Advances in Parasitology Volume 10

Advances in PARASITOLOGY

V O L U M E 10

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Advances in

PARASITOLOGY Edited by

BEN DAWES Professor Emeritus, University of London

V O L U M E 10

1972

ACADEMIC PRESS London and New York

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road, London NWl United States Edition published by ACADEMIC PRESS INC. 111 Fifth Avenue New York, New York 10003

Copyright @ 1972 by ACADEMIC PRESS INC. (LONDON) LTD.

AII Rights Reserved No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers Library of Congress Catalog Card Number: 62-22124 ISBN: 0-12-03 1710-9

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CONTRIBUTORS TO VOLUME 10 JOHNR. BAKER,Molten0 Institute, University of Cambridge, England (p. 1) GORDON F. BENNETT,Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada (p. 1)

GLENW. CLARK, Department of Biological Sciences, Central Washington State College, Ellensburg, Washington, U.S.A. (p. 1)

ALEXANDER FLETCHER, Department of Tropical Medicine, Liverpool School of Tropical Medicine, Liverpool, England (pp. 31 and 49) DONALDHEYNEMAN, Hooper Foundation, University of California, San Francisco, California 94122, U.S.A. (p. 191)

LAIRD,Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada (p. 1)

MARSHALL

*DONALD L. LEE,Houghton Poultry Research Station, Houghton, Huntingdon, England (p. 347)

HOK-KAN LIM, Hooper Foundation, University of California, San Francisco, California 94122, U.S.A. (p. 191) Department of Tropical Medicine, Liverpool School of Tropical Medicine, Liverpool, England (pp. 3 1 and 49)

BRIAN MAEGRAITH,

ZBIGNIEW PAWLOWSKI, Clinic of Parasitic Diseases, Przybyszewskiego 49, Poznan, Poland (p. 269) C. PEARSON, Department of Parasitology, University of Queensland, St. Lucia, Brisbane 4067, Queensland, Australia (p. 153)

JOHN

tKLAus ROHDE,Department of Parasitology, University of Queensland, St. Lucia, Brisbane 4067, Australia (p. 78)

MYRONG. Scnurrz, Center for Disease Control, U S . Department of Health, Education and Welfare, Atlanta, Georgia, U.S.A. (p. 269)

* Author in the section “Short Review” Present address: Department of Pure and Applied Zoology, University of Leeds, Leeds, England. t Present address: Department of Zoology, University of Khartoum, Sudan.

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PREFACE At the end of a decade and with ten volumes of Advances in Parasitology published I may be permitted to make an appraisal of a series of books which, because of a regular annual addition, has been regarded wrongly in some biological bibliographies as a scientific journal. Now that Volume 10 has appeared, 68 contributors have produced 57 original reviews and (since 1968) 15 short updated reviews, a vast summation of modern biological information and ideas in the field of parasitology. Thirty-one contributors lived in Great Britain, nine in Australia, ten in the U.S.A., six in Canada, three in Brazil, two each in Czechoslovakia, Poland, South Africa and Japan, and one in Israel. At my request, these writers found the time to record their thoughts and research experiences in well documented reviews. This may have cost innumerable man-hours, as have my editorial duties, but the benefit gained by stimulating teachers and researchers to greater effort in parasitology could hardly be reckoned in such units. Sixteen reviews deal with Protozoa, 13 with Trematoda, five with Cestoda and Acanthocephala, 12 with Nematoda, five with “helminths” and six with various parasitological topics. In the entire sequence of reviews, those on the Protozoa are concerned with avian and mammalian malaria, Chagas’ disease, coccidiosis, entamoebiasis, leishmaniasis, toxoplasmosis and trypanosomiasis. Trematode reviews are linked similarly with clonorchiasis, fascioliasis, paragonimiasis, paraimphistomiasis and schistosomiasis, cestode reviews with cysticercosis, echinococcosis and taeniasis, nematode reviews with ancylostomiasis, dracontiasis, filariasis, onchocercosis, parasitic bronchitis and trichiniasis. In vitro and in vivo culture of Protozoa come into the picture, and also similar methods of handling schistosomes and other trematodes and cestodes. There are reviews about snail vectors of trematodes and their control, about small but significant groups such as Aspidogastrea and Acanthocephala, about the evolution of parasites and their hosts, and about dynamic parasitic equilibrium in nematodes. There are reviews on larval Monogenea, on the life history sequence of digenetic trematodes and on intra-molluscan and intertrematode antagonism. There are reviews on parasitism and symbiosis in Turbellaria, on tissue reactions in the hosts of nematodes and on anthelmintic treatment and its results. Electronmicroscopy often comes into the picture and biochemistry plays some part in reviews. Much groundwork has been covered but it must be extended and many lacunae remain to be filled in. However, it is my hope to achieve much of this during another decade, if health permits. In the present volume, John R. Baker, Gordon F. Bennett, Glen Clark and Marshall Laird deal with what were once considered to be protozoan parasites of doubtful status and are here termed avian blood coccidians. They make a bold attempt to unravel tangled taxonomic confusion regarding two distinct groups of blood-dwelling stages of coccidia: (1) small non-pigmented nucleophilic parasites of mononuclear cells in the viscera and peripheral circulation; and (2) typical adeleine haemogregarines such as parasitize the vii

...

Vlll

PREFACE

blood cells of vertebrates during the course of the life cycle. Publications on avian blood coccidians of both groups of parasites are listed chronologically in Table I and a check-list of avian hosts of both groups of parasites is given in Table 11.These parasites are considered in separate sections as atoxoplasms and adeleine haemogregarines. It is unnecessary here to follow the detailed discussions of this team of professional protozoologists, who also give in Table I11 a classified host-list indicating atoxoplasm materials in thin Giemsastained blood films which are available for study in the WHO International Reference Centre for Avian Malaria Parasites at the Memorial University of Newfoundland. That there has been much confusion about the taxonomic status of these enigmatical blood parasites of birds is evident to the reader and the summarized information presented provides, in the words of the authors, “a fascinating basis for speculation”, and also “suggests hypotheses meriting early testing” by controlled experimentation to determine the true nature of atoxoplasms, and whether or not all atoxoplasms belong to the genus Zsospora and belong to one or more than one widespread species. Another point of wonderment is whether or not the few records of adeleine haemogregarines from birds represent simply accidental infection with parasites of reptiles which share the avian host environment. Alexander Fletcher and Brian Maegraith have not attempted to write a comprehensive review of literature on the metabolism of the malaria parasite but instead discuss some considerations and some current resarch trends which seem to advance our knowledge of fundamental biochemical processes which occur in parasites and are involved in the host-parasite relationship. After making some general considerations about the hazards presented by most types of cell preparation, they focus attention on the pentose phosphate pathway (PPP), for which the malaria parasite could have an absolute requirement, because it is probably the principal pathway for the production of pentose sugars necessary for nucleic acid synthesis. There is now evidence that most of the malaria parasites studied up to the present are dependent on the glucose-6-phosphate dehydrogenase (G-CPD), the initial enzyme of the PPP, the parasites being unlikely to thrive in enzyme-deficient cells and thus unable to produce an overwhelming infection. Further metabolism remains enigmatical, but current trends in research with mammalian species are discussed. Other topics considered are carbon dioxide fixation by malaria parasites, aerobic mechanisms, and the metabolism of chloroquine-resistant malaria parasites. These writers then turn to consider the metabolism of the host during infection, namely biological changes in erythrocytes, the effects of acute infection on host-tissue metabolism and host lipid metabolism. Such studies are enabling some advances to be made in our understanding of the pathological process involved in malaria. In another review Brian Maegraith and Alexander Fletcher concern themselves with the pathogenesis of malaria and they commence by indicating that the hypothesis of the physiopathological pattern of malaria is essentially inflammatory and non-specific in nature has long been upheld and is substantially correct. Research has been applied mainly on the effects of malaria infection on vascular membranes and vasomotor mechanisms of the host

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ix

and on the links between the parasite in its erythrocytic environment and the host. Two features are examined: the pathophysiology of malaria as an “inflammation”, and the possible initiating and maintaining factors involved. Attention is drawn to local and general responses in malaria which are similar to those which occur in inflammation and are demonstrable in bacterial infections and in malaria, babesiasis and other protozoan infections. Changes in endothelial permeability in malaria are next considered at some length, followed by statements on vasomotor changes in malaria in the hepatic, renal and intestinal circulations, which have been specially studied at the Liverpool School of Tropical Medicine. The kinin complex and other pharmacologically active agents are discussed fully, and it is shown that the kinin complex plays a significant part in disturbances that develop in permeable membranes, and is concerned with physiological change and resultant ultimate structural patterns. Another topic discussed is intravascular coagulation in malaria, the importance of which remains to be resolved in future research, one interesting region of which is the possible relationship between the peptide-peptidase which has been demonstrated in the kinin complex and the peptidase reactions involved in coagulation. Other topics discussed are anoxic anoxia (asphyxia), cytotoxic factors, and particularly mitochondria1 respiration inhibitors, and the physiological chain reaction which is set up and then leads to local or general disturbances that may be reversible but in time may become irreversible, leading to tissue death and the appearance of characteristic patterns of pathology. It is deemed helpful in visualizing the effects of a developing plasmodia1 infection to have this simple concept of initiating factors setting off a chain reaction of interlinked and interacting pathophysiological processes which may eventually involve changes in the local and general circulation of the blood, membrane permeability, hormone balance, and perhaps other as yet undisclosed effects. Klaus Rohde has not spared himself in writing the long review on the Aspidogastrea, especially Multicotyle purvisi Dawes, 1941. His researches on this trematode have occupied him throughout the last decade and it is noteworthy that we now know more about M . purvisi than we do about any other single aspidogastrid species. None of this research was completed when in 1961 I visited Kuala Lumpur as Examiner to the University of Malaya, and the review is of special interest to me because I described and proposed the erection of the new species and genus more than 30 years ago (Parasitology 33 (1 949, 300-3 15) from two well-preserved specimens collected on 25 July, 1932 from the river turtle Siebenrockiella crassicollis at Alor Star (north of Penang) by Mr G. B. Purvis, F.R.C.V.S., who was then serving as District Government Veterinary Surgeon in Malaya. I hasten to add that Klaus Rohde has given the name of this trematode fully not out of deference to any wish of mine but solely because of what I will call his teutonic insistence on precise delineation, and I am delighted to mention this record on the account of Mr G . B. Purvis. The review is divided into an introduction and eight other parts, which are subdivided. The introduction draws attention to the fact that the Aspidogastrea is the smallest of the three groups of Trematoda but of great interest because it shows a combination of the characters of Monogenea

X

PREFACE

and Digenea and contains forms ill-adapted to a parasitic mode of life and likely therefore to throw some light on the origin of parasitism in the flatworms. One section deals with general characters and leads on to another section giving a very detailed account of adult structure under the headings of tegument, digestive tract, protonephridial, genital and nervous systems, receptors and details of the characteristic ventral adhesive disc occupying most of the ventral part of the body. Rohde prefers to call the outermost layer of the body the “tegument”, which other writers designate by other names; the essential point is that it is not a hard resistant cuticle as once supposed but a cytoplasmic layer of much greater complexity, as revealed by electron-microscopy. It contains mitochondria and ovoid bodies, in some areas vacuoles and lamellated bodies, and the surface membrane forms elevations between which there is a mucoid layer of variable thickness. In some areas rib-like elevations of the surface support a thick mucoid layer consisting of a reticulum of fibres with electron dense bodies of various sizes. This “tegument” is syncytial, and apparently it is not formed by fusion of epithelial cells in M . purvisi but originates in its definitive form. Other organs of the adult-digestive tract, protonephridial, genital and nervous systemshave their structures elucidated with the help of the electron-microscope, and the details of the nervous system are revealed in their exquisite complexity. The sensory receptors are more complex and varied than has generally been supposed, and much more numerous. The structure of the free-living larva is described in the same thorough manner, in terms relating to general structure, tegument and ciliated tufts, glands and caudal appendage, digestive protonephridial and nervous systems, and sense receptors. As far as possible the details are integrated with what is known about other aspidogastrid larvae. Development is then considered in terms of the egg, cleavage and larval development, hatching, development of the parasitic stage and relative (allometric) growth. The portrayal of changing relative size in various organs during growth approaches and surpasses what I first attempted more than 30 years ago for Styphlodora elegans Dawes, 1941 (Parasitology, 33 (1941), 445-458) and more recently for Fasciola hepatica (J. Helminthology 36 (1962), 11-38). The biology of aspidogastrids is closely considered in respect of life span, behaviour and infectivity of free larvae, route of invasion in the mollusc, localization and sexual maturation in the intermediate host, infectivity in the vertebrate host and growth, localization in this host and specificity of infection, with some information on the survival of adult forms outside this host. Rohde then discussed the phylogenetic position of the Aspidogastrea, which have some archaic features. Reasons are given for placing aspidogastrids in a separate group, and for believing that they are closely related to the Digenea. An attempt is made to derive the digenetic life cycle from that of the aspidogastrid and to discuss some unresolved problems. It is likely that many parasitologists will agree that Rohde’s review greatly enhances our knowledge of trematode structure development and biology. John C . Pearson has chosen to write on a phylogeny of life cycle patterns of digenetic trematodes, a theme that has long invited speculation. After an Introduction giving details of earlier ideas and some recent views, this writer

PREFACE

xi

details what he considers to be the “singular features” of digenetic life cycles, adding what he believes any scheme of phylogeny should take into consideration, namely, alternation of generations, alternation of molluscan and piscine hosts, the existence of a tailed larva (the cercariae) and methods of transfer that include penetration of the free-swimming miracidium into some snail, the escape from the snail in many instances, and the ingestion of a cercarial or metacercarial stage by a definitive host, except in schistosomes. He is generous in his comments about these minimal considerations before passing on to his detailed scheme. After considering the adoption of parasitism and a one-host cycle, he goes on to deal in turn with the origin of an alternation of generations, the addition of a vertebrate host and a redial generation in the parasite, the addition of a metacercarial stage, the acquisition of a second intermediate host and the three-host cycle and its modifications. He then notes phylogenetic implications and summarizes his findings. In order to explain the “ubiquity” of the cercaria he postulates that the present first intermediate host was the original host of the proto-digenean and that escape from this host is primitive. To explain the occurrence in many life cycles of a free-swimming miracidium he further postulates that the proto-digenean was an ectoparasite of the molluscan host. Assuming that it became a visceral parasite that escaped from the host as an adult in order to lay its eggs, he indicates that the known life cycles of contemporary Digenea may be interpreted in an order of acquisition of hosts as follows: vertebrate definitive (two-host cycle) and invertebrate second intermediate (three-host cycle). More than this it is not necessary to state here, except that the three-host cycle is the commoner and has arisen several times over from two-host cycles, and has been secondarily reduced in some groups of the Digenea through loss of the definitive host or of the second intermediate host, or possibly the loss of both hosts. There are no simple cycles amongst the Digenea and speculation is hypothetical but this thoughtful effort by John Pearson will help us to classify the multifarious life cycles and also point the way to further study in other and future researches. The review by Hok-Kan Lim and Donald Heyneman is concerned with intramolluscan inter-trematode antagonism. It has been known for some years that echinostome larvae within a snail host may inhibit and disrupt or prevent the development of other trematode larvae but only recently was this phenomenon demonstrated in laboratory experiments carried out in San Francisco and Kuala Lumpur, Malaya, basically to study single or double trematode infections within a molluscan host. After an Introduction, Lim and Heyneman give much information about the maintenance of certain snails and trematodes, mainly the snail Biomphalaria glabrata and the trematodes Paryphostomum segregatum (an echinostome) maintained as adults in a Brazilian black vulture (uruba) and Schistosoma mansoni, maintained as adults in the golden hamster. The term direct antagonism was used to denote predatory or physical activities by rediae on other trematode larvae within the snail. By study and analysis it was hoped to recognize, evaluate and “quantitate” the conditions that predispose one trematode to dominate another trematode within the same snail. Various trematode interaction

xii

PREFACE

patterns are noted-rediae dominating sporocysts, predatory activity of rediae which feed upon sporocysts but may devour other larvae, e.g. mother rediae devouring daughter rediae or young cercariae. Redial morphology is considered carefully, the efficiency of predation and a triggering mechanism as well. Non-predatory effects are considered for both rediae and sporocysts as inhibitory or degenerative changes and possible mechanisms such as snail immunity, direct toxicity, and competition for nutrients or oxygen. An important section of the review is concerned with the parameters of intramolluscan inter-trematode antagonism. In the quest for a suitable antagonist against Schistosoma mansoni six species of echinostomes were tested in the laboratory, and Paryphostomum segregatum was the most effective. Consequently, much research was carried out on antagonism using the snail Biomphalaria glabrata, the trematode Schistosoma mansoni (target) and the echinostome Paryphostomum segregatum (predator). This model gave examples of miracidial penetration in either trematode species superimposed on the other species, the establishment of infection in B. glabrata, delayed redial migration, delayed germinal development within the redia, the attraction of predaceous rediae to sporocysts, the speed at which larval domination is completed and the unusual appearance of what appear to be third-generation sporocysts of S. mansoni. The phenomenon of trematode antagonism is then discussed in a separate section, in its relation to biological control. Trematode diseases such as fascioliasis and schistosomiasis have called forth the use of parasites, pathogens and predators of snails-protozoa, nematodes, leeches, fishes, ducks and geese-and reference is made to the discoveries with the molluscicide larvae of sciomyzid flies by C. 0. Berg (Advances in Parasitology 2 (1965), 259). Intramolluscan and inter-trematode antagonism is a new approach to biological control, and it is claimed that encouraging results so far obtained justify field trials against human schistosomes in endemic areas. Echinostomes are at present favoured for control of such trematode disease but other rediae-producing species must be isolated and tested, although trematode biocontrol may be applicable only in local or regional approaches. Success of control by trematode antagonism depends on strong infectivity of the antagonist in the snail that harbours the target species, and this entails heavy infection by a single miracidium in all ages of host snail in the full range of natural habitats, followed by rapid growth and development despite preceding trematode infection. It may therefore be that a single trematode species cannot be so adaptive and powerful an antagonist as to serve in biological control in other than endemic areas where it is already adapted to local snails. A plea is made therefore to develop trematode biocontrol locally. The snail is the limiting environment, imposing real barriers to a newly-introduced trematode species. Once inside the snail and successfully multiplying it is not unlikely that the antagonist would dispatch the prey trematode. Much that cannot be mentioned here is discussed in this review and in a summary the point is made that this formof biocontrol “offers limited but possibly important usefulness, especially if teamed with other control methods, such as molluscicide, sanitary and therapeutic”. In the review by Zbigniew Pawlowski and Myron G. Schultz we are

PREFACE

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XI11

reminded that cestodes have been known since ancient times and also told that in recent years several reviews dealing with cestode diseases have appeared. Nevertheless, there have not been any recent reviews which deal in a comprehensive manner with infection by Taeniu suginuta. Therefore, these writers have tried to summarize all significant matters concerning this cestode and its cysticercus, i.e. nomenclature, host-relationships, structure and biology, clinical and therapeutic features, epidemiology and epizootology, and the prevention of infection. After a brief Introduction, they consider some taxonomic problems in one section and the hosts of T. saginata in another. Man appears to be the sole definitive host of the adult of this cestode species, but because larval cestodes are much less specific than corresponding adults, the list of intermediate hosts is long and constantly extending. Man may serve as intermediate host, 12 instances having been described, one in Chile concerned 59 patients, another in Rhodesia 62 patients. In India 450 instances of cysticercosis concerned soldiers, but T. saginata was not reported. A section of this review deals with structure and biology of the adult worm, egg, onchosphere and cysticercus in turn. One interesting point about the living adult tapeworm is that it is by no means passive but often moves against the peristaltic movements in the host’s intestines. The usual site seems to be the jejunum, but radiologists have found the worm in the terminal ileum, which is said to be the part best shown by radiological examination. The entire scolex and strobila has been recorded in several unusual locations such as the appendix and the gall bladder. The idea that the worm occurs singly has been refuted, although multiple infections occur in less than 1 % of cases, except in Mexico (nearly 573, but much greater infections have been observed in the southern republics of U.S.S.R. Many other interesting points arise in this part of the review. The number of eggs in one proglottis of T. saginata has been put at about 80,000, the daily output 720,000. Elsewhere the clinical aspects of taeniasis are dealt with in terms of symptomatology, clinical pathology, diagnosis and treatment, which cannot be considered here, except to say that there is much to interest clinicians. The matter of treatment by chemotherapy has been dealt with in two reviews during the 1960s, both from the Wellcome Research Laboratories, and they are detailed here. Yomesan is the drug of choice for T. saginata infection in Man at present and some suggestions are made for treatment with this and other drugs. A sixth section deals with epidemiology and concerns transmission between animals and man, a seventh with losses due to taeniasis and cysticercosis. These sections are very informative. It is stated that taeniasis and cysticercosis (T. saginata) are cosmopolitan in distribution and have become more prevalent in recent years. During the past 25 years the world population has increased by about 50% and that of cattle by about loo%, so that it is safe to assume that infections greatly exceed the 39 million estimated by Norman Stoll in 1947. Losses are difficult to estimate, because infection is rarely fatal, but some figures are available for European, African and American areas. Meat inspection is dealt with as a means of prevention, likewise serological diagnosis and the immunization of cattle. Sanitation is a matter discussed, its improvement, expensive but connected with higher standards of living.

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The solitary updated review by Donald L. Lee lays emphasis on new work rather than on research that does not break new ground, and much more space is now devoted to the outer coverings of larval helminths and the development of this covering in adults. The following groups and species are considered : Turbellaria (Kronborgia amphipodicola) ; Monogenea (various species); Digenea (Fasciola hepatica and various other species); Cestodaria (Gyrocotyle urna) ; Cestoda (various species) ; Nematoda (various species); Acanthocephala (Polymorphus minutus). A summary is provided. At the end of one decade and the beginning of another I am happy once more to express my gratitude to friends and colleagues who have worked hard and long at compiling these reviews and who have helped to produce what I referred to above as a vast summation of modern biological information and ideas in the field of parasitology. I am equally pleased to thank the staff of Academic Press who have ironed out innumerable difficulties and smoothly brought out the tenth volume in this series, and to hope that our cooperation can continue far into a second decade, to say the least. “Rodenhurst” 22 Meadow Close* Reedley Drive REEDLEY, Nr Burnley Lancs BBlO 2QU England

* Note modified address.

BENDAWES Professor Emeritus: University of London January 1972

CONTENTS ............................................................

v vii

CONTRIE~UTORS TO VOLUME 10 PREFACE.......................................................................................

.

. . .

Avian Blood Coccidians .

.

JOHN R BAKER. GORDON F BENNETT. GLEN W CLARK AND MARSHALL LAIRD

I Introduction ........................................................................... I1 Atoxoplasms ........................................................................... 111 Adeleine Haemogregarines .........................................................

1 3 14 16 17 17

IV. Conclusions ........................................................................... Acknowledgements .................................................................. References ..............................................................................

The Metabolism of the Malaria Parasite and its Host ALEXANDER FLETCHER A N D BRIAN MAEGRAITH

I. The Metabolism of the Malaria Parasite ....................................... I1. The Metabolism of the Host During Infection .............................. References ..............................................................................

31 41

44

The Pathogenesis of Mammalian Malaria

. .

BRIAN MAEGRAITH AND ALEXANDER FLETCHER

I Introduction ........................................................................... 11 Local and General Responses in Malaria Similar to those which Occur in Inflammation .................................................................. 111. Changes in Endothelial Permeability in Malaria .............................. IV. Vasomotor Changes in Malaria ................................................ V. The Kinin Complex and Other Pharmacologically Active Agents ...... VI. Intravascular Coagulation in Malaria .......................................... VII Anoxic Anoxia ........................................................................ VIII. Cytotoxic Factors: Mitochondria1 Respiration Inhibitors .................. IX. The Chain Reaction ............................................................... References ..............................................................................

49 51 51

58 60 67 68 70 71 72

.

The Aspidogastrea, Especially Multicotyle purvisi Dawes, 1941 KLAUS ROHDE

I. Introduction ........................................................................... 11. General Characteristics ............................................................ I11. Structure of the Adult ............................................................... IV Structure of the Free Larva ...................................................... V. Development ........................................................................... VI . Biology ................................................................................. VII Phylogenetic Position of Aspidogastrea ....................................... VIII Derivation of Digenean Life Cycles from Aspidogastrean Cycle............ IX. Some Unresolved Problems ...................................................... References ..............................................................................

.

. .

xv

78 78 79 107 120 134 140 143 144 145

xvi

CONTENTS

A Phylogeny of Life-cycle Patterns of the Digenea . .

J C PEARSON

I . Introduction ........................................................................... I1. Adoption of Parasitism: One-host cycle .......................................

111. Origin of Alternation of Generations

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

IV . Addition of Vertebrate ............................................................... V . Addition of Metacercarial Stage ................................................ VI . Acquisition of Second Intermediate Host: The Three-host Life.cyc1e ... VII . Three-host Life-cycle ............................................................... VIII . Modification of the Three-host Life.cyc1e ....................................... IX . Phylogenetic Implications ......................................................... X . Summary .............................................................................. References ..............................................................................

153 156 158 160 165 167 170 173 178 179 181

Intramolluscan Inter-trematode Antagonism: a Review of Factors Influencing the Host-Parasite System and its Possible Role in Biological Control HOK-KAN LIM AND DONALD HEYNEMAN

I . Introduction

........................................................................... ............................................................ 111. Trematode Interaction Patterns ................................................... IV. Parameters of Intramolluscan Inter-trematode Antagonism ............... V . Trematode Antagonism in Biological Control ................................. VI. Summary .............................................................................. References .............................................................................. I1. Materials and Methods

192 193 199 225 244 251 253

Taeniasis and Cysticercosis (Tueniu suginata) .

ZBIGNIEW PAWLOWSKI AND MYRON G SCHULTZ

I . Introduction ........................................................................... I1. Nomenclature ........................................................................ 111. Hosts of Taenia saginutu ............................................................ IV. Structure and Biology of Taenia saginatu ....................................... V. Clinical Aspects of Taeniasis ( T. suginata) .................................... VI . Epidemiology and Epizootiology ................................................ VII . Prevention ..............................................................................

269 270 271 275 282 295 304

C 0N T E N T S

xvii

SHORT REVIEW Supplementary Contribution of Previous Volume

The Structure of the Helminth Cuticle D . L . LEE

I . Introduction ........................................................................... 11. Turbellaria .............................................................................. 111. Monogenea ........................................................................... IV. Digenea ................................................................................. V . Cestodaria .............................................................................. VI . Cestoda ................................................................................. VII . Nematoda .............................................................................. VIII . Acanthocephala ........................................................................ IX . General Summary ..................................................................... References.............. .................................... .........

347 348 348 352 357 357 360 369 370 312

AUTHOR INDEX ................................................................................. SUBJECT INDEX.................................................................................

381 401

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Avian Blood Coccidians* JOHN R. BAKER?, GORDON F. BENNETT!:, GLEN W. CLARK5 A N D MARSHALL LAIRD!:

I. Introduction. ................. 11. Atoxoplasms ...................................................... I l l . Adeleine Haernogregarines ........................................................... IV. Conclusions .................. Acknowledgements ....................... References ............... .........................................

1.

14 11

INTRODUCTION

The “Pseudovermiculi sanguinis” that Danilewsky (1 889) found in the blood of certain birds (particularly magpies and owls) were said by him to resemble drepanidia and haemogregarines on the one hand, and coccidians from the kidneys of frogs on the other. He thus regarded them as gregarines, probably Coccidia. Labbe afterwards variously ascribed them (in part) to Drepanidium and Lankesterella, his (1 899) work on the Sporozoa listing Lankesterella ( = Drepanidium) avium (LabbC, 1894) from Danilewsky’s hosts and other birds too. Labbe had demonstrated his familiarity with Proteosoma (=Plasmodium) and Halteridium (= Huemoproteus)from avian hosts at this time, and moreover was well aware of the appearance of a diversity of haemogregarines of reptiles and amphibians in both living and fixed material. We wonder, therefore, whether amidst all the taxonomic confusion relating to avian blood parasitology in the last decade of the nineteenth century adeleine haemogregarines had not in fact already been recognized in birds at this time. Nevertheless, the earliest recognizable account of a blood coccidian from birds appeared-conveniently enough for the tidy-minded bibliographer-in the first year of this century (Laveran, 1900). The parasite of Padda oryzivora (the Java sparrow) described therein, was duly designated Haemogregarina paddae by Aragilo (1911). It was soon afterwards transferred to the genus Toxoplasma by Marullaz (1913), there to remain until Zasukhin et a/. (1956)

* Studies in Biology from Memorial University of Newfoundland No. 295. Contribution from the World Health Organization’s International Reference Centre for Avian Malaria Parasites No. 2. Molteno Institute, University of Cambridge, England. $ Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada. 9: Department of Biological Sciences, Central Washington State College, Ellensburg, Washington, United States of America. 1

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J . R . B A K E R , G. F. B E N N E T T , G . W . C L A R K A N D M . L A I R D

recognized it as referable to the recently established Atoxoplasma. Just three years later, Lainson (1959) claimed that the organism was really a member of the genus Lankesterella, while Laird (1959) argued that Atoxoplasma paddae (Aragilo) is the valid type species of Atoxoplasma-Garnham (1950) having so designated a synonym of this species, A. avium (Marullaz, 1913). Box (1970) has now suggested that the same or a closely related parasite of Serinus canaria and Passer domesticus is in fact a species of Isospora. This sequence of events eloquently summarizes the taxonomic confusion which has reigned over these organisms since their first discovery. Although they have been relegated to that repository for insufficiently known forms, “Protista Incertae Sedis” from time to time, they are nowadays rather generally held to be bloodinhabiting stages of coccidia, referable to two distinct groups. Members of the first group resemble the organism described by Laveran (1900) and are seen in the peripheral circulation and (more often) the viscera, as small, unpigmented nucleophilic parasites, usually of mononuclear cells. Where one organism is present it generally lies in the cytoplasm of the host cell, which stains much more brightly with Giemsa than its own. It then appears as a conspicuously pale haemogregarine with a large and rather diffuse nucleus and a decidedly faint outline, and is often pressed into a depression of the host cell nucleus. Where (as is commonly the case) two organisms are present, they are round, oval or pyriform. They stain as do the larger single forms, and quite deeply indent the host cell nucleus, chromatin matter of which often appears to be pinched up between them. We are inclined to question the apparent homogeneity of this first group. Nevertheless, in order to defer commitment to the use of any particular generic name, all such intraleucocytic blood coccidians considered in this literature review will be referred to as “atoxoplasms”. The second group are typical adeleine haemogregarines (i.e. members of the sub-order Adeleina, order Eucoccida, sub-class Coccidia, class Telosporea, sub-phylum Sporozoa, which inhabit the blood cells of vertebrates at some stage of their life cycle). There has, however, been much dispute about their generic position. They occur in erythrocytes of the circulating blood, and their intense red-and-blue staining reaction with Giemsa is characteristically haemogregarine. Table I lists chronologically publications dealing with avian blood coccidians of both groups. The relevant literature will be reviewed separately for each. Papers where it is either impossible to tell whether the author was or was not dealing with Toxoplasma, or where there is a probability of confusion with haemoproteids, babesioids, etc. (e.g. de Mello’s 1937 record of an intraleucocytic organism from an Indian Buzzard-eagle) are left out of this Table; so are various purely secondary authorities such as Bhatia (1938). These omissions notwithstanding, it is still impossible to be quite certain to which of the two groups some reports refer, and the author’s views have not always been followed. Table II lists the recorded hosts of both groups. Other check-lists of avian haemogregarines (amongst other parasites) have been published by Wenyon (1926), Lucena (1941 ; Neotropical Region), Herman (1944; North America), Levine and Kantor (1959; Columbiformes) and Bray (1964; West Africa). The last-mentioned author was the only one of these to

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attempt a critical reassessment of authors’ identifications of the parasites. In the year when Bray’s paper appeared, Berson (1964) published a review of the protozoa parasitizing avian blood cells. The latter review mentions only a few of the records of blood coccidians from birds. Also, it retains Atoxoplasma as well as Lankesterella with A . argyae Garnham and L. argyae (Garnham) both listed under the appropriate headings. 11. ATOXOPLASMS As already mentioned, the first report of members of this group was made by Laveran (1900). He saw them in mononuclear cells in spleen and bonemarrow smears of Padda oryzivora also infected with “Haemamoeba danilewskyi”. The latter designation might refer to a true Plasmodium, but data from the World Health Organization’s International Reference Centre for Avian Malaria Parasites (Memorial University of Newfoundland) indicate that haemoproteids are much more characteristic of this host. Laveran thought that the small non-pigmented organisms might be stages in the life-cycle of “ H . danilewskyi”, but did not exclude the possibility that they were “parasites nouveaux”. Novy and MacNeal (1904, 1905) described similar organisms from Passer domesticus as Haemoproteus rouxi (“rouxii” in the second paper). Adie (1907, 1908) observed parasites in mononuclear cells of P . domesticus in India (recommending-1908-their study to fellow expatriate Englishmen in India as one of “a few things to amuse oneself with in the long days of the hot weather. . .”). He sent specimens to Laveran, who recognized their similarity to the parasite of P . oryzivora but thought they probably represented a different species. Adie (1908) was perhaps the first author to remark on the characteristic nucleophily of the atoxoplasms, which frequently lie apparently pressed into a notch in the host cell’s nucleus. (See Plate I, Figs 1 and 2. The white arrow in Fig. 1 points towards the inner margin of this notch, the black arrow to the rather brightly staining parasite nucleus.) Noller (1920) thought that some of the stages of this parasite might have been close to Toxoplasma, and some of his stages illustrating “Schizogonie” in the spleen might indeed have been. However, the birds were obviously infected with Plasmodium, either alone or together with Huemoproteus, since erythrocytic schizonts were described. It is thus possible that some at least of the atoxoplasm-like forms were exo-erythrocytic schizonts of Plasmodium. Araggo (191 1) named the organisms described by Laveran (1900) and Adie (1908) Haemogregarina paddae and H . adiei respectively. He also described what he thought were seven other species of Haemogregarina from mononuclear cells of as many species of birds in Brazil. Hoare (1924) transferred five of these species to the genus Hepatozoon (see p. 14 below), regarding the other two as avian toxoplasmas (i.e. atoxoplasms). The latter two (which to modern eyes may well be conspecific) had already been named Haemogregarina sporophilae and H. sicalidis by AragFto (191 I), who discussed them in Sporophila albogularis and Sicalisflaveola respectively. Marullaz (1913) afterwards studied (in Padda oryzivora and other passerines) the organism first reported by Laveran (1900). Marullaz tried, but failed, to

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FIGS1, 2. “Lunkesterellu garnhumi” from fledgling Passer domesticus (spleen impression smear) (J. R. Baker, England, 15 June 1960). Note marked indentation of host-cell nucleus, and deeply staining atoxoplasm nucleus. FIGS3-6. “Atoxoplasma corcothraustis” from Coccothruustes corcothraustes (lung impression smear) (A. Corradetti, Italy, ref. Corradetti and Scanga, 1963). Note faintly staining periphery of parasite and of the associated shrinkage zone outline. FIGS7, 8. “Lankesterella corvi” from Corvus fvugilegus (thin blood smear) (J. R. Baker, England, 30 June 1960). Note clearly marked periphery of intraerythrocytic sporozoites. FIGS9-16. Adeleid haemogregarine (of lizard origin?) (thin blood smear of Loomelaniu meluniu) ( G . W. Clark, San Benito Island, N. Mexico, 10 May 1967). Note pyknotic appearance of parasites as, e.g., in Figs 13 and 14, also typically reptilian-type “tailed” haemogregarine (Fig. 16). See pp. 15-16. All figures x 825 approx.

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infect mice with the parasite (which he named Toxoplasnza avium); he concluded, on morphological grounds, that it belonged to the genus Toxoplasma, described only a few years previously by Nicolle and Manceaux (1908, 1909). As pointed out by Noller (1920), this specific name is a junior synonym of AragBo’s (191 1) Haemogregarina paddae. Attempts to transmit it to other birds were inconclusive. The following year, Laveran and Marullaz (1914) described another species-T. liothricis [sic]-from Leiothrix lutea. They mentioned that the parasite was often pressed into the host cell’s nucleus. This feature seems to be almost diagnostic of the atoxoplasms (Figs 1 and 2), as does the remarkably indistinct periphery of the parasites themselves and the only faintly staining boundary of the host-cell cytoplasm with the parasites-or with a slight empty space perhaps indicating shrinkage of the latter during the preparation of the Giemsa-stained slides (Figs 3-6): it should be added that the periphery of intraerythrocytic atoxoplasms-Baker et al’s (1959) “sporozoites of Lankesterella corvi”-is usually well demarcated (Figs 7 and 8). Mine (1914) described a so-called Leucocytozoon of Passer montanus in Japan. Certain stages of his parasite (in particular, schizonts from the spleen) could well have been atoxoplasms (as suggested by Noller, 1920). Plimmer (1 9 15, 1916) recorded “Toxoplasma” from a Ducula (= Carpophaga) concinna* (fruit pigeon) dying in the London Zoological Gardens. Carini (1909) had shown that “pigeons” (presumably Columba livia) were susceptible to Toxoplasma gondii (“T. cuniculi”) and (Carini, 1911) had recorded a spontaneous infection in the same species of bird. The parasites of the latter pigeon were named T. columbue by Yakimoff and Kohl-Yakimoff (1912). Noller (1920) suggested that the organisms reported by de Mello (1913) and de Mello et a]. (1917) from Indian pigeons-again presumably C. livia-belonged to this species. Unfortunately, de Mello’s organism is impossible to categorize. It could just as well have been an atoxoplasm or even an adeleine haemogregarine (see p. 14 below). Plimmer (I 9 1 5) also recorded “To.uoplasms” from Saxicola (=Pratincola) caprata in India; this may have been in fact an atoxoplasm. Under the heading “Toxoplasmes (ou HCmogregarines?)”, Carini and Maciel (19 16) referred to finding parasites resembling those described by AragBo (191 I ) and others in various unspecified birds in Brazil. For these, they adopted Marullaz’s (1913) name Toxoplasma avium. I t is likely that some at least of their organisms were atoxoplasms. Fantham (1919, 1924) described what was probably an atoxoplasm from the mononuclear cells of Amadina erythrocephala in South Africa. Naming it Leucocytogregarina amadinae, Fantham ( 1924) remarked that the genus in question “has by some been termed Hepatozoon”. Noller (1920) discussed the “Vogeltoxoplasmen” at some length, citing unpublished work by Mayer, continued by himself, in which these parasites were studied in naturally infected siskins, Carduelis (= Chryson7itris) spinus, obtained from a Hamburg dealer. Stages in the intestinal wall of infected birds were identified as schizonts (or sporonts) and gametocytes of the eimeriine *When the host name used by the original author differs from that accepted by Peters (193 I et seq.) the former is shown in parentheses thus.

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type. The macrogametocytes were said to be significantly smaller than those of Zsospora though the schizonts were similar in size. The lymph spaces of the intestinal wall were filled with “toxoplasms” which were thought to be the progeny of the neighbouring schizonts. Other, larger “merozoites” (10-12 pm long) were also seen. Attempts to transmit the parasite failed, which would have been an unlikely result had the organism been Toxoplasma gondii. Noller concluded that while atoxoplasms bear a close morphological resemblance to both Toxoplasma and intraleucocytic haemogregarines, they differ considerably from the former in their pathogenicity and transmissibility. A further account of what was thought to be the parasite found by Mayer in siskins was given by Walzberg (1923), who concentrated upon the pathology of birds which appeared to be dying from the infection. Although the parasites themselves were not described in detail by Walzberg, his illustrations show organisms more resembling T. gondii than atoxoplasms. Perhaps Mayer and Walzberg were not in fact dealing with the same organism. Thus Walzberg’s birds might conceivably have acquired accidental laboratory infections with T. gondii. In a footnote added to the proof of Walzberg’s paper (1923; p. 32), Noller referred to work by Nitsche, and stressed the importance of differentiating the gut stages of atoxoplasms from those of Zsospora lacazei (see also Noller and Nitsche, 1923). Various other authors reported small intraleucocytic parasites of birds under the general designation Toxoplasma. Some of these reports were accompanied by little or no descriptive or illustrative material. Thus it is not always possible to decide whether the organisms concerned were atoxoplasms, adeleine haemogregarines or even, perhaps, sometimes Toxoplasma gondii itself. Nevertheless, the first of these identifications often seems the most probable. These reports include references 9, 10, 13, 16, 20, 22, 25, 30, 31, 33, 34, 36, 37, 38,40,41, 43,46, 54, 55, 57, 61, 62 and 64 in Table 1. They are not further referred to in this review apart from the inclusion of relevant host records in Table IT. Table I1 also includes numerous records concerning material that has been deposited in the World Health Organization’s International Reference Centre for Avian Malaria Parasites, which is in the Department of Biology, Memorial University of Newfoundland (Bennett and Laird, 1971) (this material is listed in more detail in Table 111). References by Neiva and Penna (1 91 6) and Primio (1925) to “Haemogregarina” of birds, which may refer to atoxoplasms, are discussed on pp. 14 and 15. Correa (1928) recorded as “Haemogregarina” parasites from three species of birds in Brazil : “H.” pessoai nsp. from Poospiza thoracica, “H.” paulasousai” nsp. from Stephanophorus diadematus (= S. leancocephalus) and “H.” sp. indet. from Molothrus b. bonariensis. These forms are perhaps more likely to be atoxoplasms than adeleine haemogregarines. Noller (1 93 l), briefly reviewing the

* Also spelt “puulusouzui” in the original paper. Since the dedication was to Professor de Paulo Souza, and the “z” was used after the first usage, the first spelling is probably a lupsus culumi. However, since the “s” spelling was used by Lucena (1941), the “first reviser” in the terms of article 32b of the International Code of Zoological Nomenclature (ed. 2, 1964; International Trust of Zoological Nomenclature, London), this must presumably stand as the “correct original spelling”.

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atoxoplasms of cagebirds, thought that transmission occurred most probably through the mite Dermanyssus uvium. AragiXo (1933) repeated his assertion that parasites of this type were all members of the genus Haemogregarina (see pp. 3 and 14), and criticized their identification as Toxoplusma. He gave the name H. serini to the species found by himself and other workers in Serinus canaria. Wolfson (1 937, 1938) recorded “Toxoplasma” or “Toxoplasma-like bodies” from S. canaria and P. domesticus. She later (1940) concluded that the organisms identified in her first paper (Wolfson, 1937) were in fact exo-erythrocytic schizonts of Plasmodium. In her second paper (Wolfson, 1938), though, she described parasites resembling atoxoplasms from birds in which malaria was not demonstrable. Kikuth and Mudrow (1938) described “Einschlusse” (inclusions) in mononuclear cells of S. canaria used in the experimental study of avian malarias. One of these inclusions illustrated by Kikuth and Mudrow (1938, Plate 1, Fig. 6 ) looks like an atoxoplasm, and was so identified by Wolfson (1940). Manwell (1939) discussed at length the confusion between “avian Toxoplasma” and exo-erythrocytic schizonts of Plasmodium in birds. He concluded that the two forms were distinct, those which were not Plasmodium being “most probably Toxoplasma”. However, he also pointed out that “there is no doubt that the Toxoplasma commonly found in birds and that isolated from mammals (. . .) are different species, and it is indeed possible that they should be placed in different genera. . . . Morphologically the bird and mammalian types are quite distinct. . . and they affect the host cell rather differently. . . .” Manwell (1 939) drew attention to the characteristic “notching” of the host cell’s nucleus by “avian Toxoplusma”, a feature which, as already mentioned, is almost diagnostic of the atoxoplasms. He also remarked that he had seen the “Einschlusse” (Kikuth and Mudrow, 1938; see above) in “numerous wild birds”, implying that they were unrelated to either Plusmodium or “avian Toxoplasma”. Six years later he (Manwell et al., 1945) suggested that they “might be due to light infections with coccidia”. Hewitt (1940) described, under the wisely non-committal term “unclassified intra-leucocytic parasites”, what were certainly atoxoplasms from two birds (Curpodacus mexicanus frontalis and Molothrus sp.) in Mexico. He was unable to transmit the parasites to S . canaria by blood inoculation. Hewitt ( 1940) concluded that the organism “resembles Hepatozoon in many respects although no sporogonic stages have ever been described”. In the same year, Wolfson (1940) discussed in detail the identity of “organisms described as avian Toxoplusma”. In what was perhaps the greatest understatement of that year, she wrote that “there exists a certain amount of confusion in the literature.. . .” Wolfson’s excellent account (1940) reviewed the literature up to that time. She pointed out that there were three main periods in the recorded history of the “avian Toxoplasmas” ; ( I ) pre-1909, when they were generally confused with the haemoproteids; (2) 1909-1937, when they were usually regarded as species of Toxoplusma (sensu stricto); and (3) the “recent” period during which this identification began to be doubted and in which some confusion arose between the organisms in question and the newly discovered exoerythrocytic stages of avian malaria parasites (cf. Manwell, 1939).

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Wolfson concluded that, in the past, at least three types of parasite had been included in the “avian Toxoplasmas”: ( I ) exo-erythrocytic stages of Plustnodium; (2) a “distinct parasite which resembles certain stages of leucocytic haemogregarines” ; and (3) organisms “perhaps identical with the mammalian Toxoplasma” ; all three types were illustrated by line drawings. Type 2, which corresponded with what are here referred to as atoxoplasms, had been seen by Wolfson in “over 25% of laboratory canaries” (S. canaria). Type 3, she studied in slides provided by Rosenbusch (cf. Rosenbusch, 1932). Wolfson concluded that Rosenbusch’s organism was probably true Toxoplasma. A study of his paper’s illustrations (1932) suggests that he may have been observing mixed infections including atoxoplasms as well (for this reason the paper has been included in the list in Table I). Wolfson further pointed out that the “type 2” organism differed from true Toxoplasma in that it “cannot bc transmitted to mammals nor even to birds of the same or different species” and also the two parasites in canaries “can be distinguished morphologically”. Finally, Wolfson considered the life-cycle of her “type 2” parasite. She observed stages in the intestinal epithelium of canaries which resembled merozoites and schizonts of “Coccidium” (presumably Isospora) in birds with and without patent infections with “type 2” and attempted to establish a ielationship between these two organisms. “Some evidence” for such a relationship was obtained by the apparent infection of two birds with “type 2” after introducing into their crops epithelial scrapings from a bird showing intestinal forms. However, she rightly pointed out that more work was needed before any such relationship, or the reality of this transmission, could be accepted. The first report of atoxoplasms in erythrocytes was published by Manwell (1941), who recorded seeing “avian Toxoplasma” in many red blood cells, as well as mononuclear cells, of an infected Passer domesticus. Plasmodium was excluded, for the parasites were unpigmented and sub-inoculation of blood did not produce infection in the recipient. This appears to be the only record of atoxoplasms of the sparrow inhabiting any circulating cells other than those of the lymphoid-macrophage series. Indeed there were, as far as we know, only two other records of intraerythrocytic atoxoplasms, one from Corvus frugilegus in England (Baker et al., 1959) and one from Climacterus picumnus in Australia (Mackerras and Mackerras, 1960). To these, we now add two (of 55) examples of the white-breasted swiftlet Collocalia esculenta, from Malaysia (Table 111). Thin blood films from both birds showed occasional small intraerythrocytic Lankesterella-like haemogregarines as well as the more typical pale-staining intraleucocytic stages of atoxoplasms. Also, only one of the fcw descriptions of avian adeleine haemogregarines refers to intraerythrocytic parasites (Franchini, 1923, 1924; see p. 14 below). Coulston (1942), in a paper unfortunately published only as an abstract, resurrected Noller’s (1920) and Wolfson’s (1 940) idea of a possible relationship between atoxoplasms and intestinal coccidia of the Isospora type. Coulston thought that the “so-called Toxoplasrna of English sparrows” ( P . domesticus) was “probably the result of a coccidial infection of the intestine having no apparent relationship with classical Toxoplasma”. He suggested that merozoites were carried in circulating lymphocytes, monocytes and macrophages

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from the intestine (where they were present in large numbers) to the spleen, liver, lungs, brain and kidneys. Unfortunately it is not clear what, if any, precautions Coulston took to avoid possible confusion due to mixed infections in his birds. This proposed identification of atoxoplasms with intestinal coccidia was also adopted by Manwell et al. (1945), who wrote that “avian Toxoplasma . . . is probably of coccidian nature”. Later, however, Manwell (1957) had changed his mind sufficiently to write that Coulston’s suggestion was “indeed possible, b u t . . . unlikely”, and he rightly drew attention to the possibility of confusing two distinct parasites in birds infected with both. Wood and Herman (1943) recorded “Hepatozoon” and “intraleucocytic parasites” from monocytes, lymphocytes and (in one bird) thrombocytes of nine species of birds in south-western United States of America (specific names of the hosts are included in the check-list, Table 11). The two types o f parasite were differentiated mainly on the grounds of shape and rather minor morphological features (nuclear shape, presence or absence of cytoplasmic granules and vacuoles), though there were also distinct size differences. Some of the so-called Hepatozoon spp., as well as the intraleucocytic parasites, resembled atoxoplasms. However, the “Hepatozoon” from Parus (= Baeolophus) inornatus transpositus was appreciably larger than atoxoplasms usually are. This parasite alone of those described by Wood and Herman we provisionally query as a true adeleine haemogregarine, with the reservation that it also may in fact be only a larger form or stage of an atoxoplasm. Garnham (1950) sought to clarify the rather confused situation by separating the small intraleucocytic parasites into a new genus, Atoxoplasma. The species A . avium (Marullaz, 1913) was designated as the type species but, as pointed out by Laird (1959), this name is a synonym of A . paddae (AragBo, 19 1 I ) and hence (under Article 67(e) of the International Code of Zoological Nomenclature, ed. 2: London, 1964) the latter must be the type species. In establishing this genus, Garnham (1950) summarized the previous work on the group and concluded that, “although the systematic position of these organisms remains unknown, for the sake of convenience it is desirable to given them a new generic name.. . .” The parasites were recorded from Lanius collaris and Turdoides (= Argya) rubiginosa in Kenya, and the species from the latter bird was named Atoxoplasnm argyae. Reichenow (1953), while accepting Garnham’s (1950) name Atoxoplasma, classified these organisms “probably” (“vermutlich”) with Schellackia and Lankesterella-i.e. in the subfamily Cryptosporidiinae of the family Eimeriidae. This suggestion was later apparently substantiated by Lainson (1 959see below). Rousselot (1953) described, under the name “Hepatozoon sperniesti n. sp.”, a parasite in the mononuclear cells of Lonchura (= Spermestes) c. cucullarus and Passer griseus in West Africa. He believed that he had succeeded in transmitting the parasite by inoculation of blood or liver homogenate to other L. c. cucullatus and P. griseus. The possibility of a pre-existent infection in the recipient birds was not adequately excluded, though Rousselot’s ( 1 953) description and illustrations indicate that these parasites were probably atoxoplasms. His remark that the organisms were sometimes “inclus dans le

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noyau” of the host cell is particularly suggestive in this context. Bray (1964) transferred this species to Lankesterella. Rousselot (1953) also reported “Toxoplasma passeris” from Passer griseus and Bray (1964) suggested that this, too, may have been an atoxoplasm. Zasukhin et al. (1956) reviewed the status of the small avian intraleucocytic parasites, concluding that they fell into two groups-Toxoplasma (sensu stricto), recorded from “pigeons, hens, capercaillie, blackcocks and some other birds”* and Garnham’s (1950) Atoxoplasma, recorded mainly from Passeriformes. They described and illustrated schizogony of “Atoxoplasma”, and recorded its presence in five species of birds in the U.S.S.R., including the ubiquitous Passer domesticus (see check-list, Table 11). Zasukhin et al. (1957) published a further description of the schizogony of “Atoxoplasma danilewskii” in Carduelis (= Spinus) spinus and recorded their failure to transmit the organism experimentally. They regarded the genus as probably belonging to the Sporozoa, but thought that further study of its systematic position was necessary. Manwell (1957) wrote that “Atoxoplasma is perhaps actually the most common blood parasite of the English sparrow” (P. domesticus), having observed it in at least 20% of the young birds he had examined in the U.S.A. He inoculated brain homogenates from over 250 P. domesticus to mice, but failed to isolate a single strain of Toxoplasma gondii-a most valuable piece of negative evidence. However, Manwell’s experimental inoculation of forty P. domesticus showed that this bird is susceptible to T. gondii, none of the inoculated animals recovering from the infection. This paper also briefly recorded the isolation of true Toxoplasma from a “crow”. Manwell (1957) now thought it “unlikely” that “Atoxoplasma” was a stage in the life-cycle of a coccidian intestinal parasite, as suggested by Coulston (1942) (but cf. Manwell et al., (1945), p. 9 herein). Mohammed (1958) recorded atoxoplasms (under the term “intra-leucocytic parasites”) from 13 of 176P. domesticus niloticus from Egypt, reserving judgement as to their correct generic name. Lainson (1958a) recorded atoxoplasms from P. domesticus in England and differentiated the parasites from Toxoplasma. Later in the same year he (Lainson, 1958b) described schizonts of this parasite within lymphoid-macrophage cells of the spleen, liver and bone-marrow of naturally infected fledgling P. domesticus. All the birds examined (adult and fledgling) were found to be infected, some of the fledglings “very heavily”. The infection appeared to be pathogenic, killing many of the fledglings under the presumed additional stress of captivity. Lainson (1958a) suspected transmission by Dermanyssus gallinae (red mite), but was unable to prove this because of the impossibility of obtaining experimental birds known to be uninfected. In the following year, Lainson (1959) described this organism’s full life-cycle including schizogony (as reported earlier), gametogony and sporogony. He claimed that the latter two processes occurred within lymphoid-macrophage cells of the liver, kidney and lung. Gametogony was said to be “of the typical Eimeria type”, the oocyst developing after fertilization of the macrogametocyte having a prominent cyst wall, and the zygote within the oocyst dividing repeatedly to produce

* Quoted from the English summary.

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many small sporozoites without the intervention of any sporoblastic or sporocystic stages. It was indicated that these sporozoites then enter lymphocytes or monocytes of the circulating blood. All these stages were fully and convincingly described, and illustrated by means of line drawings and photomicrographs. On the basis of these studies, Lainson (1959) agreed with Reichenow (1953) that atoxoplasms belong to the family Eimeriidae, sub-family Cryptosporidiinae. Within this sub-family there were only two genera into which atoxoplasms could be placed, as they possessed asporous, polyzoic oocystsLankesterella and Eleutheroschizon. The latter was parasitic in annelids, while the former haemogregarine genus was best known from amphibia (but had also been reported from Passer domesticus italiae by Raffaele, 1938). Lainson (1959) could “find no reason why they [atoxoplasms and Lankesterella] should be separated as two distinct genera”. He therefore proposed that the name Atoxoplasma Garnham, 1950, be regarded as a synonym of Lankesterella Labbt, 1899. Lainson (1959) provisionally regarded the forms from P. d. domesticus and Serinus canaria as distinct species, naming them L. garnhami (Figs 1 and 2) and L . serini respectively. He retained Raffaele’s (1938) specific name L. passeris for the form from P. domesticus italiae, but stated that the previously described species “may well be all included under the single specific name of L. paddae in the future”. Unfortunately, although quoting LabbC (1 899), Lainson (1 959) overlooked this author’s designation of Lankesterella avium. Laird (1959) also missed this point which clearly gives priority to Lankesterella (= Drepanidium) avium (Labbe, 1894) over names later proposed by AragBo (191 1) and Marullaz (1913). Lainson (1960) later reduced L. serini to a junior synonym of L. garnhami, the transmission of which he demonstrated, using Dermanyssus gallinae (a mite which he had earlier-1958b-suspected of being a vector) to infect Serinus canaria from unparasitized stock, after acquiring the organism from other canaries and house sparrows. No development of L. garnhami occurred in Dermanyssus, infection taking place when mites containing sporozoites were ingested by the recipient birds. Writing before the publication of Lainson’s later work, Laird (1959) had stressed the affinities between atoxoplasms and haemogregarines, emending the definition of the genus Atoxoplasma Garnham, 1950, to take into account its pathogenicity, demonstrated by Lainson (1958b), and the probability that it would prove to be less host-specific than had been thought when the natural means of transmission was discovered. Laird (1959) regarded all the species of atoxoplasm then described, on morphological grounds, as inseparable from Laveran’s (1900) original material. As already indicated he pointed out that the correct name for this organism was A . paddue (AragBo, 1911), not, as stated by Garnham (1950), A. avium (Marullaz, 1913). In so doing, though, he failed to notice LabbC’s (1894) description of Drepanidium avium, later emended to Lankesterella avium by LabbC (1899) himself. Laird (I 959) also recorded atoxoplasms from several species and sub-species of birds in New Zealand and tropical South Pacific islands (see Table 11). Two other species of Lankesterella, with life cycles resembling that des-

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I . R. BAKER, G . F . BENNETT, G . W . CLARK A N D M . LAIRD

cribed by Lainson (1959; 1960) for L. garnhami, were now described. The hosts were Corvusf.jrugilegus in England (Baker et a/., 1959) (Figs 7 and 8) and Acridotheres tristis melanosternus in Ceylon (Dissanaike et a/., 1965; Dissanaike, 1967). The parasites were named L. corvi and L. lainsoni respectively. Mackerras and Mackerras (1960) described L. picumni from Climacteris picumnus and recorded L. paddae-following Laird’s (1950) usage-from Passer domesticus and Zosterops lateralis, in Australia. They pointed out, however, that L. picumni (which inhabits erythrocytes and, both in this respect and morphologically, resembles L. corvi Baker et al., 1959) differs considerably in appearance from the “atoxoplasms” and that “it may well be that two different genera occur in birds”. Levine (1961) reduced L. garnhami Lainson. 1959, to a junior synonym of L. adiei (AragBo, 191 I)* since the hosts of both were merely different subspecies of P. domesticus. Lainson (1959) had in fact thought it “especially likely” that these species would be found to be conspecific. Corradetti and Scanga ( I 963) described a parasite of the atoxoplasm type from mononuclear cells of Coccothrausks coccothraustes in Italy (Figs 3-6), suggesting that only parasites of this type which inhabit erythrocytes should be placed in Lankesterella LabbC, 1899. They regarded the difference in host-cells of even a single stage in the life cycle as having generic value. However, it must be pointed out that this contention had been expressly denied by Lainson (I 959) on the grounds that (a) some species of Heputozoon and Schelluckia inhabit erythrocytes while others live in white cells and (b) sporozoites of Lankesterella corvi (see p. 12 above) were seen by Baker et a/. (1959) in erythrocytes (Figs 7 and 8), thrombocytes, lymphocytes, monocytes and polymorphonuclear leucocytes. Bray ( I 964) recorded “Lankesterella sp.” from Ploceus ( = Plesiositagra) cucullutus in Liberia. In the same paper, he transferred the species Hepatozoon spermesti Rousselot, 1950, to Lankesterella (see pp. 9-10). It now seemed that the problem of the “intra-leucocytic parasites” or atoxoplasms of birds was largely solved-they were in fact eimeriine haemogregarines of the genus Lankesterella (though Mackerras and Mackerras, 1960, sounded a warning that more than one genus might be involved), Box (1966) then began studying these parasites (which she referred to as Lankesterella) in Passer domesticus in Texas, U.S.A. She later dropped a bombshell into the midst of this relatively quiescent situation by reviving the suggestion made by Manwell et a/. (1945) and, earlier, by Coulston (1942), Wolfson (1940) and Noller (1920), that the atoxoplasms are in fact stages in the life cycle of an intestinal coccidian. Initially, Box (1967) found that the feeding of Isospora oocysts to P. domesticus resulted in increased parasitaemia and mortality due to atoxoplasms. Exposure to, or administration of, mites (Ornithonyssus bursa) did not have this effect. Neither did administration of tissue homogenates containing atoxoplasms. Although she explained these observations “as a suppression of premunition to the former infection [atoxoplasmosis] by the coccidial infection”, Box did permit herself the speculation that “the identity of Lankesterella and Isospora would fit my observations with [one] exception. . . .”

* Levine (1961) gave incorrectly the date of publication of L. rrdieias

1933.

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Taking scrupulous care to avoid previous infections of experimental birds (Serinuscanaria)with lsosporu or atoxoplasms, or infestations of Ornithonyssus, Box (1970) was later able to show that feeding such birds on oocysts resembling those of I . lacazei gave rise to tissue infections with organisms morphologically indistinguishable from atoxoplasms. Similarly, she showed that transfer of liver from birds infected with atoxoplasms (but not uninfected liver), produced infections of I . lacazei in the recipients. Much of the life-cycle was elucidated and is described in her paper (Box, 1970). After infection with oocysts, parasites are first seen in the core of the duodenal villi; they then apparently spread posteriorly along the small intestine, enter the blood stream in monocytes and parasitize other viscera such as the liver, spleen and lungs. It is impossible at present to reconcile with any certainty the conflicting results obtained by Lainson (1959, 1960) and by Box (1970). The work of both authors was careful and convincing, and Lainson’s observations were at least partially confirmed by Dissanaike (1967). Further study is clearly necessary, using atoxoplasms of as many different host species as possible and under the most rigorous conditions to exclude previous infection (a very difficult thing to do, as both Lainson and Box record). The use of tissue cultures might be worth investigating. Meanwhile, there seem to be two possible explanationseither that the two studies were made on parasites which, though morphologically identical at certain stages of their life-cycle, are in fact distinct at the familial level or (perhaps and) that one, or both, investigators was (or were) using birds with mixed infections in spite of the care taken to avoid this. While the first explanation seems less probable, it is considered likely, as predicted by Mackerras and Mackerras (1960), that more than one genus has been included under the name Lankesterella in recent years (see Khan and Desser, 1971). There are considerable morphological differences between the atoxoplasms seen in avian monocytes and the classical Lankesterella minima, which inhabits erythrocytes of frogs (see Wenyon, 1926). There have been three earlier reports of avian atoxoplasms from erythrocytes (Manwell, 1941 ; Baker et af., 1959; Mackerras and Mackerras, 1960). These are now supplemented by a further record from Malaysia (p. 8). Perhaps, as suggested by Corradetti and Scanga ( I 963), only these forms are truly members of the genus Lankesterella, the other “intraleucocytic parasites” requiring to be placed in a separate genus. Nevertheless, we agree with Lainson (1959) that the type of host cell inhabited by the sporozoites is not, of itself, a sufficient criterion for generic distinction (as proposed by Corradetti and Scanga, 1963). Were this generic separation valid, it would be conceivable that Lainson’s birds were chronically infected with the “true” Lankesterella as well as with atoxoplasms; although the fact that he never saw parasites in their erythrocytes would then be difficult to explain (Manwell, 1941, had found atoxoplasms in both leucocytes and erythrocytes of P. domesticus). Alternatively, it is also possible that, in spite of all her care, the birds used by Box were harbouring chronic, sub-patent infections of lsospora (their parents were known to be infected with this parasite).

14

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F.

B E N N E T TG, . w .

CLARK AND M. LAIRD

The recent demonstration that Toxoplasma (sensu stricto) of mammals is itself an isosporan coccidian (or even a species of Zsospora; see Overdulve, 1970) undergoing metastatic schizogony throughout the viscera of a wide range of warm-blooded hosts but, apparently, able to complete its typical isosporan sexual development only in the cat (Hutchison et ul., 1970; Frenkel et al., 1970; Sheffield and Melton, 1970), has, as noted by Box (1970), made especially relevant the latter’s findings and tends to support her suggestion that the “life-cycles of some parasites classified as Isospora may be quite different from that of the typical coccidian” (Box, 1970). Atoxoplasms have been studied by electron microscopy (Lainson, 1961 ; Garnham et al., 1962; Ludvik, 1963; Biittner, 1968; Khan andDesser, 1971). The ultrastructure revealed is markedly similar to that of Toxoplasma (e.g. SCnaud, 1967) and the other einieriid coccidia such as Eimeria spp. (e.g. Snigirevskaya, 1969; Strout and Scholtyseck, 1970) and Isospora (Schmidt et al., 1967). 111. ADELE~NE HAEMOGRECARINFS

Eleven years after Laveran’s (1900) clear description of undoubted atoxoplasms, Aragilo (I 9 11) described seven species of what he regarded as Haeniogregarina from mononuclear blood cells of seven species of South American birds. He also named the parasites described by Laveran (1900) and Adie ( I 907, 1908) Haemogregarina paddae and H. adiei respectively. Hoare ( I 924) believed that, of Aragiio’s seven species, five-H. atticorae from Notiochelidon ( = Attica [sic])gyanoleucus, H. rhamphoceli from Ramphocoelus bresilius, H. poroariae [sic] from Paroaria?dominica (= P . lavata [sic]),H. tanagrae from Thraupis ( = Tanagra) palmarium and H. brachyspizae from Zonotrichia (= Brachyspiza) capensis-were true adeleine haemogregarines of the genus Hepatozoon, probably representing only a single species, while the other two ( H . sporophilae and H. sicalidis) were not; the latter two almost certainly being atoxoplasms (see p. 3 above). Reports of adeleine haemogregarines from birds are rather rare. Todd and Wolbach (19 12) described an organism almost certainly belonging to this group from the mononuclear cells and, rarely, eosinophils and neutrophils, of a Necrosyrtes (= Neophron) monachus (vulture) in Gambia. They named the parasite Leucocytogregarina neophrontis. Their species has since been transferred to the genus Hepatozoon by Bray (1964). The parasite reported by de Mello (1915) and de Mello et al. (1917) from a pigeon (presumably Columba livia) in India, and named Haemogregarina francae by de Mello (1915), and that reported as a “leucocytogregarina” of Belonopterus chilensis lampronotus ( = B . cayennensis) by Neiva and Penna (1916), may belong in this group; they are impossible to categorize (see pp. 5 and 6.) Franchini (1923 and 1924) recorded (with no detailed description) haemogregarines within the erythrocytes and free in the plasma of Anas (= Querqueduula) crecca and A . (= Q.) circia in Italy. Hoare (1924) described a parasite from mononuclear leucocytes of an unidentified Indian eagle, and named it Hepatozoon adiei. A review by

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Mohammed and Mansour (1960) makes brief reference to this species. Primio (1 925) described three species of “Haemogregarina” from birds in Brazil“H.” aragaoi from Paroaria c. capitata, “H.” pintoi from Cathartes aura ruficollis and Coragyps atratus foetens, and “H.” travassosi from Taraba m. major. We have not been able to refer to Primio’s publication and it is therefore not possible to criticize his taxonomic ascriptions of the parasites. However, as their hosts are vultures (family Carthartidae, order Falconiformes), a group from which other undoubted adeleine haemogregarines have been reported, it is very probable that “H.” pinioi belongs to this group-perhaps to the genus Hepatozoon. Lucena (1938) briefly recorded “Haemogregarina” from four passerine birds in Brazil (Certhiaxis cinnamomea russeola, Passerina (= Cyanocompsa) c. cyanea, Saltator similis and Tachyphonus coronatus). In the absence of any description of the parasites or their host-cells, though, it is impossible to be certain of the group to which these organisms belonged. Huff (1939), also, recorded briefly, without description, a “haemogregarine?” in unspecified host cells of one Zenaidura macroura carolinensis out of nearly 200 examined during a ten-year period. Wood and Herman (1943) recorded “Hepatozoon” from six species of birds in the south-western United States of America. As discussed above (p. 9) most of these records are thought to refer to atoxoplasms. However, that from Parus (=Baeolophus) inornatus transpositus is provisionally regarded as referring to a true adeleine haemogregarine. Bray (1954) recorded Hepatozoon from a Pandion haliaetus of unspecified provenance. Other reports of “haemogregarines”, “Hepatozoon”, etc. from avian hosts are considered probably to have referred to atoxoplasms and have been reviewed in the previous section. In fact, Bray (1964) regarded only three species of adeleine haemogregarines from birds as “relatively authenticated”. These are H. monachus (Todd and Wolbach, 1912), H. adiei Hoare 1924, and his own record from P. haliaetus (Bray, 1954). Bray (1964) pointed out that these three “authenticated” records are all from large birds of prey. He continued, “it is at least possible that these birds when consuming their prey also swallow the mites, ticks, leeches, etc., which are the vectors of the haemogregarines and thus become accidentally infected by their prey’s haemogregarines”. It is also possible that Franchini’s (1923, 1924) ducks (Anas spp.) (see p. 14 above) had fed upon leeches serving as vectors of some species of adeleine haemogregarine of amphibia or fishes. These possibilities were strengthened by a very interesting discovery on the part of Clark and Swinehart (1969), who reported “Hepatozoon” from one of 15 frigate birds (Fregafa magniJicens) and one of five black storm-petrels (Loomelania melania) from offshore islands of northern Mexico. Neither host was previously known to harbour haematozoa, which are remarkably uncommon in marine birds as a whole (except for certain gulls locally parasitized by Plasmodium spp., e.g. in Australia and the U S A . and penguins, in the northern part of their range and when maintained in zoos in the presence of infected vectors). Further examination of the slide from L. melania showed the abundant presence of adeleine haemogregarines in erythrocytes only (Figs 916). The large parasites occupied the greater part of the free space on one side of the erythrocyte’s nucleus, which was crammed against the opposite cell 3

16

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G . W. CLARK AND

M. L A I R D

membrane. These organisms immediately brought to mind similar adeleine haemogregarines of reptiles. In some instances, especially where the host red blood corpuscle was partly disintegrated, the haemogregarine could clearly be seen to have a sharply reflected “tail” (Fig. 16). This was the more thoughtprovoking in that the particular island from which the parasitized petrel was collected (San Benito) abounded with terrestrial lizards. Add to this the fact that like other storm-petrels L. melaniu nest in burrows and crevices where encounters with terrestrial lizards (and their ectoparasites) are likely to occur with some frequency, and the possibility must be entertained that the bird in question was harbouring not a true avian haemogregarine but a species properly referable to a lizard host or hosts and accidentally acquired in this instance by, e.g. the ingestion of infective acarines, if not the bite of a mosquito or sandfly (see note on p. 22). Such an explanation is further supported by the fact that every one of the many intraerythrocytic haemogregarines examined was margined by a rim staining red with Giemsa; exhibited poorly defined chromatin/cytoplasm separation; and had irregular reddish masses distributed about (especially at the periphery of its body). These indications of unsuccessful capsule formation(?) and advancing pyknosis would be in accord with the presence of a haematozoon in an altogether foreign host to which it was failing to adjust. It is also worth mentioning in this context that Ayala (1970) has recently demonstrated that Hepatozoon-like parasites discovered as oocysts in the haemocoele of Californian sandflies, Phlebotomus vexator occidentalis, are capable of infecting hosts as diverse as garter snakes and western fence lizards when inoculated as sporocysts. At all events, there are various known field situations where the hypothesis of accidental infection of birds by reptilian adeleine haemogregarines could be tested. One that comes to mind besides the Baja Californian Islands is Stephens Island in Cook Strait, one of a number of New Zealand’s offshore islands where the only surviving rhynchocephalian, the tuatara (Sphenodon punctatus), is still not uncommon. This reptile very often shares burrows with the petrels and shearwaters which occupy the island in great numbers-and its extremely large erythrocytes are parasitized by an adeleine haemogregarine (Laird, 1950b).

IV. CONCLUSIONS It is evident that much confusion persists as to the proper taxonomic status of avian blood coccidians. The existing information, summarized herein, certainly provides a fascinating basis for speculation. It also suggests hypotheses meriting early testing by carefully controlled laboratory and field experimentation, to settle such questions as : Are all atoxoplasms really referable to Zsospora? If they are, do they really belong to more than a single widespread species? If there are indeed two different groups currently lumped together as atoxoplasms, are these two groups monospecific or not? Last but not least, are the relatively few probable records of adeleine haemo-

AVIAN BLOOD COCCIDIANS

17

gregarines from birds simply due to accidental infection with parasites of reptiles sharing the avian hosts’ habitats? ACKNOWLEDGEMENTS

We are very grateful to the Librarians and staffs of the London School of Hygiene and Tropical Medicine (University of London) and the Scientific Periodicals Library, Cambridge, for their cooperation and to Mr. C. W. Benson of the Department of Zoology, University of Cambridge, for invaluable assistance generously given, in checking the names of birds. Mr. Roy Ficken, Biology Department Photographer, Memorial University of Newfoundland, prepared the plate of photomicrographs (Plate I, Figs 9-16 of which were made available by one of us; G.W.C.), and our thanks are due to him for this. The work was in part supported by a grant to two of the authors (G.F.B. and M.L.) from the National Research Council of Canada, and the services of the World Health Organization’s International Reference Centre for Avian Malaria Parasites proved very helpful. REFERENCES Adie, J. R. (1907). A plea for scraps. Indian med. Gar. 42, 250-256. Adie, J. R. (1908). Note on a parasite in the sparrow. Indian med. Gat. 43, 176-180. Anschutz, G. (1909). Ueber den Entwicklungsgang des “Haemoproteus orizivorae” nov. spec. Zentbl. Bakt. ParasitKde (Abt. I, Orig.) 51, 654-659. Araggo, H. de B. (1911). ObservaG6es sobre algumas haemogregarinas das aves. Mems Inst. Oswaldo Cruz 3, 54-64. Aragiio, H. de B. (1918). Classifica~iiodos hemosporidios. Mems Inst. Butantan 1, 167-185. Aragiio, H. de B. (1933). Considbrations sur les himogregarines des oiseaux. C. r. SPanc. Sac. Biol. 113,214-215. Ayala, S. C. (1970). Hemogregarine from sandfly infecting both lizards and snakes. J. Parasit. 56, 387-388. Baker, J. R., Lainson, R. and Killick-Kendrick, R. (1959). Lankesterella corvi n. sp., a blood parasite of the English rook, Coruusf: frugilegus L. J. Protozool. 6, 233-238. Bennett, G. F. and Laird, M. (1971). Reference centre for avian malaria parasites. WHO Chronicle 25, 17-1 9. Berson, J. P. (1964). Les protozoaires parasites des hematies et du systbme histiocytaire des oiseaux: essai de nomenclature. Rev. Elev. Med. vet. Pays trop. 17, 43-96. Bhatia, B. L. (1938). Protozoa : Sporozoa. In “The Fauna of British India”. Taylor and Francis Ltd., London. Box, Edith D. (1966). Blood and tissue protozoa of the English sparrow (Passer domesticus domesticus) in Galveston, Texas. J. Protozool. 13,204-208. Box, Edith D. (1967). Influence of Isospora infections on patency of avian Lankesterella (Atoxtplasmu,Garnham, 1950). J. Parusit. 53, 1140-1 147. Box, Edith D. (1970). Atoxoplusma associated with an isosporan oocyst in canaries. J. Protozool. 17, 391-396. Bray, R. S. (1954). A Hepatozoon sp. of the osprey. Trans. R. SOC.trop. Med. Hyg. 48, 1.

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Bray, R. S. (1964). A check-list of the parasitic protozoa of West Africa with some notes on their classification Bull. Inst. fr. Afr. noire 26 (series A), 238-315. Biittner, D. W. (1968). Das cytostom von Lankesterella garnhami. Z . Zellforsch. mikrosk. Anat. 88, 126-137. Carini, A. (1909). Reproduction expkrimentale de la toxoplasmose du lapin. BUN. SOC.Path. exot. 2,465-469 and 524-525. Carini, A. (1911) Infection spontanke du pigeon et du chien due au “Toxoplasrna cuniculi”. Bull. SOC.Path. exot. 4, 518-519. Carini, A. and Maciel, J. (1916). Quelques hernoparasites du Brdsil. Bull. SOC.Path. exot. 9,247-265. Clark, G. W. and Swinehart, B. (1969). Avian haematozoa from the offshore islands of northern Mexico. Bull. Wildlife Disease Assoc. 5 , 11 1-1 12. Clark, G. W., Lee, M. A. and Lieb, D. E. (1968). Avian haematozoa of central Washington. Bull. Wildlqe Disease Assoc. 4, 15. Corradetti, A. and Scanga, M. (1963). Atoxoplasma coccothraustis n. sp., parassita del frosone (Coccothraustes coccothraustes). Parassitologia 5, 61-72. Correa, C . (1928). ContribuiCBo ao estudo das hemogregarinas do Brasil. Reuta Biol. Hyg. 1 (3), 75-81. Cory, C. B. and Hellmayr, C. E. (1927). “Catalogue of Birds of the Americas and the Adjacent Islands in Field Museum of Natural History” (Ed. W. H. Osgood), Vol. 5. Field Museum of Natural History, Chicago. Coulston, F. (1942). The coccidial nature of “avian Toxoplasma”. J . Parasit. 28, Suppl. 16 (Abstract only). Danilewsky, B. (1889). “La Parasitologie compark du Sang. I. Nouvelles recherches sur les parasites du sang des oiseaux.” D a d , Kharkov. Dissanaike, A. S. (1967). Lankesterella lainsoni sp. nov. from the Ceylon Mynah bird Acridotheres tristis melanosternus. Ceylon J. Sci. biol. Sci. 6, 225-229. Dissanaike, A. S., Nelson, P., Fernando, M. A. and Niles, W. J. (1965). Studies on haemosporidia of Ceylon birds with special reference to plasmodia. Ceylon vet. J. 13, 65-75. Fantham, H. B. (1919). Some parasitic protozoa found in South Africa-11. S. Afr. J. Sci. 16, 185-191. Fantham, H. B. (1924). Some parasitic protozoa found in South Africa-VII. S. Afr. J. Sci. 21,4344l4. Franchini, G. (1923). Hkmatozoaires de quelques oiseaux d’Italie. Bull. SOC.Path. exot. 16,118-125. Franchini, G . (1924). Observations sur les hematozoaires des oiseaux d‘Italie. Annls Inst. Pasteur, Paris 38,470-51 5. Frenkel, J. IS.,Dubey, J. P. and Miller, N. L. (1970). Toxoplasma gondii in cats: fecal stages identified as coccidian oocysts. Science, N. Y. 167, 893-896. Garnham, P. C. C. (1950). Blood parasites of East African vertebrates, with a brief description of exo-erythrocytic schizogony in Plasmodium pitrnani. Parasitology 40,328-337. Garnham, P. C. C., Baker, J. R. and Bird, R. G. (1962). The fine structure of Lankesterella garnhami. J. Protozool. 9, 107-1 14. Hamerton, A. E. (1936). Report on the deaths occurring in the Society‘s gardens during the year 1935. Proc. zool. SOC.Lond. 659-686. Hart, J. W. (1949). Observations on blood parasites of birds in South Carolina. J. Parasit. 35, 79-82. Hegner, R. and Wolfson, Fruma (1938). Toxoplasrna-like parasites in canaries infected with Plasmodium. Am. J. Hyg. 27,212-220.

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Herman, C. M. (1937). Toxoplasma in North American birds and attempted transmission to canaries and chickens. Am. J. Hyg. 25, 303-312. Herman, C. M. (1938). The relative incidence of blood protozoa in some birds from Cape Cod. Trans. Am. microsc. SOC.57,132-141. Herman, C. M. (1944). The blood protozoa of North American birds. Bird-Banding 15,89-112. Hewitt, R. (1940). Studies on blood protozoa obtained from Mexican wild birds. J . Parasit. 26, 287-295. Hoare, C. A. (1924). Hepatozoon adiei, n. sp. A blood parasite of an Indian eagle. Trans. R. SOC.trop. Med. Hyg. 18, 63-66. Huff, C. G. (1939). A survey of the blood parasites of birds caught for banding purposes. J. Am. vet. med. Ass. 94, 615-620. Hutchison, W. M., Dunachie, J. R., Siim, J. C. and Work, K. (1970). Coccidian-like nature of Toxoplasma gondii. Br. med. J. i, 142-144. Khan, R. A. and Desser, S. S. (1971). Avian Lankesterella infections in Algonquin Park, Ontario. Can. J. Zool. 49, 1105-1110. Kikuth, W. and Mudrow, Lily (1938). Die endothelialen Stadien der Malariaparasiten in experiment und theorie. Zentbl. Bakt. ParasitKde (Abt. I, Orig.) 142, 113132. Labbb, A. (1894). Recherches zoologiques et biologiques sur les parasites endoglobulaires du sang des vert6brCs. Arch. 2001.expkr., Ser. 3,2, 55-258 (158-160). LabbC, A. (1899). Sporozoa. In “Das Tierreich”. Friedlander, Berlin. Lainson, E. (1958a). Atoxoplasma Garnham, 1950, in an English sparrow (Passer domesticus domesticus Linn.). Trans. R. SOC.trop. Med. Hyg. 52, 15-16. Lainson, R. (1958b). Some observations on the life-cycle of Atoxoplasma, with particular reference to the parasite’s schizogony and its transmission by the mite Dermanyssus gallinae. Nature, Lond. 182, 1250-1251. Lainson, R. (1959). Atoxoplasma Garnham, 1950, as a synonym for Lankesterella LabbC, 1899. Its life cycle in the English sparrow (Passer domesticus domesticus, Linn.). J. Protozool. 6, 360-371. Lainson, R. (1960). The transmission of Lankesterella (= Atoxoplasma) in birds by the mite Dermanyssus gallinae. J. Protozool. 7, 321-322. Lainson, R. (1961). [Discussion to paper by Goldman, M. Classification of Toxoplasma, pp. 700-720.1 Surv. Ophthal. 6, 71 3-71 6. Laird, M. (1950a). Some blood parasites of New Zealand birds. Victoria University College, N.Z., Publs Zool. no. 5, 1-20. Laird, M. (1950b). Haemogregarina tuatarae sp. n., from the New Zealand rhynchocephalian Sphenodon punctatus (Gray). Proc. zool. SOC.Lond. 120, 529-533. Laird, M. (1959). Atoxoplasma paddae (AragBo) from several South Pacific silvereyes (Zosteropidae) and a New Zealand rail. J. Parasit. 45,47-52. Laird, M. (1962). Malayan protozoa 5. Two avian malaria parasites. J. Protozool. 9, 21-26. Laird, M. and Laird, Elizabeth (1959). In “The Natural History of Rennell Island, British Solomon Islands”, 2,213-234. Roy. Danish Museum, Copenhagen. Laveran, A. (1900). Au sujet de I’hCmatozoaire endoglobulaire de Padda oryziuora. C. r. SLanc. SOC.Biol. 52, 19-20. Laveran, A. and Marullaz, M. (1914). Sur deux htmamibes et un toxoplasme du Liothrix luteus. Bull. SOC.Path. exot. 7 , 21-25. Lawrence, J. (1946). Some observations on the plasmodia and other blood parasites of sparrows. Proc. Linn. SOC.N.S. W. 71, 1-5. 38

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J . R . BAKER, G . F . B E N N E T T , G . W . C L A R K A N D M . L A I R D

Levine, N. D. (1961). “Protozoan Parasites of Domestic Animals and Man.” Burgess Publishing Company, Minneapolis. Levine, N. D. and Kantor, S. (1959). Check-list of blood parasites of birds of the order Columbiformes. Wildl. Dis. no. 1, 1-38 [Microcard]. Lucena, D. (1938). Haemoparasitas de algumas aves de SBo Paulo. Revta Biol. Hyg. 9, 158-161. Lucena, D. T. (1941). Lista dos protozokrios hemoparasitas de aves da regiao neotr6pica. Anais SOC.Biol. Pernamb. 2 (2), 3-61. Ludvik, J. (1963). Electron microscopic studies of some parasitic protozoa. Int. Congr. Protozool. 1, 387-392. Mackerras, M. Josephine and Mackerras, I. M. (1960).The haematozoa of Australian birds. Aust. J. Zool. 8, 226260. Manwell, R. D. (1939). Toxoplasma or exoerythrocytic schizogony in malaria? Riv. Malar. 18,7688. Manwell, R. D. (1941). Avian toxoplasmosis with invasion of the erythrocytes. J. Parasit. 27, 245-250. Manwell, R. D. (1957). Blood parasitism in the English sparrow, with certain biological implications. J. Parasit. 43,428-433. Manwell, R. D. and Herman, C. M. (1935). Blood parasites of birds of the Syracuse (N.Y.) region. J. Parasit. 21,415-416. Manwell, R. D., Coulston, F., Binckley, Ellen C. and Jones, Virginia P. (1945). Mammalian and avian Toxoplasma. J. infect. Dis. 76, 1-14. Marullaz, M. (1913). Au sujet d’un toxoplasme des oiseaux. Bull. SOC.path. exot. 6, 323-326. de Mello, F. (1915). Preliminary note on a new haemogregarine found in the pigeon’s blood. Indian J. med. Res. 3,93-94. de Mello, F. (1937). Further contributions to the study of the blood parasites of the Indian birds, together with a list of the hemoparasites hitherto recorded. J. roy. Asiat. SOC.Bengal, Science, 1936 2,95-122. de Mello, F., de Sa, B., Bras, de Sousa Loreto, Dias, A. and Moroha, R. (1917). Hematozoaires et pseudo-hematozoaires de 1’Inde portugaise. Annais scient. Fac, Med. Porto 3,5-24. Mine, N. (1914). Beitrage zur kenntnis der Blutparasiten der Vogel in Japan. Arch. Protistenk. 34, 198-21 1. Mohammed, A. H. H. (1958). “Systematic and Experimental Studies on Protozoal Blood Parasites of Egyptian Birds”, Vol. 1. University Press, Cairo. Mohammed, A. H. H. and Mansour, N. S. (1960). The haemogregarine complex. Bull. Fac. Sci. Cairo Univ. no. 35, 39-51 (dated 1959). Neiva, A. and Penna, B. (1916). Viajem cientifica pel0 Norte da Bahia, sudoeste de Pernambuco, Sul do Piauhi e de norte a sul de Goiaz. Mems. Inst. Oswaldo Cruz 8 (3), 74-224. Nicolle, C. and Manceaux, L. (1908). Sur une infection A corps de Leishman (ou organismes voisins) du gondi. C . r. hebd. Se‘anc. Acad. Sci. 147,763-766. Nicolle, C. and Manceaux, L. (1909). Sur un protozoaire nouveau du gondi. C. r. hebd. Skanc. Acad. Sci. 148,369-372. Noller, W. (1920). In “Handbuch der pathogenen Protozoen” (Eds S. von Prowazek and W. Noller), Vol. 2, pp. 907-918. J. A. Barth, Leipzig. Noller, W. (1931). In “Tierheilkunde und Tierzucht” (Eds V. Stang and D. Wirth), vol. 9, pp. 424-440. Urban and Schwarzenberg, Berlin and Vienna. Noller, W.and Nitsche, 0. (1923). Ueber einige verbreitete Erkraukungen unserer einheimischen Sperlingsvogel. Berl. tierartzl. Wschr. 39,443-447 and 455-458.

A V I A N BLOOD C O C C I D I A N S

21

Now, F. G. and MacNeal, W. J. (1904). On the trypanosomes of birds. J . infect. Dis. 2, 256-308. Novy, F. G. and MacNeal, W. J. (1905). Trypanosomes and bird malaria. Am. Med. 8, 932-934. Overdulve, J. P. (1970). The identity of Toxoplasma Nicolle and Manceaux, 1909 with Zsosporu Schneider, 1881 (I). Proc. K . ned. Adad. wet., C 73,129-151. Pessha, S. B. and Conga, Clovis (1929). Nota sobre toxoplasmas dos passaros. Anaispaul. Med. Cirurg. 20, 103-106. Peters, J. L. (1931 et seq.). “Check-list of Birds of the World” (continued by various authors and editors). Harvard University Press (continued by Museum of Comparative Zoology), Cambridge, Mass. Plimmer, H. G. (1915). Report on the deaths which occurred in the zoological gardens during 1914, together with a list of the blood parasites found during the year. Proc. zool. SOC. Lond. 123-130. Plinimer, H. G. (1916). Notes on the genus Toxoplasma, with a description of three new species. Proc. R. SOC.,B 89,291-296. Primio, R. di (1 925). “Contribuiqiio para o Estudo das Hemogregarinas Brasilieras”. Typ. Lenzinger, Rio de Janeiro. [Not seen; cited by Lucena (1914).] Raffaele, G. (1932). Sulle cosidette toxoplasmosi dei passeri. Riu. Malar. 11, 825838. Raffaele, G. (1938). Evoluzione di Plasmodium, Toxoplasma ed altri microrganismi negli organi interni dei vertebrati. Riu. Malur. 17, 85-100. Reichenow, E. (1953). “Lehrbuch der Protozoenkunde”, Vol. 2, pp. 820-966. Gustav Fischer, Jena. Rosenbusch, F. (1932). Toxoplasmosis avium en 10s canarios. Reun. SOC.argent. Patol. reg. N. (7), 904-906. Rousselot, R. (1953). “Notes de Parasitologie Tropicale”, Vol. 1, pp. 62-64. Vigot, Paris. Schmidt, K., Johnston, M. R. L. and Stebhens, W. E. (1967). Fine structure of the schizont and merozoite of Zsospora sp. (Sporozoa : Eimeriidae) parasitic in Cehyra uariegata (Dumeril and Bibron, 1836) (Reptilia: Gekkonidae). J. Protorool. 14,602-608. SBnaud, J. (1967). Contribution A 1’6tude des sarcosporidies et des toxoplasmes (Toxoplasmea). Protistologica 3, 167-232. Sheffield, H. G. and Melton, Marjorie L. (1970). Toxoplasma gondii: the oocyst, sporozoite, and infection of cultured cells. Science, N. Y. 167, 892-893. Snigirevskaya, E. S. (1969). Electron microscopic study of the schizogony process in Eimeria intestinalis. Acta Protozool. 7, 57-70. [Russian with English summary.] Stauber, Mabel F. and Stauber, L. A. (1942). Bird malaria in southern New Jersey. Proc. New Jers. Mosq. Exterm. Ass. 29,4546. Strout, R. G . and Scholtyseck, E. (1970). The ultrastructure of first generation development of Eimeria tenella (Railliet and Lucet, 1891) Fantham, 1909 in cell cultures. 2.ParasitKde 35, 87-96. Taddia, L. (1938). Plasmodidi e corpi Toxoplusma-simili nei passeri del Veneto. Riu. Malar. 17,239-241. Todd, J. L. and Wolbach, S. B. (1912). Parasitic protozoa from the Gambia. J. med. Res. 26, 195-218. Uegaki, J. (1930). Untersuchungen iiber die Blutprotozoen von Vogeln der Siidsee. Arch. Protistenk. 72,7490. Walzberg, U . (1923). Zur pathologischen Histologie der naturlichen Toxoplasmose des Zeisigs. Z . Infektkrankh. parasit. Krankh. Hyg. Haustiere 25, 19-33.

22

J . R . BAKER, G . F . B E N N E T T , G . W . C L A R K A N D M . L A I R D

Wenyon, C. M. (1926). “Protozoology”, Vol. 2. Ballikre, Tindall and Cox, London. [Reprinted 1965 by Ballikre, Tindall and Cassell, London.] Wetmore, Psyche W. (1941). Blood parasites of birds of the District of Columbia and Patuxent Research Refuge vicinity. J. Parasit. 27, 379-393. Wohnus, J. F. and Ryerson, D. L. (1941). Hematozoa from California birds. J. Parasit. 27, 540-541. Wolfson, Fruma (1937). Experimental transmission of Toxoplasma in canaries. J. Parasit. 23, 553. Wolfson, Fruma (1938). Two types of Toxoplasma-like bodies in canaries. J. Parasit. 24, Suppl. 22 [Abstract only]. Wolfson, Fruma (1940). Organisms described as avian Toxoplasma. Am. J. Hyg. 32, C 88-99. Wood, S. F. and Herman, C. M. (1943). The occurrence of blood parasites in birds from southwestern United States. J. Parasit. 29, 187-196. Wood, Fae D. and Wood, S. F. (1937). Occurrence of haematozoa in some Californian birds and mammals. J. Parasit. 23, 197-201. Yakimoff, W. L. and Kohl-Yakimoff, N. (1912). Toxoplasma canis (Mello). Arch. Protistenk. 27, 195-206. Zasukhin, D. N., Vasina, S. G . and Levitanskaya, P. B. (1956). [Atoxoplasma and Toxoplasma of birds]. Zool. Zh. 35, 1799-1808. [English summary in Zool. Zh. 35 (12), Summaries, 6.1 Zasukhin, D. N., Vasina, S. G . and Levitanskaya, P. B. (1957). [On the problem of the atoxoplasmas of birds.] Trudj Leningr. Obshch. Estest. 73,117-120. [German summary on p. 120.1 Papers not included in Tables I and 11: Bax, Edith D. (1971). Lankesterella (Atoxoplasma). In “Infectious and Parasitic Diseases of Wild Birds” (Eds. J. W. Davis, R. C. Anderson, L. Karstad and D. 0. Trainer), pp. 309-312. Iowa State University Press, Ames, Iowa. Oda, S. M., Chao, J. and Ball, G. H. (1971). Additional instances of transfer of reptile haemogragarines to foreign hosts. J. Parisit, 53, 1377-1378. Poelma, F. G., Zwart, P. and Strick, W. J. (1970). [Lankesterella infections in birds in the Netherlands.] Tijdschr. Diergeneesk. 95, 1163-1 169. [In Dutch; English version (1971) in Nerh. J. vet. Sci. 4, 43-50.]

ADDENDUM.

NOTE ADDED IN PROOF. Oda et al. (1971; see addendum above) have recently reported the experimental transfer of a Hepatozoon from a snake to a lizard, and state that “There is now a considerable body of evidence that at least some haemogregarines of reptiles can be transferred experimentally to foreign hosts”.

23

AVIAN BLOOD COCCIDIANS

TABLEI Chronological list of publications on avian haemogregarines (including atoxoplasms)*

1 2 3 4

Laveran, 1900 Novy and MacNeal, 1904 Novy and MacNeal, 1905 Adie, 1907 5 Adie, 1908 6 Anschiitz, 1909 7 AragBo, 1911 8 Todd and Wolbach, 1912 9 Marullaz, 1913 10 Laveran and Marullaz, 1914 11 Mine, 1914 12 de Mello, 1915 13 Plimrner, 19151 14 Carini and Maciel, 1916 15 Neiva and Penna, 1916 16 Plimmer, 19161 17 de Mello et al., 1917 18 AragBo, 1918 19 Fantham, 1919 20 Noller, 1920 21 Franchini, 1923 22 Noller and Nitsche, 1923 23 Walzberg, 19232 24 Fantham, 1924 25 Franchini, 1924 26 Hoare, 1924 27 Primio, 1925 28 Wenyon, 1926 29 CorrEa, 1928 30 Pessoa and CorrEa, 1929 31 Uegaki, 1930 32 Noller, 1931 33 Raffaele, 1932 34 Rosenbusch, 1932 35 AragBo, 1933 36 Manwell and Herman, 1935 37 Hamerton, 1936 38 Herman, 1937 39 Wolfson, 19373 40 Wood and Wood, 1937 41 Hegner and Wolfson, 1938 42 Kikuth and Mudrow, 1938 43 Herman, 1938 ~

44 Lucena, 1938 45 46 47 48 49 50 51 52 53 54

Raffaele, 1938 Taddia, 1938 Wolfson, 1938 Huff, 1939 Manwell, 1939 Hewitt, 1940 Wolfson, 1940 Lucena, 1941 Manwell, 1941 Wetmore, 1941 55 Wohnus and Ryerson, 1941 56 Coulston, 1942 57 Stauber and Stauber, 1942 58 Wood and Herman, 1943 59 Herman, 1944 60 Manwell et al., 1945 61 Lawrence, 1946 62 Hart, 1949 63 Garnham, 1950 64 Laird, 1950 65 Reichenow, 1953 66 Rousselot, 1953 67 Bray, 1954 68 Zasukhin et al., 1956 69 Manwell, 1957 70 Zasukhin et al., 1957 71 Lainson, 1958a 72 Lainson, 1958b 73 Mohammed, 1958 74 Baker et al., 1959 75 Lainson, 1959 76 Laird, 1959 77 Laird and Laird, 1959 78 Levhe and Kantor, 1959 79 Lainson, 1960 80 Mackerras and Mackerras, 1960 81 Mohammed and Mansour, 1960 82 Lainson, 1961 83 Levine, 1961 84 Garnham et al., 1962 85 Corradetti and Scanga, 1963 86 Ludvik, 1963

~

~~

~

* Authors publishing in the same year are listed alphabetically. Possibly refers to Toxoplasma sensu siricto. Mostly refers to Toxoplasma but record from Chrysomiiris spinus on p. 20 may refer to atoxoplasms. 3 Said by Wolfson (1940) to be a misidentification of exoerythrocytic forms of PIasmodium. 1 2

24

J . R. BAKER, G . F. BENNETT, G . W. CLARK A N D M . LAIRD

TABLEI (continued) 87 Bray, 1964 88 Dissanaike et al., 1965 89 Box, 1966 90 BOX,1967 91 Dissanaike, 1967 92 Biittner, 1968

93 94 95 96 97

Clark et al., 1968 Clark and Swinehart, 1969 Ayala, 1970 Box, 1970 Khan and Desser, 1971

TABLEI1 Check list of avian hosts of haemogregarines (including atoxoplasms)* Acanthis cannabina cannabina (L.)[Cannabinalinota] Acridotheres tristis tristis (L.) Acridotheres tristis melanosternus Legge Agelaius phoeniceus (L.) Amadina erythrocephalassp. Ammodramus sandwichensis savanna (Wilson) [Passerculus sandwichensis savanna] Anas crecca ssp. [Querquedulacrecca] Anas querquedula L. [Q. circial Aramides cajanea cajanea (P. L. s. Miiller) Belonopterus chilensis lampronotus (Wagler) [B. cayennensis] Carduelis chloris ssp. [Chlorischloris] Ibid. [Ligurinuschloris] Carduelis cucullata Swainson ( ?) [Coryphospinguscucullatus] Carduelis spinus (L.) [Chrysomitrisspinus] Ibid. [Spinusspinus] Carpodacus mexicanus ssp. Carpodacus mexicanusfrontalis (Say) Cathartes aura ruficollis Spix Certhiaxis cinnamomea russeola (Vieillot) Chamaeafasciata ssp. Chamaea fasciata henshawi Ridgway Climacterispicumnus ssp. Coccothraustes coccothraustes coccothraustes (L.) Collocalia esculenta (L.) Columba livia ssp. ["pigeon"] Columba rufina sylvestris Vieillot Columbigallinatabacoti talpacoti (Temminck) Copsychus saularis ssp. Coragyps atratusfoetens (Lichenstein) Corvusfrugilegusfrugilegus L. Cyanocitta cristata ssp. Dendroica coronata auduboni (Townsend) Ibid. [D. auduboni] Ducula concinna ssp. [Carpophaga concinna] Dumetella carolinensis (L.) Emberiza citrinella ssp. Erythruraprasina ssp.

22,23 WHO/IRC 88,91 89 19,24 43 21 H 25 H 44 15 68 22,23 44 201 68,70 WHO/IRC 40, 50, 58 27 H 44H WHO/IRC 55,58 80 85, WHO/IRC WHO/IRC 12, 17, 20, 28 44 44 WHO/IRC 27 H 74 2 WHO/IRC 58 13,16 38,43, 54, 57 68 31

25

A V I A N BLOOD C O C C I D I A N S

TABLE I1 (continued) Euphagus carolinus nigraus Burleigh and Peters Euplectus orix franciscanus (Isert) [Pyromelanafranciscana] Fregata magnificens Fringilla coelebs coelebs L. (?) [Serinus balearicus] Fringilla coelebs ssp. Gallirallus australis australis (Sparrman) [G.a. scotti] Gallusgallus ssp. (var. domesticus) Garrulax erythrocephalus ssp. Hesperosiphonia v. vespertina (Cooper) Hirundo rustica erythrogaster Boddaert Icterus galbula ssp. Lagonosticta senegala ssp. Lagopus Iagopus ssp. Lanius collaris ssp. Leiothrix lutea ssp. Lonchura cucullatus cucullatus (Swainson) [Spermestes c. cucullatus] Lonchura maja (L.) [Munia maja] Lonchura malacca atricapillu (Vieillot) [M.atricapilla] Lonchura m. malacca (L.) [M. malacca] Lonchura malabarica ssp. [Aidemosyne malabarica] Lonchura punctulata topela (Swinhoe) [M. topela] Macronus ptilosus ssp. Mimus polyglottus leucopterus (Vigors) Molothrus ater ater (Boddaert) Molothrus ater obscurus (Gmelin) Molothrus bonariensis ssp. Molothrus bonariensis bonariensis (Gmelin) Molothrus sp. Muscicapa narcissina ssp. Necrosyrtes monachus ssp. [Neophronmonachus] Notiochelidon cyanoleuca ssp. [Attica cyanoleucus] Oceanodroma meania (Bonaparte) [Loomelania melania] Pachycephala cinarea ssp. Padda oryzivora (L.) Ibid. [“Spermestes oryzivora oder Orizornis oryzivora”] Ibid. [“Oryzornisoryzivora (Padda oryzivora)”] Ibid. [Reisvogel] Pandion haliaetus ssp. Paroaria capitata capitata (d‘orbigny and Lafresnaye) P. ? dominicana (L.) [P. lavata] Parus inornatus ssp. Parus inornatus transpositus (Grinnell) [Baeolophusinornatus transpositus] Passer domesticus indicus Jardine & Selby [Sparrow] Passer d. italiae (Vieillot) [P. italiae] Ibid. [Sparrow] Ibid. [P. italicus] Passer d. niloticus Nicolle & Bonhote

WHO/IRC 9 94 H 37 9 76 44 WHO/IRC 97 93 2, 43 9 WHO/IRC 63 10

66 31 31 31 31 31 WHO/IRC 58 43, WHO/IRC 58 29 H 44 50 WHO/IRC 82 H 7H 94 H, WHO/IRC WHO/IRC 1,9 6 31 65 67 H 27 7H WHO/IRC 58 H 4, 5 25 33,46 45 73

26

J . R . BAKER, G . F. B E N N E T T , G.

W. C L A R K A N D M. L A I R D

TABLE I1 (continued) Passer domesticus ssp. [“Sparrow”] Ibid.

Passer griseus ssp. Passer montanus ssp. Passer flaveolus ssp. Passerina cyanea (L.)[Cyanocompsacyanea cyaneal Petrochelidonpyrrhonota ssp. Pheucticus melanocephalus ssp. I bid. [Hedymeles rnelanocephalus] P. ludovicianus (L.) Pipilo erythrophthalmus erythropthalmus (L.) Pitta brachyura ssp. Ploceus cucullatus ssp. [Plesiositagracucullatus] Ploceusphilippinusphilippinus (L.)[P. bayal Pycnonotus goiavier ssp. Pycnonotus jocosus ssp. Pycnonotus xanthopygos (Ehrenberg) Poospiza thoracica (Nordman) Quelea erythrops (Hartlaub) Quiscalus mexicanus ssp. Quiscalusquiscula quiscula (L.) Ramphocoelus bresilius ssp. Rhipidurajavanica ssp. Saltator similis ssp. Saxicola caprata ssp. Serinus canaria (L.)

Sicalis flaveola ssp. Spizella passerina ssp. Ibid. [Chipping sparrow] Sporophila albogularis (Spix) Sporophila caerulescens ssp. Stachyris leucotis ssp. Stachyris nigricollis (Temminck) Stephanophorus diadematus (Temminck) [S. leancocephalus] Sturnella magna ssp. Sturnus vulgaris ssp. Tachyphonus coronatus (Vieillot) Ibid. [Techyphoceuscoronatus] Taraba major major (Vieillot) Thraupispalmarum palmarum (Wied.) [Tanagra palmarum]

3,85 22, 232, 36,38, 43,47,49, 53, 54,56, 58, 61, 62, 68, 69,71, 72, 75, 79, 80, 82, 833, 84, 89, 90,92,96 66 11,25 WHO/IRC 44 H WHO/IRC WHO/IRC 58 97 38,43 WHO/IRC 87 31 WHO/IRC WHO/IRC WHO/IRC 29 H 9 89 54 7H WHO/IRC 44H 16 34, 35, 36, 38, 3g4,41,42,47 51, 68,72, 75, 79,90,96, WHO/IRC 7 38,43 69 7

44 WHO/IRC WHO/IRC 29 H 89 38,43 44H 30 27 7H

27

AVIAN BLOOD COCCIDIANS

TABLE I1 (continued) ~

Thraupis sayaca sayaca (L.) [Tanagra sayaca] Toxostoma redivivum redivivum (Gambel) Trichostoma abbotti ssp. Trichostoma bicolor (Lesson) Trichostoma restratum ssp. Turdoides rubiginosa ssp. [Argya rubiginosa] Tyrannus tyrannus (L.)5 Uraeginthusbengalus bengalus (L.) [Estrildaphoenicotis] Volatiniajacarina ssp. Woodfordia superciliosa North Zenaidura macroura carolinensis(L.) Zonotrichia capensis matutina (Lichenstein) [Brachyspizacapensis matutinal Zonotrichia capensis ssp. [Brachyspiza capensis] Zonotrichia georgiana ssp. [Melospizageorgiana] Zonotrichia melodia melodia (Wilson) [Melospiza melodia melodia] Zonotrichia melodia ssp. [songsparrow] Zosterops flavifrons flavifrons (Gmelin) Zosteropsf. majuscula Murphy & Mathews Zosterops lateralis ssp. Zosterops 1. griseonota Gray Zosterops rennelliana Murphy “Indian eagle”

30 58, WHO/IRC WHO/IRC WHO/IRC WHO/IRC

63 38,43 9 44 76,17 48 H 44 7H 38,43 38,43 69 76 76 64,80 76 76,77 26 H

* (i) Host names have been checked with Peters (1931 et seq.) and, where this differs significantly, the nomenclature used by the author of the paper cited is given in square brackets. Where a subspecific determination is neither given nor self-evident from the context, “ssp.” (indicating “subspecies unknown”) follows the specific name without indication of authorship. (ii) Numbers refer to Table I, and “WHO/IRC” to material deposited in the World Health Organization’s International Reference Centre for Avian Malaria Parasites separately listed in more detail in Table 111. (iii) Records marked “H” refer, or are believed to refer, to adeleine haemogregarines. All others (and the great majority) refer to atoxoplasms, or organisms believed to be atoxoplasms. 1 Citing personal communication by Mayer. a Footnote by W. Noller on p. 32. 3 Citing unpublished work by D. D. Myers. 4 Probably misidentification of Plasmodium-see ref. 51. 5 Name checked in Cory and Hellmayr (1927).

TABLE I11

h, 00

Atoxoplasmmaterial(thin Giemsa-stained bloodfilms)availablefor study in the collections of the WorldHealth Organization’s International Reference Centrefor Avian Malaria Parasites (Department of Biology, Memorial Universityof Newfoundland,St. John’s, Newfoundland, Canada)

Systematic position APODIDAE Collocalia esculenta (L.) CHAMAEIDAE Ckamaeafasciata ssp. EMBERIZIDAE Pkeucticus melanocephalus spp. FRINGILLIDAE Carpodacus mexicanus ssp. Coccotkraustescoccotkraustes coccotkraustes (L.) Serinus canaria (L.) ICTElUDAE Euphagus carolinus nigraus Burleigh and Peters Molotkrus ater ater (Boddaert)

MIMIDAE Toxostoma redivioum rediviuum (Gambel)

Common name

WHO/IRC accession number

Country where collected

Date of collection

LI

? m

>

No. sampled for WHO/IRC up to July 1971

No. infected

55

2 1

2 2

*z

White-breasted Swiftlet

2680 2679)

Malaysia

-14161

Pallid Wren-Tit

1036

U.S.A.

15/8/37

1

Black-headed Grosbeak

379

U.S.A.

22/8/37

48

House Finch

1446 1382} 276

,lb n ? m

rn

U.S.A.

17/6/36

Hawfinch

1506 16922

Italy

26/11/61

1

Canary

16920

Madagascar

1/4/66

1

1

Rusty Blackbird

16660

Canada

14/7/70

10

1

1168

U.S.A.

21/5/37

55

1

U

3 r

Cowbird

2 0

U

California Thrasher

880

U.S.A.

6/8/37

14

1

MUSCICAPIDAE Muscicapa narcissina ssp. Pachycephala cinerea ssp. Rhipidurajavanica ssp.

Narcissus Flycatcher Mangrove Whistler Pied Fantail

4701 4846 4468

Malaysia Malaysia Malaysia

4471 PARIDAE Parus inornatus ssp. PARULIDAE Dendroica coronata auduboni (Townsend) PIl7-IDA.E Pitta brachyura ssp.

San Diego Titmouse Audubon’s Warbler Blue-winged Pitta

2017161 5/8/62 15/6/61 31/5/61

U.S.A.

1 1

6

4

2

U.S.A. 957

13 54

20112/37

47

3332 3333} Malaysia

(8i)

20

3334 Pitta megarhyncha ssp.

PLOCELDAE Passer fIaveolusssp. PYCNONOTIDAE Pycnonotus goiavier ssp. Pycnonotusjocosus ssp. Pycnonotus xanthopygos (Ehrenberg) STURNLDAE Acridotheres tristis tristis (L.)

TETRAONIDAE Lagopus lagopus ssp.

Greater Bluewinged pitta

Malaysia

15/6/60

0

3

Pegu Sparrow

12615

Thailand

27/7/67

122

Yellow-vented Bulbul Red-whiskered Bulbul Black-capped Bulbul

7875 12376

Malaysia Thailand

25/4/65 15/1/68

561 22

1 1

19449

Tanzania

21/11/70

30

1

Indian Mynah

16915 16916)

Madagascar

3

2

188

1

Willow Ptarmigan

8932

Canada

0

a

N \D

Systematic position TIMALIIDAE Minla strigula malayana (Hartert) Macronusptilosus ssp. Stachyris leucotis ssp. Stachyris nigricollis (Temminck) Trichostoma abbotti ssp. Trichostoma bicolor (Lesson) Trichostoma rostratum ssp.

TABLE I11 (continued) WHO/IRC Country accession where Common name number collected

w

0

Date of collection

No. sampled for WHO/IRC up to July 1971

No. infected

p m

Chestnut-tailed Siva

4313

Malaysia

2013162

Fluffy-backed Tit Babbler White-eared Tree Babbler Black-necked Tree Babbler Abbott’s Jungle Babbler Ferruginous Jungle Babbler Blyth’s Jungle Babbler

3866

Malaysia

3015162

32

3987

Malaysia

9/5/63

3

3732

Malaysia

2911163

19

3708

Malaysia

24/8/62

24

Malaysia

3577

34

16/2/61 Malaysia

1 119

3589 TURDIDAE Copsychussaularis ssp. Garrulax erythrocephalus ssp.

Magpie Robin Red-headed Laughing Thrush

5271 4087

Malaysia Malaysia

* Listed as “Lunkesteria” (in double infection with Plasmodium rouxi) by Laird (1962).

