Advances in Parasitology Volume 11

Advances in PARASITOLOGY

V O L U M E 11

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Advances in

PARASITOLOGY Edited by

BEN DAWES Professor Emeritus, University of London

VOLUME 1 I

1973

ACADEMIC PRESS London and New York A Subsidiary of Hurcoicvf Bruce Jovcinovich, Publishers

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road London NW1 United States Edition published by ACADEMIC PRESS INC. 111 Fifth Avenue New York, New York 10003

Copyright 01973 by ACADEMIC PRESS INC. (LONDON) LTD.

AN 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 : 0-1243171 1-7

PRINTED IN GREAT BRITAIN BY ADLARD AND SON LTD, BARTHOLOMEW PRESS, DORKING

CONTRIBUTORS TO VOLUME 11 J. E. BARDSLEY, Biology Department, Queen’s University, Kingston, Canada

(P. 1) WILLIAM N. BEESLEY, Sub-Department of Veterinary Parasitology, University of Liverpool, Liverpool School of Tropical Medicine (p. 1 15) LEONARD J. BRUCE-CHWATT, The Ross Institute, London School of Hygiene and Tropical Medicine, London, England (p. 75) REINO S. FREEMAN, Department of Parasitology, School of Hygiene, University of Toronto, Toronto, Canada M5S 1A1 (p. 481) *P. C. C. GARNHAM, Department of Zoology, Imperial College of Science and Technology, London, England (p. 603)

R. HARMSEN, Biology Department, Queen’s University, Kingston, Canada (p. 1) D. J. HOCKLEY, National Institute for Medical Research, Mill Hill, London, England (p. 233) *LEONJACOBS,U S . Department of Health, Education and Welfare, Bethesda, Maryland, U.S.A. (p. 631) K. M. LYONS,Zoology Department, King’s College, University of London, England (p. 193)

* W.

L, NICHOLAS, Department of Zoology, Australian National University, Canberra, Australia (p. 671)

J. H. ROSE,Central Veterinary Laboratory, Weybridge, Surrey, England

(P. 559) JAROSLAVSLAIS,Institute of Parasitology, Czechoslovak Academy of Scierices, Prague, Czechoslovakia (p. 395) J. D. THOMAS, School of Biological Sciences, University of Sussex, England (P. 307) *MARIETTA VOGE,Department of Medical Microbiology and Immunology U.C.L.A. School of Medicine, Los Angeles, California, U.S.A. (p, 707)

* Authors in the section “Short Reviews”

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PREFACE This book, the eleventh of the series, contains nine full reviews by ten writers. who deal with various kinds of parasites (trypanosomes, monogenetic and digenetic trematodes, acanthocephalans, cestodes and nematodes) and also with some broad parasitological problems such as imported parasitic disease, the control of arthropods of medical and veterinary importance, and the effects of population density on the growth and reproduction of snail vectors of a schistosome parasite of Man. Short updated reviews deal with malaria in mammals excluding Man, Toxoplasma and toxoplasmosis, the biology of the Acanthocephala, and the post-embryonic development of cestodes. The writers of these reviews live in Britain, Czechoslovakia, Canada, U.S.A. and Australia. John E. Bardsley and Rudolph Harmsen have made a detailed study of the trypanosomes of tailless Amphibia (Anura), an area of research in which knowledge can be gained about the evolution of trypanosomes. The fact that arthropods serve as vectors in a group predominantly “vectored” by leeches may be an example of “evolution in action”. Ten main sections of the review deal with taxonomic and phylogenetic considerations, morphology, electron microscopy and cytology, life cycles, distribution, the vertebrate host and pathogenesis, physiological processes, the invertebrate host, and media, physiology and biochemistry applied to methods of culture. Three useful tables are provided: one is a list of all published specific and subspecific names of anuran trypanosomes, and their authors are named; another is an even more formidable list indicating the distribution of anuran trypanosomes by geographical regions and hosts; and the third is a list of Hirudinid vectors of anuran trypanosomes. Research on anuran trypanosomes was intensive during the first two decades of this century but then declined, to be revived only during the 1950s. In the sense that modern concepts of taxonomy, genetics, cytology, cell physiology and biochemistry have not yet been applied fully, the classification of anuran trypanosomes is still relatively undeveloped. The same is true of life cycles, reproductive patterns and polymorphism. A reliable classification is badly needed but to construct this is a difficult and even arbitrary task. However, a dynamic model of classification of anuran trypanosomes is proposed which can grow without needing regular total revisions. The phylogeny of the genus Trypanosoma can hardly be considered unless we can understand more fully the relationships between the trypanosomes of “higher” and ‘‘lower’’ vertebrates. The electron microscope has not yet been put to significant usage in this field, but we know that anuran trypanosomes are large and complex in structure, whereas mammalian trypanosomes are smaller and less complex, which may be examples of primitive complexity and secondary reduction of size and complexity. Study of the fine structure of anuran trypanosomes, may reveal much about the rather inscrutable structures of mammalian trypanosomes. Study of the vii

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literature indicates that trypanosomes are ubiquitous in nearly all populations of Anura, perhaps denoting ancient origin. An effort has been made to integrate existing knowledge of anuran physiology and ecology, together with the ecology of the invertebrate host and trypanosome physiology and behaviour, into a dynamic model of host-parasite relationship. Another benefit of studying anuran trypanosomes will show in a broader context of pathogenicity. Possible endogenous adrenergic control systems have survived in the adaptation of mammalian trypanosomes to their homiothermic hosts. Leeches are regarded as the primary vector in anuran trypanosomiasis, but various insect vectors add further interest, underlining the important place occupied by anuran trypanosomes within the genus. Culture has been possible now for many years but generally only to provide experimental subjects for the study of nutrition, reproduction and metamorphosis. The writers hope that some effort in this area may be drawn away from the study of human trypanosomiasis with ultimate advantage. Leonard J. Bruce-Chwatt deals with world problems of imported diseases. He shows that we must have knowledge of communicable disease if protective measures are to be established, and then states that the present international control established by W.H.O. has sufficed up to a point but that the enhanced amount and the heightened rapidity of international travel and trade has revealed danger of the spread of cholera, plague, smallpox, yellow fever and many other diseases of only slightly lesser importance. We must realise that tropical countries are not the only reservoirs of infectious diseases and that rapid urbanization and industrial development make for widespread redistribution of disease. The recognition of imported disease is now becoming the responsibility of medical practitioners, who must collaborate with higher authorities to protect travellers by means of vaccines, provide them with international certificates, advise on simple measures of protection whilst travelling abroad, and deal by diagnostic means with imported diseases when they return. Six major sections of this review have a rich content of information about the past and present of international health, international health regulations and increase of world air transport, the importation of animal diseases and vectors of disease, the major imported diseases, the common diseases of travellers, and prevention of imported disease by means of immunization. Major imported diseases include cholera, smallpox, yellow fever, plague and relapsing fever, and typhus. Common diseases of travellers include gasto-intestinal infections, malaria, trypsanosomiasis, leishmaniasis, schistosorniasis, filariasis and other helminthiases, rabies, arthropod-borne encephalitis, dengue, haemorrhagic fever, poliomyelitis and leprosy. One section is devoted to medical puzzles and diagnostic fallacies, which is highly significant because the commonest symptoms of disease may be present in a wide variety of infections. Some medical puzzles can be solved by means of simple, well-directed investigation, but many other cases may call for great circumspection. The traveller should become familiar with health problems and dangers when travelling abroad, and improvement of conditions must be continued and safety maintained by airline authorities, charter companies and travel agencies as well as by relevant health authorities.

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William N. Beesley’s very detailed review deals with methods and materials for the control of arthropods of medical and veterinary importance, which are sometimes parasites and just as often vectors of serious parasitic diseases. Control depends largely on the use of insecticides but good hygiene or animal husbandry is essential for its success. In the Introduction mention is made of benefits accruing from the use of insecticides: in malaria alone, eradication programmes may have saved more than 2000 million infections of the disease during a single recent decade. Difficulties are notable: more than 3000 species of mosquitoes exist; 350 species of Anopheles include 60 species that are known to be vectors of human malaria. Culicine mosquitoes include more than 500 species of Aedes and 300 species of Culex. Aedes aegypri is the vector of urban yellow fever, several types of dengue, virus and mosquitoborne haemorrhagic fever, and species of Culex transmit some types of arbovirus, encephalitis and filariasis. Most insecticides are synthetic chemicals (many of them with imponderable names: see the List on pp. 180-182) and they involve techniques of dusting, spraying or dipping, although other and more exotic means include the use of insect juvenile hormone, insectivorous fishes and viruses or fungi. Ten sections of this review trace out methods of arthropodan vector control : one deals with mosquitoes (anopheline and culicine), insecticide resistance, new insecticides and repellents, and genetic control; another section deals with blackflies and midges, and Onchocercu in animals; and other sections are concerned with domestic flies, tsetse flies, blowflies and screwworms, keds, oestrid flies, lice and fleas, and ticks and mites. There is also a section dealing with the future of arthropod control, summing up the situation existing after remarkable successes, but also indicating where further effort is required in the future. Epidemics of malaria, louse-borne typhus, plague, yellow fever and other diseases can flourish “despite all the paraphernalia of modern insect control programmes”. Many millions of South Americans of all ages still suffer from Chagas’ disease, many millions of Africans are victims of onchocerciasis and even more millions are victims of one or another form of filariasis. However, vast amounts of data are now available which bear on the distribution of pathogens and vectors on the face of the earth, greatly improved insecticides and methods of administration have led to hitherto unsuspected results. Micromethods in insect physiology linked with chromatographical analysis have indicated that minute amounts of some insecticides can affect insects at some or all stages of development. Insecticides resistance has been shown in the field of genetics to be due to single principal genes, and biological considerations are dependent on specialist laboratories of various kinds. In the future, we are told, vector control will for some time continue to depend on chemical insecticides, increasingly based on new types of chemicals, and there will be much more and closer integration of biological and chemical control techniques giving greater effect for least cost. Few vectors will be completely eradicated, but reduction of vector populations to minimal levels, with few parasites to transmit, will help to provide for a demanding and ever-increasing world population. Kathleen M. Lyons has been concerned with the fine structure of the

X

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“epidermis” and sense organs of some Turbellaria, Aspidogastrea and Monogenea. Following Donald L. Lee (vols 4 and 10 of this series of books) she has adopted the term “epidermis” for the outer covering of the body in Platyhelminthes, although some other writers have preferred “tegument” (see Hockley, pp 233-234) or “integument” for this living protoplasmic layer once commonly known as “cuticle” and wrongly regarded as a non-living, protective outer covering of the body. Turbellaria considered are members of the Acoela (e.g. Convoluta), Rhabdocoela (Kronborgia and Syndesmis), Temnocephalida (Temnocephala), Tricladida (Dugesia) and Polycladida (Kaburakia). Mention is made of two genera of Aspidogastrea (Aspidogaster and Multicotyle); the latter genus was considered in great detail by Klaus Rohde in vol. 10 of this series (1972). One major concern was the embryology and structure of the larval epidermis and the epidermis of adult Monogenea. The range of variability and of conformity in epidermal fine structure is considered in a number of monogenetic trematodes such as species of Entobdella, Acanthocotyle, Rajonchocotyle and Polystoma. In addition to regional differentiation, microvilli on the body surface of some Monogenea and the terminal webs of Polystoma and Rajonchocotyle, the secretory and lamellate inclusions of the “epidermis” and the plasma membrane and surface coat of adult Monogenea are considered, along with syncytial nature and surface differentiation. Finally, some general evolutionary considerations are made. The sense organs of Monogenea taken into consideration include eyes, organs ending in cilia, uniciliate and compound multiciliate receptors. In final conclusion, there are indications of where in this field of study there is most promise of interesting results. David J. Hockley’s very detailed review of ultrastructure of the tegument of Schistosoma mansoni has four main sections, dealing respectively with the cercaria, the schistosomulum, the adult trematode and miracidium and sporocyst. The cercaria section concerns the development of the tegument, the cercarial surface coat and associated structures of the tegument in developed cercariae. A syncytial tegument connected to subtegmental cells occurs in all adult digenetic trematodes that have been examined and this unusual structure involves unusual cytoplasmic inclusions that are also related to the host-parasite interface. As schistosome cercariae penetrate the host directly, the tegument of the larva eventually becomes the tegument of the adult worm, not becoming involved in the formation of a metacercarial cyst as in Fasciola hepatica. The tegument of schistosomula is considered within 30 min to 3 h of penetration into the host and up to two weeks after penetration, after which this surface region is examined closely in adult worms, where sexual dimorphism necessitates noting differences in the tegument and associated structures in males and females, notably cytochemical differences. Specialized regions of tegument occur in the oesophagus and the uterus, which are regarded as tegumental structures but differ in structure. The oesophagus is primarily concerned with digestion, it is suggested, and the function of the intestinal caecum is absorption and egestion. Destruction of the tegument is specially considered in respect of the effects of hypo- and hypertonic media, drugs and host immunity. Finally, the epithelium and

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associated structures of the miracidium are considered and the contrasting tegument of the daughter sporocyst, which has the syncytial tegument and nucleated subtegumental cells of the typical digenetic trematode. All these and other matters are considered in such intricate detail as is necessary at this time, when ultrastructural detail can improve our understanding of the hostparasite relationship in established schistosomiasis. John D. Thomas’s review is a contribution to our understanding of the epidemiology of schistosomiasis, a parasitic disease that has probably surpassed malaria in prevalence and continues to increase in some parts of the world despite expensive efforts to institute control measures. His review has four principal parts: a historical section deals with the rationale for focusing attention on the molluscan hosts, control of molluscs either by means of chemical molluscicides or by manipulation of environmental factors in various ways, and an alternative solution of parasite control by reducing the success of miracidia, sporocysts and cercariae, and of adult worms. The mathematical models that have been used to predict the probability of success of such possible control measures are said to lack precision and generality because certain facts are overlooked or wrongly interpreted, for instance, the immune response of the definitive host, the longevity of the adult parasite and parasite-induced mortality of the snail host, not overlooking the timescale of various events. Here in this review are described the results of experiments designed to show how various environmental factors such as contacts resulting in copulatory behaviour, resources of food and ions in the external medium, and substances added to the medium either by snails or their plant food receive expression in growth and natality rates of individual snails (Biomphalaria glabrata; host of Schistosoma mansoni). Attention is given to the possibility that snails may produce specific inhibitory pherones and to other considerations, such as the effects of chemical conditioning and several effects were observed and are summarized in the final section of the review. Jaroslav Slais has produced a finely-detailed review on the functional morphology of cestode larvae, which show great variability during postembryonic development. Following a brief Introduction there are seven sections and ultimate Conclusion. One section deals with the oncospheral stage and its development, i.e. formation of embryonic envelopes and the development of the penetration glands, the hooklets and their muscles. Then, in another section there is consideration of the structure of the oncosphere (hexacanth), its functional capability at the infective stage and subsequent metamorphosis. Other sections consider post-oncocercal development of larvae that do not form a cavity, e.g. the procercoid and plerocercoid of Pseudophyllidea and the larvae of Tetraphyllidea and Caryophyllaeidae, the tetrathyridium of Mesocestoides and larval stages of other genera, and similar stages of development of larvae which form a cavity, e.g. various cysticercoids, and the cysticercus. Special treatment is given to the functional morphology of the strobilar tegument of adult stages, histogenesis of the calcareous corpuscles and morphogenesis of the mature plerocercoid to the adult stage. The scolex of larval Taeniidae is examined in the cysticercoid and cysticercus, also the cyst wall of the cysticercoid and the cysticercus bladder. Excystment

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of the cysticercoid and evagination of the cysticercus are noted. A sexual multiplication and abnormal growth are other topics dealt with. In conclusion we are told that successful cultivation of the oncosphere and other larval stages has produced much of this new information about the physiology of larval cestodes with the help of histochemistry and electron microscopy, information that facilitates more precise placing of these cestode parasites in the whole zoological system and improves our understanding of hostparasite relationships. Jaroslav Slais believes that demonstration of the course of infection in the intermediate host, the establishment of the larva in its definitive host and the causes and forms of standard and abnormal growth of larval stages are perhaps the most significant advances made. Reino S. Freeman points out that in the more recent systems of cestode classification there is wide disagreement on the limits, relationships and validity of various taxons, particularly at the level of orders. He doubts that there is even general agreement on six of the major orders, Tetrarhynchidea, Tetraphyllidea, Pseudophyllidea, Protocephalidea, Caryophyllaeidea and Cyclophyllidea.The systems are based mainly on adult morphology, especially scolex structure, and to a lesser extent on the uterus, position of the genital pores and the nature of the vitellaria but he places great emphasis on host specificity. For the most part these systems ignore cestode ontogeny, which has received little attention, and the need for taxonomic revision is now evident. Freeman discusses a basic cestode development cycle in an attempt to develop a unified system of naming the various stages of cestode development which suggests the course of evolution of cestode life cycles and may help to delineate the taxons in cestodes with a six-hooked larva, or oncosphere (hexacanth). The cestode life cycle is usually regarded as either two-host or three-host and rarely one-host, with a free-swimming stage only when in aquatic hosts, never in terrestrial hosts. Moreover, there is little agreement on the definition of the coracidium, procercoid, plerocercoid and cysticercoid forms. The review has four main sections between Introduction and Conclusions. One section deals with the basic cestode life cycle, its stages and ecology; another with variations in cestode ontogeny, the adult types of eggs and oncospheres. A section is then devoted to the evolution of life cycles and another to phylogenetic relationships. In conclusion, we are told that more data are required before a complete pattern of cestode ontogeny can emerge. This is true of early ontogeny and especially the origin, development and final disposition of the primary lacuna, cercoma, invaginal canal and excretory system of the metacestode. There is also a need for data bearing on growth patterns of metacestodes in the alimentary canals of vertebrates. However, data already available show that metacestodes follow recognizable patterns of growth, which may help to establish taxonomic relationships between cestodes. John H. Rose tells us that although lungworms of sheep and pigs are less important and have been studied less intensively than other helminths of these hosts, they are being studied in many parts of the world and our knowledge of them has increased sufficiently in recent years to warrant this review. Four species of these nematodes infect pigs, namely Metastrongylus elongatus

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and M . pudendotectus, which are cosmopolitan and have been well studied, and M. salmi and M . madagascariensis, which are little known. At least fourteen species of lungworms occur in sheep but only four of them are widely distributed and have been extensively studied: these are Dictyocaulus filaria, Muellarius capillaris, Protostrongylus rufescens and Cystocaulus ocreatus. The first three named species are cosmopolitan but C. ocreatus is restricted to parts of Europe, U.S.S.R.,North Africa and the Middle East. The two main sections of the review deal with pig and sheep lungworms respectively and each of these sections is concerned with geographical distribution, incidence of infections, life cycle, pathology in the definitive host, immunity, treatment and control. Pig lungworms have been surveyed in pigs slaughtered at abattoirs and bacon factories and M . elongatus and M . pudendotectus occur, sometimes in almost equal numbers, although M . elongatus usually predominates. Incidence varies according to the age of pigs. The life cycle of these two species is similar; adult worms Jive in the bronchi and usually in secondary branches of the bronchioles. Eggs pass up the trachea, are swallowed and pass through the alimentary canal, to be thrown out in faeces. Pigs are infected by devouring earthworms of any one of a score of species which contain larvae derived from eggs swallowed. The life histories of sheep lungworms are treated separately and space does not permit mention here, except that individual land and freshwater molluscs serve as the intermediate hosts, a formidable list appearing in Rose’s TabIe I. In considering treatment and control, only the more recently developed anthelmintics are referred to and control may depend on preventing pigs from ingesting infected earthworms and modifying methods of sheep husbandry, such as keeping sheep off pastures in the early morning and evening when the molluscan hosts are active. The four short, updated reviews in this volume are concerned respectively with malaria in mammals excluding man, Toxoplasma and toxoplasmosis, the biology of the Acanthocephala, and the post-embryonic developmental stages of cestodes. Percy Cyril Claude Garnham’s review on malaria has an introductory section and then sections dealing with taxonomic problems and new species, life cycles including exoerythrocytic and sporogonic stages, pathogenesis and culture, host susceptibilities and affinities, and fine structure. The most important discoveries of the last five years, we are told, are probably in the field of immunology, but dramatic research results concern the response of New World monkeys to the human species of Plasmodium. Taxonomic investigations leave unsettled the status of parasites beneath species level, but one major advance has been the use of isoenzyme analysis in the identification of species and subspecies in rodents. Numerical taxonomy is being pressed into service. Studies of ultrastructure are progressing and the use of scanning microscope techniques has given useful results, especially in relation to surface membranes after freeze-etching. Cytochemistry has not helped much in the determination of organelle functions but autoradiography may soon give useful clues. Two great problems that remain unsolved are the nature of anopheline susceptibility and resistance to various species of malaria, and the mechanism of relapses. These and other problems are discussed as fully as is possible.

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Leon Jacobs’s review (based on more than 200 from over 2,000 papers) deals with the life cycle and morphology of Toxoplasma, epidemiology, animal toxoplasmosis, human toxoplasmosis, serology and immunology, biology, chemotherapy, new knowledge of Sarcocystis and conclusions. The most important advance during the last six years is that much has been learned about the life cycle of T. gondii. However, other important studies have added to our knowledge of immune mechanisms in toxoplasmosis and how these relate to other intracellular infections and to tumours. In his previous review, Leon Jacobs cited instances in which transmission of T.gondii was obtained from cat’s faeces which did not contain Toxocura cati eggs. This has stimulated other researchers interested in the contaminative route of infection of Toxoplasma gondii, and has led to the discovery that the nematode egg is not necessary in the life cycle of the protozoan. In this review research in some areas had to be neglected in favour of developments concerning the life cycle of the parasite and their implication in respect of epidemiology. In the future the balance may shift to immunology and the physiology and biochemistry of the parasite, hopefully to successes in chemotherapy and in the diagnosis of chronic disease. Warwick L. Nicholas has given very full treatment of the biology of the Acanthocephala, indicating in his Introduction that interest in this group has increased and diversified since his previous review was written. One section of his updated review deals with morphology, functional anatomy and histology in respect of proboscis, trunk, uterine bell, acanthor and characteristic nuclei and nucleoli. In other sections there is consideration of development both in the intermediate and definitive hosts, fine structure of the tegument in adult and larva (acanthor), development and ultrastructure of spermatozoa, physiological matters including osmotic regulation and hatching of the acanthor, biochemical matters including intermediary metabolism, and hostparasite relationship. In a final section of the review there is a summary with conclusions. It is unnecessary in this place to go into details about all these topics, but readers will note that the nature of the “tegument” and its growth and development have been considered; other interesting topics are the mode of action of the uterine-bell apparatus and the movement of the acanthor. The relationship between Acanthocephala and Cestoda can now be better understood as a result of advances in knowledge of biochemistry and fine structure. Other comparisons between these two groups are made in relation to intermediary metabolism, and finally there is a phylogenetic explanation of peculiarities of acanthocephalan embryonic and larval development. Marietta Voge’s review on post-embryonic stages of cestodes has seven sections. Following the Introduction, one section deals with life cycles and larval growth in the orders Tetraphyllidea, Pseudophyllidea and Cyclophyllidea. The next section is concerned with histology, histochemistry and fine structure in Lecanicephalidea, Pseudophyllidea and Cyclophyllidea. Another section deals with host-parasite relationships in invertebrate hosts, vertebrate intermediate hosts and final hosts, with consideration of immunity. Finally, there are sections dealing respectively with metabolism and growth in vitro, followed by conclusions drawn. The author explains that recent trends

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in research have shifted towards fine structure, metabolism and immunology, although life histories and morphological features have not been neglected. She has given many interesting developments, e.g. female hosts are more resistant to infection than male hosts, strain differences occur in Taeniu crassiceps, and methods used can be useful for the detection of mutations. However, our ignorance of different (internal) environments available to the parasite in the host is great, likewise the composition of host body fluids (blood, serum) used in culture of parasites. After completing my work on this book and at the beginning of a second decade of publishing Advances in Parasitology I am grateful to and thank friends and colleagues who have contributed to volume 11 of this series of books and who have thus helped to further my aim to organize and edit precious information and ideas that will assist progress in the modern biological field of parasitology. I am equally pleased to say thank you to other friends and colleagues on the staff of Academic Press for continued assistance in producing this book and thus helping what I regard as a worthy cause. It is a privilege to be able to continue production of this series of books, further volumes of which are assured. “Roden hurst” 22 Meadow Close Reedley, BURNLEY Lancs BBlO 2QU England

BEN DAWES Professor Emeritus: University of London June 1973

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CONTENTS CONTFUBUTORS TO VOLUME 1 1 ............................................................

PREFACE ..........................................................................................

v vii

The Trypanosomes of Anura . . . ........................................................................... J E BARDSLEY AND R HARMSEN

I . Introduction I1. Taxonomic and Phylogenetic Considerations ................................. 111. Morphology and Cytology ......................................................... IV. Life Cycles .............................................................................. V . Distribution ........................................................................... VI. The Vertebrate Host ............................................................... VII Physiology .............................................................................. VIII. The Invertebrate Host ............................................................... IX. Culture ................................................................................. X Conclusion.............................................................................. Tables .................................................................................... References .............................................................................. Addendum ..............................................................................

.

.

1

2 11 14 24 24 34 35 38 42 45 58 72

Global Problems of Imported Disease

.

LEONARD J BRUCE-CHWATT

.

1 Introduction ........................................................................... 75 I1. Past and Present of International Health ....................................... 77 111. InternationaI Health Regulations and the Increase of World Air Transport ................................................................................. 78 IV . Importation of Animal Diseases and of Disease Vectors..................... 82 V. Major Imported Diseases ......................................................... 84 VI . Common Disease of Travellers ................................................... 94 VII. Prevention of Imported Disease by Immunization ........................... 110 Acknowledgement .................................................................. 112 References ..............................................................................112

Control of Arthropods of Medical and Veterinary Importance WILLIAM N . BEESLEY

I. Introduction ........................................................................... I1. Mosquitoes ........................................................................... 111. Blackflies and Midges (Simrrli~imand Ciilicoides) ...........................

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115 120 134

xviii CONTENTS IV. Domestic Flies and “Fly Worry” ................................................

V . Tsetse Flies (Glossina spp.) ......................................................... VI . Blowfly and Screw-worm ......................................................... VII . Keds (Melophagus ovinus) ......................................................... VIII . Oestrid Flies ........................................................................... IX. Lice ....................................................................................... X . Fleas .................................................................................... XI . Ticks .................................................................................... XI1. Mites .................................................................................... XITI. The Future of Arthropod Control ................................................ References ..............................................................................

138 142 146 151 152 157 161 163 171 176 183

The Epidermis and Sense Organs of the Monogenea and Some Related Groups . I . Introduction ........................................................................... I1. Turbellaria .............................................................................. 111. Aspidogastrea ........................................................................ IV. The Epidermis of Monogenea...................................................... V . Sense Organs of Monogenea ...................................................... VI. Conclusion.............................................................................. References .............................................................................. K M . LYONS

193 194 200 201 218 227 228

Ultrastructure of the Tegument of Schistosoma

..

D J HOCKLEY

I . Introduction ........................................................................... I1. The Cercaria ........................................................................... 111. The Schistosomulum ............................................................... IV. The Adult Worm ..................................................................... V . The Miracidium and Sporocyst ................................................... VI . Conclusion.............................................................................. References ..............................................................................

233 234 250 257 289 296 297

Schistosomiasis and the Control of Molluscan Hosts of Human Schistosomes with]Particular Reference to PossiblelSelf-regulatorylMechanisms .

J . D THOMAS

1. Introduction ........................................................................... 1 I Historical ..............................................................................

.

I11. Materials and Methods ............................................................ 1 V. Results ................................................................................. V . Discussion .............................................................................. V I . Summary .............................................................................. Acknowledgements .................................................................. References ..............................................................................

307 208 323 327 359 382 384 384

C 0 N T E N TS

xi x

Functional Morphology of Cestode Larvae JAROSLAV iLAlS

I. Introduction ........................................................................... I1. Morphogenesis of the Oncospheral Stage, and its Development ......... 111. Functional Morphology of the Oncosphere (hexacanth) .................. IV Post-oncospheral Development of Larvae which do not Form a Cavity V. Post-oncospheral Development of Larval Stages which Form a Cavity VI . Functional Morphology of Post-oncospherai Development ............... VII Specific Larval Organs of Taeniids ................................................ VIII Proliferation During the Larval Stage.......................................... IX Conclusion .............................................................................. Acknowledgements .................................................................. References ..............................................................................

.

. . .

Ontogeny of Cestodes and its Bearing on their Phylogeny and Systematics REIN0 S . FREEMAN I. Introduction ........................................................................... I1. The Basic Cestode Life-cycle ...................................................... I11. Variations in Cestode Ontogeny ................................................... IV . Evolution of Cestode Life-cycles ................................................

. .

V Phylogenetic Relationships ......................................................... VI Conclusion .............................................................................. Acknowledgements .................................................................. References ..............................................................................

396 396 400 406 415 436 445 458 466 466 466

481 483 490 531 543 547 547 548

Lungworms of the Domestic Pig and Sheep J . H . ROSE I. Introduction ........................................................................... I1. Pig Lungworms ........................................................................ I11. Sheep Lungworms .....................................................................

559 559 570

SHORT REVIEWS Supplementing Contributions of Previous Volumes

Recent Research on Malaria in Mammals Excluding Man P. C . C . GARNHAM

. . .

I Introduction ........................................................................... I1. Taxonomic Problems and New Species.......................................... I11 Life Cycles Including Exoerythrocytic and Sporogonic Stages............ N Pathogenesis and Culture ............................................................ V . Host Susceptibilities and Affinities ................................................ VI. Fine Structure ........................................................................ References ..............................................................................

603 605 609 617 619 621 626

xx

CONTENTS

New Knowledge of Toxoplasma and Toxoplasmosis LEON JACOBS

I. Life Cycle and Morphology ...................................................... I1. Epidemiology ........................................................................ 111. Animal Toxoplasmosis ............................................................... IV. Human Toxoplasmosis ............................................................... V. Serology and Immunology ......................................................... V1. Biology ................................................................................. VII . Chemotherapy ........................................................................ VIII . New Knowledge of Sarcocystis ................................................... IX . Conclusion .............................................................................. References ..............................................................................

631 639 642 644 651 656 657 658 659 659

The Biology of the Acanthocephala W . L . NICHOLAS I . Introduction

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

I1. Morphology, Functional Anatomy and Histology ...........................

.

111 Development ...........................................................................

IV . Fine Structure ........................................................................ V . Physiology .............................................................................. VI . Biochemistry ........................................................................... VII . Host-Parasite Relationship ......................................................... VIII . Summary and Conclusion ......................................................... Acknowledgements .................................................................. References ..............................................................................

671 672 677 680 685 688 696 699 701 701

The Post-Embryonic Developmental Stages of Cestodes MARIETTA VOGE

I . Introduction ........................................................................... I1. Life Cycles and Larval Growth ................................................... I11. Histology, Histochemistty and Fine Structure ................................. IV . Host-Parasite Relationships ...................................................... V . Metabolism ........................................................................... VI . Growth in vitro........................................................................ V1I . Conclusions ........................................................................... References ..............................................................................

INDEX ................................................................................. AUTHOR SUBJECT INDEX ................................................................................. CUMULATIVE LISTOF AUTHORS ............................................................ CUMULATIVE LISTOF CHAPTER TITLES...................................................

707 708 711 714 718 720 722 723 731 757 775 777

The Trypanosomes of Anura J . E. BARDSLEY* AND R . HARMSEN Biology Department. Queen’s University. Kingston. Canada

........................................................ ............................ .......................................... A. Taxonomic Problems..................................................................... B. A Dynamic Model of Classification ................................................ C. Phylogeny of the Genus Trypanosoma ............................................. 111. Morphology and Cytology .................................................................. A. Morphology .............................................................................. B. Cytology .................................................................................... I . Introduction

11. Taxonomic and Phylogenetic Considerations

IV

.

V.

VI.

VII. VIII. IX .

X.

Life Cycles ....................................................................................... A. Reproduction .............................................................................. B. Polymorphism ........................................................................... Distribution....................................................................................... The Vertebrate Host ........................................................................... A. The Adult as an Environment ......................................................... B. The Tadpole as an Environment...................................................... C. Pathogenesis .............................................................................. Physiology ....................................................................................... The Invertebrate Host ........................................................................ Culture............................................................................................. A . Media ....................................................................................... B. Physiology and Biochemistry ......................................................... C. Comments ................................................................................. Conclusion ....................................................................................... Tables ............................................................................................. References ....................................................................................... Addendum ......................................................................................

1 2 2 5 9 11 11 13 14 14 20 24 24 24 32 32 34 35 38 38 40 41 42 45 58

72

I . INTRODUCTION This review had its origin several years ago when the authors took an interest in the relatively neglected field of amphibian trypanosomiases . An extensive review of the literature revealed that. although anuran trypanosomes were discovered over a century ago (Gluge. 1842) and given the present generic name Trypanosoma a year later (Gruby. 1843). their biology remains little studied. Many of approximately 300 papers published in this field deal solely with host records. Moreover. most of the early papers concentrate on the concepts of taxonomy and polymorphism. and from such a restricted viewpoint that more confusion than illumination is generated . Proportionately fewer papers have been devoted to other areas of investigation. for example

* Address for correspondence: 57-B Lundy’s Lane. Kingston. Ontario. Canada. until July. 1974; thereafter. as above. 1

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J. E. B A R D S L E Y A N D R . H A R M S E N

the ecology of the parasite/hosts systems, life cycles and reproduction. The only review article in the field is by FranCa and Athias (1906b), and although this is an excellent summary and discussion of the older works, it was written at a time when little existed to be reviewed. The overall paucity of continuous and extensive studies may be due partly to the fact that most researchers who have studied amphibian trypanosomes did so for a short period, especially for a thesis topic, and then abandoned the field for other areas, particularly mammalian trypanosomiasis. Thus, the Iiterature is characterized by one or a few papers by an author who, within a few years, disappears from the field (e.g. Ayala, Barrow, Diamond, Lauter, Mason, Woo), with the resultant unfortunate loss of their unpublished information, techniques and theories. However, recently there have been certain authors who have used anuran trypanosomes on a long-term basis in studying various aspects of the biology of trypanosomes (e.g. Steinert and co-workers). This review, then, is written with several objectives in mind, the first being to fill the absence of an up-to-date review. As well as this basic objective, we want to concentrate on recent advances in this field from an integrative perspective in order to reveal the relevance and importance of studying anuran trypanosomes. For example, this field has much to contribute to the study of the evolution of trypanosomes. The fact that there exist arthropodborne parasites in a group predominantly vectored by leeches may be an example of “evolution in action” and gives us some possible clues concerning the development of mammalian trypanosomiases (see Sections 11, VIII). Undoubtedly, the morphology and fine structure (Section 111), physiology (Section VII) and life cycles (Section IV) of this intermediate group also have something to contribute to the general study of trypanosomes. The study of the eco-physiological relationships among host, parasite and vector in this group adds a great deal to our understanding of the dynamics of hostparasite systems in general (see Section VI). These studies are especially practicable since the trypanosomes can be maintained readily in culture (see Section IX), and various Anura can be easily and inexpensivelymaintained in the ordinary laboratory (Bardsley and Harmsen, 1972). Parasite distribution, speciation and pathogenicity (see Sections V, VIC) may add to current concepts of the evolution and dispersal of the host Anura. And so it goes. We would like this review not only to serve as a synopsis of published material, but also to develop interest in this fascinating group of parasitic organisms. We hope that our approach to the various topics, and our opinions and hypotheses on them, will serve to add some direction to future research and to stimulate healthy controversy.

II. TAXONOMIC AND PHYLOGENETIC CONSIDERATIONS A.

TAXONOMlC PROBLEMS

To date, the taxonomy of anuran trypanosomes has suffered from much hasty and arbitrary work. Despite the sixty-odd species names recorded for anuran trypanosomes (see Table I at the end of review), there is not one

THE T R Y P A N O S O M E S O F ANWRA

3

clearly distinct species which has been shown to be consistently separable from all other anuran trypanosomes. No consensus exists on which species names are valid. Furthermore, it is impossible to say for sure what the trypanosomes that most early researchers described really looked like (e.g. Mayer, 1843; Gruby, 1843). As a result, most modern authors conveniently ignore the taxonomic background, and either use one of the more commonly used names, hoping that they are indeed dealing with that species, or circumvent the problem by naming yet more new species. It is the intention of this section to establish the criteria that should guide the taxonomist in naming anuran trypanosomes, and examine both the theoretical and practical reasons that have led to the present unreliable state in their taxonomy. Finally, we shall attempt to extract the reliable classification that does exist, and point out the direction that taxonomic research should take in order to arrive at a consistent and reliable classification. In order to classify a group of related organisms into different species, the taxonomist uses observable, and preferably measurable criteria, to recognize consistently and reliably separable categories of individuals, and each such category can then be considered a separate species. When this job is done well, the taxonomist’s species will coincide exactly with the ecological and genetic species. This means that a species can be defined as a persisting group of organisms that can be separated from all other such groups by: (1) its morphological, physiological and behavioural characteristics, (2) its ecological niche, and (3) the gene pool to which it belongs. It must, of course, be recognized that criterion 1 is the only practically available one in most cases, but it must also be recognized that if criteria 2 and 3 are not met, the resulting classification may well become the source of much confusion and redundancy in research effort. Sexually reproducing, biparental species maintain their integrity through an interplay of genetic and ecological selection. Uniparental (clonal) species obviously cannot be restricted around a breeding norm, so that in such cases genetic isolation plays no part in the maintenance of the species integrity. For these species it is still possible that “fossilized” norms of development remain, dating back to sexually reproducing ancestors, but that explanation seems an unlikely one. It seems much more likely, when we find taxonomically discrete species among asexually reproducing organisms, that we are dealing with what has been referred to as an “agamospecies”*: a group of back-related clonal individuals that, through the action of normalizing natural selection alone, maintains a body form and function describable within defined, relatively narrow limits. For example, such a situation has been described and analysed in considerable detail for the bdelloid rotifers and some other groups of organisms by Hutchinson (1968), who also analysed the theoretical problems involved in such a system. Trypanosomes are generally recognized as asexually reproducing organisms. The most critical question, therefore, is: do trypanosomes occur in an environment which can be separated into distinct, non-overlapping niches resulting in the trypanosome clones occurring as distinct, peristent and

* See Stebbins, J.

H., “Variation and Evolution in Plants”, p. 411.

4

J. E. B A R D S L E Y A N D R. H A R M S E N

non-overlapping agamospecies, or, is the environment a multidimensional cline of adaptive situations containing a fluid species-complex? An example of a well-studied group of trypanosomes is the Trypanozoon subgenus. Here we find three tsetse-vectored, polymorphic species, plus a number of direct-transmission monomorphic species (Hoare, 1967). The three polymorphic species are separated basically by their pathological effects on man, as well as their geographical distribution. However, there is no guarantee that the three species (clonal aggregates) are each of independent, single-event origin. For instance, the question as to whether a strain of Trypanosoma gambiense could evolve under the right circumstances into Trypanosoma rhodesiense within a limited number of generations remains an unanswered question with only negative evidence available at this time. The monomorphic species of this group (evansi, equinum, equiperdum, etc.) are considered recent clonal offshoots that have evolved under circumstances where transfer in the absence of tsetse flies became a frequent occurrence. Some of these “species” occur under ecological circumstances sufficiently different and distinct from the others that a species notation is easily assigned and maintained (e.g. equiperdum). Others are more easily interpreted as local and temporarily abundant stages in a spacial and/or temporal continuum (e.g. equinum and hippicum). I t is, therefore, highly likely that at least some of these species are in fact polyphyletic in origin; a situation which theory would predict in many tropical agamospecies. In the Trypanozoon subgenus the distinct hosts and/or general ecological conditions seem to lead to distinct species in most cases. This is not necessarily the case for all trypanosomes. The trypanosomes of Anura inhabit mostly aquatic and semi-aquatic cold-blooded hosts that offer a less species-specific environment than do warm-blooded vertebrates. This means that the environment inhabited by anuran trypanosomes is on the one hand vastly more variable, and on the other hand much less discrete, than that inhabited by avian or mammalian trypanosomes. Such an ecological environment can be met in a variety of ways. One would expect the development of polymorphism and/or a ffexibie phenotype. In view of the asexual reproduction of trypanosomes, one would expect the polymorphism to be sequential and strictly phenotypic; in fact, a contagious type of phenotypic flexibility. The alternative would be parallel polymorphism, which in this case would be indistinguishable from a large number of sibling species. After this rather lengthy theoretical introduction, we must now look at anuran trypanosomes as described in the literature, and decide whether we are dealing with: (1) a large number of distinct agamospecies, (2) one unseparable polymorphic species complex, or (3) a situation intermediate between these two extremes. For a group of trypanosomes to be classified into a species, and for this species to be a useful and reliable characterization, all the individuals so classified must belong to a clonal aggregate that, in time and niche utilization is separated widely from all other such clonal aggregates. As a product of this first (theoretical) criterion, one must state as a series of secondary, practical criteria the following points.

5 The members of a species of trypanosome must be persistently, and therefore predictably, recognizable and separable from other species on at least a combination of some of: THE T R Y P A N O S O M E S OF A N U R A

1. vertebrate host@) 2. invertebrate host(s) 3. geographical locality 4. morphology and cytology 5. biochemical composition (enzymes, metabolic intermediates, etc.) 6. physiology (phenology, tolerances, culture media, pathology) 7. behaviour 8. reproductive pattern (life cycle) Moreover, the differences must be proven (preferably experimentally) to be part of neither a polymorphic system nor a geographic cline (e.g. Scorza and Dagert, 1958). The last statement is extremely important even though it is the biggest hurdle for practical taxonomists-we must realize that what may appear in one geographic area as two totally separate species may be separate clinal extensions of a polymorphic species in a geographically removed locality, where the ecosystem is more continuous. I t must be stressed that organisms such as anuran trypanosomes which can be highly polymorphic in one locality and yet be found as morphologically identical specimens on separate continents cannot be classified on morphological criteria alone. This opinion has been stated repeatedly in the literature (Acanfora, 1939; Hegner, 1921; Lehmann, 1952; Nigrelli, 1945; Plimmer, 1912; Senn, 1902; Vucetich and Giacobbe, 1949), but unfortunately more often ignored. Of the listed criteria to be used, 1, 2 and 3 are the best indicators of true separation in time, with 1 and 2 also being a strong indication of separate selective environments. Criteria 4-8 are indications of the extent of the effectiveness of the separate evolutionary pathways followed by separate clonal aggregates (species, subspecies or strains). Especially important here is the last criterion: reproductive patterns and life cycles. I t is, therefore, unfortunate, yet indicative of the state of anuran trypanosome taxonomy, that a large number of “species” have been named in the absence of such information (Brumpt, 1906a, 1923c, 1936; Diamond, 1950,1958;DuttonandTodd, 1903; FranGa, 1911a; FranGa and Athias, 1906b; Grassi, 1881; Gruby, 1843; Johnston, 1916; Kudo, 1922; Lankester, 1871 ; Laveran, 1904; Lehmann, 1959b; Lieberkuhn, 1870; Marchoux and Salimbeni, 1907; Mathis and Ltger, 1911a, b; Mayer, 1843; Mazza et al., 1927; Nabarro, 1907; Nigrelli, 1944; Patton, 1908; PCrez-Reyes, 1968; Ptrez-Reyes et al., 1960; Pittaluga, 1905; Sergent and Sergent, 1905; Woo, 1969b). B. A DYNAMIC MODEL OF CLASSIFICATION

In his monograph on the taxonomy of anuran trypanosomes, Diamond (1958) recognized 26 species within a worldwide distribution. Unfortunately,

in the mammoth task of extracting an orderly classification from the literature,

G

J. E. B A R D S L E Y A N D R. H A R M S E N

Diamond has not scrutinized his predecessors’ work very critically, and his resulting classification does not seem warranted on the basis of the available evidence. Despite his highly organized, comparative study of qualitative as well as quantitative morphology and cytology, and his synopsis of geographical, historical and ecological data for each species, his classification is probably not a representation of reality, and should be used with considerable caution. The only species of anuran trypanosome which is adequately described (for at least one region), and which consequently comes close to fulfilling all the requirements established in Section IIA, is Trypanosoma pipientis (Diamond, 1950; 1958). Two other species which Diamond (1958) deals with in some detail, T. ranarum and T. chattoni, appear to be consistently separate species in Minnesota, but whether his ranarum is the same species as the one originally described by Lankester (1871) from Europe is not at all certain. Since other authors (e.g. Noller, 1913a, b; Kudo, 1922; Bailey, 1962) have considered the ranarum-like trypanosomes that they studied to be part of a polymorphic species, we feel that at this point it is safer to treat the name ranarum with some reservation. The species T. chattoni, as described by Diamond (1958), resembles quite closely many other rounded forms which are probably transient, reproductive forms of other species. Diamond’s evidence concerning the life cycle of this unique species makes us cautiously accept chattoni as a separate species. Again, however, it is doubtful that the species described by Diamond from ranids of Minnesota is the same as the original T. chattoni described by Mathis and LBger (1911b) from Vietnamese toads. The one other specific name for anuran trypanosomes that must be retained at this stage is rotatorium. This name is an adaptation from Mayer’s (1843) name Amoeba rotatoria, and is the most widely used name for anuran trypanosomes. Unfortunately, it is impossible to say with any certainty what Mayer actually observed, although it probably was a mixed population of trypanosomes. All other names are best treated as synonymous with rotatorium (Scorza and Dagert, 1958) except for a few species with a known insect vector. FranCa (1911a) described very cursorily a new species of trypanosome from Bufo regularis of the Congo and Nile watersheds in Central Africa, which he named T. bocagei. Two very similar trypanosomes were later described in more detail, one by Mathis and LCger (1911a) for Bufo melanostictus from Vietnam, the other by Feng and Chung (1940) for Bufo bufo from Northern China: both collections were assumed by the above authors to be of T. bocagei. What is of particular interest is that Feng and Chung (1940) showed quite convincingly that their trypanosome did not have the usual leech invertebrate host (see Section VIIZ) but was vectored by the sandfly, Phlebotomus squamirostris. Recently Anderson and Ayala (1968) and Ayala (1970, 1971) have described a very similar situation from California: a trypanosome very much like bocagei which Ayala (1970) named T. bufophlebotomi was found in Bufo boreas and vectored by Lutzomyia (= Phlebotomus) vexatrix. Ayala (1970)

THE T R Y P A N O S O M E S O F A N U R A

7

makes an interesting phylogenetic comment: “. . . Bufo boreas belongs to a Holarctic complex of toads which invaded the New World from Eurasia during the Pliocene and early Pleistocene. Populations of B. boreas still occur in Alaska, north of the 60th parallel. The nearest Eurasian relatives of the complex include B. bufo. The trypanosome of B. bufo is the only other sandfly-transmitted trypanosome yet reported from anurans. New and Old World sandflies are placed in separate genera by some authors, but the New World sandflies show evidence of recent speciation from relatively few Old World ancestral stocks.” The predominantly terrestrial habit of Bufo spp. and their very short aquatic larval stages would make a leech vector unlikely; the three or four species of Trypunosoma found in Bufo are probably restricted to a totally different ecological niche from other anuran trypanosomes. This different niche has probably supplied these species with the necessary isolation and selective pressure to cause sufficiently consistent morphological change to allow the taxonomist to separate them clearly from other anuran trypanosomes. Whether or not the species from Africa, Vietnam, China and California are of single event origin is impossible to say, and whether they differ sufficiently to warrant specific separation from one another depends on further coIlections and a more detailed study of their biology. A similar situation may exist for some of the trypanosomes found in various tree frogs. Nigrelli (1944, 1945) described T. grylli from Acris gryllus in Georgia and a very similar trypanosome has recently been collected by us from Hyla versicolor in Ontario. Again, it is possible that these trypanosomes represent a clonal aggregate that has evolved away from the main line of anuran trypanosomes because of the predominantly terrestrial habit of its host, and consequently a possible insect vector. The unique reproduction by binary fission in the peripheral blood of the host (Nigrelli, 1945) may be an indication of a divergence from the rotatorium complex (see also p. 72). One other trypanosome species complex obviously of common ancestry with T. rotatorium is the one found in Caudata, as represented by T. diemyctyli in Triturus (Barrow, 1953, 1954). Here again, we find that a different ecological niche (vertebrate host in this case) has produced sufficient adaptive isolation to result in the formation of a taxonomic species recognizably separable from the rotatorium complex, even though vectored by leeches. It is now possible to recognize a phylogenetic development rather similar to the one described for the Trypanozoon subgenus: one widely distributed polymorphic species, including a number of rather arbitrary races or “species”, shows evidence of having speciated along its geographical and/or ecological periphery into more easily recognizable species of more restricted distribution and/or niche utilization. It is desirable for practical purposes to have, at all times, a usable classification for any group of related organisms, and Diamond (1958) has produced such a classification. We question, however, the lasting usefulness of his classification for the reasons discussed above. We prefer to approach the classification of anuran trypanosomes as a dynamic model which can grow in complexity and accuracy as further data

8

J. E. B A R D S L E Y A N D R. H A R M S E N

are reported, and one which does not change essentially in the hands of either “splitters” or “lumpers”. The main feature of our classification (see Fig. 1) is a worldwide Trypanosoma rotatorium species complex composed of a number of clonal aggregates of varying degrees of separateness. One or two of these aggregates can be recognized as separate species (e.g. T. pipientis and T. ranarum as described by Diamond for Minnesota); the status of the others is at the moment uncertain. Branching from the main species complex are a number of species of less ubiquitous occurrence, of more closely defined appearance, and of a narrower niche (T. bocugei, T. bufophfebotomi, perhaps T. grylli). The position

California

East Asia

parasites of

Africa

higher vertebrates

insect vector?

I

Trypanosoma rotatorlum spp.co”1pler

I 1-li

FIG.1. A dynamic model of classification for the anuran trypanosomes. A central, highly polymorphic, leech-vectored species complex with worldwide distribution has given rise to a large number of recognizably distinct agamospecies specific to certain regions or hosts. Some of these are insect vectored, and may eventually speciate further upon entry into reptiles or higher vertebrates. It is not certain whether the caudatan trypanosomes belong to this complex or not.

of T. chattoni is questionable: it may be a less polymorphic offshoot of the rotatorium complex, or it may be a much older, parallel development, as is probably the case for T. diemyctyli. If future collections of anuran trypanosomes are screened carefully, and studied along the lines of Diamond’s (1958) work with T. pipientis, but preferably using collections from wider geographical origin, it will be possible to unravel step-by-step the rotatorium complex into a number of not-so-arbitrary species. We must, however, be prepared for the possibility of finding one or more central cores of highly variable, polymorphic species which may have to remain as unresolved species complexes in the eyes of all except the most ardent “splitters”. Unfort~inately,many of the older names (e.g. runarum, sanguinis) and some

T H E T R Y P A N O S O M E S OF A N U R A

9

of the more recent ones (e.g. canudensis, schmidti) are based on either so poorly described material, or on such minor collections, that it will be very difficult in the future to set acceptable and useful limits of description and distribution for these species.

c.

PHYLOGENY OF THE GENUS Trypanosoma

In his extensive review of the phylogeny of the Trypanosomatidea, Baker (1963) has proposed a diphyletic origin of the genus Trypanosoma. As Baker himself pointed out, the remaining problem is to decide where exactly to draw the boundary between the two branches of the genus, or where to spIit the genus as has been suggested by some authors (Jacono, 1935; Acanfora, 1939). Baker (1963) prefers to consider the trypanosomes of fish, Amphibia, most reptiles and some birds, and the vivax, congolense and brucei groups of mammalian trypanosomes as one branch of the genus, while placing all other mammalian trypanosornes, most bird parasitic species, and some of the reptilian ones, in the other branch. Hoare (1967) in his considerations of the evolution of mammalian trypanosomes considers all species to have evolved from monogenetic insect parasitic trypanosomatids, and presumably considers the boundary to lie somewhere among the reptilian parasites. More recently, Woo (1970) joins Baker, and on rather speculative evidence envisages a recent evolution of the vivax, congolense, and brucei groups from tropical reptilian trypanosomes. Several other authors have speculated on the possible origin of trypanosomes as parasites of aquatic invertebrates and therefore envisage an evolutionary development via lower, aquatic vertebrates (e.g. G r a d , 1952; Nicoli and Quilici, 1964). In order to appreciate the important position of the anuran trypanosomes, especially with regard to the possible evolutionary pathways that various branches of the “genus” have followed, it is necessary to review briefly the evidence for both a monogenetic insect parasite origin, and a monogenetic leech parasite origin. A monogenetic trypanosomatid ancestor parasitic of insects is supported by : (1) the striking similarity between certain developmental stages of trypanosomes in insects, and monogenetic insect parasites such as members of the genus Blastocrithidiu (Hoare, 1967); (2) the observation that T. cruzi can pass from one reduviid bug to another via cysts, as well as pass from bug to mammal via metatrypanosomes (Silva, 1965); (3) Blastocrirhidiu is found in many non-haematophagous insects, especially in Heteroptera related to the Triatominae (Wallace, 1966). Further evidence is often sought in degrees of “completeness” of adaptation of trypanosomes to either vertebrate or insect host. Such evidence is weak at best, and is often based on poor understanding of the dynamics of biological adaptation. The above arguments are particularIy convincing for the neotropical trypanosomes and their close relatives (Hoare, 1967), and least convincing for the African salivarian trypanosomes (Baker, 1963), although we feel that Baker overstresses the importance of anterior or posterior development and passage. The ability of T. brucei to complete posterior passage via Muscu spectandu (Thompson and

10

I. E. B A R D S L E Y A N D R . H A R M S E N

Lamborn, 1943) certainly indicates that the anterior development in brucei is not a hard and fast rule. A close look at anuran trypanosomes reveals very convincing evidence for considering their ancestry closely related with leeches, not with insects. (1) The anatomically complex, large and often highly polymorphic trypanosomes of lower vertebrates are generally considered more “primitive” (Lavier, 1943), and most, if not all of these species are leech vectored. (2) The highly complex and intricately balanced relationship between anuran trypanosomes and both the vertebrate and leech (see Sections VIA and VIII) must be considered an old relationship. (3) Leech trypanosomes can pass from leech to leech as well as from leech to vertebrate (Barrow, 1953; Brumpt, 1907). Indeed, Brumpt considered T. inopinatum a leech parasite which only opportunistically entered a frog. (4) The near ubiquitous distribution of leech-vectored trypanosomes among lower vertebrates virtually excludes a recent invasion. ( 5 ) Anuran trypanosomes appear metabolically more complete(1essspecialized) than the insect-vectored mammalian trypanosomes (see Section VII). The acceptance of both sets of evidence forces one to agree with Baker (1963) on a diphyletic origin of the genus Trypanosoma. In all the theorizing on this topic, most authors have been hesitant to introduce rapid (evolutionarily speaking) readaptations of trypanosomes to new hosts and/or vectors. In the anuran and reptilian trypanosomes, however, we find several examples of such readaptations (see also Section IIB). The most striking examples are T. bocugei (Feng and Chung, 1940) and T.bufophlebotomi (Ayala, 1970,1971), toad trypanosomes vectored by a sandfly. These trypanosomes appear to be typical anuran parasites, yet, the invertebrate hosts are insects, and their development takes place in the sandfly’s hindgut. Sufficient evidence is lacking for a conclusive opinion on the phylogenetic background of these trypanosomes, but it is probable that their origin lies within a leech-vectored rotatorium-like species which has become secondarily adapted to the toadsandfly host systems. Sandflies feed not only on toads; could a secondary readaptation introduce T. bufophlebotomi into lizards* or mammals ? Have similar sequences of readaptations taken place in the past? What significance can we attach to the observation that Glossina tachinoides will feed on Anura in its natural habitat, and that T. rotatorium will undergo at least partial development in the fly’s midgut (Lloyd et al., 1924)? How many reptilian, avian or even mammalian trypanosomes have their origin, directly or indirectly, in anuran trypanosomes? And finally, one can envisage readaptations in the opposite direction too; have typical mammalian trypanosomes invaded some of the lower vertebrates? It is not fruitful at this stage to scrutinize all species and, on the basis of whatever scant evidence is available, classify them into one or the other branch of the genus. As evidence on such topics as metabolic requirements (see Section IX) and biochemical composition becomes available in the future we will be able to draw the dividing line with increasing accuracy, and we * T. thecadacfyli has recently been described from geckoes in Panama; it is sandflytransmitted, and has the appearance of a typical anuran trypanosome (Christensen and Telford, 1972).

THE TRYPANOSOMES O F A N U R A

I1

will be able to discover how general a phenomenon readaptations to new host systems has been in the “genus” Trypanosoma.

111. MORPHOLOGY AND CYTOLOGY A.

MORPHOLOGY

Light microscopic examination of trypanosomatids is at best a frustrating experience. The size, motility and lack of consistent structure makes it virtually impossible to extract realiable generalizations out of most such studies. In his review of the morphology of Trypanosomatidae, Wallace (1 970) recognizes this fact. The amphibian trypanosomes are usually larger and have more complex structure than mammalian trypanosomes ; but unfortunately little recent work utilizing modern cytochemical processes has been done. Most of the earlier published papers give extensive but unreliable descriptions of trypanosomes, indeed, the undulating membrane was not recognized for what it was until 1850 (von Siebold), and had been described as mobile teeth or protruberances by earlier authors. One of the earliest, accurate sets of drawings can be found in Kruse’s (1890) paper. From his drawings it is obvious that both slender trypanosomes with pointed posterior ends and broad, leaf-shaped forms could be found in mixed populations. Kruse (1890) shows the shape of the nucleus, depicts a clear free flagellum, an undulating membrane, posterior kinetoplast, and in the broad form, distinct longitudinal striations. During the first two decades of this century a large volume of work on anuran trypanosomes was published (e.g. Broden, 1905; Dutton and Todd, 1903; Franca, 1915; Franca and Athias, 1906b; Laveran and Mesnil, 1904, 1907, 1912; Machado, 1911; Mathis and LCger, 1911a, b; Ogawa, 1913). The criteria by which the various trypanosomes were described by these authors were mainly overall shape and size, shape, size and relative position of the nucleus, position of the kinetoplast, length of flagellum, and such descriptive qualities as intensity of cytoplasmic granulation and striation. Although much of this more or less detailed descriptive work failed to adequately categorize and/or classify anuran trypanosomes, certain outstanding “types” or “morphs” appear regularly from diverse regions of the world, and certain morphological traits are reported that appear to be typical of anuran trypanosomes. In overall shape and size, a large variability is the dominating feature in the literature (see Fig. 2). Besides the normal, slender trypanoform appearance, as represented by such “species” as T. inopinatum and T. belli, a variety of much broader, yet typical trypanoform species were described (e.g. T. elegans, T. ranarum, T. sanguinis and T. mega). Also under the name of T. inopinatum or under the catch-all name T. rotatorium are described very large, broad trypanosomes without free flagella. Finally, a number of authors have reported medium sized, to very large round, oval or irregularly shaped “trypanosomes” with either no flagellum at all, or only a small internal

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I.

I_‘.

IJARDSLITY AN11 It. H A R M S C N

FIG.2. Typical types of anuran trypanosomes. A, Type A, ranarum-like; B, Type B, large inopinatum-like; C, Type C, chaftoni-like; C (lower), multi-kinetoplastic Type C ; D, Type D, rotatoriurn-like; X, Type X, small inopinatum-like, typical of tadpoles, early infections and Ram pipiens.

remnant. More recent work on overall shape and size has added little to the general picture which is represented for instance by Laveran and Mesnil in 1912. The problem as to whether all differently structured trypanosomes are different species, or morphs of a polymorphic sequence, is dealt with in Section 11A. Many of the older publications which deal with the morphology of anuran trypanosomes in culture or in the invertebrate host use vague descriptive terms such as “leptomonad” or “crithidial” (see also Section IVA). It is usually not possible to correlate these terms with their modern counterparts, promastigote and epimastigote respectively (Hoare and Wallace, 1966; Brack, 1968), with any certainty. More recent work, however, usually depicts clearly the relationships between nucleus, kinetoplast and flagellar pocket. Unfortunately, inany developmental forms found in the invertebrate gut fail to fit neatly into the Hoare and Wallace (1966) categorization. Such intermediate forms led Ayala (1971) to write: “. . . the new terms serve as useful categories, but should not be too rigidly interpreted”. Several unique structures of anuran trypanosomes have received attention. The longitudinal or spiral striations or “costae” are one of the most striking features of many (but not all) anuran trypanosomes. Jirovec (1933) using a silver reduction technique showed that these striations were neither myonemes (as had been suggested) nor ribs in the pellicle or cell membrane, but tubular

rijL T R Y P A ~ O S O MsI

or

ANURA

13

structures underneath the pellicle. N o further evidence is available concerning the structure or function of these striations, although electron microscopy of culture forms of both T. rotatorium and T.mega has revealed a network of sub-pellicular tubules (see Section IIIB). Many anuran trypanosomes have long, slender, highly mobile posterior extensions. It appears that these extensions enable the trypanosome to attach themselves to solid objects such as red blood cells. We have often observed T. ranarum-like (type A) trypanosomes attached to the microscope slide or coverslip by the tip of the posterior extension. Stevenson (1911) published a drawing of a ranarum-like trypanosome attached to an erythrocyte, and PCrez-Reyes (1967) shows a photograph of T. grandis thus attached. It is possible that the same form of attachment is used by the trypanosomes when they withdraw from the circulating blood into organs such as the liver and kidney, where they appear to be attached to the capillary endothelium (Bardsley and Harmsen, 1969,1970; Machado, 1911). Whether the attachment extensions of anuran trypanosomes are homologous to the “filopodia” or “plasmanemes” of certain mammalian trypanosomes is not at all certain (Wright et al., 1970). B.

CYTOLOGY

No ultrastructural work has been published as yet on the large blood-stream forms of any of the anuran trypanosomes. The culture forms appear to be typical trypanosomatids. Steinert (1960) and Steinert and Novikoff (1 960) reported on the fine structure of culture forms of T. mega, and Creemers and Jadin (1966) have published an electron-microscopic survey of the promastigote culture form of T. rotatorium. In both cases the surface membrane appears as a normal unit membrane, but some evidence suggests that an approximately 100 A thick electron-lucid pellicle surrounds the outside of the organism. Underneath the membrane a network of helically arranged, parallel 200 8, thick microtubules can be seen. It is possible that similar tubules are part of the structural mechanism responsible for the striations or “costae” of the larger blood stream forms, and may be involved in an osmoregulatory function, Slightly to one side of the anterior apex of T. mega, Steinert and Novikoff (1960) observed a depression in the surface membrane which forms a channel leading posteriorly into the interior of the cell. The sub-pellicular tubules curve inward around this channel. Making use of ferritin as a marker molecule, Steinert and Novikoff (1960) showed that macromolecules can be taken into the flagellate by micropinocytosis at the site of the depression leading into the channel. They considered this structure a typical cytostome. The vacuoles formed at the external end of this cytostome migrate inward and posteriad along a precise pathway and become closely involved with lysosomelike structures in the posterior part of the cell. The flagellum leaves the anterior portion of the trypanosome in a typical flagellar pocket, below which it originates from a blepharoplast which is always closely attached to the kinetoplast (Creemers and Jadin, 1966). The kinetoplast has two distinct zones, a dense, DNA-containing area adjoining 2

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the blepharoplast (Steinert, 1964; Steinert et al., 1958) and a posterior portion, which appears to be a typical mitochondrion. The kinetoplast is surrounded by a double membrane, the outer one of which is continuous with the mitochondrial membrane, and the inner one appears to be continuous with the cristae. In T. mega the cristae are either irregularly arranged or present in concentric bundles (Steinert, 1960). The intimate arrangement of the kinetoplast-mitochondrion complex has led Steinert (1960) to speculate that the kinetoplast synthesizes the mitochondrion. In the later paper on T. mega Laurent and Steinert (1970) consider the kinetoplast to be a specialized part of the mitochondrion. In this paper, the authors report on the structure and molecular size of kinetoplastic DNA. The low yields of kinetoplastic DNA reported previously for trypanosomatids (Riou and Delain, 1969; Simpson, 1969) can possibly be explained by the finding of two types of DNA-a large rosette-structured fraction closely attached to various insoluble membranes, and a smaller, much more easily soluble circular molecule. Previous work had only disclosed the smaller, soluble fraction. DNA synthesis in the culture form of T. mega takes place synchronously in the nucleus and the kinetoplast, during a specific 7 h period of the 18.9 h division cycle (Steinert and Steinert, 1962). During this period thymidine and adenine are incorporated into nucleic acids, but glycine and formate are not (Bonk and Steinert, 1956; Steinert et al., 1958). This may mean that T. mega is dependent on an exogenous source of purine and/or pyrimidine. The Golgi apparatus, ribosomes, nucleus (IvaniC, 1936) and intracellular membranes appear to be not significantly different from those of the better described mammalian trypanosomes (e.g. Vickerman, 1971). IV. LIFECYCLES A.

REPRODUCTION

1. In the vertebrate host During the many years of research on anuran trypanosomes, relatively little concrete evidence has been obtained on the type, or types, of reproduction occurring in the vertebrate host. Many authors who have studied these haemoflagellates have found no detectable signs of reproduction, either specifically in the peripheral blood (Doflein, 1910, 1913; FranCa, 1907a, c, 1908a, 1915; Kudo, 1922; MacFie, 1914; Noller, 1913a) or in the frog as a whole (Gaule, 1880; Jorg, 1933; Kruse, 1890; Seed, 1970). The lack of detectable signs of division has even prompted one researcher (G. Mason, personal communication) to postulate that no reproduction occurs in the adult frog, the high levels of infection being due simply to reinoculation by the vector. However, several authors have reported various types of division in anuran trypanosomes, and our own experience supports this. Other than a few reports resulting from confusion between the life cycles of certain Sporozoa and trypanosomes (Billet, 1904b; Carini, 1910), and the odd unsubstantiated hypothesis of sexual reproduction (Carini, 1910, 1911; Lebedeff, 191Ob; Machado, 191l), all reports of reproduction are confined

THE TRYPANOSOMES OF A N U R A

15

to various types of fission. Binary fission has been reported as the sole means of reproduction in T. ranarum (Diamond, 19581, T. lavalia, T. gaumontis and T. montrealis (Fantham et al., 1942), T. rofatorium (Lebedeff, 1910a, b ; Noller, 1913b; Tanabe, 1931), T. clamatae (Stebbins, 1907) and T. montezumae (Pkrez-Reyes et al., 1960). On the other hand, multiple fission of rounded, amastigote stages is the only type reported in T. parroti (Buttner, 1966), T. leptodactyli (Carini, 191l), T. rotatorium (FranGa, 1907a; IvaniE, 1936), and T. aurorae (Lehmann, 1959a). A combination of multiple and binary fission, occurring at different stages of the life cycle in the vertebrate host, has been reported for T. inopinatum (Buttner and Bourcart, 1955a; FranGa, 1915), T. ranarum (Danilewsky, 1889), T. pipientis and T. chattoni (Diamond, 1958), T. rotatorium (Fantham et al., 1942) and T. loricatum (Dutton et al., 1907). Our experience supports a combination of multiple and binary fission, having witnessed binary fission in types A and B and a multikinetoplastic state in type C (see Fig. 2). Differing reports exist on the site of reproduction, including the conjunctiva for T. inopinatum (Brumpt, 1906b) and T. leptodactyli (Carini, 1907), the bone marrow for T. inopinatum (Buttner and Bourcart, 1955b) and T. rotatorium (Tanabe, 193l), predominantly the heart for T. leptodactyli (Carini, 191l), T.pipientis (Diamond, 1958) and T. montezumae (Perez-Reyes, 1969b), the kidney for T. rotatorium (IvaniE, 1936), T. aurorae (Lehmann, 1959a) and T. galbae (Perez-Reyes, 1967), the lungs and spleen for T. unduluns (FranCa, 1912) and T. inopinatum (FranCa, 1915), in a variety of internal organs, occasionally including the peripheral blood, for T. inopinatum (Buttner and Bourcart, 1955a), T. ranarum and T. chattoni (Diamond, 1958), T. loricatum (Dutton et al., 1907) and T. rotatorium (Fantham et al., 1942). To our knowledge, only one researcher has reported moderately high levels of reproduction in the peripheral blood (T. grylli by Nigrelli, 1945), and the only intra-cellular development is reported for T. inopinatum in liver and bone marrow (Buttner and Bourcart, 1955a, b). The fact that many authors have reported very low levels of reproduction in the peripheral blood (our own observations included) may explain in part, the negative reports previously cited (see also Section IVA, 3). Finally, the timing of reproductive activity, where such activity has been observed, also remains nebulous. Some authors report that reproduction occurs only during the initial stages of the cycle in the vertebrate host: T. inopinatum (Brumpt, 1906b; FranCa, 1912), T. rotaforium (Noller, 1913b) and several species (P6rez-Reyes, 1967). Diamond (1958) states that in T. ranarum the initial stages witness the heaviest degrees of reproduction, but that division is a continuous process. Buttner and Bourcart (1955a) report for T. inopinatum, and IvaniE (1936) for T. rotatorium, that binary fission occurs during the initial stages of infection, but multiple fission ensues later, accounting for the permanent infection in the adult anuran. Our experience would favour this latter view. The number of positive reports concerning multiplication of trypanosomes in the adult anuran host indeed establishes that such a phenomenon does exist. In addition, reports of both binary and multiple fission in the verte-

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brate host indicates that these two forms of reproduction both occur. The questions as to when, where, under what conditions and in what species of trypanosome each type of reproduction does occur can not be answered with any certainty as yet. The contradictions in the literature may be based on a number of factors. The most obvious one is that each author has worked with a different isolate, and many may have worked with different species, especially when the species used were referred to under one of the loosely applied names such as rotatorium or inopinatum. Reproductive rates are easily subject to pressures of natural selection and very closely related agamospecies may have markedly different reproductive patterns. A second reason for the conflicting reports in the literature may be the dependence of reproduction on a complex set of environmental conditions, both current and preceding, as well as on such factors as maturity and density of the population. Indeed, Galliard et al. (1953) have established that the growth hormone somatotrophin (STH) will affect mitosis in T. inopinatum, raising the interesting question as to the effects of other hormones and metabolites on the trypanosomes, especially since the levels of such agents in the blood of Amphibia fluctuate on a seasonal basis. Certainly, we have shown that a variety of biochemical agents appears to affect the metabolism and behaviour of trypanosomes in the vertebrate host (see Section VII). The only species of anuran trypanosome which comes close to having received sufficient attention to warrant the formulation of a reliable opinion concerning its reproductive pattern in the vertebrate host is T. pipientis (see Diamond, 1958). This species appears to pass through an extensive phase of reproduction immediately after the injection of culture forms into uninfected frogs. From Diamond’s work (1958) it is not possible to say which type(s) of culture form (epimastigote, amastigote or metacyclictrypomastigote) is involved in the burst of reproduction, and his data are unfortunately too scanty to define the site@) of reproduction any closer than “the spleen and possibly also other organs such as the heart, kidney and lung.” The pattern of reproduction appears to involve a multiple fission of small amastigote forms. Diamond also mentions a much less frequent binary fission of trypomastigote forms in the circulating blood, but does not indicate at what stage in the infection this occurs. Diamond’s observations are restricted to the first 24 h after infection, but he did describe an increase in population density in the circulating blood up to 7 or 8 days after infection. Whether further bursts of reproduction may occur under certain circumstances (e.g. breaking of hibernation) or other cyclic conditions in the host is not known. However, it appears unlikely that T. pipientis represents the general reproductive pattern of the rotatorium complex, if indeed such a general pattern exists. 2. In the invertebrate host Billet (1904b) successfully infected Helobdella algira with T. rotatorium and T. inopinatum and noted reproduction of the trypanosomes in the leech gut. FranGa (1907a, b, 1908a) noted the diversity of developmental forms in the

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leech gut, describing what appear to be both small trypomastigote and epimastigote or promastigote forms. Noller (1913b) recognized a cyclical development in Hemiclepsis marginata with trypomastigote blood forms dividing rapidly into “crithidial” (probably epimastigote) forms which later metamorphose into metatrypanosomes, still in the gut of the leech. The very complex reproductive patterns observed by Noller (1913b) and others (see Sections IX and IVA, 3) for various culture environments may not at all mimic stages of natural development in the leech or in various frog organs. Buttner and Bourcart (1955a) working with T. inopinatum and H . algira noted that only some of the vertebrate blood forms (“Ies grandes formes adultes”) will develop when ingested by the leech; the others degenerate soon after ingestion. The ones that do develop undergo unequal binary fission approximately 4 h after ingestion, followed by a series of divisions leading to a large number of small trypanosomes. During this period the percentage of epimastigote forms rapidly increases and eventually no trypomastigote forms remain. The subsequent formation of metacyclic trypomastigote forms is the resuIt of a slow forward movement of the blepharoplast along the surface of the nucleus. Maintenance of the infected leech at low temperatures results in cessation of reproduction, and instead of metatrypanosomes, large persistent epimastigote forms appear. Observing T. pipientis in PIacobdella phalera, Diamond (1958) described a situation differing from that described by Buttner and Bourcart (1955a) in only minor detail, except for the occurrence of a second type of reproduction. Diamond noted that besides the common unequal binary fission, a process of rounding-up followed by multiple fission could occur. This latter process is probably the same as described by Brumpt (I 9 14) for T. leptodactyli in Placobdella brasiliensis. Consequently, the trypanosome population as observed by Diamond 24 h after ingestion by the leech is more varied, including not only epimastigote forms, but also a number of amastigote and transitional forms. The onset of reproductive behaviour in trypanosomes ingested by a leech appears to be triggered by a factor (or factors) which is independent of the size of the blood meal, probably chemical and released by the leech. Consequently, due to dilution of the factor(s), trypanosomes in large meals undergo their first division much later than do the ones in smaller blood meals (Barrow, 1953; Diamond, 1958; see also Section IVB, 3). Diamond (1958) described in older, established infections in the leech, short and long epimastigote forms very similar to the ones described by Buttner and Bourcart (I 955a), but Diamond also described short and long metacyclic trypanosomes. Unfortunately the most interesting observation of Buttner and Bourcart, relating the appearance of certain forms to the temperature regime has not been followed up by Diamond, who does not even report the temperatures at which his experimental leeches were kept. Feng and Chao (1943) give a detailed account of the reproduction of T. bocagei in the sandfly Phlebotomus squamirostris. The similarity between their observations and the ones observed for typical anuran trypanosomes in the leech is remarkable. Both multiple fission involving amastigote and

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sphaeromastigote forms, and binary fission involving small “crithidial” (probably promastigote) forms occur. One striking difference, however, is the absence of a final transition to a trypomastigote. The trypanosomes are excreted by the sandfly as stumpy “crithidia” or amastigote bodies. A virtually identical situation is described by Ayala (1971) for T. bufophlebotomi in the sandfly Lutzomyiu vexafrix. He stresses the absence not only of trypomastigote forms but also of epimastigote forms, in that none of the flagellates developed an undulating membrane (see also Section 111). The above-described situations seem to indicate a general trend. From a polymorphic vertebrate blood stream population only a limited number of trypanosomes are in a competent state for development in the invertebrate gut. These “transmission” forms when entering the invertebrate host start a cyclical development when triggered by a set of specific environmental stimuli. The nature, source and pathway of the triggering stimuli are as yet unknown. Obviously, development will take place in culture media (see Section IVA, 3) but the patterns are different, indicating that the controlling stimuli are probably a balance of excitatory and inhibitory units. Different temperature regimes affect the reproductive patterns and the resulting types of trypanosome. The reproductive pathways of the trypanosomes involve binary or/and multiple fission. Certain ecological circumstances lead to the production of metatrypanosomes, whereas other circumstances produce some type of “dauer-epimastigote”. It appears a likely speculation that the metatrypanosomes are produced only when the environment will induce feeding behaviour in the invertebrate host. In more specialized “species” (see above) of anuran trypanosome some of the complexities may be absent as a result of readaptations to new host systems where the right set of triggering mechanisms was absent. Such a loss of diversification is seen as monomorphic blood stream populations, simplified reproductive patterns, the loss of metatrypanosome formation and probably also as other, less obvious phenomena. Clearly, much more research is needed in this area before the various isolated findings will start to form a pattern, leading us to an understanding of the functional interactions of the trypanosomes and their invertebrate host.

3. In culture media It is impossible to conclude with any accuracy what reproductive and developmental patterns were observed and described by the early workers who cultured anuran trypanosomes. Each author used a different medium and cultured trypanosomes from different sources and under different conditions (see Section IX) ; the sequences of developmental forms were characterized by different criteria and the nomenclature used was often arbitrary. The drawings of the various forms are usually incomplete, inaccurate and lacking in essential detail (e.g. Bouet, 1906; Doflein, 1910; FranCa, 1911c; Lebedeff, 1910b; Noller, 1913b; 1917). Despite this lack of rigour in the early work it is possible to extract a general pattern of growth and development of anuran trypanosomes in culture media. The present interpretation has gained reliability by incorporating a number of accurate, though isolated, observations of modern

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researchers (e.g. Steinert, 1958a; Lehmann, 1962, 1963b, c; Lehniann and Sorsoli, 1962; Ayala, 1971; Diamond, 1958). When bloodstream forms are transferred into one of the standard culture media (see Section IXA), the most frequent pattern of growth and development involves an initial rounding up of the bloodstream form, followed by rapid nuclear division, somewhat slower cell division, and little or no growth. The culture at this stage, therefore, consists of clusters or “rosettes” of sphaeromastigote or amastigote forms, many of them multinucleated. After a period of time the cells in these clusters elongate, the clusters fall apart, and the individual cells appear to be free-swimming “crithidia”, although modern microscopic examination and standardization in nomenclature makes it appear likely that the earlier flagellated forms are in fact promastigote. Only later in the culture do epimastigote forms appear. Most species of cultured anuran trypanosomes will eventually produce a certain percentage of trypomastigote metatrypanosomes, although that depends on the composition of the medium (Steinert and Bond, 1956) and other environmental factors such as temperature (Buttner and Bourcart, 1955a). Equal or unequal binary fission among amastigote, promastigote and epimastigote forms occurs as well, and this type of reproduction persists for long periods in the cultures, long after typical multiple fission has ceased. Under certain conditions this latter type of reproduction seems to be the only type present (Diamond, 1958; Lehmann and Sorsoli, 1962). A similar situation of combined phases of binary and multiple fission is reported for two species of trypanosomes from urodeles (Lehmann, 1959~). Robertson (1912) reported a division-stimulating effect of hypotonic media on fish trypanosomes. A similar phenomenon was later established also for T. inopinatum (Ponselle, 1923b; Galliard, 1929) and Diamond (1958) found that T.pipientis will only start a cycle of reproduction in a medium hypotonic to frog’s blood. Other species of anuran trypanosomes do not need a temporary lowering in osmolarity to initiate reproduction. Whether the actual stimulus triggering the onset of reproduction in the invertebrate host is the lowered osmolarity of the environment is yet to be proven; it could be that the effect of a hypotonic substrate merely mimics some other situation in the gut of the invertebrate host. The fact that several species will start a burst of reproductive activity in isotonic media after a long period without reproduction in the vertebrate host suggests other control factors, probably biogenic ones, especially since other environmental factors have occasionally been associated with the onset of reproduction in culture, such as low pH (Ponselle, 1917) or high pH (Lehmann, 1963~). The other end of the reproductive sequence, the production of metacyclic, trypomastigote forms, has been studied in more detail. FranCa (1911~)first reported experimentally induced morphogenesis at this stage: epimastigote forms of T. rotatorium taken from the leech gut would transform into trypomastigotes when placed in agitated and oxygenated frog blood. His observations were not followed up by further experimentation until the work of Steinert and Bond (1956) and Steinert (1958a). These authors kept T. mega for over 50 subcultures in a serum-free medium, and observed that under

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these conditions tio inetatrypanosonies were formed. The addition of large amounts of toad or calf serum to the culture caused 100% mortality, but small additions of serum or additions of “deagglutinated” serum resulted in transformation. Dialysis and fractionation of the serum led Steinert and Bonk (1956) to the conclusion that the morphogenetic activity was linked with the globulin fraction. In a subsequent publication, however, Steinert (1958a) attributed the cytodifferentiation potential to urea, a substance which is often tightly adsorbed to globulin molecules. Steinert and Steinert (1960) published experimental data which show that urea inhibits DNA synthesis in culture forms of T. mega. The normal division cycle of epimastigote culture forms takes on the average 18-9 h, DNA being synthesized during a 7 h period only (Steinert and Steinert, 1962). These authors observed, however, that cultures exposed to physiological doses of urea not only showed transformation and cessation of DNA synthesis, but further cell division was arrested even in those cells where DNA synthesis was already completed. Steinert (1965) has suggested that in those species of Trypanosoma where low, non-pathogenic populations may persist in the vertebrate host at constant levels, the small percentage of “dividing forms” reported are in fact trypanosomes whose cell division was abruptly arrested, probably under the influence of urea, when they entered the host (see Section IVA). In this publication Steinert reports experimental evidence for this suggestion, and further shows that transformation from epimastigote to the trypomastigote stage can in fact take place in partly divided epimastigotes. A strict physiological connection between DNA synthesis and cell division does not seem to exist in T. mega, since such substances as acriflavine (Steinert and van Assel, 1967) and ethidium bromide (Steinert, 1969) do inhibit kinetoplastic DNA synthesis, but do not inhibit subsequent cell division, leading to the formation of akinetoplastic trypanosomes. When transformation takes place in a trypanosome population it rarely affects more than 10% of the population, the fraction varying with the age and conditions of the culture. Steinert (1958b) believes that a previous pattern of growth and development had to be completed before the organisms acquired a competence for transformation. As pointed out by Guttman (1963) an alternative explanation may be that the population is of mixed genetic stock, since the subcultures were not clonally initiated. The work of Steinert and co-workers shows very clearly that morphogenetic studies with cultures of anuran trypanosomes can lead to a much better understanding of the entire process of cyclical development in trypanosomes. Some exciting questions remain. For instance, is urea the normal, natural transforming agent at work in the vertebrate host; and to what extent is the diversity in growth patterns in culture media and in living hosts caused by intrapopulation genetic diversity, and to what extent by a delicate balance between growth and transformation stimulating and inhibiting factors ? B.

POLYMORPHISM

1. Phenomenon and terminology It is unfortunate that the term “polymorphism” has different meanings in

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different biological subdisciplines. The term as used by population geneticists is very difficult to apply to agamospecies (see Section 11) and consequently is of no use to the student of trypanosomes. The polymorphism of trypanosomes is a sequential (developmental) and perhaps in some cases also a parallel phenotypic expression of one genotype. In order to use the term meaningfully, the multiple phenotypes must present themselves multimodally in samples, that is, intermediate forms must be much rarer than the typical morphs. Using this rather broad definition, there can be no disagreement that the trypanosomes found in Anura are polymorphic. The startling dissimilarity between the forms found in the invertebrate and vertebrate hosts will attest to that. Moreover, the series of developmental morphs found in each of the vertebrate and invertebrate hosts also adds credence to this statement. Many authors, however, place a much more restricted definition on polymorphismas a morphological variation in the “mature”, “adult” or trypanoform stage as it occurs only in the vertebrate host: monomorphism being the lack of such variation. However, what under one set of circumstances and/or at one time may be of one morphological type, could conceivably be a different type under a different set of circumstances, and/or at another time; this would render a system that may appear monomorphic, really polymorphic. For instance, in the caudatan trypanosome, T. diemyctyli, maintenance of the vertebrate host (Triturus viridescens) at temperatures about 20-25°C results in a monomorphic population of “short form” trypanosomes; at 15°C the population is polymorphic, with one group of “short forms” as found at 20-25”C, and another statistically separable group of “long forms” (Barrow, 1954). In a later paper, Barrow (1958) found that the populations could be made purely “short” or “long” by varying the temperature, the latter being a pathogenic infection, the former not. These experiments establish the critical role that just one factor in the host’s environment may play in affecting the morphological state of a population of amphibian trypanosomes. Also, Galliard and co-workers (1953, 1954) have shown that injections of growth hormone (STH) into Rana esculetzta will alter the morphology of T. inopinatum. There is no reason to believe that other factors which affect the vertebrate host’s physiological state may not also have similar effects, especially in the light of recent findings on the effect of certain hormones on the metabolism of various anuran trypanosomes (Bardsley, 1972). The morphology of the trypanosome appears to be affected in certain cases by the host species in which it is found as well. PCrez-Reyes (1967) shows that infections of T. montezumae in three different species of ranid frogs have three different and separable sets of measurements (unfortunately not statistically analysed). If these three sets of data are statistically separable it would be a good example of polymorphism dependent on the host species parasitized. Scorza and Dagert (1958) add credence to this phenomenon in that inoculations of T. leptodactyli crithidia into various hosts gave rise to different morphs or “species”. For example, inoculations into Hyla crepitans gave rise to T. borrelli infections ; into Leptou’actylusbolivianus, to T. costatuin followed by T. leptodactyli, with intermediates between the two; and into Phyllomedusa

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bicolor, to rounded aflagellate forms followed by T. arcei, and this followed by the series reported from L. bolivianus. The above observations raise several cautionary points. First, the decision as to whether an amphibian trypanosome species is monomorphic or polymorphic (sensu strictu) can only be made after subjecting the parasite and its vertebrate host to a variety of conditions found in their normal environments, including a seasonal study. Second, the use of the “mature” trypanoform stage as the most important criterion for separating species of anuran trypanosomes (Diamond, 1958) must be recognized as an arbitrary measure full of possible pitfalls. That is, the trypanosomes under comparison must be subjected to identical conditions, including presence in the same species of host of the same age and sex, at the same seasonal time, etc. Of course, this information must be combined with the other criteria for separating species (see Section 11). Third, this raises the question of the usefulness of the strict definitions of poly- and monomorphism in describing the trypanosomes of Amphibia (and possibly of poikilotherms in general). If, as is the case with T. diemyctyli (Barrow, 1954, 1958), rearing the organisms in a homogeneous environment (i.e. like those found in laboratory infections) results in a monomodal measurement distribution, then one might be misled into describing the trypanosome as monomorphic, when indeed it may not be so in the fluctuating environment found in nature. Examples can be found in the literature which reveal the confusion that has been caused by the use of the restricted concept of monomorphism and polymorphism. For example, T. inopinatum as originally described by Sergent and Sergent (1904) appeared to be a small monomorphic trypanosome infecting Rana esculenta of Europe. Two other trypanosomes named T. elegans and T. undulans by Franqa and Athias (1906b) also appeared to be monomorphic species from the same European host. However, Franca (1911b, 1915) later found that the morph originally described as T. inopinatum was the youngest stage in a sequence of morphs including T. elegans and finally T. undulans. Franqa (1915) thus concluded that T. elegans and T. undulans were not good species, the whole complex being T. inopinatum Sergent and Sergent, 1904. This finding was later substantiated by Buttner and Bourcart (1955a) and Buttner (1966). This confusion points out the danger of naming new species by assuming, but not establishing, the real “mature” stage in the life cycle. A second example of a different sort is also revealing. Diamond (1958) indicated that T. ranarum (Lankester, 1871) Danilewsky, 1885 is a polymorphic species with two “adult” forms. In our local species of ranid frogs we also find these two morphs (assuming we are dealing with the same species, and, indeed, that it is a good species). However, we have never seen a pure infection of small type A’s (Diamonds’ type I adult), and moreover, only find this type during a restricted period seasonally (June, July). We interpret this as indicating that the small type A is a developmental morph in a type A infection, since we do find pure large type A infections (i.e. without small ones) and find them continually throughout the seasons. This view is supported by PCrez-Reyes (1970b). However, it may be that, under the

T H E T R Y P A N O S O M E S OF A N U R A

23

laboratory conditions that Diamond’s trypanosomes were grown, the trypanosome was polymorphic (sensu strictu), but, under field conditions, the type I adult is merely a developmental morph. It could be also that the geographic and/or host differences has rendered the trypanosome polymorphic (sensu strictu) in Minnesota, and otherwise here in Ontario. I n any event, this example reveals the confusion that can result from the usage of such terms. In the light of the foregoing, maybe it would be better to discard the limited definitions of the terms monomorphic and polymorphic with respect to amphibian trypanosomes, and concentrate more on functional terminology such as reproductive stage, transmission stage, etc., wherever possible. Where such functional connotations cannot be made, well-documented descriptive terms for each morph can be used. It is extremely difficult to know which is indeed the end stage (or end stages) of development in the vertebrate host, especially if this end stage may vary with the environment of the parasite. Also it has never been established that an “adult” or “mature” form cannot revert to another form (or forms) which may be construed as more “immature”. Perhaps the whole concept of the “mature” or “adult” stage is a relative phenomenon. Certainly, it has led to much confusion and conflict in the literature on anuran trypanosomes, especially between the pure monomorphists such as Pkrez-Reyes (1970b) and the polymorphists such as Noller (1913), Tanabe (1931), Vucetich and Giacobbe (1949). Finally, while considering terminology, two other terms used for multiple phenotypes of one genome, pleomorphism and allattomorphism, might best be dropped from use. Pleomorphism appears to be totally synonymous with polymorphism, while allattomorphism as used by Diamond (1958) is a far more restricted term which only makes sense when contrasted with Diamond’s more restricted use of polymorphism. 2 . Functional considerations Within the field of anuran trypanosomiasis virtually no work, and very little speculative thinking has been devoted to the problem of the functional significance of the observed polymorphism. The most frequently held view considers the various morphs specific adaptations to subenvironments found within the host (Mendeleef-Goldberg, 1913; Hegner, 1921; etc.). A more detailed approach was voiced by Barrow (1958) who suggested a connection between the appearance of different morphs of T. diemyctyli and the host’s antibody spectrum. There seems to be considerable evidence for recognizing that certain endemic developmental sequences occur (e.g. Franqa, 1915; Buttner, 1966). This is particularly true for growing populations in the invertebrate host or in culture media (see Section IVA), and may well be the case for new infections in the vertebrate host too. Such morphs as appear in sequence during a developmental period may be morphological reflections of the different functions the trypanosomes perform, or one could say adaptations to temporal niches rather than to spacial niches within the host. The “slenderstumpy” sequence in a typical T. brucei infection can be interpreted best in

24

J. E. BARDSLEY A N D R. HARMSEN

such words (e.g. Vickerman, 1971). The two morphs here have different functions and perform metabolically differently. I n anuran trypanosomes only developmental polymorphic sequences can be correlated to some functional parameter. Other sequences have as yet no functional interpretation. Some evidence, on the other hand suggests that exogenous influences may result in the appearance of various morphs, with transformations possible in different directions (Barrow, 1954, 1958; Scorza and Dagert, 1958). Such morphs may well be adapted to various spacial niches within the host, or between hosts. These morphs are probably morphological reflections of functional relationships with certain factors of the environment. At present nothing is known about the nature or mechanisms of these functional relationships. V. DISTRIBUTION Trypanosomes have been found in Anura from all the major life zones of the world (see Table ll), and most anuran species examined have been found to be infected. There are exceptions, of course, one being Rana clamitans from Newfoundland, Canada. The lack of infection in this case can be explained as being due to the absence of appropriate leech vectors on the island (Bennett, personal communication). It is also important to note that this frog has only recently been introduced into Newfoundland. HOST VI. THEVERTEBRATE

A.

THE ADULT AS AN ENVIRONMENT

1. Seasonal cycle in peripheral parasitaemia That the intensity of the peripheral parasitaemia in Anura varies on a seasonal basis is an observation as old as the discovery of frog trypanosomes itself. Indeed, Gruby (1843), who named the genus Trypanosoma, noticed that trypanosomes could be found in the peripheral blood of frogs only during spring and summer. Since that time, although there has been the odd report to the contrary (Koniiiski, 1901; Lauter, 1960) the seasonal fluctuations in the percentage of the anuran populations infected with trypanosomes have been well documented (Bardsley, 1969; Bollinger et al., 1969; Bouet, 1906; Brandt, 1936; Franqa, 1907a; Lebedeff, 1910b; Lewis and Willaims, 1905; PCrez-Reyes et al., 1960; Seed, 1970; Sergent and Sergent, 1905; Shalashnikov, 1888; Tobey, 1906). As well as percentage infected, average infections for each member of the anuran population are also reported to vary on a seasonal basis (Bardsley, 1969; Bollinger et al., 1969). Similar seasonal fluctuations have long since been reported for trypanosome infections in the Caudata (Nigrelli, 1929; Pearse, 1932). Even though the general phenomenon has been amply reported, few authors have surmised causes for such seasonal variations. Those who have, usually correlated the trypanosomes’ cycles with general fluctuations in the host’s environment, i.e. with climate (Lebedeff, 1910; Pearse, 1932) or more particularly with temperature changes (Brandt, 1936; Bollinger et al., 1969; Shalashnikov, 1888). However, a quick glance at

THE TRYPANOSOMES OF A N U R A

25

the reported data reveals that: ( I ) spring (seasonally not the time of highest temperatures), not summer, is the usual time of peak parasitaemias (Bardsley, 1969, 1972; Bollinger et al., 1969; Nigrelli, 1929; PCrez-Reyes et al., 1960); (2) there is a dip in the reported curves in August (see Figs 2 and 3 of Bardsley, 1969, 1972, and Fig. 2 of Bollinger et al., 1969) a period of seasonally high temperatures; and (3) hosts retained in the laboratory under constant temperatures show the same spring increase in parasitaemia as those captured from the field (Bollinger et al., 1969; Nigrelli, 1929). These observations suggest that some other environmental factors, such as photoperiod, or endogenous seasonal rhythms, may be involved as well. The effect of environmental factors on the trypanosomes is probably indirect, through alterations of the host’s physiology. Indeed, several authors have postulated a relationship between the host’s physiology and the seasonal oscillations in parasitaemia (Bardsley, 1969; Bollinger et al., 1969; Gruby, 1843). Moreover, it is possible to correlate certain reported seasonal cycles in the physiological state of the host with the cycles in the extent of the peripheral parasitaemia of the bullfrog (Rana catesbeiana). Since the environment of the parasite is supplied entirely by the host’s blood, alterations in the latter are probably directly responsible for the variations in the peripheral parasitaemia, acting as transducers of stimuli in the host’s environment. Since temperature is involved so critically in the cyclicity of anuran physiology (Dierickx et al., 1960; Kepinov, 1941; Long and Johnson, 1952), it undoubtedly has at least such an indirect role to play in the cyclicity in the parasitaemia. Other host environmental factors are probably likewise involved. Injections of hyperglycaemic agents (e.g. catecholamines, glucagon and glucose) into the host effect an increase in the peripheral parasitaemia (Bardsley, 1972; Bardsley and Harmsen, 1970). Furthermore, since the reported seasonal cycles in blood glucose and in the activity of the glands controfling it (especially the adrenals), correlate with the seasonal cycle in parasitaemia, these authors surmise that the catecholamines and glucose are probably responsible for the seasonal cycle of the flagellates, especially since injections of other agents which are also seasonally cyclic in the Anura, did not effect a significant increase in the peripheral parasitaemia. Finally, it must be recognized that certain environmental factors, such as temperature, may act directly on the trypanosomes, and that other systems such as the host’s immune response may be involved. 2. Short-term variations in peripheral parasitaemia As well as a seasonal cycle, the trypanosomes of the southern greenfrog display a circadian rhythm in peripheral parasitaemia, showing the highest levels during periods of light (Southworth et al., 1968). These authors observed that the number of flagellates in the kidney fluctuated in a fashion opposite from that in the peripheral blood under normal photoperiod, but in constant dark remained at a high 1evel.h conditions of constant light the cycle persisted as normal. They postulated that the pineal andfor chromatophores may be involved in the regulation of this rhythm. A continuation of this work was done by Mason (1970) who showed that under a 14 : 10 photoperiod one

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J. E. B A R D S L E Y A N D R. H A R M S E N

morph showed a circadian rhythm with a peak between 10 a.m. and 2 p.m. She was able to reverse this cycle by changing the photoperiod, or, in constant light, by changing the thermoperiod. A search for the host’s photoreceptor revealed that neither the eyes nor “stirnorganpineal” complex were involved. In attempting to discover the nature of the physiological mediator of this rhythm, Mason found that noradrenaline would cause a quick increase in peripheral parasitaemia, but only at 12.00 hours. Injections of 3’ monoiodoL-tyrosine prevented the evening fall in peripheral parasitaemia. Injections of acetylcholine, adrenaline, insulin and serotonin were reported to be without effect, as were long-term treatments with serotonin and melatonin. From this work Mason concluded that: (1) environmental factors, such as photoperiod and temperature are acting as cues to an endogenous clock, and (2) noradrenaline is a mediator in this cycle, but not the primary one. Mason’s data reveal that insulin increases the perpheral parasitaemia within 30 min after injection, an effect which she dismissed as due to the fact that the suspending medium overrides the effects of insulin. Since she did not get a lower parasitaemia with insulin injections than with the control she stated: “It seems highly unlikely that this parasitaemia cycle is dependent on the glucose levels of the blood”. However, she examined the parasitaemia only 30 min after injection and it takes at least 6 h for insulin to effect hypoglycaemia in amphibians (Cori and Buchwald, 1930b; Smith, 1953; Wurster and Miller, 1960). Finally, in a personal communication, Mason has revealed that thermoperiod can override normal photoperiod in its effects on this circadian rhythm. Laveran and Mesnil (1907) found that lowering the host’s temperature to 0°C caused a rapid depletion of T. inopinatum from the peripheral blood of R . esculenta. More recently, it has been disclosed that variations in the temperature of the host have directly proportional effects on the peripheral parasitaemia in the bullfrog (Bardsley, 1969; Bardsley and Harmsen, 1969) both over long-term periods (e.g. 3 weeks) and immediately (e.g. within 60 min). The parasitaemia also increases in response to excitation and injections of adrenaline (Bardsley and Harmsen, 1970a). In a more comprehensive piece of work, Bardsley (1972) has shown that the peripheral parasitaemia will also increase within 60 min in response to injections of physiological doses of noradrenaline, histamine, glucagon, and dopamine. An opposite effect was reported with tyramine, aminophylline and sodium nitrite, and no effect with a variety of other agents, including such hormones as serotonin, thyroxine and steroids. In the light of these and other results, and the physiological effects of these agents reported in the literature, Bardsley postulated that the catecholamines are the prime effectors of the short-term increase in peripheral parasitaemia, and that they act via venoconstriction, hyperglycaemia and a direct effect. (The last mentioned is dealt with in Section VII.) 3. Sex of host The effects of sex of the host on the extent and composition of the parasitaemia has been studied only by a few authors. Gruby (1843) reported that female frogs had more trypanosomes than males, but Konidski (1901)

THE TRYPANOSOMES OF ANURA

27

and Tobey ( I 906) reported just the opposite. However, two recent reports involving statistical validation have shown no difference in the infection levels between the sexes (Bardsley, 1969; Bollinger et ul., 1969). Moreover, Bardsley (1969) found no difference in the composition of the infection. 4. Age ofhost Gruby (1843) reported that young frogs have no detectable parasitaemias, a finding supported by some workers (e.g. Lloyd et al., 1924) but not by others (e.g. Koninski, 1901; Machado, 1911). Our experience has been that the recently metamorphosed frogs have no detectable parasitaemia, the infection appearing and increasing with the age of the host. The composition of the trypanosome population is reported to alter with age as well (Bardsley, 1969; Vucetich and Giacobbe, 1949). Bardsley (1969) has shown that younger frogs have high levels of type B (= T. inopinatum ? see Fig. 2) and low levels of type A ( = T . ranarum?), the ratio reversing itself with age. Type D ( = T . rotutorium ?) on the other hand remained at fairly constant levels throughout the age spectrum. These observations indicate that either types A and B are morphs of a polymorphic system which respond to changes in the environment by changes in morphology, or, that the different age groups are more susceptible to the one species than to the other. Certainly this area, as with the ones concerning taxonomy and polymorphism (see Sections I1 and IV) needs more attention before anything definite can be said about them. 5. Subenvironments in the host Various reports from different sources have been made concerning the internal organs and tissues of the host where concentrations of trypanosomes could be found. The amassing of reproductive stages in various sites has been dealt with in Section IV. Other than this, certain authors have found varying types (here, for convenience, we include named species as well) in specific internal sites. For example, the large rounded types without undulating membrane and with or without flagellum have been reported predominantly in the heart (Fantham et al., 1942; Galliard et al., 1953; Bardsley and Harmsen, unpublished) and the kidney (Berestnev, 1902; Diamond, 1958). Concentrations of other morphs and/or named species have been reported from such organs as bone marrow (Brumpt, 1923c; Tanabe, 1931), heart (Brumpt, 1924, 1928b; Wasielewski, 1908), kidneys (Danilewsky, 1889; Finkelstein, 1907; Grassi, 1881; Kudo, 1922, 1966; Mason, 1970; Southworth et al., 1968; Wasielewski, 1908), liver (Brumpt, 1928a; Buttner and Bourcart, 1955a; Lebredo, 1903; Machado, 1911; Pkrez-Reyes, 1967; Pittaluga, 1905), spleen (Buttner and Bourcart, 1955a; Fantham et al., 1942; FranCa, 1912; Jorg, 1936; Machado, 1911 ; Pittaluga, 1905), conjunctiva (Carini, 1907), lymphatic tissue (Jorg, 1936), nerve tissue, especially brain (ShaIashnikov, 1888; Wedl, 1850), and, very strangely, in the gut lumen by Gourvitsch (1 926). Our experience in this matter has been that we find type C (Fig. 2) concentrated in heart with a type E being restricted almost exclusively to the liver and kidney. We have never really found an obvious organ of concentration

28

J. E. B A R D S L E Y A N D R. H A R M S E N

for types A, D, and X, but the liver and kidney show fairly high levels. Type B appears to be found concentrated in the kidney. All types seen are found frequently in the peripheral blood (except E) depending on the time of year. Numerous searches have failed to reveal more than just a few trypanosomes in the spleen, bone marrow, nervous tissue, muscle and other organs, with lung being moderately infected with most types. The foregoing observations seem to indicate that some morphs and/or species are adapted to the particular microenvironments found in various tissues of the vertebrate host. In this context it would be interesting to study the factors in these microenvironments that render them unique, and to see if these factors could be responsible for possible transformation from one morph to another, or for the selection of one species as opposed to others. 6. The environment of the host The effects of photoperiod and temperature are already discussed (Section VIA, 1 and 2). Moreover, many authors correlate changes in the host's environment with changes in the composition and density of the parasitaemias (Brandt, 1936; Lauter, 1960; Vucetich and Giacobbe, 1949). The most precise piece of work along this line was done by Odening (1955) in correlating biotopes with the quality and quantity of infection. However, none of these studies is detailed enough to form any conclusions on whether the differences are due to the presence of specific parasites and vectors in the areas studied, or due to an effect on the morphology and reproduction of the trypanosome population. More and detailed work is necessary in this area. Finally, a few authors have correlated other aspects of the host/parasite/ vector relationship (e.g. length of host larval period, behaviour of the members of the relationship, etc.) with the extent of the parasitaemia (Bardsley, 1972; Lauter, 1960). 7 . A proposed model We have reviewed extensively the literature on amphibian physiology and find that blood glucose does indeed vary on a seasonal basis with peak values in the spring at the breeding time (Fluch et al., 1935; Houssay, 1949; Lesser, 1913; Mazzocco, 1938; Mizell, 1965; Smith, 1950, 1954; etc.) that is, at a time when it is apparently unopposed by the effects of insulin (Pfeiffer, 1968). After the breeding period, the blood glucose levels fall during the summer months (Miller, 1960) to rise again just before hibernation (Mizell, 1965; Smith, 1950) and ultimately fall to the seasonal ebb during the winter (Miller and Wurster, 1959; Smith, 1950, 1954; Suzuki, 1935). These low winter values are apparently caused by high blood levels of insulin during the autumn (Carter, 1933). In addition, Smith (1954) has shown that the effectiveness of catecholamines in mediating hyperglycaemia during excitement varies on a seasonal basis, with a peak at the breeding period, declining thereafter, to reappear later in the summer phase, eventually falling off as autumn progresses. A similar seasonal cycle in hyperglycaemic effects, but in this case in response to injections of adrenaline, was found by Smith (1960) and Lee (1936a, b). These curves correlate well with the curves for seasonal variations in the

THE TRYPANOSOMES OF A N U R A

29

peripheral parasitaemia (Bardsley, 1969, 1972; Bollinger et a/., 1969). Smith (1 954) also reported that an active thyroid gland was required for the hyperglycaemic effects of the catecholamines. The glycogenolytic effects of the catecholamines also require that there is an active hypophysis (Houssay, 1949; Fluch et al., 1935; Kepinov, 1941). Since both the thyroid (Carter, 1933; Wurster and Miller, 1960) and hypophysis (Carter, 1933; Holzapfel, 1937) are seasonally active, with peaks in the breeding season, their activity would potentiate the hyperglycaemic effects of the catecholamines in a fashion synchronous with their activity cycles. A comparison of these cycles with those of the peripheral trypanosome parasitaemia cited above also shows a positive correlation. Concerning short-term effects, injections of physiological doses of glucagon (see p. 26) effected a dramatic hyperglycaemia in the bullfrog, showing a peak within 20 min (Wright, 1956, 1957, 1959). Jauregui and Goldner (1954) showed that histamine also elevated blood glucose by a direct effect on glycogenolysis in bullfrog hepatocytes, showing a peak effect 60 min after treatment. Mathews and Zaentz (1963) and Lee (1936a, b) found that frogs displayed high blood sugar levels when exposed to high temperatures, the reverse being true at low temperatures. This effect may be mediated by catecholamines since Tindal (1956) has shown that blood glucose quickly increases when cold R . temporaria are warmed, glycogenolysis being regulated by the release of an endogenous sympathomimetic substance from the liver above 12-14°C. Also, catecholamines otherwise effect hyperglycaemia in Amphibia (Cori and Buchwald, 1930b; Houssay, 1936a; Jauregui and Goldner, 1954; Tindal, 1956; etc.) This effect apparently is mediated by a beta receptor site in the bullfrog (Wright, 1957; Wright et al., 1958), and reaches its peak within 30 min (Jauregui and Goldner, 1954; Mathews and Zaentz, 1963). The adrenal tissue of Amphibia secretes catecholamines in response to excitation (Burgers et al., 1953; Schlossmann, 1927). Finally, Lee (1936a, b) has shown that glucose injections effect hyperglycaemia. All of these agents effect an increase in the peripheral trypanosome parasitaemia within comparable times under similar conditions in the bullfrog (Bardsley, 1969, 1972; Bardsley and Harmsen, 1969, 1970a, b), and some are reported to do likewise in the southern greenfrog (Mason, 1970). In the light of this review and the foregoing reports on the seasonal and short-term (i.e. within hours) variations in the peripheral parasitaemia, including observations not as yet published, we propose the following model of the system controlling the distribution of trypanosomes in anuran hosts (Fig. 3). Anuran trypanosomes have been reported as having attachment points (see Section 111) and we have found them frequently attached to erythrocytes and to the cover glass and slide in fresh preparations. Machado (191 1) found that T. rotatorium attached itself by this means to the capillary endothelium in Leptodactylus ocellatus. Thus, it appears that these flagellates may attach themselves to the endothelium of capillaries, venules and other vessels in storage centres such as the kidney and liver in a fashion analogous to the margination of leucocytes in mammals (Athens et al., 1961). In fact, to pursue

30

J. E. B A R D S L E Y A N D R. H A R M S E N

am

u response

-

patliaay I .-.-..,pathwny 2

..,.......

pathway 3 pathways

FIG.3. A diagrammatic representation of the control system (and its pathways) postulated to regulate the distribution, composition and density of populations of trypanosomes in their anuran hosts. The main pathways of this system are:

Pathway A-A long-term, seasonally induced and partly internally regulated change in hormonal balance which leads to release of the trypanosomes into the peripheral blood. 1. The secretion of ACTH by a temperature-activated pituitary gland, causing secretion of glucocorticoids from the interrenal glands which seasonally regulate blood glucose over the long-term, commencing in the spring. 2. The secretion of growth hormone from the pituitary which (among other effects) raises blood glucose levels over the long-term, commencing in the spring. 3. Secretion of unknown hormone from a seasonally active pituitary gland. Synergizes the the hyperglycaemic effects of the catecholamines (see pathway B). Most effective in spring. 4. Secretion of TSH from a seasonally active pituitary gland. Causes synthesis and release of thyroid hormones which synergize the hyperglycaemic effects of the catecholamines. Most effective in spring 5 Hyperglycaemia and the resultant increasing effects of it on the peripheral parasitaemia. The seasonal cycle of long-term blood glucose regulation is under the control of pathway A. 6. Increased motility of the trypanosome leading to a release from storage sites into the circulating blood. Pathway B-Of short-term effect; induced by stimulation of the CNS by various agents, such as excitation (e.g. leech-feeding), thermoperiod, photoperiod, endogenous rhythms, etc. Acts via the adrenergic system, causing release of trypanosomes from storage sites into the peripheral circulation. 7. Secretion of catecholamines from chromaffin tissue (especially the adrenal glands, which are active in the spring and summer). Raises blood glucose over the short-term in response to excitation, photoperiod, temperature, etc. Most active in the spring. 8. Short-term fluctuations in hyperglycaemia in response to 7, but synergized by 3 and 4 during the frog’s breeding season.

THE T R Y P A N O S O M E S O F A N U R A

31

this analogy further, it is interesting to note that the shift of leucocytes between the marginal granuloctye pool (MGP) in various storage centres and the circulating granulocyte pool (CGP) can be effected through such manipulations as exercise (Athens et al., 1961), injections of adrenaline (Athens, et al., 1961; Bierman et al., 1952; Lucia et al., 1937), hypothermia (Villalobos et al., 1958) and injections of histamine (Bierman el al., 1953). The actual final stimulus to demargination of leucocytes has not been established, but physiological alterations in blood flow and redistribution have been postulated (Lucia et al., 1937; Vejlens, 1938). In this context, noradrenaline and glucose have a direct metabolic effect on the trypanosomes in vitro (see Section VII) and it is possible that this effect, coupled with that of various accompanying physiological alterations are responsible for the release from attachment in various internal organ “storage centres” resulting in an increase in the peripheral parasitaemia. Thus, we postulate that when catecholamines are released from chromaffin tissue through such stimuli as excitation (e.g. leech feeding, which causes extreme excitation of various ranid frogs), thermoperiod, photoperiod and endogenous rhythms, the trypanosomes release themselves from attachment in various storage centres (e.g. kidney and liver). Concomitant with this direct effect are the effects of hyperglycaemia and various physiological alterations (e.g. venoconstriction) leading to increased blood flow through the viscera, all ultimately resulting in an increase in the peripheral parasitaemia. These mechanisms appear responsible for the short-term variations (Pathway 2, Fig. 3). They are probably responsible for part of the long-term variations as well, but seasonal fluctuations in glucose under the control of other hormones are probably also involved (Pathways 1 and 2 of Fig. 3), as are other factors (see other pathways in Fig. 3). For example, the immune response is reported to by cyclical in amphibians (Barrow, 1958; Kaplan and Crouse, ~

~

9. The other effects (direct and/or vascular) of the catecholamines, especially noradrenaline, on the trypanosomes. Pathway &The role of pancreatic secretions in control over blood glucose levels, effecting the dispersal from storage sites and possibly the re-entry into such sites. 10. The secretion of glucagon from alpha cells of the pancreas. Raises glucose levels in the blood. Role uncertain. 11. The secretion of insulin from the pancreas, apparently active in autumn and winter, when it lowers blood glucose levels. 12. Hyper- or hypo-glycaernia affecting respectively the detachment and attachment behaviour of the trypanosomes. Other pathways-probably of importance, but little evidence available. 13. Primary or secondary infections-established by the vector. 14. The effects of the host’s immune response on the trypanosomes; probably most

15.

16. 17. 18.

effective in summer and late autumn, and thereby acting in a permissive fashion in the spring. Reproductive activity of the trypanosomes. Metamorphosis of trypanosomes within polymorphic populations. Mortality due to factors other than 14. Direct temperature effects. May cause increased metabolism in trypanosomes, and thus be synergistic with the other factors causing detachment behaviour.

32

J. E. B A R n S L E Y A N D R . H A R M S E N

10.56) with low levels of antibodies in the early spring and during other times of low temperatures (Jackson et af., 1969). Thus it may act in a permissive fashion allowing a high spring parasitaemia. Temperature may be involved in a similar direct fashion, enabling higher metabolic activity in the warmer months, and thus permitting an enhanced response to the physiological factors just mentioned. A detailed schematic representation of this model can be found in Fig. 3. B.

THE TADPOLE AS A N ENVIRONMENT

Tadpoles have not been studied very extensively with respect to trypanosomiasis. Many of the existing reports are simply to the effect that tadpoles are infected (Kruse, 1890; Lebedeff, 1910b; Senn, 1902; Walton, 1950c), and only in two papers is the tadpole morph adequately described (Noller, 1913a, b). With the exception of one report (Diamond, 1958), the morphology of the trypanosomes found in the tadpole is reported to be different from that found commonly in the adult anuran (Creemers and Jadin, 1966; Doflein, 1913 ; Mendeleef-Goldberg, 19I3 ; Noller, 1913a, b) and our observations would support these reports (Fig. 2). This difference has been accredited to the difference in environments offered by the tadpole and adult, with the changes occurring during metamorphosis of the host being ultimately responsible for a change in morphology of the parasite (Creemers and Jadin, 1966; Doflein, 1913; Mendeleef-Goldberg, 1913; Noller, 1913b), since recently metamorphosed frogs are reported to have the tadpole morph, whereas mature adults do not (Creemers and Jadin, 1966; Noller, 1913b). Various reports attest to the fact that the environments of the two host stages are indeed at least partially different. First, the serum of adults was found to have a Iytic effect on cultures of T. rofatorium, whereas that of tadpoles did not, and second, tadpoles could be easily infected with cultures in contrast to adults (Doflein, 1913; Mendeleef-Goldberg, 1913). IvaniC (1936) stated that the environment offered by the tadpole is more conducive to division of the trypanosomes. This may be due to the fact that ablastin has been reported from adult frogs (Jackson et al., 1969) but not from larvae. In the light of the foregoing, it would be most revealing to study the biochemical differences that exist between the larval and adult stages of the host, and study the effects of these factors on the trypanosomes. It would be particularly interesting, in the light of the stress placed on host metamorphosis, to study the effects of thyroxine and triiodo-thyronine on the morphology of the tadpole morph. Further research is also indicated along the lines of observing changes that occur in clonal infections of tadpoles through metamorphosis, to see if the tadpole morph does indeed develop into the variety of adult forms as postulated by Noller (1913b). C.

PATHOGENESIS

The pathogenic effect of trypanosomes in Anura has been studied in detail only for T. inopinatum. Brumpt (1906b) discovered that the Algerian

THE TRYPANOSOMES OF A N U R A

33

strain of this trypanosonie was lethal to European greenfrogs after introduction by inoculation of infected blood or by leech feeding. Autopsy revealed agglutinated trypanosomes in the heart. The pathogenic effects were studied later by the same author (1924) revealing localized haemorrhages with swollen lymph glands and anaemia, the lymph fluid containing rosettes. Still later, Brumpt (1936) concluded that the Algerian strain was not lethal for Algerian Rana esculenta, but was lethal for both European R . esculenta and R. temporaria. Galliard and associates (1953, 1954) studied the effects of injections of growth hormone (= somatotropic hormone) on the pathogenicity of this parasite, and found that the hormone would reduce the fatality, dependent on dose and the diet of the host. They postulated that this effect was due to the hormone abetting phagocytosis. Buttner and Bourcart (1955a, b) did similar work, and stated further that death of the host was ultimately due to destruction of the reticulo-endothelial system. They postulated that the Algerian greenfrogs were resistant to the trypanosome because of their diet, and that T. inopinatum was pathogenic due to the retention of young reproductive forms. A similar reason for pathogenicity was given by Noller (1917) but he stated further that the species T.inopinatum as originally described (Sergent and Sergent, 1904) was merely these young reproductive forms found in the initial stages of infection. This may serve to explain the report by FranCa (1912) on the occasional pathogenicity of T. undulans (a later developmental stage of T. inopinatum). A variety of authors report that T. rutaturium is non-pathogenic (Creemers and Jadin, 1966; Lauter, 1960; Lebedeff, 1910; Mazza et al., 1927) and Doflein (1913) stated that it is so in adult hosts, but may be pathogenic in tadpoles. However, Noller (1917) reported that heavy infections may be pathogenic, especially in superinfections, which resulted in death with distinct amassing in the kidneys. A similar report is given by Reichenbach-Klinke and Elkan (1965) for Canadian frog infections, the pathogenicity manifesting itself as listlessness, food refusal and ultimate death. Other “species” of trypanosome have been reported as non-pathogenic, including T. sanguinis (Gruby, 1843), T. karyozeuktun (Lauter, 1960), and T. Ieptodactyli (Mazza et al., 1927). However, the latter has been reported as pathogenic by Brumpt (1928b), who also stated that T. hylae and T. parroti may also be pathogenic to specific hosts. Diamond (personal communication) has reported that he found a trypanosome from R . sphenocephala of Florida which proved lethal to R. pipiens of Minnesota. We have never noticed any pathological signs in our local frogs which could be correlated to trypanosomiasis in the frog. Pathogenicity is an exceedingly difficult phenomenon to establish, requiring repeated correlative and other studies. Most of the above reports are just casual observations, and thus could be confused with the symptoms of other common pathology (e.g. redleg). The pathogenicity of T. inopinatum on the other hand, seems well established. However, even this pathogenicity is in a non-endemic host, and it is conceivable that in a host previously unexposed to the trypanosome, and thus without the rapid anamnestic immune response, the parasite would be pathogenic. On the other hand, a previously exposed

34

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E.

B A R D S L E Y A N D R. H A R M S E N

frog would handle the infection without any such manifestations. This also would apply to hosts reared in the laboratory. The use of this character for the separation of species is debatable on these grounds. Finally, it does seem logical that the rapidly reproducing stage of the life cycle in the vertebrate host would be the pathogenic stage, and this does seem to occur most often during the initial stages of infection (see Section IV).

VII. PHYSIOLOGY Brumpt (1908) was the first to study the physiology of the blood stream forms of anuran trypanosomes. Noticing that T. inopinatum infections in hosts kept at low temperatures remained at low levels, he stated that cold inhibited reproduction in this species. A lack of a similar effect was reported in T. rotatorium. Later, Brumpt (1923a, b) found that treatment with Bayer 205 (Antrypol, Suramin) of frogs infected with the pathogenic strain of T. inopinatum resulted in complete remission, whereas no effect was reported on culture and leech forms of T. rotatoriurn. We have tried three trypanocides (“Antrycide” Quinapyramine Sulphate, “Antrycide” pro-salt and “Antrypol” Suramin B.P.”) on frogs infected with our types A, B and D (Fig. 2), all with negative results at even twice the recommended dose. What these results mean in terms of the physiology of the flagellates is difficult to determine in view of the wide range of inhibitory activity accredited to these substances (Hutner, 1964) but it seems probable that the selective effect on T. inopinatum is due to the high rate of division in this “species” as opposed to the apparent lack of such division in our morphs. Galliard et al. (1954) reported that growth hormone (= somatotropic hormone) inhibited mitosis in T. inopinatum. Mason reported that the DNA inhibitor mitomicin had no effect on the infection levels of T. rotatorium, whereas, as one might expect, puromycin and actinomycin did decrease the levels. Rattig (1875) studied the effects of a variety of common chemicals (e.g. NaCI) on frog trypanosomes. Ptrez-Reyes and Streber (1968) reported that T. montezumue and T. gulbae possessed glycogen reserves and alkaline phosphatase activity, both increasing in extent with the age of the flagellates. Doflein (1913) reported the presence of lipid inclusions in the bloodstream forms of T. rotatorium. This report has been substantiated by Bardsley (1972). These lipid inclusions proved to be metabolically active reserves in several morphs of trypanosomes from the bullfrog (Bardsley, 1972), being increased in relative size and number by a 1 h incubation in a glucose-Ringer medium, and decreased after similar treatment without the glucose. Bardsley also found that adding noradrenaline (in concentrations comparable to those reported in the blood) to the glucose medium resulted in a decrease in the extent of lipid inclusions, as did adding aminophylline. From these results Bardsley postulated that these anuran trypanosomes possess an adrenergic control system of lipolysis, possibly involving cyclic AMP as a second messenger, and that this system appears to function to ensure an adequate nutritional supply to the trypanosomes during

* The trypanocides were kindly supplied gratis by Imperial Chemical Industries Ltd., Cheshire, England.

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35

adverse conditions, for example, hibernation of the host. These direct effects of noradrenaline also fit into the model of the system regulating variations in peripheral parasitaemia (Section VIA), the catecholamines released seasonally, and by excitation, temperature, etc., affecting release from attachment in storage centres. This seems to be a good example of a parasite evolving as an allosomatic cell in a host and responding to its hormones and metabolites along with the host’s own cells. Another example may also come from this work. Bardsley (1972) showed that insulin effected a decrease in the extent of the lipid inclusions when added to the glucose medium. These results may substantiate those of Harvey (1948) on the inhibition of glucose uptake by insulin in T. hippicum. We are presently examining these inclusions to find if there is any seasonal cyclicity in the extent of these reserves similar to that seen in Opalina ranarum (von Brand, 1952), and if so, if there is any correlation between that cycle and the seasonal cycles in the physiology of the host. Certainly the finding of reserves in anuran trypanosomes explains why we can retain these flagellates in vitro without a suitable exogenous energy source for more than 36 h, a virtual impossibility with mammalian trypanosomes.

VIII. THEINVERTEBRATE HOST One of the most prevalent sanguivorous ectoparasites of the Anura are the leeches (Hirudinea). Many species of these annelids feed preferentially on poikilotherms, especially on the softbodied Amphibia (Mann, 1962; Moore, 1901; Nachtrieb et al., 1912; Sawyer, 1972). The first researcher to link the leech with anuran trypanosomiases was Billet (1904). He observed that T. inopinatum developed readily in the digestive cavity of the leech HeIobdella (=Batracobdella) algira. Noting also that this leech was a common ectoparasite of Rana esculenta in Algeria, Billet postulated that it was the natural vector of T. inopinatum. Brumpt (1906b) confirmed Billet’s theory by successfully transmitting the trypanosome to R. esculenta using this leech. Since then, several different species of leech have been identified as the invertebrate hosts of anuran trypanosomes (see Table 111). Many other leech species have been reported from various anurans by Walton, including the Bufoninae (1946a), the Hylidae (1946b, 1947a) and the Ranidae (1947b, 1948a, by 1949b, c, e, 1950a), but unfortunately have never been examined for trypanosomes. Finally, leeches have been established as vectors of various species of trypanosomes from different species of Caudata (Lehmann, 1952, 1958; Nigrelli, 1929). The development of ingested trypanosomes in the leech was studied by Buttner and Bourcart (1955a) for T. inopinatum in H. algira. They found development in the caeca, followed by an anteriad migration into the proboscal sheath, where metacyclic forms could be found. The presence of metacyclic trypomastigote forms has also been described for T. leptodactyli in Placobdella brasiliensis and in Placobdella catenigera (Brumpt, 1914), and for T. rotatorium in Placobdella ceylonica (Pujatti, 1953). Pujatti (1953) also found trypomastigote forms in the gut. Diamond (1958) described two distinct

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types of metacyclic trypanosome for T. pipientis in the crop of Placobdella (= Batracobdella) phalera. For a further discussion of reproduction and development in the invertebrate host see Section IVA, 2. The necessity for a cyclic development in the leech is well attested to by the failure of most attempts to infect frogs with trypanosomes taken from other frogs (e.g. Lebedeff, 1910b). Barrow (1953) in his work on T. diemyctyli of the newt established that Batracobdella picta was the invertebrate host of this system. He found that the high infection rate within the leech population was the result of more than simply leeches feeding on randomly encountered, infected hosts. Two other behavioural patterns of the leech were involved: (I) parasitism of one leech on another, and (2) the mothers carrying their young to a newt which would become infected by the mother, the infection in the newt subsequently being taken up by the young which remained attached to the host for 7-14 days. In most instances, the young become infective from the trypanosomiasis in the newt caused by the mother. Barrow could find no evidence of transovarial transmission like that reported by Brumpt (1907) for T. inopinaturn in H. algira. The feeding behaviour of B. picta on R . catesbeiana is restricted to a limited period of the year, this period coinciding with both the aquatic stage of the host and the annual peak in trypanosome parasitaemia (Bardsley, 1972). It is also at this time of year that stimulation of the frog (such as that caused by leech feeding) appears to have the greatest effect in releasing trypanosomes from the liver and other storage organs into the circulating blood (see Section VII). The parasitic relationship between leeches, trypanosomes and aquatic Anura appears to be far more complex and intricate in its physiological and behavioural adaptations than the host-parasite systems of mammalian trypanosomes, probably indicating a very old relationship (see Section IT). Support for this is found in the hypothesis that transmission can only be effected to the tadpole stage (Mendeleef-Goldberg, 1913 ; Noller, 19I 3). This hypothesis is based partly on the inability of several researchers to infect adult frogs using infected leeches (Creemers and Jadin, 1966; Fantham et al., 1942; Noller, 1913; and our own results included) while achieving success with tadpoles (Creemers and Jadin, 1966; Noller, 1913; Pujatti, 1953). Our results of studying our local suspected leech vector indicates that this system could be extremely complex, involving an uptake of infection from adult frogs, tadpoles or other leeches with the transmission to new tadpoles being effected only by juvenile leeches which contract the infection indirectly via their mother through the adult frog (Barrow, 1953). However, this system has certainly not been established definitively, and indeed, certain authors have infected adult frogs with culture forms (Diamond, 1958; Lebedeff, 1910; Noller, 1913b; our own results) and using infected leeches (Diamond, 1958). These may be specific examples different from the hypothesis under consideration, and thus, the latter deserves further study. The fact that leeches are usually restricted to aquatic habitats, and the fact that many mainly terrestrial Anura are heavily infected with trypanosonies, raises the possibility that other invertebrates may be capable of

THE TRYPANOSOMES

or

ANURA

37

sustaining anuran trypanosomes as well. A logical suspect would be haematophagous arthropods, and several authors have hypothesized their existence as vectors (Bardsley and Harmsen, 1969; Barrow, 1953; Mazza et al., 1927; Scorza and Dagert, 1958; etc.). In fact, Phlebotomus squumirostris has been established as a vector of T. bocagei of Bufo gargarizans in China (Feng and Chao, 1943; Feng and Chung, 1940), and more recently, Phlebotomus vexator occidentis has been shown to be the vector of T. bufophlebotomi of Bufo boreas in California (Anderson and Ayala, 1968; Ayala, 1971). In both of these cases the sandflies become infected by taking a blood meal from the toad, and subsequently transmit the infection by being ingested by an uninfected toad. However, sandflies are not the only arthropods that have been reported to feed commonly on various Anura. For example, we have seen mosquitoes feeding on basking frogs, and certainly this is not a new finding (Burgess and Hammond, 1961; Shannon, 1915; Walton, 1947b, 1949a, c, d ; Woke, 1937). Moreover, certain authors have established infections in various mosquitoes for up to 72 h (Bailey, 1962, in Aedes aegypti; PCrez-Reyes, 1967, in Culex quinquefasciatus). However, in both of these cases, and in another instance (Fantham et al., 1942), no transmission has been effected. This has prompted PCrez-Reyes (1970b) to dismiss the culicids as vectors of anuran trypanosomes. On the other hand, we concur with Barrow (1953) and Bailey (1962) that culicids cannot be disregarded as potential vectors, especially since the common, and sometimes exclusive, amphibian feeders have not been studied.* Other haematophagous Diptera have also been reported to feed on anurans (Walton, 1948a, 1949b; Noller, 1913a) and thus should be examined as possible invertebrate hosts. Of especial interest are those known trypanosome vectors such as the triatomid bug, Triatoma sanguisuga which has been reported feeding on hylid frogs (Walton, 1947b), and the tsetse fly Glossina tachinoides which has been observed feeding on anurans during those periods of the year when reptiles were not abundant (Lloyd et al., 1924). Indeed, G. tachinoides has been infected with T, rotatorium after feeding on Bufo regularis (Lloyd et al., 1924). PessBa (1969) has shown that the triatomid bug, Rhodnius prolixus would sustain T. rotatorium for 24 h, and T. leptodactyli for 72 h, after feeding on Leptoductylus ocellatus, although multiplication did not take place. The above-mentioned observations on insects show that some (Phlebotomus spp.) do indeed function as normal vectors of some species of anuran trypanosomes. The ease with which a variety of insects pick up and harbour anuran trypanosomes would suggest that under the right ecological circumstances a population of trypanosomes could establish a new relationship with a mainly terrestrial anuran(s) and an insect vector. Such ecological isolation of one clone from the main population, coupled with the new environmental demands, could lead to rapid speciation (see Section 11). By virtue of the frequency with which they are found on the Anura (Hoffmann-Mendizabel, 1965; Mazza et al., 1927; Parry and Grundmann, 1965), one other potentially important group are the larval Acarina. Walton

* See addendum on page 72.

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has published detailed lists of these parasites as found on the Bufoninae (1946a), the Hylidae (1946b, 1947a), the Ranidae (1947b, 1949a, c, d) and other Amphibia (1942, 1944). Their frequency as ectoparasites on toads makes them an important suspect, along with various arthropods, as vectors of the trypanosomes of terrestrial Anura. Ducceschi (1913) suggested that these arthropods should be studied as possible intermediate hosts for T. leptodactyli of Leptodactylus spp. in Argentina. In this respect it is interesting to note that the data collected by Brandt (1936) show an interesting correlation between the trypanosome parasitaemia and presence of the ectoparasitic mite Hannemania penetrans on Rana catesbeiana, Bufo fowleri and possibly Rana sphenocephala (as well as the correlation that he does draw between the parasitaemia and the presence of leeches on some of these anuran species). Hubert (1927) reported a seasonal cycle in the presence of H . penetrans in the skin of frogs. To the authors’ knowledge, larval Acarina have only been examined once for the presence of trypanosomes (Ducceschi, 1913) and a more extensive study is required. Although the bulk of the literature on the invertebrate host for anuran trypanosomes points to the leech as the principal vector in most instances, there are certain cases where this is not the case. For the aquatic anuran species an aquatic vector seems the most important ecologically, the leech being the most logical. However, for the more terrestrial species, especially those sustaining very high parasitaemias, a terrestrial vector seems more likely. Logical suspects here are the haematophagous arthropods, especially in the light of the work cited establishing Phlebotomus spp. as vectors of two species of toad trypanosomes. T. grylli from Acris gryllus (Nigrelli, 1944) and T. sp. that we find in dense infections in our local Hyla versicolor are strongly suspected as having arthropod vectors, since the ecology of these two situations is not really conducive to the leech being the principal vector. Moreover, the trypanosomes found in these two species are morphologically so distinct that they may be separate species (see Section 11) and thus may have a vector distinct from the leech. The existence of arthropod-borne trypanosomes in a group largely vectored by leeches may be an example of evolution in action (see Section 11).

1X. CULTURE A.

MEDIA

1. T. rotatorium

The first attempt to culture anuran trypanosomes met only with limited success (Lewis and Williams, 1905). The anuran blood-agar medium employed supported only feeble growth and only one subculture was successful. The following year, Bouet (1906), using essentially the medium of Novy and MacNeal for mammalian trypanosomes (= NN), easily cultured T. rotatorium from Rana esculenta. Bouet was the first to describe the development of the trypanosome in culture using both fresh and stained preparations. Mathis (1906), using a modification of the NN medium also got luxuriant cultures of T. rotatorium, as did Lebedeff (1910b) and Tobey (1906) using anuran,

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instead of rabbit blood in the medium. Doflein (1910, 1913) and MendeleefGoldberg (1913) did extensive work with cultures of T. rotatorium using the basic NN medium, describing the development of the culture forms in intricate detail. Noller (1913b, 1917) using a sheep’s blood modification of the NN medium has also done extensive work on the culturing of T. rotatorium. The basic N N medium was also successfully employed for T. rotatorium by Packchanian (1934). Ponselle (1917, 1923b) was the first to culture T. rotatorium on a medium distinct from NN. Cleveland and Collier (1930) describe 12 media, several of which supported growth of T. rotatorium. A brain-heart infusion medium was employed successfully by Creemers and Jadin (1966) whereas Ruiz and Alfaro (1958) found the basic Rugai leishmania1 medium satisfactory. Ptrez-Reyes (1966) grew cultures on a modification of Diamond and Herman’s (1954) SNB-9 medium (=SNB-T) using tryptone instead of neopeptone. Finally there is the comprehensive work of Fromentin on the culture requirements of T. rotatorium. In her earlier papers (1967, 1969) she developed a semidefined liquid medium which she later (1971), using Parker’s 199 as a base, converted to a defined medium which would support the trypanosomes for 21 days. 2. T. ranarum Diamond (1958) found T. ranarum easily maintained on either his own SNB-9 medium (Diamond and Herman, 1954) or Nicolle’s modification of the basic N N medium (=NNN). Wallace (1956) reported similar results but also found the diphasic medium superior to the monophasic. Using a lysed human red cell modification of SNB-9 (=SNBL) Lehmann (1966a) got enhanced growth of T. ranarum as compared to SNB-9. Halevy and Gisry (1964) developed a lactalbumin hydrolysate medium for this flagellate. Nakamura (1967a) found that his protein-free dialysate medium for T. cruzi would also support T. ranarum, as would his totally autoclavable one (1967b). Guttman’s (1963) defined medium for monogenetic trypanosomatids will also support T. ranarum (Taylor and Baker, 1968). 3. T. mega The culture requirements of a strain of T. mega isolated from Bufo regularis have been worked out. The initial medium for this trypanosome was developed by Bond and Steinert (1956) with a more detailed description of the preparation given by Steinert (1958b). Williams et al. (1966) cultured T. mega on a slight modification of this medium. Bonk et al. (1964) report that they have synthesized a defined medium which would support T. mega for at least a year, although the constituents are not given. Guttman (1967) has developed a defined medium for this trypanosome as well, using a modification of her defined medium for monogenetic trypanosomatids (1963).

4. Miscellaneous Ponselle (1923a) synthesized a hypotonic medium for T. inopinatum. This medium was successfully used by Galliard (1926), who later (1929) showed

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that the hypotonicity and acid pH (5.5) were essential for this trypanosome. T. inopinatum was also cultured by PCrez-Reyes (1966) using Rugai's potato medium. Brumpt (1928a) failed to culture T. neveu-lemairei of Rana esculenfa on either NNN or Ponselle's hypotonic media, even though T. costatum and T. rotatorium did grow on the former. He did succeed in culturing T. parroti, but the medium used is not disclosed. As well as T. parroti, Galliard (1929) also cultured T. sergenti on Ponselle's hypotonic medium. A modification of this latter medium was used by Diamond (1950) to culture T. pipientis. Diamond (1958) also cultured T. schmidti and T. chattoni on both monophasic and diphasic SNB-9 media, the latter trypanosome growing more readily on the monophasic form. PCrez-Reyes (1 967) successfully cultured T. montezumae and T. galbae on a tryptone modification of SNB-9 (= SNB-T), also achieving variable results with Rugai's potato medium for both, and blood-agar for T. galbae. He also cultured T. diamondi, T. grandis, T. Ioricatum and T. sp. (=T. rofatoriun??) on both blood-agar and SNB-T; he could not culture T. chattoni on any medium tried, including the SNB-9 with which Diamond (I 958) achieved such ready success. The toad trypanosome, T. bocagei, was cultured by Lebailly and Caillon (1919) on NNN medium. Similar results were obtained by Ayala (1971) with T. bufophlebotomi, but he has also achieved good results with Senekjie's leishmania1 agar. Finally, we have cultured our local trypanosomes on a dried beef blood modification of the SNB-9 medium, and on a prepared trypticase soy broth medium. We have never been able to culture any trypanosomes on the Bonk and Steinert (1956) medium for T. mega. B.

PHYSIOLOGY A N D BIOCHEMISTRY

Temperature has been shown to affect the cultures of anuran trypanosomes. Lehmann (1962) found that the cycle of T. runarum was normal from 9 to 20°C, with lower temperatures causing a compression of the cycle, and higher ones eventually leading to a decrease in numbers and finally death. Fromentin (1970) found that she could maintain cultures of T. rotatorium at 35°C if glycerol was added to the medium. Along this line, Galliard (1929) found that T. inopinatum could withstand alterations in pH and salt concentrations if glucose was added to the medium. Doflein (1913) reported that various modifications in the medium growing T. rotatorium would alter the morphology of the culture forms, but the modifications and the alterations that they create are not specified. Anuran trypanosomes apparently do not possess all the enzymes required for their survival in culture (and probably thus in the invertebrate host as well). Fromentin (1967, 1969) found that T. rofaforiumneeded erythrocytes in the medium, and postulated that the substances supplied were enzymes. She later (1971) narrowed down the requirements to enzymes utilized at about the level of glucose-6-phosphate dehydrogenase in the first part of the glycolytic cycle, probably involving NAD or NADP. T. ranarum has been found to possess succinic, malic, and lactic dehydrogenases (Lehmann and Claflin, 1965). I t appears that enzyme requirements may also vary with the

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morphology of the flagellates. Lehmann and Sorsoli (1962) found that the non-dividing “slender” forms of T. ranarum had a 47% higher oxygen consumption than the dividing “pear” forms, and that inhibition of succinic dehydrogenase in the latter caused only a 27% depression, as compared to 61 % in the former. They concluded that the “slender” forms utilize Krebs’ cycle the most. Oxygen consumption in both types was dependent on the presence of the glucose component of the SNB-9 medium employed. However, oxygen consumption of several species of trypanosomes from Mexican frogs was increased by adding tryptose to the same medium instead of neopeptone (PCrez-Reyes, 1967). The latter author also found that all species tested were both cyanide- and iodoacetate-sensitive. Concerning substrate utilization, Lehmann (1963a) found that T. ranarum metabolized galactose most readily, but would also use a variety of other polysaccharides to varying degrees. These observations appear in contrast to those of Noguchi (1926) who found that T. rotatorium would not ferment any of the carbohydrates supplied. The biochemical and histochemical characterization of anuran trypanosomes is in its first throes. Doflein (1913) was the first to notice that culture forms of T. rotatorium contained discrete lipid “granules”, the extent depending on composition of the medium. Lipid droplets were subsequently demonstrated in T. mega by Steinert (1964). This species has also been shown to possess both ergosterol and cholesterol (Williams et al., 1966). Halevy and Gisry (1964) found that T. ranarum culture forms also contained a variety of lipids, including sterols (mostly ergosterol), free fatty acids, and monoand tri-glycerides. In contrast to the blood stream forms, PCrez-Reyes and Streber (1968) could find no glycogen or alkaline phosphatase in the culture forms of several species of anuran trypanosomes. On the other hand, Lehmann (1963b) found both alkaline and acid phosphatases in T. ranarum culture forms. It is obvious that the composition of the culture media and conditions of culturing will have a considerable effect on reproductive patterns displayed by the cultural trypanosomes. For a discussion of reproduction in culture media see Section IVA, 3. For a description of morphological and cytological aspects of culture morphs see Section IIIB. C.

COMMENTS

The cultural requirements of various anuran trypanosomes, in terms of nutritional needs and other factors such as pH, osmolarity, etc., appear to be readily available adjuncts to morphology and other properties in the establishment of species status (see Section 11). For example, T. mega has markedly different cultural requirements from most other anuran trypanosomes, and this fact, coupled with its original isolation from African toads and its distinct morphology indicates that this may indeed be a good species (and also suggests looking for a possible terrestrial invertebrate host). However, whether the relatively minor cultural differences between media for T. inopinatum and T. rotalorium is indicative of separate species status, or merely indicates the differences required by various stages in a poly-

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morphic life cycle, is open to question. On the other hand, similarities in culture requirements (e.g. like those between T. inopinatum and T. pipientis) is not necessarily indicative of species congruity. Thus, although this is a valid informational unit, it is only truly constructive when taken with other equally important criteria for species separation. By the same token, the cultural similarities and differences may tell us something of the relationship of anuran trypanosomes to other vertebrate and invertebrate flagellates. For example the identical cultural requirements for urodelan and anuran trypanosomes (Lehmann, 1959b) may obviously be taken as indicating a close phylogenetic relationship. However, do the slight differences in the defined media for Crithidia, Blastocrithdia, T, ranarum and T. mega indicate a close relationship between anuran trypanosomes and insect flagellates? Also, what does the fact that T. cruzi and T. ranarum can be cultured on identical media indicate (Lehmann, 1966a)? This is an especially germane question when one considers that T. rotatorium has recently been sustained in a reduviid bug (Pessaa, 1969). Further research in this area, including biochemical comparisons among the various flagellates would be most revealing as to the possible phylogeny of the flagellates.

X. CONCLUSION Research on anuran trypanosomiasis witnessed a height of activity during the first 15 years of this century; after that period interest in the field declined, to revive during the 1950s. Unfortunately, the early work, however voluminous, lacks the descriptive precision to be of much value today, and the more recent work tends to be of a highly specialized nature. Modern concepts of taxonomy, genetics, cytology and cell physiology and biochemistry, for instance, have yet to be fully applied to the study of anuran trypanosomes. As a consequence of this state of affairs, the classification of anuran trypanosomes is still in its infancy, and their relationship to other trypanosomes is not understood. The morphology and cytology of these organisms is the best documented area of their biology, but even here we find too many conflicting reports to synthesize generally acceptable generalizations. The same can certainly be said for their life cycles, reproductive patterns and their polymorphism. Recent work on the physiology and dynamics of host-parasite relationships has greatly extended our understanding of anuran trypanosomiasis, but the physiology and biochemistry of the individual trypanosomes is still very much a closed book. A number of general conclusions and pointers towards identifiable problems can be recognized, and have been stressed in the foregoing pages. In short, the following points of particular interest have emerged from this review of the field: 1. A reliable taxonomic classification of anuran trypanosomes is badly needed, but constructing such a classification will be a very difficult, and at best, an arbitrary task, because of the asexual, clonal reproduction of these organisms. We have proposed a dynamic model of classificationwhich can grow as further research data come in, without the need of regular total revisions.

T H E T R Y P A N O S O M E S OF A N U R A

43

2. Considering the phylogeny of the genus Trypanosoma without fully understanding the relationship between the trypanosomes of higher and lower vertebrates appears to be a rather futile exercise. A number of phylogenetic proposals are discussed in Section II, and we have tried to bring the various opinions together after stressing the important place anuran trypanosomes occupy in the genus. 3. It is surprising that the powerful eye of the electron microscope has not yet been focused on the fine structure of the blood-stream forms of anuran trypanosomes. The large size and cellular complexity of these trypanosomes is probably a primitive complexity; the simpler structure of mammalian trypanosomes a secondary reduction. A study of the fine structure of anuran trypanosomes could very well reveal much about the origin of the less well understood structures of mammalian trypanosomes. 4.Reproductive patterns appear to be very complex. This complexity, as well as the existing taxonomic muddle, must be responsible for the total lack of consensus on the topic of reproduction. We feel that a general pattern will emerge if and when adequate research will seek the correlations between environment, timing and genetic background. 5. Polymorphism is another controversial phenomenon among anuran trypanosomes. The term has been diversely defined in the past, and again, the taxonomic muddle has not helped in this matter. Indeed, no work to date has attempted to provide experimental evidence for or against the various theories on the function of this polymorphism. 6. It appears from extracting the literature that trypanosomes are found in nearly all populations of Anura, in all regions of all the major life zones. This ubiquitous distribution is interpreted as an indication of a very old age for this group of organisms. 7. The dynamics of the relationship between the trypanosomes and the vertebrate host has recently become a well documented and well modelled topic of research. We have attempted to integrate existing scientific knowledge of anuran physiology, anuran ecology, the ecology of the invertebrate host and the behaviour and physiology of the anuran trypanosomes into a dynamic model of host-parasite interrelationship. We have derived great encouragement from our comparing this model with a similar model constructed by Dr D. F. Mettrick of Toronto (personal communication) based on parasitic helminths. The similarity between the two models is striking. 8. Past studies of pathogenicity and physiological behaviour of anuran trypanosomes have been performed and interpreted too much in isolation. We hope that the above-mentioned model will lead to a greater co-ordination of future research data, and will stimulate further research workers to study such topics as pathogenicity within a broader context. 9. The recent report of a possible endogenous adrenergic control system in an anuran trypanosome is a most interesting finding. Not only does it tie in neatly with the above-mentioned host-parasite model, it also points out the advantages of a broad, integrating approach in parasitic protozoology. More comprehensive research in this system in anuran trypanosomes in the

44

J. E. B A R D S L E Y A N D R. H A R M S E N

direction of the biochemical and physiological studies by Dr J . J . Bluni of Duke University on Tetraliymenaand Crithidia is indicated. We wonder to what extent such endogenous control systems have survived the adaptation of mammalian trypanosomes to their warmblooded hosts. 10. Leeches must be considered the primary vector in anuran trypanosomiasis. Yet, the reports of various insect vectored species are of particular interest, in that these incidences underline the important place anuran trypanosomes occupy in the genus. 11. Culturing of anuran trypanosomes in artificial media has been successfully done for many years. Only recently, however, have such cultures been used as experimental subjects for a study of reproduction, nutrition, and metamorphosis. Many outstandingly important questions may be approached in this way. We hope that this review of the trypanosomes of Anura will stimulate more intensive and more effective research in this field. It would be particularly rewarding for us if we could feel, sometime in the future, that drawing the attention of the students of human trypanosomiasis away from their own immediate subject had resulted in eventual advances in that subject.

TABLEI List of all published specific and subspecific names of anuran trypanosomes with hosts and authors w

Name T. tumida T. bufophlebotomi T. somalense T. sergenti T. parroti T.neveu-lemairei T. ocellati T. celestinoi T. leptodactyli T. ranarum T. pipientis T. schmidti T. mega T. karyozeukton T. loricatum vel costaturn T. lavalia T. gaurnontis T.montrealis T.hylae T. bocagei T. undulans T. elegans T. loricatum T. costatum T. rotatorium Paramecioides costatus T. sanguinis T. clelandi T. parvum Undulina ranarum

Host Rana nutti Bufo boreas halophilus B. reticulatus Discoglossus pictus Discoglossus pictus R. esculenta Leptodactylus ocellatus Leptodactylus ocellatus L. ocellatus R. esculenta R. pipiens R. sphenocephala Frog Frog Anura B. americanus B. americanus B. americanus Hyla arborea B. regularis R. esculenta R. esculenta R. esculenta R. esculenta R. esculenta Anura Frogs Lymnodynastes spp. R. clamitans R. esculenta

Author Avkrinzev, 1916 Ayala, 1970 Brumpt, 1906a Brumpt, 1923c Brumpt, 1923c Brumpt, 1928a Brumpt, 1936 Brumpt, 1936 Carini, 1911 Danilewsky, 1889 Diamond, 1950 Diamond, 1958 Dutton and Todd, 1903 Dutton and Todd, 1903 Dutton et al., 1907 Fatham et al., 1942 Fatham et al., 1942 Fatham et al., 1942 Franqa, 1908b Franqa, 1911a FranCa and Athias, 1906b Franqa and Athias, 1906b Franqa and Athias, 1906b Franqa and Athias, 1906b Franqa and Athias, 1906b Grassi, 1881 Gruby, 1843 Johnston, 1916 Kudo, 1922 Lankester, 1871

TABLEI (continued) Name

T. nelspruitense T. aurorae T. boyli Monas rotatoria T. borrelli T. bocagei var parva T. bocagei var magna T. chattoni Paramaecium loricatum Paramaecium costatum Amoeba rotatoria T. arcei T. belli T. grylli T. hendersoni T.galba T.grandis T. diamondi T. montezumae T. innominatum T. inopinatum T. rotatorium var nana T.sanguinis ranarum T. clamatae Trypanosomata rotatorium T. varani T. rotatorium major T. striatum T. rotatorium Iiscia T. rotatorium striata T. canadensis

Host

R. angolensis R. aurora R. boyli boyli R. esculenta ? H. sp. B. melanostictus B. melanostictus B. melanostictus R. esculenta R. esculenta R. esculenta L. ocellatus R. esculenta ? Acris gryllus R. spp. R. spp. R. pipiens R. pipiens R. spp. Anura? R. esculenta R. esculenta R. and H. spp. R. clamata R. clamata B. regularis H. raddiana R. esculenta Frogs Frogs R. pipiens

Author Laveran, 1904 Lehmann, 1959a Lehmann, 1959b Lieberkijhn, 1870 Marchoux and Salimbeni, 1907 Mathis and Gger, 1911a Mathis and Lkger, 1911a Mathis & Gger, 1911b Mayer, 1843 Mayer, 1843 Mayer, 1843 Mazza et al., 1927 Nabarro, 1907 Nigrelli, 1944 Patton, 1908 Pkrez-Reyes, 1968 PCrez-Reyes, 1969a Pkrez-Reyes, 1969a PCrez-Reyes et al., 1960 Pittaluga, 1905 Sergent and Sergent, 1904 Sergent and Sergent, 1905 Shalashnikov, 1888 Stebbins, 1907 Tobey, 1906 in Walton, 1946 in Walton, 1947 in Walton, 1947-50 in Walton, 1951 in Walton, 1951 Woo, 1969b

TABLE II Distribution of anuran trypanosomes by geographic region and host Species of Trypanosoma

Palaearctic Region bocagei bocagei costatum costatum hylae innominatum inopinatum inopinatum inopinatum neveu-lemairei parroti ranarum rofatorium rotatorium rotatorium rotaforium rotatorium rotatorium rotaforium rofatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium

Host species

Bufo mauritanicus B. bufo Rana esculenta Rana esculenta Hyla arborea R. sp. R.esculentu R.esculenta R. esculenta R. esculenta Discoglossuspictus R. esculenta Frog R. esculenta R. esculenta R. esculenta H. arborea meridionalis R. ridbunria R. esculenta R. esculenta H. arborea H. aborea R.esculenta R. esculenta R. temporaria R. rugosa

Locality Tunisia N. China Portugal Corsica Italy Spain Algeria Portugal Algeria Corsica Algeria Germany France Caucasus Portugal Corsica

Europe

U.S.S.R. Italy Italy Italy Danube Valley Italy Japan Japan Japan

Author Lebailly and Caillon, 1919 Feng and Chung, 1940 Franpa, 1908a Brumpt, 1928a Franqa, 1908b Pittaluga, 1905 Sergent and Sergent, 1904, 1905 Franm, 1908a Billet, 1904 Brumpt, 1928a Brumpt, 1923c Lankester, 1871 Bouet, 1906 Finkelstein, 1907 Franqa, 1908a Brumpt, 1928a Franqa and Athias, 1907 Glushchenko, 1961 Acanfora, 1939 Babudieri, 1931 Babudieri, 1931 IvaniE, 1936 Jacono, 1935 Koidzumi, 1911 Koidzumi, 1911 Koidzumi, 1911

ei

2 ei

w

*:

w

9

z

z

0

5

v1

0 +rI

's

z

C

*!=

t

TABLEI 1 (continued) ~

Species of Trypanosoma

~~

Host species

Locality

Palaearctic Region (continued) rotatorium rotatorium rotatorium rotatorium

R. esculenta R. temporaria Toad B. regularis

QYPt

rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sanguinis sergenti

R. esculenta R. nigromaculata R. esculenta B. bufo 3.viridis R. terrestris R. esculenta R. temporaria H. arborea R. esculenta R. esculenta H. viridis 3.vulgaris R. temporaria R. esculenta H. arborea B. viridis R. sp. R. esculenta R. temporaria H. arborea D. pictus

Germany Japan Algiers Europe Europe Europe Russia Russia Russia France Italy Italy Italy Hungary Hungary HumY Hungary s. Italy N. Russia N. Russia N. Russia Algeria

Central Russia Central Russia Central Russia

Author Lebedeff, 1910b Lebedeff, 1910b Lebedeff, 1910b Mohammed and Mansour, 1959a, b, 1966 Noller, 1913a, b Tanabe, 1931 Sergent and Sergent, 1904, 1905 Walton, 1946 Walton, 1946 Walton, 1947-50 Danilewsky, 1885 Danilewsky, 1885 Danilewsky, 1885 Dollfus, 1961 Grassi, 1881, 1883 Grassi, 1881, 1883 Grassi, 1881, 1883 Koninski, 1901 Koninski, 1901 Koninski, 1901 Koninski, 1901 Kruse, 1890 Shalashnikov, 1888 Shalashnikov, 1888 Shalashnikov, 1888 Brumpt, 1923c

? m W

9

21

: P

m z

Indian Region SP.

SP. SP. SP.

SP. SP.

SP. SP.

belli bocagei magna bocagei parva borreli chattoni elegans elegans hendersoni henakrsoni rotatorium rotatorium rotatorium rotatorium rotatoriwn rotatorium rofatorium

R. tigrina R. Iimnocharis R. tigrina R. temporaria Polvpedates Ieucomystax Pobpedates Ieucomystax Pobpedates kucomystax Microhyla pulchra R sp. B. melanostictus B. mehnostictus R. tigrina B. melanostictus R. Iimnocharis R. guntheri R. tigrina R. hexidactyla R. esculenta R. plancyi R. cyanophlyctis R. tigrina R. escuienta R. tigrina Frog

India India Ceylon Hong Kong Ceylon, India Java, Sumatra Indochina China, Indochina Hong Kong Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Formosa Formosa S. India S. India Hong Kong Vietnam Vietnam

Berestnev, 1902 Berestnev, 1902 Dobell, 1910 Nabarro, 1907 Walton, 1950 Walton, 1950 Walton, 1950 Walton, 1950 Nabarro, 1907 Mathis and LKger, 191la Mathis and Uger, 1911a Mathis and Uger, 191lc Mathis and Uger, 1911b Mathis and Eger, 191lc Mathis and Uger, 1911c Patton, 1908 Patton, 1908 Ogawa and Uegaki, 1927 Ogawa and Uegaki, 1927 Pujatti, 1953 Pujatti, 1953 Hunter, 1908 Mathis and Leger, 191l b Mathis and Uger, 1911c

B. regularis B. regularis Hylambates murmoratm B. regularis B. regularis

Fr. O d d . Afr. Transvaal Afr.

Bouet, 1909 Fantham et al., 1942 Walton, 1950 Wenyon, 1908 Franrp, 1911a

Ethiopian Region SP. SP. SP.

SP.

bocagei

Sudan Port. Guinea

0 -J

3-

2:

C

w

9

cn

TABLEII (continued) Species of Trypanosoma

Ethiopian Region (continued) elegans karyozeukton karyozeukton karyozeuk ton karyozeukton karyozeukton karyozeukton karyozeukton loricatum loricatum loricatum mega mega mega mega mega mega mega mega mega mega nelspruitense nelspruitense nelspruitense rotatorium rotatorium rotatorium

Host Species

B. regularis R. spp. B. regularis Frog Frogs R. oxyrhynchus B. regularis R. mascarensis R. galamensis R. oxyrhynchus R. mascarensis Frogs R. spp. B. regularis Frog B. regularis B. regularis Frogs R. oxyrhynchus B. regularis R. tuberculosa R. angolensis R. angolensis R. theileri Toad B. obstetricans Xenopus laevis

0

Locality Congo Senegambia Angola Congo Congo Congo Congo Congo Senegambia Gambia Gambia Congo Senegambia Angola Congo Nigeria Congo Congo Congo Congo Afr. Transvaal Transvaal Transvaal Sudan Afr. S. Africa

Author Martin et al., 1909 Dutton and Todd, 1903 Francp, 1925 Martin et al., 1909 Rodhain, 1907 Schwetz, 1930 Schwetz, 1930 Schwetz, 1944 Dutton et al., 1907 Dutton et al., 1907 Dutton et al., 1907 Broden, 1905 Dutton and Todd, 1903 FranGa, 1925 Kerandel, 1909 MacFie, 1914 Martin et al., 1909 Rodhain, 1907 Schwetz, 1944 Schwetz, 1944 Walton, 1947-50 Laveran, 1904 Nabarro, 1907 Nabarro, 1907 Balfour, 1908 Chaussat, 1850 Fantham et al., 1942

?

P td 9 td

b v) P

m

4 9

2: U

3: 9 Y

3 v)

m

21

rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium sanguinis somalense tumida

s.Africa

R. fuscigula Hyperolius sp. Ptychadena parroti Frog B. regularis B. regularis Frogs B. regularis R. fuscigula Frogs R. occipitalis R. albilabris R. occipitalis R. oxyrhynchus Leptopelis sp. R. mascarensis B. regularis R. trinodis B. reticulatus R. nutti

Bugala Island Sp. Guinea Congo Nigeria Nigeria Nigeria Congo Congo Congo Afr. Congo Congo Congo Congo Congo Sudan Senegambia Somalia Usmabara

Fantham et al., 1942 Hoare, 1932 Holberton, 1966 Kerandel, 1909 MacFie, 1914 Lloyd et a[., 1924 Lloyd et al., 1924 Martin et al., 1909 Wrez-Reyes, 1967 Rodhain, 1907 Rousselot, 1953 Schwetz, 1930 Schwetz, 1930 Schwetz, 1930 Schwetz, 1930 Schwetz, 1944 Stevenson, 1911 Dutton and Todd, 1903 Brumpt, 1906a Avkrinzev, 1916

H. nasuta H. lesueurii Limnodynastes ornatus L. tasmaniensis H. infrafrenata Asterophrys spp. Oreophryne sp. Platymantis papuensis R. papua L. tasmaniensis

Queensland Queensland Queensland Queensland New Guinea New Guinea New Guinea New Guinea New Guinea Queensland

Bancroft, 1891 Cleland and Johnston, 1910 Cleland and Johnston, 1910 Cleland and Johnston, 1910 Ewers, 1968 Ewers, 1968 Ewers, 1968 Ewers, 1968 Ewers, 1968 Johnston, 1916

Australian and Oceanic Region SP. SP.

SP. SP. SP.

SP. SP. SP.

SPclelandi

1

J: m

TABLE XI (continued) ~

Species of Trypanosoma

Australian and Oceanic Region (continued) clelandi L. orantus clelandi clelandi rotatorium

~

Host species

L. tarmaniensis L. ornatus L. tasmaniensis

Locality Queensland Austr. Austr. S. Austr.

~

Author Johnston, 1916 Mackerras and Mackerras, 1961 Mackerras and Mackerras, 1961 Cleland, 1914

Nearctic Region SP. SP.

SP.

SP. SP. SP. SP.

