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 While many such review articles encourage further research, some of them soon become out of date.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