7/5/61 -/-/61

107 17

3

r >

The Metabolism of the Malaria Parasite and its Host ALEXANDER FLETCHER AND BRIANMAEGRAITH Department of Tropical Medicine, Liverpool School of Tropical Medicine, Liverpool, England I. The Metabolism of the Malaria Parasite ................................................... A. Introduction ................................................................................. B. General Considerations .................................................................. C. Pentose Phosphate Pathway Activity in Malaria-infected Erythrocytes ...... D. Further Metabolism of Glucose ......................................................... E. Carbon Dioxide Fixation by Malaria Parasites .................................... F. Aerobic Mechanisms in Mammalian Malaria Parasites ........................... G. The Metabolism of Chloroquine-resistant Malaria Parasites.. ................... H. Concluding Remarks ..................................................................... 11. The Metabolism of the Host During Infection ............................................. A. Biochemical Changes in Erythrocytes ................................................ B. Effects of Acute Infection on Host-tissue Metabolism ........................... C. Host Lipid Metabolism .................................................................. D. Concluding Comments ..................................................................

I. THE METABOLISM OF A.

THE

31 31 31 33 35 31 38 39 41 41 41 43

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MALARIA PARASITE

INTRODUCTION

This review is not intended as a comprehensive account of recent literature in this field. Instead, we shall discuss certain considerations which are necessary in metabolic investigations of Plasmodia and some current research trends which we think are advancing our understanding of the fundamental biochemical processes present in parasites and involved in the host-parasite relationship. The mode of action of drugs and mechanisms of drug resistance will be mentioned only when necessary, in order to present a logical account of the selected subject matter; these topics have recently been well reviewed by Peters (1970). B.

GENERAL CONSIDERATIONS

Metabolic studies of malaria parasites are usually performed either on infected blood, parasitized erythrocytes or on parasites freed from their host erythrocytes. When infected blood is examined biochemically, the tacit assumption is often made that any changes in the biochemical parameters being studied 31

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ALEXANDER FLETCHER A N D BRIAN MAEGRAITH

are due directly to the activities within parasitized erythrocytes. The contribution of the non-infected cells in the infected blood is generally assumed to alter very little from its pre-infection state. Some workers do make reservations regarding this assumption but others do not mentioned this point at all. It is becoming increasingly obvious that biochemical changes occur in uninfected cells as well as in their parasitized counterparts. This will be referred to in a later section. It must be borne in mind that such biochemical changes may not be in the same direction. As a result the apparent changes in parasitized cells may either be minimized or magnified, depending on the stage of parasite development and other related factors. Many workers have attempted to overcome such problems by attempting to isolate suspensions of parasitized erythrocytes. This is relatively easy when there is a synchronous development of asexual parasites, as shown by the pioneering work of Christophers and Fulton (1938), working with P.knowlesi. Other workers have extended their method of differential centrifugation by the use of gradients, sometimes employing polymers; an example of this approach is the albumin flotation method used by Fulton and Spooner (1956) to concentrate P. berghei-infected rat reticulocytes. Such methods employing large molecules or sucrose gradients (Williamson and Cover, 1966), while being suitable for the isolation of parasitized erythrocytes with the aim of isolating certain parasite fractions, may not necessarily be suitable for studying metabolic processes in the host-erythrocyte complex because of associated osmotic effects. The introduction of zonal centrifugation for the separation of marginally different populations of cells or cell organelles offered a new approach to the isolation of parasitized erythrocytes. Sucrose gradients have commonly been used for this procedure but because of possible osmotic effects, large molecules, such as synthetic polysaccharides, have to be considered. Using this technique and the synthetic polymer Ficoll (Pharmacia, A.G., Uppsala, Sweden) Ali and Fletcher (1971b) have recently described promising results for the concentration of P.knowlesi-infectedmonkey erythrocytes. Preliminary results with P.berghei-infected mouse erythrocytes are also promising. Leucocyte and platelet contamination in separated cell suspensions, a problem which should always be considered and the steps taken to eliminate them described, can be reduced to a minimum. There are several examples in the literature where this factor does not appear to have been satisfactorily controlled. This may distort the findings considerably. We consider that it is generally necessary to think of parasite metabolism as being so integrated with that of the host erythrocyte that the system must be studied as a metabolic complex; however, there are occasions when it is useful to study the parasite in isolation. Methods of isolation have mainly employed the use of haemolytic anti-serum and complement or surface-active agents such as saponin. Recent electron microscopic studies (Cook et al., 1969; Killby and Silverman, 1969) have revealed the possible hazards of these methods of isolation. Host-cell contamination of parasites is a frequent occurrence and can be a potential source of error when differences between parasite and host-cell biochemical and physio-chemical characteristics are being determined. The effects of these liberating agents may be far reaching

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and may even produce changes in those biochemical features of the host cell mentioned above, with the result that grossly misleading information may be obtained. Rupture of, or damage to the liberated parasites may make such preparations of questionablevalue in metabolic studies. It is therefore important that such possible consequences are considered when this kind of approach is necessitated. Thus it is clear that work with most types of cell preparation present their particular hazards. These should, however, not deter workers from choosing the preparation which they consider is the best means of giving them the information they require, but it must be emphasized that adequate steps should be taken to overcome the problems outlined. It is equally important to convince others that such steps have been taken.

C.

PENTOSE PHOSPHATE PATHWAY ACTIVITY IN MALARIA-INFECTED ERYTHROCYTES

Considerable attention has been focused on this metabolic pathway since it was suggested, by analogy with the sickle-cell gene, that genetically determined deficiency of human erythrocytic glucose-6-phosphate-dehydrogenase (G-6-PD), the initial enzyme of the pathway, could afford some degree of protection against infection with P. falciparum (Motulsky, 1960; Allison and Clyde, 1961). Results of field and hospital studies to investigate this hypothesis have been conflicting (see Gilles et al., 1967). Metabolic studies to investigate whether malaria parasites utilize this pathway appear to have also produced conflicting results. On theoretical grounds, it seemed probable that the malaria parasite could have an absolute requirement for the pentose phosphate pathway (PPP), even if considered only from the point of view that it is probably the principal, if not the only, pathway for the production of the pentose sugars necessary for nucleic acid synthesis. Bowman et al. (1961) used specifically labelled 1%-glucose to determine the extent of the participation of the PPP in the breakdown of glucose by P. berghei-infected mouse erythrocytes and by freed parasites. They came to the conclusion that this represented only a minor route of glucose utilization. Fletcher and Maegraith (1962) found that levels of G-6-PD and 6-phosphogluconate-dehydrogenase (6-PGD) in erythrocytes from monkeys infected with P. knowlesi rose with increasing parasitaemia, presumably reflecting increased PPP activity. Increases in the dehydrogenases were mainly restricted to the parasitized erythrocytes. They could find no dehydrogenase activity in parasites liberated by a variety of methods, suggesting that the parasite is dependent on the PPP of the host erythrocyte. A similar conclusion could be drawn from the radioactive studies of P. berghei by Bryant et al. (1964). Herman et al. (1966) used specifically labelled glucose in their studies on P. gallinaceum-infected chicken erythrocytes and found that PPP activity increased on erythrocyte infection. At the same time no activity could be detected in liberated parasites. Langer and his colleagues (1967) also studied the dehydrogenases of the

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ALEXANDER FLETCHER A N D B R I A N MAEGRAITH

PPP, and three pentose cycle enzymes, in P. berghei infections in mice; on the basis of electrophoretic and kinetic observations they suggested that P. berghei itself has an active pentose phosphate pathway. Their electrophoretic evidence is debatable because although they were able to show bands of G-6-PD activity with different mobilities from uninfected erythrocytes and from extracts of liberated P. berghei, it is significant that they were able to show only one band with infected erythrocytes with a mobility similar to that obtained from normal erythrocytes. Attempts in this laboratory to reproduce these electrophoretic findings in P. berghei and also P . knowlesi infections have so far been unsuccessful, only one zone of G-6-PD activity being demonstrable in each case. It could be argued that all the biochemical approaches mentioned above are open to some criticism,because, just as it is possible to get host-cell contamination of parasite preparations, it could be claimed that soluble enzymes (as G-6-PD is reputed to be) may be leached out of parasite preparations by washing procedures. In an effort to overcome some of these difficulties Theakston and Fletcher (1971a, b) adopted a different approach. They used a cytochemical procedure at the electron microscope level to determine the localization of G-6-PD activity in infected erythrocytes ( P . gallinaceum, P. berghei, P. knowlesi and P.fakiparum in Aotus). Activity was demonstrable only in the host erythrocyte, none being present in any parasite except in food vacuoles with recognizable host-cell contents. Here again, it could be suggested that diffusion of enzymes out of the parasite may have taken place or that the reaction components did not gain access into the parasite. It seems reasonable to suggestthat the incubation procedure necessary for demonstration of enzyme activity would maintain a metabolically viable system in almost a native state. Moreover, some demonstrable enzyme activity within food vacuoles indicates that parasite membranes were not impermeable to reaction components. Further indirect support for the above observations that malaria parasites are completely dependent on the G-6-PDYand possibly the 6-PGD, of the host erythrocyte is provided by the recent work of Luzzatto et al. (1969). They used a cytochemical method at the light microscope level to demonstrate that G-6PD-deficient human erythrocytes were rarely infected with P. falciparum while erythrocytes with a normal enzyme content in the same individual (female heterozygotes) were found to be frequently infected. This indicates clearly the dependence of the parasite on the G-6-PD of the host erythrocyte. The studies of Pollack et al. (1966) who investigated the effects of chemical agents which simulated the metabolic limitations of G-6-PD-deficient red cells in P . berghei malaria have some relevance in this respect. It would seem therefore that whereas field and hospital studies are always likely to be open to criticism, if only on statistical grounds, there is now a substantial amount of evidence to indicate that most, if not all, of the malaria parasites studied to date are dependent on the G-6-PD, and probably the PPP, of the host erythrocyte; parasites are unlikely to thrive in enzyme-deficient cells, at least not to the extent where they are able to produce an overwhelming infection.

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D. FURTHER METABOLISM OF GLUCOSE

Although the central role of glycolysis in the breakdown of glucose by plasmodia has been firmly established for many years, the nature of further metabolism beyond this metabolic sequence remains enigmatic, particularly in the case of the mammalian species of malaria parasites studied. Early work established that there was malonate inhibition of the aerobic oxidation of various substrates by avian species, indicating the presence of an active Krebs cycle, whereas no inhibition by malonate of glucose oxidation by P.knowlesi was observed (see Moulder, 1948). The following discussion therefore refers mainly to current trends of work with mammalian species. Recent work indicates that in P. knowlesi no homolactate breakdown of glucose occurs (Scheibel and Miller, 1969; Ali and Fletcher, 1971a). The preliminary report of the latter workers states that for each micromole of glucose utilized, one micromole of lactate accumulates and one microatom of oxygen is consumed. Pentose phosphate pathway activity could account for no more than 20 % of this oxygen uptake. This suggests that glycolytic intermediates are being diverted, perhaps to oxidative steps, or that there is further metabolism of pyruvate. Cenedella et al. (1969), working with P. berghei in rats, found that a significant amount of tritium from 6-labelled glucose was incorporated into parasite phospholipids exclusively via a-glycerol phosphate, no tritium label being found in the total fatty acids of the parasite. This suggests that triose phosphates are being utilized either via dihydroxy-acetone phosphate and glycerol-phosphate dehydrogenase activity, or via glyceraldehyde phosphate, and the action of glyceraldehyde kinase, glycerol dehydrogenase and glycerol kinase. Both sequences involve reductive steps using reduced nicotinamide-adenine-dinucleotide(NADH or NADPH), so that there is no possibility of oxidative mechanisms being involved. However, any surplus of glycerol phosphate formed by the second sequence, or otherwise, could be oxidized aerobically by glycerol-phosphate dehydrogenase to dihydroxyacetone-phosphate which could with glyceraldehyde-3-phosphate, then complete an aerobic glycerol cycle. Alternatively, a situation somewhat similar to that observed in some trypanosomes (Grant el al., 1961), may be present in malaria parasites. It is interesting to note that early work on the carbohydrate metabolism of several parasites showed that glycerol as substrate alone would maintain, and in the case of P. knowlesi apparently enhance, their oxygen uptake (see McKee, 1951). It cannot be ruled out that glyceraldehyde-3-phosphate dehydrogenase can also operate aerobically in parasites. Another explanation for the heterolactate degradation of glucose could be that 2,3-diphosphoglycerate, which is present in red cells in relatively high concentrations and is intimately concerned with haemoglobin in oxygen transport (Benesch and Benesch, 1969), accumulates in infected erythrocytes. No evidence for this was found in P.berghei-infected mouse erythrocytes (Ali, Fletcher and Maegraith, 1971). Usually, decreases in the amounts of this phosphate ester occurred during infection. Firm information on the further metabolism of pyruvate by mammalian

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parasites is still lacking, although more recent work is shedding new light on the possibilities. As already stated, the work of Cenedella et al. (1969) with P. berghei showed that there was no significant labelling of fatty acids from tritiated glucose, indicating that if acetyl-CoA is formed from pyruvate little, if any, is diverted towards de novo synthesis of fatty acids. Various workers have shown that there is little radioactive COZproduction by malaria parasites from 6-14C-glucoseindicating that there is insignificant Krebs cycle activity either in free parasites or infected erythrocytes. P.berghei and P. knowlesi particularly have been examined in this respect (Bowman et al., 1961; Scheibel and Miller, 1969; Ali and Fletcher, 1971a). It should however always be borne in mind that the possibility of dilution of radioactive intermediates between glucose and any oxidation in the Krebs cycle means that the yield of any radioactive COz from 6-14C-glucose is always likely to be relatively low compared with the evolution of labelled COz from l-14C-glucose in the pentose phosphate pathway. In this case only two or three enzymatic steps take place prior to the oxidative decarboxylation of 6-14C-giucose. Scheibel and Miller (1969) also used 3,4-14C-glucose in their studies on liberated P. knowlesi and showed some evolution of radioactive COz which indicates decarboxylation of pyruvate to a Cz-compound. This suggests the presence of pyruvic dehydrogenase activity, and the subsequent formation of acetyl-CoA. These workers did not consider that the addition of CoA increased the yield of l4COZin the above system although their figures could be interpreted as indicating the reverse. Much of the earlier work, particularly on mammalian parasites (see Moulder, 1948) showed that lactate as substrate maintained oxygen uptake at a level similar to that with glucose. The recent work by Ali and Fletcher (1971a) indicates that lactate and pyruvate will stimulate the endogenous respiration of P. knowlesi-infected erythrocytes, the respiration in the presence of lactate exceeding that of glucose. They also reported that malonate does not inhibit glucose-, lactate- or pyruvate-stimulated respiration. In these circumstances it is not easy to explain this respiration, if it is accepted that there is insignificant Krebs cycle activity, unless it is envisaged that there is a very active pyruvic dehydrogenase operating aerobically. There seems to be little possibility of oxidation of any lipid substances formed from acetyl-CoA and apart from a reversal of glycolysis leading to oxidation of glycerol phosphate, as outlined earlier, explanation of this stimulation of respiration by lactate is difficult. The only other possibility is that pyruvate undergoes transamination with glutamate to alanine and that alanine then undergoes oxidative deamination back to pyruvate, in a reaction analogous to that catalysed by glutamate dehydrogenase. Obviously, this would produce no net removal of pyruvate and is therefore unlikely to be a mechanism of any significance. The above account shows that, in the absence of a Krebs cycle, which is discussed in a later section, the respiration of mammalian malaria parasites is still inexplicable, although the possibility of an active and aerobic pyruvic dehydrogenase is currently under investigation in our laboratory. In this context it is worthwhile bearing in mind the work of Trager (1954, 1966) and Bennett and Trager (1967) on P. lophurae which indicated the dependence of

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this parasite, at least, on supplies of co-enzyme A, necessary for the above enzyme reaction, from the host erythrocyte. It is feasible that in mammalian parasites generally, supplies are limited and impose some restrictions on this reaction and any other which are co-enzyme A-dependent. E.

CARBON DIOXIDE FIXATION BY MALARIA PARASITES

The mechanism of C02 fixation by Cs-compoundsin certain micro-organisms and mammalian tissues is well established (see Wood and Utter, 1965). It is only relatively recently that this mechanism has been demonstrated in plasmodia, first in P. lophurae-infected duck erythrocytes (Ting and Sherman, 1966) and then in P.knowlesi-infected monkey red cells by Sherman and Ting (1968). Siu (1967) showed that C02 fixation occurred in liberated P. berghei and also demonstrated the presence of two of the enzymes involved, phosphoenolpyruvic carboxylase and carboxykinase in cell-free preparations. He showed that both enzymes could be inhibited by chloroquine and quinine. C02 fixation by liberated P. berghei and infected rat erythrocytes was also demonstrated by Nagarajan (1968a). The products of C02 fixation were shown in each case to be C4-dicarboxylic acids which are normally associated with the Krebs cycle. The studies of Ting and Sherman (1966) and Sherman and Ting (1968) on the kinetics of radioactive labelling of C4-compounds in P. lophurae- and P. berghei-infected erythrocytes respectively following incubation with 14C-bicarbonateindicated that oxaloacetic and a-ketoglutaric acids were the initial products of this reaction, with the amino acids, aspartate and glutamate appearing as stable secondary products. Their work also indicated that radioactivity appeared rapidly in other dicarboxylic and also tricarboxylic acids normally present in the Krebs cycle. The rapid labelling of these acids suggested to these workers that C02 fixation by the parasites was serving an anaplerotic function. Kornberg (1966) described anaplerotic sequences (from the Greek for “filling up”) as “routes ancilliary to the (central) cycles which must operate to maintain the levels of intermediates in these cycles”. This is necessary so that, when intermediates are tapped off from the central cycles to be built into anabolic products, the cycles will not be interrupted. The importance of this mechanism is not easily understood, particularly in mammalian parasites in which the majority of workers think that a functional Krebs cycle is unlikely to be present. In this respect it is interesting to note that the labelled amino acids, the major products of COz fixation in P.lophuraeinfected erythrocytes, were not incorporated into parasite protein. It is also worthwhile to note in all the studies mentioned above that the incorporation of radioactive C02 into either free parasites or infected erythrocytes amounted to about 1% of the added radioactive bicarbonate. In these studies it is relatively easy to explain the rapid labelling of aspartic acid by transamination of oxaloacetate, an initial product of C02 fixation. The rapid labelling also of a-ketoglutarate and glutamic acid is more difficult to explain. Either oxaloacetate is rapidly converted via citrate and iso-citrate to a-ketoglutarate in a conventional Krebs-cycle manner with subsequent

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transamination to glutamic acid or an as yet unknown COz-fixing mechanism gives rise to x-ketoglutarate directly. The first alternative would of course require the participation of acetyl co-enzyme A and citrate synthase. It is worthwhile noting that, in the studies using P. lophurae- and P. knowlesiinfected erythrocytes, significant amounts of labelled citrate and iso-citrate were reported to be found.

F. AEROBIC MECHANISMS I N MAMMALIAN MALARIA PARASITES

Many electron microscopic studies of the asexual stages of malaria parasites have shown that whereas the avian species studied all possess typical protozoan mitochondria with tubular cristae, the mammalian species do not appear to possess recognizable mitochondria, although trophozoites of P.falciparum in Aotus monkeys have structures which resemble mitochondria (Smith et al., 1969). Smith and Theakston (1970) in a short study of P. malariae-infected blood from two patients, observed structures which appeared to be typical protozoan mitochondria. Apart from these two exceptions, the ultrastructural studies agree with the biochemical evidence so far available on these species. For instance, the classical studies of Speck et al. (1946) showed quite clearly that P. gallinaceum fulfilled all the biochemical criteria necessary to show that it possesses a Krebs cycle. On the other hand, the work of Bowman et al. (1961) on P. berghei and more recently that of Scheibel and Miller (1969) and Ali and Fletcher (1971a) with P.knowlesi shows that these mammalian parasites do not possess a Krebs cycle as judged by the standard criteria. It is interesting to note that Ladda (1969) and Howells (1970) reported the presence of mitochondria in trophozoites of certain strains of P. berghei, which they considered to contain villus-like cristae. However, the latter worker found that these cristate mitochondria were rare and stated that such parasites usually possessed acristate mitochondria, unlike the erythrOcytic sexual and sporogonic stages of P.berghei in which typical protozoan-type mitochondria could be recognized. It may seem a little premature at this stage to consider that organelles without recognizable morphological features can be described as mitochondria, although the general hypothesis proposed by Howells (1970) of mitochondria1 metamorphosis during the life cycle of P. berghei is attractive. In this respect it is worthwhile to note that the electron cytochemical study of Theakston et al. (1969) showed that cytochrome oxidase activity in trophozoites of P.berghei were more frequently associated with the concentric-membraned organelle than with other membranes of the plasma, nuclear or food vacuole membranes or the “acristate structures”. Similar studies of NADH- and NADPH-dehydrogenases in the erythrocytic stages of P. berghei also showed that this “oxidative enzyme complex” was again mainly associated with the concentric membraned organelle. In neither case was enzyme activity particularly concentrated in the relatively structureless organelles termed “acristate mitochondria”. Moreover, Howells and his colleagues (1970) could not detect succinate dehydrogenase activity in normal drug-sensitive P. berghei trophozoites in contrast to chloroquine-resistant

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strains. These biochemical considerations,therefore, are another factor making it difficult to describe these organelles as mitochondria at this stage. Biochemical studies by Nagarajan (1968b) on blood from P . bergheiinfected rats, however, demonstrated progressive stimulation of oxygen uptake by free parasites by increasing concentrations of succinate and also inhibition of this stimulated uptake by malonate. This is in contrast to the findings of other workers already mentioned. The host cells in this infection were reticulocytes which can often present problems particularly with regard to contamination of free parasite preparations. It is interesting to note that this worker, like Howells et al. (1970) working with P. berghei-infected mouse erythrocytes, was unable to demonstrate succinate dehydrogenase activity in cell-free extracts of free parasites. The only demonstrable enzymes normally associated with the Krebs cycle were malate dehydrogenase and fumarase, two cycleenzymeswhich are found in mature human erythrocytes(see Prankerd, 1961). The presence of malate dehydrogenase has previously been demonstrated in extracts of P. berghei by Sherman (1966), who also reported that the electrophoreticand kinetic characteristics of the parasite and host-erythrocyte enzyme were different. It is still not possible, therefore, on the evidence available to date to get a clear picture of aerobic mechanisms in mammalian species. What is clear, however, is that if COZkation by these species takes place at a significantlevel it must be accepted at present that the Krebs-cycle sequence from oxalacetate to a-ketoglutarate through citrate does function, since a reversal of the steps of the conventional Krebs cycle sequence from malate to a-ketoglutarate via succinate is extremely unlikely, certainly on thermodynamic grounds. Moreover, co-enzyme A participates in one of the intermediate steps of the reverse sequence and may be limited in supply even for the metabolism of pyruvate, as mentioned in an earlier section. If the enzyme sequences normally associated with the Krebs cycle are restricted to a shuttle operating at the most to between malate and a-ketoglutarate, it is difficult to envisage a significant role for the cytochrome oxidase activity (Theakston et al., 1969; Scheibel and Miller, 1969) and NADH- and NADPH-dehydrogenases (Theakston et al., 1970), and co-enzyme Q (ubiquinone) system (Skelton et al., 1969) already demonstrated in mammalian parasites. The possibility remains that these are associated with aerobic mechanisms operating in the glycolytic sequence, further metabolism of pyruvate or side reactions of glycolysis, as discussed earlier. G . THE METABOLISM OF CHLOROQUINE-RESISTANT MALARIA PARASITES

To date, only a limited number of reports on the aerobic and carbohydrate metabolism of drug-resistant parasites have appeared. These have all been carried out on chloroquine-resistant strains of P. berghei in the mouse. Cho and Aviado (1968) used an oxygen-electrode system to compare the oxygen uptake of control mouse erythrocytes with those parasitized with drugsensitive P. berghei and a chloroquine-resistant strain. Comparison of the oxygen uptakes by the sensitive and resistant strains is difficult because of

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differences in the respective erythrocyte infection rates. A lack of data on reticulocyte levels in the two infections also makes the results difficult, if not impossible, to interpret. The latter is an important consideration since it is well established that a preference for immature red cells is a feature of chloroquine-resistant strains of P . berghei (see Peters, 1970). Preliminary investigations in our laboratory have attempted to take this factor into consideration. Table I compares the figures for the oxygen consumption of erythrocytes infected with a drug-sensitive and a drug-resistant strain of P . berghei with those for mouse blood in which an approximately equivalent level of reticulocytes to that occurring in the resistant strain had been induced by bleeding. TABLEI The oxygen consumption of mouse erythrocytes infected with chloroquine-sensitiveand chloroquine-resistantP. berghei, and with elevated reticulocyte levels (Boonyun and Fletcher, unpublished observations)

Cell suspension N strain P. berghei,

drug-semitive RC strain P. berghei, chloroquineresistant Reticulocyte-rich mouse blood

Erythrocyte infection rate (%) 72 60 66 67 0 0

Reticulocyte level (%)

Oxygen consumption (patoms/lOecells/h)

<2 <2

2.6 1.8

37 40 35

4.3 3.5 0.9

51

1.1

Leucocyte levels were reduced to c lOOO/mm3; reticulocyte levels were elevated by daily bleeding for 9-12 days.

Simple examination of the figures shows that the consumption of the resistant strain cannot be accounted for by the presence of a parasite with an oxygen consumption equivalent to that of the sensitive strain residing in a reticulocyte. It is an open question whether the resistant parasite can in some way stimulate the aerobic mechanisms of the host reticulocyte or whether there are additional aerobic mechanisms in the resistant parasite itself. The recent cytochemical evidence, at the light microscopic level, of Howells et al. (1970) would supported the latter alternative. They report the appearance of succinate dehydrogenase in the chloroquine-resistant parasite and suggest that resistance is acquired by the parasite’s ability to switch its respiratory pathway to a Krebs cycle. Succinate dehydrogenase activity was also demonstrated in reticulocytes, which these workers stated differed in distribution from that observed in reticulocytes infected with the resistant parasites. Such a switch to a tricarboxylic acid cycle by resistant parasites is an attractive theory and further support for it is claimed by the demonstration of iso-citrate lyase activity in extracts of chloroquine-resistant P. berghei by Howells and Homewood (1971). Whether the possession of these two enzymes necessarily indicates a functional Krebs cycle, or merely mechanisms normally associated with this cycle, is still a matter for conjecture. Convincing cytochemical

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localization of succinate dehydrogenase activity at the electron microscope level would be of value in clarifying this situation, but efforts in this laboratory and by Howells (personal communication) have so far proved unsatisfactory. It is worthwhile noting that Howells et al. (1969) in their electron microscopic studies could not detect any significant differences in cytochrome oxidase activity between a drug-sensitive and a chloroquine-resistant strain of P. berghei. Comparative studies such as those described above are obviously a fruitful area for investigation and it is to be hoped that clarification of the apparently fundamental differences in metabolism can be achieved. Such knowledge would obviously be of great value in our understanding of the phenomenon of drug resistance, and even of the action of drugs in normal parasites. H.

CONCLUDING COMMENTS

It is now more generally appreciated that there are probably certain fundamental differences in metabolism between avian and mammalian species of malaria parasite. This means that only certain analogies can be made, and with some reservations. For instance, qualitative differences in metabolism are likely to be accompanied by quantitative differences in similar metabolic pathways used by the respective parasites. It is also worth remembering that when certain biochemical pathways are envisaged in the parasite for the metabolism of various substrates, it is usual, but useful, to think only in terms of known pathways elucidated in other biological situations. It is possible that there are as yet unknown metabolic pathways or reactions operating in malaria parasites, and perhaps in parasitized erythrocytes. Progress in this field of biochemical studies is likely to be relatively slow because of the many specific complications present, for example the influences of the host cell and its environment. Despite these difficulties, however, progress is being made and it is hoped that the momentum can be maintained. OF THE HOSTDURING INFECTION 11. THEMETABOLISM A.

BIOCHEMICAL CHANGES IN ERYTHROCYTES

Overman (1948) first showed convincingly that significant ionic changes occur in non-parasitized erythrocytes as well as parasitized ones during acute P . knowlesi infections in monkeys. These findings have recently been extended, again using P. kriowlesi infections, by the studies of Dunn (1969a, b), who showed that cellular sodium increased with parasitaemia and that equivalent decreases in cellular potassium occurred. Similar findings were obtained in P . falciparum, P . coatneyi and P . berghei infections in the chimpanzee, rhesus monkey and hamster respectively. Dunn further demonstrated that the ouabain-sensitive sodium outflux rate was significantly depressed during infection. The main component of this active transport (pumping) of sodium

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out of the erythrocyte is ATP-dependent (Hoffman, 1966). Dunn’s results also show that the defect is quite clearly not just restricted to parasitized erythrocytes but that changes of a similar magnitude occur in non-parasitized cells as well. He concluded that the parasite or the infected host may produce a circulating toxin which affects erythrocytic membrane cation transport, whereas Overman et al. (1949) considered that changes in adrenal cortical hormones were responsible. Dunn (1969b) also showed in a single P . coatneyi infection of a rhesus monkey that the increase in red cell sodium was accompanied by an increase in the erythrocytic ATP level. Both increases persisted for about seven days after eradication of parasites with chloroquine therapy, again showing that the defects in cation membrane transport are not restricted to parasitized erythrocytes during infection. Fletcher et al. (1970) studied adenosine phosphate levels in erythrocytes during P . knowlesi infections. They found that, as well as fluctuations in ATP levels which reflected mainly changes in infected erythrocytes due to the metabolic activities of the parasites, appreciable increases occurred in the nonparasitized cells. These findings complement those of Dunn (1969b) and indicate that changes similar to those reported by him in a P. coatneyi infection occur in P . knowlesi infections. Brewer and Coan (1969) examined the erythrocyte ATP levels during P. berghei and P . vinckei infections of rats. Although they found mainly decreases in ATP levels during infection, they considered that uninfected cells must also be affected to explain the magnitude of the changes they found. Another compound concerned in maintaining the structural and functional integrity of erythrocytes is reduced glutathione, and its levels in P. berghei and P . knowlesi infections were examined by Fletcher and Maegraith (1970). On the basis of their results they concluded that a reduction of this substance may contribute to lysis of parasitized cells in P. knowlesi infections but not in P. berghei malaria. They did not, however, consider that any significant changes in the reduced glutathione levels of non-parasitized erythrocytes occurred. This substance, therefore, does not appear to be involved in the lysis of uninfected cells, unlike the defect in cation transport. It may be relevant in relation to erythrocyte membrane changes during malaria that Angus et al. (1971d) reported results which indicate that changes in the lipid content of non-parasitized erythrocytes occur in P . knowlesi infections of rhesus monkeys, although not to the same extent as those occurring in parasitized cells. It is also interesting to note that Seed and Krier (1969), on the basis of their studies of P. gallinaceurn infections of chickens, suggest that membrane lipids of parasitized erythrocytes become exposed during infection and with lipid material from red-cell destruction adsorbed by non-parasitized cells may act as antigenic sites during autoimmune lysis of erythrocytes during infection. The above studies draw attention to the fact that biochemical changes are likely to occur in all cells of the erythrocyte population during malaria infection and are therefore advancing our understanding of the associated lytic processes. The possibility of such changes, therefore, should never be discounted or ignored when biochemical studies of parasitized blood are being made.

M A L A R I A : M E T A B O L I S M OF T H E H O S T B.

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EFFECTS OF ACUTE INFECTION O N HOST-TISSUE METABOLISM

Further clarification of the factors described by Maegraith (1968) which produce these biochemical changes and associated cell damage has been made by a recent series of papers by the Liverpool group of workers. The factors inhibiting mitochondrial oxidative processes in vitro, which can be detected quite readily in the blood of P. knowlesi-infected rhesus monkeys and P. berghei-infected mice, can be resolved into several components by ultrafiltration. The ultrafiltrable components have been found by Thurnham et al. (1971b) to be small molecules, with molecular weights of probably less than 1000. Lipids, polysaccharides and large polypeptides are not involved. A proportion of this inhibitory activity appears to be due to weakly anionic compounds. Further studies by Thurnham et al. (1971~)on the ultrafiltrable components used Sephadex gel columns to separate the activity. A large portion of the inhibitory activity was associated with lactic acid. This compound is increased in the blood of infected animals but was excluded as a cause of inhibition. Thurnham et al. (1971a) also showed that although serum bilirubin levels were increased in both infections, they were not increased sufficiently to inhibit in vitro mitochondrial respiration and oxidative phosphorylation, unlike the results produced by Zetterstrom and Ernster (1956) when using high levels of non-conjugated bilirubin and rat-liver mitochondria to investigate the role of bilirubin in the pathogenesis of kernicterus in infants. Non-esterified fatty acids (NEFA), shown by Angus et al. (1971b) to be increased in the plasma of P. knowlesi-infected monkeys, were also investigated as substances capable of producing in vitro inhibition of mitochondrial metabolism. These acids are normally bound at certain sites on plasma albumin (Fredrickson and Gordon, 1958) and in this form are incapable of causing any inhibition. Addition of CIS-fatty acids to serum from control rhesus monkeys did not produce any inhibition whereas their addition to serum from infected animals increased the level of inhibition ; this suggests that few binding sites on the albumin in serum from infected animals were still available. Furthermore, it was shown that NEFA do not appear in ultrafiltrates of serum from infected animals. These findings indicate that NEFA are not involved in the in vitro inhibition of mitochondrial oxidative processes, although they could play a contributory role when they are discharged into the liver cell. Maskrey et al. (1971) estimated that from 30% upwards of the inhibitory activity of serum from P. knowlesi-infected monkeys is non-ultrafiltrable. The possibility of these inhibitory substances being bound to plasma protein is unlikely because such binding would probably result in a loss of their inhibitory activities, as in the case of NEFA. Electron microscopy has been carried out on liver and kidney slices from control mice incubated with serum from P. berghei-infected mice and P. knowlesi-infected monkeys (Theakston el al., 1971). General cellular damage was usually visible and considerable mitochondria1 damage is often an early feature. Combined cytochemical studies to demonstrate the location of certain oxidative enzymes showed that these are released from mitochondria,

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A L E X A N D E R FLETCHER A N D B R I A N MAEGRAITH

probably as a result of damage to the mitochondria1 membranes. Acid phosphatase, normally restricted to the lysosomes of cells, also appears to be released into the cell cytoplasm, suggesting that the lysosomal membrane may be similarly affected. This could result in cellular autolysis. Such changes are being correlated with the effects of serum on in vivo mitochondrial oxidative metabolism. It is interesting to note that the effects on mitochondria of intact cells appear as marked as those on the isolated organelles. C.

HOST LIPID METABOLISM

Fatty degeneration of the liver in acute malaria is a well documented pathological change (Maegraith, 1948, 1954; Mercado and von Brand, 1957; Ray, 1958), but its genesis has not been well understood. The recent findings of Angus et al. (1971a, b) shed much light on the processes involved. These workers studied the serum lipid changes in acute P . knowlesi malaria and found significant increases in all lipid components, including the NEFA. Increases in NEFA are believed to arise from stimulation of adipose tissue lipase as a result of the sympathetic hyperactivity known to occur in acute P. knowlesi malaria (Skirrow et al., 1964). Although there is a post-hepatic hyperlipaemia caused by the increased discharge of lipids from the liver as lipoproteins, the variable increase in liver lipids (Angus et al., 1971c) mainly reflects the inability of the liver to handle the increased influx of NEFA. The damage to the oxidative metabolism of the liver-cell mitochondria (Maegraith et al., 1962) probably results in impaired oxidation of excess fatty acids and/or an inability of lipoprotein synthesis to keep pace with the increased lipid influx as a result of diminishing energy supplies. Lipids released by the lysis of parasitized and non-parasitized erythrocytes may also contribute to the overall lipid changes, depending on the severity of the lytic process. There could also be some inhibition of lipoprotein lipase at the adipose tissue face which would maintain the post-hepatic hyperlipaemia even though lipoprotein secretion by the liver was diminishing as the infection progressed. D. CONCLUDING COMMENTS

Although biochemical studies, such as those described above, are enabling some advances to be made in our understanding of the pathological processes involved in malaria, more progress, as in most fields of medicine, can be made by a multidisciplinary approach. This is illustrated to some extent by Maegraith and Fletcher in the next review of this volume dealing with the pathogenesis of mammalian malaria.

REFERENCES: I AND I1 Ali, S. N., Fletcher, K. A. and Maegraith, B. G . (1971). 2 : 3 Diphosphoglyceratein P. berghei-infected blood. Trans. R . SOC.trop. Men. Hyg. 65, 8. Ali, S. N. and Fletcher, K. A. (1971a). Further studies on the carbohydrate metabolism of malaria parasites. Trans. R. SOC.trop. Med. Hyg. 65, 419.

REFERENCES

45

Ali, S. N. and Fletcher, K. A. (1971b). Zonal centrifugation as a technique for the separation of malaria-infected erythrocytes. Trans. R. SOC. trop. Med. Hyg. 65,4. Allison, A. C. and Clyde, D. F. (1961). Malaria in African children with deficient erythrocyte glucose-6-phosphate dehydrogenase. Br. med. J. i, 1346. Angus, M. G. N., Fletcher, K. A. and Maegraith, B. G . (1971a). Studies on the lipids of Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta). I. Changes in serum lipids. Ann. trop. Med. Parasit. 65, 135. Angus, M. G. N., Fletcher, K. A. and Maegraith, B. G. (1971b). Studies on the lipids of Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta). 11. Changes in serum non-esterified fatty acids. Ann. trop. Med. Parasit. 65, 155.

Angus, M. G. N., Fletcher, K. A. and Maegraith, B. G . (1971~).Studies on the lipids of Plasmodium knowlesi-infectedrhesus monkeys (Macaca mulatta). 111.Changes in liver lipids. Ann. trop. Med. Parasit. 65, 419. Angus, M. G. N., Fletcher, K. A. and Maegraith, B. G . (1971d). Studies on the lipids of Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta). 1V. Changes in erythrocyte lipids. Ann. trop. Med. Parasit. 65, 429. Benesch, R. E. and Benesch, R. (1969). Intracellular organic phosphates as regulators of oxygen release by haemoglobin. Nature, Lond. 221,618. Bennett, T. P. and Trager, W. (1967). Pantothenic acid metabolism during Avian malaria infection: Pantothenate kinase activity in duck erythrocytes and in Plasmodium lophurae. J . Protozool. 14 (l), 214. Bowman, I. B. R., Grant, P. T., Kermack, W. 0. and Ogston, D. (1961). The metabolism of Plasmodium berghei, the malaria parasite of rodents. Biochem. J . 78, 472. Brewer, G. J. and Coan, C. C. (1969). Interaction of red cell ATP levels and malaria and the treatment of malaria with hyperoxia. Milit. Med. 134 (Special Issue). 1056. Bryant, C., Voller, A. and Smith, K. J. H. (1964). The incorporation of radioactivity from W-glucose into the soluble metabolic intermediates of malaria parasites. Am. J. trop. Med. Hyg. 13,515. Cenedella, R. J., Jarrell, J. J. and Saxe, L. H. (1969). Lipid synthesis in vitro from 1-1%-oleic acid and 6-3H-glucose by intraerythrocytic Plasmodium berghei. Milit. Med. 134 (Special Issue), 1045. Cho, W. C. and Aviado, D. M. (1968). Pathogenic physiology and chemotherapy of Plasmodium berghei. IV. Influence of chloroquine on oxygen uptake of red blood cells infected with sensitive or resistant strains. Exp. Parasit. 23, 143. Christophers, S. R. and Fulton, J. D. (1938). Observations on the respiratory metabolism of malaria parasites and trypanosomes. Ann. trop. Med. Parasit. 32, 43. Cook, R.T., Aikawa, M., Rock, R. C., Little, W. and Sprinz, H. (1969). The isolation and fractionation of Plasmodium knowlesi. Milit. Med. 134 (Special Issue), 866. Dunn, M. J. (1969a). Alterations of red blood cell sodium transport during malarial infection. J. clin. Invest. 48, 674. Dunn, M. J. (1969b). Alterations of red blood cell metabolism in simian malaria: Evidence for abnormalities of nonparasitized cells. Milit. Med. 134 (Special Issue), 1100. Fletcher, K. A. and Maegraith, B. G . (1962). Glucose-6-phosphate and 6-phosphogluconate dehydrogenase activities in erythrocytes of monkeys infected with Plasmodium knowlesi. Nature, Lond. 196, 1316. Fletcher, K. A. and Maegraith, B. G. (1970). Erythrocyte reduced glutathione in malaria (Plasmodium berghei and P. knowlesi). Ann. trop. Med. Parasit. 64,48.

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Fletcher, K. A,, Fielding, C. M. and Maegraith, B. G. (1970). Studies on the role of adenosine phosphates in erythrocytes of Plasmodium knowlesi-infectedmonkeys. Ann. trop. Med. Parasit. 64,487. Fredrickson, D. S. and Gordon, R. S. (1958). Transport of fatty acids. Physiol. Rev. 38, 585. Fulton, J. D. and Spooner, D. F. (1956). The in vitro respiratory metabolism of erythrocytic forms of Plasmodium berghei. Expl Parasit. 5, 59. Gilles, H. M., Fletcher, K. A., Hendrickse, R. G.,Lindner, R., Reddy, S. and Allan, N. (1967). Glucose-6-phosphate dehydrogenase deficiency, sickling and malaria in African children in South Western Nigeria. Lancet i, 138. Grant, P. T., Sargent, J. R. and Ryley, J. F. (1961). Respiratory systems in the trypanosomidae. Biochem. J. 81,200. Herman, Y.F., Ward, R. A. and Herman, R. (1966). Stimulation of the utilization of l-14C-glucosein chicken red blood cells infected with Plasmodium gallinaceum Am. J. trop. Med. Hyg. 15,276. Hoffman, J. F. (1966). The red cell membrane and the transport of sodium and potassium. Am. J. Med. 41, 666. Howells, R. E., Peters, W. and Fullard, J. (1969). Cytochrome oxidase activity in a normal and some drug resistant strains of Plasmodium berghei-a cytochemical study. 1. Asexual erythrocytic stages. Milit. Med. 134 (Special Issue), 893. Howells, R. E., Peters, W., Homewood, C. A., Warhurst, D. C. (1970). Theory for the mechanism of chloroquine resistance in rodent malaria. Nature, Lond. 228. 625. Howells, R. E. (1970). Mitochondria1 changes during the life cycle of Plasmodium berghei. Ann. trop. Med. Parasit. 64, 181. Howells, R. E. and Homewood, C. A. (1971). Modifications of the respiratory enzymes associated with chloroquine resistance in P. berghei. Trans. R. SOC. trop. Med. Hyg. 65, 10. Killby, V. A. A. and Silverman, P. H. (1969). Isolated erythrocytic forms of Plasmodium berghei. An electron-microscopical study. Am. J. trop. Med. Hyg. 18, 836. Kornberg, H. L. (1966). Anaplerotic sequences and their role in metabolism. In “Essays in Biochemistry” (Eds. P. N. Campbell and G .D. Greville), Vol. 2, p. 1 . Academic Press, London. Ladda, R. L. (1969). New insights into the fine structure of rodent malarial parasites. Milit. Med. 134 (Special Issue), 825. Langer, B. W. Jr., Phisphumividhi, P. and Friendlander, Y.(1967). Malaria parasite metabolism: The pentose cycle in Plasmodium berghei. Expl Parasit. 20, 68. Luzzato, L., Usanga, E. A. and Reddy, S. (1969). Glucose-6-phosphate dehydrogenase deficient red cells : Resistance to infection by malarial parasites. Science 164,839. Maegraith, B. G . (1948). “Pathological Processes in Malaria and Blackwater Fever”, Blackwell, Oxford. Maegraith, B. G. (1954). Some physiological and pathological processes in Plasmodium berghei infections in white rats. Indian J. Malaria 8,281. Maegraith, B. G . (1968). Liver involvement in acute mammalian malaria with special reference to Plasmodiumknowlesi malaria. In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 6, p. 189. Maegraith, B. G . , Riley, M. V. and Deegan, T. (1962). Changes in the metabolism of liver mitochondria of monkeys infected with Plasmodium knowlesi and their importance in the pathogenesis of malaria. Ann. trop. Med. Parasit. 56,483.

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Maskrey, P., Fletcher, K. A. and Maegraith, B. G. (1971). Non-ultrafiltrable inhibitory substances in serum from P . knowlesi-infected monkeys. Trans. R. SOC. trop. Med. Hyg. 65 (in the press). McKee, R. W. (1951). Biochemistry of Plasmodium and the influence of antimalarials. In “Biochemistry and Physiology of Protozoa”, Vol. 1. Academic Press Inc., New York. Mercado, T. I. and von Brand, T. (1957). The influence of some steroids on glucogenesis i n the liver of rats infected with Plasmodium berghei. Am. J. Hyg. 66, 20. Motulsky, A. G. (1960). Metabolic polymorphisms and the role of infectious diseases in human evolution. Hum. Biol. 32,28. Moulder, J. W. (1948). The metabolism of malarial parasites, Ann. Rev.of Microbiol. 2, 101. Nagarajan, K. (1968a). Metabolism of Plasmodium berghei. 111. Carbon dioxide fixation and role of pyruvate and dicarboxylic acids. Expl Parasit. 22, 33. Nagarajan, K. (1968b). Metabolism of Plasmodium berghei. I. Krebs cycle. Expl Parasit. 22, 19. Overman, R. R. (1948). Reversible cellular permeability alterations in disease. In vivo studies on sodium, potassium and chloride concentrations in erythrocytes of the malarious monkey. Am. J. Physiol. 152, 113. Overman, R. R., Bass, A. C., Davis, A. K. and Golden, A. (1949). The effect of lipo-adrenal extract on ionic balance in fatal simian malaria. Am. J. clin. Pathol. 19, 907. Peters, W. (1970). In “Chemotherapy and Drug Resistance in Malaria”. Academic Press, London. Pollack, S., George, J. N. and Crosby, W. H. (1966). Effects of agents simulating the abnormalities of the glucosed-phosphate dehydrogenase-deficient red cell on Plasmodium berghei malaria. Nature, Lond. 210, 33. Prankerd, T. A. J. (1961). “The Red Cell”. Blackwell, Oxford. Ray, A. P. (1958). Experimental studies on liver injury in malaria. Part I: Pathogenesis. Indian J. med. Res. 46, 359. Scheibel, L. W. and Miller, J. (1969). Glycolytic and cytochrome oxidase activity in plasmodia. Mifit.Med. 134 (Special Issue), 1074. Seed, T. M. and Kreier, J. P. (1969). Autoimmune reactions in chickens with Plasmodium gallinuceum infection : The isolation and characterization of a lipid from trypsinized erythrocytes which reacts with serum from acutely infected chickens. Milit. Med. 134 (Special Issue), 1220. Sherman, I. W. (1966). Malic dehydrogenase heterogeneity in malaria (Plasmodium lophurae and P. berghei). J. Protozool. 13 (2), 344. Sherman, I. W. and Ting, I. P. (1968). Carbon dioxide fixation in malaria. 11. Plasmodium knowlesi (monkey malaria). Comp. Biochem. Physiol. 24, 639. Siu, P. M. L. (1967). Carbon dioxide fixation in Plasmodia and the effect of some antimalarial drugs on the enzyme. Comp. Biochem.Physiol. 23,785. Skelton, F. S., Lunan, K. D., Folkers, K., Schnell, J. V., Siddiqui, W. A. and Geiman, Q. M. (1969). Biosynthesis of Ubiquinones by Malarial Parasites. I. Isolation of [14C] Ubiquinones from cultures of rhesus monkey blood infected with Plasmodium knowlesi. Biochemistry 8, 1284. Skirrow, M. B., Chongsuphajaisiddhi, T. and Maegraith, B. G. (1964). The circulation of malaria. 11. Portal angiography in monkeys (Macaca mulatta) infected with Plasmodium knowlesi and in shock following manipulation of the gut. Ann. trop. Med. Parusit. 58, 502.

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Smith, D. H., Theakston, R. D. G. and Moore, G . A. (1969). The ultrastructure of Plasmodium falciparum in splenectomized Aotus trivirgatus monkeys. Ann. trop Med. Parasit. 63,433. Smith, D. H. and Theakston, R. D. G . (1970). Comments on the ultrastructure of human erythrocytes infected with Plasmodium malariae. Ann. trop. Med. Parasit. 64, 329. Speck, J. F., Moulder, J. W. and Evans, E. A. Jr. (1946). The biochemistry of the malaria parasite. V. Mechanism of pyruvate exidation in the malaria parasite. J. Biol. Chem. 164, 119. Theakston, R. D. G. and Fletcher, K. A. (1971a). An electron cytochemical study of glucosed-phosphate dehydrogenase activity in malaria-infected erythrocytes. Trans. R. SOC. trop. Med. Hyg. 65,4. Theakston, R. D. G . and Fletcher, K. A. (1971b). An electron cytochemical study of glucose-6-phosphate dehydrogenase activity in erythrocytes of malaria-infected mice, monkeys and chickens. Life Sci. 10,701. Theakston, R. D. G., Fletcher, K. A. and Maegraith, B. G . (1970). Ultrastructural localisation of NADH- and NADPH-dehydrogenases in the erythrocytic stages of the rodent malaria parasite, Plasmodium berghei. Life Sci. 9,421. Theakston, R. D. G . , Fletcher, K. A., Maskrey, P. and Maegraith, B. G . (1971). Ultrastructural studies of the effects of malarial toxic factors on normal mouse tissues. Trans. R . SOC. trop. Med. Hyg. 65, 3. Theakston, R. D. G., Howells, R. E., Fletcher, K. A., Peters, W., Fullard, J. and Moore, G . A. (1 969). The ultrastructural distribution of cytochrome oxidase activity in Plasmodium berghei and P. gallinaceum. Life Sci. 8, 521-529. Thurnham, D. I., Fletcher, K. A. and Maegraith, B. G. (1971a). The inhibition of mitochondrial respiration and oxidative phosphorylation by serum from malariainfected animals. I. The contribution of bilirubin and non-esterified fatty acids to the inhibitory activity in Plasmodium knowlesi and P. berghei malaria. Ann. trop. Med. Parasit. 65,275. Thurnham, D. I., Fletcher, K. A. and Maegraith, B. G . (1971b). The inhibition of mitochondrial respiration and oxidative phosphorylation by serum from malariainfected animals. 11. The inhibitory activity of serum ultrafiltrates from Plasmodium knowlesi-infected monkeys. Ann. trop. Med. Parasit. 65, 287. Thurnham, D. I., Fletcher, K. A. and Maegraith, B. G . (1971~).The inhibition of mitochondria1respiration and oxidative phosphorylation by serum from malariainfected animals. 111. Sephadex separation of inhibitory serum ultrafiltrates from Plasmodium knowlesi-infected monkeys and P. berghei-infected mice. Ann. trop. Med. Parasit. 65,297. Ting, I. P. and Sherman, I. W. (1966). Carbon dioxide fixation in malaria. I. Kinetic studies in Plasmodium lophurae. Comp. Biochem. Physiol. 19, 855. Trager, W. (1954). Coenzyme A and the malaria parasite Plasmodium lophurae. J. Protozool. 1, 231. Trager, W. (1966). Coenzyme A and the antimalarial action in vitro of antipantothenate against Plasmodium lophurae, P. coatneyi and P. falciparum. Trans. N. Y. Acad. Sci. Ser. 11,28, 1094. Williamson, J. and Cover, B. (1966). Separation of blood-cell-freetrypanosomes and malaria parasites on a sucrose gradient. Trans. R. SOC. trop. Med. Hyg. 60,425. Wood, H. G . and Utter, M. F. (1965). The role of COz fixation in metabolism. In “Essays in Biochemistry” (Eds. P. N. Campbell and G . D. Greville) Vol. 1, p. 1. Academic Press, London. Zetterstrom, R. and Ernster, L. (1956). Bilirubin, an uncoupler of oxidative phosphorylation in isolated mitochondria. Nature, Lond. 178, 1335.

The Pathogenesis of Mammalian Malaria BRIAN MAECRAITH AND ALEXANDER FLETCHER

Department of Tropical Medicine, Liverpool School of Tropical Medicine, Liverpool, England

I. Introduction .................................................................................... 11. Local and General Responses in Malaria Similar to those which Occur in Inflammation.. ............................................................................... 111. Changes in Endothelial Permeability in Malaria ....................................... A, Autoradiography ........................................................................ B. Fluorescence. ............................................................................. IV. Vasomotor Changes in Malaria ............................................................ A. Hepatic Circulation in Malaria ...................................................... B. Renal Circulation.. ...................................................................... C. Intestinal Circulation .................................................................. V. The Kinin Complex and Other Pharmacologically Active Agents. ................. VI. Intravascular Coagulation in Malaria ................................................... VII. Anoxic Anoxia ................................................................................. VIII. Cytotoxic Factors: Mitochondria1 Respiration Inhibitors ........................... IX. The Chain Reaction ........................................................................... References.......................................................................................

49 51 51 56 57 58 59 59 59

60 67 68 70 71 72

I. INTRODUCTION “Increase in endothelial permeability to protein with associated loss of fluid, stasis of red cells and diapedesis of red cells through the vessel walls ... are ... characteristic of acute inflammation resulting from infective agents or trauma. . . In the former case the phenomena are primarily induced by the presence of the agent and its products, which in certain instances have been found to include polypeptides specifically active in increasing endothelial permeability (Chain and Duthie, 1939). “The circulatory phenomena of malaria are in many ways so similar to those of acute general inflammation (Cannon, 1941) that it would not be surprising to find similar substances were produced during malaria infections and acted as the factors initiating the vascular damage. “The vascular changes of malaria are generalized, although they may be manifested differently in the various organs, depending on the anatomical and physiological type of the microcirculation present. There is ample evidence of damage to the vascular endothelium and indirect evidence in some 49

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organs of local and sometimes general fluid loss from the vessels, resulting in retardation or obstruction to blood flow from stasis. In organs such as the brain, in which the vessels are normally relatively impermeable to protein, these latter factors predominate. In others, such as the liver, where the vessels are normally relatively permeable and free escape of protein and fluid to the tissues is physiological, they are not much in evidence. “The vascular phenomena of malaria are, however, so widespread through the tissues that some kind of common initiating factor must be postulated . . . The identification of some substance in malaria capable of initiating generalized leakage of fluid from the blood vessels would make the whole concept of the disease much more intelligible. “. . .Tissue damage appears to arise in the final analysis from anoxia due to a combination of local and general circulatory disturbances, generalized anoxaemia arising from changes in erythrocytes and their destruction and histotoxic effects of metabolites and possibly ‘toxins’ . . . the initiating factor in this complicated chain of events. , . may be some non-specific substance, like polypeptides of inflammation, which exerts its effect upon vascular endothelium probably by producing a state of histotoxic anoxia and allows local escape of protein and fluid leading to stasis in some organs and ultimately to generalized loss of blood volume and circulatory derangements.” The above is quoted from “Pathological Processes” (Maegraith, 1948). After an interval of twenty-three years, it can be said that the views expressed have proved to be substantially correct. The hypothesis that much of the physiopathological pattern of malaria is essentially inflammatory and nonspecific in nature has been examined experimentally over this period, in which work has been concentrated largely on (i) the effects of malaria infection on the vascular membranes and vasomotor mechanisms of the host, and (ii) the links between the parasite in its erythrocyte environment and the host, which may initiate and maintain the pathophysiological changes in the host and associated changes in the parasites. Recent work has shown that, as anticipated, many of the pathophysiological processes of acute malaria are primarily “inflammatory” in nature. The evidence indicates that this is a common background to processes developing in many other acute medical states, for example, the synthesis of renal and hepatic functional and structural lesions (Maegraith and Desowitz, 1967). In mammalian malaria the Liverpool work has gone some way towards determining the manner in which some of the processes are initiated, by demonstrating chemical messengers in the serum which are of considerable metabolic significance and which may be capable of initiating and maintaining many of the processes involved. The work has been slow since many of the techniques and, indeed, many of the active substances concerned were not developed or understood when it was begun as a study of anuria in British West Africa during World War I1 (Maegraith, 1940-1941 ; Maegraith and Findlay, 1944). We will here examine two principal features: (a) the pathophysiology of malaria as an “inflammation”, and (b) the possible initiating and maintaining factors involved.

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RESPONSES IN MALARIA SIMILAR 11. LOCALAND GENERAL THOSE WHICH OCCURIN INFLAMMATION

TO

In local inflammation produced by trauma or by infection certain changes of significance are found near the periphery of the lesion where the tissue is not completely necrosed. Here the major phenomenon is increased permeability of the vascular endothelium, particularly in the very small blood vessels. This increased permeability results in the passage of protein through the normally impermeable vascular endothelium and with this protein passes water. The result is a viscous concentration of the fluid of the blood within the vessels concerned giving rise to local inflammatory “stasis”. This may be associated with obstruction to blood flow through the vessel and accumulation of excess fluid in the tissue surrounding the vessel, leading to oedema. In addition there is some escape of leucocytes and diapedesis of erythrocytes through the vascular endothelium. In general inflammation following bacterial infection similar changes in endothelial permeability may occur, the effects depending on the function and properties of the specific endothelial lining membrane involved. The whole picture is modified also by dynamic vasomotor responses, local and general, leading to vasodilatation in one area and vasoconstriction in another, depending on the vessels involved. General medical shock may result, in which some visceral vessels dilate and others constrict. The variation between these extremes represents Franklin’s so-called “order of sacrifice”, by means of which the blood flow through certain tissues is restricted, so increasing the volume of venous blood available for return to the heart. Organs involved in this vasoconstrictive sacrifice include the liver and the kidney. These phenomena are demonstrable in bacterial infections. There is ample evidence that they are also involved in protozoal infections, especially in acute malaria (and babesiasis). 111. CHANGES IN ENDOTHELIAL PERMEABILITY IN MALARIA

It is well to note at this point that the blood vessels of an organ or a tissue are specific to that organ or tissue and vary greatly amongst themselves in their potential of dynamic dilatation and constriction and in the functional permeability of the endothelium. In some organs, including the liver, the endothelial linings are in general highly permeable. In others, as in the brain, the endothelium of most of the vessels is normally very impermeable, allowing the passage of water and small molecules only. This is a generalization which does not apply to all areas of the brain, since in the normal animal some vessel walls are sufficientlypermeable to allow a limited amount of protein to pass through, so providing the normal protein content of the cerebrospinal fluid. Maegraith (1967) pointed out the importance of distinguishing cause and effect in pathological patterns and processes. In commenting on the basic cause of obstruction to cerebral circulation in falciparum malaria and on the hoary discussion about the role of the parasitized cells which sometimes

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appear to block the vessels, he says: “If a man is found at the bottom of a lift shaft with his neck broken I would accept the cause of death as a broken neck but I would also try to find out who has pushed him down”. This should be the line of thought in studying any pathological lesion. In 1948 Maegraith reviewed the then current descriptions of cerebral changes observed at autopsy in malaria particularly in relation to the blood vessels. There has been general agreement from the earliest observations to the present time that the cerebral circulation (cerebral being used in the sense of the circulation through the brain) is impeded in some way. In descriptions of brains in fatal cases of falciparum malaria it has been commonly pointed out that in patients who have not been treated the vessels are often “packed” with parasitized erythrocytes in which the parasites are in the same stage of the life cycle, usually late schizogony. Most pathologists have drawn the obvious conclusions that this collection (“plug”) of parasitized cells is responsible for the interruption of blood flow. It will be demonstrated later that this obstruction is, in fact, post hoc rather then propter hoc and that the processes involved in slowing and possibly stopping the circulation through these vessels are, in fact, not primarily dependent on the presence of the parasites per se but on the inflammatory processes mentioned above. Intravascular coagulation has often been blamed for the obstruction of the cerebral circulation in falciparum malaria. In fact clotting has rarely been demonstrated and there is very little evidence that coagulation thrombosis or embolus, play significant roles in producing the vascular lesions (as distinct from being associated with them). When they occur they represent developments following and not preceding the reduction of blood flow. The same is probably true of Knisely’s “sludge” (Knisely et al., 1941). A better explanation of the slowing down or the stoppage of cerebral circulation is the production of inflammatory stasis in the same manner as it occurs in an inflammatory lesion. The obstruction of the vessels by “plugged” infected erythrocytes or by clotting or embolus would represent permanent interference with local blood flow. In view of the absence of collaterals in the cerebral circulation it is difficult to see how such obstruction could be rapidly removed. Nevertheless, on administration of an anti-malarial drug the comatose patient usually recovers quickly if he is going to recover at all, often in a matter of a few hours, indicating that the obstructed or almost obstructed vessels must somehow reopen. Such rapid reversibility of the obstruction would be very unlikely in vessels which were completely blocked mechanically by the parasitized erythrocytes or by coagulum (which is seldom visible, in any case). The so-called “plugging” of the small cerebral vessels by parasitized erythrocytes continues to be the common pathological pattern described at autopsy findings in fatal P.falciparum cases. It has been perhaps not unfairly described as an idea more fixed in the mind of the pathologist than in the brain of the patient. The alternative view, that the plugging follows the pathological obstruction to blood flow and does not cause it, is well substantiated.

THE PATHOGENESIS O F MAMMALIAN MALARIA

53

In the comatose falciparum patient treated with chloroquine or quinine (or with cortisone), the release from coma (if it is to occur) normally takes only a few hours, i.e. long before the drugs have any obvious effect on the parasites. Recovery from coma can therefore be presumed to be physiological and dependent on restoration of the cerebral blood flow. It is difficult to see how this would be achieved other than by some pharmacological action exerted by the cortisone or the antimalarial drugs (both of which have strong antiinflammatory activity). If the patient remains comatose and dies, say, the following day, it is usual to find that parasites have mostly disappeared from the blood and few are to be found in the brain vessels, in which, nevertheless, the circulation is presumably slowed. In the falciparum case in which “plugged” vessels are visible the parasites are usually in the same stage of development, almost always in the schizont phase. This suggests that a large number of the erythrocytes in the “plug” have been infected with merozoites which had escaped from a parasite undergoing schizogony in the vicinity. If this parasite were packed in with masses of other erythrocytes in stasis, most of the merozoites extruded would find and infect erythrocytes. The parasites would develop in the static cells so long as the stasis continued and, at any one time, would all be in the same phase of life-cycle. This a quite different picture from the usual events in the circulating blood in which relatively few merozoites find a new erythrocyte home. Alternatively, the blocked vessel could have locally concentrated or filtered out infected erythrocytes to the exclusion of the non-infected and with no regard to the degree of parasitization of the circulating blood. This is extremely unlikely. An interesting point in support of this post hoc view of local infection of stased erythrocytes is the fact that the erythrocytes which are found surrounding damaged vessels or malarial “granulomata” and which have escaped by diapedesis, are seldom heavily parasitized (even when the relevant vessel is apparently “plugged” with parasites) and when they are, they may contain parasites at any stage of the life-cycle, even when the plugged vessel is full of schizonts. If stasis occurs, it results from loss of protein and accompanying water. There is a local concentration of the blood plasma and the circulating erythrocytes which coalesce into a more or less homogeneous mass. These masses are easily seen in P. knowlesi infection, in which “plugging” with large numbers of infected erythrocytes is very uncommon. The same is true in P. berghei infection where “plugging” is also rare. We have examined the function of the endothelial membranes of the brain in the laboratory in P. knowlesi and P . berghei infections. Using these infections as models, we have been able to show that excessive movements of protein and of water do occur across the membranes during infection. We have also been able to extract from the blood of infected animals active substances which can produce this effect themselves in vitro and in vivo. The membranes of the brain of Macaca mulatra are essentially the same as those of the human brain. They include the vascular endothelium of the small blood vessels of the brain substance and cortex, the choroid plexus and the walls of the lateral ventricles. This complex (the blood brain barrier) is made

54

B R I A N MAEGRAITH A N D A L E X A N D E R FLETCHER

up of several kinds of membrane which are basically resistant to the passage of heavy molecules, except in certain areas where more permeable membranes allow the passage of a limited quantity of protein molecules which account for the protein content of the cerebrospinal fluid. The circulation is completed by return of the fluid over the arachnoid villi to the blood. Migasena and Maegraith (1967) studied the movement of albumin during P. knowlesi infection (1) from the blood into the cerebrospinal fluid and (2) from the cerebrospinal fluid into the blood, using radioactive human serum albumin tagged with Iodine-131 (R.H.S.A. 1311). The normal flow was estimated both ways in normal Macaca mulatta and in the same animal at various stages of P. knowlesi infection. Very considerable changes in permeability were observed during the last 24 h of the infection, when the animal was clinically ill and sometimes semicomatose. The net movement of albumin was increased. We interpret this as an indication of increased permeability of the membranes which allowed the passage of protein into the cerebrospinal fluid and also out of it back into the bloodstream. This change of permeability is, of course, very similar to that which occurs in the endothelium of normally impermeable vessels in inflamed tissue. In the infected monkey the movement of the tagged protein from blood to C.S.F. was somewhere between five and ten times as great as in the normal

s ,,,6 -

O'I-f

D

D

.$

.-

'lo

P

@ -a *'-/

Mean of 8 normal

Mean of 3 Infected f~----~.of+blood

Dose injected into blood Iml II3' R.H.S.A. IOOpc

Dose injected into cisterna 0 . 5 ml II3l R.H.S.A. 5Opc

.4-

0

Time after odministrotion of isotoDe-hours

FIG.1. The movement of R.H.S.A. 1311 from blood into C.S.F. and vice versa in normal and P. knowlesi-infectedmonkeys.

Five hours after injection into the C.S.F. the concentration of the protein in the plasma was about the same in the normal and in the infected animals. Measurements of plasma concentrations were therefore made at shorter intervals after injection of the marker into the C.S.F., i.e. at 10,20,30,60 min and thereafter hourly. The results showed that transference from C.S.F. into

THE PATHOGENESIS OF MAMMALIAN MALARIA

55

the blood was very much faster in the infected animal than in the normal, i.e. about ten times in the first 10 min, the rate slowing until by the end of an hour it was only about twice as fast. By 5 h the transfer rate was about the same as in the normal animal.

I-

Dose injected into c i s t e r n 0 0 . 5 m l I ~ ~ ~ R . H . S50pC .A.

M794 2 0 m g ChloroquineQ Ih before isotope MI103 2 0 m g Chloroquine MI103 2 0 m g Hydrocortisone

111

I

10 30

2 0 m g Hydrocortisone

MI131

60

I

90

I

120

lo

Ih before

@ Ih before isotope

I

180

Time after odministrotion of isotope -minutes

FIG.2. The effect of chloroquine and chloroquine with hydrocortisone on the movement of R.H.S.A. passing from cerebrospinal fluid into blood in P . knowhi-infected monkeys.

Thus in Plasmodium knowlesi infection in M . mulatta the tagged albumin moves faster from the blood to the cerebrospinal fluid and faster from the C.S.F. to the blood. The result is a small increase in the protein concentration of the C.S.F. but this is limited by the speed of the return of the albumin from the C.S.F. back into the blood. It is interesting to compare this with meningitis in which the rate of protein escape from the meningeal vessels into the C.S.F. is accelerated, but not the speed of return via the villi, with the result that the protein content of the C.S.F. increases. If the demonstrated escape of protein across the blood brain barrier in P.knowlesi malaria were due to reduction in impermeability of the membranes as a result of an inflammatory process, then the giving of drugs which have an anti-inflammatory effect should eventually restore normal flow. We examined the effect of anti-inflammatory drugs on the movement of albumin across the blood brain barrier in P. knowlesi malaria in both directions, i.e. from the blood to the C.S.F. and from the C.S.F. to the blood. Chloroquine, mepacrine and hydrocortisone rapidly reduced the excessive flow of protein in both directions, indicating restoration of the blood brain 4

56

B R l A N M A E G R A I T H A N D A L E X A N D E R FLETCHER

barrier resulting from the anti-inflammatory activity of the drugs, which would finally end in the resolution of stasis. Thus anti-malarial drugs act in P.knowdesi infection by restoring the cerebral circulation as a result of anti-inflammatory activity. This has nothing to do with their anti-parasitic activity and must be clearly separated from it (Maegraith, 1969). The importance of this in the clinical field is illustrated by the work recently done in S.E. Asia in which a considerable acceleration has been demonstrated in the clinical effect (as distinct from the anti-parasitic effect) of Sulphometoxine in the treatment of chloroquine-resistant P. fulcipurum infection (Harinasuta; McKelvey : personal communications, 1970). Tracing the distribution of albumin tagged with I3lI by direct radio-isotopic measurement is a complicated procedure and limited in application for a particular animal. We therefore used other methods to demonstrate the escape of protein across the cerebral vascular endothelium, choroid plexus, ventricular walls and other parts of the brain. In these experiments we examined the situation in both P . knowlesi and P. berghei infections, using autoradiography and fluorescence techniques. We were also able to measure the movement of water across the membrane by using a water soluble dye. A.

AUTORADIOGRAPHY

Using R.H.S.A. 1311 we examined the distribution of the isotope in normal and infected animals in and around vessels in the brain tissue and in the

FIG.3. Autoradiograph of a brain section taken from a monkey in an advanced stage of

P. knowlesi infection. The animal was killed 5 h after intravenous administration of the radioactive albumin. Moderate radioactivity is seen in the choroid plexus, the ependyma of the ventricle and around the blood vessel wall. Diffused dark spots of radioactivity are seen in brain tissue in the area prostema.

THE PATHOGENESIS OF MAMMALIAN MALARIA

57

cerebrospinal fluid. The results indicated the movement of the protein across the blood brain barrier. This was particularly evident in the prostema, a region of the brain in which the vessels are normally sinusoidal and where it is believed some protein escape occurs under normal conditions. In this area in the normal animal there was very little distribution of isotope visible in the autoradiographic slides but in the infected animal there were masses of particles visible in the brain substance. This indicated a substantial escape of protein from the vessels. B.

FLUORESCENCE

Fluorescin isothiocyanate labelled with Human albumin (F.I.T.C.) was injected into the carotid artery. Five hours later the animal was killed. Frozen sections of the brain were examined by fluorescent microscopy without fixation. The results clearly indicated the passage of albumin through the membranes of the small vessels, the vessels of the meninges, and clumping of the fluorescent material in the walls and outside the walls of the small vessels and in the choroid arachnoid plexus and the walls of the ventricles. This movement was very much greater in the infected than in the normal animal.

FIG.4. The cerebral blood capillaries in the normal rhesus monkey. Note the relatively insignificant agglutination of fluorescent material within the capillaries.

It was to be expected that water would move with the protein and, to test this, disulphine blue was used. Because this is a soluble dye its transfer was regarded as indicating the movement of water. When the dye was introduced into the blood of mice, blueing of the brain substance occurred in animals

58

THE PATHOGENESIS OF MAMMALIAN MALARIA

infected withP. berghei but not in normal animals. Some of the blueing reniained even after washing. This movement of water was confirmed by measuring the nitrogen per nig of brain substance. It was found that oedema was substantial. The general picture was also seen in subsequent work in which kallikrein extracted from the blood of infected animals was injected intracerebrally (see p. 65).

FIG.5. The cerebral blood capillaries in the terminal stage of P. knawlesi infection in a rhesus monkey. (A) Large clumps of fluorescent material within the capillaries and (B) clumps lie along and outside the vessel. I

The evidence thus showed that in Plasmodium knowlesi albumin moves abnormally fast across the membranes of the blood brain barrier indicating that the membranes have temporarily become abnormally permeable to protein and that in P . berghei infections water also moves across the membrane with the large molecules.

1V. VASOMOTOR CHANGES IN MALARIA There is no need to go into details for the evidence of shock in clinical P. ,fakiparum or P. knowlesi malaria. This is now well known (Maegraith, 1948). Skirrow and his colleagues (1964) showed quite clearly that in P . knowfesi malaria in the late stages the sympathetic nervous system becomes

T H E P A T H O G E N E S I S OF M A M M A L I A N M A L A R I A

59

hyperactive. This accounts for most of the vascular phenomena which can be observed locally, for example in the liver, the kidney and the intestines and generally, in the failure of return of blood to the venous side of the heart and the development of shock. This hyperactivity of the sympathetic nervous system was not unexpected as it was indicated by the work of Ray and Sharma (1958) who showed that the characteristic centrilobular lesions in the liver could be prevented by total sympathectomy before infection of M . mulatta with P. knowlesi. Vasomotor changes occurring in malaria have been studied in Liverpool in the hepatic, renal and intestinal circulations. A.

HEPATIC CIRCULATION IN MALARIA

In the hepatic area we have shown that in the last two days of P. knonjesi infection there occurs a steady and continuing constriction of the small vessels of the portal venous tree. This results in reduction of blood flow through the liver with a consequent rise in portal pressure which in turn may lead to increase in size of the spleen (Andrews et al., 1949; Maegraith et a/., 1949; Skirrow et al., 1964). The constriction of the portal venous tree was unexpected, as we had anticipated that the major constriction would occur in the hepatic venous tree. The constriction could be relieved immediately by the administration of phenoxybenzamine (Dibenzyline). This drug was extremely effective in restoring the liver circulation not only in malaria but also in various forms of shock initiated by such methods as handling of the viscera under anaesthetic. The fact that the adrenolytic drug would relieve the situation offered a ready explanation of the results obtained by Ray and Sharma (1958) following sympathectomy (Skirrow er a/., 1964; Chongsuphajaisiddhi, 1966). B.

RENAL CIRCULATION

In the kidney we were able to demonstrate constriction of the renal arteries and arterioles which developed during the last 48 h of P. Itnowfesi infection particularly in animals in shock. The vascular constriction within the kidney could be relieved by phenoxybenzamine. C.

INTESTINAL CIRCULATION

The very striking changes in local circulatory flow in the liver and kidney were paralleled in the micro-circulation of the small intestine. The absorption of 14C-tagged cc-amino isobutyric acid was greatly restricted in P.knowlesi infection. The same was true of D-xylose. In both cases phenoxybenzamine restored the absorption curve towards normal (Migasena and Maegraith, 1969). In order to determine the meaning of this acute reduction of intestinal absorption we examined the effect of pitressin and phenoxybenzamine on the absorption curve of the amino acid and of xylose in normal animals. It was

60

BRIAN MAEGRAITH A N D ALEXANDER FLETCHER

FIG.6. Relief of extrahepatic and intrahepatic portal constriction during acute malaria (P. knowlesi infection in M. mulatta) following adrenergic blockade with Dibenzyline. (Courtesy Dr. M. Skirrow.) Left: before, right : 15 min. after injection of Dibenzyline.

found that, as suspected, the administration of pitressin, an active vasoconstrictor, reduced and altered the absorption curves and that normal curves could be drawn for the animals given pitressin if phenoxybenzamine were subsequently administered. The explanation of this effect of pitressin on absorption is probably that the depression resulted from vasoconstriction induced by the pitressin of the small vessels which lie just beneath the epithelial surface and behave somewhat like the glomeruli of the kidney (Migasena and Maegraith, 1969). The overall stimulation of the visceral sympathetic nervous complex demonstrated in P. knowlesi infection and in shock would presumably lead to the vasoconstriction in the areas described, although local changes may occur without general vascular failure (Maegraith, 1948, 1966, 1968).

v.

THEKININCOMPLEX

A N D OTHER PHARMACOLOGICALLY

ACTIVE

AGENTS The changes in membrane permeability and some of the circulatory disturbances noted above could be induced by physiologically active peptides such as kinins. We therefore over the last decade have examined the role of such peptides in malaria. We have followed aspects of the standard system in which the kininogens formed by the liver are converted by kininogenases into kinins, and the kinins in turn are converted into inactive peptides by kininases. The kininogens studied have been bradykininogen and kallidinogen; these are converted respectively into bradykinin and kallidin which have identical pharmacological activity. Using established pharmacological techniques and improved extraction

THE PATHOGENESIS O F M A M M A L I A N M A L A R l A

61

methods, we have examined in particular the kinin-complex at all stages in P. knoizYesi infection in M. rnu/uttu (Tella, 1962; Tella and Maegraith, 1963, 1966a, b; Onabanjo and Maegraith, 1970a, 1971). First attempts to demonstrate increased quantities of kinin in the blood in acute P. knowlrsi infection were not successful. However, it was demonstrated that on the fourth or fifth day of the infection a dramatic fall in bradykininogen occurred, the levels remaining low thereafter, until death (Tella and Maegraith, 1966a). A similar fall was later demonstrated in kallidinogen content at the same stage of infection (Maegraith and Tella, 1968). 0-0

X----

t

Bradykininogen X Parasitaernia

Days after infection

no infection

FIG.7. Bradykininogen levels in normal rhesus monkeys ( M . tnuluffa) and in monkeys infected with P. knowlesi. (i) Note the rapid fall in bradykininogen or bradykinin potential levels after day 3 of the infection. Each point represents the estimate on one monkey. (ii) Note that the fall in the bradykininogen level occurs over the same period as the rapid increase in parasitaemia.

This fall of concentration of the kinin precursors, which was a constant feature of the middle stages of the infection, could result from failure ofproduction, probably in the liver or from breakdown of the kininogens by kininogenases producing active kinins. Sudden failure of production is unlikely in view of the otherwise normal hepatic synthesis proceeding at the time, as instanced by the stable albumin production. We therefore examined the blood of animals in which the kininogen levels had fallen sharply with a view to detecting the expected increase in kinin content parallel to the fall in kininogen. Using the standard crude methods requiring very large volumes of blood, we were unable to detect any difference in kinin content in the infected and in the normal animals (Onabanjo et a/.,

62

B R I A N MAEGRAITH A N D ALEXANDER FLETCHER

1970). New methods had to be developed using kininase inhibitors and requiring as little as I n d of plasma (Onabanjo, 1970). With these techniques we have been able to demonstrate a considerable rise in kinin in the second half of the clinical infection, following the dramatic fall in kininogen (Onabanjo and Maegraith, 1971). At about the same time the urinary kinins are considerably increased (Tella and Maegraith, 1963). At the same time as the fall of kininogen (and the rise of kininogenases) there is a very considerable increase in the quantity of circulating kininases; these enzymes in some animals increase three to fourfold (Onabanjo el al., 1970). This means that the kinins liberated from the kininogen by the activity of the kininogenases are themselves rapidly rendered inert by the kininases. It was because this reaction continued in the blood during its relatively slow removal from the subject that the earlier attempts to demonstrate increase in kinin content in the blood of infected animals failed. The rapid production and destruction of kinins in malaria indicates a quick turn-over of the active peptides in the circulating blood. Unfortunately, it does not indicate what is happening in the peripheral tissues, where the changes in endothelial permeability are occurring. We will have to wait for better techniques before we can determine this. In the meantime, it is interesting to speculate on whether the kinin produced from the kininogen by the kininogenase remains long enough in the peripheral tissue circulation to exert an effect before it is itself destroyed by the kininases. It is possible that kinin may be able to exert an effect in such circumstances, even though its half life may be a matter of only milliseconds. This concept of very fast reactions, perhaps series of reactions, seems to be somewhat new in pharmacological thinking, but it is helpful at the present stage when we are trying to translate the more static situation in the serum into what is happening at the tissue face. The rise in kinin level during the late stages of P. knowlesi infection has a parallel in trypanosomiasis. Goodwin and Richards (1 960) demonstrated pharmacologically active kinins in the urine of mice infected with T. brucei, with Babesia rodhaini and with P . bergliei. Boreham (1 968a) subsequently reported massive release of kinin in the second peak of parasitaemia in T. brucei infection in cattle at the time when fluorescent antibodies first appeared. Boreham (1970) has also reported kinin release in human trypanosomiasis. Goodwin (1 970) noted a similar release of kinins about the eighth day of acute T. brucei infection in rabbits. Boreham (1 968b) has suggested that in trypanosomiasis, the parasites may in some way activate the precursors of the kininogenases which act on the kininogens to release kinins and that this activation follows an antigenantibody reaction. He noted that, as in malaria, the blood kininogen level fell in acute T. brucei infection and that treatment of the infection by Berenil was followed by a rise of kininogen concentration to above normal. These observations indicate a rapid turn-over of kinins in acute trypanosomiasis similar to that occurring in malaria. The effect of treatment on kininogen level in P. knowlesi infection has not yet been ascertained. At the moment, there is no information regarding a possible link between the production of antibodies

THE PATHOGENESIS OF MAMMALIAN MALARIA

63

in acute P. knowlesi malaria and the precipitous fall of kininogen (and associated rise in kinins and kininogenases) which occur about the fifth day of the overt blood-transmitted infection. This is obviously a useful field for further investigation. The evidence in P . knowlesi malaria is that the balance of kinin to kininase in the later stages of the infection allows the circulation of active kinin at above normal levels. It can be presumed, therefore, that these peptides exert their activity on endothelial membranes despite their rapid destruction by the increase in kininases. We have at the moment no means of determining the real significance of the increase in serum kinin in malaria, as against its potential significance. This will remain uncertain until new techniques make it possible for us to study the situation in the peripheral circulation. There is, however, evidence of another source of peptide pharmacological activity, namely the big increase in serum kininogenases, since these enzymes themselves are as active as the kinins in causing increase in endothelial permeability and, in certain circumstances, vasodilatation and constriction. Onabanjo and Maegraith (1 970a, b, c) and Onabanjo ef al. (1 970) examined the active peptides and relevant peptidases present in protein fractions obtained by column chromatography from serum of normal M . mulutta and of M . muluttu infected with P . knowlesi (in the last 2 4 4 8 h of the infection). Peaks 1-3 of the chromatograms from both normal and infected animals contained the y-globulins. Peak 4, which was present in normal and infected serum, contained mixtures of slow-moving y-globulins and (3-globulins; peak 4a, present only in the infected animals, contained similar proteins. Peak 5, present in both normal and infected animals, contained albumin, with a1 and a2 globulins. Kallikrein was eluted from fractions 1-3 in both normal and infected animals. This kininogenase was absent from fractions 4 and 5 in normal animals, but was present in fraction 4a (not found in normal animals) and sometimes in fraction 5. As expected, bradykininogen was present in fraction 5 in normal animals but was absent from the corresponding fraction in the infected monkeys. This latter finding was in keeping with that of Tella and Maegraith (1966a) who observed a sharp fall in the kininogen after the fifth day of the overt infection, and later showed that the behaviour of kallidinogen runs parallel to that of bradykininogen in P. k n o i i h i infection (Maegraith and Tella, 1968). The kallikrein content of the protein fractions was estimated by incubating them with freshly prepared plasma edate stable substrate (PESS), followed by assay on the isolated rat uterus (Onabanjo and Maegraith, 1970a). Using this method, it was demonstrated that there was a mean total increase of 4.5 pg bradykinin equivalents per pg protein (about 30% increase) in the blood of the P. knowlesi infected animals as compared with normal animals. The kallikrein fractions obtained in this way increased capillary permeability on intradermal injection into guinea-pigs and rabbits. These effects were blocked by protense inhibitors but unaffected by histaminases. The fractions also caused local lymphocytic infiltration at the injection sites. The

64

BRIAN MAEGRAITH A N D ALEXANDER FLETCHER

Tube number

FIG.8. Chromatograph of normal monkey ( M . mdutta) serum on a DEAE-anion exchange cellulose column. Protein fractions 1-5 eluted.

Tube number

FIG.9. Chromatography of serum from a P. knowlesi-infected monkey on DEAE-cellulose. Note protein fraction 4a which is present only in infected monkeys.

fractions from the infected animals were more active pharmacologically than were those from normal animals. Kallikrein from fraction 4a, which was present only in the infected animals, was very active in initiating the inflammatory responses. The kallikrein fractions from normal and infected animals produced hypotension when injected intravenously into rabbits. Thus the kallikrein fractions behaved pharmacologically in much the same way as kinins.

THE P A T H O G E N L S I S O F M A M M A L I A N M A L A R I A

65

The significance of the observed increase of kininogenases in the blood in the late stages of P . knorrlesi infection lies in the possibility that the enzymes could behave as active pharmacological substances in viw in the infected animals and cause the increased permeability of endothelium demonstrated in the blood-brain barrier by Migasena and Maegraith (1967). To test this possibility the effects of intracerebral injections of samples of kallikrein, extracted from blood protein fractions as described above, were studied in mice and guinea-pigs (Onabanjo and Maegraith, 1970b, c). Intracerebral injection of kallikrein extracts from fractions 1,2 and 3 caused blueing of the brain substance and some dilatation of the small vessels of the cortex and the meningeal vessels with some leucocytic perivascular infiltration. The effect was much more marked with extracts from blood of infected animals, and with fraction 4a, which was not present in normal animals. The changes in the brains induced by the kallikrein fractions closely resembled those induced in P . knowVesi and P . berghei infections, as described by Migasena and Maegraith (1967). The changes in malaria followed “inflammatory” increase in vascular permeability, with escape of protein and water, and were reversible by antiinflammatory drugs such as cortisone and chloroquine. It was concluded that the lesions induced by kallikrein were induced by similar changes in vascular permeability, caused by the peptide enzyme and demonstrable by intradermal injection into guinea pigs (Onabanjo and Maegraith, 1970a). Kallikrein may act directly or indirectly through the production of kinins from the precursors. In the presence of kininogen inhibitors when kinin is not produced, kallikreins are still active. A reasonable interpretation of the experimental observations noted above is that both kinins and kallikrein act as non-specific inflammatory agents in P. knowlesi malaria, causing increase in the permeability of endothelial membranes which are normally relatively impermeable (as in the brain), with associated progressive loss of large molecules including albumin and other proteins, together with water, from the blood circulating in the local blood vessels. This escape of fluid leads eventually to inflammatory “stasis” and obstruction of local circulatjon. That this view may be correct is indicated by the reversal of the protein and water loss from the brain vessels in P . knowlesi and P . berghei infections in the presence of strong anti-inflammatory drugs such as cortisone, chloroquine and quinine (Migasena and Maegraith, 1967). It will be noted that there is substantial evidence that in malaria and trypanosomiasis the kinin complex plays a significant role in the disturbances which develop in permeable membranes, and is thus concerned with the physiological change and ultimate strutucral patterns that result. It is probable that other factors are involved. Desowitz and Pavanand (1967) have, for instance, described an uncategorized permeability factor in the serum of gibbons infected with P . coatneyi. This could well be related to the peptides under discussion. Other substances may also be concerned with these processes, amongst them histamine, which exhibits very similar activity on permeable membranes and is also a powerful vasodilator (Spector, 1951). It is, moreover, known

66

B R I A N M A E G R A I T H A N D A L E X A N D E R FLETCHER

to be liberated by antigen-antibody reactions. Little study seems to have been made on this subject in protozoal infections. Richards (1965) demonstrated an increase in histamine in the blood of mice infected with T. brucei but this was not confirmed by Yates (1970) who could find no increase in histamine content or in the enzyme which forms it in the blood of mice infected with T. brucei. On the other hand, Maegraith and Onabanjo (1970) have reported an increase in histamine concentration in the blood of M . muluttu in the late stages of P . knowlesi infection. Clearly, more research is needed to define the role of histamine in these acute protozoal infections. If an increase can be established, it may well be that histamine will join the group of pharmacologically active substances which take part in the chain reactions of the host. It will be interesting to see if this is the case and whether any increase in histamine that occurs is the result of enhanced production or of depression of circulating histaminases. In normal blood, histamine is rapidly destroyed by histaminases. Billings and Maegraith (1938), for instance, showed that large doses of histamine injected intravenously into rabbits disappeared completely in 20 min. If the rise in histamine content in malaria is confirmed, therefore, it could indicate enormous overproduction or, more likely, a fall in the histaminase activity. The same is true of adenosine and other derivatives of adenylic acid with vasomotor activity which have been demonstrated in blood returning from ischaemic, hyperaemic or inflamed tissues. Billings and Maegraith (1938) demonstrated a rise in concentration of these substances in venous blood returning from an ischaemic hind limb within a few hours of tying the femoral artery. It is inviting to speculate that adenosine and similar compounds may increase and be consequently active in malaria in the tissues where there are circulatory disturbances, especially reduction in blood flow. However, Billings and Maegraith also demonstrated that adenosine is removed very rapidly from the blood by enzymes and the same argument applies as in considering the histamine of the circulating blood. It is clear therefore that any study of adenosine should be paralleled with studies on the enzymes that destroy it. Maegraith ef al. (1956) demonstrated a dramatic temporary recovery from shock in P . knowlesi infected M . mulatta following intravenous injection of nor-adrenaline. They later reported similar recovery in shocked puppies infected with Bubesiu cunis (Maegraith et a/., 1957). They believed that the recovery from shock was due to redistribution of blood which had been pooled peripherally as the result of the vasomotor response of the host to pharmacologically active soluble substances derived from the parasites or host-parasite reactions. It seemed reasonable to assume that the kinin complex might be involved in these reactions, as was subsequently proved to be the case. Goodwin (1970) has pointed out that the activity of nor-adrenaline under these circumstances could equally indicate that catecholamine metabolism might have been upset in the shocked animals. If this is so, it may well be a further example of the complex interacting patterns of the chain reaction of pathogenesis since interference with adrenal function (which in malaria may arise from local vascular derangements and changes of blood flow) may in itself be responsible for cardiovascular failure. Such a possibility in malaria was suggested by Flosi

THE PATHOGENESIS O F M A M M A L I A N M A L A R I A

67

( I 944) and there is ample evidence of changes in the adrenal medullary-cortical hormonal balance (Maegraith, 1948; Devaltul and Maegraith, 1958). v1.

INTRAVASCULAR

COAGULATION

IN

MALARIA

Devakul et a/. (1966) injected 1251-Iabelled fibrinogen intravenously into Thai patients infected with local chloroquine-resistant P . falciparum parasites. In two severely ill patients with high parasite counts the fibrinogen was removed very rapidly from the circulation. In three moderately ill patients the removal of fibrinogen proceeded at the normal rate. The authors suggested that the rapid loss of fibrinogen in the severely ill patients might indicate widely disseminated intravascular coagulation. Dennis et al. (1967) later claimed that heparin was of some value in the treatment of complicated infections with chloroquine-resistant P. falciparum. They suggested this effect might be related to control of clotting, but noted also that heparin has a static effect on P. knowlesi parasitaemia (Dennis and Conrad, 1968). They were able to demonstrate coagulation defects in P. knowlesi infections in M . niulatta which could be interpreted in terms of disseminated intravascular coagulation. Edington and Gilles (1969) stated that “stasis with local anoxaemia” ( ? anoxia) “causing changes in the endothelial cells would adequately explain the histological changes and clinical symptoms” (of cerebral malaria). They suggested that the lesions might be secondary to mitochondrial damage in the brain capillary endothelium, with consequent loss of fluid into the pericapillary tissues. It is interesting to note here that mitochondria1 damage similar to that demonstrated in the epithelial cells of the liver and the kidney has been recorded by the Liverpool workers in the mitochondria of capillary endothelium. This damage probably originates from the activity of the serum factor which inhibits respiration and oxidative phosphorylation (see p. 70). The fact remains that extensive intravascular clotting has not been consistently demonstrated at autopsy in P .falciparum infections or in mammalian malaria in laboratory animals. Edington and Gilles (1969) have however reported a few strands of fibrin in brain capillaries heavily “plugged” with parasitized cells in patients dead from falciparum malaria. Jaroonvesema ( I 969) recently studied the effect of P. berghei infection on coagulation in mice. She recorded a small increase in fibrinogen breakdown products, as have other authors, and a slight increase in plasminogen, indicating that fibrinolysis was active and intrinsic, and probably secondary to intravenous coagulation. There was also some decrease in prothrombin and Factor X . The small increase in fibrinogen degradation products was presumed to indicate that clotting had occurred in the past, not that it was current. There was a normal range of values of fibrinogen in the infected animals, suggesting continuing synthesis despite the (presumed) increased usage. This would apply also to other coagulation factors, except for Factor X, which remained in low concentration in the infected animals. On the whole, the picture was one of low grade consumption coagulopathy. On the other hand, the platelet numbers were increased and there was no aggregation of platelets. In this respect there was thus apparently less tendency to clotting (the

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reverse of the findings of Dennis et a/. ( I 967) and Dennis and Conrad (1 968) in P. fakiparum and P . knowlesi infections respectively) in the P . berglieiinfected animals. The failure of aggregation of platelets is interesting in light of the demonstration by Onabanjo and Maegraith (1970d) that adenosine (which inhibits aggregation) increases in the blood of monkeys infected with P . knowlesi. Thus the findings in P . berghei infection are indicative of some form of consumption coagulopathy but do not give much of a clue to the role of intravascular clotting as such. It is difficult to believe that any increased coagulation would be exactly neutralized by increased fibrinolysis (which has not been detected in P . knowlesi infection). For the time it seems wise to take the old-fashioned view that if intravascular coagulation is a factor in the pathogenesis of tissue lesions in malaria, evidence of clotting should be regularly visible. “Instant” micro-clotting countered by equally fast fibrinolysis, which has been suggested as a pathogenic phenomenon in shock, is difficult to credit under the circumstances, and equally difficult to incriminate as a cause of obstruction of the circulating blood in the small vessels. We believe that if extensive intravascular coagulation is a pathogenic factor, the fibrin should be present in the vessels for the pathologists to find. It seems more likely to us that inflammatory stasis is the chief pathogenic process and that the occasional clot observed at autopsy is secondary to the failing circulation and not the cause of it. There is the possibility that the consumption coagulopathy reported by the authors quoted above may be interpreted in terms of production failures, but the evidence for this is unconvincing. It is also possible that the complicated pathways of the clotting mechanisms may be changed or become incomplete in malaria infection and that the demonstrated increase in fibrinogen degradation products represents some form of breakdown of this protein which is unrelated to the coagulation processes. Kniseley’s “sludge” is a late phenomenon. It has been suggested that the constituent erythrocytes in the circulating “sludge” mass as seen in shock may be cemented in fibrin (Knisely et a/., 1945). This could account for some of the increase in fibrinogen degradation products found by later workers (see above). The whole question of coagulation in malaria, so far as it may be a factor in initiating circulatory disturbances, is still open-ended. No satisfactory demonstration of its importance has yet been offered. It is an interesting area of research to pursue, however, since there is clearly a possible close relation between the peptide-peptidase reactions demonstrated in the kinin complex and the peptidase reactions involved in coagulation.

V11. ANOXIC ANOXIA Leakage of protein and loss of water through the vascular endothelium similar to that we have demonstrated in malaria can also be induced in some tissues by anoxic anoxia. Florey (1926) demonstrated this effect in the mesenteric vessels of the mammalian omentum in which he produced stasis by

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removing the oxygen supply; he then reversed the process by restoring oxygenation. Landis (1927) described similar experiments on the vessels of the frog mesentery, and concluded that local “asphyxia” could account for the changed permeability and consequent stasis; resolution of the stasis on reoxygenation indicated restoration of normal impermeability. It is possible that anoxic anoxia (or “asphyxia”) as such may have some part in inducing the demonstrable increase in permeability in the brain vessels in P. knodesi infection, but there is no direct evidence of this. In certain areas, for example, in those parts of the liver lobule fed by the “ladder-rung’’ cross sinusoids, circulation may stop completely, as it may in the brain if stasis is not resolved. In such circumstances anoxic anoxia will develop and produce its lethal effect on local tissue. This process, however, is terminal and not likely to be a factor in initiating the cell damage which is seen in other areas where the circulation is still functioning. The hypotheses that the properties of haemoglobin with regard to oxygen carriage and discharge may be intrinsically changed in malaria, or affected by concurrent changes in plasma pH, have not been substantiated. So far as is known, the unaltered haemoglobin of the infected erythrocytes and that of uninfected cells behaves normally, accepting oxygen in the lungs and discharging it at the tissue face (Maegraith et al., 1951). The recorded changes in pH however, would assist rather than retard the release of oxygen from the oxyhaemoglobin. Haemozoin, produced from haemoglobin by the parasite undergoing its life-cycle in the erythrocyte, is inert so far as repiration is concerned and is otherwise non-toxic (Deegan and Maegraith, 1956a, b). Oxygen carriage by the residual haemoglobin in the infected cells in unaffected even when haemozoin is present. Anaemia, with consequent anoxaemia, is also not a major factor in initiating an anoxic environment in the tissues in malaria. lntravascular lysis is a major factor in P.,falciparum malaria (Devakul et a/., 1969) but, except in theextreme haemolysis of severe blackwater fever, the loss of erythrocytes is insufficient to reduce oxygen carriage to levels below those needed for basic metabolic processes (Maegraith, 1948, 1966, 1967). There thus seems to be no general anoxaemia of real significance in malaria, but there is the possibility that the circulatory disturbances described above, particularly the vasoconstriction and reduced blood flow in the liver and the kidney, may in themselves induce a local anoxic state. Other factors may also be involved here at times, including mechanical obstruction from parasitized cells, macrophages, etc., or from Knisely’s “sludge”. As pointed out above, there is some evidence of consumption coagulopathy developing in malaria, but it is difficult to interpret this in terms of coagulum acting as a mechanical obstruction to flow, since clots are only very rarely demonstrable in fatal P. .falciparum cases or in mammalian malaria in the laboratory. Moreover, “coagulopathy” as such indicates merely the presence of residues of fibrinogen and minor changes in the coagulation cascade which, at the time of examination of the blood, actually indicates a relative inability to clot,

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rather than current coagulation. The suggestion that the clots are cleared by fibrinolysis as soon as they are formed is not supported by the experimental findings, since the evidence is that fibrinolysis is not accelerated and may even be retarded in malaria. VIII. CYTOTOXIC FACTORS : MITOCHONDRIAL RESPIRATION INHIBITORS The evidence we have at the moment suggests that many factors are involved in creating the environment which leads to cellular damage in malaria. Since anoxic anoxia as such is not generally involved, except perhaps in particular areas (as suggested above, for example, in some of the sinusoidal pathways of the liver) some other basis for cellular damage must be sought which could provide the link between the parasite in its erythrocyte environment and the host tissues. We have evidence of certain factors circulating in the blood which are capable of disturbing the host-cell metabolism by inhibiting the respiration and oxidative phosphorylation of the mitochondria. It is probable that these reactions may be additive in relation to other environmental situations arising from local circulatory disturbances, the resulting imbalance of vasomotor hormones, and the many biochemical changes described elsewhere in this review. Evidence of biochemical tissue damage can be demonstrated in P. knowlesi and P . berghei malaria in which the respiration and oxidative phosphorylation of mitochondria isolated from infected animals are inhibited to varying degrees. This biochemical lesion is paralleled by certain structural changes in the organelles, seen in the liver cells (Fig 10). The mitochondria become swollen and irregularly wavy in outline, with an apparently intact but convoluted double membrane. Many are surrounded almost completely by endoplasmic reticulum. The cristae are blurred and may disappear. Later the mitochondria disintegrate and are replaced by vacuoles. Other vacuolation develops as a result of lysosomal disruption and eventually the cell is destroyed (Maegraith, 1966, 1967). The mitochondria1 damage becomes visible later in the infection. About this time a soluble factor appears in the serum of the infected host which depresses respiration and phosphorylation process in suspensions of normal monkey or mouse liver-cell mitochondria (Riley and Maegraith, 1961, 1962). The inhibiting factor has recently been found to produce changes in mitochondria in tissue slices, sometimes at a somewhat earlier stage than can be detected biochemically in suspensions of mitochondria. We believe this factor acts as a chemical messenger and may be one of the direct links we have been seeking between the parasite in the erythrocyte and the host animal. It would seem to be a more significant factor than the agent in the serum of the P. knowlesi-infected monkey which induces fatty changes in the parenchymal liver cells on intravenous injection into normal monkeys (Ray and Sharma, 1958). We regard this action on mitochondria as being largely non-specific,

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71

somewhat resembling that of carbon tetrachloride. Its mechanism of action seems primarily concerned with damage to the mitochondria[ structure, involving loss of co-factors. We have so far not fully categorized it chemically (see Fletcher and Maegraith in this volume).

Fig. 10. Electron micrograph of part of a biopsy specimen taken on the 7th morning of infection of M . mularta with P . knowlesi, Changes in the nucleoplasm and in the nuclear membrane ( n m )areevident. Mitochondria1 swelling and distortion are marked and proliferation of the endoplasniic reticulum elements ( e n ) is apparent around these particles. Their cristae (c) are less numerous and distinct than normal. Large vacuoles (Vu) probably represent simple lipid deposition. Disorganization of the vesicles of the E R. ( e r i ) is evident. Note the presence in the cytoplasm of several abnormal particles (pa). x 20,000.

1X. THECHAINREACTION Our studies have convinced us that physiological and pathophysiological responses by the host to the infection are largely non-specific and are thus in some respects applicable to other acute medical estates. At some stage in the development of a malaria infection the pathogenic processes must be initiated by the parasite, either during its development in

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the erythrocyte or at schizogony. One possible link between the parasite and the host may be the soluble factor we have demonstrated, which is capable of inhibiting mitochondria1 respiration and phosphorylation. This and possibly other factors may be involved, acting at various strategic points in the host and influencing its physiological and biochemical balance. These in turn may be modified by factors such as the nutritional status of the host, which may react equally on the parasite, for example, by retarding its development (Maegraith et al., 1952). In the long run, the overall result is the “disease”, malaria. We have shown that in the later stages of P. knowlesi infection the visceral sympathetic nervous system becomes hyperstimulated so that the dynamics of the circulation of blood in many organs are disturbed, with resultant metabolic deviations. The endocrine balance alters. Pharmacologically active substances including kinins and kininogenases exert their effects on membranes and blood vessels. Protein and water escape through damaged endothelium and “inflammatory” stasis may obstruct blood flow in the brain and other areas of the body where the vessels are normally relatively impermeable. In this way a physiological chain reaction is set up, leading to local or general disturbances which are at first reversible, but, with time, may become irreversible and lead to tissue death and the appearance of characteristic pathological patterns. Unchecked, the chain reaction expands; more processes pass to the stage of irreversibility and general circulatory failure or some other catastrophe finally eliminates the host (Maegraith, 1966). The simple concept of initiating factors firing off a chain reaction of interlinked and interacting pathophysiological processes, which involve local and, eventually, general circulation of the blood, membrane permeability, hormone balance and so on, is helpful in visualizing the effects of a developing infection, plasmodia1 or otherwise, on the host. We believe it is also useful for planning and promoting research which will eventually provide the details that are still missing (Maegraith et al., 1952; Maegraith, 1968). REFERENCES

Andrews, W. H. H., Maegraith, B. G. and Wenyon, C. E. M. (1949). Studies in the liver circulation. 11. The microanatomy of the hepatic circulation. Ann. trop. Med. Parasit. 43,229. Billings, F. T. and Maegraith, B. G . (1938). Chemical changes in tissues following obstruction of the blood supply. Q . Jl exp. Physiol. 27, 249-269. Boreham, P. F. L. (1968a). Immune reactions and kinin formation in chronic trypanosomiasis. Br. J. Pharmac. Chemother. 32, 493. Boreham, P. F. L. (196813). I n vitro studies on the mechanism of kinin formation by trypanosomes. Br. J. Pharmac. Chemother. 34, 598. Boreham, P. F. L. (1970). Kinin release and the immune reaction in human trypanosomiasis caused by Trypanosoma rhodesiense. Trans. R . Soc. trop. Med. Hyg. 64, 394. Cannon, P. R. (1941). Some pathological aspects of human malaria. Symposium on human malaria 214. Amer. Ass. Adv. Sci., Washington.

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Chain, E. and Duthie, E. S. (1939). A polypeptide responsible for some of the phenomena of acute inflammation. Br. J. exp. Path. 20. 41 7. Chongsuphajaisiddhi, T. ( I 966). Circulatory changes in Plasmodirrrn hnowlesi malaria. Ph.D. Thesis, Univ. Liverpool. Deegan, T. and Maegraith, B. G. (l956a, b). Studies on the nature of malarial pigment (haemozoin). I. The pigment of the simian species Plasmodium knowlesi and Plasmodium cynomolgi. 11. The pigment of human species Plasmodium falciparum and P. malariae. Ann. trop. Med. Parasit. 50, 194, 212. Dennis, L. H., Eichelberger, J. N., Inmin, M. M. and Conrad, M. E. (1967). Depletion of coagulation factors in drug-resistant Plasmodium falciparum malaria. Blood 29, 713. Dennis, L. H. and Conrad, M. E. (1968). Anticoagulant and antimalarial action of Heparin in simian malaria. Lancet i, 769. Desowitz, R. S. and Pavanand, . (K1967). A vascular-permeability increasing factor in the serum of monkeys infected with primate malarias. Ann. trop. Med. Parasit. 61, 128. Devakul, K. and Maegraith, B. G. (1958). Blood sugar and tissue glycogen in infections in Macuca mirlatta with the Nuri strain of Plasmodium knowlesi. Ann. frop. Med. Parasit. 52, 366. Devakul, K., Harinasuta, T. and Reid, H. A. (1966). 125“Ilabelledfibrinogen in cerebral malaria. Lancet ii, 886. Devakul, K., Harinasuta, T. and Kanakakorn, K . (1969). Erythrocyte destruction in Plasmodium falcipurum malaria : an investigation of intravascular haemolysis. Ann. trop. Med. Parasit. 63, 317. Edington, G . H. and Gilles, H. M. (1969). “Pathology in the Tropics”. Edward Arnold, London. Florey, H. W. (1926). Observations on the resolution of stasis in the finer blood vessels. Proc. R. Soc. Ser. B, 100, 269. Flosi, A. Z. (1944). “ContribuiCBo para o estudo da insuficiencia supra-renal paludica”. Editora Renascanca, SBo Paulo. Goodwin, L. G. and Richards, W. H. G. (1960). Pharmacologically active peptides in the blood and urine of animals infected with Eabesia rodhaini and other pathogenic organisms. Br. J . Pharmac. Chemother. 15, 152. Goodwin, L. G . (1970). The pathology of African trypanosomiasis. Trans. R . Soc. trop. Med. Hyg. 64, 797. Jaroonvesema, N. (1969). Pathophysiological phenomena in the host infected with normal and drug resistant malaria parasites. Ph.D. Thesis, Univ. Liverpool. Knisely, M. H., Stratman-Thomas, W. K. and Eliot, T. S. (1941). Observations on circulating blood in the small vessels of internal organs in living Macacus rhesus infected with malarial parasites. Anat. Rec. 79,Suppl. p. 90. Knisely, M. H., Eliot, T. S. and Bloch, E. H. (1945). Sludged blood in traumatic shock. I. Microscopic observations of the precipitation and agglutination of blood flowing through vessels in crushed tissues. Archs Sirrg., Chicago 51. 220. Landis, E. M. (1927). Microinjection studies of capillary permeability. 111. The effect of lack of oxygen on the permeability of the capillary wall to fluid and to plasma proteins. Am. J . Physiol. 83, 528. Maegraith, B. G. (1940-1941). Reports to War Office (unpublished). Maegraith, B. G . and Findlay, G . M. (1944). Oliguria in blackwater fever. Lancet ii, 403.

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Maegraith, B. G. (1948). “Pathological Processes in Malaria and Blackwater Fever”. Blackwell, Oxford. Maegraith, B. G., Andrews, W. H. H. and Wenyon, C. E. M. (1949). Studies on liver circulation. I. Active constriction of the hepatic venous tree in anaphylactic shock. Ann. trop. Med. Parasit. 43, 225. Maegraith, B. G., Sherwood Jones, E. and Andrews, W. H. H. (1951). Pathological processes in malaria: progress report. Trans. R. Soc. trop. Med. Hyg. 45, 15. Maegraith, B. G., Deegan, T. and Jones, E. S. (1952). Suppression of malaria (Plasmodium berghei) by milk. Br. Med. J. ii, 1382. Maegraith, B. G., Gilles, H. M. and Devakul, K. (1957). Pathological processes in Babesia canis infections. 2. Tropenmed.Parasit. 8, 485. Maegraith, B. G., Devakul, K. and Leithead, C. S. (1959). The terminal stages of Plasmodium knowlesiinfectionin Macaca mulatta. I. Theclinical state: Resuscitation by L-Noradrenaline. Ann. trop. Med. Parasit. 53, 358-368. Maegraith, B. G. (I 966). Pathogenic processes in malaria in the pathology of parasitic diseases. 4th Symposium, Brit. SOC.Paiasit. Blackwell, Oxford. pp. 15-32. Maegraith, B. G. (1967). Biochemical and physiological host: parasite relationships in mammalian malaria. J . Protozool. 2, 65. Maegraith, B. G. and Desowitz, R. S. (1967). The comparative pathophysiology of malaria: report of a symposium held at the School of Tropical Medicine, Bangkok. Ann. trop. Med. Parusit. 61, 515. Maegraith, B. G. and Tella, A. (1 968). Kallidin-a probable factor in the pathogenesis of malaria. Br. J . Pharmac. Chemother. 34, 235P. Maegraith, B. G. (1968). Zusammenhang zwischen Ernahrung und Infektion bei der Malaria. Kongr. Ber. 3. Tag. dt, tropenmerl. Ges. Hamburg. 26. Maegraith, B. G. (1969). Complications of falciparum malaria. Bull. N . Y. Acad. Med. 2nd ser., 45, 1061. Mdegraith, B. G. and Onabanjo, A. 0. (1970). The effects of histamine in malaria. Br. J. Pharmac. Chemother. 39,755. Migasena, P. and Maegraith, B. G. (1967). Pharmacological action of antimalarial drugs. Action of chloroquine and hydrocortisone on blood: brain barrier in Plasmodium knowlesi malaria. Trans. R. SOC.trop. Med. Hyg. 61, 6. Migasena, P. and Maegraith, B. G. (1969). Intestinal absorption in malaria. I. The absorption of an amino acid (AIB-I-I4C) across the gut membrane in normal and in Plasmodium knowlesi infected monkeys. Ann. trop. Med. Parasit. 63, 439. Onabanjo, A. 0. and Maegraith, B. G. (1970a). Kallikrein as a pathogenic agent in Plasmodium knowlesi infection in Macaca mulatta. Br. J . exp. Path. 51, 523. Onabanjo, A. 0. and Maegraith, B. G. (1970b). Inflammatory changes in small blood vessels induced by kallikrein (kininogenase) in the blood of Maraca mulatta infected with Plasmodium knowlesi. Ann. trop. Med. Parusit. 64, 227. Onabanjo, A. 0. and Maegraith, B. G. (1970~).Pathological lesions produced in the brain by kallikrein (kininogenase) in Macaca mulatta infected with Plasmodium knowlesi. Ann. trop. Med. Parasit. 64, 237. Onabanjo, A. 0. and Maegraith, €3. G. (1970d). The probable pathogenic role of adenosine in malaria. Br. J. exp. Path. 51, 581. Onabanjo, A. 0.(1970). A simple micro-method for the estimation of plasma kinins. Pharmacol. Research Comm. 2, 165. Onabanjo, A. O., Bhabani, A. R., and Maegraith, B. G. (1970). The significance of kinin-destroying enzyme activity in Plasmodium knowlesi malarial infection. Br. J. exp. Path. 51, 534.