SP. aurorae boyli bufophlebotomi canadensis chattoni chattoni

diomondi gabae galbae

galbae gawnontis gawnonfis grandis

B. boreas R. pretiosa H. versicolor H. andersoni R. pipiens H. crucifer H. arenicolor H. sp. R. aurora R. boyli B. boreas R. pipiens R. pipiens R. pipiens R. pipiens R. montezumae R. pustulosa R. palmipes B. americanus R. pipiens R. pipiens

California N.W. U.S.A. Georgia Georgia Georgia Georgia Utah Louisiana Oregon California California

Ontario Minnesota Mexico Mexico Mexico Mexico Mexico Quebec Ontario Mexico

Anderson and Ayala, 1968 Clark et al., 1969 Nigrelli, 1945 Nigrelli, 1945 Nigrelli, 1945 Nigrelli, 1945 Parry and Grundmann, 1965 Schmidt, 1878 Lehmann,1959a Lehmann, 1959b Ayala, 1970 Woo, 1969 Diamond, 1958 Perez-Reyes, 1967 Perez-Reyes, 1967 Pkrez-Reyes, 1967 Perez-Reyes, 1967 Perez-Reyes, 1967 Fantham et al., 1942 Woo, 1969 Perez-Reyes, 1967

>

z

c)

v1

rn 2

grylli inopinatwn inopinatum karyozeukton lavalia loricatwn montezuntae montezumae montezumae montrealis parvwn pipientis pipientis pipientis pipientis ranarum ranarwn rotatorim rotatoriwn rotatorium rotatorium rotatorium rotatoriwn rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatoriwn

Acris gryllus R. pipiens R catesbeiana A. gryllus B. americanus R. pipiens R. montezumae R. pustulosa R. palmipes B. americanus R. clamitans R. pipiens R. sylvatica R pipiens R. sylvatica R. clamitans R. pipiens R. clamitans R. catesbeiana R. sphenocephala Pseudacris brimleyi H. crucifer B. woodhouseii H. versicolor R. catesbeiana R. clamitans R. clamitans R. pipiens R. catesbeiana R palustris R. areolata R. catesbeiana

Georgia Quebec Quebec Louisiana Quebec Mexico Mexico Mexiw Mexiw Quebec U.S.A. Minnesota Minnesota Ontario Ontario Ontario Minnesota Louisiana N. Carolina N. Carolina N. Carolina N. Carolina Virginia Virginia Virginia Virginia Quebec Quebec Quebec Maryland Louisiana Louisiana

Nigrelli, 1945 Fantham et al., 1942 Fantham et al., 1942 Lauter, 1960 Fantham et al., 1942 Piez-Reyes, 1967 P&-Reyes, 1967 Pkrez-Reyes, 1967 Pkrez-Reyes, 1967 Fantham et al., 1942 Kudo, 1922 Diamond, 1950, 1958 Diamond, 1950 Woo, 1969 Woo, 1969 Woo, 1969 Diamond, 1958 Bollinger et al., 1969 Brandt, 1936 Brandt, 1936 Brandt, 1936 Brandt, 1936

cf cf

P

*a

>

z

0 rA 0

E rA

0

w

Campbell, 1968 Campbell, 1968 Campbell, 1968 Campbell, 1968 Fantham et al., 1942 Fantham et al., 1942 Fantham et al., 1942 Laird, 1951 Lauter, 1960 Lauter, 1960

VI

w

VI

TABLE II (continued) Species of Trypanosoma

Host species

Nearctic Region (continued) rotatorium rotatorium rotatorium rotaforium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium sanguinis schmidti

H. cinerea H. crucifer P. nigrita R. catesbeiana R. clamitans H. lafrentzi Phyllomedusa dacnicolor B. compactilus B. fowleri Ensatina eschscholtzii R. cutesbeiana Rappia marmota R. mugiens R. sphenocephala

Neotropical Region SP. SP. arcei borelli celestinoi leptodactyli

Frog B. marinus L. oscellatus H. luteristriga L. ocellarus L. ocellatus

R. clamitans R. palustris R. sphenocephala A. g r y l h H. avivoca

P

Locality Louisiana Louisiana Louisiana Louisiana Louisiana Louisiana Louisiana Louisiana Georgia Georgia Mexico Mexico U.S.A. U.S.A. U.S.A. Ontario ? Quebec

Author

FIOrida

Lauter, 1960 Lauter, 1960 Lauter, 1960 Lauter, 1960 Lauter, 1960 Lauter, 1960 Lauter, 1960 Lauter, 1960 Nigrelli, 1945 Nigrelli, 1945 PLrez-Reyes, 1967 PCrez-Reyes, 1967 Walton, 1946 Walton, 1946 Walton, 1963 Woo, 1969 Kudo, 1922 Osler, 1883 Diamond, 1958

Cuba Peru Argentina Brazil Brazil Brazil

Lebredo, 1903 Lehmann, 1966b Mazza et al., 1927 Marchoux and Salimbeni, 1907 Brumpt, 1936 Brumpt, 1914; Carini, 1911

leptodactyli mega ocellati rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatoriwn rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium rotatorium

L. ocellatus H. venulosa L. ocellatus H. raddiana L. gracilis L. ocellatus L. ocellatus H. venulosa R. pipiens R. warschewitschii H. crepitans L. bolivianus Phyllomedusa bicolor H. rubra Ceratophrys ornata L. bufonis P. sauvagii B. arenarum H. raddiana Lepidobatrachus asper L. ocellatus

Argentina Venezuela Brazil Argentina Argentina Brazil Argentina Argentina Costa Rica Costa Rica Venezuela Venezuela Venezuela S.A. Argentina Argentina Argentina Argentina Argentina Argentina Argentina

Mau;i et al., 1927 Scorza and Dagert, 1955 Brumpt, 1936 Jorg, 1933, 1936 Jorg, 1936 Machado, 191 1 Mazza et al., 1927 Plimmer, 1912 ? ?

Scorza and Dagert, 1955, 1958 Scorn and Dagert, 1958 Scorza and Dagert, 1958 Walton, 1947a Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949 Vucetich and Giacobbe, 1949

TABUIII Hirudinid vectors of anuran trypanosomes (Post.-postulated* ;Est.-established vectorship; Exp.-experimental (non-natural) infection)

Species of leech

Species of Trypanosome

Helobdella algira Helobdella algira Placobdella brasiliensis

T. inopinatum T. inopinatum T. leptodactyri

P. catenigera

T. leptodactyli

H. algira H. algira

T. costatum T. rotatorium T. inopinatum T. inopinatum

H. algira

T. inopinatum

Hemiclepsis marginata and Piscicola geometra Leech sp.

T. rotatorium

Hirudo lnedicinalis Clepsina sp.

T. rotatorium

T. rotatorium

T. rotatorium

species of vertebrate host involved

Gmgraphic locality

Post. Est.

Exp. Reference -

Billet, 1904 Brumpt, 1906b Brumpt, 1914

Rana esculenta Rana esculenta Leptodactylus ocellatus Leptodactylus ocellatus R. esculenta

Algeria Algeria Brazil

Discoglossus pictus

Algeria

R. esculenta R. temporaria Tadpoles

Portugal Europe

X

Noller, 1913a

R. esculenta R. temporaria R. esculenta

Central Russia Europe

X

Lebedeff, 1910b

Rana tigrina R. hexidactyla

India

X X X

Brazil

X

Portugal

Brumpt, 1914

Frantp, 1908a, c

X

Galliard et al., 1953

X X

X X

LabM (Noller, 1913b) Patton, 1908

Placobdella ceylonica

T. rotatorium

Macrobdella sp. Macrobdella ditetra Placobdella marginata Placobdella sp. Placobdella phalera Batracobdellapicta

T. rotatorium T. rotatorium T. rotatorium T. canadensis T. pipientis T. rotatorium (sp. complex)

Haementeria lutzi Glossiphoniacomplanata

T. rotatorium T. rotatorium

Glossiphonia complanata

T. leptodactyli

Frogs and tadpoles of Southern India R. tigrina R. cyanophlyctis R . catesbeiana North America R. catesbeiana North America Anura North America R. pipiens Ontario R. pipiens Minnesota Rana pipiens Ontario Rana catesbeiana Rana clamitans

x

x

Pujatti, 1953

(frogs) (tadpoles) X X

X X X X

X

Hyla crepitans

Venezuela

X

Leptoductylus bolivianus

Venezuela

X

Nigrelli, 1945 Brandt, 1936 Kudo, 1966 Woo, 1969 Diamond, 1950 Bardsley, 1972 Pinto, 1921 Scorza and Dagert, 1958 Scorza and Dagert, 1958 0

Postulated vectors are based on infections being found in the leeches and other supporting data, but without de6nitive proof of transmission.

5

M

0 +I7

> z

C

;d

>

58

J. E. B A R D S L E Y A N D R. H A R M S E N

REFERENCES Acanfora, G. (1939). Sul Tripanosoma rotatorium. Archo ital. Sci. med. colon. Parassit. 20, 625-636. Anderson, J. R. and Ayala, S. C. (1968). Trypanosome transmitted by Phlebotomus: First report from the Americas. Science, N. Y. 161 (3845), 1023-1025. Athens, J. W., Raab, S. O., Haab, 0. P., Mauer, A. M., Ashenbrucker, H., CartWright, G. E. and Wintrobe, M. M. (1961). Leukokinetic Studies, 111. The distribution of granulocytes in the blood of normal subjects. J. clin. Invest. 40, 159-164. Avirinzev, S . (1916). [Concerning parasites of Rana nutti Blgr.] Zool. W s t . 1, 519. Ayala, S. C. (1970). Two new trypanosomes from California toads and lizards. J. Protozool. 17, 370-373. Ayala, S. C. (1971). Trypanosomes in wild California sandflies and extrinsic stages in Trypanosoma bufophlebotomi, J. Protozool. 18, 433436. Babudieri, B. (1931). Emoprotozoi parassiti di vertebrati italiani. Annuli Ig. Sper. 15,620-636. Bailey, J. K. (1962). Aedes aegypti as a possible new invertebrate host for frog trypanosomes. Exptl Parasit. 12, 155-163. Baker, J. R. (1963). Speculations on the evolution of the family Trypanosomatidae Doflein, 1901. Exptl Parasit. 13, 219-233. Balfour, A. (1908). Blood parasites of the common Khartoum toad. Rep. Wellcome trop. Res. Labs. 3, 59. Bancroft, T. L. (1891). Two apparently new infusorian parasites in the blood of a frog, Hyla nasuta. Proc. R. SOC.Queensland 8, 8 . Bardsley, J. E. (1969). “A Preliminary Study of the Trypanosoma rotatorium Complex in the Bullfrog, Rana catesbeiana Shaw”. M.Sc. Thesis, Queen’s University, Kingston, Canada. Bardsley, J. E. (1972). “An Investigation of the Endocrine Control System Regulating the Distribution of Trypanosomes in the Bullfrog”. Doctoral Thesis, Queen’s University, Kingston, Canada. Bardsley, J. E. and Harmsen, R. (1969). The trypanosomes of Ranidae. I. The effects of temperature and diurnal periodicity on the peripheral parasitaemia in the bullfrog (Rana catesbeiana Shaw). Can. J. Zool. 47 (3), 283-288. Bardsley, J. E. and Harmsen, R. (1970a). 11. The effects of excitation and adrenalin on the peripheral parasitaemia in the bullfrog (Rana catesbeiana Shaw). Can. J. 2001.48 (6), 1317-1319. Bardsley, J. E. and Harmsen, R. (1970b). The effects of various stimuli on the peripheral parasitaemia of the Trypanosoma rotatorium complex in the bullfrog (Rana catesbeiana Shaw) of eastern Ontario. J. Parasit. 56, 20-21. Bardsley, J. E. and Harmsen, R. (1972). A simple and inexpensive methodology for the care and maintenance of experimental laboratory frogs. Lab. Anim. 6, 95-104. Barrow, J. H. (1953). The biology of Trypanosoma diemyctyli (Tobey). I. Trypanosoma diemyctyli in the leech, Batrachobdella picta (Verrill). Trans. Am. microsc. SOC. 72, 197-216. Barrow, J. H. (1954). The biology of Trypanosoma diemyctyli, Tobey. 11. Cytology and morphology of Trypanosoma diemyctyli in the vertebrate host, Tritrirus v. viridescens. Trans. Am. microsc. SOC. 73, 242-257. Barrow, J. H. (1958). The biology of Trypanosoma diemyctyli, Tobey. III.Factors influencing the cycle of Trypanosomadiemyctyli in the vertebrate host Triturus v. viridescens. J. Protozool. 5 , 161 -170.

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ADDENDUM Since this review was written, Desser et al. (1973), found that Culex territans (reported by Crans (1970) to be an almost exclusive amphibian feeder) will readily take up “T.rotatorium” from R. clamitans and sustain complete development of the trypanosome. Development entails transformation of the blood stream forms with subsequent reproduction in the midgut; this followed by the appearance of sphaeromastigotes which migrate to the hindgut about 72 h after ignestion. Epirnastigotes appeared in the hindgut at about 85 h when the midgut was becoming rapidly depleted of trypanosomes. Trypanosomes could not be found in the salivary glands at any time, nor were any obvious metacyclic forms found in the hindgut. Unfortunately, no transmission experiments have been carried out at the time of this writing. The observations of Desser et al. (1973) are almost indentical to those of Feng and Chao (1943) and Ayala (1971) on the development of toad trypanosomes in phlebotomids (see Section IVA, 2). This raises the likely possibility that the mosquito transmits the trypanosomes in a similar fashion to the phlebotomids, that is, by infected mosquitoes being ingested by the anuran host (and possibly by the caudatan as well). Culex territans and other such species would thus be logical suspects as the vectors of such trypanosomes as T. grylli of Acris gryllus and T. sp. of Hyla versicolor (see Section VIII). If indeed this were the case, it may explain the morphological distinctness of these two trypanosomes by permitting the trypanosomes to evolve as clonal aggregates in unique environments away from the main line of anuran trypanosomes (similar to that seen in the phlebotomid-borne toad trypanosomes) (see Section IIB). This recent report not only co&s our suspicions concerning the importance of mosquitoes as vectors of anuran trypanosomes, but also adds further to our hypotheses on one of the mechanisms of speciation in anuran trypanosomes, (and the evolution of at least some members of the genus Trypanosoma (see Section IIB and Fig. 1). The polyhospitalic nature of anuran trypanosomes makes the study of this group of tantamount importance in any significant work on the evolution of the genus.

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ADDITIONAL REFERENCES Crans, W. J. (1970). The blood feeding habits of Culex territans Walker. Mosquito News 30,445447. Desser, S. S., McIver, S. B. and Ryckman, A. (1973). Culex territans as a potential vector of Trypanosoma rotatoriurn I. Development of the flagellate in the mosquito. J. Parasit. (in press).

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Global Problems of Imported Disease .

LEONARD J BRUCE-CHWATT

The Ross Institute. London School of Hygiene and Tropical Medicine.

London. England I. I1. I11. IV. V.

Introduction ....................................................................................... 75 Past and Present of International Health ................................................ 77 International Health Regulations and the Increase of World Air Transport ...... 78 Importation of Animal Diseases and of Disease Vectors .............................. 82 Major Imported Diseases ..................................................................... 84 A . Cholera ....................................................................................... 86 B . Smallpox .................................................................................... 88 C Yellow Fever .............................................................................. 92 D. Plague ....................................................................................... 93 E. Relapsing Fever and Typhus ............................................................ 94 Common Disease of Travellers ............................................................... 94 A . Gastro-intestinal Infections ............................................................ 95 B. Malaria ....................................................................................... 97 C. Trypanosomiasis ........................................................................... 103 D. Leishmaniasis .............................................................................. 104 E. Schistosomiasis, Filariasis and Helminthiasis....................................... 105 F. Rabies ....................................................................................... 106 G . Arthropod-borne Encephalitis, Dengue, Haemorrhagic Fever, Poliomyelitis, Leprosy.............................................................................. 107 H . Medical Puzzles and Diagnostic Fallacies .......................................... 109 Prevention of Imported Disease by Immunization ....................................... 110 Acknowledgement .............................................................................. 112 References ....................................................................................... 112

.

VI .

VII .

Never in history has distance meant less. Never have man’s relationships with places been more numerous. fragile and temporary ... We are witnessing a historic decline in the significance of place to human life. We are breeding a new race of nomads. and few suspect quite how massive. widespread and significant their migrations are Alvin Toffler (1970)

.

I. INTRODUCTION Knowledge of the incidence of communicable disease is fundamental to the establishment of protective measures and all countries with organized public health services have developed systems of notification relevant to the existing epidemiological. social and administrative conditions . 75

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LEONARD J . BRUCE-CHWATT

The present international control of communicable diseases established by the World Health Organization over 20 years ago is based on the notification of cases of certain diseases of global importance and the results have been satisfactory on the whole. However, the increased volume and speed of international travel and trade have altered the previous situation and the past decade has given us a warning of the persisting danger of the spread of cholera, plague, smallpox, yellow fever and other infections from their endemic foci in tropical areas. Moreover, new aspects of previously less important diseases such as dengue, several viral encephalitides, leptospirosis, brucellosis, rabies, amoebiasis, have drawn attention to the continued menace of the spread of pathogens related to activity of man. Tropical countries are not the only reservoirs of infectious disease; diphtheria, pertussis, measles, tuberculosis can be introduced from the developed into developing countries with dire results. Moreover, the rapid urbanization of many tropical areas and the large scale industrial development projects such as “man-made lakes” for provision of hydroelectric power or for irrigation are powerful factors in the re-distribution of disease even if they also create new networks of communications and expand the availability of medical services (Bruce-Chwatt, 1968, 1970a, b; Smith, 1972). The World Health Organization recognized the limitations of various national systems of notification of communicable disease and proposed a new approach to epidemiological surveillance aiming at developing in each country a continuous collection of accurate data, follow-up of the distribution and spread of infections and prevention of dangerous outbreaks. This decentralized concept may be the only one feasible in the present circumstances but it has a number of gaps due to the uneven quality and coverage of public health services i n the 140 countries of today’s world (Dorolle, 1968, 1972). The responsibility for recognizing an imported disease has now passed from the airport health medical authority to the general practitioner. He and the medical adviser of any travel agency, commercial enterprise or cultural group now have three duties towards their customers: (a) To protect them by vaccines and provide them with appropriate international certificates ; (b) to advise them on simple measures of prevention while abroad; (c) to be aware of the possibility of imported disease on their return and to diagnose, treat the patient and if necessary refer him to a special centre (Maegraith, 1970). In the meantime an increasing number of travellers are exposed to infectious diseases with which the medical practitioner in their home country is not familiar. The disease seen in the initial stages is usually misdiagnosed and the consequences of delay of proper treatment are often tragic for the individual concerned and serious for the country should the communicable disease spread (Schneider, 1962; Maegraith, 1965; Cahill, 1964). The doctor is frequently asked to advise prospective travellers on the protective measures which they should take either before or after travelling abroad and more knowledge of main infectious diseases likely to be encountered in various parts of the world is essential. Recognition of diseases unusual in temperate

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climates depends to a large extent on awareness of the possibility of their occurrence. Should any suspicion exist of the presence of a serious case, expert opinion and appropriate laboratory investigation sought without delay may be vital for the patient and for the community. 11. PASTAND PRESENT OF INTERNATIONAL HEALTH

The historical developments of the concept of protection of a community from an epidemic go back to the Middle Ages when outbreaks of plague often followed the arrival of ships from the East. The first regulations were introduced in Venice and in Rhodes in the twelfth century and regularly practised from the fourteenth century onwards (Gear and Deutschman, 1956). Ships arriving into European ports were kept at a distance and travellers were detained in isolation for 40 days (quaranta giorni) before they were allowed to proceed to their final destination. This law first imposed in 1377 by the Venetian Republic was the origin of the concept of quarantine. In the course of the next five centuries the fear of foreign disease invading European countries led to the establishment of many vexatious measures which interfered with travel and trade. Eventually, after several unsuccessful attempts a conference of 12 powers met in Paris in 1851 to regulate the prevention of imported pestilential diseases. That date coinciding with the year of the first great International Exhibition in London inaugurated a new period of international action in public health. It was accompanied by a number of other changes each of them of momentous importance for future generations. Abolition of the slave trade, prison reforms, educational progress, organized labour movements, growth of basic sanitation and above all an enormous increase of travel and trade due to the technical improvement in transport by land and by sea. This first conference was convened under the shadow of three diseases which menaced the new freedom of movement; these were cholera, plague and yellow fever. Pandemics of cholera which hit western Europe during the first half of the nineteenth century were disastrous. Although plague disappeared from Europe during the previous century it was still common in Turkey and the Levant and the existence of it in India and China was a constant threat. Yellow fever in the West Indies, in the U.S.A., Mexico and Central America hampered the economic growth, decimated the military expeditions and outbreaks of this disease kept occurring on board ships and in ports such as Brest, St. Nazaire, Swansea, Southampton, Rio de Janeiro, New Orleans, Charleston, Baltimore, Philadelphia and elsewhere (Goodman, 1971). The first International Sanitary Conference of 1851 reached some provisional agreement on the regulation of quarantine although this was ratified by only three countries. Over the next 50 years ten international sanitary conferences followed but the lack of knowledge of the transmission of cholera and plague as well as various political manoeuvres impeded the real progress of any serious collaborative effort. In fact, the creation of a permanent international body to collect the epidemiological information and to use it 4

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for practical action materialized only in 1902 when the Pan-American Sanitary Organization was set up in Washington. Five years later the first world-wide international health organization was formed in Paris and received the name of Office International d’Hygitne Publique. It lasted for 40 years and together with the Health Organisation of the League of Nations (created in 1921) was eventually absorbed into the World Health Organization conceived in 1946 under the charter of the United Nations but formally established in 1948. One of the most important achievements of the World Health Organization during the first decade of its existence was the replacement of obsolete and often confusing sanitary conventions by a single code based on modern epidemiological principles and which made its application more flexible (World Health Organization, 1958, 1968). After a great deal of preparatory work the text of the new international sanitary regulations was drawn up, revised in the light of comments received from various governments and finally adopted by the Fourth World Health Assembly in 1951. These regulations covered all forms of international transport by land, sea and air. They dealt with the sanitary conditions to be maintained and measures to be taken against communicable diseases at international seaports and airports, including measures on arrival and departure, sanitary documents and other details. Special provision related to each of the six “quarantinable” diseases (cholera, plague, smallpox, louseborne typhus, relapsing fever, and yellow fever) and outlined the vaccination requirements, disinsectization, isolation or surveillance. The aims of sanitary measwes permitted by the regulations represented the maximum strictures compatible with the least interference of traffic of passengers or goods (World Health Organization, 1967a).

111. INTERNATIONAL HEALTH REGULATIONS AND THE INCREASE OF WORLD AIR TRANSPORT The international regulations came into force in 1952 and in spite of some difficulties were generally successful for over 15 years. The basic principles of the initial International Sanitary Regulations were: (a) exchange of information on major communicable diseases, (b) specifications of maximum measures that could be imposed on ships and aircraft from infected areas or with disease aboard, (c) action with regard to possibly infected persons. They have been useful in promoting a large degree of international co-operation and in establishing a method for notification of diseases. Although the network of data collection has not been complete nevertheless it has provided much valuable information. During the past decade a number of important changes have occurred both in the pattern of disease as well as in the attempts to prevent their spread. Many of these changes are due to geographical, social, economic and political factors that characterize our today’s “runaway world”-a term used to describe the events that are almost beyond our control. Among these events the increase of world population, pressure on land and all natural resources, rapid and uncontrolled urbanization, fragmentation of large

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territories into small semi-independent nations, political disruption and military intrusion and above all the phenomenal expansion of international trade and travel play a major role. It is true that the new methods of control and the eradication programmes have limited in many countries the incidence of several major communicable diseases such as yaws, malaria, smallpox, yellow fever, tuberculosis, measles and typhus but on a world scale the menace of plague, several arthropod-borne virus infections and above all cholera have never been more real (Dorolle, 1968). The appraisal of the magnitude of the problem of imported diseases may become easier if we realize the impact of air travel on the mobility of human population over the past 20 years. In 1950 the number of passengers on international scheduled airlines was 5 million, it reached 7 million in 1951, 12 million in 1955, 31 million in 1963, 46 million in 1966, 51 million in 1967 and nearly 70 million in 1970 (Organization for Economic Co-operation and Development, 1970).But this is only one fifth of the total number of passengers on all domestic and international airlines: the overall number of passengers travelling by air was 289 million in 1970 and is expected to increase to 325 million in 1973. The number of tourists arriving by air, sea and land into the 98 countries that belong to the International Union of Official Travel Organizations amounted in 1970 to 150 million. TABLE I Comparison of the speed and volume of international air travel in 1950 and 1970

Air travel London-Hong Kong: Duration of travel Mean number of passengers per aircraft Mean cruising speed per hour

1950

1970

5-6 days

8-10 h 200-300

20-40 300-350 miles

(480-560 km) Overall air travel : Total annual distance flown by aircraft Number of passengers on international scheduled airlines

550-650 miles (890-1050 km)

6000 million miles

200 000 million

5-7 million

about 70 million

miles

Table I shows the increase between 1950 and 1970 of the speed and volume of air travel. Some additional points indicate the continuous growth of this extraordinary technical and social phenomenon of our times. There was one transatlantic flight every 2 h in 1950, while today there are about 10 flights every hour. In 1972 the total traffic across the North Atlantic rose to 16 million passengers, an increase of 16% in comparison with 1971. Charter flights which now amount to 25% of scheduled airline flights will soon double their intake of passengers and by 1980 the North Atlantic traffic alone should reach 30 million passengers or nearly 110000 million passenger-miles. In the Pacific area which extends from the Indian sub-continent in the

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west, to North America in the east, including Japan, the countries of SouthEast Asia, Australia, New Zealand and Hawaii, Hong Kong is now the busiest centre for international tourists in this area and is linked with Tokyo, Taiwan, Bangkok and Singapore. The heaviest flow of passengers to Australia and New Zealand goes through Singapore (now the fourth busiest airport in the world) and from the North American continent through Fiji. The annual number of air passengers travelling across the Pacific in 1970 was about 6 million of which about 5 % were charter passengers. But the charter traffic has been growing much faster than the scheduled air traffic and by 1980, with the opening up of China, one could expect that it will reach 320000 million passenger miles. The package tour holiday industry so conspicuous in Europe is now extending to other continents. Long-term charter agreements for the next 3-5 years indicate that tourists who previously were content to go to the Mediterranean, now fly to India, Ceylon, Thailand, East and West Africa and further afield. It is easy to foresee groups of 500 being transported to holiday centres while the smaller aircraft with 150-200 passengers will be travelling to new and more distant places. The rapid development of air transport especially during the past 30 years has abolished one of the main criteria of quarantine, namely the possibility of the symptoms of infectious disease appearing during the voyage or on arrival. The speed of air travel is such that the infected persons may arrive at their destination long before the end of the incubation period and thus may spread the disease before its symptoms are apparent. The baffling problem of processing the passengers at some major airports for checking their health certificates may become more obvious when one remembers that Heathrow, the main London airport, together with Gatwick and Stansted registered in 1971 some 530000 airline movements with nearly 24 million passengers, an increase of 15% in comparison with the previous year; by mid-1973 this figure may reach 30 million-nearly four times the population of the capital of the United Kingdom. The International Sanitary Regulations placed main emphasis on air and sea transport as possible causes for the spread of disease. Land transport by rail or road was given little attention because of the sheer impossibility of adequate health control. And yet, epidemiologically speaking, large numbers of people crossing national frontiers by land may be just as important. This is the situation in Europe today. There are over 7 million migrant workers in all the countries of western Europe. It is estimated that there are about 2.5 million temporary foreign workers in West Germany, over 1 million in France and at least 2 million in Austria, Switzerland, Sweden, Belgium and Holland. Some 50 % of these foreign workers come from southern and southeastern Europe. It is obvious that the traditional system of protection of national health based on the former International Sanitary Regulations is no longer practicable and a new approach is needed, especially as the number of independent countries has nearly doubled over the two decades. The new approach became particularly important because under the previous system which had many aspects of enforced notification many

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outbreaks of serious communicable diseases were not reported under the pressure of political authorities of the country concerned. Dorolle (1972) pointed out several reasons for this regrettable attitude: among these national prestige certainly played some part but there was also fear of excessive and unreasonable restrictions from other countries, especially when such reaction hurt the national pride or the economic interests of the infected country. Many examples of this could be quoted: in the case of cholera one country insisted that the mail coming from infected countries should be sterilized, other countries prohibited the import of lemons, tinned food and even mineral ore. It became obvious in the 1960s that the whole approach to International Sanitary Regulations would have to be changed from the sanitary police and protective barrier concept to a more positive system based on full and frank dissemination of information, epidemiological understanding of it and provision of assistance for the prevention and control of infectious disease (Gelfand, 1971). This involves the problem of surveillance as a key to new international rules. Surveillance in general is the systematic practice of detection of disease, evaluation, consolidation and interpretation of collected data, dissemination of information and application of corrective measures. (Langmuir, 1963; Raska, 1966). The new International Health Regulations which came into force at the beginning of 1971 recognize that the detection and control of communicable disease at the borders of each country will be of little value. Their purpose is to ensure the speediest possible detection and notification of infectious disease combined with the least possible interference with world traffic and with the reasonable degree of security to prevent the spread from endemic foci (World Health Organization, 1969b). These Regulations indicate the new, more flexible and less legalistic approach to international action for prevention of imported disease. The omission of terms “sanitary” and “quarantinable disease” from the title and text of the new document is symbolic of the present thinking. The main features of the new regulations are as follows: (a) Greater attention placed on the prevention of departure of infected persons or contaminated goods from their country of origin; (b) Improvement of port and airport environmental health conditions ; (c) Epidemiological surveillance of diseases likely to threaten other countries and W.H.O. participation in any measures at the request of the country concerned; (d) Recognition of a term “infected area” of a country irrespective of the administrative boundary of such an area: (e) Application within each country of appropriate steps to adapt the health regulations to new techniques of transport of goods such as containers; (f) Maintenance of special international surveillance of four infections (cholera, smallpox, plague, yellow fever) previously classified as “quarantinable diseases”. Generally speaking, the new regulations attempt to strike a balance between technical requirements of preventive medicine on an international scale and social, economic and political realities (Gelfand, 1971). The weakest point of the new system seems to be that countries which have the major responsibility because of the existence of endemic foci are

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also the countries with inadequate public health organization and poorer resources. It would not be surprising if the attempted system were to break down in the detection of communicable disease, or in its notification or in its control; conceivably breakdowns may occur at all these levels. The new health regulations lay great stress on the fact that the conditions in the developing countries will affect the rest of the world; thus the technical assistance given by the rich part of the globe to our poorer neighbours may be the best long-term protection. Nevertheless at the present time and for an unforeseeable number of years the problem of imported exotic human disease will haunt the public health authorities of many countries.

IV. IMPORTATION OF ANIMAL DISEASES AND OF DISEASE VECTORS This important chapter of veterinary public health so closely related to human welfare can be given only scant attention here. There is good evidence that a few years ago an epizootic of foot and mouth disease of cattle in Canada was related to the arrival of an immigrant worker from Central Europe. A few outbreaks confirmed that although man is rarely affected he can act as mechanical carrier of the virus and can be a source of infection. A virus infection of pigs causing “swine vesicular disease” was known to exist in several countries in isolated foci. Thus in 1971 Hong Kong was hit by a serious outbreak of this disease the symptoms of which are not unlike those of foot and mouth disease. In December 1972 this infection previously unknown in the United Kingdom appeared in a few isolated foci in the Midlands. The origin of this infection has not been traced but there is a possibility that food waste from airliners arriving in Britain may have been responsible for it. About 100 tons per week of discarded food from about 60 airlines were previously collected by farmers and sterilized by boiling before feeding it to pigs as swill. Recently, the food waste together with wrappings, plastic crockery, cutlery, etc. has been compacted into large metal containers and emptied unsterilized on open ground. It is likely that seagulls and other birds as also rats and dogs have had access to this food and that piggeries were contaminated with infected meat from countries where “swine vesicular disease” and foot and mouth disease are common. Other animal diseases associated with swill feeding have been described in California in the 1930s; this led to a tight legislation concerning movement of animals and compulsory slaughter before the infection was eliminated. Obviously the same but even stricter controls will be necessary at the present time to prevent the introduction of animal diseases resulting from the increase and mobility of human populations. The ever growing volume of international traffic has greatly increased the danger of importing new diseases, not only through their human or animal hosts but also through the infected vectors. The introduction of Anopheles gambiae into Brazil from Africa in the late 1930s was followed by a spectacular epidemic of malaria. The presence of a new huge lake formed by the Aswan dam at the SudanEgyptian border has created an artificial bridge for this African mosquito that could now easily invade the lower Egypt. Much vigilance will be needed

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to prevent this occurrence which in the 1940s led to serious epidemics of malaria in the Nile valley. Recently five new species of mosquitoes, potential transmitters of dengue and filariasis, have been introduced into Guam. A potentially dangerous situation exists with regard to the Anopheles punctulatus group of mosquitoes in South Pacific and Anopheles stephensi transported from Iran, Iraq and Saudi Arabia to north-east Africa (BruceChwatt, 1970a). Obviously, the possibility of introducing infected Anopheles mosquitoes into a country (e.g. Mauritius) where malaria eradication has been successful is of great concern to several national health authorities. The same can be said about the possibility of importation of Aedes aegypti, or other insect vectors and especially those that have become resistant to some of the generally used insecticides (World Health Organization, 1967~).Ticks may also be introduced into new areas on domestic animals and by migrant birds (Hoogstraal et al., 1961). The introduction of disease vectors by air is not a hypothesis but a well proven fact. During the period 1964-1968 not less than 373 various insects (including 65 mosquitoes) were recovered from spot checks in aircraft arriving at Hawaii (World Health Organization, 1971). The procedures for vector control in international health have been developed by the World Health Organization since the 1950s but the increasing complexity of the situation calls for constant improvement of methods acceptable to the aircraft industry, the International Civil Aviation Organisation (ICAO) and the International Air Transport Association (IATA). Insect quarantine measures take two forms: airport sanitation and the disinsectization of aircraft. Effective vector control at and around the airport is the first line of defence while aircraft disinsectization aims at eliminating insects that have entered the aircraft in spite of control measures. The earliest method used for disinsectizating aircraft was the application of an aerosol spray during flight or after the aircraft landed at its destination. This was replaced by the “blocks away” system in which the insecticide is applied between the time the doors are shut and the take-off. Further research resulted in the development of an automatic vapour disinsectization system using dichlorvos. This method encountered some difficulties (World Health Organization, 1971) because of possible corrosive effect of dichlorvos on plastics and metals used for construction of modern aircraft. For the present time the former “blocks away” procedure and aerosol disinsectization are being used but new methods are being developed. It appears that most of such methods will face some problems and in the long run the only acceptable approach will be by providing a zone around each airport in which insect vectors cannot survive. Insects may be introduced into a country not only accidentally but through sheer ignorance of possible consequences. In March 1971 live African Anopheles gambiae were brought to Palawan province in the Philippines by a research team from the University of Nagasaki. This caused much concern to the Philippine health authorities and gave rise to a diplomatic incident, Fortunately the mosquitoes were destroyed before they could possibly escape from the cage and gain a foothold in a new tropical country. One can only

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hope that the lesson learned by too enthusiastic scientists will serve as a general warning. Cities in Asia, Africa and South America are expanding rapidly to keep pace with increases in the human population, while hitherto virgin lands are cleared for food production. Thus domestic rats and mice are brought into contact with various wild rodents and the inevitable exchange of parasites may then produce unpredictable results to human health. The role of wild and domestic rodents in the transmission of plague, cutaneous leishmaniasis, leptospirosis, scrub typhus (Tsutsugamushi fever), Venezuelan equine encephalitis, hydatid disease (echinococcosis), murine typhus etc. is well established. There is no doubt that the activity of man contributes to the spread of some zoonoses (Mackenzie, 1972). A problem related to the spectacular growthof container transport by seaand cargo service by air has been considered by the W.H.O. Committee of International Surveillance of Communicable Diseases held in 1970 (World Health Organization, 1971). Several instances of containers infested with rats and insects were notified but this does not yet call for any specific action. However, if the large scale use of containers extends to countries where rodents and insects are abundant the situation may require new international regulations. Among animals that may be the source of newly introduced human diseases monkeys deserve special attention because of their increased use in scientific research and also on account of their biological closeness to man. In 1970 imported laboratory monkeys were responsible for 28 cases and 7 deaths from Marburg disease in Germany and Yugoslavia (Smith, 1971).

V. MAJORIMPORTED DISEASES The most common error in diagnosing an exotic disease lies in the failure to think of the possibility of its occurrence. The knowledge of the geographical distribution of various important diseases of tropical countries is of substantial assistance for the diagnosis of imported infections. As pointed out by Maegraith (1963, 1965) the doctor should be conditioned to inquire whether the patient has been exposed to any tropical infection. It is obvious that a suspicion of malaria or African trypanosomiasis will be aroused if the patient with fever and unusual symptoms admits that he recently spent 2-3 weeks in tropical Africa. On the other hand, haematuria in a patient who lived in some countries of the Middle East points to the probability of urinary schistosomiasis. Table I1 shows the geographical distribution by continents and regions of some of the most important communicable diseases. It is true that some diseases of world-wide distribution may nevertheless be more common in certain countries and are more easily acquired by the traveller when abroad, while he is exposed to unusual circumstances. This would be relevant to such infections as poliomyelitis, infectious hepatitis, typhoid fever and paratyphoid, or to some intestinal helminths. A large number of tropical diseases can be identified by the discovery of the pathogenic agent involved, such as the eggs of Schistosoma in the urine or faeces, or trypanosomes in the blood, or Leishmania from the edge of an

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TABLE I1 Distribution in 1971 of some of the most important communicable diseases

*

Central and West Africa East and South-East Africa North Africa Ethiopia and Sudan South Africa Near East Africa Southern Arabia and the Yemen Pakistan and North India Southern India and Ceylon South-East Asia Central Asia Far East Asia and Indonesia Australia and New Zealand S.W. Pacific Islands Central America TropicalSouth America Temperate South America North America Northern Europe Southern Europe

3

2

3

1

3

1

3

1

1

3

3

2

3 2 3 2 3

2 0 2 0 0

3 2 3 2 3

2 1 3 0 3

3 2 3 1 2

1 0 1 1 1

3 2 3 1 2

1 1 2 1 1

1 0 3 1 0

3 2 3 2 3

3 0 1 0 0

2 1 3 1 2

3

0

3

3

2

1

3

1

0

2

0

2

0

3

2

3

2

3

1

2

2

3

0

0

2

0

3 3 3

2 2 2

3 3 3

3 1 2

2 3 1

1 3 2

3 3 2

1 0 1

2 0 1

0 0 0

0 0 0

1 2 1

0 0 0

3

2

3

1

2

1

3

0

2

0

0

3

0

1 2 3 3

0 0 0 0

1 3 3 3

0 0 3 3

0 2 2 3

0 0 1 2

1 1 3 3

0 0 1 1

0 0 0 0

0 0 1 2

0 0 2 2

1 2 2 2

0 0 1 1

2 1 1 2

0 0 0 0

2 2 2 2

0 0 0 1

0 0 0 0

0 1 0 0

1 1 1 1

0 1 0 1

0 0 0 0

0 0 0 0

0 0 0 0

1 1 1 1

0 0

2

0

0

-

The figures 0, 1, 2 and 3 indicate the degree of endemicity of the disease. O= indigenous disease absent. 1=indigenous disease uncommon. 2 =indigenous disease common in some areas. 3 =indigenous disease widespread. Diseases marked by an asterisk are covered by the International Health Regulations.

ulcer. The recent development of serological techniques applied to parasitic diseases made so much progress that even when the pathogen is difficult to detect an additional clue may be given by the study of the immune response

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of the host. The degree of the immune response depends on the pathogen, on the tissue invaded by it, on the quantity of the antigen, and on the previous exposure to infection. In exotic diseases where parasites play the dominant role, the specificity and sensitivity of the immune response varies considerably and the precise interpretation of serological tests must be cautious. Generally, immunodiagnosis is of value when the parasites are very scanty or not accessible but a positive test may indicate at least a previous exposure to the infection. The presumed or confirmed diagnosis of an exotic disease should be followed by a timely and appropriate treatment. In both the diagnosis and the therapy, specialist advice should be sought from institutes and departments of tropical medicine. In cases where the exotic disease can be easily transmitted by direct contact or indirectly departments of public health must be notified and closely involved in the follow-up of the situation. While theoretically many exotic infections may be rapidly imported into any country open to air communications, in practice only a few communicable diseases deserve special attention because of their high incidence, severity and especially infectivity. The latter characteristic determines the possibility of rapid spread of the disease in the new environment. A.

CHOLERA

Cholera which in the past swept through Asia and Europe in six pandemics became once again a dominant public health problem (Cvjetanovic and Barua, 1972). During the nineteenth century cholera spread in successive waves from its hotbed in the Indian peninsula and reached many parts of Europe though for most of the time it has been confined to Asia. However, after 1961 the seventh pandemic of cholera, due to the El Tor vibrio", started its westward advance from South-East Asia and within 5 years reached India and the Middle East. In 1970 it appeared in southern Europe, in North, East and West Africa for the first time this century and became a dominant public health problem (Fig. 1). In 1971 cholera outbreaks were reported in 33 countries, one-third of them experiencing the disease for the first time. In that year not less than 150000 cases were reported; the incidence in Asia doubled (some 50000 cases occurred in the West Bengal refugee camps) while it increased 6-fold in Africa (World Health Organization, 1972b). In most of the newly affected countries the disease caused severe outbreaks with a fatality rate of 40 % or more. However, when the emergency assistance provided by the World Health Organization and some governments got into its stride the fatality was drastically reduced. Intravenous rehydration, fluid, antibiotics such as tetracycline, laboratory media and antisera and vaccines, have most successfully dealt with local epidemics but have not solved the problem of cholera establishing itself in

* This causative agent of the present pandemic of cholera received its name after the El Tor quarantine camp on the Sinai peninsula where it was first isolated in 1905 from the intestines of pilgrims returning from Mecca. Today it is generally agreed that the El Tor vibrio has some slightly different characteristics from that of classical cholera vibrio but can cause epidemics of nearly equal severity and rapid spread.

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new parts of the world where it may remain in an endemic state for years to come (Barua and Cvjetanovic, 1970). The main reason for this is the ability of the El Tor vibrio to cause mild or asymptomatic infections so that longterm carriers can spread the disease through contamination of food or water. The present pandemic has been certainly due to the extent of travel between the islands of its previous focus and subsequent increase of international travel. In South-East Asia the standards of sanitation have been impaired by overpopulation of urban peripheries, by military operations and other disturbances. In some of these areas cultural habits (such as cleansing by hand after defaecation) and the use of human excreta in agriculture contribute to the spread of infection. The possibility of cholera establishing itself more or less permanently in Africa and some countries of the Middle East causes much concern. The future spread of the disease to Central and South America is not unlikely and the consequences of such a calamity are unpredictable (Lapeyssonnie, 1972). On the other hand the appearance of a few cases of cholera in European or American countries with a high standard of sanitation and medical services is of limited importance. Modern treatment of the disease is most successful while an early detection, notification and isolation of confirmed or suspected cases will prevent the spread of the infection to the community (Gangarosa and Faich, 1971). Nevertheless,to the modern traveller cholera remains a potentially serious menace, The recent episode of over 40 persons travelling by air from London to Australia and infected with cholera from contaminated food taken on board at Bahrain underscores the dangers of exotic disease. Only the strictest measures of sanitation combined with active immunization of people exposed

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to unusual or continued risk can limit the occurrence of such outbreaks. It should be stressed that in a few cases of cholera imported into the United Kingdom (from Spain or North Africa) the symptoms of the disease were not very severe and some patients had a concomitant infection with Shigella. Currently available vaccines whether prepared from classic strains or El Tor strains (Inaba and Ogawa serotypes) are of limited usefulness as they provide only 60-70% protection for a period of 3-6 months. Full primary immunization requires the subcutaneous or intramuscular injection of two doses of vaccine given at an interval of 1 4 weeks. Single “booster” doses should be given every 6 months and in endemic areas where cholera outbreaks are periodic the vaccine should be given before the start of the cholera season. However, we must remember that cholera vaccine does not preclude the individual concerned from becoming a carrier. The systematic detection of carriers among millions of tourists is impossible and thus the vaccination certificate giving a false sense of security is of little public health value even if it protects the vaccinated person from severe clinical disease. The obligation for a traveller coming from an infected country to have a valid certificate of vaccination against cholera has now been abolished in the United States and will probably be abolished by other countries. But a long term effective measure is the improvement of standards of living and of sanitation in those areas of the world where cholera is still present (Araoz et al., 1970). Individual travellers will be reasonably well protected from cholera when they follow the general precepts valid for prevention of all gastro-intestinalinfections. Avoidance of drinking water or milk of suspect or unknown quality, or not eating raw vegetables, salads and other similar food are the most important step when visiting a country with low standards of hygiene. B. SMALLPOX

Smallpox, one of the great scourges of mankind, is known to have existed since the dawn of history in India and Egypt; it was most probably introduced into Europe by the returning crusaders and as sea communications developed it was carried to the New World by the Spanish and Portuguese conquerors and settlers. Pandemics of smallpox which were so common in the eighteenth and nineteenth century stopped with the introduction of compulsory vaccination, with the port health inspection of immigrants and seafarers and with the improvement of medical knowledge (Dixon, 1962). Until recently smallpox has been endemic in many countries of South America, on the subcontinent of India and South-East Asia, and in large areas of tropical Africa*. However, the remarkable effort aiming at eradication of smallpox, promoted over the past few years by the World Health Organization, has already achieved substantial success (World Health Organization, 1968b).

* It may be useful to distinguish between an “endemic” and an “infected” smallpox

area. In the first case one refers to transmission of indigenous cases of smallpox within a

population. An “infected area” refers to secondary cases of smallpox following an imported case.

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During 1971,the fifth year of intensified programme of smallpox eradication, the area of endemic disease decreased still further. In Brazil, the only country of the Americas where endemic smallpox was still present in 1970, only a small localized outbreak was seen in 1971 and since then no cases have been detected (World Health Organization, 1972b). In western and Central Africa no case of smallpox has been discovered since May 1970 (with the exception of Botswana where an outbreak occurred in 1971) while in eastern Africa the disease is endemic only in Ethiopia and Sudan. Foci of endemic smallpox exist in Afghanistan, Bangladesh, India, Nepal, Pakistan and Indonesia (Figs 2 and 3).

FIG,2. Incidence of endemic smallpox in 1967.

Although the number of cases of smallpox has been decreasing steadily from about 200000 in 1967 to 33000 cases in 1970, the year 1971 showed an increase to over 50000 cases mainly from Ethiopia and 64000 cases were recorded in 1972. The increased number of reported cases is due to improved notification. The striking progress of smallpox eradication has been ascribed to the emphasis placed on detection and isolation of cases, to the widespread use of potent lyophilized vaccine and to the excellent international collaboration of systematic vaccination programmes. The discovery in Africa of monkeypox, an infection closely resembling smallpox, suggested at first the existence of a potential animal reservoir of the human disease; further studies showed that the transmission of this virus to man is an exception rather than a rule and that eradication of human disease is based on sound principles. However, as long as endemic foci of human infection exist the introduction of smallpox into countries where it is unkown remains a constant possibility. Nearly 30 episodes of introduction of smallpox into non-endemic countries (mostly into Europe) have been reported during the past 10years and in 1972 in Yugoslavia

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FIG.3. Incidence of endemic smallpox in 1972.

an outbreak affecting 175 people originated from one single traveller with a mild infection contracted in the Middle East. There were 34 deaths and 15 million people had to be vaccinated. Nearly all introductions of smallpox into non-endemic areas were due to persons coming from infected areas (mostly in Asia) and in spite of a fair degree of control of individual vaccination certifications. Twenty years ago the majority of imported cases travelled by sea but since 1950 nearly all infected individuals have come by air. The resulting outbreaks have sometimes been widespread and severe. Wherever they have taken place extensive epidemiological follow-up and mass vaccinations have been needed. In the 18 episodes of importation of smallpox into various European countries between 1961 and 1965 there were an average of 16 cases and 2.4 deaths per outbreak. In 1966-1970 the 10 outbreaks averaged 10 cases and 0.5 deaths per outbreak. Nearly one half of these cases were seen in medical and auxiliary health personnel (Center for Disease Control, 1972b). The solution of the problem of smallpox depends on the elimination of the disease from all its endemic foci, by repeated mass vaccination campaigns combined with surveillance, isolation of the few remaining cases and revaccination of contacts. This is the basis for the global eradication programme. In the meantime, the strategy of protection of non-endemic areas from imported smallpox is still largely based on checking of vaccination certificates of persons possibly exposed to the infection and on preventive vaccination of the community. The fallacies of the first of these measures is obvious (Dorolle, 1968, 1972). Vaccination certificates are often unchecked at busy airports; when checked they may be false or past their validity and even when valid they give no guarantee that the vaccine used was fully potent and the vaccinated person is immune. Nevertheless, the principle of a valid smallpox vaccination certificate required from individuals arriving from international

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travel is sound especially if the traveller has been in a country where the disease has been reported with the preceding 2-4 weeks. With regard to the second measure the opinion is divided. Generally speaking countries have had various legislation to enforce vaccination against smallpox in relation to the epidemiological state of smallpox in their region, the probability of importation, the state of vigilance of recognition and control of an imported case and the knowledge of contraindications of vaccination. Several countries (e.g. France and Germany) maintain that systematic smallpox vaccination of certain age groups results in a level of collective immunity that prevents a major outbreak of smallpox. Other countries as the United States and the United Kingdom have discounted routine vaccination because the value of it as a collective measure requires that not less than 90 % of the population should be immune and also because the small risk of vaccination is still greater than the likelihood of the importation of smallpox into the country. This policy is justified only in countries with a highly developed health service where access to medical care is universally available so that there is every opportunity for early recognition of an infection and for taking immediate preventive measures. Admittedly it presents an element of calculated risk. Fortunately, the global eradication programme of smallpox is making satisfactory progress and one can certainly hope that the human disease will be eliminated within the next few years. When it comes to individual protection, effective vaccination before exposure prevents the disease. In the U.S.A. and the U.K., primary vaccination is recommended during the end of the first year of life with revaccination every 3-5 years. This is particularly important for persons at special risk such as international travellers, doctors and nurses. In countries where smallpox is endemic, primary vaccination during the neonatal period or during infancy has been advocated with re-vaccination after 1 year and then at usual intervals. It is known today that a potent vaccine and proper technique guarantee a 90-100% “take” of primary vaccination with high immunity of at least 3 years duration. Re-vaccination has a rapid booster effect and maintains a degree of immunity for 5 years and perhaps even longer. An important though at times forgotten point is the certainty that the primary vaccination has been successful. This is shown by the presence of a typical local reaction seen 6-8 days later at the site of the application of the vaccine. Otherwise the vaccination should be repeated, if necessary with a different batch of vaccine. Among the common errors resulting in unsatisfactory vaccination is the use of glycerinated vaccine stored under improper conditions of refrigeration. Lyophilized vaccine is preferred since it has a far better stability. Another important point is the correct entry on the international vaccination certificate. Omission of some details may result in the holder not being allowed to land in some countries or being compelled to undergo another vaccination by the airport’s health authorities. Most countries require all travellers arriving from endemic or infected

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areas to produce International Certificates of Vaccination, valid for 3 years after successful vaccination. In the United States this is now the only international vaccination certificate required for entry, if the traveller has been in a country reporting smallpox during the preceding 2 weeks. C. YELLOW FEVER

Yellow fever, the third communicable disease covered by the International Health Regulations, presents a serious problem because its virus is harboured by a number of forest animals, especially monkeys, and is transmitted by various species of jungle mosquitoes. In urban areas the infection is transmitted from man to man by the ubiquitous Aedes aegypti. Thirty years ago it seemed that the development of the yellow fever vaccine-one of the most successful methods of prevention-together with the discovery of potent insecticides spelled the end of yellow fever. However, the eradication of the disease or its vectors has not been achieved and has proved to be unlikely in the present conditions. During the past two decades several large outbreaks of yellow fever in Africa and the occurrence of a number of yellow fever foci in the Americas showed that the disease remains a constant menace. In South America there were 48 cases in 1969, and 86 cases in 1970 mainly from Peru but also from Bolivia, Brazil, Colombia and more recently from Venezuela. The disease has not spread into Central America since the mid 1950s though the animal foci of transmission are known to persist. The fact that 31 countries in the New World are still infested with A2de.s aegypti stresses the hidden transmission potential of the infection (Reeves, 1972). In Africa the history of yellow fever could be divided into four phases: pre-1940,1940-1957,1958-1966 and from 1967 onwards (Hamon et al., 1971; Brks, 1972). During the past decade more or less extensive epidemics were seen in Ethiopia, Senegal, Togo, Mali, Ghana, Upper Volta, Nigeria, Angola and Zalre. The notified cases are far below the actual numbers of victims and it is likely that as many as 100000 cases may have occurred during the past few years. It appears that on the African continent a permanent silent transmission in densely forested areas produces periodic large outbreaks of human disease. The reason for this periodic activity is not fully known but certainly related to seasonal rainfall in the savannah areas and to human activity and movements of populations. As elsewhere the rapid growth of peri-urban slums provides opportunities for increased breeding of mosquitoes including Aedes aegypti (Simpson, 1972). Much of the epidemiology of yellow fever in West Africa still remains a mystery and there may be areas with unkown cycles of transmission between animals and men (Hamon et ul., 1971). The fact that the West African populations show serological cross reactivity between the yellow fever virus and many other viruses of the B group increases the difficulty of interpretation of some surveys (Downs, 1972). Fortunately, the yellow fever vaccine is of outstanding efficiency and prevention of human disease requires immunization of all persons at risk. Two

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types of vaccines are known: the French neurotropic Dakar vaccine is prepared from brains of mice infected with the attenuated strain of the virus and administered by skin scarification; the 17D type of vaccine is prepared from a virus cultivated in chicken embryos and injected subcutaneously. Because of the high incidence of meningo-encephalitic reactions following the Dakar vaccine only the 17D type is generally used but even with this vaccine infants below 6-9 months of age should not be vaccinated unless for some special reason. The degree of protection from the infection is very high and although the vaccination certificate is valid for 10 years the persistence of protective antibodies is much longer (Center for Disease Control, 1972a). The control of yellow fever depends on vaccination, reduction of the vector density in urban areas and surveillance. Large epidemics can be prevented by an early detection of cases, rapid control activities and compliance with International Health Regulations. These provide for the compulsory vaccination of travellers going from infected to receptive areas and to disinsectization of aircraft. In spite of all these measures it is generally recognized that the problem of yellow fever remains unsolved and the frightening possibility of the disease being brought into Asia by an unvaccinated traveller who was infected only a few days ago in the Brazilian forest always exists in the mind of every public health man (Dorolle, 1972). No-one can explain convincingly why yellow fever has never invaded southern Asia where Aedes aegypti is common and where an epidemic among receptive human population might assume cataclysmic proportions. It cannot be said that the present International Health Regulations governing the protection of Asia against yellow fever are adequate though they are the best that can be devised. The increased volume of international air communications between Africa and Asia constitutes a real danger in this respect. In spite of present trends in favour of liberalization of International Health ReguIations it is likely that the requirements for the yellow fever vaccination certificate will be maintained in most of the receptive countries. Constant vigilance comprising a speedy detection of suspected cases, immediate notification and appropriate public health measures are permanently needed in receptive areas. D. PLAGUE

This is the fourth “quarantinable” disease covered by the International Health Regulations. Although transmissible from man to man in some instances, it has all the aspects of a true zoonosis since in its sylvatic form it is common in wild rodents from which it may be transmitted by fleas to domestic rats in rural and urban areas. In the not too distant past plague was common in southern Africa, large areas of South America, the western part of the U.S.A., Central and SouthEast Asia. During the past decade the incidence of plague has markedly declined in many parts of the world except for South Vietnam where several thousands of cases were reported. In 1971-1972 there were in South Vietnam

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over 6000 cases with a 25 % fatality rate. However, it should not be forgotten that during the period 1960-1969 about 5000 human cases of plague have been notified in the Americas. Bubonic plague is the most common clinical form with an acute inflammation of lymph nodes and secondary septicaemia. Modern antibiotics have spectacularly improved the cure even in serious cases. Most of the foci of endemic plague show varying degrees of activity but large outbreaks are always possible whenever war or a natural disaster create conditions suitable for greater contact of rats with men (World Health Organization, 1970). The risk of transmission of plague by rodents has traditionally been related to sea-borne trade. Rat-proofing and desinsectization of ships together with continuing anti-rat measures in harbours have decreased the danger but today cargo-planes and airports have created new habitats for rats. The latter problem has not been given everywhere the attention it deserves. Large airports produce as much refuse and garbage as small cities and create potentially serious problems of sanitation. Methods of rat control combined with surveillance of suspected cases of plague are the best collective method of protection. Immunization with plague vaccine prepared from Yersiniu pestis grown in artificial media and inactivated provides some degree of protection for 6-12 months and is recommended for persons exposed to contact with wild rodents and to visitors travelling to Cambodia, Laos and Vietnam. E.

RELAPSING FEVER AND TYPHUS

Two other communicable diseases, namely relapsing fever and typhus transmitted by infected lice or ticks, persist in small, limited foci in Africa, Asia and America. Sizeable outbreaks of these diseases are usually connected with war, natural disasters, famine when impoverished, overcrowded conditions of life contribute to the multiplication of the insect vectors. Transmission of these infections through international traffic is less likely and they have now been removed from the list of “quarantinable diseases” covered previously by the International Regulations. VI. COMMON DISEASES OF TRAVELLERS

Cholera, smallpox, yellow fever and plague deserve special attention of public health authorities because of the facility with which they can spread in the community. However, a number of other diseases can easily be acquired in countries where the environmental conditions are not fully satisfactory or where the tropical climate maintains the presence of certain endemic infections. Because of their frequency these diseases are of considerable importance to the individual traveller and, naturally enough, to his doctor. An investigation of 1000 patients in a London hospital who had visited or resided in tropical or sub-tropical areas revealed that 33% of them were infected with pathogenic or potentially pathogenic helminths or protozoa (Allen and Ridley, 1971).

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GASTRO-INTESTINAL INFECTIONS

Gastro-intestinal infections are certainly by far the most common complaint. The term “travellers’ diarrhoea” designates usually an onset of loose or watery, frequent stools with nausea, vomiting, abdominal pain and acute discomfort. This condition lasts for one to three days and is due to several combined factors suchas fatigue,unusual or excessive food, bacterial contamination etc. Outbreaks of diarrhoea in a group of people are invariably due to some infective agents such as bacteria of the Salmonella group, Clostridia, Staphylococci, Escherichia coli etc. ; viruses (e.g. Coxsackie) may also produce simple diarrhoea though the pathogens are not easily identifiable. Diarrhoea lasting more than 2-3 days and associated with fever may conceal a variety of more serious conditions such as amoebic dysentery, infections due to Salmonella (typhoid or paratyphoid fever) or Shigella (bacillary dysenteries) as also several protozoan parasites (Giardiu lamblia, Balantidium coli, etc.) and helminths. The severity of the disease dominated by the symptom of diarrhoea may range from a transient indisposition to fatal Shigella dysentery, typhoid fever or cholera. Amoebiasis though seldom a cause of acute diarrhoea may present a wide variety of symptoms related to intestinal tract with complications ranging from skin ulceration to liver abscess. Chronic amoebiasis is a particularly awkward medical problem as the patient may be susceptible to other intestinal infections. On the other hand, some infected individuals with trivial complaints may pass amoebic cysts in their excreta for years and may undergo long and ineffectivetreatments that could lead to mental depression (Woodruff and Nelson. 1970). The whole problem of amoebiasis is of considerable complexity (Cahill, 1972) and it appears that although foreign travel has not greatly increased the overall incidence of clinical amoebiasis the causal agent (Entamoeba histolytica) is being more commonly found in Americans returning from overseas. The diagnostic value of modern serological methods should be noted in this respect. Typhoid fevers (including paratyphoid infections) due to Salmonella typhi and Salmonella paratyphi A, By C are of great public health importance. They have a world-wide distribution and are often associated with travels in the Mediterranean. In the United States about 400-500 cases are reported every year; the relevant average figure in the United Kingdom is 200 (Department of Health and Social Security, 1972b). Out of 128 cases of typhoid fever notified in England and Wales in 1971 some 75% occurred on return from summer holidays. A combination of fever, headache, abdominal tenderness, cough with signs of bronchitis and diarrhoea or constipation in a traveller returning from abroad should always be suspect. The disease may present all degrees of severity and there is still a mortality of 5-10% in untreated typhoid fever; any undue delay of the diagnosis must be avoided. Paratyphoids are generally milder.

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Most of the enteric infections due to Salmonella typhi or paratyphi have as their source patients with mild symptoms or chronic carriers handling food. Transmission is mainly by ingestion of food (including milk and ice cream) contaminated by human hands and flies ; waterborne outbreaks are less common but possible even in some highly advanced countries (e.g. Zermatt epidemic in Switzerland) when sewage has an accidental contact with water supply. However, the Aberdeen outbreak of 1964 in Scotland, affecting 500 people, was due to meat contaminated during processing in South America and insufficiently sterilized. Oysters and shellfish are often infected in polluted tidal waters. Acute gastro-intestinal infections of human or of animal origin are usually due to eating meat contaminated with Salmonella typhimurium and other bacteria of this group, through contact with uncooked carcasses of cattle or chickens. Greater use of factory processed foods, restaurants and other collective feeding places employing casual and often immigrant workers has increased the possibility of such “food poisoning” episodes. In the United States the isolations of Salmonellae from human infections averaged 20 000 every year in the 1960s but are showing an increase of 16% between 1969 and 1970 and 6 % between 1970 and 1971. In the United Kingdom some 5000 cases are reported annually but the actual number is much higher. Although generally mild, an outbreak of “food poisoning” may cause serious problems in elderly people with cardio-vascular deficiencies. In 1970 a group of tourists from California developed symptoms of gastro-enteritis on their return from Asia. Twenty-four persons became acutely ill in flight necessitating a diversion of the aircraft. One person died and another with coronary insufficiency had to be hospitalized. The pathogen isolated was Clostridium perfringens and Vibrio parahaemolyticus. Another outbreak of acute gastro-enteritis caused by Vibrioparahaemolyticus occurred on an international charter air flight from Bangkok to London (Peffers, et al., 1973). Nine passengers were affected, five of them seriously; three members of the cabin crew were also ill. The food item responsible for the outbreak was traced to cooked crab meat contaminated from raw crabs after preparation. The pathogen isolated in this case is common in fish and shellfish in the Far East. This outbreak stresses the growing importance of monitoring of food and water supplies to safeguard against imported disease and the need for prompt action by the airlines and public health authorities. Bacillary dysentery due to the Shigella group of micro-organisms has a sudden onset after an incubation period of 1-5 days and a wide range of severity of diarrhoea1 symptoms. Shigella dysenteriae is a common pathogen in Asia while Shigella sonnei is prevalent in countries where personal contact with carriers rather than poor sanitation is responsible for the spread of disease. A recent outbreak of dysentery due to Shigella sonnei on board a large British ship during a cruise to the tropics affected 300 passengers and crew; it was due to the presence of carriers among the catering staff recruited in a foreign country. In all cases of diarrhoea lasting for more than 3 days only laboratory

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diagnosis can clinch the clinical presumption. Specimens should be examined soon after they have been voided to speed up the correct treatment and prevent the spread of the infection. Prevention of intestinal infections depends on taking elementary precautions with regard to food and drink. Unless it is known on good authority that the local supply of water is safe, the best course is to boil all water used for drinking or cleaning the teeth. Filters for domestic use are available but it is unwise to rely on them in hotels or guestbouses. Milk should be boiled or pasteurized and stored in a refrigerator. Food should be protected from flies and cooked shordy before eating; underdone meat, fish or raw shellfish should be avoided. Raw vegetables, salads and ice cream are common vehicles for pathogens; fruit is reasonably safe when washed and peeled. Investigation of convalescent or chronic carriers among persons handling food should form an important part of preventive measures. All persons going abroad are well advised to be effectively immunized against typhoid and paratyphoid fevers (TAB vaccine) with a vaccine of high antigenicity given in a primary series of 2 injections separated by 3-4 weeks; protection is fairly high for up to a year. Single periodic booster doses every 2-3 years are indicated for individuals exposed to possible infection while travelling. Some authors prefer to use a monovalent typhoid vaccine. One must admit that none of the parenteral vaccines against enteric bacterial infections is wholly satisfactory. The duration of the immunity they confer is relatively short, they tend to cause side reactions and the cost of using them on a large scale is high. It is hoped that oral vaccines against typhoid, shigellosis, cholera and Escherichia coli enteritis will eventually be developed but despite many advances the induction of immunity via the intestinal mucosa is still far away (World Health Organization, 1972a).

B.

MALARIA

The frequency of malaria contracted overseas may be lower than the cumulative incidence of gastro-intestinal infections but the consequences of an unrecognized or incorrectly treated plasmodia1 infection are much more serious. Malaria deserves the greatest attention since numerous deaths due to infection with P. falciparum are reported every year among travellers who were not aware of or underestimated the danger of contracting the infection and consequently failed to take the advocated protective measures. Moreover, malignant tertian malaria caused by P. fulciparum may simulate a number of diseases; thus the unwary doctor who fails to inquire from the febrile patient if he travelled in tropical or semi-tropical countries may miss an early diagnosis, often vital to the patient’s recovery. The successes of malaria eradication have been striking over the past 15 years but this disease is still the most common infection of the tropical world. Recent reports of the World Health Organization have shown that malaria as an indigenous disease has disappeared from Europe and from the U.S.A.,

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from most of the U.S.S.R., from Israel, Lebanon, large parts of India, from Singapore, Hong Kong, Taiwan, South Africa, Australia, Chile, most of Argentina and Venezuela, most of the islands of the Caribbean, and from Mauritius. Nevertheless, the whole of tropical Africa, parts of North Africa and Central America, many countries of South America, some areas of the Middle East, and most of the countries of South-East Asia and of the South-West Pacific are still malarious (Fig. 4). At the end of 1971 of the estimated 1844 million people living in the originally malarious areas of the world at least 472 million people were still exposed to endemic malaria (World Health Organization, 1972~).Thus, despite immense gains from malaria eradication campaigns, very large parts of the world are still sources and reservoirs of plasmodia1 infection (BruceChwatt, 1971b; World Health Organization, 1973b). The term “imported malaria” has a definite epidemiological meaning; it refers to cases of infection acquired outside the area where it has been found. “Introduced malaria”, on the other hand, is due to a local transmission by mosquitoes, subsequent to a case imported from outside the country concerned. Malaria may also be transmitted intentionally (“induced”) as a therapeutic measure (malariatherapy for neurosyphilis) or it may be seen as an accidental disease following blood transfusion or (as it happens with some drug addicts) due to the sharing of syringes which have been contaminated with infected blood. In the U.S.A. alone during the 5 years 1967-1971 there were nearly 17000 cases of malaria, with a peak in 1970 of 4239 cases. Most of these cases (95 %) occurred in military personnel returning from South-East Asia; the remainder were seen in teachers, students, merchant seamen, tourists, and Peace Corps volunteers (Center for Disease Control, 1972b). The number of cases of malaria imported into 25 European countries and notified to health authorities has been steadily increasing from 2966 in 1969, to 3412 in 1970 and to 4987 in 1971. About 90% of these cases occurred in Portugal and were related to military activities in Africa. Over three-quarters of cases of malaria were due to P. falciparum, with P. vivax amounting to 21%. There were 54 deaths during the reviewed period-a P . falciparurn fatality rate of 0-6% (World Health Organization, 1973b). Some 2500 cases of imported malaria have been recorded in the United Kingdom over the past 15 years. About half of these infections originated in Africa, while the other half (mainly Plasmodium vivax) came from Asia (Bruce-Chwatt et al., 1971). In 1971 the number of reported cases of malaria in Britain was 294 (twice as high as during the previous years) but this official figure is probably below the actual incidence. Among the 2000 cases of malaria notified in the United Kingdom between 1954 and 1969, 360 patients were infected with P. falciparum; many of the 58 deaths could have been avoided if the diagnosis had been made without delay and the correct treatment instituted promptly. This very high fatality rate of 16 % compares unfavourably with that of less than 2.0 % reported from the U.S.A. However, in American merchant seamen the fatality rate was twice as high.

FIG.4. Status of malaria eradication in 1971.

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Numerous cases of imported malaria with high fatality rate in P .falciparum infections were reported from France, Germany and Holland (Bruce-Chwatt, 1969, 1970b; Bruce-Chwatt et al., 1971). The incubation period in malaria covers the time between the infection and the first appearance of clinical signs, of which fever is most common. For mosquito-transmitted infection this period varies according to the species of Plasmodium. Usually about 12 days are required for the development of P.falciparum infection, 13-15 days for P. vivax or P. ovale, and up to 1 month for P. malariae. With some strains of P. vivax the incubation period may be delayed for several weeks or months; this may also occur in persons who have been taking suppressive antimalarial drugs. Preceded by various premonitory signs such as headache, malaise and nausea, a classical attack of malaria consists of several short febrile paroxysms preceded by a rigor and recurring every 2 days (tertian periodicity of vivax or ovale malaria), or every 3 days (quartan malaria) ; falciparum malaria produces either daily or irregular fever and rigors are not a common feature. Typical paroxysms consisting of a “cold stage, hot stage, and sweating stage” are far from constant ; variations of the classical clinical course are common, particularly in children. There are three potentially serious aspects of imported malaria in any country where the relative absence of tropical diseases has not prepared the medical profession to appreciate their significance and consequences. The first is the severity of some P. fakiparum infections in nonimmune persons; the second is the complication by a relapsing latent malaria infection of any acute disease, delivery, or surgical intervention (particularly splenectomy) occurring in a person who previously inhabited an endemic area; the third is the possibility of transfusion malaria when the donor of whole blood is an asymptomatic carrier of plasmodia. Because of the high rate of multiplication of P. falciparum the symptoms of this infection may appear with dramatic suddenness and severity. Four groups of complications are generally recognized though many symptoms may overlap : (a) cerebral malaria; (b) gastro-intestinal malaria ; (c) hyperpyrexia with delirium and convulsions; and (d) algid malaria, with low blood pressure and cardiovascular: failure. Anaemia and hepatic symptoms, with jaundice, renal, respiratory, and other involvement, develop often when the parasites are numerous in the peripheral blood, but exceptionally also when they are scanty, because of their accumulation in the internal organs. Rapid diagnosis and immediate, adequate treatment are necessary in every case of falciparum malaria, especially in non-immune persons. Malaria due to P. vivax or P. malariae is rarely fatal by itself but can be serious, especially in children (Bruce-Chwatt, 1971). Recurrences of acute falciparum malaria incompletely treated are generally milder than the initial attack and disappear, often spontaneously, within 12-1 8 months; however, in immune persons from endemic areas, parasites may persist in the blood much longer, with very mild if any clinical symptoms. It is unusual for vivax and ovale malaria to relapse after 3 years following the primary attack; on the other hand, quartan malaria is much more persistent and relapses may be seen 5, 10, or even 40-50 years later. In fact, some persons with untreated or poorly

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treated quartan malaria may become asymptomatic carriers of plasmodia for life and can transmit the infection whenever they serve as blood donors. Transfusion Malaria. Accidentally induced malaria as a result of blood transfusion is not uncommon in several countries and may become more frequent as the demand for blood increases. The available records covering the period 1950-1971 and based on data gathered in 49 countries gave the figure of nearly 1300 cases of malaria as an accidental complication of blood transfusion. Some 65% of these infections were due to P. malariae with P. vivax and P . falciparum following in that order (Bruce-Chwatt, 1972a). Prevention of transfusion malaria depends on the elimination as a wholeblood donor of anyone who ever had malaria in the past or who has ever been exposed to malaria during a specified period prior to blood transfusion. This type of screening is quite effective when strictly applied but detection of malaria infection in a suspected donor is notoriously difficult. The only certain proof of the infection is the finding of malaria parasites in the peripheral blood and this should never be neglected. Malaria should be suspected in every febrile patient who has ever been in a malarious area or who has had blood transfusion up to 3 months before the start of his symptoms. Although the finding of parasites in the blood clinches the diagnosis, one or two negative blood slides must not eliminate the possibility of malaria. Several thick blood films should be taken in any suspected case and examined as rapidly as possible by a competent microscopist. The correct identification of the species of the malaria parasite gives much guidance for the treatment of the patient. The parasites are usually in greatest numbers just before or soon after the rigor. Serological methods of diagnosis are now of increasing value. Among these methods the indirect fluorescent antibody test (IFA test) is now widely used, In clinical practice the test is of value in retrospective diagnosis-for example, in a person who has been exposed to malaria, has suffered a febrile illness, but in whom antimalaria treatment has made conventional parasitological diagnosis impossible. It has proved to be of great value in identifying the responsible donors in cases of transfusion malaria, where the parasitaemia may be low and intermittent. The diagnosis of malaria should not depend solely on a laboratory report but requires sound clinical judgement. While the finding of plasmodia in the blood provides a criterion of infection, parasites may be so scanty that they escape detection in a patient who took small doses of antimalarial drugs before the blood slide was taken. Protection against malaria is based on three traditional principles : (a) prevention of transmission by bites of mosquitoes; (b) reduction of the sources and of numbers of mosquitoes; and (c) chemoprophylaxis by the use of antimalarial compounds. In malarious areas, all sleeping and living quarters should be mosquitoproofed, and mosquito nets should be used over the beds. Insect-repellent lotions, such as dimethylphthalate may be applied to the exposed parts of the body of persons remaining out of doors after dark to protect them for

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3-4 h from bites of mosquitoes. Wearing of long trousers and long-sleeved dresses after dark decreases the chance of mosquito bites. Wherever insecticidal control of malaria is indicated this can be carried out by spraying the inside walls of inhabited houses with a residual insecticide at the proper dosage (1-2 g of DDT to each m2 of inside surface of walls and ceilings). This should be repeated twice a year or more often if necessary. Other insecticides such as hexachlorocyclohexane (HCH), dieldrin, malathion, carbamates, etc., may be preferable to DDT in some conditions, especially if the local malaria vector has developed resistance to DDT or other compounds. A degree of control of vectors can also be achieved by frequent indoor application of quick-acting preparations of pyrethrum as atomized spray ;aerosol formulations for outdoor fogging or “space-spraying” are also used. Volatile dichlorvos compounds incorporated into synthetic wax strips may be useful (World Health Organization, 1971). For prevention of malaria in adult individuals visiting or residing in malarious areas, one of the following drugs should be taken regularly during the possible exposure and also for at least 1 month after leaving the endemic area: (1) Pyrimethamine (Daraprim): 1 or 2 tablets (25 mg each) once a week, preferably on Sundays, to ensure regular taking. (2) Proguanil hydrochloride (Paludrine): 1-2 tablets (100 mg each) every day. (3) Chloroquine diphosphate or sulphate (Aralen, Avloclor, Nivaquine, Resochin, etc.): 2 tablets (150 mg base each) once a week, or 1 tablet twice a week. (4) Amodiaquine dihydrochloride or base (Camoquin, Flavoquine, Basoquin, etc.): 2-3 tablets (200 mg base each) once a week. In areas where malaria is highly endemic and exposure to infection is high, once-weekly administration of drugs 2 and 4 does not give a sufficiently wide margin of safety and higher dosage of these drugs, e.g. up to 600-700 mg of chloroquine base over the week has been advised ; proguanil (chlorguanide), 2 tablets daily has also been widely used in these conditions. Some persons may have mild gastro-intestinal or other symptoms with any of these drugs; a change to a different drug is then indicated. Children should be given proportionally lower doses of drugs. Various potentiating oral combinations of pyrimethamine with sulphonamides or with sulphones are currently being promoted. At the time of writing few data are available to indicate how effective these combinations are for individual or collective prophylaxis. They cannot, in the absence of further evidence, be generally recommended but may be necessary where resistance of malaria parasites to standard drugs exists. Some strains of P . falciparum have become resistant to Caminoquinolines and have shown lessened response to chloroquine and other drugs. This has been reported from certain parts of Brazil, Colombia, Panama, Thailand, Laos, Cambodia, Vietnam, Western Malaysia, Singapore, Burma and a small area in the Philippines (World Health Organization, 1967c;Peters, 1969). The problem of which drug to recommend for prophylaxis in countries where strains of P .falciparum are known to be resistant to 4aminoquinolines

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is difficult to answer at present. While proguanil or pyrimethamine may still give good protection, they cannot be completely relied upon. Chloroquine will protect against P . vivax, P. ovule, or P . malariae as well as against most strains of P . falciparum. Even increased dosage, however, may not always prevent or cure infections with resistant strains. It is against these strains that combinations of long-acting sulphonamides with pyrimethamine or trimethoprim may well prove the most useful prophylactic available at the present time (Peters, 1969; Bruce-Chwatt et al., 1971).

C. TRYPANOSOMIASIS

Trypanosomiasis is not far behind malaria among the major endemic tropical diseases. The infections caused by haemoflagellates can be divided into two main groups : American trypanosomiasis (Chagas’s disease) due to Trypanosoma cruzi and African trypanosomiasis (sleeping sickness) due to Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense. Both diseases are essentially infections of animals transmissible to man (zoonoses). The vectors of American trypanosomiasis are several species of blood sucking Triatoms or “cone-nosed” bugs (Reduviidae) while in the case of African trypanosomiasis tse-tse flies (Glossina sp.) are the usual vectors. American trypanosomiasis occurs within a wide belt of Latin America from Mexico to southern Brazil. It is estimated (World Health Organization, 1969a) that some 35 million people are exposed to the disease and about 7 million are infected. The triatomid bugs involved in the transmission are commonly found in rural houses built of mud bricks or poor masonry; infection occurs through infected bites but also through skin abrasions or through mucous membranes contaminated with faeces of infected bugs. Much attention has been drawn recently to the possibility of accidental transmission of Chagas’s disease by blood transfusion. It has been found that in some parts of South America up to 10% of blood donors have serological tests indicative of previous infection (Bruce-Chwatt, 1972a). The incubation period of American trypanosomiasis is 7-14 days after the bite of an infected bug but 30-40 days after an infection by blood transfusion. The acute stage of the disease starts with fever, headache and peri-orbital oedema (if the contamination occurred by a bite on the face); the fatality rate in children may be as high as 10%. Serious cardiac and other complications may follow the chronic infection. Early diagnosis can be arrived at by finding trypanosomes in blood films or by inoculation of cultures, by experimental infection of clean reduviid bugs from the suspected patients (xenodiagnosis) or by serological methods. Systematic control of reduviid bugs by effective use of residual insecticides, avoidance of contaminated dwellings and use of bed nets for protection against bites are the only effective methods of protection. Screening of suspected blood donors or addition of gentian violet to the blood used for transfusion is necessary in endemic areas.

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African trypanosomiasis which was in the decline since the days of the widespread epidemics in the 1930s has not only maintained its hold in tropical Africa but even increased in some countries of that continent in the wake of political disturbances and economic hardships. It has been estimated recently that the annual number of new cases of sleeping sickness in Africa is 330 per 100000; as many as 10% of some populations may be infected. A few disquieting findings indicate that not only wild animals, but also cattle may act as reservoirs of human disease. The transmission of disease through Glossina paIpalis the main vector in Gambia, Liberia, Sierra Leone, Ghana, Congo, Angola occurs mainly along water courses ; GIossina morsitans transmits the infection over dry savannah areas in Malawi, Mozambique, Kenya, Rhodesia, Tanzania, Uganda, Zambia. Although the danger of infection with trypanosomiasis in short-term visitors must not be exaggerated,neverthelessthe infection may occur in tourists visiting African game reserves and national parks, as the tse-tse fly is attracted to moving vehicles, which it often enters to bite the passengers. Several instances of trypanosomiasis subsequent to “safari-holidays” in East Africa have been reported and about one case of imported infection is notified in the U.S.A. every year. An inflamed local lesion may appear at the site of the infective bite 1-2 weeks later. Within a month or less there are symptoms of fever, adenopathy, headache, insomnia and sometimes erythematous rash. After weeks or months various neurological or mental symptoms signal the advanced stage of the disease (Lumsden, 1972). Diagnosis in the early stage is by recognition of trypanosomes in the blood film or in the aspirated fluid from enlarged lymph nodes (especially posterior-cervical). In later stages there are characteristic changes in the cerebrospinal fluid. Adenopathy and fever in anyone who visited Africa must always raise a suspicion of incipient trypanosomiasis. While general methods of control of African trypanosomiasis are based on destruction of habitats of tse-tse flies and on spraying with insecticides individual protection is usually limited to prevention of bites by avoiding certain heavily infected zones, wearing appropriate clothing or application of repellent creams. Persons particularly exposed may be given prophylactic injection of a single dose of 250 mg pentamidine isethionate which protects for 3-6 months. D.

LEISHMANIASIS

Leishmaniasis whether visceral (Kala-azar) or cutaneous (tropical sore, chiclero ulcer, etc.), occurs not only in tropical areas of Africa, Asia and Latin America but also in some sub-tropical countries of the Mediterranean, Imported infections have occasionally been notified. Long-lasting skin ulcers or fever with hepato-splenomegaly in residents in these areas are a good guide to correct diagnosis easily confirmed by demonstration of the pathogen (either directly or by culture) from the edge of the ulcer or from the material obtained by puncture of the spleen, lymph node or sternal marrow (Lumsden and Marsden, 1971; Bray, 1972).

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E. SCHISTOSOMIASIS, FILARIASIS AND HELMINTHIASIS

A group of diseases now increasingly frequent in persons returning from abroad after prolonged stay but which are occasionally seen also in holiday travellers comprises schistosomiasis, filariasis and intestinal helminthiasis. Schistomiasis or bilharziasis, one of the major endemic diases in warm climates, affects over 200 million people. Of the three species of the trematode (blood fluke) that occur in man, Schistomma haematobium (which gives rise to urinary symptoms) is found mainly in Africa and the Middle East. The two other species give rise to intestinal and related manifestations : Schistomma mansoni is found in Africa, the Arabian peninsula, eastern and north-eastern South America and the Caribbean ; Schistosoma japonicum is prevalent in South-East Asia, western Indonesia (Sulawesi), Philippines, Japan and China. The transmission of disease is related to the presence of water and to several species of snails that act as intermediate hosts. Endemic schistosomiasis is a disease of rural communities and its recent increase is due to the introduction of large scale irrigation schemes whenever water-filled canals become the breeding sites of snails. This has happened recently after the construction of the Aswan dam in Egypt and the Volta dam in Ghana. After an involved cycle of development in the snail, freeswimming larvae of the worm (cercariae) penetrate the skin of persons swimming or wading in contaminated waters. They enter the bloodstream, develop to maturity and migrate to veins of abdominal organs (Jordan and Webbe, 1969). Symptoms of infection appear some 6-8 weeks after the infection. Painless haematuria may be one of the first symptoms in urinary schistosomiasis. In the intestinal form of the disease intermittent diarrhoea is not uncommon. Diagnosis of both forms is clinched when the characteristic eggs of the parasitic worm are found in urine or faeces; serological tests are of increasing value. Treatment with modern drugs is increasingly successful (Wilcocks and Manson-Bahr, 1972). Various methods of control include chemotherapy of the infected population and molluscicidal measures but they have not been outstandingly successful except for some small areas (Jordan, 1972). Personal protection depends on avoidance of contact with fresh surface water that is possibly contaminated (sea water is safe). Provision of filtered or boiled water for drinking is nesessary wherever the piped water supply is suspect. Filariasis is a composite name for a group of infections with small nematode worms transmitted by mosquitoes. The most common of these are Wuchereria bancrofti and Brugia malayi; the first occurs in Latin America, Asia and some Pacific islands, the second is endemic in India, South-East Asia, South Korea and a few Indonesian islands. Transmission occurs by bites of serveral species of mosquitoes harbouring infective larvae. Early acute symptoms include fever, lymphadenitis and various allergic reactions but chronic infections may cause swellings of leg, scrotum and more rarely forearms and breast (in females). Many cases may remain virtually symptomless.

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The incubation period varies between 3 and 9 months. Diagnosis can be easy when the examination of blood for microfilariae coincides with their periodic appearance (e.g. W. bancrofti from Asia, Africa and S. America appears in the blood at night, while in case of bancroftian filariasis contracted in the Pacific the periodicity may be diurnal). Unless this possibility is remembered the diagnosis may be difficult. Individual protection depends on the avoidance of mosquito bites by mechanical means (bed nets, mosquitoproof homes, repellent creams, mosquito control measures). Other filarial diseases such as onchocerciasis or loiasis while endemic in Africa or parts of Latin America are less common in short-term visitors and need not be described in detail, but comprehensive surveys of epidemiology and control of filariases are available (World Health Organization, 1967a, 1967b; Duke, 1972; Edeson, 1972). Intestinal helminths are not regarded as a serious public health problem in comparison with other diseases that are often imported from abroad. Nevertheless, they may undermine the health of individuals (especially pregnant women) afflicted with one or several species of such parasites. Symptoms of intestinal helminthiasis may range from pruritus, insomnia, urticaria, nausea, abdominal pain, constipation, diarrhoea, loss of weight, to iron deficiency anaemia, bowel obstruction and pulmonary or cerebral complications. The commonest intestinal helminths imported from abroad are : the hookworms (Ancylostoma duodenale and Necator americanus), the whipworm (Trichiuris trichiura), the large roundworm (Ascaris lumbricoides), the small intestinal nematode (Strongyloides stercoralis), the tapeworms (Taenia solium and Taenia saginata), and the threadworm (Enterobius vermicularis). Many other intestinal helminths have been reported. Diagnosis is often obvious from eggs or segments of worms found in the faeces but precise recognition of the species of the parasite is important as it dictates the correct treatment with modern antihelminthic drugs of high activity. Intestinal helminths may be responsible for serious complications of abdominal surgery and a stool examination should be an essential preliminary step before any intestinal operation on those who have travelled abroad (Woodruff and Nelson, 1970). A number of rarer diseases are occasionally seen in travellers returning from various unconventional or adventurous travels abroad ; anthrax, brucellosis, infectious hepatitis and leptospirosis may be mentioned though poliomyelitis and rabies certainly deserve even more attention (Sikes, 1970).

F.

RABIES

There is evidence of an upsurge of animal rabies in many parts of the world. The situation is kept under constant review by the international agencies which publish an annual world survey of the incidence of rabies and of preventive measures adopted (World Health Organization, 1966). The countries free of rabies are Australia, Great Britain, Hawaii, Ireland,

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Japan, New Zealand, Norway and Sweden. Control measures adopted in several other countries have reduced the number of human cases but wild life rabies continues to spread and it has been estimated that in Central Europe the infection extends westwards at the rate of about 40 km every year. Foxes appear to be the principal vector. In Latin America where bats transmit rabies by bite only a small proportion of them (less than 1 %) excrete rabies virus in the saliva. Airborne spread of virus in caves where bats are roosting may exceptionally occur. Rabies is enzootic in wolves (Canada, Eastern Europe, Iran, Turkey), jackals and mongooses (Caribbean islands, India South Africa), foxes, coyotes, racoons, skunks (U.S.A.). In urban areas a number of domestic animals including cows, horses, donkeys or cats may become infected but dogs are by far the commonest animals responsible for human rabies. Stroking or attempting to feed by hand a stray dog encountered on holiday in a foreign country is a dangerous pastime. Attempting to break the quarantine regulations imposed on dogs and cats by countries where rabies is unknown borders on criminal irresponsibility. At times emotional pleas are made to relax the regulations by animal lovers but such moves are misguided and should be strongly resisted. The frequency of rabies in man following exposure varies in relation to the part of the body in contact with the virus and to the amount of the infective material; the incubation period may range from 10 days to nearly a year. Prevention depends on the avoidance of contact with any possible source of the virus and on the eradication of rabies from a country by elimination and control of entry of infected or suspected animals. Persons in professional contact with dogs and cats should be effectively immunized by avian embryo vaccine. Post-exposure treatment comprises thorough cleansing of the wound and (if the suspicion of rabies is warranted) administration of specific vaccine supplemented if necessary by injection of hyperimmune serum.

G. ARTHROPOD-BORNE ENCEPHALITIS, DENGUE, HAEMORRHAGIC FEVERS, POLIOMYELITIS, LEPROSY

Arthropod-borne virus diseases deserve a mention at the present time because of their resurgence in Africa, Asia and the Americas (Reeves, 1972; Simpson, 1972). Several mosquito-borne viruses have been responsible for most human epidemics in the New World. These are St. Louis encephalitis, Western equine encephalomyelitis, Eastern equine encephalomyelitis, California encephalomyelitis, Venezuelan equine encephalitis, dengue fever, yellow fever, and finally the Mayaro, Oropouche and Uruma fevers (in Bolivia, Trinidad and Brazil). The first four have caused more than 2000 human cases in the U S A . in the past decade. The presence of these viruses has been confirmed in many parts of Central and South America but human epidemics in these areas are exceptional while epizootics in horses are common. A wide range of birds and

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mosquitoes are associated with these infections. It is possible that increased urbanization of Latin America together with major irrigation projects will favour future epidemics. The prevention of human disease depends on vector control or on individual protection from mosquitoes; there is no vaccine for human use (Reeves, 1972). The recent history of Venezuelan equine encephalitis (VEE) may illustrate the possibility of extension and the change from a purely animal infection to an important human disease. It was in the 1960s that VEE which was previously considered as exclusive to horses was recognized as a disease of man. The 1962-1964 outbreak of Venezuelan equine encephalitis caused in Venezuela over 23000 human cases with 156 deaths, in 1967 in Colombia it affected at least 200 000 people and killed nearly 100000 horses and donkeys. Since then many thousands of cases were seen in other parts of Latin America but only one human case in the U S A . Like other encephalitis viruses VEE is maintained in a silent cycle between small mammals and birds with mosquitoes acting as vectors; horses are probably amplifying hosts during epizootics and contribute to the mutation of the virus increasing its effects on the host. Dengue. Among the arthropod borne viruses dengue is now of particular interest because a disease that seemed to have been conquered in the Americas 10 years ago has made its sensational reappearance since 1963 in coastal Venezuela and in the Caribbean islands where an estimated 100000 people have been affected by it. The exact explanation of this outbreak is not fully known but it may be related to the fact that after a period of intensive control of Aedes uegypti (one of the main vectors of urban dengue) the eradication programmes of this mosquito have become less effective. Importation of dengue into the southern United States from the Caribbean is very likely. A warning given by Reeves (1972) may be quoted here: “It would be extremely short-sighted if we did not recognize that the dengue epidemics may be an early warning of a possible resurgence of epidemics of urban yellow fever.” The clinical course of classical dengue consists of one or two sharp febrile attacks of short duration, with headache, joint and muscular pains and an occasional maculopapular or erythematous rash. Normally the disease is not serious even though it may be followed by fatigue or depression. However, the recent occurrence of haemorrhagic fevers caused by dengue viruses is causing much concern. Classical dengue has been known to occur in the Indian peninsula, Pakistan, the whole of South-East Asia, the Philippines and Indonesia but haemorrhagic symptoms associated with dengue were first seen in the 1950s as severe epidemics in Manila, Bangkok and Singapore; other outbreaks were reported from India, Malaysia and South Vietnam. In 1969 there were 9000 cases in Thailand mainly in the urban area with a high proportion of affected children and a fatality rate of 10-1 5 %. The hypothesis that the disease had spread from the Philippines to Thailand and India through the increase of air transport has not been proved (Simpson, 1972).

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Hueinorriiugic fevers have receiitly beeii reported from Argentina and Bolivia and caused by new viruses (Junin, Machupo). These viruses are found in various wild rodents and are expelled in urine; transmission occurs through contamination of food by rodents and the infection is related to the extension of human activities into uninhabited pampas of South America (Mackenzie, 1972). The control of dengue and haemorrhagic fevers depends on the application of intensive anti-mosquito and anti-rodent measures and protection of individuals from mosquito bites. No vaccines are available.

Poliomyelitis, one of the important enteroviruses, has a worldwide distribution but its epidemiological picture has changed during the past 25 years. Its incidence has decreased in advanced countries owing to immunization of children and generally high socio-economic level. However, it has become more common in adults who had no previous exposure to the virus. At the same time there is evidence that both asymptomatic and symptomatic infections are extremely common in crowded and insanitary urban areas of the tropical world (Beale, 1969). The mode of infection is faecal-oral and flies play an important part in the transmission. The incubation period varies between 6-20 days and the clinical symptoms may range from a minor illness to spinal, bulbar or combined paralysis. Effective immunization by live oral poliomyelitis is emphatically advised for any traveller likely to be exposed to the infection and this means nearly everyone. Leprosy, a tropical disease affecting between 10 and 15 million people, of whom only about 2 million are under active treatment, is of low infectivity and while single imported cases occur among immigrants from Asia or Africa it presents no problem for a short-term visitor. Small numbers of patients seen in Europe or the United States have contracted the disease during prolonged residence abroad. H. MEDICAL PUZZLES AND DIAGNOSTIC FALLACIES

It is obviously impossible to enumerate all communicable diseases that could be acquired when travelling in many parts of the world. The commonest symptoms of disease may be present in a wide variety of infections (Wilcocks and Manson-Bahr, 1972). Thus fever in a returning traveller may be due to malaria in the first instance. But illness may also be due to typhoid or other Salmonella organisms, to trypanosomiasis, typhus, relapsing fever, bacillary or amoebic dysentery, arthropod-borne viruses, influenza, infectious hepatitis, brucellosis, leptospirosis etc. Some of the latter infections may have been contracted anywhere, and the origin of the disease need not be in a foreign country. Cough with sputum may be the symptom of any cosmopolitan bronchial or pulmonary infection but may also be due to hookworm and ascaris larvae migrating through the lungs or to paragonimiasis. Such clinical pointers have been presented by Maegraith (1965). 5

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As a source of comprehensive, reliable and admirably concise information on the epidemiology and control of a large number of communicable diseases the latest booklet of the American Public Health Association (Benenson, 1970) is unparalleled. One should also remember that at times “medical conundrums” or “clinical puzzles” can be easily solved by one simple well directed investigation. A painless ulcer on the face may be due to cutaneous leishmaniasis; a skin “patch” with a loss of pigment and loss of pain may be an early symptom of leprosy; vague abdominal symptoms with anorexia need a stool examination rather than a psychoanalyst’s couch; inexplicable pyrexia in a patient who had a serious accident weeks or even months before the onset of symptoms may be due to malaria induced by blood transfusion from an infected donor. Persistent diarrhoea labelled as “ulcerative colitis” should be carefully screened to exclude amoebiasis. “Many patients have been snatched from the surgeons at the last minute with their colons still intact while other patients have been less fortunate” (Wright, 1967). It may be surprising how often the clue to the recognition of the disease depends on the simple question so strongly advocated by Maegraith (1963); “Where have you been and when ?,’ VII. PREVENTION OF IMPORTED DISEASE BY IMMUNIZATION Under the International Health Regulations which have been accepted by most (but not all) countries of the world, national health authorities may require certificates of immunization against cholera, smallpox and yellow fever. However, such requirements may vary not only from country to country but may also depend on circumstances of local or wider outbreaks of infectious diseases. Moreover, changes occur when there are some unusual conditions in a country in which the traveller disembarks for a short period before continuing his voyage or flight. A list of national requirements concerning vaccination certificates for international travel is published annually by the World Health Organization (1973a) but in the case of epidemics the embassies or consulates of relevant countries should have up-to-date information of any drastic changes. The validity periods for International Certificates are shown below (Table III); each such certificate must be issued on a special form and is strictly individual. Unlike passports they cannot be issued collectively to cover the wife of the holder and his children. Risks of vaccinations against these three diseases are small but should be understood. In smallpox the majority of complications occur during primary vaccinations. In the United States in 1968 the over-all incidence of complications was 10.8 per million of primary and re-vaccinations (153/14.2 million) with nine deaths but surveys have shown that more than 50 % of these complications might not have occurred if known contraindications had been heeded. In the United Kingdom during the period 1951-1970 the incidence of complications was 40 per million vaccinations (651/15.5 million) with 77 deaths. The main contraindications are inflammatory or allergic skin diseases

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TABLE ITI Validity periodi of International Certificates of vaccination against communicable diseases covered by International Health Regulations

Vaccination against :

Type of Vaccination

Period of

Smallpox

primary

3 Y-

Cholera

Revaccination Primary Revaccination

Yellow Fever

PrillWy

Validity

(if successful)

Revaccination

3 years 6 months 6 months 10 years 10 years

Validity begins 8 days after vaccination Immediately 6 days after

Immediately 10 days after

Immediately

(especially eczematous conditions in infants) and any natural or drug-induced alterations of immune responses (dysgammaglobulinela, administration of steroids). If vaccination in such persons is mandatory, vaccinia immune globuline WIG) should be given at the time of vaccination. Foetal vaccinia following vaccination of a pregnant women is extremely rare and since 1932 only 20 cases have been reported out of many millions of women. There is no evidence that vaccination in pregnancy increases the risk of teratogenic effects or abortion. As the case-fatality of smallpox in pregnant women is of the order of 70 % primary vaccination (and even more so, revaccination) are justified if there is any possibility of exposure to infections. Serious reactions following cholera vaccinations are rare but they do occur. Revaccination in such cases may not be advisable and the individuals concerned must be in possession of a medical certificate. Yellow fever vaccine prepared from a live attenuated virus (17D strain) grown in chicken embryos is remarkably free of any after-effects. A small proportion of people may have low fever, headache and muscle pains. Individuals hypersensitive to eggs may occasionally show various allergic reactions. As in the case of smallpox, persons with an abnormal immune response should not be vaccinated against yellow fever. Pregnancy is not a contraindication but it may be prudent to avoid administration of yellow fever vaccine to pregnant women. It is also advisable not to administer the yellow fever vaccine and the smallpox vaccine on the same day but to leave 2-3 weeks interval between them. This is more important for infants than for other age groups. Apart from vaccinations required by international health regulations immunization against typhoid and paratyphoid is advisable for everyone going outside northern Europe and North America. The same goes for poliomyelitis and an oral booster dose before departure is recommended. Naturally, children proceeding abroad should have completed their standard course of immunization against tuberculosis (BCG vaccine), diphtheria, pertussis, measles and poliomyelitis. It may be a wise precaution

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to have children protected against tetanus and this can be easily done by using the combined diphtheria, tetanus toxoid and pertussis (DTP) vaccine. Infectious hepatitis (Hepatitis A) is common in tropical countries but the passive protection afforded by a specific immune serum globulin (ISG) is short-lived. Ordinary travellers are not exposed to the infection any more than in their own countries but long-term visitors to the tropics may be protected by ISG and single injections of 5 ml (for adults) repeated every 4-6 months may be indicated for them. General and specific recommendations concerning immunization procedures are periodically reviewed by the U.S. Public Health Service Advisory Committee on Immunization Practices (ACIP Recommendations); the recent comprehensive review refers to the use of vaccines and other biological products for prevention of 18 communicable diseases (Center for Disease Control, 1972a). Similar periodic documents are published in other countries (U.K. Department of Health and Social Security, 1972a) and the general practitioner asked to advise prospective travellers on various preventive measures should possess all necessary information (Cannon, 1969; Ross Institute, 1971). However, in the final account it is the traveller himself who must be aware of health problems when travelling abroad. Main airlines are usually fully informed of their responsibilities but this cannot be said about various small charter companies and travel agencies. Improvement of the present situation is needed and one can only hope that the relevant national health authorities will show greater interest in this respect. ACKNOWLEDGEMENT Figures 1-4 are reproduced with the permission of the World Health Organization. REFERENCES

Allen, A. V. K. and Ridley, D. S. (1971). J. trop. Med. Hyg. 74, 83. Araoz, J. de, Barua, D., Burrows, W., Carpenter, Jr., C. C. J., Cash, R. A,, Cvjetanovic, B., Feeley, J. C., Gallut, J. C., Mahalanabis, D., Mondal, A., Mosley, W. H., Mukerjee, S., Nalin, D. R., Pierce, N. F., RaSka, K., Sack, R. B., Sakazaki, R., Subrahmanyan, D. V., Verwey, W. F. and Watanabe, Y . (1970). “Principles and Practice of Cholera Control”. Public Health Papers No. 40, WHO, Geneva. Barua, D. and Cvjetanovic, B. (1970). WHO C h o n . 24, 41. Beale, A. J. (1969). Br. med. Bull. 25, 148. Benenson, A. S. (Ed.) (1970). “Control of Communicable Diseases in Man”, 11th edition. American Public Health Association, New York. Bray, R. S. (1972). Br. med. Bull. 28, 39. BrCs, P. (1972). I n “Vector control and the recrudescence of vectorborne diseases”. PAHOlWHO Scientific publication No. 238, Washington. Bruce-Chwatt, L. J. (1968). E. Afr. med. J. 45, 266. Bruce-Chwatt, L. J. (1969). Br. med. J. 2, 237. Bruce-Chwatt, L. J . (1970a). Misc. Puhl. !?/it. Soc. Am. 7,7. Bruce-Chwatt, L. J. (I970b). Br. med. .I. 64, 201.

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Bruce-Chwatt, L. J. (1971a). Malaria, In “Cecil-Loeb Textbook of Medicine” (Eds P. B. Beeson and W. McDermott). Saunders Company, Philadelphia and London. Bruce-Chwatt, L. J. (1972a). Trop. Dis.Bull. 69, 825. Bruce-Chwatt, L. J. (1972b). Bull. SOC.Path. Exot. 64, 782. Bruce-Chwatt, L. J., Draper, C. C. and Peters, W. (1971). Br. med. J. 2, 91. Cahill, Kevin M. (1964). “Tropical Diseases in Temperature Climates”. Lippincott, Philadelphia. Cahill, Kevin M. (1972). “Clinical Tropical Medicine”, vol. 11. University Park Press, Baltimore, London and Toronto. Cannon, D. A. (1969). Trans. R. SOC.trop. Med. Hyg. 63, 867. Center for Disease Control (1972a). Collected Recommendations of the Public Health Service Advisory Committee on Immunization Practices. Morb. Mort. Weekly Rep. 21, No.25. Center for Disease Control (1972b). “Malaria surveillance, 1971 Annual Report”. US . Dept. of Health, Education and Welfare, Atlanta, Ga. Cvjetanovic, B. and Barua, D. (1972). Nature, Lond. 239, 137. Department of Health and Social Security U.K. (1972a). “Immunization Against Infectious Disease”. D.H.S.S., Alexander Fleming House, London. Department of Health and Social Security (1972b). On the state of public health. Annual Report of the Chief Medical Officer for 1971 OHMS, London. Dixon, C. W. (1962). “Smallpox”. Churchill, London. Dorolle, P. (1968). Br. rned. J. 4, 789. Dorolle, P. (1972). Lancet 2, 525. Downs, W. (1972). Discussion In “Vector Control and the Recrudescence of Vector Borne Diseases”. PAHOlWHO Symposium Scient. Publ. No. 238, Washington. Duke, B. 0. L. (1972). Br. med. Bull. 28, 66. Edeson, J. F. B. (1972). Br. med. Bull. 28, 60. Gangarosa, E. J. and Faich, G. A. (1971). Ann. Znt. Med. 74, 412. Gear, H. S . and Deutschman, Z . (1956). WHO Chroiz. 10,273. Gelfand, H. M. (1971). An. Esc. nac. Saud. Publ. (Lisboa) 5, 1933. Goodman, N. M. (1971). “International Health Organizations and their Work”, 2nd edn. Churchill Livingstone, London. Hamon, J., Pichon, G. and Cornet, M. (1971). Cahiers OASTOM 9, 3. Hoogstraal, H., Kaiser, M. N., Taylor, M. A., Gaber, S. and Guindy, E. (1961). Bull. Wld. Hlth. Org. 24, 197. Jordan, P. (1972). Br. rned. Bull. 28, 55. Jordan, P. and Webbe, C. (1969). “Human Schistosomiasis”. Heinemann, London. Langmuir, A. D. (1963). New Engl. J. Med. 268, 182. Lapeyssonnie, L. (1972). “Le cholera a vibrions El Tor”. Thesis, University of Montpellier. Lumsden, W. H. R. (1972). Br. med. Bull. 28, 34. Lumsden, W. H. R. and Marsden, P. D. (1971). Practitioner 207, 181. Mackenzie, R. B. (1972). Bull. Wld, Hlth. Org. 47, 161. Maegraith, B. G. (1963). Lancet 1, 401. Maegraith, B. G. (1965). “Exotic Diseases in Practice”. Heinemann, London. Maegraith, B. G. (1970). Trans. R . SOC.trop. Med. Hgy. 64, 199. Organization for Economic Co-operation and Development (1970). “Tourism in OECD Member Countries”. Paris. Peffers, A. S . R., Bailey, J., Barrow, G. T. and Hobbs, B. C. 11973). Lmret 1, 143.

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Peters, W. (1969). “Chemotherapy and Drug Resistance in Malaria”. Academic Press, London and New York. Ralika, K. (1966). WHO Chron. 20, 315. Reeves, W. C. (1972). In “Vector control and recrudescence of vectorborne diseases”. PAHOlWHO Scientific publication No. 238, Washington D.C. Ross Institute of Tropical Hygiene (1971). “Preservation of personal health in warm climates”. London. Schneider, J. (1962). “Les Maladies Tropicales dans la Pratique Mtdicale Courante”. Masson, Paris. Sikes, R. K. (1970). Am. J. Publ. Hlth. 60, No. 6. Simpson, D. I. H. (1972). Br. med. Buff.28, 10. Smith, C. E. G. (1971). Truns. R. Soc. trop. Med. Hyg. 65, Suppl. 73. Smith, C. E. G. (1972). Brit. med. Bull. 28, 3. Toffler Alvin (1970). “Future Shock”. Random House, New York. Wilcocks, C. and Manson-Bahr, P. E. C. (1972). “Manson’s Tropical Diseases”, 17th Edition. Bailliere Tindall, London. Woodruff, A. W. and Nelson, G. S. (1970). Practitioner 207, 173. World Health Organization (1958). “The First Ten Years of the World Health Organization”. Geneva. World Health Organization (1966). Expert Committee on Rabies, Fifth Report, Technical Report Series No. 321, Geneva. World Health Organization (1967a). Expert Committee on Filariasis, Second Report, Techn. Report Series NO. 359, Geneva. World Health Organization (1967b). Expert Committee on Onchocerciasis, Second Report. Technical Report Series No. 335, Geneva. World Health Organization (1967d). International Sanitary Regulations, WHO, Geneva. World Health Organization (1967~).“Chemotherapy of Malaria”. Techn. Report Series No. 375, Geneva. World Health Organization (1968a). “The Second Ten Years of the World Health Organization”. Geneva. World Health Organization (1968b). Expert Committee on Smallpox, Second Report, Technical Report Series No. 393, Geneva. World Health Organization (1969a). Comparative studies of American and African trypanosomiasis. Technical Report Series No. 411, Geneva. World Health Organization (1969b). International Health Regulations, WHO, Geneva, World Health Organization (1970). Expert Committee on Plague, Fourth Report. Technical Report Series No. 447, Geneva. World Health Organization (1971). WHO Chron. 25, 236. World Health Organization (1972a). “Oral enteric bacterial vaccines. Techn. Rep. Series No. 500, Geneva. World Health Organization (1972b). “The work of WHO in 1971”: Annual Report of the Director General to the World Health Assembly and to the United Nations. Official Records No. 197, WHO, Geneva. World Health Organization (1972~).WHO Chron. 26, 485. World Health Organization (1973a). “Vaccination Certificate Requirements for International Travel”. (Situation as on 1 January 1973). WHO, Geneva. World Health Organization (1973b). Wkly. epidem. Rec. 48, No. 1, 1, No. 3, 25. Wright, F. J. (1967). Scoft. med. J. 12, 279.

Control of Arthropods of Medical and Veterinary Importance WILLIAM N . BEESLEY Sub-Department of Veterinary Parasitology. University of Liverpool. Liverpool School of Tropical Medicine. England I . Introduction .................................................................................... I1. Mosquitoes .................................................................................... A Anopheline Mosquitoes ............................................................... B Culicine Mosquitoes ..................................................................... C Insecticide Resistance .................................................................. D. New Insecticides and Repellents ................................................... E Genetic Control ........................................................................ 111. Blackflies and Midges (Simulium and Culicoides) .................................... A Simulium ................................................................................. B. Culicoides ................................................................................. C Onchocerca in Animals ............................................................... IV. Domestic Flies and “Fly Worry” ......................................................... V Tsetse Flies (Glossina spp.) .................................................................. VI Blowfly and Screw-worm ..................................................................... VII Keds (Melophagus ovinus) .................................................................. VIII. Oestrid Flies .................................................................................... IX . Lice ............................................................................................. X . Fleas ............................................................................................. xr. Ticks ............................................................................................. A Cattle Ticks .............................................................................. B Resistance .............................. ............................................... C. “Pasture Spelling” (Rotational Grazing) .......................................... D The Use of Tick-resistant Cattle ................................................... E. Sheep Ticks .............................................................................. XI1 Mites ............................................................................................. A Animal Mites ........................................................................... B. Poultry Mites .............................................................................. XTII The Future of Arthropod Control ......................................................... References .......................................................................................

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115 120 120 123 125 127 131 134 134 137 137 138 142 146 151 152 157 161 163 163 167 170 170 171 171 171 175 176 183

I. INTRODUCTION This review deals with current and developing methods and materials for the control of arthropods of medical and veterinary importance. Some of these are parasites in their own right. while others act chiefly as vectors of one or several diseases. The control measures considered concern mainly the use of insecticides. but it cannot be stressed too strongly that good hygiene 115

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(or good animal husbandry) is a pre-requisite: the use of pesticidal chemicals is an integral part of good hygiene, but is not a substitute. Indiscriminate use of insecticides may actually unmask formerly unrecognized pests, themselves tolerant to the chemicals, but previously held in check by predatory insects. The benefits which have accrued from the use of insecticides can hardly be questioned: in the field of malaria control, for example, it has been estimated that in the period 1955/1965some 15 million people were saved from death by this disease, and the malaria eradication programmes have probably saved the occurrence of well over 2000 million cases of the disease in 1960-1970 (Bruce-Chwatt, 1971). The importance of insecticides is very plain in the control of arboviruses, plague, louse-borne diseases and onchocerciasis, to name but a few, and this makes nonsense of the hysteria that has been generated in some quarters about the dangers of pesticides, properly applied. An insecticidal material will be unusual if it is harmful only to the target insect, and someone will usually be able to demonstrate that a very large dose of a particular insecticide can kill at least some harmless species. By 1945, a few scientists were already concerned about possible chronic effects arising from the use of new persistent organo-chlorine compounds, but the public outcry really began with the publication of Rachel Carson’s “Silent Spring” (1963), fortunately and sensibly mellowed by the more balanced “Pesticides and Pollution” of Kenneth Mellanby (1967). In Britain, wild birds were found dead after they had eaten grain dressed with pesticides, and official reports on pesticide residues followed one another in rapid succession (Cook, 1964; Frazer, 1964; Wilson, 1969), while overseas many agencies were also studying the problem intensively (World Health Organization, 1971a; Menzies, 1972). It was shown, for example, that during the peak period for the time of use of dieldrin in sheep dips for the control of the sheep blowfly, English mutton kidney fat contained about 2.4 ppm (parts per million) of this insecticide, while the corresponding figures for AustralianNew Zealand and Argentinian imports were 0.03-0.05ppm. About the same time, residues of DDT/DDE in Australian butter reached a level of 0.28 ppm and of benzene hexachloride (BHC) in Danish butter 0.02 ppm (Cook, 1964). Apart from the general withdrawal of dieldrin in the mid-1960s (which was happening in several countries because of the development of insecticide resistance) there has been little significant change in the type of insecticides used in public health, and on animals, that was not due to the natural process of improvement and need to counter insecticide resistance. The general awareness of just which compounds are most suitable, from every point of view, has led to periodic lists of recommended insecticides (Knipling, 1968). Several countries have special arrangements for the acceptance into general use of new insecticides and new formulations of existing insecticides. In Britain, for example, the Ministry of Agriculture runs an extremely efficient notification scheme, known as the Veterinary Products Safety Precautions Scheme (VPSPS), in which details of new products are considered in confidence by committees of scientists, with particular attention to supporting evidence from animal screening tests (acute and chronic toxicity, carcinogenicity, teratogenic effects, dermal absorption, residues in various tissues,

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etc.). Additional material may be requested, although commercial organizations usually provide fully adequate data on each product. After a chemical has been cleared, the Ministry issues a special recommendations sheet, in which appropriate details are provided, with instructions as to the advisability for the operator to wear a mask, overall, respirator, etc., especially while handling concentrates. Quite apart from the VPSPS arrangements in Britain, there is also an “approval” scheme, in which the efficiency as distinct from the safety of the product is evaluated, so that the purchaser will know from the “A” on the pack that the product will in fact do the job claimed for it. Overall liaison between companies is greatly assisted by their membership of the British Agrochemical Association (the former Association of British Manufacturers of Agricultural Chemicals). While most insecticides are synthetic chemicals, and involve some form of spraying, dusting or dipping, other materials include insect juvenile hormone, insectivorous fish, bacterial spores (Bacillus thuringiensis), viruses, fungi or nematodes (Burges and Hussey, 1971 ; World Health Organization, 1970). Some form o f displacing competitor may be introduced, as has been attempted with certain natural populations of disease-carrying mosquitoes, or genetic control may prove feasible, as in the very successful swamping of local populations of screw-worm flies with sterile, irradiated male flies, specially produced in millions each week to clear the south-eastern United States of this pest. Despite these rather exotic means of controlling arthropods,research investigation is mainly directed at finding even more active members of existing groups of insecticidal compounds, such as organo-phosphorus, carbamate and pyrethroid chemicals, from which have come such insecticides as difenphos, butacarb, resmethrin and kikuthrin. Great efforts are directed to the elaboration of the modes of action of insecticides, the ways in which they may affect other fauna and flora, and particularly to increasing the gap between the levels of toxicity to mammals and insects. O’Brien (1966) suggested that the study of chemical pathways in insects might lead to the development of novel and much more specific insecticides, as by the examination of trehalose enzymes, the mode of action of the different insect acetylcholinesterases, a study of the peripheral inhibitory neurones, and detailed investigation of the structure of the layers of the cuticle. It has now been realized that organo-phosphorus and carbamate insecticides exert their effects primarily by a cholinergic action on the central nervous system, whereas effects on respiratory enzymes are probably in general of much less importance in insects, which rely on the near passive diffusion of oxygen from the tracheal system to the internal tissues. An interesting development in the study of the mode of action of insecticides has been the finding that in some triatomid bugs a diuretic hormone is released by the Malpighian tubules during the paralytic stage of poisoning by several types of insecticides (Maddrell and Casida, 1971). The hormone causes a very large increase in the volume of rectal fluid and a corresponding reduction in the volume of the haemolymph. The disturbances in water distribution and cationic balance resulting from the diuresis are thought to contribute to the eventual death of the treated insects. In addition to fluid loss

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a plasticizing factor is secreted which allows the plates of the exoskeleton to expand-this may be due to a pH or ionic change which pushes the cuticular proteins offtheir isoelectric point, thus allowing the microfibrils to slide over one another. In studies on carbamates it has been suggested that these show insecticidal activity because they are structurally complementary to the active site of cholinesterase: in effect they behave as neurohormones and interrupt the normal action of the enzyme so that acetylcholine accumulates at the synapses (Metcalf, 1971). It is also now apparent that one of the most vital sites of action of carbamates and other insecticides is the peripheral area of the thoracic ganglion. Work on insecticide synergists indicates that these block the enzymic detoxification of the insecticides with which they are combined, and that they can act either as analogue synergists or as inhibitors of microsoma1 oxidation (Wilkinson, 1971). Synergists for carbamate insecticides include aryloxyalkylamines, propylaryl ethers, thiocyanates and 1,2,3-benzothiadiazoles : the action of synergists such as piperonyl butoxide (which can potentiate both carbamates and organophosphorus insecticides) helps to prove the common basis for the observed cross-resistance between these two groups of insecticides (Plapp, 1970). Studies by Holan (1971) on DDT analogues have shown that certain members of the series are 25 times as active as the parent insecticides, one of the best being p-chloro-phenyl-substituted3,3-dimethyl oxetane (a spatial form reminiscent of the molluscicide metaldehyde). The main feature of these new insecticides is that the central area of the molecule should optimally be about 0.55-0.65 nm in diameter; the corresponding measurement for the particular compound mentioned is 0.57 nm. Such elegant experiments point the way to the design of further insecticides, and Metcalf et al. (1971) have synthesized many biodegradable DDT analogues. The original paper should be consulted for details of his studies with methiochlor, methylchlor, ethoxychlor and propoxychlor. Despite considerable efforts, however, certain details of the mode of action of even quite commonly used insecticides remain vague. For example, there is confusion about what happens when very small amounts of DDT are incorporated in the diet of housefly larvae: Walker (1970) claimed that at a dose rate of 10 ppm development to the adult stage proceeds quite normally, and the females have rather more follicles than usual. Sherman and Sanchez (1964), and several other workers, on the other hand, believed that there is a “delayed lethal effect”, in which many abnormal follicles are produced, and a proportion of the dosed individuals die, Walker suggested that factors such as continuous inbreeding and high-density rearing of laboratory colonies may be very important in the evaluation of insecticides, and this is probably particularly true when attempting to correlate results from different countries. The fact is hardly ever discussed that a strain which has been maintained in isolation since, say, 1950, is today physiologically different from its ancestors, although it is readily acknowledged that an insecticideresistant strain changes during continuous laboratory culture, depending upon the presence or absence of monitorable insecticide pressure. In addition to killing insects with chemicals, it may be possible to repel

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them from humans or animals with substances such as dimethyl phthalate, indalone or N,N-diethyl m-toludmide (“deet”); the latter compound was unfortunately discovered too late for use in the last war against scrub typhus mites. Modern repellents steer the attacking insect away from the intended victim more by neutralizing his naturally attractant odours than by providing a strongly repellent odour of the type obtained with oil of citronella. The threshold quantity for sex attractants, or pheromones, can be as little as about 30 molecules (10-14pg). Examples of these compounds come mainly from agricultural pests, such as methyl eugenol which attracts the tropical fruit fly, but studies are in progress on the pheromones of arthropods of public health and veterinary importance, since these could be incorporated with insecticides in self-dosing traps. One feature to come from the research is that one compound may be able to attract insects from a great distance, while another may cause the insect to stop flying and settle on the bait point. Some reference has already been made to the development of insecticide resistance, optimum conditions for which are provided in circumstances where tiny amounts of toxicant persist for a very long time, as in the case of low residues of dieldrin in the wool of dipped or sprayed sheep. The first case of insecticide resistance by an arthropod of medico-veterinary importance was that recorded in South African cattle ticks which evolved tolerance to arsenic. Details of the development of insecticide resistance appear in this review in mainly the sections on mosquitoes, blowflies and ticks. The mechanisms of insecticide resistance may be morphological, e.g. in thickened or spiny cuticle, behavioural, e.g. the avoidance by recently engorged mosquitoes of sprayed walls as resting places, or biochemical-the most common form, in which a by-pass of the normal physiological processes is achieved, e.g. the dehydrochlorination of DDT to DDE, or its reduction to DDD. It is an indication of the complexity of the subject that DDT resistance is usually recessive in anopheline mosquitoes, dieldrin resistance is nearly always intermediate, but resistance to organo-phosphorus insecticides is usually dominant (Brown and Pal, 1971). Again, in the housefly, Musca domestica, the gene responsible for the appearance of DDT-dehydrochlorinase lies on chromosome 2, along with one of the genes for carbamate resistance, while gene R2, concerned with the delayed penetration of DDT and organophosphorus insecticides, lies on chromosome 3; gene R3 is concerned with the development of microsomal mixed function oxidases and lies on chromosome 5, together with a second gene for carbamate resistance. Dieldrin resistance in M. domestica depends upon a gene of uncertain precise function on chromosome 4. Because of the practical problems of standardizing techniques for the evaluation of insecticide resistance in different parts of the world, the World Health Organization (1 970) has published detailed descriptions of techniques and necessary apparatus for testing the following: susceptibility or resistance to insecticides of adult and larval mosquitoes, human body lice, adult bed-bugs, reduviid bugs and fleas. Tentative or provisional instructions are given for the examination of larval and adult biting midges and blackflies, and also for adult sandflies, houseflies, stableflies, blowflies, ticks and cockroaches.

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Details are included for the bioassay of insecticidal deposits on walls, the bioassay of persistent fumigants, and for the determination of the irritability of adult mosquitoes to deposits of insecticide. Alternatives and additions to chemical control are discussed in the sections of this review which deal with mosquitoes and blowflies; such methods include the use of synthetic juvenile hormone (Wigglesworth, 1970) or of parasites and predators, and various forms of genetic manipulation such as the release of sterile males and “booby-trap” females. The latter has been discussed by Whitten and Norris (1967) in relation to the proposed control of the Australian sheep blowfly, Lucilia cuprina. It is suggested that certain species of insects are particularly suited to the booby-trap technique if they can be cultured easily and cheaply in the laboratory, have a low natural population density, are not attracted to humans (in case of accidental contamination when highly toxic insecticides are used on the fiies), but are considerably attracted to one another so as to ensure maximum bodily contact. In the case of L. cuprina the females would be dosed with a chemosterilant-which would sterilize them and the males which mated with them, or (using a resistant strain) with a suitable insecticide-which would not kill them but would prove lethal to males of a non-resistant strain.

11. MOSQUITOES There are over 3000 species of mosquitoes, including the vectors of malaria, yellow fever, dengue fever, filariasis and most of the arthropod-borne (arbor) types of encephalitis. There are over 500 species of Aedes, which is the dominant genus in temperate and cold areas, occurring right up into Alaska, northern Canada and the U.S.S.R. Culex is represented by about 300 species, most of which are in the tropics and subtropics, and include many of the socalled house mosquitoes. Approximately 350 species of Anopheles have been described, some 60 of which are known vectors of human malaria; most species are found in the tropics and subtropics, like Culex, but they also occur through most temperate zones and up to an altitude of at least 2500 m in South America, Africa and Asia. The control of anophelines and culicines will be considered separately. A.

ANOPHELINE MOSQUITOES

The control of anopheline mosquitoes is inextricably bound up with programmes for the control of malaria, so that it is convenient to begin with a brief consideration of the present status of eradication of this disease. The effectiveness, ready availability, cheapness and apparent safety of DDT led to the conception just after the Second World War of malaria eradication by the complete control of anopheline mosquitoes in all the important malarious areas of the world. It was believed by many that the residual application of the new “cure-all” insecticide would result in extermination of the vector and thus complete interruption of transmission in endemic areas.

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It was hoped that 8- 10 year eradication progranirnes would be organized, carried through in four stages : preparatory, attack, consolidation and maintenance. These four phases are now established in the literature and correspond as follows : (a) preparatory-the epidemiological and geographical reconnaissance, establishment of services and training of staff; (b) attack-a total coverage of the target area with the appropriate anti-malarial measures; (c) consolidation-active, intense and complete surveillance with the object of eliminating any remaining infections ; and (d) maintenance-the period which begins when the criteria of malaria eradication in the area are met, and which continues until world-wide eradication has been achieved. By 1959 malaria had been reported as completely eradicated from new “maintenance”areas with a population of some 280 million, including the United States, five republics of the U.S.S.R., the Netherlands, Singapore, Italy, Corsica, Cyprus, Chile and parts of the Caribbean. By this time also 60 eradication programmes were in operation, and up to 1966 there was a steady increase in the numbers of people living in areas from which malaria has been eradicated (Wright et al., 1972). Thereafter, progress was less rapid but in late 1970 the corresponding figure in maintenance areas was about 710 million, and the fact that a total of 1000 million people now live in areas which have been freed from the threat of endemic malaria (maintenance and consolidation stages) is acknowledged as an achievement unique in the field of public health, reflecting great credit on the governments concerned. The “attack” phase is now in action in countries and states which have a combined population of about 350 million. There has been a move in the strategy of malaria control from preparation and attack to consolidation, in other words a change from attempts at I00 % eradication to programmes of extensive control. This has usually been for technical, administrative or financial reasons, although the dual problems of resistance to insecticides by Anopheles and to drugs by Plasmodium have gradually become more significant. Another difficulty is the movement of people from one area to another, as in Afghanistan, sometimes complicated by national disaster, as in Pakistan. It has been felt that the employment of the different available methods of control and their adaptation to local conditions should be more fully considered, and malaria control programmes have been replanned to take into account good control as a valid and indispensable interim step where complete eradication may not at present be feasible (Wright et al., 1972.) A complete review and map of the present malaria control measures in Africa, Europe, the Mediterranean, the Americas and South-East Asia are presented in a recent World Health Orginization publication (1971a). In 1970 a total of 18 countries had been placed on the official W.H.O. register of areas where malaria eradication had been achieved, with a further 19 for which eradication had been claimed but not yet registered. W.H.O. was helping 43 countries with their programmes, seven were carrying out programmes without assistance, and 28 countries were helped with miscellaneous antimalarial investigations, e.g. a survey of the situation in West Iran. Newly laid eggs of Anopheles usually require incubation for 2-3 days before

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they hatch, although those of A . albimanus and A . balabacensis may remain dormant for at least 23 weeks on mud, hatching in 3-4 min when flooded. Some species, such as A . walkeri, can overwinter in the egg stage. Domestic utensils such as tins, pots and bottles are generally unsuitable for anophelines, although the Indian A . stephensi can breed in a variety of man-made receptacles. Eggs are usually not laid in large open areas of water so much as in small pools or pockets of still water along rivers and lakes. Most species prefer fresh water, but some are found in brackish water, e.g. A . albimanus, A . gambiae melas, A. labranchiae atroparvus, A . sacharovi, A . stephensi and A. sundaicus. Some species will breed in polluted as well as clean water, e.g. A . stephensi, A. punctipennis, A . albimanus and A . vagus. Potential anopheline breeding sites of still or slowly moving water should not be regarded as unsuitable for breeding, or cleared of larvae, unless frequent and continuous surveys have produced negative results. Larvicidal chemicals which are employed against mosquitoes include oils, 5 % Paris Green pellets (840 g/hectare), malathion (224-672), fenitrothion (224-336), DDT (224), BHC (112), heptachlor (1 12), dieldrin (1 12), fenthion (22-112), difenphos (56-1 12) and Dursban (11-16): W.H.O. (1972). All new insecticides found to have activity against mosquito adults are tested against larvae, and hundreds of new compounds are under examination at any one time. Many of these are vapiations of existing active organo-phosphorus and carbamate groupings, but some are of novel form, such as benzene sulphonamide (Beesley, 1972). With the current anxiety about the contamination of the environment with potentially toxic insecticides, there has been some re-examination of the use of oiling for mosquito larva control. In one test kill, the speed of entry of coloured oils into the tracheal system and the surfacing habits of the larvae were compared during exposure to severaI petroleum derivatives at a dose rate of 0.5 gal per acre (Micks et al., 1972). Ae. taeniorhynchus was shown to be more affected than was Ae. aegypti although, as the effects were mainly due to asphyxiation, death did not usually occur for several days. Adult anophelines normally rest during the day time in vegetation, rot holes in trees, crevices in rocks and soil, etc., but many species rest on the walls in barns, animal shelters and houses. Their flight range may vary from only a few hundred metres to as much as 29-45 km, e.g. A . pharoensis. Anophelines, like other arthropods, may also be transported long distances in cars, trucks, trains, ships and aircraft. Resting places used inside houses tend to be the most humid and dark areas, such as the lower parts of the walls, undersides of pieces of furniture and backs of pictures. The adult female often takes her blood meal during the night following her emergence from the pupal stage, resting in or near dwelling houses. Many anophelines feed during the day, but this tends to be in shaded woodland or in the dark interiors of houses. Aestivation is another important feature of mosquito biology, and A . gambiae can survive for 9 months in the semi-desert region of the Sudan (Omer and Cloudsley-Thompson, 1970). All these characteristics are of great importance in control, and the habits of the vector species must be well understood by the organizing personnel engaged in a control programme.

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Adult mosquitoes are most commonly controlled by the application of residual insecticides applied by spray to the interiors and, where convenient, exteriors of houses. DDT is in practice the insecticide of choice in many countries and standard doses of 1-2 g m-2 applied 1-3 times annually are used, usually applied as a water suspension of 75 % water-dispersible powder. Malathion (2 g m-2), and to a lesser extent dieldrin and BHC (both 0.5 g m-2), are next in importance as residual insecticides, especially in areas where resistance to DDT has occurred (see below). The use of dieldrin has been abandoned in most countries because of the possible hazard to man and domestic animals. For space spraying, fogs, dusts and mists are employed, with residual doses in g per hectare as follows: carbaryl (224-1120), DDT (224), malathion (112-560), BHC ( I 12-224), fenthion (1 12), and dichlorvos (56-280). Ultra-low volume aerial sprays of phoxim, fenthion and Dursban have also proved effective. Smoke from insecticidal coils is widely used in South-East Asia, Japan, South America and parts of Africa, as a means of abating the mosquito nuisance. Mosquito coils commonly contain pyrethrum, and may include 20-40 % of 1.3 % pyrethrum powder, 25-30 % water-soluble glue, 3040 % of filters, 0.2-0.5% benzoic acid (as fungistat), and perhaps a dye such as malachite green. They are commonly prepared by extrusion from a belt of wet paste, and smoulder for about 8-10 h. Hudson and Esozed (1971), in Tanzania, examined several types of coils, including some which had been made in China and contained 74-13 % DDT. Against A . gambiae and Mansonia uniformis it was shown that coils containing DDT were more repellent and deterrent than those containing pyrethrins, although this may have been related to the large amount of insecticide in the DDT coils. DDT produced very high mortalities in 24 h, probably due mainly to its attachment to the mosquito netting of sleepers in the experimental huts. Not more than about 80 % of the pyrethrins are present in the smoke, and these originate by volatilization from the hot part (170400°C) of the coil, the glowing tip (700°C) being sufficient to destroy this insecticide.

B.

CULICINE MOSQUITOES

Ae. aegypti is recognized as the vector of urban yellow fever, four types of dengue, Chikungunya virus and mosquito-borne haemorrhagic fever, while species of Culex transmit a number of arthropod-borne types of viral (arbovirus) encephalitis and filariasis. Sylvatic (jungle) yellow fever is normally a disease of monkeys, and is transmitted by Haemogogus spegazinii falco, Ae. simpsoni, Ae. africanus and Ae. leucocelaenus; it occasionally passes from the sylvatic cycle to the urban cycle when men in the forest are bitten by infected culicines. Aedes breeding places are extremely diverse and an important feature is that the egg shell is very resistant to external agents and can survive several years. Larvae may be found usually in temporary pools where the water level rises and falls, in floodland along the coast, in irrigation ditches, paddy

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fields, etc. The flight range is very variable, from less than t m to at least 20 miles (32 km). It seems likely that Ae. aegypti originally bred in tree rot holes, logs, etc., but has gradually become more or less domesticated, so that larvae of this species are found in all manner of objects that can contain water: jars, old tyres, tin cans, pots, coconut husks, ant traps, underground cisterns, gutters, etc. Surveys of Ae. aegypti may be carried out on the basis of collecting larvae from breeding containers, and Tonn and Bang (1971), in Bangkok, used a single-larva-per-container technique to demonstrate changes in the incidence of this mosquito in domestic housing areas and a market. Just as a “houseindex” is used to indicate the presence or absence of, especially sparse, populations of adult Aedes, so the “Breteau Index” is employed to show the number of positive larval breeding containers per 100 houses (World Health Organization, 1972). After drying for over a year, Aedes eggs will hatch in a few minutes, and in parts of the South-Eastern United States it has been shown that eggs laid in the spring and early summer, then dried, hatch on first submersion in water, while later in the year there is a progressive fall in the number of eggs that hatch at first contact with water. Eggs can also hatch successfully after exposure to a temperature of 48°C (1 18°F) for 5 min, or to - 17°C (2°F) for 1 h (World Health Organization, 1971a, 1972). Heavy infestations of C. pipiensfatigans in towns may be the result of poor surface water drainage and pollution (Brown, 1972), while C. tritaeniorhynchus and other vectors of encephalitis multiply when irrigation and rice-field waters are allowed to accumulate. Domestic infestations of Ae. aegypti are frequently eliminated by the installation of a piped water supply. The elimination of aquatic vegetation is necessary where filariasis is transmitted by Mansonia mosquitoes, although the deliberate planting of larvicidal aquatic plants such as Utricularia may have a place in mosquito abatement. There is far too little information on the hibernation of mosquitoes, especially for the culicines in relation to the persistence of viruses; some of these have been taken from hibernating mosquitoes, such as Western Equine Encephalitis (WEE) from Culex tarsalis and Tahyna virus from C. modestus, but the role of the many species of mosquitoes that might maintain a virus through the winter months cannot yet be defined. Some species, such as C. pipiens, take no blood meals either before or during their hibernation. The sugar on which they feed enables them to amass fat stores so that their ovarian development does not begin until the following spring. Other mosquito species go into semi-hibernation with gonotrophic dissociation, and continue to feed throughout the winter at reduced levels; this happens in Europe with A . labranchiae atroparvus, for example, and can result in the transmission of malaria during the hibernation period. However, the nutritive requirements of most species, particularly those that hibernate in vegetation, are largely unknown, and this basic knowledge is vital for a fully effective programme of control. Chemical control of Aedes and Cirlex is by larviciding or by treating resting places of the adults with residual insecticides. Suitable larvicides include suspensions, solutions and granules of DDT, dieldrin, malathion, fenthion,

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tetrachlorvinphos (Gardona), difenphos or Dursban (Brown, 1972). At BoboDioulasso, in the Upper Volta, alarge-scale trial with the two latter compounds in pits and latrines indicated that 1 ppm of difenphos and 0.5 ppm of Dursban gave good control of Culex pipiens fatigans for at least 3 weeks (Subra et al., 1970); Dursban at 0.75 ppm remained active for 14 weeks. Controlled release formulations of difenphos, iodofenphos and phoxim have been used for the control of Ae. aegypti in drinking water storage drums (Taylor et al., 1969). The insecticides were carried on sand granules, neoprene rubber granules or in foam or solid polyvinyl chloride, and were effective against dieldrin-resistant mosquitoes for up to 20 weeks. The residues of insecticides used against the larvae should also kill adults which alight on the water to rest or lay eggs. Dichlorvos dispensers, containing 20% of the fumigant insecticide in resin, can be hung in the roof space and remain effective for about 6 weeks. The application of insecticides against adult mosquitoes should include treatment of eaves, the undersides of stilted dwellings, etc., using insecticides such as 1 % BHC, 1 % dieldrin, 1 % fenthion, 2.5 % DDT or 2.5 % malathion, as emulsions or suspensions. The treatment should be to run-off point, with an overall coverage aimed at about 05-2 g m-2. Residues last for about 4-5 months on surfaces protected from the weather, but perhaps only 1-3 weeks if exposed to rain or strong sun. Complete eradication of Ae. aegypti from a limited area is much preferable to 80-90 % control over a very large area, which rapidly becomes re-infested, and vector control programmes involving a sea port or airport, for example, should include a mile (I km) surrounding belt. Recent epidemics of yellow fever in West Africa have emphasized the need for means of rapid intervention in emergencies of this kind. Where the vector is Aedes simpsoni, which breeds in plants occurring over very large areas, such intervention may not be economically feasible. However, if the epidemic is confined to towns and villages where the vector is domestic Ae. aegypti, treatment can speedily be carried out by means of insecticidal fogs or the ultra-low-volume application of malathion from the air. Fogging machines and the appropriate insecticidal formulations have been purchased by the World Health Organization and are stored in West Africa for rapid dispatch to wheresoever they may be needed. Ae. aegypti adults are not attracted to light, and therefore light traps are useless in assessing the value of control programmes; a suitable technique currently suggested is to use glass oviposition traps (“ovitraps”), each of which contains 1 cm of water and an upright “paddle”, made from compressed fibre-board, measuring 13 x 2 cm. The traps are inspected weekly, the paddle removed, the jar cleaned, and a fresh paddle inserted. The collected exposed paddles are examined in the laboratory for eggs, which are hatched out so that the larvae may be identified (World Health Organization, 1972).

+

C.

INSECTICIDE RESISTANCE

From the original solitary observation in Greece in 1950 of resistance to DDT by A . sacharovi, the present tally of anopheline species resistant to DDT

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has risen to 15, with at least 37 instances of dieldrin resistance, and one of “increased tolerance” to malathion ( A . gambiue). Of the culicine mosquitoes 16 species have developed resistance to DDT, 12 to dieldrin and 9 to organophosphorus insecticides (Brown and Pal, 1971). In practical terms, resistance has occurred in the principal malaria vectors in many places during malaria eradication programmes. In temperate regions such as Europe, where malaria does not attain hyperendemic levels, resistance has not prevented the achievement of eradication. In these countries dieldrin resistance has generally been more intense and DDT has been used to complete the campaigns. In warmer countries the situation is very different, and eradication compaigns in, for example, Mexico and other parts of Central America have run into difficulties because of simultaneous resistance to DDT and dieldrin. In Africa considerable difficulties face the large scale effective control of malaria mosquitoes: DDT is not completely satisfactory, and there has been widespread resistance to dieldrin (which also means BHC, as resistance to both is genetically linked). Similar cross-resistance has occurred with Culex fatiguns, although in this case control can be carried out with organo-phosphorus insecticides as alternatives. The same situation has occurred with non-vector species of mosquitoes in South-East Asia and the United States. Between 1965 and 1971 the West African A . funestus, A . nili and A . rufipes developed dieldrin resistance, with DDT resistance in A . hyrcanus simensis (Okinawa) (Brown and Pal, 1971). It has been realized that DDT resistance in both anophelines and culicines is sometimes associated with DDTirritability : in Mexico, for example, a population of A . pseudopunctipennis appears to have developed an increased ability to escape alive from sprayed houses. As a result, an experimental technique has been described for determining the irritability of mosquitoes to test papers impregnated with 2 % DDT (World Health Organization, 1970). Among culicine mosquitoes during the 6-year period ending 1971 the number of instances of organophosphorus resistance increased from five to nine, including C. pipiens fatiguns to malathion in West Africaand Okinawa, and C. tritaeniorhynchus to malathion in Okinawa. There have also been new records of organo-chlorine resistance by C. pipiens in North Africa and Korea, and widespread resistance to DDT and/or dieldrin by Ae. aegypti, which also shows increased tolerance to malathion in Venezuela, the Congo and South-East Asia. Georghiou and Hawley (1971) have suggested that in a strain of mosquitoes treated in the larval stage with propoxur the larvae are more resistant than the adults to this insecticide, whereas the reverse is true when the adults are selected from a sister strain. When both larvae and adults are selected at the same time resistance develops to a similar extent in both stages. The identification of detoxication mechanisms with particular genes assists us in understanding the complexity of resistance problems in different countries, and indicates the probable outcome of the chemical control measures which are being applied. Resistance is nearly always due to single principal genes, and genes have been discovered that produce resistance to dieldrin in 17 species of insects, to DDT in 12, to organo-phosphorus compounds in four and to carbamates in one species (Musca domestica). The genes

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have been located on certain chromosomes by studies with linked mutant markers of which many are now recognized (Brown, 1967). Examples include white-eye plus malathion resistance in Culex tarsalis and white stripes on the dorsum of the adults and immature stages in dieldrin-resistant Anopheles albimanus. The actual mechanism of dieldrin resistance remains uncertain, but in the case of DDT resistance (by the DDT-dehydrochlorinase enzyme system) several genes may be involved. Still greater efforts are needed to find out more about cross-resistance, the speed of development of resistance (and the extent to which it develops in different species), the use of synergists, and the relationships between the probably hydrolytic detoxication of organo-phosphorus compounds and the probably oxidative detoxication of carbamates. D.

NEW INSECTICIDES AND REPELLENTS

Insecticidesare screened in accordance with a seven-stage evaluation scheme organized by the World Health Organization (Fig. 1). Stage I consists in establishing dosage and mortality curves for known susceptible and resistant mosquitoes, including dieldrin-resistant A . albimanus. Promising compounds are then tested in Stage I1 for neural toxicity, chronic toxicity, residual effectiveness on various types of building materials, etc. Stage 111 takes the candidate compound into simulated field trials, using rooms which have been sprayed with different formulations. In Stage IV field tests are carried out in experimental huts in tropical Africa and the United States, and further studies are conducted on insecticidal activity against susceptible and resistant mosquitoes. Stage V, carried out in Kaduna, Northern Nigeria, involves the spraying of up to 400 huts by the W.H.O. Anopheles control Research Unit No. 1 (ACRU I). Careful observations are also made on people concerned in the application of sprays, and on the rate of breakdown of the deposits on the walls of the houses. Another important feature is the degree of breakdown, settling and compaction of various formulations under tropical conditions. In Stage VI, 8000-10 000 houses are sprayed, with an associated human population of up to about 25 000. At least four rounds of spraying are carried out so as to take into account the full seasonal cycle. In the final stage, VII there is a full epidemiological evaluation of the compound in the most suitable formulation(s) which have been proved in the preceding work. By the end of 1970 more than 1400 compounds had been screened, and 300 reached Stages I1 and 111; 41 compounds were recommended for tests against anophelines in Stage IV, and in 1971 20 from this category reached Stage VI. Cost figures highly when considering the use of new insecticides as most of the countries that are operating malaria control programmes can hardly afford to use even DDT, so that those compounds which are more expensive than DDT may be too costly to use without heavy financial support from outside. The present cost of spraying operations with DDT for the control of malaria mosquitoes is estimated at about $60 million, whereas the annual costs if this insecticide were to be replaced by malathion would be over $180 million (Wright et al., 1972).

Structure of the evaluation programme and roles of collaborating laboratories source

I

Pesticide Manufacturers k Univeraitv Laboratories

1 1

Stage I (screening Tests) Stages I 1 k 111 (Laboratory h Simulated Field Tests) Stage IV (Field Tests)

W

m rn

Stage V (Village Trial)

r A

r m ?-I

Stage 1'1' (operational Field Trial)

Stage V I I (large-scale Tri a1 ) arc0 13?tj

FIG.1. The W.H.O. programme for the evaluation of insecticides.

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Because of the success of DDT and the organo-chlorineinsectides, it is not surprising that many DDT analogues have been investigated for the control of susceptible and resistant mosquitoes and other insects. In ane study (Metcalf and Fukuto, 1968) 120 analogues of DDT were examined, and it was shown that the relative effectiveness of these chemicals against DDTresistant Culex pipiens quinquefasciatus and A . albimanus is correlated with the susceptibility of the molecule to attack by DDTase at the benzylic hydrogen. The most effective compounds blocked this detoxication mechanism by o-chlorination, alpha-fluorination or by altering the aliphatic part of the molecule, as in nitrophenyl, neopentyl, dichlorocyclopropyl and trichlorobenzanilide derivatives. Several of these new compounds show practical possibilities for the control of DDT-resistance mosquitoes. Similar studies by Bailenger et al. (1970) showed that the compound 1,1,1-trichloro-2,2-bis(2methyl-4-hydroxy-5-isopropyl-phenyl)-ethaneis more toxic to adult A . stephensi than is DDT (LD 50 of 0.25 pg m-z), although this insecticide shows little activity against blowff ies or cockroaches, for example. The new pyrethroid insecticides prothrin and kikuthrin have been described from Japan and show good effectiveness against Culex pipiens pallens, houseflies and cockroaches. Prothrin is 5-propargyfurfuryl ( k )cis, transchrysanthemate (Ogami et al., 1970), and kikuthrin 5-(2-propynyl)-2-methyl3-furynethyl chrysanthemate (Nakanishi et al., 1970). Both insecticides are at least as effective as allethrin against mosquitoes. A new group of mosquito repellents has recently been described by Quintana et al. (1972). These are dihydroxyacetone mono-esters, characterized by branching, cyclization and unsaturation in the carboxylic component. It was found that, especially in perspiring human volunteers, compounds with a single branch in the chain were the more active, e.g. dihydroxyacetone mono(4-methyl-pentanoate) and dihydroxyacetone mono-(2-methyl-hexanoate). The new repellents parallelled the efficacy of the standard N,N-diethyl-mtoluamide (deet) only at high concentrations, These repellents may be added to the existing dimethyl phthalate (“Dimp”) and dibutylphthalate. It is a measure of the efforts made in this field that Smith et al. (1972) examined 1700 candidate repellent substances against Ae. aegypti, using impregnated mosquito bed-nets and head-nets ; the most effective chemicals included diamyl tartrate, hexyl-p-isopropyl mandelate, N,N-dibutyl-o-ethoxybenzamideand o-ethoxy-N,N-diethylbenzamide.Further studies on repellents (including repellent insecticides)may help us to understand why certain substances have this property, for example why female anopheline mosquitoes are more irritated by methoxy-DDT and DDD than by DDT, and the reason for the apparent decline of the irritating effect of DDT with the age of mosquito (Kaschef, 1970). Considerable confusion may follow the incorrect quotation of “proprietary” names (of which there may be be more than one in each country), “common” names (officially approved, but which are not necessarily identical in, for example, Britain and the United States, and “chemical” names (of which there are usually at least two for each compound, especially among the organo-phosphorus insecticides). The World Health Organization has there-

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fore coded all insecticides with an “OMS” number (Organization Mondiale de la SantB), e.g. malathion: 1, dichlorvos: 14, propoxur: 33, Dursban: 1155. The sense of the system becomes very obvious when arprocarb is considered, as this “common” name has unfortunately been applied to two different insecticides, or in the case of trichlorphon which has the trade names “Dipterex”, “Dyvon”, “Dylox” and “Neguvon”. The various names of the insecticides are listed at the end of this review. The many practical techniques used in the application of insecticides for the control of mosquitoes were considered in some detail by a W.H.O. Expert Committee on Insecticides (World Health Organization, 1971b), e.g. new modifications in hand-operated sprayers-the mainstay of most control operations, improved nozzle tips to reduce abrasion from the carrier in waterdispersible powder formulations, and new disc flow regulators. Some interesting details were that a droplet of 17.5-20pm, depending upon the insecticide, will kill one Aedes taeniorhynchus, and that the optimal droplet size for ground aerosols is 5-10 pm, while for ultra-volume space sprays it is 30-60 pm. Useful definitions were also provided for the sizes of the particles in various types of insecticidal applications e.g. aerosols (up to 50 pm), mists (50-100 pm), fine sprays (100-400 pm) and coarse sprays (over 400 pm). For residual spraying the particle size should be 10-20 pm, even as small as 5 pm for some organo-phosphorus insecticides. Although aerial application of insecticides is rarely employed for the control of anopheline mosquitoes, this W.H.O. booklet is intended for consultation by interested workers in other fields such as tsetse control etc., and there is a statement of the main characteristics of the 25 types of aircraft which have been found useful in ground treatment. Although the practical control of mosquitoes is usually carried out by treating the breeding areas with chemicals in order to kill the larvae, it may be that the water containing the larvae has economic or aesthetic value, in which case it may be possible to introduce mosquito-fish such as Gambusia afinis and Poecilia (Lebistes) reticulatus (Bay and Self, 1972). In one trial with G. ufinis in California, the fish were seine-netted in bulk from farm ponds, passed through mesh screens in order to select mature females, and released in rice fields at rates of 50-300 fish per acre, about 24 days after the rice had been planted (Hoy et al., 1971). A . freeborni comprised 95 % of the larvae in the later phases of the growth of the rice crop; Culex tarsalis was also present, and proved the more easily controlled of the two species. The higher fish densities led to a decrease in larval numbers to about 0.1 per dip compared with 0.45-0-6 larvae per dip in the fields that did not have fish placed in them. In some areas of Thailand the houses stand on stilts over accumulations of stagnant water into which passes a heavy load of faecal pollution. The pools are a favoured breeding site for the filarial vector C. pipiens fatigans, but may be controlled by the addition to the water of Lebistes reticulatus: one fish can eat 100 mosquito larvae in a single day. Once the larvae have been reduced in numbers the fish feed on the human faeces, and may consume 0.25 g faeces per hour (Gillett, 1971). Sometimes it may prove possible to use fish and

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insecticides together in integrated control. Predatory mosquitoes, e.g. Toxorhynchiies, Mucidus and Lutzia have also been used in mosquito control : Toxorhynchites brevipalpis and T . splendens were reared in the laboratory and released in Samoa in 1952; they became established after about 3 years, and it was hoped that their larvae would attack those of Ae. pseudoscutellaris (Peterson, 1956). There is also a possibility that parasitic nematodes (e.g. Romanomermis spp.), microsporidea (e.g. Nosema stegomyiae), fungi (e.g. Coelomomyces macleayae : Pillae and Rakai, 1970), bacteria (e.g. Bacillus thuringiensis) and viruses such as those of nuclear or cytoplasmic polyhedrosis (Bird et al., 1972) may become very useful in mosquito control, perhaps especially in integrated programmes. A polyhedrosis virus discovered by Bird et al. (1972), in An. stephensi was found to be present also in the sporogonic stages of the rodent malaria parasite Plasmodium berghei yoelii, transmitted in the laboratory by this species of mosquito. A “mosquito iridescent virus” which has recently been isolated from some African and American species of Aedes and from Psorophora ferox, is apparently very lethal against the larval stages of Ae. detritus (Hasan et al., 1971). Signs of the disease become evident 10 days after infection, with a blue iridescence in the sides of the abdomen and thorax. Death occurs 2 weeks later; infection can occur through the eggs of infected females or by larvae feeding on the bodies of their infected fellows. E. GENETIC CONTROL

Genetic control has been defined as “The use of any condition or treatment that can reduce the reproductive potential of noxious forms by altering or replacing the hereditary material” (World Health Organization, 1964); it is not limited to the use of insects that have been sterilized by irradiation or chemicals, and may include other mechanisms such as cytoplasmic incompatibility, hybrid sterility, meiotic drive, distorted sex ratios and lethal factors. The basic principle is to utilize factors that will lead to reproductive failure, but (to take a field example) the numbers of vigorous and competitive sterile males which are introduced into the population must greatly exceed that of the fertile males so that the chance of a sterile male mating with a native fertile female is correspondingly very high. For example, a population of a monocoitic species (Nelson et al., 1969) which contains 100 normal males, 100 normal females and lo00 sterile males should result in a 91 % reduction of the natural population, and the continued introduction of 1000 sterile males into each new generation should theoretically lead to the complete elimination of the population with 4 5 generations. The principle has been very successful in the control of the screw-worm fly, tropical fruit flies, tobacco hornworm, codling moth and other species of economic importance. Knipling et al. (1968) prepared an excellent review of the genetic control in insects of public health importance, in which past and suggested future studies are discussed in detail. Methods for the chemosterilization of insects by dusting, dipping, baiting or the use of females “booby-trapped” with chemosterilants all require much more investigation.

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Attention must also be paid to the degree of degradation of potentially dangerous mutagens. Careful studies are being made, using cell culture techniques etc. in order to identify the precise effects of chemosterilants on mammalian chromosomes. The exact dose of sterilant may be rather critical, as a dose which causes 100 % sterility may also make the flies less competitive in seeking their mates in the field, so that, in the long-term, a level of sterility of 95-99 % may in practice be most suitable. The sterile male technique may be used either by the addition to the natural population of overwhelming numbers of known sterile flies (treated usually in the pupal stage), or by treating the wild population with a sterilizing chemical, the object in either case being to damage the DNA in the chromosomes of the spermatozoa without impairing the mating activity of the males. The technique is potentially some ten times as effective as the use on one occasion of an insecticide which gives 90 % control, because the genetic technique becomes increasingly effective as the size of the population diminishes. Costwise, too, the method is attractive: the cost of eradicating the screw-worm fly from the south-eastern United States was only about U.S.$8 million, while the accrued saving to the livestock industry is estimated at U.S.$ 100-200 million. The development and experimental use of chemosterilant materials is a very active research field. This type of chemical may act by: (a) causing a failure in the production of ova or sperm; (b) causing the death of ova or sperm after these have been produced; or (c) producing multiple dominant lethal mutations or otherwise severely injuring the genetic material in the reproductive organs, so that the zygotes, if formed, cannot complete their development. The main problem with chemosterilants is that the target insect may not be suitable for large-scale laboratory culture (and consequent on-site sterilization) and the chemical may be insufficiently safe to humans to permit its general distribution. Immediately obvious alternatives include self-dosing with chemosterilants following attraction with pheromones or light traps. Of the chemosterilant compounds, one of the earliest to be discovered was tepa, (1,2-tris(1-aziridinyl) phosphine oxide, which has also been employed in the treatment of fabrics for flame resistance, crease resistance and in the curing of textile printing inks. It has been concluded that aziridine derivatives constitute the most promising series of compounds, the aziridine ring itself being the centre of activity, with the substituents considerably influencing overall activity (Smith et at., 1964). The aziridines are particularly active at high temperatures and low pH. The alkylating aziridine chemosterilants, such as tepa, thiotepa and apholate, and the non-alkylating aziridines, such as hempa, have been investigated in considerable detail: Grover and Pillai (1 969), for example, have shown the effectiveness of hempa against larvae and adults of Ae. aegypti and C. pipiens fatigans. Other chemicals which possess some degree of sterilizing activity include colchicine, piperonyl butoxide, riboflavine, tyrosine, uracil, methionine, s-triazines, phosphoramides and derivatives of acetamide, thiourea, glutamic acid and butyric acid (Smith et al., 1964; Grover and Pillai, 1969).The interest in phosphoramides and s-triazines, which affect the fertility of C. pipiens

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and Ar. urgypti, among other species, centres on the fact that these compounds are physiologically less reactive, thermally more stable and have low toxicity to mammals; their residual activity is also greater than that of the aziridines. Grover and Pillai (1969) showed that adult C. pipiens fatigans are sterilized following the exposure of the second instar larvae to as little as 10 ppm of certain s-triazines. One compound, 2,4-diamino-6-morpholino-striazine hydrochloride, coded as ENT-51143, had an LD 50 to 2nd stage larvae of less than 1.0 ppm, but was not as effective a chemosterilant as some other s-triazines. Both the phosphoramides and s-triazines that were tested produced a greater degree of sterility in the females than in the males. Experiments in the control of mosquitoes by the release of large numbers of sterile males have not been very successful (e.g. Morlan et al., 1962), but in the village of Okpo near Rangoon the release of males of a genetically incompatible strain resulted in the virtual elimination of C. pipiensfatigans (Laven, 1967a). Chemosterilized C. pipiens quinquejusciatus were employed in a successful eradication project on an island off the coast of Florida (Patterson et ul., 1970). Various other experimental control schemes have failed or had inconclusive results, but in India the World Health Organization, the Indian Council of Medical Research and the United States government have jointly established a Unit on the Genetic Control of Mosquitoes. The unit will study the general operational feasibility of genetic control and the economics of various types of schemes. Among certain species of mosquitoes, the cytoplasm of the eggs of one population is to a greater or lesser extent incompatible with the spermatozoa of another, so that matings are partially or completely sterile. For example, in strains of C. pipiens from different geographical areas, matings may result in normal numbers of progeny (compatible matings), few (partial compatability) or none (incompatible matings) (Laven, 1967b). In the incompatible matings a cytoplasmic factor in the ova prevents gamete fusion, resulting in a haploid condition and death of the eggs during embryogenesis. This form of sterility is inherited through the females. It has been suggested by Yen and Barr (1971) that the mysterious factor in the ovum may be a rickettsia-like organism, possibly identical with the Wolbachiu pipientis described from C. pipiens and Ae. aegypti. The organism was found in adults, eggs and embryos, but not in mature sperm. It was also considered that “partial incompatibility” may be due to destruction of sperm by some substancewhich is associated with ova only at a particular stageof development. An example of sterile mating is that of’ Ae. scutellaris and C . pipiens and several studies have been made on the feasibility of using such hybrids in the elimination of a vector species. Gubler (1970) showed that males of Ae. albopictus readily mate with females of Ae. polynesiensis, and in so doing compete strongly with males of the latter species, the major vector of aperiodic Bancroftian filariasis. The presence of the Ae. albopictus sperm and the coital plug in the spermathecae apparently physically blocks re-insemination by Ae. polynesiensis males, effectively preventing embryonation of the hybrid eggs. The barrier to hybridization appears to be much stronger in the cross described than in the reciprocal mating. In addition to the genetic competition,

fiitigans

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W I L L I A M N. B E E S L E Y

the larvae of the more dominant species have a higher survival potential than those of Ae. polynesiensis. Field trials of the method as a practical means of eliminating the vector species are now under active consideration. Two laboratory colonies of morphologically identical “ A . farauti” (within the A . punctulatus species complex), one from New Guinea and the other from Queensland, Australia, were crossed and produced only sterile male and female progeny (Bryan and Coluzzi, 1971). The parent strains had very similar karyotypes, but there were differences in the gene arrangement on chromosome 2, and in gene homology. In studies on the A . gambiae complex, five related “sibling species” have been identified: A, B, C, melas and merus, of which the first two are efficient vectors of human malaria. Crosses between these forms produce sterile males and fertile females, and Davidson (1970) introduced into a natural population in the Upper Volta sterile males bred in the laboratory from crossing A . gambiae “B” males with A . melas females. The sterile males mated well in preliminary experiments, but in the field the level of mating between them and the normal females of an “A” form was so low as to have a negligible effect on the main population. 111. BLACKFLIES AND MIDGES (Simulium and Culicoides) A.

Simulium

In many hyperendemic areas of West and Central Africa, 4 1 0 % of the population may suffer from serious ocular lesions and blindness, and the rate of onchocercal blindness in some villages may reach 36%. Control of the vectors of human onchocerciasis in Africa and the New World is being achieved with the use of larvicides and adulticides. The species of Simulium concerned in Africa are damnosum, by far the most important, the neavei complex, and in Central and South America S. metallicum, S. ochraceum and S. callidum (there may also be secondary vectors such as S. gonzaleze, S. veracruzanum and S. haematopotum). A knowledge of the resting places of these vectors is important in order to obtain population indices, to select appropriate chemical control measures, and to collect specimens for blood meal analysis, determination of infection rates, age-grading, and other population analyses. Ground application is used for larviciding, while aerial treatment is used for both larviciding and adulticiding, but on balance ground Iarviciding tends to achieve the best results, at less cost, in African onchocercal foci where the vectors commonly breed in densely wooded streams rather than alongside large open rivers (McMahon, 1967). Aerial larviciding is more applicable in extensive breeding areas such as may be found in some parts of Canada and the United States. Emulsifiable concentrates, containing up to 33 % DDT, appear to give the most effective control of Simulium larvae and are applied at 0.03-0.5 ppm per 30 min flow. Several of the papers which deal with the control of Simulium in Africa appear under the name of McMahon, who has been personally responsible for the detailed organization of many of the surveys and control programmes.

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Total eradication of a vector simuliid has in fact been achieved in only one country, Kenya, where McMahon eliminated S. neavei from 6000 square miles, having previously surveyed every stream and river within an area of 15000 square miles. As discussed by Nelson (1970), the work was to some extent facilitated by the following features: (a) the local vector, S. neavei, had a very limited flight range and did not cross open country between watersheds; (b) the area concerned was geographically isolated ; (c) breeding took place mainly in terrain between 3500 and 7000 ft (1000-2100 m) which was also well supplied with access roads; (d) the streams were all perennial so that the spraying campaign could continue throughout the year; and (e) it had been shown that S. neavei has an obligatory association with crabs, so that the examination of crabs for larvae and pupae of SimuIium was a better index of the degree of control than would have been the extremely laborious process of searching for the immature stages on vegetation. It is interesting that S. nyasalandicum, which does not transmit Onchocerca, is also associated with crabs. Eradication of S. damnosum is much more difficult than that of S. neavei. The flight range of damnosum is at least 100 km (62-5 miles), and in the savannah areas of Africa the flies may survive throughout the dry season, when many rivers dry out completely, Because wide-scale eradication may be impossible in practice, the best thing may be to limit larviciding to those areas which have a high incidence of blindness, as distinct from the larger areas where biting is only a nuisance. Eradication of S. damnosum may be possible simply by interfering with the environment in which the immature stages live, as by the flooding of rapids by the waters backing up from a new dam (Owen Falls, Uganda), although of course the vector flies may continue, or resume, breeding downstream from the dam where fords, spillways, etc. make new rough water (Nelson, 1970). More commonly the application of insecticide may be carried out more easily with the construction of the dam, as the water flow is then being carefully controlled, and the DDT may be added quite precisely. The standard example of this is at the Jinja dam, Uganda, where the breeding of S. damnosum for more than 50 miles downstream has been controlled by the regular application of DDT. DDT was used first as an aerial spray along 8 km of the banks of the Nile, but the good results obtained were thought to be due to the coincidental larviciding of the banks, and 10 weekly treatments were then applied, each of 1 part DDT in 2 million of river water. Eradication appeared to follow, but re-infestation from an unknown source meant that further treatments had to be applied in 1952, 1956, 1961 and 1964, using as little as 0.036 ppm DDT per 30 min. The overall picture seems to be one of virtually complete eradication with periodical re-infestations, and the situation is now that some 225 000 people in an area of about 1600 square miles (4150 kmz) are protected from S. damnosum (see McMahon, 1967). The scheme has also meant that a large area of fertile land, which has been virtually empty for many years because of onchocerciasis and the nuisance biting of the fly, has now been opened up. It must be mentioned that the Jinja situation was unusual because there was no breeding above the treated area, nor could there be massive re-infestation

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from downstream, whichever of these alternative foci constituted the source of the occasional contaminations. The importance of the potential 100 km flight range of S. damnosum cannot be over-emphasized. In the commoner type of focus, there are vast numbers of infested small streams, and eradication is then very difficult indeed. The few successes have included the Murchison Nile, Mayo Kebbi (Chad), Abuya and some minor schemes in Kainji (Nigeria), Sierra Leone (Tonkolili River) and the Congo at Kinshasa (Leopoldville). Depending upon the area of the control scheme and the personnel available, the preliminary entomological survey could take about 12 months to 3 years, and would then be followed by aerial or ground treatment of the larval habitats perhaps every 7-10 days, with monitoring of the fly densities (which might have originally reached 30-40 or even up to 250 flies per man hour and been reduced to less than 1.0 flies per man hour) and correlated monitoring of the larval populations. It may or may not be feasible to assess the interruption of transmission of onchocerciasis, as indicated by means of skin snips. At Abuja, Nigeria, for example, an incidence of 83% was recorded prior to larviciding, and 3 1% afterwards; the disease was still being contracted by children under 5 years. The optimal time for treatment was found to be in the early rainy season-May-July. The greatest decrease occurred in villages situated at least 6 miles (9 km) from the treated rivers, indicating that at and beyond this distance the parasite densities may fall below the threshold required for the transmission of the disease (Davies et al., 1962). However, the prevalence rate was not affected in some other parts of the area, suggesting that transmission can continue in the presence of low densities of the vector. S. neavei was virtually eradicated from Kenya in 1956, save for a small area which is more or less continuous with a large focus on the western slopes of Mt. Elgon, Uganda. Before the control programme adult densities of up to 300 flies per man hour were recorded, and the outstanding degree of control was a reflection of the comparatively easy accessibility to treatment of the rivers and streams, the costs of the operation being reasonable, at U.S.$ 1.02.4 km-2. S. neavei is not always found in such convenient foci, and in these instances modified control measures may have to be used. Control of onchocerciasis in Mexico, Guatemala and Venezuela has proved difficult because of the several vector species concerned, and no widespread eradication schemes have been carried through in these areas. The terrain is extremely difficult, with ranges of steep-sided mountains, and many swift rivers rushing through the thick forest. Both DDT and dieldrin have been used by ground treatment, with only moderate success despite great efforts. The programmes in Venezuela are having some success, but are most unlikely to result in anything like complete control in face of the natural difficulties, unavoidably high costs and incomplete information on the identity and biology of the vectors. None of the vectors of onchocerciasis to man appear to have developed resistance to insecticides, with the possible exception of S. damnosum to DDT on the Lower Volta River in Ghana. Resistance in non-vector species of Sitnulium has been reported since 1963 from Japun. ( S . uokii, S. ortiutim),

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Canada (S. venustum) and the United States ( S . venustum, Prosimulium fuscum) (Brown and Pal, 1971). The possible development of resistance in vectors of Onchocerca is being monitored very carefully in view of the importance of this crippling disease. The problem of the possible contamination of the environment by the application of insecticides to simuliid breeding areas has been discussed at some length; it has been suggested that the use of oil solutions containing 0.01-0.02 ppm of DDT or 0.1 ppm of methoxychlor (more biodegradable than DDT) would kill prepupae as well as larvae, and would also be less harmful to the other fauna in the water (Jamnback et al., 1970). In this connection it is of interest that Rivosecchi (1969) demonstrated the disappearance of simuliids from foci in the Apennine range of Italy by various forms of pollution and human activity, not specifically aimed at the destruction of the larvae. B.

