortality spread to neighboring oyster beds and eventually destroyed shellfisheries in all of the principal producing areas of Prince Edward Island, reaching Enmore River in 1933 and the important Charlottetown area in 1936. During the 1930s another devastating mortality of Ostrea edulis occurred in Dutch waters and lasted until the 1940s. The cause has been investigated by Korringa (1947, 1951c) who believes that a fungus was responsible. Furthermore, he has expressed the opinion that the fungus may have reached the oysters from the shells of Cardium and Crepidula that had invaded the oyster beds in large numbers during this period. The oyster industry along the Atlantic and Gulf coasts of the United States has suffered two severe blows during the last two decades and which are continuing. Crassostrea virginica along the south Atlantic and Gulf coasts are being killed by a disease caused by a fungus named Dermocystidium marinurn* by Mackin et al. (1950). Even more severe, oysters in both the Chesapeake and Delaware Bays and adjacent waters, two major producing areas, were, and are, continuing to be killed off by a sporozoan originally designated as MSX and recently named Minchinia nelsoni by Haskin et al. (1966). In addition to these major mortalities, numerous others have been reported and several zooparasites have been found in oysters and other commercially important marine pelecypods. Although the cause-andeffect relationship has not been established in most cases, there is a legitimate concern over such parasites as possible lethal agents. Another reason for the interest of shellfish biologists in parasites of marine molluscs lies in the possibility that certain parasites of

* Dermocyatidium marinum has been transferred by Mackin and Ray (1900) to the fungal genus Labyrinthornyxa as L. marina.

12

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

predatory molluscs such as the oyster drills, Urosalpinx cinerea, Eupleura caudata and Thais haemastoma, may be utilized as biological control agents. Until now, only limited and preliminary studies of this nature have been conducted (Cooley, 1958, 1962), and hence the effectiveness of these parasites as control agents remains unknown for for the most part (Carriker, 1955). A third reason why parasites of marine molluscs are of importance to fisheries biologists is because many of them, especially the helminths, develop to their infective larval forms in molluscs and later, as adults, are parasitic in various fish, including the economically important species. Although many of these parasites are essentially non-injurious to their piscine hosts, they can be utilized as biological markers to trace their hosts' migrations.

11. IMPORTANCE TO PUBLIC HEALTH I n both North America and Europe there has been a concern over the possibility of shellfish serving as carriers of human pathogens. The correlation between typhoid and the eating of raw or poorly cooked shellfish from polluted waters in China and elsewhere in Asia is a wellknown public health problem. If viruses and bacteria can be transmitted by shellfish, there is no reason not to believe that zooparasites could also be transmitted. It is known, for example, that in the Philippines the intestinal trematode Echinostoma ibocanum is transmitted to man through the ingestion of metacercariae-harboring snails and corbiculid pelecypods. It is also known that in the Lake Lindoe district of the Central Celebes the population is heavily infected ( 2 6 9 6 % ) with Echinostoma lindoensis which is contracted through the ingestion of the clams Corbicula lindoensis and C. subplanata, and other metacercariae-harboring molluscs. Relative to zooparasites of marine molluscs transmissible to man, Vogel (1933) has reported that a German became infected with the intestinal trematode Himasthla muehlensi after eating several raw " littlenecks " (Mercenaria mercenaria) on the half-shell in New York. Recently, Cheng and Burton (1965a) have demonstrated that the nematode Angiostrongylus cantonensis can develop to the infective third-stage larva in both Crassostrea virginica and Mercenaria mercenaria, and hence these pelecypods, which are commonly eaten raw, could serve as potential transmitters. Although Knapp and Alicata (1967)have reported that they were unable to infect Crassostrea virginica and the clam Venerupis philippinarum experimentally, as will be discussed later (p. 270), their negative results are questionable in view of their experimental procedures, especially in the case of the

3. UNDERSTANDING SYMBIONTS OF MARINE MOLLUSCS

13

oyster. Angiostrongylus cantonensis is the metastrongylid rat lungworm that is believed to be the causative agent of one type of eosinophilic meningoencephalitis in man in Asia and certain Pacific islands, including Hawaii (see review by Alicata, 1965). Another aspect of the role of parasites of shellfish which is of potential importance to public health is the possible role of parasites, protozoa, helminths and arthropods, as carriers of pathogenic bacteria, viruses and other microorganisms. This concept is not as far-fetched as it would appear. Recently Moewus (1963) has reported that a holotrichous hymenostome ciliate, later named Miamiensis avidus by Thompson and Moewus (1964), a facultative parasite associated with skin tumors of seahorses, was experimentally found to be able to harbor polio viruses of the Mahoney strain. Furthermore, it is well known that the so-called “ salmon-poisoning fluke ”, Nanophyetus salmincola, can harbor the rickettsia Neorickettsia helminthoeca, which is the causative agent of salmon-poisoning disease in canines that have ingested raw salmon parasitized by Nanophyetus salmincola. I n addition to serving as intermediate hosts of zooparasites, which in turn may act as vectors for microorganisms, marine molluscs, as the result of parasitization by otherwise medically unimportant parasites, could cause temporary gastrointestinal disturbances resulting from the presence of butyric and other toxic short-chain fatty acids that accumulate in molluscs resulting from the degradation of the molluscs’ neutral fats by parasite-secreted enzymes (Cheng, 1966a). Thus, a more thorough understanding of both the types of parasites found in commercially important and potentially important marine molluscs and their metabolic effects on hosts are of importance to medical and public health workers. Another reason why certain parasites of marine molluscs are of public health significance is associated with the ability of a number of marine avian schistosomes to cause human cercarial dermatitis, commonly referred to as “ clam-diggers’ itch ” or “ swimmers’ itch ”. These schistosome trematodes develop to the cercarial stage in various littoral gastropods that commonly share the same habitat as certain commercially important shellfish. For example, the bird schistosome Austrobilharxia variglandis is found along the Atlantic coast of the United States in Nassarius obsoletus which shares the same mud flats with the soft clam, M y a arenaria, and the quahaug clam, Mercenaria mercenaria. Thus, individuals digging for clams in these areas may be subjected to cercarial attack resulting in dermatitis on exposed skin. Another group of marine trematodes that is of potential importance in public health is represented by certain members of the genus

14

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

Philophthalmus, such as P. lucipetus, found in gulls in Europe, P.skrjabini found in gulls in Russia and P. lachrymosus found in gulls in Brazil. These, like P. hegneri in the United States, undergo larval development in marine snails. I n the case of P. hegneri, Penner and Fried (1961, 1963) have reported that the molluscan intermediate host is the marine snail BatilEaria minima found along the Gulf of Mexico from Dunedin to Key West, Florida. Cercariae escaping from parasitized snails encyst ectopically and develop into metacercariae. When these metacercariae are fed to certain birds, migration to the eyes occurs and mature trematodes can be recovered from under the nictitating membrane. Although Philophthalmus hegneri, P. lucipetus and P. skrjabini have not been reported from mammals, P. lachrymosus has (Dissanaike and Bilimoria, 1958). Furthermore, another related freshwater species, P.gralli, has been shown experimentally to be able to infect rabbits and white rats if cercariae or excysted metacercariae were placed in their eyes (Alicata and Ching, 1960). According to these authors : I‘ These results indicate possible methods of human infection as reported in the literature.” It is thus possible that individuals bathing in Philophthalmus-infested waters could become infected if cercariae enter the eyes. Philophthalmus is an example of a marine trematode of potential public health importance. Since this area of parasitology, i.e. the potential importance of marine parasites developing in molluscs, has received practically no attention up until now, it is not onIy conceivable, but very possible, that other zoonotic parasites of marine origin will be found. With the world looking increasingly more to the ocean for food substances, medical or public health marine parasitology will undoubtedly receive increased attention.

111. IMPORTANCE TO BIOLOGY A thorough understanding of relationships between symbionts and marine molluscs can aid in answering various questions fundamental to biology and a t the same time serve to resolve many of the problems of the fisheries biologist and public health worker. By analyzing these associations, it is possible to seek and obtain answers to such questions as these. Are symbionts attracted to their hosts, and, if so, what are the attracting forces! What are the nutritional requirements of symbiotic protozoa, mesozoa, helminths, molluscs, annelids, arthropods and other categories of symbionts, and what are the sources of these requirements? Do differences exist between aspects of the metabolism of ectophoretic and endoparasitic symbionts, and, if so, do these differences suggest mechanisms involved in the adaptation to endopara-

3. UNDERSTANDING SYMBIONTS O F MA RIN E MOLLUSCS

15

sitism? The number of questions that can be asked are numerous. Many already have been investigated and answers are available ; however, in comparison with what potentially can be done, we have barely begun. I n the following chapter some of the information now available is reviewed.

CHAPTER 4

A N ANALYSIS OFTHE FACTORS INVOLVED I N SY MBlOSlS If the duration of any symbiotic relationship is examined, one can, with some justification, divide the association into three phases, and as the result focus critically on each. The phases I am proposing are: (1) the period of initial host-symbiont contact ; (2) the establishment of the symbiont on or within the host, and (3) the eventual escape of the symbiont or its progeny so as to effect other similar associations. AS outlined in Table I, each of these major phases can be subdivided into several factors, each of which can be subjected to experimental or observational analysis. TABLEI. FACTORS WHICH MAY INFLUENCE THE PRINCIPAL PHASES OF HOST-SYMBIONT RELATIONSHIP

A Host-symbiont contact

B Establishment of the symbiont

1. Accidental contact

1. Successful attachment 2. Developmental stimuli contributed by host 3. Host’s defense mechanisms

2. Contact dependent on host’s feeding mechanisms 3. Contact influenced by chemotaxis 4. Contact influenced by other taxes 5. Selectivity by symbiont 6. Influence of nature of substrate

4. Symbiont’s nutritional

requirements 5. Role of host’s digestive enzymes 6. Host’s control of symbiont’s maturation 7. Pathological changes induced by symbiont

C Escape of the symbiont 1. Active escape 2. Involuntary escape

3. Passive escape 4. Cellular escape

Briefly, at least five factors could be considered as possible influences on host-symbiont contact : (1) the contact could be accidental ; (2) it could be governed by the host’s activities during feeding or some 16

4.

17

ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

other behavior pattern ; (3) it could be influenced by chemotaxis ; (4)it could be influenced by other tactic responses of both the host and the symbiont, or (5)it could be influenced by combinations of some or all of these factors. Two additional factors may well be involved when one considers ectosymbionts : (6) for example, for hydroids that become attached to the shells of marine molluscs, selectivity on the part of the settling larvae could also influence the contact; (7) furthermore, in those associations in which the symbiont has to be in continuous contact with a solid or semi-solid substrate while approaching the host, the chemical and physical nature of the substrate could be of importance. At least seven factors could be considered subordinate to the major heading “ establishment of the symbiont ”. (1) For commensalism and phoresis, successful attachment at a suitable site is of prime importance, although this is also important for the establishment of parasitic and mutualistic relationships. (2) I n many instances even prior to successful attachment, the symbiont must overcome the host’s defense mechanisms, be these in the form of physical defense or a5 internal immunity. (3) I n the case of parasites and mutualists, the availability of sufficient and proper nourishment, derived from the host, as well as (4) digestive enzymes for those unable to synthesize their own, are essential. I n the case of commensals and phoronts, the availability of nutrients, although not directly of host origin, is also necessary. (5) The role of developmental stimuli contributed by the host as well as (6) factors that control maturation are of great importance. The occurrence of developmental stimuli and control of maturation implies metabolic dependence, and hence occurs only during mutualism and parasitism. (7) Finally, a tolerable level of pathological change in the host appears to be a prerequisite for successful establishment over a satisfactory period of time. This is particularly true during parasitic relationships. For example, if a parasite is highly pathogenic and causes the death of the host within a relatively brief period, the relationship, and therefore the establishment, from the standpoint of the parasite, could hardly be considered a biologically successful one. Eventually either the symbiont or its germ cell-bearing progeny must successfully escape from the host to perpetuate a similar association with another or the same host. From what is known about the severance of symbionts from hosts, various mechanisms could be involved to effect the escape. The conceptual factor analysis of the phases involved in symbiosis presented above serves as the guide line along which the following review is based. A.M.B.--S

3

18

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

I. HOST-SYMBIONT CONTACT

As Davenport (1955) has pointed out, symbiosis may be considered as an example of specialized behavior, grading from casual association to relationships in which the symbiont makes an active search for its host. This implies that the factors influencing host-symbiont contact range from none to one of very definite attraction, although even in the case of “ casual associations ” there may or may not be the involvement of certain factors, other than the physical properties of the ambient environment, which influence the contact. A. Accidental contact The first type of host-symbiont contact may be thought of as a casual and accidental one. There is no reason to believe that many epiphoretic organisms found clinging to the shells of marine molluscs had actively sought out and became attached to their hosts, nor is there experimental evidence to suggest that there is host-specificity in the choice of hosts by all epiphoronts. Although Dales (1957) has chosen to interpret the association of the hydroid Clytia bakeri with certain molluscs on the surf-swept beaches of southern California as reported by Torrey (1904) to be beneficial for the survival of the hydroids, and probably correctly so, there is no reason to believe that C. bakeri has a specific affinity for the clam, Donax gouldi, the Pismo clam, Tivela stultorum, or the olive shell, Olivella, on all three of which species it has been found. The abundance of these molluscs, coupled with their availability when the planula larvae were settling, most probably accounts for their relationships with these more or less sedentary molluscs. Because of the sedentary habits of these molluscs, the hydroids are prevented from being swept away with the tide. I n addition, Dales has suggested that the hydroids probably benefit from their position near the feeding currents of the clams. When found on Olivella, the hydroid gains mobility and its usual position near the host’s siphon may further improve its chances for food. If the hydroids indeed do share the hosts’ food, then the relationship should be considered as commensalism, but at this time no direct evidence is available. A list of some hydroids known to be symbiotic on marine molluscs is given in Table 11. B. Contact dependent upon hosts’ feeding mechanisms The second type of mechanism involves the active feeding habits of the hosts. I n the case of sedentary filter-feeding molluscs, smaller organisms, comprising the zooplankton, are drawn into the host and if these successfully pass through the selective process of the host’s gill

4. ANALYSIS OF FACTORS INVOLVED I N SYMBIOSIS

19

apparatus (Nelson, 1938, 1960) could become established as endosymbionts or, if they do not, certain species could become attached to the exterior of the soft tissues as ectosymbionts. The thigmotrichous ciliates, often found in the mantle cavity or on the gills and palps of estuarine pelecypods, belong to the second group, while the ciliate Trichodina myicola, found in the alimentary canal of M y a arenaria, belongs to the first. If the host, as in the case of many marine gastropods, is an active detritus feeder, various symbionts could be included in the food. Various protozoa and helminths, including cysts and eggs, could be introduced into the host in this manner. Although examples have not yet been found among marine molluscs, it is possible that cannibalistic molluscs could become parasitized while feeding on other molluscs that are parasitized. Cheng and Alicata (1965) have reported that the transfer of the third-stage larvae of Angiostrongylus cantonensis from one land snail, A c h t i n a fulica, to another can be effected by this method. C. Contact inpuenced by chemotaxis In addition to the two methods of host-symbiont contact given above, many symbionts, ranging from ectocommensals to endoparasites, in varying degrees, seek and contact their hosts. It is primarily with such active symbionts that specific attraction to the host is suspected. Unfortunately, information pertaining to the chemotactic response of symbionts to marine molluscs is extremely sparse. Although specially designed studies have been carried out to determine the attraction for symbionts, primarily commensal polychaetes, to nonmolluscan hosts (Davenport, 1950 ; Davenport and Hickok, 1951), the attraction of the sea anemone Stoichactis for the pomacentrid fish Amphiprion percula (Davenport and Norris, 1958), the attraction of the east coast pinnotherid crab, Dissodactylus mellitae, to its echinoid host, Mellita (Johnson, 1952), and the attraction between Anodonta implicata glochidia and the alewife, Pomolobus pseudoharengus (Davenport and Warmuth, 1965), parallel studies involving marine molluscan hosts are few (see reviews by Davenport, 1955, 1966). Among commensalistic relationships, Ross (1960), who studied the relationship between the actinian anemone, Calliactis parasitica, and the hermit crab, Eupagurus bernhardus, with the latter within the shell of the whelk, Buccinum undatum, has demonstrated that Calliactis parasitica will readily settle on shells of living Buccinum undatum in the laboratory and will not desert these for shells occupied by crabs. Similarly, Calliactis parasitica will with equal frequency become

TABLE11. SOMESYMBIOTIC CNIDARIA WHICH HAVEBEEN F o m ASSOCIATED WITH MARINE M o ~ ~ n s c s (Compiled by Dr Cadet Hand; after Dales, 1957, with later additions)

Host Pelecypoda Donax gouldi Tivela stultorum

Nuculana pwtulosa Nucula nucleus Nucula tumida Craasostrea rhizophorae

Tapes decwraatwr

Hydroid Clytia bakeri

Locality

Remarks

Reference

E

Mereschkowsky(l877) ; Fraser (1918); Hand (1957)

&

Southern California

An unmodified form which lives on D o m in the surf on sandy beaches ; a similar hydroid lives on species of D o m on Texas and Louisiana coasts Monobrachiurn Puget Sound, A one-tentacled hydroid living on paraaitum Southern small bivalves. A species of MonoCalifornia, brachium also occurs in about White Sea 1OOfm off coast of Baja and Southern California Perigonimus Arctic and A minute Perigonimus with reduced abyssi North Atlantic numbers of tentacles. Also found on Dentaliurn sp. Eugymnanthea Puerto Rico A solitary hydroid which lives as a ostrearum commensal in the oyster’s mantle cavity. Produces only one medusa a t a time Eugymnanthea Italy A commensal in the mantle cavity inquilina

Ritchie (1913)

a

8 21

2 2

Mattox and Crowell (1951) Palombi (1936s)

Ei

F a m mm

TABLE11.-continued Host Mytilus galloprovincialis Crassostrea gigas

Gastropoda Naasarius obsoletus

Hyalaea trispinosa

Phyllirhoe bucephala

Hydroid

Locality

Remarks

Eugymnanthea ltaly polimantii Eugymnanthea Japan japonica

A commensal in the mantle cavity

Stylactis hooperi

On shells of living snails ; an obligate relationship for the hydroid

Kinetocodium dame

Mnmtra parasites = Zanclea coslata

New England

Reference P

Cerruti (1941a) Yamada (1950); see also Crowell (1957) Sigerfoos (1899); Fraser (1944) for

literature & (1921); also withremarksonthree others of five known species found only on pteropods Atlantic and This medusa is commonly considered Ankel (1952) good Mediterranean t o be on the nudibranch. Howillustrations ; ever, evidence indicates the oppoMartin and site-that the nudibranch begins Brinckmann (1963) as a commensal inside the bell of life hist'ory Zanclea, and when this habitat is outgrown it attaches itself to its manubrium which it later devours with its tentacles North and South Large, few-tentacled, naked polyps. Atlantic, Gonophores produced from hydroIndian Ocean rhizal net

Ei

5w m

0

w w

! i e

2

2

Q Fn

!LE 0

wm

22

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

attached to empty shells. Subsequently, Ross and Sutton (1961), as the result of a series of tests designed to determine the frequency and speed of the clinging response of C. parasitica under various conditions, have confirmed that Buccinum shells that had never been occupied by crabs evoked the clinging response most consistently and rapidly. Ross and Sutton (1961), while reinvestigating Ross’ (1960) earlier observation that C. parasitica is not attracted to Buccinum shells that have been boiled in strong alkali, found that removal of the periostracum, either by boiling in alkali or by mechanical means, will reduce, but not abolish, clinging by the anemone. Furthermore, the anemone does respond to isolated strips of periostracum as to untreated shells. In addition, C . parasitica does not cling to inactivated shells when these become reoccupied by crabs, nor does it cling to dummy shells or to shells coated with a thin plastic layer. From these experiments it was concluded that the clinging response depends entirely on a general molluscan “ shell-factor ” associated with the periostracum. An extension of this work by Davenport et al. (1961) has revealed that C. parasitica attaches itself to Buccinum shells partially by tentacular nematocyst activity. Moreover, the threshold of nematocyst discharge changes markedly in accordance with the attachment behavior of the animal. The discharge threshold is lower in free animals than in animals already attached by their pedal disks to a shell. Thus it would appear that an anemone receives information from the contact of the pedal disk with the shell which in turn influences the discharge threshold. It has been suggested that chemoreceptors in the pedal disk may be responsible for initiating this reaction after responding to some organic material in the periostracum. Although the studies cited above strongly suggest chemotaxis of C. parasitica to the periostracum of Buccinum undatum in the laboratory, Davenport (personal communication) has related a still unexplained phenomenon that occurs in nature. According to him, one can dredge up numerous Buccinum in the English Channel, but one almost never finds Calliactis parasitica on these. It is only on empty shells harboring hermit crabs that C. parasitica is found, or on empty shells alone. Since it is known that the hermit crab, Eupagurus bernhardus, does not aid the anemone in becoming attached (Ross, 1960), the question arises as to whether the reaction of the anemone to the periostracum of Buccinum undatum is a conditioned reflex that only appears after the molluscs vacate their shells. This remains to be determined. Another study which demonstrates chemotaxic attraction of a symbiont to its molluscan host was carried out by Muriel A. Wikswo

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

23

(unpublished). I n brief, she has been able to demonstrate that the polynoid polychaete Arctonoe wittata is stimulated when exposed to sea water in which its host, the keyhole limpet, Diodora aspera, had been placed. Wikswo has also shown that the nature of the substrate does influence the attraction of Arctonoe vittata to Diodora aspera. If both organisms are placed in a glass-bottomed bowl the polychaete readily moves towards the host, but if placed in a sandy bottom bowl it does not. Similarly, if the bottom of the bowl is lined with cheese cloth, Arctonoe vittata rarely moves towards its host. Wikswo has also demonstrated that Arctonoe vittata is repelled by dead Diodora aspera. This observation confirms the finding of Davenport and Hickok (1951) who have demonstrated that commensals, Arctonoe fragilis in their case, are repelled by water from an aquarium that contained injured starfish, Ewasterias, its natural host. It would thus appear that the attractant can be either destroyed or masked by substances produced by dead or damaged hosts. This would undoubtedly be of advantage to symbionts in nature, since they would actively migrate from a dead or moribund host. Among parasites of molluscs, studies designed to determine the attraction or non-attraction of parasites are primarily limited to those concerned with the attraction of trematode miracidia to freshwater gastropod intermediate hosts. Since the results of such studies, which are still highly controversial, may contribute further insights into the nature of host-secreted attractants (host factors), the available information is briefly reviewed herein. The pitfalls and misinterpretations by those working with freshwater molluscs, as well as their concrete findings, can no doubt serve as guards against similar mistakes and act as profitable guides in future work with marine molluscan hosts. The subject at hand has been reviewed by Wright (1959a). My review incorporates, but extends beyond, his. Wesenberg-Lund (1934), expounding on the behavior of trematode miracidia, has stated: “ If we try to gain some knowledge from the literature of the behaviour of the miracidia in their relation to the different mollusc species, it is very difficult to get a clear idea of the real facts.” Strange as it may seem, in 1967 we are faced with the same problem although studies published during the last few years have tended to alleviate this situation. I n brief, two schools of thought are in existence relative to the mechanisms involved in miracidiummollusc, primarily gastropod, contact. There are those who champion the concept that a host-elaborated attractant or stimulant exists, and there are those who believe that host contact is strictly a random process.

24

MARINE MOLLUSCS AS HOSTS F OR SYMBIOSES

Many earlier workers believed that miracidia are attracted to gastropod hosts by a chemotactic substance(s) secreted in the host’s mucus or “ juice.” Faust and Meleney (1924), Faust (1924) and Faust and Hoffman (1934), who studied the behavior of the miracidia of three species of human-infecting schistosomes, Xchistosoma mansoni, S. haematobium and S. japonicum, are among the first, if not the first, to support the “ attraction theory.’’ I n the initial study by Faust and Meleney, they observed that S. japonicum miracidia, when in the vicinity of the snail Katayama nesophora, show powerful response to the snail as well as to the mucus tract left by it. The response to K. nesophora does not occur until the swimming miracidia come “ within a few millimeters of the range of the snail.” Although no detailed study of the specificity of this behavior was made, it was reported that no response was elicited by two common snails from northern China, Vivipara quadrata and Lymnaea plicatula, both of which are not compatible hosts for these schistosomes. Barlow (1925),who studied the miracidia of Pasciolopsis buski, and Tubangui and Pasco (1933),who studied those of Echinostoma ilocanum, were also among the first to support the ‘‘ attraction theory.” Barlow reported that Fasciolopsis buski miracidia chose only two snails, Segmentina nitidellus and Planorbis schmackeri, if these hosts were presented among a number of other species. These are among the first experimental demonstrations of the manner in which specificity may be determined by precise behavior of a parasite under host influence. As to the exact behavior pattern, the following quotation from Faust and Hoffman (1934) gives an account of the behavior of Schistosoma mansoni miracidia in the presence of Australorbis glabratus: When active miracidia of Schistosoma mansoni, swimming rapidly through the water, come within a few millimeters of the appropriate molluscan host, they become stimulated almost immediately and head ” €or the snail. The exact attraction-mechanism is unknown, but the work of Barlow on Fasciolopsis buski and of Tubangui and Pasco on Echinostoma ilocanum miracidia indicates that it is some fraction of the tissue juice of tho appropriate snail. The secretion of this substance into the immediate vicinity of the snail provides the stimulus which directs the miracidium t o the snail and ‘‘ notifies ” it that such snail is its suitable intermediate host. cL

Recently, Davenport et al. (1962), utilizing the flying-spot microscope technique, reported that Schistosoma mansoni miracidia, when in the proximity of filtered extract of whole ground Australorbis glabratus, frequently exhibit a “ whirling dance ” upon initial contact with the extract before heading towards the site of greatest concentration. Following the early proponents of the “ attraction theory ”, various

4. ANALYSIS OF FACTORS INVOLVED I N SYMBIOSIS

25

other workers have presented supporting evidences. Briefly, Mathias (1925), working with the miracidia of Strigea tarda (= Cotylurus cornutus),has reported that these demonstrate a preference for Lymnaea stagnalis but that development would occur in both L. limosa and L. palustris. Wesenberg-Lund (1934) was convinced by his field observations that the miracidia of a species of trematode demonstrate a pronounced preference for a distinot species of mollusc within a given locality. Neuhaus (1941), who observed the behavior of Fasciola hepatica miracidia, has suggested that these ciliated larvae are initially attracted by the ciliary currents maintained by the epithelia of Lymnaea spp., but later, when they become drawn within a certain range of the gastropod, the attraction is converted to one purely chemical in nature, with the effective range varying with the species of Lymnaea used. In an extension of his earlier observations, Neuhaus (1953) reported a definite chemotaxis between various species of Lymnaea and F . hepatica miracidia, with the attraction being most strong with Lymnaea trunculata, the species generally accepted as the normal host in Europe (see Kendall, 1950). I n the same paper, Neuhaus stated that Wirniewski’s observations on Parafasciolopsis fasciolaemorpha miracidia in the presence of its snail host also suggests chemotaxis. I n more recent years, several investigators have designed and carried out more elaborate experiments to prove or disprove the occurrence of attraction between trematode miracidia and molluscs. Those whose results favored the “ attraction theory ” are reviewed at this point. Kloetzel (1958) has carried out a series of carefully controlled experiments with Schistosoma mansoni miracidia and the snail Australorbis glabratus. I n the initial experiment he placed a single snail in a dish containing a known number of miracidia. He made counts of the number of miracidia in the immediate vicinity of the snail and a t other points in the dish at known time intervals. Thus he was able to demonstrate that the number of miracidia around the snail was significantly higher than at a point diametrically opposed to it after 15 min. In the second series of experiments he removed the snail from the miracidia-containing dish after 15 min and, after washing it to remove adhering larvae, replaced it at the opposite side of the dish for an additional 15 min. The difference in larval densities at the snail’s original position and where it was replaced was no longer so significant. This suggests that some substance was left behind at the initial site which continued to attract miracidia. Subsequently, Kloetzel has found that miracidia are even more strongly attracted to a snail squashed on filter paper than to a living snail and that their attraction to an empty shell is not significantly more than random. These findings

26

MARINE MOLLUSUS AS HOSTS FOR SYMBIOSES

led to another series of experiments during which the extract from a snail was added to a dish with a known number of miracidia before a healthy snail was placed therein. It was found that in such a preparation the number of miracidia which aggregated around the snail is markedly reduced when compared with control snail and miracidia preparations to which no snail extract had been added. Comparable evidences were again reported by Kloetzel (1960). His studies certainly give strong support to the belief that some degree of chemical attraction exists between Austrabrbis glabratus and Schistosoma mansoni miracidia although the attraction appears to be effective only over short distances.

FIG.3. Diagram of Y-shaped chemotrometer for testing reactions of miracidia. Size in mm. I n chamber A is placed the attractant (snails or other substances), in chamber B any substance, and in chamber C the miracidia. (Redrawn after Kawashima et al., 1961a.)

Another study of attraction between miracidia and molluscs, one which has been often overlooked by European and American workers, was contributed by Kawashima et al. (1961a). These Japanese investigators studied the attraction between the miracidia of one of the mammalian lung flukes, Paragonimus ohirai, and three species of snails of the genus Assiminea-A . parasitologica, A. japonica and A . latericea miyazakii. These snails are found in brackish waters at the mouths of rivers but at different salinities. By using a modified Y-tube choice apparatus (Fig. 3), they demonstrated that Paragonimus ohirai miracidia are attracted to all three species of snails. Furthermore, the miracidia are also attracted to homogenates of Assiminea

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

27

japonica and to an amino acid mixture encapsuled in a sheet of cellophane. Kawashima et al. were uncertain whether the “ host factor ” or attractant is chemical or physical. It was also pointed out that the host-preference of the miracidia occurs without any relation to the suitability of the snails as hosts since it is known that A . parasitologica is the common natural host. I n fact, studies have shown that A. parasitologica can be readily infected experimentally and that this snail is naturally infected in endemic areas. On the other hand, A. latericea miyazakii appears to be an incompatible host while A . japonica can be infected experimentally but the level of infection is always low. It is thus apparent that some additional factor must be operative in nature to bring about the selection by the miracidia for A . parasitologica. I n a later paper (Kawashima et al., 1961b), it was found that thesalinities occurring at the intertidal habitats of these three species of snails influence the survival of the miracidia, with that existing at the habitat of A. parasitologica being most favorable to the miracidia. While studying the locomotive speed and survival of Paragonimus ohirai miracidia in various concentrations of NaCl, it was found that the lower the salt concentration is, the more active the miracidia become. I n fact, the authors stated that : “ It seems to be indispensable for the eggs to hatch and for the larvae to get the host, to be kept in the solution of less than 0.25% NaCl.” Concurrent studies on the “ population activities ” of the three species of snails at various salinities revealed that the optimum salinity for Assiminea parasitologica was 0.25%, that for A . latericea miyaxakii was 0.4%, and that for A . japonica was 0.6%. These findings explain the preferred habitats of the snails, since A . parasitologica is usually found in the proximity of the high tide line while the other two species are found near the low tide line. These also serve to explain why A . parasitologica is a compatible host from the ecological viewpoint. Thus these investigators not only have demonstrated an instance where the influence of the host’s attractant can be superimposed by an ambient environmental factor but also have demonstrated that attraction of miracidia to molluscs need not mean successful subsequent development, i.e. specific attraction. Furthermore, they have demonstrated how the salinity tolerances of the parasite and the host can serve as natural mechanisms responsible for host specificity. Another study of mollusc-miracidium attraction was contributed by Etges and Decker (1963). These investigators employed a cast-iron maze consisting of a central cylindrical chamber (4 x 8 cm diam.) with four cylindrical side arms (each 3.5 x 1 cm diam.). To the free end of each side arm was attached a vertical terminal cylindrical

28

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

chamber ( 3 x 1.5 cm diam.). The inner surface of the maze was coated with inert waterproof lacquer to prevent contamination and to facilitate cleaning between trials. I n the first series of experiments, two specimens of Australorbis glabratus, the shells of which had been crushed, were placed in two of the terminal chambers, one in each. I n each of the other two terminal chambers was placed a sham snail modelled out of inert aquarium cement. About 100-200 Xchistosoma mansoni miracidia were placed in the central chamber in conditioned water after the crushed and sham snails had been permitted to stand in the terminal chambers for 1 h, during which time substances of crushed snail origin had entered the water. After the central chamber was covered, the entire apparatus was placed under a strong light source to prevent miracidia which had reached any of the terminal chambers from returning to the central chamber. This was carried out since it is known that certain schistosome miracidia are positively phototactic. Etges and Decker were careful to state that with the sham snails in pIace, the amounts of light reflected into the central chamber through the four arms were essentially equal. After 1 h 80 min, 80% of the miracidia had entered the side arms which were then stoppered at the center chamber-arm junctions. Counts of the number of miracidia in each of the terminal chambers and adjoining arms during nine runs revealed that the number of miracidia in the terminal chambers and arms associated with crushed snails was significantly greater. As a result, these authors stated: “Such a great degree of significance strongly indicates positive chemotaxis of S. rnansoni miracidia toward A. glabratus under these experimental conditions.” Since it was observed that crushed A . glabratus gave a slight reddish turbidity to the water resulting from released hemoglobin, thus decreasing the amount of light transmitted to the center chamber from the two arms leading t o the real snails, and it was feared that some of the miracidia which had reached the crushed snails had entered the hosts’ tissues and had thus been missed in the counting, a second series of experiments, involving uncrushed snails restrained by loosely wrapped nylon mesh, was conducted. Again, the live snails attracted significantly more miracidia. I n addition to using A. glabratus, two other groups of gastropods, Helisoma anceps and a mixture of Bulinus (Bulinus) truncatus and B. (Physopsis) sp., both crushed, were employed in identical experiments. The results revealed that the miracidia were distributed in all cases in favor of the sham snails. Rather than interpreting this to mean that repulsion occurred between Bulinus spp. or H . anceps and the miracidia, the authors offered the explanation that the condition resulted from less transmitted light from the arms

4. ANALYSIS OF FACTORS INVOLVED I N SYMBIOSIS

29

associated with the crushed snails due to increased turbidity caused by released respiratory pigments. Control experiments with four sham snails revealed no statistically significant difference in miracidial distribution. Thus, Etges and Decker have quite convincingly dernonstrated chemotactic attraction of miracidia to their normal host, although these workers expressed their uncertainty as to whether such a stimulus is operative under natural conditions. They further maintained that both light and gravity are far more powerful influences in determining the orientation of Sch.istosoma rnansoni miracidia than the molluscan host’s chemotactic attraction. S. mansoni miracidia are known to be negatively geotactic in addition to being positively phototactic. The fact that Etges and Decker have found that crushed snails attracted miracidia 1 h after the death of the snails is of interest since

END VIEW

SIDE VIEW

FIG.4. Standard truncated pyramids used in miracidia chemotaxis studies. (After Machnis, 1965.)

Davenport and Hickok (1951) have shown that the commensal polychaete Arctonoe fragilis is repelled from water which had contained injured starfish, its natural host. These seemingly opposing results indicate that the response of Arctonoe fragilis to injured host is different from that of Schistosoma mansoni miracidia, with the former showing definite repulsion. MacInnis (1965), using another set of procedures involving agar pyramids (Fig. 4) and Australorbis glabratus, has been able to demonstrate not only chemotaxis between Schistosoma mansoni and substances from the snail host, but has also given some indication as to the nature of the attractants. He constructed the experimental apparatus in the following manner. I n order to test the reactions of miracidia to various amino acids, short-chain fatty acids, sugars, and various salts (Table 111),two types of agar pyramids were used. I n the first type, referred to as “ impregnated pyramids ”, distilled water-agar pyramids

30

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

were pre-soaked for 2 h in filtered river water, blotted, and allowed to soak overnight in the specific chemical solution. I n the second type, referred to as " integral pyramids ", the agar pyramids incorporated an aqueous solution of the chemical to be tested. These were not subsequently soaked in river water. TABLE111. EFFECTIVENESS OF CERTAIN CHEMICALSAS ATTRACTANTS FOR Schbtosoma mansoni MIRACIDIA All experiments conducted in 50rnl river water except those indicated by asterisks which were in phosphate buffer. (After MacInnis, 1965.) N o . of miracidia

Test chemical

Test chemical cone. or p H

10 n-Butyric 2.5 r n M n-Butyric 30 5.5 r n M Uric 25 Sat. A&. 15 Mannose 5.5 r n M 16 Galactose 5.6 mM 25 DL-Aspartic 1.0 r n M 25 DL-Valine 1.0 r n M 25 L-Cysteine-HC1 1.0 r n M 25 L-Asparagine 1.0 r n M DL-Leucine 25 1.0 r n M DL-Methionine 25 1.0 r n M 25 L-Phenylalanine 1.0 rnM L-Histidine 1.0 r n M 25 L-Proline 1.0 r n M 25 DL-Tyrosine 1.0 r n M 26 Glycine 1.0 r n M 25 25 DL-Serine 1.0 r n M L-Cystine 1.0 r n M 25 25 DL-Alanine 1.0 r n M L-Lysine 1.0 r n M 25 L-Arginine 1.0 m M 25 5.5 r n M 25 Acetic 25 NH, acetate 6.5 r n M 1-0r n M 25 HCl 25 NaH,PO, 4.9 pH 25 Na,HPO, 7.0 pH 25 Na ,HPO 8.9 pH 6.5 rnM *n-Butyric 25 25 **n-Butyric 6.5 mM 25 ***Miracidia killed Controlt -

Length of exposure (min)

Total conlacts

15 10 15 15 15 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 6 6 6 6 6 6 6 6 -

7 7 16 0 7 12 8 7 12 12 14 8 6 7 6 2 4 3 2 9 3 16 7 8 7 2 3 2 6 -

Per cent contact with return 86 100 6 0 100 76 75 86 68 75 68 75 66 28 33 60

0 0 0 0

0 68 0 88 15 0 0 0 0 2

* Phosphate buffer pH 7.0. ** Phosphate buffer p H 8.9. *** Phosphate buffer p H 4.8. t Control is the average of fifteen experiments, each for 15 min with twenty-five miracidia in 50 ml water.

4. ANALYSIS OF FACTORS INVOLVED IN~SYMBIOSIS

31

The observations were conducted in Pyrex Petri dishes containing 50 ml of water or in finger bowls with 100 ml of water and observed under a stereomicroscope appropriately illuminated so that the diffuse light did not influence the miracidia. A known number of miracidia was added to each dish or bowl and a pyramid containing the test chemical, or a control pyramid, was placed in the center of the container. I n addition, Australorbis gbbratus tissues were also used in another series of observations. Uninterrupted observations were recorded for 10-15 min during the experiments.

FIQ.6. Responses of Schistosoma rnansoni miracidia to test pyramids (contact without return). A, Normal change of direction upon contact with an obstruction; B, increased random turning ;C, directed turn at a distance ;D, encircling at a distance ; E, 180" turnback; F, circus movements. (After MacInnis, 1966.)

Schistosorna rnansoni rniracidia normally swim with a uniform speed in a straight line while rotating clockwise or counterclockwise along the longitudinal axis. If such a miracidium should encounter a surface, including the surface film, it changes its direction of movement so as to by-pass the obstruction and continues to swim normally (Fig. 5A). MacInnis reported that if miracidia enter the vicinity (0-5 mm) of a snail or impregnated pyramid, they portray a variety of behavior patterns. These can be summarized as follows. (1) They sometimes increase their speed of swimming. This, according to the definition of Fraenkel and Gunn (1961), is known as " chemo-orthokinesis" (i.e. increased linear velocity in response to a chemical

32

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

stimulus). (2) Other miracidia, when in the same situation, turn abruptly back and forth in a " wigwag " manner (Fig. 5B). This movement appears to be an exaggeration of the rotation of the miracidium along its longitudinal axis and subscribes to the definition of " chemo-klino-kinesis" of Fraenkel and Gunn (i.e. change in the rate of random turning or angular velocity in response to a chemical stimulus). According to MacInnis, this type of movement, which may commence at 7 mm or more from the pyramid, appears to aid in locating a gradient of diffusing chemicals, and thus the source. (3) A third type of behavior involves miracidia not swimming directly towards the test object. I n these instances, if a miracidium is attracted by the pyramid it suddenly turns and swims toward test object (Fig. 5C). This response can be considered as Fraenkel and Gunn's " chemo-tropo-taxis " in part (i.e. locomotion straight towards or away from the source of the chemical stimulus ; the result of simultaneous comparison by two receptors). MacInnis has reported, however, that the directional turn is often accompanied by increased speed (chemo-ortho-kinesis) and increased " wigwagging " (chemo-klino-kinesis). The combination of these three behavior patterns had been reported by Campbell and Todd (1955a) who referred to the condition as " excitement ". (4) A fourth type of reactional behavior involves a complete or incomplete circling 3-5mm away from the test object (Fig. 5D). This behavior pattern can be considered as " chemo-klino-taxis " (i.e. movement directly to the stimulus, or locomotion along the line to the stimulus, modified by regular symmetrical deviations ; only one receptor is needed). MacInnis has suggested that this behavior might be explained as a reaction occurring at a boundary of the moving front of the diffusing chemical, or at one place in the gradient where the concentration of the chemical is at the threshold of intensity for a positive reaction. ( 5 ) A fifth type of behavior is displayed by miracidia moving away from the source of the chemical stimulant. These execute a 180" turn, thus pointing themselves toward the source of the stimulus (Fig. 5E). This type of behavior is considered by Fraenkel and Gunn (1961) as " chemo-tropo-taxis ". (6) The last type of behavior occurs in the zone of a diffusing chemical where some miracidia change from the normal swimming behavior to swimming in a small circle (Fig. 5F). This type of behavior appears to be identical with the " whirling dance " of Davenport et al. (1962). It has also been described by Campbell (1961) for the miracidium of Fascioloides magna, and according to Campbell was found by Hugghins for the miracidium of Hysteromorpha triloba, a strigeid trematode. Earlier, Fraenkel and Gunn (1961) had reported this behavior among

33

4. ANALYSIS OF FACTORS INVOLVED I N SYMBIOSIS

free-living flatworms and called it “ circus movement ”. According to these investigators, the “circus movement” is a consequence of a “ chemo-tropo-tactic ” type of response. It appears to reflect the response of only one of a pair of bilateral receptors, causing a complete turning towards a favorable stimulus or away from an unfavorable one. I n addition to utilizing the behavior patterns described above, MacInnis also has recorded six types of behavior portrayed by miracidia upon contact with a test object containing a chemical stimulus. The detailed descriptions of these can be consulted in his paper. For the purpose of this review, they are designated as A, B, C, D, E and F and depicted in Fig. 6.

FIG.6. Responses of Schistosoma mansoni miracidia t o test pyramids (contact with return). A, One loop; B, several loops; C, dipping response; D, body parallel; E, body perpendicular ; F, beeline drive, attachment, and partial penetration. (After MacInnis, 1965.)

Utilizing these two series of behavior patterns and agar pyramids which contained various test chemicals, MacInnis has demonstrated that short-chain fatty acids, some amino acids, and a sialic acid (Table 111) are attractive to Schistosoma mansoni miracidia and also stimulate attachment to and penetration of the agar. Among the amino acids tested, the dibasic forms showed little or no attracting ability. It was also demonstrated that the solvent action of distilled water, ethyl ether, acetone, and ethyl alcohol can remove attracting substances from Australorbis glabratus. Furthermore, subsequent addition of butyric A.M.B.-5

4

34

MARINE MOLLUSOS AS HOSTS FOR SYMBIOSES

or glutamic acids to snail tissue from which the attracting substances had been removed by solvents restores the capacity of the snail tissue to attract and stimulate miracidia to attempt penetration. There is little doubt from MacInnis’s results that chemotaxis does exist between miracidia and the test agar pyramids. MacInnis has stated that : ‘ I Some of the chemicals investigated were considered possible components of snail mucus, or on other grounds were considered as possible attractants.’’ Nevertheless, it would appear that further studies are necessary to demonstrate that the attractants do occur in the mucus or body fluids of compatible molluscs. Although there is some indication that amino acids and amino sugars are present in snail mucus (Wright, 1959b), these substances have not been shown conclusively to be present. Relative to short-chain fatty acids, there is no evidence at this time that they occur in snail mucus although they may be end-products of snail metabolism (von Brand et al., 1955). Furthermore, the question may be asked if the attraction of miracidia to butyric acid and glutamic acid, as has been demonstrated by MacInnis, is meaningful in nature, especially when one considers host-specificity which may be governed, at least in part, by the chemotactic material@) (Faust and Meleney, 1924 ; Barlow, 1925 ; Neuhaus, 1953 ; Etges and Decker, 1963). If butyric acid is as commonly found as an end-product of molluscan metabolism as suggested by von Brand et al. (1955), it certainly would not serve to direct miracidia to a specific species of snail host. Similarly, glutamic acid, which is a commonly occurring amino acid, could not again be expected to serve as a selective guide. On the other hand, MacInnis’s results may indicate that chemotaxis is not always associated with host-specificity. On the other side of the fence, there are evidences suggesting that symbiont selection of hosts, specifically miracidia-mollusc contact, is strictly a random phenomenon without the occurrence of attracting factors. Mattes (1926, 1936) has reported that Fasciolu hepatica miracidia find their host randomly and will attack almost any softbodied animal ; Stunkard (1943), in one of the very few studies of this nature involving a marine mollusc, has reported that the miracidia of Zoogonoides laevis are not noticeably attracted to their molluscan host Columbella lunata (= Mitrella lunata) and that contact between the miracidia and snails appears to be merely accidental. Similarly, Griffiths (1939) and LaRue (1951) believed that miracidia-mollusc contact is primarily a random phenomenon. LaRue, however, has indicated that, although under laboratory conditions trial and error appears to be much more important than chemotactic response in bringing miracidium and snail together, in nature the possibility of the

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

35

occurrence of some chemical stimulus operating over very short distances may exist. Abdel-Malek (1950), who specifically looked for attraction, reported that Schistosoma mansoni miracidia will attack any object including empty snail shells and particles of fine gravel. When in the presence of Biomphalaria boissyi, a suitable intermediate host, Abdel-Malek reported that the miracidia’s movements are random and that it appears to find its host largely by chance. Similarly, Stirewalt (1951) working with Schistosoma mansoni and Australorbis glabratus, Chu and Cutress (1954) working with the marine avian schistosome believed to be Azcstrobilharzia variglundis and the marine snail Littorina pintado, and Najim (1956) working with the freshwater avian schistosome, Bigantobilharxia huronensis, and its molluscan host, Physa gyrina, have all reported the lack of any apparent attraction. It should be mentioned that, except in the instance of Abdel-Malek, the other earlier reports were incidental observations not subjected to critical analyses. More recently, Sudds (1960) has examined for the presence or absence of chemotaxis between four different species of schistosome miracidia (Trichobilharzia elvae, T . physellae, Schistosomatium douthitti and Schistosoma mansoni) and a number of normal and generally considered incompatible molluscan hosts. It is unfortunate that Sudd’s thorough experiments were marred by the assumption that it is only in the case of normal or compatible hosts that specific chemotaxis occurs. It is my opinion that attraction of symbiont to host should be considered a distinct operation from successful establishment of symbiont in or on its host, although there are suggestionsthat host-symbiont contact may influence morphogenetic changes. For example, Campbell and Todd (1965b)have reported the in vitro metamorphosis of Fascioloides magna miracidium into a sporocyst after a short contact with snail tissue. At any rate, Sudds discovered that of nineteen incompatible host-parasite combinations studied, the miracidia in six displayed “ a determined effort to penetrate the tissues of the snails ”. According to his data, the “ determined effort ” was in the form of either “ miracidia sticking to snail upon contact and appear to be penetrating ” (Sudds’ type 4 behavior pattern) or “ miracidia contacting snail and moving rapidly over the surface, sticking intermittently, swimming away, returning, etc.” (Sudds’ type 3 behavior pattern). I n eleven other incompatible host-parasite combinations, the miraoidia were observed making brief attempts to penetrate. As defined elsewhere in his paper, this represents Sudds’ type 3 behavior pattern described as “ miracidia sticking briefly to snail, swimming away, returning, etc.”. I n the remaining two incompatible host-parasite combinations, the miracidia did not appear

36

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

t o be aware of the presence of the snails, even when contact was made (Sudd’s type 1 behavior pattern). Of the seven normal host-parasite combinations, all demonstrated either type 3 or type 4 behavior patterns. From these studies, Sudds has concluded that : It is clear, therefore, that under the controlled conditions of this study there is little support for the “ attraction theory ”, since only 2 of the 19 combinations gave results of the type expected under the operation of a specific chemical stimulus. On the other hand, 17 of the 19 combinations yielded results supporting the view that contact with snail hosts occurs by chance, and that, once contact is made, the miracidia are stimulated to attempt penetration. Although Sudds’ subsequent histopathological studies on the reaction of compatible and incompatible snails to schistosomes are t o be commended, it is not at all clear that his “ attraction” studies proved what he concluded. The reasons for this doubt are discussed below. Chernin and Dunavan (1962), in discussing a series of experiments designed to test the influence of host-parasite dispersion on the capacity of Schistosoma mansoni miracidia t o infect Australorbis glabratus, stated that they found nothing to support the “ attraction theory ”. They further stated that: . . . nor do the experiments and rationalizations which have been published in support of it (the attraction theory) seem entirely convincing. This is not to say that ‘‘ attraction ” between snail and schistosome miracidium does not exist, but rather that the fact of its existence has not yet been demonstrated unequivocally. I n critically analyzing the data by Chernin and Dunavan, I failed t o see how their experiments in any manner either support or deny the existence of a chemotactic factor. Their experiments were designed t o determine whether a t a constant water level, regardless of volume, the capacity of miracidia to infect snails was influenced. They found that there is no significant difference in the different volumes used. They also studied the preference of miracidia for different depths and distances in reaching hosts. They found that miracidia do not demonstrate a preference for any specific depth when placed in 20 cm of water and that some miracidia traverse a t least 86 cm horizontally or 33 cm downwards to reach molluscan hosts. Their other experiments demonstrated that miracidia do not follow a linear course and travel a t a velocity of 2.1 mm per sec (range 1.7-2.8 mm per sec) during the first 15 min after emergence and average 1.9 mm per sec (range 1.3-2.5 mm per sec) after about 1 h of free life. Their studies on miracidial taxes disclosed that negative geotaxis has a stronger influence on their

4. ANALYSIS OF FACTORS INVOLVED I N SYMBIOSIS

37

behavior than positive phototaxis, and their observations on the natural dispersal of A. glabratus and 8. mansoni miracidia in vessels revealed that in the case of both organisms the perimeter of a vessel is the preferred site. It is of significance to note that Chernin and Dunavan did state that “ . . . the tropisms (negative geotaxis and positive phototaxis) do not elicit an absolute response for all miracidia even when acting together ”. I n addition to the evidences reviewed above, unfortunately there have appeared in the literature ‘‘ evidences ” which cannot be critically analyzed as the result of misunderstandings or misidentifications of the taxonomy of either the molluscs or the parasites. For example, Cort (1918)) to illustrate his point that Schistosoma haematobiurn lacks host specificity and indirectly denying the existence of specific hostelaborated chemotactic agents, stated that in Egypt X. haematobiurn utilizes both Bulinus contortus and B. dydowski as intermediate hosts while in South Africa Physopsis africana is implicated. More recent studies have shown that the two Egyptian snails are synonyms for Bulinus truncatus. Furthermore, the investigations of McCullough (1957) and Le Roux (1958) have suggested that the schistosomes utilizing Bulinus (Physopsis)spp. as intermediate hosts are specifically distinct from those which utilize B. truncatus. In view of the work of Newton (1952, 1953, 1954) on Australorbis glabratus infected with Schistosoma mansoni, it is apparent that successful establishment of the parasite, which must in most cases be considered distinct from the initial host-parasite contact, is dependent upon the genetic strain of the host, and perhaps also of the parasite, beside other factors. Another example of confusion resulting from misidentification of molluscan hosts has been cited by Wright (1960) who pointed out that Stunkard (1957)) in presenting evidence to indicate that a high degree of host-specificity need not exist in trematode-mollusc relationships, stated that the intermediate hosts of S. mansoni in Africa are species of Planorbis, Physopsis and Isidora, while in the West Indies and South America the snails concerned belong to the genera Australorbis and Tropicorbis. However, Wright has pointed out that Isadora is a synonym for Bulinus and Physopsis is a subgenus of Bulinus. He indicated that the Planorbis referred to by Stunkard is the African genus Biomphalaria since Planorbis does not occur in the Ethiopian region. Furthermore, Hubendick (1954) had shown that African Biomphalaria is congeneric with the New World Australorbis and Tropicorbis. Thus it would appear that the confirmed natural intermediate hosts of Schistosoma mansoni all belong to one genus. The fact that these snails are closely related has been demonstrated by

38

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

Barbosa and Carneiro (1957) who reported that under laboratory conditions some of the South American and African species hybridize freely and produce viable young. The type of confusion which exists relative to schistosomes and their molluscan hosts has not yet plagued studies on marine parasites and their molluscan hosts. This does not mean that such problems will not arise, especially since the taxonomy of marine molluscs shows indications of becoming just as complex, but the lack of such confusion merely reflects the less intensive nature of research in marine parasitology. Another factor which evidently has an important influence on miracidium-mollusc contact but which, in my opinion, has not received sufficient attention by those interested in the presence or absence of chemotactic response has to do with the age of the miracidium. Differences in the ability of miracidia to make contact with their hosts as a function of age was discovered by Campbell and Todd (1955a). These investigators studied contact between Fascioloides magna miracidia and Xtagnicola refZexa. They divided their miracidia into three age groups. The young ” group included those ranging from 10 to 40 min post-hatching, the medium ” group included those ranging from 92 to 104 min post-hatching, and the members of the old ” group ranged from 8 h 40 min to 8 h 43 min post-hatching. A total of 102 miracidia and forty-eight small snails of approximately the same size were used. Each snail was exposed to a known number of miracidia of a specific group. The snail was examined at time intervals and the number of miracidia not attached to the snail was recorded. I n some instances a miracidium became detached from the snail, hence the number of detached miracidia at any time may have been slightly more than at the previous count. It was also noted that in each series of exposures more miracidia became attached to snails during the first 15 min than during the succeeding 55 min. Campbell and Todd’s results are tabulated in Table IV. From these they concluded that (‘the miracidia are more infective when between 1.5 and 2 h old than when either very young (less than one hour) or very old (eight hours) ”. Their results also suggest that while the members of the medium ” group are the most effective attackers, members of the ‘I young ” group are more effective than those of the ‘( old ”. It is of importance to note that Campbell and Todd have stated that : The effect of age on the attacking power of miracidia may help to explain inconsistencies found among previous reports on the behavior of miracidia in the presence of a snail.” Although Campbell and Todd found no consistent behavior pattern which suggested chemotaxis, they did state that : ((

((

((

((

((

4. ANALYSIS O F FACTORS INVOLVED IN SYMBIOSIS

39

It seems probable that the total period of incubation of F . magna eggs may influence the ability of the miracidia to establish contact with their molluscan hosts. The age of the miracidia at the time of hatching perhaps controls their ability to respond to the presence of a snail. It is possible that miracidia which hatch as soon as their development permits, have not yet acquired a sensitivity to whatever stimulus (chemical or otherwise) the snail may provide. Since miracidia have been observed to exhibit excitement [italics added] in the presence of a snail, it is felt that under certain conditions, not yet understood, a marked attraction on the part of a snail for a miracidium may exist. Since the age of the parasite does influence the efficiency of contact with its host, it appears feasible that other factors, such as the age of the host, may also be influential but this has not yet been tested. TABLE Iv. DISAPPEARANCE O F Pascioloides magna MIRACIDIAIN THE PRESENCE OB Stagnicola rejlexa EXPRESSED AS PERCENTAGES Miracidia of series A were 10-40 rnin post-hatching,those of series B 92-104 min post-hatching, and those of series C 520-523 min post-hatching. (After Campbell and Todd, 1955a.) Time interval (min)

‘‘ Young ”

‘‘ Medium ’’

(series A )

(series B )

8-15 15-35 35-40 40-50 50-70

54 86.5 84 -

76 88 82 -

86.5

-

“ Old ” (aerie8 C )

47

75 78

Campbell and Todd have also compared the percentages of contact between F.magna miracidia and two species of molluscs. The parasites used were between 41 and 56 min old. It was noted that : No difference was detected between the swimming behavior of the miracidium in the presence of Xtagnicola re$exa and Fossaria modicella rustica but the results of this experiment suggest that the rniracidia,attack the latter species more readily. The significance of this last study, in my opinion, rests with the fact that the miracidia did become attached to two species of molluscs. Although both of these happento be compatible hosts (Krull, 1933; Campbell and Todd, 1955b),it is interesting that both snails “attracted” miracidia and hence indicate that the “ attraction ” is not speciesspecific. A large part of this controversy pertaining t o the existence or nonexistence of an attractant of host origin, as I interpret it, is based on several fallacies.

40

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

(1)Those who expect to find evidence for the attraction theory ” and have been disappointed have erroneously expected an immediate, dramatic, and all or none ” manifestation of attraction. As the statistically analyzed studies of Kloetzel (1958, 1960), Etges and Decker (1963) and MacInnis (1965) have shown, chemotaxis is not an “ all or none ’’ phenomenon, nor is it dramatic. If it exists, as these data suggest, it is a very subtle process which is operative only within short ranges and can be only fully appreciated upon analysis of quantitative data. Various studies of this nature in related areas have convincingly shown that chemotaxis does occur. For example, host-localization among parasitic insects as reported by Salt (1935)and Laing (1937),host attraction of the annelid Acholoe as reviewed by Davenport (1955, 1966), chemoreception and responses to chemical stimuli in free-living flatworms as reported by Pearl (1903), Koehler (1932), Hyman (1951), and Fraenkel and Gunn (1961) are all now widely accepted discoveries. These and other authors have suggested that even prior to the operation of chemotaxis the symbiont is initially attracted to a certain type of environment and, if such an environment coincides with the natural habitat of the host, the first stage of host-symbiont contact is accomplished. This is what Wright (1959a, 1960) has also proposed. (2) The problem has been oversimplified by those who expect specific chemical attraction and host-specificity to be parts of the same process. Thus Sudds (1960), for example, expected the attraction of schistosome miracidia to natural hosts to be distinctly more apparent than attraction to incompatible molluscs, and a part of his argument against the I ‘ attraction theory ” is based on comparable attraction to at least some unnatural hosts as determined later by histopathological studies of host reactions to invading parasites. Although the observations of Faust and Meleney (1924),Barlow (1925),Neuhaus (1953), and the data of Etges and Decker (1963) suggest specific attraction of S. mansoni miracidia to A . glabratus, there is no reason to believe that host-specificity is always manifested in the initial contact. The finding by Cheng (1963a) that the plasma” of five different species of molluscs will activate the quiescent cercaria of Gorgodera amplicava but in varying degrees, depending on the molluscan species, and the report of Cheng et al. (1966b)that the plasma and tissue extracts of seven different species of marine pelecypods have similar effects on the cercaria of Himasthla quissetensis, but again in varying degrees depending on the ‘(

* The fluid portion of molluscan blood has been referred to in the literature as serum, plasma or hernolymph. As molluscan blood does not clot, this fluid is technically not serum, but because of common usage these three terms are used interchangeably in this review.

4. ANALYSIS OF FACTORS INVOLVED IN SYMBIOSIS

41

molluscan species, suggest that the stimulant given off by the molluscan hosts may be of a general rather than a species-specific nature. Thus in the case of Sudds’ work, the more apparent attraction of such unnatural hosts as Bulimnaea megasoma and Fossaria abrussa for Trichobilharzia elvae miracidia could very well represent non-specific attraction, divorced from subsequent successful or unsuccessful development of the parasite in the host. Only additional studies of this nature will reveal whether specific and general attractions both occur in nature. Direct evidences which indicate that molluscs do secrete substances to the exterior where they can influence symbionts in the immediate proximity is still in need of confirmation. Suggestive evidences, however, are available. Wright (1959b),for example, has demonstrated by paper chromatography that there are what he considers species-specific substances in the body-surface mucus of a number of species of snails. It is unfortunate that later (1959a) he should imply that such substances may serve as specific attractants, since no proof of this exists as far as I can determine. Another example of the ability of molluscs to secrete parasite-effecting substances has been demonstrated by Cheng et al. (1966a,b). It was found that the plasma (hemolymph) seeping to the exterior from the soft tissues of two species of oysters, Crassostrea virgirtica and C. gigas, will stimulate the cercariae of Himasthla quissetensis to encyst and thus immobilize them and render them unable to penetrate these pelecypods. It is well known that oysters and perhaps other pelecypods continuously lose blood during diapedesis (see review by Galtsoff, 1964). It is not known, however, if the diapedetic rate fluctuates and, if it does, what ambient or physiological factors influence this. Thus the secretion of chemical substances from a mollusc, which may result in chemotaxis, could also be influenced by both the environment and the host’s physiological state. An evaluation of presently available data leads me to believe that chemotaxis, operative within short distances and probably not speciesspecific, does exist in the case of mollusc-symbiont relationships. Although earlier authors who have specifically studied miracidiamollusc relationships have attributed chemotactic attraction to mucus, there is no direct evidence for this. I n fact, misquotes and failures to check the original literature have misled others to single out mucus as the only attractant-incorporating exudate. Faust and Hoffman (1934) used the loose term “ juice ”. MacInnis’s (1965) elaborate study with known chemicals is a commendable beginning in attempting to characterize the attractant; however, characterization of natural chemotactic agent(s) is still wanting.

42

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

If my conclusion that chemotaxis does occur is correct, it is necessary to examine the possible importance of this type of attraction in aiding the symbiont to find its molluscan host. Chernin and Dunavan’s (1962) investigation, which in essence expresses in quantitative terms what has been known about taxes of Schistosoma mansoni miracidia, is an excellent example demonstrating that negative geotaxis appears to be a more powerful determinant of miracidial behavior than positive phototaxis. I n nature these two taxes may be assumed to reinforce each other in the host-seeking process, although Chernin and Dunavan have carefully stated: “ However, it is now evident that these responses of the miracidium should be thought of more as tendencies than an (sic) inviolable characteristics of their total behavior.’’ The question is, are these taxes dominant over the host’s chemotactic stimulation if the forces are opposed? Etges and Decker (1963) have stated: “ There is no doubt that both light and gravity are far more powerful stimuli in determining the orientation of S. mansoni miracidia than the chemical ones produced by their molluscan host.” From these statements, and from personal experience, it would appear that if the taxes, plus other environmental factors, direct the miracidium away from its host, the chemical attractants emitted from molluscs are definitely insufficient to bring about host-symbiont contact. It is only under optimum conditions, for example in the case of S. mamoni miracidia when the combined forces of positive phototaxis and negative geotaxis bring the miracidia into the intimate proximity of Australorbis gbbratus, that the occurrence of the chemotaxis favors host-symbiont contact. The detailed analysis of schistosome-snail relationship presented above is not meant to deter the reader from marine biology. Rather, it is meant to stimulate work along these lines using marine mollusctrematode combinations. I n the marine environment, especially the estuarine, where large numbers of semi-sedentary pelecypods serve as intermediate hosts of trematodes, the presence or absence of chemotactic forces may well be academic. For example, trematodes such as Cercaria myae, which develops to the cercarial stage in N y a arenaria (see Uzmann, 1952), or Cercaria milfordensis, which develops in M y t i l w edulis (see Uzmann, 1953), would undoubtedly be swept into their molluscan hosts by the strong in-current if these possess a freeswimming miracidial stage. I have observed the uptake of Himasthla quissetensis cercariae by actively pumping oysters. The cercariae are swiftly and unquestionably carried in beyond the boundaries of the oysters’ valves by the in-current. On the other hand, if the mollusc, like oysters, does not possess a siphon, mere penetration beyond the valves need not mean successful penetration into the host’s tissues.

4. ANALYSIS OF FACTORS INVOLVED

IN SYMBIOSIS

43

Indeed, as stated earlier, cercariae of H . quissetensis, stimulated by the plasma of Crassostrea virginica or C. gigas, encyst on the exterior, specifically on the gill surfaces, and are thus prevented from penetrating the host’s tissues. A theoretical point which may be raised is concerned with the effectiveness of chemical attractants in the marine environment. Sea water, at least in certain areas, comparatively speaking, appears to be richer in organic molecules than fresh water (Duursma, 1961; Sutcliffe et al., 1963 ; Riley et al., 1964 ; Wangersky, 1965 ; and others). Much of the organic materials have resulted from the plant and animal excretions and secretions and the degradation of decaying biota. It is possible that these organic molecules may act in competition with the attractants of molluscs and thus diminish the effectiveness of the latter. It is also known that various marine animals engage in extraintestinal digestion, i.e. proteolytic secretions are poured over the food to reduce it to a semi-liquid form. For example, to cope with large food masses, many echinoderms evert their stomachs and pour proteases over the food. Similarly, the Portuguese man-of-war, Physalia, discharges ferments through the gasterozooids which adhere to the prey, the polyclad Leptoplana initiates digestion outside its body by exuding proteases through its everted pharynx over the food mass, and octopi are known to predigest their prey by discharging a protease into them (see Nicol, 1960). Thus, if the chemotactic substance is in the form of a large protein or protein-containing molecule, there is the possibility that it may be digested by various extra-intestinal proteases in sea water if the secretion of these enzymes occurs in the proximity of molluscs and during periods when symbionts are vulnerable to attraction. If such occurs, attractants would be enzymatically altered and rendered ineffective. These theoretically possible chemical influences, plus the continuous flow of sea water that is particularly noticeable in estuaries, could render chemotactic substances ineffective or at least reduce their efficiency. Hence, in considering the role of chemotaxis in the marine environment, comparable ambient chemical and physical factors should be taken into consideration when working models are designed in the laboratory. Dwelling on the question of host attraction a bit longer, it appears appropriate to raise the question as to whether some type of attraction exists when larger symbionts, such as the commensalistic and parasitic crabs of the genus Pinnotheres, find and enter the mantle cavities of marine pelecypods. Observations on the invasion of Crassostreu virginica by Pinnotheres ostreum suggest that it is not a “ hit or miss ”

44

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

process. If the invasive stage of this crab is placed in an aquarium with several species of pelecypods, it chooses Crassostrea virginica. No experimental evidence is yet available that would indica
FIQ.7. Y-tube choice apparatus used to study chemotaxis of commensal crabs. I n aquarium A is placed either the water from the glass aquarium alone or the water and host ; B contains fresh sea water. The commensal crab is put in the stem of the Y-tube a t C and the stopper is replaced. At the initiation of the test, clamps 1 and 2 are opened and so is clamp 3, thus allowing a continuous flow from each aquarium to pass over the crab. (After Johnson, 1952.)

he found an attraction between the pinnotherid crab, Dissodactylus mellitae, and the sand dollar Mellita quiquesperforata, its natural host. Johnson’s apparatus consisted of a Y-shaped tube, each arm of which was attached to an aquarium (Fig. 7). The aquarium marked A contained either the water from a glass aquarium in which the host had been maintained or the water and the host. Aquarium B contained fresh sea water. Sand was placed at the bottom of each aquarium. The commensal was placed in the stem of the Y-tube at point C after which stoppers 1, 2 and 3 were opened to allow a continuous flow of the water from tanks A and B. Each time the crab entered the stem leading to A, the trial was recorded as a positive, and if it entered the stem leading to B, it was considered negative. If the crab did not move

4. ANALYSIS OF FACTORS INVOLVED IN SYMBIOSIS

45

within 10 min, the trial was recorded as a (‘no run ”. Because of the nature of this apparatus, it would be of interest to know whether response could be elicited if the crabs were placed in closer contact with the hosts. In the case of a related species of Pinnotheres, P. rnaculatus, Sastry and Menzel(1962), by employing the circular choice apparatus of Bartel

U

FIG.8. Test apparatus dcsigned by Bartel and Davenport. Plan (below) and in section (above). (After Bartel and Davenport, 1956.)

and Davenport (1956) (Fig. 8), have demonstrated that the commensal crab is (‘capable of recognizing its hosts, Aequipecten irradians and Atrina rigida ”, i.e. they are attracted to them. It is of importance to note that Sastry and Menzel state that : The attraction of commensals t o the host scallops decreased when the hosts were not directly introduced into the radial chamber of the choice apparatus. This suggests that perhaps a spatial proximity of hosts to commensals is

46

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

necessary for demonstration of attraction [italics added] under experimental conditions, The decreased attraction could have resulted either from a gradient or a highly diffusible nature of the attractant. Sastry and Menzel have also demonstrated that if Pinnotheres macubtus is given a choice between the bay scallop, Aequipecten irradians, and the penshell, Atrina rigida, no preference for one host over another occurs. Furthermore, the crabs are not attracted to empty A. irradians shells with epiphoronts attached, thus indicating that the source of the attractant is the soft parts of the scallops. It was also found that adult male crabs are as equally attracted to their molluscan hosts as females. It is of interest to emphasize that Pinnotheres maculatus is not attracted by the empty shells of the scallop, thus differing from the relationship between Calliactis parasitica and Buccinum undatum as reported by Ross and Sutton (1961). It would thus appear that the " host factor '' or attractant can differ, depending on the specific host and symbiont. It should be mentioned at this point, although briefly, that whereas host attractants are generally believed to be chemical as the result of available evidences, there are some indications which suggest that certain physical properties may be attractive to certain symbionts. Specifically, Sasa et al. (1960) have demonstrated that the larvae of several species of parasitic nematodes will respond positively to certain concentrations of COz in the air, and Kawashima et al. (1961a) have reported the attraction of Paragonimus ohirai miracidia to slightly acidic solutions.

D. Contact injluenced by other natural taxes Since chemotaxis, as has been pointed out, is only operative within short distances, i t would appear possible that other mechanisms exist that tend to bring the symbiont and its host into the proximity of one another from greater distances. I n this regard, Laing (1937) appears to have been the first to establish the principle that symbionts may be attracted to the environment in which their hosts are found. She stated : The conclusion t o be drawn . . . is that some parasites do first seek out a particular environment, in which they afterwards proceed t o seek their hosts. The analysis of the process by which the parasite finds its host into two parts-the finding of the environment and the finding of the host in that environment-is, therefore, not merely a convenient theoretical division, but corresponds to an actual differencein the behavior of the parasite.

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47

Laing further stated : Not only, then, do some parasites find environments first and hosts later, they may often use quite different senses for the perception of the two and make quite different movements t o reach them. What those senses and movements are, however, will differ greatly with different parasites, and must be especially determined in each particular case, Wright (1959a, 1960), in considering host-finding by trematode miracidia, reiterated Laing’s theory. He suggested that three stages are involved of which the first is concerned with the location of the hoethabitat by response to physical stimuli. Subsequent to this, random movement, which ends when the parasite encounters a chemotactic zone, was also postulated. The third stage involves chemotaxis. Thus it would appear that the ‘‘ location ” of the host-habitat by a symbiont occurs prior to the operation of any chemotactic force. Not all of the mechanisms involved in host-habitat location are yet known; however, the natural taxes of both symbiont and host, where such exist, undoubtedly serve as major forces in bringing the partners into each other’s proximity. Thus, for example, the negative geotaxis and positive phototaxis of Schistosoma rnansoni miracidia mentioned in the preceding section serve to bring these ciliated larvae to the habitat of their molluscan host which a~ a rule is found in the subsurfacial region, clinging to the underside of vegetation. Although other instances of taxes among miracidia are known (Takahashi, 1927; Suzuki, 1931; Yasuraoka, 1954; Ingalls et a,?., 1949; and others), such information is lacking for most marine species. Similarly, phototaxis among certain larval parasitic nematodes has been reported (Veglia, 1915; Croll, 1965; and others) but not among marine species. Furthermore, natural taxes among other categories of marine symbionts remain essentially unexplored. It is of interest at this point to mention the studies of Welsh (1930, 1931) on tactic responses which revealed that once hostsymbiont contact is made the original response may become altered and serve to further advantage in permitting successful establishment of the symbiont. Working with the commensal ( 2 ) mite, Unionicola ypsilophorus var. huldemani, that inhabits the mantle cavity and gills of the freshwater mussel, Anodonta cataracta, he has found that the tactic responses of the mite could be reversed by a host factor. Specifically, Welsh has demonstrated that if Unionicola ypsilophorus, removed from its host, is washed free of a factor of host origin, it is positively phototactic. If the host factor is added to an aquarium containing positively phototactic mites, these immediately reversed to negative photo-

48

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

taxis. He has found that this taxis-reversing factor is relatively stable and that it resists boiling and putrefaction. As the result, Welsh has proposed that it may be a decomposition product of mucus or some other protein from the gills. Welsh (1931) has demonstrated that the taxis-reversing factor is specific. By testing materials from eight species of bivalves (Anodonta cataracta, Lampsilis radiata, Elliptio

complanatus, E. dilatatua, Sphaerium sulcatum, Cyclonais tuberculata, Eurynia iris and Ligumia fasciola) on three species of commensal mites (Unionicola ypsilophorus from Anodonta cataracta, Unionicola fossulata from Cyclonais tuberculata, and an unidentified species from Lampsilis radiata), he found that only materials from natural hosts cause tactic reversal. These interesting findings by Welsh indicate that the natural taxes of a symbiont may be altered once it makes contact with the host. I n the case of the mites studied, Welsh has suggested that: “ This reversal may be considered adaptive, for, aided by a positive chemotropism and stereotropism, it enables the mites to enter and remain within the host.” Furthermore, he considered the positive phototaxis to be an adaptive and secondarily acquired characteristic. Whether this last postulation is true or not is a moot point since Davenport (1955), speculating on this phenomenon, has stated : We have little detailed information about the early stages of these inquiline mites studied by Welsh. Obviously they must move from host to host;

otherwise, their spread would never be accomplished. One may wonder whether during larval life the mites exhibit that positive response t o light which enables them to wander more widely. If so, one may suppose that subsequently internal changes take place which “ trigger ” the reversal of response under the influence of the host-factor. This mechanism, plus a positive chemotaxis appearing at the same time either t o the same or t o another host-factor, would insure colonization of hosts when the larvae are in proximity t o a mussel bed. Thus, if Davenport’s reasonable postulation is correct, then the positive phototaxis of the mites should not be considered as a secondary adaptation but as a primary characteristic present since larval stages. On the other hand, the negative phototaxis brought about by the host-factor could be considered a later adaptation which enables the adult to live in a commensalistic relationship with the molluscan host. However, Davenport (1955), in my opinion, has correctly pointed out that the rapid reversal from positive to negative phototaxis suggests that “ this ability to change behavior suddenly at the first experience of external stimuli from the host may be firmly fixed in the hereditary constitution of symbionts ”.

49

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

It still remains unknown just how widely spread such host-factor stimulated reversals of taxes are in nature although Welsh (1930) has stated : (‘Doubtless many parasitic animals become conditioned to stimuli from the host and show adaptive reactions quite different from their primitive responses.” E. Xelectivity of symbiont Although host-selection by marine symbionts remains t o be studied, the principle of selectivity of hosts by symbionts has been established in studies concerned with other types of symbiotic relationships. As Davenport (1955) has pointed out, true predators among arthropods and vertebrates with specialized food habits may locate their prey by highly precise behavior involving, a t least in part, olfactory recognition of the prey. Although this type of precise recognition during predation has been demonstrated among certain molluscs (Stehouwer, 1952 ; Kohn, 1961 ; Prings and Frings, 1965) and nudibranchs (Braams and Geelen, 1953), its importance in symbiotic relationships has been little studied except in the case of a few parasitic insects. Such insects, often termed parasitoids, are in fact parasites that more often than not gradually kill their hosts. For this reason, some have chosen to refer t o them as ‘(internal predators ”. However, in following my earlier interpretation (Cheng, 1964b), I have chosen to consider them as true parasites since they are obligatorily dependent upon their hosts. That they do cause the death of their hosts should be considered as a secondary factor which they share with other pathogenic and/or lethal parasites. That host-selection does occur implies that the symbiont is capable of recognizing its natural host prior to establishment. As discussed above (Section I, C) doubt still remains as to whether the selection process occurs with all symbionts. Certainly the results of Sudds (1960) with trematode miracidia indicate that selection prior to penetration does not necessarily occur. Nevertheless, there is evidence to support the belief that among certain symbionts of more complex organization, particularly among the arthropods, host-selection does occur. In addition, there appears to be host-selection, a t least host-preference, among certain marine polynoid polychaetes (Davenport, 1950, 1953a,b, 1954; Davenport and Hickok, 1951),and sea anemones (Davenport and Norris, 1958 ; Ross, 1965). Among parasitic insects two classical examples may be cited. Laing (1937), by employing a choice-apparatus, has demonstrated that the chalcid Alysia manducator may be attracted by olfactory means t o the environment of its host which is the blowfly larva. The parasite is attracted, as the result of a powerful chemotaxis, to some factor(s) A.M.B.-5

5

50

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

in decomposing meat. Thus, although A . manducator is not directly attracted t o its host, its selective preference for rotting meat, which is its host’s habitat, indirectly aids in its selection of the preferred host. Another example of host-preference among parasitic insects has to do with the parasitic wasp Nerneritis. Thorpe and Jones (1937) have demonstrated that if the eggs of Nemeritis are artificially introduced into an unnatural host, the wax moth, Meliphora, instead of its natural host, Ephestia, the resultant adults give a strong olfactometric response to Meliphora when given a choice between it and a blank. These workers, however, were not successful in attempts to demonstrate a preference of conditioned Nemeritis for Meliphora over Ephestia. It is nevertheless of interest to point out that wasps which develop in the unnatural host become sufficiently conditioned so as to recognize the new host. This model is useful in considering the origin of new hostsymbiont associations. It is conceivable that many such associations arose when during a brief free developmental period in the life of the symbiont it happened to encounter a potentially new host in some niche different from that of its natural host. If the new host produces some factor(s) related to, but not identical with, that with which the symbiont has been associated in the natural host, it is conceivable that an effective conditioning to the new host factor(s) may bring about a change in host-preference, aided perhaps by a shortening or loss of the free stage, and resulting in subsequent generations becoming genetically isolated in or on their new host. From then on, the course of evolution of the symbiont would be controlled by that of its new environment, the new host (Davenport, 1955). The mechanisms underlying the natural combination of new associations may not always be so simple; nevertheless, this hypothetical model as presented by Davenport is worthy of serious consideration. It is of interest t o mention a t this point that Dethier (1954),in considering the feeding preferences of insects, has stated : The crucial problem is whether a phenomenon like olfactory conditioning can bring about sympatric splitting of a single population into two nonbreeding populations. It is clear that in the absence of effective isolating mechanisms there would be free hybridization between individuals with different . . . preferences so that the preferences fostered by olfactory conditioning could never become sorted out genetically. . . . Thus, in evaluating the significance of olfactory conditioning as an effective factor in establishing populations with new . . . preferences, a question of fundamental importance arises, i.e. whether olfactory conditioning by itself can lead to any kind of isolation . . . or whether the isolation must be interjected from another quarter.

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51

Since in Davenport’s model several additional favorable pressures have been introduced, Dethier’s reservations can be removed. The examples and theoretical considerations presented above are again not meant to detract from our main theme of marine symbioses. On the contrary, these are presented in hopes of stimulating thinking and perhaps suggesting new avenues of approach in its study. One of the few studies concerned with host-preference by a marine symbiont for a mollusc was reported by Ross (1965), who studied the relationship between the sea anemone, Stomphia coccinea, and the ribbed mussel, Modiolus modiolus. Specimens of Stomphia coccinea collected by dredging in San Juan Channel of Puget Sound usually are found attached to shells of Modiolus modiolus. This suggests that the epiphoretic anemone prefers settling on these pelecypods. This led to experiments in which eighteen anemones were induced to swim by placing them in contact with the starfish Dermasterias imbricata. This swimming reaction of Stomphia coccinea to Dermasterias imbricatn and other starfish had been discovered earlier by Yentsch and Pierce (1955) and Sund (1958). After the anemones ceased swimming, each specimen was placed in a separate bowl containing flat stones and a live Modiolus modiolus, with an ample area of glass in between. The anemones were arranged so that they lay on the glass, with the tentacles of six and the pedal disks of another six touching the molluscs’ shells. The remaining six anemones were touching stones. One hour later, eleven of the twelve anemones touching shells had settled on these. Of those remaining, five had settled on glass and two remained unattached. Sund (1958) has reported that when Stomphia comes out of its “ post-swimming torpor ”, usually it quickly settles on any available surface by attaching its pedal disk little by little to that surface. ROSS has found that those which settle on M . modiolus behave differently. Some bent over shells with their tentacles and oral disk extended outwards like a n umbrella (Fig. 9A). Others leaned on the shells with a few tentacles in contact. This tentacular-oral activity was followed by movements of the basal region. The latter was either extended horizontally like a huge plate which was turned towards the shell or swelled up into a hemisphere which was pushed towards the shell. The events beginning with the tentacular response and ending with the movement of the basal disk to the shell only took about 1-2 min. It was also noted that once the pedal disk became attached, the anemone generally slides across the shell surface towards a final resting position (Fig. 9B). Subsequent experiments revealed that once the pedal disk makes contact with the shell, the tentacles and oral disk become detached and the animal straightens itself. If the pedal disk contact is not made

52

MARINE MOLLUSCS A S HOSTS FOR SYMBIOSES

FIG.9. Reartion of Stowkphia coecineu t o Modiolus rnodiolus. A , Stomphiu bent over shell ; B, Stomphia in resting position on Modiolus shell. (Drawings of photographs by Ross, 1965.)

4. ANALYSIS OF FACTORS INVOLVED IN SYMBIOSIS

53

during the initial attempt, subsequent trials are made until successful. It has also been discovered that S. coccinea, which had settled on other surfaces up to about 3 h earlier, transferred readily to M . modiolus shells. During this process, the tent,acles and oral disk again make the initial contacts with the shell, followed by release of the pedal disk from the other surface and its attachment to the mussel shell. Anemones which had settled on other surfaces for more than 3 h revealed no tendency to transfer to shells. Ross also has determined that the behavior is not specific for M . modiolus. The shells of other pelecypods, including Pecten, will evoke the same behavior. Furthermore, empty shells also will evoke the same response. Ross considers this behavior as ‘‘ purposive ” and indicative of preferential settling. Although the nature of the attraction remains undetermined, the finding by Ross (1960) and Ross and Sutton (1961) that the periostracum of Buccinum will attract Calliactis (Section I, C) should be borne in mind.

F. Injlucnce of nature of substrate I n the case of those symbionts which can only arrive a t their hosts via migration across a solid or semi-solid substrate, the nature of the substrate is often influential in determining the host-symbiont contact. The results of Wikswo relative to the influence of glass versus sand as a substrate for the migration of Arctonoe vittata has been cited in Section I, C. I n brief, she has found that the nature of the substrate does influence the symbiont’s migration. Studies of this nature involving marine molluscan hosts, or any mollusc for that matter, as far as I can determine, are still wanting. I n addition to influencing the symbiont’s migration towards its host, the nature of the substrate may in some instances influence the symbiont’s metamorphosis. This principle was first recognized by Mortensen (1921) while working with echinoderms. He noticed that metamorphosis may not always take place a t a fixed stage during development and that the presence of certain factors in the environment, however, will stimulate larvae to metamorphose. Thus it follows that in the case of certain symbionts the physical and chemical nature of the substrate governs metamorphosis and thus indirectly governs the successful contact with their hosts, since in most instances a specific stage of development must be reached before the symbiont can establish itself on or in its host. This has been convincingly demonstrated by Bourdillon (1950) who studied the settling and metamorphosis of the commensal mussel M o d i o l a k and its subsequent contact with its

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

ascidian host. Again, parallel studies involving marine molluscan hosts are still not available. It should also be mentioned a t this point that the nature of the substrate may influence not only metamorphosis but also the initial settling of the larvae of marine symbionts. The investigations of Wilson (1937, 1948, 1952) on Notomastus, Ophelia, and other marine larvae, as well as the findings of Knight-Jones (1963) relative to Balanus balanoides and other barnacles, serve as examples to illustrate how the nature of the substrate can influence the settling of marine invertebrate larvae. These investigators have demonstrated that settling larvae of special habit are sensitive to minute differences in their environment. Furthermore, they may actually sample the substrate before settling, being either attracted or repelled by minute physical or chemical differences.

11. ESTABLISHMENT OF TIIE SYMBIONT Successful establishment of a symbiont in or on its host is defined as the ability of the symbiont to become located a t a suitable site and to succeed in continuing its normal physiological and reproductive processes. This implies that the symbiont, has entered an oftentimes delicate but nevertheless physiologically balanced state with its host. I n the case of commensals and epiphoretic organisms, successful establishment depends upon what the host environment (the microenvironment) can provide to shelter it from the surrounding environment (the macroenvironment). I n the case of parasites and mutualists, the relationship is more complicated since it involves overcoming the natural internal defense mechanisms of the host. I n other words, itJ has to overcome the resistance of the host to an invading organism. I n the earlier literature, a host's resistance was considered as the reciprocal of its susceptibility to the invading organism (Falk, 1928). A highly susceptible organism is thus one lacking in resistance. More recently Schneider (1951) and Read (195810) have correctly pointed out that this is too simple an expression. Susceptibility and resistance, from the modern viewpoint, must be considered distinct and separate biological attributes. Schneider has chosen to define " susceptibility " and " resistance " in nutritional terms. Specifically, he has defined as " susceptible factors ') those factors in the nutrition which, when withheld from the host,decrease the extent or effect of an infection and, when supplied increase the effect. He has defined " resistance factors " as those nutritional factors which, when withheld, increase the extent of the infection and, when supplied, decrease the effect. Although these are satisfactory definitions, they are only useful when considering the

55

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

relationship between nutrition and invasion by mutualists, or even more so by parasites. Read has defined susceptibility as “ a physiological state of the host in which the parasite is supplied with its life needs ” and insusceptibility as “ the state in which these life needs are not satisfied.” He has defined resistance as “ those alterations of the physiological state of the host which represent a response to previous or present experience with the parasite, or a chemically related entity.” According to the broader definitions given by Read, we are really concerned with susceptibilityinsusceptibility and varying degrees of resistance when we consider establishment of a symbiont. When one deals with relationships between symbionts and hosts, invariably the terms “ normal ” or “ compatible ” hosts and “ abnormal ” or “ incompatible ” hosts are mentioned. Since “ normal ” and “ abnormal ” connote meanings beyond that implied within the realm of host-symbiont relationships, the alternative terms compatible 7 7 or “ natural ” and “ incompatible ” or “ unnatural ” are preferred. In using these adjectives, host-specificity is implied although the degree of specificity may vary. Host-specificity7 in the light of Read’s broad definitions, may be expressed as a manifestation of both susceptibility and/or resistance, primarily the former. The role of resistance in determining host-specificity naturally excludes acquired resistance, since this type of resistance, for example, the development of specific antibodiesas the result ofan initial contactwithaparasite, may bring about complete immunityto a homologous parasite in asnbsequent contact, and hence cannot be considered as naturally occurring incompatibility. On the other hand, innate resistance may be in whole or in part responsible for incompatibility. It is thus apparent that one of the first, if not the initial, confrontation facing a symbiont which has successfully made contact with its host is to overcome resistance. It is generally recognized that basically resistance can be of two types : physiological, including genetical, and behavioral, including mechanical. ((

A. Physiological resistance This type of resistance is the one defined by Read (1958b) and has been cited above. In order to analyze critically what is known about physiological resistance in marine molluscs, in all molluscs for that matter, it is necessary to divide this type of resistance into two subcategories. (1) I n defining parasitism it has been stated that the parasite is metabolically dependent on its host and that the metabolic dependency

56

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

may be in the form of nutritional requirements, digestive enzymes, development stimuli, or control of maturation. Among these, the contribution of developmental stimuli by the host is known to be of critical importance during the initial stages of successful establishment by a parasite. Thus alterations in the quality or quantityof potentially important stimulatory substances could effectively serve as a form of physiological resistance, I n conjunction with this, the secretion of inhibitory substances by the host could prevent establishment by the parasite. (2) I n addition to physiological factors which may inhibit or deter normal development of the parasite, another set of factors may have the same effect. These could be considered as “ physiological factors ” in the broad sense ; however, these are generally considered as distinct and are termed “ defense mechanisms.” I n order to facilitate the presentation of a clearer review, defense mechanisms are considered as distinct from other types of physiological resistance although without doubt this is to some degree artificial. 1. Developmental stimuli

The development of the science of symbiosis has been such that those concerned with commensalism and phoresis in the marine environment have independently followed one avenue of development and most of their published results have been of a descriptive and ecological nature, while those concerned with parasitism and mutualism have examined primarily the relationship between definitive vertebrate hosts and their parasites or mutualists. The studies of Cleveland, Hungate, Kirby, and others on the mutualistic flagellates of insects are exceptions as are the studies of Richards and Brooks on the bacteroid mutualists of roaches. As a result, practically nothing is known about developmental stimuli contributed by marine molluscs, or any mollusc, to their symbionts. Nevertheless, such must exist. It is known from classical life history studies of parasites that molluscs, except in a few rare cases, serve as the first or only intermediate host of digenetic trematodes and in some instances of certain cestodes and nematodes. It is also known that trematodes undergo both morphogenetic change and delayed polyembryony (asexual reproduction) and nematodes undergo molts while in their molluscan hosts. Practically nothing is known about host factors which govern or influence these essential developmental phases other than certain aspects of nutritional requirements which are considered in a later section (p. 80). Thus information comparable to what is known about the influence of vertebrate hosts on the excystment of larval tapeworms (see review by

4. ANALYSIS OF FACTORS INVOLVED IN SYMBIOSIS

57

Read and Simmons, 1963) or that of vertebrate hosts on the molting of nematodes (Rogers, 1960, 1962) remain unexplored. There is some evidence that molluscs do provide some type of stimulus which causes the shedding of trematode miracidial epidermal plates. Campbell and Todd (1955b), for example, have reported that the miracidium of Pascioloides magna will shed its ciliated epidermal plates after a short contact with tissues of its molluscan host and will metamorphose into a sporocyst in vitro. As Dawes (1960) has pointed out, other trematode miracidia also shed their ciliated epidermis prior t o penetration of their molluscan hosts, and it is possible that it is the stimulus contributed by the mollusc which initiates the shedding process. It should be pointed out, however, that certain species of trematodes, such as Schistosoma mansoni, apparently do not shed their ciliated epidermis prior to penetration. Maldonado and Matienzo (1947) have demonstrated that, in the case of S. mansoni, shedding of the plates does not occur until the miracidium has penetrated the snail host. According to Lengy (1962), discharge of the contents of the penetration glands and apical gland of Schistosoma bovis miracidia is also stimulated by contact with the molluscan host. Similarly Cheng (1967) has demonstrated that when Fasciola gigantica miracidia are exposed t o the plasma of Galba ollula, the natural molluscan host in Hawaii, and Helisoma duryi normale, an incompatible host, not only is the secretion of some substance, perhaps the lytic enzyme, observed, but the apical papilla of many of the miracidia also becomes invaginated to form a terminal cup. This finding suggests that the stimulatory factor(s) is not limited to the plasma of compatible hosts. Although the chemical nature of the stimulants contributed by the host which elicit shedding or glandular secretion is not yet known, these fragmentary pieces of information suggest that molluscs do contribute morphogenetic factors. It is also known that the plasma of certain molluscs will enhance the establishment of certain trematode larvae (Cheng, 1963a). Although in this instance the sera (plasma or hemolymph) do not bring about morphogenetic changes, they do serve to activate infectj ve cercariae. Specifically, it has been shown that the sera (plasma or hemolymph) of five species of freshwater molluscs, Viviparus malleatus, Helisoma trivolvis, H . anceps, Physa gyrina and Musculium partumeium, will activate the normally quiescent cystocercous cercaria of Gorgodera amplicava. The activation has been shown not t o be due to the pH of the plasma but to some component fraction. I n nature, once the quiescent cercaria becomes ingested by the molluscan second intermediate host, it is believed that the plasma seeping into the mollusc’s

TABLEV. COMPARISON OF

Molluscan species

THE

No. of specimens pooled

STIMULATORY EFFECTS OF UNTREATED PLASMA OF SEVEN SPECIES OF MOLLUSCSON CYST FORMATION OF Himasthla quissetensis (After Cheng et al., 1966b.) Size of molluscs (length of shell

in cm) T . philippinarum M . demissus M . edulis M . mercenaria M . arenaria C . virginica c. gigas

5.1-5.8 7.4 4.7-6.5 6.5-7.2 4.1-5.2 7’0-12.0 12.1-14.0

Plasma protein

Plasma

COnCn

PH

(g/100 m u 1.95 1.85 1.80 2.05 2.05 2.25 2.00

6.55 6.65 7.31 7.40 6.70 6.35 6.51

No. of

trials

No. of cercariae used in each trial 70 50 52 20 100 46 20

Mean time at which 5004 of cercariae encysted (min after contact) 16 40 30 53 12 21 9

f 3.2 & 2.5 & 3.0 4.2 & 2.5 3.4 & 2.5

*

cz z

M

4. ANALYSIS O F FACTORS INVOLVED IN SYMBIOSIS

59

alimentary tract activates it, thus enabling it to migrate to the appropriate site, become attached, and encyst as a metacercaria. Relative to marine molluscs, it is known through the work of Cheng et al. (1966b) that the plasma of several species of pelecypods, namely Tapes philippinarum ( = T . semidecussata), Mytilus edulis, Modiolus demissus, Mercenaria mercenaria, M y a arenaria, Crassostrea virginica and C. gigas, will induce the encystment of the cercaria of Himasthla quissetensis. It is believed that in nature once the cercariae succeed in penetrating one of these molluscan hosts, contact with plasma within the open circulatory system induces encystment, i.e. the secretion of

FIG.10. Cercaria of Himasthla quissetensis encysted between the gill and palp of Crassostrea. C, Enveloping cyst.

cystogenous material, and thus the cercaria develops into a metacercaria. It should be pointed out, however, that encysted metacercariae were not found within the body of C. gigas. A possible explanation for this is that the plasma of this oyster has the fastest stimulatory effect on cercariae (Table V), and that plasma seeping into the mantle cavity of C. gigas causes the encystment of cercariae, thus immobilizing them, before they successfully penetrate the soft tissues. Indeed, metacercariae were found encysted on the gill and palpal surfaces of this oyster (Fig. 10). Another aspect of growth and developmental stimuli of host origin has t o do with the possible role of the host’s hormones. Although still

60

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

uninvestigated among parasitized marine molluscs, studies on freshwater gastropods parasitized by larval trematodes suggest that such hormones may influence the growth and differentiation of the intramolluscan trematode larvae (Szidat, 1959 ; Meade and Pratt, 1966). 2. Internal defense mechanisms

Our understanding of internal defense mechanisms of molluscs is a t a relatively primitive state as compared to what is known about insect defense mechanisms among the invertebrates. This is particularly true in the case of humoral factors (see reviews by Huff, 1940 ; Bisset, 1947 ; Stauber, 1961 ; Cheng and Sanders, 1962 ; Tripp, 1963). It is not surprising that much of what is known about defense mechanisms in molluscs has come from studies on commercially important marine species, especially the American oyster, Crassostrea virginica. As the result of the late Dr Thurlow C. Nelson's interest in oyster research a t Rutgers University, many of his students have continued this line of investigation. Among these, Dr Leslie A. Stauber, Dr Harold H. Haskin and their students have pioneered in studying defense mechanisms in C. virginica, especially the role of leucocytes. In order t o present a more complete picture of defense mechanisms in molluscs, it is necessary that pieces of information gained from investigations on fi-eshwater species be interjected where appropriate to suggest some specific mechanism or improve upon our understanding of the total picture. Such may in addition serve to stimulate comparable studies on marine species. Furthermore, in order to facilitate the presentation, molluscan defense mechanisms, in following the general pattern of Stauber (1961) and Cheng and Sanders (1962), have been divided into subcategories, namely, phagocytosis, encapsulation, leucocytosis, nacrezation, and humoral factors. a. Phagocytosis The role of phagocytes in defense reactions of invertebrates and vertebrates has been recognized ever since the original studies of Metchnikoff and others (Metchnikoff, 1901). Actually Haeckel (1862) was the first to discover phagocytosis. He injected particulate dyes into molluscs so that the distribution pattern of their vascular systems might be determined. I n so doing, he accidentally discovered that leucocytes" phagocytized the dyes. Following these studies were those of De

* Since the various types of leucocytes found in the majority, if not all, molluscs remain uncertain, the terms " leucocyte ", '' amoebocyte " and " phagocyte " me used interchangeably in this paper. They all scrve to designate the free leucocytes in molluscs which are amoeboid and are capable of phagocytosis.