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Onabanjo, A. 0. and Maegraith, B. G. (1971). Circulating plasma kinins in malaria. Trans. R . SOC.trop. Med. Hyg. 65, 5 . Ray, A. P. and Sharma, G. K . (1958). Experimental studies on liver injury in malaria. 11. Pathogenesis. Indian J. med. Res. 46, 367. Richards, W. H. G. (1965). Pharmacologically active substances in the blood, tissues and urine of mice infected with Trypanosoma brucei. Br. J. Pharmac. Chemother. 24, 124. Riley, M. V. and Maegraith, B. G. (1961). A factor in the serum of malaria-infected animals capable of inhibiting the in vitro oxidative metabolism of normal liver mitochondria. Aim. trop. Med. Parasir. 55, 489. Riley, M. V. and Maegraith, B. G. (1962). Changes in the metabolism of liver mitochondria of mice infected with rapid acute Plasmodium berghei malaria. Ann. trop. Med. Purasit. 56, 473. Skirrow, M. B., Chongsuphajaisiddhi, T. and Maegraith, B. G. (1964). The circulation in malaria. 11. Portal angiography in monkeys (Macacu mulatta) infected with Plasmodium knowlesi and in shock following manipulation of the gut. Ann. trop. Med. Parasit. 48, 502. Spector, W. G. (1951). The role of some higher peptides in inflammation. J. Path. Bart. 63, 93. Tella, A. (1962). Pathogenic processes involved in protozoan infections, including the study of pharmacologically active substances in host-parasite relationships. Ph.D. Thesis, Univ. Liverpool. Tella, A. and Maegraith, B. G . (1963). Further studies on bradykinin involvement in P . knowlesi infection. Trans. R . SOC. trop. Med. Hyg. 57, 1. Tella, A. and Maegraith, B. G. (1966a). Studies on bradykinin and bradykininogen in malaria. Ann. trop. Med. Purasit. 60, 304. Tella, A. and Maegraith, B. G. (1966b). Bradykinin and bradykininogen in the blood of the rhesus monkey (Macaca mulatta). Ann. trop. Med. Parasit. 60,423. Yates, D. B. (1970). Pharmacology of trypanosomiasis. Trans. R. Soc. trop. Med. Hyg. 64,167.

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The Aspidogastrea, Especially Multicotyle purvisi Dawes, 1941 KLAUS ROHDE

Departnieiit of Parasitology, University of Queenslatid, St. Lucia, Brishane 4067, Australia 1. Introduction ...... 11. General Characteristics .............. .. 111.

Structure of the Adult A. Tegument .........

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

.............. F.

Sense Receptors

H. Marginal Bodies .... .

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IV.

78 78 79 79 81 82 90 93 99 103 103 107 107 108 111

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V.

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VI.

of Infection

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VII. VI". Derivation of Digenean Life Cycles from the Aspidogastrean Cycle ............ ................................................... 1x. Some Unresolved Problems ........................................................... ...... Acknc3wledgments References .... , , ...................., .............., .... ........ ..,, ........... ......................

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111 112 1I4 1 I8 120 120 121 124 131 134 134 135 135

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I. INTRODUCTION Of the three trematode groups-Digenea, Monogenea and Aspidogastreathe last is the smallest and least significant in respect of disease in man and animals. From a biological point of view, however, the Aspidogastrea have great interest, combining characteristics of the Digenea and Monogenea and appearing to be only ill-adapted to a parasitic way of life. A closer examination of the Aspidogastrea, therefore, could throw light upon the phylogenetic relationship of the various trematode groups and the origin of parasitism in the platyhelminths. Previous, more or less extensive discussions of the Aspidogastrea are those by Braun (1879-1893), Dawes (1946, 1947), Skrjabin (1952), Baer and Joyeux (1961), and Yamaguti (1963). Of great significance, the monograph by Dollfus (1958a) includes most relevant data then known. The present review deals mainly with recent work; for older data on distribution, host records, generic and species characteristics, the reader is referred to Dollfus’ work, to Skrjabin and Yamaguti and to the “Index Catalogue of Medical and Veterinary Zoology”. Such data are discussed herein only as far as is necessary to give a coherent account of the group. 11. GENERAL CHARACTERISTICS (Figs 1, 2)

The Aspidogastrea are trematodes with a ventral disc (Baer’s disc) divided into one to four longitudinal rows of alveoli, or represented by a single row of ventral suckers. A septum of connective tissue and muscle fibres separates the ventral part of the body and its ventral disc from the dorsal part of the body(at leastanteriorly). Betweenthe marginal alveoli thereare marginal bodies or else tentacles between alveoli, but both are sometimes absent. Hooks are never present. The mouth may be surrounded by lip-like processes or lobes, and an oral sucker may or may not be present. The pharynx is well developed, and the intestine is simple and sac-like. Single or double testes lie behind the ovary. A cirrus and its pouch may be present and the genital pore is ventral near the anterior end of the body. Sometimes there are separate male and female pores. The ovary is always single and a seminal receptacle may be present. Laurer’s canal is usually present and it may end blindly. The uterus contains from a few to many operculate eggs without polar filaments. The vitellaria are paired or unpaired, extending throughout the length of the body or else are less extensive. The oviduct is septate and partly ciliated. The excretory pore or pores are usually dorso-terminal. Development is direct, without alternation of generations. The Aspidogastrea are endoparasites or may live on the surface of molluscs (as ectoparasites or ectocommensals). Their hosts are bivalves, gastropods, (ascidians ?), elasmobranchs, teleosts and turtles. The larva of one species was found encysted in the intestinal wall of marine crustaceans. The species Zonocotyle bicuecatu Travassos and Aspidocotyle mutabile Diesing (= Aspidocotylus cochleariformis Diesing) which are sometimes included in the Aspidogastrea, resemble other species of this group only superficially, having either some transverse ridges or numerous gland- or

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(1)

(2)

FIG.1. Multicotyle purvisi, compressed mature specimen. SN-alveolus, C--caecuni, CS&cirrus sac, Go-genital opening, LK-Laurer's canal, AM-marginal body, Mmouth, 0-ovary, P-pharynx, H-testis, U-uterus with eggs, SS-ventral disc, VD-vas deferens, DS-vitellaria (Rohde, 1971a). FIG.2. Multieotyle purvisi, diagrammatic sagittal section. D-caecum, OGT-Mehlis' gland and genital ducts, OV-ovary, RS-receptaculum seminis, SEPT-septum, HODtestis, UT-uterus, SS-ventral disc, DOT-vitellaria, VS-vesicula seminalis (Rohde, 1971a).

sucker-like structure. Both species have paired caeca and are certainly not Aspidogastrea. 111. STRUCTURE OF THE ADULT A. TEGUMENT* (Figs 3, 4) The ultrastructure of the tegument has been examined in Multicotyle purvisi only (Rohde, 1971d). It corresponds in all details to the tegument of Digenea.

* See footnote on p. 80.

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The basal cell membrane is located above a well-developed basal lamina and has extensive invaginations which, however, never reach the surface and are partly lamellar. The tegument contains mitochondria and numerous ovoid bodies, in some areas also vacuoles and lamellated bodies. The surface membrane forms many regular elevations between and partly above which extends a mucoid layer of variable thickness. In some areas there are rib-like elevations

FIG.3. Multicotyle purvisi, adult. Dorsal tegutr.ent in middle of body (Rohde, 1971d).

of the surface which serve as supporting structures of an excessively thick mucoid layer, consisting of a reticulum of fibres with electron dense bodies of various sizes. The tegument is syncytial, connected by processes to sub-tegumental cells, which are very rich in Golgi complexes. The tegument of the posterior part of the body (i.e. of the region where new alveoli are formed during growth) has the same structure as the tegument in the more anterior parts of the body. This implies that the tegument in adult Multicaryle originates

* Symbols in electron micrographs and diagrams based on electron micrographs. Aampulla (marginal body) of marginal gland; AK-external chamber of flame cell; AL--external leptotriches; AR-xternal rib; AX-axon; AXF-axial filament complex; BI-connective tissue sheath around protonephridial vessel ; BK-basal granule; BL-basal lamina; BZ-basal cell membrane; CI--cilia; DW-thick-walled upper portion of flame cell; EBZ-invaginations of basal cell membrane; EK-close cell contacts; EP-invaginations of parenchyma; EX-xcretory body; EXZ--excretory bladder cell ; FA-fibres; FADfascia adhaerens; 1K- internal chamber of flame cell ; IL-internal leptotriches; I R-internal rib; K--capillary of protonephridial system; L-cords of weir membrane; LA-lamellae (of nerves) or lamellar evaginations of surface membrane (of protonephridia); LI-lipoid droplet; LU-lumen of caecum; MC-mucoid layer; MF-microfila; MI-mitochondria; MO-macula occludens; MT-microtubuli; MU-muscle cells; MV-microvilli; N and NU-nuclei; NT-neurotubuli; 0-ovoid bodies; RM-weir membrane; S-distal processes of ciliary membranes; SE-secretory droplets; U-irregular inclusion ; V-vesicles ; VA-vacuoles; W-rootlets of cilia; W M d i a r y membrane; X-tubuli; ZA-zonula adhaerens; ZF-cytoplasmic processes of flame cell.

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81

FIG.4. Mulficoiylepurvisi, adult. Basal part of tegument (Rohde, 1971d).

in its definitive form and does not arise by fusion of primarily separate epithelial cells. B.

DIGESTIVE TRACT

(Figs 5, 6)

The intestinal tract of aspidogastrids is a simple tube, consisting of mouth cavity, pharynx and caecum. In Stichocotyle nephropis and Macraspis elegans, Nickerson (1895) and Brinkmann (1958) founda valve-like constriction behind the pharynx. There are no ultrastructural studies on the anterior part of the intestinal tract. The caecum of adult Mulficotyle appears by light-microscopy as a tube surrounded by circular and longitudinal muscles; the epithelial cells of the caecum pass apically over into an irregular reticulum of processes. Occasionally, nuclei are found in the lumen of the caecum. In semi-thin Araldite sections, the lumen appears to be filled with a vacuolar substance and nuclei. The material is less dense near the centre of the lumen. The following layers can be distinguished in ultrathin sections: ( I ) muscles, ( 2 ) epithelium, (3) cells and detritus of cells in the lumen (Rohde, 1970d, 1971g). The muscle layer is formed of longitudinal and circular muscles. Often a layer of longitudinal muscles below the epithelium is followed by a layer of circular muscles and by one or more layers of longitudinal muscles, an arrangement which, however, is not constant; thus, the inner longitudinal muscles may be absent in some sections or the circular layers may be doubled. Between the muscle fibres and not far below the basal lamina of the intestinal epithelium are fibres of the nerve plexus surrounding the caecum. The epithelium rests on a basal lamina; it is formed by numerous cells which are separated by lateral, deeply convoluted cell membranes. The epithelial cells are rich in Golgi complexes, granular endoplasmatic reticulum, mitochondria and invaginations of the basal cell membrane. Occasionally, lipoid droplets, and very often concentric bodies are formed in spaces (cisternae) of the granular endoplasmatic reticulum. The epithelial cells make contact at the surface by means of zonulae adhaerentes. The surface is enlarged by numerous lamellae repeatedly interconnected. Free in the lumen are found-parts of the surface lamellae and of the epithelial cells, including concentric bodies and lipoid bodies; complete

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KLAUS ROHDE

FIG.5. Multicotylc purvisi, adult. Transverse section through caecum (Rohae, 19719).

cells rich in granular endoplasmatic reticulum and containing concentric bodies and lipoid droplets; and cells without these inclusions. These cells have probably been given off into the lumen and disintegrate there.

c.

PROTONEPHRIDIAL SYSTEM

(Figs 7-1 I)

At the light microscopic level, the protonephridial system of Aspidogastrea has been examined carefully only in Aspidogaster conchicola (cf. Faust, 1922) and A . indica (cf. Rai, 1964). The flame cell formula for both species, as given by these two authors, is 2 [ ( 3 ~ 3 x~ 3)+(3 x 3 x 3 ~ 3 ) + ( 3x 3 x 3 x 3)]=2[34+34+34]=2[34

x 3]=486

Contrary to this, Syogaki (1936) gave a formula of 34 x 10for the former species. Moriya (1944) mentioned for Lophotaspis corbiculae Moriya, 1944, the rather

T H E A S PI D 0G A S T R E A

83

FIG.6 . Multicotj4e purvisi, adult. Cells and detritus in lumen of caecum (Rohde, 1971g).

aberrant flame cell formula of 2(35 x 6 x 2). His description is very brief and no drawings of the protonephridia are given. The excretory pore (or, exceptionally, pores) are located dorso-terminally or, in few species, terminally. In some cases, lateral flames (non-terminal ciliary flames) have been demonstrated in addition to the terminal flame cells (for instance Stichocotyle, see Nickerson, 1895; Aspiclogaster, see Zacharias, 1895; Stafford, 1896; Cotyluspis, see Osborn, 1903; Najarian, 1961 ; Hendrix and Short, 1965). I n Multicotyle, the dorso-terminal excretory pore joins a small excretory bladder, which

FIG.7. Multicoryle purvisi, adult. Sagittal section through flame cell (Rohde, 1970a).

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85

FIG.8. Multicotyle purvisi, adult. Oblique T.S. (left) and L.S. (right) through tip of “flame” (Rohde, 1970a; R, orig.)

receives two large excretory ducts that run forwards and turn back near the anterior extremity. The ducts branch repeatedly and the numerous small capillaries terminateinflamecells. Theelectron microscope reveals that thesecells of Multicoryle have the same structure as the flame cells of all other parasitic platyhelminths examined (Rohde, 1970a, e, and in preparation). The main difference is, that the ciliary membranes continue beyond the tips of the cilia forming an irregular honeycomb-like pattern of membranes which anchor the flame apically in the cytoplasm of the flame cell. The internal chamber of the flame cell does not directly communicate with the surrounding parenchyma, i.e. there are no nephrostomes. The ribs of the flame cell form the so-called weir apparatus and are connected by a weir membrane which forms a complete sheath around the internal chamber. Furthermore, the terminal portion of a capillary with the weir apparatus and the flame consists of a single cell and not of two cells, as claimed by some authors for other parasitic platyhelminths. In the parenchyma surrounding the flame numerous filaments are arranged in a predominantly concentric pattern. Possibly, these filaments are contractile and play a part in pressing the parenchymal fluid through the weir membrane

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KLAUS ROHDE

FIG. 9. Mulricotyle purvisi, adult. Section through capillary. Parenchymal cell (top) and cell of protonephridial system (bottom) (original).

into the internal chamber of the flame. The distal lumen of the flame cell opens into a strongly convoluted capillary with a surface enlarged by numerous small microvilli (or very narrow lamellae). The larger capillaries and excretory ducts have, instead, numerous lamellar evaginations of the surface membrane.

FIG.lO(a) 5

KLAUS ROHDE

FIG. 10. Multicotyle purvisi, adult. (a) Section through protonephridial duct with lateral (non-terminal) flames. (b) Part of (a) at X enlarged (original).

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89

FIG.1 1 . Mirltirotyle purvisi, adult. Section through anteriorly running portion of main protonephridial canal in middle of body. At X section throueh bundle of tubules (original)

All capillaries (except perhaps parts of the smallest) and excretory ducts have a sheath of connective tissue containing many filaments, penetrated by processes of the parenchyma. The larger capillaries and ducts have numerous

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KLAUS ROHDE

invaginations of the basal cell membrane, Golgi complexes, mitochondria, microtubuli and vacuoles. The largest ducts also contain tubuli which occur usually in bundles and consist of peripheral and central tube-like structures. The tubuli resemble axial filament complexes but are niuch smaller. All larger capillaries and ducts, except the anteriorly directed part of the largest ducts, have lateral flames (non-terminal flames) consisting of loosely packed cilia not forming a functional unit of tightly packed cilia as in flame cells. The terminal portion of the main excretory canals has a spongy structure due to numerous invaginations of the basal cell membrane and irregular outgrowths at the surface. The distribution of mitochondria, Golgi complexes, vacuoles and invaginations of the basal cell membrane indicates that excretion and probably reabsorption are mainly limited to the larger capillaries and ducts, while in the flame cells filtration takes place through the weir membrane. D.

GENITAL SYSTEM

(Figs 12-14)

The feature distinguishing the genital system of Aspidogastrea most clearly from that of other trematodes is the structure of the oviduct. In all species that have been thoroughly examined, the oviduct is divided into compartments by numerous septa, and part of it is ciliated. Septa were demonstrated in Corylogaster occidentalis (by Nickerson, 1902),Aspidogaster conchicola(by Voeltzkow, I888a; Stafford, 1896), Cotylaspis insignis (by Osborn, 1903, 1905; Hendrix and Short, 1965), Stichocotyle nephropis and Macraspis elegans (by Odhner,

1/ LC

0c

FIG. 12. Mu/ticat.de purvisi, adult. Diagram of female genital system near ovary. DGvitelloducts, LC-Laurer’s canal, MD-Mehlis’ gland, OD-oviduct, UT-uterus. Arrow points at horizontal section through septum of oviduct (Rohde, 1971a).

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91

FIG. 13. Aspidogaster ronchrtolu. Diagram of genital system ACL-triangular space, CEJ-ductus ejaculatorius, CL-Laurer’s canal, D C A a e c a of ductus ejaculatorius, GM-Mehlis’ gland, M-metraterm, OG-genital pore OO-ootype, OV-ovary, OVDoviduct, P-parenchyma, PCE--circular folds, PCL-longitudinal muscles of cirrus pouch, PIC-wall of inner ductus ejaculatoritis, PP-axial papilla of copulatory organ, RSUuterine seminal receptacle, RV-yolk reservoir, T-testis, U-uterus, VS-seminal vesicle, VT-longitudinal vitelloduct, VTI-unpaired yolk duct (from Dollfus, 1958a, after Stafford, 1896).

FIG. 14. Mu/Iicot.v!e purrisi. Diagrammatic section through spermatozoon behind nucleu(Rohde, 1971h).

92

KLAUS ROHDE

THE ASPIDOGASTREA

93

1910). In all except the last two species mentioned, the authors also refer to ciliation of the oviduct. In Multicotyle purvisi, septa are present in the oviduct between the ovary and the point where Laurer's canal arises. The distal half of the septate portion is also ciliated (Rohde, 1971a). A septate oviduct is not known to exist in other parasitic platyhelminths. Laurer's canal sometimes opens into the terminal portion of the excretory system (Aspidogaster Iimacoidev and Cotylogaster occidentulis) (see Bychowsky and Bychowsky, 1934). In most respects, the female genital system of Aspidogastrea corresponds closely to that found in Digenea. The single and paired vitelline ducts of Multicotyle purvisi show an extent of ciliation which varies in different specimens, possibly depending on their age. In Multicotyle as well as in Cotylogaster occidentalis (cf. Nickerson, 1902), C. michaelis (cf. Monticelli, 1892), and Aspidogaster conchicola (cf. Stafford, 1896) the terminal portion of the uterus is surrounded by clusters of cells which are probably glandular. The ultrastructure of the genital system of Aspidogastrea has not been examined. However, in a critical study of the spermatozoa of Multicotyle pzrrvisi by Rohde ( I 971f), each spermatozoon was found to contain an elongate nucleus, one large elongate mitochondrion (or some large mitochondria arranged in a row?), microtubules below the plasma membrane running parallel with the longitudinal axis of the spermatozoon, and two axial filament complexes. The microtubules in some portions of the spermatozoon are arranged in several rows. The axial filament complexes have the 9 f 1 structure which is typical of the platyhelminth spermatozoon. At the posterior end, the axial filament complexes continue for some distance as free cilia. E.

NERVOUS SYSTEM

(Figs 15-22)

There are few data on the complex nervous system of Aspidogastrea. Most details are given by Monticelli (1892) for Cotylogaster michaelis Monticelli. This writer found a dorsal cerebral commissure and paired anterior dorsal internal, anterior dorsal external, anterior ventral and anterior lateral nerves, and paired posterior ventral, ventro-lateral and dorso-lateral nerves. All the nerves and commissures are figured only for the anterior part of the body. Fewer details for other species were given by Voeltzkow (1888a), Nickerson (1895), Osborn (1903, 1905) and Stunkard (1917). In no instance were special methods used for demonstrating elements of the nervous system. Various methods of impregnating serial sections of Multicotyle pirrvisi with silver compounds showed that this species has a remarkably complicated -

FIG.15. Multicotyle purvisi, adult. Diagram of nervous system in anterior part of body. I-nervus medialis, 2-n. lateralis, 3-n. mediodorsalis (rami lateralis and intermedius), 4-n. mediodorsalis (ramus medialis), 5-n. dorsalis anterior, 6-commissura terminalis anterius, 7-commissura circumoralis externa, S-commissura circumoralis interna, 9-n. ventralis internus, 10-n. ventralis externus, 1 1&cornniissura infrapharyngealis majus, 12-41. genitalis, 13-n. ventralis posterior (ramus ventralis anterior), 14-11. ventralis posterior (ramus ventralis posterior), 15-11. ventralis posterior (ramus dorsalis), 16-n. dorsalis posterior, 17-n. dorsolateralis posteriw, 18-n. pharyngealis, 19-nervi septi (Rohde, 1971~).

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KLAUS ROHDE

FIG. 16. Multicotyle purvixi, adult. Dorsal view of extraplantar nerves at posterior end. Arrows point at junctions with the posterior dorsal nerves (Rohde, 1971~).

It 12 13

1

2

3 4

27 8

9

FIG.17. Mulrieotyle purvisi, adult. Diagram of nerves in middle of body. D-caecum, DOT-vitelline glands, DR-vitelline reservoir, HOD-testis, I-nervus intraplantaris lateralis, 2-opening of marginal gland, 3-ampulla (marginal body) of marginal gland, 4-n. extraplantaris lateralis, 5 and 6-connections between extra- and intraplantar commissures, 7-n. extraplantaris intermedius, 8--commissura extraplantaris, 9-n. extraplantaris medianus, 10 and 13--commissura intraplantaris, lateral part (10) and middle part (1 3), 1I-n. intraplantaris intermedius major, 12-n. intraplantaris intermedius minor, 14-n. intraplantaris medialis, 15-11. ventralis posterior, 16-n. dorsolateralis posterior, 17-11. dorsalis posterior (Rohde, 1971~).

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FIG.18. Mulficotyle purvisi, adult. Main extraplantar nerves dorsal to anterior part of ventral disc; dorsal view. I-nervus ventralis posterior (ramus ventralis posterior), 2 and 8-roots of extraplantar comrnissures shifted anteriorly, 3-n. extraplantaris l a t e r a h 4 - n . ventralis posterior (ramus ventralis anterior), 5-n. extraplantaris medianus, &penetration of posterior ventral nerve into ventral disc, 7-n. extraplantaris anterior (Rohde, 1971c).

FIG.19. Multicotyle purvisi, adult. lntraplantar nerves i n anterior part of ventral disc. Arrows point at penetration of rami ventrales anteriores of posterior ventral nerve into ventral disc (Rohde, 1971~).

96

KLAUS ROHDE

nervous system, which appears to be more characteristic of free living than of parasitic forms (Rohde, 1968a, d, 1970b, 1971~).The anterior part of the system consists of the following components (Rohde, 1971~):(i) internal circumoral commissures beneath the tegument of the mouth cavity, (ii) external circumoral commissures beneath the tegument of the body surface, (iii) longitudinal medial and internal ventral nerves connected by the internal circumoral commissures, (iv) anterior dorsal, anterior dorso-lateral, lateral and anterior external ventral nerves connected by the external circumoral commissures, (v) medio-dorsal nerves dividing into three branches, the rami lateralis, interinedius and medialis between the internal and external circumoral commissures and the longitudinal nerves connected by them. These commissures and connectives (longitudinal nerves) possess numerous additional transverse connections. At the anterior end, the larger longitudinal nerves are connected by a well-developed anterior terminal commissure located in the dorsal lip above the oral opening.

FIG.20. Multicotj*lepurvisi, adult. Nervi intraplantares in posterior part of ventral disc. Ventral view (Rohde, 1971~).

The brain (cerebral commissure) appears to be the dorsal portion of an internal circumoral commissure. Posterior longitudinal nerves are the pharyngeal nerve, which passes along the pharynx wall and enters the pharynx from behind, the posterior dorsal nerve, the posterior dorso-lateral nerve and the posterior ventral nerve. All these nerves are paired, as are the anterior connectives. There are many transverse commissures at the level of the pharynx,

THE A S P I D O G A S T R E A

97

the largest the ventral part ofa commissureat the anterior margin ofthe pharynx (commissura infrapharyngealis majus). Behind this commissure is the genital nerve, which innervates the margin of the genital opening. The posterior ventral nerve is the best developed of all posterior nerves; after leaving the brain, it

FIG.21. Multicofylepurvisi, adult. T.S. through posterior ventral nerve with nerve sheath (Rohde, 1971~).

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K L A U S ROHDE

soon divides into single dorsal and ventral branches which reunite but then divide again; both branches penetrate the septum; the ventral branch divides into anterior and posterior ventral rami; the anterior ventral ramus enters the ventral disc between the second and third transverse row of alveoli, where it forms a rather regular pattern of connectives and commissures in the walls of the alveoli. This system of intraplantar (derived from latinpluntu = sole) nerves communicates by means of numerous connections with similarly

FIG.22. Multicotyle purvisi, adult. T.S. through intraplantar nerve (Rohde, 1971~).

arranged nerves above the ventral disc, i.e. with the extraplantar nerves. The dorsal and the posterior ventral rami of the posterior ventral nerve unite again at the level of the ovary. One complete transverse commissural ring in the dorsal part of the body corresponds generally to each transverse row of alveoli, though there may be irregularities. The posterior ventral nerve divides near the hind end of the body into two terminal branches, the dorsal and ventral terminal rami.

THE ASPIDOGASTREA

99

The septum separating the ventral part of the body from the dorsal part, the intestine, pharynx, prepharynx, cirrus pouch, uterus, genital and excretory openings are innervated by plexuses. The intestinal plexus is connected to the pharyngeal nerve and the posterior dorsal nerve. A tendency to form plexuses can also be recognized in the internal and external circumoral commissures as well as in the posterior connectives and commissures of the dorsal part of the body. Most of the transverse and longitudinal nerves, including the cerebral commissure, lie free in the parenchyma. However, in sections variously stained, and with the electron microscope, it is seen that parts of the well-developed posterior ventral nerve are surrounded by a distinct sheath (Rohde, 1970b, 1971~).This sheath consists of lamelfae of connective tissue with numerous filaments, concentrically arranged around the axons. The lamellae are often in close contact with one another. They branch, branches of the innermost lamellae forming the walls of the axons. The axons contain neurotubuli of about 200 A diameter, mitochondria, a few lamellated bodies of various sizes and irregular inclusions (Rohde, 1971~).Some axons have accumulations of ovoid electron dense bodies measuring up to 0.21 x 0.44 pm. There are also vesicles showing all transitions from empty and small ones to larger vesicles filled with electron dense materials. The small empty vesicles are 300-500 A in diameter, the largest ones about 740 A. The diameter of the largest filled vesicles is generally 800 A, exceptionally 1200 A. Nuclei are usually found in the periphery of the nerves, sometimes also in the interior, between the axons. In some areas synapses are abundant. They are characterized by an enlarged space between two adjacent membranes, the space containing electron dense material, and by thickenings on both sides, especially of the postsynaptic side. While the presynaptic side contains numerous vesicles, the postsynaptic side is rich in granular material. In serial sections stained with paraldehyde fuchsin, clusters of stained cells occur at the intersections of the longitudinal and transverse extraplantar nerves. Similar clusters are present along the transverse extraplantar nerves between the ventral nerve cord and the marginal bodies. Material stained in the same way was sometimes seen along paths corresponding to the lateral portions of the extraplantar nerves and to fibres connecting the extra- and intraplantar nerves. I n some instances, the secretion appeared to enter the alveoli of the ventral disc along these paths. Possibly, the paraldehyde fuchsin-positive material represents neurosecretion, which is transported in the extraplantar nerves. Not all cells in the clusters are paraldehyde fuchsin-positive, indicating that they are perhaps not always active. Electron microscopic studies of thew cells have yet to be made. F.

SENSE RECEPTORS

(Figs 23, 24)

Of the older authors, Nickerson (1895) described sensory papillae in Sticliocotyle nephropis. Similar papillae, which correspond in their light microscopic structure to those of digenetic trematodes, were found by Osborn ( I 903, 1905) in Cotyluspis insignis.

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KLAUS ROHDE

Serial sections of Multicotyle yurvisi impregnated with silver compounds revealed not only a great number but also a great variety of sense receptors (Rohde, 1966a, 1968b). Because of the similarity of connective tissue fibres impregnated with silver, to nervous elements, the connection of a fibre to a nerve was used as the only criterion of its nervous nature. The least specialized sensory elements are the free nerve endings found below the tegument in most parts of the body. They are most common in the alveoli of the ventral disc, on the walls separating them, and around the oral opening. Their endings are often branched in a T-like fashion. Processes of the nerve fibres penetrate sometimes into the tegument, occasionally forming loops or coiled structures. They sometimes reach the surface, forming free sensory hairs. However, these structures are rare and may be artefacts. There is also a variety of small and large sensory capsules. Commonly around and

FIG.23. Multicotyle purvisi, adult. Most common types of sense receptors. Left to right, top: large capsule with thick fibre and without terminal plate, large capsule with thick fibre and terminal plate, small capsule with thin fibre; bottom: large sensory disc on ventral disc, small subtegumental capsule, free T-shaped nerve fibre ending (original).

behind the oral opening(but not in the mouth cavity) are large, solidly stained bodies sometimes capsulate, measuring 6 x 3.6 pm. Small capsules are dispersed throughout the region below the tegument of the main body; they can be regarded as T-shaped nerve endings surrounded by a capsule. Their maximum size is 5.4 x 3.6 pm, but usually they are only about 2.5 x 2.5 pm, and the terminal solid plate is often absent. Occasionally, the capsules penetrate for a short distance into the tegument. Large capsules related to these small subtegumental capsules are found in most parts of the body, often located in an elevation of the tegument and varying greatly in size, reaching a maximum of 7.0 x 7.4 pm. They are largest and most common in the regions dorsal and lateral to the oral opening and they rarely occur on the adhesive disc. Typically, the capsule is pyriform, with a long projection reaching below the tegument. A thick nerve fibre crosses the capsule, forming an S-shaped figure in its widest part. Terminally, it either forms a solid disc sometimes bearing a hair-like process or else it has no such plate, becoming very thin and passing over into the process at the surface. A modification of this kind of capsule is a smaller capsule found on the ventral disc, in the lumen of the mouth cavity, on the

T H E AS PIDOGASTKEA

101

FIG.24. Multicofyle purvisi, adult. Sense receptors (original).

lips surrounding the oral opening, and in the region ventral and lateral to the mouth opening. Its maximum size is 3.4 x 3.6 pm and there is no thick S-shaped nerve fibre in the capsule; instead, the thin fibre passes straight through the capsule and continues as a long sensory hair at the surface. Normally, there is a small swelling of the fibre at the base of the hair. Large bodies with very thick terminal portions and without sensory hairs and fibres passing through them, are very conspicuous and common on the walls separating the suckerlets of the ventral disc (maximum size 6.0 x 7.2 pm).

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K L A U S ROHDE

The number of sense receptors is greatest around and behind the mouth and on the ventral disc. In a specimen 6.1 mm long, the numbers counted were: in the prepharyngeal region with a length of about 0.3 mm, 360 dorsal, 260 ventral sense receptors; in the mouth cavity 140 receptors; the numerous free nerve endings below the tegument were not counted. In another specimen 3.1 mm long, the hind part of the prepharyngeal region (about 0.1 mm long and excluding the mouth) had about 160 sense receptors, while a portion of the body 0.3 mm long and beginning 0.6 mm behind the anterior extremity, contained more than 200 sense receptors, not counting free nerve endings. The numbers given are probably minimal, because of difficulty in counting all receptors, especially in areas where they are closely packed. Nickerson ( I 895)

FIG.25. Multicotyle purvisi, adult. T. S. through marginal alveolus. M-mucous cells, MU-muscles, P-parenchymal cell, RD-marginal gland cells, SC-subtegumental cells (Rohde, 1971e).

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103

counted large numbers of sense receptors in the suckers of Stichocotyle nephropis. G. VENTRAL DISC (Fig. 25) The ventral disc of Multicotyle purvisi is innervated by the intraplantar nerves described above (Ill.E). The lateral rows of alveoli on the disc contain the marginal gland complexes (see 111. H). The ventral disc is rich in transverse and radial muscle fibres. Below the ventral tegument a thick layer of cells is deeply “insunk” in the parenchyma (Rohde, 1971e). The cytoplasm of these cells stains strongly granular and pinkish orange with azan. The parenchyma of the ventral disc is formed by cells with long cytoplasmic processes and large vacuoles. Scattered between them are cells with cytoplasm which stains deep blue with azan.

H.

MARGINAL BODIES

(Figs 25-30)

Peculiar round to oval, so-called marginal bodies are located between the marginal alveoli of the ventral disc of most Aspidogastrea. Some authors consider them to be glands (Stafford, 1896; Looss, 1902; Osborn, 1905; Najarian, 1961; Dollfus, 1958a); but according to other authors they are of a sensory nature (Voeltzkow, 1888a; Stunkard, 1917). Nickerson (1901, 1902) considered them to have both sensory and glandular functions. Reasons given for the supposed sensory function are: parts of the organ can actively move and nerve fibres and sensory cells have been found near them. Voeltzkow (1888a) found that the neck-like part of the marginal bodies of Aspidogaster conchicola which is located near the surface, can be projected and “voluntarily” moved. Nickerson (1901, 1902) observed in Cotylogaster occidentalis that the terminal parts of the marginal bodies can be protruded and bipolar cells near the marginal bodies and fibres were considered “without doubt” to be sensory cells and nerve fibres respectively. Stunkard (1917) assumed that the marginal bodies of Cotylaspis cokeri have a sensory function because nerve fibres surround the marginal bodies terminating near them, and because the marginal bodies are protrusible. The supposed nerve structures were never stained by special methods used for demonstrating nervous tissue. Serial sections of Multicotyle purvisi impregnated with silver showed that some fibres and a nerve pass along the marginal bodies. This, however, does not permit the conclusion that the bodies are sensory. The innervation is much poorer than that of the septum, which certainly does not have a sensory function. Nor does the fact that the terminal parts of the marginal bodies can be protruded indicate a sensory function. Protrusion could help in secretion or deposition of secretion (perhaps mucus) on the substratum. The marginal bodies of Multicotyle purvisi are ampulla-like structures surrounded by connective tissue and muscle fibres (Rohde, 1966b, 1970f, 1971e). They open through a narrow terminal duct lined with circular muscle fibres at the outer side. The variability of the shape of the duct suggests that they are responsible for peristaltic movements of the duct. The terminal duct contains sphincter like invaginations of the tegument. Near the invaginations are

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KLAUS ROHDE

sense receptors with sensory hairs which probably play a part in regulating the outflow of secretion. Towards the interior of the body, the ampulla opens into a large transverse duct connected to the ampulla on the opposite side of the body. Transverse ducts at different levels are connected by up to six smaller longitudinal ducts in the marginal alveoli. These ducts are connected to gland cells located in the dorsal part of the marginal alveoli. The clusters of gland

*

.-.

UM-. *.

FIG 26. Multicotyle privvisi, adult. Sagittal section through marginal bodies. A-ampulla (marginal body), DG-glandular duct, M-mucous cell, M U-niuscles, N-nerve, SKsubtegumental cells (Rohde, 1971e).

FIG. 27. Multicotyle puvvisi, adult. Diagram of arrangement of marginal gland cells (coarse stipple) and of gland ducts (fine stipple) (Rohde, 1971e).

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105

cells are sometimes so extensive that they fuse with adjacent clusters forming a continuous band. The marginal gland cells contain abundant granular endoplasmatic riticuluni. The cells are not always in an active secretory stage, but those which are contain many electron-dense secretory droplets which do not appear to be surrounded by a membrane. The droplets originate in the numerous Golgi complexes and pass through the secretory ducts to the ampulla, where they fuse to form a homogeneous dark mass.

FIG.28. Multicofyle puvvisi, adult. Section through ampulla of marginal gland (marginal body) and terminal duct (inset). Arrows point at sense receptors (Rohde, 1971e).

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K L A U S ROHDE

These findings show that the marginal bodies of Multicotyle are parts of glands consisting of the following parts: marginal gland cells in the ventral disc, secretory ducts, ampulla-like marginal body for storing secretion, and terminal secretory duct. There is no evidence that the marginal bodies have a sensory function and as the marginal bodies of other species are morphologically similar to those of Multicotyle, it is assumed that they, too, are parts of glands. The same refers to the tentacles in the genus Lophotaspis, where histological structure suggests a glandular function, and these also are probably modified marginal organs. Burt ( I 968) found that the marginal organs of Macraspis elegans consist of funnels into which numerous unicellular glands open. Internal to the tip of the funnel and surrounded by these gland cells is a body of deeply staining cells, among and probably through which ramify many nerve fibres. Silver nitrate

FIG.29. Multicotyle purvisi, adult. Section through active marginal gland cells (Rohde, 19714.

THE ASPIDOGASTREA

107

impregnation revealed that they are connected to a nerve net dorsal to the alveoli. Burt does not state whether he considers the marginal organs of Macraspis primarily as sensory or glandular, or both sensory and glandular.

FIG.30. Lophotuspis vu//ei, adult. Sagittal section through tentacle on ventral disc (from Dollfus, 1958a, after Looss, 1902).

1v. STRUCTURE A.

OF THE

FREELARVA

GENERAL MORPHOLOGY

(Fig. 31)

All larval Aspidogastrea examined have a mouth cavity, prepharynx, pharynx, simple caecum and a postero-ventral disc* without alveoli. The larva of Corylogasrer (= Corylogasteroides) occidenralis differs from those of other species in having an intestine divided into a thin-walled ventral sac and a larger thick-walled dorsal portion (Wootton, 1966). While some larvae are not ciliated (Macraspis elegans, cf. Brinkmann, 1958; Aspidogaster conchicolt, cf. Voeltzkow, 1888a, and Williams, 1942; A . i d i c a , cf. Rai, 1964), others have a number of ciliary tufts. According to Manter ( 1 932), Lophotuspis vallei has one posterior ciliary tuft at the posterior end and two lateral ciliary tufts slightly behind the middle of the body. According to Nickerson (1900, 1902), the larva of Cotylogaster occidentalis has an incomplete band of cilia forming dorsal and lateral tufts near the pharynx and another tuft at the posterior end. The absolute number of tufts was not determined. Wootton (1966) found in the same species four ventral and four dorsal tufts anteriorly and two ventrolateral and two dorso-lateral tufts terminally. The free larva of Mulricotyle

* For simplicity, the larval postero-ventral sucker-like haptor which is destined to undergo drastic change is referred to as a “ventral disc” throughout development (Ed.).

I08

KLAUS ROHDE

purvisi has four ciliary tufts near the anterior margin of the ventral disc and six dorso-terminal ciliary tufts arranged in a circle (Rohde, 1968c, 1970c, 1971a).

FIG.31. Mitlticotylepurvisi, free larva. Ventral view (Rohde, 1968~).

All larval Aspidogastrea examined were about the same size. The larva of Multicotyle is 0.15-0.20 mm long, that of Lophotaspis vallei, according to Manter ( I 932), 0.15-0.21 mm long, and that of Aspidogaster indica, according to Rai (1964), 0.10-0.14 mm long. Various authors (Voeltzkow, 1888a; Stafford, 1898; Williams, 1942) respectively gave lengths of the larvae of Apidogaster conchicola as 0.13-0.1 5 mm, 0.17 mm and 0.16 mm. The free larva of Cotylogaster occidentalis is about 0.17 mm long (Wootton, 1966). B.

TEGUMENT AND CILIARY TUFTS

(Figs 32, 33)

Electron microscopy showed that the tegument of the free larva of Multicotylepurvisiis the syncytial outer layer ofcells sunk in the parenchyma (Rohde, 1971d). The basal cell membrane rests on a well-developed basal lamina and forms relatively few invaginations, some of which look like lateral cell membranes in close contact with one another. They do not reach the surface, but it cannot be denied that they may represent the remnants of lateral cell

THE ASPIDOCASTREA

I09

FIG.32. Multirotyle prrrvisi, free larva. Transverse and oblique sections through microfila (top) and section through tegument (bottom) (Rohde, 1971d).

110

KLAUS ROHDE

membranes of separate cells originally forming the outer layer. The tegument also contains many electron dense ovoid bodies, and in this respect the free larva of Mulricotyle corresponds closely to that of other parasitic platyhel-

FIG. 33. Multicofyle purvisi, free larva. Diagrammatic sagittal section through ciliary tuft and tegument (Rohde, 1971d).

minths. Unknown in other platyhelminths are long processes, 120-180 8,thick, which arise singly or in pairs from minute elevations of the tegumental surface. They have one central filament and several (usually 9-12) peripheral filaments between which radial connecting structures may be seen. They differ from microvilli in not having a cytoplasmic core and were named microfila. The layer of microfila, which covers most of the body (excepting the mouth and pharyngeal walls and most of the lumen of the ventral disc), is 6 pm thick at the anterior end of the body and may aid the ability of the larva to float (see VI .A). Electron microscopy showed that each ciliary tuft has a strongly lobed nucleus located in an invagination of the cell into the adjacent tegument.

FIG.34. Aspidogusrev conchicola, free larva with head glands. CGC-gland cells. (From Dollfus, 1958a, after Faust, 1922.)

duct, GC-gland

111

THE ASPIDOGASTREA

Nickerson (1900, 1902) stated that in the free larva of Cotj iogaster occiu’etitalis the sockets of the tufts may contain two nuclei, but as the exact number of tufts was not determined, he probably referred to nuclei of a ciliary band formed by several tufts (see IV.A), though in his figure, the nuclei seem to belong to one tuft. Tuft and tegument are in contact by large desmosomes, close contacts, and near the surface, by a septate zonula adhaerens. The cilia of the tuft are anchored by long striated rootlets running at an oblique angle to the axis of the cilia. Between the basal granules, elevations of the surface cell membrane are several times as large as those of the tegument, with a reticular mucoid layer extending between then1 and the basal granules. Electron dense fibres extend from the surface into the cell. The basal cell membrane and the cell membrane below the zonula adhaerens form many tubular invaginations. Mitochondria are dispersed throughout the cell and are especially large near the nucleus. Processes of the distal basal lamina anchor the tuft between the underlying muscles and parenchyma. The cilia are often thrown off in the process of fixation, but basal granules and rootlets of the cilia remain intact. This may correspond to the natural process of shedding the cilia after infection of the snails. C.

GLANDS AND CAUDAL APPENDAGE

(Fig. 34).

The presence of head glands and of a caudal appendage in Aspidogaster has been used often as evidence of a close phylogenetic relationship between the Aspidogastrea and larvae of the Digenea. However, head glands and caudal appendages are not present in all larval Aspidogastrea; they are absent in the larva of Muiticotyle, which, instead of head glands, has a number of scattered gland cells which open near the anterior, middle and posterior end of the body (Rohde, 1971a). D.

DIGESTIVE TRACT

(Figs 35, 36)

The digestive tract has been examined with the electron microscope only in the larva of Multicotyiepurvisi (Rohde, 1971g). The mouth, the prepharynx and the pharynx are lined by tegument similar to that of the body surface, except that microfila are absent. In addition to the ovoid bodies found in the tegument at the surface, there are inclusions with an irregular reticular structure. The caecum, on the other hand, is lined by a single layer of epithelial cells, which are in contact at their surface by means of zonulae adhaerentes. The surface of the epithelial cells is enlarged by numerous lamellae, which are interconnected repeatedly. The cytoplasm of the cells contains numerous vacuoles which are either empty, or contain a little or a dense mass of dark material. Some cells contain a well-developed endoplasmatic reticulum. The epithelial cells rest on a basal lamina which at irregular intervals forms shallow invaginations with thin muscle fibres surrounding the caecum. Nuclei of the parenchymal cclls and muscle fibres running in various directions are distributed below the epithelium. The mouth cavity is surrounded by longitudinal and radial muscles. In the

112

KLAUS ROHDE

pharynx, which also contains circular muscles, the radial muscles are especially well developed.

FIG.35.-Mu/ricoty/epurvisi, free larva. Horizontal section through caecum (Rohde, 19719).

E. PROTONEPHRIDIAL SYSTEM (Figs 37-39)

All free larvae of Aspidogastrea examined have three flame cells, two separate excretory bladder cells and two separate dorso-terminal excretory

THE ASPIDOCASTREA

113

FIG.36. Mulricotyle puuvisi, free larva. Section through surface of caecal epithelium, part of Fig 35 (Rohde, 1971g).

openings (cf. Nickerson, 1902, for Cotylogaster occidentalis; Osborn, 1903, 1905, for Cotylaspis insignis; Rai, 1964, for Aspidogaster iridica; Faust, 1922, for A . conchirola; Rohde, 1968c, 1970c, 1971d, in preparation, for Multicotyle purvisi). However, in Cotylogaster occidentalis, Wootton (1 966) found, contrary to the findings of Nickerson, two pairs of flame cells lateral to the intestine and two pairs of flame cells “associated” with the excretory bladder cells, A full account with figures has not yet been published and there is a possibility that some of the flame cells are lateral flames. In Multicotyle, a number of lateral flames (non-terminal ciliary flames) was found. In other species, such flames have not been seen, though they are probably present, as indicated by their presence in some adult aspidogastrids. (In the account given by Zacharias, 1895, for Aspidogaster, it is not clear whether he means flame cells or lateral flames.) Examined with the electron microscope, the excretory system of the larva of Mzrlticotyle is very similar to that of the adult, though the distal portion of the flame cells, which in the adult has membranes anchoring the top of the flame in the cytoplasm, was not examined.

114

KLAUS ROHDE

I

0.05 mm

FIG.37. Multicotyle purvisi, free larva. Protonephridial system. Lateral flames not shown (Rohde, 1971a).

The capillaries and ducts have surface lamellae, as in the adult. The cells forming the ducts contain numerous microtubuli. In contrast to the adult, vacuoles, inclusions or invaginations of the basal cell membrane were not found. The surface enlargement by the lamellae is less than in the adult. The terminal excretory cells (bladder cells) have strongly vacuolated cytoplasm. In each cell the terminal excretory duct forms two branches which run along the sides of the nucleus and reunite at the apical pole of the cell, forming a short unpaired duct opening to the exterior. The terminal excretory ducts in these cells possess numerous irregular surface lamellae which are sometimes interconnected (Rohde, in preparation).

F.

NERVOUS SYSTEM

(Fig. 40)

The anterior part of the nervous system of larval Multicotyle purvisi is very similar to that of the adult (Rohde, 1968a, 1971~).However, there are fewer anterior comniissures. The posterior part of the nervous system differs markedly from that of the adult, apparently due to different degrees of development of the ventral disc, which is still without alveoli. The larva differs from the adult in the following: (i) the intestinal and pharynx nerves are sometimes still separate; (ii) there are additional posterior internal and external ventral nerves; (iii) nerves dorsal to the posterior ventral nerve cannot clearly be correlated with the dorsal ramus of the posterior ventral nerve and the posterior dorso-

THE ASPIDOGASTREA

115

FIG.38. Mubicotyle purvisi, free larva. T. S. through flame cell (top) and capillaries with and without lateral flames (original).

116

K L A U S ROHDE

FIG.39. Multicotyle purvisi, free larva. Parasagittal section through excretory bladder cell (original).

THE ASPIDOGASTREA 3

2

117

I

!

0.05m m

FIG.40. Multicoryle purvisi, free larva. Diagram of nervous system. ILcompact body (probably sense receptor), 2 ~ o m m i s s u r aterminalis anterius, 3-nervus dorsalis anterior, 4-n. lateralis, 5-n. dorsolateralis anterior, 6-8-11. mediodorsalis (6-ramus lateralis, 7-r. intermedius, 8-r. medialis), 9-n. medialis, 10-n. ventralis internus anterior, 1 I-n. ventralis externus anterior, 12-n. pharyngealis. penetration into pharynx, 13-n. ventralis posterior, dorsal branches?, 14-n. intestinalis, 15-n. dorsalis posterior, 16-n. ventralis posterior, penetration into ventral disc, 1 7 ~ c o m p a c tbody, probably sense receptor (Rohde, 1971~).

118

KLAUS ROHDE

lateral nerve of the adult; (iv) the ventral disc is innervated by a commissure surrounding the lumen and two posteriorly connected longitudinal nerves i n its dorsal part. Best developed among the transverse nerves is a dorsal commissure in front of the ventral disc and this may correspond to the terminal ramus dorsalis of the posterior ventral nerve cord. (Figs 41, 42) In the free larva of Multicotyle, sensory capsules with a long sensory hair were demonstrated in vivo, in total preparations impregnated with silver, and in serial sections stained with urea-silver nitrate (Rohde, 1966a, 1968b). In total preparations, the area of the sensory capsules close to the surface appears SENSE RECEPTORS

G.

,...... .

,,..

'..

-___-FIG.41. Multicotyle purvisi, free larva. Papillae of two specimens. Ventral (top) and dorsal view (bottom) (Rohde, 1968b).

T H E A S P I DOGA S T R 8 A

119

FIG.42. Mulricoryle p w v i s i , free larva. Sections through approx 1 : 20000 one sense receptor (original). 6

120

K L A U S ROHDE

as small, ring-shaped papillae, which have an approximately symmetrical arrangement dorsally. On the ventral side, number and variability are greater, especially at the anterior end and on the ventral disc. It is often difficult to recognize a symmetrical distribution at all. Near the anterior end of the larva there are dorsal, ventral and lateral swellings of the tegument on each side of the body which may contain up to seven papillae. The number of papillae in two specimens was 115 and 1 1 1 . Sometimes structures other than sensory capsules may be impregnated with silver, e.g. particles on the tegument and secretory openings, etc. (cf. Rohde, 1968b). In addition to the sensory capsules, paired dark bodies occur in serial sections stained with urea-silver nitrate. In seven specimens, one pair (2 pin maximum dia.) was present dorso-laterally to the oral opening, one pair (2.5 pm) laterally in the mouth cavity, one pair in the middle ventral wall of the ventral disc (2.4 pm), and one pair (2.4 pm) near the posterior end of the disc lumen. In three of the seven specimens, there were additionally 1-2 pairs of less distinctly stained bodies near the most posterior pair. These bodies possibly represent sensory structures. A connection to nerve fibres has not yet been found. Under the electron microscope, the sensory capsules of the larva of Multicotyle were shown to have a complete zonula adhaerens at the surface. The capsules contain numerous vesicles with some electron dense material. A cilium arises from the capsule and penetrates through an opening of the tegument. It may be assumed that the ring-like papillae stained with silver nitrate are sediments of silver in the surface openings of the tegument. V. DEVELOPMENT A.

EGG,

CLEAVAGE AND LARVAL DEVELOPMENT WITHIN THE EGG

(Figs 43-46) Multicotyle, like the other species examined, has an ectolecithal egg. However, the yolk cells do not lie at one pole as in Cotylogaster and Macraspis, but surround the egg cell located in the centre of the egg as in Cotylaspis (Rohde, 1970c, 1971a). The eggs of Multicotyle are laid at the 1-3 cell stage, whereas those of Aspidogaster conchicola and A . indica contain already a completely formed larva (see Voeltzkow, I888a; Rai, 1964). In Aspidogaster and Cotylogaster, the developing embryo is surrounded by a membrane which is formed by special cells; such cells and membrane were not demonstrated in Multicotyle. A certain variability in the arrangement of the blastomeres in Multicotyle is already found at the 3-cell stage, and embryos with 5 or 8 cells are very variable. Here is a description of development of the egg of Multicotyle at 21-28°C (mean 25°C): The zygote divides into a large and a small cell. The 3-cell stage consists of cells of approximately the same size, though occasionally one or two of the cells may be slightly larger. Further cell division leads to an irregular cluster of cells, whose outline may vary. At a later stage, the embryo becomes round with a regular smooth surface but gradually becomes ovoid. The first pigment

THE ASPIDOCASTREA

121

FIG.43. Mirhicotyle purvisi. Lateral and ventral views of two eggs with 38- and 29-day-old embryos (Rohde, 1971a).

spots are seen 24 days after laying of the egg and at 27 days the ventral disc is advanced but not yet completed, and the pharynx and mouth are being formed. On the 29th day the pharynx is well developed and the rudiment of the intestine can be seen. In the lumen of the intestine and pharynx there are always 3-4 nuclei without visible cytoplasm. These nuclei disappear after hatching of the larva. The dorsal wall of the mouth cavity has a distinct swelling. 33 days after laying, the ventral disc, oral cavity, pharynx, intestine and ciliary tufts are well developed. The first flame cells were seen in a round embryo which had developed for 47 days at 20°C and corresponded approximately to a 20 days old embryo at 25°C. The yolk cells are gradually consumed by the embryo and a short time before hatching only few small yolk fragments remain. B.

HATCHING

The larvae of Multicotyle purvisi first hatch 25 days after laying of the egg (at 28-29°C; Rohde, 1968c, 1970c, 1971a). The eggs of Aspidogaster conchicola and A . indica are laid with fully developed larvae (Voeltzkow, 1888a; Rai, 1964) which hatch after about 24 h and 1 h respectively. Eggs of Lophotaspis vallei hatch “readily”, i.e. probably soon after laying (Manter, 1932) while those of L. orientalis contain early cleavage stages (Faust and Tang, 1936). The eggs of Cotylogaster occidentalis and Stichocotyle nephropis contain embryos before they leave the uterus (see Odhner, 1910; Dickerman, 1948); hence, the larvae probably also hatch soon after laying. On the other hand, Cotylaspis insignis and Multicalyx cristatus lay eggs with an undivided egg cell or only few cells (Osborn, 1903, 1905; Faust and Tang, 1936; Najarian, 1961), and here, therefore, a longer time to hatching can be assumed. In Multicotyle purvisi, the ciliary tufts beat already before hatching occurs and the larva turns round in the egg in such a way that the anterior end comes repeatedly to lie at the pole opposite theoperculum, though duringdevelopment

I22

KLAUS ROHDE

DEL . CC.

cv .

VIT

FIG.44. Aspidogaster conchicolu, development in egg. CC-calotte cells, C P - c a u d a l appendage, CV-vitelline balls, DEL-delimited part, E+mbryo, EB-oral sucker, Nnucleus, OA-ventral disc, S-rudiment of protonephridial system, VIT-yolk (from Dollfus, 1958a. after Voeltzkow, 1888a).

most larvae (41 of 43 larvae examined) are located with their anterior end towards it. All larvae hatch anterior end first, and all six larvae observed swam away after hatching. In one case it was observed that a larva could not completely escape through the operculum because of fungal growth on it, and tried to creep out of the egg. However, ciliary movements continued when the creeping movements ceased after I5 min. The releasing stimulus for hatching is light but if a culture containing mature eggs is kept in the dark, the larvae hatch without such a stimulus. The time taken to escape from the egg is usually only a few minutes whereas larvae of Aspidogaster indica need 1-3 h for the escape, apparently because they lack ciliary tufts and therefore are less motile (Rai, 1964). Manter (1932) observed that the larva of Lophotaspis vallei may hatch ventral disc first.

123

THE ASPIDOGASTREA

cv I

HM FIG.45. Cotyloguster rtiichaelis, development in egg. CVI-yolk membrane (from Dollfiis, 1958a. after Monticelli, 1892).

-

cells, HM--embryonic

0.1 rnm

FIG.46. Macvaspis elegms. Embryo and larva in egg (after Brinkmann, 1958).

124

KLAUS ROHDE

c.

DEVELOPMENT OF PARASITIC STAGE

(Figs 47-53)

Multicotylepurvisi develops in the snail during the first 3-4 weeks at 2CL23"C without forming alveoli. There is a simple increase in size. Later, alveoli are formed in the anterior part of the ventral disc. For some time, the original cavity of the disc remains behind the alveoli. New alveoli are being formed continuouslybehindtheexistent series and then enlarge; growth is by apposition and stretching. The rate of formation of alveoli depends on temperature and host species. In specimens with few (about 4) transverse rows of alveoli, the two excretory openings fuse; in larvae with about 9 transverse rows (the first

'.

I

.

.

.

.

.

.

.

I

,

.

.

30 Lo 50 60 70 80 90 100 110 T a p FIG.47. Multicotylepurvisi. Growth of larvae in snails. Abscissa: age of infection in days. Ordinate: number of transverse rows of alveoli. 0 , larvae in Pilu scututu at 23-30°C. 0, larvae in P.scututa at 20-23°C. A, larvae in Tuiapolyzonutu at 20-23°C. larvae in Bithynia (Digoniostomu) siumensis at 20-23°C (Rohde, 1971a). 10

20

+,

FIG.48. Multicotyle purvisi. Young worm with fused excretory pores; from snail (Rohde, 1971a).

THE ASPIDOGASTREA

125

alveolus is counted as a row of its own), the wall of the hind part of the intestine begins to thicken and this thickening leads to the formation of the genital rudiments and the genital duct rudiments, which extend alongside the intestine. In specimens with 17 rows of alveoli, the rudimentary genital organs and ducts are visible. The rudiment of the male duct extends from a cell cluster at the anterior end of the pharynx and this later develops to the tissue surrounding the genital opening and into the anterior part of the cirrus pouch. Extending

FIG.49. Mulficoryle purvisi. Young worms (17 and 18 transverse rows of alveoli) with rudiments of genital ducts (Rohde, 1971a).

along the wall of the intestine, it crosses the intestine and unites with the rudiment of the female genital duct, which passes along the other side of the intestine. The rudiments of the ducts at this stage are still connected with the intestinal wall. At a slightly later stage (about 18 rows of alveoli), the rudiments of the ducts begin to separate from the intestinal wall. However, they are still solid and follow a wave-like course. The rudiment of the cirrus pouch can be seen as a distinct swelling for some distance behind the pharynx. In specimens with 21 rows of alveoli, the testes, ovary, oviduct and Mehlis’ gland are visible as separate bodies connected to the solid genital ducts. The cirrus pouch is of definitive length as a solid cluster of cells, and the thick anterior part of the sperm duct is separated from the thinner posterior part. Laurer’s

126

K L A U S ROHDC

FIG.50. Mulricofyle puvvisi. Young worms with developing genital ducts and organs (Rohde, 1971a).

canal and the transverse vitelloduct are present as cords of cells. In specimens with 27 rows of alveoli, the ducts have lumina and all ducts and organs of the mature worm are present, though not yet of definitive size. In specimens with 33 rows of alveoli, the transverse vitelloduct has branched to form the paired ducts and the first vitelline follicles are formed. The vitellaria in the mid-body and fore-body are formed first. Eggs are first seen in the uterus in specimens with 34 rows of alveoli. The genital organs and the vitellaria, which in the fully mature worm are united posteriorly in a U-like formation, attain full size only in specimens with 44 or more rows of alveoli. The greatest count of rows was 50. As few fully mature specimens were examined, some variability in alveoli numbers cannot be discounted. The hind end of the completed ventral disc is the mirror image of the front end. In Stichocotyle nephropis growth is somewhat similar. According to Nickerson (1895), the suckers are formed at the hind end from a germinal mass of cell, and the hindmost ones are smallest. Cunningham (1887) and Nickerson (1895) mentioned that the number of alveoli varies with size of the body. In Cotylogaster occidentalis, alveoli are formed in the anterior part of the ventral disc as in Multicotyle (see Wootton, 1966). The data for Aspidogaster

THE ASPIDOCASTREA

127

FIG.50(b)

conchicola are contradictory. According to Voeltzkow ( I888a), the transverse

walls of the alveoli are formed first, beginning in front. After completed transverse division of the ventral disc the median and lateral longitudinal walls develop. Subsequently, the disc extends. However, the figures given by Voeltzkow show that the smallest animal without longitudinal alveolar walls had only 10 and two larger ones with such walls 15 and 17 transverse rows. This seems to support the data of Rai (1964) that two specimens of A . indica 53 days old and 1.2 mm long had 38 rows, whereas ten specimens 68-99 days old and 1.0-1.4 mm long had 50-54 rows. Two animals 134 days old and 1.7-2.2 mm long with eggs had the same number of alveoli. Thus, although in mature Aspidogaster there seems to be some variability in the number of alveoli (cf. Bychowsky and Bychowsky (1934, 1940) for A . limaroides and other species of Aspidogaster; Rai (1964) for A. indica; and Yamaguti (1963)

128

KLAUS ROHDE

for A . conchicola) there is nevertheless an increase in the number of alveoli during development (see also Shipley and Hornell, 1904). According to Michelson (1970), there is also a variability in the arrangement of the most posterior alveoli in A. conchicola. It is not clear whether the differences described are due to the age of the worms. Bychowsky and Bychowsky (1934) claimed that the number of alveoli is not correlated with body size in A. limacoides, but these authors did not examine very small specimens from molluscs. Possibly, there is growth by apposition at the hind end in Aspidogaster as in the two species mentioned. Williams (1942) found that the lateral alveoli in A . conchicola are formed before the median ones and according to Steinberg (1931) there is at first a longitudinal constriction in the ventral disc. Mucraspis eleguns grows, according to Jagerskiold (1 899) mainly between the ovary and the testes, a region which contains the uterus. This is probably a secondary effect due to the many eggs in the uterus, superimposed on the primary growth. Manter (1954) found that in this species the rudiments of alveoli appear first posteriorly and that the number of alveoli increases with age. At the posterior end, there is a blastema from which the alveoli differentiate (Jagerskiold, 1899; Burt, 1968). Because of the striking similarity of certain other characteristics, it seems unlikely that growth is basically different in various genera, but further studies are needed to ascertain whether there is a posterior growth zone in Aspidogaster and other genera. Williams (1942) studied the development of Aspidogaster conchicola and distinguished the following developmental stages : 1st larval stage with anterior, middle and posterior regions of the body set off from one another by external grooves, with all the features of the free larva, and with very little locomotory ability; 2nd larval stage with length increased from 0.15 mm to 0.275 mm and correspondingly larger anterior sucker and ventral disc with loss of body constrictions and greater powers of (leech-like) locomotion; 3rd larval stage with further increase in size (0.88-0.96 mm) and the disc almost filling the posterior one-quarter of the worm, but not yet divided into alveoli; 4th larval stage 1-2-1.44 mm long with ventral disc like that of the adult, but alveoli not fully formed, and with a slit-like opening to a shallow pit, the vestige of the opening of the larval disc. The development of the genital system in aspidogastrids is little known. Steinberg (1931) found the ovary rudiment in 0.4mm long specimens of Aspidogaster conchicola, 6-8 cells at the postero-ventral end of the intestine. The testis develops slightly later and ventro-lateral to the ovary rudiment. In specimens 0.8 mm long the rudiments of the male and female genitalia are developed and the ducts differentiate from cells located between the rudimentary testis and ovary. According to Voeltzkow (1888a), the genital ducts in Aspidogaster conchicola are at first solid. However, contrary to the observations of Steinberg, the vas deferens and the so-called “oviduct” (uterus) are supposed to grow as solid cell cords from the rudiments of the vulva and penis behind the testes and ovary, which are formed by an external unicellular layer of connective tissue and internal germinal cells. The rudiments of the vitellaria appear later. Cells of the ectoderm grow inwards and form the “receptaculum

THE ASPIDOGASTREA

129

130

K L A U S ROHDE

131

THE ASPIDOGASlREA

vitelli” (Laurer’s canal). Further studies are needed to clarify the contradictory descriptions of Steinberg and Voeltzkow. According to Brinkmann (1958), the genital organs of Macraspis elegans become visible in the following sequence: (i) testis, vas deferens and uterus; (ii) ovary; (iii) vitelline glands; (iv) genital aperture. D.

ALLOMETRIC GROWTH

(Figs 52-55)

According to Rohde (1970g, 1971b), three growth phases can be distinguished in the development of Multicotyle purvisi: 1. Growth of the free larva to the first developmental stages in snails, which are about twice or thrice the size of the free larva and have a ventral disc without alveoli. During this phase, pharynx, ventral disc and body grow with only minor shifts in their proportions. 2. Growth of intermediate developmental stages: the ventral disc grows relatively faster than the whole body and divides into alveoli; the part of the body in front of the disc becomes relatively and probably absolutely shorter; the rudiments of the genital organs are formed around the terminal portion of the intestine. 3. Growth of older developmental stages; more and more rows of alveoli are formed at the hind end of the disc (growth by apposition) and enlarge subsequently (growth by stretching); the genital organs and ducts develop.

The changes in the relative growth of the various organs and parts of the body lead to complicated allometric shifts. Thus, during the first growth phase, the ventral disc grows with negative allometry (allometric exponent 0-6), in the second with positive allometry (exponent 1*4), and in the third isometriA positive allometry cally to slightly negative-allometrically (exponent 1 of the anterior part of the body corresponds to the negative allometry of the ventral disc during the first phase. In the second phase, this part of the body does not increase in size at all, owing to strong forward growth of the ventral disc. Connected with this is a forward growth of the extraplantar nerves, and of the anterior ventral ramus of the posterior ventral nerve cord which supply the antero-dorsal wall of the ventral disc. In the third growth phase, the anterior part of the body shows a strongly negative allometric growth. The pharynx grows with slight negative allometry during the first two phases, with very strong negative allometry during the last phase. The growth of the genital organs (testes and ovary) is (almost?) exclusively limited to the last phase. Hence, they have a straight allometric line as known from other trematodes (Rohde, 1966~).Their allometric exponent is 2.4, i.e. they grow with strong positive allometry. For the first time, allometric curves composed of several straight lines with differing allometric exponents have been determined in a trematode. This may indicate that in taxononiic studies the question whether or not two trematode populations of different body size and varied proportions of organs and body belong to one species, cannot always be decided on the basis of a simple extrapolation of allometric gradients in one or both populations. In e l ) .

c

1 I

2

I

3 I

t

:

6 I

?

s

w

9

h)

iprnrn

\

FIG.53. Mulficofyle purvisi. 3rd growth phase. Numbers in top row indicate length of worms, those in vertical row indicate the position of rows of alveoli (Rohde, 1971b).

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THE ASPIDOGASTREA

rnm 10

f

7 5

3

1

0.5

0.2

01

I

0.1

I

0.2

0.5

1

t

3

I

1

5

,

I

7

I

I

I

tom

FIG.54. Multiroryle puuvisi. Growth of ventral disc. Abscissa: length of body. Ordinate: length of ventral disc. 0 , free larvae, not or lightly pressed. A, larvae from snails, not pressed. A, larvae from snails, pressed. 0,worms from turtles, pressed. 0 , worms from turtles, not pressed (Rohde, 1971b).

cases like Mulricotyle, all intermediate forms have to be known to establish beyond doubt whether or not two populations, which differ only in body size and relative sizes of organs, belong to one species. Allometric studies require material treated as uniformly as possible. Heat fixation is not always sufficient; in thickened forms such as Multicotyle and most amphistonies, light pressure during fixation can hardly be avoided, but this should be as well standardized as possible. Comparison of pressed and unpressed specimens of Mzrlticotyle showed that the allometric exponents in both are approximately the same, though there may be a parallel shift in the position of the allometric curve. The possibility must also be noted that populations of one species from different hosts have different allometries i.e. a parallel shift of (a different constant b in the allometric formula y = ha,

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KLAUS ROHDE

the allometric curves) and that different species have identical allometries (see the discussion by Rohde, 1971b). It should also be stressed that apart from the points mentioned (different allometries in same species from different hosts, similar allometries in different species) the use of allometries for taxonomic studies in trematodes is limited by the lack of a rigid body shape in this group. This tends to alter the variability of body and organ measurements and makes an exact mathematical comparison of populations more difficult. 1.0

-

0.5

-

0.2-

01

-

FIG.55. Multicoryle purvisi. Growth of testes. A and 0,pressed; not pressed. On the ordinate, the average diameter of the testes (length+ width/2) is given (Rohde, 1971 b).

VI. BIOLOGY A.

LIFE SPAN AND BEHAVIOUR O F FREE LARVAE

The free larva of Multicotylepurvisi may survive in water from several hours to approximately I + days. According to Voeltzkow (1888a), the larva of Aspidogaster conchicola never stays alive for more than two days. The larva of Multicotyle purvisi swims in the water or creeps on the bottom, as does the free larva of Lophotaspis vallei, which “swims rapidly” or creeps “extending its anterior end, releasing its posterior sucker, then pulling the posterior end up near the mouth” (Manter, 1932). When swimming, the larva of Multicotyle rotates around its longitudinal axis, with a screw-like forward movement. The average speed in rain water at 20°C is 0.22 mmjsec. The movements are sometimes undirected, or they are along the bottom, or straight up to the surface or down to the bottom. The larva often adheres to the surface with its anterior extremity, while the body is slowly rotating. Sometimes it sinks to the bottom with the hind end directed downward, and it may drift thus for some time in the water. Long flotation indicates that the specific gravity of the larva is similar to that of the water. This may be due partly to water

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trapped between the microfila (see IV. B). On the bottom or at the surface, the larva often seems to “explore” the substratum with its anterior end. It does not actively swim towards the host, but is carried into the mantle cavity by the inhalent current of the snail, often while it is drifting in the water. The larva shows positive phototaxis. B.

INFECTIVITY OF FREE LARVAE

Voeltzkow (1888a) infected bivalves with larvae of Aspidogaster conchicola, and Rai (1964) infected various species of bivalves with larvae of A. indica, which could not be transmitted to vertebrates. Similar results were obtained in infection experiments with Multicotyle purvisi (Rohde, 1968c, 1970c, 1971a). Feeding of large numbers of larvae and eggs containing mature larvae to 11 specimens of tortoises (Siebenrockiella crassicollis Gray and Cuora amboinensis Daud.)-natural hosts of the parasites-did not lead to infection. When a tortoise was kept in a small container with numerous mature eggs, the results were negative likewise. On the other hand, four species of gastropods became infected after exposure to free larvae of Multicotyle purvisi. The species concerned are : Pila scutata (Mousson), Ampullariidae; Taia polyzonata Frauenfeld, and Sinotaia martensi (Frauenfeld), Viviparidae; Bithynia (Digoniostoma) siamensis kintana De Morgan, Bithyniidae. It seems safe to conclude that vertebrates are not receptive to free larvae of aspidogastrids, and that there is no strict host specificity with regard to the mollusc host. C.

ROUTE OF INVASION IN MOLLUSC

There are no empirical data on how the larvae migrate from the mantle cavity of molluscs into their tissues. Voeltzkow (1888a) discussed two possibilities-by way of the kidney funnel or the intestine. He considered the first route improbable because the ciliary beat in the funnel is directed outwards and because the opening of the funnel is very small. Furthermore, he never found young stages of Aspidogaster conchicola in the kidneys or near their opening. He considered the second route to be more probable because 8-14 days after infection he once found a single worm and once two young worms in the intestine. The parasites are most common in the pericardium and the pericardial gland, near which the intestinal wall is particularly thin. Apparently the latter route is also taken by Cotylogaster (Cotylogasteroides) occidentalis. According to Wootton (1966), larvae of this species are ingested by mussels and begin their development in the stomach region. D.

LOCALIZATION IN MOLLUSC AND SPECIFICITY OF INFECTION

All aspidogastrids which are well known, i.e. found repeatedly, occur in a variety of molluscs and in a number of different sites. Thus, Aspidogaster conchicola has been found in various genera of freshwater bivalves (Unionidae, Mutelidae, Sphaeriidae, Corbiculidae) and snails (Prosobranchia) (Baer, 7

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1826, 1827; Dujardin, 1845; Aubert, 1855; Leidy, 1858; Voeltzkow, 1888a; Linstow, 1889; Monticelli, 1892; Sonsino, 1892; Stafford, 1896; Kelly, 1899; Kofoid, 1899; Sinitzin, 1905; Stunkard, 1917; Faust, 1922; Steinberg, 1931 ; Williams, 1942; Van Cleave and Williams, 1943; Dollfus, 1953, 1959; Hendrix and Short, 1965; Gentner and Hopkins, 1966; Hendrix, 1968; Pauley and Becker, 1968; Michelson, 1970; see also “Index Cat.”). The worms live in the pericardial cavity, liver (doubted by Voeltzkow, 1888a), pericardial gland, intestine, on the mantle, on or in the kidneys of bivalves, and in the testes, liver (digestive gland) and “visceral mass” of snails, although the pericardial and kidney cavities of bivalves seem to be the most common sites of infection (Kelly, 1899; Hendrix and Short, 1965). Michelson (1970) found the parasite in the ducts of the digestive gland of snails and noted tissue reactions around the worms and throughout the tissue of the gland. Occasionally, the worms are found encapsulated in the tissues (Stafford, 1896; Kelly, 1899; Pauley and Becker, 1968). According to Baer (1827), Redi often found worms resembling Aspidogaster in the gill chamber of ascidians (A. ascidiae Diesing, 1858), but this finding has yet to be verified. Bychowsky and Bychowsky (1934) found Aspidogaster sp. in the pericardium of marine bivalves (Cardium, Adacna) and A . antipai Lepsi was found in Unionidae (Dollfus, 1959). Rai (1964) demonstrated A . indica in the pericardial cavity and on the gills of various freshwater bivalves after experimental infection. Cotylaspis insignis occurs on the foot, on the internal gills, in the mucus of the host’s surface, in the gills and in the mantle and suprabranchial cavities of freshwater bivalves (Leidy, 1857, 1858; Osborn, 1898, 1903; Kelly, 1899; Kofoid, 1899; Stunkard, 1917; Hendrix and Short, 1965; Hendrix, 1968). Kelly (1899) found this species in Anodonta sp, most frequently “adherent to the surface of the host in the angle between the inner gill and the visceral mass”, and only in rare heavy infections “the flukes extended well out upon the inner surface of the gill” or “were crowded down upon the abdominal surface”, whereas in another species, they also infected the “tubes of the inner gills” and in Lampsilis ellipsis “occasionally even . . . those of the outer gills”. One Cotylaspis was found in the pericardium. According to Stromberg (1971 and personal communication), this species lives predominantly in Anodonta and is most commonly found in the mucus of the visceral mass and suprabranchial space, but sometimes also on the gills, mantle and labial palps; but he never found specimens inside the molluscs. Similarly Osborn (1903, 1905) stressed that the species never occurs in the pericardial and kidney cavities and that it should be regarded as a commensal and not a parasite, keeping the surface of the host clean, not feeding at the host’s expense. Najarian (1955, 1961) on the other hand, mentioned as sites of infection for a species, which he first tentatively identified as C. insignis and later described as C. reelfootensis (which was regarded as identical with C . insignis by Hendrix and Short, 1965), not only foot and gills, but also the surface of the heart and kidneys, and inside the gill filaments.Apparently, different populations of this speciesbehave differently with regard to species of host and sites of infection. Thus, in the populations studied by Stromberg (1971 and personal communication, compare above) Cotylaspis insignis lives predominantly on (but never in) Anodonta though it is

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occasionally found in other species, while in the populations examined by Kelly (1899) several genera of Unionidae show a bigh frequency of infection and a species of Anodonta has “usually” some worms in the gills. Both authors examined many specimens of several species of Unionidae and, thus, a comparison of their data is justified (see also Osborn, 1903; Najarian, 1955; Hendrix and Short, 1965). Cotylogaster occidentalis was found by Dickerman (1948) in freshwater snails and by Kelly (1927) and Wootton (1966) in the intestine of freshwater mussels. According to Wharton (1939) Lophotaspis vallei occurs in marine snails and L. macdonaldi (Monticelli) in the siphon of a marine species of prosobranch (Macdonald, 1877). The related species L. corbiculae Moriya was found in the pericardium of the bivalve Corbicula leana Prime and L. margaritiferae (Shipley and Hornell) in the pericardium of marine bivalves (Shipley and Hornell, 1904; Moriya, 1944). Only one immature specimen of Lobatostoma sp. was collected from more than one thousand bivalves of the species Donax variabilis (see Hopkins, 1958). Records of these species are few and, thus, it is likely that they have a wider range of hosts and sites of infection than is known. Lissemysia ovata Tandon has been recorded from the gills of freshwater snails and bivalves (Tandon, 1949). Stichocotyle nephropis differs from all other aspidogastrids in respect of sites of infection and hosts. Immature specimens were found encysted in the intestinal wall of the marine crustaceans Nephrops norvegicus L. (see Cunningham, 1884, 1887) and Homarus americanus H. M. Edwards (see Nickerson, 1895; Linton, 1940). In the latter species the parasites were found only in the posterior part of the alimentary tract. E.

SEXUAL MATURATION I N MOLLUSCS

Mature specimens of the following species were found in molluscs : Aspidogaster conchicola (see Baer, 1827; Aubert, 1855; Voeltzkow, 1888a; Stafford, 1896; Steinberg, 1931 ; Williams, 1942), A . indica (see Rai, 1964), Cotylaspis insignis (see Osborn, 1898, 1903, 1905; Hendrix and Short, 1965), C. reel.footensis ( ?insignis) (see Najarian, 1961) ; Lissemysia ovatu (see Tandon, 1949), Cotylogaster occidentalis (see Dickerman, 1948), and Lophotaspis corbiculae (see Moriya, 1944). Aspidogaster indica produces the first eggs about 129 days after infection of bivalves (Rai, 1964). For technical reasons, the development of Multicotyle purvisi in snails could not be traced until sexual maturity was reached. The evidence available at present indicates that aspidogastrids can complete their life cycle in molluscs. Lophotaspis vallei has not been proved to be an exception, though Wharton (1939) found the immature stage (“nymph”) in the conch Fasciolaria gigas (L.) and the adult in the turtle Caretta caretta (L.). Of the former, only six specimens from “some” snails were examined and of the latter, only two specimens from one turtle. A possible exception is Stichocotyle (see section IX). There is no evidence for the assumption that the vertebrates were originally obligatory hosts and that aspidogastrids have only

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secondarily acquired the ability to reach sexual maturity in molluscs due to progenesis. There is an interesting seasonal change in the activity of the genital glands of Apidogaster conchicola. Steinberg (1931) found specimens in bivalves from the Bay of Newa (Baltic Sea); they had been sexually active in the preceding year, and the testis enlarged and became active only at the beginning of June. The first spermatozoa were produced in July. In the summer, two types of large cells appeared in the testis, one type with a small nucleus and much chromatin and another indistinguishable from oocytes, which degenerated at the end of the summer and in autumn. In September, fewer spermatozoa were formed and in October, the testis became smaller as its cells began to degenerate, its cavity becoming filled with loose embryonic tissue. During winter, most of this tissue degenerated. The spermatozoa in the seminal vesicle survived and served for fertilization in spring. Connected with the changes in the germinal tissue was a change in the structure of the sheath around the testis. Ventrally glandular cells existed in autumn which hardly changed during winter but became more active in spring. These cells seemed to decrease in number when spermatogenesis began and they were much fewer in July and August. Such changes in the sheath were seen also in immature specimens. Oogenesis began in May. Few oocytes entered the oviduct in June. In the middle of July the ovary reached maximum size and produced many oocytes. In September, fewer oocytes were formed and at the beginning of October activity ceased. The ovary became smaller and degeneration of its cells occurred in winter. Immature worms showed similar changes. In Cotylaspis insignis the testis was inactive in May and active in July (see Osborn, 1903, 1905). F. INFECTIVITY TO VERTEBRATE HOST AND GROWTH THEREIN

As far as is known, free aspidogastrid larvae are not infective to vertebrates (see V1.B). However, young and adult worms can be transferred to them. Dickerman (1 948) infected five small fishes (Aplodinotusgrunniens Rafinesque"sheephead") with Cotylogaster occidentalis. Nine days after the first of five feedings, five worms were found in the terminal portion of the intestine of each fish. The fish Ambloplites rupestris could not be infected. Van Cleave and Williams (1943) found one specimen of Aspidogaster conchicola in the stomach of a tortoise (Pseudemys troosti) 14 days after feeding six mature parasites to it. Two specimens of the tortoise Graptemys sp. remained negative after injection of worms into their cloaca. None of these writers determined whether or not worms may grow in the vertebrate host and how long they can survive. In my experiments (Rohde, 1968c, 1970c, 1971a) two snails with 28-day-old infection were fed to each of two tortoises of the species Siebenrockiella crussicollis. Six of seven remaining snails of the same population had 1-3 larvae with the first alveoli each but dissection of the tortoises 15 days after infection did not reveal any parasites. Two further specimens of Siebenrockiella were given respectively one or two 105-day-old Multicotyle from a snail. Three and 14 days after infection one specimen was found in each tortoise.

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The speciniens had 10 and 11 transverse rows of alveoli, as compared with 10, 13 and 13 rows in the remaining smallest and largest specimens from the same snail, i.e. there was no demonstrable growth. Three specimens of Siebenrockiella were fed with one immature Multicotyle each from other tortoises. The specimens had 28, 32 and 40 rows of alveoli; 14-15 days after infection no worms were found in the animals. The number of animals used in these experiments is small, and further experiments are necessary to establish whether or not only certain developmental stages are infective to vertebrate hosts, and whether or not the worms can grow in them. It seems probable, however, that very young worms from snails cannot be transferred to vertebrates, because specimens of Multicotyle from naturally infected tortoises had never less than 17 or 18 transverse rows of alveoli (counting the first single alveolus as a row of its own).

G . LOCALIZATION IN VERTEBRATE AND SPECIFICITY OF INFECTION

The range of vertebrate hosts is as wide as that of molluscs. Hosts are sharks, rays, holocephalans, teleosts and tortoises in the sea and in freshwater. The best known species, i.e. Aspidogaster conchicola, has been found in a number of genera belonging to several families of freshwater teleosts and in freshwater tortoises (Kofoid, 1899; Faust, 1922). A . limacoides occurs in many genera of freshwater and marine teleosts (Diesing, 1834,1835;Voeltzkow, 1888b; Monticelli, 1892; Popov, 1926, Bychowsky and Bychowsky, 1934, 1940; Osmanov, 1940; Agapova, 1956; Dollfus, 1959). Lobatostoma ringens (Linton) lives in various genera of marine teleosts (Linton, 1905, 1907, 1910; MacCallum and MacCallum, 1913; Manter, 1931, 1947). For data on other species see Olsson, 1869, 1896; Poirier, 1886; Monticelli, 1892, 1906; Odhner, 1898, 1910; Jagerskiold, 1899; Stossich, 1899; Looss, 1901, 1902; Nickerson, 1902; MacCallum and MacCallum, 1913; Barker and Parsons, 1914; Stunkard, 1917; Kelly, 1927; Rumbold, 1928; Simer, 1929; Manter, 1931, 1932, 1940, 1954; Eckmann, 1932; Ward and Hopkins, 1932; Sinha, 1935; Faust and Tang, 1935, 1936; Wharton, 1939; Linton, 1940; Araujo, 1941; Dawes, 1941; Dayal, 1943; Rausch, 1947; Dickerman, 1948; Rawat, 1948; Chauhan, 1954; Sogandares-Bernal, 1955; Brinkmann, 1958; Dollfus, 1958b, 1959 and especially the review in Dollfus (1958a); Siddiqi and Cable, 1960; Pavlovskii (1962); Rohde, 1963; Rai, 1964; Yamaguti, 1968. Most aspidogastrids occur in the intestinal tract of vertebrates. Exceptions are Macraspis elegans in the gall bladder and bile ducts of Chimaera monstrosa L. and the gall bladder of Callorhynchus rnilii St. Vincent (see Olsson, 1869, 1896; Manter, 1954; Brinkmann, 1958), Macraspis sp. in the gall bladder of sharks (Manter, 1954), Macraspis (Multicalyx) cristata (Faust and Tang) in the spiral valve of rays (Faust and Tang, 1936, one specimen sent for identification) and in the gall bladder of sharks and rays (Dollfus, 1958b; Stunkard, 1962),and Stichocotyle nephropis in the bile ducts of rays (Odhner, 1898,1910; Linton, 1940). Immature Macraspis sp. were also found in the intestine of marine teleosts (Menticirrhus arnericanus (L.), see Manter, 1931).

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KLAUS ROHDE H.

SURVIVAL OF THE ADULT OUTSIDE A HOST

Aspidogaster conchicola Baer may, according to Baer (1827) stay alive in “pure water” for up to a fortnight. According to Dujardin (1845), it survives in water more than 12 days, but according to Aubert (1855) usually 5-6 days (in one case however more than 20 days). Voeltzkow (1888a) kept this species alive for up to 5 weeks in a mixture of water and saline solution. Were it not for an “accident”, the specimens might have survived longer. Van Cleave and Williams (1943) kept specimens active for at least 20 days in 0.75 % saline in a refrigerator. One specimen died at the 37-38th day. Somewhat different data were given by Rai (1964) for Aspidogaster indica Dayal. Adults of this species remained alive only up to 36 h in N-NaCl solution changed every 6 h. The number of animals used is not given and it is likely that with large numbers of worms and with different saline concentrations some worms at least may survive longer. In the experiments of Osborn (1903, 1905), Coiylaspis insignis Leidy survived for more than 3 weeks in water, N-NaCl solution, or equal parts of both liquids. Najarian (1955) kept the same species alive for several days in refrigerated tap water. Stichocotyle nephropis Cunningham was still alive after 24 h in sea water but the survival time was not determined. Adult Lissemysia ovata Tandon, kept in water at room temperature and fed twice a day on gills of freshly dissected snails survived for 6-11 days 17 h, although eggs were not laid (Tandon, 1949). Adult Multicofyle purvisi remain active for up to 8 days in 0.7% saline solution at 19-20°C (Rohde, 1971a). Four out of five species of aspidogastrids examined have a survival time outside a host in simple media, which is much greater than that of most Monogenea and Digenea (for details see Rohde, 1971a). It is also relatively easy to keep aspidogastrids alive in more complex media. Thus, Van Cleave and Williams (1 943) kept Aspidogaster conchicola alive for various periods in the following media: (i) mussel Ringer solution at 2-9°C (survival time 21-39 days); (ii) Hedon-Fleig’s solution at 2-9°C (s. time 29-38 days), at room temperature (s. time 20 days); mussel blood at 2-9°C (s. time approximately 75 days). Michelson (1 970) kept Aspidogaster conchicola axenically in glass roller tubes (20 x 150 mm) containing 5 ml of a modified Unionid Ringer’s solution with 5 % sterile clam blood from Anodonta implicata, 100 units/ml penicillin G, 100 pg/ml streptomycin sulphate and sufficient sodium bicarbonate to yield pH 7.9. The tubes were sealed but opened every 10 days. Mean survival times were 92, 65 and 49 days at 4”, 20” and 25”C, respectively, maximum survival times 135, 78 and 60 days. Many worms laid eggs, most of which hatched, the larvae staying alive for up to 28 days, without developing to adults.

VIT. PHYLOGENETIC POSITION OF ASPIDOGASTREA The Aspidogastrea seem to combine features of the Monogenea and Digenea. While their development is direct, without an alternation of generations and hosts as in Monogenea, they agree with the Digenea in the nature of their hosts (molluscs and vertebrates), and in several morphological character-

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istics. The structure of the caecum and the presence of head glands in some species and of separate excretory pores at least in young worms led some authors even to the assumption that the Aspidogastrea are highly differentiated, progenetic rediae (Leuckart, 1879-1901 ; Osborn, 1903, 1905; Faust, 1922). On the other hand, the aspidogastrids are distinctly different from all other platyhelminths, especially in the development of the ventral disc and a septate oviduct, as well as in their direct development in molluscs. Accordingly, the Aspidogastrea was often regarded as a sub-group of the Digenea (see Monticelli, 1888; Odhner, 1902; Fuhrmann, 1928; Faust, 1932; Linton, 1940; Skrjabin, 1952) or as a suborder, order or even subclass (see Burmeister, 1856; Monticelli, 1892; Pratt, 1900; Faust and Tang, 1936; Williams, 1942; Dawes, 1946, 1947; Dickerman, 1948; Hyman, 1951; Chauhan, 1954; Dollfus, 1956, 1958a; Yamaguti, 1963; Rai, 1964; Price, 1967). Some authors considered Aspidogastrea as intermediate between Digenea and Monogenea (e.g. Faust and Tang, 1935, 1936). These writers (Faust and Tang, 1935) even considered returning the group to the Monogenea, as did some older authors, e.g. Cunningham (1 884, 1887), who put these trematodes in the polystomes. Baer and Joyeux (1961) regarded them as an archaic group more closely related to the Digenea than to the Monogenea. Stunkard (1946, 1962, 1963, 1970), examined the historical data, concluding that aspidogastrids show a clear relationship to the Digenea, are primarily parasites of molluscs and represent a primitive and ancient group with the archaic features of a simple caecum, direct development and adults occurring in molluscs. The Digenea, according to Stunkard, acquired polyembryonic asexual reproduction in molluscs and their sexual maturation was progressively shifted to stages in vertebrates. The data presented in this review make it feasible that the Aspidogastrea are ill-adapted to parasitism, have many archaic features, form a group of their own, and are closely related to the Digenea. Their direct life cycle without alternation of generations and the use of molluscan hosts are primitive characteristics. Poor adaptation of the aspidogastrids to parasitism is indicated by a relatively long survival time outside a host in simple media, by their low host and organ specificity, by an extremely complex nervous system with a great variety of sense receptors, and by the small number of species. Long survival time outside a host and low specificity show that the parasites depend on their hosts only for very unspecific factors. Furthermore, it is generally accepted that a strict host specificity is usually the result of a long phylogenetic adaptation of a parasite to its host, though exceptions may occur (“secondary host range expansion”, see Osche, 1957, 1962). A complicated nervous system and many and varied sense receptors are more characteristic of free-living animals than of parasites. Exceptions to this rule are very rare (see Rohde, 1968a). Finally, the small number of aspidogastrid species (fewer than 40) is evidence of relatively unsuccessful adaptation of this group to a parasitic mode of life. Archaic features are the low degree of oligomerization (reduction in the number of identical parts) of the nervous system (Rohde, 1968a, 1971c), of the microtubuli of the spermatozoon (Rohde, 1971f),of the number of papillae on the free larva (Rohde, 1968b), and, in Srichocotyle, of the number of suckers.

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A low degree of oligomerization is usually an indication of a low phylogenetic status (see Rensch, 1954). The occurrence of oligomerization in trematodes has been shown by Ginetzinskaya (1963, 1968) and Lim (1968). The simple sac-like caecum is probably an archaic characteristic derived from the turbellaria-like ancestors of the trematodes. The following reasons can be given for putting the Aspidogastrea in a group of their own: the presence of a ventral disc or (exceptionally) a row of suckers; the presence of a septum separating ventral and dorsal parts of the body; growth by apposition and stretching-or in other words the presence of a posterior germinal zone; the presence of a septate oviduct which is partly ciliated; the presence of a simple sac-like caecum; development without alternation of hosts and generations. A sac-like caecum occurs in other parasitic platyhelminths but only in rediae and some adult Monogenea and Digenea. In the latter, however, paired caeca may have been reduced, as partially indicated by intermediate forms with short paired caeca (Lutztrerna), or an unpaired caecum opening on the ventral side of the body (Gasterostomata). Development without alternation of generations occurs also in the Monogenea and many Cestoda. However, the hosts are different and there are no reasons to assume a closer phylogenetic relationship of the Aspidogastrea with one of these groups. The Aspidogastrea are most closely related to the Digenea, as shown by the presence in both groups of a sucker or a ventral disc which divides to form alveoli or multiplies in adult aspidogastrids, a Laurer’s canal and posterior excretory pores. The ultrastructure of the protonephridial system is very similar in the Digenea and Aspidogastrea. In adult Multicotyle purvisi and Fusciolu hepatica, the flame cell opens into a capillary with many microvilli, while all the larger capillaries and ducts have lamella-like evaginations of the surface in their lumen. The capillaries and ducts are surrounded by a connective tissue sheath, through which parenchymal processes penetrate, In the Monogenea and Cestoda examined, such a sheath is absent. The capillaries and ducts in the Monogenea have a reticular surface and those of cestodes are covered by numerous microvilli (Rohde, 1971h; in preparation). The ultrastructure of the tegument is similar in all groups of parasitic platyhelminths examined. Only the cestodes differ significantlyin the presence of tegumental microvilli, obviously a consequence of their particular way of absorbing food through the surface. In Monogenea, such microvilli are not consistently present (Rohde, unpublished); their tegument agrees in all essential details with that of Digenea and Aspidogastrea. Hence, the ultrastructure of the tegument cannot be used for establishing phylogenetic relationships among the parasitic platyhelminths. Whether the ultrastructure of the spermatozoon can be used for this, is still open to doubt (see Rohde, 1971f, h). That molluscs must be considered the original hosts of the Aspidogastrea, can be deduced from the findings of sexually mature worms of several species in molluscs. In no case has it been demonstrated that sexual maturity may be reached in vertebrates or even that the worms can grow in these hosts (see Rohde, 1971a). There is no evidence that maturation in molluscs is due to progenesis. This excludes quite clearly the possibility that aspidogastrids are

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neotenic rediae. The presence of a highly complicated nervous system and numerous sense receptors in Multicotyle as against a very simple nervous system and few receptors in rediae is further proof against this assumption. Evidence for an alternation of generations has not been found in any of the species examined. VIII. DERIVATION OF DIGENEAN LIFE CYCLES ASPIDOGASTREAN LIFECYCLE

FROM THE

The foregoing discussion shows that the Aspidogastrea have many archaic features, are ill-adapted to a parasitic way of life and are closely related to the Digenea. This suggests that they stand close to the root of the Digenea, i.e. to the hypothetical Prodigenea. If this be so, it is probable that the Prodigenea also had a simple one-host life cycle without alternation of generations, that molluscs were their only hosts, and that they could survive in vertebrates when ingested along with the molluscan hosts. Based on this premise, Rohde (1971h) derived the complicated life cycles of Digenea in the following way, not considering obviously secondary complications such as the various types of threehost cycles, and not excluding the possibility that some of the steps given were phylogenetically taken in a different sequence. The Prodigenea, like the Aspidogastrea today, were able to survive in a vertebrate host when ingested. The fact that most Digenea are parasites of the intestine or organs communicating with it indicates that the oral route of infection is primitive, and since quite a few vertebrates feed on molluscs, it is more probable that they first acquired the Prodigenea by eating molluscs than by accidentally swallowing free-living worms. Furthermore, if free-living cercariae are ancestral forms, one would expect that the primitive mode of infecting the final host was through the skin, because cercariae have readily acquired the means of penetrating into their intermediate hosts, including vertebrates, while they are only very rarely ingested by them. The Prodigenea probaply became more and more dependent on vertebrates and had to find ways to increase the number of their offspring and disperse them in order to secure transmission to these hosts. Fecundity was achieved by multiplication of the larvae derived from one egg; this was possible only by transferring larval development from body and organ cavities into the tissues of the molluscs, where more nutrients were available. That such a transfer is possible, is indicated by the fact that Aspidogaster sometimes occurs in the tissues of molluscs. Dispersal was achieved by “inventing” cercariae, which are not relicts of originally free-living forms, but purely stages for dispersal, as is indicated by the loose attachment of their tail, which is easily cast off. The findings that Cotylaspis can live on the surface of molluscs and that several Aspidogastrea can survive outside a host for long periods, also suggests that primitive digeneans could escape from the molluscs. Intercalation of the cercarial stage also made possible infection of vertebrates which do not feed on molluscs. As many vertebrates are predators, encystment of many cercariae was transferred from the free environment into a second intermediate host which

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is eaten by the final host. Progenesis in the second intermediate host and omission of the final host occurred in those flukes which infect their “final” host not by ingestion but by penetration through the skin (e.g. schistosomes). The scheme propounded has the advantage over all other hypotheses on the origin of life cycles in Digenea, that it proceeds from a group close to the hypothetical ancestors of the Digenea; most of the features characterizing Digenea (development in molluscs tissues, escape from mollusc, survival in vertebrate) are represented in a rudimentary form already in this group, thus offering something upon which selection can act. No evidence has yet been found in the aspidogastrids for multiplicative stages in molluscs, for a tail in dispersal stages (though the larvae of Aspidogaster conchicola and A . indica have short caudal processes), or for sexual maturation in vertebrates. The assumption that maturation was gradually transferred to vertebrates, would explain how selection pressure (to enhance the probability of reaching vertebrate hosts) would gradually lead to the development of multiplicative stages in the molluscs and to dispersal stages. As in all groups, not all intermediate forms have survived for the reason that radically new features often originate in small, phylogenetically unstable populations. A rapid transformation leads to a stable group such as the Digenea which undergoes adaptive radiation, with numerous species in many habitats. IX. SOMEUNRESOLVED PROBLEMS

Of all the Aspidogastrea, only Stichocotyle has been found in the pre-adult stage encysted in crustaceans, while mature forms are known from rays. Possibly, crustaceans are purely accidental hosts in which an abnormal parasite is encapsulated (as Aspidogaster conchicola is sometimes en.capsulated in the tissues of molluscs-see Stafford, 1896; Kelly, 1899; Pauley and Becker, 1968). The possibility cannot be ruled out that the life cycle of Stichocotyle differs from that of other aspidogastrids, including perhaps molluscs as first host, crustaceans as transport hosts and rays as definitive hosts. This implies that a life cycle intermediate between that of most Aspidogastrea and that of the Digenea occurs in an aspidogastrid, but further elucidation of the development of Stichocotyle is highly desirable. It has been shown for some species that free larvae of aspidogastrids are not infective to vertebrates, while mature worms or worms at an intermediate stage of development from molluscs can be transferred to them. For an understanding of the adaptation of aspidogastrids to parasitism it would be important to find out at what stage the worms become infective, and in what physiological features non-infective and infective stages differ. For clarifying how well adapted the worms are to life in vertebrates, it would be important to demonstrate whether they can grow and how long they may survive in them. Long survival time outside a host in simple media and low degree of organ and host specificity indicate that aspidogastrids depend on their hosts for very unspecific and as yet undemonstrated factors. A knowledge of these factors (osmotic pressure, acidity, food?) would be important in a study of the evolution of physiological adaptation to parasitism.

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Assuming that the Aspidogastrea are close to the root of the Digenea, i.e. primitive trematodes derived from rhabdocoelidan stock, an elucidation of the route of infection in the mollusc might throw light on the mode of entry of the original free-living platyhelminths into their hosts. Study of this route is a highly desirable matter for the near future.

ACKNOWLEDGEMENTS I wish to thank Prof. J. F. A. Sprent for working facilities at the Department of Parasitology, University of Queensland, Brisbane, where this review was written. Prof. Dr. W. Stockem, Bonn, kindly checked the labels of the electron micrographs published in Zool. Jahrb. Anatomie. Prof. Ben Dawes improved the style of the manuscript in many places. The electron micrographs were taken at the Institut fur Cytologie und Mikromorphologie, Bonn, and the Institut fur Topographische Anatomie, Essen. I thank Prof. Dr. Wohlfarth-Bottermann and Prof. Dr. Brett-Schneider for permission to work there. Figs. 1-6, 12, 14-17, 18, 20-22, 25-29, 32, 33, 35-37, 40, 43, 47-50, 52-55 were reproduced with the permission of VEB Gustav Fischer Verlag, Jena; Figs. 7, 8 (left), 19, 31 with the permission of Springer Verlag, Berlin, Heidelberg, New York; Figs. 13, 30, 34, 41, 44-46, 51 with the permission of Dr. R. Ph. Dollfus, Paris. REFERENCES (References marked with an asterisk do not deal with Aspidogastrea.) Agapova, A. I. (1956). Parasites of fish in reservoirs of western Kasachstan Trudy Znst. Zool. Akad. Nauk Kasakhst. SSR 5 (Paras.)5-60 (in Russian). Araujo, T. L. (1941). Nota sobre un Trematoide Aspidogastrea de tartaruga marinha. Bol. Znd. animal. N.S. 4, 184-186. Aubert, H. (1855). Uber das Wassergefassystem, die Geschlechtsverhaltnisse,die Eibildung und die Entwicklung des Aspidogaster conchicola mit Beriicksichtigung und Vergleichung anderer Trematoden. Z. wiss. Zool. 6, 349-376. Baer, J. G. and Joyeux, Ch. (1961). “Classe des Trkmatodes”. In Grass6, “Trait6 de Zoologie”, 4. Baer, K. E. v. (1826). Sur les Entozoaires ou vers intestinaux. Bull. SOC.Nut. GPoI. 9, 123-1 26.

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Rohde, K. (1970e). The ultrastructure of the protonephridial system of the adult and the free larva of Multicotyle purvisi. Proc. 2nd Int. Congr. Paras. J. Paras. 56, 289 (Abstract). Rohde, K. (1970f). Ultrastructure of the marginal bodies of Multicotyle purvisi. Proc. 2nd Int. Congr. Paras. J. Paras. 56,289 (Abstract). Rohde, K. (1970g). The growth of Multicotyle purvisi Dawes (Aspidogastrea). Proc. 2nd Int. Congr. Paras. J. Paras. 56,290 (Abstract). Rohde, K. (1971a). Untersuchungen an MulticotylepurvisiDawes, 1941 (Trematoda : Aspidogastrea). I. Entwicklung und Morphologie. Zool. Jahrb. Anat. 88, 138187. Rohde, K. (1971b). Untersuchungen an MulficofylepurvisiDawes,1941 (Trematoda : Aspidogastrea). 11. Quantitative Analyse des Wachstums. Zool. Jahrb. Anat. 88, 188-202. Rohde, K. (1971~).Untersuchungen an MulticofylepurvisiDawes,1941 (Trematoda : Aspidogastrea). 111. Licht- und elektronenmikroskopischer Bau des Nervensystems. Zool. Jahrb. Anat. 88,320-363. Rohde, K. (1971d). Untersuchungen an MulficofykpurvisiDawes,1941 (Trematoda : Aspidogastrea). IV. Ultrastructur des Integuments der geschlechtsreifen Form und der freien Larve. Zool. Jahrb. Anat. 88,365-386. Rohde, K. (1971e). Untersuchungen an MulticotylepurvisiDawes, 1941 (Trematoda : Aspidogastrea). V. Licht- und elektronenmikroskopischer Bau der Randkorper. 2001. Jahrb. Anat. 88,387-398. Rohde, K. (1971f). Untersuchungen an MulficofylepurvisiDawes,1941 (Trematoda : Aspidogastrea). VI. Ultrastruktur des Spermatozoons. Zool. Jahrb. Anat. 88,399-405. Rohde, K. (1971g). Untersuchungen an MulticotylepurvisiDawes,1941 (Trematoda : Aspidogastrea). VII. Ultrastruktur des Caecums der freien Larve und der geschlechtsreifen Form. Zool. Jahrb. Anat. 88,406-420. Rohde, K. (1971h). Phylogenetic origin of trematodes. In Odening ed. “Perspektiven der Cercarienforschung”. Paras. Schrijtenreihe 21. Rohde, K. (in preparation). Untersuchungen an Multicotyle purvisi Dawes, 1941 (Trematoda : Aspidogastrea). VIII. Elektronenmikroskopischer Bau des Exkretionssystems. Infernat.J. Parasit. 1. Rumbold, D. W. (1928). A new trematode from the Snapping Turtle. J. Elisha Mitchell Sc. SOC.43, 195-198. Shipley, A. E. and Hornell, J. (1904). “The parasites of the Pearl Oyster”. Report to Government Ceylon on Pearl Oyster Fisheries of Gulf of Manaar (Herdman). Part 2,77-106. Siddiqi, A. H. and Cable, R. M. (1960). Digenetic trematodes of marine fishes of Puerto Rim. Sc. Surv. PorfoRico and VirginIslands, N. Y. Acud. Sci. 17,257-369. Simer, P. H. (1929). Fish trematodes from the lower Tallahatchie River. Am. Mid. Nut. 11, 563-588. Sinha, B. B. (1935). Morphology of a new genus of trematode, family Aspidogastridae Poche, 1907, from the intestine of a tortoise, Lissemys punctafa, together with a key for the identification of the known genera. Proc. Indian Acad. Sci. 1, 677-685. Sinitzin, D. F. (1905). Materials on the natural history of trematodes. Distomes of fish and frogs in the vicinity of Warsaw. (in Russian). Skrjabin, K. I. (1952). Trematodes of animals and Man, 6. Zsd. Akud. Nauk SSSR, Moscow (in Russian). Sogandares-Bernal, F. (1955). Some helminth parasites of fresh and brackish water fishes from Louisiana and Panama. J. Parasit. 41, 587-594.

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Sonsino, P. (1892). Studi sui parassiti di molluschi di acque dolce nei dintorni di Cairo in Egitto. Festschr. 70. Ceburtstag RudolfLeuckarts. Leipzig. Stafford, J. (1896). Anatomical structure of Aspidogaster conchicola. Zool. Jahrb. Anat. 9,477-542. Stafford, J. (1 898). The post-embryonic development of Aspidogaster conchicola. Rep. Br. Ass. Adv. Sci. 67, 698 (Abstract). Steinberg, D. (1931). Die Geschlechtsorgane von Aspidogaster conchicola Baer und ihr Jahrescyclus (Vorlaufige Mitteilung). Zool. Anz. 94, 153-1 70. Stossich, M. (1899). Appunti die Elmintologia. Boll. SOC.adriatica Sci. nut. Trieste 19, 1-6. Stromberg, P. C. (1971). Aspidobothrean trematodes from Ohio mussels. Ohio J. Sci. 70, 335-341. Stunkard, H. W. (1917). Studies on North American Polystomidae, Aspidogastridae, and Paramphistomidae. Illinois Biol. Monogr. 3, 1-1 14. Stunkard, H. W. (1946). Interrelationships and taxonomy of the digenetic trematodes. Biol. Rev. 21, 148-158. Stunkard, H. W. (1962). Taeniocotyle nom.nov. for Macraspis Olsson, 1869, preoccupied, and systematic position of the Aspidobothria. Biol. Bull. 122, 137-148. Stunkard, H. W. (1963). Systematics, taxonomy, and nomenclature of the trematoda. Q . Rev. Biol. 38, 221-233. Stunkard, H. W. (1967). Platyhelminthic parasites of invertebrates. J. Parasit. 53, 673-682. Stunkard, H. W. (1970). Trematode parasites of insular and relict vertebrates. Proc. 2nd Int. Congr. Paras. J. Paras. 56, 334-335 (Abstract). Syogaki, K. (1936). Some observations on Aspidogaster conchicola Baer. Zool. Mag., Tokyo 4 8 , 5 6 5 9 . Tandon, R. S . (1949). A new trematode, Lissemysia ovata n.sp., of the family Aspidogastridae Poche, 1907, from freshwater molluscs. Indian J. Helminth. 1, 85-92. U S . Department of Agriculture (1963-1968). “Index Catalogue of Medical and Veterinary Zoology. Trematoda and Trematode Diseases”. Washington. Van Cleave, H.-J. and Williams, C. 0. (1943). Maintenance of a trematode, Aspidogaster conchicola, outside the body of its natural host. J. Parasit. 29, 127-130. Voeltzkow, A. (1888a). Aspidogaster conchicola. Arb. zool. zootom. Inst. Wiirzburg 8, 249-289. Voeltzkow, A. (1 888b). Aspidogaster limacoides. Arb. zool. zootom. Inst. Wiirzburg 8,290-292. Ward, H. B. and Hopkins, S. H. (1932). A new North American aspidogastrid, Lophotaspis interiora. J. Parasit. 18, 69-78. Wharton, G. W. (1939). Studies on Lophotaspis vallei (Stossich, 1899) (Trematoda : Aspidogastridae). J. Parasit. 25, 83-86. Williams, C. 0. (1942). Observations on the life history and taxonomic relationship of the trematode Aspidogaster conchicola. J. Parasit. 28,467475. Wootton, D. M. (1966). The cotylocidium larva of Cotylogasteroides occidentalis (Nickerson, 1902) Yamaguti 1963 (Aspidocotylea-Trematoda). Proc. 1st Znt. Congr. Parasit. Rome, 1964, 547-548. (Abstract). Yamaguti, S. (1963). “Systema Helminthum. IV. Monogenea and Aspidocotylea”. Interscience Publ., New York, London. Yamaguti, S. (1968). “Monogenetic trematodes of Hawaiian fishes. Appendix”, 193. University of Hawaii Press. Zacharias, 0. (1895). Faunistische Mitteilungen. (c) Beitrage zur Histologie von Aspidogaster conchicola Baer. Forschungsberichte biol. Stat. Plon 3, 83-96.

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A Phylogeny of Life-cycle Patterns of the Digenea . .

J C PEARSON

Department of Parasitology. University of Queensland. St Lucia. Brisbane 4067. Queensland. Australia I. Introduction

.................................................................................... 153 ................................................ 156 158 Origin of Alternation of Generations ...................................................... Addition of Vertebrate ........................................................................ 160 A . Two-host Life-cycle ..................................................................... 160 B. Addition of Redial Generation ...................................................... 162 C . General Tendencies in Further Evolution .......................................... 163 D. Suppression and Replacement of Generations .................................... 164 165 Addition of Metacercarial Stage ............................................................ Acquisition of Second Intermediate Host :The Three-host Life-cycle ............ 167 Three-host Life-cycle ........................................................................... 170 A . Acquisition of Penetration Glands ................................................... 170 B . Modification of the Cyst Wall ......................................................... 171 C . Growth and Differentiation of the Metacercaria .................................... 172 173 Modification of the Three-host Life-cycle ................................................ A . Loss of Definitive Host .................................................................. 174 B. Loss of SecondIntermediate Host ...................................................... 176 C. Increase to Four Hosts .................................................................. 177 Phylogenetic Implications ..................................................................... 178 Summary.......................................................................................... 179 References ....................................................................................... 181

I1. Adoption of Parasitism: One-host Cycle

.

111

N.

V. VI . VII.

VIII

.