Culicoides

Standfast and Dyce (1972) recently reported the results of a survey of the insects biting cattle during the 1968 epizootic of ephemeral fever in eastern Australia. A total of 21 collections were made, yielding about 18000 Culicoides (mainly C. brevitarsis), with 190 Austrosimulium pestilens. Viruses were isolated from the Culicoidesbut they did not include the causative agent of ephemeral fever, and the vector or vectors remain unknown, although C. brevitarsis is suspected. Control could be carried out by treatment of the breeding areas, which can be associated with wet cow dung, and by the use on the cattle of suitable repellents and insecticides. Although Culicoides probably does not travel as far as S. damnosum there is little doubt that it can be blown considerable distances on the wind, this perhaps explaining the rapid spread of ephemeral fever in certain years. Further studies are in progress on the identification of the blood-meals of the Culicoides which attack cattle in Australia, using precipitin and haemagglutination inhibition (HI) techniques (Murray, 1970). Blood from a single feed can be tested against 30-40 antisera, using wells of 0.001 ml in the Ouchterlony test or 0.025 ml of extract for the HI test. Although in Australia the problem of identification of the source of blood meals is not complicated by a large native ungulate fauna closely related to potential mammal hosts, it includes about 150 species of marsupials, and 650 species of birds. The development of this investigation in the next few years will help to indicate which species of Culicoides are most important in the transmission of diseases, and hence against which control measures should be directed. C.

Onchocerca IN

ANIMALS

It is very likely that Onchocerca in animals is much more common than is generally believed, and the infection rate of cattle with 0. gutturosa (0. lienalis) in Britain may be as high as 50% (Nelson, 1970), and with 0. urmillata in India even 100"/o. The vector of 0.gutturosa is Simuliirm oriiatum,

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of 0. cervicalis in the horse C. nubeculosus, of 0 .gibsoni in cattle C . pungens, but that of 0. armillata is not known, although it is suspected to be one or other of the subgenera. Control of the vector flies is usually by means of repellents or sometimes by the application of larvicides to the breeding places. There have been no instances of resistance to insecticides by Culicoides of medical (e.g. C. grahamii) or veterinary (e.g. C . pungens) importance, but in the United States and Panama resistance has occurred in C . furens to heptachlor, dieldrin, endrin and chlordane, but not to DDT or to organophosphorus insecticides; malathion was therefore chosen as the alternative for control (Brown and Pal, 1971). Elsewhere control with DDT or dieldrin is satisfactory, but usualIy in limited areas (Service, 1968). Two of the principal species which cause annoyance are C . impunctatus and C . obsoletus, which are responsible for considerable distress by their persistent biting activities. Control of the muddy or marshy breeding areas is difficult as complete surveying of all sites may present considerable practical problems, and some species tend to oviposit in rotting vegetation and garbage. The Bodega Bay gnat, Leptoconops kerteszii, a biting midge which breeds along the shoreline in California, developed resistance to DDT in 1961 after about 10 years’ use of this insecticide. It is not intended to discuss in detail the control of such non-vector “midges,” but these would include Psychoda alternata and Chironomus zealandicus, which cause general annoyance by swarming around people.

FLIES A N D “FLYWORRY” Iv. DOMESTIC In this catagory we may include the house fly Musca domestica, Australian bush fly M. vetustissima, face fly M. autumnalis, lesser house fly Fannia canicularis, head fly Hydrotaea irritans, black garbage fly Ophyra leucostoma, horn fly Haematobia irritans and stable fly Stomoxys calcitrans. These are all muscid flies, some sucking up fluids through non-piercing mouth-parts, others taking blood from the coarse wounds that they inflict. The latter species are of course closely related to the tsetse flies, Glossina spp., a muscid genus which has been allocated a separate section of this review. Some species of the group trouble animals hardly at all, e.g. M. domestica and F. canicularis, and even then only indoors, as in piggeries and poultry houses. Others, such as S. calcitrans, can take considerable amounts of blood, especially as they may attack in large numbers. Several species, such as Hydrotaea irritans, cause much distress by feeding on the natural secretions from the eyes, mouth and teats, giving rise to general irritability, restlessness and poor grazing. The life history of some of these pests remains uncertain, again as in the case of Hydrotaea irritans, the so-called “head fly” of sheep in Britain. The adults attack existing raw and sore places, including head wounds in rams and horned sheep, gorging themselves on blood; this species can also transmit the micro-organisms which cause summer mastitis in dairy cattle, but the favoured oviposition site of the adult fly has not been recorded. Related Russian species, H. pandelli and H. meteorica, actively

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scratch the skin surface of the host in order to Lake blood (Makhano, 1972). Control measures directed against adult flies include residual and space spraying inside farm buildings, and the use of baits and impregnated cords. Fly populations are subject to sudden and rapid increases, and control methods should be applied just before the seasonal renewal of the main breeding period. Suitable insecticides include pyrethrum, DDT, methoxychlor, BHC, dieldrin, chlordane, and such organo-phosphorus compounds as trichlorphon, coumaphos, fenthion, fenchlorphos, dimethoate, malathion and diazinon. DDT (2 g m-2) and dieldrin (0.5 g m-2) can give good control for 3-6 months, but the corresponding period for organo-phosphorus insecticides tends to be rather shorter, e.g. 2-34 months for fenthion and dimethoate. The addition of sugar to formulations of organo-phosphorus insecticides, at 2-3 times the strength of the active ingredient, improves their effectiveness. Cotton cords, spongy plastic bands, gauze or felted cellulose, impregnated with insecticide, may be installed at the rate of 1 m of cord per m2 of floor space, and can provide reasonable control for up to 6 months. For this type of control technique the temperature should not rise above about 32”C, nor should the relative humidity be less than about 50 %. Poison baits may be used at suitable sites in and around farms and places where food is handled. Dry baits may contain 1-2 % of insecticide in a carrier such as sand or ground maize cobs, with sugar added as a sweetener; alternatively, the sugar itself may be used as the carrier. Liquid baits would contain 0 . 1 4 2 % of insecticide and 10% sugar or other sweetener. Baits are sprinkled at the rate of about 60 g per 100 m2, or sprayed at 4 1 per 100 m2. Baits can remain effective outdoors for at least 2 months, but may need replenishment every 2-3 weeks, depending upon the weather. Cattle can be sprayed out of doors, or treated with various types of selfapplicators, including showering devices, “dust bags” and “back-rubbers” (placed so that the animals treat themselves on their way to feed or shelter). In one trial with dust bags, cattle had to pass between five 90 cm bags each filled with 1.8 kg 3 % crotoxyphos, while parallel tests were conducted with rubbers impregnated with toxaphene in used motor oil (Kessler and Berndt, 1971). Both techniques gave very good control of Haematobia irritans, but were much less effective against the wider-ranging M . autumnalis. Repellents and insecticides applied to the skin have generally not been very successful, because although some very effective repellents are available, these have to be applied fairly frequently, particularly as flies are most active and numerous during the warmer parts of the summer, when the animals are perspiring profusely. Repellents which have been used to help control “fly worry” on cattle, horse and sheep include dimethyl phthalate, dibutyl phthalate, diethyltoluamide (“deet”), and the butyl ester of 3-methylcinchonic acid (Yeoman and Warren, 1968). Crotoxyphos has proved effective in the protection of sheep against Hydrotaea irritans. Any accumulation of moist organic matter is a potential site for the breeding of some species of domestic flies, including those which irritate man and farm animals. M. domestica commonly breeds in refuse, M . sorbens, M . domestica vicina and Chrysomyia putoria in human and animal faeces,

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F. canicu1uri.s in animal and poultry excreta, and S. calcitrans in horse dung, wet straw and marsh grass. Farm manure heaps can therefore be a serious source of flies, but proper composting and regular turning of the fermenting mass can be as effective in controlling fly larvae as can the use of expensive insecticides. If the heap does become infested with larvae it may then have to be treated with an insecticide, such as diazinon, malathion, DDT, or methoxychlor. The importance of good hygiene, however, cannot be overemphasized, especially in the disposal of animal carcasses and offal: burning or burial is far better than saturating such waste with insecticide. Modern channelled liquid manure systems cannot support the development of stable flies, house flies, etc., although they may lead to an abundance of midges. Apart from the problem of resistance, fly larvae are not difficult to control if they can be reached by the operator, although the larvae of certain species, such as M . autumnalis, tend to be less susceptible than others to the usual concentrations of insecticides employed. A novel method of fly control is to feed insecticides to cattle, so that the insecticide eventually becomes intimately mixed with the dung. This technique gives good control of larvae of M . autumnalis and Haematobia irritans hatching from eggs laid in the droppings of the host. A recently published modification has been to encapsulate the fed insecticides so that a much smaller proportion is acted upon by the metabolic processes of the host. In one test 99.7 % of non-encapsulated tetrachlorvinphos and 85 % of the encapsulated insecticide were broken down in the tissues of experimental cattle (Miller and Gordon, 1972). The first indication of DDT resistance in M . domestica was reported from Arnas, Sweden, in 1946 (Brown and Pal, 1971), only 2 years after the first introduction of DDT into the country. First reports of DDT resistance in some other countries include: Denmark 1946, U.S.A. 1947, New Zealand 1948, Britain, West Germany, U.S.S.R. and Brazil 1949, Switzerland, East Germany and Australia 1950, Japan 1953 and India 1959 (Brown and Pal, 1971). Resistance may develop against one or more of the insecticide groups I (DDT, methoxychlor), I1 (BHC, dieldrin, etc.), I11 (analogues of group I that have no chemical groupings capable of dehydrochlorination, e.g. Prolan and Bulan), IV (organo-phosphorus insecticides, which in turn may be grouped into the diazinon-parathion and malathion subgroups), V (pyrethrins), VI (thiocyanates) and VII (carbamates). More research has been carried out on the physiology and genetics of insecticide resistance in M . dornestica than in any other species of insect, and it has shown that crossresistance may be negatively correlated between various insecticides. For example, phenyl isothiocyanate is negatively correlated with diazinon and dichlorvos, phenylthiourea with DDT (Ogita, 1958) and cetyl bromoacetate with DDT (Ascher, 1957). Wilson (1962) found that DDT-resistant M . dornestica can be killed more easily when DDT is reinforced by “WarfAntiresistant” (named from the Wisconsin Alumni Research Foundation) : N,N-di-butyl-p-chlorobenzenesulphonamide. There are numerous reports in the literature on the efforts which have made to control resistant flies by changing from one insecticide to another, e.g. from DDT to malathion, then

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to diazinon and fenchlorphos. A practical point is whether resistant strains of flies lose their ability to tolerate insecticides after release of pressure by the appropriate toxicant, and it appears that resistance against organo-chlorine compounds is more stable than is that against organo-phosphorus insecticides. However, reversion to a high level of resistance occurs soon after pressure has been re-applied (Brown and Pal, 1971). S.calcitrans developed DDT resistance in Sweden by 1949, but this did not appear to be paralleled in the United States, although dieldrin resistance was reported. Resistance to a variety of insecticides has been reported in F. canicularis, Haematobia irritans and 0. leucostoma. Very serious problems are currently presented by the spread of resistance against most of the organo-phosphorus insecticides. The multiplicity of defence mechanisms now know to be available to the common housefly, such as phosphatases, carboxylesterases,mixed-function oxidases, and reduced penetration, alone or in combination, appears to provide the field population of this species, and probably several others, with the ability to resist simultaneously a far greater variety of organo-phosphorus insecticides that was ever expected (Georghiou and Hawley, 1971). Chemosterilization has been used experimentally as an alternative means of controlling the housefly ; the compounds tested include those already discussed under Section I1 of this review. Hormones and hormone-like substances are also being tested for their effects on the development and fertility of houseflies, and Morgan and LaBrecque (1971) showed that both sexes were sterilized after treatment of either larvae or adult flies with 4-(2(2-butoxyethoxy)ethoxy-1,2-(methylenedioxy)benzene or with (E)-4-((6,7epoxy-3, 7-dimethyl-2-octenyl)oxy)-1,2-(methylenedioxy)benzene. Integrated control of M . domestica by the combined use of dimethoate and the chemosterilant hempa was attempted on two farms by Meifert and LaBrecque (1971) and proved superior to the use of either material on its own. Simple biological control by the use of pests and parasites of flies has been reported on a number of occasions and Vilagiova (1968)found the nematode Heterotylenchus autumnalis in 51 of 6217 individuals of M . autumnalis he dissected in Czechoslovakia. A particularly interesting form of biological control of flies was demonstrated by Norris (1966)in Australia: he showed that the dung beetle Onthophagus gazella, by burying cattle faeces, deprives the bush fly, M , vetustissima of much of its preferred breeding medium, so that fewer and smaller flies develop from the remaining dung pats. Dung beetles are now being released in southern areas of Australia and may well prove capable of reducing the menace of this insect to cattle. A development in the control of F. canicularis and Haematobia irritans has been the discovery that many flies are attracted to ultraviolet light, and this has been exploited by placing electrically-charged wire grids in front of “black-light” lamps. The light traps can be placed in alleyways in cowsheds, etc., and the insects land on the deadly screens as they fly towards the light source. This device works best against the larger and more robust species, such as blowflies, but is also very effective in the control of muscids in farm buildings, bakehouses, etc. (Tarry et a/., 1971). 6

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Surely one of the ultimate experiments in integrated control must be that described by Wicht and Rodriguez (1970), in which were employed predator mites (Macrocheles muscae-domesticae and Fuscuropoda vegetans), fly larvicides and poison baits! One of the insecticides used was a preparation made from Bacillus thuringiensis; it was shown that diazinon gave consistent reduction of the fly larvae with comparatively little effect on the mites. A parasitic nematode was ineffective, however, largely because of its susceptibility to desiccation.

V. TSETSE FLIES(Glossina spp.) The overall loss in potential income in Africa because of trypanosomiasis represents an area of about 10 million km2 (4.9 million square miles), with a lack of a potential cattle population of about 125 million (World Health Organization, 1969). The disease constitutes the greatest single impediment to the development of the livestock industry in Africa; in West Africa, for example, the Zebu cattle are bred north of the tsetse line and are then driven down through fly belts to the consuming areas, literally beginning to die as they start to travel. It would hardly be possible for an animal that had walked from the north of Nigeria to survive long enough to complete the return journey, were that necessary. It is estimated that 20% of the human population and 25 % of cattle in Nigeria are at risk from trypanosomiasis, although the number of clinical human cases is small. A joint F.A.OJW.H.0. Expert committee has stressed that further work should be done on the role of tsetse as vectors; studies should be made on its attacking behaviour in relation to its physiological state and investigations carried out on why one species of Glossina may have very different feeding preferences in different localities (World Health Organization, 1969). The species of vector can no longer be regarded as a valid criterion for determining the type of trypanosome that causes a given infection in man: rhodesienselike infections are known to result from bites by pallipides-group tsetse, and gambiense-like infections may be transmitted by flies of the morsitans group, but it would be unwise to assume that all species of Glossina are equally efficient as hosts to the protozoan or as vectors to the vertebrate. The basic method for surveying tsetse populations is by fly-rounds, in which “fly boys” walk along pre-determined paths at regular time intervals. The paths may be laid out so as to include various types of locality, or may be straight tracks cut along compass bearings. In this way detailed information is gained about the distribution of the different species and how this changes seasonally. Different species of Glossina have different diurnal activity rhythms, so that the catches are recorded as the “apparent density” traversed, say the number of flies per 10000yards (9140 m). For epidemiological studies both sexes should be included; indices used may be based on sex, pregnancy, age, state of hunger, etc., and this type of data may yield very significant results in the assessment of insecticides. Traps may be useful for catching tsetse, and of course economize on human or cattle bait; they can be improved

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by the incorporation of attractants such as extracts of pig skin or pig hair. Resting flies must be sought at night as well as in daytime, using €lies released after they have been dusted with a fluorescent powder. Resting flies are particularly significant in the case of the fusca group, which does not readily come to man. The identification of the types of resting sites for each species is essential for the most effective use of residual insecticides (Glover, 1967; MacLennan, 1967). In the case of G. morsitans, for example about 70 % of the flies resting during the daytime are fully or recently engorged, and they rest on tree trunks, quite close to the ground. Unengorged flies show a diurnal change of preference for resting places: tree trunks during the day, and the upper surfaces of leaves at night. Some species of tsetse are also associated with particular types of vegetation, as G. morsitans with isoberlinia woodland in northern Nigeria (MacLennan, 1967). Bush clearing was the first method of control to be employed against the tsetse, used originally at river crossings and watering places, then later to help develop the cattle industry. It is now realized that the degree and size of clearings must vary with the type of area concerned, and clearing is not now practised so widely as in the past. The time for this indirect method of control to take full effect may not be felt for several years, meanwhile backed up by manual or herbicidal maintenance of cleared areas, but clearing is nevertheless a valuable ancillary contribution to tsetse/trypanosomiasis control. One of the associated problems in a primitive community is that there may be a tendency to overstock and overgraze, leading to deterioration in the condition of the land, and eventually to erosion. Game destruction was formerly used as a method of tsetse control in East Africa, but it has now been realized that this is no longer necessary (Glover, 1967). Game barriers, on the other hand, may be used to limit the movement of large animals during insecticidal control operations, and one such obstacle, in Zambia, consists of two high fences separated by a gap of 2+ miles (Park et al., 1972). It is not difficult to sterilize tsetses in the pupal stage, and it would be possible to launch a control programme based on the release of laboratoryreared sterile males. The most promising approach might be one based upon auto-chemosterilization in the field, using some attractant selective to the tsetse, plus apholate or metepa (Chadwick, quoted by Glover, 1967). Attempts are being made to produce a suitable chromosome translocation in Glossina austeni. If a colony of fertile homozygotes bearing such a translocation could be cultured and released in the field, matings with wild types would give rise to heterozygotes, which would lead to a fall in the numbers of the target species. One necessary investigation would be to show that the translocation homozygotes were not more readily infected by trypanosomes than were the existing wild tsetse stock. Another biological approach to the control of Glossina has been based on the fact that the tsetse has a number of parasites and predators. This had an unexpected effect in the course of the successful insecticidalcontrol programme in Zululand, when the parasites were more affected by the chemical than was the tsetse, so that there was a resurgence in the numbers of the target insect

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(Fiedler et al., 1954). This type of experience indicates the need for careful studies on the natural host-parasite and host-predator balance, so the biological and insecticidal control methods can be fully integrated. Only the adult stage is available for insecticidal attack, as the fly produces a late third instar larva which rapidly pupates and hides in the soil or under logs etc., and at least one half of any tsetse population is at any given time in the pupal stage (Burnett, 1970). The insecticides so far used against Glossina have all operated by direct contact with residues, and it is interesting that insecticide control in West Africa began only in 1953. Out of the large number of insecticides available, only about seven have been employed in practice: DDT and dieldrin (the most usual insecticides), benzene hexachloride endosulfan, isobenzan, fenthion and pyrethrum, with a possibility that three others may be used presently : chlorbicyclen, bromocyclen and arprocarb, a carbamate. MacLennan (1967) and Burnett (1970) present details of these insecticides, with suitable formulations and usage for tsetse control. There is little doubt that many of the new organo-phosphorus insecticides are very effective against tsetse (Hadaway, 1972), but they are usually more expensive and less persistent than the organo-chlorine compounds, DDT, dieldrin, endosulfan and isobenzan. Lack of good persistence means more frequent application, with a consequent rise in labour costs. One answer to the problem is to use ultra-low-volume (ULV) application of insecticide concentrates (Lofgren, 1970), but despite this the costs of operations are likely to remain high. In areas with a lengthy intense dry season, DDT wettable powder can be used, and is effective for a long period, but in areas with a higher rainfall and shorter dry season, it is necessary to use an emulsifiable concentrate, usually of DDT or dieldrin, and to apply the insecticide more frequently. As examples of techniques of successful ground to ground treatment the World Health Organization (1969) cites the following: the use in the Sudan vegetation zone of Nigeria of a single application of 2.5% w.p. DDT to selected plant species in forest islands to control G. morsitans and G . tuchinoides. In Uganda “blanket” spraying of all vegetation at a height of 0.6-3.7 m is used against G . morsitans throughout the year. In Zambia, Rhodesia, Mozambique, Botswana and southern Angola DDT and dieldrin spraying is confined to the dry season, when the fly habitat is limited. In Kenya non-riverine habitats are treated by “block” spraying along lanes 37 or 46 m apart, with four monthly applications with DDT or dieldrin, while riverine habitats are sprayed in the same way along paths cut along river banks or along lake shores. All these techniques give good results. The costs of ground to ground treatment may be about U.S.$45-270 km-2, compared with aerial spraying at about U.S.$ 100-160 km-2. Before commencing any tsetse eradication scheme it is essential to ensure that the area is fully isolated and adequately surveyed (Burnett, 1970). The whole operation may be ruined by the immigration of flies from neighbouring areas. A survey may cost 20-25% of the total expenditure of the scheme, but may save perhaps 35% of the proportion of sites that would otherwise have been indiscriminately sprayed with residual insecticide (Gledhill and Caughey, 1963). An aerial spraying operation may cost E130-175 mile-2, but

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a great deal depends upon individual conditions and hourly flying charges, which may range from E8.75 to 639 per flying hour (Park et al., 1972). Aerial treatments must be carried out on complete areas, not piecemeal, but this is not usually too difficult as aircraft can spray large areas very rapidly, ensuring that the majority of the infective flies will be killed on the first run. The expense of cutting tracks is of course saved, and the aircraft fly on marker beacons, hydrogen-filled balloons or flares (Park et al., 1972); eventually a refinement of the Decca Navigation system may be used. To the normal aerial armament of Ansons, Pawnees, Cessnas, etc., may be added helicopters, which have now been used experimentally in the Niger Republic: a 15 km2 area of wood sprayed twice in 3 weeks with 5 1 of dieldrin/DDT remained completely free from G . tachinoides and G . morsitans for at least 4 months (Spielberger and Sivers, 1970). A 3-week interval between spray treatments is commonly recommended (Ye0 and Simpson, 1960). Aircraft normally apply insecticidesas a formulation in diesel oil, naphtha or a mixture of kerosene (paraffin) and diesel; concentrations of 1-21b insecticide per gallon are used, applied at 0-012-0.25 gal per acre, yielding 6 (isobenzan) to 14 (dieldrin) or 78 (DDT) g active ingredient per acre (Burnett, 1970). Good application of insecticide is difficult without careful consideration of the local weather conditions, with the overriding feature that the best time for flying will normally be the early morning, say from 15 min before sunrise, before the emergence of strong rising thermal currents. Using the ULV technique it can be calculated that a large (100 pm) droplet hardly drifts at all, while one a third this size moves considerably in only a very slight air current (Thompson, 1953). The small and large droplets could carry similarly lethal quantities of endosulfan and DDT, respectively, but the differences in efficiency of coverage could be considerable. So far there has been no substantiated report of insecticide resistance in tsetse flies (Brown and Pal, 1971), but this may merely reflect the common use of chemicals in what are virtually large-scale field experimental programmes. The situation may change considerably if insecticides are used on a really wide or even a national scale. The low density of tsetse populations is probably unfavourable to the development of insecticide resistance, because the smaller the number of individuals the smaller will be the pool of resistant genes. A 99.9% reduction of a housefly or mosquito population still leaves many flies in a small area, while in the case of Glossina this might result in only one or two insects in a similar small area in the field. It may be added that because of the comparatively slow kill of tsetse by certain insecticides, such as dieldrin and endosulfan (Hadaway, 1972), standard W.H.0.-type tests of susceptibility have to be run for up to 72 h ; it is also useful to employ for these tests only male flies, as this will at least reduce any inaccuracies due to the inclusion of pregnant female flies (Burnett, 1970). Lack of personnel, especially of trained field officers, is the most serious obstacle to the control of trypanosomiasis in Africa. Technical aid is needed as much as ever before in order to discover cheaper and more effective means of control, whether biological, chemical or integrated.

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VI. BLOWFLY AND SCREW-WORM The sheep blowfly problem is most severe in Australia and South Africa (L. cuprina), and Europe and New Zealand (Lucilia sericata). In warmer countries Lucilia tends to be replaced by the screw-worms, Cochliomyia (Callitroga) hominivorax and Chrysomya macellaria. Dieldrin emerged as the best of the large number of organo-chlorine insecticides produced in the 1940s and 1950s, and 0.05% dieldrin gives adequate protection to sheep for at least one complete summer season. Hill farmers were able to handle 800-1000 or more sheep on the fells, knowing that they would be safe from sheep strike. The situation in many countries by 1960 was that dieldrin, followed by DDT, benzene hexachloride, arsenic and tar-acid dips, were in common use. Around this time two things happened: first, the realization that some of the organochlorine insecticides were much more persistent than anyone had imagined, and were leading to the accumulation of high residues in animal products such as mutton, butter and milk; secondly, the discovery, in Australia, at least, that insecticide resistance at had last overtaken dieldrin. One or other circumstance has now led to the withdrawal of dieldrin from use as a sheep dip in many parts of the world. The replacement of dieldrin by the organo-phosphorus and carbamate insecticides was related as much to the activities of the anti-pesticides lobby as to the development of resistance. The publication of “Silent Spring” (Carson, 1963), “Pesticides and Pollution” (Mellanby, 1967), and similar books brought home to the public the potential risks involved in the then widespread and sometimes indiscriminate use of DDT, dieldrin and other organo-chlorine insecticides. By the early 1960s official reports had been published in various countries recommending changes in the ways in which the more persistent insecticides were employed. Following the publication of the Cook (1964) and Frazer (1964) Reports the insecticide dieldrin was, more or less voluntarily, withdrawn from use in Britain in sheep dips at the end of 1965. One of the common dip preparations in use until the withdrawal of dieldrin was a mixture of 0.016 % gamma-benzene hexachloride and 0.05 % dieldrin, and it was shown that, in addition to the immediate kill of blowfly larvae which took place when they were exposed to wool soon after and within a few months of dipping, there was also a so-called “delayed lethal effect.” It was shown that sufficient insecticide remained in the fleece to affect the older larvae, pupae and adults that developed from the first instar larvae exposed to the wool (Beesley, 1960). In the adults the effect took the form of the development of a large proportion of half-emerged flies, a lack of eggs or the production of infertile eggs, depending upon the length of the period from dipping to exposure of the larvae. A completely normal cycle of development was not obtained until the wool had weathered on the sheep for about 36 months. Similar sub-lethal effects have been recorded for other insecticides, but few studies have so far been made on this aspect of sheep blowfly control, and further research is in progress. The existence of extremely persistent tiny residues of dieldrin on treated sheep has certainly contributed to the development of resistance against this insecticide.

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Resistance to dieldrin was first observed in New South Wales, Australia, by Shanahan (1958), who reported the failure of high pressure jetting with this insecticide on some 15 properties. The LD50 of adult females was 1.25-2.5 pg per fly, compared with the normal 0.025 pg. By 1959 dieldrin was failing to protect jetted sheep for more than 3 weeks, whereas it had previously given at least 7 weeks’ protection. The organo-phosphorus insecticide diazinon was therefore recommended, but resistance to this in turn was eventually reported (Shanahan, 1966) and various other alternative compounds have since had to be used. The situation has become so serious that additional publicity has been now given to the alternative use of biological control of blowfly by surgical interference with the conformation of the skin of the sheep (Richardson, 1971). This old technique, known as the Mules operation, consists of the removal of a crescentic slice of skin on either side of the urino-genital area in the Merino. The operation is carried out before the onset of the fly season by efficient and experienced workers, and the scarring tissue flattens the natural folds of skin in this breed; there is therefore much less chance of accumulation of excretions, which would otherwise provide the main attractant stimulus for the ovipositing blowfly. The operation does not affect the rate of weight gain of the sheep (Richardson, 1971). In 1959, dieldrin resistance also developed in L. sericata in New Zealand and South Africa (Snelson, cited by Brown, 1961), and a single case of resistance was reported near Dublin, Irish Republic, by Shaw et al. (1968). It is surprising that in California, where houseflies had become resistant to DDT, L. sericata remained susceptible to this insecticide (Brown and Pal, 1971). It will be appreciated that although these findings were of interest, they were by now of only slight practical importance, as most sheep farmers had already begun to use organo-phosphorus insecticides. There would also have been little point in changing from dieldrin to BHC, as resistance to these two insecticides is genetically linked. Commercial trials of insecticides which have already been shown useful in preliminary laboratory tests are run with various wash and spray concentrations, perhaps 0-01-0.1 % for the dips and up to 0.25 % for jet sprays (high pressure) or tip sprays (low pressure). Groups of about 100-500 sheep, preferably including both ewes and lambs, are commonly used, and these should be located on farms in different parts of the country so as to include several breeds of sheep and to allow for local variations in the standard application technique. The size of the sheep dip bath may well vary from 150800 gal (700-3600 1). In order to parallel likely practical conditions, low pressure spraying may be carried out by a crop sprayer, knapsack machine or adapted weed sprayer, as well as by purpose-built spray races (Harrison, 1965). The efficacy of the insecticide will be evaluated by implanting first instar larvae weekly, and by examining wool samples in the laboratory, as well as by watching for natural strike (Beesley, 1960; Wood et al., 1965). The length of the fleece at the time of dipping should be as long as conveniently possible, say3-4weeks aftershearing, at least. If lambs of lowland flocks are dipped in late May or early June, it is usually possible to delay the main summer dipping of the whole flock until several weeks after the ewes have been shorn.

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Some larvicides are more active than others, and degrade more slowly, but the period of residual protection afforded is proportional to the amount deposited in the fleece at the time of application. Thus, if an insecticide has a half-life of 5 weeks as a result of degradation and dilution in the fleece, then a doubling of the amount deposited can result in a further 5 weeks’ protection. This increase in the amount of deposit may be brought about by frequent replenishment of the wash, which should be topped up with concentrate before it has lost more than 20% of its original volume. Even better is a system of continuous replenishment, which ensures a steady concentration of insecticide and facilitates handling of the sheep since the bath is always full and this avoids delays from the periodic replenishment. Sheep should be immersed for at least 30 sec. Organo-phosphorus insecticides currently in use include : bromophos (Harrison, 1965); chlorfenvinphos (Wood, et al., 1965); dichlofenthion (Brown, et al., 1965); carbophenothion (Treeby, 1967); pyrimithate (Ryley, 1969); fenchlorphos (Marquardt and Hawkins, 1958); coumaphos (Thomas, 1962); malathion (Riches and O’Sullivan, 1957); diazinon (O’Neill and Hebden, 1968);and the carbamate butacarb (Harrison, 1969); most of which are employed in this country as dips at concentrations of about 0.02-0.05%. The number of sheep dip preparations available in Britain at the end of 1970 was 85, of which 67 contained gamma-BHC, 5 arsenic and 13 tar acids (Ministry of Agriculture, Fisheries and Food, 1971). Ryley (1969) discussed some of the factors which affect the persistence of insecticides used for the control of Lucilia. He concluded that laboratory in vitro tests with serum, wool samples and larvae may not have any absolute significance, but are of comparative value. Under practical conditions the insecticide must be picked up from the dip wash or spray, be retained on the wool and not removed by weathering, yet it must not be so tightly bound that it is not available to kill eggs and larvae. In order to give extended protection it should be capable of diffusing into new wool growth, but on the other hand it must not diffuse into the tissues of the host and so be lost from the fleece, perhaps then giving rise to undesirable residues in the sheep. The candidate compound must also, of course, have a low mammalian toxicity. At least in some breeds, the outer wool has less lanolin on it than does the deeper wool; this is probably due to weathering, as the fleece of housed sheep is very greasy. In turn this affects the amount of insecticide that can be taken up by the grease, although in the case of dieldrin a certain proportion of the insecticide withstands several hours’ Soxhlet extraction with solvents such as acetone and petroleum ether, indicating possible adsorption by the keratin (Beesley, 1960). The duration of protection will also depend upon the actual length of the staple: a spray on sheep with short wool will easily reach down to the skin, whereas on a long-wooled sheep the insecticide will have spread too thinly to give protection near the skin. Some insecticides, such as dieldrin and chlorfenvinphos, have a marked ability to diffuse down the staple, and this again may considerably affect the degree of protection. In the extreme case of dieldrin the insecticide not only passes down the staple, but into the skin through the tissues, and up into growing adjacent wool; the phenonemon of “translocation” means that even rather patchy spraying of the surface of

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the fleece can give a very good final result. Unfortunately, translocation at the rate observed with dieldrin does not appear to occur with any other insecticides, despite their good protective effect. Ryley (1969) investigated the temperatures at different levels of the fleece, the lanolin content of the wool and the length of the staple, in relation to the season of the year. It seems quite possible that the lanolin content of the wool will vary with the time of year, and this is one of the factors that may be very important in explaining the variability in protection from fly observed with the same insecticidal formulations tested by experienced workers in different countries. Another important feature is the variation in wax content from one breed to another with corresponding different degrees in the uptake insecticide (O’Neill and Hebden, 1968). Several new types of compounds have been evaluated as possible insecticides, for example the organo-tins; Hall and Ludwig (1972) showed that trimethyltin chloride, distributed over wool clips, then artificially weathered and tested against larvae of L. sericata, began to fail only after about 5-6 months. The insecticide was then jetted on sheep at 0.025% and gave protection against natural strike for at least 13 weeks, whereas diazinon began to break down under the conditions of the test after about 2 months. Further studies are in progress, including a detailed examination of the mammalian toxicity, especially as no antidote has been named in cases of accidental overdose. A further example of the search for new insecticides to control blowfly was the investigation by Greenwood and Harrison (1965) of over 80 benzene and toluenesulphonamides and disulphonamides. Some of the substituted benzeneI , 3-disulphonamides were quite active against blowfly at concentrations as low as 0.003%, but none persisted in the wool of sprayed sheep for longer than 1 week. It was reported that the activity of these compounds was specific to blowfly larvae, and that they had no effect on adult L. sericata or any stages of the mosquito Aedes aegypti. Other sulphonamides had already shown activity against houseflies, chicken mites (Furman and Stratton, 1963) and mosquitoes (Beesley and Peters, 1968, 1971). An interesting development from the treatment of sheep with dieldrin for protection against L. sericata was the finding that the wool retained sufficient of this very persistent insecticide to give further protection against clothes moth and beetle in the finished wool. Although dieldrin has now been largely superseded for the control of blowfly on animals, the search for new mothproofing agents, as well as blowfly insecticides, continues. In a recent study in the United States, Bry et al., (1972) showed that “Ciba C-9491” (0-(2,5dichloro-4-iodophenyl) 0,O-dimethyl phosphorothioate) applied to woollens protected these against attack by larvae of the black carpet beetle, Attagenus megatoma. The residual activity of the insecticide lasted for at least 6 months on cloth that had been dry-cleaned once. Although Callitroga (Cochliomyia) hominivorax, the primary screw-worm fly of cattle, sheep and goats in the southern United States has now been eradicated from that area owing to the intensive “sterile-male” programme in 1958-1 959 (Knipling, 1960), the pest persists across the Mexican border, and

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considerable effort has gone into finding the minimum suitable width for the 250-mile buffer zone which has been created within northern Mexico; United States entomologists have also gone into Mexico and helped to keep back C. hominivorux behind the frontier. In addition to sexual sterilization of laboratory-bred flies, insecticides were used in certain stages of the eradication scheme, and work on new insecticides has continued (Drummond, 1967), as have studies on chemical sterilants (Demilo and Crystal, 1972). One of these, N,N‘-tetramethylenebis (1-aziridinecarboxamide) was applied to emerging adult screw-worm flies by allowing them to migrate through polystyrene foam strands coated with a 5 % solution of the chemical, or by immersing adults in 0.1-1-0% solutions. Good sterility was induced in the males which had been treated by either method, and the eggs of females mated with the treated males were much less fertile than those from normal matings (Crystal, 1971). The success of the sterile-male technique for the control of screw-worm naturally led to the consideration of this method for the eradication of other fast-breeding species, such as the sheep blowfly. Because of the concern in Australia that available insecticides may be “used up” quite soon, detailed investigations with L. cuprina have been carried out. As part of the project it would be very useful to be able to recover male puparia rapidly from a mixture of both sexes in artificial culture, because in the screw-worm programme all pupae had to be irradiated indiscriminately, despite the known differences in the most suitable doses of gamma rays for sterilization of the males and females in that stage of development. Whitten (1969) has recently described a genetic marker technique whereby the male puparia develop the normal brown colour, while the females become black (due to the deposition of melanin on the inner surface of the epicuticle of the pupa case). By using a machine that sorts the puparia at the rate of 7000 per hour, large numbers of puparia of known sex can quickly be made available. Among advantages to a control programme through an ability to sex pupae are that: (a) female pupae could be discarded to reduce expenditure on irradiation and liberation of redundant and possibly harmful sterile females ; (b) female pupae could be suitably parasitized before liberation ; (c) female pupae could be retained for “booby-trapping” (in which these pupae are dosed with a tiny amount of chemosterilant or insecticide, which sterilizes the emerging fly and also any males with which she comes into contact) as an adjunct to the release of sterile males; (d) females and males could be sterilized and released in separate areas. The latter possibility is now being investigated for L. cuprinu. The release of sterile females can also result in the failure of normal females to mate effectively. Sterile females remain attractive to males and, in their presence, the mating ability of normal males becomes limiting. These experiments suggest the release in separate areas of sterile males and females as a means of enhancing efficiency of the sterile insect method of control; this is being investigated further by Whitten and Taylor (1970), who have demonstrated that when a normal population is swamped 1OO:l with sterile females this leads to ineffective insemination, or a failure to inseminate, by the normal males, because these are limited to approximately ten matings each. Thus in nature this should lead to a decline in the overall size of the

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population. A further factor is that in normal populations the female L. cuprina mates only once, but attempts are being made to produce sterile polygamous females, which-if introduced in a control programme-would considerably tax the available amount of spermatozoa from the natural males. Further genetic manipulation might even lead to the development of a strain of sheep blowflies that oviposited exclusively on carrion or rubbish (Whitten, 1970), an interesting suggestion in view of the likelihood that at an early stage in their evolution these flies laid eggs on decaying organic material. It has been known for some time that many insects are attracted to light, and traps have been designed incorporating a light source and insecticide. The ultraviolet (u.v.) region of the spectrum is particularly attractive, and this phenomenum has now been used in the design of an electrocution fly trap. In a recent experiment, 600-1000 individuals of each of various species of flies were released 3 m from two parallel 20 W “black-light” lamps (wavelength 3100-4400 nm), placed immediately in front of a 3500 volt 10 mA grid (Tarry et al., 1971). About 60% of Phormia terraenovae, 40% of Lucilia sericata and 30 % of Calliphora erythrocephala were caught during the first 5 min after release. Corresponding figures for Stomoxys calcitrans, Fannia cancicularis and Musca domestica were 19, 17 and 12 %. It was concluded that the larger flies of the family Calliphoridae travel more directly to a U.V. source than do the smaller muscid flies. 90 % of the Lucilia and Phormia were recovered within 200 min while corresponding recoveries took over 100 min for Calliphora and Fannia. The energy available for flight apparently becomes depleted more rapidly in heavier species of flies, which soon tend to enter a short resting phase, while smaller flies may keep on the wing for much longer periods.

VII. KEDS(Melophagus ovinus) Keds tend to be most common on lambs, which become infested by direct contact with the ewes. Nelson and Slen (1968) showed that ked-free lambs each gained on average 3.6 kg more than infested lambs, while ked-free ewes produced 11 % more wool than infested ewes. The differences become apparent only after the development by the sheep of a skin immunity against the keds. The immunity is of a temporary nature, and this may help to account for the observed summer decrease in the parasite population on the adults, while ked numbers gradually increase on the lambs. Adult sheep also have slightly different levels of infestation, depending upon the time at which they originally became infested (Nelson, 1962). In the winter months the immunity of both adult and immature sheep wanes, probably in view of their poorer nutrition, and ked numbers rise to a peak in the late winter. Despite their superficial leathery-skinned resemblance to ticks, populations of keds are usually surprisingly easy to reduce in numbers, although not necessarily to eradicate completely. Most insecticides are effective, e.g. arsenic/lime sulphur, rotenone, DDT, BHC, toxaphene, methoxychlor, dieldrin, diazinon, fenchlorphos and coumaphos (Nelson, 1962). They may be

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employed in dips, sprays or (in the United States) dusts, while a novel alternative is to sprinkle the fleece with a formulation of an insecticide, such as diazinon, which has a high vapour pressure (Matthysse, 1967). Like dieldrin, diazinon also has the ability to “translocate” down the fleece to the skin surface following even light spraying of the wool. Pressure spraying at 200-400 Ib in -2 is effective, using & in nozzle discs for emulsions and & in discs for wettable powders (Nelson, 1962). 1-2 quarts (1.2-2.4 1) of wash per sheep are needed, depending upon the length of the coat. Spraying is favoured in many countries because of its speed and ease of operation. The question naturally arises as to why Melophagus has not been eradicated from Britain: one would have thought that the compulsory dipping which preceded the disappearance of sheep scab would also have led to the complete control of keds. It seems likely that the mandatory dipping arrangements were relaxed at about the same rate as the scab disappeared, first in the lowlands and then in the hills and fells. It may well be that the rapid relaxation of compulsory dipping with BHC in marginal areas where scab was never or no longer present, but where keds were a problem, may have led to a rising incidence of this parasite (Page, 1969). There was then probably a decrease in ked populations during the period when dieldrin was used against blowfly, but now ked numbers appear to have again increased. No surveys have been carried out, but it is possible that the highest incidences of ked infestation may be correlated with areas where there is little dipping against blowfly, ticks and lice.

FLIES VIII. OESTRID This group of Diptera includes several of the best known causes of myiasis: Hypoderma (ox warble or cattle grub), Gasterophilus (horse bot) and Oestrus (sheep nasal fly). The species of Hypoderma include the virtually host-specific H. lineatum and H . bovis of cattle, and H. diana of European red deer. Other species occur in goats, yaks, etc., and some of these have now been placed in separate genera. It is difficult to estimate the economic losses due to the activities of parasites, especially ecto-parasites (Gibson, 1964); many workers have studied the effects of warble infestation on the daily weight gain, milk yield, and restlessness of cattle (Bishopp et al., 1926), but results have often been conflicting. A recent study by Rich (1970) in Alberta, showed an annual loss of over $654000 from the activities of Hypoderma, i.e. weight of flesh trimmed from the carcasses, extra labour involved, and devaluation of the carcass and hide. The mean loss per carcass in Alberta ranged from $0.55 in the south to $3.18 in the north, while the cost of treatment with systemic insecticides was only $0.65 per head. The cattle warble flies should be remarkably susceptible to eradication by insecticides, as they are virtually host-specific, produce a single generation each year, and it is easy to see if an animal is infested with the mature grubs. Control of the larvae was formerly possible only by the application of derris

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to the skin of the back, after the third-instar maggots had made their breathing holes in that area. The treatment was satisfactory, but of course failed to prevent damage to the skin which seriously reduced its value or made it completely worthless to the leather industry (hides being graded solely on the presence or absence of fleshing cuts and open or blind warble holes). About 20 years ago the new class of systemically active organo-phosphorus insecticideswas discovered, and the compounds fenchlorphos and trichorphon were found capable of killing all the migrating first stage larvae within the host before they could reach the skin of the back. This was a great advance, and it pointed the way to possible eradication of the pest, because virtually all larvae in the cattle in one region can be killed by applying the insecticide soon after the last flies have laid their eggs on the hosts. Other systemic insecticides were developed, and the current list includes trichlorphon, crufomate and fenthion. Suitable methods of application have been investigated in some detail. Cattle may be dosed orally: by single application of a drench or bolus, as a feed additive over a period of several days, or allowed free access to medicated mineral licks (Drummond and Graham, 1965). The individually calculated dose is tedious to work out, only approximately correct in many cases, and may from time to time give rise to drenching pneumonia, especially if the animals are unused to handling. It is obvious that with feed additives or licks some animals may take more than others, leading to possible overdose and toxicosis or underdosing. Intramuscular dosing is slow, and may be attended by necrosis. The present standard technique is to cover (“pour-on”) the skin of the back with a systemic insecticide, which then passes into the blood and tissues of the host. A further technique for the application of systemic insecticides is to dress or spray the skin of the back. Dressing was formerly always carried out in the spring, using the contact insecticide derris (rotenone) to kill the third-instar larvae which had recently penetrated the skin from below. Most or all of the larvae would die, but because the insecticide failed to persist for more than a week or two, further dressings were needed to cope with the larvae which appeared later. The systemic property of the new insecticides meant that they could be used once, in the autumn, to kill all the first-instar larvae that had penetrated the host from eggs laid during the summer. The modern systemic insecticides are used in concentrations of 6 8 % and administered, by “pouron”, to give dose rates of 25-50 mg per kg body weight. Sprayed systemic insecticides have been used in concentrations of about 0-05-1-5 %, with about 1 - 1 5 gal (4.5-6.8 1) per animal at a pressure of 300-400 lb in-2 (Khan, 1964). From time to time, dust bags have also been used for the control of warble larvae, and 10 % phosmet has recently given good results in Wyoming, where the cattle were made to pass under the bags as they went to obtain drinking water (Lloyd, 1971). A considerable “screening” programme is undertaken in the United States (Drummond et al., 1971), where the standard technique involves the oral or subcutaneous dosing of potential insecticides to guinea-pigs which have previously been infested with larvae of the black blowfly, Phormia regina, secondary screw-worm, Cochliomyia (Callitroga) macellaria, and nymphal

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lone star ticks, Amblyomma americanum, and then exposing the dosed test animals to the bites of Stomoxys calcitrans. The test procedure formerly utilized larvae of C. hominivorax, but C. macellaria is now employed due to the eradication of the primary screw-worm fly from the southern United States. Of 1078insecticideswhich passed through the screen in 1953-1967,299 were found to be systemically active, and were then put through suitable tests in large animals; 41 of these insecticides (mainly organo-phosphorus compounds) were tested in cattle for warble control but only three were effective orally, and none of these was active by the “pour-on” technique. Few organo-phosphorus insecticides are systemically active, malathion being an obvious exception, nor is the systemic effect limited to organophosphorus compounds, alternatives being such insecticides as dieldrin and benezene hexachloride, but in practice the “systemics” are confined to phosphorothioates (coumaphos, fenthion, fenchlorphos), phosphorodithioates (phosmet), phosphoramidates (crufomate) and phosphonates (trichlorphon). In an attempt to find some suitable experimental animal for the investigation of potential systemic insecticides larvae of Hypoderma have been cultured in vitro or used to infect calves or rabbits (Beesley, 1962), but there has been no really satisfactory alternative to the artificial infestation of cattle with flies bred out in the laboratory from collected mature larvae, an extremely time-consuming process. It has been known for some time that certain oestrids parasitize small rodents, and Baird (1971) has shown that the larvae of Cuterebrajellisoni will develop to maturity in rabbits and six other species of rodents. The use of a natural oestrid parasite would be a useful addition to the guinea-pig screen, as a preliminary to field studies with Hypoderma in cattle. Although many papers have been published on the general suitability of new insecticides for cutaneous application, much less work has been done on the actual, as distinct from the presumed, abilities of such insecticides to pass through bovine skin. The use of a labelled organo-phosphorus compound, such as trichlorphon, indicates that, following pour-on application, peak levels occur in the blood within 12-30 min (Dedek and Schwarz, 1968). The rate at which the insecticide appears in the blood depends very much, as one would expect, upon the particular solvent employed, best results being obtained with a mixture of mineral oil and alchohol, while fatty oil yields 50 % less of the insecticide in the blood, and water hardly any at all, despite the high water solubility of trichlorphon. A rather similar experiment was carried out by OBrien and Dannelley (1956), who used intact rat skin to show that the penetration rate of dieldrin and malathion exceeds that of DDT, famphur and carbaryl, but varies considerably depending upon the solvent used, the rate being greater with acetone than vegetable maize oil. These workers analysed the extent to which the insecticide passed into the outer skin, rather than the rate at which they passed through the skin into the blood, and showed that an early rapid penetration might be followed by a slow diffusion of one or more primary degradation products, such as p-dimethylsulphamoylphenolin the case of famphur.

155 Corticosteroids, dexamethasone and flumethasone appear useful in the treatment of the side effects that are occasionally encountered following the administration of organo-phosphorus systemic insecticides. The use of corticosteroids for shock (as distinct from atropine sulphate therapy for the control of symptoms of poisoning) has given good results in Eire, where a total of some 20 million doses of systemically active insecticides have now been administered ( H. Thornberry, personal communication). Attempts at the complete extermination of warble from large areas or from entire countries have seldom been successful, although Hypoderma has been cleared from Cyprus, Sweden and Clare Island, off the coast of Ireland. These successes followed the manual expression of grubs from the backs of cattle in the spring and summer, or the use of derris insecticide dressings. Later, under the impetus of the systemically active compounds, virtual eradication was accomplished on Santa Rosa Island, California (Riehl et al., 1965). The island had a cattle population of about 3000, and dressing with crufomate was carried out during each of the 4 years of the trial. At the end of the period, only a single grub was seen in one animal, as against an average of 22-7 in a group of untreated calves. Near-extermination of warble has been reported on many occasions, from the time of Peter (1912), who gained a reduction from 10to 0.1 larvae per animal during a 9-year programme in Denmark, to various projects in North America by Teskey (1961), Rich and Kahn (1964) and Graham and Drummond (1967). In Arkansas, Walton and Lancaster (1965) attempted control in a herd of 380, situated 2 miles from any other cattle. Various systemic treatments were used, and were virtually 100 % effectivein the 1 year of the programme, but in the following summer moderate numbers of larvae began to appear, and it was concluded that several further applications might be needed in order to effect complete eradication. Rich (1965) studied the effects of a systemic insecticide upon an isolated population of Hypoderma, in lo00 beef cattle in British Columbia, separated from other cattle by some 10 miles of rugged mountainous country. Fenchlorphos or crufomate was applied each year from 1957 to 1962. In the same area the incidence of warbles in untreated calves, which were manually dewarbled, fell from 30.2 to 0.2 per animal, but in 1963-1964 and 19641965 rose to 1.7 and then to 10.2, respectively, indicating the existence of a very resilient population of warble flies. As it seemed very unlikely that infested cattle could have strayed into the area from outside, it may be that the fly can travel further than the maximum of 300-3500 yd which has sometimes been quoted (Gregson, 1958). During the course of several warble eradication campaigns it has thus become plain that the final few Hypoderrna individuals are extremely difficult to exterminate, despite the lack of alternative or reservoir hosts. A modification in the planning of a control scheme might be to treat cattle both in the autumn and the following spring, and this procedure has been adopted in the Republic of Ireland, where the incidence of warble has fallen from about 40-60 % to a present 1-5 % (depending upon the part of the country surveyed). The 5 million cattle of Eire were all dressed in the autumn of 1965 and 1966, CONTROL OF A R T H R O P O D S

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then these treatments were discontinued and the 6OOOO warbled cattle which were found by inspectors were dressed in the following spring. The condition of warble infestation has also been made notifiable, so that visibly infested cattle must now be treated when seen on the farm or if they pass through a market, port or border station (H. Thornberry, personal communication). In Canada, Khan (1968) attempted the eradication of warbles from a 5 x 10 mile range area, which was bordered by a 5 mile wide “buffer zone”. All 17518 cattle, with the exception of a few controls, were treated with 0.5% coumaphos or 0.5 % crufomate. The mean number of larvae in the untreated “bait” cattle fell from 13.7 to 4.8 to zero in the 3 years of the programme, but after a further 5 years the number had risen to 0.8 grubs per animal. This resurgence of a population of Hypoderma is all the more interesting because of the accepted high mortality rate of the different stages in the development of this insect. Semenov (1970) has suggested that H. bovis lays about 300 eggs on the host but that 40% of the eggs and larvae die, with an even higher death rate in older cattle, perhaps because of an increasingly strong immune reaction. An average mortality among the pupae on the ground of a further 55 % was also suggested, leaving only about six adult flies from each female that laid on adult cattle, and 13 from calves. It may be relevant here to quote from the recent 17th Report of the W.H.O. Expert Committee on Insecticides, in which it is recommended that special attention be paid to small populations of insects that survive control measures in isolated foci. Such populations may differ considerably in their dispersion and other behaviour patterns from the original population-individuals may move much greater distances than normal and they may reproduce at a higher rate, resulting in a rapid invasion of nearby areas in which control programmes have been successful. The detection of such changes requires continuing study of all aspects, especially as any change in the structure of behaviour of populations may indicate a corresponding change in techniques for control or in the overall strategy of the control plan (World Health Organization, 1970). In addition to control measures directed against the larvae, it may be possible to find and kill the adult flies, for some progress has been made towards delineating the aggregation sites of the male flies (Catts et al., 1965). The adult flies do not feed and are probably short-lived; the females must mate and find a host soon after they emerge from the puparium, and one would think that interference at this stage could be a very effective means of control, possibly by suitably insecticide-baited sex attractant chemicals such as have been used to help in the control of some other agricultural pests; attractants coupled with chemosterilants might also be employed (Graham and Drummond, 1967). Weintraub et al. (1968) examined the hybridization of H . bovis and H. lineaturn and found that none of 28 apparently successful matings was fertile; it is most unlikely, however, that any methods based on the use of sterile or incompatible matings will be appropriate to the control of warble fly, because of the technical difficultiesin breeding out sufficientflies. The use of repellents, such as aromatic oils, dimethyl phthalate and dibutyl phthalate, has been found of no value in the protection of cattle

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from the attacks of warble flies, although Knapp (1972) has shown that twice daily self-treatment of dairy cows with 1 oz (28 g) of 2 % of the non-systemic insecticide crotoxyphos gave 74-96 control of larvae. The self-sprayer was actuated by an antenna wire hanging down in the centre of a spray corridor which led away from the milking area; spray was automatically released for 3-4 sec after contact with each cow. Control of warbles was probably achieved by the action of the insecticide as a combined fly repellent, ovicide and larvicide (against newly-hatched larvae on the skin and hair of the cattle). Up to 58 larvae were found in individual untreated cattle. Resistance to insecticides has not been reported in Hypoderma, or in any other oestrid flies, but it is possible that this could occur in association with the very low levels of pesticide that could remain in the hair and skin during the spring following the application of these materials the previous autumn. The insecticides that are now in use break down quite rapidly in the tissues, and are not used with the intention of long persistence in the hair, so that the appearance of resistance seems very unlikely. Systemic insecticides are also effective against larvae of the sheep nasal fly, Oesfrus ovis. In South Africa, Fiedler (1967) injected a 4 % formulation of the organo-phosphorus insecticide bromophos into the nostrils of lambs and adult sheep and had 100% success with a single treatment, without toxic signs in the hosts. In the United States, Pfadt (1967) used 3 % mineral oil sprays of dichlorvos and oral drenches of 50% fenthion at the rate of 30 mg kg-1; the fenthion gave a complete kill of larvae, and the dichlorvos was about 90% effective. Drummond and Graham (1965) found an injection of dimethoate at 25 mg kg-1 effective against nasal bots. Three species of Gasterophilus infest horses in many parts of the world: G. intestinalis, G . haemorrhoidalis and G . nasalis. Their larvae rarely cause serious harm, despite their occurrence in very large numbers in the stomach and intestines. Treatment may consist of an oral dose of carbon disulphide or carbon tetrachloride, but systemics have also been employed, e.g. dichlorvos (17-75 mg kg-I), crufomate (50 mg kg-1) and trichlorphon (25-100 mg kg-1) (Drummond and Graham, 1965).

IX. LICE

The human lice include the body louse, Pediculus humanus corporis, the head louse, P. humanus capitis, and the pubic (crab) louse, Phthirus pubis. Body lice can transmit epidemic typhus (Rickettsia prowazeki) and one form of relapsing fever (Borrelia recurrentis). The itching caused by the lice may lead to impetigo. Infestations of body lice can be common in developing countries, and may be a serious problem where standards of hygiene are poor or during times of great disorganization, such as wars or major natural disasters. In economically developed countries infestations of body lice are normally infrequent but may be found among people lacking good personal hygiene; head lice and public lice tend to be the more common, especially in underprivileged children. In Britain it has been suggested that some I

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1 million people may be infested, including at least 200000 children. Long hair is undoubtedly a good habitat for the head louse, and the high incidence of pediculosis among schoolchildren must be in part a reflection of the current hair styling, or lack of it, such that half the pupils in a class may be infested. Control of body lice on small groups of people presents a different sort of public health problem from that of general infestations among refugees during a war, for example, when rapid reduction of lousiness is essential in view of the threat of louse-borne disease, whereas under normal conditions the danger of disease is nil or negligible. In all cases the hair, head covering and clothing must be treated, using 10% DDT dust-unless there is an insecticide resistance problem, when dusts containing 1 % BHC or malathion are generally effective. Other insecticides which have been employed include carbaryl, pyrethrin and allethrin. Powders and dusts are easily and rapidly applied, and about 30 g of DDT powder should be used to treat the clothing of each person (World Health Organization, 1972). In mass treatments 40-50 g of powder is shaken or blown into the clothing, the classical case of typhus control in Naples in 1943 having been carried out with 10%DDT dust at 28-42 g per person (Brown and Pal, 1971). DDT also halted a severe epidemic of louse-borne relapsing fever in Kenya in 1945 (Garnham et al., 1947), where there were I500 cases of disease, 380 of them fatal. 100000 Africans were disinfested with 5 % DDT powder, and the weekly case incidence fell from almost 100 to 55, then to 12, within 1 month. Control of lice on infested clothing and bedding has been obtained by (a) soaking these in four times their weight of 1 % DDT emulsion, (b) applying 5 % DDT emulsion by brush so as to leave a deposit of about 2 g DDT per m2, (c) marking fabrics in a cross-hatched pattern with a special wax crayon containing 30% paraffin wax, 63 % DDT and 7 % BHC, (d) distributing to each family a 500 g cake of soap containing 3 % DDT, sufficient for personal hygiene and laundry for 2 weeks, and (e) washing clothes with 7 % DDT soap. Infestations are usually eradicated by one treatment, but re-treatments may be required at intervals of 3-4 weeks. It should be noted that repeated weekly treatments with BHC, pyrethrin or allethrin may be required because of the limited residual activity of these insecticides. The treatment of infrequent infestations, where there is little chance of re-infestation, can be carried out by heating (stoving) clothes and bedding to a temperature of 70°C for 1 h, or by fumigating with ethyl formate in a plastic bag or metal bin, using 2 ml 1-1 for 1 h. Although head lice can be treated with DDT dusts, liquid shampoos are generally preferred for aesthetic reasons. Suitable insecticides are DDT, BHC and pyrethrum; DDT stimulates the activity of the lice, which become extremely irritating for a short time. Examples of formulations used for the control of head lice are NBIN (68% benzyl benzoate, 6 % DDT, 12% benzocahe and 14% Tween-80, diluted 1 : 5 with water prior to use), 0.5 % deodorized malathion, and tinctures of derris root (rotenone) and delphinium flowers. After application treated persons should not bathe or shampoo for at least 24 h, and persistent infestations should be re-treated at intervals of 1-2 weeks.

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Pubic lice are not known to transmit disease, but can give rise to localized eczematous conditions in the pubic or axillary regions. Phthiruspubis responds to the treatments that have already been described, together with creams, lotions and shampoos containing 1 ”/, BHC. Resistance in Human Lice DDT louse powder was successfully used in many parts of the world from the early 1940s until the winter of 1950-1951, when it was found that the application of 10% DDT to a large group of Korean military personnel served only to increase the degree of infestation, while in the laboratory barely half the lice died following exposure to DDT at a rate equivalent to 68 g per uniform (Hurlbut et al., 1952). Strangely enough, head lice from persons who harboured resistant body lice were quite susceptible to the insecticide. At about the same time, resistance to DDT was recorded in Japan, Syria, Jordan and Egypt, and by the mid-1950s the problem was occurring in many parts of the Middle East, some coastal areas of W.Africa, South Africa, Chile, Peru and Hong Kong (Brown and Pal, 1971). A W.H.O. survey in 1965 showed an improvement in the position in some countries, but there was nevertheless an overall increase in the numbers of reports of DDT resistance, e.g. in the Sudan, Afghanistan, France, and the Balkans (Brown and Pal, 1971). Resistance to BHC was experienced from 1956 onwards, in countries as widely separated as Norway, England, Iran and Hong Kong, and tolerance to pyrethrum was reported at about the same time. Laboratory studies on resistance indicated that DDT and BHC resistance could be developed in only 3-7 generations (Kitaoka, 1952). It was fortunate that the DDTresistant strains of body lice were quite susceptible to malathion and other organo-phosphorus compounds (Cole and Burden, 1956), although a strain from Freetown, West Africa, that was selected for BHC-resistance simultaneously developed strong resistance against carbaryl. Very little that is new has been concluded from a study of the biochemistry of resistant lice, apart from the fact that in lice (as in houseflies) piperonyl cyclonene acts as a DDT synergist against the resistant strains. Animal Lice In the main cattle-rearing areas of the world, heavy infestations of sucking and chewing lice can cause severe unthriftiness, loss in weight, anaemia, abortion and even death (Shemanchuk et al., 1963), so that control measures assume considerable practical importance. In addition to the use of insecticides, attention has been focused on the ways in which louse populations change during the year in response to climatic and other factors. In one Australian experiment a group of ten heifers was split into one sub-group maintained on good rations, the other on poor rations; in addition the cattle were each infested with 5000 larvae of Boophilus microplus twice weekly. After about 10 months the cattle on the poor rations became infested with chewing lice, Damulinia (Bovicolu) bovis, and also had more ticks, a lower haematocrit and lower haemoglobin count than the other group. Self-

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grooming is an important way in which cattle control their lice and ticks, but there may be a different response to grooming by the two types of parasite, as one remains attached to the skin while the other moves over the hairs during feeding or egg-laying. It appears that the more delicate lice are vulnerable to self-grooming, and that reduced grooming in animals on a low nutritional plane can lead to an increase in lousiness. A further effect may stem from the tendency of the winter coat to remain in poorly fed animals, while in normal cattle this is shed-together with lice and many of the louse eggs-in the late spring or early summer (Utech et al., 1969). There is a similar loss of sheep lice and keds at shearing time. Quite apart from the effects of grooming, it has become plain that many herds of cattle, and flocks of sheep, include “carrier” animals, that may remain heavily infested throughout the year, withstanding the application of insecticides in concentrations that eliminate infestations from the other animals. The problem can be sufficiently serious to persuade the farmer that it is economically best to cull the carrier animals than to single them out for special treatment (Khan, 1964). Insecticides that are recommended for the control of lice on cattle include synergized pyrethrins, rotenone, DDT, BHC, toxaphene, malathion, diazinon, crufomate, crotoxyphos, dichlorvos, carbaryl, coumaphos and fenchlorphos. Most of these are employed as sprays or dusts, but crufomate may be used as a 6-8 ”/, “pour-on” preparation, as for the control of warble larvae. There is an interesting public relations lesson in the use of systemically active organo-phosphorus insecticides for the control of warbles, in that the farmer witnesses the unseen death of migrating Hvpoderma larvae deep within the body of the host, so that success is correlated with a non-appearance of warbles (which may not have been there in the first place); however, the same application of insecticide gives excellent control of lice, showing the sceptical farmer that something really is happening! A single treatment with a systemically active insecticide can give good control of lice for at least 3 months (Shemanchuk ef a/., 1963). Generally similar insecticides are recommended for the control of lice on sheep and goats as for use against cattle lice (Heath and Millar, 1970), but Chamberlain and Hopkins (1971) controlled Damalinia (Bovicola) limbata on Angora goats by spraying them three times with 0.1 ”/, of a juvenile hormone analogue, (mixed isomers of methyl 10, 1 I-epoxy-7ethyl-3, 1 I-dimethyl-2, 6-tridecadienoate). Atypical forms of the parasites were seen during the tests. The synthetic juvenile hormone is identical with the natural substance found in insects, and it was suggested that its use in the control of lice on meat animals may offer a solution to the problem of residues in tissues. Insecticide resistance has been reported in cattle lice in North America and South Africa, and this has unfortunately included tolerance to malathion (Brown and Pal, 1971). Resistance to BHC in the sheep louse Damalinia (Trichodectes) ovis was observed in the north of England by Barr and Hamilton (1965). The immediate possibility arose that “stripping” (exhaustion) of the dip wash may have occurred, so that the last-dipped sheep had received less insecticide than that necessary to control the lice, but Treeby (1966a) showed

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that 0.072 % gamma-BHC (compared with the usual 0.016 %) was required for the control of these lice; an alternative was the use of 0.021 carbophenothion, which gave protection from re-infestation for about 5 months (Treeby, 1966b). No other species of animal lice have been reported resistant to insecticides. For the control of lice on poultry malathion, crotoxyphos, carbaryl and coumaphos may be used, also dichlorvos strips. Some residue and taint problems attend the use of DDT and BHC. Recent trials with insecticides for the control of Menacanthus straniineus (Hoffman and Hogan, 1972) indicated that volatile compounds such as dichlorvos gave a good initial kill but could not provide protection for more than 4 weeks, whereas control for at least this period was obtained by as little as 0.050/0of “Sectran” (=the 4-(dimethylamino)-3, 5 xylyl ester of methyl carbamic acid), “Hooker HRS - 1422” (=the 3,5-diisopropylphenyI ester of methyl carbamic acid), formothion or certain phosphorus dithioates. The direct mama1 dusting of birds is much too slow for large operations, when a crank type rotary hand duster is more useful. In conventional poultry houses the easiest way is to dust the litter, while dust bath boxes provide a means of self-treatment. Comparatively coarse sprays may be applied by knapsack machines or power sprayers and insecticide may also be applied by mist or fog machines, depending upon the type of formulation to be applied (Matthysse, 1971). It is not easy to obtain adequate coverage of massed birds, and many treatments fail because of this; birds loose on the floor of a house may be treated by misting them as they first crowd into a corner and then run past the machine. Care has to be exercised because the sudden panic of several thousand birds may lead to packing in one part of the house, with possible death from asphyxiation for a few.

X. FLEAS Fleas are the primary vectors of bubonic plague and murine typhus, both of which have made a tremendous impact on the history of man. Fleas are also important enzootic vectors of disease in wild or commensal rodent populations. Widespread epizootics of plague periodically decimate rodent populations in parts of Asia, Africa, and the Americas, and directly or indirectly affect man. In addition to their capacities as vectors of disease, some fleas associated with man and domestic animals are serious pests, causing considerable distress. For example, following its introduction from South America into Africa in the late nineteenth century, Tunga penetruns, a flea that burrows into the skin, caused thousands of human deaths as a result of secondary infection and this species remains a serious pest in parts of Africa and the Americas. Dog and cat fleas may also transmit the dog tapeworm, Dipylidium caninum, and rodent tapeworms Hymenolepis diminuta and H. nana. Relatively few species of fleas can initiate and maintain the murine plague epizootics that lead to widespread epidemics of human bubonic plague. The important vectors of bubonic plague likely to be found in ports and ships,

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for example, are Xenopsyllu cheopis (worldwide), X . brusiliensis, X . ustia, X . vexabilis, Pulex irritans (which can be confused with P. simulans in North America) and Nosopsyllus fasciutus (World Health Organization, 1972). Long-term control of rodent fleas is best achieved by methods of basic sanitation aimed at reducing or eradicating hosts by eliminating their habitats and sources of food. Chemical control should be considered more as an emergency measure for the control or prevention of outbreaks of epidemic disease, as a means of temporarily alleviating a pest problem or as a preliminary stage in the application of rodent control measures, although in some instances chemical treatments may be the only means of preventing human exposure to plague. Problems in the control of fleas with insecticides are most likely to be associated with practical difficulties of getting the toxicant to the parasite than with any intrinsic tolerance to an insecticidal material, and an important feature in the evaluation of insecticides for the control of these pests is that re-infestation may occur from other infested animals and from developing eggs, larvae and pupae in dust, carpets, floor cracks, etc. Insecticides in use range from knock-down compounds, such as pyrethrins, which are very effective but have little residual activity, to the more persistent insecticides such as DDT, BHC, malathion and diazinon. In the treatment of houses against P . irritans, insecticides are often used in the form of dusts, e.g. 4 1 0 % DDT, or aerosols and sprays, e.g. 0.5% diazinon, 1.0% BHC and 2.0% malathion. Dichlorvos resin strips have shown promise in the control of rat fleas in enclosed spaces such as cargo holds and domestic premises. A similar technique has been employed for the control of fleas on pets : plastic collars impregnated with dichlorvos have been successfully used to control Ctenocephalides canis on dogs (Parkinson and Ketterer, 1969). The collars remain lethal to fleas for several months, but very occasionally dogs which wear these collars may suffer an inflamed exudative dermatitis. The causes of the irritation may include excessive perspiration under a too-tight collar, allergic reactions to the insecticide or to the polyvinyl chloride carrier, or self-trauma, with subsequent bacterial invasion, in dogs which have not previously worn a collar. Fleas on cats and dogs are more normally controlled by the use of BHC, derris or pybuthrin (pyrethrin synergized with piperonyl butoxide); BHC may cause toxicity problems in cats, partly because of their self-grooming habits. Bromocyclen aerosol and dusting powder have been used with success against heavy infestations with C. felis on short-haired and Persian cats (Butt, 1971). One of the cats had previously shown clinical signs of flea allergy but 7 days after treatment its eczematous lesions were heaIing. Resistance to DDT eventually developed in X.cheopis, X. brasiliensis and wild rodent fleas, first in South America and then in South-East Asia and the Middle East. Insecticide resistance now occurs in many additional species of fleas, including P. irritans, Ct. canis and Ct. felis (Brown and Pal, 1971), so that before using an insecticide in a field control programme it is essential to test the local flea population for susceptibility. An interesting variation on the usual insecticide resistance picture has

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been the possible explanation for the persistence of small pockets of plague in Madras and Mysore. These were areas which had been treated with insecticide during a large-scale malaria eradication programme, and it is thought that exposure of the fleas to insecticide may have induced the development of resistance, so that elimination of one disease hindered rather than assisted the elimination of a second (World Health Organization, 1970). XI. TICKS The Ornithodoros ticks that transmit relapsing fever and the Ixodes ticks that transmit encephalitis and other diseases are controllable with BHC, Dursban and, to a lesser extent, DDT. A degree of personal protection may also be achieved by the use of repellents such as deet and di-butyl phthalate. Virtually no other means are available for the control of these ticks other than the application of insecticides to the areas in which they are found. The ticks that attack cattle and sheep are of great importance and are considered in separate sections below. A.

CATTLE TICKS

It has been estimated that ticks affect 80 % of the world cattle population, causing apparent losses of about f200 million-the result of unthriftiness, general unproductivity, anaemia, transmitted diseases and death (Gresty, 1971). They present the most serious problem in South-Central Africa, Australia, Argentina, Brazil, Uruguay, Colombia and Mexico. In Australia and South America the dominant species is the one-host tick Boophilus microplus, which also transmits Anaplasma and Babesia; the tick is relatively easy to kill (because it is on a single host) but has developed resistance to several acaricides. In Africa and the Near East, on the other hand, the multihost ticks are of greatest importance ; Rhipicephalus, Hyalomma, Amblyomma and to some extent Zxodes-again as vectors of protozoan and other diseases, in addition to causing anaemia and paralysis (Dermacentor andersoni, Ix. holocyclus, Ix. rubicundus) (Wilkinson, 1968). Toxicosis may be a serious complication arising from infestations with B. microplus and some species of Rhipicephalus (O’Kelly et al., 1971). Ticks may be controlled either by chemicals or by some form of alteration of the environment, usually rotational grazing (“spelling”) or clearance of the vegetation. Repellents are hardly ever used on cattle, although deet, dimethyl phthalate and dibutyl phthalate would offer some slight protection. The original paraffin sulphur and 0.32% arsenic formulation used at the turn of the century gave way to organo-chlorine and organo-phosphous acaricides. These are usually applied by dipping, although spray races are often preferred by dairy farmers because the cattle can be treated quickly and with virtually no risk of injury. The form of acaricide most suitable for use in one area may change with the time of the year, as for example in those parts of Africa where B. decoloratus is the main problem in the dry season,

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but is superseded by multi-host ticks at the start of the rains. Dipping is always a less precise technique for the application of acaricide, but it is simple and effective. If the target tick is a species of Boophilus (one-host type, with all the parasitic stages on the one animal for a period of about 3 weeks), then good control should be possible by dipping or spraying every 10-20 days, and treatment every 7 days should ensure that no animal carries stages more mature than larvae. With the multi-host ticks, however, there is a relatively short feeding period, especially in the case of the adult female, so that treatment at intervals of about 7 days is necessary in order to “catch” the ticks. If there is a severe infestation with R. uppendiculutus (Fig. 2) it may be necessary to treat as often as every 2-3 days in order to avoid the transmission of East Coast Fever.

FIG.2. The result of combined attack on the ear of a cow by the tick Rhipicephulus uppendiculntus and screw-worm maggots. (Reproduced by permission of Cooper, McDougall and Robertson Ltd., Beckhamsted, Herts.)

During the dipping of cattle it is essential that the animal swims through the tank for 20-30 sec, so that the head, ears and area under the tail are thoroughly wetted, as these are the parts particularly liable to attack by hungry ticks (Fig. 3). Even so, there will be little retention of acaricide on the less hairy regions of the body, and to improve the residual protection it is

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FIG.3. A dipping bath in South Africa. (Reproduced by permission of Cooper, McDougall and Robertson Ltd., Berkhamsted, Herts.)

often necessary to dress these with an acaricidal grease, particularly for protection against ear ticks such as R. uppendiculutus. The dip tank is usually constructed from concrete, although wood, metal, stone and brick are also used. A roof is a useful addition, as this prevents dilution of the wash by rainwater and evaporation from the heat of the sun. It has been calculated that each animal may remove from the bath about 9 1 of wash, of which 2.5 1 remain on it and the remainder drains off (Gresty, 1971). As with sheep dipping, a dip concentrate should be added to take account of the loss due to “stripping” or “exhaustion” (i.e. the removal of insecticide by the hair of the coat, etc. at a greater rate than would be expected from the diminution

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in volume of the wash). In the case of chlorfenvinphos, for example, a replenishment concentration of 16CL175 % of the original strength is recommended (Gresty, 1971). Mixing instructions should be carefully followed, especially those relating to the premixing of liquid dip concentrates, because this premixing or creaming procedure will ensure the production of a fine emulsion wash, whereas lack of creaming may produce a coarse, “high exhausting” wash. The volume of water and dip concentrate which have been added from time to time and details of analyses of the wash should be noted in a dip record book, so that a proper history of the particular tank is available. A suitable dipside analytical kit may be provided, or alternatively the farmer may have to send samples to a distant laboratory; dips are too expensive to be discarded at frequent intervals, but one of the problems is that stale dip may eventually become fouled with a thick deposit of faeces etc. and a scum which contains potentially pathogenic micro-organisms. Modern dips are stable for 3-4 years. Because of fouling of the wash, especially in wet weather or in areas with clay soils, a bath with a capacity of 2000-2500 gal (9000-1 1 250 1) should be cleaned out after the passage of 20000-25000 cattle, and a 3000-4000 gal (13 500-1 8 000 1) bath after about 30 000-35 000 head of cattle, according to the instructions of one of the main dip manufacturers, Cooper, McDougall and Robertson Ltd., Berkhamsted, England. There are many practical additional points to be borne in mind before and during the dipping operation such as watering the animals and allowing them to cool off before immersion, and not dipping weakly or full-term pregnant cattle. The main alternative method to dipping for the application of acaricide is by some form of sprayer, preferably a spray race, in which the cattle pass through a tunnel of tubes carrying wash under pressure, sprayed at the animals from both sides, above and below. Normal spray pressures are of the order of 15-20 lb in-1 (1.CL1.4 kg cm-a), Under these conditions the average animal will remove perhaps one-third to one-half of a gallon (1.52-25 1) of wash, so that this, plus a spare 70-120 gal (315-540 1) will be the volume of wash to be prepared. The wash remaining at the end of the operation is pumped out into a cesspool. Modifications of the dipping tank are the use of the Machakos dipbath in East Africa; here each animal is yolked in the 500 gal (2250 1) bath while buckets of wash are manually poured over it. The method economizes in dipwash and is useful to the farmer who owns only a few cattle. Spraying can also be carried out perfectly well by treating individual cattle, but the spraying of groups of say 6-10 cattle is unlikely to be effective because it will be difficult to see and treat all parts of all the animals, especially the shoulders, ears and underside of the body. Hand-dressing of cattle is very effective, but laborious; It may be necessary in areas where the degree of infestation is high. The cattle may be treated with a brush, swab or knapsack sprayer, and the technique is particularly valuable against ear ticks which may not have been affected in the bath and can be seen and sprayed immediately after dipping. Treatment of the ground in order to kill ticks rarely has much effect: in any case it would have to be done on a large scale, with suitable barrier areas and

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careful attention to possible contamination of the environment, so that it is likely that only a government-backed programme could succeed. Tick eradication is more likely to be attempted in association with a thorough dipping or spraying scheme, as in Australia. Especially when a dipping programme has been running successfully for some time, farmers may consider that the trouble of walking their cattle some distance to the dip is no longer worthwhile. This happened in New South Wales, Australia, where systematic compulsory dipping was followed by eradication of B. microplus from 99.7% of 6300 herds. Many owners of tick-free animals then began to think that there might be a loss in milk or beef production through the nervousness of their animals and continuous interruptions in feeding. However, in one test a trek to the dipbath of up to 4.4 miles was involved, and it was shown that walking with or without dipping had no adverse effect (McCulloch and Barrow, 1970). It would be a considerable task to produce a comprehensive table listing the ranges of concentrations of all known acaricides against all ticks of veterinary importance, but some of the best materials at present in use include bromophos ethyl, chlorfenvinphos, coumaphos, fenthion, dioxathion, diazinon, ethion, carbaryl, trichlorphon and carbophenothion. Suitable concentrations would probably be in the range of 0.0545% for dips and 0 . 1 4 2 % for sprays. In areas with large populations of ticks protection from re-infestation might be about 90-98% after 1-2 days to 7 5 4 5 % after 1 week for the better materials. The subject of control, including techniques, was covered in a monograph by Barnett (1961), but there are many recent papers, especially in relation to acaricide resistance, as indicated in the next section (Shaw, 1971; Baker et al., 1969). B. RESISTANCE

Several reviews of the overall subject have been published recently (Newton, 1967; Wharton, 1967; Brown and Pal, 1971; Jones-Davies, 1972). Ticks, particularly Boophilus species, can develop resistance to many types of acaricides, and since Australian and South African B. microplus and decolorarus became resistant to arsenic in 1937-1938, resistance has occurred to DDT, BHC, toxaphene, carbaryl and many of the organo-phosphorus acaricides. Unfortunately, strong acaricide resistance by ticks has been reported from intensive beef areas of Australia and South America, and a point has been reached in some parts of the world at which high-level tick control is not possible (Shaw, 1971). In Australia there were originally two important strains of acaricideresistant B. microplus, from Ridgelands (Rockhampton) and Biarra (Brisbane Valley). The former became resistant (70% kill) to dioxathion in 1963 after only 4 years’ exposure, and then showed cross-resistance to carbophenothion, diazinon and to the carbamate carbaryl ; they remained susceptible to Dursban, bromophos ethyl, crotoxyphos, coumaphos and ethion, which were therefore available for control. The Biarra strain was then detected ; this

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could not be controlled by ethion, crotoxyphos, or coumaphos (Roulston and Wharton, 1967), although it could be successfully treated with Dursban and bromophos ethyl (Wharton, 1967). Only lmidan (phosmet) gave good control against both Biarra and Ridgelands ticks, but this acaricide appears to lack sufficient dipbath stability to feature as a real alternative to the other organo-phosphorus compounds. Ridgelands-type resistance almost certainly occurs elsewhere in Australia, but is still apparently rather limited geographically, and dioxathion remains in common practical use. Biarra-type resistance, first reported in 1966, is limited to the Brisbane Valley. The two strains of ticks exhibit differences in their cholinesterase systems from normal strains of B. microplus, the 3iarra form being rather less sensitive to the organo-phosphous (= cholinesteraseinhibiting) acaricides. This suggests that the most useful acaricide for the future will not be a cholinesterase inhibitor, and one such compound has been discovered, a formamidine named chlorphenamidine, closely related to one of the babesicides. Like phosmet this compound at first seemed unlikely to prove sufficiently stable in the dip tank to be of much value, but recent work has shown that the addition of 0.01 chlorphenamidine to 0.0250,075 organo-phosphorus (or 0.2 ‘%, arsenic) acaricides gives very good results (Roulston et al., 1971). Further strains of ticks resistant to organo-phosphorus acaricides were discovered in 1968 at Mackay, in 1970 near Mount Alford, and in 1971 at Gracemere (O’Sullivan and Green, 1971). It was demonstrated and advised to farmers that the Ridgelands strain can be controlled by coumaphos, crotoxyphos, bromophos ethyl, phosmet, Dursban and ethion, the Gracemere strain by the same with the exception of Dursban, the Biarra and MacKay strains by bromophos ethyl, phosmet and Dursban, but the Mt. Alford ticks only by bromophos ethyl and phosmet. This extremely complex situation is summarized in Fig. 4, reproduced by permission from an anonymous article “Mechanisms of tick resistance” which appeared in the Australian publication “Rural Research in CSIRO” no. 75, (1972) pp. 28-30. It seems possible that there may have been a n evolution from the sensitive-cholinesterase normal strain along two paths (a) to the still rather sensitive MacKay strain and (b) to the Ridgelands-Gracemere type and then on to the insensitivecholinesterase Biarra-M t. Alford strains. While the resistance problem is most severe and widespread in the Boophilus species of ticks, it exists to a lesser degree in other genera. None of these, fortunately, have become resistant to the organo-phosphorus or carbamate insecticides, but it is reasonable to suppose that this will occur in due course. If a situation were to arise with R . appendiculatus, the vector of East Coast Fever, such as now exists with the Biarra and other strains of B. microplus, it would be very difficult to control this dangerous disease and this might well make cattle farming untenable in several areas of Africa (Shaw, 1971). Workers in South Africa have been able to show that a mixture of the two organo-phosphorus insecticides dioxathion and chlorfenvinphos is highly effective against B. decolorattu, B. microplus, R . evertsi, R . appendiculatus and A . hebraeurn (Baker et al., 1969). In some of the tests 0.02%

1 69

CONTROL OF A R T H R O P O D S Strain of cattle tick Ridgelands

Acaricide

0

sdsceptible

-

parlial resistance overcome by increased dosage

a __v

Biarra

Mackay

MI. Alford Gracemere

enhanced resistance

FIG.4. The resistance spectra of five strains of Australian cattle ticks

dioxathion plus 0.025 % chlorfenvinphos was shown to give better results than either of its constituents at 0.050/0.However, the potentiation which occurred in experiments with Biarra-type resistant B. microplus was not great enough to suggest that the mixture would control these ticks under practical field conditions. Tests for the investigation of suspected insecticide resistance have been described by Baker et a/., (1969), Shaw (1971) and Brown and Pal (1971). Some of the complications which may arise were discussed by Darrow and Whetstone (1972), working with the lone-star tick, Ambfyomma americanum. It was shown that over a 21 week period the susceptibility of starved nymphs increased 13004)-fold to DDT, 150-fold to dioxathion and malathion, and 10-fold to carbaryl. However, when the laboratory pre-treatment conditions included a combined warm-and-light and cool-and-dark cycle, susceptibility increased less rapidly than when there was no cycling. It was concluded that changes in susceptibility can take place so rapidly that nymphs which have

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been collected in the field cannot be used to measure resistance because of their completely unknown age and previous environmental situation. Reasons for the appearance of insecticide resistance in cattle ticks include the ineffective treatment of the animals, leaving too long an interval between treatments, or using weak wash in the dip or sprayer (Shaw, 1971). They are also discussed in considerable detail by Smith (1968), who suggests a thorough investigation of the conditions on the suspect farm before making any assumption that resistance is actually present, the alternative being to switch to another insecticide quite unnecessarily. Some of the ways in which ticks may appear on farms from which they have supposedly been eradicated (so giving the impression of the development of tolerant ticks) include importation of ticks on grazing and fodder reserves from nearby stands of high grass, on goats and sheep, or by cattle that have strayed through broken fences into tick-infested pasture and then returned to the farm with ticks on their bodies. Efforts are being made in several countries to contain the spread of resistant ticks by stringent quarantine regulations, and by allowing only tick-free cattle to move from one farm to another.

c.

“PASTURE SPELLING” (ROTATIONAL GRAZING)

Apart from the use of acaricides, cattle may be kept free from ticks by removing them from the most heavily infested grazing, using two fields alternately. In northern Queensland a modified “spelling” technique was recently outlined by Harley and Wilkinson (1971). The system is based on the movement of infested cattle into disinfestation paddocks, in which their B. microplus ticks fall off but where insufficient time is allowed for the progeny of these ticks to hatch and re-infest the cattle. During a 2-year period it was shown that a herd of 15 steers which were allowed to drop their ticks into a special paddock before moving on had consistently fewer ticks on them and required treatment with acaricide (0.075 % dioxathion) only seven times; in a herd of control animals that were not moved, a total of 23 treatments were required in the same period. Four paddocks were used, two each for disinfestation, one for standard grazing. The method could be equally suitable for acaricide-resistant and susceptible ticks, but the application of this particular variation on the technique is unlikely because of the need for increased fencing and movement of animals. The practical farmer, despite the acknowledged value of integrated spelling and chemical treatment, may nevertheless find that control programmes are difficult to carry out. The tick season and control measures may coincide with the calving season, and some systems of pasture spelling are not practicable. D. THE USE OF TICK-RESISTANT CATTLE

Tick-resistant cattle, such as Brahmans or Brahmancrosses (Droughtmaster), may be the ultimate answer to the B. microplus problem in Australia. One study in Queensland has shown that during a 12-month period Herefords were dipped three times and Shorthorns four times, while Droughtmasters were dipped only twice in 3 years (Johnston and Haydock, 1971). There

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were no differences between the tick infestations in pregnant and nonpregnant or between lactating and dry cows, even though they were under severe nutritional stress. It was suggested that some form of tick control on breeding Brahman-cross cows, aimed at reducing tick numbers in July, August and September may be of value in North Queensland. Research in Queensland has also shown that Zebu cattle carry only 40% of the tick load of British cattle, the difference tending to be greater when only female animals are considered although male cattle carry significantly more ticks than females (Seifert, 1971). Wilkinson (1 968) has summarized the present status of pest-management concepts, with particular reference to the British Columbian problem of human and animal tick paralysis due to D.undersoni. Drawing on published work from other countries where tick paralysis occurs, Wilkinson discussed, in addition to the treatment of cattle with insecticides, the role of hyperimmune serum in dogs, replacement of a paralysing strain of the tick by a non-paralysing strain, the stimulation of resistance in cattle after ticks have climbed on to them, allowing only the less active cattle (cows and calves) to graze in tick areas, the chemical treatment of the burrows of rodent reservoir hosts in order to reduce the numbers of ticks, and the liberation of effectively sterilized male ticks. The future of cattle tick control is uncertain, but there is a need for much more investigation of completely new types of insecticides (such as chlorphenamidine), of rotational grazing techniques, of tick-resistant cattle (studies on which are prone to considerable error by novice observers under commercial conditions) and of sterile male genetic control methods. E.

SHEEP TICKS

Most of the features of the control of ticks on sheep are generally similar to those which apply in the control of cattle ticks, e.g. dipping and spraying methods, types of insecticides and frequency of treatment, but there are a number of differences, stemming from the overall lesser importance of tickborne diseases in the sheep industry, the fortunate failure of sheep ticks to develop insecticide resistance, and-perhaps correspondingly-the smaller amount of research time spent on the problem.

XII. M~TES A.

ANIMAL MITES

In 1800 it was reported that Lord Summerville dipped sheep in Norfolk, England, in a bath containing arsenic, soft soap and water in order to control sheep scab (caused by Psoroptes cornrnunis ovis); this was the first recorded case of sheep dipping in Britain (Page, 1969). Nicotine, sulphur, mercurial compounds, coal tar creosote, cresylic acid and rotenone were also used in the early days of scab control, and by the turn of the century some countries were able to claim scab eradication; in Australia complete control was aided by the judicious use of the rifle and knife (Seddon, 1964)! Scab in Britain became notifiable in 1870, but this had no effect on the high, and increasing,

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incidence of the disease, which is associated with acute pruritus, broken fleeces, loss of wool, and secondary infection of mite lesions, which in turn may even lead to death from general debility and exposure. In 1900, for example, there were I939 outbreaks of sheep scab in Britain, excluding Ireland. Sheep scab Orders were made during the first 20 years of this century and as a result sheephad to be dipped twice each year under strict veterinary supervision. Double-dipping was necessary because of the lack of ovidical action of the dips, and their limited persistence. Dip concentrates were formulated which were decreasingly toxic to the sheep, but more effective against the mites, but it was not until 1948 that a new group of dips, based on gamma-benzene hexachloride (BHC), were introduced. Tests at the Central Veterinary Laboratory of the Ministry of Agriculture at Weybridge, and in the field showed that the minimum effective concentration of BHC against P.communis ovis was 0.016% and this has remained the standard level of BHC dips until the present day, despite the eradication of the mite in 1952 after only 4 years’ use of the new dips.” The need for the annual approval of sheep dips in Britain has receded and there is now a virtual absence of local authority statutory dipping requirements. In 1970, for example, only one new dip of the “single-dipping” type (gamma-BHC) was approved, while there were 30 revocations of approvals-I 7 of the “double-dipping” type dips, i.e. tar acid or arsenic, and I3 of single-dipping type dips. By the end of 1970,67 sheep dips remained as approved under the orders: 49 of the single-dipping type and 18 of the double-dipping type. The dips were made by a total of 23 companies and sold under 297 different names (Ministry of Agriculture, Fisheries and Food, 1971). In other countries the situation has been more complicated, and rather reminiscent of the problems associated with the control of sheep blowfly in the late 1950s. By 1952, a strain of scab mite resistant to BHC appeared in Buenos Aires province, Argentina, possibly because of inadequate dipping technique in some areas, where stray infected sheep may in due course have re-infected dipped sheep, themselves perhaps dipped in BHC washes stripped out (“exhausted”) of sufficient acaricide to control the disease properly. The current feeling at this time against residues of organo-chlorine insecticides in animal products led to the banning of BHC from all dips in the Argentine, and these have now been replaced by organo-phosphorus compounds, such as diazinon (Page, 1969). Rosa and Lukovich (1970), however, had difficulty in controlling P. oilis with either diazinon or BHC, needing four dippings in 0.0087 % BHC, two dippings in 0.015 % BHC, or two treatments with 0.01 ”/, diazinon, for satisfactory control. In Eire there are still a few outbreaks of sheep scab each year, despite the operation of a notification scheme and the twice-yearly use of BHC dips (1 June-7 August and 15 September-30 November). The reasons for the continuing incidence of the disease may include inadequate skilled supervision ‘Note added in proof: An outbreak of sheep scab which occurred in north-west England in December 1972 involved some 30 farms, but has apparently been eradicated following the use of BHC dipping.

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at the dipping bath and the natural reluctance of farmers to retain sheep for three weeks after clipping so as to allow sufficient wool to grow to hold the insecticide; the sheep are dipped, but in the circumstances there is little residual effect (Thornberry, 1969). Ninety outbreaks of scab were reported in the Irish Republic in 1949, 40 in 1957 and 34 in 1965. The other important mite condition of large animals in Britain was formerly “Parasitic Mange of Equines”, which could involve Psoroptes, Sarcoptes or both genera. The last case of sarcoptic mange in horses in Britain was recorded on 23 June, 1939, and the Psoroptes on 12 February, 1948, in the Midlands. The most common form of mange on horses has always been that caused by Chorioptes, usually in the form of “itchy leg”, and this mite is also a frequent parasite on cattle. Although it would have been expected previously, psoroptic mange of the goat was not described from Britain until the clinical report by Littlejohn (1968) who concluded that the condition is rare in this country. The remaining ectoparasitic conditions of large animals in the British Isles are sarcoptic mange, especially common in pigs, and demodectic mange in cattle, which is said to be uncommon as it is rarely reported by the tanners. Sarcoptes scabiei in the pig causes the formation of thick crusty lesions along the back and on the ears, and infection rates of over 40% may occur, particularly in weaners (Sheahan, 1970). With the marked exception of Demodex, infections with none of the above mites are difficult to eradicate, the best acaricides probably being gamma-BHC and diazinon. It is usually necessary to employ 0.1 % gamma-BHC to eradicate S. suis, whereas 0.016% is quite adequate to deal with P. ovis, the difference reflecting the skinburrowing habit of the Sarcoptes (Brownlie and Harrison, 1960). The organophosphorus insecticide trichlorphon is also very effective. Although BHC is very active against sheep scab, Littlejohn (1968) reported that two thorough applications of this compound failed to give complete cure of the 16 cases of goat psoroptic mange that she inspected. Sarcoptes is also likely to be found from time to time on dogs, where it tends to attack the edges of the pinnae. The burrowing activities of the mites provoke an intense pruritus and there may be alopecia and extensive excoriation. It seems unlikely that Sarcopfes persists in a carrier-stage, so that the finding of a single mite indicates that there is a definite mange condition (Baker, 1970a). The mite is accessible to treatment with 0.02 % gamma-BHC, rotenone, diazinon, benzyl benzoate or a sulphide soap of the type recommended for Demodex (see below). Although a burrowing mite like Sarcoptes, Demodex has always proved far more difficult to control, particularly on the commonest host, the domestic dog (Colglazier et al., 1960; Baker, 1970a). Puppies become infected from their dams, with lesions at first peri-orbitally and on the muzzle, then spreading over the body (“red mange” and “pustular mange”). The mites penetrate the primary hair follicles when the puppy is very young, so that access is gained to the numerous daughter follicles which develop directly from these without the mites necessarily having no return to the skin surface. The follicles become mechanically distended, the hairs are loosened and

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alopecia follows (Baker, 1970a; 1970~).Because of the deep sites in which the mites live, the condition is obviously one that should be amenable to the use of organo-phosphorus systemic insecticides, and compounds such as fenchlorphos have been used for the purpose. Several veterinarians have reported good results, but others have been concerned about the occasional side effects and the potential dangers to children of having such preparations about the horse. Among the many prescribed treatments may be mentioned soap containing tetramethyl thiouram-monosulphide (“Tetmosol”), and bathing or dressing with 0-02% gamma-BHC or benzyl benzoate (Baker, 1970~).The latter is rather toxic and should be used on only small areas of the skin each day, in rotation, or every 3-6 days. A recent preparation which has been used with success in Australia is thiacetarsamide (Heath and Morton, 1966). It is unfortunate that some practitioners give a hopeless prognosis when confronted with a severe case of demodicidosis in the dog, as many will recover if treatment is persevered with (Baker, 1970~).It is well known also that a large number of cases will resolve without any attention, just as some of those which have apparently been treated successfully may later relapse. Pre-natal infections do not occur, so that it would be possible to eradicate demodicidosis from pedigree dogs by the caesarean removal of puppies from infected bitches of more valuable lines; this would prevent any opportunity of infection during suckling, which is apparently when virtually all transmission takes place. Demodex in cattle may be more common in Britain than formerly supposed (Reid and Lauder, 1966), but it does not produce the considerable damage seen in bovine skins in some countries. In severe cases the lesions take the form of split-pea nodules, from which a thick caseous material may be expressed. It may well be that, as with the canine form of the disease, some animals can act as carriers, and mites may be seen in the material expressed from the Meibomian glands of the eyelids (Kirkwood and Kendall, 1966). BHC and coumaphos give satisfactory control of Demodex in cattle, pigs and sheep (Kirkwood, 1969). Notoedres and Otodectes are common on cats, causing “face and ear mange” and “canker” respectively. Notoedres gives rise to persistent scratching of the head and neck, but is fairly easy to cure with, for example, gammaBHC. A good alternative is benzyl benzoate, although this may be toxic to cats, perhaps especially where the skin has been damaged by the mites and by the scratching of the host. Orodectes cynotis is extremely common and Baker (1970b) found it in most cats presented for clinical examination in Dublin. The condition may not be obvious, and lesions are not always irritant to the host; they may, however, lead to trouble in the external auditory meatus, self trauma, haematoma and secondary bacterial otitis of the external ear. The meatus may be almost sealed with hardened wax, exudate and debris, and this must be removed before treating with, for example, 0.05-0-1 %, gamma-BHC or piperonyl butoxide. In long-haired dogs the tip of the tail may harbour mites from contact with the ears during rest, so that it may also be useful to treat this additional site. The larva of the harvest mite, Trombicula autumnalis, is an occasional

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parasite on dogs and cats in rural areas; only the orange six-legged larvae are parasitic, attaching to sites where the skin is thin and for about 3 days sucking serous fluid and exudate. The mite doubles in size and may reach a length of 0.4 mm, and it is at this stage that it is most likely to be visible. There may be a good deal of irritation and scratching, continuing after the mites have left the skin; this may be coupled with an allergic reaction, so that in the absence of the parasites precise diagnosis may be difficult. Treatment consists of the application of insecticides (gamma-BHC or pybuthrin), and a repellent such as dimethyl phthalate or dibutyl phthalate may be useful. Cats may also harbour Cheyletiella, a tiny mite which was originally known from the rabbit as the “fur mite” C. parasitivorax. It was supposed that this species also infects the dog, but it has now been demonstrated that the dog Cheyletiella is a different species, namely C. yasguri. It appears that none of the dog material which has been re-examined belongs to the species parasitivorax (see Gething and Walton, 1972) and it will be interesting to see if yasguri or, as is supposed, parusitivorax, is the cat species. Infested cats may not have very obvious lesions, but dogs and man may suffer considerably from the condition. Pet owners not infrequently contract a n infestation with Cheyletielfa from dogs and cats (Baker, 1970b; Hewitt et al., 1971), and may have urticaria1 weals of the trunk and arms, with intense pruritis. BHC is a good acaricide for this form of mange, but should preferably be applied two or three times, at intervals of about 7-10 days. B. POULTRY MITES

The most serious are the red mite, Dermanyssus gullinae, and the northern mite, Liponyssus (Ornithonyssus) syhiarum, the latter being the more common of the two in Britain (Kirkwood, 1969). Formerly the standard acaricidal materials employed for the control of these species were nicotine sulphate or pyrethrum for the birds and paraffin or hot soda washes for the houses. These were followed by BHC, which is very effective but may give rise to the accumulation of residues of the acaricide in the tissues or, from thermal vaporizers, in the feathers (Whitehead, 1971, Matthysse, 1971). Birds or their litter may be dusted directly, or allowed access to dust boxes containing insecticide; spraying, misting or fogging may also be used, although it is preferable to allow the mist cloud to drift towards the birds (if this is a suitable insecticide) rather than to soak-spray them directly (Matthysse, 1971). Typical recommendations include sprays of 0.25 % coumaphos, 0.5 % carbaryl or 2 % malathion, or dusts of 4% malathion or 5 % carbaryl. Dichlorvos in the familiar yellow resin-bonded polyvinyl chloride strips will volatilize and kill exposed mites, but fails to kill those that are hiding in cracks and crevices, as in the case of non-feeding stages of D . gallinae (see Kirkwood, 1969). Malathion is extremely effective as a spray or dust against both mites and lice on poultry, and fenchlorphos has also been used for this purpose. The carbamate carbaryl is a broad-spectrum pesticide which is effective as a spray, dust or systemically, a daily dose of 100 mg kg-1 body

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weight killing all the D. gullinue on treated birds in a period of 3 weeks (Kirkwood, 1969). A standard daily dose of 100 mg k g l would be difficult to guarantee for birds in a commercial house, but the general technique may have promise, just as coccidiostats are incorporated in the feed for the control of Eimeria spp.

XIII. THEFUTURE OF ARTHROPOD CONTROL The arthropods of greatest medico-veterinary importance are the vector mosquitoes, tsetse flies, ticks, etc., and the ectoparasites responsible for scabies and myiasis; infestations by calliphorids and oestrids, for example, lead to serious losses for the meat and hide industries in many parts of the world, while scabies and louse infestations cause much human misery. In this review, I have traced the development of modern insecticides with notes of some quite controversial moves, such as the withdrawal of dieldrin from most of its former veterinary uses. Despite this, progress has been impressive, with the continuing synthesis of very potent organo-phosphorus and pyrethroid insecticides (Ogami et al., 1970); several persistent but biodegradable analogues of DDT have also been made (Metcalf et al., 1971), and studies have continued with slow-release plastic insecticidal strips, micro-encapsulation formulations, and insecticide-baited pheromone and light traps. Despite remarkable successes, however, further efforts are needed. Recent epidemics of malaria (Ceylon) and louse-borne typhus (Burundi), and the resurgence of plague (Viet-Nam), dengue haemorrhagic fever (South-East Asia) and yellow fever (Africa) show just how easily serious vector-borne diseases can flourish given suitable conditions, despite all the paraphernalia of modern insect control programmes. Insect-borne diseases in some parts of the world have virtually unchanged effects on human populations: as for example the millions of South Americans who still suffer from Chagas’ disease, the 40 million African victims of onchocerciasis and the 250 million people who have Bancroftian or Malayan filariasis. On the credit side a great deal of data is now available on the geographical distribution of vectors and pathogens; new standard W.H.O. test methods indicate the degree of susceptibility of vectors to insecticides (and so reveal the areas in which significant resistance is arising); the W.H.O. 7-stage programme for the evaluation of new insecticides (World Health Organization, 1971c) is also developing safer, more naturally degradable and “resistanceproof” chemicals. All this is also of mutual interest to the Food and Agricultural Organization (F.A.O.) of the United Nations Organization, so that many of the chemicals used for the control of medically important mosquitoes and ticks are being simultaneously employed in the control of ectoparasites of animals. Mechanisms of insecticide action are being examined with increasing interest, and with vastly improved physical and chemical micro-techniques. For example, the demonstration by Maddrell and Casida (1971) that certain pesticides can paralyse the target insect and cause it to evacuate large volumes

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of fluid is a typical recent development. These workers have shown that contact between insecticide and insect for only a brief period can lead to a hitherto unsuspected result; this suggests even more relevance for the new non-persistent biodegradable insecticides. The use of micro-methods in insect physiology is now being linked with gas chromatographic analysis to show the minute amounts of insecticides that can affect insects in the various stages of their development. Multi-residue analysis is now possible following recent improvement in extraction and clean-up procedures. Current research on the subject of insecticide resistance has made most progress in the field of genetics. Resistance has usually been found to be due to single principal genes, with DDT resistance usually recessive, organophosphorus resistance dominant and dieldrin resistance intermediate. In the housefly, for example, it is now known that one gene determines the resistance mechanism of detoxication by DDT-dehydrochlorinase, another is associated with either reduced cuticular penetration or decreased nerve sensitivity (or both), and a third with microsomal oxidation of this insecticide. These findings have an important bearing on future measures that will be taken to counter resistance to DDT by the use of enzyme-inhibiting synergists and analogues that do not undergo dehydrochlorination. The mechanism of dieldrin resistance remains unknown, and does not appear to involve detoxication. The World Health Organization has made available marker strains in order to further research into this vital topic. Basic research on insecticide resistance has become concentrated within a few specialist laboratories, so that certain investigators have a considerable familiarity with the biochemistry and genetics of the subject. Future studies should include work with the organo-phosphorus and carbamate insecticides, now used in such large quantiy. We do not, for example, understand the relationship between organo-phosphorus detoxication (which seems to be hydrolytic) and carbamate detoxication (which seems to be oxidative)yet both mechanisms are associated with the same gene in the housefly. Laboratory selection experiments give a useful lead on the likely field performance of an insecticide, e.g. human body lice can be induced to develop a 300-fold resistance to the carbamate carbaryl, but only two-fold resistance to malathion. Valid baseline data for each new compound becomes necessary as more and more insecticides come into general use. Information on the development of cross-resistance is also needed, as between the organo-phosphorus and carbamate insecticides. This will again provide advance data for plans to combat the resistance which may follow the use of new insecticides or insecticide combinations, including new synergists. Studies on insect nutrition are also valuable, as specialists in this subject can be called on for advice in unravelling the complexities of the mode of action of an insecticide. Current vector control is achieved very largely by chemical insecticides, but biological techniques have also been used in a few programmes, as with viruses, bacteria, nematodes, mosquito fish and irradiation. Most of these non-chemical methods have been directed against screw-worms, houseflies and mosquitoes, but genetic control has also been considered for use against

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blackflies, triatomid bugs, tsetse flies and ticks. The main practical problem has been to devise suitable mass-rearing techniques and the most likely solution may lie in auto-sterilization methods directed against the natural populations, perhaps as in the booby-trap procedure (Whitten and Norris, 1967). Future biological control is likely to continue in a secondary role, with genetic techniques probably offering the greatest promise. However, with populations of tropical mosquitoes up to 200000 per hectare (as with Culex p. farigam) a really vast release programme would be required in order to cover even a small area of territory-and even then there is the ever-present likelihood of re-invasion. The need is for a genetic control scheme in which the primary agent is inoculated into a large population and then perpetuates and intensifies itself in each succeeding generation. The answer may lie with strains that have particular chromosome translocations, as are now available for Ae. uegypti. Whatever the outcome of these investigations, the target insect population will have first to be reduced very considerably by previous treatment with insecticides. In the end, practical field programmes will always need to link purely hygienic measures with the use of chemical or biological control techniques. Man himself is often responsible for the conditions suitable for disease transmission : inadequate drainage and poor sanitation are prime causes of the huge populations of C.p . fatigans, while the storage of water in jars, etc., produces the urban Ae. aegypti problem, with the hazards of yellow fever and dengue haemorrhagic fever. The whole basis of future vector control therefore lies not only in vector ecology but human ecology, which again focusses attention on personnel. It is still a fact that the personnel concerned in control operations often lack suitable education and training. The continuous surveillance of vector sources and densities requires people who not only know their own area very thoroughly, but keep good records, make proper use of maps, and can carry through their own insecticide susceptibility tests. They must be able to choose the insecticide or combination of insecticides that has the greatest chance of success in their own programme, so that they will not continue to be committed to some standard method developed years previously without modification. In those countries that eventually develop facilities for the production of genetic or other biological material, key local workers must be able to say how this will be employed-perhaps as the final blow in a project where the original population of insects have been decimated by a conventional insecticide. Looking ahead, it seems that vector control 10 years hence will continue to rely heavily upon chemical insecticides. DDT will probably remain for the control of malaria in Africa, with perhaps the large-scale application elsewhere of organo-phosphorus and carbamate insecticides (provided that these become cheaper than they are at present). For the emergency control of adult mosquitoes there will probably be available several safe biodegradable insecticides, applied in high concentrations but in ultra-low-volume (ULV) from aircraft or the ground. As resistance develops to most of the compounds

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we know today, it may be that control will come to be increasingly based on the use of completely new types of chemicals, synthetic juvenile hormones and new petroleum larvicides. There will certainly be far more integration of biological and chemical control techniques to give maximum effectiveness at minimum cost. Few vectors will ever be eradicated, but a realistic goal is to provide for our increasing world population in the midst of a vector population which has been reduced to low levels, perhaps much of it with no longer any parasites to transmit.

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WILLIAM N. BEESLPY

List of common, chemical and trade names of chemicals used for the control of insects and acarines. More extensive lists appeared in 1965 and 1971 in the Review of Applied Entomology (Series B) 53, 1-9 and 59, 1-12 Common or other name in general use

Apholate

..

Chemical names or definitions

Other definitions

2,2,4,4,6,6-hexa(1-aziridinyl)-2,4, 6,-triphospha-l,3,5-triazine

ENT-263 16

2,3,4,4,5,6-hexahydro-2,2,4,4,6, 6-hexakis(l-aziridinyl)-l,3,5,2 4,6Arprocarb BHC

.. ..

Bromocyclen

.. ..

..

..

Bromophos

Bromophos-ethyl

Bulan Butacarb Carbaryl

..

.. ..

.. ..

..

Carbophenothion

..

Chlorbicyclen

..

t riazatriphosphorine see propoxur

1,2,3,4,5,6-hexachIorocyclohexane HCH, 666 benzene hexachloride (BHC is used for a mixture of isomers; the British Standard requires that the percentage of BHC be stated.) 5-bromomethyl-l,2,3,4,7,7hexachloro-(2,2,1)bicyclohept-2ene O,O-dimethyl0-4-bromo-2,5dichlorophenyl phosphorothioate 4-bromo-2,5-dichlorophenyl dimethyl phosphorothionate 0,O-diethyl 0-(-bromo-2,5dichlorophenyl) phosphorothioate 4-bromo-2,5-dichlorophenyl diethyl phosphorothionate 1,l -di(p-chlorophenyl)-2nitrobutane 3,5-di-tert.-butylphenyl met hylcarbamate I-naphthyl methylcarbamate 0,O-diethyl S-p-chlorophenylthiomethyl phosphorodithioate 5,6-di(chloromethyl)-1,2,3,4,7,7-

Bromodan Nexion, Cela S-1942, OMS-658 Cela S-2225

CS-674A

Sevin, Union Carbide 7744 Trithion, Stauffer R-I303 Alodan

hexachlorobi-cyclo(2.2.1)

Chlorfenvinphos

.,

Coumaphos

..

heptene-2 1,2,3,4,7,7-hexachIoro-5,6-di (chloromethyl)-(2.2.1) bicyclohept-2-ene diethyl 1-(2,4-dichloropheny1)-2chlorovinyl phosphate 0,O-diethyl 0-3-chloro-4-methyl-7coumarinyl phosphorothioate

GC 4072, SD 7859, Supona Co-Ral, Bayer 21/199, ENT-17957

181

CONTROL OF ARTHROPODS

Common or other name in general use Crotoxyphos

..

Crufomate

..

..

DDD

..

..

p,p’DDD

.. ..

..

..

..

Dichlofen thion

DDE

Dxt Diazinon

..

..

..

Dichlorvos

.,

.. ..

Difenphos

..

..

Dimethoate. .

..

.. ..

.. ..

.. ..

Dioxathion

Dursban Ethion

Fenchlorphos

*.

Fenitrot hion

..

Fenthion

..

..

Hemel

..

Hempa

..

.. ..

Chemical names or definitions

Other definitions

Ciodrin dimethyl 1-(alpha-methylbenzyloxycarbony1)- 1-propen-2-yl phosphate dimethy lcis- 1-methyl2 4 -phenylethoxy carbony1)vinyl phosphate O-methyl)4-tert. butyl-2-chloroRuelene phenyl methyl-phosphoramidate a complex chemical mixture in TDE which p,p’DDD (q.v.) predominates 1,l -di(p-chlorophenyl)-2,2dichloroethane 1,1 -di(p-chlorophenyl)-2,2dichloroethylene N, N-diethyl-m-toluamide Diethyl toluamide O,O-diethylO-2-isopropyl-4-methyl Basudin 6-pyrimidinyl phosphorothioate O,O-diethyl)-(2,4-dichlorophenyl) Nemacide, V-C 1-13, phosphorothioate vc-I 3 dimethyl 2,2-dichlorovinyl DDVP, Vapona, phosphate ENT-20738 O,O,O’,O’-tetramethyl 0,O-thiodi-p- Abate phenylene diphosphorothioate 0,O-dimethyl S-methylcarbamoyl- Rogor, Am.Cyanamid methyl phosphorodithioate 12880 2,3-p-dioxane S,S-bis(0,O-diethyl DeInav, ENT-22897. phosphorodithioate 1,4-dioxan-2,3-ylidene S,S-bis (0,O-diethyl phosphorodithioate 0,O-diethyl 0-3,5,6-trichIoro-2pyridyl phosphorothioate tetra-0-ethyl S,S‘-methylene Nialate bisdithiophosphate O,O,O,O’tetraethyl S,S’-methylene bisphosphorodithioate 0,O-dimethyl 0-(2,4,5-trichloroRonnel, Dow ET-57, phenyl) phosphorothioate Korlan, Trolene, Etrolene, Nankor 0,O-dimethyl 0-3-methyl-4Sumithion, Bayer nitrophenyl phosphorothioate 41831, Folithion, Agrothion 0,O-dimethyl 0-3-methyl-4Bayer 29493, methylthiophenyl phosphoroBaytex, Lebaycid, thioate Tiguvon

2,4,6-tris(dimethylamino)-striazine (as hydrochloride) hexamethylphosphoric triamide

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WILLIAM N. BEESLEY

Common or other name in general use Heptachlor

..

..

Chemical names or definitions

Other definitions

1(or 9),4,5,6,7,10,10-heptachloro4,7,8,9-tetrahydro-4,5-endomethyleneindene l(or 3a),4,5,6,7,8,8-

heptachloro-3a,4,7,7a-tetrahydro-4, Indalone

7-methanoindene

,.

Iodofenphos Isobenzan

..

*.

2,2-dimethyl-6-carbobutoxy-5,6-

..

dihydropyrone 0,O-dimethyl 0-2,5-dichloro-4iodophenyl phosphorothioate

..

Ciba 9491, OMS-121 1, jodofenphos 1,3,4,5,6,7,8,8-octachloro-3a,4,7, Telodrin, WL-1650

7a-tetrahydro-4,7-methanophthalan 1,3,4,5,6,7,8,8-octachloro-l,3,3a,4, 7,7a-hexahydro-4,7-methanoisoMalathion

..

.,

.. ..

Methotrexate Phosmet

..

Piperonyl butoxide

Prolan

,

.

..

Propoxur

..

..

..

Pyrimithate

.. Tepa .. Tetrachlorvinphos . . Toxaphene .. Trichlorphon .. Resmethrin

a .

WARF Antiresistant

benzofuran Mercaptothion 0,O-dimethyl S-( 1,2-di(ethoxycarbonyl) ethyl) phosphorodithioate karbofos (USSR) N-(p-((2,4-diamino-6-pteridinyI) Amethopterin methyl-aminobenzoy1)glutamicacid 0,O-dimethyl S-phthalimidomethyl Imidan, Prolate, phosphorodithioate Stauffer R-1504 product containing as its principal constituent ~~-(2-(2-butoxyethoxy)ethoxy)-4,5-methylenedio-xy-2propyltoluene 1,l -di(p-chlorophenyl)-2nitropropane 2-isopropoxyphenyl methylBayer 39007, Baycarbamate gon, Ortho IMPC, Unden, arprocarb, OMS-33 O,O-diethyI-O-[2-(dimethylamino)-Diothyl, ICI 29, 661 4-methy1-6-pyrimidinyll phosphorothioate 5-benzyl-3-furylmethyl(k )-cis, trans-chrysanthema te tris( 1-aziridiny1)phosphineoxide Aphoxide 2-chloro- 1-(2,4,5-trichlorophenyi) Gardona, SD-8447, vinyl phosphate, trans isomer Rabon chlorinated camphene having a chlorine content of 67-69 % dimethyl 2,2,2-trichloro-lTrichlorfon, hydroxyethylphosphonate metriphonate Dipterex, Dylox, Neguvon N,N-di-n-butyl-p-chlorobenzene- WARF anti-resistant sulphonamide

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183

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Brown, P. R. M., Ferguson, D. A. M., Page, K. W., Smith, M. S.and Wood, J. C. (1965). The development of dichlofenthion for the control of sheep maggot fly in the United Kingdom. Vet. Rec. 77, 920-921. Brownlie, W. M. and Harrison, I. R. (1960). Sarcoptic mange in pigs. Vet. Rec. 72, 1022-1023. Bruce-Chwatt, L. J. (1971). Insecticides and the control of vector-borne diseases. Bull. Wld Hlth Org. 44, 419-424. Bry, R. E., Bowman, M. C., Crumley, F. G., Lang, J. H. and Cail, R. S. (1972). Evaluation of Ciba (2-9491 as a mothproofing agent. J. econ. Ent. 65, 584590. Bryan, J. H. and Coluzzi, M. (1971). Cytogenetic observations on Anopheles farauti Laveran. Bull. Wld Hlth Org. 45, 266-267. Burges, H. D. and Hussey, N. W.(1971). “Microbial Control of Insects and Mites”. Academic Press, London and New York. Burnett, G. F. (1970). In “The African Trypanosomiases”. (Ed. H. W. Mulligan), pp. 464-520. Allen and Unwin, London. Butt, K. M. (1971). The use of Bromocyclen for the control of the cat flea (Ctenocephalides felis). Vet. Rec. 88, 253-254. Carson, R. (1963). “Silent Spring”. Hamish Hamilton, London. Catts, E. P., Garcia, R. and Poorbaugh, J. H. (1965). Aggregation sites of males of the common cattle grub, Hypoderma lineatum (De Villers) (Diptera: Oestridae). J. med. Ent. 1, 357-358. Chamberlain, W. F. and Hopkins, D. E. (1971). The synthetic juvenile hormone for control of Bovicola limbata on Angora goats. J. econ. Ent. 64, 1198-1199. Cole, M. M. and Burden, C. S. (1956). Phosphorus compounds as ovicides and adulticides against body lice. J. won. Ent. 49, 747-750. Colglazier, M. L., Enzie, F. D. and Wilkens, E. H. (1960). Some chemotherapeutic trials in canine demodectic mange. Proc. Helm. SOC.Wash. 27, 139. Collins, R. C. and Dawhirst, L. W. (1971). The cattle grub problem in Arizona. 11: Phenology of common cattle grub infestations and their effects on weight gains of preweaning calves. J. econ. Ent. 64, 1467-1471. Cook, J. (1964). Review of the persistent organochlorine pesticides. Rept Advisory Committee to Min. Agric. Fisheries and Food. pp, 68. London, M.A.F.F. Crystal, M. M. (1971). Sexual sterilization of screw-worm flies by N,N’-tetramethylenebis (1 -aziridinecarboxamide) : further studies on influence of route of administration. J. med. Ent. 8, 304-306. Darrow, D. I. and Whetstone, T. M. (1972). Age and susceptibility of nymphal lone star ticks to selected ixodicides. J. econ. Ent. 65, 156-158. Davidson, G. (1970). Prospects for the control of Anopheles gambiae by genetic means. I n “Health and Disease in Africa”. Session 11, pp. 155-1 60. Proc. 1970 E. Afr. med. Res. Council Sci. Conf., Nairobi. Davies, J. B., Crosskey, R. W., Johnston, M. R. L. and Crosskey, M. E. (1962). The control of Simulium damnosum at Abuja, Northern Nigeria, 1955-60. Bull. Wld Hlth Org. 27, 491-516. Dedek, W., and Schwarz, H. (I 968). “Percutaneous absorption of S2P-labelled organophosphorus compounds”. Soc. chem. Ind. Monograph no. 29, 120-1 33. S.C.I.,London. Demilo, A. B. and Crystal, M. M. (1972). Chemosterilants against screw-worm flies. J. econ. Ent. 65, 594595. Drummond, R. 0. (1967). Control of larvae of screw-worm in cattle with insectidical sprays. J. econ. Ent. 60, 199-200. Drummond, R. 0. and Graham, 0. H. (1965). Systemic insecticides in livestock insect control. Vet. Rec. 77, 1418-1420.

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Drummond, R. O., Darrow, D. I. and Gladney, W. J. (1971). Further evaluation of animal systemic insecticides, 1970. J. econ. Ent. 64, 11661170. Fiedler, 0. G. H. (1967). The use of bromophos on livestock. Agric. vet. Chem. 8, 57-58.

Fiedler, 0. G. H., Du Toit, R. and Kluge, E. G. (1954). The influence of the tsetse fly eradication campaign on the breeding activity of Glossinae and their parasites in Zululand. Onderstepoort J. vet. Res. 26, 389-397. Frazer, A. C. (1964). Report of the Research Committee on Toxic Chemicals, Agricultural Research Council, pp. 38. London, A.R.C. Furman, D. P. and Stratton, V. S. (1963). Control of northern fowl mites, Ornithonyssus sylviarum, with sulfaquinoxaline.J . econ. Ent. 56, 904-906. Galley, R. A. E. (1967). World Review of Pesticide Use. Proc. 3rd British Pest Control Conference (series I, paper I), pp. 1-6. Garnham, P. C. C., Davies, C. W., Heisch, R. B. and Timms, G. L. (1947). An epidemic of louse-borne relapsing fever in Kenya. Trans. R. See. trop. Med. Hyg. 41, 141-170. Georghiou, G. P. and Hawley, M. K. (1971). Insecticide resistance resulting from sequential selection of houseflies in the field by organo-phosphoruscompounds. Bull. Wid Hlth Org. 45, 43-51. Gething, M.A. and Walton, G. S. (1972). Possible host specificity of Cheyletiella mites. Vet. Rec. 90, 512. Gibson, T. E. (1964). The cost of animal parasites. Span. 7, 2-5. Gillett, J. D. (1971). “Mosquitoes”. World Naturalist Series, Weidenfeld and Nicolson, London. Gledhill, J. A. and Caughey, W. (1963). Report on a field trial in the use of dieldrin for the control of Glossina morsitans in the Zambesi Valley, 1961. Int. Sci. Commis. Tryp. Res. 9th meeting, 239-251. Glover, P. E. (1967). The importance of ecological studies in the control of tsetse flies. Bull. Wld Hlth Org. 37, 581-614. Graham, D. H. and Drummond, R. 0. (1967). The potential of animal systemic insecticides for eradicating cattle grubs, Hypoderma spp. J. econ. Ent. 60, 1050-1053.

Gregson, J. D. (1958). Recent cattle grub life-history studies at Kmloops, British Columbia and Lethbridge, Alberta. Proc. X Int. Ent. Congress (Montreal, 1956), 3, 725-734. Greenwood, D. and Harrison, I. R. ( I 965). The search for a veterinary insecticide. I: Sulphonamides and disulphonamides active against sheep blowfly. J. Sci. Fd Agric. 16, 293-299. Gresty, R. H. C. (1971). Control of cattle ticks by dipping. Span. 14, 16-18. Grover, K. K. and Pillai, M.K. K. (1969). Chemosterilization of Culex pipiens fatigans Wiedemann by exposure of larval stages. Bull. Wld Hlth Org. 41, 929-93 6. Gubler, D. J. (1970). Competitive displacement of Aedes (Stegomyia) polynesiensis Marks by Aedes (Stegomyia) albopictus Skuse in laboratory populations. J. med. Ent. 7 , 229-235. Hadaway, A. B. (1972). Toxicity of insecticides to tsetse flies. Bull. Wld Hlfh Org. 46,353-362. Hall, C. A. and Ludwig, P. D. (1972). Evaluation of the potential use for several organotin compounds against the sheep blowfly (Lucilia spp.). Vet. Rec. 84, 29-32.

Harley, K. L. S. and Wilkinson, P. R. (1971). A modification of pasture spelling to reduce acaricide treatments for cattle tick control. Aust. oef. J. 47, 108-1 I I .

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The Epidermis and Sense Organs of the Monogenea and Some Related Groups K. M. LYONS Zoology Department, King’s CoUege, University of London, England 1. Introduction ..........................................................

11. Turbellaria .......................................................... 111. Aspidogastrea ........................................................

1V. The Epidermis of Monogenea .......................................... A. Embryology and Structure of the Larval Epidermis .................. B. Adult Monogenea ................................................ V. Sense Organs of Monogenea.. .......................................... A. Eyes (including possible ciliary eye). ................................. B. Sense Organs other than Eyes .................................... VI. Conclusion .......................................................... References ..........................................................

193 194 200 201 201 206 218

219 220 227 228

1. INTRODUCTION As the structure of helminth surfaces in general has been reviewed by Lee (1966, 1972) this review aims to provide a more specialized and detailed account dealing mainly with the epidermis* and sense organs of Monogenea and their larvae, and also with the epidermis of some Turbellaria and Aspidogastrea. The Turbellaria contains free-living forms but they are considered here because their epidermal fine structure, with its function and development, may be said to relate particularly to the free-living stages of the larvae of monogeneans. In addition, study of the turbellarians as a whole adds considerably to our knowledge of the range of epidermal structure open to the Platyhelminthes, allowing the constancy of epidermal cytomorphology in various groups and its possible use in taxonomy to be assessed, and fostering a dawning appreciation of plasticity upon a basic structural plan in conformation to functional requirement. In reviewing work on some of these groups it has been necessary to draw on unpublished observations, some of which are of a preliminary nature.

* Following Lee (1966, 1972) the term “epidermis” is used for the living cytoplasmic covering of Turbellaria and Monogenea, and their larvae. This is equivalent to what in trematodes and cestodes was once called the “cuticle”, by some writers more recently, the ”tegument” or “integument”. 193

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In general, owing to the interesting phylogenetic position of the monogeneans ris-d-iis the “rhabdocoeles” on the one hand and cestodes on the other (Llewellyn, 1965) and the largely ectoparasitic habit of the Monogeneans, knowledge of epidermal structure, development and function in this group provides useful comparisons with the endoparasitic Digenea and Cestoda. The Monogenea is a group with a fairly well worked out taxonomy, which has been investigated in regard to epidermal fine structure so that the cytoarchitecture of this layer is now known in most of the major groups.

11. TURBELLARIA The epidermis of several species of Acoela was studied by Dorey (1965) (see Lee 1966). In addition, the fine structure of the surface of Convolutu rosroflensis has been studied by Pedersen (1964) and Oschman (1967). Dorey found that in all the Acoela he examined, the epidermal cells were not separated from the muscle layers by any kind of basement lamina and that in some Acoela, e.g. C. roscoflensis, the epidermal cells have nucleated processes situated amongst the muscle fibres of the peripheral parenchyma. This may not be true of all Acoela because Dorey (1965) stated that in Aphanostoma diversicoh, most of the epidermal nuclei lie superficial to the integumentary muscle, although even here lobes of the epidermal cells penetrate the muscle layers. In Amphiscolops lungerharisi (kindly provided and embedded by Dr D. L. Taylor) Lyons (unpublished observations) showed that the epidermis of this acoelan resembles that of Convoluta in having nucleated portions “insunk” into the muscle layers. The epidermal cells are apparently secretory, as Colgi bodies in the nuclear region bud off small vesicles condensed into “multivesicular” bodies which are the commonest secretion found in the epidermis. Secretions of a similar nature occur in the epidermis of other Acoela (Dorey, 1965). Short branching microvilli (0.4-0-5 pm long) are present on the epidermis of Amphiscolops and on the epidermis of other Acoela, where they may fuse at their tips to form “fences” running between the cilia (Dorey, 1965). Like many other free-living invertebrates (see Southward and Southward, 1972) Co/?i~olutu has been said to absorb amino acids across the epidermis (Read, cited by Lumsden, 1966) and the microvilli may assist in this-although they are much shorter than, for instance, those on the surface of the neorhabdocoel Kronborgia amphipodicola (see Bresciani and K~rie, 1970), which lacks a gut and must therefore absorb nutrients through the

FIG.1. Electron micrograph of a section through the body wall of the dalyellioid “rhabdocoel” Syndesmb ecltinorum. The epidermis is cellular and nucleated and small microvilli are present between the cilia. The distal edge of epidermal cytoplasm forms a thickened terminal web-like zone. bl, basement lamina; cb, cell boundary; m, mitochondria; mv, microvilli; nu, nucleus; tw, terminal web. FIG.2. Epidermis of Syndesmis erhinorum showing the junction between a microvillous cell and a ciliated cell. The microvilli have a ring-like internal thickening. mv, microvilli; tw, terminal web.

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epidermis. The ciliary rootlet system of Amplziscolops langerliansi resembles that of the acoelans described by Dorey (1965) in that the main rootlet gives off two lateral rootlets each of which makes contact with the ends of adjacent rootlets on each side (Lyons, unpublished observations). This arrangement may be characteristic for Acoela. Comparatively little attention has been paid to the epidermis of those turbellarians loosely classed together as “rhabdocoels” (see Ax, 1963, for more detailed taxonomy) which show endocommensal or parasitic associations with various invertebrate hosts and are said to have ancestors which gave rise to the two major parasitic lines, the digenean and the monogeneancestode lines (Llewellyn, 1965). The epidermis of the endoparasitic species K. amphipodicola, however, was studied by Bresciani and Klaie (1970), and Burt and Phillips (1969) gave a brief account of the epidermis of Urastoma cyprinae, a parasite of certain oysters (Crassostrea tirginicaj. In both these forms the epidermis is cellular (though possibly “lateral” cell boundaries break down in mature female Kronborgia-see Bresciani and Ksie, 1970) and in Kronborgia rests on a well developed basement lamina. The epidermis of both Kronborgia and Urastoma is microvillous and ciliated. The fine structure of the dalyellioid “rhabdocoela” Syndesmis echinorum from the gut of Echinus esculentus (Plymouth, England) has been investigated (Lyons, unpublished observations) for comparison with these “rhabdocoeles” and with the Temnocephalida which are said by Ax (I 963) to be related to the dalyellioids. Like Kronborgia, S. echinorum has a single layered epidermis which rests on a basement lamina (Fig. 1). The epidermis is cellular but the cell boundaries become highly convoluted and interlocking in the basal third of the thickness of the cell where the nuclei are situated. Apically, the “lateral” boundaries of cells are joined by septate desmosomes. Microvilli occur between the cilia on the surface of Syndesmis but are much shorter than those of Kronborgia and measure only 0.20-0-25pm. They are unbranched and also lack the club-shaped, vesicular ends present on the long microvilli of Kronborgia. The microvilli of Syndesmis have an internal cylinder of thickening which is bilaterally rather than radially symmetrical (Fig. 2) ; this thickening recalls that in the microvilli of cestodarians (Lyons, 1969a) and the microtriches of cestodes (Jha and Smyth, 1969). The surface of Syndesmis is not completely ciliated and some cells bear only short microvilli and have fewer mitochondria than the ciliated cells. A particularly dense terminal web-like thickening is present in the distal cytoplasm of the microvillous cells but a terminal web-like zone is also present at the free surface of the ciliated cells

FIG.3. Electron micrograph of the body wall of Temnocephala novae-zealandiae, The epidermis appears to be largely syncytial although the deeply folded basal plasma membrane makes this difficult to establish with certainty. It contains prominent nuclei and rod-like inclusions. Uniciliate receptors penetrate the epidermis and ducts containing rhabdite-like inclusions. bl, basement lamina; bpl, folds of basal plasma membrane; ep, epidermis; mu, muscle; nu, nucleus; rh, rhabdite-like inclusion; so, sense organ (By kind permission of Kirstin Clark Nichols).

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(Fig. 2). The functions attributed to terminal webs are reviewed later (p. 210). The epidermal cells of Syndesmis appear to be polarized into an outer zone concerned with the energetics of the ciliary movement and perhaps secretion and an inner synthetic zone. The outer terminal web zone contains the striated ciliary rootlets and mitochondria; the basal nucleated region contains ribosomes, a folded endoplasmic reticulum and Golgi bodies. The major “secretory” inclusions in these cells are small electron-opaque vesicles. Rhabditelike inclusions similar to those described for Kronborgia have not been found in the epidermis of Syndesmis and gland cells opening into the epidermis have not been observed. Very little is known about the development and regeneration of the “rhabdocoel” outer layer, although this would be of great interest. Cell division in the epidermis of the “rhabdocoel” Stenostomum was reported by Pullen (1957). The Temnocephalida may have fairly close relationships with the dalyellioid neorhabdocoels (see Ax, 1963). Clark (1968) examined Temnocephala norae-zealandiae Haswell 1888, an ectocommensal of the New Zealand crayfish Pnranephrops neozealanicus; with the aid of the electron microscope (EM), she found that the outer covering is a non-ciliated cytoplasmic layer 4-7 pm thick resting on a basement lamina and containing conspicuous nuclei and Golgi bodies (Fig. 3). Thr epidermis appears to be largely syncytial but septate desmosomes are present where nerve endings and gland ducts penetrate it. The basal plasma membrane of the epidermis is deeply infolded. The free surface of the epidermis bears short, irregular microvilli about 0.3 p m long. Rod-like inclusions in the epidermis resemble those seen in the epidermis of adult Digenea and Cestoda. The tentacular epithelium of Temnocephala is specialized and secretory and at its outer edge contains a zone of vesicles with fibrous contents which may be some kind of mucous secretion (Fig. 4). Gland ducts lined with microtubules enter the epidermis of the body and contain dense “rhabdite-like” inclusions 3 pm long. The epidermis of the posterior sucker is highly modified and, except at its outer edge, is penetrated by membrane folds. Long regularly arranged microvilli about 0.6 pm long are present on the sucker (Fig. 5). Little work has been done on epidermal ultrastructure in Tricladida since that of Skaer (1961, 1965). but MacRae (1967) and Best et a/. (1968), working on Dugesia tigrina and D.dorotocephala, confirmed Skaer‘s findings that the epidermis of triclads is generally a single cell layer with foot-like processes resting on a basement lamina and bearing cilia and short microvilli. It is penetrated by many gland ducts. The epidermis of triclads seems to show -.

.

FIG.4. Epidermis of the tentacle of Temnocephala novae-zealandiae showing the, apparently secretory, modified distal surface. mc, mucus-like inclusions. (By kind permission of Kirstin Clark Nichols). FIG.5. Epidermis of the sucker of Tenmocephala novae-zealundiae which is penetrated by membrane folds and has comparatively long microvilli on its free surface. mv, microvilli. (By kind permission of Kirstin Clark Nichols).

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some surface differentiation, because ciliation is largely confined to the ventral body surface, and the pharynx epithelium of Polycelis nigra is densely microvillous (Skaer, 1961). Epidermal cell nuclei are not always superficial in triclads and the pharynx of Polycefis is lined by an epithelium described as having “insunk” nuclei (Skaer, 1961). The close integration of the outer epidermis with the parenchymal mesenchyme is shown by gland cells that differentiate in the parenchyma migrate out and establish contacts with the epidermis, through which their secretions are extruded. This contact may break down when the gland cells exhaust their secretions. In addition, regeneration of the epidermis in adult triclads occurs by differentiation and replacement from a pool of parenchymal neoblasts (Sakuma, 1969) and replacement of embryonic epidermal cells by parenchymal cells also occurs during a short period of embryonic development in Polycefis tenuis (see Skaer, 1965). Preliminary observations on a species of Polycladida, Kaburakia excelsa Bock, 1925, collected from the San Juan region of Puget Sound, U.S.A. show that the epidermis is cellular, with columnar cells packed tightly together above a thick multilayered basement lamina with an orthogonal fibre arrangement in successive layers. The free surface bears branched microvilli between the cilia and these lack an internal thickening and are much longer than those of Syndesmis (2 pm compared with 0-2 pm), Gland ducts penetrate the epidermal layer and extend beyond the general surface level.

111. ASPIDOGASTREA

Rohde (1972) has reviewed his own researches on the fine structure of Aspidogastrea and especially adult and larval Mufticotyfe purvisi. The larval epidermis is largely non-ciliated syncytial cytoplasm, which adjoins parenchymal “cell-bodies”. There are also ten ciliary tufts, each of which contains a single nucleus situated in a socket-like extension of the ciliated cell into the neighbouring superficial syncytial tegument. The cilia of the tuft-cells have long striated “rootlets” situated at an acute angle to the surface plasma membrane, rather like those of the ciliated cells of larval Entobdellu. The syncytial (interciliary) tegument contains electron-dense ovoid bodies and bears at its free edge unusual long fine processes (microfila), which arise from knob-like projections of the surface, are much more slender than microvilli and may be some kind of flotation device. They are about 6 pm long at the anterior end of the larva, only 12-18 nm in thickness and contain internal filaments (Rohde, 1971, 1972). The epidermis of adult Multicotyle purvisi probably derives from the interciliary tegument of the larva. It is again syncytial and has secretory “cell” bodies situated in the parenchyma. There are some regularly arranged microtubercles or “rib-like elevations” (Rhode, 1972) at the free surface of the tegument; these support a layer of mucus and are probably equivalent to the rounded basal regions of the tegumental microfila of the larva, the long processes having broken off. Electron-dense ovoid or disc-shaped granules occur in the surface syncytium and are secreted by the “cell” bodies.

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The epidermis of adult Aspidogaster conchicola was studied by Bailey and Tompkins (1971) and Halton and Lyness (1971). The epidermis of adult Taeniocotyfe sp. from the gall bladder of Hydrofagus coffiei (collected in Puget Sound, U.S.A.) has been studied by Lyons, Laurie and Whitfield (unpublished observations). As in Mufficotyfe,the epidermis of Aspidogaster and Tueniocotyle is syncytial, with connections to parenchymally situated ‘‘cell’’ bodies secreting disc-shaped dense inclusions into the outer syncytium and regularly arranged processes or microtubercles (0.08-0*1 pm in A. conchicofu;see Halton and Lyness, 1971) on the free surface. If the microtubercles of adult Multicutyfe do represent the basal processes of larval microfila it would be of interest to know whether or not microfila occur in the larvae of these other Aspidogastrea. The outer level of the epidermis of Aspidogaster and Tueniocotyle is thickened into a fibrous terminal web-like zone (0.5 pm thick in A. conchicolu: Halton and Lyness, 1971) and this, like surface microtubules and disc-shaped epidermal inclusions, may prove to be a typical feature of the aspidogastrean epidermis. Thus, the epidermis in three genera of Aspidogastrea studied is similar, and although it resembles the epidermis of Monogenea, Digenea and Cestoda in general, it differs from all these groups in detail. Adult Digenea typically have an infolded channel containing the epidermal surface ; Monogenea and Cestoda tend to have microvilli or microtriches on their free surfaces, these being unlike microtubercles of Aspidogastrea. Secretory gland cells occur in the ventral sucker of A. coplchicolu and elsewhere over the body, and in the adhesive organ gland cells may be involved in extra-corporeal digestion of host tissue, resulting products then being absorbed through the epidermis (Bailey and Tompkins, 1971).

Iv. THEEPIDERMIS OF MONOGENEA A.

EMBRYOLOGY A N D STRUCTURE OF THE LARVAL EPIDERMIS

1 . Embryos A more detailed account of the work briefly recorded by Lyons (1968) (Lyons, 1973a) and reviewed by Lee (1972) on the embryology and fine structure of the larval epidermis of Entobdelfu soleue is in preparation and the results are summarized here. Development was studied in embryos of different ages dissected from eggs after brief initial glutaraldehyde fixation and processed for EM study. At first (in 7-8 day embryos incubated at ISOC) the embryo is covered with a nucleated primary epidermis of flattened cells which are closely associated with the vitelline cells inside the egg capsule and may take up nutrients from them. This layer is either replaced by or develops into the secondary epidermis which consists of ciliated cellular regions joined by an apparently syncytial interciliary cytoplasmic layer (Figs 6 and 8). Both the ciliated cells and the interciliary cytoplasm are nucleated at 9-13 days (at 15°C) and the interciliary cytoplasm contains Golgi bodies and rod-shaped granules (Figs 8 and 6). Ciliated cells were seen apparently migrating out of

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the parenchyma in 9-10 day embryos whilst other ciliated cells were apparently being lost, so that there may be a turnover of ciliated epidermal cells at the surface of young embryos similar to that occurring during the early embryology of Polycelis tenuis as described by Skaer (1965). However, ciliated replacement cells of the kind found by Dorey (1965) in the regenerating acoelan C . roscoflensis, with cilia directed into a central vacuole, were not observed in the parenchyma with certainty. The basement lamina and muscle layers start to differentiate about days 12-13 and may inhibit further migration of ciliatedreplacement cells. At days 16-20 (at 15”C),a discontinuous “presumptive adult” epidermis appears beneath the ciliated cells. This has connections to cell bodies lying in the parenchyma which have a peripheral ribosome-rich cytoplasm and areas of electron-lucent cytoplasm which may contain glycogen, also granules and vesicles of the type found in the “presumptive adult” layer. This layer fuses with the (syncytial?) interciliary regions which by this stage have lost their nuclei. Despite this apparent fusion of “presumptive adult” and interciliary regions the interciliary cytoplasm continues to contain dense crystalline granules which are much larger than granules present in the “presumptive adult” layer (Fig. 8c). The ciliated cells are nucleated at this stage and between days 16-20 pits develop around the bases of the cilia and dense granules, that previously had been situated centrally to basally in the cells, migrate up into the walls of these pits. 2. The hatched larva The structure of the epidermis in the hatched larva is shown in Figs 7 and 8c. There are three main regions of ciliated cells, as described by Kearn (1963, 1971), and a modified Robinow silver osmium method has been used to stain the boundaries of these cells (Lyons, 1973a). The anterior band contains about 30 cells, the posterior body band 23 and the haptor bears at least 11 ciliated cells. The ciliated cells measure about 20 pm in diameter, are very thin (1.5-2.0 pm) and contain droplets of triglyceride which appear to constitute the main energy store for these cells, because although glycogen was found in the parenchyma of hatched larvae, little was located in the ciliated cells. The cilia are 16 pm long and situated in shallow pits, the walls of which contain dense secretion bodies and have long anteriorly directed striated rootlets set at about 13” to the cell surface. The mitochondria occupy

FIG.6. Electron micrograph of a section through the epidermis of a 9-10 day embryo of Entobdellu soleae which has been dissected from the egg. Part of the embryo is ciliated and is covered by nucleated ciliatedcAls, the remainder of the embryo is covered by an apparently syncytial interciliary cytoplasm which is also nucleated at this stage. cil, cilia; ic, (syncytial ?) interciliary cytoplasm; I, lipid droplets; nu, nucleus. FIG.7. Epidermis of the hatched larva of Entobdellu soleue. A discontinuous layer of “presumptive adult’’ epidermis has come to lie beneath the ciliated cells and is connected to “cell’ ’bodies lying in the parenchyma. The ciliated cells have lost their nuclei and contain large mitochondria and lipid stores. cil, cilium; m, mitochondrion; mu, muscle; p, pits around cilia; pal, presumptive adult epidermis;.r, ciliary rootlets.

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FIG.8. Diagram showing the series of events in the embryogenesis of the epidermis in Entobdella saleae. (a) 9-10 day stage showing nucleated ciliated cells and (syncytial?) interciliary region containing a nucleus and Golgi body. (b) 13-16 day stage. The ciliated cells have flattened greatly and cell bodies of the presumptive adult layer are differentiating in the parenchyma. The basement lamina and muscle layers are almost completely formed. Nuclei in the interciliary cytoplasm project right out from the surface and may be lost. (c) Hatched larva. The ciliated cells have lost their nuclei. Cells in the parenchyma have sent cytoplasmic processes beneath the epidermis to form a “presumptive adult” layer that is discontinuous but seems to make contact with the interciliary cytoplasm (now anucleate). cb, “cell” body; ic, interciliary cytoplasm; nu, nucleus; pal, presumptive adult epidermis.

a zone between the ciliary rootlets and are enormously developed, with many cristae. The epidermal cells of the hatched larva lack nuclei; these are lost about days 20-24 (at 15”C), when hatching occurs. The ciliated cells of this larva are thus unlike the nucleated cells of the miracidium of Fusciofu

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hepatica as described by Wilson (1969) and Southgate (1970), and unlike those of the miracidium of Schisrosoma mansoni where the nuclei of the ciliated epidermal cells lie in extensions of the cell inside the body wall muscles (Brooker, in Wright, 1971). The syncytial(?) interciliary cytoplasm contains crystalline rod-like inclusions and is joined to a discontinuous layer of “presumptive adult” cytoplasm underlying the ciliated cells. The interciliary cytoplasm and “presumptive adult” cytoplasm connect to “cell” bodies lying in the parenchyma as described. This is similar to what was found in the hatched miracidium of Fasciola hepatica, where a discontinuous layer of cytoplasm lies beneath the ciliated epidermal cells and has connections with parenchymal “cell” bodies. This layer is continuous with the intercellular ridges lying between the ciliated epidermal cells (Wilson, 1969; Southgate, 1970). In the hatched monogenean larva the discontinuous epidermis has infiltrated between the junctions of the ciliated cells and sends long processes up into them, although these are separated by their apposed plasma membranes and an intercellular gap. Shedding of the ciliated cells normally occurs when the larva infects the head of a sole (Solea solea) but can be induced experimentally by exposing larvae to host secretions (see Kearn, 1967a). Complete shedding of the ciliated coat takes only 30 sec (Kearn, 1967a, 1971). Observations made on ultrastructure of larvae shedding and having shed their ciliated cells (Lyons, 1973a) show that the discontinuous presumptive adult cytoplasm spreads out as a continuous syncytium (as far as can be judged) to cover the post-larva. As shedding of the ciliated cells occurs in response to host stimuli, it is presumably under nervous control and not caused merely, for instance, by the spreading of the presumptive adult cytoplasm beneath the overlying ciliated cells. What appear to be gap junctions (Gilula and Satir, 1971) are present both between ciliated cells and between the presumptive adult epidermis and the ciliated cells. These have been implicated as low resistance pathways for ion flow and electric coupling and could be involved both in the co-ordination of the ciliary beat in the larval epidermis and to provide pathways for information about when the ciliated coat is to be shed. Wilson et al. (1971) have investigated the effects of various enzymes, chelating agents and basic solutions on the shedding of the ciliated cells of the miracidium of Fasciola hepatica and found that only a pancreatin-trypsin mixture had any effect and this after only 14-2 h. These writers also discuss the possibility that acid phosphatase which is present in both the ciliated cells and underlying layer) may digest the desmosomal cement binding the epidermal cells to the ridge cells, or that changes in ion or water content of this cement may lead to cell shedding. Southgate (1970) also reviewed various possibilities and suggested that changes in conformation of the plasma membrane of the ciliated cell causing its expansion might be important. Although monogenean larvae were both fixed and observed casting their ciliated cells (Lyons, 1973a) giving few clues as to how this occurred; large vacuoles appeared between the two layers and the ciliated cells gradually separated, aided no doubt by the active beating of their cilia. Loss of the nuclei of the ciliated cells might conceivably be associated with the programmed life of these cells.

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As regards the development of the larval epidermis in E. soleae, the epidermis is a mosaic of nucleated ciliated and non-ciliated regions from very early (9-10 day stage at 15°C) (Figs 6 and 8a). Why ciliated epidermal cell replacement should occur for a short period in embryogenesis and why loss of nuclei from the interciliary cytoplasm and subsequent coupling with parenchymal “cell” bodies should occur is not clear. This latter phenomenon is very similar to that recorded during the development of cercaria by Matricon-Gondran (1969) and Hockley (1972) who describe how the “tegument” of developing cercariae is at first nucleated but loses its nuclei and establishes connections with parenchymally situated “cell” bodies (the subtegumental cells). Other authors (e.g. Bils and Martin, 1966; Dixon, 1970) suggested that the superficial nuclei of the primitive “epidermis” of developing cercariae sink into the parenchyma during tegument formation. In view of the considerable evidence for the outward migration of cells from the parenchyma to the epidermis and the pushing out of connections (presumably) from parenchymally situated epidermal or gland cells to join the superficial layer, 1 am sceptical about the view that epidermal nuclei sink inwards. It would be interesting to compare the embryogenesis of the epidermis of Acanthocotyle sp., a monogenean ectoparasite of rays, with that of Entobdella because Acanthocotyle has a non-ciliated larva (Kearn, I967b). The development of the epidermis of Gyrodactylus would bear further investigation. (Lyons 1970a), found that the epidermis of fully formed but unborn Gyrodactylus consisted of a nucleated apparent syncytium and suggested that this persists into the adult, perhaps with loss of the nuclei, because these were not found in adults, nor any connection to parenchymal cell bodies. The epidermis of Gyrodactylus may be equivalent to the first-formed primary epidermis of flattened blastomeres found in the larva of E. soleae and in the embryo of the triclad Polycelis tenuis described by Skaer (1965). Cell fusion to form a multinucleate syncytium--as may occur in the embryonic interciliary epidermis of E. soleae before this loses its nuclei and makes connection with the “cell” bodies and as apparently occurs in development of the epidermis of Gyrodactylus-has also been reported to occur in the formation of the outer envelope and inner envelope (=embryophore) of cestodes (Rybicka, 1966). The embryophore of some coracidia appears to be a ciliated syncytium, which is more unusual (see Rybicka, 1966). B.

ADULT MONOGENEA

We can now review the range of variation and of conformity in epidermal fine structure that occurs throughout the Monogenea, because representatives of many of the major groups have now been studied by electron microscopy. The Monogenea are divided into two main groups, the Monopisthocotylea, which have a single, undivided opisthaptor armed with hooks, and the Polyopisthocotylea, which have a subdivided opisthaptor that may either be armed with several hook-sucker units (as in the hexabothriids, including the rajonchocotylids) or by clamps as in the Mazocraeoidea (including the diclidophoriids), or be equipped with unarmed suckers as in

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the polystomatids and sphyranurans (see Llewellyn, 1970). The Monopisthocotylea mainly parasitize the skin of fishes and are probably nearer to the ancestral type of the Monogenea than the more specialized Polyopisthocotylea, which use their multiple attachment organs for clinging to the gill lamellae of the fish host (Llewellyn, 1963). Until recently, most work on the epidermal fine structure of Monogenea centred on the Monopisthocotylea. Lyons (1968) gave a brief description of the epidermis of Leptocotyle minor and Dictyocotyle coeliaca (an endoparasite of Raia naevus). A detailed comparative account of the epidermis of the two marine skin parasitic Monogenea, Entobdella soleae and Acanthocotyle elegans, was given by Lyons (1970h) and a description of Gyrodactylus by Lyons (1970a). The fine structure of the epidermis of the juvenile of Amphibdella flarolineafa,which lives in the blood system of the electric ray Torpedo nobiliana, was compared by Lyons (1971) with that of the gill-living adult in order to see whether or not the surface of the juvenile showed any ultrastructural modifications to life in the blood of its host. Of the few polyopistocotylean gill parasites that have been investigated most are mazocraeoidean gill parasites of teleost fishes. Before investigating the uptake of nutrients through the tegument of Diclidophora merlangi, Halton et al. (1968) published an account of the ultrastructure of the gut of this worm, Morris and Halton (1971) described the tegument of Diclidophoru merlangiand Halton and Arme (1971) outlined methods of detecting damage to the tegument in D. merlangi due to ligatures applied to seal off the gut during uptake experiments using a labelled amino acid. Preliminary observations on Gastrocotyle frachuri were briefly mentioned by Lyons (I 968) and a more detailed investigation on Plectanocotyle gurnardi has been reported (Lyons, 1972a). Two non-mazocraeoidean polypisthocotylea have so far been investigated in respect of ultrastructure, the hexabothriid Rajonchocotyle emarginata from the gills of Raia clavata (see Lyons, 1972a) and the polystomatid Polystoma integerrimum, a parasite in the bladder of the frog Rana temporaria (see Bresciani, 1972). A brief account of the covering of Udonella caligorum has been given by Clark (1968). In Friday Harbour, Washington, U.S.A. this hyperparasite infects the caligid copepod Lepeoptheirus longipm which lives on Sebastes maliger. There is some controversy as to whether or not Udonella is a monogenean, and Ivanov (1952) proposed to create a new group, Udonelloidea, to accommodate this genus. The main reasons for this decision seem to be that Udonella lacks haptoral sclerites and cements itself on to the smooth carapace of its host and that the larva/juvenile emerging from the egg is non-ciliated. However, both these features are within the scope of the class Monogenea; Leptocotyle minor lacks haptoral sclerites as an adult (Kearn, 1965) and Acanthocotyle spp. have unciliated larvae (Kearn, 1967h). In this review Udonella will be placed in the Monogenea.

I. Regional diferentiation, microvilli and terminal webs The covering layer of all these monogeneans (including Udonella) is a cytoplasmic epidermis which appears to be syncytial (see later) and which,

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except in the case of Gyraduactylus sp., connects by means of microtubule-lined processes to nucleated regions (inaccurately termed “cell” bodies) lying beneath the integumentary muscle layers in the parenchyma. The epidermis of Gyrodactylus does not seem to have parenchymally situated “cell” bodies and the superficial epidermal nuclei found in late unborn juveniles appear to have been lost in the adult (Lyons, 1970a). The epidermis of most adult monogeneans bears microvilli (as has been confirmed using scanning electron microscopy on Acanthocotyle and Gyrodactylus-Lyons, unpublished observations) and is therefore unlike the folded or channel-containing epidermis described for many Digenea (see Lyons, 1971 ; Lee, 1972). The epidermis of Rajonchocotyle emarginata (see Lyons, 1972a) and Polystoma integerrimum (see Bresciani, 1972) is however specialized in unusual ways. Over the general body surface the microvilli are usually short and scattered like those present on the epithelia of some vertebrates and invertebrates (e.g. Dictyocotyle coeliaca (see Lyons, I968), Udonella caligorum (see Clark, 1968) (Fig. 9), Amphibdella jlavolineata (juvenile and adult: Lyons, 1971), Gyrodactylus sp. (see Lyons, 1970a), Diclidophora merlangi (see Morris and Halton, 1971) and Plectanocotyle gurnardi (see Lyons, 1972a). The dorsal surface of Entobdella soleae and Acanthocotyle elegans bears unusually long microvilli (9-1 1 pm) whilst the ventral surface of these worms is smooth, with no villi. The epidermis of the opisthaptor of Entobdella is “digenean” in the sense that it is folded into crevices and channels (Lyons, 1970b); this is true also of the ventral haptoral surface of Diclidophora merlangi (see Morris and Halton, 1971). There is in general fairly obvious regional differentiation of the epidermis. Adhesive regions are for instance densely microvillous even in worms that have short, scattered microvilli over the remainder of the body. This has been shown for the anterior adhesive surface of Entobdella and Acanthocotyle by Lyons (l970b) and for Gyrodactylus by Lyons (1970a). Clark (1968) found that the posterior glandular haptor of Udonellu is covered with a thin epidermis bearing densely arranged microvilli. The microvilli in adhesive regions may help to spread and mix the sticky secretions of different gland cells (Lyons, 1970b). The long dorsal microvilli of Entobdella and Acanthocotyle must be specializations, though for what function is not clear. They may support a mat of mucus which might be protective in a mechanical, osmoregulatory or antibacterial sense or provide an extended area for respiratory exchanges, or possibly nutrient or ion uptake (see later). Vertebrate kidney microvilli have been observed (Tilney and Cardell, 1970) to pulsate; if this were true of the microvilli of these monogeneans they could set up a circulatory current. In Diclidophora merlangi the epidermis of the

FIG.9. Electron micrograph of a section through the body wall of Udonella caligorum which is like that of other monogeneans, syncytial with connections to secretory “cell” bodies. The epidermis is packed with dense granules and bears short microvilli. bl, basement lamina; c, connection linking outer epidermis to “cell “body; gr, granules; m, mitochondrion; mv, microvilli. (By kind permission of Kirstin Clark Nichols)

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mouth and covering the buccal suckers bears “bristled” protuberances which may have an abrasive or adhesive function (Morris and Halton, 1971). The epidermis of Rajonchocotyle emarginata (see Lyons, 1972a) and Polystoma integerrimum (see Bresciani, 1972) is not microvillous. In Polystoma the epidermis is thrown into ramifying folds which enclose shallow pits, as has been demonstrated with both the transmission and scanning EM (Bresciani, 1972). The surface of Rajonchocotyle is smoother than that of Polystomu, is not elevated into folds but is also pitted, the outer cytoplasmic surface being penetrated by numerous shallow pores measuring 0-3-0.4 pm dia where they open to the surface but tapering to a point proximally (Fig. 10). Incubation of living and fixed worms in thorium and lanthanum nitrate (respectively) indicated that these pores extended 0.7-0.8 pm into the epidermis, which has a total thickness of 5-6 pm, also that thorium was not pinocytosed by the surface. These pores may be sites of secretion because mucus on the general surface was most obvious in the pores. The outer layer of cytoplasm is also specialized, forming a dense terminal web-like region containing microfilaments. In Rajonchocotyle this is 0-34.4 pm thick and is sharply demarcated from the remainder of the epidermal cytoplasm, appearing as a dense outer band in sections (Fig. 10) (Lyons, 1972a). A less well developed terminal web is also shown in micrographs of Polystomu epidermis (Bresciani, 1972). This terminal web is not dissimilar from the dense sub-surface epidermal cytoplasm of Aspidogastrea (Bailey and Tompkins, 1971 ; Halton and Lyness, 1971; Rohde, 1972) although the dense cytoplasm in the aspidogastrean epidermis is far less obviously fibrous than the terminal web of Rajonchocotyle. A terminal web is usually characteristic of microvillous surfaces such as those of the intestinal epithelial cells and proximal convoluted kidney cells of vertebrates and microvillar microfilaments may be inserted into the terminal web. There is accumulating evidence that terminal web microfilaments of vertebrate tissues may be actin-like since they bond with heavy meromyosin ; they may therefore be contractile (Ishikawa et al., 1969). The terminal web of Rajonchocotyle may be either merely supportive, or by analogy could have contractile properties. If the surface of Rujonchocoryle were contractile this could provide a pumping mechanism, perhaps aiding discharge from the pores or flushing of the surface, in association with ion or metabolite exchange. For a more critical account of these possibilities see Lyons (1972a). With regard to mazocraeoideans, a much reduced fibrous terminal web is present in association with the distal plasma membrane of Plectanocotyle epidermis (Lyons, 1972a) (Fig. 7) and Morris and Halton (1971) report that “a thin

F~G.10. The body wall of Rujonchocotyle emarginafashowing the smooth-surfacedepidermis developed into shallow pores, the dense terminal web and a Golgi body and ribosomes in the superficial epidermis. g, Golgi; m, mitochondria; mu, muscle; PO, pores; ri, ribosomes; tw, terminal web. FIG.11. (inset) Detail of a Golgi body in the outer epidermis of Rajonchocotyle showing its association with ribosomes. g, Golgi; PO, pores; ri, ribosomes.