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61

Bruyne ( 1 893, 1895) who also investigated phagocytic activity by molluscan leucocytes. Cuenot (1914), in connection with his report on phagocytosis in molluscs, particularly the amphineuran Acanthochites discrepeus, reported that ink particles introduced into the blood-stream are removed via phagocytic activity of blood cells. His primary concern, however, was with the immediate removal of the ink particles rather than with their ultimate disposal. Other investigators (Vonk, 1924; Yonge, 1926; Takatsuki, 1934; Nelson, 1933) have described the role of leucocytes in the digestive functions of various species of oysters. Modern experiments on the role of molluscan amoebocytes began with Stauber (1950) who experimentally injected India ink intracardially into Crassostrea virginica and traced the fate of the ink particles. He found that, starting as soon as 15 min after injection, the ink suspensions will agglomerate and produce emboli which virtually occlude the arterial vessels of the viscera, mantle and adductor muscle. Subsequently, the ink particles are phagocytized by mobile phagocytes and are distributed to all parts of the oyster with concomitant resolution of the emboli. Eventually, around the 8th day after injection, the ink particles are eliminated from the mollusc by the migration of ink-laden phagocytes through the epithelial layers of the alimentary tract, digestive diverticula, palps, mantle and pericardium from whence the.y are voided to the exterior. It is of interest to note that the epithelia of the gonaducts, nephridia, and shell-forming mantle were not found to be routes of elimination. Subsequent studies by Tripp (1958a,b, 1960), who experimentally injected normal erythrocytes of the rabbit, weakfish, duck, duck erythrocytes infected with the avian malaria parasite Plasmodium lophurae, bacterial spores, vegetative bacteria, and yeast cells into Crassostrea virginica, showed essentially the same pattern of phagocytic activity. However, Tripp, as the result of employing a greater variety of foreign particles, has been able to show that digestible particles (erythrocytes, vegetative bacteria, some yeast cells) are eliminated via the migration of particle-laden phagocytes across epithelial barriers as well as being digested intracellularly in phagocytes. Non-digestible particles (malarial pigments, most yeast cells, bacterial spores) are eliminated almost completely by migration of phagocytes across epithelial barriers. He also has demonstrated that the plasma (hemolymph) of the oyster is bactericidal. Another of Stauber’s students, Feng (1959, 1965a), also worked on this problem. He has found that soluble starch, bovine hemoglobin, human serum albumin, diphtheria antitoxin, and rhodamine-labelled proteins, all nonparticulate materials, will become pinocytized and are either digested

62

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

intracellularly in phagocytes or removed through epithelial layers. Later, Tripp (1961b), exploring the existence of these phenomena in the only gastropod studied to date, Australorbis glabratus, introduced by injection or implantation, yeast cells, bacteria, chicken erythrocytes, carmine particles, willow pollen, polystyrene spheres, and homologous and heterologous tissue implants into the cephalopedal sinus of the snail. He reported that particles small enough to be phagocytized are removed from the tissues by migration of host amoebocytes to the exterior through epithelial layers, by intracellular degradation if the particles are digestible, or by retention within tissue phagocytes. Particles too large to be phagocytized (pollen and polystyrene spheres) are enca-psulated in walls of fibroblasts. I n the case of tissue implants, fresh homologous tissues elicit no response, fixed homologous tissues are encapsulated, and fresh heterologous tissues are a t first also encapsulated but progressive atrophy of cells ensues and after 26 days only a poorly organized fibrotic mass remains. The implant was by then only surrounded by scattered groups of fibroblasts and the implant tissue itself becomes invaded by fibroblasts and muscle cells which had migrated from adjacent host connective tissues. Other instances of phagocytosis in molluscs are known as the result of histopathological studies. I n brief, Mackin (1951) has reported phagocytosis of Dermocystidium marinum, a fungal parasite of oysters ; Mackin et al. (1952) have reported phagocytosis of the flagellate Hexamita by oyster leucocytes ; and Haskin (personal communication) has found phagocytized MSX, now known as Ninchinia nelsoni, a parasite of Crassostrea virginica which is believed to be the major mortality factor among oysters along the mid-Atlantic coast of the United States. The fates of these phagocytized parasites are not known. I n addition to these observations, Mikhailova and Prazdnikov (1961, 1962) have noted the occurrence of phagocytosis as an internal defense mechanism against invading materials in Mytilus edulis. Bang (1961) has contributed an interesting study of the mechanics of phagocytosis by studying C. virginica leucocytes in vitro using both phase-contrast and electron microscopy. By placing various freshly grown marine bacteria in a drop of freshly obtained oyster blood, he observed the process of phagocytosis. He discovered that although phagocytosis of marine bacteria is readily demonstrable in most instances, it is by no means an invariable phenomenon. When it occurs, actual phagocytosis is usually preceded by a massive sticking of the bacteria to the amoebocyte so that the amoebocyte resembled a ‘< porcupine ”. This is followed by migration where the bacteria become trapped between the fine filamentous pseudopods of the amoebocyte

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

63

and gradually are engulfed as the web-like leucocytic ectoplasm flowed and filled in between the pseudopods. The curious anchoring or sticking of bacteria to amoebocytes prior to actual phagocytosis is explained by Bang as the result of a fortunate electromicrograph. He has demonstrated the unipolar flagellum of a bacterium wrapped around an amoebocyte’s filamentous pseudopodia. This explains the continual tugging and jerking at an invisible “ anchor ” when observed under the light microscope. Thus, at least in this case, the sticking of the foreign particle (bacterium) to be engulfed to the amoebocyte appears to be a mechanical process. It is of interest to note that Bang has found that not all bacteria are phagocytized. I n some instances amoebocytes were observed to approach bacteria with their filamentous pseudopods but then they either reversed their flow or turned aside. This process was observed for several hours and no phagocytosis was observed. The cause of this unusual behavior pattern remains unknown. Bang stated that “ . . . it remains likely that there is an undiscovered factor important in phagocytosis which is responsible for this variation.” The studies cited above, particularly those by Stauber’s group at Rutgers University, convincingly demonstrate the efficiency of molluscan phagocytes as an internal defense mechanism. However, it should be pointed out that a few reports are available which suggest the ineffectiveness, i.e. non-destruction, of phagocytes against certain invading organisms. For example, Michelson (1961) has stated that in planorbid snails, acid-fast bacteria can multiply within phagocytes and presumably can be carried by them to uninfected tissues. Later (Michelson, 1963b), he reported the absence of cellular response in aquatic pulmonate snails parasitized by certain microsporidea. Pan (1956) has also shown the apparent inability of Australorbis glabratus phagocytes to cope with yeast-like organisms found in the nerve cells and amoebocytes of naturally infected hosts, and Mackin (1962) has reported the lack of cellular reaction in Crassostrea virginica, including phagocytosis, to a mycelial parasite and to the sporozoan Nematopsis although in the latter case Feng (1958) has suggested that C. virginica can rid itself of this parasite. Feng based his conclusion on experiments in which he transplanted oysters with high and low initial infections to areas of low and high infections respectively and found that the transplanted oysters attained the characteristic level of infections of native oysters in that area. He concluded that presumably “ a dynamic equiIibrium of elimination and reinfection of the parasite was reached. There is no direct evidence that Nematopsis spores can be eliminated by phagocytes.

64

M A R I N E MOLLUSCS AS HOSTS FOR SYMBIOSES

Little is known about environmental parameters which influence molluscan phagocytic activity. As Stauber (1950) has pointed out, it is to be expected that in molluscs (he referred specifically to the oyster, but the same undoubtedly holds true for all molluscs), which are poikilothermic animals, the degree of activity of phagocytes would be affected by temperature changes. I n his experiments with India ink cited above, he demonstrated that a t temperatures ranging from 12 to 2 1"C, ink-laden phagocytes were observed migrating across epithelial layers after 8 days. With the temperature maintained as 21"C, Tripp (1958a) has shown that phagocytes enclosing introduced erythrocytes migrated through epithelia in 2 days. It is thus evident that temperature could influence the rate of phagocytosis and discharge ; however, critical studies, utilizing identical introduced material, should be conducted to study the influence of various environmental parameters. b. Encapsulation Besides phagocytosis, encapsulation appears to be a rather common form of molluscan defense mechanism. This mechanism is defined as the surrounding of the foreign material by connective tissue elements and/or cells which may or may not include leucocytes (Fig. 11). The occurrence of encapsulation around endoparasites of molluscs has

FIG.11.

Metacestode of Tylocephalurn encapsulated in Leydig tissue of Grassostreci wirginica. M , Metacestode. (After Cheng, 1966a.)

65

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

been randomly reported in the literature, more or less as afterthoughts. A survey of the literature has revealed the following reports. Mackin ( 1951) has reported that in Dermocystidium-infected Crassostrea virginica abscesses are formed of which the outer margins are composed of phagocytes, other hemocytes (?), and some fibrous connective tissue. Newton (1952, 1954), working with experimental infections of Puerto Rican and Brazilian strains of Australorbis glabratus by Puerto Rican Schistosoma mansoni, has found that the Puerto Rican snails, when exposed to miracidia, yield 95%infection while none of the Brazilian snails become infected. Histological examination of the Brazilian snails has revealed a rapid tissue reaction, occurring within 48 h, where there are marked cellular infiltrations around the miracidia. This is followed by a fibrotic type of encapsulation and degeneration of the parasites. Studies on the susceptibility of the progenies, resulting from crossing the Puerto Rican and Brazilian strains of Australorbis glabratus, have revealed that the susceptibility, i.e. the presence and efficiency of the encapsulation phenomenon, is genetically controlled, involving several factors. Inheritance, however, does not appear to be the sole determining factor, since Newton (1953) has also been able to demonstrate that young snails (1-4 weeks old) of the Brazilian strain are susceptible and will sustain the growth of Schistosoma mansoni larvae of the Puerto Rican strain. Subsequently, El Gindy (1954) and Moore et al. (1953)have confirmed that the susceptibility of snailsis influenced by age although apparently this does not hold true in all species (see Stunkard, 1946 ; Abdel-Malek, 1950 ; Kagan et al., 1954). Brooks (1953) has also reported the occurrence of encapsulation in unnatural hosts. It was reported that 8. mansoni miracidia will penetrate A . glabratus, its normal host, and Tropicorbis havanensis, an unnatural host, in approximately the same number ; however, in T. havanensis, encapsulation of the majority of the miracidia, involving cellular proliferation and infiltration, occurs within 24 h and the parasites die within 3 days. As the intramolluscan stages of trematodes are generally believed not to elicit encapsulation in their natural hosts (see review of pathological changes in trematode-parasitized molluscs by Cheng and Snyder, 1962a), Sudds (1960), utilizing this assumption as his working hypothesis, has tested the compatibility or incompatibility of four species of schistosomes (Trichobilharzia elvae, T. physellae, Schistosonaatium douthitti and Schistosoma mansoni) with twenty-six species of gastropods. He has found that the four species of miracidia will attempt to penetrate all of the natural hosts and the majority of the unnatural hosts. Subsequent histological examinations revealed that miracidia in natural hosts develop normally, without A.M.B.--G

0

66

M A R I N E MOLLUSCS AS HOSTS FOR SYMBIOSES

host cellular reactions. However, in the four species of unnatural hosts into which specific miracidia successfully penetrated, severe host-tissue reactions occurred in certain host-parasite associations. These were in the form of encapsulation by cells and fibers. I n others, no cellular responses were apparent but the parasite did not develop normally, eventually dying, thus suggesting the occurrence of some type of innate humoral response. Mackin (1961), in an abstract, has reported that the entire encapsulation complex, i.e. host cells and foreign material, in oysters may be discharged through the external epithelium. He did not present any experimental data for this, and until convincing evidence becomes available, this type of mass elimination must be doubted. I n the same abstract, Mackin has reported that leucocytes which contribute t o encapsulation will later form epithelial tissue surrounding the zone of infection. Again no experimental evidence was provided, and since this concept appears to be in direct contradiction to the evidences of others cited below, it cannot be accepted a t this time. The works of Newton, Brooks, and Sudds mentioned above wouid suggest the complete absence of the encapsulation process surrounding larval trematodes in their natural hosts. This is true if conspicuous massive encapsulations are sought for ; however, Cheng and Cooperman (1964) have demonstrated that if Helisoma trivolvis, the natural host, becomes heavily infected with Glypthelmins pennsylvaniensis larvae, sporocysts and cercariae escaping from the hepatopancreas will invade and continue to develop in the host’s reproductive system and auxiliary glands. When found in the albumin gland, encapsulation will occur although the capsule is generally weak, being comprised of a thin layer of connective tissue fibers. Similarly, escaping cercariae trapped in the snail’s foot musculature will become encapsulated by myofibers and connective tissue fibers. I n neither instance, however, are the parasites killed. The lack of host cellular responses elsewhere in the host’s body caused Cheng and Cooperman t o state : From the instances of encapsulation observed, it is now evident that only certain types of host tissues are capable of encapsulation. In this instance, encapsulation occurs only when connective tissue and myofibers are present. This observation is upheld by the fact that host-elaborated envelopes surrounding the larvae of G. pennsylvaniensis are only in the albumin gland, which possesses a connective tissue tunica, and in the foot, which includes muscle fibers and connective tissues. This concept has been strengthened by the results of Cheng et al. ( 1966a) who experimentally introduced known numbers of Himasthla quissetensis cercariae into eight species of marine pelecypods and

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

67

studied both metacercarial recovery rates a n d host responses. It was determined t h a t t h e nature of the outer metacercarial cyst wall, which actually represents host tissues responding t o the presence of the parasite, differs, depending upon the site of infection, although in each instance leucocytes are present (Fig. 12). We concluded t h a t : These data suggest that the composition of the outer wall, which represents the host’s reaction t o the parasite, was dependent upon the availability and nature of the surrounding host tissues.

FIG.12. Uimasthla quissetensis metacercaria encysted in foot musculature of Ensis directus. Note the host’s leucocytes surrounding the parasite. (After Cheng et al., 1966a.)

Furthermore, we stated : This is in contrast to the finding of Bogitsh (1962) who stated that in fish parasitized by the metacercariae of Posthodiplostomum minimum (the reaction) cells arise from a common source. . . (and) are derived from the reticulo-endothelial system of the host, and that this phase of the encapsulation process follows the typical vertebrate response to inflammation. This difference is most probably due to differences in the reaction mechanisms between a vertebrate (fish) and an invertebrate (mollusc). It should be pointed out t h a t among the eight species of pelecypods used by Cheng et al., at least one, M y a arenaria, is generally considered t o be the natural host, yet encapsulation, although not resulting in the parasite’s death, does occur. In the same paper, the stimulatory agent(s) for the host’s reactions was sought for a n d evidence indicates

68

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

that the inner metacercarial cyst wall, secreted by the parasite, is that agent. The chemical basis remains to be determined. The concept that the absence or presence of a t least the fibrous elements (connective tissue fibers and myofibers) of the capsule is dependent only on their availability in the immediate environment is not contradictory t o Pan’s (1963) and Tripp’s (1963) belief that leucocytes may come from more distant sources and even that under certain conditions, fibroblasts and epithelial cells may transform into leucocytes (Haughton, 1934 ; Wagge, 1951, 1955). Although such transformations have yet to stand experimental testing, it is possible. However, no one, up until this time, has convincingly demonstrated the transformation of phagocytes into fibrous elements in molluscs. As stated, Mackin’s belief that phagocytes can transform into epithelial cells is in doubt. Similarly, LabbB’s (1928) report of transformation of amoebocytes to connective tissue fibers in Doris tuberculafa is in need of confirmation. In this connection,it is of interest to note that Shrivastava and Richards (1965), who traced the fate of tritiated thymidine-labelled blood cells (hemocytes) by autoradiography, have found that blood cells are not involved in the formation of connective tissue in the wax moth larva, Galleria mellonella. Several earlier workers have experimentally induced encapsulation in molluscs by introducing relatively large foreign materials or through injury. Drew and De Morgan (1910) have demonstrated this in Pecten maximus; LabbB (1928, 1929, 1930) has reported this in Doris tuberculata; and Jullien ( 1940) has demonstrated encapsulation in cephalopods. To emphasize that encapsulation of parasites is known to occur in so-called natural hosts, although definitely less conspicuously, the findings of Probert and Erasmus (1965) may be cited. These British workers have found encapsulation of Cercaria X in the blood vessels and tissues of the mantle of Lymnaea stagnalis, the natural host of this trematode. The observations of Canzonier (personal communication) on the reactions of the oyster, Crassostrea virginica, to sporocysts of Bucephalus should also be mentioned. The oyster is the natural intermediate host for this trematode. Generally no appreciable reactions occur in oysters parasitized by Bucephalus (see Cheng and Burton, 1965b) ; however, Canzonier informed me that encapsulation does occur around sporocysts which are moribund or disintegrating*. The question is : did the

* It should be noted that Pauley and Sparks (1965) have reported that Stauber (personal communication) informed them that : ‘‘ The oyster parasite, Bucephalus sp., is not encysted and even when resolution of dead masses of it occurs, it is usually without encapsulation.”

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69

encapsulation cause the death of the parasite or is the host reaction the result of stimulation by dead or dying parasites? This question should be tested. Another incidence of encapsulation within Crassostrea virginica that provides some information pertaining to its role in the destruction of incompatible parasites has been reported by Cheng ( 1966a). Specimens of C. virginica that had been introduced to Hawaiian waters were examined during 1965. It was found that a relatively large number harbored metacestodes of the lecanicephalid cestode Tylocephalum sp. This cestode was originally described from the pearl oyster, Margariti-

FIG.13.

Healthy and resorbed Tylocephalum metacestodes in Crassostrea virginica. Notice lack of host’s cellular reaction around healthy rnetacestode ( M ) and conspicuous reaction surrounding resorbed metacestode (R).(After Cheng, 1966a.)

fera vulgaris, in Ceylonese waters. Its complete life cycle, including its definitive host, remains unknown ; however, the ciliated coracidium and its modes of penetration into C. virginica were reported by Cheng (1966a). Thus in Hawaii, C. virginica, introduced from the Atlantic seaboard of the continental United States where Tylocephalum metacestodes have only recently been reported, is believed not to be the natural intermediate host. The cestode larvae will undergo what appears to be normal development in C. virginica (Fig. 13), but when it is well developed a delayed but nevertheless conspicuous encapsula-

70

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

tion occurs, with the capsule comprised of leucocytes and connective tissue fibers (Fig. 11). Concurrent with the completion of encapsulation, the larvae commence to atrophy and are eventually resorbed (Fig. 13). Thus it would appear that in the unnatural host, encapsulation, although delayed, does occur and the capsules, including the cells and cell products involved, contribute in some yet unknown way to the death and resorption of the parasites. The origin and chemical nature of the encapsulating fibers surrounding Tylocephalum metacestodes in C. virginica have been studied by Rifkin and Cheng (1967). We have found that the fibers, except for those comprising the innermost layer, are not transformed from leucocytes or fibroblasts but are concentrically deposited thickened intercellular material originating from between the Leydig cells located on the periphery of the parasite. These fibers are reticular rather than collagenous and include glycoproteins and/or mucoproteins and neutral mucopolysaccharides. The innermost layer of each capsule is comprised of fibroblast-like cells which possess a y-metachromatic and acid mucopolysaccharide-rich matrix. The positive acid mucopolysaccharide histochemical reaction is due to the occurrence of chondroitin sulfate B. The origin of these cells is uncertain. It is also uncertain as to what stimulates the intracellular material between surrounding Leydig cells to thicken and become fibrous or what attracts leucocytes to migrate into the area of the fibrous capsule. It has been noted that extremely thin periodic acid-Schiff-positive strands are commonly found connecting the surface of each metacestode with the surrounding capsule. These strands may represent some parasite-secreted material which serves as the stimulatory agent for the host tissue reactions. Direct proof of this, however, is currently unavailable. Another interesting aspect of this study is that the capsules surrounding metacestodes situated in the zone surrounding the oyster’s alimentary tract are consistently thicker than those surrounding metacestodes located in the interdiverticular spaces of the digestive gland. Since it has been shown that most of the fibers forming each capsule originate from intercellular reticular fibers, one possible explanation for this difference in capsule thickness is that there is less Leydig tissue in the digestive gland than in the area surrounding the alimentary tract, and hence less intercellular material. This interpretation lends further support to our earlier contention (Cheng and Cooperman, 1964; Cheng et al., 1966a) that the presence or absence of fibers in molluscan encapsulating cysts is dependent on the availability and nature of the surrounding host tissues. Other instances of encapsulation in molluscs are known. For

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71

example, Chernin (1962) has found that the eggs of the nematode Daubaylia potomaca do become encapsulated in the snail Helisoma caribaeum. c. Leucocytosis Very little is known about the cause and function of this type o internal defense mechanism. I n fact, it remains to be clarified as to whether this should be considered a distinct type of defense mechanism or should be considered as a manifestation of early stages of phagocytosis, encapsulation, or both. Leucocytosis is manifested as an increase in the number of amoebocytes. Although this phenomenon is well known among vertebrates and certain invertebrates, it has not been studied to any extent among molluscs. Stauber (1961) reported that Feng and Canzonier (unpublished data) have found that the number of leucocytes in C. virginica is increased within 24 h after intracardial injection of even sterile sea water. He stated that “ the proportions of the various types of blood cells may also change after the injection for foreign materials (ink, bacteria) or even after hemorrhage.” Cameron (1934) has described a similar phenomenon in the larvae of the wax moth caterpillar. The difficulty in evaluating and explaining leucocytosis in oysters, or in any mollusc for that matter, rests with the fact that the normal leucocyte count of molluscs is difficult t o ascertain due to difficulties in sampling and normal fluctuations. Furthermore, except for the report by George and Ferguson (1950) on Busycon carica, B. canaliculatum and Fasciolaria tulipa, and a few earlier workers (cited by George and Ferguson), the types of leucocytes present and their normal ratios have not been studied. Recently, Feng (1965b) has published his data. He has found that the number of circulating leucocytes from heart blood samples increases linearly with rising ambient temperatures a t which the oysters are held. Concurrent with the rise in leucocyte count, the corresponding heart rates increase (Fig. 14). He concluded that the fluctuation of leucocyte counts in oyster blood is probably associated with the intensity of agitation exerted by the heart beat, which in turn is influenced by the ambient temperature. Feng has also demonstrated that repeated bleedings do not effect the heart rate nor the number of leucocytes, while injections of sea water and spinach chloroplasts reduce, rather than elevate, the heart rate and leucocyte count significantly for a period of 2 to a t least 4 h. His findings need not contradict Stauber’s original reference to this work, although it may. Actually, Feng counted free circulating blood cells which by no means represent all

72

MARINE MOLLUSCS AS H O S T S FOR SYMBIOSES

the Ieucocytes present in the heart since it is known that there is some sludging of leucocytes induced by temporary heart failure. Similar cell clots have been reported by Bang (1961). Since leucocytosis obviously suggests the rapid formation of leucocytes within the mollusc’s body, the question arises as to where the hemopoetic sites are within a mollusc? Surprisingly, such sites have yet to be convincingly demonstrated, especially in pelecypods. It is noted, however, that Haughton (1934) and Wagge (1951, 1955) have expressed 35

30

25

20

; c L

0

15

f 0

i 10

- 5

. . . . . . . . . . . 0

2 4

6

8 10

140

Temperature

18

-0 24

c

FIG.14. Tho effect of temperature on leucocyte number and heart rittr of oysters, Crnssostrea virginiccc. Each point rcprosents the average hoart rate of three determinations on ten oysters. The vertical lines are ranges of the means. (After Fang, 1965b.)

the opinion that molluscan leucocytes arise via the differentiation of fibroblasts and epithelial cells, a concept which cannot be accepted without critical testing. Pan (1958) has reported that in Australorbis glabratus the hemopoetic sites are the walls of the saccular portion of the kidney and the blood sinuses. Certainly further studies on both the types of blood cells and hemopoetic sites in molluscs should be encouraged. It is evident that eventual better understanding of all types of cellular reactions in molluscs, including leucocytosis, phagocytosis

4. ANALYSIS OF FACTORS INVOLVED I N SYMBIOSIS

73

and encapsulation, can only be brought about with a clearer understanding of these aspects of molluscan hematology. d. Nacrezation This is a new term which is being coined to describe a type of molluscan defense mechanism which does not subscribe to any of the foregoing categories. Nacrezation involves a series of processes which result in the walling off of foreign materials by a layer or layers of nacre, thus forming a pearl. This type of defense mechanism against zooparasites has been known since the report of Worm in 1655 that pearly formations occur in the mantle of Mytilus edulis collected in Sweden. This original observation was followed by the studies of Dubois (1901, 1907), Jameson (1902), Giard (1907), and others. It is now known that certain trematode metacercariae, especially those of Gymnophallus margaritarum, that are found between the shell and mantle of marine pelecypods, will stimulate the mantle to secrete nacre which becomes deposited around the parasites. Nacrezation is not limited to protection against trematode larvae. It has long been known that polychaetes of the genus Polydora, commonly referred to as mud blister worms, are prevented from reaching the soft tissues of oysters by the laying down of nacre by the underlying mantle epithelium as soon as the annelids break through the internal shell surface. e. Humoral immunity Most, if not all, of our information on the existence of humoral immunity in molluscs is based on indirect evidences. For simplicity in presentation, evidence for the occurrence of humoral immunity in molluscs is being classified as natural (innate) and acquired.

Natural factors. The earlier literature pertaining to naturally occurring humoral factors is difficult to analyze critically since the molluscs studied were all caught in the wild, hence their histories were not available. Couvreur (1923) has reported the occurrence of a natural antitoxin in Helixpomatia. Chahovitch (1921) has reported the presence of a natural hemagglutinin in the cephalopod Sepia. More recently, Tyler (1946) has reported natural agglutinating activity when the blood cells and spermatozoa of a number of non-molluscan invertebrates were placed in the body fluids of five marine gastropods, Acmea digitalis, Lattia gigantia, Tegula galena, Astraea undosa and Megathura crenulata, and one pelecypod, Mytilus californianus. I n no instance did the fluids affect blood cells or spermatozoa of animals of the same taxonomic class,

74

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

thus suggesting phylogenetic significance. It should be pointed out that Tyler made no attempts to determine whether the agglutinations were brought about by a protein or proteins in the molluscan fluids. Along similar lines, Johnson (1964) has reported that saline extracts of the butter clam, Saxidomus giganteus, will agglutinate human erythrocytes of the phenotypes A, and A,B only. Adsorption experiments revealed that the naturally occurring hemagglutinin can be completely adsorbed by A, and A,B cells, partially adsorbed by A, cells, but is unaffected by B and 0 cells. This agglutinin is nondialyzable and is thus probably a large molecule. Moreover, it is inactivated when the extract is heated to 70°C for 20min, thus indicating that it is most probably a protein. A similar natural agglutinin has been reported by Boyd and Brown (1965) in the body fluids of the land snail Otala (Helix) lactea. This agglutinin, when tested against various phenotypic human erythrocytes, only agglutinates those of the A type. Cushing et al. (1963) have demonstrated that the plasma of Octopus bimaculatus will not agglutinate human erythrocytes carrying A, B, 0, M and N antigens; however, it will partially inhibit commercial anti-A serum with respect to its reaction with type A cells. On the other hand, the plasma will not inhibit anti-B, anti-M, anti-N, and anti-H sera. It would thus appear that the naturally occurring molluscan agglutinins cited above all portray some degree of specificity. The possible value of these agglutinins to the molluscs possessing them is unknown. The most recent evidence suggesting the possible existence of innate humoral factors has been contributed by Heyneman (1966) who has succeeded in demonstrating that larval trematodes transplanted from natural host to natural host will continue to develop normally, while larvae transplanted into incompatible hosts will fail to become established. These studies suggest the occurrence of a factor or factors in incompatible hosts which act against the establishment of the parasites. Furthermore, as Heyneman has stated : “The conclusion reached is that resistance t o larva1 trematode development in the incorrect snail host is a physiological rejection within snail tissues distinct from factors responsible for failure of miracidia t o attach t o or penetrate the body wall of the nonadapted host.”

All the information cited above suggests the existence of innate humoral factors in molluscs ; however, an equal number of investigators have reported the absence of such factors in other molluscan species. For example, Dungern (1903) and Cantacuzene (1923) have reported that natural precipitins and complements cannot be detected in

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75

Eledone. CantacuzAne (1915) also reported the absence of agglutinins, precipitins and hemolysins in the blood of Helix pomatia ; moreover, he could not detect the occurrence of a natural complement. Chahovitch (1921) has reported that neither natural nor acquired agglutinins can be detected in the cephalopod Sepia ; likewise, acquired precipitins cannot be detected. Similarly, Cantacuzene (1912) has reported that natural complements cannot be detected in Sepia and Eledone. I n recent years, Tripp (1958a) has reported that the blood o f . C. virginica is bactericidal against certain bacteria but could not detect specific agglutinins and precipitins. However, in a later abstract, he (1958b) and Feng (1959) have found some indications of both agglutinating and properdin-like properties in C. virginica plasma. Continuing along this line of investigation, Tripp (1960, 1961a,b) has further suggested the occurrence of innate humoral bactericidal factors in C. virginica and Australorbis glabratus. These factors are believed to be present since bacteria inoculated into these molluscs are rapidly killed off extracellularly. More recently, Tripp (1966), continuing his studies on oyster blood, has reported that the " shell liquor" (mantle fluid), plasma, and pericardial fluid of Crassostrea virginica will agglutinate erythrocytes of the mouse, chicken, guinea-pig, cow, sheep, rabbit, horse and man. I n the case of human blood cells, oyster fluids will agglutinate cells of types A, B, AB, and 0, thus indicating a lack of specificity. Tripp has noted that intracardially acquired plasma is more reactive than both the pericardial and mantle fluids. This, as he has indicated, is because these fluids represent plasma diluted with sea water. Tripp has also shown that the agglutinin exerts an opsonic effect on rabbit erythrocytes in vitro and probably influences the rate of phagocytosis of similar erythrocytes and their disposal when experimentally injected into oysters. The agglutinin in C. virginica plasma is not dialyzable and the hemagglutination titer is directly proportional to the protein concentration. This proteinaceous agglutinin is also heat-labile, being partially inactivated a t 60°C and totally inactivated after heating at 70°C or above. The first attempt to characterize a naturally occurring humoral factor in a mollusc was performed by Cheng and Sanders (1962). We have found that the sera (plasma) of Viviparus malleatus includes a naturally occurring hemagglutinin which is specific for rabbit erythrocytes among five types of vertebrate erythrocytes tested (Coturnix quail, Rana pipiens, rat, mice and rabbit). Electrophoretic fractionations of pre- and post-rabbit erythrocyte-adsorbed sera have revealed that the agglutinating property of the plasma is associated with all

76

MARINE MOLLUSCS AS HOSTS F OR SYMBIOSES

five of the plasma1 fractions (Fig. 15). Confirmation of the proteinaceous nature of the agglutinin was in the establishment of a linear correlation between the agglutination titers of the plasma samples and their total protein concentrations. I n the same paper we postulated that this natural humoral factor may be responsible for the apparent parasitefree condition of Viviparus malleatus, with the factor acting as a natural defense mechanism. (point of application)

A . Normal, unadsorbed serum

c5

(point of application) I

8. Adsorbed serum (point of application)

C. Diluted serum

FIG.15. Electrophoretic patterns of plasma (sera) of Viwiparus malleafus. The systern ernployed was paper electrophoresis (Beckman Durrum type) with Verona1 buffer a t pH 8.6. (After Cheng and Sanders, 1962.)

At this point it should be mentioned that naturally occurring humoral factors can prevent establishment of a parasite by means other than killing the penetrating parasite if such occurs. The findings of Cheng et al. (196613) relative to the effects of marine pelecypod plasma, especially the plasma of Crassostrea gigas cited earlier, may be interpreted t o reflect the occurrence of an innate humoral factor which prevented parasitization by Himasthla quissetensis metacercariae by preventing the cercariae from penetrating through immobilization.

Acquired resistance. I n molluscs the importance of phagocytosis and encapsulation as forms of internal defense mechanisms is well established but acquired antibodies have not yet been demonstrated.

77

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

The belief that antibodies do occur in molluscs stems primarily from the studies of Winfield (1932) and Nolf and Cort (1933) who have indirectly suggested that acquired immunity may be present in gastropod intermediate hosts of larval trematodes. They reported that the presence of sporocysts of Cotylurus JEabelliformis, enclosing developing cercariae, in varieties of Lymnaea stagnalis prevents almost all of the cercariae of this trematode from successfully penetrating and encysting as metacercariae, even when the snails are exposed to large numbers of cercariae. Cort et al. (1945) have repeated their studies and have reported that the same phenomenon occurs in Stagnicola emarginata angulata infected with Cotylurus Jlabelliformis sporocysts They did add that the few cercariae which do succeed in penetrating are inhibited from developing into metacercariae unless they enter sporocysts and are thus presumably protected from the host’s antibodies. On the basis of these reports, Culbertson (1941) has generalized that “ . . it is clear that snails acquire an immunity after infection by trematodes.” This sweeping statement is premature since, as stated, antibodies in molluscs have not yet been demonstrated. Other indirect pieces of evidence of acquired immunity in molluscs exist. Dye (1924), for example, has observed that if the snail Melania nodocinca in Africa is naturally infected with a ‘‘ single-tailed cercariae”, infection with Schistosorna haematobiurn cannot be superimposed. Gordon et al. (1934) have considered Dye’s results to indicate the occurrence of acquired immunity ; however, Michelson (1963a, 1964) has correctly pointed out that Dye’s observations should be accepted with reservation since it has since been shown that only one species of Bulinus serves as the intermediate host for S. haematobiurn in Africa. Benex and Lamy (1959) discovered that tissue extracts from the planorbid snail Planorbis corneus (which is not an intermediate host for mammalian schistosomes) will immobilize Schistosonza mansoni miracidia. Based on this observation, these French workers have suggested that species of snails found refractory to schistosome infection may possess “ immune-like ” immobilizing substances. It is extremely difficult, if not impossible, to evaluate Benex and Lamy’s results since, although Planorbis corneus is not a known host for mammalian schistosomes, it can serve as a host for non-mammalian schistosomes. Thus, it cannot be determined whether the “ immunelike immobilizing substances are acquired as the reault of previous experience with non-mammalian schistosomes which share common antigens with mammalian schistosomes, or whether it is an innate humoral factor similar to the cercaricidal substance found by Cheng

.

”

78

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

et al. (196613) in the tissue extracts of certain marine pelecypods. Certainly the occurrence of ‘( immune-like ” substances in molluscs which act against incompatible parasites is not widespread. I have tested many such combinations and have found nothing suggestive of innate immunity except that reported by Cheng et al. (196610). Another observation which suggests acquired immune factors in molluscs has been reported by Barbosa and Coelho (1956). These workers have demonstrated that Australorbis glabratus previously ‘( cured ” of Schistosoma mansoni infection could be reinfected ; however, some tissue reaction is evoked in reinfected snails, a phenomenon not usually found in initial infections (exceptions reported by Cheng and Cooperman (1964), Probert and Erasmus (1965) and Cheng et al. (1966a) have been cited earlier). Barbosa and Coelho’s observations could mean that some type of acquired incomplete immunity exists in A . glabratus after the initial infection and becomes manifested during the reinfection as cellular response. These workers, however, have found that the sera (plasma) of infected A. glabratus has no effect on S. mansoni miracidia. Recently, Michelson (1963b, 1964) has reported the existence of S. mansoni miracidia-immobilizing substances in tissue extracts of A. glabratus infected with this trematode.* I n 1964 he stated that seven species of gastropods were used ; furthermore, in the case of Helisoma caribaeum, six strains ” were employed. Although the extracts of all the species, including uninfected Australorbis glabratus extract and water, gave positive miracidial-immobilization, that brought about by infected A . glabratus extract was significantly greater. Michelson (1963b) reported his results on whether the miracidia-immobilizing substances were specifically associated with S. mansoni. To this end, he tested extracts of (1) snails infected with an acid-fast bacillus, (2) snails infected with an echinostome metacercaria, (3) snails infected with the nematode Daubaylia potomaca, (4) snails inoculated with bovine albumin, ( 5 ) snails inoculated with Schistosoma mansoni eggs, and (6) snails inoculated with polystyrene spheres. Immobilization of miracidia was observed only in extracts of snails infected with D. potomaca or lyophylized S. mansoni eggs. I n neither case, however, did the percentage of immobilization reach the level observed in extracts of S. mansoni-infected snails. From the review presented above, it is evident that there are evidences which support the concept that acquired humoral immunity does exist, a t least in certain species of molluscs, although, needless t o state, more direct evidences are required. Relative to the miracidia((

*See note on p. 389.

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

79

immobilizing substance in Australorbis glabratus, Michelson (1963a) has cautiously stated that : Although the suggestion that the immobilizing phenomenon might be associated with an antigen-antibody is an appealing one, data are lacking t o substantiate this hypothesis. The possibility that the immobilizing substance(s) might be related either t o parasite-produced toxins or t o products resulting from alterations in the snail’s metabolism cannot be excluded. It is of interest, nevertheless, to speculate as to why Michelson’s miracidia-immobilizing substance is only found in tissue extracts and not in plasma. One possible explanation is that these factors are either located intracellularly, or, more likely, adhere to the surfaces of cells and can only be detected after the cells are disrupted or ruptured during the extraction process. As a final word, Michelson’s speculation that it may be a parasite-produced toxin or a product resulting from alterations in the snail’s metabolism certainly cannot be overlooked, especially since it is known through the works of Faust (1917), Agersborg (1924), Hurst (1927), Cheng and Snyder (1962a), and Cheng and Burton (1965b) that there is, among other pathological changes, an increase of so-called ferment cell secretions in molluscs parasitized by trematodes. Hurst has attributed a “ lytic ” activity to these secreted globules. Although Cheng and Burton (1965a) have pointed out that there is no direct evidence for Hurst’s contention, a t the same time there is no direct evidence to the contrary. Thus, these globules, among many possibilities, may represent a “ toxin ” which could be manifested as an immobilizing factor. At any rate, much remains to be studied relative to both cellular and humoral defense mechanisms in molluscs. I n addition to the small yellowish ferment cell secretions, another type of cell, commonly referred to as “ brown cells ” (Takatsuki, 1934), “ das rotbraunes organ ” (Keber, 1851), or cells of “ Keber’s organ ” (Fernau, 1914), are known t o increase in number in parasitized oysters and other pelecypods. These cells are believed to have their origin in the darkened areas on the mantle, in the pericardium, in connective tissues, but primarily on the surface of the auricles (see reviews by White, 1942 ; GrassB, 1960). They are also known to undergo mitotic division in tho Leydig tissues of oysters (Cheng and Burton, 1965b). The function of “ brown cells ” has not been demonstrated experiment d l y although Mackin (1951, 1962) and Stein and Mackin (1955) have suggested that their function is associated with the internal defense mechanisms of oysters since they have found a correlation between the increase in number of “brown cells” and infection by the fungus

80

MARINE MOLLUSCS AS H O S T S FOR SYMBIOSES

Dermocystidium marinum. Similarly, increased numbers of ‘‘ brown cells ” have been observed in Crassostrea virginica parasitized by MSX, certain nematodes (unpublished), and Bucephalus sp. (Cheng and Burton, 1965b). Recently Pauley and Sparks (1966) have reported the occurrence of similar cells, although vacuolated and with as many as three or four nuclei, in Crassostrea gigas experimentally injected with turpentine. Chemically, the smaller and larger “ brown cells ” have been shown by Cheng and Burton (1966) not to include acid mucopolysaccharides but the medium-sized ones, i.e. those that measure between 0.006 and 0.012 mm in diameter in C. virginica, are partially or totally positive for acid mucopolysaccharide. Moreover, slight metachromasia and sphingomyelin are also found in certain “ brown cells ” of parasitized oysters. 3. Nutritional requirements If a symbiont, in this case primarily a parasite, is successful in overcoming the initial or delayed confrontation with the host’s resistance, successful establishment may then proceed. Success now is primarily dependent upon the parasite’s ability to cope with the host’s relative susceptibility or partial insusceptibility. By Read’s ( 1958b) definition stated earlier, a totally insusceptible host is one in which the parasite’s life needs cannot be satisfied and therefore successful establishment cannot occur. Information pertaining to the needs of zooparasites of molluscs is conspicuously scanty. This is especially true in the case of marine molluscs. As the result of the lack of information pertaining to the metabolism, particularly intermediary metabolism, of intramolluscan parasites and mutualists, there is no way to estimate what their enzymic, coenzymic, and specific nutritional requirements are. Moreover, it is generally not known if special metabolic modifications exist which reflect their adaptations t o a symbiotic existence. I n this section is reviewed the available information about the nutritional requirements of intramolluscan symbionts. The nature of such information by necessity limits the discussion to what is known concerning the nutritional aspects of larval trematodes in molluscs. Cheng (1963d) has reviewed what is known about the biochemical composition and requirements of intramolluscan trematode larvae. Since then, a few additional facts of this nature pertaining to marine molluscs have been contributed. Again, to make the review presented below more meaningful, the general area of mollusc-parasite interactions as related to nutritional requirements must be consulted.

81

4. ANALYSIS OF FACTORS INVOLVED IN SYMBIOSIS

Although in some instances artificial, the nutritional requirements are being discussed under the headings of carbohydrates, lipids, and proteins. a. Carbohydrates The occurrence of stored glycogen in the soma of a number of intramolluscan parasitic helminths, especially pre-adult stages of trematodes, is known (Axmann, 1947 ; Ginecinskij, 1960 ; Ginecinskij and Dobrovalskij, 1962 ; Snyder and Cheng, 1961; Cheng and Snyder, 1962a; Cheng, 1963b,c; Palm, 1962a,b; Cheng and Burton, 1966; and others). The source of this polysaccharide theoretically could be from the metabolism of fatty acids via acetyl CoA or from amino acids via pyruvic acid or acetyl CoA, with both processes involving reversal of the Embden-Meyerhof glycolytic pathway, and some probably is synthesized via these pathways. There is evidence, however, that most of the glycogen is synthesized from glucose obtained from the molluscan host. The series of studies which have led to this conclusion was initiated by Faust (1920) who detected a reduction in stored glycogen in trematode-infected snails. Hurst (1927) has confirmed this finding. Von Brand and Files (1947), employing biochemical techniques, have concurred by reporting a significant reduction in the amount of stored glycogen in Australorbis gbbratus infected with Schistosoma mansoni. These investigators stated that “whether this disease is due to an impaired carbohydrate digestion and resorption, or to a toxic action by the parasite is not clear ”. Furthermore, they have stated: “ The possibility that it is due to the food consumption of the parasites has not been ruled out completely.” Similarly, Zischke and Zischke (1965) have reported a reduction in host glycogen in trematode-parasitized snails. An explanation for this reduction in host glycogen has been given by Cheng and Snyder (1962a) who studied this aspect of the relationship between the freshwater pulmonate Helisoma trivolvis and the trematode G l y ~ € h e l m ~ pennsylvaniensis. ns We have demonstrated that concurrent with the reduction of glycogen in the host’s hepatopancreatic cells, there is an increase in the amount of stored glycogen in the developing cercariae. This led to the postulation that the parasites utilize the host’s glycogen and to further search for evidence. By employing histochemistry, Cheng and Snyder (1963) have demonstrated that the gradual reduction of glycogen in the mollusc’s hepatopancreas is due to the breakdown of this polysaccharide to glucose which in turn permeates the host’s hepatopancreatic cell membrane and the sporocyst wall and becomes incorporated in the developing A.P.B.--~

7

82

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

cercariae, thus serving as a source for glycogen synthesis. Later, Cheng and Burton (1966) examined for a similar process in Crassostrea virginica parasitized by Bucephalus sp. and found an identical pattern. It is of importance to state at this point that both Glypthelmins pennsylvaniensis and Bucephalus sp. include sporocyst but not redial stages in their life cycles, hence the uptake of nutrients by the intramolluscan sporocysts, which are devoid of a mouth, must be by absorption from the micro-environment. COMPARISON OF TOTALCARBOHYDRATESAND REDUCING SUGARS IN NON-PARASITIZED Nassarius obsoletus AND THOSE PARASITIZED BY Stephanostomum tenue

TABLE VI.

The figures represent mean values of the per cent of wet tissue weight per snail 5 one standard error of the mean. The difference of the means of roducing sugar values is not significant at the 5% level but the difference for total carbohydrates is significant a t the 1%level. (After Blumenthal, unpublished.)

Categories of snails

Infected Uninfected

Reducing sugars

Total carbohydrates

No. of snails

Percent of total weight

No. of snails

Percent of total weight

11 19

0.0413 & 0.0054 0.0392 f 0.0029

18 18

1.59 -& 0.29 2-55 0.22

I n another marine mollusc, Nassarius obsoletus, Alan B. Blumenthal, one of my former students, has examined the quantitative changes in total carbohydrates and reducing sugars when this snail is parasitized by the rediae of another trematode, Xtephanostomum tenue. A summary of his data is presented in Table VI. From these it is evident that a significant reduction in totaI carbohydrates occurs but no significant increase in reducing sugars. The reduction of total carbohydrates, probably mostly glycogen and other polysaccharides since the amount of reducing sugars in infected and control snails is low (about 0.04% of the total weight), reaffirms the loss of glycogen by the hosb. I n fact, both Blumenthal and von Brand and Piles (1947) have noted that differences in polysaccharide levels in uninfected and infected snails are probably greater than those reported since the glycogen stored within the bodies of enclosed parasites is also reflected in the total polysaccharide figures reported for infected snails. The absence of a significant difference between the levels of reducing sugar in infected and control Nassarius obsoletus c m be explained by the nutrient acquiring

4. ANALYSIS O F FACTORS INVOLVED I N SYMBIOSIS

83

behavior of the larvae of Stephanostomum tenue. Earlier, Cheng (1962, 1963c) had shown that in the case of Helisoma trivolvis infected with another trematode, Echinoparyphium sp., no dramatic reduction of glycogen occurs in the host’s intact hepatopancreatic cells. Echinoparyphium sp., like Stephanostomum tenue, includes redial rather than sporocyst generations in its life cycle. Since rediae possess a functional mouth, it has been shown that the intramolluscan rediae acquire their carbohydrate requirements by ingesting glycogen-containing cells primarily, leaving the intact cells essentially unaltered as far as their glycogen content is concerned. Thus in the case of 8. tenue, the rediae presumably also ingest cells directly without the necessity of converting the host’s stored glycogen t o glucose, hence one can expect the levels of reducing sugars to remain at a comparable level in infected and uninfected Nassarius obsoletus. It should be noted that rediae do not acquire their nutrient requirement exclusively by ingestion. The electron microscope studies by G. Rees (1966), Bils and Martin (1966), Krupa et aE. (1966) and Cheng and Hamamoto (unpublished) have revealed the occurrence of microvilli on redial surfaces which suggests that rediae may also absorb certain nutrients. I n sporocyst-containing hosts, it still remains uncertain what causes the breakdown of stored glycogen to glucose. Preliminary experiments reported by Clieng (1963d) indicate that the hydrolyzing enzyme is not of parasite origin,which appears reasonable since evidences on membrane permeability have indicated fairly conclusively that large protein molecules, such as a carbohydrase, could not permeate the cell membrane of the host’s hepatopancreatic cells. It is thus necessary to speculate that the presence of sporocysts in some way triggers the host’s native enzymes to increase the rate of glycogen degradation. As stated, it is known that glucose will pass through both the sporocyst wall and the body surface of developing cercariae ; however, no direct evidence is yet available which indicates whether the glucose enters by simple diffusion or by some other mechanism. The finding by Cheng and Snyder (196213) and Cheng (1964a) that there is alkaline phosphatase activity associated with the body surface of cercariae may be suggestive that glucose, or perhaps some other substance, is conducted across the body wall by phosphorylative transport. This mechanism has been considered to be possible by Danielli (1952). Certainly the presence of microvilli on the surface of sporocysts, as demonstrated by the electron microscope studies of Bils and Martin (1966), suggests that the sporocyst wall is an absorptive surface. Similarly James et al. (1966), who have also studied the fine structure of the sporocyst wall, have suggested that it is absorptive.

84

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

The requirement of a carbohydrate source of host origin among intramolluscan trematode larvae appears to be one of absolute necessity. Simoeo and Coelho (1955) have demonstrated that when Australorbis glabratus is removed from water, the enclosed larvae of Schistosoma mansoni stop developing but development is resumed when the snail is returned to water. This finding is extremely significant when the work of MagalhBes and de Almeida (1956) is considered. These workers have reported that the hepatopancreatic glycogen of A . glabratus drops to 50% of the normal content when the snails are kept out of water and to 10-15% just before death in 75 days. These data suggest that the inhibition of normal development of the trematode larvae is correlated with the amount of glycogen in the host’s digestive gland. This hypothesis appears to be supported by the results of Sindermann el al. (1957) who have found that the emission of the cercariae of Austrobilharzia variglandis, the marine dermatitis-causing trematode, by Nassarius obsoletus is reduced if the gastropod is starved. But upon reinitiation of feeding, massive cercarial emergence follows within 48 h. Similarly, Brackett (1940) has reported that starved snails do not emit cercariae until they are fed. These data may well be interpreted to mean that the decrease in cercaria production in starved snails has resulted from a decrease in stored glycogen as mentioned above, and with the reinstitution of feeding there is an accumulation of stored glycogen which in turn becomes available for developing cercariae. Another piece of evidencc which lends weight to this hypothesis is the work of Kendall (1949) pertaining to the development of Fasciola hepatica in Lymnaea trunculata. He has found a correlation between the amount of food intake and the number of developing cercariae reaching maturity. Other than trematodes, practically nothing is known about the carbohydrate contents or requirements of symbionts of molluscs. Among mesozoans, Lameere (1916) has reported that the rhombogens of Pseudicyema truncatum in the nephridium of the squid, Xepia oflicinalis, include somatic cells packed with glycogen. These cells often projected conspicuously on the body surface and are termed “ verruciform ” cells. The origin of this polysaccharide remains unknown, although presumably it is synthesized from sugars of host origin and is utilized as a source of energy. b. Lipids Little is known about lipid metabolism in intramolluscan parasites. Even the lipid contents in those species of protozoa, mesozoa, and arthropods found in marine molluscs remain uninvestigated. The same can be said of the nematodes and cestodes found in molluscs. The

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85

only available information is that the verruciform cells of the mesozoan Pseudicyema truncatum are said to include lipoproteins (Hyman, 1940a). .As in the case of carbohydrates, only the larval trematodes have been studied to any extent; then again, the information pertains primarily to a few species parasitic in freshwater gastropods although the findings of Cheng (1965b) on Bucephalus sp. in the American oyster, Crassostrea virginica, suggest that the general pattern is the same in marine and freshwater species. For this reason, the literature is briefly reviewed at this point with emphasis placed on function. The occurrence of lipids in intramolluscan larval trematodes is known for a few species. I n miracidia fats occur, although not in as large a quantity as glycogen (Axmann, 1947 ; von Brand, 1952). No information is yet available as to whether the stored fats in miracidia, which are generally the pre-molluscan, free-swimming stage or are found in the alimentary tract if eggs are ingested intact, are employed as an energy source. Purely from the speculative standpoint, it would appear that among the free-swimming species the availability of oxygen would suggest the utilization of lipids as among free-swimming cercariae. Ginecinskij (1961) has studied the occurrence of lipids in eight species of freshwater larval trematodes. It is of interest to note that she has found that the quantity of lipids present in sporocyst walls is directly correlated with the sites which they occupy within the molluscan hosts. Those situated on the surfaces of the host’s intestine are almost completely devoid of lipids as are those located between organs. However, the sporocysts of Cotylurus brevis and Cercaria spinulosa, which are embedded in their hosts’ hepatopancreas, are rich in fats. Cheng and Snyder (1962c),while studying the lipid composition of Glypthelmins pennsylvaniensis, have found fatty acids adhering to and on the interior of the sporocyst wall, thus suggesting the permeation of fatty acid molecules. For this reason, Cheng (1963d) questioned whether the 1ipid.s found by Ginecinskij in sporocyst walls were stationary or transient. That fatty acids occur in sporocyst walls has been confirmed by Cheng (1965b)who found them in the sporocyst walls of Bucephalus sp. parasitizing oysters. The question raised in 1963 appears to be partially answered in 1965 when I found neutral fats in the sporocyst wall of Bucephalus, since although fatty acids are believed to be capable of permeating sporocyst walls, neutral fats are not. Hence, the neutral fats found in Bucephalus sporocyst walls most probably are not transient. It is unfortunate that Ginecinskij’sstudies only involved the use of Sudan black B which does not permit differentiation between neutral fats and fatty acids.

86

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

I n the case of rediae, as far as I can determine, Ginecinskij’s (1961) report of the occurrence of lipids in the rediae of members of the Echinostomatidae and Notocotylidae, embedded in the hepatopancreas of their respective molluscan hosts, is the only one available. The occurrence of lipids in cercariae has been more frequently studied (Ivanov, 1950; Deiana, 1954a,b; Ginecinskij, 1961 ; Ginecinskij and Dobrovalskij, 1962; Lutta, 1939; Palm, 1962a,b; Cheng and Snyder, 1962c ; Cheng, 196513). Lipids as a rule are found limited to the excretory (osmoregulatory) system and to the parenchyma. Cheng and Snyder (1962b), thus far the only ones to employ more specific and differentiating histochemical tests, have pointed out that in Helisoma trivolvis parasitized by Glypthelmins pennsylvaniensis there i s an initial increase in the amount of neutral fats stored in the host’s hepatopancreatic cells. The neutral fats, however, are gradually degraded to fatty acids. Much of these short-chained fatty acids are believed to pass through the wall of the sporocysts. This hypothesis is based on the finding of fatty acid droplets adhering to the walls and in the brood chambers of sporocysts. Concurrent with the loss of fatty acids from host cells, leaving conspicuous cytoplasmic vacuoles in these cells, there is the appearance of fatty acid deposits in older developing cercariae. Cheng and Snyder have stated that it is not surprising that fatty acids, rather than neutral fats, are found deposited in the body of cercariae, basing this on the fact that it is generally the unsaturated higher fatty acids, rather than fats, which are found in adult parasitic platyhelminths. It was not until Cheng’s (1965b) later work with Crassostrea virginica parasitized by Bucephalus sp. that neutral fats were discovered in certain cells comprising older germ balls and in developing cercariae as detected by the oil red 0 technique of Lillie (1944). No explanation is yet possible as to why neutral fats, in addition to fatty acids, are present in the intramolluscan larvae of this parasite of a marine pelecypod. I n addition to this difference, another has also been reported. I n parasitized oysters there is an increase in total fats in the digestive diverticular (hepatopancreatic) cells. This, upon further histochemical analysis, has been found to reflect increases of both neutral fats and fatty acids. The increase in fatty acids is believed to be due to hydrolysis of the neutral fats by lipase of parasite origin. This enzyme was detected in cercarial tissues, in brood chambers, and adhering to sporocyst walls. No explanation is yet available as to why there is no subsequent decrease in fatty acids as in the case of Helisoma trivolvis infected with Glypthelmins pennsylvaniensis. Moreover, no explanation is again yet available as to why the level of neutral fats in the host’s cells increases in spite of their degradation by lipase

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87

activity other than the possibility that the presence of Bucephalus continuously stimulates their synthesis. Unlike the situation found in digestive diverticular cells, there is a significant drop in the amount of total fats stored in the Leydig cells of parasitized Crassostrea virginica. This reflects decreases of both neutral fats and fatty acids, thus paralleling the condition noted earlier in Helisoma trivolvis. Increases in total lipids in trematode-parasitized mollusca had been reported earlier by Faust (1917) and Hurst (1927),although von Brand and Files (1947) found that the fat content in Australorbis glabratus parasitized by Xchistosoma mansoni larvae remains unchanged. The increases in total lipids reported by Faust and Hurst could be attributed to either the initial increase of neutral fats as found in parasitized Helisoma trivolvis or to the continuous increase as found in the digestive diverticular cells of Crassostrea virginica. I n any event, information is still too scanty to justify any speculations as to what occurs in molluscs parasitized by trematodes other than to state that there is definitely a difference between the changes that occur in parasitized C. virginica and Helisoma trivolvis as far as the lipids found in the hepatopancreas are concerned. There is another aspect of lipid metabolism in intramolluscan trematode larvae which bears mentioning. Bullock (1948) has reported that among acanthocephalans fats are synthesized from products of carbohydrate metabolism by phosphatase activity and that the site of lipid synthesis is immediately beneath the body surface. I n the case of Glypthelmins pennsylvaniensis, Cheng and Snyder (19624 have found that fatty acids f i s t appear in developing cercariae at the same site, thus suggesting a parallel situation, especially since a phosphatase system is now known to occur in the subcuticular zone of developing cercariae (Cheng and Snyder, 1962c ; Cheng, 1964a). Indeed Lutta (1939) has suggested, as I have at the beginning of this section (p. 81), that there may exist an interdependence between lipid and carbohydrate metabolism. Lutta’s hypothesis is based on his finding that the greatest amount of lipids in cercariae are found at sites where the greatest accumulation of glycogen also occurs. Little is known about the function of lipids in the intramolluscan stages of trematodes. Their role in the metabolic production of energy does not appear to be an important one, since a t these essentially anaerobic microenvironments lipids do not lend themselves to the internal oxidation-reduction characteristic of anaerobic processes (Cheng and Snyder, 1962c ; Cheng, 1963d). Evidences contributed by Ginecinskij (1961) appear to support the hypothesis that lipid meta-

88

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

bolism as an energy source occurs primarily during the post-molluscan, free-swimming phases of the life-span of cercariae. Specifically, Ginecinskij has found that stored lipids are completely exhausted during the free-swimming phase of several species of cercariae, namely Opisthioglyphae ranae, Cotylurus brevis and Cercaria spinulosa. I n other species, namely Cercaria helvetica XXII and two unidentified xiphidiocercariae, the stored lipids are only slightly diminished. It is of interest to point out that Ginecinskij has reported in the same paper that there are practically no stored lipids in non-swimming cercariae, small quantities in gliding furcocercous cercariae, and relatively large quantities in active swimmers, thus suggesting a correlation between the degree of activity during the post-molluscan phase and the amount of stored lipids. Ginecinskij has proposed to explain this condition in terms of the type of cercarial motility. She postulated that the stored lipids may play a hydrostatic role, i.e. in lightening the body weight, since fatty droplets are absent in non-swimmers, present in small amounts in gliders, and in relatively large quantities in active swimmers. Perhaps her interpretation is justified and the lipids do serve a dual function. However, in my opinion the correlation between active swimming and the abundance of Iipids and their metabolic utilization is the more important one.

c . Proteins From the broad viewpoint, there can be no doubt that intramolluscan parasites, and perhaps mutualists, especially those embedded in their hosts’ tissues, must derive some, if not all, of their proteins and amino acid requirements from the host. Even among those symbionts which live near or within the mantle cavity of such pelecypods as oysters, it is more than likely that they derive certain proteins and amino acids from the host’s blood seeping into the mantle cavity. Furthermore, some of these molecules may be released during diapedesis which is a naturally occurring phenomenon among these molluscs (Galtsoff, 1964). Specific data pertaining to specific parasites are in most instances wanting. Studies on amino acid uptake comparable to those of Stephens (1962a,b, 1964) and Stephens and Schinske (1961) on freeliving estuarine invertebrates or on the amino acid compositions and metabolism of molluscs (reviewed by Allen, 1961a) are not yet available for organisms associated with marine molluscs. Evidences, however, are available which suggest that trematode larvae in freshwater molluscs do utilize free amino acids derived from their hosts’ adjacent tissues and plasma (Cheng, 1963d). Such data may well have their counterparts

4. ANALYSIS O F FACTORS INVOLVED M SYMBIOSIS

89

in marine parasitic relationships. Indeed, some of my unpublished data on Crassostrea virginica parasitized by Bucephalus sporocysts suggest this. For this reason, what is known about protein and amino acid utilization by parasites of freshwater molluscs is briefly reviewed below. The uptake of amino acids in vivo and in vitro by adult trematodes is known as the result of the studies of Robinson (1961),Senft and Senft (1962) and Senft (1963). Little is known about the intramolluscan stages. Friedl (1961c) has analyzed the constituents of the free amino acid pool of Lymnaea stagnalis jugularis to determine whether there is any correlation between prevalent amino acids and those found earlier (Friedl, 196la,b) to favor the in vitro survival rate of the rediae of Fascioloides magna which utilize this gastropod as the intermediate host. Of the three amino acids (hydroxyproline, proline and serine) previously found to favor survival, only serine has been found to be a major constituent in the mollusc’s serum (plasma). I n fact, proline has been quantitatively determined to be present in the least amount of seventeen amino acids detected while hydroxyproline has not been found. Generally speaking, however, alanine, glycine, serine and threonine, all prominently present in plasma, have been shown to extend longevity in vitro. These indirect evidences could be interpreted as suggesting that the potential for uptake of certain amino acids by intramolluscan trematode larvae exists. Dusanic and Lewert (1963) have studied alterations in the protein and amino acid compositions in the plasma of Australorbis glabratus following infection by Schistosoma mansoni. Although their primary concern was to detect changes which might reflect molluscan response in the form of humoral immunity, it is of interest to comment on their results, especially their findings on changes in the amino acid composition. Using two dimensional paper chromatography involving butanol, acetic acid, and water (4 : 1 : 1) as the first irrigant and a phenolsaturated solution of 6.3% sodium citrate and 3.7% monobasic sodium phosphate as the second, these workers detected only three amino acids, lysine, glycine and proline, in the plasma of uninfected snails. I n 45 min post-miracidial penetration, they detected an increase in all three of these amino acids but, in addition, they detected the appearance of two additional amino acids, threonine and leucine. This condition persists until 2 h post-infection at which time there is a decrease of all five amino acids, with the disappearance of leucine by the 14th h. By the 24th h post-infection, the amino acid composition returns to normal. These workers have inferred that the initial increase in amino acids may be correlated with the appearance of humoral factors although they did not state this as a fact. Their findings are of interest since evidence for

TABLEVII. BOUNDAND FREEA M I N O ACID COMPOSITIONS OF SERA (PLASMA) O F UNINFECTED AND INFECTED Physa gyrina, HEPATOPANCREAS OF P. gyrina, AND SPOROCYSTS AND CERCARIAE OF Glypthelmim quieta (After Cheng, 1963d.) Uninfected P. gyrina sera (plasma)

Infected P. gyrina sera (plasma)

Uninfected P. gyrina hepatopancreaa

Bound

Bound

Bound

Cystine Lysine Histidine Asparagine Serine

-

Free Cystine Lysine Histidine Asparagine Serine

-

Glutamic acid

Glutamic acid

Threonine Alanine Proline Cysteine Tyrosine Valine Methionine

Threonine Alanine Proline

Leucine Isoleucine

Free

Cystine Lysine Histidine Asparagine Serine Arginine Aspartic acid Glutamic acid Glycine Threonine Alanine Proline Cysteine Tyrosine Valine Methionine Tryptophan Leucine Isoleucine

Cystine Lysine Histidine Asparagine Serine -

Glutamic acid

Threonine Alanine Proline Cysteine Tyrosine Tyrosine Valine Methionine Tryptophan Tryptophan Leucine Leucine Isoleucine Isoleucine I

No. of amino acids: 16 13

16

1

19

Free Cystine Lysine Histidine Asparagine Serine Arginine Aspartic acid Glutamic acid Glycine Threonine Alanine Proline Cysteine Valine Tryptophan Leucine Isoleucine 17

G. quieta spwocysta Bound Cystine Lysine Histidine Asparagine Serine Arginine Aspartic acid Glutamic acid Glycine Threonine Alanine Proline Cysteine Tyrosine Valine Methionine Tryptophan Leucine Isoleucine 19

G. quieta cereariae

Free Cystine Lysine Histidine Asparagine Serine Arginine Glutamic acid Glycine Threonine Proline

Valine

-

Bound Cystine Lysine Histidine Asparagine Serine Arginine Aspartic acid Glutamic acid Glycine Threonine Alanine Proline Cysteine Tyrosine Valine

-

Free Cystine Lysine Histidine Asparagine Serine Arginine

-

Glutamic acid Glycine Threonine

-

Proline

-

Valine

Tryptophan Tryptophan Tryptophan Leucine Leucine Leucine Isoleucine Isoleucine Isoleucine 14

18

14

T u r n VIII. BOUND AND FREE&NO ACID COMPOSIT~ON OF SERA(PLASNA) OF UNINFECTED AND INFECTED Musculium partumeium, GILL TISSUESOF M . partumeium, AND SPOROCYSTS AND CERCARIAE OF Gorgodera amplicava (After Cheng, 1963d.) Uninfected M. partumeium ,sera ( p l m a ) Bound Cysteine Arginine Aspartic acid Threonine Glutamic acid Alanine Tyrosine Valine Tryptophan Isoleucine Cystine Lysine Serine Asparagine Proline

Free Cysteine Arginine Aspartic acid Threonine Glutamic acid Alanine Tyrosine

-

-

-

Infected M. partumeium sera (plasma) Bound

Free

Cysteine Argin in e Aspartic acid Threonine Glutamic acid Alanine Tyrosine Valine Tryptophan Isoleucine

-

Cystine Lysine Serine Asparagine Asparagine Proline Proline Cystine Lysine

-

Uninfected M. partumeiurn gill tissues Bound Cysteine Arginine Aspartic acid Glycine Threonine Glutamic acid Alanina Tyrosine Valine Tryptophan Isoleucine Leucine Cystine Lysine Serine

Free Cysteine Arginine Aspartic acid

-

Threonine Glutamic acid L

L

Valine

x

L

Cystine

-

-

15

7

L

G. amplicava sporwyst8 Bound Cysteine Arginine Aspartic acid Threonine Glutamic acid Alanine Tyrosine Valine Tryptophan Isoleucine Cystine Lysine Serine Asparagine Proline

Free

G. amplicava cercariae Bound

Cysteine Arginine Aspartic Aspartic acid acid Threonine Threonine Glutamic Glutamic acid acid Alanine Alanine Tyrosine Tyrosine Valine Tryptophan Isoleucine Cystine Lysine Serine Serine Asparagine Asparagine Proline Proline -

-

Free Aspartic acid

-

Threonine Glutamic acid Alanine Tyrosine -

-

-

Serine

-

Proline

No. of amino acids : 15

11

15

0

15

8

15

7

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

an initial increase of amino acids is now available and as these authors have stated: “ . . . (this) may be related to a change in the protein metabolism of the snails or may be indicative of enzymatic activity of the miracidia during penetration ”. Cheng (1963d), also using chromatographic techniques, has studied alterations in the compositions of both the bound and free amino acids in the tissues and plasma of three host-parasite associations ; Physa gyrina parasitized by Glypthelmins quieta sporocysts, the pelecypod Musculium partumeium parasitized by Gorgodera amplicava sporocysts, and the gastropod Helisoma trivolvis parasitized by Echinoparyphium rediae. My findings are summarized in Tables VII, V I I I and IX. From these data it is apparent that there is a decrease in detectable free amino acids in the sera of infected molluscs. The similarity between the bound and free amino acids in the trematode larvae, plus the finding that there is a significant decrease in the plasma protein concentrations in all three species of infected molluscs, together with conspicuous decreases in the hosts’ amino acids, strongly suggest that the parasites do utilize the hosts’ amino acids. It has been stated, however, that some of the loss of amino acids may be accounted for by the synthesis of some still undetermined humoral factor. The requirement of relatively large quantities of nitrogen, most probably in the form of essential amino acids, by intramolluscan trematodes is not surprising since such are needed not only for growth but also for the prolific asexual multiplication which occurs. It is known, for example, that 10 000 cercariae may be derived from a single miracidium of Schistosoma japonicum and more than 200 000 cercariae may arise from a single miracidium of 8. mansoni (Paust and Hoffman, 1934). Meyerhof and Rothschild (1940) have reported that one parasitized Littorina may emit as many as 1 300 000 Cryptocotyle lingua cercariae during the course of a year. My studies on alterations in the amino acid composition of parasitized molluscs do not reveal the initial increase as reported by Dusanic and Lewert, but then I did not investigate the molluscs shortly after infection. I n fact, the infected molluscs used in my studies were naturally infected and presumably for a considerable time since the enclosed parasites were plentiful and well developed. A note of caution should be interjected at this point for those anticipating studies on the amino acid composition of uninfected and infected marine molluscs, particularly estuarine species. Allen (196lb), working with the brackish water clam, Rangia cuneata, has shown that the concentrations of individual amino acids increase as the saliriity increases. Hence, when determining quantitative differences, the in-

TDLE IX. BOUNDAND FREEAMINOACID COMPOSITIONOF SERA(PLASMA) OF UNINFECTED AND INFECTED Helkoma trivolvk, HEPATOPANCREAS OF H . trivolvis, AND REDIAEAND CERCARIAE OF Echinoparyphiurn sp. (After Cheng, 1963d.) Uninfected H. trivolvis sera (plasma) Bound Cystine Lysine Asparagine Arginine Serine Aspartic acid Threonine Glutamic acid Glycine Alanine Cysteine Tyrosine Valine Methionine Tryptophan Leucine Isoleucine

Free Cystine Lysine Arginine Serine Aspartic acid Threonine Glutamic acid Cysteine Tyrosine Tryptophan Leucine Isoleucine

Infected H. trivolvis sera (plasma) Bound

Free

.. Bound

Cystine Cystine Lysine Lysine Asparagine Asparagine Arginine Arginine Serine Serine Aspartic Aspartic acid acid Threonine Threonine Threonine Glutamic Glutamic acid acid Glycine Glycine Alanine Alanine Cysteine Cysteine Tyrosine Tyrosine Valine Valine Methionine Methionine Tryptophan Tryptophan Leucine Leucine Isoleucine Isoleucine

Free Cystine Lysine Asparagine Arginine Serine Aspartic acid Threonine Glutamic acid Alanine Cysteine Valine -

Leucine Isoleucine

Bound Cystine Lysine Asparagine Arginine Serine Aspartic acid Threonine Glutamic acid Glycine Alanine Cysteine Tyrosine Valine Meth ion in e Tryptophan Leucine Isoleucine

r;cninoparypmum cercariae

E

Free

Bound

Cystine

Cystine Lysine Asparagine Arginine Arginine Serine Aspartic Aspartic acid acid Threonine Threonine Glutamic Glutamic acid acid Glycine Glycine Alanine Alanine Cysteine Cysteine Tyrosine Valine Valine Methionine Methionine Tryptophan Tryptophan Leucine Leucine Isoleucine Isoleucine

-

Free Cystine

-

-

Arginine Aspartic acid Threonine Glutamic acid Glycine Cysteine -

Valine Methionine Tryptophan Leucine Isoleucine

No. of amino acids : 17

12

17

1

17

13

17

13

17

12

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

fluence of the ambient sea water must be taken into consideration. From the information presented above, it is obvious that a great deal remains to be learned about amino acid uptake by parasites and TABLEX. TAURINECONTENTIN VARIOUS MOLLUSCS These data indicate that taurine is only present in marine species. (After Allen, 1961b.)

Species Gastropoda Lymnaea paluatrk Marisa cornwlrietk Pomucea bridgesi Rumina decollata Otala lactea Mesodon thyroidua Bulimulua alternatua Murex fulvescens Littorina irrorata Oliva sayana Polinices duplicata Busycon perversum Siphonaria Eineolata Fmciolaria distans Thais haemmtoma haysae Pelecypoda Anodonta grandis Quadrula quadrula Lampsilia sp. Elliptio sp. Rangia cuneata Brachidontea recurvua Crmsostrea virginica D o m variabilis Venus mercenaria Dosinia discus Area ineongrua Area campeehiensis Noetia ponderosa Cephalopoda Loliguncula brevis

Environment

Taurine

Fresh water Fresh water Fresh water Terrestrial Terrestrial Terrestrial Terrestrial Marine Marine Marine Marine Marine Marine Marine Marine Fresh water Fresh water Fresh water Fresh water Brackish-fresh water Brackish-marine Brackish-marine Marine Marine Marine Marine Marine Marine Marine

mutualists. Direct evidence of uptake is still wanting. As a speculation, it would be interesting to determine whether the decarboxylation product taurine, which has been identified from nearly every marine mollusc examined (Table X), is taken up by their parasites since its presence

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95

has not been recorded in any protozoa, mesozoa, helminth or, to my knowledge, arthropod. It has been found in the conjugated bile acids of mammals as taurocholic acid, and in other tissues as well, having been metabolized from cysteine, usually via cysteine sulfenic acid -+ cysteine sulfinic acid -+ cysteic acid -+ taurine, although alternate pathways may occur. 4. Digestive enzymes

Nothing is known at this time about the role of digestive enzymes of molluscs in the biology of parasites and mutualists. From the reviews of digestion in molluscs, primarily pelecypods and gastropods, presented by Galtsoff (1964), van Wee1 (1961) and Reid (1966), it would appear that considerably more needs to be learned about the nature and function of digestive enzymes in molluscs. 5. Control of maturation

Again, nothing is known at this time as to what host factors may be involved in the control of maturation of parasites and mutualists of molluscs other than the possible role of sex hormones on larval trematodes (Szidat, 1959). With our increasing knowledge concerning hormones and neurosecretions in molluscs (Charniaux-Cotton and Kleinholz, 1964; Simpson et al., 1966), it may be possible in the not too distant future that the influence of hosts’ hormones on the maturation of parasites and mutualists will be explored. 6. Resultant pathology I n the past, prior to the advent of a physiological or biochemical approach to understanding host-symbiont relationships, that aspect of symbiosis known as parasitism was often defined as a relationship during which the parasite inflicts some injury to its host. Although this definition has now been rejected, it still remains true, nevertheless, that parasites, in varying degrees, do very often inflict “ injury ” to their hosts. “ Injury,” unfortunately, is a difficult term to define without involving anthropocentric judgmenh. It is primarily for this reason that the older definition of parasitism is being rejected. On the other hand, pathological change, especially histopathological manifestations, expressed from a functional viewpoint, can be a rewarding and fruitful avenue of research. A certain number of studies of this nature have been made on marine molluscs in connection with parasitism, and these are reviewed below. I n considering parasite-caused pathology, it should be emphasized that evaluation of the lethality of such changes must be approached

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with great caution. It is well known that even extremely drastic histopathological changes, such as those produced by pre-adult trematodes in molluscs, need not necessarily indicate impending death. It is in many ways unfortunate that in the past molluscan pathologists, especially those interested in the pathology of shellfish, have tended to equate histopathological change with imminent mortality and thus in many instances have confused the biological significance and economic importance of certain host-parasite relationships. Despite many arguments to the contrary, there is still no perfectly satisfactory substitute for the “ cause and effect ” approach to understanding lethal agents. I n other words, despite the importance of epizootiological evidences, which may appear to be most convincing, the demonstration of Koch’s postulates still remains as the one indisputable method of proving the lethality of a parasite. The story of Hexamita being erroneously incriminated as a lethal pathogen of oysters a t one time serves as a good example. I n this section I have chosen not to discuss all aspects of histopathological change in molluscs due to zooparasites. Comments on changes related to specific parasites are given in Chapters 5-9. I n this section, only types of changes, together with examples, will be dealt with. The position of the parasite is as good a criterion as any to use in subdividing the following information. For this reason, the subheadings of endoparasitism and ectoparasitism will be employed. The following outline indicates the subcategories to be considered.

\:. * --. , ,-A’

Ectoparasitism

On the shell I n the mantle cavity

Endoparasitism

Intracellular Intercellular in tissues In the alimentary canal I n other organ systems

a. Ectoparasitism Under this heading are considered those changes associated with organisms found intimately associated with the outer surfaces of the shell as well as within the shell but not in the soft parts or in the alimentary tract.

On the shell. As far as I can determine, no true parasites have been reported from the exterior of the shells of marine molluscs; in fact, most of the animals found at these sites have been described as

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‘‘ epifauna ” or “ epizoites.” However, as Korringa (1951a) and others have pointed out, the habitats of certain populations of marine molluscs, such as oyster beds, are particularly rich in invertebrates, many of which are actually attached to the shells of oysters. Korringa has expressed the opinion that ‘‘ shelter ” is a major factor which accounts for the densities of the large number of species of organisms found associated with oyster beds. Relative to the possibility that these associated invertebrates also derive nutrients from the oysters, Korringa has stated : We wonder whether or not the oyster itself contributes to the food of its epifauna. Though I investigated this possibility very carefully, I found no evidence that any of the epibionts can be considered as true parasites feeding on the living tissues of the oyster itself. The possibility remains, however, that faeces and pseudo-faeces (dejecta and rejecta) produced by the oyster may contribute to the fertility of the habitat. However, after studying the ‘‘ epifauna ” of dummy oysters, Korringa has concluded that both the feces and pseudo-feces are not of great importance in the ecology of these associated animals. Another possible source of nutrients which Korringa has failed to consider is the “shell liquor” or mantle fluid of oysters which is continuously being exuded to the exterior during active pumping. This protein- and amino acid-rich fluid may well serve as a nutrient source. Needless to say, a great deal remains to be studied relative to the associations between oysters and associated organisms. I n addition to Korringa’s (1951a) extensive study, the ‘‘ epifauna ”, or preferably the epiphoronts, of oysters have been studied by Mdbius (1893) in German oyster beds. These investigations had led Mdbius, who also recognized the richness of such communities, to formulate the concept of “ biocoenosis ”. I n addition, Zernov (1913) has studied the fauna of the natural oyster beds of the Black Sea, Verrill (1873) has included a list of animals found on oyster beds in Vineyard Sound, Massachusetts, and Miyazaki (1938) has studied the organisms fouling the shell-strings hung from rafts in some Japanese oyster farms. Similarly, Schodduyn (1927, 1931) and FerroniAres (1901) have studied the fauna associated with oysters in France, Leloup (1940) has studied the flora and fauna in oyster beds at Ostend, Belgium, and BytinskiSalz (1935) has included notes on the epiphoronts of oysters in Rovigno d’Istria, Italy. Although undoubtedly the majority of these animals are epiphoronts, some may be commensals while others may become facultative parasites. An example of the last category is in the form of the flagellate Hexamita, probably H . in$ata. This protozoan is commonly A.M.B.-6

8

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found as a saprobic member of the community of organisms normally living at the mud-water interface in coastal areas. It has also been found as a facultative parasite in the stomach and tissues of oysters. This is especially true when anaerobic conditions and other environmental factors in oyster bed sediments reach such a level that the oysters are placed under stress. The frequency with which Hexamita is found in moribund and dead oysters has caused Mackin et al. (1952), Stein et al. (1961) and Laird (1961) t o consider it as a pathogenic parasite. However, as Shuster and Hillman (1963) have pointed out, other evidences (see under Hexamita in Chapter 5, pp. 138-142) indicate that Hexamita is not a pathogen and its presence in oysters in poor condition is the effect rather than the cause. Nevertheless, an important ecologicalparasitological principle is exemplified, that is, certain free-living organisms can become facultative parasites when unfavorable environmental conditions cause physiological stress in the host and thus render it more susceptible to parasitism. The pathology associated with Hexamita is discussed later in this section and in Chapter 5.

I n mantle cavity. Although a variety of microscopic and macroscopic animals are known to live within the mantle cavities of marine molluscs, especially pelecypods, there is no reason to believe that all of these are obligatory parasites. As the mantle cavity of molluscs, particularly pelecypods, is naturally bathed in a mixture of sea water and plasma (a mixture termed (‘shell liquor ” by the shellfishermen), it is conceivable that certain facultative invaders do become temporarily dependent upon ‘(shell liquor ” as a source of proteins and amino acids. Among the best known examples of symbionts found in the mantle cavities of marine molluscs are the grapsoid crabs belonging to the genus Pinnotheres. I n her monograph of members on t,his genus, Rathbun (1918) has compared the numerous species which have been collected from the Americas in association with various tunicates, annelids, sea urchins and molluscs. Like most authors, she has considered the majority of these crabs to be commensals. As to whether all pinnotherids are commensals, or whether any of them are, in light of their possible dependency on their molluscan hosts, must remain a moot point until further evidence becomes available. At least two species, P. pisum, the pea crab, and P. ostreum, the oyster crab, are believed to be true parasites (Dean, 1892; Orton, 1920; Stauber, 1945; Christensen and McDermott, 1958). Basing his definition of parasitism on doing harm to the host ”, Dean considered P . ostreum a parasite because this crab evidently annoys the oyster since the latter’s palps

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sometimes portray thickened out-growth or are malformed and stunted in size. Stauber and Christensen and McDermott have noticed gill injuries in Crassostrea virginica caused by Pinnotheres ostreum. Stauber has described two types of gill damage: the small-crab type, characterized by local, sharply delimited erosion of one or more demibranchs ; and the large-crab type, characterized by an extensive shortening of one of more demibranchs, reaching from the anterior end of the gills to a point usually ventral to the adductor muscle. Such gill damage in oysters harboring P. ostreum is very common. Christensen and McDermott have reported that : Nearly all infested oysters show some gill damage. Examination of 1502 oysters, all of the 1955 year class, collected from January 6th t o August 1st in 1956 revealed that about 50% had light damage, about 40% had moderate gill damage, about 9 % had heavy gill damage, and only about 1% had no discernible gill damage. Relative to the consequences of such crab-inflicted damage, these investigators have reported : Among older oysters we found a few extreme cases of heavy gill damage where there was hardly anything left of the gills, and such oysters were usually also very poor in condition. Earlier, Stauber, who has studied tissue regeneration in injured oysters, stated : Rapid healing and regeneration of gill tissue almost keeps pace with destruction and probably saves many oysters from death. He further noted, however, that crab-enclosing oysters do not keep well when maintained out of water. This could either have been due to irritation by the crab or from greater loss of ‘ I shell liquor ” resulting from an enlarged mantle cavity created by the presence of the crab or crabs. From the viewpoint of the commercial oysterman, this is a serious matter. Despite the well-documented accounts of injury to pelecypods inflicted by P. pisum and P. ostreum, ironically the gill lesions are not caused by the crabs eating the hosts’ tissues. If this is so, these crabs would qualify as parasites by every definition. It has been shown by Coupin (1894), and later confirmed by Orton (1920) and others, that P. pisum feeds on particulate food filtered from the water by the host’s filtering mechanisms. Orton, who observed this crab’s feeding behavior through “ windows ” cut in the oyster’s valves, has reported that the pea crab picks food strings from the gill margins with its chelipeds. Stauber (1945) has reported that P. ostreum feeds in the same manner

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but in addition will catch newly formed mucus-food masses with its walking legs, then reach beneath its abdomen with its chelipeds, comb the legs, and pass the food on to the mouth. It is while engaged in this type of commensalistic feeding that the crabs injure their hosts mechanically. Since the " metabolic dependency " concept of parasitism is believed to be the most objective and appears to be a far more useful one, further physiological studies on Pinnotheres must be forthcoming before we can definitely establish these crabs as parasites. The only evidence available at tliis time that suggests metabolic dependency of pinnotherid crabs on their hosts is the apparent hostspecificity of certain species (Pearce, 1962). Exceptions, however, exist. For example, Pinnotheres pugettensis, which most frequently occurs in the large tunicate Haloc~nthiaigaboja, has been found by Pearce to occur in both H . aurantium and Ascidia paratropa. Also, while adults of the mussel crab, Fabia subquadrata, are typically found in horse mussels, Modiolus modiolus, in the waters of Puget Sound, the immature crabs, according to Pearce, are frequently found in a number of smaller bivalve species. The latter situation, however, may well reflect a change in host during development rather than non-host specificity. Again, while adult pinnixid crabs, Pinnixa faba and P. littoralis, almost invariably occur only in the horse or giant calm, Schizothaerus capax, juveniles of these species are ubiquitous in 5t wide range of bivalves and larger limpets. It should be pointed out that even in the instances of Pabia subquadrata, Pinnixa faba and P. littoralis, the fact that the adults are host-specific indirectly suggests rather specific metabolic dependency. Pearce (1962) has reported that differences, especially in the integument, as seen with the light microscope and in electron micrographs, between P. faba and P. littoralis and free-living brachyurans exist and has suggested that these differences " are the result of adaptation to a protective, symbiotic environment ". Various other invertebrates have been reported from within the mantle cavities of marine molluscs. For example, in Southern California, a shrimp of the genus Betaeus is found in abalones. According to Hart (1964), this hooded shrimp is B. harfordi and its natural range extends from Maddalena Bay, Mexico, to Fort Bragg, California. It is most commonly found as a commensal in the pink abalone, Haliotis corrugata, but is also found within the red abalone, H . rufescens, the black abalone, H . cracheridii, and the green abalone, H . kamtschatkana. I n addition, it has been found in H . walbalensis, fI. sorenseni and H . assirnilis. It is also known that the so-called boring clam, Diplothyra smithii, occurs on and in the shell of Crassostrea virginica. Galtsoff (1964) has

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reported that in 1926 oysters from Matagorda Bay, Texas, were extremely heavily infested with Diplothyra smithii, with one adult harboring over 200 clams. Relative to this relationship, it is of interest to note that as the cavity bored by the clam increased and approached the nacrous layer, the oyster reacted by depositing layers of conchiolin over the nearly perforated areas (nacrezation). Thus, rarely does one find boring clams in contact with the mantle. Another example of commensals which live in the mantle cavities of marine pelecypods are hydroids of the genus Eugymnanthea. As in practically all relationships categorized as commensalism, the exact nature of the relationships between the above mentioned hydroids and their hosts remain unexamined. Another large group of arthropods which have been found within the mantle cavities or elsewhere on the surface of the soft tissues of marine molluscs is the copepods. Again, little is known about the relationships of these with their hosts although the individuals who have studied these most extensively, Wilson (1921, 1935), Yamaguti (1936), Humes (1953, 1954a, 195813) and Humes and Cressey (1958, 1960) have considered them to be parasites. On the other hand, Bocquet and Stock (1957), who have also studied them extensively, referred to these copepods as commensals. Among the pelecypods, which according to Humes (1958a) are associated with more copepods than any other molluscan class, representatives of the copepod genera Modiolicola, Tisbe, Ostrincola, Pseudomyicola, Conchyliurus, Myocheres, Paranthessius, Myicola, and others are found living in their mantle cavities where they cling to the surfaces of gills. These ectosymbionts, unlike endoparasitic copepods, are relatively unmodified in form. Little is known about the pathology, if any, caused by these copepods. Among the gastropods, the prosobranchs are hosts for a few genera of copepods, including Trochicola, Ceratocheres, Panaietis and Monstrilla. The opisthobranchs, on the other hand, are hosts to many genera among which the most common are Lichomolgus, Splanchnotrophus, Briarella, Artotrogus and Anthessius. Some, like Lichomolgus, are found on the surfaces of the gills or mantle. Others are endosymbiotic, probably parasitic, Again, the surface-dwelling species are little or not modified, while the endosymbiotic forms are generally greatly modified. Nothing is known about pathological changes associated with these copepods. RAOCL IFCP Among the cephalopods, only a few copepods have been reported. Most of t,hese are ectoparasites. For example, Cholidya occurs on the arm membranes of Benthooctopus, Anchicaligus lives in the mantle

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cavity of Nautilus, Lichomolgus is found on the gills of Sepia, and Octopicola lives on the mantle of Octopus. These, like the other ectosymbiotic copepods of molluscs, are relatively unmodified. Histopathological alteration, if any, associated with these have also not been studied. Copepod symbionts are not known from the remaining three classes of molluscs, namely, Monoplacophora, Amphineura and Scaphopoda. It is of interest to note that Humes (1958a) has stated that based on our present knowledge, host-specificity among copepod symbionts of molluscs is not very strict. Furthermore, freshwater molluscs have no known copepod symbionts. b. Endoparasitism Under this category, pathological changes associated with intracellular parasitism, intercellular parasitism, alimentary tract, and for convenience, parasitism of other organ systems, will be considered separately.

Intracellular parasitism. Although pathological changes in vertebrates, especially mammals, associated with such intracellular parasites as Plasmodium spp. have been studied extensively (see reviews by Trager, 1960a)b; Moulder, 1962), comparable studies on molluscs harboring intracellular parasites are wanting. This is especially true in the case of marine molluscs. A number of sporozoans are known to parasitize cells of marine molluscs ; however, in almost every instance investigations have been concerned with the morphology and life cycles of these parasites and not with the associated pathological changes. Intercellular parasitism. Most of the known parasites of marine molluscs fall into this category, including the large number of trematodes which commonly are found in the intertubular spaces of their hosts’ hepatopancreas. Despite the wealth of helminth fauna known from marine molluscs, few studies of pathological changes have been reported. Furthermore, many of the studies have been primarily concerned with cellular defense mechanisms, a topic already reviewed (Section 11,A, 2). Among known studies, Sparks (1963) has reported on the effects of the metacestode of Tylocephalum on Crassostrea virginica. He has noted the presence of a fibrous cyst wall surrounding each larva situated in the host’s Leydig tissues although he has stated : Whether the cyst wall is produced by the helminth or the oyster is not known, but it is assumed that the extensive cysts are the result of a fibrosis of the host tissues in response to the invasion of the parasite. He has further stated :

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Whether a pathogenic effect on the oyster resuIts from infection is not known, with no histopathological indications noted other than the extensive cyst formation, but the Condition Indices of oysters from numerous locations in Pearl Harbor (Hawaii)were consistently much lower than normal for the species for the season of the year. After commenting on the availability of nutrients and the crowded growing conditions, Sparks has concluded that it remains unknown whether the lower Condition Index (a measure of fatness) is caused by the parasite or crowding. I n a subsequent study of the same larval cestode, Cheng (1966a) has demonstrated that the cyst is of host origin and that delayed encapsulation, resulting in the death and resorption of the parasite, occurs. The fibrous encapsulation of another larval cestode, Echeneibothrium sp., in Venerupis staminea has been reported by Sparks and Chew (1966). These authors have suggested that the encapsulating fibers are collagenous, but since they have only examined sections stained with hematoxylin and eosin one cannot be certain of the chemical nature of the fibers. Recently Rifkin and Cheng (1967), using histochemistry, have demonstrated that these fibers are reticular rather than collagenous. Digenetic trematodes comprise the largest group of known parasites of marine molluscs (see Fretter and Graham, 1962). Damage to the tissues of molluscs caused by trematode larvae range from slight, involving only the dislocation of the surrounding hepatopancreatic tubules (Pratt and Lindquist, 1943), to rather severe changes, involving mechanical destruction of cells coupled with autolysis and parasiteinduced lysis (Faust, 1917, 1920; Faust and Meleney, 1924; Hurst, 1927 ; I?. G. Rees, 1934 ; W. J. Rees, 1936a; Fatham and Porter, 1936 ; Agersborg, 1924; La1 and Premvati, 1955; Cheng and James, 1960; Cheng and Snyder, 1962a; Cheng and Burton, 1965b). The types of histo- and cytopathological changes have been reviewed by Cheng and Snyder (1962a).* Such alterations are tabulated below. 1. 2. 3. 4. 5.

6. 7. 8.

Fatty bodies accumulate in hepatopancreatic cells. Vacuoles appear in the cytoplasm of hepatopancreatic cells. Karyolysis occurs. Sloughing of tissues occurs. Formation of fibromata and granulomata occurs. The tunica propria surrounding the hepatopancreas may be ruptured and penetrated by foreign bodies. Granular substances are secreted by host cells. Release of cell pigments occurs.

* For another general review of pathologic changes in gastropods due to helminths, see Wright (1966).