IX. X.

I. INTRODUCTION The origin of the digenetic life-cycle has invited speculation for over 100 years. following the experimental corroboration by many workers of Steenstrup’s (1845) shrewd interpretation of the life-cycle as an alternation of generations. Earlier ideas on the nature and origin of the digenetic life-cycle are to be found in the historical accounts of Leuckart (1886). Baylis (1938) and Dawes (1946). and will not be summarized again here . Instead. particular ideas are referred to where relevant throughout the text . More recently. three schemes have been advanced by Heyneman (1960). James and Bowers (1967) and Ginetsinskaya (1968). to account step by step for the origin of digeneans from free-living turbellarians . Additionally. both Heyneman and Ginetsinskaya propose a sequence. or phylogeny. of life-cycle patterns . Heyneman (1960) proposes four phases in the development of the cycle. Tn the first phase. the adult is free-living. earlier stages commensal in the 153

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mantle cavity of the mollusc, and many of the elements of the digenetic cycle are foreshadowed. Thus, a planuloid larva (miracidium) hatches from the egg and enters the mantle cavity, where it develops into a sporocyst-like stage that undergoes metamorphosis into a tailed pre-adult (cercaria) that, in turn, escapes and often over-wintering in an encysted state (metacercaria), becomes a free-living adult. In the second phase, the sporocyst-like stage becomes a visceral parasite of the mollusc, and in the third, adds asexual multiplication by polyembryony in what now becomes the sporocyst, and in a later stage of development that becomes the redia. In the fourth phase, it would seem that the definitive host and the second intermediate host are added at the same time to give a three-host life-cycle which Heyneman considers to be the most primitive pattern among contemporary digeneans, all other patterns being derived from it by reduction or addition. James and Bowers (1967), following their convincing argument for interpreting reproduction in sporocysts and rediae as parthenogenesis, and the digenetic life-cycle as a cyclical alternation of homologous generations, propose as the earliest parasitic phase, a succession of three adult generations, two viviparous and parasitic in the intestine of the mollusc, and the third oviparous and free living. Following the adoption of visceral parasitism by the viviparous generations, these become the mother sporocyst and the redia/daughter sporocyst. Later, the oviparous generation becomes parasitic in the gut of a vertebrate, but James and Bowers do not consider the sequence in which definitive and second intermediate hosts are added, nor that of cercarial and metacercarial stages. Ginetsinskaya (1968), in a lengthy section in her most interesting book on the digeneans, devotes some five chapters to phylogeny, comprising origin of alternation of generations, and sequence of addition and method of acquisition of the various hosts. In brief, her scheme supposes the following sequence: (i) adult free-living and larva ectocommensal in mantle cavity of mollusc, (ii) larva becomes visceral parasite, but escapes and develops into free adult, (iii) larva matures within mollusc but escapes to lay eggs, (iv) larva matures and lays eggs within mollusc; eggs hatch and larvae from them escape to become free-living adults, giving two generations, (v) parasitic generation becomes parthenogenetic and viviparous (mother sporocyst), and precocious development of free-living generation gives rise to a second parasitic generation (redia), (vi) acquisition of vertebrate host through ingestion of escaped larva of free-living generation, (vii) addition of metacercarial stage, and (viii) acquisition of second intermediate host following encystment on and then within animals eaten by the definitive host. The phylogeny proposed herein is the result of an attempt to combine in a single scheme those ideas and suggestions of others that accord with what I consider to be the singular features of digenetic life-cycles. Thus, I believe that any phylogeny must account for, or at least take into consideration, the following features : (i) alternation of generations, (ii) alternation of hosts, in which

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(a) first intermediate host a mollusc, usually a gastropod, and (b) definitive host a vertebrate, typically a fish, (iii) larval stage (cercaria) of adult generation has (or can be shown to have had) a tail, and (iv) methods of transfer: (a) free-swimmingmiracidium penetrates snail host in some life-cycles, (b) cercaria actively escapes from snail in many life-cycles, and (c) cercaria/metacercaria is ingested by the definitive host (except schistosomes). Before selecting those elements of known life-cycles on which the phylogeny is based, by reference to these features as criteria, some comment is called for on the individual items. Alternation of generations is the most singular feature of digeneans, but the account of its origin and development is the least satisfactory part of the present phylogeny, although it was the starting point of speculation. It may appear that alternation of generations and alternation of hosts are inextricably linked in digenetic cycles, but there is no apriori reason why they should be, and they may therefore be considered separately. Indeed, in the schemes of Sinitsin (1931a), Heyneman (1960), Cameron (1956), Stunkard (1959), Ginetsinskaya (1968) and James and Bowers (1967), alternation of generations arises before alternation of hosts. Although the larval first-stage of the adult generation is named cercaria, or tail-bearer, not all cercariae have tails. Those without, named cercariaeum, belong to a number of families, all (or most) of which also contain species with tailed cercariae. Is absence of the tail primary (primitive) or secondary? The law of parsimony suggests that it is secondary, especially as tail-less cercariae are scattered among a number of unrelated families. More convincing, however, is the evidence from cercarial embryology, as exemplified in the studies of Hussey (1941, 1943) and Kuntz(1950, 1951,1952),that the single posterior excretory pore characteristic of adult digeneans is a consequence of the moulding and growth of the posterior portion of the cercarial embryo to form the tail. Thus, the single excretory pore of tail-less forms is an indication of the former presence of a tail. With regard to the methods of transfer from one host to the next it is noteworthy that in contrast with life-cycles in other helminth groups (nematodes and cestodes) ingestion of a host with its contained parasite is not the only, or even the commonest, method of transfer to the definitive host in two-host life-cycles, or to the second intermediate host in three-host life-cycles. The phylogenetic significance of penetration of the snail by a free-swimming miracidium, and active escape from it of the cercaria will be discussed below. With the exception of Sinitsin (1931a) who would derive digeneans from insects, and Leuckart (1886), Janicki (1923) and Pigulevskii (1958) who would derive digeneans from monogeneans, authors agree that digeneans were derived from dalyellioid rhabdocoeles (Hyman, 1951; Stunkard, 1959; Heyneman, 1960; Baer and Joyew, 1961; Ginetsinskaya, 1968) or at least from a rhabdocoele-like ancestor (Cameron, 1956; Llewellyn, 1965;James and

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Bowers, 1967). Features common to rhabdocoeles and digeneans are discussed by Hyman (1951) and Heyneman (1960). The case against the view that digeneans arose from monogeneans can be best argued after the discussion of the order in which intermediate and definitive hosts were probably added to the life-cycle. 11. ADOPTION OF PARASITISM: ONE-HOST CYCLE Assuming, then, that digeneans arose from free-living dalyellioid rhabdocoeles, and that the hosts were added one at a time, which host was added first, and what were the relations of the proto-digenean withthis host? Most authors, beginning with Leuckart (1886), have postulated the present first intermediate host, the mollusc, as the original host, arguing that the following features indicate a longer association with the mollusc: (i) high host-specificity (=long association) for the (first) intermediate host as opposed to a lower hostspecificity (=more recent association) for the definitive host (Stunkard, 1959; Wright, 1960;Heyneman, 1960;Baer and Joyeux, 1961); (ii) the more complex phase, alternation of generations, occurs in the mollusc (Baer and Joyeux, 1961); (iii) the first intermediate host is usually a gastropod, less often a lamellibranch, rarely a scaphopod, and very rarely an annelid, whereas the definitive host may belong to any of the five classes of vertebrates (Heyneman, 1960). To this argument it may be added that if, as has been suggested, the present vertebrate definitive host was the original host, then one would expect that, with the addition of the molluscan intermediate host, infection of the vertebrate would be through ingestion of the mollusc. But, although there are present-day life-cycles in which this occurs (e.g. cyclocoelids), it can be argued from the ubiquity of the cercarial tail that this is secondary and that primitively the cercaria emerged from the snail, a habit difficult ifnot impossible to account for if one assumes that the definitive host was the original host, but not if one assumes the converse. Returning, now, to the assumption that the mollusc, or more specifically a snail, was the first host, how did a predaceous rhabdocoele become a visceral parasite of a snail? Two routes have been suggested, namely (i) an endocommensal in the intestine (cf. the rhabdocoele Puruvortex) becoming a visceral parasite (James and Bowers, 1967),and (ii) an ectocommensal in the mantle cavity becoming a visceral parasite (Leuckart, 1886; Sinitsin, 193la; Heyneman, 1960; Ginetsinskaya, 1968). Among present-day life-cycles there are two routes of infection of the snail host, namely, via the intestine through ingestion of an egg, and via the integument through penetration by a free-swimming miracidium. Thus, both proposals would appear to gain confirmation from present cycles ;however, it is much easier to picture modificaof entry through the integument into entry through the intestine, than it is to postulate the steps by which the reverse might have taken place. It might be argued that the original mode of entry into the body of the snail by the ectoparasitic proto-digenean, was via a natural body opening into the mantle cavity, such as the uterus, rectum or kidney, in view of the occurrence of azygiid rediae in the uterus (Wootton, 1957b) and cyathocotylid daughter sporocysts in the rectum (Sewell, 1923). However, it is probable that this is

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secondary, as the miracidium in these cases penetrates and early development takes place in the haemocoele. These unusual sites may represent an adaptation to aid the escape of large-tailed cercariae, viz. azygiids. Similarly, the essentially ectoparasitic sporocysts of gorgoderoids (Schell, 1967) may represent a comparable adaptation. If, then, in this proto-digenetic life-cycle, entry was effected by penetration of the integument and the parasite grew and matured in the viscera, how did the eggs escape to the outside? The question can perhaps best be answered by looking at the cercaria, the larval form unique to the Digenea. Whereas the cercarial tail, or evidence of its former existence in the form of a single posterior excretory pore, is the most striking morphological feature of the cercaria, even more important phylogenetically is its singular behaviour, namely active escape from its molluscan host. If, as has been proposed by Pigulevskii (1958), early vertebrates acquired digeneans, or proto-digeneans, through the ingestion of infected molluscs, then it is difficult to conceive how the cercarial stage was interpolated, and why it is now of universal occurrence. Stunkard (1959) has suggested that the cercaria arose from immature individuals expelled from the mollusc as a result of population pressure and forced to hunt for new hosts; the cercaria attained maturity in these new hosts, presumably in the tissues, as Stunkard suggests that progenetic metacercariae are a relict of this stage in the development of the digenetic life-cycle. A similar hypothesis is advanced by Jamieson (1966).

hatches EGG-

/

free, hermaphroditic ADULT

swimming JUVENILE

penetrates

SNAIL

gravid ADULT with tail

FIG.1. Hypothetical one-host life-cycle.

However, Stunkard does not say how the parasite got from one snail to another in the one-host stage in this phylogeny, nor does he consider how it got back to the snail in the two-host stage. It is difficult to imagine immature forms expelled from a host surviving long enough to invade other animals, nor can one visualize such a form as able to penetrate into the tissues of the new host. A more plausible explanation advanced, apparently independently, by Sinitsin (193la) and Baylis (1938), contends that primitively the larval stages were parasitic in the snail but that the sexually mature adult was free-living (or at least free). More recently, Heyneman (1960), Cable (1965) and Ginetsinskaya (1968) have endorsed this view. As argued above, the ubiquity

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of the cercaria among digeneans can be accounted for by assuming that the cercarial stage was present before the vertebrate host was added. The elements of the earliest parasitic cycle may now be combined in the following scheme, representing a one-host, pre-digenetic life-cycle (Fig. 1). It may be appropriate to comment here on the position of the Aspidogastrea, a small and strange group assigned, together with the Digenea, to the class Trematoda (Dollfus, 1958; Baer and Joyeux, 1961 ; Stunkard, 1963; Cable, 1965), although considered distinct on morphological grounds by Faust and Tang (1936). It has been suggested by Llewellyn (1965) that Aspidogastrea may represent an offshoot from an early stage in the evolution of digeneans, and by Smyth (1966) that they illustrate how the digenean life-cycle became two-host. Among Aspidogastrea, transfer from the molluscan host to the vertebrate (fish or tortoise) is by ingestion of an infected mollusc, and as pointed out above, this cannot have been the method used by digeneans as it does not account for the cercaria. If, then, Aspidogastrea are an offshoot from the digenean line, then they must have split off very early, before the origin of the cercaria. Indeed, their remarkable ventral attaching organ and casual relationships with molluscs, suggest that they are largely ectoparasitic, and so must have arisen before the proto-digeneans became visceral parasites. If, as assumed above, the mollusc was the first host, then monogeneans, which are one-host parasites of vertebrates, cannot have given rise to digeneans. 111. ORIGINOF ALTERNATION OF GENERATIONS

Before going on to consider the modification of this one-host cycle into a two-host cycle, it is necessary to consider the origin of that most singular feature, alternation of generations. Most authors (Stunkard, 1959 ; Heyneman, 1960; James and Bowers, 1967) have assumed that this arose during the one-host stage; some (Sinitsin, 1931a; Heyneman, 1960) have proposed that it arose while the predigenean was still an ectoparasite in the snail’s mantle cavity. But it may equally well have arisen in the two-host stage, in response to the added difficulties of a more complex cycle. Indeed, it might be argued that the mother sporocyst did not lose the intestine until after the vertebrate host was added. One can imagine an efficient one-host life-cycle in which the pre-adult (cercaria) had lost the intestine, feeding instead by absorption through the body wall, and finally leaving its snail host as a non-feeding, gravid adult, in much the same way as is seen in the dalyellioid fecampiids (Christensen and Kanneworff, 1965). But such an acoelous adult could not be the ancestor of the Digenea. There are two schools of thought on the origin of new generations, but these need not be mutually exclusive. One school has it that the present sexually reproducing adult is the original adult (Baylis, 1938; Cameron, 1956; Cable, 1965) and that the intra-molluscan generations are derived from homologous adult generations (James and Bowers, 1967), or are paedogenetic larvae of such adults (Ginetsinskaya, 1968), characterized in both cases by viviparity and parthenogenesis. The other school would derive new generations by paedogenesis from ontogenetic stages of the original generation through the

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acquisition of asexual reproduction (Sewell, 1923 ;Stunkard, 1959; Heyneman, 1960), or through the addition of new stages (cercaria, metacercaria, adult) to the ontogeny of the original adult (mother sporocyst) (Leuckart, 1886). Of the two possibilities, the first, termed cyclical alternation of homologous generations by James and Bowers (1967), is supported here. In either case the essential features of the new generation are regression of the reproductive system, parthenogenesis and viviparity. James and Bowers (1967) have suggested that this arose before the proto-digenean became a visceral parasite, but it would seem more likely that it arose after the transfer to the viscera. The steps in the modification of the added generation are suggested in Fig. 2. (a) EGG

swimming LARVA

SNAIL

parthhogenetic SNAIL viviparous ADULT

free, oviparous ADULT

(proto-mother

As with hosts, so with new generations, it is considered that these have been added one at a time. This notion gains support from the great differences in morphology and method of feeding of mother sporocyst and redia. Further, these differences suggest that the mother sporocyst was added first, as it is more highly modified from the adult condition, which is here assumed to approximate to the primitive condition. Once the new generation is established, the following changes can be envisaged: (i) loss or suppression of male reproductive system, and regression of female system resulting in fragmented ovary (=scattered germinal cells) which lies in the body cavity, (ii) adaptation to feeding through body wall, followed by loss of gut after addition of vertebrate host (see below), (iii) loss of gut in parthenita leads to its loss in larval stage (the parthenita is now a

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mother sporocyst and its larva a miracidium), (iv) retention of developing adult generation in body cavity of parthenita until it is ready or almost ready to leave the snail. The final stage, in which the cercaria is ready to escape from the snail at birth from the parthenita, is seen today in a number of groups, viz. echinostomatoids, strigeatoids, and plagiorchioids. An earlier stage, in which a partly developed cercaria emerges from the parthenita and develops further in the haemocoele before escaping, is seen in the paramphistomoids, notocotyloids, azygioids and transversotrematoids, groups that on other grounds are held to be more primitive than those listed above. It is also seen in opisthorchioids, a group with many advanced features. Jt is possible that the primitive habit of early escape has been retained in forms with large-tailed cercariae in order to facilitate escape from the redia/ daughter sporocyst. This is not the case in gorgoderids, a group characterized by magnacercous cercariae, in which escape of cercariae is facilitated by protrusion of the daughter sporocyst into the mantle cavity of the lamellibranch host (Rankin, 1939). It is perhaps easier to imagine loss of the gut in the parthenita after the acquisition of the vertebrate host (Fig. 3); otherwise, how can one account for its loss in one stage, the parthenita, and not in the other, the sexual adult, when the latter develops as a visceral parasite, and in the one-host life-cycle does not feed after escaping from its molluscan host? It may be argued that the free adult was, in fact, free-living and fed predaceously in the ancestral rhabdocoele fashion, as suggested by the scheme of James and Bowers (1967), but this is not supported by what little is known of endoparasitic rhabdocoeles. IV. A.

ADDITION OF VERTEBRATE TWO-HOST LIFE-CYCLE

Just as the cercarial tail may originally have aided in the dispersal of eggs laid by the sexual adult, so may accidental ingestion by an early vertebrate, such as a cyclostome. It may be envisaged that the proto-digenean, already parasitic in its developmental phase, became progressively better adapted to feeding in the vertebrate intestine, thus prolonging the period of egg-laying and thereby enhancing the chances of reaching the molluscan host. The two-host life-cycle is outlined in Fig. 3. Such a life-cycle, lacking a metacercarial stage (Cable, 1965; Pearson, 1968) is seen in azygiids, bivesiculids and transversotrematids, although the last-named are ectoparasites (Angel, 1969). Although primitive in outline, the life-cycles of azygiids and bivesiculids exhibit specialized features. Thus, in both groups the body of the cercaria is withdrawn into the base of the tail, an adaptation for protecting the cercarial body from damage during ingestion. It may be remarked in passing that the cysticercous condition has arisen in three distinct lines,* azygiids, gorgoderids *Or four times, if the striking differences between azygiids and bivesiculids in the morphology of the miracidium, a conservative stage in other groups, are interpreted as indicating a distant relationship between the two groups.

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and hemiurids; in all cases the cercaria is eaten, but in gorgoderids and hemiurids by the second intermediate host. Cercariae of azygiids and Bivesicula spp. are conspicuous by virtue of their swimming habits and large tails, the attractiveness of the latter being enhanced by pigmentation in Bivesicula (LeZotte, 1954; Cable, 1956) thereby inviting ingestion by the predaceous definitive hosts. The cercaria of the bivesiculid,Paucivitellosusfragilis, attaches to surfaces grazed by the definitive host, and awaits (presumably accidental) ingestion (Pearson, 1968).

hatches (a 1

PROTO -MIRACIDIUM

in faeces

penetrates

PROTO-MOTHER SPOROCYST

SNAIL

-

PROTO CERCARIA

(mature)

(b)

EGG

hatches

~

A

VERTEBRATE

‘\I

MIRACIDIUM

(aut lost) penetrates MOTHER SPOROCYST

(gut lost), ISNAIL (immature)

swims

FIG.3. Addition of vertebrate host.

It is tempting to speculate on whether the oral and ventral suckers are primitively absent in the adults of all bivesiculids, especially as the life-cycle is primitive. Further, if it is assumed that these attaching organs arose in response to the problem of maintaining position in the intestine of the newly acquired definitive host for increasingly longer periods as the adult adapted to endoparasitism, then one can picture the early parasitic adult as being without suckers. The need for attaching organs was probably greater, the larger the adult became; thus we find the minute adult bivesiculid without suckers but the large, to very large, azygiid adult with both oral and ventral suckers.

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B. ADDITION OF REDIAL GENERATION

As a life-cycle comprising three generations is widespread, and apparently primitive, it would seem likely that the second, or redial, intra-molluscan generation was added at an early stage. James and Bowers (1967) suggest that it arose very early, during the one-host stage, but if so, it is difficult to see why it should retain the gut when the mother sporocyst has lost it. Indeed, it has been argued above that the mother sporocyst generation did not lose the intestine until after the vertebrate host was added. It appears likelier that the redial generation was added in response to the increased difficulties of a two-host life-cycle. Further, it is proposed, following on Cable’s (1965) suggestion, that the redia is derived from a cercaria in the same fashion as are the “parthenitae” of Parvatrema homoeotecnum as described by James (1964). Two objections may be raised to such an interpretation, namely (i) that the redia has a single caecum whereas adult digeneans, with few exceptions, have two caeca, and (ii) that the redia lacks a tail (and, related to this, has separate excretory pores). The first objection may be met by assuming that the redia arose before the adult modified its rhabdocoele gut into a pair of caeca, a development paralleled in monogeneans and some parasitic turbellaria, and probably arising in all three from increased metabolic demands resulting from increased egg-production, together with hypertrophy of the female system (Baer and Joyeux, 1961). Alternatively, the redial gut may have become secondarily simplified following the acquisition of feeding by absorption through the body wall. It is of interest that recent studies on the fine structure of the body wall suggest that rediae and daughter sporocysts have similar absorptive teguments (Bils and Martin, 1966). Thus, rediae feed both by absorption through the tegument and ingestion into the caecum. This change in manner of feeding leads to regression, culminating in the loss of the pharynx and caecum, and results in the modification of the redia into a daughter sporocyst, a modification repeated in the ontogeny of azygiid (Szidat, 1932; Sillman, 1962; Rai, 1963) and allocreadiid (Anderson et al., 1965) daughter sporocysts, and thus affording corroborative evidence for the origin of this stage. Similarly, among hemiuroids there is in a few species evidence of the regression of the redial gut (Jamieson, 1966; Chabaud and Campana-Rouget, 1959; Cable and Nahhas, 1963). The case of Philophthalmus, as described by Cable (1965), is of particular significance, as the mother-sporocyst stage of the first generation is suppressed and the miracidium delivers into the snail host a single, precocious but undifferentiated, offspring. Depending apparently on the level of differentiation attained, the offspring may either develop a pharynx and caecum, becoming a typical redia, or remain undeveloped, a “sporocyst”, yet give rise to a new generation. The remarkable “brood mass” in Dusymetra conferfa, described by Byrd and Maples (1964), may be comparable. Evidence in four groups, azygiids, allocreadiids, hemiurids and philophthalmids, of the alteration of the redia into a daughter sporocyst strongly

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suggests that such a change has occurred several times over in different groups. It follows from this that the form of the second generation is not a fundamental character, as suggested by Lebour (1912), Dubois (1929), Dollfus (1949) and Heyneman (1960), to be interpreted at the higher levels of the classification, as has been done by Odening (1961). Indeed, it would appear that this character may not be constant at even the family level in some groups. A strange, apparently invariable, and to date unexplained difference between redia and daughter sporocystis that the former may produce another generation of rediae (La Rue, 1951) whereas the latter never repeats itself but gives rise only to cercariae. Production of rediae by rediae may be facultative as in fasciolids (Dinnik and Dinnik, 1956) and paramphistomes (Durie, 1953) or apparently obligatory as in diplodiscids (Van Der Woude, 1954), troglotrematids (Ameel, 1934), echinostomes (Cort et al., 1948; Donges, 1963) and clinostomes (Edney, 1950) in which one or more mother rediae give rise to daughter rediae, and these in some cases to grand-daughter rediae, that in turn give rise to cercariae. Redia and daughter sporocyst appear to be mutually exclusive as there is no convincing account of a life-cycle with both forms. Noda (1959) has described daughter sporocysts giving rise to rediae in Stellantchasmusfalcatus, but recent studies do not bear him out (Pearson, unpublished). The second objection, lack of a tail, finds its counter-argument in the suggestion of Cable (1965) and James and Bowers (1967) that the bivesiculid redia (LeZotte, 1954), and by extension other rediae, may be derived from the cercaria/adult generation. If the cercarial tail was not lost, but instead became an extension of the hollow body of the redia, then the two halves of the excretory system would remain separate. C.

GENERAL TENDENCIES IN FURTHER EVOLUTION

A number of general tendencies may be discerned among the descendants of this two-host life-cycle, such as: (i) change from early escape of cercaria from redia and completion of development in haemocoele, to full development of cercaria within redia/daughter sporocyst ;(ii) progressive retardation of sexual development of cercaria; (iii) small, short-lived, primitive mother sporocyst becomes larger and lives longer, producing more offspring; and (iv) an opposite tendency toward reduction of the mother sporocyst stage and production by it of a single redia; (v) change from large eggs that embryonate and hatch in water releasing short-lived miracidia, to small ( ? more numerous) embryonated eggs that hatch on ingestion by the molluscan host, thus increasing the infective life of the miracidium, and in the opposite direction; (vi) change to large, embryonated eggs that hatch on reaching the external environment. The first of these, (i), has already been dealt with, but it may be added that early escape of a partially developed cercaria, here considered primitive, occurs only from rediae, whereas only fully developed cercariae escape from daughter sporocysts. This affords another argument, perhaps suffering from circularity, in favour of considering the redia more primitive than the daughter sporocyst. In the degree of development of the reproductive system, (ii), cercariae range

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from sexually mature and gravid (Proterometru-Anderson and Anderson, 1963; Dickerman, 1934) through sexually mature but not gravid (Succucoeliodes-Cable and Isseroff, 1969), advanced but sexually immature forms (Puucivitellosus-Pearson, 1968), and forms with recognizable “anlagen” of the reproductive organs (plagiorchioids and echinostomes), to forms with an undifferentiated genital rudiment (strigeatoids, opisthorchioids). Anderson and Anderson (1963) have interpreted the life-cycle of Proteroinetru dickermuni as beng primitively one-host ; but the presence of welldeveloped oral and ventral suckers in the cercaria indicates that it is secondarily reduced from a two-host cycle. Of all stages in the life-cycle, the mother sporocyst is the least well known; indeed, it is unknown for several groups, including such large ones as opisthorchioids and such phylogeneticallyimportant ones as bivesiculids. Nevertheless, there would appear to be a rough series (iii) (not a strict phylogeny) running from small mother sporocysts in relatively primitive groups (paramphistomes, Durie, 1953; fasciolids, Dinnik and Dinnik, 1956; azygiids, Stunkard, 1956) to large mother sporocysts in more advanced groups (plagiorchioids and schistosomes, Cort et ul., 1954) at least some of which are long-lived (strigeids-Pearson, 1956), and some branched zoogonids, ( ? some) bucephalids (Ciordia, 1956), and possibly in haplorchine heterophyids (Pearson, unpublished)). Elimination of the mother sporocyst stage of the first generation as a result of the precocious development of a single mother redia within the miracidium, (iv), is found in the paramphistomid, Stichorchis (Bennett and Humes, 1939), in philophthalmids (Cable, 1965), and in cyclocoelids (Timon-David, 1955). This condition is approached in clinostomids; thus in Clinostomum gigunticum the mother sporocyst produces a small number of rediae (Agarwal, 1963), and in C. murginutum a single redia (Cort et ul., 1954). In his scheme indicating the stages in the elaboration of the digenetic life-cycle, Sewell (1923) considers this to be the most primitive condition. But it is difficult to envisage any selective advantage in the acquisition of a generation that produces a single offspring. Rather, it would appear that in these few cases the first (or miracidium/mother sporocyst) generation has been simplified, and that it is not a relict retained in a few unrelated groups (a position difficult to justify), but a separate and parallel development in each group, at a stage when the mother sporocyst was small and perhaps short-lived. D. SUPPRESSION A N D REPLACEMENT OF GENERATIONS

In addition to suppression of the mother sporocyst, there are a few examples of suppression of the redia/daughter sporocyst generation, and even of suppression of both of these generations and their replacement by modified stages of the adult generation. Thus the mother sporocyst gives rise directly to cercariae in Heronimus chelydrue (Ulmer and Sommer, 1957; Crandall, 1960), and possibly in some bucephalids (Ciordia, 1956). In discussing the sequence in which generations were added, it was argued

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165

that the mother sporocyst generation was added before the redia/daughtersporocyst generation. Later, it was pointed out that most digenetic life-cycles manifest three generations. In the remarkable life-cycle of Heronimus chelydrae (=Heronimus mollis), one of many unusual features is the production of cercariae by the mother sporocyst (Ulmer and Sommer, 1957; Crandall, 1960). Is this a primitively two-generation cycle, comparable with the hypothetical stage proposed above, or is it secondarily simplified through loss of a generation (the redial)? This fluke is highly aberrant and specialized, as indicated by (i) an anterior and dorsal excretory pore, (ii) location in the lungs of the definitive host, (iii) a mother sporocyst with lateral outgrowths, and (iv) a cercaria, with a functional tail, that does not emerge from the snail host, and with a functional ventral sucker that disappears as the adult develops. But, despite the singular features of this species, it has been assigned (Cable and Crandall, 1956; La Rue, 1957; Crandall, 1960) to the superfamily Paramphistomoidea, a primitive group by virtue of the form of germinal multiplication, namely absence of persistent germinal masses and limited reproduction (Cort et al., 1954), early escape from the redia of partly developed cercariae (vide supra), free-swimming miracidium (vide supra), and non-epithelial bladder (La Rue, 1957). Although the branching form of the mother sporocyst is not primitive, its muscularity and limited power of reproduction are. It may well be, then, that the redial generation is primitively absent in Heronimus. Even more remarkable is the life-cycle of Par vatrema homoeotecriutn elucidated by James (1964). Although the earliest phase in the mollusc is not clear, it may represent an evanescent mother sporocyst that gives rise to a single offspring, the primary germinal sac of James (1964). But this latter is not a redia, as it possesses such adult features as oral and ventral suckers, pharynx and bifid gut, and single posterior excretory pore. However, it is not a sexual adult as it reproduces parthenogenetically in a manner comparable with that of a redia. The primary germinal sac gives rise to offspring that look at first like developing cercariae, but that lose their tails and develop into a form similar to that of the primary germinal sac. This, the daughter germinal sac of James (1964), gives rise to proper cercariae. Here, we have suppression of both mother and daughter sporocyst generations common to other fellodistomoids (Mehra, 1963), and their functional replacement by a succession of two modified adult generations. Perhaps here we have a contemporary parallel with the proposed ancient origin of the redial generation from the adult at an early stage in the evolution of the life-cycle.

v.

ADDITION OF METACERCARIAL STAGE

In the primitive two-host life-cycle described above, transfer from snail to vertebrate must be accomplished during the brief free life of the cercaria. The metacercaria is an adaptation for prolonging the infective life of the cercaria, thereby enhancing the chances of ingestion by the definitive host. The manner in which this stage was added may be suggested as follows. On escape from the snail, cercariae disperse by swimming, then settle to the bottom and remain quiescent, thus conserving their limited resources of

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stored food while awaiting ingestion by a bottom-feeding vertebrate (e.g a cyclostome). Comparable behaviour is exhibited by the cercaria of Paucivitellosus frugilis (Pearson, 1968). Just as free-living rhabdocoeles secrete a protective envelope under harsh environmental conditions (Hyman, 1951), so the quiescent cercaria secreted the forerunner of the complex metacercarial cyst, and in the process pinched off the no-longer-useful tail. At this stage, the metacercaria is an encysted cercarial body that undergoes little if any change and is immediately, or almost immediately, infective for the definitive host (Donges, 1969). The two-host life-cycle with a free metacercaria is remarkably successful, and by relatively simple modifications in cercarial behaviour affecting the site of encystment, has become adapted to the changing food habits of the evolving vertebrate hosts (Fig. 4), as these became herbivores, predators, plankton-feeders, etc. NON-SPECIFIC ENCYSTMENT

Paramphistomidae

Notocotylidoe

Fosciolidoe

Pronocephalidae

Haploporidae

Haplosplanchnidae

Philophtholmidoe Echinoslomatidae

FIG.4. Adaptation of two-host life-cycle to food habits.

One interesting modification of this type is seen in notocotyloids, a group with a primitively two-host life-cycle in which the cercaria typically escapes and encysts on hard surfaces, such as the shell of the molluscan host. In three species, Cututropis verrucosa described by Odening (1965), Purapronocephulum symmetricum described by Chubrik (1954), and Notocotyloides petusaturn described by Dollfus (1966), the cercaria, which has lost its eyespots and has a reduced tail, fails to emerge and instead encysts within the first intermediate host. A two-host life-cycle such as this, in which the definitive host eats the molluscan intermediate host containing the metacercaria, can also arise through abridgement of a three-host life-cycle (see Section WIT). Thus, the pattern seen in cyclocoelids (Timon-David, 1955) could have arisen either way. However, as the cercaria lacks evidences, such as penetration glands or stylet, suggestive of a three-host life-cycle, it would appear that the original cycle may well have been two-host. Heyneman (1960) postulated that the metacercarial stage was added while the proto-digenean was a one-host molluscan parasite. His interpretation of the metacercaria as a resistant over-wintering form presupposed a temperate and presumably freshwater origin, and a free-living (i.e. feeding) as opposed to a

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free (non-feeding) adult. All three of these features would seem highly unlikely in the light of the arguments advanced earlier. VI.

ACQUISITIONOF SECOND INTERMEDIATE

HOST:

THETHREE-HOST LIFE-CYCLE There are two views on the position of the second intermediate host, which is primitively an invertebrate; one holds that it was originally a definitive host harbouring the sexual adult acquired either by ingestion of the infected snail host or by invasion of the invertebrate by the cercaria (Stunkard, 1959); the other holds that it was interpolated beteewn the molluscan intermediate and thevertebratedefinitive host (Cameron, 1956; Ginetsinskaya, 1968; Heyneman, 1960; Baer and Joyeux, 1961).

2-HOST

Step I

encyst in open

Step 2

encys; on outside of animal (shell or carapace) Hirnasthla, Phi/ophthalrnus

TRANSITIONAL

Step 3

3-HOST

Siep 4

I

I

1

CERCARIA large ; METACERCARIA

infective immediately

enter natural body opening (e.g kidney) and encyst

J.

Echinostorna penetrate tissues and encyst A canthoparyphiurn

CERCARIA Smaller

and more numerous ; METACERCARIA grows, not infective immediately

FIG.5. Addition of second intermediate host.

The first view can be rejected on the grounds that it cannot explain the cercaria; and Stunkard’s view on the grounds that the mainspring of his argument, precocious sexual maturity in the tissues of the second intermediate host (i.e. progenesis) is clearly secondary (vide injia). If, as Stunkard claims, progenesis is a relict of a two-host, mollusc-invertebrate cycle, why does the sexual phase have suckers, organs of attachment that are scarcely required by a visceral parasite? If it is accepted that the second intermediate host has been interpolated, how has this come about? More especially, how is it that this host becomes infected not by eating the first intermediate host, as is characteristic of threehost life-cycles in other helminth groups, but by active entry of the cercaria after its escape from the snail host? 1 believe that the various life-cycle patterns of echinostomes illustrate one way in which the second intermediate host was probably added (Fig. 5). 8

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J . C. PEARSON

In this scheme, stage 1 and stage 2 are typical two-host life-cycles, as exemplified for stage 2 by Himasthla (Adams and Martin, 1963). In the transitional phase (stage 3), the cercaria enters without penetrating into the kidney of another snail (or of a tadpole) and there encysts, but derives no benefit from this beyond a more secluded and better protected site for the metacercaria. Thus, the metacercaria is a largely unaltered cercarial body, showing no advance in development, and is quickly infective for the definitive host. Life-cycles of this type are known in Echinostoma (Beaver, 1937; Lie and Umathevy, 1965a; Lie, 1967), Echinoparyphium (Johnston and Angel, 1949; Najarian, 1954; Odening, 1962; Lie and Umathevy, 1965b), and Euparyphium (Beaver, 1941). Heyneman (1960), convinced that in contemporary digeneans the three-host life-cycle is the most primitive and that two-host life-cycles are derived, would read this series the other way around, as indicating loss of the second intermediate host. Entry into the tissues would not be difficult from the pericardial sac of the snail, the end-point of migration via the reno-pericardial duct which arises from the kidney. And once within the tissues, it is envisaged that by degrees the metacercaria became adapted to feeding by absorption through the thinning cyst wall, and in consequence, developed toward the adult condition. An early stage in this development is seen in Acanthoparyphium spinulosurn in which the cercaria penetrates into a snail and grows “to several times the cercarial size” as a metacercaria (Martin and Adams, 1961). A similar life-cycle is seen in Puryphostomum (Johnston and Angel, 1942), Petasiger (Johnston and Angel, 1941 ; Abdel-Malek, 1952; Beaver, 1939a), Echinochasmus (Johnston and Simpson, 1944; Nasir and Diaz, 1968), and Sfephanoprora (Nasir and Scarza, 1968). There may well be variation in the level of development attained by the metacercaria of any one species in different hosts and in different geographical localities, such as PrCvot (1 965) has called attention to in Proctoeces maculatus, or in hosts at different temperatures (Buttner, I951 b). Indeed, this may be the case in Acanthoparyphium spinulosum in Australia (Bearup, 1960) and in California (Martin and Adams, 1961). However, I do not feel that the occurrence of such variation invalidates the hypothesis that there is among the echinostomes a tendency toward penetration into the tissues and growth of the metacercaria. Growth of the metacercaria probably accelerated the tendency referred to earlier, towards more, smaller, and less well-developed cercariae. As echinostomes are, as adults, a rather specialized group, characterized by a collar of spines, it seems unlikely that they are ancestral to all three-host digeneans. Hence, it seems probable that a second intermediate host has been added in at least two, and possibly more than two, distinct evolutionary lines. A parallel series of stages can be seen in the life-cycles of the psilostomes. Thus, in Psilotrema spiculigerum the cercaria forms a thick-walled cyst in the open or on the shell of a snail (Mathias, 1925); in Sphaeridiotrema globulus the cercaria forms a thick-walled cyst between the mantle and the shell of a snail (Szidat, 1937); and in Ribeiroia ondatrae, the cercaria forms a thin-walled cyst in cavities such as the lateral line, nostrils, and cloaca of fish and the cloaca

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169

of tadpoles (Beaver, 1939b; Riggin, 1956). If Martin (1968) is correct in assigning Cercaria gorgonocephala to the Psilostomidae, then it probably represents a further step in which the cercaria encysts after ingestion, as it is an aggregating form. Psilotomes, however, are closely related to echinostomes, and their life-cycles may reflect a tendency common to the whole of the echinostomatoid group. A possible second evolutionary line is suggested by the life-cycles of fellodistomoids and brachylaimoids, two groups considered by Cable (1965) to be closely related. The simplest life-cycle, of Burnellus trichofurcatus, a tandanicoline fellodistomoid (Angel, 1971) is a two-host one, comparable with that of bivesiculids, in which an advanced cercaria is ingested by the definitive host and develops directly to the adult. Other fellodistomoids have three-host cycles with a metacercaria, but of one group, the gymnophallines, Cable (1965) says that they live as metacercariae in loose even superficial association with their hosts. The cercariae of most brachylaimoids do not penetrate the molluscan second intermediate host but enter the uterus via the female pore (Allison, 1943), or the kidney (Krull, 1935; Ulmer, 1951 ; TimonDavid, 1953; Joyeux et al., 1932) and develop in the lumen into advanced metacercariae. In at least one species, the cercaria enters the tissues (Sinitsin, 1931b). As well as suggesting the way in which the second intermediate host was added, an analysis of the fellodistomoids and brachylaimoids appears to indicate that three-host life-cycles have been arrived at separately in them. A relationship between the two groups was first suggested by Allison (1943), agreed to by Cable (1953), and formalized by La Rue (1957) in his classification. Among fellodistomoids, both two- and three-host life-cycles are found, and although some of the two-host life-cycles, such as Ching (1965) describes, clearly show reduction (vide hfra), in at least one case, that of Burnellus trichofurcatus cited above, the life-cycle is primitively a two-host one. Among brachylaimoids, only the three-host life-cycle is known. From this it would appear that brachylaimoids cannot have given rise to fellodistomoids. Comparison of the excretory systems in the two groups shows that the paranephridial system ( =reserve excretory system) is well developed in brachylaimoids but reduced or absent in fellodistomoids, as pointed out by Cable (1953). The paranephridial system appears to be a primitive feature, as it is more widely occurring and better developed among anepitheliocystidians than among epitheliocystidians. Accepting Cable’s (1965) view that anepitheliocystidians are the more primitive group, as indicated especially by their life-cycle patterns, it follows that fellodistomoids without welldeveloped paranephridial system cannot have given rise to brachylaimoids with well-developed paranephridial system. It would appear, then, that fellodistomoids and brachylaimoids are separately derived from a common ancestor, a view advanced without support by Cable (1965). But to account for the life-cycle patterns of fellodistomoids, it must be assumed that this ancestral group had a primitive two-host life-cycle. And from this it would follow that a second intermediate host was acquired separately in each of the two groups.

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J . C. P E A R S O N

Another way in which a second intermediate host may have been added, and one that accords with the method proposed for cestodes (Llewellyn, 1965) and nematodes (Chabaud, 1955), namely ingestion by a wrong host, may be foreshadowed in certain azygioid life-cycles. Szidat (1932) and Sillman (1962) both suggest that cercariae of Azygiu may be transported, without development, in the gut of small fishes to the large predaceous fishes that act as definitive hosts. By analogy, Pearson (1968) has suggested the same mode of infection for the larger definitive hosts of Bivesiculu spp. After the above hypothesis on the mode of origin of the second intermediate host was worked out, it was found that Ginetsinskaya (1968) had advanced a very similar hypothesis, also based on life-cycle patterns of echinostomes, thus lending further weight to this interpretation.

VII. THREE-HOST LIFE-CYCLE Changes consequent on encystment in the viscera may be discussed under three headings: A, acquisition of penetration glands; B, modification of the cyst wall; and c , growth and differentiation of the metacercaria. A.

ACQUISITION OF PENETRATION GLANDS

Before advancing a purely hypothetical origin, it is necessary to point out that there exists in the literature a great deal of confusion on the nature and function of pre-orally opening gland-cells in cercariae, metacercariae, and adults, confusion that is reflected in the variable and inconsistently applied nomenclature of these gland cells. Some order may be enforced upon the plethora of vague terms by considering these cells in terms of the three major roles they are known to play in the various stages of the adult generation. The first of these is in escape of the cercaria from the snail. Although surprisingly little attention has been paid to the route and manner of escape, it is now clear that cercariae actively leave the snail host (Smyth, 1966). Gland cells, usually one pair, that are used up in escaping, have been described in the cercariae of schistosomes (Price, 1931 ; Stirewalt, 1963) and gorgoderids (Fischthal, 195I). The second role, penetration into the second intermediate host, is so well attested and the literature on it so abundant, that it would be invidious as well as unnecessary to cite references. Almost all digeneans with a three-host life-cycle in which the metacercaria occurs in the tissues, exhibit penetration glands in the cercaria. Evidence for the third role, feeding (external digestion) in the adult, is recent (Halton, 1967), although Looss (1894) pointed out many years ago that pre-orally opening, unicellular gland cells (here termed frontal glands) were widespread among adult digeneans. Indeed, such gland cells are probably present in all digeneans that feed on tissues, but are apparently absent in such as paramphistomes that feed on gut contents. Bivesiculids and transversotrematids may be exceptions to this, as they apparently lack such gland cells. They also lack an oral sucker, possibly primitively, and it may be that oral

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171

sucker and pre-orally opening gland-cells go together as elements of a new way of feeding. In all three roles, the function of the glands is histolytic. Are they, then, homologous, and in what order might they have appeared? Are the gland cells used by the cercaria in penetrating carried over in the adult to be used in feeding? Answers to these questions may be suggested by reference to the phylogeny herein proposed. In the hypothetical one-host life-cycle, the “cercaria” (=tailed, mature adult) escapes from its snail host; thus escape glands would be the first type added, possibly by modification of the homologue of the frontal gland of turbellarians. Following the addition of the vertebrate host and adaptation to feeding on the intestinal mucosa, escape glands or their equivalent would undergo hypertrophy to become the adult frontal glands. Both escape glands and frontal glands antedate penetration glands, and either, or both, may have given rise to them. On the whole, it appears more probable that they arose from frontal glands, as these in an early cycle, with advanced cercaria, would be developed before entry into the definitive host, and so available to aid in penetrating. Such early development is seen in Burnellus tvichofurcarus, a fellodistomoid with a primitive two-host life-cycle (Angel, 1971) in which the cercaria is eaten directly by the definitive host. Here, pre-orally opening glands seen in the cercaria (Johnston and Angel, 1940) persist in the adult (Pearson, unpublished), and are certainly not penetration glands. Again, in some plagiorchioid cercariae (Pearson, unpublished), there are both penetration glands opening close to the stylet that disappear following penetration, and frontal glands opening in a transverse row anterior to the stylet that persist in the metacercaria. 6. MODIFICATION OF THE CYST WALL

To accommodate feeding and growth of a cercarial body encysted in the tissues, certain modification of the cyst itself would be expected. For example, the cyst wall would become thinner and less complex to facilitate absorption of nutrients and growth of the metacercaria, and because a stout wall is no longer required in the fastness of the tissues. These changes are borne out by comparison of cysts formed in the open with those formed in tissues, but the latter have not been studied in the same detail as have the former, as for example, by Dixon and Mercer (I 967). As the metacercaria became larger and larger, that is more nearly adult, and, perhaps, the cercaria smaller, as suggested above, the cyst would require altering to accommodate a large metacercaria derived from a small cercarial body. This requirement has been met in two, or possibly three ways, viz. (i) by having the cercarial body secrete an inflated cyst, large enough for the future metacercaria, as in heterophyids (Pearson, unpublished); (ii) by having the cyst increase in size as the metacercaria grows, as in at least some plagiorchioids (Pearson, unpublished); and (iii) by delaying formation of the cyst until the metacercaria has reached full size, as in some strigeids (Olivier, 1940; Ulmer,

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1957; Cort et al., 1941) and in some plagiorchioids, e.g. Prosthogonimus and Acanthatrium (Komiya, 1965). Progressive delay in encystment probably led to loss of the ability to encyst as seen in clinostomoids (Singh, 1959; Pearson, unpublished), some strigeoids (Dubois, 1938), and some microphallids (Rothschild, 1937; Belopol’skaya, 1962; Sogandares-Bernal, 1962). Other groups lacking a metacercarial cyst are hemiurids (Cable, 1965) and some brachylaimoids (Allison, 1943), but in these it is not clear whether a cyst was ever present.

C.

GROWTH A N D DIFFERENTIATION OF THE METACERCARIA

It was suggested above that once the second intermediate host had been added and the developing metacercaria had learned to feed in the tissues of this host, then two concomitant changes followed. By degrees the metacercaria developed further and further toward the fully adult condition, thereby shortening the time taken to mature in the definitive host; and at the same time the cercaria became “younger” and more specialized as a larva, and smaller, thereby allowing a larger number to be produced. But whereas in two-host life-cycles and in transitional stages of three-host life-cycles, the metacercaria is quickly infective for the definitive host, in three-host life-cycles the metacercaria may take weeks, or even months, to become infective (Donges, 1969). The disadvantage of delayed infectivity is presumably outweighed by the production of more cercariae and the advantage of arriving in the definitive host in a more advanced state. It has, perhaps, been generally assumed that a metacercaria is not infective until it completes its development in the intermediate host. That this is not necessarily so, is shown by the observation of Goodchild (1943) on Phyllodistomum solidum that although the metacercaria is infective in four days, it continues to grow in the intermediate host. The end-point of the first trend is the attainment of sexual maturity, while in the tissues of the second intermediate host. Such precocious development is usually called progenesis (Baer and Joyeux, 1961), an unfortunate choice as progenesis is a synonym of neoteny (De Beer, 1958), and as Buttner (1 95 1 b) has so clearly pointed out, the sexually mature individuals are adults, not metacercariae as is so often held. Recent authors have used “progenetic” in the sense of “precocious”; thus Smyth (1969), defines progenesis as “advanced development of genitalia in a larva without maturation”, whereas the phenomenon that the term progenesis was borrowed to describe, is the attainment of full maturity, sexual and somatic, in an unusual site, the tissues of an intermediate host. It does not really matter if the term progenesis becomes blurred as long as it is remembered that its most advanced state is not neoteny in a metacercaria but precocious sexual maturity in an adult. As will be described in the next section, attainment of sexual maturity in the second intermediate host has led in some cases to the elision of the definitive host and the reduction of the life-cycle from three hosts to two.

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VIlI. MODIFICATION OF THE THREE-HOST LIFE-CYCLE The three-host life-cycle is both common and widespread, occurring in at least some members of I2 of the 18 major superfamilies (Fig. 7). Of these 12 superfamilies, seven exhibit another pattern in some members, viz. secondarily two-host in five, although in these the three-host pattern is commonest. It was probably in part this abundance that led La Rue ( 1 95 I ) and Heyneman ( 1 960) to suggest that the three-host life-cycle is the most primitive pattern extant and that all other patterns are derived from it. The other factor that no doubt influenced them is the clear indication that many abridged life-cycles are derived from this three-host cycle. These abridged cycles will now be considered (Fig. 6).

7t wT CERCARIA

DEFINITIVE HOST

FIRST INTERMEDIATE HOST

penetrates

ingested

METACERCARIA

loss of second intermediate host

SECOND INTERMEDIATE

I

loss o f original definitive host

I DEFINITIVE

fo \I

1

ingested

CERCARIA

penetrates

ADULT

FIRST INTERMEDIATE HOST METACERCARIA

FIG.6. Loss of hosts.

There appears to be only one way in which a three-host life-cycle can be shortened, namely loss of a host as the result of combining the functions of two hosts in a single host. There are three ways in which this has come about: A, growth to sexual maturity in second intermediate host and loss of definitive host; B, utilization of the definitive host as both second intermediate and definitive hosts; and c, development of metacercaria in first intermediate host and loss of second intermediate host. Yet another way, elimination of the first intermediate host, has been proposed by James and Bowers (1967) to explain the host succession in the life-cycles of Parvatrema homoeotecnum, as described

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I . C . PEARSON

by James (1 964), the two-host life-cycle of bivesiculids, as described by LeZotte (I 954), and the rare phenomenon of annelid first-intermediate hosts, summarized by Martin (1952). But, in the life-cycle of P. homoeotecnum, it is the miracidium that enters the first host, although this is a snail rather than a clam, as is usual for the family, and so, despite the remarkable intramolluscan development in this species, there is no indication of loss of the first intermediate host, but only of a change, albeit a great one, in the species of host used. A.

LOSS OF DEFINITIVE HOST

Actual or potential loss of the definitive host is exhibited in varying degrees by a number of species, but rather than attempt a catalogue of instances (Buttner, 1950, 1951~)some examples are given below to suggest the steps by which this host is deleted. As pointed out earlier, once cercariae entered the tissues of second intermediate hosts, they exploited this site by feeding, thereby allowing both growth and differentiation to proceed toward the adult condition. The level of development reached would appear to depend, at least in part, on the balance struck between the advantage of arriving in the definitive host in an advanced state and the corollary disadvantage in not being immediately infective for the definitive host. Related to this may be size and number of cercariae. To take two extreme cases, Echinostoma produces relatively small numbers of large cercariae which become metacercariae that do not advance toward the adult condition but are infective for the definitive host within hours of the cercaria entering; whereas Clinostomum produces enormous numbers of minute cercariae that develop into metacercariae that are nearly sexually mature, but that take some months to become infective. According to the phylogeny advanced herein, a metacercaria approaching sexual maturity is advanced, whereas one that changes little if any from the cercarial body is primitive. Except in the case where regression of metacercarial development is indicated, it follows that if in two groups a series from “primitive” to “advanced” metacercaria can be discerned, then the two have presumably been separated for a long time and can be related phyletically only at an early stage. I t also follows that a group with a “primitive” metacercaria cannot be derived from a group with an “advanced” metacercaria. Such considerations may help, in conjunction with other features, to illuminate the interrelationships of digenetic groups. Advanced metacercariae are well known among all of the three-host groups listed in Fig. 7, and range in development from (i) those with rudimentary gonads, such as the strigeoids, and some plagiorchioids, hemiuroids, allocreadioids, lepocreadioids, and opecoeloids, to (ii) those with a fully formed reproductive system but without gametogenesis, such as clinostomes, brachylaimoids, and (some) fellodistomoids, some bucephaloids, plagiorchioids (especially microphallids; Belopol’skaya, 1963), opisthorchioids (haplorchine heterophyids), opecoeloids (Dobrovolny, 1939a and b), and beyond to (iii) those with sperm in the seminal vesicle, such as some bucephalids and microphallids (Belopol’skaya, 1963; Rausch, 1947) and finally (iv) to gravid adults

Subclass DIGENEA

-1 superorder

superorder

ANE~ITHELIOCYSTIDIA

order ECHINOSTOMIDA Echinostomatoidea *m, :. Paramphistomoidea *m, * Notocotyloidea *m

r O 0

EPlTHELIOCYSTIDIA

I

I STRIGEATOIDEA

Strigeoidea :., : : Clinostomoidea :. Schistosomatoidea 4 Azygioidea *, Transversotrematoidea* Cyclocoeloidea 4 or *rn Brachylaimoidea :. , 4 Fellodistornoidea :. , 4,* Bucephaloidea .:

+

PLAGIORCHIIDA

+ +

Plagiorchioidea :. , Allocreadioidea :. , Lepocreadioidea :. Opecoeloidea :. , 4 (f. Megaperidae *m)

n

z OPISTHORCHIIDA

Opisthorchioidea ;. , 4 Hemiuroidea :., 4,

+

O n

cn

m , 0

-e r m

0

a

>

-1 4

n

KEY:

;cl

*

2-host, without metacercaria. *m 2-host with metacercaria. .*. 3-host. 4 Secondarily 2-host. Possible 1-host. : : 4-host.

z

r A

0, -1

+

3

n

FIG.7. Distribution of life-cycle patterns among superfamilies.

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J . C. PEARSON

(not, or no longer, metacercariae) as in some plagiorchioids (Buttner, 1950, 1951a), opisthorchioids (Buttner, 1951b), opecoeloids (Hickman, 1934; Short and Powell, 1968), hemiuroids (Manter, 1967 ; Dollfus, 1954), allocreadioids (Wootton, 1957a), and monorchids (Stunkard, 1959). The life-cycle of Pleurogenes medians, as described by Buttner (1951 b), is of particular interest as, in this species, attainment of sexual maturity in the second intermediate host is variable, depending on a combination of genetic and environmental factors. The life-cycle of Ratzia,joyeuxi,as described by Buttner (l952), appears to be intermediate between a three-host and an abridged two-host life-cycle, as the adult will live, and even mate, in the snake host. Derogenes varicus, with its puzzling wide host-range (Yamaguti, 1958), is probably no longer a parasite of the many fishes in which it is found, as it can mature in intermediate hosts (Dollfus, 1954), but is simply freed to lay its eggs in the intestines of the fish host, no doubt to the fluke’s advantage in disseminating the eggs (Manter, in press). Indeed, the wide host-range of other hemiurids, such as Sterrhurus musculosus, probably results from the precocious development of sexually mature adults in the second intermediate host. Strictly two-host life-cycles have been demonstrated experimentally for Paralepoderma brumpti by Buttner (195 I a), Asymphylodora amnicolae by Stunkard (1 959), and Allocreadium alloneotenicum by Wootton (1 957a), and proposed on ecological grounds for Coitocaecum anaspidis by Hickman ( I 934). Further reduction, resulting from precocious development in the first intermediate host, has occurred in Parahemiurus bennettae, as described by Jamieson (1966), probably resulting in a one-host life-cycle adapted to singularly difficult ecological conditions. As suggested by La Rue (1951), the schistosomatoids have the ear-marks of a group that has lost its original definitive host. B.

LOSS OF SECOND INTERMEDLATE HOST

The second intermediate host has been suppressed through assimilation of its function-development of the metacercaria-by the first intermediate host or by the definitive host. The former is commoner. By the simple expedient of remaining and developing into the metacercaria in the first intermediate host, the second intermediate host is lost, and the life-cycle reduced to two hosts. Such reduction may be facultative as described by Thomas (1958) for Phyllodistomum simile, by Cort and Brackett (1937) for Diplostomum Jlexicaudum, by McMullen (1937, 1938) in Plagiorchis spp., by Burns and Pratt (1953) for Metagonimoides oregonensis, by Pearson (unpublished) in Sigmapera cincta, or facultative and seasonal as shown by Loos-Frank (1969) in Gymnophallus choledochus. In others, encystment in the first intermediate host is obligatory; for example, in some microphallids (Belopol’skaya, 1962), brachylaimids (Kagan, 1951 ; Krull, 1935; Byrd, 1940), and hemiuroids (Jamieson, 1966). A curious feature of some abridged life-cycles, is the escape from the snail

A PHYLOGENY O F LIFE-CYCLE PATTERNS OF THE D I G E N E A

177

host of the daughter sporocyst-never, it seems, the redia-containing metacercariae that have encysted in situ. This condition, seen in some phyllodistomes (Ssinitzin, 1901), and opecoeloids (Dobrovolny, 1939a), must have arisen separately as in single genera, Plagiopours and Phyllodistomum, a stylet is present in those species that escape and enter a second intermediate host, but not in those that encyst within the daughter sporocyst which itself escapes and is eaten by the definitive host. In this case, escape of the daughter sporocyst and loss of the stylet are both secondary, and escape cannot be a relict of an earlier phase comparable with escape of the cercaria. Rather it would appear to be an extension of the habit of entry from the haemocoele into the rectum or uterus of the fully developed daughter sporocyst, most probably to facilitate the escape of cercariae. Examples of this are cyathocotylid daughter sporocysts in the rectum (Sewell, 1923), daughter sporocysts of Plagioporus lepomis, a three-host species, in the rectum (Dobrovolny, I939b), and “daughter germinal sacs” of Proterometra dickermani in gonoducts (Anderson and Anderson, 1963). A similar condition is seen in the three-host life-cycles of some dicrocoeliids (Timon-David, 1957, 1960) and ptychogonimids (Palombi, 1942), but here the cercariae do not encyst within the daughter sporocyst, but within the second intermediate host after it has eaten the daughter sporocyst. Utilization of the same animal as both second intermediate and definite host is not uncommon, that is one may find metacercariae in the tissues and adults in the intestine, but it is most uncommon to find that these adults are derived from metacercariae from the same individual of the host species. Indeed, such a combination of host functions leading to loss of an intermediate host is known in only four genera, Glypthelmins, Cephalogonimus, Opisthioglyphe and Alaria. In two species of Glypthelmins, cercariae penetrate and encyst in the skin of the frog, and the metacercariae are swallowed with the shed skin (Rankin, 1944; Leigh, 1946; Cheng, 1961). The life-cycle of Cephalogonimus is similar (Lang, 1968, 1969). In a third species of Glypthelmins, G . hyloreus, there are two routes of infection of the definitive host. In addition to ingestion in the shed skin of the frog, metacercariae that develop in the coelom of the tadpole migrate into the lumen of the intestine, following metamorphosis of the host (Martin, 1969). In two species of Opisthioglyphe, cercariae encyst on or in the buccal mucosa of tadpoles, and the metacercarial cysts soon enter the gut where excystment and growth to the adult occur (Grabda-Kazubska, 1969). The case of Alaria is discussed below as it exhibits both increase and reduction. C.

INCREASE TO FOUR HOSTS

Modification in another direction is seen in certain strigeoids in which a new stage, the mesocercaria, is interpolated between the cercaria and the metacercaria, and an additional host, the third intermediate, is acquired in which the metacercaria now develops (Strigea spp.-Pearson, 1959; Odening, 1967). In what appears to be a parallel development, a comparable four-host

178

1. C . P E A R S O N

life-cycle has been reduced to three by adding the function of the third intermediate host to the definite host (Alaria spp., Pearson, 1956; Odening, 1968). Addition to a three-host life-cycle may also have taken place in didymozoids. Although no complete life-cycles are known, it is likely, as argued by Cable and Nahhas (1962), that three hosts at least are involved-a snail, a crustacean, and a fish-and that the immature forms from the intestine of a variety of small fishes, assigned to Torticaecum and Monilicaecum, are juvenile didymozoids Manter, 1934; Cable, 1956). Transport in the intestine of small fishes may be an integral link in the life-cycle of didymozoids of pelagic fishes, such as tuna and mackerel.

IX. PHYLOGENETIC IMPLICATIONS Some implications of the phylogeny developed above may be illustrated by reference to the renicolids. The renicolids are a small group with, it seems, little variation in the adults, but with two very different types of cercariae, one large, pigmented, with excretory bladder of adult form-the rhodometopa type-and the other in Cable’s (1965) words, “a rather prosaic xiphidiocercaria”, small, and with simple Y-shaped bladder. That both types of cercariae belong to the renicolids seems to be indisputable. The evidence for identifying rhodometopa cercariae with renicolids is strong, although circumstantial, based as it is primarily on the form of the excretory bladder in cercaria, metacercaria, and adult (Wright, 1956). The identity of the “prosaic xiphidiocercaria” as renicolid was revealed by experimental demonstration of the life-cycle of Renicola thaidus by Stunkard (1964). Cable (1965) believed that cercariae described by himself (1956, 1963) and by Holliman (1961) form an almost continuous series of linking forms between the rhodometopa type and the cercaria of Renicola thaidus. Martin (I 971) has recently demonstrated experimentally the life-cycle of a renicolid with cercaria of intermediate type. Rhodometopa cercariae are large, and the level of development of the excretory bladder in them is not reached by Renicola thaidus until the adult stage. Thus it might seem that rhodometopa cercariae, which lack a stylet and approach the adult condition in development of the bladder, have advanced over the more primitive type exemplified by Renicola thaidus. But, in view of the changes postulated in life-cycles following acquisition of a second intermediate host, another possibility must be considered, namely that the rhodometopa type is more primitive than the xiphidiocercarial type. Earlier, it was postulated that following the acquisition of the second intermediate host, the cercaria, formerly in an advanced stage of development toward the adult condition on leaving the molluscan host, became by degrees less well developed as the metacercaria adapted to feeding and growing in the tissues of the second intermediate host. Rhodometopa cercariae are in an advanced stage of development and do not develop significantly as metacercariae. The cercaria of Renicola thaidus is considerably less advanced, and does develop toward the adult condition in the metacercarial stage. That renicolids have plagiorchioid affinities is clear from the form of the

A PHYLOGENY OF LIFE-CYCLE PATTERNS OF THE DICENEA

179

cercaria of Renicolu thuidus. It is suggested by the form of the excretory bladder, with its numerous outgrowths, that they are primitive, and near the origins of the plagiorchioid line. Such outgrowths constitute what has been named the reserve excretory system, but is perhaps more appropriately named the paranephridial plexus or system (Reisinger and Graack, 1962). This system is both commoner and better developed among anepitheliocystidians, which on other grounds, such as life-cycle patterns, are thought to be more primitive than epitheliocystidians (including plagiorchioids) among which the paranephridial system is both rare and seldom well developed. A second feature of the excretory bladder suggests that the rhodometopa type is primitive, namely the occurrence of numerous excretory corpuscles in the excretory bladder of the cercaria. These corpuscles are absent from the bladder of the cercaria of Renicolu thuidus, as indeed they are from the bladders of most cercariae with three-host cycles. They are, on the other hand, present in the bladders of cercariae with primitively two-host life-cycles, such as paramphistomes and notocotylids, and with transitional three-host life-cycles, such as some echinostomes. A corollary of this interpretation of renicolid cercarial types is that the stylet has not been lost in rhodometopa cercariae but gained in the Renicolu thaidus type. Comparison of anteriorly opening gland cells in the two types perhaps lends weight to the argument. In the cercaria of Renicolu rhaidus, such gland cells are large and few, and at least some of them open close to the stylet, and are clearly penetration glands, whereas in rhodometopa cercariae such gland cells are small and numerous (Wright, 1956; Rothschild, 1935), and persist in the metacercaria and early adult (Wright, 1956). The gland cells in rhodometopa cercariae may function in penetration into the second intermediate host; but if so, this cannot be their only function as they persist in both metacercaria and early adult. Unfortunately, it is not known whether they are present in the sexually mature adult, but even without knowing this, their size, number, disposition of duct mouths in a transverse row, and persistence into the adult stage together suggest a strong resemblance to frontal glands. And as suggested earlier, cercarial penetration glands may have been derived from adult frontal glands. Loss of the stylet has undoubtedly occurred in some groups, but where it has, as in the cases cited earlier of gorgoderids and opecoelids, it is clearly related to a change in the life-cycle from three hosts to two such that the cercaria no longer penetrates into a second intermediate host. Absence of the stylet in rhodometopa cercariae cannot be accounted for in this way as both they and Renicola thuidus have three-host life-cycles. In summary, it is suggested that renicolids may be a primitive plagiorchioid group in which the cercarial stylet makes its appearance.

X. SUMMARY A comparison of helminth life-cycles reveals a number of singular features in the digenean pattern. Thus, in addition to that most singular feature, alternation of generations, there is the ubiquity of the cercaria, a stage designed

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J . C. PEARSON

for escape from the molluscan first host and for swimming, two patterns of behaviour that are not called for in many life-cycles. In order to explain the ubiquity of the cercaria, it is postulated that the present molluscan first intermediate host was the original host of the proto-digenean and that escape of the cercaria from this host is primitive. And to explain the occurrence in many life-cycles of a free-swimming miracidium that penetrates the integument of the molluscan host, it is postulated that in an earlier stage the proto-digenean was an ectoparasite of the mollusc. Assuming that the proto-digenean was a visceral parasite of a mollusc and that it escaped from its host as an adult, presumably tailed, to lay its eggs, then the known life-cycles of contemporary digeneans may be interpreted as suggesting the following order in the acquisition of hosts: (i) vertebrate definitive (two-host life-cycle), and (ii) invertebrate second intermediate (three-host life-cycle). It is further suggested that a second intermediate host has been added several times in different groups. From the scheme proposed, it would appear that the most primitive life-cycle extant is one with two-hosts, mollusc-vertebrate, in which the cercaria escapes from the snail and is ingested by the fish. The metacercarial stage, absent in this life-cycle, is a later addition albeit a successful one as attested by the number of groups exhibiting a two-host life-cycle with a metacercaria. The three-host life-cycle, the commonest pattern among the digeneans, has arisen several times over from two-host life-cycles, and has been secondarily reduced in a number of groups through telescoping of host roles resulting in loss of the definitive host or of the second intermediate host, or, possibly of both of these hosts. With regard to the succession of generations, it is postulated that the present adult was the original adult, that the mother sporocyst generation was the first of the new generations, that the redia was the second, and that daughter sporocysts have been derived from rediae several times. Corollaries of the postulates advanced include the following general statements : (i) large cercariae with advanced reproductive systems are primitive, whereas small cercariae with rudimentary reproductive systems are derived ; (ii) in three-host life-cycles, metacercariae with rudimentary reproductive systems are primitive, whereas metacercariae with advanced reproductive systems, and sexually mature adults in intermediate hosts, are derived ; (iii) three-host life-cycles need not imply close relationship; (iv) daughter sporocysts occur in unrelated groups; (v) small, short-lived mother sporocysts are primitive, whereas both miracidia containing a single redia and large, long-lived mother sporocysts are derived ; (vi) a metacercarial stage is not present in all life-cycles; and (vii) gland cells opening pre-orally in cercariae (and metacercariae) are not all penetration glands, for some may be adult structures formed early in development. There are no simple cycles among the Digenea; all, including such aberrant forms as Parvatrema homoeotecnum, are digenetic in the original sense of Van Beneden (1853). Hence, all speculation on origins is hypothetical. But, whatever the faults of the scheme proposed, it does help in ordering, or classifying, the multifarious life-cycles of the Digenea and in suggesting lines for further study, such as multiple origin of the second intermediate host and

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181

the nature and relationships of pre-orally opening glands in the various stages of the adult generation. REFERENCES Abdel-Malek, E. T. (1952). Cercaria chandleri, a new echinostome species from the snail Helisoma corpulentum in Lake Itasca, Minnesota. Trans. Am. microsc. SOC. 71,277-281. Adams, J. E. and Martin, W. E. (1963). Life cycle of Himasthla rhigedana Dietz 1909 (Trematoda: Echinostomatidae). Trans. Am. microsc. Soc. 82, 1-6. Agarwal, S. M. (1963). Further observations on the life history of Clinosromum giganticum Agarwal, 1955, ’59. Indian J . Helminth. 15,85-90. Allison, L. N. (1943). Leucochlorirfiomorphaconstantiae (Mueller) (Brachylaemidae), its life cycle and taxonomic relationships among digenetic trematodes. Trans. Am. microsc. Soc. 62, 127-168. Ameel, D. J. (1934). Paragonimus, its life history and distribution in North America and its taxonomy (Trematoda: Troglotrematidae). Am. J . Hyg. 19,279-317. Anderson, M. G. and Anderson, F. M. (1963). Life history of Proterometra dickermani Anderson, 1962. J . Parasit. 49, 2755280. Anderson, G. A., Schell, S. C. and Pratt, I. (1965). The life cycle of Bunoderella metteri (Allocreadiidae : Bunoderinae) a trematode parasite of Ascaphus truei. J . Parasit. 51, 579-582. Angel, L. M. (1969). Prototransversotrema steeri gen. nov., sp. nov. (Digenea : Transversotrematidae) from a South Australian fish. Parasitology 59, 719-724. Angel, L. M. (1971). Burnellus gen. nov. (Digenea: Fellodistomatidae), the life history of the type-species, B. trichojiircatus (Johnson & Angel, 1940) and a note on a related species, Tandanicola bancrofti Johnson, 1927, both from the Australian freshwater catfish, Taridanus tandanus. Parasitology 62, 375-384. Baer, J. and Joyeux, Ch. (1961). Classe des Trematodes (Trematoda Rudolphi). In “Trait6 de Zoologie Anatomie, Systematique, Biologie” (P. P. Grasse, director). Vol. 4, pp. 561-692. Masson et Cie, Paris. Baylis, H . A. (1938). Helminths and evolution. I n “Evolution, Essays on Aspects of Evolutionary Biology” (ed. G. R. de Beer), pp. 249-270. Clarendon Press, Oxford. Bearup, A. J. (1960). Life history of Acarrthoparyphium spit~ulosumJohnston, I91 7 (Trematoda : Echinostomatidae). Aust. J . Zoo]. 8, 21 7-225. Beaver, P. C. (1937). Experimental studies on Echinostoma revolutiim (Froelich), a fluke from birds and mammals. Univ. of Ill. Bull. 34, 1-96. Beaver, P. C. (1939a). The morphology and life history of Petasiger nitidirs Linton (Trematoda : Echinostomidae). J . Parasit. 25, 269-276. Beaver, P. C. (1939b). The morphology and life history of Psilostomum ondatrae Price, 1931 (Trematoda : Psilostomidae). J . Parasit. 25, 383-393. Beaver, P. C. (1941). Studies on the life history of Euparyphium melis (Trematoda : Echinostomidae). J. Parasit. 27, 35-44. Belopol’skaya, M. M. (1962). The life cycles of the trematodes of the family Microphallidae Travassos 1920. Vest. leningr. Univ. 17,45-53. (ln Russian.) Belopol’skaya, M. M. (1963). Progenesis of trematodes in the family Microphallidae Travassos, 1920. Helminths of man, animals and plants and a conflict with them, for 85th Anniversary of Akademician K. I . Skrjabin, pp. 208-210. (ln Russian.) Bennett, M. J. and Humes, A. G . (1939). Studies on the pre-cercarial development of Stichorchis subtriquetrus (Trematoda : Paramphistomidae). J. Parasit. 25, 223-23 1.

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Rothschild, M. (1937). Note on the excretory system of the trematode genus Muritrema Nicoll, 1907, and the systematic position of the Microphallinae Ward, 1901. Ann. Mag. nut. Hist. 19 (10th Series), 355-365. Schell, S. C. (1967). The life history of Phyllodistomum stafordi Pearse, 1924 (Trematoda : Gorgoderidae Looss, 1901). J . Parusit. 53, 569-576. Sewell, R. B. S. (1923). “Cercariae indicae,” Indian J. med. Res. 10, Supplementary Number, 370 pp. Short, R. B. and Powell, E. C. (1968). Mature digenetic trematodes from New Zealand octopuses. J. Parasit. 54, 757-760. Sillman, E. I. (1962). The life history of Azygia longu (Leidy 1851) (Trematoda : Digenea), and notes on A . acuminutu Goldberger 191 I . Trans. Am. microsc. Sac. 81,43-65. Singh, R. N. (1959). Studies on the morphology and life history of Clinostomum piscidium Southwell and Prashad, 191 8 (Trematoda : Clinostomatidae). Proc. nutn. Acad. Sci. India. Section B, 29, 12-33. Sinitsin, D. (1931a). Studien iiber die Phylogenie der Trematoden. IV. The life histories of Plagioporus siliculus and Plugioporus virens, with special reference to the origin of Digenea. Z . wiss. Zool. 138,409-456. Sinitsin, D. (1931b). Studien iiber die Phylogenie der Trematoden. V. Revision of Harmostominae in the light of new facts from their morphology and life history. Z. PurasitKde 3, 786-835. Smyth, J. D. (1966). “The Physiology of Trematodes”, 256 pp. Oliver and Boyd, Edinburgh. Smyth, J. D. (1969). “The Physiology of Cestodes”, 279 pp. Oliver and Boyd, Edinburgh. Sogandares-Bernal, F. (1 962). Microphallus progeneticus, a new apharyngeate progenetic trematode (Microphallidae) from the dwarf crayfish, Cumbarellus puer, in Louisana. Tulane Stud. Zool. 9, 3 19-322. Ssinitzin, D. Th. von (1901). 4. Einige Beobachtungen iiber die Entwicklungsgeschichte von Distomum folium Olf. (Vorlaufige Mittheilung). Zool. Anz. 24, 689-694. Steenstrup, J. J. Sm. (1845). “Alternation of Generations.” Trdnsl. G. Busk from the German version of C. H. Lorenzen. Ray Society, London. 130 pp. Stirewalt, M. A. (1963). Chemical biology of secretions of larval helminths. Ann. N . Y. Acad. Sci. 113, 36-53. Stunkard, H. W. (1956). The morphology and life-history of the digenetic trematode, Azygiu sebago Ward, 1910. Biol. Bull. 111, 248-268. Stunkard, H. W. (1959). The morphology and life-history of the digenetic trematode, Asymphylodora amnicolae n. sp. ; the possible significance of progenesis for the phylogeny of the Digenea. Biol. Bull. 117, 562-581. Stunkard, H . W. (1963). Systematics, taxonomy, and nomenclature of the trematoda. Q . Rev. Biol. 38 221-233. Stunkard, H. W. (1964). Studies on the trematode genus Renicolu: observations on the life-history, specificity, and systematic position. Biol. Bull. 126,467-489. Szidat, L. (1 932). Uber cysticerke Riesencercarien, insbesondere Cercaria rnirubilis H. Braun und Cercaria splendens n. sp. und ihre Entwicklung im Magen von Raubfischenzu Trematoden der Gattung Azygiu L0oss.Z. ParasitKde4,4 77-505. Szidat, L. (1937). Uber die Entwicklungsgeschichte von Sphueridiotrernu globulus Rud. 1814 und die Stellung der Psilostomidae Odhner im natiirlichen System. I. (1) Die Entwicklungsgeschichte von Sphaeridiotrernu globulus Rud. Z . ParasitKde 9, 529-542.

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Thomas, J. D. (1958). Studies on the structure, life history and ecology of the trematode Phyllodistomum simile Nybelin, I926 (Gorgoderidae : Gorgoderinae) from the urinary bladder of brown trout, Salmo truttu L. Proc. 2001. Soc. Lond. 130,397435. Timon-David, J. (1 953). Recherches sur les trematodes de la Pie en Provence. Annls Parasit. hum. comp. 28,247-288. Timon-David, J. (1955). Cycle evolutif d’un trematode cyclocoelide: Pseudhyptiusmus dollfusi Timon-David 1950 Recherches experimentales. Annls Parasit. hum. comp. 30,43-61. Timon-David, J . (1957). Recherches sur le developpement experimental de Bruchylecithum alfortense (A. Railliet) R. Ph. Dollfus 1954, Trematode dicrocoeliide parasite des voies biliaires de la pie. A i d s Parasit. hum. comp. 32, 353-368. Timon-David, J. (1 960). Recherches experimentales sur le cycle de Dicrocoelioides petiolatum (A. Railliet 1900) (Trematoda, Dicrocoeliidae). Annls Parasit. hum. comp. 35, 251-267. Ulmer, M. J. (1951). Postharmostomum helicis (Leidy, 1847) Robinson, 1949, (Trematoda), its life history and a revision of the subfamily Brachylaeminae. Part I. Trans. Am. microsc. Soc. 70, 189-238. Ulmer, M. J . (1957). Notes on the development of Cotylurus~abellifbrmis tetracotyles in the second intermediate host (Trematoda : Strigeidae). Trans. Am. microsc. SOC.76, 321-327. Ulmer, M. J. and Sommer, S. C. (1957). Development of sporocysts of the turtle lung fluke, Heronimus chelydrae MacCallum (Trematoda : Heronimidae). Proc. Iowa Acad. Sci. 64,601-613. Van Beneden, P. J. (1853). La generation alternante et la digenese. Bull. Acad. R. Sci. Belg. 20, 10-23. Van Der Woude, A. (1954). Germ cell cycle of Megalodiscus temperatus (Stafford, 1905) Harwood, 1932 (Paramphistomidae : Trematoda). Am. Midl. Nut. 51, 172-202. Wootton, D. M. (1957a). Studies on the life-history of Allocreadium alloneotetiicum sp. nov. (Allocreadiidae-Trematoda). Biol. Bull. 113, 302-3 15. Wootton, D. M. (1957b). Notes on the life-cycle of Azygia ucuminatu Goldberger, 191 1 (Azygiidae : Trematoda). Biol. Bull. 113, 488498. Wright, C. A. (1956). Studies on the life-history and ecology of the trematode genus Renicola Cohn, 1904. Proc. zool. Soc. Lond. 126, 1-49. Wright, C. A. (1960). Relationships between trematodes and molluscs. Ann. trop. Med. Parasit. 54, 1-7. Yamaguti, S . (1958). “Systema Helminthum. Vol. I. The Digenetic Trematodes of Vertebrates”, Parts 1 , 2. Interscience Publishers, Inc., New York.

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Intramolluscan Inter-trematode Antagonism: a Review of Factors Influencing the Host-parasite System and its Possible Role in Biological Control HOK-KAN LIM AND DONALD HEYNEMAN

Hooper Foundation. University of California. San Francisco. Califbrnia 94122 1 . Introduction ....................................................................................... I 1 . Materials and Methods ................................................................ A . Maintenance of Bio ria glubrata Snails .................................... B . Maintenance of Schistosoma mansoni ................................................ C . Maintenance of Paryphostomum segregatum ...... D . Maintenance of Other Echinostomes ................................................ E . Exposure of Snails ........................................................................ F. Sporocyst and Redial Counts ......................................................... G . Histological Methods .................................................................. 111. Trematode Interaction Patterns ......................... ........................... A . General Considerations .................................................................. B . Review of Intramolluscan Dev-lopmental Stages ................................. C . Characteristics of Redial Predation-Direct Antagonism ..................... I . Redia dominant over sporocyst ................................................ 2. Predatory activity of redia ......................................................... 3 . Redial morphology and efficiency of predation .............................. 4 . Triggering mechanism for predatory activity ........ D. Characteristics of Sporocyst or Redial Nonpredato Antagonism ................................................................................. 1 . Inhibitoryeffect ..................................................................... 2. Degenerative changes ............................................................... 3 . Possible mechanisms ............... a . Snail immunity ............................................................... b . Direct toxicity .................................................................. c . Competition for nutrients and oxygen .................................... d . Other mechanisms ............................................................ E. Assessment of Adaptation of the Trematode to the Snail Host ......... 1. Infection rate ........................................................................ a . General considerations ...................................................... b . Miracidial penetration ...... ................................. c . Successful development ...... ................................. 2 . Size of the redial population .............. .................................. 3 . Adaptation index .......................... .................................. IV . Parameters of Intramolluscan Inter-trematode Antagonism ........................... A . Stages of Antagonism Based upon the Biomphalaria glabrata-Schistosoma mansoni-Paryphostom segregutum Model .......................................... 1. Some general aspects ...............................................................

191

192 193 193 197 197 198 198 199 199 199 199 201 203 203 204 205 205 206 206 207 207 201 213 214 215 215 215 215 220 222 223 224 225 225 225

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2. Miracidial penetration ............................................................ a. P.segregatum superimposed on S. mansoni .............................. b. S. mansoni superimposed on P. segregatum .............................. 3. Establishment of the infection in B. glabrata ................................. 4. Delayed redial migration ......................................................... 5 . Delayed germinal development within the redia ........................... 6. Attraction of predaceous rediae to sporocysts.. ............................... 7. Speed of completion of dominance ............................................. 8. Third-generation sporocysts of S. mansoni .................................... B. Histological Observations ............................................................... 1. P.segregatum superimposed on S. mansoni ................................. 2. S. mansoni superimposed on P.segregatum ................................. V. Trematode Antagonism in Biological Control .......................................... VI. Summary .......................................................................................... References .......................................................................................

229 229 229 23 1 234 235 235 237 237 238 238 239 244 25 1 253

I. INTRODUCTION The presence of echinostome larvae within their snail hosts has been shown to inhibit or preclude development of larvae of other trematode species. This originally was suggested by field observations, such as those of Cort et al. (1937); Martin (1955); Boray (1964, 1967), Basch and Lie (1965). Lie and co-workers demonstrated this phenomenon in a series of laboratory experiments (cf. review 1968b) and Boray knew of it (cf. review 1969). These studies on trematode antagonism and inhibition appear to have opened a new approach to biological control of snail-transmitted trematode diseases, such as schistosomiasis and fascioliasis. Much more basic information is required, however, to evaluate and measure the importance of factors that determine the outcome of various patterns of infection or degrees of dominance shown in interspecific antagonism studies. We have tried in the present review to analyse some of these factors based on published information and suggested by new research data that indicate lines of research we feel would be fruitful. Discussion of double infection will focus on data from our laboratory studies, which began with the work of Lie, Basch, and Umathevy (1965) and have continued actively since then in Kuala Lumpur* and San Francisco.? Primary responsibility for developing this program lies with Professor Lie Kian Joe, who initiated the study and has carried it through each of its developmental phases. To Dr Lie, for his innovative and significant contributions, we wish to dedicate this discussion. Earlier work on intramolluscan inter-trematode antagonism was concerned chiefly with establishing that such interactions did occur and discovering the dominant and subordinate species in the various combinations being tested (Table I). Twenty-four combinations of 19 species and strains of Digenea in

* University of California International Center for Medical Research and Training at the Institute for Medical Research, Kuala Lumpur, Malaysia; funded under a United States Public Health Service, National Institutes of Health (USPHS-NIH) grant A1 10051. t University of California, Hooper Foundation, Department of International Health, San Francisco. Studies funded by a USPHS-NIH research grant No. Al-07054 from the National Institute of Allergy and Infectious Diseases.

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193

5 species and strains of snail hosts have been studied so far by Lie and coworkers. Boray (1 967) established that an unidentified echinostome is dominant over Fasciola hepatica in Lymnaea tomentosa. From these reports one can discern the broad spectrum of interaction involved, covering all degrees of interaction between the parasites themselves and between them and their snail hosts. We shall attempt here to define some of the criteria used to evaluate these interactions and to discuss possible mechanisms responsible for them. As our laboratory model, we shall rely chiefly on the system consisting of the NIH laboratory strain of albino Biomphalaria glabrata (Say, 1818) and on the interaction of an NIH laboratory strain of Schistosoma mansoni (Sambon, 1907) and the echinostome Paryphostomum segregatum Dietz, 1909 from Belo Horizonte, Brazil, where it parasitizes B. glabrata. Cercariae of the echinostome encyst on small fish and tadpoles, and adult worms are found in the intestines of the common black vulture (Urubu), Coragyps atratus foetens (Lichtenstein).

TI. MATERIALS A N D METHODS Methods of maintaining host snails, of handling the parasites used in our model infection system and conducting the infections will be described briefly, as these procedures may significantly alter the conditions that affect the host-parasite interaction. Techniques for cultivation of B. glabrata and maintenance of S. mansoni in the laboratory have been reviewed by several workers, most recently Pellegrino and Katz (1968), Webbe and James (1971) and Bruce and Radke (1971). A.

MAINTENANCE OF

B. glabrata SNAILS

The laboratory-reared snails we have used and maintained since 1964 are derived from an albino strain developed at the National Institutes of Health, Bethesda, Maryland, from a cross between Brazilian and Puerto Rican B. glabrata (Newton, 1953, 1955). The relatively non-pigmented shell facilitates observation of parasites through the intact animal. Each strain or species is maintained in a series of glass or plastic aquaria in a separate room (Fig. I). Banks of shelves, enclosed by curtains of heavy polyethylene sheeting, permit each worker to keep experimental material isolated. This also reduces the danger of accidental mixing of separate snail strains, which may be morphologically inseparable. Space heaters within each such shelving unit allow reasonable temperature control (25-27°C) and permit laboratory work to be carried out elsewhere in the room at a more suitable temperature (22-25°C). The higher temperature for these tropical snails simulates their tropical environment, and from our observation, produces a healthier colony with optimal rates of growth and reproduction. The snails are separated and maintained in several size-classes: eggs, under 3 mm, 3-7 mm, 7-10, and over 10 mm. Groups above 3 mm are maintained in glass aquaria with an overflow siphon filtration system, holding about 4 litres of

TABLE I Intramolluscan inter-trematode antagonism. Combinations studied at the Hooper Foundation (UC-ICMRT)laboratories in Kuala Lumpur (Malaysia)and San Francisco (U.S.A.)

Snail host

Dominant parasitea

Lymnaea rubiginosa Michelin, 183 1 Lymnaea rubiginosa Michelin, 183 1 Lymnaea rubiginosa Michelin, 1831

(R)b Echinostoma audyi Lie and Umathevy, 1965 (R)b Echinostoma audyi Lie and Umathevy, 1965 (R)b Echinostoma audyi Lie and Umathevy, 1965

Lymnaea rubiginosa Michelin, 1831 Lymnaea rubiginosa Michelin, 1831 Indoplanorbis exustus Deshayes, 1834 Biomphalaria straminea Dunker, 1834 Biomphalaria straminea Dunker, 1834 Biomphalaria glabrata Say, 1818 (St. Lucia) Biomphalaria glabrata Say 1818 (St. Lucia)

(R)b Echinostoma audyi Lie and Umathevy, 1965 (R) Echinoparyphium dunni Lie and Umathevy, 1965 (R)Echinostoma malayanum Leiper, 191 1 (R) Paryphostomum segregatum Dietz, 1909 ( R )Paryphostomum segregatum Dietz, 1909 (R) Paryphostomum segregatum Dietz, 1909 (R) Ribeiroia marini Faust and Hoffman, 1934

Subordinate parasite

Reference

(S)b Unidentified strigeid

Lie, Basch and Umathevy, 1965

(S) Unidentified xiphidiocercaria (S) Trichobilharzia brevis Basch, 1966

Lie, Basch and Umathevy, 1965

(R) Fasciola gigantica

Lie, Basch and Umathevy, 1965 and Basch and Lie, 1966a, b; Owyang, Lie and Kwo, 1970 Kwo, Lie and Owyang, 1970

0

x x 3z

t

I

>

z W 0

0

(S) Unidentified xiphidiocercaria ( S ) Schistosoma spindale Montgomery, 1906 (R) Echinostoma barbosai Lie and Basch, 1966 (R) Ribeiroia marini Faust and Hoffman, 1934 (R) Ribeiroia marini Faust and Hoffman, I934 (S) Schistosoma mansoni Sambon, 1907 (St. Lucia)

Lie, Basch and Umathevy, 1965

z

3-

*

b

Heyneman and Umathevy, 1968; Lie, Kwo and Owyang, 1970, 1971 m < Lie, Basch and Hoffman, 1967 z

n

Basch, Lie and Heyneman, 1970 Basch, Lie and Heyneman, 1970 Basch, Lie and Heyneman, 1970

' 32

Biomphalaria glabrata Say, 1818 (NIH) Biomphalaria glabra ta Say, 1818 (NIH) Biomphalaria glabrata Say, 1818 (NIH) Biomphalaria glabrata Say, 1818 (NIH) Biomphalaria glabrata Say, 1818 (NIH) Biomphalaria glabrata Say, 1818(NIH) Biomphalaria glabrata Say, 1818 (NIH) Biomphalaria glabrata Say, 1818 (NIH) Biomphalaria glabrata Say, 1818 (NIH)

( R )Echinostoma barbosai Lie and Basch, 1966 (R) Paryphostomum segregatum Dietz, 1909 ( R )Paryphostomum segregatum Dietz, 1909 (R) Paryphostomum segregatum Dietz, 1909 (R) Paryphostornum segregatum Dietz, 1909 (R) Paryphostomum segregatum Dietz, 1909 ( S ) Schistosoma mansoni Sarnbon, 1907 (NIH) (R) Ribeiroia marini Faust and Hoffman, 1934 (R) Echinostoma liei rns. sp.

( S ) Schistosoma marrsorii Sarnbon, 1907 ( S ) Schistosoma mansoni Sarnbon, 1907 (R) Echinostoma lindoense Sandground and Bonne, 1940 (R) Echinostoma paraensei Lie and Basch, 1967 (R) Ribeiroia marini Faust and Hoffman, 1934 (R) Echinostoma lie; ms. sp. ( S ) Cotylurus lutzi Basch, 1969 ( S ) Schistosoma mansoni Sambon, 1907 (NIH) ( S ) Schistosoma mansoni Sarnbon, 1907 (NIH)

Lie, 1966 Lie, 1967, 1969c Lie et al., 1968c Lie, Basch and Heyneman, 1968a Basch, Lie and Heynernan, 1970 Heynernan, Lirn and Jeyarasasingarn, in press Basch, Lie and Heynernan, 1969 Basch, Lie and Heynernan, 1970 Heynernan, Lirn and Jeyarasasingam, in press

a The following combinations of echinostomes were studied in Lymnuea rubiginosa Cie, Basch and Umathevy, 1966): Echinosroma uudyi with Echinoparyphium dunni, E. audyi with Hypoderueuni dingeri Lie, 1964, H. dingeri with Echinostomu hystricosum Lie and Umathevy, 1966, H . dingeri with E. dunni, and E. dunni with E. hystricosum. The first infections were naturally acquired. Challenging infections with the heterologous species did not develop in most cases. Microsporidiawere found and probably did interfere with antagonistic interactions between the echinostome larvae. No dominancy could be determined in these cases. h Letters in bracket indicate (R)=redia stage present, (S)= only sporocyst stage present.

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water (Figs 1, 2). The number of snails per tank does not exceed 20 for the larger snails and 50 for the 3-7 mm class. Newly hatched snails may number up to several hundreds in a plastic aquarium containing about 4 litres of water, but are transferred to glass tanks as soon as they reach 3 mm. Eggs, collected weekly, are maintained in plastic aquaria with continual gentle air-stone aeration. Water used for the snails is passed through a standard Barnstead demineralizer. Water pH, checked every 2 weeks, is kept between 6 and 7 by addition of sodium bicarbonate solution. Pieces of chalk are added and red leaf lettuce (Lactuca sativa) is broken up and fed to the snails daily (Fig. 2). Aquaria are cleaned once a week, more frequently when needed or for snails to be kept under special observation. Water control is of particular importance for the health and normal behaviour of the snails. We use a small plastic tank, holding about 1+ litres of water and up to 10 snails for our experimental

FIGS1-4. ( I ) , Maintenance of uninfected B. glabrata; (2), Glass aquarium (a, chalk; 6 , lettuce); (3), Maintenance of experimental snails (n, plastic aquarium holding about 4 litres water; b, smaller plastic tanks inside larger aquarium; c, air tubing); (4),Glass finger bowls for observation of individual snails

The following symbols are used for the figures: albumen gland C cercaria DS daughter sporocyst FH foot-head organ GB germball I intestine AG

L MC MS OT R UE

liver mantle collar mother sporocyst ovotestis redia unidentified embryo

INTRAMOLLUSCAN INTER-TREMATODE ANTAGONISM

197

material (Fig. 3). Holes drilled through the side walls and bases of some of these tanks enable them to be sunk in larger aquaria to share a common water supply. Two such tanks placed in the same larger aquarium permit water conservation or exposure of several experimental lots to identical conditions. Individual snails kept isolated for experimental observation are housed in 350 ml glass finger bowls (Fig. 4). Experimental snail rooms are lighted with fluorescent ceiling light for about 10 h during weekdays and varying periods on weekends. B.

MAINTENANCE OF

s. mansoni

The strain used for most of our studies in the San Francisco laboratory was one originally received from the National Institutes of Health at the same time we received the NIH albino snails. Adult worms are maintained in golden hamsters (Mesocricetus auratus auratus Waterhouse), infected by subdermal injection of about 200 cercariae. Hamsters are killed 8 weeks later and their livers removed. Worm eggs are usually harvested from several heavily infected livers typically marked with whitish fibrous surface spots. The liver tissue is chopped with a single-edged razor blade for 2-3 min after tissue fragments have been examined to ensure presence of sufficient fully embryonated eggs. After the tissue has been blended for 45 sec at high speed, it is rinsed in 0 . 9 % saline for 4 or 5 washings. The sediment is poured into a volumetric flask painted black with the top 10cm left unpainted. The flask is then filled with demineralized water, placed under a 100-watt lamp, and within a few minutes miracidia can be seen collected in the clear upper portion. They are easily collected by pipette and placed in a petri dish, where counted numbers can be isolated in small drops and used as needed. Miracidia more than 1 h old were not used for experimental study. Mother sporocysts of S . mansoni, which develop in the anterior region of the snail, appear as small, colourless swellings in the host's epithelial surface and contain numerous second-generation sporocysts. Developing S . mansoni mother sporocysts that chance to develop in the tentacle form very conspicuous swellings, which are useful as areas for histological study of trematode interaction. Daughter sporocysts migrate via blood sinuses to the posterior organs of the snail, usually the hepatopancreas, where they appear in 2 weeks as elongated, whitish bodies. Cercarial shedding begins during the 4th week of infection. Emergence of larvae is stimulated by placing snails in individual beakers with 30 ml water under a 100-watt lamp (28-29°C). Exposure for 2 h during the afternoon provides optimal numbers of cercariae. The time of day for maximum emergence is a well-defined characteristic of most of the trematode species with which we have worked.

c.

MAINTENANCEOF

P.segregatum

The adult stage is maintained in the urubu or Brazilian black vulture (Coragyps atratusfoetens Lichtenstein). Five infected birds were brought from

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Brazil to San Francisco by Dr Paul Basch in 1966. Infections have been maintained in 3 of these birds since then, 2 having died during that period. Egg production is sustained for 3-6 months in the vulture’s faeces, then can be revived by a fresh feeding of metacercariae. N o evidence of host resistance to periodic reinfection has been noted over the 5 years we have maintained the infection. Throughout this period an unidentified strigeid infection has sustained itself without addition to the adult worm population. We have observed that egg output of the strigeid is inversely correlated with that of the echinostome, but no other evidence of interaction is apparent between the two populations of worms, one self-sustaining, the other artificially maintained by feeding metacercariae. Eggs are collected daily from urubu faeces, washed several times with demineralized water, then incubated at 28°C. Some miracidia hatch after one week but usually 2-week-old eggs are used. These eggs, placed under a 100watt lamp at a I-foot distance in the morning hours, hatch rapidly, and the miracidia swim rapidly at a uniform rate in a characteristic straight line, punctuated by sharp changes of direction. They penetrate the snail host in an exposed area, chiefly the head-foot epithelium. Sporocysts develop in the anterior region of the snail, near the point of miracidial entry. Rediae migrate to the posterior portion via the blood sinuses and occupy the ovotestis. Cercariae appear in the water in greatest numbers in the early morning. To check cercarial shed, snails were isolated in glass bowls and left overnight. Cercariae encyst on a variety of fish species and on tadpoles. We usually reinfect the vultures with about 200 metacercariae 2 or 3 days after the larvae have encysted on goldfish. Metacercariae are collected from the fish and placed in 0.45% sodium chloride solution. Counted dosages then can be pipetted into the vulture’s mouth or given as infected fish tails and fins (where the cysts usually congregate). Metacercariae also may be localized in the fish lateral line, periorbital or supracranial canals. For a detailed study of the life history and morphology ofP. segregatum, see Lie and Basch (1967a). D.

MAINTENANCE OF OTHER ECHINOSTOMES

Other species of echinostomes are maintained and worked with in our San Francisco laboratory and studied by similar methods, largely developed by Dr Lie. These can be found in the original descriptions and life history reports: E. barbosai (Lie and Basch, 1966), E. paraensei (Lie and Basch, 1967b), E. lindoense (Lie, 1968), and E. Iiei sp. nov. (Jeyarasasingam et a/., in press). E.

EXPOSURE OF SNAILS

Glass vials, 5 cm in height by 2.2 cm inner diameter, with 5 ml demineralized water delivered by a Cornwall automatic pipette were found to be optimal for infection. Snails to be exposed are washed twice, cleaned with a fine brush, then placed individually into a vial. The required number of miracidia are added with a small glass pipette. Freshly hatched miracidia are first placed in small drops on glass to facilitate accurate counts and correct dosage.

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Exposed snails are kept for 2 h at room temperature (27°C water temperature), then returned to the appropriate aquarium. F.

SPOROCYST A N D REDIAL COUNTS

Dissection of snails is initiated by crushing the shell gently with a glass vial and separating the columellar muscles from it, after which the whole snail can easily be extracted. To study sporocysts and rediae of P . segregatum, the snail body is cut with iris scissors at a point slightly in front of the atrium. Anterior and posterior halves of the animal are then placed in separate dishes for examination. For study of P . segregatum sporocysts alone, only the anterior part is examined. The foot-head organ, tentacles, mantle collar, and kidney region are separated and examined under the compound microscope. P . segregatum rediae in well-developed infections are usually found in the ovotestis region and lymph spaces surrounding the mantle cavity. Rediae are carefully dissected out of these tissues for study or counted under a dissecting microscope with a hand tally counter. In 12-day old infections rediae usually are localized in the anterior blood sinuses. G.

HISTOLOGICAL METHODS

The body of a snail to be examined histologically is first separated from its shell as described in the preceding section. Bouin’s or Carnoy’s solutions are the preferred fixatives. Care to remove air bubbles trapped inside the pulmonary cavity is needed to ensure proper penetration of fixative. Use of a fine camel-hair brush or, in the case of larger specimens, injection of fixative through the pneumostome into the pulmonary cavity has proved helpful. All shell fragments must be separated and whenever possible the stomach also removed during fixation to prevent damage to the microtome and disruption of sections by sand grains and other hard objects. The snail is then dehydrated in alcohol series, cleared in xylene and embedded in paraplast. Sections, cut at 6 or 8 pm, can be stained with hematoxylin and eosin or Gomori’s trichrome (AFIP, 1960), and mounted in piccolyte. Each snail provides between 500 and 1000 serial sections. Terminology used to describe the histology follows Pan (1958), while description of pathological processes follows Robbins (1967). 111. TREMATODE INTERACTION PATTERNS

A.

GENERAL CONSIDERATIONS

Direct antagonism of Lie (1966) was used to describe predatory or physical activities by rediae on other trematode larvae within a snail. This he contrasted with indirect antagonism, an inclusive and non-specific term for noncontact physiological interactions that included inhibition or modification of development (even of a predatory species) and cytolysis of germinal contents 9

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within an affected trematode. “Indirect” in this sense represents a variety of responses, and, equally important, a variety of sources of toxic or inhibitory factors. Therefore, though the term direct antagonism is confined to trematode-trematode predatory interactions, indirect antagonism should not be construed to involve only trematode-trematode interactions between physically separated antagonists within a snail, or restricted to processes mediated through the snail host. It includes all sources of physiological effects involving no direct trematode-trematode physical interaction. In microbial interactions the term “direct antagonism” is used to imply direct action on the host cell; “indirect antagonism” alludes to metabolic products produced by one organism injurious to others (Waksman, 1945). For interspecific and intraspecific interaction among insect parasitoids, the comparable terms “physical attack” and “physiological suppression” have been used (Salt, 1961; Fisher, 1961). With these analogous phenomena in mind, we will use direct antagonism synonymously with physical or predatory action between different trematode species within a snail, involving a redial stage as the predator. For indirect antagonism, we include all other interspecific trematode interactions, or physiological reactions, including host-mediated ones. The latter term, one is tempted to note, is admittedly a “can of worms”, but none the less it serves a useful, if nondescript, role. Both classes of interaction can involve the same trematode species (interacting with another species), and, at different times, may even involve the same individual larva. Knowledge of the mode of action or identification of stimuli leading to these various forms of inter-trematode rivalry are still very little understood. Along with the strictly inter-trematode interactions are those expressions of indirect antagonism that involve the hast as an active participant. We have evidence that host reaction is an important contributory factor as well, as in the tissue response against S. mansoni sporocysts previously injured by predatory activity of P. segregutum rediae, to be reviewed subsequently. By analysis of the processes involved in snail-trematode-trematode interactions, we hope to recognize, evaluate and ultimately to quantitate conditions and characteristics that predispose a trematode species to dominate another trematode in the same snail, to affect it to some lesser degree, or to become subordinate to it. This should represent an initial stage in the development of concepts as well as tools to enable us to study the nature of these interactions and to predict the probability of a specific outcome. For this purpose, previous studies on insect and insect-plant pathology are especially useful. Antagonistic interactions or competition among insects, whether termed parasitoids or predators of various types, have been studied in many laboratories and in field operations for over 50 years, work that has developed the theoretical base and the practical application in biological control projects against insect pests (DeBach and Sundby, 1963; Doutt and DeBach, 1964; DeBach, 1966). Physical attack or competition among first instar larvae of parasitoids for occupation of the insect host has been summarized by Salt (1961). Fisher (1961) has studied the mechanisms of non-contact antagonism. The relevance of Fisher’s studies to our work with trematodes will be discussed in a following section.

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REVIEW OF INTRAMOLLUSCAN DEVELOPMENTAL STAGES

Immediately after penetrating the snail, the miracidium changes directly into a mother sporocyst, whose germinal cells produce one daughter sporocyst generation consisting of several or many daughter sporocysts, or a series of rediae which represent at least two generations, frequently more. Daughter sporocysts or rediae continue the germinal proliferation, producing numerous cercariae, which mark the end of the intramolluscan proliferative phase, and beginning of the brief free-swimming extramolluscan phase. In some groups, rediae and cercariae may be produced simultaneously in the same redia (Wesenberg-Lund, 1934; Dinnik and Dinnik, 1956, 1964; Zischke, 1966; Lie, 1968; Lie and Basch, 1966, 1967b; Lim, 1970b; Jeyarasasingam et al., in press). The cercariae, a true larval or pre-adult freeliving stage, undergoes no further multiplication, though in most instances (schistosomes being the major exception) cercariae first pass through an encysted or metacercarial stage in or on a transport host or agent before final development to the adult worm in the vertebrate final host. The mother sporocyst, as noted above, is little more than a miracidium that has penetrated the snail epithelium and lost its outer ciliated covering. Two patterns of loss of this ciliated coat have been observed. For Fasciola (Dawes, 1959, 1960a, b, c) and related forms such as the echinostomes (Lie, 1969b), the ciliated plates are lost during penetration and remain outside of the snail, shed like the protein coat of a bacteria-invading phage. The other pattern is that of the schistosomes, in which the epidermal plate is not shed during miracidial penetration, but shortly afterwards, as reported by Faust and Meleney (1924) for S.japonicum, by Faust and Hoffman (1934) and Maldonado and Acosta-Matienzo (1947) and Wajdi (1966a) for S . mansoni, and by Lengy (1962) for S. bovis. The miracidial stage, being so abbreviated within the snail, is of little direct significance in the inter-trematode interaction within the snail tissues. It may, however, be a target of host resistance to its penetration or early development, or it may be subjected to inter-trematode inhibition. The cercaria also appears to be relatively little involved in inter-trematode rivalries, since it is not a proliferative stage undergoing continual growth and intimate metabolic exchange with the host. None the less many cercariae complete their growth in the snail tissues after premature passage from the sporocyst or the redial parent stage and may be subject to inhibitory repression. Direct predation and ingestion of echinostome or schistosome cercariae by echinostome rediae have been observed (Lie et a/., 1967; Heyneman and Umathevy, 1968). We have also observed capture and ingestion of S. mansoni cercariae by an Egyptian echinostome, E. liei (Figs 5, 6). These observations were made during dissection of the infected snail, and we can only presume that the same process also occurs within the intact snail. Though we have little precise information, modification of the host’s physiological state by cercariae would certainly seem probable, especially when large numbers are held within the snail for some time. The interval between birth from the sporocyst or redial parent and escape from the snail may vary from a very

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short period to many days. In certain species of echinostomes the cercariae require a maturation period in the snail tissues outside the redial body before they can pass from the snail host (Wesenberg-Lund, 1934; Beaver, 1939,1941 ; Stunkard, 1960,1966; Etges, 1961). Changes produced during this period or induced in the host may have an important effect on development of a competing species.

FIGS5-8. (5) and (6), Capture of S. mansoni cercaria by E. liei redia, lOOx (5) and 250 x (6); (7) and (8), Double infection in B. glabrafa: S. mansoni (day 120) and P . segregaturn (day 90). Arrows indicate degenerative changes in S. mansoni sporocysts. From saccular portion of kidney, 125 x (7), and from liver, 260 x (8).

The period of cercarial shed is generally a critical one for the snail. High mortality commonly occurs at this time, though this varies markedly with the species of trematode involved (Heyneman et al., in press). Pan (1965) reported severe histopathological changes associated with S. mansoni cercariae. He postulated that secretions from cercariae may serve as stimuli to alert host cellular defenses. This remains an interesting but untested hypothesis. Cercariae of many echinostomes return and encyst in their own host or in other snails, usually with little or no host specificity. Theeffect of metacercariae, no development and establishment of a competing trematode is not known though presence of these cysts appears able to do considerable damage to the snail (Bayer, 1954; La1 and Baugh, 1955). The larvae themselves are little affected, however. Presumably this is due to the protective cyst wall, known to be resistant to highly predaceous larvae (Lie et al., 1967, 1968c; Basch et al., 1970), and to the relatively passive state of pre-adults within the cyst enclosure, which have completed larval growth and, except in progenetic forms (Buttner,

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1951), remain quiescent until the vertebrate final host initiates the stimulus for excystment, growth, and maturation. The proliferative, rapidly growing, and metabolically demanding stages within the snail are those with which we are primarily concerned: the sporocysts and rediae. The sporocyst, essentially a germinative sac of limited mobility, feeds only by diffusion through its body wall. Cheng and Snyder (1 962) suggest that Glypthelmins pennsylvaniensis Cheng, 1961 sporocysts secrete enzymes that hydrolyse glycogen; simpler sugars could then be absorbed by the sporocyst. The sporocysts of Cercaria doricha Rothschild, 1935, in Turritella communis Risso, appear able to obtain nourishment by use of a placenta-like region of the sporocyst wall which is in contact with the gonadal tubule of the host (Negus, 1968). A comparable feeding mechanism appears to permit the rootlike microvilli of Hymenolepis diminuta cysticercoids (which Cheng (pers. commun.) has shown to be branched) to feed on wandering cells in the host beetle, Tribolium conjiusum (Collin, 1970; Ubelaker et al., 1970a, b; Heyneman and Voge, 1971). The sporocyst stage can exert a profound indirect effect on other parasites as well as on the host, as we will discuss shortly. The redia on the other hand, is actively mobile, equipped with a well-developed mouth, a muscular pharynx, and a saccular gut of varying length. It feeds directly on host tissue and, at particular times, on rival trematodes as well. It is this latter aspect which we shall next consider. C.

CHARACTERISTICS OF REDIAL PREDATION-DIRECT

ANTAGONISM

1. Redia dominant over sporocyst In ten combinations of experimental double infections by Lie, Basch and colleagues using redia-producing species (various echinostomes or the psilostome Ribeiroia marini) and a non-redia producer (strigeids, schistosomes, xiphidiocercariae), the first group has always developed into the dominant form. Lie (1969~)stressed that predatory activity by the redial stages represents an efficient mechanism for one trematode species to eliminate a competing species for host sustenance. It also provides a rich food resource, judging by the rapid growth of P. segregatum rediae in a doubly infected host, compared with the same species in a host with no other parasite species present (Basch et al., 1970). Paperna (1967) in Ghana, collected Bulinus truncatus rohlfsi snails shedding xiphidiocercariae, and exposed them to S . haematobium miracidia from infected children. Only 7 snails out of 52 shed both types of cercariae. In a control singly infected group, 66 of 87 snails became infected with S . haematobium. He dissected 20 experimental snails and found no signs of schistosome sporocysts, but noted that: “the entire digestive gland was occupied by the dominant prior infection, accompanied by heavy deposits of deep orange-red pigment”. Field studies, as mentioned earlier, indicate that double infections involving redia-producing species, especially echinostomes, are rare (Cort et a/., 1937; Martin, 1955; Boray, 1964, 1967; Vernberg et a/., 1969). However, interpretation of such field data often is difficult. The duration of interaction between the competing species is unknown, as is the sequence of infection.