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layer of moderately dense cuticular matrix is occasionally visible” immediately beneath the surface membrane of D. merlangi. The terminal web may possibly be characteristic of or at least best developed in Polyopisthocotylea because it has not been found in the epidermis of those Monopisthocotylea so far investigated. 2. Secretory and lamellate inclusions The epidermis of all the monogeneans studied is secretory and various kinds of granular and vesicular inclusions are produced in the “cell” bodies which are rich in ribosomes and often have conspicuous Golgi bodies, before being transported into the outer syncytial layer of cytoplasm. It has generally been assumed that comparatively little synthetic activity occurred in the outer level of the epidermis because this contains few ribosomes and was thought to lack Golgi bodies; however, Golgi bodies have been found in the outer epidermis of Diclidophora by Morris and Halton (1971) and they also occur in association with ribosomes in the outer epidermis of Rajonchocotyle (see Lyons, 1972a and Figs 10 and 11). Apparent Golgi bodies also occur in the epidermis of Gyroaizcrylus where they are associated with lamellate bodies composed of microtubule stacks (Lyons, 1970a). It has not so far been possible to detect what kind of synthetic activity superficial Golgi bodies might be involved in. Bogitsh (1971) found Golgi bodies in the superficial epidermis of the digenetic trematode Haernatoloechus niedioplexus which can hydrolyse thiamine pyrophosphate (TPP) and ATP; (TPP) is considered to be a reliable marker for Golgi bodies in vertebrate cells. The function of these superficial Golgi complexes is presumably the same as those in the perinuclear cytoplasm. The secretory inclusions recorded in the epidermal cytoplasm of Monogenea are of two main kinds: membrane-bound, electron-dense granules which are rod-shaped to round and membrane bound vesicles with electronlucent contents. In Entohdella and Udonella dense granules are the dominant kind of epidermal inclusion ;in Acanthocotyle both kinds are present in similar proportions. Rajonchocofyle has three kinds of epidermal inclusion (see below). As very little is known about the chemical nature of these inclusions, the homology of secretions of similar appearance in different species or even in different regions of the same worm must be open to speculation. The dense granules may be mucoproteinaceous as they give a slight positive diastase-fast reaction with the PAS test in Entobdella, Acanrhocotyle and Amphibdella (Lyons, 1970b, 1971). The mucus coat in Entobdella, which may derive from dense granules, also gives metachromasia with toluidine blue which is said to be a property of mucoproteins (Lyons, 1970b). The dense granules of monogeneans measure 0-1-0-2 pm x 0 . 1 4 4 pm in the different worms studied. They are produced by the Golgi region of the epidermal “cell” bodies. In Acanthocotyle two kinds of cell body are present; one kind produces dense gianular epidermal inclusions and the other produces vesicles with electronlucent contents (Lyons, 1970b). Morris and Halton (1971) suggest that two morphologically different kinds of Golgi zone in the same epidermal “cell” body may produce the two different kinds of inclusions found in the epidermis of Diclidophora. In Plectanocotyle the folds of the basal plasma membrane

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into the epidermis enclose dense material and have sacculate ends and might be secretory (Lyons, 1972a). Rajonchocotyle emarginata has three kinds of epidermal inclusion: irregularly shaped vesicles with electron-lucent contents but electron-dense walls, round vesicles with coarsely fibrous contents and, these occurring more in frequently, small dense granules. All three kinds of inclusion occur in one type of “cell” body (Lyons, 1972a). Regional differentiation occurs in the distribution of various kinds of secretion. Morris and Halton (1971) find that dense granules in Diclidophora occur in the ventral epidermis more than in the dorsal and Lyons (1970b) found that membraneous inclusions are present in the ventral epidermis only of Entobdella. Lamellate inclusions of various kinds have been found in the epidermis of many monogeneans apart from Entobdella soleae ;in Amphibdella, compact lamellate inclusions were found in adults and loose lamellate inclusions in juvenile worms (Lyons, 197I). They are most highly developed in Gyrodactylus where they take the form of stacks of microtubules and may be associated with Golgi bodies (Lyons, 1970a). Vesicles containing smaller vesicles in Diclidophora have been compared to multivesicular bodies or phagolysosomes by Morris and Halton (1971), but this is contradicted by the absence of acid phosphatase from the tegument of this worm. Glycogen is present in the epidermal cell bodies of Amphibdella and Rajonchocotyle (see Lyons, 1971, 1972a) and may be a carbohydrate reserve for the production of secretory inclusions. There is obviously scope for the application and further development of cytochemical and biochemical methods that will help to elucidate the nature of these secretions. In addition to secretory “cell” bodies, gland cell ducts open via channels that penetrate as far as the epidermal surface. In Entobdella these are present not only in specialized regions such as the adhesive head gland area, but scattered glands open over much of the body surface (Lyons, unpublished observations). 3. Plasma membrane and surface coat The plasma membrane of adult monogeneans is a simple trilaminar unit membrane. This is not true of all parasitic platyhelminths; the schistosomula of S. mansoni may have a double unit membrane and adult S. mansoni may have a seven-layered membrane, this perhaps indicating continuous membrane recycling associated with transport activities or with membrane damage due to host immune reactions (Hockley, 1970; Clegg, 1972). The limiting membrane of the microthrix shafts of the cestode Echinococcus granulosus may also be multiple and have been described as consisting of two closely opposed plasma membranes (Jha and Smyth, 1969; Smyth, 1972) although other cestodes appear to have a unit membrane with a thickened outer leaflet (Lumsden et al., 1970a). Lamellate bodies in the epidermis of monogeneans may be phospholipid or lipoprotein and have been associated with membrane production and maintenance (Lyons, I370a, 1971). The so-called surface coat has many names, being referred to as a glycocalyx, the fuzzy coat, filamentous coat or amorphous coat. There exists some confusion over what can properly be considered as a surface coat and this is partly due to confusion between PAS-positive glycoproteins containing

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acidic groups and acid mucopolysaccharides, which are PAS-negative. Acid mucopolysaccharides are not usually considered to form part of the surface coat proper but this does consist of PAS-positive mucoproteins or glycoproteins usually rich in acidic groups which therefore also stain using methods for acid mucopolysaccharides (reviewed in Rambourg, 1971). The acidic groups may be carboxyl (e.g. those of sialic acid), sulphate or phosphate anions. A surface coat is present in monogeneans (Lyons, 1970b, 1971; Morris and Halton, 1971; Bresciani, 1972) as in the endoparasites (see Rothman and Elder, 1970; Oaks and Lumsden, 1971). This is thought to consist largely of mucoprotein because only a slight diastase fast reaction is given with the PAS test; also this layer gives B but not a! metachromasia with toluidine blue in Entobdella, as mentioned. The mucus mat overlying the tips of the dorsal microvilli of Entobdella soZeae binds thorium dioxide used as a stain for acid carbohydrates but this mucus could represent contamination from the host. Slight binding of colloidal thorium to the general surface coat occurs, so probably only few acid groups are present here (Lyons, 1970b). This is at present being further investigated using ruthenium red staining at neutral pH. Densification of the plasma membrane of the dorsal surface of E. soleae was produced using ruthenium red but there was no pronounced staining of the surface coat and this could have been an artefact due to post fixation in 2 % 0 s 0 4 (Lyons, unpublished results). Rothman and Elder (1970) found that a ruthenium red staining method at neutral pH gave unreliable results for acidic groups in the surface coats of Moniliformis dubius, Hymenolepis citelli and Haematoloechus medioplexus. Polyanionic groups of sialic acid in the surface coat have been implicated as important in absorption of cationic colloids in tapeworms (Lumsden et al., 1970a, b; Lumsden, 1972). This predominantly negative electrostatic charge associated with the surface coat may enable the glycocalyx of cestodes to act as a cation exchange resin, binding cations critical to the functioning of surface enzymes prior to uptake in the case of amino acids bearing a positive charge (Lumsden et al., 1970b). The paucity of acidic groups in the surface coats of the monogeneans studied may indicate that ion or amino acid binding associated with the transport of nutrients is less important in these ectoparasites, which may feed predominantly via the gut. Evidence is accumulating that the vesicular or granular inclusions produced in the Golgi zones of the “cell” bodies of digenean trematodes and cestodes migrate into the superficial epidermis and are discharged, contributing to the surface coat (see Oaks and Lumsden, 1971). The precise structure of the inclusion that carries carbohydrate-rich surface coat material to the epidermis remains to be identified. Bogitsh ( I 968) and Shannon and Bogitsh (1971) found that in Megalodiscus temperatus, rod-shaped vesicles with electron-opaque contents which stain using the PATCO technique for macromolecular diglycols, contribute neutral mucosubstance to the surface coat. Hockley (1970) suggests that spherical membraneous bodies may contribute to the outer membrane of S. mansoni adult whilst dense granules (elliptical bodies) may contribute to the ground substance of the epidermis. Bennett (1970) found that the surface coat material

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of duodenal cells in young rats was transported in electron-lucent vesicles rather than as dense granules. Dense granules in the tracheal epithelium of vertebrates are said to contribute to surface mucus layers rather than to the surface coat (quoted in Lyons, 1971). Oaks and Lumsden (1971) found, by pulsing tapeworms with radiogalactose, that the surface coat is renewed every 6-8 h. Nothing is known of the dynamics of surface coat secretion in monogeneans and very little of its function. Protective roles in a mechanical, antibacterial and osmotic or ion regulatory sense have been attributed to the surface coat and mucus layers of Entobdellu soleue (see Lyons, 1970b). By analogy with the schistosomes, the surface coat of the juvenile Amphibdella Jlavolineata could protect against possible immune reactions caused by the ray host by incorporating host-like antigenic determinants into itself (see Clegg, 1972) but this has not been investigated and comparatively little is known about the immune responses of lower vertebrates. The surface coat of Entobdella and Gyrodactylus does not play any part in the pinocytosis of colloidal thorium dioxide and ferritin; in the case of Gyrodactylus, surface mucus appeared to skim off ferritin from the surface of the body (Lyons, 1970a). Incubation of living specimens of Rajonchorotyle in thorium dioxide produced an accumulation of the colloid at the surface of the worms but this was not subsequently incorporated into the epidermis. It has not been demonstrated convincingly that monogeneans incorporate labelled nutrients across the epidermis, and this is due partly to difficulties of leakage caused by damaging the epidermis by ligaturing the gut (Halton and Arme, 1971). Morris and Halton (I 97 1) have suggested that the opisthaptor of Diclidophora merlangi, which is intimately associated with host gill tissue, may be a potential site where nutrient uptake occurs. In adult Amphibdella Jlavolineata the haptor is actually buried in host gill tissue and might be interesting to investigate in this respect.

4.Syncytiu and surface diferentiatian The extent to which the epidermis of monogeneans and other parasitic platyhelminths is syncytial is not known, but it is generally assumed that the surface cytoplasm is more or less continuous and without internal membrane boundaries, except where the epidermis is penetrated by gland ducts or sense endings, over the whole body of these worms. Septate junctions have in fact been seen joining two kinds of epidermis in the head region of E. soleae (see Lyons, 1970b). It would be desirable to reinvestigate this supposition using lanthanum nitrate or some other marker of intercellular space, although if boundaries are present but few they would be very difficult to pick up, especially using transmission electron microscopy. It may prove quite a problem to decide whether, for instance, a tapeworm is really covered with a continuous sheet of cytoplasm from scolex to end of strobila. Regional differentiation of this syncytium plainly occurs, as has been discussed for instance regarding microvillosity or in the distribution of different kinds of epidermal inclusion. The way in which this differentiation arises and is maintained in a syncytium situated some distance from the epidermal “cell”

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bodies poses interesting questions. The effects of a single cell body may extend through a considerable volume of superficial cytoplasm if the observations of Clark (1968) on Udonellu are substantiated, since epidermal “cell” bodies are said to be localized in the anterior dorsal third of the body but dense granules produced in these “cells” extend throughout the epidermis covering the main body. In the miracidium of Fasciola hepatica and the encysting cercaria of Fasciola and other digeneans, local differentiation in the tegument occurs by epidermal “cell” bodies pumping out specialized secretions into the overlying syncytium. In the miracidium of Fasciola the anterior vesiculated and apical gland “cells” (=specialized epidermal cell bodies ?) contribute cytoplasm and characteristic secretory inclusions, which remain fairly localized in the apical papilla, to the developing sporcyst wall (Wilson, 1969; Wilson et al., 1971). During cyst formation in Acunthoparyphiurn spinulosum and Fasciola hepatica parenchymal cystogenous cells connected to the epidermis empty secretions into this layer which are eventually released to the exterior, then the connections break down (Bils and Martin, 1966; Southgate, 1971). Hockley (1972) stated that the epidermal cell bodies of Schistosoma rnansoni may also be only temporarily connected to the epidermis, cells emptied of secretion being replaced by newly differentiated parenchymal cells. This kind of renewal has not been observed for monogeneans. In the case of Amphibrlellajlavolineata, however, juvenile worms have multinucleate epidermal “cell” bodies, so mitosis may occur in a differentiated epidermal blastema here rather than new epidermal elements differentiating de n o w from the parenchyma. Adult Aniphibdella has uninucleate “cell” bodies. An attempt has been made to investigate regeneration of the epidermis of adult E. sofeae after wounding (Lyons, unpublished results) but the results proved difficult to interpret, partly due to the difficulty or locating any “cell” bodies in this worm. Acanthocotyle would be a better subject for regeneration studies. Despite its syncytial arrangement, the epidermis of parasitic platyhelminths is obviously highly differentiated. In hymenolepid tapeworms differentiation of the microtriches appears to be partly a function of age, these becoming larger in more mature proglottids (Berger and Mettrick, 1971). Surface differentiation probably has its culmination in cestodes where the surface plasma membrane may be associated with different anionic groups over the shaft and tip of the microtriches (Lumsden, 1972) and carrier molecules and enzymes probably have a highly patterned arrangement in the surface membrane. This is not a particularly profound observation because biological membranes tend to carry highly organized functional units, e.g. the electron transport units in mitochondria1 membranes. It has been suggested that the incorporation of Golgi membrane into the outer plasma membrane during secretion of surface coat material provides a mechanism for specifying precisely differentiated membrane areas on the surface of the epidermis (Oaks and Lumsden, 1971). The advantages of having a syncytial layer rather than a cellular epidermis in the ectocommensal Temnocephalu (see p. 198) and in the parasitic platyhelminths are not obvious. It has been suggested that this peculiarity may be the result of the way the epidermis arises in embryology, a primary, cellular layer

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being shed and replaced from cells differentiating in the parenchyma which do not complete an outward migration because of the presence of a basement lamina but send out cytoplasmic processes which fuse into a surface syncytium (Lyons, 1970b, 1972a). As Hockley (1972) has pointed out, cell fusion is a somewhat unusual process if one bears in mind the antigenically incompatible surfaces of most differentiated cells. It occurs normally, however, in the phagocytic cells of vertebrate inflammatory lesions (see Hockley, 1972). Further work needs to be done on the temnocephalans to investigate the extent to which the epidermis is truly syncytial and the way in which, if syncytial, this condition arises during development and is maintained by regeneration after damage. If the outer nucleated layer of Temnocephala and Gyrodactylus is syncytial it would seem that this condition has been produced by breakdown of “lateral” cell boundaries, similar to that recorded for ageing female Kronborgia amphipodicola (Neorhabdocoela) by Bresciani and K ~ i e (1970) and in the formation of the embryonic envelopes of cestodes (Rybicka, 1966). Syncytia may therefore originate in more than one manner in the Platyhelminthes, i.e. by fusion of cells in a superficial layer or by fusion of the outer regions of parenchymal “cell” bodies only. The formation of epithelia into syncytia is not unique to the parasitic and commensal platyhelminths; parts of the hypodermis of nematodes, e.g. Nippostrongylus brasiliensis, are multinucleate (Bonner and Weinstein, 1972) and the body wall of adult acanthocephalans is syncytial, as indeed is the cortex of the early acanthella (Butterworth, 1969). In vertebrates the trophoblast of embryos is syncytial and it would be interesting to learn in what ways this condition is related to its mode of formation, its function as a covering and absorptive surface and its ability to penetrate and maintain contact with tissues of a different immunological character.

5 . General evolutionary considerations Superficially it might seem that the structural organization of the acoele epidermis was closest to that of the helminths, but the acoeles, although primitive in some ways (e.g. sperm ultrastructure), are also highly specialized and Ax (1963) has suggested that loss of the basement lamina and development of parenchymally situated nucleated epidermal lobes are probably secondarily acquired features. This writer suggests that a cellular epidermis with superficial nuclei overlying a basement lamina is the primitive condition in the platyhelminths. It is to the “rhabdocoeles” that we should look for relations of the digenean, monogenean and cestodan progenitors and all too few rhabdocoeles have been investigated using the EM. There is a particular need to investigate the embryology and regeneration of the “rhabdocoele” epidermis to see whether the relationship between the covering layer and the parenchyma is as intimate as in the triclads. The observations of Bresciani and Karie (1970) and Clark (1968) on the syncytial nature of the epidermis of the neorhabdocoels Kronborgia (adult females only: an observation that needs checking?) and Temnocephala respectively suggest that the propensity for developing syncytial coverings may be well marked in this group. It might be expected that the cellular ciliated epidermis of “rhabdocoels” is, if anything,

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equivalent to the ciliated coat of larval parasitic platyhelminths but the phenomenon of embryonic epidermal replacement from the parenchyma that seems to occur during the development of the one larval monogenean studied (see p. 202) may complicate homologizing of this kind. The cytomorphology of the epidermis in several of the major groups dealt with here seems to be remarkably constant throughout each group and suggests that this could be a useful feature in taxonomy. The arrangement of the main and lateral ciliary rootlets and the type of vesicular inclusions encountered in the epidermis of Acoela are very similar throughout the group. The peculiar basal attachments of the epidermal cells of planarians may also prove to be characteristic. In the Aspidogastrea a morphologically similar kind of disc-shaped epidermal inclusion occurs in members of three genera so far investigated and the terminal web and microtubercles are a constant feature. The epidermis of those adult monogeneans so far investigated (except for Rajonchocotyle emarginata and Polystoma integerrimum) is basically covered with short scattered microvilli, but these may be either longer and denser (dorsal surface of Entobdella and Acanthocotyle) or absent (ventral surfaces of Entobdella and Acanthocotyle). The epidermis of digeneans is in general smoothly avillous with channel-like infoldings (see Lee, 1966, 1972) although microvilli occur on some larval stages and, in adults, on the epidermis of specialized adhesive regions. Cestodes have quite characteristic microtriches which are regularly arranged, have a thickened internal dense cylinder and are capped with a dense terminal spine. A similar kind of microvillus, but lacking the thickened tip, occurs in cestodarians of G-vrocotyle spp. (Lyons, I969a; Laurie and Lyons, unpublished observations). The epidermis of adult monogeneans, digeneans and cestodes-cestodarians have very different and particular kinds of surface developments, but only when these are considered generally. In view of this difference between the epidermis of monogeneans and digeneans, it is tempting to suppose that the microthrixcovered epidermis of cestodes is more likely to have arisen from the microvillous epidermis of a monogenean-like ancestor than from an (adult) digenean-like ancestor, and this would follow the view substantiated by Llewellyn (1965) that the cestodes are in fact more closely related to monogeneans than is either group to the digeneans (see Lyons, 1971). The microtubercles of aspidogastreans are microvillous-like, yet fall into a class of their own not easily related to the surface structures of the other groups. The finding that the small microvilli covering the cellular epidermis of the “rhabdocoele” Syndesmis echinorum contain an internal thickening similar to the quite characteristic thickened internal cylinder of cestode microtriches is interesting and deserves further attention.

v. SENSE ORGANS OF MONOGENEA The kinds of presumed sense organs so far found in Monogenea have been reviewed (Lyons, 1972b) but an account of the haptorial papillae of E. soleae has been produced recently (Lyons, 1973b3.

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EYES (INCLUDING A POSSIBLE CILIARY EYE)

Most Monogenea have pigmented eyes, which tend to be most conspicuous in the free-living larval stage and are often reduced or absent in adults. The larvae of Monopisthocotylea have four eyespots each equipped with a “crystalline” lens, the anterior pair being the smaller. This is probably the primitive condition in Monogenea. Most larvae of Polyopisthocotylea have only a single pair of pigmented eyes (reviewed in Lyons, 1972b). The eyes of Monogenea consist of a pigment cup which encloses a sensory cell or cells and may or may not be associated with a lens. Kearn (unpublished results) and Lyons (unpublished results) have found that the pigmented eyes of E. soleae are rhabdomeric, consisting of retinular cells ending in microvillar stacks and are therefore similar to those of free living platyhelminthes and larval Digenea. The small anterior eyespots of Enrobdella have only a single retinular cell, whilst the posterior eyes have two receptor cells (Kearn, unpublished results). The possession of two pairs of eyespots with an anterior postero-laterally orientated pair and a posterior antero-laterally oriented pair (possibly the primitive condition in Monogenea) increases the acceptance angle for light on each side of the body and may enhance directional sensitivity to light (Lyons, 1972b). Little is known about the behaviour of monogeneans in response to light, but light activates the larva of Diplozoon paradoxum at hatching (Bovet, 1967) and the larvae of some Monogenea show an initial positive phototaxis, which may be a dispersive phase, before becoming negatively phototactic (see Lyons, 1972b). In addition to the four pigmented eyespots in larval Entobdella soleae, a new kind of sense organ, which may be a non-pigmented ciliary eye has been found on each side of the “head” of this larva (Lyons, 1972b). These “ciliary” eyes” arc rounded or ovoid structures measuring about 6 pm x 4 pm and lie about 7 pm anterior and slightly ventral to the pigmented eyes. They are intracellular structures which do not appear to open to the outside but contain a central cavity lined by a thin rim of nerve cytoplasm (Figs 12 and 13) containing the basal bodies of aberrant 9 + 2 cilia. These project for a short distance into the central lumen and their ends appear to be modified into lamellar bodies composed of spiralling intracellular layers bound by unit membranes which are extensions of the outer ciliary membranes. At the anterior and posterior poles of this organ the cytoplasmic wall is considerably thickened and contains microtubules (or neurotubules) and many closely packed mitochondria (Figs 12 and 13). The anterior pole connects with a nucleated region but the “innervation” of this organ has not been traced further, despite staining of larvae using the indoxyl acetate method for nonspecific esterases. Paired intracellular structures containing 9+0 cilia have been described in the miracidium of Fmciola hepatica by Wilson (1970) and structures also similar to the ciliary lamellate head organs of Entobdella have been found in the miracidium of Schistosoma mansoni by Brooker (1972). The reason for attributing a photoreceptive function to these organs is that they resemble proven ciliary photoreceptors of other invertebrates (e.g. the ciliary eyes of Pecten irradians and Cardium edule) and the pineal eyes of

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vertebrates (see Brooker, 1972; Lyons, 1972b; Oksche et al., 1972). The ciliary eyes of Pecten and Cardium produce “off”responses to light and mediate a protective shadow response on the part of the animal (Land, 1968; Barber and Wright, 1969); it is therefore possible that the ciliary eyes of Entobdella may also, in contrast to the pigmented rhabdomeric eyes, signal the presence of shadows. Alternatively they might act in a manner more similar to pineal eyes, perhaps as a long-term light receptor setting some diurnal or nocturnal activity phase. Kearn ( I 973) has demonstrated a diurnal hatching rhythm for the larva of E. soleae which is set by previous exposure to alternating periods of darkness and light and does not occur if embryos are incubated in continuous light or continuous darkness. This rhythm synchronizes a dawn larval hatch with the return of nocturnally feeding soles to the sea bed and is presumed to facilitate infection of the host (Kearn, 1973). The ciliary organs of E. soleae larva might be involved in setting the time of hatching and this is perhaps a more likely hypothesis than the shadow receptor hypothesis, because strong shadows are unlikely to be cast in deep water and actual infection of the sole appears to be governed by chemotaxis rather than phototaxis; “accurate” experimental infections have been achieved in total darkness (Kearn, 1967a, 1971). B.

SENSE ORGANS OTHER THAN EYES

1. Ending in cilia The Monogenea are fairly richly endowed with ciliated presumed sensilla. At the EM level these are of three main types: (a) single receptors, consisting of a nerve bulb and terminal, non-motile 9+2 cilium with no rootlet (Halton and Morris, 1969; Lyons, 1969b, 1972b; Bresciani, 1972); (b) compoud (uniciliate) receptors consisting of a number of associated nerve terminals each of which bears only a single terminal cilium (Lyons, 1969b, 1969c, 1972b); (c) compound (multiciliate) receptors made up of one or a few nerve endings only, each of which bears many modified cilia (Lyons, 1969c, 1972b). (a) Single receptors. These are the most frequently observed sensilla in Monogenea. They consist of a nerve bulb lined by a thickened collar region and sealed into the epidermis by septate desmosomes, and have been observed to connect to bipolar neurones. They end in a 9+2 cilium 9-1 I pm long which lacks a basal body (Fig. 14B) (Lyons, 1969b). This arises as a free structure from the nerve bulb in Entobdella soleae adult and larva, Gyrodactylus sp., the juvenile of Amphibdella jlavolineata, Leptocotyle minor and Diclidophora merlangi (reviewed in Lyons, 1972b). In Acanthocotyle lobianchi the terminal

FIG.12. Electron micrograph of a section through a “ciliary photoreceptor” of the larva of Enfobdellu soleae; it is about 7 pm long and is lined with a thin rind of cytoplasm in which are embedded the basal regions of 9 1-2 cilia. The ends of the cilia are developed into rnembraneous whorls. b.b., basal bodies of cilia; c., cilia; lam., lamellate whorl; m., mitochondria. (From Lyons, (1972h). I.Linn.Soc.(Zool.)51.)

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cilium of certain “peg organs” is surrounded by a cone of epidermis which projects 7-10 pm beyond the general surface of the body and the enclosed cilium projects for a further 2-5 pm beyond this (Fig. 14C) (Lyons, 1972b). These “peg organs” are extremely numerous and occur over the whole body of both dorsal and ventral surfaces, but are concentrated on the head and in

FIG.13. Reconstruction of the supposed ciliary eye of the larva of Enrobdella soleae. b.b., basal bodies of cilia; c., cilia; lam., lamellate whorls; m., mitochondria; n.p., nerve process; nrt., neurotubules; nu., nucleus. (From Lyons (1972b). J.Linn.Soc.(Zool.) 51.)

FIG.14. Single sensilla and compound uniciliate receptors of Monogenea. A and B. Single sensillum of monogeneans consisting of a nerve bulb lined with a strengthened collar and ending in a 9 +2 cilium. C. Peg-like sense organ of Acanthocotyle (diagrammatic) showing how the epidermis is formed into a projection around a nerve process terminating in a superficial nerve bulb. This kind of receptor connects to a bipolar neurone in the parenchyma. D. Compound uniciliate receptors from the “head” of adult Entobdelh soleae. The cilia have a 9+2 structure and rootlets seem to be absent. E. Compound uniciliate receptors from the “head” of larval Entobdellu soleae. The cilia contain supernumerary microtubules; only a small rootlet is present. F.Spike sensilla from the head of Gyroducrylussp. The edges of the nerve bulb are thrown into a deep collar region around the modified cilia. Long striped rootlets are present and dense (neurosecretory?) granules in the innervation processes. b.b., basal body; bi., bipolar; c., cilium; ep., epidermis; e.m.v., epidermal microvilli; g., (neurosecretory?) granules; m.c., modified cilia; n.b., nerve bulb; n.c., nerve collar; n.p., nerve process; n.t., neurotubule; s.d., septate desmosome; s.r., striped rootlet; t.f., transitional fibres; v., vesicles. (From Lyons (1972). J.Linn.Soc.(Zml.)51.)

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a region just anterior to the pseudohaptor. Single sensilla may be mechanoreceptors and could be tangoreceptors or rheoreceptors. This supposition is based on morphological similarity of these organs to proven mechanoreceptors in other invertebrates (Lyons, 1972b). The thickened nerve collar region lining the nerve bulb is highly specialized and might, if undeformable, play a part in constraining cytoplasm affected by the shear force produced in the base of the cilium during, for instance, mechanoreception. Interestingly the dense collar of single sensilla in the two Polyopisthocotylea studied is very similar and, in both Diclidophora merlangi (see Halton and Morris, 1969) and Polystomu integerrimum (see Bresciani, 1972), consists of a number of ring-like or helical thickenings. These differ from the more continuous thickening found in the nerve bulbs of the skin parasites studied so far, although the functional reasons for this difference are not clear. (b) Compound uniciliate receptors. These consist of a number of associated nerve endings each sealed separately into the epidermis and each terminating in a single cilium which may have a 9+2 tubule arrangement or be modified and contain supernumerary tubules (Fig. 14D,E, F). The precise innervation of these endings is not known and it seems possible that their processes may unite on to a single nerve cell under the epidermis. Compound (uniciliate) receptors have been found only on the “heads” of Monogenea and three different types have been described. These are the head-organs of the adult Entobdellu soleue, grouped receptors on the “head” of larval E. soleue (=cone sensilla) and the spike sensilla of Gyrodactylus sp. (see Lyons, 1969c, 1972b). Because such compound receptors are often surrounded by single sensilla which are thought to be tangoreceptors, it has been suggested that they may be chemoreceptors (Lyons, 1969~). ( c ) Compound mirlticiliufe receptors. This kind of receptor is made up of 1-5 associated nerve endings each of which is equipped with many highly modified cilia (Fig. 15) The edges of each multiciliate terminal are formed into a nerve collar; there is no nerve collar around the individual cilia in these receptors (Lyons, 1969c, 197213). The ciliated pits on the “head” of larval E. soleae and the hillock sensilla on the “head” and general body of Acanthocotyle elegans and A . lobianchi are of this type (Lyons, 1972b). There are paired groups of three ciliated pits on each side of the “head” of larval E. soleue. Each set of three pits contains one large (2 pm across) and two smaller pits (1 pm across) and the larger pits contain 15-18 highly modified cilia. The hillock sensilla of Acanthocoryle were originally described from around the feeding organ of A. elegans but in fact have a much wider distribution over the body (Lyons, 1969c, 1972b). At the light microscope level these structures correspond with bump-like structures 5 pm broad and 3 pm tall bearing 3-5 bunches of immobile cilia 5-7 pm long, which are highly modified and contain supernumerary microtubules inside and outside the triplet ring. A long striated rootlet is present. The lack of the 9+2 arrangement and bunching of the cilia in these receptors suggests they would not be very sensitive to directional mechanical stimulation; they may therefore be chemoreceptors, but without electrophysiological recording apparatus this would be difficult to establish.

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FIG. 15. Diagram showing compound (multiciliate) receptors from two monogeneans. A. ciliated pit on the “head” of larval Enrobdeila. B. compound multiciliate receptors on the body of Acanthocotyle lohianchi. Annotations as in Fig. 14. (From Lyons (1972). J . Linn.Soc.(Zool.) 51.)

2. Lacking cilia or ciliary structures The ventral surface of the haptor of Entobdella soleae has been shown, using the stereoscan EM, to be covered with more than 800 papillae ranging in size from 2.5 to 19 pm dia. and about 6-8 pm high (Fig. 16). They are packed with one or more fine nerve endings which are doubled over and piled stack-like one on top of another (Fig. 17). The nerve stacks are separated by thin layers of mainly amorphous connective tissue. No basal body or ciliary structure is associated with them and the papillae do not open to the outside. A capsule of connective tissue invests the papilla under the folded epidermis and the proximal wall of connective tissue is very thick except where it is penetrated by a connection from the haptoral nerves (Lyons, 1973b). The most likely function for the haptoral papillae would seem to be that they may be mechanoreceptors, either contact receptors signalling direct contact with the host or strain receptors signalling tensions developed in the sucker wall during adhesion. Information about the pattern of contact points during attachment or detachment of the haptor could form part of a proprioceptive system affecting the co-ordination of hook muscles or general musculature (Lyons, 1973b). It is remarkable that so many of these papillae should be present (800 on a

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FIG.16. Scanning electron micrograph of the haptor of Enrabdellu soleae showing the papillae which are thought to be sensory. The large sheathed hooks in the centre of the haptor are the accessory sclerites and long hamuli open at the posterior edge of the sucker.

surface measuring 1 m m x 1 mm). This could indicate that each papilla or each papillar array feeds information directly to a particular localized motor system (Lyons, 1973b). The papillae may be peculiar to the genus Entobdellu where they occur on most species. They are absent from the larvae of E. soleae.

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FIG.17. Transverse section through a haptor papilla showing that this contains a nerve process which is much folded over itself. The epidermis covering the papilla is smooth and is folded into crypt-like channels. bl. basement lamina (thick); gr, dense granules in nerves; np, nerve process penetrating papilla.

V1. CONCLUSION After more than a decade of work on the nature of the external surface of parasitic platyhelminthes we are entering a new phase of investigation. Whereas in the past work has concentrated largely on a straightforward ultrastructural approach linked with histochemistry at the light microscope level, recent developments in cytochemical and macromolecular labelling techniques in general promise a more detailed probing of tegument functions. Much remains to be elucidated about the precise nature of the glycocalyx and mucus coat of monogeneans and other platyhelminthes; the chemical nature of the secretions produced by the epidermal “cell” bodies and the relation of these secretions to the surface coat; on the nature and function of the terminal web of polyopisthocotyleans; on the permeability of the tegument to ions and possibly nutrients (not only in monogeneans but also in turbellarians for

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comparison) and 011 the functions of the infolded epidermal basal plasma membrane processes. In addition there is great scope for work on regeneration, in particular of the epidermis, in these ectoparasites and for additional studies on the embryology of this layer perhaps in a monogenean such as Acanthocotyle, which has a non-ciliated larva; also for more work on various kinds of hatched larva. An investigation into the ways by which separated larval ciliated plates or tufts are controlled and co-ordinated and on the mechanism of ciliated cell shedding would also be of great interest. At a general level more ultrastructural work needs to be done on the epidermis of dalyellioid Rhabdocoela and the Temnocephala, and within the Monogenea, on adult representatives of, in particular, the hexabothriid, diclybothriid, chimaericolid and polystomatid lines. Investigation of the fused body regions of Diplozoon paradoxum, and on epidermal permeability of Polystoma to gonadotrophin-like hormones could produce interesting information. Regarding sense organs and nervous systems : the ranges of neurotransmitter types and neurosecretory material need more detailed characterization and the function of neurosecretory products linked with the biology of these worms. More accurate methods for mapping sense organs on marine adult and larval worms, perhaps utilizing specific staining of nerve endings and stereoscan investigations involving critical point drying to preserve ciliary endings, are called for. The electrophysiological response of accessible sense organs (e.g. the haptor papillae of entobdellids) also needs to be studied. In all cases these investigations should be closely related to the overall behaviour and biology of the stage under consideration. REFERENCES

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Dorey, A. E. (1965). The organization and replacement of the epidermis in acoelous turbellarians. Q. J I microsc. Sci. 106, 147-172. Gilula, N. B. and Satir, P. (1971). Septate and gap junctions in molluscan gill epithelium. J. Cell Biol. 51, 869-872. Halton, D. W. and Arme, C. (1971). I n vitro technique for detecting tegument damage in Diclidophora merlangi: possible screening method for selection of undamaged tissues or organisms prior to physiological investigation. Exptl. Parasit. 30, 54-57. Halton, D. W. and Lyness, R. A. W. (1971). Ultrastructure of the tegument and associated structures of Aspidoguster conchicola (Trematoda: Aspidogastrea). J . Parasit. 57, 1198-1210. Halton, D. W. and Morris, G. P. (1969). Occurrence of cholinesterase. and ciliated sensory structures in a fish gill-fluke, Diclidophora rnerfungi (Trematoda: Monogenea). Z . Parasitenkde 33, 21-30. Halton, D. W.,Dermott, E. and Morris, G. P. (1968). Electron microscope studies of Diclidophora merlungi (Monogenea: Polyopisthocotylea). I. Ultrastructure of the cecal epithelium. J . Parasit. 54, 909-916. Hockley, D. J. (1970). Ultrastructure of the outer membrane of Schistosomu mansoni, In Proc. 2nd Int. Cong. Parasit. J. Parasit. 56 (2), 151. Hockley, D. J. (1 972). Schistosoma mansoni: the development of the cercarial tegument. Purusitology 64, 245-252. Ishikawa, H., Bischoff, R. and Holtzer, H. (1969). Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. J. Cell Biol. 43, 312328.

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Ultrastructure of the Tegument of Schistosorna D. J. HOCKLEY

National Institute for Medical Research, Mill Hill, London, EngIand I. Introduction ..........................................................

........................................................ The Development of the Tegument ................................ The Cercarial Surface Coat ........................................

11. The Cercaria

111.

IV.

V. VI.

A. B. C. The Tegument and A.ssociated Structures of Fully Developed Cercariae. . The Schistosomulum .................................................. A. The Tegument of Schistosomula 30 min-3 h after Penetration .......... B. The Tegument of Schistosomula 24 h-2 weeks after Penetration ........ The Adult Worm .................................................... A. The Tegument and Associated Structures of Normal Worms ............ B. Comparison of the Tegument and the Caecum.. ...................... C. Destruction of the Tegument ...................................... The Miracidium and Sporocyst ........................................ A. The Epithelium and Associated Structures of the Miracidium. ........... B. The Tegument of the Daughter Sporocyst ............................ Conclusion .......................................................... References ..........................................................

233 234 234 237 243 250 250 254 257 25 7

280 282 289 290 293 296 297

I. INTRODUCTION Schistosonia niansoni was the first digenetic trematode to be examined with the electron microscope (EM) (Gannert., 1955a; Senft, 1959) and the ultrastructure of 5’.munsoni has since been examined more than that of any other digenean. This continued interest reflects not only the availability of S. mansotii in the laboratory but also the medical importance of the worm, and the continuing advances in electron microscopical techniques. It was with Fusciola hepatica, however, that Threadgold (1963) first found that the trematode tegument was a living, anucleate, cytoplasmic structure continuous with nucleated cell bodies located beneath the superficial muscle fibres. To emphasize that the tegument was not an inert structure secreted by the cell bodies, as it was formerly thought to be, he proposed that cuticle was an inappropriate name and that only the term tegument should be used. This proposal has become generally accepted even though cuticle is derived from cuticula (Latin), the diminutive of cutis, meaning skin, whereas tegumentum (Latin) simply means, a cover or covering. Several other names have been suggested for both the tegument and the cell bodies. Integument is commonly used in the U.S.A. (Burton, 1964) with internuncial processes connecting the 233

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integumental epithelium to integumental cytons (Smith et al., 1969). The suggestion by Lee (1966) to use the simpler name epidermis has not been generally adopted, possibly because it does not indicate the unusual structure of the tegument. The syncytial tegument connected to the subtegumental cells, has been found in all adult Digenea that have been examined and it was first reported to occur in S. mansoni by Lee (1966). Not only does the tegument have this unusual gross cytological structure but it also has unusual cytoplasmic inclusions. These unique features of the trematode tegument are presumably related to its role as part of the host-parasite interface and ultrastructural studies of the tegument should, therefore, increase our understanding of the host-parasite relationship. At the present time it is mostly the normal ultrastructure of the tegument that has been studied, this being a necessary preliminary to experimental studies. This review summarizes the observations on the normal tegument and therefore, hopefully, provides a basis for future experimental work. Comparisons are made, where possible, with the teguments of other Digenea in order to show which features are unique to the tegument of Schistosoma and which features are shared with other trematodes. 11. THECERCARIA A.

THE DEVELOPMENT OF THE TEGUMENT

Schistosome cercariae penetrate the vertebrate host directly and the cercarial tegument, therefore, eventually becomes the tegument of the adult worm without being involved in the formation of a metacercarial cyst. This makes it possible to study the origin of the adult tegument in developing cercariae and knowledge of the development of the tegument must help in the understanding of its unusual ultrastructure. The resolution of the light microscope (LM), however, has severely limited this type of study and surprisingly few EM observations have been made on developing cercariae. The two published ultrastructural studies of developing S. mansoni cercariae are brief and conflicting (Rifkin, 1970; Hockley, 1972). 1. The primitive epithelium The observations of Hockley (1972) show that from an early stage in development the cercarial germ ball is surrounded by a thin, nucleated cytoplasmic layer, which confirms the observations of Gordon et al. (1934) and Dusanic (1959). This type of temporary, larval tegument has been seen on several species of developing cercariae and has been called a primitive epithelium by Dubois (1929). The origins of the primitive epithelia of Acanthoparyphium spinulosum, Cercaria dichotoma and Cercaria bucephalopsis haimeana have been studied by Bils and Martin (1966) and James and Bowers (1 967). By comparison of his observations with these studies Hockley (1972) concluded that the primitive epithelium of S. mansoni is derived from the embryo rather than from the sporocyst. This conclusion is supported by the

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results of a recent LM study of developing S. munsoni cercariae (Cheng and Bier, 1972). The function of the schistosome primitive epithelium is unknown. In Fasciolu hepatica the primitive epithelium has an important function in cyst formation and it is also thought to function in the absorption of food and in the attachment of the embryo to the redia (Dixon and Mercer, 1967). In S. munsoni, however, there is no morphological evidence that the epithelium has an absorptive or attachment function and it presumably acts simply as a protective tegument until the true tegument is formed. Possibly a temporary tegument is necessary to allow for growth of the embryo. 2. The tegument Figure 1 summarizes diagrammatically the development of the tegument of S. rnunsoni as described by Hockley (1972). The true tegument appears beneath the primitive epithelium which then degenerates and is lost. The tegument is presumably formed from cells of the germ ball. At first, it is similar to the primitive epithelium in that it is a thin, nucleated syncytial layer containing a few ribosomes and mitochondria. The tegument can always be distinguished from the primitive epithelium by its thickened outer membrane and by the basal lamina beneath its basal membrane. Early in the development of the cercaria the tegumental nuclei become pycnotic and are lost from the tegument. At about the same time, it appears that nucleated subtegumental cells become continuous with the tegument. Thus the typical trematode tegument of an outer, anucleate, syncytial part connected to nucleated subtegumental cells is formed. As development proceeds, the tegument becomes filled with electron-dense, granular material and crystalline spines appear. The mechanism of spine formation remains unknown but, as in other species of cercariae, the spines are associated with a thickened part of the basal membrane of the tegument (Bils and Martin, 1966; Belton and Belton, 1971). Dense, spherical bodies also appear in the tegument; they are formed from Golgi complexes in the subtegumental cells and passed into the tegument. The production of vesicles in subtegumental cells and their passage into the tegument through microtubular-lined connections is reminscent of the process of encystment in some cyst-forming cercariae (Bils and Martin, 1966; Southgate, 1971); after the cystogenous material has passed from the cells into the tegument, the tegumental connection usually degenerates. Hockley (1 972) suggested that the subtegumental cells of S. mansoni cercariae may also be only temporarily connected to the tegument for the passage of the bodies from cell to tegument. The development of the tegument of S. munsoni cercariae as described in a brief abstract by Rifkin (1970) is very different. He reported that, after the cercarial germ ball has begun to differentiate, it is surrounded by a number of elongate sporocyst cells. These cells form a syncytium and become the tegument of the cercaria. Ultimately the syncytium becomes connected to nucleated cell bodies located beneath the cercarial musculature. Rifkin apparently did not observe a primitive epithelium. Clearly, further studies are required to confirm the presence of the primitive epithelium and to determine whether

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FIG.1 . Diagram summarizing three stages in the formation of the tegument during cercarial development (based on Hockley, 1972). A, germ ball covered with a primitive epithelium (pe) and the tegument (t) which has a thickened outer membrane (tm) and a n underlying basal lamina (bl). B, young cercaria with a degenerating primitive epithelium (dpe) and a pycnotic tegumental nucleus (n). C, cercaria nearly ready to emerge from the sporocyst; the primitive epithelium has been lost and the tegument (t) is connected to nucleated subtegumental cells (stc).

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the true tegument arises from the embryo or the sporocyst. Although types of primitive epithelia derived from sporocyts have been described (James and Bowers, 1967; Belton and Belton, 1971) it does seem unlikely that the final tegument would be formed in this way. Rifkin (1970) and Hockley (1972) do agree, however, that the subtegumental cells originate separately from the tegument. The manner in which the subtegumental cells originate in S. munsoni is different from the methods of tegument and subtegumental cell formation described for A. spinulosum (Bils and Martin, 1966), F. hepatica (Dixon and Mercer, 1967; Dixon, 1968) and Cloacitrema narrabeenensis (Dixon, 1970). In A. spinulosum the nucleated parts of the tegument migrate down into the body of the cercaria. F. hepatica cercariae are covered only by a primitive epithelium which is lost during the process of cyst formation. At the end of encystment, the final tegument is formed from processes of the keratinforming cells which have migrated to the surface while the nucleated parts of the cell remain deep in the body of the cercaria. C. narrabeenensis is intermediate between A. spinulosum and F. hepatica in its methods of tegument formation. A primitive epithelium is present, beneath which the cercarial tegument is formed. The nuclei of the tegument sink into the body but remain connected to the tegument and at about the same time the primitive epithelium is shed. The tegument is lost during encystment and, as in F. hepatica, cytoplasm from some of the cystogenous cells forms the final tegument. Notocotylus atrenuatus is similar to S. mansoni in that subtegumental cystogenous cells connect with the tegument (Southgate, 1971). The different methods of formation of the trematode tegument all result in the typical anucleate, syncytial tegument which is connected to nucleated subtegumental cells. In S . munsoni the tegument and the cells arise separately and it is possible that the cells are only temporarily connected to the tegument. B.

THE CERCARIAL SURFACE COAT

1. The origin of the surface coat The fully developed tegument of the schistosome cercaria is covered by a surface coat which consists of a layer of diffuse granular and fibrous material about 1 pm in thickness (Fig. 2) (Kruidenier and Stirewalt, 1955; Lumsden and Foor, 1968; Inatomi et ul., 1970). Surface coats have been identified by LM cytochemistry on a variety of species of cercariae (Kruidenier, 1951, 1953a, b, c; Ito and Watanabe, 1959); they are thought to be formed by secretions from unicellular glands in the cercariae and possibly adhere to the tegument because of a change in viscosity after secretion (Kruidenier, 1953a). Kruidenier and Stirewalt (1955) suggested that the surface coat of S. munsoni cercariae is secreted by the post-acetabular glands and Dixon and Mercer (1967) suggested that the coat might be secreted into the primitive epithelium and thus be retained on the surface. Hockley (1972), however, found that the primitive epithelium of S. munsoni is lost before the coat is formed. Rifkin (1970) and Hockley (1972) both made observations on the origin of the cercarial surface coat in their ultrastructural studies of developing cercariae.

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Rifkin (1970) used cytochemical methods which indicated that the coat is synthesized within sporocyst cells, but Hockley (1972) observed that the coat is specifically attached to the cercarial surface and that it is different from the material in the lumen of the sporocyst. Hockley (1972) suggested that the surface coat is an integral part of the outer membrane and that it is produced by the tegument itself, although he also observed that the contents of the penetration gland ducts are continuous with the surface coat. Surface coats are found on a wide variety of animal cells and are generally thought to be produced by the cells themselves rather than having an extraneous origin from separate gland cells (Bennett, 1963; Ito, 1965, 1969). Thus it seems most probable that the schistosome cercarial surface coat is produced by the tegument but the exact mechanism of formation remains to be determined. Possibly it is formed from some of the dense bodies that originate in the subtegumental cells. Kruidenier (1953a) found that surface coats are particularly prominent on cercariae that are in the snail host and suggested several possible functions for the coat in this environment. The coat might act as a lubricant to assist the escape of the cercaria from the snail host and it might also protect the cercaria from enzymes of the snail digestive gland and the enzymatic secretions of the cercarial glands. He also suggested that the development of the coat might prevent further absorption of nutrients by the cercaria and it might therefore act as a stimulus for emergence from the snail host. Possibly the formation of the schistosome surface coat can be correlated with the cercarial tegument becoming dense and granular and apparently inactive. 2. The fully developed surface coat of free-swimming cercariae The surface coat is prominent on free-swimming schistosome cercariae which have escaped from the snail host (Figs 2 and 3). Kruidenier and Stirewalt (1955) made the first EM observations on the surface coat of free-swimming S. mansoni cercariae; this work was reported only in abstract and without micrographs. The surface coat of fully developed S. mansoni cercariae consists of short, branched and interconnected filaments and dense granules, both approximately 1-5 nm dia (Fig. 3) (Hockley, 1970; Morris, 1971). The filaments form a diffuse, matted layer over the tegument, filling all the irregularities of the surface and some of the filaments appear to be continuous with the outer membrane of the tegument (Morris, 1971). Smith et al. (1969) and Morris (1971) described the coat as being 02-0.5 pm in thickness but Hockley (1970) described it as being up to 1 pm in thickness and Kemp (1970) also figures a 1 pm thick coat. There is probably considerable variation in thickness of the coats of different individual cercariae which may be related to the age or condition of the cercariae. The coat is always thinner on the tail than on the body (Hockley, 1970; Morris, 1971) and also thinner on the ventral sucker (Hockley, 1970). The coat is absent over the openings of the penetration gland ducts (Robson and Erasmus, 1970) and over the tegumental urinary bladder epithelium (Smith et al., 1969). Morris (1971) states that the coat is absent over the sensory cilia and tegumental spines, but Hockley (1970) found that the coat is present over the spines. Since the coat appears

FIG.2. Low power electron micrograph of cercadal tegument (t) and surface coat (sc). FIG.3. Higher magnification micrograph showing the fibrous and granular nature of the surface coat.

FIG.4. Electron micrograph of the dense, granular cercarienhiillenreaktion (CHR).

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D. J. HOCKLEY

to be intimately associated with the outer membrane it is to be expected that the spines should be covered by the surface coat. Conversely, the duct openings and cilia are not covered by the tegumental outer membrane and therefore should not be covered by the coat. It is possible that the urinary bladder epithelium is not part of the cercarial tegument and thus would not have a surface coat, since Krupa et al. (1969) state that the epithelium of the excretory vesicle of Cryptocotyle lingua is probably not continuous with the tegument. Hockley (1970) observed that secretions from the penetration glands, produced by free-swimmingcercariae, do not appear to mix with the surface coat; this is possibly further evidence that the coat is part of the tegumental outer membrane and does not originate from the glands. Schistosome cercariae are unusual in having a thick surface coat on the free-swimming cercariae which have escaped from the snail host. Kruidenier (1953a) fcund by the LM that surface coats of opisthorcoid cercariae are most obvious while the cercariae are still in the snail. The EM has disclosed a thick surface coat on the free-swimming stage of only one other species of cercaria, Zoogonoides vhiparus (Kerie, 1971a). The coat is very similar to that of the schistosome cercaria; it has a maximum thickness of 2 pm at the anterior end of the cercaria. 3. Cytochemistry of the surface coat The cytochemical staining properties of the surface coat of S. mansoni cercariae are summarized in Table I. Strongly positive staining with the PAS technique has been found consistently, but variable results have been obtained with methods to detect acidic groups. The conclusion generally drawn from cytochemical staining is that the cercarial surface coat is a mucosubstance, possibly a glycoprotein or a neutral or slightly acidic mucosubstance. It is probably best simply to use the name mucosubstance until a biochemical analysis of the surface coat has been performed, since the cytochemical techniques do not permit identification of glycoproteins or mucopolysaccharides according to their biochemical definitions. The schistosome cercarial surface coat appears to be cytochemically different from the surface coats of other species of cercariae. Kruidenier (1953a, b,c) and Ito and Watanabe (1959) identified surface coats on various species of cercariae by their strongly positive toluidene blue and alcian blue staining. Kraie (1971a) found that the surface coat of Zoogonoides vivipurus does not stain with the PAS method but stains vigorously with alcian blue and toluidine blue. Thus acidic groups are more prominent in other species of cercariae. 4. Functions of the surface coat

Kruidenier (1953b) has suggested a number of possible functions for the surface coat of the free-swimming cercaria. It might stick cercariae together and to the host during penetration. This could be advantageous because a number of cercariae might enter the host by the same route thus facilitating their entry (Griffiths, 1953). The coat may also protect the cercaria from its own enzymatic secretions during penetration and act as a lubricant. Stirewalt (1963) has suggested that the surface coat of S.rnansoni cercariae is modified

TABLE I Cyrochemical staining of the surface coat of S . mansoni cercariae Techniques

To demonstrate

--

Results

References

+++ +++

Kruidenier and Stirewalt (1955) Kemp (1970); Hockley (1970) Smith et al. (1969) Kemp (1970) Kemp (1970)

(1) Periodic acid-Schiff (PAS) LM

Carbohydrates

(2) Diastase Digestion + PAS ( 3 ) PA-Silver Methenamine (4) Alcian blue (5) Toluidine blue metachromasia (6) Colloidal iron

LM EM LM

Carbohydrates other than gylcogen Carbohydrates other than gylcogen Acidic groups

LM LM

Acidic groups Acidic groups

(7) Colloidal iron

EM

Acidic groups

(8) Colloidal Thorium

EM

Acidic groups

(9) Ruthenium Red

EM

Acidic groups

(10) Coupled Tetrazonium (1 1) Dihydroxy-dinephthyl

LM

Protein

0 0

disulphide (DDD)

LM

Sulphydryl-disulphide bonds

0

LM =light microscopy; EM =electron microscopy;

+++ 0

0

++ 0 + 0 + 0 +

Kemp (1970) Smith ei al. (1969) Kemp (1970) Hockley (1970) Kemp (1970) Hockley (1 970) Kemp (1970) Hockley (I 970) Kemp (1970) Smith ei ul. (1969) Smith el al. (1969)

-

+ = positive reaction, arbitrarily graded; 0 =negative reaction.

-I

0

-I

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D. J. H O C K L E Y

or lost during penetration and has shown that after penetration the cercariae are unable to survive in water; possibly the coat is essential for the survival of the schistosome cercaria in water. Since the cercarial surface coat is probably analogous to the surface mucosubstance component found in many cell membranes, some of the suggested functions for these surface coats may also apply to cercariae. Bennett (1963) suggested that surface coats might influence the composition of the environment close to the plasma membrane by means of filtration and selective binding of ions and Morris (1971) suggested, therefore, that the coat might be involved in permeability control in the cercaria. Kent (1967) stated, however, that glycoproteins have no ion exchange properties, but he did suggest that surface coats could act as gel filtration devices and thus they may protect cercariae from large particles which might damage the surface. Other suggested functions of cell surface coats, such as the binding of particles prior to phagocytosis (Brandt and Pappas, 1960) or the provision of a large surface for enzyme-substrate interactions (Wright and Lumsden, 1968) probably do not apply to cercariae. 5. The Cercarienhiillenreaktion (CHR) The CHR occurs when cercariae are incubated in serum from an infected animal. The reaction was first described from the LM by Vogel and Minning (1949) as the formation of a membraneous envelope round the cercaria. Several degrees or stages of the CHR have been described ranging from a barely detectable reaction to the formation of a thick sheath, separated from the cercaria and from which the cercaria can emerge. Kemp (1 970) and Hockley (1970) have observed stages in the development of the CHR with the EM. Kemp described an increase in thickness of the surface coat and an accumulation of material exterior to the coat. Cercariae which break free of the CHR only lose the material exterior to the coat. Hockley found that a moderately vigorous reaction appears as an increase in the amount of material in the surface coat so that the coat increases in thickness to about 2 pm and becomes dense and granular (Fig. 4) instead of having the normal fibrillar appearance. In a more vigorous reaction, the enlarged surface coat becomes separated from the cercaria and very little material remains attached to the tegument. The CHR is clearly a reaction between the antiserum and the surface coat; whether the whole or only the outer part of the surface coat is involved probably depends on the conditions of the reaction. Hockley (1970) examined a CHR produced with a ferritin conjugated gamma globulin fraction of an antiserum. The ferritin molecules are located throughout the whole surface coat and the appropriate controls showed that this is an antigen-antibody reaction and not just the accidental trapping of ferritin molecules. The enlargement of the surface coat, however, does not appear and Evans et al. (1955) have shown that both the gamma and alpha fractions of the antiserum are necessary to give the CHR. The mechanism of the swelling of the surface coat has not been investigated. The CHR is very similar to the enlargement (quellung) of the capsules of bacteria in homologous antiserum (Baker and Loosli, 1966) which was explained by Johnson and Dennison (1944) as hydration of the capsularpolysaccharidegel. The “quellung” phenomenondiffers from the CHR

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FIG.5. Diagram summarizing the basic features of the tegument of the free-swimming schistosome cercaria. bm =trilaminate basal membrane. dl = dense layer beneath outer membrane. eb = elongate body. m =mitochondrion. s = spine. sb = spherical body. sc = surface coat. tm = trilaminate outer membrane. ts = trilaminate structure in the surface coat. v = vesicles in tegumental pocket.

in that it is reversible (Nungester and Kempf, 1940) and non-specific swelling can also be produced (Jacox, 1947). C.

THE TEGUMENT AND ASSOCIATED STRUCTURES OF FULLY DEVELOPED CERCARIAE

The most complete ultrastructural study of the tegument and associated structures of the free-swimming cercaria of S. mansoni is that made by Morris (1971) but brief mention of the cercarial tegument is also given by Smith et al. (1969), Hockley (1970) and Hockley and McLaren (1973). Inatomi et al. (1970) have studied the cercaria of S..japonicum. The following account is based on the observations of Morris (1971). 1. The tegumeni Figure 5 summarizes diagrammatically the basic features of the cercarial tegument. The tegument is a single, continuous, cytoplasmic structure over the body and tail of the cercaria. It is approximately 0.5 pm in thickness on the body and approximately 0.2 pm in thickness on the tail. There is considerable variation in the thickness of the tegument since both the outer and inner surfaces are very irregular. The outer membrane is trilaminate and has been reported to measure approximately 7 nm in thickness (Morris, 1971), 8.5 nm (Hockley and McLaren, 1973) and 10 nm (Smith et al., 1969). Variations in measurements of this order can probably be attributed to different fixation and staining procedures and to the use of different microscopes, and membrane can be considered as a typical trilaminate membrane. Measurement of the thickness of the membrane is made more difficult by the

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presence of an electron-dense layer, approximately 15 nm in thickness, immediately beneath the inner leaflet of the outer membrane. This layer merges into the dense, granular cytoplasm of the tegument. The tail tegument appears less electron-dense than the body tegument since the cytoplasm consists of less closely packed granules. According to Morris (1971) the basal membrane of the tegument is 3 4 nm in thickness and rarely appears trilaminate. Smith et ul. (1969) and Hockley and McLaren (1973), however, both described the basal membrane as trilaminate and of similar thickness to the outer membrane. The basal membrane is usually even more folded and invaginated than the outer membrane which makes observation and accurate measurement difficult. The tegument of schistosome cercariae is basically the same as, but thinner and more electron-dense than the teguments of the other species of cercariae that have been examined by the EM. A variety of inclusions have been identified in the tegument of S. mansoni cercariae. The spines are enclosed by the outer and basal membranes and are completely within the tegument. Morris (1971) described the spines as being composed of parallel longitudinal elements 5 nm in diameter and separated by a 5 nm electron-lucent gap. Transverse sections show that each group of four elements form an equilateral parallelogram with internal angles of 60" and 120'. This type of spine structure has been observed in the cercariae and adult worms of several species of trematodes and has been interpreted as a crystalline lattice (Burton, 1964). Morris (1971) observed small dense bodies associated with the spines but otherwise found nothing further to indicate the method of spine formation. Smith et al. (1969) found that the tegument can be stained cytochemically for protein and that the spines are similarly but even more intensely stained. Possibly the spines are a crystalline part of the cytoplasmic ground substance with which they are continuous. Mitochondria are sparsely distributed throughout the tegument of S. munsoni cercariae; they are small and contain only two or three cristae and in these respects they are similar to the tegumental mitochondria of other cercariae (Belton and Harris, 1967; Rees, 1971). Kaie (1971a) found large numbers of mitochondria in the tegument of the cercaria of Zoogonoides viviparus. In contrast to the tegumental mitochondria of schistosome cercariae, the mitochondria of the muscle fibres are large and contain many cristae. The characteristic tegumental inclusion of the fully developed schistosome cercaria is a spherical body approximately 100 nm in diameter; it has a trilaminate limiting membrane and contains a peripheral electron-lucent part and a central dense, granular mass. These inclusions are very numerous in the body tegument but do not occur in the tail tegument. Somewhat similar inclusion bodies have been found, in large numbers, in the tegument of Acanthutrium oregonense cercariae by Belton and Harris ( I 967). The contents of these bodies are more fibrous than those of schistosome cercariae and they probably contain mucosubstances which are released to the exterior (Belton and Belton, 1971). There is no evidence that the contents of the spherical bodies of schistosome cercariae are released to the exterior and it is unlikely that they contribute to the surface coat since the inclusions are only present in the body tegument whereas the coat is present over both body and tail.

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245

A second type of tegumental inclusion body is also found in schistosome cercariae. These bodies are elongate or rod-shaped, approximately 100 nm in length and 10-15 nm in width; they have a trilaminate membrane and dense granular contents. The elongate bodies are present in both the body and tail tegument but are never as numerous as the spherical bodies. Similar, dense elongate bodies have been seen in several other species of adult trematodes and cercariae and it has usually been suggested that they contribute to the dense ground substance of the tegumental cytoplasm (Burton, 1966). Kerie (1971 ) has observed, however, that elongate bodies are extruded from the tegument of young cercariae of Zoogonoides viviparus when the cercariae are in an abnormal situation. No evidence of extrusion has been seen in schistosome cercariae. Two other minor constituents of the cercarial tegument were found by Morris (1971). They are lipid-like vacuoles and pockets containing small, trilaminate membrane-bounded vesicles. The pockets open to the exterior and the small vesicles could possibly give rise to the trilaminate structures observed in the surface coat by Hockley and McLaren (1973). Lipid-like inclusions have been seen in the tegument of moribund cercariae by Hockley (1970). The functions of the tegumental inclusions bodies of schistosome cercariae are unknown, Cytochemical staining methods have failed to localize any substances within the inclusions, Sodeman et al. (1968) examined the distribution of acid and alkaline phosphatases in schistosome cercariae with the LM; they could not find any staining in the tegument but they did find a continuous band of subtegumental staining for acid phosphatases. Ebrahimzadeh ( I 970), however, detected alkaline phosphatase activity in the tegument of S. munsoni cercariae and Hockley (1970) obtained the same result at the ultrastructural level. The precipitate of lead phosphate is specifically localized in the tegument but is not associated with any of the inclusions or tegumental membranes. The two major types of inclusion bodies almost certainly arise from subtegumental cells during cercarial development, but Morris (1971) and Hockley and McLaren (1973) were unable to find any subtegumental cells connected to the tegument in fully developed cercariae. Morris (1971) did find processes of the tegument which extend into the underlying muscle fibres and he also identified cells in the cercariae containing large numbers of dense bodies. Possibly this is further evidence that the subtegumental cells are only temporarily connected to the tegument. Schistosome cercariae have relatively few tegumental inclusions, both in type and number, compared with species of cercariae that produce a metacercarial cyst. In some of these species the tegument of the free-swimming cercaria becomes filled with bodies of cyst-forming material (Bils and Martin, 1966; Southgate, 1971). The schistosome cercarial tegument is, presumably, mainly a protective layer. The cercaria is a short-lived, larval stage, probably non-feeding, and there is no morphological evidence that the tegument is an absorptive structure. Microvilli have been identified on the cercariae of Parorchis acanthus and Z. viviparus (Rees,1971; Kerie, 1971a) but none has been seen on the surface of S. mansoni cercariae. The dense, granular cyto-

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FIG.6. Diagram showing the relationship between the cercarial tegument and its associated structures. aup = anterior sensory organelle; uniciliated papilla with short, ensheathed cilium. bl = basal lamina. cdb =cell containing dense bodies. cmf =circularly arranged muscle fibres. hd =hemidesrnosome. hgd =head gland duct. im = interstitial material. Imf = longitudinally arranged muscle fibre. mp = multiciliated pit sensory organelle. nmf = nucleus of muscle fibre. pgd = penetration gland duct. sd = spetate desmosorne. t = tegument. tb = tadpole-shaped bodies in tegumentary vacuole adjacent to head gland (Morris, 1971). tf = tegumentary folds. tr = tegurnentary rim round penetration glad aperture. up =uniciliated papilla sensory organelle.

plasm of the tegument, the dense layer beneath the outer membrane, the lack of nuclei and ribosomes and the small mitochondria, all suggest that the tegument of the schistosome cercaria is an inactive and resistant structure. 2. Basal lamina, interstitial material and muscle jibres Beneath the tegument of the fully developed schistosome cercaria there are consecutively a basal lamina, a thick layer of interstitial material, circularly arranged muscle fibres and inner, longitudinal muscle fibres (Fig. 6). The basal lamina appears early in cercarial development and persists in the freeswimming cercaria as a thin layer of 7 nm dia granules immediately beneath the basal membrane of the tegument. The layer is about 20 nm in thickness and closely follows the irregular basal membrane although it does not extend into the narrower invaginations. The interstitial material consists of diffuse, irregularly arranged fibres approximately 6 nm in diameter. The material is present as a thin layer between the muscle fibres and round most of the cells of the cercaria. Beneath the tegument, the interstitial material forms a layer of about the same thickness as the tegument. The basal lamina and the layer of interstitial material together have been referred to as the basal lamina by some authors (Smith et al., 1969; Hockley, 1970) but it is more appropriate to distinguish them as Morris (1971) has done, because in the young cercaria, only the thin, granular basal lamina is present. The two structures probably

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function together, however, as a supporting structure (Threadgold and Gallagher, 1966). Small hemi-desmosomes on the basal membrane of the tegument form points of attachment of the tegument to the basal lamina and interstitial material (Morris, 1971). Hockley (1970) examined dead and dying cercariae and found that the basal lamina and interstitial material persist long after the tegument has been lost which suggests that they may form a tough, skeletal structure. The two layers of muscle fibres are typical of soft-bodied invertebrates. The filaments are irregularly arranged in the body muscle fibres, thus the fibres do not have a striated appearance. The longitudinal tail muscle fibres, however, are striated, and have a more extensive sacroplasmic reticulum. Lumsden and Foor (1 968) have correlated the striated fibres and their large glycogen reserves and numerous mitochondria with the rapid and sustained movements of the tail during swimming. In comparison with the tail, the movements of the body are slower and less frequent. The skeletal function of the interstitial material is again demonstrated by the apparent attachment of muscle fibres to the layer of material beneath the tegument. 3. The suckers The scanning EM has proved to be useful for demonstrating regional variations in the surface appearance of the cercarial tegument (Hockley, 1968; Rees, 1971; Ksie, 1971a). S. rnansoni cercariae have been examined in this manner by Hockley (1968) and Race et ai. (I 971), and have been shown to be evenly covered with spines which point in a posterior direction. The tail has a more irregular surface than the body with fewer spines and between the body and tail there is a smooth, tegumental collar. The ventral sucker is covered with spines which are slightly larger than those on the rest of the body. A detailed study of the oral sucker has been made with the scanning EM by Robson and Erasmus (1970). They found that the oral sucker is represented by the anterior quarter of the body. It is differentiated from the body by a shallow constriction in the surface of the tegument at its posterior margin and by the presence of a greater concentration of spines. The openings of the penetration gland ducts are situated at the anterior tip of the oral sucker on a disc-like, spineless area; they are linearly arranged in two lateral crescents each containing five separate apertures. Each aperture is surrounded by a tegumentary rim and by larger, tegumentary folds which are longer on the outer, convex side of the crescent than on the inner side. Transmission EM studies of the suckers have been made by Hockley (1970) and of the duct apertures by Robson and Erasmus (1970), Morris (1971), Dorsey and Sitrewalt (1971), Ebrahimzadeh and Kraft (1971) and Hockley (1970). The tegument of the suckers is basically the same as the rest of the cercarial tegument. Schistosome cercariae thus differ from A. oregonense cercariae which have a ventral sucker with a greatly thickened tegument containing large numbers of inclusions (Belton and Harris, 1967). The shape of the suckers of schistosome cerariae is mainly due to the arrangement of interstitial material and muscle fibres beneath the tegument. Both suckers are separated from the other cells of the body by a thick layer of interstitial

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material. This layer is joined to the interstitial material beneath the tegument and the site of the junction in the oral sucker is marked by the surface constriction described by Robson and Erasmus (I 970). The layer is cone-shaped in the oral sucker and projects posteriorly into the body of the cercaria. Numerous large muscle fibres are associated with the interstitial layers of both suckers. The two groups of five ducts of the penetration glands pass through the muscle fibres and interstitial layer of the oral sucker. The ducts are lined with microtubules, an arrangement commonly found in trematode glands (Halton and Dermott, 1967; Southgate, 1971) and the walls of the ducts have numerous, small projections into the surrounding interstitial material. A few small muscle fibres partly encircle the bundle of ducts. At their apertures the ducts are joined to the tegument by a circular septate desmosome beneath which there is a space and below this space projections from the duct wall and the tegument come close together again. Round the opening of the duct, adjacent to the desmosome, there is the tegumentary rim and outside the rim there are the surrounding tegumentary folds as seen with the scanning EM (Robson and Erasmus, 1970). In sections there usually appear to be three concentrically arranged folds. The rim and the folds cover the duct opening when secretion is not being released (Hockley, 1970; Dorsey and Stirewalt, 1971). No mechanism for opening or closing the duct has been recognized but Archibald and Marshall (1932) stated that the anterior tip of the sucker can be withdrawn into the body of the cercaria which may effectively close the duct apertures. When the ducts are open the large globules of secretion break down at the aperture to form a granular mass attached to the aperture and the tegumental folds. The secretion does not spread over the surface of the cercariae (Hockley, 1970). Light microscopists have stated that the ducts open through papillae or spines (Archibald and Marshall, 1932; Faust and Russell, 1964) but these structures probably correspond to the tegumental folds. Robson and Erasmus (1970) suggested that the folds may abrade the surface of the host during penetration. Although abrasion does occur it seems more likely that it is caused by the enzymatic secretions of the glands and by the spines on the oral sucker and body. The folds may possibly restrict the spread of secretions during penetration. Ebrahimzadeh and Kraft (1971) observed the escape glands in the cercariae of S. mansoni before emergence from the snail host; these glands have basically the same structure as the penetration glands. Hockley (1970), Ebrahimzadeh and Kraft (1971) and Morris (1971) have observed the head gland which is located entirely within the oral sucker. The head gland contains large numbers of electron-dense, homogeneous bodies and it differs from the penetration glands in that the contents are passed into the tegument and not directly to the exterior. The gland has at least two microtubular-lined connections to the tegument situated adjacent to the penetration gland apertures. The secretion bodies are only seen in the tegument in the region of the connections. Morris (1971) has also observed vacuoles and surface invaginations containing tadpole-shaped bodies in this region of the tegument. He suggested that the electron-dense bodies are transformed and released to the

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exterior but no function was suggested for this secretion. The passage of secretion bodies into the tegument and then to the exterior commonly occurs in encysting cercariae (Southgate, 1971). Details of the connection of the penetration and head gland ducts with the tegument are summarized in Fig. 6. 4. Sensory organelles

Structures presumed to be sensory receptors are found in the tegument of both the body and tail of schistosome cercariae; they have been described in detail by Morris (1971) and Nuttman (1971). They were also identified with the scanning EM by Robson and Erasmus (1970) who found seven sensory organelles around the outer edges of each of the two crescents containing the penetration gland apertures. Light microscopists have described papillae bearing long flagellae on the body arid tail of schistosome cercariae (Gordon et al., 1934). Wagner (1961) made a detailed LM study of the papillae on S. mansoni cercariae; he found 53 symmetrically arranged papillae on the body, including the suckers, and more on the tail which were not regularly arranged. The following ultrastructural description of the sensory organelles is based on the observations of Nuttman (1971) who has classified the organelles as three types. The first two types are uniciliated papillae. They consist of a bulbous nerve-ending, 1-1.5 pm dia, situated at the level of the tegument so that they cause a surface protrusion of the overlying tegument. The cilium projects from the apex of the bulb through an aperture in the tegument and the tegument is attached to the bulb by a circular, septate desmosome round the cilium. The cilium is 2-8 pm in length and has the usual 9 2 arrangement of microtubules and a large basal body which extends into the middle of the bulbous nerve ending. The nerve ending also contains a small amount of peripheral granular material, a few strands of which connect to the ciliary basal body and, adjacent to the desmosome, there is a granular, electron-dense ring which encircles the basal body. The two types of uniciliated papillae are distinguished by the length of the cilium. The organelles on the anterior tip of the oral sucker have short cilia, approximately 2 pm in length, which are partly ensheathed by tegument, the cilia of the papillae on the remainder of the body are up to 8 pm long and are unsheathed. The third type of sensory organelle is a multiciliated pit. The pits are situated anteriorly and laterally; they are much fewer in number than the papillae. The pits are 1.5 pm dia and are situated beneath the tegument. The rim of the pit is connected to the tegument by a circular, septate desmosome and the pit opens to the exterior through a pore in the tegument. The wall of the pit, like the papillae, consists of a nerve ending. It contains electron-lucent vesicles and the basal bodies of five or six cilia which project into the cavity of the pit. The cavity also contains small, dense vesicles. Details of the sensory organelles and their association with the tegument are diagrammatically summarized in Fig. 6. Hockley (1970) and Nuttman (1971) suggested that the papillae might be mechanoreceptors and the pits might be chemoreceptors. Tactile and chemical stimuli are probably important to the cercaria during penetration of the host. Light microscopists have shown that other species of cercariae are well

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provided with sensory receptors and EM studies of cercariae have shown that the uniciliated papilla is the most commonly occurring type of sensory organelle (Dixon and Mercer, 1965; Chapman and Wilson, 1970; MatriconGondran, 1971 ; Bibby and Rees, 1971a). 111. THESCHISTOSOMULUM

Once the free-swimming schistosome cercaria has penetrated the vertebrate host it is referred to as a schistosomulum. The schistosomulum has lost the cercarial tail and evacuated the pre- and post-acetabular penetration glands. Unlike the cercaria, the schistosomulum is saline- and serum-adapted and water-intolerant; it has also lost the precise cercarial shape and the ability to form the CHR (Stirewalt et al., 1966). Specimens continue to be called schistosomula until the second stage of development described by Clegg (1965). This stage occurs about 14 days after penetration, by which time the schistosomula have migrated from the skin to the lungs and then to the liver. The 14-day old schistosomula have gut caeca filled with pigment and the caeca are joined together posterior to the ventral sucker. They have increased only slightly in volume compared with cercariae but they are about to grow and develop rapidly into adult worms. Schistosomes thus omit the metacercarial stage, which usually involves encystment in a second intermediate host, and through which the cercariae of most Digenea must pass before they become infective to the final host. The schistosomulum is interesting, however, because at this stage the transition occurs from the fresh water environment to life in the blood stream of the vertebrate host. Furthermore, it is probably useful to compare schistosomula and metacercariae since both must exhibit stages in the development of the adult tegument. A.

THE TEGUMENT OF SCHISTOSOMULA

30 MIN-3 H AITER PENETRATION

Comprehensive light microscopical studies of newly penetrated schistosomula have been made by Stirewalt (1963) who suggested that the changes in permeability of the specimens after penetration are due to the loss or modification of the cercarial surface coat. Surprisingly, this suggestion of surface changes has led to only three EM studies of the tegument of young schistosomula (Bruce et al., 1970; Rifkin, 1971; Hockley and McLaren, 1973). Bruce et al. (1970) were concerned as much with changes in the host skin as with the tegumental changes of the schistosomulum. They examined schistosomula in mouse tail skin at intervals of up to 2 h after penetration. The cells of the skin appear to be destroyed by lytic secretions from the schistosomulum, as has been suggested from other studies of the penetration gland secretions (Stirewalt and Fregeau, 1966). A surface coat is still present on the newly penetrated schistosomulum but it is thinner and more dense and granular than the cercarial coat. The tegument of the schistosomulum contains a variety of inclusions which are not seen in the cercaria. There are vacuoles, osmiophilic bodies and bodies consisting of concentric laminations round a central,

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granular core. Some of the laminated bodies appear to be extruded from tegumental invaginations and it was suggested that they represent secretions. Subtegumental cells are more prominent in the schistosomulum than in the cercaria and they are connected to the tegument by tortuous, microtubularlined connections. The cells contain laminated bodies similar to those in the tegument. Bruce et al. (1 970) also observed the tegumental uniciliated papillae, which they chose to interpret as locomotory organelles for propelling the schistosomulum through fluid media such as blood or tissue fluid. Although the cilia or flagella have the 9 + 2 arrangement of microtubules, which is usually associated with locomotion, there are three features which suggest that this is not their function. The features are (a) the limited number of cilia, (b) their concentration at the anterior end of the cercaria and (c) their association with what are almost certainly nerve endings, all of which indicate a sensory rather than a locomotory function. Rifkin (1971) examined schistosomula in mouse skin 20 min after penetration. Again, he was interested in the interaction between the schistosomulum and the host and he, also, observed that the damage to the host cells is inversely related to the distance from the parasite. This was interpreted as evidence of lytic secretions from the schistosomulum. Rifkin (1971) found the altered surface coat and laminated tegumental inclusions as described by Bruce et ul. (1970). He suggested that the coat is of host origin and that the bodies might be involved in repair of the tegumental outer membrane which may be abraded during penetration. Hockley and McLaren (1973) examined schistosomula in mouse skin 30 min and 1 h after penetration and also schistosomula recovered 1 h and 3 h after passing through a mouse skin preparation in vitro (Clegg and Smithers, 1972). They observed similar changes in the tegument, compared with the cercaria, to those described by Bruce et al. (1970) and Rifkin (1971). They also made some additional observations which permitted a more comprehensive interpretation of the results. Hockley and McLaren (1973) found that the surface coat on the 30 min schistosomulum is reduced in amount but otherwise is similar to the cercarial coat. Unlike the cercaria, the 30 min schistosomulum typically has numerous subtegumental cells connected to the tegument. Few of the cercarial inclusion bodies are present in the tegument but the tegument adjacent to the subtegumental cells is filled with laminated bodies, as described by Bruce et al. (1970) and Rifkin (1971). Usually these bodies have two trilaminate limiting membranes and a central electron-lucent area containing a small amount of granular material. The 1 h schistosomula are strikingly different to the 30 min specimens in that parts of the tegumental outer membrane are multilaminate. The membrane has the appearance of two closely applied trilaminate membranes; thus it has seven layers (heptalaminate) with a total thickness of approximately 17 nm. Hockley and McLaren (1973) used for their specimens a uranyl acetate fixative which is particularly good for preserving membranes (Terzakis, 1968). Bruce et ul. (1970) did not use this fixative, and this may explain why they did not observe the heptalaminate outer membrane. There is no surface coat on the heptalaminate membrane but the regions of tri-

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laminate outer membrane do have a thin surface coat. Examination of specimens recovered from skin preparations 1 h after penetration showed that there are microvillus-like projections from the surface of the tegument. The microvilli are long and thin, approximately 1500 nm in length and 50 nm in width, and they always have a trilaminate membrane with a thin surface coat. The laminated inclusion bodies, seen in the tegument of the 30 min schistosomulum, are also present in the 1 h specimens but, in addition, there are larger vacuoles which have a heptalaminate limiting membrane and which contain a few, small, membrane fragments. Some of the larger vacuoles are joined to the surface of the tegument, so that they open to the exterior; in this manner, the heptalaminate membrane of the vacuole becomes part of the outer membrane of the tegument. In the 3 h schistosomulum the tegumental outer membrane is almost entirely heptalaminate. The microvilli are no longer present but there are some additional small pieces of membrane on the surface. The large vacuoles with the heptalaminate limiting membrane are still present in the 3 h specimens but they occur less frequently than in the 1 h specimens. Another type of smaller, membraneous body is present in the 3 h schistosomulum; it consists of tightly packed, concentrically arranged membranes. The elongate bodies which are found in the cercarial tegument are also present in the 3 h schistosomulum. The observations on 30 min, 1 h and 3 h schistosomula are summarized in Fig. 7. Hockley and McLaren (1973) offered the following interpretation of these observations. They suggested that penetration is possibly a stimulus for the connection of subtegumental cells to the tegument. Thus the bodies, which are presumably formed in the cells from Golgi complexes, are passed from the cell to the tegument. The bodies appear to join together in the tegument to produce larger vacuoles with heptalaminate limiting membranes. The membrane of the vacuoles then becomes the new tegumental outer membrane and the original trilaminate cercarial membrane is formed into microvilli which are cast off from the schistosomulum. The reduced cercarial surface coat is finally lost with the cercarial membrane. By about 3 h after penetration the schistosomula are completely covered by the new heptalaminate outer membrane. The connection of subtegumental cells to the tegument and the formation of the new outer membrane in Schistosoma has some similarities to the process of cyst formation in F. hepafica and Notocotylus attenuatus as described by Dixon and Mercer (1967) and Southgate (1971). In these species cystogenous cells connect to the tegument and bodies of cyst-forming material are passed into the tegument and then to the exterior. Further studies on species of cercariae that encyst only after penetration of an intermediate host would provide a more direct point of comparison with Schistosoma. The cercarial tegument of F. hepatica and also of C. narrabeenensis, is lost during encystment and a new tegument is formed at the end of encystment (Dixon and Mercer, 1967; Dixon, 1970). This is perhaps comparable to the loss of the cercarial membrane and the formation of the new, heptalaminate membrane in S. mansoni.

FIG.7. Diagram summarizing changes in the tegument of schistosomula after penetration of the vertebrate host. 30 min after penetration the tegument has a thin surface coat (sc) and a trilaminate outer membrane (tm). Subtegumental cells (stc) containing laminated bodies (Ib) are connected to the tegument. The tegument contains groups of laminated bodies adjacent to the cell connections and a few spherical, cercarial, inclusion bodies (sb). 1 h after penetration parts of the tegument have a heptalaminate outer membrane (hm) and there are microvilli (mv) with a trilaminate membrane and a thin, surface coat. The microvilli appear to be cast off from the tegument. The tegument contains laminated bodies (Ib) and large vauoles (lv) with a heptalaminate limiting membrane. Some large vacuoles open to the tegumental surface. 3 h after penetration most of the outer membrane is heptalaminate (hm) but parts are pentalaminate (pm) and there are fragments of membrane (fm) on the tegumental surface. The tegument contains large vacoules (Iv), also smaller, membraneous bodies (mb) and elongate bodies (eb).

10

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The heptalaminate membrane of S. mansoni is presumably an adaptation to survival in the blood stream of the vertebrate host. Smithers et al. (1969) demonstrated host or host-like antigens on the surface of adult worms and suggested that these antigens enable the worms to evade the host’s immune response. It is not yet known how soon after penetration schistosomula acquire host antigens but a large proportion of schistosomula are known to be protected within 24 h against damage caused by antibodies in vifro (Clegg and Smithers, 1972). This rapid development of protection may be associated with the formation of the heptalaminate membrane; possibly the formation of the heptalaminate outer membrane is a necessary change prior to the attachment of host antigens to the surface.

B.

THE TEGUMENT OF SCHISTOSOMULA AFTER PENETRATION

24 H-14 DAYS

Examination of schistosomula after they have left the skin is difficult because of the problem of recovering specimens while they are migrating through the host. Hockley and McLaren (1973) examined schistosomula that had passed through a mouse skin preparation in vitro and had then been maintained in vitro for 24 h, 48 h, 4 days or 7 days. It has been shown by Clegg and Smithers (1972) that, under the correct conditions, the growth rate of schistosomula in vitro is identical with the rate in vivo for at least 12 days. Smith er al. (1969) examined 7-day old schistosomula obtained from compression preparations of mouse lung and Hockley (1970) examined 14-day-old schistosomula recovered from the liver of a mouse by perfusion. Compared with 3 h schistosomula the 24 h and 48 h schistosomula have a more folded and pitted surface. The heptalaminate outer membrane is prominent over most of the tegument but regions with a tri- or pentalaminate membrane are also present. The cytoplasm of the tegument is granular but the granules are arranged in discrete groups so that the tegument appears less electron-dense than in the cercariae and younger schistosomula. A thin layer of more closely packed granules forms a dense layer immediately beneath the outer membrane. The membraneous bodies, which are found in the 3 h schistosomulum, are the most numerous tegumental inclusions in the 24 h and 48 h specimens and a few of the elongate bodies are also present. The 4 day and 7 day schistosomula are essentially similar to the 24 h and 48 h specimens except that the surface is more regularly and deeply pitted. The heptalaminate outer membrane and the two types of tegumental inclusions are present and a third type of inclusion is also found in the 7 day schistosomula. These bodies are of about the same size as the membraneous bodies, approximately 150 nm in diameter; they have a trilaminate limiting membrane and homogeneous or finely granular contents. The homogeneous bodies occur as frequently as the membraneous bodies and they both appear to be derived from the same subtegumental cells. Smith et al. (1969) first described the homogeneous bodies in 7 day schistosomula. They also found that mitochondria are less numerous in the tegument of these specimens than

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in the tegument of cercariae. Spines also appear to be less numerous than in cercariae. Possibly this is because the schistosomulum has increased in length, compared with the cercaria (Clegg, 1965), so that the spines become more dispersed. There is no evidence of the formation of new spines. The tegument of the 14 day schistosomulum shows a marked increase in thickness compared with the cercaria. The surface pits, however, are deeper than in the younger schistosomula so that the thinnest parts of the tegument are still only 0.3 pm in thickness (Fig. 8). The great increase in surface area produced by the pits is well illustrated in scanning electron micrographs (Fig. 9). It is possible to calculate that the surface area of a 14 day schistosomulum is approximately 4 x that of a 7 day schistosomulum (Hockley, 1970). Small depressions in the surface membrane, which may be stages in the formation of phagocytic vesicles, have been seen by Hockley (1970). Similar small, electron-lucent vesicles are also present in the tegument but Hockley (unpublished results) was unable to obtain any evidence of the uptake of ferritin by phagocytosis. The membraneous and elongate bodies are present in the tegument in approximately equal numbers. The homogeneous bodies seen in the 7 day specimens are absent at 14 days. The basal membrane of the tegument also increases in surface area; the fold-like invaginations of the membrane into the tegument are longer and more numerous in the 14 day schistosomulum than in the cercaria and younger schistosomula. The basal lamina beneath the basal membrane is thicker and more dense and granular than in the cercaria but the thick layer of fibrous interstitial material, which was present beneath the cercarial tegument, is absent in the 14 day schistosomulum. The observations on schistosomula 24 h-14 days after penetration have shown that, during this time, the major change in the tegument is the formation of folds and pits in the surface. The increase in surface area produced by the pits must require the formation of new outer membrane. Hockley and McLaren (1973) suggested that the membraneous bodies in the tegument might be involved in the formation of outer membrane. Possibly the homogeneous bodies in the 7 day schistosomula also have a similar function since no changes in the spines or other constituents of the tegument have been detected in which they might be involved. The significance of the increase in surface area is unknown but, together with the heptalaminate outer membrane, it must be an adaptation to life in the vertebrate host. A large surface area is often associated with an absorptive function and it is possible that the growing schistosomulum absorbs material through the tegument. Bibby and Rees (1971b) concluded from autoradiography that the metacercariae of Diplostomurn phoxini absorb glucose through the tegument. The small vesicles in the tegument of the schistosomulum and the basal membrane invaginations may also be associated in some way with transport of material through the tegument. There is, however, no definite evidence, as yet, of the uptake of material into the tegument of schistosomula and, apart from the large surface area, the ultrastructural features of the tegument would suggest that it is not an important site of food absorption. Furthermore, in the 14-day schistosomulum, the gut caeca are filled with pigment which results from the digestion

FIG.8. Electron micrograph of the pitted tegument (t) of a 14-day-old schistosornulum. FIG. 9. Scanning electron micrograph of the surface of a 14-day-old schistosomulum showing the pits and spines.

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of haemoglobin and most of the food utilized during the rapid growth of the worms after 14 days is probably absorbed through the caeca. Following the increase in surface area, the tegument itself starts to increase in thickness at about 7-14 days after penetration. Further studies on the changes during growth of the tegument would help in understanding the ultrastructure of the adult tegument. At present the only ultrastructural feature noted during growth is the increase in number of elongate bodies. Possibly these bodies contribute to the granular ground substance of the tegumental cytoplasm. The mechanism of growth is itself not understood. s of the original The tegument of the metacercaria of N . a t t en u a ~ ~consists cercarial teguemnt plus cytoplasm from the cystogenous cells (Southgate, 1971). Possibly the tegument of Schistosoma, and othcr trematodes, increases in thickness by the contribution of cytoplasm from subtegumental cells. IV. THEADULTWORM A.

THE TEGUMENT AND ASSOCIATED STRUCTURES OF NORMAL WORMS

The adult schistosomes are unusual digenetic trematodes in two respects: (a) they inhabit the bloodstream of the vertebrate host and (b) the sexes are separate and dimorphic. Nearly all other Digenea are hermaphrodite and they mostly inhabit the gut and associated body cavities of the host. Both male and female schistosomes are elongate and are thus adapted to their environment. The lateral margins of the male are curved ventrally and overlap to form the gynaecophoric canal. The mature female is cylindrical and is held in the gynaecophoric canal of the male.

I . External appearance Light microscopists have described the different appearances of male and female worms. Gonnert (1949) described the arrangement of the tegumental spines in the two sexes of S. mansoni and in particular the regional variations in spination in the male worm. Recently the scanning EM has provided dramatic illustrations of the external appearance of S. mansoni adult worms (Hockley, 1970; Silk et al., 1970; Race et al., 1971) (Figs 10-15). The inner surfaces of the oral suckers of both sexes are covered with small spines, which point towards the mouth. The ventral sucker or acetabulum of the male worm is larger and more prominent than the female’s acetabulum; both have spines and sensory papillae on the inner surface and a peripheral ring of larger spines. Spines are absent on the outer surfaces of the oral and ventral suckers. The gonopores of both sexes are situated just posterior to the acetabulum. Large papillae or tubercles are present on the dorsal surface of the male worm, posterior to the ventral sucker. The sides of the tubercles are covered with spines which point towards the spine-free tubercle apex. The tubercles become fewer in number towards the lateral and posterior margins of the dorsal surface. In these regions and between the tubercles, the surface of the worm is ridged and in the depressions between the ridges there are numerous distinct pits

FIGS10-15. Scanning electron micrographs of adult male worms: 10, oral and ventral suckers. 11, High magnification of mouth and surrounding spines. 12, Ventral sucker and anterior end of gynaecophoric canal. 13, Opened gynaecophoric canal. 14, Dorsal surface of anterior end showing tubercles posterior to the suckers. 15, Contracted specimen showing dorsal tubercles and area of spines at lateral margin of gynaecophoric canal.

FIG.16. Scanning electron micrograph of a tubercle and spines on a male worm. FIG.17. Higher magnification micrograph showing the ridged and pitted surface between the tubercles.

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(Figs 16 and 17). A few individual spines and sensory papillae are also present between the ridges. On the lateral margins of the male worm, which overlap to form the gynaecophoric canal, there are bands of large spines. These spines interlock to hold the female worm in the canal. The surface of the gynaecophoric canal, which is in the ventral surface of the male worm, is ridged and covered with many small, irregularly arranged spines. There are comparatively few spines on the female worms and the surface is similar to the ridged and pitted surface of male worms. Spines are most numerous on the posterior end of the female worm. Scanning electron microscopy has confirmed details of the spination of S. mansoni and provided a valuable new view of the surface pits. The technique is a useful aid in interpretation of the functional morphology of worms and for accurately recording surface specializations of the tegument such as occur in strigeoid trematodes (Erasmus, 1970). 2. The tegument There have been numerous transmission EM studies of S. mansoni adult worms, but many of the eatlier observations are now of little value because of recent improvements in techniques. The following account is based on the observations of Morris and Threadgold (1968), Smith et al. (1969). Silk et al. (1969) and Hockley (1970), and also on the observations of Inatomi et al. (1969) on S. japonicum. Even these more recent studies have only provided a general description of the tegument and few differences between male and female worms or between the various regions of male worms have been recorded. Figure 18 is a diagram of a typical part of the tegument and its associated structures. The tegument of the adult worm is approximately 4 pm in thickness (Fig. 19) but it is thinner on the suckers and on the ventral surface and tubercles of male worms. Measurement of the thickness is difficult because of the irregularity of the outer surface. The schistosome tegument is slightly thinner than that of some other Digenea. The thickness of the digenean tegument possibly depends on the environment and the size of the worm. F. hepatica, which is a relatively large worm, has a tegument 15-20 pm in thickness (Threadgold, 1963). The tegumental surface pits seen with the scanning electron microscope are shown in sections to be deep, tortuous channels which may be branched and interconnected (Fig. 19). Incubation of fixed worms in colloidal iron or thorium has shown that the channels all open to the exterior, thus providing a large surface area. It can be calculated that the pitted surface has an area of about l o x that of a flat surface of the same basic size. The channels are a development of the simple pits in the surface of the schistosomula; they are present over the whole worm but are less well developed in the thinner parts of the tegument. A similarly irregular but less pitted surface has been found on most Digenea that have been examined with the EM although specialized regions of the tegument of strigeoid worms have regularly folded or microvillous surfaces (Erasmus and Ohman, 1965; Erasmus, 1967). The functional advantages of pits, rather than microvilli, for increasing the surface

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FIG. 18. Diagram of a typical part of the adult schistosome tegument and its related structures. bl = basal lamina. cmf = circularly arranged muscle fibres. dr = peripheral dense region of muscle fibre. eb =elongate body. g = Golgi region. hm = heptalaminate outer membrane. i = invagination of basal membrane. im = interstitial material. jc =junctional complex with parenchymal cell. Imf= longitudinal muscle fibre. m=mitochondrion. mb= membraneois body. mt = microtubule. n = nucleus. r = ribosomes. sp = surface pits. sr = sarcoplasmic reticulum. stc = subtegumental cell. t = tegument.

area of Schisfosoma is unknown. Spence and Silk (1970) suggested that the sponge-like structure of the tegument would increase its flexibility as compared with a solid tegument but also that pits would provide a more rigid structure than microvilli. In worms examined while still within host blood vessels, blood plasma is present in all the surface channels (Bruce et al., 1971). Possibly the plasma is trapped in the pits, which may facilitate the absorption of food material. (a) The tegumental outer membrane. The outer membrane of S. mansoni

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D. J. H O C K L E Y

extends over the whole of the pitted surface of the tegument. It has usually been described as trilaminate in structure and approximately 10 nm in thickness, although Smith et al. (1969) described the outer membrane of the gynaecophoric canal as pentalaminate and 16 nm in thickness. Hockley and McLaren (1973), however, examined specimens of S. mansoni that had been fixed with uranyl acetate and they found that the tegumental outer membrane is basically heptalaminate with a total thickness of approximately 17 nm (Fig. 20). Clearly the heptalaminate membrane, which is formed in the schistosomulum, persists in the adult worm. As in the schistosomulum, there are some small regions of the outer membrane that are trilaminate and there are also regions with one or two additional layers of trilaminate membrane which therefore have an 1 1- or 15-layered structure. The significance of the heptalaminate outer membrane has been discussed in the section on the schistosomulum. Whether or not the heptalaminate membrane is unique to Schisfosoma can only be revealed by fixation of other trematodes with uranyl acetate. There is no morphological evidence of a surface coat on the outer membrane but, as will be described in the section on cytochemistry, the outer membrane stains specifically with colloidal iron and thorium, ruthenium red and with silver methanamine after periodic oxidation (Morris and Threadgold, 1968; Hockley, 1970; Reissig, 1970). (b) The tegumental matrix. The ground substance of the tegumental cytoplasm consists of electron-dense, granular material, as has been seen in the younger stages of the worm. This material forms a dense layer, approximately 50 nm in thickness, immediately beneath the outer membrane although it is less conspicuous beneath the outer membrane which forms the surface pits. The granular material also tends to be more abundant in the outer part of the tegument than in the basal regions and thus the outer parts are more electrondense. There is some regional variation in the distribution of the tegumental ground substance; the ventral tegument of the male worm is more electrondense than other regions. A dense, granular tegumental matrix has been described for several species of Digenea. The tegument of F. hepatica has a superficial dense zone (Threadgold, 1963) and Haplometra cylindracea, Cyathocotyle bushiensis and Gorgoderina sp. have a dense, particulate layer beneath the outer membrane (Threadgold, 1968b; Erasmus, 1967; Burton, 1966). This layer is prominent in Gorgoderina and Burton (1966) suggested that it has a protective function. (c) The basal membrane of the tegument. The basal membrane is trilaminate and approximately 10 nm in thickness (Fig. 21). The membrane closely follows the contours of the underlying muscle fibres but it also invaginates into the tegumental cytoplasm in the form of numerous, long (approximately 0.5 pm), thin (approximately 25 nm) folds (Fig. 21). The invaginations have usually been described as tubular (Morris and Threadgold, 1968; Smith et al., 1969) but the absence of circular profiles in sections and the fact that they are sometimes looped and branched means that they are more accurately interpreted as folds. The ends of the folds are occasionally swollen to form large, empty vacuoles or channels. This appearance is commonly found in damaged worms and may simply represent a fixation artefact in normal worms. The

FIG.19. Low magnificationelectron micrograph of part of the body wall of an adult male worm. t = tegument. sp= surface pits. mf= muscle fibres. im= interstitial material. FIG.20. High magnification micrograph of part of the tegument showing the heptalaminate outer membrane (hm) of a surface pit (sp), and elongate body (eb) and a membraneous body (mb).

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D. J. H O C K L E Y

folds increase the surface area of the basal membrane and also effectively reduce the thickness of the tegument since they reduce the distance between the closest parts of the outer and basal membranes. These facts have been taken as evidence that the invaginations are involved in the passage of substances through the tegument. Fawcett (1962) has correlated the basal membrane invaginations of vertebrate kidney and salt gland cells with the movement of fluid or ions, and Shannon and Bogitsh (1969) suggested a similar function for the schistosome basal membrane invaginations. Alternatively, it is possible to suggest that the invaginations are involved in the attachment of the tegument to the underlying tissues and that they may be necessary for flexibility of the tegument as has been suggested for the infoldings of the outer membrane. ( d ) The spines. Spines in the adult tegument are basically the same as those in the teguments of cercariae and young worms except that they are larger. They are enclosed within the tegumental outer and basal membranes but the pointed tip projects beyond the general level of the tegument and the base of the spine is surrounded by a large invagination of the basal membrane. The spines appear to have a crystalline lattice structure (Fig. 22) of the same type and dimensions as in the cercariae. The variety of appearances of the spines produced by sections in different planes have led to various descriptions of spine structure. Further studies would probably provide a more accurate interpretation of the lattice structure. The spines or scales of other Digenea, where present, almost certainly have a crystalline lattice structure, possibly with subunits of different dimensions from those of Schistosoma. Only a relatively small part of the spine is in direct contact with the tegumental matrix because of the invaginations of the outer and basal membranes which surround the two ends of the spine. Smith et ul. (1969) described this middle portion of the spine as being surrounded by elongate bodies and they suggested that the bodies break down to form the spines. This association of bodies with the spines has not been observed by other workers and the adult worm has not really provided any further information on the mechanism of spine formation and growth. Spence and Silk (1970) suggested that the tegumentary matrix crystallizes to form spines where subtegumental cells are absent and where the tegument extends down between the underlying circular muscle fibres to come in contact with longitudinal muscle fibres. Longitudinal and transverse muscle fibres are often closely associated with the base of spines. Spence and Silk (1970) also argued, somewhat circularly, that spines are formed as a protective device to reduce the surface area of tegument available for exchange of substances in regions where few subtegumental cells are present to utilize such materials. This suggests a rather negative function. Morns and Threadgold (1968) described the spines as being flexible; they observed that the spines on the male worm are bent when in contact with the female tegument whereas normally the spines are straight. Bending of spines has not been observed by other workers. Hockley (1970) examined worms in host blood vessels and found that the tegument of the worm may be distorted by host blood cells but that spines are straight and often deeply embedded in

FIG.21. invagination (i) of the trilaminate basal membrane (tbm) into the tegument (t). Underlying basal lamina (bl) and transverse sections of fibres of interstitial material (m). FIG.22. Transverse section of the tegumental spine (s) with surrounding invagination of the basal membrane (i) and associated dense bodies (db). FIG.23. Typical tegumental mitochondria (m).

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the endothelial cells of the blood vessel. The spines do not appear to rupture the plasma membrane or damage the cell; they probably hold the worm against the flow of the blood by being embedded within the wall of the vessel. If the spines are movable within the tegument, rather than flexible, then the worm would be able to change its position in the blood vessel without causing extensive damage to the endothelial cells. Hockley (1970) found small, dense bodies between the spines and the basal membrane which may represent points of attachment to the membrane (Fig. 22). Possibly the spine is only attached to the basal membrane and the long invagination of the membrane around the base of the spine might, therefore, allow the spine to be moved in the tegument. (e) The mitochondria. Mitochondria occur very sparsely in the tegument of adult schistosomes; they are all very small and often contain only a single crista (Fig. 23). Morris and Threadgold (1968) observed a group of mitochondria adjacent to the ventral sucker of S. mansoni in male worms and Smith et al. (1969) found that the mitochondria are more abundant in the dorsal tegument of male worms than in other regions of either the male or female tegument. Tegumental mitochondria of other species of trematodes are often smalland morphologically simple but they are usually morenumerous than in Schistosoma. Gorgoderina sp. is the only digenean in which numerous, large mitochondria have been found in the tegument (Burton, 1966). In this species the mitochondria are closely associated with the outer membrane and its underlying dense layer; Burton suggested that they provide energy for a protective function. Morris and Threadgold (1968) concluded that the tegument of S. mansoni requires little energy either for the uptake of substances or for a protective function. (f) Elongate and membraneous inclusion bodies. Two characteristic inclusion bodies have been recognized in the tegument of adult worms by most investigators ; they are the elongate and membraneous bodies (Fig. 20) which have also been recognized in the schistosomulum. The elongate bodies are extremely numerous in the adult worm; they are approximately 40 nmx 200 nm in size (about twice the size of cercarial elongate bodies) with a trilaminate limiting membrane and dense granular contents. The bodies exhibit a wide variety of shapes but they are mostly elongate and often appear elliptical. Morris and Threadgold (1968) and Smith et al. (1969) suggested that these bodies are disc-shaped in three dimensions. The very rare occurrence of bodies which appear circular may be because the bodies are thinner than the sections in which they are observed and thus do not appear clearly, or it may be because the bodies are mostly elliptical in shape. There is also some variation in the contents of the bodies. Mostly they are more electrondense than the surrounding tegumental matrix but occassionally they are less dense. The tegument also contains some small, empty vesicles which might be derived from the elongate bodies. The elongate bodies are distributed throughout the tegument of both male and female worms and they are often arranged with their long axes at right angles to the outer and inner surfaces of the tegument. Electron-dense, elongate bodies have been found in the teguments of several

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species of Digenea; they are often larger than those of Schistosoma, biconcave and again, probably disc-shaped. In Haplometra cylindracea and Cyathocotyle bushiensis the bodies are regularly arranged beneath the outer membrane (Threadgold, 1968b; Erasmus, 1967) and it has been suggested that they give rise to the dense, granular layer beneath the membrane or possibly to a surface coat. The bodies in Schistosoma are not closely associated with the outer membrane. Burton (1966) suggested that dense bodies in the tegument of Gorgoderina may contribute to the tegumental matrix or ground substance and a similar function might apply in Schistosoma. If the spines represent a crystalline part of the ground substance, then the elongate bodies would contribute indirectly to the spines. The membraneous bodies in the tegument of adult worms appear identical to those which are present in the schistosomulum tegument; they are 150200 nm in diameter. The membraneous appearance of the bodies has only been revealed with uranyl acetate fixative (Hockley and McLaren, 1973). Other workers have described the inclusions as spherical or multilaminate bodies, since with conventional fixation the bodies have irregularly arranged contents which appear lamellate at high magnification. With uranyl acetate fixative the bodies appear either as a mass of tightly packed, concentrically arranged membranes or as a mass of more irregularly arranged membranes. The membraneous bodies are distributed throughout the tegument but are much less numerous than the elongate bodies. The bodies are occasionally found close to the tegumental outer membrane although only where the membrane forms the surface pits (Smith et al., 1969; Hockley and McLaren, 1973). Bodies close to the outer membrane usually have irregularly arranged contents. Very occasionally, membraneous bodies appear to be incorporated in the outer membrane so that they cause small surface protusions (Hockley and McLaren, 1973). Membraneous bodies have not been described in the teguments of other digenetic trematodes although a variety of dense, spherical, so-called secretion bodies have been observed. An obvious suggestion for the function of membraneous bodies of Schisfosoma is that they contribute to the outer membrane, since some membraneous bodies are closely associated with the membrane and the membrane itself has a multilaminate structure. It is perhaps possible that themultilaminate outer membraneis continually breaking down and being replaced by the membraneous bodies. Some evidence for the lability of the membrane is the fact that it has a variable structure, sometimes being trilaminate and occasionally having 11 to 15 layers. Smith et al. (1969) suggested that the membraneous bodies might be concerned with resorption of the membrane as well as regeneration. They found that horseradish peroxidase is present in membraneous bodies after worms have been incubated in the enzyme. Further evidence of resorption is required from studies of the fate of labelled outer membrane. Some further evidence of formation or regeneration of the membrane from the membraneous bodies comes from the origin of the bodies in subtegumental cells and their fate during destruction of the tegument, both of which are discussed in later sections. The outer membrane of cells such as amoebae and bladder epithelium are known to be

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formed from bodies derived from Golgi regions in the cells (Stockem, 1969; Hicks, 1966), and a similar process may occur in Schistosoma. Silk et al. (1 969) recognized the two characteristic tegumental inclusions of Schistosoma but also described three further types of inclusions, namely, ringlike elements, circular inclusions with uniform granular contents and small, circular vesicles. These inclusions are mainly found in the subtegumental cell bodies and they probably represent stages in the formation of the elongate bodies. In a later paper (Spence and Silk, 1970) it was suggested that some of these bodies are simply elongate bodies cut in different planes of section. It was also suggested that the membraneous bodies can form elongate bodies by a process of dehydration in the subtegumental cells and can then be rehydrated to form membraneous bodies in the tegument. These speculations need to be tested and at present it is simpler to regard the tegumental inclusions as two types, elongate and membraneous bodies. 3. Cytochemistry of the adult tegument A summary of conclusions drawn from cytochemical staining of the body tegument of S. mansoni is presented in Table 11. Different workers using different techniques to demonstrate the same substances have obtained results that are mostly in agreement. Some techniques have been applied both light and electron microscopically; EM permits a more precise localization of the reaction product. The results of cytochemical staining of the schistosome tegument are very similar to those obtained with other digeneans. There is evidence from several species that the tegument is basically proteinaceous (MonnC, 1959; La1 and Shrivastava, 1960; Lee, 1962; Bjorkman et al,, 1963; Slais and iddrsk8, 1967). Lipids do not appear to be a common component of the trematode tegument (La1 and Shrivastava, 1960; Bjorkman et al., 1963; Thorpe, 1968) and this is in agreement with the ultrastructural morphology of Schistosoma. Glycogen appears to be absent from the trematode tegument (Axmann, 1947) and it has never been identified in the schistosome tegument with the EM, although abundant glycogen granules have been observed in the muscle cells of worm (Silk and Spence, 1969a; Reissig, 1970). Carbohydrates other than glycogen have been identified in the tegument of several species of Digenea by PAS staining with the appropriate controls (MonnC, 1959; Thorpe, 1967). It has been suggested that these substances are acid mucopolysaccharides which enable the worm to inhibit or resist the action of host enzymes (MonnC, 1959; Crompton, 1963). EM cytochemistry has shown the presence of non-glycogen, PAS-positive material and acidic groups on the surface membrane of S. mansoni (Reissig, 1970; Morris and Threadgold, 1968; Hockley, 1970). There is some evidence that this material is removed by the action of neuraminidase (Hockley, 1970), which would indicate that it contains sialic acid. Glycoproteins containing sialic acid are common components of cell membranes (Cook, 1968) and it has been suggested that these substances mask the antigens of tumours so that the immune system of the animal does not recognize the tumour as foreign material (Currie, 1968). Masking of antigens by surface glycoproteins would be of great

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value to Schistosoma and might explain how the worm is able to live for long periods in the blood stream of the host. An alternative mechanism to prevent the worm from being attacked from the host’s immune defences has been suggested by Smithers et al. (1969). They found that S. mansoni has a coating of host antigens and proposed that 1 he coating effectively disguises the worm as host. The host antigens are almost certainly glycolipids derived from host red blood cells (Clegg, 1972). Possibly some of the PAS-positive staining of the schistosome surface is due to glycolipids. Complex carbohydrates are the only substances that have been identified in the two characteristic tegumental inclusion bodies (Reissig, 1970). The observation is compatible with the suggestion that the membraneous bodies contribute to the tegumental outer membrane because a small amount of similar material has been identified in the outer membrane (Reissig, 1970). The small amount of this material in the membrane makes it unlikely that the elongate bodies also contribute to the membrane and cytochemical staining provides no further evidence that the elongate bodies produce tegumental ground substance. Reissig (1970) did find that the interstitial material of the worm has similar staining properties to the tegumental inclusion bodies and suggested that the bodies might give rise to this material. Cytochemical staining to demonstrate phosphatase activity in S. mansoni has been performed with a variety of substrates and over a wide range of pH and has shown that there are probably several different alkaline phosphatases at the surface of the worm (Nimmo-Smith and Standen, 1963; Bogitsh and Krupa, 1971). In this respect S. mansoni is different from Haematoloechus medioplexus in which the tegumental phosphatases are only capable of hydrolysing nucleoside di- and triphosphatases (Bogitsh and Krupa, 1971). Alkaline phosphatases may be completely absent from the tegument of some digeneans: Nollen (1968) and Bogitsh and Krupa (1971) found no nucleoside diphosphatase activity in the teguments of Megalodiscus temperatiis and Gorgoderina attenuata. Electron microscopy has shown that the alkaline phosphatases of Schisrosorna are present over the whole surface of the female tegument but in the male worm they are restricted to the outer membrane of the dorsal tegument. The presence of alkaline phosphatase at the surface of the trematode tegument has usually been correlated with the metabolic uptake of glucose by the tegument from the surrounding medium. In vertebrate tissues, high concentrations of alkaline phosphatases have been found at the sites of active transport of glucose (Roche, 1950) and it has been suggested that phosphorylation of hexoses occurs at their site of entry into the epithelial membrane followed by hydrolysis of the ester inside the cell. A similar mechanism may possibly operate in the trematode tegument. Bueding and Mackinnon (1955) have shown that S. mansoni contains four distinct hexokinases which phosphorylate glucose, fructose, mannose or glucosamine. Fripp ( I 967a) found that the uptake of radioactive glucose into S. haematobium is prevented by p-chloromercuribenzoic acid, an inhibitor of the glucokinase reaction, and by sodium arsenate, an alkaline phosphatase inhibitor. Thus there is some evidence for a phosphorylation mechanism of uptake of glucose into the

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HOCKLEY

TABLE 11. Cytochemisfry of the tegument

Outer membrane Substances demonstrated PROTEIN SULPHYDRYL-DISULPHIDE GROUPS TRYPTOPHANE NEUTRAL LIPID GLYCOGEN CARBOHYDRATES INCL. GLYCOGEN CARBOHYDRATES EXCL. GLYCOOEN ACIDIC GROUPS

Tegumental cytoplasm

ElonMale Male Male Male gate dorsal ventral Female dorsal ventral Female bodies

++

++

+ + 0 + + + + +++ +++ +++

+++ + + + +++ ALKALINE PHOSPHATASE

+++ +++

0

+

++ +++

ACID PHOSPHATASE

CHOLINESTERASE PEROXIDASE

~GLUCURONDASE AMINO-PFF'T'IDASE

(S. rodhaini)

+ =positive

* =electron

0

0

0

++ ++ + + 0 +

++ ++ + + 0

+ ++++

+

+

+

0

0

0

+ +++ +++

+ +

+ +++ +++

++ 0 ++ + +++ ++ ++ 0

+ ++ ++ +

+++ 0 ++++ +

+++ +++

0

0

0

0

0

0 0

result, arbitrarily graded. 0 =negative result. Blank = not examined. microscopical cytochemistry.

tegument of Schistosoma. Evidence that glucose is taken up by Schistosoma through the tegument has been provided autoradiographically by Fripp (1967a) and from the ability of headless worms to survive for some time in vitro (Senft and Senft, 1962). Glucose is an important food for many trematodes but the gut may often be a more important site of absorption than the tegument, particularly in species which do not have tegumental phosphatases (Bogitsh and Krupa, 1971). Philophthalamus megaluris, however, appears not to have any tegumental

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of adult worms of S. mansoni

Membraneous Mitobodies chondria

Tubercles= Subtegunontegumental Subtegumental cell con- mental parenchymal Spines nections cells cells

++++ ++++ +++

+

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

0

0

++++

References Smith er al. (1969) Smith et al. (1969) Smith et al. (1969) Smith et al. (1969) Axmann (1947) Smith et QI. (1969) Reissig (1970)*

++ 0

0

0

++

0

0

0

+-t+

0

+

0

++ + ++

+ +++

0

Morris and Threadgold (1968)* Smith et al. (1969) Hockley (1970)* Dusanic (1959) Robinson (1961a) Nimmo-Smith and Standen (1963) Halton (1967) Morris and Threadgold (1968)* Bogitsh and Krupa (1971)* Nimmo-Smith and Standen (1963) Halton (1967a) Morris and Threadgold (1968)* Fripp (1967b) McLaren (unpublished)* Fripp (1966) Fripp (1967~)

phosphatases although glucose is taken up very rapidly through the tegument (Nollen, 1968). It is possible that the tegumental phosphatases are not associated with the uptake of glucose. Read (1966) and Threadgold (1968a) suggested that the phosphatases may prevent the uptake of phosphorylated compounds, an excess of which might upset the normal metabolism of the worm.The part played by acid and alkaline phosphatases in glucose transport and in other possible roles in the tegument of Schisrosoma remains to be fully determined.

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The functions of cholinesterase, peroxidase and /3-glucuronidase in the trematode tegument are also unknown. Fripp ( I 967b) found that the cholinesterase activity in Schistosoma is most marked on the dorsal surface of male worms and with F. hepatica, Halton (1967b) found the strongest acetylcholinesterase activity in those regions of the tegument which are in most intimate contact with the host. Both workers suggested that the enzymes may be involved in the transport of substances through the tegument. Fripp (1967b) found no evidence of secretion of the enzyme. Peroxidase has been found only in mitochondria in trematodes (Rothman, 1968; Halton, 1969; Threadgold and Read, 1968) and is therefore presumably connected with respiratory processes. It has been suggested, however, that the staining reaction is due to the mitochondria1 cytochrome system (Lumsden et al., 1969;Bogitsh and Shannon, 1970)./3-Glucuronidasehas a similar distribution in Schistosoma to the phosphatases; Fripp (1966) suggested that this enzyme might have a digestive function. Strong leucine aminopeptidase activity occurs in the tegument of S. rodhaini but the enzyme is absent from the gut and other organs of the worm (Fripp, 1967~).This enzyme hydrolyses peptides which have a free a-amino group on a terminal leucine or other related amino acid. Fripp (1967~) suggested, therefore, that the enzyme breaks down serum proteins and that free aliphatic amino acids ai-e absorbed through the tegument. Timms and Bueding (1959), however, were unable to find any enzyme in homogenates of S. mansoni that would break down serum proteins. Schistosomes have remarkable powers of protein synthesis, as shown by egg production, and they live in an environment rich in protein but their methods of protein digestion and absorption are not fully understood. Robinson (1961b) found that S. mansoni is able to utilize free amino acids from serum and Senft (1968) found that radio-proline is taken up into the tegument of male worms. Amino acids are transferred in both directions across the tegument of F. hepatica (Kurelec and Ehrlich, 1963) and Senft (1969) suggested that, in Schistosoma, amino acids may pass from the male ventral tegument into the female tegument. There is also evidence, however, that amino acids are absorbed through the gut of trematodes (Pantelouris, 1965; Nollen, 1968). Alicyclic and aromatic amino acids, which are derived from globin, may be absorbed through the gut of schistosomes (Fripp, 1967c) although Senft (1969) believes that the enzymes in the gut only produce peptides. The assimilation of amino acids by Schistosoma and the part played by the tegument and its enzymes remains to be fully elucidated. Indeed, the cytochemical observations on the schistosome tegument have led to speculations on tegument function but in most cases definite proof of these ideas is still awaited. 4. Subtegumental cells

The subtegumental cells have been described by Morris and Threadgold (1968), Smith et al. (1969) and Silk et al. (1969). These authors regarded the cells as the nucleated part of the tegument since they contain a nucleus and are continuous with the tegument. As has been discussed for the cercaria, however, the cells probably arise separately from the tegument and it is possible that they are only temporarily connected to the tegument even in the adult worm.

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Figure 18 shows a subtegumental cell and its relationship to the tegument in diagrammatic form. The subtegumental cells form a clearly recognizable type of cell in the parenchyma of the worm (Reissig, 1970). The cells are situated below the muscle fibres, deep in the body of the worm; they are irregular in shape but are readily identified by the electron-dense appearance of the nucleus and cytoplasm. Some of the cells are multinucleate; the dense appearance of the nuclei is due to the presence of several prominent nucleoli and patches of condensed chromatin. A small amount of granular endoplasmic reticulum is present in the peripheral parts of the cell and the remainder of the cytoplasm typically contains a large number of free ribosomes or polysomes, which contribute to the electron-dense appearance of the cell. The cells also contain one or more well developed Golgi regions and a few mitochondria which are usually larger than those in the tegument. The subtegumental cells can easily be recognized by the presence within their cytoplasm of the two types of characteristic tegumental inclusion bodies. There is considerable variation in the type and number of the inclusions within the cells. Morris and Threadgold (1968) found that either elongate or membraneous bodies might predominate in a cell but rarely to the total exclusion of one type. In some cells the cytoplasm is filled with inclusion bodies and in other cells only a few bodies are present. Amongst the large masses of elongate bodies in the cells there are some less-distinct, circular bodies which is further evidence that the elongate bodies, are, in fact, disc-shaped, Both the cells that are full of inclusion bodies and those that mainly contain ribosomes have been seen to be connected to the tegument (Morris and Threadgold, 1968). The connections are long and tenuous and they intertwine and branch between the muscle fibres and other cells of the body. Each cell is probably connected at more than one place to the tegument. The narrowest part of the connection, which is approximately 0.1 pm in diameter, occurs at the junction with the tegument. In this region the connection has a lining of about 20 longitudinally arranged microtubules. These junctional regions, which are readily recognized by their microtubules, are found quite frequently but complete connections from the tegument to the subtegumental cells are rarely observed because of their length and tortuous course (Smith et al., 1969). For this reason it is difficult to know if all the cells are connected to the tegument. The subtegumental cells of Schistosoma are similar in all respects to those of other digeneans. The abundant ribosomes and the well-developed Golgi complexes in the cells, together with the presence of tegumental inclusion bodies in the cells and in the connections, clearly suggest that the bodies are formed in the cells and passed up into the tegument. A similar function has been suggested for the subtegumental cells of other species. Threadgold (1967) found that F. hepatica differs slightly from Schistosoma in that it has two types of subtegumental cells, each of which produces a different type of tegumental inclusion body. The cells occur in groups together. Also in this species, Gallagher and Threadgold (1 967) found that there are regions of the subtegumental cells that are in close contact with other parenchymal cells.

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They suggested that these regions may be sites of adhesion or possibly of transport between the cells. Similar junctional complexes have been found at the surfaces of S. nzansoni subtegumental cells by Silk et a/. (1969).

5 . Basal lamina, interstitial material and muscle fibres These structures have the same basic form and arrangement in the adult worm as in the cercaria and schistosomulum (Fig. 18). The basal lamina is well developed in the adult worm; it consists of a granular layer about 50 nm in width and it is separated from the basal membrane of the tegument by about the same distance (Fig. 21). The fibrous interstitial material forms a thick layer beneath the basal lamina in regions of the worm which have tegumental spines. In regions where spines are absent the interstitial material is mainly located between or beneath the outer, circularly arranged muscle fibres. The fibres of the interstitial material are approximately 5 nm in diameter and they are more regularly arranged in the adult worm than in the cercaria (Fig. 21). Similar microfibrils have been seen in the extracellular space of vertebrate tissues (Haust, 1965) but the nature of the interstitial fibres is unknown. The basal lamina and interstitial material probably have a skeletal function as has been described for the cercaria. No desmosomes have been reported which might attach the basal membrane of the tegument to the underlying tissues. The musculature of the adult worms has been studied by Silk and Spence (1969a) and Reissig (1970). The muscle fibres are larger in the adult worm than in the cercaria and schistosomulum and they are also better developed in the male worm than in the female. The fibres consist of irregularly arranged thick and thin filaments, as in the body of the cercaria. At the periphery of the fibres, there are numerous electron-dense regions which are often associated with a small invagination of the sarcolemma, these regions probably represent the site of attachment of the thin filaments to the sarcolemma. Adjacent to the dense regions, there are tubular elements of sarcoplasmic reticulum. The muscle fibres are typically arranged in circular and longitudinal layers with some additional radially arranged fibres, particularly in the male worm. The radial fibres are prominent in the suckers and are often closely associated with spines. The non-contractile, nucleated part of the muscle cell is joined to the fibrillar portion by a long, tenuous connection, similar to the subtegumental cell connections. The fibre may be sub-divided into several fibrils which are all connected to one cell body. Reissig (1970) found that there are two types of muscle cell bodies. One type is large and has a clear nucleus with a single nucleolus, many free ribosomes and characteristic, large, membrane-bounded vacuoles in the abundant cytoplasm. The second type of muscle cell body is smaller, has a nucleus with clumped chromatin and has no cytoplasmic vacuoles. Reissig (1970) did not suggest any functional difference between the two types of muscle cells. The muscle cells contain numerous, large mitochondria with well-developed cristae. Most of the glycogen in the worm is located in the muscle cells. There are particles of p-glycogen between the muscle filaments and also in sac-like extensions of the sarcoplasm adjacent

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to the filaments. Some or-glycogen particles are found in the deeper sarcoplasm. Silk and Spence (1969a) and Reissig (1970) have shown that the cells beneath the tegument in the dorsal tubercles of male worms are sarcoplasmic processes which probably form a glycogen store. 6 . Sensory organelles The sensory organelles at the surface of adult worms have been described in detail by Morris and Threadgold (1967) and Silk and Spence (1969b) and mentioned briefly by Smith et al. (1969) and Hockley (1970). The organelles are all of one type and they have the same basic structure as the uniciliate papillae of the cercaria. Figure 24 is a diagram of the adult sensory organelle. Sensory organelles are present on all parts of male and female worms but are particularly abundant at the anterior end of the male gynaecophoric canal. The bulb-like nerve ending of the organelle is larger than in the cercaria (approximately 2 pm in diameter); it is completely embedded in the tegument but causes a slight protrusion. Unlike the cercaria, the apical cilium is also completely covered by a thin layer of tegument even though it projects at least 2 pm beyond the general body surface. Smith et al. (1969) reported that the cilium reaches the exterior through an opening in the tegument, as in the cercaria, but this has not been confirmed. The circumferential septate desmosome and two associated dense, reticulate rings are present round the base of the cilium. Silk and Spence (1969b) described satellite bodies connected to the basal body by radiating arms but these structures have not been seen by other workers. The bulbous nerve ending of the adult organelle differs from the cercarial nerve ending in that it contains numerous granules resembling glycogen, one or more large, well-developed mitochondria and at its base, invaginations from the tegument. It also contains granular material, vesicles and microtubules. The sensory organelles of schistosome adult worms are similar in structure to the sensory nerve endings that have been found on other digenetic trematodes. The adult organelles probably function as mechanoreceptors as has been suggested for the cercaria. The tegumental covering of the cilium would probably not prevent mechanical movement of the cilium but it would seem to preclude a chemoreceptive function. Simple contact stimuli are probably important to worms which must become paired before they mature and which must migrate into small blood vessels to lay their eggs. Morris and Threadgold (1967) suggested that the organelle might detect the direction of flow of a fluid medium but the radial symmetry of the organelle is not compatible with directional sensitivity. If, however, the ring-like structures round the ciliary basal body are, in fact, semi-circular as described by Silk and Spence (1969b) then this might be a mechanism to provide directional sensitivity. Silk and Spence (1969b) have demonstrated that the sensory receptors are continuous with nerve processes. In their study of the nervous system they found nucleated cell bodies only in the circumoesophageal commissure. Reissig (1970), however, examined the types of cells in the parenchyma and found nerve cells which are connected to nerve fibres. The presence.or absence of nerve cells in the body of the worm needs to be confirmed since Reissig

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FIG. 24. Diagram of a sensory organelle of an adult worm. bb=basal body. c=cilium. gl =glycogen. hm = heptalaminate tegumental outer membrane. m = mitochondrion. mt = microtubule. np = nerve process. rr = reticulate dense ring. sb= satellite body. sd = septate desmosome. t = tegument. ti = tegumental invagination. v = vesicles.

suggested that the only cells in the parenchyma are nerve, muscle and subtegumental cells and that there is no true parenchymal cell. 7 . Specialized regions of the tegument-the oesophagus and the uterus Two important structures in the adult worm (a) the oesophagus and (b) the uterus are formed from the tegument. Spence and Silk (1970, 1971) stated that the entire gut and female reproductive system are extensions of the tegument. There are, however, well defined limits to the oesophagus and uterus and both these structures have fundamental differences from the

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remainder of the alimentary tract and reproductive system. Thus, for the purposes of this review, only the oesophagus and uterus will be regarded as tegumental structures. These regions of the tegument have a different structure from the normal body tegument. The oesophagus of S. mansoni has been described in detail by Dike (1971) and has been mentioned briefly by Morris and Threadgold (1968) and Spence and Silk (1970). The thin, spiny tegument of the oral sucker of both male and female adult worms extends into the oral opening where it becomes more deeply pitted and folded. The anterior part of the oesophageal tegument has the same basic structure as the body tegument; it has a heptalaminate outer membrane, dense, granular cytoplasm and the basal membrane has the associated lamina and muscle fibres. The surface pits and folds, however, became extremely elaborate so that the whole oesophageal lumen appears to be filled with long, branched and anastomosing tegumental folds (Fig. 25). Often there are small amounts of extracellular, vesicular material trapped between the folds. Long invaginations of the basal membrane extend into the folds. Occasionally the invaginations are expanded to form vacuole-like structures which contain small granules or vesicles. Membraneous bodies are very numerous in the cytoplasm but there are comparatively few elongate bodies. Small vesicles are also present. The luminal tips of the folds have the form of large microvilli and they contain many small vesicles and numerous mitochondria. Dike (1971) found that, in places, the small vesicles are so numerous that they form a reticulum in the cytoplasm. The subtegumental cells of the anterior region of the oesophagus have the same contents and basically the same structure as the subtegumental cells of the body tegument. They are, however, relatively larger and more numerous and the connections are shorter and broader. In the middle region of the oesophagus the tegumental folds are more uniform and regular in size and have few branches (Fig. 26) and they are very closely and regularly arranged. Spence and Silk (1970) suggested that the tegument in this region is formed into loops, rather than simple folds but Bruce et al. (1971) examined 27-day-old worms, in which the tegumental folding in the oesophagus is less pronounced and they found no evidence of loops. Small amounts of lamellated or crystalline material are present between the folds, in the oesophageal lumen. The folds have a heptalaminate outer membrane. The cytoplasm is less electron-dense than in the anterior oesophagus. A long invagination of the tegumental basal membrane extends almost to the tip of each of the folds. Elongate bodies, small vesicles, mitochondria and microtubules are present within the folds. Membraneous bodies are not present but a third type of dense, spherical or ovoid inclusion is found in this region of the oesophagus. These bodies are about the same size as the membraneous bodies and they often cause a surface protrusion of the tegumental folds. They are membrane-bounded and have homogeneous contents, although Spence and Silk (1970) observed a banded appearance. Shannon and Bogitsh (1969) examined the digestive tract of Schistosomatium dourhirri and found a characteristic muhivesicular body in the oesophageal tegument. The subtegumental cells of the mid-oesophagus of S. rnansoni

FIG. 25. Extremely folded tegument (tf) of the anterior region of the oesophagus. FIG.26. More regular tegumental folds of the middle region of the oesophagus. The folds contain long invaginations of the basal membrane (i), spherical bodies (sb) and elongate bodies (eb). Between the folds there are small amounts of lamellated material (lm). FIG. 27. Irregular folds and microvilli with short, blunt projections in the posterior oesophagus. The long cell junction (cj) with the caecal syncytium (cs) has an apical septate desmosome (sd). Thin folds of the caecal syncytium project into the caecal lumen (cl).

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are large; they are multinucleate and contain an extensive endoplasmic reticulum and also the homogeneous or banded bodies. Morris and Threadgold (I 968) suggested that these cells represent what is classically known as the oesophageal gland. Dike (1971) described a side pocket in the middle region of the oesophagus. The pocket is lined with the same type of tegumental folds as those found in the main part of the oesophagus. The folds are continuous from one side of the pocket to the other and are themselves folded in an angular pattern. This angular folding is also seen in the more posterior part of the mid-oesophagus and in this region the luminal tips of the tegumental folds are swollen. In the most posterior part of the oesophagus the tegumental folds are less regularly arranged and they progressively become more like microvilli (Fig. 27). The folds and microvilli are long and thin with numerous, short, blunt projections. The tips of the microvilli are often swollen. The homogeneous, dense bodies are present in the folds. The oesophageal lumen is wider in the posterior region and often contains large masses of laminated material and also membraneous and homogeneous material. The junction between the oesophagus and the gut caecum is marked by a septate desosome which extends for several microns along the distal parts of the apposed tegumental and caecal syncytium membranes. The tegument lies over the caecal syncytium so that a long cell junction is formed. Spence and Silk (1970) have detected gaps in this cell junction so that the tegumental and caecal cytoplasm appear to be continuous. Similarly, Shannon and Bogitsh (1969) found no clear demarcation of the oesophagus and caecum in S. dourhitti. Hockley (19701, however, described the membranes as forming a complete junction in S . mansani and other workers have assumed that it is a true cell junction (Dike, 1971 ;Morris and Threadgold, 1968). This conclusion seems reasonable in view of the major differences between the caecal syncytium and the tegument which will be described later. The elaborate tegumental specializations of the schistosome oesophagus presumably function in the ingestion and digestion of host blood cells. Host erythrocytes and leucocytes are found in the lumen of the oesophagus, usually in various stages of digestion. The elaborate folds of the anterior oesophageal tegument appear to be capable of completely occluding the oesophageal lumen and, together with the underlying musculature, they may assist in the uptake of blood cells. There is also some evidence that the anterior oesophagus produces a secretion (Dike, 1971). The characteristic tegumental inclusion bodies of the middle region of the oesophagus are almost certainly secretion granules which are produced in the large subtegumental cells, or oesophageal glands, and passed into the tegument from where they are secreted. The lamellated material in the lumen of the oesophagus may represent secretion derived from the bodies, as suggested by Spence and Silk (1970) or it may possibly be a product of haemoglobin digestion (Dike, 1971). Bogitsh and Shannon (1971) examined the oesophagus of S. mansoni and S. dourhitti for acid phosphatase activity. Some reaction product was found in the basal membrane invaginations and also in large vesicles which occur in the oesophageal tegument and particularly in the subtegumental cells. No

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activity was found in the characteristic inclusion bodies. Bogitsh and Shannon (1971) concluded that the enzyme is located in autophagic vesicles which are known to occur in synthetically active cells. From their study of 27-day-old worms of S. munsoni, Bruce et ul. (1971) suggested that the oesophagus is primarily concerned with digestion and that the role of the caecum is primarily absorption and egestion. Whether or not the whole of the important digestive function is restricted to the comparatively small though complex oesophagus remains to be determined. Comparative studies on the pharynx and oesophagus of other digeneans might help in answering this question. The second specialized region of the adult tegument to be described is the uterus of the female worm. The uterus has not been examined in such detail as the oesophagus and it does not represent such a modification of the body tegument. Spence and Silk (1971) in a preliminary report on the female reproductive system state that the narrow tegument of the body broadens and invaginates to form the gonopore. The tegument is extensively folded in this region and continues posteriorly to form the uterus. The surface folds and pits of the uterine tegument are characteristically much larger than those of the body tegument, particularly in the posterior uterus. Unpublished observations (Hockley) suggest that, compared with the body tegument, the cytoplasm of the uterine tegument is more electron-dense and that inclusion bodies occur more sparsely. The basal lamina and associated interstitial material are less well developed. Subtegumental cells connected to the uterine tegument are present (Spence and Silk, 1971). From these observations it would appear that the uterus is a relatively inactive structure simply for the storage or passage of eggs and sperm. The large surface folds may indicate that the lumen is capable of considerable changes in volume. Spence and Silk (1971) suggested that, although there is a prominent septate desmosome connecting the uterine tegument to the ootype syncytium, the cytoplasm of the two structures is continuous and therefore the whole female reproductive system can be considered as a tegumental structure. However, the syncytial linings of the ootype and oviduct contain nuclei and have no connections to deep cell bodies, unlike the tegument. For the present, this basic difference is perhaps a justification for regarding the reproductive system as non-tegumental. B.

COMPARISON OF THE TEGUMENT AND THE CAECUM

It has often been suggested that the schistosome tegument is an absorptive surface. Schistosomes, like other trematodes, however, have a large intestinal caecum which is almost certainly an absorptive structure. Comparison of the tegument and the caecal epithelium might reveal features of the tegument concerned with absorption and also indicate the relative importance of the two structures as absorptive surfaces. The ultrastructure of the caecum has been described by several authors (Morris, 1968; Senft, 1969; Spence and Silk, 1970; Bruce et af., 1971 ; Dike, 1971) and there is general agreement in the results although the speculations

TABLE I11 Comparison of the tegument and the caecum TEGUMENT

SUBTEGUMENTAL CELLS

CAECAL SYNCYTIUM

Surface area increased by thin folds Surface area increased by pits approximately approximately 120 x (Halton, 1966) 10 x Thin surface coat on lumenal membrane No distinct surface coat on outer membrane Trilaminate lumenal membrane 10 nm in Heptalaminate outer membrane 17 nm in thickness thickness Cytoplasmic matrix not prominent Dense, granular, cytoplasmic ground substance A few crystalline inclusions Crystalline spines Numerous, well-developed mitochondria Few, small mitochondria Numerous, characteristic, membrane-bounded inclusions Some vacuoles with granular contents Nuclei only in subtegumental cells Numerous, large nuclei Full of ribosomes with extensive Ribosomes only in subtegumental cells; few organized on endoplasmic endoplasmic reticulum reticulum Golgi bodies only in subtegumental cells Some Golgi bodies with associated vacuoles Long invaginations of the basal membrane Invaginations of the basal membrane Underlying basal lamina, interstitial Underlying basal lamina, interstitial material and single layer of muscle fibres material and muscle fibres Junctional complexes between base of Junctional complexes between syncytium and parenchymal cells subtegumental cells and other parenchymal cells

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about functional aspects are more controversial. The observations on the caecum will not be described in detail but the main points of similarity and difference between the caecal epithelium and the tegument, including the subtegumental cells, are listed in Table I11 and then discussed below in relation to absorption. The major similarity and difference can be stated first; both structures are syncytial but the anucleate tegument is connected to nucleated subtegumental cells, whereas the caecal syncytium consists of a single layer of cytoplasm which contains nuclei. The relatively greater surface area of the caecal syncytium indicates that there is more transport of material through the caecal surface than through the tegumental outer membrane. The different meachanism of increasing the surface area may be related not only to the amount of increase but also to the differences between the external environment of the worm and the internal environment of the caecal lumen. The significance of the surface coat on the caecal membrane is unknown but surface coats are prominent on the intestinal epithelium of many organisms. The typical trilaminate membrane is also found on intestinal cells and these two features can thus be related to an absorptive function. Conversely, the thickened tegumental outer membrane is an unusual structure and is certainly not a feature associated with absorptive cells. Thickened membranes, together with electron-dense, cytoplasmic ground substance, are prominent in inactive, resistent cells such as keratinizing epithelium (Farbman, 1966). The caecal syncytium contains large mitochondria, nuclei, Golgi bodies, many ribosomes and an extensive endoplasmic reticulum which is often swollen; it is clearly, therefore, a metabolically active structure although the nature of the metabolism is unknown (Senft, 1969). These organelles are absent from the tegument and are only present in the subtegumental cells so that the total volume of active cytoplasm is much less than that of the caecal syncytium. The subtegumental cells appear to have the specific function of producing inclusion bodies which are passed into the tegument; there is no evidence that the cells receive any material from the tegument. The basal membrane invaginations are present in the tegument and in the caecal syncytium but they are usually longer and straighter in the caecum. If the invaginations are involved in the transport of substances then it is to be expected that they would be well developed in the caecal syncytium. Morphologically the caecal syncytium appears as a metabolically active structure with a large surface area which is indicative of an absorptive function. The tegument appears as a more resistant and inactive structure and is therefore probably less important than the caecum as an absorptive surface. The mechanisms of digestion and absorption in the caecum are not understood, but Fripp (1967a, c) and Senft (1969) have suggested that proteins are digested in the caecum and that amino acids or peptides are taken up by pinocytosis while free amino acids in the blood and other small, diffusible molecules, such as glucose, may be taken up through the tegument. C. DESTRUCTION OF THE TEGUMENT

One of the major functions of the tegument must be to protect the worm against unfavourable aspects of the environment. Some of the protective

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mechanisms of the tegument might be revealed by examination of worms subjected to unusual conditions and the appearance of the damaged tegument might effectively demonstrate further details of the normal structure. As yet there have been few ultrastructural studies of the tegument under severely artificial or pathological conditions. 1 . Efect of hypotonic and hypertonic media Smith et al. (1969) incubated worms in vitro for periods of up to 90 min in isotonic phosphate buffered saline, normal human serum and hyperimmune serum. All three media result in similar degenerative changes. There is a progressive disappearance of the tegumental membraneous bodies and the surface pits in the tegument become enlarged. There is also a profound extracellular oedema in the parenchymal tissues which straightens the connections between the subtegumental cells and the tegument and so produces a better demonstration of the connections than is possible in normal worms. Hockley (1970) examined worms maintained in hypotonic and hypertonic media. In the hypertonic medium the tegument becomes shrunken. The normally circular and open channels of the surface pits appear slit-like and folded and the cytoplasm becomes very electron-dense, so that the inclusion bodies are barely recognizable. There are, however, large, round, empty vacuoles in the basal region of the tegument and these appear to be formed from swollen basal membrane invaginations. In a hypotonic medium the surface pits remain as large, open channels but again the cytoplasm becomes very electron-dense and the basal membrane invaginations are swollen. The invaginations become so enlarged that the tegument is almost separated from underlying tissues and in some regions of the worms, the tegument is lost and the muscle fibres are exposed to the exterior. The increase in density of the damaged tegument and the corresponding disappearance of the tegumental elongate bodies may be further evidence that the bodies give rise to the tegumental ground substance. The basal membrane appears to be particularly sensitive to osmotic stress. Even in so-called normal worms the invaginations are occasionally enlarged at their ends (Morris and Threadgold, 1968). This may be a normal appearance or it may be a result of the procedure that is necessary to recover worms from the host. The response of the invaginations to the osmotic conditions of the environment can, perhaps, be correlated with the suggestion of Shannon and Bogitsh (1969) that the invaginations are associated with the movement of fluid or ions.

2. Efect of drugs The few observations on the effects of drugs on the tegument of schistosomes have mostly been made with the light microscope. Vacuolation of the tegument of S. mansoni caused by Miracil D was observed in GiSnnert (1955b). Bueding et al. (1967) found that a subcurative dose of tris (paminophenyl) carbonium chloride administered to mice infected with S. mansoni produces a reduction in the amount of glycogen in the dorsal tubercles of male worms. This results in the flattening and subsequent

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disappearance of the tubercles but there is no direct effect on the tegument. Standen (1962) examined the effect on 1 :7-bis (p-aminophenoxy) heptane on 5’.mansoni in mice and found marked changes in the tegument. The earliest effect is the formation of small, surface outgrowths from the tegument followed by increasing tegumental vacuolation and larger, balloon-like, surface exudates. Phagocytic cells become associated with the exudates and, as degeneration of the tegument continues, the phagocytes penetrate the tegument and eventually invade all the tissues. Standen (1962) interpreted these observations as showing that the drug exerts a profound effect on the permeability and physical structure of the tegument. This, he suggested, affects the uptake of nutrients by the parasite and leads to the rapid senescence of the worm. The eventual breakdown of the tegument, he suggested, alters the status of the host-parasite relationship and permits the invasion of host phagocytic cells. EM examination of these tegumental changes would provide further details of the tegumental exudates and their association with phagocytic cells. A preliminary EM examination of worms recovered from a monkey 1 h after treatment with Triostam (sodium antimonygluconate) has shown balloon-like swellings of the tegument at its outer surface (Hockley, 1970). The swollen parts of the tegument are 3-4 pm in diameter and have a narrow junction with the more normal tegument beneath. The outer membrane of the tegument is continuous over the swollen parts but the tegumental inclusions do not extend into the swellings. Possibly the balloon-like exudates observed by Standen (1962) have a similar structure. Vacuolation of the tegument of F. hepatica has been observed after treatment of the host with Bithionol (Dawes, 1966a, by 1967) or hexachlorophane (Thorsell and Bjorkman, 1966). However, Dawes (1968) concluded finally that such vacuolation was due to the moribund condition of injured drugtreated flukes. Dawes (1963, 1964) also showed that invasion of the tegument by phagocytes occurs in specimens of F. hepatica weakened by X-irradiation and suggested that this may be the typical fate of injured trematodes. Further ultrastructural studies on the effects of drugs on the schistosome tegument would be of interest, in particular the effect of dibenzylamines which are known to interfere with glucose transport or absorption (Bueding, 1962). 3. Eflect of’host immunity The effects of the host on the ultrastructure of the schistosome tegument have received little attention even though the worms live in the blood stream where they are constantly exposed to the immune defences of the host. In fact, S. mansoni is able to live in this environment for a considerable period of time apparently unaffected by host immunity (Smithers and Terry, 1967, 1969). A possible mechanism to explain this evasion of the immune response has been suggested by Smithers ef al. (1969). They found that when worms grown in mice are transferred into the hepatic protal systems of normal monkeys, they survive well, but when “mouse” worms are transferred into monkeys that have been previously immunized against mouse erythrocytes, then the worms are rapidly destroyed. This indicates that “mouse” antigens

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become closely associated with worms grown in mice and that, on transfer to an anti-mouse monkey, the “mouse” worms are destroyed by the ensuing immunological reaction. Tt was suggested that the host-type antigens serve to mask the parasite antigens of the worm thus enabling the worm to escape recognition by the host. The location of the host antigens was studied with the EM using ferritinlabelled monkey anti-mouse serum (Smithers et al., 1969). The labelled antibodies became attached evenly over the whole surface of mouse worms indicating that the host antigens are either in or on the tegumental outer membrane. The experiments were not capable of revealing if the host antigens are also present within the tegument or other cells of the worm. Further unpublished experiments (McLaren and Hockley) using fragments of worms and antibodies labelled with 125 iodine or peroxidase have indicated that host antigens are restricted to the tegumental outer membrane. The destruction of mouse worms in anti-mouse monkeys was also studied with the electron microscope by Smithers el al. (1969) and Hockley (1970). Mouse worms were transferred to monkeys immunized against mouse tissues and recovered after 2h, 7 h, 25 h or 44 h. Damage is confined to the tegument at least until parts of the tegument have been destroyed. The observed appearances of the damaged tegument can be placed in a probable sequence of occurrence. There is, however, considerable variation in the timing of events since some small regions of the tegument are lost as early as 2 h and even in extensively damaged worms there are some regions of the tegument that appear normal. One of the earliest signs of damage is vacuolation of the basal region of the tegument. This appears to be followed by another characteristic damaged appearance : the tegument becomes full of vacuoles which contain numerous, small, spherical bodies. The later stages of damage consist of breaks in the outer membrane which leads to gross tegumental vacuolation and loss of tegumental material, so that finally whole portions of tegument are lost and the underlying muscle fibres are exposed to the surface. It is only at this final stage of damage that host phagocytic cells become closely associated with the worms in the regions where the tegument is lost. Per& and Terry (1973) have examined the effect of monkey anti-mouse erythrocyte serum on mouse worms in vifro. The tegument shows many of the features seen in worms damaged in vivo but there are also characteristic changes in the tegumental outer membrane, which may be one of the earliest stages of destruction. The surface of these worms is covered with a thin coat of precipitated material and there is some evidence that the heptalaminate membrane is delaminating. The most marked change, however, is the formation of microvilli or projections of the tegumental outer membrane (Figs 28 and 29). The projections appear to be cast off from the tegument but this does not immediately result in breaks in the outer membrane. I n these specimens the tegumental membraneous bodies are often closely associated with the outer membrane and are even incorporate3 in the membrane (Fig. 30), whereas in normal worms the bodies are only occasionally found close to the outer membrane. This is further evidence that the membraneous bodies contribute to the outer membrane and replace lost or damaged membranes. 11

Fig. 28. Adult worm from a mouse maintained for 8 days in monkey anti-mouse erythrocyte serum. The tegument (t) is covered with microvilli (rnv) and fragments of membrane. FIG.29. As above: higher magnification of microvilli (mv) showing the heptalaminate outer membrane and the thin coat of precipitated material (pm). FIG.30. As above: niembraneous body (mb) between the two halves of the heptalaminate outer membrane.

FIG.3 I. Adult worms transferred to a hyperimmune monkey and recovered after 48 h. The ends of the basal membrane invaginations (i) are swollen to form basal vacuoles (bv) in the tegument (t). FIG.32. As above: tegument breaking down to form small, membrane-bounded, spherical bodies (sb).

FIG.33. Mouse worm maintained in monkey anti-mouse erythrocyte serum. The tegument has completely broken down leaving the basal lamina (bl), interstitial material (im) and muscle fibres (mf) exposed to the exterior. FIG.34. Host leucocyte ( I ) on part of the surface or a worm that has lost the tegument.

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Eventually the tegument of the worm breaks down, as observed in vivo, and invasion of phagocytes occurs (Figs 33 and 34). The effect of naturally acquired immunity on adult worms has been investigated by Hockley and Smithers (1970) who used the rhesus monkey which becomes naturally immune to reinfection after a primary infection. Adult worms were transferred into the portal system of a hyperimmune monkey (Smithers and Terry, 1967) and recovered 48 h later. All stages of damage from a near normal appearance to the localized loss of the tegument can occur in one worm. The mildest recognizable form of damage is an increase in density of the tegumental matrix in the outer part of the tegument. This is followed by an increase in density of the whole tegument. Basal vacuolation is very prominent; the vacuoles are formed from the basal membrane invaginations (Fig. 31). A few small, spherical bodies occur in the basal vacuoles and in vacuoles in the apical tegument. These small bodies are similar to those seen in the tegument of mouse worms recovered from a monkey immunized against mice. The bodies have a limiting membrane and granular contents which are derived from the tegumental matrix (Fig. 32). The small bodies are released to the exterior where there are breaks in the outer membrane and in these regions the tegument is extensively vacuolated. Host leucocytes are also associated with the breaks in the outer membrane and are most numerous where the tegument is completely lost; they are closely attached to the underlying muscle fibres and also appear to migrate under the tegument adjacent to the damaged region. Damage appears to be confined to the tegument of the worms transferred to a hyperimmune monkey for at least 48 h. Worms transferred to a normal monkey for 48 h and then recovered have a few small swellings of the basal membrane invaginations but otherwise appear normal. There are certain features in common between the forms of tegumental damage produced by the transfer of mouse worms into anti-mouse monkeys or the maintenance of mouse worms in monkey anti-mouse serum in v i m (i.e. by an anti-host antigen host) and those produced by a hyperimmune host. The antigens involved are probably all located in the tegumental outer membrane and the initial damage may be due to changes in the membrane resulting from the antigen antibody reaction. The EM techniques of negative staining and freeze-cleaving might be employed for further study of the effects of antibodies on the outer membrane. After the initial changes in the outer membrane (Figs 28-30) it is probable that the basal region of the tegument becomes vacuolated (Fig. 31). This is followed by breakdown of the tegumental cytoplasm (Fig. 32), loss of portions of the tegument (Fig. 33) and, finally, invasion at these points by host leucocytes (Fig. 34). It is only after the loss of the tegument that the host phagocytic cells are capable of invading the worm, which indicates that the tegument is important in maintaining the relatively benign host-parasite relationship. V. THEMIRACIDJUM AND SPOROCYST Miracidia are covered with discrete ciliated cells and the surface layer is therefore referred to as an epithelium rather than a tegument. It is of interest

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to compare this epithelium with the syncytial tegument of the other stages of the life cycle and to examine the possible reasons for the difference and the subsequent fate of the cells in the sporocysts. A.

THE EPITHELIUM A N D ASSOCIATED STRUCTURES OF THE MIRACIDIUM

The published ultrastructural studies on schistosome miracidia are all brief reports on small aspects of the organism. Kinoti (1971) examined the anterior end of S. mansoni and S. mattheei miracidia and discussed the ultrastructure in relation to attachment and penetration. Brooker (1972) has described the sense organs of S. mansoni miracidia and has also examined the apical papilla and the ciliated epithelium of the body (quoted by Wright, 1971). Jamuar and Lewert (1967) examined the effect of immune serum on the surface of the miracidium of S. japonicum. The epidermal cell covering the apical papilla has an elaborate surface of branched and anastomosing folds. Kinoti (197 I ) described these processes as microvilli and suggested that they might make a close fit only with the surface of the normal snail host; thus the microvilli would aid attachment and penetration of the correct host and might, therefore, be a factor in determining host specificity. Brooker (Wright, 1971) however, recognized that the microvilli are, in fact, folds and he suggested that the interconnected folds form numerous sucker-like cups which assist in the attachment of the miracidium to the snail. He found no evidence of a more specific interconnection between the papilla surface and the snail epidermis. Beneath the epidermal cell of the apical papilla there are the anterior ends of the flask-shaped apical gland and penetration glands. Figure 35 is a diagram of the body wall of the schistosome miracidium based on Wright (1971). The large ciliated cells over the body of the miracidium are separated from each other by smaller non-ciliated cells. The cells are joined by septate desmosomes. The cilia are arranged in rows and have the typical 9 + 2 arrangement of microtubules and a basal body with a welldeveloped rootlet. The rootlet has cross striations and lies at an angle to the shaft of the cilium (Jamuar and Lewert, 1967). Both types of cells are covered with microvilli which, on the ciliated cells, lie between the rows of cilia. Beneath the epithelial cells there are circularly and longitudinally arranged muscle fibres. The nucleated parts of both types of epithelial cells lie beneath the muscle fibres in a manner similar to the subtegumental cells of schistosomula and adult worms. The nucleated cell bodies are joined to the outer parts of the epithelial cells by narrow cytoplasmic connections; they contain numerous mitochondria. The sensory organelles of S. mansoiii miracidia are mostly associated with the apical papilla and the non-ciliated epithelial cells. Brooker (1972) has recognized a variety of types of organelles. Around the apical papilla there are simple ciliated nerve endings and ciliated pits which may be tangoreceptors and chemoreceptors respectively. Laterally on the body there are multiciliated papillae and non-ciliated papillae which contain numerous small

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FIG.35. Diagram of the epithelium and some associated structures of the miracidium of S. mansoni.c = cilium. ce =ciliatedepithelial cell. mv = microvilli.nce = non-ciliatedepithelial cell. ne= nucleated parts of epithelial cells. r=rootlet.

vesicles. The vesicular papillae are c0vere.l by an epithelial cell and have no direct contact with the environment, but they are always associated with a ciliated papilla and the two papillae may possibly function together. S. rnansoni miracidia respond to light but they do not have the eyespots which are found in other species of miracidia (Isserhoff and Cable, 1968) including other schistosomes. There is, however, a cell situated below the body wall which contains a large vacuole. Several cilia project into the vacuole and long evaginations of the ciliary membranes form stacks of lamellae in the vacuole. Brooker (1 972) suggested that these organelles are photoreceptors which may be particularly sensitive to low levels of illumination. Sensory organelles similar to all these types have been found in various other species of miracidia (Brooker, 1972) and the miracidial sensory papillae and pits are almost identical in structure to the sensory organelles of cercariae (MatriconGondran, 1971). Jamuar and Lewert (1967) found that, when S. juponicurn miracidia are exposed to the serum from an infected rabbit, the outer parts of the epithelial cells become swollen and covered with a coat of amorphous material. The cilia also become swollen and the microtubules become extremely bent and coiled. These results confirm and extend light microscopical observations on the immobilizing effect of immune serum (Senterfit, 1953) and they also suggest that there might be a mucosubstance at the surface of the miracidium which reacts with immune serum in a similar manner to the surface coat of the cercaria. Senterfit (1953) found that the immobilizing factor appears in serum at the same time as CHR activity and Oliver-GonzBlez el al. (1955) suggest that the cercarial component of the CHR is also present in the egg. In several respects the epithelium of the miracidium is similar to the cer-

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carial tegument, as might be expected since they both live in the same environment, Both probably have a surface coat and they both have subepithelial or subtegumental cells. They also have similar associations with sensory organelles and with the underlying muscle fibres. The main differences between the miracidium and the cercaria are the cilia and the cell boundaries which are only present in the epithelium of the miracidium. The cilia are required for locomotion and possibly the cellular structure is related to the presence of the cilia. The cellular epithelium may be necessary for coordination of the cilia and to provide a firm anchorage for the powerful ciliary movements. The miracidium of F. hepatica has been studied in detail by Wilson (1969) and Southgate (1 970) and this species provides an interesting comparison with Schistosoma. The apical papilla has a corrugated surface, unlike the complex folds on the apical appilla on the schistosome miracidium, and the contents of the apical gland are passed into the surface layer of cytoplasm. The accessory glands, which probably correspond to the penetration glands of schistosome miracidia, secrete their contents directly to the exterior. Wilson (1969) found that the surface of the miracidium can be stained with alcian blue and deduced that there is a thin acid mucopolysaccharide surface coat. The major difference between the two species is that the ciliated epithelial cells of F. hepatica contain a nucleus and are not connected to nucleated cell bodies beneath the muscle fibres. The ciliated cells have no microvilli but there are a few knob-like projections at the surface. A non-ciliated, cytoplasmic ridge is present between the ciliated cells of F. hepatica but, unlike Schistosoma, the ridge is a syncytium. It contains membraneous vesicles and is connected to nucleated cell bodies which also contain membraneous vesicles. In addition to the connections to the intercellular ridge, the cell bodies have processes which extend between the muscle fibres and terminate beneath the ciliated epithelial cells. Wright (1971) believes that the differences between the epithelial cells of Schistosoma and F. hepatica are fundamental rather than an adaptation to special conditions. Related to this difference is the fact that schistosome miracidia do not lose their ciliated cells when they penetrate the snail host (Wajdi, 1966) whereas the fasciolid larvae cast off their ciliated epithelial cells during penetration (Mattes, 1949; Dawes, 1960). The schistosome epithelium with its subepithelial part is clearly not designed to be shed (Wright, 1971). There have been no published ultrastructural studies of the outer layers of the schistosome mother sporocyst but Southgate (1970) and Wilson et al. (1971) have described the loss of the ciliated cells of F. hepatica miracidia and the formation of the mother sporocyst tegument. At first, vacuoles appear beneath the basal membrane of the ciliated cells and they enlarge so that cell junctions are broken and the cells are eventually separated from the underlying tissues. The cells are lost sequentially from the anterior end as the miracidium penetrates the snail epidermis (Wilson el al., 1971). After the loss of the ciliated cells, the non-ciliated intercellular ridges enlarge and spread out over the surface of the miracidium. At the same time more membraneous vesicles are passed up to the surface cytoplasm from the subepithelial cell

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bodies. Southgate (1970) suggested that the vesicles may form new outer membrane. Within 23 h after penetration the ridges fuse together and to the other processes of the subepithelial cell bodies to form a thin syncytial tegument covering the mother sporocyst. The tegument finally increases in thickness, mitochondria increase in number and the surface becomes covered with small, branched folds. Southgate (1970) called the outer covering of the fasciolid mother sporocyst a tegument because it is syncytial and has the same basic structure as the tegument of the adult worm. Possibly the epithelium of the S. mansoni miracidium also forms a syncytial tegument on the mother sporocyst. The cilia are probably lost since they are no longer necessary for locomotion and the junctions between the epithelial cells may break down since firm attachment between the cells will be less important once the cells have lost their locomotory junction. The microvilli on the surface of the miracidium almost certainly will remain to increase the surface area of the mother sporocyst. Further ultrastructural studies will ascertain whether or not the two different types of miracidial epithelium of the schistosomes and fasciolids result in similar teguments on the mother sporocyst. B. THE TEGUMENT OF THE DAUGHTER SPOROCYST

Daughter sporocysts of Schisrosorna have not been extensively examined with the EM. A few observations on the sporocysts of S. mattheei and S. bovis have been published by Kinoti et a/. (1971) and Rifkin (1970) and Hockley (unpublished) made some observations on S . mansoni sporocysts while studying developing cercariae. Hansen and Perez-Mendez (I 972) used the scanning EM to examine S. mansoni daughter sporocysts; they recorded the presence of a few posteriorly projecting spines at the anterior end of the specimens. The spines decrease in number along the length of the body and are absent at the posterior end. The scanning EM also shows that the daughter sporocysts have an extremely irregular surface. The transmission EM studies have shown that the surface is covered with microvilli. The microvilli have a variable appearance and are irregularly arranged; they often form a thick (1 pm) sponge-like layer on the surface of the sporocyst. The microvilli arise from a continuous surface layer of cytoplasm, which will be called the tegument. The tegument is approximately 0.5 pm in thickness and again variable in appearance: it usually contains a few mitochondria and scattered groups of ribosomes. Beneath the tegument there is a thin basal lamina and a few small muscle fibres. Rifkin (1970) recorded an extensive muscular system of outer longitudinal and inner circular layers and he also found an excretory system of flame cells. There are also present in the body wall of the daughter sporocyst large cells containing granular endoplasmic reticulum, prominent Golgi complexes and large nuclei. Some of these cells are continuous with the tegument. The observations on the schistosome daughter sporocyst are summarized in Fig. 36. The daughter sporocyst has the typical syncytial trematode tegument connected to the nucleated subtegumental cells. The variable appearance of the

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FIG.36. Diagram of the tegument and some associated structures of the daughter sporocyst of S.munsoni. mv= microvilli. stc= subtegumental cells. t = tegument.

sporocyst tegument probably depends on the age or degree of degeneration of the sporocyst. The wall of older sporocysts, which contain fully developed cercariae, simply consists of a thin tegument and a few thin underlying cells. The younger sporocysts probably have a thicker tegument with numerous microvilli and an extensive muscular system. Observations on daughter sporocysts within the mother sporocyst, would provide details of the normal structure and, in addition, information about the development of the daughter sporocysts. Further studies are also required on the relationship between the daughter sporocyst and the developing cercariae contained within it, as has been mentioned in the section on cercarial development. Dusanic (1959) and Kinoti et ul. (1971) performed light microscope histochemical staining experiments on Schistosomu daughter sporocysts. They both found alkaline phosphatase activity either in or on the body wall. Krupa and Bogitsh (1972), using EM cytochemistry, located alkaline phosphatase activity on the outer surface of the tegumental microvilli. Kinoti (1971) suggested that the alkaline phosphatase might be associated with the uptake of glucose through the surface. Kinoti et al. (1971) found no evidence of esterase activity in the sporocyst. EM study of the sporocysts and rediae of other species of trematodes also provides evidence of the uptake of substances at the surface of the tegument. Other species of sporocysts generally have a tegument of the same basic structure and with a large surface area, as in the schistosome sporocysts, but the form of the microvilli varies in different species. Cercuriu buccini sporocysts have long, uniform microvilli (Knrie, I971b) whereas an unidentified strigeid has long but irregular, bent and twisted microvilli (Bils and Martin, 1966) and C . buchununi has similar but shorter microvilli (Bils and Martin, 1966). Bibby and Rees (1971a) described the surface of Diplostomum phoxini sporocysts as having blunt, irregularly branched projections. Bucciger bacciger sporocysts have tubular invaginations of the surface membrane (MatriconGondran, 1967, 1969). Thus, all sporocysts appear to have an increased

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surface areawhich probably indicates that absorption takes placeat the surface. Kerie (1971b) found evidence of micropinocytosis at the surface of the tegument of C . buccini and a high degree of acid and alkaline phosphatase activity and smaller amounts of esterase and aminopeptidase activity. Alkaline phosphatase activity is also associated with the tubular invaginations of the surface of B. bacciger sporocysts (Matricon-Gondran, 1967). Kaie (1971 b) found histolysis of the snail tissue surrounding the sporocysts and suggested that extracorporeal digestion takes place followed by absorption. James et al. (1966) described a tegument on Cercaria bucephalopsis haimeana daughter sporocysts which is very different to the tegument of Schistosoma and other sporocysts. The tegument is 8-12 pm in thickness and has an inner and outer part. The inner part has a structure very similar to the tegument of other sporocysts in that it consists of a cytoplasmic layer with a microvillous outer membrane. Beneath the inner part of the tegument there are a basal lamina, muscle fibres and somatic cells. External to the microvilli, however, there is another thick, nucleated, syncytial layer of diffuse, reticulate cytoplasm, which contains a variety of dense bodies. James et al. (1966) also examined the daughter sporocyst of Cercaria dichotoma and found that the thick, outer, syncytial layer is shed early in development so that the elongate, unbranched microvilli o f this species become the outer surface of the sporocyst. James et al. (1966) suggested that C . b. haimaena is unusual in retaining the outer syncytial layer on the sporocyst throughout its life. Another possible interpretation is that this species is, in fact, like Schistosoma and other sporocysts and that the outer layer is simply degenerating host cells on the surface of the sporocyst. Studies on the development of the daughter sporocysts would resolve this question. The teguments of rediae are basically the same as sporocyst teguments and, although rediae have a small intestine, there is considerable evidence that substances are taken up through the tegument. The rediae ofkanthoparyphium spinulosum, Cloacitrema narrabeenensis and Parorchis acanthus have microvilli on the surface of their teguments (Bils and Martin, 1966; Dixon, 1970; Rees, 1966) but the redia of Neophasis lageniformis has an irregularly folded surface ( K ~ i e1971~) , and Cryptocotyle lingua rediae have broad folds on their surface which are connected by numerous, regularly arranged lamellae (Krupa et al., 1967). In N . lageniformis rediae there are pinocytotic vesicles in the tegument (Ksie, 1971c) and Matricon-Gondran (1967) also found evidence of pinocytosis in the teguments of echinostome and hemiurid rediae. Krupa et al. (1970) demonstrated pinocytosis in the tegument o f C . lingua rediae with colloidal thorium and Dixon (1970) described a similar uptake o f horseradish peroxidase into the tegument of C . narrabeenensis. Acid phosphatase activity, which might be associated with pinocytotic vesicles, has been found in the teguments of N.lageniformis and C. lingua (Kaie, 1971~;Krupa et al., 1968). Alkaline phosphatase activity has also been found in N. lageniformis and C. narrabeenensis (Ksie, 1971; Dixon, 1970). The uptake of glucose by C . narrabeenensis, P . acanthus and C. lingua has been demonstrated biochemically by Dixon (1970) and McDaniel and Dixon (1967) and this has been correlated with the presence of alkaline phosphatase activity in the tegument.