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9. Cells are altered from the columnar to the squamous type, especially hepatopancreatic cells. 10. Displacement of hepatopancreatic tubules occurs. 11. Abnormal mitosis in the form of multipolar spindle fibers may occur. 12. Veins in the intertubular spaces in the hepatopancreas may be ruptured. 13. Changes in the carbohydrate, lipid, and protein compositions of cells occur (discussed under Section 11, A, 3). Apparently the amount of damage inflicted by trematode larvae is in most instances directly proportional with the number of parasites present (Kikuth and Gonnert, 1948)and is also correlated with the type of larva present. Cheng (1962), for example, has pointed out that rediae, because of their capacity to actively ingest host cells, inflict comparatively more damage than do sporocysts. Similarly, Brown (1926) has shown that echinostomes, which possess a redial stage or stages, cause more damage than certain other species. Brown has also pointed out that stylet-bearing cercariae cause more damage. Little is known about the lethality caused by trematode-induced pathology. F. G. Rees (1931) has expressed the opinion that in spite of the damage done to the hepatopancreas, the vitality of the mollusc does not appear to be greatly reduced. SimilarlyKendall(1964)considers that infected molluscs “ suffer ” only moderately, at least during the initial stages of infection, Faust and Hoffman (1934) are also of the opinion that negligible damage occurs during the initial stages of infection since the first generation larvae are generally very few; however, they believe that as the result of asexual multiplication and rapid cercarial development, the tunica propria of the host’s hepatopancreas may become ruptured, which commonly leads to death. Working with Australorbis glabratus infected with Schistosoma mansoni, Schreiber and Schubert (1949) went as far as to quote a ‘‘ half-life ” for infected snails and have suggested that the frequent stimuli to shed cercariae is most probably what shortens the life-span. Similarly Pan (1965) has indicated that the high mortality of parasitized snails coincides with the heavy emergence of cercariae and the appearance of severe generalized tissue reactions. It is generally agreed that parasitized molluscs are more prone to die if they are maintained under adverse conditions. Kendall(l964) has suggested that parasitized snails exposed to stress, e.g. excessive heat, have shorter life-spans. Olivier et ul. (1953) have reported that S. mansoni-infected A. glabrutus are less able to withstand long periods under anaerobic conditions than uninfected ones. At least some of the infected snails included much less polysaccharides than uninfected ones.

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These investigators have suggested that this may be the explanation for their inability to withstand long periods under anaerobic conditions since polysaccharide metabolism is the main source of energy under anaerobic conditions. This finding may be significant when considering parasitized marine pelecypods. It has been suggested by Korringa and others that marine pelecypods, especially oysters, live in essentially an anaerobic condition when their valves are closed. Thus, the life-span of parasitized pelecypods, which are continuously closed for one reason or another, could be expected to be shorter. There are other aspects of trematode-induced pathology in molluscs which are for the most part unexplained. For example, Hurst and Walker (1933, 1935) have reported that the body temperature of parasitized Lymnaea stagnalis is 2.7 times higher than that of uninfected snails. No explanation for this is yet available other than that the temperature rise most probably reflects some metabolic change. Duerr (1965) has studied the effect of trematode parasitization on the respiratory rate of the same pulmonate gastropod. By employing a Warburg constant volume respirometer maintained at 25°C) he has demonstrated that snails infected with trematodes (germinal sacs of Cotylurus Jlabelliformis, metacercariae of C . jlabelliformis, and an unidentified xiphidiocercaria) not only have a lower oxygen consumption slope (logarithm of body weight plotted against the natural logarithm of oxygen consumption) but as a group are more variable when compared to laboratory reared uninfected snails. Duerr’s results again indicate marked physiological effects of parasitism. Vernberg and Vernberg (1963) have demonstrated another physiological manifestation of parasitization by trematodes. The investigators have found that the upper lethal thermal limits uf the mud-flat snail, Nassarius obsoletus, heavily parasitized by trematodes (Lepocreadium ovalis or Zoogonus rubellus) are significantly lower than those of uninfected snails. Another study, this one by Kendall (1964), also has revealed yet unexplained pathological alterations. It has been pointed out earlier (Section 11, A, 2) that there is generally considerable host cellular response when trematodes are introduced into unnatural molluscan hosts. Kendall has reported that if young Lymnaea palustris, L. glabra and L. stagnalis, all semi-suitable hosts, are successfully infected with Fasciola hepatica, some become stunted, their shells ar0 distorted, and the snails show signs of “ ill-health ”.

Wound healing. Little is known about wound healing following parasite-inflicted damage. F. G. Rees (1931) and Fretter and Graham (1962) have indicated that if the infection is terminated, the host

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tissues will gradually recover, undergo regeneration, and finally become normal. Cort (1941) has stated that the recovery from a trematode infection would have to involve the walling off of old sporocysts and rediae and the regeneration of tissues. He has commented that in all his examinations of snails infected with trematode larvae, he has never seen one suggesting recovery from an old infection. Not enough is known about environmental and physiological conditions which favor or inhibit the loss of infections in molluscs to warrant evaluation of these seemingly opposed viewpoints. It should be pointed out, however, that molluscs can lose their trematode infections (Standen, 1963 ; Kendall, 1964)) especially if maintained a t less than optimum conditions, such as at lower temperatures (Stirewalt, 1954). From the speculative viewpoint, there is no reason to believe that molluscs are not capable of wound healing. I n Crassostrea virginica, Mackin (1961) has suggested, although experimental proof has not been provided, that epithelial encapsulation of injured tissues could occur and the entire lesion may be discharged through the external epithelium. Pauley and Sparks (1965) have contributed a study of acute inflammatory reactions in the Pacific oyster, C. gigas. Although their experiments were not carried out long enough after injection of turpentine into the tissues to suggest wound healing, in their discussion they suggested that healing may occur by " (1) fibrous encapsulation of the injury and eventually fibrous infiltration of the wound to form a scar ; (2) the normal Leydig cells surrounding the injured area will grow together once the injurious substance is removed;" and (3) the epithelial encapsulation and discharge process suggested by Mackin discussed earlier. Pauley and Sparks (1966) have given a more detailed account of this and similar experiments in a more recent paper in which they pointed out the importance of amoebocytes in inflammatory reactions. I n the preceding paragraphs, pathological changes in molluscs in general caused by essentially intercellular parasites have been reviewed. Several of the contributors cited worked with marine molluscs. Their results are given further detailed consideration at this point. F. G. Rees (1934) has studied pathological changes in the marine gastropod Patella vulgata parasitized by the rediae of Cercaria patellae. She has demonstrated that the presence of rediae in the intertubular spaces within the hepatopancreas results in profound damage, involving local histolysis of the glandular epithelium. This is brought about by the following set of circumstances. The rediae, according to F. G. Rees, most probably reach the digestive gland via the blood-stream. While lodged between the hepato-

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pancreatic tubules, they grew rapidly, both in size and number, thus causing the lumina of the hepatopancreatic tubules to be completely obliterated and a t the same time stretching the tunica propria which covers the entire gland. The excretory wastes of the parasites greatly tax the absorptive cells of the hepatopancreatic tubules. Although some of these wastes may have initially become dissolved in the host’s plasma (hemolymph) and are passed back to the kidneys, as the infection progresses the intertubular spaces become blocked (Fig. 17) and the metabolic wastes of both the host and parasites must then be eliminated via the absorptive cells which comprise each tubule. Rees has stated : “ If they (the waste products) cannot be removed sufficiently rapidly the accumulation of acids brings about histological alterations

Fro. 16. Three hepatopancreatic cells of Patella wulgata parasitized by Cercaria patellae, showing formation of a transverse partition across each cell resulting in squamous rather than columnar cells. The luminar portion of each original columnar cell is destroyed. L , Lysed portion of cell; T P , transverse partition.

in the epithelium lining the diverticula.” Such alterations are first appreciated when the nucleus of each absorptive cell shifts towards the luminal border from its typically basal position. This is followed by increased vacuolization, followed by cytolysis of these cells a t the luminal borders where the greatest concentration of toxic products occurs. Concurrently, the regular arrangements of the cells become disrupted. Subsequently, cytoplasm, food vacuoles and excretory products are extruded into the lumen of each hepatopancreatic tubule and the cells become degenerate and undergo total or partial lysis. F. G. Rees has pointed out that not all of the cells become completely lysed. In some a new transverse partition grows across the base of each cell, enclosing the nucleus, thus giving the ultimate appearance of being squamous rather than columnar (Fig. 16). She has also pointed out that secretion

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cells, especially their membrane, are more resistant to lysis than the absorptive cells. Although the cytoplasm of the secretion cells contract and collect around their respective nuclei, the membranes remain intact except for some breakdown at the luminal borders. She is of the opinion that this difference is due to the fact that the secretion cells are not influenced by acid wastes from within since they are not involved in the process of elimination. I n addition to the hepatopancreas, F. G. Rees has also reported that the gonads become parasitized in instances of heavy infections. This phenomenon has been verified by Cheng and Cooperman (1964) who have pointed out that the reproductive system of heavily infected gastropods generally serves as a site for secondary invasion when the hepatopancreas becomes completely destroyed. Rees has also reported that the rediae increase both in size and number in the intertrabeculate spaces in the ovary and thus cause the germinal epithelium to atrophy and ultimately become destroyed. She believes that the cell destruction is due to a combination of deprivation of nutrients and mechanical pressure. Subsequently, secondary transverse partitions of the germinal epithelium become reduced to a layer of flattened cells which cover the connective tissue of the trabeculae. I n the case of extremely heavy infections, even the trabeculae break down and are devoured by the emerging cercariae. It is of interest to point out at this time that F. G. Rees is of the opinion that both physiological and mechanical factors contribute to the breakdown of the ovarian germinal epithelium. Cheng and Cooperman (1964))who have studied the invasion of the female reproductive system of Helisoma trivolvis by the sporocysts and cercariae of Glypthelmins pennsylvaniensis, interpreted the atrophy of germinal epithelium to be due primarily, if not exclusively, to mechanical pressure exerted by the parasites. F. G. Rees has also reported that the male reproductive system of Patella vulgata is invaded by the rediae and cercariae of Cercaria patellae. Again, Rees believes that the destruction and atrophy of the germinal epithelium and developing germinal cells are due to physiological degradation, specifically due to starvation, since the parasites presumably consumed the food-material intended for the developing male cells.” Her finding that C. patellae will invade the male reproductive system is of interest, as is the similar finding by W. J. Rees (1936a)summarized at a later point, since Cheng and Cooperman (1964) have reported that in Helisoma trivolvis parasitized by Glypthelmins pennsylvaniensis, the parasites do not invade the male reproductive system except accidentally through the male genital pore and become ((

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lodged in the preputium. We believe that some factor or factors associated with the male reproductive tract, perhaps the pH, discourages the parasites from invading. Thus it would appear that differences do exist which influence the suitability or unsuitability of the male reproductive system of molluscs as a site for larval trematode development. Obviously additional investigations of this nature on both freshwater and marine gastropods must be forthcoming before any postulation can be justifiably offered to explain the difference between the findings of F. G. Rees (1934), W. J. Rees (1936a) and Cheng and Cooperman (1964). In addition to invading the hepatopancreas and gonads, larval trematodes have occasionally been reported from other sites. For example, F. G. Rees (1934) has found the rediae of Cercaria patellae in the salivary glands of Patella vulgata, and W. J. Rees (1936b) has found the sporocysts of Cercaria littorinae in the blood and kidney of Littorina littorea. Apparently little injury is caused at these sites. The second extensive study of pathological changes which occur in a marine snail, Littorina littorea, as the result of parasitization by larval trematodes has been reported by W. J. Rees (1936a). This investigator has compared the effects of five species of larval trematodes on the hepatopancreatic cells and gonads of the molluscan host. The trematodes studied include Himasthla leptosoma ( = Cercaria himasthh secunda) which develops in rediae, Cercaria emasculans which develops in large sporocysts, C. lophocerca which develops in rediae, what may have been C. ubiquita ( = C. ubiquitoides) although doubtful (but will be referred to as such in this review) which develops in small sporocysts, and G. littorinae which develops in active sporocysts. He has concluded from his studies that the amount of pathological change which occurs in Littorina littorea is dependent upon three major factors: (1) whether the cercariae develop in sporocysts or rediae; (2) the sizes of the sporocysts or rediae; and (3) whether inactive sporocysts form a " blocking layer ". W. J. Rees has reported that the primary target tissue destroyed or injured by rediae, and therefore by both Himasthla leptosoma and Cercaria lophocerca,,is the gonad. It was implied that this is true in all instances in which the cercariae develop in rediae. Reports since then have shown this not to be true. Rediae, according to W. J. Rees, actively ingest gonadal tissues and germinal cells and complete destruction generally occurs. In the case of parasitization by sporocysts, and therefore by C. emasculans, C. ubiquita and C. littorinae, destruction of gonadal tissues and gametic cells may also take place. Differences, however, occur in tho. amount of damage inflicted, with both C. ubiquita

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

and C. littorinae causing far less damage than C. emascubns. The relatively mild injuries caused by C. ubiguita and C. littorinae are believed to be primarily caused by starvation resulting from competition with the parasites. I n snails infected by the former, he has reported that the starved condition is reflected in the smaller size of the eggs. I n addition to starvation, toxicity from the parasites’ waste products and mechanical pressure also contribute to the destruction and atrophy of some of the germinal cells. In the case of C. emascuZans, comparatively more severe atrophy occurs. This is believed to have resulted from the less motile sporocysts which form a “ blocking layer ” at the proximal end of the lymph channels (blood vessels) supplying the gonad. The “ blocking layer )’ is believed to prevent nutrients from reaching the gonadal tissues and hence these starve and atrophy. I n addition, waste products from the parasites are believed to contribute to the rapid disintegration of the host’s gonadal cells. I n the cases of C. emmculans, C. ubiquita and C. littorinae, the hosts’ hepatopancreas is the primary target tissue although €3. leptosoma and C. lophocerca, both of which develop in rediae, also invade the hepatopancreas secondarily. I n the mollusc’s hepatopancreas, the size and motility of the sporocysts or rediae have been reported to be important factors which govern the amount of damage inflicted. Since rediae are generally motile, those of H . leptosoma and C. Zophocerca become evenly distributed throughout the coiled gland. Of these two species, H . Eeptosoma causes more damage. W. J. Rees has expressed the opinion that the atrophy and lysis of the host’s hepatopancreatic cells, particularly the secretory cells, are due to a combination of starvation resulting from blockage of the intertubular blood channels and the accumulation of toxic waste products. Thus physiological factors, rather than direct mechanical damage, are believed to be more important. I n the case of C. lophocerca, no serious blocking of blood channels occurs. Moreover, no increased mechanical pressures are found. As the result, Rees has stated that the host is more able to cope with the excess waste products accumulated by the rediae. Cytologically, his findings are comparable to those reported by F. G. Rees (1934), including regeneration of the luminal borders of secretory cells which had been partially destroyed. It is of interest to note that W. J. Rees has stated that : ‘‘ Snails parasitized by this cercaria (C. Zophocerca) probably recover from the infestation, as the efiects produced are not very dangerous as compared with C. cmasculans.” Of the three species of larval trematodes which develop in sporo-

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cysts, C. emasculans produces the most profound pathological changes. This is because the relative larger sporocysts are little or non-motile and thus form “ blocking layers ” in the proximal portion of the hepatopancreas. These “ blocking layers ” prevent the flow of nutrients towards the cells at the distal end and at the same time prevent waste products from being conducted to the host’s nephridium. For these reasons, according to W. J. Rees, not only are the hepatopancreatic cells in the immediate vicinity of the sporocysts lysed or atrophied, but the entire organ distad to the “ blocking layer ” becomes atrophied. I n the case of C. ubiquita, the small sporocysts cause little damage. Even in fairly heavily infected snails, only local cytological changes, similar to those reported by F. G. Rees (1934), occur. Again, W. J. Rees has postulated that infected snails probably would recover from their infections. The active sporocysts of C. littorinae migrate throughout the intertubular spaces in the host’s hepatopancreas, and are relatively evenly distributed. Damage to the host’s cells is rather severe. Again this investigator believed that such is primarily due to physiological pressures resulting from excess wastes and starvation. Although the studies of F. G. and W. J. Rees are exceedingly interesting and serve as baselines for future research, direct modern biochemical evidences of the toxicity of the parasites’ excreta on host cells, as well as direct evidences for the uptake of the hosts’ nutritions, should be attempted to confirm their interpretations. Cooley (1958, 1962) has given a brief account of the histopathology in the southern oyster drill, Thais haemastoma, parasitized by the rediae of Parorchis acanthus. This trematode, in its adult stage, parasitizes marine birds, primarily gulls and terns, and utilizes a number of marine gastropods, including Urosalpinx cinerea, Thais lapillus, T . haemastoma, Littorina planaxis, L. pintado, Melogena sp. and Cerithium sp., as the intermediate host. Hopkins (1957b) had stated earlier that Parorchis acanthus larvae will castrate Thais haemastoma. Cooley has been able to confirm this. I n addition, he has reported extensive destruction of the snail’s digestive gland, which he believed to be the result of ingestion by rediae. Approximately 70-90% of the hepatopancreas is destroyed. Of the remaining tubules, most, if not all, are mechanically compressed so that their lumina are obliterated. Cooley has also reported the presence of basophilic cytoplasmic inclusions in large “ triangular cells ” of the hepatopancreas of parasitized T . haemastoma, the significance of which is not understood. He has also reported the grouping of amoeboid cells, believed to be phagocytes, around some of the rediae in the hepatopancreas. Relative to damage

au.d.

I

'.

au. b.

1

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4. ANALYSIS O F BACTORS INVOLVED I N SYMBIOSIS

to the gonads, he has reported that the damage is ‘‘ directly related in extent to severity of infection, massive infections resulting in severe to total destruction due to ingestion of host tissue by rediae.” I n my opinion, the most extensive and detailed study of cytopathological effects on a marine snail caused by larval trematodes is that recently contributed by James (1965). This investigator has studied structural and chemical changes in the hepatopancreatic cells of Littorina saxatilis tenebrosa parasitized by five species of trematodes, Cercaria ubiquita, C. Eittorinae rudis, C. roscovita, C. lebouri and C. parvatrema homoeotecnum. I n addition, he has compared the changes which occur in the host’s cells in the presence of parasites with those that occur in uninfected but starved snails. James has reported that starvation autolysis does take place in hepatopancreatic cells if the number of parasites present is sufficiently large to block off the lumina of the tubules (Fig. 17). However, if the tubules are not completely blocked, there are still reductions in the amounts of glucose, glycogen, glycoproteins, and lipids in the host’s cells accompanied by a compensatory increase in the number of food vacuoles in the digestive cells. He has also noted an increase in the number of secretory cells which he believes are sufficiently effective in removing the excretory products of the parasites from the intertubular spaces. Thus, James is of the opinion that damage to host cells results primarily from starvation and not from the lytic properties of the parasites’ wastes. I n analyzing the factors which may have influenced the amount of damage inflicted on the host’s hepatopancreatic cells, James has concluded that (1) the rate of development of the parasite in relation to the life-span of the host and the time of initial infection, (2) the mobility of the germinal sacs (sporocysts or rediae), and (3) the physiological resistance of the host, are all important. I n considering the first factor, James has noted that the normal lifespan of Littorina saxatilis tenebrosa at Twr Gwylanod, off the coast of Fxa. 11. Section of hepatopancreas of Littorina saxatilia parasitized by Cercaria Zittorime rudis sporocysts. ( 1 ) Sporocysts compressing and blocking the tubule lumen; (2) the tubules distal to the block undergoing starvation autolysis; (3) tubules proximal to the block with few food storage globules, many food vacuoles, and many secretory cells; (4)hedthy tubules, uninfected by parasites, with many food storage globules, few food vacuoles, and few secretory cells. (Redrawn after James, 1966.) au. a-j,Hepatopancreatic tubules showing various stages of starvation autolysia ; c.t., connective tissue ; di. h., healthy hepatopancreatic tubule ; di. p., hepatopancreatic tubule showing physiological effects of parasitism ;Zu., lumen of hepatopancreatic tubule ; m, sporocyst containing cercarisa ; s.c., secretory cell ; u.h., visceral hemocoelic space. A.I.B.-5

8

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

Wales, is 16 months during which it undergoes two reproductive cycles. He has found that Cercaria parvatrema homoeotecnum infects only juvenile snails shortly after birth. Thus, in theory, this trematode has practically the entire 16 months in which to affect its host. However, because of the rapid rate of the parasite’s growth and proliferation, some 80% of the host’s digestive gland is destroyed prior to its death in 5 months. C. Zebouri, C. ubiquita, C. roscovita and C. littorinae rudis, on the other hand, infect only spent adults and therefore only have from 2 to 8 months to affect their hosts, depending on whether the snails become parasitized after the first or second reproductive cycle. I n the case of C. lebouri, the rediae develop slowly and consequently, even if given 8 months, only about 15% of its host’s hepatopancreas is destroyed. Thus this parasite apparently does not destroy its host. I n fact, both parasitized and non-parasitized specimens are reported to die at approximately the same time. I n the case of C. ubiquita, the sporocysts develop at a relatively moderate rate. If its host becomes infected after the second reproductive cycle, it dies from senility 4 months later at which time 80% of its hepatopancreas is found to be destroyed. But if it is infected after the first reproductive cycle, the entire hepatopancreas is destroyed, causing its premature death in about 6 months. I n the instances of C. roscovita, C. Zittorinae rudis and C. parvatrema homoeotecnum, the development of the germinal sacs is so rapid and prolific that James has stated: “ . . . the intensity of infection and the extent of the damage to the digestive gland is limited by the damage the host can withstand before being killed and not by the available time for development.” The longevity of Littorina saxatilis tenebrosa parasitized by Cercaria parvatrema homoeotecnum has been mentioned. Those parasitized by C. roscovita and C. littorinae rudis have their digestive glandular tissues completely destroyed within 2 and 4 months, respectively, thus leading to their deaths. James has noted that if parasitization occurs in juvenile snails, such as those parasitized by C. parvatrema homoeotecnum, the hosts are prevented from reproducing completely. If the hosts are parasitized after the first reproductive cycle, as in the case of the other four trematodes studied, they do not reproduce during the second cycle. Relative to the motility of the germinal sacs, James has stated that the occurrence or non-occurrence of motility not only influences the nature of the initial infection but also the progress of the infection. Thus he is in general agreement with the earlier findings of W. J. Rees (1936a) in that if the germinal sacs (sporocysts) are only located in the proximal region of the hepatopancreas, the “ blocking layer ” formed

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would cause the destruction of the cells distad to the site of blockage. James, however, as noted earlier, disagrees with W. J. Rees who has claimed that the destruction of these cells results from the effects of accumulated parasite waste products. James is of the opinion that it is primarily due to starvation autolysis. Among the five species of trematodes compared, only C . roscovita forms a “ blocking layer.” James’s hypothesis on physiological resistance is of considerable interest and differs from any of the factors discussed earlier (Section 11,A, 2), although up to a point it is similar to the finding of Heyneman (1966). This hypothesis is briefly mentioned here. C. ubiquita will parasitize three species of Littorina 1 L. saxatilis, L. littoralis and L. littorea (Lebour, 1911; Stunkard, 1932; W. J. Rees, 193613). James has established the incidence of infection of C. ubiquita in all three species of Littorina on College Rocks, Aberystwyth, Wales, during 1958-59. These data are tabulated in Table XI. From these it is evident TABLEXI. NUMBER AND PERCENTAGE OF PARASITIZATION OF THREESPECIES OF Littorina BY Cercaritc ubiquita AT COLLEGE ROCKS,ABERYSTWYTH, WALES, DURING 1958-59 (Data from James, 1965.) Littorina littoralis Littorina saxatilis Littorina littorea

Host ~

~

No. examined No. parasitized Per cent parasitized

~

~~

523 383 73.2

~

~~

~

4 009 1093 45.25

237 1 0.42

that both L. littorulis and L. saxatilis are the more favored hosts. Upon comparing histological sections of infected snails, he has found that there is considerable pathological change associated with C. ubiquita sporocysts in L. littoralis and L. saxatilis tenebrosa, with the entire hepatopancreas almost completely destroyed. On the other hand, James has confirmed W. J. Rees’ (1936a) finding that relatively few sporocysts develop in L. littorea and hence there is little damage. He has expressed the opinion that L. littorea is more resistant. Similarly C. lebouri will parasitize the same three species of Littorina and is more frequently found in L. littorea. James is of the opinion that the comparatively little damage done in L. saxatilis tenebrosa “ may reflect the difficulty this parasite has in developing in this host and the ‘ resistance ’ of the host.” The difference between James’s observations and those of earlier workers rests in the fact that he did not report the occurrence of encapsulation around parasites in the less frequently infected hosts.

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Sudds (1960) and others, it is recalled, have found that incompatible parasites are commonly encapsulated in molluscs but need not be, although the non-encapsulated parasites do not develop normally and eventually die. Thus James’s finding suggests a third type of manifestation of resistance, where the parasites do not elicit encapsulation or die from abnormal development but develop in less numbers. Considerably more investigations of this nature must be forthcoming before any conclusions may be drawn. However, the findings of Sudds, Heyneman and James suggest the presence of some innate humoral factor(s) which is responsible for at least partial host-specificity. Another interesting aspect of James’s study is that he does not agree with W. J. Rees (1936a) that the size of the germinal sacs, especially sporocysts, influences the degree of damage inflicted. In fact, he has pointed out that the small sporocysts of C. ubiquita and C. roscovita cause more damage than the larger germinal sacs of C. parvatrema homoeotecnum and C. lebouri. He does agree that the occurrence of rediae, rather than sporocysts, especially if they possess strong pharynges, would contribute to mechanical ingestion of hepatopancreatic cells. A t this point it is of interest to comment on whether larval trematodes could cause the death of their molluscan hosts. Prom the observations of James (1965) and others, it would appear that lethality is dependent upon (1) the number of parasites present, (2) the rate of development and proliferation of the parasite as correlated with the host’s life-span, hence a specific phenomenon, and consequently (3) the amount of damage inflicted. Even then, some still unknown factor appears to be involved since Cheng and Snyder (1962a), engaged in similar studies, have stated: “ . . . the survival of Helisoma trivolvis does not appear to be impaired by Glypthelmins pennsylvaniensis even in instances of extremely dense infections where only a few isolated liver tubules were retained.” Yet during an earlier study, Cheng and James (1960) have reported that the bivalve Xphaerium striatinurn is killed when heavily infected with Crepidostomum cornutum rediae. Similarly Hopkins (1957a) and others have claimed that Bucephalus cucuZus will not kill Crassostrea virginica, its oyster host, but Millar (1963) has reported that an undetermined species of bucephalid trematode will kill oysters. Again, Cheng and Burton’s (1965b) study on the histopathology of C. virginica parasitized by Bucephalus sp. has failed to incriminate this trematode as a lethal agent. It is thus evident that critically controlled laboratory studies, involving known numbers of parasites and knowledge of their rate of proliferation, controlled nutritional and ambient factors, chemical knowledge of

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toxicity, coupled with intense studies of population fluctuations in nature, must be forthcoming before we can fully understand this aspect of host-parasite relationship. The only detailed study to date on the histopathoIogy of a marine pelecypod caused by larval trematodes is that of Cheng and Burton (1965a). We have studied the effects of the dendritic sporocysts of Bucephalus sp. on Crassostrea virginica in Rhode Island. Our findings are summarized below. In lightly infected oysters, the sporocyst branches of the parasite are confined primarily to the intertubular spaces in the digestive gland, in the space normally occupied by Leydig cells. These sporocysts appear merely to exert mechanical pressure on the digestive diverticula, pushing them towards the periphery, against the gonadal tissues. As the result, many of the diverticula are collapsed and their characteristically T- or X-shaped lumina are no longer apparent. In addition to invading the interdiverticular spaces, a number of sporocyst branches have also been observed among the Leydig cells surrounding the gut. The presence of sporocysts in this region causes the disruption and destruction of the normally loosely arranged Leydig cells and connective tissue fibers. In moderately infected oysters, the spaces normally occupied by the digestive diverticula and surrounding Leydig cells are almost completely taken over by sporocyst branches. The branches are tightly packed and what remains of the diverticula are in the form of isolated tubules and fragments. We have indicated that their appearance suggests that their destruction is the result of both mechanical and physiological pressures, comparable to those that have been described by F. G. Rees (1934) and James (1965) in marine gastropods. Again, as in lightly infected oysters, sporocysts in moderately infected ones are not confined to the region of the digestive gland. Branches infiltrate the connective tissue tunica surrounding the digestive tract also. This tunica is reduced to a thin compressed layer enveloping the gut, apparently resulting from physical pressure exerted by sporocysts extending from the area of the digestive gland. On the external periphery of the digestive gland a few sporocyst branches have been observed to infiltrate the interlobular spaces of the gonads. I n heavily parasitized oysters the digestive gland is completely obliterated, and it is of interest to note that in spite of this the oysters are not killed. Furthermore, the sporocyst branches completely infiltrate the surrounding gonadal tissues. The parasitized oysters studied were all in the female reproductive phase, hence the ovaries were destroyed. The Leydig cells normally occurring between the

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

ovarian follicles appear degenerate and most are no longer visible. The appearance of the few remaining Leydig cells suggest physiological degradation. The integrity of the ovarian lobes is completely disrupted. Moreover, the majority of the remaining ova appear degenerate and smaller, averaging 0.028 x 0.021 mm instead of the normal average of 0.053 x 0-030 mm. This finding serves to confirm the earlier observation of W. J. Rees (1936a) that the ova of Littorina littorea parasitized by Cercaria ubiquita are smaller. It is of interest to note at this point that subsequent histochemical studies by Cheng and Burton (1966) have revealed that the amount of stored glycogen in the ova of infected oysters is markedly reduced. Whether this is the result of utilization by surrounding sporocysts or due to starvation resulting from blockage of the glucose-transporting blood vessels, as suggested by W. J. Rees (1936a), remains undetermined. Resorption of ova, however, does occur. There is no doubt that parasitic castration occurs in Crassostrea virginica parasitized by Bucephalus sp. Affected tissues in extremely heavily parasitized oysters are not limited to the circumenteral connective tissue tunica, the digestive gland, and the gonads. Young sporocyst branches, as determined by the polarity of their parenchymal cells (Cheng, 1966b), do infiltrate the matrices of the palps, gills and mantle. I n addition, sporocysts are found in blood vessels, suggesting that this is a possible route of tissue infiltration, and in water tubes situated at the base of gill lamellae, which undoubtedly serve as the avenues for the infiltration of the gills. Most of the damage that occurs in the gills, palps and mantle appears to be mechanical in nature, although reduction in stored carbohydrates at these sites does occur (Cheng and Burton, 1966). The species of Bucephalus studied by us in Rhode Island is different from B. cuculus reported by McCrady (1873) and others farther south along the Atlantic coast of North America. Furthermore, it appears to be different from B. haimeanus commonly found in European oysters and clams. According to Hopkins (1954a, 1957a), B. cuculus is also different from B. haimeanus and not a synonym as has been suggested by Tennent (1905, 1906, 1909), Dawes (1946) and Yamaguti (1958). Unfortunately, detailed histopathological changes associated with both B. cuculus and B. haimeanus have not yet been studied. I n the case of B. cuculus, however, McCrady (1873), Tennent (1906) and Hopkins (1954a), as the result of gross observations, have all pointed out that the sporocysts are concentrated primarily in the gonads, and only in extremely heavy infections, resulting from prolific growth of the sporocysts, do the digestive gland and other parts of the oyster's anatomy become infiltrated. I have been able to confirm this in

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histological sections of parasitized oysters from Maryland, Virginia, and Texas kindly donated by both Dr S. H. Hopkins and Dr V. Sprague. Thus the primary site of infection between B. cucuZus and our Bucephalus sp. is different. The reason for this remains undetermined.

Parasitic castration. By comparing what is known about the primary sites of infection by trematodes in molluscs, it is quite evident that in the majority of cases the hepatopancreas is the preferred site. Cheng and Cooperman (1964) have found that larval trematodes may invade the reproductive system secondarily after completely filling the spaces enclosed by the tunica propria of the hepatopancreas, thus confirming the findings by F. G. Rees (1934),Cooley (1962) and Cheng and Burton (1965a). On the other hand, apparently among certain other species such as Himasthla leptosoma and C. lophocerca, as reported by W. J. Rees (1936a), and Bucephakus cuculus, the gonads are the primary sites of infection, followed by secondary invasion of the hepatopancreas after the gonads become completely destroyed. The invasion and destruction of the mollusc’s gonads, especially the germinal epithelium, is commonly referred to as parasitic castration. This aspect of molluscan host-parasite relationship is believed by some to be of practical importance in the biological control of medically and economically important species (Nagano, 1927 ; Brumpt, 1941 ; Bayer, 1954; Michelson, 1957). Relative to interference with gamete formation in trematodeparasitized molluscs, gametogenesis need not be completely inhibited. For example, Coelho (1954), Najarian (1961), Pesigan et al. (1958), Etges and Gresso (1965), Zischke and Zischke (1965), and others have all reported that there may be a decrease in but not necessarily complete elimination of egg production in parasitized snails. Furthermore, the percentage of sterile eggs is greater among those produced by parasitized snails (Coehlo, 1954 ; Zischke and Zischke, 1965;and others). Complete sterilization does occur also (Pan, 1965). Another aspect of parasitic castration which has interested biologists has to do with gigantism. Wesenberg-Lund (1934) appears to be the first to have reported that molluscs parasitized by larval trematodes become abnormally large. Specifically, he found that parasitized Radix auricularis ( = Lymnaea auriculata) become larger. He has offered the explanation that gigantism results from excessive growth induced by the presence of the parasites, suggesting an excessive consumption of food to meet the demands of the parasite. The classic observations on gigantism are those by Rothschild (1936,

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

1938, 1941a,b) and Rothschild and Rothschild (1939). Rothschild (1936) has reported that Hydrobia (= Peringia) ulvae parasitized by Cercaria oocysta and C. ubiquita are larger. She has expressed the opinion that the abnormal increase in size is not due to the ingestion of greater quantities of food, as suggested by Wesenberg-Lund, but " it seems more likely that this is brought about by the destruction of the gonads and other glands " thus suggesting that gigantism is a manifestation of castration. In 1938 Rothschild extended her observations on parasitized Hydrobia ulvae and was convinced that gigantism does occur. Moreover, she has concluded after studying the correlation between host size and number of cercariae produced, that : The gigantism of the host, which involves an increase in the soft parts o. the body as well as the shell, is thus of great advantage to the parasite The faculty of producing this increase in size is a character which is presumably most susceptible t o selection and its widespread occurrence among the Trematoda is therefore not surprising. Rothschild and Rothschild (1939) reported that the sizes of H . ulvae raised in the laboratory varies with the habitat, either in glass bowls or glass tubes, with those maintained in bowls growing considerably faster. They have also pointed out that uninfected females grow faster than uninfected males. Relative to the effects of parasitism, they found that parasitized snails grow faster than uninfected ones. M. Rothschild (1941a) has further confirmed her earlier finding that parasitized H . ulvae demonstrate gigantism. These grow to lengths of 9-10 mm while uninfected snails of comparable age measure 6.75 mm long. M. Rothschild (1941b) has considered three additional factors which might have accounted for the correlation between the higher incidence of parasitism in larger Littorina neritoides in addition to gigantism due to parasitism. She postulated that (1)young snails are " unattractive " to miracidia, (2) infections may be lethal to young snails and hence these are killed off, and (3) the growth rate of snails may be so greatly slowed down after attaining a certain size that the time factor alone could have accounted for the greatly increased percentage of infection in the larger size groups. She considered all three of these to be feasible auxiliary factors but, nevertheless, she considered parasite-induced gigantism to be real. By plotting the infection rate in L. neritoides which serve as the first intermediate host (cercarial production involved) and in specimens which serve as the second intermediate host (metacercarial encystment), Rothschild has demonstrated two different curves. The first reveals a low rate of infection in the small size groups, but with a steep upward slope rising to 91% in the large size groups. The second curve reveals an increasing

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uniformity up to 87% infection (Fig. 18). From these data she has concluded that the difference is probably due to the fact that primary infections cause accelerated growth of the host. It should be mentioned at this point that Hoshina and Ogino (1951) have reported that specimens of Crassostrea gigas which serve as the second intermediate host of the trematode Gymnophalloides tokiensis do not grow as fast as uninfected ones. But then, the metacercariae of G . tokiensis are encysted in the mantle of the oyster and do not effect the gonads. Most of Rothschild’s work was summarized by Rothschild and Clay (1952).

100CERCARIA B ~T0r~Ll-

SIZE GROUPS IN MILLIMETERS

FIG. 18. Percentages of infections in a population of Littorina neritoides. (Data from Lysaght, 1941; after Rothschild, 1941b.)

Lysaght (1941) has also found that the larger specimens of Littorina neritoides in nature are more frequently infected by trematodes. However, she has stated: The data as they stand do not, on first inspection, suggest that the parasites stimulate growth ;but no conclusions can be drawn, since the real information required, namely comparison of growth-rates in infected and uninfected specimens of the same initial size, is not available. Such data have been recently contributed by Pan (1965) who has

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found that the mean shell diameter of parasitized adolescent snails is larger than that of non-parasitized snails during the f i s t few weeks after infection but parasitized snails eventually become stunted. Similarly, Menzel and Hopkins (1955a,b) basing their conclusion on the growth rate of a single specimen of Crassostrea virginica, have suggested that parasitic castration by Bucephalus cuculus causes unusually fast growth during the first year of its life, followed by a lag slightly behind that of uninfected oysters. Eventually it fails to gain in length or height during the last two months of the experiment. Although gigantism in the form of weight gain has been reported in a number of parasitized hosts, including sheep infected with Fasciola hepatica (Neumann, 1905), lambs infected with trichostrongylid nematodes (Whitlock, 1949), Australorbis glabratus infected with Schistosoma mansoni (Chernin, 1960 ; Pan, 1962), rats infected with Trypanosoma lewisi (Lincicome and Shepperson, 1961;Lincicome et al., 1960, 1963 ; Lincicome, 1963), and mice ( M u s domesticus), deer mice (Peromyscus sp.) and hamsters (Cricetus sp.) experimentally inoculated with the plerocercoid (spargana) larvae of Spirometra mansonoides (Mueller, 1962, 1963, 1965a,b),the exact causes of the increased growth in all these hosts remain uncertain except that in the instances of parasitic obesity in rodents caused by S. mansonoides spargana, Mueller ( 1965a) has hypothesized that some worm-produced hormone may leak out into the host system and in some manner influence the host. Even in the detailed studies by Rothschild, the explanation offered, i.e. parasitic castration, leaves a great deal to be desired since this presumes the existence of a hormonal system in molluscs comparable to that in birds and mammals where caponization or castration is known t o bring about increased growth. It has only been in recent years that gonadal hormones have been demonstrated in gastropods (Abeloos, 1943 ; Laviolette, 1954, 1956) but their influence on growth has not yet been conclusively determined (Charniaux-Cotton and Kleinholz, 1964). Even less is known about the gonadal hormones of pelecypods. Recently, Zischke and Zischke (1965), in a preliminary note, have reported that observations on the growth of experimentally infected snails, specifically Stagnicola palustris infected with Echinostoma revolutum, indicate that the infected ones grew more slowly during the 60 days post-infection than the uninfected control snails, and Cheng et al. (1966) have found no significant differences in the shell and soft tissue weights of Nitrocris dilatatus parasitized by Prosthodendrium (Acanthatrium) anaplocami. Thus it would appear that gigantism in trematode-infected snails is not a consistent phenomenon.

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flex reversal. Also related to invasion of the molluscan hosts’ gonads by parasites is the interesting phenomenon of sex reversal. Pelseneer (1906, 1928) appears to have been the first to point out that the penes of infected snails are reduced, due, in his opinion, to the partial or complete castration of the host. It was Wesenburg-Lund (1931), however, who first proposed the theory that changes in sex in molluscs might be directly due to the effect of larval trematodes. His belief is based on his observations on the hermaphroditic snail Succinea putris in which, after the female organs are completely destroyed, the production of spermatozoa occurs for some time before complete castration. As the result of his observation, he stated : “ With some right it may be maintained that many parasitized snails pass through a male stage, or in other words that the parasite alters a hermaphrodite organism into a male.” Wesenberg-Lund’s report was followed by that of Krull (1935) who studied the morphology of a number of snails, including Hydrobia ulvae. This German investigator has reported that a reduced penis is present in parasitized H . ulvae which actually represent “ sexreversed ’’ females. He believed that these were originally females, since during the examination of internal organs which were only partially destroyed he found what he considered to be unquestionably female structures. The small penis was non-functional although there is no question that it had developed in these females. Krull did not suggest that these snails had ever functioned as males; therefore, as Rothschild (1938) has pointed out, ‘‘ sex-reversal ” is an unsatisfactory term in this instance. W. J. Rees (1936a), as the result of his study on the effects of parasitism on Littorina littorea, has reported that there are definite reductions in the size and prominence of the accessory sex organs (penis and vas deferens in males, oviduct in females) resulting from invasion of the gonads by parasites. This appears t o indicate that the sizes of these accessory sex organs are dependent upon gonadal activity. The dependence of the development of accessory reproductive organs in gastropods has since been experimentally shown to be indeed dependent upon a gonadal hormone (see review by Charniaux-Cotton and Kleinholz, 1964). Rees has found that if the snails are parasitized by either Himasthla leptosorna or Cercaria lophocerca, both of which completely destroy the host’s gonads, it is very difficult, if not impossible, to determine their sex since the accessory organs become invisible. In Littorina littorea parasitized by C. emasculans, the accessory glands are reduced but are still visible. According to W. J. Rees, this is because C. emasculans never completely destroys the host’s gonads. Although

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Rees has found no transitional stages from male to female or vice versa he did suggest that there is the possibility of sex-reversal, since among the individuals recovering from parasitization all were found to be females while among the older non-parasitized snails there was only a slight preponderance of females. As to whether this indirect evidence is reliable is discussed below in connection with the preference of host sex by larval trematodes. Rothschild (1938) has also studied manifestations of gonadal destruction in Hydrobia ulvae. She has found that the penes of all infected snails are abnormal, usually more or less reduced in size. She believed that some specimens without a penis are caused by the complete destruction of the testes. I n one infected female, she found a small penis ; however, she has stated that it is highly improbable that all the infected snails with reduced penes are sex-reversed females. From the above, it is concluded that true sex reversal in molluscs caused by trematodes has yet to be convincingly demonstrated. Krull’s findings, which have been confirmed by Rothschild, clearly indicate that females whose ovaries are completely or mostly destroyed could develop a small non-functional penis but these individuals have not been shown ever to function as males. The only direct evidence is that of Wesenberg-Lund (1931) ; however, it should be recalled that Succinea putris is a hermaphroditic snail, hence the spermatozoa observed by him may well represent gametes produced by the still functional testicular tissues. His concept that a hermaphrodite can be altered into a male can hardly be considered as true sex reversal. Moreover, it should be recalled that Cheng and Cooperman (1964) have demonstrated in Helisoma trivolvis parasitized by Glypthelmins pennsylvaniensis that the parasites prefer the female reproductive tract, thus sperm in the male tract may remain undamaged for some time. It should also be mentioned that Laviolette (1954, 1956) has shown in various arionid and limacid molluscs that castration of young animals is followed by regeneration of the gonad and thus no secondary effects are noticed. This information may be of importance in considering alterations of secondary sex organs in molluscs that are temporarily parasitized during youth.

Other effects. Still other yet unexplained or partially explained pathological changes in parasitized molluscs are known. WesenburgLund (1934) has reported that parasitized Lymnaea auriculata are often thinner and corroded and certain whorls of their shells are “ ballooned”. There are also color changes. On the other hand, Etges (1961) has reported that specimens of Helisoma anceps parasitized by the rediae of

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Cercaria reynoldsi possess thicker and more heavily calcified shells than uninfected ones. Rothschild (1936) has reported abnormal shell development in Hydrobia ulvae parasitized by Cercaria oocysta and C. ubiquita. Relative to the “ ballooning ” of shell whorls and abnormal shell development, Rothschild (1936) has suggested that such could be due to pressure exerted from the interior by the parasites. Unpublished observations of mine on several species of gastropods, including Helisomu anceps, Littorina Eittorea, H . trivolvis, Physa gyrina and Nassarius obsoletus, infected with trematode germinal sacs have revealed that abnormal growth of the shell is more common in thin-shelled freshwater species than in thick-shelled marine species. Furthermore, my observations tend to confirm Rothschild’s belief that it is the pressure exerted from within by parasites which causes the abnormal whorling and “ ballooning ”. It thus follows that abnormal shell development occurs more frequently in extremely heavily parasitized gastropods in which the sporocysts or rediae exert pressure against the mantle. It might be noted that examination of thousands of parasitized pelecypods, both marine and freshwater, has not yet revealed any abnormal shell formation. A possible explanation for this is that endoparasites of pelecypods do not as a rule exert as much pressure, if any at all, on the mantle, even in very heavy infections, since the large mantle cavity serves to “ cushion ” the exerted pressure. Relative to shell calcification in parasitized molluscs, Cheng et al. (1966) have demonstrated that the amount of ionic calcium (0.570 f 0.170 mg) in each gram of soft tissue of Nitrocris dilatatus parasitized by Prosthodendrium (Acanthatrium) anaplocami is significantly greater at the 5% level than that found in each gram of soft tissue of nonparasitized snails (0.409 f 0-242 mg) of the same age and clone. Subsequent histological and histochemical studies have revealed that the number and size of the hepatopancreatic calcium spherites in parasitized snails are increased and, when host cells become ruptured by the parasites, the release of ionic calcium stored in calcium cells, plus the breakdown of calcium phosphates which are incorporated in the spherites, both contribute free ionic calcium to the tissues. It is believed that some of the ionic calcium may eventually become incorporated in the shell, much in the same manner as during shell repair as reported by Aboling-Krogis (1960)) thus accounting for the heavier shell of certain parasitized snails (Etges, 1961). Similarly, one of my former students, Alan H. Anderson (unpublished),has found that the shells of Nassarius obsoletus parasitized by Austrobilharzia variglandis or Himasthla quissetensis sporocysts are significantly larger and heavier than those of uninfected ones.

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Another manifestation of trematode infection in molluscs was originally reported by Willey a n d Gross (1957). During the process of seeking some visible characteristic which would permit the separation of uninfected Littorina littorea from those infected with Cryptocotyle lingua, these investigators noticed that the foot of parasitized periwinkles consistently portray a dark yellow brown color while the foot of uninfected ones remains white. I n addition to C. lingua, it has been found that periwinkles infected with two other species of trematodes (unidentified) also portray the characteristic brown color. I n attempts to identify the pigment(s), the following characteristics have been

400

450

500

550

600

650

700

WAVELENGTH^^)

PIQ. 19. Absorption spectra of alcoholic extracts of Littorina Zittorecc. LI, Liver (hepatopancreas) extract ; I F , extract of foot of parasitized snail ; N P , extract of foot of non-parasitized snail. (After Willey and Gross, 1957.)

established: yellow to orange color in tissue; insoluble in water; soluble in various organic solvents, following denaturation of tissue protein ; yellow in ethanol and chloroform solution, but rose-red in carbon disulfide ; chloroform solution rendered blue on mixing with concentrated H,SO, ; pigment soluble in concentrated mineral acids and in glacial acetic acid ; turns blue or green in sulfuric acid, depending on concentration of pigment ; dissolved in CS,, the pigment shows a broad absorption band in the visible region of the spectrum. The absorption spectra of absolute alcoholic extracts have been studied, along with extracts of the liver and foot of uninfected snails (Fig. 19). It is indicated by these spectra that the pigment has an absorption peak near 450 mp, another a t 600 mp (not plotted in the figure). These correspond to the absorption peaks of alcoholic extracts of the yellow

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pigment present in the hepatopancreas of Littorina littorea since concentrated extracts of this pigment possess absorption peaks at 420, 450 and 660mp. Furthermore, as the result of comparing the characteristics of this pigment with those of known pigments (Fox, 1953), the snail pigment has been identified as a carotenoid. Willey and Gross have concluded that the pigment in the foot of parasitized snails must be released through the destruction of hepatopancreatic cells and is conducted, probably via the circulation, to the foot musculature. In connection with the release of carotenoid pigments from molluscan hepatopancreatic cells, James (1965) has commented that their release from histolyzed glands in the haemocoel may be an explanation for gigantism rather than parasitic castration, since Deuel(l957) has shown that some of the carotenoids are precursors of the growth-promoting vitamin A. It should be noted that at least two earlier workers, W. J. Rees and F. G. Rees, have reported changes in the coloration of the hepatopancreas of parasitized marine molluscs. Both of them have noticed a decrease in the amount of pigmentation in the glands of infected snails except in the instances of infection by Himasthla leptosoma in which case the parasitized gland becomes bright orange. This could mean that the rediae concentrate the released pigments during their active feeding and their presence in the gland gives the latter its bright orange color. Pigments are not limited to rediae. Nadakal (1960a,b), who studied the nature and origin of pigments found in ten species of trematodes in the marine snail Cerithidea californica, has reported that p-carotene is present in eight of these species. Furthermore, a ketocarotenoid has been identified from one of the species of sporocysts. On the other hand, only three species of rediae and none of the sporocysts contain chlorophyll derivatives. Nadakal is of the opinion, and probably correctly so, that the carotenoid and chlorophyllic pigments are passed along to the snail via its food chain and the parasites absorb these pigments from the snail host. He has also noted that there appears to be selective absorption of only the hydrocarbons from several kinds of carotenoids available in the snails. No significant metabolic alteration of pigments in the trematode larvae has been found. Thus the intake of pigments from the molluscan host can be accomplished by selective absorption or, as in the case of Himasthla leptosoma rediae, most probably by active ingestion. Ewers and Rose (1965) have reported another interesting, although still not completely understood, manifestation of parasitism in marine gastropods. They have found that a small proportion of individuals of

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many populations of the Australian mud whelk, Velacumantus australis, which occurs in large numbers along the eastern and southern coasts of Australia, possess banded shells. Each band is white, about 1-2 mm wide, and is present at the lower part of each whorl. Examination of 25 155 snails of all ages has revealed that 3.92% are banded. Upon examining 815 banded and 1 5 5 5 unbanded snails of various ages (Table XII), it was found that the incidence of larval trematodes in banded snails (5.64%) is significantly less than that in unbanded snails (9.00%). Ewers and Rose have concluded that : Banding is probably genetically controlled, and it would appear that a lowered susceptibility to larval trematode infection is associated with it. . . . Other evidence suggests that banding may be a balanced polymorphism, if this is so, parasitism is partly responsible for its maintenance. TABLEXII. NUMBERS OF BANDED AND NON-BANDED PARASITIZED AND NON-PARASITIZED Velacumantus australis (After Ewers and Rose, 1965.) Condition

Parasitized Non-parasitized Total

Banded

Non-banded

46 769 815

140 1415 1555

As mentioned in Section 11,A, 6, a (p. 97), under certain conditions the free-living saprobic flagellate Hexamita can become a facultative parasite in the gut and tissues of oysters. Various investigators, including M a c h et al. (1952), Medcof (1959), Stein et ab. (1961) and Laird (1961), have incriminated this flagellate as a pathogenic parasite. Among these investigators, Mackin et al. (1952) and Stein et al. (1961) have reported histopathological changes associated with Hexamita. Such changes are particularly evident in oysters which include moderate to high levels of Hexamita. I n these, the blood vessels portray inflammation of the vascular epithelium and at times Hexamita trophozoites are numerous enough to occlude the vessels. Along the gastrointestinal tract, necrosis of the intestinal mucosa frequently occurs when Hexamita trophozoites are common therein. Trophozoites have been reported also to invade the areas normally occupied by Leydig connective tissue cells. When such occurs, the Leydig cells are lysed and/or disarticulated. Trophozoites are also commonly found in the gill branchia. When such occurs, gill tissues frequently manifest various stages of decomposition. What remains unknown is : do these histopathological changes

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represent the effect or cause of the presence of Hexamita? If these changes are caused by the facultative parasite, the secretion of some toxic and/or lytic substance(s) is suggested since the histopathological changes indicate more than mechanical damage. But if one compares the photomicrographs of Stein et a2. (1961: Pig. 6) of the pathological intestinal mucosa with those of Sparks and Pauley (1964 : Figs. 5-8) of intestinal mucosa undergoing " normal )' post-mortem changes, it is certainly suggestive that the " histopathological changes " associated with Hexamita are not caused by the flagellates but are " normal " post-mortem necrotic changes. The same can be said of the changes in the Leydig tissues (viz. compare Sparks and Pauley's Figs. 10 and 11 with Fig. 7 of Stein et al.). Thus it may be that Hexamita, a saprobic organism, are attracted to necrotic tissues in dead or dying oysters.

Parasitization of the alimentary tract. Although a number of symbionts, particularly parasites, have been reported from the alimentary tract of marine molluscs, for example the amoeba VahlkamJiapatuxent reported by Hogue (1921) in the gut of oysters, there is essentially no information concerning their pathogenicity. Furthermore, there is no reason to believe that most of these are pathogenic, at least seriously so, or that they cause any mechanical damage. When one considers parasitization of the alimentary tract of molluscs, it should be borne in mind that a number of parasites, such as the post-egg stages of certain helminths, are temporarily found within the alimentary tract. Although these transient parasites, such as the coracidium of Tylocephalum sp., commonly do cause lesions of varying severity while penetrating the host's gut (Cheng, 1966a), they are not herein considered as intestinal or gastric parasites. Examples of what appear to be true intestinal parasites of a number of species of marine molluscs are the cyclopoid copepods of the genera Mytilicola, Piratasta and related forms. Steuer ( 1902) first described such a copepod, M . intestinalis, from the intestine of the mussel, Mytilus galloprovincialis, in the Gulf of Trieste. Since then, Mytilicola intestinalis has been repeatedly found and studied. (Other references to this parasite are given in Chapter 9, Section I, A.) It appears to be confined to European waters where it has been reported in the Adriatic Sea, the Mediterranean Sea, from Germany, the northern coast of France, Great Britain and Ireland. It was believed to be of sufficient importance as a mortality factor among commercially important marine pelecypods to form the subject of a conference held in Paris. In the proceedings of that conference published in 1951, several papers (Korringa (b) ; Korringa and Lambert ; Lambert ; Heldt ; A.M.B.-5

10

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Leloup ; Cole (a) ; Meyer and Mann ; Havinga ; Dollfus) dealt with the ecology, host mortalities, distribution, and morphology of the parasite. Unfortunately, detailed histopathological findings were apparently not available a t that time and as far as I can determine are still unavailable. Mori (1935) has described a second species, Mytilicola orientalis, in the gut of Crassostrea gigas from the Inland Sea of Japan. He has stated that the same parasite also occurs in Mytilus crassitesta in the same region. Wilson ( 1938) apparently overlooked Mori’s description since he has described representatives of Mori’s copepod as Mytilicola ostreae from Crassostrea gigas in Puget Sound, Washington. These oysters had been introduced to Puget Sound from Japan. Basing his interpretation on the older definition of a parasite, that is, one which inflicts injury to its host, Wilson did not consider Mytilicola orientalis as a parasite since, in his opinion, it does not harm the host and its mouthparts are not suited for sucking blood or biting tissues. He did point out, however, that this copepod maintains its position within the oyster’s gut by holding on with the distal segments of the second antennae which are provided with two spine-like setae and terminate in a stout curved claw. Humes) (1958a) belief that M . orientalis is a true parasite is shared by me. The fact that this copepod is consistently and frequently found in the gut of oysters and other pelecypods suggest some type of obligatory metabolic dependency. Certainly the structure of their second antennae suggests adaptation to symbiosis, most probably parasitism. Furthermore, Odlaug ( 1946), who studied the physiological state of Ostrea lurida infected with Mytilicola orientalis, has found that such oysters display a subnormal physiological condition (lower Condition Index), thus indicating a metabolic drain on the host. It should be noted that Odlaug has found that this copepod is more numerous in Mytilus edulis than in Ostrea lurida and has concluded that the mussel is the normal host, thus again suggesting some degree of obligatory relationship. Similarly Rankin (personal communication) has studied the effects of Mytilicola orientalis on Ostrea lurida. He has found that if more than five copepods parasitize one oyster, the host becomes weak and watery, and if twelve or more copepods are present, death generally ensues. I n one instance he noted erosion of the host’s gut wall caused by ingestion by the copepod. This is the k s t , although limited, observation of histopathology. Sparks (1962) has contributed a study of Mytilicola orientaliscaused histopathology in Crassostrea gigas in the State of Washington. He has reported that during light and initial infections the ciliated columnar gut epithelium remains unaltered ;however, if large numbers

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of copepods are present or if prolonged parasitization occurs, the cilia become depressed or lost and the cells are reduced to low cuboidal or squamous types. Furthermore, the mucosa may become completely eroded and, in one instance, an appendage of a copepod has been observed to protrude into the underlying connective tissue. Although the cause and effect process has not been demonstrated, Sparks did report that the cellular metaplasia described above is particularly conspicuous in gapers ”, i.e. dead oysters. It is of interest to note that no increase of leucocytes has been found in any of the parasitized oysters, even when the parasites perforate the gut. The significance of this remains unclear, although in this connection it is of interest to note that both Pillars (1921) and Griffiths and O’Rouke (1950) have reported that inflammatory lesions are absent during the initial stages of “ scaly-leg ” infections of fowls caused by the mite Knemidokoptes mutans and, similarly, Yunker and Ishak (1957) have reported that there is an absence of any inflammatory reaction in budgerigars, Melopsitta undulatus, parasitized by Knemidokoptes pilae after the initial perforation of the skin. It is believed that the arthropod exoskeleton may have served as an antigenically neutral shield that does not elicit host cellular response (see Cheng, 1964b). Sparks did report that beneath the areas of epithelial metaplasia there appears to be a trend towards fibrosis of the underlying connective tissue, with the cells becoming more densely packed and containing less cytoplasm. Sparks has stated : ‘‘ This condition suggests an attempt by the organism to protect the underlying tissue by encapsulation of the copepod, and may be a cellular aspect of resistance to a Iarge mass of foreign material as suggested by Stauber (1961).” It has also been found that Mytilicola orientalis, while occupying a large portion of the intestinal lumen, will cause the distension of the gut and in one instance extrinsically occluded a nearby blood vessel. Prom what is known about parasites which penetrate the gut wall of molluscs, it is not surprising that cellular reaction does not occur in the intestinal epithelium. Cheng (1966a)has shown this to be true when the coracidium of Tylocephalum penetrates the gut wall of Crassostrea virginica. The lack of cellular reaction even after the wall is completely perforated, as stated, may be explained by the antigenically inert property of arthropodan cuticles. This hypothesis is in need of further testing. A third species of Mytilicola, M . porrecta, has been described by Humes (1954b) in several species of marine pelecypods. Pathological changes associated with it, if any, remain unstudied. The same is true of a number of other copepods parasitic in molluscs. ((

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B. Behavioral and mechanical resistance Nothing is known at this time about behavioral and mechanical resistance or avoidance patterns among molluscs to approaching symbionts. 111. ESCAPE OF THE SYMBIONT When one discusses the escape of symbionts from marine molluscs, a definition of the verb " escape " is necessary. The term is used to designate the processes involved during the physical departure or severance of a symbiont or its germ-cell bearing progeny from the host. It is thus evident that the medusa buds, discharged from the gonangia of ectophoretic (epizootic) hydroids of molluscs, or the adult animal itself in the case of commensalistic arthropods, are the escaping forms. Similarly it i s the cercaria that escapes from trematode-parasitized molluscs, the infusoriform that escapes from mesozoa-infected gastropods and cephalopods, and presumably an encysted or pre-cystic form of an intestinal amoeba that escapes. I n instances where either the progeny or the symbiont itself effects the escape, the process may be termed active escape. I n the case of intestinal fauna which are egested in feces, the process may be termed involuntary escape. The examples cited above all indicate the departure of the mature animal or a germ-cell bearing stage. I n either case, they leave the host intact and eventually enter into a similar relationship with another host. However, still another method exists which permits the symbiont to continue its life cycle. This involves primarily those relationships during which there is metabolic dependence, that is, mutualists and parasites, primarily the latter, although not all parasites escape in this manner. This method requires the ingestion of its host by the subsequent host. Thus the escape is a passive process, herein designated as passive escape, which does not involve active behavior on the part of either the escaping form and/or the parental form, i.e. active escape, nor does it involve elimination via egestion by the host, i.e. involuntary escape. Examples of this include the encysted metacestodes of Tylocephalurn in marine pelecypods, or the encysted metacercariae of such trematodes as Himasthla spp. also in marine pelecypods. Although from experience with closely related species it is generally possible to predict at what stage in the life cycle of a symbiont it escapes from its host, there is practically nothing known about the mechanisms involved. This is especially true of parasites and mutualists. I n the case of intestina1 protozoa, it is assumed that these can be expelled by involuntary escape. Similarly, those species, such as the sporozoan Aggregata eberthi in the cuttlefish, Sepia oficinalis, which

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form spores are expelled by involuntary escape despite the fact that they undergo one phase of their development (sexual reproduction in the case of A. eberthi) in the cells lining the host’s gut. I n the case of another sporozoan, Nematopsis ostrearum, which is found in the mantle, adductor muscle, heart, gills, labial palps, and perhaps other organs of oysters as vegetative spores or sporozoites (Prytherch, 1938, 1940; Sprague and Orr, 1955), the exact mechanism of escape remains undetermined. Feng (1958) has demonstrated that this parasite does escape by transplanting oysters, Crassostrea virginica, with high and low initial infections into areas of low and high infections, respectively. Subsequent checks have revealed that the transplanted oysters attain the characteristic level of infections of oysters native to that area. Thus a dynamic equilibrium of elimination (escape) and reinfection appears to exist. Feng has postulated that the spores may be phagocytized by the oyster’s leucocytes and are eliminated across epithelial surfaces like experimentally introduced India ink and vertebrate erythrocytes (Stauber, 1950; Tripp, 1958a) but this has yet to be demonstrated. If his postulation is correct, then the escape of Nematopsis ostrearum from oysters is effected not by its own motility or behavior, but by the host’s cells. This represents another possible method of escape, herein designated as cellular escape. Our present knowledge concerning the escape of trematode cercariae, which is the best understood among zoosymbionts, is based mainly on schistosome cercariae. Leiper (1915)) working with Schistosoma rnansoni, has observed that cercariae are discharged in puffs and quite independently of the passage of feces. Lutz (1919)) who also studied S. mansoni, has suggested that all channels on the mollusc’s body surface are used and has even reported emergence to take place via the intestinal canal. Faust and Meleney (1924) maintain that S. japonicurn cercariae accumulate in the spaces between the hepatopancreatic tubules and cause the tunica propria which surrounds the entire gland to burst, thus permitting the cercariae to escape in swarms between the body wall and the shell of the snail. Faust and Hoffman (1934) have reported the same for X. mansoni cercariae. Brumpt (1941) has demonstrated S. mansoni cercariae within the eggs of Australorbis glabratus, a phenomenon verified by Etges and Gresso (1965), and has concluded that escape occurs via the snail’s genital ducts. Duke (1952)) also working with 8. mansoni, has shown that cercarial exodus takes place chiefly at the pseudobranch and collar and has suggested that they travel with the main flow of the venous blood, which passes down alongside the rectum, before being returned to the heart via the mantle. Furthermore, he regards the escape as an

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

active process, with the cercariae utilizing glandular secretions combined with mechanical activity. Richards (1961) has confirmed Duke’s observations. The escape mechanism among non-schistosome ceroariae is known only for three species. Kendall and McCullough (1951), working with Fasciola hepatica cercariae, have reported that these travel to the site of emergence, which is in the region contiguous with the snail’s anus, and that emergence is a passive (involuntary) process, at least in the later stages. The force behind the ejection is in the form of pressures set up in the host’s mantle cavity which in turn cause pressures to be exerted on the perivisceral spaces containing the cercariae. Pearson (1961), working with Neodiplostomum intermedium cercariae in Pettancylus assirnilis, has observed cercariae free within the visceral haemocoel and has stated that “ cercariae crawled about the visceral hnemocoel, probing vigorously with the anterior end, and entered the first part of the peri-rectal sinus and mantle vessels ”. He has further observed that cercariae escape from the mantle via a fixed “escape pore ”. According to him, a similar phenomenon has been observed in seven other species of strigeid cercariae. Finally, Probert and Erasmus (1965) have contributed a detailed study of the migration and escape of the strigeid cercaria, Cercaria X, from Lymnaea stagnalis. By studying serial sections of infected snails, they have found that the main pathway along which migrating cercariae travel is the blood vascular system. The cercariae enter the main visceral haemocoel after the tunica propria of the host’s hepatopancreas ruptures, and from there enter the main venous vessels, deploying through the rectal sinus, sub-renal sinus, lung, and heart. The cercariae then travel from the heart via the mantle arteries and become localized in the mantle sinuses prior to emergence. These investigators are of the opinion that while in the circulatory system the cercariae are partially active. The inner surface of the mantle, particularly the leading edge, is said to be the main site of escape. Unlike the cercariae of F . hepatica as has been reported by Kendall and McCullough (1951), Probert and Erasmus believe that the final escape of Cercaria X is an active (voluntary) process. From the above, it would appear that cercariae can escape from their molluscan hosts via different routes and can be either actively motile, passively carried along in the blood, or pushed by pressures.

CHAPTER 5

PARASITES OF COMMERCIALLY IMPORTANT MARINE MOLLUSCS THE PHYLUM PROTOZOA What is a commercially important marine mollusc? Presumably the answer is that it is one which is harvested commercially and used on a large scale as a food source. Thus on the east coast of the United States, the American oyster, Crassostrea virginica, the soft clam, Mya arenuria, the quahaug, Mercenaria mercenaria, the bay scallop, Aequipecten irradians, and the surf clam, Xpisula solidissima, would certainly qualify. From Virginia to Texas, Mercenaria campechiensis, the southern quahaug, is harvested in large quantities. Similarly, along the Atlantic coast of Canada, Crassostrea virginica would be considered the major marine economic mollusc. Along the Pacific coast of the United States, the native Pacific oyster, Ostrea lurida, the Japanese oyster, Crassostrea gigas, the soft clam, M y a arenaria, the so-called Japanese little neck clam, Tapes semidecussata (= Venerupis semidecussata, V . philippinarum, Paphia philippinarum, Tapes japonica), the razor clam, Siliqua patula, the pismo clam, Tivela stultorum, a number of species of abalone, Haliotis spp., as well as several other species of pelecypods would qualify. In British and northern European waters, the European oyster, Ostrea edulis, the cockles, Cardium spp., the mussel, Mytilus edulis, and the buckie, Buccinum undatum, are harvested commercially, while in the Mediterranean, conchs of the genus Busycon, the pelecypods Tapes decussatus, Mytilus galloprovincialis, as well as other molluscs are of commercial importance. I n the Far East, commercial shellfisheries in Japan are primarily limited to the Japanese oyster, Crassostrea gigas, and the clam, Tapes semidecussata, although other species are also harvested. As one travels throughout the world and investigates the species of molluscs incorporated in the diets of various ethnic groups, a number of species of marine molluscs must definitely be considered to be of economic importance since not only are these frequently used as food, but are regularly found in markets. Among the Japanese and Chinese, for example, various species of squids and octopi form a part of the 136

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common diet. I n Hawaii and other islands of the Pacific, the limpet, Patella hawaiiensis, and related species are served in a traditional dish eaten a t Polynesian banquets or “ luaus ”. Similarly, on the continental United States, various estuarine molluscs are utilized as food by various ethnic groups. For example, although the “ Yankee ” New Englander seldom eats the blue mussel, Mytilus edulis, this pelecypod is harvested and shipped to the markets of New York City where it is considered a delicacy by people of Italian descent. Again, the common periwinkle, Littorina littorea, has been eaten in England for hundreds of years. For the year 1867, according to Zinn (1964), 76 000 baskets of this snail, weighing 1 900 tons and worth upwards of E50 000 were consumed in London alone. Littorina spp. are also eaten in Scotland, Ireland, China, Japan, and in the United States, where they are considered a delicacy by people of southern European descent. One could cite numerous other examples of marine molluscs which are used in relatively large quantities throughout the world as food. Indeed, molluscs represent one group of marine animals which is already serving to fulfill the need for proteins by a large percentage of the world’s population. As the result of the large number of species of marine molluscs that are used as food, some globally, others in restricted areas, the major difficulty encountered during the compilation of the following information was to decide which hosts to include and thus limit the number of parasites to be considered to a reasonable number. The decision was made to include those species mentioned in the preceding paragraphs except for the cephalopods, periwinkles and limpets, and a few other related species. Information pertaining to the parasites of Littorina spp., especially the helminths of those occurring in British waters, can be found in Fretter and Graham’s (1962) review. If no parasites are listed for certain of the other species mentioned, it is because none is yet known. As stated in Chapter 3, as a consequence of the economic importance of shellfisheries and possible public health implications, the parasites of economically important molluscs have been receiving increasingly more attention although a great deal remains to be learned. It is with the shellfishery biologist in mind that the following information has been compiled. Professional parasitologists, who generally have access to more adequate library facilities, may find the information elementary, but even so some may find the information useful, especially those being introduced to marine parasites for the first time. Relative to the public health importance of the parasites considered herein, there is no information available on the majority of these. The

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absence of statements concerning the public health significance of a species indicates that nothing is known.

I. Phylum PROTOZOA Although many species of protozoa are known to be parasites of vertebrates and invertebrates, including marine animals, comparatively few have been reported from commercially important marine molluscs. Those which have been reported are briefly described and information pertinent to these, including their life cycles, ecology, physiology, pathology, and other types of information are given under appropriate subheadings. The omission of specific subheadings indicates the lack of information in these areas. The classification employed herein for the higher taxa (order and higher) is that recommended by Honigberg et al. (1964) who comprise the Committee on Taxonomy and Taxonomic Problems of the Society of Protozoologists. The familial designations used are those of Kudo (1966) or, if not listed in that treatise, those of the original authors. A. Subphylum

SARCOMASTIGOPHOREA

Superclass Mastigophora Although several species of flagellates have been reported as symbionts, primarily as parasites, of molluscs (cf. Dujardin, 1841 ; Kozloff, 1945; Reynolds, 1936)) as far as I can determine, only one species, Hexamita injlata, has been reported as a parasite of commercially important marine molluscs, specifically the oysters, Ostrea edulis and Crassostrea virginica. A second organism, originally named Trypanosoma balbianii, was described by Certes (1882, 1883) from the stomach contents of oysters, Ostrea edulis and Crassostrea angulata, taken a t Cancale and Marennes, on the west coast of France. Certes believed that this “ spirillum-like ” organism, which measures from 0.04 to 0.12 mm long x 0.001 to 0.003 mm thick, is related to one of the true trypanosomes, T . sanguinis, a blood parasite of frogs, and also to the so-called “ Trypanosoma eberthi ” described by S. Kent from the intestine of a duck. Certes’s report was followed by that of Mobius (1883) who stated that he had observed T . balbianii in 1869 in oysters taken a t Schleswig-Holstein, Germany. I n the same year Ryder (1883), working at Woods Hole, Massachusetts, noted that T . balbianii is probably similar to a spiral organism which he had observed in the stomach of Crassostrea virginica and named Spirillum ostrearum. According to Ryder, this organism is not a flagellated protozoan but a schizomycetous fungus which can be 10.

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MARINE MOLLUSCS AS HOSTS F O R SYMBIOSES

found in large numbers in the stomach and around the crystalline style of oysters. The present concensus of opinion is that Certes’s “ Trypanosoma balbianii ” and Ryder’s ‘‘ Spirillum ostrearum ” represent the same or closely related spirochaetes. It should be noted that Ryder has stated ‘‘ Yet, upon eating these same individuals (oysters) known to be infested with parasites, no inconvenience was experienced, showing that these organisms, whatever they may be, are probably harmless t o man.” Hexamita injlata is considered herein as the only known zooflagellate parasite of oysters. 1. Hexamita injlata Dujardin, 1841. (Fig. 20) (Class Zoomastigophorea; order Oxymonadida; family Hexamitidae)

Hexamita in$ata, a common free-living saprobic flagellate, was originally observed in the stomach of oysters, Ostrea edulis, by the French biologist Certes (1882) who considered it to be a commensal.

FIG.20. Hexamita inJlatu trophozoite. (Redrawn after Cheng, 1964b.)

Since then, this flagellate has been repeatedly found in both 0. edulis and Crassostrea virginica in both Europe and North America. The opinion now generally held is that this protozoan is a facultative parasite when found in oysters. Description. Body broadly oval, 13-25 p x 9-15 p, posterior terminal truncate; two nuclei near anterior end, one on each side of body, clearly visible in properly stained specimens only; eight flagella present, six anterolateral and two trailing ; two inconspicuous axo-

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139

styles extending along length of body, not readily visible in living specimens ; one to two contractile vacuoles in free-living forms ; cytostome obscure ; endoplasm with refractile granules. Life cycle. The complete life cycle of H . inJlata is not known but that of H . salmonis, a related species found in the intestine of various species of trout and salmon, is known (Moore, 1922, 1923 ; Davies, 1925). I n H . salmonis, schizogony is said to occur in the lining epithelium of the piscine host's pyloric caeca and intestine although this observation is in need of verification. Encystment occurs, with the cyst serving as the infective form which is transmitted from fish to fish via the ingestion of contaminated water. Similarly, in the case of H . intestinalis, another related species found in the intestine of frogs and of Trutta fario and in the rectum of Motella tricirrata and M . mustela in European waters, cysts are known to exist (Alexeieff, 1912). As mentioned earlier, Hexamita inJlata is usually a free-living saprobic species commonly found in the proximity of oyster beds. It is only under unfavorable ambient conditions, when the oyster is placed under physiological stress, that H . inJlata invades the pelecypod and becomes a facultative parasite. Although so-called " intracellular stages ') of H . inJlata in oysters have been reported by Mackin et al. (1952), these have not been conclusively implicated as representing stages in the life cycle of this flagellate. I n fact, these authors have stated that " an effort . . . to piece the various intracellular elements of the intestinal epithelium and the leucocytes into a cohesive life cycle (was) without success.)' Scheltema (1962) has infected Crassostrea virginica experimentally. He placed fifteen 2-year-old oysters in each of two aquaria containing 7 liters of sea water maintained at 5.8 f 0.8"C. To each aquarium was added high concentrations of Hexamita trophozoites. It was found after 33 days that a very dense population of Hexamita grew in the aquaria water with the numbers observed in both aquaria equaling those found in heavily infected oyster stomachs (ca l o 4 organisms per ml). Of the thirty oysters, only one died and among the survivors, only 37.9% were infected. Furthermore, the infections that did occur were not heavy. Scheltema concluded that: " Thus even under exposure to extraordinarily high concentrations of Hexamita, no significant mortality occurred and the index of incidence remained relatively low." From this and similar unpublished reports by others, it appears quite definite that H . inJlata is not a highly lethal parasite as has been claimed by Mackin et al. (1952) and Stein et al. (1961), although there is some indication that the mortality rate of parasitized oysters maintained a t low temperatures is greater than that of controls (Stein et al., 1961).

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Ecology. It is known from studies along the Atlantic coast of North America that the occurrence of Hexamita in Crassostreu virginicu fluctuates with the season and hence with the environmental temperature. Scheltema (1962), in one of the few published reports, although ample evidence has been collected by Dr. H. H. Haskin of Rutgers University, has demonstrated that in oysters dredged from Delaware

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FIG.21. Incidence of Hexarnita in the stomach of dredged samples of living oysters, Crassoatrea virginica, taken from Delaware Bay between May 1959 and April 1960. Solid bars denote per cent incidence, white bars denote index of weighted incidence. Numbers above bars give the sample size on which the data from each month are based. Points on the temperature curve indicate single measurements of bottom temperature taken a t the time oyster collections were made. (After Scheltema, 1962.)

Bay during May 1959 to April 1960 the incidence of flagellates is highest during the winter months and lowest during the summer and early fall (Fig. 21). Scheltema has also studied the reproductive rate of Hexamita grown in cultures a t different temperatures. He has found that starting with an equal volume of inoculum, the number of flagellates in cultures maintained at 14-22°C multiply rapidly, reaching concentrations of 10 x 104 organisms per ml within 2 or 3 days. I n one experiment at

5 . THE PHYLUM PROTOZOA

141

22"C, concentrations of more than 50 x lo4 organisms per ml were reached in 3 days. Cultures maintained a t 14-22°C decline rapidly, however, and trophozoites cannot be found within a week. I n cultures maintained a t - 0.2 to 50"C, the organisms grow slower. The number of flagellates reaches concentrations equal to those grown a t 14-22°C in 3-4 weeks. It has also been determined that temperatures above 25°C are lethal to Hexamita. Knowing the thermal death point of Hexamita, Scheltema has proposed the following hypothesis to explain the seasonal fluctuation of this flagellate in oysters. During the winter, when the oyster's metabolism is very low, Hexamita is apparently capable of reproducing rapidly enough so that it is found in a large percentage of the oysters. On the other hand, during the late spring and early summer months, although the flagellate reproduces more rapidly, the processes within the oyster which remove Hexamita, i.e. phagocytosis by leucocytes, exceed the flagellate's reproductive rate and hence its number declines. However, if the oyster's metabolic rate is for some reason lowered, the increased divisional rate of the flagellates results in the occurrence of large numbers of flagellates, hence the large numbers of Hexamita in physiologically stressed or moribund oysters. During the summer months the temperature approaches or even reaches the thermal death point of Hexamita and hence it is seldom found in large numbers in oysters during this period.

Physiology. Practically nothing is know about the physiology of H . in$ata other than that its reproductive rate is dependent upon the ambient temperature. Thus far H . inflata has been cultured in two types of media. (1) It has been successfully cultured in " shell liquor " (mantle fluid) from oysters filtered through No. 42 Whatman paper and subsequently through a 0.8 p Millipore filter. Streptomycin is added to 15 ml samples of the filtrate in standard bacteriological culture tubes as a bacteriastatic agent. ( 2 ) Hexamita has also been cultured in a medium consisting of 15 ml of filtered sea water in a bacteriological culture tube to which is added one small shucked oyster. Oysters to be used for this purpose are first held a t 30°C for about 1 h. Streptomycin is added to each tube to inhibit bacterial growth (Scheltema, 1962). Pathology. A detailed account of the histopathological changes which occur in oysters associated with H . inflata has been given earlier (see Chapter 4,Section 11,A, 6). Although rather severe alterations are sometimes found in infected oysters, such as severe atrophy of the surrounding Leydig tissues (Fig. 22), it is not known whether such

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changes are caused by Hexarrhita trophozoites or represent necrotic tissues of moribund oysters which in turn attract saprobic flagellates. I n any case, experimental evidences support the concept that H . inJlata is not a serious pathogen or a lethal agent.

FIG.22. Section of Crnssostrea virginiea parasitized by Hexamita showing destruction of surrounding Leydig cells.

B. Subphylum

SARCOMASTIGOPHOREA

Superclass Sarcodina As is the case with flagellates, although many species of amoebae have been reported as symbionts, including parasites, of vertebrates and invertebrates, as far as I have been able to determine, only t,wo species, Vahlkampjia calkensi and V . patuxent, have been described from a commercially important marine mollusc, the American oyster, Crassostrea virginica."

* Sawyer (1966) has reported in an abstract that '' Studies on G. virginica from Chesapeake Bay, Md. have revealed 4 different amoeboid organisms of which two show affinities to the Amoebidae and two to the Labyrinthulidae and Vampyrellidae ". The identities of these amoeboid protozoa are still unknown. I n addition, Sawyer has noted that Amoeba radiosa sp. inq. (see Bovee, 1951, 1953, 1964), a free-living amoeba commonly found in fresh water, has been found in Crassostrea virginica. The occasional occurrence of A . radiosa in the alimentary tract of oysters undoubtedly represent instances of an accidental transcient relationship rather than symbiosis.

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143

1. Vahlkampjia calkensi Hogue, 1915*. (Figs. 23-25) (Class Rhizopodea; order Amoebida; family Amoebidae) According t o Hogue (1915), who originally described this amoeba, Vahlkampjia calkensi was originally found in the liquid content of the digestive tract of oysters, Crassostrea virginica, found on a tramp steamer a t Woods Hole, Massachusetts, in 1912. Subsequently, it was redis-

FIGS.23-26. Vahlkampfia calkensi. (23) Trophozoite showing typical broad forward moving ectoplasmic pseudopodium and elongated trailing portion, notice rounded karyosome surrounded by clear zone in nucleus ; (24) formation of endogenous buds in multinucleated form ; (25) typical cyst, showing three irregular cyst wall layers. (Redrawn after Hogue, 1915.)

covered in most of the oysters from the New York City area while oysters from Cape Cod were found t o be particularly free of this amoeba. Among the infected oysters examined, some included numerous amoebae while others included only a few.

* The validity of Vahlkampfia calkensi and the related species, V . patuxent, has been questioned by Schaeffer (1926). Although Hyman (1940) continues to recognize Vahlkampfia as a valid genus of ‘‘ small limax-type amoebas. . . inhabiting the intestine, distinguished mainly by their type of mitosis. . . ”, as do Kudo (1966) and others, Schaeffer (1926) transferred both species to the genus FlabelZulu. If this interpretation is accepted, then V . calkensi and V . patuxent should be designated as F. calkensi and P. patuzent respectively. Sawyer’s (1966) statement that Bovee has transferred both species to the genus Vannella is in error. Bovee (1966 and personal communication) has followed Schaeffer’s interpretation in considering these species as member of Flubellula, the characteristics of which genus he has emended.

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Description. Small amoebae, specimens removed from oysters measure 10-3Op in greatest diameter (average 15p), those grown in culture may measure 45 p in greatest diameter (average 26.3 p) ; forward moving surface comprised of large ectoplasmic pseudopodium which may be multi-branched ; posterior portion of body slightly elongated, frequently with many small fine ectoplasmic pseudopodia ; ecto- and endoplasms distinct ; conspicuous refractile granules in specimens freshly taken from host ; large spherules visible in endoplasm when stained with methylene blue GG or methylene blue R : normally without contractile vacuoles but vacuoles will appear in specimens maintained in hypotonic media ; nucleus round or oval, in endoplasm, with chromatin granules associated with circumkaryosomic zone ; undergo exogenous as well as endogenous budding (Fig. 24). Cyst opaque, dorsoventrally flattened, round but with irregular edges, consists of three layers (Fig. 25).

Life cycle. The life cycle of V . calkensi remains incompletely known although both trophozoites and cysts are known to occur. It is not known for certain whether oysters become infected through the ingestion of cysts although this is possible. Ecology. Nothing is known about the ecology of V . calkensi except that Hogue (1915))during culture studies, has noted that it will thrive not only on isotonic media but also will adapt readily to a hypotonic one. Physiology. Hogue (1915), has been successful in culturing V . calkensi on a semi-solid medium comprised of 1.5% agar prepared with either sea water or a 0.7% NaCl solution. While on such a medium, the amoebae thrive on the accompanying bacteria but gradually become smaller. However, when oyster “ broth ” (body fluids) is added, the amoebae increase in size. Hogue also has been successful a t culturing this amoeba on the same agar with 0.4% Witte’s peptone, on agar containing “ ova mucoid ” and a little egg yolk, and in depression slides to which sterile oyster “ broth ” is added daily. Pathology. Apparently V . calkensi does no damage to Crassostrea virginica. Since it will thrive on bacteria, it is assumed that this amoeba normally feeds on the intestinal bacterial flora of its host, although it may be dependent on the host for other requirements. It is not known whether its relationship with the oyster is obligatory although the same amoeba has never been found free-living in estuarine waters, but then the protozoan fauna of oyster beds, as Korringa (1951a) has pointed out, has not been thoroughly studied.

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It is of interest t o note t h a t Kudo (1966) has speculated " that Vahlkampjia,Hydramoeba, Schizamoeba a n d Endamoeba, are the different stages of the course t h e intestinal amoebae might have taken during their evolution ." Basing his definition of parasitism on t h e criterion of infliction of injury t o the host, Kudo has proposed t h a t : Obviously endocommensalism in the alimentary canal (as in the case of Vahlkampjia) was the initial phase of endoparasitism. When these endocommensals began to consume an excessive amount of food or to feed on the tissue cells of the host gut, they became the true endoparasites. H e has further stated : Destroying or penetrating through the intestinal wall, they became first established in the body of organ cavities and then invaded tissues, cells or even nuclei, t,hus developing into pathogenic Protozoa. 2.

Vahlknmpjia patuxent Hogue, 1921." (Figs. 26-28) (Class Rhizopoda; order Amoebida; family Amoebidae)

This amoeba was originally found in the contents of the alimentary tract of Crassostrea virginica b y Hogue (1921) collected from the Patuxent River, Maryland.

FIGS.26-28. Vahlkampjn patuxent. (26) Trophozoite ; (27) greatly elongated trophozoites grown on agar slides to which a drop of 0.7% NaCl had been added; (28) trophozoitos crawling along mould hypha. (Redrawn after Hogue, 1921 .)

*

See footnote on p. 143 relative t o Vnhlkampja spp,

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

Description. Body usually broad and fan-shaped, with large clear ectoplasmic pseudopodium a t forward moving end ; posterior end commonly pointed, usually with small fine pseudopodia extending from it ; specimens taken from plate culture during initial 3-4 days are more or less uniform in size, measuring 21 p long x 13 p wide, after that many small amoebae, resulting from division and measuring 1-6 p in greater diameter, are present in addition to giant multinucleated forms which may be as large as 143.3 p in greatest diameter ; ecto- and endoplasms distinct ; with small dense and large refractile granules in endoplasm ; small granules may enter ectoplasm in moribund specimens but large ones are limited to endoplasm ; no contractile vacuoles present ; some food vacuoles, filled with bacteria and other finely granular materials, present ; nucleus generally spherical, with large slightly eccentric karyosome; large and small chromatin granules on inner surface of nuclear membrane ; reproduction by binary fission and exogenous and endogenous budding. Cyst spherical, thin, transparent and with regular outline ; believed to be semipermeable ; varies greatly in size, those found on surface of agar cultures measure 11-1 8 p in diameter, those found in hanging-drop cultures measure 4-12 p in diameter ; single nucleus present ; cytoplasm with one or more vacuoles.

Life cycle. The life cycle of 8.patuxent is only known from laboratory observations on cultures (Hogue, 1921). Trophozoites reproduce by binary fission and by budding, both exogenously and endogenously. Occasionally encystment occurs but excystment has not been observed. How oysters become infected is not known although presumably cysts, and even trophozoites, if the latter is ever free-living, could be ingested. Ecology. Hogue (1921) has studied the influence of the environment on cultures of V . patuxent. Earlier (Hogue, 1917) she had demonstrated that the density of the culture medium influences the shape of amoebae. This was confirmed in 1921. If V . patuxent is cultured on agar to which no moisture is added, the amoebae group together and even overlap. When a 0.7% NaCl solution is added to the agar surface, the amoebae move about freely and those a t the edge become greatly elongated (Fig. 27). Hogue has proposed that the reason for this is the one given earlier by Uhlenhuth (1915) who claimed it is due to the fact that the desired relation between the consistency of the medium and the consistency of the cell plasma has been reached resulting in greater freedom of movement.” Hogue also has demonstrated that if amoebae are contained in a drop of agar medium contaminated with moulds, they immediately stretch out along the fungal hyphae if contact is made (Fig. 28). I‘

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V . patuxent, according to Hogue, is also influenced by temperature. When amoebae cultured on agar plates are maintained for 24 h at 35OC and later removed, numerous trophozoites encyst. Similarly, trophozoites cultured in a hanging drop of sterile 0.7% NaCl solution are influenced t o round up or encyst if maintained a t 35°C. At low temperatures, 10-15"C, the amoebae multiply more slowly than if maintained at 18-23°C. Physiology. Physiological processes of V . patuxent in the modern sense have not been studied. There is some information on the cultivation of this amoeba. Hogue (1921) has reported successful growth and division in both solid and liquid media. One type of suitable solid medium is composed of 1.5% agar in 0.7y0, 1.0% or 1.5% NaCl solution. The amoebae grow equally well on all three. The second type of suitable solid medium consists of: Agar Sodium chloride Calcium chloride Potassium chloride Sodium bicarbonate Dextrose

g 1.5 0.9 0.024 0.042 0.02 0.25

The liquid medium used consists of Locke's solution to which ovomucoid " is added. The latter is made by adding 200 ml of 1.0 or 1.5% NaCl solution to the albumin of an egg. This is cooked for 30 min after which it is filtered. To each tube of Locke's solution is added one loop full of the " ovomucoid " and the tubes are autoclaved before use. Hogue also has attempted to use a stab culture comprised of 1.5% agar, 0.4% peptone, and 1.0 or 1.5% NaCl ; however, in this medium trophozoites only grow on the surface, thus suggesting a requirement for oxygen. "

Pathology. Like V . calkensi, V . patuxent does not appear to be pathogenic to Crassostrea virginica. It appears to feed primarily on the bacterial flora in its host's digestive tract, although its other growth requirements have not been defined. I n experiments designed to test the pathogenicity of V . patuxent to mammals, Hogue (1921) introduced trophozoites into kittens both per 0s and per rectum. She failed to establish infection in any of the kittens and concluded that this amoeba is not infective to kittens and probably also not to man.

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C. Subphylum

SPOROZOA

Class Telosporea The sporozoan class Telosporea includes several species which are known t o parasitize commercially important marine molluscs. These parasites belong t o the genera Nematopsis, Porospora, Pseudoklossia and Hyaloklossia. 1. Nematopsis ostrearum Prytherch, 1938. (Figs. 29 and 30)

(Subclass Gregarinia; order Eugregarinia; suborder Cephalina; family Porosporidae) Nematopsis ostrearum was first described by Prytherch in 1938. Later (Prytherch, 1940), he redescribed this parasite and reported its life cycle. This gregarine is found in the tissues of moribund and dead oysters, Crassostrea virginica, along the Atlantic coast of the United

FIG.29. Nematopsis ostrearum spores in labial palp of Crassostrea virginica. two spores are located within phagocytes.

Upper

States, extending from Mobjack Bay, Virginia, t o Lake Barre and vicinity in Louisiana. Prytherch, as the result of experimental infection experiments, has reported that : fatal effects of N . ostrearum were demonstrated and depended upon the intensity of the infection, the age and general condition of the host, and the

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energy requirements of the adductor muscle under natural or artificial conditions. On the other hand, Landau and Galtsoff (1951), who have found this parasite t o be widespread along the Atlantic and Gulf coasts of the United States, have found no evidence to support the contention that N . ostrearum is destructive to oysters. Similarly, Sprague and Orr (1955) have found no evidence to indicate that N . ostrearum causes large scale mortalities of oysters. Description of stages in mollusc. Rounded gymnospores, 3-4 p in diameter, comprised of a group of sporozoites ; spores, formed when each sporozoite secretes a resistant wall, each measures 10-16 p x 11-12 p, generally found within phagocytes (Fig. 2 9 ) ; each spore encloses a single sporozoite. Life cycle (Fig. 30). The complete life cycle of N . ostrearum has been demonstrated by Prytherch (1938, 1940). This parasite utilizes two hosts during its life cycle. The definitive host, according to Prytherch (1940), Sprague (1950) and Kent (unpublished), is one of four species of xanthid crabs, Panopeus herbsti, Eurypanopeus depressus, Eurytium limosum or Neopanope texana. The crab becomes infected when it ingests oyster leucocytes enclosing spores. This is generally accomplished when infected oysters are ingested by the predatory crab ; however, Feng (1958) has presented evidence that spore-enclosing leucocytes may be discharged from oysters and these may serve as the source of infection for crabs. When an ingested spore reaches the crab’s intestine, the enclosed sporozoite emerges and becomes attached to the gut wall by means of a small, globular epimerite and develops into a motile sporadin (also known as a gregarine or trophozoite). The next stage in its development involves the end-to-end coupling of a number of sporadins. These soon break up and the young sporadins again become fastened to the host’s intestinal wall. After a short period of attachment and growth, the sporadins develop into short cylindrical sporonts (LBger’s sporadins) which measure 18-30 p in length. These sporonts become detached and actively migrate about the intestinal lumen for 2-3 weeks during which they increase in size until each one attains a length of 220-342 p. Some may penetrate through the host’s gut epithelium. As these sporonts approach maturity, two individuals of unequal length become longitudinally attached in syzygy. Such a couple becomes attached to the crab’s rectal wall and transforms into a gametocyst. Within the gametocyst the nucleus of each sporont, now known as a gametocyte, undergoes repeated divisions followed by

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

endogenous budding (Sprague, 1954) resulting in numerous minute gametes. The gametes resulting from one gametocyte are smaller and

C Y C LE

A = = = = t= d R

\N MUD C R A B

CYCLE

"

.-

I

OYSTER

PIG.30. Stages in the life cycle of Nematopsis ostrearum. A, Spore in oyster containing a sporozoite ; B, sporozoite escaping from spore in intestine of crab ; C, attachment t o host's intestinal epithelium ; D, development into trophozoite or sporadin ; E, coupling of sporadins; F, temporary reattachment to intestinal wall ; G, mature sporont ; H-J, sporonts in syzygy followed by attachment to crab's rectum and formation of gametocyst ; K, sporozoites escaping from ruptured gametocyst ; L, single gymnospore; M, engulfment of gymnospore by host's phagocyte and breaking apart of sporozoites ; N-Q, growth of sporozoites within phagocyte and formation of resistant spores. (Redrawn after Prytherch, 1940.)