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Ewers (1960) reported that double infections were unexpectedly high between Stictodora sp. (a redia-producing heterophyid species) and Austrobilharzia terrigalensis (a schistosome sporocyst producer) in the estuarine snail Valacumantus (Pyrazus) australis. The heterophyid in this instance may not have had sufficient time to eliminate the schistosome, microsporidia infection may have damaged or killed the rediae (as with E. hystricosum observed by Lie et al., 1966), the rediae may not have reached its predatory stage of development, or, we may, in fact, be dealing with a truly non-predatory redia. El-Gindy (l965), studying the monthly prevalence rate of S . haematobium in B. truncatus in Central Iraq, stated that paramphistome infection in snails of all sizes over most of the year partially interrupts schistosome infection in the same snails. He felt that this was indicated by initial appearance of paramphistome infections in smaller snails two months earlier than the appearance of schistosomes, and the scarcity of double infections, which were found only in older snails, 10 mm or larger. An exception has been found to the usual observation that rediae will always prove dominant in any combination of a redia- with a sporocystproducer (Owyang and Lie, 1971).! In this study Trichobilharzia brevis (a sporocyst producer, and strong indirect antagonist) is dominant over Hypoderaeum dingeri (a redia producer, and weak direct antagonist, perhaps a non-predatory form). This finding is discussed subsequently (p. 249) with respect to additional experimental complications being explored with this interaction system. 2. Predatory activity of redia Redial direct antagonism against sporocysts is associated with possession of a well-developed feeding apparatus and a pair of locomotor appendages. The redia can move about within the mollusc body, feed upon host tissues or, when properly stimulated, seek out, attack, and feed upon susceptible parasite larvae. The ability of predatory rediae to seek out and locate sporocysts will be discussed and exemplified in section IV .6. Predatory activity of rediae was first reported by Wesenberg-Lund (l934), who observed “daughter rediae in the mother rediae devouring either other daughter rediae or young cercariae where the rediae or young cercariae are present simultaneously in the same redia”. Nasir (1962) also reported cannibalism in Echinostoma nudicaudatum rediae, and speculated that this phenomenon occurs under adverse conditions, as when insufficient food supply is present for the parasites. Interspecific predation by rediae was observed by Lie et a]. (1969, then described in more detail in succeeding publications. The following, from Lie et al. (1968c), describes the predatory activity of P. segregatum rediae towards rediae of E. lindoense: “The attacking P. segregatum redia attached its mouth to its prey and began sucking vigorously, using its large muscular pharynx as a suction organ, assisted by the powerful contraction and expansion of the anterior third of its intestine. The cuticle or tegument of the attacked redia soon gave way and was drawn into the pharynx of the attacker, forming a narrow cuticular or tegumentary tube visible when the attacking redia

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detached from its prey. The dead redia and its contents were gradually sucked in and consumed. Cercariae were small enough to be swallowed whole, and we often saw several P . segregatum rediae consuming one E. lindoense redia.” 3. Redial morphology and efficiency of predation Lie et al. (1968b) speculated that rediae with a longer intestine may have a greater capacity for predation. They observed that as much as the anterior half of the gut of a P. segregatum redia can act as a powerful pump, apparently more powerful than the action of the pharynx. Unfortunately, available descriptions of rediae of trematode species do not lend themselves to comparison. Differences in techniques may result in differences in observed size, identification of successive redial generations often is difficult, careful morphological study of rediae is seldom done, and analyses of redial gut contents are even more rare. The relationship between redial gut length and predatory vigor therefore must still be considered conjectural o r applying only to those forms with a heavily muscular gut wall. Among insect parasitoids, inherent superiority of one species by means of physical attacks has been described by various authors (Pemberton a n d Willard, 1918 ; Van Steenburgh and Boyce, 1937 ; Ullyett, 1943; Simmonds, 1953). Effectiveness of physical attack is generally associated with better developed tissues and more effective use of specialized mouthparts used in aggressive o r defensive activities as reviewed by Salt (1961). The following excerpt describes such a n interaction between Diachasma tryoni and Opius humilis larvae, both parasitic in fruit-fly larvae (Pemberton and Willard, 1918): “0.humilis is killed purely by wounds and lacerations inflicted upon it by the long, curved, sickle-like mandibles of the newly hatched larva of D. tryoni. . . . Its mandibles open wide and snap into the body of the attacked larva spasmodically and with remarkable quickness. “. . . Besides possessing unusual powers for inflicting injury to other parasitic larvae about it, it may avoid counterattack through ability to move quickly and through the protection afforded the entire ventral surface of the body by a thick mass of serosal, cellular material that accompanies the larva when it emerges from the egg, and which remains with it during its entire life in the first instar. “The newly hatched larva of Opius humilis possesses mandibles which are also long and pointed. . . . These may be used to good advantage when the larva is successful in bringing them in contact with individuals of its own or of other species of parasites. The larva, however, is sluggish. . . and is protected ventrally by a much thinner, less adhesive mass of serosal cells, is much less capable of quick and powerful movement of the mandibles, and usually holds the body in a somewhat horizontal and exposed position. These deficiencies seem to explain its inability to avoid destruction by the larvae of D . tryorii or to offer successful counterattack when the two are lodged within the same host larva.”

4. Triggering mechanism for predatory activity Rediae of different species (e.g. P . segregatum and E. lindoense; P. segregaturn and E. paraensei) were observed in close proximity in B. glabrata without apparent antagonism o r mutual predatory response. Suddenly the dominant rediae proceed towards the subordinate ones, whose rediae at the time may

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be many times larger. They quickly attack them, even penetrating the prey rediae to clear out their germinal contents, eventually eliminating the entire population of the subordinate species (Lie et al., 1968a, c). These workers postulated that a stimulus originated from the subordinate species, perhaps at a specific growth or metabolic stage, to trigger the sudden predatory activity of P. segregatum. Apparently this response to a stimulus works in only one direction. The predatory species from a specific infection sequence is always the aggressor, always the dominant species-though the subordinate one may well be dominant in a different combination or timing sequence. A battle between competing rediae in a snail has not been seen to occur. Only the stimulus of one species to attack, and the quite helpless or passive non-resistance by the other have been observed. D.

CHARACTERISTICS OF SPOROCYST OR REDIAL NON-PREDATORY EFFEClINDIRECT ANTAGONISM

Various aspects of direct antagonism can be studied by observing the live snail, by dissection, and by histological section. Somecharacteristics ofindirect antagonismcan be studied by the same methods. A delay in the normal development occurs, and degenerative changes are found that cannot be associated with redial attack. These are briefly described below (D1 and 2). Possible mechanisms to account for these effects are discussed in the following section (D3), one of considerable interest but distressingly little hard data. In Section E we outline the preliminary stages ofaquantitative approach to these findings.

1. Inhibitory effect Under this heading we include the partial or total inhibition of development of one trematode by presence of developing forms of another species within the same snail, a process that does not involve direct physical attack or predatory activity. This effect is of particular interest because it can involve sporocysts against rediae, even cases where the latter, though slowed in development, survive and subsequently destroy the inhibitory sporocysts. The phenomenon occurs, then, between sporocysts and rediae, sporocyst stages only, or between rediaproducing species during early development when the chance for direct antagonism is minimal. Lie (1 967) challenged S . mansoni-infected snails with P. segregatum miracidia and found that the future dominant rediae were delayed in their development, which was extended 50% over the normal period. We have since found that the inhibitory effect is expressed during each of the developmental phases: delay in migration of first-generation rediae, delay in development of germballs within the rediae, and delay in cercarial development and shed. Histological sections show that the initial effect probably occurs early in the sporocyst stage. A more detailed review of these observations will be found in Section I V . B. Basch et al. (1969) reported that with S. mansonif Cotylurus Iutzi in B. glabrata, a combination that involves sporocysts only, S . mansoni proved to be the dominant species. N o strigeid infection could be found after the

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schistosome had been 15 days or longer in the snail. With shorter intervals between the two infections, double infection was possible. Yet even in these cases dissection and histological studies showed that degenerative changes had occurred in the subordinate species. An interesting aspect is the fact that, as with direct antagonism this appears to be a one-way impact, with the same species responsible for a given response with each combination and infection interval. There is, however, the possibility that one effect is cancelled by another or an effect may not be recognizable in the face of a more obvious one, such as an indirect effect of a dominant predator being masked by the latter’s direct antagonism. Our studies with the less aggressively predaceous echinostome, E. liei, interacting with S. mansoni, suggest that the direct antagonist can also induce an indirect as well as a direct effect against the schistome. This was observed as a rapid degeneration of sporocysts that escaped echinostome predation and began to develop in the snail hepatopancreas (Heyneman et a/., in press). 2. Degenerative changes In B. glabrata snails harbouring both S. mansoni and C. lutzi sporocysts, Basch et al. (1969) found that the subordinate strigeid sporocysts were thin, shrunken, granular, yellowish, dull and relatively slow-moving. In singleinfection controls, these strigeid sporocysts were “plump, white, glistening, and actively motile”. Histological sections of C. lutzi sporocysts in doubly infected snails showed degenerative changes had taken place in the strigeid germballs, in which there was an almost total absence of mitotic figures. In the combination P. segregatum S. mansoni in B. glabrata, degenerative changes of germinal material within schistosome sporocysts were also seen (Figs 7, 8). Although no amoebocytic infiltration could be seen, the affected sporocysts frequently were encapsulated in a host tissue granuloma. The degenerative changes could have been the result of prior redial attack on the sporocyst wall, permitting toxic material to leak through broken or altered portions of the sporocyst. Another example, referred to in the preceding section, is the degeneration observed in S. mansoni sporocysts in the presence of E. h i rediae. Again, this might have been due to prior injury of the sporocyst by predatory activity, followed by a host response, but the suggestion of a toxic or degenerative factor causing the result remains a significant, if unproved, possibility.

+

3. Possible mechanisms a. Snail immunity. Various aspects of snail immune mechanisms have been reviewed recently (Cheng, 1967, 1970; Brooks, 1969; Tripp, 1969, 1970, 1971). More general aspects were discussed as part of the problem of invertebrate immunity in several symposia : “Defense Reactions in invertebrates” (Bang, 1967b); “Reticuloendothelial Aspects of Invertebrate Pathology” (Bang, 1970) ; “Phylogeny of Transplantation Reactions” (Hildemann and Cooper, 1970). Many authors agree that a host tissue response can be stimulated in molluscs, but that it is a relatively non-specific cellular response, not

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homologous with a vertebrate antibody-based system of defense. No experimental evidence suggesting a specific blood-borne immunity with an antibody basis is available. Recent work in our laboratory, however, suggests that a specific “antigen-binding” substance can be demonstrated in the hemolymph of infected snails that is absent in non-infected snail hernolymph, using E. lindoense in B. glabrata (Faulk et al., in preparation). Further work must be done to isolate and characterize this substance before it can be considered definitive evidence of an invertebrate antibody. Anatural immunity to parasitic attack in which snail cellular responses play a primary role is also present (cf. reviews Tripp, 1963, 1969, 1971; Cheng, 1967, 1970; Cheng and Rifkin, 1970; Cheng et al., 1970). This is similar to a natural resistance described in other molluscs, in insects, and in several other invertebrate groups (cf. reviews Huff, 1940; Bisset, 1947; Stauber, 1961; Stephens, 1963; Salt, 1963, 1967,1970; Feng, 1967; Poinar, 1969; Cheng and Rifkin, 1970; Cheng et al., 1970; Rabin, 1970; Tripp, 1970, 1971). Tissue transplantation experiments have also demonstrated the importance of cellular mechanisms in invertebrate immunity (Hildeman and Cooper, 1970). BCnex and Lamy (1959) obtained rapid immobilization of Caribbean S. mansoni miracidia in extracts of Planorbis corneus snails and a resistant Brazilian strain of B. glabrata, but not by the normal host strain of Caribbean B. glabrata. They considered that a widely distributed immobilizing substance may be responsible for the resistance of many snails to S. mansoni infection. Michelson (1963, 1964a) reported a miracidia-immobilizing substance(s) from extracts of B. glabrata infected with S . mansoni. Miracidia of the same species were more strongly affected than were those of other species. He felt that the presence of an antibody was only suggested, not proved, by these experiments and that parasite-produced toxins or products from alterations in the snail’s metabolism cannot be excluded. In another study, Dusanic and Lewert (1963) demonstrated changes in amino acid composition and electrophoretic mobility patterns of host hemolymph shortly after S. mansoni infection, seen after the first hour and lasting only about 20 h. They observed that the motility of S. mansoni miracidia is affected by hemolymph from snails with an early S. mansoni infection, a response seen only during the brief period of altered amino acid composition. The authors concluded that these changes could as readily be accounted for by miracidial penetration and migration as by induction of a snail protective response. Two small-scale experiments we have performed appear to add to the evidence that there is a factor or agent in the blood of infected snails that may account for some expressions of physiological antagonism. In one experiment (Lim, 1970b) snails were parabiosed according to the technique described by Heyneman et al. (I 971b). The parabiotic twins consisted of one snail harbouring a well-developed S. mansoni infection joined to a partner uninfected snail. After 24 h the twins were separated and the uninfected snail was exposed to P. segregatum miracidia. The results, shown in Table 11, indicate that the delay in development of P. segregatum in the snail that had been briefly joined to the S . mansoni snail is comparable to the delay produced when the 2 parasites are in the same snail. This is a rather remarkable demon-

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stration of a n indirect antagonism operating from a distance, with the host(s) acting either as active participants o r as a transmission medium. I n the second experiment (Lim, 1970b), P. segregatum miracidia were incubated in hemolymph from S. mansoni snails for 30min at 30°C. The miracidia were then allowed to penetrate B. glabrata snails. Table I11 indicates that this treatment resulted in a lower infection rate in miracidia exposed to infected as opposed to normal snail hemolymph. On the other hand inoculation of either infected o r uninfected hemolymph into irradiated B. glabrata failed to restore the resistance normally found in larger snails (over 1 0 m m diameter). This was demonstrated with the E. lindoense-B. glabrata combination (Jeong, unpublished observations). These and other data on effects of radiation on snail resistance will be published elsewhere. However, the fact that radiation can break down natural resistance to infection by certain snails (Heyneman and Lim, 1971 ; Faulk et a/., in preparation) does suggest that a cell system vulnerable to radiation is involved in the normal pattern of snail resistance to trematode infection. Winfield (1932) reported that lymnaeid snails harbouring sporocysts of Corylurus flabell(formis became highly resistant to penetration of cercariae of the same species. Anteson ( I 970) found that Lymnaea catascopium pallida TABLE I1 Result of challenging unirlfected snails joined parabiotically for 24 h with S. mansoniinfected snails, separated, and then exposed to 10 P. segregatum miracidia

P.s. rediae in ovotestis

Exp. -

-

snails surviving 3

~~

4

5

6

4

~~~

1 . Parab.a

Non-parab. P.s. only 2. Parab. Non-para b. P.s. only 3. Parab. Non-parab. P.s. only Totals Parab. Non-parab. P.s. only

at week

No. snails

No.

Onset P.s. a r c . shed at week

43 43

-

61 61 32-61 32-61

5

6

~~

3 3 6 10 10 8 4 4 8

3 2 5 9 6 8 4 2 7

0 0 2 0 0 3 0 0 2

0 0 4 2 1 6 1 1 5

2 2 4 5 3 6 3 2 5

2 2 4 7 4 6 3 2 5

17 17 22

16 10 20

0 0 7

3 2 15

10 7 15

12 8 15

0 0 2 1 0 5 0 1 5 0 0 2

7

8

~

0 0 4 3 1 6 2 2 5

2 2 4 6 4 6 3 2 5

I 5 II 1 3 8 12 15 15

12 8 15

a Parab.=Snail joined 24 h with S.m.-infected snail, then separated and challenged with 10 P.s. miracidia. Non-parab.=Snail not parabiosed, same size class as experimentals, with same S.m. infection and same P.s. challenge. P.s. only= Control snails, same age class as above, exposed to P.s. only.

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TABLE 111 Infectivity of’P. segregatum miracidiu when exposed to B. glabrata after 30 miti incubation in various test solutions (1 miracidium per snail)&

Incubation media: ~

Diluted hemolymph Diluted hemolymph from uninfected from B. glabrata with S. munsoni B. glabratu

__

~~

-~

Demineralized water

Pond water

-

Infection results: ~ _ _ _ _ _

~~

~

~~

~~

~~

No.

No.

No.

No.

No.

No.

No.

snails exam.

snails pos.

snails exam.

snails pos.

snails exam.

snails

snails exam.

~

~~

103

7 (7 %)

135

pos.

No. snails pos.

~

~~~

27 (20 79

142

68 (47 %)

120

47 (39 %)

&Texton p. 209.

in Quebec were infected with a strigeid, 2 Plagiorchis species, and an echinostome-but only single infections were found in field collections. Experimental exposures of laboratory-raised L. catascopium to Diplostomum Jlexicaudum and Cotylurus Jlabelliformis miracidia simultaneously or sequentially failed to demonstrate dual infections. Anteson (1970) suggested that the first species to penetrate destroyed the snail’s chemotaxis to the second, or that a true immunity was responsible. Data are insufficient to establish either alternative. The 50 days wait for shed of cercariae from a challenge exposure may have been insufficient, as in our long-delayed shed of S. mansoni in snails previously infected with P. segregatum, Nolf and Cort (1933) and Cort et al. (1945) suggested that the homologous immune reaction discovered by Winfield against C. jlabelliformis cercariae was induced by a chemical change in the snail tissue, causing it to inhibit the process of cercarial encystment. Non-specific immunity was also considered as a possibility, seen as the resistance of snails harbouring sporocysts of other trematode species to development of C. jlabelliformis metacercariae. These authors also made the interesting observation that cercariae of this strigeid are able to penetrate and to develop in abnormal snail second intermediate hosts, if and only if these snails already harbour other trematode larvae (Cort et al., 1945). Snails not previously infected permit cercariae of C. Jlabelliformis to penetrate in large numbers, but these rarely developed. When other larval trematodes were present, however, many C. jlabelliformis penetrated their germinal sacs and developed within them into normal encysted metacercariae. Cort and his associates suggested that when the strigeid cercariae penetrated abnormal hosts they sought out and entered sporocysts or rediae of other trematode parasites and thereby escaped the immune reactions of the abnormal host. Intrigued by these findings, Basch (1970) did a series of experiments with

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21 1

C. Iutzi and with P. segregatum in B. glabrata. Using P. segregatum as a predator against C. lutzi, he first cleared snails of their Cotylurus sporocysts, leaving only the echinostome rediae. He then challenged the strigeid-cleared snails with C. Iutzi cercariae. Hyperparasitic C. lutzi metacercariae developed in P. segregatum rediae just as readily in the snails cleared of their strigeid parasites as in snails never previously infected with C. lutzi and only parasitized with the echinostome rediae. He therefore concluded that Cort’s hypothesis was invalid and a snail tissue response could not be considered responsible for the protective effect. Instead, Basch suggested that the protective response or aversion may more likely be an intraspecific trematode interaction, a crowding effect of some type, rather than a host tissue response. He termed it the “Winfield effect”. Acquired cellular immunity has been difficult to demonstrate with certainty in snails, in part owing to the technical problems involved, such as elimination of the immunizing infection. Basch (1970) managed to do this in an ingenious fashion by utilizing the predatory activity of P. segregatum against the strigeid as noted above. Earlier studies in our laboratory (Heyneman, Lie and Basch, unpublished observations) used homologous and heterologous infection and challenge experiments with the various echinostomes then available to us (E. lindoense, E. barbosai, E. paraensei, P. segregatum). For those species developing as mother sporocysts in the snail heart, initial and challenge infections could be distinguished by examining this organ at appropriate intervals after the test infection. In most cases the infected heart can be seen through the shell of the albino host snails. All of the echinostomes tested were unable to reinfect experimental B. glabrata snails or showed drastically reduced challenge infections, beginning about the third day. Homologous challenges were more effective than heterologous ones in inducing a host resistance to the challenge infection. However easily monitored, these studies still represent a premunition, not a clear case of acquired sterile immunity. Chemical removal of the prior infection is therefore a necessary concomitant study. This has been made possible by Warren’s (1967) finding that amethopterin effectively clears B. glabrata of S. mansoni. We currently have experiments under way using this approach to retest our earlier demonstration of host resistance to echinostome reinfection. Other workers have also reported failure to reinfect at a high level snails harbouring the homologous species (El-Cindy, 1950; Chowaniec, 1961). However, successful homologous reinfection of B. glabrata or B. sudanica with S. mansoni has been reported by Kagan and Geiger (1964), Pan (1965), and Purnell (1966a), a finding frequently reconfirmed in our laboratory with our strain of NIH B. glabrata. Though a partial resistance to schistosome reinfection may develop, the degree of protection appears to be substantially less than that developed against an echinostome challenge reinfection. Several workers, using histological methods, have reported an enhanced cellular response in B. glabrata and B. nasutus after re-exposure of these snails to schistosome miracidia (Barbosa and Coelho, 1956; McClelland, 1965). However, Wajdi (1966b) was not able to confirm these results using the B. truncatus-S. haematobium and the B. glabrata-S. mansoni combinations.

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FIGS9-16. (9) and (lo), Effect of lucanthone HCI on schistosomiasis mansoni in B. glabrata: S. mansoni (day 50) exposed to lucanthone HCI, 1 ppm (9) and 5 ppm (10) and fixed 5 days later. From liver area, 140x ; (11) and (12), Double infection in B. glabrata: S. mansoni (day 34) and P. segregatum (day 4). Arrows indicate P . segregatum sporocysts. Mantle collar, 140x (11) and 350x (12); (13) and (14), Penetration ofP. segregatum miracidia into B. glabrata foot epithelium, 75 x (13) and 750x (14); (15), Foot epithelium of B. glabrata, one week after irradiation with 5000 rads, 200x ; (16), Foot epithelium of non-irradiated B. glabrata, 215 x .

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213

Donges et al. (1969), using the redia implantation technique (Heyneman, 1966; Chernin, 1966, 1967; Cheng, 1970) showed that L. stagnalis harbouring Isthrniophoru rnelis rediae could be reinfected with miracidia of the same species, although the challenge infection rate was lower than that of the control or primary infection. Whether this indicates a partial immunity or was caused by a parasite-parasite interaction cannot be determined. After the frustrating inability to demonstrate mammalian-type antibody in invertebrates, some workers have suggested that invertebrate resistance is a fundamentally distinct process and should be measured by different parameters (Bang, 1967a; Chadwick, 1967; Salt, 1970). An analogous situation has developed among plant pathologists who have discovered and explored the significance of phytoalexins, a group of inhibitory substances (also known as pisantin, ipomeamarone, orchinol, phaseolin, isocoumarin, etc.) produced by infected plants. These compounds apparently can inhibit development of plant parasites, especially fungi (cf. reviews by Allen, 1959; Miiller, 1959; Cruickshank, 1963; Yarwood, 1967; Wood, 1967; Rohringer and Samborski, 1967; Van der Plank, 1968; Metlitskii and Ozeretskovskaya, 1968; Matta, 1971). Miiller (1966), one of the original investigators of phytoalexins, considers them “biologically comparable to animal antibodies”. They are not homologous substances to classical mammalian antibodies, but, in terms of biological activity, rather more like interferon. Perhaps snail humoral factors play a similar role and a review of approaches used by plant pathologists may help us develop a new approach to the study of humoral substances in snails as well as in other invertebrates. b. Direct toxicity. The inhibitory and cytolytic or degenerative changes collected under the term indirect antagonism are assumed to be caused, at least in part, by injurious chemical substances, toxins, or antimetabolites originating from the antagonist trematode. However, we have no direct evidence to support this possibility, nor are we able as yet to determine whether these effects are from the trematode or via a host response. The injurious effect of antibiotics, antimetabolites, and molluscicides on growth and development of intramolluscan stages of S. rnansoni have recently been studied by Warren and Weisberger (1 966), Warren (1967), Massoud and Webbe (1969). We have repeated their observations with lucanthone and amethopterin and followed the effects histologically (Lim et a f . , in preparation). These chemicals do have a damaging effect on the tissues of both the snail and the schistosome. The snail’s response to injury to its parasites is especially interesting. After an initial period of disorganization, the host is able to mobilize its cellular defenses and encapsulate the injured parasite (Figs 9, 10). Comparative studies on antagonism among other parasites or free-living soil organisms may be helpful in the study of these intramolluscan effects. Among fungi and bacteria, substances produced by one species exert injurious effects on others, which is the classical description of natural antibiosis (Waksman, 1945). Chemical substances also play a role in antagonistic interaction between fungi and nematode parasites in plants (Powell, 1971). h i vitro cultivation and rigorous biochemical analyses of metabolic products

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have been the basic tools in these studies. However, until host and parasite metabolic products can be measured separately (e.g. by in vitro culture in known media) we are limited to qualitative studies of the results of intramolluscan interaction. Available information, summarized in such monographs and reviews as those by Cheng (1963), Von Brand (1966), and Taylor and Baker (1968) cannot yet be used to help determine the specific agents and mechanisms involved in indirect antagonism among trematode larvae. For example, Wright (1966b) noted that increases in blood lipids occur commonly with invertebrate parasitism, or Lie et al. (1968b) observed that such lipoids may injure one of the competing species in the snail; but these observations still do not enable us to determine whether these substances were produced by the host or by one of the competing parasites. Competition among insect parasitoids may also involve toxicity by secretions from one parasitoid affecting a competitor. Timberlake (1910) found aphiidine larvae dead in their hosts with no mark of physical attack, and concluded that they were killed by a secretion from older larvae present in the same host. Salt (1 961), however, emphasized some key unanswered puzzles. How do secretions affecting competitors fail to injure the individuals secreting it? And, how is the battle of secretions decided? Fisher (1961) could find no evidence of toxic substances that play a role in the antagonistic interaction between parasitoids of the moth Ephestia serecarium. Instead, his data suggest that competition for oxygen is an important factor. c. Competition .for nutrients and oxygen. Interaction among soil bacteria competing for nutrients and oxygen plays an important role in their survival (Waksman, 1945). Competition for nutrients is important in the antagonistic interaction among insect parasitoids (Salt, 1961 ; DeBach and Sundby, 1963; DeBach, 1966) or relationships among parasitic nematodes and fungi within the same plant host (Powell, 1971). Our knowledge of the role of oxygen as a limiting factor for nematodes in the micro-environments of the vertebrate gut was pioneered by Rogers and Sommerville (reviewed in Rogers, 1962). Fisher (1 961) demonstrated that competition for oxygen was important in the competition between larvae of the ichneumonids Horogenes chrysostictos and Nemeritis cunescens within their host, the larva of the flour moth Ephestia serecarium. In the antagonism between P . segregatum and S. mansoni in B. glabrata snails, the inhibitory effect of the schistosome against the echinostome begins, as was reviewed above, early in the sporocyst stage. In older, well-established infections of S. mansoni, various host tissues react strongly to the presence of the schistosome cercariae. When P . segregutum miracidia infect such snails their sporocysts develop in their normal location, near the site of penetration, but often are pocketed in protruding (partially rejected ?) bumpy projections along the lining of the head-foot organ. This region is already marked by a disrupted architecture from the earlier presence of first-generation schistosome sporocysts and by entrapped, degenerating cercariae (Figs 1 I , 12). This modification of the host environment may be responsible for the modification of the normal growth pattern of the echinostome, but in deeper areas of the foot and mantle, where integrity of the tissues is retained, there still is an

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inhibitory response to challenging echinostome sporocysts. We therefore feel that the mechanism responsible for delayed growth and development of P. segregatum is of a systemic nature, in spite of manifestations of resistance in some areas that may suggest a locally restricted nutritive or competitive factor. Our studies on redial populations in the snail indicate that parasite population size or parasite mass is dependent on biomass of the snail host and not size of the infecting dose (Lim and Lie, 1969; Lim, 1970b). A host that already supports one species of parasite to its maximum capacity offers a diminished biomass for support of a second parasite, although some compensatory hypertrophy of the host may occur (Rothschild, 1936, 1941;Pan, 1965). This growth phenomenon is commonly observed in field collecting, when one finds that the largest snails are most frequently infected. Although vital requirements of two parasites in a given host are not necessarily the same, we assume that nutrition for the second parasite to arrive would be less favourable than that in snails without a competing trematode. d. Othermechanisms. From analysis of double infections in the common mudflat snail, Vernberg et al. (1969) found that the two most common trematodes, Lepocreadium setiferoides and Himasth la quissetensis (an echinostome), were never found together in the same host. This was also the case with the two most common parasites of Cerithidea californica (Martin, 1955). Vernberg et al. (1969) suggested that a biochemical alteration of host tissue by one parasite makes it unsuitable for the other species. This idea probably is based on their previous studies demonstrating that larval trematodes alter the thermal acclimation patterns of cytochrome c oxidase in the host tissue, and that this alteration is distinctive for each species (Vernberg and Vernberg, 1968). While such a mechanism is an intriguing one that might account for some cases of single-speciesoccupancy in a host, H . quissetensis is an echinostome and very likely double infections involving this species revert to single ones by redial predatory activity. However, unrecognized metabolic alterations of a host by the first occupant might be an important adaptive attribute, one that protects the host from over-parasitism and the parasite from competition by another parasite or from its own excessive multiplication, a mutually destructive process if it destroys the host snail. E.

ASSESSMENT OF ADAPTATION OF THE TREMATODE TO THE SNAIL HOST

1. Infection rate

a. General considerations. Degree of adaptation between snail and trematode is often reflected in the infection rate. A natural rate, based on samplings of snails in natural waters, obviously is more an ecological than an intrinsic measure of host-parasite adaptation. To suggest host-parasite adaptation, the rate must be based upon laboratory-controlled, standardized experimental infections. A useful discussion of these snail trematode relationships, especially in their evolutionary perspective, is found in Wright (1966a). We have therefore standardized our infection procedures and techniques (reviewed in Materials and Methods) to be able to compare infection rates in single 10

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TABLE N Influence of snail size (B. glabrata, NZH) on infection with varying numbers of P. segretatum miracidia

Initial snail size (m)

2f0.5

5f1

No. mirac.

15+1

20+1

8% (2/24)

29 % (l5/5l)

Snail infection rate (no. +/no. exam.)

1 1

10+1

Normal Range (2-25)

63% (22/35) 41 % (21/51)

2

5

64% (48/78) 71 % (40/56) 80 %

44% (16/36) 50% (28/56)

41 % (19/46)

(44/55) 89 % (68/76) 100% (39/39)

10 100

TABLE V Influence of snail size (B. glabrata, NZH) on infection with varying numbers of E. liei miracidia

Initial snail size

(-1

2f0.5

26 % (34/129)

2

44%

5

(35/80) 67 % (26/39)

10 20

10f 1

15f 1

20f 1

12% (11/92) 14% (13/90)

6% (2/36)

Snail infection rate (no. +/no. exam.)

No. mirac. 1

5fl

31 % (119/377) 36 % (38/105) 37 % (10/27) 67 %

13% (18/137) 20 % (18/88) 26 % (13/49)

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infections of various snail-trematode combinations to develop some degree of ability to predict the outcome of double infections. The following factors directly influence rate of infection under our laboratory conditions : number of miracidia per snail, water temperature during snail exposure and maintenance, age of miracidia, age and size of snail. The snail infection rate, though generally rising with increased number of miracidia exposed per snail, may not always reflect a precise relationship, even at lower miracidial levels. Lengy (1962) exposed B. truncatus with local S. bovis miracidia, and found that at 5, 10, 15 and 20 miracidia each, the rate ranged between 36 and 58-6%, but was not consistently related to the dosage. At a higher level (60 miracidia) the rate was 87%. Kinoti (1968), with S. mattheei in bulinid snails, found no significant difference between snails exposed to 5, 10, 12 or 15 miracidia. Bruce and Radke (1971) used 6, 9, 10, 11, 15 and 25 miracidia per snail and also found no significant differences. In our echinostome and schistosome single-species infections, the infection rate TABLE VI Influence of snailsize (B. glabrata, NZH) on infection with varying numbers of S . mansoni (NZH)miracidia

Initial snail size

(-1

2f0.5

5+1

lo+ 1

15f 1

20+ 1

Snail infection rate (no. +/no. exam.)

No.

mirac.

~~~

1 2 5 10 20 50

19% (7/37)

21 % (24/114) 32 % (W6)

18% (7/39)

13% (5/39)

5% (2/W

44% (42/94) 60% (69/116) 81 % (78/96) 90% (33/37)

does not increase proportionately with miracidial number, but rises at a diminishing rate to a saturation or asymptote level (Tables IV, V, VI). This may reflect increased parasite competitionat higher miracidial levels, modification of host susceptibility at these higher levels, or some alteration in snail behaviour when the host is attacked by larger numbers of miracidia. Host resistance by increased mucus production or withdrawal of the head-foot may offer some protection, but we find that mass-exposedB. glabrata show very little evidence of avoidance behaviour against parasites to which they are

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highly susceptible, such as Zndoplanorbis exustus in response to E. malayanum, or B. glabratu (NIH) either to S. mansoni (NIH) or various echinostomes (Figs 13, 14). We have, however, seen avoidance and withdrawal behaviour among maladapted (abnormal) host-miracidia combinations. Competition among miracidia for penetration sites or for sporocyst development sites may also be of importance. This factor is now under investigation in our laboratory. A primary factor that is often neglected (or unknown) is the genetic background of both snail and trematode. McQuay (1948) suggested that an enhanced susceptibility occurred in the progeny of susceptible Louisiana Tropicorbis havanensis exposed to Puerto Rican S. mansoni. Newton’s studies (1954,1955) drew attention to the inheritance of snail susceptibility to schistosome infection, a line of investigation repeated and enlarged by other workers, partly in the hope of finding a new method for biological control (Paraense and Correa, 1963; Moose and Williams, 1963; Richards, 1970). In the organization of a snail colony in the laboratory such routine variables as aquarium size, temperature, or water conditions may quite unintentionally favor selectivelyeither the susceptible or refractory variants within the population. Crowding, feeding methods, removal of certain size classes from the breeding population and other biological variables may also introduce a strong selective pressure in the colony. Cram et al. (1947), for example, noted a decreasing susceptibility in their laboratory snails to S. mansoni miracidia over a period of 3 years and an increase in the prepatent period of successful infections. They suggested that a change in the snail as well as the parasite had occurred after their prolonged series of infections in laboratory animals. Kagan and Geiger (1965) compiling infection data in 3 strains of B. glabrata and 2 strains of S. mansoni, found over a 3-year period fluctuations in infection rates in certain combinations. They attributed this to a change in susceptibility of the snail populations, but also suggested that some type of selection may have taken place in the genetic constitution of the trematode, and that the miracidia became more infective for the same snail population. In our B.glabratu colony, Kuris (unpublished observations) made a statistical analysis of infection rates in B. glabrata (NIH) to varying numbers of Brazilian E. lindoense miracidia. He showed that there appears to be a dichotomy within the same interbreeding population, a bimodal rather than a normal distribution of susceptible and resistant individuals, The overall rate of infection of the mixed population has remained fairly constant owing to standardized infection procedures, but the snail heterogeneity has been sustained. It is likely that minor variations in maintenance or infection procedures could unwittingly affect selection pressure on the snail or trematode populations, creating otherwise inexplicable changes in infection rate or in other important infection parameters. This intrinsic heterogeneity in infectivity was demonstrated in our colony by selection of individual snails for infectivity vs. non-infectivity to E. lindoense over several generations, which showed that both highly resistant and highly susceptible populations could be segregated, thereby unmasking the genetic heterogeneity or “balanced polymorphism” that had been present but unrecognized (Heyneman, unpublished observations). The importance of using a controlled genetic constitution in snails for experimental purposes is

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self-evident. But only recently has knowledge of snail hereditary make-up begun to provide the tools and morphological markers needed, as in the recent studies by Richards (1969a, b, 1970, 1971), Burch (1967, 1969), Burch and Lindsay (1970) and Lo (1969). Not only are such genetic and cytogenetic investigations essential for life-cycle and other infection experiments in the laboratory, but they should lead to a better understanding of the great infectivity range observed in nature as well, particularly the sharp geographical strain differences that have been the subject of many investigations in the post WW-I1 period. Cross-infection studies with various geographic strains of schistosomes and their hosts, have been particularly favoured by investigators (as, for example, experimental studies by Stunkard, 1946; Cram et al., 1947; Files and Cram, 1949; Abdel-Malek, 1950, 1967; Files, 1951; Kuntz, 1952; McQuay, 1952, 1953; DeWitt, 1954; Hsii and Hsii, 1967; Barbosa and Baretto, 1960; Coelho, 1962; Moose and Williams, 1963; Saoud, 1965, 1966; Kinoti, 1968; Cridland, 1968, 1970; Chi et al. 1971). Water temperature influences infection rate during exposure, but only when extreme are these differences of major importance (Purnell, 1966b; Ubelaker and Olsen, 1970). Within ordinary laboratory conditions (25 to 30"C), infection rate does not differ significantly. In a test involving 90 snails divided into three groups exposed to P. segregatum miracidia at 15, 25 and 35"C, rates were 27%, 87% and 80% respectively (Lim, 1970b). Many of the miracidia were sluggish at 15°C and most of the exposed snails lay motionless at the bottom of the vials, some of them fully retracted. Within sublethal temperatures permitting normal activity, however, infection rates were little affected. The impact of temperature was noticeable chiefly at lower ranges when motility (and infectivity) were reduced. Temperature for maintaining snails also influences the rate of infection (Standen, 1952; Stirewalt, 1954; DeWitt, 1955), though, again, we found the range 25-30°C to be optimal. Infectivity of schistosome miracidia decreases markedly with age after several hours (Maldonado and Acosta-Matienzo, 1948; Purnell, 1966c), falling to negligible levels after 8 h. We found that miracidia less than 1 h from hatching, exposed for 2 h, give maximal results. In most species of trematodes we have studied, penetration of B. glabrata occurred during the first 30 min. Snail age expressed by size has a major influence on infection rate, although reports with schistosomes are mush less clear. As in so many aspects of their biology, the schistosomes here, too, are unusual. Humphreys (1932), Moore et al. (1953), Newton (1953), Lengy (1962) mentioned that younger snails were more susceptible to schistosome infection in the laboratory. McQuay (1953) and Milward De Andrade (1962) found the opposite. Other workers considered age unimportant for snails exposed to schistosome infection (Gordon et al., 1934; Stunkard, 1946; Maldonado and Acosta-Matienzo, 1947; Abdel-Malek, 1950). We have also found snail size to be unrelated to infectivity with S. mansoni. Chu et al. (1966) reported that the infection rate of S. haematobium in day-old B. truncatus from Iran was significantly lower than in snails 1-5 weeks old. With a B. truncatus-S. haematobium combination from Egypt,

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1-7 day old snails did not differ significantly in infection rate, but snails from l&-5 weeks old showed a decreasing rate of infection. With fasciolid trematodes exposed to normal or susceptible hosts, the snail host apparently can be infected at all ages, whereas with abnormal combinations, only the very young snails can be infected (Sasanov, 1957; Kendall, 1964,1965,1970; Boray, 1969). With the Brazilian and Egyptian echinostomes used in our laboratory (P.segregatum, E. lindoense, E. barbosai, E. paraensei and E. liei), which readily infect the NIH strain of B. glabrata, larger snails are very much less susceptible and, in some experimental series, are quite intractable to infection. Using single-miracidiumexposures, our most sensitive barometer of infectivity, previously unexposed snails over 20 mm in diameter were uniformly difficult or impossible to infect with these echinostomes. Maximum size that most of our echinostome species are able to infect is 20 mm. With others, such as E. lindoense, 15 mm is the maximum, while species such as E. barbosai, less well adapted to B. glabrata, may not be able to infect hosts larger than 10 mm. The maximum size of host that can be infected and the rate of infection by single miracidia exposed to a series of snails of a standard size are two distinct and important indicators of host-parasite adaptation. The presence of annelids parasitic on the snail may have an influence on infection rates. Several workers have observed capture of miracidia by these worms (Ruiz, 1951; Coelho, 1957; Khalil, 1961; Boray, 1964; Michelson, 1964b). Boray (1964) in Australia, very commonly found 60-100 oligochaetes attached to a singleLymnaea tomentosa from semi-stagnant pools or from small dams. In one snail, 7 mm long, he found more than 500 wriggling worms. Michelson’s (1964b) experiments showed that these worms interfere with penetration of miracidia into the snail or with entrance of echinostome cercariae. In our laboratory we do find a small number of our snails infested with these annelids, especially in unattended aquaria. Generally in younger snails and in snails used for infection studies, annelids are absent. b. Miracidial penetration. Miracidia gain access to the snail tissue after penetrating the epithelial lining, most commonly in the head-foot and mantle areas. The process of penetration through the foot and mantle tissues has been described by a number of authors, e.g. for S. mansoni (Faust and Hoffman, 1934; Maldonado and Acosta-Matienzo, 1947; Wajdi, 1966a), S. bovis (Lengy, 1962), Fasciola hepatica (Mattes, 1949; Dawes, 1959, 1960c; Saint-Guillain, 1968; Wilson, 1971; Wilson et al., 1971); F. gigantica (Dawes, 1960a, b) E. malayanum (Lie and Owyang, 1962; Lie, 1969b). Both mechanical penetration and chemical lysis are involved, though their relative importance varies with the trematode group. The cytolytic action of the apical gland (“primitive gut”) and importance of the accessory gland cells with ducts opening at the tip of the apical papilla were emphasized by Dawes (1960c), a view we find fits both morphological evidence and our observations of miracidia in the act of penetration. We have observed that attachment of the apical papilla plays a primary role. Separation of the miracidium from its attachment site during the initial phase of penetration results in failure of the miracidium to penetrate its host. Some workers have assumed that this early attachment is by suction or an adhesive fit, either by

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221

mechanical means or aided by a mucoid plug (Dawes, 1959, 1960b; Lie and Owyang, 1962). Kinoti (1971) in an electron microscope study of the anterior end of S. mattheei suggests that the anterior papilla serves to attach the miracidium to the snail surface, hypothesizing that the degree of “fit” between organ and snail microsurface determines success or failure of miracidial penetration. Mattes (1949) considered that the apical papilla, aided by the accessory gland and so-called vesiculated cells, forms an adhesive plug by which the miracidium attaches to the snail surface. Southgate (1970) offers the view that the vesiculated cells of the miracidial body, presumably including the apical portion, retain their contents and contribute them to the body wall of the sporocyst. The relative importance of epithelium thickness and its integrity as a barrier to miracidial penetration has not been critically studied, although Boray (1966) considers this factor as one aspect of specificity. Perhaps the length of time for complete miracidial penetration could be used as an indicator of snail-trematode compatiblity. Penetration of S.japonicum miracidia in its usual intermediate host is completed in a few minutes (Faust and Meleney, 1924); S. mansoni miracidia penetrates its normal snail host in 2-15 min, depending on size of the region of penetration and the energy reserve left in the miracidium (Maldonado and Acosta-Matienzo, 1947). In their usual intermediate hosts, complete penetration of miracidia occurs in 30-45 min for S. bovis (Lengy, 1962), 30 min for F. hepatica (Dawes, 1959), 45 min for E. malayanurn (Lie and Owyang, 1962). Rao (1966) compared the susceptibility of Lymnaea natalensis from West Africa with L. rufescens from Pakistan when both were exposed to West African I;. gigantica. He observed that it took the miracidia longer to begin to penetrate the abnormal host than it did the normal snail. A delayed response by the miracidia to the abnormal host may also introduce an additional factor-miracidial ageing, which can rapidly reduce infectivity. Heyneman (1965, 1966) transplanted echinostome miracidia directly into snail hosts to circumvent possible host cellular reactions against the invading miracidium and to determine whether or not loss of the ciliated coat during penetration was necessary for parasite survival. He found that both transplanted miracidia and rediae survived, grew, and multiplied-but only in hosts to which the parasites were already adapted. Transplantation of miracidiae or rediae into the “wrong” species of snail resulted in parasite death. He concluded that resistance to larval trematode development in a maladapted host is a physiological rejection distinct from failure of miracidia to attach or to penetrate the host epithelium. Similarly, successful development can occur without loss of the ciliated coat during the process of penetration. No attempt has yet been made to study histologically the thickness of epithelium and its efficiency as a barrier. Perhaps “age immunity” of larger snails is related to a thicker host epithelium, not simply as a greater mechanical obstacle, but as an increased concentration of host amoebocytes or other cells that may be involved in an organized cellular defence response. In a preliminary study to test this concept, using 137Cs radiation, we observed some damage to

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B. glabrata epithelium of the head-foot when it was irradiated with 5000 and 15 000 rads (Figs 15, 16). Disruption of the integrity of the lining may permit the miracidium to penetrate or cytolyse its way in more readily, either by epithelial disruption or inhibition of host cellular defenses. We observed in another study that irradiated snails, even of a normally resistant strain of B. straminea, become considerably more susceptible to S, mansoni in comparison with non-irradiated controls. Snail infectivity was increased 10- to 20-fold rising from about 1 % to 20% for single-miracidium exposures, and to comparable levels at higher dosages (Heyneman and Limy 1971; Faulk et al., in preparation). In another study we observed a significantly higher number of E. lindoense sporocysts developing in the heart of irradiated compared with non-irradiated B. glabrata. Twenty-five snails, 5 1 mm in diameter, were irradiated at 5000 rads, and five days later exposed to 10 E. lindoense miracidia. A comparable group of non-irradiated snails were similarly exposed on the same day to 10 miracidia each. After 5 days, the snails were examined in the heart for E. lindoense sporocysts. All irradiated snails were infected, harbouring a total of 175 sporocysts; non-irradiated controls were infected with a total of 19 sporocysts. This reduction in rate and intensity of infection, whether by easier penetration of miracidia in the irradiated snails, easier tissue migration, or reduced natural inhibition in the site ofestablishment, adds to the evidence of a radiosensitive cell-based natural resistance. Sudds (1960) compared host-parasite relationships of four species of miracidia in “normal” and “abnormal” snail hosts. He described four types of miracidial behaviour, but basically he showed that miracidial penetration occurs easily in the normal host but only with great difficulty in abnormal hosts. We have observed the extraordinary lack of attraction of Lymnaea rubiginosa snails for miracidia of Echinostoma malayanum (in contrast with miracidia of Schistosoma spindale). Hundreds of E. malayanum may swim actively around the snail and none or few will attempt to penetrate, and then only for a few seconds. In dramatic contrast, the host-seeking, host-finding, and penetration pattern of a “normal” type is very different. Lie (1963) graphically described the behaviour of E. malayanum miracidia that were pipetted into a petri dish in which Indoplanorbis exustus had been placed: “ numerous miracidia swarm[ing] on the head and the tentacles of the snail within a few minutes, when they soon attach, then remain motionless, the body being held stiff at a right angle to the surface of the snail. The miracidia gradually sink into the skin of the snail, taking about 45 minutes to penetrate completely.” He added: “The snail itself seems to be undisturbed by the penetration of numerous miracidia.” Although the host snail may react less to the penetration of host-adapted miracidia, this will vary with number of miracidia, age of the snail, and the particular host-parasite system. c. Successful development. Cases of miracidia penetrating (as opposed to developing) in non-susceptible hosts do occur, as often has been noted. Newton (1952) observed penetration of both susceptible and non-susceptible strains of B. glabrata by S. mansoni miracidia. Destruction of the parasites in maladapted hosts took place within 48 hours by cell infiltration followed by fibrosis and walling off. With Fasciola hepatica, the same thing occurs,

*

...

I N T R A M O L L U S C A N INTER-TREMATODE A N T A G O N I S M

223

though parasite destruction or restriction may develop after varying periods (Kendall, 1964). Brooks (1953) showed that S. mansoni miracidia penetrated both susceptible and non-susceptible hosts in equal numbers, but sporocysts did not develop in the non-susceptible host. Identical results were reported by Barbosa and Barreto (1960) inBrazil. They tested S. mansoni from Pernambuco in B. glabrata snails from San Salvador, Bahia, and from Paulista, Pernambuco. Pan (1965) exposed a strain of B. glabrata to S. mansoni miracidia. After one day he found 14 degenerating mother sporocysts among 257 in three snails (5.4%). The next day 32.2% of 227 sporocysts in three snails had degenerated. Dissections of 42 snails at various intervals from day 0 to 35 showed 21 % degenerating mother sporocysts among the 1142 counted. Apparently host tissue reaction was not the important factor responsible, as only 4 % showed a cellular reaction. Complete development may occur in an abnormal host, but with greatly reduced cercarial output compared with the normal host, as Swales (1935) described for Fascioloides magna in Fossaria parva, the preferred host, and in Stagnicolapalustris nuttalliana, the unsuitable host. Successful penetration is obviously only the first of a series of conditions that must be met before infection can be established. Cheng (1967, 1968) mentions several factors that influence successful establishment of the sporocyst, e.g. reaching a suitable site, overcoming the host’s internal defense mechanisms, effect of host-elaborated growth or other stimulatory factors, and ability to obtain required nutrients. None of these factors are understood much beyond our ability to name and describe them. General aspects of various influences on the host-parasite interaction are discussed by Kendall (1964, 1965, 1970) and by Schwabe and Kilejian (1968). 2. Size of the redial population The expression of parasite adaptation to its host as indicated by the redial population has been examined in B. glabrata for several of the echinostome parasites with which we work. The maximum number is chiefly determined by the size of the snail host and not by the number of penetrating miracidia (Lim and Lie, 1969). The time period required to reach saturation population varies with each trematode species. For P. segregatum in B. glabrata (NIH) it is the fifth week of infection (Lim and Lie, 1969). The absolute redial number is therefore not so much a measure of host compatibility as it is the size, or volume, in which growth and multiplication can occur. To determine intrinsic capacity to produce rediae under comparable conditions, we exposed snails to a single miracidium and examined each infected host within a limited, identical period. B. glabrata (NIH) of varying size were each exposed to one miracidium of P. segregatum, E. lindoense, E. paraensei, E. barbosai and E. liei. Infected snails were dissected and their rediae counted during the fifth week after exposure. The results show that snails measuring 10-21 mm and exposed to E. lindoense (18 snails), E. paraensei (14 snails), E. barbosai (27 snails) and E. liei (28 snails) contained no more than 700 rediae per snail. B. glabrata of the same size range exposed to one P. segregatum miracidia after the same

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H O K - K A N LIM A N D D O N A L D H E Y N E M A N

period developed rediae ranging from 700 to 2400 per snail (47 snails), while 17 snails had redial counts under 700. These figures can be considered estimates of the rate and level of saturation with redia. Other factors must still be incorporated, however, such as differences in the snail’s growth rate as affected by infection with different trematode species, differences in stimulation of a host response-positive or negative-as well as the parasite’s intrinsic germinative capacity. Redial number does not necessarily determine the total larval production. A smaller number of rediae, if individually larger, may outproduce a larger number of small rediae. Cercarial output represents a final estimate of the total larval reproductive power, but as a measure of host adaptation, this figure must be evaluated with regard to the external ecology and probability of infecting the next host and the effect of cercarial production on the snail. Survival time of the shedding snail is a significant determinant of total reproductive output. The total adaptation of the parasite depends not only on its success in the snail, but on its adaptive response to the ecological requirements for cercarial output, whether this is met best by a short-term flood or a continuing low-level output, whether by a massive total production or by a smaller number. If saturation of available transport hosts is readily reached, or if the probability of finding the proper vertebrate host is relatively good, higher-than-necessary cercarial production would put both parasite and host at a disadvantage. An energy-waste seldom survives long in nature, and most parasite life cycles appear to represent a remarkable adaptation so that their huge fecundity does not far exceed their ability to meet demands on this production for successful completion of the life cycle. TABLE VII Adaptation indexes of several trematodes in Biomphalaria glabrata (NZH) snailsa

Trematode species P. segregatum S. mansoni E. lindoense E. paraensei E. barbosai a

No. infected

snails

i

tm

te

A1

48 38 31 26 20

64 21 19 21 10

133 63

18 28 24 24 22

465 47 47 35 25

60

40 55

Text on p. 225.

3. Adaptation Index The following simple relationship, or Adaptation Index, is a preliminary working tool with which to suggest relative adaptation of a trematode parasite to a given snail host under conditions made as standard or comparable as possible. ixtm AI=to ’

-

I N TR A MO L L U S C A N INTER TR EM A T O D E A N T A G O N I SM

225

in which i=infection rate (using 1 miracidium exposed to each of a standard number and size of young adult snails for 2 h in 5 ml demineralized water at 27°C) x 100 tm = time in days for 50% mortality of the infected snails tc= time in days for 50 % of the infected snails to shed cercariae. Using B. glabrata (NIH), of 51t 1 nim, we have calculated A1 levels for P . segregatum, E. lindoense, E. paraensei, E. barbosai and S. mansoni (Table VII). P . segregatum has the highest rank according to this calculation. This echinostome also is the dominant species in combinations with each of the other four trematode species. Unfortunately, not all possible combinations among the other four species have been tried. If such a simple ranking procedure does prove to have some predictive value for rating trematode-snail adaptation, perhaps a similar expression could be used to measure relative efficiency of antagonism, given sufficient standardized data, e.g. a Competition Index. Initially, such an approach toward quantifying several biological variables can only be done under standardized laboratory conditions. Ultimately, one might hope to see a far more sophisticated expression compare infectivity, competitive ability and host-parasite balance in nature.

OF INTRAMOLLUSCAN INTER-TREMATODE ANTAGONISM IV. PARAMETERS

A. STAGES OF ANTAGONISM BASED UPON THE Biomphalaria glabrata-

Schistosoma mansoni-Paryphostomum segregatum MODEL

1. Some general aspects After intramolluscan inter-trematode antagonism had become well established in our laboratory experiments, we began some small-scale attempts to test its application in the field. Preliminary studies by Heyneman and Umathevy (1967, 1968) indicated that it was possible to control S. spindale infection in a small pond by release of E. malayanum eggs. This study was repeated on a larger scale in an abandoned tin mining pond in Malaya by Lie et al. (1970). Their results were confirmatory, although considerably modified by the presence in both parasites of a nosematid microsporidian (probably Perezia helminthorum Canning and Basch, 1968). In a subsequent review of these results, Lie et al. (1971) and Lie (1971) emphasized that 3 factors were interacting in this system : trematode antagonism, microsporidial infection of both antagonists, and parasitic castration of the snails serving as hosts of this inter-parasite hyperparasite-host complex. The net result was a dramatic decline in the population of infected snails. In another field site in Malaya, Lie et al. (personal communication) are studying the Echinostoma audyi YS. Trichobilharzia brevis system with highly promising results. These field control studies currently are being continued, both in Malaysia and Thailand. More general discussion of biological control by trematode antagonism is found in Lie et al. (1968b) and Lie (1969a) and considered in section V of the present report.

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HOK-KAN LIM A N D D O N A L D HEYNEMAN

In our search for a suitable antagonist against S. mansoni, six species of echinostomes have been laboratory tested so far: P. segregatum, Ribeiroia marini, E. lindoense, E. paraensei, E. barbosai and E. liei (Lie, 1966, 1967, 1969a; Basch et al., 1970; Heyneman et al., in press). P . segregatum appears to be the most effective antagonist of S. mansoni, E. barbosai the least. However, these experimentswere designed primarily to test for the existence of an antagonistic interaction and not to measure the efficiency or degree of antagonism. Miracidial dosage of the second or challenging infection and the time interval between first and second infections were two variables not held constant among the various combinations tested, so that quantitative comparison is not possible, though we can establish a relative ranking for the combinations. Since more species of trematodes from widely varying ecological conditions will have to be tested before wide scale application of predatory trematodes can be employed as possible antagonists against schistosomes, fasciolids, or other flukes, development of standardized criteria of measurement and evaluation are essential. Usiiig the S. mansoni+ P. segregatum infection system in B. glubrata as a model, we have attempted to develop one such set of procedures for evaluation of trematode antagonism. However, for each set of antagonists in a given host species, time intervals between infections and appropriate dosages must be adapted to the testing laboratory or to local conditions in the field. Across-the-board standardization is unlikely for these complex and variable biological systems, but adherence to general conditions of standardized infections would be very helpful. It should then be possible to quantify results in a given region or with a given host-trematode system. The term “infection rate” should be employed only if the distinction between “take” and completion of the cycle is made clear. If development stops at sporocyst or redial stages, use of an initial infection rate to indicate “take” may mask subsequent suppression or destruction of the cercariae. In a normally adapted snail-trematode combination, developing sporocyst and redial stages can be expected to proceed to cercarial shed. Under a dual infection there may be a partial development of one species and its gradual replacement by the other, as in the E. liei-S. mansoni system (Heyneman et al., in press). In the latter case, schistosome daughter sporocysts may reach the host hepatopancreas, but cercarial production is absent or very sharply curtailed. We therefore use cercarial shed as the basic criterion for infection rate in our laboratory studies of the antagonistic interaction. Generally the initial infection rate of a dominant species in a snail with a pre-existing subordinate infection is not modified by the interaction. For example, 135 B. glabrata, 5 & 1 mm, were each exposed to 10 S. mansoni miracidia. Four weeks later, 114 of the 125 live snails were infected, of which 67 had begun to shed cercariae. Two days later, 60 of the 67 shedding snails were each re-exposed to 20 P. segregatum miracidia. The other 44 snails with S. mansoni infections were maintained as schistosome single-infection controls, while P. segregatum single-infection controls were obtained from 60 snails of the same size individually exposed to 20 miracidia, and 40 uninfected snails were kept as controls to be examined histologically. On the basis of presence of rediae and on cercarial shed, all doubly-exposed snails as well as singly-exposed controls developed P. segregatum infections.

I N T R AMOLLUSC A N INTE R-TREMATOD E A N T A G O N I S M

227

A useful measure of suppression of one species by another is the effect of a pre-existing infection of the dominant species on the infection rate of a challenging subordinate one. Table VIII indicates that in our studies no subordinate species succeeded after the dominant parasite had been allowed to develop in the snail host beyond a critical time period. However, the timing of infection rate determination may be important, and, as in the following experiment, surprising. Fifty B. glabrata, 5 & 1 mm, were individually exposed to 10 P. segregatum miracidia. After 4 weeks, 30 shedding snails were selected and individually exposed to 20 S. mansoni miracidia. No double infections developed in this group for 6 weeks when the experiment was terminated, whereas among single-infection S. mansoni controls, 15 of 24 were by this time shedding schistosome cercariae. The experiment then was repeated with a heavier challenging dose of S. mansoni miracidia and a longer waiting period. Fifty snails shedding P. segregatum cercariae were individually exposed after 39 days to 50 S. mansoni miracidia. Several snails were taken out periodically for histological study. Of 25 snailsin the experimental group, 8 (32 %) eventually shed schistosome in addition to echinostome cercariae. These 8 cases of delayed schistosome shed involved 2 at day 29, 2 at day 43, and the final 4 only after 116 days post exposure. Among single-infection schistosome controls, 95 % shed cercariae within the normal developmental period (Table IX). The count of schistosome cercariae from the doubly-exposed snails was strikingly reduced, TABLE VIII

Minimum period required for selected experimentally tested dominant trematode species to prevent establishment of a challenging subordinate infection

Snail host

Interacting trematodes Mirac. dose Minimum Dominant Subordinate (no. mirac. interval (2nd inf.) in chall.) (days) (1st inf.)

B. straminea P. segregatum E. barbosai L. rubiginosa E. audyi T.brevis B.glabrata

S. mansoni

C. h t z i

B.glabrata B. glabrata

P. segregatum E. lindoense P. segregatum E. paraensei

10

5-1 5 20 20

17-59

+ 15+

20

14+

9-28

Reference Lieetal., 1967 Owyang et al., 1970 Basch et al.,

1969 Lieetal., 1968c Lie et a/., 1968a

usually to fewer than 20 per test per snail. One snail shed 26 and 34 cercariae on 2 occasions, and 4 of the 8 shed twice, then stopped shedding. Nevertheless, some schistosomes had succeeded in penetrating the echinostome barrier, though appearance of their cercariae was delayed up to 4 months, suggesting the continual battle under way. Had the preceding group of 30 doubly-exposed snails been held longer than 6 weeks, some of the challenging schistosomes might eventually have developed and reached cercarial production. This extreme delay in schistosome development with greatly reduced cercarial

228

H O K - K A N LIM A N D D O N A L D HEYNEMAN

output suggests that both forms of antagonism are expressed against the schistosomes, but that a few sporocysts somehow escape predation and, despite strong indirect inhibition, complete development and produce cercariae. This “escape”, even from a highly predaceous trematode antagonist such as P. segregatum, may prove to be epidemiologically important for future field trials. It also emphasizes the unpredictability of the system and our need of criteria to measure “strength” of trematode antagonism. In this combination and infection sequence, the S. mansoni infection remained hidden for a period longer than most of the earlier experiments had been conducted, hence the true infection rate remained undetected until the experimental period was greatly prolonged. In order to follow this interaction and learn more of the precise points of contact and mechanisms responsible, we examined the P. segregatum-S. mansoni interaction in greater detail, with an emphasis on TABLE IX

Schistosoma mansoni cercarial counts from snails with and without prior infection with Paryphostomum segregatum Days after S. mansoni

exposure

No. cercariae from previously

Average no. cerc. from uninfected snailsb

infected snails& Snail no :

26 29 32 38 43 58 72 92 116 118 126 133 140

144 147 151 156 165

1 2 3 0 0 0 0 8 1 0 10 0 0 26 0 2 34 0 4 0 0 15 0 0 0 0 Died

4 0

5 0

0

0

0 0 3 0 0 12 2 1 11

0 0 0 0 0 0 5 2 1

0

Died

6 2 2 0 1

6 0 0

7 8 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0

6 5 2 22 0 2 0 8 7 5 0 2 3

2 2 0 0 0 0 0 0

0 0 0 0 0

0 6 0

>loo > 100 > 100 > 100 > 100 > 100 > 100 > 100 >loo > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100

a B. glubrutu (NIH) infected with P. segregutum 39 days before exposure to 50 S. munsoni miracidia. b B. glubrufa of same age as experimental snails, not previously exposed to P. segregutum. Each count averaged from 14 snails. Infection rates, based on cercarial shedding: for double infection (P.segregatum S. munsoni)= 32 % (8/25); for single infection (S.munsoni)=95 % (19/20).

+

-

I N T R A M 0 L L U S C A N 1N T E R TR E M A T ODE A N T A G 0 N I SM

229

histological study at key time intervals. General findings and some of the histological observations are summarized below (1V.A. 2-8 and B), divided into stages of infection, with the subordinate species (S. mansoni) both preceding and following the dominant species ( P . segregatum).

2. Miracidialpenetration a. P. segregatum superimposed on S . mansoni. Ten snails shedding schistosome cercariae from a 53-day-old infection were each exposed to 100 P . segregatum miracidia. Ten other uninfected snails were exposed in a similar manner and served as controls. On days 2, 4, 6 , 8 and 10 after exposure, 2 snails from each group were fixed for histological serial examination of the anterior part of each snail. Observations from days 2 and 4 are summarized in Table X. It is difficult to distinguish developing P. segregatum sporocysts from migrating rediae in sections taken after 6, 8, and 10 days. Nonetheless, a study of these sections indicate that P. segregatum miracidia penetrate snails already harbouring a well-developed S. mansoni infection as readily as they do uninfected snails. The penetration phase of the snail-echinostome relationship is apparently not affected by presence of an earlier schistosome infection. X TABLE Comparison of P. segregatum miracidia or sporocyst counts in snails with and without prior infection with S . mansonia B. glabrata with Days after 53-day-oldS . mansoni, P. segregatum exp. to 100 P . segregatum exposure miracidia

No. snails

exam. 2 4 Totals 2-4 Av. per snail

2 2 4

B. glabrata exp. to 100 P. segregatum miracidia

No. mirac.

No. snails

No. mirac.

or spor.

exam.

or spor.

36 42 78 20

2 2 4

41 47 88 22

* Counts based on study of serial sections of anterior portion of B. glabrata. b. S . mansoni superimposed on P. segregatum. Ten B. glabrata snails with a 61-day P. segregatum infection were re-exposed individually to 100 S. mansoni miracidia. Ten uninfected control snails of the same size were exposed in a similar manner. All snails were fixed for histological study after 1, 3, 6, 7 and 16 days and serially sectioned to determine the count of penetrating S. mansoni miracidia and condition of developing sporocysts. The results (Table XI) show that S. mansoni miracidia penetrate previously infected and uninfected snails

230

H O K - K A N L I M A N D D O N A L D HEYNEMAN

in equal numbers. These results are similar to those from the reverse experiment in which P. segregatum miracidia were not impeded in penetrating a host with an established S. mansoni infection. However, the developing schistosome mother sporocysts soon were attacked by rediae of the pre-existing echinostome infection (Figs 17 and 18). Many disappeared or were so damaged as to become unrecognizable. The number of visibly degenerating mother sporocysts increased during the period of observation. In single infections, some degenerating mother sporocysts were also seen. During the first week this number averaged up to 20 % of the total larval count. In double infections during the first week, the number of degenerating schistosome sporocysts rose to about 75 % of the total. However, no severe host cellular response accompanied the presence of damaged sporocysts during this short period of observation, in contrast with observations made over a longer time span. These injuries appear to be directly attributable to the presence of attacking echinostome rediae. TABLE XI

Comparison of S. mansoni miracidia or mother sporocyst counts in snails with and without prior infection with P. segregatuma

Days after S. mansoni

exposure

1 3

6 7 16 Totals 1-16 8

B. glabrata with 61-day-old P. segregatum, exp. to 100 S. mansoni miracidia

B. glabrata exp. to 100 S. mamoni

miracidia

No snails

No. mirac. or

No. snails

No. mirac. or

exam.

mother spor.

exam.

mother spor.

2 2 2 1 1 8

41

2

51 22 9 3

2 2 1 1 8

49 53

126

57

26 16 201

Counts based on study of serial sections of anterior portions of B. glabrata.

Some workers feel that the inability of miracidia to penetrate the snail epithelium is one factor to help explain failure of infection by non-adapted species. Sudds (1960), working with 2 bird schistosomes and 2 mammalian schistosomes and 26 species or varieties of snails, found that no miracidial penetration occurred in 12 “abnormal” host snails. These “abnormal” snail hosts produced a variety of behaviour patterns. Only a few snail species aroused no miracidial response. When contact was made on these hosts, the miracidia immediately swam off. In other encounters, miracidia stuck briefly at the point of contact, spun about with a rapid, head-on, boring motion, withdrew and swam away, then returned, stuck again briefly, swam away, and repeated the

I N T R A M O L L u sc A N I N T E R- T R E M A T O D E A N T A G O N I s M

23 1

pattern. Histological examinations showed that the miracidia failed to penetrate most of these “abnormal” hosts. We previously described the apparent inviolability of Lymnaea rubiginosa, quite ignored by hundreds of circling E. malayanum miracidia. It seems clear that miracidia are far more capable of detecting snail strain differences than are their human observers. We have, in fact, been led to seek strain differences between morphologically identical snails because of differences in miracidial response to them. On the other side of the coin, distinction between similar snail hosts may also lead to an explanation of their parasite differences as well (Jeyarasasingam et a/., in press). In the present infection model, failure ofparasite infection did not occur at the level of miracidial penetration, but at a subsequent developmental stage. 3. Establishment ojthe infection in B. glabrata Both S. mansoni and P . segregatum develop at the site of miracidial entry, e.g. the head-foot, mantle collar, or tentacles. Single infections of P . segregatum usually infect the snail at a higher rate than do S. mansoni, and, as was noted above, snails with single infections of S. mansoni still showed some degenerating mother sporocysts, whereas most of the single-infection echinostome sporocysts appear to develop normally. These are among the criteria for determining host-parasite adaptation given by Cheng (1968). The overall effect suggests an adaptation by the echinostome to B. glabrata that is superior to that of the schistosome. With S. mansoni superimposed on a well-developed P . segregatum infection, a fairly high dose of miracidia appears able to overcome any host-associated epithelial inhibition, judging from the ready entry of schistosome miracidia and early development of the sporocysts. However, the echinostome rediae rapidly interrupt further schistosome development, and most of the sporocysts are quickly attacked and destroyed. In the longer-term experiment previously reviewed, however, some daughter sporocysts escaped predation and after a long delay finally completed their development. This suggests the presence of a previously undetected indirect antagonism or inhibition of development by P . segregatum rediae. Previous evidence of an indirect as well as direct effect of P. segregatum against S. mansoni was clouded by subsequent discovery that very small predatory rediae were attacking the sporocysts to produce the germinal cytolysis originally thought to have been caused by a distant toxic effect (Lie, 1967, 1969c). Redial predation is not a permanent or constant attribute, but one that varies with redial age and, perhaps, with the condition (stimulus?) of the target trematodes. P . segregatum rediae quickly and easily dispatch S. mansoni mother sporocysts, but are less able to pierce the heavier body wall of daughter sporocysts (Lie, 1969~).On the other hand P. segregatum rediae readily devour all redial stages of the other echinostomes with which we have tested it. E. nzalayanunz in Malaya can feed on S. spindale sporocysts, or even on fullyformed cercariae, swallowing them head or tail first(Heyneman and Umathevy, 1968). These degrees of predatory capacity and response to the stimuli needed to express them probably vary with each species combination. 11

FIGS17-18. Double infection in B. glabrata: P . segregatum (day 46) and S. mansoni (day 7), foot. S. mansonimother sporocyst (arrow) attacked by several rediae, 75 x (17); Enlargement of redia from Fig. 17 and one mother sporocyst (arrow), 300 x (18).

FIGS22-27. (22), Embryonic stages in P . segregatum redia, 18 days after infection, 7 0 x ; (23), Double infection in B. glabrata: S. mansoni (day 48) and P.segregatunz (day 18), redia arriving in liver, 25 x ; (24) and (25), Double infection in B. glabrata: S. mansoni (day 120) and P. segregatum (day 90), third generation sporocysts of S. mansoni visible inside encapsulated daughter sporocysts, liver, 280 x ; (26) and (27), S. mansoni daughter sporocysts (day 161, arriving in liver area, 300 x (26) and 750 x (27).

I00

c

I00

,i nfection

80

80

e

c

I b

i

x

.-

60

c

F

60

C

Double

; infection

U

0)

9

0

e

f ._ 3

f

.g 40

2

40

--

u1

0

a-"

u)

8

20

20

I

0 -

4 0

1

1

I

I

I

0

6 9 I I 1416 18 22 25 29 Doys, P segregatum infection

1

1

1

1

1

I

1

1

9 I1 141618 22 25 29

0

Doys, P segregatum infection

FIG.19

FIG.20 LrvecI or =.manson/ on emergence of P segregorum cercorioe

100 r

0)

.p

8 0 - Single

infection

0

v

u

$ rn

60-

I.'

: Double

; infection

b

/

i

#

!, t Doys, P segregotum infection

FIG.19. Double infection in B. glabrata: Delayed presence of P. segregatum mother rediae in snails harbouring well-developed S. mansoni infection. FIG.20. Double infection in B. glubruru: Delayed migration of P.segregatum rediae in snails harbouring well-developed S. mansoni infection. FIG.21. Double infection in B. glabrata: Delayed P. segregatum cercarial shed in snails harbouring well-developed S. mansoni infection.

234

H O K - K A N LIM A N D D O N A L D HEYNEMAN

S. mansoni successfully established itself against a pre-existing P . segregatum infection only with the greatest difficulty. The reverse sequence occurs quite readily, with about the same success that the echinostome has in single-infection controls. Yet pre-existing sporocysts of S. mansoni can still exercise a strong influence on developing echinostomes, as will be reviewed in a subsequent section (IV. 5). The snail-sporocyst-sporocyst interaction studied by Basch et al. (1969) ( S . mansoni-Cotylurus lutzi interaction in B. glabrata) is a model system in which only indirect antagonism can occur. A surprisingly strong inhibitory and cytolytic response enables the schistosome to dominate and eliminate its rival, whereas the indirect antagonism against the schistosome exerted by Cotylurus was weak and relatively ineffectual.

4. Delayed redial migration Appearance and location of P . segregatum rediae in snails already infected with S. mansoni was checked 6, 9, 11, 14, 16, 18, 22, 25, and 29 days after exposure to the second, or echinostome infection. From 20 to 50 doublyinfected experimental and 20-60 singly-infected echinostome control snails were examined at each of these times. Percentages of snails showing mother rediae are shown in Fig. 19. In snails with a double infection, P . segregatum mother rediae appeared about 1 week later than in singly-infected snails. This delay is approximately 25 % of the usual intramolluscan developmental period, and a doubling of the time required for the early, pre-migratory redial development. Arrival of rediae in the ovotestis area is therefore delayed as well, as is the first appearance of cercariae (Figs 20,21). TABLE XI1 P. segregatum redial contents at different ages in single infections and in double infections with S . mansoni

Days after ~

P . segregatum

B. glabrataa with S. mansonib,

exposure

exp. to 20 P . segregatum mirac.