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Thus there is much evidence that the teguments of sporocysts and rediae are absorptive surfaces. VI. CONCLUSION The basic ultrastructure of the schistosome tegument at all stages of the life cycle has been established. The tegument is similar to other digenean teguments and, at present, only a few features have been recognized which may be unique to Schistosoma. The results have come from several different laboratories but there is little controversy over the observations, which testifies to the present day reliability of EM techniques. Without doubt, many further details will be added to the ultrastructural description of the tegument. Particularly interesting would be further observations on the development of the tegument in the larval stages and more accurate descriptions of the different regions of male worms and of the differences between male and female worms. The simple observations on normal ultrastructure are an essential preliminary to any further work on the tegument and the observations themselves immediately pose numerous questions. It is the resulting speculation about properties and functions of the ultrastructural features of the tegument that gives rise to controversy. More valuable results will undoubtedly be obtained when the initial observations are used to form hypotheses which can be tested experimentally. For example, it is suggested, from the preliminary observations on the normal ultrastructure, that the tegumental inclusion bodies are formed in the subtegumental cells and are passed into the tegument through the microtubular lined connections. A means of testing this hypothesis might be to disrupt the microtubuleswith colchicinewhich, possibly, would prevent the passage of bodies into the tegument. The appearance of subtegumental cells filled with inclusion bodies and the decrease of inclusions in the tegument over a period of time could be readily and quantitatively recognized with the EM. With this type of experimental approach the properties of the ultrastructural features will be revealed and EM users will avoid the criticism that their methods are unquantitative and unsuitable for elucidating dynamic processes. From their studies of normal ultrastructure, Schistosome EM experts are aware of the importance of the tegument in the host-parasite relationship and, in particular, of the protective and absorptive functions of the tegument. The further understanding of these functions will require ultrastructural studies to be performed in conjunction with immunological and biochemical investigations of these aspects of Schistosorna. I n this manner the ultrastructural observations can be directly correlated with the observations obtained by other methods and the two types of results will be mutually beneficial. An example is the contribution that ultrastructural studies have made in demonstrating the nature of the CHR and of the effects of host antibodies on adult worms. Examination of damaged worms has, at the same time, revealed further details of the normal properties and functions of the tegument. By using these experimental methods, with the basic data on the normal ultrastructure of theschistosome tegument that is now available,ultrastructural

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studies will surely add t o our understanding of the host-parasite relationship which results in the disease of schistosomiasis. REFERENCES Archibald, R. D. and Marshall, A. (1932). A descriptive study of the cercaria of Schistosoma mansoni in the Sudan. J. trop. Med. Hyg. 35, 257-259. Axmann, M. C. (1947). Morphological studies on glycogen despoition in schistosomes and other flukes. J. Morph. 80, 321-343. Baker, R. F. and Loosli, B. G . (1966). The ultrastructure of encapsulated Diplococcus pneumoniae type 1 before and after exposure to type specific antibody. Lab. Invest. 15, 716-730. Belton, J. C. and Belton, C. M. (1971). Freeze-etch and cytochemical studies of the integument of larval Acanthatrium oregonense (Trematoda). J . Parasit. 57, 252-260. Belton, C. M. and Harris, P. J. (1967). Fine structure of the cuticle of the cercaria of Acanthatrium oregonense (Macy). J . Parasit. 53, 715-724. Bennett, H. S. (1963). Morphological aspects of extracellular polysaccharides. J. Histochem. Cytochem. 11, 14-23. Bibby, M. C. and Rees, G . (1971a). The ultrastructure of the epidermis and associated structures in the metacercaria, cercaria and sporocyst of Diplostomum phoxini (Faust, 1918). Z . ParasirKde 37, 169-186. Bibby, M. C. and Rees, G . (1971b). The uptake of radioactive glucose in vivo and in vitro by the metacercaria of Diplostomitm phoxini (Faust) and its conversion to glycogen. Z . ParasitKde. 37, 187-197. Bils, R. F. and Martin, W. E. (1966). Fine structure and development of the trematode tegument. Trans. Am. microsc. SOC.85, 78-88. Bjorkman, N., Thorsell, W. and Leinert, E. (1963). Studies on the action of some enzymes on the cuticle of Fasciola hepatica L . Experientia. 19, 3-5. Bogitsh, B. J. and Krupa, P. L. (1971). Schistosoma mansoni and Haematoloechus medioplexus: Nucleosidediphosphatase localization in the tegument. Exptl Parasit. 30, 418425. Bogitsh, B. J. and Shannon, W. A. (1 970). Haematoloechirs medioplexus: enzymatic oxidation of 3,3’diaminobenzidine in mitochondria. Exptl Parasit. 28,186-1 93. Bogitsh, B. J. and Shannon, W. A. (1971). Cytochemical and biochemical observations on the digestive tracts of digenetic trematodes: Acid phosphatase activity in Schistosoma mansoni and Schistosomatium doouthitti. ExptlParasit. 29, 337-347. Brandt, P. W. and Pappas, G . D. (1960). An electron microscopic study of pinocytosis in amoeba I. The surface attachment phase. J. biophys. biochem. Cytol. 8, 675-687. Brooker, B. E. (1972). Sense organs in trematode miracidia. I n “Behavioural Aspects of Parasite Transmission” (Eds E.U. Canning and C. A. Wright), pp. 171-180. Academic Press, London. Bruce, J. I., Pezzlo, F., McCarty, J. E. and Yajima, Y . (1970). Migration of Srhistosoma mansoni through mouse tissue: Ultrastructure of host tissue and integument of migrating larva following cercarial penetration. Am. J. trop. Med. Hyg. 19, 959-981. Bruce, J. I., Pezzlo, F., Yajima, Y . and McCarty, J. E. (1 97 I ). An electron microscopic study of Schistosomu mansoni migration through mouse tissue: ultrastructure of the gut during the hepatoportal phase of migration. Exprl Parasit. 30, 165-133.

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Stockem, W. (1969). Pinocytose und bewegung von amoben 111. Die funktion des Golgi apparates von Amoebaproteus und Chaos chaos. Histochemie 18,217-240. Terzakis, J. A. (1968). Uranyl acetate, a stain and a fixative. J. iiltrastrvuct. Res. 22, 168-184. Thorpe, E. (1967). Histochemical study with Fasciola hepatica. Res. Vet. Sci. 8, 27-36. Thorpe, E. (1968). Comparative enzyme histochemistry of immature and mature stages of Fasciola hepatica. Exptl Parasit. 22, 150-159. Thorsell, W. and Bjorkman, N. (1966). In vitro studies on the effect of hexachlorophene and its dimethylether on the liver fluke Fasciola hepatica L. Z . ParasitKde 28, 116-124. Threadgold, L. T. (1963). The tegument and associated structures of Fasciola hepatica. Q . J . niicrosc. Sci. 104, 505-512. Threadgold, L. T. (1967). Electron microscopic studies of Fasciola hepatica 111. Further observations on the tegument and associated structures. Parasitology 57, 633-637. Threadgold, L. T. (1968a). Electron microscope studies of Fasciola hepatica IV. The ultrastructural localization of phosphatases. Exptl Parasit. 23, 264-276. Threadgold, L. T. (1968b). The tegument and associated structures of Haplometra cylindracea. Parasitology 58, 1-7. Threadgold, L. T. and Gallagher, S. S. E. (1966). Electron microscope studies of Fasciola hepatica I. The ultrastructure and interrelationship of the parenchymal cells. Parasitology 56, 299-304. Threadgold, L. T. and Read, C. P. (1968). Electron-microscopy of Fasciola hepatica V. Peroxidase localization. Exptl Parasit. 23, 221-227. Timms, A. R. and Bueding, E. (1959). Studies of a proteolytic enzyme from Schistosoma mansoni. Br. J. Pharmacol. 14, 68-73. Vogel, H. and Minning, W. (1949). Weitere beobachtungen uber die cercarienhiillen -reaktion eine seroprazipitation mit lebenden Bilharzia-cercarien. Z . Tropenmed Parasit. 1, 378-386. Wajdi, N. (1966). Penetration by the miracidia of S . mansoni into the snail host. J. Helminth. 40, 235-244. Wagner, A. (1961). Papillae on three species of schistosome cercariae. J. Parasit. 47, 614-618. Wilson, R. A. (1969). Fine structure of the tegument of the miracidium of Fasciola hepatica L. J. Parasit. 55, 124-133. Wilson, R. A., Pullin, R. and Denison, J. (1971). An investigation of the mechanism of infection by digenetic trematodes: the penetration of the miracidium of Fasciola hepatica into its snail host Lymnaea truncatitlata. Parasitology 63, 491-506. Wright, C. A. (1971). “Flukes and Snails”. George Allen and Unwin, London. Wright, R. D. and Lumsden, R. D. (1968). Ultrastructural and histochemical properties of the acanthocephalan epicuticle. J. Parasit. 54, 1 1 11-1 123.

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Schistosomiasis and the Control of Molluscan Hosts of Human Schistosomes with Particular Reference to Possible Self-regulatory Mechanisms J. D. THOMAS

Scl~oolof Biological Sciences, University of Sussex, England I. Introduction ..........................................................

11. Historical ............................................................

111.

IV.

V.

VI.

A. The Rationale for Focusing Attention on the Molluscan Hosts ........ B. Control of Molluscs by Chemical Molluscicides ...................... C. Control of Molluscs by Manipulation of Environmental Factors ........ D. Control of Parasite: an Alternative Solution.. ........................ E. Objectives of Present Work ........................................ Materials and Methods ................................................ Results .............................................................. A. Experiments Carried out in Closed Systems .......................... B. Influence of Varying Concentrations of Calcium and Ammonia on Growth and Reproduction in Snails ........................................ C. Influence of Heterotypically and Homotypically Conditioned Water on Growth Rates of Assay Snails in Open Systems ...................... Discussion .......................................................... A. Classification of Effects.. .......................................... B. Formulation of Hypotheses to Account for Effects on Growth and Reproductive Rates .............................................. Summary ............................................................ Acknowledgements .................................................. References ..........................................................

307 308 308 313 314 321 323 323 3 27 327 350 3 54 359 359

362 382 384 384

I. INTRODUCTION Schistosomiasis was considered to be the second most important parasitic disease by the World Health Organization (1965). More recently it was stated by Brown (1971) to have surpassed malaria and become the most prevalent parasitic disease in the world. According to Jordan (1972) the prevalence of the disease is still continuing to increase in many areas despite the fact that a great deal of costIy research has been undertaken on various aspects of the epidemiology and possible control procedures. The purpose of undertaking the work described in this review is to contribute to the understanding of the epidemiology of the disease. Particular attention is paid to the mechanisms involved in controlling the population 307

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growth of Bio/qdiuiuriu gluhruru (Say), oiie of the molluscan hosts of Schistosomu mutzsoni Sambon. It is hoped that the information thus gained can be utilized in devising means of controlling the population growth of the snail hosts and thus preventing transmission of the schistosome parasites to the mammalian host.

11. HISTORICAL A.

THE RATIONALE FOR FOCUSING ATTENTION ON THE MOLLUSCAN HOSTS

It has long been known that it is obligatory for larval stages of Schistosoma species to develop in molluscan hosts and that the larvae show a high level of specificity (Jordan and Webbe, 1969). Recent studies have shown that both the species of schistosome parasites and their snail hosts appear to exist as a mosaic of genetic races, the latter differing in their susceptibilities to attack by the various strains of parasite (Wright, 1962, 1966; Paperna, 1968; Webbe, 1971). These observations will, however, have limited practical application until more information becomes available on how these spatial patterns change in time as a result of physiological adaptation, genetic changes and dispersionary activity of the host populations. It is evident from these observations that the transmission of the parasite could, theoretically, be prevented by complete eradication of the appropriate snail host. Experienced field ecologists are agreed, however, that it would be extremely difficult to eradicate the snails completely in most areas because of their high intrinsic rate of increase, dispersionary capabilities and genetic variability. Therefore, the alternative possiblity that transmission could be prevented by reducing the snail population density below a certain critical threshold has to be considered. This approach receives some support from the mathematical models developed by Macdonald (1965), Hairston (1962, 1965a) and Goffman and Warren (1970) to describe the epidemiology of schistosomiasis. Thus the Macdonald (1965) model predicts that the break point can be achieved in about 20 years by reducing the snail density by a factor of 15 and in 4-5 years if snail density is reduced by a factor of 3 and combined with therapeutic measures which reduce the mean longevity of the adult worms by a factor of 5. Hairston (1965a) also advocates snail control and states that if this were being attempted it would be a simple matter to estimate the numbers of snails necessary to maintain the critical level of miracidial success from his model. A lowering of snail numbers below this level would then be known to represent satisfactory snail control. Goffman and Warren (1 970) applied the Kermack-McKendrick theory to the epidemiology of schistosomiasis and reached the following conclusion. If the density of the susceptible snails is above the critical level an epidemic will occur; if the density of the snails (susceptible and infective) is above the critical level but the number of susceptible snails is at or below it the process will be stable (endemic state) and if the density of the snails falls to or below the critical level then the disease will decline. The main function of these models is to suggest interrelationships worthy of

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study. They can be criticized on the grounds that they lack realism and are, therefore, deficient in precision and generality. It is important, therefore, that they should be looked at critically and not taken at face value because they have influenced the planning of costly research and control programmes including those involving snail eradication. The following brief critical review has, therefore, been attempted. The models developed by Macdonald (1 969, Hairston (1965a) and Goffman and Warren (1970) all have one common feature, namely that their derivation has been influenced by mathematical models which have been successfully applied to malarial epidemics (Macdonald, 1957). There are, however, fundamental differences between these two diseases. Thus the sexual stages of the schistosome parasite occur in man and have a much higher life expectancy than is the case with the corresponding stages of Plasmodium in the mosquito. The asexual stages of schistosomes occur in snails and those of Plasmodium in vertebrates. The asexual stages are much more pathogenic and cause a higher mortality than is the case with sexual stages. However, the higher mortality caused by the asexual stages is compensated for by their high biotic potential. In consequence some of the basic assumptions in the schistosomiasis model are considered to be of doubtful validity. These include the estimated longevity of the adult parasite and the implicit assumption that the definitive host does not develop an immunity to the parasite. Some doubt is cast on the latter assumption by experimental studies recently reviewed by Smithers and Terry (1969) which show that after the initial infection of certain mammalian hosts including primates a new factor, namely concomitant immunity, may come into play making further infection difficult or impossible. There are also some field observations which provide some support for the hypothesis that acquired resistance based on concomitant immunity may in fact be important in human hosts living under natural conditions (Jordan and Webbe, 1969). The various theories are discussed briefly below :

I . The Macdonald (1965) model This model was a promising, pioneering approach which was unfortunately cut short by the death of the author. The main purpose of the model was to determine how the break point, or the mean adult worm load below which transmission cannot be continued, could be achieved by perturbating certain key factors. These include the longevity of the adult worms, the rate of contamination measured by the proportion of eggs reaching the water, the exposure factor determined by the frequency and duration of contacts with the water by the mammalian host and finally the snail factor. Macdonald also placed a great deal of emphasis on the fact that schistosomes are bisexual. He did not consider the possibility that reinfection of the definitive host might be difficult or impossible as a result of an immune response because the facts about concomitant immunity were not then generally known. The estimated mean length of life of 3 years which was used in the calculations was based on values given by Hairston (1962). It will be shown below that there are reasons for believing this to be an underestimate. According to the Macdonald model, control of the disease can best be

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achieved by reducing the longevity factor in combination with the exposure factor or the snail factor. Decreasing contamination by a factor of 15 which is equivalent to a high standard of hygiene has virtually no effect. Macdonald (1965) and Jordan and Webbe (1969) have used the failure of sanitation campaigns as evidence for the correctness of these conclusions. Hairston (1971) has, however, pointed out that the conclusions derived from these models are based on the following erroneous assumptions. Firstly, it is assumed that nearly all the snails shed cercariae. Empirical evidence on the other hand indicates that only a small percentage (5-10% or less) of snails normally shed cercariae at transmission sites (Berrie, 1969; Jordan and Webbe, 1969). Secondly, he assumes that only 1/1000 of the excreta reaches the water when there is no sanitation. Hairston (1971), therefore, argues that the failure of sanitation campaigns are more likely to be due to sociological or economic considerations rather than to any mathematical property peculiar to the epidemiology of schistosomiasis. 2. The Hairston (1962, 1965a) model Hairston’s model is based on a life table of the parasite: Net reproparasite in snail population

infecting a definitive host

the definitive host

rate of parasite

The model is the first rigorous quantitative approach to the problem of schistosomiasis, and it makes use of empirical data obtained from both the field and laboratory. It assumes that the parasite population is in equilibrium and that its net reproductive rate is approximately 1.0. As stated by Hairston (1965a), a mere solution of the terms in the equation does not yield any predictions. Before it can be used for this purpose it is necessary to relate accurate values of the net reproductive rate of the parasite to the combined population densities of the snail and the definitive hosts from three or more contrasting areas. The cost of obtaining certain of the values is high and despite the remarkable ingenuity used in estimating others they are at best approximations with errors that are difficult to measure. As indicated by Hairston (1965a) both his model and that of Macdonald (1965) can be critized on the grounds that they assume randomization of the biotic components whereas in reality contagion is the rule. Another, perhaps more serious, criticism is that it does not take into account the possibility of the immune response in the definitive host when calculating the probability of success of the cercariae in infecting the definitive host or in estimating the death rate of female worms. One method of calculating the latter involved measuring the rate of loss of active infections from age prevalence data. The catalytic model (Hairston, 1965b) used for making the estimate assumes constant rates of acquisition and loss, thus ignoring the possible effects of the immune response. In consequence the mortality rate tends to be overestimated and the longevity of the worms underestimated. The actual estimates of

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31 I

death rate gave values ranging from 0.336 to 0.10 for S. mansoni and 0.30 to 0.09 for S. haematobium. When the mean lengths of life are calculated from these they give values ranging from 3.47 to 10.51 and 3.86 to 11.61 years for S. mansoni and S. haematobium respectively. The higher estimates are considerably in excess of those normally quoted in the literature (Jordan and Webbe, 1969) and used by Macdonald (1965) in his model. Hairston (1965a) used two other models to estimate the death rate of the adult parasite in the definitive host. The first method involved the use of experimental laboratory animals including mice and gave very erratic results. The second method is based on the assumption that male parasites have a negligible death rate compared with female worms. It should, therefore, be possible to calculate the death rate of female worms by observing changes in the sex ratio of parasites in experimental animals. This method gave values of 0.253 and 0.27 per year for S. japonicum in rats and dogs respectively. These latter estimates can be criticized on the grounds that the assumption on which the method is based is not supported by any empirical evidence. It is also clearly very dangerous to extrapolate estimates based on laboratory animals to another animal such as man with a different physiology and a much higher expectation of life. Empirical data quoted by Jordan and Webbe (1969) indicate that the adult S. mansoni can live for up to 26 years and S. haematobium for 26-30 years in the definitive host. It is not easy to reconcile these with the lower values of 3 or 4 years for the mean expectation of life obtained by using the catalytic model. Further efforts should clearly be made to obtain more empirical data on the longevity of the adult worm by investigating immigrants who have moved from endemic areas. This information is clearly of the utmost importance in planning the control and eradication of the disease.

3. The Goffman and Warren (1970) model This has already been criticized on the grounds of oversimplification by Voors (1971). For example they assume that the rates of infection, removal and influx are constants and that the rate at which people become infected is proportional to the number of infected snails. However, the high rate of cercarial production by infected snails would make this unlikely. Like the other models it ignores factors such as immunity, age, dependence of exposure rates in the definitive host and the death and birth rates of infected and noninfected components of both the definitive and intermediate hosts. There are experimental data which indicate that both the natality and survival rates of infected snails may be significantly less than is the case with non-infected snails (Berrie, 1970). This factor should, therefore, be also taken into account. Consequently, it is unlikely that quantitative predictions from this model will be reliable although it may have some qualitative relevance. 4. The Coutinho and Coutinho (1968 and in press) model The following assumptions are made in their model. Firstly, the population of adult parasites quickly reaches a steady state in which all the definitive hosts in contact with the water body become infected and immune to further

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infection. This p r o w s is fxilitated by the comparativcly long life span of the adult parasite, the development of concomitant immunity and the high intrinsic rate of increase of the parasite. Secondly, when the steady state is reached the supply of miracidia to the water body becomes constant. Finally, they assume that larval helminths including schistosomes are a major factor in causing mortality in the snail population. As already stated this is supported by empirical evidence (Berrie, 1970). The parameters in their model include the biomass density of snails that can be supported by the water body, A, the death rate of the snail, and p , the proportion of infected snails which is assumed to be proportional to X. Therefore, h=a p + p where a and ,t3 are constants. They conclude that the snail population cannot be maintained if the value of p increases beyond a certain threshold. The relevance of this model to biological control will be discussed later. Goffman and Warren (1970) stated that as yet there was no conclusive laboratory or field evidence for or against the validity of the mathematical deductions regarding critical snail densities presented in their paper. Up to the present moment it appears that the position has remained unchanged and despite an enormous expenditure of effort it is not known to what level a snail population must be reduced at a particular site in order to prevent transmission. Due to spatial and temporal heterogeneity it is likely that threshold values will vary a great deal and for this reason it may not be possible to determine them. According to Chernin and Dunavan (1962) there is evidence to suggest that in an area of high endemicity the threshold below which snails will not become infected is probably close to complete eradication of the snail population. Studies carried out by Webbe (1962) and Paperna (1968) in arid savanna areas in East and West Africa respectively tend to support this statement as they both found that snails could become infected and thus allow transmission to occur when the absolute or crude snail density was very low during the dry season. However, the ecological or effective density of the snail population and the frequency with which the human population comes into close proximity with them increases at this time. It is important to note that if the adult parasites are as long-lived as some of the clinical data would suggest it will be necessary to keep the snail population in check for a period of 20 years or more. Some encouragement is given to those who advocate snail control or eradication by the fact that there is empirical evidence, based on sound data, to indicate that significant reductions in the prevalence of schistosomiasis have been achieved in the Philippines, Rhodesia and Egypt through the application of methods directed against the molluscan hosts (Wright et al. 1958; Pesigan and Hairston, 1961 ; Clarke et af., 1961; Ayad, 1961; Farooq et al., 1966). A combination of snail control and chemotherapy in Puerto Rico also resulted in a rapid decline in the transmission of schistosomiasis (Ferguson et al., 1968;Jobin et al., 1970).The success of the control programme was attributed mainly to snail control. Other control methods, in general, appear to have been less successful and Hairston (1962) was able to state that up to that date every single case of

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success in stopping transmission had been through snail control and that no other method had ever been shown to interrupt transmission. This generalization appears to hold to the present day and control of transmission by reducing snail populations is still considered the point in the parasite life cycle most easily attacked. This view is reinforced by the fact that it is the only method of limited control which has public health significance (Macdonald, 1965). In consequence there is a general lack of interest on the part of most schistosomiasis workers in the possibility of breaking transmission of the disease by improving water supplies and sanitation. In Puerto Rico (Ferguson et al., 1968; Jobin et al., 1970) it was shown, however, that there was a decline in prevalence even in the control areas and it is uncertain, therefore, to what extent the decline in the experimental sites was also attributable to a reduction in frequency of water contact and contamination resulting from a general increase in the level of education and hygiene. It could be suggested, therefore, that it would be beneficial to integrate schistosomiasis control into a wider programme of environmental sanitation which would bring many other side benefits to the community. Properly designed field experiments to compare the relative effectiveness of the various methods that may be used to break transmission including reductions in contamination, contact, longevity of the parasite by chemotherapy, and snail control, are also indicated as good quantitative data seems to be lacking. Such information is necessary before objective decisions regarding the allocation of resources for control purposes can be taken. B. CONTROL OF MOLLUSCS BY CHEMICAL MOLLUSCICIDES

At the present stage of our knowledge it must be recognized that the only effective method of reducing snail population density to a significant extent, at least in the short term, is by using chemical molluscicides. There are certain advantages to control by this method including the relative ease of administration and the fact that public cooperation is not obligatory. On the other hand there are considerable disadvantages in using chemical molluscicides. These include the high cost, the difficulties of treating large bodies of water including man-made lakes, the damage that might be done to aquatic organisms including economically important species of fish and organisms which may be involved in the natural control and regulation of the molluscs and the need for repeated application over a long period. The latter is necessitated by the high intrinsic rate of natural increase, genetic plasticity and dispersionary power of the snails, the high biotic potential and life expectancy of the parasite and the high dispersionary activity of the human host. The high life expectancy of the parasite, indicated by the clinical data, is particularly important, because as a result of this the adult parasite population can survive for long periods almost independently of the snail hosts unless molluscicide application is accompanied by chemotherapeutic treatment of the definitive host. In view of the problems that have resulted from the application of broad spectrum insecticides the need to investigate the ecological consequences of

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using molluscicides, the development of resistant strains, the accumulation of stable residues and toxicity to other organisms has been stressed by Berg (1964) and by members of an informal consultation on snail ecology (World Health Organization, 1970). There are reasons for doubting the effectiveness of the present generation of broad spectrum molluscicides unless they are applied almost constantly because they will also probably cause mortality among potential predators and competitors. In the field of entomology it has been shown that under certain circumstances insecticide application may eventually result in an increase in pest populations probably due to the detrimental effects on predators as predicted by the Lotka-Volterra model (Newson, 1967; Dempster, 1968). Mead (1961) has given some qualititative data from the malacological field which supports this hypothesis. In view of problems associated with broad spectrum molluscicides it would clearly be advantageous to have a molluscicide which could kill snails selectively in the most economical way possible. Some current research is, therefore, being directed towards the production and testing of matrices which will release the molluscicide slowly into the water (Cardarelli, 1972). An alternative approach would be to retain the molluscicide in an edible matrix which releases specific snail attractants into the medium. This work is, however, still in the exploratory stage and extensive field trials have yet to be undertaken. The difficulties of evaluating the effects of molluscicides in the field have been discussed by Hairston (1961) and Jordan and Webbe (1969). Efficient application would necessitate the location of transmission sites and the acquisition of spatio-temporal information on the snail hosts. Before the full potential of the method can be assessed it will be necessary to obtain precise information on the behavioural, physiological and genetical responses of the snails to the released substances, including sublethal concentrations of the molluscicide, and also on the effects of environmental components including currents on the latter. C.

CONTROL OF MOLLUSCS BY MANIPULATION OF ENVIRONMENTAL FACTORS

In view of the grave disadvantages of using chemical molluscicides it is clearly important that serious consideration should also be given to the possibility of controlling snail populations by manipulating appropriate environmental factors. To be able to do this efficiently it would clearly be advantageous to know which environmental factors determine the population density of snails in nature. The various environmental factors that may affect growth, development, reproduction and spatial location of individual organisms and also the natality, mortality, emigration and immigration rates at the population level of organization are listed in Fig. 1. The term life system is used for the entity comprising a population and the components in its environment (Clark et al., 1967). The relevance of these is discussed below. Much of the research effort in ecology has been directed at identifying and measuring the effects of the key environmental factors which are mainly

Feed forward

I 1

I

I

I

Envkwmental foctws

Cllmotlc EtmThose wrth h@ predictive value eg oscillatory signals such as diurnal, l u l w and SWSOMI

dange in photoperiod

Those with lower predciive value e g rainfall, tmpemtwe

e g energy ncluding food,oxygen. minerok

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Orgonisds)

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Other sex

Different s p e c 2 Competlfor

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FIG.1. The environmental factors which may influence growth, development, reproduction, and spatial location of the individual and growth, mortality and dispersion of the population.

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involved in influencing the growth and survival at the individual or population level of organization either favourably or unfavourably. In recent years the process has been aided by analysis based on the change of logarithm of population size, a key factor analysis (Varley and Gradwell, 1960; Klomp, 1966) and statistical procedures including correlation, regression and multiple factor analysis (Williamson, 1972). These methods have made it possible to identify factors that may act in a density-dependent or regulatory manner, as opposed to those that also influence growth and survival but in a density independent or non-regulatory manner. The results of correlation analysis can be used to generate hypotheses which can then be tested. The view that all persistent populations are frequently subjected to density-dependent regulation is now generally accepted by nearly all biologists (Lack, 1966; Wilson, 1968a; Solomon, 1971) including those who formerly believed that environmental and genetic heterogeneity adequately explain the persistence of many species (Andrewartha and Birch, 1954; Birch, 1962, 1971). No well developed unifying theory has emerged because of the biological uniqueness of species and the complexity of the interactions involving the environmental components (Thompson, 1956; Reynoldson, 1958). Perhaps the best general synthesis is that of Nicholson (1958). He postulated that populations are self-governed systems which regulate their densities in relation to their own properties and that of the environment by depleting and impairing essential things to the limit of favourability or by maintaining reactive inimical forces such as natural enemies to the limit of tolerance. Recent work by Jobin and Michelson (1967) and Eisenberg (1966, 1970) suggests that snail populations are regulated to a large extent by availability of food resources. The various strategies for controlling snail populations by natural means are as follows:

I. Control by permanently altering or perturbating climatic or physicochemical factors Climatic factors are particularly important to poikilothermic animals such as snails. Temperature is clearly a key factor in temperate zones but is probably not as important in the tropics. It affects fecundity and survival in Biomphalaria glabrata and Bulinus globosus. The optimum temperature for fecundity was the same for both species, namely 25°C (Michelson, 1961; Shiff, 1964). Another important factor is water velocity. Some measure of success in controlling snails has been achieved by increasing water velocity beyond certain thresholds in water channels and by fluctuating water levels in reservoirs (Jobin and Michelson, 1969; Jordan and Webbe, 1969; Jobin, 1970 and McJunkin, 1970). In view of the fact that the snail hosts of S. mansoni and S. kaematobium are aquatic, phytophilous species that occupy marginal areas they tend to be particularly vulnerable to this strategy. However, in consequence of the genetic and physiological plasticity of molluscs, ability to survive desiccation and high intrinsic rate of increase, numbers are likely to be rapidly restored once the physicochemical factors return to normal. For this reason methods of control which involve pertur-

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bating physiocochemical factors are not likely to be as effective as biological factors acting in a density-dependent manner unless they can be operated on a permanent basis. The direct effects of climatic factors on growth and natality rates of snails are well reviewed by Jordan and Webbe (1969). It is possible, however, that the snails may also use some of the environmental factors with well defined oscillatory trends such as photoperiod and rainfall in the tropics as feed forward signals to enable growth, development and reproductive activity to occur when food resources are abundant (Lees, 1966; Strumwasser et al., 1967). Circadian rhythms in response to cycles of light have been demonstrated in Aplysia californica by Strumwasser (1965) but no work on the effects of predictive signals appear to have been carried out on snail hosts of schistosomes. 2. Control by food Recent investigations by Eisenberg (1966, 1970) and Jobin and Michelson (1967) show that food is an important density-dependent factor involved in regulating growth of snail populations. There is also evidence based on quantitative field observations that intraspecific competition for food resources may provide the basis for a regulatory mechanism in the case of planarians (Reynoldson, 1966), insects (Morris, 1963; Readshaw, 1965; Dempster, 1968; Wilson, 1968) and in birds (Lack, 1966). Andrewartha and Birch (1954) drew attention to the important distinction between absolute and relative shortage of food caused by heterogeneity in time and space and in recent years the importance of food quality has been stressed (Eisenberg, 1970). 3. Control by use of competitor species There has been a great deal of discussion regarding the definition of interspecies competition (Milne, 1961 ; Odum, 1971 and Williamson, 1972). The last author argues strongly that ecological interactions should be classified by their e$ects on the population level of organization. These effects can be classified as being positive (+), neutral (0), or negative (-), depending whether the population density increases, is unaffected or decreases. Competition is normally symbolized by (- -) and indicates that the population growth of both species is impaired when they are together. It is possible, however, to envisage the growth of one species being seriously affected and the other hardly at all. In such a case the effects of the interaction would be classed as ammensalism which is depicted by the symbols (0, -). It has been suggested that competitive interactions of this kind could be used to exclude the undesirable snail hosts of schistosomes. There is evidence that this in fact has been achieved, because the introduction of the snail Marisa cornuarietis into ponds containing B. glabrata is often followed by the exclusion of the latter species, although this is not invariably the case (Jobin et al., 1970). It is, however, necessary to test the hypothesis that competition does in fact occur between the two species. If this can be demonstrated it would be interesting to determine how these effects are caused. The possibilities that need to be considered are that both species are depleting essential

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resources such as food and calcium in the water, that physical interference involving one or both species occurs, that both species are releasing nonspecific inhibitory substances such as ammonia into the medium and that one or both species are producing specific antibiotics which act on the other species. This kind of strategy has been adopted by micro-organisms and plants (Muller, 1966) and has far-reaching ecological consequences. Should such a system occur in snails it would have interesting possibilities for snail control. Other species which may also compete with B. glabrata are Tarebia granifera and Pomacea haustrum (Reeve, 1856) according to Ferguson et al. (1968) and De Andrade (1971) respectively. The extent to which inhibition of population growth by Marisa is due to egg predation also needs to be investigated. 4. Control by using predators or parasites Although there are many potential predators and parasites of snails (Michelson, 1957; Malek, 1962; Chernin et al., 1956, 1960; Michelson, 1961, 1963; Berg, 1964; Jordan and Webbe, 1969; Lim and Heynemann, 1972) none of them as yet has been shown conclusively to be effective in controlling snails under field conditions (Jordan and Webbe, 1969; McJunkin, 1970). In marked contrast Huffaker (1971a, b) has referred to several hundred examples of successful or partially successful biological control involving insect predators. Complete biological control commonly means a density of 1/25 or 1/100 of that prevailing without the controlling agents. References to a large number of examples of biological control occurring naturally have also been listed by Huffaker (1971a, b). The lack of success in the malacological field can be mainly attributed to the dearth of workers and to the fact that few are population biologists. Further work on biological control of molluscs is clearly indicated. Potential predators such as sciomyzid flies should be screened by using the experimental approach outlined by Hassell (1966) and Huffaker and Kennet (1969) which includes the “check method” of evaluation. A promising start has recently been made by Geckler (1971). The feasibility of using particular agents could also be tested by means of a mathematical model before embarking on field trials. More recently Coutinho and Coutinho (1968 and in press) have assumed in their model that larvae of helminths, including schistosomes, are important in regulating snail populations because they decrease survival and fertility rates of snails. This assumption does in fact receive some support from observations undertaken in the laboratory and in the field (Olivier et al., 1954; Teesdale, 1962; Etges, 1963; Barbosa, 1963; Etges and Gresso, 1965; McClelland, 1965, 1967; Chu et al., 1966; Pan, 1965; Sturrock, 1966, 1967). It can, therefore, be suggested that control of schistosomiasis might be achieved by the introduction of a suitable number of vertebrate hosts containing trematodes, provided the larvae can develop in the snail hosts of schistosomes and cause heavy mortality amongst them. The larval trematodes have some of the attributes of a successful biological control agent because, in addition to causing mortality and decreased fertility in the snail population, they have high reproductive and host-searching capacities. The feasibility of

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319

using trematode parasites as control agents should be tested experimentally and also by constructing a model. The results of some experiments recently carried out in Malaya give some support to the possibility of using helminths as control agents as Lie et al. (1971) found that the introduction of echinostome eggs into a small pond was followed by a considerable reduction in snail population due to parasitic castration and high mortality among infected snails. For this kind of control to be successful it is necessary to have a biological agent capable of causing considerable mortality. Not all trematode larvae are highly pathogenic to snails. Thus it has been shown that some individual B. glabrata produce cercariae for up to 8 months while others survive their infection and subsequently become reinfected. (Ritchie et al., 1963; Pan, 1965; Pitchf'ord and Visser, 1965; Chu et al., 1966; Wajdi, 1966). It has also been suggested by Webbe (1962) that his field data can also be interpreted as indicating a high survival rate of infected Biomphalaria. It could also be argued that the selective pressures in such a system would favour the development of resistance in the snail population and a decrease in parasite pathogenicity. There is some circumstantial evidence in support of both of these hypotheses (Wright, 1971). Ewers and Rose (1966) and Ewers (1967) have also speculated that snail populations might be genetically polymorphic with respect to resistance to attack by parasites and also to other inimical forces in the environment. This hypothesis appears to be supported by their observations on the littoral prosobranch Velacumantus australis, as individuals that are resistant to parasites are more susceptible to predation. Consequently when predation pressure is heavy the parasitized snails are at a selective advantage. 5 Control by using substances of plant origin The molluscan hosts of schistosomes are aquatic, phytophilous species that tend to occupy marginal areas in ponds, lakes and slow-flowing streams and rivers. Macrophytes are important components in the snail's environment because they may serve as oviposition sites, a food source, a substrate for epiphytic organisms which may serve as another food source, a means of reaching the air-water interface to replenish their air supply and as a refuge against predators. The majority of snails feed on living plants. Although some are found in association with decaying plant material they still act as herbivores because they graze on bacteria and fungi present in the decaying material. In spite of their innate tendency to increase, populations of herbivores, including molluscs and insects, eat only a relatively small percentage of available plant food and seldom destroy it. The fact that plants are not normally overexploited by herbivores can be attributed to anatomical, physiological and chemical adaptations to resist attack combined with strong powers of regeneration which have evolved as a result of natural selection (Pimentel and Soans, 1971). Chemicals that may be implicated in the defence mechanisms include substances which were previously thought to be waste products of plant metabolism including a heterogeneous assemblage of alkaloids,

320

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I).

THOMAS

tannins, cyanogenic glycosides which repel slugs, toxic amino acids as well as analogues of insect juvenile hormones and phytoecdysones (Harbone, 1972). Many chemical substances which serve as defence mechanisms in plants such as pyrethrum have been developed successfully to control insect pests. The possibility of using analogues of insect, juvenile hormones and the phytoecdysones as a new generation of insecticides is also being actively investigated (Williams, 1970). In contrast very little has been done to develop plant factors as molluscicides. One exception is the work carried out by Lemma (1970) on the fruit of endod, Phytolacca dodecandra. This was shown to have strong molluscicidal properties and the active factor has now been characterized (Powell and Whalley, 1969). It is also possible that the complex ester with a molecular weight of 360 isolated by Berrie and Visser (1963) from pond water and shown to be toxic to snails was of plant origin. Research work in the entomological field has shown that plants release substances which serve as attractants, arrestants, repellents, mating stimulants and maturation inducers to insects (Carlisle et al., 1969; Riddiford, 1967; Wood et al., 1970). Considerable efforts are being made to utilize certain of these substances in procedures designed to control and regulate insect pests (Wood et al., 1970). Unfortunately it appears that no comparable work is being undertaken in the malacological field. 6. Control by using inhibitory pheromones, if these are in fact produced Many biologists including Thompson (1956), Chitty (1960), WynneEdwards (1962) and Richards and Southwood (1968) have come to the conclusion that the depletion of energy resources and natural enemies are rarely the ultimate sanctions against animal populations studied by them. In consequence several theories have been advanced incorporating the idea that individuals possess certain properties, evolved as a result of natural selection, which make it possible for populations to be self regulatory before environmental resources become limiting; these theories are due to Chitty (1960), Pimentel (1961), Pimentel and Soans (1971), Wynne-Edwards (1962) and Christian et al. (1964). Most of the theories were developed in the first instance to explain population changes in small mammals or birds and have been critically reviewed by Clark et al. (1967). It has also been proposed that self-regulatory mechanisms which operate before resources become limiting occur in some invertebrates. Thus interindividual interactions in aphids and locusts appear to result in physiological changes involving the endocrine glands which result in dispersive behaviour (Uvarov, 1966; Way and Cammell, 1971). It has also been suggested that population growth is inhibited by pheromones in snails (Wright, 1960; Berrie and Visser, 1963), Hydra (Davis, 1966b), social insects (Butler, 1967), Culex larvae (Ikeshoji and Mulla, 1970), fish (Yuand Perlmutter, 1970) and mice (Wilson, 1970). Growth inhibitors have also been postulated to occur in populations of somatic cells (Stoker, 1967) and in epidermal tissues (Bullough, 1969).

SCHISTOSOMIASIS A N D C O N T R O L O F M O L L U S C A N HOSTS D.

CONTROL OF

PARASITES: A N

321

ALTERNATIVE SOLUTION

A considerable amount of research effort in the schistosomiasis field is aimed at control of the parasite rather than the snail. This approach is attractive to ecologists because destruction of molluscs, even without the use of molluscicides, could be harmful to the aquatic ecosystems. The various strategies that are being considered are discussed below. 1. Control by reducing the probability of success of the miracidium a. By using snails that are resistant to the parasite. It is well known that snails of the same species vary in their susceptibility to infection for either physiological or genetical reasons (Newton, 1953; Moore et al., 1953; Paraense and Corrsa, 1963). In consequence it has been suggested by Richards (1970) that transmission of schistosomiasis might be prevented by the introduction of snails resistant to the strain of schistosomes occurring in a particular area. He succeeded in raising three kinds of snails classified with respect to their susceptibility to the Puerto Rican strain of S. mansoni; those susceptible at any age; juvenile susceptible but adult refractory and those that are refractory at any age. It can be argued that if parasitism by schistosomes is one of the key factors involved in density-dependent regulation of the snails in a given area, introduced refractory snails would be at a competitive advantage and would eventually replace susceptible snails. However, the following considerations seem to indicate that this method might not succeed. First, the genetically heterogenous, indigenous snail population would have been subjected to the selection pressures in that area for a long period and might, therefore, have a selective advantage over the introduced snails even though the latter were refractory to schistosome parasites. Second, a shift in the spatio-temporal, genetic mosaics of either the snails or the parasite could result in the previously refractory snails being attacked by a schistosome parasite. As indicated earlier it is important that more should be known about the rate of change of the genetic patterns in space. b. By using decoysfor the miracidia. Recent investigations by Chernin (1968) and Chernin and Perlstein (1969, 1971) have shown that miracidia of schistosomes are commonly attracted to non-host snails and some other invertebrates. They also attempt to penetrate them but fail to develop because they are the wrong hosts. It is possible, therefore, that the break point in transmission of the disease might be achieved by encouraging the growth of nonhost snails and appropriate invertebrates. 2. Control by reducing the probability of success of the sporocysts The possibility of reducing the population density of larval schistosomes in their molluscan hosts by making use of antagonistic reactions or predation by other larval helminths capable of living in the same snail hosts has been discussed by Heyneman and Umathevy (1968) and Lie et al. (1968). Preliminary field trials described by Lie et al. (1971) have produced some interesting results. They showed that the introduction of large numbers of Echinostoma malayanum eggs at one site was followed by the eventual

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disappearance of Schislosutizu spindufe infections in snails. In contrast an infection rate of approximately 30% was recorded in the control area. The loss of S. spindale infections was attributed to the combined effects of trematode antagonism, a microsporidian epidemic among the trematode larvae leading to a considerable suppression of cercarial production, and a marked reduction in the snail population due to parasitic castration and high mortality among infected snails. It was considered that the main factor responsible for the eradication of S. spindale was trematode antagonism as it preceded the other two effects. The importance of snail mortality caused by the echinostome larvae was not fully discussed although this may prove to be of greater practical value than the effect attributed to larval antagonism. Unfortunately the experiment described by Lie et al. (197 1) which involved one control and three treatments, was carried out, without replication. It is clearly dangerous to attribute an effect to the treatment on the basis of one result only, particularly as Schistosoma spindale continued to coexist with Echinostoma malayanum at two treatment sites including the control. The fact that the percentage of snails shedding Schistosomu spindale cercariae continued to increase during the period of the investigation at both of these sites despite the presence of Echinostoma malayanum is clearly not very encouraging. The results are, therefore, equivocal and before any firm conclusions can be drawn it seems clear that the experiment should be repeated with sufficient replication. The limitations of this method were recently discussed by Lim and Heynemann (1972). They conclude that although this method might be locally applicable, effective biocontrol as a self-sustaining life cycle appears improbable as is the expectation that this method can be adopted on a wide scale with the same controlling agent. 3. Reducing the probability of success of the cercaria a. By predation. It has been shown that predation by fish, such as the guppy Lebistes reticulatus, and also copepods and annelid worms can be effective in reducing cercarial numbers (Michelson, 1964; Courmes et al., 1964; Rowan, 1965; Pellegrino and De Maria, 1966). Preliminary experiments carried out with guppies under field conditions by the latter authors indicate that they may reduce the infection rate in mice. b. By using cercarial traps or decoys. This method does not appear to have been considered. c. By immunizing or vaccinating the definitive host. In the present state of our knowledge the immunization or vaccination of the human host is not a practical proposition. The possibility of inducing concomitant immunity by using male worms of the same species or of inducing heterologous immunity (Nelson et al., 1968) by using male worms of a different species is perhaps worth considering. Experimental work has shown that male worms survive well in the absence of females (Smyth, 1962). Further research is clearly needed in the immunological field. 4. Reducing the probability of success of the adult worms

The problems associated with chemotherapy are well covered by Jordan and

S C H I S T O S O M I A S I S A N D C O N T R O L O F M O L L U S C A N HOSTS

323

Webbe (1969). Unfortunately little research is currently being undertaken in the field of chemotherapy because high costs and difficulties associated with drug administration have reduced the demand from the developing countries where the disease is endemic. E.

OBJECTIVES OF PRESENT WORK

From the preceding review it is evident that the control of populations including those of the snails cannot be readily achieved in the present state of our knowledge. The probability of being able to control and regulate them should, however, be increased by a better understanding of the interactions that are involved in generating population changes in molluscan populations. The purpose of the work described in the present paper was to contribute to our knowledge of the interactions that affect the snail populations, particularly those which might result in self-regulation. It is hoped that precise formulation of the laws governing the population dynamics of snails will make it possible to construct a mathematical model which could then be tested by comparing its predictions with observations in the field. As suggested by Jobin and Michelson (1967) such a model might eventually serve for preliminary evaluation of various methods that could be used for snail control including the manipulation of various environmental factors and the introduction of molluscicides. In the present investigation particular attention has been focused on the manner in which various environmental factors receive expression in the growth and natality rates of individual snails. The important work of Eisenberg (1966, 1970) indicates that population regulation of Limnaea elodes, living under natural conditions, is achieved mainly through control of growth and fecundity. He found no evidence of regulation through adult mortality. The specific questions that were asked in the present investigation are as follows: (i) Is there any evidence of a density-dependent, negative feedback mechanism which could regulate growth, reproduction, metabolism or spatial location of the individual and hence population growth when resources such as food and oxygen are not apparently limiting? (ii) If such a density-dependent, negative feedback mechanism exists how does it work? To what extent does it depend on the following: accumulation of excretory products, depletion of essential ions in the culture medium, the production of inhibitory pheromones or of growth factors which may act directly on internal target organs ? (iii) To what extent are the observed effects related to age, snail biomass, time and volume? 111. MATERIALS AND METHODS A.

STOCK CULTURES

The Venezuelan, albino strain of Biomphalaria glabrata (Say) used in these experiments was maintained in stock colonies kept in aquaria with aerated and filtered tapwater, kept at a temperature of 26 2 1°C and a constant photo-

324

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period of 12 h light and 12 h dark. These were fed with lettuce in excess of requirements. B. EXPERIMENTAL PROCEDURES

I . Media a. Aquatic medium. The preliminary experiments were carried out in natural tap water and subsequent ones in a defined media prepared in two different ways. All of these media resembled each other closely in ionic composition. The two defined media differed only in the methods used for preparation and had the following composition expressed in mequiv 1-1 (Thomas and Benjamin, in preparation): Ca, 4.1920; Mg, 0,2795; Na, 0.5916; K, 0.0282; (HCO3)2, 4.0089; S04,0.2415; CI, 0.6796 and NOS, 0.0805. The pH of 8.3, conductivity values of 430 pmho cm-2 were very similar to those of natural tap water. The defined medium will be referred to as standard snail water (S.S.W.). The S.S.W. was normally changed at three day intervals unless otherwise stated. b. Food. Each experimental animal was provided with standard daily rations of washed lettuce. In later experiments including those in theflow system the lettuce was provided in the form of discs of varying sizes. When used in the open systems they measured 2-8 cm dia and weighed 171-05 11.5 mg. The food was always provided in excess of requirements and any uneaten remains removed at the end of each 24 h period. 2. Apparatus The experimental snails were kept in both closed and open systems: a. Closedsystem with no partitioning. The snails were kept in covered beakers in which either the number of snails, the volume of aquatic medium or the time allowed for changing the medium were varied (Thomas and Benjamin, in preparation). b. Closed system with partitioning. This system was designed to investigate the effects of the chemical environments of snails subjected to different density treatments on the growth, reproductive output and metabolism of individual snails. Each assay snail was kept in isolation in 200 ml of medium in one of ten compartments of an inner tank made of white perspex and were subjected to six treatments, namely the chemical environment produced in outer tanks containing 0, 10, 20, 40, 80 and 160, 300-500 mg snails, in 4 1 of aerated tap water. Each compartment containing an assay snail was connected to the outer tank by four circular apertures, 1 cm dia, covered with a double layer of fine tygan mesh. The aeration system ensured rapid mixing and in consequence the assay snails could be influenced by the chemical environment of the outer tanks but not by tactile or visual signals from the snails in the outer tank. This arrangement was designed to make allowance for the possibility that any chemical factor produced by the snail might be short-lived. Four tanks were used for each of the six treatments and as each of the inner tanks contained ten snails, a total of 40 snails was used to assay each treatment. The water was replaced at weekly intervals.

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325

c. Open systcnt. This apparatus, which was designed to produce a constant flow of test medium, also prevented depletion of resources such as calcium and the accumulation of exogenous materials produced by the assay snails. It was placed in a small environmental unit kept at a temperature of 27°C and a photoperiod of 12 h light, 12 h dark. The test substances were pumped by a Multichannel, Watson-Marlow, Delta, peristaltic pump with six tenchannel modules at a rate of 5 ml h-1 through 60 stoppered, air ventilated containers each holding an assay snail. This arrangement made it possible to assay concurrently up to ten treatments, each with six replicates. The volume of each cell containing an assay snail was 25 ml in the first four experiments and 5.5 ml subsequently. The level of each cell was kept constant by overflow arms protected by Tygan mesh barriers to prevent the assay snails from escaping. The containers delivering the test substances and the tubes collecting effluents from the assay cells were placed in water baths cooled at 5°C placed underneath the environmental unit. Two kinds of media were tested: (i) Heterotypically conditioned water (Het. C . W.) produced by lettuce-fed snails kept at a density of 100 ml per snail under either sterile or non-sterile conditions. (ii) Homotypically conditioned water (Hom. C. W.) produced by snails fed on pure cellulose and maintained under non-sterile or sterile conditions.

3. Temperature and light All the experimental snails were maintained at a temperature of 27°C and a photoperiod of 12 h light and 12 h dark. This was made possible by the construction of large environmental units which will be described in a later paper.

4. Measurements undertaken a. Measurement of snail growth (i) Weight. The change in wet weight over measured time intervals was used to measure the rate of growth of snails in the majority of the experiments in preference to change in length because it is a more sensitive measure. This is due to the fact that volume and hence mass will increase with the cube of the length if the shape and density of the organism remains constant during growth. A standard weighing procedure was adopted. This involved removing the snails from their containers, placing them carefully on tissue paper, removing the excess water from the aperture and shell with tissue paper and finally weighing them after 10 min to the nearest 0.1 mg on a Mettler balance. The 10 min time interval was chosen because a plot of percentage loss of weight against time revealed that the former measure was tending to reach an asymptotic value at this time. The change in weight can be expressed in a variety of ways: (i) Absolute growth, represented by plotting W (=weight in mg) against time. (ii) Absolute growth rate represented graphically by plotting d W or dW/dt against t. (iii) The relative or specific growth, the growth of unit mass of the system represented graphically by plotting either log W or d W/ W against t. (iv) The relative or specific growth rate, the growth of unit mass per unit time repre-

326

J. D. T H O M A S

sented graphically by plotting d W/Wd6 or d log Wjdt against t . The relative growth rate can also be expressed as a percentage; mg/100 mgjday or

(ii)Length. The diameter of the shell measured along the axis running through the centre of the spire and the junction between the aperture and the body whorl was taken as a measure of change in length. The length measurements were subjected to the methods of analysis already outlined for the weight measurements. b. Measurements of natality rate of the snails The number of egg masses in each container was counted daily and removed. In some cases the number of eggs per mass was also counted. The oviposition rate was expressed as the number of egg masses or as the number of eggs per snail per day. The results were expressed as mean values per treatment. c, Monitoring of factors in the media The following factors were monitored : pH, conductivity, nitrogenous substances, protein, total carbohydrate, calcium, magnesium, sodium, potassium and ammonia. (i) Nitrogenous substances were measured as total ninhydrin-positive substances. These were measured by the spectrophotometric method of Rosen (1957). This method measures the a-amino nitrogen in amino acids and small peptides by the reduction of the a-amino group to ammonia which then forms a blue colouring with the ninhydrin. Ammonium compounds released by the snails and plant food also give a positive colouring with ninhydrin. The results were expressed in terms of nitrogen. (ii) Total ammonium nitrogen was measured by a modification of the Conway diffusion method (Conway, 1962) and later by an E.I.L. ammonia electrode. (iii) Protein was measured by the Folin phenol reagent method described by Lowry et al. (1951). (iv) Total carbohydrate was measured by the anthrone method (Chaykin, 1966). (v) Lipids were measured by the method of Bragdon (1951) after they had been extracted from the medium by an acidified mixture of chloroform/ methanol. (vi) Other factors. pH was measured with a Pye model 78 pH meter, specific conductivity with an Electronic Switchgear conductivity meter in pmho cm-2 and the ions with an Ependorff flame photometer and an Unicam SP90A Ser. 2 atomic absorption spectrophotometer. (vii) Exogenous cellulase was measured by the fluorimetric method described by Robinson (1956).

SCHISTOSOMIASIS A N D CONTROL OF M O L L U S C A N HOSTS

327

IV. RESULTS A. EXPERIMENTS CARRIED OUT IN CLOSED SYSTEMS

1. The influence of volume and number of snaiIs on rates of growth and reproduction The various density treatments to which the snails were subjected are summarized in Table I. The 400 ml, 16 snail treatment was replicated five times and all the other treatments ten times. At the commencement of the experiment the snails weighed approximately 40 mg. The wet weight of each snail was determined on the first, third and twelfth day but on the sixth and ninth day the snails in each treatment were weighed together. The growth is expressed as absolute growth or as absolute growth rate in mg/snail/day.

TABLE I Treatmenfs used in experiment to investigate the influence of voltme of snails on rates of growth and reproduction

atid

number

Vol. S.S.W.a No. of snails and vol. per snail (latter in parenthesis)

(ml) 25 50 100 200 400 a

1 (200 ml)

1 (400 ml)

2 (200 ml)

1 (100 ml) 2 (100 ml) 4 (100 ml)

1 (50 ml) 2 (50 ml) 4 (50 ml) 8 (50 ml)

1 (25 ml) 2 (25 ml) 4 (25 ml) 8 (25 ml) 16 (25 ml)

S.S.W.=standard snail water.

Natality rate was monitored over a period of fifteen days in the case of snails kept in 25 ml per snail treatment and over a period of 21 days in all the other treatments. The S.S.W. was changed every third day. a. Growth. The snails selected for the various treatments shown in Table 1 were all within a narrow size range initially as indicated by the small standard errors of 1.5-6.7 % of the mean (Fig. 2). Analyses of variance undertaken on the absolute growth data for the treatments in which volume per snail treatment is varied and numbers stabilized indicate that despite the high variances, there are statistically significant treatment effects at P < 0.05 in the two-, four- and eight-snail treatment by the twelfth day (Fig. 2; Table 11). Similar, though not statistically significant trends at P>O*O5also occur in the onesnail treatment. These results taken as a whole indicate that over a 3 day period the optimum volume for growth is about 50-100 ml per snail. At volume per snail treatments smaller and greater than this there is a tendency for the absolute growth to increase and decrease, respectively, with increase in volume.

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J. D. T H O M A S

220-

T

200 -

F

180160-

$1

c ._

140-

'B

E

T

II

'20:

f" loo-

:8 0 - 9 60c Q

a 0

40 20 -

0No.snails , Volume/snoil (mll

'

4

1 2 4

16,

25

Fro. 2. The mean absolute weights of snails with standard errors on days 0, 3 and 12. The treatments are given in Table I.

When volume per snail is stabilized and number varied there is a general tendency for the absolute growth to increase when the number of snails is increased to two and then to decrease with further increase in snail numbers (Table 11; Fig. 2). Analysis of variance carried out on the data indicate, however, that statistically significant treatment effects at P <0.05 are only found in the 25 ml per snail treatment. It can be concluded, therefore, that there is a TABLE I1 Absolute growth achieved by snails subjected to treafrnents in Table I on /he twelfth day aster commencement of experiment together with probability ( P ) values for the various treatments

VOl. per snail

(ml) 25 50 100 200 400 P values

1

2

No. snails 4

P 8

16

138.4k 5.8 150.9k 6.6 134.9& 5.9 128.8k 3.4 124.0& 3.7 175.7 k 235 202.4 k 16.9 189 -9 8.3 183.0k 4.7 178.1 k21.2 195.0k 13.3 178.4k 10.1 162.6k28.2 142.9k 17.8 136.5k20.2 >0.05 <0.05 <0*001 10.001 -

values

< 0.05 > 0.05 > 0.05 > 0.05 -

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329

TABLE I11 Absolute growth rates expressed as daily increments in mg per snail achieved by snails subjected to treatments in Table I by the twelfh day together with probability ( P ) values for the various treatments

Vol.

per snail (ml)

1

No. snails 4

2

P

16

8

8.82f0.22 8.62 f 0.31 8.65 f0.41 7.73 f 0.33 7.15 f0.23 11*51f1*9113.64f0.60 12.78f0.60 11.92izO.45 11.62+ 1.78 13.29f 1.27 11*80+1-12 10.01 f2.38 8.73 f 1.36 400 8.11f1.76 P values >0.05 <0.01 < 0.01 < 0.001 25 50 100 200

values < 0.01 > 0.05 > 0.05 > 0.05 -

significant tendency for the absolute growth to decrease with increase in snail numbers of the 25 ml volume treatment. The trends shown by measurements of absolute growth are also reflected in the data for absolute growth rates. Thus Table I11 and Fig. 3 show that when the number of snails is stabilized and volume per snail varied there is a tendency for absolute growth rate to be highest at between 50 and 100 ml per snail. At volumes per snail less and greater than this the incremental growth rate tends to increase and decrease respectively with increase in volume. Analysis of variance undertaken on these data shows that there is a significant treatment effect for the two-, four- and eight-snail treatments by the twelfth

f z 4

-6

3

I r

25

50

L

100

> &

2 3

400

FIG.3. The absolute growth rate, expressed as mean daily increments per snail, achieved over a period of 12 days. The treatments are as outlined in Table 1. Standard errors are given.

330

J. D. T H O M A S

day. Stabilization of volume per snail and variation of number results in a statistically significant tendency at P <0.01 for the incremental growth achieved by snails in the 25 ml treatment to decrease with increase in number. As was the case with the data for absolute growth there is also a tendency, although it is not statistically significant, for the two snail in 50 and 100 ml per snail treatments to be optimal for absolute growth rates. b. Reproductive rate. The snails began to oviposit in some of the 50, 100 or 200 ml per snail treatments only five days after the commencement of the experiment. Table IV shows the mean number of eggs per egg mass for the first egg masses laid by snails in the various density treatments. When numbers are stabilized and volumes per snail varied there is a tendency for the values to be higher in the 50-100 ml treatments. With the exception of the treatment containing four snails, however, there are no statistically significant effects. Conversely when volumes per snail are stabilized and numbers of snails varied there is a tendency for the values to be lower for the treatments containing the isolated snails. In fact none of the isolated snails in the 25 ml per snail treatment oviposited. Analyses of variance revealed that statistically significant treatment TABLE IV The mean number of eggs per egg mass for the first egg masses laid by snails subjected to treatments in Table I together with probability ( P ) valuesfor the various treatments Vol.

per snail

No. snails

(mu ~~~~

1

2

P 8

4

16

values

~

14.17k2.97 10.86f 1-40 19.46f 1-60 13.93f 1.08 8.83 k 2.05 18.00f 1.99 17.78 k 2.68 16.58 t 2.03 10.50f 3.69 19.13k2.12 21.00f2.86 5.45f 1-63 12.20f 2.32 5.67 f0.67 P values >0.05 < 0.05 0.05 > 0.05 25 50 100 200 400

<0*01 < 0.05 > 0.05 > 0.05

-

TABLE V The mean absolute weights of snails subjected to treatment in Table I at commencement of egg laying No. snails

per container

Mean wt in g

1 2 4 8 16

0.2090k 0.0233 0.1729 f0.0098 0.1591 f0.0064 0.1337 +OQ095 0.1161 k0.0161

Vol. per snail in ml 400 200 100 50 25

Mean wt in g 0.2129 0.1529? 0.0336 0.1786f 0.0114 0*1756+00062 0.1286 k 0.0048

SCHIS'TOSOMIASIS A N D C O N T R O L O F M O L L U S C A N H O S T S

331

T

lor 09t

08-

.= 07-

f

e

06-

3

05-

VI

Ec"

z

3

04-

c

03-

T

02-

0 I0-

1 2 4 8 1 6 L--7--J

25

LL!

J

3

400

(mll

FIG.4. The mean daily rate of production of egg masses per snail during the period of oviposition covered in the experiment. The treatments are as outlined in Table I. Standard errors are given.

effects occurred only in the 25 and 50 ml per snail treatments. Table V shows that isolated snails tend to be larger and older than the grouped snails when they start to oviposit. The analyses of natality rates for the entire period are summarized in Fig. 4 and Table VI. When numbers are stabilized and volumes per snail varied there are statistically significant treatment effects for the one-, two-, four- and eight-snail treatments. The optimum volume for natality rates is again between 50-100 ml per snail. At volume treatments less and greater than this there is a tendency for natality rates to increase and decrease respectively with increase TABLE VI The mean natality rate, expressed as mean number of eggs per snail per day, achieved by snails subjected to treatments in Table I during the period of oviposition covered by the experiment together with probability ( P ) values for various treatments VOl. per snail (ml)

25 50 100 200 400 P values

1

2

No. snails 4

8

16

0 0.162L-0.0320*136+0.0300.085+0*0200.057+0.017 0.552+0.121 0.887+0.0880.935k0.089 0.641rt0.060 0.569+012 0.497k0.050 0554+0.058 0.481f 0.069 0214k0.033 0227+0*057 < 0.05 <0.001 <0.001 < 0~001 -

P values <005

< 0.05 > 0.05 <002

-

332

J.

I). TIHOMAS

in volume. When numbers are varied and volumes per snail stabilized there is a statistically significant tendency for natality rate to decrease with increase in snail numbers in the 25 and 200 ml treatments. It must be noted, however, that isolated snails in the 25 ml per snail treatment do not oviposit. There is also a statistically significant treatment effect in the 50 ml per snail treatment and a suggestion that the two or four snail treatment is optimal for natality rates. Table VII gives the mean number of egg masses laid by snails subjected to the various treatments on the first, second and third days after renewal of the S.S.W. The values given are based on all the data gathered during the course of the experiment. When numbers are stabilized and volume per snail treatments varied, analyses of variance show that with the single exception of the TABLE VII Mean daily rate of egg mass production per snail deposited on successive days after renewal of S.S. W . by snails subjected to the treatments in Table Z together with probabiIity ( P ) values for the various treatments First Day Vol.

per snail (ml)

1

2

No. snails 4

8

16

P values ~~

25 50 100 200 400 P values

0.325 f 0.153 0.125 k 0.056 0,262k 0.087 0.154 k 0.065 0.956f0.284 0.912k0.173 0.994f0.164 0.672k0.112 0526 k 0.207 0.350+ 0.067 0.425 f 0.086 0.280 f0.070 0.08 k 0.043 0.200 k 0.088 <0.01 < 0.001 < 0.001 < 0.05 -

> 0.05 > 0.05 > 0.05 > 0-05 -

No. snails 4

P values

-

Second Day

Vol. per snail (ml)

1

0.333 f 0.130 0.526f0.207 200 0.504 f 0.1 34 400 0.225 f 0.084 P values <0.05

25

50 100

2

8

16

0.167 f 0.079 0.167 f 0.059 0.075k 0.026 0.041 k 0.014 0.850+ 0.138 0.925 f 0.148 0.662 f 0.105 0.567k0.092 0.675k0.122 0.292 + 0.070 < 0.001

<0.001

< 0.001

-

2

No. snails 4

8

16

< 0.05 > 0.05 > 0.05 > 0.05 -

Third Day Vol. per snail (ml)

1

~

25 50 100 200 400 P values

0.075f 0.041 0.1 17k 0.043 0.006 f0.006 0.227+0.091 0*900+0147 0.887k0.153 0.594f0.096 0.630 k 0.208 0.55 f 0.050 036k 0.094 0.600 k 0.133 0-27 0.069 0.300f0*114 >0.05 < 0~001 <0.001 < 0.001

P values ~

-

< 0.05 > 0.05 > 0.05

> 005

-

-

SCHISTOSOMIASIS A N D C O N T R O L OF M O L L U S C A N HOSTS

333

one-snail treatment on the third day there are statistically significant treatment effects for the one-, two-, four- and eight-snail treatments on all three days. It can thus be seen that even when the data is broken down the optimum volume for the rate of egg mass production is 50-100 ml.At volume per snail treatments less and greater than this egg mass production tends to increase 12

0Isnail 2snails 09

6 08-

P0

4

07-

T

In

50

T

TI

e 200

I 2 3

400

FIG.5. The change in rate of egg mass production per snail per day on successive days after renewal of S.S.W. Standard errors are given. The treatments are as outlined in Table I.

and decrease respectively with increase in volume. Stabilizing volume per snail and increasing snail numbers also results in a statistically significant tendency for the rate of egg mass production to decline with increase in snail numbers in the 25 mI per snail treatment on the second and third days. Inspection of Table VII also shows that there is a tendency for egg mass production to decrease with time in both the 25 and 50 ml treatments and to increase with time in the 100, 200 and 400 ml treatments. These trends are shown more clearly in Fig. 5, but none is statistically significant. A detailed account of this work is being prepared for publication (Thomas and Benjamin, in preparation). 2. The influence of weights of snails involved in interactions on rates of growth and reproduction In the experiments described in this section attempts were made to determine the extent to which the effects on growth and reproduction of snails are 13

334

J. D. T H O M A S

influenced by the biomass and degree of sexual maturation of the snails involved in the interactions. In the first series of experiments the snails in the various weight categories were placed at densities of 0, 1, 2, 4, 8 and 16 in 200 ml of S.S.W. whereas in the second series snails in the same or different weight categories were placed either singly or in pairs in 200 ml of S.S.W. These experiments were described below. a. Experiments with 100,300,500and 700 mg snails at densities of 0, 1,2,4,8 and 16 in 200 mi of medium. Each treatment was replicated five times with the exception of the “0” treatment which was only duplicated. The experiment was carried out twice. Previously aerated tap water was used in the first experiment and S.S.W. with the same chemical formula in the second experiment. Although the ages of the snails was not known other studies have shown that snails in the 100, 300, 500 and 700 mg weight categories are approximately 7,9, 10.5 and 14 weeks old when fed on lettuce in S.S.W. The majority of the snails reach sexual maturity after they reach a weight of 100 mg. The growth and reproductive rates of the snails were monitored at three day intervals for up to 15 days. Figure 6, which summarizes the mean absolute growth rates achieved by the snails over the entire period, shows that there are statistically significant differences between some of the means in all the treatments involving snails of different weights. In the case of sexually mature snails in the 300 and 500 mg categories increasing the densities from one to two in the former and from one to four in the latter resulted in significant growth promotion. Further increases in density beyond the optimum resulted in a significant depression of absolute growth rates. Inspection of the data shows that there are no significant differences between the mean absolute growth rates achieved by 300 mg snails kept at densities of one and four per 200 ml of medium or between those kept Initial weight of sirs categories IOOrna

300rng

500rng

11 1 2 4816

7001110

T T

I 2 4 8 2 4

No.of snails in 200ml S.S.W.

Fro. 6. The mean absolute growth rates achieved by snails in various weight categories. Standard errors are given and probability values (F’) based on analyses of variance were < 0.001 in each case.

SCHISTOSOMIASIS A N D C O N T R O L O F MOLLUSCAN HOSTS

335

at densities of eight and sixteen per 200 ml of medium. Similarly there are no significant differences in the absolute growth rates achieved by 500 mg snails kept at densities of 1, 4 or 8 snails in 200 ml of medium. In contrast, the summarized results (Fig. 6 ) do not reveal a significantgrowthpromoting effect with increase in density of 100 and 700 mg snails. Analyses of the detailed results show, however, that the absolute growth rates achieved by paired 100 and 700 mg snails in 200 ml of medium were significantly higher (P c 0.05) than the controls containing one snail per 200 ml of medium on two occasions in the former and on one occasion in the latter. The detailed results reveal that on several occasions the single snails in 200 ml of medium had achieved higher absolute growth rates than paired snails. However, the differences in this experiment were not statistically significant at P < 0.05. Increasing the densities of the 100 mg and 700 mg snails beyond eight and four per 200 ml of medium, respectively, resulted in significant depression of the absolute growth rate. So far as the summarized results are concerned there are no statistically significant differences between the absolute growth rates achieved by 100 mg snails kept at densities of one, two and four per 200 ml or between the absolute growth rates of 700 mg snails kept at densities of one and two per 200 ml of S.S.W. Thus the summarized results indicate that under the conditions of this experiment the optimum volumes for growth of snails in the 100, 300, 500 and 700 mg weight categories are 100-200 rnl, 100 ml, 50 ml and 100-200 ml respectively. The mean rate of egg mass production achieved by snails in the various weight categories is summarized in Fig. 7. Analyses of variance carried out on these data indicate that there are significant differences between some of the treatment means in all the weight categories. There is a general tendency

5Wmg

TI 700mg

T

300mg 101

T

T

IOOmg

11 b 2 4 8 1 6

1 2 4 8 1 6

LD ! 4 8 1 6

4 8

No of snails in 200ml S S W.

FIG.7. The mean daily rate of egg mass production by snails of various weights. Standard errors are given and probability values (P) were < 0.001 in each case.

336

J. D. T H O M A S

for the rate of egg mass production to show an inverse relationship to density in all four cases. Analysis of the more detailed results taken at three-day intervals revealed optima in the two snail per 200 ml treatments for snails in the 500 and 700 mg weight categories. However, the differences were not statistically different at P < 0.05. b. Experiments with 100 and 300 mg snails at densities of 1 or 2 per 200 ml S.S. W. In this experiment attention was focused on the 100 mg snails because of the conflictingresults in the previous experiment. The results of the treatments which are summarized in Fig. 8 show that isolated 100 mg snails in 200 ml S.S.W. achieve significantly higher absolute growth rates than paired snails in the same volume of S.S.W. There was no significant difference between the absolute growth rates achieved by snails in 100 ml and those kept at a density of 100 ml per snail in 200 ml of medium. In contrast to the 100 mg snails paired 300 mg snails achieve significantly higher absolute growth rates than isolated snails in the same volume of S.S.W. c. Experiments with single and paired snails in the 25, 50, 100 and 150 mg weight ranges in 200 ml S.S. W. The results of this experiment which are summarized in Fig. 9 show that in all cases paired snails tended to achieve higher absolute growth rates than isolated snails. However, none of the differences between single and paired snails in the 50 mg weight category was significantly different at P < 0.05, possibly because the standard errors tended to be higher I I-

-

10

-

0" 9 -

3

._

K .8ii! 7-

4

' 6-

+

f

z

g

5-

2

3n

4-

i :: I-

OL Initial weight in mg No. snails VOl. s s w. Vol /snail

1

.OC #OO 100 I 1 2 200 DO 200 200 100 200

300 300 1 2 200 200 200

too

FIG.8. The mean absolute growth rates achieved by 100 and 300 mg. snails subjected to density treatments of one and two snails per 200 ml. S.S.W. Standards errors are also given.

337

SCHISTOSOMIASIS A N D CONTROL OF MOLLUSCAN HOSTS

than in other weight categories. Figure 9 shows that statistically significant differences were observed in all the other weight categories. The data for egg production given in Fig. 10 show that none of the 25 mg snails had laid eggs by the end of the experiment and that the paired snails in the other weight categories tended to lay more numerous and larger egg masses than solitary snails. Figure 10 shows, however, that not all the differences are statistically different. In general there is a tendency for the differences to be more marked in the case of snails in the 100 and 50 mg weight categories which were beginning to mature at the commencement of the experiment. Figure 11 shows that the natality rate tends to increase with the time allowed for conditioning. This effect was more marked in the treatments involving snails in the 50 and 100 mg weight categories. 100mq

50mg T

150mg

._

i

81

25mg snails

I

3

6

9

1

2

3

6

9

1

2

3

6

9

1

,

3

6

9

1

2

Days from start ofexperiment

FIG.9. The mean absolute growth rates achieved by snails in various weight categories when kept at densities of one and two per 200 ml. S.S.W. Probability values (P) based on “t” tests areindicated by asterisks, P
d. Experiments with snails in the same or diferent weight categories placed either singly or in pairs in 200 ml of S.S. W. Because the results for the 100 mg snails in the previous experiments conflict, the present experiment was designed to test the hypothesis that larger snails might promote the growth of snails smaller than themselves whereas small snails had either a neutral or negative effect on the growth of snails larger than themselves. The treatments used and the results of the experiment are given in Fig. 12. Analyses of variance carried out on these data showed that statistically significant differences occurred between some of the means for snails in the 25 and 50 mg categories but not for those in the 150 mg weight category. It would appear from these results that pairing of 25 mg snails with snails of the same size or with larger snails in the 100 or 150mg weight categories resulted in growth promotion in all cases. Further statistical analyses showed that the absolute growth rate of snails in this weight category was significantly correlated with the biomass of the donor snails (correlation coefficient 0.6154; P
338

J . D. T H O M A S

FIG.10. The mean daily rate of egg and egg mass production per snail for snails in 50, 100 and 150 mg weight categories subjected to density treatments of 1 or 2 per 200 ml. Standard errors are given.

FIG.11. The mean daily rate of egg production per snail for single and paired snails in the 50, 100 and 150 mg weight categories on successive days after change of 200 ml. S.S.W. Standard errors are given.

S C H l S T O S O M l A S l S A N D C O N T R O L OF M O L L U S C A N HOSTS

339

snails with others of the same weight resulted in a slight, but statistically insignificant, inhibition of growth rate compared with the control. On the other hand pairing of 50 mg snails with 150 mg snails resulted i n significant growth promotion of the former. Correlation analysis carried out on these data gave a coefficient of 0.3583; P <0.001. The pairing of 150 mg snails with others of the same weight and with snails smaller than themselves had no statistically significant effect on their growth rate. The absence of a growth effect in the case of snails in the 150 mg weight category could, however, be masked by the losses resulting from egg production. The fact that there is a reciprocal relationship between natality and absolute growth rates of snails gives some support to this 1413 -

12

9p .E

5

-

II10-

F

2

I50 mg

~

T ** ***

98

-

p!

e 7-

f

60 a

51

-s zi

5432I-

0Wtcafegory

25 25 25 25

50 50 50 50 0 25 50 150

150 150 150 150 0

25 50150

in pair

FIG.12. The mean absolute growth rate achieved by single and paired snails in the same and different weight categories in 200 ml. S.S.W. Results of analyses of variance are indicated thus: P < 0.05=*, P < 0.01 = **, P < 0.001= ***. Standard errors are given.

hypothesis. Thus “r” values based on regressions involving these two quantities give values of -0,4386; P0,05 for paired and single snails respectively. Figure 13 shows that the natality rate is very much higher for paired than it is for single snails. Isolated snails in the 25 mg weight category did not oviposit. e. Experiments involving pairing of 50 nzg snails with others in the 25, 50, 15, 100 and 150 mg weight range. The apparent lack of competence of the 50 mg snail to respond to pairing is of interest and it was, therefore, decided to reinvestigate the effects of pairing them with snails in the same and different weight

340

J. D. T H O M A S

. . 07

I

-

c

1-2

2

1.0 -

5 0.8-

z

-

0.6

040.2

-

nWt.cotego6 25 50 150 25 25 25 50 50 I50

Initialwt Of 0 other mil in

0 0 25 50 150 50 150 150

pair

FIG.13. The mean daily rate of egg mass production per snail achieved by single and paired snails in the same and different weight categories during the entire period of the experiment. Standard errors are given.

::1

12

50mg snails P < 0.001

IIx

10 -

.3 9-

<

E

8-

._ 7 2 0,

6-

f

I 5 -

K

f

2I -

-

n-

0 25 50 75 100 150 lnitiol weights of other s d in pair n 200ml

FIG.14. The absolute growth rates achieved by snails in the 50 mg weight categories, at the end of a 12-day period, when they were kept isolated or paired with snails in the 25, 50, 75 and 150 mg weight categories. Standard errors are given.

SCHISTOSOMIASIS A N D CONTROL OF MOLLUSCAN HOSTS

341

ranges. The results in Fig. 14 show that pairing with snails up to 100 mg had no statistically significant effect on growth rate. There was a tendency although it was not statistically significant for the absolute growth rate of 50 mg snails to be depressed after pairing with 75 mg snails. Once again, however, the paired 50 mg snails laid more numerous and larger egg masses than single snails. Thus the mean values in this experiment were 0.14 egg masses per snail and 8.50 eggs per mass and 0.10 egg masses per snail and 2.00 eggs per mass for paired and single 50 mg snails respectively. It is possible, therefore, that the lack of a growth promotion effect following pairing in this weight category may be due, in part, to the resultant increase in natality rate. A fuller account of this work is being prepared for publication (Thomas and Benjamin, in preparation).

3 . Conditioning of media by snails The snails may alter the chemical composition of their aquatic environment by removing certain components including ions, amino acids and oxygen or by adding substances including ammonia, carbon dioxide, ions, mucopolysaccharides and other organic substances from their excretory systems, alimentary canals and body walls. Plants and micro-organisms if present will also influence the chemical environment either directly or indirectly as a

-0.1 25 ml per snail 50 1L II

.---a 0-0

~-~1~011 I1

II

0. 0. 0.5

-

0.6 -

-

0.7

0.80.9

-

1.0-

1.11.2-v-

Days

1.3-

1 1.4

0

2

I

20

d0

3

4

I

l

60

50

5 l

I

6 I

100 120 140

7 I

i

160 180

Hours

FIG.15. Mean pH time profiles for isolated 100 mg, lettuce-fed snails placed in various volumes of S.S.W. for a period of up to 180 h.

342

J. D. T H O M A S

result of interaction with the snails. The following changes were observed to occur following the introduction of snails. a. p H . The pH-time profile (Fig. 15) shows that the pH of aerated S.S.W. containing lettuce discs, which was initially 8-3-85, declines rapidly for a period of approximately 48 h following the introduction of individual 100 mg snails into various volume treatments. After this, as the pH approaches neutral values, the rate of decline becomes much slower. The rate of decline is influenced by volume and it is interesting to note that it tends to be slower in the 25 ml treatment than in the others. It has been observed in a separate experiment that aerated media have higher pH values than non-aerated controls. Some of the pH-time profile studies have revealed small but consistent increases in the pH values of heterotypically conditioned media. It is possible that these are caused by the removal of carbon dioxide by the lettuce discs as a result of photosynthetic activity. The possibility that the snails themselves also photosynthesize is receiving consideration as recent studies have shown that certain molluscs contain chloroplasts of plant origin and are, therefore, capable of photosynthesis (Naidu, 1971; Hinde, 1972; Trench et al., 1972). Figure 16 shows the pH values in non-aerated 200 ml volumes which had been heterotypically conditioned by lettuce-fed snails of different initial weights kept at various densities ranging from 1 to 16 per 200 ml for 3 days. The pH of the S.S.W. in the control treatment conditioned only by lettuce discs remains almost constant. In contrast the introduction of snails results in a decline in pH, the extent of which is intluenced by numbers, biomass and growth rate. With the exception of the 700 mg snails which are approaching the stationary phase of somatic growth the rate of decrease in pH is positively correlated with biomass. In all four weight treatments the pH continues to

8 5.c

3

0-0

8oR=, c

100mg snails

0--030011

iJ\

16

'Xu-p/x

Number of snails in 200ml

Re. 16. Mean pH values after S.S.W. had been conditioned for three days by lettuce-fed snails in 100, 300, 500 and 700 mg weight categories kept at various densities.

SCHISTOSOMIASIS A N D C O N T R O L OF M O L L U S C A N HOSTS

343

decrease with increasing snail numbers until a density of 4-8 snails per 200 ml (50 or 25 ml per snail) is reached. Further increase in snail density beyond this level results in slight but consistent increase in pH. pH profile studies similar to the ones described above except that they involved the use of aerated, heterotypically conditioned media with food and non-aerated, homotypically conditioned media without food also produced basically similar patterns. There were, however, two noteworthy differences. First, the pH declined less in the aerated media than in the non-aerated media. Second, the pH in the pH-density profiles of the aerated media continued to decline with increasing density. Scatter diagrams show that there is a tendency for absolute growth rate and the rate of decline in pH to be positively correlated. Regression analyses were, therefore, carried out using the absolute growth rates of snails that were initially in the 100, 300, 500 and 700 mg weight categories and the corresponding rates of decline in pH values over the same period. It was found that the regression coefficients were significant, at P <0.001 only for the first two weight categories. Regression analysis based on data from subsequent growth studies involving juvenile snails weighing approximately 40 mg also failed to yield statistically significant values. The final pH achieved after three days of conditioning need not necessarily be a good guide to growth conditions. Thus optimum growth was achieved by the 500 mg snails in the 50 ml per snail treatment where the pH stabilized at around 6.2. In contrast the best growth was achieved by the 300 mg snails at a volume per snail treatment of 100 ml and a final pH of 7.1. b. Conductivity and the concentration of cations present in the defined medium (S.S.W.). The introduction of snails into a defined medium containing lettuce discs is followed by a consistent, slight increase in the conductivity of the medium which lasts for a period of approximately 5 h. The extent of the increase is a function of biomass and a 300 mg snail in 100 ml can cause the conductivity to increase by as much as 15-18 pmho cm-2. This is followed by a rapid decline in conductivity and in calcium ion concentration (Figs 17, 18). These changes are accompanied by the rapid increase in hydronium ion concentration noted in the previous section. The factor mainly responsible for the decline in conductivity in the external medium is C a + + which is removed by the snails and utilized for growth. It is not unexpected, therefore, to find that the rate of decrease of both C a + + and conductivity is a function of biomass and absolute growth rate. The analyses of a large number of regressions involving incremental growth rate of the snails and the rate of depletion of calcium from the external medium have shown that significant positive relations invariably exist between these. Similar analyses involving incremental growth rate of the snail and the rate of decrease in conductivity also show in the majority of cases that a significant positive relation exists between these two. The fact that significant positive relations are not always found between decreases in conductivity and incremental growth rate can probably be attributed to the tendency for the conductivity to increase towards the end of the conditioning period as a result of the accumulation of the ammonium ions from the snails and of other ions from the faeces and plant material macerated by the snails.

344

J. D. T H O M A S

520 480560

t

= DqO

0 :

.= 0 X = A =

I " 2

"

"4 "6 " 7

E

2

440-

+-+-t-t-t-+

E Y.

._ c 400._ .xz >,

'

t 360-

3

320280 -

240 -

Number of snails in 61itres

Fro. 17. Mean changes in conductivity of media conditioned by 500 mg lettuce-fed snails at densities of 10, 20, 30, 50, 90 and 170 in 6 1.

Calcium is depleted rapidly by growing snails and under optimum conditions when the calcium concentration in the external medium was 80 pg ml-1 a maximum net uptake rate of 11-12 pg mg -1 wet weight of snail per day has been recorded. It is not, therefore, surprising to find that under high density conditions the snails can reduce the calcium levels in the external medium to the minimum equilibrium concentration of approximately 1.5 pg ml -l (Fig. 18). At concentrations below this the snails cannot achieve a positive calcium balance. When compared with the calcium ion the concentrations of the other cations present in the defined medium change very little after the introduction of snails. In the case of the sodium ion, net uptake rates of 13-318 and 34-316 pg g -1 wet weight of snail per day have been observed for lettuce- and cellulosefed snails respectively. These values range from 1 to 6 % of the uptake values for calcium. However, a positive relation exists between the net uptake rate of sodium and the incremental growth rate for the snail as was the case with the calcium ion. This low uptake rate makes very little impression on the sodium concentration of 13.6 pg ml-1 in the external medium even when the snails are maintained under high density conditions of 16 per 200 ml. The concentration of potassium and magnesium in the external medium, although they change very little, show a net increase unlike calcium and sodium. The rate of increase of potassium expressed in pg per g wet weight of snail varies from 13 to 84 and from 4 to 51 for lettuce- and cellulose-fed snails respectively. These values range from 0.05 to 4.6% of the uptake values for

SCHISTOSOMTASIS A N D C O N T R O L O F M O L L U S C A N HOSTS

345

IOOl-

‘0O0 L Number of snoils in 6litres

FIG.18. Mean changes in calcium concentration in media conditioned by 500 mg snails at densities of 10, 20, 30, 50, 90 and 170 in 61.

calcium under the same conditions. The changes in magnesium are equally small and sometimes erratic. In general the magnesium values in the external medium show a net increase of up to 67 pg 8-1 wet weight of snail per day although in some cases small decreases of up to 13 pg g -1 wet weight of snail per day were observed when snails were fed on pure celluIose. c. Organic substances. The values given in Table VIII are based on aliquots taken from media held under bacteriostatic conditions to reduce the possibility of bacterial degradation of the end products. The methods used to achieve these conditions involved pretreating the snails, food, S.S.W., the containers and the air supply and will be described in detail in a later paper. It can be seen that appreciable quantities of lipids, carbohydrates, proteins and ninhydrinpositive nitrogenous substances are released into media which are being heterotypically conditioned. The rate of release of all the substances, except for N.P.N., when expressed in mg per g wet weight of snail, shows an inverse relation to snail density. The values for the rate of production of N.P.N. and F.P.S. are very similar to those obtained under non-bacteriostatic conditions in other experiments. The rates of release of carbohydrates, proteins and ninhydrin-positive substances into homotypically conditioned media are positively correlated with snail density and are considerably less than was the cage in heterotypically conditioned media. This is particularly marked in the case of the carbohydrates where the values for homotypically conditioned media are only 0.8917.8 % of those in heterotypically conditioned media.

346

J. D. T H O M A S

Other investigations have shown that there is an inverse relationship between the rates of N.P.N. and F.P.S. production and the weights of the snails. Thus the snails in the 100, 300, 500 and 700 mg weight categories produce approximately 500, 220, 200 and 100 pg N g-1 wet weight of snail per day and 1.85, 1-35, 2.1 and 1.0 mg 8-1 wet weight of snail per day of F.P.S. when kept at densities giving volumes af 100-12.5 ml per snail. There is a strong tendency for the rate of N.P.N. production to be positively correlated with growth rates of the snails. As already shown the optimum treatments for growth in closed systems are those in which snails are allowed to condition 100 or 50 ml for a period of three days. The highest values of N.P.N. per g of snail per day are also normally found in these treatments and at volume per snail treatments smaller and greater than this the rate of N.P.N. production tends to be less. In constrast the amount of F.P.S. per g wet weight of snails tends to be positively correlated with snail density. Estimates of the rates of N.P.N. and ammonia N production in the same media indicate that approximately 93.0 % of the N.P.N. is ammonia nitrogen. Ammonia is, therefore, the main excretory product. Other constituents of N.P.N. are amino acids including glycine, aspartic acid, serine, histidine, lysine, valine, glutamic acid and polypeptides. Qualitative tests for urea, creatinine and uric acid carried out on concentrated samples of heterotypically conditioned snail water proved negative. A fuller account of this work is being prepared for publication (Thomas, Powles and Benjamin, in preparation). It has also been shown in this laboratory that snails release exogenous cellulase into the medium. The amount produced is a function of biomass and metabolic activity of the snails. Thus deprived snails previously fed on Bemax and pure cellulose under bacteriostatic conditions and weighing approximately 120, 250 and 500 mg, produced 12.96rf: 3-12, 4.89 k 2.02, 5.64 rf: 1.11 pg of cellulase per g snail per day. It is to be expected that plantfed snails would produce more exogenous cellulase because of their higher metabolic rate and also because they would release cellulases of plant origin (Whitehead, unpublished). 4. Influence of conditioned media on growth of snails in closed systems In the previous experiments carried out in closed systems without partitioning the stimulatory and inhibitory effects could have been caused by competition for feeding sites and by density-dependent stimulation of tactile and visual sensory organs. By using a closed system with partitioning as described in the methods section (111) these possibilities are excluded and any observed effects can only be due to the chemical conditioning by the snails kept at various densities that were used to produce the heterotypically conditioned media tested. a. Growth. Figure 19 shows mean specific growth (loge W) of assay snails at weekly intervals. The mean initial weight of these snails was approximately 20 mg and at this time they were sexually immature. Each mean value is based on measurements made on 40 snails. Analyses of variance carried out

TABLE VIII The net rate of release of carbohydrate as anthrone positive substances (A.P.S.), lipid, protein as Folin positive material (F.P.S.), nitrogenous substances as Ninhydrin positive nitrogen (N.P.N.)produced by snails with plant food (Het. C.W.) and snails withoutfood (Horn. C.W.) kept at various densities. The results are expressed as mg per g wet weight of snails per day Density of 5o(Moo mg snails in 2 1. Substance

measured A.P.S. (as glucose) A.P.S. (as glucose) A.P.S. (as glucose) Lipid (as lipid) Lipid (as lipid) F.P.S. (as protein) F.P.S. (as protein) F.P.S. (as protein) F.P.S. (as protein) N.P.N. (as N) N.P.N. (as N) N.P.N. (as N)

Nutritional state of snails Lettuce-fed Lettucefed Deprived % of lettuce-fed Lettuce-fed Lettuce-fed Lettuce-fed Lettuce fed Lettuce-fed Deprived % of lettuce-fed Lettuce-fed Lettuce-fed Deprived % of lettuce-fed

A

I

5

10

20

40

80

0.41 f0.160 0.46 k 0.096 0.80f0.087 1-05+0.107 0.51 f0.020 0.39 f0.010 0.83 0057 0073 & 0.01 5 0~016f0*010 0.088 f0.026 0.083 f0.023 (1-62) (18-04) (17.80) (11 0.29 & 0.07 0-02f0.001 0.03 f0-002 0.12f0.03 0.01 f0.003 0-01k 0.001 0.27 f0.09 0.01 f0.003 0.04 5 0.02 0.005 f0.001 1-74f0.05 1.55 f0.09 1.37f0.03 3.78 +- 0.22 1.51 f0.07 1*35k 0.06 0.74+ 0.01 2.31 k 0.09 0.88 k 0-02 0.66? 0.01 0.49 f0.02 1.30 f0.08 0.42 f0.02 1.76 & 009 0.60& 0.01 0.12k0.004 0.19&0.004 0.36 5 0.03 0.1 8 k0.01 0-04 (24.9) (28.08) (31.17) (42.65) 0.196 f0.007 0.1 14f0.022 0.1 70 5 0-017 0.109 f0.022 0.213 5 0015 0-253f0.084 0.112k0.037 0.217 k 0.105 0.193 50.062 0-237f0.108 0.1 11& 0.010 0.032 f0.007 0.065 f0.010 0-085k0-005 f 0.005 (14.72) (43.73) (33.42) (35.78) 1.80f0.333 1.70 f0.020

>

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t

348

J. D. T H O M A S ,Week

.-*-*\*-. -0 . 10

I

20

t 1

1

I

30

50

90

0

I

170

Number of snails in Glilres

FIO. 19. The mean specific growth (loge W.)achieved by isolated assay snails in 200 ml when subjected to media conditioned by lettuce-fed snails at densities of 10, 20, 30, 50, 90 and 170 snails in 6 1.

on these data show that there is a statistically significant difference P < 0.05 or P < 0.01 between the treatment means from the first week until the end of the experiment nine weeks later. The highest specific growth was achieved by snails treated with water conditioned by 50 snails in 6 1. Treatment with water conditioned by snails kept at lower and higher densitites than this resulted in poorer growth. Further analyses carried out with the variances indicates that the treatment effect is mostly due to the linear C1 and quadratic C2 components; the cubic component C3 contributes little if at all. It can be concluded, therefore, that there is only one optimum treatment for growth. The optimum growth achieved by snails subjected to the 50 snail per 6 1. treatment can be attributed to the relatively high specific growth rate achieved by the snails in the first three weeks of the experiment.

SCHISTOSOMIASIS A N D C O N T R O L OF M O L L U S C A N HOSTS

349

b. Reproduction. Two snails, one in the 30 snail per 6 1 and the other in the 70 snail per 6 1treatment began to oviposit 11 days after the commencement of the experiment when they were 10and 9 mm in diameter respectively. The mean weight of the assay snails at this time was approximately 100 mg. The percentage of snails ovipositing increased steadily, until by the sixth week of the experiment nearly all the snails were ovipositing, at least once a week (Fig. 20). The treatment resulting in the highest percentage of ovipositing snails was the water conditioned by the snails kept at a density of 50 per 6 1. Water conditioned by snails kept at densities higher and lower than this resulted in lower percentages of ovipositing snails. Figure 21 shows the mean number of egg masses produced each week by the snails subjected to the various density treatments. Analyses of variance carried out on these data shows that there is a statistically significant difference between treatment means. During weeks 3-5 most egg masses were laid by snails in the 50 snails per 6 1 treatment. The optimum shifted later to the 20 snails per 6 1 treatment in weeks 6 and 7, and to the 30 snails per 6 I treatment in week 9. Density treatments above and below these optima resulted in a lower production of eggs and egg masses. Egg production in all the treatments increased up to the sixth or seventh week and then declined. A fuller account of this work is being prepared for publication (Thomas, Goldsworthy and Benjamin, in preparation). . I

8

9

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s

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(I)

:g 2

.+

4.

B * .=

E

8

,*---•

6o -

40-

20

21 10

20

30 50 90 Number of snails in 6litres

170

FIG.20. The percentage of the isolated assay snails ovipositing each week when subjected to media conditioned by lettuce-fed snails at densities of 10, 20, 30, 50, 90 and 170 snails in 6 1. (Shell diameter in parentheses.)

3 50

J. D. THOMAS

B. INFLUENCE OF VARYING CONCENTRATIONS OF CALCIUM AND AMMONIA ON GROWTH AND REPRODUCTION OF SNAILS

I. The influence of ammonia on growth rate The main excretory product of the snail is ammonia which, in an alkaline solution, may exist as the ionized or non-ionized form:

NH~+H~O+NHI++OHThe extent of ionization is influenced by the pH of the solution. Figure 22 shows that the p H value for the ammonium ion at 25°C is 9.27. At this pH the concentration of the ammonium ion is equal to that of free ammonia. All the ammonia exists as the ionized form at 6.5 pH and below, but at pH 84-10.0 the percentage of free ammonia increases rapidly. By the time pH 12.0 is reached all the ammonia exists as the non-ionized form. Any

I

10

I

I

I

I

20 30 50 90 Total number of snail / 6 litres

I

170

FIG.21. Mean number of egg masses laid per week by isolated assay snails in 200 ml when subjected to media conditioned by lettuce-fedsnail at densities of 10,20, 30, 50,90 and 170 snails in 6 1.

S C H I S T O S O M I A S I S A N D C O N T R O L O F M O L L U S C A N HOSTS

351

experiments designed to investigate the effect of ammonia must, therefore, take pH into account. Recent work has shown that the effects of ammonia on aquatic organisms can also be influenced by the concentration of dissolved oxygen (Ball, 1967). The experiments designed to investigate the effects of ammonia concentration on growth are still in progress. In the preliminary experiments 20 mg snails were placed in 50 ml beakers containing 0, 1, 10, 25, 50 and 100 units of ammonium chloride in unbuffered S.S.W. Each treatment was replicated 10 times. The media were changed and the weights of the snails monitored at three day intervals. The ammonia unit is 41.4 pg of ammonia N per 50 ml as this is the estimated rate of production of ammonia by actively feeding 20 mg snails per day. These varying concentrations of ammonia were also tested in the following media: S.S.W. buffered to pH 9.0 with borate buffer, S.S.W. buffered to pH 7.0 with tris buffer and unbuffered S.S.W. to which equivalent amounts of calcium chloride had been added instead of calcium bicarbonate. The results of the experiments involving ammonia added to unbuffered S.S.W. are given in Fig. 23. The addition of one unit of ammonia results in a slight but statistically insignificant increase in specific growth rate on days 3,6 and 9. In later experiments, however, this promoting effect was found to be statistically significant at P < 0.05. Further increases in concentration of ammonia result in a progressive decrease in specific growth rate. The reduction in specific growth rate compared with the controls was, however, only statistically significant at P < 0.05 when the concentration was 25 units or more on days 3 and 6 and at concentrations of 100 units or more on day 9.

90 -

100

80 -

70

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13

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FIO.22. The relationship between pH and ammonia.

3 52

J. D. T H O M A S

The pH of the unbuffered medium, which was initially 8.3, was influenced very little by the introduction of the small 20 mg snails. Previous experiments have shown that the pH declines at less than 0.2 pH per day when small snails of this size are introduced into 50 ml of S.S.W. In general the growth rates in the treatments containing small concentrations of ammonia tended to be less in the buffered media than in those that were not buffered. Increasing the concentrations of ammonia in media buffered to pH 7.0 with tris buffer and in the unbuffered media containing calcium chloride instead of calcium bicarbonate had much less marked inhibitory effects than was the case in unbuffered medium. In contrast snails kept in media buffered at pH 9.0 with borate buffer suffered heavy mortality rates and pronounced growth inhibition, particularly in treatments containing high concentrations of ammonia. A more detailed account is being prepared for publication (Thomas, Powles and Benjamin, in preparation). 2. The influence of calcium concentration on growth and reproduction It has been shown in previous experiments that the growth rate of snails is significantly correlated with the rate at which the snails remove calcium from their aquatic medium. The following experiment was, therefore, designed to investigate the effects of varying the volume of medium per snail, the C a f f concentration and total available calcium on the growth and reproductive rates of the snails. The snails used in the experiments had an initial weight of 25 mg and were placed individually in 6.25, 12.5, 50, 100, 200 and 400 ml

w3-w0/W0.

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16-

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080604 02

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102550 75100

Ili I 3 25 50 75 100

00

Units of ammonia (see text)

FIG.23. The mean specific growth rates achieved by 20 mg snails subjected to various concentrations of ammonia. Standard errors are given.

SCHISTOSOMIASTS A N D C O N T R O L O F M O L L U S C A N HOSTS

353

in a flow system with a volume of 5.5 ml, and a flow rate of 5 ml h-1. The calcium concentrations used in each of the volume treatments were 0, 2.5, 5 2 0 , 40 and 80 pg ml-1. Each treatment was replicated five times. The snail weights, egg output, calcium concentration and pH were monitored at threeday intervals for a period of up to 21 days. The experimental routine was similar to that already described. The rate of Ca + + uptake was investigated graphically by plotting the mean rate of uptake, v (in pg mg-1 wet weight of snail per day) against the initial calcium concentration ( S ) in pg per ml. The accumulation ratio is linear for small snails in the 25-50 mg weight range when they are placed in small volumes of 50 ml per snail. At volumes of 100 ml the rates begin to level off and become non-linear. With further increases in volume to 200 and 400 ml there was evidence that the uptake mechanism was becoming saturated at concentrations of 40-80 pg ml-1. However, towards the end of the experiment when the snails were larger, complete saturation was not observed. It is possible, therefore, that the calcium concentration in the external pool is a limiting factor for snail growth even in the larger volumes. In the case of the 400 ml treatment it was found by plotting (1/ v) against (1/S) that the relationship of the rate of uptake of Ca + to the external concentration resembled the +

16

c

0 5 20, 25 10

Vol /snail

Flow

400ml

0 5 20 0 5 2 0 I 2 5 10

200

*

100

50

5 2 0 8 0 0 5 2080 2 5 10 40, 2 5 10 40

0

,

125

6 25

FIG.24. The mean absolute growth rates achieved by snails subjected to various calcium concentrationsin different volumes of S.S.W.Standard errors are given.

Michaelis-Menton model for enzyme substrate kinetics. This suggests that a limited number of sites or carrier molecules are involved in calcium uptake. To determine the relationship between total available calcium and the mean rate of uptake, v, all the values for the various volume treatments for a particular three-day period were pooled and the mean values plotted against the total available calcium per three days. It was found that v increased with

354

J. D. THOMAS

total available calcium until it reached an asymptotic value of 11.2 pg g wet weight per day when the value of the latter was 8 mg per 3 days. Not unexpectedly the mean daily incremental growth rate of the snails also reached an asymptote after a value of 8 mg of available calcium per 3 days had been reached. Figure 24 shows that for the 200, 100 and 50 ml treatments there was a tendency for incremental growth rate over a selected three-day period to be positively correlated with Ca + + concentrations. This tendency was less well marked in other volume treatments although in all cases, except for the flow system, there were significant increases in growth rate when the calcium concentration was increased from 0 to 2.5 pg per ml. The snails show a positive calcium balance in media containing more than 1.5 pg of Ca++ per ml. Figures 24 and 25 show that there are optimum volumes for growth when the calcium concentrations are at the highest values of 80 pg per ml. From Fig. 24 it can be seen that the highest incremental growth rate for snails over a selected three-day period was achieved by those in the 100 ml treatments. However, when the mean overall specific growth rates over the entire period of investigation were plotted against the volume per snail, optimum growth was achieved by those in the 50 ml treatment. The next highest specific growth rate was achieved in the 100 ml treatment. Both the incremental and specific growth rates are less at volumes smaller and greater than these optima. Figure 26 shows that the rate of egg production also tends to be correlated positively with the concentration of calcium in the medium. The egg production rate, like the growth rate, also increases with total available calcium until a value of 8 mg available calcium per three days is reached. Increasing the amounts of calcium beyond this level results in a slight decrease in the rate of egg production. Figure 26 also shows that when the initial concentration is 80 pg calcium per ml the optimum volume for egg production is also I00 ml, as was the case with incremental growth rates. A fuller account of the work is being prepared for publication (Thomas, Benjamin, Cocks and Lough, in preparation).

C.

INFLUENCE OF HETEROTYPICALLY AND HOMOTYPICALLY CONDITIONED WATER ON THE GROWTH RATES OF ASSAY SNAILS IN OPEN SYSTEMS

The experiments carried out to investigate the effects of conditioned water on growth of isolated assay snails in closed systems have shown that optimum growth was achieved when the assay snails were treated with heterotypically conditioned media, produced by snails fed ad libitum with lettuce, at a density of from one snail to 50 or 100 ml for a period ranging from 3 to 7 days. Heterotypically conditioned media produced by snails kept at densities higher and lower than this resulted in slower growth rates by the assay snails. As a working hypothesis it can be postulated that the increased growth rate resulting from decreasing the volume from 400 ml per snail to the optimum

S C H I S T O S O M I A S I S A N D C O N T R O L O F M O L L U S C A N HOSTS

355

volume is due to the concentration of growth-promoting factors released either by the snail itself or as a result of its action on the plant food whereas the decreased growth rate at volumes less than the optimum is due to depletion

Vol /snail

FIG.25. The mean specific growth rates achieved by snails subjected to various volume per snail treatments when the calcium concentration was 80 pg

ml-l.

I .--

12-

I

2 II-

8 2P

1098-

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FIG.26. The mean daily rate of egg production per snail achieved by snails subjected to various calcium concentrations in different volumes per snail treatments.

356

J. D. T H O M A S

of essential ions such as calcium or to the addition of specific or non-specific toxic substances. Attempts were, therefore, made to ascertain whether such a growthpromoting factor(s) exists and to characterize it. A series of experiments was designed to answer the following questions: (1) Is the activity a function of concentration ? (2) What is the approximate molecular weight? (3) What is the chemical nature of the substance? (4) Does it originate from the snail or the plant or from both of these? (5) Does it act directly on the snail or indirectly on the snail through its food? To make it possible to characterize the growth-promoting factors it is necessary to isolate and purify them and to assay them in circumstances which preclude the possibility that the effects on growth can be attributed to the effects of resource depletion or to factors produced by the assay snails themselves. In the first series of experiments heterotypically conditioned S.S.W. was used. This was prepared by maintaining 40 mature snails of approximately 500mg in weight in 4 1 of aerated standard snail water and feeding them with fresh lettuce discs in excess of requirements for a period of 72 h. To achieve bacteriostatic conditions the containers and food were previously sterilized, the S.S.W. filtered through 0.22 pm Sartorius filters, the air supply filtered through packed sterilized cotton wool, and the snails pretreated by placing them in sterile S.S.W. containing streptomycin (50 units per ml) for 60 min and sterile S.S.W. containing penicillin G sodium salt (200-400 units per ml) for three days. The heterotypically conditioned water (Het. C.W.) produced by snails fed on plant food and homotypically conditioned water (Hom. C.W.) produced by snails fed on pure cellulose were filtered through 0.22 pm filters before being finally fractionated. The fractions were then concentrated and dialysed by means of 160 mm diameter Amicon, Diaflo, ultrafiltration membranes with various macrosolute retention characteristics located in magnetically stirred 1-2 1 perspex and later steel ultrafiltration cells pressurized to 20 p.s.i. with nitrogen. The concentrated fractions were dialysed with the aid of viscin tubing and carbowax in the first four experiments and subsequently by using the Amicon filters. The various concentrated fractions were made up to the desired volumes with S.S.W. The fractions were assayed in the flow apparatus described in the methods section (111). The results are summarized below: 1. Heterotypically conditioned water In the first experiment assay snails were subjected to the four treatments given in Fig. 27. The Het. C.W. was assayed at one unit strength which is the estimated amount of growth-promoting factor released by feeding 500 mg snails into the S.S.W. over a three-day period. Each treatment was replicated ten times. Analyses of variance carried out on these data show that there were statistically significant treatment effects from the sixth day onwards. The results of these experiments and subsequent ones make it possible to draw the following conclusions. The Het. C.W. prepared using the above method

S C H I S T O S O M I A S I S A N D C O N T R O L O F M O L L U S C A N HOSTS

357

and diluted to unit strength retains its activity and promotes the absolute growth rates of the snails to a significant extent compared with controls. Removal of molecules larger than 1 x 105 and 100 000-500 mol. wt resulted in partial and complete loss of activity compared with controls. The growthpromoting factors are, therefore, between 500 and 105 mol. wt. Subsequent experiments made it possible to conclude that most of the growth-promoting factors are contained in the molecular weight fraction 500-104 and that they are resistant to boiling and pronase treatment. Increasing the concentration of the 500-104 mol. wt fraction up to 2 and 4 unit strength resulted in significant growth promotion by day 9 in the open system and day 6 in the closed system (Fig. 28). Dilutions of 0.125 and 0.25 of unit strength Het. C.W. resulted in a reduction in absolute growth rate compared with controls in S.S.W. When expressed as specific growth rates these reductions proved to be significantly different compared with controls in S.S.W. (Fig. 29). It appears, therefore, that there are both growth inhibitory and growth stirnulatory factors present in Het. C.W. In this experiment the treatment in which the 500-104 fraction was diluted to unit strength did not produce a statistically significant growth-promoting effect. One possible source of the growth factor is the plant food as it has been shown that lettuce homogenates added to S.S.W. can induce statistically significant growth-promoting effects at concentrations of 50 mg of homogenate per 100 ml S.S.W. (Benjamin, in preparation). It is also possible that the faeces of lettuce-fed snails may be a source of the inhibitory factor, as homogenized faeces introduced at one unit strength (the amount produced by one 500 mg lettuce-fed snail per three days) caused a reduction in absolute

Treatment 81- a = ssw b =CSW c = C S W (-XM100) d = CSW(-UM05)

P-=O 001

L

5

g

4-

f 3-

P>O 05

n 8

4

2-

I-

Days

I

PCO 001

P
T

T

rli. 3

- a b c d 3

a b c d

6

a b c d 9

> C d 12

+

FIG.27. The mean absolute growth rates of assay snails treated with S.S.W., Het. C.W., Het. C.W. filtered through XMlOO Amicon filters (cut-off, 105 mol. wt), Het. C.W. filtered through UMO5 Amicon filters (cut off, 500 mol. wt).

358

J. D. T H O M A S

growth rate compared with controls. However, the differences were not statistically significant at P < 0.05. Subsequent experiments on snail faeces undertaken in this laboratory indicate that the absolute growth rate and specific growth rates of lettuce-fed snails with access to their faeces was significantly higher than snails in the control which were denied access to the faeces by means of partitions. However, the addition to the medium of faeces from lettuce-fed snails resulted in a statistically significant growthinhibitory effect. In contrast the faeces of snails which had fed on pure cellulose over a sufficientlylong period for all the faeces containing plant material to be evacuated had no statistically significant effects on growth rates (Cocks, unpublished). A full account of the work on the growth factors in heterotypically conditioned media is being prepared (Thomas, Aram and Goldsworthy, in preparation).

2. Homotypical!y conditioned water (Hom. C .W.) The experiments on Hom. C.W. have shown that media produced by snails fed on pure cellulose tend to have a more potent growth promoting effect than medium prepared by deprived snails previously fed on lettuce. As with the Het. C.W. most of the growth-promoting activity appears to reside in the 500-10-4 mol. wt fraction. The active factors are resistant to

t

P .z 0.01

F

-

).I0-1250.2

4

8.50

2

Units of k t C W

FIG. 28. The mean absolute growth rates achieved by assay snails subjected to S.S.W., and Het. C.W. in molecular weight range 500-104 mol. wt at concentrationsof 0-125,0*25, 0 5 0 , 1.0, 2.0 and 4.0 unit strength. (One unit strength is amount of material in mol. wt range 500-104 produced by 500 mg snails in 100 ml S.S.W.when fed on lettuce over a 3-day period). Standard errors are given.

SCHISTOSOMIASIS A N D C O N T R O L O F M O L L U S C A N HOSTS

359

boiling and acid hydrolysis. Dilution of Hom. C.W. did not result in growth inhibition but in some cases it was necessary to have fractions concentrated to two or four unit strength before significant growth promotion could be achieved. Attempts at separating active components of boiled and unboiled UM05 retentate, containing the molecular weight fractions of 500-10 O00 mol. wt by the use of activated charcoal, and anion and cation exchange columns did not succeed. A full account of the work on Hom. C.W. is being prepared (Thomas, Aram and Whitehead, in preparation). V. DISCUSSION A.

CLASSIFICATION OF EFFECTS

The various effects observed on growth and fecundity of juvenile snails have been shown to be a function of numbers, biomass, volume or space, and time, and may be classified as follows: 1. Positive feedback or Allee eflect

This refers to an increase in growth and natality rates achieved by individual snails when snail numbers or biomass in a given volume are increased, volume 16-

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P c 0~001

> 12$ II10-

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4-

32-

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0.25 0.50

0,125

4

2

Units of Het.C.W.

FIG.29. Specific growth rate achieved by assay snails subjected to the treatments given in Fig. 28 between the third and sixth day of the experiment. Standard errors are given.

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per snail decreased and the time allowed for conditioning increased up to certain critical thresholds. The experimental results indicate that snails of all sizes are competent to respond to the growth-promoting effects of conditioned media but that the effects are less well marked in the case of larger snails, weighing 700 mg because they are in the post-logarithmic phase of slow growth. In general, there is a positive relation between snail biomass and the ability to produce conditioned media favourable to growth and reproduction. It would appear that small, juvenile snails in the lag or early logarithmic phase of growth are less capable of producing a conditioned medium with growth promoting properties than larger, sexually mature snails in the logarithmic phase of growth. In some cases increasing the density of small juvenile snails by only one to two per 200 ml S.S.W. resulted in a reduction in growth rate. The Allee effect has been observed previously in molluscs including Menetus dilatatus buchanensis (Lea) by Fried and Goodchild (1963), Ampullaria sp. and Physa sp. by Rose and Rose (1965) and Biomphalaria glabrata (Say) by Pereira and Deslandes (1954), Chernin and Michelson (1957a,b) and Jobin and Michelson (1967). Previous workers on B. glabrata all achieved the Allee effect by reducing the volume of the container and keeping the number of snails constant, thus effectively reducing the volume available to each snail. By using this method Chernin and Michelson (1957a, b) found that the optimum treatments for both growth and natality rates were 50 snails per 5 1 (100 ml per snail) on three occasions and 25 snails per 5 1 (200ml per snail) in one case. It is of interest that these optima are very similar to those found in the present investigations although there were differences in the nature of the food plant, the chemical composition of the aquatic media and time allowed for the conditioning process to occur. Jobin and Michelson (1967) were mainly interested in natality rates and found that there was an inverse relationship between the natality rate of B. glabrata and volume of water. Thus when they decreased the volume of water in the containers from 7.6 to 4.5 1 the mean, daily fecundity rate of the snail increased from 10-95 to 15.5 eggs per snail despite the fact that this was accompanied by a slight numerical increase in the average number of snails per container from 7 to 9.5. According to Baily (1939) and Chernin and Michelson (1957a,b) increasing the numbers of snails in a given volume is deleterious to growth. Thus the latter authors state, “Authorities are not agreed as to how a greater volume causes greater growth but all are agreed that it does.” This view also appears to be supported by Jobin and Michelson (1967); they state that in laboratory sized habitats of constant volume, temperature and food density the birth and growth rates of snails vary inversely with the number of snails in the population. The present work shows clearly that increasing the numbers of snails in a constant volume of 200 ml and even increasing the numbers of snails when the volume per snail is stabilized can result in enhancement of growth and reproductive rates. The statements made by the above authors are, therefore, oversimplifications because they overlook the Allee effect. The Allee effect is not unique to snails, It has been reported in a variety of other organisms including small mammals where it is known as the Whitten

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effect (Wilson, 1970), birds (Allee et al., 1949; Wynne Edwards, 1962), insects (Uvarov, 1966; Wharton et al., 1968; Hodjat, 1969; Murdie, 1969), Daphnia (Frank et al., 1957), bacterial populations (Dean and Hinshelwood, 1966) and for populations of somatic cells (Stoker, 1967). 2. Growth inhibition or negative feedback efects observed when snails in the lag or early logarithmic phase of growth have their density increasedfrom one to two per 200 ml per 3 days This effect was in marked contrast to the positive feedback effects observed when larger snails, and sometimes other snails of the same size, were subjected to the same treatment. It is comparable to the inhibition of maturation of adult desert locusts by inhibitory pheromones which appear to be produced by immature larvae and adults (Richards and Mangoury, 1968).

3 . Negative feedback effects or inhibition of growth and reproduction observed under conditions of high density (or small volumes per snail < 50 ml per snail per 3 days) This effect is obtained by decreasing the volume per snail below 50 ml either by reducing the volume or by increasing the numbers of snails or by increasing the conditioning time beyond a certain threshold. Over this range growth and natality rates are inversely related to density and positively related to volume per snail. At the extreme end of the range growth of somatic and reproductive tissue may cease. This result known as the crowding effect has been widely reported in laboratory populations of snails including Limnaea stagnalis by Hogg (1 854), Semper (1874), Limnaea palustris elodes by Forbes and Crampton (1942), Limnaea columella by De Witt and Sloan (1958), Bulinus globosus by Shiff (1964), Biomphalaria angulosa by Sturrock (1965), Oncomelania spp. by Van der Schalie and Davis (1965), Bulinus forskali by Wright (I 960), Biomphalaria glabrata by Chernin and Michelson (1957a,b), Sturrock and Sturrock (1970) and Gazzinelli et al. (1970), Bulinus africanus ouoideus and Bulinus (B.) obtusispira by Webbe and James (1971). The two latter species seem to be particularly sensitive to crowding and breeding ceases when numbers exceed three per litre in the former and one per litre in the latter. There is some circumstantial evidence that crowding effects are commonly encountered under natural conditions particularly in areas subject to marked seasonality in rainfall (Sturrock and Sturrock, 1970; Jobin and Michelson, 1967) despite some views to the contrary (Shiff, 1964). Thus Berrie (1969) found there was a strong positive relation between growth and natality rates of Biomphalaria sudanica and the volume of a small pond in Uganda. In contrast Eisenberg (1966, 1970) varied numbers of Limnaea elodes in experimental field enclosures and found that there was a significant inverse relationship between snail density and their mean size and fecundity. Varying density had no apparent effect on the survival of adult snails. The density dependent responses observed were powerful enough to restore the numbers to the original equilibrium levels within a year.

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B. FORMULATION OF HYPOTHESES TO ACCOUNT FOR EFFECTS OBSERVED ON GROWTH AND REPRODUCTIVE RATES

1. Positivefeedback or Allee efects The concept of an optimum population density contributing to the survival of the individual genotype by increasing the probability of success of the individual in searching for a mate, food, a favourable physico-chemical environment and avoiding unfavourable environmental factors such as predators, parasites, pathogens and competitors has been extensively developed by Allee et al. (1949) and by later ecologists including Wynne Edwards (1962). The possible interactions involving snails which may have resulted in positive feedback are discussed below: a. Increased frequency of mating behauiour. Increasing the population density of bisexual organisms, in which male animals fertilize females, up to a certain threshold, will increase the probability of females receiving sperms. Allee et al. (1949) have shown that the optimum initial population density of Tribolium for natality rate per female per day was four individuals in 32 g of flour. At lower densities the frequency of copulation was below that which resulted in maximum fecundity. The influence of density on frequency of copulation is clearly not as important for a facultative, self-fertilizing hermaphrodite such as B. glabrata. The experiments described in this paper show, however, that paired snails normally achieve higher growth and natality rates than single snails. It is possible, however, that the increased natality rates observed in these experiments were a consequence of the increased growth rates. Subsequent experiments carried out on virgin snails in this laboratory show, however, that copulation over periods ranging from 2 to 24 h can result in significant increases in natality rates. A full account of this work will be published in a later paper. An increase in natality rate resulting from copulation alone has also been reported in other molluscs including Physa and Heliosoma species (De Witt and Sloan, 1958) and in insects (Englemann, 1970). b. Increased density related stimulation of chemical, tactile or visual receptors of individual snails. It has been shown that stimulation of growth and reproductive rates of isolated, assay snails can be achieved by both Het. C.W. and Hom. C.W. and by fractions of these in the molecular weight range 500-104 mol. wt. It is not, therefore, necessary to invoke density-dependent stimulation of tactile and visual receptors to explain the positive feedback effects, although the possibility that they may also be implicated, to some extent, when snails are allowed to interact freely, cannot be eliminated. The involvement of the eyes is considered unlikely, however, in view of their primitive anatomy (Hyman, 1967), their dorso-lateral location behind the tentacles and the absence of melanin in the albino snails used in the present study. The following questions are clearly important in connection with the chemical substances involved in inducing positive feedback effects : (i) Where are the possible sites of production ? In the case of Het. C.W. possible sites of production include the plant food, microbial organisms such as symbionts and also glands located externally or within the alimentary

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canal of the snail. The following observations lend some support to the hypothesis that some of the positive feedback effects at least are caused by substances which originate from plants. First, it has been shown in this laboratory that a strong positive relationship can be demonstrated between the growth of assay snails and the concentration of homogenized lettuce introduced into the medium when the latter was varied from 50 to 800 pg per 100ml of S.S.W. (Benjamin, unpublished). Second, it has been shown that the addition of indole acetic acid (I.A.A.), a plant hormone, into S.S.W. at concentrations of 10-3 or 10-4 M, results in significant growth promotion of small, juvenile snails. The experimental evidence indicates that the effects are caused by the I.A.A. or possibly its breakdown products acting directly on the snail. Other plant hormones including kinetin and gibberellic acid had no statistically significant effects on snail growth rates when they were introduced into S.S.W. (Cocks, unpublished). The exogenous plant factors as well as the products of decomposition may, therefore, be important to the snail in its natural environment. The present investigation indicates that although plant food on its own releases relatively little lipid, carbohydrate, ions and nitrogenous organic material including proteins into the medium the rate of release is increased enormously by the grazing action of the snail. The extent to which plant hormones are released by the action of the snail and the effective concentration which can be found in the proximity of feeding snails have yet to be measured. Third, Jobin and Michelson (1967) found that there was a strong positive correlation between snail growth rates and density of plant food although the latter was apparently in excess of requirements in all treatments. It is possible, therefore, that these effects were caused by exogenous plant factors as their concentrations would be positively correlated with plant biomass in a given volume. The fact that Hom. C.W. obtained under bacteriostatic conditions and its 500-10000 mol. wt fraction can promote growth indicates that the snails themselves are also releasing a growth-promoting factor. The site of production has not yet been identified. There is very little evidence to support the hypothesis that it is released from the alimentary canal in faeces. Preliminary experiments carried out in this laboratory indicate that the absolute growth rates of lettuce-fed snails with access to faeces are significantly higher than was the case with control snails denied access to faeces by partitioning. However, the addition to the medium of faeces from lettuce-fed snails resulted in a statistically significant growth inhibition in assay snails. In contrast the faeces from snails fed on cellulose over a sufficiently long period for all the plant material to be evacuated had no statistically significant effect on the growth rate of assay snails (Cocks, unpublished). These experiments will however be repeated before any firm conclusions are drawn. The other possibilities, that growth-promoting factors are released in the urine or from external exocrine glands including those secreting mucus, have yet to be investigated. Recent experiments in this laboratory provide no support for the hypothesis that growth factors might be retained in slime trials left by lettuce-fed snails and maintained at densities ranging from one to sixteen per 200 ml in S.S.W. for three-day periods. In contrast the work of

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Best et al. (1969) suggests that presliming by crowded Planaria may have slightly suppressed the fissioning rates of assay animals. It is clear that further efforts should be made to identify the sites of production of the growth factors. (ii) What is the nature of the growth-promoting factor? One exogenous factor released into the medium by snails is cellulase (Whitehead, unpublished). However, the question of whether it is produced by endosymbionts or by the snails has not yet been resolved. The other factors occurring in the 500 to 104 mol. wt fraction have not yet been identified. It has been shown, however, that the active fraction is heat stable and resistant to pronase treatment and hydrolysis. Some of the activity, at least, is retained for up to a month in the aquatic medium. Substances which might be implicated are polypeptides, glycoproteins and small growth factors such as I.A.A. which may be bound to the larger molecules. It has been suggested that lipoproteins enhance the growth of chick embryo cells (Rubin, 1966). The fact that the growth potency of the active fractions from Het. C.W. is not directly proportional to concentration indicates that both growthstimulatory and growth-inhibitory factors might be present in the same medium. Two simple models can be advanced to explain the results. (a) The mature snails are producing one factor which has inhibitory effects at low concentration and stimulatory effects at high concentration. It can be postulated that at low concentrations receptors which have a low threshold are activated and that these initiate a chain of physiological events which inhibit growth. At higher concentrations the factor activities other receptors with higher thresholds which trigger off physiological events resulting in faster growth. (b) In the other hypothesis it is suggested that two factors, one promoting and the other inhibiting growth, are produced. It is postulated that the growth inhibitory factor activates receptors with low thresholds which initiate a chain of physiological events resulting in growth inhibition. The growthpromoting factor, on the other hand, activates receptors with higher thresholds which initiate a chain of events resulting in increased growth rates. It is possible that the physiological events involved in growth promotion might themselves produce factors which block the receptors involved in initiating the inhibitory effects. If two antagonistic factors are implicated it will clearly add to the difficulty of identification. Unfortunately it is also possible that several factors are involved. Recent studies on pheromones indicate that they commonly occur in complex mixtures in mammals and even in some insects. Thus the sex attractant of the Californian ips, Ips confusus, is a mixture of three simple terpenes which are released into the faeces. It may act as a mating stimulant to both males and females (Wilson, 1970). In contrast only single pheromones appear to be involved in some insects. This also appears to be the case with the aquatic pulmonate Helisoma duryi, which releases a polypeptide pheromone of approximately 104 mol. wt from damaged tissue. This causes snails of the same species to bury themselves in the substrate (Snyder, 1967; Wilson, 1970). (iii) What are the modes of action ? Pretreatment of plant food with both

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Het. C.W. and Hom. C.W. and with 500-104 mol. wt fractions of these had no discernible effects on the growth rates of assay snails. It can be concluded, therefore, that the growth-promoting factors are acting directly on the snail. The possible modes of action are as follows: (a) Stimulation of external chemoreceptors which activate the endocrine system to produce factors which accelerate growth of somatic and reproductive tissues. The existence of such a mechanism has been postulated in mice by Whitten (Wilson, 1970) and in locusts by Richard and Mangoury (1968). Whitten demonstrated that an odorant found in the urine of male mice induces and accelerates the oestrus cycle in female mice. Mature male desert locusts also produce a volatile pheromone but in this case it accelerates the maturation of both immature males and females. The locust pheromone appears to be able to induce both releaser and primer effects (Richards and Mangoury, 1968). Investigations on the behaviour of B. glabrata have shown that the 500-104 mol. wt fractions of both Het. C.W. and Hom. C.W. serve as attractants to the snails. It is possible, however, that the attractants are different chemicals from those which promote growth. (b) Direct stimulation of metabolism resulting in increased growth of somatic and reproductive tissues without the intermediate involvement of the central nervous system. It is possible that the growth factors may enter through the body wall or through the mouth by trophallaxis and act directly on the target tissues as specific metabolic stimulators. (c) Removal or inactivation of toxic factors. An undefined aquatic medium may contain toxic ions such as Zn++ and Cu++ which inhibit growth and this appears to have been the case with the media used by Chernin and Michelson (1957a,b) and Fried and Goodchild (1963). Other potential sources of growth inhibitors or toxins are the food plants and micro-organisms. It can be postulated, however, that the snails can produce substances which inactivate the inhibitors either directly, or indirectly, by interacting with the food plant. Such a mechanism would become effective when the concentration of the anti-inhibitors exceeded that of the inhibitors. This could be achieved by inducing individual snails to increase the rate of production of antiinhibitors, or by increasing snail density, above critical thresholds. No unequivocal evidence has so far been produced to demonstrate the existence of an Allee type, density-dependent, detoxifying mechanism in snails but it appears to have been demonstrated in E. coli populations by Hofsten (1962). This author showed that the copper ion inhibited growth of E. coli under anaerobic conditions in small inocula but not in large ones. However, it is not necessary to invoke this mechanism to explain the Allee effect observed in the present investigation because a defined medium was used and increasing food density resulted in growth promotion rather than inhibition. (d) Increasing the concentration of leaky metabolites necessary for growth. It has been shown that aquatic molluscs lose certain metabolites including amino acids through the urine and body wall (Potts, 1967; Hammen, 1968) and that they may also accumulate them from dilute solutions (Stephens, 1972). These observations may possibly provide an explanation for the present results. Thus it can be postulated that the reduction in growth rates of snails 14

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in larger volumes or in flow systems is caused by the fact that such conditions favour the efflux rather than the influx of metabolites necessary for growth. In contrast the influx rate and hence growth would be favoured by increasing snail density and by reducing the volume. It is possible that the conditioned media and the active fractions favour growth because they contain the essential metabolites in higher concentration. Eagle and Piez (1962) have shown that such a mechanism may be involved in influencing the growth of somatic cell populations. When the cell density is low nutritional requirements such as serine and asparagine leak out at such a rate that growth fails unless a compensatory exogenous source becomes available. Increasing the cell density above a critical level does, however, make it possible to establish a favourable equilibrium between the intra- and extracellular pools of metabolites. Plans are being made to investigate the effects of free amino acids and other metabolites on the metabolism and growth of snails in the laboratory. (e) Facilitation of entry of metabolites and ions through the body wall by exogenous factors produced by snails. It would also be of interest to investigate the possibility that growth factors might act by facilitating the uptake of essential ions such as Ca++ from the medium. If such factors do exist they would resemble those recently discovered by Hodges et al. (1971). Apparently these factors called ionophores, which may also act as antibiotics, form complexes with cations thus facilitating their uptake by plants. (f) Facilitation of ingestion and assimilation of food. Observations in this laboratory have revealed that B. glabrara is a highly social animal commonly occurring in large aggregates both on food and on uniform substrates. As a working hypothesis it is suggested that the evolution of this social habit has been influenced, to some extent, by the fact that the snails release into the water enzymes which help them to dislodge and predigest food plants including epiphytes. It would then follow that the increased concentration of enzymes resulting from the social habit would increase the efficiency of the ingestion and assimilatory processes. Exogenous enzymes are known to be released by insect larvae which live socially in liquid or semi-liquid food. Thus blowfly larvae release proteolytic enzymes into the excreta so that the meat on which they live is partially liquefied before it is eaten and there is some evidence for the existence of an Allee effect (Chapman, 1969). This hypothesis receives some support from the following observations: First, it has been noted that the extent to which lettuce discs in the medium become predigested is positively correlated with snail density in S.S.W. Second, it is known that snails release cellulase into the medium (Whitehead, unpublished). Third, the addition of cellulase to S.S.W. at a concentration of 6.4 pg ml -1 causes significant growth promotion in assay snails compared with controls in S.S.W. and S.S.W. containing boiled cellulase. The pH of the media used in these experiments was approximately 7-0. However, the pH at the site of action of the cellulase on the plant food was probably near the optimum of 5.4 for cellulase because the snails condition the substrate with glycoproteins which have a pH of about 5.0. Unfortunately recent work in this laboratory has revealed that the necessary concentration of cellulase is

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not apparently achieved in media conditioned by snails. Thus deprived snails previously fed on bemax and cellulose paper release only about 1.5 % of the required amount of cellulase over a three-day period. It is to be expected that the amount released into Het. C.W. by feeding snails would be higher and this is currently being measured (Whitehead, unpublished). It is possible, however, that other enzymes, in addition to cellulases, are implicated or that the snail cellulases are more potent than those used in the growth assay experiment. 2. Negative feedback effects or inhibition of growth and reproduction observed under conditions of high density The evidence for the possible involvement of certain environmental factors in the causation of these effects is discussed below.

a. Depletion of food resources In all the experiments involving snails in free communication in closed systems the possibility that the negative feedback effects at high density were caused by an absolute shortage of food can be ruled out because food was provided in excess of requirements. There is a possibility, however, that they could have been caused by a relative food shortage resulting from competition for the most favourable feeding sites on the surface of lettuce discs. It could be argued that the statistically significant decline in growth rate of snails kept at a constant volume of 25 ml per snail when numbers were increased provides support for this hypothesis. The following facts, however, detract from this hypothesis. First, the food ration per snail is the same in all treatments; second, the lettuce discs provided are reasonably homogeneous ; third, the food ration per snail is in excess of requirements. Another possibility, namely that the effect might be related to the reduction in surface area of the air water interface and the amount of dissolved oxygen available to each snail, is discussed below. The concept of relative food shortage may, however, provide a partial explanation for the apparent paradox in the Chernin and Michelson (1957a,b) experiment. They observed that when snail number was stabilized and volume decreased their growth and natality rates increased, whereas when volume was kept constant and numbers of snails increased the converse effects were observed. As suggested earlier, a possible explanation for the first observation is that a reduction in volume results in a concentration of factors which promote growth and reproduction. The results of the second observation can be attributed at least in part to the fact that increasing the number of snails without altering the volume or surface area results in a greater density of snails in the watercress in the vicinity of the air-water interface where the majority of the snails feed. As observations in this laboratory have shown that water-cress is a poor and rather heterogenous food, this method of concentration may result in a relative food shortage. In contrast when the number of snails and the surface per snail are kept constant, the effective density and hence competitive pressure remains the same although the volume is reduced. As already indicated some aquatic pulmonates such as species of Bulinus

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studied by Webbe and James (1971) require a relatively large volume of medium before maximum growth is achieved. It would be of interest to undertake experimental work to identify the limiting factor. It is possible, however, that it is food as these snails are more stenophagous than B. glabrata and appear to feed mainly on contagiously distributed epilithic algae growing on the surface of aquaria. Growth may, therefore, be regulated by the population density of these algae and the surface area of the substrate rather than the volume. Lagrange (I 957) and Vieira (1967) have demonstrated that snails, like other organisms, have rather specialized dietary requirements. In consequence even when snails are surrounded by what appears to be an abundance of food they may suffer from an effective shortage either because some food items are inedible or because the edible plant food substances lack the necessary quantities of the right nutrients. That this may be the case in nature has been well demonstrated by the work of Eisenberg (1966, 1970); he showed that the natality rate of Limnaea elodes in experimental pens could be greatly increased by providing them with high quality food. Boray (1964) has stressed the importance of plant food in determining the distribution and abundance of freshwater molluscs. It will be noted that the amount of food was stabilized in all the experiments involving isolated assay snails and any effects observed must be attributed to the chemical conditioning. b. Depletion of chemical factors (i) Oxygen. It has been shown that the total oxygen consumption of B. glabrata, in which cutaneous respiration plays a part, falls with decreased oxygen tensions in the medium (Von Brand and Mehlman, 1953). It is possible that this observation may provide an explanation for the decline in growth rates of snails when numbers are increased in the 25 ml per snail treatment in experiment I. In this treatment the density of snail per unit area of the airwater interface, where the snails spend much of their time, increases progressively with increase in snail numbers although the volume per snail is stabilized. The dissolved oxygen in this area could, therefore, become depleted and limit the growth of the snails in a density-related manner. The dissolved oxygen should not, however, become a serious growth-limiting factor as the snails can also utilize aerial oxygen. The oxygen concentrations were stabilized in all the assay systems. In the closed system with partitioning this was achieved by aeration. (ii) Zons. The introduction of snails into an aquatic medium containing either lettuce discs or pure cellulose as food is followed by small net increases, at least in the short term, in concentrations of magnesium and potassium and large and small net decreases, respectively, of calcium and sodium in the external pool. It appears, therefore, that B. glabrata obtains its potassium and magnesium requirements from its plant food rather than from the aquatic medium and it can be concluded, therefore, that they cannot become resourcelimiting factors provided the same food and aquatic media are used. This is also the case with sodium because although B. glabrata has a high affinity

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for sodium the amount removed from the external pool is quite small. As the changes in the concentration of the external medium are negligible even when the snails are kept at high densities the uptake rate will not be affected to any significant extent. The net uptake rate of sodium covers a wide range of from 13 to 318 pg g-1 per 24 h and can be correlated with the growth rate of the snails. The estimated net uptake rate of 0.22 p~ g-1 h-1 or 120 pg g-1 day-1 for L. stagnalis in a medium with sodium at a concentration of 0.50 mM 1-1 is intermediate in value (Greenaway, 1970). Actively feeding, growing snails would probably have higher uptake rates. In contrast to the other major cations the concentration of calcium in the external medium declines rapidly when actively growing snails are present. It has been shown that the rate at which calcium is depleted from the medium is positively correlated with absolute growth rates. The conductivity of the medium also declines and it seems probable that this is caused mainly by the depletion of calcium. The strong positive correlations between the decline in conductivity and the absolute growth rates of the snails is not, therefore, unexpected. The lack of significant correlation in all cases can be attributed to the fact that the conductivity often increases towards the end of a threeday interval as a result of the accumulation of ions including Mg + +, K + and NH'; from the plant food or the snail. In consequence of the uptake of calcium by snails it can be postulated that it may become a resource limiting factor in closed systems. The experiment in which calcium concentration and volumes were varied provides corroborative evidence for this hypothesis. This shows that the absolute growth and natality rates continue to increase with calcium concentration until the net uptake rate reaches an asymptotic value of 11.2 pg g -1 day -1. This value is obtained when the concentration in the external medium is 80 pg ml-1 of C a + + in 100 ml of S.S.W. per snail, which is equivalent to a total available calcium value of 8 mg over a three-day period. Further increases in total available calcium did not result in further increases in growth and natality rates and there was evidence that increasing the volume beyond 100 ml per snail when the calcium concentration was 80 pg ml-1 resulted in growth inhibition. As already suggested this effect may be caused by the dilution of growth promoting factors at the larger volumes. It was also found that the snail cannot achieve a positive calcium balance unless the calcium concentration exceeds 1.5 pgml-1. The values obtained for Limnaea stagnalis by Greenaway (1971) are very similar. Thus the latter species has an uptake mechanism which shows a positive calcium balance in media containing more than 0.062 mM Ca 1-1 (= 2.5 pg ml-1) and is half saturated and near saturated in external media containing 0.3 mM 1-1 (= 12 pg ml-1) and 14-13 mM 1-1 (= 40-60 pg ml-1) respectively. If it were assumed that snails are dependent on the calcium in the external pool for their growth requirements it would be predicted that growth would slow down and stop when calcium concentrations reached values of less than 80 pg ml-1 and 1.5 pg ml-1 respectively. This hypothesis receives a great deal of support from empirical data. However, very small absolute growth rates of approximately 1 mg day-1 or less have been recorded in S.S.W. to which no

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calcium has been added. It is, therefore, possible that snails obtain a little calcium from the lettuce which contains 0-022-0-066mg 8-1 of calcium (Documenta Geigy Scientific Tables, 1959), either directly, or when it is released into the medium by the grazing action of the snail. The present results are, therefore, in accord with those of Van der Borght and Van Puymbroeck (1966) for L. stagnalis as they found that even when food (lettuce) is offered ad libitum over a long period about 80 % of the calcium fixed by the animal is derived directly from the water. They consider that the absorption of soluble calcium from the gut after drinking is not a likely pathway. It can be concluded, therefore, that reduction in growth and natality rates of snails at suboptimal volumes both in the present investigation and probably also in others where the dwarfing effect has been reported are to a very large extent caused by depletion of available calcium in the medium. Unfortunately previous workers in the field did not define their media, or monitor them. Chernin and Michelson (1957a,b) did, however, analyse the chemical composition of fresh medium and also media which had been subjected to conditioning for 30 days. As the conductivity of the medium had increased from 25 to 185pmho cm-zin this time it is evident that its chemical composition had been changed drastically by heterotypic conditioning. At the end of this period there were no obvious differences between the chemical composition of crowded and less crowded media. However, time profile studies would probably have shown differences in the early stages which are so critical for growth. One of the reasons why calcium is such an important resource for molluscs is that it is a major component of the shell which makes up from 52-73% of the total dry weight of B. glabrata. It is not surprising, therefore, that there is a great deal of circumstantial evidence to show that calcium concentration in the external pool is important as a density-legislative and possibly also as a density-governing factor for snails living under natural conditions (Boycott, 1936; Macan, 1950). Thus L. stagnalis, for example, does not occur in water bodies with calcium concentration less than the minimum equilibrium concentrations of 2.5 pg ml-I. In addition there is some experimental evidence to show that the natality rates of molluscs living under field conditions can be increased by the addition of calcium in available form (Harrison et al., 1966; Wareborn, 1970). c. Accumulation of chemical factors in the medium (i) Hydronium ions. The time-profile studies of pH in S.S.W. to which snails have been added show that the rate of decline in pH tends to be positively related to the absolute growth rate achieved by the snails. The decline in pH in Hom. C.W. is probably caused by the release of free carbon dioxide, amino acids and hydronium ions by the snails. It is possible that the latter are released in exchange for Ca + + the uptake of which is strongly correlated with absolute growth rate of the snails. Lettuce discs contain organic acids including nictotinic, malic, citric and oxalic acids (Documenta Geigy Scientific Tables, 1959), and these are also released into the medium as a result of grazing by the snails. The lettuce discs on their own influence the pH very

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little. In the present state of our knowledge it is not possible to specify how much the snails and the grazed food plants each contribute to the decline in pH in Het. C.W. Lettuce homogenate at a concentration of 50 mg per 100 ml and also snails in Hom. C.W. produce pH-time profiles with similar conformation although both are less steep than ones generated in Het. C.W. Whatever the origin of the factors responsible for lowering the pH, the fact that the pH can be increased in both Hom. C.W. and Het. C.W. by aeration signifies the presence of volatile components which may include carbon dioxide and organic acids. The most likely explanation for the decline in pH is that it is caused by metabolic processes involved in growth. The possible effects of p H on the growth rates of snails have not yet been studied. This would clearly be a difficult undertaking. As a working hypothesis it could be suggested that lowering the pH to 7.0 would be beneficial, under certain circumstances, because only a very small proportion of the ammonia in its toxic, undissociated, form could exist at this pH. Further lowering of the pH might, however, be harmful for two reasons. Firstly, the more acid conditions would make it possible for the calcified portions of the shell to be attacked from the outside if the periostracum was worn away. Secondly, the acid conditions might make it more difficult for the pH at the internal site of calcium carbonate deposition to be maintained at above the critical level of 8.3. In the present investigations it has been found that faster-growing snails which generate steeper pH profiles than slow-growing snails often reduce the pH to below 7.0. For example 500 mg snails which grow better at a density of 4 in 200 ml bring down the pH to 6.4 at the end of three days whereas the slower growing snails at a density of 1 and 2 in 200 ml reduce the pH to only 7.1 and 6.7 respectively. Nevertheless when they are replaced in fresh media the growth patterns remain unchanged. It would appear, therefore, that leaving snails for short periods at pH below 7.0 does no obvious harm. (ii) Organic substances. It has been shown that the rate of release of organic material into the external pool is much greater in Het. C.W. than in Hom. C.W. To what extent the reduced rate in Hom. C.W. is due to the absence of direct contributions by the plant and how much of it is due to a reduction in the metabolic rate of the snail is not possible to say with any certainty. However, in view of the fact that the deprived snails had only been without food for a day it would appear that the reduction is mainly due to the absence of material contributed either directly by the plant or as a result of the grazing action of the snail. It can be concluded, therefore, that most of the organic material in Het. C.W., particularly carbohydrates and lipids, is of plant origin. These two classes of substances are released by plants, in the absence of snails, to a much greater extent than is the case with nitrogenous substances. Nothing is known about the sites of production and release of organic substances by the snails but they may include the alimentary canal, excretory system or glands on the body wall including the foot. Wilson (1968b) has shown that mucus trails of L. truncutufu contain glucose and lipids. One of the most important classes of substances produced by B. glubratu is N.P.N.

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of which ammonia constitutes about 93%, the rest being made up mainly of amino acids and peptides. The estimated rates of ammonia production by large, 500 mg B. glabraia kept under bacteriostatic conditions, which range from 20 to 110 pg NH3 g-1 day-1, are similar to those found in other aquatic molluscs. Thus it has been estimated that L. stagnalis releases 67 pg NH3 g-l day-1 under non-sterile conditions (Spitzer, 1937) and 34 pg NHs g-1 day-1 under bacteriostatic conditions (Bayne and Friedl, 1968). Marine prosobranch gastropods studied by Duerr (1968) are also mainly ammoniotelic and release NH3 nitrogen at rates varying from 4 to 85 pg 8-1 day-1. The latter author found that the rates of production of NH3 nitrogen could be positively correlated with the weight of the snails and similar results were found in the present investigation. However, when the rate of production was expressed in pg g-1 day-1 and correlated with weight of snail it was found that the relationship was an inverse one. In other words the smaller, faster-growing snails tend to release more ammonia per unit biomass than larger, slowergrowing snails. It was also found that there was a strong tendency for the rate of ammonia production by snails in the same weight categories to be positively correlated with growth rate. These results seem to indicate as suggested by Duerr (1968) that ammonia is a true metabolic product. The present results show that it is misleading to quote a single value for the rate of ammonia production per g per day for a particular species of snail because it is clearly a function of size, growth rate and metabolic activity. Earlier published values for NH3 nitrogen production by snails may considerably underestimate the true rates of ammonia production by normally metabolizing snails because they are based on deprived snails kept under bacteriostatic conditions. It is to be expected that both of these treatments would result in a lowering of the rates of metabolism and hence ammonia production. Several studies including those by Seneca and Bergandahl (1955) and Chernin (1957) have shown that certain of the antibiotics in current use are toxic to snails and may impair their metabolism. When measuring ammonia by snails care must be taken to allow for the release of ammonia from the plant food or faeces and for losses from the water into the air. Such losses might be quite considerable under conditions of high pH and turbulence. Some of the organic substances that might provide the basis of a negative feedback are discussed below. (a) Ammonia. The experiment undertaken to determine the influence of ammonia concentration on the growth rate of young 20 mg snails indicates that at a pH of 8.3-7.9 inhibitory effects become manifested at a concentration of 8.25 pg ml -1 NH3 nitrogen and that statistically significant growth inhibition may occur at a concentration of 20.69 pg ml-1 NH3 nitrogen. If it is assumed that the rates of production of 20, 100, 300, 500 and 700mg snails are approximately 2000, 500,250,200 and 100 pg NH3 g-1 day-1 and that snails of all sizes are affected by ammonia in the same way as the juvenile snails it can be predicted that no statistically significant inhibitory effects on growth rate due to ammonia are to be expected after three days of conditioning unless the volume is reduced to approximately 5-10 ml per snail. However, before

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accurate predictions can be made it will be necessary to acquire data on the effects of varying the ammonia concentration on the growth of snails in the various weight categories. It is well established that the toxicity of ammonia to fresh water organisms is mainly dependent on concentrations of undissociated ammonia which, unlike dissociated ammonia, can pass readily through membranes (Spotte, 1970). As already indicated the relative amounts of undissociated and dissociated ammonia present are dependent on pH and the amount of the former becomes less with decreasing pH. In view of the fact that feeding snails of 100 mg or more in volumes of 100 ml or less, rapidly reduce the pH to values of approximately 7-0it is unlikely that ammonia would be toxic even if snails were crowded at volumes of 5-1 5 ml per snail because very little undissociated ammonia would exist at this pH. The hydronium ion which is being released into the medium at a rate which is proportional to absolute growth rate of the snail is, therefore, acting as an anti-inhibitor. Some of the preliminary experiments carried out in this laboratory (Powles, unpublished) support the conclusion that ammonia, even at high concentrations, would be non-toxic at a pH of 7.0, but more information is needed before the effects of ammonia can be described precisely. It is not possible to comment on the possibility that ammonia might have contributed to the crowding effects observed by other workers investigating snails under sub-optimal volume conditions because they did not define their media. It is well known, however, that ammonia can reduce the growth and survival rates of other aquatic organisms including fish (Kawamoto, 1961; Ball, 1967). Other excretory substances can also provide the basis for a density-dependent negative feedback mechanism in aquatic organisms. Thus the growth of yeast and lactic acid bacteria can be limited by alcohol and lactic acid respectively (Brock, 1966; Toennies and Shockman, 1958). In addition to being toxic, ammonia may also affect aquatic organisms in other ways. Thus Maetz (1972) has shown that the ammonium ion may interfere with the absorption of sodium. According to Berner (1968) and Speeg and Campbell (1969) there are strong reasons for believing that ammonia may function in the deposition of calcium carbonate via the following reaction : NH3 HCO; Ca + *+CaC03 +NHf;.

+

+

This reaction has been tested as a model for geochemical deposition of CaC03 (Berner, 1968). If this reaction occurs in the external environment it could be construed as being harmful as it would reduce the concentration of available calcium to the snail. On the other hand if it is implicated in the deposition of calcium in the shell it might help to promote the growth of the snails. This hypothesis receives some support from the fact that slight but consistent, statistically significant growth promotion effects have been achieved by increasing the concentration of ammonia in S.S.W. from 0 to 04328 pg NH3 ml-1. (b) Organic acids. Davis (1966a) suggested that tricarboxylic acids produced by the snails restrict their growth but gave no details regarding their sites

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of production or effective concentrations. It is also possible that some of the organic acid released from plants may inhibit growth of snails in Het. C.W. (c) Glycoproteins. Other substances released by snails which might be involved in growth inhibition are the glycoproteins, as they might bind and inactivate ions and growth factors. This phenomenon has been demonstrated in Protozoa as the glycoprotein avidin binds the growth factor biotin thus making it unavailable for growth (Lilly, 1967). (d) Specific inhibitors or pheromones. Several investigators have claimed that there is evidence to suggest that specific chemical substances which inhibit growth or reproductive rates or both are produced by certain individuals or by the entire population, and the certain individuals are competent to respond to them. It has been postulated that such factors either become effective or are released when the population density is above a certain critical threshold and that they provide the basis of a negative feedback system which can become operational before resources or non-specific excretory products become limiting (Wynne Edwards, 1962). It may be possible to distinguish between these factors and others which produce a negative feedback by stressing the endocrine system, thereby causing mortality or a reduction in fertility as postulated by Christian et al. (1964). In view of the potential importance to evolutionary theory and the practical problems of controlling population growth of a negative feedback mechanism using specific information, it is important that the evidence for its existence should be looked at critically. This is done below for different biological systems. (i) Molluscs. Wright (1960) found that although Het. C.W. produced by crowded Bulinus forskali, plants and fish caused assay snails to grow more rapidly than controls in the initial stages, the presence of a toxic factor caused them all to die suddenly in the fourth week. This author based his claim for the existence of a molluscan pheromone on the loss of the toxic factor after filtration of the Het. C.W. through activated charcoal. It was postulated that the toxic factor was a pheromone. The claim that a pheromone is implicated can be criticized for the following reasons: First, as the medium was heterotypically conditioned the source of the factors must remain in doubt. Second, the effects could have been caused by a non-specific inhibitory factor such as ammonia. Third, his experiment was not properly controlled and the possibility that the charcoal itself was producing a substance that was beneficial to growth and survival of the snail cannot be ruled out. In certain of the experiments the charcoal produced the converse effect and released substances that were toxic to the snails. Jobin and Michelson (1967) have suggested that the inhibitory effects observed by Wright (1960) were more likely to be due to the effects of gross pollution and oxygen depletion. Wright’s claim that the evidence is strongly in favour of the existence of a pheromone responsible for the inhibition of snail growth and fecundity has, therefore, to be refuted. Berrie and Visser (1963) isolated an interesting substance with a molecular weight of 350 and the structure of a complex ester from a small pool in Uganda inhabited by Biomphalaria sudanica tanganyicensis. At this time, when

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there was very little water in the pond, the growth and natality rates of the snails were negligible. The substance was found to be lethal to the snails when applied at twice the concentration occurring in the pool. As the substance was isolated from water containing plants and other organisms as well as snails its origin must remain in doubt. Although it has been described in the literature (Spotte, 1970) as a pheromone the possibility that it may have originated from plants cannot be excluded. More recently Gazzinelli et al. (1970) found that when lettuce-fed B. glubrata of approximately 5 mm shell diameter were maintained at a density of 40 per 350 ml for one week both the conditioned water and the faeces produced by them caused a marked reduction in the rate of uptake of SgFe by assay snails compared with controls. As earlier experiments had shown a strong positive correlation between growth rate of the snails and uptake rate of 59Fe they interpreted these results as showing the presence of growth inhibitory factors. Thus in conclusion they state that “whether the same or different chemical factors are responsible for inhibition of growth and of iron uptake respectively is unknown but the presence of such a factor is firmly established”. There is a possibility, however, that the poor growth achieved by snails in the conditioned media was caused to some extent by the removal of essential ions such as calcium. Unfortunately the ionic composition of the tapwater used as a medium was neither defined nor monitored. The growth inhibitory effects caused by faeces are interesting and cannot be explained on the basis of resource depletion. The results of the present investigation also indicate that growth inhibitory factors may occur in the faeces of lettuce-fed snails. The situation is, however, by n o means simple as the experiment on snails deprived of faeces indicate that faeces may aIso contain growth promoting factors. Further experiments need to be undertaken to clarify the position. When designing these consideration should be given to the possibility that the competence of the snails to produce and to respond to such factors may vary with age and diet. The source of the inhibitory factors in the faeces is not known. One possibility is that they are released from glands in the alimentary canal of the snail. The absence of inhibitory factors from the faeces of cellulose-fed snails militates against this hypothesis although it could be argued that they are not producing the inhibitor because of the inadequacy of their diet. The other possibility is that the inhibitor is of plant origin. The fact that strong positive relationships have been demonstrated bet ween the growth rate of snails and the concentration of lettuce homogenate added to the medium would appear to undermine this hypothesis. It can be postulated, however, that the plant material contains both growth-promoting and growthinhibitory factors. When these are added to the medium they result in growth promotion because the effects produced by the growth promoting factors are in ascendancy. Passage of these factors through the alimentary canal may, however, result in removal of the growth-promoting factors, leaving relatively greater amounts of the inhibitory factors in the faeces. Other organisms which appear to release factors in the faeces which influence growth and develop-

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ment rates include Daphnia and insects (Akin, 1966; Nolte et al., 1970; Englemann, 1970). (ii) Other aquatic organisnzs. It has been suggested that specific factors which reduce growth, reproduction and survival rates of other organisms of the same species are also produced by other aquatic organisms including Hydra (Davis, 1966b), Planaria (Best et al., 1969), Culex pipiens quinquefasciatus Say (Ikeshoji and Mulla, 1970) amphibian tadpoles (Rose and Rose, 1965; Akin, 1966) and fish (Yu and Perlmutter, 1970). The evidence for the existence of these specific factors is, however, equivocal for the following reasons : (a) The causation of the negative effect might not necessarily be due to the addition of an inhibitory factor. In the case of the work carried out on Planaria by Best et al. (1969) it would appear that inhibition of budding was induced when they were crowded together. As the medium was not carefully defined or monitored it is possible that the effects could have been caused by the depletion of essential resources, including oxygen and ions, in the high density cultures. (b) Certain of the treatments may have resulted in the addition of a beneficial factor. The loss of the inhibitory properties in conditioned media following filtration through methyl chloroform has been cited as evidence for the existence of inhibitory factors by Yu and Perlmutter (1970). In this case it is possible that the treatment may have resulted in an increase in the concentration of growth-promoting or detoxifying factors. Although Yu and Perlmutter (1970) did not monitor all the major components in the medium they demonstrated that filtration through methyl chloroform did result in changes in the chemical composition including an increase in pH and ammonia concentration and a decrease in nitrate concentration compared with controls. It is, therefore, possible that these changes and others that were not monitored may have resulted in the addition of beneficial factors to the filtered medium. (c) There is doubt regarding the site of origin of the growth inhibitors. Factors which reduce either the rates of growth, reproduction or survival have been isolated from media conditioned by Hydra (Davis, 1966b), third instar larvae of Culex (Ikeshoji and Mulla, 1970), older tadpoles (Rose and Rose, 1965; Akin, 1966) and fish (Yu and Perlmutter, 1970). According to Davis (1966b) the results suggest that the inhibitory factor produced by Hydra is a protein and that a high degree of integrity is necessary for its activity. The toxic, overcrowding factors produced by mosquito larvae were separated by chromatographic techniques (Ikeshoji and Mulla, 1970). In the case of water conditioned by crowded Rana pipiens tadpoles the inhibitory factors appear to be associated with a certain type of algal cell (Akin, 1966). This author proposed that the inhibitory agent is produced by growing tadpoles but that it becomes associated with a certain type of ingested algal cell as it passes through the posterior part of the gut. When the cells are egested they then release a factor which is inhibitory to young tadpoles into the medium. Yu and Perlmutter (1970) extracted the inhibitory factors by using activated charcoal and organic solvents including methyl chloroform.

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It is clear that further work needs to be undertaken to elucidate both the nature and the source of these substances. As yet there is no unequivocal evidence to indicate that they are in fact produced by the organisms themselves. Possible sites of production are the aquatic medium, the food, microorganisms or the experimental animals. Unfortunately the first possibility cannot be excluded in these investigations because none of the media was defined. There is a possibility that the toxic factors isolated from media containing mosquito larvae and their food may have originated from the latter. Thus Ikeshoji and Mulla (1970) showed that infusions of larval food without larvae, under both septic and aseptic conditions, also produce some toxic factors. It may be suggested, therefore, that the toxic factors have their origin in food and that the passage of the food through the alimentary canal of third instar larvae results in the removal of beneficial factors and the density related release and activation of factors which are toxic to first instar larvae. The latter may have a lower threshold of response than the older larvae. That two antagonistic factors might be involved receives some support from the fact that the less crowded culture medium results in better survival than the control. The hypothesis that the substances originate from the food is further supported by the fact that ether extracts of larval homogenates are free of toxic overcrowding factors. As yet there is no proof that the inhibitory factors present in tadpole cultures originate from the tadpoles. Recent work has shown that certain algal cells produce antibiotics and in some cases these have been characterized (Martin and Chatterjee, 1970). Consideration should, therefore, be given to the possibility that the growth of the juvenile tadpoles is inhibited by the algal cells either because they produce antibiotics or because they compete with the tadpoles for essential ions. (iii) Organisms living in terrestrial systems. The best documented examples of inhibitory chemicals forming the basis of a negative feedback mechanism in biological systems are those demonstrated in colonies of social insects. Thus the inhibition of oogenesis and queen rearing in workers of the honey bee Apis mellqera is normally caused by the queen substance 9-keto-2decanoic acid produced by the mandibular gland of the queen. The presence of another inhibitory scent, 9-hydroxy-decenoic acid may also be necessary (Wilson, 1970). In colonies of Kalotermes each member of a royal pair produces pheromones which inhibit the development of reproductives of its own sex (Englemann, 1970). Pheromones also appear to be implicated in inhibiting reproductive output of small rodents which live in a more open biological systems than is the case with social insects. The various effects in small mammals are named after their discoverers. The Bruce effect refers to the failure of implantation and rapid return to oestrus of a recently impregnated female mouse when exposed to the odour of a strange male. The suppression of oestrus and the development of pseudopregnancies in female mice when they are grouped together in the absence of males is known as the Le Boot effect. The Ropartz effect refers to the increase in the size of adrenal glands and in the rate of production of corticosteroids which result in a decrease in fertility when mice are sub-

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jected to odours from other mice. Although other species of mammals use pheromones in communication none of them appears to produce pheromones which inhibit growth and reproduction like the ones in mice (Wilson, 1970). (iv) Cellular and tissue levels of organization. It is of interest that negative feedback mechanisms involving growth inhibitors also occur in the cell and tissue levels of organization. Thus Stoker (1967) in reviewing contact and short range interactions involving growth of animal cells in cultures states that since the evidence is against nutritional deficiency it can be assumed that reduced growth and macromolecular synthesis is due to the presence of one or more hypothetical inhibitory factors. At the tissue level of organization, Bullough (1969) has produced evidence for the existence of factors called chalones which are produced by newly formed epidermal cells. Low concentrations appear to stimulate mitotic activity of the basal cells while high concentrations have the converse effects and thus serve to regulate the recruitment of new cells. Such factors have, however, proved difficult to assay because the effects they induce may vary with concentration, the competence of the target tissue and the presence of other factors including cofactors and inhibitors which may interfere with their mode of action. The results of the present investigation provide no support for the existence of a negative feedback mechanism based on information received from speciesspecific inhibitory pheromones produced by the snails themselves when they are living under high density conditions. On the contrary there is good evidence for the existence of a positive feedback mechanism involving factors produced by the snails. Although mice produce inhibitory pheromones there is no evidence that they are implicated in causing negative feedback effects in natural populations, although this possibility cannot be excluded. It can be concluded, therefore, that with the exception of social insects, there is no evidence that any animal population is regulated by the action of inhibitory pheromones. The social insects are, however, unique biologically because either the genetic mechanism of sex determination as in the Hymenoptera (Hamilton, 1964) or the intensity of competition between sibships as in the Isoptera (Williams and Williams, 1957) have made it possible for them t o develop “altruism” including the formation of sterile castes. “Altruism” as used here refers to the sacrifice of fitness of the individual to increase that of others. The development of “altruism” is also possible in the cellular and tissue level of organization because the cells are genetically identical. It is perhaps significant that they are the only other biological systems where there is good evidence for the existence of specific inhibitors which can provide the basis of a negative feedback mechanism. If inhibitory pheromones do occur in other biological systems it is more likely that they will be important to organisms, with low dispersive power, living in closed systems such as small pools or in subterranean burrows rather than to organisms living in open systems such as streams, rivers and other aquatic or terrestrial systems because under such conditions the pheromones would be removed by currents. It is unlikely that they will prove to be toxins as postulated by Wright (1960) because it is difficult to envisage how such factors could have evolved by natural selection.