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pyriform and are the microgametes, while those resulting from the other are macrogametes. The micro- and macrogametes fuse to form zygotes which enlarge and become surrounded by a thin wall. Each zygote then divides to form eight or sometimes sixteen sporozoites. When the gametocyst wall ruptures, hundreds of sporozoites are released into the water through the host’s anus. Clumps of sporozoites, known as gymnospores, enter an oyster in one of two ways. After gymnospores make contact with the oyster’s ctenidial or mantle surface, they penetrate the epithelium by means of a pseudopod projecting from the central cell. Gymnospores can also become phagocytized by oyster leucocytes and taken into the body. If the second mechanism is employed, the gymnospores are commonly transported to various parts of the host’s body in the circulatory system. Gymnospores, whether in leucocytes or in other tissues, break apart and the individual sporozoites increase in size. This is followed by the formation of a resistant spore wall around each sporozoite. These spores occur most abundantly in mantle tissues but are also found in the adductor muscle, heart, gills, labial palps (Figs. 29 and 30), and perhaps other organs (Prytherch, 1940 ; Sprague and Orr, 1955). The spores are the infective form to crabs. It should be noted that Prytherch (1940) has reported the occurrence of spores of two distinct sizes in oysters, but Sprague (1949) has demonstrated experimentally that the larger spores represent those of another species, N . prytherchi. Ecology. Little is known about the ecology of N . ostrearum. Landau and Galtsoff (1951), as the result of extensive surveys, have reported that this pa,rasite is widely distributed along the Atlantic and Gulf coasts of the United States. Pathology. As has been pointed out, although Prytherch (1938, 1940) has considered N . ostrearum to be a cause of mass mortalities of Crassostrea virginica, his views have not been confirmed by Landau and Galtsoff (1951) and Sprague and Orr (1955). Histopathological changes in infected oysters have not been studied in detail. According to Sprague and Orr, the increase in the number of spores in oysters is the result of repeated infections and not of intramolluscan reproduction. 2 . Nematopsis prytherchi Sprague, 1949

(Subclass Gregarinia; order Eugregarinia; suborder Cephalina; family Porosporidae) Nematopsis prytherchi was originally described by Sprague ( 1949) who demonstrated experimentally that the larger Nematopsis spores

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

found primarily in the gills of Crassostrea virginica are not those of N . ostrearum but those of a distinct species, N . prytherchi.

Description of stages in mollusc. The complete description of N . prytherchi has never been published by Sprague. I n an abstract published in 1949, however, he has pointed out that the main diagnostic feature of N . prytherchi in oysters is its larger spores, measuring approximately l o p x 1 9 p , which have a special affinity for the host’s gill tissues.

Life cycle. Again, the complete life cycle of N . prytherchi has not been published by Sprague although he has cited the salient features in two abstracts (Sprague, 1949, 1954) and has made a detailed account of the life cycle available in mimeographed form (Sprague, 1962). The cycle is essentially the same as that of N . ostrearurn. The definitive host of N . prytherchi is the crab Menippe mercenaria which becomes infected when oyster tissues, primarily gill tissues, enclosing spores are ingested. Upon reaching the crab’s gut, a vermiform sporozoite emerges from each spore through a terminally located micropyle. Such sporozoites become attached by their anterior ends to epithelial cells lining the host’s gut and a small globose epimerite is formed. This is followed by detachment during which some sporozoites lose their epimerites, and the joining of two or more sporadins in linear or bifurcated syzygy. These associated sporadins are found free in the host’s midgut where they increase in size. During this period all of the satellites (i.e. those syzygynous individuals other than the anteriormost one, which is the primite) fuse to form a single multinucleated compartment. When this developniental stage is reached, the associated primite and satellite migrate to the host’s rectum where they become a rounded gametocyst which adheres t o the chitinous wall. Cytological changes now occur, resulting in the formation of numerous gametes. Sexual reproduction has not yet been reported for N . prytherchi although bodies, tentatively identified as zygotes, have been reported by Sprague. Each of these zygotes gives rise to eight sporozoites which are arranged in clumps as gymnospores and which are released when the gametocyst ruptures. Exactly how oysters become infected remains uncertain. It is known that sporozoites comprising each gymnospore are phagocytized by the oyster’s leucocytes in small numbers, but it is not clear whether the phagocytized sporozoites are taken into the host’s body through the surface epithelium or are first ingested and later taken in through the gut epithelium. Both methods have been reported for certain species found in Europe (see Hatt, 1931). Within leucocytes, the sporozoites (termed sporoblasts by Sprague) are spherical or oval and

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each measures 3-4p in diameter. These become fusiform and a protective spore wall is laid down around each, forming a spore. Mature spores, found primarily in the oyster’s gills, are ovoid, slightly pointed at each end, and measure 15-20 p x 9-11 p. Each spore encloses a sporozoite and represents the infective form for crabs. Ecology. Nothing is known about the ecology of N . prytherchi except that concurrent infections with N . ostrearum commonly occur in Crassostrea virginica. Physiology. Nothing is known about the physiology of N . prytherchi except that Sprague (unpublished) has reported that “ highly refractive fat droplets occur in young sporozoites comprising gymnospores ”. These are no longer apparent in mature sporozoites, thus suggesting that these lipid droplets are utilized during maturation. Pathology. Again, specific observations on pathological changes in oysters and crabs parasitized by N . prytherchi have not been made. Superficial observations, however, suggest that this parasite does not injure its hosts to any great extent. 3. Nematopsis legeri (de Beauchamp, 1910). (Figs. 31-40) (Subclass Gregarinia; order Eugregarinia ; suborder Cephalina; family Porosporidae) Nematopsis legeri is a common parasite in a number of species of marine molluscs including pelecypods, chitons, and gastropods. Porospora galloprovincialis, described by LBger and Duboscq ( 1925), is a synonym. According to Hatt (1931), who has critically evaluated the then known species of Nematopsis and those of the related genus Porospora, N . legeri has been reported to parasitize the following molluscs in European waters : Mytilus galloprovincialis, M . minimus,

Lasea rubra, Cardita calyculata, Chiton caprearum, Trochocochlea turbinata, T . articulata, T . mutabilis, Phorcus richardi, Gibbula divaricata, G. rarilineata, G. adamsoni, Pisania maculosa, Cerithium rupestre, Columbella ristica and Conus mediterraneus. N . legeri has not been reported from either North or South America. It has also not been reported from Africa, Asia or Australia. Description of stages in mollusc. Gymnospores, 7 p in diameter, comprised of few large sporozoites ; spore, averaging 14-15 p long, with distinct one-piece shell (endospore) and a less conspicuous epispore, circular in cross-section. Life cycle. The life cycle of N . legeri involves a crustacean definitive host, Eriphia spinifrons (see Hatt, 1931), and a molluscan intermediate host which may be one of the several species listed above.

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The developmental pattern and stages are the same as those of N . ostrearum and N . prytherchi except for minor morphological differences. The sporadins (also known as gregarines or trophozoites) found in the

4

5

FIQS.31-38. Stages in the life cycle of Nematopsis Zegeri. (31,32) Sporadins or trophozoites engaged in bifurcated and linear syzygy ; (33) associated sporadins attached to gut epithelium of crustacean host; (34) gymnospore; (35) gymnospore after entering molluscan host; (36) young sporozoite; (37) cyst in mollusc enclosing six spores ; (38) germination of spore in gut of crustacean host. (Redrawn after Hatt, 1931.)

gut of Eriphia spinifrons each measures from 75 to 7 5 0 p in length. These engage in either linear or bifurcated syzygy (Figs. 31 and 32).

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Gametocysts found in the crustacean host’s rectal region measure about 80 p in diameter.

FIGS.39 and 40. Ponetration of mollusc by Nematopk Zegeri gymnospore. (39) A gymnospore penetrating gill lamella of host ; (40) two gymnospores penetrating gill lamella of host. Notice the formation of minor lesions in Fig. 40. (Redrawn after Hatt, 1931.)

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Ecology. N . legeri is widely distributed in both northern and southern Europe (Hatt, 1931). Nothing is known about the physiological aspects of its ecology. Pathology. Hatt (1931) has reviewed and contributed some new information concerning histopathological changes in molluscs, primarily pelecypods, parasitized by N . legeri. From what is known, there appears to be little damage associated with this parasite. There is the formation of minor lesions on the surfaces of gill lamellae during the penetration process of gymnospores (Figs. 39 and 40) which may or may not involve the complete destruction of cells in the immediate area or the sloughing of cilia. The gymnospores, as a rule, enter via the intercellular spaces. There is little or no host cellular reaction to gymnospores that have entered regions of the host’s body which are packed with connective tissues.

4. Porospora gigantea (van Beneden, 1869). (Figs. 41-46) (6ubclass Gregarinia; order Eugregarinia; suborder Cephalina; family Porosporidae) Porospora gigantea was the first porosporid gregarine t o be described. It was found by van Beneden (1869) in the intestine of a lobster and named Gregarina gigantea. I n a series of subsequent papers (van Beneden, 1871, 1872a,b) he has described the various stages in its life history. It was later placed in the genus Porospora by Schneider (1875). P. gigantea, like the other members of this genus, differs from the members of the closely related genus Nematopsis by the fact that its sporozoites are found in molluscan phagocytes without a protective spore wall (Hatt, 1931). Description of stages in mollusc. Gymnospores spherical, 8 p in diameter, comprised of approximately 1 5 0 0 merozoites (Figs. 41 and 42) ; naked sporozoites 17 p long (Figs. 43 and 44)) usually grouped within phagocytes. Life cycle. The life cycle of P. gigantea is essentially the same as that of members of the genus Nematopsis (see Hatt, 1931). Two hosts are involved, a crustacean definitive host, Homarus gammarus, and a molluscan intermediate host, either Mytilus minimus or Trochocochlea mutabilis. The sporadins, found in the gut of Homarus gummarus, measure up to 10 mm long (Fig. 45). The gametocysts, found in the lobster’s rectal region, measure from 3 to 4 mm in diameter. The gymnospores invade the molluscan host via the intercellular spaces of the gill filaments (Fig. 46). It is while in the process of working its way through the gill surface that the gymnospores become phagocytized by the mollusc’s blood cells. It is still not completely under-

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stood how the individual merozoites comprising each gymnospore become dispersed and subsequently become engulfed by other phagocytes. It is within phagocytes that the naked sporozoites are formed. Other information. Although P. gigantea has been observed in the process of attempting to penetrate the molluscan host’s gill epithelium, no serious histopathological changes occur (Hatt, 1931). While within phagocytes, no changes in the surrounding tissues are known to occur.

FIGS.41-46.

Stages in the life cycle of Porospora gigantea. (41) Echinulate form of gymnospore; (42) smooth form of gymnospore; (43, 44) developing sporozoites in mollusc ; (45) sporadin in gut of Hornarus; (46) phagocytized gymnospore beginning to penetrate gill filament of mussel. (Redrawn after Hatt, 1931.)

The absence of severe pathological changes in the mollusc may be attributed to the fact that the intramolluscan stages of P. gigantea are almost continuously found within phagocytes and thus prevented from making direct contact with the host’s tissues. 5. Pseudoklossia glomerata LBger and Duboscq, 1915. (Figs. 47-49)

(Subclass Coccidia; order Eucoccida; suborder Eimeriina; family Aggregatidae) Pseudoklossia glomerata is the type for the genus. It was originally described by LBger and Duboscq (1915) from the nephridia of Tapes Jloridus and T . virgineus collected from the Mediterranean Sea.

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Description of stages in mollusc. Anisogamy and sporogony occur in molluscan host ; oocyst or zygote contains numerous spores ; each spore includes two sporozoites, each measuring 4-5 p in diameter (Fig. 47).

FIGS.47-49. stages in the life cycle of Pseudoklo.%sia glomernta. (47)Oocyst enclosing spores, each in turn containing two sporozoites; (48) gametocysts in process of entering epithelial lining of nephridium of Tapes jloridus, one (a) in renal excretory canal, second (b) in hypertrophied host cell, and third (c) in lumen of excretory canal ; (49) aggregate of parasites in host’s renal cavity. H N , nucleus of host cell. (Redrawn after LBger and Duboscq, 1915.)

Life cycle. LBger and Duboscq (1915) have given a detailed account of the formation of micro- and macrogametes from gametocytes in addition to a detailed cytological account of sporogony. Schizogony

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does not take place in the mollusc and presumably occurs in another host which is still unknown. Other information. I n addition to being found in the nephridia of clams, LBger and Duboscq (1915) have reported that P. glomerata also occurs in visceral ganglia and in ducts leading from the nephridia. At these sites, the macro- and microgametocytes are found either penetrating or within individual cells. When a young gametocyte first enters a host cell, the latter becomes enlarged and extends beyond the normal cell level (Fig. 48) ; however, in time the parasite-enclosing cell is retracted into position although it is hypertrophied and its larger size causes the compression of adjacent cells. The space normally occupied by the cytoplasm in parasitized cells is entirely occupied by the parasite. The nucleus becomes hypertrophied and gradually disappears. If the infection is intense, individual parasites, enveloped by their host cell, will join with one another and thus five to ten parasites can be seen aggregated in the host’s renal cavity (Fig. 49). ItJis for this reason that the specific name “glomerata” was proposed to describe such aggregates. There is no information available relative to the fate of parasitized clams; however, there is no reason to believe that P. glomerata is lethal to Tapes. Similarly, there is no information available on the infectivity of P. glomerata to man, although there is no reason to believe that this parasite is of any public health significance. 6. Pseudoklossia pectinis LBger and Duboscq, 1917. (Figs. 50-52) (Subclass Coccidia; order Eucoccida; suborder Eimeriina; family Aggregatidae) Pseudoklossia pectinis is the only other species of this genus known to parasitize a marine pelecypod. The other two known species, P. patellae and P. chitonis, parasitize the hepatopancreas of the marine prosobranch Patella and the digestive tract of the chiton Acanthochites fascicularis respectively (Debaisieux, 1922). A fifth, but doubtful, species, P. legeri, was reported by Harant (1931) from the pyloric gland of the ascidians Styela and Polycarpa. P. pectinis was reported by LBger and Duboscq (1917) from the nephridia of the scallop Pecten maximus in France. Description of stages in mollusc. Anisogamy and sporogony in molluscan host ; young microgametes elongate, 10-1 1 p long ; macrogamete pyriform, 36-38 p long, 24 p wide ; oocyst (or zygote) spherical, 32-35 p in diameter, includes elongate sporozoites typically grouped in pairs (Fig. 50) ; spore membrane not seen ; each sporozoite measures 3-5 p in diameter.

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Life cycle. The complete life cycle of P . pectinis is not known. Presumably there is a schizogonic phase which occurs in some other host. Within Pecten, fusion of the macro- and microgametes occurs after these cells have entered a host cell (Figs. 51 and 52). Physiology. Nothing is known about the physiology of P. pectinis except that LBger and Duboscq (1917) have reported that very young

PIGS.50-52. Stages in the life cycle of Pseudoklossia pectinis. (50) Two sporozoites of a spore; (51) macro- and microgametes in renal cell of Pecten nmximus; ( 5 2 ) completion of fusion of macro- and microgametes in host cell. (Redrawn after LBger and Duboscq, 1917.)

gametes, measuring 10-1 1p long, include several paraglycogen spherules in their cytoplasm. These workers believe that these inclusions are paraglycogen although the test employed was the classical iodine test, hence the inclusion bodies may well be glycogen. Pathology. Nothing is known about the pathology in infected Pecten except that renal epithelial cells enclosing parasites are enlarged and the space normally occupied by cytoplasm is almost completely taken over by the parasite. Furthermore, parasitized cells appear to undergo degeneration.

1G1

5 . THE PHYLUM PROTOZOA

7. Hyaloklossia pelseneeri LBger, 1897

(Subclass Coccidia; order Eucoccida; suborder Eimeriina; family Aggregatidae) Hyaloklossia pelseneeri was reported by LBger (1897) from the nephridia of the marine pelecypods Tellina sp. and Donax sp. Although Kudo (1966) lists Hyaloklossia as a valid genus, Grass6 (1953) is of the opinion that it should be transferred to the genus Pseudoklossia since it appears to be more closely related to the known species of that genus in marine pelecypods than it is to H . lieberkhilhni, a renal parasite of frogs which has since been shown by Laveran and Mesnil (1902) to be a species of Diplospora. Some minor differences, however, do exist between H . pelseneeri, Pseudoklossia pectinis and P. glomerata, the most conspicuous one being that there are two types of spores within the oocysts of H . pelseneeri (see Lbger, 1897). For this reason, until further evidence to the contrary becomes available, I am in agreement with Kudo that H . pelseneeri should be considered to be generically distinct from Pseudoklossia.

Description of stages in mollusc. Oocysts, 75-8Op in diameter, in renal cavity of molluscan host ; each oocyst includes two types of spores, the smaller, 8 p in diameter, includes spirally coiled sporozoites, while the larger, 11-12 p in diameter, includes four to six sporozoites.

Life cycle. Sporogony occurs in the molluscan host. How the pelecypods become infected and where schizogony occurs remain unknown. Other information. Pathological changes in pelecypods parasitized by H . pelseneeri have not been studied; however, there is no reason to believe that this parasite is seriously detrimental to its host.

D. Subphylum

SPOROZOA

Class Haplosporea" Included in the sporozoan class Haplosporea are two parasites of Crassostrea virginica and one of Mytilus californianus. Minchinia costalis is believed to cause a disease in oysters which may lead to death while the mysterious MSX, now known as Minchinia nelsoni, which continues to evade the protozoan taxonomist, is believed to be the primary mortality factor among oysters along the mid-Atlantic coast of the United States. The third species, Haplosporidium tume-

* For another interpretation of the taxonomic position of the Haplosporea, see Sprague (1966). A.M.B.-5

11

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facientis, is a pathogenic parasite found in the digestive gland and nephridia of Mytilus californianus. It is of considerable interest since it is the only known protozoan parasite of marine molluscs which causes conspicuous swellings in the parasitized tissues. 1. Minchinia costalis (Wood and Andrews, 1962). (Figs. 53 and 54)

(Class Haplosporea; order Haplosporida; family Haplosporidiidae) During 1959-61, while studying the mortality of Crassostrea virginica along the Virginia coast, Wood and Andrews (1962) discovered a sporozoan parasite which they believed to be the cause of an oyster disease that may lead to death. This parasite was originally referred to as SSO (seaside organism) a t informal conferences. In 1962, Wood and Andrews formally described it as Haplosporidium costale. Later, Sprague (1963), as the result of a revision of the genus Haplosporidiurn, restored the genus Minchinia, which was originally erected by Labbe in 1896 and later suppressed by Debaisieux in 1919, and placed H . costale therein as M . costalis. The genus Minchinia includes four other species that parasitize molluscs. M . chitonis, originally named Klossia chitonis by Lankester ( 1885), later transferred to the genus Haplosporidium by Debaisieux (1919), but considered the genotype of Minchinia by Labbe (1896) and Sprague (1963), is a liver parasite of various European chitons ; M . dentali, originally named Haplosporidium dentali by Arvy (1949), is found in the scaphopod Dentalium ; M . piclcfordae, originally named Haplosporidium piclcfordae by Barrow (1961), is found in the hepatopancreas of freshwater snails of the genera Helisoma, Physa and Lymnaea in North America; and M . nelsoni is a parasite of the oyster, Crassostrea virginica. According to Sprague, Minchinia spores possess " a relatively small orifice in the typically rounded anterior end but differ conspicuously in having a lid over the orifice " (Fig. 53). Furthermore, he has stated that : The lid is not homologous with that of Haplosporidiurn; it originates from the outer gelatinous spore envelope instead of being a portion of the spore membrane. Because of its separate origin, the lid lies over the orifice, not in it, and has a free margin extending beyond the orifice and overlapping the end of the spore. Description of stages in mollusc. Parasitic in connective tissues of Crassostrea virginica. Plasmodia in connective tissue, each measuring 6.1 x 7.8 p i n diameter (Fig. 54) ; spores obovate, truncate, uninucleate, with operculum like that described by Sprague (1963) (Fig. 53) ; each

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spore measures 2.42-4-20 p (average 3.09 p) x 2-14-3.26 p (average 2.58 p), without projections ; sporocysts 7.014 p (average 9 p ) in diameter, each containing twenty to fifty spores. Life cycle. Although Wood and Andrews (1962) have given certain aspects of the life cycle of M . costalis, its complete life cycle remains undetermined. According to Wood and Andrews, the earliest stage observed in the oyster is a small multinucleated plasmodium which is more or less irregular in outline. Its cell membrane is at first definite but becomes less definite with age. As each plasmodium increases in size, its nuclei multiply in number and the cytoplasm becomes vacuolated. The multinucleated plasmodium eventually cleaves into uninucleated portions each of which develops into a characteristic spore. A t this point the

FIQS.53 and 54. Minchinia costalis. (53) A spore; (54) a young plasmodium. (Redrawn after Wood and Andrews, 1962.)

wall of the plasmodium, which encloses the spores, is known as the sporocyst. The fate of fully developed spores remains unknown as is the method by which oysters become infected. Ecology. Wood and Andrews (1962) mentioned that a t Seaside, on the eastern shore of Virginia, M . costalis first becomes evident in oysters in February. The prevalence gradually increases until mid-May when as high as 39% of the oysters may be infected. During June, the death rate, believed to be caused by this parasite, is extremely high. The mortality is sharp but of a short duration after which the parasite lapses into obscurity for another year. In a later paper (Andrews et al., 1962), these earlier observations were expanded and reported in detail. It was reported that M . costalis is found in live oysters during March to July and in a high proportion of dying or dead oysters during May and June (Fig. 55). This seasonal fluctuation of M . costalis is

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not unique since it has been reported for certain species of Haplosporidium (Granata, 1914 ; Debaisieux, 1920 ; Jirovic, 1936 ; CEtullery, 1953 ; Ganapati and Narasimhamurti, 1960 ; Barrow, 1961). Pathology. Although the cause-and-effect relationship between M . costalis and the death of Crassostrea virginica has not been experi1959

1960

H O G I S L A N D BAY

JAN

FEB MAR

APR

MAY JUN J U L AUG

SEP

OCT NOV DEC

FIG. 55. Death rates of native seaside oysters, Crassostrea virginica, in trays resulting from parasitism by Minchinia costalw. Each point represents the monthly death rate for the preceding interval. (After Andrews et al., 1962.)

mentally demonstrated, the high mortality data presented by Andrews et al. (1962) among parasitized oysters strongly suggest that this parasite is a pathogen. These data are particularly convincing since the number of deaths is remarkably greater during the warm months than during those months when M . costalis cannot be found in oysters. Moreover, the mortality rate is significantly higher when M . costalis is present than those associated with other endemic pathogens.

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Detailed histopathological and histochemical changes associated with M . costalis infections have not yet been studied although Farley (1965), who stained sections of infected oysters with a modification of Ziehl-Neelsen’s carbol fuchsin stain, has reported that the spores (sporoplasm) of M . costalis are acid fast. 2. Minchinia nelsoni. (Figs. 56 and 57)

(Class Haplosporea; order Haplosporida; family Haplosporiidae)* It is generally agreed among those interested in parasites of commercially important molluscs that the organism originally referred to as MSX (multinucleated sphere unknown) and now known as Minchinia nelsoni is by far the most destructive of the known parasites and is believed t o be the causative agent of the mass oyster mortalities which began in the Delaware and Chesapeake Bays during 1957 and have been continuing and spreading ever since (Haskin et al., 1965). Although considerable information is known about the distribution, seasonal fluctuations, and morphology of the intramolluscan stages of MSX, such information for the most part has not appeared in published form but has been circulated informally among oyster biologists in the United States. As the result of this situation, I am forced to present the following information without being able to cite the publications or in most instances the investigators.

Description of stages in mollusc. Multinucleated spherical stage (plasmodium) found in gills, palps, suprabranchial chambers and Leydig tissues of Crassostren virginica (Figs. 56, 57A-F) but may be found in other sites as well. Each plasmodium, 4-30 p (occasionally as large as 50 p ) in greatest diameter, includes variable numbers of nuclei (one to more than sixty); each nucleus, 1.5-7.5 p in diameter, possesses a distinct endosome resting against a thin but sharply defined nuclear membrane; intranuclear bar in all except smallest nuclei?; nuclei frequently lying in central cytoplasmic zone surrounded by a less densely staining cytoplasmic zone (Fig. 57A-C). According to Haskin et al. (1966), two types of nuclei are recognizeable: (1) small nuclei, 1.5-1.6 p in diameter, with densely staining “ caps ” (Fig. 57E), found in plasmodia generally concentrated in and under epithelium of gills, palps and epibranchial chambers of recently infected oysters;

* MSX was only tentatively assigned to the genus Minchinia as M . nelsoni by Haskin et al. (1966), and therefore its assignment t o the Haplosporiidae of the Haplosporide is also tentative. t The nuclear details are visible in both fresh preparations stained with 0.1% methylene blue and in sectioned materials fixed in Zenker’s or Davidson’s fluids and stainecl with Harris’ or iron hematoxylin.

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FIG.56. Minchinia nelson;. (A) Multinucleated plasmodium in Leydig tissue of Crassostrea wirginica (10 x ocul., 40 x obj.) ; (B) slightly older plasmodium in Leydig tissue of C. airginica (10 x ocul., 90 x obj.). Infected oysters from New Jersey.

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167

(2) larger nuclei, usually 2.5-3.0 p in diameter, with intranuclear bar (Fig. 57A and B), in plasmodia located throughout blood spaces of oysters within 10 days after infection. The larger nuclei persist throughout the year in plasmodia with frequently vacuolated and granular cytoplasm. Frequently interspersed with these throughout the year are plasmodia with very large ovoid or spherical nuclei, ranging up to 7.5 p in diameter, and invariably with prominent intradesmose (Fig. 57B). Occasionally large and very large nuclei are found in the same plasmodia. Couch et al. (1966) have described what they believe to be the sporulating and spore stages of M . nelsoni from the digestive diverticula of naturally infected oysters. Brief descriptions of these extemely rarely seen stages are given below. Sporulation and spore. With the onset of sporulation, small plasmodia and their nuclei increase in size. When the nuclei in enlarged plasmodia reach three to four times their initial size (Fig. 57F), karyokinesis commences. A conspicuous bar or spindle appears within the nucleus during division and the endosome becomes associated with the spindle and divides (Fig. 57G). Daughter nuclei resulting from initial divisions are relatively large (3-4 p ) but diminish in size in subsequent stages. Sporonts (Fig. 57H), each measuring 18-30 p, formed when dividing nuclei develop into sporoblasts. Sporoblast nucleus, 3-4 p in diameter, surrounded by cytoplasmic condensation, often includes remnants of spindle; rarely do sporoblasts include more than one nucleus. Early sporocysts (Fig. 57I), each measuring 28-54 p, filled with variable numbers of incompletely formed spores. Immature spores, each measuring 6 - 8 p in diameter, round, with an orifice, but rarely with fully formed operculum. Definitive sporocysts, each measuring 28-54 p , contain from eight to about fifty spores. Mature spores (Figs. 57J and K), ranging from 5.3-10-7p x 4.87-5 p (mode 7.5 x 5.4p), surrounded by refractjle capsule, with or without projections or appendages; sporal operculum, approximately 1 p high, extends laterally beyond margin of orifice to margin of capsule; sporoplasm nucleus small, 1.5-2-0 p, with peripheral endosome. Since sporulation and mature spores are extremely rarely found in oysters (Haskin et al. (1966) have reported finding only two out of 10 000 infected oysters harboring such spores), and since the spores described by Couch et al. (1966) were from naturally infected oysters, some doubt exists as to whether the spores which they have described are those of M . nelsoni. Relative t o this point, Haskin et al. (1966) have stated:

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

This extremely low incidence of spores leads us to suspect, however, that they are not M . nelsoni spores but rather indicate double infections as discussed by Canzonier (1963). Since late 1958 all attempts to transfer infection to healthy oysters in the laboratory have failed. Failures in effecting transmission and rarity of oysters with sporulation stages point strongly to an alternate or reservoir host for the parasite. The primary reason why t h e sporulating a n d spores stages given above are being given serious consideration herein is because Barrow

FIG.57. Stages of Minchinia nrlsoni in Crassostrea virginica. (A) Young plasmodium with small nuclei; (B) older plasmodium with one large nucleus on each side of very large nucleus; (C) and (D) forms of plasmodia commonly seen in parasitized Delaware Bay oysters ; (E)young plasmodia from connective tissue ; (F)plasmodium with enlarged nuclei ; (G) nuclear division within enlarged plasmodium (early sporont) ; (H)sporont containing sporoblasts ; (I)early sporocyst ; (J)fresh spore ; (K) fixed and stained spores. Upper scale refers to A-D, lower scale to E-K. (A-D redrawn after Haskin et al., 1966; E-K redrawn after Couch et al., 1966.)

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and Taylor (1966) have successfully demonstrated that the postplasmodial stages described by Couch et al. (1966) are antigenically similar to the plasmodia1 stage generally recognized as that of M . nelsoni and hence, until further evidences indicate otherwise, are suspected to be stages in the life cycle of M . nelsoni. What Barrow and Taylor did was to prepare a fluorecein-conjugated antibody from TABLEXIII. REACTIONS OF VARIOUS PARASITES AND DISEASE ENTITIES WITH FLUORESCENT ANTIBODY TO Minchinia nelsoni

+ ~++ +,

++ +

Maximum fluorescence, brilliant yellow green; , slightly less fluorescence; , definite fluorescence, yellow green, less brilliant,; -, no fluorescence. (Data after Barrow and Taylor, 1966.)

++

Test organism Protozoa Hexamita sp. Nematopsis ostrearum Minchinia costalis Minchink nelsoni M . nelsoni from Delaware Bay M . nelsoni from Chincoteague Bay M . nelsoni from Chesapeake Bay M . nelsoni from James River, Va. Haplosporidium sp. (A hyperparasite of Bucephrtlus sp. in C. virginica) Ancistrocoma sp. Platyhelminthes Bucephalus sp. Tylocephalum sp. Thallophyta " Actinomycetc " disease " Mycelial " disease Dermocystidium marinum Disease of unknown etiology " Rickettsia1 " disease " Necrosis " of the gut " Lymphocystosis "

T y p e of reaction __

-

++++ ++++ ++++ ++++ +++ +f -

-

-

-

-

-

horse serum using homogenates of M . nelsoni-infected oysters as the antigen. Non-specific antigens in this homogenate had been previously extracted by reacting the homogenate with horse antisera prepared by using M . nelsoni-free powdered oyster antigen. Oyster antibodies were also adsorbed from the anti-M. nelsoni antisera by using the M. nelsoni-free oyster antigen. The fluorecein-conjugated anti-M. nelsoni antiserum proved t o be highly specific for all known stages of

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this parasite, including the sporulating and spore stages. To test its specificity, Barrow and Taylor reacted the conjugated antiserum against fifteen disease-causing entities in C. virginica (Table XIII) and found it to be highly specific.

Life cycle. The complete life cycle of Minchinia nelsoni remains unknown. Various postulated life cycle patterns have been advanced from time to time but until concrete experimental evidence becomes available, I have chosen not to recognize the majority of these. Experimental studies carried out at several distinguished laboratories, especially a t the New Jersey Oyster Research Laboratories of Rutgers University under the directorship of Dr. H. H. Haskin, have indicated that M . nelsoni cannot be transmitted from oyster to oyster when placed in the same aquarium or through the transplantation of infected tissues. Relative to the developmental stages within the oyster, although plasmotomy and sporulation have been observed, these findings have been made in histological sections of naturally infected oysters from areas where other species of Haplosporida are known to occur and hence cannot be accepted without question. According to the most popular hypothesis, the development of M . nelsoni within its molluscan host is similar to that of other members of the genus Minchinia, with the mature spore produced within the host serving as the infective stage for others. Ecology. Continuous surveys conducted by the personnel of various laboratories along the New Jersey, Delaware, Maryland, and Virginia coasts have revealed the continuous spread of M . nelsoni in C. virginica along these shores from 1957 until the present. Its current distribution is represented by a continuous zone encompassing this region of the Atlantic coast of North America, extending from Long Island Sound to Virginia. For some reason, most probably the inability of M . nelsoni to withstand lower salinities, this parasite will disappear from oysters maintained in waters with salinities lower than 20%,. Hence in the past, oysters living in estuaries with low salinities were essentially free of M . nelsoni; however, as the result of the severe droughts which affected the mid-Atlantic coasts during 1961-66, there has been a marked increase in salinity in normally low salinity areas and the spread of M . nelsoni, as a consequence, has been conspicuous and disastrous t o the oyster industry in these areas. Temperature, on the other hand, does not appear to affect the survival of M . neboni in oysters but does alter the rate of pathological effects, with warm summer temperatures being most deleterious to parasitized oysters. According to information from H. H. Haskin’s laboratory and from

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J. D. Andrews’ laboratory a t the Virginia Institute of Marine Science, the appearance and persistence of M . nelsoni in oysters are not seasonal. Andrews and Wood (1967) have given a detailed account of the history and distribution of M . nelsoni in oysters in Virginia.& Evidences collected thus far appear to indicate that M . nelsoni is specific for C. virginica and is limited to the mid-Atlantic coast of the United States. However, histological examination of C. gigas from Taiwan by U.S. Bureau of Commercial Fisheries personnel and by me have revealed the presence of multinucleated plasmodia similar to M . nelsoni. Thus far no mass oyster mortalities have been reported from Taiwan. Pathology. Although Minchinia nelsoni is generally believed to be highly lethal to C. virginica as based on epizootiological evidences, there is comparatively little histopathological change in parasitized oysters. When few plasmodia are present, slight lesions in the surrounding connective tissue (Leydig cells) generally occur. Furthermore, there may be some leucocytes surrounding the plasmodia but their number is seldom large and definitely does not compare with the more typical encapsulation phenomenon. When large numbers of plasmodia occur, relatively large number of Leydig cells may be ruptured. Here again, leucocytosis and encapsulation are not particularly pronounced. In addition, Haskin et al. (1965) have reported frequent and extensive sloughing of the gill, palpal and suprabranchial epithelia during invasion by M . nelsoni. Relative to the lethality of this parasite, Haskin et al. (1966) have reported that epizootic kills begin within 3 weeks after first appearance of plasmodia in gills. I n lightly infected oysters, however, death may be delayed. It is being mentioned at this point that Parley (1965b) has proposed that histopathological changes in C. virginica can be categorized into five stages. According to him, initial infections, most commonly encountered during July through early September, are characterized by localized infection in the gills. Intracellular plasmodia first appear in the ctenidal columnar epithelia, each surrounded by a clear zone which is believed to have resulted from lysis. From these locations, the plasmodia migrate to the underlying basement membrane where plasmotomy occurs and thence into the connective tissue and blood sinuses of the gills. During this initial stage, Parley has reported a concentration of “ lymphocyte-like cells ” surrounding the parasite. Each of these cells measures 8 p in diameter and includes a large nucleus that measures 5 p in diameter. * See note on p. 389.

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

Farley’s intermediate infection stage either follows the initial infection stage or occurs as ‘‘ the result of relapse of chronic infection.” Intermediate infections that follow initial infections occur most frequently during July through September while relapses occur most commonly during June. This stage is characterized by localized lesions, with accompanying host response, in the gills, gonad, digestive diverticula, and Leydig tissues adjacent to the gut and mantle epithelia. Furthermore, recessive shell growth and shell-pustule formation may occur. According to Farley, the intermediate infection phase resuIting from relapse is characterized by the occurrence of lesions in the diverticula and connective tissue while that resulting from a continuation of the initial infection phase is characterized by the appearance of lesions in the gill, gonad, and gut epithelium. The third phase, termed the advanced infection stage, is reportedly most prevalent from September through December. It is characterized by massive infection and host response throughout the oyster, resorption of gametes, shrinkage of gonadal tubules, and a marked tendency toward recessive shell growth. This is followed by the terminal infection stage which occurs in the fall and winter. Oysters at this stage include massive invasion of tissues ” by the parasite but, surprisingly, Farley has stated that “ host response is usually light or absent ” but pycnosis of host and parasite cells often occur. Furthermore, recessive shell growth is usually present. Death of oysters generally follows the terminal infection stage. I n some instances, Farley is of the opinion that recovery, the fifth stage, may occur. Oysters a t this stage are recognized by the deposition of yellowish brown conchiolin-covered shell pustules which is preceded by the formation of localized fibrotic lesions in the tissues situated near or comprising the mantle epithelium, Moribund M . nelsoni plasmodia are said to occur in both the lesions and shell pustules but rarely elsewhere within the host’s body. Recovery, if it occurs, takes place most commonly in the summer and fall. Farley’s progressive stages of infection may represent a natural sequence of events in parasitized oysters; however, since thesc stages have been described from naturally infected oysters rather than at time intervals during experimental infections, it may be questioned whether these actually form a sequence or merely reflect different intensities of infection. This doubt becomes even more serious, especially that pertaining to recovery, when one considers the report by Haskin et al. (1966) that the onset of oyster mortalities occurs within three weeks after the initial infection and the ‘‘ incidence of gapers (recently dead oysters) during the epizootics is commonly 100 per cent.” <(

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3. Haplosporidium tumefacientis Taylor, 1966. (Figs. 58-65) (Class Haplosporea; order Haplosporida; family Haplosporiidae) Since Sprague’s (1963) revision of the genus Haplosporidium during which he transferred those species found in molluscs to the genus Minchinia, only one species, H . tumefacientis, is now known to be a parasite of a mollusc. This parasite was described by Taylor (1966) from the digestive gland and nephridium of the California musscl, Mytilus californianus, collected at Corona del Mar, California.

2

3

Frm. 58-65. Stages in the life cycle of Huplosporidium tumefacientis. (58-61) Mature spores showing variable surface sculpturing ; (62) mature spore ES seen in sectioned material, notice nucleus with three small ‘‘ endosomes ” and vacuole enclosing ‘‘ body ”; (63) mature spore with internal “ rod ” which is refractory to all stains used; (64) young plasmodium with four nuclei a t metaphase; (65) young plasmodium with nuclei at late telophase and containing endosomes which reappear during this phase. (Redrawn after Taylor, 1966.)

Description of stages in mollusc. Multinucleated plasmodia in mussel (Figs. 64 and 65) ; spores obovate, truncate, operculate, 5-8 p x 8-1 1 p ; minucleate ; with inner thick wall and outer thin sculptured wall, sculpturing as thin filament wrapped around spore (Figs. 58-61) ; spore wall thickest a t anterior truncatsd and operculated end ; oper culum 2-3 p in diameter, attached by “ hinge ” when opened; nucleus

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

1.5-2.5 p in diameter, with one to four small endosomes each measuring 0.5 p in diameter; a vacuole, 1-5-2.5 p in diameter, at interior end of spore in which a small “ body ” is sometimes seen (Fig. 62) as is a rod within wall (Fig. 63) ; sporocyst spherical, 30-80 p in diameter, enclosing variable number of spores, from sixteen to over 100.

Life cycle. The complete life cycle of H . tumefacientis is not known. According to Taylor (1966), the invasive form for mussels is most probably an amoebula which gives rise to a plasmodium through repeated division of its nucleus coupled with an increase in size. Spores are formed within each fully developed plasmodium (now known as a cyst) when spore walls develop around each nucleus. At first each spore only possesses a single thin membrane and a centrally located nucleus enclosing a single large endosome. It is only in the older spores that the delicate outer membrane appears. It is composed in part of one or two compactly wrapped filaments. It is also in the older spores that the endospore fragments into as many as four smaller bodies. The number of spores within each sporocyst (i.e. the membrane of the original plasmodium enclosing spores) varies from sixteen to over 100. Upon reaching maturity, the sporocyst wall ruptures and the spores are released. These are set free in the lumina of the digestive gland tubules and are believed to be expelled from the molluscan host via the excretory products or may reinfect the same host although it is highly doubtful. Taylor has attempted to infect both Mytilus californianus and M . edulis by injecting tumefaction brei. His experiments were unsuccessful. Ecology. Of the 1 114 M . californianus examined by Taylor, twenty-three, or 2.1 yo,were infected.

Physiology. Nothing is known about the physiology of H . tumefacientis except for certain histochemical observations contributed by Taylor. The walls of nearly mature spores include a PAS (periodic acid-Schiff)-positive but diastase resistant thickened portion which is believed to be the developing operculum but the definitive operculum is PAS-negative. The cytoplasm of the mature spore is strongly PASpositive but diastase resistant and hence may be assumed not to represent glycogen. It most probably includes certain neutral mucopolysaccharides, muco- or glycoproteins, or a complex of these. The cytoplasm is acid fast when stained with Farley’s (1965a) modification of Ziehl-Neelson’s carbol fuchsin. On the other hand, the cytoplasm of immature spores is only faintly or not acid fast. Taylor also has attempted to culture this haplosporidan. He employed thioglycollate broth as given by Ray (1952a,b), potato-

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dextrose agar, nutrient agar, and thioglycollate agar. All his attempts have been unsuccessful although mature spores maintained their jntegrity in these media for 3 months. Pathology. H . tumefacientis causes tumefactions (swellings) in its host’s tissues. These swellings vary in size and are light brown or tan in color. Microscopically, parasites in the digestive gland tubules tend to be subepithelial, situated towards the peripheral margin of each cell. As they develop, they displace the epithelial cells and may inhibit their growth. Despite these histopathological alterations, no necrosis of mussel tissues or neoplasms occur. The fate of infected mussels is not known.

E. Subphylum CNIDOSPORA Class Microsporidea The microsporidean genus Chytridiopsis includes two species known to parasitize commercially important marine molluscs. These protozoa, at one time considered to be sporozoans, are now considered to be members of a distinct subphylum, the Cnidospora. 1. Chytridiopsis mytilovum (Field, 1924). (Figs. 66-76) (Order Microsporida; family?)

Chytridiopsis mytilovum was originally superficially described by Field (1924) who was making a comprehensive study of the host, Mytilus edulis. Although he admitted little familiarity with the (6 Sporozoa,” he felt obliged to report the occurrence of this parasite in the ova of the mussel. He stated that: “ It was finally identified for the author through the kindness of Dr. Gary N. Calkins, of Columbia University, as a probably new species of the genus Haplosporidium.” As the result, Field briefly described the parasite, namedit H . rnytilovum, and promised to investigate it further. Unfortunately, he died soon thereafter and no additional studies were made. Sprague rediscovered H . mytilovum in the ova of the same host, Mytilus edulis, collected at Ocean City, Maryland. After critical study, he (Sprague, 1963) stated that it does not belong to the genus Haplosporidium and later (Sprague, 1965a) transferred it to Chytridiopsis, a genus the systematic position of which remains uncertain. I n his discussion, Sprague pointed out that Chytridiopsis has been thought of as either resembling or being related to the Chytridiales (Schneider, 1884; TrBgouboff, 1913), the Microsporidia (LBger and Duboscq, 1909 ; LBger and Hesse, 1921), the Mycetozoa (LBger and Duboscq, 1909), and

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MARINE MOLLUSCS A S HOSTS FOR SYMBIOSES

the Haplosporida (Caullery, 1953 ; Caullery and Mesnil, 1905 ; Minchin, 1903). Sprague is of the opinion that this genus should be considered as subordinate to the Microsporida since: ‘‘ The spores, having an anterior ‘ vacuole ’, look essentially like those of certain Microsporida which have an anterior ‘ vacuole ’ containing an unextruded polar

1

Stages in the life cycle of Chytridiopsis mytilowurn. (66) M y t i l u s egg containing cysts in five different stages of development ; (67) cyst containing spores not fully mature; (68, 69) lateral and polar views of mature spores; (70) mature spore showing pair of nuclei and spore membrane; (71, 72) lateral and polar views of typical mature spores showing details of structure; (73, 74) mature spore almost in polar view showing a n inclusion body in anterior vacuole and a similar, but uncommon, spore showing minute chromophilic polar granule ; (75, 76) mature spores showing elongated form and internal structure. (Redrawn after Sprague, 1965a.)

FIGS.66-76.

filament ”. Furthermore, he has demonstrated the occurrence of a PAS-positive mass in the anterior pole of the spore which appears to be characteristic of microsporidean spores (Huger, 1960 ; VBvra, 1959, 1960 ; Sprague, 1964, 196513). I n following him, C. mytilowurn is considered herein as a member of the Microsporida.

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5. THE PHYLUM PROTOZOA

Description of sta,ges in mollusc. Cyst, 14-20 p in diameter, most commonly found in egg cytoplasm abutting nucleus (Fig. 66) but is also found within nucleus or in undifferentiated gonadal cells ; cyst spherical or ovoid, with smooth membrane measuring 0 . 3 ~thick (Fig. 67); immature cysts may include one, two, four, eight or more nuclei ; mature cysts enclose thirty-two to sixty-four spores which tend t o clump together near the center ; each spore (Figs. 68-76), 3-5 p in diameter, rarely 6-8 p, spherical or elongate and commonly with large vacuole a t one pole in cytoplasm ; vacuole contains inclusion body ; one or two, typically two, chromatin granules present in each spore. Other information. Although the effect of C. mytilovum on M . edulis, especially its ova, has not been studied in any detail, there is no reason to believe that this parasite is economically important. Sprague (19658) has stated that : “ I n most (infected) specimens the proportion of infected eggs to normal ones was quite small and considerable searching was usually required to find the parasite.’’ 2. Chytridiopsis ovicola LBger and Hollande, 1917

(Order Microsporida; family?) Chytridiopsis ovicola, the only other species of Chytridiopsis known to parasitize a mollusc, the European oyster, Ostrea edulis, was originally described by LBger and Hollande (1917) in France. It is quite similar to C. mytilovum. It too is generally found within its host’s ova. I n fact, as Sprague (1965a) has pointed out, the morphological differences between the two species are slight ; however, “since they seem to have some slight morphological differences, it would be premature to conclude that they belong to the same species.” Description of stages in mollusc. Cysts, 14-16p in diameter, in cytoplasm of host’s ova, closely associated with nucleus ; cyst membrane smooth and identical in shape with that of C. mytilovum ; spores within mature cysts very similar to those of C. mytilovum except that they are slightly smaller, measuring 2.3 p in diameter ; each spore with one large chromatin granule surrounded by a clear zone located at the pole opposite that occupied by vacuole.

F. Subphylum

CILIOPHORA

Class Ciliatea Among the ciliates, a number of species belonging to the order Thigmotrichida have been reported associated with marine molluscs, including some which are commercially or semi-commercially important. A.kI.B.--L

12

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MARINE MOLLTJSCS AS HOSTS FOR SYMBIOSES

Some of these ciliates have been reported to be true parasites while others are believed to be commensals ; however, in no instance has the physiological relationship between the host and symbiont been studied except that some have been reported to feed on their hosts’ cytoplasm. Considered below are those thigmotrichous ciliates which are known to be associated with the more economically important molluscs. For a listing of the known species of the Thigmotrichida, the reader is referred to the extensive studies of Issel (1903)) Jarocki and Raabe (1932))Chatton and Lwoff (1922a,b, 1923, 1924, 1926, 1939, 1949, 1950), Kidder (1933a-d, 1934))Uyemura (1937) and Corliss (1961). 1. Ancistrum mytili (Quennerstedt, 1867). (Figs. 77-79) (Subclass Holotrichia; order Thigmotrichida; family Hemispeiridae) Ancistrum mytili was originally described by Quennerstedt ( 1867) from the mantle cavity of Mytilus in Sweden and named Opalina mytili. Kent (1882) transferred it to the genus Anoplophrya. Maupas (1883) in redescribing this ciliate called attention to the fact that a cytostome is present. He established the genus Ancistrum with A. mytili as the genotype. Strand (1928) erroneously believed that Ancistrum was preoccupied and as the consequence proposed Ancistruma as a replacement, but as Corliss (1961) has pointed out, this was not necessary. According to Kidder (1933c), A. mytili is found in abundance in the mantle cavity of the mussel Mytilus edulis a t Woods Hole, Massachusetts, and at Brighton Beach, Manhattan Beach, and Port Washington, Long Island, New York. According to Issel (1903), it occurs on the gills of Modiola ba,rbata in the Gulf of Naples. More recently, Pauley et al. (1966), in an abstract, have reported the occurrence of ciliates which closely resemble A . mytili and a related species, A . caudatum, free in the branchial chambers of the gills and attached to the ciliated epithelium of the gills of Mytilus edulis in Humboldt Bay, California. Description. Body oval, 52-74 p (average 67 p ) x 20-38 p (average 31 p ) , dorsoventrally flattened ; dorsal surface convex, ventral surface concave ; cilia on both surfaces ; dorsal edge of peristome curves around cytostome which is located near posterior extremity ; peristomal floor folded and protruding ; three rows of cilia on peristomal edges ; macronucleus elongate, kidney-shaped ; compact micronucleus anterior to or a t same level as anterior portion of macronucleus; many food vacuoles, filled with bacteria and small particles, in posterior portion of body ; one contractile vacuole on left of body near posterior terminal.

5 . THE PHYLUM PROTOZOA

179

For detailed accounts of the ciliature, fibrillar system, and nuclear division, see Kidder (1933c), Raabe (1936), and MacKinnon and Hawes (1961).

Life cycle. The complete life cycle of A . mytili is not known although certain aspects have been noted. Raabe (1936) has described its cyst

FIGS.77-79. Ancistrum rnytili. (77) Ventral view; (78) dorsal view, mouth is seen t o bc slightly dorsal near posterior end; (79) lateral view of right side, notice tuft of straight tactile cilia at anterior end. C V , Contractile vacuole; P V , food vacuole; M A , macronucleus ; M I , micronucleus. (Redrawn after Kidder, 1933a.)

which he found during the winter. These cysts are attached to the host’s gill surfaces. The cyst wall is intimately associated with the encysted trophozoite but is drawn out to an anterior point by which it is attached to the host’s ctenidial tissue. MacKinnon and Hawes (1961) have reported that trophozoites placed in a Petri dish will become

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

attached by their thigmotactic cilia but will leave the substratum constantly and flutter up to the surface in a series of jerky somersaults and then return to the bottom. These investigators have expressed the opinion that : " These free-swimming intervals may account for crossinfections (between hosts) ". Ecology. Certain cilia of A . mytili, like those of the other thigmotrichs, are thigmotactic and are employed for attachment. Kidder (1933~)has reported that while swimming, this ciliate engages in " jerky flights ", a phenomenon which has also been noted by MacKinnon and Hawes (1961). Physiology. Nothing is known about the physiology of A. mytili trophozoites. Raabe (1936) has found that encysted forms will excyst in 7-8 h when cysts are maintained a t room temperature. Pathology. As far as is known, A. mytili causes no serious damage to its host, although Pauley et al. (1966), in an abstract, reported that: It appears that ciliates ( A .mytili and A . caudatum) are potential pathogens, capable of infecting a large number of individuals (Mytilis edulis) within a shellfish population and causing pathological changes in the hosts, under certain adverse conditions when the bivalves are weakened. Proof of this remains to be contributed. 2 . Ancistrum cylidioides Issel, 1903. (Figs. 80-82)

(Subclass Holotrichia; order Thigmotrichida; family Hernispeiridae) Issel (1903), in his paper dealing with thigmotrichs collected in the Gulf of Naples, described five new species of Ancistrum. Among these, A . cylidioides was described from the gill surfaces of the amphineuran Chiton olivaceus, the gastropod Natica habraea, and the pelecypods Tellina exigua, Capsa fragilis, Donax trunculus and Tapes decussatus. Since T . decussatus is of some commercial importance, this ciliate is being included herein. I n addition to A. cylidioides, Issel described A. compressum from C q s a fragilis, A. tellinae from Tellina exigua, A. subtruncatum from Tapes decussatus, and A . barbatum from the gastropods Murez trunculus and Fusis syracusanus. Description. Body ovoid when viewed laterally, slightly pointed posteriorly, right side of body strongly convex, left side almost straight, dorsal and ventral surfaces slightly convex, 27-33 p long x 13-18 ,u wide ; seven ciliary rows on right surface, eleven t o twelve on left surface ; with long sweeping cilia on right side of body, cytostome as marginal invagination a t anterior limit of posterior one-

181

5 . T H E PHYLUM PROTOZOA

fourth of body ; single contractile vacuole near posterior body terminal ; numerous food vacuoles in posterior half of body ; macroiiucleus large, occupying most of anterior half of body; micronucleus, 2-5-3 p in diameter, neat anterior body terminal, adjacent to macronucleus.

' \

/

I

I

I

I I

FIGS.80-85. Ancistrum spp. (80) A . cylidioides right view; (81) A . cylidioides left view; (82) A . cylidioides ventral view; (83), A . subtruncatum right view; (84) A . subtruncatum left view; (85) A . subtruncatum ventral view. CS, Cytostome; C V , contractile vacuole ; P V , food vacuole. (Redrawn after Issel, 1903.)

Issel has given a short account of nuclear Other information. division in A . cylidioides.

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M A R I N E MOLLUSCS A S HOSTS F O R SYMBIOSES

3. Ancistrum subtruncatum Issel, 1903. (Figs. 83-85) (Subclass Holotrichia; order Thigmotrichida; family Hemispeiridae) This species was described by Issel (1903) from the gill surfaces of Tapes decussatus collected in the Gulf of Naples. Description. Body ovoid when viewed from lateral aspect, slightly more blunt a t posterior terminal, laterally flattened ; 37-46 p long x 20-25 p in greatest width ; with nine longitudinal ciliary rows on right side and twenty-one to twenty-two rows on left side; cytostome as invagination on left margin for almost one-fourth body length ; rapidly pulsating contractile vacuole in posterior one-fourth of body ; smaller food vacuoles in posterior half of body ; elongate ovoid macronucleus diagonally arranged in anterior half of body ; micronucleus, approximately 4.5 p in diameter, near anterolateral margin of macronucleus. Other information. According to Issel, A. subtruncatum swims by gyrating like a pinwheel and changes direction in a bizarre fashion. It is non-motile when attached to its host. 4. Ancistrum isseli Kahl, 1931. (Figs. 86-88) (Subclass Holotrichia; order Thigmotrichida; family Hemispeiridae) I n his paper on thigmotrichous ciliates from the Gulf of Naples, Issel (1903) described an Ancistrum from the mussel Modiola barbata and, although he found certain differences between his specimens and the earlier descriptions of A . mytili, he considered his specimens as representative of that species. Kahl (1931), however, considered Issel’s ciliates to represent a new species and named it Ancistruma isseli (which should be designated as Ancistrum isseli). Kidder (1933c), who found large numbers of this ciliate in the mussel Modiolus modiolus at Woods Hole, Massachusetts, and Pelham Bay, New York, is in agreement with Kahl that A . isseli is distinct from A . mytili. I have found this ciliate intimately associated with the gill surfaces of Modiolus modiolus collected in Narragansett Bay, Rhode Island (Fig. 88). Description. Body oval, 70-88 p (average 77 p ) x 31-54 p (average 42 p ) , more pointed at both ends than A. mytili, dorsoventrally flattened, concave ventrally, convex dorsally, with both surfaces ciliated ; peristome similar to that of A. mytili but with less prominent flap under cytostome ; enormous macronucleus occupying center of body ; micronucleus small and spherical, in>anterior quarter of body ; numerous food vacuoles in posterior third of body, filled with yellow pigments; contractile vacuole same as in A . mytili. For a detailed description of the body ciliature and fibrillar system, see Kidder (1933~).

5. THE PHYLUM PROTOZOA

183

Life cycle. The complete life cycle of A. isseli is not known but conjugation and nuclear reorganization in this species have been demonstrated by Kidder (1933~). Ecology. Nothing is known about the ecology of A . isseli except that it too possesses thigmotactic cilia. Kidder (1933~)has reported that while swimming this ciliate tends to do so in one direction.

,MN

F

87. Ancistrum isseli. (86) Ventralview; (87) dorsal view. C V , Contractile vacuole ; F V , food vacuole; M N , macronucleus. (Redrawn after Kidder, 1933c.)

FIQS.86 and

Physiology. Kidder has demonstrated that the food vacuoles of A . isseli are filled with the yellow pigment of its host, Modiolus modiolus, but it is not known whether the ciliate actively ingests its hoat’s pigment-bearing tissues.

Ancistrunz japonica Uyemura, 1937. (Fig. 89) (Subclass Holotrichia; order Thigmotrichida; family Hemispeiridae) Ancistrum japonica was described by Uyemura (1937) from within the mantle cavities of several species of marine pelecypods including

5.

184

M A R I N E MOLLUSCS AS HOSTS F O R SYMBIOSES

Meretrix meritrix, Paphia philippinarum (= Tapes semidecussata), Cyclina sinensis, Mactra veneriformis, M . sulcataria and Dosinia bilunulata. Description. Body oval or elongate pyriform, 55-76 p (average 67 p ) x 14-29 p (average 20 p), blunt anteriorly, dorsoventrally flattened, ciliated on both surfaces ; conspicuous macronucleus subspherical, in center of body ; micronucleus spherical, anterior to macronucleus ; single contractile vacuole in posterior portion of body as are numerous food vacuoles. Other information. Nothing else is known about this species.

FIG. 88.

Section through gills of Modiolus modiolus collected in Narragansett Bay, Rhode Island, showing Ancistrum isseli on gill surface.

6 . Boveria subcylindrica Stevens, 1901. (Figs. 90 and 91) (Subclass Holotrichia; order Thigmotrichida; family Hemispeiridae) This species was originally described by Stevens (1901) in California, but Issel(l903)has since found it on the gills of a number of pelecypods in the Gulf of Naples including Tapes decussatus. It is definitely not host-specific as its other hosts include Tellina nitida, T . planata, T .

exigua, Capsa fragilis, Donax politus, Venus gallina, Loripes lecteus, Cardita sulcata and Pinna nobilis. Issel believed that his specimens were sufficiently different from those described by Stevens to justify the establishment of a new variety, concharum.

185

5 . THE PHYLUM PROTOZOA

Description. Body more or less conical, with rounded anterior tapering end, 27-169 p long (average 81.2 p ) x 20-34 p wide (average 27.8 p ) ; cytostome a t posterior end ; peristome spirals posteriorly ; cilia of approximately same length except for peristomal long trailing cilia; definite aciliate girdle between anterior portion of body and peristomal area; macronucleus oval, in anterior half of body; micro-

-cv

- cv

FIGS.89-91. Thigmotrichous ciliates. (89) A ncistrum japonica (redrawn after Uyemura, 1937); (90) Boweriu subcylindrica, left view; (91) B. subcylindrica, ventral view (redrawn after Issel, 1903). C V , Contractile vacuole; F V , food vacuole; M N , macronurleus ; N , micronucleus.

nucleus, 3.5-4.5 p in diameter, anterior to macronucleus ; contractile vacuole near posterior margin of body ; cytoplasmic “ corpuscles ” scattered throughout ; food vacuoles limited to posterior half of body. Other information. According to Issel, this ciliate is sedentary on its host’s gills with its cytostome held against the host’s tissues. This is suggestive that it may be a true parasite feeding from the apposed tissues although some consider it to be a commensal.

186

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

7. Ancistrospiru veneris Chatton and Lwoff, 1926. (Fig. 92)

(Subclass Holotrichia; order Thigmotrichida; family Hemispeiridae) This ciliate is found on the gill surfaces of Venusfasciata in European waters. Its relationship with its host has not been examined although some consider it t o be a commensal. Description. Body ovoid but with pointed anterior end, 50-6Op x 22-28 p ; ciliary rows meridional ; thigmotactic cilia concentrated on left side of body, sharply marked from body ciliature ; peristome with right spiral. Other information. Nothing else is known about this species.

Conchophthirus mytili De Morgan, 1925. (Fig. 93) (Subclass Holotrichia; order Thigmotrichida; family Conchophthiridae) This ciliate was originally described by De Morgan (1925) on the foot of Mytilus edulis collected a t Plymouth, England. Its morphology and nuclear cytology have been studied by Kidder (1933a,b) who also considers it a member of Conchophthirus. However, Raabe (1933), in a review of this and related ciliates, erected the genus Kidderia with K . mytili as the type. Similarly, Kahl (1934) erected Morgunia with M . mytili as the type. Although MacKinnon and Hawes (1961), who recognized the great similarity between Conchophthirus and Kidderia, referred to this ciliate as K . mytili, Corks (1961) has correctly pointed out that both Kidderia and Morgania are junior homonyms and that the correct designation is Conchophthirus mytili. This ciliate can be found in relatively large numbers creeping on the surface of the foot of Mytilus edulis. A single large specimen of M . edulis may yield seventy-five ciliates although five to thirty ciliates per host is more likely. The relationship of C. mytili with its mussel host has not been studied. It may be a commensal, an epiphoront, or an ectoparasi te , Description. Body reniform, 130-220 p x 76-161 p ; dorsal body surface convex; ventral surface flat except posteriorly where it is slightly concave ; numerous trichocysts located in pellicular ridges, more numerous ventrally than dorsally; cytostome on right edge of body ; buccal cavity shallow, with fundamentally similar ciliature ; body cilia slightly longer posteriorly ; cilia on ventral body surface generally used to adhere to host but no special thigmotactic area visible ; numerous food vacuoles scattered throughout body ; macronucleus large and oval, in anterior half of body; two, rarely three, micronuclei present. 8.

5 . THE PHYLUM PROTOZOA

187

Ecology. MacKinnon and Hawes (1961) have noted that the motility of C. mytili is not limited to creeping over its host’s foot. It can also swim with a rotating movement and thus could infect other hosts in the vicinity. Beers (1959))who has examined aspects of the ecology of C. mytili, has found that in general each mussel has from ten to thirty ciliates and about 5 % of these are regularly in division. This is true of ciliates on mussels collected at low tide (mussels out of water) and a t high tide (mussels submerged), hence no correlation exists between the activities of the ciliates and the cycles of water propulsion. As a relatively high percentage of ciliates on any mussel are dividing, yet the total number of ciliates remains relatively constant, it is believed that ciliates are probably continuously being lost in the excurrent stream. That normal reproduction of C . mytili is dependent upon its host, a strong argument in favor of considering these ciliates to be parasites, has also been demonstrated. Beers has reported that although ciliates cultured in vitro feed principally on bacteria and colorless flagellates, they will only grow and divide in sea water t o which a small amount of Mytilus blood (about 0.25 ml per 10 ml sea water) is added. The contribution supplied by blood has not been identified. Beers has also demonstrated that contrary to earlier observations (Kidder, 1933a) C. mytili has considerable tolerance for sea water when removed from its host. It is able to survive for 48-72 h at 14°C. Thus it is well adapted t o live for short periods apart from its host and hence ensures its maintenance and dissemination in a Mytilus population. Relative to host-symbiont contact, Beers has suggested that ciliation is an important physical property of the substratum on which C. mytili lives. I n addition, he has stated that : “ . . . though chemotaxis no doubt affects significantly its choice of a substratum ”. Physiology. The physiology of C. mytili has not been studied. It is of interest to note that MacKinnon and Hawes (1961) have reported that this ciliate has a marked affinity for neutral red, even in weak dilutions. 9.

Crebricoma koxlofji Chatton and Lwoff, 1950. (Figs. 94 and 95) (Subclass Holotrichia; order Thigmotrichida; family Ancistrocomidae)

Kozloff (1946a), while studying ancistrocomid ciliates attached to the palps and ctenidia of the mussel Mytilus edulis in San Francisco Bay, California, found a ciliate which he believed to be identical with Raabe’s (1934) Hypocomina carinata. Certain features of this ciliate

188

MARINE MOLLUSC'S AS HOSTS FOR S Y M B I O S E S

A

FIGS.92-100. Some thigmotrichous ciliates. (92) The infraciliature of Ancistrospira showing oral ciliature on right of animal with typical counterclockwise pattern (redrawn after MacKinnon and Hawes, 1961); (93) Conchophthirus mytili, ventral view (redrawn after Kidder, 1933a) ; (94) Crebricoma kozlofi, ventral view; (95) anterior end of C . kozZo$i showing dorsal origin of some left-hand kineties (redrawn after Kozloff, 1946a); (96) C. carinata, right view; (97) C . carinata, ventral view (redrawn after Raabe, 1934); (98) Raabella helensis forma major, ventral view; (99) R.helensis forma minor, dorsal view (redrawn after Raabe, 1933) ; (100) Isocomidea mytili, ventral view (redrawn after Chatton and Lwoff, 1922a).

5. THE PHYLUM PROTOZOA

189

prompted Kozloff to erect a new genus, Crebricoma, and to designate C. carinata as the type. However, later, Chatton and Lwoff (1950) pointed out that the ciliate studied by Kozloff was not Raabe’s organism but a new species. They named it Crebricoma kozlofi. These French workers, nevertheless, considered Raabe’s ciliate to be a member of Kozloff’s genus Crebricoma and designated it as C . carinata. C. kozloffi is found attached to the ctenidia and palps of Mytilus edulis. Its relationship to its host is not clearly understood. MacKinnon and Hawes (1961), in following Kozloff, have suggested that it may be a true parasite “ and probably feeds on the tissues of its host ”. Description. Body pear-shaped, dorsoventrally flattened, 58-7 1 p (average 64 p ) long, 27-39 p (average 31 p ) wide, and 22-31 p (average 25 p ) thick, with anterior end pointed and posterior end swollen ; with a shallow concavity on anterior portion of ventral surface containing thigmotactic cilia ; dorsal surface and part of ventral surface behind thigmotactic area both convex ; suctorial tentacle, by which the ciliate is attached to host, a t anterior body terminal ; nearly all cilia contained in thigmotactic depression on ventral body surface, arranged in thirtytwo to thirty-six rows; cilia on right, about 10-11 p long, slightly longer than those on left of body ; large contractile vacuole present ; macronucleus ovoid ; micronucleus spherical. Life cycle. Nothing is known about the life cycle of C. kozlofi. It may be speculated, however, that since this ciliate is not continuously attached to its host but is capable of swimming, it could be actively passed from host to host. Pathology. Kozloff (1946a) and MacKinnon and Hawes (1961) have stated that C. k o z b f i probably feeds on its host’s tissues. Actual histopathological changes resulting from its feeding habit have not been studied although it has been stated that “ the damage may be inconsiderable ”. 10. Crebricoma carinata, (Raabe, 1934). (Figs. 96 and 97)

(Subclass Holotrichia; order Thigmotrichida; family Ancistrocomidae) It has been explained above in connection with C . kozlofi that Chatton and Lwoff (1950) considered Kozloff’s C. carinata to be distinct from Raabe’s Hypocomina carinata and have designated the former as C . kozlofli. On the other hand, Chatton and Lwoff, in following Jarocki (1935), believed that Raabe’s ciliate does not belong to the genus Hypocomina but to Kozloff’s genus Crebricoma and designated it as C . carinata. This is a relatively uncommon ciliate found on the ctenidial surfaces of Mytilus edulis in Europe.

190

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

Description. C. carinata resembles C. kozlofi in general size and shape except that its anterior end is more pointed and there is a pronounced ventral curvature of the entire body. A keel is said to occur on the convex dorsal body surface along the anterior half. Details of its thigmotactic ciliature are given by Raabe (1934, 1938) under the name Hypocomina carinata. Other information. Nothing else is known about this species. 11. Raabella helensis Chatton and Lwoff, 1950. (Figs. 98 and 99)

(Subclass Holotrichia; order Thigmotrichida; family Ancistrocomidae) This small and active ciliate was originally described by Raabe (1938) from the gill surfaces of Mytilus edulis collected at Hel, on the Gulf of Danzig. Raabe believed it to be Hypocomides mytili (although it is not the same animal as Chatton and Lwoff’s (1922a) H . mytili which is now known as Isocomides mytili). I n 1950, Chatton and Lwoff established the genus Raabella with R. helensis, the new designation for Raabe’s H . mytili, as the genotype. Kozloff (1964b) found R. helensis on the ctenidia and palps of 80% of the mussels, Mytilus edulis, collected from San Francisco Bay, California. Description. According to Raabe (1938), two forms of R. helensis, major and minor, exist, the latter being consistently smaller. Body of both forms ovoid, slightly dorsoventrally compressed, narrows to pointed anterior terminal which bears suctorial tentacle by which the ciliate is attached to host; major form measures 26-36 p x 19-21 p (Kozloff’s specimens measured 34-38 p long), minor form measures 17-26 p x 15-17 p ; anteriorly located tentacle hollow and continuous with canal in endoplasm which leads half way along body length; tentacle withdrawn while swimming ; anterior portion of central body surface flattened as thigmotactic area ; body cilia, arranged in longitudinal rows, about 8.5 p long ; macro- and micronuclei spherical ; contractile vacuole posterior (Kozloff’s specimens possessed an ovoid macronucleus and the contractile vacuole was centrally located). Ecology. As stated, R. helensis has been found on Mytilus edulis both in the Gulf of Danzig and in San Francisco Bay. There is some doubt whether the specimens from these two areas are identical. MacKinnon and Hawes (1961) have expressed the opinion that Kozloff’s specimens may represent a new subspecies. Although the feeding habit of R. helensis has not been studied, the occurrence of an anterior suctorial tentacle, a feature shared by all the members of the families Hypocomidae and Ancistrocomidae, suggests that it may feed on the host’s tissues as in the case of Crebricorna kozlofi.

6. THE PHYLUM PROTOZOA

191

12. Isocomides mytili Chatton and Lwoff, 1922. (Fig. 100)

(Subclass Holotrichia; order Thigmotrichida; family Ancistrocomidae) This ciliate was originally described by Chatton and Lwoff (1922a) as Hypocomides mytili. It was later (Chatton and Lwoff, 1950) transferred to the genus Isocomides. This ciliate is found on the ctenidia of Mytilus edulis. Description. Body oblong, 57-63 p x 20-22 p, tapering anteriorly to a process which bears suctorial tentacle ; thigmotactic field longitudinally subdivided by a keel of cilia ; cytoplasm on each side of thigmotactic field devoid of cilia ; vestigial kinety, running transversely in groove posterior to thigmotactic field, bears very long cilia. Other information. Nothing else is known about this species. 13. Ancistrocoma pelseneeri Chatton and Lwoff, 1926. (Fig. 101) (Subclass Holotrichia; order Thigmotrichida; family Ancistrocomidae) Ancistrocoma pelseneeri was originally described in a preliminary manner by Chatton and Lwoff (1926) from the surfaces of the gills and

FIG. 101. Ancistrocoma pelseneeri, ventral view. (Redrawn after Kozloff, 1946c.)

palps of Macoma balthica. Since then a more detailed description together with illustrations, has been contributed by Raabe (1934, 1938). I n 1936, Kofoid and Bush described Parachaenia myae from the pericardial cavity and excurrent siphon of Mya arenaria. Kirby (1941) noted that this ciliate is quite similar to A. pelseneeri in several respects. Kozloff (1946c), as the result of studies on ciliates associated with several species of marine pelecypods in San Francisco and Tomales Bays, California, namely Mya arenaria, Cryptomya californica, Macoma inconspicua, M . nasuta, M . irus and M . secta, has concluded that Parachaenia myae is not specific for Mya arenaria as claimed by

102

MARINE MOLLUSCS A S HOSTS FOR SYMBIOSES

Kofoid and Bush (1936), but is associated with all six of the species of molluscs examined. Furthermore he has pointed out that i t is identical with A . pelseneeri. Mackin (1962) has reported the occurrence of Ancistrocoma pelseneeri in Crassostrea virginica " distributed over the Atlantic and Gulf coasts of the United States ". According to him, the ciliate reported by Richardson (1938)) in an unpublished in-house report of the Fisheries Research Board of Canada, from oysters from Charlottetown, Prince Edward Island, Canada, and placed in the genus Orchitophrya, is most probably A . pelseneeri. Relative to parasitization by this ciliate, Mackin has stated: This parasite is usually rather rare in healthy oysters, but in diseased oysters it is often found in large numbers in the stomach or intestine, and may penetrate the ducts and tubules of the digestive gland. It may become a complicating factor in Dermocystidium marinum infections but there is no reason to think that it has much independent effect on the host.

Description. Body 50-83 p (average 62 p ) long x 14-20 p (average x 11-16 p (average 12.5 p ) thick, elongate, somewhat flattened dorsoventrally, anterior terminal more or less attenuated, banana-shaped, with incurved ventral surface when observed in lateral view ; fourteen ciliary rows distributed on ventral, lateral and dorsolateral body surfaces ; usually with five approximately equal rows of cilia about two-thirds of body length on ventral surface, bounded on right by three progressively longer and more widely spaced rows and on left by six similar rows of cilia ; cilia forming outer row on each side extend to near posterior body terminal ; retractable suctorial tentacle at anterior end, with canal leading into cytoplasm ; macronucleua, 11-16 p x 4-7 p, usually sausage-shaped, rarely ovoid, typically situated dorsally near middle of body ; micronucleus, 1.22.1 p x 3.03-3.2 p, ovoid, fusiform, or sausage-shaped, usually on right of macronucleus ; cytoplasm with numerous refractile lipoid granules ; typical food vacuoles in posterior part of body. Ecology. According to Kofoid and Bush (1936), A . pelsenewi appears to be a commensal, feeding on bacteria and cellular debris associated with its host's body surface. On the other hand, Kozloff (1946~)has stated : " I believe that they are primarily branchial parasites which feed by means of the suctorial tentacle." At a later point in his paper Kozloff further stated : " In the posterior part of the body there are in addition t o typical food vacuoles containing ingested fragments of epithelial cells one or more large vacuoles containing globular masses usually of a dense, homogeneous character. ') Thus it 16 p ) wide

193

5 . THE PHYLUM PROTOZOA

would appear that A . pelseneeri is dependent on or a t least can utilize its host’s cells as a nutrient source and hence should be considered a parasite. Pathology. Since A . pelseneeri is known to ingest its host’s cells, one would expect to find some degree of pathological change on those surfaces t o which the ciliates are attached ; however, such changes have not been studied. 14. Ancistrocoma. sp. (Fig. 102)

(Subclass Holotrichia; order Thigmotrichida; family Ancistrocomidae) During a survey of zooparasites of oysters, Crassostrea virginica, in the Maryland portion of Chesapeake Bay, Burton (1961) reported the occurrence of an unidentified species of Ancistrocoma in four out of

FIG. 102.

Section through digestive gland of Grassostrea wirginica from Maryland showing sections of Ancistrocoma sp. in intertubular spaces.

312 oysters. He has noted that this ciliate is found, not as an ectoparasite, but in the hosts’ tissues. Furthermore, except in one instance, this ciliate was found only in moribund oysters. Subsequent examination of oysters from the same area by me revealed the occurrence of this ciliate in the tissues of two out of 120 healthy looking oysters. The ciliates were situated primarily in the intertubular spaces, intermingled among connective tissue elements, in the digestive gland (Fig. 102) A.M.B.-6

13

194

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

although a few also occurred in the gonads. No dramatic histopathological changes were observed by me although Burton (1961) has reported observing abnormal histological appearance of the hosts’ tissues and unusual concentrations of leucocytes which he has attributed to the presence of this ciliate among other parasites. It would appear that this ciliate is a true endosymbiont. It may or may not be identical with the ciliate identified as Ancistrocoma pelseneeri by Mackin (1960). Unfortunately, because of its endosymbiotic nature, intact specimens were not available for description. It should be noted that the possibility exists that this is not a species of Ancistrocoma, but the dimensions and what can be seen of its morphology in histological sections suggest that it is. 15. Hypocornides mytili Chatton and Lwoff, 1922. (Figs. 103 and 104) (Subclass Holotrichia; order Thigmotrichida; family Ancistrocomidae) Chatton and Lwoff (1922a) erected the genus Hypocomides to include two new species of ancistrocomid ciliates, H . rnytili from Mytilus edulis, and H . modiolariae from Modiolaria, marmorata. Subsequently (Chatton and Lwoff, 192213, 1924), brief mentions were made concerning the morphology of H . mytili but they never illustrated it. Similarly, Chatton and Lwoff (1926) published a sketchy diagnosis of a third species, H . zyrphaeae from Zirfaea crispata. Basing his decision primarily on differences between H . zyrphaeae and ciliates which he found on the gills of Mytilus edulis, Raabe (1938) reported the finding of H . mytili but he was not able to detect the structure referred to by Chatton and Lwoff as “ vestige de frange adorale ” which is supposed to be bettex developed in H . modiolariae than in H . mytili. Later, Kozloff (194613) found ciliates on Mytilus edulis from San Francisco Bay, California, which conformed to Raabe’s description of H . mytili. He too was unable to detect a vestige de frange adorale Furthermore, Kozloff stated : The position of the “ vestige de frange adorale ” in relation to other ciliary structures in these species of Hypocomides ( H . mytili, H . modiolariae and H . zyrphaeae) is entirely unclear . . . and it is altogether possible that, as Raabe has pointed out, these authors (Chatton and Lwoff) used the term “ vestige de frange adorale ” t o indicate only a short segment of the distal portion of one of the longer ciliary rows on the right side of the body. . . ((

”.

The following description of H . mytili is based on the descriptions of Raabe (1938) and Kozloff (1946b). Description. Body elongate, somewhat flattened dorsoventrally, 34-48p (average 40p) long x 16-22p (average 1 8 p ) wide x 13-18p

5. THE PHYLUM PROTOZOA

195

(average 14-5 p) thick; cilia disposed for the most part on shallow concavity occupying anterior three-fifths of central body surface ; central ciliary complex of seven rows which are one-third to one-half length of body and which become progressively longer towards left side ; right complex of two rows, each about one-half body length ; left

MI MA

FIGS.103-109. Some thigmotrichous ciliates. (103) Distribution of ciliary rows on Hypocornides mytili, d'brsal view ; (104) distribution of ciliary rows on ' H . mytili, ventral view (redrawn after Kozloff, 1946b) ; (105) adult Gargariua gargariua, dorsal view; (106) a. gargariua adult impregnated by silver (there are no cilia), ventral view ; (107) ciliated larva of B. gargarius (redrawn after Chatton and Lwoff, 1950); (108) right aspect of Thigrnophrya macomae showing torsion of kineties and displacement of cytostome; (109) ventral aspect of T. macomae with anterior suture (redrawn after Raabe, 1936). M A , Macronucleus ; M I , micronucleus.

complex of eight closely set rows, each about one-third to one-half body length ; dorsal body surface and that portion of ventral surface posterior to ciliary area are convex; short contractile tentacle a t anterior end continuous with internal canal which leads to center of body ; macronucleus, 9.0-13.2 p x 4-4-6.9p, usually ovoid, sometimes sausageshaped, rarely spherical (Raabe stated that the macronucleus is typically

196

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

spherical), dorsally situated in posterior half of body ; micronucleus, 2.7-4-0 p in diameter, spherical, near middle of body, anterior t o or to one side of macronucleus ; contractile vacuole centrally located, opens to exterior on ventral body surface ; cytoplasm with numerous lipoid droplets ; several food vacuoles in posterior part of body. Ecology. Kozloff (194613) has found H . mytili on the gills and palps of about 80% of the specimens of Mytilus edulis from various localities in San Francisco Bay. It may be the only ciliate present on these mussels or, more commonly, it is associated with two other ciliates, Ancistrum mytili and Crebricoma koxlofi. According to Kozloff, H . mytili is a true parasite since it is nutritionally dependent upon its host. He states : “ The anterior end of the body is provided with a short contractile tentacle which enables the ciliate to attach itself to the epithelial cells of the gills and palps of the host and to suck out their contents.” Pathology. Although H . mytili is known to feed on the contents of the epithelial cells of Mytilus edulis, the actual pathological changes associated with this feeding mechanism, at both the cellular and organismic levels, have not been studied. 16. Gargarius gargarius Chatton and Lwoff, 1934. (Figs. 105-107)

(Subclass Holotrichia; order Thigmotrichida; family Sphenophryidae)

Gargarius gargarius was originally discovered by Chatton and Lwoff (1934) attached to the gills of Mytilus edulis. It was redescribed by Raabe (1938) and Chatton and Lwoff (1950). Description. Body ovoid and dorsoventrally flattened, tapering a t anterior and posterior ends, more so anteriorly, 25-60 p long, devoid of cilia ; distinct suctorial tentacle continuous with canal leading to interior of body ; lateral borders of body lined with blunt, pointed, or capitate fringed papillae of unknown function ; papillae inserted on marginal filaments encircling body behind tentacle and further supported by filaments which pass into endoplasm; macronucleus large and elongate ; micronucleus large, nearly half the size of macronucleus ; no contractile vacuole present. Life cycle. G . gargarius reproduces by budding off a ciliated larva posteriorly (Fig. 107). The cilia of the daughter arise from its kinetosomes. The suctorial tentacle develops when the daughter is severed from the parent as are the marginal papillae. It still remains undetermined how individuals are passed from host to host. It is possible that a free-swimming stage, possibly the larva, exists.

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Other information. According to Raabe (1938), G. gargnrius is only found on hosts which harbor few or no other ciliates. 17. Thigmophrya macomae Chatton and Lwoff, 1923. (Figs. 108 and 109)

(Subclass Holotrichia; order Thigmotrichida; family Thigmophryidae) Thigmophrya macomae, described by Chatton and Lwoff (1923) on the gills of Macorr~abalthica, is briefly mentioned here since this clam, but particularly the closely related species M . nasuta and M . secta, are harvested as food (Hedgpeth, 1962). Description. Body elongate ovoid, averaging 110 p x 40 p, dorsoventrally flattened, with ventral surface slightly concave ; cytostome in posterior third of body ; oral funnel opened ; contractile vacuole opens a t bottom of cytopharynx ; with numerous rows of cilia.

CHAPTER 6

PARASITES OF COMMERCIALLY IMPORTANT MARINE MOLLUSCS THE PHYLA PORIFERA, CNlDARlA AND PLATYHELMINTHES I. Phylum PORIFERA Boring sponges of the genus Cliona were first mentioned in the scientific literature by Giard (1881) who indicated that they were well known to French oystermen as the causative agent of “ spice bread disease ”. This disease has been claimed to cause the mass destruction of beds of oysters. Since Giard’s original report, over 100 scientific papers dealing with boring sponges have been published. Among the more recent, the most useful is that of Old (1941) who has contributed a taxonomic study of those species found along the Atlantic coast of North America, and those of Hopkins (1956a,b, 1962) who has demonstrated that the distribution of the species, a t least the common ones, i.e. C. elata, C. vasti$ca, C. lobata and C. truitti, are governed by the ambient salinity. None of the boring sponges are believed to be true parasites. Galtsoff (1964), in his summary of the biology of boring sponges, has stated : “ It is not known whether boring sponges use the organic component (conchiolin) of the shell, but it is obvious that they do not draw their nutrients from the body of the oyster.” I n most instances boring sponges inhabit tunnels in the molluscs’ shells. These tunnels are bored by the sponges by what is believed to be mechanical action, although the secretion of acids to aid in the fragmentation of the shell may occur (Galtsoff, 1964). These tunnels do not as a rule perforate the shell, hence the sponges do not make contact with the oyster’s soft tissues. It is only in instances of old and heavy infestations that the tunnels perforate the entire thickness of the shell. Even then, the holes are rapidly mended by a deposition of conchiolin. If this mending process is delayed by adverse conditions, the sponges will make direct contact with the mantle and produce lysis of the epithelium and underlying connective tissue. Grossly, areas on the mantle in contact with sponges appear as dark pigmented pustules. 198

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11. Phylum CNIDARIA Most of the cnidarians known to be associated with marine molluscs are epiphoronts" ; however, four species of the genus Eugymnanthea have been reported as " commensals " found within the mantle cavities of commercially important pelecypods (see Crowell, 1957). Until more information on the relationship between these hydroids and their hosts becomes available, these are only briefly mentioned herein. The first of the fourt species, E . inquilina, was described by Palombi (1936a) from within the mantle cavity of Tapes decussatus collected a t Lago Fusaro, near Naples, Italy. The second, E . polimantii, was originally described as Mytilhydra polirnantii by Cerruti ( 1941b) from the mantle cavity of Mytilus galloprovincialis a t Taranto, Italy. The third species, E. ostrearum, was described by Mattox and Crowell (1951) from within the mantle cavity of Crassostrea rhizophorae from Boqubron Lagoon, Puerto Rico. The fourth, E . japonica in Crassostrea gigas in Japan, was described by Yamada (1950) in a new genus Ostreohydra, and later transferred to Eugymnanthea by Crowell (1957). All four species possess a conspicuous rounded basal disk by which the polyps are enabled t o remain attached to their host's tissues. Furthermore, both a perisarc and stolons are absent. These features, according to Mattox and Crowell (1951), may be considered as adaptive to commensalism. Within their hosts, the polyps are attached to the inner surface of the mantle but in heavily infested pelecypods, they may also be attached on the foot, gills and labial palps.

111. Phylum PLATYHELMINTHES A. Class Turbellaria Several species of turbellarians have been reported associated with oommercially important marine molluscs. Among these, several belonging to the genera Stylochus and Pseudostylochus are known to feed on oysters and other marine invertebrates. I n accepting the definition of predation given on pp. 8-9, these turbellarians should be considered as predators rather than parasites. For this reason, these turbellarians will not be considered a t any length herein but, for the convenience of those interested in invertebrates associated with commercially important marine molluscs, the more common species are briefly mentioned along with citations to the pertinent literature. * F o r a review of the types of relationships occurring between cnidarians and molluscs, see W. J. Rees (1967). t Crowell (1957) thinks that E. inquilina and E. polimantii are the same species.

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1. Stylochus ellipticus (Girard, 1850)

(Order Polycladida; suborder Acotylea; family Stylochidae) This turbellarian, one of the so-called oyster " leeches )', was originally described by Girard (1850) as Planocera elliptica but it has since been transferred to the genus Stylochus as S. ellipticus by Hyman (1939). According t o Hyman (1940b), its synonyms include Planocera nebulosa Girard, 1853, Stylochopsis littoralis Verrill, 1873, Stylochus littoralis Lang, 1884, Eustylochus ellipticus Verrill, 1892, and E . meridionalis Pearse, 1938. S . ellipticus is a littoral species, living among barnacles, oysters and shells, on pilings and under rocks. It feeds on oysters, barnacles, and perhaps other invertebrates, especially pelecypods. It apparently has no difficulty in entering oysters, especially spat, through slightly opened valves. According to Loosanoff (1956), this turbellarian was responsible for the destruction of large numbers of young oysters on the flats a t Milford, Connecticut. Its geographic range extends from Texas to Prince Edward Island, Canada, being very common south of Massachusetts. A good description of this species has been given by Hyman (1940b). 2. Stylochus frontalis Verrill, 1892 (Order Polycladida; suborder Acotylea; family Stylochidae) This gray or yellowish gray turbellarian, also known as an oyster " leech ", was originally described by Verrill (1892a). It has been referred to as Stylochus inimicus by Palombi (1931b), Pearse (1938) and Pearse and Wharton (1938), and as S . tenax by Palombi (193613). This is also a littoral species, being found among living oysters and oyster shells. Pearse and Wharton (1938), who investigated the cause of the oyster mortalities in Apalachicola Bay, Florida, could not definitely state that the destruction was due to S . frontalis, but have suggested that the oysters are first weakened by some unknown cause after which this polyclad readily invades those unable to close their valves. According to Hyman (1940b), S . frontalis feeds primarily on oysters. It is distributed on oyster beds from Florida t o Texas and northward to the Carolinas. Its biology has been studied by Pearse and Wharton (1938) and a good description is given by Hyman (1940b). 3. Stylochus oculiferus Girard, 1853 (Order Polycladida; suborder Acotylea; family Stylochidae) This comparatively rare polyclad is primarily found on oyster beds although it is not known whether it feeds on oysters. Originally

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described as Imogine oculifera by Girard (1853), it has since been transferred to the genus Stylochus as S. oculiferus by Diesing (1861). According to Hyman (1940b)) Pearse’s S. Jloridanus is a synonym. S. oculiferus has been found along the coasts of Florida and North Carolina. A good description has been given by Hyman (1940b). 4. Pseudostylochus ostreophagus Hyman, 1955

(Order Polycladida; suborder Acotylea; family Calliophanidae) During 1953, shellfish biologists in the State of Washington suspected that a polyclad turbellarian imported from Japan was responsible for the destruction of the spat and young adults of Ostrea lurida. Specimens of this polyclad were sent to Dr. L. H. Hyman who later (Hyman, 1955) described it as representing a new species, Pseudostylochus ostreophagus. That this turbellarian was imported from Japan was confirmed when Mr. C. E. Woelke visited three localities in Japan, the Liyagi Prefecture, the village of Oginshama on the Osika Peninsula, and the village of Sabusawa in the Urato Islands, and found the identical worm. The relationship of P. ostreophagus to Ostrea lurida in addition to Crassostrea gigas and C. virginica was studied by Woelke (1957). He reported that destruction of the oysters is accomplished when the turbellarian penetrates the shell and inserts the edge of its pharyngeal folds by the use of which it severs the adductor muscle from the right valve. Then the worm crawls between the gaping valves and ingests the entire living oyster. Thus, P. ostreophagus must be considered a predator rather than a parasite. The actual method of shell penetration by P. ostreophagus is not well understood, although it is believed to be a chemical process since Woelke has observed the presence of gelatinous mucoid droplets on the incomplete shell perforations of attacking worms.” Unpublished experiments by Smith ( 1955),however, have indicated that homogenates of whole worms will not dissolve oyster shells. ((

RADCLiFFe 5 . Hoploplana inquilina Wheeler, 1894

(Order.Polycladida; suborder Acotylea; faniily Hoploplanidae) This turbellarian, originally described by Wheeler (1894) as Planocera inquilina, has been referred to the genus Hoploplana by Bock (1913). It has since been studied by Hyman (1939, 1940b). It is being briefly mentioned here since this sluggishly creeping worm is said to be a commensal in the mantle cavity of Busycon.

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6. Hoploplana inquilina thaisana Hyman, 1940

(Order Polycladida; suborder Acotylea; family Hoploplanidae) Originally described as Hoploplana thaisana by Pearse (1938), this polyclad, which has been found in Apalachicola Bay, Florida, was given its present designation by Hyman (1940b) who stated: Insofar as can be determined from t he available data and material, H . thaisana differs from H . inquilina only in its smaller size and in its commensalism with a different snail host. I am therefore of the opinion that it is at best a geographical subspecies and hence should be named H . inquilina thaisana.

It is being briefly mentioned here since this turbellarian, generally a <<

commensal ” of the snail Thais Jloridana Jloridana, has also been found associated with oysters and barnacles in Florida. 7. Paravortex gemellipara (Linton, 1910)

(Order Rhabdocoela; suborder Lecithophora; family Graffillidae) This viviparous rhabdocoel was originally described by Linton (1910) as Grafilla gemellipara. He found it on the gill surfaces of the ribbed mussel, Modiolus demissus. The relationship between this turbellarian and its host is not clear although Linton inferred in the title of his paper that it is a commensal. Ball (1916), who studied the development of this worm, demonstrated why it properly belongs to the genus Paravortex. According to Linton (1910), P. gemellipara is generally found in mussels which are exposed t o fairly free tidal currents rather than in those confined t o coves. Furthermore, large mussels appear t o harbor more specimens than smaller ones. First discovered in the Woods Hole, Massachusetts, area, it has been also found in mussels from New Haven, Connecticut. Although Linton has stated that it is only found associated with Modioks demissus in Massachusetts, he did point out that a form found in Cardium edule by Nicoll (1906b) “ . . . and identified as a sporocyst is very near, if not identical with this species ”. Furthermore, Uzmann (unpublished) has reported it in M y a arenaria on the Atlantic coast of the United States. It would thus appear that P.gemellipara is not host-specific.

B. Class Trematoda A comparatively large number of larval stages of digenetic trematodes have been recorded from commercially important marine molluscs. These have been either in the form of germinal sacs (rediae and sporocysts) enclosing cercariae or as metacercariae, either encysted or

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non-encysted. From what. is known about the life cycles of the Digenea, those molluscs which harbor the germinal sacs and cercariae are first intermediate hosts while those which harbor metacercariae are second intermediate hosts. Instances are known, however, where cercariae and metacercariae of the same species are found in the same mollusc. In such cases, the mollusc is serving both as the first and second intermediate hosts. Given in this section are those species of trematodes which have been recorded from commercially important marine molluscs. Unfortunately, in attempting an adequate description of each species, it was found in many instances that the original descriptions were rather inadequate by modern standards and, as the result, the descriptions given below reflect the inadequacies of certain of the original descriptions. I n still other instances, for example in Gutsell's (1930) report of parasites of the scallop Aequipecten ( =Pecten) maximus, the descriptions of the trematodes encountered are so inadequate that these are not even being considered here. The familial designations used are those of the original author unless more recent evidences suggest otherwise. The higher taxonomic designations are those of LaRue (1957). 1. Bucephalus haimeanus Lacaze-Duthiers, 1854. (Figs. 110 and 111)

(Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Bucephaloidea; family Bucephalidae) Bucephalus haimeanus was originally described by Lacaze-Duthiers (1854) based on sporocysts and cercariae found in the oyster, Ostrea edulis, and the cockle, Cardium tuberculatum (=C. rusticum), from the Mediterranean Sea. Nicoll (1914) renamed this parasite Bucephalopsis haimeanus, basing his decision on Tennent's (1906) report that the adult of Bucephalus haimeanus (actually B. cuculus)is without a cephalic hood or papilla like that which occurs in Bucephalus polymorphus, the genotype. But, as Hopkins (1954a, 1957a) has correctly pointed out, the life cycle of B. haimeanus, as reported by Tennent, was in error due to a lack of validly controlled experiments and what Tennent considered to be stages in the life history of his parasite may well represent two or more bucephalid species. For this reason, until further indisputable evidences become available, the designation Bucephalus haimeanus is being retained. This larval trematode appears to be the common one in Ostrea edukis and Cardium tuberculatum in European waters (Huet, 1888~1, 1893 ; and others). It has been repeatedly reported in the European literature as " Bucephalopsis gracilescens ", '' Gasterostomum graci-

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MARINE MOLLTJSCS AS HOSTS FOR SYMBIOSES

lescens or as B. haimeanus in marine pelecypods. For example, Johnstone (1904) has reported its occurrence in Cardium edule from the Lancashire coast, Pelseneer (1906) has reported it in Syndosmya alba from the north of France, and Lebour (1911) has found it occurring throughout the tissues of Cardium edule collected a t Fenham Flats, Northumberland, and at Ensworth, Hampshire, both in England. It is still uncertain whether all these earlier investigators found B. haimeanus or the sporocysts of some other species of bucephalid trematode, since from first hand experience it is known that it is extremely difficult, if not impossible, to distinguish one species of bucephalid sporocyst from another and inasmuch as a t least 145 species of adult bucephalids have been described from fishes (Yamaguti, 1958), and many from marine fishes, it is highly probable that the sporocysts of various other species of bucephalids have been confused with B. haimeanus. Recently, James et al. (1966) have reported finding this parasite in Cardium edule from Llanrhidian Sands, Gower Peninsula, Wales. They designated it as Cercaria bucephalopsis haimeana. ”)

Description of stages in mollusc. Sporocysts long, tubular, multibranched, and tangled, closely packed in all body tissues of host except foot; cercariae a t various stages of development in brood chamber (Fig. 110). Cercaria (Fig. 11 1) with characteristic forked tail, with each furca attached to prominent triangular stem ; tail extremely contractile and may be many times longer than body when extended ; body proper approximately 0.26 mm long, covered with cuticular spines ; mouth, in center of ventral sucker (pharynx of some authors), leads into simple sac-like intestine ; anterior holdfast (rhynchus of some authors) armed with secretory glands ; oval excretory vesicle posterior t o ventral sucker. James et al. (1966) have studied the fine structure of the sporocyst wall of Bucephalus haimeanus. Their electron micrographs have revealed that the sporocyst wall is comprised of an external syncytial tegument, lying on a basement lamella, and an internal cellular subtegument which surrounds the brood chamber. The syncytial tegument has areas of dense cytoplasm alternating with sparse reticulate cytoplasm. The dense cytoplasm contains nuclei, a few mitochondria and secretory products.