~~~

~~

~~

~

C ~~~~~~

16 18 20 23 25 27 30

40

-

+ + +

~~~~~~

B. glabrataa exp. to 20 P. segregatum mirac. ~~

DR

+ + + + + +-

GB ~

+ + + + + + + +

~

DR

C

~~

~

-

+ + + + + + +

+ + +-

In each observation, 2 snails dissected and 20 rediae examined per snail. S. mansoni infection 30 days old at time of exposure to P . segregutum. C=cercariae:DR = daughter rediae; GB=germballs and other embryonic stages. a

GB ~~

+ + + + + + + +

INTRAMOLLUSCANINTER-TREMATODE ANTAGONISM

235

5. Delayed germinal development within the rediae Another group of 40 snails already shedding schistosome cercariae was exposed to P. segregatum at the level of 20 miracidia per snail. At day 16 after exposure to P. segregatum, and at various intervals thereafter, 2 experimental and 2 singly-infected control snails were dissected, and 20 mature rediae were removed from each snail. These rediae, placed singly on a glass slide in a drop of half-strength saline, were dissected with fine forceps and germinal stages counted. Embryonic stages were classified as cercariae, daughter rediae, germballs and undetermined embryos (Fig. 22). The daughter rediae remained within their mother rediae up to day 27 in doubly-infected snails, but redial production ceased after 20-23 days in controls (Table XII). Young cercariae were seen in rediae in the single-infection control group as early as day 18, but in doubly-infected snails, cercarial embryos were seen only after 27 days.

6. Attraction of predaceous rediae to sporocysts By using a large infecting dose of 50 S. mansoni miracidia per snail, the chances for tentacle infections were considerably increased. In this site the developing mother sporocysts form a conspicuous tumor. In doubly-infected snails, P. segregatum rediae were seen to concentrate near the developing schistosome sporocysts, and their concentration in infected tentacles was easy to observe and to compare with controls. The number of rediae in tentacles of singly-infected snails compared with those in tentacles of doubly-infected snails (Table XIII) provides a striking measure of the attraction of echinostome rediae to developing schistosome mother sporocysts (Heyneman and Lim, 1970). Rediae frequently could be seen through the dissecting microscope attacking these sporocysts. Usually the predators were very small, almost TABLE XI11

Comparison of P. segregatum rediae localized in tentacles of snails with single injections and in snails doubly infected with S. mansoni and P. segregatum

B. glabrata with P. segregatum

Age of S. mansoni infection (days)

B. glabrata with P. segregatum and S. mansoni

_____

Exp. l a 6

17

No. tent. exam. No. rediae in tent. No. tent. exam. No. rediae in tent.

64

No. tent. exam. No. rediae in tent.

20

11

42 7

82 69 60 18

Exp. 2” 6

a

3

12 26

Snails with 39-day-old P.segreguturn infection exposed to 50 S. munsoni miracidia. Snails with 61-day-old P. segregutltrn infection exposed to 100 S. munsoni miracidia.

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colorless rediae, with light brown caeca, rather than the more strongly pigmented gut seen in older rediae. Occasionally a larger redia, with light yellow body color and brown gut, also was in the neighborhood of a schistosome mother sporocyst. After 10-14 days of this interaction, the schistosome larvae decreased in size, became opaque, or disappeared completely. The number of P. segregatum rediae in the vicinity of these dead sporocysts in the tentacles also diminished. These observations were repeated in 6 B. glabrata harbouring 61-day P . segregatum infections. Each snail was then re-exposed to 100 S. mansoni miracidia. Ten snails with the same age of P. segregatum infection were also examined for their tentacle rediae. Results (Table XIII) were similar to those in the preceding section. Similar attraction of rediae toward daughter sporocysts developing in the host hepatopancreas was also seen both by live dissection and histological studies (Fig. 23). This differential concentration was accompanied by a delayed appearance of P . segregatum rediae in their normal site, the ovotestis area in doubly-infected snails. In part we attribute this to an inhibitory indirect effect, and in part to a diversion of rediae toward the liver area harbouring most of the schistosome daughter sporocysts. A similar set of observations has been made in work currently in progress in our laboratory (Jeyarasasingam, unpublished observations) using another double-infection model. B. glabrata, harbouring a well-established infection of Echinostoma liei, can still be infected by S. mansoni(Heyneman etal., in press). However, developing schistosome mother sporocysts are quickly devoured and

TABLE XIV Comparison of S. mansoni cercarial shed after single infection and afrer double injection with P. segregatum

Days after exp. to S.m.

P.s.

Doubly-infected snails" No. exam.

Days after Singly-infected snails exp. to Est. shedb S.m. No. exam. Est. shed"

~

33

3

45 60 71 83

15 30 41

105 112

53 60 68 75 82

120

90

90 98

-

~~

10 10 10 10

9 7 7 3 3 3

+++ +++ +++ ++ + ++ + + + +

33 45

60 71 83 90 98

10 10 10 10

105

9 9 7 7

112 120

5 3

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++

a B. glabrata harbouring a 30-day S. mansoni infection, exposed to 20 P. segregntum miracidia. < 20 cercariae per snail per observation; + + 20-100 cercariae; + > 100.

+

++

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rediae seek out the sporocysts, following them even into the tentacles. In 27 tentacles of 60 doubly-infected snails 33 mother sporocysts were seen after 6 days and rediae were already found in 12 tentacles (compared with a single redia found in 1 tentacle among the singly-infected echinostome controls). 7. Speed of completion of larval dominance Manifestation of dominance is expressed by disappearance or significant decrease in larval stages of the subordinate species, with particular emphasis on cessation or prevention of cercarial shed. The speed of such end-stages is doubtless influenced by the time interval between the paired infections and number of miracidia in each experimental combination. The studies conducted in our laboratories in Kuala Lumpur and San Francisco demonstrate the broad spectrum of intensity and strength of the interaction. A remarkable example of highly effective predation is the case of E. audyi studied in Malaysia by Lie (personal communication). He obtained cessation of Fasciola gigantica cercarial shed in laboratory tests in as short a period as 12-13 days, and of Trichobilharzia brevis in only 18-26 days. On the other hand, it takes over 60 days for E. malayanum to cause S. spindale to stop shedding cercariae. By this criterion E. malayanum is a weak antagonist, although the final measure of strength is the total interaction of the various parameters of the interaction plus the effect it has on the host snail. In the S. mansonifP. segregatum model studied in B. glabrata, 10 doublyinfected and 10 singly-infected snails with S. mansoni controls were maintained in small aquaria within the same larger tank, using a common circulation as previously described. Snails were examined over 90 days for shedding of schistosome cercariae, beginning 3 days after exposure to P. segregatum. Table XIV indicates the diminution of schistosome cercariae shed by the doubly-infected snails. After about 6 weeks of interaction, the average schistosome cercarial shed per snail fell below 100. Subsequently, only a few cercariae were shed. Cercarial output from the S. mansoni controls remained at a high level and undiminished throughout the study. 8. Third-generation sporocysts of S . mansoni In histological sections of double infections with P. segregatum and S. mansoni, we frequently have seen unusual structures within the schistosome daughter sporocysts that resembled young daughter sporocysts (Figs 24, 25,26,27). Lie (1969a, c) described these bodies as third-generation schistosome sporocysts. They appear to have developed from germinal cells in an injured or modified daughter sporocyst. When many third-generation sporocysts were found inside an injured second-generation larva, the germinal cells along the wall were observed to have been sharply decreased in number. This unusual sporocyst generation has only rarely been observed in singly-infected S. mansoni snails (Lie, 1969c). Apparently certain sporocysts, whether from injury or other trauma, or normal variability, are able to cease producing cercariae and to produce instead additional, albeit modified daughter sporocysts. Whether this is simply an injury reaction or an adaptive change to replace lost sporocysts from redial attacks, we have no way to determine. Dinnik

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and Dinnik (1964) demonstrated with Fasciola giguntica that the usual succession of redia followed by cercaria production can be manipulated to produce only redial stages when the temperature is lowered, and can produce mixed generations at an intermediate temperature range. E. barbosai rediae infected with microsporidia appear to develop additional redial stages, in addition to the cercarial one (Lie, 1969b). Gordon et a/. (1934) in their early description of the intramolluscan stages of S. mansoni and S. haematobium observed that the first stage of development is a local multiplication of sporocysts near the site ofpenetration by the miracidium, followed by migration of motile sporocysts, which they called “Type I sporocysts”. After these sporocysts arrived at the liver they became motionless and developed into “Type I1 sporocysts”, which are found only in the liver. Type 11 sporocysts were said to vary greatly in size, owing to the fact that some were recently formed by multiple splitting off from others. Further change later took place in the liver and then “Type TI1 sporocysts” developed, which eventually gave rise to cercariae. Maldonado and Acosta-Matienzo (1947) considered Type 111 sporocysts to be the bodies that broke away from Type 11 sporocysts. Pan (1965), assuming Type 1 sporocysts to be mother sporocyst stages, rejected the thesis of Gordon et al., because mother sporocysts ordinarily do not migrate. Gordon’s views of sporocyst formation by multiple splitting involving 3 generations, were not accepted by subsequent workers, such as Faust and Hoffman (1934), Maldonado and Acosta-Matienzo (1947), Olivier and Mao (1949), Pan (1965). Yet, the possibility of a third sporocyst generation in the schistosome life-cycle remains an interesting challenge as to whether it is a rare, an abnormal, or an injury response. Recent work with sporocyst transplantation, pioneered by Chernin (1 966) and repeated by DiConza and Hansen (1972) may add to a better understanding of the normal range of sporocyst development. The latter workers have demonstrated, by inoculation of individual daughter sporocysts into digestive gland tissue of E. glabrata, that these implanted sporocysts produce additional sporocysts, which produce normal cercariae. DiConza and Basch are currently investigating the nature of this multiplication by in vitro studies (personal communication). Hansen (personal communication) has been able to show the presence of daughter sporocysts within daughter sporocysts in vivo in the liver of E. glabrata kept at 28”C,over a period of more than 2 months after inoculation. Presence of young daughter sporocysts freely active in the liver in these older infections strengthens the view that sporocyst multiplication of S. mansoni is not so rigidly restricted to 2 generations as has formerly been believed. B.

HISTOLOGICAL OBSERVATIONS

1. P. segregatum superimposed on S . mansoni Snails harbouring 30-day S. mansoni, then re-exposed to 20 P. segregaturn miracidia, were prepared for histological study. Forty-six snails with double infections were fixed 1, 3, 6, 9, 1 1 , 13, 16, 18, 20, 25, 27, 34, 38,41,46, 53, 60 and 90 days after exposure to P. segregatum. Forty-five snails with a single P. segregatum infection, 3 1 single-infection S. mansoni snails, and 36 uninfected

INTRA MOLL USCAN INTER-TREMATODE ANTAGONISM

239

snails served as controls, being fixed along with each pair of experimental snails. The P . segregatum miracidia penetrated and developed in schistosomeinfected snails in their usual locations. Though their sporocysts were smaller during the first week of infection than in controls (Figs 28-31), no host cellular response was seen against them. Initial appearance of mother rediae in the snail tissues was delayed about a week, as was migration of daughter rediae to the ovotestis area (Figs 32-36). Daughter rediae within rediae, along with germballs and cercariae, could still be seen after 26 days (Fig. 37). In doubly-infected snails, rediae were concentrated in the hepatopancreas where the S. mansonidaughter sporocysts were located, rather than in the usual site in the ovotestis, as in the control infections. Rediae reached the liver as quickly as the 18th day in double infections. Considerable damage to the organ was done by the time a number of rediae had crowded into it (Figs 38,39,40). The ovotestis region was very lightly parasitized until about the seventh week, owing to the diversion of echinostome rediae from their normal site in the ovotestis, presumably until the food source in the liver was exhausted. After rediae had been among the schistosome daughter sporocysts for several weeks, the latter began to show injury owing to the combination of redial attack and a strong host tissue reaction (Lim, 1970a; Figs 41,42, 43,44). S. mansoni daughter sporocysts usually retained the integrity of their body wall, a remarkably resistant structure, though an occasional redia broke through and entered the sporocyst body. Striking concentrations of amoebocytes around the injured daughter schistosome larvae appeared to press in on the weak spots of the unbroken but doubtless injured sporocyst body wall (Figs 45, 46). The larvae appeared edematous, with depositions of host fibrous tissue visible on them. Encapsulated schistosome sporocysts occasionally showed lysis of their germinal contents, though direct infiltration of encapsulated sporocysts by host amoebocytes seldom was seen (Figs 7,s). Occasionally a schistosome daughter sporocyst escaped severe injury and retained well-developed cercariae (Fig. 47). As noted previously, this explains the irregular shedding of small numbers of schistosome cercariae from a few doubly-infected snails often after very long periods. By the time most schistosome larvae had been encapsulated by the host tissue response, the echinostome rediae migrated to their normal ovotestis location. The host gonads were gradually consumed by these echinostome larvae, as occurs in single-speciesP . segregatum infections (Figs 48,49). 2. S . mansoni superimposed on P. segregatum B. glabrata, about 5 mm diameter, were exposed to 10 P . segregatum miracidia. After 39 days, 50 of these snails shedding P . segregatum cercariae were exposed to 50 S. mansoni miracidia. Controls were previously noninfected snails of the same age, also exposed to 50 S. mansoni miracidia. P . segregatum single-infection and non-infected controls were also established.

FIGS28-34. (28), Single infection with 2-day P. segvegutum sporocyst in B. glubrutu foot. Arrow indicates an eye-spot, 300 x ; (29), Double infection in B. glubrufu: 2-day P. segrezut ~ sporocyst m in snail previously infected with 32-day S. munsoni infection (not visible), foot. Arrow indicates eye-spot, 175 x ; (30), Single infection with 5-day P.segregurum sporocyst in B. glubrufu foot. Arrow indicates wall of sporocyst, note well-developed redia, 280 x ; (31), Double infection in B. glubruru: 5-day P. segregufum sporocyst in snail with 35-day S. munsoni infection, note repressed development compared with (30), foot, 250 x ; (32) and (33), Double infection in B. glubrutu: S. munsoni (day 50) and P.segregufirm (day 20). Crosssections of liver area, 80 x (32) and foot-head, ovotestis-liver areas, 70 x (33). Note absence of rediae in ovotestis region, but presence in anterior region (arrows); (34), Double infection in B. glubratu: S. munsoni (day 64) and P. segregutum (day 34). Observe absence of rediae in the ovotestis, the usual site in single infections at this age (Figs 35,36).

FIGS35-42. (35) and (36), Single infection of P. segregafum in B.glubrufu:arrival of redia in ovotestis (day 9), 7 5 x (35); and complete destruction of this organ in a well-developed infection (day 77), 17.5 x (36); (37)-(42), Double infections in B. glubruta. Daughter rediae inside a first-generation redia (S. mansoni, day 56, and P. segregufum, day 26), 300 x (37); Damage to liver area and encapsulated daughter sporocysts (arrows), (S. mansoni, day 90, and P . segregatum, day a), 17.5 x (38); Damage to liver area, showing redia attacking sporocyst (arrow), (S. mansoni, day 83, and P. segregatum, day 531, 30 x (39); Enlargement of portion of Fig. 39, t o show one redia in vicinity of sporocysts, 70 x (40); Encapsulated S. mansoni daughter sporocysts (S. munsoni, day 120, and P . segregufum, day YO), 70 x (41) and (42).

FIGS43-49. Double infections in B. glabraia. (43), S. mansoni (day 71) and P . segregairrrn (day 41), predatory activity of redia inside daughter sporocyst, liver, 7 0 x ; (44), S. mansoni (day 120) and P . segregatum (day 90), redia inside schistosome sporocyst, enclosed by host reaction, liver, 300x ; (45) and (46), S. mansoni (day 120) and P . segreguium (day 90), host tissue reactions (arrows) to injured schistosome sporocysts, liver, 188 x ; (47), S. mansoi (day 120) and P . segregaium (day 90), showinguninjured S. mansoni sporocyst, note absence of host tissue response, liver, 70 x ; (48), S. mansoni (day 90) and P . segregatum (day 60), rediae filling in ovotestis, 10 x ; (49), S. mansoni (day 120) and P. segregatum (day 90), showing delayed migration of rediae into usual site (ovotestis), after mass destruction of sporocysts in liver area, 5 x

.

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243

FIGS50-55. Double infections in B. glabrata. (501, P. segregarum (day 40) and S. mansoni (day I), schistosome mother sporocyst in tentacle (arrow) shortly after penetration, 188 x ; (51), P . segregatirm (day 45)and S. mansoni (day 6 ) , uninjured schistosome mother sporocyst in tentacle, 75 x ; (52) and (53), P. segregatum (day 60) and S. mansoni (day 21), massive proliferation of rediae and single still surviving sporocyst (arrow), liver, 17.5 x (52); enlarged portion near arrow of Fig. 52, showing strong host tissue response around daughter sporocyst, 175 x (53); (54), P . segregarum (day 196) and S. mansoni (day 157), encapsulated schistosome sporocyst, liver, 75 x ; ( 5 9 , P . segregatum (day 204) and S. mansoni (day 165), encapsulated schistosome sporocyst, liver, 280 x .

Samples of experimental and control snails were fixed from the first day after the schistosome challenge exposure, and at various intervals thereafter for 165 days. Schistosome mother sporocysts began to develop in a normal fashion at the miracidial site of entry (Fig. 50) until the P. segregurum rediae appeared and began to consume them. This happened as quickly as 3 days after the S. munsoni exposure. Three or four small rediae usually attacked a single developing mother sporocyst, which rapidly degenerated (Figs 17, 18). Occasionally, a

244

HOK-KAN LIM AND DONALD HEYNEMAN

schistosome mother sporocyst survived and went on to produce daughter sporocysts (Fig. 5 1). One or more daughter sporocysts might subsequently reach the normal site in the hepatopancreas and develop cercariae (Figs 52,53), but usually most daughter sporocysts were sufficiently damaged by rediae (though seldom killed) to stimulate a strong encapsulation response by the host (Figs 54, 55). This aspect proved to be of particular interest. It was one not fully appreciated until we had undertaken a histological study of the interaction process. That is, the contribution of the host’s cellular response, leading to suppression of the subordinate parasite, S. mansoni. Though the predatory rediae seldom succeeded in destroying the schistosome daughter sporocysts, they frequently damaged them, or released stimulating material, resulting in the host’s encapsulation response. In single S. mansoni infection, daughter sporocysts generally develop and grow in the liver with no apparent host cellular recognition or reaction. After cercarial shed begins, as Pan (1965) has observed, some cellular response is manifested, especially against cercariae trapped in host tissues. He suggested that the generalized tissue response is incited primarily by the cercariae and that precercarial stages “sensitize” the snail tissue, causing it to respond promptly and intensively to the stimulus provided by cercariae after they have escaped from the daughter sporocysts. Contents of sporocysts that leak out during cercarial breakthrough may have elicited this generalized tissue response. In the case of S. mansoni daughter sporocysts attacked by P. segregatum rediae, the host tissue response is indeed severe. Frequent redial sucking on the daughter sporocyst body wall may have caused leakage of the larval contents, which may have served to stimulate the host’s amoebocytes and result in the fibrotic response. The possibility of the intact parasite surface serving as a camouflage against detection either by absorption or by incorporation of host material into the larval body wall to deceive the host’s defense mechanism was considered b$Heyneman et al. (1971a). Salt (1960,1968) demonstrated the role of the parasite surface among insect parasitoids as a defensive mechanism against host hemocytes. In our system, redial attacks on the schistosome wall may have broken this mimicry. Experiments are under way in our laboratory to explore this possibility further.

v.

TREMATODE ANTAGONISM IN BIOLOGICAL CONTROL

The relatively new field of biological control has been developed to achieve a highly specific form of control of agricultural insect, weed, or any other undesirable pest organism by use of natural predators or pathogens (see general reviews in Sweetman, 1958;DeBach, 1964). Discussion of the principles of competitive displacement and coexistence can be found in DeBach and Sundby (1963), and DeBach (1966), one of the founders of this field. Results have at times achieved spectacular success, as in the control of citrus scale by parasitic wasps in California, or of the feral European rabbit in Australia with myxomatosis. Most applications of biological control of fluke diseases, such as schistosomiasis and fascioliasis, have, prior to the development of trematode inter-

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action as a possible approach, focused on the snail intermediate host. Michelson (1957) has summarized these efforts to develop effective parasites, pathogens, and predators of snails, which vary from haplosporidia (Barrow, 1965), to amoebae (Richards, 1968), Tetrahymena ciliates (Michelson, 1971), through the full range of nematodes (Mengert, 1953; Chernin et a]., 1960; Chernin, 1962), leeches (McAnnally and Moore, 1966), fish (Davidie and Metge, 1965; Mvogo and Bard, 1964) to ducks and geese. Johnson (1968) has assembled a valuable compendium of references to the pathology of invertebrates, a major portion of which is devoted to pathogenic agents and pathology of molluscs. Stephenson and Knutson (1966) have reviewed the associates of slugs, a good example of the number and variety of potential mollusc biological control agents. Berg (1964) reviewed data on parasitoids of snails, with particular reference to his remarkable discoveries with the molluscicidal larvae of sciomyzid flies. Important biological background studies are included in Foote (1 959) ; Neff (1966); Neff and Berg (1 966); Knutson and Berg ( I 967); Bratt et al. (1969). Population dynamics were studied in the laboratory by Geckler (197 1). Competition between Marisa cornuarietis and B. glabrata has been studied and field experiments attempted in Puerto Rico with varying success by Ferguson et al. (1958); Oliver-Gonzalez and Ferguson (1959); Demian and Lutfy (1966); Ferguson and Butler (1966); Butler et a/. (1969); Ruiz-Tiben e f a/, (1969); Jobin and Berrios-Duran (1970); Jobin et a/. (1970). Another approach to biological control of trematode diseases would be by controlling the free-swimming larval stages. Reports have appeared on predatory activity or toxic effects against trematode miracidia and cercariae by rhabdocoele turbellaria (Holliman and Mecham, 1971); hydrozoans (Mattes, 1949); oligochaete annelids (Backlund, 1949; Ruiz, 1951 ; Khalil, 1961 ; Wajdi, 1964; Boray, 1964); mosquito larvae and planarian exudates (Chernin and Perlstein, 1971); Cyclops, Daphnia, and Cypridopsis crustacea (Courmes et al., 1964); guppy, Lebistes reticulatus (Oliver-Gonzalez, 1946; Pellegrino el a]., 1966; Knight et al., 1970), and aquatic plants (Gibson and Warren, 1970). Biological misdirection and other forms of interference with miracidial hostfinding capacity were tested experimentally by Chernin (1968) and by Chernin and Perlstein ( I 969). Hyperparasitism of the intramolluscan trematode stages by microsporidia, summarized in Dollfus’ monographic review (1946), has been described by Martin (1936), Dissanaike (I 957a, b), Schaller (1 959, 1960), Cort et al. ( I 960a, b), Canning and Basch (1968), Lie and Basch (1970). These sporozoan parasites show a distinct specificity for the trematode and not the mollusc host tissue, although degrees of specificity to the trematode larvae vary widely. The Nosenza species described by Cort et al. (1960a) infects strigeoid parasites, whereas Perezia helnzinthorurn Canning and Basch, 1956 can infect all of the trematode larvae to which we have exposed it in many snail hosts, both aquatic and terrestrial (and a variety of other aquatic organisms as well, such as oligochaetes and mosquito larvae-Lie, personal communication). However, the role of microsporidial hyperparasitism in biological control is probably not one that can be employed as an independent tool. I t often, however, may become

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an important adjunct to other control measures. In the Malaysian field trials, Perezia appeared abundantly in the larvae of most trematodes once high infection levels of the controlling echinostome agent had been reached. This proved to be one of the major factors in the total infection complex in these field tests (Lie et al., 1970, 1971), and similarly in other regions may be an important and unanticipated parameter or limiting factor in future biocontrol effectiveness of trematode antagonism. Other protozoan or microbial infections, such as parasitic or facultative amoebae, may also add an unexpected factor in the intramolluscan biome, as we have discovered, on occasion, to our dismay. The unusual type of specificity in which only parasite and not snail tissue is involved appears confined to the microsporidia. The trematode antagonists-the chief focus of our attention for the past six years-select trematodes as food or affect them by other means, but also consume mollusc tissues and severely debilitate the snail hosts. Intramolluscan inter-trematode antagonism as a new approach to biological control (Wright, 1968; Berrie, 1970) began with the laboratory studies already reviewed. It was followed by the small-plot field efforts in Malaysia to control S. spindale with E. malayanum in Indoplanorbis exustus, also discussed in this review and by Heyneman and Umathevy (1968), Lie (l969a, b, 1971), Lie e t a / . (1970, 1971). These studies are continuing with the E. audyi-T. brevis system in L. rubiginosa snails and E. audyi-Fasciola gigantica in the same hosts by Lie and co-workers. A very rapid and complete degree of control has been achieved with E. audyi, particularly against T. brevis (Owyang and Lie, personal communication). Comparable studies with local strains of dominant echinostomes are being carried out in Thailand by Lie and colleagues. The encouraging results achieved to date would appear to justify field trials against human schistosomes in various endemic areas, where the problems faced and lessons learned undoubtedly will differ very greatly. At the present time, echinostomes are favored for control of schistosomiasis or fascioliasis, but a variety of other redia-producing predatory forms must be isolated and tested for use in other areas if trematode biocontrol is to be broadly applied. This, in fact, is a problem that probably will keep this a local, or at least a regional approach. In biological control of insects, local strains of controlling agents generally are of little value, and extensive search must be made for imported biocontrol agents. With trematodes, the opposite condition appears to be true. A key factor in success of control by trematode antagonism is strong infectivity of the competing antagonist in the snail harbouring the target species. This entails (1) a high rate of infection by a single miracidium, in (2) all ages of host snail, in (3) its full range of natural habitats, followed by (4) rapid growth and development in spite of a preceding trematode infection. Though a sufficiently strong or infective species may overcome local strain differences among various snail populations, such differences (of both snails and parasites) may greatly alter the characteristics of host-parasite adaptation from region to region. It is therefore unlikely that we can expect to find a single trematode species so broadly adaptive and so powerful an antagonist that it could serve as a biocontrol agent in areas other than those in which it is endemic and already adapted to the local snail or strains. Perhaps a few

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powerful or active predators (such as P. segregatum and E. audyi) will prove to be exceptions to this generalization. But we feel that the main effort to develop trematode biocontrol should be made locally, by study of the regional trematode fauna in the infected area in order to discover endemic rediaproducing candidate agents that are not infective to (or pathogenic in) man or his domestic animals. The selected agents should then be tested against the target trematode in the local snail strain, followed by larger-scale field pond experiments, as test results and local conditions permit. This raises an obvious objection : if local host-adapted antagonists are most effective, why hasn’t control already occurred naturally? It reminds one of the equally challenging query made to the stockbroker: “If you’re so smart, why ain’t you rich?” Perhaps the answer, too, is analogous: egg, concentration of host and available capital. It is necessary to seed selected disease transmission hostspots with large numbers of eggs of the antagonist in order to raise the infection level to one far higher than ordinarily would occur in nature. The inevitable adaptive interplay of numerous ecological factors would be expected to keep natural infection levels low. Unless a high proportion of host snails become infected, control by trematode interaction cannot occur. Only under artificially crowded, man-created conditions can one ordinarily expect to find snailinfection levels much above 25 %. The more usual condition is an infection rate ranging from less than 1 % to about 5 % of the snail population. Since a relatively small number of snails infected with a disease-producing trematode usually can still sustain transmission, we must artificially maintain a very high infection level to control the target trematode in the entire snail population. There are therefore two important distinctions between biological control by introduced insect parasites (or parasitoids) and trematode antagonism. Both characteristics are possible disadvantages to trematode biocontrol. For the latter to occur, we must (1) use a predator already adapted (or preadapted) to the host snail, and we must (2) artificially raise and sustain a high snail infection rate with the dominant parasite. Insect biocontrol depends upon introduction of an agent against which the local target species is not adapted, so that little resistance is offered to an epidemic spread of the controlling agent. The external environment, presumably, offers few obstacles to the agent’s spread. The important limiting factors are those that affect host numbers, concentration, and natural resistance to infection or predation. Trematode biocontrol, on the other hand, depends upon an agent that is adapted to the local host, the snail. Lack of such adaptation may mean failure to respond to host signals or enzymes, lack of tissue compatibility, or imbalance of any of the little-understood characteristics that determine specificity of infection. The controlling agent must clear the snail hurdle before it can even approach its target. The snail is therefore the limiting environment, which imposes very real barriers to a newly introduced trematode species. Once inside the snail and successfully multiplying, there is little reason to believe that the antagonist would not dispatch the prey trematode, providing it is a strongly predaceous, rapidly-growingform. The other obvious obstacle to a self-sustainingcycleis the necessity for a vertebrate final host. Although specificity for the final host is seldom as extreme as for the intermediate host, the extra steps of a transport 12

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host or some means to carry the metacercaria to the final host, and the various limitations associated with the final host make it highly unlikely that a selfsustaining life cycle can be counted on to achieve control of the disease agent. For the echinostomes we have tested, snails and other aquatic organisms are readily available and easily infected transport hosts. In most of these species, water birds and rodents serve as final hosts, easily maintained and reinfected. The key for field use is a sufficient concentration of infective eggs to sustain the high snail infection levels required for biocontrol. The life cycle may well be self-maintaining without such an effort, but then usually at a low or sporadic level, unsuitable for biocontrol. The cycle must be maintained artificially, then, in order to keep up the required snail infection level. In a life cycle that requires a rare final host or a host difficult to sustain, practical field use of such a trematode is not possible. P . segregatum, a powerful, effective control agent, is an example. Its only known vertebrate hosts are vultures and other raptorial birds-not especially suitable for mass-rearing of eggs, though vultures are easily cage-adapted and relatively long-lived for experimental purposes. This requirement imposes other pre-conditions for a trematode antagonist suitable for biocontrol: (1) an appropriate number of the required vertebrate final host, (2) readily procured and inexpensively maintained, and (3) easily infected and reinfected, to allow (4) a high continuous production of hardy eggs that ( 5 ) can be frequently harvested and stored without hatching under laboratory or controlled conditions (Lie and Owyang in preparation). The comparison between insect and trematode models of biocontrol would be more apt (and accurate) if it compared hyperparasitoid-parasitoid-host insect with trematode antagonism. In this more biologically complex mode1 of insect biocontrol, the hyperparasite depends for survival upon suitability of its parasitoid host, much as the trematode antagonist depends upon suitability of its snail host. An important difference, of course, is that the hyperparasite, to the degree it reduces the predator population or efficiency, is a negative value to the control system. Development of theoretic models and comparisons between these systems is currently under way in our laboratory by A. Kuris (unpublished observations). The trematode model has still other biological levels of complexity : microsporidial infection of both the predatory and target trematode; reduced life span of snails that serve as hosts of the dual infections; and the castration of host snails by echinostome rediae and cercariae in the snail ovotestis. Differential mortality and cessation of egg production by infected snails has been a significant factor in all of the biocontrol field tests we have so far undertaken. Equally important has been the presence (or absence) of a high infection rate with Perezia helminthorum. Lie et al. (1970) found that microsporidia appeared spontaneously once the echinostome infection reached a high level, but that apparently the echinostomes were more affected than were the schistosomes. As a result, a number of examples were found of snails simultaneously shedding both species of cercariae, a three-way interaction in which the microsporidia tipped the scales in favor of the schistosomes. Eventually, however, a combination of snail death and successful echinostome antagonism eliminated the schistosomes. In a subsequent field trial in the same pond, a microsporidial

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epidemic did not occur, or at least was delayed until schistosome control by the echinostome predators was nearly complete. Echinostome superinfection developed normally, r ' dual cercarial shed occurred, and control of the subordinate parasite was readily achieved (Lie et al., 1971). These interacting factors in trematode biocontrol-echinostome infection level, microsporidial epidemic, and snail death-are intimately related. Both the microsporidial epidemic and snail castration and death reached significant levels only after the echinostome level was artificially raised to a very high level, perhaps 70 % or more of the snail population. The combination may serve the same ends-control of the disease agent, whether by death of the host or the target trematode. But the complexities they pose and possible perturbations of the interaction cannot be predicted, as in the preceding example, and each infection system selected must be evaluated for its specific area. To do so will require careful laboratory study of the underlying parameters of hostparasite relationship surveyed in the present review. Perhaps the most unusual example of biological layering of complexity is one just now being studied in Malaya (Owyang and Lie, personal communication). They have developed a three-way trematode infection model consisting of Hypoderaeum dingeri, a relatively ineffectual echinostome antagonist, Trichobilharzia brevis, a bird schistosom'e,and E. audyi, a powerful echinostome antagonist. T. brevis sporocysts, interacting with H . dingeri rediae, most remarkably, can destroy the echinostome or reduce its rediae to granule-filled sacs, acting entirely by indirect antagonism (Owyang and Lie, 1971). This is the first example reported of domination of sporocysts over rediae (indirect over direct antagonism). These workers readily reinfected a group of snails, already shedding H. dingeri cercariae, with T. brevis. The schistosome was somewhat delayed in its early development, but succeeded in producing normal cercariae, while the echinostome rediae soon ceased shedding cercariae. The rediae lost their germinal contents or developed a third redial generation in place of cercariae, much as we described for S. mansoni in the presence o f P .segregatum. Eventually, schistosome domination was complete. The reverse sequence, T. brevis followed by H . dingeri, was an even more pronounced sporocyst domination. The echinostome miracidia penetrated freely and reached the site of mother sporocyst development in the snail heart, but no further development took place (Owyang and Lie, personal communication). However, when snails were exposed first to H . dingeri, and, after an appropriate period, to T. brevis and then to the highly predatory E. audyi, a remarkable interaction took place. H . dingeri, itself repressed by T. brevis, nonetheless exerted an inhibitory effect on development of the E. audyi, which thereby was blocked from destroying the schistosome. A three-way interaction interfered with the expected two-way interactions that would have occurred in the absence of the third species. An uneasy balance was produced, with the target schistosome being protected by a preceding infection with a subordinate species, which added enough inhibitory or indirect influence to block the direct antagonism of the predator, Addition of a microsporidial infection to this conglomerate of parasites would add a further complexity we hestitate to contemplate!

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These interaction possibilities are suggestive of problems that may well appear in field applications of biological control by trematode antagonism. Successful use of the technique in one area therefore cannot assure us that application elsewhere will be equally successful, however similar the conditions may appear to be. Obviously, one success does not preclude a wider ranging success, but the method is sufficiently sensitive to local ecological conditions to suggest that a prior survey and testing of local trematode strains should precede initial field trials. Nonetheless, it is our view that such efforts should be made in endemic areas and that even the most modest chance for success must not be ignored, in view of the growing urgency of schistosomiasis spread. If the antagonist life cycle can be sustained easily and inexpensively and eggs produced in large numbers, local application of biocontrol may be quite feasible in selected areas of high transmission. Procedures not yet developed may make the process far more efficient and easily sustained at the local level. At present we seed with eggs that have been isolated from laboratory host faeces and then incubated under carefully managed conditions. It would be far simpler and less costly to use raw animal faeces or perhaps a filtered faecal product kept for a week and then applied within a day or two of normal hatching. Simple floating trays or screens of faecal matter might prevent eggs from sinking into the mud or being washed from areas where snail infection is desired. Relatively small numbers of laboratory-infected animals currently provide great numbers of eggs. Some 20 or 30 pigeons for E. audyi and 30 rats for E. malayanurn support the studies now under way in Malaysia (Lie, personal communication). Larger-scale technology can easily be developed once the basic usefulness of the approach is established for a given area. The potential advantages of biocontrol are sufficiently promising to justify the initial effort to consider and evaluate it where conditions appear to make a trial feasible. The initial steps required to locate a trematode antagonist, isolate and test it in the laboratory, and select the appropriate strain or species among eligible candidates (outlined in Fig. 56) will have to be done at a major laboratory with appropriate facilities and, most important, experienced personnel. Such laboratories do exist or could be activated in most university medical centres in endemic areas. An important factor simplifying setting up these studies is the possibility of using local snail and trematode strains, without costly search in other areas, as has traditionally been done with insect parasitoids. Furthermore, the sensitive problem of importing “foreign” parasites is avoided, though of course the responsibility is nonetheless important to select an appropriate antagonist species, one non-pathogenic to domestic animals or man. Once the process of selecting the parasite predator has been completed, maintenance of the cycle, standardization of procedures, and longterm application of eggs should be feasible and quite inexpensive, even in regions distant from the developmental laboratory. If trematode antagonism could be teamed with other means of control, chemical, biological, clinical, and sanitary, the chance for long-term success would be greatly enhanced (as proposed in a Societyfor Invertebrate Pathology symposium, Levine, 1970a, with special reference to the review of integrated control of snails-Levine, 1970b). It might prove especially useful to tie

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FIG.56. Suggested sequence in search for local schistosome antagonists in known snail hosts.

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molluscicide application, with its high kill ratio but brief duration, to biocontrol of the few remaining snails and of the newly hatched snails, highly vulnerable to infection, that escape chemical death. Rapid recolonization of chemically decimated snail colonies, soon reinfected by continued recontamination of the water with egg-laden human faeces, is the often-repeated fate of molluscicide campaigns without concomitant sanitary control and clinical treatment. In such areas of continuous high-level transmission, biocontrol may help bridge the time interval between control of cercarial shed by infected snails and of egg-shedding by man (especially infected children). Sustained, frequently repeated seeding with eggs of an effective trematode antagonist in limited areas of high transmission danger might eliminate schistosomes from the few remaining infected snails (and in all probability eliminate the snails as well). More important, the eggs could infect newly hatched and newly arrived snails at a high enough level to provide a buffer zone of pre-infected snail hosts, intractable to infection with schistosomes or other target trematodes.

VI. SUMMARY The study of intramolluscan single and double trematode infections has proved to be a useful approach to a better understanding of this ancient hostparasite relationship. Modification and disruption of snail infection with trematodes is reviewed in terms of our laboratory studies, particularly those of Dr Lie Kian Joe, to whom this review is dedicated. The parameters and characteristics of various infection combinations are described. Possible application of this knowledge to trematode biological control by introduction of rival parasites into snail populations infected with a disease agent remains a major source of interest in this work. Biologically, however, the inter-trematode reactions and the responses of the snail host offer remarkable opportunities to examine and isolate various aspects of this host-parasite relationship. The basic system used as a reference model is the interaction between Schistosoma mansoni (NIH strain) and the echinostome Paryphostomum segregatum in Biomphalaria glabrata (NIH albino strain). The single species infection patterns differ widely between the pure sporocyst type, exemplified by S. mansoni, and the redial type, typified by P. segregatum. Characteristics of each are reviewed. Their interaction is then reviewed, based on original data and comparison with other studies. Redial predation (“direct antagonism”) is discussed in terms of relative activity, efficiency, and predatory response to triggering stimuli in the snail. Sporocyst influence (a purely “indirect antagonism”, whereas rediae can show both) is viewed as a repressive influence on rate of development and time of migration of rival stages, and a cause of cytolysis of germinal material within these stages. This indirect effect may be a direct toxic or competitive inhibition or be acting as a stimulus to a tissue response by the snail. Possible mechanisms of indirect antagonism are reviewed, with a fuller consideration of snail immunity as induced by these infections and measured experimentally. Quantitation of these interactions is not yet possible, but the parameters or

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characteristics of single-species infection are described and related to one another in a preliminary fashion as an “adaptation index” (AI), which includes such parameters as infection rate, period for 50 % snail death, and period to cercarial shed. A species with a higher A1 is presumed to possess greater dominance in an interaction with a species having a lower index in a specific host snail. Other factors that influence predatory capacity must eventually be incorporated in any measure of potential value of a trematode for use as a controlling agent. These include: (1) localization in the snail, (2) vigor and speed of onset of predatory response, (3) pharyngeal size and gut length of predatory redia, (4) number of rediae produced, (5) toughness of body wall of the prey species, (6) relative adaptation or degree of success of each species in the snail host species (or geographical race). Initial data and background are reviewed for determination of an “interaction index” using the P. segregatum-S. nzansoni model in B. glabrata. Successive steps in the interaction are described, both for superinfection of the echinostome on the schistosome and the reverse sequence, as suggested by experimental data. Modification of infection rate and developmental period and of the redial migratory pattern are useful indicators of the intertrematode influence. Schistosome daughter sporocysts under attack will produce third-generation sporocysts instead of cercariae, a particularly interesting indication of an indirect effect by rediae. Intertrematode injury will initiate a tissue response by the host snail that is often well marked and suggestive of a well organized defensive response by the host. Questions of the capacity of host snails to demonstrate an immune response are reviewed in some detail. These findings are then evaluated for possible application in the biological control of human schistosomiasis and other trematode diseases of man and domestic animals. Preliminary field trials, already completed and still underway in Malaysia by Lie and colleagues, are reviewed. Results are promising, but important limitations and uncertainties of the approach are evident. Basic differences, advantages and disadvantages are explored between trematodetrematode-snail interaction and the insect prey-parasitoid approach to biological control. We conclude that the trematode method is locally applicable, using indigenous strains supplied and sustained at a high level by continual reseeding with eggs of the controlling trematode (or processed faeces from the appropriate final hosts). Effective biocontrol as a self-sustaining life-cycle appears highly improbable, as is the expectation that this method can be adopted on a wide scale with the same controlling agent. Yet, for intensive use in restricted areas of high infection transmission, the approach may well prove to be valuable and research should be initiated to develop improved methods and isolate additional species in endemic areas that might be useful as antagonists. Biocontrol, in our view, offers limited but possibly important usefulness, especially if teamed with other control methods, such as molluscicide, sanitary, and therapeutic. REFERENCES

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Berg, C. 0. (1964).Snail control in trematode diseases: the possiblevalue of sciomyzid larvae snail-killing diptera. In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 2, pp. 259-309. Academic Press, London and New York. Berrie, A. D. (1970). Snail problems in African schistosomiasis. In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 8, pp. 43-96. Academic Press, London and New York. Bisset, K. A. (1947). Bacterial infection and immunity in lower vertebrates and invertebrates. J. Hyg. 45, 128-135. Boray, J. C. (1964). Studies on the ecology of Ljlmnaea tomentosa, the intermediate host of Fasciola hepatica. I. History, geographical distribution, and environment. Aust. J. Zool. 12,217-230. Boray, J. C. (1966). Studies on the relative susceptibility of some lymnaeids to infection with Fasciola hepatica and Fasciola gigantica and on the adaptation of Fasciola spp. Ann. trop. Med. Parasit. 60, 114-1 24. Boray, J. C. (1967). Host-parasite relationship between Iymnaeid snails and Fasciola hepatica. Vet. med. Rev. 132-140. Boray, J. C. (1969). Experimental fascioliasis in Australia. In “Advances in Parasitology” (Ed. Ben Dawes), VoI. 7, pp. 95-210. Academic Press, London and New York. Bratt, A. D., Knutson, L. V., Foote, B. A. andBerg, C. 0.(1969). BiologyofPherbellia (Diptera: Sciomyzidae). Memoir 404. New York State College of Agriculture, New York. Brooks, C. P. (1953). A comparative study of Schistosoma mansoni in Tropicorbis havanensis and Australorbis glabratus. J. Parasit. 39, 159-163. Brooks, W. M. (1969). Molluscan immunity to metazoan parasites. In “Immunity to Parasitic Animals” (Eds G. J. Jackson, R. Herman and I. Singer), Vol. 1 , 149-171. Appleton-Century-Crofts, New York. Bruce, J. I. and Radke, M. G. (1971). Cultivation of Biomphalaria glabrata and maintenance of Schistosoma mansoni in the laboratory. In “Culturing Biomphalaria and Oncomelania (Gastropoda) for Large Scale Studies of Schistosomiasis. Part I.” Biomedical report number 19, 406th Medical Laboratory, U.S. Army Medical Department Activity, Japan, pp. 1-84. Burch, J. B. (1967). Chromosomes of intermediate hosts of human bilharziasis. Malacologia 5, 127-135. Burch, J. B. (1969). The chromosome number of Bulinus sericinus from Ethiopia. Malacol. Rev. 2, 113-1 14. Burch, J. B. and Lindsay, G. K . (1970). An immune-cytological study of Bulinirs s.s (Basommatophora : Planorbidae). Malacol. Rev. 3, 1-1 8. Butler, J. M., Ferguson, F. F. and Olivier-Gonzalez, J. (1969). A system for mass producing the snail, Marisu cornuarietis, for use as a biological control agent of schistosomiasis. Caribb. J. Sci. 9, 135-139. Buttner, A. (1951). La progenese chez Ies trematodes digenetiques. La signification. Les manifestations. Contributions a I’etude de son determinisme. Ann. Parasit. 25, 376-434. Canning, E. U. and Basch, P. F. (1968). Perezia helminthorum sp. nov., a microsporidian hyperparasite of trematode larvae from Malaysian snails. Parasitology 58, 341-347. Chadwick, J. S. (1967). Serological responses of insects. Fed. Proc. 26, 1675-1679. Cheng, T. C. (1963). Biochemical requirements of larval trematodes. Ann. N.Y. Acad. Sci. 113, 289-320.

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McClelland, W. F. J. (1965). Development of S. haematobium in B. (Ph.) rzasutus. In “East African Institute for Medical Research, Annual Report 1963-64”, pp. 15-17. Mwanza, Tanzania. McQuay, R. M., Jr. (1948). Susceptibility of two species of Louisiana Tropicorbis to Schistosoma mansoni. J. Parasit. 34 (Suppl.), 24. McQuay, R. M., Jr. (1952). Susceptibility of a Louisiana species of Tropicorbis to infection with Schistosoma mansoni. Expl Parasit. 1, 184-188. McQuay, R. M., Jr. (1953). Studies on variability in the susceptibility of a North American snail, Tropicorbis havanensis, to infection with the Puerto Rican strain of Schistosoma mansoni. Trans. R . SOC.trop. Med. Hyg. 47, 56-61. Mengert, H. (1953). Nematoden und Schnecken. Z . Morphol. Oekol. Tierre 41, 311-349. Metlitskii, L. V. and Ozeretskovskaya, 0. L. (1968). “Plant Immunity. Biochemical Aspects of Plant Resistance to Parasitic Fungi”. Plenum Press, New York. Michelson, E. H. (1957). Studies on the biological control of schistosome-bearing snails. Predators and parasites of fresh-water Mollusca: a review of the literature. Parasitology 47,413-426. Michelson, E. H. (1963). Development and specificity of miracidial immobilizing substances in extracts of the snail Australorbis glabratus exposed to various agents. Ann. N. Y.Acad. Sci. 113,486-491. Michelson, E. H. (1964a). Miracidia-immobilizing substances in extracts prepared from snails infected with Schistosoma mansoni. Am. J. trop. Med. Hyg. 13, 36-42. Michelson, E. H. (1964b). The protective action of Chaetogaster limnuei on snails exposed to Schistosoma mansoni. J. Parasit. 50,441444. Michelson, E. H. (1971). Distribution and pathogenicity of Tetrahymena limacis in the slug Deroceras reticulatum. Parasitology 62, 125-1 31. Milward De Andrade, R. W. (1962). Ecologia de Australorbis glabratus em Belo Horizonte, Brazil. I11: Indices de infeccao natural por Schistosoma mansoni segundo 0s diametros dos caramujos. Revta Bras. Biol. 22, 383-390. Moore, D. V., Thillet, C. J., Carney, D. M. and Meleney, H. E. (1953). Experimental infection of Bulinus truncatus with Schistosoma haematobium. J. Parasit. 39, 21 5-22 1. Moose, J. W. and Williams, J. E. (1963). Susceptibility of Oncomelaniu formosana from three different areas of Taiwan to infection with Formosan strain of Schistosoma japonicum. J. Parasit. 49,702-703. Miiller, K. 0.(1959). Hypersensitivity. In “Plant Pathology. An Advanced Treatise” (Eds J. G . Horsfall and A. E. Dimond), Vol. 1, pp. 469-519. Academic Press, New York. Miiller, K. 0. (1966). How plants fight disease. Sci. J. 2,57-61. Mvogo, L. and Bard, J. (1964). Seconde note d’information sur I’Astatoreochromia alluaudi, poison malacophage utilisable dans la prophylaxie de la bilharziose. Bull. SOC.Path. exot. 57,21-23. Nasir, P. (1962). Further observations on the life cycle of Echinostoma nudicaudatum Nasir, 1960 (Echinostomatidae: Trematoda). Proc. helm. SOC.Wash.29,115-127. Neff, S. E. (1964). Snail-killing sciomyzid flies: application in biological control. Verh. internat. Verein. Limnol. 15,933-939. Neff, S. E. and Berg, C. 0. (1966). “Biology and immature stages of malacophagous Diptera of the genus Sepedon (Sciomyzidae).” Bulletin 566, Virginia Agricultural Experiment Station, Blacksburg.

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Negus, M. R. S. (1968). The nutrition of sporocysts of the trematode Cercaria doricha Rothschild, 1935 in the molluscan host Turritella communis Risso. Parasitology 58, 355-366. Newton, W. L. (1952). The comparative tissue reaction of two strains of Australorbis glabratus to infection with Schistosoma mansoni. J. Parasit. 38, 362-366. Newton, W. L. (1953). The inheritance of susceptibility to infection with Schistosoma mansoni in Australorbis glabratus. Expl Parasit. 2,242-257. Newton, W. L. (1954). Tissue response to Schistosoma mansoni in second generation snails from a cross between two strains of Australorbis glabratus. J. Parasit. 40,352-355. Newton, W. L. (1955). The establishment of a strain of Australorbisglabratus which combines albinism and a high susceptibility to infection with Schistosoma mansoni. J. Parasit. 41, 526-528. Nolf, L. 0.and Cort, W. W. (1933). On immunity reactions of snails to the penetration of the cercariae of the strigeid trematode, Cotylurus jlabelliformis (Faust). J. Parasit. 20, 38-48. Oliver-Gonzalez, J. (1946). The possible role of the guppy, Lebistes reticulatus, on the biological control of schistosomiasis mansoni. Science 104,605. Oliver-Gonzalez, J. and Ferguson, F. F. (1959). Probable biological control of schistomiasis mansoni in a Puerto Rican watershed. Am. J. trop. Med. Hjtg. 8,5659. Olivier, L. and Mao, C. P. (1949). The early larval stages of Schistosoma mansoni Sambon, 1907 in the snail host Australorbisglabratus (Say, 1818). J. Parasit. 35, 267-275. Owyang, C. K. and Lie, K. J. (1971). Interaction between Hypoderaeum dingeri and Trichobilharzia brevis in the Lymnaea intermediate host. Proc. Twelfth Pac. Sci. Congr., Canberra, Australia, 18 August-3 September 1971, Vol. 1, p. 203. Owyang, C. K., Lie, K. J. and Kwo, E. H. (1970). The interspecific competition between Trichobilharzia brevis and Echinostoma audyi in the snail intermediate host. S.E. Asian J. trop. Med. Pub. Hlth 1,160-161. Pan, C. T. (1958). The general histology and topographicmicroanatomy of Australorbis glabratus. Bull. Mus. comp. 2001. 119, 237-299. Pan, C. T. (1965). Studies on the host-parasite relationship between Schistosoma mansoni and the snail Australorbisglabratus. Am. J. trop. Med. Hyg. 14,931-976. Paperna, I. (1967). The effect of pre-existing trematode infection on the establishment of Schistosoma haematobium larvae in bulinid snails. Ghana med. J. 6,89-90. Paraense, W. L. and Correa, L. R. (1963). Variation in susceptibility of populations of Australorbis glabratus to a strain of Schistosoma mansoni. Revta Inst. Med. trop. S . Paul0 5 , 15-22. Pellegrino, J. and Katz, N. (1968). Experimental chemotherapy of Schistosoma mansoni. In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 6, pp. 233-290. Academic Press, London and New York. Pellegrino, J., De Maria, M. and De Moura, M. F. (1966). Observations on the predatory activity of Lebistes reticulatus (Peters, 1859) on cercariae of Schistosoma mansoni. Am. J. trop. Med. Hyg. 15, 337-341. Pemberton, C. E. and Willard, H. F. (1918). A contribution to the biology of fruitfly parasites in Hawaii. J. agric. Res. 15,419-460. Poinar, G. 0. (1969). Arthropod immunity to worms. In “Immunity to Parasitic Animals” (Eds G . J. Jackson and I. Singer), Vol. 1, pp. 173-210. Appleton, New Kork.

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Taeniasis and Cysticercosis (Tueniu suginutu) ZBIGNIEW PAWLOWSKI

Clinic of Parasitic Diseases. Przybyszewskiego 49. Poznah. Poland AND

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MYRON G SCHULTZ

Center for Disease Control. U S. Department of Health. Education and Welfare. Atlanta. Georgia. U.S.A. I . Introduction .................................................................................... I1. Nomenclature .......................................... ......................................

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111 Hosts of Tueniu suginutu ........................... ...................................... IV . Structure and Biology of Tueniu suginutu ................................................ A Adult ....................................................................................... B. Egg ..................... ................................................................. C. Onchosphere .............................................................................. D Cysticercus ................................. ................................. V . Clinical Aspects of Taeniasis (T suginutu) ................................. A Symptomatology ........................................................................ B. Clinical Pathology ..................................................................... C. Diagnosis ................................................................................. D . Treatment ................................................................................. VI . Epidemiology and Epizootiology ......................................................... A . Transmission Between Man and Animals .......................................... B Epidemiological and Epizootiological Data ....................................... C. Losses Due to Taeniasis and Cysticercosis ....................................... VII . Prevention ....................................................................................... A . Meat Inspection .............................. ..................................... B Serological Diagnosis and Immunization of Cattle .............................. C Sanitation ................................................................................. References .................................................................................

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“NO animal has been responsible for more hypotheses, discussions and errors than the tapeworm.” C . Davaine, 1860

I. INTRODUCTION Tapeworms have been known since prehistoric times-it is difficult indeed for the host to ignore the discharge of proglottides or fail to wonder about their origin. Tapeworms are referred to in the Papyrus Ebers. in Indian literature. in Chinese literature. and by Graeco.Roman. Byzantine and Arabian authors (Hoeppli. 1958). Many theories were held about the nature of tapeworms . 269

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They were viewed as products of spontaneous generation, as transformed strips of intestinal mucosa, or as a union of separate animals in a chain invested by a membrane formed in the intestine. The term cucurbitini was applied to proglottides not only because they look like seeds of the pumpkin (Cucurbita sp.) but also because pumpkin seeds were one of the earliest, effective treatments for tapeworm infection. Taenia saginata was recognized as a distinct species by Goeze in 1782. The relationship between the adult parasite and bladder worms of cattle was established by Leuckart in 1861 who fed gravid proglottides to a calf and obtained the larval stage. Eight years later Oliver reversed the procedure and infected man with cysticerci of cattle. In the century that has since passed a great deal of knowledge has been developed about this parasite, although work on the adult stage has been impeded by the inability to infect experimental animals. Taeniasis of man and cysticercosis of cattle have also been recognized as important public health and economic problems. In recent years several reviews have appeared dealing with selected aspects of cestodiasis but none has reviewed Taenia saginata infection in a comprehensive manner. Our aim in preparing this review is to summarize advances in all aspects of T. saginata taeniasis and cysticercosisnomenclature, host relationships, structure and biology, clinical and therapeutic features, epidemiology and epizootiology, and prevention, in the hope that our work will stimulate further inquiry and lead to better control of this zoonotic disease.

11. NOMENCLATURE At the outset we must indicate that there is considerable confusion about the nomenclature and the taxonomy of the parasite that is the subject of this monograph. At present, some unarmed tapeworms occurring in man are poorly understood (Huang, 1967) or doubtfully named. Indeed, in the last few years two different opinions have been expressed concerning the generic name of the unarmed tapeworm of man acquired from the consumption of beef. Both Taenia saginata and Taeniarhynchus saginatus are generic and specific names used for this parasite. The choice of name depends on whether unarmed human tapeworms belong to a genus separate from other tapeworms. The two opposite opinions on this point are best expressed by Abuladze and Verster. Abuladze 1964 (Principles of Cestodology, Vol. 4, Taeniata, p. 163) said “Perrier (1 897) and a number of other workers regarded Taeniarhynchus as a sub-genus of Taenia, and Holl (1919) used it as a synonym of Taenia, we cannot agree with these opinions because the presence or absence of hooks in teniid should be respected as a generic criterion. We and most present day working helminthologists regard Taeniarhynchus as a separate genus.” Abuladze’s opinion is in agreement with that of Wardle and McLeod (1952) who used the generic name Taeniarhynchus proposed by Weinland in 1859. Wardle and McLeod’s textbook probably popularized the generic name Taeniarhynchus, especiallyin Europe. On the other hand, Verster (1969) in a paper titled “A taxonomic revision of the geuns Taenia (Linnaeus, 1758)” disagreed with the use of the generic name

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Tueniarhynchus and says “The genera Tueniurhynchus Weinland (I 858) and Monordotaenia Little (I 967) (synonym : Fossor Honess 1937) are differentiated from Taenia oiily on the absence of rostellar hooks in the former and on a single row in the latter. A single character may justify the creation of a new species but it cannot be the sole criterion for the erection of a new genus. If the practice of basing a genus on a single character were to be consistently followed, it would necessitate the erection of four more genera to accommodate the eight species in which the genital ducts pass the longitudinal excretory vessels ventrally to cross into the cortex. This is, however, clearly unwarranted and the continued use of Tueniurhynchus and Monordotaeniu as well as Multiceps, Hydutigeru, and Tetrutirotaenia at the generic level would only lead to further confusion.” Venter’s opinion agrees with the earlier opinion of Joyeux and Baer (1929). Abuladze and Verster also disagree on the number of species of unarmed tapeworms of man. Abuladze (1964) distinguished T. africuna (Linstow, 1900), T. confusa (Ward, 1896) and T, hominis (Linstow, 1902) from Taeniarhynchw suginutus. However, Verster (1969) stated that these species are no longer valid and considers them all synonyms of Taeniu suginutu. The final verdict remains for the taxonomists to decide. In the meantime we have chosen, without prejudice, to use the generic name Taeniu suginatu for the common, unarmed tapeworm of man, and for human infection we use the term taeniasis (T. saginutu). We do have strong convictions about the term ‘‘Cysticercus bovis”. It is illogical to give a separate generic and specific names to the larval stage of a parasite that already has a distinctive name, therefore, the term “Cysticercus bovis”, although it enjoys common usage, should be cast into oblivion. For the term “Cysticercus bovis” we use “Taeniu saginata cysticercus” and for infection with “Cysticercus bovis” we use “Taenia saginata cysticercosis”. 111. HOSTSOF TAENIA SAGINATA

Man is the only definitive host of Taenia suginuta. Laboratory animals, including monkeys (Calrenburg, 1932, cit. Nelson et ul., 1965)failed to develop adult Taenia suginatu infections when fed with the intermediary stage. A very extensive search for adult Taenia saginata in Kenya carried out by Nelson et ul. (1965) in 271 wild primates revealed tapeworms of at least six different species, but none was Tueniu saginutu. Southwell (1921) described tapeworm proglottides (from a goat in Accra) which closely resembled Tuenia saginuta segments, however, no scolex was found and this observation is inconclusive. Cestode larvae are less host specific than adult cestodes, therefore, the list of intermediary hosts of Taeniu suginatu is considerable and expands as each year goes by. The main intermediary hosts for Taenia saginafu are domestic Bovidue. These include Bos tuurus, Bos bufellus, Bos indicus and Bos grunniens (see Abuladze, 1964). In recent years the reindeer (Rungifer turundus) has been added to the list of intermediary hosts. According to Abuladze (1964) raw reindeer meat was first implicated in 1956 as the source of T. suginuta infection in the Jamalsko-Nienecki region of the U.S.S.R. In 1960, Safronov

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found cysticerci in ten reindeer carcasses in the Oleniekski region of the U.S.S.R. and suggested that they might be cysticerci of T. suginutu because there are many individuals with tapeworm infection in the region (59 of 200 school children examined) and cattle are not bred in this area. In 1961 Krotov confirmed this observation in reindeer breeders at a state farm in the U.S.S.R. There are virtually no data about wild intermediary hosts of T. suginutu in the Americas. The one exception is a report in 1906 of infection in the llama and pronghorn antelope. However, in Africa unhooked cysticerci are sporadically found in wild animals: wildebeeste (Gorgon tuurinus) (Nelson et ul., 1965), bush-buck (Tregefuphus scriptus) (Tremlett, personal communication to Nelson et ul., 1965)and the tame oribi (Ourebiu ourebi) (see LeRoux, 1957). According to Nelson et ul. (1965) there are only a few records of T. suginutu cysticerci from African animals in zoological gardens ; they include giraffes (Railliet 1885, Schwartz 1928, Buckley 1947) and lemur (Hamerton 1934). In addition Maczulski (1941) (according to Abuladze, 1964) found cysticerci, probably of T. suginutu, in Guzeffugutturosuand Taylor (1958) found cysticerci of Tueniu suginuru in the liver of an onyx-antelope kept in a zoo. To the contrary Graber (1959) found cysticerci of Tueniu suginutu in cattle, camels, sheep, Dorcas gazelle (Guzeffudorcu), red fronted gazelle (Guzeffurusifrons) and other unspecified antelopes in the Chad Republic. This finding should be confirmed and further work is necessary to understand the role of wild animals in the potential spread of T. suginutu in Africa. There is one striking report concerning wild animals as intermediary hosts of T. suginutu in Asia. Among aborigines in Wulai District of Taiwan, Huang (1967) found 28 tapeworms with scoleces which he identified as T. saginutu. He successfully infected newborn calves with eggs of these tapeworms and produced cysticerci which differed in some respects from normal cysticerci of T. suginutu. Because there are no cattle in this area he suggested that wild goats might be a natural intermediary host of T. suginutu in this region. Boev (1960) pointed out that only experimental cross infections could prove a particular wild animal as the intermediary host of T. suginutu. For example, he corrected the improper finding of T. suginutu cysticercosis in roe-deer. Sometimes cross infection experiments raise further questions. Price (1961), for example, infected himself with some cysticerci from the liver of a giraffe and found that the adult worms differed from normal T. suginutu. He proposed the name of Tueniu suginutu var. giruffae. It has been suggested that this may be an example of morphologic variation caused by the transmission of a parasite to an abnormal host. There will probably never be a final list of all the intermediary hosts of T. suginutu. The most controversial question about these hosts is the role of man himself. Two different opinions have been expressed in recent years concerning cysticercosis of man. Nelson et ul. (1965) said that some cysts reported in man may be cysts of Tueniu saginutu or small hydatids. On the other hand, Verster (1967) commented on the discrepancy between the incidence of human cysticercosis and the incidence of adult Tueniu solium infection. She said that Tueniu solium infection may be not as rare as is generally assumed. As far as we are able to determine, 12 cases of Tueniu suginutu cysticercosis

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273

of man have been described in the literature. The older reports include: Arndt (1867, according to Meggitt, 1924), Nabias and Dubreuilh (1889, according to Meggitt, 1924), Heller (according to Abuladze, 1964), Fontan (1919), Watkins and Pitchford (1924, according to Brumpt, 1936), and Castellano et al. (1928). The more recent reports include: Naumov (1929), de Rivas (1937), Tanasescu and Repciuc (1939), Asenjo and Bustamente (1950), Niiio (1950), Bacigalupo and Bacigalupo (1956) and Goldsmid (1966). Naumov in 1929 reported the first case of human T. suginuta cysticercosis in the U.S.S.R. (Abuladze, 1964): the patient was a 40-year-old man who at autopsy had nine cysticerci discovered in his heart and one in the meninges and an adult T. saginata in the intestine. The cysticerci were hookless and were diagnosed by Naumov as cysticerci of T. saginata. A case in Pennsylvania was reported by de Rivas in 1937. Autopsy examination revealed numerous cysticerci distributed throughout all the muscles and the author’s description and drawing depict cysticerci with scoleces each with four suckers but without hooks. Tanasescu and Repciuc (1939) reported a 59-yearold Romanian woman with a tumor in the mammary region. Their paper presents seven photographs of cross sections through the unarmed scolex. They believed it was a cysticercus of T. saginata because it had “five suckers instead of four characteristic of Taenia solium larvae”. Since the photographs show rugae in the wall surface the diagnosis is probable. The patient also had tumors in other areas of the body. This case and a very similar case of Fontan’s (1919) probably came to the attention of the authors because of the suspicion of carcinoma in the mammary region. Asenjo and Bustamente (1950) in discussing 59 patients with cysticercosis in the Santiago de Chile Neurological Clinic mentioned that all but one were due to cysticerci of T. solium and stated that the other was due to cysticerci of Taeniasaginutu. No other details were given. Nifio (1950a, b) reported a case of C. bovis in a lymph node of the meso-appendix. Goldsmid (1966) in discussing human cysticercosis in Rhodesia mentioned that of 62 cases at Harari Central Hospital in Salisbury between 1955-1965 one was diagnosed as T. saginata cysticercosis but no proof was found in the records indicating the absence of hooks. In the report by Dixon and Lipscomb (1961) of 450 cases of cysticercosis in soldiers from India no mention is made of T. saginata cysticercosis. There is no doubt that hookless scoleces in cysticerci have been found in the human body. Also, there are instances where adult Taenia saginata infection occurs contemporaneously with human cysticercosis (Fontan, 1919; Naumov, 1929). It would be interesting to know, however, how many cases of cysticercosis due to T. solium in man or swine have cysticerci with unarmed scoleces, as well as what percentage of scoleces in each case are unarmed. From the theoretical point of view a parasite may find itself in an unexpected host or unusual tissue in very special circumstances, i.e. malignancy. These special conditions can occur in man but in the cases previously described none were noted. Cysticercosis of man due to T. saginata is still an open question. Its resolution is dependent on careful morphological examination of cysticerci removed from human patients.

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Z B I G N I E W PAWLOWSKI A N D M Y R O N G . SCHULTZ

Iv.

STRUCTURE AND

BIOLOGYOF Taenia saginata

A. ADULT

Work on the morphology of adult T. saginata has attracted little interest during the past 20 years. However, there are two recent, important papers dealing with morphological aspects of T. saginata (Verster 1967,1969) and one observation by Rivero (1952) that described a new sphincter in the cirrus pouch of the adult T. saginata. Morphological studies of T. saginata have two important aspects; they often demonstrate abnormalities of structure and they reveal taxonomic variants of T. saginata which may be of epidemiological importance. Elsdon-Dew and Proctor (1965) and Verster (1967) claimed that the apparent scarcity of T. solium, at least in Africa, may be due to a misdiagnosis of the proglottides of T. suginata. For this and other reasons, it is worthwhile to present the important diagnostic features of T. saginata which will serve to differentiate it from T. solium. We have chosen four of the many works on this subject that cite the important diagnostic features of T. saginata. They are listed in Table I. Table I shows how different four authors’ criteria can be when considering the diagnosis of T. saginuta. Verster (1967) carefully points out those features that are unquestionably diagnostic of T. saginata. They are: (1) the presence or absence of an armed rostellum, (2) the number of ovarian lobes, and (3) the presence or absence of a vaginal sphincter. She completely discards the diagnostic value of the number of lateral uterine branches in the gravid proglottis. These differentiating features are important in doubtful cases. However, in most cases where the lateral uterine branches number more than 20-25 there is no need to complicate the routine diagnostic procedures by making permanent slides to look for the vaginal sphincter. Morphological abnormalities of T. saginata that have been observed for the past two centuries are presented in Clapham’s paper (1939). In a more recent summary of abnormal morphology of T. saginata, Burrows and Klink (1955) discussed abnormalities of the entire strobila (tri-radiation, tetra-radiation, penta-radiation, pigmentation and bifurcation) ; abnormalities of one or more proglottides (bifurcation, fenestration, fusion, variation in segmentation, supernumerary or intercalary segments); and variations in reproductive organs. Other more recent papers on morphological abnormalities of T. saginata are Pezenburg and Oleck (1955). Thlice and PBrei Moreira (1955), Starkoff (1956), Voge! (1961), Larrougy and Sardou (1963), Merdivenci (1964) and Kuimicki (1970). Owing to the introduction of new techniques, i.e. histochemistry and electron microscopy, knowledge of cestode structure and physiology has grown very rapidly and has been summarized in recent reviews by Read and Simmons (1963), von Brand (1 966) and Smyth (1 969). However, the physiology of adult T. saginata has not been as well investigated as other more readily available cestodes. For example, the ultrastructure of the tegument of nine different species of Cyclophyllidea has been examined but not that of T. saginata (see Srnyth 1969).

275

TAENIASIS AND CYSTICERCOSIS

TABLEI Specific morphological features of Taenia saginata that di.#er jkom Taenia solium

du Noyer and Baer Brumpt (1 928) (1949) ~

~

Entire body Length

Verster (1 967)

~~

4-8 ni

412 m

Maximal breadth 12-14 m m Approx. 2000 No. of proglot tides Scolex Diameter Shape Diameter of suckers Hooks Rostellum

Abuladze (1964)

1 ‘5-2 nim 1 ‘9-2 mni Quadrangular 0.7-0.8 mm Absent A bsen t Absent

Mature proglottides Musculature Calcareous bodies 300-400 Testes

Up to 10 m, exceptionally more 12-14 mni

-

1 5-2 inn1

Absenl

Well developed Very numerous 800-1 200 not confluent posterior to vitellarium

Terminal vesical Present Cirrus pouch

Absent

Ovary Vaginal sphincter Present Present Course of vagina Straight Irregular Genital atrium Alternate

2 lobes Present

Gravid proglottides Breadth to length 1 : 3 4 ratio No. of uterus 18-25 branch Way of leaving host

Absent Absent

15-30 18-32 Dichotomic Single and spontaneously

Does not extend to excretory vessels 2 lobes Present

Spontaneously

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Z B I G N I E W P A W L O W S K I AND M Y R O N G . S C H U L T Z

According to Lunisden et al. (1970) the tapeworm body surface can no longer be considered “an inert cuticle”. It has a major role in integrating the parasite’s physiological activities with the immediate environment. Very little is known about the chemical constitution of the tegument of T. saginata, but it is known to show alkaline phosphatase activity (Chowdhury, 1955). According to Arme and Read (1970) the tegument of T, saginata, like that of other tapeworms, is probably a digestive-absorptive structure showing some morphological and functional similarities to the luminal mucosal structures of vertebrates. Apart from the works of Logachev (1953a, b) and Chowdhury et al. (1956a) little attention has been paid to the mesenchymal cells of T. saginata. These studies may provide some interesting information about the differentiation of cells in the hind part of the “neck” region of T. saginata which would help to explain details in the growth of the parasite (Logachev, 1953b). Much information has arisen about the calcareous corpuscles in T. saginata due to the work of Chowdhury et al. (1956d, 1962) and von Brand (1960, 1965, 1967) but their function still remains a mystery. Possibly they act to buffer anaerobically produced acids (von Brand et al., 1960)or as a reservoir of phosphates (Brand, 1966). Very little is known about the uptake of tagged substances into the body of T. saginata. In comparison to Diphyllobothrium latum, T. saginata absorbs only a small amount of vitamin B-12 (Nyberg, 1958) and it does not absorb CoB0(Scudamose et al., 1961). The presence of cholinesterase and acetylcholine-like substances (Artemov and Lure, 1941; Schardein and Waitz, 1955; Pylko, 1956) suggests that they may be transmitter substances in the nervous system of the tapeworm. The presence of glucose-6-phosphate and glucose-6phosphate dehydrogenase suggests that in carbohydrate metabolism a pentose phosphate pathway is active besides the pathway of Embden-Meyerhof (Ley and Vercruysse, 1955). The finding of purinolytic enzymes is difficult to interpret (Read and Simmons, 1963): a search for some antigenic substances is another source of information about proteins (Machnicka-Roguska, 1965) and mucopolysaccharides which appear to be concentrated in the scolex and first part of the strobila of T. saginata (see Marzullo et al., 1957). The biochemical analysis of lipids resulted in the finding of saturated and unsaturated fatty acids (cmelik and Bartl, 1956), some lipid-like lecithins, inositol phosphatides (von Brand, 1966) and cholesterol and other sterol-like material (C‘melik and Bartl, 1956). Due to the fine studies of PrBv6t et al. (1952) the normal site of attachment of T. saginata was found to be 40-50 cm below the duodenojejunal flexure. Only three of 53 patients with T. saginata infection had the scolex situated further down in the jejunum. Their observations indicate that the tapeworm is by no means passive or resting, but is itself often moving against intestinaI peristaltic motion in the host. Most radiologists find the tapeworm body in the ileum, some even located it in the terminal ileum. However, PrCv6t et al. (1952) pointed out that only the hind part of the tapeworm body may reach the ileum and this is the part most easily shown by radiological examination. The observations of PrBvBt et al. have been confirmed by Hornbostel and Dorken (1952).

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277

The entire body of T. saginata, including the scolex, has been found in several unusual locations such as the appendix, gall-bladder, and adenoid tissue of the nasopharynx (Shakhsuvarli et a/., 1964). In the latter case a 14-year-old girl more or less regularly discharged T. saginata proglottides from her nose. This condition started several days after a bout of vomiting and a feeling of some foreign body in her nasopharynx; it lasted, with some remittent periods, for more than one year. Part of the tapeworm body was found in material taken out by adenectomy and gravid proglottides were found in the posterior part of the nasopharynx. Prior to surgery she expelled 14 proglottides during a 4-day period, providing an example of a most unusual adaptation of the adult tapeworm to a luminal region other than the intestinal tract. The idea that T.saginataoccurs only in single infections is no longer upheld. This topic was discussed by Andrews and Ogilvie (1944), Vogelsang and Fernandez (1943, Mazzotti et al. (1947), Vieira (1954), Pawlowski and Rydzewski (1958), Altmann and Bubis (I959), Donckaster and Donoso (1960), Lee e t a / . (1966), and Strikovsky (1970). Donckaster and Donoso (1960) summarized five authors’ observations of 2020 Taenia sp. infections and found that the percentage of multiple infections is below 1 %: P e r u 4 . 2 9 % (Castillo, 1958), United Kingdom--O.46% (Jopling and Woodruff, 1959), Poland-456 % (Pawlowski and Rydzewski, 1958), C h i l e 4 . 8 5 % (Donckaster and Donoso, 1960). However, in Mexico the percentage of multiple infections was 4.9 % (Mazzotti et al., 1947) and it is very high in some endemic foci in the southern republics of the U.S.S.R., e.g. in Azerbaidjan multiple infection reached 40 % and in Armenia 67 % with an extraordinary maximum of I50 tapeworms in one person (Podyapolskaya and Kapustin, 1958). Evidently, the “crowding effect” operates in multiple T. saginata infections. For instance, in one patient with 16 tapeworms the lengths of the strobila was 50-80 cm (Altmann and Bubis, 1959). At present, the factors that account for single infections with T. saginata are not known and general belief is that superinfection does not occur. However, Hornbostel (1959) described a case in which three adults of T. saginata developed after the sequential ingestion of three cysticerci, the second administered 1 week after the first and the third 2 months after the first. The coexistence of T. saginata with other intestinal parasites is a most interesting biological phenomena. T. saginata can coexist with T. solium (Vogelsang and Fernandez, 1945; Mazzotti et al., 1947; Donckaster and Donoso, 1960; Strikovsky, 1970) or Diphyllobothrium latum (O’Connor, 1944). T, saginata infection is often found in association with Giardia intestinalis infection (Junod, 1967; Batko and Kacka, 1969). The specific reasons why tapeworms can withstand digestion during their life in the small intestine is not understood but we know that the ability to withstand digestion is a general attribute of all living membranes. The time of development from the ingestion of a cysticercus to a fully grown tapeworm has been established by Penfold et al. (1937a) as 100 days, and by Sztrom (1938) as 91 days; however, in recent years with the use of modern taeniacides the regeneration time appears to be up to 4 months (Frolova

278

ZBIGNIEW PAWLOWSKI A N D MYRON G . SCHULTZ

1970). Proglottides are expelled irregularly. As many as 34 proglottides may be expelled in 1 day but the mean daily output was found to be 6.6 by Belyaev and Monisov (1967) and nine by Penfold and Penfold (1936b). In a patient with artificial anus as many as 40 proglottides were discharged daily (BonillaNaar, 1946). Most proglottides are passive; but they become motile most commonly between the hours of 1-8 p.m. The length of life of an adult T, saginata appears to be limited only by that of the host. Spontaneous cure may occur but this is exceptional. B.

EGG

During the last 10 years the eggs of T. saginata have attracted much more interest than any other stage of the parasite. The development of taeniidae eggs has been summarized by Rybicka (1966). An interesting phenomenon pointed out by Kamalova (1953), and Silverman (1954b) is the uneven maturation of ova within proglottides. According to Silverman (1954b) the gravid proglottides of T. saginata and T. pisifbrmis contain 50 % mature ova, 40 immature ova, and 10 % infertile ova. The mature ova are present only in the terminal 30-50 proglottides. Immature ova are present in distal as well as proximal gravid proglottides. Some immature ova can mature outside the host within 2 weeks, whereas others have failed to mature after 2 months. There are a considerable number of recent publications concerning the membranes of T. saginata ova (Silverman, 1954a; Chowdhury et al., 1955a, c; 1956b, c; Lee et al., 1959; Morseth, 1965; Slais, 1970a). According to Slais (1970a) each egg consists of an outer shell, chorionic membrane, thick and striated embryophore, basement embryophore membrane, and two oncospherol membranes. There are two anatomical features of T. saginata eggs that are of epidemiological interest. The remnants of yolk masses, which rest on the outer membrane clot the embryophores together and enable them to remain attached to the host skin for some time. Also, the complicated membrane structure of T. saginata embryophores may in some way explain their resistance to chemical and physical factors. Some authors (Noyer and Baer, 1928; Kamalova, 1953; Verster, 1967) believe that there are morphological differences between T. saginata and T. solium embryophores; the former are said to be more ovoid, the latter more spherical. In fact, the difference is about 1-2 pm, and can hardly be regarded as an essential one. Other authors (Maplestone, 1937; Miretski, 1948) believe that the embryophores of T. solium and T. saginata cannot be distinguished by shape or measurement. An interesting way of differentiating T. saginata and T. solium embryophores is by staining with Ziehl-Nelsen stain (Brygoo er al., 1959; Capron and Rose, 1962). The stain is positive in T. saginata embryophores and negative in those of T. solium: this selective staining phenomenon depends on acid-alcohol resistance of egg membranes as suggested by Dubos (1947). The number of eggs in a single proglottis was determined by Penfold (1937a), as about 80 OOO, and the mean daily output of eggs as approximately 720 000. The number of eggs in an active proglottis varies a great deal according to

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279

the patency of the uterine branches and the activity of the proglottis. Most eggs leave the proglottis through an opening called the proctostoma, which is developed by damage of the thysanus, a bundle of parallel uterine branches, with club-shaped endings that touch the anterior margin of the proglottis (Mazzotti, 1944b; Rijpstra et al., 1961). Rijpstra et al. (1961) observed that of 16 proglottides discharged in the feces only two were “fully” gravid (77 700 and 82 430 eggs); in five, the number was 1000-6000, and in nine, the number was only 20G800. According to the very precise studies of these authors there is an active egg laying mechanism effected by pressure exerted by large masses of ova in the uterus and later by muscular activity of the motile proglottides. They observed a single proglottis lay 15 490 eggs within a few minutes, then 48 070 eggs were deposited in a track 6.5 mm long during creeping, and 18 870 eggs were left in the dried proglottis. The number of eggs remaining in a proglottis is usually less than 500 (Rijpstra et al., 1961). The eggs can be released only after maceration of the proglottis (Gonnert et al., 1968). C.

ONCHOSPHERE

This stage of T. saginata has not often been invesligated. Some information about the morphology and development of the onchospheral stage was summarized by Voge (1967). Hatching involves two processes : digestion of the embryophore and activation of the hexacanth embryo. A 13h action by gastric juice, then a 45 min action by intestinal juice seems to be necessary to digest the embryophore (Silverman, 1954a). Peptic and then tryptic digestion in uitro will also cause a rapid disintegration of the embryophore. Hyaluronidase, peptic digestion alone, chymotrypsin, carboxypeptidase B, and tryptic digestion alone, have little effect on distintegration of the embryophore (Silverman, 1954a; Gonnert et al., 1967; Gonnert and Thomas, 1969). A stimulus to activate the onchosphere must penetrate through lipoid and scleroprotein membranes after the embryophore has disintegrated. Since 1922 (Isobe according to Silverman, 1954a) it has been known that bile is necessary to activate the tapeworm embryo but it is not yet known how bile works and how important bile is in creating selective host specificity (Smyth, 1969). Voge (1967) stated that the physiological factors that affect host specificity or susceptibility are very complex and not yet fully understood. The free, activated onchosphere penetrates the intestinal mucosa by means of hooks and penetration glands. Hooks appear to be used for the initial attachment to the mucosa. The secretion of penetration glands in some tapeworms other than T. saginata has been observed by Reid (1948). Silverman and Maneely (1955) found secreting glands in the onchospheral state of T. saginata which play some part in penetration by cytolysis of the mucosal cells. The cytolytic effect of the onchospheral stage of some Taenidae has been shown in the rabbit’s intestines by Heath (see Smyth, 1969). Smyth pointed out, that the secretion of onchospheral penetration glands may produce an immunological effect. Very little is known about the migration of the onchospheral stage of T. suginata to the final location in the intermediary host’s tissues. Intravenous 13

280

ZBIGNIEW PAWLOWSKI A N D M Y R O N G. SCHULTZ

administration of onchospheres to 6-day-old calves failed to produce cysticercosis. Onchospheres given subcutaneously or intramuscularly do not migrate but develop in the site of inoculation (Froyd and Round, 1960; Wikerhauser et al., 1970). D. CYSTICERCUS

There are two reviews that summarize recent knowledge on the postembryonic development of cestodes and the morphology of the cysticercus stage of T. saginata and T. solium (Voge, 1967; Slais, 1970a). In addition, there are a number of original papers on the morphology of the cysticercus (Holz and Petzenburg, 1957; Voge, 1963; Siddiqui, 1963; $lais 1966a, 1966b, 1970a, b). The structure of cysticerci appears to be more complicated than originally believed. According to Voge (1963) the external tissue of the T. saginata cysticercus consists of hairlike processes, a peripheral collagenous-fibrous layer, below which are a group of ovoid cells, muscle bundles, a duct system, flame cells, and fine fibres. The internal tissue fold consists of tegument, a peripheral fibrous layer, two muscle layers, peripheral cells, calcareous corpuscles, flame cells, a duct system embedded in a loose fibrous net, and a central band of muscles. Slais (1970a) pointed out that the histological structure of the bladder portion of the T. saginata cysticercus differs from that of the scolex portion. It is too early to say how far a knowledge of structure may influence knowledge of the functions of the cysticercus. Slais suggested a morphological analogy between the bladder wall of the cysticercus and the trophoblast of mammalian embryos. There are different opinions about the possibility of differentiating between the wall structure of T. solium and T. saginatu cysticerci. Voge (1963) said “in spite of minor apparent differences the structure of the two species is very similar and their specific differentiation cannot be guaranteed when the scolex is not available”. On the other hand, Slais (1970a) stated that “even if scoleces are not present, a differential diagnosis of C . cellulosae, C. bovis and Coenurus cerebralis can be made on the grounds of a detailed analysis of the histological structure of the bladder wall of the cysticercus”. We are reproducing Slais’s valuable table on the diagnostic characters of some cestode larvae which occur in man (Table 11). The development of T. saginata cysticerci has been studied by McIntosh and Miller (1960) in 34 infected steers. The first cysticerci visible by naked eye were found on day 11 and the size was about 0.13 x 0.1 mm, the surrounding connective tissue 3 x 2 mm. Three weeks after infection a cavity and immature scolex were found and at 5-6 weeks the scolex with suckers was fully developed. At 10 weeks an invaginated neck is visible. Cysticerci that are 10-12 weeks old are believed to be the youngest stage capable of infecting the definitive host. Throughout the experimental period of 55 weeks no absorption of dead cysts was observed. The detailed histological studies of Silverman and Hulland (1961) showed that the growth rate and development of T. saginuta cysticerci is variable and depends on the host response and the tissue invaded. Varying results by different authors working on the development, viability, and longevity of T. suginata is therefore understandable.

28 1

T A E N I A S I S A N D CYSTICERCOSIS

TABLEI1 Diagnostic characters of some cestode larvae which may be found in man (after slais (1970a)) Coenurus cellulosae -

C. bovis

C. cerebralis

______.____~_

One

Scolex :

____

~.

~~

One

...

+

Hooks :

____ 0

Several

Many

-~

+

+

Bladder : _

_

Echinococcus granulosus

.

. _

~

-

.

~~

~

Cuticle

Surface

Cuticle -

~~~

Ranging from 3-6 pm below 1 pm to 2.5 pm

Subcuticular

+

groups of muscles

~ _ ~ _ _ _ _ _ _

-

Cuticle

_ _ _ _ ~

Superficial hairlike cuticular extensions

~~

Stratified hyaloidine membrane -

1-2 pm

0

0

0

0

Make-up of wall

Wartlike processes

Rugae

Smooth and Smooth also rugose

Base of superficial protuberances

27-38 pm

50-70 pm

28-46 pm

0

Height of superficial protuberances

15-27 pm

23-27 pm

15-22 pm

0

A point of particular epidemiological interest is the question of the longevity of T. saginata cysticerci, which may not be uniform even in the same animal and depends on the tissue invaded (Van den Heever, 1967; Soulsby, 1963). In the liver, lung and heart some cysticerci degenerate as early as 20 days after infection (Soulsby, 1963). It is not unusual to find living and dead cysticerci in the same host (Penfold et al., 1937b; Friedrich, 1961; Dewhirst et al., 1963). Calves may differ from cattle in the maximal survival time of cysticerci (Koudela, 1967b). In Froyd’s experiment (1964b) cysticerci survived 21-30 months. Dewhirst et al. (1963) infected 4-6 month old steers with one million eggs and viable cysticerci were found on day 639. According to Urquhart and Brocklesby (1965) cysticerci survive for 21 months in lightly infected animals. Van den Heever and Tustin (1967) found viable cysticerci in an Afrikander crossbred cow 3 years after an experimental infection. In an experiment done by Leikina et al. (1964) in 14 young calves, all cysticerci were degenerated

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within 11 months. In Penfold’s classic experiment (1937b) carried out in 30 oxen, no cysticerci survived 9 months. Peel (1953) and Leikina et aZ. (1964) suggest that the variable longevity of cysticerci may be referable to different strains of T. saginata in Africa and elsewhere in the world. Discussing this point, Smyth (1969) stated that the differences might be attributable to various strains of cattle rather than to differences of tapeworms. It is an oversimplification to expect that this complex phenomenon is dependent only on differing parasite or host strains. Unlike the adult worm, which is weakly immunogenic, the larval stage of T. saginata produces an active immunological response when it invades the intermediary host, as Weinmann (1966, 1970) has shown. According to Froyd and Round’s (1960) suggestions and Gemmell’s (1967) observations of tapeworm occurring in sheep there are at least two immune responses against larval taeniid tapeworms in the intermediary host. One, which is probably species specific, occurs in the small intestine and is directed against penetrating oncospheres. The other interferes with growing larval forms in the muscle. It is responsible for strong, life-long resistance of cattle to reinfection, approaching a level of absolute immunity. Also, the life span of primary cysticerci can be shortened by secondary exposure (Leikina et al.,1964). It is generally assumed that T. saginata cysticercosis does not alter the state of health of the intermediary host. The only exceptions are Dewhirst et aZ.’s observation (1960) of a small decline in the haemoglobin level of infected animals, the finding of Evranova and Mosina (1966) that glycogen synthesis in the liver and skeletal muscles of infected calves is depressed, and Taylor’s report (1958) of a death of an onyx-antelope because of pericarditis and coronary vessel embolization secondary to intensive cysticercosis. The physiological factors that stimulate evagination of the cysticercus once it is ingested by the definitive host were investigated by Hornbostel (1959). The most important factors are the action of gastric and intestinal juice and the speed of intestinal passage. The stimulation of cysticercus evagination by surface-active agents was observed by Campbell and Richardson (1960) and by Campbell (1963). V. CLINICAL ASPECTS OF TAENIASIS (T. saginata) In a paper titled “Troublesome Tapeworms” in Lancet (i, 1953) Asher, who worked at the Central Middlesex Hospital in London, wrote, “Tapeworms in this country are often considered as a joke, and regarded as more suitable for examination questions than for consideration of their clinical importance”. Even in the United Kingdom, with its high standards of meat inspection, Taeniasaginatainfection can no longer be considered risible for it is encountered by clinicians there as well as in most other parts of the world. A.

SYMPTOMATOLOGY

Taenia saginata like all other human helminths may provoke symptoms or may cause an unrecognized infection. However, an asymptomatic T. suginata infection may, within a short time, change into a life-threatening

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condition when a proglottis is vomited and aspirated or when a proglottis enters the appendix. Short of these dramatic events, the difference between a symptomatic and an asymptomatic T.saginata infection can be very obscure. There are no pathognomonic signs and even the most experienced clinician cannot say for sure that a particular sign or symptom is due to a tapeworm infection. The literature on the symptomatology of T. saginata infection is widely scattered ;we have summarized eight of the more important reports. The most frequent symptom of T. suginata infection is the discharge of proglottides (98.3 %). This is a distinctive symptom that can hardly go unnoticed by the patient, although it is well tolerated by some individuals, For a period of 5-10 min the patient feels a sensation in the rectum and then the passing of the proglottis through the anus associated with a crawling sensation in the perianal area (Belyaev and Monisov, 1967). In addition to the passage of proglottides approximately three of every four patients infected with T. saginata experience one or more other symptoms. From eight studies including observations of 31 10 patients the symptoms are arranged in decreasing order of frequency (Penfold et al., 1937a; Swartzwelder, 1939; Mazzotti et al., 1947; Dzicciolowski and Kuimicki, 1953; d’Alessandro Bacigalupo, 1956a; Hornbostel, 1959; Beier, 1963; Pawlowski and Chwirot, 1970). Symptom Abdominal pain Nausea Weakness Loss of weight Increased appetite Headache Constipation Dizziness Diarrhea Pruritis ani Excitation

Per cent 35.6 34.4 24.8 21 .o 17.0 15.5 9.4 8.2 5.9 4.5 3.4

The above mentioned symptoms may vary widely in character. Abdominal pains are usually vaguely localized in the midline of the epigastrium or umbilical region. They vary in intensity from dull, aching, gnawing, burning to intensive colic-like sharp pain. This pain is probably due to a distension or spasm of the intestinal wall in reaction to the movement of the tapeworm. This is true visceral pain. A characteristic feature of this abdominal pain is its prompt relief by taking some food. The same feature applies to the symptom of nausea. Both abdominal pain and nausea are usually more intensive in the morning. Nausea is the third most common symptom of T.saginata infection. Nausea might be explained as the result of extension or spasm of the duodenum or jejunum as is also true of abdominal pain. An alternate explanation is that it is

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caused by gastric hyposecretion, a phenomenon that commonly occurs in Taenia saginata infection. A common symptom of T. saginata infection is alteration of appetite (Beier, 1963; Pawlowski and Chwirot, 1970; Hennemann and D’Heureuse, 1958). Some patients have an increase of appetite, others have a decreased appetite, and still others have an alternating increase and decrease in their appetite. The hunger frequently associated with tapeworm infection is difficult to explain. It might be due to hypoglycemia. Hornbostel(l959) noted a low blood glucose value of 64 mg % during an “attack” of hunger in a patient with T. suginata infection. On the other hand, Abuladze (1964) said that hunger “attacks” might be the result of irritation of the ileocaecal valve by tapeworm proglottides. A characteristic feature of increased appetite associated with T. saginata infection is its satisfaction by an inordinately small amount of food. This increased appetite rarely results in weight gain, a phenomenon that has been observed in only 2 % of 2200 patients by Pawlowski and Chwirot (1970). Decreased appetite is as frequently observed as increased appetite (12 and 13% respectively; Pawlowski and Chwirot, 1970). It seems to be more strongly influenced by the psyche of patients than by any other factor. Loss of weight was observed in 15% of 2200 patients (Pawlowski and Chwirot in press). Weight loss correlates well with the symptom of decreased appetite, but weight loss can also occur in individuals with no change in appetite or even in those patients who report that they eat more. The tendency to lose weight as a result of tapeworm infection is not a dependable phenomenon and is surely a poor rationale for allowing tapeworm infections to survive in obese patients in order to correct their body weight. Vomiting is an infrequent symptom of T. saginata infection. It occurs most commonly in children and in emotionally labile individuals. At times proglottides are vomited. Penfold (1937a) reported two such cases, one in an individual undergoing anaesthesia and another in a patient with pneumonia. Alterations in bowel movements due to T. suginata infection are usually temporary in nature. Some individuals experience both constipation and diarrhea, whereas others experience one or the other of these symptoms, which may be due to irritation of the intestinal wall by the parasite. Some cases of severe diarrhea due to T. saginata infection have been reported (Loeper, 1931 ; Hurst and Robb-Smith, 1942; Kaufman, 1961). The mechanism of this diarrhea is obscure; it may occur with or without an associated eosinophilic response. Although the most common symptom of T. suginata infection is the passage of tapeworm proglottides only a minority of patients developed pruritis ani. Some authors believe that pruritis ani has an allergic background (Burckhardt, 1945). Mazzotti et al. (1947) and d’Alessandro Bacigalupo (1956a) found that pruritis ani was a common symptom, but many of their patients were infected with other parasites at the time of examination. Urticaria is seldom reported in patients with T. saginata infection (da Franqa, 1952; Wigand and Warnecke, 1953; d’Alessandro Bacigalupo, 1956a; Link and Cassorla, 1964). Other skin disorders such as prurigo

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nodularis and allergic skin pruritis are even less commonly reported than urticaria (Rollier, 1956; da FranCa, 1958). There are a few reports of syncope associated with T. saginata infection, usually in young patients. Although the mechanism is obscure two possible explanations are vaso-vagal disorders or hypoglycemia. Another uncommon symptom is the sensation of a lump in the throat (globus hystericus). This phenomenon is ill-understood (Lockhart, 1961). Tt was reported by Pawlowski and Chwirot (1970) in 3% of 2200 patients, most commonly in middle-aged women. In addition to specific characteristics of T. saginata infection in individual patients there are group characteristics that are dependent on age, sex, and co-existing conditions. Infected children more frequently manifest change of appetite, abdominal pain, epileptic-like seizures and syncopal episodes (Shah and Joshi, 1965; Karnaukhov, 1967). In 30 children who were 5-14 years of age Kluska et al. (1969) reported that the mean gain in weight within 8 weeks after successful treatment of T. saginata infection was 1-2kg. Signs of infection in infants (Perez, 1955; Mossmer, 1955; Link and Cassorla, 1964) are usually quite pronounced and consist of vomiting, diarrhea, fever, weight loss, and irritability. Elderly patients usually manifest fewer symptoms than young or middle-aged patients except for hypersalivation. There is a slightly higher frequency of symptoms in women than in men (79.3% vs. 74.9%)). Change in appetite, loss of weight, nausea, vomiting, constipation, and headache are more pronouned in women. Two symptoms, globus hystericus and urticaria, do not occur in men. Pawlowski and Chwirot (1 970), found a slight correlation between asymptomatic infections and duration of infection. 21 % of patients discharging proglottides for no more than 2 weeks were asymptomatic whereas 31 % of patients discharging proglottides for more than 3 years were asymptomatic. The symptomatology of T. saginata infection depends not only on the host but also on certain parasite factors. In unusual cases of multiple infection or bizarre location, the parasite can cause various acute conditions or complications. These complications are as follows: intestinal obstruction (Christopherson and Izzedin, 1918, according to Brumpt, 1949; Ferracani, 1941); perforation (one case reported by Stieda, 1900, and by Nauwerck, 1900, Kaan, 1941); perineal abscess (Gombarros, 1943); hepatic abscess (Negre, 1957); cholecystitis (Arnell, 1949; Talice and Ptrei-Moreira, 1954; Ardao et al., 1956; Logan, 1960; Adamiya and Gogotishvili, 1968); and appendicitis (Letulle and Lagane, 1908; Boveri, 1939; Richard, 1943; Niiio, 1944; Clark, 1946: Deschiens and Bablet, 1948; Upton, 1950; Berry and Burrows, 1955). These complications, of course, have their own unique symptomatology. B.

CLINICAL PATHOLOGY

Rees’ review (1967) gives an admittedly general account of the pathogensis of adult cestodes in man and animals. The paper by Hornbostel (1959) is more medically oriented, however, but the portion dealing with pathogensis is limited. The pathogenesis of T. suginuta infection will be reviewed here in terms of its traumatic, irritative, toxic, allergic, local and systemic actions.

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Whenever the parasite changes its natural environment it may cause traumatic or irritative action in its new location. For example, during vomiting T. saginata proglottides may be aspirated and cause obstruction of the respiratory tract (Shahin, 1932; Bruning, 1933; Verdiev, 1958); the worm may enter the middle ear through the eustachian tube (Shahin, 1932); it may localize and grow in the adenoid tissue of the nasopharynx (Shakhsuvarli et al., 1964) and it has been found in the uterine cavity (Schacher and Hajj, 1970). Cases have been reported of the migration of T.saginata proglottides into the common bile duct (Logan, 1960; Benedict, 1926). A perforation of the intestinal tract by T. saginata was reported 72 years ago (Stieda, 1900; and Nauwerck, 1900). A more common complication is appendicitis. Until 1950 there were 55 reported cases of appendicitis caused by Taenia sp. (Upton, 1950). Berry and Burrows (1955) cited cases of appendicitis caused by various cestodes; amongst them were 42 cases of T. saginata, ten of T. solium, 25 of Taenia sp., one of Hymenolepsis nana, and five of Echinococcus granulosis. As many as four proglottides have been found in the appendix but usually only one or two are present and sometimes only abundant eggs are found. In three cases the scolex was recovered. The tapeworm may elicit a wide range of reactions in the appendix from very slight inflammatory reaction to a chronic, subacute, or acute appendicitis of catarrhal, phlegmatic, or follicular type (Berry and Burrows, 1955). The traumatic and irritative action of T. saginata on the intestinal wall is ill-understood. One report describes a piece of intestinal mucosa in the tapeworm sucker (see Hornbostel, 1959); however, Hornbostel (1959) has not confirmed this in the examination of 16 scoleces. Intestinal mucosa taken by biopsy in patients with T. saginata infection show slight subacute inflammatory reaction in many cases (Gasparov et al., 1962; Kubicki and Karlinska, 1967). The symptomatology of T. saginata infection such as abdominal pain and nausea suggest that there is an irritative action of the tapeworm which may result in a distension or spasm of the intestine. A study of intestinal absorption in T. saginata infection by El-Mawla et al. (1966) in 20 Egyptians failed to demonstrate any abnormalities in d-xylose absorption or fecal fat excretion but Ciauri and Mastrandrea (1960) observed lower fat absorption in patients with T. saginata infection. The suggestion of Hennemann and d’Heureuse (1958) that hypochromic anemia due to diminished iron absorption occurs in children with T. saginata infection has not been proved. Separate cases of agranulocytosis (Thiodet et al., 1953) and hyperchromic anemia (Tronchetti and Cartei, 1948) have been reported. Moderate eosinophilia has been reported in from 5 % (d’Alessandro Bacigalupo, 1956a) to 46% (Adonajto and Bonczak, 1961) of patients. A higher level of eosinophilia, 20-30 %, is observed sporadically. Lapierre (1953) reported an unusual case of increasing eosinophilia with a maximum of 53 ”/, 14-2 months before discharge of T. saginata proglottides and 36 76 at the time of the appearance of proglottides. Talyzin (1949) in a self-experimentproduced eosinophilia up to 16.5 % by injections of extracts of T. saginata. The evidence for an allergic action of the parasite includes not only the finding of eosinophilia but also the symptoms of urticaria, pruritis and asthma (Burckhardt, 1945; Blamoutier, 1952; da Franca, 1952; 1958; Rollier, 1956).

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There is little evidence that tapeworms induce a bacterial infection in their intestinal environment; however, when proglottides migrate to unusual locations, i.e. appendix, biliary ducts, they may carry intestinal bacteria with them. On the other hand, it is known that cestodes possess some bactericidal properties (Rees, 1967). There is an interesting association between T. saginata infection and lowered gastric secretion (Hornbostel, 1959; Todorov, 1966; Chodera et al., 1967). C.

DIAGNOSIS

Since there is no characteristic clinical picture of T. saginata infection the diagnosis must be based on laboratory findings. Fecal tests, anal swabs, serological and skin tests determine whether a tapeworm infection exists; however, the exact species diagnosis of T. saginata infection is made by the finding and examination of the scolex or those proglottides that show typical species characteristics. Questioning of the patient about discharge of proglottides is an important part of the diagnosis (Podyapolskaya, 1942; Artikov and Safarov, 1964). The value of the medical history is dependent on the degree of cooperation between the physician and the patient. Monisov (1966) showed how frequently a false answer would be obtained by failure to ask detailed questions. The demonstration of T. saginata proglottides and brief questioning of a group of 842 people elicited 54 positive replies including 26 (48.2 %) false positive answers which were not confirmed by laboratory examination. People who gave a false positive answer had either a T. saginata infection in the past or an infection with Ascaris or Enterobius or other inappropriate reasons. In another group of 920 people more detailed questions about the number, size, color, activity of actually discharged proglottides, together with their demonstration, gave much better results. Fifty patients answered affirmatively and only three (6 %) proved to be false positive replies. In another group of 986 people to whom Ascaris and Enterobius adult €orms were demonstrated, in addition to the detailed questioning, 116 gave positive replies and four (3.9%) proved to be false positive. The inadequacy of fecal examination for detecting T. saginata infection has been known for many years (Kouri and Basnuevo, 1933; Mazzotti, 1944a, b). Nevertheless, in the past decade some authors have pointed out that thick fecal smears are valuable. Rijpstra et al. (1961) arranged the following coprological methods in decreasing order of efficiency: thick smear according to Hein; concentration technique according to Teleman ; thin fecal smears repeated six times; and flotation according to Faust-Bijlmer. Miiller (1968) stated that the efficacy of the Hein thick smear technique for a single examination was 77 %, for two examinatioiis 91 %, and for three examinations 97 %, in 35 cases. He indicated that the Fiilleborn flotation technique was 20% effective, the direct thin smear 57 %, the Teleman concentration method 66 %, and simple sedimentation 71 % effective, Scraping of the anal region as an effective means of diagnosing T. saginata infection was reported as early as 1927 by Oleinikov and 1929 by Bogojavlenski and Lewitski. Anal swabs have become more and more popular as a means of

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detecting T. saginata infections (Bacigalupo, 1940). Podyapolskaya ( I 942) reported that scrapings of the wall of the lower part of the rectum gave 76 % positive results whereas the Fulleborn technique gave only 57 % positive results. Mazzotti (1944a, b) found the Graham method superior to fecal examination (85 % versus 73 %). The usefulness of anal swabs has been confirmed by many authors; Pipkin and Rizk (1948), Garaguso (1954), Doby et al. (1957), Hsieh (1960), Roman (1961), Petru and VojtEchovskB (1963a). Roman (1961) preferred the Graham Scotch tape method over the Schiiffner-Swellengrebel (1943) pestle method which was advocated by Petru and VojtEchovskB (1963a). Eggs found on perianal skin are more likely to be T. saginata. T. saginata eggs may be distinguished from T. solium eggs by special stains (Capron and Rose, 1962). The differential species diagnosis of mature proglottides requires the use of fixation and staining methods. Gravid proglottides are usually prepared by simply pressing them between two glass slides and examining the number of lateral uterine branches in strong transmitted light. This method has been accepted everywhere for many years. In those cases where the diagnosis is uncertain or where there are unusual features of the tapeworm body, it would be desirable to send a portion of the tapeworm pressed between two glass slides and fixed in 10% formalin to a helminthologist. Staining of the specimen and examination according to Verster's criteria should differentiate T. saginata from T. solium in these doubtful cases. Examination of the scolex is a classic means of differentiating T. saginata from T. solium. There is, however, a case report (Hussey 1963) in which the scolex was unhooked and the proglottides resembled T. solium having only 4-12 paired uterine branches. Serological tests are more useful for the detection of human cysticercosis than for adult tapeworm infection. Although the precipitin reaction with T. saginata antigen has long been used (Langer, 1905), the complement fixation test since 1909 (Weinberg, 1908) and the skin hypersensitivity test since 1927 (Ramsdell, 1928), their value in the diagnosis of intestinal infection has been in doubt (Deschiens and Renaudet, 1941; Podyapolskaya and Kamalova, 1942; Gaehtgens, 1943). Nevertheless, in the past decade considerable progress has been made in the serological diagnosis of T. saginata infection (Dobrowolska, 1950; Roguska and Zwierz, l964,1966a, b; Machnicka-Roguska, 1965; Sokolovskaya, 1968, 1969; Zapart et al., 1969). Machnicka-Roguska and Zwierz (1964, 1966a, b), using the hemagglutination test of Middlebrook-Dubos, obtained 60 % positive results in 125 patients infected with T. saginata. Best results were obtained with a purified polysaccharide fraction of the antigen. 15% of sera still gave a positive reaction 5-19 months after successful treatment. Sokolovskaya (1968) used a latex agglutination test with an homogenate of T. saginata body as an antigen. She obtained 96.3 % positive results with sera of 285 T.saginata patients, but she also obtained 15.6"/o positive results with sera from 96 patients who formerly had T. saginata infection 2 months to 2 years in the past and 3.6% positive results with sera of 55 individuals who had no prior exposure and were free from infection. The latex agglutination test with a protein fraction homogenate from the body of T. saginata gave even better results. In patients

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infected with T. saginata 99.2% were positive, but positive results were obtained in patients who formerly had T. saginata infection (20.8%) and in uninfected persons (10.9 %). Zapart (1968) using a complement fixation test with an antigen fractionated according to Melcher’s method found 73.7 % of patients positive and when he used a ring precipitation test found 82.5 % of patients positive. Three months after successful treatment 12.5% reactions were found using the CFT and 10% using the ring precipitation test. The efficacy of intradermal tests using similar antigens was much lower (Zapart et al., 1969). Attempts to develop more specific serological tests have been made by fractionating antigens (MachnickaRoguska, 1965), by preparing antigens from different parts of the tapeworm’s body (Mukhin, 1969), by using a cysticercus antigen (Cramer and Dewhirst, 1965) by finding a special substance (C-type) in antigens (Biguet et al., 1965), and by purification of antigens by physico-chemical methods (T. solium) Morris et al., 1968). In sporadic cases where T. saginata infection is suspected but the patient is not passing proglottides, a radiological examination of the intestinal tract may be helpful. In distinction to Ascaris lumbricoides which gives a sharply outlined radiolucent shadow and linear traces of barium in the worm’s intestine, the tapeworm body, despite its great length, usually remains concealed from the radiologist. The front part of the body is so extremely narrow as to preclude its demonstration by radiological technique (Monroe and Norton, 1962). The broader hind parts of the tapeworm body give a ribbon-like radiolucent defect but in most cases this is visible only in the ileum (Hamilton, 1946; PrCv6t et al., 1952; Benassi, 1954; Monroe and Norton, 1962; Fetterman, 1965). D. TREATMENT

In the past decade, two valuable reviews of the chemotherapy of cestode infections have appeared ;both are from the Wellcome Research Laboratories. The review by Standen (1963) deals mainly with experimental chemotherapy, and Keeling’s review (1968) deals with advances in chemotherapy in the intervening 5 years. With these valuable reviews of the chemotherapy of cestode infections as a background we have concentrated our attention on the clinical aspects of the therapy of T. saginata infections. Despite the advances that have been madeit is still true that precise knowledge of the mode of action of even well-known taeniacides is scanty (Hatton, 1970). Experimental chemotherapy has been based exclusively on cestodes other than T. saginata (Steward, 1955; Pawlowski, 1964b; Mattila and Takki, 1966; Cavier and Notteghem, 1968; Saz and Lescure, 1968; Scheibel et al., 1968; Saz, 1970) and does not satisfactorily explain the mode of action of the common taeniacides. Some observations have been made of the T. saginata body expelled after treatment (Mustakallio and Saikkonen, 1954; Makhmudova, 1958; Izmailova-Guseinova, 1959; Rusak, 1964; Gonnert, 1968) but they do not provide conclusive information. Individual clinical reports concerning efficacy and safety of taeniacides are also inadequate in most cases. This is true because methods are not uniform, the number of patients observed is usually inadequate and follow-up examination is frequently

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deficient. Despite these shortcomings, there is a wealth of publications about drugs for the treatment of tapeworm infection and cumulatively they provide much useful information on which to base the choice of drug. The choice of drug depends on two paramount factors-efficacy and safety. The common taeniacides are at least 80 % effective (Dufek and Kalivoda, 1969; Petrosyan et al., 1969; Pawlowski, 1970a); therefore, any new compound must be at least this effectivein order to replace the ones that are in current use. Because taeniasis (T. saginata) is not a life-threatening infection, taeniacides that are toxic should not be used. I t is generally true that the newer taeniacides are less toxic than the older taeniacides and this is the main reason that they have won a place in the therapeutic armamentarium. Keeling (1968) suggested that the following drugs be used for T. saginata infection in man: niclosamide, dichlorphen, aspidium oleoresin, and mepacrine hydrochloride. There is no doubt in our minds that niclosamide is now the drug of choice, but in the place of dichlorophen and aspidium oleoresin we would substitute tin compounds. Other taeniacides that will be discussed briefly are paromomycin, bithionol, pumpkin seed extract, and hypertonic magnesium sulfate solution. Niclosamide (Yomesan) [N-/2’-chloro-4’-nitrophenyl/-5-chlorosalicylamide] is undoubtedly the drug of choice for T. saginata infection of man at the present time. During the past 3 years the following authors reported on their experience with Yomesan: Gherman (1968) 93.6% cure rate; Ahkami and Hadjian (1969) 95.5% cure rate; Khalil (1969) 84.6% cure rate; Pawlowski (1970a) 90% cure rate; Perera et al. (1970) 97.0% cure rate. The efficacy of Yomesan based on 3 3 publications from 1960 to 1966 which included 766 cases of T. saginarainfection was 88.5 % (Schultz, 1968). There are some reports which give a much lower efficacy of niclosamide: Donckaster et al. (1961) 53 % cure rate; Nitzulescu et al, (1962) 58%; Karnaukhov and Stromskaya (1966) 7% cure rate; Krotov et al. (1968) 74% cure rate. Pawlowski (1970a) stated that there were some batches of drug (Yomesan produced in 1962) or products (Vermitin produced in 1964) that had much lower efficacy. This is also true of Phenasal produced in 1962 to 1963 (Karnaukhov and Stromskaya, 1966). It seems that the particle size of these compounds was outside the range of 2-6 pm which is necessary for the drug to be active (Krotov et al., 1968). The following are some suggestions for treating patients with T. saginata infections with Yomesan. No pretreatment is necessary except for those patients with constipation who should receive a purge or enema one day before treatment. The drug is taken on an empty stomach or just after a light meal. The tablets should be crushed or chewed and followed by a small amount of water. It is best to keep the patient on a light diet during the day of treatment. As a rule, it is not necessary to use any purgative drug, but it might be helpful for a patient when he does not tolerate the treatment well. A saline purge would help to eliminate the worm more easily, avoid some adverse reactions and perhaps relieve the anxiety in waiting for the worm to be passed. The dosage of Yomesan has remained the same for the past 10 years. For adults it is four tablets which are each 0.5 g, thus the total dose is 2 g. Although reported side effects of niclosamide are mostly minor ones, syncope has been

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reported (Beier, 1963, 1966; Pawlowski and Chwirot, 1970). There are no strict contraindications to this drug. Although Yomesan is widely used throughout the world, knowledge of its mode of action is quite limited. Mattila and Takki (1966) did pointout that niclosamide uncouples the oxidative phosphorylation in rat liver mitochondria as do other salicylate compounds. Scheibel et al. (1968) indicated that niclosamide inhibits the phosphorylation mechanism of mitochondria. This may account for the anthelmintic action of niclosamide. Gonnert (1968) summarized knowledge of Yomesan’s mode of action as follows: “The drug inhibits the uptake of oxygen and glucose. The decomposition of glycogen is increased, whereas the activity of the lactodehydrogenase is inhibited. Taeniacidal activity is concentrated upon the upper part of the strobila, whereas the microscopic and histological investigation of gravid segments T. saginata and T. solium proves that no significant differences exist in the state of maceration between proglottides of untreated and Yomesan treated cases.” According to Gonnert (1 968) “the investigations with radioactive labeled and unlabeled compound prove that 25-30 % of orally administered compound are excreted with the urine (the greater part being metabolites) and the remainder is excreted with the feces. No accumulation of compound or its metabolites take place in the whole body or in single organs. The compound is rapidly and quantitatively excreted.” These undoubtedly are the reasons for the good tolerance and safety of niclosamide. Standen (1963) briefly mentioned inorganic tin compounds and Keeling (1968) did not, although he devoted a chapter to organic tin compounds, which are promising veterinary taeniacides, especially for poultry. Tin as a taeniacide was used by Paracelsus (Cavier, 1953) and was still in usein Europe in the early nineteenth century (Hirte, 1951, 1957); however, it fell into disuse, possibly due to toxic reactions from lead, antimony, and arsenic contamination (Deschiens et al., 19.56). The efficacy of metallic tin for taeniasis was rediscovered in 1943by Poey-Noquez (Cavier, 1953)and confirmed by some French authors in the 1940s (LeGac, 1947) and Basnuevo (1947, 1948). In the early 1950s some German investigators introduced tin compounds into broader medical use (Hirte, 1951; Kuhls, 1953). The efficacy of tin compounds varies according to the particular drug used. Taenifuge is 72% effective and Stannotaen 98% (Chodera et al., 1970). Storage of the drug for longer than 2-3 years will result in a loss of efficacy (Pawlowski, 1959,1970a). The efficacy of various tin compounds based on the treatment of 868 outpatients with T. saginata infection was 88.8 % (Dufek and Kalivoda, 1969; Pawlowski, 1970). The drug is taken two or three times a day for a period of 5 days irrespective of the fact that the strobila may be evacuated early in the treatment course. At the third day and at the end of treatment, a saline purgative is advocated. The exact dosage depends on the particular product used sincethe ingredients in each product differ. For example, one tablet of Cestodin contains 580mgmetallic tin, 150 mg tin oxide, and 32.5 mg tin chloride (Hirte, 1957). The total number of tablets vary from Cestodin-15, Stannotaen-10, Taenifuge-90. The drug should be well pulverized since it probably acts by covering the cuticle with a

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thin layer of active tin particles and renders the strobila susceptible to digestion (Harant et al., 1957). The partially digested distal part of the strobila is usually expelled on the second or third day of treatment. Sometimes no part of the strobila is visible. Pure metallic tin and tin oxide particles seem to be virtually non-toxic but the irritative action of tin chloride is probably responsible for the side effects observed in patients treated with tin compounds (Dadlez et al., 1954). The percentage of side effects noted by several authors was 36.6% (Dufek and Kalivoda, 1969), 46.6 % in outpatients (Pawlowski and Chwirot, 1970) and 5-37 % in inpatients depending on which drug was used (Chodera et al., 1970). The side effects are mostly gastrointestinal disorders and rarely fever and syncope (Pawlowski and Chwirot, 1970). Malformation of a newborn child (agenesis of the right hand with aplasia of the radius and ulna) was reported by Notter et al. (1963) after treatment of a 28-year-old woman in the tenth week of pregnancy with stannous oxide. The contraindications for this group of drugs are pregnancy, gastrointestinal disorders that produce increased drug absorption and severe liver and renal disease (Bojanowicz and Pietrowa, 1968). Despite the risk of intolerance and overdosage Acridine derivatives are still in common use. At the present time they should be reserved for refractory cases of T. saginata infection (Keeling, 1968). Acridine derivatives other than quinacrine (Mepacrine, Atabrine), i.e. Acrichin (Krotov and Rusak, 1964) and Acranil (Beier, 1963; Pawlowski, 1970a), are now in use. The usual dose of one of these drugs for an adult is 0-6-0.8 g. The most effective way of giving the treatment is by intraduodenal tube followed by a saline purgative 1 h later. Treatment by mouth and treatment of infected children have become less popular because of vomiting, with consequent loss of a portion of the dose. Although the acridine derivatives have been used since 1939 (Neghme, 1951) their mode of action is as yet not understood (Standen, 1963). According to Mattila and Takki (1966) Atabrine uncouples the oxidative phosphorylation of Taenia taeniaformis but this fact does not fully explain its anthelmintic activity. Beier (1965a) observed that these drugs intensify both the motility of the tapeworm and the peristaltic movements of the host intestine resulting in the expulsion of the parasite. It is highly probable that the anthelmintic action is based on interference with the sucking action of the tapeworm. The affinity of the acridine derivatives for the suckers of the tapeworm has been confirmed by fluorescent microscope observations (Mustakallio and Saikkonen, 1954; Saikkonen and Mustakallio, 1963). It is possible that these relax as a result of some shrinking of mucous membranes producing a partial vacuum between the scolex and the intestinal wall (Lepes, 1956, cit. Beier, 1965a). Following the observations of Wagner in Ethiopia in 1960, Ulivelli (1968) presented a more detailed study on the eficacy of paromomycin for T. saginata infection. He treated 208 cases of T. saginata infection, 32 cases of T. solium infection, and eight cases of Hymenolepis nana infection with an overall efficacy of 98-100%. Doses of 20-30 mg/kg/day divided into four parts for four consecutive days were given. No preliminary preparation or subsequent treatment was necessary. The treatment was well tolerated even in children.

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Gastrointestinal side effects occurred infrequently. The early observations of Ulivelli have been confirmed by Salem and Al-Allaf (1967), Garin et al. (1970) and Botero (1970). In a group of patients with amebiasis treated by Salem and Al-Allaf (1967) 47 patients also had T. saginata infection. All patients were cured with a daily dose of 50 mg/kg of paromomycin administered for 5 days. The strobila was evacuated on the second or third day of treatment. Reexamination of patients was done between the fourth and eighth month. Eight of the 47 patients complained of abdominal discomfort or diarrhea during treatment. Botero (1970) used two dose schedules of paromomycin: 40 mg/kg/day for 5 days in 15 patients and a single dose of 75 mg/kg (max. 4 g) in another 15 patients. One treatment failure occurred in each group. Side effects were less common when a single dose was given. Garin et al. (1970) tested four oligosaccharide antibiotics in patients infected with T. saginata. Paromomycin was the most effective and cured all of 20 patients. The mode of action of paromomycin is unknown. Cavier and Notteghem (1968) were unable to correlate their clinical results with the results of their experiments in animals. Garin et a/. (1970) suggested that the action against T. saginata may be similar to the drug’s action against bacteria, i.e. by changing ultrastructure of the basic membrane. In tapeworms this would make the parasite susceptible to the host’s digestive mechanisms. These reports make paromomycin a promising alternative treatment for T. saginata infection. Dichlorophen, 5 :5’-dichloro-2:2’-dehydroxydiphenylmethane,has been the subject of some favorable early reports (Lassance et al., 1957; Adams and Seaton, 1959; Schneider, 1959; Shafei, 1959; Seaton, 1960; Guilhon and Graber, 1960) but this has not been verified in practice (Turner, 1963; Alterio, 1968; Dufek and Kalivoda, 1969; Pawlowski, 1970a; Chodera er al., 1970). For some time this drug has been used in the U.S.S.R. for mass treatment but more recently intolerance to standard doses (6-9 g in adults) have relegated dichlorophen to use only as an alternative drug (Krotov et al., 1968). Following the suggestion of Krotov et al. (1968) dichlorophen has been used in a very low dose (1.0 g) together with niclosamide (2.0 g) under the name of Dichlosal. In the U.S.S.R. Dichlosal is believed to be the most effective drug for the mass treatment of T. saginata infection. Cure rates of 100 % have been reported by Suvorov (1966), 98-100% by Monisov and Niezbekov (1966) and 97.2% by Petrosyan et al. (1969). The report of Doroshchak and Kite1 (1968) concerning very serious side effects in two hospitalized patients raises doubt whether the mixture of these drugs is safe. Bithionol (2,2’-thiobis 9/4,6-dichlorophenol) for the treatment of taeniasis was introduced by Yokogawa et al. (1962), Miyakoda et al. (1963) and Nagahana et al. (1966). Kaliszewicz et al. (1967) on the basis of treating four patients stated that bithionol is a promising drug. Dufek and Kalivoda (1969) administered bithionol to 20 patients and cured 18 of them. They advocated 40-55 mg/kg usually in two doses separated by a 1 h interval and followed in 2 h by a saline purgative. The maximum total dose used was 3.4 g. Gastroenteric side effects were more frequent than those that occur with tin compounds or niclosamide. At the present time these limited observations are insufficient

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to warrant the introduction of bithionol for broader use as an alternative drug for the treatment of T. saginata infection. The discovery of pumpkin seeds as a taeniacide has been reviewed by Rybaltovsky (1966). The bulk of pumpkin seeds limits their use; however, refined extracts of pumpkin seeds have overcome this problem. Junod (1964) showed that a concentrated extract of pumpkin seeds is an effective taeniacide. Twenty-two of 26 patients were cured when the drug was given orally and 100% of 54 patients were cured when the drug was given by gastric or duodenal intubation. Bailinger and Sequin (1966) found two extracts from the seed coat of Cucurbitapepo active against T.saginata. There is much more to be done in the evaluation of the active substances of pumpkin seeds to determine their safety and eficacy. Extracts of male fern or related synthetic chemicals are still in use for the treatment of T. saginata infections (Kovalev, 1960; Dodion, 1962; Shah and Joshi, 1965; Ditzel and Schwartz, 1967; Alterio, 1968; Petrosyan et al., 1969), but the high toxicity of these drugs should exclude them from further use. Reports from Europe have cited unexpected fatalities from the use of these regimens. For example, Hanel (1950) summarized the experience with 22 000 treatments for tapeworm infection with extract of male fern in Germany. Permanent blindness occurred in four patients and at least 20patients were temporarily blinded. From other statistics cited by Hiinel (1950) among 121 reported cases of intoxication from extract of male fern, 47 resulted in permanent blindness and 17 were fatal. Since male fern and related compounds are relatively effectual taeniacides, work is still going on to develop further knowledge of their chemistry and toxicology (Oelkers and Ohnesorge, 1954; Heikinheimo, 1963; Nosslin, 1963; Hargreaves, 1966; Takki, 1967; Takki et al., 1968). As early as 1932 de Rivas suggested the use of hypertonic solutions given by duodenal tube for the treatment of tapeworm infection. In more recent years some authors rekindled this idea and used hypertonic solutions of magnesium sulfate as a taenifuge (Furst, 1951; Rosen and Kiefer, 1958; Donckaster et al., 1960). Fatal reactions from intraduodenal doses in excess of 60 g due to magnesium intoxication have been reported from Germany (Rosler, 1952) consequently this treatment is no longer advisable. It is evident that considerable progress has been made in the treatment of T. saginata infection. This point is illustrated by data from Poland during the past 15 years (Kalawski and Pawlowski, 1970) which show that the average TABLE I11 Treatment of 1750patients with T.saginata infection Poznah, Poland, 1953-1968* Years

Mean treatments required for cure

1953-56 1957-60 1961-65 1966-68

1.9 1.4 1.4 1.1

Drugs used

Pumpkin seeds, mepacrine, male fern

Tin compounds, pumpkin seeds, mepacrine Tin compounds, niclosamide Niclosamide, tin compounds

* Kalawski and Pawlowski (1970).

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number of treatments required for each patient dropped from 1.9 to 1.1 (Table 111). There is still need for further progress as there is no taeniacide that may be given without medical supervision or for mass chemotherapy. VI. EPIDEMIOLOGY AND EPIZOOTIOLOGY A.

TRANSMISSION BETWEEN MAN AND ANIMALS

T. saginata infection is a true zoonosis according to Sprent (1969b) who states that it is an “anthropozoonotic helminthiasis” in which “man is the usual or essential definitive host and disseminator of infection; vertebrate animals act as intermediate hosts”. This is in agreement with Garnham’s 1958 definition of “euzoonoses” in which man is an essential link in the life-history of the parasite. The fact that man is the only definitive host of T. saginata simplifies the epidemiology of this infection. On the other hand, the varied relationships between man, his animals and his environment makes for complex factors affecting the transmission of this parasite. In the most general terms transmission from man to animals may be either direct or indirect. Direct transmission is uncommon. It can occur when hands contaminated with T. saginata eggs feed and handle calves (Urquhart, 1961;Goulart et al., 1966). Much more common is the indirect method of transmission. This can occur through contamination of cattle feed, soil, sewage, spread by birds or flies, etc. In general, when there is close contact between an infected human and susceptible animals heavy infections result, whereas, when eggs are widely distributed in the environment most infections are light. A variety of ecologic factors influence the viability of eggs during indirect transmission. Knowledge of these factors is rather limited but some general statements can be made. Taeniid eggs will withstand the action of unfavorable external factors quite well. According to Laws (1967) and Mackie and Parnell (1967) taeniid eggs survive the action of most chemical disinfectants. They also resist a variety of physical factors. Laws (1968) did a variety of in vitro experiments with Echinococcus granulosus, Taenia pisiforrnis, T. ovis and T. hydatigena eggs. He concluded that desiccation is the dominant factor affecting the survival of taeniid eggs under natural conditions. At a temperature of 38°C desiccation is accelerated and leads to a rapid breakdown of the eggs. He demonstrated that desiccated eggs carry an electrostatic charge which may in part account for their adhesive properties. Under natural conditions it is generally accepted that moisture is the most important factor controlling the survival of T. saginata eggs (Penfold et al., 1937b; Jepsen and Roth, 1952; Silverman, 1956b; Suvorov, 1965). Silverman (1956b) pointed out that eggs do not survive longer than 14 days in the absence of surface moisture. Lucker and Douvres (1960), stated that small numbers of eggs survived in hay stored for 22 days but none survived in hay stored for 10 weeks. At a temperature of 4-5”C, T. suginata eggs can survive for at least 168 days (Froyd, 1962). Silverman (1956b) pointed out that some could be “activated” after 335 days. He stated that eggs could be activated when kept in saline at

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room temperature for 60 days and are inactivated by at least 10 min exposure at 59”C, but not by 4 h exposure at 45°C. According to Suvorov (1965) the optimum temperature for survival of T.saginata eggs is -4°C at which temperature they survive for 62-64 days. When the temperature is decreased to - 30°C the survival time is decreased to 16-19 days. These data confirm the observations of Lucker (1960) that at -4.5”C T. saginata eggs survived in significant numbers for 12 days but few kept at this temperature for 76 days were able to infect cattle. Jepsen and Roth (1952) found that T. suginata eggs survived at least 16 days when stored at 18°C in a dish filled with liquid manure. The survival time in liquid manure in an underground cistern was up to 71 days. In Denmark during the months of June-July 1947 the eggs survived for 58 days in grass and for 159 days during the months of February-July 1948. According to Penfold et al. (1937b) T. saginata eggs survive in Australia long enough to permit a protracted contamination of fields, water and crops. According to Duthy and van Someren (1948) the eggs can survive for about 1 year in the highlands of Kenya. A considerable number of experiments on the survival of T. suginata eggs in natural field conditions were performed by various authors in the U.S.S.R. They are summarized by Suvorov (1965). These studies, which were done in continental climates, show that under conditions where there are great differences between summer and winter temperatures, T. saginuta eggs will withstand cold winter conditions much better than they will withstand hot summer conditions. This is probably due to the desiccation that occurs during the summertime. It is important to note that under most conditions eggs survive better when they are free than when they are within proglottides (Suvorov, 1965). Sewage is important means of spreading T. saginata infection between human populations and cattle (Profi, 1934; Sinnecker, 1955; Liebmann, 1963). The increase of urban populations with consequent increased usage of water and overloading of sewage works lead to the breakdown of formerly reliable sewage treatment systems and the spread of T. saginata infection (Silverman and Griffiths, 1955b). Furthermore, the increased use of detergents interferes with the sedimentation, putrefaction and oxidation processes and enables a greater portion of parasite eggs to survive these processes (Silverman and Griffiths, 1955b). The passage of T. suginata ova through a variety of sewage installations has been observed by Vasilkova (1944); Newton et al. (1949); Jepsen and Roth (1952); Wang and Dunlop (1954); Silverman (1955b); Silverman and Griffiths (1955b); Kabler (1959); Menschel (1964); and Amirov and Salamov (1967). Greenberg and Dean (1958) summarized the situation by stating that “conventional sewage treatment is inadequate to completely eliminate Tuenia sp. eggs”. They state that treatment to eliminate T. suginata eggs adequately from sewage effluent can only be done by very slow sand filtration as described by Newton et ul. (1949) or by microstraining as described by Silverman and Griffiths (1955b). Sewage sludge has to be heattreated or left to dry for at least a year in order to be safe. Silverman and Guiver (1960) suggested that storage for 20 days at 35°C or retention for 1-5 days under mesophilic anaerobic conditions suffices to inactivate T.

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saginata eggs. Liebman (1963) stated that in order to assure the destruction of Taenia sp. eggs the time of digestion of the sludge in unheated septic tanks must be at least 3 months whereas in heated septic tanks it must be at least 2 months. When sewage is improperly treated, the final effluent is an important source of infection. This has been noted in a variety of circumstances. For example, in the United Kingdom in 1955 Silverman stated that 50% of cattle drank sewage-polluted water; in the United States in 1956 Miller commented that changing farming and irrigation practices increased the likelihood that livestock would be exposed to contaminated sewage. Epizootics of T. saginata infection attributed to sewage have also been noted by Schultz etal. (1969) in the U.S.A., and Sinnecker (1955) and Denecke (1966) in Germany. Even when sewage is channeled to the sea or rivers the natural purification process is slow. This phenomenon was noted by Vasilkova (1944) in the Moskva river in 1940 to 1941 where there were 4500 helminth eggs per m3 of sewage and 3 % of these eggs were Taenia sp. eggs. 2-5 km below the main sewage outlet there were 263 eggs per m3 of river water and 32 km below there were still 91 helminths eggs per m3. Also Amirov and Salamov (1967) found Taenia and Ascaris eggs in sea water and on the sea shore near Baku (Azerbaijan, U.S.S.R.) where the sewage outlet was 250 m out into the sea. These facts have led to the opinion that sewage effluent should be very carefully used in agriculture if indeed it should be used at all (Miller, 1956; Greenberg and Dean, 1958). Liebman (1963) even suggested an inverse correlation between the degree of sewage purification and the spread of cysticercosis in some areas in middle Europe. Gotzsche (1951), seeking an explanation for the uniform dissemination of T. saginata infection in Denmark, suggested that T. saginata eggs were transmitted by gulls and possibly other birds fed on proglottides. He failed to find viable Taenia eggs in herring-gull’s feces but he was able to infect some calves by feeding them the droppings of gulls. Gulls were also implicated by Silverman and Griffith (1955), Guildal (1956), Crewe (1967) and Crewe and Crewe (1969). Silverman and Griffiths (1955b) were also able to pass Taenia eggs through the intestinal tract of young chickens (before they developed a crop). They failed to demonstrate transmission through pigeons. Guildal (1956) found Taenia sp. eggs in the intestinal tract of six of 96 blackheaded gulls (Larus ridibundus), one of 34 common gulls (L. canus) none of 15 herring-gulls (L. argentatus) or lesser blackheaded gull (L.fuscus). In one Larus ridibundus he found as many as 28 000 eggs in the intestinal tract. The transmission of helminth eggs by flies was discussed early in the twentieth century by Nicolle (191 1) and Shircore (1916). In Mombasa, where 29%of natives had Taenia sp. eggs in their feces, Shircore (1916) observed that eight of 270 houseflies (species not described) had helminth eggs in their intestinal tracts. In Uzbek, U.S.S.R., Sycevskaja and Petrova (1958) observed that flies transmitted Taenia saginata eggs. Round (1961) examined filth flies, Chrysomyia albicans, Ch. chloropyga and Sarcophaga sp. in Kenya. He stated that T. saginata eggs can be passed by these flies for periods up to 11 days after ingestion. However, the majority are passed within 3 days and during

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that time the eggs are viable, He concluded that filth flies in Kenya may play an important role in the transmission of Taenia sp. eggs. Nadzhafov (1967) found that seven out of eight species of flies experimentally infected with T. saginata had ova in their feces and regurgitations. The greatest number of eggs were found in Lucilia sericata and L. caesar. According to Nadzhafov (1967) in Azerbaijan, U.S.S.R., an area endemic for T. saginata infection, 4.8% of 4372 flies belonging to 20 species had Taenia sp. eggs on their external surfaces or in their intestinal tracts. The highest infection rates (8.8 %) were observed in Sarcophagidae. The degree of contamination of flies depended substantially on the extent of fecal contamination of the soil in four village territories that were investigated. On the basis of these experiments and observations Nadzhafov (1967) stated that synanthropic flies definitely play a role in the dissemination of T. saginata infection. Other species of insects, e.g. Periplaneta americana (Macfie, 1922) and BIatta germanica (Round, 1961) are also able to disseminate T. saginata eggs but their role in natural conditions seems to be of little importance because they do not feed on feces. The role of other coprophagic invertebrates as disseminators of T. saginata eggs has not been investigated yet despite some suggestions that they may be effective in spreading the infection. The finding of mature cysticerci in calves a few weeks old would indicate that transmission by the intrauterine route is possible (Kolbe, 1937; Canhan, 1946; McManus, 1960; Urquhart, 1961,1966). This phenomenon seems to be rather frequent in endemic areas. For example, McManus (1960) stated that 3.07% of 14855 calves in Kenya in 1957 to 1958 were condemned for T. saginata cysticercosis. The majority of calves (80%) were 2-21 days old. The author was able to find mature T. saginata cysticerci in 28 calves that were 2-10 days old. Prenatal transmission of T. saginata infection is of epidemiological importance because thorough sanitation may not suffice to eradicate the disease when this alternate means of transmission occurs. Also Soulsby’s experiments (1963) show that very young animals are immunologically unresponsive to T. saginata cysticercosisinfection. Thus, prenatal infection may confer little immunity to exogenous infection. At the present time it is too early to evaluate the importance of intrauterine transmission in maintaining the disease in enzootic areas. Infection of man occurs by the simple act of eating raw or partially cooked infected beef. However, the factors that favor the eating of raw meat are very complex. They are discussed in their ecological, economic and ethnological contexts. Ecological factors are more important in the transmission of T. saginata from man to animals than they are in the transmission from animals to man. Since the cysticercus has no contact with the external environment, ecological factors act only on the host. Man is the only known definitive host of T. saginata; the only geographical limitation of this infection is the regions of the world inhabited by man. Ecological factors do have an important role in determining the cattle breeding areas of the world. Also, wild animal reservoirs of this infection have been found in Taiwan (Huang, 1967), and the northern zones of the U.S.S.R. ;their role in Africa is not clear (Nelson et al,, 1965).

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From the economic standpoint, world beef production has doubled in the 20 years following World War 11.In many areas there is greater contact between cattle and man both as a breeder as well as a consumer. In the Poznan region of Poland a direct correlation has been found between increased beef consumption and incidence of T. saginata infection in the urban population (Pawlowski, 1970b; Kalawski and Pawlowski, 1970). Unfortunately similar data are not available from other countries. Animal-to-man transmission of T. saginata infection occurs both in developed and developing countries but the socioeconomic reasons for this are ironically opposite-infection is prevalent in developed countries because they are rich, and in developing countries because they are poor ! Transmission between animal and man also depends on ethnological factors, i.e. human habits, behavior, religion and beliefs. They influence the type of food man consumes and the manner of its preparation. Some practices are based on hundreds of years of tradition. Some are conducive to the transmission of T. saginata and it is doubtful that they can be changed rapidly. In the Transcaucasus and Buriat regions of the U.S.S.R. slices of meat cooked on spits such as “shashlik” or semi-raw meat used as stuffing for regional dishes are responsible for transmitting T. suginata (Abasov, 1957; Kovalev, 1965; Abdullaev, 1968). In Egypt, Turkey and Middle East countries, a beef dish known as “basterma” or kebab-like dishes are suspected (Nagaty, 1946). In East and Central Africa pieces of beef briefly roasted in an open fire are an important source of infection (Carmichael, 1952). In Thailand a raw beef dish, “larb” is responsible for group infections (Chularerk et al., 1967). In areas such as Lebanon taeniasis is caused by adulteration of raw mutton dishes with beef which is cheaper (Schwabe, 1963). The reasons why raw mutton meat, steak tartar, and a variety of semi-raw beef products are so popular in western European countries is not well understood (Pawlowski, 1970b) but they are responsible for the increase of T. saginata infections in this region. The chance of being infected by tasting meat during cooking is probably exaggerated because the amount of meat taken in this manner is very small compared to the meat taken as a dish. It is worthwhile to point out Hornbostel’s suggestion (1954) that T. saginata cysticerci easily attach themselves to the hands and can be readily transmitted to the mouth; it is doubtful that this fact has epidemiologic importance. Studies of the social and ethnological aspects of taeniasis are only beginning; they should be included in future epidemiological investigation. Studies in the Poznah region of Poland have shown that they are very important in evaluating the epidemiological situation in this area (Kalawski and Pawlowski, 1970; Pawlowski, 1970b). B.

EPIDEMIOLOGICAL AND EPIZOOTIOLOGICAL DATA

Epidemiological data on the prevalence of human taeniasis (T. saginatu) are grossly defective. Frequently the data that do exist are inadequate because laboratory procedures are not standardized, or only some part of a population is examined, e.g. children, hospital patients, or else the number of people examined is too small to give an objective picture of the true prevalence.

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Moreover, much of the data is dispersed in time, covering the last 20 years. The exceptions are some mass surveys in the U.S.S.R. and Poland. Knowledge of the incidence of taeniasis is also scanty. Compulsory notification of new cases is the exception rather than the rule for most countries in the world. Even if notification were introduced in some countries it would not cover all the diagnosed or treated cases. Therefore, it is possible only to point out some figures from the world literature which may not be representative of the particular region or country and to provide bibliographies on the world-wide distribution of T. saginata cysticercosisand taeniasis. Data on T. saginata cysticercosis originate from meat inspection reports. Meat inspection is carried out in many parts of the world but not in a uniform comparable way, Therefore, the data can be discussed only in a general manner. Data in FAO/WHO/OIE Animal Year Book (1970) shows where cysticercosis is found frequently, rarely, not at all, or where no data exists. The most recent papers that deal with the incidence of T.saginata cysticercosis are Merle (1958), Urquhart (1961), and Froyd (1965b). The following discussion deals with the incidence and prevalence of T. saginata taeniasis and cysticercosis in various parts of the world. The most detailed data come from theU.S.S.R. and they were summarized by Prokopenko (1968). Based on stool examination of 14.2 million people in 1950, the mean prevalence of human T. saginata infection was 0.6 %. The respective data for the years 1960 and 1966 are 46.4 and 65.8 million examined and prevalences of 0.3 % and 0.075 %. The sharp decrease in prevalence was due to extensive mass examination followed by mass treatment. For example, in 1960more than 97 000 cases were treated and in 1966 more than 120 000 cases were treated. The prevalence was not equal in the different republics of the U.S.S.R. The endemic foci are in Caucasian area (southern Dagestan, western Azerbaijan, northern Armenian, eastern Georgian) and in the south-central Asian republics (Uzbek, Kirghiz and Kazak). The highest prevalences were in Dagestan20.4% (Kovalev, 1960), in Azerbaijan-45-2% (Abasov, 1957) and 29.1 % (Nadzhafov, 1966), in Armenia-7.7 % (Avakyan, 1961) and 20 % (Martikyan, 1963), in Uzbek-43.5 % (Magdiev, 1966, 1968), in Kazak-7.9 % (Ghenis, 1968). The incidence of cysticercosis for the whole of the U.S.S.R. was about 1 % in the 1960s. In some areas of Armenia it was as high as 20.7 % (Avakyan, 1961). In contrast to data on human infection the animal data are very scanty. In Poland the prevalence of taeniasis in 1954 to 1956 was 259/100 000 based on stool examination of more than 100 000 adults (Zembrzuski, 1965). The incidence in 1965 was six per 100 000 and in 1967 it was nine per 100 000 (Adonajlo et al., 1969). The corresponding figures for T. saginata cysticercosis were 0.56% and 0.58% in the years 1965 and 1967 (Adonajlo et al., 1969). In the city of Poznan the prevalence in 1967 was 40.1 per 100 000 for men and 63.3 per 100 000 for women (Kalawski and Pawlowski, 1970). In Czechoslovakia in 1964 the incidence of T.saginata cysticercosis was 1.385% in the Czech region and 0.312% in Slovakia (Koudela, 1965b). The incidence of cysticercosis of cattle in Hungary was 0.213% (Takics et al., 1967) and in Bulgaria it was 0.07416% in the years 1937 to 1942

TAENIASIS AND CYSTICERCOSIS

30 1

(Pavlov, 1944) and 0.62% in 1960 to 1964 (Petkov and Jodorov, 1970). In East Germany T. saginata cysticercosis was found in 1946 in 0.49% of cattle, in 1952 in 2.2% (Kupey, 1954), in 1963 in 2.01 %, and in 1964 in 2.63 % of examined cattle (Hiepe et al., 1967). Cysticercosis was as high as 7.0% in 1962 at Halle (Ockert, 1965) and 7.32% in 1966 at Frankfurt (Oder) (Mielke, 1969). According to Raschke (1957) the percentage of cysticercosis found in Germany was 0.32% in 1904, 0.37% in 1910, 0.16% in 1920, 0.33% in 1930, and 0.30 % in 1940. In West Germany it was 0.38 % in 1951 and 2.06 % in 1964. The incidence of human 7'.saginata infection is calculated at 0.024 in Frankfurt (Oder) (Mielke, 1969). In Yugoslavia, in the areas of Bosnia, Kosmet, Sandzak, and Montenegro more than 15% of the inhabitants were carriers of T. saginata and more than 30% of cattle were infected with cysticercosis (Lepes, 1954). In other parts of Yugoslavia the incidence of cysticercosis was 10% or more, e.g. 24.64% in Pljevla (Nenadic, 1958), and approximately 10% in Bosnia and Hercegovina (Grujic, 1960). In the United Kingdom T. saginata was seldom found in man and cattle before World War 11. However, after the war many authors observed a sharp increase in the incidence of T. saginata cysticercosis (LeRoux, 1949; Marsden, 1950; Priestly, 1950; Griffiths, 1950; Seiler and Norval, 1950). Silverman in 1955a reported an incidence between 0.21 % and 0.58 % from 200 slaughterhouses but he suggested that the real percentage is between 0.81 % and 3.47 %. Logan (1967) gave the official percentages of 2.3% and 2.8% for Ireland in 1961 and 1963, but pointed out that the percentage may differ in various abattoirs from 0.1 % to 10%. In Holland, Belgium, Sweden, Denmark, the incidence of T. saginata cysticercosis is about 1 % (Tarnaala, 1941 ; Grigoire et al., 1956; van Keulen, 1959; van Gils, 1963; Honer, 1963; Enequist, 1965; de Vries, 1968). These data indicate that in many parts of Europe the prevalence of human T. saginata infection does not exceed 0.5 % except for some endemic foci in U.S.S.R. and Yugoslavia. This is far below the prevalence noted before the introduction of meat inspection in Europe when, for example, 5 % of the population was infected in Germany in 1855 (cited by Beier, 1963). Also, the prevalence of T. saginata cysticercosis is below 1 %, but in some areas it may exceed 5 % (Blackpool, 1948; Belfast, 1948; Genoa, 1953; Pljevla, 1958; Frankfurt (Oder), 1962; Halle, 1962; Berlin, 1965). Despite improvements brought about by meat inspection T. saginata taeniasis and cysticercosis appears to be increasing in Europe as shown by an analysis of 85 publications in the post-World War 11 period (Pawlowski, 1971). The following figures from European slaughterhouses illustrate this point: Prague 0.34 % (1945) to 1.6% (1955) and 3.1 % (1964); Berlin approx. 1 % (1945-1959) to 5.5 % (1965); and Poznan 0.5 % (1955) to 2-3% (1962). Cysticercosis and taeniasis have decreased, however, in the U.S.S.R. and Bulgaria (Petkov and Todorov, 1970) where mass control measures have been introduced. In the United States comprehensive data on T. saginata cysticercosis and taeniasis has only recently become available (Schultz et al., 1970). Prior to

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1959, only sporadic reports of cysticercosis of cattle were available; in 1912 the incidence was 0*14%,in 1930 it was 0*37%,and in 1942 it was 0.6%. Since 1959 annual data from the U.S. Department of Agriculture has shown that 0.04-0.08 % of cattle slaughtered in federally inspected abattoirs have cysticercosis. The rate for cattle slaughtered in California is 20 times greater than for cattle slaughtered in the remainder of the United States. A review of 1.8 million stool examinations from 43 states has shown that 23/100 OOO are positive for Taenia sp.; since other evidence indicates that T. solium is rare in the United States, virtually all of these identifications are believed to be T. saginata. Thus, transmission of T. saginata, albeit at a low level, occurs even in a highly developed country-the United States. In the latter part of the nineteenth century T. saginata infections were common in Abyssinia, Sudan, Algeria and Senegal (Hoeppli, 1969). Now, with more intensive grazing of cattle in the African continent the incidence is quite high in comparison to other parts of the world. Prevalances of human taeniasis above 10% have been reported from Kenya (Froyd, 1965a; McKinnon, 1957), the Congo (van Grunderbeeck and Penson, 1954), and South Africa (Elsdon-Dew, 1964). No infections, however, have been found in Congo pygmies (Price et al., 1963) and there is a low prevalence in children living in forest regions of Cameroun (Doby et al., 1957) and in coastal tribes in Kenya (Froyd, 1965a). An incidence of T. saginata cysticercosis greater than 10 % has been reported from Sudan (Eisa et al., 1962), Eritrea (Coceani, 1949), Kenya (Ginsberg, 1954, 1955; Ginsberg et al., 1956; Froyd, 1960, 1965a), Rhodesia (Rhodesia report, 1969), Uganda (Mitchell, 1967), Urundi (Marsboom et al., 1960; Biche and Thienpont, 1959), Chad (Graber and Thome, 1966), Congo (Versyck and Jacob, 1958; Urquhart, 1961) and Portuguese Guinea (de Oliveira Lecuona, 1956). Urquhart (1961) added to this list Bechuanaland, Cameroun, Ethiopia, French Guinea, Madagascar, Nigeria, Oubangui, Sierra Leone and Tanganyika, and Froyd (1965b) added Libya. An incidence of cysticercosis less than 10% has been reported from Egypt (Abdou, 1959; El-Afifi et al., 1961), and the Republic of South Africa (Canhan, 1946; Verster 1966; van den Heever, 1969). The conclusion is inescapable that cattle breeding Africa is full of T. saginata. There is very limited and outdated information on taeniasis and cysticercosis in the world’s two largest populations-China (Wu, 1939) and India (Mukerji and Bhaduri, 1944). Froyd (1965a) stated that the incidence of T. saginata cysticercosis in India was 1.4% and reports by Peatt (1950) and Shah and Joshi, (1965) show that T. saginata taeniasis and cysticercosis are common in some parts of the country. India is better known as the source of T. solium cysticercosis (Dixon and Lipscomb, 1961). In Asia the endemic foci of T. saginata are in the Near Eastern countries (Bottom, 1945; Thornton, 1957; Interdept. Committee, Lebanon, 1962; Froyd, 1965a; Witenberg, 1968). There are also foci in Thailand (Interdept. Committee, Thailand, 1962; Chularerk et al., 1967); Burma (Tu and Hkun-Saw-Lwin, 1968); Taiwan (Hsieh, 1960; Huang, 1967); Mongolia (Froyd, 1965b); South Korea (Lee et al., 1966; Seo et al., 1969); and Japan (Morishita et al., 1964).

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There is little information available from Australia except for Penfold’s (1936a) remark that T. saginata is very common in people of Syrian origin. According to Fewster (1967) cysticercosis was suspected in 0.21 % of one million slaughtered cattle and proved in 57% of suspected cases. Shortridge (1966) reported T. suginata infection in cattle in New Zealand. Most of the information on T. suginata taeniasis and cysticercosis in South America comes from Brazil. Pessoa (1967) stated that the prevalence of T. saginutu in Sao Paolo is 1-1.5 %. Elsewhere in Brazil, DeFreitas (1964) cited a prevalence of 1.6 %, Paoliello (1965) 2.0 % and Dias (1967) 0.37 % of children. Cysticercosis has been reported in approximately 1 % of Brazilian cattle (Ribeiro, 1949; Pardi et al., 1952; Costa and Brant, 1964; Brant et al., 1965). suginatu) in other Central and South American The prevalence of taeniasis (7‘. countries are: Cuba-0.16 % (Alvaro-Diaz, 1967), Panama Canal Zone0.16 % (Cosgrove, 1960), Guatemala-1.72 % (Acha and Aguilar, 1964), Ecuador--O.7% (Lopez, 1969), Chile-1.6 % (Delard et al., 1958) and Argentina-062 % (Niiio, 1964). Since these data are so fragmentary it is difficult to compare them with Stoll’s assessment in 1947 that 39 million of the world‘s population was infected with T. saginata. One can, however, state that T. suginata taeniasis and cysticercosisare cosmopolitan in distribution ;that they have become more prevalent in many areas of the world where comparative data exist; that since 1947 the world’s population has increased by approximately 50 % and the cattle population has increased by about 100%, so that it seems safe to conclude that more people are infected with T. suginatu now than the 39 million estimated by Stoll in 1947. C.