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Neither is there any good empirical evidence that other species specific information based on visual, olfactory and tactile signals can provide the basis of a negative feedback mechanism in populations of free-living animals. The laboratory experiment carried out to test the hypothesis of Christian et al. (1964) can be criticized on the grounds that the density treatments are abnormally high. It has also proved difficult to interpret changes in the endocrine system of organisms in the field because other factors including age, sex and diet as well as the direct effects of interindividual interaction may alter them. Many others including Pitelka, mentioned in Odum (1971), have failed to find significant correfations between changes in population density and the state of the endocrine systems. Although Wynne Edwards (1962) suggested that epideictic display could serve as a basis for reducing the fertility rates there is no empirical evidence that this is the case. Both Wynne Edwards (1962) and Christian et al. (1964) find it necessary to invoke group selection to account for the evolution of such a feedback mechanism because in their view some individuals in the population behave altruistically. Thus Christian et al. (1 964) state, “The endocrine functions, therefore, have survival value for the species even when the effects on the individual may be deleterious”. It is difficult to support the group selection hypothesis because there is no good evidence that organisms behave altruistically. Examples of so-called “altruistic” behaviour are best explained on the basis of kin or individual selection (Maynard Smith, 1964; Hamilton, 1964). However, even if informational feedback mechanisms of the kind envisaged by Wynne Edwards (1962) do occur it is not necessary to invoke group selection. It could be argued that under certain circumstances a reduction or delay in the onset of reproductive activity might increase the fitness of the individual living at the lower end of a social hierarchy or under conditions of high population density. Information regarding the population structure could thus act as a feed forward signal. It is well known that many organisms make use of signals with a high predictive value such as photoperiod change to induce diapause when either metabolic or reproductive activity is slowed down. It must be noted, however, that although it is not necessary to invoke group selection it does not follow that this does not occur. Recent work has shown that there is some evidence to indicate that population extinction may take place at a sufficiently high rate to allow for group selection to occur (Odum, 1971). Chitty (1960) proposed that populations of field voles and possibly also other animals are numerically self-regulatory owing to genetically determined changes in quality including the average vitality and aggressiveness of individuals associated with changes in population density. It has proved impossible to formulate this theory precisely in mathematical terms or to test it empirically. Krebs (1971) found only a weak correlation between population increase and aggressiveness in male Microtus sp. Although genetic change could be correlated with changes in population density there was no evidence that it was the causal factor. Another more effective strategy which may be used in the control of population density involves predispersive aggregation. Such behavioural patterns have been described in organisms as diverse as slime moulds and locusts and

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they may well occur in other organisms. It is interesting to note that pheromones appear to be involved in causing the aggregative responses and the resultant physiological adaptations to dispersion that occur in slime moulds (Bonner, 1970), aphids (Way and Cammell, 1971) and locusts (Norris, 1964; Thomas, 1970; Nolte et al., 1970). Dispersionary behaviour resulting from chemical communication under high density conditions may also occur in solitary animals such as the pine looper, Bupalus bipinnarius (See Klomp, 1966; Gruys, 1971). Various attempts have been made to ascertain to what extent negative feedback effects observed under field conditions are caused by resource depletion and informational feedback when the effects of inimical forces can be ruled out (Watson and Moss, 1970). In view of the complexity of the interactions this is clearly a very difficult problem. To help resolve the question it is best to simplify the situation by regarding resources as being a function of time, space, energy and other components including oxygen, essential ions and organic substances. The degree of integration between space and other components of resource including energy have increased during the course of evolution. The consequent development of territorial and hierarchical systems has increased the probability of individuals obtaining all their requirements for growth and reproduction. As pointed out by Nicholson (1958) the replacement of a scramble for resources by a conventional contest has resulted in a more efficient utilization of resources. In a closed system it is clearly impossible to determine by observation alone whether a negative feedback effect is being caused by resource depletion or by informational feedback because population density and resources tend to interact with each other in a reciprocal manner. Correlations tell us nothing about causation but they do make it possible to formulate hypotheses which can be tested by experiments. One way of determining whether negative feedback effects can be induced by specific informational signals would be to vary the strength of stimuli suspected of acting as specific informational signals in closed systems and to observe their effects. An alternative approach which can be used with suspected specific chemical signals or pheromones is to assay them in a flow or chemostat system as was done in the present investigation. Before a factor can be described as being a pheromone it is necessary to purify and isolate it, identify its site of production and show that it can induce a primer or releaser effect by acting on specific receptors in another organism of the same species. 3. Growth inhibition or negative feedback effects observed when snails in the lag or earIy logarithmic phase of growth have their density increased from one to two per 200 ml Although statistically significant growth inhibition, compared with controls, was observed on several occasions when snails were subjected to this treatment this was not invariably the case. On some occasions there was either no statistically significant treatment effect or else the converse effect was obtained. Such contradictory results have been found in growth studies involving species of Protozoa and can be attributed to the extreme complexity of growth re-

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sponses of organisms to a wide variety of agents which may either stimulate or inhibit growth (Lilly, 1967). It is perhaps significant that they only occur in experiments involving snails in the lag or early logarithmic phase of growth. The fact that growth inhibition occurs as a result of this treatment is of interest and it is important that the causative mechanism should be elucidated. There are two possible causes, namely resource depletion or an increase in concentration of inhibitory factors. The former possibility is unlikely because growth of larger, 300 mg snails is invariably enhanced when they are subjected to the same treatment despite the fact that their absolute growth rates and demands on calcium are roughly equal to those of smaller snails. However, it is possible, though unlikely, that the ionic requirements of juvenile snails may be different from those of larger, mature snails and that their growth may be limited by the supply of trace elements. It seems more likely, therefore, that the effects are caused by a specific or non-specific inhibitory factor. As already indicated there is empirical evidence that larger molluscs in the logarithmic phase of growth can release both inhibitory and stimulatory substances into Het. C.W. when they are fed on lettuce and that juvenile snails are competent to respond to both of these. It can be postulated that the juvenile snails produce relatively more of the inhibitory and less of the growth stimulatory or anti-inhibitory factor than larger snails. One hypothesis that can be formulated is that ammonia is the inhibitor and that hydronium ion is the anti-inhibitor. The latter is released into the medium at a relatively slow rate by the small juvenile snails because of their slow growth. This hypothesis has to be discarded, however, because it has been shown that ammonia is not toxic to the snails under these conditions. At the present stage of our knowledge no other inhibitory factors which might be implicated have been identified. It is possible that in those cases where no effectsare observed following pairing of juvenile snails they are producing roughly equal amounts of the growth inhibitory and stimulatory substances. In contrast it can be postulated that snails in the treatments where growth promotion occurs as a result of pairing are producing relatively larger amounts of the factors which can generate positive feedback effects including possibly the growth-promoting factor in the molecular weight range 500-104, ammonia, cellulase and faeces factors. The fact that the snails are highly social, usually occurring in close proximity to or in contact with each other, would facilitate the negative and positive feedback effects that have been observed. The occurrence of factors that inhibit and stimulate growth and development offers an interesting parallel with locusts. In the case of the desert locust, Schistocerca greguriu, it has been shown that mature males produce a pheromone which accelerates maturation in other males and females. This pheromone is not produced by alletectomized males, immature adults or the larvae of both sexes. Instead immature males and larvae appear to exert an inhibitory influence, possibly by another pheromone, on the development of immature males (Norris, 1964; Richards and Mangoury, 1968). There is evidence that in the African migratory locust, Locusta migratoria, the duration of the period when the inhibitory pheromone is being produced is longer than

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the maturation time of isolated pairs, whereas in S. gregaria it is shorter (Norris, 1964). The following tentative explanation is offered for the adaptive significance of the occurrence of both growth stimuIatory and inhibitory factors. 3. glabrata is a highly social animal which tends to aggregate either in the presence of food or in its absence. It can be argued that if it is selectivity advantageous for the animals to aggregate in space it would also be advantageous for them to synchronize their growth and development. The mechanism for synchronization involves the sexually mature snails producing more growth stimulators and the smaller, slow-growing juvenile snails producing more growth inhibitors. These factors when working together would promote synchronization of growth and maturation. The experiments which have been carried out in this laboratory to test this hypothesis have produced some evidence in support of it. However, some of the results are equivocal and the investigation is to be continued. The possible advantage of aggregation and of synchronization of growth may be ranked as follows: (1) they result in physiological advantages making it possible for the snails to utilize resources including food, growth factors and ions more efficiently; (2) they increase the probability of snails being able to cross fertilize thus increasing genetic heterogeneity and increasing the probability of survival of the genotype; (3) they facilitate synchronization with other key environmental factors such as photoperiodic changes; (4) they might help to reduce the probability of inimical agents harming or killing individual snails. It is possible that the bright red colour of the albino B. glubruta is aposematic. As pointed out by Klomp (1966) aposematic species such as Diprion pini and Neodiprion sertger are gregarious whereas cryptically coloured species such as the pine looper, Bupaluspiniarius, are solitary. VI. SUMMARY The various strategies that might succeed in preventing transmission of schistosomes,including the control of snail hosts, and reducing the probability of success of miracidia, sporocysts, cercariae and adult parasites are discussed and evaluated. The mathematical models that have been used to predict the probability of success of possible control measures are shown to lack realism and hence precision and generality because certain facts including the immune response of the definitive host, the longevity of the adult parasite, the parasiteinduced mortality of the snail host and the time scale of the various events are either overlooked or wrongly interpreted. The need for more accurate data on the immune response and the longevity of the adult parasite is stressed in view of their importance in constructing mathematical models and planning control measures. Here are described the results of experiments designed to show how various environmental factors including contact by other individuals resulting in copulatory behaviour, resources including food and ions in the external pool, and substances added to the medium either by the snails or their plant food receive expression in the growth and natality rates of individual Biomphalaria

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glubratu. Particular attention is given to the possibility that the snails may be producing specific inhibitory pheromones. The effects of chemical conditioning or of particular chemicals were assayed in both closed and open systems using the principle of the chemostat. The two types of media used were heterotypically conditioned media (Het. C.W.) produced by interactions between the snail, food and the medium and homotypically conditioned media (Hom. C.W.) produced by interaction between the snails, the aquatic medium and a pure cellulose food source. The following effects were observed. First, a positive feedback or Allee effect was manifested by an increase in growth and natality rates of individual snails when snail number of biomass in a given volume was increased, volume per snail decreased and the time allowed for conditioning increased up to a certain threshold. Second, a negative feedback effect was observed when small snails in the lag or early logarithmic phase of growth had their density increased from one to two per 200 ml. Third, a negative feedback effect was observed when the volume per snail was reduced to below 50 ml per three days per snail. The causes of these effects are summarized as follows: (1) Allee effectThis may be caused by mating behaviour over a period of 2-24 h and by chemical factors including substances in the molecular weight range 500-104 mol. wt, ammonia, faeces factors and possibly by cellulases under certain circumstances. With the exception of indole acetic acid and possibly also the faeces factor these originate from the snails themselves. Possible modes of action of these various factors are discussed. (2) Negative feedback effects under high density conditions-These may be caused by an absolute or relative food shortage, a depletion of resources such as oxygen and calcium from the medium and possibly also by the addition of inhibitory factors which appear to be of plant origin as their presence could only be demonstrated in heterotypically conditioned medium (Het. C.W.) or in the water conditioned by faeces of lettuce-fed snails. The rate of depletion of calcium from the medium and the rate of addition of ammonia and hydronium ions are correlated strongly with absolute growth rate of the snails. It is considered unlikely that ammonia could produce inhibitory effects in the volume treatments used in the experiment. There is no evidence that snails or other aquatic organisms produce specific inhibitory pheromones. Although there is evidence that specific chemical inhibitors are produced by mice, social insects and tissues, only in the two latter cases is there any evidence that they are effective in regulating population growth. The conditions under which such specific inhibitors might work in natural populations are outlined. (3) The negative feedback effects observed when juvenile snails have their density increased from one to two in 200 ml. It is considered that these effects are caused by inhibitors, possibly of plant origin, rather than by resource depletion. It is suggested that the adaptive significance of the inhibitory growth factors produced by juvenile snails and the stimulatory growth factors produced by larger, faster growing snails is that they may help to synchronize growth of snails living in social groups. As B. glubratu is highly social, it could beargued that if it is selectively advantageous for them to aggregate in

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space it would also be advantageous for them to synchronize growth and development.

VII. ACKNOWLEDGEMENTS I am grateful to the Overseas Development Administration for financial aid which has made the work possible, to my friend F. A. B. Coutinho for valuable and stimulating discussions and to members of our research group mentioned in the text.

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Functional Morphology of Cestode Larvae JAROSLAV

LAI IS

Institute of Parasitology. Czechoslovak Academy of Sciences. Prague. Czechoslovakia I. Introduction ........................................................

.

I1

396

Morphogenesis of the oncospheral stage. and its development .............. 396 396 A . Formation of the embryonic envelopes............................ B. Development of the penetration gland. the hooks and their musculature . . 399

111. Functional morphology of the oncosphere (hexacanth) .................. 400

. Anatomy of the fully developed oncosphere ........................ B. Functional capability of the infective oncosphere ....................

400 403

IV . Post-oncospheral development of larvae which do not form a cavity ........ A Procercoid of Pseudophyllidea.................................... B Plerocercoid (sparganum) of Pseudophyllidea...................... C Larval stages of Tetraphyllidea .................................... D Larval stages of Caryophyllaeidea.................................. E Tetrathyridium of Mesocestoides .................................. F Larval stages of Catenotaenia, Paruterina, and Cladotaenia............

406 407 408 411 412 412 414

A

.

. . . . .

V

.

Post-oncospheral development of larval stages which form a cavity .......... A Larval stages of Linstowiidae ...................................... B. Post-oncospheral development of cysticercoids...................... C Post-oncospheral development of the cysticercus ....................

. .

VI. Functional morphology of post-oncospheral development ................ A The strobilar tegument of the adult stages .......................... B. Histogenesis of calcareous corpuscles .............................. C Morphogenesis of the mature plerocercoid to the adult stage .......... D. Scolex of Taeniidae at the larval stage .............................. VII . Specific larval organs of taeniids ...................................... A Cyst wall of the cysticercoid ...................................... B. Cysticercus bladder .............................................. C Excystment and evagination of the cystic larva ...................... VIII . Proliferation during the larval stage .................................... A. Asexual multiplication .......................................... B. Abnormal growth ..............................................

.

.

.

.

IX. Conclusion

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

Acknowledgements ................................................ References ........................................................

395

415 415 416 420 436 436 438 439 440 445 445 448 456 458 458 463 466 466 466

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SLAIS

I. INTRODUCTION

Larval stages of cestodes show considerable variation during the second stage of post-embryonic development and this has been used for identifying individual species. Classical species have been known for a long time, but the complete life cycle of other less well known species is often unknown. The morphological pattern of the larvae is not always clear-cut and the correct identification of the individual larval types is sometimes difficult. This applies particularly to metacestodes with variable morphological patterns. Verster (1959), in view of the instability of the larval morphology, refrained from using the larval type as a criterion in the taxonomic revision of the genus Tueniu, despite the fact that larval stages of this group show much variability. Larval morphology is greatly affected by the individual host species, and by the different sites of location in the organs of the host, this being particularly marked if the incidence of infection is high. Age is another factor changing the appearance of the metacestode, because the differentiation of the scolex organs, after which infection of the definitive host is possible, occurs relatively early. The morphology of the larva is also greatly affected by continuous growth and, eventually, by a life-long stay in the intermediate host. In my opinion it may be possible to place the larva in the appropriate higher taxonomic group, and to identify the species, if comprehensiveknowledge of the morphology of the larva is supported by knowledge of other biological factors. Much progress has been made in our knowledge of larval morphology since the introduction of modern research methods. The results have suggested that sound knowledge of the morphology of the larva may be more useful for its identification than standard diagnostic data. On the basis of literature and my own results I have tried to present a survey of comparative and functional morphology of larvae of the subclass Eucestoda developing from the oncosphere, i.e., a larva bearing 3 pairs of embryonic hooks (hexacanth). 11. MORPHOGENESIS OF THE ONCOSPHERAL STAGE AND

ITS

ENVELOPES

In her review of the embryonic development of cestodes, Rybicka (1966) differentiated three phases, i.e. that of cleavage and the preoncospheral and oncospheral phases. Of these, the preoncospheral phase is most important, because it leads to the formation of the oncosphere. Oncospheral development is closely associated with the formation of the embryonic envelopes, particularly that of the embryophore. Differentiation of somatic and germinative cells, development of glands, hooks and their musculature occur in the embryo itself.

A.

FORMATION OF THE EMBRYONIC ENVELOPES

In the developing oncosphere, four embryonic envelopes can be distinguished: the capsule, the outer envelope, the inner envelope and the onco-

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spheral membrane. Eggs of pseudophyllideans have a thick operculate capsule, because the larvae mature in water. Eggs of proteocephalids have a thin, non-operculate capsule. The development of the capsule shows considerable variation and is not fully understood in Cyclophyllidea. Often, the capsule is so thin that it is rarely seen after normal fixation procedures. Swiderski (1 968) described its fine structure for Cafaenofaeniapusilla. It consists of finely granulated material, and is bounded by a distinct outer membrane with a thickness much greater than that of an ordinary plasma membrane. Tn several cyclophyllideans, the thicker coat appears to be formed by a thickening of the thin egg capsule. Pence (1967) called it the outer capsule in infective eggs of Dipliyllidium caninum. Electron microscope studies revealed that the outer layer of this capsule consists of granular vitelline material which is separated from the inner zone of material by a thin lamina adjoining the cytoplasm of the egg. According to this author, this is not formed by a sclerotin. The same author (Pence, 1970) found in eggs of Hymenolepis diminuta that the outer coat was cellular in origin and consisted of an inner zone of electron-dense material. Its surface was irregular with bud like projections extending into the outer zone of granular material. Histochemically, the outer coat was formed by a structural mucopolysaccharide or mucoprotein similar to that in D . caninum. The outer envelope of pseudophyllideans forms two or four macromeres and vitelline cells. In cyclophyllideans, Swiderski (1968) observed by the electron microscope (EM) a transformation of macromeres with a characteristic cellular ultrastructure on the outer envelope of C. pusilla. Pence (1967) found additional cellular fragment i n the ultrastructure of the outer envelope of infective eggs of D. caninum. In most eggs of H . diminuia, Pence (1970) found only a clear space with signs of lipid accumulations instead of an outer envelope. lncreased attention has been given to the inner envelope because from it originates the principal and most resistant protective cover of the oncosphere, i.e. the embryophore. This envelope originates by cells separating from the surface of the developing embryo. In a pseudophyllidean cestode developing in water cilia arise from the surface of the embryophore. The cells forming the embryophore remain as a syncytium on the surface of the embryo leaving the operculated capsule. For a certain period, the embryo inside its embryophore lives free in the water and represents the coracidium. The cell cytoplasm is heavily vacuolized. The embryophore of the coracidium containing a motile larva represents a special embryonic organ safeguarding the temporary existence of the embryo in the water. Studies on the ultrastructure of the coracidium of Triaenophorus nodulosus by Timofeev and Kuperman (1 967) disclosed the cellular structure of the embryophore. These authors observed in the cilia a fine structure typical of a kinocilium, but differing from it in that the kinetosomal part was not submerged in the superficial layer of the cytoplasm; in contrast, part of the cytoplasm of the cells was raised above the cellular surface and formed the base of the cilia. The well developed mitochondria in the cytoplasm 15

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of the embryophore of the coracidium were i n close contact with lipoid granules. According to the authors, this arrangement of cytoplasmic structure suggested that energy was obtained under aerobic conditions. In cyclophyllideans, the inner enveloped originates by detachment of several cells from the embryo. Swiderski (1968) observed by the EM that the inner envelope of C. ptwilla consisted of two layers. The external portion, the inner envelope proper, was formed by a different type of blastomere called the “third macromere”. The internal portion, the embryophore, was formed by typical mesomeres. The structure of the embryophore of cyclophyllideans shows variation. Rybicka (1 966) distinguished with the light microscope (LM) three types of embryophores but EM studies added new knowledge of the fine structure of the individual types. For the first group of embryophores, this envelope was described in detail in the mature egg of H . diminufa. Pence (1970) found remnants of the outer cellular zone in the inner envelope separating the homogeneous, finely granular material from the embryophore. It was composed of a structural protein. The embryophore proper consisted of electron-dense homogeneous material without morphological subunits composed of a structural protein, but not keratin. The special composition of this type of embryophore appears to be associated with the solid outer coat of the egg. According to Rybicka, the second type of embryophore appears to be homogeneous by the LM; the ratio of egg envelopes was similar to that in Procephalidae. The cellular structure of the inner envelope disappeared entirely during formation of this type of embryophore. Pence (1967) observed in D. caninirm that the fine structure of the embryophore was not homogeneous in nature, but consisted of two layers of rods at right angles to one another. In cross section, the rods measured approximately 300 A in diameter and gave a positive reaction for keratin. The third type comprised striated embryophores secreted in block form typical of members of the family Taeniidae. The ultrastructure of these blocks was first described by Morseth (1965) who drew attention to the circular bodies around which the embryophoric blocks developed, and which remained enclosed in the block substance. Nieland ( I 968) identified circular bodies in Taenia taeniacformis as mitochondria of the cell cytoplasm which formed the original inner envelope. lnitial block formation was characterized by the appearance of discrete granules on the outer membrane of the inner envelope which enlarged without coalescing. Upon completion of embryophore development, the remnants of the inner envelope formed its basement membrane. Morseth (19664 concluded that a keratin-type protein was the principal component of the embryophore of T. Iiydatigena, T. ovis and T. pisiformis. h i s (1970) confirmed a strong reaction of these blocks for tyrosine, but not for cystine and tryptophan; using several histological methods he demonstrated the similarity of this substance to keratin of vertebrate hair. The cement substance, seemingly a protein, was not found to be keratinous. The oncospheral membrane appears at a later stage of oncosphere development; at first, it represents a basal limiting membrane of the embryophore. In the final stage, its character is that of an independent, detached membrane

F U N C T I O N A L MORPHOLOGY OF CESTODE L A R V A E

399

with a complicated ultrastructure showing two pairs of laminae separated by closely spaced vesicles (Nieland, 1968). Coil (1967, 1968) observed a different situation in the origin of the egg envelopes of Diplophallus polymorphus and Infula macrophallus. Conditions of the outer egg envelopes were similar in all cyclophyllideans. The embryophore, however (the “inner capsule”), is very complex. It consists of two parts, the cylinder and the plugs. The first constitutes the principal cover; it is elongate and passes into the plugs at both poles. The two components of the embryophore differ in their histochemical structure although both involve proteins.

S.

IIEVELOPMENT OF THE PENETRATION GLAND, THE HOOKS A N D THEIR MUSCULATURE

During the development of the oncosphere, the penetration gland changes, mainly in the fine structure of specific granules and bodies. In the preoncosphere of H. diminutu (see Pence, 1970), these granules surrounded by a unit membrane show slight variation in electron density. The cytoplasm of the gland cells is rich in glycogen. The cytoplasm of the penetration gland of the preoncospheral D. caninum contains oval-elongate dense bodies, which swell in the mature oncosphere; their contents disappear and the granular cytoplasm becomes loose. Also in H . diminuta granule density shows variation. Numerous granules are broken up into finely granular material. Earlier studies with the light microscope on hook development in special cells, oncoblasts, were reviewed in detail by Rybicka (1966), the same as the development of contractile structures in two regions of the embryo (mainly on the basis of Orgen’s studies). In the late preoncospheral stage, the cortical region produces a marked cuticle and the majority of superficial fibres. The medullar region forms intercellular material of a contractile nature, The fibres of both regions interconnect and become attached to the hooks. New knowledge of hook origin has been derived from Nieland’s (1968) EM study. The superficial layer of the cytoplasm (“cuticle”) forms sheaths directed towards the centre of the oncospheral body. Each sheath is joined by a desmosomal formation called midpiece which is in direct association with the hook-forming cell (oncoblast). The basal lamina separating the superficial layer of the cytoplasm from the body of the oncosphere is extended to the oncoblast. First, the hook originates intracellularly, and is surrounded by characteristic mitochondria. Later, the distal portion called the hook shaft (blade) projects through the surface of the body into the sheath, the cytoplasm of which forms numerous folds and contains characteristic dense bodies. During appositional growth of the “adage” of the oncospheral hooks, the sheath plays a role similar to that of the hypertrophic tegument during the histogenesis of the rostellar hooks in the cysticercus (see Mount, 1970). The proximal portion of the hook developing in the oncoblast is called the shank. The muscles develop in the so-called somatic cells rich in glycogen,

400

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SLAIS

and insert in the basal lamina near the collar and the base (the end of the shank) of the hooks. 111.

FUNCTIONAL

MORPHOLOSY OF

THE

QNCOSPHERE (HEXACANTH)

The results of recent extensive EM and histochemical studies on the genus Hymenolepis have added new knowledge to the cellular organization of the oncosphere. These studies dealing mainly with completely developed or artificially hatched infective oncosphere confirmed that there are two polarities in embryonic cestode development. The primary polarity in the immature hexacanth is somatic and leads to the origin of basic cell pittern and differentiation inside the infective oncosphere. The second polarity is germinative and establishes the pattern for further post-oncospheral development (Ogren, 1968b).

A.

ANATOMY OF THE FULLY DEVLLOPkI) ONCOSPHERE.

Numerous studies on oncospheres (mainly in whole mounts or histological sections) revealed the presence of two penetration gland cells and embryonic cells in the region behind the hooks. Ogren (1967) found 5 pairs of these cells in H. diminuta and arranged them into two classes: Class I cells with very distinct chromosomes and a compact cytoplasm: Class I1 cells with large nuclei, indistinct chromosomes, but prominent nucleoli. Later, Ogren (I 968a) determined that Class I embryonic cells are plastic, germinative cells responsible for post-oncospheral development : Class IT cells are somatic cells of the oncospheral stage associated with the differentiation of the musculature and specialized similarly to the oncoblasts and gland cells of preceding developmental stages. An excellent EM study on the cellular organization of the hatched oncosphere of H. citelli was presented by Collin (1969) (Fig. I). Reconstructing the larva on the basis of ultrathin serial sections, he demonstrated that the pattern of the oncosphere was bilaterally symmetrical, and that the anterior region was situated opposite the hook end (posterior region). The gland cells abutted on the so-called ventral face, the muscle system adjoined the dorsal face with the hooks. Inside the body towards the ventral side he found 13 cells which he called “cells of undetermined function”. These appear to coincide with the germinative cells described by the various authors, and could be subdivided into three types on the basis of their fine structure. The muscle system of H. citelli consists of 29 cells: of these, 16 form the somatic muscle system, 13 are hook muscle cells. The muscle cells are, generally, typical of cestode muscles as described by Lumsden and Byram (1967). The body muscle system forms wide bands of transverse encircling muscles situated close to the basal lamina of the outer oncospheral coat. They are underlaid by a dorsal and ventral pair of longitudinal muscles. Hook musculature is complex. The hook muscles insert in the collar and base of the hooks and in the basal lamina of the outer coat. Their pattern for H . citelli was

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FIG. I . Reconstruction of a hatched oncosphere of Hymenolepis citelli (ventral view). A-reconstruction of 13 cells of “unidentified function” (germinative cells) and their orientation in the organism: B-reconstruction of the gland. Schematically redrawn from Collin (1969).

described by Collin (1968). Ogren (1972) demonstrated the exact anatomical pattern of the basic hook musculature in H. diminuta (Fig. 2). He distinguished three muscle systems in each pair of hooks: (a) the protraction system extending the hooks; (b) abduction system drawing the hooks together toward the midline; (c) retraction system pulling the hooks back in the body, The general pattern of the hooks is similar in all developed oncospheres. The oncoblasts have disappeared and all that remains of the histogenetic process is the dense matrix of the collar encircling the hook below the desmosome. The circular desmosome lies at the site of projection of the hook through the peripheral cytoplasm. The hooks are composed of three layers

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(Collin, 1968; Pence, 1970). The outer granular layer surrounding the shaft is continuous with a zone of connective nature which joins the hook with the musculature. The granular layer becomes a wide band near the basal lamina of the outer coat, and forms the hook collar. This layer is absent from the

FIG.2. A-schematic

side view of basic musculature for a lateral hook on the left and a medium hook on the right in the invasive oncopshere of Hymenolepis diminutu; Bschematic ventral view of the oncosphere showing hook musculature as though on one plane. 1-protractors; 2, 10-tensors; 3, I 1-levators; 4,12-short adductors; 5 , 13-long muscle; 8-short abductor; 9-median adductors; 6, Irl-retractors; 6A-interhook abductor. Schematically redrawn from Ogren (1972).

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blade of the hook. An electron-dense middle layer of fibrous material surrounds the central crystalloid core of laminated structure. The composition of the oncospheral hook is not known. Positive tests for keratin in the outer layers of the hook observed with the light microscope correspond to the middle fibrous layer seen in the electron microscope. Although the size of the fibre is smaller than that normally present in vertebrate keratin, the middle layer of the hook should be considered to be a substance with a high concentration of cystine, i.e. a keratin-like substance. Histochemical tests were positive for phospholipids in the inner (central) core, but its basic substance is not known. Dvorak (1969a,b) found by interference microscopy of the lateral embryonic hooks of H. microstoma that the middle fibrous layer (sheath) is probably richer in disulphide bonds than the core. The outer granular zone differed in composition from either of the two other layers of the hook. Of interest is the outer cytoplasmic layer covering the oncosphere. Numerous evaginations on the surface form folds and ballooning regions. Below this outer cytoplasmic coat lies a basal lamina of connective tissue nature with numerous infoldings into the outer cytoplasm. Collin (1969) observed gland ducts in H . citelli oncospheres at the site where they passed through the basal lamina to the exterior. The secretion of the penetration gland containing two types of secretory granules formed typical blebs above this opening in the superficial cytoplasm. Histologically, the secretion reacted positively for basic proteins, and contained glycogen. The U-shaped gland of a developed oncosphere represented a syncytium with two cell nuclei in each of the two lobate arms. N o evidence of syncytial organization was found in the remaining cells of the oncosphere. The excretory glands, formed by two cells and placed in symmetrical arrangement below the lateral hooks, were described for pseudophyllidean oncospheres only. In a coracidium moving freely in water, these glands are, in fact, protonephridia, and their function is osmoregulatory rather than excretory. Studies on their fine structure are not available as yet. B.

FUNCTIONAL CAPABILITY OF THE INFECTIVE ONCOSPHERE

The infective oncosphere, having entered its first intermediate host, harbours a complex of germinative cells from which the next post-oncospheral stage develops. Somatic differentiation of the oncosphere enables it to escape from the embryonic envelopes and is responsible for its activities and motility leading to the infection of the intermediate host and location at the typical site where development continues. 1. Release from the embryonic envelopes The term “hatching”, generally used for both Pseudophyllidea and Cyclophyllidea, denotes a different process in each of the two groups. In the pseudophyllidean egg, hatching refers to the liberation of the coracidium from the thick capsule through the opened operculum. Apart from comprehensive studies on the influence of light on the hatching of the coracidium,

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little is known of the structure of the operculum, which differs from the resistant capsule formed by a quinone-tanned protein, or of the mechanism of liberation. It is evident that the structure of the operculum must be labile and highly susceptible to biochemical and mechanical effects, but it is still unknown whether the oncosphere itself plays an active role in opening the operculum. After ingestion by the intermediate host, the active oncosphere escapes rapidly from the coracidial embryophore which has no solid structure. Most authors concluded that liberation of the oncosphere was due to the effect of digestive juices. In eggs of the remaining cestode species, the term hatching refers to the complete liberation of the larva from the embryonic envelopes in the intermediate host. Smyth (1963,1969b) distinguished two processes during hatching the release of the hexacanth from its envelopes, and activation of the embryo. Release from the envelopes is greatly influenced by the mechanical activity of the embryo, the lytic effect of the oncospheral gland and enzymatic activity of the host stimulating the embryo and digesting the embryonic envelopes. In Proteocephalidae, the oncosphere leaves the embryophore by its own activity at a very early stage and remains free in the outer egg envelopes i n the water. In Hymenolepididae, mechanical damage of the outer egg envelope occurs frequently and is often facilitated by the host's digestive substances. Generally, the oncosphere escapes from the embryophore and the oncospheral membrane by its own activity. A survey of information on artificial hatching of numerous cestode species was given by Rybicka (1966). In D. polymorphus and Infula macrophallus (Coil 1967, 1968), hatching occurs without oncospheral activity. After the removal of the outer capsule in a standard hatching solution with trypsin, the plugs were lysed, and the oncosphere was released passively from the egg. In taeniids, the outer envelope is delicate, readily lost and seldom seen in faecal eggs. Hatching from the taeniid embryophore preassumes disintegration believed to occur mainly through digestion of the cement substance. Laws (1968) found that a two-stage swelling of the embryophore attending hatching causes doubling in volume of the eggs at each step. His experimental results suggested an active function during hatching for the cement substance between the embryophoric blocks, and of the blocks themselves, Both structures are converted by enzymatic action to a hydrophilic colloid and expanded by imbibition of water. After disintegration of the outer embryophoric membrane, the swollen embryophoric blocks are released and the expanding colloid of the cement substance causes the sudden disintegration of the embryophore. According to Smyth's contention (1963), the nature of the cement substance is different in the various species, and plays a major role in determining host specificity. Webbe (1967) demonstrated in eggs of T. solium and T. saginaza that the presence of protease is necessary for the disintegration of the embryophore and hatching of the hexacanth embryo prior to its activation by tryptic digestion and the influence of bile salts in the intestine. The oncospheral membrane is lipoidal in nature. Heath (1971) observed in several taeniid species that the oncospheres initially become motile within the

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oncospheral membrane after artificial hatching and activation. This appeared to result in the extrusion of the penetration gland contents which, in turn, appeared to soften the oncospheral membrane. The membrane showed ballooning, and the oncosphere escaped from the membrane by tearing the softened region by vigorous action of the hooks. This indicates also that the secretion of the penetration gland contains a specific enzyme enabling the embryo to escape from the oncospheral membrane. The secretion of the penetration gland was produced also by oncospheres cultured in vitro during the post-oncospheral stage (Heath and Smyth, 1970). 2. Infection of the first intermediate host Numerous authors employed copepods for experimental infection of the intermediate host. Oncospheres of pseudophyllideans rapidly penetrated the intestinal wall of the arthropod host by means of their hooks, and the first oncospheres appeared in the body cavity within several minutes. The fact that infection of juvenile copepods occurred more frequently than that of the adults indicated that the greater thickness of the intestinal wall restricted the possibility of infection. The presence of a penetration gland has not been disclosed as yet in the pseudophyllid hexacanth, which appears to penetrate the gut wall mechanically by means of its hooks. By contrast, in cyclophyllidean oncospheres including those developing in an arthropod host, the route through the gut is facilitated by penetration glands. Heath (1971) described penetration of T. pisformis, T. serialis and Echinococcus granirlosus oncospheres in mammalian intermediate hosts. After penetrating the epithelium of the intestinal mucosa, the matrix cells of the villus are rapidly lysed by the oncosphere, which produces two enzymes: a mucopolysaccharidase for activation and penetration, and a protease or hyalurinidase for further migration through the tissues. This observation coincides with EM findings of different types of granules in the penetration gland (see p. 399). It depends on the anatomical structure of the villi of the intestinal mucosa, and on the size of the oncosphere, whether the larva enters the lymphatic lacteal or a venule of the villus. During migration, oncospheres are capable of distorting into various shapes. Heath (1971) observed the passage of T. serialis oncospheres through lung capillaries. Changes in shape suggest that the oncospheres are very motile; this is in accord with a welldeveloped muscular system occupying most of their body. Ogren (1969) studied motility of H. diminuta oncospheres. He divided the complete cycle of motility comprising extension, holding, retraction and relaxation, into time intervals supposing a coordination of these rhythmic movements. The factor or mechanism responsible for arresting oncospheral motility at the site of its typical location in the intermediate host, and initiating post-oncospheral development is unknown. 3. Metamorphosis of the oncosphere The oncosphere, having reached the predilection site in the intermediate host, undergoes changes involving degeneration of one developmental stage

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and its replacement by another stage of different morphology. Ogren (1968b) described this late stage for H.diminutu as a stage of oncospheral development. Shults and Gvozdev (1970) suggested that post-embryonic development starts with the release of the oncosphere from the embryonic envelopes. Voge (I 967a) included in post-embryonic development all forms habitually referred to as “larval” stages which develop in the intermediate host. Smyth (1963) used the term larval development but after successful completion of in v i m cultivation of larval cestodes proposed the term post-oncospheral development (Heath and Smyth, 1970). I feel that the latter term is suitable for designating the complete development of the cestode regardless of the fact that one or several intermediate hosts are involved. It covers not only the period ending with the differentiation of the infective larva known otherwise as the metacestode, but also includes the period of a prolonged stay of the larva in the intermediate host associated with continued growth and development which is not essential for the completion of its normal life cycle. As suggested by culture in v i m , cessation of oncospheral activity marks an important turning point in post-oncospheral development. It initiates the growth of primary germinative cells and their division. Reorganization continues with the atrophy of the oncospheral musculature, the multiplication of the daughter germinative cells and their further differentiation. It is possible to distinguish two types of larvae during the initial stage of larval development. Larvae of the first type (procercoid and plerocercoid) are those which do not form a cavity and which utilize generally two different intermediate hosts. This type is typical of pseudophyllidean larvae. The second type comprises larvae forming a body cavity and differentiating into a scolex (the “anlage” of the future cestode) and a temporary larval organ. The morphology of these larvae shows considerable variation. They are called cysticercoid and cysticercus, and are typical of cyclophyllideans. Little is known of larvae of Tetraphyllidea, Diphyllidea, Trypanorhyncha and of several groups of Cyclophyllidea; unless their histology is studied, they are difficult to arrange in either of these two groups. In the following section an attempt has been made to analyse larvae for which knowledge of their morphology during the life cycle is available.

1v. POST-ONCOSPHERAL DEVELOPMENT OF LARVAE WHICH DO NOT FORM A CAVITY Considerable progress has been made in our knowledge of this type of post-oncospheral development in pseudophyllideans, and exact descriptions are available of the morphology of the procercoid and plerocercoid. These terms, however, have frequently been used for larvae of other groups (Freeman, 1957, 1959) which were not fully homologous with the pseudophyllidean plerocercoid larva although their shape was similar to it. Larval stages of several Trypanorhyncha have also been referred to as “plerocercus” or “plerocercoid”, but descriptions of the larva of the best known species,

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Lucistorl7ync4us lenuis, are incomplete. This indicates the need for obtaining comprehensive knowledge of the morphology of typical larval forms. A.

PROCERCOID OF PSEUDOPHYLLIDEA

The procercoid develops in the body cavity of copepods; Vogel (1930) described the metamorphosis of the oncosphere to the larval stage for Diphyllobotl7rium latum. The oncosphere penetrates the stomach wall of the host by means of its hooks, attaches itself to the outer wall and begins to round off. No considerable changes of the oncosphere occur during the first 24 h. On the following day, the germinative cells start to multiply intensively and shift from the centre of the body to its periphery and also to the posterior end with the hooks, whereby the somatic cells are forced towards the centre of the larval body. On day 7, the shape of the larval body is elongate-oval, its length up to 100 pm. The somatic cells being pushed to the background do not participate in the formation of the procercoidal body. On day 11, a ridge separates the posterior portion of the body with the hooks, the cercomer, leaving only a thin stalk to connect it with the larval body. The procercoid completes development in the intermediate host from day 16-18 p.i. The cells forming the cercomer originate from the germinative cells; later, its tissue degenerates and the cercomer separates generally at the time when the procercoid infects the second intermediate host. At the anterior end of the procercoidal body, an invagination is formed which changes its shape due to muscular activity. A cuticle and a layer of subcuticular cells originate; the cuticle is covered with fine spines except the frontal invagination, the caudal stricture of the cuticle and the cercomer, which narrows towards the caudal end. The separation of the cercomer is accompanied by the shedding of the old oncospheral “cuticle”, indicating that moulting occurs during procercoidal development. The newly formed musculature lies underneath the cuticle and differentiates into circular and longitudinal layers. Calcareous corpuscles appear on day I0 of development and increase in size. Frontal glands differentiate in the central axis of the larval body; their ducts are directed towards the frontal invagination. From the two primitive protonephridia a complicated excretory system originates with 30-50 flame cells, and anastomosing canals collecting in the 3-4 main canals and opening to the exterior at the posterior end of the body after the shedding of the cercomer. Even the finer branches of the canals open through secondary pores to the surface. A similar morphological development of similar timing was described by Mueller (1965) for the procercoid of Spirometra mansoides. According to the description of T. nodulosus by Fuhrmann (1931), the cercomer of this species is larger than that of D . Iatum and other Diphyllobothriidae, but development of the procercoid is similar in all pseudophyllideans. An exception is Ezrbotkrium crassum, in which the cercomer is longer, the frontal invagination and the cuticlar spines are absent. These morphological differences may be associated with the fact that the procercoid of Amphicotylidae does not leave the intestine of its second intermediate host, but changes in it to a plerocercoid. The spines on the cuticle of the procercoid of

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Diphyllobothriidae are believed, together with the secretion of the frontal glands, to facilitate penetration of the larva through the gut wall of the second intermediate host on its way to the body cavity in which further development occurs. Braten (1968a) inferred from studies on the body covering of D. laturn that it represents a tegument formed by the syncytial distal cytoplasm of the subtegumental (subcuticular) cells typical of cestodes. The distal part of the microtriches is bent and well developed, its ends are tapering and it may constitute an almost continuous covering of the larva. Also Timofeev and Kuperman (1968) found large microtriches on the tegument of the T. nodulosus procercoid, which were of remarkably sound structure; the wall of their basal portion was supported by a highly osmiophilic substance under the superficial membrane. Fine microtriches were also present on the tegument to which the authors ascribed a trophic function. The tegument covering the anterior end of the body was thin; no coarse microtriches were present on the tegument of the cercomer. The fine structure of the cercomer suggested tissue degeneration. The coarse microtriches of the tegument of T. nodulosus, and the massive microtriches on that of D. laturn may have a protective function particularly for a procercoid penetrating the intestinal wall of its second intermediate host. B.

PLEROCERCOID (SPARGANUM) OF PSEUDOPHYLLIDEA

Not much work has been done on the morphogenesis and histogenesis of the plerocercoid during the initial period of its differentiation from a procercoid in the body cavity of its intermediate host. The procercoid of D. laturn loses its cercomer when penetrating the intestinal wall. It starts to grow rapidly in the body cavity of the host; its cuticle thickens and gradually loses the fine hairs on its surface. Bothria are formed at the sides of the frontal invagination at the tip of the body. Calcareous corpuscles increase in numbers and development of the excretory and nervous system continues. The conversion of the procercoid to the plerocercoid proceeds smoothly. The larva has no appendix and its change to the adult stage occurs in the definitive host mainly by subsequent differentiation of the genital organs. In some groups (e.g. Ligulidae) an advanced differentiation of the rudiments of the genital organs and strobilization occurs within the intermediate host. Wikgren (1964b) observed spontaneous invagination of the scolex in plerocercoids of D. laturn more than 5 mm in length. In several members of the Triaenophoridae, hooks develop on the scolex at this stage, but their histogenesis is unknown. Such unknown data could be compared with the development of the rostellar hooks of Cyclophyllidea. Pseudophyllidean plerocercoids, since they are of a similar pattern and often utilize a large number of different host species, have been studied extensively, often with the purpose of identifying the species under consideration. As plerocercoids of the Diphyllobothriidae are difficult to identify, increased attention has been given to their microscopic anatomy. Chizhova and Gofman-Kadoshnikov (1 959) recognized two types of plerocercoids by

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diagnostic features: length, presence of metameric folds dividing the body, shape of the scolex and presence of frontal glands, thickness of the cuticle and length of cuticular bristles. Wikgren (1964b) used similar criteria for identifying plerocercoids of D . dendriticum and D. osmeri. According to the length of the bristles (also hairs, filaments) and their presence or absence, this author distinguished 3 groups of plerocercoids. The bristles are, in fact, microtriches of the larval tegument. D . latum represented the group without bristles, this being consistent with BrAten’s ( I 968b) EM finding inferring that microtriches of this plerocercoid are few and short. Their proximal part is only 0-2 pm long, the distal part 1-1.2 pm, this being the reason for not having observed them under the LM. The microtriches of other diphyllobothriids were not studied with the EM. However, it was confirmed by EM studies on the plerocercoid of T. nodulosus (see Timofeev and Kuperman, 1968),of Schistocephalus pungitii (see Timofeev, 1965) and of S.solidus (see Morris and Finnegan, 1969) that the long bristles of these larvae are typical larval microtriches with a short base (0.25-0.50 pm) and a long distal part (up to 5 pm); the diameter of the cross section of the base (0.1-0.15 pm) decreases towards the point. The microtriches of the plerocercoid are similar to those of the cysticercus bladder wall (see p. 449), this being in accord with the mode of life of both larval types in the tissues of their vertebrate hosts. In plerocercoids of T. nodulosus and, particularly, of S. pungirii and S. solidus, the distal cytoplasm forms characteristic processes enlarging the tegumental surface. The distal cytoplasm of the plerocercoid contains inclusions, in part ovoid (lamellated bodies) in part rod-shaped (disc-like bodies) which are typical of the larval stage. Kwa (1972%b, c) showed alkaline phosphatase and protease to be present in the region of the tegument of the sparganum scolex of S. erinacri. Although histological study showed the absence of any visible gland cells, studies on the fine structure of the scolex tegument revealed the presence of two organelles. The first was shaped as a pit in the surface of the tegument with a ciliary ring at its mouth and dark-staining granules at the base. The second organelle appeared as a packet enclosing membrane-bound transparent granules. Both opened at the surface apparently for releasing the contents to the exterior. The morphological structures may be the explanation of the sparganum’s efficacy in penetrating the gut of the intermediate host. Wikgren and Gustafsson (1965) observed in D. larum, D. osmeri and D . dendriricum that the karyotypes of the somatic cells of these species are identical. Mitotic activity of all tissue plerocercoid cells (Wikgren, 1964a) showed variation in types of cells, and the author confirmed the existence of a relationship between mitotic activity and glycogen distribution. Cestode mitosis is of short duration: Wikgren (1966) recorded a period lasting from 13 to 17 rnin at 38°C for D.latum; but the process was prolonged at lower temperatures. Cell mitosis was not seen in the subcuticular layer, in the flame cells or in cells of the calcareous corpuscles, seemingly because of their functional differentiation. Populations of parenchymal cells were heterogeneous and only a fraction contained mitotically active cells. Wikgren and Gustafsson (1967) reported that these strongly basophilic cells represented more

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than 50 of the cell population of the inner parenchyma. They demonstrated (Wikgren and Gustafsson, 1971) in D. dendriticum plerocercoids a pool of free basophilic cells identical with embryonic cells and amounting to more than 32% of the entire cell population. These were germinative cells from which various specialized cells and the primitive genital rudiments originated. Migration and division of these cells was responsible for the growth of the subtegumental tissue (Wikgren and Knuts, 1970). v. Bonsdorf et al. (1971) demonstrated cell composition of the plerocercoid of D. dentriticum on the basis of different findings with the LM and EM. The various tissue components were distinguishable in cross-section, i.e., the tegument, the outer (cortical) parenchyma, the parenchyma, the parenchymal muscle layer composed of outer longitudinal and inner transverse muscle fibres, the inner (medullary) parenchyma, the nerve trunks and the excretory ducts. The type

FIG.3. Part of the histological section through a diphyllobothriidnodule on the wall of the mid-gut of Thymallus urcticus baicalensis. In the centre of the plerocercoid which is coiled in a ball, the scolex portion with developed bothria. The increased number of cells in this part is responsible for intensive staining. Weigert-van Gieson (25 x ). Material by courtesy of Dr. E. Ergens.

of structure of the tegument is typical of cestodes (p. 436). The germinative cells mentioned earlier in the text are distributed throughout the body; they are most numerous along the inner border of the parenchymal muscle layer showing a tendency to adhere to one another and form cell aggregates. They contain numerous ribosomes and dark cytoplasm. Parenchymal cells are large; the nucleus is surrounded by a slightly basophilic cytoplasm radiating

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in strands and sheaths, and their processes contain glycogen. These cells are specialized for glycogen and storage supportive in nature. The cell bodies of the dorsoventral muscles lie in the inner parenchyma. Their muscle fibres extend to the tegument or to the parenchymal muscle layer. The cells of the transverse and longitudinal muscles are arachniform. Types of cells supposed to be nerve cells were found. Calcareous corpuscle cells were numerous in the parenchyma, but absent from the tegument and the muscle sheath (Fig. 3). C. L A R V A L STAGES OF TETRAPHYLLIDEA

Extensive knowledge is available of the larval stages of Proteocephalidea. A description of the life cycle of Proteoc.ephaluspirviatilis was given recently by Fischer (1968). He observed in the oncosphere a structure consisting of two parts connected with a bridge, and 4 bodies suggesting cell nuclei. The secretion of this gland was produced by the oncosphere mainly during liberation from the oncospheral membrane. The larva developing in the body cavity of the copepod was similar to the procercoid of Pseudophyllidea and so was the period of differentiation. The cercomer originating on day 10-1 1, however, disappeared and larval development in the first host continued by a differentiation of the suckers and the rostellum, the excretory system and the calcareous corpuscles. In accord with Freeman ( I 964), the author called this larva a “plerocercoid I”, it continued growing in the intestine of the fish host as “plerocercoid 11”. attaining the adult stage without leaving the intestinal lumen. Similar small plerocercoids of Tetraphyllidea from marine fishes and invertebrates were described by Anantaraman ( 1963a). The remarkable behaviour of parenteral plerocercoids of P. ambloplitis was described by Fischer and Freeman (1969). These larvae are similar to the sparganules of S. mansoides, obtained by Mueller (1965) after 9 weeks of in vitro culture of the procercoid; they left the viscera if the temperature of the water was raised, and penetrated the intestinal lumen of the fish host. A substance secreted by the differentiated large end organ of this larva apparently aided penetration. After attachment to the mucosa, this organ disappeared rapidly. The larval stage of the lecanicephalid cestode Tyloceplialum sp. encysted in the American oyster Cravsostrra virginica, is neither a true procercoid nor a plerocercoid. Rifkin et a/. (1970) referring to it as metacestode observed that the tegument of this larva consisted of a distal cytoplasm and of subtegumental cells similar to those of other cestode larvae. In contrast, however, there were also many membrane-lined vesicles extending into the distal cytoplasm, in which were embedded minute superficial hooks. A surprising feature of the tegumental surface were special rnicrovilli. The fine structure of their stem was similar to that of typical microtriches, but they were not differentiated into a basal and distal part and terminated as a spherical vesicle; sometimes they were intertwined. Apparently, they prevented any intimate contact with the encapsulating fibres; the fluid of the host was kept in motion by microvillar movement.

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LARVAL STAGES OF CARYOPHYLLAEIDEA

The larval stages of Caryophyllaeidea develop in oligochaetes or other annelids. The oncosphere develops in operculated eggs and changes into a typical procercoid with a cercomer in one intermediate host only. Calentine (1965) studied the development of Biacetabulum macrocephalum and B. infreguens; their oncosphere penetrated the intestinal wall of the tubificids and migrated through the coelom to the gonad area. The scolex of thedeveloped procercoid was similar to that of the adult form, but only the primordia of the gonads were present. The larva lost its cercomer in the intestines of the definitive host and matured therein. A similar development was observed by Calentine (1 967) in four different species of caryophyllaeid cestodes. In members of the genus Archigrtes, the genital “anlage” of the parasite completed differentiation into a procercoid representing the neotenic larva. In A. iowensis, Calentine (1964) confirmed experimentally that development of the procercoid lasted 70 days in oligochaetes; embryonic hooks were present in the posterior end of the cercomer. Recent information on the histogenesis of these larvae has not been available as confirmed in the reviewing article by Mackiewicz (1972). In another paper Mackiewicz et al. (1972) described the attachment of the scolex to the intestinal mucosa for 15 species of caryophyllaeid cestodes. The pathogenic effect of these larvae on the host depends largely on the morphology of the scolex and its organs. E.

TETRATHYRIDIUM OF MESOCESTOIDES

Larval stages, similar to the plerocercoid, from the body cavities of vertebrates, known as plerocercus or dithyridium, represent a parenchymal larva with a scolex invaginated deeper into its body than that of the cysticercus. In this larva, however, the cystic cavity, the rostellum and the hooks are absent. This larvae, nowadays called tetrathyridium, is the second larval stage of Mesocestoides. According to Yamaguti (1959), the first larval stage of this group is not known. Voge (1967b), and Voge and Seidel (1968) first described the development of Mesocestoides grown in vitro, from the oncosphere to the fully developed tetrathyridium. Yoge’s studies disclosed that the first developmental stages are essentially similar to those of the procercoid in the formation of the cercomer and the differentiation of the cone at the anterior end, first protruded out of the body and later withdrawn as a frontal invagination. After separation of the cercomer “hairs” start to differentiate on the cuticle. Young tetrathyridia 0-5 mm long have a developed apical organ with signs of excretory functions, outlines of 4 suckers and an excretory system opening to the exterior at the pointed end of the body. Later, a completely developed tetrathyridium was obtained from culture in vitro; it was 1.5 mm long, had lost its apical organ, and had fully developed suckers. In the viable tetrathyridium, the scolex is generally withdrawn deep into the anterior portion of the body, and the suckers open into the invaginated canal, the tegument of which is similar to that of the cysticercus. Similar

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observations were made by Voge and Berntzen (1 963) in a histological section through the tetrathyridium. The structure of the tegument was similar to that of the invaginated scolex of the cysticercus except that the subtegumental muscle layer was not differentiated into a circular and longitudinal layer. According to Voge’s illustration of Mesocestoides (Voge 1967a, Fig. lo), this represented an uninvaginated larva lying in a cyst in the liver, although the fibrous strands allowing withdrawal of the anterior portion of the body were clearly visible in the section. The tetrathyridia seen are either invaginated or in plerocercoid form. The microscopical anatomy of the invaginated tetrathyridium is similar to that in Voge’s illustration (Voge, 1967a, Fig. 1 I ) showing a tissue section of Ooclioristica sp. from Tribolium confcwm. The histological structure of tetrathyrid larvae with a withdrawn canal leading to the invaginated suckers (many were found in the subcutaneous cavity of the muskrat) did not differ from that of the parenchymal portion of T. crassiceps and T. pisiformis cysticerci (&is, 1966a). lnstead of the caudal

FIG.4. Three differently orientated sections through a tetrathyridium from the subepidermal pseudocyst of a muskrat. The invaginated canal leading to the scolex is similar to that of the cysticercus.In the body portion resemblinga tail, lies the main excretory canal. Weigert-van Gieson (30 x). Material by courtesy of Dr. J. ProkopiC.

bladder of the cysticercus, these tetrathyridia had a pointed process like that described by Fuhrmann (1931). Its centre consisted of loose fibrous tissue interspersed with granules of different size. In this process the wider excretory canals collected for discharging at a single excretory pore in the caudal process. The wall of this outlet showed histological differentiation (Fig. 4). In larvae encapsulating in connective tissues from the pericardiac cavity of a fox, and identified as Mesocestoides litteratus, the cephalic end was less markedly differentiated from the remaining parts of the body (Slais, unpublished). The invaginated surface leading to the suckers was similar to the remaining parts of the highly folded body surface. The morphological appearance and the size of the tetrathyridia (width 2 mm, length 5 mm) suggested that the larvae had aged under anomalous conditions in the body of the definitive host (Fig. 5).

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FIG.5. Tetrathyridium of Mesocesfoides litteratus from pseudocysts on the pericardium of a fox showing proliferation of the larval tissue, particularly that of the tail, and loss of the typical histological structure in view of its occurrence in the definitive host. Haematoxylin eosin (35 x ). Material by courtesy of Dr. J. Kh. Irgashev.

F.

LARVAL STAGES OF Catenotaenia, Pariiterina AND Cladotaenia

The types of larval stages of the suborder Anoplocephala are most varied. One larval stage of the plerocercoid type of Catenotaenia called the merocercoid or merocercus, was found in tyroglyphoid mites. It represents a solid-body stage with an apical sucker apparently homologous with the rostellum. This is resorbed when the typical scolex develops. The identification of the larval type and the cestode species concerned is difficult with larval stages of Cyclophyllidea obtained from the liver and lymphatic nodes of various small mammals. Freeman (1957) used the term plerocercoid for the larval stages of Paruterina rauschi and P . candelabraria, although in most of these larvae the scolex end and, hence, the suckers, were withdrawn deeply in the parenchymal body. In studies on the larval stages of Cladotaenia globifera and C. circi, Freeman (1959) found stages with an invaginated rostellar cone which resembled a plerocercoid. The author referred also to an invaginated scolex and to an external similarity of evaginated larvae to the cysticercus, but he examined only whole mounts and larval stages from day 8 or 10 of development onwards. Hence, the initial developmental stages of oncospheral metamorphosis are not known. The author found no cavity in the larval body of these parenchymal stages. The description of these larvae suggested that they differ from the fully developed tetrathyridium described in the foregoing chapter in only the presence of the rostellar sucker with the hooks. However, neither the form-

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ation of the cercomer nor the shedding of the embryonic hooks were observed, and there was no developmental stage that could be compared with the procercoid. Abuladze (1964) used the term “cladothyridium” for a similar larval stage of taeniids to denote the parenchymal larva with the invaginated scolex which as such transforms into the adult stage. This indicates that exact information on this type of larval development in Cyclophyllidea can be obtained only from histological studies on the complete post-oncospheral development in situ, because sometimes, e.g., in D. caninurn, a cavity starts to be formed in the growing oncosphere, and becomes occupied by parenchymatous tissue before other differentiation occurs.

v.

POST-ONCOSPHERAL DEVELOPMENT OF LARVALSTAGES WHICH FORM A CAVITY

This type of post-oncospheral development typical mainly of Cyclophyllidea, involves the formation of a cavity in the parenchymal body of the larva. It originates usually in the developed larval body at an early larval stage allowing the invagination of the scolex differentiated at first i n an everted position. This form of development, the cysticercoid, occurs generally in an invertebrate intermediate host. The second developmental form involves the very early origin of the cavity in a developing larva; the cavity is spacious and gives the larva the appearance of a bladder. The scolex differentiates in an invaginated condition inside the cavity. This type of larva, the cysticercus, is typical mainly of mammalian intermediate hosts. In both the cysticercoid and the cysticercus, the wall enclosing the cavity differentiates into a specific larval organ. Also, larval stages of several Tetrarhynchidea (Rliynchobothrium, Eutetrarhynchus, Gymnorhynchus) and Diphyllidea have a cystic character. Anantaraman (I 963b), describing these larval stages for Ecliinobothrium from Bullia sp., called them “cysticercoids”, thus using the same designation as for the remaining larvae of Tetrarhynchidea. Their morphology, however, is not known and their similarity to the typical cysticercoid is negligible. A.

LARVAL STAGES OF LlNSTOWllDAE

Experimental studies on the development of several members of this family (in several species of the genus Tribolium) offered general information on morphogenesis, but not histogenesis, of their particular larvae. Gallati (1 959) demonstrated in experiments the complete post-oncospheral development of Atriotaenia procyonis, Millemann (1955) that of Ooclioristica deserti, and Widmer and Olsen (1967) that of 0. osherofl. The oncosphere has a marked penetration gland. After entering the intestine of its intermediate host, the oncosphere changes into a spherical larva; its embryonic hooks, initially dispersed in the posterior portion of the body, disappear, but no portion of the larval body becomes separated. A cavity is formed inside the body and four suckers differentiate at the anterior end. The shape of the larva becomes moderately ovoid; the anterior end of the

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body with the suckers invaginates into the cavity which has now been reduced to a mere slit. After invagination, the larva regains its spherical shape, and its diameter does not exceed 0-5 mm. Although the histological structure of these larvae has not been described as yet, they evidently do not develop a special larval organ such as the cyst of the cysticercoid or the bladder of the cysticercus. There is a certain likeness to the tetrathyridium and we can agree with Voge (1967a) in that the term “cysticercoid without a capsule” is unsuitable for these larvae, as well as for those of Thysanosovna. B.

POST-ONCOSPHERAL DEVELOPMENT OF CYSTICERCOIDS

When the oncosphere has reached the haemocoele of the invertebrate host, the germinative cells start to grow and multiply rapidly. Their increasing numbers can be demonstrated within the first three days of development as shown by Rybicka (1961) in Diorchis ransomi from an ostracod (intermediate host). The larva, measuring approximately 100 pm, maintained its specific shape. Schiller (1959) confirmed this finding in experiments with H . nana from Tribolium confusum. Later, the larva changes to an ovoid shape; growth continues and a cavity is formed in the posterior half of the body. Elongation of the larval body continues and the body differentiates into three parts: the anterior parenchymal part attenuating slightly towards the anterior end of the body; the central part with the cavity; the posterior part with the embryonic hooks. The attenuated posterior part homologous with the cercomer of the procercoid increases in length. Histogenetical differentiation has started during this phase, The primitive cavity (lacuna) becomes occupied by a cellular network (Voge, 1960d). Collin (1970) distinguished in H . citelli on day 5 of post-embryonic development a praescolex, a central cavity, the future wall of the cysticercoid, and a tail. He observed by E.M. that the anterior praescolex region is composed of cells with a scant cytoplasm and electron-pale nuclei. These cells represent the primordium of the future scolex and are, probably, undifferentiated proliferating germinative cells. Myofibres were typical of adult muscle cells. The central cavity was lined with cells having well differentiated cytoplasm with glycogen granules in rosette or alpha form, and lipid inclusions; their fine, long, cytoplasmic projections lined the central cavity. The tegumzntal zone with specific microvilli was differentiated as early as the third day of development. The differentiated interstitial cells were characterized by large nuclei and a large amount of cytoplasm with differentiated cytoorganelles. These cells lay close to the larval periphery and dominated in the tail (cercomer). Ubelaker (1970) described the ultramicroscopic character of the nucleoli of germinative cells of H . diminuta. Of interest is the early differentiation of the muscle fibres during the initial development of the cysticercoid after having entered the haemocoele of the intermediate host and attached itself to the outer gut wall, where at first it remains. This early development of the muscular system is associated with the developmental phase of invagination into the cavity of the differen-

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tiating cysticercoid. Although fully developed cyst icercoids are similar in appearance, their morphogenesis shows considerable variation. 1, Cysticercoids with ajully difkrenfiated scolex prior to invagination (Fig. 6)

During the initial developmental stages, these cysticercoids are similar to the procercoid of Pseudophyllidea. Later, their larval body differentiates into three parts. The anterior portion soon outgrows in volume the central portion with the primitive cavity, Rybicka (I 957) described morphogenesis but not histogenesis of these larvae for Diorchis ransomi. A rostellar cone differentiated at the bottom of an invagination formed at the anterior end of the developing scolex, and hooklets were formed. The histogenesis of the definitive hook has not been described but this may not differ from the mode of hook formation in the cysticercus (see p. 442). The well developed rostellar invagination formed a rostellar sac equipped with circular and loiigitudinal muscles which enabled a long protrusion as well as a deep withdrawal of the rostellum with the hooks.

FIG.6. Development of a cysticercoid with a fully differentiated scolex prior to invagination. Schematically redrawn after Rybicka ( I 957).

The middle portion of the scolex is widest and the differentiation of the four suckers is completed in it. The anterior portion of the body, attenuated caudally, passes into the central portion with the cavity. The attenuated area is the neck region of the adult cestode. The morphogenesis of the scolex is accompanied by the differentiation of the wall of the central portion, which forms the cyst wall of the cysticercoid. The third, caudal, portion of the body, the cercomer, elongates and forms the tail. Kotecki (1964, 1967) described a similar development for the cysticercoid of Purubissacanthes philuctes which forms a long tail, and for that of Wardoides nyrocue. The complete development of Fimbriaria fasciolaris cysticercoids in copepods, and the process of invagination was studied by Neradova-Valkounova (1 97 I).

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Invagination of the fully differentiated scolex into the cyst proceeds in that its cavity extends into the neck in the form of its central fission. The neck, resembling an empty cylinder with the scolex on top, starts to invaginate. The inner surface of its wall abuts the cyst wall and the scolex is drawn into the cavity without changing the normal position of the suckers; it is solid and therefore not reversed down to the rostellum as in the cysticercus. Only the rostellar sac of the cysticercoid with the rostellum and the hooks withdraw deep into the inside of the scolex. After invagination, a rim is formed at the site where the neck wall passes into the cyst wall. This, generally, is closed up by a mucus plug and represents the anterior pore (anterior canal) through which the scolex excysts in the definitive host. A similar morphological differentiation of the larval body into three parts occurring before invagination was observed in Anoplocephalidae, in which the fully developed larva is also a cysticercoid. The morphogenesis and differentiation of the spherical scolex with suckers, but without a rostellum, as well as the cyst with a differentiated wall and the cercomer (a short tail) was described before invagination by Freeman (1952) for two species of Monoecocestus from oribatid mites, by Melvin (1952) for M. sigmodontis. 2. Cysticercoids with a partly diiflerentiated scolex prior to invagination (Fig. 7 ) In some cysticercoids, a plug of cells differentiates between the neck and the central portion of the body, the cavity of which is filled with loose tissue.

FIG.7. Development of a cysticercoid with a partly differentiated scolex prior to invagination. Schematically redrawn after Voge and Heyneman (1957).

The formation of the plug occurs after the tripartite body division. Voge (1960d) observed it in H.diminuta at 5 days of development. A central slit (fission) was formed in the neck; the scolex invaginated at day 6 of development although the rudiments of its suckers were only just being formed.

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Further scolex differentiation proceeds inside the cyst as was confirmed by Voge and Heyneman (1957) for H . nmtu and If. diminuru, and by Schiller (1959), for H . nunu. In H . diminuru, differentiation of the cyst wall is very complex; in H . nana, the tail remains short and thick, like that of H . cifelli (Voge, I96 I a). 3. Cysticercoids with a scolex differentiating afferinvagination (Fig. 8) In H.microstoma, the larval body elongates, divides into two parts, and invagination of the anterior body portion occurs rapidly. The anterior end, the future scolex, contains an aggregation of cells at the site of the suckers: the posterior portion has two cavites originating from the separation of primitive lacunae. A recent detailed histological description of the morphogenesis of this species by Goodchild and Stullken (1970) completes Voge’s (1964) morphological study. The larva, by invaginating its anterior portion forms the invaginated scolex canal, which opens by a canal to the exterior. Differentiation of the rostellar suckers starts at the bottom of the invagination.

FIG.8. Development of a cysticercoid with scolex differentiation after invagination. Schematically redrawn after Goodchild and Stullken (1 970).

The initial developmental phase is similar to the primordial rudiment of the cysticercus scolex after withdrawal from the bladder wall to the inside (see p. 422), but the next step in the development of the rostellum and the scolex of the cysticercoid is different. The rostellar bud continues to elevate during differentiation and, hence, the suckers differentiate on the primitive scolex protruding into the scolex cavity. This type of development involves the formation of occlusions of the anterior canal visible also in the fully developed cysticercoid (Voge, 1963a). The scolex rudiment continues its growth until the scolex arises on ii short, solid, neck-like region from the bottom of the scolex cavity like the secondary growth of the scolex in

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invaginated overage cysticerci (see p. 423). Also of interest is the development of the tail of If,microstoma; its large size is apparently due to a cavity (Voge, 1963a). A cysticercoid of a similar type with a remarkable tail was described by Rysavq and ProkopiE (1965) for Staphylocystis furcata. 4. Cysticercoids without a tail

The tail of cysticercoids of Raillietinu cesticillus is replaced by a relatively large cavity which opens to the exterior, incorrectly referred to as the “excretory bladder”. Voge (1960~)called it the “posterior fold”. Its origin is unknown, but the histological structure is similar to that of the anterior canal. Little information is available on the morphogenesis of the complete post-oncospheral development of cysticercoids without a tail. Although studies on the scolex of Choanotaenia crassiscolex cysticercoids (Rawson and Rigby, 1960) gave some details of the very early stages resembling a plerocercoid in having bothria and a tail, the precise mode of development is unknown. Voge (I961 b) found in the superficial hyaline coat of developed Ch. infundibulum cysticercoids a minute, separated, cellular formation adjoining the external membrane of the relatively thin cyst wall. She suggested that this may be the reduced tail. Tailless cysticercoids with a very thick-walled cyst extracted from the body cavity of oligochaetes (monocercus) were identified by Karmanova (196 1, 1962) as Aplopuraxis filum and A . furrigera respectively. Recently, four developmental stages of Clt. estavarensis and Ch. crussiscolex cysticercoids from arionid hosts were studied by Jourdane (1972). The oncosphere changed into a solid, cellular ball (0.5 m m dia) on day 10 of experimental infection; this differentiated into two parts. The anterior part with the rudiments of the suckers and rostellum joins the appendix-like posterior part, the histological structure of which was analogous to that of the preceding stage. The author called this portion the cercomer. According to his drawings the fully developed scolex was overgrown by the cercomer and did not invaginate because there was no cavity in the posterior portion. Specific differentiation of the cyst surface was similar to that of H. brusutae cysticercoids from fhlebotomus species described by Quentin e t a / . (1971). C.

POST-ONCOSPHERAL DEVELOPMENT OF THE CYSTICERCUS

Hatched and activated taeniid oncospheres carried by the blood stream to the site of primary location in the host may be faced with a difficult migration through the organ. This requires the rapid development of the muscle system and the capability of producing lytic ferments for damaging the tissues and facilitating the passage. As regards nutrition, the larvae depend on the uptake of food from the host tissues, which necessitates the differentiation of resorptive, osmoregulatory and excretory structures. The larva attains its cystic stage very early, the bladder (primordial bladder, vesicule, Mutterblaschen). According to the literature, the cysticercus bladder is formed within a week of exceeding a length of 0.1 mm, but larvae of T. tae-

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niaejormis, E. grunirlosus and Alveococcus nrultilociiluris form it even when smaller than this. D t v t (1916) found in a 4-day-old echinococcus larva with a plasmodia1 mass 25-30 p m dia, a distinct vacuole. At 7 days development the larva (60-70 pm) had changed into a typical bladder with a germinative layer (8-10 pm thick) and a distinct cuticular membrane on its surface. Mankau (1957) found a vesicle (60 pm in diameter) in a 7-day-old alveococcus; this was covered by a thin germinal membrane which measured 1.5 pm at 16 days of larval development, i.e. at a time when the bladder was already alveolar. Orihara (1962) observed in T. taeniuejormis larvae that bladders with a thin wall (50 pm in diameter) were present at the third day of development. These findings indicate that histogenetical processes occur in the initial stage of larval development: the cells reorganize and multiply, a cavity is formed, the bladder wall starts to differentiate, a muscle system promoting motility develops, the bladder grows; the first stage is concluded with the origin of the scolex rudiment. Young (1908) drew attention to the indistinct cells of a T. pisformis oncosphere during the formation of the bladder. Aligon (1955) distinguished two types of cells in the larva of this species on days 8-10 of development, i.e. after the formation of the central cavity. At first, both types of cells were found at one pole of the elongate larva only. Crusz (1948~)found hair-like processes and differentiating cells in the subcuticle, parenchyma and muscles of a 10-day-old T. tueniaeforrnis strobilocercus. Rausch (1954) described cells of the germinative layer in the primary vesicle of an alveococcus measuring 30 pm. In an alveococcus at the same stage of development Sakamoto and Sugimura (1970) revealed by EM two types of cells in the germinative layer, i.e. immature syncytial cells and lightly-stained differentiated cells with many polysomes showing mitosis. Bortoletti and Ferretti (1972) observed microtriches of the larval type in a 14-day-old T. taeniaeformis larva; these were curled, grouped in bundles and penetrated the deep folds of the cytoplasm of adjacent host cells. The superficial syncytial cytoplasm contained conspicuous vacuoles and was underlaid by well-developed muscle fibres. Heath and Smyth (1970) first succeeded in cultivating a cystic larva from the oncosphere and contributed to our understanding of post-oncospheral development of a number of taeniids. The results were most satisfactory with T. pisformis in which larval development progressed from polar differentiation to the formation of the scolex rudiment with the hooks. In larvae 0.1 mm long, a well defined zone of tegumental cells with drops of secretion on the surface was observed at the anterior end. According to histochemical reactions the cells of this zone, from which the scolex rudiment developed, contained a substance with characteristics of an acid mucopolysaccharide, suggesting a secretory nature. A scolex rudiment also developed in a culture of activated T. ovis oncospheres; drops of secretion were found on the periphery of numerous bladders. Larvae of T. hydatigena, T. serialis and E. granuloslrs grown in vitro developed only to the cystic stage. Heath and Elsdon-Dew’s (1972) results of culture experiments with T. taeniuejormis and T. suginara oncospheres were analogous to a typical early cystic larva

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at 9-10 days of developmenl. Successful cultivution of the complete postoncospheral stage of both species may solve the problem of hook formation in T. saginata-these may be temporary hooks which become reducedand the mode of metamorphosis of the invaginated T. tueniaeformis larva to the strobilocercus. The time of appearance of the first signs of active movement in vivo recorded in the literature was consistent with that of motility in vitvo. These larvae are represented by a simple bladder with a histological structure in accord with its functional requirements. The differentiation of the histological structures of the parenchyma has advanced to such a degree that it can be demonstrated in necrotic larvae. This was confirmed in larvae from calcified foci in the liver of man by Slais (1965b). In three cases the author succeeded in identifying them on the basis of differential diagnoses as primordial bladders of T. hydutigena. I . Development of the invaginated scolex in the cysticercus The primordial bladder wall of the larva starts to thicken very early, generally at one of the poles only. The thickening is due to a group of cells with proliferation properties; these act as a morphogenic organizer. Cell multiplication proceeds at speed and a cellular wedge extends into the bladder cavity. The tegument infolds into the primitive scolex rudiment and the bottom of the fold widens. Later, the rostellum, suckers and hooks of the scolex originate at this site. This process has received increased attention in numerous studies explaining the histogenesis of the muscle tissue of the rostellar cone and the suckers, this being of primary importance for the movement of the hooks and for the attachment of the scolex to the intestinal

FIG.9. Schematic illustration of the structure of the invaginated portion of C . bovis. aiiivaginated scolex; b-spiral canal ; c-bladder wall. After Slais (1970).

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mucosa of the definitive host. Bilquees and Freeman (1969) gave a detailed picture of the histogenesis of the rostellum of T. crussiceps in relation to earlier data in the literature. By the time the rostellum and the hooks have completed their differentiation, the remaining tissues of the scolex have developed to a degree that enables evagination and attachment. Growth of the invaginated canal continues at a slower rate and it coils and forms the spiral canal (Zwischenstuck) (Fig. 9). After evagination of the cysticercus, this portion forms the neck region and establishes the connection with the bladder. This portion is digested in the intestine of the definitive host. The cells acting as a morphogenic organizer concentrate under the tegument close below the suckers and, in the adult cestode, are responsible for the growth and differentiation of the proglottids (Slais, 1966b, 1970) (Fig. 10).

FIG.10. A---Spiral canal with accumulation of subtegumental cells in the terminal portion near the suckers (e) of an invaginated C . bovis (80 x ). B-Proliferation zone of subtegumental cells (a) below the suckers in an evaginated scolex of C. bouis (150 x ). Weigert-van Gieson. After Slais (1966b).

The proliferation centre of an invaginated cysticercus, which has remained for a prolonged period in its intermediate host, becomes reactivated and the scolex grows out of the invaginated canal on an elongating neck. Authors once believed this to be true evagination of the cysticercus in its intermediate host preceding death and resorption by intensive tissue reaction of the host (Fig. 11). Slais (1966b) found that this phenomenon observed in various species and types of cysticerci does not lead to the differentiation of the larva into a n adult in the intermediate host, but involves only initial organic

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FIG.1 1 . A-Scolex

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growing on a short neck into the invaginated canal of C. bovis ( I 3 x ),

B-C. hovis with a scolex on a neck protruding from the invaginated canal far above the surface of the bladder ( 5 x ). After Slais (1966b).

growth similar to that of a cysticercus cultivated in vitro in an unsuitable medium. Similar observations were made by Freeman (1 962); repeatedly injected parent budders into the peritoneal cavity of mice lead to a pronounced elongation of the solid body proper on the scolex end. Sometimes, pseudosegmentation occurred in this region which was more than 20 mm long and such a budder resembled a strobilocercus. Serial inoculation experiments with C. crussiceps (strain ORF) into the body cavity of mice showed that continued growth of the scolex region was also possible outside the gut of the definitive host, because proliferation of the inffammatory tissue was negligible. The author did not test the infectivity of these forms. Similar viable forms of C. bovis were found by $lais (1966b); these forms may be capable of attachment to the intestine and of continuing in their development. A similar proliferation of the scolex zone of growth occurs apparently in the development of the strobilocercus, but in this case it is one of the stages of normal larval development. In the coenurus larva, the primary bladder has to grow to a larger size before a number of primitive scolex rudiments can originate in it. Wagner (1939) found bladders measuring 1-2 mm dia without scolex rudiments on the brain surface of lambs on day 8-14 of experimental infection. In addition, he found bladders 1-4 mm dia without scolex rudiments in the brain grooves of a lamb dying on day 18 of experimental infection; these showed peristaltic movements. Bondareva (1963) found bladders 7 mm dia with a basic number of almost uniformly distributed rudiments in lambs on day 28 of infection

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FIG.12. Part of a group of scoleces in a large bladder of Curriitrits skrjuhini. On t3c right, two invaginated canals united to discharge at a single pore. Haematoxylin eosin. Material by courtesy of Dr. P. P. Vibe. Inset: primary “adage” of two scoleces from a 35-day-old bladder of a C. cerebralis measuring 8 mm in diameter. Weigert-van Gieson (I I x ). Material by courtesy of Dr. V. I. Bondareva.

with T. multiceps. Scolex rudiments in 30-day-old bladders of a similar size represented groups of minute infoldings of the bladder wall with a formed rostellar cone situated at the base of the fold ($lais, unpublished) (Fig. 12). Larsh et al. (1965) recovered thin-walled cysts (2 mm dia) from the brain and from under the skin of mice on day 30 of experimental infection with T. serialis oncospheres. They observed by E.M. typical thin and long microtriches on the bladder tegument. In large coenurus larvae, new scolex rudiments originate on the growing bladder; these are formed in the initial parts of the invaginated canals of the primary scolex rudiments and explain the typical local concentration of invaginated scoleces on the otherwise smooth surface of these large bladders (Kunsemiiller, 1908). Sometimes, the bladder wall regresses close to the opening of the invaginated scolex canal, and the scolex rudiment with the parenchymal tissue arises like a bud from the bladder surface. The external scoleces grow continuously, their corpus being joined to the main wall by an attenuating stalk (Fig. 13). The tegument of these scoleces loses the microscopic character of that of the bladder wall and resembles the surface of the invaginated canal. These scoleces have sometimes been mistaken for evaginated scoleces (Wagner, 1939) and sometimes described as atypical larval forms, especially when all scoleces of the cyst looked alike (Rukavina et ul., 1957-in T. serialis from a hare). The echinococcus scolex differentiates in a slightly different way. Generally, the rudiments are not formed from the original germinative cyst layer, but in special brood capsules originating as many small nuclear masses of

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FIG.13. Outer scoleces on the bladder wall of T. nirtlticeps coenurus. Schematically redrawn after Wagner ( I 939).

the germinative layer. These form small vesicles joined to the main wall by a delicate pedicle. Sakamoto and Sugimura (1970) observed by the EM that the brood capsule of the alveococcus at first forms a mass of dark-stained undifferentiated cells. The cavity of the brood-capsule originating at a later stage is surrounded by syncytial cells with microvilli on their surface, The ultrastructure of the surface of the cavity is thus consistent with that of the wall of the primordial bladder. The final differentiation of the rostellum, hooks and suckers of the echinococcus scolex is similar to differentiation of these organs in the remaining cysticerci, but no satisfactory explanation has been given as yet of the development of the primary local thickening of the wall of the brood capsule. The first complete description of all stages of scolex formation in brood capsules since Goldschmidt (1900) was given by Dissanaike (1962) from cysts found in Indian goats. The rudiment originated like a local thickening of the wall which grew continuously into the brood capsule. Its peak, the future rostellum, withdrew into this solid cone and the invagination deepened during hook differentiation as in the rostellar sac of the cysticercoid. The suckers differentiated on the wall of the invagination. Early development of the muscle system in the scolex rudiment was responsible for changes in its relationship to the wall of the brood capsule, because the complete rudiment protruded towards the outer side of the wall. In the final phase, the differentiated protoscoleces withdrew again to the inner side of the brood capsule wall and contact with the wall was maintained by means of fine stalks only. A protoscolex actually represents a scolex with a deeply withdrawn rostellum and suckers covered by a tegument of the minute post-sucker region passing into the stalk. They lack a larval portion homologous to the invaginated canal present in all other cysticerci.

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The brood capsule of the alveococcus generally contains one scolex only. According to Lukashenko (1964), the sucker and rostellum differentiate on a conical bud growing into the capsule. This becomes a stalk in contact with the capsule wall. Invagination of the protoscolex occurs after the formation of the hooks in the thickened cuticle on the rostellum. Differences in the histogenesis of the echinococcus protoscolex are associated with the minute space in the brood capsule and with the minute size of the vesicles of the alveococcus. Lukashenko (1966) found in various hosts that bladders of a certain size divide into two by a partition growing from the wall into the cavity. This exogenous mode of division is responsible for the origin of the alveolar complex of this parasite. 2. Relationship of the scolex to the blacider in the morphology of the cysticercus The relationship of the scolex to the bladder changes after primary differentiation in a mode typical of the various larval types. This basic course of development is retained in the T. crussiceps cysticercus, in which development of the invaginated scolex rudiment transforms the original thin-walled mother bladder. The location of this larva mainly in the body cavities, and its existence in exudative fluid, frequently without an encapsulating reaction of the host, or with the formation of extensive inflammatory pseudocysts (Bondareva, 1968) is responsible for considerable mutability of this larva. Development of C . crassiceps starts with the formation of a primary bladder with the withdrawn scolex rudiment; this was described in detail in Mount’s (1970) EM study. The larva changes into an ageing larva if it remains for a prolonged period in the intermediate host. Under these conditions, the

FIG.14. Morphological change in a T. erussieeps larva during ageing in the intermediate host. a-typical infective larva; &considerable reduction of the caudal bladder due to the development of the parenchymatous portion and the spiral canal (Slais, 1966a).

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invaginated canal elongates, the parenchyma with the calcareous corpuscles multiplies and, finally, the larva has a solid scolex portion and a reduced caudal bladder (Fig. 14). This development covering the complete morphogenesis of C. crussiceps, is only the first developmental stage in other cysticerci (Slais, 1970). Important histological signs of the larval body during differentiation are changes in the tegument covering the growing parenchymal psrt of the larva; this loses the specific character of a bladder surface (p. 425). In the body of the host, advanced larval forms are fewer than young ones and the finding of all developmental stages in one host is very rare (Baer and Scheidegger, 1946; Freeman, 1962). In most findings of C. crussiceps, asexual reproduction of new individuals occurred by budding at the terminal pole of the bladder (p. 460). 3 . Larval stages of Taenia polyacantha and T. inartis The abdominal and thoracic body cavities of small mammals offer suitable conditions for mutability in the morphology of larval cestode stages. Although the adult T. crussictps is very similar to the adult T. polyacant/w, the larval stage of the latter is long, flattened, shows pseudosegmentation, and its scolex is always withdrawn deeply into the anterior portion (Rausch, 1959a). The initial larval stages of T. poljiacuntha are similar to those of the differentiated C. crassiceps, but after a prolonged stay of the former in the intermediate host, its body elongates and takes on bizarre forms. The adult stage, however, is attained only by the invaginated scolex. Abuladze (lY64) used the term “armatetrathyridium” suggested by Baer for these larvae. A larva of similar appearance is formed by T. murtis (syn. T. intermediu), if its site of location in the intermediate host is similar to that described. Shakhmatova (1964) confirmed in experiments that the initial development of this larva is similar to that of C. crassiceps. At the age of 2 months, and a length of 10 mm, the shape of this larva is like that of C. pisiformis. At 100 developmental days, the larva is 30 mm long and shows no resemblance to the cysticercus. It is of interest to compare the histological structure of these old forms with initial stages (Slais, unpublished) (Fig. 15). The armatetrathyridium of T. polyucunthu (from the thoracic cavity of Sciurus rulguris) divided into an anterior portion with the scolex, and a flattened body portion with folded margins during a more advanced stage. The maximum length of these forms was 70 mm, maximum breadth 10 mm. The body of the larvae was formed by loose parenchymal tissue; the structure of several folds of the body covering was like that of the racemose cysticercus with an abnormally proliferating bladder wall. Remnants of the bladder cavity could be distinguished histologically at the caudal end of armatetrathyridia measuring more than 30 mm. In a different set of armatetrathyrid material, bladders measuring 1.5 x 2.5 mm contained a differentiated scolex; in parenchymal elongate larvae (width 1 mm, length 6-7 mm) the scolex was protrusible and armed with hooks typical of this species. Several of the armatetrathyrids from T. murtis were recovered from the thoracic cavity of S . vulgaris, Mus rnusculus, Apodemus sylvuticus and Evotornys glureolus. Although the parenchymal portion of the body with the scolex was at different

FIG.15. Armatetrathyridium of T. polyacantha. (a)-Larva of tetrathyrid appearance; the invaginated scolex shining darkly through the body, inside the body cavity. (b)-Parenchyma1 larva with a small caudal bladder and the completely differentiated scolex (after artificial evagination). (c), (d)-Similar parenchymal larvae of different appearance. (e)-Anterior body portion with the invaginated scolex in over-age larvae; size of this portion similar to that of early developmental stages. Armatetrathyridium of T. marfis. (f)-Over-age larva divided into an anterior parenchymal portion with the scolex, and a large posterior portion shaped as a conical bladder. (g), (h)-Manifestation of proliferation during ageing of the larva restricted to the anterior body portion ( 5 x ). Material of different small rodents by courtesy of Dr. B. Horning. 16

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FIG.16. Subsequent differentiationof scoleces of a T.endothoruckus larva from the surface of the primordial bladder (from the thoracic cavity of Rhombomys opimus). (a)-(c)-differdisappearanceof the bladder the scoleces entiating scoleces reducing the bladder; (d-fter (right) remain joined by long degenerating bodies. (Scale: mm.) Material by courtesy of Dr. Ye. V. Gvozdev.

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stages of development, the remaining part of the body was always cylindrical with a conical ending and not flattened as that of T. po(yacunt/ra. Histological examination showed a cavity extending throughout this portion and a wall similar to that in the cysticercus bladder.

FIG.17. Schematic illustration of histological differentiation of scoleces on the bladder surface of a T. endothoracicus larva. Phases (c), (d), (el, consistent with photographs (a), (b), (c) of Fig. 16. (Slais, unpublished.)

4. Polyccphalic larvae from small mammals, mainly rodents Larvae developing in the thoracic and abdominal cavities of small rodents are often of the coenurus type. Rausch (1952) described the passage to the pleural cavity for the T. twitchelli larva and later (Rausch, 1959b) studied experimentally the compIete development of this species. After penetration of the lung tissue, fine bladders develop in the pleural cavity, elongate to about 10 mm and produce buds and primordia of scoleces as in the cysticercus. Later, the bladders become spherical and regress simultaneously with the development of the invaginated scoleces. However, in the true, large, coenurus the bladders grow extensively. The scoleces remain withdrawn in the growing parenchymal portion which attenuates towards the centre of the degenerating bladder. The surface of these formations changes in a very characteristic way (p. 425). The polycephalic bladders of T. twitchelli are of irregular shape also at an advanced stage of development, while in T. endofhoracicus these larval complexes ale of a centrally regular shape as described from an experimental infection by Gvozdev and Agapova (1963). A similar polycephalic form was described by Dollfus and Saint Girons ( I 958) from the abdominal cavity of Apodemus Jlavicollis. Gvozdev and Agapova ( 1 963) encountered T. endothoracicus at an advanced stage of development in a naturally infected Rhombomys opimus; the original bladder had disappeared, having been divided into the individual scolex formations. Their posterior portion pointing towards the centre showed bladder-like degenerative changes, and their connection comprised a thin band of easily severed connective tissue (Figs 16, 17). This may explain

FIG.18. Continuing proliferation of Hydutigeru krepkogorski scoleces from the abdominal ~ ~ ~ y s(a), (b), (c)-scoleces growing on the bladder surface showing cavity of R / t o n ~ / ~ oopimts. similarity to the strobilocet-cus; (d)-scoleces passing into long degenerating bodies are placed separately in a common inflammatory encapsulation after the original bladder has been reduced completely. (Scale: mm.) Material by courtesy of Dr. Ye. V. Gvozdev.

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the finding of separated T, twitchelli scoleces (Rausch, 1959b) in a number of spontaneously infected animals. Polycephalic larvae of similar appearance were found by Bernard (1963) in the abdominal cavity of Muridae, and identified as larvae of T. parva. Also polycephalic in nature are larval stages originally described as Hydatigera krepkogorski. These scolex formations are similar to a strobilocercus with the scolex in a normal position. Agapova (1950) first found various developmental stages of these larvae in the abdominal cavity of R. opimus and called them “coenurostrobilocercus”. The morphology of their development is similar to that of T. endothoracicus in that the larvae separate and the central ,bladder degenerates. Different, however, is the early development of the uninvaginated scoleces occurring as rapidly as the strobilocercus of T. tueniueformis (Slais, unpublished) (Fig. 18). Freeman (1956) found polycephalic larvae in liver cysts from small North American rodents and identified these as larvae of T. mustelae. The early bladders were similar to those of T. twitchelli and T. endotlioracicus, but the more advanced bladders were different, from compact to almost branched bladders. In several animals with experimental infection, the author found larvae with one and several scoleces in single animal, but never monocephalic larvae in one host only. Although the factor determining the larval type is unknown, the author assumed that monocephalic larvae may occur also in nature. The number of calcareous corpuscles present even in the bladder wall, exceeded by far the numbers observed in other cysticercus species. Recently, McKeever and Henry ( 1 971) observed monocephalic and polycephalic larvae of T. mustelae in several organs of a cotton rat with massive infection. The histological structure of these larvae, as illustrated by the authors, was consistent with that observed in monocephalic larvae of T. mustelae (syn. T. tenuicollis) from liver cysts of small rodents from Czechoslovakia and Austria (ProkopiE and Mahnert, 1970). According to Slais (unpublished) advanced age and the development of an invaginated canal connecting the completely differentiated withdrawn scolex with the surface is responsible for changes in the morphology of these larvae (Fig. 19). The increase in the number of calcareous corpuscles was marked in the enlarging parenchymal portion and in the wall of the reducing bladder. Thus, this cysticercus can always be distinguished by the histological structure of its bladder wall from early developmental stages of T. tueniueformis which have a thin bladder wall. Older stages of both species can easily be distinguished by the size of their hooks. The coenurus of T. bruuni has no predilection site in the organs of wild rodents. In Lemniscomys striutus, for instance, this coenurus can be found in the body cavities, under the skin and in the brain. The parasite is believed to cause brain coenurosis of man in the Congo and Ruanda Urundi (Fain, 1956). In view of its morphology, the T. brauni coenurus is intermediate between the small and the large coenurus because, according to Fain (l952), it forms smooth bladders with internal scoleces, bladders with scoleces on the surface as well as branched forms with scoleces both inside and outside the bladder.

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FIG. 19. Comparison of scolex “anlage” of T. mustelae and T. tueniaeforrnis larvae from pseudocysts in the liver of Microtus arvulis. (a)-Infective T. musrelue larva in inflammatory encapsulation-longitudinal section; (b)-the same larva in transverse section stained selectively for calcareous corpuscles. Note the complete differentiation of the scolex (length 50 pm)of the T. mustelae larva and, by contrast, the primordial anlage (of the same length) of the T. tueniaeforrnis larva (c) at the initial stage of growth. The scolex of the T. laenheformis larva with differentiating hooks (d) is of the same length (230 pm) as the longer diameter of the developed T. niusfelue cysticercus. (a)-Giemsa, (b)-Kossa, (c)-Weigertvan Gieson, (d)-haematoxylin eosin (340 x ). Material by courtesy of Dr. J. ProkopiE.

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The picture of advanced regression of the original bladder of polycephalic larvae, and the separation of the individual scoleces, is in remarkable accord with the picture of the brood capsule with scoleces of an echinococcus grown in viiro (Sniyth, 1962: text Fig. IB). The origin of these formations, however, is different. 5. Relationship ojthe location in the organ to the morphology of the cysiicercus

Slais (1966a) demonstrated proliferation of the caudal bladder around the developed scolex of T. solium in swine, and in brain cysticercosis of man

FIG.20. Subsequent growth of the bladder around the parenchymal portion of C. cellidosae from the muscles of swine. (ab-lnitial growth of the bladder folds around the invaginated canal. (bk-Transverse and (c)-longitudinal section through the invaginatedcanal in fully developed cysticerci. Goldner (12 x ). After Slais (1970).

(1965a, 1968). (Fig. 20.) In the typical host, however, location in the muscles is responsible for the morphological pattern of this cysticercus. The completely invaginated scolex with the long spiral canal lies inside the oval bladder, in the middle of its longer wall. A situation typical of C. cellulosae and C. bovis was observed by Sweatman and Henshall (1962) in T. ovis and, hence, applies to species recently placed in synonymy with T. ovis, i.e. T. cervi, T. djerani (Tazieva, 1964) and T. krabbei (Brzheskiy, 1963). The location of T.pisiformis and T,hydatigena cysticerci in the omentum and mesentery after their passage through the liver is responsible for the

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formation of pedunculate pseudocysts. It is typical of these cysticerci that the parenchymal portion with the scolex lies at one pole of the elongate bladder. The invaginated canal is less spirally coiled (Voge, 1962) than that of cysticerci located in the muscles. Of similar shape is the larva of T. parenchymatosa. The independent taxonomic status of this species was confirmed by Brzheskiy (1963) who rejected that of T. krabbei and its larva C. tarandi. In the T. parenclzymatosa cysticercus, however, a large number of calcareous corpuscles is produced in the scolex portion and the bladder during early developmental stages. An increased number of calcareous corpuscles is a typical feature of all cysticerci developing in the liver (this applies also to T. mustelae and T. taeniaeformis). From the comparative point of view, interesting results may be obtained from a comparative study on the histological structure and ultrastructure of the so-called posterior bladders originating under certain conditions in protoscoleces of E. granulusus cultured in vitro (Smyth, 1962; Yaniashita et al., 1962; Pauluzzi et al., 1965; Benex, 1968a). These protoscoleces are similar to a cysticercus with a caudal bladder. MORPHOLOGY OF POST-ONCOSPHERAL DEVELOPMENT v1. FUNCTIONAL

The oncosphere carries in its body the rudiment of the second larval stage of the cestode and the larva carries the rudiment of the adult. This is represented by the differentiating scolex which, in the final stage of its morphogenesis, forms the attachment organs, the muscles, the nervous and excretory systems, and a specific tissue component, the calcareous corpuscles. The only undifferentiated part of this developmental stage is the tegument, because the external environment of the larval stages differs from that of the adult forms which live in the alimentary canal of the definitive host. The existence of the larval stages inside the body cavities and organs of invertebrate and mammalian intermediate hosts is responsible for the specific adaptation of the body surface as in pseudophyllidean larvae, or the direct differentiation of specific larval organs, as in cyclophyllideans. A.

THE STROBILAR TEGUMENT OF THE ADULT STAGES

In order to evaluate and compare the degree of differentiation of the tegument during larval development, a survey should be made of all recent knowledge of the surface of the adult forms, which is mostly resorptive in nature. The most recent advances in knowledge of the structure of the body covering of these larval cestodes have come with the application of EM, histochemistry and biochemistry to the study of these worms. Lee (1 966) surveyed and evaluated the results of the first step of these investigations, which indicated that the cuticle and its subcuticular layer are special ectodermal coverings of syncytial character. The cuticle is a symplasmic border of the distal cytoplasm without cell nuclei, connected by cytoplasmic extensions with the perinuclear cytoplasm lying below the muscle layer in the parenchyma. The distal cytoplasm rests on a fibrous basal layer (zone) composed of fine fibrils; these surround and separate the individual fibres of

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the muscle layer and represent the finest fibrous network of the parenchyma, the basal membrane of classical histology. Slais ( I 965a) demonstrated this membrane with Gomori’s method for reticulin, disclosing also minute pores through which the cytoplasmic extensions of the subcuticular cell-body pass into the continuous plasmic surface. The proximal limiting membrane of the distal cytoplasm is joined to the muscle bundles with fine strands crossing the basal fibrous zone. The basal portion of the distal cytoplasm contains vesicular inclusions and mitochondria. The superficial layer of the cuticle, according to the description in classical histology, is the superficial layer of the cytoplasmic projections of the type of cellular microvilli. In view of their complicated structure, they are called microtriches. Morseth (1966b) studied their ultrastructure in the adults of E. granulosus, T. hydatigena and T. pisiformis, Race et al. (1966) in T. mtrlticeps. The fine structure of the microtriches is well known: a wide shaft (called also basal region, proximal half, proximal zone) with a thick electron-dense wall and a central core of considerably lower electron density than that of the wall, but consistent with that of the distal cytoplasm, arises from the distal cytoplasm of the tegument. The distal zone of the microtrix attenuates into a marked electron-dense conical tip (cap; spike; “shaft” is used by Jha and Smyth, 1969) placed aslant on the shaft; the transitional area between the central core and the electron-dense tip is marked by two dark lines, Precise electron micrographs showed that these lines are a pentalaminate base of desmosomal-like structure. The apical plasma membrane forms shallow crypts within the microtrix base. A relationship of these crypts with certain vesicles in the distal cytoplasm could be found. Several authors considered these vesicles to be pinocytotic. The plasma membrane passing as an outer membrane onto the surface of the microtrix, is complicated in structure and frequently referred to as being triple-layered. Jha and Smyth (1969) distinguished in it an outer and inner double membrane and found gaps which Morseth (1966b) and Lumsden et al. (1970) regarded as artifacts. Microtubules, however, were demonstrated in the electron-dense core of the tip. The microtriches were closely spaced and the ratio of proximal to distal zone differed in the various species. In H. diminuta, for example, the electron-dense tips were very short. Featherston (1972) described similar microtriches from the tegument of mature and gravid proglottids of T. hydatigena. He pointed out the tube-like membranes extending into the distal cytoplasm, and the close association of their distal end with the mitochondrion. This coincides with Jha and Smyth’s (1971) hypothesis on the biogenesis of the mitochondria. Berger and Mettrick (197 1) observed polymorphic microtriches in scanning electron micrographs of three Hymenolepis species. They observed marked differences between short tubular microtriches (approx. I pm) with a conical tip, and long flattened microtriches (approx. Spm) with spatulated tips. According to these authors, the microtriches are not important for nutrient absorption by the enlarged surface of the tegumental area. The larger microtriches may play a locomotory role in site-selection and intra-luminal migration of these worms. They also confirmed the existence of the pore canals, tubular structures penetrating the distal cytoplasm, but not forming cellular bound-

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aries. They open to the exterior and form the direct connection of the surface with the basal fibrous zone under the distal cytoplasm. Sometimes they are described as evaginations of this zone which, in earlier studies, has been regarded as a subcuticular canal, because the fine fibrils inside it could not be demonstrated by contemporary techniques. The mitochondria1 character of cytoplasmic formations in the distal cytoplasm is suggested also by the enzymatic activity in the tegument (Lee, 1966). A resorptive function is indicated by the high content of enzymes and the microvillous surface. Evidence of the absorptive surface of cestodes indicates that proteins of the tegument are synthesized in the subtegumental cell body and then transferred to the syncytial cytoplasm (Lumsden, 1966b). Carbohydrate constituents of the surface membrane appear to be added in quanta elaborated by the synthetic apparatus, particularly in the cisternae of Golgi and the vesicles of the perikarya (Oaks and Lumsden, 1971). In studies on the hydrolysis of phosphate esters, Lumsden et al. (1968) inferred that phosphatase activity may be localized largely at the external surface of the plasmalemma bordering the microtrix surface of the strobilar tegument. It is possible that surface phosphatases facilitate absorption of nutrients from the parasite’s microenvironment. The outer membrane of the microtriches contains a layer of acid anions capable of absorbing cations at neutral pH with a topical differentiation between the proximal zone and the distal tip. The new observations (Lumsden, 1972) are consistent with the presence at the tapeworm surface of a relatively intense negative electrostatic field potentially capable of repelling similarly charged particles. King and Lumsden (1969) confirmed assimilation of linoleic acid in H . diminuta grown in vitro. The transport of colloidal particles directly through the tegument of this helminth, described by Rothman ( I 967) under the term “transmembranosis”, was not confirmed by Lumsden et al. (1970). The study of Dike and Read (1971) supports the concept that the microtrix border of tapeworms should be viewed as a digestive-absorptive surface with a specialized arrangement of various functional components. B. HISTOGENESIS OF CALCAREOUS CORPUSCLES

Much progress has been made in our knowledge of the origin and importance of calcareous corpuscles, but no evidence was found that those of cestodes have an excretory role. This was confirmed by von Brand (1966) in his survey of the pertinent literature. Their large carbonate content may serve to neutralize the acid products of metabolism, or protect the larval stage as it passes through the stomach of the definitive host. It may explain also why only the invaginated scolex and not the bladder wall contains calcareous corpuscles. These also play an active role in the metabolic processes of the organism and constitute, as one of their functions, a phosphate reservoir for metabolic needs of the tapeworm (von Brand and Weinbach 1965). Kegley et al. (1970) investigated the mechanism of incorporation of inorganic cations. von Brand et a/. (1969) found much variation in the size

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and shape between corpuscles of various species. Differences in the inorganic composition of the corpuscles, however, may be a product of the parasite’s environment and not a typical feature of a certain species (Kegley et al. 1969). By EM study of cestode calcareous corpuscle formation, Nieland and von Brand (1969) inferred, in contrast with histological studies, that these originate in the cytoplasmic cavity of the corpuscle-forming cell. The corpuscle enlarges by the accretion of an organic homogeneous matrix and of granular inorganic materials in concentric layers with progressive compression of the surrounding cytoplasm. C.

MORPHOGENESIS OF THE MATURE PLEROCERCOID TO THE ADULT STAGE

Plerocercoids capable of infecting the definitive host are at different stages of differentiation in pseudophyllideans. This does not apply to the scolex which, at this stage, has already completed its morphogenesis. The most frequent changes were found in the fine structure of the tegument. This is apparently due to the change of hosts, i.e. from the cold-blooded intermediate host to the warm-blooded definitive host. BrAten (1968b) observed in D. laturn that rapidly developing microtriches cause a considerable thickening of the body surface. While in the plerocercoid the microtriches are loose and short (1.5 pm long), those on the surface of the adult worm are densely packed, and more than 4 pm long. In a plerocercoid entering its definitive host, the distal cytoplasm of the tegument loses its lamellar corpuscles and the mitochondria in the tegument of the adult form increase remarkably in number and size. Also, enzymatic activity in the scolex increases, In relation to the close host-parasite interface maintained by the bothria (Ohman-James 1968-for D . dendriticum). General enzymatic activity of the plerocercoid, however, is lower in view of the lower temperature of the cold-blooded host. Arme (1966) demonstrated biochemically in Ligdu intestinalis that acid phosphatase dominates in the larva, alkaline phosphatase in the adult. The primordial genital rudiments of the big plerocercoid of D. dendriticum coincide, according to Wikgren et al. (1971), with the small rudiments in the neck of the adult both of which are formed by an aggregate of germinative cells. In the plerocercoid of Schistocephalus, advanced differentiation and strobilation occur within the body cavity of the piscine intermediate host, because the span of life in the intestines of the definitive host is very short. Morris and Finnegdn (1 969) observed in the plerocercoid of S. solidus that advanced differentiation occurs only in the thick superficial tegument of each segment. The tegument of the velum remains thin and apparently undifferentiated like the surface of small worms. Plerocercoidal development is accompanied by changes in the composition of the tegument the surface of which contains a sulphomucin-basic protein complex (Morris and Finnegan, 1968). Subsequent growth of the tegument and the origin of microtriches typical of the adult worm were observed in S. solidus grown in vitro (McCraig and Hopkins, 1965). Changes in the ultrastructure of the tegument of S.pungitii at the stage of transition, from the plerocercoid to the adult form, were described by

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Timofeev ( I965), who did not observe finger-like outgrowths (cuticular processes) on the high tegument. In T. nodulosus, however, Timofeev and Kupernian ( I 968) did not observe any substantial differences in the fine structure of the tegument of this worm during the various developmental stages. D.

SCOLEX OF TAENIIDAE AT THE LARVAL STAGE

The scolex of taeniids, except that of several early stages of some cysticercoids, develops from the beginning in protective larval envelopes. The differentiated scolex of the future tapeworm is separated from the tissue of the intermediate host and enclosed in specific larval organs, ensuring its protection and nutrition.

I . Scolex of the cysticercoid The microscopical anatomy of the cysticercoid scolex has been described in detail in the earlier literature. The scolex proper lies in its normal position at the end of the invaginated neck region and is separated from the cyst wall by the scolex cavity which extends into the anterior canal. The rostellar sac is withdrawn deeply into the scolex. The rudiment of the adult cestode is separated from the more solid and more complicated wall of the cystercoid by the cysticercoid cavity. Rawson and Rigby (1960) described the functional anatomy of the scolex proper of a Ch. crassiscolex cysticercoid. Baron (1971) described the ultrastructure of the scolex of a R . cesticillus cysticercoid. The fine structure of the scolex tegument did not differ from that of the cysticercus, and neither did the typical microtriches with their electron-dense tips. The surface of the invaginated neck facing the surface of the scolex proper with its reversed side, consisted of a layer of distal cytoplasm only 0.25-0.7 pm in width. Its fine microtriches, with a diameter of approximately 0.05 pm, were called “bore microtriches” by the author. The distal cytoplasm, however, lacked mitochondria and other organelles and resembled the germinal membrane of the hydatoid cyst of E. granulosus (Morseth, 1967a). 2. Scolex of the cysticercus Slais (1966a, 1970) described in detail the histological structure of the invaginated portion for the cysticercus of C. cellulosae and C. bovis. The tegument, the muscle-, excretory- and nervous-system of the invaginated scolex coincide in their anatomy with that of the adult helminth. This histological structure comprises the complete invaginated canal up to the site of its invagination into the bladder, while after evagination, not all parts of the canal portion participate in the organisation of the adult form. LM studies revealed on the high tegument a low brush border of short microtriches (microvilli) similar to that described by Lumsden (1 966a) for H . diminuta. Baron (1968) observed at the same sites in C . crassiceps microtriches with electron-dense tips similar to those of the adult cestode, and nerve endings which were presumably sensory in and just below the surface of the invaginated scolex tegument. These appeared to be similar to the sensory endings described by Morseth (1967b) for the adult E. granulosus. Except for a brief

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reference to excretory ducts (Baron, I968), no information is available on the fine structure of the invaginated portion of the cysticercus. Morseth (l967a) found medullated microtriches on the surface of the distal cytoplasm in the anterior, invaginated portion of the echinococcus protoscolex which were present also on the surface of the suckers, while posterior to the suckers microtriches started to develop after scolex evagination. Distal caps were formed on the medullated projections of the distal cytoplasm covered with a protective film of PAS-positive material. Medullated microtriches, pore canals and a very thick layer of distal cytoplasm were found also i n the larval scolex of T. tnulticeps (Race et al., 1965). These findings indicate the possibility of determining in studies on the ultrastructure of the invaginated cysticercus scolex which portion of the invaginated canal remains and which is digested in the intestine of the definitive host. Similarly, knowledge may be obtained of the functional activity or inactivity of the invaginated scolex. This may be a helpful contribution to the knowledge of the physiology of the cysticercus.

FIG.21. A certain similarity, in transverse section, of the microscopical structure of the differentiated body of a T. tueniueformis strobilocercus (left), to the body of a DiphyNuhufhriurn sp. plerocercoid (right). Weigert-van Gieson (left 325 x ; right 650x). Slais (unpublished).

Bartels (1902) and especially Rees (1951) provided data on the functional anatomy of the differentiated locomotory organs of the T. farniuejiirmis strobilocercus. N o satisfactory explanation has yet been given of the way in which the invaginated scolex with fully differentiated suckers and rostellum is turned to a normal position in alarva in which the bladder is still its principal

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structure. Some such information was provided by Hutchison (1958), but no histological confirmation. The distortion of scolex shape after handling the material was demonstrated by de Rycke (1963). According to histological sections illustrated by Orihara (1962, Figs 6, 7), the scolex of T. taeniaeformis larvae arising from the bottom of the invaginated canal remains, at first, in this canal, i.e. the process involved is not a true evagination of the invaginated scolex from the bladder, as was confirmed also by a finding of larvae at the transitory stage in spontaneously infected M.arvalis (Slais, unpublished). A striking morphological similarity was found also between the outer segmentation of a strobilocercus at an advanced stage of outgrowth (Waltz, 1963) and the plerocercoid with premature segmentation in the intermediate host described for members of the genus Schistocephalus (p. 439). The question is still being discussed, whether or not true segments are formed in the strobilocercus in which only the rudiments of the genital organs develop rapidly after attachment in the definitive host. Studies on the origin of the genital organs in the various diphyllobothriid plerocercoids and data from the literature suggest that at least several of the segments under the scolex attain the adult stage. Cross sections through the body of very old T. taeniaeformis larvae from liver cysts showed a marked similarity in the pattern of the various tissue components to the histological arrangement of the Diphyllobothrium plerocercoid (p. 410). (Fig. 21 .) 3. Development of the rostellar hooks The functions of the tegument participating in the formation of the rostellar hooks during the development of the scolex rudiment are not only absorptive in nature as has been confirmed at large, but may also be secretory. According to Crusz (1948c), the blade of the hooks shows evidence of the presence of chitin. The base is non-chitinous, chemically and optically different from the blade and, hence, more liable to variation in shape and size. h i i s (1970) disclosed in histochemical studies on the hooks of C. cellulosae that the substance forming the hook is a scleroprotein with the typical presence of SS groups similar to the keratin of the cortex layer of vertebrate hair; he was unable to confirm the presence of chitin in the blade. Reactions for tyrosine, tryptophan and arginine were negative. The basophilic layer on the surface of the hook base was complicated in nature. The protein in it was different in composition mainly in that it contained tyrosine (but not tryptophan), cystein and arginine. Metochromasia and other methods suggested the presence of a mucopolysaccharide. The layer communicated with the parenchymal tissue sheath of the hook, in which a network of argyrophylic fibres was demonstrated. Biochemical and physico-chemical analyses of the strobilocercus hook (Dvorak, 1969c,d, e) revealed the dominant component of the hook as a keratin-like protein similar to bovine hoof-keratin and markedly different from insect chitin. The hooks were polymer-like structures with cystin disulphide Iinkage of the heterogeneous protein subunits. The antigenic potential of this structure was reduced by masking or neutralizing chemically active sites on the hook protein, and also by covering the structure with an antigenitally less active lipid.

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Scott (1969) studied the development of hooks of the Puricferotuenia paradooxa cysticercoid on a well developed rostellum deeply withdrawn into the rostellar sac. The distribution of the small hooks covering the rostellum in two rows changed finally to a single row of definitive hooks, changes which may be of phylogenetic significance. Disagreement on the origin of the primary hooklets in the furrow between the rostellar cone and the invaginated cavity of the developing scolex rudiment was resolved by Mount’s (1970) EM study. The hooklets originate by transformation of the slender, undifferentiated microtriches which grow to form the electron-dense distal cap; this enlarges and is surrounded by hypertrophied tegument termed “hook organ” by Bilquees and Freeman (1969). The tegument is modified by the presence of clusters of ribosomes which may function in the production of protein for hook formation. Hook protein is deposited along the edge of the hooklets to form the blade of the definitive hook. The handle and guard are formed by a deposition of hook protein along the base of the blade. As the hook matures the tegument surrounding the basal region develops numerous hemi-desmosomes. These connect with the fibrillar layer beneath the tegument anchoring the hook in position. The intensive secretory activity of the tegument during hook formation was confirmed by Smith et al. (1972b) who found the highest rate of RNA synthesis in the hook organ of larval T. crussiceps. 4. Esfablislmtenf of the scolex in the definitive host Not much is known of the mode of evagination and attachment of the scolex of a cystic larva in the intestine of the definitive host. In the cysticercoid, encystment of the uninvaginated scolex is mainly involved; in the cysticercus, evagination of the spiral canal (sometimes of considerable length) with the invaginated scolex at its end. Cysts of the echinococcus broken up mechanically by the definitive host along with the food, facilitate evagination of the protoscolex in the intestine. In other cases, all parts of the larval body except the scolex are digested mainly by pepsin, but also by trypsin and pancreatin. Bile salts play a major role in the evagination of the scolex. Numerous features concerning the attachment of the echinococcus have been explained. Smyth (1964) first drew attention to the special rostellar glands important for scolex function after evagination and attachment to the intestinal mucosa. Jha and Smyth (1971) observed in studies on the ultrastructure of the rostellar tegument of this cestode that the cells of the rostellar glands are already differentiated in the protoscolex. Secretory activity of the nuclei of these cells was observed after evagination in the intestine or in vitro. The authors associated this activity with the biogenesis of the mitochondria which are absent from the rostellar tegument in a freshly evaginated protoscolex. They described possible steps in the process of this biogenesis starting with the evagination of the basal membrane and the dumb-bell shaped membranous bodies. In a protoscolex grown in vitro, secretion droplets of the distal cytoplasm were observed ;these contained vesicles, mitochondria and vacuoles of varying sizes.

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Contact with the host tissue is maintained only by the top of the rostellum of the adult form. Featherston (1972) observed in T. lrydatigena that a major part of the rostellar surface was covered by a type of microtrix with a thin base and long spike. In this area mitochondria were not restricted to the basal layer of the distal cytoplasm, but were found close to the microtriches. The distal cytoplasm was extensively vacuolated. This indicates that the morphological character of this region is close to that of the bladder wall of the cysticercus. Similar observations were made by Jha and Smyth (1971) on the rostellum of E. grunulosus. The tegumental microtriches were long and thin, and often branched, hooked, barbed or curved. This pattern probably provides firmer adhesion to the host gut and increases the absorption area. It has been suggested by Smyth (1969b) that the scolex is an organ of attachment but it may also have a “placental” function and be capable of absorbing nutrients from the mucosal wall. According to Featherston (1971) evagination of in vivo T. hydatigena took place in the small intestine where much larval tissue was shed almost immediately. Marked differences between a freshly evaginated T. hydatigena cysticercus grown in vitro, and a 3-day-old scolex from the gut were pointed out by Featherston (1969). The rapid activation of the proliferation centre in the post-sucker region in the host’s gut was indicated by the speedy development of microtriches in this area as demonstrated by Smyth ( I 967) in E. grunu/osus on the third day following evagination in vivo. Differences in the structure of the surface of evaginated cestodes attached to the intestines (of different age and at different degrees of proglottid development) were observed by Featherston (1972). At the site where the larval tissue separated from the distal end in a 3-day-old cestode, a distal cytoplasm originated; its hair-like microtriches lacked the complete differentiation between the base and spike. In areas anterior to this site, this type of microtrix was replaced by the more typical form. Typical microtriches were found also on the terminal proglottid in 15-day-old worms. The findings suggest that freshly differentiating microtriches are similar to those of the bladder wall. Microtriches in the neck and sucker regions of 7-day-old worms were similar to those on the surface of the proglottid with certain variations in the electron-dense spikes. de Rycke (1966a) observed in H. microstomu that encysted larvae reached the bile duct of white mice on day 4 p.i.; the rudiments of the genital organs originated as early as day 3 p.i. External segmentation was seen on days 4 5 p.i. Bolla and Roberts (1971) showed in their extensive study on the differentiation of the genital rudiments of H. diminutu, that a marked proliferation of the germinative cells occurred 200 pm posterior to the apex of the scolex. Tissue reaction to infestation with H. microstomu in the bile duct of white mice within the first week post-exposure to cysticercoids was studied by Lumsden and Karin (1970) with the EM. Excystment of H. nafyi cysticercoids, and attachment of the scolex to the mucosa was studied by Hunkeler (1969). The rostellum was fixed in the dilated crypts of Lieberkuhn. Deep penetration of the rostellum in the intestinal mucosa of the shrew was facilitated by rostellar glands which atrophied later. Anchorage of the parasite deep in the mucosa was performed by the hyper-

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trophic rostellar sucker, while the scolex suckers of the adult form were completely non-functional. Of interest is the complete development of the genital organs in the unsegmented minute forms of E. granulosus grown in vitro (Smyth, 1971). The author, calling these forms monozoic forms, inferred that somatic growth and differentiation had been largely suppressed. Studies on this phenomenon in relation to neoteny of various larval cestode stages may offer interesting results.

VII. SPECIFIC LARVAL ORGANS

OF TAENIIDS

Specificallydifferentiated organs are well developed only in cyclophyllidean larvae, although these larvae sometimes exist under conditions similar to those of larval pseudophyllideans, and frequently utilize similar intermediate hosts. Sometimes, the shape of the cyclophyllidean larva is similar to that of the pseudophyllidean larva such as the strobilocercus of T.taeniaeformis. The metacestode resembles a cysticercus during its initial stage of development; later it lives free in the inflammatory liver pseudocyst similar to numerous plerocercoids in their specific hosts. Increased resistance of the scolex covering of the cysticercoid seems to be associated with the species of invertebrate intermediate host utilized. The protective function of the bladder is marked in cysticerci developing inside the host tissues; its structure and function are most specific in the echinococcus. A.

CYST WALL OF THE CYSTICERC013

The course of development of different cysticercoid species shows variation, and so does the degree of histological differentiation of their cyst wall. Not much work has been done on their histogenesis which would help in clarifying the complicated structure of the superficial layers of a fully differentiated cysticercoid. I . Cyst wall of Choanotaenia and Raillietina cysticercoids The structure of the wall of tailless cysticercoids of these cestodes is simple. According to Voge (1 961b), the cyst wall of Ch. infundibulum is differentiated into basic layers only. The hyaline coat on the surface ;the external membrane underlying the coat and consisting of two layers revealed by histological staining methods-the outer layer formed by a polysaccharide, and the internal layer formed by a protein. This is followed by a fibrous layer, mainly of longitudinal fibres with circular fibres on the surface. The internal lining layer is composed of undifferentiated parenchymal cells. The structure of the cysticercoid of R. cesticillus, also described by Voge (1960c), is slightly more Complicated than that of the foregoing species. The hyaline coat is underlaid by an external membrane of a polysaccharide nature ; beneath it lies a more noticeably differentiated layer of circular fibres organized in bundles. Better developed is the longitudinal fibrous layer with a central portion of cellular character representing the so-called intermediate cell layer. It separates the outer fibrous layer from the inner fibrous layer, which contains many cell nuclei. Fibrous processes extend among the cells

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of the intermediate layer, and connect both fibrous layers. The wall facing the cavity of the cysticercoid is covered by a thin lining layer. Baron (1971) confirmed the existence of the hyaline coat and the underlying external membrane in his EM study on the R . cesticillus cysticercoid. The external membrane, however, was not a solid membrane, but was symplasmic in nature; its base contained large numbers of electron-dense globules and he therefore called it the “globular layer”. This layer may be homologous with the distal cytoplasm of the body covering of larval and adult cestodes. Its surface had microtrix-like projections which passed into the hyaline coat as undulate tubules. The cytoplasm of the globular layer was connected with the intermediate symplasmic zone situated in a layer of inner longitudinal fibres, by large cytoplasmic projections (intrusions), which passed through the thick layer of the outer circular fibrils. The thin layer below the globular layer consisted of outer longitudinal fibrils. The intermediate layer also contains structures resembling a duct system. The internal lining of the cyst wall was formed by a layer of very electron-dense membranes. Muscle fibres were not found in the wall of the cysticercoid; only fibrils of connective nature measuring 100-150 A in diameter and sometimes 0.2-2 pm in length, were present in it. The fine structure of the cysticercoid surface with the hyaline coat is similar to that of the cyst wall of the echinococcus (p. 455). The cellular syncytium of the intermediate layer produces secretory globules into the distal cytoplasm which form the hyaline coat. The function of this type of cyst wall is mainly protective. The resorptive component of its function is indicated by the modified microtriches extending into the hyaline coat. The general composition of the cyst wall of the cysticercoid does not differ from that of the bladder wall of the cysticercus except for the presence of a large number of fibrils arranged functionally, and suppressing the cellular component, and for the absence of muscle fibres. 2. Cyst wall of several hymenolepidid cysticercoids

Cysticercoids have a number of features in common. These are: the absence of a hyaline coat and the two-layered external membrane demonstrable with standard histological staining methods. The dominant element of the wall below the external membrane of H.nana (Voge and Heyneman, 1960), and H . microstoma (Voge, 1963a) is the fibrous component divided into an outer layer of circular fibres, and an inner layer of longitudinal fibres. In H, citelli, there is an intermediate layer situated between the outer and inner fibrous layer (Voge, 1961a). The same author observed in the H . diminufacysticercoid that the composition of its wall was very complicated (Voge, 1960d). The elements of the intermediate cell layer were conspicuously large and a special regular line of cells of the peripheral layer was differentiated at the inner side. The fibres of these cells extended radially to below the external membrane. The outer fibrous layer was not mentioned in the author’s original description. Bogitsch (1967) found that acid phosphatase activity at pH 5.0 was typical of the intermediate cell layer.

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Neradovl-Valkounovi (1971) gave a detailed description of the wall layers of a Fimhriaria fasciolaris cysticercoid. She distinguished from the surface downwards the hyaline, homogeneous, basal, outer fibrous, intermediate parenchymatous and inner fibrous layer. The general division of the wall was in accord with that given by Voge, but it would be extremely difficult to homologize the superficial layers in particular. The histogenesis of the cyst wall of the H . diminuta cysticercoid was studied by Voge (1960d) who observed in a 5-day-old, not yet invaginated cysticercoid that the cavity of the central body portion was filIed with loose tissue. The central slit in the scolex neck was separated from this “cavity” by a plug of cells. These cells adjoined the cyst wall after the invagination of the neck and formed the principal inner fibrous layer. This caused the origin of a slit called cysticercoid cavity being formed from the cavity of the invaginating scolex neck region. The outer layers of the scolex differentiated from the socalled peripheral cells of which several formed elongating hairy processes under the external membrane, while others were situated deeper below the cells with the hairs, and attenuated towards the surface. The author observed also the differentiation of the canals with the flame cell in the fibrous layer. A similar developmental stage to that studied histologically by Voge in H. diminuta, was studied by Ubelaker et al. (1970a) with the EM; the cyst wall was covered with the distal cytoplasm which formed a marked superficial layer of branched microvilli. These showed distended regions, often at the tip, of completely different ultrastructure than that described from the body coverings of larval (p. 440) and adult (p. 437) cestodes. Near the surface of the distal cytoplasm there were pinocytotic vesicles followed by a terminal web of filaments and mitochondria at the base. The basal lamina often projected into the distal cytoplasm. Cytoplasmic bridges extended from the subtegumental syncytial layer to the distal cytoplasm through the basement layer. These bridges appear to be homologous with the hairy processes observed by Voge. The second type of peripheral cells disclosing their excretory nature also by EM may be important only in the differentiation of the cysticercoid cyst wall. The structure and differentiation of the well developed hairy processes described as the final stage by Voge in a 5%-month-oldH. diminuta cysticercoid requires further study. In the older cysticercoid these processes form a layer with marked radial striation called the peripheral or basal layer. The differentiation of this layer seems to be due to the advanced age of the cysticercoid; this was inferred in the older literature by referring no findings of peculiar cysticercoids with a very thick cyst wall of irregular shape and a peripheral rim of very well developed fibrils with radial orientation (Mrlzek, 1896). ProkopiE and Groschaft (1961) identified this cysticercoid as the larva of Vampirolepis hamanni. A tegument with special microvilli was observed in larvae at the initial stage of post-oncospheral development. Collin (1970) first observed host cells attached to the microvillar zone in a 3-day-old of H . citelli. Ubelaker et al. (1970b) compared this microvillar surface of the cysticercoid to similar microvilli of truly secretory function in Tylocephalus metacestodes (Rifkin et al.,

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I970), and speculated on the defence mechanism and possibly resorptive capabilities of the tegument of the cysticercoid cyst wall. In order to discover the functional possibilities of the microvillar surface, Heyneman and Voge (1971) studied host response of the flour beetle to infection with H. dimiizuta, H . microstoma and H . citelli. They concluded that absorption may be the primary function of this surface which, however, may also be capable of damaging the cells of the host during the initial stages of cysticercoid development. The latter possibility was demonstrated particularly in the early response of the host to infection with H . citelli but did not lead to the encapsulation of the cysticercoid. The first biochemical report on the nutrient absorption of H . diminuta cysticercoids was presented by Arme and Coates (1971). Allison et al. (1972) studied the inner wall of an 8-day-old H . diminuta cysticercoid with the EM and found fibroblastic cells and extracellular fibrils in the fibrous layer of the inner capsule wall. The primitive lacunae were lined with bipolar squamous epithelial cells. 3 . Functional importance of the cysticercoid tail

In many cysticercoids the tail loses the original character of a cercomer separating the embryonic hooks from the body of the developing larva. The tail grows to a considerable length in a number of species. In other cysticercoids, particularly in those developing in Lumbricidae, the tail branches and appears to be overgrowing the cyst (HrabE, 1957). A similar process appears to occur in the origin of the second covering of the cysticercoid separated from the cyst wall by a slit. Generally, these forms are referred to as diplocyst or monocercus. The histological structure of the tail tissue differs from that of the cyst wall, and its degenerative appearance is a frequent feature. Variation is shown in the size and shape of the individual species. Voge (1963a) confirmed considerable variation in the tail of H . microstoma cysticercoids and found numerous fibres proliferating from the longitudinal fibrous layer into the tail. In another paper (Voge, 1963b) changes in the morphology of the tail in temperature-stressed cysticercoids are described. This indicates that the body temperature of the intermediate host may be responsible for changes observed by Weinmann (1969) during larval development of H . nana in different vertebrate classes. Cysticercoids with a thick-walled tail were found in coldblooded hosts, those without a tail in warm-blooded hosts. The morphogenesis of these differing forms described earlier for H . nana in connection with the second intermediate host is unknown. V. CYSTICERCUS BLADDER Differentiation of the remaining parts of the bladder into a definitive cysticercus bladder occurs after the formation of the primary scolex rudiment from the primordial bladder. This process involves growth and enlargement of the bladder, and changes in the structure of the subtegumental layer. An irregularly arranged row of freely distributed subtegumental cells, typical of

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the bladder wall, is formed from the originally densely packed cells of the embryonic type with their long axis in vertical position to the tegumental surface. The cells of the parenchymal layer differentiate into duct (capillary) cells, flame cells, muscle cells, neuro-muscular cells which form contacts and anastomoses. Earlier descriptions of the complicated histological differentiation of the bladder wall were given by Young (1908) for T. pisiformis larvae, and by Rossler (1902) for T. taeniaeformis and T. hydatigena larvae. The measurements of the cells and of other structures of the cysticercus could not be assessed with the LM. Recent evidence on the structure and function of the bladder wall has been obtained with the EM. Slais et a f . (1971) gave a comprehensive account of the submicroscopic structure of the bladder wall of C. bovis drawing attention to the simple histological character of the tissues and the great importance of structures of specific differentiation. 1. Tegument of the bladder

Much work has been done on the surface of the bladder. Race e f al. (1965), Nieland and Weinbach (1968) and Baron (1968) demonstrated in the various cysticerci that microtriches of the bladder tegument differ greatly from those on the surface of the adult form. Similar observations were made by Slais

FIG.22. Tegument of the bladder of C.bovis. Distal cytoplasm (dc) with receding microtriches (mi) seen in the figure to about half of their actual length. Note typical attenuation under the basal portion (bm). Cytoplasm showing disc-shaped vacuolar structures (v) and mitochondria (m).The basement layer (bl) appears as a light strip under the cytoplasm. The two arrows point to its evagination and to the limiting membrane proper of the distal cytoplasm. lm-subtegumental muscle bundles; ce-extensions of the subtegumental cells; m-mitochondria; c-cytoplasm (17 O00 x ). Inset: detailed view of the basal portion of the microtrix (34000~ ). After Slais et al. (1971).

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et a/. (1971) in C. bovis larvae (Fig. 22). The microtriches are slender, elongate

and their division into basal and distal portions is less marked than in the adult, lacking the typical electron-dense tip. The distal portion is needleshaped and decidedly longer in proportion than the basal zone. The microtriches are surrounded by a double membrane covering the external surface of the tegument. A bilaminated membrane lies at the junction of the basal and distal portion. Bortolletti and Ferretti (I 971) found similar microtriches in 14-day-old H . taeniaeforrnis larvae, while in 30-day-old larvae 2-4 microtriches were grouped together in the basal portion and a plasma membrane enveloped the whole group. These authors suggested that multiplication of the microtriches during larval growth was due to longitudinal division rather than to gemmation. There was remarkable similarity, in electron-density and shape of the developing tips of the primitive hooklets of the rostellum originating by the transformation of the microtriches and the production of a proteinlike keratin, to the electron-dense tips of microtriches of the adult form. It is conceivable that a protein similar to that of the developing hooklets may be present also in the microtrix tips of the adult form, and that these serve -contrary to larvae-as protection against the digestive enzymes of the host. 2. Wall of the bladder Microscopical and anatomical relationships between the individual tissue components of the cysticercus bladder are extremely complicated (Slais, 1970). A large amount of ground substance with characteristic microfibrils separates the individual cells with the nuclei, and connection between the cells of the parenchyma, the excretory system, the muscles and the tegument is established by means of long plasmic extensions. There is no such intimate cell relationship as that described by Threadgold and Read (1970) for the adult H . diminutu. Typical of the parenchyma of the bladder wall of the cysticercus are electron-dense structures containing abundant glycogen and forming a complicated system with fine branching distributed throughout the subtegumental and parenchymal wall layers and bounded by a membrane. The branches form a continuous system in the ground substance, the sparse cellular nuclei of which were disclosed by Schramlovh and Slais (unpublished). The cells, evidently, are of a special type and not specifically differentiated extensions from the subtegumental cells containing glycogen, as suggested by Btguin (1966). The system appears to originate from cells similar to the so-called glycogen-storing cells observed by Sakamoto and Sugimura (1 970) in the EM study on the histogenesis of the alveococcus. Sometimes, these structures contain either individual or ductile-like tubular structures measuring from 0-1-0.4 pm dia. The pattern of these intraplasmic tubules is similar to that of ductules of all types in the nephridial system. The long extensions of the ductules are in intimate contact with the glycogen-containing structures (Nieland and Weinbach, 1968; Slais et al., 1971). The nephridial system is developed from cells termed reticular interstitial cells by the two Japanese authors. It may well be that all types of cells in the parenchyma of the cysticercus bladder form a syncytium detectable only with the electron microscope.

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The fact that glycogen-containing structures are most numerous close to the inner surface of the bladder wall is important when assessing the function of the bladder. In the histological demonstration of glycogen globules of different size in T. lzydatigena larvae (Thakur et a/., 197 I), the storage form of glycogen isolated from the bladder wall represented a mixture of two separate moieties of different molecular weight. The polymorphism of glycogen deposits was confirmed in EM studies; single granules (alpha particles) and rosettes (beta particles) were distinguished. The nephridial system of the invaginated cysticercus differs from that of the adult form, and also from that of the strobilocercus, the latter being the system of the adult form. Bartels (1902) and Rees (1951) observed in grown larvae of T. taeniaeformis dorsal (medial) and ventral (lateral) excretory vessels (ducts) at each side of the strobila extending from a complicated

FIG.23. ( a t v e r y oblique section through the duct with a distinct, dark, wall and extensions in the bladder wall of C. hovis. Note the circular internal ribs of the wall (x) and the close communication of the extensions of the wall with the branches of the pale elements (arrows). (6 500 x ). (b)-The duct wall (a) in higher magnification. Rod-shaped bodies in the granular cytoplasm with a paler superficial layer (1 3 000 x ). (c)-Nodular bleb-like formations on the surface of the duct lumen with separating electron-lucid spherical blisters (arrows). The rod-shaped bodies in the granular cytoplasm show the lighter superficial layer (30 000 x ). After Slais et al. (I 97 I ).

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network forming the cephalic ring. At the posterior end of each segment, a transverse vessel joined the ventral vessel of one side with that of the other. Having entered the terminal portion of the caudal bladder, the larger ventral vessel formed a branching network close to the bladder surface, the medial vessel a network close to the bladder cavity. Both networks were confluent at the posterior end of the bladder. The dorsal and ventral vessel network communicated, according to Bartels, with the exterior by a number of small irregularly placed excretory pores only. Howells (1969) observed in the adult form of M . espunsa that, posterior to the neck region, the dorsal longitudinal duct is smaller in diameter and has thicker walls than the ventral duct. The dorsal and ventral ducts of each side were confluent in the scolex, but all four ducts opened separately to the exterior at the posterior end of the worm. Logachev (1958) observed differences in the histological structure of both canals in Thysanieziu ovillu and concluded that the structure of the median canal suggests an active periodical contractility of its wall. A similar structure was demonstrated in the collecting ducts of the bladder wall of C. bovis by Slais (1970) with histological methods, and by Slais et al. (1971) with the EM (Fig. 23). Two similar systems of branching canals to those of the strobilocercus bladder are components of the bladder of all cysticercus types. In C. celldosue and C. bovis, the two systems join at the site of transition of the bladder wall into the invaginated canal, and form two ducts which anastomose in the rostellum region (Slais, 1970). A similar situation occurs in the scolex of the adult M . expansa except that all terminal ducts appear to connect by a trans-

FIG.24. Fine branching of the contractile elements (br) close to the flame cell at the inner surface of the bladder wall of C. hovis. The flame cell encountered in the oblique longitudinal section: pf-projection of flame cell; ch-chromatin patches; n-nucleus; r-rootlets of cilia; pc-pack of cilia; Ic-limiting cytoplasm of terminal ductule; fd-fine ductules. 7 300). After Slais et ul. (1972).

FUNCTIONAL MORPHOLOGY OF CESTODE LARVAE

453

verse duct. The fine structure of the flame cell and of the terminal ductules of C. bovis is similar to that of the adult M. expansa (Slais et al., 1971) (Fig. 24), but in these forms the terminal ductiles join in the primary collecting ducts and form a network close to the bladder cavity which is similar to the median network of the strobilocercus. In the bladder wall of C. bovis, the flame cells lie remarkably close to the inner side of the wall lined by a layer of loosely arranged parenchymal cells; in the invaginated portion they lie close under the layer of tegumental cells. This indicates their functional relationship to the liquid in the bladder cavity.

FIG.25. A-Transverse (x) and longitudinal (y) section through the primary contractile lamellae in the ground substance (rf) of the bladder wall of C. bovis (6 000 x ). B-Transverse section through the subtegumental muscle fibre. Dark, thick myofilaments (arrows) surrounded by thin myofilaments. x4ytoplasmic connections with the muscle fibres. (25 000 x ). After Slais et at. ( I 972).

The muscle system of the scolex portion becomes reduced upon entrance into the bladder wall. The subtegumental muscle bundles in the bladder wall are too loosely arranged to form a compact muscle system; their course is circular and longitudinal. Fine muscle fibres extend also irregularly in a transversal direction in the parenchymal layer of the wall. Flat branches separate from them at many points so that each fibre now has a large number of extensions as this was first described by Rossler (1902). EM study disclosed that microfibrils of the ground substance extended parallel to the course of these fibres and formed their sheath-like covering (Slais et al., 1972) (Fig. 25). The ultrastructure of all myofibres in the bladder wall, however, is similar to that of the musculature of the adult cestode as described by Lumsden and Byram (1967). The considerable reduction of the muscle system in the

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cysticercus bladder indicates that locomotion is of secondary importance in this larval organ. In situ, muscle contraction may stimulate circulation of the fluid in the canal system and, later, may facilitate the process of evagination. 3. Functional morphology a j the cysticercus bladder The fine structure of the duct cells in the bladder wall suggests their considerable metabolic activity. Nodular bleb-like formations on the surface of the duct lumen are differentiated from “excretory blebs” described by Race et al. (1966) from the lumen and along the wall of the excretory canal for gravid T. multiceps proglottids. The nephridial system may function in the movement of material, for example glucose, from one part of the worm to another (Howells, 1969). Nieland and Weinbach (1968) found that bladder fluid of T. taeniaejormis larvae contained more glucose than the plasma of the respective host animal. The tegument with the microtriches of the bladder appears to be adapted for absorption of nutritive substances from the host, e.g. in C. cellulosae and C . bovis at first from the dilated lymphatic space, later from the transudate and detritus of exudative cells in the inflammatory encapsulation. This has been observed also in other cysticercus species. The site of initial tissue reaction to the presence of C. hovis is of interest. It occurs in the tissue opposing the opening of the invaginated canal to the exterior, i.e. on the bladder surface. No irritation was observed on the opposite site where the bladder wall abutted the endothelial cells. zdhrski (1973) showed in this cysticercus a high activity of alkaline and acid phosphatase in the subtegumental cells at the site of entrance of the invaginated canal into the bladder wall (entrance zone), and in a wide zone of the bladder wall surrounding this opening. No activity occurred in the remaining parts of the wall. Optimal physiological conditions for respiration and glucose absorption for C. crussiceps are present in the peritoneal fluid from which glucose is transported by a mediated process (Murrell, 1968). Also absorption of amino acids by larval T. crassiceps occurs by a mechanism of active transport (Haynes, 1970). As regards the larval structure, differences were observed between young T. crassiceps cysticerci with a dominant bladder and a more solid parenchymatous portion, and more advanced larval forms; this refers particularly to the absorption of valine and methionine (Haynes and Taylor, 1968). This shows that there exist considerable differences in the metabolism of a larva with a progressively differentiating scolex. The nephridial system may play a role in the transport of nutritive substances to the developing scolex and, therefore, the functions assigned to it may not be only excretory, osmoregulatory and hydrostatic in nature. The bladder of a cysticercus ageing in situ enlarges continuously, apparently under the influence of intercalary growth along the periphery. Communication of the invaginated canal with the surface is maintained, in that its transitory portion elongates by a structural transformation of the adjoining bladder wall. The histological structure of this entrance canal differs from that of the bladder wall (Slais, 1966a) primarily in the altered degenerative parenchyma. EM studies should help to obtain more knowledge of the morphology and functions of its histological elements.

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4. Morphological differentiation of the echinococcus cyst I n the E. granulosus larva the original wall of the larval organ differentiates

into a germinal (proligerous) membrane which functions as a larval organ (cyst) and represents also the germinative centre from which the numerous protoscoleces of the parasite originate. In the principal wall of the cyst, the germinal membrane produces a special superficial laminated layer; towards the inside of the cyst it forms specialized formations-brood capsules-in which the scoleces originate. The superficial protective layer of the echinococcus cyst, called the cuticular of hyaloid membrane, has a most characteristic lamination which is particularly marked in old cysts showing signs of regression. The importance of this lamination as regards its use as a diagnostic sign is similar to that of the brownish-black pigment which is deposited in the degenerating germinal membrane and is a lipid-containing autolytic pigment (Freese, 1963). The hyaloid membrane which is often considered to be chitinous, is formed by a mucopolysaccharide with a characteristic infrared spectrum; it contains only galactose and glucosamine as sugar units (Kilejian ef al., 1962). According to Campesi (1961) maturation of the echinococcus cyst is characterized by a prevalence of neutral mucopolysaccharides which exclusively constitute the outer layers. Sakamoto (1961) found with histochemical methods that the hyaloid membrane contains a neutral polysaccharide, an acid polysaccharide (hyaluronic acid), a protein, and alkaline and acid phosphatase. Slais (1964) observed that lamination of the membrane was due to an interchange of layers in which either neutral or acid mucopolysaccharides dominated. The proteinaceous substance was overlaid by polysaccharides and was demonstrable only in a laminated membrane damaged by autolysis. Apart from the protective function, little is known about other functions of this membrane, Cysts of E. granulosus show hydrostatic overpressure as a result of permeability control and osmoregulation of the germinal membrane. Pressure is relatively higher in small cysts. De Rycke (1966b) concluded that overpressure in E. granulosus was the result of increasing water imbibition and of the strength of the slowly growing wall. The ultrastructure of the germinal membrane (Morseth, 1967a) was similar to that of the cysticercus bladder wall. A similar structure was observed also in the wall of the brood capsule. This, however, is very thin as compared with the thickness of the cyst wall. The distal cytoplasm of the germinal membrane contains numerous vacuoles and finger-like extensions penetrating the laminated layer; these seem to be differently shaped microtriches. The laminated membrane is made up of layers of particulate material with a background of fine, irregular fibrils and it is at these sites that separation of the layers is seen. Lamination of the hyaloid membrane of the echinococcus was shown by Freese (1963) in the electron microscope. Studies on the ultrastructure of the alveococcus by Sakamoto and Sugimura (1969) did not reveal substantial differences from the echinococcus, but the authors failed to notice the lamination of the extracellular, fine fibrous material in the cuticular layer of the cyst. This may be explained by the fact that the cuticular layer of the alveococcus in the definitive host is never as distinct as that of the echinococcus.

456

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c. EXCYSTMENT AND

LAI IS

EVAGINATION OF THE

cxmc

LARVA

Attachment of the larval stage to the intestine is preceded by the liberation of the larva from the pseudocyst and inflammatory encapsulation enclosing the larva when it enters its definitive host. This process will not be discussed i n this section. Conditions responsible for the excystment of the cysticercoid and evagination of the cysticercus in vitro have been investigated. Smyth (l963,1969a, b) and Voge (1967a), reviewing recent studies on this subject, give the most detailed accounts available in the literature. Unfortunately, no correlation is available as yet of these physical and chemical conditions with the anatomical structure and morphological readiness of the larval stage for the process of establishment in the definitive host. I . Excystment of the cysticercoid The scolex of the cestode situated in the cysticercoid in its normal position inside the cyst is ready to leave. The resistant structure of the cyst wall indicates that excystment of the cysticercoid and the evagination of the cysticercus are influenced by different factors. This was pointed out by Campbell (1963) who observed that surface-active agents had no effect on the liberation of the cysticercoid scolex. This accords with the histological structure of this type of larva. Release of the scolex from the cyst is stimulated by the reducing volume of the cysticercoid cavity forcing the scolex to excyst on the evaginated neck through the anterior canal. Goodchild and Harrison (1961) presented a clear picture of H . diminuta excystment. Reduction of the central cavity may occur primarily by contraction of the fibres which are the main component of the histological structure of the cyst wall. A detailed account on their functional arrangement was given by Voge and Heyneman (1960) for H. nana. The relation of fats in the wall of the cysticercoid cavity after excystmpnt of the H . diminutu cysticercoid has only briefly been mentioned by Voge (1960a). Studies on the mechanism responsible for the excystment of the cysticercoid have been started recently by various authors. Bogitsch (I 967) found that the effect of bile salts played a major role in the release of acid hydrolases and the disruption of the cyst tissues surrounding the larva. He observed in H. diminuta that bile salts disturbed the integrity of the intermediate cell layer in which vacuolation of the cells occurs and, hence, the process of excystment is stimulated. 2. Evagination of the cysticercus The process of evagination and the morphological steps were described by Slais (1966b) for cysticerci with invaginated scoleces deeply withdrawn into the bladder cavity. In this larval type, the bladder is of major importance because it increases pressure inside the bladder cavity by contraction of its wall, and forces the invaginated canal with the scolex to evaginate. In cysticerci with a bladder attached to the caudal body end, its function in the process of evagination is less important than in the foregoing type, although the fluid of the diminished bladder cavity is affected by periodical waves of contraction

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of the bladder wall and exerts a one-way pressure on the parenchymal portion with the invaginated scolex. It has been observed in all cysticerci that in the invaginated canal under the undulated tegument there is a layer of subtegumental muscles and main ducts which extend beyond the opening of the canal into the outer wall of the parenchymal portion of the cysticercus. The principal functional tissue is represented by a layer of parallel fibrous tissue forming the so-called true receptaculum typical of the cysticercus. The fibrous tissue constitutes the main central portion of the scolex and neck after evagination (Fig. 26). The invaginated spiral canal with the scolex is pressed into this fibrous tissue and

FIG.26. A-Longitudinal section through the parenchymatous portion of C. hovis with a meandering, invaginated spiral canal on the bladder wall. Its opening is not seen in the section. Note the layer of well-arranged connective tissue forming the lining of this portion opposite the bladder cavity. After Slais (1966b). B-longitudinal section through the evaginated scolex and the connecting portion. Note the well-arranged connective tissue in the centre of the formation. After Slais (1966b). Weigert-van Gieson (20 x ).

this forms the bed in which the invaginated portion lies. The complete mass of fibrous tissue is extended in the invaginated cysticercus and is under constant tension as the result of fibril contractibility. If, in microdissection experiments with C. pisijormis (Slais, unpublished) the anterior parenchymal portion surrounding the opening of the invaginated canal is cut off, the severed portion forms a tube with turned-out margins. Evagination occurs normally except that a ringlike defect remains on the surface of the

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tegumental and subtegumental area in about half the length of the cysticercus body. The missing portion coincides with the severed and now independent tube. Evagination of the cysticercus is thus independent of the undisturbed continuity of the subtegumental muscle layer. If the tissue separating the parenchymal portion from the bladder cavity is damaged, false evagination of the reversed spiral canal with the scolex occurs caudally. The sequence of layers of the invaginated canal remains unchanged, but the disturbance of the enclosing tissue releases internal tension in the fibrous layer, which straightens into the conical formation protruding directly into the empty space (Fig. 27). This explains why surface active agents responsible for the slight decrease of surface tension are of such importance in the evagination of the cysticercus. These experimental results explain also the remarkably positive effect obtained by Campbell (1963) and by de Rycke and van Grembergen (1966) with C. pisiformis only. As regards the so-called partial evagination of T. hydatigena larvae described and figured by Featherston (1971, Fig. 6) it seems that the scoleces under consideration grew only partly into the invaginated canal (Slais, 1966b). Evagination of the echinococcus protoscolex is simple because it is small; a functional state of internal tension is evoked in these protoscoleces by the relationship of their muscle system. Coutelen et al. (1952) observed in studies on these scoleces, apart from other functional muscles, groups of deep muscles which invaginate the rostellum and suckers and remain contracted until evagination, while the subtegumental muscles of the minute post-sucker neck region are dilated and function in the withdrawal of the scolex organs to this portion of the body. Release of the invaginating muscles and contraction of the subgetumental muscle fibres of the neck region lead to the evagination. The importance of different osmotic pressures and their changes in a medium containing E. grunulosus protoscoleces in the process of evagination was pointed out by de Rycke (1968) and de Rycke and van Grembergen (1968).

WIT. PROLIFERATION DURING THE LARVAL STAGE A common feature of larval cestode development is the proliferation of the larval tissue during the post-oncospheral stage. Sometimes, this is in harmony with the life cycle of the cestode and leads to asexual multiplication of the larval stage; sometimes, it is abnormal and responsible for extensive autonomous growth resembling tumorous growth of vertebrate tissue in several of its aspects. A.

ASEXUAL MULTIPLICATION

It is difficult to distinguish typical asexual multiplication from abnormal growth because histogenetical processes are often very similar in both types and there are also transitory forms of proliferation. Larval proliferation in cestodes involves a form of growth similar to regenerative processes in turbellarians.

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1 . Multiple scolex formation in larvae o j the coenurws and ecliinococcus type

Asexual multiplication of these types of larvae is a normal development feature comprising the differentiation of either a series or masses of scolex rudiments from the bladder wall. Sometimes, many scoleces originate primarily and their numbers do not increase during larval development. This type of development is characteristic of larvae with multiple scolex formation, which develop in small rodents (p. 430). 'The larva demonstrated by Voge (1967a, Fig. 2) from the body cavity of a Peruvian rodent seems to belong to this group of larvae with multiple scolex formation and not to the group of larvae forming new scoleces by secondary budding. By contrast, the large coenurus and echinococcus larvae show continuous formation of scolex rudiments associated with the growth of the bladder and ageing. It is unknown to what extent the cystic metamorphosis of the scoleces can be considered to be part of the normal developmental process. In large larvae of T. multiceps and T. serialis (Hamilton, 1950) and T. geigeri (Bondareva, 1963), however, cystic metamorphosis is extensive and degenerative in nature.

(a)

--1

FIG.27. Schematic illustration of two operations on C. pisiformis. (a)-Severance of the top of the parenchymal portion with the opening of the invaginated canal; (b)--severance of the bladder and disturbance of the base of the parenchymal portion. Dots= the tegument of the invaginated canal ; hatching= the fibrous tissue. Slais (unpublished).

460

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I t was shown by Coutelen (1929) that protoscoleces of T. seriulis grown in virro became vesicular. The factors influencing vesiculation were discussed

by Smyth (1962), Smyth et al. (1966), Pauluzzi et al. (1965), Benex (1968a) for protoscoleces of E. granulosus grown in vitro, and by Yamashita et al. (1962) for protoscoleces of A . multilocularis. Two alternatives are involved: the first concerns development, differentiation and growth of the so-called caudal bladder at the site of the stalk of the protoscolex; the second concerns direct cystic metamorphosis of the protoscolex itself and this is accompanied by the gradual regression and lysis of the tissues of the rostellum and the suckers. In experiments in vitro, bladders with a differentiated, iaminated, hyaloid membrane were obtained with both alternatives. Webster and Cameron (1963) pointed out that the local internal thickening of the germinal membrane of bladders grown in vitro may indicate the origin of a new brood capsule or scolex. Lubinsky (1 960) and other authors used intraperitoneal implantation for vegetatively propagated strains of larval A . multilocularis in small rodents. A similar method used in experiments with E. granulosus showed that the cystic metamorphosis of the protoscoleces in the intraperitoneal cavity leads to secondary echinococcosis. Pennoit-de Cooman and de Rycke ( I 970) and, particularly, Heath (1970) confirmed the origin of fertile cysts in experimentally induced echinococcosis. According to Sweatman et al. (l963), transformed scoleces seemed to be the only source of secondary cysts which in their experiments remained sterile. Benex (1968a) suggested on the basis of his experiments in vitro that the so-called secondary bladder originated from fragments of the germinal membrane near the stalk of the protoscoleces. Later (Benex, 1968b), he observed in E. granulosus, in vivo, that the histogenesis of the secondary exogenous cyst originated from the germinative cells of the germinal membrane, which he confirmed in experiments in virro. Cells from fragments of the germinal membrane without protoscoleces produced secondary cysts similar to those grown in vivo. Experiences from in vitro experiments suggested that the so-called caudal bladder is capable of developing into a fertile cyst. According to this author, cystic transformation of the protoscolex is degenerative in nature and may lead to the origin of sterile secondary cysts only. 2. Asexual multiplication by proliferation of’the bladder wall The type of asexual multiplication which occurs by exogenous budding of the new bladders from the wall of the primary bladder is typical of the T. crassiceps larva. Over 70 years ago, the histogenesis of this mode of proliferation of the bladder wall, the morphogenesis of the scolex in the bud, growth and separation of the bud was studied by Bott (1898). This subject was taken up by Freeman (1962) who studied proliferation of the bladder wall of T. crassiceps also on repeated intraperitoneal infection of the larvae. Bondareva (1968) found massive multiplication of T. crassiceps larvae in the beaver. This mode of reproduction seems to be normal in this cestode species, but the rate of reproduction shows variation in the different intermediate hosts. Attention should be given to the outer morphological similarity of the

F U N C T I O N A L M O R P H O L O G Y O F CESTODE L A R V A E

46 I

FIG.28. (a)-Various initial developmental stages and budding of daughter bladders of T.crussiceps larvae from pseudocyst located under the skin of a beaver. (b)-Cystic degeneration of liberated inner scoleces of a large T. geigeri coenurus. (Scale: mm). Histological sections show that in T. cuassiceps larvae a true cysticerciis bladder (C) is formed, while the scoleces of the coenurus (D) form a degenerative widening of the invaginated canal leading to the scolex. Weigert-van Gieson ( 2 4 0 ~ ) Material . by courtesy of Dr. V. I. Bondareva. 17

462

JAROSLAV SLAlS

individual developing C. crassiceps, and to scoleces of the large coenuri detached from the wall with signs of massive cystic degeneration (Slais, unpublished) (Fig. 28). Budding, or more likely the endogenous origin of cystic formations on the bladder wall in which the scoleces do not differentiate, has also been observed in C. crassiceps. Voge (1962) drew attention to a proliferation zone in the tail-like extension of connective tissue in the receptaculum of the scolex of a T. hydutigenu larva; in C. tenuicoffis,this extends into the bladder cavity. Crusz (1948a) observed endogenous budding in C. tenuicollis occurring simultaneously with the origin of daughter cysticerci which were armed with a scolex. He described also the histogenesis of a transverse fission in C.pisiformis leading to the origin of an almost separate bladder (Crusz, 1948b). Heath and Smyth (1970) found in oncospheres grown in vitro that abnormal development resulted frequently in the origin of a bifid larva with two scolex rudiments. This referred to T. pisiformis. The authors also found triple bladders with three scolex rudiments. All these forms developed from oncospheres with 6 hooks (hexacanths). Lubinsky and Galauther (1966) recovered repeatedly from the cotton rat abnormal scoleces of A. multilocularis with 6 suckers and 32 hooks. These scoleces occurred together with normal scoleces in contradiction to earlier assumptions that abnormal forms originate from oncospheres with 12 hooks, i.e. from two merged oncospheres. Webster and Cameron (1969) described similar protoscoleces for E. grunulosirs and demonstrated that they originated from eggs with double oncospheres. Prolonged artificial asexual multiplication of larval cestodes may lead to genetic mutations. According to Smith et al. (1972a,b) the morphological, antigenic and reproductive abnormalities described for the ORF strain of larval T. crassiceps are the result of aneuploidy. The histogenesis of asexual multiplication of cysticercoids similar to the type of budding described by Kisielewska (1960) for Pseudodiorchis prolifer has not been described in detail. Apart from this urocyst (Urocystis prolifer) similar larval forms have been described under the name staphylocyst and cercocyst. A similar morphogenesis may occur in abnormal cysticercus larvae such as that described for T. taeniaeformis by Bernard (1959). 3. Asexual multiplication of the parenchymatous larva Multiplication by branching and budding was observed in plerocercoids of the Sparganum prolifer type. Similarly proliferating forms were found by Mueller (1965) in S. mansoides grown in vitro. Specht and Voge (1965) reported this type of asexual multiplication for the tetrathyridium ; this was obtained from the body cavity of white mice infected orally or intraperitoneally with larvae from the liver or body cavity of a lizard. The larvae multiplied by binary or multiple fission and their mature forms were identified as Mesocestoides corti. James and Ulmer (1967) failed to produce this type of asexual multiplication in tetrathyridia in similar experiments. A normal tetrathyridium, however, capable of both asexual proliferation and strobilation, can originate by direct regeneration of fragments containing either one

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or several suckers (Hart, 1968). Regeneration is similar to that of Turbellaria suggesting the presence of undifferentiated germinative cells in the larva as mentioned earlier by Wikgren (p. 410). The presence of such proliferation zones has been confirmed in asexual multiplication of M . corti as late as the strobila stage (Eckert et al., 1969).

B.

ABNORMAL GROWTH

Abnormal growth of the bladders has generally been observed in the coenurus. Angulo and Roque (1949) attempted at identifying the various types of larval coenuri by the appearance of their bladders and the mode of branching. Both authors based their conclusions on their findings (1948) of differently branched cysts recovered from inflamed encapsulating tissue under the skin and in the muscles of Capromys pilorides. Several of the cysts contained scoleces. Voge and Berntzen (1963) found similar cysts in the abdominal and thoracic cavity, and also in the liver and lung tissues of a dog; these cysts did not contain scoleces. The histological structure of these cysts was similar to cysts of T. serialis. Smyth (1969a) observed the development of abnormally big bladders in scoleces of T. serialis grown in vifro;thesedeveloped in the so-called stalk region even if the scolex was separated from it by enzymes. Generally, proliferation did not continue in these bladders; most scoleces in the medium became segmented and only a few showed abnormal development, became vesicular and their wall differentiated into secondary bladders. The portion of the evaginated scolex of T. serialis (called “stalk” by Smyth) coincided with the invaginated canal leading to the invaginated scolex. These experimental results reconfirmed that the wall of this canal of the coenurus is capable of proliferation, the formation of secondary bladders and, under normal conditions, the formation of new scolex rudiments (p. 425); these growth properties of the bladder wall of the cysticercus are important in solving the origin of the “racemose” form of brain cysticercosis. In Europe, this form is considered to occur as the consequence of abnormal proliferation of the bladder of C. cellulosae with an either undeveloped or extinct scolex. This concept was confirmed by a literary review and histological study on autopsy and biopsy material (Slais, 1967, 1970). In Africa, where coenurosis is frequent in man, several authors identified the presence of cysts with no scolex in the brain as coenurosis. Goldschmid (1966) found in post mortem of 62 cases of brain cysticercosis from Rhodesia caused by C. cellulosae, two abnormal bladders; in one of these he even observed the bilocular nature of the cysts and the presence of daughter cysts. The histological origin of new cysts accompanying the proliferation and growth of the racemose cysticercus at the base of the brain is not well known. Talice and Gurri (1949, 1950) recovered minute buds from the bladder wall. Their description of a brush-like covering of the inner surface of the bladder wall is in contradication to the knowledge of its histological structure. Slais (1967) described the bioptic finding of an exceptional grape-like formation consisting of numerous buds, many of them vesicular; their wall did not

464

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LAI IS

FIG. 29. A-Typical bladders of the racemose cysticercus in histological section. Haematoxylin eosin. After Slais (1967). B-Bladder of the racemose cysticercus with a daughter bud on the wall. The formation lies in fluid on glass balls. Slais (unpublished). (8 x .)

differ from that of C. cellulosue. In a recent case of racemose cysticercosis Slais (unpublished) found bladders with secondary buds at various stages of development. Detailed studies will have to be performed in order to reach conclusions on the morphogenesis of this fatal disease, now rare in Europe (Fig. 29). Abnormal proliferation of the germinal membrane of the echinococcus (minute lobate formations-chitinom or cuticulom) has been reported in man. Benex (1968b) obtained similar formations by explanation in vitro of the germinal membrane of E. grunulosus. In all cases it was possible to identify these formations histologically by their laminated hyaloid membrane even in the absence of a scolex. Over-age larval forms of Mesocestoides, T. murtis, and particularly T. polyucuntha, are also forms of abnormal growth of larval tissue (Fig. 30). It is evident that age is one of the factors responsible for proliferation and subsequent degenerative changes in these larvae. Proliferation of the larval tissue may dominate and attain an autonomic character under exceptional conditions only. Of interest is an analogy of autonomic growth of larval tissue to exceptionally abnormal proliferation of placentary tissue in mammalian embryos (Molu I~ydddosu)(Slais, 1966d).