Life cycle. The complete life cycle of B. haimeanus is not known. Although various authors (Williamson, 1911 ; Johnstone, 1904 ; Dawes, 1946 ; and others) have reported what they consider to be the metacercaria of this trematode encysted in the nerves, mainly the cranial but also in the spinal nerves near the tail of gadoid fishes, commonly

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the cod, Gadus callarias, and the whiting, G. merlangus, it is not known with certainty that this is the metacercarial stage of B. haimeanus although it may very well be. The adult is believed t o be an intestinal parasite of the angler, Lophius piscatorius, which feeds on gadoids.

FIGS.110-114.Bucephalus spp. (110) Portion of dendritic sporocyst of B. haimeanus en&osing cercariae at various stages of development ; (111)fully developed cercaria, of B. haimeanus (redrawn after Lebour, 1911) ; (112) young sporocyst of B. cuculus showing initiation of branching (redrawn after Tennent, 1906) ; (113) sections of tlendritic sporocysts of B. cuculus showing developing cercariae within brood rhambers; (114) fully developed B. cuculus cercaria (redrawn after Hopkins, 1954). E V , Excretory vesicle ; B P , genital primordium ; H , haptor (rhynchus); OS, oral sucker.

Ecology. According t o Hopkins ( 1954a), B. haimeanus sporocysts are found in more saline oceanic waters where its most common host,

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Ostrea edulis, lives and is thus different from the American species, B. cuculus, in Crassostrea virginica, which is found in more landlocked estuarine waters of comparatively low salinities. Physiology. The histochemical studies by James et al. (1966), together with electron microscope studies, have revealed that the dense cytoplasmic areas of the syncytial tegument of Bucephalus haimeanus sporocysts include a neutral mucopolysaccharide which may be the precursor of the acid mucopolysaccharide found in the sparse reticulate cytoplasmic areas of the same zone and on the sporocyst surface. James et al. have also stated : Although there is no evidence of pinocytosis (on the outer surfaye of the external syncytial tegument) in the electron-micrographs, the peculiar distribution of glucose, glycogen, fatty acids and neutral lipids, in the tegument and subtegument, suggests that these substances pass through the outer plasma membrane and the sparse reticulate cytoplasm of the middle region to accumulate above the inner plasma membrane of the tegument. They then pass through this and the basement membrane into the extensions of the giant somatic cells, which form such a conspicuous part of the subtegument. This represents the first published interpretation of the mechanism by which sporocysts acquire their nutrients and perhaps other materials as based on electron microscope studies. Pathology. Although the sporocysts of B. hairneanus are known to infiltrate practically every organ in the body of its molluscan host, a detailed histopathological study has not yet been made. I t s effect on the mollusc has not been investigated other than that this parasite is known t o cause parasitic castration. It could be noted that Millar (1963) has reported that a significant percentage of Ostrea lutraria shipped to Great Britain from Faveaux Strait, New Zealand, parasitized by an unidentified bucephalid, was killed. 2. Bucephalus cucuZus McCrady, 1873. (Figs. 112-1 14) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Bucephaloidea; family Bucephalidae) What is generally believed t o be the American species of Bucephalus, B. cuculus, was originally found and named by McCrady (1873) from the American oyster, Crassostrea virginica, in Charleston, South Carolina, although his description was far from adequate by modern standards. Since then, what is presumed to be the same species has been reported by Nelson (1890, 1903, 1915) in C. virginica from New Jersey, and Glaser (1904) and others from the same oyster at Cameron,

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Louisiana. It was most probably this species that Tennent (1905, 1906, 1909) found in C. virginica taken from the Rappahannock River in Virginia and a t Beaufort, North Carolina, although he designated his specimens as B. haimeanus. Although B. cuculus is extremely difficult to distinguish from B. haimeanus morphologically, I am in agreement with Hopkins (1954a) that : “ it would be very difficult t o believe that two forms with such striking ecological and physiological differences could belong to one species.” B. haimeanus is found in molluscs in waters of high salinity, while B. cuculus is confined to the least saline parts of the range of Crassostrea virginica.

Description of stages in mollusc. The tubular and dendritic sporocysts of B. cuculus are extremely similar to those of B. haimeanus (Fig. 113). Tennent (1906), under the name B. haimeanus, has given a good description of the developing stages of the sporocyst of B. cuculus. The most characteristic features of the sporocyst are that its diameter is rather uniform, measuring 0.1 mm, and the growing end is rounded and somewhat greater in diameter than the older portions (Fig. 112). The following description of the cercaria is based on the descriptions given by Tennent (1906) and Hopkins (1954a). Cercaria (Fig. 114) with characteristic forked tail, with each furca measuring 0-04-0.20 mm long x 0.07-0.17 mm wide in contracted state, it may be as long as 5-10 mm when extended ; each furca attached to globose, more or less bilobed stem a t its anteroventral borders ; body proper elongate, 0.15-0.40 mm long x 0-04-0.12 mm wide, covered with cuticular spines anteriorly ; mouth a t middle of body, surrounded by ventral sucker which measures 0.026-0.029 mm in diameter ; mouth leads into simple blind sac caecum ; anterior holdfast (rhynchus) surrounded with gland cells as is ventral sucker ; genital anlagen posterior t o pharynx and intestine ; subcuticular glands opaque, scattered over entire body surface, also along posterior edge of tail 2 2) (2 2 a)]. stem and furcae; flame-cell pattern 2[2

+ + + + +

Life cycle. Tennent (1906) has reported what he assumed t o be the life cycle of B. cuculus (referred t o by him as B. haimeanus). He reported the finding of encysted metacercariae in the silverside, Menidia menidia, and adults in the intestine of Strongylura marina. Thus the life cycle is similar in pattern to what has been assumed for B. haimeanus. Hopkins (1954a), however, has correctly pointed out that Tennent has not proven beyond doubt that there is any connection between B. cuculus from Crassostrea virginica and the stages found in Menidia and Xtrongylura. Furthermore, as the result of examining bucephalid trematodes in these two species of fish, Hopkins has convincingly

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pointed out that a t least some of the trematodes which Tennent believed to be B. cuculus metacercariae and adults could have been those of a species of Rhipidocotyle. It should be pointed out, however, that Tennent (1909) did prove that eggs or miracidia from an unknown adult bucephalid in the gar, Lepasdosteus osseus, could infect Crassostrea virginica and develop into sporocysts which grow for a t least one month. However, whether this parasite of the gar is B. cuculus is not known. Ecology. According to Hopkins (1954a), B. cuculus, from what is known, occurs in oysters from New Jersey to Texas, but its distribution is not continuous throughout this range. It is also known that the incidence of infection in Crassostrea virginica is highest in small landlocked estuaries. Physiology. Nothing is known about the physiology of 3. cuculus except that it apparently cannot survive high salinities. Pathology. A detailed histopathological study of Crassostrea virginica parasitized by B. cuculus has not yet been made although certain pathological changes are known. It is known that B. cuculus sporocysts utilize the oyster's gonads as the primary site of infection, and in so doing usually cause parasitic castration. As the sporocysts increase in length and undergo further branching, practically all of the organs of its host, except the foot, become infiltrated. Whether the parasite will eventually kill the oyster has not been studied under controlled conditions. Hopkins (1957a), who has had long experience with this parasite, has stated : " Presumably the parasite would eventually kill the host, if the oyster did not die of old age or something else first, but this has not yet been proved." The effects of B. cuculus in the growth of oysters as reported by Menzel and Hopkins (1955a,b) have been reviewed in Chapter 4, Section 11, A, 5. 3. Bucephalus sp. (Fig. 115) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Az ygiata; superfamily Bucephaloidea; family Bucephalidae) During a survey of zooparasites of Crassostrea virginica in Rhode Island, U.S.A., we (Cheng and Burton, 1965b; Cheng, 1965c) found the long dendritic sporocysts of a species of Bucephalus in oysters from Ninigret Pond in Washington County. The cercariae produced within these sporocysts are very similar to those of B. cuculus but two distinct differences between this form and 3. cuculus are evident. Firstly, the primary site of infection by the Rhode Island form is the digestive gland rather than the gonads. Secondly, the growing terminal

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of Bucephalus sp. is not rounded and larger in diameter than the preceding section. Rather, it is smaller in diameter and more or less knobshaped. Perhaps some physiological reason for the difference in the primary site of infection is possible. However, until further detailed studies are made, it would appear appropriate to refer to the Rhode Island form as Bucephalus sp. rather than designate it as representing a new species.

FIG. 11 5 . Section through digestive gland of Crassostrea viryinica from Rhotle Island parasitized by sporocysts of' Bucephalus sp.

The histopathological changes in Crassostrea virginica parasitized by Bucephalus sp. have been studied by Cheng and Burton (1965b) and have been reviewed in Chapter 4, Section 11,A, 5 .

4. Bucephalus mytili Cole, 1935. (Figs. 116 and 117) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Bucephaloidea; family Bucephalidae) This cercaria was described by Cole (1935) from Mytilus edulis from Conway, Wales. According to him, Atkins has also found this or a similar species in M .edulis from Padstow, England. A.M.B.--8

14

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D e s c r i p t i o n of stages in mollusc. Xporocyst (Fig. 116) long, threadlike, and branched, tangled together in host’s digestive gland, gonad and mantle ; pale yellow in color. Cercaria (Fig. 117) typical of bucephalids,

FIas. 116-1 18. Bucephalus spp. (116) Portion of B. mytili sporocyst enclosing developing cercariae ; (117) fully developed B. mytili cercaria (redrawn after Cole, 1935); (118) fully developed Cercaria noblei (redrawn after Giles, 1962). C , Cirrus; CO, cystogenous organ ; C P , c m u s pouch ; DC, developing cercaria ; E V , excretory vesicle ; GS, genital sinus ; H , haptor ; 08, oral sucker ; 0 V , ovary ; T,testis.

with long furcae, nearly twice as long as body, attached t o anterior surface of tail stem; tail stem comprised of median and two lateral lobes ; body proper about 0.26-0.27 mm long, anterior holdfast (rhyn-

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chus) about 0.04 mm x 0.03 mm ;ventral sucker, 0.025 mm in diameter, situated behind middle of body, 0.16-0.17 mm from anterior end; intestinal caecum simple and sac-like ; body with few rows of minute cuticular spines a t anterior end; stem of tail covered with minute spines ; two testes well developed, spherical, one behind the other, just posterior to ventral sucker ;vas deferens leads from testes to prominent cirrus opening a t posterior tip of body ; no excretory vesicle seen. Other information. From gross descriptions given by Cole (1935), it would appear that the sporocysts of B. mytili, like those of other known bucephalids, could cause parasitic castration in the molluscan host. 5 . Cercaria noblei Giles, 1962. (Fig. 118)

(Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Bucephaloidea; family Bucephalidae) This bucephalid cercaria was described by Giles (1962) from Mytilus californianus collected a t Dillon Beach, Marin County, California. Description of stages in mollusc. Sporocyst typically bucephalid, orange in color, occupying principally the gonads, may occur in other tissues, including kidney, packed with cercariae at various stages of development. Cercaria (Fig. 118) with blunt cylindrical body, tapering slightly a t posterior end, 0.155-0.230 mm (average 0.185 mm) long x 0.032-0.058 mm (average 0.040 mm) wide; tail stem of three lobes, median lobe 0.066-0-073mm (average 0-070mm)long x 0~015-0~018mm (average 0.017 mm) in diameter, lateral lobes 0.051 mm long x 0.016 mm in diameter ; long filamentous furcae highly contractile, 2-7.5 times length of body when extended, generally directly anteriorly ; sucker-like structure a t free end of each lobe of tail stem; anterior holdfast (referred t o as a haptor by Giles) eversible and cup-shaped, 0.045 mm long, covered with small spines distally ; butterfly-shaped cytogenous gland, 0.040 mm long x 0.035 mm wide, immediately posterior to haptor; ventral oral sucker in posterior half of body, 0.014-0-016 mm in diameter ; intestinal caecum simple, sac-like, 0.031 mm long; two testes posterior to ventral sucker, right testis 0.018 mm x 0.010 mm, left testis 0.017 mm x 0.017 mm; cirrus pouch, ventral to left testis, continuous with genital atrium on ventral body surface immediately anterior to posterior body terminal ; ovary, 0.015 mm x 0.008 mm, dorsal to right testis and extends slightly anterior to i t ; excretory vesicle extends from body-tail junction to posterior edge of ventral sucker. Other injormation. Nothing else is knowB abmf C, n~bZ..i

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

6. Mefacercaria (Gymnophallus)margaritarum (Dubois, 1901). (Figs. 119-121)

(Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family Fellodistomatidae) The majority of gymnophallid trematodes known from commercially important marine molluscs are only known in their metacercarial forms. These are usually found between the mantle and shell of pelecypods. From what is known, it may be said that the cercariae of these trematodes develop in spherical or subspherical sporocysts in pelecypods. The cercariae possess eye-spots which may be reduced or wanting. Furthermore, they are usually without a tail by the time they escape from the sporocyst ; however, a small forked tail does develop in certain species (Fig. 127), thus suggesting their relationship to the furcocercous cercariae, but the tail is usually lost prior to emergence. I n at least three species, Cercaria dichotoma, C. fulbrighti and C. myae, the tail is retained by the emerging cercaria. Stunkard and Uzmann (1958) have given an excellent review of the gymnophallid trematodes. As they have stated, very often in the past individuals working on these trematodes have mistaken metacercariae for cercariae. Furthermore, there is little information on the asexual and adult stages among this group and the descriptions of the metacercariae in most instances are far from adequate by modern standards. As the result, to quote from Stunkard and Uzmann : ‘‘ The situation is chaotic and one of utter confusion.” Nevertheless, so that this section may be more complete, a brief account of each of the gymnophallids known from commercially important molluscs is given. I n many instances the diagnosis is far from satisfactory, even after I have attempted to supplement the original descriptions by describing features shown in the illustrations of the earlier workers. Perhaps this undesirable situation will stimulate others to re-examine this group of trematodes. Relative to the systematic position of the gymnophallids, although Dawes (1946) and Yamaguti (1958) have continued to consider them as members of the family Microphallidae, Cable (1953, 1954a,b, 1956) has shown that they should be included in the family E’ellodistomatidae. I am following Cable in placing these trematodes in the latter family. The trematode referred to as Gymnophallus margaritarum is still not known in its adult form although various individuals (Jameson, 1902 ; Lebour, 1911 ; Palombi, 1924) have postulated, and most probably correctly so, that it is a parasite of certain shore birds which feed on Mytilus edulis. Our present knowledge concerning this parasite dates back to a t least 1655 when, according to Giard (1907), Olaus

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Worm reported the occurrence of pearly formations in the mantle of Mytilus edzilis collected a t Roeskild, near Copenhagen. The information available on worm-associated pearl formation has been reviewed in Chapter 4, Section 11, A, 2. Interest in this phenomenon led Dubois (1901) to study the parasit&esof M . edulis a t Billiers. He found trematodes (now known to be metacercariae) measuring 0-4-0.6 mm in length in reddish brown spots which are the loci of pearl formation. He named the parasite Distomum margaritarum. He also found the same or a very similar parasite in M ytilus galloprovincialis from the coast of Provence. Later (Dubois, 1907))he placed this trematode in the genus Gymnophallus. Since Dubois' initial study, various individuals have examined this parasite. Jameson (1902), who has also examined the parasites of Mytilus edulis at Billiers, described and depicted worms which he believed to closely resemble Gymnophallus ( = Distomum) somateriae, the adult of which was described by Levinsen (1881) from the intestine of the eider duck, Somateria mollissima, captured near Egedesminde, Greenland. I n following Stossich (1899), Jameson, however, relegated his specimens to the genus Lecithodendrium as L. somateriae. I n addition to Mytilus edulis from Billiers, Jameson has also reported the occurrence of similar larvae (now known t o be cercariae)* in sporocysts in Tapes decussatus from the same location, larvae (metacercariae) in Mytilus edulis from Piel, Barrow, Lancashire, England*, sporocysts in Cardium edule from Piel, and adult worms from the intestine of the scoter, Melanitta ( = Oidemia) nigra, a t Billiers. Although he described and depicted what he considered the adult of L. somateriae to be 0.2-0.55 mm long, which is about one-half the size of the larvae (metacercariae) in Mytilus edulis, he concluded that morphological similarity between the metacercaria in M . edulis and adult Lecithodendrium somateriae and the fact that the adults were found in two species of mussel-eating birds were sufficient proof that the metacercariae are the larvae of L. somateriae. His conclusion, of course, is not acceptable.* Lebour (1911) has designated the cercariae found in Cardium edule and Tapes decussatus as Cercaria margaritae sp. inq. ( = the Pearl Trematode of Jameson (1902)).* She also has reported metacercariae encysted in Mytilus edulis. Lebour (1911) pointed out that she had earlier found the same sporocyst in Cardium (Lebour, 1907a) as had Nicoll (1906a), but both of them had found the sporocystst just beneath the umbo where they were embedded in a jelly-like mass frequently embedded with " chalky spots rather than in the muscular or connective tissue of the mantle edge, particularly dorsally, close t o the * See note to p. 218 on p. 390. t See note on p. 389. ))

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siphonal muscles of Tapes and in the mantle of Cardium as Jameson (1902) had indicated. Nicoll (1907a) has expressed the opinion that these larvae are those of Gymnophallus dapsilis, a parasite of scoters, Melanitta nigra and M . fusca,, but experimental evidence is wanting. The identification of this parasite, a t least the larval stages, appears to have been clarified by Palombi (1924) who has demonstrated that Jameson’s (1902) “ pearl trematode ”, which he believed to be L. somateriae from Mytilus edulis, is identical with Dubois’ Gymnophallus margaritarum, as is Lebour’s Cercaria margaritae’. I n addition, Palombi has stated that the metacercaria named Metacercaria (Gymnophallus) duboisi by Dollfus (1923a) from Mytilus galloprovincialis is the same as G . margaritarum, as is the metacercaria (reported as a cercaria) found by Sinitsin (1911) in Mytilus edulis and Venus sp.? from Sebastopol on the Black Sea and named Adolescaria perla. He has also demonstrated that the sporocysts and cercariae in Tapes and Cardium are the progenitor stages of the metacercaria in Mytilus. 7 Description of stages in mollusc. Sporocyst (Fig. 119) simple, more or less spherical and colorless, size varies greatly. Cercaria (Figs. 120 and 121) without tail, pear-shaped, more pointed posteriorly, 0.150.3 mm long, covered with cuticular spines ; a pair of brown eye-spots a t anterior end, one on each side of anterior sucker ; ventral sucker in posterior half of body ; ratio of anterior to ventral sucker 3 : 2 (Lebour (1911) reported it to be 2: 3, but, as Cole (1938) has pointed out, this is probably a misprint); prepharynx absent; pharynx and short esophagus present ; globose intestinal caeca reaching midlength of body or slightly beyond; according to Nicoll (1906a), anlagen of two testes are situated posterior to ventral sucker (Fig. 121) ; excretory vesicle large, Y-shaped, with arms reaching anteriorly t o as far as level of pharynx. Life cycle. The complete life cycle of G . margaritarum is not known although, as stated, mollusc-eating shore birds have been postulated to be the definitive hosts. According to Lebour (1911) : The cercaria presumably leaves its first host by aid of currents or by crawling on the ground by aid of its suckers, reaches its (second) intermediate host, Mytilus edulis. Here it settles down between the mantle and shell, feeds and grows and finally makes its way into the tissues of the mantle, curls itself up and rests. The metacercaria does not secrete its own cyst, rather the mantle grows around it and forms an enclosing epithelial sac. The cells of this sac eventually secrete a pearly layer around the parasite. If such a pearly layer is formed, the metacercaria generally dies, forming the * See note to p. 218 on p. 390. i See note on p. 390.

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nucleus of the pearl. But if it is ingested by the avian host before the pearly wall is formed, it will attain maturity. Other information. Nothing is known about the ecology of G. margaritarum except that it is apparently widely distributed throughout Europe. Nothing is known about its physiology. Histopathological changes in Cardium or Tapes resulting from the presence of sporocysts have not been studied. Similarly, other than Jameson’s (1902) account of pearl formation around the metacercaria, other changes, if any, in M . edulis are not known. 7. Metacercaria (Gymnophallus)strigata Lebour, 1908. (Fig. 122) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family Fellodistomatidae) This gymnophallid parasite was originally described as Cercaria strigata by Lebour (1908, 1911) but there is no doubt that the form described by her is a metacercaria. It is found between the mantle and shell of Tellina tenuis and Donax vittatus a t Alnmouth, Northumberland, England, and in Tellina tenuis a t Millport, Scotland. The sporocyst has since been found by Rees (1939a) in Cardium edule collected a t the Dovey estuary, Wales. Giard (1907) is of the opinion that Metacercaria strigata is the larva of Gymnophallus somateriae but this has not been proven. Description of stages in mollusc. Sporocyst (in digestive gland of Cardium edule) thin-walled, colorless, ovoid, contains cercariae at various stages of development. Cercariae removed from sporocysts “ show the same structure as the metacercaria ” (Rees, 1939a). Metacercaria (Fig. 122) (on mantle of Tellinn tenuis and Donas vittatus) broadly ovate, tapers slightly to posterior extremity, 0.17-0.40 mm long, 0.30-0.396 mm? wide (Rees reported the maximum width to be 0-095 mni which is obviously a misprint), armed with cuticular spines; anterior sucker 0.09 x 0.075 mm, ventral sucker, 0.05 x 0.048 mm, situated slightly posterior to midlength of body ; prepharynx absent ; pharynx 0.025 x 0.021 mm ; esophagus short ; intestinal caeca bulbous, capable of expanding and contracting, extending to anterior border of ventral sucker ; large number of glands a t anterior extremity around anterior sucker with ducts opening by a series of pores along dorsal border of mouth ; excretory vesicle, lyre-shaped (Y-shaped with lateral projection off of arms), with arms reaching level of anterior sucker; (2 a ) ] ; two oval testes, each measurflame-cell pattern 2 [ ( 2 2) ing 0-05 x 0-024 mm, one on each side midway between ventral sucker and posterior body terminal ; single ovoid ovary, 0-03 x 0.02 mm, in front of right testis.

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Life cycle. The complete life cycle of Xetacercaria strigatu is not known. Giard ( 1 9 0 7 ) , who found this parasite in the same second intermediate hosts but did not name or describe it, has suggested that

FIGS.119-124. Larval gymnophallids. (119) Sporocyst of Metncercaria (GymnophnZZus) margaritarum ; (120) Metacercaria (G.) margaritarum (redrawn after Lebour, 1911); (121) Metacercwia (C.) margaritarum showing distended excretory vesicular arms and developing testes (redrawn after Nicoll, 1906a) ; (122) Metacercaria (C.) strigata showing distended intestinal caecum and flame-cells on one side and contracted caecum, cephalic glands, and excretory vesicular arm on the other (redrawn after Rees, 1939a) ; (123) Metacercaria (G.) megalocoela covered with thick gelatinous coat; (1 24) Metacercuria ( G . ) rnegalocoela removed from gelatinous coat (redrawn after Paloinbi, 1934a). C’C, C‘ephalir glands ; DOV, developing (wary ; DT,(leveloping testis ; E S , eye-spot ; El.’, excretory vesicle ; GE, gelatinous coat embedded with calcium particles; I N T , intestinal caecum ; I’S, ventral sucker.

it may be the larval form of Gymnophallus oedemiue; but, as F. G. Rees (1939a) has pointed out, conspicuous differences in structure make

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this unlikely. Rees has suggested that it may be the larva of either G . macroporus, an intestinal parasite of Melanitta (=Oidemia) nigra, or G. deliciosus, a parasite of Larus argentatus, L. fuscus and L. canus, but experimental data are wanting. Relative to the infection of the second intermediate host, either Tellina tenuis or Donax vittatus, Rees (1939a) has suggested that the cephalic glands found in cercariae (and metacercariae) " are probably histolytic in function, and enable the cercaria to bore its way out of the digestive gland of its first intermediate host, and also to bore through to the outside of the mantle of its second intermediate host ". This, although possible, remains to be demonstrated. 8. Metacercaria (Gymnophallus) megalocoela Palombi, 1934. (Figs. 123

and 124) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family Fellodistomatidae) Only the metacercarial stage of Gymnophallus megalocoela is known. It was described by Palombi (1934a) from between the shell and mantle of Tapes decussatus (=Amygdala decussata) collected in the Gulf of Naples. The metacercaria is said to be covered by a thick ellipsoid gelatinous coat of host origin impregnated with calcium particles (Fig. 123). This coat appears to be comparable to the " jelly-like mass " described by Lebour (1911) in which G. margaritarum metacercariae are found.

Description. Gelatinous cyst (Fig. 123) fairly transparent, 0.36 mm in diameter. Metacercaria (Fig. 124) ovoid, 0.21-0.29 mm long, 0.220.23 mm wide ; body covered with minute cuticular spines ; anterior sucker 0.07-0-08 mm in diameter ; ventral sucker, 0.03 mm in diameter, in posterior half of body ; prepharynx absent ; pharynx and extremely short esophagus present ; pharynx averages 0.02 mm in diameter ; intestinal caeca large and globose, 0-11-0-13 mm long x 0.08-0.09 mm wide, reaching slightly past level of ventJral sucker; two testes present, one on each side of stem of excretory vesicle ; ovary anterolateral to right testis ; excretory vesicle Y-shaped, large, with arms reaching level of anterior sucker. Other information. Nothing else is known about 0. megalocoela. From what is known about the life cycle pattern among gymnophallids, Tapes decussatus is undoubtedly the second intermediate host while some other pelecypod is the first. The definitive host is most probably one or more species of shore birds.

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9. Cercaria (Gymnophallus) cambrensis Cole, 1938."

(Fig. 125) (Superorder Anepitheliocystida; order Strigeatoidea; suborder Azygiara; superfamily Fellodistomatoidea; family Fellodistomatidae) This cercaria was originally described by Cole (1938) from Cardizm edule brought t o the Conway Mussel Cleansing Station, collected on the Menai Straits, and collected by W. J. Rees from the Dovey estuary,

FIGS.125-128. Larval gymnophallids. (12.5) Cercaria (Gymnophallus) cambrensis showing arrangement of flame-cells on one side (redrawn after Cole, 1938) ; (126) sporocyst of Cercaria (G.) fulhrighti with snout-like projection and enclosing daughter sporocyst and developing cercariae ; (127) young fork-tailed cercaria of Cercaria ( G . )fulbrighti; (128) fully developecl tailless cercaria of Cercaria (G.) f u l brighti. (Redrawn after Hutton, 1952.) A S , Anterior (oral) sucker; CG, cephalic gland; DS, daughter sporocyst,; DT, developing testis; E V , arm of excretory vesicle ; V S , ventral sucker.

Cardigan Bay, Wales. Within the host, the sporocysts appear as brown masses located immediately beneath the hinge within the small wedgeshaped cavity found a t that site. Descriptim of stages in mollusc. Sporocyst more or less oval, thickwalled, colorless or slightly straw-colored, non-motile, containing * See note 011 p. 390.

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varying number of cercariae and considerable amount of granular brown material. Cercaria (Fig. 125) 0.27-0.32 mm long x 0.14-0-16 mm wide, spindle-shaped, moderately active, covered with fine cuticular spines ; anterior sucker 0.03-0.04 mm in diameter ; ventral sucker, 0.02-0.04 mm in diameter, in posterior half of body; prepharynx absent ; pharynx 0.015 mm in diameter ; esophagus short, intestinal caeca large, extending to level of anterior border of ventral sucker; with a group of penetration glands closely packed on each side of anterior sucker, opening by four ducts on each side at anterior margin of sucker ; excretory vesicle Y-shaped, with large arms extending anteriorly to level of pharynx, filled with dense excretory granules each measuring 0.005 mm in diameter. Life cycle. Cole (1938) has expressed the opinion that C. cambrensis may be the cercaria of Metacercaria (Gymnophallus) margaritarum but this remains to be proven. 10. Cercaria (Gymnophallus)fulbrighti Hutton, 1952. (Figs. 126-129)

(Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family Fellodistomatidae) Cercaria fulbrighti was described by Hutton (1952) from Cardium edule collected a t Millbrook, Plymouth, England. Description of stages in mollusc. Sporocyst (Fig. 126) somewhat irregularly ovoid, 1.416 mm x 0.768-0-880 mm, capable of independent movement by means of projecting parts of body wall ; definite snoutlike structure situated anteriorly ; with relatively few fully developed cercariae in any one sporocyst, five being the maximum number observed ; both daughter sporocysts and cercariae develop within mother sporocyst ; in gonad, digestive gland, and upper part of foot of Cardium edule. Fully developed cercaria tailless (Fig. l28), uniformly covered with cuticular spines, body 0-258-0.408 mm (average 0.328 mm) long x 0.152-0.196 mm (average 0-160 mm) wide; anterior sucker 0.068-0.082 mm (average 0.072 mm) wide ; ventral sucker, 0.0620.076 mm (average 0.070 mm) wide, in posterior half of body; prepharynx absent ; pharynx 0.020-0.024 mm (average 0.022 mm) wide ; intestinal caeca bulbous, reaching beyond anterior level of ventral sucker.; excretory vesicle Y-shaped with arms extending to midlength 2) (2 2)]. Young cercariae of esophagus ; flame-cell pattern 2[(2 within some sporocysts with forked tail (Figs. 127 and 129). Life cycle. The complete life cycle of Cercaria fulbrighti is not known. Hutton (1952) has pointed out that there is a “remarkable similarity between this larva and the adult stage of G(ymnophallus) choledochus

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Odhner ”, a parasite in the gall-bladder of the common sheld duck, Vulpanser tadorna, and other shore ducks, including A ythya ferina, Clangula hyemalis, Somateria mollissima and S . spectabilis, in Europe. Other information. Nothing else is known about C. fulbrighti except that Hutton has reported that dual infections in Cardium edule with this cercaria and Bucephalus haimeanus and triple infections with C . fulbrighti, B. huimeanus and C . cambrensis do occur.

c 003

c

FIG. 129. Development and subsequent degeneration of the tail of Cercaricz (Gymnophallus)fulbrighti. These changes occur within the brood chamber of the sporocyst. The escaping cercaria is tailless. (Redrawn after Hutton, 1962.)

11. Metacercaria (Gymnophallus)scrivenensis sp. inq. Lebour, 1911.

(Fig. 130) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea;family Fellodistomatidae) This Gymnophallus metacercaria was described by Lebour (1911) as a cercaria in a single specimen of Tapes pullastra collected from Loch Striven, Firth of Clyde, Scotland. She found the parasite free between the mantle and the shell without an enveloping gelatinous cyst as in the case of G. rnegalocoela or G. margaritarum. From what is known about the life cycle pattern among gymnophallid trematodes,

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Lebour’s specimens represent metacercariae in the second intermediate host. Description. Metacercaria (Fig. 130) elongate, more pointed posteriorly, averaging 0.4mm long x 0-24 mm wide ; covered with cuticular spines ; anterior sucker 0.09 mm in transverse diameter ; ventral sucker, 0.3 mm in transverse diameter, in posterior half of body ; prepharynx absent ; pharynx 0-03 mm long ; esophagus extremely short or nonexistent ; intestinal caeca reaching slightly beyond level of ventral sucker ; excretory vesicle Y-shaped with short stem, vesicular arms reaching level of pharynx. Other information. Nothing else is known. It is most probably a parasite of some shore bird.

Metacercaria (Gymnophallus)macomae Lebour, 1908. (Fig. 131) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea;family Fellodistomatidae) This parasite, found between the mantle and shell of Tellina solidula (=Macoma balthica) was described by Lebour (1908) as a cercaria, C . macomae. The hosts were collected a t Fenham Flats, England. Description. Metacercaria (Fig. 131) 0.70 mm long; covered with cuticular spines ; anterior sucker 0.22-0.26 mm in diameter ; ventral sucker, 0.06-0.07 mm in diameter, in posterior half of body; prepharynx absent ; small pharynx and short esophagus present ;intestinal caeca reaching between levels of anterior margin and midlength of ventral sucker ; excretory vesicle said to be horseshoe-shaped, actually Y-shaped with extremely short stem ; two testes, each slightly larger than ovary, situated posterior to ventral sucker, each overlapping one arm of excretory vesicle; ovary in front of right testis; vitellaria as two masses posterolateral and partially overlapping ventral sucker. Other information. No other information is available on this species. Again, Macoma balthica is undoubtedly the second intermediate host and the definitive host is most probably some shore bird. 12.

13. Metacercaria (Gymnophallus)perligena Palombi, 1940. (Fig. 132)

(Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea;family Fellodistomatidae) This metacercaria was described by Palombi (1940) from specimens provided by A. Cerruti from the mantle of Mytilus galloprovincialis collected in the Gulf of Taranto, Italy. The mantle of parasitized mussels is commonly covered, with encysted metacercariae (Fig. 132).

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Description. Body subspherical or slightly ovoid, 0-16mm in greatest transverse diameter, covered with uniformly distributed minute spines ; anterior sucker 0.06 mm in diameter ; ventral sucker extremely small,

FIGS.130-136. Larval gymnophallids. ( 1 30) Metacercaria (Gymnophallus) scrivenensis; (131) M . (G.) maromae (redrawn after Lebour, 1911); (132) M . (G.) perZigena surrounded by gelatinous wall (redrawn after T'alomhi, 1940); (133) (f. bursicola metarerraria enveloped within thin grlatinous tunira (redrawn after Stunkard and Uzniann, 1958) : (134) Metace?carm (Gym~rophaZZoitles)tokimsis (redrawn after Fujita, 1925); (135) spororyst of Gercutia dichotomu enclosing developing rercariae ; (136) fully developed fork-tailed C. dichotoma (redrawn after Lebour, 1911). CG, Cephalic gland; E , eye-spot ; G P , genital pore ; 0, developing ovary ; T,developing testis; V I T , developing vitelline gland ; V S , ventral sucker.

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about one-sixth size of anterior sucker according to Palombi (although that of the specimen which he depicted (Fig. 132) appears to be considerably smaller), situated in posterior half of body between caeca ; numerous glands surround and empty into anterior sucker ; prepharynx absent ; pharynx large, with its diameter larger than that of ventral sucker ; esophagus short ; intestinal caeca large and bulbous, occupying middle portion of body; excretory vesicle Y-shaped with minute stem, arms reaching level of anterior sucker ; immature testes ovoid, one on each side of body near posterolateral margins, posterior to caeca; small immature ovary t o the left and overlapping ventral sucker; genital pore immediately anterior to ventral sucker.

Other information. The cyst wall of this metacercaria is in the form of a gelatinous capsule within which the metacercaria is not doubled over. I t s presence on the mantle of Mytilus galloprovincialis causes the laying down of nacrous material in pearl formation. 14. Gymnophallus bursicola Odhner, 1900. (Fig. 133) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family Fellodistomatidae) Stunkard and Uzmann (1958), as a part of their extensive study on gymnophallids of northeastern United States, reported the occurrence of a gymnophallid metacercaria on the mantle of Mytilus edulis collected a t Milford, Connecticut, and Newburyport, Massachusetts. This metacercaria was found in 20% of the mussels examined, with one t o ten per mussel. They fed these metacercariae to hamsters and young eider ducks, Somateria mollissima. Adults were recovered from both experimental hosts but those from the hamster had not reached maturity in 7 days while one specimen from an eider duck had just commenced producing eggs in what was believed to be 10 days. The adults were tentatively identified as Gymnophallus bursicola, a species described by Odhner (1900) from the bursa Fabricii of Somateria mollissima in Sweden, and later reported to occur in Melanitta fusca and M . nigra in Japan (Yamaguti, 1939). Its life cycle had been reported by Giard (1907).

Description of metacercaria (the form found in economically important molluscs). Metacercaria ovoid, 0.4-0.6 mm long x 0.2-0.3 mm wide ; body covered with cuticular spines ; anterior sucker 0.088 mm in diameter ; prepharynx absent ; pharynx, 0.030 mm in diameter, and short esophagus present ; intestinal caeca extending to near anterior margin of ventral sucker ; ventral sucker slightly smaller than anterior

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sucker ; testicular anlage in posterior half of body, one on each side of ventral sucker ; ovarial anlagen, if present, immediately anterior to left testis; with cephalic glands (exact number not known) on each side of pharynx ; genital pore near anterior margin of ventral sucker ; genital atrium, if present, tubular ; excretory vesicle Y-shaped, with arms reaching level of pharynx anteriorly.

Other information. Stunkard and Uzmann (1958) have noted that the metacercariae produce lesions in the mantle and body wall of Mytilus edulis which may result in the deposition of nacrous material. 15. Metacercaria (Gymnophalloides) tokiensis Fujita, 1925. (Fig. 134)

(Superorder Anepitheliocystidia;order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea;family Fellodistomatidae) This metacercaria was described by Fujita (1925) on the external surface of the mantle of Crassostrea gigas from Hokkaido, Matsushima Bay, Tokyo Bay, Hamana Lake and Ise Bay, Japan.

Description. Body elongate ovoid, pointed posteriorly, 0.36 mm long x 0.23 mm wide, covered with sparsely arranged minute papillalike projections ; with two eye-spots on ventral surface overlapping intestinal caeca ; anterior sucker 0.13 mm x 0.09 mm ; ventral sucker, 0.05 mm x 0.06 mm, in posterior one-third of body; prepharynx absent ; pharynx 0.02 mm long ; esophagus short ; intestinal caeca bulbous, measuring 0.08 x 0.04 mm, reaching midlength of body or slightly beyond ; two testes, each measuring 0425mm ingreatest length, one on each side of ventral sucker ; ovary slightly larger than testes, anterior to left testis and lateral to intestinal caecum ; seminal vesicle, seminal receptacle, ootype, Laurer's canal, and cirrus (ejaculatory duct) present ; genital pore on ventral surface, approximately midway between ventral sucker and caecal bifurcation ; vitelline glands scattered in parenchyma on each side of body, extending along entire length of body ; excretory vesicle Y-shaped, with extremely short stem and large arms which reach level of anterior sucker. Physiology. Fujita (1925) has shown that this metacercaria is very sensitive to changes in the density (" densiti: ") of the surrounding water. At 20°C, if the water has a density of 1.024, the parasites will live for 5 h but if the density is increased to 1.026, the longevity of the metacercaria is reduced to 30 min. Similarly, at water densities of 1.028 and 1.030, the survival periods are reduced to 15 and 10 min respectively.

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16. Cercaria dichotoma La Valette, 1855. (Figs. 135 and 136) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family Fellodistomatidae) Cercaria dichotoma was first found by Muller free in the Mediterranean near Nice, hence the designation C. dichotoma Miiller as used by Lebour (1908, 1911), Stunkard and Uzmann (1958) and Hutton (1952). But it was described by La Valette (1855) from the marine pelecypod Scrobicularia tenuis. Villot (1878) believed that it was identical to La Valette’s Cercaria Jissicauda but, as Pelseneer (1906) has pointed out, this could not be since C. Jissicauda was described from a freshwater gastropod, Lymnaea stagnalis. I n the same paper, Pelseneer reported C. dichotoma from Tellina solidula (= Macoma balthica) from deep water near Boulogne-sur-Mer,France, and recognized that C. dichotoma is extremely similar to an unnamed cercaria described by Huet (1888b) from Cardium edule collected along the coast of Normandy. But, as indicated in the following section, Huet’s cercaria cannot be C . dichotoma. Johnstone (1904) has reported C. dichotoma from Cardium edule from the Lancashire coast and Lebour (1908, 1911) has also reported its occusreiice in Scrobicularia tenuis collected at Fenham Flats, England. Thus it would appear from what is known that this cercaria utilizes a marine pelecypod as the first intermediate host but is not restricted to a single species. Furthermore, if Johnstone’s identification is correct, it has been found in a commercially important species, Cardium edule. Description. Sporocyst (Fig. 135) long and irregular, about 1 mm long, more pointed at one end than other, very transparent, filled with cercariae a t all stages of development. Cercaria (Fig. 136) with bifurcated tail, 0.20 mm long with tail; tail slightly shorter than body; anterior sucker 0.033 mm in transverse diameter ; prepharynx absent ; pharynx 0.016 mm long ; esophagus long ; intestinal caeca reaching slightly beyond anterior level of ventral sucker; ventral sucker in posterior half of body, 0.029 mm in transverse diameter; excretory vesicle said to be lyre-shaped, actually Y-shaped with arms slightly convoluted, occupying most of posterior half of body, arms reaching midlength of body anteriorly, stem extending into tail, bifurcating, and terminating at tips of furcae. Other information. No other information is available on C. dichotoma except for the statement by James et al. (1966) that:

We have made some unpublished histochemical and electron-microscope observations on the tegument of the daughter sporocyst of Cercaria dichomata Lebour, 1911 (James et al. have erroneously credited this species t o Lebour), which suggest that the outer region and the middle nucleated and secretory A.X.B.--6

16

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MARINE MOLLUSCS A S HOSTS FOR SYMBIOSES

region of the tegument are shed early in development, only the narrow inner dense cytoplasm, with the now external fingerlike projections of cytoplasm, being retained throughout life. An uninterrupted basement membrane is also retained. The external fingerlike projections of protoplasm are very elongate and unbranched in C . dichotoma and are thus remarkably similar in appearance to vertebrate microvilli. 17. Cercaria hueti sp. inq.

(Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatidea; family Fellodistomatidae) As mentioned above, Pelseneer (1906) has expressed the opinion that the unnamed cercaria described by Huet (188823) in Cardium edule from Normandy is Cercaria dichotoma. But, as Stunkard and Uzmann (1958) have pointed out, Huet’s figures indicate that this cercaria, although a gymnophallid, is not C. dichotoma since the latter possesses a bifid tail at maturity while Huet’s cercaria loses its tail prior to escaping from the sporocyst. Furthermore, Huet’s description of the sporocyst generation, as far as I have been able to determine, differs from any of the known gymnophallid sporocysts, and hence I am designating Huet’s cercaria as Cercaria hueti sp. inq. until further information indicates otherwise.

Description of stages in mollusc. Sporocyst short, compact, spherical to pyriform, with anterior end elongated to form a “ neck ”, covered with ‘‘ cilia ”, and free swimming. Immature sporocyst 0.2 mm long, grayish ; older sporocysts 0-3 mm long, yellowish, with birthpore at anterior end surrounded by five tubercles ; crown of “ verrucosities ” below tubercles ; intervening area covered with fine still bristles ; mature sporocyst, 0.5 mm long, yellow, contains few distomate cercariae with bifid tails; mature cercariae shed their tails prior to emergence from sporocysts. Other information. No other information is available on this species. 18. Metacercaria 1 of Stunkard and Uzmann, 1958. (Fig. 137) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata;

superfamily Fellodistomatoidea; family Fellodistomatidae) Stunkard and Uzmann (1958) reported the occurrence of a species of metacercaria which they designated as Metacercaria I in about 25% of the Mya arenaria collected at Boothbay Harbor, Maine, and Woods Hole, Massachusetts. The soft clams were uniformly IightIy infected, each harboring one to twelve metacercariae. The metacercariae are unencysted and are found on the mantles of their hosts. Their morphology suggests that they are gymnophallids.

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Description. Metacercariae (Fig. 137) vary greatly in size, 0.120-20 mm long, width ordinarily about one-half length but length and width may be subequal when contracted while the width may be onefourth the length when extended ; body covered with cuticular spines ; anterior sucker 0.147-0.160 mm in diameter (in large specimens); ventral sucker in posterior half of body, 0.13-0.14 mm in diameter (in large specimens); anterior body region with several small papillae, each tipped with short stiff bristle ; large number of unicellular glands in region of and opening into anterior sucker ; prepharynx absent, pharynx and esophagus of median length present ; intestinal caeca reaching level of anterior margin of ventral sucker, lined with large cells with yellow granules intracellularly and intraluminally ; excretory vesicle Y-shaped, with arms extending to posterior level of pharynx ; flame-cell pattern 2[(2 2 2) (2 2 2)]. Life cycle. Stunkard and Uzmann (1958) have fed this metacercaria to mice, hamsters, eider ducks, and herring gulls but failed to recover adults. The life cycle of this metacercaria remains undetermined. Other information. It is known that Metacercaria I actively ingests materials from the host’s mantle. Furthermore, Stunkard and Uzmann have stated : “ The yellow material (in caecal cells and lumen) appears like that in the digestive gland of the clam and may be taken by the parasite from the vascular fluid of the mollusk.”

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19. Cercaria myae Uzmann, 1952. (Fig. 138) (SuperorderAnepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family Fellodistomatidae) Cercaria myae was originally described by Uzmann (1952) from the soft clam, Mya arenaria, collected in the vicinity of Newburyport, Massachusetts. It has been subsequently found in the same host collected at Boothbay Harbor, Maine (Stunkard and Uzmann, 1958), and I (unpublished) have found it in the same clam in Narragansett Bay, Rhode Island. Description of stages in mollusc. Sporocyst motile, unpigmented, clavate, occupying interfollicular spaces of gonad and interlobular spaces of digestive gland of host, few throughout haemocoel, with thin wall and birthpore at apical end; 0.21-0.60 mm long. Cercaria (Fig. 138) small, elongate ovoid, furcocercous ; body proper 0.12-0.25 mm long ; tail stem short, about one-third body length, slightly shorter than furcae ; body covered with minute spines, each 0.001 mm long ;delicate setae on posterior surfaces of furcae ; anterior sucker 0.039-0-052 mm in diameter ; ventral sucker 0-040-0.046 mm in diameter ; prepharynx

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absent ; pharynx 0.020-0.025 mm in diameter ; esophagus short ; intestinal caeca large, with walls comprised of polygonal cells, reaching level of anterior one-third of ventral sucker; small deeply staining spiral coil maintains a constant lumen between pharynx and esophagus ; two pairs of cephalic glands at posterior level of pharynx, ducts leading to anterior body terminal ; excretory vesicle Y-shaped with extremely fine stem (V-shaped according to Uzmann) and arms recurved around intestinal caeca ; stem extends and enlarges in tail, bifurcates, branches terminating at tips of furcae; flame-cell pattern 2[(2 2 2) (2 2 211. Life cycle. The life cycle of C. myae remains unknown. The cercariae are capable of swimming. While swimming, the posterior portion of the body is bent ventrally in apposition to the anterior portion. The tail lashes vigorously and the spring-like action produced when the body straightens out makes this cercaria a very active swimmer. It does not encyst ectopically and becomes moribund after about 12 h. Uzmann (1952) has suggested a similarity between this cercaria and Sinitsin’s Cercaria discursata which Allison (1943)has suggested may belong to the family Brachylaemidae but Stunkard and Uzmann (1958) have expressed the opinion that C. myae is a gymnophallid. Ecology. C. myae is a rare parasite. Uzmann (1952) found only five out of over 1 000 clams at Newburyport, Massachusetts, to be infected. Stunkard and Uzmann (1958) only found three out of over 1 000 clams from Boothbay Harbor, Maine, to be infected, and I found one infected M . arenaria out of 800 examined in Rhode Island. Pathology. Uzmann (1952) has stated that : “ There is histological evidence of partial to complete sterilization (of parasitized clams).” I have been able to confirm this in addition to finding almost complete destruction of the digestive gland tubules.

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20. Bacciger bacciger (Rudolphi, 1819). (Figs. 139 and 140) (SuperorderAnepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family B’ellodistomatidae) The adult of Bacciger bacciger was originally discovered by Rudolphi (1819)in marine fish. Nicoll(l914) redescribed the adult and placed it in the genus Bacciger. Although considered a member of the family Cryptogonimidae by Yamaguti (1958),I am in agreement with Dawes (1946), Cable (195413) and Skrjabin (1957) that it is a member of the Fellodistomatidae. The definitive host includes Atherina hepsetus, A. presbyter and A. boyeri. Its cercaria was first found by Lespes (1857) who, unaware of its relationship with the adult, named it Cercaria lata.

FIGS.137-142. Larval fellodistomatids. (137) Metacercaria I of Stunkard and Uzmann (redrawn after Stunkard and Uzmann, 1958) ; (138) fully developed Cercaria myae (redrawn after Uzmann, 1952); (139) sporocyst of Bacciger bmcigor enclosing cercariae ; (140) anterior end of fully developed B . bacciger cercaria (redrawn after Palombi, 1934a) ; (141) cercaria of Proctoeces rnmulatus (redrawn after Uzmann, 1963); (142) Metacercaria (Proctoecee) ostreae (redrawn after Fujita, 1925). CG, Cephalic gland; CP,cirrus pouch ( s a c ) ; CYQ,cystogenous gland; DOV, developing ovary; DT, developing testis; EV, arm of excretory vesicle; E V T , extension of stem of excretory vesicle into tail ; 0 V , ovary ; P , bristle-bearing papilla ; T , testis ; V I T , vitelline follicle.

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Later, Qiard (1897a)described the same cercaria as C. lutea. It has also been referred to as C . pectinata by Huet (1891), Pelseneer (1896, 1906), Fujita (1906, 1907),Kobayashi (1922),Dollfus (1911, 1925)and Palombi (1932, 1933, 1934a). Palombi (1934b) finally was able to associate this cercaria with the adult in the intestine of Atherina spp. The sporocysts of B. bacciger are found in the visceral mass and reproductive organs of Tapes decussatus (= Amygdala decussata), Tapes pullastra, T . semidecussata, T . aureus, Donax vittatus, Pholas candida and Tellina exigua. It has been found in the Mediterranean, the Adriatic and the Atlantic in European waters, and in Japanese waters. Description of stages in mollusc. Sporocyst (Fig. 139) simple, elongate, and sacculate, 0.5-3.0 mm long, 0.18-0-28 mm in diameter, enclosing numerous cercariae at approximately the same stage of development (synchronized development). Cercaria (Fig. 140) trichocercous, i.e. with setiferous tail; body ovoid, 0.22 mm long, 0-lOmm wide ; anterior sucker 0.041 mm in diameter ; ventral sucker at or near middle of body, 0.040 mm in diameter; no prepharynx; pharynx 0.021 mm in diameter ;esophagus fairly long ;intestinal caeca terminating immediately below level of ventral sucker ; two cephalic (penetration) glands on each side of esophagus, emptying to exterior anteriorly each by its own duct ; two testes (non-functional),one on each side at postero-lateral margin of ventral sucker ; small ovarial anlage situated between testes ; excretory vesicle Y-shaped, with arms reaching level of caecal bifurcation; tail, 0.47 mm long, 0.034 mm wide, with lateral setae along entire length; each seta measures 0.12-0.20 mm long. Life cycle. According to Palombi (1934b), the cercariae, after escaping from the pelecypod first intermediate host, penetrate and encyst as metacercariae in Erichthonius difformis, which, when eaten by Atherina spp., develop into adults in the alimentary tract. Other information. Sporocysts in the first intermediate host are known to cause parasitic castration. 21. Proctoeces maculatus Looss, 1901. (Fig. 141)

(SuperorderAnepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family Fellodistomatidae) Uzmann (1953) described a microcercous cercaria, Cercaria milfordensis, which develops in sporocysts found in the blood vessels and sinuses of Mytilus edulis collected from Mill Neck, Long Island, New York, and Milford and Bridgeport, Connecticut. It was reported to be absent from the Massachusetts coast ; however, Stunkard and Uzmann

6. THE PHYLA PORIFERA, CNIDARIA AND PLATYHELMINTHES

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(1959)have since reported its occurrence inMytilus edulis taken in the Woods Hole, Massachusetts, area. It is of interest to note that Stunkard and Uzmann have not only found two generations of sporocysts in addition to cercariae in the molluscan host but also found sexually mature adults in the mollusc, thus indicating the occurrence of a telescoped life cycle. The adults were identified as Proctoeces maculatus, a species previously reported in a number of species of bottom-feeding labroid fishes and porgies (Labrus merula, Crenilabrus pavo, C. griseus and Blennius ocellaris) from the Mediterranean and Black Sea (Looss, 1901 ; Odhner, 1911), and from Sparus macrocephalus, S. aries, Pagrosomus auratus, Epinephelus akaara and Semicossyphus reticulatus from the Inland Sea of Japan, and Duymeria JEagellifera from Hamazima, Japan (Yamaguti, 1938, 1953). The ability of members of the genus Proctoeces to develop to sexual maturity in molluscs had been reported earlier by Freeman and Llewellyn (1958) and Fujita (1925), and more recently by Dollfus (1964). Freeman and Llewellyn have reported that P. subtenuis, a species normally parasitic in the hindgut of labrid and sparid marine fishes, can attain sexual maturity in the renal organ of the pelecypod Scrobicularia plana. Fujita has reported what he considered to be progenetic metacercariae of P. ostreae in the gonads of Crassostrea gigas, and Dollfus has found progenetic metacercariae of P. progeneticus in the prosobranch Gibbula. I n the case of P. maculatus, sporocysts, cercariae, metacercariae (juveniles), and adults can all occur in Mytilus edulis. Description of stages in mollusc. Mother sporocysts globose and sacculate, 0.34 mm long (according to Stunkard and Uzmann’s illustration), contains few daughter sporocysts. Daughter sporocyst simple, sacculate, motile, orange in color, 0.857-1.652 mm (average 1.246 mm) long, primarily in blood sinuses and lymph spaces in gonad and digestive gland of host, may contain 100 or more cercariae. Cercaria (Fig. 141) 0.147-0.525 mm long (living) or 0.221-0.284 mm (average 0.254 mm) long (fixed) x 0-072-0.084 mm (average 0.075 mm) wide ; anterior sucker, usually longer than wide, 0.040-0.045 mm (average 0.043 mm) long x 0.038-0.041 mm (average 0.038 mm) wide; prepharynx 0.002-0.004 mm long; pharynx subspherical, 0.020 mm x 0.024 mm ; esophagus short ; intestinal caeca extend to level posterior to bifurcation of excretory vesicle; caecal walls of large polygonal cells ; ventral sucker 0.045-0-051 mm (average 0-048 mm) in diameter, slightly larger than anterior sucker ; approximate anterior suckerpharynx-ventral sucker ratio being 2 : 1 :2 ; small spherical tail, 0.0100.013 mm in diameter, attached to slight invagination at posterior end

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of body ; tail may or may not be lost prior to emergence ; excretory vesicle Y-shaped, arms longer than stem, extending anteriorly to level of caecal bifurcation ; unicellular cystogenous glands present throughout body; three pairs of cephalic gland ducts emanate from postpharyngeal region and terminate in six distinct pores on dorsal lip of anterior sucker (actual glands not seen by Uzmann (1953) but Stunkard and Uzmann (1959) have depicted glands located on each side of esophagus immediately anterior to caecal bifurcation. I n addition, they reported two additional pairs of ducts arising from cell bodies located lateral to the preacetabular area) ;flame-cell pattern 2[(2 2) (2 211. Metacercaria progenetic, elongate, 2.0 mm long, 0.8 mm wide; anterior sucker 0.18 mm x 0.22 mm; ventral sucker 0.34 mm x 0.37 mm; pharynx 0.125 mm x 0.150 mm; esophagus short; intestinal caeca reaching beyond level of testes to near posterior end of body; testes obliquely arranged in posterior one-third of body, each testis measures 0.17 mm x 0-15 mm; ovary between anterior testis and ventral sucker, 0.15 mm x 0.14 mm; cirrus pouch enclosing cirrus anterior to ventral sucker. Adult (from Mytilus edulis) 2.4 mm-3.2 mm (average 2.74 mm) long, 0.6 mm-0.92 mm (average 0.81 mm) wide; aspinous; anterior sucker 0.24 mm x 0.21-0.32 mm (average 0.30 mm) ; pharynx 0.160.20 mm (average 0.18 mm) in diameter; extremely short or no esophagus ; intestinal caeca reaching posterior one-fifth of body ; testes in posterior half of middle one-third of body, anterior testis 0.15-0.20 mm (average 0.18 mm), posterior testis 0.16-0.23 mm (average 0.19 mm); sterile and abnormal, 0.055 mm x 0.026 mm.

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Life cycle. Normally the metacercaria of P. maculatus found in Mytilus edulis is ingested by the definitive fish host within which it develops to maturity. But, as has been demonstrated by Stunkard and Uzmann (1959) for P. maculatus and Freeman and Llewellyn (1958) for P. subtenuis, the progenetic metacercariae of Proctoeces are capable of developing to sexually mature adults in the molluscan intermediate host although these produce small and abnormal sterile eggs. Ecology. The production of cercariae in P. maculatus sporocysts appears to be temperature influenced. Uzmann (1953) has shown that the smaller germ-ball-bearing sporocysts (most probably mother sporocysts) dominate in number during July and August while during the relatively cooler months of April, May and June, the cercariae-bearing daughter sporocysts are more plentiful. Moreover, in Mytilus edulis found in subtidal regions, the numerical dominance of daughter sporo-

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cysts enclosing cercariae may extend beyond June. Uzmann is of the opinion that this occurs " presumably as a direct result of the lower prevailing temperature." According to Stunkard and Uzmann (1969) the release of cercariae in greatest numbers occurs in the later winter and spring, thus suggesting that cercarial development is enhanced by lower temperature. Uzmann (1953) has also examined the effect of ambient temperatures on cercariae. Those maintained in filtered sea water at 21°C will only survive for 24-48 h. Those maintained at 14OC are healthy until the fourth day but all are moribund or dead by the seventh day. Among those maintained a t 7°C) twenty-nine of the seventy-five cercariae tested are still active after 12 days. The obvious endurance of cercariae at the lowest temperature tested is meaningful when the natural developmental cycle is considered. Uzmann has stated : While it has not been established that spontaneous emergence of cercariae occurs more frequently at any particular temperature, it is important to note that maturation of cercarial generations occurs during late winter and spring when the annual temperature is at its lowest (2"-8"C.).

It is known that P. maculatus cercariae will undergo periodic contraction and elongation after escaping from Mytilus edulis and " effects a serpentine but random progress upon the substratum " (Uzmann, 1953). Although it has well-developed suckers, it seldom utilizes these. Uzmann has also reported that they " do not manifest any pronounced taxes. . . " but the specific experiments performed were not reported. Physiology. Nothing is known about the physiology of P. maculatus other than their better survival rate at low temperatures. Pathology. The ability of Mytilus edulis to survive parasitization by P. maculatus has not been examined under controlled conditions although Uzmann believes that their longevity is impaired. He has stated that : " The severe intensity of the parasitism suggested that C. milfordensis (= P. maculatus) infections are probably lethal under temporary or sustained periods of ecological conditions unfavorable to the host. " The reproductive system of Mytilus edulis consists of a racemose complex of follicles and ducts which ramify throughout the visceral mass and mantle lobes. Just prior to spawning, the system is greatly swollen and occupies the mantle lobes. I n parasitized specimens, what Uzmann refers to as a blocking layer of sporocysts develops in the vascular system and causes a serious reduction in the efficiency of circulatory system, including that of the mantle, and thus precludes '(

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normal gametogenesis. Thus follicular development is either seriously impaired or totally precluded. I n addition, ‘‘ the vascular system of the mantle eventually loses its organized continuity through the rupturing of the lymph bearing vessels and appears finally as a more or less continuous hemocoel with sporocysts pervading throughout.” 22. Zetacercaria (Proctoeces) ostreae Fujita, 1925. (Fig. 142) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family Fellodistomatidae) This metacercaria was described by Fujita (1925) from the gonads of Crassostrea gigas collected at Hiroshima, Japan. Description. Unencysted, progenetic, with eggs measuring 0-04 mm X 0.02 mm; body elongate, aspinous, approximately 3 mm long, 0.97 mm wide ; anterior sucker 0.27 mm in diameter ; ventral sucker, 0.47 mm x 0.42 mm, in anterior half of body ; prepharynx extremely short, pharynx 0.18 mm x 0.15 mm; no esophagus; intestinal caeca, each 2-35 mm long, extending to near posterior margin of body ; testes, each approximately 0.17 mm x 0.13 mm, tandemly arranged in posterior one-third of body; ovary, 0.11 mm x 0.07 mm, anterior to testes ; ootype, Mehlis’ gland, cirrus pouch, seminal vesicle and internal prostate glands present ;follicular vitelline glands scattered throughout parenchyma, extending along entire length of body, limited primarily to two lateral fields but may converge along midline; genital pore immediately anterior to ventral sucker, posterior to caecal bifurcation ; excretory vesicle Y-shaped, with tubular arms extending to level of caecal bifurcation. 23. Cercaria brachidontis Hopkins, 1954. (Fig. 143) (Superorder Anepitheliocystidia; order Strigeatoidea; suborder Azygiata; superfamily Fellodistomatoidea; family Fellodistomatidae) Cercaria brachidontis, a parasite of Brachidontes recurvus (= Mytilus recurvus, M . hamatus), was described by Hopkins (195413) from hosts collected at Barataria Bay, Louisiana. He compared this cercaria to Uzmann’s Cercaria milfordensis, which is now known to be the larva of Proctoeces maculatus, among other similar cercariae and concluded that “ it is not improbable that C. brachidontis also belongs to that family (Fellodistomatidae).’ ’

Description of stages in mollusc. Sporocyst orange pigmented, in gonads, mantle and other tissues of host. Cercaria (Fig. 143) elongate oval, 0.33-0.43 mm long, 0.14-0.18 mm wide ; anterior sucker averaging 0.075 mm in diameter ; ventral sucker, 0.09 mm in diameter, slightly

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posterior to midlength of body ; body covered with fine cuticular spines ; no tail in fully developed form ; immature cercaria with small knob-like tail ; no stylet or other structures specialized for penetration at anterior end ; prepharynx not usually visible in frontal view ; pharynx muscular, averaging 0.045 mm long x 0.035-0.040 mm wide ; esophagus approximately of same length as pharynx ; intestinal caeca reaching to near posterior body terminal; six small cephalic glands on each side of pharynx, with ducts running dorsally over anterior sucker to anterior end ; anlage of cirrus pouch immediately anterior to ventral sucker ; anlager of testes and ovary immediately posterior to ventral sucker ; excretory vesicle Y-shaped, with sac-like stem terminating anteriorIy behind gonadal anlagen ; club-shaped arms extend anteriorly to level of caecal bifurcation; flame-cell pattern 2[(2 2) (2 2)]

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Other information. Nothing else is known about C. brachidontis except that Hopkins (1954b), as the result of not finding metacercariae in forty Brachidontes recurvus and hundreds ” of oysters from the area where the infected mussels were found, concluded : “ Therefore it seems improbable that Cercaria brachidontis uses bivalve molluscs as second intermediate hosts.’’ ((

24. Himasthla leptosoma Creplin, 1829. (Fig. 144) (Superorder Anepitheliocystidia; order Echinostomida; suborder Echinostomata; superfamily Echinostomatoidea; family Echinostomatidae) Nicoll (1906a,b) reported the occurrence of an echinostome metacercaria encysted in Cardium edule, Mytilus edulis and Mactra stultorum which is morphologically sufficiently similar to adults found by him in the oyster catcher, Haematopus ostmlegus, and herring gull, Larus argentatus, for him to consider it as the larva of the adult in these birds. He named this trematode Echinostomum secundum, a designation adopted by Lebour (1906, 1909, 1911). Although Holliman (1961) still lists E. secundum as a distinct species, in recent years several investigators (Sprehn, 1932 ; Palombi, 1934a ; Dawes, 1946 ; Hutton, 1952 ; Yamaguti, 1958) have designated, and probably correctly, E. secundum as a synonym of Himmthla leptosoma, a species described as Distoma Zeptosomum by Creplin (1829) and later placed in the genus Himasthla as H . leptosoma by Dietz (1909). Not only has Nicoll’s metacercaria been found in the three pelecypods listed above, but Lebour (1911) has also reported it in Mya arenaria and Tapes pullastra. I n addition, Cubnot (1892, 1917) has found what he believed to be H . leptosoma metacercariae encysted in the circumoral tentacles of the holothurian Leptosynapta inhoerens in

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addition to Arenicola marina and Scrobicularia tenuis plana, and Villot (1875, 1878) has reported it in 8. tenuis and in isopods.

Description of metacercaria (the stage found in economically important molluscs). Cyst (Fig. 144) spherical, 0.21-0.25 mm in diameter, commonly found in foot of pelecypods. The metacercaria has not yet been described but the cercaria and adult, and therefore presumably the metacercaria, bear twenty-nine collar spines arranged in an unbroken row, with the last lateral ones being slightly smaller and situated slightly lower than the remainder. Other information. Lebour (1909) has been able to infect Mytilus edulis with cercariae emerging from the first intermediate host, Littorina littorea. Although the other aspects of the life cycle of H . leptosoma have not been demonstrated, there is no reason to believe that its life cycle should be different from that of H . quissetensis given below. The avian definitive host, which is also known to include Tringia aZpina, T . canutus, Limosa and Arenaria, undoubtedly acquires its infection by ingesting metacercariae-harboring pelecypods. 25. Himasthla quissetensis Miller and Northrup, 1926. (Fig. 145)

(Superorder Anepitheliocystidia; order Echinostomida; suborder Echinostomata; superfamily Echinostomatoidea; family Echinostomatidae) During a survey of larval trematodes in the mud flat snail, Nassarius obsoletus, Miller and Northrup (1926) examined 8 875 specimens from Quamquisset Harbor, Massachusetts, and as the result described a new echinostome cercaria, Cercaria quissetensis. Subsequently, Stunkard (1934, 1937, 1938), following successful life cycle studies, has reported that C. quissetensis is the larva of Himasthla quissetensis, an up until then undescribed species. Stunkard has demonstrated that cercariae escaping from Nassarius obsoletus will penetrate and encyst in the mantle, gills and foot of M y a arenaria, Modiolus modiolus, Mytilus edulis, Cumingia tellinoides, Aequipecten irradians, Ensis directus and Crepidula fornicata. Of these, Uzmann (1951), as the result of a survey in Merrimack Bay, Plum Island Sound, and Annisquam River, Massachusetts, and examination of clams from St. Andrews, New Brunswick, Canada, has reported that M y a arenaria is a commonly parasitized second intermediate host in nature. Cheng et al. (1966a)have extended the list of known second intermediate hosts to include Modiolus demissus, Mercenaria mercenaria and Tapes semidecussata. I n addition, we have reported the finding of metacercariae in the blood vessels and heart of experimentally infected Crassostrea virginica and on the gill

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surfaces of C. gigas although none were found in the tissues. Among the eight marine pelecypods experimentally infected, M y a arenaria has been demonstrated to be the most efficient host, i.e. the greatest percentage of metacercariae can be recovered from it, thus supporting Uzmann’s concept that this clam is an important and heavily parasitized one in nature. The natural definitive host of H . quissetensis is most probably some shore bird that feeds on pelecypods. Stunkard (1934, 1937, 1938) has been able experimentally to infect the tern, Sterna hirundo, and the herring gull, Larus argentatus.

Description of metacermria (the stage found in economically important molluscs). Cysts spherical to oval, 0.14-0-19 mm in diameter, enclosing metacercaria which is doubled over ventrally (Fig. 145). A detailed description of the metacercaria is not available ; however, cercariae and young adults, hence presumably also the metacercariae, bear thirty-one spines on a ventrally interrupted reniform collar ; spines arranged in a single row except at the two terminals where two spines are situated between and behind the others. When cysts are observed in histological section, the inner parasite-secreted wall is usually surrounded by an outer wall comprised of the host’s connective tissue or muscle fibers, depending on the site of the cyst, and leucocytes. Life cycle. As stated, Stunkard has been able to infect Sterna hirundo and Larus argentatus by feeding them metacercariae. He also has compared the adults recovered from his experimentally infected birds with those of Linton (1928) collected from four species of naturally infected Larus and from Nycticorax nycticorax which had been identified as Himasthla elongata. Although the redial stage in Nassarius obsoletus, the cercaria, metacercaria, and adult are known, the exact manner in which N . obsoletus, the first intermediate host, becomes infected remains to be demonstrated. Other information. Nothing is known about the ecology of this parasite, except Miller and Northrup’s (1926) report that in Massachusetts cercariae-bearing rediae predominate during July through October while immature rediae are more abundant during November and December. The effects of the plasma of pelecypods on cercariae have been reviewed in an earlier section (see Chapter 4, Section 11, A, 1). Furthermore, the reactions of various pelecypods to encysting metacercariae, as reported by Cheng et al. (1966a), have also been reviewed above (see Chapter 4, Section 11, A, 2).

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26. Himasthla muehlensi Vogel, 1933 (Superorder Anepitheliocystidia; order Echinostoniida; suborder Echinostomata; superfamily Echinostomatoidea; family Echinostomatidae) Adults of H . muehlensi were originally described by Vogel (1933) from a human who had eaten many raw small quahaugs (cherry stones), Mercenaria mercenaria, in New York and later, upon arrival in Germany, complained of gastrointestinal disturbances. The worms were passed after treatment with an anthelmintic. Since it is now known that the definitive hosts of Himasthla spp. acquire their infections from ingesting metacercariae-harboring pelecypods, it can be assumed that the metacercaria of H . muehlensi occurs in Mercenaria mercenaria from the east coast of the United States although it has yet t o be found. Stunkard (1938), in reference t o H . muehzensi, has stated : The first experimental demonstration of the life cycle in the genus Himasthla (that of H . puissetensis) supports the opinion of Vogel that the human species, H . muehlensi, is acquired by eating raw or insufficiently cooked mollusks. Examination of large numbers of Venus ( =Memenaria) mercenaria, purchased in the New York market, has failed, however, to disclose the metacercaria of this species. Whether or not it is distinct from H . quissetensis remains to be determined. Later (Stunkard, 1939), he stated that in a letter t o Dr. Vogel from Dr. H. Mendheim of Munchen, Germany, it was suggested that H . muehlensi might be a synonym of H . elonqata. However, Stunkard, after examining Vogel’s specimens of H . muehlensi, stated : Although they are very similar, specimens of H . muehlelzsi may be distinguished from those of H . puissetensis. The principal difference is the shape of the testes; in the former species the contour is always smooth whereas in the latter one, these organs are invariably notched or lobed. It seems unlikely that such a character would be dependent upon development in a particular host. Moreover, there are important reasons for considering H . muehlensi distinct from H. elongata. The two species are very different in size and in measurements of particular organs. The number of cephalic spines is regarded as one of the best specific criteria, and H . elongata bears twenty-nine of these spines whereas the number of H . muehlensi is certainly greater. Reexamination of the material has led both Dr. Vogel and myself to the opinion that the specific number of spines in H . muehlensi is probably thirty-one, rather than thirty-two as stated previously. Since H . quissetensis also bears thirty-one collar spines, it still remains uncertain, despite Stunkard’s observations on the shape of the testes, as t o whether H . muehlensi should be considered a distinct species, but until further evidence becomes available, i t will be considered as such.

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Another aspect of the importance of Himasthla spp. to public health has been briefly stated (Cheng, 1965a). Back in the 1930s and 1940s, Dr. N. Hathaway," the university physician at Yale University, conducted a clinical and epidemiological study on the cause of a series of short-termed (24-36 h) but severe gastrointestinal disturbances in students who shared one common experience, that of having eaten raw Mercenaria mercenaria purchased from the New Haven, Connecticut, market, and presumably from the Connecticut coast. Bacteriological tests performed on samples of this clam and on victims failed to reveal any bacteria which could be responsible. On the other hand, it wm discovered that M . mercenaria from the Connecticut coast harbored large numbers of H . quissetensis metacercarial cysts. Subsequent feeding of isolated cysts to volunteers failed to establish infections or produce the specific clinical symptoms and thus the mystery remained unsolved. I n 1965, I reported that the tissues of Mercenaria mercenaria surrounding H . quissentensis cysts undergo chemical change. Among these changes is the accumulation of short-chained fatty acids, provisionally identified as butyric acid among others. It was postulated that if sufficient number of clams which include toxic short-chained fatty acids in their tissues are ingested, gastrointestinal disturbances, such as that found at Yale, could occur. Although chemical changes in pelecypods harboring other species of Himasthla have not yet been examined, there is no reason to believe that a parallel phenomenon should not occur. 27. Himasthla ambigua Palombi, 1934

(Superorder Anepitheliocystidia; order Echinostomida; suborder Echinostomata; superfamily Echinostomatoidea; family Echinostomatidae) Only the metacercaria of H . ambigua is known. It was described by Palombi (1934a)encysted on the branchial lamellae of Tapes decussatus collected in the Gulf of Naples. The morphology of the metacercaria suggests that it is a member of the genus Himasthla. Description. Cyst (Fig. 146) transparent, 0.20 mm in diameter. Metacercaria (Fig. 147) elongate, with ventrally interrupted collar near anterior end beset with thirty-two spines ; twenty-eight of these, each measuring 0.20 mm long, arranged in a curved row, but two small terminal spines are found on each end behind the others ; body 0.72 mm long, 0-19 mm wide; anterior sucker 0.065 mm in diameter; ventral sucker posterior to midlength of body, 0.093 mm in diameter; pre-

* I am deeply grateful to Dr. Hathaway for sharing his yet unpublished findings with me during a visit to New Haven, Connecticut, in 1966.

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pharynx, pharynx, and long esophagus present ; pharynx 0.025 mm long x 0-016mm wide ;intestinal caeca long, reaching to near posterior end of body ; excretory vesicle Y-shaped, with arms reaching anteriorly

FIas. 143-150. Fellodistomatid and echinostomatid trematode larvae. (143) Cercaria brwhidontis (redrawn after Hopkins, 1954b) ;(144) Himasthla leptosoma metacercaria within cyst (redrawn after Lebour, 1911); (145) H . quissetensis metacercaria within cyst; (146) H . ambigua metacercaria within cyst ; (147) H . ambigua metacercaria removed from cyst (redrawn after Palombi, 1934s) ; (148) unidentified echinostome metacercaria of Nicoll (redrawn after Nicoll, 1906a) ; (149) encysted Parorchis acanthus metacercaria, frontal view ; (150) encysted P. ancanthus metacercaria, lateral view showing characteristic flat surface (redrawn after Stunkard awd Cable, 1932).

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to near posterior level of pharynx, with conspicuous lateral secondary tubules, stem subovoid.

Other information. Nothing else is known about H . ambigua; however, from what is known about the life cycle pattern in this genus, Tapes decussatus is undoubtedly the second intermediate host, while the first is most probably a gastropod and the natural definitive host is one or more species of shore birds. 28. Himasthla compacta Stunkard, 1960

(Superorder Anepitheliocystidia; order Echinostomida; suborder Echinostomata; superfamily Echinostomatoidea; family Echinostomatidae) Stunkard (1960), in a paper in which is given a concise but excellent review of the genus Himasthla, also described a new species, H . compacta, from laboratory-reared Larus argentatus which had been fed tissues of Mya arenaria, collected near Boothbay Harbor, Maine, which included an unknown species of metacercaria. He also recovered specimens of H . cornpacta from Lurus argentatus fed metacercariae from Mya arenaria which in turn had been exposed t o echinostome cercariae from the gastropod Hydrobia minutu collected at Woods Hole, Massachusetts. Since Mya arenaria, and possibly other commercially important marine pelecypods, are involved in the life cycle of H . compacta, it is being included therein.

Description of metacercuria (the stage found in economically important molluscs). Unfortunately, although Stunkard described the adult, redia, and cercaria of H . compacta, he did not give a description of the metacercaria. However, since both the cercaria and adult possess twenty-nine collar spines, it may be assumed that the encysted metacercaria also possesses twenty-nine spines. These are located on a ventrally interrupted reniform collar. Twenty-seven of the spines are arranged in a curved linear row while two smaller spines, one at each end, are located behind the terminal large spine at each end. Furthermore, since the body proper of the cercaria measures 0.300.60 mm long and 0.08-0.19 mm wide while a very young adult specimen recovered from Larus argentatus measures 0.46 mm long and 0.12 mm wide (extrapolated from Stunkard’s drawing), it can be assumed that the body dimensions of the metacercaria are between these two sets of limits, most probably closer to that of the very young adult in which the reproductive organs had not yet developed.

Life cycle. Stunkard (1960) was able to infect juveniles of Hydrobia minuta by exposing them to eggs from experimentally infected Larw A.M.B.--G

16

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argentatus, although the rediae recovered 4 weeks after infection were small and he was unable to determine whether these were mother or daughter rediae. Cercariae escaping from naturally infected H . minuta will penetrate and encyst in the palps and gills of Mya arenaria. Tissues of naturally and experimentally infected M . arenaria were fed to eider ducks, Somateria mollissima, herring gulls, Larus argentatus, terns, Sterna hirundo, white mice and golden hamsters. Adults were recovered from the intestine of the gull only. I n the case of eider ducks, the metacercariae were said to have excysted “ b u t did not persist more than about 10 days.” 29. Himasthla littorinae Stunkard, 1966

(Superorder Anepitheliocystidia; order Echinostomida; suborder Echinostomata; superfamily Echinostomatoidea; family Echinostomatidae) In his report of the life cycle of Himasthla littorinae, a trematode which will develop to maturity in experimentally infected herring gulls, Larus argentatus, Stunkard (1966) stated that this parasite will encyst, as metacercariae, in the palps, gills and mantle of Mytilus edulis, Mya arenaria and in species of Littorina. Description of metacercaria (the stage found in commercially important molluscs). Cysts average 0.26 mm in diameter, with laminated wall which measures 0.005-0.006 mm in thickness. A complete description of the metacercaria was not given by Stunkard ; however, since the body of the cercaria is 0.45-0-85mm long x 0.20-0-30 mm wide, and the adult is 2.8-5.4 mm long x 0.41 mm wide, the dimensions of the metacercaria must fall between these limits. Furthermore, since both the cercaria and the adult possess twenty-nine collar spines which are similarly arranged, it can be assumed that the metacercaria also possesses twenty-nine spines arranged in a single curved row except at the ventral ends where the last spine is flanked on each side by a smaller spine set slightly more posteriorly. The larger collar spines of the metacercaria are 0.029 mm long and 0.006 mm wide at their bases. Life cycle. According to Stunkard, the cercariae-bearing rediae are found in Littorina saxatilis and L. obtusata taken near Woods Hole, Massachusetts. Cercariae escaping from the gastropod first intermediate host will penetrate and encyst in the molluscs mentioned, including specimens of Littorina spp. from which the cercariae had emerged. Metacercariae from the molluscan second intermediate host will develop into adults in Larus argentatus but not domestic ducklings.

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30. Unidentified Echinostome of Nicoll, 1906. Himasthla sp.? (Fig. 148)

(Superorder Anepitheliocystidia; order Echinostomida; suborder Echinostomata; superfamily Echinostomatoidea; family Echinostomatidae) During a survey of the parasites of Cardiurn edule collected a t the mouth of the Eden, northern England, Nicoll (1906a) found an echinostome metacercaria encysted along the inner mantle fold and in the foot musculature. He did not name this larva but did give a brief description. According to him, the adult is an intestinal parasite of the oyster-catcher, Haernatopus ostralegus, although experimental proof was not provided. Neither his description of the adult nor his illustration of what is assumed to be a very young adult are sufficient to attempt a definite identification although the occurrence of a conspicuous spinous collar and the positions of its suckers definitely establish it as an echinostome, probably a member of the genus Himasthla. Moreover, the morphology of the metacercaria and the fact that it encysts in a pelecypod suggest that it is a species of Himasthla or some closely allied genus. Description of metacercaria. Cyst transparent, slightly brownish, 0.21-0.25 mm in diameter ; cyst wall comprised of three layers which collectively measure around 0.13 mm in width. Metacercaria (Fig. 148) 0.6-0.8 mm long x 0.19 mm a t its greatest width; cuticle armed with thirty to forty alternate rows of conspicuous spines in anterior half of body ; anterior sucker, 0.095 mm in diameter, smaller than ventral sucker ; slender prepharynx, pharynx (0.05 mm x 0.03 mm), and long slender esophagus present ; termination of intestinal caeca undetermined ; ventral sucker immediately posterior to midlength of body ;reniform collar armed with twenty-nine spines each measuring 0.025 mm long except for last three on each end which are smaller; excretory vesicle with long lateral collecting tubules, with side branches, reaching to posterior margin of collar. 31. Parorchis acanthus Nicoll, 1906. (Figs. 149 and 150) (Superorder Anepitheliocystidia; order Echinostomida; suborder Echinostomata; superfamily Echinostomatoidea; family Philophthalmidae) The adult of Parorchis acanthus was originally described by Nicoll (1906b) from the bursa Pabricii and cloaca of the herring gull, Larus argentatus, under the name Zeugorchis acanthus. Later (Nicoll, 1907a), he erected tho genus Parorchis and placed P. acanthus therein. Linton (1914) described P. avitus also from Larus argentatus, but since then Cable and Martin (1936) have demonstrated that it is a synonym of P . acanthus although earlier Stunkard and Cable (1932), who carried

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out experimental life cycle studies, believed that P. acanthus and P. avitus are distinct and that the cercaria described by Stunkard and Shaw (1931) as Cercaria sensifera is the larva of P. avitus. P. acanthus is mentioned in connection with parasites of commercially important marine molluscs since its cercaria, originally described as Cercaria purpurae by Lebour (1907c, 1911) from Nucella ( = Purpura) hpillus from British waters, was reported by Lebour and Elmhirst (1922) to penetrate and encyst in the mantle and foot of either Cardiunz edule or Mytilus edulis. Their observations have been criticized by Stunkard and Shaw (1931) and Stunkard and Cable (1932) who have shown that the cercaria of P. acanthus does not penetrate pelecypods but encysts on the body surface. The observation by Stunkard and his co-workers has been strengthened by the observation of F. G . Rees (1939b, 1940) who has studied the early stages of P. acanthus in great detail. She has demonstrated that cercariae maintained in aquaria will encyst on the outside of Nucella shells and on other objects. Similarly, Xtunkard and Cable (1932) have found that the cercariae would encyst on the shells of Mytilus which are placed in a container with cercariae. They also have reported that : In one case, three cysts were found on the surface of the foot and mantle, and a few others were seen in masses of mucus in the mantle cavity. In the same specimen, large numbers of cercariae encysted on the outside of the shell, most of them near the incurrent siphon. Many of them struck the shell around the incurrent opening, adhered, and immediately encysted.

It is now known that P. acanthus cercariae will encyst on practically any surface and that mechanical stimulation, such as stirring or shaking, accelerates encystment (Stunkard and Cable, 1932). I n nature, it is possible that birds become infected with the ingestion of Cardiunz edule, Mytilus edulis, or other molluscs with encysted metacercariae attached to their surfaces.

Description of metacercaria (the stage which may encyst on the body surface of commercially important molluscs). Cyst fairly transparent, comprised of two walls, an outer thick and brittle one, and an inner thin and tough one; flat on the attached side; fluid in space between metacercaria and host sometimes contains small granules which, according to Xtunkard and Cable (1932), probably represent materials discharged from the metacercaria’s excretory system. Metacercaria with collar armed with forty-eight spines ; doubled over ventrally ; motile for several hours after encystment. A detailed description of this metacercaria is not yet available.

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Life cycle. Aspects of the life cycle of P. acanthus have been examined by Linton (1914), F. G. Rees (1939b, 1940), Lebour (1911) and Stunkard and Cable (1932). The adults may and commonly do include free miracidia in the terminal portion of their uteri although eggs are also laid containing fully developed miracidia. These hatch in 0ea water after being voided in the avian host’s feces. The miracidium, either born free or hatching from the egg, penetrates the first intermediate host, Nucella lapillus in British waters, Urosalrpinx cinerea in addition to Nucella lapillus in northeastern United States, and Littorina pintado in Hawaii.* Each miracidium contains a mother redia. Within the snail, mother rediae give rise to daughter rediae which in turn give rise to cercariae. Cercariae, once out of the first intermediate host, will encyst on practically any surface and hence a second intermediate host is not really necessary. Shore birds (Larus argentatus, L. canus, Nycticorax nycticorax hoactili) most probably become infected with the ingestion of shellfish with metacercariae attached to their surfaces. Attempts by Stunkard and Cable (1932) to infect guinea-pigs, rats and mice were unsuccessful. 32. Neophasis pusilla Stafford, 1904. (Figs. 151-154)

(SuperorderEpitheliocysticlia; order Plagiorchiida; suborder Plagiorchiata; superfamily Allocreadioidea; family Allocreadiidae) While examining specimens of the buckie, Buccinum undatum, brought in by the fishing boats from Cullercoats, northeast England, Lebour (1911) found that 7 % were parasitized by a species of larval trematode which she associated with an adult form found in the catfish Anarrhichm lwpus. She designated this trematode aa Acanthopsolus lageniformis. I n addition, she found one specimen of B. undatum collected at Holy Island, northeast England, parasitizedbylarvaeof this trematode. Description of stages in mollusc. Older rediae (Fig. 151) pale yellow, measuring from 0.50 mm long (one specimen depicted by Lebour measured 3 mm) ; without collar or ambulatory buds ; containing thirty or more cercariae. Smallest rediae (Fig. 152) about 0.30 mm long, with pointed posterior terminal, pharynx, and intestinal caecum ; caecum short and inconspicuous in older rediae. Young cercaria (Fig. 153) with straight tail which is slightly longer than body, apparently lost prior to emergence from redia. Fully grown cercaria (Fig. 154) about 0.5 mm long, width less than half body length; oval or flaskshaped, with anterior end more pointed ; body covered with spines which are more sparse posteriorly ; with two eye-spots, one on each side *The Hawaiian species of Parorchis has only been tentatively identified as P . acalzthua. It may be a subspecies or another species.

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of prepharynx ; suckers about the same size, 0.06 mm in diameter, with anterior sucker slightly larger ; small glands along posterior margin of anterior sucker ; ventral sucker in center of body ; prepharynx, pharynx, and short esophagus present ; intestinal caeca reaching to near posterior end of body ; excretory vesicle large and pear-shaped, sometimes bilobed ; two testes, one on each side, midway between ventral sucker and posterior end of body ; ovary in front of right testis. Life cycle. According t o Lebour (1911), The final host is the catfish Anarrhichas lupus and it is very probable that there is no (second) intermediate host, the fish swallowing the Buccinum undatum with the contained rediae, the cercaria thus getting into the intestine without aid of another host. I have never seen any trace of cysts in the catfish. Whether this is the case remains to be determined experimentally. Lebour was apparently unaware that Stafford (1904) has described what she considered t o be the adult of her cercaria in Anarrhichas lupus under the name Neophasis pusilla, which, according t o the rules of nomenclature, is the valid one. Other information. Lebour (191 1) has reported that the cercariae (probably also the rediae) are found during the spring and summer but never in the winter. This suggests that parasitized Buccinum undatum lose their infections preceding or during the cold months and a t the same time are not reinfected. Reinfection would appear t o occur during later winter or early spring. 33. Cercaria neptuneae sp. inq. Lebour, 1911. (Figs. 155 and 156) (Superorder Epitheliocystidia; order Plagiorchiida;suborder Plagiorchiata; superfamily Allocreadioidea; family Allocreadiidae) Cercaria neptuneae develops in rediae found in the digestive gland of Neptunea antiqua and Buccinum undatum “brought in by the Cullercoats [northeast England] fishing boats. . . in July ” (Lebour, 1911).

Description of stages in mollusc. Redia (Fig. 155) colorless, 2 mm or more in length; with pharynx, 0.04 mm long, and very short intestinal caecum, not longer than pharynx ; no collar or ambulatory buds ; contains cercariae a t various stages of development. Cercaria (Fig. 156) covered with small cuticular spines ; 0.4 mm long (without tail), 0-10 mm wide (highly variable) ; tail straight, exceedingly large and thick, about 0.26 mm long x 0.049 mm wide; anterior sucker 0-033 mm in diameter; prepharynx 0.06 mm long, pharynx 0.03 mm long, rest of alimentary tract not seen ;two large eye-spots immediately

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anterior to and on each side of pharynx; large penetration glands (Lebour depicts four on each side) on each side of ventral sucker and which empty t o the exterior near anterior sucker via four long ducts ;

FIGS.151-158. Larval allocreads. (151) Older redia of Neophasis ptailla enclosing developing cercariae; (152) young redia of N. pusilla; (153)young N . pusiEla oercaria with tail; (154) fully developed tailless N. pusilla cercaria (redrawn after Lebour, 191 1); (155) sporocyst of Cercaria neptuneae enclosing developing cercariae ; (166) fully developed C . neptuneae (redrawn after Lebour, 1911) ; (167) sporocyst of Cercariu ophicerca ;(1 68) fully developed C . opkicerca (redrawn after Palombi, 1934s). DO, Developing ovary ; DT, developing testis ; ES, eye-spot ; E V , excretory vesicle ;I N T , intestinal caecum ; PB,penetration gland; P H , pharynx.

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stylet not seen (but may be present) ; another group of smaller glands with long ducts immediately in front of ventral sucker (Lebour considered these to be cytogenous glands but this is doubtful); ventral sucker near middle of body, about same size as anterior sucker or slightly smaller ; excretory vesicle epitheloid and oval.

Life cycle. The life cycle of C. neptuneae has not been determined although Lebour (19ll)hasstated that it maybe the larva of anAZlocreadium or Lebouria (= Plagioporus), both of which are known to include representatives which are intestinal parasites of fish. Pathology. Lebour (1911) has pointed out that the entire whorl of both species of molluscan hosts is packed with rediae and that the reproductive organs are destroyed. 34. Cercaria ophicerca Palombi, 1934. (Figs. 157 and 158) (SuperorderEpitheliocystidia;order Plagiorchiida; suborder Plagiorchiata; superfamily Allocreadioidea; family Allocreadiidae?) Cercaria ophicerca, developing in sporocysts, was described by Palombi (1934a) from the visceral mass of Tapes decussatus and T . aureus in the Gulf of Naples. He tentatively placed it in the family Allocreadiidae.

Description of stages in mollusc. Sporocyst (Fig. 157) sacciform, semi-transparent, whitish in color, 1.07 mm long, 0.13 mrn wide. Cercaria (Fig. 158) with elongate body, averaging 0.34 mm long, 0.09 mm wide, 0.08 mm thick ; cuticle spinous ; anterior sucker averaging 0.05 mm in diameter; ventral sucker averaging 0’05 mm in diameter ; cephalic glands (referred to as “ glandole cistogene ” by Palombi) on each side of esophagus (Palombi depicts four glands on each side) which empty to exterior at anterior end of body via separate ducts ; one pair of eye-spots present, one on each side of prepharynx ; prepharynx, pharynx, and long esophagus present ; intestinal caeca terminating at posterior end of body ; two testes, tandemly arranged in posterior half of body, between ventral sucker and ovoid epitheloid excretory vesicle; tail 1.20 mm long, 0.03 mm wide, straight and simple tapering, to point posteriorly. Other information. Palombi (1934a) has reported the occurrence of a metacercaria in a single specimen of Tapes decussatus collected at Naples which is morphologically similar to C. ophicerca and which, according to him, may be the metacercaria of this cercaria but experimental evidence is wanting.

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35. Lepocreadium album Stossich, 1890. (Figs. 159 and 160) (SuperorderEpitheliocystidia; order Plagiorchiida;suborder Plagiorchiata; superfamily Allocreadioidea; family Allocreadiidae)

Adults of Lepocreadium album were originally described by Stossich (1890) from the intestine of the marine fish Cantharus orbicularis

collected at Trieste. Later (1904), he found adults of the same species in C. Zineatus and Oblata melanura and in that report placed this trematode in the genus Lepocreadium. This trematode has been shown by Palombi (1931a, 1932, 1937) to be the adult of Cercaria setifera which had been described by Monticelli (1888) in Nassa mutabilis and has since been reported by Odhner (1914), Dollfus (1926) and Palombi (1931a). Palombi (1934a, 1937) has shown that this cercaria utilizes Tapes decussatus, T . aureus and Aplysia punctata as the second intermediate host. Experimental infection of Blennius gattoruginae and other fishes by feeding them encysted metacercariae has been successfully accomplished. Description of metacercaria (the stage found in commercially important molluscs). Metacercaria (Fig. 159) encysted in cutaneous tissue of Aplysia punctata and in gill and mantle tissues of Tapes decussatus and T . aureus. The cyst may be surrounded by a gelatinous outer coat of host origin which is impregnated with calcium carbonate granules. Cysts without this outer wall average 0.30 mm in diameter. Metacercaria elongate, oval, measurements not available ; anterior sucker connected to prepharynx which in turn is connected to large pharynx ; esophagus short ; intestinal caeca reaching to near posterior end of body; cercarial eye-spots on each side of pharynx; excretory vesicle tubular, extending to level of ventral sucker ; ventral sucker smaller than anterior sucker ; genital primordia, situated in intercaecal space, in posterior half of body. Life cycle. Within the first intermediate host, Nassa mutabilis, the straight-tailed cercariae (not the trichocercous cercaria referred to aB C. setijera by J. Miiller) develop in rediae located in the gastropod's hepatopancreas. The escaping cercariae encyst in the superficial tissues of Aplysia punctata, Tapes decussatus or T . aureus. When the second intermediate host is ingested by a suitable fish, the adult stage (Fig. 160) is attained in its intestine (Palombi, 1937). 36. Metacercaria acherusiae Palombi, 1934. (Fig. 161) (SuperorderEpitheliocystidia; order Plagiorchiida; suborder Plagiorchiata; superfamily Allocreadioidea; family Allocreadiidae) Palombi (1934a) described Metacercaria acherusiae based on one specimen found encysted in Tapes decussatus collected at Fusaro.