LOSSES DUE TO TAENIASIS AND CYSTICERCOSIS

T. suginatu infection causes two types of losses-the intangible losses due to the medical complications caused by the adult tapeworm and the tangible economic losses to agricultural societiesdue to cysticercosis. The medical costs of T. saginatu taeniasis are difficult to establish. The infection is rarely fatal unless the tapeworm body is in an unusual location. Symptomatology is quite varied and probably causes some lowering of productivity in infected populations. There is no information concerning the debilitating effects of taeniasis in a population living on a protein-deficient diet, but it is probably not a serious problem becauseinfected peopleare usually meat eaters. In Poland more than 20% of patients with T. saginata were treated in hospitals (Adonajlo and Gancarz, 1967) and the proportion seems to be the same in other countries. The cost of treating patients has been estimated to be between 1/20 (Mielke, 1969) and 1/13 (Logan, 1967) of the annual loss due to cysticercosis. There is considerable information about the financial losses due to T. saginata cysticercosis. In West Germany it has been said to be as high as 5 million DM (Friedrich, 1961); in East Germany 121 000 DM (Mielke, 1969); in Yugoslavia 6 million dinars (Winterhalter, 1965) and 87 million old dinars (Diinleski et al., 1963); in Belgium 25 million Belgian francs (Granville et al.,

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1958); in the United Kingdom 2100 000-500 000 (Silverman and Griffiths, 1955a); in Ireland €100000 (Logan, 1967), in Kenya €500000 (Mann, personal communication, 1971); and in the United States about $500 000 is lost each year by the condemnation and freezing of infected carcasses. These figures are impressive but they cannot be generalized to other regions. A better way is to estimate the average loss per animal and multiply it by the number infected for each particular region. According to Logan (1967) the mean loss for an infected animal is €10 (approximately $25). Silverman and Griffiths (1955a) counted the decreased value of meat, loss on weight (about 279, cost of additional handling, refrigeration and transport and an estimated mean loss as 230 ($75). According to Costa and Brant (1964) an infected animal is worth about one-third less than an uninfected animal. In the U.S. the cost of freezing infected carcasses is approximately $75 (condemned carcasses, of which there are few, represent a loss of approximately $300). Thus, a reasonable estimate of the losses due to cysticercosis is $25 per animal in developing countries and $75 per animal in industrialized countries. Apart from the direct losses there are other important consequences of cysticercosis. This is especially true in East Africa where the development of a profitable beef industry is inhibited by the high prevalence of cysticercosis. VII. PREVENTION A. MEAT INSPECTION

The history of meat inspection has recently been described by Dolman (1957) and Schwabe (1969). Meat inspection for cysticercosis has been in existence for 70 years and it is the foremost public health measure for the prevention of T. saginata transmission. However, meat inspection alone, particularly as it is now practised, cannot be expected to eradicate T. saginata infection (Thieulin et al., 1963). At the present time the important issues in the inspection of beef carcasses for cysticercosisare the question of the localization of cysticerci, the thoroughness of the inspection, and the use of new diagnostic techniques. Knowledge of the localization of cysticerci in beef carcasses is essential for proper meat inspection. There have been numerous papers dealing with this subject; nevertheless, there are still many opinions about “predilection sites” and the subject is controversial. Some authors report that the heart is the organ most frequently involved (LiGvre, 1933; Penfold et al., 1938; Silverman, 1956a; Varges, 1957; Marsboom et al., 1960; El-Afifi et al., 1963; Plaschke and Kramm, 1966; Dewhirst et al., 1967; Fewster, 1967); other authors believe that the examination of masseters, introduced by Hertwig in 1889 (cit. Desprts and Ruosch, 1961), is the most effective means of detection (Lazzaro, 1961; Koudela, 1967a), and still others believe the shoulder muscle incision, advocated by Viljoen in 1937 is most effective (El-Afifi et al., 1963; Cironeau and Popovici, 1968). There is probably no “predilection site” which would be acceptable for all cattle. There may be differences due to geographic area, breed of cattle, age, and activity of muscle groups (Kearney, 1970). For example, cysticerci tend to settle in the deeper muscles of the body,

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which have a higher level of metabolic activity and a dense branching distribution of blood vessels. The choice of muscles examined by meat inspectors should be based on studies done within each country or region. Another aspect of meat inspection is its efficacy. This is discussed by Marsboom et al. (1960), El-Afifi et al. (1963), Urquhart (1966), Koudela (1967a), Takics et al. (1967) and Dewhirst et al. (1967). They found that examination of the masseters, heart, tongue and shoulder muscles was important but routine and found 27.7%-68*9% of all inspection of these organs is insufficient for detecting light infections. According to Ginzel(l961) routine meat inspection can be improved by 2 %; Varges (1957) said from 1.5% to 14-2%, Kleibel (1961) from 2 to 8.6%, Desprts and Ruosch (1961) from 1.05 % to 5.3 %, Takbcs et al. (1967) from 0.2 to 0.7%. The improvement was brought about by better lighting, better timing of processing, and better training and rewarding of inspectorspsychological factors that are as important as physical factors. Luminescence under ultraviolet light of cysticerci was first described by Derrier (1927) (cit. after Marazza and Persiani, 1960) and first put to practical use by Koller (1943) (cit. Gibson, 1969). The efficacy of meat inspection with ultraviolet light was studied by Brandes (1958), Lerche and Elmossalami (1958), Marazza and Persiani (1960, 1961a, 1961b), van Gils (1963), Pirkl (1964), Franssen (1 964), and Koudela (1966a). These authors agree that ultraviolet light increases the probability of finding cysticerci but they disagree about the practical value of this technique. There are two aspects of treatment of infected meat: first to decide to what degree the meat is infected, and second, to determine the disposition of the meat. The regulations dealing with “measIy” beef are usually old ones (Antipin et al., 1956; Liegeois, 1956; Schmid, 1957; Ahrens and Aedtner, 1964; Gibson, 1969). The exceptions are the regulations in Australia (Fewster, 1967) and recent changes in United States regulations. The old regulations adhere to two archaic concepts. The first is the belief that cysticerci exist singly in carcasses and the other is the belief that there is a uniform life span of cysticerci. There is much information which shows that living cysticerci coexist with dead ones in the same carcass. Furthermore, removal of a single visible cysticercus on the assumption that the carcass then becomes safe for consumption is unjustified, even in light infections, because many more cysticerci are undoubtedly present in the carcass. Discarding all infected carcasses is not feasible economically, unless they are heavily infected. The carcasses are usually frozen or boiled. Gamma radiation as proposed by Pawel and Janikk (1963a, b) has not been put to practical use. Boiling meat to a temperature above 56”C, the thermal death point of T. saginata cysticerci (Allen, 1949, is effective provided the heat penetrates to the center of the meat. Freezing of meat at - 10°C (1 5°F) for 10 days effectively kills cysticerci (Landi and Monzini, 1954; Malheiro et al., 1966; Hajduk et al., 1969). Shorter time periods would reduce the expense of freezing carcasses, but studies dealing with various time and temperature requirements to kill cysticerci are generally out of date (Zunker, 1935; Keller, 1937, 1938a; Terhorst, 1938).

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B. SEROLOGICAL DIAGNOSIS AND IMMUNIZATION OF CATTLE

Ante-mortem diagnosis of cysticercosis of cattle has been attempted by skin serological and biochemical examination (Dewhirst et al., 1960; Dewhirst and Cramer, 1965). Skin tests performed in the caudal fold of cattle by Bugyaki (1961) gave 83.1 % positive reaction in infected animals but false positive reactions were frequent. In the detailed studies of Froyd (1963) various antigens were not specific enough. Similar results were obtained by Kosminkov (1965) and Leikina et al. (1966). Serological tests such as the hemagglutination and latex agglutination tests (Leikina et al., 1964, 1966; Kosminkov and Filipov, 1967; Sokolovskaya, 1968; Alferova, 1969) as more specific. According to Sokolovskaya (1968) the latex agglutination test with antigen from lyophylized cysticerci is 98.7 % sensitive and 99.7 % specific. According to Alferova (1969) the indirect hemagglutination test using Boyden’s technique and Kent’s antigen successfully diagnosed cysticercosis in 18 calves. No cross reaction with Echinococcus or Fasciola was observed. Two peaks of antibody response were observed, one was between 18-24 days and due to migration and development of larvae, and the other was observed between 81-101 days and probably due to destruction and release of somatic antigen. Soulsby found a substantial level of non-specific globulins in the serum of infected cattle and Mosina (1965) noted an increase of gamma globulin by paper electrophoresis on the 25th day of infection. This area deserves further work, Penfold and Penfold (1937) described complete immunity to reinfection of oxen that were infected with T. saginata cysticerci. The existence of immunity has been proved both experimentally (Urquhart, 1961) and in practice (Peel, 1953; Froyd, 1960; Graber and Thome, 1966). This immunity seems to be lifelong (Urquhart, 1961) except for light infection of calves (Soulsby, 1963). This lifelong immune response has led to attempts to immunize cattle as originally suggested by Penfold and Penfold (1937), as a means of controlling cysticercosis. Immunization can be carried out in three ways: per os infection with irradiated eggs (Urquhart et al., 1963; Urquhart, 1966), intramuscular infection with onchospheres (Wikerhauser et al., 1970), or by induction of heterologous immunity with T. hydatigena (Wikerhauser et al., 1970) but all of these methods are still experimental. Froyd’s (1964a) attempt to induce passive immunity in calves was unsuccessful. Further studies on passive and active immunization is warranted. C.

SANITATION

Schoop’s statement (1962) that no proper control methods for T. saginata taeniasis and cysticercosis have been found is too pessimistic. The control measures used in the U.S.S.R. have significantly reduced, but not eradicated, human taeniasis (Prokopenko, 1968; Sergiev, 1966; Magdiev, 1968; Abasov, 1968) and the changing sanitary and economic conditions in modern Israel have virtually eliminated T.saginata from that country (Witenberg, 1968). Various authors have expressed different views on the best ways to control

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this zoonosis. Some have put the major emphasis on man as the carrier of the adult parasite whereas others have emphasized animals as the disseminator of infection to man. This is really a moot point since successful control depends on a variety of measures that are aimed at all parts of the life cycle of the parasite. The need for close cooperation between medical and veterinary services has been pointed out by Ginsberg (1954), Kupey (1954), Magdiev (1968) and Popov et al. (1970). The control measures proposed by various authors fall into the following categories ; mass diagnosis and treatment, sanitation and sanitary education, ante-mortem testing and post-mortem inspection of cattle, and immunization (the latter two subjects have been discussed in the previous section). Mass diagnosis of populations in endemic areas and compulsory treatment of all patients infected with T. saginata has been carried out in U.S.S.R. and Bulgaria (Kovalev, 1960; Nadzhafov, 1966; Magdiev, 1968; Popov et al., 1970; Todorov, 1970). In Poland there is also compulsory treatment of all diagnosed cases and compulsory mass-treatment has been carried out in Kenya (Ginsberg, 1954) and the Congo (Versyck and Jacob, 1958). Compulsory diagnosis and treatment of cattle farm workers has been advocated by Sussman and Prchal (1950), Miller (1956), McIntosh and Miller (1960), Mattes (1962), Ahrens and Aedtner (1964) and Schultz et al. (1970). Although notification of Taenia cases is not a new idea (Krueger, 1934) it is rather the exception than the rule even in countries with high levels of preventive medical services. Long-term reduction in transmission depends on improved sanitation and sanitary education. The need for sanitation in rural endemic areas has been pointed out by Kovalev (1960), Enequist (1965), Logan (1967) Abasov (1968), Magdiev (1968), Schwabe, (1969) and Popov et al. (1970). The need for proper treatment and disposal of sewage has been stressed by Silverman (1955b), Mattes (1962), Miller (1956), Logan (1967) and Friedrich (1961) and proper sanitation in campgrounds and recreation areas has been discussed by Kleibel (1961) and Mattes (1962). Improvement of sanitation is expensive and inevitably connected with a generally higher standard of living. Without social acceptance of control measures through programs of education they will be less effective; the importance of sanitary education has been pointed out by Sussman and Prchal (1950), Gregoire et al. (1956), Kovalev (1960) and Hermus (1961). At the 19th WorId Veterinary Congress in Mexico during 1971, the veterinary profession virtually declared war on cysticercosis and taeniasis caused by Taenia solium and T. suginata (communication of Dr I. Mann). In our opinion, any success gained in that war will depend on the promotion of adequate research, better cooperation between workers in the veterinary and medical professions in both the field and the laboratory, and the acceptance and understanding of control measures by authorities deciding matters of economic importance and meat-eating peoples.

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TABLE IV World-wide distribution of taeniasis and cysticercosis (T.saginata) : References

General Stoll (1947); Greenberg and Dean (1958); Merle (1958); Urquhart (1961); Froyd (1965b); FAO/WHO/OIE Animal Health Yearbook (1970). Europe Bachlechner (1941); Ginzel (1961); Kleibel(l961) Gregoire et al. (1956); Granville et al. (1958) Pavlov (1944); Ghenov (1964); Avlavidov and Kovchazov (1965); Denev (1965) ; Petkov and Todorov (1970) CZECHOSLOVAKIA Hovorka (1963); Petrh and Vojtechovskti (1963b); Petrd and Gregor (1963); Koudela (1965b, 1966b) DENMARK Emsbo (1938); Jepsen and Roth (1950); Thorp (1950); Gotzsche (1951); Jepsen and Roth (1952) FINLAND Tarnaala (1941); Huhtala (1950) FRANCE Reuter (1944); Fonteneau (1950) GERMANY EAST Kupey (1954); Sinnecker (1955); Hermus (1961); Schulze (1964); Grumbach (1965); Ockert (1965); Plaschke and Kramm (1966); Hiepe et al. (1967); Hajduk et al. (1969); Mielke (1969) GERMANY WEST Bishop (1952); Summa and Steppe (1956); Raschke (1957); Lerche and Elmossalami (1958); Friedrich (1961); Beier (1963); Schlachter (1963); Ahrens and Aedtner (1964); Beier (1965b) HOLLAND Hofstra (1954); van Keulen (1959); Honer (1963); Van Gils (1963); D e Vries (1968) HUNGARY Taktics et al. (1967) IRELAND Logan (1967) ITALY Pellegrino (1954); Pellegrini (1958); Schmid (1958); Masellis (1960); Ricci (1961); Marrenghi (1962); Gallo and Anello (1967); Pennisi et al. (1967); D e Carneri et a/. (1968); Corso et al. (1969) POLAND Trawinski (1957); Pawlowski and Rydzewski (1958); Pawtowski (1959); Adonajlo and Boliczak (1961); Lutyhski and Wasowa (1963); Pawtowski (1964a); Zembrzuski (1965); Adonajto and Gancarz (1967); Piqtkowska (1967); Adonajlo et al. (1969); Kalawski and Pawlowski (1970) RUMANIA Ureche (1965); Cironeau and Popovici (1968) SOVIET WONAbasov (1957); Mamedov (!958); Kovalev (1960); Avakyan (1961); Mukvoz (1961); Topuriya and Matikashvili (1961); Merkushev et al. (1962); Martikyan (1963); Pugachevskaya (1965); Ma diev (1966); Nadzhafov (1966); Prokopenko(l966); Sergiev et af. (1966); Shekhovtsov 8967); Ghenis (1968); Magdiev (1968); Prokopenko (1968) SPAIN Orduna (1957) SWEDEN Enequist (1965) SWITZERLAND Despres and Ruosch (1961); Despres (1962); Boch (1965) UNITED KINGDOM LeRoux 1949); Griffiths (1950); Hardwick (1990); Marsden (1950); McCleery and Blamire l1950); Priestly (1950); Seiler and Norval (1950); Silverman AUSTRIA BELGIUM BULGARIA

(1955a)

Lepes (1954); Simitch and Nevenitch (1955); Winterhalter and Stuparid (1957); NenadiC (1957,1958); ZelejkoviC and BokoviC (1959); Grujic 1960); NeCev and ACkov (1960); NenadiC (1960); Vujic et al. (1961); AlagiC eta/. (1965; DZinleski et al. (1963); Mijatovif (1964); Winterhalter (1965)

YUGOSLAVIA

Africa

ALGERIA Pampiglione et al. (1965) EGYPT Nor El Din and Baz (1949); Abdou ETHIOPIA Diesfeld (1965) CAMEROON Doby et al. (1957)

(1959); El Afifi et al. (1961)

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TABLEIV World-wide riistributio~iof cysticercosis (T. saginata) : References

Graber and Thome (1966); Graber and Tab0 (1968); Shabelnik and Chechugo (1970) CONGO van Grunderbeeck and Penson (1954); Versyck and Jacob (1958); Urquhart (1961); Price et a/. (1963); Roberts (1970) KENYA Daubney and Carman (1928); Mann and Mann (1947); Ginsberg (1954, 1955); Ginsberg eta/.(1956a, b); McKinnon (1957); Froyd (1960); Ginsberg(l960); McManus (1960); Froyd (1965a, 1966) MALAWI Fitzsimmons (1966) NIGERIA Okpala (1961); Hinz (1966) PORTUGUESE GUINEA de Oliveira Lecuona (1956) RHODESIA Goldsmid (1968); Rhodesia report (1969); Thornton and Goldsmid (1969) SOUTH AFRICA Canhan (1946); Elsdon-Dew (1964); Verster (1966); van den Heever (1969) SUDAN Sankale et al. (1958); Eisa et al. (1962); El Afifi et a/. (1963) UGANDA Mitchell (1967) URUNDI Biche and Thienpont (1959); Marsboom et nl. (1960) CHAD

ARGENTINA NiAo (1964) BRAZIL Ribeiro de Assis

Americas

(1949); Pardi et al. (1952); Costa and Brant (1964); De Freitas (1964); Brant et a/. (1965); Paoliello (1965); DaSilva et a/. (1966); Alonso (1967); Dias (1967); Pessoa (1967); Roiter (1968); Lima et a/. (1970) CHILE Delard et a/. (1958) CUBA Alvaro-Diaz Artidle (1967) ECUADOR Lope2 o l ? i Z (1969) GUATEMALA Acha and Aguilar (1964); Zapatel et al. (1965) PANAMA CANAL ZONE Cosgrove (1960) UNITED STATES Marx (1942); Sussman and Prchal (1950): Miller (1956); Schultz et al. (1970) VENEZUELA del Corral and Vogelsang (1943)

Asia BURMA Tu and Hkun-Saw-Lwin (1968) CAMBODIA Brumpt and Kong-Kim-Chuon (1965) CHINA Wu (1939); Chin (1959) I ~ I A Mukerji and Bhaduri (1944); Peatt (1950); Shah and Joshi (1965) IRAN Endrejat (1938); Sabbaghian and Arfaa (1970) IRAQ Thornton (1957) ISRAEL Witenberg (1968) JAPAN Masdaki (1960); Morishita et a/. (1964) KOREA Lee et a/. (1966); Seo et a/. (1969) LEBANON Interdept. Committee (1962) MONGOLIA Froyd (1965b) PAKISTAN Yaqub (1953); Muazzam and Ali (1961); Haleem el al. (1965) PHILIPPINES Refuerzo and Albis (1949) SIKKIM Mitra (1970) SYRIA Bottom (1945) TAIWAN Hsieh (1960); Bergner (1964); Huang (1967); Wen (1969) THAILAND Interdept. Committee (1962); Chularerk et a/. (1967); Papasarathorn

(1967) YEMEN Felsani (1959)

Australia AUSTRALIA Fewster (1 967) NEW ZEALAND Shortridge (1966)

et a/.

310

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Taylor, D. C. (1958). Cysticercosis in an onyx. (Clinical note.) Vet. Rec. London 70, 1207. Terhorst, H. (1938). Untersuchungen uber die Invasionstuchtigkeit der Rinderfinne nach Aufbewahren finnigen Fleisches in Kiihlraumen. Dissertation, Giessen, 32 PP. Ter-Icarapetiants, N. N. (1967). [Treatment of taeniasis patients under conditions of rural district hospital.] Ter. Arkh. 39,99. Thieulin, G., Pantaleon, 3. and Rosset, R. (1963). La cysticercose bovine: recherche d'une prophylaxie rationelle. Int. vet. Congr., 17th, Hannover, August 14-21, 1963. Proceedings, Vol. 11, pp. 881-884. Thiodet, J., Fourrier, A. and Babeau, P. (1953). Syndrome agranulocytaire et Taenia saginata. Alga. mid. 57, 165-1 66, 169. Thornton, H. (1957). The incidence of Cysticercus bovis in Persia. (Correspondence.) Ver. Rec. 69, 102. Thornton, H. and Goldsmid, J. M. (1969). Taeniasis and cysticercosis with special reference to Rhodesia. Cent. Afr. J. Med. 15,47-52. Thorp, W. T. S. (1950). World public health problems. J. am. Vet. Med. Ass. 117, 374-378. Todorov, R. D. (1966). [Study of the quantitative deviation of gastro-intestinal enzymes in taeniid infections.] Zzv. tsent. khelmint. Lab., Sof. 11, 173-175. Todorov, R. D. (1970). [Development of medical parasitology and organization of control of parasitic diseases in the People's Republic of Bulgaria.] Medskaya Parazit. 48, 144-148. Topuriya, I. I. and Matikashvili, T. S. (1961). [Means of eradicating Taenia saginafa in the Georgian SSR.] Medskaya Parazit. 30, 276-277. Totterman, G. (1938). Uber die Pathogenese der Wurmanamie. Acta med. scand. 96, 268-288. Totterman, G .(1939). Uber Sternalmark undBlut bei Wurmstragern. (Bothriocephalus latus, Taenia mediocanellata). Acta med. scand., Suppl. 104, 176 pp. Trawiriski, A. (1957). La cysticercose chez les animaux et chez I'homme et spicialement la cysticercose du cerveau. Bull. Of.int. Epizoot. 48,191-198. Tronchetti, F. and Cartei, S. (1948). Anemia iperemolitica perniciosiforme da Taenia saginata. Rass. Fisiopatol. Clin. Terap. Pisa 20, 27-38. Tu, M. and Hkun-Saw-Lwin. (1968). Intestinal parasitism in the Inthas. Un. Burma J. Life Sci. 1,255-256. Turner, P. P. (1963). The treatment of tapeworm infestation with Anthiphen. J. trop. Med. Hyg. 66, 259-260. Ulivelli, A. (1968). Paromomycin sulfate in tapeworm infection in man. Znt. Congr. trop. Med. Malar. (8th) Teheran, 1968,1089-1091. Upton, A. C. (1950). Taenial proglottides in the appendix. Possible association with appendicitis. Report of cases. Am. J. din. Path. 20, 11 17-1 120. Ureche, L. (1965). Cercetkri cu privire la cunoa$terea frecventei cisticercozei bovine. Revta Zooteh. Med. vet. Buc. 15, 71-74. Urquhart, G. M. (1961). Epizootiological and experimental studies on bovine cysticercosis in East Africa. J . Parasit. 47, 857-869. Urquhart, G. M. (1965). Parenteral production of cysticercosis. J. Parasit. 51, 544. Urquhart, G. M. (1966). Bovine cysticercosis. Znt. Congr. Parasit. (1st) Rome, Sept. 21-26,1964. Proceedings, Vol. 11, pp. 829. (Discussion p. 839). Urquhart, G . M. and Brocklesby, D. W. (1965). Longevity of Cysticercus bovis. J. Parasit. 51, 349.

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Urquhart, G. M., Jarrett, W. F. H. and Mulligan, W. (1962). Helminth Immunity. Adv. vet. Sci. 7 , 87-129. Urquhart, G. M., McIntyre, W. I. M., Mulligan, W., Jarrett, W. F. H. and Sharp, N. C. C. (1963). Vaccination against helminth disease. Int. vet. Congr. (17th), Hannover, August 14-21, 1963. Proceedings, Vol. I, pp. 769-774. Valverde, A. (1948). Un caso de teniasis curado con solitaricida. (Correspondence). Revta Kuba Med. trop. Parasit. 4, 130. Van den Heever, L. W. (1967). On the longevity of Cysticercus bovis in various organs in a bovine. J . Parasit. 53, 1168. Van den Heever, L. W. (1969). The degree of cysticercosis infestation of cattle in terms of standard meat inspection procedures. JIS. Afr. vet. rned. Ass. 40,4749. Van den Heever, L. W. and Reinecke, R. K. (1963). The significance of the shoulder incision in the routine inspection of food animals for cysticercosis. Znt. vet. Congr. (17th), Hannover, August 14-21, 1963. Proceedings, Vol. 11, pp. 909-910. Van den Heever, L. W. and Tustin, R. C. (1967). A note on intracerebral Cysticercus bovis in a calf. JI. S . Afr. vet. med. Ass. 38, 309-310. Van Gils, J. H. J. (1963). Some data about cysticercosis in the Netherlands. h t . Vet. Congr. (17th) Hannover, August 14-21, 1963. Proceedings, Vol. 11, pp. 901907. Van Grunderbeeck, R. and Penson, D. (1954). La taeniase en Ituri. Recherche d’une mkthode de dkparasitation massive adaptke A I’Ituri. Effects taenifuges de la camoquin. Annls SOC.belge MeV. trop. 34, 98 1-997. Van Keulen, A. (1959). Epidemiology of cysticercosis bovis. Znt. vet. Congr. (16th), Madrid, May 21-27, 1959, Vol. 11, pp. 753-754. Varges, W. (1957). Intensivierung der Finnenschau-ein Beitrag zur Losung des Rinderfinnenproblems. Monatsheffe1: Vet. Med. 12,320-321. Vasilkova, 2.G. (1944). The problem of the purification of the water of the River Moskva from the eggs of helminths. Medskaya Parasit. 13, 11-16. Vegors, H. H. and Lucker, J. T. (1971). Age and susceptibility of cattle to initial infection with Cysticercus bovis. Proc. Helminth. SOC.Wash. 38, 122-1 27. Verdiev, G. Y. (1958). [Acute cholecystitis caused by tapeworms.] Khirurgiya, Moskva 34, 106-107. Verster, A. (1966). Cysticercosis hydatidosis and coenurosis in the Republic of South Africa. J. S. Afr. vet. med. Ass. 37, 37-45. Verster, A. (1967). Redescription of Taenia solium Linnaeus, 1758 and Taenia saginata Goeze, 1782. Z . ParasitKde 29, 313-328. Verster, A. (1969). A taxonomic revision of the genus Taenia Linnaeus, 1758. Onderstepoort J. vet. Res. 36, 3-58. Versyck, M. and Jacob, H. (1958). La lutte anti-thia dans I’Ituri. Bull. agric. Congo belge 49, 155-164. Vieira, C. B. (1954). InfestaGao mutiple por Taenia saginara e ulcera duodenal cronica. Consideraceoes sobre urn caso. Folia Clin. Biol.Sao Paul0 21, 3-6. Viljoen, N. F. (1937). Cysticercosis in swine and bovines, with special reference to South African conditions. Onderstepoort J . vet. Res. 9, 337-570. Viljoen, N. F. (1939). Suggestions for the eradication of cysticercosis-taeniasis. J . S . Afr. vet. rned. Ass. 10, 115-1 25. Voge, M. (1963). Observations on the structure of cysticerci of Taenia solium and Taenia saginata (Cestoda: Taeniidae). J. Parasit. 49,85-90. Voge, M. (1967). The post-embryonic developmental stages of cestodes. In “Advances in Parasitology”. (ed. Ben Dawes) 5,247-297. Academic Press. Voge, M. (1969). Systematics of cestodes-present and future. In “Problems in Systematics of Parasites” ( G . D. Schmidt). University Park Press, Baltimore.

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Vogel, H. (1961). Uber eine unsegmentierte, eingeschlechtliche Taenia saginata. J . Helminth., R. T. Leiper. Suppl., pp. 191-198. Vogelsang, E. G. and Fernandez, A. J. (1945). Caso de poliparasitismo simulthneo por T. saginata y T. solium, Trabajos del Centro de Investigaciones Cientifcas, Caracas, No. 3, pp. 1 1 1-1 15. von Bfand, T. (1966). “Biochemistry of Parasites.” Academic Press, New York and London. vonBrand,T., Mercado,T.I.,Nylen, M. U. andScott,D. B.(1960). Observationson function, composition and structure of cestode calcareous corpuscles. Expl Parasit. 9,205-214. von Brand, T., Scott, D. B., Nylen, M. U. and Pugh, M. H. (1965). Variations in the mineralogical composition of cestode calcareous corpuscles. Expl Parasit. 16, 382-391. von Brand, T., Nylen, M. U., Martin, G. N. and Churchwell, F. K. (1967). Composition and crystallization patterns of calcareous corpuscles of cestodes grown in different classes of hosts. J. Parasit. 53,683-687. von Harnack, G. A. (1959). Bandwurmbehandlung im Kindesalter mit Cestodin. Dt. med. Wschr. 84, 865-866. Vujic, B., Nikolic, P. and Anic, N. (1961). Neka pitanja epizootiologije Taenia saginata i Cysticercus inermis u jednom delu Sandfaka. Vet. Glasn. Belgrade 15,65-69. Wang, W. L. and Dunlop, S. G. (1954). Animal parasites in sewage and irrigation water. Sewage Works J. 26, 1020-1032. Wardle, R. A. and McLeod, J. A. (1952). “The Zoology of Tapeworms.” The University of Minnesota Press, Minneapolis, Minnesota. Webbe, G. (1967). The hatching and activation of taeniid ova in relation to the development of cysticercosis in man. Z . Tropenmed. Parasit. 18, 354-369. Wegmann, T. (1965). Diagnostik, Klinik und Therapie von Echinococcus and Taeniases. Schweizer Arch. Tierheilk. 107, 244-265. Weinberg, M. (1908). Valeur comparee de deux prockdbs de laboratoire (dkviation du complement et prkcipito-diagnostic) au vue du diagnostic de l’bchinococcose. C . r . Seanc. SOC. Biol. 66,133-135. Weinland, F. (1859). Observations on a new genus of Taenioides. Proc. Boston SOC. Natur. History, Vol. 6. Weinmann, C. J. (1966). Immunity mechanisms in cestode infections. In “Biology of Parasites” (Ed. E. J. L. Soulsby). Academic Press. Weinmann, C. J. (1970). Cestodes and acanthocephala. In “Immunity to Parasitic Animals,” Vol. 2 (Ed. G. J. Jackson, R. Herman and Ira Singer). AppletonCentury-Crofts, New York. Wen, Y. F. (1969). [A survey on helminthic infections among aborigines in Chien-Shih district of Hsin-Chu County, Taiwan.] J. Formosan Med. Ass. 68,445-452. Wigand, R. and Warnecke, W. (1953). Uber Bandwurmkuren (Taenia saginata). Dt. med. Wschr. 78, 1493-1494. Wikerhauser, T., Zukovic, M. and Diakula, N. (1970). Vaccination against bovine cysticercosis.J. Parasit. 56, 369. Winterhalter, M. (1965). IkriCavost goveda u toviligtima stoke. Vet. Glasn. Belgrade 19, 779-783. Winterhalter, M. and Stuparic, D. (1957). IkriCavost goveda. Vet. GIasn. Belgrade 11,458-465. Witenberg, G . G. (1968). Helminth fauna in man and domestic animals in Israel. Israel J. Med. Sci. 4, 1069-1073.

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Wojciechowska, L. and Wachowska, M. (1964). [Specific antibodies in sera of patients infected with Taenia saginata.] Medycyna dosw. Mikrobiol. 16, 343-346. Wolff, F., Schanabel, R. and Moldenschardt, H. (1959). [A fatal case of mepacrine poisoning during the treatment of tapeworm infection by the intraduodenal route. Z . arztl. Fortbill. 53, 683-687. Wu, L. S. (1939). Taenia infection. Report based on stool examinations of 56,286 patients in the Peiping Union Medical College. Chin. med. J . 55, 561-565. Yaqub, M. (1953). Cysticercirs bovis in a bullock. (Abstract.) Proc. Pakist. Sci. Conf, 5th 1953, Medicine a. Veterinary Science Section, p. 13. Yokogawa, M., Yoshimura, H., Okura, T. and Saito, M. (1962). [Treatment of Taeniu saginata with bithionol.] Jap. J. Parasit. 11, 3 9 4 4 . Lapart, W. ( 1 968). Zastosowanie antygenu frakcjonowanego w serologicznej diagnostyce tasiemczyc u ludzi. Wiad. Parazyt. 14, 203-209. Zapart, W., Adonajlo, A. and Gancarz, Z. (1969). Proby srodskorne z alergenami frakcjonowanymi w diagnostyce tasiemczyc u ludzi. W a d . Parazyt. 15,77-81. Zapatel, J., Ubieto, A. and Martinez, M. (1965). Cysticerci in processed meat in Guatemala. Am. J. trop. Med. Hyg. 14, 1 13-1 16. ZelejkoviC, S. and Bokovic, T. (1 959). Istraiivanje bobicavosti kod goveda i teladi zaklanih u banjaluEkoj klaonici 1957 godine. Vet. Glusn. Belgrade 13, 308-310. Zembrzuski, K . (1965). Materialy do epidemiologii tasiemczyc. Wiad. Parazyt. 11, 161-164. Zunker, M. (1935).DieAbtotung der Rinnderfinen durchKuhl-und Gefrier-verfahren. Z . Fleisch-u. Milchhyg. 45, I 2 1 -I 26. Zwierz, C. (1 963). Skojarzone leczenie inwazji Taenia saginata. Bull. Inst. Mar. Med. Gdat'isic, 14, 289-29 1 . Zwierz, C. (1964). Modyfikacjaleczenia inwazji Taetiiasaginuta preparatem Yomesan. [A modified treatment of Taenia saginata invasion with Yomesan.] (Abstract.) Wial. Parazyt. 10,454-455. ADDITIONAL REFERENCES ADDED IN PROOF

Brandes, H. (1958). Untersuchungen zur Feststellung der Finnigkeit beim Rind unter besonderer Beriicksichtigung der Untersuchung mit filtrierten U.V.Strahlen. Archiv Lebensmittelhjg. 9, 241-243. Graber, M. (1959). La cysticercose bovine. Son importance dans les zones sahaliennes d'elevage de la Republique du Tchad. Revue Elev. MPd, vkt Pays trop. 12, 121-143. Naumov, N. A. (1929). A case of cysticercosis of heart and meninges. Perm. Med. Zh. 7, 1-2.

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SHORT REVIEW Supplementing Contribution of Previous Volume

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The Structure of the Helminth Cuticle D. L. LEE*

Houghton Poultry Research Station, Houghton, Huntingdon, England

. .

............ I. Introduction ....................... ..... ...... 11. Turbellaria .................................................................................... A. Structure ............................................. ............................. B. Function ....... .................................................... 111. Monogenea ............................................... .................................... .................. A. Structure .................................. B. Function ................................................................................. .......... IV. Digenea ............................................ ............. A. Structure ................................................................................. B. Function ................................................................................. ................. V.

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.................................. v11. VIII.

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341 348 348 348 348 348 352 352 352 356 351 357 357 357 351 359 360 360 368 369 369 369 369 370

I. INTRODUCTION This short review supplements the earlier one (Lee, 1966b) and should be read in conjunction with it. Because of the limit on size of these up-dated reviews emphasis will be placed on completely new work and less attention will be paid to work which consolidates, or is very similar to, previously described work. When the earlier review was published little attention had been paid to the outer coverings of larval helminths. This has now changed and much of the work described in this review is on larval forms and on the development of the outer covering of the adult.

* Present address: Dept. of Pure and Applied Zoology, University of Leeds. 347

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11. TURBELLARIA

A.

STRUCTURE

Kronborgia amphipodicola is an endoparasitic turbellarian which lives in the body cavity of certain amphipods. It is dioecious and both sexes lack mouth, pharynx, intestine and excretory system. The female has a single-layered epidermis” which rests on a thin basal lamina. Sub-epidermal gland cells are numerous. The epidermis is covered with numerous cilia and long microvilli. The cilia are arranged in longitudinal rows and have a cross-striated rootlet running from the basal body of the cilium into the epidermis. The microvilli have globular expanded tips and narrow bases but show no internal differentiation. A glycocalyx covers the epidermis. Rhabdite-like structures occur close to the outer surface of the epidermis. Nuclei lie close to the base of the epidermis; Golgi bodies, poorly developed endoplasmic reticulum, ribosomes, large lipid droplets, mitochondria and an irregular network of microtubules are also present (Bresciani and Karie, 1970). An interesting aspect of this epidermis is the apparent breakdown of the lateral membranes of the epidermal cells. Lateral membranes are seldom continuous; in most places only the basal part of the lateral cell membrane remains. The outer regions of some epidermal cells are connected by desmosomes but these disappear during development. The sub-epidermal gland cells have long slender processes which pass through the epidermis to the surface. Desmosomes connect the process to the epidermis at the surface. After the female has left the host and has completed spawning the epidermis changes in character, apparently as a result of histolysis (Bresciana and K&e, 1970). B.

FUNCTION

K . amphipodicola possesses no alimentary tract and grows considerably in 10 months and it must therefore absorb nutriment from the host through the epidermis. The epidermis of free-living turbellaria possesses short microvilli but that of K . amphipodicola possesses numerous long microvilli; it would appear that these enlarged microvilli are an adaptation to parasitism as they increase the absorptive area of the parasite. The sub-epidermal glands are believed to function during escape of the parasite from the host and also during construction of the cocoon. 111. MONOCENEA A.

STRUCTURE

When the earlier review was written there were no papers published on the ultrastructure of the outer covering of the Monogenea. Brief descriptions of the adult epidermis of Lepfocotyle minor, Rajonchocotyle emarginata,

* The term “epidermis” is used throughout this review to denote what in trematodes and cestodes was once commonly known as the “cuticle” and, by some writers more recently, the “tegument” or “integument” (see Rohde’s review in this volume-Ed.).

FIG.1. Electronmicrograph of T.S. body wallof the unusual juvenilemonogenean Anzphibclella flavolimwtu, from the blood system of an electric ray (Torpedo nobiliuna). The outer epidermis is a cytoplasmic syncytium bearing small microvilli and it contains dense granules and mitochondria. The nucleated epidermal “cell” body which connects with this layer lies beneath the basement lamina and muscle layers and secretes dense granules into the superficial epidermis. bl, basement lamina; dg, dense granules; ep, epidermis; epc, epidermal “cell” body; mu, muscle; mv, microvilli; nu, nucleus. (From Lyons (1971). Parasitology 62. By kind permission of the author and Cambridge University Press.)

FIG.2A. Electronmicrograph of the syncytial epidermis of Acunthocotyle eleguns from the dorsal surface of Raiu cluvafu.The epidermis bears microvilli on the dorsal surface of the worm and contains two kinds of granular inclusions as well as mitochondria. FIG.2B. Electronmicrograph of L.S. body wall of Acunthocotyle eleguns showing a large epidermal “cell” body in the parenchyma which contains many Golgi bodies secreting the epidermal granules. Several processes are given off from this “cell” and penetrate the muscle layers and basement lamina to connect with the superficial cytoplasmic, syncytial epidermis. bl, basement lamina; ep, epidermis; epc, epidermal “cell” body; g, Golgi bodies; mu, muscles; mv, microvilli ; nu, nucleus; pr, processes of epidermal “cell”. (By kind permission of Dr. K. M. Lyons.)

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Gastrocotyle trachuri and Dictyocotyle coeliaca have been given by Lyons (1968). More recently, fuller descriptions of the epidermis of adult Entobdella soleae, Acanthocotyle elegans and Gyrodactylus sp. and of the unborn juvenile of Gyrodactylus sp. have been given (Lyons, 1970a, b). The outer covering of all of these species is a cytoplasmic epidermis which is syncytial(Figs 1and2),exceptinthe head region of E.soleae. Nuclei do not occur in this syncytial epidermis but, in E. soleae and A . elegans, they are situated in nucleated extensions of the epidermis that lie in the parenchyma beneath the outer muscle layers (Fig. 2B) (Lyons, 1970a).Nuclei are apparently absent from the epidermis of adult Gyrodactylus and no sunken nucleated regions were found (Lyons, 1970b). The dorsal surface of E. soleae and A . elegans, including the dorsal surface of the haptor, bears long microvilli but the ventral surface, including the ventral surface of the haptor, is smooth. The epidermis which covers the adhesive head glands is densely covered with microvilli. An amorphous surface coat, probably glycocalyx, covers all of the epidermis. In the epidermis of E. soleae small mitochondria with few cristae lie near the base, and electron-dense inclusions are scattered in the cytoplasm. The epidermis is attached to the basement lamina by half-desmosomes. The cytoplasm of the nucleated cell-like structures contains more ribosomes than the outer epidermis, Golgi complexes, granular endoplasmic reticulum and mitochondria. The epidermis of A . elegans (Fig. 2) is similar to that of E. soleae but it contains two types of secretory inclusion and also has two types of nucleated cell-like structure sunk in the parenchyma. The epidermis of Leptocotyle minor, Rajonchocotyle emarginata and Gastrocotyle trachuri is rather similar to that of E. soleae and A . elegans. The epidermis of the endoparasitic monogeneans, Dictyocoryle coeliaca and juvenile Amphibdellaflauolineata, is not obviously modified as an adaptation to endoparasitism (Lyons, 1968). Diclidophora merlangi has an epidermis which is more like that of adult Digenea than of the other Monogenea which have been described. The syncytial epidermis is connected by cytoplasmic processes to nucleated celllike structures in the parenchyma; a few finger-likeprotrusions extend from the surface and bristle-like structures occur on the epidermis of the buccal region (Morris and Halton, 1971). The epidermis of Gyrodactylus sp. has short microvilli scattered over the surface and contains small mitochondria, lamellate inclusions, Golgi complexes and membrane-bound vesicles with fibrous contents, some of which open to the exterior. The epidermis which covers the ventral adhesive head glands bears many long microvilli and contains denser vesicular inclusions. The epidermis which covers the posterior haptor is similar to, but thinner than, the epidermis of the main body region (Lyons, 1970b). The late-stage juvenile of Gyrodactylus sp., which lies within the parent, has no microvilli, or other epidermal processes. Unlike the epidermis of the adult, that of the juvenile worm is packed with ribosomes. Nuclei are present in the epidermis of the juvenile but these apparently disappear in the adult worm (Lyons, 1970b).

352

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A study of the early embryos of E. soleae dissected from the egg suggests that the original ciliated cells are replaced by other ciliated cells which arise in the parenchyma (Lyons, 1968), as described by Skaer (1965) for the embryos of the free-living turbellarian Polycelis tenuis. The unciliated adult epidermis, which initially has superficial nuclei, appears under these ciliated cells. Later, the epidermal cell bodies differentiate but remain sunk in the parenchyma. In the hatched oncomiracidium the ciliated cells overlie the thin adult epidermis which, although at first discontinuous, may eventually cause shedding of the superficial ciliated epidermal cells by spreading beneath them (Lyons, 1968). B.

FUNCTION

Lyons (1970a) has suggested that the function of the microvilli on the epidermis of the Monogenea may be to support a layer of mucus or, alternatively, to increase the surface area and thus facilitate respiratory exchanges. The role of the mucus layer may be to protect the epidermis from abrasion and to discourage the growth of bacteria and other micro-organisms on the body surface. Neither ferritin nor thorium dioxide were taken up by E. soleae, A . elegans or Gyrodactylus sp. Non-specific esterases, acid and alkaline phosphatases were not detected in the epidermis of these three worms (Lyons, 1970a, b). Lyons (1970a) suggested that the surface coat of mucoprotein may bind ions such as Ca2’,prior to uptake by the worm. The microvilli which cover the epidermis of the adhesive head glands of E. soleae and A . elegans may assist in spreading the adhesive secretion of the head glands over the skin of the host into a thin “tacky” film and may also help to mix the products of different gland cells (Lyons, 1970a). Morris and Halton (1971) found that inclusion bodies in the epidermis of D.merlangi are continually secreted on to the surface of the worm and could have a protective function. They also found evidence that the epidermis may be involved in absorption of nutrients, especially through the opisthaptor. The bristle like protuberances on the epidermis of the buccal region suggest an abrasive or adhesive function; it is possible that they are responsible for rupturing blood vessels of the host tissues as the parasite is sanguinivorous. Lyons (1970a) has proposed an interesting hypothesis about the origin of the cuticle in some other invertebrates. Microvilli are characteristic of the epidermal and epithelial layers of both invertebrates and vertebrates and Lyons suggests that the extra cellular covering layers, such as earthworm cuticle, pogonophoran cuticle (see Gupta and Little, 1970) and even insect cuticle, could have arisen from ancestors which had a body surface similar to that of the monogeneans described above if the mucoprotein which lies over and between the microvilli were to become stabilized and perhaps collagenous (or chitinous). 1V. DICENEA A.

STRUCTURE

1. Miracidia Wilson (1969) and Southgate (1970) described the ultrastructure of the outer covering of the miracidium of Fasciola hepatica, and Southgate (1970) has

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described the changes which occur during development of the sporocyst from the miracidium. The epidermis of the miracidium of F. hepatica is made up of 21 ciliated cells which are arranged in five tiers (Dawes, 1960). These are separated from each other by a syncytial non-ciliated ridge of material formed by an extension of the sub-epidermal layer to the surface. The ciliated cells are attached to the ridges by prominent desmosomes. The cilia and their tapering rootlets have been described by Wilson (1969). The nucleus of the ciliated cells lies near the posterior border of the cell. In the miracidium of Schistosoma mansoni the nuclei of the ciliated epidermal cells lies in extensions of the cell below the muscles of the body wall (Brooker, in Southgate, 1970). Numerous cytoplasmic projections are present between the cilia of the epidermal cells in F. hepatica and large numbers of mitochondria, membrane-bound vesicles and large amounts of glycogen are present in the cells. The surface of the miracidium is coated with a thin layer of acid mucopolysaccharide (Wilson, 1969). The intercellular ridges contain mitochondria, elongate membrane-bound vesicles and granules. Nucleated cells, which lie below the muscle layers of the body wall, are in cytoplasmic continuity with the syncytial ridges and form the sub-epidermal layer (Southgate, 1970). As the ridges are syncytial and are in continuity with the sub-epidermal cells then the whole complex forms a syncytium. The apical papilla has no ciliated epidermal cells; the surface is covered with a thin corrugated layer of cytoplasm and is connected by cytoplasmic processes to the apical gland (Wilson, 1969).

2. Sporocysts Dawes (1960) has shown that when the miracidium of F. hepatica penetrates the snail the ciliated epidermal cells are shed. This has been confirmed by Wilson (1969) and Southgate (1970). According to Wajdi (1966), the miracidia of S. mansoni does not shed these cells during penetration of the snail. Southgate (1970) described the changes which occur in the outer layer of the trematode during penetration of the snail and during consequent development of the sporocyst stage. During penetration large vacuoles appear beneath the ciliated cells and these are eventually cast. The intercellular ridges then flow over the exposed muscles of the body wall to form a thin cytoplasmic covering. The membranous vesicles of the ridges apparently contribute their membrane to this rapidly forming layer and material from the sub-epidermal cells also contributes to the formation of the layer. It is complete within 2-3 h after penetration. This syncytial layer then increases in depth and develops the characteristic structure of the outer covering of the sporocyst. No cytoplasmic connections between the epidermis of the sporocyst and sub-epidermal cells were detected but these may be broken and reformed depending upon the requirements of the epidermis. Thin folds extend from the surface of the epidermis; it contains mitochondria, small amounts of glycogen, and a few Golgi complexes but no nuclei (Southgate, 1970). The sporocysts of other trematodes which have been investigated are rather similar to that of F. hepatica in that they have an outer syncytial epidermis,

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covered with cytoplasmic folds or microvilli, which is usually in cytoplasmic continuity with nucleated cell-like bodies in the parenchyma. Examples are Posthodiplostonium cuticola and an unidentified strigeid (Ginetsinskaya et al., 1966), and the sporocysts of Cercaria pectinata (Matricon-Gondran, 1969), Cercaria buchanani and an unidentified strigeid (Bils and Martin, 1966). According to Matricon-Gondran (1969) the sub-epidermal cell-like structures originate in the parenchyma and later make contact with the outer epidermis: also, the epidermis of the redia and cercaria is apparently derived from the primary syncytial epidermis of the embryo. According to James et al. (1966) the outer covering of the daughter sporocysts of Cercaria bucephalopsis hairnaena consists of a syncytial cytoplasmic layer in which nuclei are present and are not in sunken portions in the parenchyma. 3. Rediae The outer covering of rediae appears to be similar in the few species which have been studied. There is a cytoplasmic syncytial epidermis which has no cytoplasmic connection with cells in the parenchyma. The outer surface is thrown into folds or microvilli; mitochondria are present in the epidermis but nuclei have not been detected. Rees (1966, 1971a) described the redia of Parorchis acanthus; Bils and Martin (1966) described that of Acanrhoparyphium spinulosum; Ginetsinskya et al. (1966) that of Petasiger neocommense, Krupa et al. (1967, 1968) that of Cryptocotyle lingua and Dixon (1970) that of Cloacitrema narrapeenensis.

4.Cercariae An understanding of the early development of cercariae is essential if one is to determine the origin of the outer layer of cercariae, metacercariae and adults but surprisingly little attention has been paid to this aspect of the life cycle. Bils and Martin (1966) described the development of the cercarial epidermis in Acanthoparyphium spinulosum. In rediae, the germ cell is enveloped by one or more supporting cells; later, cell membranes of this supporting layer disappear so that the germ cell is surrounded by a syncytial layer. The germ cell then divides to form the germinal ball. Once this germinal ball is a multicellular mass, the enveloping syncytial layer, which originated from the redia, is shed. Prior to this, peripherally located cells of the germinal ball form a syncytial outer layer which is the epidermis of the cercaria. The cell bodies of the epidermis remain within the parenchyma but retain cytoplasmic connection with the epidermis. Dixon and Mercer (1967), however, stated that the outermost layer of the cercariae of F. hepatica within the redia is a cellular embryonic epithelium, which accumulates mucopolysaccharide, mucoprotein and tanned protein granules from secretory cells within the parenchyma. This embryonic epithelium apparently forms the outer covering of the cercariae and is shed when the cyst of the metacercaria is formed. Hockley (1970a) stated that the cercariae of Schistosoma mansoniare covered at an early stage of development with an epithelium of flattened, nucleated cells.

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The outer covering of the cercaria appears beneath this epithelium as a similar thin, cytoplasmic, nucleated, syncytial layer. The epithelium is lost before much differentiation of the embryo has occurred and at about the same time the nuclei of the cercarial epidermis degenerate and disappear. The surface coat on the cercaria is produced by the epidermis once the primitive epithelium is lost. The sub-epidermal cells are only temporarily connected to the epidermis. According to Rifkin (1970), however, the development of the cercaria ensues as germinal cells enter the lumen of the sporocyst, divide, and produce a germ ball. After the cercarial cells begin to differentiate, a number of elongate daughter sporocyst cells, which line the lumen of the sporocyst, surround the cercarial germ ball. These cells form the cytoplasmic syncytium which will become the epidermis of the cercaria. Development of the epidermis of the cercaria of Cloacitrema riarrabeenensis is similar to that described by Hockley (1970a) for S. mansoni (see Dixon, 1970). The epidermis of emerged cercariae is fundamentally the same in all species which have been examined. There is a syncytial cytoplasmic epidermis, containing mitochondria but no nuclei, which is in cytoplasmic continuity with nucleated cell-like structures in the parenchyma, i.e. a typical sunken epidermis except that it is syncytial. Microvilli are rarely present on the surface. The structure of the epidermis varies in different regions of the body (Hockley, 1968; Rees, 1967, 1971a, b). The epidermis of the main body of the cercaria contains a variety of secretory granules, some of which appear to produce mucopolysaccharides, as in Acanthatriunz oregonense (see Belton and Harris, 1967). An outer mucus-like covering on the cercaria of Schistosoma mansoni has been described by several authors (see Kemp, 1970). Descriptions of the epidermis of the cercaria and schistosomula of S. mansoni have been given by Smith et al. (1969), by Bruce et al. (1970) and by Morris and Halton (1971) (cercaria only). The epidermis of the schistosomula is similar to that of the cercaria but has fewer mitochondria. Inatomi et a/. (1970) gave brief descriptions of the epidermis of Metagonimus takahoshii, S. japonicum and S. spindale.

5. Adults Several papers on the ultrastructure of the outer covering of a variety of adult digeneans have appeared since the last review but it is impossible to describe them in detail in the space allotted. The epidermis of S. niansoni was studied by Morris and Threadgold (1968), Smith et al. (1969) and Silk et al. (1969, 1970) and they extend the brief description given by Lee (1966b). Very few mitochondria are present in the epidermis and the structures originally described as large vacuoles are probably channels which extend down into, but not through, the epidermis. Hockley (1970b) has shown that the outer membrane of the epidermis of S. mansoni usually consists of four dense layers separated by three less dense layers and that some of these layers are lost by delamination. Dense elliptical bodies and spherical membranous bodies, both of which are formed in the sub-epidermal cells, pass into the epidermis where the elliptical bodies probably contribute to the ground substance of the epidermis while the spherical membranous

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bodies may contribute to the outer membrane. It is suggested that this outer membrane is continuously delaminated and is reformed by the membranous bodies. The epidermis of S. japonicum appears to be similar to that of S. mansoni (Inatomi et al., 1969, 1970). Burton (1966) found that the epidermis of Gorgoderina sp. was similar to that of Haplometra but it lacked spines. The following digeneans have been shown to possess the basic epidermal structure, with minor modifications: Dicrocoelium dendriticum (see Reznik, 1966); Ornithobilharzia turkestanicum (see Logachev, 1964); Clonorchis sinensis (see lnatomi et al., 1968a, 1970); Haplometra cylindracea (see Threadgold, 1968); Posthodiplostomum minimum (see Bogitsh and Aldridge, 1967) ;Megalodiscus temperatus(see Bogitsh, 1968);F. hepatica(see Threadgold, 1967); Metagonimus yokogawai takashii (see Inatomi et al., 1968b, 1970); Opisthorchis viiierrini and Paragonimus ohirrai (see Inatomi et al., 1970). Erasmus, in an excellent series of papers (Erasmus, 1967, 1968, 1969a, b, c, 1970a, b, c) has studied the structure of the adhesive organ and the outer covering of strigeid trematodes (see also Ohman, 1965, 1966). The outer layer of these trematodes is essentially the same as those of other digeneans but the adhesive organ is modified as a secretory and absorptive epithelium. Erasmus has also studied the surface of the lappets of these strigeids. It is apparent from the work done by Erasmus and others that there is surface differentiation in some trematodes. This is especially so in the case of S. mansoni (Smith et al., 1969). B.

FUNCTION

The ciliated epidermal cells of the miracidia are undoubtedly locomotory in function and are used to bring the niiracidium into contact with the next host. They may also assist in initial penetration of the mollusc. The outer coverings of the sporocyst and redia are apparently adapted to an absorptive function because their extensively folded epidermis will greatly increase the surface area of the parasite. Presumably it is involved in the uptake of nutrients from the molluscan host (see Dixon, 1970). The epidermis of the cercaria probably performs different functions at different stages in the life of this particular form. Within the redia it will probably be used in the uptake of nutrients through the body surface (Rees, 1971a). It may also play a part in avoiding the defence reactions of the host. Outside the mollusc it may serve in osmoregulation and ionic regulation. In a secondary intermediate host it may assist in the evasion of the host’s defence reactions or in the uptake of nutrients. The epidermis of cercariae is very important in the formation of the metacercarial cyst as the various granules which go to form the various layers of the cyst wall pass into and then out of the epidermis. According to Dixon and Mercer (1967) the granules which form the outer cyst wall are shed together with the outer layer of the cercaria of F. hepatica. Other granules then pass through the new epidermis to form the remaining layers of the cyst. The new epidermis is formed from the cells containing the keratin-type granules which migrate from deep within the cercaria to the external surface. Southgate (in press) has studied the changes which occur in the epidermis of the cercaria

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of Notocotylus attenuatus during the formation of the metacercarial cyst. The original epidermis is retained in an altered form, after the various components which form the cyst wall have been extruded, and becomes the epidermis of the metacercaria and of the adult worm. The epidermis of adult digeneans is still thought to play a part in the uptake of nutrients but this may vary from species to species. In S. mansoni horseradish peroxidase was taken up only by the dorsal epidermis of the male (Smith et a/., 1969). The outer surface of S. mansoni also plays an important part in “disguising” the parasite in the host (Smithers and Terry, 1969; Smithers et al., 1969; Clegg et al., 1971). In strigeids the adhesive organ apparently plays a part in bringing about histolysis of host tissue and in the subsequent uptake of nutrients (see papers by Erasmus).

V. CESTODARIA A.

STRUCTURE

The Cestodaria have, until recently, been classified as a separate group of cestodes. Recently it has been suggested that either they are monogeneans or that they occupy a phylogenetic position between the monogeneans and the cestodes (see Llewellyn, 1965). Lyons (1969) has studied the fine structure of the outer covering of Gyrocotyle urna, which resembles that of the cestodes more than that of the monogeneans. The cytoplasmic epidermis has numerous microvilli and is in cytoplasmic continuity with nucleated cell bodies situated in the parenchyma. The microvilli lack the dense tip which is so characteristic of the microvilli of adult cestodes, but terminate in a fine spike composed of an extension of the apposed apical plasma membranes. The epidermis contains rod-shaped bodies and they are apparently released at the surface to form the mucoprotein substance which overlies the surface. B.

FUNCTION

Of the various functions which the epidermis performs one of the most important will be the uptake of nutrients.

VI. CESTODA A.

STRUCTURE

I . Laroal cestodes The outer surface layer of oncospheres of Taenia taeniaeformis and of Hymenolepis citelli is a thin dense cytoplasmic layer which has long thin cytoplasmic evaginations on its surface but no nuclei or connections with nucleated cell-like bodies deeper in the body wall (Nieland, 1968; Collin, 1969). Nieland (1968) has described the formation of the various outer layers of the developing onchosphere of T. taeniaeformis. The cyst wall of Multiceps serialis invaginates to form the protoscoleces and

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is covered with a cytoplasmic layer, which is very similar in structure to that described for adult cestodes except that the outer border of microvilli lack the dense tip found on microvilli of adult cestodes (Race et al., 1965), The epidermis contains mitochondria and connections join it to cells in the parenchyma. In Echinococcusgranulosus the germinal “membrane” is continuous with the wall of the brood capsule and with the outer covering of the protoscolex (Morseth, 1967). Finger-like processes extend from this membrane into the cyst wall. The rest of the “membrane” is somewhat similar to the epidermis of the protoscolex but it contains nuclei within it. The wall of the brood capsule is a vacuolated layer which has nuclei within it at irregular intervals. The epidermis of the protoscolex is similar in structure to that of adult cestodes. The outer walls of the suckers are formed by the epidermis and have spined microvilli like the rest of the epidermis. After 42 days in culture the sunken cells of the epidermis were multinucleate and it has been suggested that this, togethei with an increase in number of mitochondria and of Golgi complexes, may be related to the rapid growth necessary for segmentation which occurs about this time (Morseth, 1967). The protoscolex of E. multilocularis has an epidermis similar to that of E. granulosus (Sakamoto and Sugimura, 1969). Development of larval E. multilocularis, including formation of the outer layers, has been well described by Sakamoto and Sugimura (1970). The bladder wall of Cysticercus jasciolaris consists of an outer epidermis and an inner parenchyma (Nieland and Weinbach, 1968); the epidermis is similar in structure to that of adult cestodes. That of C. longicollis is also like the epidermis of adult cestodes but the tips of the microvilli are not dense. The epidermis of the scolex is continuous with that of the bladder wall (Baron, 1968). The wall of C. bovis seems to be similar ($lais, 1970). During budding and scolex differentiation of the bladder wall of Taenia crassiceps the sub-epidermal cells dominate budding and it is suggested that they are multipotential cells, capable of migration and derived from undifferentiated parenchyma cells (Bilqees, 1970). The outer surface of the 3-day precysticercoid of Hymenolepis citelli in the beetle is a thin epidermis which is covered with long, thin microvilli. After 5 days in the beetle the epidermis bears shorter and fewer microvilli and host cells attached to these microvilli are frequently cytolysed (Collin, 1970). The epidermis of the cysticercoid of Raillietina cesticillus is covered with short microvilli (Baron, 1971). The surface layer of Tylocephalum sp. from the oyster is a non-nucleate syncytium connected by occasional cytoplasmic bridges to internal cell bodies. Long coiled microvilli are present on the surface (Rifkin etal., 1970). The epidermis of the procercoid and plerocercoid stages of Diphyllobothrium Zatum is similar to that of the adult worm except that lamellated bodies occur in the larval stages but not in the adult, and they have fewer microvilli. The larval epidermis also contains fewer mitochondria (BrAten, 1968a, b) Similar results were obtained by Timofiev and Kuperman (1968) for the procercoid and plerocercoid of Triaenophorus nodulosus. Charles and Orr (1968) studied the epidermis of the plerocercoid of Schistocephalus solidus and of Ligula intestinalis and found that they have the basic structure as found

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in adult cestodes. The microvilli have long tapering electron-dense spines but there are differences in the nature of the microvilli of the two species. Morris and Finnegan (1969) found that in the differentiating plerocercoid of S . solidus portions of the epidermis retain an apparently primitive or undifferentiated morphology during growth. The epidermis of the velum remains thin and similar to the epidermis of small worms but the superficial epidermis increases greatly in depth, develops basally situated mitochondria and elaborates numerous microvilli and other protrusions. Pore canals were found. 2. Adult cestodes Work published since 1965 on the structure of the outer covering of adult cestodes has confirmed the basic plan of the cestode epidermis. Lumsden (1966) carried out a thorough study of the outer covering of Hymenolepis diminuta, Lacistorhynchus tenuis and Calliobothrium verticillatum and clarified many points. The epidermis consists of an anucleate cytoplasmic layer covered with microvilli with dense spine-like tips and is in cytoplasmic continuity with nucleated cells in the parenchyma. These cells appear to synthesize proteins which are then transported to the outer epidermis. Morseth (1966) showed that the epidermis of Echinococcus granulosus, Taenia hydatigena and T. pisformis is similar to that of other adult cestodes but he found structures resembling pore canals in the epidermis. The epidermis of Diphylbothrium latum is similar to that of other cestodes. The microvilli on the plerocercoid increase in length, are slimmer and are more numerous in the adult and there is also an increase in the number of mitochondria present from the plerocercoid stage to the adult (Briten, 1968b). The basic plan of the epidermis has been demonstrated in T. multiceps (see Race et al., 1966); Triaenophorus nodulosus (see Timofiev and Kuperman, 1968); Caryoplzyllaeus laticeps, C. fennica and Anomotaenia constricta (see Biguin, 1966); Ligula intestinalis and Schistocephalus solidus (see Charles and Orr, 1968); Diphylidium caninum and Taenia saginata (see Inatomi et al., 1970). According to Howells and Erasmus (1969) the interproglottidal glands of Moniezia expansa consist of clusters of cells surrounding crypt-like invaginations of the epidermis. Three main types of epidermis were found covering the apex of the scolex, the suckers and the rest of the surface of the scolex; on the scolex it resembles normal cestode epidermis; on the apex of the scolex it resembles that on the posterior face of the mature proglottides, and on the acetabula it is thinner and the microvilli are short. B.

FUNCTION

The function of the bladder wall of larval cestodes is probably partly protective against the defence reactions of the host, partly nutritive and, in cases such as E. granulosus, partly to retain the numerous scoleces in one area of the host. Slais (1966) made an interesting comparison between the trophoblast of the mammalian embryo and the bladder of the cestode cysticercus. The epidermis of procercoids and plerocercoids probably plays an important

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role in the nutrition of these larval stages as well as serving as a protective and packing layer. There has been a lot of work published on the distribution of enzymes and on the uptake of nutrients by adult cestodes since the earlier review appeared, and all confirm the important role of the epidermis in the uptake of nutrients. Lumsden et al. (1970b) have shown that adult Hymenolepis diminuta do not take up thorium dioxide, carbon particles, ferritin or the 14C-labelled protein fraction from Chlorella vulgaris and claim that transmembranosis of colloids by tapeworms does not occur. The glycocalyx on the surface of tapeworms is capable of adsorbing cations at neutral pH (Lumsden et al., 1970a) and it also plays an important part in contact digestion (Taylor and Thomas, 1968).

V11. NEMATODA A.

STRUCTURE

Since the earlier review appeared a number of papers which describe the structure of the cuticle of a number of larval nematodes have been published and the subject has been reviewed by Bird and Bird (1969). Wisse and Daems (1968) published an excellent paper on the second stage larvae of Heterodera rostochiensis. Four different layers were distinguished in the cuticle: (1) a dark layer 250 A thick; (2) a fibrillar layer 0.25 pm thick; (3) an electron transparent layer about 0.15 pm thick, which is thought to be fluid filled and which is crossed by small columns of material; (4) a striated layer, 0.25 pm thick, which has characteristic striations with a periodicity of 200 A. This fourth layer is lined on its inner side by a thin dark layer. The striations are perpendicular to the surface of the cuticle. The lateral alae consist of three ridges of cuticle in which the layers remain the same except that the striated layer becomes fibrous, thicker and two-layered. This basic plan of the cuticle has been found, with modifications (usually one or more fibrous layers beneath the striated layer and a membrane or membrane-like structure on the surface of the cuticle) in a number of larval nematodes and also in a few adults. Examples are : the third-stage larva of Nippostrongylus brasiliensis (see Lee, 1966a; Janiuar, 1966; Inatomi et al., 1970); the third-stage larva of Necator americanus, Ancylostoma duodenale, A . caninum, Trichostrongylus orientalis, Strongyloides stercoralis, S. fulleborni and S. ratti (see Inatomi et al., 1963), S. myoptonii (see Colley, 1970) and Haemonchusplacei(see Smith, 1970); the fourth-stage larva and adults of Panagrellus silusiue (see Samoiloff and Pasternak, 1968, 1969; Yuen, 1968); third-stage larva of Neoaplectana glaseri (see Jackson and Bradbury, 1970); the second-, third- and fourthstage larvae and adults of Hemicycliophora arenaria (these nematodes are unusual in that they also possess a multi-layered sheath), adults and fourthstage larva of Hirschmaniella gracilis and H. belli and all stages of Aphelenchus avenue (see Johnson et al., 1970a, b); developmental stages of Heterodera schachtii (see Gunther and Kampfe, 1967); the second-stage larva of Meloidogyne javanica (see Bird, 1968) and of M . haplu (see Ibrahim and Hollis, 1967); adults of Ditylenchus dipsaci (see Yuen, 1967); adults of Tylenchorhynchus

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martini (slight differences between swarming and non-swarming individuals) (see Ibrahim, 1967) ; adults of Capillaria hepatica and Trichuris myocastoris (see Wright, 1968) and T. suis (see Bogoyavlenski, 1965; Jenkins, 1969); adults of Rhabditispellio (no striated layer mentioned) (see Beams and Sekhon, 1967); and the microfilaria of Dirojlaria immitis (see Kozek, 1970b). The cuticle of the second-stage larvae of Ascaris lumbricoides has been briefly described by Thust (1966) and that of the third-stage larva of A . mum illustrated, but not described, by Morseth and Soulsby (1969). In these there does not appear to be a striated layer. The cuticle has an outer membrane, a thin dense outer cortex, a thicker less dense layer and then what appears to be a fibrillar layer. A rib-like structure is present in the lateral alae (based on the plates in Morseth and Soulsby, 1969). Bogoyavlenski has studied the structure of the cuticle of a number of different nematode groups. In the Spirurata he studied Crassicauda crassicauda, Ascarops strongylina, Habronema muscae and Echinuria uncinata. The following features were typical of these species : (1) a single cortical layer; (2) two homogeneous layers between the fibrous layers (in all except E. uncinata);(3) one or two well-developed basal layers (Bogoyavlenski, 1961). The cuticle of Dictyocaulus iiuiparous, Metastrongylus elongatus, M . salmi and M . pudentotectus are similar to one another, but differ considerably from that of intestinal strongylids and ascarids. The cuticle is very thin and consists of a cortex, a homogeneous layer, a fibre layer and a basal layer (Bogoyavlenski, 1963). The cuticles of Oesophagostomum columbianum and of Delafondia vulgaris, which are intestinal species, have eight layers (Bogoyavlenski, 1964a). The cuticle of Syngamus skrjabinomorpha has five layers (Bogoyavlenski (1964b) ; that of Amidostomum anseris, Epomidiostomum orispinum and Ostertagia ostertagi have six, six and four layers respectively. Differences in the cuticles of 0. ostertagi and Mecistocirrus digitatus are related to the fact that one lives in the mucosa, and the other in the lumen of the abomasum (Bogoyavlenski, 1964~). The cuticles of two metastrongyloid nematodes, Crenosoma vulpis and Perostrongylus pridhani, consists of a cortex, a matrix and a basal layer. The so-called teguminal sheath of these nematodes is not a true sheath but corresponds to the cortical layer of the cuticle. The very wide matrix layer, which separates the cortex from the basal layer, is thought to contain a fluid (Stockdale et al., 1970). The cuticle of the third-stage larvae of Contracaecirm multipapillaturn is rather similar to that of adult Ascaris lumbricoides but has one, not three, fibre layers. There is no striated layer present (Larsh et al., 1968). Bruce (1970) has described the formation of the first cuticle and the cuticles of the pre-infective and infective larvae and of the adult of Trichinella spiralis. The embryo in utero is covered by a thin sheet of mucus-like material and by the egg-shell. The early post-embryonic larvae is covered by this mucoid-looking material. Internal to this is a thin dense layer (precursor of the outer membrane) and a fine homogeneous layer attached to the hypodermis by half-desmosomes at crests in the hypodermis. The cuticle of the late-stage larva in utero has an outer membrane, a cortex and an inner fibrillar layer. The pre-infective larva,

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11-12 days after parturition, in the muscles of the host has a cuticle consisting of an outer membrane, a cortical layer, a medial layer and a fibril layer. That of the infective larva has an outer membrane, an outer and inner cortex, a thin dense medial layer and two fibril layers (see also Despommier et al., 1967). In the cuticle of the adult worm the medial layer is striated and the orientation of the fibrils in the two fibril layers is more clearly shown. The cuticle of the microfilaria of Litomosoides carinii has an outer and an inner membrane separated by a thin amorphous layer (Schardein et al., 1968). The cuticle of the fourth-stage larva of Nematospiroidesdubius is divided into six layers. The outer membrane is covered by fine filamentous material to which Thorotrast adheres. There is an outer and inner cortex, a matrix and two fibre layers. Dense bars are embedded in the matrix layer and extend into the longitudinal ridges of the cuticle. The outer membrane and the outer cortex of the adult cuticle are similar to those of the fourth-stage cuticle, but the rest of the cuticle is not clearly layered. Bars, which support the longitudinal ridges, are present (Bonner et al., 1970). The cuticle of the fourth-stage larva of Nippostrongylus brasilirnsis is similar to that of the adult (Lee, 1970b). Tsubota (1966) and Inatonii et al. (1970) have shown that the cuticle of Enterobius vermicularis is very similar in structure to that of Aspiculuris tetrupteru (Anya, 1966). The cuticle of Syphacia obveluta consists of cortical, matrix and basal layers in the larva, young female and mature male (Dick, 1970). There is a striated zone in the cortex; tubules were observed in all regions of the cuticle, except the cortex. The gravid female has a thicker cuticle and the matrix layer contains a lot of striated material. Regional differentiation of the cuticle gives rise to alae, annulae and striae. The cuticle of the cephalic regions of Trichodorus christiei consists of an outer and an inner cuticle with a space between. The outer cuticle is covered by a membrane and has an outer fibrous layer followed by an inner electron dense homogeneous layer. The inner cuticle consists of a homogeneous material (Hirumi et al., 1968). The cuticle of Xiphinema index has been re-described by Roggen et al. (1967). There is an outer membrane, a structureless layer, a layer consisting of regularly spaced longitudinal ribbons, three fibre layers, a layer of variable thickness consisting of a number of sub-layers, and a basal layer. The cuticle of Longidorus macrosoma has a similar structure (Aboul-Eid, 1969). Wright and Hope (1968) have shown that the punctations and pore complexes in the cuticle of Acanthonchus duplicatus are due to the presence of dense material within the middle layer of the cuticle in the form of rods and collar-like rings. The cuticle is three-layered. The pore passes through the cuticle into the hypodermis. They concluded that these pore complexes are not campaniform sense receptors. Lee (1970a) described the cuticle of adult female Mermis nigrescens. There is an epicuticle of muco-protein-like material on the surface membrane. The cortex is penetrated by canals which extend from the surface to the matrix of the layer beneath the cortex (Fig. 3). Two layers of giant fibres, which spiral

FIG.3A. Electron micrograph of L.S. outer layers of the cuticle of Mermis nigrescens to show the canals in the cortex and the outer layer of giant fibres. x 29 O00. FIG.3B. Electron micrograph of H. (horizontal) S . cortex of the cuticle of Mermis nigrescens to show the canals cut in cross section. x 36 000. c, canal; cl, cortical layer; gf, giant fibre.

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around the nematode, lie beneath the cortex (Fig. 3A) and beneath these is a thick layer containing a network of fibres and a basal layer. The canals in this cuticle are much smaller than the pore canal complex described by Wright and Hope (1968) for Acanthonchus. Riding (1970) has made the startling discovery that adult females of Bradynema sp., which live in the haemocoel of the mushroom fly Megaselia halterata, lack a cuticle. The hypodermis has a microvillous surface and that is the outer covering of the nematode. Deladenus siricidicola also has scattered clusters of microvilli on its surface. Green (1967), de Grisse and Lagasse (1969) and Ogden (1971) have used the scanning electron microscope to study the cuticular structure of nematodes. This technique is very useful in the study of surface configuration but does not reveal internal structure.

Moulting A number of important papers describing moulting and cuticle formation have appeared since the earlier review (1966) and that of Bird and Bird (I 969) were published. Roggen et al. (1967) briefly described moulting in Xiphinema index. Cyclical fluctuations of leucine aminopeptidase occur in the hypodermis and it is thought that this enzyme could be involved in the breakdown of the old cuticle and the synthesis of the new cuticle. Samoiloff and Pasternak (1969) described the formation of the new cuticle at each of the postpartum moults of Panagrellus silusiae; it is similar for each moult. At the beginning of the moult a fibrous material accumulates adjacent to the hypodermis. Eventually a new cuticle forms beneath thepre-existing one; this new cuticle lacks organization during the early stage of the moult. Shedding of the old cuticle varies at different moults. In the second and third moults and in the moult to the adult female, the cuticle is discarded gradually. At the moult to form the adult male the old cuticle splits and is shed in one piece. Resorption of the old cuticle does not occur. Johnson et al. (1970b) studied cuticle formation and moulting in Hemicyliophora arenaria, Aphelenchus avenue and Hirschrnanniella gracilis. In all parasitic stages of H . arenaria the moult commenced with the separation of the cuticle from the hypodermis. The new sheath and cuticle were then formed by the hypodermis. Most of the old cuticIe is apparently absorbed before ecdysis. Fourth larval stage males undergo a final moult-a double moult during which a sixth cuticle is formed. The sheath is produced at each moult and must be regarded as an integral part of the whole cuticle and not as a residual cuticle. Moulting in A. avenue and H. gracilis was less complex. After cuticle separation the hypodermis gave rise to a new three-layered membrane, the future cortex, the matrix and striated layers. The old cuticle broke down as moulting neared completion. Observations on the last moult of the developing female of Syphacia obvelata indicate that during formation of the new cuticle striated regions are laid down between projections of the hypodermis in close association with the cell surface (Dick, 1970). It is suggested that striated material is deposited at

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the cuticle-hypodermis interface and moves through spaces within the basal layer to the matrix layer where it is deposited. Thust ( I 966) stated that in the second moult of Ascaris lumbricoides, which occurs within the egg, the cortex separates from the middle layer and a new membrane forms on the outside of the middle layers. Subsequently the new cortex develops between the new membrane and the middle layer; all that remains of the moulted cuticle is the external cortical layer (Crandall and Arean, 1967). During cuticle formation in Nematospiroides dubius the outer hypodermal membrane becomes the outer membrane of the new cuticle. The cortex and matrix layers of the adult cuticle form by differentiation of hypodermis cytoplasm underneath the cuticle/hypodermis membrane. The fibre layers form under the matrix layer by secretion from within the hypodermis (Bonner et al., 1970). In the final moult of Nipposrrongylus brasiliensis the old cuticle separates from the hypodermal membrane and the new cuticle is secreted between the outer hypodermal membrane and a new hypodermal membrane which forms beneath it. The first layers to be formed under the outer membrane of the new cuticle are the two fibre layers (Fig. 4). Formation of the outer cortex and of the struts is associated with an increase in rough endoplasmic reticulum in the hypodermis. The struts separate out from the fibrillar and granular components of the outer cuticle (Fig. 5). Formation and secretion of* the cuticular substances by the hypodermis is similar to secretion of collagen by fibroblasts. There is no resorption of the old cuticle (Figs 4, 5) (Lee, 1970b). In Trichinellu spiralis the moulting infective larva in the intestine of the host forms a new cuticle beneath the old cuticle and this old cuticle is shed at an early stage in development of the new cuticle. The cast cuticle consists of the outer membrane, the medial layer and parts of the fibril layers; the rest of the old cuticle is apparently digested, possibly by digestive enzymes in the host’s intestine. A cast cuticle has not been observed around the encapsulated larva in the muscles of the host although a moult probably occurs there 12 days after parturition (Bruce, 1970). Kozek (1970a), however, states that all four moults occur in the intestine of the host. During formation of new cuticle the material of the cuticle is secreted into trough regions of the outer hypodermal membrane under the previously formed outer membrane of the cuticle (Bruce, 1970). According to Kan and Davey (1 968) the adult cuticle of Phocanema decipens is deposited at the edge of the hypodermis as three discrete layers of protein. The cortex is keratinized at the periphery as soon as it appears and later differentiates into two layers. The middle layer differentiates into three bands, apparently by the secretion of more material from the hypodermis. The larval cuticle becomes loosened from the developing adult cuticle as soon as the second primary layer is formed, but ecdysis does not occur in culture until the new cuticle is fully differentiated. Ecdysis is also described by Davey and Kan (1968). According to Kampfe (1966) the last moult of various cyst-forming

FIG.4. Electron micrograph of L.S. body wall of a moulting fourth-stage larva of Nippostrongylus brasiliensis (middle stages of moulting). x 26 000. h, hypodermis; mu, muscle; ncu, new cuticle; ocu, old cuticle.

FIG.5. Electron micrograph of L.S. body wall of a moulting fourth-stage larva of Nippostrongylris brusiliensis (late stages of moulting). x 20 OOO. h, hypodermis; ncu, new cuticle; ocu. old cuticle.

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nematodes may be a partial one in that some layers of the cuticle are retained while a new basal layer is formed. It would appear that moulting differs in different types of nematode and sometimes in different stages of the life cycle. Some species break down and resorb much of the old cuticle whereas others discard it almost intact. This may be related to the environment of the nematode. For example, it is to the advantage of a moulting larva inside an egg to reduce the thickness of the old cuticle, as it has to live within this cast cuticle in the confines of the egg until it is released from the egg. A thin flexible cuticular remnant allows greater growth and freedom of movement than a thicker intact cast cuticle. The larva also depends upon stored foods in the egg for its development and will therefore have a greater need for the amino acids and carbohydrates of the broken down cuticle than will nematodes which are able to feed freely on exogenous nutrients. Similarly, nematodes which are fixed in one place in the tissues of the host animal or plant, e.g. Meloidogyne and infective larvae of Trichinella spiralis, will obtain an advantage if they greatly reduce the thickness of the old cuticle; the cuticle will be more pliable and, being thinner, will be easier to break by their restricted body movements. Actively moving nematodes will be able to free themselves of thicker cuticles more easily and, especially i n nematodes parasitic in the alimentary tract of animals, the old thick cuticle will also protect the nematode while the new cuticle is thickening and becoming resistant. B.

FUNCTION

There is little to add to the comments given in the earlier review on the function of the cuticle of nematodes. The lack of a cuticle and the presence of microvilli on the body wall of some insect parasitic nematodes is obviously related to the uptake of nutrients through the body wall of these nematodes (Riding, 1970). The function of the pores in the cuticle of Acanrhoncus (Wright and Hope, 1968) and of the small canals in the cuticle of Mermis (Lee, 1970a) is obscure; it may be that in Mermis they secrete the epicuticle. It has been suggested by Wright (1968) that the striated layer, which occurs in the cuticle of many nematodes, provides the tensile strength given by fibre layers in the cuticle of other species of nematode. Lee (1969) has suggested that in those nematodes which have broad, blunt lateral alae, such as the third-stage larvae of Nippostrongylus brasiliensis, the shape of the alae is such that they give the larva a stable base during movement, as nematodes lie on their sides and move by means of dorso-ventral undulations. The alae, especially the longer, sharper type, will also act as a fin during swimming. The fluid-filled layer in the lateral alae of the third-stage larva of N . brasiliensis may reduce the effect of shearing forces set up in this part of the cuticle when the larva moves in thin films of moisture by increasing the elasticity of that part of the cuticlewhichlies between the substratum and the contracting muscles of the nematode. Lee (1969) has also suggested that the fluid-filled layer of the cuticle of adult N . brusiliensis may play an important role in locomotion.

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VIII. ACANTHOCEPHALA A.

LARVAL FORMS

Butterworth (1969) has described the development of the body wall of Polymorphus minutus in its intermediate host. The cortex of the early acanthella is a syncytium which contains giant nuclei, mitochondria, Golgi bodies, endoplasmic reticulum and a few lipid droplets. The cuticle, which is similar to that of the adult, is penetrated by pores which open into vesicles in a vesicular region below the cuticle. The pores in the cuticle increase in number as the animal grows. A pore canal layer develops beneath the pores in the cuticle towards the end of development of the late acanthella stage. The outer cortex of the cystacanth has a cuticle and pore canals similar to that of the adult. The body wall of the acanthor of Moniliformis dubius is composed of several components. The outer three-layered membrane is associated externally with an amorphous material. Two felt-like layers, separated by a cytoplasmic area, lie beneath the surface membrane. The surface membrane is unfolded to form intra-hypodermal crypts; these crypts are lined by a membrane which is extensively evaginated to form microvilli within the crypt. The crypts and labyrinths associated with them open at the surface by means of pores (Wright and Lumsden, 1970). B.

ADULT Acanthocephala

Hammond (1967) described the fine structure of the trunk and praesoma wall of Acanthocephalus ranae. It is similar in many ways to that of other adult acanthocephala described in the earlier review. The wall of both the trunk and of the praesoma are similar. There is an external membrane, a cuticle, a striped layer, a canal layer, central layers containing the so-called lacunar system (see Butterworth, 1969) and a folded basement membrane. The wall of the praesoma is much thinner than that of the trunk. Hammond (1968) also used the scanning electron microscope to study the body surface of A . ranae, Echinorhynchus truttae and Pomphorhynchus laevis. Pores were clearly seen on the trunk surface of A. ranae but were not seen in the other two species, possibly because they were masked with mucus. Most of the pores on the proboscides were associated with discrete masses of material which was apparently exuded from the pores. Wright and Lumsden (1969) redescribed the body wall of Moniliformis dubius and also the epicuticle (1968). The epicuticle is a filamentous coat external to the superficial membrane of the body wall and intimately associated with the structure. It is similar cytochemically and morphologically to a glycocalyx and, as such, is an integral part of the surface of the parasite. C.

FUNCTION

The epicuticle probably serves as a protective layer and possibly for the adsorption of enzymes for contact digestion. The pore-canal complex is probably a stable, structural specialization for increasing the free surface of the

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parasite and for chemical exchange with the environment (Wright and Lumsden, 1969). Rothman (1967) has localized alkaline and acid phosphatases on the membranes lining the canals of Moniliformis dubius and these may be involved in the uptake of materials by the worm.

IX. GENERAL SUMMARY In the earlier review I indicated that work on the ultrastructure of the Monogenea, the Temnocephalida, the larval stages of Platyhelminthes and freeliving nematodes would help to fill many gaps in our knowledge of the structure and origins of the outer covering of helminths. In this up-dated review much of the space has been devoted to work carried out on the Monogenea, the larval stages of Platyhelminthes, the larval stages of nematodes and moulting in nematodes, but work is still needed on the Temnocephalida, on more freeliving nematodes, on more Monogenea and Turbellaria and on the embryology and development of the outer covering of all of the Platyhelminthes. The description of the outer covering of an endoparasitic turbellarian has been of particular interest because although the epidermis is ciliated it is also covered with many long microvilli and the epidermis appears to become syncytial by the breakdown of the lateral membranes of the epidermal cells. On present evidence it would appear that the outer covering of the Monogenea may vary in structure. The epidermis of some monogeneans is apparently very similar to that of the Digenea in that it is a syncytial epidermis with cytoplasmic connections to nucleated cell-like bodies sunk in the parenchyma, whereas in others, notably Gyrodactylus, nuclei and sunken nucleated celllike bodies are not associated with the epidermis. In the one species which has been studied the original ciliated cells of the early embryo are replaced by other ciliated cells which arise in the parenchyma, as in the embryo of free-living turbellaria. The unciliated adult epidermis initially has superficial nuclei and appears under these ciliated cells. In the Digenea the change from miracidium to sporocyst also involves shedding of the ciliated epidermal cells and the formation of a syncytial epidermis from cells which lie below the muscles. The epidermis of the sporocyst is covered with cytoplasmic folds or microvilli and is usually in cytoplasmic continuity with nucleated cell-like bodies in the parenchyma, although in one example nuclei are present in the epidermis. The epidermis of the redia is similar in the few species which have been studied. The syncytial epidermis appears to have no cytoplasmic connection with cells in the parenchyma; nuclei have not been detected but mitochondria are present. The outer surface is thrown into folds or microvilli. There is still confusion about the origin of the outer layer of the cercaria. Some authors claim that the outer layer of the cercariae developing inside the redia is formed by cells of the redia and that once the germinal ball is a multicellular mass then this layer is shed. The layer which is to be the epidermis of the cercaria is formed from peripherally located cells of the germinal ball. Other authors state that the outermost layer of the cercaria within the redia

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is an embryonic epithelium, that this forms the epidermis of the cercaria and that it is shed when the cyst of the metacercaria is formed. Other authors claim that during cyst formation the epidermis is altered but not shed and that it becomes the epidermis of the metacercaria and then the adult. Whatever its origin, the epidermis of emerged cercariae is very similar in all species which have been examined. There is a syncytial epidermis, containing mitochondria but no nuclei, which is in cytoplasmic continuity with nucleated cell-like bodies in the parenchyma. There are variations in structure of the epidermis in different regions of the body, especially on the tail. Recent work on the epidermis of adult digeneans has been briefly summarized as it has confirmed descriptions of the epidermis of other species described in the earlier review. One important point which is emerging from this work is the fact that surface specializations of the epidermis do occur in different regions of the adult worm, especially in the strigeids. Recently it has been suggested that the Cestodaria are either monogeneans or that they occupy a phylogenetic position between the monogeneans and the cestodes. Ultrastructural work on one species has shown that the epidermis resembles that of cestodes more than that of monogeneans but this could be adaptation to the environment and does not invalidate the theory. The cytoplasmic epidermis bears numerous microvilli on its surface and is in cytoplasmic continuity with nucleated cell-like bodies in the parenchyma. The microvilli lack the dense spine-like tip which is so characteristic of the microvilli of adult cestodes. The development of the epidermis of adult cestodes still needs much work to clarify the situation, especially from the embryological point of view. The epidermis of adult cestodes has been confirmed as an outer cytoplasmic syncytial epidermis which is covered with microvilli bearing electron-dense spine-like tips, and in cytoplasmic continuity with nucleated cell-like bodies in the parenchyma. A characteristic feature of most of the parasitic Platyhelminthes, including many of the larval stages, is the presence of a syncytial epidermis which is in cytoplasmic continuity with nucleated cell-like bodies in the parenchyma, There is accumulating evidence that these sunken cells are not permanently in contact with the epidermis but that they migrate from within the parenchyma to make contact with the epidermis and then perhaps lose contact once their function at the epidermis is complete. The numbers associated with the epidermis could be related to the activity or degree of growth of the epidermis. If this is so then it could help to explain why these cells have not been found, or are only rarely found, associated with the epidermis of some species or stages in the life cycle. The cuticle of many different stages and species of nematode have now been examined with the electron microscope and a certain type of cuticle structure appears to be common to most larval nematodes and also to some adults. There is an outer layer which is similar to, and may be, a thick unit membrane; a cortical layer which is usually sub-divided; a striated layer; and a thin fibrillar (as distinct from a fibre) layer. The cuticle of adult nematodes appears to vary considerably from species

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to species and can usually be related to the nature of the environment. The most extreme cases are in some insect-parasitic nematodes where the cuticle is almost completely absent and the hypodermis has developed microvilli. Several examples of moulting in nematodes have been described and it would appear that moulting differs in different types of nematode and sometimes in different stages of the life cycle. Some species resorb much of the old cuticle whereas others discard it almost intact. It has been suggested that this may be related to the environment of the nematode. The body wall of some larval stages of two acanthocephalans has been described by means of electron microscopy and shown to be rather similar to that of the adult. More work has also been done on the body wall of adult acanthocephalans and has extended previous descriptions.

REFERENCES

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Baron, P. J. (1971). On the histology, histochemistry and ultrastructure of the cysticercoid of Raillietinu cesticillus (Molin, 1858) Fuhrmann, 1920 (Cestoda, Cyclophyllidea). Parasitology 62, 233-245. Beams, H. W. and Sekhon, S. S. (1967). Fine structure of the body wall and cells i n the pseudocoelom of the nematode Rhabditispellio. J. ultrastr. Res. 18, 580-594. Begiun, F. (1966). etude au microscope electronique de la cuticle et de ses structures associees chez quelques cestodes. l h a i d’histologie comparee. Z . Zellforsch. 72, 3 0 4 6 . Belton, C. M. and Harris, P. J. (1967). Fine structure of the cuticle of the cercaria of Acanthatrium oreganense (Macy). J. Parasit. 53, 71 5-724. Bilqees, F. M. (1970). Histological study of external budding in Taenia crassiceps. Aust. J. ZOO^. 18, 1-7. Bils, R. F. and Martin, W. E. (1966). Fine structure and development of the trematode integument. Trans. Am. microsc. Soc. 85, 78-88. Bird, A. F. (1968). Changes associated with parasitism in nematodes. 111. Ultrastructure of the egg shell, larval cuticle, and contents of the subventral esophageal glands in Meloidogyne javanica, with some observations on hatching. J. Parasit. 54,475489.

Bird, A. F. and Bird, J. (1969). Skeletal structures and integument of Acanthocephala and Nematoda. In “Chemical Zoology”, Vol. 3, 253-288. Academic Press, New York. Bogitsh, B. J. (1968). Cytochemical and ultrastructural observations on the tegument of the trematode Megalodiscus temperatus. Trans. Am. microsc. SOC.87, 477-486.

Bogitsh, B. J. and Aldridge, F. P. (1967). Histochemical observations on Posthodiplostomurn minimum. IV. Electron microscopy of tegument and associated structures. Expl Parasit. 21, 1-8.

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Bogoyavlenski, Y. K. (1961). [Comparative histological study of the cuticle structure in different groups of Spirurata.] Helminthologia 3 , 3 8 4 6 . Bogoyavlenski, Y . K. (1963). [Structure of the cuticle and hypodermis of strongylata parasitic in the trachea and bronchi of mammals.] Helminthologia 4, 89-94. Bogoyavlenski, Y . K. (1964a). [On the microscopical structure of the integuments of some intestinal Strongylata.] Helminthologia 5, 167-1 72. Bogoyavlenski, Y. K . ( I 964b). [Comparative histological analysis of the covering tissues of pulmonary nematodes of the suborder Strongylata and some observations on their phylogeny.] Trudy gel’mint.Lab. 14,80-86. Bogoyavlenski, Y . K. (1964~).[New data on the histological structure o f the covering tissues of some nematodes of the suborder Strongylata.] Trudygel’mint. Lab. 14, 87-92. Bogoyavlenski, Y. K. (1965). [Comparative histological and histochemical study of the cuticle and hypodermis of trichuroids.] Trudygel’mint.Lab. 15,45-54. Bonner, T. P., Menefee, M. G. and Etges, F. J. (1970). Ultrastructure of cuticle formation in a parasitic nematode, Nematospiroides dubius. Z . Zellforsch. 104, 193-204. BrAten, T. (1968a). An electron microscope study of the tegument and associated structures of the procercoid of Diphyllobothrium latum (L). Z . ParasitKde 30, 95-103. Briten, T. (1968b).The fine structure of the tegument of Diphyllobothrium latum (L.). A comparison of the plerocercoid and the adult stages. Z . ParasitKde 30,104-1 12. Bresciani, J. and Kgie, M. (1970). On the ultrastructure of the epidermis of the adult female of Kronborgia amphipodicola Christensen & Kanneworff, 1964 (Turbellaria, Neothabdocoela). Ophelia 8, 209-230. Bruce, J. I., Pezzlo, F., McCarty, J. E. and Yajima, Y. (1970). Migration of Schistosoma mansoni through mouse tissue. Ultrastructure of host tissue and integument of migrating larva following cercarial penetration. Am. J. trop. Med. Hyg. 19, 959-98 I . Bruce, R. G. (1970). Trichinella spiralis: fine structure of body wall with special reference to formation and moulting of cuticle. ExplParasit. 28,499-51 1. Burton, P. R. (1966). The ultrastructure of the integument of the frog bladder fluke, Gorgoderina sp. J. Parasit. 52, 926-934. Butterworth, P. E. (1969). The development of the body wall OfPolymorphus minutus (Acanthocephala) in its intermediate host Gammarus pulex. Parasitology 59, 373-388. Charles, G. H. and Orr, T. S. C. (1968). Comparative fine structure of outer tegument of Ligula intestinalis and Schistocephalus solidus. Expl Purasit. 22, 137-149. Clegg, J. A., Smithers, S. R. and Terry, R. J. (1971). Concomitant immunity and host antigens associated with schistosomiasis. Int. J. Parasit. 1 , 4 3 4 9 . Colley, F. C. (1970). Strongyloides myopotomi: fine structure of the body wall and alimentary tract of the adult and third-stage larva. Expl Parasit. 28, 420434. Collin, W. K. (1969). The cellular organization of hatched oncospheres of Hymenorepis citelli (Cestoda, Cyclophyllidea). J. Parasit. 55, 149-166. Collin, W. K. (1970). Electron microscopy of postembryonic stages of the tapeworm, Hymenolepis citelli. J. Parasit. 56,1159-1 170. Crandall, C. A. and Aredn, V. M. (1967). Electron microscope observations on the cuticle and submicroscopic binding of antibody in Ascaris swum larvae. J. Parasit. 53, 105-109. Davey, K. G. and Kan, S. P. (1968). Molting in a parasitic nematode, Phocanerna decipens. IV. Ecdysis and its control. Can.J. Zool. 46,893-898.

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Dawes, B. (1960). A study of the miracidium of Fasciola hepatica and an account of the mode of penetration of the sporocyst into Lymnaea triincatula. In: “Libro Homenage al Dr. Eduardo Caballero y Caballero.” Jubileo 1930-1960, 95-1 I I . Escuela Nacional de Ciencias Biologicas, Mexico. Despommier, D. D., Kajima, M. and Wostmann, B. S. (1967). Ferritin-conjugated antibody studies on the larvae of Trichinella spiralis. J. Parasit. 53,618-624. Dick, T. A. (1970). Observations of the fine structure and development of the cuticle of the mouse pinworms, Syphacia obvelata and Aspiciiluris tetraptera. In Proc. 2nd Int. Congress Parasit. J. Parasit. 56, Section 2, 80. Dixon, K. E. (1970). Absorption by developing cercariae of Cloacitrema narrabeetietisis (Philophthalmidae). In Proc. 2nd Int. Cong. Parasit. J. Parasit. 56, Section 2, 416-417. Dixon, K. E. and Mercer, E. H. (1967). The formation of the cyst wall of the metacercaria of Fasciola hepatica. Z . Zellforsch. 77, 345-60. Erasmus, D. A. (1967). The host-parasite interface of Cyathocotyle bushiensis Khan, 1962 (Trematoda: Strigeoidea). 11. Electron microscope studies of the tegument. J. Parasit. 53, 703-714. Erasmus, D. A. (1968). The host-parasite interface of Cyathocotyle bushirnsis Khan, 1962 (Trematoda: Strigeoidea). 111. Electron microscope observations on nonspecific phosphatase activity. Parasitology 58, 371-375. Erasmus, D. A. (1969a). Studies on the host-parasite interface of strigeoid trematodes. IV. The ultrastructure of the lappets of Apatemon gracilis minor Yamaguti, 1933. Parasitology 59, 193-201. Erasmus, D. A. (1969b). Studies on the host-parasite interface of strigeoid trematodes. V. Regional differentiation of the adhesive organ of Apatemon gracilis minor Yamaguti, 1933. Parasitology 59, 245-256. Erasmus, D. A. (1 969c). Studies on the host-parasite interface of strigeoid trematodes. VI. Ultrastructural observations on the lappets of Diplostomum phoxini Faust, 1918. Z . ParasitenKde 32,48-58. Erasmus, D. A. (1970a). The host-parasite interface of strigeoid trematodes. VII. Ultrastructural observations on the adhesive organ of Diplostomurn phoxini Faust, 1918. Z . ParasitenKde 33, 21 1-224. Erasmus, D. A. (1970b). The host-parasite interface of strigeoid trematodes. VI11. Surface specialization of the adhesive organ of Cardiocephaloides physalis (Lutz, 1926). Experientia 26, 439-441. Erasmus, D. A. (19704. The host-parasite interface of strigeoid trematodes. IX. A probe and transmission electron microscope study of the tegument of Diplnstomumphoxini Faust, 1918. Parasitology 61, 3541. Ginetsinskaya,T. A., Mashanski, U. F. andDobrovolski, A. A. (1966). [Ultrastructure of the wall and method of feeding of redia and sporocysts (trematodes).] Dokl. Akad. Nauk SSSR 166, 1003-1004. Green, C. D. (1967). Preparation of nematodes for examination under the Stereoscan electron microscope. Nematologica 13, 279-282. de Grisse, A. and Lagasse, A. (1969). L‘utilisation du microscope dlectronique a balayage dans I’etude des nematodes. J. Microsc. 8,677-680. Gunther, B. and Kampfe, L. (1967). Bau und Veranderung des Integumentes im Entwicklungszyklus cystenbildender Nematoden. Verh. dt. Zool. Ges. Year 1966, 152-1 66. Gupta, B. L. and Little, C . (1970). Studies on Pogonophora. 4. Fine structure of the cuticle and epidermis. Tissue & Cell 2, 637-696. Hammond, R. A. (1967). The fine structure of the trunk and praesoma wall of Acanthorephalus ranae (Schrank, 1788), Liihe, 1911 . Parasitology 57,475-486.

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Hammond, R. A. (1968). Observations on the body surface of someacanthocephalans. Nature, Lond. 218, 872-873. Hirumi, H., Chen, T. A., Lee, K. J. and Maramorosche, K. (1968). Ultrastructure of the feeding apparatus of the nematode Trichodorus christiei. J. ultrastr. Res. 24,434-453. Hockley, D. J. (1968). Scanning electron microscopy of Schistosoma mansoni cercariae. J. Parasit. 54, 1241-1243. Hockley, D. J. (1970a). The development of the tegument of Schistosoma mansoni. In Proc 2nd Int. Cong. Parasit. J. Parasit. 56, Section 2, 150-1 51. Hockley, D. J. (1970b). Ultrastructure of the outer membrane of Schistosoma mansoni. In Proc 2nd Int. Cong. Parasit. J. Parasit. 56, Section 2, 151. Howells, R. E. and Erasmus, D. A. (1969). Histochemical observations on the tegumentary epithelium and interproglottidal glands of Moniezia expansa. Parasitology 59,505-51 8. Ibrahim, I. K. A. (1967). Morphological differences between the cuticle of swarming and nonswarming Tylenchorhynchus martini. Proc. helminth. SOC. Wash. 34, 18-20. Ibrahim, I. K. A. and Hollis, J. P. (1967). Cuticle ultrastructure of the Meloidogyne hapla larva. Proc. helminth. SOC.Wash. 34, 137-1 39. Inatomi, S., Sakamoto, D. Itano, K. and Tanaka, H. (1963). [Studies on the submicroscopic structure of body surface of larval nematodes.] Jap. J. Parasit. 12, 16-39. Sakamoto, D., Itano, K., Suguri, S. and Ito, Y.(1968a). [The Inatomi, S.,Tongu, Y., ultrastructure of helminths. I. The body wall of Clonorchis sinensis (Cobbold, 1875) Looss, 1907.1Jap. J. Parasit. 17, 395-401. Inatomi, S., Tongu, Y.,Sakamoto, D., Suguri, S. and Itano, K. (1968b). [The ultrastructure of helminths. 2. The body wall of Metagonimus yokagawi takahashii Suzuki, 1930.1Jap. J. Parasit. 17,455-460. Inatomi, S . Tongu, Y.,Sakamoto, D., Suguri, S. and Itano, K. (1969). [The ultrastructure of helminths. 3. The body wall of Schistosoma japonicum.] Jap. J. Parasit. 18, 174-181. Inatomi, S., Sakumoto, D., Tongu, Y.,Suguri, S. and Itano, K. (1970). “Electron Micrograph of Helminth.” The 20th ann. Publ., Dept. of Parasitology, Okayama University Medical School, Okayana, Japan. Jackson, G. J. and Bradbury, P. C. (1970). Cuticular fine structure and molting of Neoaplectana glmeri (Nematoda), after prolonged contact with rat peritoneal exudate. J. Parasit. 56, 108-1 15. James, B. L., Bowers, E. A. and Richards, J. G. (1966). The ultrastructure of the daughter sporocyst of Cercaria bucephalopsis haimaena Lacaze-Duthiers, 1854 (Digenea: Bucephalidae) from the edible cockle, Cardum edule L. Parasitology 56,752-762. Jamuar, M. P. (1966). Electron microscope studies on the body wall of the nematode Nippostrongylusbrasiliensis.J. Parasit. 52,209-232. Jenkins, T. (1969). Electron microscope observation of the body wall of Trichuris suis, Schrank, 1788 (Nematoda: Trichuroidea). 1. The cuticle and bacillary band. Z. ParasitenKde 32, 374-387. Johnson, P. W., Van Gundy, S. D. and Thomson, W. W. (1970a). Cuticle ultrastructure of Hemicycliophora arenaria, Aphelenchus avenue, Hirschmaniella gracilis and Hirschmaniella belli. J. Nematol. 2, 42-58. Johnson, P. W., Van Gundy, S. D. andThomson, W. W. (1970b). Cuticle formation in Hemicycliophora arenaria, Aphelenchus avenue and Hirschmaniella gracilis. J. Nematol. 2, 59-79. 16

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Kampfe, L. (1966). Okologische und histologische Bemerkungen zur Cystenbildung bei Nematoden. Mitt. biol. Bund Anst. Ld-u. Forstw. 118,5470. Kan, S . P. and Davey, K. G. (1968). Molting in a parasitic nematode, Phoconema decipens. 111. The histochemistry of cuticle deposition and protein synthesis. Can. J. Zool. 46, 723-727. Kemp, W. M. (1970). Ultrastructure of the cercarienhiillen reaktion of Schistosoma mansoni. J. Parasit. 56, 713-723. Kozek, W. J. (1970a). Molting pattern in the larval stages of Trichinella spiralis. In Proc. 2nd Int. Cong. Parasit. J. Parasit. 56, Section 2, 192. Kozek, W. J. (1970b). Ultrastructure of the microfilaria of Dirofilaria immitis. J . Parasit. 56, Section 2, 192-193. Krupa, P. L., Bal, A. K. and Cousineau, G. H. (1967). Ultrastructure of the redia of Cryptocotyle lingua. J. Parasit. 53,725-734. Krupa, P. L., Cousineau, G. H. and Bal, A. K. (1968). Ultrastructural and histochemical observations on the body wall of Cryptocotyle lingua rediae (Trematoda). J. Parasit. 54, 900-908. Larsh, J. E., Huizinga, H. W., Race, G . J. and Martin, J. H. (1968). A study of the body wall of the third-stage larva of Contracaecum multipapillatum by electron microscopy. J. Elisha Mitchell Scient. SOC.84, 285-292. Lee, D. L. (1966a). An electron microscope study of the body wall of the third-stage larva of Nippostrongylus brasiliensis. Parasitology 56, 127-1 35. Lee, D. L. (1966b). The structure and composition of the helminth cuticle. “Advances in Parasitology”, Vol. 4 (Ed. Ben Dawes), 187-254. Academic Press, London. Lee, D. L. (1969). “Nippostrongylus and Toxoplasma.” Symposia of the British Society for Parasitology 7,3-16. Blackwell, Oxford. Lee, D. L. (1970a). The ultrastructure of the cuticle of adult female Mermis nigrescens (Nematoda). J . Zoo. SOC.Lond. 161,513-518. Lee, D. L. (1970b). Moulting in nematodes: the formation of the adult cuticle during the final moult of Nippostrongylus brasiliensis. Tissue & Cell 2, 139-1 53. Llewellyn, J. (1965). “Evolution of Parasites.” Symposia of the British Society for Parasitology 3,47-78. Blackwell, Oxford. Logachev, E. D. (1964). p h e functional histology of Ornithobilharzia turkestanicum.] In “Parasites of Farm Animals in Kazakhstan” (Ed. S . N. Boev). Alma-Ata: Izdatel. Akad. Nauk Kazakh. SSR 3, 104-107. Lumsden, R. D. (1966). Cytological studies on the absorptive surfaces of cestodes. I. The fine structure of the strobilar integument. Z . ParasitKde 27, 355-382. Lumsden, R. D., Oaks, J. A. and Alworth, W. L. (1970a). Cytological studies on the absorptive surfaces of cestodes. IV. Localization and cytochemical properties of membrane-fixedcation binding sites. J. Parasit. 56,736-747. Lumsden, R. D., Threadgold, L. T., Oaks, J. A. and Arme, C . (1970b). On thepermeability of cestodes to colloids : an evaluation of the transmembranosis hypothesis. Parasitology 60, 185-193. Lyons, K. M. (1968). A comparison of the adult epidermis of some monogeneans: the development of the outer layer of Entobdella soleae. Parasitology 58,14 pp. Lyons, K. M. (1969). The fine structure of the body wall of Gyrocotyle urna. Z. ParasitenKde 33,95-109. Lyons, K. M. (1970a). The fine structure and function of the adult epidermis of two skin parasitic monogeneans, Entobdella soleae and Acanthocotyle elegans. Parasitology 60, 39-52. Lyons, K. M. (1970b). Fine structure of the outer epidermis of the viviparous monogenean Gyrodactylus sp. from the skin of Gasterosteus aculeatus. J. Parasit. 56.1110-1117.

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Lyons, K. M. (19711. Comparative electron microscope studies on the epidermis of the blood living juvenile and gill living adult stages of Amphibdella fiavolineata (Monogenea) from the electric ray Torpedo nobiliana. Parasitology 63, 18 1-1 90. Matricon-Gondran, M. (1969). Btude ultrastructurale du syncytium tegumentaire et de son evolution chez des Trematodes Digenetiques larvaires. C . r. Acad. Sci. Hebd. Paris skrie D 269,2384-2387. Morris, G. P. (1971). The fine structure of the tegument and associated structures of the cercaria of Schistosoma mansoni. 2.ParasitenKde 36, 15-3 1. Morris, G. P. and Finnegan, C. V. (1969). Studies of the differentiating plerocercoid cuticle of Schistocephalus solidus. 11. The ultrastructural examination of cuticle development. Can. J. Zool. 47,957-964. Morris, G . P. and Halton, D. W. (1971). Electron microscope studies of Diclidophora merlangi (Monogenea; Polyophisthocotylea). 11. Ultrastructure of the tegument. J. Parasit. 57,49-61. Morris, G. P. and Threadgold, L. T. (1968). Ultrastructure of the tegument of adult Schistosoma mansoni.J. Parasit. 54, 15-27. Morseth, D. J. (1966). The fine structure of the tegument of adult Echinococcus granulosus, Taenia hydatigena, and Taenia pisiformis. J. Parasit. 52, 10741085. Morseth, D. J. (1967). Fine structure of the hydatid cyst and protoscolex of Echinococcus granulosus.J. Parasit. 53, 312-325. Morseth, D. J. and Soulsby, E. J. L. (1969). Fine structure of leukocytes adhering to the cuticle of Ascaris mum larvae. 1. Pyroninophils. J. Parasit. 55,22-31. Nieland, M. L. (1968). Electron microscope observations on the egg of Taenia taeniaeformis.J. Parasit. 54, 957-969. Nieland, M. L. and Weinbach, E. C. (1968). The bladder of Cysticercusfasciolaris: electron microscopy and carbohydrate content. Parasitology 58, 489-496. Ogden, C. G. (1971). Observations on the systematics of nematodes belonging to the genus Syphacia Seurat, 1916. Bull. Br. Mus. nut. Hist. (Zool.)20, 255-280. Ohman, C. (1965). The structure and function of the adhesive organ in strigeid trematodes. Part 11. Diplostomum spathaceum Braun, 1893. Parasitology 55, 481-502. Ohman, C . (1966). The structure and function of the adhesive organ in strigeid trematodes. Part 111. Apatemon gracilis minor Yamaguti, 1933. Parasitology 56,209-226. Race, G. J., Larsh, J. F., Esch, G. W. and Martin, J. H. (1965). A study of the larval stage of Multiceps serialis by electron microscopy. J. Parasit. 51, 364-369. Race, G. J., Larsh, J. F., Esch, G. W. and Martin, J. H. (1966). A study of the adult stage of Taenia multiceps (Multiceps serialis) by electron microscopy. J. Elisha Mitchell Scient. SOC.82, 44-57. Rees, G. (1966). Light and electron microscope studies of the redia of Parorchis acanthus Nicoll. Parasitology 56, 589-602. Rees, G. (1967). The histochemistry of the cystogenous gland cells and cyst wall of Parorchis acanthus Nicoll, and some details of the morphology and fine structure of the cercaria. Parasitology 57,87-110. Rees, G . (1971a). The ultrastructure of the epidermis of the redia and cercaria of Parorchis acanthus, Nicoll. A study by scanning and transmission electronmicroscopy. Parasitology 62, 479-488. Rees, G. (1971b). Locomotion of the cercaria of Parorchis acanthus, Nicoll and the ultrastructure of the tail. Parasitology 62,489-503. Reznik, G. K. (1966). [New data on the structure of the wall of Dicrocoeliumdendriticum from the liver of animals.] Dokl. Akad. Nauk SSSR 171,758-759.