FIG.30. Three T. polyucunthu armatetrathyridia of different size from the thoracic cavity of rodents. Apart from age, the size of the host influences abnormal larval growth. The biggest larva was obtained from Sciurus vulgaris, the smaller from Apodemrrs sylvaticus, the smallest larva from Evotomys glureolus. (Scale: mm.) Material by courtesy of Dr. B. Homing.

466

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Ix.

SLAIS

CONCLUSION

Many advances have been made in recent years in our understanding of the physiology and biochemistry of cestode larvae and adults. Successful cultivation of the oncosphere and of other larval stages has produced new information concerning the physiology of the larva and facilitating studies on laboratory-raised strains. The same applies to cestode immunology and its application in practice. The fact that a correlation between this information and the morphology of larval cestodes is inevitable, has been demonstrated in recent comparative studies on larval cestode morphology which could not have been made without the use of the modern tools of histochemistry and electron microscopy. These studies have brought to light new facts facilitating the determination of the exact systematic position of these organisms in the zoological system, and contributed also to our understanding of host-parasite relationships. Perhaps the greatest advance, because of its many implications, is the elucidation of the course of infection in the intermediate host which, until recently, has been unknown, and the causes and forms of normal and abnormal growth of larval stages. All this new evidence will have its impact on veterinary and human medicine.

ACKNOWLEDGEMENTS I wish to thank all colleagues from the various institutions, and their directors, who so generously lent material of larval stages for comparative study. These are: The Institute of Parasitology, Czechoslovak Academy of Sciences, Prague; Vsesoyuzniy Institut Gelrnintologii im. K.T.Skrjabina, Moskva; Gelmintologichskaya Laboratoria AN SSSR, Moskva; Zoologicheskiy Institut AN kazakh. SSR., Alma-Ata; Department of Parasitology, Veterinarniy Institut, Samarkand; Veterinarbakteriologischesund parasitologisches Institut, Zurich. Thanks are given to my willing helpers (all from the Institute of Parasitology, Czechoslovak Academy of Sciences, Prague) for assisting in- histological work, checking the references (Dr. Hulinska, Dr. Scharmlova, Dr. StBrba), and giving technicalassistance (Mrs. Vavrova, Mrs. Kiidova. Mrs. Svobodova,Mrs. Zakavcova). I am grateful to Professor Ben Dawes for his valuable advice and for improving the style of the manuscript, and to Mrs. E. Kalinovk for translating the text into English.

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NOTE ADDEDIN PROOF Addendum to p . 462

The possibility of asexual proliferation by budding in cysticercoids is of significance especially with H. nana. The massive infection with this helminth in man is mostly explained as a superinfection, but some findings resembling the budding of this cysticercoid have already been reported before. Astaphiev (1970) described similar forms of H. nana larvae in lymphatic nodes of white mice obtained by experimental infection. His observation, however, was based on whole mounts only and further study, especially from the histological viewpoint, is necessary. Astaphiev, B.A. (1970). [On the budding of larvae of Hymenolepis nana in tissues.] Medskaya Parazit. 39, 299-302.

Ontogeny of Cestodes and its Bearing on their Phylogeny and Systematics* REIN0 S. FREEMAN

Department of Parasitology, School of Hygiene, Universityof Toronto, Toronto, Canada M5S 1A1 I. Introduction. ......................................................... 11. The Basic Cestode Life-cycle .......................................... A. Stages in the Cycle ................................................ B. Ecology of the Lifecycle .......................................... 111. Variations in Cestode Ontogeny ........................................ A. The Adult ........................................................ B. Types of Eggs and Oncospheres .................................... C. Development of the Metacestode.. .................................. IV. Evolution of Cestode Life-cycles ........................................ A. The Precestode Cycle ............................................... B. The Protocestode Cycle.. .......................................... C. Evolution of Modern Taxa .......................................... V. Phylogenetic Relationships ............................................ VI. Conclusion .......................................................... Acknowledgements .................................................. References ..........................................................

481 483 483 487 490 490 493 496 531 531 536 538 543

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I. INTRODUCTION Examination of more recent systems of cestode classification (Spasskii, 1951, 1963a; Mathevossian, 1963; Abuladze, 1964; Freze, 1965; Artyukh, 1966; Wardle and McLeod, 1952; Yamaguti, 1959; Joyeux and Baer, 1961; Schmidt, 1970) reveals wide disagreement on the limits, relationships, and validity of various taxa. This is apparent particularly at ordinal levels. For example, Spasskii (1963b) prefers a restricted number of orders with many suborders, whereas others consider that most of these suborders and superfamilies should stand at the ordinal level, but each lists some orders not accepted by the others (Wardle and McLeod, 1952; Yamaguti, 1959; Joyeux and Baer, 1961; Schmidt, 1970). It is questionable whether there is even general agreement on the six major orders (Tetrarhynchidea, Tetraphyllidea, Pseudophyllidea, Proteocephalidea, Caryophyllaeidea, Cyclophyllidea) indicated by Voge (1 969). These systems, based primarily on “Supported i n part by grant A-I969 from National I<esenrch Council of Canada

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adult morphology, especially the scolex, and less so on the uterus, position of genital pores and nature of the vitellaria, also place great emphasis on host specificity, i.e. the types and numbers of hosts infected (e.g. Spasskii, 1951, 1963a, b; Riser, 1955; Joyeux and Baer, 1961; Stunkard, 1967). In large measure these systems ignore data based on cestody ontogeny. Apparently this is based on acceptance of the position taken by Joyeux and Baer (1937) who stated: “Ces variations de la morphologie et du comportement biologique des larves de Cestodes rendent donc illusoire la possibilite d’une classification rationelle parallkle A celle des Vers adultes. I1 s’ensuit que les genres crtds pour les designer n’ont aucune valeur systematique: tout au plus peuvent-ils &tre considerks comme des termes commodes A employer dans le langage courant, mais sans pritention taxinomique [sic].” Recently it has been asserted that proper evaluation of patterns of ontogeny may suggest relationships (Stunkard, 1962; Voge, 1969), but this has received scant attention among most taxonomists. WiSniewski (I 97 1) showed, however, that two genera, formerly the basis for the order Aporidea, have a developmental pattern in copepods which indicates a relationship with the hymenolepids. This supports Spasskii (1963b), who maintained earlier that aporideans should be retained within the order Cyclophyllidea. In contrast, Joyeux and Baer (1945) discovered that Catenotaenia ptisilla developed to what they called a merocercoid in a tyroglyphoid mite. This cestode never undergoes the more complicated development of an anoplocephalid in oribatid mites (Freeman, 1952). However, the fact that C. pusilla grew in a mite was sufficient justification for these authors to remove C. pusilla from the family Dilepididae and place it in the Anoplocephalidae. When life-cycles are considered, the morphology of the developmental stages frequently receives superficial attention (Spasskii, 1951, 1963a, b; Joyeux and Baer, 1961; Olsen, 1967; Baer, 1971). This is inevitable as long as oversimplified concepts prevail concerning the morphology of the various types of ontogenic stages, and possibly more importantly how they grow (e.g. see the “Key to Larval Tapeworms” by Schmidt, 1970; definitions by Yamaguti, 1959; figures by Joyeux and Baer, 1961, Olsen, 1967, and Baer, 1971). Small wonder that such data are largely ignored by taxonomists, since similar, although better, data for trematodes were not utilized effectively for classification at the ordinal level until adopted by LaRue (1957). There is increasing awareness of the need for taxonomic revision and during the past few years suggestions have been made (Freeman, 1957, 1970; Voge, 1967; Jarecka, 1970c, d). The concept of the cestode life-cycle encountered in most texts almost precludes understanding relationships among cestodes. The cycle is presented as either two-host or three-host, rarely reduced to one-host, which if in aquatic hosts “typically” has a free-swimming ciliated stage which is lacking in those cestodes developing in terrestrial hosts (Stunkard, 1962; Jarecka, 1970d). But, as Jarecka (1970d) points out, there is little agreement on the definition of coracidium, procercoid, plerocercoid, or cysticercoid. Leiby and Dyer (1971) oversimplify to the other extreme by stating that “. . . larval cestodcs . . . essentinlly . . . are miniature adults.” Nevertheless, the

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statement has been made that the essential information for understanding cestode life-cycles is known (Mueller, 1965). The existing confusion is well exemplified by the fact that recently Katansky et al. (1969) suggested that a ciliated animal found in the intestine of a marine clam probably was the first stage of the tetraphyllidean genus Echeneibothrium. Yet no ciliated stage has been demonstrated for this order let alone the genus (Wardle and McLeod, 1952; Euzet, 1959), and apparently their oncospheres are no more than one-third the size of the stage from the clam (Williams, 1966). Probably the same holds true for the “coracidium” Cheng (1966) found in an oyster and which he attributed to the genus Tylocephalum, since no coracidia are known from other lecanicephalideans (Wardle and McLeod, 1952; Euzet, 1959). Much is known about the ontogeny of cestodes, but the data require serious reexamination (Voge, 1969 : Mackiewicz, 1972), particularly from the standpoint of the cestodes themselves, including a more precise delineation of the various stages and phases of growth, rather than placing primary stress on the hosts involved as done presently. Transfer from host to host is an ecological phenomenon which cestodes have mastered very well. All transfer from host to host when eaten as food, or with food. It follows, therefore, that the cestode fauna in the host’s gut may change as the host’s diet changes unless the cestode accommodates to these changes in diet. Such changes in fauna are particularly obvious in anadromous fish, or often when the diet of the young differs from that of the adult. Changes may be obvious also among animals introduced into totally new environments. Throughout this presentation the role of the host will be oversimplified intentionally and hosts in large measure will be treated as sites where cestodes can function, rather than, as frequently presented, the prime determinants of the cycle. A review of literature on the ontogeny of cestodes reveals a diversity of terminology and divergent views as to the significance of the forms described. A basic cestode cycle and variations in it will be discussed to develop a unified system of naming the various stages and phases of cestode development. This in turn suggests the course of evolution of cestode life-cycles, which may help delineate modern taxa. The discussion will be limited to the “true” cestodes, namely those with a six-hooked larva, the oncosphere, or an obvious derivative, and excludes the interesting amphilinids and gyrocotylids. 11. THEBASICCESTODE LIFE-CYCLE A.

STAGES IN THE CYCLE

The basic sequence of cestode development consists of: (a) the monoecious (rarely dioecious) adult which produces, (b) an ovum, that presumably requires fertilization (Rybicka, 1966) before cellular differentiation and development to, (c) an oncosphere, a six-hooked (hexacanth) larva (hooks rarely lacking), which migrates to a suitable parenteral site where it, (d) metamorphoses and grows as a metacestode that has completed development

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wliea it has (i) a fully dilkrentialed scolex of adult size and (ii) a body showing proglottidation (= proglottisation of Wardle and McLeod, 1952), or first signs of approaching sexual maturation, and (e) the sexually reproducing adult in the enteron (rarely elsewhere) (Fig. I). Stunkard (1962) considered that repetition of semi-independent sexual units by proximal growth in cestodes is strobilization, and not metamerism, the repetition of body units by distal growth; he used interchangeably the terms segment and proglottid [term now in common usage rather than the original Greek proglottis (-ides)*]. Hyman (1951) accepted that cestodes exhibit true mesodermal segmentation ( = metamerism). Wardle and McLeod (1 952) showed, however, that proglottidation, the repetition of sexual units, can occur without segmentation, the division of the body into repetitive units, and that segments may contain one or more proglottides. Accordingly both terms are necessary for full comprehension of the process of sexual maturation.

FIG.I . The basic cestode life-cycle,

Diversity is imposed on this basic plan (Fig. l), but without exception the oncosphere invades a parenteral site (invertebrate or vertebrate) where metamorphosis follows (Stunkard, 1962; Ogren, 1968). Almost as constant is the completion of sexual maturation in the gut, or some diverticulum such as the bile duct, of a vertebrate; exceptions occur among a few unique cestodes, which may reproduce sexually in a parenteral site in an invertebrate (e.g. Archigetes sp. in an aquatic annelid, see Mackiewicz, 1972, for review; and possibly some spathebothriideans, see WardIe and McLeod, 1952; Sandeman and Burt, 1972). Overlooking such exceptions, then most adult growth occurs characteristically in a vertebrate host’s gut, and most, although frequently not all, metacestode growth occurs outside the gut, primarily in an invertebrate; the larva (oncosphere), once eaten, invades the latter site

* Which is preferred (Ed.).

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(Fig. 1). The metacestode enters the vertebrate gut either by being eaten, or by migration from tissues of the same vertebrate (Fischer and Freeman, 1969). By convention the host in which sexual maturation occurs is called the final (or definitive) host. All other hosts are termed either intermediate, if the metacestode undergoes physiological development, or paratenic (transport), if the host functions as a storage depot for the metacestode (Baer, 1951). The demonstration of physiological dependency may require careful experimentation, except when morphological change is evident. Hence, it is suggested that hosts be labelled first, second, third . final host, depending on the minimum number of hosts required to complete the cycle. Again by convention, it is assumed that the minimum number of hosts required to complete the cestode cycle under controlled conditions is “the life-cycle”, although some species, particularly in aquatic habitats, apparently may not reach the final host without an intervening host, which by standards given above, would be labelled as paratenic. The terms larva and metacestode as used here are not universally accepted, and require further clarification. The term larva is restricted to the oncosphere and not used for subsequent phases of development, whether in parenteral or enteral sites. This concept, first proposed by Rosen (1918), is theoretically necessary if the function of the oncosphere is to invade a parenteral site where it dedifferentiates, resorbing most structures used during the process of invasion, and differentiates into a new form. This is the usual concept of metamorphosis for insects and other invertebrates (Clements, 1968). The oncosphere obviously undergoes metamorphosis, because the next stage not only is totally different but includes little of the anatomy of the oncosphere. Even the two flame cells of the coracidium of Diphyllobothriurn spp., although functional in the developing metacestode, apparently are not incorporated into the new protonephridial system of the differentiated metacestode (Malmberg, 1971). The change is so complete that the functional anterior end of the oncosphere is incorporated into the functional posterior, i.e. abscolex, end of the metacestode (Ogren, 1968). Others have described this change as metamorphosis (Villot, 1883; Leuckart, 1886; Baer, 1951; Stunkard, 1962, 1967; Ogren, 1968; Malmberg, 1971). Ogren (1968) considers, however, that the metacestode growth is embryonic, “ . . . a metamorphosis involving degeneration of one embryonic stage and its replacement by another of different morphology.” However, the analogy with the metamorphosis of larvae of insects is obvious, and has been discussed by others, e.g. Wardle and McLeod (1 952). Metamorphosis is completed when the recognizable systems, other than the hard oncospheral hooks and flame cells when present, have been resorbed. Subsequent growth of the metacestode is direct, although there may be one or more interruptions, even including asexual reproduction (polycephalism in one body, binary fission, various forms of “budding”). Special structures may develop, such as a modified end organ on the scolex, or a specialized cyst-like bladder on the abscolex end, which permit further exploitation of the environment (slais, 1966; Fischer and Freeman, 1969). In all cases each deviator, no

..

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matter how extreme the apparent deviation, ultimately returns to the basic plan and the ultimate adult includes most of the tissues and organ systems of the scolex and “neck ” of the metacestode. In short, as will be shown more fully below, the metacestode immediately preceding proglottidation usually is a plerocercoid. Wardle and McLeod (1 952) originally suggested the term metacestode (i.e. changing cestode) as a collective term to cover all phases of parenteral growth. They also endorsed the concept of metamorphosis but then apparently contradicted themselves by saying (p. 58): “Where a succession of metacestode stages occurs, the stages may be referred to as larval phases of the metacestode stage.” Freeman (1970) extended the definition of a metacestode to include all growth phases between oncosphere and first evidence of sexuality, i.e. proglottidation, whether this occurs parenterally or enterally. It could be argued that the dedifferentiation phase which the oncosphere undergoes is, or is not, part of metacestode growth. This period usually is short, and is recognized as an enlarging ball of cells. Once the distribution of these cells begins to take on a recognizable pattern, however, growth and differentiation of the metacestode certainly has begun. The term metacestode is appropriate, accurate, and avoids the connotation of another or a continuing metamorphosis implied by using the term larva for postoncospheral forms, although the idea that there is a second metamorphosis between the metacestode and adult has been in the literature for a long time (Leuckart, 1886; Cheng, 1964). The term metacestode has received some acceptance, and will be used here in the above sense, and the term larva will be restricted to the oncosphere, the stage preceding metamorphosis. Metacestode growth ends when the scolex achieves full or nearly full adult size, and the presence of reproductive primordia indicate that sexual reproduction has begun. The presence of segments also may suggest the onset of sexuality, although it is less precise. Obviously the cestode is immature until the first evidence of gametes. Apparently the scolex in many groups of cestodes does not achieve adult size while in the parenteral site and must complete its growth in the gut lumen of the vertebrate. This is contrary to what one might expect knowing that the rostellar hooks, on those cyclophyllideans that have hooks, usually are fully formed on the parenteral metacestode. That a part of the abscolex metacestode body, e.g. cercomer or bladder, may be lost during transfer to the next host does not indicate another metamorphosis, any more than eclosion of the oncosphere from its eiiclosing “egg membranes” indicates metamorphosis. Therefore, continuing to use the term larva, or such combinations as larval cysticercoid, larval plerocercoid, or larvocyst to describe parenteral cestodes (Waxdle and McLeod, 1952; Stunkard, 1962, 1967; Smyth, 1962; Abuladze, 1964; Smyth and Heath, 1970; Noble and Noble, 1971), is misleading since in a strict sense it implies that these “larvae” will again undergo metamorphosis when they reach the enteron, and, as already indicated, this does not occur. In contrast to Ogren (1968), Jarecka (1970c, d) refers to parenteral forms as the “second larval stagc”, and suggests the collective term cercoid for

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them, recognizing the oncosphere as the “first larval stage”. She considers that adult development has begun once the cercomer or equivalent is lost. This assumes that all cestodes produce cercomers, which is not true, as will be elaborated later. If, as is common with certain pseudophyllideans, e.g. the plerocercoid of Diphyllobothrium latum, further parenteral development occurs after loss of the cercomer, then Jarecka considers such plerocercoids to be metacercoids, the “larva of the adult”. By this reasoning there may be three larval stages in a cestode life-cycle: (a) the oncosphere, (b) the cercoid, and (c) when present, the larval adult. The term larva in the last case is used in the sense of any recognizable nonadult “phase” or “stage” provided it occurs parenterally, whereas in the first two cases the larva loses first the egg membranes, and second the cercomer or equivalent. This system apparently is based on an acceptance of a correlation between the number of “stages” and the number of hosts in any given life-cycle, and of the idea that all parenteral growth is “larval” whereas growth in the enteron is “adult”. This goes counter to the position taken here, namely, that cestodes have only three stages: the oncosphere (larva), metacestode (stage following metamorphosis), and the sexual stage usually recognized as immature, mature, and gravid. Furthermore, regardless of the number of developmental sites, any distinct interruptions within each stage will be considered phases. For clarity the terms will not be used interchangeably, as is done frequently (Smyth, 1969). Shedding the cercomer may have phylogenetic significance, but it is doubtful that this act per se represents termination of a major stage of development, consequently a special term, such as cercoid (Jarecka, 1970c), is deemed unnecessary. Furthermore, such a term is not applicable with species which produce no cercomer. It may be that the discovery of the lifecycle of Diphyllobothrium latum by Janicki and Rosen (1917), before that of other pseudophyllideans or proteocephalideans with simpler and probably more typical life-cycles became better known, was an historic accident which retarded comprehension of cestode life-cycles. It represented a reasonable model, therefore subsequent workers deemed it necessary to fit their observations into the proposed system. However, the procercoid of D . latum in a copepod is quite different from any known proteocephalidean metacestode in a copepod, or for that matter many metacestodes of what usually are considered either more primitive pseudophyllideans or caryophyllideans and presumably other groups as well. In fact Baer (1940) pointed out that procercoids of most cestodes are seldom as well delimited as those of D. latum. Yet it is precisely this unusual procercoid, and the shedding of its cercomer, which frequently is taken as the base for explaining the evolution of cestodes today (Llewellyn, 1965; Jarecka, 1970~). B. ECOLOGY OF THE LIFE-CYCLE

The number of hosts in a cestode cycle varies for individual species as well as groupings of species (genus, family, etc.) and is related to the fact that cestodes move both up and down through the trophic levels in the

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macroenvironments, or ecosystems, utilized by the hosts in which the cestodes live. This suggests that a cestode might move up the food chain through an infinite number of steps, being dependent only on the number of times it can be eaten. Actually the maximum number of transfers is restricted to four or five, since that is the number of trophic levels in most food webs (Odum, 1971). For example, in an aquatic cycle the cestode egg may masquerade as plankton (first level: many species-Jarecka, 1961), be eaten by a copepod plant eater or primary consumer (second level: most proteocephalideans and pseudophyllideans), which in turn may be eaten by a small fish secondary consumer (third level: some protocephalideans and pseudophyllideans), followed by a large fish tertiary consumer (fourth level : some Proteocephalus spp. and Diphyllobothrium spp.), which finally may be eaten by a bird, mammal or other final consumer (fifth level: some Diphyllobothrium spp.). Although the examples used all occur in fresh water, there is reason to believe that the same occurs in the sea (Euzet, 1959, p. 237). For the cestode to recycle, it is equally important, furthermore, that it has an efficient system for moving some stage back down the food chain to lower trophic levels. This usually occurs as the “egg”, i.e. the ovum or oncosphere with surrounding membranes, which is discharged from the adult parasite in a host at a higher trophic level. Such stages have adaptations making them available to hosts at a lower trophic level. There are other potential, albeit almost totally uninvestigated, mechanisms for moving within the food web, such as plerocercoids in an aquatic ecosystem passing through the gut of an unsuitable carnivore, e.g. large fish, and being ingested by a more suitable host, e.g. small fish, lower down the food chain (Fischer, 1972). Euzet (1959) suggested that the same may apply for tetraphyllideans in the sea. Conceivably metacestodes which escape from living or dead aquatic hosts as described for procercoids of Spirometra sp. (see Mueller, 1959), or metacestodes of Proteocephalus spp., Diphyllobothrium sp. and Ligula sp. (personal observations) also are available for transfer to other hosts. Some freed metacestodes are remarkably hardy, surviving for hours and even days outside the host. Whether this occurs naturally has not been investigated. At one time such statements were not pertinent to higher cyclophyllideans, but observations by Rybavjr (1961) and Lesinsh and Klyavinsh (1966) suggest that they may pertain to certain aquatic hymenolepids as well. Another feature usually not considered in most discussions of cestode life-cycles is the movement of cestodes from one site to another at the same trophic level: the metacestode first enters one parenteral site, but after suitable time and with appropriate stimulation it then moves to another site (e.g. Proteocephalus ambloplitis and Ophiotaenia Jilaroides: Fischer and Freeman, 1969; Mead and Olsen, 1971). Possibly this latent ability for lateral movement permitted Hymenolepis nana to develop from the typical arthropod-vertebrate cycle to one where parenteral and enteral development can be completed in a single vertebrate host. In fact, it has been demonstrated that H. nana not only can develop in a variety of vertebrate hosts, but also in several parenteral sites in addition to the intestinal wall (Weinmann, 1969).

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Conceivably metacestodes from deeper in the viscera, as well as those in the gut wall, migrate to the gut lumen. The preceding comments generally are more pertinent to those metacestodes that have most of their cycle in aquatic hosts, and if there is a terrestrial host in the cycle it is a vertebrate that acquires the cestode from an aquatic host. Transfer between exclusively terrestrial hosts presents more severe problems owing to danger from desiccation. Generally it is assumed that most cestodes with terrestrial cycles are restricted to two trophic levels, i.e. most metacestode development in a parenteral site in a host on a lower trophic level, and all subsequent development in the gut lumen of a host at a higher trophic level. However, some cyclophyllideans require three hosts (Gupta, 1970; Jarecka, 1970a, b), as presumably does Mesocestoides sp. At the present time it must be assumed that paratenic or “accumulator” hosts are less important in cyclophyllidean cycles, although they must not be overlooked (RySavjr, 1961 ; Lesinsh and Klyavinsh, 1966). Movement back down to the lower trophic level by most cyclophyllideans is via a well protected oncosphere either with its own resistant shell, or within a modified capsule, paruterine organ, or similar structure produced within the parent body. The latter structures are comparatively rare among cestodes with aquatic cycles, although capsules with more than one egg are known (Euzet, 1959; Jarecka, 1961). In some species the gravid segment may have a brief free-living migratory phase (e.g. Davainea sp., Raillietinu sp., Choanotaenia sp., Taenia sp., Oochoristica vacuolata, Echinococcus sp. ; see Wetzel, 1936; Hickman, 1963; Poole and Marcial-Rojas, 1971 ; Distoichomefra sp., according to Whitcomb, R. unpublished). The crucial point arising from the preceding discussion is that cestodes may be more dependent for their development on various sites at various trophic levels than on hosts per se, a fact frequently overlooked when one thinks only in terms of one-host, two-host, or three-host cycles. The last concept implies that a cestode either gets into an appropriate host, frequently a very specific one, and “succeeds”, or if it enters an inappropriate one it “fails”. It is well known, however, that hosts thrust into new or unusual environments acquire cestodes which are new to them. For example, Kotecki (1970) reported 21 new host records for adult cestodes of anseriform birds confined to one zoo in Poland. Paratenic hosts are not uncommon and may be of great importance, i.e. a host may not be suitable for further differentiation of a cestode but it may survive if it finds and remains in an appropriate site. RySavjr (1964) proposed the term “reservoir habitationalism” for the relationship between hymenolepid metacestodes and snails which acquire them by eating the copepods in which the original metacestode development occurs. It is remarkable, however, how well segregated the parasites are within particular hosts in nature, even when hosts superficially appear to have similar feeding habits and occupy similar macroenvironments. Conceivably this segregation reflects interspecific competition within the tissues of these hosts, and those best adapted for a particular host and site survive, with the others surviving in other hosts and sites for which they in turn are better adapted. These remarks are particularly applicable to cestodes in

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aquatic hosts, but may also prove true of metacestodes in terrestrial hosts. Such host-cestode relationships merit more investigation. Theoretically then, a metacestode might move from one parenteral site to another an infinite number of times, depending on the vagaries of the food web in which it occurs, although if it can move only up the food chain and not down or within trophic levels then presumably it is restricted to one to three or four transfers (e.g. Diphyllobothrium latum). Similarly some metacestodes may move from one gut to another within the food web without being lost from the population, although this may be unnecessary for adult cestodes since once they are in a gut suitable for the final adult stage they continue the cycle by producing eggs. Thus cestode cycles, as shown graphically in Fig. 1, have unity. A cycle may be completed in a matter of a few weeks, or may take some years. Furthermore the length of time spent in any stage may occupy little or most of the time in a given cycle, but each species passes through the same three stages in each cycle. Generally, the ovumlarval stage is short-lived for cestodes in aquatic environments, but may be longer in others which overwinter (Abuladze, 1964, p. 429). It should be stressed also that once a metacestode enters the gut, even in a suitable host, adult growth may not ensue immediately. Some proteocephalideans, for example, have extensive growth periods as metacestodes within the gut of suitable hosts before proglottidation begins (Freeman, 1964; Fischer, 1972; Befus and Freeman, 1973). This probably occurs with cyclophyllideans as well (Goodchild and Harrison, 1961;Hickman, 1963). On the other hand, adult growth, i.e. proglottidation of pseudophyllideans and similar worms, may occur while the worms are in parenteral sites. Archigetes Iimnodrili is an extreme example, as viable eggs are produced while in the haemocoel of annelids (for review, see Mackiewicz, 1972). It frequently is stated that such cestodes, e.g. the genera Ligula, Schistocephalus, Archigetes, Bothrimonus, Cyathocephalus, etc., are progenetic or neotenic (Wardle and McLeod, 1952; Mackiewicz, 1972; Sandeman and Burt, 1972), suggesting that earlier there was a more typical adult stage which now is lost. However, do the strobilate metacestodes of certain cyclophyllideans (e.g. Taenia sp. and Schistotaenia sp : Abuladze, 1964, and Boertje and Ulmer, 1965) present equivalent development? In these cases the adult scoleces and bodies are typical of other closely related species which supposedly do not initiate such segmentation while in the parenteral site. Conceivably cestodes which begin sexual maturation in parenteral sites are, as Rosen (1918) maintained, neither progenetic nor neotenic. This will be discussed further when the evolution of cestodes is considered. 111. VARIATIONS IN CESTODE ONTOCENY A.

THEADULT

This review will stress differentiation of the metacestode rather than morphological variations associated with growth of adult cestodes. However, since some cestodes initiate segmentation or even proglottidation while in the parenteral site, a site usually considered primarily suitable only for

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metacestode development, it is appropriate to consider briefly whether the onset of segmentation and of proglottidation are valid criteria for separating the metacestode stage from the adult stage. Usually the body, or strobila, of an adult cestode has one reproductive unit in each segment. Wardle and McColl(1937), Wardle and McLeod (1952) and later Kuhlow (1953) showed, however, that the strobila of Diphyllobothrium spp. may be of two morphological types, which they called primary and secondary. The terminal segments in a primary strobila supposedly are elongate and may be multiproglottate (i.e. with several reproductive units) and the body is widest near mid-region and tapers toward both ends (Fig. 2, D). In contrast the secondary strobila 1

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FIG.2. Types of strobilae and segments. Haplobothrium sp.: A. Primary strobila with primary and secondary segments; B. Secondary strobila with secondary and tertiary segments. Diphyllobothriunz sp. : C. Plerocercoid with primary segments; D. Transitional strobila with primary and secondary segments; E. Secondary strobila with secondary segments. Scales= 1 mm (original).

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does not narrow toward the abscolex end, and typically all segments are broader than long and contain a single proglottid (Fig. 2, E). Why this difference? Markowski (1949) assumed that the elongate segments were degenerate; Meyer (1966) considered them teratological; and Graham (1970) and Jamieson (1972) found elongate multiproglottate terminal segments a typical feature of young Diphyllobothrium dendriticum developing in gulls, men, and dogs. Apparently the answer is that much of the plerocercoid body of such species may be incorporated into the developing adult (Archer and Hopkins, 1958), and segments produced from the plerocercoid body (primary) (Fig. 2, C) differ morphologically from those budded later from the "neck" (secondary) (Fig. 2, D, E). Since secondary segments are produced by budding from the neck before the primary segments are lost by apolysis from the other end, it follows that strobilae may be primary only for a short period until the first secondary segments are produced. Archer and Hopkins (1958) reported a remarkable drop in growth rate of strobilae of Diphyllobothrium sp. at the time the primary segments became gravid. Differences in primary and secondary segments probably are associated with the distribution of the germinative cells which produce the gonads. In plerocercoids of Diphyllobothrium dendriticum they are distributed along the length of the body, whereas in the adult they are abundant only in the neck (Wikgren and Gustafsson, 1971; Bonsdorff et al., 1971). Since strobilae cannot become secondary until primary segments are lost, it follows that they are transitional, i.e. contain both primary and secondary segments. A transitional strobila may last for several weeks in the case of Diphyllobothrium dendriticum in gulls, dogs, and man (Graham, 1970; Jamieson, 1972). This apparently is not true for all plerocercoids, as Joyeux and Baer (1938a) observed both in vitro and in vivo that those of Diphyllobothrium erinacei europaei shed the posterior part of the plerocercoid on entering the gut of the final host. This is known for other pseudophyllideans as well (Mueller, 1938, 1972; Berntzen and Mueller, 1964). Other pseudophyllideans, e.g. Eubothriuni salvelini, or Haplobothrium sp. (Fig. 2, A, B), or the marine genus Diplogonoporus, produce tertiary segments from segments well down the body of an adult worm (Freeman, unpublished; Rausch, 1964). With certain cyclophyllideans which have no deciduous cercomer, the entire metacestode body probably is incorporated into the terminal few segment(s) (Wardle and McLeod, 1952; Freeman, 1957, 1959; Hickman, 1963). Presumably these are primary segments as well. The same is true of the remaining body of certain cyclophyllideans which lose the cercomer, e.g. Dipylidium caninum (see Venard, 1938; Marshall, 1967). Conceivably incorporating the entire metacestode body into the young adult may be more typical than realized previously, although with various cyclophyllideans (e.g. Hymenolepis spp., Taenia spp.) it is well established that some of the metacestode body is discarded. In the latter, after discarding the posterior cyst-like part, a new terminal excretory* vesicle and pore is developed and *There is a disagreement whether the flame cells, longitudinal and other ducts, and the terminal vesicle and pore should be considered as parts of an excretory, osmo-regulatory, or protonephridial system. Excretory system is used most commonly.

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these are incorporated into the terminal segments of the developing adult (Leuckart, 1886; Freeman, personal observation). Obviously most if not all of the remaining adult body will consist of secondary segments. The same is true for Taenia taeniaeformis in which the scolex-bearing part, the “true larval strobila” according to Hutchison (1959), still contains numerous primary segments after it separates from the bladder. The terminal segment of the scolex-bearing segment generates a “new” terminal excretory vesicle and pore (Joyeux and Baer, 1938c), and the body immediately begins growth at the rate characteristic of the adult (Hutchison, 1959), without a growth lag as observed when the unsegmented plerocercoid of ~ ~ p h y Z ~ ~ b o ~ sp. ~zriu}n reaches the final site (Archer and Hopkins, 1958). Proglottidation, with or without concurrent segmentation, is a form of asexual reproduction and is associated with the adult stage of the cycle since it produces the units which reproduce sexually. Thus proglottidation indicates that metacestode development has ceased and adult development has begun. Whether it is valid to consider forms which undergo proglottidation parenterally as progenetic or neotenic (Wardle and McLeod, 1952; Macy and Berntzen, 1971; Mackiewicz, 1972) is another matter. This is particularly so since parenteral segmenting metacestodes occur in such cyclophyllidean genera as Taenia, Schistotaenia, and Lateriyorus (see Wardle and McLeod, 1952; Boertje and TJlmer, 1965; Denny, 1969, respectively) and these are not considered progenetic or neotenic. B.

TYPES OF EGGS AND ONCOSPHERES

Growth and development of the oncosphere with its surrounding membranes, envelopes, coats or shells as they are variously called, has received some attention (Ogren, 1957a, b, 1968; Rybicka, 1966; Collin, 1969; Pence, 1970; Coil, 1972). Egg is the term usually used to describe the fertilized ovum or oncosphere and the surrounding membranes, although Hyman (1951) for technical reasons used “capsule”. Development in utero or e x utero results in different types of eggs among different groups of cestodes, and according to Loser (1965a, b) and Ogren (1968) some result from fundamentally different types of growth. The purpose here is to examine whether different types of eggs relate to different patterns of oncospheral metamorphosis and growth. The term embryo or hexacanth embryo occasionally is applied to the oncosphere (Ogren, 1957a, b). Ogren (1957a) stated: “The mature oncosphere represented the termination of one phase of embryonic development. . upon entering the intermediate host the oncosphere began a new period of differentiation.” Ogren (1968) continued this contradictory position and maintained the metacestode is the result of a “ . . metamorphosis involving degeneration of one embryonic stage and its replacement by another of different morphology.” Voge (I 967) and others refer to metacestode development as postembryonic. However, the oncosphere is a fully differentiated, functional unit; therefore calling it an oncosphere or hexacanth larva would seem more appropriate, acceptable, and less confusing than using the term

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embryo. Embryogeny may be completed in utero, or ex utero, either inside or outside the host body (Wardle and McLeod, 1952; Mackiewicz, 1972); the oncosphere of the caryophyllidean Khawia sinensis may undergo embryogenesis after ingestion by the first host (Mackiewicz, 1972, p. 443). The oncosphere with its surrounding membranes is usually well adapted for transfer to the next host. Frequently eggs are modified to float, sink, cling, resist desiccation, and may mimic foods of suitable hosts (Jarecka, 1961), or in some cases the larva escapes and swims freely; all apparently are adaptations which increase the availability of particular oncospheres to suitable hosts. Secondarily the uterus may be modified into capsules or a paruterine organ for protection and dispersal of the eggs. The oncospheres may activate spontaneously once removed from the uterus or paruterine organ (Mesocestoides sp. and Oochoristica sp. : personal observations). Some require mechanical rupture and trypsin (Coil, 1972), whereas others require seemingly specific trigger substances produced in the gut of suitable hosts to activate oncospheres (Silverman, 1954). Quite obviously the oncosphere with its membranes is highly specialized and well suited for passive transfer to the first host, where by active migration the larva enters a suitable parenteral site. Although it is assumed that the oncospheral hooks are used to “claw through” the tissues, in fact the few species studied suggest that these hooks may be used more for attachment to the gut wall, and that secretions facilitate a more passive less disruptive penetration than that resulting from “clawing”. Such “penetration glands” have been demonstrated in proteocephalidean and cyclophyllidean oncospheres (Reid, 1948; Hickman, 1963; Bilqees, 1968; Fischer, 1968; Befus and Freeman, 1973). In copepods the process can be followed directly, and here penetration occurs with no apparent rupture of the cells. The oncosphere is a highly specialized larva. Ogren (1972) points out, for example, that: “Oncosphere musculature, thus, represents a high degree of differentiation that suggests evolutionary and developmental specialization.” Another characteristic feature of this highly specialized stage is, “. . . that some somatic cells degenerate after their differentiation [within the oncosphere] whereas the germinative cells remain to multiply in a later stage of [metacestode] development” (Ogren, 1968). The evolutionary process that led to development of the modern-day oncosphere is a mystery, but that the precursor was adapted for crawling, clinging, as well as digging would seem logical. Understanding the evolution of the oncosphere is important for understanding the evolution of cestodes. For a comprehensive review of cestode embryogenesis the reader is referred to Rybicka (1966); for more recent information see particularly Ogren (1968), Collin (1969), Smyth (1969), Pence (1970), Coil (1972), Mackiewicz (1972). Commonly it is assumed that operculate eggs with ciliated coracidia, such as the well known Diphyllobothrium latum, must be characteristic not only for other pseudophyllideans but also for trypanorhynchs and tetraphyllideans, and furthermore that probably such eggs are the primitive type best adapted for an aquatic habitat (Stunkard, 1962; Smyth, 1969, p. 109). This seems most improbable. In fact, operculate cggs which liberate ciliated

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coracidia are relatively rare and occur only among certain, but not all, pseudophyllideans and trypanorhynchs (Wardle and McLeod, 1952; Mudry and Dailey, 1971). They are unknown from tetraphyllideans (Euzet, 1959), proteocephalideans (Freze, 1965), and the array of other aquatic cestodes. Jarecka (1970c) maintains that some eggs, e.g. pseudophyllideans, are “oviparous”, i.e. do not embryonate until after discharge from the parent, and are basically more primitive than eggs of proteocephalideans and cyclophyllideans, whch she considers “viviparous” since they develop in utero. Rybicka (1966, p. 124) apparently agrees, although she does not use the terms viviparous and oviparous. Presumably it is the deposition of a sclerotinized capsule from vitelline secretions from the parent, as occurs with pseudophyllideans and presumably tetraphyllideans and trypanorhynchs, as well as inclusion of numerous nurse cells, which permit ex utero development (for review see Smyth, 1969). Whether ex utero development is primitive (Jarecka, 1970c) is questionable, however, since such pseudophyllideans as amphcotylids, haplobothriids, ptychobothriids and others, e.g. caryophyllideans, which on other grounds are considered primitive, include species which (a) complete embryogeny in utero (genera Haplobothrium, Eubothrium, Cephalochlamys, some caryophyllideans : Essex, 1929; Vik, 1963; Thurston, 1967; Mackiewicz, 1972, respectively), (b) have non-operculate eggs (Eubothrium, Cephalochlamys), (c) do not produce a ciliated coracidium (Eubothrium, some caryophyllideans), and (d) have oncospheral emergence after eggs are ingested by suitable hosts. In these respects they functionally resemble eggs of proteocephalideans and cyclophyllideans. Once cleavage has begun, it has not been demonstrated conclusively for any group, however, that nutrients are acquired by the oncosphere directly from the parent (Smyth, 1969, p, 118), a true sign of viviparity. It would appear, therefore, that using the terms oviparous and viviparous, which refer to the nutrition of the embryo in the uterus and its condition at the time of discharge from the uterus, confuses rather than clarifies. Furthermore if the various layers within the outer shell of pseudophyllideaii eggs are products of cells produced by the embryo and are homologous to similar layers in proteocephalideans and cyclophyllideans, as Rybicka (1966) contended, then where are the fundamental differences in their development ? Moreover, in some, presumably more primitive, pseudophyllideans, e.g. Eubothrium sp., “fewer vitelline cells are associated with one oocyte” (Rybicka, 1966, p. 122). Therefore, except for the thicker proteinaceous shell produced from products of vitelline glands in certain pseudophyllideans (and less so in other groups, e.g. tetraphyllideans and trypanorhynchs), the other layers surrounding the oncospheres apparently are of similar origin, albeit secondarily highly modified, except possibly some cyclophyllidean eggs (e.g. Stilesia sp. ; Loser, 1965b). Ogren (1957b) did in fact distinguish seven groups of cestodes based on formation of these layers, and Loser (1965a, b) recognized four groups, but Rybicka (1966) questioned whether such differences are of phylogenetic significance. Ogren (1968) summarized the process succinctly: “Thus, it is clear that oncosphere morphogenesis results from a developmental system programmed to produce an invasive oncosphere and a few stem

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germinative cells c:ipable o f multiplying to provide embryonic mesenchyme of the early cysticercoid [met:icestode].” Regardless of the number of membranes, envelopes, shells, coats, layers, macromeres, micromeres, or mesomeres, the end result is a hexacanth larva, the oncosphere, although there are exceptions where oncospheral hooks are absent (Anonchotaeniu sp., Cyathocephalirs spp. ; Wardle and McLeod, 1952). The oncosphere is a bilaterally symmetrical animal (Freeman, 1964; Ogren, 1972), with three pairs of hooks, the medial, medio-lateral, and latero-lateral pairs. Each pair of hooks, although small, may be characteristically different in shape, stoutness or length (Hickman, 1963; Freeman, 1964; Fischer, 1968). The medial hooks are ventral, usually long and slender. The lateral pairs are dorso-lateral according to Ogren (1972), but Hickman (1963) maintained the stouter lateral hooks are ventro-lateral, and the others dorso-lateral. The U-shaped “penetration glands”, if present, usually stain well with vital stains; they have a pore on each side of the hooked end between the median and medio-lateral hooks (Collin, 1969). In the oncospheral body there are a limited number of nuclei, numerous muscle fibers, but no recognizable nerve fibers (Collin, 1969; Pence, 1970; Ogren, 1972). Apparently some, but not all, oncospheres which escape from the egg enclosed in a ciliated embryophore have two flame cells, in contrast to the consistent absence of flame cells in oncospheres which do not have this short free-living existence. Possibly flame cells are required for survival in the hypotonic environment of fresh water (e.g. Diphyllobothrium sp.), since according to Riser (1956) the free-swimming coracidia of the marine trypanorhynch Lacistorhynchus tentiis lack flame cells. Do flame cells in an oncosphere suggest a primitive or more recently evolved condition? Malmberg (1971) showed that the oncospheral flame cells do function early in the developing metacestode of Diphyllobothrium spp. This he called the primary protonephridial system. Apparently, however, the primary system is not incorporated into the secondary protonephridial system, which becomes the system utilized by the advanced metacestode and ensuing adult. This suggests that flame cells in the coracidium may be a recent adaptation which has permitted one stream of cestode evolution, culminating in the higher pseudophyllideans such as Diphyllobothrium spp. and Triaeriophorus spp. to have a short free-living existence in fresh water. C.

DEVELOPMENT OF THE METACESTODE

1. An overview The oncosphere always metamorphoses and the resulting metacestode usually completes most differentiation in a parenteral site(s), commonly the haemocoel of an invertebrate. Within a few days after reaching the site, coordinated movement of oncospheral hooks diminishes and the body begins to enlarge due to cellular proliferation. The cells undergoing division have not been studied well, but presumably most if not all growth stems from relatively few precursors (Ogren, 1968). Apparently little or none of the musculatrwe of the oncosphere, or the penetration glands if originally present,

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is incorporated into the developing metacestode. Flame cells, if present, continue to function, but an entirely separate excretory system develops (Malmberg, 1971). The body remains Spherical as it enlarges, and initially the cells appear similar and uniform in size; there may be feeble waves of contraction suggesting that some contractile fibers are present. Voge (1960b) found “few definite cells’’ but numerous nuclei in this phase of development of Hymenolepis diminuta. Ultimately the sphere begins to elongate, and usually a thickening outer wall of the metacestode and a fixed pattern of underlying cells is apparent. Contractions become more marked suggesting functional contractile fibers in the subtegument as well. Presumably metamorphosis, i.e. resorption of oncospheral structures, now has been completed. The disposition of the oncospheral hooks varies among different metacestodes. The hooks may remain surficial or become embedded in the developing matrix. If surficial, they may become widely dispersed, either individually or in pairs. When deeply embedded, or if confined to the future cercomer, they may remain tightly clustered. There is no single pattern of hook distribution, however, and occasionally some end up on the surface of the future scolex, or a compact cluster may be well up the body of the metacestode. Surficial hooks may be so loosely attached that they fall off soon after the metacestode begins marked contractions. The hooks may be lost early in development, or at the opposite extreme they may become incorporated into the terminal segment of the young adult (Hickman, 1963). In all cases, however, they are relict reminders of the brief migratory role of the oncosphere; they have no further role in subsequent development of the cestode. Up to this point most metacestode development is remarkably similar, with incipient differentiation of the integument, but internally only an increase in the number of relatively similar cells. Many metacestodes continue growth as a compact mass of cells, but others develop a distinct cavity, the “primitive lacuna” (Fig. 3, A, E). Here this cavity will be called the primary lacuna, because it will be shown later that metacestodes with such a cavity have a more advanced type of development. Such cavities have been known for a long time (Leuckart, 1886, p. 360), but their formation must be distinguished from abnormal cavities which occasionally develop. For example, Kuczkowski (1925) describes the “lacuna primitiva” in Proteocephalus fificolfis.Subsequent study suggests, however, that proteocephalideans which develop cavities are abnormal (Freeman, 1964; Befus and Freeman, 1973). A primary lacuna may occur before or approximately at the time that body elongation begins. Such cavity formation, which may result from migration of cells to the periphery (Scott, 1965a), occurs among cyclophyllideans that mature sexually in reptiles, birds, or mammals, although not all cyclophyllideans have a primary lacuna. The metacestode body elongates and enlarges, and by this time irregular refractile calcareous granules of various sizes and numbers are usually evident. Their function is unknown (Smyth, 1969). They usually lie in the cortex of the fully differentiated metacestode, and may be so abundant they obscure the internal anatomy. Whether with or without a primary lacuna, differentiation may occur at either, or both, end@) of the metacestode.

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One end, usually the narrower, may ultimately become set off from the remainder of the body by a constriction and develop into a cercomer, or tail (Fig. 3, ByC , F). Presumably the cercomer is on the posterior end of the metacestode, since the future scolex or holdfast develops on the end opposite. This poses a conundrum, however, since the end of the oncosphere with the hooks and, when present, the openings of the “penetration glands” was the functional anterior end during locomotion, yet now the “head” develops on the opposite end. Such “reversal of polarity” (Stunkard, 1962, 1967) has precipitated much philosophical discussion (Mrhzek, 1916; Venard, 1938). Ogren (1968) concluded after extensive study of cestode embryogenesis that there is a true reversal of the metabolic gradient. He stated, however: “The primary embryonic polarity in the immature hexacanth is somatic, establishing basic cell patterns and differentiation needed for invasiveness. The secondary embryonic polarity in the invasive oncosphere is germinative, establishing the pattern for further development into the metacestode.” Thus according to him the reversal of metabolic gradient, hence polarity, has occurred before the oncosphere is activated and begins its migration. It follows that the oncosphere moves with the posterior end forward, and the end opposite, where the scolex will develop, is anterior. If one were very concerned about this matter, one might state that some cestodes reverse polarity twice, since, for example, the coracidium of Diphyllobothrium sp. exhibits a distinct antero-posterior axis during movement, and the hooked end of the oncosphere is posterior (Wardle and McLeod, 1952, p. 53; Freeman, personal observation), yet the oncosphere when liberated moves in the opposite direction, and finally when the scolex is developed the worm reverses direction of movement again. Voge and Heyneman (1957) discussed the development of Hymenolepis nana and H. diminuta and concluded : “To speak about polarity and reversal of polarity because of the direction of movement of the relatively undifferentiated oncosphere, as is done by Venard (1 938), is meaningless.” Development of the scolex end varies considerably. During exogenous development frequently the first sign of scolex differentiation is a single structure at the extreme tip. It may develop into a true sucker, or a highly glandularized structure, or to something with barbs, hooks, finger-like projections, or other features. This structure, called an end or apical organ, myzorhynchus, rostellum, or other names, may remain and function on the adult scolex, but it also may become highly developed and functional on the metacestode, only to be resorbed leaving little recognizable trace on the adult scolex, e.g. one or more species in the following genera: Catenotaenia, Testudotaenia, Mesocestoides, Proteocephalus, Acanthobothrium, Parachristianella, Corallobothrium (see respectively, Joyeux and Baer, 1945; Freze, 1965; Voge, 1967; Fischer and Freeman, 1969; Mudry and Dailey, 1971; Befus and Freeman, 1973). In some genera such a structure is the adult scolex (e.g. Nippotaenia, Litobothrium; Schmidt, 1970, p. 117). The scolex may complete all development without invagination or withdrawal into the body, or withdrawal may occur when the scolex is partly or completely differentiated. The scolex on most taeniid metacestodes differentiates

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at the end of an invaginal canal. In some genera, e.g. Echinococcus, Amoebotaenia, Paricterotaenia, the scolex develops endogenously (DCvC, 1949; Mathevossian, 1963; Scott, 1965a). A recognizable scolex frequently develops while the metacestode is in the site invaded by the oncosphere. Scoleces (preferred spelling, though “scolices” is commonly used in North American literature) may increase in size or modify in various ways in subsequent parenteral sites invaded by the developing metacestodes. Frequently scoleces do not reach the ultimate size and development recognized on the adult strobila until after the metacestode has entered a suitable gut lumen (e.g. Cladotaenia sp., Paruterina sp., Proteocephalus sp., and Echinococcus sp. : Freeman, 1957, 1959, 1964; Smyth, 1969), although others attain full size in the final parenteral site (e.g. Diglzyllobothrium spp., other pseudophyllideans, and Taenia spp. Kuhlow, 1953; Wardle and McLeod, 1952). Usually rostellar hooks develop to adult size and morphology in the first parenteral site (Wardle and McLeod, 1952), but not always (Jarecka, 1958). If the metacestode develops in more than one parenteral site, rostellar hooks may continue growth in each site (Jarecka, 1970b). It is uncertain whether scoleces increase in size after segmentation or proglottidation has begun, but it may occur occasionally, e.g. see Smyth (1969). As a rule scoleces of metacestodes which initiate development in invertebrates continue growth to adult size and form in a vertebrate, be it in a parenteral or enteral site. There are few experimental data on growth and development of marine forms, although numerous metacestodes have been recorded (Euzet, 1959; Dollfus, 1964b, 1967). Conceivably scoleces on metacestodes in the gut lumen of cephalopods and other large marine invertebrates approach adult size while still in such hosts. Other features of the metacestode body may change also. One obvious feature is the number and distribution of calcareous corpuscles, which are absent from the scolex. Another is the appendage-like cercomer which, if produced, is recognizable before scolex differentiation. It may be produced and quickly shed as in some proteocephalideans and cyclophyllideans. Such cercomers usually are compact, although some have cavities. In a few species the cercomer elongates considerably after the constriction separating it from the remainder of the body has formed. Rarely it forms another complete layer around the remainder of the metacestode (Fuhrmann, 1931, p. 388). When fully differentiated the cercomer may be attached to one side or dorsal to the excretory pore (Fig. 3, C), and occasionally it is attached at the inner end of the developing excretory vesicle. The cercomer may contain excretory ducts, e.g. in some hymenolepids and anoplocephalids (Braun, 1894-1900, p. 1614; Freeman, 1952), but usually it does not. In other species (e.g. Hymenolepis nana; Voge and Heyneman, 1957) there is no obvious excretory vesicle and pore at the point where the cercomer is attached to the body (Fig. 3, G , H). Between the cercomer and the scolex, the remainder of the body may remain a compact single unit, and within it may develop the characteristic parenchyma, musculature, nerve fibers, flame cells with associated ducts, gland cells, and calcareous corpuscles so typical of cestodes. The integument is much the same as on the remainder of the body. In other forms there may

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be noticeable thickening and other changes of the integument in the body anterior to the cercomer, and the parenchyma may be more sponge-like but without a cavity (e.g. Fig. 3 , Corallobothrium sp.; Essex, 1928). The body may be clearly divided into three parts which in Hymenolepis spp., Voge and Heyneman (1957) call the fore-body, mid-body, and hind-body or tail (Fig. 3), and the mid-body may incorporate the primary lacuna and remain hollow. Similar body divisions are apparent in Corallobothriirrn sp. (Fig. 3), but the mid-body is not hollow because there is no primary lacuna. The body wall surrounding

FIG. 3. Metacestode development of Coraflobothriunz sp. with cercomer and without primary lacuna (after Befus, 1972, and original), compared with Hymenolepis sp. with cercomer and with primary lacuna (after Voge, 1960b and Voge and Heyneman, 1957). Corallobothrium sp.: A. Elongate, solid ball of cells; B. Early cercomer formation; C . Three-part body: fore-body with scolex before withdrawal, mid-body with terminal excretory vesicle and pore, and cercomer; D. Scolex fully invaginated into fore-body and mid-body, and cercomer gone. Hymenolepis sp.: E. Hollow ball of cells; F. Elongate body with incipient cercomer; G . Three-part body: fore-body with scolex, mid-body modified but no excretory vesicle and pore, and cercomer; H. Scolex retracted and invaginated fore-body withdrawn into hollow mid-body, no excretory vesicle and pore, cercomer attached to mid-body; I. Longitudinal section showing retracted scolex invaginated into fore-body, and cyst-like nature of mid-body wall. Scales: A=50 pm; same for B through H; I=50 pm.

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50 I

the mid-body may thicken and become multilayered, taking on a “cyst-like” appearance (Voge and Heyneman, 1957) after the scolex is retracted within this cavity. It is apparent, however, that only the “tail” of Hymenolepis sp. is equivalent to the cercomer of Corulfobothriumsp. (Fig. 3). In yet other groups of metacestodes a recognizable cercomer is not delimited (Catenotueniu sp.; Joyeux and Baer, 1945), and frequently in these the oncospheral hooks are clustered on the end opposite the scolex (Cladotuenia sp., Marsipometra sp., Oochoristica sp. : Freeman, 1959; Meyer, 1960; Hickman, 1963), although the hooks may be either dispersed or clustered elsewhere (e.g. “Cysticercus urionis” ; see Liihe, 1910, p. 146). Cercomerless metacestodes which develop without a primary lacuna have a scolex on a compact body and present the appearance of an unsegmented cestode (Proteocephalus sp., Cladotaenia sp.). Others may have a primary lacuna in the end opposite to the scolex, in which case the body wall may become modified and “cyst-like” ; when fully differentiated such metacestodes may have the scolex within this cavity (Oochoristica sp., Tueniu sp.). It is selfevident that there is no cercomer on such metacestodes, yet unaccountably

C

D

PIG. 4. Various positions assumed by scolex of Ophiotueniusp. (after Herde, 1938). A. With cercomer before scolex withdrawal into compact mid-body. B. With cercomer and with scolex invaginated into fore-body and fore-body withdrawn into mid-body. C. Without cercomer, scolex withdrawn (retracted) into mid-body. D. Without cercomer, scolex invaginated into fore-body and mid-body. Scales: A, B=50 pm; C, D=50 pm.

502

R E I N 0 S. F R E E M A N

some workers (e.g. Mrazek, 1916; Jarecka, 1970c, d) consider that since the cyst-like portions of those metacestodes are shed, e.g. Taenia sp., they must be cercomers. This will be discussed more fully below. Retraction (withdrawal) of the scolex (Davainea sp., Choanotaenia sp. : Abdou, 1958; Voge, 1961) or invagination (Oochoristica sp., Taenia sp. : Hickman, 1963; Voge, 1967) frequently is considered of phylogenetic importance. The scolex is, however, quite labile, and there is increasing evidence that in some species the scolex of the metacestode may evert and retract as need be (e.g. corallobothriins; Befus and Freeman, 1973), or assume variable positions (Ophiotaenia sp.; Herde, 1938 (Fig. 4,A, B, C, D)), and that scoIeces of metacestodes of some higher cyclophyllideans such as hymenolepids presumably evert, function, and withdraw again (Ryzavj, 1961). The development, growth, and final disposition of the excretory ducts, vesicle and pore(s) on the differentiated metacestode have received little consideration by previous workers. Part or most of such a system may be differentiated before the scolex is recognizable, e.g. Diphyllobothrium sp., Catenotaenia sp., Acanthobothrium sp., and Lacistorhynchus sp. (see Vogel, 1930; Joyeux and Baer, 1945; Mudry and Dailey, 1971, respectively); or it may not be evident on the fully differentiated parenteral metacestode (e.g. Oochoristica deserti; Millemann, 1955). The system may be seen best, however, on the metacestode immediately prior to segmentation or proglottidation especially in a living metacestode in a hypotonic medium. The longitudinal ducts vary in number (two to many) and distribution, but frequently there are a dorsal and a ventral pair. One pair may be medullary and the other cortical in position; the latter may be in the form of a network. Commonly the system empties to the outside via a posterior vesicle and pore. Frequently only the ventral ducts open into the excretory vesicle (e.g. Proteocephalus sp.), but sometimes the dorsal ducts do. likewise, or a complex of ducts may appear to attach to the vesicle. There may be secondary ducts and pores opening to the outside elsewhere on the body from the cortical network (Diphyllobothrium sp.). In one genus at least (Corallobothrium; Befus and Freeman, 1973) such secondary ducts and pores appear on the final metacestode and replace the terminal vesicle and pore characteristic of the penultimate metacestode form. In others (Taenia spp.; Rees, 1951) there may be more of a network of ducts and secondary pores in the wall of the metacestode bladder, which are replaced by a single terminal vesicle and pore on the cestode body after the bladder is shed in the gut of the final host. The ducts may be difficult to see and follow. The ultimate metacestode in the gut lumen of the final host usually has a terminal excretory vesicle and pore when sexual maturation begins. This vesicle and pore may develop on the posterior end, the primary position, of such metacestodes as Catenotaenia sp., Cladotaenia sp., Marsipometra sp., and Oochoristica sp. (Joyeux and Baer, 1945; Freeman, 1959; Meyer, 1960; Hickman, 1963), between the cercomer and mid-body, the secondary position, as in Diphyllobothrium sp. or Proteocephalus sp. (Kuhlow, 1953; Freze, 1965), or further up the body, frequently at the juncture of the fore-body and midbody, the tertiary position (Goodchild and Harrison, 1961 ; Freeman and Webb, personal observations). Adult cestodes shed the excretory vesicle and

O N T O G E N Y O F CESTODES

503

pore at first apolysis (loss of segments), which may occur soon after segmentation begins, or considerably later. The terminal part of some metacestodes, e.g. Spirometra sp., may be shed before regular segmentation is evident (Mueller, 1972). This may be equivalent to first apolysis, since, as with the adult in the vertebrate gut, there is no evidence that a new excretory vesicle and pore regenerates (Mueller, 1972). This may, however, represent a pattern of growth not typical for most cestodes, although apparently somewhat similar shedding of the posterior end occurs with certain other pseudophyllideans (Joyeux and Baer, 1938a, b).

2. The terminology of cestode ontogeny The argument developed so far is that there are three major stages in the life-cycle of each species of cestode: the oncosphere, metacestode, and sexual stage. Furthermore, the time required to complete each stage varies with species and with the site in which they develop. The only constant is the entry of the oncosphere into a parenteral site where metamorphosis occurs, The metacestode differentiates and grows in one or a series of hosts; ultimately proglottidation indicates the onset of sexuality. Sexuality may be initiated in the final parenteral site, although it usually begins in the gut of a suitable vertebrate. Sexual maturation and a new generation of eggs follow (Fig. 1). Metacestodes of different species show various degrees of development in particular hosts. For example, Archigetes limnodrili may complete metacestode and sexual development parenterally in the polychaete first host (Kennedy, 1965). Hymenolepis nana, although superficially similar in that it can complete the entire life-cycle in one host, nevertheless has a typical cycle, since parenteral development first is completed in the intestinal wall or elsewhere outside the gut, followed by sexual maturation in the gut lumen of the same host (Voge, 1967; Weinmann, 1969). At the other extreme, limited development occurs in each of a sequence of four hosts, e.g. Diphyllobothrium spp. (see Vik, 1964). Freeman (1970) recognizes two types of metacestode development, viz. primitive when complete ontogeny occurs without a primary lacuna, and neoteric, i.e. recent, when a primary lacuna develops, whether it ultimately is incorporated into the fully differentiated metacestode or is resorbed. These are roughly equivalent to the gymnosomic and cystosomic developmental patterns proposed by Jarecka (1970~). As might be expected, a complete gradation of neoteric metacestodes can be demonstrated, from those in which the cavity is resorbed before the metacestode is fully differentiated (e.g. Dipylidium sp.; Marshall, 1967) to the opposite extreme where the cavity enlarges and the ensuing bladder becomes the dominant feature of the metacestode (e.g. Tuenia spp.). Metacestodes of trypanorhynchs, as far as is known now, lack a primary lacuna during early development (Riser, 1956; Mudry and Dailey, 1971), but some advanced trypanorhynchan metacestodes may have a cavity called the blastocyst (Wardle and McLeod, 1952). This will be considered a secondary lacuna until it is known how and when it develops. Obviously, to describe the sequence of metacestode development, it is necessary to decide whether each phase of development should have: (a) a different iiame i n each host in which the metacestode occurs; or (b) a distinct

TABLE I Nanzes used fo describe ontogeny of cestodes with primitive developnient

VI

0 P

Hosts and Names Order and Species

Egg

First

Second

Third

Fourth

Author

PSELDOPHYLLIDEA

Diphyllobothriirtn sp. hfarsiponietra hastafa Bothriocephaliis spp. Eirbothrium salveliiii

Operculate Coracidium

Procercoid Procercoid Coracidium Plerocercoid Coracidium Procercoid 7Procercoid Nonoperculate Coracidium-Procercoid Oncosphere -Plerocercoid -I

-

~

Plerocercoid Plerocercoid I Adult Adult Young tapeworms-Adult

Adult Plerocercoid XI Adult

Kuhlow, 1953 Vik, 1964 Meyer, 1960 Jarecka, 1959, 1964 Thomas, 1937

Adult Plerocercoid 11-Adult

Vik, 1963 Present study

Plerocercoid-Adul t Plerocercoid 11-Adult Plerocercoid 11, a,b,c -Adult Procercoid-Plerocercoid Plerocercoid-Adul t

Freze, 1965 Fischer, 1968 Fischer and Freeman, 1973 Hunter, 1928 Thomas, 1941

PROTEOCEPHALlDEh

Nonoperculate Pmteocephahis filicollis Oncosphere Procercoid Proreocephalrisfliiviati/is Oncosphere Plerocercoid IProteocephaliis middop/itis Oncosphere -Plerocercoid 1-

-

Ophiotaenia spp.

CYCLOPHYLLIDEA

Oncosphere

Procercoid Procercoid Caudate procercoid Caudate cysticercoid Ecaudate cysticercoid-Plerocercoid-Adult Plerocercoid Plerocercoid

7

~

Nonoperculate Plerocercus b'ulipora canpylancristrota Oncosphere -Cercoscolex Oncosphere -PlerocercoidPlerocercoid-Adul t Clndotaenia spp. Cladothyridium -Adult MerocercoidPlerocercoid-Adul t Oncosphere Catenotaerriapusilla

-

Adult

Adult Adult

Freze, 1965 Mead and Olsen, 1971 Jarecka, 1970a Freeman, 1959 Abuladze, 1964 Joyeux and Baer, 1945

ONTOGENY OF CESTODES

505

name, regardless of their morphology to correspond with different orders of cestodes; or (c) a name reflecting the type of host, e.g. invertebrate versus vertebrate; or (d) the same name for various developmental phases which are basically similar regardless of the type of host, classification, or sequence of development. Ideally, the name should indicate all these aspects, although indicating the type of host is less meaningful since, with the possible exception of the bladder-like taeniids, the major types of metacestodes apparently occur in a wide range of vertebrates and invertebrates. (a) Primitive development. The jumble of names used to describe primitive development is illustrated for a selected number of life-cycles from three orders of cestodes (Fig. 5 and Table I). The oncosphere of all these cestodes invades a parenteral site, metamorphoses, and grows and differentiates as a metacestode. Some move to the gut of the final host, after development in the initial site, others move through one or more parenteral sites with varying degrees of growth in each site. Thus, little or great change and growth is necessary to complete metacestode development in the gut of the final host. Many workers virtually ignore the fact that most metacestodes complete their growth in the gut lumen, apparently assuming that once the metacestode is in the final site the cestode quickly initiates “adult” growth; some do (Hutchison, 1959), others do not (Archer and Hopkins, 1958). Actually identification of certain tetraphyllidean plerocercoids is difficult because the plerocercoid scolex modifies greatly in the definitive site (Wardle and McLeod, 1952). This is also evident with certain proteocephalideans (Befus and Freeman, 1973). In the examples (Fig. 5), the fully differentiated metacestode would have an adult scolex on an unsegmented body with a terminal excretory vesicle and pore. This is a plerocercoid ready to mature sexually, i.e. begin proglottidation. A plerocercoid is recognizable in the first host with all but Diphyllobothrium sp. and Cutenotueniu sp., of the species illustrated (Fig. 5). The scolex of Diphyllobothriurn sp. is not recognizable until after development in the second parenteral site, and the metacestode may grow further in at least one more parenteral site before final development, if any, to a full-sized plerocercoid in the gut of the final host. Catenotueniu sp. apparently is the opposite extreme, since the metacestode completes major development in the gut of the second host. These examples suggest the extremes at which a metacestode may become competent to establish in the gut lumen of the final host. Development of both Diphyllobothrium sp. and Cutenotaenia sp. is arrested in the first host before an identifiable adult scolex develops. The metacestode may develop frontal glands permitting it to leave the gut of the next host in order to continue development parenterally (Diphyllobothriurnspp. ; Vik, 1964), or it develops a sucker-like organ of attachment permitting it to remain in the gut of the final host (Catenotaeniu sp.; Joyeux and Baer, 1945). This initial development of the scolex dictates the events that can follow, whereas the cercomer, when present, is not involved in any obvious way. Metacestodes which undergo only limited development in the first site occur in some genera in all the major orders indicated by Voge (1969). It is proposed that such primitive metacestodes, which develop neither n primary cavity nor a scolex

HOSTS AND

Genus

First

(authority1

Parenteral

SITES md

Parenteral

Enteral

I I

Diphyllobothrium sp. (Kuhlow, 1953; V1k,1964)

I

Third Parenteral

1

I

Enteral

Fourth Enteral

I

,+

Marsipometra sp.

...

(Meyer, 1960)

Bothriocepholus sp. (Thomas, 1937; Jorecka, 1959,1964:

Eubothrhm sp. (Vik, 1963; present study)

Proteocephalus filicollii (Freze, 1965)

-*Lo- .+

Proteocqhalus fluwatiis (Fischer, 1968) Proteoephalus amblopiitis ( Fischw, 1972)

-?

0

E

Ophiotaenia sp. (lhornus,1941; Mead &Olse 1971

Volipora sp, (Jorecka, l970a) Cladotaenio sp. (Freeman, 1959)

Catenoioenio sp. (JoyeuxB BaerJ945)

I

ONTOGENY OF CESTODES

507

recognizably similar to that of the adult in the first parenteral site, collectively be called procercoids in the sense of the term originally proposed by Janicki and Rosen (1917). A descriptive prefix can be added to indicate the nature of the anterior end, and the adjective caudate or acaudate would indicate presence or absence of a cercomer. Thus caudate uniacetabuloprocercoid describes Mesocestoides sp. (see Voge, 1969) and acaudate uniacetabulo-procercoid is appropriate for Cateizotaenia sp. (Fig. 5 ; Joyeux and Baer, 1945), Acanthobothrium sp., and Lacistorhynchus sp. (see Mudry and Dailey, 1971). Caudate glando-procercoid describes Diphyllobothrium sp. (Fig. 5 ; Kuhlow, 1953), and acaudate glando-procercoid is appropriate for Tylocephalum sp. (see Cheng, 1966). If, however, the metacestode continues development in the first site until a recognizable scolex develops, then there is little point of referring to intervening development as other than that of a differentiating metacestode. If procercoid is the appropriate term for a metacestode which does not develop a scolex identifiable with that of the adult in the first site of development, then obviously a metacestode that does develop such a scolex requires another name. Furthermore, procercoids also grow and produce a scolex in the next site, and they require another name to indicate the new level of development. In the examples given (Fig. 5), the metacestodes all are plerocercoids as defined above, yet a variety of names are used to describe them (Table I). Each has a scolex with suckers or bothria; others may have bothridia, or tentacles (=proboscides) (Wardle and McLeod, 1952), and some may retain a cercomer. The tip of the scolex may have no obvious structure, or it can be a swelling, or there may be an end organ such as a sucker, gland, rostellum with hooks, or conceivably as many or more variations as described for adults (Wardle and McLeod, 1952). The scolex or its parts may be extended as in the adult, or invaginated, or withdrawn (i.e. partly invaginated and retracted) into the anterior end of the body (Fig. 5). Fuhrmann (1931) uses, albeit inconsistently, the term plerocercoid if the scolex is everted, and plerocercus or cysticercoid for morphologically similar metacestodes with invaginated or withdrawn scoleces; Freze (1965) maintains that proteocephalideans in the first host are procercoids regardless of morphology (also the position of Wardle and McLeod, 1952, pp. 64-65), unless the scolex is pulled into the body, in which case Freze calls it a cysticercoid (Table 1). Plerocercoids, Freze maintains, develop in subsequent hosts. Jarecka (1970a) proposes the name cercoscolex for forms similar to what Freze calls cysticercoids, provided they have a cercomer, but whether or not the scolex is withdrawn into the body. Jarecka uses the term plerocercus once the cercomer is lost and if suckers are present, retaining the term plerocercoid only for those forms which have a scolex with bothria; Abuladze (1964) uses cladothyridium or possibly armatetrathyridium for the metacestode which Jarecka (1970a) FIG.5. Diagrammatic presentation of ontogeny from egg to proglottidation of 11 representative cestode genera or species, from three orders, which undergo primitive development; sequence of sites, and significant stages and phases of development are shown. Authorities for information are in left-hand column. Drawings are free-hand, scales where indicated- 100 wm.

508

R E I N 0 S. F R E E M A N

calls plerouerciis and t'recman ( I Y57) calls plerocercoid (Table 1). Obviously there is disagreement as t o what these various names signify! Consequently a more consistent but descriptive pattern of naming will be suggested below, It was shown above that the scolex, particularly on primitive metacestodes, is an active, mobile structure which may assume various positions (Fig. 4, A, B, C , D; Herde, 1938), and may evert and function, and may or may not be TABLE I1 Genera based primarily on metacestodes Primitive Development

Neoteric Development

Acnnthorhynchus Diesi ng Anthocephaliis Rudolphi Balanophorus Briganti Bothriorhynchus Lidth de Jeude Cephalocotyleum Diesing Cestoscolex Parona Corynesomn F. S. Leuckart Dithyridium Rudolphi Dubium Rudolphi Floriceps Cuvier a , b ~ d Glossocercirs Chandler Gryporhynchus Nordmannaeb,c,'l Cymnorhynchus Rudolphiash*c Gymnoscolex Diesing Hepatoxylon B o s c ~ . ~ . ~ , ~ Hydatis Larnarck Ligula Blocha,b.c,d Merocercus Petathyrus Cobbold Piestocystis Diesing Plerocercoides Neumann PIerocercus Braun Pseudoscicus Pol. Pterobothrium Diesinga~h~cJ Scolex Miiller Slossia Meggitt Sparganum Diesing Spirometra Diesing a,b Steganobothrium Diesing Stenobothrium Diesing Tetrastoma Forb, and Goods Tetrathyridium Rudolphi Tetrathyrus Creplin

Acanthotrins Weinland Acephalocystis Ludersen Alveococcus Abuladzee Astoma Gairdner and Lee Cercocystis Villot Coenurus Rudolphi Cryptocystis Vil lot Cysticercoides Blanchard Cysticercus Zeder Cystotaenia Leuckart Discostoma Gairdner and Lee Echinococcus Rudolphia8bBcJ.e Finna Werner Hydatigena Bloch Hydatigera Lamarckalb,e Hydatula Abildgaard Hygroma Schrank Monocercus Villot Multiceps G0ezea.b.e Neotaenia Sodero Polycephalus Zeder Polycercus Villot Reditotaenia Sambon Staphylocystis Villotb Stenotaenia Gervais Tetratirotaenia Abuladzee Trachelocamphylus Frtidault Urocystidium Beddard Urocystis Villot Vesicaria Miiller

BConsidered valid by Wardle and McLeod, 1952. b Considered valid by Yarnaguti, 1959. C Considered valid by Joyeux and Baer, 1961. * Considered valid by Schmidt, 1970. e Considered valid by Abuladze, 1964.

ONTOGENY OF CESTODES

509

withdrawn again (Fig. 5; Fischer and Freeman, 1969, 1973; Jarecka, 1970b). Freeman (1957) stated earlier that the degree of invagination or withdrawal, or whether this occurs at all among cestodes undergoing primitive development, is less important that the morphology of the body in which this occurs. This conclusion still appears valid. The confusion associated with the use of names is rooted in the history of their origin and application. Initially as metacestodes were discovered, many were considered as new, and frequently new genera were created to receive them. By the turn of the present century approximately 70 names already were proposed for various types of metacestodes (Braun, 1894-1900). Since then at least another 25-30 names have been proposed. Some names were and still are used in the generic sense (Table 11), others as collective names for groups (Table IV). Braun (1894-1900, p. 1568) maintained that until more is known it probably would be simpler to relate intermediate types to plerocercoid, plerocercus, cysticercoid, cysticercus or coenurus, although he did hold that when more is known it may be necessary to classify further the different kinds of metacestodes according to their development. These names, other than plerocercoid, have been used quite loosely, but usually in association with metacestodes which develop a primary or secondary lacuna, which may or may not be apparent in the differentiated metacestode. Accordingly it is suggested that the term plerocercoid be restricted to metacestodes which undergo primitive development, including all examples in Table I and Fig. 5, whether they occur in parenteral or enteral sites. Since it is the scolex which differs most characteristically, it is reasonable to use the root word plerocercoid for the type of body, with suitable descriptors as prefixes to describe the scolex. Whether the scolex is invaginated or withdrawn and the presence or absence of a cercomer can be indicated by appropriate adjectives. Thus, acetabulo-, bothrio-, bothridio-, tentaculo- would indicate an ordinal relationship, and other prefixes, possibly gland(o)-, acanth(o)-, or culcit(o)(=pad or cushion) etc., are suitable descriptors of the scolex, e.g. caudate uniacetabulo-plerocercoid for the genus Nippofaenia (see Yamaguti, 195I), acaudate invaginated acanthacetabulo-plerocercoidfor the genus Cladotaenia, or acaudate invaginated glandacetabulo-plerocercoid for differentiated Proteocephalus ambloplitis in the copepod. The metacestode of Diphyllobothrium sp. becomes an acaudate bothrio-plerocercoid, and invaginated may be used if appropriate. A parenteral diphyllobothriid metacestode which is segmented probably has initiated sexual maturation, but to indicate its parenteral location the term bothrio-strobiloplerocercoid is suggested for it. A single name may not, however, account for those metacestodes that move through a sequence of sites in one or more hosts. If there is a significant change in the scolex, such as growth of, or resorption of, some structure during such movement, this can be indicated. For example, Corallotaenia minutia is an acaudate invaginated glandacetabulo-plerocercoidwhen fully differentiated in the haemocoel of a copepod (Fig. 3), but changes to an invaginated acetabuloplerocercoid in tissues of a fish, and in the gut of the definitive host becomes a metacetabulo-plerocercoid (i.e. develops a metascolex and modified excretory system) before proglottidation (Befus and Freeman, 1973). Such names 19

510

R E I N 0 S. F R E E M A N

are more meaningful than the convention of using plerocercoid I and I1 as done previously (Befus and Freeman, 1973). One also faces the problem that plerocercoids of other species may occupy one or more sites i n sequence, with little obvious morphological change. In such cases the convention of using I, 11, etc. may be necessary (Table I, acaudate culcitacetabulo-plerocercoid I and I1 for Proteocephalus parallacticus and P . Jluviatilis; see Freeman, 1964, and Fisher, 1968, respectively; or bothrio-plerocercoid I and I1 for Diphyllobothrium spp. in one or more fish; see Vik, 1964).P . ambloplitis has yet another sequence of metacestode forms; acaudate invaginated glandacetabuloplerocercoid in the copepod haemocoel, invaginated glandacetabuloplerocercoid I1 in the tissues of one or more fish, including the final host; finally in the gut lumen of the final host it becomes an acetabulo-plerocercoid before proglottidation (Fischer and Freeman, 1973). Apparently none of the acetabulo-plerocercoids initiates segmentation or proglottidation while in parenteral sites. Metacestodes which undergo primitive development generally produce a single scolex from each oncosphere, although instances of asexual reproduction, i.e. polycephalism or several individuals from one oncosphere, are known. This is true of Echinobothrium sp., which develops a number of buds from a central body, and at the tip of many a scolex retracted into a solid body is evident (Dollfus, 1964a). Recently Specht and Voge (1965) and Novak (I 972) described asexual reproduction by acetabulo-plerocercoids of Mesocestoides sp., in which an individual divides by longitudinal binary fission beginning at the scolex end. This is a regular process since few individuals produce a large population in a matter of a few months. Another well documented example is that of Spirometra sp. (=Sparaganurn sp. ; see Mueller, 1938), in which bothrio-plerocercoids produce lateral buds. These buds can separate and in turn develop more buds, but apparently true scoleces have not been found on them and they cannot mature sexually. Mueller (1972) stated some 34 years later that Sparganum proliferum, “. . . is essentially a budding and fragmenting ‘tail’ in which the scolex is lacking.” It is of interest that Mesocestoides sp. also produces budding fragments which lack scoleces (Novak, 1972). The above scheme can be used to name all known metacestodes which undergo primitive development, and which develop a difossate (e.g. pseudophyllidean) or tetrafossate (e.g. proteocephalidean, tetraphyllidean, and cyclophyllidean) scolex on the adult, except possibly the poorly known trypanorhynchs. Evidence to date (Riser, 1956; Heinz and Dailey, 1971; Mudry and Dailey, 1971) suggests that trypanorhynchs do not develop a primary lacuna. Presumably the blastocyst, i.e. the posterior cavity, which may form in some (Wardle and McLeod, 1952, p. 296) develops later. The term plerocercus is relatively widely accepted for such metacestodes, although not by Fuhrmann (1931) or Jarecka (1970a). Plerocercus would include most or all forms in Cystidea Guiart, 1927 (see Schmidt, 1970) or Thecophora Dollfus, 1942 (see Wardle and McLeod, 1952). Again the same system of naming proposed for plerocercoids could be used to delineate more precisely the type of plerocercus. There is reason to believe that, unlike many plerocercoids,

O N T O G 8 N Y O F CESTODES

51 I

during transfer to a new site (or sites) the trypanorhynch plerocercus may shed the blastocyst part of its body and develop a new excretory pore and vesicle (Wardle and McLeod, 1952, p. 297). Such a new secondary plerocercoid, although morphologically similar, is not equivalent to the primary plerocercoids described earlier. Therefore to avoid confusing the two, the term neoplerocercoid is suggested for it. Suitable descriptors again can be used to describe the scolex end. Whether or not some tetraphyllideans shed part of the initial metacestode body and develop a new posterior excretory vesicle and pore is not certain, although this is implied by Wardle and McLeod (1952, p. 197). Should this occur, such metacestodes would be neoplerocercoids as well. Cestodes usually included in the orders Caryophyllidea (see Mackiewicz, 1972), Spathebothriidea and Nippotaeniidea (see Wardle and McLeod, 1952) are difficult to classify and relate to other cestodes. Yamaguti (1959) placed the caryophyllideans, which lack a typical scolex and are monozoic, i.e. with only a single proglottid, in the subclass Cestodaria. In all other respects, however, they appear more closely related to the Eucestoda, and frequently are placed close to the spathebothriideans or pseudophyllideans, depending on the authors. The scoleces on the adults of all these forms are neither obviously di- nor tetrafossate (Fig. 6), although some caryophyllideans may

FIG.6. A. Caudate postplerocercoid of Caryophyllaects sp. B. Caudate monozoic adult of Archigefes sp. C. Caudate strobilate adult of Cyathocephalus sp. (A and B after Mackiewicz, 1972; C. after Wiiniewski, 1932). Scales: A, C= I mm;B-0.1 mm.

512

R E I N 0 S. F R E E M A N

have acetabula, loculi, bothria, etc. (Mackiewicz, 1972). Nippotaeniideans and some spathebothriideans have a single or modified terminal sucker or funnel as a scolex; the genus Spathebothrium has no obvious scolex. Caryophyllidean and spathebothriidean eggs are operculate, but the larvae are non-ciliated and do not escape until the eggs are ingested. Oncospheres of Cyathocephalus sp. may lack hooks (WiSniewski, 1932). Nippotaeniidean eggs resemble those of proteocephalideans and tetraphyllideans, and the oncosphere develops into elongate “procercoids” in Diaptomus sp. The oncospheres of caryophyllideans invade and develop in the coelom or tissues of aquatic annelids, whereas those of spathebothriideans develop in the haemocoel of amphipods (Cyathocephalus sp. and D@locotyle sp. : WiSniewski, 1932; Stark, 1965). In both groups some metacestodes develop in the first site until gonads and even eggs are present; they produce and retain a cercomer (Fig, 6). Several species of the caryophyllidean genus Archigetes can complete their life-cycles without a stay in the vertebrate gut, although most caryophyllideans apparently require at least a short stay in the gut of a fish for final maturation and egg production. There is considerable controversy whether such caryophyllideans and spathebothriideans are “procercoids” (Wardle and McLeod, 1952; Mackiewicz, 1972) or “plerocercoids” (Stark, 1965). Kennedy (1965) and most workers assume that such a metacestode is neotenic or progenetic, i.e. a “sexually mature larva” (see Mackiewicz, 1972, p. 490), which presupposes that the true adult stage has disappeared. Rosen (1918), on the other hand, considered such cestodes primitive and not neotenic adults. Rosen will be followed here, and the spathebothriideans and caryophyllideans which develop functional gonads while in the parenteral site (WiSniewski, 1932; Mackiewicz, 1972) will be considered caudate adults. For closely related species, e.g. some caryophyllideans (see Mackiewicz, 1972), or for some pseudophyllideans, e.g. Ligula sp., which develop genital primordia but do not mature fully while in the parenteral site, the term postplerocercoid is suggested, since they must move to the gut of a vertebrate for gametogenesis. Again suitable descriptors can be used as prefixes should they be required. The importance of these cestodes will be discussed further when cestode evolution is considered. (b) Neoteric development. Apparently all cestodes which undergo neoteric development, i.e. develop primary lacunae early during cestode differentiation, belong to the Cyclophyllidea, although not all cyclophyllideans undergo neoteric development. Most neoteric forms initiate development in either freshwater or terrestrial invertebrate hosts. A few poorly known species of marine trypanorhynchs have a posterior cavity, the blastocyst, which might be construed as a primary lacuna, but in no case has development of such a cavity been followed to establish its origin. A trypanorhynch for which data are available, Parachristianellu monomegacantha, initiates development, without a primary lacuna, in the haemocoel of a copepod (Mudry and Dailey, 1971). Nevertheless the most advanced metacestode they found had an “immature scolex” within an “anterior capsule-like portion”. The scolex-bearing body was attached by its posterior end to the inner posterior end of the capsule, which suggests that the “scolex” may have developed in a typical fashion and

O N T O G E N Y OF CESTODES

513

then been drawn into a compact body. If true, then this is similar to primitive forms already described. The report of Heinz and Dailey (1971) that the metacestode of the closely related Christianella sp. develops to a “plerocercoid” in a ghost shrimp supports this interpretation. Whether other trypanorhynchs have different ontogenic patterns remains to be determined. Endogenous budding in some marine metacestodes (tetraphyllideans ?) has been reported (Southwell and Prashad, 1918).This suggests that they may have a cavity as in neoteric metacestodes to be described below, although the illustration indicates that the buds grow within the parenchyma. This requires verification. Metacestodes which undergo neoteric development can be arranged to reflect increasing order of complexity, particularly if the sequence shows how the primary lacuna is incorporated into the fully differentiated metacestode. A representative series of such cyclophyllideans is shown in Fig. 7. The problem arises, how much of the metacestode is cercomer? It was established above that only some primitive metacestodes have a cercomer, and in some cases this is best determined during early ontogeny. Jarecka (1970c, d) maintains, however, that all parts of primitive and neoteric metacestodes which are not incorporated into the maturing strobila, including the “tail” and mid-body of metacestodes such as Hymenolepis sp. (Fig. 3; Voge and Heyneman, 1957), are ‘Lcercomer”.However, when the ontogeny of the primitive Corallobothrium sp. is compared with neoteric Hymenolepis sp. (Fig. 3), it is evident that the cercomer of the former and “tail” of the latter are homologous, and that the mid-body is part of the metacestode proper. With Corallobothriutn sp. the mid-body, the posterior part of the plerocercoid, is incorporated into the maturing strobila, whereas the equivalent part of Hymenolepis sp. is discarded and does not become part of the adult (Figs. 3, 7). Furthermore, the similarity between primitive metacestodes which develop no cercomer (Fig. 5; Marsipontetra sp., Cladotaenia sp., Catenotaenia sp.), and neoteric metacestodes with no cercomer (Fig. 7; Taenia sp., Oochoristica sp., Amoebotaenia sp.) is obvious. Again with primitive forms the entire metacestode becomes part of the maturing strobila, whereas with neoteric forms the posterior bladder may (Taenia sp.), or may not (Ooclzoristica sp.), be discarded before the strobila begins to mature (Fig. 7). This supports Fuhrmann’s, view (1931, p. 396) that only the tail-like appendage of certain neoteric metacestodes initiates and continues development, as does the cercomer of primitive cestodes. It is accepted here, therefore, that the cyst-like portion (mid-body of Voge and Heyneman, 1957) is part of the body proper and not the cercomer. Recently Goodchild and Davis (1972) called the tail-like appendage of Hymenolepis microstoma a cercomer, although earlier it was called a tail (Goodchild and Stullken, 1970). During early development of neoteric metacestodes the primary lacuna may divide, with part in the cercomer and part in the mid-body (Fig. 3; Monoecocestus sp., Hymenolepis sp. : Freeman, 1952; Goodchild and Stullken, 1970). Goodchild and Stullken (1970) refer to the resulting cavity in the mid-body where the scolex resides as the “primordial cavity”. The relationship of the developing scolex to the primary lacuna also varies. The scolex may difl’erentiatc partly (H~wicnolepistnirrostoiiia; Goodchild

514

R E I N 0 S. F R E E M A N

Davaenfu sp (Wetzel, 1932) Rufllfetma sp (Weizel, 1934.Sawod0, 1959, Voge,1960)

Monoecocestus (Freeman, 1952)

Hymenolepis sp (Voge 8 Heyneman, 1957; Schiller, 1959)

0

a Anornofaenin sp Aploparaksa sp ( Luhe, l9l0,Fuhrmann2 1931

Dfpylidfurn sp (Venard, 1938, Marsho11,1967

FIG. 7. Diagrammatic presentation of ontogeny from egg to proglottisation of 13 representative cyclophyllidean genera or species which undergo nwteric development; sequence of sites, and significant stages and phases of development are shown. Authorities for information are in left-hand column. Drawings are free-hand and not to scale.

IA e

IA

516

R E I N 0 S. F R E E M A N

and Stullken, 1970)or completely (hfonoccocrstusspp., H . diminutu: Freeman, 1952; Voge and Heyneman, 1957) on one end of the body, as during primitive development, before withdrawal into the body. This scolex withdrawal may be a complete invagination, so that the scolex becomes inside out (Oochoristica sp.; Hickman, 1963), or a partial invagination with the partly invaginated scolex then withdrawn into the body (Figs 3, 7; Hymenolepis sp.). Now the posterior part of the neck extends over the scolex producing a “reflected layer” (Goodchild and Davis, 1972), often containing calcareous corpuscles (Fig. 3). The resulting invaginal canal may remain patent (Oochoristica sp., H. diminuta) or be obliterated subsequently (H. microsroma). The body into which the scolex is taken may be: (a) “solid”, if the primary cavity has filled with tissue (Dipylidium sp.); or (b) a cavity with the body wall only slightly modified (Oochoristica sp.); or (c) the body wall may be more highly modified, so that when the scolex is fully withdrawn, the body wall is multilayered and the primary lacuna may or may not be evident (Fig. 7; Monoecocestus sp., Hymenolepis sp., Choanotaenia sp. : Freeman, 1952; Voge, 1960a, b, 1961 ; Goodchild and Davis, 1972). Such scoleces (Fig. 7) develop from teguniental and subtegumental tissues at one end of the metacestode, regardless of the extent of invagination. If subsequent function of the scolex is considered, obviously a fully invaginated scolex within the neck (the future scolex body) requires an invaginal canal in order to evaginate, e.g. Oochoristica sp., Taenia sp. (Fig. 7).If most of the scolex and neck within the reflected layer is not invaginated, and the invaginal canal is short, consisting primarily of the wall of the mid-body, then evagination of the scolex and body can occur within the cavity of the mid-body, as Schiller (1959) reported for Hymenolepis nana. He found that the fore-body moved freely within the mid-body while in the beetle (Fig. 7). Conceivably the fore-body of H. nuna does the same in parenteral metacestodes in mouse and man, and such reorganized metacestodes excyst, enter the host’s gut, and maturation follows. The existence of the primary lacuna and its involvement in differentiation of the metacestodes frequently is difficult to determine by study of the fully differentiated metacestodes alone, as pointed out for Choanotaenia crassiscolex by Rawson and Rigby (1 960). In many metacestodes, longitudinal excretory ducts are evident in the neck posterior to the scolex. The ducts extend posteriorly through this region, then loop into the reflected layer, extend anteriorly, and ultimately enter the outer cyst wall and continue posteriorly, terminating with an excretory vesicle and pore on the posterior end (e.g. Cysticercus arionis, Monoecocestus sp., Lateriporus sp. : Braun, 1894-1900; Freeman, 1952) (Figs 7, 8). In others the ducts may loop and enter the reflected layer but become difficult to follow, obviously not entering the outer body wall (e.g. Hymenolepis nana: see Voge and Heyneman, 1957). The fate of the cercomer also varies among caudate neoteric metacestodes. It may (a) be small and detach early (e.g. Raillietina cesticillus, Davainea proglottina: Wisseman, 1945; Abdou, 1958) (Fig. 7); or (b) be relatively short and broad, not well delimited from the remainder of the metacestode body (e.g. Hymenolepis nana; Voge and Heyneman, 1957) (Fig. 7); or (c) be

ONTOGENY O F CESTODES

517

extremely long (e.g. Drepanidotaenia bisacculina; Jarecka, 1960); or (d) even develop into a second envelope encircling the mid-body containing the typically invaginated and retracted fore-body (Fig. 7; Aploparuksis (=Haploparaksis=Haplopuruxis) sp.: Liihe, 1910, p. 143; Fuhrmann, 1931, p. 388). Undoubtedly other variations occur also. In another type of development somewhat modified from that just described, the scolex is again on one “end” of the differentiating metacestode, but the scolex develops while invaginated (e.g. Taenia sp., Fig. 7). In these forms the primary lacuna increases in size and modifies to a “bladder”, which becomes the dominant feature of the metacestode. The excretory system consists of four longitudinal ducts at the scolex end of the metacestode, but these ducts apparently form a network along each side of the bladder wall. A “terminal” excretory vesicle and pore, if present, cannot be differentiated from numerous other openings on the ends of short ducts extending to the tegument from the network of excretory vessels in the bladder wall (Rees, 1951; Taenia crassiceps: Freeman, personal observation). The bladder wall remains “single-layered”. As with certain primitive metacestodes, the body immediately posterior to the scolex may segment while still in the parenteral site (e.g. Taenia taeniaeformis, Lateriporus sp., Fig. 8). That the segmented fore-body of T. tueniueformis has begun adult development is suggested by growth studies reported by Hutchison (1959). Some neoteric metacestodes go further since they have several foci of scolex development. Hymenolepis cuntaniuna (see Jones and Alicata, 1935) has bud-like outgrowths along the length of a primary solid body, and on each bud a characteristic terminal scolex develops. There is no primary lacuna, but the parenchyma is a loose tissue so that a partly developed scolex partially invaginates and withdraws into the end, where differentiation is completed. These differentiated buds readily separate from the parent stem, and apparently are indistinguishable from other hymenolepid metacestodes. Pseudodiorchis prolifer develops buds in a similar fashion, but each bud produces a lacuna, and scolex development and withdrawal proceed as for typical hymenolepids (Kisielewska, 1960). It would seem to be a matter of semantics to argue that such lacunae are not primary lacunae, albeit of multiple form. Similarly, metacestodes with a large bladder and an invaginated scolex may increase in size, and numerous invaginated scoleces, rather than a single scolex, develop in the wall and extend into the enlarged primary lacuna, e.g. Taenia mustelae (Fig. 7), T. mirlficeps (see Wardle and McLeod, 1952, p. 84). Such multicephalic bladders may have from two to several hundred scoleces, in part depending on the species. Another modification is the production of numerous exogenous buds from the abscolex end (Tueniu crassiceps; Freeman, 1962) (Fig. 7) with a characteristic scolex developing on the distal end of each bud. The primary lacuna of the parent is not contiguous with the lacuna developing in each bud (Mujib, 1966). These new individuals may remain attached to the parent or detach and lead a separate existence as new budders, unlike the scoleces of Taenia mustelae. The latter metacestode bodies may fragment into smaller parts (Freeman, 1956) but

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this probably is not typical. These types of development suggest that some metacestodes retain truly embryonic cells which are multipotential. In all the preceding examples the scoleces, whether they develop while everted, after withdrawal, or while invaginated, are exogenous, i.e. oriented to the external surface of the metacestode. Many are ready to function merely by everting the scolex. In some, the wall of the metacestode must be breached before the scolex can function and continue growth. There are other types of neoteric development, however, where germinative tissue lines an enlarged primary lacuna, and an endogenous scolex, or scoleces, develops from it. In such forms (e.g. Amoebotaenia sp., Paricterotaenia paradoxa, Fig. 7 : Mathevossian, 1963; Scott, 1965a, b; and Echinococcus granulosus, Fig. 7; Smyth, 1967), growth and orientation of the scolex is into the lumen of the cavity, and occurs either while attached to the bladder wall or after the developing form is freed into the bladder lumen. Clearly the primary lacuna no longer is incorporated into the body of either type of daughter metacestode, yet each daughter develops in a fashion characteristic of species which do not undergo such asexual reproduction, but which otherwise appear related. When the little metacestodes of Paricterotaenia paradoxa are released from the parent cyst and the scolex is induced to evert (Fig. 7; Scott, 1965b), they are cercomerless metacestodes with a posterior bladder. Excretory ducts extend from the scolex through the wall of the bladder to a terminal excretory pore. The scolex-bearing fore-body quickly separates, however, and presumably a new terminal excretory vesicle and pore regenerate. Such bladders develop from secondary lacunae. Echinococcus granulosus presumably is somewhat similar, except that the daughter scolex and body (protoscolex) is compact. Although cell lines have not been followed, asexual reproduction by these two genera and Taenia crassiceps could be construed as polyembryony, in which multipotential germinative cells occur in various foci. Furthermore, the multipotentiality of some cells is retained even after a scolex is formed, since apparently some metacestodes with scoleces, e.g. Echinococcus granulosus, can dedifferentiate into a bladder under appropriate conditions (Smyth, 1967). Budding may continue for many generations (e.g. Taenia crassiceps; Freeman, 1962). A largely ignored aspect of neoteric development is that which the metacestode undergoes preceding maturation in the gut lumen of the final host. That the scolex excysts, attaches to the gut wall, and begins to mature, is taken for granted. Yet it is well known that for one or more days after ingestion, the pattern of growth is unpredictable (Goodchild and Davis, 1972). In part this is because the posterior end of the excysted and freed fore-body of the metacestode must reorganize and develop a new excretory vesicle and pore, whether development is like that of a hymenolepid (Goodchild and Harrison, 1961) or a taeniid (Leuckart, 1886; Joyeux and Baer, 1938c) (Fig. 7). This may prove typical of many neoteric metacestodes, albeit not all (Fig. 7). Obviously once the new excretory vesicle and pore are complete, the resulting metacestode has become a solid plerocercoid (Fig. 7). Before proglottidation the body or scolex may increase in size (e.g. five-fold with Monoecocestus sp. : Freeman, 1952).

TABLE III Names used to describe ontogeny of cestodes with neoteric development

Host and Names Species of Cyclophyllidea

First

Second

Author 0

Echinococcus sp. Taenia mustelae Taenia crassiceps

hydatid (with scolex or echinocoques) uniscolex and multiscolex larvae (bladders) cysticercus (budder)

adult adult adult

Oochoristica sp. Paricterotaenia sp. Amoebotaenia sp. Davainea sp. Raillietina spp. Monoecocestur spp. Hymenolepis spp.

cysticercoid mother cyst with cysticercoids cysticercoid cysticercoid cysticercoid cysticercoid cysticercoid

immatureadult adult adult adult adult juvenile-adult adult

Anomotaenia sp. Aploparaksis sp. Dipylidium sp.

cysticercus (= monocercus) cysticercoid or cysticercus cysticercoid

adult adult adult

D&6, 1949; Smyth, 1964 Freeman, 1956 Freeman, 1962; Bilqees and Freeman, 1969 Hickman, 1963 Scott, 1965a, b Mathevossian, 1963 Wetzel, 1932 Wetzel, 1934; Voge, 1960a Freeman, 1952 Voge and Heyneman, 1957; Schiller, 1959 Liihe, 1910 Liihe, 1910 Venard, 1938; Marshall, 1967

5d

n rn

z

0

a

520

R E I N 0 S. F R E E M A N

The names cysticcrcoid and cysticercus continue to be used most often in coiljunction with monocephalic neoteric metacestodes (Table HI), the latter for the family Taeniidae and the former for most other families (Voge, 1967). Multicephalic forms related to these basic types may have such names as urocystis, staphylocystis, polycercus, coenurus, hydatid, among others. The first three are considered to be related to cysticercoids, the latter two to cysticerci. The problem of names used for neoteric cestodes is the reverse of that for primitive cestodes. With the latter a jumble of names is used for fundamentally morphologically similar forms (Fig. 5, Table I), whereas with the former the same name frequently is used to describe widely differing morphological forms. Voge (1967) indicates, for example, that more than one basic type of metacestode is grouped under the broad term “cy~ticercoid~~. This is obvious for H . nana, Oochoristica vacuolata, and Dipylidium caninum (Fig. 7, Table 111), all of which are called cysticercoids (Voge and Heyneman, 1957; Hickman, 1963; Marshall, 1967). “Clearly, the comparative aspects of development have been neglected and further studies would be most helpful” (Voge, 1967), remarks which are reminiscent of Braun (18941900) ! The neoteric metacestodes just described occur only in certain families in the order Cyclophyllidea. They differ from primitive metacestodes described earlier by developing: (a) a primary lacuna; (b) an exogenous or endogenous scolex which typically is invaginated or withdrawn until further development in the vertebrate gut; (c) a body wall surrounding the scolex which frequently is modified into a multilayered structure or a voluminous bladder; and (d) an excretory vesicle and pore which may be primary, secondary, but frequently is tertiary in position on the differentiated metacestode in the vertebrate gut. Asexual reproduction is relatively common among neoteric metacestodes. Cestodes are most common and diverse among aquatic vertebrates, especially fishes (Wardle and McLeod, 1952; Yamaguti, 1959). It is reasonable to assume therefore that a changing pattern of morphological forms may be traced, progressing from those in fishes through amphibians and reptiles to birds and mammals. There would seem to be a logical sequence, for example, from a straightforward caudate or acaudate culcitacetabulo-plerocercoid (Fig. 5; Proteocephalusjlicollis; Freze, 1965, p. 42), through a caudate or acaudate invaginated glandacetabulo-plerocercoid (Fig. 5 ; Batrachotaenia ranae; Freze, 1965, p. 467; or Ophiotaeniaperspicua; Thomas, 1941), to a caudate or acaudate acanthacetabulo-plerocercoid(Fig. 7; D@yZidium canfnum; Marshall, 1967), and finally to the metacestode of Monoecocestus spp. or Hymenolepis spp. (Fig. 7; Freeman, 1952; Voge and Heyneman, 1957). However, a significant innovation has occurred in the latter two, namely, the development and incorporation of the primary lacuna into the metacestode so that the body wall provides additional protection to the scolex, and yet permits the future scolex to function whether the invaginal canal remains patent or not. As a consequence, unlike typical primitive plerocercoids, a significant part of the metacestode body, as well as cercomer when present, is discarded, and an excretory vesicle and pore regenerate at the end of the fore-

ONTOGENY OF CESTODES

52 1

body i n the tertiary position. The well established root term cysticercoid should be restricted to those metacestodes which undergo this type of neoteric development, and the term neoplerocercoid is suggested for the metacestode which develops from the fore-body in the definitive site. Cysticercoids are acetabulate, with or without a patent invaginal canal; some lack a rostellum (Anoplocephalidae; Wardle and McLeod, 1952); some have an unarmed or armed rostellum (Hymenolepis dirninuta, H . nana; Voge and Heyneman, 1957); in the examples given (Fig. 7) all have prominent cercomers. Formerly such a metacestode, if identified further, was called a cercocystis. It is proposed that descriptors can be used to define more appropriately such cysticercoids. They could be, respectively, a caudate arostello-cysticercoid, a caudate anacanthorostello-cysticercoid, and a caudate acanthorostello-cysticercoid. The cercomer may be very prominent, e.g. long, thick, or reflected back around the body proper as a sleeve, as in Aploparaksis sp. (Fig. 7; Fuhrmann, 1931, Fig. 420, p. 388), and these features also can be indicated by appropriate adjectives, e.g. longicaudate, megacaudate, oncericaudate, or even peri- or circumcaudate. The proper prefixes should be proposed by those most familiar with the groups. Using such adjectives and descriptors would define “cysticercoid” more precisely, and provide a mechanism of recognizing and comparing those of different kinds. In other neoteric forms the cercomer is small or transient (Fig. 7 ; Raillietina sp., Davainea sp.: Wisseman, 1945; Abdou, 1958) and the primary lacuna may be filled with “loose parenchymatous tissue” (Wisseman, 1945). Apparently there may be remnants of a secondary excretory vesicle and pore (Wisseman, 1945), but since no excretory ducts attach to such a structure, Voge (1960a) called it a “posterior fold”. Seemingly there is no concrete evidence that the ultimate davaineid metacestode is a neoplerocercoid with a tertiary excretory vesicle and pore, but by analogy with previous forms this seems likely (also see illustrations by Wisseman, 1945, and Sawada, 1959). In the past, such a metacestode, if identified further, was called a cryptocystis. However, to show the fundamental similarity between it and the one described above, the same basic terminology should be used with appropriate prefixes, e.g. brevicaudate micracanthorostello-cysticercoid. The metacestodes of various dilepidids (Fig. 7; Dipylidiurn spp., Choanotaenia spp.) and linstowiids (Fig. 7; Oochoristica spp.) which have a primary lacuna early in development also have been called “cysticercoids”, although differing from cysticercoids described above. The primary lacuna fills with a parenchymatous matrix during early development of Dipylidiurn caninum (see Marshall, 1967). According to Horsfall and Jones (1937) the same occurs with Choanotaenia infundibulurn, although Voge (1961) found a patent lacuna in the fully differentiated metacestode of this species, as Rawson and Rigby (1960) did for C. crassiscolex. D . caninum has a deciduous cercomer, as does C. infundibulurn (see Marshall, 1967; Horsfall and Jones, 1937), although Rawson and Rigby (1960) did not observe one with C. crassiscolex. After loss of the cercomer, the metacestode of D . caninum has a scolex invaginated or evaginated on one end, with four ducts associated with an excretory vesicle and pore on the other end, i.e. in the secondary position. Obviously

522

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this is a true plerocercoid (Fig. 7). All of the metacestode begins adult development, but apolysis occurs soon after segmentation begins (Venard, 1938). To indicate both the transient primary lacuna and the basic plerocercoid morphology, this metacestode is named a neoteric, acaudate, invaginated acanthacetabulo-plerocercoid. Some apparently closely related metacestodes (Fig. 8, B, C ; Lateriporus sp.; Denny, 1969; also see Liihe, 1910, p. 146) have a segmented fore-body with a well developed excretory vesicle and pore with associated ducts at the posterior end of the bladder. It is not known if there is a cercomer or how much of the metacestode becomes the adult. Therefore it cannot be determined whether such a metacestode is a cysticercoid. Provisionally for such metacestodes, including those of the genera Tatria and Schistotaenia which seemingly are similar (Liihe, 1910, p. 147; Boertje and Ulmer, 1965), the term strobilo-cysticercoid may be proposed (but see precysticercus below).

L

I

A

FIG.8. A. Strobilo-cysticercus of Tueniu tueniueformis (original); Lateriporus sp. (original, based on specimens collected by Denny, 1969). B. Evaginated strobilo-cysticercoid (or strobilo-precysticercoidorstrobilo-precysticercus?).C.Same, invaginated.Scales: A = lOmm; B = 1 mm; C=0.5 mm.

Both Choatiotaenia infundibulum and C. crassiscolex partially invaginate the scolex end into the neck and withdraw this fore-body into a hollow midbody. The body wall following scolex withdrawal is “two-layered”, but otherwise shows relatively little further layering (Voge, 1961), unlike most cysticercoidsdescribed earlier. Presumably the invaginal canal remains patent. Apparently there is a terminal excretory vesicle and pore at the posterior end of the bladder (Rawson and Rigby, 1960), which suggests that a tertiary excretory vesicle and pore may not develop when the metacestode establishes in the vertebrate gut. If so, then these are not true cysticercoids and the term precysticercoid is suggested for them. These become then either caudate or acaudate acanthorostello-precysticercoids. The metacestode of Lateriporus sp. (see above) and the genus Thysanosoma (see Allen, 1959) also might be a precysticercoid. As more details are established for similar precysticercoids, suitable descriptors can be used to describe them more fully. With Oochoristica spp. there is no trace of a cercomer; the scolex develops exogenously before it completely invaginates into a primary lacuna (Fig. 7).

ONTOGENY O F CESTODES

523

A primary posterior excretory vesicle and pore with associated ducts may be present (Hickman, 1963) or absent (Millemann, 1955). Here again the entire metacestode body initiates adult development. This suggests an early experiment in the evolution of a cysticercus, for which the term precysticercus is proposed. As far as is known now, all precysticerci are unarmed linstowiids, but conceivably this type of metacestode may occur amongst some dilepidids also, e.g. Lateriporus sp. (Fig. 8 , B, C). With most taeniids the scolex is exogenous, notwithstanding that it develops while invaginated. The posterior body becomes a relatively voluminous bladder, but the body wall is not multilayered as with the cysticercoids (Fig. 7). Rather than a terminal excretory pore and vesicle, the dorsal and ventral longitudinal ducts on each side extend from the scolex into the bladder wall and form a network of vessels with numerous secondary ducts and pores opening to the outside (Rees, 1951). In the vertebrate gut the fore-body separates from the bladder and develops a tertiary excretory vesicle and pore, thus becoming a neoplerocercoid. Such a bladder-worm is a cysticercus. All cysticerci develop without a cercomer. Most cysticerci have two rings of dissimilar rostellar hooks, being heteracantho-cysticerci. At least one species, Taenia ( =Fossor = Monordotaenia) taxidiensis, has a single ring of similar rostellar hooks, thus being an homacantho-cysticercus. The metacestode of T. saginata is an anacantho-cysticercus. Obviously if the fore-body has initiated segmentation, this is astrobilo-cysticercus (Fig. 8, A; T. taeniaeformis). The term coenurus has become ambiguous, and does not fit the convention proposed here (Esch and Self, 1965). The term multicephalo-cysticercus can be substituted; this would serve for all forms with two or more scoleces invaginated into a common bladder (Fig. 7). If the scoleces are distinctive, a suitable descriptor can be added. For the exogenously budding metacestode of Taenia crassiceps, multigermino-cysticercus is appropriate. The terms monocercus, polycercus, and hydatid (or Echinococcus) have been used since the last century for metacestodes which produce one or more endogenous scoleces (Fig. 7; Amoebotaenia sp., Paricterotaenia sp., and Echinococcus sp.). Presumably polycerci and hydatids represent convergent evolution. The tiny metacestode within a monocercus or polycercus develops with a secondary lacuna, and is an homacanthorostello-neocysticercoid which sheds its cyst-like mid-body in the vertebrate gut, becoming an homacanthorostello-neoplerocercoid (Mathevossian, 1963, p. 498; Scott, 1965a, b). The individual “protoscoleces” in a hydatid are invaginated heteracantho-neoplerocercoids or modified multicephalo-neocysticerci (Fig. 7 ; Smyth, 1967). However, unless more of these types of metacestodes are discovered, the terms monocercus, polycercus, and hydatid are adequate to describe parent cysts. Alveohydatid, for the metacestode of Eclzinococcus multilocularis, differs from an hydatid in that it produces “numerous exogenous vesicles” in addition to the metacestode with the primary lacuna developing from the oncosphere (Rausch, 1954). The names proposed here for the various types of primitive metacestodes may be compared with the more common names used by various authors (Tables I, 111, and 1V). Generic names based on primitive and neoteric

TABLE N Sequence of metacestode development with proposed names contrasted with names used preciously Metacestode sequence and names -

Order and species Proposed here

Used previously

Authority for previous names

TRYPANORHYNCHA

Lacistorhynchus tenuis

acaudate uniacetabulo-procercoid ?tentaculo-plerocercoid

caudate ?invaginated tentaculoParachristianella monomegacantha plerocercoid ?neoplerocercoid Numerous forms recorded, but difficult to name without knowing their ontogeny

procercoid ?plerocercoid procercoid or plerocercus (not known) plerocercoid or plerocercus

Wardle and McLeod, 1952; Mudry and Dailey, 197 1 Mudry and Dailey, 1971

scolex (not known) merocercus (not known) metacestode (not known) procercoid ?plerocercoid ?plerocercoid plerocercus (Scolex pleuronectis) Usually plerocercoid

rn

I

See.Fuhrmann, 1931 ; Joyeux and Baer, 1936; Wardle and McLeod, 1952; Dollfus, 1964b, 1967

TETRAPHYLLIDEA

Calliobothriumsp. bothridio-(or acetabulo-) plerocercoid (not known) Lecanicephalum sp. glando-procercoid (not known) Tvlocephalum sp. glando-procercoid (not known) Acanthobothrium acaudate uniacetabulo-procercoid ?plerocercoid olseni Acanthobothrium (not known) bothrio-plerocercoid coronaturn Numerous forms recorded

w

Fuhrmann, 1931 Fuhrmann, 1931 Cheng, 1966 Mudry and Dailey, 1971 Reichenbach-Klinke, 1956, 1957 See.authors under Trypanorhyncha

z

0

SPATHEBOTHRIIDEA

Cyathocephalus truncatus Bothrimonus (= Diplocotyle) sturionis

caudate adult acaudate adult acaudate adult acaudate adult

procercoid plerocercoid (= sexually mature) progenetic plerocercoid progenetic plerocercoid (sexually mature)

caudate adult

progenetic procercoid (sexually mature) caudate procercoid progenetic procercoid (sexually mature) caudate procercoid progenetic procercoid (sexually mature)

Wiiniewski, 1932 Sandeman and Burt, 1972

CARYOPHYLLIDEA

Archigetes limnodrili Archigetes iowensis

caudate postplerocercoid acaudate adult

Caryophyllaeus sp. caudate postplerocercoid acaudate adult

Kennedy, 1965 ; Mackiewicz, 1972 Mackiewicz, 1972 0

Mackiewicz, 1972

0

n rn

z

NIPPOTAENIIDEA

Nippotaenia chaenogobii

caudate (acaudate) uniacetabulo-plerocercoid procercoid (+ or - cercomer) (not known) (not known)

Yamaguti, 1959

caudate bothrio-plerocercoid acaudate bothrio-plerocercoid bothrio-plerocercoid bothrio-plerocercoid caudate bothrio-plerocercoid acaudate bothrio-plerocercoid caudate bothrio-plerocercoid acaudate bothrio-plerocercoid caudate glando-procercoid tentaculo-plerocercoid caudate glando-procercoid acaudate bothrio-plerocercoid I acaudate bothrio-plerocercoid II ?acaudate bothrio-plerocercoid I11

procercoid immature plerocercoid

Vik, 1963

procercoid immature procercoid acaudate larva procercoid plerocercoid procercoid plerocercoid I plerocercoid I1

Thurston, 1967

.e 0

a

PSEUDOPHYLLIDEA

Eubothrium salvelini Marsipometra hastata Cephalochlamys namaquensis Bothriocephalus claviceps Haplobothrium globiforme Diphyllobothrium laium

z4

Meyer, 1960

c1

rn La

4 0 J rn v,

Jarecka, 1964 Wardle and McLeod, 1952 Vik, 1964 ul N ul

IJI

N Q\

TABLE IV (continued) -~

Metacestode sequence and names Order and species

--

Proposed here (continued) Diphj,lIobothrium caudate glando-procercoid drndriticum acaudate bothrio-plerocercoid

Used previously

Authority for previous names

PSEGDOPHYLLIDEA

Spirometra mansonoides

strobilobothrio-plerocercoid caudate glando-procercoid acaudate bothrio-plerocercoid I acaudate bothrio-plerocercoid 11 ?acaudate bothrio-plerocercoid I11

procercoid plerocercoid I plerocercoid II procercoid sparganum ?sparganum

Graham, 1970 Mueller, 1938; Wardle and McLeod, 1952

Pro teocqhaIus filicollis Proteocephalus pnmllacticus Proteocephalus ambloplitis Corallobothrium jinibrintum

?acaudate culcitacetabulo-plerocercoidI culcitacetabulo-plerocercoidI1 ?culcitacetabulo-plerocercoidIII caudate culcitacetabulo-plerocercoid I culcitacetabulo-pierocercoidI1 caudate culcitacetabulo-plerocercoidI culcitacetabulo-plerocercoidI1 acaudate invaginated glandacetabuloplerocercoid I invaginated glandacetabulo-plerocercoidTI acetabulo-plerocercoid acaudate invaginated glandacetabuloplerocercoid I invaginated glandacetabulo-plerocercoidI1 (and 111) metacetabulo-plerocercoid

2

0

PROTEOCEPHALIDEA

Proteocephahs fluciatilis

I

plerocercoid I plerocercoid I1 ?plerocercoid 111 procercoid plerocercoid plerocercoid I plerocercoid 11 plerocercoid I

Fischer, 1968

plerocercoid cysticercoid plerocercoid

a

a m Freze, 1965 Freeman, 1964 Fischer and Freeman, 1969, 1973

plerocercoid IIa, b, c procercoid

I”

Freze, 1965

m

F2

PROTEOCEPHALIDEA

Corallotaenia minutia Batrachotaeriiu ranae

Ophiotaenia filaroides

(continued) acaudate invaginated glandacetabuloplerocercoid invaginated acetabulo-plerocercoid metacetabulo-plerocercoid caudate invaginated glandacetabuloplerocercoid acaudate invaginated (or not) glandacetabulo-plerocercoidI invaginated (or not) glandacetabuloplerocercoid I1 caudate invaginated (or not) glandacetabulo-plerocercoid acaudate invaginated glandacetabuloplerocercoid ?acetabulo-plerocercoid

plerocercoid I plerocercoid I1 caudate procercoid

Befus and Freeman, 1973 Freze, 1965

caudate cysticercoid acaudate cysticercoid plerocercoid invaginated (or not) plerocercoid plerocercoid

Mead and Olsen, 1971

0

2:

-1

d

n rn

z

.e 0

a

CYCLOPHYLLDEA

caudate invaginated acanthacetabuloValipora cutnpylancristrota plerocercoid invaginated acanthacetabulo-plerocercoid

acanthacetabulo-plerocercoid caudate invaginated acanthacetabuloParudilepis plerocercoid scolecina, and Neogryporhynchus invaginated acanthacetabulo-plerocercoid acanthacetabulo-plerocercoid cheilancristrotus caudate uniacetabulo-procercoid Mesocestoides invaginated acetabulo-plerocercoid corti acetabulo-plerocercoid acaudate invaginated acetabulo-plerocercoid Metroliasthes acetabulo-plerocercoid lucida

cercoscolex

Jarecka, 1970a

plerocercus immature cercoscolex

Jarecka, 1970b

plerocercus immature procercoid-like tetrathyridium immature cysticercoid immature

Voge, 1969 Wardle and McLeod. 1952

TABLE N (continued) Metacestode sequence and names Order and species Proposed here (continued) neoteric acaudate invaginated acant hacetabulo-plerocercoid acanthacetabulo-plerocercoid Cladotaenia acaudate invaginated (or not) globifera acan thacetabulo-plerocercoid acanthacetabulo-plerocercoid Paruterina invaginated (or not) acanthacetabulocandelabraria plerocercoid acanthacetabulo-plerocercoid Catenotaenia acaudate uniacetabulo-procercoid pusilla acetabulo-plerocercoid Choanotaenia caudate (or not) acanthorostelloinfundibulum precysticercoid immature Thysanosoma ?acaudate arostello-precysticercoid actinoides immature Davainea brevicaudate micracanthorostello-cysticeroid proglottina micracanthorostello-neoplerocercoid Raillietina brevicaudate micracanthorostello-cysticercoid cesticillus micracanthorostello-neoplerocercoid Monoecocestus caudate arostello-cysticercoid americanus acetabulo-neoplerocercoid Cittotaenia caudate arostello-cysticercoid ctenoides acetabulo-neoplerocercoid

Used previously

Authority for previous names

CYCLOPHYLLIDEA

Dipylidium caninum

cysticercoid immature plero-cysticercoid (or cysticercoid) immature plerocercoid

Venard, 1938; Marshall, 1967 Joyeux and Baer, 1961

Freeman, 1957 immature merocercoid plerocercoid cysticercoid (tailless) immature cysticercoid immature cysticeroid immature cysticercoid immature cysticercoid immature cysticercoid immature

Joyeux and Baer, 1945 Horsfall and Jones, I937 Allen, 1959 Wetzel, 1932 Wetzel, 1934 Freeman, 1952 Wardle and McLeod, 1952

(continued) Hymenolepis caudate anacanthorostello-cysticrcoid diminuta acetabulo-neoplerocercoid Hymenolepis caudate acanthorostello-cysticercoid nana acanthacetabulo-neoplerocercoid Drepanidotaenia longicaudate acanthorostello-cysticercoid bisacculina acanthacetabulo-neoplerocercoid Aploparaksis sp. circumcaudate acanthorosteuo-cysticercoid acanthacetabulo-neoplerocercoid Hymenolepis mu1t igermino-cysticercoid cantaniana acanthacetabulo-neoplerocercoid(s) Pseudodiorchis multigermino-cysticercoid prolifer acanthacetabulo-neoplerocercoid(s) Oochoristica precysticercus uacuolata immature Oochoristica precysticercus ?precysticercus osherofi immature Lateriporus sp. ?strobilo-precystircus ?immature Taenia anacantho-cysticercus acetabulo-neoplerocercoid saginata Taenia heteracantho-cy sticercus solium heteracantho-neoplerocercoid Taenia homacantho-cysticercus homacantho-neoplerocrcoid taxidiensis Taenia strobilo-cysticercus taeniaeformis immature

CYCLOPHnLIDEA

Taenia mustelae

multicephalo-cysticercus heteracantho-neoplerocercoid(s)

cysticercoid imm at ur e cysticercoid immature cysticercoid immature cysticercoid immature cysticercoids (staphylocystis) immature urocystis immature cysticercoid immature cysticercoid ?cysticercoid immature cysticercoid ?immature cysticercus immature cysticercus immature cysticercus immature strobilocercus immature multiscolex larva immature

Voge and Heyneman, 1957 Voge and Heyneman, 1957 Jarecka, 1960 Liihe, 1910; Fuhrmann, 1931 Jones and Alicata, 1935 Kisielewska, 1960 Hickman, 1963 Widmer and Olsen, 1967 Denny, 1969

0

z

--I

0

n n

z

.C

0

a

0

m v1

Wardle and McLeod, 1952 Wardle and McLeod. 1952 Wardle and M c k o d , 1952 Joyeux and Baer, 1938c; Hutchison, 1959 Freeman, 1956

--I

o

U m

VI W

TABLE IV (continued)

0

Metacestode sequence and names Order and species Proposed here

Authority for previous names

Used previously

(continued) coenurus multicephalo-cysticercus heteracantho-neoplerocermid(s) immature cysticercus (budder) mul tigermino-cysticercus heteracantho-neocysticercus(i) cysticercus (budder) heteracantho-neoplerocercoid(s) immature alveohydatid Echinococcus hydatid pro toscolex(es) invaginated heteracantho-neoplerocercoid(s) multilocularis (or) multicephalo-neocysticercus(i) protoscolex(es) heteracantho-neoplerocercoids immature Echinococcus hydatid hydatid granulosus invaginated heteracantho-neoplerocercoid(s) protoscolex(es) (or) multicephalo-neocysticercus(i) protoscolex(es) immature heteracantho-neoplerocercoids Amoebotaenia monocercus cysticercoid (=monocercus) sphenoides homacanthorostello-neocysticercoid immature homacantho-neoplerocercoid Paricterotaenia polycercus (mother cyst polycercus paradoxa homacant horostello-neocysticercoid(s) cysticercoids) immature homancantho-neoplerocercoid(s)

CYCLOPHYLLIDEA

Taenia multiceps Taenia crassiceps

+

Wardle and McLeod, 1952 Freeman, 1962 Rausch, 1954

Smyth, 1964

Mathevossian, 1963 Scott, 1965a

ONTOGENY OF CESTODES

53 I

metacestodes are given in Table 11. Of the 61 generic names included, only seven or eight are considered valid today by most authors and they are of little value for understanding relationships among cestodes. It is suggested, however, that the sequences of primitive and neoteric metacestodes (Figs 5, 7) given above, and the descriptive names proposed here (Table IV) to reflect these sequences indicate fundamental relationships among cestodes. It is suggested, furthermore, that they include clues which lead to a somewhat different, more realistic interpretation of the patterns of cestode evolution and classification than generally accepted, as will be described below.

Iv. EVOLUTION OF CESTODE LIFE-CYCLES Fundamental similarities between cestodes and free-living platyhelminths suggest that cestodes evolved from some free-living or parasitic platyhelminth, rather than directly from free-living or parasitic protozoa. Most theories assume that the original parasitic precestode was the adult worm (Stunkard, 1962), although Stunkard (1967) suggested, “When certain turbellarians became parasitic in invertebrates, the adult stage may have remained freeliving. . . ” A.

THE PRECESTODE CYCLE

The most commonly accepted theory relating to cestode evolution suggests that the precestode ancestor developed to the adult in invertebrates, conceivably as Archigetes sp. does today. Stunkard (1 962, 1967) and Cameron (1964) held that some such pattern of parasitism arose by the Cambrian time before vertebrates evolved; Baer (1951) and Joyeux and Baer (1961) disagreed. They believed that cestodes are more specific to their vertebrate rather than invertebrate hosts, which persuaded them to believe that the cestodeinvertebrate relationship is more recent than the cestode-vertebrate relationship. Joyew and Baer (1961) suggested therefore that adult Archigetes spp. probably are neotenic procercoids, i.e. the original vertebrate host has been lost and sexual maturation, now occurring in a larval body in an invertebrate, is secondarily acquired. They suggested, furthermore, that cestodes probably evolved from a turbellarian-like “larva” which invaded the body cavity of a primitive freshwater fish after it had eaten the larva. The larva matured and its eggs escaped from the host via the abdominal pores. Presumably the egg hatched and the larva had a free-living phase. Ultimately, however, the eggs escaping from the vertebrate were ingested by invertebrates wherein the larva now developed. Presumably such infected invertebrates were ingested by the fish, and a two-host cycle was established, apparently without sexual maturation in the gut of either host. Ultimately this or another fish, now with the cestode in either the coelom or developing in the gut lumen, was eaten by yet another fish host and the sexually mature stage developed in its gut. This type of three-host cycle, similar to that of some modern-day pseudophyllideans, Joyeux and Baer (1961) maintain, is primitive and from it present-day cycles evolved. As support for their theory they point to Haplnbothriuni globifornzc, an unusual and presumably primitive cestode

532

R E I N 0 S. F R E E M A N

that has such a three-host cycle and that infects the primitive freshwater fish Amia calva. Finally, their theory is based on another assumption, namely that cestode life-cycles evolve from multihost cycles to simpler two-host or onehost cycles, which according to them predominate today. Llewellyn (1965) and Price (1967), accepting proposals originated by Bychowsky (1957), who in turn was influenced by Janicki (1920), see a strong relationship between the monogeneans on the one hand and cestodarians on the other, particularly the form and function of the characteristic hooks on the monogenean haptor and those on the cestodarian lycophore and the cestode oncosphere. According to Llewellyn (1965) the precestode was a monogenean-like ectoparasite attached to the host with a six-hooked haptor. This gradually evolved into a gut-inhabiting form which matured within the gut while attached by a haptor presumably much like the hook-bearing cercomer on certain pseudophyllidean procercoids today, e.g. Diphyllobothrium sp. The eggs, probably quinone-tanned, escaped in the host’s feces and, following delayed embryogenesis, hatched into free-swimming coracidialike larvae. Presumably these moved directly to the next vertebrate host. Invertebrate hosts were added to the cycle later, and in them the haptor functioned as a penetrating organ. Thus the precestode cycle involved a single vertebrate in which the adult developed in the gut. Price (1967) took this concept further, evolving cestodes from vertebrate-coelom-dwelling amphilinideans, which evolved from more primitive gyrocotylideans in the intestine of vertebrates. The latter evolved from more primitive ectoparasitic monogeneans, which evolved from free-living turbellarians. The basis for Price’s concept apparently is a progressive reduction in the number of hooks from 14 on the monogenean haptor, to 10 to 6 + 4 hooks on the lycophore, to 6 hooks on the oncosphere. How hooks developed in primitive monogeneans is not stated. Obviously if precestodes evolved directly or indirectly from monogeneans, this must have occurred after, and possibly long after, primitive vertebrates appeared. Recently Malmberg (197 I) studied the early ontogeny, especially the protonephridial systems, of three species of Diphyllobothrium. On the basis of similarity in development between these cestodes and trematodes, he concludes that digeneans and cestodes evolved from a common rhabdocoeloid stem. He has the amphilinids and cestodes separating from this stem together; the gyrocotylideans and monogeneans he has further removed. Unfortunately he assumes that atypical oncospheres of Diphyllobothrium sp. with flame cells are characteristic for all cestodes, and shows them developing into cyclophyllideans (Malmberg, 1971, p. 54, Fig. 7). He also considers that it was an adult “rhabdocoelan hexacanthoid” that invaded the vertebrate intestine and gave rise to cestodes, and that subsequently one or more intermediate hosts were added to the cycle. Was it necessarily the adult precestode that was the first parasite? The single universal, and therefore probably most primitive, feature of cestodes is the need for the oncosphere to activate in the gut of the first host, enter a parenteral site, and there metamorphose and develop to the metacestode. Without exception all cestodes infecting aquatic hosts, where precestodes

ONTOGENY OF CESTODES

533

undoubtedly first appeared, have invertebrates as first hosts; the relatively few species of cestodes whose oncospheres do infect vertebrates develop in terrestrial hosts. Furthermore, invertebrates preceded vertebrates in geological time, which suggests that the opportunity for precestodes to infect invertebrates existed before vertebrates appeared. All these facts suggest that precestodes probably invaded invertebrates first. However, only Archigetes sp. is known to mature sexually in the site invaded by the oncosphere, i.e. parenterally in an oligochaete. This suggests, therefore, that the adult precestode probably was not parasitic. As has been demonstrated, the parenteral site has been exploited to both extremes: (a) either relatively little development in the first parenteral site followed by much development in one or more subsequent parenteral sites (Diphyllobothriunz sp.) or in the vertebrate gut (Carenoraeniasp.); or (b) more typically, much development in the first parenteral site, frequently with the adult scolex and even development of genital primordia, or rarely approaching sexual maturity (Cyathocephafus sp.), to the extreme where Archigetes sp. does not require further development in a vertebrate gut. Archigetes sp. is an egg-laying adult while free-living, or at least after death of the host (Kennedy, 1965). Asexual cestode reproduction in invertebrates is almost as rare as sexual reproduction, notwithstanding the examples cited above. Another interesting fact is that no cestodes are known which initiate development, let alone mature, in the gut lumen of invertebrates, although fully differentiated metacestodes are common in the digestive tract of many marine invertebrates (Dollfus, 1964b, 1967). Obviously the invertebrate gut lacks some factor(s), or conversely possesses some inhibitory factor(s), making it unsuitable for sexual maturation of cestodes. The need of the oncosphere to initiate development in a parenteral site, and the fact that this always occurs in invertebrates for cestodes in aquatic hosts, strongly suggests that the initial precestode life-cycle was analogous to that of mermithid nematodes today. In such cycles the haemocoel or similar site in the invertebrate is a rich culture chamber where the preadult grows, differentiates, and accumulates most or all energy reserves necessary before emerging for a relatively nontrophic, free-living, sexual life. Acceptance of this hypothesis requires only that the larva of the precestode ancestor when it emerged from the egg had a capacity to penetrate the gut wall. This certainly occurred, and there is as much justification to assume this occurred before the adult became parasitic, as after. Quite likely it was the evolution of such a gut-penetrating preoncosphere that separated the precestodes from their free-living ancestors, as well as from trematodes. One issue that has clouded understanding of the origin of effective aquatic life-cycles among cestodes is the presumed need for an operculate egg and delayed embryogenesis resulting in a free-swimming coracidium (Joyeux and Baer, 1961 ; Stunkard, 1962, 1967; Cameron, 1964; Llewellyn, 1965). In fact, neither operculate eggs nor coracidia are found in : (a) any modern tetraphyllideans (Riser, 1956; Euzet, 1959; Williams, 1966) [excluding the poorly known genus Tetracampos Wedl, 1961 which is considered a tetraphyllidean by some and pseudophyllidean by others (Wardle and McLeod, 1952, p. 269)];

534

R E I N 0 S. F R E E M A N

(b) any proteocephalideans (Freze, 1965); (c) several minor orders which are totally aquatic; or (d) some trypanorhynchs (Mudry and Dailey, 2971) or some pseudophyllideans (Wardle and McLeod, 1952) or those cyclophyllideans that utilize aquatic invertebrates as first hosts (Jarecka, 1961). The first hosts of these cestodes vary from bottom-feeding oligochaetes, molluscs, copepods and other arthropods, to invertebrates which crawl about vegetation, and to others which are strictly planktonic. On the other hand, freeswimming ciliated coracidia, such as are known for a few trypanorhynchs and some pseudophyllideans, are highly specialized since they utilize planktonic copepods as intermediate hosts. Furthermore if an operculate egg and coracidium were characteristic of precestodes, it follows that a relatively simple egg and embryo, as might occur with primitive ancestors just described, had to evolve into an operculate egg with coracidium as in higher pseudophyllideans and certain trypanorhynchs, and later evolve back to the nonoperculate egg with oncosphere characteristic of the majority of known cestodes. This position seems most untenable. If, as proposed herein, the oncosphere of precestodes initially penetrated and parasitized invertebrates, this could, and probably did, occur before vertebrates evolved, as has been suggested (Cameron, 1964; Stunkard, 1967). Conceivably metacestode growth in parenteral sites with adequate nutrients could be equally successful with or without a gut, particularly for small organisms. This could account for the lack of a recognizable enteron in any cestode today. Furthermore, such precestodes probably remained small in size until a suitable larger environment rich in nutrients, such as the vertebrate gut, was available. The type of parasitism with a free-living non-feeding adult, and a parasitic, energy-storing preadult is known for gordiacian worms, as well as the aphasmidian nematodes ; warble flies and some chalcid wasps also are parasites as larvae and free-living and essentially non-feeding as adults. Inglis (1965) suggested that probably parasitism arose in certain phasmidian nematodes in a similar manner. The digenetic trematodes, most unlike cestodes, universally reproduce asexually and even sexually, albeit rarely (Stunkard, 1959; Heyneman, 1960; Anderson and Anderson, 1963), in the invertebrate. It might seem valid to assume, therefore, that sexually reproducing digenetic trematodes parasitized invertebrates first and only later moved to vertebrates (Stunkard, 1967). However, Heyneman (1960), speculating on the origin of the life-cycles of digenetic flukes, concluded : “The morphological, ecological, embryological, and physiological evidence, therefore, appears to favor an evolutionary sequence from larval forms in a specific snail with free-living adult worms to a subsequent phase with adult worms maturing in the gut of a vertebrate host . . . ” Pearson (1970, 1972) apparently is of much the same opinion since he suggests that the pretrematode ancestor was a carnivorous rhabdocoel which became ectoparasitic on snails. Eggs it produced fell to the bottom and liberated a miracidium which moved to another snail and developed to an ectoparasitic adult. The platyhelminth subsequently invaded and grew in the snail haemocoel emerging as a tailed free-swimming egglaying adult, Miracidia emerged from the eggs and penetrated another snail. Ultimately asexual reproduction developed in the snail, and adult flukes began

O N T O G E N Y O F CESTODES

535

to mature in the vertebrate gut. The important point is that the juvenile in all of these groups successfully invaded and exploited an environment dissimilar to that utilized by the adults. Conceivably during the early period of cestode evolution the cestode cercomer, or tail, was used for swimming as Hyman (1951) suggested. It is generally accepted that the cercomer must have evolved early in the history of cestodes because today this structure apparently is vestigial, although secondarily it may produce a protective envelope. Frequently the oncospheral hooks are in it, and consequently the shedding of these hooks along with the cercomer has been considered of phylogenetic significance; e.g. Riser (1955) uses this as one character for separating two new superorders Trixenidea and Dixenidea, and Jarecka (1970c, d) considers this the basis for separating the second from the third stage in her concept of the basic cestode life-cycle. Llewellyn (1965), arguing that the cercomer was an organ of attachment and against the concept that the cercomer could be used as a swimming organ as in digenetic trematodes, stated: “. . .it should be borne in mind that the procercoid cercomere is an organ that completes its task early in larval development, but that the cercarial tail is a late ‘pre-adult’ organ.” And later he reiterates: “. . . they are so different in function that there is no real correspondence, the cercomere being a very early ontogenetic adaptation, and the cercarial tail a very late one.” Certainly the oncospheral hooks on presentday cestodes attach the larva to the gut wall during its penetration into the parenteron, as Llewellyn maintained. Contrary to what he said, however, the cercomer occurs late in the ontogeny of many pseudophyllideans, except Diphyllobothrium sp. and closely related forms, as well as in most if not all proteocephalideans and cyclophyllideans which have one, since typically by the time the cercomer is shed the scolex is well developed. In other words, Llewellyn’s argument (as with Malmberg, 1971) was based on the erroneous assumption that the genus Diphyllobothrium is primitive, and furthermore, that the type of development it undergoes is characteristic of cestodes in general. In fact caryophyllideans and spathebothriideans may have functional reproductive organs before the cercomer is shed. One can readily assume, therefore, that as with cercariae so with metacestodes, the cercomer typically is found late in cestode ontogeny. Thus the hypothesis that the cercomer was used originally for swimming is plausible. It could be asked how the ancestral metacestode escaped from the haemocoel or tissues of the invertebrate. This may have been less of a problem than it seems. Mueller (1959), for example, used the natural emergence of plerocercoids of Spirometra sp. from dead copepods as a method of harvesting large numbers of these organisms. Similarly the author has observed plerocercoids of Proteocephalus sp. emerging from dead copepods. In fact, it is becoming apparent that some metacestodes at least are not passive, rather they are quite capable of reacting to stimuli and not only moving within the host but leaving it. This aspect of cestode biology has been virtually ignored up to the present time. Conceivably a tailed, free-swimming stage was the mechanism by which the organism reached the site occupied by the free-living adult. Some such adults

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could have been relatively sessile forms attached to the substrate by the abcercomer end. This suggests a function for the single, terminal sucker-like structure which appears early in the ontogeny of various genera of cestodes in almost every order recognized today (see above). In fact, such a structure is the only organ of attachment on some adults (e.g. genera Nippotaenia, Litobothrium, Cyuthocephalus). Could it be that the scolex end originally functioned as a “primitive foot”? If so, subsequent development could have led to elaboration of an enlarged neural mass and an increase in the number of sensory receptors associated with an expanded locomotory function. By analogy with higher animals, this end could be the “head”, but even today a primary function of the scolex is locomotion and attachment, a function more of a foot than of a head. Consequently some such structure, and not a hookbearing haptor, might be well preadapted for attachment to the gut wall when the opportunity arose for the adult to remain in the vertebrate gut. Furthermore was there a true reversal of polarity as postulated by some workers ? A precestode cycle with (a) a free-living adult, (b) penetrating preoncosphere, and (c) major differentiation and increase in size while parasitic in the tissues of a suitable invertebrate leading to (d) a free-living adult, with or without a tail, could have evolved long before vertebrates evolved. Presumably such worms were small, primarily monozoic, although evolutionary experiments with polyzoic bodies, as with coelenterates, could have occurred. Probably they were eaten by carnivorous invertebrates, but could not establish in the gut. The pattern changed once predatory vertebrates evolved and began to prey on the free-swimming preadult or the more sedentary adult, as well as the invertebrates in which preadults developed. The protoscolex was a suitable mechanism for attaching to the gut wall. Conceivably those precestodes that survived in the vertebrate gut and successfully exploited this new habitat thrived, whereas those that continued with the need for free-living existence were less successful competitors and disappeared. Conceivably, Archigetes sp. or some of the spathebothriideans continue a modified form of the original type of life-cycle, although their free-living adults have not been found. Or is it that they are unknown because they have not been sought? Regardless, such species may have survived by retaining a relatively short free-living adult stage. This stage such as is known now, occurs brieffy following death of the host, e.g. Archigetes sp. At the other extreme, necessary changes associated with evolution of the oncosphere into a more effective organism adapted for invading a parenteral site, led to greater deviation from the primitive pattern of development. Ultimately deviation became so great that the most successful mechanism for returning to the basic developmental pattern was the process of metamorphosis. This suggests that the perfection of the hexacanth larva was the major evolutionary thrust originally separating cestodes from other platyhelminths. B.

THE PROTOCESTODE CYCLE

If, as proposed by several people (Fuhrmann, 1931 ; I-Iymnn, 1951 ; Wiirdle and McLcod, 1052; Stunkard, 1967; Malmberg, 1971), the protocestodes

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arose from rhabdocoel-like turbellarian ancestors which tended toward elimination of the mouth and digestive tract so necessary for holozoic nutrition, then the precestode cycle already suggested would select in favor of this feature. Absorption of nutrients through the tegument and distribution by diffusion throughout the body would be simpler, at least initially, in the smaller metacestode. As the tegument increased its capacity in this function the animal could grow in size. Sirnultaneously a digestive system would become less essential for a free-living, relatively nontrophic adult. The adult tegument became adapted for saprozoic existence, possibly functioning in a suitably rich organic environment, such as bottom sediments, but becoming fully efficient later in the gut of early vertebrates. The latter acquired precestodes by feeding either on infected invertebrates or on the cercomerbearing, swimming forms as already suggested. Predatory invertebrates undoubtedly ate other invertebrates, as they do today, and undoubtedly parenteral precestodes were ingested. Yet no cestodes are known to mature in the gut lumen of invertebrates, suggesting that ancestral cestodes never matured there either. The specificity of sexual maturation to the gut of vertebrates, when it occurs enterally, is almost as universal as the need for the oncosphere to initiate metacestode development in a parenteral site. This suggests that once regular access to the vertebrate gut was obtained, this rich medium might become an acceptable site for sexual development of some preadapted precestodes. Presumably those becoming established as parasites in a vertebrate gut were more successful than those with a continued need for a free-living sexual phase; the latter disappeared, the precestode cycle being replaced by the protocestode cycle. Conceivably, as suggested by Claus (1889), the vertebrate gut provided more abundant and suitable nutrients as well as space than parenteral sites in invertebrates, and one way the evolving, gutless cestode had for increasing in size, numbers, and total egg production was by proglottidation. A two-host cycle still is the rule rather than the exception among most aquatic cestodes, including proteocephalideans (Freze, 1965) and probably tetraphyllideans (Riser, 1955; Williams, 1966), as well as numerous species in other orders including certain pseudophyllideans (Vik, 1963; Jarecka, 1964). Another interesting feature about present-day cestodes which appears to suggest that direct alternation between parenteral and enteral sites may be most primitive, is the fact that cestodes always transfer passively, being eaten either as food (prey) or with food. This argues against the thesis that such endoparasitism evolved gradually from an ectoparasitic condition, i.e. active invasion, as is necessary for evolving cestodes from ectoparasitic protomonogenean ancestors with a hooked posterior organ of attachment (Llewellyn, 1965). Conceivably this passive means of infecting the invertebrate host by the oncosphere is what set the cestodes on the path of evolution diverging from the ancestral digenetic trematodes, since according to Heyneman (1960), with whom Pearson (1972) agrees, the first stage of endoparasitism by trematodes probably was penetration of the foot or mantle of a snail by the mi racidi um . Llewellyn (1 965) believed the evolution of the quinone-tanned egg by

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ancestors common to monogeneans and cestodes probably permitted an original single-host gut-inhabiting cestode precursor to function. According to him such an adult laid resistant eggs in the gut of primitive fish. These passed out, fell to the bottom, “. . . to pass any resting or incubating period”, then were eaten by microphagous animals and originally were not digested. According to Llewellyn such eggs might: (a) pass through the gut of such microphages “. . . and so maintain the reproductive potential. . .”; (b) be in the gut when the microphage was eaten by a primitive fish where the oncosphere hatched “. . . directly into the micro-habitat occupied by the adult parasite. . .”; or (c) hatch “. . . precociously in the gut of the ‘carrier’ and then attaching itself by its haptoral hooks to the gut wall” remain until eaten by the fish; or (d) “. . . by deeper attachment the larva might have penetrated the gut . . [and] continued development . . .”, which would establish the two-host cycle. Such a two-host cycle had “. . . an adult in the gut of a specific vertebrate host a n d . . . an invertebrate as a non-specific intermediate host.” Evolution of the system proposed by Llewellyn does not seem in accord with the facts when it is considered that: (a) host specificity for the vertebrates probably is just as varied as for the invertebrates in most cestode life-cycles; (b) without exception all aquatic cestodes have an oncosphere which activates in an invertebrate and develops parenterally; and (c) relatively few aquatic cestodes have operculate, thick-shelled tanned eggs, and they are most common in the higher pseudophyllideans (e.g. diphyllobothriids) and some trypanorhynchs. Finally, how can an egg be resistant to digestion and yet be digested so that it can function? It should be emphasized that, whereas cestodes may develop reproductive organs in parenteral sites in invertebrates, not one oncosphere develops directly to a sexually mature cestode in the gut lumen of a vertebrate.

.

C.

EVOLUTION OF MODERN TAXA

Whether cestodes originated in the sea or in fresh water is open to argument. Wardle and McLeod (1952) and Joyeux and Baer (1961) argued in favor of their origin in fresh water. The argument depends in part on where the first suitable vertebrate hosts evolved, although the precestode cycle suggested above could have arisen in either the sea or fresh water. Yamaguti (1959) recognized 10 orders and 51 families of cestodes, of which 8 orders and 34 families are in fishes, with 6 of these orders only in fishes. This indicates that cestodes were well established in fish long before other vertebrates evolved, since most cestode diversity and radiation occurs among fishes. Absence of unique cestodes in lampreys and hagfishes suggests that the primitive vertebrate filter feeders (Romer, 1967) were not parasitized regularly by primitive cestodes. The evolution of predatory, jawed fishes presumably produced the first suitable vertebrate hosts. According to Romer (1967) jawed ancestors of present-day fish originated in fresh water possibly 350-400 million years ago, although admittedly this hypothesis of freshwater origin is not universally accepted. A major problem in determining the evolution of cestodes arises from lack

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of understanding of the morphology of primitive adults. Most present-day adult cestodes are either difossate or tetrafossate, or are related to one or the other, although the scoleces of caryophyllideans, cyathocephalids, and a few others may differ. Usually it is assumed, therefore, that the ancestral cestodes were either tetrafossate, like the Tetraphyllidea or Proteocephalidea (see Wardle and McLeod, 1952), or difossate like the Pseudophyllidea (see Joyeux and Baer, 1961). Wardle and McLeod (1952, p. 147) thoroughly examined the association of tetra- and difossate types of cestodes with their vertebrate hosts and stated: “On the basis of host distribution, the conclusion seems inescapable that tetrafossate tapeworms are more primitive than difossate forms, and that the most primitive of present-day tapeworms are the tetraphyllidean forms and the collared proteocephalans, which appear to be the results of divergent evolution from a common ancestral tetraphyllideanproteocephalan stock.” Later (p. 187) they stated: “It may be accepted that they [Proteocephala] are on the whole very primitive tapeworms. The tetraphyllidean and trypanorhynchan tapeworms of selachians would seem to be primitive, too, and the deep-seated resemblances between these forms and Proteocephala may therefore be of deep significance.” Elsewhere (p. 154) they stated : “The difossate tapeworm are neotenic, persistent larval forms of the proto-cestode stock. Several lines of evolutionary divergence are represented. Caryophyllidea and Spathebothridea came off before suckers or bothridia or even bothria evolved. Haplobothriidae may represent neotenic larvae of the proto-Trypanorhyncha. Amphicotylidae, Bothriocephalidae, and Dibothriocephalidae may be descendants of proto-tetraphyllidean forms, the four-lobed apical disk of the former two families being reminiscent of the proto-bothridia.” These views of Wardle and McLeod (1952) expand those of Fuhrmann (1931), who considered that parasitic rhabdocoels gave rise to the Tetraphyllidea from which modern cestodes arose along two paths; the first were the Tetrarhynchidea (= Trypanorhyncha) from which in turn evolved the Pseudophyllidea; the second path included the Proteocephalidae from which evolved the Cyclophyllidea. Stunkard (1967) is less definite when he states: “The Tetraphyllidea are apparently the most generalized existing cestodes, and together with the tetrarhynchidean and pseudophyllidean worms, have been derived from a common ancestral stock.” He did not mention the proteocephalideans. Apparently he accepted Riser’s (1955) argument, but not his new superorders, because Stunkard attributed to these “generalized” cestodes the three-host type of life-cycle including a coracidium which is eaten by a crustacean, as in certain Pseudophyllidea. Unfortunately lumping the tetraphyllideans and all trypanorhynchs with pseudophyllideans is not in accord with the facts, since they differ both morphologically and in their life-cycles. Eggs of all tetraphyllideans and at least some trypanorhynchs are nonoperculate and do not contain coracidia (Southwell, 1925; Joyeux and Baer, 1936, 1961; Riser, 1955,1956; Euzet, 1959; Yamaguti, 1959; Williams, 1966; Mudry and Dailey, 1971). This supports Wardle and McLeod (1952, p. 143) who stated when discussing this problem: “It might be supposed, too, that if the coracidium is an ancestral phase it would occur in the majority of tapeworm life cycles, but

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actually it is known mainly from four pseudophyllidean families-Bothriocephalidae, Haplobothriidae, Triaenophoridae, and Di bothriocephalidae. It is significantly absent from the life cycles of Caryophyllidea, Spathebothridea, and the pseudophyllidean families Amphicotylidae and Ptychobothriidae. Ruszkowski . . . it is true, demonstrated a coracidial embryo in the life cycle of the trypanorhynchan tapeworm Grillotia erinaceus, and it may well be that the coracidial phase is more prevalent among tapeworms than seems at present to be the case; but it certainly cannot be widespread.” The life-cycles of several freshwater proteocephalideans, pseudophyllideans, and cyclophyllideans are known. Presumably these reflect the basic life-cycle patterns in these orders, although undoubtedly other variations exist. There is, however, only a smattering of information on the life-cycles of marine cestodes. This includes information on three tetraphyllideans (all in the genus Acanthobothrium), although even these life-cycles are not known fully (Reichenbach-Klinke, 1956, 1957; Riser, 1956; Mudry and Dailey, 1971). Apparently all have a nonoperculate egg which is eaten by a copepod in which the oncosphere hatches and develops parenterally to an acetabuloprocercoid. Presumably transfer to the final host may occur directly or via one or more hosts in which a plerocercoid develops and remains in the gut lumen. Tetraphyllidean plerocercoids are known from plankton, mainly crustaceans, and there are numerous records of other tetraphyllidean plerocercoids from the gut lumens of cephalopods, teleosts and other hosts (Joyeux and Baer, 1936; Dollfus, 1964b, 1967). Williams (1966) speculates following extensive study of the genus Echeneibothrium from rays (Raja spp.), however, “. . . it is reasonable to assume from these observations on the food of the host and from the size and form of the eggs. . . that the eggs may be eaten by an arthropod which is most likely to be eaten by the final host.” Thus the similarity of the life-cycle to that of proteocephalideans is unmistakable. Apparently there are at least two types of trypanorhynch life-cycles, Grillotia sp. being similar to Diphyllobothrium sp., and Parachristianella sp. and Christianella sp. having a tetraphyllidean-proteocephalidean pattern (Wardle and McLeod, 1952; Heinz and Dailey, 1971; Mudry and Dailey, 1971). Again the complete life-cycle is not known with certainty for any of them. Cestode ontogeny suggests that probably the protocestodes were neither di- nor tetrafossate, having rather a single apical sucker-like holdfast, much like spathebothriideans, nippotaeniideans, or possibly caryophyllideans. Placement of these cestodes has always presented a problem to taxonomists. Joyeux and Baer (1961) consider that the caryophyllideans and cyathocephalids belong to separate families which they place in the Pseudophyllidea ; Wardle and McLeod (1952) consider that they belong respectively in separate orders Caryophyllidea and Spathebothridea of the class Cestoda; Schmidt (1970) follows Wardle and McLeod, but restricts the orders to the subclass Eucestoda; Yamaguti (1959), however, places the Caryophyllidea with the subclass Cestodaria and the Spathebothridea with the subclass Eucestoda! Obviously there is little agreement as to the significance of these morphological features. Are these forms aberrant or primitive ? It is suggested that cestodes as known today evolved from similar unifossate protocestodes.

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54 1

Conceivably, unifossate protocestodes arose in fresh water where the lifecycle was largely restricted to bottom-inhabiting invertebrates and vertebrates. Experiments with di- and tetrafossate scoleces may have begun there. When primitive vertebrates began to colonize the sea they took with them uni-, di-, and tetrafossate precursors of cestodes living today. Cestodes diversified in morphology and in life-cycles in both the sea and fresh water, and ultimately no longer were restricted to bottom-inhabiting invertebrates and vertebrates. During this time coracidia probably appeared. Along the way some metacestodes began to infect more than one invertebrate host in sequence depending on the ecology of the hosts and the development of the metacestode; i.e. the end organ became glandular on some and could be used for penetrating into successive hosts, invertebrates and vertebrates. In others the metacestodes lacked effective “penetrating” end organs but had organs for attachment, and so were capable of surviving for varying periods within the gut lumen of invertebrates and vertebrates, even when they were not normal hosts. Those host-parasite relationships which had survival value for the cestodes were retained, and the numbers of intermediate hosts in a cycle reflected both ecological opportunities and the adaptations cestodes had to utilize these opportunities. The preceding suggests that most primitive adult cestodes probably had a single apical sucker-like or glandular holdfast (Fig. 9). Some forms experimented with polyzooty, others remained monozoic. From the unifossate monozoic stalk evolved modern caryophyllideans with their diverse types of holdfasts, but a single set of reproductive organs in each body. The caryophyllideans are exclusively freshwater forms, although there are a few, presumably accidental, records in bottom-feeding, estuarine fish (Mackiewicz, 1972). From the polyzoic stalk (Fig. 9) evolved: (a) a unifossate stalk which gave rise to modern spathebothriideans (marine and freshwater) and possibly nippotaeniideans (freshwater) ; (b) a difossate pseudophyllidean ancestor, without a coracidium, from which evolved modern difossate cestodes with and without coracidia (marine and freshwater); (c) a di- and tetrafossate stalk with proboscides, or tentacles, from which came the trypanorhynchs and possibly some less well known forms (exclusively marine); and (d) a tetrafossate tetraphyllidean-proteocephalidean ancestor from which modern tetraphyllideans and other less well known forms (marine), proteocephalideans (freshwater), and cyclophyllideans (freshwater and terrestrial) evolved. Probably di- and tetrafossate stalks evolved more or less simultaneously rather than in sequence as frequently suggested (Wardle and McLeod, 1952). All five stalks may be very old, since representatives of each occur in such diverse natural bodies of water inhabited by such diverse aquatic vertebrates. Obviously cestodes rose, diversified, and radiated most among the fishes. Nevertheless some cestodes have adapted to terrestrial hosts. Presumably this occurred by parallel evolution of the life-cycles of cestodes and that of their vertebrate hosts, and probably co-evolution of both populations (Odum, 1971). Only three major orders of cestodes occur as adults in bony fishes and tetrapods : the Pseudophyllidea in fishes and higher vertebrates, Proteocephalidea in amphibians and reptiles as well as fishes, and Cyclophyllidea in all classes 20

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above fishes (Yamaguti, 1959). This suggests that possibly pseudophyllideans are best adapted for transferring to the final host, with proteocephalideans next best. In fact, all species in both orders require aquatic first hosts; none is completely terrestrial. Those pseudophyllideans and proteocephalideans that do mature in terrestrial vertebrates have in common the migratory plerocercoid. Such plerocercoids occurring in aquatic vertebrates, the second or third hosts in the cycle, may mature in the gut of a terrestrial vertebrate (e.g. Diphyllobotlzrium spp. in birds and mammals, Vik, 1964; Ophiotaenia spp. in reptiles, Freze, 1965). There is little doubt that the Cyclophyllidea evolved from the Proteocephalidea (Fuhrmann, 1931; Wardle and McLeod, 1952; Jarecka, 1970d), and that some adaptation(s) as well as the migratory plerocercoid probably made the terrestrial cestode life-cycle possible. The fact that cyclophyllideans are relatively rare among amphibians and reptiles, except for the nematotaeniids in the former and linstowiids in the latter, suggests that possibly something associated with the biology of birds and mammals may be as important as adaptations of the cestodes themselves. Quite likely they are associated with overcoming potential dangers of desiccation that oncospheres encounter on land during transfer from the feces of the final host to the first host. Terrestrial cestodes have two modifications which apparently lessen such danger. First are changes in the gravid segment to produce an auxilliary protective covering for the eggs, e.g. packaging eggs in capsules as in a paruterine organ in nematotaeniids, mesocestoidids and dilepidids. The other is producing a protective covering on individual eggs, e.g. Taenia spp. The hosts themselves probably produced the most important modification, however, and this relates to the formed fecal mass which cestodes of terrestrial hosts may use as a vehicle for reaching the first host. Feces produced by poikilothermic aquatic hosts usually are not in a well formed bolus, in contrast to those of most mammals. In water, desiccation is no problem, and a distinct advantage of an unformed stool could be rapid scattering of eggs. On land, in contrast, scattering may be a disadvantage except when the eggs themselves can withstand desiccation. Therefore the formed stool, characteristic of most mammals, may be more important for transmission of relatively unprotected cestode eggs in a terrestrial setting than previously realized. In fact, it is remarkable how soon after feces are dropped they are visited by various consumers (personal observation). Obviously success of cyclophyllideans indicates that many eggs are ingested before harmful desiccation occurs. In addition to these mechanisms, some terrestrial cestodes may show behavioral modifications which increase the likelihood that their eggs are available to a first terrestrial host, particularly when the latter is not a dung feeder. Probably the most obvious adaptation is migration of gravid segments away from the fecal mass and the subsequent scattering of relatively resistant eggs, or contraction of the gravid segment into a somewhat protective capsule. Metacestodes of some cyclophyllideans do develop in more than one host, but most such cycles also are at least partly aquatic (Jarecka, 1970a, b). Apparently some Dipylidiinae may move through three terrestrial hosts in a fully terrestrial cycle (Gupta, 1970). Generally, however, most successful

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cyclophyllideans alternate between two hosts, usually a first invertebrate host and a final host. Second paratenic hosts may be involved in some life-cycles, especially when the final hosts are major carnivores (Gupta, 1970). Most cestodes which mature in large carnivores, e.g. the Taeniidae, are adapted to infect primary vertebrate consumers, such as rodents and ungulates. Undoubtedly at one time such cestodes also had invertebrate first hosts. Thus it may be that the formed stool of homiothermic vertebrates led to use of vertebrates as first hosts in terrestrial life-cycles, and lack of such stools may have forestalled such cestode life-cycles among aquatic vertebrates. Regardless, the basic two-host cycle continues to be as common among cyclophyllideans as among many cestodes in aquatic hosts. V. PHYLOGENETIC RELATIONSHIPS It is generally accepted that, “Organs formed during the course of the life history of the individual are related to stages in the life cycle of the species and probably were functional in progenitors, so the life cycle portrays a succession of forms adapted to the life of the animal at corresponding previous stages” (Stunkard, 1962). This has been the approach taken here. The oncosphere, and structures associated with it, apparently are among the most conservative cestode features. Next are those associated with development of the metacestode. If the data from preceding pages are evaluated from this viewpoint, and related to the taxa on the ordinal level recognized today, the pattern shown in Fig. 9 results.

FIG.9. Pattern of evolution from rhabdocoel-like aiicestors to present-day orders of cestodes.