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Italy. He assigned it to the family Allocreadiidae. As far as I can determine, it has not been found since. Description. Cyst (Fig. 161) ovoid, transparent, 0.32 mm x 0.25mm. Metacercaria (Fig. 161) elongate, 0.66 mm long x 0.20 mm wide, covered with fine cuticular spines ; anterior sucker 0.1 1 mm in diameter ; ventral sucker 0.092 mm in diameter ; relatively long prepharynx present ; pharynx 0.063 mm long x 0.038 mm in diameter ; esophagus approximately same length as prepharynx ; intestinal caeca reaching into posterior one-fourth of body ; group of cephalic glands (Palombi depicts nineteen) on each side of prepharynx, pharynx, and esophagus, with ducts leading to margin of anterior sucker ; testicular primordia, two in number, tandemly arranged in intercaecal space in posterior one-third of body ; ovarian anlage situated between testes ; excretory vesicle tubular, reaching posterior margin of ventral sucker. Other information. If M . acherusiae is an allocread trematode, then from our knowledge of the life cycle pattern within this family, the definitive host is most probably a fish. 37. Metacercaria mytili sp. inq. Lebour, 1906. (Fig. 162)

(Taxonomic position undetermined) This metacercaria, erroneously identified as a cercaria and originally named Cercaria mytili, was described by Lebour (1906) encysted in the digestive gland of Mytilus edulis from Northumberland and Cardium edule from the west coast of Scotland, primarily in the former. It is not possible even to attempt an identification of this larva from Lebour's description and figure except that the two conspicuous lateral main collecting tubules of the excretory system, which are filled with granules, is suggestive of certain echinostome metacercariae, but Lebour did not report the occurrence of a collar with spines which is characteristic of echinostomes. Description. Metacercarial cyst (Fig. 162) spherical, transparent, 0.20 mm in diameter. Metacercaria 0.46 mm long, covered with spines ; posterior end pointed, anterior end rounded ; anterior and ventral suckers about the same size, 0.06 mm in diameter; ventral sucker immediately posterior of center of body ; two lateral tubules of excretory system conspicuous, full of clear granules. 38. Cercaria buccini sp. inq. Lebour, 1911. (Figs. 163 and 164)

(Taxonomic position undetermined) Cercaria buccini is a microcercous cercaria described briefly by Lebour (1911) from Buccinum undatum " brought in by the fishing

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FIGS.169-167. Larval allocreads and trematodes of undetermined taxonomic positions. (169) Metacercaria of Lepocreadium album enveloped by gelatinous coat ; (160) young adult of L. album recovered from intestine of definitive fish host; (181) Metacercaria acherusiae escaping from cyst (redrawn after Palombi, 1934a); (162) Metacercaria mytili within cyst wall ; (163) sporocyst of Cercaria buccind; (164) fully developed C. buccini (redrawn after Lebour, 191 1 ) ; sporocyst of Cercaria tenuans; fully developed C. tenuans (redrawn after Cole, 1936) ; unidentified “ aporocyst ” of Nicoll (redrawn after Nicoll, 1906b). C, Cyst; C I R , cirrus; DO, developing ovary ; DS, ‘‘ daughter sporocyst ” ; D T , developing testis ; ES, eyespot; E V , excretory vesicle; BP, genital pore; I N T , intestinsl caecum; MC, metacercarial cyst ; PO, penetration (cephalic) gland; P H , pharynx; ST,stylet.

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

boats ” in England. Its systematic position cannot be determined from the available information. Description of &yes in mollusc. Sporocyst (Fig. 163) long, 1.43.0 mm in length, sausage-shaped, pale yellow or colorless ; may contain twenty to thirty cercariae. Cercaria (Fig. 164) 0.33 mm long including tail ; tail, 0.06 mm long, elongate cup-shaped ; anterior sucker 0-046 mm in transverse diameter ; stylet with four points a t anterior terminal of body, with middle pair of points long and outer pair short and bluntly rounded behind ; ventral sucker posterior to midlength of body, same size as anterior sucker ; penetration glands (Lebour depicts two groups of six) situated between suckers, close to ventral sucker ; three ducts from each group of glands open to exterior in area of stylet: excretory vesicle epitheloid and oval. 39. Cercaria tenuans sp. inq. Cole, 1935. (Figs. 165 and 166)

(Taxonomic position undetermined) During 1933, Cole (1935) investigated an unusual mortality which occurred among the mussels, Mytilus edulis, brought to Conway, Wales, for purification. Among the 900-1 000 mussels which had died, he examined 300-400 and found six to be infected by a larval trematode. Since the mantle of parasitized M . edulis revealed “ a vivid marigold to blood orange ” color, Cole referred to the condition as the “ orange sickness of mussels ”. Although the percentage of infection found by Cole was not large, he stated: That this parasite was much more prevalent in the early years of the Conway installation seems certain, as Dr. Dodgson tells me that at one time it was possible t o obtain specimens by inspecting the few dead mussels which are picked out of the tanks during the normal process of purification. Cole also mentioned that Atkins (1931) had found but did not describe this parasite in 2.16% of the mussels she had examined from Padstow, Devon. Description of stages in mollusc. Sporocysts (Fig. 165) in all tissues of host’s body, particularly mantle, but not in foot; bright orange and thin-walled, oval, moderately contractile, vary considerably in size ; those containing fully developed and active cercariae averaging 1.1 mm X 0.45 mm; orange pigment spotty, rest of sporocyst wall colorless; thickened grayish rim at anterior end. Cercaria (Fig. 166) 0 .3 mm when fully extended ; without tail ;anterior sucker 0.05 mm in diameter ; ventral sucker 0-07 mm in diameter, with its center being 0-18 mm from anterior body terminal ; pharynx present, esophagus non-existent or extremely short ; intestinal caeca short and bulbous, terminating in

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anterior one-third of body; two testicular anlagen globular, one on each side of pyriform structure (identity unknown) , behind ventral sucker; duct leading from pyriform structure to cirrus situated immediately anterior to ventral sucker ; excretory vesicle Y-shaped with arms extending to between caecal tips and ventral sucker, stem pearshaped. Other information. Nothing else is known about C. tenuans. Although Cole appeared to be convinced that this parasite caused the death of Mytilus edulis, in my opinion this hypothesis should be accepted with caution since he only found six out of 300-400 dead mussels to be infected. 40. Unidentified

‘ I Sporocyst ” of Nicoll, 1906. (Fig. 167) (Taxonomic position undetermined) During an examination for parasites in Cardium edule from the vicinity of St. Andrews, Scotland, Nicoll(1906b) reported the occurrence of a most unusual “ sporocyst ” in the hepatopancreas and on the adjacent intestine. From his description, which is summarized below, it would appear that he had found miracidia in the process of metamorphosing into rediae rather than sporocysts since it contains a pharynx ; however, the fact that the “ daughter sporocysts ” (rediae) within globular “ cysts ” within ciliated ‘‘ mother sporocysts ” (rediae) are also ciliated and bear eye-spots is indeed puzzling. I n addition to finding these unusual sporocysts ” in Cardium edule, Nicoll reported that they also encyst in the foot of Mytilus edulis and Mactra stultorum. Description. Body elongate oval, with pointed anterior end and blunt posterior end, covered with cilia, 0.2-0.8 mm long x 0.38 mm at greatest width ; with two crescentric eye-spots near anterior end ; small pharynx anteromedial to eye-spots ; with six to eight globular ‘‘ cysts ” in body; with each mature “ cyst ” containing two to four ‘‘ daughter sporocysts ” (rediae); immature “ cysts ” generally situated near body wall at anterior end of ‘‘ mother sporocyst ” (rediae); fully formed “ daughter sporocysts ” (rediae) within “ cyst ” appear as miniature “ mother sporocysts ” (rediae), including two eye-spots and cilia. Other information. From Nicoll’s description it would appear that a second miracidial generation is formed within the “ cysts ” found in the parenchyma of mother rediae which in turn most probably represent miracidia which have not lost their ciliated coat. This most unusual succession of generations may represent a misinterpretation on Nicoll’s part, but then it may represent an aberrant life cycle pattern somewhat

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similar to that of Parvatrema homoeotecnum as reported by James (1964) and speculated on by Cable (1965) and Cheng (1967). It is certainly in need of verification. It should be pointed out that this unusual sporocyst ” may not be a larval trematode at all, but is a viviparous rhabdocoel turbellarian as suggested by Linton (1910). According to Nicoll, this parasite is commonly found in Cardium edule and Mytilus edulis but not as frequently in Mactra stultorum. There is rarely more than six in each cockle. ((

41. Acanthoparyphium spinulosum Johnston, 1917

(Superorder Anepitheliocystida; order Echinostomida; suborder Echinostomata; superfamily Echinostomatoidea; family Echinostomatidae)

Since the completion of this section, Mr. John W. Little of the Texas A & M University has informed me that he has found the larval stages of Acanthoparyphium spinulosum in Crassostrea uirginica near Port Isabel, Texas. A research note (Little et aZ., 1966) reporting this finding has since appeared. In this note, the authors reported that A. spinulosum metacercariae have been found in nearly 1 0 0 ~ ’of the oysters examined, embedded along the mantle. Of the fifty oysters examined in detail, each contained an average of forty-five metacercariae. Metacercariae were used to infect chicks and sexually mature adults were recovered on the eighth day. The adult of A. spinulosum is normally an intestinal parasite of shore birds and has been reported from Australia, Japan and California. Its life cycle has been studied by Martin and Adams (1961) and Bearup (1960) who found metacercariae encysted in marine gastropods.

C. Class Cestoidea Molluscs as a group are not common hosts for cestode parasites. In the exceptional cases where molluscs have been reported to serve as hosts for cestodes, their role is that of intermediate hosts. As far as I can determine, only three orders of cestodes, members of the subclass Eucestoda, include members which have been found in molluscs. Specifically, the order Tetraphyllidea includes two genera, Phyllobothrium and Pelichnibothrium, which in turn include species the larvae of which have been reported from cephalopods. The larva of P. speciosum has also been reported from teleost fishes. What has been identified as the larva of a species of a third tetraphyllid genus, Echeneibothrium, has been found by Sparks and Chew (1966) in Venerupis staminea, a pelecypod. The second order which includes ‘( species ” with larvae found in molluscs is the Lecanicephala. Among the members of this order, the

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genus Tylocephalum includes several species ” the larvae of which have been reported from pelecypods. According to Fujita (1943), a cestode tentatively identified as a member of the third order, Trypanorhyncha, has been found in “ oysters ’’ from Belgium. In addition, Herdman and Hornell (1903) were of the opinion that pearl-formation in Nargaritifera vulgaris in Ceylonese waters was caused by another trypanorhynch cestode designated as Tetrarhynchus zcnionifactor. However, later, Herdman (in Southwell, 1924) has stated : ‘(

I am now inclined t o think that the globular cysts in the liver of the Ceylon Pearl Oysters which Hornell and I found in 1902 and regarded as larval stages of a Tetrarhynchus and which were formally described by Shipley and Hornell in 1904 under the name Tetrarhynchus unionifactor, are-as Mr. Southwell says in the present paper-more probably to be referred to the genus Tylocephalum.

Thus the occurrence of this trypanorhynch in pearl oysters is no longer accepted. From the practical viewpoint, interest in cestode parasites of commercially important molluscs has centered primarily around the question: ‘‘ Can these parasites cause sufficient injury so as to result in death or a severely unhealthy state?” Nosatisfactory answers are yet available resulting from controlled experiments. From the standpoint of public health, again no definite answers are available. However, since the natural definitive hosts of those larval cestodes known from marine molluscs are all elasmobranch fishes, the possibility of their becoming established in humans is doubtful. Reviewed below are the three genera of cestodes which have been reported from commercially important marine molluscs. The classification employed is that of Yamaguti (1959). 1. Tylocephalum spp. Linton, 1890. (Figs. 168 and 169) (Order Lecanicephalidea; family Lecanicephalidae)

The presence of larval lecanicephalid cestodes in oysters and other pelecypods has been known since Herdman and Hornell (1906), Shipley and Hornell (1906), Jameson (1912) and Southwell (1924) reported their occurrence in Ceylonese waters, especially in the pearl oyster, Margaritifera vulgaris, and Seurat (1904, 1906) reported a species in M . margaritifera cumingi in the South Pacific. The original interest in these larval cestodes stems from Herdman’s (1903-06) hypothesie that they are one of the causes of pearl formation in oysters and that remains of such larvae could be found in the center of pearls. Although South-

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well (1924) supported this theory to some extent, Jameson (1912), in whose detailed study a complete account of the controversy is given, has pointed out the lack of evidence to support Herdman’s theory, including the demonstration of the absence of any structures remotely resembling tapeworm larvae in the center of pearls. It is now generally agreed that Jameson is correct since these globose larvae are found in the soft tissues on the interior of bivalves and not on the mantle, as are gymnophallid trematode metacercariae which do stimulate pearl formation, and hence could not stimulate the nacre-secreting mantle to form pearls. But, as Southwell (1924) has contended, if moribund Tylocephalum larvae fail to penetrate the oyster, they may stimulate pearl formation on the mantle surface. Proof of this, however, is wanting. At least four “ species ” of Tylocephalum larvae have been described from pelecypods. T . Zudificans was described by Jameson (1912) encysted in the connective tissue of Margaritifera vulgaris in Ceylon ; T . margaritiferae was reported by Seurat (1904, 1906) encysted in Margaritifera margaritifera cumingi from the Gambier Islands ; T . minus was reported by Jameson (1912) in M . vulgaris from Ceylon; and T. unionifactor was reported by Herdman and Hornell (1906) in M . vulgaris from Ceylon. In addition, Dollfus (1923b) has reported a Tylocephalum larva in Meleagrina occa and M . irradians from Nossi-BB, offthe coast of Madagascar. Among these, Southwell (1930)regarded T. 1udiJicans and T . minus as the larvae of and hence synonyms of T. dieranha, a species described by Shipley and Hornell (1906) from the elasmobranch Myliobatis maculata in Ceylonese waters. This has never been demonstrated experimentally. As Yamaguti (1959) has pointed out, the original descriptions of these larvae are so incomplete that they are of no value in identification. As the result, I have no choice but to refer to them collectively as Tylocephalum spp. It should be pointed out that the genus Tylocephalum was erected by Linton (1890) for T . pingue, an adult lecanicephalid found in the elasmobranchs Rhinoptera quadriloba and R. bonasus caught at Woods Hole, Massachusetts. Although it is generally agreed that the encysted larvae found in pelecypods are the immature forms of one or more species of Tylocephalum, attempts to recover adult tapeworms from sharks which had been fed these larvae have all failed (Jameson, 1912); hence, despite a morphological similarity between the holdfasts of the larvae and adults, doubt still exists as to whether the larvae are those of Tylocephalum. Relative to the designation applicable to the globose larvae found in marine pelecypods, Cheng (1966a) has stated :

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The intramolluscan larva of Tylocephalum could be considered a procercoid although a distinct myzorhynchus, a holdfast organ, is present and hence it is further along in its development than the typical procercoid. On the other hand, this larva develops from the coracidium and hence is not a plerocercoid according to the definition of Wardle and McLeod (1952). Until the complete life cycle of this cestode is known, the intra-molluscan larva is referred to herein as the metacestode according to the definition of Wardle and McLeod (1952). I n recent years, Tylocephalum metacestodes have been reported from the American oyster, Crassostrea virginica, from Pearl Harbor, Oahu, Hawaii (Sparks, 1963 ; Cheng, 1966a), and in C . virginica trans-

FIG. 168. Coracidium of Tylocephalum sp. removed from the gill surface of Crassostrea wirginica fom Oahu, Hawaii. (After Cheng, 19661%) GC, Germinal cells; M Z , myzorhynchus; PG, penetration gland ; Y , yellow spherules.

planted from Apalachicola Bay, Florida, t o Chinoteague Bay, Maryland (Burton, cited in Sparks, 1963 ; Burton, personal communication). It has recently also been found in Tapes semidecussatus in Hawaii (Cheng and Rifkin, 1967) . While examining washings of the body sufaces and histological sections of parasitized Crassostrea virginica from Hawaii, Cheng (1966a) has found and described the coracidium of this cestode. Description of stages in or on mollusc. Coracidium (Fig. 168) elongate, ovoid, slightly more blunt anteriorly than posteriorly, 0.24-0.27 mm (average 0.264 mm) long, 0.145-0.165 mm (average 0-160 mm) wide; covered with a layer of ciliated epithelium; cilia of uniform length, measuring 0.01 mm long ; prominent invagination a t anterior terminal, A.M.B.-5

17

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representing developing myzorhynchus ; few elongate flask-shaped unicellular glands a t anterior end, in region of invagination ; parenchyma filled with globular pale yellow bodies, each measuring 0.004-0.015 mm x 0.004-0-01 1 mm ; few large granular cells (germinal cells) scattered along periphery in posterior portion of body ; no typical cestode larval hooks present. The detailed histology of this coracidium has been given earlier (Cheng, 1966a). Metacestode (Figs. 11 and 169) primarily

FIG. 169. Metacestodes of Tylocephalrm teased from tissues of parasitized Crassostrea virginica from Oahu, Hawaii. Living material. (After Sparks, 1963.) (Photomicrograph courtesy of Dr. A. K. Sparks, University of Washington.)

within capsules of host tissues in region surrounding the alimentary tract but may occur elsewhere in the host’s body, particularly in digestive gland and gill matrices ; body pear-shaped, broader anteriorly, approximately 0-7-1.5 mm long x 0-5-1.0 mm wide; with globose protrusible muscular myzorhynchus a t anterior end. Life cycle. As free-swimming coracidia have been reported to occur in the alimentary tract and on the gill surfaces of Crassostrea virginica (Cheng, 1966a), it can be assumed that the eggs laid by the adult and passed out from some still undetermined definitive host, possibly an

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elasmobranch, hatch in water. The ciliated coracidium is swept up with other planktonic organisms during the oyster’s feeding process and is ingested or becomes attached to the gill surfaces. It sheds its ciliated coat prior to penetrating through either the alimentary wall or through the gill surface, develops into a metacestode, and eventually is immobilized when the host forms a capsule around it. The remainder of its life cycle remains undertermined. Other information. Both Sparks (1963) and Cheng (1966a) have studied pathological changes in parasitized Crassostrea virginica (see Chapter 4, Section 11, A, 2). Since it has been found that encysted metacestodes are eventually resorbed in C. virginica, I have concluded that this oyster is probably not the natural intermediate host. Further evidence for this has recently been uncovered while examining parasitized Tapes semidecussatus. I n this clam, the host’s cellular reaction is even more severe than in Crassostrea virginica, and resorption of the parasite is commonly observed (Cheng and Rifkin, 1967). 2 . Echeneibothrium sp. van Beneden, 1850. (Figs. 170 and 171)

(Order Tetraphyllidea; family Phyllobothriidae) The genus Echeneibothrium was erected by van Beneden (1850) to include E. minimum, a parasite found in the spiral valve of elasmobranchs. Since then, a t least thirty-five species of adults have been described, all from elasmobranchs (Yamaguti, 1959). Hyman (1951) has depicted the larval stage (plerocercoid) of an undetermined species of Echeneibothrium encysted in the foot of a “ clam ” collected in Puget Sound. This larva may be the same as that reported by Sparks and Chew (1966) encysted throughout the tissues of the littleneck clam, Venerupis staminea (Figs. 170 and 171), collected in Humboldt Bay, California, which they have indicated to be similar to E. myxorhynchum although minor differences exist. Description of plerocercoid (the stage found in molluscs). Body elongate, tapering towards posterior terminal ; with apical sucker (or opening) on presumably extensible muscular rostellum (referred to by some as a myzorhynchus) and four protruding broad and trumpet-like bothridia ; each bothridium with ten loculi (or areolae) (according to Hart, 1936; Sparks and Chew, 1966) or eighteen loculi formed by a single longitudinal and eight transverse septa plus one locule a t each terminal (according to Riser, 1955) ; pedicels and abothrial surfaces of bothridia covered with small hooks. L(fe cycle. The life cycle of Echeneibothrium has not been demonstrated. It is believed that plerocercoids in molluscs are taken in by

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the elasmobranch definitive host when the latter feeds on clams (Wardle and McLeod, 1952 ; Sparks and Chew, 1966). Sparks and Chew have suggested that the bat stingray, Holoyhinus californicus, may be the definitive host. Pathology. According to Sparks and Chew, in histological section, each plerocercoid larva is surrounded by " a compact network of fine

FIG. 170. Cross-section of entire clam, Venerupia staminea, from Humboldt Bay, California., showing numerous encysted Echeneibothriuln sp. throughout tissues. (After Sparks and Chew, 1966.) (Photograph courtesy of Dr. K. K. Chew, University of Washington.)

collagenous fibers and numerous leucocytes ". Since these investigators examined sections stained with hematoxylin and eosin, it cannot be certain whether the fibers are of collagen. Between the cyst wall and parasite are found amorphous materials and occasional clumps of host leucocytes. Grossly, cysts appear as small yellowish bodies which are most conspicuous in the mantle and foot. Parasitized clams are said to be exposed on gravel beds rather than buried and thus presumably rendering them more vulnerable to predation by elasmobranchs.

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PIG. 171. Single encysted Eeheneibothrium sp. in the clam Ven,erupis stamilzetl from Humboldt Bay, California, showing fibrous encapsulating cyst of host origin. (After Sparks and Chew, 1966.) (Photomicrograph courtesy of Dr. K. K. Chew, University of Washington.)

3. Eutetrarhynchus ruficolle Eysenhardt, 1829‘2 (Order Trypanorhyncha; suborder Acystidea; family Eutetrarhynchidae)

Fujita (1943) reported : “ From the internal organs of oysters from Belgium oyster beds the cestode Tetrarhynchus rujicollis Eysenhardt has been reported, but it is uncertain whether this identification is correct.” From the writings of Joyeux and Baer (1936) and Dollfus (1942), it would appear that this identification is in error since T. ruficollis, now known as E. rujicolle, which is found in the elasmobranchs Mustelus, Acanthias and Raja, utilizes crustaceans, Eupagarus, Hyas, Portunus, Carcinus, Inachus, Cancer, Stenorhynchus and Pilurnnus, as the intermediate host. The possibility exists, however, that the Belgian “ oysters ” are aberrant hosts.

CHAPTER -7

PARASITES OF COMMERCIALLY IMPORTANT MARINE MOLLUSCS THE PHYLA NEMERTINEA, ASCHELMINTHESAND ANNELIDA I . Phylum NEMERTINEA A number of nemerteans belonging to the genus Malacobdella have been reported from the mantle cavities of clams. Malacobdella is the sole genus in the order Bdellonemertea and to date six species have been described although, as Coe (1945) has pointed out, two of these, M . obesa and M . rnercenaria, both described by Verrill (1892b), are specifically identical with M . grossa. Hence only four recognized species exist (Table XIV).

TABLEXIV. SPECIESOF NEMERTEANS KNOWN FROM MARINE MOLLUSCS P, reported from Pacific coast of North America; A, reported from Atlantic coast of North America; E, reported from Europe; J, reported from Japan; C , reported from Chile.

Nemertean species

Host

Malacobdella grossa

Siliqua patula (P)

Malacobdella japonica Malacobdella auriculae Malacobdella minuta

Mactra secta (P) Mactra stultorum (E) Mercenaria mercenaria (A, E ) Mercenaria campechiensis (A) Venus praeparca (A) Venus ezoleta (E) M y a arenaria (A, E ) M y a truncata (E) Pholas crispata (E) Cyprina islandica (E) Cardium aculeatum (E) Isocardia cor (E) Mactra sachalinensis (J) Chilina dombeiana (C) Yoldia cooperi (P) 286

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Although earlier workers (van Beneden and Hesse, 1863 ; Semper, 1877 ; Verrill, 1892b ; Burger, 1895) have regarded these as parasites, subsequent workers (Burger, 1907 ; Guberlet, 1925 ; Coe, 1943, 1945 ; Hyman, 1951; Barnes, 1963; Cheng, 1964b) have stated that these nemerteans are definitely commensals which feed on food brought in by the molluscs’ incurrents. Furthermore, Guberlet (1925) has pointed out that the lesions sometimes found on pelecypods harboring Malacobdella, as has been reported by McMillin (1924) and others, cannot be attributed to these nemerteans. There is no reliable evidence to indicate that Malacobdella spp. are in any way injurious to their hosts.

11. Phylum ASCHELMINTHES Class Nematoda Among the aschelminthes, only a few species of nematodes have been reported from commercially important marine molluscs although they have been recorded, either as larvae or as adults, from a number of terrestrial, freshwater, and a few commercially unimportant marine molluscs (see Pelseneer, 1928 ; Gerichter, 1948). Relative to those nematodes sometimes encountered during the routine examination of shellfish, the question invariably arises as to whether these are true parasites or are free-living species which have become temporarily attached. That nematodes are fairly commonly found on the surfaces and in the mantle cavity of marine molluscs is beyond doubt. The possibility also exists, as with certain protozoa and arthropods which have been reported from moribund pelecypods, as to whether the nematodes found in unhealthy molluscan shellfish are obligatory parasites, facultative parasites, or predators which have invaded unhealthy, moribund and dead molluscs. The sparsity of reports on nematodes parasitic in commercially important marine molluscs would suggest that nematodes rarely occur in these hosts. As to whether this is true or not cannot be stated since perhaps the situation merely reflects the lack of competent nematode taxonomists among marine parasitologists. For example, various workers (personal communication) have found nematodes in histological sections of certain pelecypods, especially oysters ; however, no attempts have been made to identify or study the biology of these. My former associate, R. W. Burton (1961), has found cross-sections of an unidentified nematode in several specimens of Crassostrea virginica collected at Marumsco Bar, Pocomoke Sound, Clay Island Bar, and Malls Bar, Tangier Sound, all in Worcester County, Maryland, during 1960. I n addition, Burton (unpublished) has found unidentified

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nematodes in C. virginica collected in St. Mary’s Creek, St. Mary’s County, Maryland. I n all instances, the nematodes were tightly coiled in tissues in the region of the digestive diverticula (Fig. 172). When observed in cross-section, each worm measures approximately 0.075 mm in diameter. There appears to be little doubt that these are true endoparasites ; however, the obligatory or facultative nature of their association with oysters remains undetermined.

FIG. 172. Section of the oyster Grassostrea wirginica from Maryland showing cross- and partial longitudinal sections of an unidentified nematode in Leydig tissue. Notice leucocytosis in area surrounding nematode. (Section from which the photomicrograph was taken was kindly loaned by Mr. R. W. Burton, Rhode Island Fish and Game Division.)

Another known instance of an unidentified nematode parasite of a commercially important marine mollusc is the one encountered by Vadel (1855) in the scallop Pecten (=Aequipecten). Vadel has given a rather incomplete description of this nematode so that positive identification is not possible. He has merely stated that it was a larva, 3.5 cm long and 0.5 mm wide, tapering a t both ends but with a blunt anterior and a pointed posterior extremity. A coiled punctate organ, barely visible to the naked eye, is present near the anterior extremity. When observed in the living state, this nematode executes a variety of movements while rapidly coiling and uncoiling.

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The few definitely identified species of nematodes from commercially important marine molluscs have all been juveniles or larvae. These are reviewed below. 1. Echinocephalus uncinatus Molin, 1858(‘1)

(Subclass Phasmidia; superfamily Spiruroidea; family Gnathostomatidae) Molin (1858)erected the genus Echinocephalus to include E. uncinatus the larvae of which are found in the spiral valves of the ray Trygon brucco in the Adriatic Sea. The original description was based on two distinct types of specimens. Molin (1858, 1861)designated one specimen, 7.0 mm long with six rows of cephalic hooks, as the female, and two others, 24-35 mm long with thirty rows of hooks, as males. Baylis and Lane (1920) considered Molin’s males t o be identical with another species, E. spinosissimus, and the female t o be distinct and selected it as the true E. uncinatus. More recently, Millemann (1963), who has contributed a thorough study of this genus, stated : It is now clear, as shown in the present study, that the early juvenile stages of E. pseudouncinatus, and probably of other species as well, have 8 rows of of hooks (the first 2 rows are easily overlooked unless en face preparations are studied and this would account for the reports of juveniles with only 6 rows of hooks). The definitive number of rows is attained at the third-stage molt. This evidence, and t h e fact that adults of E. uncinatus have never been found, strongly suggests that Molin’s “ female and all other specimens with 6 (8Z) rows of hooks assigned to the species uncinatus by different authors. . . are actually early juvenile stages of known or unknown echinocephalids. ”

Furthermore, Millemann has designated Molin’s male as the true E. uncinatus and considered E . spinosissimus to be its synonym. Earlier, von Linstow (1904) had found what he assumed to be the larva of E . uncinatus encysted in the adductor muscle of the pearl oyster, Margaritifera vulgaris, in Ceylonese waters, and Baylis and Lane (1920) had reported this larva in the pelecypod Pinna sp. and immature adults in the sting-ray, Myliobatis nieuhoji, in Ceylon. I n addition, Hopkins (1935) has described what he believed to be the larva of E . uncinatus collected by Van Cleave in the gonad of the sea urchin, Arbacia punctulata, a t Woods Hole, Massachusetts, and Johnston and Mawson (1945a) have reported this larva from Polinices conica and Katylesia scalarina from Australian waters. As the result of Millemann’s (1963) studies, it may now be said that the larval echinocephalids found by von Linstow, Baylis and Lane, Hopkins, an Johnston and Mawson cannot be definitely designated as E. uncinatus

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Adults of the four currently recognized species, E. uncinatus, E. multidentatus, E . southwelli and E. pseudouncinatus, have all been reported from elasmobranchs (see Millemann, 1963). Description of larva in mollusc (of Echinocephalus spp.). Body 10-0-14.5 mm long, coiled, approximately 0.4-0-45 mm in diameter in anterior portion, 0.7 mm in diameter in posterior portion; lips not developed in Baylis and Lane’s specimens from Pinna sp. but with six lips a t anterior terminal (trilobed in adults) in von Linstow’s specimens from Margaritifera vulgaris; with six (eight 2 ) rows of forty to fifty hooks per row on slightly bulbous head region with each row forming a complete circle ; hooks of each row of approximately same size, measuring 0.047-0.057 mm long ; distances between hooks range from 0.058 to 0.068 mm ; cuticular striation fine ; esophagus, approximately one-sixth of body length, and long well-developed intestine, occupying almost all of pseudocoelom ; anus ventral and subterminal. (For descriptions of adults, see Millemann (1963).) Life cycle. The life cycle of E. uncinatus remains undetermined experimentally. Von Linstow (1904) and Baylis and Lane (1920) have expressed the opinion that the larvae which they found in pelecypods would develop to maturity when parasitized molluscs are ingested by some elasmobranch definitive host. Since the life cycle of the related species, E . pseudouncinatus, is known, a t least as determined from collections of developmental stages from naturally infected hostjs (Millemann, 1963), and the pattern is essentially that postulated by von Linstow and Baylis and Lane, it appears that only one intermediate host, a mollusc, is involved. Earlier reports by von Linstow (1904) and Johnston and Mawson (1945b) of larvae in teleost fishes suggest that these fishes may serve as transport (paratenic) hosts. The occurrence of larvae in sea urchins most probably represents an unnatural condition. Furthermore, since what appears to be second-stage larvae have been reported from elasmobranchs (Molin, 1958 ; von Linstow, 1904 ; Baylis and Lane, 1920; MacCallum, 1921), this may be indicative that infection of the definitive host can be acquired from the ingestion of second-stage larvae. 2. Echinocephalus pseudouncinutusMillemann, 1951. (Figs. 173 and 174) (Subclass Phasmidia; superfamily Spiruroidea; family Gnathostomat)idae) As the result of examining pink abalones, Haliotis corrugata, from Pyramid Cove, San Clemente Island, in southern California, Millemann (1951) described the larva of another species of Echinocephalus, E. pseudouncinatus, encysted in the foot of this gastropod. Later (1963),

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he reported its life cycle and included descriptions of the larval and adul t stages. Description of stage in mollusc. Millemann only found second-stage larvae in Haliotis corrugata. Second stage larva, 18-21 mm (average 20 mm) long, 0.614-0.704 mm (average 0.651 mm) wide; with eight rows of hooks on head (Fig. 173 and 174), first two rows incomplete, with lateral separation of these small rudimentary hooks ; third row almost complete ; two hooks per side in first row, six or seven per side in second row ; last six rows with twenty-seven, thirty-nine, forty-four, forty-four, forty-four and forty-four hooks respectively ; hooks increase

FIGS.173 and 174. Echinocephalus pseudouncimtus. (173) Anterior end of second-stage larva, lateral view ; (174) en face view o f head of second-stage larva. (Redrawn after Millemann, 1963.)

in size from first to eighth row, smallest hooks 0.008-0.014 mm (average 0.0082 mm) long, largest hooks 0.0336-0.0464 mm (average 0.0406 mm) long ; distance between rows of hooks 0.016-0.033 mm (average 0.025 mm) ; dorsal and ventral hooks in each row slightly smaller than lateral ones ; two lips present (Fig. 174), each with two papillae, not divided into lobes; no teeth; head, 0.270-0.331 mm (average 0.307 mm) long by 0.380-0.442 mm (average 0.411 mm) wide, with four ballonets connected to four cervical sacs; tail blunt, 0.152-0.235 mm (average 0.185 mm) long, with small papilla a t terminal ; cervical papillae, caudal alae, and caudal papillae absent ; reproductive organs absent. It is of interest to point out that Millemann has stated that : These juveniles (second-stagelarvae) are similar to third-stage juveniles in the number of rows of hooks, the number of hooks per row, and the arrangement of the hooks. They differ in being slightly larger, the hooks are longer [italics mine], the lips are not trilobed and have two papillae and no teeth, and in the shape of the tlail.

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As to why the second-stage larva should be larger and its hooks longer than the older third-stage larva is puzzling. Life cycire. Millemann (1951, 1963) has described the second stage larva of E. pseudouncinatus from Haliotis corrugata, and (1963) the third- and fourth-stage larvae plus the adult from the horned shark, Heterodontus francisci and the bat sting-ray, Holorhinus ( = Myliobatis) californicus. Thus, although morphological and survey data strongly suggest that the larva in the abalone is the second-stage larva of the stages found in the elasmobranchs, the life cycle pattern reported by him is not based on experimental feeding experiments but from piecing together stages collected from naturally infected hosts. The elasmobranch hosts from which Millemann’s specimens were collected were caught a t San Francisco Bay and Puerto Refugio, Angel de la Guarda Island, Gulf of California, Mexico. It is not known whether E. pseudouncinatus is an ovoviviparous or an oviparous nematode, although the pattern in the family Gnathostomatidae, as exemplified by Gnathostoma spp., includes oviposition and a free-swimming first-stage larva that hatches after the eggs remain in water for a period of time (Miyazaki, 1960). It is known, however, that abalones become infected, not through the ingestion of eggs or first-stage larvae, but when what is assumed to be first-stage larvae burrow into the foot. Ecology. Millemann (1951) has reported that only old abalones are parasitized. It is not known if this indicates that younger ones are refractile or if the parasite for some unknown reason prefers older abalones. Pathology. Millemann (1951) has demonstrated that the larvae burrow into the foot of Haliotis corrugata where they encyst in the ventral portion, producing a blister-like effect on the exterior of the foot. Furthermore, he has noted that the vesicatory effect of encysted larvae in the host’s foot, coupled with the burrowing of the larvae prior to encystment, apparently weakens the foot musculature and decreases its efficacy as a hold-fast structure. As the result, parasitized abalones can be removed from rocks with ease while non-parasitized ones hold on more rigidly. Other information. Although Millemann (1951) did not find encysted larvae in the few specimens of the southern green abalone, Haliotis fulgens, which he examined, he did report that : However, reliable reports from the divers and the processing plants indicate that the green abalone is as susceptible to this parasite as the pink abalone, an indication that the parasite is not host specific.” (I

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3. Porrocaecum pectinis Cobb, 1930. (Figs. 175 and 176)

(Subclass Phasmidia; superfamily Ascarididea; family Heterocheilidae) Cobb (1930) described a new nematode, Paranisakis pectinis, based on a single immature specimen found in the visceral mass of a scallop, Pecten ( = Aepuipecten) collected a t Beaufort, North Carolina. Gutsell (1930) found what he considered to be the same nematode in Aequipecten mazimus collected from the same location. Hutton (1964), while examining the bay scallop, A . gibbus, collected off the east coast

FIGS.176 and 176. Porrocaecum pectinis. (175) Lateral view of anterior portion of larva; (176) en face view o f anterior terminal of larva. (Redrawn after Cobb, 1930.) A M , Amphid ;C, caecum ;CC, cephalic constriction ;ES, esophagus ;I N T , intestine ; N R , nerve ring; P A , papilla; S P , submedian papilla; V E N , ventriculus.

of Florida, found numerous specimens of an immature nematode which he believed to represent Cobb’s species. He noted, however, that a short anteriorly projecting caecum is present and for this reason he transferred P. pectinis to the genus Porrocaecum. The genus Porrocaecum was erected by Railliet and Henry (1912) to include P. crassum found in the small intestine of various birds, Anas, Numidn, DaJla, Cairina and Grus, in Europe. Since that time, various other species have been described. Yamaguti (1961) lists thirty-nine recognized species, one a parasite of amphibians, two of reptiles, thirty-one of birds, and five of mammals.

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Description of stage in mollusc. Cobb (1930) has stated that his description " is probably inadequate for recognizing the nema. . . ,, This is especially true of the measurements since it is not known at what stage the larva is in (M. Chitwood, personal communication); however, as Cobb has pointed out, the description may be of value to call attention to the fact that such a nematode has been found parasitic in Aequipecten. Body (Fig. 175) 16.3 mm long, 0.11 mm wide a t level of mouth, 0.20 mm wide a t level of nerve ring, and 0.13 mm wide a t level of anus ; nearly colorless ; cuticle with regular transverse striations, with striae 0.008 mm apart a t base of " neck " ; nerve ring 0.36 mm from anterior end; distance from anterior end to base of esophagus 1.61 m m ; esophagus delimited from ventriculus by collum ; with short anteriorly projecting caecum connected to posterior terminal of ventriculus ; with three lips (Fig. 175); distinct wings (alae?) beginning on head, near base of lips, and extending along length of body; excretory pore at base of lips ; anus 16-02 mm from anterior end. Other information. No other information is available on P.pectinis. 4. Angiostrongylus cantonensis Chen, 1935. (Figs. 177 and 178)

(Subclass Phasmidia; superfamily Strongyloidea; family Metastrongylidae) Angiostrongylus cantonensis is a metastrongylid lungworm of rats. Mackerras and Sandars (1955) have shown that this nematode utilizes slugs as the intermediate host. Since then, a number of molluscs, primarily gastropods, have been shown to be suitable intermediate hosts (see Cheng and Alicata, 1965 ; Alicata, 1965). Normally marine pelecypods are not infected by this parasite, a t least surveys of clams and oysters from endemic areas have not revealed natural infections. 1 However, under certain ambient conditions, Cheng and Burton ( 1 965a) have shown that both the young quahaug, M e r c e n a r i a mercenaria, and the American oyster, Crassostrea v i r g i n i c a , can be infected experimentally although the percentage of recovery of third-stage larvae is very low (Cheng, 1966b). Again, this is not a natural parasite of salt-water clams and oysters, but it is briefly mentioned here since these pelecypods are known to be suitable experimental intermediate hosts. A review of the literature pertaining to the role of A . cantonensis as a causative agent for one type of meningoencephalitis in man in the Pacific and Southeast Asia has been given by Alicata (1965). It has been noted (p. 12) that recently Kiiapp and Alicata (1967) have reported that they were unable to infect Crassostrea virginica and Venerupis philippinarum (= T a p e s semidecussuta) from Hawaii

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with Angiostrongylus cantonensis larvae. Considering their experimental procedure, their negative results are not surprising. Relative to their attempt to infect C. virginica, their failure to appreciate the ecology and functional biology of oysters as related to their feeding habits most probably was responsible for their negative results. It has been shown that infection of C. virginica from New England only occurs if first-stage A . cantonensis larvae are ingested, followed by successful penetration of the host’s alimentary wall (Cheng, 196613). I n the trials by Knapp and Alicata, they failed to appreciate this and did not

FIG. 1 7 7 .

Section of the oyster Crassostrea v i r g i n k a with larva of Angiostrongy~ue cnntoiaensis in blood vessel. The oyster was experimentally infected with first-stage larvae and examined on the third day post-infection.

provide adequate precautions to ensure pumping on the part of the oysters. Experiences by various investigators working with oysters, and other marine pelecypods for that matter, have indicated that it is extremely difficult to maintain estuarine pelecypods, particularly oysters, in small aquaria under artificial conditions and expect pumping, especially if the molluscs had been subjected to traumatic treatment. It is well known to marine biologists that pumping activity is an essential part of the oyster’s feeding mechanism. Furthermore, oysters almost invariably must rest on their cup-shaped or left valve as a prerequisite to pumping. I n our earlier experiments carried out

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in Rhode Island, great care was taken to ensure that the natural sea-water system in which the oysters were maintained was condusive to normal pumping and actual pumping was observed. It is not known whether the failure by Knapp and Alicata to infect Tapes semidecussata reflects true incompatibility or another example of failure to appreciate the biology of marine molluscs. T . semidecussata is normally buried in sand in its natural environment, generally oriented

FIG. 178. Anterior portion of third-stage larva of Angiostrongylus cantonensis showing characteristic sclerotized stomatorhabdions (8)a t anterior end. This is the oldest stage found in the molluscan host and is the one infective for mammals. (Photomicrograph by courtesy of Dr. J. E . Alicata, Agricultural Experiment Station, University of Hawaii.)

in a more or less anterior-posterior upright position, and when actively feeding its siphon is protruded and directed upwards. I n their experiment, Knapp and Alicata maintained their specimens horizontally in a glass aquarium without sand, thus feeding, if it occurred at all, was impeded. I n addition to the experimental errors cited, a further explanation for their failure to infect C. virginica should be considered. It is well known that different strains of this oyster exist (Coe, 1934, 1938; Loosanoff and Engle, 1942 ; Nelson, 1947 ; Loosanoff and Tommers,

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1948; Stauber, 1947, 1950a; Loosanoff and Nomejko, 1951 ; Hillman, 1964 ; and others), even along a relatively similar zoogeographical range such as the Atlantic coast of North America. Since strains, varieties, genetic races, or physiological species, terms commonly employed to designate morphologically similar but functionally different organisms (see Bates, 1940), are generally accepted as possible manifestations of speciation, and since geographic isolation is recognized as an important factor in the origin of new species, it would not be a t all surprising, I think, to find that the Hawaiian “ variety ” of C . virginica is physiologically distinct from the New England “ variety ”. Thus, it is possible that if the incompatibility of Hawaiian C . virginica is real, it may be due to strain differences. Indeed, differences in susceptibility to zooparasites in C. virginica, for example to Minchinia nelsoni, is well known. Relative to this point, Stauber’s (1950) statement pertaining to observed differences among strains of marine molluscs should be heeded with great attention. He has stated : The presence of such physiological species, each the result of selection over long periods of time in situations almost completely isolated from each other and with quite different hydrographical conditions, might explain, for example, some of the other differences already reported between oysters from different areas. Caution is suggested in generalization on the basis of observations on a single variety under study until the nature of these temperature (and other) variants is adequately disclosed.

111. Phylum ANNELIDA Several species of polychaetous annelids are known to live in association with marine molluscs (see Clark, 1956). Among these, some have been found associated with molluscs of commercial importance. Relative to the nature of the relationship between these annelids and their hosts, Clark has pointed out : Although some worms are almost always, or even exclusively, t o be found in more or less intimate association with other animals, very little structural adaptation appears to have taken place t o suit them t o this mode of life. Whether or not physiological adaptations have been evolved remains unknown for the most part. Loosanoff and Engle (1943) are more definite in their statement. They are of the opinion that at least the members of the genus Polydora are definitely not parasites. Until more precise information becomes available, these annelids are only briefly mentioned herein. The best known of the polychaetes associated with marine molluscs of commercial importance are the so-called mudworms of the genus A.l-1.B.-5

18

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Polydora. A number of species have been reported in U-shaped burrows beneath the nacrous layer of the pelecypods’ shells, primarily oysters. The so-called blister, which is actually a large excavation over which a thin layer of nacre has been laid down, is almost always filled with TABLEXV.

POLYCHAETES KNOWN TO BE ASSOCIATED WITH COMMERCIALLY IMPORTANT MARINE MOLLUSCS The references listed by no means include all the published works on these annelids but should be sufficient in initiating a search of the literature. (Data partially after Clark, 1956.)

Polychaete spp. Capitellidae Capitella ovincola

Capitomastus minimus Polynoidae Arctonoe vittata Eunicidae Eunice harassi Spionidae Polydora ciliata

Molluscan host

Loligo vulgaris (in egg masses)

Hartman, 1947 McGinitie and McGinitie, 1949 McGowan, 1954 Harant and Jecklin, 1933

Haliotis kamtschatkana

Clark, 1956

Ostrea edulis

Gravier, 1900

Ostrea edulia Mytilus edulis

Clark, 1956 Lebour, 1907b Field, 1922 Lunz, 1940, 1941 Kavanagh, 1940 Whitelegge, 1890 Roughley, 1922, 1926 Clark, 1956 Clark, 1956 Takahashi, 1937 Haswell, 1886 Loosanoff and Engle, 1943 Owen, 1957 Davis, 1967 Mortensen and Galbsoff, 1944

Loligo opalescens (in egg masses)

Crassostrea virginica Crassostrea gigas Oysters (Australian) Polydora hoplura

Reference

Polydora pacijca Polydora polybranchia Polydora websteri

Ostrea edulis Mytilus spp. Pinctada margaritqera Oysters Crassostrea virginica

Polydora ligni

Mesodema deauratum Crassostrea virginica

mud in which the polychaete is found. Commonly small channels lead from each main blister, via which the polychaete migrates within the shell. The mud blisters almost invariably continue to the outer edge of the shell and communicate with the exterior. The mechanisms

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involved in blister-formation have been reported by Whitelegge (1890), Kavanagh (1940) and Lunz (1941). I n addition, Lankester (1868) has suggested that the polychaete can secrete a strong acid which accounts for the tunnels which they burrow in the shell. Relative to the effects of Polydora on oysters, Whitelegge (1890) and Roughley (1922, 1925), who worked in Australian waters, have reported that these polychaete pests can cause heavy mortalities among oysters. On the other hand, Lunz (1941) and Loosanoff and Engle (1943), working along the east coast of the United States, have reported that even heavy infestations do not affect the growth or health of oysters, although severe shell markings, abnormal appearance of the mantle, and occasional damage to the adductor muscle may occur (Mackin and Cauthron, 1952). These apparently opposing viewpoints may well reflect species differences. Whitelegge and Roughley studied P. ciliata although Wilson (1928) has pointed out that the polychaetes examined by Whitelegge may be P. ligni. Loosanoff and Engle studied P. websteri. On the other hand, Korringa (1951a) has reported that P. ciliata does not inflict serious injuries on Ostrea edulis in Dutch waters and is of the opinion that the injurious species are P. websteri and P. hoplura. Obviously then, the effect of Polydora on oysters is still in need of further investigation. The species of polychaetes known to be associated with the shell of commercially important molluscs are listed in Table XV.

CHAPTER 8

PARASITES OF COMMERCIALLY IMPORTANT MARINE MOLLUSCS THE PHYLUM MOLLUSCA I. Phylum MOLLUSCA Several species of molluscs belonging to the family Pyramidellidae have been reported as parasites of commercially important marine molluscs. It is the general opinion that more undoubtedly will be found, but progress relative to these parasites has been comparatively slow, the prime reason being that the taxonomy of these gastropods is in such a state that it has discouraged many from working with this group. Their very small sizes undoubtedly have also discouraged some. The pyramidellids are all parasites, with molluscs and annelids being the most common hosts. Others have been reported as parasites of poriferans, cnidarians, sipunculids, crustaceans, echinoderms, hemichordates and tunicates (Robertson and Orr, 1961). Although Laserson (1951, 1959), Sanders (1958) and Robertson and Orr (1961) have suggested that certain pyramidellids may not be parasites since they have been found free on piles, in cracks, under stones and rocks and on algae, Ankel and Christensen (1963) have pointed out that the majority of the species often do leave their hosts between feedings, and hence are found detached and free-living. Indeed, the majority of pyramidellids have been found detached from their hosts. Another argument raised by Sanders (1958) in favor of his belief that Turbonilla is not a parasite is that from his benthic samples taken in Buzzards Bay, Massachusetts, he found possible hosts in insufficient numbers to account for the numbers of Turbonilla found in the bottom communities. But, as Robertson and Orr (1961) and Ankel and Christensen (1963) have correctly pointed out, Sanders inferred that Turbonilla is host-specific. This need not be the case and hence Turbonilla,during its parasitic phases, could feed on many of the species of molluscs, or even other invertebrates, present. Furthermore, the specialization portrayed by the gut of pyramidellids strongly suggests that they are fluid feeders, thus they are dependent on the body fluids of hosts for nutrition. 278

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Fretter and Graham (1949) have strongly emphasized that pyramidellids are host-specific. However, various subsequent investigators (Cole, 1951b; Cole and Hancock, 1955; Allen, 1958; Ankel, 1959; Wells and Wells, 1961 ; Ankel and Christensen, 1963), as the result of finding widespread absence of host-specificity, have challenged this concept. Indeed, the currently accepted opinion is that the pyramidellids are not as host-specific as believed by Fretter and Graham, although both Odostomiu scalaris and 0. impressa, and perhaps others, do portray a certain degree of host preference (Boss and Merrill, 1965). All of the pyramidellids are ectoparasites since they are generally attached on the exterior, near the edges of the valves of pelecypods, while inserting their long proboscis into the hosts’ soft tissues and feed on blood or tissue fluids (Fig. 179). Other species, such as Udostomia

FIQ. 179. Odostomia scalaris feeding on small specimens of Mytilus edulis by inserting their probosci. (Redrawn after Fretter and Graham, 1962.)

tellinae, live within the mantle cavities of pelecypods but never within tissues. As far as is known, all of these snails possess a highly specialized feeding mechanism, with a piercing stylet and esophageal pump (Ankel, 1949 ; Fretter and Graham, 1949 ; Maas, 1965). The taxonomy of the Pyramidellidae is in such a state that numerous doubtful species exist. The description of the majority of the species has been based on shells and nothing is known of their soft parts. However, of those species whose soft parts have been examined, these show a surprising degree of uniformity. Relative to shell characteristics, there appears to be little agreement as to which represent reliable specific characteristics and which are merely infraspecific differences.

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For example, the characteristics recognized by Dall and Bartsch (1909) in their monograph on the western American pyramidellids are considerably more conservative than those employed by Laseron (1951, 1959) in his papers on the species of Australia. No attempt is being made in this paper to resolve the taxonomic problems involved. For a good account of the morphology and development of pyramidellids, the reader is referred to Fretter and Graham (1949). As the pyramidellids are now recognized not to be as host-specific as originally thought, the species included in this review cannot definitely be said to be the only parasites associated with the hosts under consideration. Other yet unknown species will undoubtedly be found associated with commercially important molluscs. The following are those species which have been reported from such molluscs. I n addition to the specific characteristics given for each species under consideration, it is noted that the species of Odostomia all possess a tooth on the columella, i.e. a tooth-shaped projection on its lower end. According to Fretter and Graham (1962)) this " tooth " is " probably due to secretion of material over the surface of the columella by a mantle which has outgrown the space available for it and which has therefore become folded." Also, the shell includes a heterostrophic apex. 1. Odostomia impressa Say, 1822. (Figs. 180 and 181)

(Class Gastropoda; subclass Opisthobranchia; order Tectibranchia; family Pyramidellidae) Odostomia impressa has been reported by Hopkins (19564, Allen (1958) and Wells (1959) t o feed on Crassostrea virginica. It is not specific for this oyster since Allen has demonstrated that it will also feed on the prosobranchs Bittiurn varium, Crepidula convexa, Triphora nigrocincta and Urosalpinx cinerea, as well as a polychaete and the tunicate Molgula. Description. About 7 mm long, although younger specimens attached to oysters may be as small as 0.7 m m ; shell milky white, elongate, consisting of six or seven rather flattened whorls ; with channeled sutures ; surface with three equally spaced spiral grooves ; aperture oval, with outer lip thin and sharp, occasionally slightly flared in older shells. Ecology. According to Morris (1947), this species ranges from southern New England to the Gulf of Mexico and is commonly found on oyster beds. Hopkins ( 1 9 5 6 ~has ) shown that, unlike 0. bisuturalis, 0. impressa prefers large oysters. Furthermore : " Rough surfaces, such

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as the outside surfaces of oyster shells, attract or retain more snails than smooth surfaces, such as the inside surfaces of oyster shells.” That the attraction of oysters for 0. impressa is by its soft tissues also has been demonstrated by Hopkins. He has found that once the soft tissues of an oyster are removed, the snails attached to the exterior fall off and replacements are not attracted. Although Allen’s results indicate that 0. impressa is not hostspecific, one observation did suggest that it may demonstrate a certain degree of preference. She has found that this gastropod will not feed on Triphora nigrocincta if any of the other compatible hosts is available. Furthermore, Hopkins (1956~)has found that 0. impressa will not feed on either Mercenaria or Volsella. Even those few snails which crawl on to these pelecypods do not stay.

FIQS.180-183.

Odostomia spp. (180. 181) 0. impressa shells; (182, 183) 0. bkutulark shells. (Redrewnfrom photographs after Morris, 1947.)

Relative to the duration of the association between 0. impressa and Crassostrea virginica, Hopkins (1956~)has reported that at Bears Bluff, Wadmalaw Island, South Carolina, 0. impressa collected in the beginning of July ranged from 3.3 to 5.2 mm long (average 4.2 mm) but these older snails soon disappear and those collected at the end of July only measured from 0.7 to 3.3 mm long (mode 1.5 mm). Thus it would appear that there is a rapid replacement of older snails on hosts by younger ones. According to Wells (1959), 0. impressa lives but one year, being spawned the first summer, then spawning and dying the second. Hence the older snails collected by Hopkins during the first part of July most probably represented snails at the terminal of their life-span while the younger specimens collected towards the end of July represented snails which were spawned during July.

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2. Odostomia bisutularis Say, 1922. (Figs. 182 and 183) (Class Gastropoda; subclass Opisthobranchia; order Tectibranchia; family Pyramidellidae) Odostomia bisutularis, also referred to as Menestho bisutularis or 0. ( M . ) bisutularis, has been reported by Loosanoff (1956) to be a parasite of Crassostrea virginica a t Milford, Connecticut. Description. About 5-7 mm long, some smaller ; shell dull white, with a thin brownish periostracum ; with seven or eight whorls ; somewhat flattened and slightly shouldered at sutures; with a deeply impressed revolving line on upper part of each volution ; aperture oval ; columella bears one oblique fold ; operculum brown and horny. Ecology. 0. bisutularis is commonly found on sandy mud flats ranging from the Gulf of St. Lawrence to Florida. Loosanoff (1956) has stated that this gastropod shows a preference for young oysters, especially those found in shallow waters. Boss and Merrill (1965) have studied host-preference by 0. bisutuEaris. By permitting specimens removed from Crassostrea virginica collected near West Dennis, Massachusetts, to choose between the oyster and Crepidula fornicata, it was demonstrated that after 2 weeks, of eighteen 0. bisutularis, three became attached to C. virginica and the remainder were free on the substrate. In another experiment during which specimens of C. fornicata were placed between 0. bisutularis and its original host, C. virginica, so that the parasite had to by-pass C. fornicata in order to reach oysters, it was found that after 2 days of the twelve 0. bisutularis tested, two became attached to C. virginica, two to C. fornicata, and eight were on the substrate. I n another series of experiments, specimens of 0. bisutularis were given a choice between

C. virginica, Mercenaria mercenaria, Aequipecten irradians, Crepidula fornicata, C. plana, Crucibulum striatum, Modiolus rnodiolus, Arctica islandica and Placopecten magellanicus in different combinations. It was demonstrated that this parasite will become attached to all of the host species except C. plana, but it showed a behavioral preference for A. irradians, sometimes even to the point of ignoring its original host, C. virginica. From these studies, Boss and Merrill have concluded that although “ trends towards host-specificity are present and detectable, but some measure of non-specificity is retained.” They also have found that 0. bisutularis does spend considerable time off of hosts. Pathology. This is one of two species of Odostomia which has been reported to cause serious damage to their hosts. As more becomes known about other species, similar deleterious effects will probably be found. Relative to 0. bisutuhris, Loosanoff has stated :

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Although these snails may not be very successful in killing the oysters after the latter reach the size of about 1.0 cm, they, no doubt, interfere with the oysters’ normal development and growth. This is often shown by the characteristically deformed shells of the young oysters that came from areas heavily infested with M . bisutularis. The shells, instead of being flat, which is normal for shells of young oysters that are not too crowded, are deeply cupped and may have thickened edges. This abnormality is apparently due to injuries caused by the activities of M . bisutularis, to the edges of the oyster mantle, the organ that secretes the shell. 3. Odostomia seminuda Adams, 1839. (Fig. 184)

(Class Gastropoda; subclass Opisthobranchia; order Tectibranchia; family Pyramidellidae) Odostomia seminuda, considered by some to be a member of the subgenus Chrysallida, appears to be another North American species. Originally described by Adams (1839) from four specimens collected on single valves of Aequipecten irradians, it was rediscovered by Hackney (1944) from the upper valves of the same scallop and presumed to be a parasite. Subsequently, it has been found as a parasite on Crepidula fornicata by Robertson (1957) and on Aequipecten gibbus by Wells and Wells (1961). Description. About 4-7 mm long ; shell white, often glossy, stoutly conical, with six or seven whorls and distinct sutures ; sculptured with several revolving ridges and cut by vertical striations so that the surface appears beaded in most instances but consistently with four rows of tubercles (beads) on each whorl ; with a strong oblique fold or plait a t base of columella and within aperture. Ecology. 0. seminuda occurs from Prince Edward Island, Canada, to the Gulf of Mexico in North America. Although direct evidence is wanting, the observation by Robertson (1957) that 0. seminuda is consistently found on or near Crepidula fornicata although numerous Aequipecten irradians occur in the immediate vicinity suggests that it demonstrates a preference for the gastropod. This parasite is active and when not feeding may move around, sometimes away from the host for a short distance. Robertson has also reported that during the first half of August a t Woods Hole, Massachusetts, adults measuring up to 4 mm in length occur concurrently with juveniles, some less than 1 mm long, thus suggesting that spawning had recently taken place. Robertson’s hypothesis that 0. seminuda prefers C . fornicata over A. irradians, and perhaps other hosts, was tested in a series of behavioral experiments by Boss and Merrill (1965). They collected specimens of

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0. seminuda attached to C. fornicata from Bass River, an estuarine habitat near West Dennis, Massachusetts. When these 0. seminuda were detached from C. fornicata and given a choice between C. fornicata and Crassostrea virginica, after 2 weeks, of the fifty-eight specimens of 0. seminuda tested, forty were found in the feeding position on C. fornicata, their original host, and eighteen were wandering on the substrate. I n a second experiment during which specimens of C. virginica were placed between 0. seminuda and C. fornicata so that in order to reach its original host, 0. seminuda had to by-pass the oysters, it was found that after 2 days, of the thirty 0. seminuda tested, twentyfour were attached to C.fornicata and two to C. virginica. The remainder were free on the substrate. From these and similar experiments, Boss and Merrill have concluded that 0. seminuda prefers its original host, C. fornicata, but will exchange hosts to some degree. Furthermore, it has been determined that this parasitic gastropod does spend considerable time off of hosts. I n another series of experiments, Boss and Merrill (1965) have demonstrated that if 0. seminuda is exposed to various hosts, including Mercenaria mercenaria, Aequipecten irradians, Crepidula fornicata, C. plana, Crucibulum striatum, Crassostrea virginica, Arctica (= Pecten) islandica, Placopecten magellanicus and Modiolus modiolus, it prefers C. fornicata but will become attached in varying numbers to all of the other molluscs except A. islandica and C. plana. Furthermore, it shows some preferential behavior towards A . irradians, C. striatum and M . modiolus, especially if C. fornicata is not available. Pathology. Although nothing is known about the effects of Odostomia seminuda on the commercially important species of molluscs which it is known to parasitize, Merrill and Boss (1964) have examined the effects of this parasite on Crepidula fornicata and Crucibulum striatum. It has been noted that the parasite’s proboscis actually penetrates the host’s mantle and in some instances it actually pierces the visceral mass. The host responds to this penetration by muscular contraction in the immediate area. I n the same paper, Merrill and Boss have pointed out that 0. seminuda only feeds intermittently on its host.

4. Odostomia scalaris Mcgillivray, 1843. (Fig. 185) (Class Gastropoda; subclass Opisthobranchia; order Tectibranchia; family Pyramidellidae) Odostomia scalaris has been found by Ankel and Christensen (1963) feeding on the prosobranchs Hydrobia ulvae, Lacuna divaricata, Littorina saxatilis and Rissoa membranacea, and on the scallop Chlamys opercularis in Danish waters. Earlier, Pelseneer (1914) and others

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have recorded it in association with Mytilus edulis. While feeding on Chlarnp opercularis, it does not orient itself on the “ ears ” of the scallop as does 0. eulirnoides, but sits near the ventral edge of the shell as it does on Mytilus edulis. The specimens of Chlamys opercularis used during this study were small and, according to Ankel’s (1959) earlier report, it is perhaps doubtful if feeding could take place from its position on the shell on larger scallops. 0. rissoides, a species reported by Pelseneer (1914) as a parasite on Mytilus, is considered a synonym of 0. scdaris by Jeffreys (1867) and several subsequent authors. Description. About 4-5 mm long; shell white and polished, subconical, with five to six whorls ; each whorl slightly ballooned and with summit with strongly tabulated shoulder ;post-nuclear whorls similarly sculptured throughout ; spiral sculpture absent or as extremely fine microscopic lines of growth or striae ; varices absent ; with outer lip at edge of aperture ; with fold at base of columella. Other information. Ankel and Christensen (1963) have noted that 0. scalaris will feed on hosts which are smaller than itself; for example, Hydrobia ulvae and Rissoa membranacea. 5. Odostomia eulimoides Hanley, 1844 ( = O . pallida Montagu)*

(Class Gastropoda; subclass Opisthobranchia; family Pyramidellidae) This species was the first pyramidellid to be reported as a parasite. Jeffreys (1867) has stated that it occurs “ chiefly (if not only) on the ears of Pecten (=Chlamys) opercularis and P. (= Aequipecten) maxirnus, in the coralline zone ; it is widely distributed and rather common. The trawl-refuse a t Plymouth and Brixham is especially productive of this shell.” It has also been reported as a parasite of Pecten islandicus by Sars (1878), of P. jacobaeus by Kobelt (1886)) of Mytilus edulis by Marshall (1899), of Turritella comrnunis by Smart (1887), and of Xaxicava rugosa by Ankel(l959). Fretter and Graham (1949) and Graham (1955) have restated Jeffreys’ observations while Cole (1951b) has reported it as a parasite of oysters (most probably Ostrea edulis) in British waters. Cole and Hancock (1955) have reported it on Mytilus edulis in addition to Ostrea edulis. On both mussels and oysters this snail is found lodged in small pockets inside the ventral margin of the shell. The pockets had resulted from the withdrawal of the mantle in response to the irritation caused

* According to Jeffreys (1867),0. eulimoides is a synonym of 0. pall& and the letter name has priority. For some reason undetermined by me, the more recent authors have all designated this snail as 0. eulirnoidea.

by the snail’s proboscis during feeding. According to Cole, 0. eulimoides commonly occurs in pairs, but three may be found within the same pocket. Description. Body 3-6 mm long ; shell white, opaque, (with faint orange or purplish tinge a t apical end of living specimens) elongate conical, covered with minute yellow specks and pale yellow line longitudinally oriented along each side ; with fine and close-set microscopical spiral striae; apex tapering to a blunt point; six t o seven

FIGS.184-187. Odostomia spp. (184) 0. aelninuda shell with enlarged drawing of apical whorls (redrawn after Wells and Wells, 1961); (186) 0. scalaris, living specimen showing head structures including proboscis ( P )(redrawn after Ankel and Christensen, 1963); (186,187) 0. trifida shells (redrawn from photographs after Morris, 1947).

rather compressed and rapidly enlarging whorls ; with distinct but shallow more or less oblique sutures; outer lip gently curved, not very prominent ; inner lip very slight, limited to upper part. Pathology. I n addition to 0. bisutularis, this is the only other species of pyramidellid known t o cause injury t o its host. According to Cole and Hancock (1955), who have examined the effects of 0. eulimoides on Ostrea edulis and Mytilus edulis, numerous thin, approximately parallel laminae of shell substance occur on the shell margins in addition t o characteristic pockets. These laminae mark the successive positions of the withdrawn mantle edge as the oyster or mussel attempts to evade the parasite’s proboscis. It has also been noted that the shell margin

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becomes thickened, sometimes as wide as 1 cm, and with double or triple lips. Cole and Hancock are also of the opinion that: “ I n the most severe cases, the constant irritation appears to interfere with the normal metabolism of the oyster,” and may even lead to death. One cause of death is the destruction of the adductor muscle resulting from the gradual withdrawal of the mantle. When the mantle edge reaches the muscle, the latter is almost completely covered by a horn-like ridge of brownish deposited shell material and atrophies. Such destruction naturally leads to gaping. 6. Odostomia tri$da Totten, 1834. (Figs. 186 and 187)

Fretter and Graham (1949) have listed Odostomia trijda as a parasite of M y a arenaria. Description. About 3-5 mm long ; shell pale greenish-white, conical, with five or six whorls, each separated by a well-defined suture ; two or three impressed lines below suture giving shell the appearance of having multiple sutures; aperture oval; outer lip simple; inner lip with a single oblique fold. Ecology. This species is often found off of its host(s) under stones and driftwood at low tide. 7. Other Pyramidellids

I n addition to the six species given above, two others have been reported associated with commercially important pelecypods. Cole and Hancock (1955) have reported Chrysallida obtusa attached to oysters, most probably Ostrea edulis, from Salcombe harbour, England, and Verrill (1873) found a species referred to by him as Turbonilla interrupta associated with ‘‘ Pecten and other shells ” in Vineyard Sound, Massachusetts. Since the shells on which Verrill found T . interrupta were empty, it cannot be definitely stated at this time whether this gastropod was a parasite of the living animals.

CHAPTER 9

PARASITES OF COMMERCIALLY IMPORTANT MARINE MOLLUSCS THE CLASS CRUSTACEA I. Class Crustacea

A. Subclass Copepoda Among the copepod crustaceans, numerous species have been reported as parasites ” or as (‘commensals ” of molluscs including several from commercially important marine species. These are reviewed in this section together with a description of each species. Since most of the earlier descriptions were rather incomplete by modern standards, there are discrepancies in the descriptions given. For an account of known copepods parasitic in all categories of molluscs, see Monod and Dollfus (1932). ((

1. Mytilicola intestinalis Steuer, 1902. (Fig. 188) (Order Cyclopoida; family Clausidiidae) Steuer (1902) described a cyclopoid copepod, Mytilicola intestinalis, from the intestine of the mussel, Mytilus galloprovincialis, in the Gulf of Trieste. Later (Steuer, 1903), he published a more detailed account of this copepod including a description of its internal anatomy. This was followed by Pesta’s (1907) description of the free-swimming nauplius, metanauplius, and first copepodid stages, and Caspers’ (1939) descriptions of the second and third copepodid stages. Cytological details of the adults have been contributed by Ahrens (1937, 1939a-c). This parasitic copepod appears to be limited to European waters. It has been reported from the Adriatic Sea (Steuer, 1902, 1903 ; Pests, 1907 ; Ahrens, 1939a), the Mediterranean (Vayssikre, 1914 ; Dollfus, 1927; Cerruti, 1932; Ahrens, 1939a; Bassedas, 1950; Monod and Dollfus, 1932) where it is primarily a parasite of M . galloprovincialis although Vayssiere (1914)has found it in M . edulis. It has been reported from Germany by Caspers (1939), from the north of France by Dollfus (1914, 1927) and Monod and Dollfus (1932), from England by Ellenby (1947), Grainger (1951), Baird et al. (1951) and Cole and Savage (1951), 286

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and from Ireland by Grainger (1951). According to Monod and Dollfus (1932), the species described by Dollfus (1914) as Trochicola enterica from the intestine of a marine snail along the coast of France is most probably identical with M . intestinalis. Pearse and Wharton (1938) found a single specimen identified as M . intestindis in an oyster in Apalachicola Bay, Florida, but, as

FIQ. 188. Mytilicolu intestinalis male adult, ventral view. (Redrawnafter Steuer, 1902.)

Humes (1954b) has pointed out, it is most probably not M . intestinalis but M . porrecta. The known natural hosts of M . intestinalis include Mytilus galloprovincialis, M . edulis, Ostrea edulis, Cardium edule, and the gastropods Zizyphinus zizyphinus, Gibbula cineraria and G. varia. I n addition, Hepper ( 1953) has successfully experimentally infected Mytilus edulis, Ostrea edulis, Crepidula fornicata and Paphia pullastra. Description. Male (Fig. 188) about 3 mm long ; female about 8 mm long ; body long and worm-like ; thoracic segments each with a pair of dorsolateral processes, more conspicuous in females than males ;

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abdominal segmentation incomplete ;median eye present ;first antenna of four segments, first segment with fourteen bristles, second with four, third with two, and fourth with five bristles ; second antenna of three segments, hook-shaped ; mandible short, strobiliform, always with two sharp bristles ; maxilla in form of triangular plate with lateral comb of teeth and medially located feeler ; f i s t maxilliped of female reduced to small chitinous thickening, that of male hook-shaped at same position; second maxilliped of male is only noticeable as a weak chitinous thickening ; two-jointed endopodite and exopodite of first four pairs of legs with double chitinous ring ; first four pairs of legs with short setae and spines and fine hairs along exterior margin ; fifth pair of legs reduced to short bristled pegs; genital openings paired; egg sac of female long and slender.

Life cycle. Studies on aspects of the life cycle of M . intestinalis have been contributed by Pesta (1907), Caspers (1939) and Grainger (1951). The nauplii escape into the host’s alimentary canal from within egg sacs when the latter rupture at their posterior ends. These nauplii are enclosed within a membrane. It is still not known how these nauplii escape from the enclosing membrane but in sea water cultures the nauplii escape from the egg sacs leaving this membrane behind. The nauplius and metanauplius are free-swimming. Although in laboratory cultures the first, second and third copepodid stages can be found freeswimming, Grainger (1951) is of the opinion that M . intestinalis can become parasitic during the first copepodid stage. Hepper (1953), in reporting his successful experimental infections of various molluscs, did not indicate whether i t was the first, second or third copepodid that invades the molluscs ; however, since Steuer (1902) has reported that the youngest forms found in naturally infected Mytilus galbprovincialis are 0.83 mm long and with bodies similar to that of the adult, it may be assumed that these represent first stage copepodids. Thus M . intestinalis does undergo further development, through the second and third copepodid stages to the adult, within the molluscan host. It has not yet been demonstrated how first stage copepodids enter molluscs but, since they are found within the gut, it may be assumed that they are ingested. Ecology. Various investigators have reported that the nauplius of M . intestinalis is very active and is positively phototactic. The metanauplius is less active and less positively phototactic. The first copepodid, like the nauplius, is an active swimmer although its tactic response to sunlight is varied. Some are positively phototactic, others are negatively phototactic, and still others do not respond to sunlight. The varied

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responses of first copepodids, which is the invasive form, to sunlight makes it doubtful if phototaxis is an important factor involved in host-searching. Grainger (1951), as the result of his maintenance studies, has reported that crowding has a serious effect on nauplii, metanauplii and first copepodids, especially among metanauplii. If ten to twenty specimens are placed in a 4-5 in. Petri dish, they are maintained quite satisfactorily ; however, if larger numbers are thus maintained, death occurs. Grainger (1951) has also studied the distribution of M . intestinalis in naturally infected Mytilus edulis. He has found that the copepods are almost exclusively limited to the recurrent portion of the intestine and in the rectum. Moreover, the largest number can be found in that portion of the recurrent intestine embedded in the digestive gland. This would suggest that perhaps some nutritional requirements are derived from the digestive gland. Grainger has also found a significant correlation between the size of the mussel host and the number of parasites. It has been shown that the larger mussels harbor the greatest number of small copepods. Relative to seasonal fluctuations in the copepod density in naturally infected M . edulis in Ireland, Grainger has reported that there is a significant decrease in the number of copepods per mussel between November to December and May to June. Furthermore, although egg-bearing females are found all year round, the lowest number of adults in mussels of the 50-69 mm group occurs during September while during November and December the number of immature stages is highest. On the other hand, the number of immature stages is lowest during May and August. I n natural populations there is always an excess of male adults throughout the year. Since M . intestinalis has been reported to be a parasite of both Mytilus edulis and Ostrea edulis among other molluscs, Hepper (1956) studied the transmission of this parasite from mussels to oysters. It is of considerable interest t o point out that although earlier (Hepper, 1953) he was able to infect 0. edulis exposed to copepodids, when 0. edulis and M . edulis are exposed to copepodids in the same aquarium the mussels are readily infected but the oysters are not. The same phenomenon has been mentioned earlier by Bolster (1954). It would thus appear that M . intestinalis demonstrates a preference for Mytilus edulis, but if this mussel is not available as a choice it will parasitize Ostrea edulis. Physiology. What is known about the physiology of M . intestinalis has stemmed from the maintenance and culture studies of Grainger A.M.R.-5

19

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(1951) and Hepper (1953), primarily the former. Grainger has shown that eggs of M . intestinalis hatched in sea water can be satisfactorily maintained in Petri dishes without aeration. When such preparations are maintained a t 13-14"C, first copepodids will develop within 40 h and will survive up to 11 days after reaching that stage. Nauplii, metanauplii and first copepodids can be maintained in sea water that has been passed through a Berkefeld filter so that all diatoms, protozoa and other microorganisms have been removed. Grainger has concluded from such observations that the development of M . intestinalis from egg through the first copepodid is independent of ambient nutrients and derives its energy from the metabolism of nutrients stored within the body. Hepper (1953) has demonstrated that eggs of this copepod will hatch and develop to what may be assumed to be the first copepodid stage maintained in sea water a t 18°C. Pathology. It is known that LW.intestinalis has an adverse effect on Nytilus edulis and may even cause mass mortalities (Cole and Savage, 1951 ;Korringa, 1951b, 1953,1954). Cole and Savage have demonstrated that among the 5.5-5.9 cm (shell length) group of mussels there is an inverse relationship between the condition of the mussels and the mean number of parasites per mussel and the mean number of parasites over 1.5 mm per mussel. The mean weight of mussels between 5 5 - 5 9 cm long is 3.206 g among parasitized ones and 5.975 g among non-parasitized ones. Personal communications with various individuals have indicated that the adverse effect of M . intestinalis on Ostrea edulis is not as severe, although casual observations indicate that the condition of parasitized oysters is also affected to some extent. According t o Chew et al. (1963), Korringa (1950a) is said t o have reported extensive mortalities in commercial mussel beds in Europe due to parasitization by this copepod. 2. Mytilicola orientalis Mori, 1935. (Figs. 189-191) (Order Cyclopoida ; family Clausidiidae)

The second species of Mytilicola, M . orientalis, was described by Mori (1935) from the digestive tract of Crassostrea gigas (Fig. 189) and Mytilus crassitesta collected from the Inland Sea of Japan. Mori, in following Steuer, believed that this species is a member of the family Dichelestiidae, a caligoid family which includes copepods parasitic in sturgeons and marine teleosts. However) I am in agreement with Grainger (1951) that the genus Mytilicola belongs in the cyclopoid family Clausidiidae.

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Wilson (1938), unaware of Mori’s M . orientalis, described M . ostreae from the intestine of Crassostrea gigas which had been imported to Puget Sound, Washington, from Japan. Wilson’s description is sufficiently similar to that of M . orientalis so that Odlaug (1946) has suggested that the two species are identical. After having analyzed both descriptions and compared the original illustrations, I am in agreement with Odlaug that M . ostreae is a synonym of M . orientalis. This parasite is widely spread in Puget Sound where it occurs in Mytilus edulis, Crassostrea gigas, Ostrea lurida, Venerupis ( =Paphia) staminea

FIG. 189. Cross-section of Mytilicola intestinalis in stomach o f Crassostrea giga.8 f r o m Washington. (Slide courtesy of Dr. Kenneth K . Chew, University of Washington.)

and Crepidula fornicnta. It has also been reported in Mytilus californianus collected in Humboldt Bay, California (Chew et al., 1963).

Description. Adult female (Fig. 190), 10-12 mm in total length, 1-33 in greatest width (fourth segment), with elongate narrow body, tapering posteriorly ; head separated from thorax, wider than long, with small dorsal carapace divided longitudinally through its center ; five thoracic segments and genital segment completely fused, with a pair of posterolateral protuberances (processes) on each of the five thoracic segments, these being more prominent than those of M . intestinalis,

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

fifth pair as large as third pair, each process is triangular and extends diagonally outward and backward and with an acute tip which sometimes curves slightly forward ; genital segment enlarged a t posterior corners but without processes ; abdomen considerably narrower and thinner than genital segment, tapers slightly posteriorly, as long as genital segment, undivided and with smooth lateral margins ; caudal rami cylindrical, longer than wide, slightly divergent, with no setae ; first antenna of four segments, basal segment large and swollen, all four sparsely armed with small spines; second antenna of two segments, distal segment in form of a curved claw, divided a t center and with each half armed with a spine-like seta ; upper lip subtriangular ; mandible cylindrical, unsegmented, extremely small ; first maxilla as elliptical mamma, armed with two short spine-like setae; second maxilla of stout basal portion and a two-segmented portion with terminal segment curved and fringed with hairs ; maxilliped lacking in female ; each of four pairs of uniramous swimming legs reduced to simple pointed knob; ovisacs elongate conical, 7 mm long; eggs minute, about 200 in each sac. Adult male (Fig. 191) considerably smaller than female, total length 4 mm, greatest width 0.55 mm ; with thoracic segments more or less separated by grooves ; thoracic processes more reduced than in female but more prominent than those of M . intestinalis except for first pair which is wanting in M . orieatalis ; legs larger than in female but still as uniramous pointed knobs; abdomen unsegmented ; caudal rami enlarged and nearly parallel ; mouth parts similar to those of female but with additional pair of stout maxillipeds behind second maxillae.

Life cycle. The life cycle of M . orientalis is not known although it is most probably of the same pattern as that of M . intestinalis. Ecology.* I n their paper in which was reported the occurrence of this copepod in Mytilus californianus, Chew et al. (1963) also reported that although the average number of copepods per mussel is not significantly different in Ostrea lurida (2*4),Mytilus edulis (2-3),and M . californianus (2.5), those in Ostrea lurida are larger, averaging 6-3 mm in length, than those in the two species of mussels, with those in mussels ranging from 3-0 to 4.0mm. Examination of these copepods has revealed that the smaller specimens (3.0-4.0 mm) are males while the larger ones (6.0 mm and longer) are females. Although their samples included only fifty-two 0. lurida (9.6% infected), twenty-four M . edulis (5893% infected) and twenty M . californianus (65yo infected), their finding of proportionally more males in 0. lurida may be significant.

* See note

011

p. 390.

293

9. T H E CLASS CRUSTACEA

Pathology. Odlaug (1946) has demonstrated that Olympia oysters, Ostrea lurida, when infected with M . orientalis, have a lower condition index (a measurement of fatness) than uninfected ones. Similarly, Chew et al. (1964) have shown that M . orientalis reduces the condition index in Crassostrea gigas." I n the area of histopathology, Sparks (1962) has shown that metaplasia occurs in the gut of Crassostrea gigas when parasitized by this copepod (see Chapter 4, Section 11, A, 6).

2

FIGS.190-193. Mytilicola spp. (190) Adult female M . orientalis, ventral view; (191) adult male M . orientalis, ventral view (redrawn after Mori, 1935); (192) adult female M . porrecta, ventral view; (193) adult male M . porrecta ventral view (redrawn after Humes, 1954b).

3. Mytilicola porrecta Humes, 1954. (Figs. 192 and 193) (Order Cyclopoida; family Clausidiidae) The third species of Mytilicola, M . porrecta, is also known to be a parasite of commercially important molluscs. It was described by * See note on p. 390.

294

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

Humes (195413) from the intestine of the quahaug, Mercenaria mercenaria, the recurved mussel, Brachidontes recurvus ( =Mytilus recurvus), and the ribbed mussel, Modiolus demissus granosissimus, collected near Grand Isle and Grand Terre, Barataria Bay, Louisiana.

Description. Adult female (Fig. 192) reddish to brownish orange ; egg sacs whitish to greenish blue (depending on age of embryos) ; total length of body (from anterior end to tips of rami) 4.017-5.850 mm (average 4.875 mm), width 0.343-0.414 mm (average 0.377 mm) a t anterior border of somite bearing second pair of legs; five pairs of dorsolateral thoracic projections of subequal length ; abdomen elongate and slender, terminating in two small caudal rami which extend posteriorly and bear four small spines ; no distinct cuticular division between thoracic and abdominal somites but segmentation suggested by arrangement of internal muscles ; first antenna with four segments, each of three distal ones armed with an aesthete and several setae or spines, basal segment large with scattered spines and a group of fine spinules along anterior border ; second antenna of four segments, the terminal one in the form of a chitinized hook curving slightly towards midline of body ; mandibles absent ; first maxilla as circular base from which two setae arise ; second maxilla with three podomeres, basal one elongated and largest, middle one short, distal one spatulate and fringed with row of slender setae ; maxillipeds lacking but with chitinous thickenings a t their positions ; head 0.342 mm long x 0.418 mm wide ; ratio of length to width is 0.924; all four pairs of swimming legs biramous; first leg with rami of two podomeres, expanded on their outer margins to form a wide lamella, first exopodite podomere with single seta on outer distal corner, secondexopodite podomere with three terminal setae, endopodites unarmed ; second leg similar to first but with outermost terminal exopodite reduced in size ;third and fourth legs with only two terminal setae; fifth leg absent; openings of oviducts strongly chitinized and with transverse rod across aperture ; egg sacs 1.28 mm x 0.15 mm, contain about forty-five to fifty-five eggs, extend beyond ends of caudal rami. Adult male (Fig. 193) reddish to brownish orange but paler than female ; total length 2.379-2.574 mm (average 2.472 mm), width, measured as in female, is 0.272-0.340 mm (average 0.319 mm) ; dorsolateral thoracic processes reduced to indiscernible humps ; abdomen elongate, without distinct segmentation, terminates in caudal rami as in female ; first and second antennae and first and second maxillae like those of female ; mandibles absent ; maxilliped well developed, consisting of large subtriangular basal podomere and terminal podomere

TABLEXVI. A COMPARISONOF

DIAGNOSTIC CHARACTERISTICSOF Mytilicola spp. After Humes, 1954b.

THE

~~

M . intestinalis Steuer Female Caudal ramus : Posterior corners of head :

M . porrectu Humes ?

Elongated (237p) and widely divergent Smoothly rounded

Dorsolateral thoracic projections : Short and rather stout Ratio of length of head to width : 0.647 Egg sac : Long, reaching far beyond caudal rami Male Dorsolateral thoracic projections : Well formed

Claw of maxilliped :

M . orientalis Mori ( = M . ostreae Wilson)

~

Elongated, not strongly hooked

Elongated (233p) but not widely divergent With a pair of slender processes Long and tapering 0.782 Long, 2.74 mm, reaching far beyond caudal rami Well formed but absent on 1st leg-bearing somite according to Mori (1935) Elongated, not strongly bent

Short ( 9 6 p ) and not divergent Smoothly rounded Short and rather stout 0.924 Short, 1.28 mm, reaching only short distance Reduced to almost undiscernible humps Short, stout, hooked

strongly

c3

i Q

F

kiR

8 kM +-

296

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

in shape of recurved hook ; four swimming legs resemble those of female except for setal armature ; endopodites of legs bear long terminal setae ; second and third legs similar ; fifth leg as erect lobe with three terminal setae and one on outer margin; sixth leg as flat rounded lobe with two setae, located on posterior area of ventral surface of sixth thoracic somite. Humes’ ( 195413) table comparing the distinguishing characteristics of the three species of Mytilicola found in commercially important molluscs is reproduced in Table XVI. Life cycle. Although the various stages in the life cycle of M . porrecta have not been found, the nauplius, metanauplius, and most probably the first copepodid stages undoubtedly are free-swimming as they are in the two related species. Parasitization of the molluscs most probably occurs when first copepodids invade these hosts. Ecology. According to Humes (1954b), the pelecypods in which M . porrecta are found occur in water with a salinity of 25 %,,. Modiolus demissus granosissimus is the most frequently infected (42 yo)followed by Brachidontes recurvus (18%). Only one Mercenaria mercenaria was examined and it was infected. Humes also has noted that 400 Crassostrea virginica were examined from the area but none was parasitized. This suggested that perhaps as in the case of M . intestinalis, the invasive first copepodid stage is selective for certain pelecypods. The largest number of M . porrecta in a single Modiolus was fifteen (five males and ten females) but the average number was 2.75. Pathology. Although pathological changes in pelecypods harboring M . porrecta have not been studied, the fact that both M . intestinalis and M . orientalis are known to cause injury suggests that this species may also be injurious. 4. Panaietis haliotis Yamaguti, 1936. (Figs. 194 and 195) (Order Cyclopoida; family Clausidiidae) Panaietis haliotis was described by Yamaguti (1936) based on one male and three females found in the mouth cavity of the abalone Hccliotis (Sulculus) gigantea from “ the Pacific coast ” of Japan. Description. Adult female (Fig. 194) with elongate body, 7.4-7-6 mm long, tapering posteriorly ; head broadened posteriorly, 0.92-0.98 mm long x 1-6-1.7 mm broad a t posterior end, with nearly truncate frontal margin 0.62-0.7 mm broad ; five thoracic segments distinct, with rounded sides, gradually narrowed posteriorly, 2.8-3.0 mm long in combined length ; genital segment 0-5-0.6 mm long x 1.2-1.8 mm broad, with prominent sides ; egg strings 2-25-2.7 mm long x 0.4-0.5

9. THE CLASS CRUSTACEA

297

mm broad ; eggs large, rounded, in several longitudinal rows ; abdomen of four segments, nearly cylindrical, 2.1-2.2 mm long, first through third segments broader than long but fourth segment is longer than broad ; caudal rami 0.83-0.85 mm long, each tapering slightly toward distal end which has one subterminal and four claw-like terminal spines ; first antenna of seven segments, 1.06-0.1 mm long, 0.25 mm wide a t base, narrowed distally, with not very numerous small spiniform setae ; terminal segment with four short setiform claws at tip and four setae on posterior margin ; second antenna stout, of three segments, first segment with a seta a t distal end, second segment with a shorter pedunculate seta on inner margin, third with a group of three small spines on inner margin and four terminal claws, one of which is stout and the others are two-segmented; mandible terminating as pointed blade with about twelve sharp teeth of which the two basal ones each have an accessory tooth and a long spiniform ramus fringed with spines on inner margin and four terminal claws, one of which is stout seta, the latter with a very small spine on each side of its base, and three spines close together near inner end; second maxilla comparatively short, with four teeth on distal inner margin and two small mediodorsal spines near base ; maxilliped with rudimentary spine at its bluntly pointed end ; first four legs biramous, each with three-segmented rami ; proximal and middle podomeres of first through fourth endopodites with short spine a t outer end of each pectinate distal margin, corresponding podomeres of exopodites with three similar spines and a comb on each outer margin; each distal podomere of endopodites of first through fourth legs with eight to nine spines, of which the outer ones form three groups of two each with a comb a t each base and the inner ones are longer, pectinate, and isolated ; corresponding podomere of exopodites with eleven to twelve spines of which the outer ones form four groups of two each and a comb a t each base and the inner ones are longer, pectinate and isolated; fifth leg uniramous, digitiform, not reaching posterior end of genital segment, with one seta and one spine a t tip and two short subterminal spines, each of which is provided with a comb a t the base. Adult male (Fig. 195) 4.1 mm long, resembling female in general shape but with differing maxilliped and genital segment ; head 0.75 mm long x 1.2 mm wide; thorax 1.75 mm long, tapering gradually posteriorly, 1 . 1 mm broad a t first segment, 0-67 mm broad a t fift,hsegment ; genital segment nearly quadrangular, 0.40 mm x 0.625 mm, with a pair of ventral prominences bearing two setae on each posterior edge ; abdomen of four segments, 1.5 mm long, first through third segments broader than long, fourth segment longer than broad; caudal rami

298

MARINE MOLLUSCS AS HOSTS F O R SYMBIOSES

0.67 min x 0.15 mm ; basal and distal segments of maxilliped subequal in length with terminal claw which is slender and 0.2 mm long, armed with a small spine on inner side of base ; fifth leg reaching to posterior end of genital segment, with one seta and two spines a t tip, and one subterminal spine on outer margin.

FIGS.194-197. Clausidiid copepods from Japan. (194) Adult female Panaietis haliotis, dorsal view; (195) adult male P . haliotis, dorsal view (redrawn after Yamaguti, 1936): (196) adult female Philoconcha amygdalae, dorsal view; (197) adult male P . amygdalae, dorsal view (redrawn after Yamaguti, 1936).

5 . Philoconcha amygdalae Yamaguti, 1936. (Figs. 196 and 197)

(Order Cyclopoida; family Clauuidiidae) Philoconcha amygdalae, the genotype, was described by Yamaguti (1936) based on two mature males, one gravid female, and some immature specimens found in Tapes semidecussata from Tiba Prefecture, Japan. Their exact location within the pelecypod was not reported. Description. Adult female (Fig. 196) up to 6.6 mm long, with greenish digestive tract ; head approximately triangular, with strongly convex sides, sharply demarcated from first thoracic segment in immature specimens but more or less fused in adults and measuring 1.2 mm wide a t its somewhat prominent posterolateral corners ; frontal margin prominent, with rostrum projecting ventrad between

9. THE CLASS CRUSTACEA

299

bases of first antennae ; nauplius eye 0-2-0.27 mm from frontal margin ; cephalothorax and free thoracic segments arched dorsally and turned over ventrally, with latter segments projecting onward beyond former, measuring 0.75 mm x 1.85 mm, 0.91 mm x 2.1 mm, and 0.9 mm x 1.9 mm respectively ; fifth segment markedly narrower, 0.33 mm long x 0.7 mm wide, with a small rounded protuberance on each side in young, with uneven but nearly parallel sides in adults ; genital segment very short, 0-225 mm long x 0.6 mm broad, with somewhat convex sides ; abdomen almost cylindrical, three- or four-segmented, 1-25 mm x 0-5-0.6 m m ; caudal rami short, cigar-shaped, 0-66-0.66 mm x 0.21-0.23 mm, each with a fine seta on outer margin behind its middle and five similar terminal setae, of which three are close together in the middle ; eggs small, rounded, 0.060-0-080 mm in diameter, multiseriated ; first antenna tapering gradually towards distal end, about 0.3 mm long, of seven segments, armed with few setae, tipped with a medianlengthed and a few shorter setae; second antenna of five segments, first segment enlarged, second the longest and with a spine on inner margin, third short and with two setae a t distal end of inner margin, terminal claw about 0.1 mm long ; first maxilla with two setae ; second maxilla stout a t base, distal segment terminating in two setiform pectinate rami, with a seta on inner margin ; maxilliped swollen, with a digitiform process and two setae a t tip; first four legs biramous, each basipodite comprised of two segments, with a seta on inner margin of proximal segment, each ramus of three segments except endopodite of fourth leg which consists of two ; fifth leg uniramous, plump, slightly curved inward, tipped with a blunt spine-like inner process and a simple outer seta. Adult male (Fig. 197) with narrow body, 2.7-2.8 mm long, grayish white, semi-transparent ;head subtriangular, with convex sides, 0.61mm wide a t posterior end, wall fused with first thoracic segment, frontal margin prominent, with rostrum projecting ventrad between bases of first antennae; nauplius eye about 0.13 mm from frontal margin; cephalothorax and free segments projecting ventrally on each side but less conspicuously than in female ; first fused segment only slightly narrower than head, wider than second ; second through fourth segments diminishing in width posteriorly, measuring 0.20-0.21 mm x 0.51 mm, 0.18-0.19 mm x 0.41-0-42 mm, and 0.225 mm x 0.338 mm respectively ; fifth segment strongly constricted off from fourth, fused with genital segment; genital segment 0.34-0-4 mm x 0-4-0-21 mm, with convex sides ; abdomen nearly cylindrical, of three segments, 0.65 mm long; first segment rounded, 0.2 mm long x 0.3 mm wide, second segment rounded but shorter, third segment 0.3-0.31 mm x 0.24-

300

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

0.25 mm, with parallel sides; caudal rami cigar-shaped, 0.45 mm x 0.12-0.13 mm, armed as in female; maxilliped of four segments, terminal claw 0.12-0.138 mm long, with slender basal spine measuring 0.010 mm long ; legs armed with spines and setae except endopodite of

fourth leg which may sometimes possess a rudimentary seta on its basal podomere. 6 . Myocheres major Williams, 1907. (Fig. 198)

(Order Cyclopoida ; family Clausidiidae) This copepod was originally described by Williams (1907) as Lichomolgus major based on male and female adults collected from the

FIG. 198. Adult female Myocheies major, dorsal view. (Redrawn after Williams, 1907.)

mantle cavity of M y a arenaria, Mercenaria mercenaria and Mactra solidissima a t Wickford and Matunuck on Narragansett Bay, Rhode Island. It was transferred to the genus Myicola by C. B. Wilson (1932) who also gave a short redescription. Later, M. S. Wilson (1950) erected Myocheres with this species as the genotype. Although Wilson placed it in the family Lichomolgidae and Pearse (1947) placed it in the family Myicolidae, in following Humes and Cressey (1958) I am assigning i t to the family Clausidiidae.