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Riding, I. L. (1970). Microvilli on the outside of a nematode. Nature, Lond. 226, 179-180. Rifkin, E. (1970).An ultrastructural study of the interaction between the sporocysts and the developing cercariae of Schistosoma mansoni. In Proc. 2nd Int. Cong. Parasit. J. Parasit. 56,Section 2,284. Rifkin, E., Cheng, T. C. and Hohl, H. R. (1970).The fine structure of the tegument of Tylocephalum metacestodes: with emphasis on a new type of microvilli. J. Morph. 130,ll-24. Roggen, D. R., Raski, D. J. and Jones, N. 0. (1967). Further electron microscopic observations of Xiphinema index. Nematologica 13,1-16. Rothman, A. H. (1967). Ultrastructural enzyme localization in the surface of Moniliformis dubius (Acanthocephala). Expl Parasit. 21,42-46. Sakamoto, T. and Sugimura, M. (1969). [Studies on echinococcosis. XXI. Electron microscopical observations on general structure of larval tissue of multilocular echinococcus.] Jap. J. Vet. Res. 17,67-80. Sakamoto, T. and Sugimura, M. (1970). [Studies on echinococcosis. XXIII. Electron microscopical observations on histogenesis of larval Echinococcus multilocularis.] Jap. J. vet. Res. 18,131-144. Samoiloff, M. R. and Pasternak, J. (1968). Nematode morphogenesis: fine structure of the cuticle of each stage of the nematode, Panagrellus silusiae (de Man 1913) Goodey 1945.Can. J. Zool. 46,1019-1022. Samoiloff, M. R. and Pasternak, J. (1969).Nematode morphogenesis: fine structure of the molting cycles in Panagrellus silusiae (de Man 1913)Goodey 1945. Can. J. ZOO^. 47,639-643. Schardein, J. L., Lucas, J. A. and Dickerson, C. W.(1968).Ultrastructural changes in Litomosoides carinii microfilariae in gerbils treated with diethylcarbamazine. J. Parasit. 54, 351-358. Silk, M. H., Spence, I. M. and Buch, B. (1970).Observations of Schistosoma mansoni blood flukes in the scanning electron microscope. S. Afr. J. med. Sci. 35, 23-29. Silk, M. H., Spence, I. M. and Gear, J. H. S. (1969). Ultrastructural studies of the blood fluke-Schistosoma mansoni. I. The integument. S. Afr. J. med. Sci. 34, 1-10. Skaer, R. J. (1965).The origin and continuous replacement of epidermal cells in the planarian Polycelis tenuis (Iijima). J. Embryol. exp. Morph. 13, 129-1 39. Slais, J. (1966).The importance of the bladder for the development of the cysticercus. Parasitology 56,707-713. Slais, J. (1970).The electron-microscopical characteristics of various components of the bladder wall of the cysticercus. In Proc. 2nd Int. Cong. Parasit. J. Parasit. 56,Section 2, 320-321. Smith, K. (1970).Electron-microscopical observations on the body wall of the third stage larva of Haemonchusplacei. Parasitology 60,411-416. Smith, J. H.,Reynolds, E. S. and von Lichtenberg, F. (1969). The integument of Schistosoma mansoni. Am. J. trop. Med. Hyg. 18,28-49. Smithers, S . R. and Terry, R. J. (1969).Immunity in schistosomiasis.Ann. N. Y. Acad. Sci. 160,826-840. Smithers, S . R., Terry, R. J. and Hockley, D. J. (1969). Host antigens in schistosomiasis. Proc. R. SOC.B 171,483-494. Southgate, V. R. (1970). Observations on the epidermis of the miracidium and on the formation of the tegument of the sporocyst of Fasciola hepatica. Parasitology 61,177-190.

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Southgate, V. R. (1971). Observations on the fine structure of the cercaria of Notocotyliis attentiatus and formation of the cyst wall of the metacercaria. 2. Zellforsch. 120, 420449 Stockdale, P. H. G., Fernando, M. A. and Gilroy, J. (1970). Ultrastructural study of the teguminal sheaths of two metastrongyloid nematodes. Can. J. Zool. 48, 423425. Taylor, E. W. and Thomas, J. N. (1968). Membrane (contact) digestion in the three species of tapeworm Hymenolepis diminuta, Hymenolepis microstoma and Moniezia expansa. Parasitology 58, 535-546. Threadgold, L. T. (1967). Electron-microscope studies of Fasciola hepatica. 111. Further observations on the tegument and associated structures. Parasitology 57,633-637. Threadgold, L. T . (1968). The tegument and associated structures of Haplometra cylindracea. Parasitology 58, 1-7. Thust, R. (1966). Elektronenmikroskopische Untersuchungen uber den Bau des larvalen Integumentes und zur Hautungsmorphologie von Ascaris lumbricoides. Zool. Anz. 177,411-417. Timofiev, V. A. and Kuperman, B. I. (1967). [Ultrastructure of the outside layers of Triaenophorus nodulosus coracidia.] Parazitolgiya 1, 124-1 30. Timofiev, V. A. and Kuperman, B. I. (1968). [Ultrastructure of the cuticle and subcuticular layer in the procercoid, plerocercoid and adult of Triaenophorus nodulosus.]Parazitologiya 2,42-49. Tsubota, T. (1966). [Ultrastructure of Enterobius vermicularis. I. Cuticle.] Jap. J. Parasit. 15,58-63. Wajdi, N. (1966). Penetration by the miracidia of 5’.mansoni into the snail host. J. Helminth. 40,235-244. Wilson, R. A. (1969). Fine structure of the tegument of the miracidium of Fasciola hepatica L. J. Parasit. 55,1241 34. Wisse, E. and Daems, W. T. (1968). Electron microscopic observations on secondstage larvae of the potato root eelworm Heterodera rostochiensis. J. ultrastr. Res. 24, 210-231. Wright, K. A. (1968). The fine structure of the cuticle and interchordal hypodermis of the parasitic nematodes, Capillaria hepatica and Trichuris myocastoris. Can. J. 2001.46, 173-179. Wright, K. A. and Hope, W. D. (1968). Elaborations of the cuticle of Acanthonchus duplicatus Wieser, 1959 (Nematoda : Cyatholaimidae) as revealed by light and electron microscopy. Can.J. Zool. 46, 1005-101 1. Wright, R. D. and Lumsden, R. D. (1968). Ultrastructure and histochemical properties oftheacanthocephalanepicutic1e.J. Parasit. 54,1111-1 123. Wright, R. D. and Lumsden, R. D. (1969). Ultrastructure of the tegumentary porecanal system of the acanthocephalan Moniliformis dubius.J. Parasit. 55,993-1003. Wright, R. D. and Lumsden, R. D. (1970). The acanthor tegument of Moniliformis dubius. J. Parasit. 56, 727-735. Yuen, P. H. (1967). Electron microscopical studies on Ditylenchirs dipsaci (Kuhn). I. Stomata1 region. Can. J . Zool.45,1019-1033. Yuen, P. H. (1968). Electron microscopical studies on the anterior end ofPanagrelhs silusiae (Rhabditidae). Nematologica 14, 554-564.

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Author Index Numbers in italics refer to pages in the References at the end of each article

A Abasov, K. D., 299, 300, 306, 307, 308, 310 Abbate, E. A., 310 Abdallah, A,, 286, 317 Abdel-Malek, E. T., 168, 181, 219, 253, 254 Abdou, A. E. H., 302, 308,310 Abdullaev, A. M., 299, 310 Aboul-Eid, H . Z., 362, 372 Abramova, I . G., 306,307,308,333,337 Abuladze, K. I., 270, 271, 272, 273, 275, 284,310 Abunagimov, Kh. Z., 327 Acha, P. N., 303, 309,310 ACkov, M., 308, 330 Acosta-Matienzi, J., 201, 219, 220, 221, 238,262 Adamiya, G., 285,310 Adams, A. R. D., 293,310 Adams, J. E., 168,181,186 Adie, J. R., 3, 14, 17, 25 Adonajlo, A., 286, 288, 289, 300, 303, 308,310, 343 Aedtner, K., 305, 307, 308, 310 Agapova, A. I., 139, I45 Agarwal, S. M., 164, I81 Aguilar, F . J., 303, 309,310 Ahkami, S . , 290, 310 Ahrens, G., 305, 307, 308, 310 Aikawa, M., 32, 45 Akram, M., 309,321 &am, S., 309,321 AlagiC, D., 308, 310 Al-Allaf, G., 293, 335 Albis, F. S., 309, 334 Aldridge, F. P., 356, 372 d’Alessandro Bacigalupo, A., 273, 283, 284, 286,310,311 Alferova, M . V., 306,310 Ali, M.T., 309, 329 Ali, S. N., 32, 35, 36, 38, 44, 45 381

AIieva, S. I., 277, 286, 337 Allan, N., 33, 46 Allen, P. J., 213, 254 Allen, R. W., 305, 310, 311 Allison, A. C., 33, 45 Allison, L. N., 169, 172, 181 Allison, V . F., 203, 267 Alonso, M. T., 309, 311 Alterio, D. L., 293, 294, 311 Altmann, G., 277,311 Alvaro-Diaz Artilde, J., 303, 309, 311 Alworth, W . L.. 360. 376 Ameel, D. J., i63, 164, 165, 181, 183, 245,257 Amirov, R. O., 296,297,311 Anderson, F . M., 164, 177, 181 Anderson, G. A,, 162,181 Anderson, M. G., 164, 177, 181 Andrews, G. W. S., 277, 311 Andrews, W . H . H., 59, 69, 72, 74 Anello, G., 308, 319 Angel, L. M., 160, 168, 171, 181, 184, 185 Angus, M. G. N., 42,43,44,45 Anic, N., 308, 342 Anon, 311 Anschiitz, G., 17, 25 Anteson, R. K., 209, 210, 254 Antipin, D. N., 305, 311 Antoniewicz, K., 287, 314 Anya, A. O., 362,372 Araggo, H. deB., 1,3,5,7,9, 11, 12, 14, 17, 25, 26, 27 Araujo, T. L., 139, I45 Ardao, H., 285,311 Arean, V . M., 365, 373 Arfaa, F., 309,335 Anne, C., 276,311, 360,376 Armed Forces Institute of Pathology, 199,254 Amell, O., 285, 311 Artemov, N.M., 276, 311 Artikov, M. B., 287,311

382

AUTHOR INDEX

Ascenti, E., 308, 333 Asenjo, A., 273,311 Asher, R., 282, 311 Astakhova, 0. O., 306,325 Atias, A., 290, 311, 317 Atienza Fernandez, M., 311 Aubert, H., 136, 137, 140, 145 Audy, J. R., 192,205,214, 225, 262 Augustine, D. L., 245,256 Avakyan, D. M., 300,308,311 Avetisyan, S. F., 290, 293, 294,333 Aviado, D. M.,39, 45 Avlavidov, T., 308, 311 Ayala, S. C., 16, 17 B Babeau, P.,286,340 Bablet, J., 285, 316 Bachlechner, K., 308,311 Bacigalupo, J., 273, 288, 311 Backlund, H. O., 245,254 Baer, J., 155,156, 158,162,167,172,181 Baer, J. G., 78, 141, 145, 169, 185, 271, 275, 278, 322,331 Baer, K. E.V., 135, 136, 137, 140, 145 Bailinger, J., 294, 311 Bairamalibekova, R. T., 277, 286, 337 Baker, J. R., 5, 8, 12, 13, 14, 17, 18, 24, 26, 214,267 Bal, A. K., 354, 376 Bang, F. B., 207, 213, 254 Barbosa, F. S., 211, 219, 223, 254 Bard, J., 245, 263 Bareto, A. C., 219,223, 254 Barker, F. C., 139,145 Baron, P. J., 358, 372 Barrow, J. H. Jr., 245,254 Bartl, Z., 276,314 Basch, P. F., 192, 193, 194, 195, 198, 201,202, 203, 204, 205, 206, 207, 210, 211, 214, 225,226,227,234, 245,254, 255, 260,261, 262 Basnuevo, J. G., 287, 291,312,324 Bass, A. C., 42, 47 Batko, B., 277,312 Batsakis, J. G., 320 Baugh, S. C., 202,260 Bayer, F. A. H., 202,254 Baylis, H. A., 153, 157, 158, 181 Baz, I, I., 308, 331 Beamer, P. R., 320 Beams, H. W., 361,372

Bearup, A. J., 168,181 Beaver, P. C., 168, 169,181, 202,254 Beck, A. J., 192, 205,214, 225,262 Becker, C . D., 136, 144,148 Beckerdite, F. W. Jr., 203, 215, 267 BeganoviC, H. A., 308,310 Begiun, F., 359,372 Beier, A., 283, 284, 291, 292, 301, 308, 312 Bekhli, A. F., 290, 293, 324 Belopol'skaya, M. M.,172, 174, 181 Belton, C. M., 355, 372 Belyaev, A. E., 278, 283,312 Benassi, E., 289, 312 Benedict, E. G., 286, 312 Benesch, R., 35,45 Benesch, R. E., 35, 45 Benetazzo, B., 312 Btnex, J., 208, 245, 254, 257 Bennett, G. F., 6, 17 Bennett, H. J., 296, 330 Bennett, M. J., 164, 181 Bennett, T. P., 36,45 Berg, C. O., 245, 255, 260, 263 Bergner, J. F., 309, 312 Berrie, A. D., 246, 255 Bemos-Durran, L. A., 245, 260 Berry, L. R., 285,286,312 Berson, J. P., 3, 17 Bertrand, D., 291, 316 Bhabani, A. R., 61, 62, 63, 74 Bhaduri, N. V., 276,278, 302,314, 329 Bhatia, B. L., 2, 17 Biagi, F. F., 308, 315 Biche, Y., 302, 309, 312 Biguet, J., 289, 312 Billings, F. T., 66, 72 Bilqees, F. M., 358, 372 Bils, R. F., 162, 181, 354,372 Binckley, Ellen C., 7, 9, 10, 12, 20 Bioy, E., 326 Bird, A. F., 360, 364, 372 Bird, J., 360, 364, 372 Bird, R. G., 14, 18, 26 Bishop, H. W.,308,3I2 Bisset, K. A,, 208, 255 Black, D. A. K., 312 Blamoutier, P., 286, 312 Blamire, R. V., 308, 328 Blanchard, R., 312 Bloch, E. H., 68, 73

383

A U T H O R INDEX

Boch, J., 308, 312 Boev, S. N., 272,312 Bogitsh, B. J., 356, 372 Bogojavlenski, N. A., 287, 312 Bogoyavlenski, Y.K., 361, 373 Bojanowicz, K., 292, 312 BokoviC, T., 308, 343 Bonczak, J., 286, 300, 308, 310 Bonilla-Naar, A., 278, 312 Bonner, T. P., 362, 365, 373 Boray, J. C., 192, 193, 203, 220, 221, 245,255 Boreham, P. F. L., 62, 72 Botero, D. R., 293, 313 Bottom, F., 302, 309, 313 Boudenes, G., 292,331 Boveri, J. L., 285, 313 Bowers, E. A., 153, 154, 155, 156, 158, 159, 160, 162, 163, 173, 184, 354, 375 Bowman, I. B. R., 33, 36, 38,45 Box, Edith D., 2, 12, 13, 14, 17, 24, 26 Boyce, H. R., 205, 267 Brackett, S., 172, 176, 183, 192, 203, 210,256, 257 Bradbury, P. C., 360, 375 Brandes, 305, 343 Bras, de Sousa Loreto, 5, 14, 20, 24 Brant, P. C., 303, 304, 309, 3B, 315 Brlten, T., 358, 359, 373 Bratt, A. D., 245, 255 Braun, M., 78, 145 Bray, R. S., 2, 10, 12, 14, 15, 17, 18, 25, 26 Bresciani, J., 348, 373 Brewer, G. J., 42, 45 Brinkmann, A. Jr., 81, 107, 123, 131, 139, 145 Brocklesby, D. W., 281, 340 Brodsky, M., 302, 304, 305, 309, 327 Brooks, C. P., 223, 255 Brooks, W. M., 207,255 Brown, P. W., 327 Bruce, J. I., 193, 217, 255, 355, 373 Bruce, R. G., 361, 365, 373 Brumpt, E., 273, 275, 285, 313 Brumpt, V., 309,313 Briining, H., 286, 313 Bruzzoni, N. R., 335 Bryant, C., 33, 45 Brygoo, E. R., 278,313 Bubis, J. J., 277, 311 Buch, B., 355,378

Buchwalder, R., 301, 308, 321 Bueding, E., 289,291,336 Bugyaki, L., 306,313 Burch, J. B., 219, 255 Burckhardt, W., 284, 286, 313 Burmeister, H., 141, 145 Bums, Wrn. C., 176,181 Burrows, R. B., 274, 285, 286, 312, 313 Burt, D. R. R., 106, 128,145 Burton, P. R., 356, 373 Bustamente, E., 273, 311 Butler, J. M., 245, 255, 258 Butterworth, P. E., 369, 373 Buttner, A,, 168, 172, 174, 176, 182, 202,255 Buttner, D. W., 14, 18, 26 Bychowsky, B., 93, 127, 128, 136, 139, 145 Bychowsky, I., 93, 127, 128, 136, 139, 145 Byram 111, J., 326 Byrd, E. E., 162, 176,182 C Cable, R. M., 139, 150, 157, 158, 160, 161, 162, 163, 164, 165, 169, 172, 178, 182 Cameron, J., 302, 309, 319 Cameron, T. W. M., 155, 158, 167, 182 Campana-Rouget, Y.,162,182 Campbell, W. C., 282,313 Canhan, A. S., 298, 302, 309,313 Canning, E. U., 225, 245, 255 Cannon, P. R., 49, 72 Caponnetto, S., 308, 315 Capron, A., 278, 288, 289, 312, 313 Carini, A., 5, 18 Carman, J. A., 309, 315 Carmichael, J., 299, 313 Carney, D. M., 219, 263 Cartei, S., 286, 340 Cassorla, E., 284, 285, 326 Castel, P., 292, 321 Castellano, T., 273, 313 Castillo, Lindley E., 277, 313 Catala, L., 315 Cavier, R., 289, 291, 293, 313 Cenadella, R. J., 35, 36, 45 Chabaud, A. G., 162, 170,182 Chadwick, J. S., 213, 255 Chain, E., 49, 73

384

A U T H O R INDEX

Chandler, A. C., 313 Chapman, K . H., 313 Charles, G. H., 358, 359, 373 Chauhan, B. S., 139, 141, 145 Chechugo, I. S., 309,337 Chen, T . A., 362, 375 Cheng, T. C., 177, 182, 203, 207, 208, 213, 214, 223, 231, 255, 256, 358, 378 Chernin, E., 213, 238, 245, 256 Chi, L. W., 219, 256 Chin, T. H., 309, 314 Ching, H. L., 169, 182 Chiriboga, J., 245, 260 Cho, W. C., 39,45 Chodera, L., 287, 291, 292, 293, 314 Chongsuphajaisiddhi, T., 44, 47, 58, 59, 73, 75 Chowaniec, W., 21 1, 256 Chowdhury, A. B., 276, 278, 314 Christensen, A. M . , 158, 183 Christophers, S. R., 32, 45 Chu, K. Y., 219,256 Chubrik, G. K., 166, 183 Chularek, P., 299, 302, 309, 314 Chularerk, U., 299, 302, 309, 314 Churchwell, F. K., 276, 342 Chwirot, E., 283, 284, 285, 287, 291, 292, 293,314,332 Ciauri, G., 286, 314 Cigala, O., 328 Ciordia, H., 164, 183 Cironeau, I., 304, 308, 314 Clapham, P. A., 274,314 Clarenburg, A., 314 Clark, G. W., 15, 18, 25 Clark, H . C., 285, 314 Clegg, J. A., 357, 373 Clyde, D. F., 33, 45 Cmelik, S., 276, 314 Coan, C. C., 42,45 Coceani, C., 302, 314 Coelho, M. D. V., 21 9, 220, 256 Coelho, M. V., 211, 254 Colley, F. C., 360, 373 Collin, W. K., 203, 256, 357, 358, 373 Combnescu, N., 290, 331 Conrad, M. E., 67, 68, 73 Conroy, D. A., 315 Cook, R.T . , 32, 45 Cooper, B., 203, 267 Cooper, E. L., 207, 208, 259 Corradetti, A., 4, 12, 13, 18, 24, 26

del Corral, P., 309, 315 Correa, L. R., 218, 264 Correa, C., 6, 18, 25, 26 Corrba, Clovis, 21, 26, 27 Corso, P., 308, 315 Cort, W. W., 163, 164, 165, 172, 176, 183, 192, 203, 210, 245, 256, 257, 264 Cory, C. B., 18, 27 Cosgrove, G. E., 303, 309, 315 da Costa, A. S., 303, 304, 309, 313, 315 Coulson, F., 7, 8, 9, 10, 12, 18, 20, 26 Courmes, E., 245, 257 Cousi, D., 315 Cousineau, G. H., 354, 376 Cover, B., 32, 48 Cram, E. B., 218, 219. 257, 259 Cramer, J. D., 281, 289, 304, 305, 306, 315.316 Crandall, C. A., 365, 373 Crandall, R. B., 164, 165, 182,183 Crane, R. K., 315 Crewe, S. M., 297, 315 Crewe, W., 297, 315 Cridland, C. C., 219, 257 Crosby, W. H., 34,47 Cruickshank, I. A. M., 213, 257 Cunningham, J. T., 126, 137, 141, 145

D Dadlez, J., 292, 315 Daems, W. T., 360, 379 Damme, E., 329 Danilewsky, B., 1, 18 Dasgupta, B., 276, 278, 314 Da Silva, A. L., 309, 315 Da Silva, W . R. K., 295, 320 Daubney, R., 309,315 Davaine, C., 315 Davey, K . G., 365, 373, 376 Davey, T . H., 219,238, 259 Davidie, J. A., 245, 257 Davis, A. K., 42, 47 Dawes, B., 78, 139, 141, 145, 153, 183, 201, 220, 221, 257, 353, 374 Dayal, J., 139, 146 Dean, B. H., 296, 297, 308,320 DeBach, P., 200, 214, 244, 257, 258 De Beer, G . Sir, 172, 183 Deberdt, A,, 301, 307, 308, 320 Deberdt, P., 303, 308, 320 Deblock, S., 288, 302, 308, 316 Debrot, S.,315

385

AUTHOR I N D E X

De Carneri, I., 308, 315 Deegan, T., 44,46, 69, 72, 73, 74 De Freitas, 0 . T., 303, 309, 315 Delard, G., 303, 309, 315 Del Gesso, L., 310 Delon, J., 315 De Maria, M., 245, 264 Demian, E. S . , 245, 257 De Morales, D. S., 295, 320 De Moura, M. F., 245, 264 Denecke, K., 297, 315 Denev, D., 308, 315 Denison, J., 268 Dennis, L. H., 67, 68, 73 De Resende, P. R., 309, 315 De Rivas, D., 273, 294, 315 Derrier, E., 305, 315 Deschiens, R., 285, 288, 291, 316 Deschiens, R. E. A., 245, 257 Despeignes, J., 293, 319 Despommier, D. D., 362, 374 Desprks, P., 304, 305, 308, 316 Desser, S. S . , 13, 14, 19, 25, 26 Desowitz, R. S . , 50, 65, 73, 74 Devakul, K., 66, 67, 69, 73, 74 De hies, J., 301, 308, 316 Dewhirst, L. W., 281,282, 289, 304, 305, 306, 315,316 Dewitt, W. B., 219, 258 D’heureuse, R., 284, 286, 321 Dias, A., 5 , 14, 20, 24 Dias, J. C. P., 303, 309, 316 Diaz, L. M. T., 168, 186 Dick, T. A., 362, 364, 374 Dickermann, E. E., 121, 137, 138, 139, 141, 146, 164, 183 Dickerson, C. W., 362, 378 Di Conza, J. J., 238, 258 Diesfeld, H. J., 308, 316 Diesing, K. M., 136, 139, 146 Dike, S. C., 276, 326 Dinnik, J. A., 163, 164, 183, 201, 238, 258 Dinnik, N. N., 163, 164, 183, 201, 238, 258 Dissanaike, A. S., 12, 13, 18, 24, 245, 258,316 Ditzel, J., 294, 316 Dixon, H. B. F., 273, 302, 316 Dixon, K. E., 171, 183, 354, 355, 356, 3 74 Dobrovolny, C. G., 174, 177, 183

Dobrovolski, A. A., 354, 374 Dobrowolska, H., 288, 316 Doby, J. M., 288, 302, 308, 316 Doby-Dubois, M., 288, 302, 308, 316 Dodion, L., 294, 316 Dollfus, R. Ph., 78, 91, 103, 107, 110, 122, 123, 129, 136, 139, 141, 146, 158, 163, 166, 176, 183, 245, 258 Dolman, C. E., 304, 316 Donckaster, R., 277, 290, 294, 317 Donges, J., 163, 166, 172, 183, 213, 258 Donoso, F., 277, 290, 317 Dorken, H., 276, 289, 321, 334 Doroshchak, 0 . F., 293,317 Doutt, R. L., 200, 258 Douvres, F. W., 295, 326 Duarte, G. G., 303, 309, 331 Dubey, J. P., 14, 18 Dubois, G., 163, 172, 183, 184 Dubos, R. J., 278, 317 Dubreuilh, W., 273, 329 Dufek, M., 290, 291, 292, 293, 317 Dujardin, F., 136, 140, 146 Dunachie, J. R., 14, 19 Dunlop, S. G., 296, 342 Dunn, M. J., 41, 42, 45 Durie, P. H., 163, 164, 184 Dusanic, D. G., 208, 258 Duthie, E. S., 49, 73 Duthy, B. L., 296, 317 Diakulla, N., 280, 306, 342 Dzhabriev, N. I., 327 Dzieciolowski, Z . , 283, 317 Diinleski, B., 303, 308, 317

E Eckmann, F., 139,146 Edington, G. H., 67, 73 Edney, J. M., 163, 184 Eichelberger, J. N., 67, 68, 73 Eisa, A. M., 302, 309, 317 El-AM, A., 302, 304, 305, 308,309,317 El-Cindy, M. El. S., 204, 211, 258 Eliot, T. S . , 52, 68, 73 El-Mawla, N. G., 286, 317 Elmossalami, E. S., 302. 304, 305, 308, 309,317,325 Elsdon-Dew, D., 334 Elsdon-Dew, R., 274,289,302, 309,317, 329,334 El Sherif, A. F., 310 Emsbo, P., 308, 317

386

AUTHOR INDEX

Endrejat, E., 309,317 Enequist, N., 301, 307, 308, 317 Enigk, K., 317 Erasmus, D. A., 356, 359, 374, 375 Ernster, L., 43, 48 Ershov, V. S., 305, 311 Esch, G. W., 358, 359, 377 Etges, F. J., 202, 258, 362, 365, 373 Evans, E. A. Jr., 38,48 Evranova, V. G., 282,317 Ewers, W. H., 204, 258 Eymmer, H. J., 305, 308, 321

F Fahmy, M. A. M., 302, 308,317 Faiguenbaum, J., 290, 317, 330 Fantham, H. B., 5,18, 24 Fao, Who, Oie, 300,318 Faraco, B. F. C., 295, 320 Farchmin, G., 301, 308, 321 Faulk, W. P., 208, 209, 222, 226, 258, 259 Fauran, P., 245, 257 Faust, E. C., 82, 110, 113, 121, 136, 139, 141, 146, 158,184, 201, 220, 221,238, 258 Fehkr, J., 300, 305, 308, 339 Felsani, F., 309, 317 Feng, S. Y.,208, 258 Ferguson, F. F., 245,255, 258,259,260, 263,264, 265,266 Fernandez, A. F., 277, 342 Fernando, M. A., 12,18,24, 361, 379 Ferracani, R. S., 285, 317 Fetterman, L. E., 289, 318 Fewster, G. E., 303, 304, 305, 309, 318 Fielding, C. M., 42, 46 Figgat, W. B., 296, 330 Files, V. S., 218, 219, 257, 259 Filipov, V. V., 306, 323 Filipovic, B., 286, 319 Findlay, G. M., 50, 73 Finnegan, C. V., 359, 377 Fischer, C., 318 Fishthal, J. H., 170, 184 Fisher, R. C., 200, 214, 259 Fitzgerald, F., 202, 204, 206, 227, 262 Fitzsimmons, W. M., 309, 318 Fletcher, K. A,, 32, 33, 34, 35, 36, 38, 39, 42, 43, 44,45, 46, 47, 48 Florey, H. W., 68, 73 Flosi, A. Z., 66, 73

Foes, 0. M., 309,326 Folkers, K., 39, 47 Fontan, C., 273, 318 Fonteneau, M., 308, 318 Foote, B. A., 245,255, 259 Fourrier, A,, 286, 340 da Franca, 0. H., 284,285, 286, 318 Franchini, G., 8, 14, 15, 18, 24, 25, 28 Franssen, J. G., 305, 318 Fredrickson, D. S., 43,46 Frenkel, J. K., 14, 18 Friedrich, J., 281, 303, 307, 308, 318 Friendlander, Y., 33, 46 Frolova, A. A., 277, 318 Froltsova, A. E., 331 Froyd, G., 280,281,282, 295, 300, 302, 306, 308, 309,318 Fudenberg, H. H., 226, 259 Fuentes, P. B., 318 Fuhrmann, O., 141, I46 Fullard, J., 38, 39, 41, 46, 48 Fulton, J. D., 32, 45, 46 Furst, O., 294, 319 G Gaehtgens, W., 288, 319 Gailiunas, P., 319 Galil, N., 286, 317 Gallo, C., 308, 319 Gancarz, Z., 288, 289, 300, 303, 308, 310,343 Gangolli, D. A,, 319 Garaguso, P., 288, 319 Garin, J. P., 293, 319 Garnham, P. C. C., 2, 9, 10, 11, 14, 18, 25, 26, 27, 295, 319 Gasparov, A., 286, 319 GavranoviC, I., 308, 310 Gazzola, E., 308, 315 Gear, J. H. S., 355, 378 Gebauer, O., 319 Geckler, R. P., 245, 259 Geiger, S., 211, 218, 260 Geiman, W. M., 39,47 Gelber, A,, 290, 331 Gemmell, M. A., 282, 319 Gentner, H. W., 136, 146 George, J. N., 34,47 Gerwel, Cz., 292, 315 Ghenis, D. E., 300, 308, 319 Ghenov, G. M., 308,319 Gherman, I., 290, 319

AUTHOR

Gibson, M., 245, 259 Gibson, T. E., 305, 319 Gilles, H. M., 33, 46, 66, 67, 73, 74 Gilroy, J., 361, 379 Ginetzinskaya, T. A., 142,146, 153, 154, 155, 156, 157, 158, 167, 170, 184, 354, 374 Ginsberg, A., 302, 307, 309, 319 Ginzel, E., 305, 308, 319 Giroud, P., 322 Gladkikh, V. F., 320 Goddard, W. B., 302, 309, 319 Godoy, M., 294, 31 7 Gogotishvili, T. G., 285, 310 Golden, A., 42, 47 Goldsmid, J. M., 273, 309, 320, 340 Gombarros Alvarez, E., 285, 320 Gomez, Garcia, V., 311 Gonnert, R., 279, 289, 291, 320 Gonzales Castro, J., 311 Gonzalez, G., 326 Goodchild, C. G., 172, 184 Goodwin, L. G., 62, 66, 73 Gordon, R. M., 219, 238, 259 Gordon, R. S., 43,46 Gotzsche, N. O., 297, 308, 320 Goulart, E. G., 295, 320 Gould, S . E., 320 Gouveia, A. L., 303, 309, 313 Graack, B., 179, 187 Grabda-Kazubska, B., 177, 184 Graber, M., 272, 293, 302,306,309,320, 343 Graham, C. F., 320 Grailet, L., 293, 325 Grant, P. R., 33, 35, 36, 38, 45, 46 Granville, A., 301, 303, 307, 308, 320 Gras, G., 292, 321 Green, C. D., 364, 374 Greenberg, A. E., 296, 297, 308,320 Grkgoire, C., 301, 303, 307, 308, 320 Gregor, O., 308,333 Grieve, J. M., 302, 309, 319 Griffiths, R. B., 296, 297, 301, 304, 308, 320,337 Grimaldi, E., 325 Grisse, A., de, 364, 374 Grott, J. W., 293, 322 Grujic, I., 301, 308, 320 Grumbach, R., 308, 320 Guarniera, D., 308, 333 Guildal, J. A., 297, 320

INDEX

387

Guilhon, J., 293, 320 Guiver, K., 296, 337 Guilisano, G., 308, 315 Gunther, B., 360, 374 Gunther, H., 320 Gupta, B. L., 352, 374 H Hadjian, A., 290, 310 Hajduk, F., 305, 308,321 Hajj, S. N., 286, 336 Haleem, M. A,, 309,321 Halterman, L. G., 297, 336 Halton, D. W., 170, 184, 351, 352, 355, 377 Hamerton, A. E., 18, 25 Hamilton, J. B., 289, 321 Hammond, R. A,, 369, 374, 375 Hanel, L., 294, 321 HanSen, E. L., 238, 258 Harant, H., 292, 321 Hardwick, E. F., 308, 321 Hargreaves, T., 294, 321 Harinasuta, T., 67, 69, 73 Harris, P. J., 355, 372 Hart, J. W., 18, 26 Hatton, C. J., 289, 321 Heath, D. D., 279, 338 Hegner, R., 18, 26 Heikinheimo, E., 294, 321 Hein, B., 324 Helander, E. V., 321 Hellmayr, C. E., 18, 27 Hendrickse, R. G., 33, 46 Hendrix, S . S., 83, 90, 136, 137, I46 Hennemann, H. H., 284,286,321 Hennessy, E., 334 Herman; C. M., 2, 9, 15, 19, 20, 22, 24, 25, 26, 27 Herman, R., 33,46 Herman, Y. F., 33, 46 Hermos, J. A., 301, 307, 309, 336 Hermus, G., 307, 308, 321 Hewitt, R., 7, 19, 24, 25 Heyneman, D., 153, 155, 156, 157, 158, 159, 163, 166, 167, 168, 173, 184, 192, 194, 195, 198, 201, 202, 203, 204, 205, 206, 207, 208, 209, 213, 214, 221, 222, 225,226,227,231, 234,236,244,246, 254, 258, 259, 260,261, 262 Hickman, V. V., 176, 184 Hiepe, T., 301, 308,321

388

AUTHOR INDEX

Hildemann, W. H., 207, 208, 259 Hinerman, D. L., 320 Hinz, E., 309, 321 Hirte, W. E., 291, 321 Hirumi, H., 362, 375 Hkun-Saw-Lwin, 302, 309, 340 Hoare, C. A., 3, 14, 15, 19, 27 Hockley, D. J., 354, 355, 357, 375, 378 Hoeppli, R., 269, 302, 321 Hoffman, J. F., 42, 46 Hoffman, M. A,, 193, 194, 201, 202, 227, 261 Hoffman, W. A., 201, 220, 238, 258 Hofstra, K., 308, 321 Hohl, H. R., 358, 378 Holliman, R. B., 178, 184, 245, 259 Hollis, J. P., 360, 375 Holz, J., 280, 321 Homewood, C. A., 38, 39, 40, 46 Honer, M. R., 301, 308,321 Hope, W. D., 362, 364, 368,379 Hopkins, S. H., 136, 137, 139, 146, ISI Hornbostel, H., 276, 277, 282, 283, 284, 285, 286, 287, 289, 299,321, 324 Hornell, J., 128, 137, 150 Hovorka, J., 308,321 Howells, R. E., 38, 39, 40, 41, 46, 48, 359, 275 Hsieh, H. C., 288, 302, 309, 322 Hsu, H. F., 219, 260 Hsu, S. Y., 219, 260 Huang, S. W., 270, 272, 298, 302, 309, 322 Huff, C. G., 15, 19, 27, 208, 260 Huhtala, A., 308, 322 Huizinga, H. W., 361, 376 Hulland, T. J., 280, 337 Humes, A. G., 164, 181 Humphreys, R. M., 219, 260 Hurst, A., 284, 322 Hussey, K. L., 155, 184, 245, 257, 288, 322 Hutchison, W. M., 14, 19 Hyman, L. H., 141, 146, 155, 156, 166, 184 I

Ibrahim, I. K. A., 360, 361, 375 Ilin, M. M., 308, 328 Tnatomi, S.,355, 356, 359, 360, 362, 375 Index Catalogue, 136, 146 Inmin, M. M., 67, 68, 73

Interdepartmental Committee on Nutrition, 302, 309, 322 Irvine. C., 322 Isseroff, H., 164, 182 Itakura, T., 293, 329 .-. Itano, K., 355, 356, 359, 360, 362, 375 Ito, Y., 356. 375 IAailova-Guseinova, R. A., 289, 322 J Jackson, G. J., 360, 375 Jacob, H., 302, 307, 309, 341 Jadin, J., 322 Jagerskiold, L. A., 128, 139, 147 James, B. L., 153, 154, 155, 156, 158, 159, 160, 162, 163, 165, 173, 174, 184, 354, 375 James, C., 193, 268 Jamieson, B. G. M., 157, 162, 176, 184 Jamuar, M. P., 360, 375 JaniEek, J., 305, 332 Janicki, C., 155, 184 Jaroonvesema, N., 67, 73 Jarpa, A., 290, 317 Jarrell, J. J., 35, 36, 45 Jarrett, W. F. H., 306, 340, 341 Jarzebski, Z., 300, 308, 310 Jenkins, T., 361, 375 Jeong, K. H., 208, 244, 259 Jepsen, A., 295,296, 308, 322 Jeyarasasingam, U., 195, 198, 201, 202, 207, 231, 236, 259, 260 Jobin, W. R., 245, 260 Johnson, P. T., 245, 260 Johnson, P. W., 360, 364, 375 Johnston, M. R. L., 14, 21 Johnston, T. H., 168, 171, 184, 185 Jones, A. W., 278,322,325 Jones, M. F., 218, 219, 257 Jones, N. O., 362, 364, 378 Jones, Virginia, P., 7, 9, 10, 12, 20 Jopling, W. H., 277, 322 Joshi, V. G., 285, 294, 302, 309, 337 Joyeux, Ch., 78, 141, 145, 155, 156, 158, 162, 167, 169, 172, 181, 185, 271, 322 Junod, C., 277, 294, 322 K Kaan, J. D., 285, 322 Kabler, P., 296, 322 Kacka, I., 277, 312

A U T H O R INDEX

Kagan, I. G., 176, 185, 211, 218, 260, 339 Kajima, M., 362, 374 Kalawski, K., 294, 299, 300, 308, 322 Kalb, J. C., 293, 319 Kaliszewicz, S., 293, 322 Kalivoda, R., 290, 291, 292, 293, 317 Kamalova, A. G., 278, 288, 322, 333 Kaminski, A., 292, 315 Kamo, H., 293,329 Kampfe, 365, 376 Kampfe, L., 360, 374 Kan, S. P., 365, 373, 376 Kanakakorn, K., 69, 73 KanneworfT, B., 158, 183 Kantor, S., 2, 20 Kapustin, V. T., 277, 333 Karle, E., 213, 258 Karlinska, A., 286, 324 Karnaukhov, V. K., 285, 290, 323 Katz, N., 193, 264 Kaufman, R. S., 284, 323 Kawanishi, K., 323 Kearney, A., 304, 323 Kebbouche, L., 308, 331 Keeling, J. E., 289, 290, 291, 292, 323 Keller, H., 305, 323 Kelly, H. M., 136, 137, 139, 144, 147 Kemp, W. M., 355,376 Kendall, S. B., 220, 223, 260 Kent, N., 323 Kermack, W. O., 33, 36, 38, 45 Kerr, K. B., 323 Khalil, H. M., 290, 323 Khalil, L. F., 220, 245, 260 Khan, R. A., 13, 14, 19, 25, 26 Kiefer, E. D., 294, 335 Kikuth, W., 7, 19, 26 Kilejian, A., 223, 266 Killby, V. A. A., 32, 46 Killick-Kendrick, R., 5, 8, 12, 13, 17, 24 Kim, C. H., 277, 302, 309, 325 King, A. C., 323 Kinoti, G., 217, 219, 221, 260 Kitel, V. S., 293, 317 Kleibel, A., 305, 307, 308, 323 Klink, G. E., 274,313 Klusaka, J., 285,323 Knight, W. B., 245, 260 Knisely, M. H., 52, 68, 73 Knorr, R., 323 Knotz, I., 323

389

Knutson, L. V., 245, 255, 260, 266 Kofoid, C. A., 136, 139, 147 Kohl-Yakimoff, N., 5, 22 K0oe, M., 348, 373 Kolbe, F., 298, 323 Komiya, Y., 172,185, 302, 309, 329 Kondo, K., 293, 330 Kondracka, H., 300, 308, 310 Kong-Kim-Chuon, 309,313 Kornberg, H. L., 37, 46 Koskowski, W., 323 Kosminkov, N. E., 306,323 Kostenko, D., 285, 323 Kotova, 0. M., 308, 328 Koudela, K., 281, 300, 304, 305, 308, 323, 324 Kouri, P., 287, 324 Kovalev, N. E., 294, 299, 300, 307, 308, 324 Kovchazov, G., 308, 311 Kozek, W. J., 361, 365, 376 Kramm, D., 304, 308,333 Kreier, J. P., 42, 47 Krotov, A. I., 272, 290, 292, 293, 324 Krueger, 307, 324 Krull, W. H., 169, 176, 185 Krupa, P. L., 354, 376 Kubicki, S., 286, 324 Kuhls, R., 291, 324 Kumzicki, R., 283,317 Kuntz, R. E., 155,185, 219,260 Kuperman, B. I., 358, 359,379 Kupey, P., 301, 307, 308, 324 Kuimicki, R., 274, 324 Kwo, E. H., 194, 225, 227, 246, 248, 249, 260, 262, 264 L Labbk, A., 1, 11, 12, 19 Ladda, R. L., 38, 46 Lagane, D. M. M., 285,325 Lagasse, A., 364, 374 Lainson, E., 10, 19, 26 Lainson,R.,2, 5,8,9, 10, 11, 12, 13, 14, 17, 19, 24, 25 Laird, Elizabeth, 19, 27 Laird, M., 2, 6, 9, 11, 12, 16, 17, 19, 25, 27, 30 Lal, M. B., 202, 260 Lamina, J., 324 Lamy, L., 208, 254 Landi, A., 305, 325

390

AUTHOR INDEX

Landis, E. M., 69, 73 Lang, B. Z., 177,185 Langer, B. W. Jr., 33, 46 Langer, J., 288, 325 Lapierre, J., 286, 325 Larrougy, G., 274,325 Larsh, J. E., 358, 359, 361, 376, 377 LaRue, G. R., 163, 165, 169, 173, 176, 185 Lassance, M., 293, 325 Laveran, A., 1, 2, 3, 5 , 11, 14, 19, 23, 25 Lawrence, J., 19, 26 Laws, G. F., 295,325 Lazzaro, D. A., 304,325 Lebedeva, M. N., 320 Lebour, M. V., 163,185 Lee, D. L., 347, 355, 360, 362, 365, 368, 376 Lee, H. G., 213, 262 Lee, H. H. K., 278,325 Lee, K. J., 362, 375 Lee, K. T., 277, 302, 309, 325 Lee, M. A., 18, 25 Lee, M. Y.,277, 302, 309, 325 Le Gac, P., 291, 325 Leidholdt, H. G., 329 Leidy, J., 136, 147 Leigh, W. H., 177,185 Leikina, E. S., 281, 282, 306, 325 Leinati, L., 325 Leithead, C. S., 66, 74 Lengy, J., 201, 217, 219, 220, 221, 261 Lennon, E. A,, 308,333 Lepes, T., 301, 308, 325 Lerche, M., 305, 308, 325 Le Row, P. L., 272, 301, 308, 325 Lescure, 0. L., 289, 336 Letulle, M., 285, 325 Leuckart, K. G. F. R., 153, 155, 156, 159,185 Leuckart, R., 141, 147 Le Viguelloux, J., 309, 335 Levine, N. D., 2, 12, 20, 26, 250, 261 Levitanskaya, P. B., 1, 10, 22, 24, 26 Lewert, R. M., 208, 258 Lewitzki, R. G., 287,312 Ley, J., de, 276, 325 Le Zotte, L. A,, 161, 163, 174, 185 Liard, F., 245,260 Lichtenberg, F., von, 355, 356, 357, 378 Lie, K. J., 168, 185, 192, 193, 194, 195, 198, 199, 201, 202, 203, 204, 205, 206,

207, 214, 215, 220, 221, 222, 223, 225, 226, 227, 231, 234, 237, 238, 245, 246, 248, 249, 254, 260, 261, 262, 264 Lieb, D. E., 18, 25 Liebman, H., 296, 297, 325 Liegeois, F., 305, 325 Lidvre, H., 304, 326 Lim, H. K., 195,198,201,202,207,208, 209, 213, 215, 219, 222, 223, 231, 236, 244,258,259, 260,262 Lim, H. W., 142,147 Lima, D. F., 309,326 Lindner, R., 33, 46 Lindsay, G. K., 219, 255 Link, A,, 284, 285,326 Linstow, O., 136, 147 Linton, E., 137, 139, 141, 147 Lipscomb, F. M., 273, 302, 316 Little, C., 352, 374 Little, W., 32, 45 Llewellyn, J., 155,158, 170,186,357,376 Lloyd, E. L., 326 Lo, C. T., 219, 262 Lockhart, H. B., 285, 326 Loeper, M., 284,326 Logachev, E. D., 276,326, 356,376 Logan, C. J. H., 285,286,326 Logan, J. S., 301, 303, 304, 307, 308,326 Loos-Frank, B., 176,186 Looss, A., 103, 107, 139, 147, 170, 186 Lopez Ortiz, R., 303, 309, 326 Lopukhina, N. G., 307,333 Lucas, J. A., 362,378 Lucena, D., 15, 20, 24, 25, 26, 27 Lucena, D. T., 2, 6,20 Lucian, O., 290, 331 Lucker, J. T., 295, 296,326,341 Ludvik, J., 14, 20 Lumme, R., 326 Lumsden, R. D., 276,326,359,360,369, 370, 376, 379 Lunan, K. D., 39,47 Luque, F., 273, 313 Lure, R. N., 276,311 Lutfy, R. G., 245, 257 Lutyfiski, R., 308, 326 Luzzato, L., 34, 46 Lyons,K. M., 349,351,352,357,376,377 M McAnnally, R. D., 245, 262 MacCallum, G. A., 139,147

A U T H O R INDEX

MacCallum, W. G., 139, 147 McCarty, J. E., 355, 373 McCleery, E. F., 308, 328 McClelland, W. F. J., 211, 263 Macfie, J. W. S., 298, 327 Macdonald, J. D., 137, 147 Macheoboeuf, M., 323 Machnicka-Roguska, B., 276, 288, 289, 326,327 Maciel, G. A., 305, 327 Maciel, J., 5, 18 McIntosh, A., 280, 307, 328 McIntyre, W. I. M., 306, 341 McKee, R. W., 35,47 Mackerras, I. M., 8,12, 13,20,24,26,27 Mackerras, M. Josephine, 8, 12, 13, 20, 24, 26, 27 Mackie, A., 295, 327 McKinnon, J. A,, 302, 309,327,328 McLeod, J. A., 270,342 McManus, D., 298, 309,328 McMullen, D. B., 176,186, 192,203,257 MacNeal, W. J., 3, 21, 24, 25, 26 McQuay, R. M. Jr., 218, 219, 263 Maegraith, B. G., 33, 39, 42, 43, 44, 45, 46,47,48,50,51, 52,56,58, 59,60,61, 62, 63, 65, 66, 67, 68, 69, 70, 72, 72, 73, 74, 75 Magath, T. B., 327 Magdiev, R. R., 300, 306, 307, 308, 327 Mahfouz, M., 323 Makhmudova, S. A., 289, 327 Maldonado, J. F., 201, 219, 220, 221, 238,262 Malheiro, D. M., 305, 327 Malko, A. T., 335 Mamedov, A. A., 308,327 Manceaux, L., 5, 20 Maneely, R. B., 279,338 Mann, E., 309,327 Mann, G. V., 302, 309,334 Mann, I., 309, 327 Mansour, N., 198, 201, 231, 260 Mansour, N. S., 15, 20 Manter, H. W., 107, 108, 121, 122, 128, 134, 139,147, 176, 178, 186 Manwell, R. D., 7, 8, 9, 10, 12, 13, 20, 26, 27 Mao, C. P., 238, 264 Maples, W. P., 162, 182 Maplestone, P. A., 278, 327 Maramorosche, K., 362, 375

39 I

Marazza, V., 305, 325, 327 Markaryants, L. A., 327 Markkanen, T., 327 Marrenghi, O., 308, 327 Marsboom, R., 302, 304, 305, 309, 327 Marsden, K. H., 301, 308, 328 Martikyan, E. S., 300, 308, 328 Martin, G. A., 297, 336 Martin, G. N., 276, 342 Martin, G. W., 177, 186 Martin, J. H., 358, 359, 361, 376, 377 Martin, W. E., 162, 168, 169, 174, 178, 181, 186, 192, 203, 215, 245, 262, 354, 372 Martinez, M., 309, 343 Marullaz, M., 1, 2, 3, 5,9, 11, 19,20, 25, 26, 27 Marx, G. W., 309, 328 Marzano, F., 309, 315 Marzullo, F., 276, 328 Masdaki, S., 309, 328 Masellis, G. de, 308, 328 Mashanski, U. F., 354, 374 Maskrey, P., 43, 47, 48 Massoud, J., 213, 219, 256, 262 Mastrandrea, G., 286, 314, 328 Mathias, P., 168, 186 Matikashvili, T. S., 308, 340 Matricon-Gondran, M., 354, 377 Matsubayashi, H., 302, 309, 329 Matsuno, K., 293, 330 Matta, A., 213, 262 Mattes, D., 220, 221, 245, 262 Matsubayashi, H., 329 Mattes, D., 307, 328 Mattila, M., 289, 291, 292, 328 Mazzotti, L., 277, 279, 283, 284, 287, 288,328 Mecham, J., 245,259 Meggitt, F. J., 273, 328 Mehra, H. R., 165,186 Meister, G., 279, 320 Meleney, H. E., 201, 219, 221, 258, 263 de Mello, F., 2, 5, 14, 20, 24 Melton, Marjorie L., 14, 21 Menefee, M. G., 362, 365, 373 Mengert, H., 245, 263 Menschel, E., 296, 328 Mercado, T. I,, 44,47, 276, 342 Mercer, E. H., 171, 183, 354, 356, 374 Merdivenchi, A., 214, 328 Merkushev, A. V., 308,328

392

AUTHOR INDEX

Merle, A., 300, 308, 328 Memll, J. M., 302, 309, 334 Metge, R., 245, 257 Metlitskii, L. V., 213, 263 Michelson, E. H., 128, 136, 140, 147, 208,220, 245, 256,263 Mielke, D., 301, 303, 308, 328 Migasena, P., 59, 60, 65, 74 Mijatovic, I., 308, 328 Mikhailyan, E. A., 290, 293, 294, 333 Milhade, J., 309, 335 Miller, D., 280, 297, 307, 309, 328, 329 Miller, J., 35, 36, 38, 39, 47 Miller, N. L., 14, 18 Mills, R. R., 326 Milward de Andrade, R. W., 219, 263 Mine, N., 5, 20, 26 Miretski, 0. Y., 278, 329 Mitchell, J. R., 302, 309, 329 Mitra, S.K., 309, 329 Miyakoda, J., 293, 329 Mohammed, A. H . H., 10, 15,20, 25 Moldenschardt, H., 343 Mollaret, C., 329 Molnir, L., 300, 305, 308, 339 Monisov, A. A., 278, 283, 287, 293, 312, 329 Monroe, L. S.,289, 329 Monticelli, F. S., 93, 123, 136, 139, 141, 147, 148 Monzini, A., 305, 325 Moore, D. V., 219, 245, 263 Moore, G. A., 38, 39, 48 Moos, W., 285,323 Moose, J. W., 218, 219, 263 Morales, I., 294, 317 Moreno, A. G., 305,327 Morishita, K., 302, 309, 329 Moriya, K., 82, 137, 148 Moriyama, S.,329 Moroha, R., 5, 14, 20, 24 Morris, G. P., 351, 352, 355, 359, 377 Morris, N., 289, 329 Morseth, D. J., 278, 329, 358, 359, 361, 377 Mosina, S. K., 282, 306, 317, 329 Moskvin, S. N., 281, 282, 306, 325, 338 Mossmer, A., 285,329 Motulsky, A. G., 33, 47 Moulder, J. W., 35, 36, 38, 47, 48 Muazzam, M. G., 309, 329 Mudrow, Lily, 7 , 19, 26

Mukerji, A. K., 302, 309, 329 Mukhin, V. N., 289, 329 Mukhtari, L., 308, 331 Mukvoz, L. G., 308,329 Miiller, K . H., 287, 305, 308, 329 Miiller, K. O., 213, 263 Miiller, R., 321 Mulligan, W., 306, 340, 341 Mustafa, A. A., 302, 309, 317 Mustakillio, K. K., 326, 335 Mustakallio, K . M., 289, 292, 329 Mvogo, L., 245, 263 N Nabias, B. de, 273, 329 Nadzhafov, I. G., 298, 300, 307, 308,330 Nagahana, M., 293, 330 Nagarajan, K., 37, 39, 47 Nagaty, H. F., 299, 330 Nahhas, F. M., 162, 178, 182 Najarian, H. H., 83, 103, 121, 136, 137, 140,148, 168,186 Naquira, F., 330 Nasir, P., 168, 186, 204, 263 Naumov, 273, 343 ' Naumova, R. P., 330 Nauwerck, C., 285, 286, 330 NeEev, T., 303, 308, 317, 330 Neff, S. E., 245, 263 Neghme, A., 292, 330 Nkgre, A., 285,330 Negrete, M. J., 318 Negus, M. R. S., 203, 264 Neiva, A., 6, 14, 20, 24 Nelson, G. S.,271, 272, 298, 330 Nelson, P., 12, 18, 24 NenadiC, M. B., 301, 308, 330 Nevenitch, V., 308, 338 Newton, W. L., 193, 218, 219, 222,264, 296, 330 Nickerson, W. S., 81, 83,90, 93,99, 102, 103, 107, 111, 113, 126, 137, 139, 148 Nicolle, C., 5, 20 Nieland, M. L., 357, 358, 377 Niezbekov, K., 293, 329 Nikolic, P., 308, 342 Nikulshina, 0. A., 308, 328 Niles, W. J., 12, 18, 24 Niiio, F . L., 273,285, 303, 309, 330 Nitsche, O., 6, 20, 24, 26

AUTHOR INDEX

Nitzulescu, V., 290, 331 Noda, K., 163, I86 Nolf, L. O., 210, 256, 264 Noller, W., 3, 5, 6, 8, 12, 20, 24, 26, 27 Nor El Din, G., 308, 331 Norton, R. A., 289, 329 Norval, J., 301, 308, 337 Nosslin, B., 294, 331 Notteghem, M. J., 289, 293, 313 Notter, A., 292, 331 Novy, F. G., 3, 21, 24, 25, 26 Nowak, S., 285, 323 du Noyer, M. R., 275, 278, 331 Nyberg, W., 276, 331 Nylen, M. U., 276, 342 0

Oaks, J. A., 276, 326, 360,376 Ockert, G., 301, 308, 331 O’Connor, N., 277, 331 Odening, K., 163, 166, 168, 177, 178, 186,187 Odhner, T., 90, 121, 139, 141, 148 Oelkers, H. A., 294, 331 Ogden, C. G., 364, 377 Ogilvie, A. C., 277, 311 Ogston, D., 33, 36, 38, 45 ohman, C., 356, 377 Ohnesorge, G., 294, 331 Okpala, L., 309, 331 Okura, T., 293, 343 Oleck, H. G., 274,333 Oleinikov, S. V., 287, 331 Olivier, L., 171, 172, 183, 187, 210, 238, 256, 264 Oliveira Lecuona, M., de, 302, 309, 331 Olivier-Gonzalez, J., 245, 255, 259, 264 Olsen, 0. W., 219, 267 Olsson, P., 139, 148 Onabanjo, A. O., 61, 62, 63, 65, 66, 68, 74, 75 Orduna Prieto, C., 308, 331 Orecchia, P., 308, 331 Orgaz, J., 273, 313 Orr, T. S. C., 358, 359, 373 Orzet-Fichtel, A., 331 Osborn, H. L., 83, 90, 93, 99, 103, 113, 121, 136, 137, 138, 140, 141, I48 Osche, G., 141, 148 Osmanov, S. O., 139, 148 Ostertag, V., 332 Overdulve, J. P., 14, 21

393

Overman, R. R., 41, 42, 47 Owen, Ch. A. Jr., 276, 336 Ow-Yang, C. K., 194, 204, 220, 221, 225, 227, 246, 248, 249, 260, 261, 262, 264 Ozeretskovskaya, N. N., 331 Ozeretskovskaya, 0. L., 213, 263 P Paggi, L., 308, 331 Palmer, J. R., 245, 259, 260, 266 Palombi, A., 177, 187 Pampiglione, S., 308, 331 Pan, C. T., 199, 202, 211, 215, 223, 238, 244,264 Panetta, J. C., 305, 327 Pantaleon, J., 304, 340 Paoliello, J. C., 303, 309, 331 Papasarathorn, T., 299, 302, 309, 314, 331 Paperna, I., 203, 264 Paraense, W. L., 218, 264 Pardi, M. C., 303, 309, 331 Park, C. T., 277, 302, 309, 325 Parnell, I. W., 295, 327 Parsons, S., 139, 145 van Parys, O., 302, 304, 305, 309, 327 Pasternak, J., 360, 364, 378 Pauley, G. B., 136, 144, 148 Pavanand, K., 65, 73 Pavlov, P., 307, 308, 332 Pavlovskii, E. N., 139, 148 Pawel, O., 305, 332 Pawlowski, Z., 291, 292, 293, 314 Pawlowski, 2. S., 277, 283, 284, 285, 289, 290, 291, 292, 293, 294, 299, 300, 301, 308, 322, 332 Pearson, J. C., 160, 161, 164, 166, 170, 177, 178, 187 Peaston, H., 219, 238, 259 Peatt, E. S. W., 302, 309, 332 Peel, C., 282, 306, 332 Peeters, E., 293, 325 Pellegrini, D., 308, 332 Pellegrino, A., 308, 332 Pellegrino, J., 193, 245, 264 Pemberton, C. E., 205, 264 Penfold, H. B., 277, 278, 281, 282, 283, 284, 295, 296, 303, 304, 306, 332 Penfold, W. J., 277, 278, 281, 283, 295, 296, 303, 304, 306, 332 Penna, B., 6, 14, 20, 24

394

AUTHOR INDEX

Pennisi, L., 308, 333 Penson, D., 302, 309,341 Perera, D. R., 290, 333 Pkref-Moreira, L., 274, 285, 311, 339 Perez Moreno, B., 285, 333 Perlstein, J. M., 245, 256 Persiani, G., 305, 325, 327 PessBa, S. B., 21, 26, 27, 303, 309, 333 Pester, F. R. N., 271, 272, 298, 330 Peters, J. L., 5 , 21, 27 Peters, W., 31, 38, 39, 40, 41, 46, 47;48 Petkov, A., 301, 308, 333 Petrosyan, N. A., 290, 293, 294, 333 Petrova, T. A., 297, 339 Petru, M., 288, 308, 333 Pezenburg, E., 274, 280, 321,333 Pezzlo, F., 355, 373 Pham-Ngoc-Thach, 333 Phillips, M., 277, 281, 283, 295, 296, 304,332 Phisphumividhi, P., 33, 46 Piqtkowska, W., 308, 333 Pietrowa, R., 292, 312 Pigulevskii, S. V., 155, 157, 187 Pipkin, A. C., 288, 333 Pirkl, J., 305, 333 Pistor, W. J., 281, 282, 306, 316 Pittaluga, J. Z., 333 Planeta-Malecka, I., 285, 323 Plaschke, W., 304, 308, 333 Plimmer, H. G., 5, 21, 24, 26 Podyalpolskaya, V. P., 277, 287, 288, 333 Poinar, G. O., 208, 264 Pokier, J., 139, 148 Pokier, M., 333 Poletaeva, 0. G., 281, 282, 306, 325 Pollack, S., 34, 47 Popov, N. P., 139,148 Popov, V. F., 307,333 Popovici, V., 304, 308, 314 Porter, A., 265 Poll, M. C., 334 Pouplard, L., 301, 307, 308, 320 Powell, E. C., 176, 188 Powell, N. T., 213, 214, 265 Powell, S. J., 334 Praderi, L. A., 285,311 Prankerd, T. A. J., 39, 47 Pratt, H. S., 141, 149 Pratt, I., 162, 176, 181 Prchal, C. J., 307, 309, 339

Prkvot, G., 168, 186 Prkvbt, R., 276, 289, 334 Price, C. E., 141, 149 Price, D., 208, 209, 222, 258 Price, D. L., 302, 309, 334 Price, E. W., 272, 334 Price, H. F., 170,187 Priestly, H., 301, 308, 334 Primio, R. di, 6, 15, 21, 24, 25, 26 Proctor, E. M., 274, 317, 329 Proctor, R. M., 289,334 Profk, 296,334 Prokopenko, L. I., 290, 293, 300, 306, 307, 308, 324, 333,334 Przyjalkowski, Z., 334 Pugachevskaya, E. F., 308, 334 Pugh, M. H., 276,342 Pujatti, D., 334 Pullin, R., 268 Purnell, R. E., 211, 219, 265 Pylko, 0. O., 276, 334 Pytel, A. Y., 334 R Rabin, H., 208, 265 Race, G. J., 358, 359, 361, 376, 377 Radke, M. G., 193, 217, 255,265 Raffaele, G., 11, 21, 25 Rai, S. L., 82, 107, 108, 113, 120, 121, 122, 127, 135, 136, 137, 139, 140, 141, 149, 162, 187 Ramsdell, S. G., 288, 334 Randriamalala, J. Ch., 278, 313 Rankin, J. S., 177, 187 Rankin, J. S. Jr., 160, 187 Rao, M. P. C., 221, 265 Rasameeprabha, K., 299, 302, 309, 314 Raschke, H., 301, 308, 334 Raski D. J., 362, 364, 378 Rausch, R., 174, 187 Rausch, R. L., 139, 149 Rawat, P., 139, 149 Ray, A. P., 44,47, 59, 70, 75 Ray, H. N., 276, 278,314 Read, C. P., 274,276,311, 334 Reddy, S., 33, 34, 46 Reed, R. E., 282, 306,316 Rees, G., 285, 287, 334, 354, 355, 356, 377 Refuerzo, P. G., 309, 334 Reichenow, E., 9, 11, 21, 25 Reid, H. A., 67, 73

395

AUTHOR INDEX

Reid, W. M., 279, 334 Reinecke, R. K., 341 Reisinger, F., 179, 187 Renaudet, R., 288, 316 Renesch, B., 142, 149 Repciuc, E., 273, 339 Reuter, F., 308, 334 Reynolds, E. S., 355, 356, 357, 378 Reznik, G. K., 356, 377 Ribeiro, P. de Assis, 303, 309, 334 Ricci, M., 308, 334 Rich, A. B., 297, 336 Richard, A., 285, 334 Richards, C. S., 218, 219, 245, 265 Richards, J. G., 354, 375 Richards, W. H. G., 62, 66, 73, 75 Richardson, T., 282, 313 Rickman, R., 271, 272, 298,330 Riding, I. L., 364, 368, 378 Riflcin, E., 208, 256, 355, 358, 378 Riggin, G. T., 169,187 Rijpstra, A. C., 279, 287, 335 Riley, M. V., 44, 46, 70, 75 Ritchie, L. S., 245, 260, 265 Rivero, E., 274, 335 Rivoalen, A., 309, 335 Rizk, E., 288, 333 Robbins, C. L., 199, 265 Robb-Smith, A. H. T., 284, 322 Roberts, C. J., 309, 335 Robert, J., 292, 331 Robson, E. M., 265 Robtser, A. N., 335 Rocha, U. F., 303, 309,331 Rock, R. C., 32, 45 Rodrigues Solis, L., 335 Rodriguez, L., 277, 283, 284, 328 Rodriguez Gonzalez, M., 334 Roels, 0. A., 302, 309, 334 Rogers, W. P., 214, 265 Roggen, D. R., 362, 364, 378 Rohde, K., 79, 80, 81, 82, 83, 84, 85, 90, 91, 93, 94, 95,96, 97, 98, 99, 100, 102, 103, 104, 105, 106, 108, 109, 110, 111, 112, 113, 114, 117, 118, 120, 121, 124, 125, 126, 130, 131, 132, 133, 134, 135, 138, 139, 140, 141, 142, 143, 149,150 Rohringer, R., 213, 265 Roiter, M., 309, 335 Rollier, R., 285, 286, 315, 335 Roman, E., 288, 335 Romand, A., 291,316

Romanenko, N. A., 335 RosB, F., 278, 288, 289, 312, 313 Rosen, S. W., 294, 335 Rosenbusch, F., 8, 21, 26 Rosler, 0 . A., 294, 335 Rosset, R., 304, 340 Rossi, A. A., 335 Roth, H., 295, 296, 308, 322 Rothman, A. H., 370, 378 Rothschild, M., 172, 179, 187, 215, 265 Round, M. C., 280, 282, 297, 298, 318, 335 Rousselot, R., 9, 10, 12, 21, 25, 26 Ruiz, J. M., 220, 245, 265 Ruiz-Tiben, E., 245, 266 Rumbold, D. W., 139, 150 Ruosch, W., 304, 305, 308, 316 Rusak, L. V., 289, 292, 324,335 Rybaltovsky, 0. V., 294, 335 Rybicka, K., 278, 335 Rydzewski, A., 277, 308, 332 Ryerson, D. L., 22, 24 Ryley, J. F., 35, 46

S de Sa, B., 5 , 14, 20, 24 Saalbreiter, R., 305, 308, 321 Sabbaghian, H., 219, 256, 309, 335 Sachs, R., 335 Safarov, G. I., 287, 311 Saikkonen, J., 289, 292, 329, 335 Saint-Guillain, M., 220, 266 Saito, M., 293, 343 Sakamoto, D., 355, 356, 359, 360, 362, 375 Sakamoto, T., 358, 378 Salamov, D. A., 296, 297, 311 Salem, H. H., 293,335 Salemme, M. A., 312 Salt, G., 200, 208, 213, 214, 244, 266 Salyaeva, V. A,, 305,311 Samborski, D. J., 213, 265 Samoiloff, M. R., 360, 364, 378 Sandars, D. F., 335 Sankale, M., 309, 335 Saoud, M. F. A., 219,266 Sardou, R., 274, 325 Sargent, J. R., 35, 46 Sarkisyan, V. A., 290, 293, 294, 333 Sasanov, A. M., 220,266 Saugrain, J.. 336 Savchenko, L. P., 336

396

AUTHOR INDEX

Saxe, L. H., 35, 36,45 Saz, H. J., 289, 291, 336 Scanga, M., 4, 12, 13, 18, 24, 26 Scarza, J. V., 168, 186 Schacher, J. F., 286,336 Schaller, G., 245, 266 Schanabel, R., 343 Schardein, J. L., 276, 336, 362, 378 Scheibel, L. W., 35, 36, 38, 39, 47, 289, 291, 336 Schell, S. C., 157, 162, 181, 187 Schlachter, H., 308, 336 Schmid, G., 305,336 Schmid, M., 308, 336 Schmidt, K., 14, 21 Schneider, H. H., 336 Schneider, I. S., 305, 327 Schneider, J., 293, 336 Schnell, J. V., 39, 47 Schoen, R., 336 Scholtyseck, E., 14, 21 Schoon, J. G., 336 Schoop, G., 306,336 Schuffner, W., 288,336 Schultz, M. G., 290, 297, 301, 307, 309, 333, 336 Schulze, U., 308, 336 Schumbert, R., 336 Schwabe, C. W., 223,266,299,304,307, 336 Schwartz, M., 294, 316 Scott, D. B., 276, 342 Scudamose, H. H., 276, 336 Seaton, D. R., 293, 310, 336 Seed, T. M., 42, 47 SBnaud, J., 14, 21 Seguin, F., 294,311 Seiler, H. E., 301, 308, 337 Sekhon, S. S., 361, 372 Sellers, T. F., 339 Seo, B. S., 302, 309, 337 Sergiev, P. G., 306, 308, 337 Sewell, R. B. S., 156, 159, 164, 177, 188 Shabelnik, V. I., 309, 337 Shafei, A. Z., 293, 337 Shah, P. M., 285,294, 302, 309,337 Shahin, H., 286 337 Shakhsuvarli, M,. A., 277, 286, 337 Sharma, G. K., 59, 70, 75 Sharp, N. C. C., 306, 341 Sheffield, H. G., 14, 21 Shekhovtsov, V. S., 308, 337

Sheldon, J. J., 304, 305, 316 Sherman, I. W., 37, 39, 47, 48 Sherwood Jones, E., 69, 72, 74 Shipley, A. E., 128, 137, 150 Shircore, T. O., 297, 337 Short, R. B., 83, 90, 136, 137, 146, 176, 188

Shortridge, E. H., 303, 309, 337 Shulman, E. S., 306, 307, 308, 333, 337 Sibileva, L. M., 331 Siddiqi, A. H., 139, 150 Siddiqui, E. H., 280, 337 Siddiqui, W. A., 39, 47 sijakov, I., 303, 308, 317 Siim, J. C., 14, 19 Silk, M. H., 355, 378 Sillman, E. I., 162, 170, 188 Silverman, P. H., 32, 46, 278, 279, 280, 295, 296, 297, 301, 304, 307, 308, 337, 338 Simer, P. H., 139, 150 Simitch, T., 308, 338 Simmonds, F. J., 205, 266 Simmons, J. E. Jr., 274, 276, 334 Simonescu, O., 290, 331 Simonffy, Z., 300, 305, 308,339 Simpson, E. R., 168, 185 Singh, R. N., 172, 188 Sinha, B. B., 139, 150 Sinitsin, D., 155, 156, 157, 158, 169, 188 Sinitzin, D. F., 136, 150 Sinnecker, H., 296, 297, 308, 338 Siu, P. M. L., 37, 47 Skaer, R. J., 352, 378 Skelton, F. S., 39, 47 Skirrow, M. B., 44,47, 58, 59, 75 Skrjabin, K. I., 78, 141, 150 Skvortsov, A. A., 338 slais, J., 278,280,281,338, 358, 359, 378 Smircic, P., 286, 319 Smit, A. M., 279,287,335 Smith, D. H., 38, 48 Smith, J. H., 355, 356, 357, 378 Smith, K., 360, 378 Smith, K. J. H., 33, 45 Smithers, S. R., 357, 373, 378 Smyth, J. D., 158, 170, 172, 188, 274, 279, 282, 338 Snigirevskaya, E. S., 14, 21 Snyder, R. W., 203, 256 Sogandares-Bernal, F., 139, 150, 172, 188

397

A U T H O R INDEX

Sokolovskaya, 0. M., 281,282,288,306, 325,338 Solarino, A., 308, 315 Soliman, K . N., 302, 309, 317 Someren, V. D., van, 296, 317 Sommer, S. C., 164, 165, 189 Sonsino, P., 136, 150 Soulie, P., 326 Soulsby, E. J . L., 281, 298, 306, 319, 338, 361, 377 Southgate, V. R., 221, 266, 352, 353, 356, 378, 379 Southwell, T., 271, 338 Speck, J . F., 38, 48 Spector, W. G., 65, 75 Spence, I. M., 355, 378 Spooner, D. F., 32, 46 Sprengers, R., 301, 307, 308, 320 Sprent, J . F . A., 295, 338 Sprinz, H., 32, 45 Squadrini, F., 276, 328 Ssinitzin, D. Th. von, 177, I88 Stafford, J., 83, 90, 91, 93, 103, 108, 136, 137, 144, 150 Standen, 0. D., 219,266, 289, 291, 292, 338 Starkoff, O., 274,338 Stauber, L. A., 21, 24, 208, 266 Stauber, Mabel F., 21, 24 Stebhens, W . E., 14, 21 Steele, J . H., 301, 307, 309, 336 Steenstrup, J . J . Sm., 153, 188 Steigler, 301, 308 Steinberg, D., 128, 136, 137, 138, I50 Stephens, J. M., 208, 266 Stephenson, J . W., 245,266 Steppe, W., 308, 339 Steward, J . S., 289, 339 Stieda, A., 285, 286, 339 Stirewalt, M . A., 170, 188, 219, 266 Stockdale, P. H . G., 361, 379 Stoll, N. R., 303, 308, 339 Stossich, M., 139, I50 Stoye, M., 317 Stratman-Thomas, W . K., 52, 73 Strikovsky, T. L., 277, 339 Stromberg, P. C., 136, 150 Stromskaya, T. F., 290, 323 Strout, R. G., 14, 21 Strufe, R., 279, 320 Stunkard, H. W., 93, 103, 136, 139, 141, 151, 155, 156, 157, 158, 159, 164, 167,

176, 178,188, 202, 219, 266, 267 Stuparic, D., 308, 342 Sudds, R. H . Jr., 222, 230, 267 Sugimura, M . , 358, 378 Suguri, S., 355, 356, 359, 360, 362, 375 Summa, H., 308, 339 Sundby, R. A., 200, 214,244, 257 Sunkes, E. J., 339 Sussman, O., 307, 309, 339 Suvorov, V. Y., 293, 295, 296, 339 Swales, W. E., 223, 267 Swartzwelder, J . C., 283, 339 Sweetman, H. L., 244, 267 Swellengrebel, N. H., 279, 287, 288, 335,336, 339 Swierstra, D., 339 Swiezawska, E., 293, 322,324 Swinehart, B., 15,18, 25 Sycevskaja, V. L., 297,339 Syogaki, K., 82,151 Szelenyi, L., 300, 305, 308, 339 Szidat, L., 162, 168, 170, 188 Sztrom, Z. K., 277, 339 Szyfres, B., 339 T Tabo, R., 309,320 Taddia, L., 21, 25 Tadros, G., 310 Takacs, J., 300, 305, 308, 339 Takki, S., 289, 291, 292, 294, 328, 339 Talavera, J., 339 Talice, R. V., 274, 285, 311, 339 Talyzin, F . F., 286, 339 Tanaka, H., 360, 375 Tanasescu, I., 273, 339 Tandon, R. S., 137, 140, 151 Tang,C.C., 121, 139, 141, 146, 158, 184 Taparelli, F., 276, 328 Tarnaala, K., 308, 339 Tarpila, S., 294, 339 Taylor, A. E. R., 214, 267 Taylor, D. C., 272, 282, 339 Taylor, E. W., 360, 379 Telkka, A., 326 Tella, A., 61, 62, 63, 74, 75 Terhorst, H., 305, 340 Thakur, A. S., 208, 256 Ter-Karapetiants, N. N., 340 Terry, R. J., 357, 373, 378 Theakston, R. D. G., 34, 38, 39, 43,48

398

A U T H O R INDEX

Thienpont, D., 302, 309, 312 Thieulin, G., 304, 340 Thillet, C. J., 219, 263 Thiodet, J., 286, 340 Thomas, H., 279,320 Thomas, J. D., 176,188 Thomas, J. N., 360, 379 Thome, M., 302, 306, 309,320 Thompson, Jr., J. H., 276, 336 Thomson, W. W., 360, 364, 375 Thornton, H., 302, 309, 340 Thorp, W. T. S., 308, 340 Threadgold, L. T., 355, 356, 360, 376, 377,379 Thurnham, D. I., 43,48 Thust, R., 361, 365, 379 Timberlake, P. EI., 214, 267 Timofiev, V. A., 358, 359, 379 Timon-David, J., 164, 166, 169, 177, 185,188,189 Ting, I. P., 37, 47, 48 Todd, J. L., 14, 15, 21 Todorov, R., 287,301,307,308,333,340 Tongu, Y., 355, 356, 359, 360, 362, 375 Topuriya, I. I., 308, 340 Totterman, G., 326, 340 Trager, W., 36, 45, 48 Tran van Ky, P., 289,312 Trautman, R. J., 282, 306, 316 Trawinski, A., 308, 340 Trevino, A., 277, 283, 284, 328 Tripp, M. R., 207, 208,267 Tronchetti, F., 286, 340 Tsubota, T., 362, 379 Tu, M., 302, 309,340 Turner, P. P., 293, 340 Tustin, R. C., 281, 341

U.S. Department of Agriculture, 78,151 Utter, M. F., 37, 48

V Valtonen, E. J., 294, 339 Valverde, A., 341 Van Beneden, P. J., 180, 189 Van Cleave, H.-J., 136, 138, 140, 151 Van den Heever, L. W., 281, 302, 309, 341 Van der Plank, J. E., 213, 267 Van Der Woude, A., 163, 164, 165,183, 189 Van Gils, J. H. J., 301, 305, 308, 341 Van Grunderbeeck, R., 302, 309, 341 Van Gundy, S. D., 360, 364, 375 Van Keulen, A., 301, 308, 341 Van Steenburgh, W. E., 205, 267 Varges, W., 304, 305,341 Vasilkova, Z. G., 296, 297, 341 Vasina, S. G., 1, 10, 22, 24, 26 Vegors, H. H., 341 Vercruysse, R., 276, 325 Verdiev, G. Y., 286, 341 Vernberg, F. J., 203, 215, 267 Vernberg, W. B., 203, 215, 267 Verster, A., 270, 271, 272, 274, 275, 278, 302, 309, 341 Versyck, M., 302, 307, 309, 341 Vieira, C. B., 277, 341 Viles, J. M., 326 Viljoen, N. F., 304,341 Villalobos, P. R., 318 Villanyi, J., 301, 307, 308, 320 Vincent, G., 293, 319 Voeltzkow, A., 90, 93, 103, 107, 108, 120, 121, 122, 127, 128, 134, 135, 136, 137, 139, 140, 151 U Voge, M., 203, 259, 279, 280, 341 Ubelaker, J. E., 203, 219, 267 Vogel, H., 274, 341 Ubieto, A., 309, 343 Vogelsang, E. G., 277, 309,314, 342 Vojtgchovskk, M., 288, 308, 333 Uegaki, J., 21, 24, 25, 26 Ulivelli, A., 292, 340 Voller, A., 33, 45 Ullyett, G. C., 205, 267 von Brand, T., 44, 47, 214, 267, 274, Ulmer, M. J., 164, 165, 169, 171, 189 276, 342 Umathevy, T., 168, 185, 192, 194, 195, Von Harnack, G. A., 342 201, 204, 225, 231, 246, 259, 261, 262 Vujic, B., 308, 342 Upton, A. C., 285,286,340 W Ureche, L., 308, 340 Urquhart, G. M., 281, 295, 298, 300, Wachowska, M., 342 Wagner, E. D., 219, 256 302, 305, 306, 308, 309,340, 341 Usanga, E. A., 34,46 Waitz, J. A,, 276, 336

A U T H O R INDEX

Wajdi, N., 211, 220, 245, 267, 353, 379 Waksman, S. A,, 200, 213, 214, 268 Walde, A. W., 323 Walzberg, U., 6, 21, 24, 26 Wang, W. L., 296, 342 Ward, H. B., 139, 151 Ward, R. A., 33, 46 Wardle, R. A., 270,342 Warhurst, D. C., 38, 39, 40, 46 Warnecke, W., 284, 342 Warren, K. S., 21 1, 213, 245, 259, 268 Wasowa, D., 308, 326 Watkins, S., 213, 262 Webbe, G., 193,213, 262,268,279,320, 342 Wegmann, T., 342 Weinbach, E. C., 358, 377 Weinberg, M., 288, 342 Weinland, F., 270, 342 Weinmann, C. J., 282, 342 Weisberger, A. S., 213, 268 Wen, Y. F., 309, 342 Wenyon, C. E. M., 59, 72, 74 Wenyon, C. M., 2, 13, 22, 24 Wesenberg-Lund, C., 201, 202, 204, 268 Western, K. A., 290, 333 Wetmore, Psyche W., 22, 24, 26 Wharton, G. W., 137, 139,151 Wigand, R., 284, 342 Wikerhauser, T., 280, 306, 342 Willard, H. F., 205, 264 Williams, C. O., 107, 108, 128, 129, 136, 137, 138, 140, 141,151 Williams, I. C., 265 Williams, J. E., 218, 219, 263 Williamson, J., 32, 48 Wilmot, A. J., 334 Wilson, R. A., 220, 268, 352, 353, 379 Winfield, G. F., 209, 268 Wisse, E., 360, 379 Witenberg, G. G., 302, 306, 309, 342 Winterhalter, M., 303, 308, 342 Wohnus, J. F., 22, 24 Wojciechowska, L., 342 Wojtak, S., 331 Wolbach, S. B., 14, 15, 21 Wolbert, B., 213, 258 Wold, N., 219, 256

399

Wolff, F., 343 Wolfson, Fruma, 7, 8, 12, 18, 22, 23, 26 Wood, Fae D., 22, 24 Wood, H. G., 37, 48 Wood, R. K. S., 213, 268 Wood, S. F., 9, 15, 22, 24, 25, 26, 27 Woodruff, A. W., 277,322 Wootton,D. M., 107,108,113,126,135, 137,151, 156, 176, 189 Work, K., 14, 19 Wostmann, B. S., 362, 374 Wright, C. A., 156, 178, 179, 189, 214, 215,246,268 Wright, K. A., 361, 362, 364, 368, 379 Wright, R. D., 369, 370, 379 Wu, L. S., 302, 309, 343 Wyant, K. D., 278, 322,325 Y Yajima, Y., 355, 373 Yakimoff, W. L., 5, 22 Yamaguti, S., 78, 127, 139, 141, 151, 176,189 Yaqub, M., 309,343 Yarwood, C. E., 213, 268 Yates, D. B., 66, 75 Yokogawa, M., 293,343 Yoshida, Y., 293, 330 Yoshimura, H., 293, 343 Youssef, L. B., 304, 305, 309, 317 Yuen, P. H., 360, 379 Z Zacharias, O., 83, 113, 151 Zapart, W., 288, 289,343 Zapatel, J., 309, 343 Zasukhin, D. N., 1, 10, 22, 24, 26 ZelejkoviC, S., 308, 343 Zembruski, K., 300, 308, 343 Zetterstrom, R., 43, 48 Zimmer, E., 317 Zingano, A. G., 309,326 Zischke, J. A., 201, 268 Zolotarev, N. A., 305, 311 hkovic, M., 280, 306, 342 Zunker, M., 305, 343 Zwierz, C., 326, 327, 343

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Subject Index Acanthatrium, 172 oregonense, cuticle structure, 355 Acanthocephala, cuticle structure and function, 369-370 Acanthocephalus ranae, cuticle structure, 367 Acanthocotyle elegans cuticle function, 352 structure, 350, 351 Acanthoncus duplicatus cuticle function, 368 structure, 362 Acanthoparyphium spinulosum, I 68 cuticle structure, 354 Acridines in taeniasis therapy, 292 Adeleine haemogregarines, 2-3, 14-1 6 Allocreadium alloneotenicum, life cycle 176 Alaria, life cycle, 177-8 Amidostomum anseris, cuticIe structure, 361 Amphibdella flavolineata, cuticle str ucture, 349, 351 Ancylostoma caninum, cuticle structure, 360 duodenale, cuticle structure, 360 Anomotaenia constricta, cuticle structure, 359 Aphelenchus avenae cuticle structure, 360 moulting, 364 Ascaris lumbricoides cuticle structure, 361 diagnosis, 289 moulting, 365 Ascarops strongylina, cuticle structure, 361 Aspiculuris tetrptera, cuticle structure, 362 Aspidocotyle miitabile, 78 Aspidocotylus cochleariformis, 78 401

Aspidocaster . protonephridial system, 83 antipai, localization in mollusc, I36 conchicola adult genital system, 90, 91, 93 marginal bodies, 103 protonephridial system, 82 biology infectivity of larvae, 136 infectivity to vertebrate host. 138 invasion into mollusc, 135 life span, 134 localization in mollusc, 135 in vertebrate host, 139 sexual maturation in mollusc, 137, 138 survival of adult outside host, 140 development egg, cleavage and larval development, 120 hatching, 121, 122 parasitic stage, 126-1 3 1 larvae, free infectivity, 135 life span, 134 structure general morphology, 107, 108 protonephridial system, 1 13 tegument and ciliary tufts, I10 indica adult, protonephridial system, 82 biology localization in mollusc, 136 sexual maturation in mollusc, 137 survival of adult outside host, 140 development egg, cleavage and larval development, 120 hatching, 121, 122 parasitic stage, 127 larvae, free

402

S U B J E C T INDEX

Aspidogaster indica (contd.)

general morphology, 107, 108 protonephridial system, 113 limacoides

genital system, 93 localization in vertebrate host, 139 parasitic stage, 127, 128 Aspidogastrea adult digestive tract, 81-82 genital system, 90-93 marginal bodies, 103-107 nervous system, 82-90 protonephridial system, 82-90 sense receptors, 99-103 tegument, 79-81 ventral disc, 103 biology infectivity to vertebrate host, 138139 invasion into mollusc, 135 larvae, free infectivity, 135 life span and behaviour, 134-135 localization in mollusc, 135-137 in vertebrate host, 139 sexual maturation in mollusc, 137138 survival of adult outside host, 140 development allometric growth, 131-134 egg, cleavage and larval development, 120-121 hatching, 121-123 parasitic stage, 124-131 larvae, free infectivity, 135 life span and behaviour, 134-5 structure digestive tract, 111-1 12 general morphology, 107-108 glands and caudal appendage, 111 nervous system, 114-1 18 protonephridial system, 112-1 14 sense receptors, 118-120 tegument and ciliary tufts, 108111 life cycle and digenean cycles, 143144 phylogenetic position, 140-143

Asymphylodora amnicolae, life cycle, 176

Atabrine in taeniasis therapy, 292 A toxoplasma argyae, 9 avium, 2, 9, 11 coccothraustus, 4 danilewskii, 10 paddae, 2, 9, 11

Atoxoplasms, 2,3-14 Austrobilharzia terrigalensis, intraniolluscan interaction with Stictodora,

204 Azygia, 170

B Babesiu canis, effect of nor-adrenaline

in shock caused by, 66 Biological control using inter-trematode antagonism 244-251 Biomphalaria glabrata

inter-trematode interactiois in, 194-195,225-238 maintenance, 193-197 straminea, inter-trematode interaction in, 194 Bithionol in taeniasis therapy, 293 Bivesicula, 170 cercariae, 161 Bradynema, lack of cuticle, 364 Burnellus, 169 trichofurcatus, 171 life cycle, 169 Calliobothrium

C verticillatum,

cuticle

structure, 359 Capillaria hepatica, cuticle structure, 361 Carbon dioxide fixation by Plasmod i m , 37-38 Caryophyllaeus fennica, cuticle structure, 359 laticeps, cuticle structure, 359 Catatropis verrucosa, 166 Cephalogonimus, life cycle, 177 Cercaria bucephalopsis haimaena, cuticle struc-

ture, 354 buchanani, cuticle structure, 354 doricha, intramolluscan development,

203 gorgonocephala, 169

pectinata, cuticle structure, 354

403

SUBJECT INDEX

Cestoda, cuticle structure and function, 357-360 Cestodaria, cuticle structure and function, 357 Cestodin in taeniasis therapy, 291 Chloroquine resistance in Plasmodium, 39-41 Clinostomum, 174 giganticum. 164 marginatum, 164 Cloacitrema narrapeenensis, cuticle structure, 354,355 Clonorchis sinensis, cuticle structure, 356 Coccidia, avian, 1-17 adeleine haemogregarines, 14-1 6 atoxoplasms, 3-14 Coenurus cerebralis, differential diagnosis, 280, 281 Coitocaecum anaspidis, life cycle, 176 Contracaecum multipapillaturn, cuticle structure, 361 Control, biological, using inter-trematode antagonism 244-251 Cotylaspis egg, cleavage and larval development, 120 protonephridial system, 83 cokeri, marginal bodies, 103 insignis adult genital system, 90 sense receptors, 99 biology localization in mollusc, 136-137 sexual maturation in mollusc, 137,138 survival of adult outside host, 140 hatching, 121 larvae, free, structure, 113 protonephridial system, 113 reelfootensis, 136, 137 Cotylogaster michaelis adult genital system, 93 nervous system, 93 hatching, 123 occidentalis adult genital system, 90, 93 marginal bodies, 103

biology, infectivity to vertebrate host, 138 localization in mollusc, 137 sexual maturation in mollusc, 137 development hatching, 121 parasitic stage, 126 larvae, free general morphology, 107, 108 protonephridial system, 1 1 3 tegument and ciliary tufts, 1 1 1 Cotylogasteroides occidentalis see Cotylogaster Cotylurus flabelliformis, snail immunity to, 209, 210 lutzi intramolluscan interactions with Paryphostomum segregaturn, 21 1 Schistosoma mansoni, 195, 206207,234 Crassicauda crassicauda, cuticle structure, 361 Crenosema vulpis, cuticle structure, 361 Cryptocotyle lingua, cuticle structure, 354 Cucurbita pep0 seeds in taeniasis therapy, 294 Cysticercosis epidemiology, 299-303 medical and economic losses, 303-304 prevention, 304-307 transmission, 295-299 Cysticercus bovis cuticle structure, 358 differential diagnosis, 280, 281 cellulosae, differential diagnosis, 280, 281 fasciolaris, cuticle structure, 358 longicollis, cuticle structure, 358

D Dasymetra conferta, 162 Delandenus siricidicola, cuticle structure,

364 Delafondia vulgaris, cuticle structure, 361 Derogenes varicus, life cycle, 176

404

S U B J E C T INDEX

Diachasma tryoni, interaction with Opius humilis, 205 Dichlorophen in taeniasis therapy, 293 Dichlosal in taeniasis therapy, 293 Diclidophora merlangi, cuticle structure and function, 351, 352 Dicrocoelium dendriticum, cuticle structure, 356 Dictyocaulus viviparirs, cuticle structure, 361 Dictyocotyle coeliaca, cuticle structure, 351 Digenea cuticle structure and function, 352356 generations alternation, 158-160 suppression and replacement, 164 evolution, 163-1 64 life cycles one host, 156-158 two host, 160-161 three host, 167-177 four host, 177-178 metacercarial stage, 165-1 67 phylogenetic implications, 178-1 79 redial generation, 162-163 Diphylidium caninum, cuticle structure, 359 Diphyllobothrium laturn, 276, 211 cuticle structure, 358, 359 Diplostomum JIexicaudum life cycle, 176 snail immunity to, 210 Dirofilaria immitis, cuticle structure, 361 Ditylenchus dispnci, cuticle structure, 360 Drepanidium, 1 avium, I , 1 1

E Echinochasmus, life cycle, 168 Echinococcus granulosus appendicitis caused by, 286 cuticle function, 359 structure, 358,359 differential diagnosis, 281

egg survival, 295 multilocularis, cuticle structure, 358 Echinoparyphium life cycle, 168 dunni, intramolluscan interaction with xiphidiocercaria, 194 Echinorhynchus truttae, cuticle structure, 369 Echinostoma, 174 life cycle, 168 auclyi biological control of trematodes with, 246 intramolluscan interaction with other trematodes, 194, 225, 237, 249 harbosai adaptation index, 224, 225 infection rate, 220 intramolluscan interaction with Paryphostomum segregatum, I 94 Schistosoma mansoni, 195 maintenance, 198 redial population size, 223, 238 snail immunity to, 21 1 liei infection rate, 216, 220 intramolluscan interaction with Paryphostomum segregatum, 195 Schistosoma mansoni, 195, 20 I , 202, 207, 226, 236 maintenance, 198 redial population size, 223 Iindoense adaptation index, 224, 225 antigen-binding substance in infected Biomphalaria glabrata, 208 infection rate, 218, 220 intramolluscan interaction with Paryphostomum segrega tum, I 95, 204-205 maintenance, 198 miracidia penetration, 222 redial population size, 223 snail immunity to, 21 I malayanum biological control of Schistosoma mansoni with, 246 infection rate, 218 intramolluscan interaction with Schistosoma spindale, 194, 225, 237

S U B J E C T INDEX

Echinostoma malayanum (contd.) miracidia penetration, 220, 221, 222 nudicaudatum, rediae cannibalism, 204 paraensi adaptation index, 224, 225 infection rate, 220 intramolluscan interaction with Paryphostomum segregatui, 195, 205 maintenance, 198 redial population size, 223 snail immunity to, 21 1 Echinuria uncinata, cuticle structure, 361 Eimeria, 14 Eleutheroschizon, I I Enterobius vermicularis, cuticle structure, 362 Etitobdella solne, cuticle structure and function, 351,352 Epomidiosfomum orispinum, cuticle structure, 361 Eiiparyphium, life cycle, 168

F Fasciola intramolluscan development, 201 gigantica biological control by Echinostoma audyi, 246 intramolluscan interaction with E. audyi, 194, 237 miracidia penetration, 220, 221 redial stage limitation, 238 hepatica cuticle, function, 356 structure, 352-354, 356 miracidia penetration, 220, 221, 222 Fascioloides magna, 223 Fern extracts in taeniasis therapy, 294 G Gastrocotyle trachuri, cuticle structure, 351 Glucose nietabolism in Plasmodium, 35-1 Glucose-6-phosphate-dehydrogenase in malaria infected erythrocytes, 33-34

405

Glypthelmins life cycle, 177 pennsylvanicus, intramolluscan development, 203 Gorgoderina,cuticle structure, 356 Gymnophallus choledochus, life cycle, 176 Gyrocotyle urna, cuticle structure, 357 Gyrodactylus, cuticle structure and function, 351, 352 H Habronema muscae, cuticle structure, 361 Haemamoeba danilewkyi, 3 Haemogregarina adiei, 3, 14 aragaoi, 15 atticorae, 14 brachyspizae, 14 francae, 14 monachus, 15 paddae, I , 3, 5, 14 paulasousai, 6 pessoai, 6 pintoi, 15

poroiae, 14 rhamphoceli, 14 serini, 7 sicalidis, 3, 14 sporohilae, 3, 14 tanagrae, 14 travassosi, 15 Haemogregarines, adeleine, 2-3, 14-16 Haemonchus placei, cuticle structure, 360 Haemoproteits rouxi, 3 Haplometra cylindracea,cuticle structure, 356 Helminths, cuticle structure and fiinction Acant hocephala, 369-3 70 Cestoda, 357-360 Cestodaria, 357 Digenea, 352-6 Monogenea, 348-352 Nematoda, 360-369 Turbellaria, 348 Hemicycliophora aretiaria cuticle structure, 360 moulting, 364 Hepatozoon, 9, 15-1 6 ndiei, 14, 15

406

S U B J E C T INDEX

Hepatozoon (contd .) nephrontis, 14 spermesti, 9, 12 Heronimus chelydrae, 164-1 65 mollis, 165 Heterodera rostochiensis, cuticle structure, 360 schachtii, cuticle structure, 360 Himasthla life cycle, 167, 168 quissetensis, 215 Hirshmaniella belli, cuticle structure, 360 gracilis cuticle structure, 360 moulting, 364 Histamine in protozoal infections, 66 Horogenes chrysosticto, oxygen cornpetition with Nerneritis canescens, 214 Hydatigera, 271 Hymenolepis citelli, cuticle structure, 357, 358 diminuta cuticle function, 360 structure, 359 intramolluscan development, 203 nana appendicitis caused by, 286 infection, parornomycin therapy, 292 Hypoderaeum dingeri intramolluscan interaction with Trichobilharziabrevis, 204 T.brevis-Echinostoma audyi, 249 I

Indoplanorbis exustus, inter-trematode interaction in, 194 Isospora, 2, 12-14 lacazei, 6, 13 Zsthmiophora melis, snail immunity to, 213 K Kallikrein in malaria, 63-65 Kinin response to malaria, 60-67 Kronbergia amphipodicola, cuticle structure and function, 348

L Lacistorhynchus tenuis, cuticle structure, 359 Lankesterella, 1, 2, 7, 10, 11, 12-13 adiei, 12 avium, 1, 11 corvi, 4, 5, 12 garnhami, 4,11,12 lainsoni, 12 minima, 13 paddae, 11, 12 passeris, 11 picumni, 12 serini, 11 Lepocreadium setijieroides,215 Leptocotyle minor, cuticle structure, 348, 351 Leucocytogregarina amadinae, 5 neophrontis, 14 Ligula intestinalis, cuticle structure, 358, 359 Lissemysia ovata localization in mollusc, 137 sexual maturation in mollusc, 137 survival of adult outside host, 140 Litomosoides carinii, cuticle structure, 362 Lobatostoma localization in mollusc, 137 ringens, localization in vertebrate, 139 Longidirus macrosoma, cuticle structure, 362 Lophotaspis corbiculae, protonephridial system, 82-83 macdonaldi, localization in mollusc, 137 margaritgerae, localization in mollusc, 137 orientalis, hatching, 121 vallei adult marginal bodies biology localization in mollusc, 137 movement, 134 sexual maturation in mollusc, 137 hatching, 121, 122 larvae, free general morphology, 107,108 life span, 134

SUBJECT INDEX

Lymnaea rubiginosa, inter-trematode interactions in, 194

407

pudentotectus, cuticle structure, 361 salmi, cuticle structure, 361 Mitochondria] respiration inhibition in malaria, 70-72 M Molluscs, biological control, 245 Macraspis Monieza expansa, cuticle structure, cristatn, localization in vertebrate, 139 359 elegans Moniliformis dubius, cuticle structure adult and function, 369, 370 digestive tract, 81 Monogenea, cuticle structure and funcgenital system, 90 tion, 348-352 marginal bodies, 106 Monordotaenia, nomenclature, 27 1 development Multiceps, 271 hatching, 123 serialis, cuticle structure, 357-358 parasitic stage, 128 larvae, free, general morphology, Multicalyx cristata see Macraspis Multicalyx cristatus, hatching, 121 107 localization in vertebrate, 139 Multicotyle purvisi Malaria (see also Plasmodium) adult mammalian, pathogenesis digestive tract, 81-82 anoxic anoxia, 68-70 genital system, 90-93 chain reaction, 71-72 marginal bodies, 103-106 cytotoxic factors, 70 nervous system, 93-99 endothelial permeability changes, protonephridial system, 83-90 51-58 sense receptors, 100-102 inflammation-like responses, 51 tegument, 79-81 intravascular coagulation, 67-68 ventral disc, 103 kinins, 60-67 biology mitochondria1 respiration inhibiinfectivity to vertebrate host, 138tors, 70-72 139 vasomotor changes, 58-60 larvae, free, hepatic, 59 infectivity, 135 intestinal, 59-60 life span and behaviour, 134renal, 59 135 Marisa cornuarietis-Biomphalaria glasexual maturation in mollusc, 137 brata competition in biological control survival of adult outside host, 140 Mecistocirrus digitatus, cuticle strucdevelopment ture, 361 allometric growth, 131-134 Megalodiscus temeperatus, cuticle strucegg, cleavage and larval developture, 356 ment, 120-121 Meloidogyne hatching, 121-122 haplu, cuticle structure, 360 parasitic stage, 124-126, 130 javonica, cuticle structure, 360 larvae, free Mermis nigrescens, cuticle structure and infectivity, 135 function, 3624, 368 life span and behaviour, 134-135 Metagonimoides oregonensis, life cycle, structure 176 digestive tract, 111-1 12 Metagonimus general morphology, 108 takahoshi, cuticle structure, 355 glands and caudal appendage, yakagawi, cuticle structure, 356 111 Metastrongylus nervous system, 114-1 18 elongatus, cuticle structure, 361 protonephridial system, 113-1 14

408

S U B J E C T INDEX

Parahemiurus bennettae, life cycle, 176 Multicotyle purvisi (contd.) Paralepoderma brumpti, life cycle, 176 sense receptors, 118-119 tegument and ciliary tufts, 108- Parapronocephalum symmetricum, 1 66 111 Paromomycin in taeniasis therapy, 292-293 Parorchis acanthus, cuticle structure, 354 N Parvatrema homoeotecnum, 162 Necator americanus, cuticle structure, life cycle, 165,173-174 360 Paryphostomum Nematoda, cuticle structure and funclife cycle, 168 tion, 36&369 segregatum Nematospiroides dubius adaptation index, 224,225 cuticle structure, 362 infection rate, 216,219,220 moulting, 365 intraniolluscan interaction with Nemeritis canescens, oxygen competition Cotylurus lutzi, 21 1 with Horogenes chrysostictos, 214 Echinostoma barbosai, 194 Neoaplectana glaseri, cuticle structure, E. liei, 195 360 E. lindoense, 195,204-205 Niclosamide in taeniasis therapy, 290E. paraensi, 195,205 291 Ribeiroia marini, 194,195 Nippostrongylus brasiliensis Schistosoma mansoni, 195, 200, cuticle 202, 206, 207, 208-209, 210, function, 368 214-215,225-244 structure, 360,362 maintenance, 197-198 moulting, 365-367 rediae Nosema, in biological control of treformation, 203,205 matodes, 245 population, 223 Notocotyloides petasatum, 1 66 snail immunity to, 21 1 Notocotylus attenuatus, cuticle structure, sporocysts, 199 357 Paucivitellosus, cercariae, 164 0 fragilis, 166 cercariae, 161 Oesophagostomum columbianum, cuticle Pentose phosphate pathway in malaria structure, 361 infected erythrocytes, 33-34 Opisthioglyphe, life cycle, 177 Perezia helminthorum, in biological Opisthorchis viyerrini, cuticle structure, control of trematodes, 245-6,248 356 Perostrongylus pridhani, cuticle strucOpius humilis, interaction with Diachasture, 361 ma tryoni, 205 Petasiger, Ornithobilharzia turkestanicum, cuticle life cycle, 168 structure, 356 neocommense, cuticle structure, 354 Ostertagia ostertagi, cuticle structure, Phenasal in taeniasis therapy, 290 361 Phenoxybenzamine, effect on vasomotor changes in malaria, 59-60 Philophthalmus, 162 P Phocanema decipens, moulting, 365 Panagrellus silusiae 6-phosphogluconate-dehydrogenase in cuticle structure, 360 malaria infected erythrocytes, 33-34 moulting, 364 Phyllodistomum Paragonimirs ohirri, cuticle structure, simile, life cycle, 176 356 solidum, 172

SUBJECT INDEX

Phytoalexins, 213 Plagioporus lepomis, 177 Piagiorchis life cycle, 176 snail irmnunity to, 210 Plasmodium, 7-8 (See also Malaria) metabolism, 3 1 4 1 aerobic mechanisms, 38-39 carbon dioxide fixation, 37-38 chloroquine resistance, 3 9 4 I glucose, 35-37 pentose phosphate pathway in infected erythrocytes, 33-34 metabolism of host, 41-44 berghei effect on permeability of brain endothelial membrane, 56 erythrocyte coalescence by, 53 intravascular coagulation in infection, 67-68 kallikrein in infection, 65 metabolism, 32 aerobic mechanisms, 38-39 carbon dioxide fixation, 37 chloroquine resistance, 3 9 4 1 glucose, 35, 36 pentose phosphate pathway, 3334 metabolism of host erythrocytes, 41, 42 tissue, 43 mitochondria, 38 mitochondria1 respiration inhibition by, 70 roatneyi host erythrocyte metabolism, 41,42 kinins in infection, 65 falciparum anoxic anoxia, 69 52-53 intravascular coagulation, 67, 68 metabolism, pentose phosphate pathway, 33, 34 metabolism of host erythrocytes, 41 mitochondria, 38 sulphamethoxine effect on cerebral circulation in infection, 56 vasomotor changes in infection, 58 gallinaceum metabolism aerobic mechanisms, 38

409

pentose phosphate pathway, 33, 34 metabolism of host erythrocytes, 42 knowlesi anoxic anoxia in infection, 69 effect of nor-adrenaline on shock caused by, 66-67 effect on permeability of brain endothelial membranes, 54-55 erythrocyte coalescence by, 53 histamine in infection, 63-65 kallikrein in infection, 63-5 kinins in infection, 61-67 metabolism, 32 aerobic mechanisms, 38 carbon dioxide fixation, 37, 38 glucose, 35, 36 pentose phosphate pathway, 33, 34 metabolism of host, erythrocytes, 41, 42 lipids, 44 tissue, 43 mi tochondrial respiration in hi bition by, 70 vasomotor changes in infection hepatic, 59 intestinal 59-60 renal, 59 Iophurae carbon dioxide fixation, 37, 38 glucose metabolism, 36-37 malariae, mitochondria, 38 vinckei, metabolism of host erythrocytes, 42 Pleurogenes medians, life cycle, 176 Polycelis tenuis, 352 Polymorphus minutus, cuticle structure, 369 Pomphorhynchus laevus, cuticle structure, 369 Posthodiplostomum cuticola, cuticle structure, 354 minimum, cuticle structure, 356 Proctoeces maculutus, 168 Prosthogonimus~ 72 Proterometra dickermani, 164, 1 77 Pseudovermiculi sanguinis, 1 Psilotrema spiculigerum, life cycle, 168 Pumpkin seeds in taeniasis therapy, 293-294

410

SUBJECT INDEX

R Raillietina cesticullus, cuticle structure, 358 Rajonchocontyle ernarginata, cuticle structure, 348, 351 Ratzia j o y e d , life cycle, 176 Renicola thaidus, life cycle, 178-179 Rhabditis pellio, cuticle structure, 361 Ribeiroia marini, interaction with Paryphostomum segregatum, 194, 195 Schistosoma mansoni, 194, 195 ondatrae, life cycle, 168 S

Saccacocoeliodes,cercariae, 164 Schistocephalus solidus, cuticle structure, 358-3 59 Schistosoma bovis infection rate, 217 intramolluscan development, 20 1 miracidia penetration, 220, 221 haematobium infection rate, 219-220 intramolluscan interaction, 203 stages, 238 japonicurn cuticle structure, 355, 356 intramolluscan development, 201 miracidia penetration, 221 mansoni adaptation index, 224, 225 biological control with Echinostoma malayanum, 246 cuticle function, 357 structure, 353, 354356, 358 infection rate, 217-218, 219 intramolluscan development, 201,202 interaction with Cotylurus lutzi, 195, 206-207, 234 Echinostoma barbosai, 195, 226 E. liei, 195, 201, 202, 207, 226, 236 E. Indoense, 226 E. piaraense, 226

Paryphostomurn segregaturn, 195, 200, 202, 206, 207, 208, 210,214215,225-244 Ribeiroia marini, 194, 195, 226 maintenance, 197 miracidia immobilization in Planorbis corneus, 208 penetration, 220, 221, 222-223 snail immunity to, 21 1 toxicity, 213 mattheei infection rate, 217 miracidia penetration, 21 1 spindale cuticle structure, 355 intramolluscan interaction with Echinostoma malayanum, 194, 225, 237 miracidia penetration, 222 Sigrnapera cincta, life cycle, 176 Sphaeridiotrernaglobulus, life cycle, 168 Stannotaen in taeniasis therapy, 291 Stellantchasmus falcatus, 163 Stephanoprora, life cycle, 168 Sterrhurus musculosus,host range, 176 Stichocotyle nephropis adult digestive tract, 81 genital system, 90 sense receptors, 99, 103 biology localization in mollusc, 137 in vertebrate, 139 survival of adult outside host, 140 development hatching, 121 parasitic stage, 126 Stichorchis, 164 Stictodora, intramolluscan interaction with Austrobilharzia terrigalensis, 204 Strigea, life cycle, 177 Strongy loides fulleborni, cuticle structure, 360 rnyoptorni, cuticle structure, 360 ratti, cuticle structure, 360 stercoralis, cuticle structure, 360 Syngarnus skrjabinomorpha, cuticle structure, 361

SUBJECT INDEX

Syphacia obvelata, cuticle structure, 362 moulting, 364-365

T Taenia africana, 271 confusa, 271 crassiceps, cuticle structure, 358 hydatigena cuticle structure, 359 egg survival, 295 immunization against, 306 hominis, 271 multiceps, cuticle structure, 359 ovis, egg survival, 295 pisiformis cuticle structure, 350 egg survival, 295 proglottides, 278 saginata egg survival, 295-299 hosts, 271-273 nomenclature, 270-27 1 structure and biology adult, 274-278 cuticle, 359 cysticercus, 280-282 eggs, 278-279 oncosphere, 279-280 solium, 272-275 appendicitis caused by, 286 cysticercus, 280 diagnosis, 288 embryophores, 278 taeniaeformis, cuticle structure, 357 Taeniarhynchussaginatus, nomenclature, 270-271 Taeniasis clinical aspects diagnosis, 287-289 pathology, 285-287 symptornatology, 282-285 treatment, 289-294 acridines, 292 bithionol, 293 dichlorphen, 293 fern extracts, 294 paromomycin, 292-293 pumpkin seeds, 293-294 tin compounds, 291-292

41 1

Yomesan, 290-291 epidemiology and epizootiology epidemiology, 299-303, 308-309 medical and economic losses, 303304 transmission between man and animals, 295-299 prevention meat inspection, 304305 sanitation, 306-307 serological diagnosis and imrnunization of cattle, 305-306 Taenifuge, 291 Tetratirotaenia, 271 Tin compounds in taeniasis therapy, 291-292 Toxoplasma, 3-10, 14 avium, 5 columbae, 5 cuniculi, 5 gondii, 5, 6, 10 liothricis, 5 passeris, 10 Trematodes, intramolluscan interaction interaction factors, 199-225 materials and methods, 193-199 parameters of antagonism, 225-244 use in biological control, 244-251 Triaenophorus nodulosus, cuticle structure, 358, 359 Trichinellaspiralis cuticle structure, 361-362 moulting, 365, 368 Trichobilharzia brevis biological control by Echinostoma audyi, 246 intramolluscan interaction with E. audyi, 194, 225, 237 E. audyi-Hypoderaeum dingeri, 249 H. dingeri, 204 Trichodorus christiei, cuticle structure, 362 Trichostrongylusorientalis, cuticle structure, 360 Trichuris myocastoris, cuticle structure, 361 suis, cuticle structure, 361 Trypanosomabrucei histamine in infection, 66 kinins in infection, 62

412

SUBJECT INDEX

Turbellaria, cuticle structure and function, 348 Tylenchorhynchus martini, cuticle structure, 360-361 Tylacephalum,cuticle structure, 358

v Vermitin in taeniasis therapy, 290

X Xiphinema index

cuticle structure, 362 moulting, 364 Y Yomesan in taeniasis therapy, 290-291

z Zonocotyfe bicaecata, 78