9. T H E CLASS CRUSTACEA

301

Description. Adult female (Fig. 198) with fairly transparent body of uniform grayish or pinkish color ; total length 1-1-1.3 mm ; metasome cylindrical ; head fused with first segment and about as wide as long ; second, third, and fourth segments slightly tapered, fifth segment abruptly reduced to half the width of the fourth segment, but about the same length ; urosome as long as metasome, tapered posteriorly ; caudal rami a little longer than anal segment, six times as long as wide; first antenna of six segments of which second segment is the longest ; second antenna of four segments the terminal one of which is tipped with two stout curved claws and three long, nearly straight setae ; labrum ending in an acute, backwardly directed spine ; labium with apparently movable terminal claw with hemispherical pad at base beset with recurved bristles ; second maxilla of two joints with one bristle midway on its posterior edge ; maxilliped absent ( 2 ) ; first four swimming legs with both rami of three podomeres, distal margin of basipodite of second leg and outer margins of both rami of first leg fringed with stout triangular spines ; fifth leg of two segments with the distal segment three times as long as wide and with long apical and two stout outer serrate spines and a stalked filiform seta on dorsal surface near tip. Adult male much larger than female, 1.75-1-9 mm in total length ; of same general shape as female; genital segment swollen and with fringe of spines and single seta on each lateral margin; antennae, mouth parts, and swimming legs as in female except that a pair of two-jointed maxillipeds is present ; maxilliped with second segment swollen and armed with two rows of tubercles and two setae on inner surface, sickle-shaped apical claw, enlarged a t base where thereis a small bristle, and serrated on inner margin. Other informahon. Although this copepod is undoubtedly usually found in the mantle cavity, on one occasion I found a single female in the stomach of a specimen of M y a arenaria collected in Narragansett Bay, Rhode Island. M. S. Wilson (1950) has reduced Myicola spinosa, described by Pearse (1947) from the gills and mantle of Tagelus gibbus, Dosinia discus and Mercenaria mercenaria from Beaufort, North Carolina, to synonymy with M . major. 7 . Other Clausidiids

I n addition to the clausidiids given above, Humes and Cressey (1958) have described five additional species from the mantle cavities of marine pelecypods from West Africa. Since these molluscs are presently of

302

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

limited commercial importance, information on their copepods wilI not be reviewed in this paper. However, since these pelecypods are of potential commercial importance, these parasites are briefly mentioned a t this point. Humes and Cressey have described Conchyliurus torosus from Mactra glabrato from Pointe-Noire, Congo ; C. lobatus from Cardita ajar dredged near Freetown, Sierra Leone ; Conchyliurus sp. from Cardium ringens dredged in the harbor of Accra, Ghana ; Myocheres scobina from Tellina nymphalis buried in the muddy sand a t Loango, 19 km north of Pointe-Noire, Congo ; and M . dentata from Macoma cumana dredged from mud in the bay of Accra, Ghana. 8 . Ostrincola gracilis Wilson, 1944. (Figs. 199 and 200) (Order Cyclopoida; family Ergasilidae)

Adults of Ostrincola gracilis were originally described by Wilson (1944) from specimens removed from the mantle cavity of Crassostrea virginica taken at Beaufort, North Carolina, and deposited in the U.S. National Museum. This species was redescribed by Humes (1953) based on specimens from the mantle cavity of C. virginica, Modiolus demissus gmnisissiwzus, Brachidontes recurvus (= Mytilus recurvus) and Mercenaria mercenaria collected fsom the Barataria Bay region of Louisiana. Description. Adult female (Fig. 199) colorless except for dark reddish-black median eye and brownish intestine : total length (from anterior end to tip of caudal ramus) averages 1-083mm, greatest width 0.274 mm ; abdomen three-segmented ; minute spines on ventral surfaces of genital segment and abdomen, dorsal surfaces without spines ; caudal ramus bears three terminal setae and three along outer edge ; first antenna of seven segments and several aesthetes, arrangement of setae on distal segment suggests a subdivision but there is no articulation ; second antenna with notch-like interruption on inner margin of claw; mouth parts greatly reduced; first, second, third, and fourth swimming legs biramous, with spine and setal formula as follows :

1st podomere 2ndpodomere 3rdpodomere

Leg 1 exp end

Leg 2 exp end

Leg 3 exp end

Leg 4 exp end

1:0 1:l 8

1:0 1:l 9

1:0 1:1 8

1:0 1:l 8

0:l 0:l 6

0:l 0:2 6

0:1 0:2 6

0:l 0:2 5

9. T H E CLASS CRUSTACEA

303

fifth leg with two podomeres, basal one small, with single outer seta, distal one expanded as broad inwardly concave disk with four marginal setae ; egg sacs attached dorsoventrally, each containing about seven eggsAdult male (Fig. 200) resembles females in color; total length averaged 0-760 mm, greatest width 0.200 mm ; abdomen of four segments ; ventral surfaces of genital segment and abdomen bear minute spines ; caudal rami similar to those of female ; first antenna of seven segments, with all but basal one having an aesthete ; second antenna

FIQS.199-202. Ostrincola spp. (199) Adult female 0. gracilis, dorsal view; (200) adult male 0. gracilis, dorsal view (redrawn after Humes, 1953); (201) adult female 0. clawator, dorsal view; (202) adult male 0. clavator, dorsal view (redrawn after Humes, 1958b).

like that of female ; four pairs of swimming legs identical with those of female ; fifth leg with two podomeres, basal one with single outer seta, second podomere longer, not expanded, with more or less parallel margins, and with four marginal setae ; sixth leg consists of two setae a t posterior corner of genital segment. Other information. Humes (1953) has reported that from one to eleven copepods are found in each of the 157 parasitized Crassostrea virginica, one to seven in each of the eighty-nine parasitized Modiolus

304

M A R I N E MOLLUSCS AS HOSTS FOR SYMBIOSES

dernissus, one to two in each of the seven Brachidontes recurvus, and the single specimen of Mercenaria mercenaria examined was parasitized and it harbored two copepods. 9. Ostrincola clavator Humes, 1958. (Figs. 201 and 202) (Order Cyclopoida; family Ergasilidae) Ostrincola clavator adults were described by Humes (1958b) from the mantle cavity of the oyster, Ostrea sp., clinging to rocks off the coast of Madagascar on the island of Nossi-BB. Description. Adult female (Fig. 201) body cyclopoid, transparent, eye brown or black, total length (from anterior end to tip of caudal ramus) 1.068-1.152 mm (average 1.098 mm), greatest width 0.3000.348 mm (average 0-318 mm) ; base of first leg indistinctly separated from head by transverse suture ; several bristles dispersed on dorsal surfaces of head and thorax; posterior margins of thoracic segments rounded ; genital segment distinct, not very swollen, slightly longer than wide, approximately 0.148 mm x 0.151 m m ; ovigerous sacs attached dorsolaterally on anterior portion of segment, each with two very small spinous processes ; ventral surface of genital segment with two transverse groups of spines, two short rows on posterolateral regions and a transverse row of very smaIl spines along posterior margin; abdomen of three segments measuring 0-070, 0-050 and 0.036 mm respectively ; transverse row of very small spines along posterior borders of first and second abdominal segments and several small spines on posterolateral borders of third ; ovigerous sac not seen ; first antenna of seven segments, measuring (from base to tip) 0.024, 0.036, 0.010, 0.012, 0.023, 0.13 and 0.016 mm in length respectively; a sensory organ on each of three basal segments; second antenna of three joints, third segment elongate and slightly curved, with short seta on medial edge, recurved hook a t terminal, and shorter bifurcated process, with small lateral spine, abutting hook ; first through fourth swimming legs biramous ; formula for spines and setae is as follows :

1st podomere 2ndpodomere 3rd podomere

Leg 1 exp end

Leg 2 esp end

Leg 3 exp end

1:0 1:l 8

1:0 1:l 9

1:0 1:l 8

0:l 0:1 6

0:l 0:2 6

0:1 0:2 6

Leg 4 exp end

1:l

0:l 0:2

8

5

1:0

first leg with medial spine on basipodite, with three spines on lateral surface of basal podomere of exopodite, exopodite also with a semigladiate terminal spine and four plumose medial bristles ; second leg

305

9. THE CLASS CRUSTACEA

without medial spines on basipodite, with three lateral spines on basal podomere of exopodite, one terminal spine, and five medial setae, also with three lateral spines and three medial setae on basal podomere of endopodite ; third leg similar to second leg but with only two lateral spines on basal podomere of exopodite and a seta on basal podomere of endopodite which transforms into a plumose seta along its basal portion but becomes blade-like distally ; fourth leg with three lateral spines and four medial setae on basal podomere of exopodite, with four spines and one medial plumose seta on basal podomere of endopodite ; fifth leg with distal podomere flattened, 0.072 mm x 0.036 mm, with a seta (0.023 mm long) on its medial border, three setae (0.018, 0-018 and 0.020 mm long) at its tip, and with small spines scattered along it ; sixth leg absent ; caudal ramus elongate, 0.093 mm x 0.017 mm, with flattened dorsal seta and remaining setae relatively strong and claviform including one slightly dorsal to midline of ramus, three long setae a t tip (longest measuring 0-024 mm), and three short setae near base. Adult male (Fig. 202) with body shape and color similar to female ; total length 0.612-0-696 mm (average 0.645 mm), greatest width 0.1680-192 mm (average 0.183 mm); base of first leg distinctly separated from head; genital segment almost as long as wide, 0.18 mm x 0.112 mm, with posteroventral suture ; ventral surface with a transverse band of spines near midlength and with row of small spines at each posterolateral margin ; two spermatophores visible, each approximately 0.090 mm x 0-042 mm; abdomen of four segments measuring (from base to tip) 0.036, 0.031, 0,024 and 0.018 mm respectively; with transverse rows of small spines along posteroventral margin of segments except the posteriormost segment which has a group of small spines near the base of each caudal ramus ; first antenna of seven segments measuring (from base to tip) 0.018, 0.024, 0.007, 0.011, 0.017, 0.011 and 0.017 mrn respectively; large sensory organ on third and fourth segment; small sensory organ on fifth and two on basal segment; second antenna like that of female ; first through fourth legs like those of female ; fifth leg with flattened distal podomere, 0.042 mm x 0.014 mm, with four setae measuring (from base to tip) 0.016, 0.016, 0.014 and 0.022 mm ; sixth leg composed of two setae, 0.018 mm and 0.017 mm long ; caudal ramus like that of female. 10. Ostrincola simplex Humes, 1958. (Figs. 203 and 204)

(Order Cyclopoida; family Ergasilidae) Ostrincola simplex adults were described by Humes (1958b)from the mantle cavity of oysters, Ostrea sp., off the coast of Madagascar on the island of Nossi-BB. A.M.D.-5

20

306

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

Description. Adult female (Fig. 203) total length (from anterior terminal to tip of caudal ramus) 0.696-0.852 mm (average 0.766 mm), greatest width 0.216-0.264 mm (average 0.230 mm) ; genital segment a little wider than long, 0.108 mm x 0-128 mm, with smooth dorsal surface but with several lateral spines ; ventral surface with two transverse rows of spines across anterior half of segment and with a row of small spines along its posterior border; abdomen of three segments, 0.045, 0.057 and 0.024 mm long, without spines dorsally but with a row of small spines ventrally along posterior border of first segment; first antenna with seven segments, measuring (from base to tip) 0.024, 0.029, 0.013, 0.010, 0.022, 0.011 and 0.019 mm respectively; a sensory organ on each of three basal segments ;second antenna of three segments, third segment long and slender, with a sensory organ and a row of indistinct bristles along interior border, a row of small spines along basal portion of exterior border, and three thin setae and large recurved claw a t its apex ; segments of first through fourth swimming legs like those of 0. cluvutor ; spines and setae of exopodites like those of 0. clavator ; terminal spine on endopodite of first leg semi-gladiate, with a row of bristles along interior margin; terminal podomere of endopodite of second leg with a short gladiate spine on external surface, a long semi-gladiate spine with bristles along internal border, and four plumose setae exteriorly ; terminal podomere of endopodite of third leg with three spines and three setae ; terminal podomere of fourth leg with three external spines, a gladiate seta on its distal half, and a plumose seta on its medial surface ; terminal spines on podomeres very long and large, that on terminal podomere of exopodite of fourth leg, for example, measures 0.056 mm long; second podomere of fifth leg flattened, 0.098 mm x 0.078 mm, with single spine, 0.022 mm long, on distal border of external edge and three spines on medial edge measuring 0.011, 0.024 and 0-014 mm respectively; edge of middle podomere without spines but with small sclerified denticles along medial surface ; groups of long spines on both edges of basal podomere ;sixth leg absent; caudal ramus, 0.096 mm x 0.017 mm basally, 0.096 mm x 0.006 mm distally, with dorsal seta 0.012 mm long and lateral seta about 0.030 mm long, lateral subterminal seta 0.005 mm long, three terminal setae 0.006, 0.013 and 0.010 mm long ; egg sacs slightly orange, 0.132 mm x 0.070 mm, containing about fifty-five eggs. Adult male (Fig. 204) with body outline like that of female ; total length 0.540-0.648 mm (average 0.602 mm), greatest width 0.1440.192 mm (average 0.160 mm); first podomere of first leg distinctly separated from head ; genital segment somewhat rounded, 0.092 mm x 0.093 mm, posteroventrally divided, with group of small spines at

9. THE CLASS CRUSTACEA

307

border of division and another group of small spines tranversely arranged in posterior half of segment; abdomen of four segments measuring 0.030, 0.034, 0.036 and 0.016 mm respectively ; posterior borders of first and second abdominal segments each with a group of small spines; dorsal surface of genital segment without spines; two spermatophores present ; first antenna with seven segments measuring (from base to tip) 0.033, 0.042, 0.015, 0.022, 0-041, 0.026 and 0.030 mm long respectively ; two large sensory organs on fourth segment and one little one on each of three basal segments ; second antenna as in female ; first through fourth legs liks those of female but medial seta of coxo-

FIGS. 203-206. Ost?%COh and Myicola. (203) Adult female 0. simplex, dorsal view; (204) adult male 0. simplex, dorsal view (redrawn after Humes, 1968b); (206) adult female M . rnetisiensis, dorsal view; (206) adult male M . metisienais. dorsal view (redrawn after Wright, 1886).

podite of fourth leg much longer than that of female ; distal podomere of fifth leg much finer than that of female measuring 0.042 mm x 0.012 mm, seta on its external border 0.025 mm long while the terminal setae measure 0.019, 0.016 and 0.032 mm long respectively; sixth leg includes two setae, 0.015 and 0.013 mm long; caudal rami like those of female. Other information. Humes (1958b) has noted that these copepods will live in a glass container in the laboratory and will swim capriciously when disturbed.

308

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

11. Myicola metisiensis Wright, 1885. (Figs. 205 and 206)

(Order Cyclopoida; family Myicolidae) Myicola metisiensis, the genotype, was originally found and described by Wright (1885) between the gill lamellae of M y a arenaria collected a t the village of Little Metis, Quebec, on the Gulf of St. Lawrence. It was redescribed by Wilson (1932) from the mantle cavity of the same host collected a t Wellfleet, Massachusetts, and placed in the family Lichomolgidae. Wright had placed this species in the no longer recognized family Corycaeidae. However, in following Pearse (1947), I am placing it in the cyclopoid family Myicolidae. Description. Adult female (Fig. 205) averaging 3 mm long, first four thoracic segments subequal, broader than long ; fifth segment smaller than first abdominal segment ; double genital segments nearly as long as remaining three abdominal segments; furcal segments as long as last two abdominal segments, with six setae of which three are apical and one subapical ; rostrum shield-shaped ; anterior antennae as long as head, with first, second, and fifth segments being the longest ; second antennae directed downwards, shorter than first pair ; labrum with lateral borders denticulated and posterior border emarginated ; mandible with two setose lobes and two setose filaments; maxillae with three setae of which the middle is the longest; two basal joints of first maxillipeds tumid, with two converging oblique patches of spines, distal joint of first maxilliped with strong seta and terminating in two curved setose filaments of which the more slender is attached like a palp ; first through fourth legs biramous ; basipodite of first leg with a row of strong spines on ventral surface which decrease in rigidity on the second and third legs and are absent from the fourth ; fifth leg uniramous, of three podomeres, two basal ones each with a seta while the distal podomere has two apical setae and a group of subequal spines ; egg sacs cylindrical, 1.0 mm x 0.5 mm. Adult male (Fig. 206) very similar to female, smaller, measuring 1-75 mm or less in length ; thoracic segments gradually decreasing in breadth anteroposteriorly ; posterior borders of genital and two subsequent abdominal segments denticulated ; second maxilliped with basal joint denticulated ; spermatophores subpyriform, 0.2 mm x 0.1 mm. Life cycle. The stages in the life cycle of this species are not known. Relative to the mode of infection of the soft clam, Wright (1885) has stated : I have found some in the suprabranchial chambers, which would seem to indicate entry through the cloaca1 siphon, while I have found others, head

9. THE CLASS CRUSTACEA

309

upward, in the gill-tubes, which would appear to argue an entry while still in the nauplius-stage, through the inhalant siphon and the water-pores of the gill-plates. Ecology. According t o Wright, female copepods found in clams during June carry egg sacs enclosing eggs a t various stages of development, but by August they have lost their egg sacs. Males have been reported to be much faster swimmers than females when teased out of their hosts. Pathology. The only information available on the effects of M . metisiensis on its host is that reported by Wright who has stated : No considerable irritation appears to be set up by the presence ofthe parasite in the gill-tubes. The claws of the posterior antennae and the setae of the various appendages are often invested by a yellowish film undoubtedly derived from the blood of the host, but no greater exudation resulting in the formation of a cyst round the foreign body is to be observed. . . . The granular contents of the intestine of the copepod have a bluish-green tint, which is most readily noticed in the wider rectum, but I must leave undecided whether these are derived from the blood of the host. 12. Pseudomyicola ostreae Yamaguti, 1936. (Pig. 207) RADGL~FF~ (Order Cyclopoida; family Pseudomyicolidae) Pseudomyicola ostreae, the genotype, was described by Yamaguti (1936) based on two female specimens recovered from the branchial cavity of Ostrea denselamellosa cultured a t the Hutami oyster bed in Hy6go Prefecture, Japan. I n following Humes and Cressey (1958), I am assigning it t o the family Pseudomyicolidae.

Description. Female (Fig. 207) 2.1-2-43 mm long, uniformly grayish in alcohol-preserved specimens ; head 0.45-0.51 mm x 0.525-0.625 mm, abruptly narrowed anteriorly, with setae on each side as in first thoracic segment ; head indistinctly demarcated from thorax ; thorax 0.9-1.25 mm long, with first four segments almost uniformly broad, 0.67 mm in maximum breadth; fifth thoracic segment about half as long as and much narrower than fourth but slightly broader than genital segment ; genital segment 0.225-0.260 mm x 0.28 mm, with two transverse rows of spines and scattered setae on ventral surface ; egg strings not seen; abdomen of three segments, first segment 0.12-0.13 mm x 0.20.21 mm, with about ten setae on ventral side near posterior margin, second segment 0.10-0.11 mm x 0-175 mm, third segment 0.0880.1 mm x 0.14-0.15 mm, with numerous small spines on ventral and outer sides near posterior end; caudal rami narrow, 0.16-0.18 mm x 0.035-0.04 mm, each with two spines on outer margin, three a t tip and

310

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

numerous smaller ones on ventral side ; first antenna of six segments, about 0.3 mm long, basal half enlarged, basal segment with slender, slightly curved spines, measuring 0.051 mm long, a t anteromedial corner and four setae a t distal anteroventral part, second segment incompletely segmented from third, approximately triangular in ventral aspect, with about twelve simple setae, third segment with nine setae one of which is on dorsal surface, fourth segment with four setae, fifth with three setae, sixth with one subterminal and six terminal

FIGS.207-209. Pseudomyicola spp. (207) Adult female P. ostreae, dorsal view (redrawn after Yamrtguti, 1936); (208) abdomen of adult female P. glabra, dorsal view; (209) abdomen of adult male P . glabra, dorsal view (redrawn after Pearse, 1947).

setae, with one much longer than others; second antenna of three segments with basal segment stout, middle segment about 0.225 mm long, arcuate, with one basal and three terminal setae, and covered with numerous minute spines ; terminal claw comparatively short with a false joint near middle; mandible terminating in two pectinate blades ; first maxilla with four setae of which the outermost is long and the two inner are difficult to see; basal segment of second maxilla armed with numerous spines along posteroventral margin of its larger proximal portion, terminal blade fringed with eight spines on inner

9. THE CLASS CRUSTACEA

311

side ; maxilliped knoblike, behind base of second maxilla ; first four legs biramous, each ramus of three segments which are fringed with spines on outer margin; each basipodite of two indistinct segments and with a seta and a row of two to five minute spines on outer side and a seta on inner side, spines also occur along posteroventral margin between bases of both rami ; basipodite of first leg with a stout pectinate spine and a group of smaller simple spines at its posteromedial corner ; fifth leg uniramous, of two podomeres, basal podomere with one seta at dorsal distal terminal, terminal podomere about twice as long as broad, with marginal setae a t distal portion, a row of four to ten setae on inner margin, and a few oblique rows of spines on ventral surface. Male unknown. 13. Pseudomyicola gbbra Pearse, 1947. (Figs. 208 and 209)

(Order Cyclopoida; family Pseudomyicolidae) This species was described by Pearse (1947) based on male and female specimens removed from the gills and mantle cavities of Modiolus demissus, Crassostrea virginica, Atrina rigida, Aequipecten irradians, Noetia ponderosa and Mytilus edulis collected at Beaufort, North Carolina. I n following Humes and Cressey (1958), it is being assigned to the family Pseudomyicolidae. Description. Adult female (Pig. 208) 2.23 mm in total length, 0.53 mm wide; head separate from first thoracic segment; second through fifth thoracic segments become narrower posteriorly, urosome half as long as metasome ; caudal rami more than twice as long as anal segment, slender, tapering, with two short terminal setae and two on the lateral and dorsal surfaces ; genital segment with five little ridges on sides near posterior margin ; anal segment with minute spinules around posterior margin ; first antenna with sharp strong spine projecting posteriorly from basal segment, entire ant,enna of six segments, the basal being the longest and the basal three being wider than distal three ; second antenna with basal segment widest, second segment shortest, and third segment longest, narrowest, and recurved, strong terminal claw present; swimming legs all biramous with each ramus being three-segmented ; fifth leg of two podomeres, broad, flat, and thickened along one margin, with four distal setae, two rows of seven and eight denticles along middle of thin margin, and eleven spines across anterior surface ; egg strings, each measuring 0.33-0.55 mm long, contain five to twelve eggs arranged in linear groups. Adult male (Fig. 209) similar to female but smaller; total length 1-78 mm, width 0.47 mm; fifth legs relatively smaller, about half as long as those of female, of two podomeres and with five denticles on

312

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

medial distal angle of terminal segment ; lobes of genital segment with a small seta on posterior margin ; denticles in posterior margin of anal segment longer than those on female. 14. Other Pseudomyicoliids I n addition to the pseudomyicolids given above, Humes (1958b) described Pseudomyicola mirabilis from the mantle cavity of Arca decussata collected a t Nossi-Bk, Madagascar. Later, Humes and Cressey (1958) reported finding the same copepod in A . senilis collected at Somone, Senegal, in Ostrea tulipa from the same locality, and in Pitar tumens a t Haan, Senegal. Since these pelecypod hosts are of limited commercial importance, P. mirabilis is being briefly recorded herein. 15. Tisbe celata Humes, 1954. (Figs. 210 and 211) (Order Harpacticoida; family Tisbidae) The genus Tisbe includes twenty-seven recognized species and several doubtful ones. Among these, only two, T . wilsoni and T . celata, have been found as symbionts, possibly parasites, while two others, T . elongata and T . furcata, which are usually free-living, have been found in association with other invertebrates. Specifically, T . wilsoni was described by Seiwell (1928) as a " commensal " from Amaroucium a t Woods Hole, Massachusetts, and T . celata, the species under consideration, was described by Humes (1954a) from the mantle cavity of Mytilus edulis collected a t St. Andrews, New Brunswick, Canada. T . elongata was recorded by Gurney (1933) and Leigh-Sharpe (1935) on the gills of Homarus vulgaris, and T . furcata was found by Aurivillius (1885, 1887) in the tunicate Molgula ampulloides. Relative to T . celata, Humes (1954a) is of the opinion that it is a parasite of Mytilus edulis. Description. Adult female (Fig. 210) colorless except for bright red eye and pale orange egg sac ; total length (from tip of rostrum to tip of caudal rami) 0.929-0.979 mm (average 0.961 mm); greatest width, a t level of first thoracic segment, 0.415 mm ; genital segment, 0.641 mm long, distinctly divided dorsally and laterally by transverse furrow; first four thoracic segments and genital segment each with minute slender seta, 0.010 mm long, located near posterolateral margin in addition to a pair of similar setae in front of transverse furrow; abdomen of three segments ; anal operculum as a smooth flap ; rostrum, 0.028 mm long, broadly rounded, with two minute setae; second thoracic segment 0.115 mm long; third thoracic segment 0-103 mm

9. THE CLASS CRUSTACEA

313

long, fourth thoracic segment 0.080 mm long, fifth thoracic segment 0.064 mm long, sixth thoracic segment 0.136 mm long; genital and abdominal segments each armed with a row of minute spines along posteroventral margin but unarmed dorsally ; caudal ramus, 0.029 mm x 0.025 mm, slightly wider than long, with longest terminal seta, which may be partially retracted, measuring 0.336 mm ; two additional setae present on ramus; egg sac dorsoventrally flattened, 0.290 mm long x 0.270 mm wide x 0-120 mm thick, reaching bases of caudal

FIGS.210 and 211. Tisbe celutu adults. (210) Female, dorsal view; (211) thorax and abdomen of male, dorsal view. (Redrawn after Humes, 1954a.)

rami, contains about forty-five eggs, each about 0-054 mm in diameter ; first antenna of eight segments measuring 0.038, 0.060, 0.044, 0.027, 0.015, 0.014, 0.010 and 0.028 mm long respectively; fourth segment with aesthetask, 0.125 mm long, extending well beyond tip of antenna; second antenna with four segments of endopodite somewhat indistinctly separated ; first swimming leg with exopodite shorter than endopodite, bent at an angle so as to cross behind endopodite, endopodite podomeres measure 0.082, 0.081 and 0.007 mm respectively, exopodite podomeres measure 0.045, 0.045 and 0.020 mm respectively ; second,

314

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

third and fourth pairs of legs similar except for certain differences in armature, stronger inner basipodite spine absent and outer basipodite spine slender in all three legs ; leg spine and setal formula as follows :

1st podomere 2ndpodomere 3rd podomere

Lag 1 exp and

Leg 2 exp and

Leg 3 exp end

1:0 1:l 6

1:l 1:l 7

1:l 1:l 8

0:l 0:l 2

0:l 0:2 5

0:l 0:2 6

Lag 4 exp end 1:l 1:l

0:l 0:2

8

5

fifth leg with three slender setae on inner expansion of basal podomere, middle seta 0.115 mm long, two shorter setae subequal, about 0.048 mm long; single seta, 0.044 mm, a t outer corner of podomere; distal podomere 3.1 times longer than wide, averaging 0.097 mm x 0.031 mm, with four terminal setae, three subequal in length (0.046-0*051 mm), fourth member of quartette, next to innermost, longer (0.067 mm), fifth seta, 0.073 mm long, on outer edge of podomere at junction of distal two quarters, with small spines along both edges of podomere ; sixth pair of legs visible on anteroventral part of genital segment, represented by three setae on either side of oviducal opening. Adult male (Fig. 211) resembles female in color and body form; total length 0.705-0-800 mm (average 0-781 mm), greatest body width 0.328 mm, a t level of first thoracic segment ; abdomen of four segments ; rostrum 0.025 mm long ; head plus somite of first leg 0-223 mm long, second, third, fourth, fifth and sixth legs measure 0.070, 0.083, 0.064 0.057 and 0.061 mm long respectively ;caudal ramus, 0-020mmlong, like that of female ;first antenna subprehensile, with nine segments measuring 0.030, 0.048, 0.023, 0.006, 0.029, 0.009, 0.021, 0.022 and 0.032 mm respectively, aesthetask on fifth segment measures 0.109 mm long ; second antenna similar to that of female ; second through fourth legs like those of female ; distal podomere of fifth leg, 0.042 mm x 0.018 mm, shorter than that of female, with few minute spines on slightly convex broad outer surface, with middle spine, 0.043 mm long, broad and with fine hair-like tip; inner expansion of proximal podomere less pronounced than in female, with two setae, one long (0.057mm) and one short (0.017 mm); sixth leg as broad lobe bearing spinose, distally naked setae, a row of small spines near bases of two inner setae ; spermatophore, 0.070 mm x 0.030 mm with slender neck.

9. THE CLASS CRUSTACEA

315

Other information. Humes (1954a) has stated that : T . celata would seem t o be a parasite of Mytilus edulis rather than a freeswimming species accidentally introduced into the mantle cavity, since it occurs only in the mussel and then in relatively large numbers, and since the presence of immature stages indicates it probably breeds in the mussel. 16. Lichomolgid Copepods Humes and Cressey (1958) described three species of the family

Lichomolgidae from the mantle cavities of pelecypods of limited commercial importance in West Africa. These are briefly mentioned a t this point. They described Anthessius sp. from Pinna rudis taken a t a depth of 1 m at Pointe-Noire, Congo; Modiolicola injlatipes from Mytilus perna attached to rocks a t Pointe-Noire ; and Lichomolgus arcanus from Ostrea tulipa collected at Joal, Senegal, and from Pitar tumens collected at Hann, Senegal.

B. Subclass Malacostraca Among the malacostracans, a number of decapods are known to live symbiotically with commercially important marine molluscs, a t least during one phase of their life cycles. Specifically, certain species of crabs of the family Pinnotheridae are known t o live within the mantle cavities of oysters and other pelecypods. Although the relationships between these crabs and their hosts are not clearly understood, a t least certain species are believed to be parasitic (see Chapter 4, Section 11, A, 6). Since the nature of the relationship between pinnotherid crabs and their hosts is a t best uncertain, the following information is limited primarily to the more common species, especially those which have been incriminated as being true parasites. For detailed information on the entire family, the monographs of Rathbun (1918) and Sakai (1965) should be consulted. 1. Pinnotheres ostreum Say, 1817. (Figs. 212-215)

(Order Decapoda; suborder Reptantia; section Brachyura; superfamily Brachyrhyncha; family Pinnotheridae) The adults and post-planktonic stages of Pinnotheres ostreum, the oyster crab, are found primarily in the mantle cavity of the American oyster, Crassostrea virginica, but they can also occur in Aequipecten and Anomia simplex (Christensen and McDermott, 1958) and in Mytilus edulis (McDermott, 1961). They need not always be associated with pelecypods since Gray (1960, 1961) has reported finding them occasionally in Chaetopterus tubes. P. ostreum is a New World species with its known range extending from Massachusetts to Santa Catarina, Brazil

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

(Williams, 1965). According to Williams, its synonyms include P. depresseum Say, 1817 and P. depressus Rathbun, 1918. The combined contributions of Birge (1882), Hyman (1924), Stauber (1945), Sandoz and Hopkins (1947) and Christensen and McDermott (1958) have made this the best understood of all the species of Pinnotheres. Due t o space limitations, it is not possible to give an exhaustive treatment of all that is known about P. ostreum. Interested readers are referred t,o the original papers cited for details.

Description of stuges in mollusc. The excellent comparative chart given by Christensen and McDermott (1958))in which the distinguishing characteristics of the stages which may be encountered in molluscs are listed, is reproduced in Table XVII. In addition, the descriptions of the mature female and so-called hard-shell male are given below. Mature female (Fig. 212) whitish or salmon pink, with carapace subcircular, 4-15 mm wide, surface mostly glabrous, smooth, shiny, membranous, yielding to touch, convex from front to back and with a broad, shallow, longitudinal depression at each side of cardiac and gastric areas ; lateral margins thick and bluntly rounded ; posterior margin broad ; front rounded, slightly produced, covering and concealing eyes ; orbits small, subcircular, anteriorly placed ; antennule large ; antenna small, flagellum not as long as diameter of orbit ; buccal mass roughly quadrangular but bent into broad crescentic arch, short anteroposteriorly ; outer maxilliped with ischium and merus fused ; TABLEXVII. POST-PLANKTONIC DEVELOPMENTAL STAGES OF Pinnotheres ostreum Data based on observations by Stauber (1945) and Christensen and McDermott (1958). (After Christensen and McDermott, 1958.)

Stage of development

Range in carapace Most important external width morphological characteristics (mm)

Invasive stage 0-69-0.73 Flattened carapace and (First crab stage) pereiopods. Posterior margins of pereiopods thickened, 3rd and 4th pairs have plumose swimming hairs. Two small, white spots on carapace and on sternum. Carapace hard around these spots

Biological factors

Free-swimming until invasion of host. After invasion it is found in all parts of waterconducting system of the host

317

9. THE CLASS CRUSTACEA

TABLEXVI1.-continued

Stage of development

Range in carapace Most important external width morphological characteristics (mm)

Pre-hard stages

Male 0*75*-2.7* Female 0.75*-2*7*

Hard stage (stage I of Stauber, 1945)

Male 1.4-4.6 Female 1.3-2.7

Stage I 1

1.3*-3.1

Stage I11

2.6-4.4

Stage I V

3.6-84

Stage V (Mature female)

4.4-15.1

* Approximate measurements.

Rounded carapace. Thin, flexible exoskeleton. Slender pereiopods. No swimming hairs. Large females practically indistinguishable from 2nd stage crabs Carapace flattened and very hard. Flattened pereiopods with posterior margins thickened and with plumose swimming hairs on 3rd and 4th pair. Two large, white spots on carapace and on sternum. Males larger on the average than females Rounded carapace. Thin flexible exoskeleton. Slender pereoipods. No swimming hairs. Abdomen wholly contained in sternal grove. No hairs on pleopods Edges of abdomen extend beyond depression in sternum. First two pairs of pleopods clearly segmented and supplied with a few hairs Relative width of abdomen larger than in preceding stage, just reaching coxae of pereiopods in most cases. Pleopods almost fully developed and well supplied with hairs Abdominal edges covers coxae of pereiopods. Pleopods fully developed. The orange gonads may be seen through the thin carapace

Biological factors

Found in all parts of the water-conducting system of the host

Found free-swimming and in all parts of water-conducting system of the host. Copulatory stage. Males die in this stage

Never free-swimming. Predominantly, possibly always, found only on the gills of the host Only found on the gills of the host

As in 3rd stage

As in 3rd stage

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MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

carpus (first article) of palp short and oblong ; propodus elongate with rounded terminal ; dactyl inserted behind middle of propodus, minute and slender ; chelipeds small ; merus and carpus slender ; palm some-

FIQS.212 and 213. Pinnotheres ostreum. (212) Adult female, dorsal view; (213) adult male, dorsal view. ( R e d r a m after Williams, 1965.)

what flattened inside, swollen outside, widened from proximal toward distal end, then narrowed ; width across base of fingers less than width of palm ; fingers stout, not gaping, tips hooked past each other, with minute teeth on opposing edges and a larger tooth near base of each ;

9. THE CLASS CRUSTACEA

319

walking legs slender, subcylindrical, last two articles with thin fringe of hair; second and third legs subequal in length but with first leg slightly stouter ; abdomen large, extending beyond carapace in all directions. Hard-shell male (Fig. 213) and female dark or brownish with two large almost circular pale white spots on both carapace and sternum ; with dorsal spots on brachial regions and ventral spots flanking abdomen and medial to first pair of legs ; with well-calcified carapace, 1.4-4.6 mm wide in males, 1.3-2.7 mm wide in females, flat dorsally, subcircular in outline, with truncate front more advanced than in mature female ; posterior margin straight ; lateral margin thin, sharply bent from dorsal side, margin marked by a raised band of short dense hair ; eyes well developed ; buccal mass crescentic, arched, broad from side to side but short anteroposteriorly ; cavity completely closed by external maxillipeds; chelipeds stout; merus and carpus not slender as in mature female ; palm slightly flattened inside, swollen outside, with both margins convex ; hands with bands of pubescence on upper and outer surfaces of palm and outer surface of immovable finger ; fingers stout, toothed proximally, tooth fitting between two protuberances on immovable finger when closed; both fingers with stiff hairs on gripping edges ; walking legs flattened, with posterior margin thickened and with plumose swimming hairs on second and third pairs; abdomen narrow, confined to sternal depression ; copulatory stylets of male well developed, first pair blade-like and hairy, second pair rodlike and almost hairless. Life cycle. The pre-symbiotic larval stages of P. ostreurn occur in the plankton. These include four zoeae followed by one megalops. Descriptions of the first zoeal stages have been given by Birge (1882) and redescribed by Hyman (1924) while descriptions of all the planktonic stages have been given by Sandoz and Hopkins (1947). The planktonic larval stages do not possess dorsal or lateral spines on the carapace. The time required from hatching to molting of the megalops to give rise t o the first crab stage is 25 days. The symbiotic (parasitic?) stages have been studied by Stauber (1945) and Christensen and McDermott (1958). The so-called '' invasive stage '' is the first crab stage (Figs. 214 and 215). It is this form that invades oysters. Six additional developmental stages ensue in the case of females, including the mature adult stage. Males do not develop beyond the hard stage. I n both sexes it is the hard stage which is the specialized stage a t which copulation occurs. The males leave their hosts a t this stage to search for females in another host. It is only during this stage that the crab is free-swimming. After copulating

320

MARINE MOLLUSCS AS HOSTS BOR SYMBIOSES

with one or more females, which usually takes place in June or July, the males disappear. Thus males only become 1 year old or less. Females become ovjgerous in their first summer but do not attain maximum size before their second summer. Some do not attain this until their third summer. Fully grown ovigerous females measure from 9.4 to 10.8 mm in width. These carry from 7 957-9 456 eggs. The

P

FIGS.214 and 215. Stage I (hard stage) Pinnotheresostreurn. (214)Female, dorsal view; (215) female, ventral view. (Redrawn after Stauber, 1945.)

exact period for which the eggs are carried is not known but it is believed to be from 3 t o 5 weeks. Females only produce one batch of eggs during the first year but may produce two batches during the second and third years. Ecology. Again, interested readers are referred to the detailed ecological observations by Stauber (1945) and Christensen and McDermott (1958). It is known that in Delaware Bay, and presumably also in other temperate regions, invasion of oysters by P. ostreum is

32 1

9. THE CLASS CRUSTACEA

seasonal. Few invasions occur before 1 August. It should be noted that the peak of oyster setting occurs during July in Delaware Bay ; thus by the time the f i s t crab stage commences to invade in large numbers, the oyster spat has grown to sufficient size to harbor one or two crabs. A single young oyster measuring 4.2 mm long may harbor two crabs while larger ones may harbor up to seven crabs. The crab appears to prefer spat (76.7% infestation) over yearling oysters (54.6 yoinfestation) and older ones (21.5% infestation), but the survival rate of crabs is better in yearlings and older oysters. Although the first crab stage (invasive stage) of P. ostreum can be found in oysters all winter, growth and development stop in Delaware Bay a t the beginning of November when the ambient water temperature begins to drop below 15°C. Pathology. During the feeding of P. ostreum in oysters, two types of gill erosions are secondarily effected. These pathological changes are discussed in an earlier section (Chapter 4, Section 11,A, 6). Resulting from a study designed to determine the effect of P. ostreum on the gross physical condition of Crassostrea virginica, Haven (1959) has demonstrated that oysters harboring crabs contain less soft tissues (meat) per unit of shell cavity volume than those without crabs but the water contents of oysters with and without crabs are not significantly different. Another aspect of crab-associated change in oysters has been postulated by Christensen and McDermott (1958). These investigators believe that P. ostreum may influence the sex ratio among oysters during their second spawning season if they retain their crab " parasites '' from the first year. Their postulation is based on the earlier finding by Awati and Rai (1931) who found that among 794 uninfested Ostrea cucullata, 41.7% were males, 56.4% were females, and 2.9% werc hermaphrodites. On the other hand, among eighty-six oysters harboring Pinnotheres s ~ . 82.6% , were males, 10.4% were females, and 7.0% were hermaphrodites. Since females can be induced to change sex in the laboratory by simple starvation, Awati and Rai concluded that the crab probably interferes sufficiently with food intake of the oyster so that it produces sperm instead of the more " expensive " eggs. That the change in sex is due to reduction in food intake and is not chemically stimulated by some crab-secreted substance appears to be supported by the findings of Amemiya (1935) and Egami (1953), who have shown that the experimental removal of a part of the gill in Crassostrea gigas will cause the number of males to far exceed that of females during the breeding season, provided that the operation is performed no later than the previous October. A.P.B.-6

21

322

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

2. Pinnotheres maculatus Say, 1818. (Figs. 216 and 217)

(Order Decapoda; suborder Reptantia; section Brachyura; subsection Brachygnatha; superfamily Brachyrhyncha ; family Pinnotheridae) Pinnotheres maculatus, the mussel crab, is the second New World species which is known t o be a symbiont of various marine invertebrates, including several commercially important molluscs. Its range,

FIQS.216 and 217. Pinnotheres maculatus. (216) Adult female, dorsal view; (217) adult male, dorsal view. (Redrawn after Williams, 1965.)

according to Williams (1965), extends from off Martha’s Vineyard, Massachusetts, to Mar del Plata, Argentina. Both fully developed males and females have been reported in the mantle cavities of a large number of pelecypods including Mytilus edulis, Modiolus modiolus, M . americanus, Mya arenaria, Aequipecten irradians, A. gibbus, Placopecfen magellanicus, Atrina serrata and oysters (species unspecified).

9. THE CLASS CRUSTACEA

323

It has also been found in the tubes of Chaetopterus variopedatus, from Molgula robusta, in the pharynx of Bostrichobranchus pilularis and on Asterias vulgaris. It was originally described by Say (1818) as inhabiting " the muricated Pinna of our coast." Descriptions. Mature female (Fig. 2 16) with suborbicular carapace, 13.7 mm long, 14.3 mm wide, thick and firm but not hard, convex; surface uneven, covered with short and deciduous tomentum ; gastrocardiac area higher than and separated by depressions from branchiohepatic area ; front slightly advanced, approximately one-fifth width of carapace, subtruncate in dorsal view, slightly bilobed ; orbits small, subcircular ; eyes spherical ; antenna longer than width of orbit ; antennule large, obliquely transverse ; buccal mass roughly quadrangular, crescentic, much broader than long ; ischium and merus of external maxilliped united ;propodus larger than carpus ; dactyl narrow, curved, spatulate, attached near middle of propodus, reaching to near extremity of propodus ; chelipeds moderately stout, articles subcylindrical, more or less pubescent ; carpus elongate ; palm thick, blunt edged, increasing in size distally ; fingers stout, fitting closely together with tips hooking past each other ; immovable finger nearly horizontal ; dactyl with tooth near base fitting into sinus with tooth at either side on immovable finger ; walking legs slender, hairy above and below ; second pair longest but shorter than chelipeds ; first three dactyls falcate, shorter than propodi; last leg shortest, turned forward and upward, with long dactyl of same length as propodus. Obscure brown in color, Young females resemble dark-colored males except in shape of abdomen and characteristics of appendages ;free-swimming,measuring up to 6.2 mm in length. More mature females symbiotic with hosts (usually pelecypods) ; light colored, measuring from 3.3 mm in length ; long hair persists on such small- and medium-sized females. Mature male (Fig. 217) with flat subcircular carapace, 9.1 mm long, 8.7 mm wide, harder than female ; regions superficially defined, more by color than by structural prominence, light areas mostly elevated, usually allowing pubescence to wear ; gastric, cardiac, and branchial regions separated by broad, shallow confluent indentations ; front broad, prominent, depressed, slightly lobed, approximately one-third width of carapace ; orbits subcircular, eyes large ; antenna somewhat longer than width of orbit; chelipeds shorter than in female, hands stouter ; walking legs wider, especially propodal articles of first three legs ; posterior surface overlaid with thin fringe of hairs attached near upper margin ; last leg relatively shorter than in female, not reaching propodus of third leg, dactyl more like third leg than in female;

324

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

abdomen at middle, approximately one-third width of sternum, gradually narrowing from third to seventh segment, sides of third convex, sides of seventh obtusely rounded ; sutures between segments of abdomen and sternum with narrow lines of dark pubescence. Strikingly light dorsal color pattern of bare spots on a background of dark pubescence consisting of a median stripe constricted on middle and behind, a subtriangular spot on each side in front of middle, and a linear spot on each side behind; chelipeds with dark pubescence on inner and upper surface of carpus, a bit on upper surface of merus and inner side of palm proximally, otherwise scattered flecks on hands and walking legs. Some males resemble mature females in coloration and structure of legs, ranging in length from about 4 mm upwards. Such males are symbiotic. Life cycle. Accounts of the life cycle of P. maculatus have been contributed by Pearce (1964) and Costlow and Bookhout (1966). As in the case of P. ostreum, the zoeal and megalopal stages are planktonic. Myman (1924)has described the first zoeal stage which, unlike that of P. ostreum, has well-developed spines in the carapace. According to Costlow and Bookhout, who have reared the larval stages from hatching to the crab stage under laboratory conditions there are five zoeal stages followed by the megalops stage. Thus, in addition to morphological differences,which have been given in detail by Costlow and Bookhout, P. maculatus differs from P. ostreum in its number of zoeal stages, the former with five and the latter with four. It may be noted that thenumber of zoeal stages is known to vary among the Pinnotheridae. For example, Lebour (1928b),working with material from the plankton, has reported two zoeal stages in the life cycle of P. veterum; Hart (1935), who has reared P. taylori, has also reported two zoeal stages in this species ; and Irvine and Coffin (1960) have reported four zoeal stages in the life cycle of Pabia subquadrata. According to Pearce (1964), ovigerous females of P. maculatus occur from late May to mid-June in the Woods Hole, Massachusetts, area. By early August the eggs are hatched and the zoeal and megalopal stages are planktonic. The megalopal stage molts during mid-September to give rise to the first true crab stage. It is at this stage that P. maculatus invades the mantle cavity of Mytilus edulis. Within the host, the crab undergoes several molts with each succeeding instar being somewhat larger but morphologically similar to the preceding. By mid-October, the crabs measure approximately 3.3 mm in carapace width. A molt follows, resulting in an anomalous juvenile with a well calcified exoskeleton (thus compamble to Christensen and McBermott’s hard stage). It is

9.

THE CLASS CRUSTACEA

325

at this stage that hard stage males and females leave their host and engage in copulatory swimming in open water. After this, females reinfest mussels and undergo four post-hard molts with each instar possessing unique characteristics but all with soft and poorly calcified exoskeletons. Male crabs in the hard stage are said to spend more time in open water during and after copulatory swimming and hence are more subject to predation; however, a few males are found in hosts following swimming. It is of interest to point out that Pearse has reported that juvenile (hard stage) females which enter hosts already inhabited by mature females beome retarded in their development and do not reach the sexually mature stage V instar. All crabs are believed to over-winter in the first post-hard (stage 11)instar, but with the advent of higher water temperatures in May the precociously inseminated crabs undergo the remaining three post-hard molts and attain the adult form (stage V).

Pathology. Detailed accounts of damage inflicted in molluscs by P. maculatus are not yet available. It may or may not damage its host's gills like P. ostreum and P. pisum although it is suspected that it does. Ecology. The seasonal periodicity manifested in the appearance of ovigerous females varies through the range. Rathbun (1918) has indicated that such females occur in January at St. Thomas, Virgin Islands, in March in Jamaica, from May to November in Florida, from June to January in North Carolina, from July to September in Rhode Island and Massachusetts (although Pearce has stated that they only occur from late May to mid-June at Cape Cod), and in June in Brazil. Relative to the swimming velocity of P. maculatus larvae, Welsh (1932) has reported that it is greatly influenced by temperature and light intensity. At temperatures between 20" and 25"C, the maximum swimming velocity is attained at light intensities between 10 and 25 meter-candles. When maintained a t different constant temperatures, it is known that there is a change in the relationship of velocity to light intensity, with the maximum possible velocity occurring at each constant temperature during the initial period of exposure to higher temperatures. Furthermore, the light intensities will bring about a marked effect on general activity. 3. Pinnotheres poisum Linnaeus, 1767. (Figs. 218-221)

(Order Decapoda; suborder Reptantia; section Brachyura; subsection Brachygnatha ; superfamily Brachyrhyncha; family Pinnotheridae) The pea crab, Pinnotheres pisum, is the common symbiont of pelecypods in northern European waters. It appears t o be most

326

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

frequently found in Mytilus edulis although it has been reported from Cardium edule, Ostrea edulis (Orton, 1920) and Cardium norwegicum

FIGS.218 and 219. Pinnotheres pisum. (218) First crab stage (invase stage); (219) prehard stage. (Redrawn after Christensen, 1959).

(Smith and Weldon, 1904). It has been known since the days of the Pharaohs and has been the subject of many colorful, although not quite accurate, legends (see Calman, 1911).

9. THE CLASS CRUSTACEA

327

Descriptions of stages I-IV females have been given by Atkins (1926) (Figs. 218-221).

FIQE.220 and 221. Pinnotheres pisum. (220) Hard stage female, dorsal view; (221) stage III female, dorsal view. (Redrawn after Atkins, 1926).

Descriptions. Adult female between 9 and 18 mm wide, carapace soft, wider than long, often quadrilateral in shape, generally without conspicuous color pattern ; front very narrow, about one-fifth width of

328

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

carapace, hardly visible from dorsal surface ; eyes feebly developed, very small and not visible from above; abdomen large, overlaps basipodite of legs laterally, completely covers mouth parts anteriorly, reaching immediately posterior to eyes ; last abdominal segment bent inwards rather sharply while feeding so that mouth is uncovered; abdomen deeply hollowed ; thorax hollowed ; numerous long hairs on edges of abdomen and sternum, between bases of cheliped and on pleopods. Smallest ovigerous female with carapace 7.5 mm wide ; numerous eggs carried in cavity formed by hollowed thorax and deeply hollowed abdomen ; space between sides of abdomen and thorax very small, well guarded by long fringing hair ; exopodite of second pair of pleopods long and blade-like, fringed with long and numerous hairs, fits along inside of gap as far forward as fifth abdominal segment and gives protection to eggs. Hard-shelled male with carapace almost circular, 1.9-6.3 mm wide, for the most part glabrous, light gray or fawn in color, with a conspicuous pattern of pale yellow areas outlined with darker yellow or yellowish orange ; in larger specimens the yellow areas increase in size and fuse to cover the greater part of dorsal surface of carapace ; pigmented lines and areas also occur on chelipeds and legs, including symmetrically placed yellow spots on their ventral surfaces ; frequently numerous black, occasionally red, chromatophores scattered over body ; chelipeds hairy, with broad and swollen palms; two rows of setae beneath chela, with one reaching from base of palm to tip of finger and other extending on inner surface only slightly beyond immovable finger, both rows converging distally ; large tooth present near base of dactylus, fits into slight notch, with small tooth a t both ends, in propodia1 finger; both biting surfaces bear stiff setae, with closely set spines towards tips of fingers; small teeth of propodial finger and curved spines from both fingers absent in some ;walking legs somewhat flattened, extremely hairy, with hairs being plumose ; second leg slightly longer than third, both extremely hairy; first leg shorter than third, fourth leg shortest ; short curved dactyli terminate in horny tips ; abdomen narrow and tapering ; two small transversely ridged chitinous nodules on fifth thoracic somite which fit into two pockets on sixth abdominal segment ; copulatory organs large, first appendage bladelike and hairy, with closed groove along inner surface, with numerous rosette glands around lower portion of groove, second appendage rodshaped with swollen base, with distal stylet almost hairless and without glands. Atkins (1958) has reported the occurrence of thin- or soft-shelled males. She states : " The thin-shelled male has a normal male abdomen,

9. THE CLASS CRUSTACEA

329

only slightly wider in relation to the width of the thorax than that of the thick-shelled male. . . thin-shelled males (measure) 1-9-6.3 mm.” Life cycle. P. pisum, like P. ostreum, includes four zoeal stages and a single megalops. The first zoea has been described, but not very effectively, by Thompson (1835) and later redescribed in detail by Lebour (1928a,b). What may have been the second zoea has been described by Lebour (19288). All of the stages, including the prezoea, first through fourth zoeae, and megalops, have been described in detail by Atkins (1955). The zoeal and megalopal stages are planktonic. The most recent and complete accounts of the life cycle of P. pisum are those given by Christensen (1959, 1962). Prior to this, Thompson (1935) had briefly described male and female adults. He stated that the males are hard-shelled while the females are soft-shelled. This concept was held until Orton (1920) demonstrated the existence of hard-shelled females which resemble males in all respects except for their genital apertures and pleopods. Atkins (1926) has described the molting stages of P. pisum in Mytilus edulis but followed Orton (1920) in designating the hard-shell stage as the first crab stage (which obviously does not correspond to the first crab stage of P. ostreum as given by Christensen and McDermott (1958)). Later Atkins (1954, 1955) realized her error and abandoned the concept that the hard stage is the first crab or invasive stage. Atkins (1955) has described the postembryonic development of P. pisum and still later (1958) has recorded the occurrence of thin-shelled males which resembles stage I1 females. Actually soft-shelled males had been found by Mercier and Poisson (1929) but these French workers failed to realize that this is a normal form. They were of the opinion that the soft-shelled condition had resulted from parasitism by an entoniscid parasite. Although Atkins (1958) has advanced our understanding of the stages in the life cycle of P. pisum by definitely demonstrating the soft-shelled males, she did not grasp their significance. She expressed the opinion that males altered between the hard- and soft-shelled forms during growth. It is now clear that the hard-shelled form, or hard stage, precedes the softshelled stages. Investigators prior to 1959 had consistently failed to find the prehard stages of P. pisum in Mytilus edulis, and it was for this reason that Atkins (1926) was originally of the opinion that the hard stage represents the first crab or invasive stage. Christensen (1959), however, has been able to correct this misinterpretation with the discovery that P. pisum changes hosts at the hard stage and hence this is the youngest form found in mussels. He has also found the true first crab or invasive stage of P. pisum in Spisula solida. Although Christensen (1962, and personal

330

MARWE MOLLUSCS AS HOSTS FOR SYMBIOSES

communication) later found an ovigerous female in S. solida and informed Houghton (cited in Houghton, 1963) that he had also found what is possibly a pre-hard stage of P. pisum in a mussel at Roscoff, he considers these to be exceptions. Thus it would appear that although the symbiotic (parasitic) stages of P. pisum are comparable to those of P. ostreum (see Table XVII), its life cycle differs in that two hosts are involved, with the second being invaded by the hard stage. It is of interest to note that Christensen (personal communication) has reported that the ovigerous female found in Spisula solida was only about 4 mm in carapace width and was obviously retarded in its growth but not its development.

Ecology. As the result of field studies in Langstone Harbour, near Portsmouth, England, and at Conway, Wales, Houghton (1963) has reported that the larger crabs are found only in Iarger mussels ;furthermore, this crab is found more frequently in the larger mussels which occur in greater numbers lower along the shore. Houghton has also reported that “ the percentage of infestation differs from one locality to another a t the same tidal level and that it increases from the middle shore to the sublittoral zone for any given size-group.” Since Christensen (cited in Houghton, 1963) has found that the first crab stage, which is the invasive stage, is photonegative, it is unlikely that it will invade a, primary host high up on the shore line or near the water surface. It thus follows that the hard stage escaping from the primary host is more likely to enter Mytilus edulis living from the middle shore to the sublittoral zone where the primary host is found. Other information. Orton (1920) has observed the feeding habits of P. pisum in Mytilus edulis through a window cut in one of the host’s valves. He has reported a feeding habit similar to that observed by Stauber (1945) in P. ostreum (see Chapter 4, Section 11,A, 6). It should be noted, however, that Orton did not observe the pea crab to inflict any injury to Mytilus edulis comparable to that observed by Stauber in Crassostrea virginica invaded by P. ostreum. Nevertheless, the presence of P. pisum in mussels is known to cause certain physiological changes. Specifically, Berner (1952) has noted that there is a partial or even complete cessation in the production of gametes in those mussels infected with P. pisum measuring 10 mm or more in carapace width. Mussels harboring smaller crabs are very seldom affected in this manner. This change in sex in Mytilus edulis is most probably the result of starvation as is the case of Ostrea ceccullata infested by Pinnotheres sp. (Awati and Rai, 1931) and not due to some crab-secreted substance.

9. THE CLASS CRUSTACEA

331

4. Pinnotheres pholadis de Haan, 1835. (Figs. 222 and 223) (Order Decapod&; suborder Reptantia; section Brachyura; superfamily Brachyrhyncha; family Pinnotheridae) This species, originally described by de Haan (1836), has been reported but not redescribed since then by Tesch (1918))Balss (1922))

i'

FIQS.222 and 223. Pinnotheres pholudia. (222) Adult female, dorsal view; (223) adult male, dorsal view. (Redrawn after Sakai, 1965.)

Yokoya (1928,1933)and Sakai (1934,1936,1939, 1963)from a number of marine pelecypods including Chlarnys nipponensis, Mytilus edulis, Crassostrea gigas, Meretrix lusoria, Tapes japonica and Mactra sulcataria. According to Sakai (1966))who redescribed both the male and female of P . pholudis, P . cardii, described by him (Sakai, 1934))is a synonym.

332

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

Description. Adult female (Fig. 222) with soft exoskeleton, carapace rounded quadrangular, 12-13 mm in length and width, with markedly deflexed front; with four pairs of ambulatory legs, with the second being the longest ; row of long hair on carpi and propodi in very young specimens, disappears in full-grown ones ; dactyli of all legs very short and uniformly hook-shaped ; dactylus of external maxilliped styliform with tip exceeding that of propodus. Adult male (Fig. 223) smaller than female, with well-calcified body ; carapace circular, about 9 mm in length and nearly 9 mm in width ; dorsal surface strikingly convex, frontal edge thick, produced anteriorly, slightly emarginated in middle ; of four pairs of ambulatory legs, the second is longest; carpi with an oblique row of very long, featherlike hairs ;propodi with a row of similar hairs along anterior border ;dactylus of external maxilliped styliform as in female. Other information. According to Sakai (1965), during the mat,ing season, which occurs in early summer in Japan, both the male and female are usually seen together within the same mollusc's mantle cavity. This would suggest that unlike the three species of Pinnotheres discussed previously, neither the male nor both the male and female leave the pelecypod. 5 . Other Pinnotherid Grabs

Rathbun (1918), in her definitive treatise of the grapsoid crabs of America which includes the Pinnotheridae, has listed twenty-seven species of the genus Pinnotheres. Of these, she lists seven which are " commensals " of commercially important pelecypods (Table XVIII). No host is listed for ten other species, while the remaining ten are listed as " commensals '' of other types of invertebrates. Among the four species of Fabia listed, one is noted as a " commensal '' of Tapes and other pelecypods, while another, F . subquadrata, is listed as a " commensal " of " bivalved molluscs ". Of the five species of Parapinnixa and seven species of Dissodactylus, she lists none to be associated with a pelecypod. Of the twenty-six species of Pinnixa, she lists two species as being " commensals '' of commercially important molluscs (Table XVIII). The monotypic genera Xcleroplax, Tetrias and Pinnotherelia are reported not to include symbionts as is the case among the three species of Pinnaxodes. The single species of Opisthopus, 0. transversus, is said to be a " commensal of Mytilus edulis ". The relationships between the eleven species of pinnotherid crabs listed by Rathbun as occurring in the mantle cavities of commercially important molluscs have not been investigated. If futuro studies should

TABLE XVIII.

SPECIESOF PINNOTHERID CRABSREPORTED BY RATEBUN(1918) AND SAKAI(1965) COMMERCIALLY IMPORTANT MOLLUSCS “Oysters” M y t i h

Pinnotheres ostreurn P . geddesi P . politus P . angelicus P . maculatus P . margarita P . guerini P . sinensis P . boninewis Fabia lowei Pinnixa faba P . littoralis Opisthoptm tranaveraua Ostracotherea subquudratwr

+ + + + + + + +

+ + +

Mya

+

Aequipecten Pinna Margaritifera

+

+

Tapes

TO BE

SYMBIOTIC IN

“Clams” Gaatropods

+

i

+

+ + +

+

+

+*

* Schizothaerus.

W

w

w

334

MARINE MOLLUSCS AS HOSTS FOR SYMBIOSES

reveal metabolic dependency, as is suspected in the case of Pinnotheres ostreum, P. maculatus and P. pisum, then these pinnotherids should be considered as parasites. For further information, especially the descriptions of these species, Rathbun’s monograph should be consulted. Sakai (1965), in his monograph on the crabs of Sagami Bay, has listed those species of pinnotherid crabs commonly found in commercially important molluscs in the Far East. These are also included in Table XVIII. References to the descriptions of these species, along with notes on their natural history, can be found in Sakai’s monograph. Since the appearance of Rathbun’s monograph, several authors have reported the occurrence of immature Pinnixa fuba and P. littoralis associated with various species of pelecypods, including some commerically important ones. Although the relationship between these two species of crabs and their molluscan hosts has not been demonstrated to be a parasitic one, such accounts are being briefly mentioned for those interested in crabs associated with commerically important marine molluscs. Pearce (1966a) has reported that the adults of both P. faba and P . littoralis are sympatrically distributed with their clam host, Tresus capax, in Puget Sound, State of Washington, but are never associated with the closely related clam T . nutalli. Furthermore, he has confirmed the observations of others that immature specimens of both of these crabs are found within the mantle cavities of a number of pelecypods. Specifically, Pearce has confirmed Rathbun’s (1918) report that immature P. faba and P. littoralis have been found in Mya arenaria, Tapes, Saxidomus, Macoma nasuta and “ cockles ”, and Wells’s (1928, 1940) reports that the young of P. littoralis can occur in Macoma nasuta, M . inquinuta, M . indentata, M . secta, Mya arenaria, Saxidomus giganteus and Clinocardium nuttalli. It should be mentioned that in addition to immature specimens, Wells (1940) has also reported the occurrence of both P.faba and P. littoralis adults in small specimens of Macoma, Mya and Cardium on rare occasions. It would appear from such observations that both P. faba and P. littoralis may undergo a change in host as is the case with Pinnotheres pisum. In another paper (Pearce, 1966b), it has been reported that the post-planktonic stages of Fabia subquadrata occurs in the mantle cavity of the horse mussel, Modiolus modiolus, collected from the San Juan Archipelago in the State of Washington. Pearce not only has given a detailed description of the ecology, mating habits, and growth of this crab, but also has considered it a true parasite, basing his opinion on the fact that F. subquadrata causes extensive damage to the gills, palps, and mantle of its host.

9. THE CLASS CRUSTACEA

335

6. Other Crabs Associated with Pelecypods Crabs of the family Xanthidae, commonly known as mud crabs, are known to include certain species which are predators of the American oyster, Crassostrea virginica, and other pelecypods. For example, according to McDermott (1960), both Chestnut (unpublished) and Smith and Richards (unpublished) have shown that mud crabs, probably Panopeus herbsti, prey on Crassostrea virginica in New Jersey waters. It has been reported that a single crab will destroy thirty oysters in a month under laboratory conditions. McDermott and Flower (1952) have reported that Panopeus herbsti is an active predator of 1- or 2-year-old oysters. Similarly, Menzel and Hopkins (1956) have reported that another xanthid crab, Menippe mercenaria, preys on oysters, and Landers (1954) has reported that in Rhode Island another species, Neopanope texana preys on young Mercenaria mercenaria. McDermott (1960) has reported that Panopeus herbsti, Neopanope texana sayi and Eurypanopeus depressus will all prey on Crassostrea virginica in nature and that under laboratory conditions Panopeus herbsti will destroy 1- to 2-year-old oysters at the rate of 0.15 oyster per crab per day. He has also demonstrated a selectivity on the part of this crab for the smaller, thin-shelled specimens. The predatory xanthid crabs are being briefly mentioned here to emphasize that these are not parasites sensu strictu but are of importance to the shellfisheries industry. For a listing of the literature pertaining to the ecology of xanthid crabs, the paper by Ryan (1956) should be consulted.

APPENDIX

A LIST OF COMMERCIALLY IMPORTANT MARINE MOLLUSCS A N D THEIR K N O W N PARASITES Host Aequipecten ( = Pecten) gibbus (Defrance).

g Aequipecten (

= Pecten)

irradians (Lamarck)

. .

Aequipecten ( = Pecten) maximus (Link)

.

Brachidontes recurvus Rafinesque

.

Buccinum undatum Pennant

.

Stage parasitic in mollusc

Parasite

Porrocaecum pectinis (Nematoda) . Odostomia seminuda (Gastropoda) . Odostomia bisutalaris (Gastropoda) (experimental) Pinnotheres maculatus (Malacostraca)

.

Larvae Adults Adults Post-planktonic larvae and adults

.

Metacercariae Adults Adults (mature and immature) Post-planktonic larvae and adults Post-planktonic larvae

.

Himasthla quissetensis (Trematoda) Odostomia seminuda (Gastropoda) . Pseudomyicola glabra (Copepoda) . Pinnotheres ostreum (Malacostraca) . Pinnothews maculatus (Malacostraca) Porrocaecum pectinis (Nematoda) Odostomia eulimoides (Gastropoda)

. .

Larvae Adults

Cercarica brachiodontis (Trematoda) Mytilicola porrecta (Copepoda) . Ostrincola gracilis (Copepoda) .

.

Sporocysts Adults Adults

Neophasis pusilla (Trematoda) Cercaria neptuneae (Trematoda) Cermria buccini (Trematoda)

.

.

Rediae Rediae Sporocysts

?

z

Cardita ajar de Blainville.

7 Cardita sulcata Bruguiere. UI

Conchyliurus lobatus (Copepoda) Boveria subcylindrica (Ciliatea)

. . . .

.

.

.

Cardium norwegicum (Schroeter)

.

E

Pinnotheres p k u m (Malacostraca)

.

Conchyliurus sp. (Copepoda)

Bucephalus haimeanus (Trematoda)

Chlamys nipponensis Kuroda .

Pinnotheres pholadia (Malacostraca)

Chlumys opercularis Philippi .

.

.

Cardium tuberculatum Pusch

.

Trophozoites

Bucephalus haimeanus (Trematoda) Gymnophallus margaritarum (Trematoda) Gymnophallus strigata (Trematoda) . Gymnophallus cambrenais (Trematoda) Gymnophallus fulbrighti (Trematoda) Cercaria dichotoma (Trematoda) . Cercaria hueti (Trematoda) Himasthla leptosoma (Trematoda) Parorchis acanthus (Trematoda) (on surface) Metucercuria mytili (Trematoda) . Unidentified echinostome of Nicoll (Trematoda) Unidentified ‘‘ sporocyst ” of Nicoll (Trematoda) Mytilicola intestinalw (Copepoda) . Pinnotheres p k u m (Melacostraca) Pinnixa faba (Malacostraca) . Pinnixa littoralis (Malacostraca) .

Cardium edule Linn.

Cardium ringena Wood

Adults

Odostomia scalar& (Gastropoda) Odostomio eulimoides (Gastropoda)

. . .

.

Sporocysts Sporocysts Sporocysts Sporocysts Sporocysts Sporocysts Sporocysts Metacercariae Metacercariae Metacercariae Metacercariae Rediae Adults Adults Adults Adults Adults Adults

.

Sporocysts Adults

.

.

Adults Adults

Host

Parasite

Crassostrea gigas (Thunberg)

.

.

Stage para&tic in mollusc

.

Gymnophalloides tokiensis (Trematoda) Proctoeces ostreae (Trematoda) Himasthla quissetenais (Trematoda) (on surface) Mytilicola orientalis (Copepoda) Pinnotheres pholadis (Malacostraca)

.

. .

Crassostrea virginica (Gmelin)

.

.

Hexamita injllata (Zoomastigophorea) Vahlhmpjia calkensi (Sarcodina) VahJhmpjia patuxent (Sarcodina) Nematopsis ostrearum (Sporozoa) .

. .

Nematopsis prytherchi (Sporozoa)

Metacercariae Metacercariae Metacercariae Adults Adults

.

.

Trophozoites

.

Minchinia costidis (Sporozoa) . Minchinia nelsoni (Sporozoa) Ancistrocoma pekeneeri (Ciliatea) Ancistrocoma sp. (Ciliatea) . Bucephalus cuculus (Trematoda) Bucephalus sp. (Trematoda) . Himasthla quissetensis (Trematoda) (limited) Acunthoparyphium spindosum (Trematoda) . Tylocephalum sp. (Cestoda) Angiostrongylus cantonensis (Nematoda) (experimental) Odostomia impressa (Gastropoda) . Odostomia seminuda (Gastropoda) (experimental) Odostomia bktularia (Gastropoda) . Ostrincola gracilis (Copepoda) . Pseudomyicola glabra (Copepoda) . Pinnotheres ostreum (Malacostraca) Pinnotheres maculatus (Malacostraca) ( 9 )

.

. .

.

.

.

.

.

%

Trophozoites Trophozoites Gymnospores, sporozoites, and spores Gymnospores, sporozoitea, and spores Plasmodia, spores, sporocysts Plasmodia, spores (?) Trophozoites Trophozoites Sporocysts Sporocysts Metacercariae Metacercariae Metacestode Larvae Adults Adults Adults Adults Adults Post-planktonic larvae and adults Post-planktonic larvae and adults

El

3 K

0

Fi

co

t: b x

4 0 5j

z

s

: M

Gn

Donax sp. Schumacher* . Donux trunculus Linn. . Donax politus Forbes and Hanley . Donax vittatus Lamarck Ensis directus (Conrad) Haliotis corrugata Wood Haliotis f d g e w Philippi Haliotis gigantea Menke Macoma balthica Linn.

. . . . . .

Macoma incompicua (Broderip and Sowerby) . Macoma n ~ - ~Conrad ~ta . Macoma irus (Lamarck) Macoma secta (Conrad)

. .

. .

Macoma stultorum Linn. Macoma cumana (Costa) Mactra glabrata de Blainville Mactra solidis.sima Dill-

.

Wyn-

Mactra a d c a t a r i a Reeve

*

.

HyaJoklossia pelaeneeri (Sporozoa) Ancistrum cylidioides (Ciliatea)

.

. .

Boveria subcylindrica (Ciliatea) Gymnophallus strigata (Trematoda) . Bacciger bacciger (Trematoda) . Himasthla quwsetensis (Trematode) . Echinocephalus pseudouncinutus (Nematode) Echinocephulus pseudouncinatus (Nematoda) Panaietis haliotis (Copepoda) Ancistrocoma pelseneeri (Ciliatea) Thigmophrya macomae (Ciliatea) Cercaria dichotoma (Trematoda) Gymnophallus macomae (Trematoda)

.

Ancistrowma pelaeneeri (Ciliatea) Ancistrocoma pelaeneeri (Ciliatea) Pinnixa faba (Malacostraca) . Pinnixa littoralis (Malacostraca) Ancistrowma pekeneeri (Ciliatea) Ancistrocoma pelaeneeri (Ciliatea) Pinnim littoralis (Malacostraca) Himasthla leptosoma (Trematode) Myocherea dentata (Copepoda)

.

Conchyliurms torosus (Copepoda) Myocheres major (Copepoda) . Ancistrum japonica (Ciliatea) Pinnotheres pholadia (Malacostraca)

.

. . . .

. . . . . .

Oocysts Trophozoites Trophozoites Metacercariae Sporocysts Metacercariae Larvae Larvae Adults Trophozoites Trophozoites Sporocysts Metacercariae Trophozoites Trophozoites Immature adults Immature adults Trophozoites Trophozoites Immature adults Metacercariae Adults

.

Adults

.

Adults Trophozoites Adults

For accounts of parasites of species of Donaz of little commercial importance, see Leidy (1879), Giard (1897a,b, 1907). Pelseneer (1896, 1928), Chatton and Lwoff (1923), Cable (1956, 1963), Loesch (1957), Hopkins (1958) and Wade (1967).

W

Stage parasitic in mollusc

Parasite

Host Mactra veneriformis Wood Margaritif era vulgaris (Leach) . Mercenaria mercenaria (Say) *

Ancistrum japonica (Ciliatea)

.

Trophozoites

Tylocephalum spp. (Cestoda) . Echinocephalus uncinatus ( ? ) (Nematoda)

Himasthla quissetensis (Trematoda) . Himasthla muehlensi (Trematoda) (indirect evidence) Angiostrongylus cantonensis (Nematoda) (experimental) Odostomia bkutularw (Gastropoda) (experimental) Odostomia seminuda (Gastropoda) (experimental) . Mytilicola porrecta (Copepoda) Myocheres major (Copepoda) . Ostrincola gracilw (Copepoda) . Ancistrum mytili (Ciliatea) . Ancistrum isseli (Ciliatea)

.

Modiolus demissus (Dillwyn) .

Modiolus modiolus (d’OrbignY) f

M y a arenaria (Linn.)

Metacestodes Larvae

. . .

Modiola barbata (Lamarck)

.

.

Himasthla quissetensis (Trematoda) Mytilicola porrecta (Copepoda) Ostrincola gracilis (Copepoda) . Pseudomyicola glabra (Copepoda)

.

b P

0

.

Metacercariae Adults Adults Adults

.

Ancistrum isseli (Ciliatea) . Himasthla quissetensis (Trematoda) Pinnotheres maculatus (Malacostraca) Fabia subquadrata (Malacostraca) Ancistrocoma pelseneeri (Ciliatea) . Metacercaria I (Trematoda) Cercuria myae (Trematoda) . Himasthla leptosoma (Trematoda)

.

.

.

Metacercariae Metacercariae Larvae Adults Adults Adults Adults Adults Trophozoites Trophozoites

.

Trophozoites Metacercariae Post-planktonic larvae and adults Post-planktonic larvae and adults Trophozoites Metacercariae Sporocysts Metacercariae

E E 3 ls

0

F

2 G:

k

m

2 r

0

0

Gn

2E M

m

Himasthla quissetensis (Trematoda) . Himasthla compacta (Trematoda) . Himasthla littorinae (Trematoda) . wiciostomia trifida (Gastropoda) . Myocheres major (Copepoda) . Myicola metisiensis (Copepoda) . Pinnotheres maculatus (Malacostraca) Pinniza faba (Malacostraca) . Pinniza littoralis (Malacostraca) . Mytilus culijornianus Con. rad .

Huplosporidium tumefucientis (Sporozoa) Cercuria noblei (Trematoda) . Mytilicola orientalis (Copepoda)

Metacercariae Metacercariae Metacercariae Adults Adults Adults Post-planktonic larvae and adults Immature adults Immature adults

. .

Plasmodia, sporocysts, spores Sporocysts Adults

.

Mytilus crassitesta (Reuss) Mytilus edulis Linn.

.

Mytilicola orientalis (Copepoda) . Chytridiopsis mytilovum (Cnidospora) Ancistrum mytili (Ciliatea) Conchophthirus mytili (Ciliatea) . Crebricoma kozlofi (Ciliatea) . Crebricomu carinuta (Ciliatea) . Raubella helensis (Ciliatea) . Isocomides mytili (Ciliatea) . Hypocomides mytili (Ciliatea) Gargarius gargarius (Ciliatea) . Bucephalus mytili (Trematoda) . . Gymnophallus margaritarum (Trematoda) Gymnophallus bursicola (Trematoda) Proctoeces rnaculatus (Trematoda) .

.

.

.

.

Hinzasthla leptosonaa (Trematoda) . Himasthla quissetensis (Trematoda) . Himasthla littorinae (Trematoda) . Parorchis acanthus (Trematoda) (on surface)

.

Adults Cysts enclosing spores Trophozoites Trophozoites Trophozoites Trophozoites Trophozoites Trophozoites Trophozoites Trophozoites Sporocysts Metacercariae Metacercariae Sporocysts, cercariae, metacercariae, adults Metacercariae Metacercariae Metacercariae Metacercariae

W b P

c

w

. . .

Metacerearia mytili (Trematoda) Cercaria tenuans (Trematoda) . Odostomia scalaris (Gestropoda) Odostornia eulimoides (Gastropoda) Mytilicola intestinalis (Copepoda) . Mytilicola orientalis (Copepoda) . Pseudomyiwla glabra (Copepoda) . Tisbe celata (Copepoda) Pinnotheres ostreum (Malacostraca) Pinnothres maculatw, (Mdacostraca) Pinnotheres pholadis (Malacostraca) .

.

Mytilus galloprovincialis Lammck .

Nematopsk legeri (Sporozoa)

.

.

Mytilus perna Dillwyn Ostrea demelamellosa Lischke . Ostreu edulis Linn.

.

.

Metacercariae Sporocysts Adults Adults Adults

.

.

Gymnophallua margaritarum (Trematoda) Gymnophallus perligem (Trematoda) Mytilicola intestinalis (Copepoda) Mytilua minimus Schroeter .

Nematopsk legeri (Sporozoa)

.

Porospora gigantea (Sporozoa)

.

rp t 9

Stuge parasitic in molluac

Parasite

Host

. .

z

Adults Adults Adults Post-planktonic larvae and adults Post-planktonic larvae and adults Adults Gymnospores, spores Metacercariae Metacercariae Adults

sporozoites

Gymnospores, sporozoites spores Gymnospores, sporozoites

Modiolicola i n w i p e s (Copepoda)

.

Adults

Pseudomyicola ostreue (Copepoda)

.

Adults

Hexamita in.& (Zoomastigophorea) Chytridwpsk ovicola (Cnidospora) . Bucephalua haimeanus (Trematoda) Odostomia eulimoides (Gastropoda)

. .

.

Trophozoites Cysts enclosing spores Sporocysts Adults

@ M F F

s 23

and

x

:e: ul

r

i3 and

2E z: M

ul

.

Ostreu lurida Solander Ostrea tulipa Lamarck Ostreu sp. Linn.

.

Pecten ( = Arctica) islandicus Linn.

..

. .

Adults Adults Adults Adults Adults Adults Adults Adults

Odoatomia eulimoides (Gastropoda) . Odostomia bisutularis (Gastropoda) (experimental) Od08tw??& eulimoides (Gastropoda) Paeudoklossia pectinis (Sporozoa) Bweria aubcylindrica (Ciliatea) . Echinocephalus uncinatus (Nematoda) (?)

Adults Adults Adults Oocysts enclosing sporozoites Trophozoites Larvae

Odostomia seminuda (Gastropoda) (experimental) Odostomia bisutularis (Gastropoda) (experimental) P ~ n m t h e r e smaeulatus (Malacostraca) . Bacciger bacciger (Trematoda) . Cercaria ophicerca (Trematoda) . Lepocreadium album (Trematoda) . Bacciger bacciger (Trematoda) . A n c i s t m m cylidioides (Ciliatea) Ancistrum subtruncatum (Ciliatea) . Boveria wbcylindriua (Ciliatea) Gymnophauue margaritarum (Trematoda) . Gymmophallus megalocoela (Trematoda) Bacciger bacciger (Trematoda) . Himasthla ambigua (Trematoda) Cercaria ophicerca (Trematoda) . . Lepocreadium album (Trematoda) Metacercaria acherusiae (Trematoda)

Adults Adults Post-planktonic larvae and adults Sporocysts Sporocysts Metacercariae Sporocysts Trophozoites Trophozoites Trophozoites Sporocysts Metacercariae Sporocysts Metacercariae Sporocysts Metacercariae Metacercariae

. . . .

.

.

. .

Pectenjacobaeus (Link) Pecten mazimus (Link) Pinna nobilis Linn.. Pinna sp. Linn. Placopecten magellanicua (Bosc)

.

.

Tapes aureua Forbes and Hanley

.

Tapes candida Helbling Tapes d w s a t u a (Linn.)

Chrysallida obtusa (Gastropoda) Mytilicola intestinalis (Copepoda) Pinmtheres p i s u m (Malacostraca) Mytilicola orientalis (Copepoda) Lichomolgua arcanus (Copepoda) . Pseudomyicola mirabihs (Copepoda) Ostrincola clavator (Copepoda) . Ostrincola simplex (Copepode)

.

.

. .

.

.

. .

.

. .

.

P

zw

2

e

W Ip

0

Host

Stage parasitic in mollusc

Parasite

Tapes Jloridus (Lamarck) .

Pseudoklossia glomerata (Sporozoa)

Tapes japonica Deshayes Tapes pullastra (Montagu)

Pinnotheres pholadis (Malacostraca) . Gymnophallus scrivenensis (Trematoda) Himasthla leptosoma (Trematoda) . Ancistrum japonica (Ciliatea) . Bacciger bacciger (Trematoda) Himasthla quissetelzsis (Trematoda) Tylocephalum sp. (Cestoda) . Philoconcha amygdalae (Copepoda) Pinniza faba (Malacostraca) . Pinniza littoralis (Malacostraca) Pseudoklossia glomerata (Sporozoa)

Tapes semidecussata Reeve

Tapes wirgineus (Fleming) Tellina exigwl Sowerby

.

Tellina nitida Perry Tellina nymphalis Lamarck . Tellina planata Linn. Tellina tenuis Montagu %llina sp. Linn.

.

.

. .

Venerupis staminea (Conrad) . Venus fasciata Donovan . Venus gallina MacGillivray . Venus sp. Linn.

.

.

Oocysts enclosing sporozoites in spores Adults Metacercariae Metacercariae Trophozoites Sporocysts Metacercariae Metacestode Adults Immature adults Immature adults Oocysts enclosing sporozoites in spores Trophozoites Trophozoites Sporocysts Trophozoites

. . . .

. .

Ancistrum cyclidioides (Ciliatea) Boveria subcylindrica (Ciliatea) Bacciger bacciger (Trematoda) . Boveria subcylindrica (Ciliatea)

.

Myocheres scobina (Copepoda) . Boveria subcylindrica (Ciliatea) Cymnophallua strigata (Trematoda) Hyaloklosaia pelseneeri (Sporozoa)

. . .

Adults Trophozoites Metacercariae Oocysts enclosing sporozoites in spores

.

Plerocercoids Adults Trophozoites

Echeneibothrium sp. (Cestoda) . Mytilicola orientalis (Copepoda) Ancistrospira veneris (Ciliatea) .

Boveriu subcylindrica (Ciliatea) . Gymnophallus margaritarum (Trematoda)

.

Trophozoites Sporocysts

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Yamaguti, S. (1939). Studies on the helminth fauna of Japan. Part 25. Trematodes of birds, IV. Jap. J. 2001.8, 129-210. Yamaguti, S. (1953). Parasitic worms mainly from Celebes. Part 1. New digenetic trematodes of fishes, 11. Acta Med. Okayama 8 , 257-295. Yamaguti, S. (1958). ‘‘ Systema Helminthum.” Vol. 1. The Digenetic Trematodes of Vertebrates. Parts 1 and 2. Interscience, New York. Yamaguti, S. (1959). “ Systema Helminthum.” Vol. 2. The Cestodes of Vertebrates. Interscience, New York. Yamaguti, S. (1961). “ Systema Helminthum.” Vol. 3. The Nematodes of Vertebrates. Parts 1 and 2. Interscience, New York. Yasuraoka, K. (1954). Ecology of the miracidium. 11. On the behavior to light of the miracidium of Faaciola hepatica. Jap. J. med. Sci. Biol. 7 , 181-192. Yentsch, C. S. and Pierce, D. C. (1955). “ Swimming ” anemone from Puget Sound. Science, N . Y . 122, 1231-1233. Yokoya, Y. (1928). Report of the biological survey of Matsu Bay. 10. Brachyura and crab-shaped Anomura. Sci. Rep. TGhoku Univ. Ser. 4 , 3, 757-784. Yokoya, Y . (1933). On the distribution of decapod crustaceans found in the vicinity of the Misaki Marine Biological Station. Jap. J . 2001.7,129-146. Yonge, C. M. (1926). Structure and physiology of the organs of feeding and digestion in Ostrea edulis. J. mar. biol. Ass. U . K . 14, 295-388. Yonge, C. M. (1957). Interrelations of organisms. C. Symbiosis. In. “ Treatise on Marine Ecology and Paleoecology,” Vol. 1. Ecology. Mem. geol. Xoc. Am. 67, 429-442. Yunger, C. E. and Ishak, K. G. (1957). Histopathological observations on the sequence of infection in knemidokoptic mange of budgerigars (Melopsittacua undulatus). J. Paraait. 43, 664-672. Zernov, S. A. (1913). [Contribution to the knowledge of the life of the Black Sea.] Zap. imp. Akad. Nauk 32, 1-299. (In Russian.) Zinn, D. J. (1964). Immigrant snail is dinner delicacy. Maritime8 8, 15-16. Zisohke, J. A. and Zischke, D. P. (1965). The effects of Echinoetoma revolutum larval infection on the growth and reproduction of the snail host Staqnicola palustria. Am. 2001.5, 707-708. NOTESADDEDIN PROOF Page 7 8 : Kagan and Geiger (1964) have shown that the miracidiaimmobilizing substance in Australorbis glabratus is immunologically ineffective in preventing reinfections in snails with patent infections. They have noted, however, that El Gindy (1950) has reported that snails are refractory to reinfection with Schistosomatium douthitti. This latter observation should be verified. Page 171: Andrews (1967) has shown that Minchinia nelsoni infection in oysters is much less affected by such ambient factors as population densities and high temperatures than the fungus Dermocystidium marinum ( = Labyrinthomyxa marina) in Virginia, and consequently M . nelsoni decimates oyster populations long before D. marinum epizootics can become established. Page 213: According to Bowers and James (1967) these are not sporocysts but a layer of host cells covering metacercariae (see note to p. 218 below).

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Page 214: Palombi’s (1924) claim that the sporocysts and cercariae in Tapes and Cardium are the progenitor stages of the motacercaria in Mytilus is confusing in view of the recent report by Bowers and James (1967) (see note to p. 218 below). Page 218: Bowers and James (1967) consider that Cercaria cambrensis as well as the larva believed by Jameson (1902) to be the immature form of Lecithodendrium somateriae (= Qymnophalloides oedemia) (see p. 213) and the larva designated as Cercaria marguritae by Lebour (1907, 1911) (see p. 213) are all three identical to the larva of Meiogymnophallus minutus, a parasite of the oyster catcher, Haematopwr ostralegus occidentalis. They have also proven experimentally that the larvae of M . minutus in Cardium edule are not cercariae within a sporocyst but metacercariae enveloped within a layer of host cells. These metacercariae are infective to the oyster catcher. Consequently Bowers and James are of the opinion that Jameson (1902), Lebour (1907) and Cole (1938) did not find cercariae enclosed within sporocysts but metacercariae in host tissue. Page 292: Recently Katkansky et al. (1967), as the result of examining Crassostrea gigas parasitized by Mytilicola orientalis over a 2-year period (1963-65) in Humboldt Bay, California, Yaquina Bay, Oregon, and Willapa Bay, Oyster Bay and Hood Canal, Washington, have reported that no shortterm cyclic effects occur in regard to the incidence or intensity of parasitization a t these sites. Page 293: Katkansky et al. (1967) have confirmed their finding that the condition index of Crassostrea gigas is reduced when parasitized by Mytilicola orientalis. In addition, they have reported that not only is the survival of parasitized oysters unaffectod but there is little evidence of reduction of shell growth. References Andrews, J. D. (1967). Interaction of two diseases of oysters in natural waters. Proc. natn. Shelljish. Ass. 57, 38-49. Bowers, E. A. and James, B. L. (1967). Studies on the morphology, ecology and life-cycle of Meiogymnophallwr minutus (Cobbold, 1859), comb. nov. (Trematoda: Gymnophallidae). Parasitology, 57, 281-300. El Gindy, M. S. (1950). Doctoral dissertation. University of Michigan. Kagan, I. G. arid Geiger, S. (1964). Susceptibilty of Australorbis glabratus to reinfection with Schistosoma mansoni. J . Parasit. 50, 474-476. Katkansky, S. C., Sparks, A. K. and Chew, K. K. (1967). Distribution and effects of the endoparasitic copepod, Mytilicola orientalis, on the Pacific oyster, Crassostrea gigas, on the Pacific coast. Proc. natn. Shellfish. Ass. 57, 50-68.

Author Index Numbers in italics refer to przges on. which the full reference is given

A Abdel-Malek, E. T., 35, 65, 345 Abelooi, M., 122, 345 Abolini-Korgis, A., 125, 345 Adams, C. B., 281, 345 Adams, J. E., 254, 371 Agersborg, H. P . K., 79, 103, 345 Ahrens, W., 286, 345 Alexeieff, A. G., 139, 345 Alicata, J. E., 12, 13, 14, 19, 270, 345, 364 Allen, J. F., 277, 278, 345 Allen, K., 88, 92, 94, 345 Allison, L. N., 228, 345 Almeida, A. M., de, 84, 370 Amemiya, I., 321, 346 Anderson, A. H., 40, 41, 58, 59, 66, 67, 70, 76, 78, 122, 125, 236, 237, 352 Andrews, J. D., 162, 163, 164, 171, 346, 388, 389, 390 Ankel, F., 276, 277, 282, 283, 284, 346 Ankel, W. E., 21, 277, 283, 346 Arvy, L., 162, 346 Atkins, D., 252, 327, 328, 329, 346 Aurivillius, C. W . S., 312, 346 Awati, P. R., 321, 330, 346 Axmann, M. C., 81, 85, 346

B Beer, J. G., 4, 261, 346, 363 Baird, R. H., 286, 346 Bal, A. K., 83, 365 Balamuth, W., 137, 361 Ball, S. C., 202, 346 Balss, H., 331, 346 Bang, F. B., 62, 72, 346 Barbosa, F. S., 38, 78, 346, 347 Barlow, C. H., 24, 34, 40, 347 Barnes, R. D., 263, 347 Barrow, J. H., Jr., 162, 164, 169, 347 Bartel, A. H., 45, 347 Bartsch, P., 278, 354

Bassedas, M., 286, 347 Bates, M., 273, 347 Bauman, P. M., 47, 363 Bayer, F. A. H., 119, 347 Baylis, H. A., 265, 266, 347 Baylor, E. R., 43, 384 Bearup, A. J., 254, 347 Beers, C. D., 187, 347 Benex, J., 77, 347 Bern, H. A., 95, 381 Berner, L., 330, 347 Besa, A. A., 119, 377 Bilimoria, D. P., 14, 355 Bils, R. F., 83, 347 Birge, E. A., 316, 319, 347 Bisset, K. A., 60, 347 Blumenthal, A. B., 122, 125, 352 Bock, S., 201, 347 Bocquet, C., 101, 347 Bogitsh, B. J., 67, 347 Bolster, G. C., 286, 289, 346, 347 Bookhout, C. G., 324, 353 Boss,K. J., 277, 280, 281,282, 347, 371 Bourdillon, A., 53, 348 Bovee, E. C.. 137, 142, 143, 348, 361 Bowers, E . A., 83, 204, 206, 225, 363, 389, 390, 390 Boyd, W. C., 74, 348 Braams, W. G., 49, 348 Bracket, S., 77, 84, 348, 353 Brand, T., von, 34, 81, 82, 85, 87, 104, 348, 375 Brinkmann, A., 21, 371 Brooks, C. P., 65, 348 Brown, F. J., 104, 348 Brown, R., 74, 348 Brumpt, E., 119, 133, 348 Bullock, T. H., 8, 348 Bullock, W. L., 87, 348 Burger, O., 263, 348 Burton, R. W., 12, 68, 79, 80, 81, 82, 103, 116, 117, 118, 119, 193, 194, 208, 209, 263, 270, 348, 351 391

392

AUTHOR INDEX

Bush, M., 192, 364 Bytinski-Salz, H., 97, 348

C Cable, R. M., 212, 228, 240, 243, 2449 245, 254, 339, 348, 349, 384 Calaprice, N. L., 74, 354 Calman, W. T., 326, 349 Cameron, G. R., 71, 349 Cameron, T. W. M., 4, 349 Campbell, W. C., 32, 35, 38, 39, 57, 349 Cantacuzhne, J., 74, 75, 349 Canzonier, W., 168, 349 Ca.nzonier, W. J., 165, 171, 360 Carneiro, E., 38, 346 Carney, D. M., 65, 373 Carriker, M. R., 12, 349 Caspers, H., 286, 288, 349 Caullery, M., 4, 6, 164, 176, 349 Causley, D., 24, 32, 355 Cauthron, F., 275, 370 Cerruti, A., 11, 21, 199, 349 Certes, A,, 137, 138, 350 Chahovitch, X., 73, 75, 350 Charniaux-Cotton, H., 95, 122, 123, 350 Chatton, E., 178, 188, 189, 190, 191, 194, 195, 196, 197, 339, 350 Cheng,T. C., 4,5,8,12,13,19,40,41,49, 57, 58, 59, 60, 64, 65, 66, 67, 68, 69, 70, 75, 76, 78, 79, 80, 81, 82, 83, 85, 86, 87, 88, 90, 91, 92, 93, 103, 104, 108, 109, 116, 117, 118, 119, 122, 124, 125, 129, 131, 138, 208, 209, 236, 237, 239, 254, 256, 257, 258, 259, 263, 270, 271, 350, 351, 352, 378, 381 Chernin, E., 36, 42, 71, 122, 352 Chew, K. K., 103, 178, 180, 254, 259, 260, 261, 290, 291, 292, 293, 352, 376, 382,390, 390 Ching, H. L., 14, 345 Christensen, A. M., 98, 276, 277, 282, 283, 284, 315, 316, 319, 320, 321, 326, 329, 346, 352 Chu, G. W. C., 35, 352 Clark, R. B., 273, 274, 353 Clay, T., 121, 379 Cobb, N. A., 269, 270, 353

Coe, W. R., 262, 263, 272, 353 Coelho, M. V., 78, 84, 119, 347, 353, 381 Coffin, H. G . , 324, 363 Cole, H. A., 209, 210, 211, 214, 218, 219, 251, 252, 277, 283, 284, 285, 286, 290, 346, 353, 390 Collier, A., 11, 370 Cooley, N. R., 12, 111, 119, 353 Cooperman, J. S . , 66, 70, 78, 108, 109, 119, 124, 351 Corliss, J. O., 137, 178, 186, 353, 361 Cort, W. W., 37, 77, 106, 353, 375 Costlow, J. D., Jr., 324, 353 Couch, J. A., 167, 168, 169, 353 Coupin, H., 99, 353 Cousineau, G. H., 83, 365 Couvreur, E., 73, 354 Creplin, F. C. H., 235, 354 Cressey, R. F., 101, 300, 301, 309, 311, 312, 315, 362 Croll, N. A., 4, 47, 354 Crowell, S., 20, 21, 199, 354, 371 Cubnot, L., 61, 235, 354 Culbertson, J. T., 77, 354 Cushing, J. E., 74, 354 Cutress, C. E., 35, 352

D Dales, R. P., 4, 18, 20, 354 Dall, W. H., 278, 354 Danielli, J. F., 83, 354 Davenport, D., 18, 19, 22, 23, 24, 29, 32, 40, 45, 48, 49, 50, 347, 354, 355 Davey, T. H., 77, 359 Davis, H. S., 139, 355 Davis, J. D., 274, 355 Dawes, B., 57, 118, 204, 212, 228, 235, 355 Dean, B., 98, 355 Debaisieux, P., 159, 162, 164, 355 De Bary, A., 4, 355 De Bruyne, M., 61, 355 Decker, C. L., 27, 34, 40, 42, 357 De Haan, W., 331, 355 Deiana, S., 86, 355 De Morgan, W., 68, 186, 355, 356 Denison, J. G., 98, 128, 129, 139, 383 Dethier, V. G., 50, 355 Deuel, H. J., Jr., 127, 355 Diesing, K. M., 201, 355

393

AUTHOR INDEX

Dietz, E., 235, 355 Dissanaike, A. S., 14, 355 Dobrovalskij, A. A., 81, 86, 359 Dogiel, V. A., 4, 355 Dollfus, R. Ph., 130, 214, 230, 231, 249, 256, 261, 286, 287, 356, 373 Drew, G. H., 68, 356 Dubois, R., 73, 212, 213, 356 Duboscq, O., 153, 157, 158, 159, 160, 175, 368 Duerr, F. G., 105, 356 Dujardin, F., 137, 356 Duke, B. 0 . L., 133, 356 Dunavan, C. A., 36, 42, 352 Dungern, E. A., von, 74, 356 Dusanic, D. G., 89, 356 Duursma, E. K., 43, 356 Dye, W. H., 77, 356

E Egami, N., 321, 356 El Gindy, M. S., 65, 356, 389, 390 Ellenby, C., 286, 357 Elmhirst, R., 244, 368 Engle, J. B., 272, 273, 274, 275, 369 Erasmus, D. A., 68, 78, 134, 377 Etges, F. J., 27, 34, 40, 42, 119, 124, 125, 133, 357 Ewers, W. H., 127, 128, 357

F Falk, I. S., 54, 357 Farley, C . A., 165, 167, 168, 169, 171, 174, 353, 357 Fatham, H. B., 103, 357 Faurot, L., 6, 357 Faust, E. C., 24, 34, 40, 41, 79, 81, 87, 92, 103, 104, 133, 357 Feder, H. M., 8, 357 Feng, S. Y . ,61, 63, 71, 72, 75, 133, 149, 357 Ferguson, J. H., 71, 359 Fernau, W., 79, 358 FerroniBres, G., 97, 358 Field, I. A., 175, 274, 358 Files, V. S., 81, 82, 87, 348 Flower, F. B., 335, 370 Fox, D. L., 127, 358 Fraenkel, G. S., 31, 32, 40, 358

Fraser, C. M., 20, 21, 358 Freeman, R. F. H., 231, 232, 358 Fretter, V., 103, 105, 136, 277, 278, 283, 285, 358 Fried, B., 14, 377 Friedl, F. E., 89, 358 Frings, C., 49, 358 Frings, H., 49, 358 Fujita, T., 222, 224, 229, 230, 231, 234, 255, 261, 358 Fukui, M., 46, 380

G Galtsoff, P. S., 41, 88, 95, 100, 149, 151, 198, 274, 358, 367, 373 Ganapati, P. N., 164, 358 Garcia, E. G., 119, 377 Geelen, H. F. M., 49, 348 Geiger, 8. 389, 390 Geiman, Q. M., 4, 358 George, W. C., 71, 359 Gerichter, C. B., 263, 359 Giard, A., 73, 198, 212, 215, 216, 223, 230, 339, 359 Giles, D. E., 210, 211, 359 Ginecinskij, T. A., 81, 85, 86, 87, 359 Girard, C. F., 200, 201, 359 Glaser, 0. C . , 206, 359 Gojdics, M., 137, 361 Gonnert, R., 104, 364 Gonor, J. J., 8, 359 Gordon, R. M., 77, 359 Graham, A., 103, 105, 136, 277, 278, 283, 285, 358, 359 Grainger, J. N. R., 286, 287, 288, 289, 290, 359 Granata, L., 164, 359 Grass6, P., 161, 359 Grass& P. P., 79, 359 Gravier, C., 274, 359 Gray, I. E., 315, 359, 360 Gresso, W., 119, 133, 357 Griffiths, H. J., 34, 360 Griffiths, R. B., 131, 360 Gross, P. R., 126, 387 Guberlet, J. E., 263, 360 Gunn, D. L., 31, 32, 40, 358 Gurney, R., 312, 360 Gutsell, J. S., 203, 269, 360

394

AUTHOR INDEX

H Hackney, A. G., 281, 360 Haeckel, E., 60, 360 Hairston, N. G., 119, 377 Hall, R. P., 137, 361 Hancock, D. A., 277, 283, 284, 285, 353 Hand, C., 20, 360 Harant, H., 159, 274, 360 Hart, J. E., 259, 360 Hart, J. F. L., 100, 324, 360 Hartman, O., 274, 360 Haskin, H. H., 11, 165, 167, 168, 169, 171, 172, 360 Haswell, W. A., 274, 360 Hatt, P., 152, 153, 154, 155, 156, 157, 360 Haughton, I., 68, 72, 360 Haven, D., 321, 360 Havinga, B., 130, 360 Hawes,R. S. J., 179, 180, 186, 187, 188, 189, 190, 370 Hedgpeth, J. W., 197, 360 Heldt, J. H., 129, 361 Henry, A., 269, 378 Henry, S. M., 4, 361 Hepper, B. T., 287, 288, 289, 290, 361 Herdman, W. A., 255, 256, 361 Hesse, C. E., 263, 386 Hesse, E., 175, 368 Heyneman, D., 74, 115, 361 Hickok, J. F., 19, 23, 29, 49, 354 Hillman, R. E., 98, 273, 361, 381 Hoese, H. D., 163, 164, 346 Hoffman, W. A., 24, 41, 92, 104, 133, 357 Hogue, M. J., 129, 143, 144, 145, 146, 147, 361 Hollande, A. C., 177, 368 Holliman, R. B., 235,361 Honigberg, B. M., 137, 361 Hopkins, S.H.,4, 62, 98, 111, 116, 118, 122, 128, 139, 198, 203, 205, 207, 208, 234, 235, 240, 254, 265, 278, 279, 316, 319, 335, 339, 361, 362, 369, 370, 371, 380 Hornell, J., 255, 256, 361, 381 Hoshina, T., 121, 362 Houghton, D. R., 330, 362 Hubendick, B., 37, 362 Huet, L., 203, 225, 226, 230, 362

Huff, C. G., 60, 362 Huger, A,, 176, 362 H u e s , A. G., 101, 102, 130, 131, 287, 293, 294, 295, 296, 300, 301, 302, 303, 304, 305, 307, 309, 311, 312, 313, 315, 362 Hunter, G. W., 47, 363 Hurst, C. T., 79, 81, 87, 103, 105, 362, 363 Hutton, R. F., 218, 219, 220, 225, 235, 269, 363 Hyman, L. H., 85, 143, 200, 201, 202, 259, 263, 363 Hyman, 0. W., 40, 316, 319, 324, 363

1 Ingalls, J. W., 47, 363 Irvine, J., 324, 363 Ishak, K. G., 131, 389 Issel, R., 178, 180, 181, 182, 184, 185, 363 Ivanov, L. I., 86, 363

J James, B. L., 83, 113, 115, 116, 117, 127, 204, 206, 225, 254, 363, 389, 390, 390 James, H. A., 103, 116, 351 Jameson, H. L., 73, 212, 213, 214, 215, 255, 256, 363, 390 Jarocki, J., 178, 189, 363, 364 Jauregui, J. J., 119, 377 Jecklin, L., 274, 360 Jeffreys, J. G., 283, 363 Jirovic, O., 164, 363 Johnson, H. M., 74, 363 Johnson, I., 19, 44, 363 Johnston, T. H., 265, 266, 363 Johnstone, J., 204, 225, 363 Jones, F. G. W., 50, 385 Jones, W. C . , 122, 369 Joyeux, C., 261, 363 Jullien, A., 68, 363

K Kagan, I. G., 65, 364, 389, 390 Kahl, A., 182, 186, 364 Katkansky, S. C . , 290, 291, 292, 293, 352,390, 390

395

AUTHOR INDEX

Kavanagh, L. D., 274, 275, 364 Kawashima, K., 26, 27, 46, 364 Keber, G. A. F., 79, 364 Kendall, S. B., 25, 84, 104, 105, 106, 134, 364 Kent, W. S., 178, 364 Kidder, G. W., 178, 179, 180, 182, 183, 186, 187, 188, 365 Kikuth, W., 104, 365 Kirby, H., 191, 365 Kleinholz, L. H., 95, 122, 123, 350 Kloetzel, K., 25, 26, 40, 365 Knapp, S. E., 12, 270, 365 Knight-Jones, E. W., 54, 365 Kobayashi, H., 230, 365 Kobelt, W., 283, 365 Koehler, O., 40, 365 Kofoid, C. A., 192, 365 Kohn, A. J., 49, 365 Korringa, P., 11, 62, 97, 98, 128, 129, 139, 144, 275, 290, 365, 366, 370 Kozloff, E. N., 137, 187, 188, 189, 190, 191, 192, 194, 195, 196, 366 Kramp, P. L., 21, 366 Krull, H., 123, 366 Krull, W. H., 39, 366 Krupa, P. L., 83, 366 Kudo, R. R., 137, 143, 145, 161, 361, 366

L LabbB, A., 68, 162, 366 Lacaze-Duthiers, F. J. H., de, 203, 367 Laing, J., 3, 40, 46, 49, 367 Laird, M., 98, 128, 367 Lal, M. B., 103, 367 Lambert, L., 129, 365, 367 Lameere, A., 84, 367 Lamy, L., 77, 347 Landau, H., 149, 151, 367 Landers, W. S., 335, 367 Lane, C., 265, 266, 347 Lankester, E. R., 162, 275, 367 Lapage, G., 4 , 367 L a Rue, G. R., 34, 203, 367 Laserson, C. F., 276, 278, 367 L a Valette, A. J. H., von, 225, 367 Laveran, C. L. A., 161, 367 Laviolette, P., 122, 124, 367

Lebour, M. V., 115, 204, 205, 212,213, 214, 215, 216, 217, 220, 221, 222, 225, 235, 236, 240, 244, 245, 246, 247, 248, 250, 251, 274, 324, 329, 367, 368, 390 LBgsr, L., 153, 157, 158, 159, 160, 161, 175, 177, 368, 390 Leidy, J., 339, 368 Leigh-Sharpe, W. H., 312, 368 Leiper, R. T., 133, 368 Leloup, E., 97, 130, 368 Lengy, L., 57, 368 Lenhoff, H. M., 6, 373 Le Roux, P. L., 37, 368 Lespes, P. G. C., 228, 368 Levine, N. D., 137, 361 Levinsen, G. M. R., 213, 368 Lewert, R. M., 89, 356 Lillie, R. D., 86, 368 Lincicome, D. R., 4, 122, 368, 369 Lindquist, W. D., 103, 377 Linstow, 0. F. B., von, 265, 266, 369 Linton, E., 202, 237, 243, 245, 255, 256, 369 Little, J. W., 254, 369 Llewellyn, J., 231, 232, 358 Loeblich, A. R., Jr., 137, 361 Loesch, H. C., 339, 369 Logie, R. R., 11, 374 Loosanoff, V. L., 200, 272, 273, 274, 275, 280, 369 Looss, A., 230, 231, 369 Lunz, G. R., Jr., 274, 275, 369 Lutta, A. S., 86, 87, 369 Lutz, A., 133, 369 Lwoff, A., 178, 188, 189, 190, 191, 194, 195, 196, 197, 339, 350 Lysaght, A. M., 121, 369

M Maas, D., 277, 370 MacCallum, G. A., 266, 370 McCrady, J., 118, 206, 370 McCullough, F. S., 37, 134, 363, 370 McDermott, J. J., 98, 315, 316, 319, 320, 321, 329, 335, 352, 370 McGinitie, G. E., 274, 370 McGinitie, N., 274, 370 McGowan, J. A., 274, 370

396

AUTHOR INDEX

MacInnis, A. J., 29, 30, 31, 33, 40, 41, 3 70 Mackerras, M. J., 270, 370 Mackin, J. G., 11, 62, 63, 65, 66, 79, 98, 106, 128, 129, 139, 165, 167, 168, 169, 171, 172, 192, 275, 360, 370, 383 Mackinnon, D. L., 179, 180, 186, 187, 188, 189, 190, 370 McLeod, J. A., 257, 260, 387 McMahon, P., 34, 348 McMillin, H. C., 263, 370 MacMullen, D. B., 47, 363 Magalhaes, N. B., 84, 370 Maldonado, J . F . , 57, 371 Mann, H . , 130, 372 Margolin, A. S., 8, 371 Marshall, J. T., 283, 371 Martin, R., 21, 371 Martin, W. E., 83, 243, 254, 347, 349, 371 Mathias, P., 26, 371 Matienzo, J. A., 57, 371 Mattes, O., 34, 371 Mattox, N. T., 20, 199, 371 Maupas, E., 178, 371 Mawson, P. M., 265, 266, 363 Meade, T. G., 60, 371 Medcof, J. C., 128, 371 Mehlman, B., 104, 375 Meleney, H. E., 24, 34, 40, 65, 103, 133, 357, 373 Nlenzel, D. W., 43, 384 Menzel, R. W., 45, 122, 208, 335, 371, 380 Mercier, L., 329, 371 Mereschkowsky, M. C., 20, 371 Merrill, A. S., 277, 280, 281, 282, 347, 371 Mesnil, F., 161, 176, 349, 367 Metchnikoff, E., 60, 372 Meyer, P. F., 130, 372 Meyerhof, E., 92, 372 Rlichelson, E. H., 63, 77, 78, 79, 119, 3 72 Mikhailova, I. G., 62, 372 Millar, R. H., 116, 206, 372 Blillemann, R. E., 265, 266, 267, 268, 372 Miller, H. M., 236, 237, 372

Minchin, E. A., 176, 372 Miura, A., 46, 380 Miyazaki, I., 26, 27, 46, 97, 268, 363, 3 72 Mobius, K., 97, 137, 372 Moewus, L., 13, 372, 385 Molin, R., 265, 266, 373 Monod, T., 286, 287, 373 Monticelli, F. S., 249, 373 Moore, D. V., 65, 373 Moore, E., 139, 373 Mori, T., 130, 290, 293, 373 Morris, P. A., 278, 279, 373 Mortensen, E., 274, 373 Mortensen, T., 53, 373 Moulder, J. W., 102, 373 Mueller, J. F., 122, 373 Muscatine, L., 6 , 373 Myhre, J. L., 165, 171, 360

N Nadakal, A. M., 127, 373 Nagano, K., 119, 374 Najarian, H. H . , 119, 374 Najim, A. T., 35, 374 Narasimhamurti, C. C., 164, 358 Needler, A. W . H., 11, 374 Nelson, J., 206, 374 Nelson, T. C., 19, 61, 272, 374 Neuhaus, W., 25, 34, 40, 374 Neumann, L. G., 122, 374 Newton, W. L., 37, 65, 374 Nez, M. M., 65, 363 Nicol, J . A. C., 43, 374 Nicoll, W., 202, 203, 213, 214, 216, 228, 235, 240, 243, 251, 253, 374, 375 Nishioka, R. S., 95, 381 Noble, E. R., 4, 5 , 375 Noble, G. A., 4, 5, 375 Nolan, M. O., 34, 348 Nolf, L. O., 77, 353, 375 Nomejko, C. A., 273, 369 Norris, K. S., 19, 49, 354 Northrup, F. E., 236, 237, 372

0 Odhner, T., 223, 231, 249, 375 Odlaug, T. O., 130, 291, 293, 375

397

AUTHOR INDEX

Ogino, C., 121, 362 Old, M. C., 198, 375 Olivier, L., 77, 104, 353, 375 Olsen, 0 . W., 4, 375 O’Rouke, F. J., 131, 360 Om, P. E., Jr., 133, 149, 151, 382 Orr, V., 276, 379 Orton, J. H., 98, 99, 326, 329, 330, 375 Owen, H. M., 11, 274, 370, 375

P Palm, V., 81, 86, 375 Palombi, A., 20, 199, 200, 212, 214, 216, 217, 221, 222, 229, 230, 235, 239, 240, 247, 248, 249, 251, 375, 376, 390 Pan, C. T., 63, 68, 72, 104, 119, 121, 122, 376 Pascoe, A. M., 24, 385 Pauley, G. B., 68, 80, 106, 129, 178, 180, 376, 382 Pearce, J . B., 100, 324, 334, 376 Pearl, R., 40, 376 Pearse, A. S . , 200, 202, 287, 300, 301, 308, 310, 311, 377 Pearson, J. C., 134, 377 Peaston, H., 77, 359 Pelseneer, P., 123, 204, 225. 226. 230. 263, 282, 283, 339, 377 Penner, L. R., 14, 377 Pesigan, T. P., 119, 377 Pesta, O., 286, 288, 377 Pierce, D. C., 51, 389 Pillars, A. W. N., 131, 377 Poisson, R., 329, 371 Porter, A., 103, 357 Pratt, I., 60, 103, 371, 377 Prazdnikov, E. V., 62, 372 Premvati, 103, 367 Probert, A. J . , 68, 78, 134, 377 Prytherch, H. F., 133, 148, 149, 150, 151, 377

Q Quennerstedt, A., 178, 377

R Raabe, Z., 178, 179, 180, 186, 187, 188, 189, 190, 191, 194, 195, 196, 197, 363, 377, 378

Rai, H. S., 321, 330, 346 Railliet, A., 269, 378 Rathbun, M. J., 98, 315, 325, 332, 333, 334, 378 Ray, S. M., 11, 174, 370, 378 Read, C. P., 4, 54, 55, 57, 80, 378 Rees, F. G., 103, 104, 105, 106, 109, 110, 111, 117, 119, 215, 216, 217, 244, 245, 378 Rees, G., 83, 378 Rees, W. J., 103, 108, 109, 114, 115, 116, 118, 119, 123, 199, 378 Reid, R. G. B., 95, 378 Reynolds, B. D., 137, 378 Richards, A. G., 68, 381 Richards, C. S., 134, 378 Richards, J. G., 83, 204, 206, 225, 363 Richardson, L. R., 192, 378 Rifkin, E., 70, 103, 257, 259, 351, 378 Riley, G. A., 43, 379 Riser, N. W., 259, 379 Ritchie, J., 20, 379 Robbins, E. J., 178, 180, 376 Robertson, R., 276, 281, 379 Robinson, D. L. H., 89, 379 Rogers, W. P., 57, 379 Rose, C. R., 127, 128, 357 Rosenfield, A., 84, 167, 168, 169, 353, 381 Ross, D. M., 19, 22, 46, 49, 51, 52, 53, 355, 379 Rossan, R. N., 122, 369 Rothschild, A., 120, 379 Rothschild, M., 92, 119, 120, 121, 123, 124, 125, 372, 379 Roughley, T. C., 274, 275, 379 Rourke, A. W., 122, 125, 352 Rudolphi, C. A., 228, 379 Ryan, E. P., 335, 379 Ryder, J. A., 137, 379

S Sakai, T., 315, 331, 332, 333, 334, 379, 380 Salt, G., 40, 380 Sandars, D. F., 270, 370 Sanders, B. G., 60, 75, 76, 351 Sanders, H. L., 276, 380

398

AUTHOR INDEX

Sandoz, M., 316, 319, 380 Santos, A, T., 119, 377 Santos, B. C., 119, 377 Sars, R. W. J., 283, 380 Sasa, M., 46, 380 Sastry, A. N., 45, 380 Sato, K., 46, 380 Savage, R. E., 286, 290, 353 Sawyer, T. K., 142, 143, 380 Say, T., 323, 380 Schaeffer, A. A., 143, 380 Scheltema, R. S., 139, 140, 141, 380 Schinske, R. A., 88, 383 Schlicht, F. G., 254, 369 Schneider, A. C . J., 156, 175, 380 Schneider, T. A., 54, 380 Schodduyn, R., 97, 380 Schreiber, F. G., 104, 380 Schubert, M., 104, 380 Seiwell, H. R., 312, 380 Semper, C., 263, 380 Senft, A. W., 89, 380 Senft, D. G., 89, 380 Seurat, G., 255, 256, 381 Seurat, L. G., 255, 256, 381 Shaw, C. R., 244, 384 Shepperson, J. R., 122, 369 Shipley, A. E., 255, 256, 381 Shirasaka, T., 46, 380 Short, R. B., 65, 363 Shrivastava, S. C . , 68, 381 Shuster, C. N., Jr., 40, 41, 58, 59, 66, 67, 70, 76,’ 78, 98, 122, 125, 236, 237, 352, 381 Sigerfoos, C . P., 21, 381 Simmons, J. E., Jr., 57, 378 Simoeo, B. F., 84, 381 Simpson, L., 95, 381 Sindermann, C., 84, 381 Sinitsin, D. F., 214, 381 Skrjabin, K. I., 228, 381 Smart, R. W. J., 283, 381 Smith, G., 326, 381 Smith, L. S., 201, 381 Smyth, J. D., 4, 5 , 6, 381 Snyder, R. W., Jr., 65, 79, 81, 83, 85, 86, 87, 103, 116, 122, 125, 351, 352, 381 Southwell, T., 255, 256, 381

Sparks, A. K., 68, 80, 102, 103, 106, 129, 130, 178, 180, 254, 257, 258, 259, 260, 261, 290, 291, 292, 293, 352, 376, 381, 382, 390, 390 Sprague, V., 133, 149, 150, 151, 152, 161, 162, 173, 175, 176, 177, 382 Sprehn, C. E. W., 235, 382 Sprent, J. F. A., 4, 382 Stafford, J., 245, 246, 382 Standen, 0. D., 106, 382 Stauber, L. A., 11, 60, 61, 64, 71, 98, 99, 131, 133, 165, 167, 168, 169, 171, 172, 273, 316, 319, 320, 330, 360, 382, 383 Stehouwer, H., 49, 383 Stein, J. E., 79, 98, 128, 129, 139, 383 Stephens, G. C . , 88, 383 Steuer, A., 129, 286, 287, 288, 383 Stevens, N. M., 184, 383 Stirewalt, M. A., 35, 106, 383 Stock, J. H., 101, 347 Stossich, M., 213, 249, 383 Strand, E., 178, 383 Strom, L., 84, 381 Stunkard, H. W., 34, 37, 65, 115, 212, 222, 223, 224, 225, 226, 227, 228, 229, 230, 232, 233, 236, 237, 238, 240, 241, 242, 243, 244, 245, 383, 384 Sudds, R. H., Jr., 35, 40, 49, 65, 116, 384 Sund, P. N., 51, 384 Sutcliffe, W. H., Jr., 43, 384 Sutton, L., 22, 46, 53, 355, 379 Suzuki, S., 47, 384 Szidat, L., 60, 95, 384

T Tada, I., 26, 27, 46, 363 Takahashi, K., 274, 384 Takahashi, S., 47, 384 Takatsuki, S., 61, 79, 384 Tanaka, H., 46, 380 Taylor, B. C . , 169, 347 Taylor, R. L., 173, 174, 384 Tennent, D. H., 118,203,205,207,208, 385 Tesch, J. J., 331, 385 Thillet, C. J., 65, 373

399

AUTHOR INDEX

Thompson, J. C., Jr., 13, 385 Thompson, J. V., 329, 385 Thorpe, W. H., 50, 385 Todd, A. C., 32, 35, 38, 39, 57, 349 Tommers, F. D., 272, 369 Torrey, H. B., 18, 20, 385 Trager, W., 102, 385 Trtigouboff, G., 175, 385 Tripp, M. R., 60, 61, 62, 64, 68, 75, 133, 385 Trump, G., 74, 354 Tubangui, M. A., 24, 385 Tyler, A., 73, 385

U Uhlenhuth, E., 146, 385 Uyemura, M., 178, 183, 185, 385 Uzmann, J. R., 42, 212, 222, 223, 224, 225, 226, 227, 228, 229, 230, 232, 233, 236, 384, 386

V Vadel, 264, 386 Van Beneden, E., 156, 386 Van Beneden, P. J., 259, 263, 386 Van Hemert, D., 43, 379 Van Weel, I?. B., 95, 386 V&vra,J., 176, 386 VayssiBre, A., 286, 386 Veglia, F., 47, 386 Vernberg, F. J., 105, 386 Vernberg, W. B., 105, 386 Verrill, A. E., 97, 200, 262, 263, 285, 386 Villot, F. C. A., 225, 236, 386 Vogel, H., 12, 238, 386 Vonk, H. J., Jr., 61, 387

w Wade, B. A., 339, 387 Wagge, L. E., 68, 72, 387 Walker, C. A., 105, 362 Wangersky, P. J., 43, 379, 387 Wardle, R. A., 257, 260, 387 Warmuth, M., 19, 355 Weiser, J., 137, 361

Weldon, W. F. R., 326, 381 Wells, H. W., 277, 278, 279, 281, 284, 387 Wells, M. J., 277, 281, 284, 387 Wells, W., 334, 387 Welsh, J. H., 47, 48, 49, 325, 387 Wenrich, D. H., 137, 361 Wesenberg-Lund, C. J., 23, 25, 119, 123, 124, 387 Wharton, G. W., 200, 287, 377 Wheeler, W. M., 201, 387 White, K. M., 79, 387 Whitelegge, T., 274, 275, 387 Whitlock, J. H., 122, 387 Willey, C. H., 126, 387 Williams, A. B., 316, 318, 322, 387 Williams, L. W., 300, 387 Williamson, H. C . , 204, 388 Wilson, C. B., 101, 130, 291, 300, 302, 308, 388 Wilson, D. P., 54, 275, 388 Wilson, M. S., 300, 301, 388 Winfield, G. F., 77, 388 Woelke, C. E., 201, 388 Wood, J. L., 162, 163, 164, 171, 346, 388 Wright, C . A., 23, 24, 32, 34, 37, 40, 41, 47, 103, 355, 388 Wright, R. R., 307, 308, 388

Y Yamada, M., 21, 199, 388 Yamaguti, S., 101, 118, 204, 212, 223, 228, 231, 235, 255, 256, 259, 269, 296, 298, 309, 310, 388, 389 Yasuraoka, K., 47, 389 Yentsch, C. S., 51, 389 Yokoya, Y., 331, 389 Yonge, C. M., 4, 61, 389 Yunker, C. E., 131, 389

Z Zernov, S. A., 97, 389 Zinn, D. J., 136, 389 Zischke, D. P., 81, 119, 122, 389 Zischke, J. A,, 81, 119, 122, 389

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Taxonomic Index A A c a n t h k , 261 Acanthocephala, 87 A canthochit# dbcrepeua, 61 fascicularb, 169 Acanthoparyphium spimdoswm, 2 64, 338 Acanthopsolua lagenijormb, 246 Achatilta fulica, 19 Acholoe, 40 Acmea digital&, 73 Acotylea, 200-202 Acystidea, 261 Adam.& palliata, 6 Adoleamria perla, 214 Aequipecten, 264, 269, 270, 316, 333 gibbua, 269, 281, 322, 336 irradhna, 46, 46, 136, 236, 280, 281, 282,311,322,336 ( = Pecten) maxhua, 203, 269, 283, 336 Aggregatu eberthi, 132, 133 Aggregatidae, 167-161 Allocreadiidae, 24Ei-260 Allocreadioidea, 246-250 Allocreadium, 248 A l y k m a n d u d o r , 49, 50 Amaroucium, 312 Amoeba radiosa, 142 Amoebida, 143-147 Amoebidae, 142, 143-147 Amphineura, 102 Amphiprion percub, 19 A m , 269 Anchicaligua, 101 A n a r r h i o h l u p , 245, 246 Ancbtrocoma, 169, 193-194, 338 pelseneem', 191-193, 194, 338, 339, 340 Ancistrocomidae, 187- 196 Ancktrospira, 188 venerh, 186, 344 Amistrum, 178, 180 barbatum, 180 mudatum, 178, 180

Ancistrum-continued compreasum, 180 cyclidioidea, 180-181, 339, 343, 344 kaeli, 182-183, 184, 340 japonica, 183-184, 186, 339, 340, 344 mytili, 178-180, 182, 196, 340, 341 mbtruncatum, 180, 181, 343 tellinae, 180 Ancktruma, 178 ieeeli, 182 Anepitheliocystida, 254 Anepitheliocystidia, 203, 246 Angio8t~ongylwcantonen& 12, 13, 19, 270-273, 338, 340 Annelida, 273-275 Anodonta cataract&, 47, 48 grandk, 94 implicata, 19 Anomia simplex, 315 Anoplophrya, 178 Antheasiua, 100, 316 Aply8ia punctata, 249 Arbacia punctukzta, 266 Arca campechiemh, 94 decuasata, 312 incongruu, 04 senilk, 312 Arotica irrdiana, 280, 282 ielandica, 280, 282,343 (= Pecten) bladica, 282, 343 Arctono5 fragilw, 23, 29 Prittata, 23, 63, 274 Arenaria, 236 Arenicola marina, 236 Artotrogua, 101 Asoarididea, 260 Aschelminthes, 263-275 Ascidia paratropa, 100 Assiminea japonica, 26, 27 kat&cea miyazakii, 26, 27 401

A.Y.B.--6

28

402

TAXONOMIC INDEX

Assiminea-continued parasitologica, 26, 27 Aster& vulgaris, 323 Aatraea undoaa, 73 Atherina boyeri, 228 hepsetua, 228 presbyter, 228 Atrina rigida, 45, 46 aerrata, 322 Auatralorbis, 37 glabratus, 1, 24, 26, 26, 28, 29, 31, 33, 36, 36, 37, 40, 42, 62, 63, 65, 72, 75, 78, 79, 81, 84, 87, 89, 104, 122, 133,389 Auatrobilharzia variglandis, 13, 84, 125 Axinopsis sericatw, 20 Aythya ferina, 220 Azygiata, 203-236

B Bacciger, 228 bacciger, 228-230, 339, 343, 344 Balanua bahnoides, 54 Batillaria minima, 14 Bdellonemertea, 262 Benthooctopua, 101 Betaeua hmfordi, 100 Bwmphalaria, 37 boisayi, 35 Bittium varium, 278 Blenniua gattoruginae, 249 ocellaria, 231 Boatrichobranchua pilularis, 323 Boveria concharum, 184 subcylindrica, 184-185, 337, 339, 343, 344 Brachidontea recurvua, 94, 234, 236, 294, 296, 302, 304, 336 Brachygnatha, 322-330 Brachyrhyncha, 3 16-332 Brachyura, 316-332 Briarella, 101 Buccinum, 22, 63 undatum, 19, 22, 46, 136, 246, 246, 260, 336

Bucephalidae, 203-21 1 Bucephaloidea, 203-2 11 Bucephalopsis grmilescem, 203 haimeanus, 203 Bucephalus, 68, 80, 82, 86, 86, 87, 89, 116, 117, 118, 119, 169, 208-209 cuculus, 116, 118, 119, 122, 203, 205, 206-208, 338 haimeanus, 118, 203-206, 207, 220, 337, 342 rnytili, 209-211, 341 polymorphua, 203 Bulimnaea megasoma, 41 Bulimulua alternatua, 94 Bulinua, 28, 37, 77 (Bulinua) truncatua, 28 contortua, 37 dydowski, 37 (Physopsis), 28, 37 truncatw, 37 Buaycon, 136, 201 Canaliculatum, 71 carica, 71 perueraum, 94

C Cairina, 269 Callkctie, 63 parasitica, 19, 22, 46 Calliophanidae, 201 Cancer, 261 Cantharus lineatua, 249 orbicularis, 249 Capitella ovincola, 274 Capitellidae, 274 Capitomastus minirnua, 274 Capsafragilis, 180, 184 Carcinus, 261 Cardita ajar, 302, 337 calyculata, 163 sulcata, 184, 337 Cardium, 11, 135, 213, 214, 215, 226, 334,390 aculeatum, 262 edzcle, 202, 204, 213, 215, 218, 219, 226, 235, 243,244, 250, 253, 254, 287, 326, 337,390

TAXONOMIU INDEX

Cardium-continued norwegicum, 326, 337 ringene, 302, 337 tuberculatum (=C. rmticum), 203, 337 Cephalina, 148, 151, 153, 156 Cephalopoda, 94 Ceratocherea, 101 Cercaricc brachidontis, 234-235, 240, 336 buccini, 250-252, 336 bucephalm haimeana, 204 cambrensis, 219, 220, 390 dichotoma, 212, 222, 225-226, 337, 339 dkcursata, 228 emmculana, 109, 110, 111, 123 fisaicauda,225 fulbrighti, 212, 219 (Gymnophallus) cambrenais, 218-219 fulbrighti, 218, 219-220 himasthla secumda, see Himz&hla leptoeoma hueti, 226, 337 bta, 228 lebouri, 113, 114, 115, 11 6 littorinae, 109, 110, 111 rudis, 113, 114 lophocerca, 109, 110, 119, 123 lutea, 230 macomae, 221 margaritae, 213, 214, 390 milfordensis, 42, 230, 233, 234 myae, 42, 212, 227-228, 229, 340 mytili, 250 neptuneae, 246-248, 336 noblei, 210, 211, 341 oocysta, 120, 125 ophicerca, 247, 248, 343 parvatrema homoeotecnum, 113, 114, 116 patellae, 106, 107, 108, 109 pectinuta, 230 purpurae, 244 guiaaetensis, 236 reynoldsi, 125 roscovita, 113, 114, 115, 116 seneifera, 244 setifera, 249 spinulosa, 85, 88 strigata, 215

403

Cercaria-continued tenuans, 251, 252-253, 342 ubipuita I( zcbipuitoides), 109, 110, 111, 113, 114, 115, 116, 118, 120, 125 X , 68, 134 Cerithidea californioa, 127 Cerithium, 111 rupeatre, 153 Cestoda, 338, 340, 344 Cestoidea, 254-261 Chaetopterm, 315 variopedatue, 44, 323 Chilina dombeiana, 262 Chiton caprearum, 153 olivacew, 180 Chlamys nipponenais, 331, 337 opercularia, 282, 283, 337 see Pecten (= Chlamys) opercularia Chlorohydra viridissima, 6 Cholidya, 101 Chrysallida, 281 obtusa, 285, 343 Chytridiales, 175 Chytridiopsia, 175 mytilovum, 175-177, 341 ovicola, 177, 342 Ciliatea, 177-197, 337, 338, 339, 340, 341, 343, 344 Ciliophora, 177-1 97 clangula h y e m a h , 220 Clausidiidae, 286-300 Clinocwdium nuttalli, 334 Cliona, 198 elata, 198 lobata, 198 truitti, 198 vasti$ca, 198 Clytia bakeri, 7 , 18, 20 Cnidaria, 20-21, 199 Cnidospora, 175-177, 341, 342 Coccidia, 157, 159, 161 Columbella lunuta (= Mitrella lunata), 34 riatica, 153 Conchophthiridae, 186 Conchophthirm, 186 mytili, 186-187, 188, 341

404

TAXONOMIO INDEZ

Conchyliurua, 101, 302, 337 lobatua, 302, 337 t o r o m , 302, 339 Conus mediterraneue, 163 Copepoda, 286-315, 336, 337, 338, 339, 340, 341, 342, 343, 344 Corbicula lindolmk, 12 subplanata, 12 Corycaeidae, 308 Coturnix, 75 Cotylumcs brevk, 86, 88 cornutua, 25 jlabell$wmis, 77, 105 Crmsostrea angulata, 137 gigas, 21, 41, 43, 58, 59, 76, 80, 106, 121, 130, 136, 171, 199, 201, 224,231,234,237, 274, 290, 291, 293, 321, 331, 338,390 rhizophorae, 20, 199 virginica, 11,12,41,43,44,58,59,60, 61, 62, 63, 64, 66, 68, 69, 71, 72, 76, 80, 82, 86, 86, 87, 89, 94, 99, 100, 102, 106, 116, 117, 118, 122, 131, 133, 136, 137, 138, 139, 140, 142, 143, 144, 145, 147, 148, 161, 152, 163, 161, 162, 164, 165, 166,168, 169, 170, 171, 192, 193, 201, 206, 207, 208, 209, 236, 254, 267, 268, 259, 263,264, 270, 271, 272,273, 274,278,279, 280, 281, 296, 302, 303, 311, 315,321, 330, 335,338 Crebricoma, 189 carinata, 188, 189-190, 341 kozlofi, 187-189, 190, 196, 341 Crenilabmcs grbeua, 231 pavo, 231 Crepidostomum cornutum, 116 Crepidula, 11 convexa, 278 fornicata, 236, 280, 281, 282, 287, 291 plana, 280, 282 Cricetua, 122 Cmcibulum striatum, 280, 282 Cmstacea, 286-335

Cryptocotyle lingua, 92, 126 Cryptogonimidae, 228 Cryptomya cali,fornica, 191 Cumingia tellinoidea, 236 Cyclina sinemis, 184 Cyclonais tuberculata, 48 Cyolopoida, 286-312 Cyprina ialandica, 262

D Dafla, 269 Daubaylia potomaca, 71, 78 Decapoda, 316-332 Dentalium, 20, 162 Dermasteriaa imbricata, 61 Dermocystidium, 66 marinum, 11, 62, 80, 169, 192, 389 Digenea, 203 Diodora aapersa, 23 Diplospora, 161 Diplothyra smithii, 100, 101 Disaodactylus, 332 rnellitae, 44 Distomum leptosomum, 235 see Gymnophallus margaritarum, aomateriae Donax, 161, 339 gouldi, 7, 18, 20 politua, 184, 339 trunculue, 180, 339 uariabilis, 94 vittatua, 215, 217, 230, 339 Doris tuberculata, 68 Dosinia bilunulata, 184 dkcus, 94 Duymeria jlagellQera, 23 1

E Echeneibothrium, 103, 264, 259-261, 344 minimum, 259 Echinocephalus, 265 multidentatua, 266 pseudouncinatua, 266, 266-268 aouthwelli, 266

406

TAXONOMIC ZNDEX

Echinocephalua-continued spinoaissimw, 265 uncimtua, 265-266, 340, 343 Echinoparyphium, 83, 92, 93 Echinostomata, 235-245, 254 Echinostomatidae, 254 Echinoatoma ilocanum, 12, 24 lindo8n&, 12 revolutum, 122 Echinostomida, 236-245, 254 Echinostomatidae, 86, 235-243 Echinostomatoidea, 235-254 Echinostomum eecundum, 235 Eimeriina, 157, 159, 161 Eledone, 76 Elliptio, 94 complanatua, 48 dilatatua, 48 Endamoeba, 145 Ensis directus, 67, 236, 339 Ephestia, 60 Epinephelw akaara, 231 Epitheliocystidia, 245-250 Ergasilidae, 302-306 Erichthoniue difformia, 230 Eriphiu spinqrona, 153, 154 Eucestoda, 254 Eucoccida, 157-161 Eugregarinia, 148-1 57 Eugymnanthea, 101, 199 inquilina, 20, 199 japonica, 21, 199 ostrearum, 20, 199 polimantii, 21, 199 Eunice haraasi, 274 Eunicidae, 274 Eupagurua, 261 bernhardus, 19, 22 prideuuxi, 6 Eupleura caudata, 12 Euryniu iris, 48 Eurypanopeua depressua, 149, 336 Eurytium l h o a u m , 149 Eustylochua elliptiom, 200 meridionalis, 200 Eutetrarhynchidae, 261 Eutetrarhynchua ruficolle, 261 Evaaterh, 23

F Fabia, 332 lowei, 333 aubquadrata, 100, 324, 332, 334, 340 Faaciola gigantica, 67 hepatica, 1, 25, 34, 84, 105, 122, 134 Faacwlaria distana, 94 tulipa, 7 1 Paacioloidea magna, 32, 35, 38, 39, 57, 89

Faaciolopais bualci, 24 Fsllodistomatidae, 2 12-235 Fellodistomatoidea, 212-235 Fieraafer, 7 Plabellula, 143 c a l k e d , 143 patuxent, 143 Possaria abruaaa, 41 modicella rwtica, 39 F w i s ayracuaanw, 180

G Gadus callarim, 205 merlangua, 206 Galba ollula, 57 Galleria mellonella, 68 Gargariua gargariua, 196, 196-197, 341 Gaateroatomum gracilescens, 203 Gastropoda, 21, 94, 278-285, 336, 337, 338, 340, 341, 342, 343

Gibbula, 231 adamsoni, 153 cineraria, 287 divaricata, 153 rarilineata, 153 varia, 287 Giganto-bilharzia huronensia, 35 cflypthelrnins pennsylvaniensis, 66, 81, 82, 85, 86, 87, 108, 116, 124

quieta, 90, 92 Gnathostoma, 268 Gnathostomatidae, 266-266 cforgodera amplicava, 40, 57, 91, 92 &a$lla gemellipam, 202 Grafillidae, 202

406

TAXONOMIC INDEX

aregarina gigantea, 166 Gregarinia, 148-167 @ma, 269 Qymnophalloides tokiensia, 121 Qymnophallua bursicola, 222, 223-224, 341 c a m b r e d , 337 see Ceroaria choledochua, 219 dapsilia, 214 deliciosua, 217 duboki, 214 fulbrighti, 337 macomae, 221, 222, 339 macroporus, 2 17 margaritarum, 73, 212-215, 216, 217, 219,220, 337, 341, 342, 343,344 megalocoela, 216, 217, 220, 343 oedemiae, 216 perligena, 221-223 scrivenaia, 220-221, 222, 344 somateriae, 213, 215 strigata, 215-217, 337, 339, 344 tokienais, 222, 224, 338

H Haematopus ostralegus, 236, 243 occidentalk, 390 Haliotia, 135 asaimilia, 100 corrugata, 100, 266, 267, 268, 339 fulgena, 268, 339 gigantea, 339 kamtachatkana, 100, 274 rujeacem, 100 sorenseni, 100 (Sulculua)gigantea, 296 wallalenaia, 100 Halocynthia aurantium, 100 igaboja, 100 Haplosporea, 161-175 Haplosporida, 162-176, 176 Haplosporidiidae, 162-175 Haplosporidium, 162, 164, 169, 173, 175 coatale, 162 dentale, 162 mytilovum, 175 pickfordae, 162 tumefacienth, 161, 173-175, 341

Harpacticoida, 3 12-3 15 Heliaoma, 162 anceps, 28, 67, 124, 125 caribaeum, 71, 77 duryi normale, 57 trholvis, 57, 66, 81, 83, 86, 87, 92, 93, 108, 116, 124, 126 Helix pomatia, 73, 75 Hemispeiridae, 178-186 Heterocheilidae, 269 Heterodontua jranciai, 268 Hexamita, 62, 96, 98, 128, 129, 169 inflata, 97, 137, 138-142, 338, 342 i.ntestimlia, 139 salmon&?,139 Hexamitidae, 138 Himmthla, 132, 235, 238, 239, 241, 243 ambiguu, 239-241, 343 compacta, 241-242, 341 elongata, 238 Zeptosoma, 109, 110, 119, 123, 127, 235236,240,337,339,340,341, 344 littorinae, 242, 341 muehlensi, 12, 238-239, 340 quksetensia, 40, 41, 42, 43, 68, 59, 66,67,76,126,236-237,238,239, 240,336,338,339,340,341, 342 Holorhinua calijorniczcs, 260 (= Myliobatis) californicus, 268 Holotrichia, 178-197 Homarus, 157 gammarus, 156 vulgaris, 312 Hoploplam inquilina, 201 thiaana, 202 thaiaana, 202 Hoploplanidae, 201-202 Hyalaea trhpinosa, 21 Hyas, 261 Hyaloklosaia, 148, 161 lieberkhiihni, 161 pelseneeri, 161, 339, 344 Hydramoeba, 145 Hydrobia minuta, 241, 242 ( 3 Peringia), 120 ulvae, 120, 123, 124, 282, 283

407

TAXONOMIC INDEX

Hypocomidae, 190 Hypocomides, 194 modiolwiae, 194 mytdi, 191, 194,136, 341 zyrphaea, 194 Hypocomina, 189 earinata, 187, 189, 190 Hypocomoides mytiti, 190 Hysteromorpha triloba, 32

I Imogine oculifera, 201 Inachus, 261 Isidora, 37 Isocardia cor, 262 Isocomides, 191 myt& 188, 190, 191, 341

K Katuyama nesopkora, 24 Katylesia scalarina, 265 Kidderia, 186 mytili, 186 Kinetocodiurn danae, 2 1 Klossia chitonis, 162 Knemidokoptes mutans, 131 pilae, 131

L Labrus merula, 231 Labyrinthomyxa marina, 11, 389 Labyrinthulidae, 142 Lacuna divarioata, 282 Larnpsilis, 94 radiata, 48 Larua, 237 argentatw, 217, 235, 237, 241, 242, 243, 246 canw, 217, 245 fuscua, 217 Laaea rubra, 163 Lattia gigantia, 73 Lecanicephala, 254 Lecanicephalidae, 255 Lecanicephalidea, 255 Lecithodelzdrium aomateriae, 213, 214, 390 (=Qz/mnophalloides oedenk), 390 Lecithophora, 202

Lepisdostew osseus, 208 Lepocreadium, 249 album, 249, 261, 343 oval&?, 106 Leptoplana, 43 Leptosynapta inhoerem, 235 Lichomolgidae, 300, 308, 315 LichomoZgua, 101, 102 arcunw, 316, 343 major, 300 Ligumb fasoiola, 48 Limoaa, 236 Littorina, 92, 136, 242 irrorata, 94 littoralis, 115 littorea, 109, 115, 118, 123, 125, 126, 127, 136, 236 neritokles, 120, 121 obtwata, 242 pintado, 35, 111, 245 plunaxk, 111 saxatilk, 113, 115, 242, 282 tenebroea, 113, 114, 115 Loligo opalemem, 214 vulgaris, 274 Lolipncula bevia, 94 Lophiua piscatorha, 205 Loripea lectew, 184 Lgmnaea, 1, 25, 162 auriculata, 124 (see also Radix) glubra, 106 limoaa, 25 p a l w t r k , 26, 94, 106 plicatula, 24 stagnalk, 26, 68, 77, 106, 134, 225 jugularis, 89 trunculata, 26, 84

M Macoma, 334 balthica, 191, 197, 221, 339 cumana, 302, 339 inconapicm, 191, 339 indentata, 334 i?lquim, 334 irua, 191, 339 nasuta, 191, 197, 334, 339 aecta, 191, 197, 334, 339 stultorum, 339

408

TAXONOMIU INDEX

Mmtra glabrata, 302, 339 sachalinensis, 262 secta, 262 solidhsima, 300, 339 stultorum, 236, 263, 254, 262 sulcataria, 184, 331, 339 veneriformis, 184, 340 Malacobdella, 262, 263 aurkulae, 262 grossa, 262 japonica, 262 mercenaria, 26 2 minuta, 262 obeaa, 262 Malacostraca, 315-335, 336, 337, 338, 339, 340, 341, 342, 343, 344 Margaritifera, 333 mrgaritifera cumingi, 266, 266 vulgar&, 69, 256, 266, 265, 266, 340 Marisa cornuarietie, 94 Maetigophora, 137-142 Megathura crenulata, 73 Meiogymmphallus mindus, 390 Melania nodocincu, 77 Melanitta fwrca, 214, 223 nigra, 214, 223 (= Oidemia) nigra, 213, 217 Melqrina irradians, 266 occa, 266 Meliphora, 60 Mellita, 19 quiqueaperf orata, 44 Melogena, 111 Melopsitta udulatwr, 131 Meneatho bisutularh, 280, 281 Menidia menidia, 207 Menippe mercenaria, 162, 335 Mereemria campeohiensis, 136, 262 mereenaria, 12, 13, 68, 69, 135, 236, 238,239, 262, 270, 280,282,294, 296,300, 301,302,304,336,340 Meretrix lusoria, 331 merit&, 184 Meaodasma deuuratum. 214 -.

Mesodon thyroidus, 94 Metacercaria achermsiae, 249-250, 261, 343 I, 226-227, 229, 340 mytili, 250, 261, 337, 342 see Gymnophallus duboisi, rnacomae, margaritarum, megalocoela, perligena, scrivensis, strigata, tokiensis; Proctocea oetreae Metastrongylidae, 270-273 Miamiensis avidwr, 13 Microsporida, 176, 176, 177 Microsporidea, 175-177 Microsporidia, 176 Minchinia, 162, 170, 173 chitonk, 162 costalia, 161, 162-165, 169, 338 dentale, 162 MSX, see nelsoni nelsoni, 11, 62, 161, 162, 165-172, 273, 338,389 pickfordae, 162 Mitrella lunata, 34 Mnestra parasites, 2 1 Modiola barbata, 178, 182, 340 Modiolaria, 63 marrnorata, 194 Modiolicola, 101 injlatipea, 316, 342 Modiolw americanua, 3 22 demissm,68,59,202,236,303,311,340

granosissimw, 294, 296, 302 modiolwr, 51, 52, 63, 100, 182, 183, 184,236, 280, 282, 322, 334, 340 Molgula, 278 ampulloidea, 312 robwrta, 323 Mollusce, 27G285 Monobrachium parasiturn, 20 Monoplacophora, 102 Monstrilla, 101 Morgania, 186 mytili, 186 MSX, see Minchinia nelsoni Motella mwrtelu, 139 trkirrata, 139 Murex fulvescens, 94

409

TAXONOMIC INDEX

MurexAontinued trunculus, 180 M u s domesticus, 122 Musculium partumeiurn, 57, 91, 92 Mustelus, 261 Mya, 333, 334 arenaria, 13, 19, 42, 58, 59, 67, 135, 191, 202, 226,227, 235, 236, 237, 241, 242, 262, 285, 300, 301, 308, 322, 334, 340 truncata, 262 Mycetozoa, 175 Myicola, 101, 300 rnetisiensis, 307, 308-309, 341 spinosa, 301 Myicolidae, 300, 308 Myliobatis maculata, 256 nieuhoj?, 265 Myocheres, 101, 300 dentata, 302, 339 major, 300-301, 339, 340, 341 scobina, 302, 344 Mytilhydra polimantii, 199 Mytilicola, 129, 290 intestinalis, 129, 286-290, 291, 292, 295, 296, 337, 342, 343 orientalis, 130, 131, 290-293, 295, 296, 338, 341, 342,343, 344,390 ostreae, 130, 291 porrecta, 131, 287, 293-296, 336, 340 Mytilus, 176, 178, 187, 214, 274, 283, 333,390 californianus, 73, 161, 162, 173, 174, 211, 291, 292, 341 eramitesta, 130, 290, 341 edulis, 42, 58, 59, 62, 72, 130, 135, 136, 174, 175, 177, 178, 180, 186, 187, 189, 190, 191, 194, 196, 209, 212, 213, 214, 215, 223, 224, 230, 231, 232, 233, 235, 236,237, 242, 244, 250, 252, 253, 254, 274, 277, 283, 284, 286, 287, 289, 290, 291, 292, 311, 312, 315, 322, 324, 326, 329, 330, 331, 332, 341 galloprovincialis, 21, 129, 135, 153, 199, 213, 214, 221, 223, 286, 287, 288, 342 hamatus, see Brachidontes minimus, 153, 156, 342 A.M.B.-5

Mytilus+ontinued perna, 315, 342 recumus, see Brachidontes

N Nanophyetus aalmincola, 13 Nassa mutabilis, 249 Nassariua obsoletus, 13, 21, 82, 83, 84, 105, 125, 236, 237 Natica habraea, 180 Nautilus, 102 Nematoda, 263-275, 336, 338, 339, 340, 343 Nematopais, 63, 148, 156 legeri, 153-156, 342 ostrearurn, 133, 148-151, 152, 154, 169, 338 prytherchi, 151-153, 154, 338 Nemeritis, 50 Nemertinea, 262-263 Neodiplostomunz intermedium, 134 Neopanope texana, 149, 335 sayi, 335 Neophasis pusilla, 245-246, 247, 336 Neorickettsia helminthoeca, 13 Neptunea antiqua, 246 Nitocrh dilaatatw, 122, 125 Noetia ponderosa, 94, 311 Notocotylidae, 86 Notomastus, 54 Nucella, 244 (= Purpura) lapillus, 244, 245 Nucula nucleus, 20 tumida, 20 Nuculana pustulosa, 20 Nycticorax nycticorax, 237 hoactili, 245

0 Oblata rnelanura, 249 Octopicola, 102 octopus, 102 bimaculatus, 74 Odoatomia, 278, 280 bisuturalis, 278, 279, 280-281, 284, 336, 337, 340, 343 27

410

TAXONOMIC INDEX

Odostomia-continued eulimoides, 283-285, 336, 337, 342, 343 impressa, 277, 21S279, 338 (M.) biauturalis, 280 pallida, see eulimoides rissoides, 283 scalaris, 277, 282-283, 284, 337, 342 seminuda, 281-282, 284, 336, 338, 340, 343 tellinae, 277 trijida, 284, 285, 341 Oliva sayana, 94 Olivella, 18 Opalina mytili, 178 Ophelia, 54 Opisthobranohia, 278-285 Opisthopus transversus, 332, 333 Orchitophyra, 192 Ostracotheres subquadratus, 333 Ostrea, 304, 305 cucullatu, 321, 330 denselamellosa, 309, 342 edulis, 11, 135, 137, 177, 203, 206, 274, 283, 284, 285, 287, 289, 290, 326, 342 lurida, 130, 135, 201, 291, 292, 293, 343 lutraria, 206 tulipa, 312, 315, 343 Ostreohydra, 199 Ostrincola, 101 clavator, 303, 304-305, 306, 343 gracilb, 302-304, 336, 338, 340 simplex, 305-301, 343 Opisthioglyphae ranae, 88 Otala (Helix)lactea, 74 lactea, 94 Oxymonadida, 138

P Pagrosomus auratus, 231 Panaietis, 101 haliotis, 296 298, 339 Panopew herbsti, 149, 335 Paphia philippinarum, 135, 184 pullastra, 287 staminea, see Venerupis

Parachaenia myae, 191 Parafasciolopsis fasciolaemorpha, 25 Paragonimus ohirai, 26, 27, 46 Paranisakis pectinis, 269 Paranthessius, 101 Parapinnixa, 332 Paravortex, 202 gemellipara, 202 Parorchis, 243 acanthus, 111,240, 243-245, 337, 341 avitus, 243, 244 Parvatrema homoeotecurn, 254 Patella, 159 hawaiiensis, 136 vulgata, 106, 107, 108, 109 Pecten, 53, 264, 269 ( = Aequipecten) maximus, 283 ( = Chlamys) opercularis, 283 islandicus, 283, 343 jacobaeus, 283, 343 maximus, 68, 159, 160, 343 see Aequipecten Pelecypoda, 20-21, 94 Pelichnibothrium, 254 speciosum, 254 Perigonimus abyssi, 20 Peringia, see Hydrobia Peromyscus, 122 Pettancylus assimilis, 134 Phasmidia, 265-273 Philoconcha amygdalae, 298-300, 344 Philophthalmus hegneri, 14 gralli, 14 lachrymosus, 14 lucipetus, 14 skrjabini, 14 Philophthalmidae, 243 Pholas candida, 230 crispata, 262 Phorcus richardi, 153 Phyllirhoe bucephala, 2 1 Phyllobothriidae, 259 Phyllobothrium, 254 Physalia, 43 Physa, 162 gyrina, 35, 57, 90, 92, 125 Physopsis, 37 africana, 37

TAXONOMIC INDEX

Pilumnua, 261 Pinctada margaritif era, 274 Pinna, 265, 266, 323, 333, 343 nobilis, 184, 343 rudis, 315 Pinnaxodes, 332 Pinnix a chaetoperana, 44 faba, 100, 333, 334, 337, 339, 341, 344 littoralis, 100, 333, 334, 337, 339, 341, 344 Pinnotherelia, 332 Pinnotheres, 43, 98, 100, 321, 330, 332 angelicus, 333 boninemis, 333 curdii, 331 depresseum, 3 1G depressus, 3 16 geddesi, 333 guerina, 333 maculatus, 45, 46, 322-325, 333, 334, 336, 338, 340, 341, 342, 343 margarita, 333 ostreum, 43, 44, 98, 99, 315-321, 324, 325, 329, 330, 333, 334, 336, 338, 342, 343 pisum, 98, 99, 325-330, 334, 337 politus, 333 pugettemis, 100 pholadis, 331-332, 337, 338, 339, 344 sinensis, 333 taylori, 324 veterum, 324 Pinnotheridae, 315-332 Piratasta, 129 Pisania maculosa, 153 Pitar mirabilis, 312 tumens, 312, 315 Placopecten magellanicus, 280, 282, 322, 343 Plagiorehiata, 245-250 Plagiorchiida, 245-250 Planocera elliptica, 200 inquilina, 201 nebulosa, 200 Planorbis, 37 corneus, 77

411

Planorbis-continued schmuckeri, 24 Plasmodium, 102 lophurae, 61 Platyhelminthes, 169, 199-261 Polinices conica, 265 duplicata, 94 Polycarpa, 159 Polycladida, 200-202 Polydora, 73, 273, 274, 275 ciliala, 274, 275 hoplura, 274, 275 ligni, 274, 275 pacifica, 274 polybranchia, 274 websteri, 274, 275 Polynoidae, 274 Polyonynx macrocheles, 44 Pomacea bridge&, 94 Pomolobus paeudoharengus, 19 Porifera, 198 Poroapora, 148 galloprovincialis, 153 gigantea, 156-157, 342 Porosporidae, 148-156 Porrocaecum, 269 crassurn, 269 pectinia, 269-270, 336 Portunus, 261 Posthod~plostomumminimum, 67 Proctoeces, 230, 232 maculatus, 229, 230-234, 341 ostreae, 229, 231, 234, 338 progeneticua, 231 subtenuis, 231, 232 Prosthodendrium (Acanthatrium) anaplocami, 122, 125 Protozoa, 137-197 Pseudicyema truncatum, 84, 85 Pseudoklossia, 148, 161 chitonis, 159 glomerata, 157-159, 161, 344 legeri, 159 patellae, 159 pectinis, 159-160, 161, 343 Pseudomyicola, 101 glabra, 310, 311-312, 336, 338, 340, 342 mirabilis, 312, 343

412

TAXONOMIC INDEX

Pseudomy icola-continued ostreae, 309-311, 342 Pseudomyicolidae, 309-312 Pseudostylochus, 199 ostreophagus, 201 Pyramidellidae, 276-285

Q Quadrula quadrula, 94

R Rmbella, 190 helensis, 188, 190, 341 Radix auricularis ( = Lymnaea auriculata), 119 Raja, 261 Rana pipiens, 75 Rangia cuneata, 92, 94 Reptantia, 315-332 Rhabdocoela, 202 Rhinoptera bonasus, 256 quadriloba, 256 Rhipidocotyle, 208 Rhizopodea, 143, 145 Rissoa membranacea, 282, 283 Rumina decollata, 94

S Sarcodina, 142-147, 338 Sarcomastigophorea, 137-147 Saxicava rugosa, 283 Saxidomus, 334 giganteus, 74, 334 Scaphopoda, 102 Schistosorna bovis, 57 haematobium, 24, 37, 7 1 japonicum, 24, 92, 133 mansoni, 1, 24, 25, 26, 28, 29, 30, 31, 33, 35, 36, 31, 40, 42, 41, 51, 65, 77, 78, 81, 84, 87, 89, 92, 104, 122, 133 Schistosomatium douthitti, 35, 65, 389 Schizamoeba, 145

Schizothaerus capax, 100, 333 Scleroplax, 332 Scro bicularia plana, 231 tenuis, 225, 236 plana, 236 Segmentina nitidellus, 24 Semicoss yphus reticu lut II s, 2 3 1 Sepia, 73, 75, 102 oficinalis, 84, 132 Siliqua patula, 135, 262 Siphonaria lineolata, 94 Somateria mollissima, 213, 220. 223, 242 spectabilis, 220 Spanis aries, 231 maerocephalus, 23 I Sphmrium striatinum, 116 sulcatum, 48 Sphenophryidae, 196 Spionidae, 274 Spirilhm ostrearum, 137, 138 Spirometra mamonoides, 122 Spiruroidea, 265-266 Spisula solida, 329, 330 solidissima, 135 Splanchnotrophus, 101 Sporozoa, 148-175, 338, 339, 341, 342, 343, 344 Stagnicola emarginata angulata, 77 palustris, 122 rejlexa, 38, 39 Stenorhynchus, 261 Stephanostomum tenue, 82, 83 Sterna hirundo, 237, 242 SWichactis, 19 Stomphia, 51 coccinea, 5 1, 52, 53 Strigea tarda, 25 Strigeatoidea, 203-235 Strongyloidea, 270-213 Strongylura marina, 201 Styela, 159 Stylactis hooperi, 21 Stylochidae, 200 Stylochopsis littoralis, 200

TAXONOMIC I N D E X

Stylochus ellipticus, 200 jloridanw, 201 frontalis, 200 inimicus, 200 littoralis, 200 oculqeerus, 200-201 tenax, 200 Siiccinea putris, 123, 124 Syndosmya alba, 204

T Tagelus gibbus, 301 Tapes, 159, 213, 214, 215, 332,333,334, 390 aureus, 230, 248, 249, 343 candida, 343 decussatus, 20, 135, 180, 182, 184, 199, 213, 217, 239, 241, 248, 249, 343 ( = Amygdala decussata), 217, 230 jloridus, 157, 158, 344 japonica, 135, 331, 344 philippinarum, 58, 59 pullastra, 220, 230, 235, 344 sernideciissata, 59, 135, 184, 230, 236, 270, 272, 344 semidecussatus, 257, 259, 298 virgineus, 157, 344 Tectibranchia, 278-283 Tegula galena, 73 Tellina, 161, 344 exigua, 180, 184, 230, 344 nitida, 184, 344 nymphalis, 302, 344 planata, 184, 344 solidula, 221, 225 tenuis, 215, 217, 344 Telosporea, 148-161 Tetraphyllidea, 254, 259 Tetvarhynchus, 255 ruficollis, 261 unionqactor, 255 Tetrias, 332 Thais jloridana jloridana, 202 haemastoma, 12, 111 haysae, 94 lapillus, 111 Thallophyta, 169

413

Thigmophrya macomae, 195, 197, 339 Thigmophryidae, 197 Thigmotrichida, 178-197 Tisbe, 101, 312 celata, 312-315 elongata, 3 12 furcata, 312 wilsoni, 312 Tisbidae, 312-315 Tivela stultorum, 7, 18, 20, 135 Trematoda, 202-254, 337, 338, 339, 340, 341, 342, 343, 344 Tresus capax, 334 nutalli, 334 Trichobilharzia elvae, 35, 41, 65 physellae, 35, 65 Trichodina myicola, 19 Tringia alpina, 236 canutus, 236 Triphora nigrocincta, 278, 279 Trochicola, 101 enterica, 287 Trochocochlea articulata, 153 mutabilis, 153, 156 turbinata, 153 Tropicorbis, 37 havanensis, 65 Trutta fario, 139 Trygon brucco, 265 Trypanorhyncha, 255, 261 Trypanosoma balbianii, 137, 138 eberthi, 137 lewisi, 122 sanguinis, 137 Turbellaria, 199-202 Turbonilla, 276 interrupta, 285 Turritelh communis, 283 Tylocephalum, 64, 69, 70, 102, 129, 131, 132, 169, 255-259, 338, 340, 344 dierama, 256 ludi$cans, 256 margaritqera, 256 minus, 256

414

TAXONOMIC INDEX

Tylocephalum-continued pingue, 256 unionijactor, 256

U Unionicola fossulata, 45 ypsilophorus, 48 var. haldemani, 47 Urosalpinx cinerea, 12, 111, 245

V Vahlkamjla calkelzsi, 142, 143-145, 147, 338 patuxent, 129, 142, 143, 145-147, 338 Vampyrellidae, 142 Vannella, 143 Velacumantus awtralis, 128 Venerupis ( = Paphia) staminea, 291 philippinarum, 12, 135, 270 semidecussatus, 135 staminea, 103,254, 259,260, 261, 344 V e n w , 214, 344 exoleta, 262

Venus-continued fasciata, 186, 344 gallina, 184, 344 mercenuria, 94, 238 see Mercenaria praeparca, 262 Vivipara quadrata, 24 viviparus malleatus, 57, 75, 76 Volsella, 279 Vulpanser tadorna, 220

X Xanthidae, 335

Y Yoldia cooper;, 262

Z Zanclea costata, 21 Zeugorchis acanthus, 243 Zirfaea crispata, 194 Zizyphinus zizyphinus, 287 Zoochlorella, 6 Zoogonoides laevis, 34 Zoogonus rubellus, 105 Zoomastigophorea, 138, 342

Subject Index A Abalones, 100, 135, 266, 268, 296 Abscesses, molluscan, 65 Accessory sex organs, reduction of, 123 Acetyl CoA, 81 Acids, in chemotaxis studies, 30, 33, 46 Adriatic Sea, 129, 230, 265, 286 Africa, 37, 38, 77, 301, 315 Agar pyramids, in chemotaxis studies, 29, 30, 31, 32, 33, 34 Age, as factor in chemotaxis studies, 38, 39 Albumin, as antigen, 78 Albumin gland, molluscan, 66 Alewife, 19 Alkaline phosphatase, 83 Allocreads, 245-251 Alternation of hosts by pinnotherids, 100, 329 Amino acids, 81, 88, 89, 90, 91, 92, 93, 97, 98 inchemotaxisstudies, 27,29,30,33,34 in molluscan plasma, 90, 91, 92, 93 in molluscan tissues, 90, 91, 92, 93 in snail mucus, 34 Amino sugars, in snail mucus, 34 Amoebae, 129, 132, 142-147 Amoebocytes (phagocytes, leucocytes), 62, 63, 68, 71 Amoebula, 174 Amphineuran, 61, 180 Anaerobism, 105 Anemones, 6, 19, 22, 49, 51, 52 Angler, 205 Annelids, 40, 44, 98, 273-275, 276 Antibodies, 77, 169 Antitoxins, 73 Arctic Ocean, 20 Argentina, 322 Arthropods, 49, 84, 101, 286-335 Ascidians, 54, 159 Asia, 13, 270 Atlantic Ocean, 20, 21, 69, 118, 135, 140, 148, 151, 161, 170, 171, 192, 198, 202, 230, 273 415

Attachment, symbiont to host, 17 Attraction, lack of in miracidium-snail relationships, 34, 35 of symbiont to host, 14, 16, 18, 23, 24, 25, 41, 44, 45 Attraction theory, 24, 25, 26, 36, 40 Auricles, 79 Australia, 128, 254, 265

B Bacteria, 12, 78 Barnacles, 64, 200, 202 Belgium, 97, 255, 261 Biological control, 12 Biology, importance of study of molluscan parasites to, 10 Black Sea, 97, 214, 231 Blockinglayer, 109, 110, 111, 113, 114, 115, 233 Blood, site of parasitization, 109, 118, 167, 230, 236 Blood sinuses, molluscan, 72, 134 Blowfly, 49 Body fluids, molluscan, 73 Body temperature, increase in parasitized snails, 105 Boring sponges, 198 Brazil, 14, 65, 315, 325 Brown cells, molluscan, 79, 80 Bucephalids, 203-21 1 Buckies, 135, 245 Budding of Vahlkampfia, 146 Budgerigars, 131

C Calcium, ionic, 125 Calcium phosphate, 125 Calcium spherites, 125 California, 18, 100, 173, 178, 184, 187, 190, 191, 194, 196, 211, 254, 259, 266, 268, 291, 292 Canada, 135, 192, 200, 236, 281, 312 Cannibalism, 8, 19

416

SUBJECT INDEX

Carbohydrate metabolism related to anaerobism, 105 related to lipid metabolism, 87 Carbohydrates, 81, 82, 118 Carbon dioxide concentration, as attractant, 46 Carotene, 127 Carotenoids, 127 Castration, surgical, 124 Catfish, 245, 246 Cephalic glands, 217 Cephalopods, 68, 73, 75, 84, 101, 102, 254 Cercaria activation of, 40, 57, 58 encystment of, 41, 59, 77 Cercariae, 202, 203, 212, 213, 217, 218, 225, 226, 228, 230, 231, 234, 236, 237, 241, 242, 244, 245, 246, 249, 252 lipid content, 88 microcercous, 230, 250 Cercarial dermatitis, 13 Cercarial emission, influence of host starvation, 84 Cercaricidal substances, 78 Cestodes, 56, 69, 84, 103, 254-261 Ceylon, 69, 255, 256, 265 Chemo-klino-kinesis, 32 Chemo-klino-taxis, 32 Chemo-ortho-kinesis, 31 Chemoreceptors, 22 Chemotaxis, 16, 19, 22, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 47, 48, 49, 187 Chemo-tropo-taxis, 32, 33 Chesapeake Bay, 165, 193 China, 136 Chitons, 159, 162 Chlorophyll, 127 Choice apparati, 26, 27, 44, 45, 49 Chondroitin sulfate, 70 Ciliary currents, attraction of miracidia by, 25 Ciliates, thigmotrichous, 19, 177, 178197 Circulatory system, molluscan, 134 Circus movement, 33 Clam boring, 100, 101

Clam-continued butter, 74 little neck, 135, 259 pismo, 18, 135 razor, 135 soft, 135, 226, 227, 228, 308, 309 surf, 135 Clam-diggers’ itch, 13 Clams, 18, 92, 100, 259, 260, 262, 270, 334 as transmitters of parasites, 12 Clinging response, 22 Cnidarians, 20, 43, 199, 276 Coccidians, 157-161 Cockles, 135, 203, 254, 334 Cod, 205 Collagen, 70, 103, 260 Columellar tooth of Odostomia, 278 Commensalism, 4, 6, 7, 8, 17, 18, 19, 54, 56, 97, 98, 101, 132, 263 Complements, 74, 75 Conchs, 135 Conchiolin, 101 Condition index, 103, 130, 293 Congo, 302, 315 Connecticut, 200, 202, 223, 230, 239, 280 Connective tissue, 70, 79 Contact accidental, 16, 18 dependent on host’s feeding, 16, 18 influenced by chemotaxis, 16, 19, 23, 24, 25, 35, 37, 42 influenced by other taxes, 16, 17, 46 influenced by substrate, 16, 17, 23, 52 influenced by symbiont selectivity, 16, 49 initial, 16 Copepodids, 286, 288, 289, 290, 290 Copepods, 101, 102, 129, 130, 131, 286-3 15 Coracidia, 69, 129, 131, 257, 258 Crab mud, 335 mussel, 100, 322 oyster, 98, 322 pea, 98-100, 325, 330 pinnixid, 100 pinnotherid, 19, 316, 334

SUBJECT INDEX

CrabGontinued xanthid, 149, 150, 335 Crabs, 43, 44, 45, 98, 99, 100, 149, 150, 151, 152, 153, 315-335 integument of, 100 Crustaceans, 261, 276, 286 Crystalline style, 138 Culture media for Haplosporidium, 174 for Hexamita, 141 for Vahlkamjla, 144, 147 Cuttlefish, 132 Cyclopoid copepods, 286-312 cysts of Ancistrum, 179, 180 of Chytridiopsis, 177 of Haplosporidiurn, 174 of Vahlkampjla, 146 Cytolysis, parasite associated, 107, 110, 111, 113, 128

D Dead hosts, repulsion from, 23 Decapods, 315-335 Defense, physical, 17 Deformed shells, associated with Odostomia, 281 Delaware Bay, 140, 165, 170, 320, 321 Denmark, 213, 282 Detritus feeding, 19 Developmental stimuli, 6, 16, 56, 59 Diapedesis, 41, 48 Digestive enzymes, 6, 16, 56 Ducks, 137, 220, 242

E Echinocephalids, 265-268 Echinoderms, 43, 44, 51, 52, 98, 276 Echinoid, 19 Echinostomes, 235-245, 254 Ectoparasitism in mantle cavity, 96 on shell, 96 Egg production, reduction in, 119 Eggs, sterile, 119 Egypt, 37 Eider ducks, 213, 223, 227, 241 Elasmobranchs, 256,259,260,261,266, 268

417

Elimination, dynamic equilibrium of, 133 Encapsulating fibers chemistry of, 70 origin of, 70 Encapsulation, 60, 64, 65, 66, 67, 68, 69, 70, 71, 73, 76, 102, 103, 106, 116, 131 stimulatory agent of, 67, 70 Endoparasitism in alimentary tract, 96, 129, 130, 131, 145 in other organ systems, 96 intercellular, 96, 102 intracellular, 96, 102 Endospore, 153 England, 136, 186, 204, 209, 215, 218, 219, 221, 225, 243, 245, 246, 250, 252, 283, 285, 286, 330 See also Great Britain Entoniscids, 329 Environment, attraction to, 40, 46, 47 Environmental factors, influence on host-parasite contact, 27, 41, 42 Epimerite, 149, 152 Epiphoronts, 54, 97, 132, 199 Epispore, 153 Epizoic animals, 7, 97 Escape active, 16, 132-134 cellular, 16, 133 involuntary, 16, 132 passive, 16, 132, 134 reactions, 8, 132 Establishment influence of developmental stimuli, 16, 17, 35 influence of host’s control of s y m biont’s maturation, 16, 17, 95 influence of host’s defense mechanisms, 16, 17 influence of host’e digestive enzymes, 16, 17, 96 influence of symbiont -induced pathology, 16, 17, 95 of symbiont, 54, 55 importance of attachment, 16, 17 Europe, 14, 25, 118, 129, 136, 138, 156, 186, 189, 203, 215, 230, 269, 286, 290, 325

418

SUBJECT INDEX

Excreta, as a factor in pathogenesis, 110, 111, 113, 115 Excretory (osmoregulatory) system, trematodes, 86 Excretory wastes of parasites, pathogenic effects of, 107 Exoskeleton, arthropod, 131 External factors, influence of, 3 Eye-spots, trematode, 212

F Facultative parasites, 97, 98, 128, 129, 139, 263 Fatty acids, 81, 85, 86, 87, 206 in chemotaxis studies, 29, 33 in snail metabolism, 34 toxicity of, 13, 239 Feeding habits of Pinnotheres, 99, 100, 330 Ferment cells, 79 Fibroblasts, 68, 70, 72 Fibrosis, 131 Filter-feeding, 18 Fishes, 248, 249, 250 as symbionts, 7 gadoid, 204, 205 labrid, 231 pomacentrid, 19 sparid, 231 teleost, 254, 266 Fisheries biologists, importance of study of molluscan parasites, to, 10 Flagellates, 56, 62, 128, 129, 137-142 Flatworms, free-living, 40 Florida, 200, 201, 202, 257, 269, 280, 287, 325 Flying-spot microscope, 24 Foot, pigmentation of, 126 France,97,129,137,204,213,225,286, 287 Fungi, 79, 137 phagocytosis of, 62 responsible for mortalities, 11

G Gambier Islands, 256 Gametes, 152, 160 Gametocysts, 149, 151, 152, 155, 166 Gametocytes, 150, 158, 159

Gametogenesis, inhibition of, 119 Gar, 208 Gastrointestinal disturbances, 239 Geotaxis, 29, 36, 37, 42, 47 Germany, 97, 129, 137, 238 Ghana, 302 Gigantism, 119, 120, 122 Gills, 128, 189, 190, 191, 196, 242 lesions caused by crabs, 99 regeneration, 99 Glochidia, 19 Glucose, 81, 83, 113, 206 Glycine, 89 Glycogen, 81, 82, 83, 84, 87, 113, 118, 206 Glycoproteins, 70, 113, 174 Gonads regeneration of, 124 site of parasitization, 108, 117, 118, 208, 230, 234 trematode-caused pathology, 108, 109, 110, 113, 117, 118 Gonangia, 132 Gonophores, 21 Great Britain, 129, 135, 206, 244, 283 Greenland, 213 Gregarines, 148-157 Guinea-pigs, 245 Gulf of Danzig, 190 Gulf of Mexico, 14, 151, 192, 278, 281 Gulf of St. Lawrence, 280, 308 Gulls, 14, 227, 235, 237, 242, 243 Gymnophallids, 212-235 Gymnospores, 151, 152, 153, 155, 156, 157

H Hamsters, 122, 223, $27, 242 Haplosporidans, 161-176 Hard stage of Pinnotheres, 317, 319, 325, 330 Harpacticoid copepods, 312-315 Hawaii, 13, 69, 103, 136, 245, 257, 270, 273 Heart, as site of parasitization, 236 Heart beat in molluscs, influence of temperature, 71 Hemagglutinins, 73, 74, 75, 76 Hemichordates, 276 Hemolysins, 75

419

SUBJECT INDEX

Hemopoesis, 72 Hepatopancreas molluscan, 81, 83, 84, 85, 86, 87, 102, 117, 118, 119, 162, 173, 208, 228, 250 pigments of, 127 trematode caused pathology, 103, 104, 106, 107, 108, 109, 110, 111, 113, 114, 115, 117, 118 Hepatopancreatic absorptive cells, 107 Hepatopancreatic calcium cells, 125 Hepatopancreatic secretion cells, 107, 108, 110, 113 Hepatopancreatic tubules, obliteration of, 107 Hermit crabs, 6, 19, 22 Heterospecific associations, 4 Histolysis, parasite associated, 106 Holothurians, 7, 235 Hormones influence of host’s, 59, 60, 95, 122 molluscan, 122, 123 of parasite origin, 122 Hosts abnormal, 55 compatible, 55 incompatible, 55, 74 intermediate, 203, 217, 241, 245, 249, 254, 261 natural, 55, 68, 74 normal, 55 paratenic, 266 unnatural, 50, 55 Host factor, 23, 27, 46, 48 Host habitat, localization of, 46, 47 Host-localization, 40 Host-preference, 51, 281 change in, 50 of Mytilicola, 289 of pyramidellids, 277, 279, 280, 281, 282 bost-specificity, 18, 24, 25, 34, 37, 40, 55, 100, 102, 116 of pyramidellids, 276, 277 Host-symbiont contact, 16, 18 Human pathogens, shellfish as carriers of, 12 Hybridization, 50 Hydroids, 7, 17, 18, 20, 21, 101, 132, 199

Hydrorhizal net, 21 Hydroxyproline, 89

I Immunity, 17, 78 humoral, 60, 66, 73, 76, 77, 78, 89 innate (natural), 73, 74, 75, 113, 116 Incompatible hosts, attraction to, 27, 35 In-current, molluscan, 42 Indian Ocean, 21 Infection by Minchinia advanced, 172 initial, 171 intermediate, 172 recovery from, 172 terminal, 172 Inflammation, 67 Infusoriform, 132 Injured hosts attraction to, 29 repulsion from, 23, 29 Inquilinism, 4 Insects, parasitic, 40, 49, 50 Insusceptibility, 55, 6.8 Internal defense mechanism, 54, 56, 60, 63, 71, 76, 79, 259 Intracellular stages of Hexarnita, 139 Intranuclear bars, 165, 167 Invasive stage (1st crab stage) of Pinnotheres, 316, 319, 321 Ireland, 129, 136, 287, 289 Isopods, 236 Italy, 20, 21, 97, 199, 221, 249, 250

J Jamaica, 325 Japan, 21, 97, 130, 136, 199, 201, 223, 224, 230, 231, 234, 254, 290, 291, 296, 298, 309, 332

K Keber’s organ, 79 Keto-carotenoid, 127 Kidney molluscan, 72 site of parasitization, 109

420

SUBJECT INDEX

L Lambs, 122 Lecanocephalids, 255, 256 Lethality of Nematopsis, 148, 149 Leucine, 89 Leucocyte count in molluscs, influence of temperature, 71 Leucocytes (amoebocytes, phagocytes), 60, 61, 64, 68, 70, 71, 131, 133, 152, 171, 194, 260 digestive function of, 61 sludging of, 72 Leucocytes, 60, 71, 72 Leydig cells, 70, 87, 117, 118, 128, 129, 171 Life-span of Odostomia, 279 Limpet, 100, 136 keyhole, 23 Lipase, 86 Lipids, 81, 84, 85, 86, 87, 88, 113, 153, 206 hydrostatic role, 88 Lipid metabolism, related t o carbohydrate metabolism, 87 Lipoproteins, 85 Long Island Sound, 170 Louisiana, 148, 207, 234, 294, 302 Lung flukes, 26, 27 Lungworms, 270 Lysine, 89

M Macroenvironment, 54 Macrogametes, 151, 158, 159, 160 Madagascar, 256, 304, 305, 312 Maine, 226, 227, 241 Male reproductive system, avoidance by parasites, 109 Malpeque disease, 11 Mantle, 79, 88, 98, 101, 183, 243, 244 Mantle fluid, molluscan, 75, 97 Marine bacteria, phagocytosis of, 62 Maryland, 119, 145, 170, 175, 193, 257, 263, 264 Massachusetts, 97, 137, 143, 178, 182, 200, 202, 223, 226, 227, 228, 231, 236, 237, 241, 242, 256, 265, 280, 281, 282, 285, 308, 312, 315, 322, 324, 325

Maturation, control of, 6, 16, 56 Mediterranean Sea, 21, 129, 135, 157, 203, 225, 230, 231, 286 Medusae, 20, 21, 132 Megalops, 319, 324, 329 Meningoencephalitis, 13, 270 Merozoites, 156, 157 Mesozoa, 84, 85, 132 Metabolic dependency, 5, 6, 7, 9, 100 Metacercariae, 59, 67, 77, 78, 121, 132, 202, 203, 205, 208, 212, 213, 214, 215, 217, 221, 223, 224, 226, 227, 230, 231, 232, 234, 235, 236, 237, 238, 239, 241, 242, 243, 245, 248, 249, 250, 254 encystment of, 58, 59, 237, 244 progenetic, 231, 232, 234 Metacestodes, 69, 102, 132, 257, 259 Metachromasia, 70, 80 Metamorphosis, influenced by substrate, 52, 54 Metanauplii, 286, 288, 289, 290, 296 Metaplasia, 131, 293 Metastrongylids, 270 Mexico, 100, 268 Mice, 122, 227, 242, 245 Microenvironment, 54 Microgametes, 151, 158, 159, 160 Micropyle, 152 Microsporidans, 175-177 Microsporidia, 63 Miracidia, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 47, 49, 57, 65, 66, 85, 120, 208, 245, 253 immobilization of, 77, 78, 79 swimming velocity, 31, 32 Miracidial epidermal plates, shedding of, 57 Mites, 47, 48, 131 Molluscan blood, bactericidal properties, 75 Molluscs, as parasites. 276 Mortality associated with Hexamita, 139 associated with Minchinia, 164, 165 associated with Polydora, 275 parasitized molluscs, 104, 114, 116, 120, 130, 162, 164, 165 MSX, 11, 80, 165

421

SUBJECT INDEX

Mucopolysaccharides, 70, 80, 174, 206 Mucoproteins, 70, 174 Mncus, as chemotactic substance, 24, 34, 41, 48 Mud blisters, 274 Mud flat snail, 236 Mudworms, 273 Musculature, molluscan, 66, 243, 253 Mussels, 47, 48, 51, 52, 100, 129, 130, 135, 136, 173, 175, 182, 187, 190, 196, 202, 221, 223, 252, 253, 289, 290, 292, 294, 315, 325, 330, 334 Mutualism, 4, 6, 7, 8, 17, 132 Mutualists, 6, 54, 88 Myzorhynchus, 257

N Nacre, 73 Nacrezation, 60, 73, 101, 198 Naples, 178, 180, 182, 184, 199, 217, 239, 248 Nauplii, 286, 288, 289, 290, 296 Necrosis, 128, 129 Nematocyst discharge threshold, 22 tentacular, 22 Nematodes, 56, 57, 71, 78, 80, 84, 122, 263-273 Nephridia, parasites in molluscan, 159, 161, 162, 173 Neurosecretions, influence of host's, 95 Neutral fats, 85, 86, 87 New England, 21, 271, 273, 278 New Jersey, 170, 206, 208, 335 New York, 143, 178, 182, 230, 238 New Zealand, 206 Normandy, 225, 226 North America, 138, 140 North Carolina, 200, 201, 207, 269, 301, 302, 311, 325 Nuclear " caps ", 165 Nudibranchs, 21, 49 Nutrition, 6, 14, 16, 54, 55, 56, 80, 81, 82

0 Octopus, 135 Olfactory recognition, 49, 50

Olive shell, 18 Oocysts, 158, 159, 161 Operculum, sporal, 162, 167, 174 Opsonic effect, 75 Orange sickness of mussels, 252 Oregon, 292 Organic molecules, in sea water, 43 Ova, as sites of parasitization, 175, 177 Oyster American, 135, 142, 148, 149, 150, 162, 163, 168, 170, 171, 193, 200, 206, 208, 254, 270, 315, 320, 321, 335 European, 135 Japanese, 135 Pacific, 135, 293 Oyster beds, 97, 139, 261, 278 Oyster catcher, 235, 243 Oyster drills, 12 Oyster " leeches ",200 Oysters, 20, 41, 59, 60, 61, 62, 68, 71, 79, 80, 85, 88, 97, 98, 99, 100, 101, 117, 118, 121, 128, 129, 130, 131, 133, 135, 137, 138, 139, 140, 141, 142, 143, 150, 151, 153, 162, 163, 168, 170, 171, 193, 201, 202, 203, 208, 235, 255, 261, 270, 271, 274, 275, 278, 280, 282, 283, 285, 287, 289, 304, 305, 315, 319 as transmitters of parasites, 12 mortality of, 11, 161, 162, 164, 165, 172, 198, 290 pearl, 255, 265 strains (varieties, races, physiological species), 272, 273

P Pacific islands, 13, 136 Pacific Ocean, 135, 255, 270 Palps, 242 lesions caused by crabs, 99 Paraglycogen, 160 Parasites, as vectors of microorganisms, 13 Parasitic castration, 111, 114, 117, 118, 119, 120, 122, 123, 206, 208, 211, 228, 230, 233, 248, 330 Parasitic obesity, 122 Parasitism, 4, 5 , 7, 8, 9, 17, 54, 95

422

SUBJECT INDEX

Parasitoids, 49 Parenchyma, trematodes, 86 Pathogenesis influence of mechanical factors, 108, 111, 117 influence of physiological factors, 108, 110, 111, 117 Pathology, 95, 103, 104, 105, 113, 117, 128, 129 trematode-caused, 105, 117 Pearl formation, 73, 213, 214, 223, 224, 255, 256 Penshell, 46 Penetration glands, miracidial, 57 Penis, reduction of, 123, 124 Pericardial fluid, molluscan, 75 Pericardium, 79 Periostracum, 22 Periwinkles, 126, 136 Phagocytes (amoebocytes, leucocytes), 60, 61, 62, 65, 68, 111, 156, 157 ineffectiveness of, 63 Phagocytosis, 60, 61, 63, 64, 71, 72, 75, 76, 133, 141 Phoresis, 4, 7, 8, 17, 56 Phosphatases, 87 Phosphorylative transport, 83 Phototaxis, 28, 37, 42, 47, 48, 288, 289, 330 Planula larva, 18 Plasma, molluscan, 40, 41, 43, 57, 58, 59, 61, 75, 76, 78, 79, 88, 89, 98, 107, 237 electrophoresis of, 75 Plasmodia, 162, 163, 165, 167, 169, 171, 172, 173, 174 Plerocercoid, 257, 259, 260 Polychaetes, 19, 23, 49, 73, 273, 274, 275, 278 Polyclads, 200-202 Polyps, 21 Poriferans, 276 Post-mortem changes, 129 Precipitins, 74, 75 Predation, 4, 8, 9, 49, 199, 260, 263 Predatory molluscs, 12 Pre-hard stages of Pinnotheres, 317, 330 Prezoeae, 329 Primite, 152

Procercoid, 257 Proline, 89 Properdin, 75 Proteins, 81, 88, 89, 97, 98 Proteolytic enzymes, in sea water, 43 Protozoa, 84, 97, 135-197, Public health, 10, 12, 136, 239, 255 Puerto Rico, 20, 65, 199 Puget Sound, 20, 51, 100, 130, 259 291, 334 Pumping of oysters, 271 Pustules on oysters, 198 Pyramidellids, detached from hosts, 276, 281 Pyruvic acid, 81

Q Quahaug, southern, 135 Quahaugs, 135, 238, 270, 294

R Rabbits, 14 Rats, 14, 122, 245, 270 Rays, 265, 268 Rediae, 82, 83, 86, 89, 104, 106, 109, 110, 111, 113, 116, 127, 202, 237, 242, 245, 246, 248, 253 microvilli of, 83 Reducing sugars, 82 Regeneration, hepatopancreatic cells, 107, 110 Reproductive rate of Hexamita, 140 Reproductive system, site of parasitization, 108, 119 Resistance, 54, 55 acquired, 55, 76 behavioral, 55, 132 genetical, 55 innate, 55 mechanical, 55, 132 physiological, 55, 115 Resorption of parasites, 70 Respiratory rate, parasitized snails, 105 Reticulum, 70, 103 Rhabdocoels, 202, 254 Rhode Island, 118, 182, 208, 209, 227, 228, 272, 300, 301, 325, 335 Rhombogens, 8

SUBJECT INDBX

Rhynchus, 210 Rotbraunes organ, 79

S Sagami Bay, 334 Salinity, influence of on host-parasite contact, 27 Salivary glands, site of parasitization, 109 Salmon-poisoning disease, 13 Salts, in chemotaxis studies, 29, 30 Satellite, 152 Scallops, 46, 136, 159,203,264,269,283 Scaphopods, 162 Schistosomes, 13, 24, 25, 26, 28, 29, 30, 31, 33, 35, 36, 37, 38, 40, 42, 47, 57, 65, 77, 78, 81, 84, 87, 89, 104, 122, 133 Scoters, 214 Scotland, 136, 215, 220, 250, 253 See also Great Britain Sea urchins, 265, 266 Seasonal fluctuation of Hezamita, 140, 141 of Minchinia, 163 of MytiZicola, 289 Senegal, 312, 315 Sera, molluscan, electrophoresis of, 75 Serine, 89 Sex ratios of oysters influenced by Pinnotheres, 321 Sex reversal, 123, 124 Sharks, 268 Shell ballooned, 124, 125 banded, 128 calcification of, 125 color change, 124 thickened, 125 Shell distortions, parasitized snails, 105, 124, 125 Shell liquor, molluscan, 75, 97, 98, 99 Shell pockets associated with Odostomia, 283, 284 Shell-factor, 22 Shellfisheries, importance of study of molluscan parasites to, 10, 255 Shelter, 54 Shore birds, 212, 214, 217, 220, 237, 245, 254

423

Shrimps, 100 Sierra Leone, 302 Silverside, 207 Sipunculids, 276 Size of trematode larva, a factor in pathogenesis, 109, 110, 116 South Africa, 37 South America, 37, 38 South Carolina, 200, 206, 279 Spargana, 122 Sphingomyelin, 80 Spice bread disease, 198 Spirochaetes, 137 Sporadins, 149, 152, 154, 156 Spores, 133, 151, 152, 153, 158, 161, 162, 163, 165, 167, 168, 170, 177 Sporoblasts, 152, 167 Sporocysts microvilli of, 83, 226 sporozoan, 163, 167, 174 tegument of, 225, 226 trematode, 35, 57, 68, 77, 82, 83, 85, 86, 104, 109, 110, 111, 114, 116, 116, 117, 118, 202, 206, 207, 208, 211, 212, 213, 215, 218, 226, 230, 231, 232, 234, 248, 252, 253 Sporocyst wall, electron microscope studies of, 204 Sporonts, 149, 167 Sporozoans, 102, 132, 133, 148-176 Sporozoites, 133, 149, 151, 152, 153, 156, 157, 158, 159, 161 Squids, 135 SSO, 162 Stages 11-V of Pinnotheres, 317, 325 Starvation, as a factor in pathogenesis, 110, 111, 113, 115 Strigeids, 134 Stunted growth, parasitized molluscs, 105, 122 Sugars, in chemotaxis studies, 29, 30 Susceptibility, 54, 55, 80 Sweden, 178, 223 Swimmers’ itch, 13 Swimming velocity of Pinnotheres, 325 Symbiology, 5 Symbiontology, 5 Symbionts, 6, 19, 40, 41, 43, 46, 47, 48, 80, 88, 98 escape of, 16

424

SUBJECT INDEX

Symbionts-continued establishment of, 16 Symbiosis, 2, 4, 7, 8, 16, 56 Sympatric splitting, 50 Syzygy, 149, 152, 154

T Tactic response, reversal of, 47, 48, 49 Taiwan, 1 7 1 Taurine, 94 Temperature, influence on cercarial development, 232, 233 Tentacles, suctorial, 189, 190, 191, 192, 195, 196 Tentacular response, 51 Terns, 237, 242 Texas, 101, 119, 135, 208, 254 Thermal death point for Hemmita, 141 Thermal limits, parasitized snails, 105 Thigmotactic cilia, 180, 183 Thin (soft) shelled males of Pinnotheres, 329 Threonine, 89 Tissue extracts, molluscan, 40, 77, 78, 79 Tissue transplants, molluscan, 62 Toxins, 79, 129 Trematode infections, loss of, 106, 110, 111 Trematodes, 56, 81, 82, 83, 84, 85, 87, 89, 92, 95, 103, 104, 106, 109, 113, 114, 116, 119, 121, 122, 123, 124, 128, 133, 202-254 pigments of, 127 Triangular cells, 111 Trichocysts, 186 Trieste, 129, 286 Trophozoites, 149, 154, 179, 180 Trypanosomes, 137 Tumefactions, 175 Tunicates, 100, 276, 278, 312

Turpentine, as experimental pathogenic agent, 80, 106 Typhoid, 12

U United States, 14, 135, 136, 223, 238, 275

V Verruciforin cells, 84, 85 Vertebrates, 49 Vestige de frange adorale, 194 Virgin Islands, 325 Virginia, 119, 135, 148, 163, 170, 171, 207 Viruses, 12, 13 Vitamin A, 127

w Wales, 114, 204, 209, 215, 218, 252, 330 See also Great Britain Washington, 130, 201, 259, 291, 292, 334 Wasps, parasitic, 50 Water tubes, as a site of parasitization, 118 Wax moth, 50, 68, 7 1 West Indies, 37 Whelks, 19, 128 Whirling dance, 24, 32 White Sea, 20 Whiting, 205 Wound healing, snails, 105, 106

Y Y-tube choice apparatus, 26, 44

Z Zoeae, 319, 324, 329 Zooplankton, 18 Zygotes, 151, 152, 158, 159