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These and other data suggest that pretrematodes, precestodes, gyrocotylids, and monogeneans all evolved from rhabdocoel-like free-living ancestors, but at different times (Fig. 9). The pretrematodes and precestodes came off in the early Paleozoic era and parasitized invertebrates; gyrocotylids and monogeneans did not come off until the Mid-Paleozoic era after aquatic vertebrates appeared. Probably the “oncosphere”, if this term is appropriate, was not as well differentiated, and possibly it had a different number of hooks than the six known today. Quite likely, metamorphosis did not occur among precestodes. Although amphilinids and protocestodes probably came off the precestode stalk, they became separated by fundamental differences in biology: protocestodes established as adults in the gut lumen of fish, and amphilinids established and matured sexually in the body cavity of other fish. Amphilinid life-cycles are incompletely known, but early amphilinids could have been parenteral parasites of invertebrates, preadapted to parenteral development in fish when the latter appeared. However, the vertebrate coelom may have proved more difficult for adult amphilinids to exploit than the vertebrate gut was for adult protocestodes, and consequently while the protocestode stalk diversified and radiated, the amphilinid stalk remained relatively small and stable. Probably amphilinids and precestodes evolved in fresh water, and amphilinids separated from the main cestode stalk before the protocestode life-cycle was well established. Ultimately the basic two-host protocestode cycle was established. Probably the oncosphere, or a recognizable precursor, also was established at this time. Quite likely protocestodes appeared in marine as well as freshwater hosts. From such protocestode ancestors evolved the main stalks of cestodes recognized today (Fig. 9). Spathebothriideans, caryophyllideans, and nippotaeniideans would appear to be closest to the protocestode stalk. If ancestors of trypanorhynchs and pseudophyllideans did not come off together, they probably arose from closely related ancestors. The tetraphyllidean-proteocephalidean stalk has proved most successful from the standpoint of diversity of species and of habitats in which they occur. Little is known about the early ontogeny of marine tetraphyllideans, lecanicephalideans, diphyllideans and trypanorhynchs; therefore speculation on their evolution from this standpoint is meaningless. For possible patterns of evolution based on other characters, see Euzet (1959). Mackiewicz (1972) gives a comprehensive discussion of the evolution of the caryophyllideans and concludes that they should stand as a distinct order. Sandeman and Burt (1972) combine the spathebothriids with the cyathocephalids and maintain that they and the closely related caryophyllids should stand as separate families in the order Pseudophyllidea as proposed by Joyeux and Baer (1961). No doubt the cyathocephalids and caryophyllids are more similar to the pseudophyllids than to the proteocephalids. However, which are closer to the haplobothriids, bothriocephalids, and higher pseudophyllids-the cyathocephalids and caryophyllids or the genera Cephalochlamys, Marsipomefra, and Eiibothrium (Fig. l o ) ? The standpoint taken here is that on the basis of adult morphology the latter genera are closer to the other pseudo-

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phyllideans, and that the Spathebothriidea and Caryophyltidea should stand as separate orders (Fig. 9). It may be, however, that the order Pseudophyllidea should be divided into two suborders: (a) one to include the more primitive families with anoperculate eggs, without delayed embryogenesis, with or without coracidia, but lacking a distinct procercoid during ontogeny, and (b) the other suborder for those families with an operculate egg, usually with delayed embryogenesis, a coracidium and usually a distinct procercoid during ontogeny (Fig. 10). The most recently evolved genera, those that mature in birds and mammals, all belong to the family Diphyllobothriidae. Diphyllobolhriidoe Ligula Schirtocapholus Diphyllobothriurn

Spiromelra

etc.

Amphicofylidae Marripomelm

primitiva preudophyllidron

FIG. 10. Phylogenetic relationships in the order Pseudophyllidea.

Freze (1965) discussed the evolution of the suborder Proteocephalata in some detail, and he emphasized its monophyletic origin. He recognized the superfamily Proteocephaloidea, which includes the Proteocephalidae and Ophiotaeniidae, and the superfamily Monticellioidea with the single family Monticelliidae. His conclusions are based on morphology of the adults, the assumption of “linked evolution” of parasites and hosts, and differences in ontogeny. As stated above, there is information on life-cycles of a number of proteocephalids and a few ophiotaeniids, but not for any monticelliids. Therefore where the latter would fit in the present scheme is uncertain. The present author agrees with Freze that the proteocephalideans are monophyletic in origin, and that the ophiotaeniids evolved from proteocephalids. He disagrees, however, if Freze assumes that there was a single path of development among the proteocephalids. In fact, the ontogenic data suggest that there are two main stems leading from the primitive proteocephalids, one via

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those proteocephalids tending to eliminate a cercomer during development, and the other where development of a cercomer is retained (Figs 7, 11). There appears to be a correlation between possession of a distinct cercomer and the subsequent development of a migratory acetabulo-plerocercoid. Further observations axe required to determine which proteocephalids are acaudate and which are caudate, to establish whether other features also can be correlated with each pattern of development. The genus Proteocephalus is large and unwieldy (Wardle and McLeod, 1952; Freze, 1965). It probably holds the key not only to understanding the Proteocephaloidea but to understanding the evolution of the Cyclophyllidea as well. It is suggested that the acaudate and caudate proteocephalids led to a diphyletic (and possibly a polyphyletic) origin of cestodes currently included in the single order Cyclophyllidea (Figs 7, 11). The acaudate stem has retained the primitive tendency of utilizing two hosts in each life-cycle, whereas the caudate stem, at least primitively, has several stems with three-host (and possibly four-host) cycles, although the davaineids, anoplocephalids, and hymenolepids appear to be exclusively two-host in pattern. If the assumptions made here prove correct, perhaps the existing order should be separated into Taeniidns

___

freshwater

terrestrial

1

birdr&mammalr

primitive ,/---’’-

profsocaphawlidaan

FIG.11. Diphyletic pattern of evolution in thc order Proteocephalidea leading to two major stems of terrestrial cestodes currently combined in the order Cyclophyllidea.

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two orders or suborders. It is suggested that the taeniid stem bear the name Taenioidea, and that the name Cyclophyllidea be retained for the other stem. Conceivably each should in turn be subdivided, with one branch for those developing with a primary lacuna and the other branch for those developing without it (Fig. 11). These groupings suggest a realignment of some families and genera. For example, the Linstowiidae, along with Amabiliidae and some Dilepidinae, are brought close to the family Taeniidae on the main taenioidean stem, since they develop with a primary lacuna; other dilepidins (Cladotaenia sp., Paruterina sp.), and the catenotaeniids and possibly the nematotaeniids, all of which develop without a primary lacuna, belong on the other taenioidean stem (Fig. 11). Similarly the now restricted order Cyclophyllidea would include on one stem the Mesocestoididae, some Dilepidinae, and some Paruterininae, all developing without a primary lacuna and usually with a migratory plerocercoid. The main cyclophyllidean stem would include the Dipylidiinae, Davaineidae, Anoplocephalidae, and Hymenolepididae. The genera formerly assigned to the order Aporidea probably merit the status of a family close to the hymenolepids (Fig. 11). The most interesting feature in all of this realignment is the break-up of the heterogeneous family Dilepididae, with some genera now assigned to each of the four major stems. Obviously many cyclophyllidean genera cannot be assigned according to this scheme until their ontogeny is established, but probably most will fall into place when the life-cycles of key genera in each group become known. Some genera will pose problems because of convergent evolution, but again other characters should help establish proper relationships. The revision of the Cyclophyllidea suggested here varies considerably from that proposed by Spasskii (1951 et seq.).

VI. CONCLUSION More information is needed, particularly for cestodes from marine habitats, before a complete pattern of cestode ontogeny becomes evident. Further detailed study of freshwater and terrestrial cestodes also is required, particularly among groups presently assigned to the Dilepididae. There is a need for data on the early ontogeny, particularly the origin, development, and final disposition of the primary lacuna, cercomer, invaginal canal, and excretory system of the metacestode, as well as for data on growth patterns of metacestodes in the vertebrate gut. Nevertheless the data already available indicate that metacestodes do follow recognizable patterns of growth, and that these patterns may be of value in establishing taxonomic relationships among cestodes. It is hoped that this study suggests new avenues of investigation for the future. ACKNOWLEDGEMENTS I gratefully acknowledge the considerable effort of my wife, Ellen, in typing and revising numerous drafts of this manuscript, the preparation of the illustrations by Mrs. Maria Staszak, and the constructive criticism and

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assistance in many ways by staff and students in the Department of Parasitology. Spirited discussions with Dr. Lena Jarecka during the summer of 1972 also were of great value. 1 thank Dr. John Holmes, University of Alberta, for the loan of metacestodes of Lateriporus sp. collected by Dr. M. Denny.

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Rawson, D. and Rigby, J. E. (1960). The functional anatomy of the cysticercoid of Choanotaenia crassiscolex (Linstow, 1890) (Dilepididae) from the digestive gland of Oxychihs cellarius (Mull.) (Stylomatophora), with some observations on developmental stages. Parasitology 50, 453-468. Rees, G. (1951). The anatomy of cysticercus Taeniae-taeniaeformis (Batsch, 1786) (Cysficercus fasciolaris Rud. 1808), from the liver of Raftus norvegicus (Erx.), including an account of spiral torsion in the species and some minor abnormalities in structure. Parasitology 41, 46-59. Reichenbach-Klinke, H. H. (1956). Die Entwicklung der Larven bei der Bandwurmordnung Tetraphyllidea Braun 1900. Abh. braunschw. wiss. Ges. 8,61-73. Reichenbach-Klinke, H. H. (1957). Artzugehorigkeit und Entwicklung der als Scolex pleuronectis Miiller bekannten Cestodenlarven (Cestoidea, Tetraphyllidea). Verh. deursch. zool. Ges. (2001.Anz., suppl. vol. 20), pp. 317-324. Reid, W. M. (1948). Penetration glands in cyclophyllidean oncospheres. Trans. Am. microsc. SOC.67, 177-182. Riser, N. W. (1955). Studies on cestode parasites of sharks and skates. J. Tenn. Acad. Sci. 30, 265-312. Riser, N. W. (1956). Early larval stages of two cestodes from elasmobranch fishes. Proc. helminth. SOC.Wash. 23, 120-124. Romer, A. S. (1967). Major steps in vertebrate evolution. Science, N.Y. 158, 1629-1 637. Rosen, F. (1918). Recherches sur Ie dCveIoppement des cestodes. 1. Le cycle tvolutif des BothriocCphales. Etude sur I’origine des Cestodes et leurs etats larvaires. Bull. SOC.neuchritel. Sci. nat. (1917-1918) 43, 241-300. Rybicka, K. (1 966). Embryogenesis in cestodes. In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 4, pp. 107-186. Academic Press, London and New York. R y l a d , B. (1961). [The problem of reservoir parasitism in Hyrnenolepididae.] Helminthologia 3, 288-293. RySad, B. (1964). Life-histories of cestodes parasitizing birds of the order Anseriformes. In “Parasitic Worms and Aquatic Conditions” (Eds R. Ergens and B. Rygavjr), pp. 107-112. Proc. of Symposium, Prague, Oct. 29-Nov. 2, 1962. Czechoslovak Acad. Sci., Prague. Sandeman, I. M. and Burt, M. D. B. (1972). Biology of Bothrimonus (= Diplocotyle) (Pseudophyllidea: Cestoda) : Ecology, life cycle, and evolution, a review and synthesis. J. Fish. Res. Bd. Canada 29, 1381-1395. Sawada, I. (1959). Experimental studies on the evagination of the cysticercoids of Raillietina kashiwarensis. Expl Parasit. 8, 325-335. Schiller, E. L. (1959). Experimental studies on morphological variation in the cestode genus Hymenolepis. I. Morphology and development of the cysticercoid of H. nana in Tribolilcm confusum. E x p l Parasit. 8, 91-118. Schmidt, G. (1970). “How to Know the Tapeworms.” Wm. C. Brown, Dubuque, Iowa. Scott, J. S. (1965a). The development and morphology of Polycercus lumbrici (Cestoda: Cyclophyllidea). Parasitology 55, 127-143. Scott, J. S. (1965b). Evagination of the cysticercoid in Polycercus lumbrici. Parasitology 55, 421-425. Silverman, P. H. (1954). Studies on the biology of some tapeworms of the genus Taenia. I. Factors affecting hatching and activation of taeniid ova, and some criteria of their viability. Ann. trop. Med. Parasit. 48, 207-215. Slais, J. (1966). The importance of the bladder for the development of the cysticercus. Parasitology 56, 707-71 3.

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Smyth, J. D. (1962). “Introduction to Animal Parasitology.” The English Universities Press, London. Smyth, J. D. (1964). The biology of the hydatid organisms. In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 2, pp. 169-219. Academic Press, London and New York. Smyth, J. D. (1967). Studies on tapeworm physiology XI. In vitro cultivation of Echinoccocus granulosus from the protoscolex to the strobilate stage. Parasitology 57, 11 1-133. Smyth, J. D. (1969). “The Physiology of Cestodes.” Freeman, San Francisco. Smyth, J. D. and Heath, D. D. (1970). Pathogenesis of larval cestodes in mammals. Helminth. Abstr. 39, 1-23. Southwell, T. (1925). A monograph on the Tetraphyllidea with notes on related cestodes. Liverpool School of Tropical Medicine, Memoir (New Series) No. 2. Liverpool University Press, Liverpool. Southwell, T. and Prashad, B. (1918). Methods of asexual and parthenogenetic reproduction in cestodes. J. Parasit. 4, 122-129. Spasskii, A. A. (1951). Anoplocephalate tapeworms of domestic and wild animals. In “Essentials of Cestodology” (Ed. K. I. Skrjabin), Vol. I. (Trans]. A. Birron and Z. S. Cole. Israel Program for Scientific TransIations, 1961). Spasskii, A. A. (1963a). Hymenolepididae. In “Essentials of Cestodology” (Ed. K. I. Skrjabin), Vol. 11. Akad. Nauk SSSR, Moscow. (In Russian.) Spasskii, A. A. (1963b). [Taxonomy of cestodes.] (Abstract). Mater. nauch. Konf. uses. Obsch. Gelmint., Part 11, pp. 107-110. Specht, D. and Voge, M. (1965). Asexual multiplication of Mesocestoides tetrathyridia in laboratory animals. J. Parasit. 51, 268-272. Stark, G. T. C. (1965). Diplocotyle (Eucestoda), a parasite of Gammarus zaddachi in the estuary of the Yorkshire Esk, Britain. Parasitology 55, 415420. 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. (1962). The organization, ontogeny, and orientation of the Cestoda. Q.Rev. Biol. 37, 23-34. Stunkard, H. W. (1967). Platyhelminth parasites of invertebrates. J. Parasit. 53, 673-682. Thomas, L. J. (1937). Environmental relations and life history of the tapeworm, Bothriocephalus rarus Thomas. J. Parasit. 23, 133-152. Thomas, L. J. (1941). The life cycle of Ophiotaenia perspicua LaRue, a cestode of snakes. Rev. Med. trop. Parasit. 7 , 74-78. Thurston, J. P. (1967). The morphology and life-cycleof Cephalochlamysnamaquensis (Cohn, 1906) (Cestoda: Pseudophyllidea) from Xenopus muelleri and X . laeuis. Parasitology 57, 187-200. Venard, C. E. (1938). Morphology, bionomics, and taxonomy of the cestode Dipylidium caninum. Ann. N. Y. Acad. Sci. 37, 273-328. Vik, R. (1963). Studies of the helminth fauna of Norway. IV. Occurrence and distribution of Eubothrium crassum (Bloch, 1779) and E. salvelini (Schrank, 1790) (Cestoda) in Norway, with notes on their life cycles. Nytt Mag. Zool. 11,47-73. Vik, R.(1964). Parasitological Review. The genus Diphyllobothrium. An example of the interdependence of systematics and experimental biology. Expl Parasit. 15, 361-380. Villot, F. C. A. (1883). MBmoire sur les cystiques des tBnias. Ann. Sci. nat. 6 Ser. Zool. 15, 1-61.

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Voge, M. (1960a). Studies in cysticercoid histology. 111. Observations on the fully developed cysticercoid of Raillietina cesticillus (Cestoda: Cyclophyllidea). Proc. helminth. SOC.Wash. 27, 271-274. Voge, M. (1960b). Studies in cysticercoid histology. TV. Observations on histogenesis in the cysticercoid of Hymenolepis diminutu (Cestoda: Cyclophyllidea). J . Parasit. 46, 71 7-725. Voge, M. (1961). Studies in cysticercoid histology. VI. Observations on the fully developed cysticercoid of Chounotaenia infundibulum (Cestoda: Cyclophyllidea). Proc. helminth. SOC.Wash. 28, 35-37. Voge, M. (1967). The post-embryonic developmental stages of cestodes. In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 5, pp. 247-297. Academic Press, London and New York. Voge, M. (1969). Systematics of cestodes-present and future. In “Problems in Systematics of Parasites” (Ed. G. D. Schmidt), pp. 49-72. University Park Press, Baltimore. Voge, M. and Heyneman, D. (1957). Development of Hymenolepis nana and Hymenolepis diminuta (Cestoda: Hymenolepididae) in the intermediate host Tribolium confusum. Univ. Calif. Publs Zool. 59, 549-580. Vogel, H. (1930). Studien iiber die Entwicklung von Diphyllobothrium. 11. Die Entwicklung des Procercoids von Diphyllobothrium latum. 2. ParasitKde 2, 629-644. Wardle, R. A. and McColl, E. L. (1937). The taxonomy of Diphyllobothrium latum (L.) in western Canada. Can. J. Res. 15, 163-175. Wardle, R. A. and McLeod, J. A. (1952). “The Zoology of Tapeworms.” University of Minnesota Press, Minneapolis. Weinmann, C. J. (1969). Larval development of Hymenolepis nana (Cestoda) in different classes of vertebrates. J. Parasit. 55, I 141-1 142. Wetzel, R. (1932). Zur Kenntnis des weniggliedrigen Hiihnerbandwurmes Dauainea proglottina. Arch. wiss. prakt. Tierheilk. 65, 595-625. Wetzel, R. (1934). Untersuchungen iiber den Entwicklungskreis des Huhnerbandwurmes Raillietina cesticillus (Molin, 1858). Arch. wiss. prakt. Tierheilk. 68, 221-232. Wetzel, R. (1936). Neuere Ergebnisse iiber die Entwicklung von Hiihnerbandwurmern. Verh. deufsch.zool. Ges. 1936, 195-200. Whitcomb, R. Unpublished information on migration of cestode segments. Widmer, E. A. and Olsen, 0. W. (1967). The life history of Oochoristica osherofi Meggitt, 1934 (Cyclophyllidea: Anoplocephalidae). J. Parasit. 53, 343-349. Wikgren, B.-J. P. and Gustafsson, M. K. S. (1971). Cell proliferation and histogenesis in diphyllobothrid tapeworms (Cestoda). Acta Acad. Aboensis (B), 31, 1-10. Williams, H. H. (1966). The ecology, functional morphology and taxonomy of Echeneibothrium Beneden, I849 (Cestoda: Tetraphyllidea), a revision of the genus and comments on Discobothrium Beneden, 1870, Pseucfanthobothrium Baer, 1956 and Phormobothrium Alexander, 1963. Parasitology 56, 227-285. WiSniewski, L . W. (I 932). Cyafhocepholus truncatus Paflas. 1. Die postembryonale Entwicklung und Biologie. 11. Allgeineine Morphologie. Bull. int. Acad. Sci. Cracocie, Cl. Sci. math. nut., S6r. B, Sci. nut. 2, 237-252 and 31 1-327. WiSniewski, R. J. (1971). Studies on the development of Nemu/oparatueniu routhwelli Fuhrmmn, 1934 and Gtr,troturnm paracygni Czapliliski et Kyiikov, 1966 (Cestoda, Hymenolepididae) in the intermediate hosts. Actii parasit. pol. 19, 49-61,

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Wisseman, C. L. Jr. (1945). Morphology of the cysticercoid of the fowl tapeworm, Raillietina cesticillus (Molin). Trans. Am. microsc. SOC.64, 145-150. Yamaguti, S. (1951). Early stages of postembryonic development of Nippotaenia chaenogobii Yamaguti, 1939 (Cestoda). Arb. med. Fak. Okayama 7 , 335-337. Yamaguti, S . (1959). “Systema Helminthum. Vol. 11. The Cestodes of Vertebrates.” Interscience Publishers, New York.

NOTEADDEDI N PROOF Addetirlum to p . 557

In an excellent, detailed article Professor G. Rees (Parasitology 66, 423-446 (1973) described the ontogeny of Tatria spp. (Amabiliidae). She shows that the metacestode develops as a precysticercus (see above pp. 522-523). However, when metacestode development within the arthroped is complete, the scolex with associated mid-body and terminal excretory pore becomes a neoplerocercoid free within the hollow hind-body. Furthermore, the neoplerocercoid initiates segmentation thus becoming a strobilo-neoplerocercoid (see above p. 521). In the gut of the definitive host this metacestode morphologically and functionally must be similar to that of Taenia taeniaeformis, at least for a brief period (see above p. 493). Such ontogenic similarity continues to suggest a close relationship between Amabiliidae and Taeniidae (p. 546, Fig. 11).

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Lungworms of the Domestic Pig and Sheep

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J H ROSE

Ministry of Agriculture. Fisheries and Food. Central Veterinary Laboratory. Weybridge. England

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Introduction ............................................ .-......................................... Pig Lungworms ................................................................................. A Species ....................................................................................... B. Geographical Distribution ............................................................... C Incidence .................................................................................... D Life-cycle .................................................................................... E Pathology in Definitive Host ............................................................ F Association with other Organisms ..................................................... G Immunity .................................................................................... H. Treatment and Control .................................................................. I11 Sheep Lungworms .............................................................................. A . Species ....................................................................................... B Geographical Distribution ............................................................... C Incidence .................................................................................... D Life Cycle.................................................................................... E Pathology in Definitive Host ........................................................... F Immunity .................................................................................... G Treatment and Control ..................................................................

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1. INTRODUCTION Lungworm infections of sheep and pigs are less important in causing disease than are other helminths which infect these animals This is undoubtedly one of the reasons why they have not been studied so extensively as some of these other helminths Nevertheless they have been the subject of investigation by workers in many parts of the world and there has been an appreciable increase in our knowledge of these lungworms in recent years so that a review of the literature is now opportune.

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I1.LUNGWORMS OF THE Pie A . SPECIES

Four species of lungworms infect the domestic pig. all belonging to the genus Metastrongylus . Two species are widely known. namely Metastrongylus 559

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elongalirs and M.puclendootectiis, and they have been the subject of investigation by numerous workers. M . salmi is restricted in its distribution and has not been the subject of much research, while the only reference to the fourth species M . madagascariensis appears to be the original description by Chabaud and Gretillat (1956). B. GEOGRAPHICAL DISTRIBUTION

M . elongatus and M . pudendotectus are cosmopolitan in their distribution. There are references to the presence of these lungworms in many parts of the world. M . salmi occurs in the Congo, Indo-China and the United States of America, Krause et al. (1969) found M . salmi in the wild boar in Germany, but not in the domestic pig; M . madagascariensis as its name implies was discovered in pigs in Madagascar but does not appear to have been found elsewhere. C. INCIDENCE

Numerous surveys have been made to assess the incidence of lungworms usually in pigs sent for slaughter at bacon factories or abattoirs. The precision with which these surveys have been carried out range from a simple assessment of infection made by incising the lung and examining for the presence of lungworms without any attempt to identify or count the worms, to thorough examinations in which the lungworm species have been identified and counted. The incidence of infection as recorded by different workers varies considerably. In Germany, Krause et al. (I 969) found M . elongatus only in 1*6% of 18 510 slaughter pigs examined and in 2.13 % of pigs which had died. Several surveys made in Great Britain have shown fairly low incidences of infection, for example, 13.08 and 25% (Robertson, 1937), 20.5% (Dunn et al., 1955) and 29 % (Whittlestone, 1957), but Jaggers and Herbert (1964) found that the incidence of infection in N. Wales varied from 8 to 65 % according to the time of year. Nevenic and Sibalic (1953) in Yugoslavia and Kelley and Sen (1959) in Nebraska, U.S.A. also found low incidences of infection. In other parts of the U.S.A. a higher incidence has been recorded. Ledet and Greve (1966) examined pigs in Iowa and found 48 % infected, while Sullivan and Shaw (1953) found lungworms in 5 1 % of a group of pigs from Oregon. Floch (1955) examined pigs sent to an abattoir in French Guiana and found that almost all of them were infected. When the species have been identified both M. elongatus and M. pudendotectus have usually been found together in mixed infestations, the former species usually being seen more frequently and in greater numbers than the latter, although Ledet and Greve (1966) found both species present in almost equal numbers. Ewing and Todd (1961) also found M. salmi present in mixed infestations but only 1 % of the infected pigs carried this species. A much higher incidence, 62.5 % of M. salmi was seen by Mattos (1943) in pigs slaughtered at an abattoir at Sgo Paulo. The incidence of infection varies according to the age of the pigs, as several workers have demonstrated. Mackenzie (l958a) made a very interesting survey

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on a single herd of pigs, examining animals of different ages which had been subjected to different management practices. Young piglets were kept indoors with the sow until they were between 7 and 14 days old; then they were put out to folding units on pasture. After weaning when 8 weeks old, pigs destined for fattening were taken indoors on to concrete floors and had no access to pasture. Gilts were selected for breeding at 5 months old and transferred to yards. All breeding stock was later grazed at pasture. Pigs were examined from two pastures, one of which (pasture A) had not been grazed by pigs for 3 years but before that time had been grazed intensively; the other (pasture B) had been free of pigs for 1 1 years. In addition a small group of pigs which had been reared indoors was examined. The pigs reared indoors were free from infection. Only one of the pigs grazed on pasture B was infected whereas most of the pigs grazed on pasture A were infected, albeit only lightly. No piglets between 2 and 9 weeks old were infected but 69.7 "/o of store pigs (10-1 5 weeks old), 59.5 of pork pigs (16-22 weeks old) and 53.2% of bacon pigs (23-30 weeks old) were infected, and an additional 13% of this last group showed residual lesions due to infection, the lungworms having been eliminated. A small number of the breeding stock was examined and had low grade patent infection. The species of lungworm present in the infected pigs were not identified. Variations in incidence related to age have been recorded also by Sivickis et al. (1958) who found an insignificant degree of infection in piglets under 2 months of age; whereas 75 % of pigs aged 4-6 months, and less than 4 % of pigs over 8 months old were infected. Neiland (1961) found lungwormsin 10.3 "/o of pigs aged up to 3 months, in 38.9 % of 6-8 months old pigs, in 55.3 % of pigs 9-12 months old and in 27.9 %of pigs over 1 year old. D. LIFE-CYCLE

The essential features of the life-cycles of M . elongatus and M . pudendotectus were elucidated by Hobmaier and Hobmaier (1929a, b, c). Subsequently other workers have confirmed their work, sometimes providing further information on points of detail. The adult worms live in the bronchi, usually in the secondary branches of the bronchioles. Eggs laid by the female worms pass up the trachea, are swallowed and pass via the alimentary canal to the external environment in the pigs' faeces. The eggs are ingested by earthworms, in which they hatch, and the larvae develop to the infective stage. The pig becomes infected by ingesting earthworms containing infective larvae. 1. Bionomics of the egg The embryonated eggs are able to survive for varying lengths of time in the free-living state depending upon the environmental conditions. Kates (1941) found that eggs in faeces on unshaded plots were destroyed in about 25 days, but when the faeces were buried at varying depths in the soil, eggs survived for a maximum of 381 days. Rose (1959a) reported that eggs in permanently moist soil survived even longer (a maximum of two years), but in soil which

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was not kept moist the longevity of the eggs varied from 5 to 12 months. Viability was tested by feeding the eggs to earthworms which were subsequently examined for lungworm larvae. The eggs are able to survive at temperatures below freezing point during the winter and they can survive in dry soil for several weeks, although they survive for a short time only when subjected to extreme desiccation (Rose, 1959a). Ustinov also (1963) has confirmed the ability of the eggs to survive for long periods. 2. Development in the intermediate host The lungworm eggs are ingested by an earthworm which acts as the intermediate host and they hatch in the anterior part of the alimentary canal. Probert (1969) studied serial sections of experimentally infected earthworms and observed first stage larvae in the crop and also in the process of penetrating the crop epithelium. From here the larvae migrate to the calciferous glands. Schuckmann and Zunker (1931) noted the presence of larvae in the lamellar sinuses of the calciferous glands. Schwartz and Porter (1938) estimated that as many as 93 % of the larvae were located in these glands. They regarded them as the preferred site of localization of the larvae and suggested that this was associated with the parasites' preference for an environment low in carbon dioxide. Probert (1969) considered that the larvae reached the calciferous glands via the peri-enteric blood sinuses and suggested that the configuration of the blood system of the calciferous glands was such that it acted as a filter for larvae carried forwards in the blood stream from the peri-enteric blood sinuses resulting in the localization of the larvae in the lamellar sinuses. He did not agree that an environment low in free carbon dioxide was a prerequisite for healthy larval development. He found that in earthworms which had been infected for 3 months and longer, third stage larvae were present in the entire length of the dorsal blood vessel of the eosophageal region, in the lateral hearts, and a few in the blood sinuses of the oesophageal wall. Larvae in the dorsal blood vessel were primarily in the region anterior to the anterior limits of the calciferous glands. Larvae were present throughout the length of these glands but most were in the main central body of the glands. Larvae were also present in the crop wall, particularly in the subepidermal blood sinuses, and a few were seen in the blood vessels of the anterior part of the intestine. In developing to the third stage the larvae increase in size and undergo two moults. Hobmaier and Hobmaier (1929a) described only one moult but Schwartz and Alicata (1929, 1931) observed two moults; they also studied the development of M. salmiin the earthworm intermediate host and found it to be essentially the same as that of M. elongatus. The first stage larvae are about 275-300 pm in length and they grow to about 650-690 pm by the time the larvae are at the third stage. The two moults occur in rapid succession and the early third stage larva retains the sheaths resulting from both moults. The rate of development of the larvae in the intermediate host varies with the temperature of the environment. Development to the infective stage takes 16 days at 22"-23"C, 21 days at 15"-16"C, and 219 days at 10°-ll"C (Rose, 1959a).

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The infective larvae can survive for long periods in the earthworm. Rose (1 959a) observed living M. elongatus larvae in artificially infected earthworms

after 18 months, and demonstrated the viability of larvae up to 11 months old by feeding them to guinea pigs. Tiunov (1967) has recorded longer periods of survival, 13 years in Octolasium lacteum, 23 years in Allolobophora calignosa, 3 years in Lumbricus rubellus and over 4+ years in Eisenia foetida. 3. Earthworm species which can serve as the intermediate host A number of different species of earthworms can serve as the intermediate host including: Eisenia foetida, E. austriaca, E. lonnebergi, E. nordenskoldi. Allolobophora nocturna, A . chlorotica, A . calignosa, Lumbricus terrestris, L . rubellus, Diplocardia spp. Dendrobena rubida, Lampito martiti Pheretima bahli, P . elongata, P. hovelli Perionyx excavatus, Nematogenia panamenis Octolasium lacteum, Bimastus tenuis.

There is, however, little information on the importance of the different species of earthworms in the transmission of infection, and the extent of infection in the field. In the Philippines a high incidence of infection has been seen in P. excavatus, N . panamensis and L . mauriti (Refuerzo and Reyes, 1958 ; Reyes and Refuerzo, 1967). Ustinov (1963) found lungworm larvae in six species of earthworms and considered Eisenia foetida to be the most important host. This earthworm inhabits dung heaps and several workers have used it in their experimental work. With modern methods of pig management this species will not be as significant in the transmission of infection as it apparently was in the area where Ustinov made his observations. 4. E#ect of lungworm larvae on the intermediate host Earthworms can be heavily infected experimentally. The larvae may become encapsulated in fibrous cysts, particularly in the oesophageal wall of the earthworm (Schwartz and Porter, 1938); Probert (1969) noted dense masses of cells forming spindle-shaped cysts in the interlamellar sinuses and considered them to represent a walling-off reaction to the presence of the lungworm larvae, although no degenerating larvae were present in the cysts. He also observed that large numbers of larvae in the blood system of the calciferous glands caused complete rupture of the lamellae and the blood sinuses became confluent, furthermore the larvae fed on the blood of the earthworm but no adverse effects were apparent.

5. Survival of the infective larvae outside the intermediate host If by some mishap the infective larvae are liberated from the intermediate host, they can survive for a surprisingly long time. Under moist conditions

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they may survive for as long as 62 weeks at 5-6°C and for 16 weeks at 10-1 1°C but they are rapidly killed by desiccation (Rose, I959a).

6. Development in the definitive host The lungworm larvae are transmitted to the pig when infected earthworms are eaten: the infective larvae are freed by digestion and penetrate the wall of the intestine to reach the lymph spaces; they migrate to the mesenteric lymph glands and pass via the thoracic duct and venous system to the lungs. They penetrate the capillary wall and pass to the alveoli; as they develop they migrate via the bronchioles to the bronchi and trachea. This phase of the life cycle was first demonstrated by Hobmaier and Hobmaier (1929b, c) and it has been confirmed subsequently by a number of workers. Schwartz and Alicata (1934) studied the development of the three species M . elongatus, M . pudendotectus and M . salmiand found no differences. The following account is therefore applicable to all three species. The larvae undergo two moults in rapid succession during the course of their migration. Schwartz and Alicata (1934) recovered advanced third stage larvae, larvae in the first parasitic moult and fifth stage larvae enclosed in two loose cuticles, from pigs killed 3 days after infection: advanced fourth stage larvae and fifth stage larvae devoid of sheaths were recovered from pigs 5 days after infection, and by 8 days after infection fifth stage larvae were present in the lungs. Mackenzie (1959) studied the development of M . elongatus in experimentally infected pigs and while confirming the earlier work added one or two additional points of detail: fourth stage larvae were obtained 24 h after infection from pepsin digest preparations of the caecum wall, and fifth stage worms were seen in the lungs seven days after infection. Mature male worms were seen on the 18th day and female worms containing embryonated eggs on the 25th day, but the full adult size was not reached until 40 days after infection. The longevity of the lungworm in the pig is not known. Dunn (1956) observed a peak egg production 5-9 weeks after infection as did Mackenzie (1959) in experimentally infected pigs. This was followed by a fall in egg output to low levels which persisted until the pigs were slaughtered at 28 weeks. The majority of lungworms are expelled during the first few weeks following infection but a small number may persist for a long time. E. PATHOLOGY IN DEFINITIVE HOST

Light to moderate infections cause no marked clinical symptoms but heavy infections can cause pneumonia and other respiratory diseases and may cause death. This has been demonstrated by Sullivan and Shaw (1953) in two pigs which they had infected with 200 and 400 earthworms respectively, each of which was infected with an average of 175 lungworm larvae. Other pigs which had moderate infections showed no obvious ill effects and there was no difference in the weight gains of these animals compared to those of uninfected pigs. Mackenzie( 1959) also reported no difference in weight gains as between infected and uninfected animals, as did Hollo (1966) who infected pigs with 50-4000

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larvae of A l . elongutus and nt.pudtrndotectuseither as single or mixed infections. The only clinical symptom was coughing. The pathological changes in the lungs consist principally of emphysema and the formation of small nodules. Several workers have described the pathology of both natural and experimental infections in varying degrees of detail (Belkin and Schletzer, 1936; Schwartz and Lucker, 1937; Casarosa, 1950; Sullivan and Shaw, 1953; Whittlestone, 1957; Mackenzie, 1958b, 1959; Ewing and Todd, 1961 ; Tamaski, 1962; Hollo, 1966; Tiunov, 1966). The work of Mackenzie (1959) is probably the most detailed account and is particularly interesting as he described the progressive pathology of experimental M . elongatus infections. 1. Macroscopic changes Macroscopic lesions were first seen in in the lungs 12 days after infection and consisted of early vesicular emphysema and small pale red areas of consolidation along the ventral margins of the lobes and occasionally on the dorsal aspect of the lungs. These changes became more extensive and by the 21st day there was marked emphysema with distinct consolidation which was most marked in infections of 35 days' or more duration. It consisted of well defined pink or plum red areas usually situated in the ventral aspect of the anterior lobes or in the lower border of the diaphragmatic lobes. Small grey sub-pleural nodules were present in lobules affected with emphysema at 4 6 6 0 days after infection. Enlargement of the bronchial lymph glands was seen 10 days after infection and persisted throughout the course of the disease. 2. Microscopic changes No significant changes occurred in the alimentary canal, mesenteric lymph nodes and other viscera as a result of migrating larvae. Changes in the lungs at $ 7 and 9 days after infection were associated with the larvae migrating through the lung parenchyma. There was an increase in the cellularity of the alveolar walls accompanied by occasional alveolar mononuclear cell exudates and some intra-alveolar haemorrhage. Phagocytosis of red blood corpuscles by macrophages was evident and some multinucleate giant cells were seen free in the alveoli. The tissue reaction around larvae consisted of a mild mononuclear cell reaction. Immature lungworms were found in the small bronchioles as well as in the parenchyma 10-12 days after infection and there was also a pronounced eosinophilia. A cellular exudate of eosinophils surrounded the larvae in the bronchioles which were ingesting eosinophils, mononuclear cells and red blood corpuscles. The increased cellularity of the alveolar walls persisted, owing to mesenchymal and small mononuclear cells and an increase in the number of alveolar wall cells. The alveoli were distended by early vesicular emphysema and microatelectasis. At the margins of cell lobes there were larger areas of collapse together with an interstitial infiltration of eosinophils causing consolidation of the lung tissue. Marked eosinophilic infiltration of the bronchial mucosa and early peri-

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bronchal hyperplasia were seen 2-3 weeks after infection. Hypertrophy of the smooth muscles of the bronchioles was seen 18 days after infection and became more distinct as the lungworms developed and emphysema became more pronounced. The lungworms in the bronchi were surrounded by a cell exudate in which eosinophils predominated. There was increased consolidation of the lungs associated with extensive pulmonary collapse, eosinophil and round cell infiltration, lymphoid hyperplasia and alveolar exudate around the developing worms. The enlargement of the lymph nodes continued and was associated with hyperplasia of the lymphoid tissue within the node and a dense infiltration of eosinophils into the sinuses and trabeculae. These changes became more severe later and persisted throughout the infection. From the 25th day onwards embryonated eggs were present in the lungworms and by the 30th day eggs were present in the bronchial exudate and occasionally in the parenchyma. Eggs and larvae aspirated into the lung parenchyma produced reactive changes, and giant cells were actively phagocytosing the parasites. A few oeosinophil granulomas were present in the lungs at about the 30th day but were more numerous after day 40, by which time the general eosinophil reaction was less severe than previously. The alveolar walls were infiltrated with alveolar exudate consisting of oedema fluid, alveolar macrophages, eosinophils, lymphocytes and some polymorphonuclear leucocytes and free giant cells. Some small giant cell granulomas were present in the alveoli. At the periphery of the lung, septa1 cells lining the alveoli formed a squamous or cuboidal epithelializing layer. As infection progressed, vesicular emphysema, lymphoid hyperplasia and smooth muscle hypertrophy all became more pronounced. At about 35 days after infection mucoid metaplasia of the bronchial epithelium commenced. There was an increase in the goblet cells in the epithelium of the small and medium sized bronchi, and subsequently many cells of the bronchi in infested areas became mucus secreting. Large quantities of mucus, lungworm eggs, mononuclear cells, eosinophils and varying numbers of polymorphonuclear leucocytes were seen in some of the bronchi. From 60 days onwards lymphoid hyperplasia, smooth muscle hypertrophy and mucoid metaplasia of the bronchial epithelium together with chronic vesicular emphysema, chronic bronchitis and persisting cellular infiltration became more pronounced. Small nodules consisting of a central focus of amorphous cellular debris, polymorphonuclear leucocytes or eosinophils were present, situated close to the pleural surface. The most pronounced changes 80 days after infection were emphysema and lymphoid hyperplasia. Infiltration and desquamation of the epithelium occurred as did bronchial ingrowths. Stenosis and partial occlusion of the bronchial lumina of the bronchi were also common. Coalescing lymphoid follicles formed nodules which are characteristic of long-standing infections. The lungworms tend to remain confined to the margins of the lobes. Mackenzie (1959) found a predilection for the upper posterior part of the lungs, thus limiting the area of pathological change; this would account for the limited harmful effects on the hosts.

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P. ASSOCIATION WITH OTHER ORGANISMS

Pig lungworms are said to act as carriers of swine influenza virus (Shope, 1941a, b, 1943a, b, 1955). The virus may infect the embryonated egg, persisting in the lungworm larvae while it is in the earthworm intermediate host. Shope suggested that the virus can persist in a latent form, being provoked into action by some stress factor such as the weather (Shope, 1955). Sen et al. (1961) confirmed Shope’s work: they demonstrated that pigs infected with lungworms reacted more vigorously to the virus than did pigs not so infected. Nayak et al. (1964) observed a gradual deterioration in the condition of pigs infected with lungworms and virus, resulting in a progressive consolidation of the lungs, whereas pigs infected with the virus alone gradually recovered and the lung lesions resolved. Kammer and Hanson (1962) however, were not so successful in their attempts to confirm Shope’s work. They were able to demonstrate propagation of the virus by the lungworm in only one of a series of experiments. Shope (1958a, b) also carried out experiments in which he demonstrated that pig lungworms can serve as a reservoir and intermediate host for hog cholera virus. He stated that the virus is normally carried in a masked form which must be provoked to pathogenicity by some stress factor, and Ascaris larvae were found to provide the necessary provocation. In his experiments only two out of 282 pigs developed hog cholera when fed earthworms containing lungworm larvae obtained from pigs with hog cholera. He maintained that the other pigs contained the virus in a masked form which became pathogenic in 13 of them either during the course of ascariasis or after recovery from this infection following the feeding of Ascaris eggs to the pigs. He also induced hog cholera by the intramuscular injection of suspensions of adult lungworms. There was a marked seasonal pattern to the infection and his experiments could be regularly reproduced during the first 4 or 5 months of the year. It has been suggested that the larvae of metastrongyles may also be reservoirs of Teschens virus (Breza and Belobrad, 1959). According to Mackenzie (1963) the clinical signs and lesions of virus pneumonia (epizootic pneumonia) were enhanced in pigs previously infected experimentally with lungworm. He noted a spread of virus pneumonia into the areas where the lungworms exerted their greatest effect. Pigs infected with lungworms when kept with pigs infected with virus pneumonia developed more extensive lung lesions than did control animals. G. IMMUNITY

There is not a great deal of information available about the immune response of pigs to infection with lungworms. EarIy work was concerned with demonstrating the development of immunity to reinfection following a previous infection. Schwartz and Lucker (1939, infected young pigs with between 500 and 2500 larvae and after the apparent termination of infection they were challenged with 500 larvae. Pigs which received the smallest initial infections did

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not appear to become immune, as at autopsy many adult worms were recovered from the lungs. Pigs which received the heaviest initial infection contained mainly juvenile worms without eggs when autopsied. It appeared that they had developed a degree of immunity which resulted in the retarded development of the worms of the challenge infection. Dixon (1968), however, produced evidence that light infections could invoke a degree of immunity. He infected young pigs with 540 lungworm larvae given in seven increasing doses over a period of 40 days, and gave challenge infections of 20000 larvae. These pigs had fewer lungworms when autopsied than did control pigs. The development of immunity resulting in the elimination of existing parasites was demonstrated by Jaggers (1965) and Jaggers and Herbert (1968). Jaggers (1965)foundlittleevidence of any inhibition of development and growth of the parasite in the immune host. As well as this acquired immunity some workers have provided evidence of an age resistance. Hollo (1966) found that 25-54% of larvae developed in 2 months old pigs whereas in pigs aged 44 months only 10 of larvae developed. A decrease in the level of infection with increase in age has also been described by Polzenhagen et al. (1969) who observed that the regular expulsion of worm eggs ceased when pigs reached the age of 8-9 months irrespective of the age at which they were infected. In recent years increasing interest has been shown in the serology of infection. Becht (1960) demonstrated that antibodies were produced 15 days after infection and that when third stage larvae were placed in immune serum in v i m , precipitates formed at the anterior end and at the excretory pore of exsheathed infective larvae. The antibodies did not, however, have any apparent ill effects, and larvae in immune serum did not die more rapidly than larvae placed in normal serum. The formation of precipitates around the larvae has also been described by Jaggers (1965). Using a complement fixation test Denev (1968) demonstrated the production of antibodies somewhat earlier, at 7-1 5 days after experimental infection, but he did not detect haemagglutinating antibodies until 20-25 days after infection. The antibodies persisted for 4-5 months and he was able to detect more infected pigs by serological tests than by faecal examination. Denev (1969) also studied other changes in the blood of experimentally infected pigs following initial infection and re-infection 30 days later. There was a rise in the phagocytic index and phagocytic number as from the first day after infection. This was followed by a fall, then a further rise which reached a maximum as from the 3rd to the 10th day, and a third rise as from the 14th day. Followingre-infection on the 30th day there was a rise in both the phagocytic index and number on the 9th day which reached maximum levels at about the 14th day. There was a slight increase in the numbers of eosinophils following both the initial infection and re-infection. Antibody production has also been demonstrated by means of an intradermal skin test (Jaggers, 1965; Jaggers and Herbert, 1968). These workers observed an increase in the serum gamma globulin after re-infection, together with a fall in serum albumin. An increase in the serum gamma globulin following infection was observed also by Tiunov (1966), who found that i n experi-

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mentally infected animals immunity was not well developed until 30 days after the initial infection. Barratt (1969) detected a homocytotropic antibody in the serum of infected pigs by the passive cutaneous anaphylactic test in recipient pigs, and recently Barratt and Herbert (1970) detected a skin-sensitizing antibody which produced immediate weal and flare reaction. The precise significanceof these various antibodies in the immune mechanism remains to be demonstrated. H. TREATMENT A N D CONTROL

Lungworm infection is not a major cause of disease in pigs. As already noted, moderate infections cause no apparent ill effects, and although experimentally, heavy infestations of lungworms have resulted in death there is no evidence to indicate that such heavy infections are common-place under farming conditions; nevertheless, a number of workers consider that lungworm infection causes sufficient ill effects to warrant treatment and control of the parasite. 1. Cyanacethydrazide In considering methods of treatment only the more recently developed anthelmintics will be referred to. At one time it appeared that cyanacethydrazide would provide an effective treatment against lungworms in pigs. Walley (1 957) reported that tests both in experimentally infected and naturally infected pigs demonstrated a high degree of efficacy against lungworm infection. Dick (1958) also reported favourably on its use, noting improvement in 95% of clinical cases. Other workers, however, were unable to confirm this degree of efficacy. Wikerhauser et al. (1959) observed clinical improvement in treated pigs but faecal examination and post mortem examination of the lungs revealed no satisfactory anthelmintic effect. Sen et al. (1960) found only slight efficacy against experimental infections in one trial ;the treatment appeared to dislodge many adult worms, but in two subsequent trials the drug had no apparent effect. Ewing et al. (1960) Colglazier and Enzie (1961) and Denev (1964) have all reported poor results with this drug. Sasaki (1963) found that the simultaneous injection of diethylcarbamate and cyanacethydrazide resulted in a 96 % reduction of lungworm eggs in the faeces and the elimination of most of the worms, but either drug alone was far less effective. Somewhat contrary results were obtained by Subramaniam et al. (1967) who found both cyanacethydrazide and diethylcarbamate citrate ineffective in treating naturally infected pigs. It would appear that cyanacethydrazide is somewhat unpredictable in its activity.

2. Tetramisole More recently Walley (1967) reported that another drug, tetramisole, was effective against lungworm infection in pigs, a dose rate of 15 mg/kg body weight given either mixed in the food or by subcutaneous injection was effective against adult worms. Subsequent work has confirmed the effectiveness of this drug. Dixon (1969) reported el’ficacies of 100 ?,; 98.7 and 98.4 against

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lungworms in experimentally infected pigs. Ueno et al. (1967) also found a single subcutaneous injection of 10 mg/kg body weight effective against both adult and immature worms, which were expelled by the pigs by coughing and vomiting. According to Lindquist et al. (1971) who used the L-isomer, levamisole, to treat naturally infected pigs, the drug acted very quickly and worms were expelled with vomitus 15 min after treatment. Ferguson (1971) has confirmed the high efficacy of L-tetramisole,administering the drug in the drinking water or feed. 3. Control Various methods of control have been advocated which aim at preventing pigs from ingesting infected earthworms, by ringing them to prevent rooting, by moving on to clean pastures or by keeping them in pens with concrete floors. In view of the long period that the lungworm larvae can survive in the earthworm, it may prove somewhat difficult in practice to find clean pastures. Keeping pigs in pens will of course reduce their chances of picking up infected earthworms but turning them on to pastures is advised as a means of controlling bacterial and virus infections which can spread in pigs kept under modern intensive systems. The control of these diseases is likely to be more important than the control of lungworms.

111. SHEEPLUNGWORMS A. SPECIES

At least 14 different species of lungworms are known to parasitize sheep, but only four species are widely distributed and have been the subject of extensive investigations. This review is concerned principally with these four species, i.e. Dictyocaulusflaria ;Muellerius capillaris ;Protostrongylus rufescens and Cystocaulus ocreatus. B. GEOGRAPHICAL DISTRIBUTION

D. $laria, M. capillaris and P. rufescens are cosmopolitan in their distribution, but present records indicate a more restricted distribution for C. ocreatus which is known to occur in various parts of Europe, the U.S.S.R., N. Africa and the Middle East. C. INCIDENCE

There are numerous records of the incidence of lungworm species from different countries. As with the pig lungworms the precision with which these records have been obtained varies from the examination of lungs, usually from healthy sheep sent to abattoirs for slaughter, and the identification of any lungworms recovered, to records based either on the presence of lesions in the lungs resulting from lungworm infection, or on the identification of lungworm larvae in the faeces. A consideration of the results of some of the surveys of the first type demonstrates variations in the incidence of the lungworm species not only in different parts of the world but sometimes in the same country. In Great Britain, Rose (1955a) examined lungs mainly from lambs sent to an abattoir for slaughter

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from farms scattered throughout the country ; M. capiffariswas the prevalent species, being present in 94.5 % of the lungs examined, C . ocreatus was present in 7 D.Ji1aria in 3.2 % and P. rufescens in less than 1 % of lungs. In the north of England, Thomas et al. (1970) found a similar incidence of M . capillaris, but a higher incidence of D. Jilaria. In young lambs the incidence of D. filaria ranged from 16.7 % t o 33-3%in June and reached apeak of 75 ”/, in the autumn, but the infections were light and only a few worms were recovered from individual lambs, with one or two exceptions. The heaviest infection was 245 worms in one animal. In older lambs and sheep D.$laria was seen in 3-10%. P. rufescens and C. ocreatus were not seen in this survey. Schanzel(l959) found D.filuriain 88.7 %, P. rufescensin 78.5 %, M. capillaris in 88-17% and C. ocreatus in 27.94 % of sheep lungs examined in Czechoslovakia. Kassai (1957a) in Hungary found 38.1 % of sheep lungs examined to be infected with D.filaria, and 55.7 % had nodules resulting from infection with protostrongylin lungworms. Examination of 305 lungs with nodules revealed C. ocreatus in 68.2 %, Protostrongylus spp. in 52-6%, M . capillaris in 19.7 % and Neostrongylus linearis in 10 %. The results of a survey carried out by Favati (1959) on 540 sheep in Tuscany are interesting in that he found N . linearis in 66-4%, although this species is generally rare; other species seen were C. ocreatus in 39.8 %, M. capillaris in 24-6%, D.filaria in 13.7 % and P. rufescens in 2.7 % of the sheep lungs. From these surveys it is evident that lungworm infection in sheep is common but the predominant species varies in different countries. An interesting feature of some of these surveys is the observation that the incidence of M. capillaris (Rose, 1955; Thomas et al., 1970) and P. rufescens (Schanzel, 1959) is higher in older sheep than in lambs, whereas the reverse is true of D.Jilaria (Olteanu, 1959a; Thomas et ul., 1970).

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D. LIFE-CYCLE

1. D. jilaria

The life-cycle of D.filaria is direct. The adult worms live in the bronchi and trachea of the host. Eggs which are embryonated when laid, pass up the trachea, are swallowed and pass into the alimentary canal. Hatching takes place in the small intestine and the first stage larvae are evacuated with the faeces. They develop to the infective stage in the external environment. Guberlet (1919) and Daubney (1920) described the morphologicaI changes involved in the development of the larvae to the infective stage. They undergo two moults but do not increase in size. The loose cuticle resulting from the first moult is retained until after the infective stage has been reached so that for a time the infective larva is enclosed in two loose cuticles, but the outer cuticle is shed. The infective larvae are ingested by sheep with herbage while grazing and develop to the adult stage within the host. (a) Bionomics of the free-living larvae. Rose (1955b) studied the bionomics of the free-living larvae of D.filaria, in particular the effects of humidity and temperature on development and longevity. Under controlled conditions in the laboratory the larvae exhibited a marked adaptation to low temperature. Infective larvae survived continuous freezing at a temperature range of - 1.1

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to - 76°C fur a maximum period of 19 days, while first and second stage larvae survived for a maximum of I2 days. At a temperature range of 3-6°C larvae survived in moist faeces for a maximum of 48 wccks although there was a heavy mortality of larvae during the first few weeks. At 23-25°C larvae survived for a maximum of only 12 weeks. When faeces dried out slowly longevity of the contained larvae was reduced to a maximum of 14 weeks at 3-6T and of 4 weeks at 23-25°C. The infective larvae were more resistant to the adverse effects of desiccation than were the first and second stage larvae. Guberlet (1919) and Daubney (1920) also, noted the ability of larvae to survive at low temperatures and Kauzal(1933) observed the adverse effects of high temperatures on the larvae. Development of the larvae can take place over a wide temperature range. Kauzal (1933) reported that development could take place at 45°C. Rose (1955b) also observed development at this low temperature: he studied rates of development at 5, 10, 15, 20 and 25°C and noted that the infective stage was reached after a minimum of 20, 8, 6, 5 and 5 days respectively. Shanzel(l958) also observed the rates of development at similar temperatures but surprisingly did not see any development at temperatures between 0 and I O T , although at higher temperatures the rates of development were similar to those recorded by Rose (1 955b). On grass plots in S.E. England larvae developed to the infective stage in five weeks during the winter, whereas in the summer development was complete in less than one week (Rose, 1955b). In the semi-desert zone of the Bukhara region, Aliev (1959) found that larvae in faeces completed their development in 14-7 days from October to May but from May to September they died before they reached the infective stage. The infective larvae are active, migrating from the faeces to the herbage. They can migrate up the grass blades but the majority of larvae tend to remain on the lower levels of the herbage, particularly during the winter months when low temperatures limit their activity (Rose, 1955b). The longevity of the larvae on pastures has been investigated by workers in different parts of the world. There are some differences of opinion regarding the ability of larvae to survive on pasture during the winter. Wetzel (1945) concluded that the infective larvae were unable to survive the winter and Borodina (1957) noted that the pre-infective larvae died during the winter. Olteanu (1959a) considered that larvae would survive the winter providing there was good snow cover. Other workers, however, have observed that infective larvae survived well throughout the winter, even in one instance when the temperature dropped to - 30°C (Borchert and Timm, 1949; Rose, 1955b, 1965a, b; Golubev, 1957a; Erhardova, 1962). It was demonstrated in field experiments carried out in S.E. England (Rose, 1965b), that larvae which have survived on pastures throughout the winter can infect lambs in the following spring. The larvae survive less effectively on pastures during the summer, particularly during dry weather. In S.E. England infective larvae survived for only 7 weeks from June to August (Rose, 1955b). In Rumania Olteanu (1959a) noted that larvae on pastures died rapidly in July and August. Davudov (1 97 I ) observed the migratory activity of larvae in soil. Second and

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third stage larvae were put into soil in pots which were kept either in the laboratory or out of doors. The larvae were most active at temperatures of 9-49-8°C and at a relative humidity of 63-90 %. Under these conditions they migrated on the surface and into the soil. On the 4th and 5th days most larvae were found at a depth of 3-8 cm in the soil and some migrated to a depth of 22 cm. Third stage larvae survived for a maximum of 59 days at a temperature range of 15-1 8°C. (b) Development in the host. Sheep become infected by ingesting infective larvae on the herbage while grazing. The development and migration of the lungworm in the definitive host were described by Hobmaier and Hobmaier (1929d), Kauzal (1933), Goldberg (1952) and Anderson and Verster (197 1). The infective larvae in the intestine exsheath, penetrate into the wall of the small intestine and pass into the lymphatic system. Eighteen hours to 6 days after infection they are present in large numbers in the mesenteric lymph glands where they undergo the third moult to become fourth stage larvae, passing via the lymph stream to reach the heart and thence to the lungs by the bloodstream. They migrate through the walls of the alveoli into the bronchioles and then move into the bronchi. Development to the adult stage is completed about 18 days after infection, and egg-laying may start a few days later, but first stage larvae are not usually seen in the faeces earlier than 26 days after infection. Anderson and Verster (1971) observed considerable numbers of larvae in the lung tissue from day 8 to day 14 after infection but from day 12 onwards there was an increase in the number of larvae reaching the bronchi and trachea. A few larvae were found in these locations as early as the 4th day after infection. Verster et al. (1971) studied in detail the development of D.filaria in experimentally infected lambs. The parasitic male 3rd stage, 3rd moult, 4th stage, 4th moult and 5th stage were reached on days 1-2,3,4,5-6 and 8 respectively after infection. The female worms developed somewhat more slowly than the males, being later by 1-2 days. By day 28 both male and female worms were sexually mature. The larvae underwent little growth on developing to the 5th stage; the main period of growth was during the maturation of the 5th stage worms and by day 28 they had not reached their maximum size. 2. M . capillaris, P. rufescens, and C. ocreatus The life-cycles of M . capillaris, P . rufescens and C. ocreatus are indirect, involving a molluscan intermediate host. Eggs laid by the female worms hatch while still in the lungs to release the first stage larvae which pass via the trachea to the alimentary canal and are evacuated with the faeces. Joyeux and Gaud (1946) and Joyeux and Baer (1951) suggested that the first stage larvae develop into second stage larvae while in the bronchioles but this view is not generally accepted. The first stage larvae infect a molluscan intermediate host wherein they develop to the infective stage. Sheep ingest the infected molluscs whle grazing and the larvae then develop to the adult stage in the definitive host. (a) Bionomics of the first stage larvae (i) M . capillaris. Rose (1 957a) studied the bionomics of the free-living first 21

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stage larvae. He found that larvae in faecal pellets could survive on pastures in S.E. England for a maximum of 44 weeks. Some larvae migrated on to the herbage and on to the soil but the majority remained in the faecal pellets; he suggested that the larvae reach the intermediate host because it is attracted to faecal pellets as a source of food. Nickel (1960) also observed long periods of survival, up to 6 months when larvae were in the soil and therefore protected from drying and from direct sunshine, but when faecal pellets were exposed in open situations during June to August the larvae died within 64 days. A number of workers have made observations on the first stage larvae in the laboratory. Morgan (1929), Pavlov (1937) and Williams (1942) all noted that the first stage larvae could withstand desiccation for several days but Hobmaier and Hobmaier (1930a) maintained that they were killed within a few hours when dried. In an attempt to resolve these conflicting views Rose (1957a) studied the effect of desiccation on first stage larvae separated from faeces and kept in atmospheres of known humidity. At a relative humidity of 25 larvae survived for a maximum of 9 days whereas at a relative humidity of 75 % larvae survived for a maximum of 14 days. They survived for much longer in faeces which were allowed to dry naturally. A few living larvae were recovered from dry faeces after a year at room temperature and at a temperature of 23-25°C larvae survived for several months. These experiments provided ample evidence of the ability of the larvae to survive under dry conditions. The first stage larvae are capable of surviving continuous freezing for several days without any mortality and a few survived for 12 days. Bright sunlight, however, proved lethal and resulted in complete mortality within l h (Rose, 1957a). Gevondian (1954) considered that there were two distinct types of M. capillaris larvae, a summer type larva which develops in active molluscs and a winter type which develops in resting molluscs. (ii) P. rufescens. The bionomics of the first stage larvae of P. rufescens have not been studied in detail. Hobmaier and Hobmaier (1930b) reported that larvae lived for a year in water, and Pavlov (1935) noted that they survived in humid conditions for 20-30 days while Davtian (1937) found that they could survive for 4-5 months and were resistant to desiccation but were rapidly killed by bright sunlight. Matekin et al. (1954) found larvae of P . rufescens and other Protostrongylus species in faeces gathered from sub-alpine pastures in Central Asia which had been on the ground for not less than 9 months, indicating their ability to survive well under cool conditions. (iii) C. ocreatus. Little information on the bionomics of C. ocreatus appears to be available. According to Schulz and Boev (1940) they can live for 2-3 months under both moist and dry conditions.

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(b) Development in the intermediate host (i) M. capillaris. The life-cycle of M. capillaris was elucidated by Hobmaier and Hobmaier (1929d, 1930a) and Hobmaier (1934). Development of the larvae to the infective stage takes place in the foot of a land mollusc. They demonstrated that species of Agriolimax, Limax, Succinea, Arion, and Monacha could serve as the intermediate host.

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This phasc of the life-cycle has been confirmed by a number of workers including Pavlov (1937), Schulz and Boev (l940), Williams (1942), Davtian (1945, 1948a), Joyeux and Gaud (1946), Gerichter (1948, 1950, 1951), Mapes (1949), Mapes and Baker (1950), Rose (1957b), Rhenova (1955), Golubev (1957b), Kassai (1958a), Egorov (1960), Rybak (l962), and Beresford Jones (1966). Most of these workers have either added to the list of molluscs which can serve as the intermediate host or have contributed more knowledge to some aspect of this phase of the life-cycle. The first stage larva, having penetrated into the sole of the mollusc’s foot, increases in size and undergoes two moults to develop into the third stage larva which retains both of the loose cuticles resulting from the moults. The rates of development as recorded by different workers vary. The Hobmaiers (1929d) and Pavlov (1937) found that development to the third stage took 12 days. Williams (1942) observed third stage larvae in Hyalina cellaria and Milax sowerbyi at 14 days after infection, whereas in Zonitoides arboreus, infective larvae were seen by Mapes and Baker (1950) after 27 days. The results of Gerichter’s (1951) observations are quite different. He noted that some mollusc species were more susceptible to invasion than were others ; furthermore there were marked differences in the extent and intensity of infection and also in the rate of development of the larvae. He classified the molluscs into appropriate and inappropriate hosts. He also observed that the third stage larvae underwent a number of changes and distinguished between pre-infective and infective larvae. In an appropriate mollusc species, development to the infective stage took 35 days at 30°C and 5 months at 48°C. Rose (1957b) studied the rates of development at 5, 10, 15, 20 and 25°C in Agriolimax reticulatus and A . agrestis and found there to be minima of 98, 37, 18, 13 and 8 days respectively to reach a stage of development comparable to the infective stage as described by Gerichter (1951). He noted that the third stage larvae underwent the changes observed by Gerichter (1 951) and investigated experimentally the effect of artificial digestion on the larvae. Those which had developed for 14, I 5 and 16 days in the mollusc and whch were comparable to Gerichter’s pre-infective larvae, were killed after 1 h whereas older larvae, comparable to the infective larvae, were alive and active after this time. He agreed with the suggestion that the third stage larvae undergo a process of development but pointed out that the only way to ascertain whether there was any difference in the infectivity of the pre-infective and infective larvae would be to try and infect the final host with them, a point reiterated by Kassai (1958a), who did not accept this distinction between pre-infective and infective larvae. Development of M . cupillaris can take place in a considerable number of species of land molluscs and also freshwater molluscs some of which are listed in Table 1. Many of them have been infected experimentally in the laboratory. Heavy infections do not appear to harm the mollusc. In those mollusc species in which development can be completed there is no obvious immune reaction and re-infection is possible. The lungworm larvae appear capable of surviving until the mollusc dies and the infective larvae can survive for a short time after death of the intermediate host (Hobmaier and Hobmaier, 1929; Pavlov, 1937; Rose, 1957b).

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The incidence of natural infection amongst molluscs tends to be low and the numbers of larvae in individual molluscs are small (Pavlov, 1937; Williams, 1942; Mapes, 1948; Mapes and Baker, 1950; Gerichter, 1951 ; Rose, 1957b; Egorov, 1960). Not all of the mollusc species in which development of M . capillaris can take place are necessarily of significance in the transmission of infection to sheep. Only those species accessible to grazing sheep will fulfil this function. In Great Britain species of Agriolimax, the common grey field slug, are important in t h s respect. These slugs are widely distributed and are likely to be found in most places where sheep graze. They are active throughout the winter in S.E. England where the winters are mild but in places where the winters are colder their activity will be reduced. Dry summer conditions also reduce the activity of the slugs during the daytime but they can be found on the pastures in the early morning and late evening. The widespread distribution of these slugs and their availability to the grazing sheep is an important factor in the high indicence of M . capillaris in Great Britain. Gerichter (1951) found Helicella barbesiana naturally infected in Israel. Abida frumentum is considered to be important in the transmission of infection in Hungary (Kassai, 1957c) while Succinea pfeifleri, S. putris, Trichia hispida (Golubev, 1957b), A . agrestis and Petinella petronella (Rybak, 1962) are regarded as important in the U.S.S.R.Mapes and Baker (1950) found Derocercas reticulaturn naturally infected in the U.S.A.;the levelof infectionwaslowwith an averageof 5 larvae per individual infected slug. (ii) P. rufescens. The development ofP. rufescens in the intermediate host was first described by Hobmaier and Hobmaier (1930b) who infected three species of Helicella, i.e. H . ericetorurn,H . obvia and H . bolli. Development was similar to that described for M. capillaris and the infective stage was reached in 12-14 days. Davtian (1937) described the development of a species which he regarded as P. rufescens, but his description differed markedly from that of the Hobmaiers, and it is now generally accepted that the species he studied was not P . rufescens. Joyeux and Gaud (1946) observed development to the infective stage in 15-20 days. Gerichter (1951) endeavoured to infect a number of mollusc species but development of the larvae to the infective stage was completed in three species only, i.e. H. barbesiana, H . vestalis joppensis and Monacha syriaca. The rate of development was very slow, the second stage was reached 35-38 days after infection and the infective stage 6 days later at an optimum temperature range of 2O-3O"C. Development did not take place at 8°C. He noted that the infective larva was enclosed in a rigid brown puparium-like casing. Other workers including Schulz and Boev (1940), Boev and Wolf (1940), Davtian (1945, 1948a, 1949), Kassai (1957a) and Zmornay and &arc (1960) have studied this phase of the life-cycle and have added to the number of rnollusc species which can serve as the intermediate host (see Table I). According to the latter workers, the larvae developed twice as rapidly in young molluscs as in old ones. There appears to be little information about the significance of different mollusc species in the transmission of infection to sheep. Pavlov (1937) considered Helicella obvia to be the most important intermediate host of P.

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rufescens in Bulgaria. Gerichter (1951) in Israel found 17.2% of 500 H. barbesiana infected with lungworm larvae. P. rufescens was present in 22% of the infected snails but the level of infection was very low. Helicella species tend to be

restricted in their distribution, preferring dry habitats, so that the area in which they can transmit infection to sheep will be similarly limited. Zmornay and Svarc (1960) infected Monochoides incarnata, Succinea putris, Zebrina detrita Cepaea vindobonensis, Derocercas re ticulatum, Euomphalia strigella, Fruticicola fruticum, H. obvia and Helicigonafaustina. They considered that one or another species occurred in practically all areas where sheep graze in Slovakia. In Hungary, Kassai (1957d) found Protostrongylus spp. larvae in Helix pomatia, Helicella obvia, Zebrina detrita, Cepaea vindobonensis, Theba carthusiana, Abida frumentum and Chondrula tridens. (iii) Cystocaulus ocreatus. The life cycle of C. ocreatus was first elucidated by

Davtian (1940) who subsequently endeavoured to infect different species of land and freshwater molluscs. Development to the infective stage was completed readily in a number of species including Pupilla spp., Chondrula spp., Helicella spp. and Levantina escheriana (Davtian, 1945).The process of development is essentially the same as that described for M. capillaris. At an optimum temperature range of 27-32°C development to the infective stage took 18-21 days and at a range of 12-17°C it took 3-5 months. Gerichter (1951) observed a similar rate of development: at a temperature range of 20-30"C, the infective stage was reached in 18-19 days whereas at 4 8 ° C the larvae developed very slowly and after 5 months they were not ready to undergo the first ecdysis. These rates of development were seen in Helicella barbesiana, H. vestalis joppensis, Monacha syriaca and Limaxflavus which he regarded as appropriate TABLE I Some molluscan intermediate hosts of the Protostrongylitieae lungworms of sheep ~~

Muellerius capillaris Agriolimax agrestis, A. reficulatus, Arion ater, A . subfuseus, A . hortensis, A. circiimscriptus,A. empiricorum, Arionaf a arbustorum, Cepaea vinrloboriensis, C. hortensis, Derocercas reticulatum, Fruticicola hispida, Gyraulus albus, G . laevis, Helix pomatia, H. hortensis, Helicella acuta, H . obvia, Hyalina cellaria, Milax sowerbyi, Zonitoides arboreus, Trichia hispida, Succinea pfeiferi, S.putris. Protostrongylusrufescens Chondrula tridens, Cepaea vindobonensis, Deroceras reticulatrim, Eucomphalia strigella, Fruticola fruticum, Helicella obvia, H. barbesiana, H. rugosiuscrrla, Helicigona faustina, Monachoidas incarnata, Theba carthusiana,Zebrina detrita, Succina putris, Monacha syriaca, Retinella nitellina. . -

_

_

~

~~

~

Cystocaulus ocreatus Agriolimax agrestis, A . reticulabas, Cepaea vindobenensis, Chondrri la fridens, Cochlicella acuta, Helix pomatia, Helicella obvia Theba carfhusiana, Zebrina detrita, Euparypha pisana, Chondrula septemdentata, Monarha syriaro, Retinella nitellina.

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intermediate hosts. In species of Levantina development to the infective stage took several months and he considered them to be inappropriate intermediate hosts. Kassai (1958a) infected Zebrina detrita, Tlieba carthusiana and Cepaea vindobonensis and development to the infective stage took 30-40 days at a temperature range of 20-21°C. Other mollusc species, some of which are listed in Table I, can serve as the intermediate host for C. ocreatus. Kassai (1958a) studied in some detail the factors influencing development of C. ocreatus in the intermediate host. He found that in artificially infected active and aestivating snails kept under identical conditions in the laboratory the rate of development was the same generally, but in naturally infected snails aestivating after being collected development was retarded, and he suggested that this was because of the reduced nutritive levels of tissue fluids in the snail which are utilized by the larvae, following a period of aestivation. Working with Heficelfa obvia, he found that the age of the snail did not affect the development of larvae. There is little information available about the incidence of natural infection in molluscs and their significance in the transmission of infection to sheep. Gerichter (195 1) found C. ocreatus in 64 % of H. barbesiana which were infected with lungworm larvae. Rose (1965a) found Agriolimax species naturally infected on pastures in S.E. England. Joyeux and Baer (1951) considered Euparyphapisana to be the main intermediate host for C. ocreatus in Morocco. In Hungary Kassai (1957d) found C. ocreatus as well as Protostrongylus spp. in seven mollusc species which were naturally infected but he considered 2. detrita, C. vindobonensis and T. carthusiana to be particularly favourable intermediate hosts (Kassai, 1958). Sheep become infected with these three lungworm species when they ingest infected molluscs while grazing. The infective larvae are released from the mollusc during digestion, and they then pass to the lungs by the route described for D.$faria. M. capiffarisand C. ocreatus develop to the adult stage within the tissues of the lungs but P . rufescens completes its development in the bronchioles and bronchi. It has been suggested that M . cupiflarisadults live for some time in the arteries and arterioles (Cameron, 1933), but no evidence of this was found by Rose (1958) in experimentally infected lambs. All stages of development from the early fourth stage larva to the adult worm were found in nodules in the lung tissue. Fourth stage larvae were present in very small nodules while adult worms were found in larger nodules. Many of the nodules contained only a single worm and it appeared that female worms isolated in this way would be unable to produce fertile eggs. Occasionally adult M . capillaris may be found in the bronchioles; they appear to have been released by the breakdown of the tissues in nodules containing first stage larvae and adult worms. Development to the adult stage takes 25-38 days for M . capilfaris and P. rufescens, and according to Sogoyan (1955) 30 days for C. ocreatus. All three species can live for a considerable time in the sheep. Rose (1959b) recovered M. capillaris larvae from an experimentally infected sheep kept indoors for a period of 2 years while Kassai (1962) observed the evacuation of larvae of P . rilfi'scms, h l . capilluii~,C'. ocrea!ii.v and N . I i t i w i Y for periods of 28, 44, 65

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and 67 months respectively. He noted that C. ocreatus could live for 2 years

after the production of larvae had ceased and suggested a life span of at least 4-7 years for M . capillaris, C. ocreaiiis and N . linearis. E. PATHOLOGY IN DEFINITIVE HOST

1. D. jilaria

D.jilaria is the most pathogenic of the four lungworm species. Sheep show distinct clinical symptoms when heavily infected. The first of these is a cough, which may occur as early as 16 days after infection but usually it occurs somewhat later, coinciding with the development of worms to the adult stage in the bronchi and trachea. Goldberg (1952) observed intermittent coughing from the 19th to the 43rd day following experimental infection of lambs. Shallow rapid breathing, lung rales, mucus drooling from the nostrils, loss of appetite and loss of weight are other clinical symptoms. Death may occur following heavy experimental infections in lambs. Michel (1954) infected lambs with 4000-100000 larvae and they all died within 21-35 days. At autopsy variable degrees of oedema were seen in the lungs. He postulated that the occurrence of the oedema was associated with the process by which the worms were eliminated from the lungs. Subsequently he recorded an outbreak of lungworm infection in lambs some of which had died without showing any symptoms of infection although at autopsy their lungs were found to be oedematous. Shortly afterwards typical symptoms developed in the remainder of the flock. Death due to lungworm infection does not appear to be common amongst naturally infected animals, however, probably because of the rapid loss of worms which prevents heavy infections from being built up. In general, sheep infected with lungworms are likely to be infected with gastro-intestinal nematodes, and D.fiIaria contributes to the general loss of condition resulting from the mixed infection. The pathological changes resulting from infection have been described by several workers (Romboli, 1953;Tsvetaeva, 1954;Panasiuk, 1959; Wandera, 1967; Ramachandran, 1967; Sedlmeier et al., 1969). The lungworm larvae cause haemorrhage and local irritation when they pass from the capillaries to the alveoli. Worms in the bronchioles and bronchi cause a bronchitis which spreads into the peribronchial tissue. The bronchi may become blocked with worms and mucus exudate causing collapse of the lung tissue which may become infected with bacteria, giving rise to a more extensive pneumonia. A compensating emphysema may occur around the collapsed areas. Histological examination shows a thickening of the alveolar walls with metaplasia, and the alveolar epithelium becomes cuboidal. Maerophages, plasma cells, giant cells, many leucocytes and eosinophils are present in the periphery ofthe alveoli. The epithelium of the bronchi shows marked desquamation and leucocytes and eosinophils infiltrate the bronchial mucosa, submucosa and muscle. The mediastinal and bronchial lymph nodes exhibit swelling and oedema.

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2. M.capillaris Infection with M . cupillaris results in no obvious clinical symptoms but experimentally infected lambs have been shown to gain weight less rapidly than uninfected lambs (Rose, 1959b). The worms live in the lung parenchyma and give rise to nodules ranging in size from less than 1 mm to several mm in diameter. A number of nodules may become confluent and cover larger areas. The form and structure of these nodules have been described in varying degrees of detail by Kassai (1957b), Rose (1958), Goto and Fujihara (1957, 1961) and Beresford-Jones (1967). The smaller nodules consist of an area of haemorrhage with capillary congestion and usually contain a fourth stage larva surrounded by eosinophils but lymphocytes and macrophages may be present also. Somewhat larger nodules contain immature or mature adult worms surrounded by an inner zone of eosinophils and an outer zone of macrophages, eosinophils and lymphocytes. Other nodules contain dead and disintegrating worms. The centre of the lesion may be necrotic due to the degeneration of the accumulated leucocytes or it may be calcified. The largest nodules contain eggs, first stage larvae and adult worms. The larvae break down the tissues and are released into the alveoli and bronchioles, but adult worms are rarely seen in the bronchioles.

3. P. rufescens Infection with P. rufescens rarely produces obvious clinical symptoms. The adult worms usually live in the small bronchioles but they may also occur in nodules together with first stage larvae. The changes in the lungs resulting from infection with P. rufescens or other Protostrongylus spp. have been described by McFadyean (1920), Pen-Lin (1 946), Kassai (1957), Ramachandran (1967) and Seidlmeier et al. (1 969). The latter workers maintain thatP. rufescens does not produce worm nodules whereas Kassai (1957b) describes and illustrates nodules resulting from infection with P.rufescens and Pen-Lin (1946) also describes nodules due to infection with a Protostrongylus sp. Changes occurring in the lung in response to the parasite involve a general cellular reaction consisting of macrophages, lymphocytes and eosinophils. There is an increase in the numbers of lymphoid cells in the sub-pleural region sometimes resulting in the formation of lymph nodules with germinal centres. Areas of consolidation may be present with undeveloped eggs and adult worms in the alveoli. Eggs and larvae may be surrounded by macrophages with the formation of pseudo-tubercles and giant cells. Near to collapsed and consolidated areas of the lungs the alveoli and terminal bronchioles may be dilated and the walls of the alveoli broken down. There may be a compensatory oedema and hypertrophy of muscle bundles in the bronchiolar ductules causing obstruction. Collapse of the lung tissue can result in so-called “muscular cirrhosis” of the lungs, usually associated with a compensatory emphysema. 4. C.ocreatus This species is found in nodules in the lung tissue and does not give rise to obvious clinical symptoms.

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58 1

Kassai (1957e) infected sheep with 1000 C. ocreatus larvae and observed decreased weight gains as compared to uninfected sheep, beginning on the 78th day after infection. Sogoyan (1950) described small nodules in the intestines of lambs 24-72 h after they were infected experimentally. Larvae reach the lungs 48 h after infection and give rise to small nodules. These have been described by Kassai (1957b). Some of these nodules are black in colour and usually contain an adult worm in a surrounding cyst-like structure. The black appearance of the nodule is due to the colour of the intestine of the lungworm. Other nodules are similar to these caused by M . capillaris, some contain adult worms and first stage larvae, others dead worms. According to Sogoyan (1955) by the 68th day after infection the tissue reaction consists of exudative and proliferative inflammatory foci with the obliteration of alveoli. The protostrongylin lungworms apparently cause little harm to the host but heavy infections probably reduce the general health of the sheep and predispose the lungs to bacterial infections. F. IMMUNITY

1. D . filaria Experimental infections of D. filaria in lambs frequently fail to become patent. Kauzal(l934) observed a marked variation in individual susceptibility to infection, lambs 5 5 7 months old were more difficult to infect than were younger lambs. Nickel (1962) found that patent infections could be established most successfully in lambs by giving them a number of small doses of infective larvae. This observation was confirmed by Michel (1968). He infected lambs and yearlings and found that there was an early reduction in the numbers of worms present in the lungs consequently the infection frequently failed to become patent. This was less likely to occur when the number of infective larvae was small, if the host was young or if it was so severely affected that it died. He suggested that the reduction in worm numbers was a consequence of an immune response by the host; however, as it appeared from his experiments that a limited decrease of worm numbers occurred when conditions did not favour a vigorous host response he suggested that there were some processes by which worms were lost or destroyed and that one of these was not due to immunity. In subsequent experiments, however, worms were found to be carried out of the Iungs in the bronchial mucus (Michel, 1969a). By inserting a tracheotomy tube into the trachea it was possible to collect the worms expelled with the mucus. Most of the worm loss occurred between the 12th and 30th day after infection. The administration of cortisone, which is known to inhibit immune mechanisms, had no effect on the rate of loss, and he concluded that an immune response was not involved in this loss of worms following the initial infection of a susceptible host. Michel suggested that this indicated that the sheep was not a good host for D.ji1aria (Michel, 1969b, c, d). Immunity does develop following an initial infection. Kauzal (1934) was unable to re-infect lambs, as was Michel(l956). By examining resistant lambs 7,9 and 14 days after re-infection Michel (1956) demonstrated that only small numbers of larvae reached their lungs, but they did reach the mesenteric lymph

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glands (Michel and Sinclair, 1963). By intravenous injection of infective larvae, the mesenteric lymph glands were by-passed, then the number of worms recovered from the lungs of resistant lambs declined sharply compared with the numbers found in susceptible lambs. Fourth and fifth stage worms which reached the lungs were quickly destroyed in resistant animals. Resistance was evoked equally by the administration of worms at different stages of development and by the termination of infections at different stages by anthelmintics, indicating that the various developmental stages of D.filuriu play a part in evoking the immune mechanism of the host. Wilson (1966) also observed larval and immature worms in the mesenteric lymph nodes of resistant animals up to 15 days after infection and in the lungs up to 49 days after infection, but both the size and number of the worms were greatly reduced. He studied the duration of immunity in both sheep which had been experimentally infected and in naturally infected ewes, by challenging them from l+ to 26 months after the initial infection with 7500-60000 larvae and killing them 4-84 days later. They remained highly resistant to re-infection although there was evidence that the resistance varied with the size of the actual exposure and decreased as the time of challenge increased(Wilson, 1970). Under field conditions Rose (1965a) observed the re-infection of ewes which developed only light patent infections for a short period of time but they did not appear to be of significance as a source of infection for their lambs. The serological changes which take place in the infected animal have been investigated by several workers. Soulsby (1952) noted that lambs infected with D. fifiluriugave a marked skin reaction to extracts of the parasite sufficient to establish a skin hypersensitivity. Following an initial infection an increase in the gamma globulin of the serum occurs while the worms are maturing and the ratio of albumin to globulin decreases, apparently in proportion to the number ofworms present. The serum proteins return to the normal range a few weeks after the production of larvae ceases. No changes in the serum proteins occur on reinfection (Wilson, 1961). Some animals do not exhibit these changes. Further work by Wilson (1 966) revealed that following an initial infection a beta 2 fraction appeared in some animals. These serological changes could not be related to immunity, however. Electrophoresis of lung fluid showed a large increase in the beta and beta 2 fractions as compared with the levels in the serum and he suggested that this might be related to immunity. There was a marked increase of eosinophils in newly infected animals and they were also found in large numbers in the lung fluid of resistant animals. Favati and Della Croce (1962) also observed similar changes in the serum albumin/ globulin ratio. Denev (1969) observed an increase in the opsonocytophagic index and an increase in the proportion of phagocytic cells in the blood following infection of lambs. Both rose to a peak between the 12th and 15th day following infection. Similar changes occurred following re-infection, a peak being reached 8 days after re-infection. Phagocytosis was observed in 1.4 of lymphocytes and in 0.6 % of monocytes as well as in neutrophil leucocytes. According to Pavlov and Denev (1970) the administration of first and second stage larvae as well as

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third stage larvae by oral, subcutancous, intratracheal or intraperitoneal routes produced an immune state in lambs as evidenced by the complement fixation test. Cf. antibodies were found on the 7th to the 30th day after infection and remained at the same level for 130-1 50 days. Specific antibodies can be detected by the fluorescent antibody technique in the sera of lambs two weeks after infection, and the reaction is said to be specific. Movsesijan and Lalic (1971) consider that the localization ofthe specific antibody/antigen complex indicates that the metabolic products rather than the somatic components of the parasite have an antigenic effect. Workers in Yugoslavia have infected sheep with infective larvae which have been attenuated with X-rays or with gamma radiation from 6OCo. The most effective radiation was from 40 to 60 kiloroentgens. When lambs were vaccinated twice with a 30 day interval between vaccinations, they exhibited a 38.1 immunity when challenged after 3 days, a 85.8% immunity when challenged after 15 days and complete immunity when challenged after 2 months. Vaccinated animals had only slight lesions in the lungs following infection (SokoliC et af., 1961; Jovanovic et al., 1961 ; Sofrenovic et al., 1961). After double vaccination with the first vaccine consisting of 1000 attenuated larvae and the second vaccine consisting of 2000 attenuated larvae, small numbers of worms were recovered from the lungs at autopsy. They were retarded in their development but a few larvae were recovered from the faeces of the vaccinated sheep (SokoliC et al., 1963). In field trials, lambs which were 2-4 months old were vaccinated before being turned out. They showed a high resistance to infection as compared with controls but patent infections developed in 29 of the vaccinated animals whereas 65 % of non-vaccinated animals became infected (Movsesijan et al., 1963; SokoliC et al., 1965). Wilson (1970) endeavoured to vaccinate sheep by infecting them with larvae of the cattle lungworm D. viviparus. These larvae developed to the fourth stage in the sheep and appeared to stimulate immunity. Vaccinated sheep infected with D.Jifaria larvae had fewer and smaller worms, lower larval production, higher gamma globulin levels and eosinophil numbers and less lung lesions than non-vaccinated sheep.

2. M. capillaris, P. rujkcens and C. ocreatus Immunity resulting from infection with M. capillaris, P . rufescens and C. ocreatus has not been investigated extensively. In naturally infected sheep, the heaviest infections are seen in older animals which suggests that a strong resistance to reinfection does not develop. In the field sheep are not likely to be subjected to sudden heavy infections with these lungworms. From such evidence as is available the molluscan intermediate hosts are usually only lightly infected with lungworm larvae so that whenever the sheep ingest an infected mollusc they will pick up only a few larvae. The heavy infestations in older sheep are therefore built up over a period of time by the frequent ingestion of small numbers of lungworm larvae. Experimental infection of lambs with small numbers of M. capillaris larvae over a period have shown that the initial infections did not invoke an immunity which completely prevented the subsequent establishment of worms in the

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lungs but it did appear to limit the number of worms which became established (Rose, 1959b). After studying the pathological changes resulting from experimental infection of lambs with M. capillaris, Beresford-Jones(1967) suggested that various immune responses were shown by the host and these checked the development of larvae at the fourth stage in the lymph glands and lungs, which inhibited reproductive activity of adult worms and which terminated the reproductive capacity of mature adult worms. Attempts have been made to immunize sheep against M. capillaris using antigens prepared either from the lungs of infected sheep or from the foot of molluscs containing infective larvae (Ozerskaia, 1960). Lambs vaccinated with the lung tissue antigen were said to develop a partial immunity manifested by the delayed development of the worms, their encapsulationin the lung tissue and by the inhibition of egg laying. Davtian and Panosian (1946) demonstrated experimentallythe development of immunity to C. ocreatus. When sheep were experimentally infected with 250-500 C. ocreatus larvae and then subsequently challenged, the rate of development of the worms of the challenge infection was slowed down and the period of egg production by the female worms was reduced. Following an initial infection of 3000-5000 larvae a stronger immunity developed so that following a challenge infection the migration of larvae to the lungs was retarded and larvae became encysted in the wall of the intestine, where they died. Attempts have been made to immunize sheep against C. ocreatus. Davtian and Schulz (1956) infected sheep with 5000 larvae either intramuscularly or intravenously. A few larvae reached the lungs and became encysted either at the third moult or as immature adults. They suggested that development was inhibited by the absence of the usual stages of migration and that this method could be used to vaccinate sheep against C.ocreahts. G. TREATMENT AND CONTROL

The treatment of lungworm infection by the intratracheal injection of a wide range of drugs was advocated for many years. Aerosol treatment was recommended also. Neither of these methods of treatment is easy to apply on the farm, and since the advent of a number of anthelmintics which can be administered either orally or by injection, interest in the older methods of treatment has waned and therefore will not be considered here. 1. Cyanacethydrazide Cyanacethydrazide was one of the first of these drugs to be recommended. Walley (1957) demonstrated in controlled trials that it was active against D. $laria and P . rufescens but not against M. capillaris and N. linearis. He recommended a dose of 15 mg kg-1 when administered orally, up to a maximum of 1 g for sheep. The drug inactivates the lungworms, thereby facilitating their removal by mucus flowing up the bronchi and trachea. Other workers have confirmed the efficacy of cyanacethydrazidein controlled experiments (Wikerhauser et al., 1959; Jordan, 1960; Vodrazka et al., 1960).

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There arc also reports of the successful treatment of lungworm infection in the field, the efficacy of the drug being assessed by criteria such as the disappearance of clinical symptoms, a general improvement of the treated sheep and sometimes by the disappearance of larvae from the faeces of treated sheep @orrington, 1958; Hiepe et al., 1959; Pierroti, 1958; O’Donoghue, 1958; Rukavina et al., 1960; Favati and Della Croce, 1961; Gibbs and Pullin, 1960). Pierroti (1958) confirmed Walley’s observation that cyanacethydrazide is ineffective against M. capillaris and N. linearis, and he found also that it was inactive against C. ocreatus. Favati and Della Croce (1961) reported lack of activity against C. ocreatus and N.linearis but deemed it to be effective against M. capillaris because larvae disappeared from the faeces of treated animals. As cyanacethydrazide has a vermifuge action on the worms it is unlikely that it would be effectiveagainst M. capillaris, which is intimately entwined within the tissues of the nodules of the lung, and even if made inactive by the drug it is unlikely that the worms could be removed from this position by the stream of mucus. The earlier reports of inactivity of the drug against M. capillaris would seem to be correct. 2. Diethylcarbamazine Diethylcarbamazine was developed to combat filariasis. It was used successfully for treating lungworms in sheep by Ozerskaia (1955). Subsequently other workers reported on its efficiency against D. $laria in sheep (Umov, 1957; Kassai, 19%; Olteanu, 1959b; Mohi-ud-din, 1959; Ozerskaia and Popova, 1959; Egorov and Morozov, 1960; Skerman et al., 1968,1970; Rhesetnikov, 1971). Kassai (19%) found it to be effective against Protostrongylus spp. as well as against D. jilaria but it was less effective against C. ocreatus. Subsequently, however, Kassai and Hollo (1963) found that repeated administration of the drug by intramuscular injection had no effect against Protostrongylus. Egorov and Morozov (1960) reported some effect on M. capillaris as there was a reduction in the number of larvae in the faeces-an unsatisfactory criterion for assessing activity against the adult worms in the lung-but they maintained that treatment of sheep in November was unsuccessful. Critical tests with diethylcarbamazine against D. vivigarus in cattle have shown that it has most effect against immature adults (Parker, 1957) so that treatment must be timed to attack the immature worms, which is difficult to determine under practical farming conditions, 3. Methyridine Methyridine,thiabendazoleand tetramisole were found to be effectiveagainst gastro-intestinal worms and subsequently their efficacy against lungworms was investigated. Methyridine has only a limited efficacy against D . Jilaria: Walley (1963) found it to have someeffect against adult worms but less effect againstimmature worms. He concluded that it was not as good as either cyanacethydrazide or diethylcarbamazine. Ozerskaia et al. (1966) also observed only a limited effect against D.jiIaria, but Sokol(1970), Thomas and George (1968) and Skerman

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rt a f .(1970) reported more favourably on its effect on D.fi1aria; Sokol(l970) also found that it removed 60 % of P. rufescens from five naturally infected sheep.

4. Thiabendazole

Gibbs and Pullin (1963) carried out critical tests with thiabendazole against gastro-intestinal worms and lungwormsin sheep and while it had a high efficacy against the former they considered it to have limited efficacy against D.ji1aria. Robinson (1966) treated sheep with thiabendazole at dose rates of 44 or 88 mg kg-1; the lower dose rate had little effect but the higher rate had some effect. Ross (1967), using 66 mg kg-1, found that 77 % of 15 day old larvae and 8 1 % of adult D.Jilaria were removed from experimentallyinfected lambs but Sokol(1970), who dosed naturally infected lambs orally with 200 mg kg-l and collected the worms expelled in the mucus, recovered an average of 58 % D. filaria and 32 % P. rufescens. The general assessment is that thiabendazole has a limited usefulness in the treatment of sheep lungworm infection. 5. Tetramisole Tetramisole is an anthelmintic which combines a high efficacy against both gastro-intestinal worms and against lungworms of sheep. Walley (1966) and Forsyth (1966a, b) described laboratory experiments and field trials which demonstrated the efficacy of the drug against D. filaria but at a dose rate of 10 mg kg-1. Walley (1966) found that it had no effect on M. capillaris. Forsyth (1966a, b) recommended a dosage of 15 mg kg-1 but Guilhon (1966) considered that a dose of 20 mg kg-1 was needed for a high efficacy against D. jilaria. Confirmation of the activity of tetramisole against D . jilaria has been provided by a number of workers who have advocated various treatment regimes (Nilsson and Sorelius, 1966; Vural et al., 1967; Pretorius, 1967; Gibson and Parfitt, 1968; Korchagin et al., 1969; Kapandze, 1969a; Walley, 1970). Forsyth (1968) demonstrated that the anthelmintic activity of tetramisole was contained in the L-isomer so that by using the pure L-form the dosage could be reduced to only half that recommended for tetramisole; this was confirmed by Presidente and Worley (1969) against 6, 12, and 45 day old D. filaria. Reineckeet al. (1971) carried out controlled tests with the h o m e r levamisole against third and fourth stage larvae in the lymph nodes and against fifth stage worms in the lungs of experimentally infected lambs. The drug was administered intraruminally at 7.5 mg kg-1 and was found to be more than 60 % effective in more than 60 % of the treated flock. Tetramisole is also effective against P. rufeeceens adults but not against first stage larvae in the parenchyma (Nilsson and Sorelius, 1966). These workers also suggested that it had a vermicidal action against M. capillaris as long as prolonged tissue levels can be maintained by repeated treatment (Nilsson and Sorelius, 1968). Kapandze (1969b) recommended 3 treatments at 24 h intervals with two more doses after an interval of 30 days for the treatment of M . capillaris and Protostrongylus spp. These recommendations were based on the

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results of experiments in which sheep naturally infected with Muellerius and Protostrongylus were injected with 15 mg kg-l of tetramisole at 24 h over 2, 3, 4 and 5 days. Halhead (1968) treated ewes with tetramisole and three which were heavily infected with M . capillaris died shortly after being treated. He suggested that death was caused by an anaphylactic reaction following the release of a larval antigen after administering tetramisole to a sensitive host. 6. Control Various methods for the control of lungworm infection in sheep have been advocated by workers in different parts of the world which relate to the methods of sheep husbandry used and to other factors which influence the transmission of infection in the particular region for which the advice is intended. There would seem to be little point in recounting these methods of control, especially as in practice the control of lungworm infection cannot generally be carried out in isolation but must form part of a general programme for the control of nematode infections, some of which may well be more damaging than lungworm disease and consequentlywill take prior considerationin the formulation of control methods. Some of the methods used in the control of nematode infections of sheep in lowland areas in Great Britain will be considered here with regard to their efficacy in controlling lungworm infections. The most damaging nematode diseases in sheep in Great Britain result from infection with gastro-intestinal nematodes and their control is of major importance. The life cycles of these nematodes are similar to that of D.filaria, being direct; furthermore the infective larvae are similar in that they can survive throughout the winter on pasture to provide a source of infection for lambs in the spring. Two distinct diseases are generally recognized : parasitic gastro-enteritis resulting from infection with several different species of nematodes, particularly Ostertagia spp., Cooperia spp. and Trichostrongylus spp., and Nematodiriasis due to Nematodirus spp. The control methods advocated for the two diseases are somewhat different, but because of the similaritiesbetween these species and 0.filaria already referred to, these control methods are applicable to the lungworm infection. Spring-born lambs can become infectedwith the nematodes causingparasitic gastro-enteritis either by ingesting larvae which have survived on the pasture throughout the winter or by ingesting larvae which have been derived from the ewe following the spring rise in worm egg production. Consequently one control method which is widely advocated is to treat the ewe with an anthelmintic prior to lambing in order to limit this source of infection. If a broad spectrum anthelmintic such as tetramisole is used any D. filaria infection carried by the ewe will also be reduced, although the ewe is not so important a source of D. jfaria infection as of gastro-intestinal nematodes. The level of infestation on pasture due to gastro-intestinalnematodes apart from Nematudirus will generally be low in the spring, as will infection with D . jilaria, so that the pasture can be grazed safely for some time. By the early summer, however, the level ofinfestation will generally be rising and so moving the lambs on to clean pasture and dosing with an anthelmintic at the same time

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is advocated. A suitable time to make this move is at weaning. This move will take the lambs away from the source of both gastro-intestinal nematodes and D.jilaria. If the lambs are not sold off until late in the autumn another move may be advocated. In areas where nematodiriasis is a problem, additional control methods are advocated. In Great Britain the disease is mainly due to N.battus and lambs become infected by ingesting infective larvae which hatch in the spring from eggs which have survived on pastures throughout the winter. Control of this disease is effected by keeping lambs off land that was grazed by lambs in the previous year; such action will be effective in avoiding D.$laria larvae which have survived the winter. Although a considerable amount of experimental work on the vaccination of sheep against D.jdaria using irradiated larvae has been carried out by workers in Yugoslavia, a vaccine is not yet available in Great Britain and so vaccination cannot yet be advocated as a control method. The protostrongylin lungworms differ from D. jiluria and the gastrointestinal nematodes in having an indirect life cycle with a mollusc intermediate host, and furthermore the infective larvae have along period of survival in some of the long-lived intermediate hosts. Because of these differences the methods of control referred to are unlikely to be effectivein controlling infection with these species. Furthermore the anthelmintics that are effective against the gastro-intestinal nematodes and D .filuriu are less effective against C. ocreatus and M. capillaris although they are effective against P.rufescens. Various general methods have been advocated for the control of the protostrongylin lungworms, such as keeping sheep off pastures in the morning and evening when the molluscs are active, and the use of molluscicides to kill off the intermediate hosts. It is doubtful whether such methods are either practicable or worthwhile. In Great Britain protostrongylin lungworms are not the cause of any significant disease in lambs and any control methods aimed specifically against these nematodes are unnecessary. M . capillaris is the commonest of these lungworms and any attempt to reduce the intermediate host population by use of molluscicides is unlikely to be practicable in view of the widespread distribution of some of the mollusc species over pastureland. REFERENCES

Aliev, A. A. (1959). Development and survival of Dictyocaulus larvae of sheep in the external environment.Bid. Nauchn-Tekh.Znf. Vses. Znst. Gelm. 5, 3-7. Anderson, P. J. S. and Venter, A. (1971). Studieson DictyocaulusfilariaII. Migration of the developmental stages in lambs. Ondersrepoorr J. vet. Res. 38, (3), 185-190. Barratt, M. E. J. (1969). HomocytotropicantibodyinpigsinfectedwithMetastrongylus spp. Vet. Rec. 85, 390. Barratt, M. E. J. and Herbert, I. V. (1970). Porcine homocytotropic antibody in response to Metasrrongyh spp. : infection.J. Parasir. 56 (4, Sect. 2), 21-22. Becht, H. (1960). Serology of Metastrongylus infestation in pigs, Dt.-iist. tierarztl. Wschr. 67, 177-180.

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SHORT REVIEWS Supplementing Contributions of Previous Volumes

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Recent Research on Malaria in Mammals Excluding Man P. C. C. GARNHAM

Department of Zoology, Imperial College of Science and Trrhnology, London, England I. Introduction.. .....................................................................................

................................................... Taxonomic Problems.. ................................................................... New Species and Redescriptions ......................................................

11. Taxonomic Problems and New Species

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IV. V. VI.

A. B. Life Cycles Including Exoerythrocytic and Sporogonic Stages ........................ A. P.berghei Complex. ....................................................................... B. Bovid Species ........... ............................................................ C. Simian Parasites (Asia and Latin America) .......................................... D. Malaria Parasites of the Higher Apes ................................................ E. Periodicity of Infectivity of Gametocytes............................................. Pathogenesis and Culture ..................................................................... A. Pathogenesis .............................................................................. B. Culture ....................................................................................... Host Susceptibilities and Mnities ......................................................... Fine Structure .................................................................................... References .......................................................................................

603 605 605 606 609 610 610 61 1 614 616 617 617 618 619 621 626

I. INTRODUCTION Research on the parasitology of malaria in the last five years has progressed steadily along the lines foreshadowed in the 1967 review. The most important discoveries are probably in the field of immunology, though the most dramatic research concerns the response of Aotus trivirgatus and other New World monkeys to the human species ofPlasmodium. Both these subjects relate largely or entirely to human malaria, but the experiments on immunity often entailed the use of simian models, and the behaviour of the human species in Aotus is casting so much light on the phylogeny of the subgenus Plasmodium as a whole that these aspects receive a little attention in the present review. Taxonomic problems remain unsettled particularly in regard to the status of parasites below the specific level. A major advance, however, has been the use of iso-enzyme analysis as a criterion for the identification of species and subspecies in rodents. Numerical taxonomy and other artificial techniques have been suggested, but it is doubtful whether they will do more than confirm the results of the classical approach. However, preliminary tests are currently being made. 603

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The study of ultrastructure has progressed, and the application of scanning microscope techniques for three dimensional observations is giving some interesting results, particularly in regard to surface membranes after freeze etching (Meszoely et a]., 1971). Unfortunately, cytochemistry has so far contributed little to the determination of function of the various organelles but autoradiography may soon provide clues. Two great problems still remain unsolved :the nature of anopheline susceptibility and resistance to various species of malaria parasites, and the mechanism of relapses. So-called relapse bodies continue to be found in the livers of mammals, up to a year or more after infection, but their origin remains as obscure as ever. The various theories on their causation are discussed by Coatney et al. (1971) in relation to the primate malaria parasites and by the writer (Garnham, 1967a) in regard to malaria parasites sensu latu. Some experimental work was carried out (see Warren and Garnham, 1970; Warren et af., 1973, and in press) on the effects of X-radiation (up to 8 krads) on the sporozoites of P. cynomolgi, the results of which were interpreted as demonstrating the existence of a heterogeneous population of sporozoites. Relapses were fewer in the irradiated group and it was postulated that the special sporozoites responsible for relapses were more sensitive to radiation and were killed. Dilution experiments, whereby diminishing numbers of sporozoites of P. cynomolgi were inoculated, tended to support this view, and it has received confirmation from other unpublished work. Two phenomena, which were first reported 6 or 7 years ago, have remained largely unconfirmed except by the authors themselves. The first was the discovery of antigenic variants by Brown and Brown (1965), and the second, of the periodicity of gametocytes (see page 616) by Hawking et af. (1966). Their theories are of fundamental importance and deserve testing on a wide scale and with different species of parasites. The study of immunity in malaria probably occupies more attention today than any other aspect of the subject. The advances which have been made since 1967 are so great that “Immunity in Malaria” deserves a review to itself in this series of Advances. Actually there are several recent reviews elsewhere on immunity in malaria as follows: I . Immunological aspects of malaria infection, by I. N. Brown (I 969) in “Advances in Immunology”. 2. “Immunity to Parasitic Animals”, Vol. 2 (1970). (a) Primate malaria, by P. C. C. Garnham. (b) Malaria of lower mammals, by A. Zuckerman. 3. Immunity to plasmodia1 infections ; consideration of factors relevant to malaria in man, by I. A. McGregor (1971) in “International Review of Tropical Medicine”, Vol. 4. It is impossible to discuss satisfactorily the subject of immunity in the present review where human, avian and reptilian forms of malaria are necessarily excluded, but a few sequels to the research problems described in the 1967 review (Garnham, 1967b) are briefly mentioned here. Brown and Brown (1969) haveextended their observations on antigenicvariants. Voller and Rossan

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(1969) examined the behaviour of P. cynomolgi bastianellii after sporozoite induction in monkeys and showed that relapse variants were produced late in the infection and that they originated directly from an exoerthrocytic source. These authors also confirmed the work of the Browns on P. knowlesi relapse variants, using in both instances cross immunity instead of schizont agglutination tests as their criteria of variants. Butcher and Cohen (1972) used still another technique and tested the action of antisera derived from different variants of P. knowlesi in their culture system; inhibitory effect of the homologous antiserum was clearly demonstrated. Three other aspects of research into immunity in non-human malaria are of particular interest today. They are firstly the problem of vaccine (see Corradetti et al., 1969; Ward and Hayes, 1972; Spitalny and Nussenzweig, 1972),of which a more optimistic view of its practical value is now taken; secondly, the extraordinary cross-immunity which exists between malaria parasites and piroplasms and other infections (see Cox, 1972), and the related question of antagonism (and on the contrary synergism), between various antigens and malaria parasites (see Wedderburn, 1970; Garnham, 1971b); and thirdly the characterization and localization of the antigens which stimulate protective antibodies (see Cohen and Butcher, 1970; Garin et al., 1972). The indirect fluorescent antibody test continues to be used extensively for practical measurements of immunity in field surveys, and for studies of its rise and fall in experimental infections (e.g. P. cynomolgi bastianellii in monkeys by Ambroise-Thomas et al., 1971). Two further International Workshops (Numbers 3 and 4) on malaria were organized by Elvio Sadun at the Walter Reed Army Institute for Medical Research in 1969 and 1972 respectively and their proceedings cover many aspects of currentlmalaria research. The report of a WHO group on the parasitology of malaria, held in Teheran in 1968, also contains material of interest.

11. TAXONOMIC PROBLEMS AND NEWSPECIES A. TAXONOMIC PROBLEMS

Separation of the genus Plasmodium into subgenera (Plasmodium, Laverania and Vinckeia) has been accepted by the majority of parasitologists and the system is used on a similar scale by Hoare (1972) in his monograph on the mammalian trypanosomes. The subgenus Vinckeia, however, needs some revision, because as at present defined the criteria are not totally applicable. Either the unsuitable species should be removed to a fourth subgenus, or Vinckeia should be redefined. Until more is known about the life cycle of most of the species (now numbering 16) within the subgenus, it would seem unwise to create a new subgeneric name. The opportunity is therefore taken here to amend the criteria of the subgenus as follows: Subgenus Vinckeia Garnham, 1964, amended 1973. Type species: Plasmodium (Vinckeia) bubalis Sheather, 1919. Malaria parasites of lemurs and other lower mammals which exhibit usually small, erythrocytic schizonts with usually 8 or fewer merozoites. Stippling of the 22

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infected erythrocytic is rarely present, or absent. Gametocytes are round and usually have scattered pigment. Exoerythrocytic schizogony takes place in the parenchyma cells of the liver with the production of a thousand or more merozoites; this stage occupies just over or just under 2 days. Sporogony occurs in anopheline mosquitoes, usually with specialized habits. The revised definition is chiefly necessary because the rodent malaria parasites often have relatively large schizonts with more than 8 merozoites. The rapidity of the exoerythrocytic cycle in the species of Vinckeia in which it is known, is in striking contrast to its much greater length in the other subgenera of Plasmodium; the absence of stippling in the infected corpuscle is also an important criterion. The whole question of subspecies of simian, rodent (and human) malaria parasites is still quite unsettled, one group of investigators being much in favour of such designations and another remaining adamant in the use of terms such as strains, races, demes, variants, etc. The chief argument in favour of formal nomenclature is that an actual name is much more useful than some awkward circumlocution, varying from author to author. The subspecific name, moreover, can always be synonymized if necessary at a later date; its current use stabilizes the nomenclature. The subspecific name must of course comply with the full requirements of the Rules. The choice of subspecific or specificnames varies greatly from group to group even within the families of the Haemosporidia. The trinomial designation is chiefly applied to parasites in the genus Plasmodium to which also the division into subgenera is confined. It has been suggested that the various haemoproteid genera should be reduced to subgenera of Haemoproteus; thus Hepatocystis kochi would become Haemoproteus (Hepatocystis) kochi; Polychromophilus murinus, Haemoproteus (Po.) murinus ; Simondia metchnikovi, Haemoproteus (S.) metchnikovi, etc. Similar treatment was also proposed for the present subgenera in the family Leucocytozoidae. Even though subgeneric divisions are not based on much experimental data, the system splits off groups in the very crowded genera (i.e. Haemoproteus and Leucocytozoon), and simplifies practical usage. They are thus terms more of convenience than of phylogenetic significance. However, malaria parasites belonging to the family Plasmodiidae alone are considered in this review, as in the earlier one (Garnham, 1967b), and the other families are omitted from consideration. B. NEW SPECIES AhD REDESCRIPTIONS

Few new species of mammalian malaria parasites have been described in the last five years. The research on simian malaria in Malaysia greatly diminished in intensity after the withdrawal of the team of investigators of the National Institute of Health, Bethesda, U.S.A., which was originally under the leadership of the late Don Eyles. An area rich in speciation of these parasites had been revealed and a great number of so-called strains had been discovered. The status of the latter remains uncertain, particularly of those belonging to the P. cynomolgi and P. inui complexes. Further data on the sporogonic cycles

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of these strains and their iso-enzyme analysis may soon clarify their respective positions. Similarly, the major work on simian malaria in Ceylon has virtually ended as far as the isolation of new species is concerned; the peculiar strain of P. cynomolgi from the langur monkey remains unnamed although its characters appear to be firm; however, its formal designation should best wait until it decision is reached about the whole complex. The strange restriction of monkey malaria in Africa to P. gonderi still holds true; parasites in the chimpanzee continue to be seen, but no fresh records exist of malaria in the highland or lowland gorilla. Malaria parasites of the New World monkeys are limited to two species, though there is a little evidence that P. brasilianum may comprise a group of closely related quartan parasites. This question is discussed in Section 111. 1. Recently described species in the orang-utan Malaria parasites from the orang-utan were first described in 1907by Halbastaedter and von Prowazek in animals imported into Germany from Borneo. They were thought to be of the quartan type and this opinion was shared by later observers who gave new but fragmentary descriptions of P.pitheci. Garnham (1966) added to this medley by including observations on some material from Wenyon’s collection. A better account is given by Coatney et al. (1971) from blood films obtained by one of the authors on a visit to Borneo. No details of the course of infection, sporogony, exoerythrocytic schizogony or infectivity to man had been obtained in these researches. In 1972, Garnham et al. studied the biocenose of orang-utan malaria in the Sepilok Forest in Borneo and elucidated most of the life history of P. pitheci and of P. silvaticum, a hitherto undescribed species which was found in the same locality. Six of the 8 orang-utans examined showed one or other, or both of the species and their infections were followed for several weeks. The periodicity of both P. pitheci and P. silvaticum was shown to be tertian. The two parasites were easily distinguished from each other by the gross enlargement of the infected erythrocyte, the presence of Schiiffner’sdots, and the disappearance of the trophozoites from the peripheral blood after 24 h in P. silvaticum, and the absence of these features in P. pitheci. Several species of Anopheles were found to be capable of supporting sporogony, but A . balabacensis was the most susceptible. Other features of the life cycle are described below. The high rate of infection in this focus suggests that, in spite of the small number (perhaps 50) of orang-utans present in the area of 10000 acres of forest, other factors, important in transmission, are operating at optimum efficiency. Plasmodium pitheci shows few affinities with the Plasmodium spp. in man, chimpanzee or gorilla, but it has some resemblance to P. youngi and P. hylobati of the gibbon. The resemblance is closer between P. silvaticum and P. eylesi of the gibbon; yet the phylogenetic relationship between the African apes and the orang-utan is thought to be greater than between the gibbon and the orang-utan. But in terms of morphology and life cycles the relationship is reversed and the gibbon and orang-utan parasites have much more in common.

608

P. C. C. GARNHAM

2. Recently described species and subspecies in rodents and other small mammals The complex of species and subspecies of malaria parasites of Grammomys surdaster and Thamnomys rutilans has gradually been sorted out in four localities in West and Central Africa, each relating to subspecies of the pair, P. berghei and P. vinckei. Table I gives the classification. TABLE I Classificationof murine malaria parasites into subspecies ~

Locality Katanga Central African Republic Congo-Brazzaville Nigeria

~-

Plasmodium berghei

Plasmodium vinckei

subspecies berghei subspecies yoelii subspecies killicki subspecies nigeriensis

subspecies vinckei subspecies chabaudi subspecies lentum subspecies chwatti

Bafort (1969, 1971,1972)in serial papers denies the validity of this interpretation, and describes experiments in which he apparently succeeded in converting one form into another. However, Carter (1970), by the analysis of isoenzymes (particularly GPI, 6GPD and LDH) clearly differentiated P. berghei subspecies from the three other P. berghei subspecies, while all four sub-species of P. vinckei appeared to possess distinctive iso-enzyme patterns. Such enzyme markers have been extremely useful, combined with a drug resistance marker, in identifying genetic recombinations (i.e. hybrids) in the mosquito host (Walliker et al., 1973). In spite of many surveys in tropical America and Asia, rodent malaria parasites seem to be confined to Africa. Misdiagnosis of a piroplasm infection is a frequent cause of error, but the absence of pigment in the parasite and of schizogony in the erythrocyte should quickly indicate the correct identity of the organism in the rodent blood. However, Landau et al. (1970) described a parasite in the blood of the spiny haired mouse (Acomys) which undergoes schizogony in the erythrocyte but produces no pigment. It was eventually placed in a new genus under the name of Anthernosoma garnhami, and Vivier et al. (1970) showed that in ultrastructure it was allied to Babesia rather than to Plasmodium. The flying squirrels of several families possess malaria parasites of the subgenus Vinckeia. In addition to P. booliati, Fong et al. (1970) reported the discovery of another species in Petaurista elegans with a different morphology, but they have yet to give it a name. It is characterized by the possession of conspicuous pigment, and by schizonts which fill the host cell and contain 12 merozoites in a rosette. Killick-Kendrick and Bellier (1972) discovered two species of Plasmodium in scaly-tailed flying squirrels from the Ivory Coast; one (from Anomalurus peli) resembled P . anomaluri of East Africa, but differed from it in that schizogony was entirely confined to the pulmonary circulation. The second species was found in A . derbianus and was characterized by the intense reddening which it produced in the host cell. Both species are as yet unnamed.

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Lien and Cross (1966) gave the name of P . watteni to a malaria parasite from the giant flying squirrel in Taiwan, which is clearly distinct from P. booliuti; it has more abundant pigment and the schizonts contain a greater number of merozoites (up to 18); also, P. watteni occurs in a different subspecies of squirrels. Garnham and Uilenberg (1973) redescribed the two malaria parasites of lemurs, from blood films taken over several months from a splenectomized Lemur macaca macaca and Lemur macacafulvus. Two specieshad been described -P. girardi by Buck et al. (1952) and P . lemurisby Huff and Hoogstraal(l963). The major difference between them is the unchanged size of the infected corpuscle in the former and the enormously distended corpuscle of the latter parasite (of which only 30 examples were found). In the new material parasites were plentiful and certainly two species of Plasmodium were present, which in many ways conformed to the descriptions of the earlier authors. However, the P. girardi schizonts showed 12-16 merozoites instead of 8-10, while the erythrocytes infected with P . lemuris were not only enlarged but markedly stippled. 3. Unnamed species in Bovids Bovids are rare hosts of malaria parasites, or rather, few observations have been made on them. The parasites of the African Duiker, however, have been well studied by Keymer (see below). The smallest species of Plasmodium so far discovered, P. traguli of the Asian mouse-deer, has attracted some attention because of its frequent detection in the mosquito host. A second species in the Malayan mouse-deer (Tragulus javanicus) has been reported by Fong et al. (1972). It is remarkable for its large size, at least four times that of the erythrocyte. This organism is as yet unnamed. Two types of oocysts were found in natural infections of A . umbrosus and it is thought that one of these may belong to the new species. On the basis of the lower number of merozoites and the different type of pigment, Dissanaike (1963) gave a subspecific name ( P . t . quadragemina) to the parasite in the mouse-deer of Ceylon. A malaria parasite of great interest is the organism discovered by Kuttler et al. (1967) in the blood of a single specimen of the Virginian White-tailed deer (Odocolius virginianus), captured in Texas. Parasites were observed 8 days after splenectomy and the infection persisted for 24 days. Three subpassages were made into splenectomized deer and pigmented trophozoites were seen. The writer was privileged to see a film and noted the presence of schizonts, brown pigment granules and gross enlargement of the corpuscle. Further blood films from deer have been examined in Georgia, but no infections were seen. 111. LIFECYCLES INCLUDING EXOERYTHROCYTIC AND SPOROGONIC STAGES The recently published monograph on primate malaria by Coatney et al. (1 971) presents, besides the magnificent coloured plates of the erythrocytic

stages, an excellent account of the life cycles of the species belonging to the

610

P. C. C . G A R N H A M

subgenera Plasmodium and Laverania. It is particularly remarkable for the description of the oocyst and sporozoite phases of each parasite, which has never been given in such detail before.

A.

Plasmodium berghei

COMPLEX

The stream of papers on P . berghei and its relatives continues unabated, primarily from the Antwerp School, where Vincke (until his premature death in 1969)’ Jadin (1971), WBry (1968) and Bafort (1969, 1971)have demonstrated the method of transmission, which was originally worked out by Yoeli and Most (1960) in collaboration with their Belgian colleagues. Yoeli’s more recent work (Yoeli et al., 1968) on the life cycle has demonstrated exoerythrocytic schizonts in parenchyma cells in isolated perfused portions of liver. The animal had previously received intravenously, sporozoites of P . berghei. This appears to be the nearest that any investigator has reached in culturing tissue stages of mammalian malaria parasites. Dunn et al. (1972) showed that the gross fatty changes produced in the liver by ethionine injections, completely inhibited the development of exoerythrocytic schizonts and that this effect could be reversed by the administration of methionine and adenosine. Of great significancehas been the continuation of the research of Landau and Chabaud (1965) on the nature of the persistent schizonts of P . berghei yoelii. They (Landau et al., 1970) showed that 5 specimens of Thamnomys rutilans out of a total of 42, captured in the enzootic area, exhibited exoerythrocytic schizonts of the chronic type (i.e. with characteristic nuclei), and that the development of these bodies could be watched in successive biopsies. Growth was shown to be very slow. Finally, Landau and Michel (1970) studied these forms in Steutomys sp. after inoculation of sporozoites, and at a low temperature; the size of the tissue forms was greatly reduced, development was retarded and the morphology was identical with that of the chronic infections found in nature. From these observations and others on various sporozoan parasites of small mammals, Landau (1973), has deduced that the phenomena represent a mechanism for assuring the ‘36rennite‘ de I’infection”.

B. BOVID SPECIES

Keymer (1969) describedin great detail the biocenoses associated with the two malaria parasites of Duikers in Malawi, viz. P . cephalothi and P . brucei. He studied the course of the blood infections in intact and splenectomized animals, and attempted to infect local species of Anopheles but without success. Both parasites proved to be non-infective to splenectomized goats, sheep and calves, Hoo and Sandosham (1968) inoculated sporozoites of P. truguli into clean mouse-deer and found exoerythrocytic schizonts in sections of the liver; they were lobulated bodies, 45 x 26 pm in size and contained merozoites. The sporogonic cycle of this parasite is now well established both in experimental infections in A . letger and in natural infection in A . umbrosus.

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61 1

C. SIMIAN PARASITES (ASIA A N D LATIN AMERICA)

Asian species : Warren (1973) has tentatively classified the species of simian malaria parasites in Asia into two groups, one of which occurs to the East and the other to the West of the Bay of Bengal; each group comprises a series of closely related pairs (Table 11). The zoogeography of simian malaria in Asia is of course primarily governed by the presence or absence of the vertebrate host, but another important factor is the occurrence of the A . Zeucosphyrus complex in the area; if this is absent, as in N.W. India and West Pakistan, monkey malaria is also absent. This may be the explanation of the negative findings of Otsuru and Sekikawa (1968) in their survey of Mucuca fuscata on Honshu and smaller islands in Japan. The monkeys (M. cyclopis) of Taiwan are, however, commonly infected with a strain of P . inui (the so-called P . inui cyclopis). TABLE II Simian species of Plasmodium East and West of Bay of Bengal

(after Warren, 1973) Eastern Group

Western Group

P. cynomolgi cynomolgi P. inui inui P. fieldi P. coatneyi P. knowlesi

P. cynomolgi ceylonensis P. inui shortti P.simiovale P. fragile Absent

The zoogeography, host distribution, and morphological and biological characters of the parasite itself are necessary criteria for the recognition of the species. The details of the sporogonic and exoerythrocyticphases are of equal or greater value. The sporogony of the Malayan group has been studied particularly by Collins and his collaborators, who began systematic observations in 1966 and summarized them in the monograph of Coatney et al. (1971). There are four main criteria of sporogonic development and all are dependent upon the i ncubation of the mosquitoes at a standard temperature. They are as follows: (a) Duration of (i) growth into mature oocysts, and (ii) appearance of sporozoites in salivary glands. (b) Size of oocyst on a given day. (c) Pattern of malaria pigment in oocyst. (d) Comparative susceptibility of different species of mosquitoes. Since 1967, the details of the exoerythrocyticschizogony of some species have been elucidated by workers at the Chamblee branch of the National Institutes of Health in the U.S.A. Work was done on the following parasites: (a) P . coatneyi was studied by Held et al. (1968); 6 day forms were 19-22 pm and the 10 day forms were 40-48 pm. Unfortunately, the time taken for the

612

P. C. C . G A R N H A M

schizont to reach maturity and the presence of merozoites in the older forms were not reported. However, this parasite is apparently a rather slow grower and matures about the 10th day. (b) P . inui. The tissue stages of two strains of this parasite were studied by the writer (Garnham, 1951, 1966) and later, of three other strains by Held et al. (1968). The latter stated that their parasites were larger than those described by the former, and it is probably that P . inui comprises a complex of closely related subspecies including P, inui shortti, which should be identifiable by sporogonic and exoerythrocytic features, and perhaps by their iso-enzyme patterns. (c) P . simiovale was investigated by Collins et al. (1972a). The tissue schizont at 7 days had a median diameter of 18 pm; at 18 days-25 pm; at 9 days29.5 pm; at 10 days-32.5 pm; at 11 d a y s 4 0 pm. The prepatent period was said to be 11 days, but it is suggested that the true period was probably 9 days as only a small dose of sporozoites was given (intrahepatically). Collins and Contacos (1971) showed that this species is accompanied by true short term relapses of the “Chesson” type; a similar phenomenon also occurs in P.Jieldi. A significant advance in our knowledge of the malaria parasites of Ceylon was made in 1971, when Nelson and Jayasuriya demonstrated that A . clegans (a member of the leucosphyrus group) was the natural vector of P . inuishorttiaiid P . fragile. A third parasite was also isolated from the mosquito, which had morphological features in the blood stages, intermediate between P.fieldi and P. simiovale. A net-trap was used to catch the infected mosquitoes; it was placed over the breeding places of A . elegans in the hope that it would trap females of this species coming to oviposit. Unlike other methods tried this technique proved very successful. Latin American species In contrast to the great proliferation ofspeciesin S.E. Asia, only two examples of simian malaria parasites exist in the New World, P . simium and P . brasilianum. Deane (1964-1972) has studied these parasites at an increasing tempo for the past 10 years and has shown that their distribution is governed by the presence of certain species of acrodendrophilic mosquitoes, A . (Kerteszia) cruzi and A . (K.) neivai and possibly by A . (Nyssorhynchus) oswaldoi, A . (Arribalzagiu) mediopunctatus and Chagasia bonneae. In Deane’s latest surveys (1 971 and 1972) he found P . simium in the States of Espirito Santo and Rio Grande do Sul to the north and south of Siio Paulo respectively, and Deane and Net0 (1969) discovered that P. brasilianum occurs in monkeys in Amapa and Maranhiio at the mouth of the Amazon as well as much further in the interior; also he found both species in the woolly spider monkey and in howlers in the southern part of Brazil. Table 111 summarizes the details of the incidence of malaria in the Cebidae of Brazil (Deane, 1972); no infections were found in over 200 marmosets. Deane makes a strong case for the division of P. brasilianum into a number of subspecies, as was first suggested by Taliaferro and Taliaferro (1934), largely on the basis of the morphoIogy of the parasite and its behaviour in different

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613

TABLE III Frequency of simian plasmodia in Brazil, by species of host, 1964-1971 (afer Deane, 1972) Species of Plasmodium

Species of primate Family CEBIDAE Cebus apella apella Cebus apella macrocephalus Cebus apella pallidus Cebus apella nigritus Cebus apella robustus Cebus apella versutus Cebus apella libidinosus Cebus nigrivittatus Cebus albifrons Alouatta f i c a fusca Alouatta f i c a clamitans Alouatta belzebul belzebul Alouatta belzebul ululata Alouatta belzebul nigerrima Alouatta seniculus straminea Alouatta caraya Ateles paniscus paniscus Ateles paniscus chamek Ateles belzebuth marginatus Lagothrix lagotricha lagotricha Lagothrix lagotricha cana Brachyteles arachnoides Chiropotes satanas satanas Chiropotes satanas chiropotes Pithecia pithecia Pithecia monacha Cacajao calvus Cacajao melanocephalus Callicebus torquatus Callicebus moloch cupreus Callicebuspersonatuspersonatus Callicebuspersonatus melanochir Aotus trivirgatus Saimiri sciureus sciureus Saimiri sciureus boliviensis

Total P. brasiwith Number lianum Plas- plasmodia exam- P. brasi- P. and modium ined lianum simium P.simium sp. No.

166 23 8

62 61

8 12 4 11 40 545 116 1 5 61 23 36 6 1 18 31 22 8

30 13 9 4

4 4 12 16 2 12 276 16

614

P . C . C. G A R N H A M

monkey hosts (Table 111). Since the discovery of P. brasilianum by Gonder and von Berenberg-Gossler (1908) in Brachyurus calvus beyond Mangos on the Amazon, the parasite had not been seen again in the type host, until Barbosa de Almeida and Deane (1970) encountered scanty infections in this monkey in the same region (670 km above Mangos). The exoerythrocytic stages of both species of parasites have been described as follows : (a) P . brasilianum. A single tissue schizont was found in Saimirisciureus after a long search by Garnham et al. (1963) and it was shown to have a most characteristic morphology. Sodeman et al. (1969) were more successful and discovered tissue forms at 14, 18 and 21 days after sporozoite inoculation, but not on the seventh day. In both instances, similar features were observed: enlarged host cell nucleus, peripheral vacuoles, clefts and slow growth (features which are also notable in P. malariae of man). (b) P . simium. Collins et al. (1969) obtained good infections of this species in A.fieeborniand A.stephensi after these mosquitoes had fed on a Saimiri monkey infected with the parasite. The oocysts became mature in 12 days and measured 45-46 pm in mean diameter (at 25°C). The infection was transmitted by bite into splenectomized Saimiri monkeys, but parasites were not detected in the blood until 22 days later, probably because of the poor quality of the sporozoites. Coatney et al. (1971) reported briefly that tissue forms were found in the liver of the owl and Saimiri monkeys, 7 days after the inoculation of sporozoites and that they resembled the 7 day schizonts of P . vivax. However, in other respects, the two parasites are dissimilar; the pigment inP. vivaxis in the characteristic “Prince of Wales’ Feather” pattern, while the enlargement of the infected erythrocyte is greater, and the trophozoite is more amoeboid than in P. simium.

D. MALARIA PARASITES OF THE HIGHER APES

Gibbon parasites The details of the life cycles of this interesting group are still incomplete and for comparison with other species, it would be useful to determine the nature of exoerythrocytic schizogony in both P. youngi and P . eylesi. P . hylobati Rodhain, 1941, was redescribed by Collins et al. (1972b) and its exoerythrocytic stages were demonstrated by Sodeman et al. (1972) from an infected Hylobatesmoloch originally sent to the writer by Hwang of WHO from Sarawak. It was splenectomized in Chamblee and the sporogonic cycle was followed in A. balabacensis. It is a tertian parasite (instead of quartan, as suspected by Rodhain), which does not enlarge the lightly stippled erythrocyte, and the mature schizont only occupies part of the corpuscle, producing 12-16 merozoites. The oocysts are considerably larger than those of P.jefleryi, but (although a complete series of measurements is not available) they appear to approximate to the size and growth rate of P. youngi. Numerous exoerythrocytic stages were found in the liver 7 days after the inoculation of sporozoites,

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615

and scanty ones at 14 days. The average diameter of the schizonts was 15 x 11 pm and of the latter 19 x 26 pm. These dimensions are much less than those of P .jefleryi. The prepatent period appeared to be 7 days. P . jesferyi. The sporogonic cycle of this parasite was determined by Collins and Orihel (1969) and presented a characteristic picture. Sporozoites were obtained on the 13th day and were inoculated into a clean gibbon. A sixth day biopsy alone was performed and oval bodies 19 x 17 pm were found in the sections of liver; the most charact.eristic feature was the presence of large vacuoles (possibly a sign of degeneration as no infection of the blood followed). P . eylesi. Data on the sporogony of this parasite are given by Coatney et al. (1971); these indicate that the cycle is almost identical with that ofP. cynomolgi (at a temperature of 27°C). The prepatent period after sporozoite inoculation was 12 days or less. P. young;. Only incomplete details of the sporogonic phase of this parasite are available, and these are referred to above.

Orang-utan parasites. The sporogonic cycle of P. pitheci and of P. silvaticum was briefly described in the report of a laboratory demonstration by KillickKendrick et al. (1973). The former parasite developed well in A . balabacensis and A . maculatus, sporogony being completed in 11 days at a temperature of approximately 27°C. P. silvaticum developed equally well in A. balabacensis (reaching maturity in 11 days) and less well in three other species of Malayan mosquitoes. The exoerythrocytic schizonts of P. silvaticum were found in the liver of a splenectomized chimpanzee 7 days after inoculation of sporozoites, and were found to bear some resemblance to those o f P .hylobati of the gibbon. P . schwetzi of the chimpanzee.The sporogoniccycle of this parasite has been described by a number of authors (see Garnham, 1966) but the most recent observations, under strictly controlled conditions, were carried out by Collins et al. (1969). They confirmed the exceptionally large size of the oocyst of this species(mean diameter of 8 1 pm). At 25°C sporozoites appeared in the salivary glands of A . balabacensis on day 14.5. The most interesting part of this investigation was the infection of man with the sporozoites (see below). Coatney et al. (1971) comment on the close resemblance of P . schwetzi to P . ovule in man and concludedthat the latter may be an adaptation of the former in the human host. This was suggested also by Languillon (1957), who had identified P.schwetzi in the blood of a child in North Cameroun; she lived in a village in the forest where chimpanzees abounded. There exist, however, striking differencesbetween the two parasites, particularlyin regard to the large size of the oocyst in P. schwetzi and the normal size of the oocyst in P. ovule. It is much to be regretted that the exoerythrocytic cycle of P. schwetzi has not been investigated, because the remarkable size of the schizont (maturing at precisely 9 days) of P. ovule should provide a significant comparison. Another

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P . C . C . GARNHAM

critical difference between these two parasites is their effect on Negroes. This race is nearly insusceptibleto P. schwetzi; just as it is to the other tertian parasites, P. vivax and P. cynomolgi; but the Negro is fully susceptible to P. ovale (see Coatney et al., 1971, pp. 180 and 181). E. PERIODICITY OF INFECTIVITY OF GAMETOCYTES

In the 1967 review, the writer referred briefly to the work of Hawking et al. (1966) on -vrariationsof the infectivity of gametocytes of a langur strain of P. cynomolgi, in particular relation to the effect of immunity at the time of crisis. In the same paper, these writers put forward the hypothesis that the parasite needs to produce forms (gametocytes) which are infective to the mosquito at its normal biting time, viz. during the night. They stated that the maximum infectivity of gametocytes of this species occurred at about midnight with a minimum at noon. Hawking et al. (1968) extended their observations to other species of malaria parasites and showed that exflagellation of the microgametocytesofP. knowlesi, P. cynomologiand P. cathemeriumoccurredmost prolificallyat midnight, around which time the gametocytes exhibited the most normal morphology. They suggested that mature gametocytes remained normal (i.e. not degenerate) for a period of only 5-12 h during the night, but the oocyst numbers that they quote do not appear to confirm the hypothesis in respect of P. cynomolgi or P. cathemerium infections and only to a limited degree to those of P. knowlesi. Hawking et al. (1971) made similar observations on P.falciparum and stated that there was clear evidence in two of five infections in Gambian children that exflagellationreached a peak at midnight, but no mosquitoes were fed to determine the infectivity of the gametocytes at different times. Hawking’s results are based on highly synchronous infections, but if there is less synchronicity in asexual development (and following such, in the gametocytes also), the figures are likely to become clouded. The chief difficulty in accepting this hypothesis lies in the results of dissections of mosquitoes by competent observers, since the time of Ross and Grassi. Nearly all such work has been performed during the day, following which heavy oocyst infections are frequently encountered. The fundamental nature of this phenomenon, if true, deserved repetition and in 1970 and 1972 Garnham and Powers (1973) carried out similar experiments on two strains of P . cynomolgi-P. c. cynomolgi and the langur strain as used by Hawking. In the first experiments (using P. c. cynomolgi in A . atroparvus) the period of high infectivity was limited to 2 days before the crisis, and during these days both the percentage ofguts infected and the number of oocysts per infected gut rose to a peak (incidentally at noon) and then fell, with no fluctuations corresponding to day and night. In the second experiment, using the langur strain and A . stephensi (as an exact replica of Hawking’s work) the infections in the mosquitoes were found to be limited to two feeds (3 days before the crisis) on two successive midnights. The infection rates were 26 % and 40 % respectivelyand the numbers of oocystswere in the sameproportions. No infections were seen in the mosquitoes fed at noon. A third experiment, conducted

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under similar conditions to the last, was even more convincing as feeding on four successive days demonstrated high infectivity at midnight and lower at noon in a quotidian periodicity. Total oocysts were as follows. First monkey: 20,200; 95,360; 186,479; 0,75; 2,O. Second monkey: 7,27; 0,58; 39,53; 50,88; 22,26. The last day was the seventh day after inoculation and at the time of crisis. (The italic figures represent results of midnight feeds on two monkeys; others are noon feeds.) Some support, therefore, is provided for Hawking’s hypothesis by these results, but the overriding influence of immunity limits the period available for extended observations. Experiments on a large scalewould be necessary to arrive at firm conclusions ;for bothHawking’s work and the writer’s were based on few animals. Moreover, these experiments were not entirely satisfactory, as they concerned infections in A . stephensi, an indifferent vector of P. cynomolgi. W. E. Collins (personal communication) obtained highly significant results in experiments in 4 monkeys infected with P. coatneyi and fed on by A .freeborni at 4-hourly intervals. For several successive days, the numbers of oocysts were in the thousands at midnight and nil at noon, and he concluded that mosquito infection is best obtained by night-time feeding. The hypothesis of Hawking et al. (1967) is valuable in drawing attention to the phenomenon of synchronicity in general; it is suggested that there is an accumulation of a “division protein”, needed for the final mitotic divisions of the nuclei in the maturing schizont. The process might be stimulated by a rise of temperature in the host leading to a periodic concentration of some unknown substance in the blood. The time signal for cell division has been considered further by Arnold et al. (1972) in experiments on mice infected with P. berghei. They showed that the cycle is very sensitive to hormones such as oestrogens, but state that these substances do not provide the actual trigger, which may come from some kind of solar phenomenon which can be screened out by conducting the experiments in a limestone cave, more than 10 m underground, and the effects of which can be restored by exposing the mice to light from a metal arc lamp at 300 foot-candles for 18 h per day.

IV. PATHOGENESIS AND CULTURE A. PATHOGENESIS

The activity of workers at the Liverpool School of Tropical Medicine on the pathogenesis of malaria has continued; it was described in some detail in my 1967 review, and in 1968 Maegraith contributed a review on the biochemical aspects in “Advances in Parasitology”, Volume 6, 1968. Maegraith (1969) summarized the traumatic effects of these changes on the different organs of the body in P. falciparum infections of man. It was shown that increased permeability of the capillary endothelium is a primary factor in pernicious malaria and further observations on the mechanism have been made by Maegraith and his co-workers (Onabanjo and Maegraith, 1970; Angus et al., 1971) using monkeys dying of P. knowlesi malaria. Kallikrein rises to a 50 % level in the plasma and this substance (and its succes-

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P. C. C. G A R N H A M

sor, kallidin) is responsible for the damage to the endothelium and loss of fluid into the surrounding tissue. There is also a simultaneous increase in kininase which destroys the kinins, and a rise in adenosine triphosphate which causes vasodilation and the condition of shock. Confirmation that a “lytic factor” was also concerned in the pathogenesis of this condition was obtained by Fife et al. (1972); they noted that a substance of low molecular weight was present in the separated parasites and that the principal components were lipids including cholesterol. This substance (a) increased the fragility of the corpuscles and (b) produced pyrexia when inoculated into hamsters. Another theory on the pathogenesis of pernicious malaria has been advanced by Miller et al. (1 972). They revert to the old, but by no means discarded, idea that the condition is caused by obstruction of the capillaries by parasitized erythrocytes, or as they term it now, “capillary trapping”. It is suggested that with certain species of Plasmodium (P. knowlesi, P. coatneyi and P. falciparum) the schizont-infected cell becomes deformed, its envelope loses its capacity to change its shape and the cell is unable to squeeze through capillaries, whose diameter is less than that of an erythrocyte; the vessels therefore become blocked. The process may be helped by the presence on the surface of such infected cells of curious knobs visible by electron microscopy. Such structures have been observed in those species which retreat to the internal organs half way through the stage of erythrocytic development, viz. P. coatneyi, P. falciparum, and as has recently been shown by Garnham et al. (1972), P. silvaticum. Both the above accounts of the pathogenesis of malaria, and descriptions of the pathology of malaria in general, are largely based on the final picture in fulminating malaria in primates; probably the pathogenesis of the usual non-lethal infections is different not only in degree but in nature. Biochemical research relating to metabolism of the normal and the drug resistant parasite has continued at a high level, but it is outside the scope of this review. It is described in detail in the monograph of Peters (1970).

B. CULTURE

The importance of culturing malaria parasites is still very apparent, but there is little new to report on the subject, particularly in regard to mammalian malaria. A thorough analysis of the present situation regarding the culture of blood stages was made by an informal consortium held in Geneva (WHO, 1972). The biochemical aspects were particularly considered and a useful list of references is provided in the report. It is interesting to note that the oldest method of all (Bass and Johns, 1912) is today giving most useful results (in the Harvard modification) in the hands of Butcher and Cohen (1971) for the study of immunity in P. knowlesi malaria. It is noteworthy that Dr Bass is still active in New Orleans at the age of 97. Ball and Chao (1971) have developed new techniques for the culture of sporogonic stages of P. gallinaceum, by introducing portions of mid-gut, with 5 day old oocysts, into a tissue culture system of Grace’s cell line of Aedes aegypti, and obtained sporozoites after 6-8 days in vitro. These techniques were originally introduced by Schneider in 1968

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and now in her hands (1972) have led to the cultivation of oocysts (with sporozoite differentiation) of P. cynomolgi in cell lines of Anopheles stephensi. The latter results are mentioned here because they may eventually open the door to large scale culture methods of sporozoites for the production of a vaccine. At present, however, growthis confined to the formation of sporozoites in a normal oocyst : the need is for the cultivation of sporoblastoid material of infinite size and the capacity (unless such material contains the suitable antigen) to differentiate into mature sporozoites. Vanderberg et al. (1972), however, demonstrated that the sporozoites ofP. bergheiin the salivary glands were more highly antigenic than those harvested from mature oocysts.

v. HOSTSUSCEPTIBILITIES AND AFFINITIES In my 1967 review, the probability of monkey malaria affecting man on any scale was considered to be fairly remote, and the observations of the last five years have confirmed this opinion. Warren et al. (1970) concluded, on the basis of negative results from the inoculation into rhesus monkeys of heparinized blood from I 117persons in an enzootic area in Pahang, Malaya, that a zoonosis must be very rare, and would arise only when a man intruded into the normal mosquito-monkey transmission cycle in the jungle. A second case from this area has, however, been reported by Fong et al. (i971) on the basis of the discovery in the blood of a North American of parasites resembling P . knowlesi, though the blood was not inoculated into a rhesus monkey. Although this review deals with “Malaria in Mammals excluding Man”, it is desirable to summarize the recent work on malaria in monkeys, derived from man, in particular reference to the relationships between P. brasilianum and P. malariae, and between P. schwetzi and P. vivax, either as zoonoses or zoonoses in reverse. The susceptibility of New World monkeys to the human malaria parasites was first demonstrated by Young et al. (1966) in Panama. The owl monkey (Aotus trivirgatus) was the experimental host and good infections of P. vivax were obtained, provided that the spleen had been removed first. Since that time, thousands of these nocturnal animals have been collected in Panama and South America and sold into captivity. The owl monkey has proved to be a remarkable host and its introduction has been of the greatest value to malaria research. The need for economy in the numbers used, and the conservation of the species has been emphasized in various conferences, but unfortunately up to now, little or no success has resulted from attempts to breed this animal in primate centres or zoos. Geiman and Meagher (1967) transmitted Plasmodium falciparum from man to Aotus trivirgatus, and Geiman and Siddiqui (1969) succeeded in transmitting P. malariae from a congenitally acquired infection in a Chinese infant, to the monkey. So far, P. ovak has eluded passage to a simian host, though it has been known for many years that this species as well as the three other human species easily infect splenectomized chimpanzees. Similarly these parasites

620

P . C. C . G A R N H A M

can be transmitted to gibbons, especially after they have been splenectomized (Cadigan et al., 1969). All the original work was carried out by the inoculation of large numbers of parasites from the blood of infected people; later, it was shown that in Aotus and other monkeys, gametocytes of P. vivax, P . malariae, and P.fakiparum were capable of infecting suitable species of mosquitoes, and the resultant sporozoites of re-infecting man. Aotus trivirgatus can also be infected with the Asian simian parasite, P. knowlesi (Geiman et al., 1969). It is most surprising that Aotus trivirgatus has never been found infected with a malaria parasite (e.g. the common P . brasih u m ) in nature, nor apparently has this monkey been tested experimentally for its susceptibilityto P. brasilianum, except in the early work of the Taliaferros (1934). They found that “frank” infections only developed if the Aotus had previously been splenectomized, and that infections could not be “established” in the intact animaI. Table IV gives a list of the New World monkeys which have been infected with the human species of Plasmodium. By adaptation, Saimiri and Saguinus monkeys will produce as good infections as does Aotus. Incidentally, Saguinus and other marmosets are easily infected with P . brasilianum. TABLE IV New World monkeys susceptible to human species of Plasmodium

Species of monkey

P.vivax

P. fakiparum

Aotus trivirgatus Alouatta illosa Saguinus geoffroyi Saimiri sciureus Ateles fusciceps A. geoffroyi Cebus capucinus

+ + + + + + +

+ + + +

P. malariae

+

-

+

Geiman et al. (1969) tried to find an explanation or a common factor for the susceptibility of this surprising range of New World monkeys (plus the chimpanzee and probably the gorilla) to the human species of Plasmodium. The presence of haemoglobin A2 in the blood of all the animals is a common factor which may be of significance, especially as it is absent in the Asian macaques which are practically unsusceptible to human malaria. A direct phylogenetic relationship between the New World primates and man seems to be out of the question. The position of P. brasilianum is of great interest. This quartan parasite, widespread in the monkeys of the New World, bears a striking resemblance in all stages of its development to P. malariae in man. In my 1967 review, I suggested that P. brasilianum was “perhaps originally the human P. malariae, brought to the New World at some remote date when it spread into the monkey

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population-in other words, the situation represents a reversed zoonosis”. Such an origin was also suggested by Coatney (1968) and has been rather widely accepted. This idea of a “zoonosis in reverse” was first put forward by Dunn (1965) on the basis of the similarity of the blood stages of P. malariae and P . brasilianum; the sporogonic and exoerythrocytic similarities present even more convincing evidence in that they concern more fundamental aspects of ontogeny. The ultrastructure of P. brasilianum was compared by Sterling et al. (1972) to that ofP. malariae and again remarkable similaritieswere noted: both species differ from other mammalian species in possessing cristate mitochondria, by feeding almost exclusively through the micropore and by the presence of a nucleolus. The evolution seems to have followed the path, P. rodhaini in the chimpanzee, to P. malariae in man, and to P. brasilianum in the New World monkeys. A similar type of origin for the tertian parasite, P . simium of Brazil, from the human P. vivax seems less likely, as the morphological resemblances are much less close. Experimental work on a large scale with P. malariae in New World monkeys and P. brasilianum in man should provide further evidence; also a comparison of the isoeiizymes in parasites in the different hosts is likely to give significant results. The affinities of P. brasilianum and P. malariae are thus seen to be close-in striking contrast to the dissimilarities between these two species and P . inui, the quartan parasite of Asian macaques. There is more divergence of opinion about the alleged relationship of P . ovule to P. schwetzi of the chimpanzee, and this subject is discussed above. Several authors (e.g. Desowitz et al., 1969) have commented on the resemblance between P.falciparum and P . coatneyi and have felt that the latter may be used as a good model of the human parasite. It is true that the infection is lethal in both and that the erythrocytic parasites complete their development in the internal organs, but the profound differences in their respective gametocytes (of a subgeneric order) make any taxonomic proximity improbable. In other fundamental respects also, e.g. exoerythrocytic schizogony, there is no resemblance between the two species. The susceptibility of the vertebrate host to foreign parasites can be broken down in many instances by removal of the spleen and the use of immunosuppressant drugs such as “imuran”. These are useful tools amongst others for the determination of the affinities between different species of Plasmodium (see Garnham, 1971a).

VI. FINESTRUCTURE As was anticipated, the use of the electron microscope has now become almost a routine procedure, and it is probably illogical to divorce the subject of “fine structure” from that of general morphology, There is one malaria parasite (P. traguli) which is so minute that the light microscope is inadequate for observations on it, and recently, Cadigan et al. (1972) in their studies on this species were forced to resort to the electron microscope (EM). This instrument of course has widened enormously our knowledge of the physiology

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of the malaria parasites as well as of their ultrastructure. In the last five years, advances have been made particularly in the following respects. 1. Anterior end

Aikawa (1971) emphasizes that the complex of structures at the anterior end of most stages of the parasite does not include the true rigid, conoid collar, which is present in all other coccidian protozoa. There are however 2 or 3 polar rings, to which are fixed the anterior ends of the subpellicular microtubules; the ducts of the paired organelles or rhoptries and convoluted rods or micronemes also terminate in this cavity. The funtion of these organelles was originally thought to be concerned in penetration; this assumption is still probably partly correct, because the structures all become dedifferentiated on entry into a host cell when they are no longer required. The so-called entry, however, is really a process of invagination, as was shown by Ladda et al. (1969) working on P. berghei.

2. Surface coat The fine structure of the curious knobs or protrusions, mentioned in page 618, has been studied by Rudzinska and Trager (1968) and by Miller et al. (1970), working on P . coatneyi and P . knowlesi respectively. Using the scanning electron microscope, Kass et al. (1971) demonstrated even stranger and much larger structures protruding from the gametocytes of P . falciparum. Beneath the two surface layers run the subpellicular microtubules which again have been shown by Vanderberg et al. (1967) to have a specific number (15 or 1 6 f l ) in P. berghei sporozoites. 3. Micropore (micropyle) This structure appears to be present in all stages of all species of malaria parasites, with the exception of the ookinete and oocyst. Recently, however, Sinden and Garnham (1973) have demonstrated that the micropore is formed fairly early in sporozoite morphogenesis in the oocyst of P. vivax, P. cynomolgi and P . gallinaceum (Table V). Its functional significance in the blood stages of the parasite is clearly related to cytostomal activity, and its dimensions vary accordingly. The structure and function of this organelle were described by TABLE V Incidence of micropore in sporozoites of Plasmodium spp. Species

Oocyst

Salivary Glands

P. uiuax P. cynomolgi P , berghei

2111617 2912121

P.uinckei P. gallinaceum

012003 2212037

3212193 10912200 012418 012396 1512135

011815

~~

Numerator-numbers of micropore Denominator-number of sections

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623

Garnham (1969) at the Third International Congress of Protozoology in Leningrad and more recent accounts are provided by Scholtyseck and Mehlhorn (1970), and by Vivier et al. (1970) whose terminology of this and of other organelles is probably the best. There are many records now which indicate that the micropore is not always single as was at first thought, and several micropores may be present at different stages in the life history. However, the dimensions of the inactive stages remain constant, and the position, on a level with the nucleus, in elongated forms of the parasite (e.g. sporozoite) is characteristic. The mouth of the micropore may be closed by the outer membrane derived from the host cell, but in extracellular stages (sporozoite) the micropore is of course open to the external environment, e.g. the haemocoelomic fluid of the mosquito. Possibly in such situations and in the salivary glands nutrients enter the parasite by diffusion through this orifice. The most remarkable features of the micropore are the thick cylinders, one inside the other, and probably derived from the outer and inner pellicular membranes respectively, though the cylinders are thicker and not always (particularly the outer cylinder) continuous with the surface membranes. The structure is more reminiscent of the conoid of other coccidians than of “desmosomes” with which Beaudoin and Strome (1972) attempted to homologize or compare it. Actually the desmosomes are thickenings on the surface of two contiguous cells which have a totally different function, that is they serve as an attachment plate. To avoid confusion with other structures, the following characteristic features of the micropore should be noted : (a) Uniformity of dimensions and location in any one stage. (b) Presence of thick double cylinder. (c) The 90” angle formed by the cylinders and the surface of the organism. 4. Nucleus Surprisingly little information about this structure was available until the last few years, when at last better techniques began to reveal some of the details of the division. These were described by Ladda et al. (1969) and Aikawa (1971) in particular reference to schizogony, by Garnham et al. (1969b) in the first division of the nucleus of the oocyst of P . berghei yoeli, by Canning and Sinden (1973) in relation to meiosis and mitosis, and by others. Aikawa and Beaudoin (1968) described the useful technique of arresting mitosis in metaphase by the administration of pyrimethamine, though the question of possible damage must be carefully appreciated in the interpretation of the structures revealed. Bahr and Mike1 (1972) were unable to discern chromosomes in dividing nuclei ofP. berghei and P . vinckei, but the DNA in the chromatin was measured by fluorescence density as a dry mass in the EM. The DNA was organized in tortuous fibres and replication forks in high orders of super-coiling. Aikawa et al. (1972) employed cytochemical techniques in an attempt to identify the new structures seen in nuclear division ofP. berghei and P. gallinaceum. They treated their preparations with DNAase and EDTA and thereby showed up more clearly the spindle fibres and centriolar plaques. Certain dense bodies also remained unaffected and these structures, hitherto thought to be chromosomes

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P. C. C . G A R N H A M

(but now presumed not to be of this nature as they do not contain DNA), are now considered to be kinetochores, i.e. the connecting rods between the chromosome and the spindle. The chromosome itself still apparently eludes definition, because present techniques are inadequate. The nuclear membrane does not disappear during division, but nuclear pores are present and bundles of nuclear microtubules radiating from centriolar plaques may be anchored to the pores during mitotic division. Small electrondense bars (in 3 subunits) were suggested by Aikawa (1971) to represent chromosomes (but see above), and the fan-shaped microtubules to represent the mitotic spindle. Similar formations were seen in the early oocyst, while Canning and Sinden (1973) showed that meiotic division of the nucleus of the P. berghei oocyst is preceded by normal spindle formation about 16 h after fertilization of the macrogamete. Subsequent divisions take place within the same nuclear membrane so that a condition which Howells and Davies (1971) term polyploidy is reached. The latter authors then showed that fragmentation of the huge reticular nucleus finally takes place some days later in sporogony (Fig. 1). Canning and Sinden (1973) studied the final phase of the process in P . cynomolgi oocysts and suggested the passage of one pole of the spindle into a sporozoite “finger” and that of the other pole into the adjoining sporozoite. These workers noted the presence of 6 kinetochores and deduced from this observation that the haploid complement of chromosomes in P . cynomolgi lies between 6 and 10.

5. Schizogony Electron microscopy should solve the vexed question of the type of division of sporozoan parasites, by showing whether the process represents endodyogeny (and endopolygeny) or schizogony proper. The presence of external budding automatically excludes the theory of endodyogeny (i.e. internal budding), and even under the light microscope, external budding is easily seen. Aikawa (197 1) provides very convincing electron micrographs of external budding (in starfish outline) of exoerythrocytic schizonts of P. gallinaceum. 6. Mitochondria Mitochondria in general are inconspicuous in mammalian species of Plasmodium; they have sometimes been incorrectly identified as such and more often acristate structures have been thought to possess a mitochondrial function (Rudzinska and Vickerman, 1968). Cytochemical studies by Theakston et al. (1969) demonstrated that the latter “multi-laminated whorls” contain cytochrome oxidase in P.berghei. The cristate mitochondria on the other hand contain succinic dehydrogenase and Howells (1970) points out that in the erythrocytic parasites, the whorls are present, but in the sporogonic phase, they are replaced by cristate mitochondria, the change reflecting different types of metabolic activity of P. berghei in the vertebrate and invertebrate hosts respectively. The only mammalian parasites which have typical cristate mitochondria in their erythrocyte phases are P . brasilianum and P. malariae (Sterling et al., 1972).

Fro.1. Diagrammatic presentation of the nuclear changes postulated to occur during oocyst development in P.berghei. 1. Ookinete-uninucleate zygote which possesses a spherical diploid nucleus; 2, Early oocyst-undergoing transformation from the ookinete. Such stages were observed 48 h after the infective blood meal; 3. Uninucleate oocyst-the nucleus is lobulate and in section shows evidence of three or more “bunches” of intranuclear microtubules which, when cut sagitally, show the typical appearance of “mitotic” spindles; 4. Growth and elongation of the single lobulate nucleus occurs, givingrise to the oocyst. Although the nucleus itself may have divided a few times the observation of irregular elongate nuclei with invaginated regions of their membranes, and of several “mitotic” spindles within a single section indicates the presence of a single, or relatively few, elongate and polyploid nuclei within the oocyst at this stage of development; 5. With the development of peripheral vacuoles between the oocyst wall and the cytoplasm of the oocyst the polyploid nuclei undergo fragmentation. The process continues with the onset of sporoblastoid formation (@ and terminates at the time of the differentiation of the sporozoite “anlagen” on the surfaces of the sporoblastoid. At this stage the nuclei are observed as a large number of spheroidal bodies (7); 8. The final stage in the process is a simple division of each nucleus; 9. Each product of this final division migrates into the base of a sporozoite bud to become the sporozoitenucleus. Nore. These diagrams are not drawn to scale and 1-4 have been adapted from electron micrographs published by Garnham er al. (1969). 4 represents a composite of observations made by Garnham et al. (1969). 5-9 represent interpretations of the observations made by HowelIs and Davies (1971). The figure is reproduced by permission of the Liverpool School of Tropical Medicine.

626

P. C. C. G A R N H A M

7. Exoerythrocytic schizogony The first observations on the ultra-structure of the tissue stages of a mammalian species of Plasmodium ( P . berghei yoelii) were made by Garnham et al. (1969) as the final paper in a series on the motile stages of malaria parasites. The motile stage in the liver is of course confined to the exoerythrocytic merozoite and an attempt was made to obtain tissue containing parasites at precisely this phase of development. A limited amount of material only was available but fortunately this was found to contain mature shizonts with merozoites, which budded off the surface of pseudocytomeres. The merozoites were characterized by striking flask-shaped paired organelles, a micropore, apical rings, and atypical mitchondria (smooth membraned organelles) ;subpellicular microtubules were not seen. Desser et al. (1972) studied the fine structure of maturing exoerythrocytic schizonts of P . berghei berghei, but only found a few mature merozoites (on which no micropores were detected). They noted that the schizont was surrounded by a vacuole (absent in the writer’s preparations) and that merozoite formation arose on the periphery of pseudocytomeres. Acristate mitochondria were seen. Nutrition of the growing parasite was thought to occur by pinocytosis. Sodeman et al. (1 970) described the tissue schizonts ofPlasmodium cynomolgi bastianellii 7 days after the inoculation of sporozoites. The schizonts had a double limiting membrane and contained multiple, irregular nuclei. The cytoplasm was packed with ribosomes; mitochondria were present as well as two types of intraparasitic vacuoles in large numbers. REFERENCES Aikawa, M. (1971). Expl Parasit. 30,284-320. Aikawa, M. and Beaudoin, R. L. (1968). J. Cell Biol. 39, 749-754. Aikawa, M., Sterling, C. R. and Rabbege, J. (1972). Proc. helminth. SOC.Wash. 39, 174-1 94.

Almeida, F. B. andDeane, L. M. (1970). Bol. Z.N.P.A. 4,1-9. Ambroise-Thomas, P., Truong, T. K. and Saliou, P. (1971). Bull. Wld HIth Org. 44, 719-728.

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Garnham, P. C. C. (1966). “Malaria Parasites and Other Haemosporidia”, Blackwell, Oxford. Garnham, P. C. C. (1967a). Protozoology 2,55-64. Garnham, P. C. C. (1967b). In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 5. Academic Press, London and New York. Garnham, P. C. C. (1969). In “Progress in Protozoology”, Vol. 8. Nauka, Leningrad. Garnham, P. C. C. (1970). In “Immunity to Parasitic Animals”, Vol. 2, 767-791. Appleton Century Crofts, New York. Garnham, P. C. C. (1971a). Ann. Zstituto Super. Sanitd 7 , 672-680. Garnham, P. C. C. (1971b). “Progress in Parasitology”, Chapter 6. Athlone Press, London. Garnham, P. C. C. and Uilenberg, G. (1973). Parassitologia 15 (in press). Garnham, P. C. C. and Powers, R. (1973). Znt. J. Parasit. 3 (in press). Garnham, P. C. C., Baker, J. R. and Nesbitt, P. E. (1963). Parassitofogia5,5-9. Garnham, P. C. C., Bird, R. G., Baker, J. R., Desser, S. S. and El Nahal, H. M. S. (1969a). Trans. R. SOC.trop. Med. Hyg. 63,194-197. Garnham, P. C. C., Bird, R. G., Baker, J. R. andKillick-Kendrick,R. (1969b). Trans. R. SOC.trop. Med. Hyg. 63,328-332. Garnham, P. C. C., Rajapaksa, N., Peters, W. and Killick-Kendrick, R. (1972). Ann. trop. Med. Parasit. 66,287-294. Geiman, Q. M. and Meagher, M. J. (1967). Nature, Lond. 215,437-439. Geiman, Q. M. and Siddiqui, W. A. (1969). Am. J. trop. Med. Hyg. 18,251-254. Geiman, Q. M., Siddiqui, W. A. and Schnell,J. V. (1969). Milit. Med. 134,780-786. Gonder, R. and vonBerenberg-Gassler, H. V. (1908). Malaria int. Arch. Leipzig 1. 47-56. Halbastaedter L. and von Prowazek, S. (1907). Arb. Kais. Gesund. 26,4041. Hawking, F., Worms, M. J., Gammage, K. and Goddard, P. A. (1966). Lancet ii, 422, 424. Hawking, F., Gammage, K. and Worms, M. J. (1967). Trans. R. SOC.frop. Med. Hyg. 61, 199-210. Hawking, F., Worms, M. J. and Gammage, K. (1968). Trans. R. SOC.trop. Med. Hyg. 62,731-760. Hawking, F., Wilson, M. E. and Gammage, K. (1971). Trans.R. SOC.trop. Med. Hyg. 65,549-559. Held, J. R. and Contacos, P. G. (1967). J. Parasit. 53,910-918. Held, J. R., Contacos, P. G., Jumper, J. R. and Smith, L. S. (1968). J. Parasit. 54, 249-254. Hoare, C. A. (1972). “The Trypanosomes of Mammals”. Blackwell, Oxford. Hoo, C. C. and Sandosham, A. (1968). Med. J. Malaya 22,299-301. Howells, R. E. (1970). Ann. trop. Med. Parusit. 64, 181-189. Howells, R. E. andDavies, E. E. (1971). Ann. trop. Med. Parasit. 65,451-459. Huff, C. G. and Hoogstraal, H. (1963). J. infect. Dis. 112, 233-236. International Panel Workshop on Experimental Malaria (1969). Milit. Med. 134, 729-1 306. International Panel Workshop on Experimental Malaria (1972). Proc. helminth. SOC.Wash.39, 1-582. Jadin, J. B. (1971). C.R. Multicolloque E u r o p h de Parasitologie 21 6-218. Kass, L., Willerson, D., Rieckmann, K. H., Carson, P. E. and Becker, R. P. (1971). Am. J. trop. Med. Hyg. 20, 187-194. Keymer, I. F. (1969). Phil. Trans. R. SOC.B255,33-108. Killick-Kendrick R. and Bellier, L. (1972). C.R. fer Multicolloque Europken de Parasitologie, 244.

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New Knowledge of Toxoplasma and Toxoplasmosis LEON JACOBS

National Institute of Health U S . Department of Health, Education and Welfare,Bethesda, Maryland, U.S.A. I. Life Cycle and Morphology

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11. Epidemiology ................................................................................. 111. Animal Toxoplasmosis .....................................................................

IV. Human Toxoplasmosis V. VI. VII. VIII. IX.

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Serology and Immunology .................................................................. Biology .......................................................................................... Chemotherapy ................................................................................. New Knowledge of Sarcocystis ............................................................ Conclusion .................................................................................... References .......................................................................................

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In the six years which have elapsed since my previous review of toxoplasmosis in “Advances in Parasitology”, a great deal has been learned about the life cycle of Toxoplasma gondii. This is the most important advance. In addition, various other important studies have contributed to our knowledge of immune mechanisms in toxoplasmosis and how these relate to other intracellular infections and to tumors. We also have advances to record in diagnosis and in regard to epidemiology of the infection. In the course of preparing this updated review, I obtained from the National Library of Medicine over 2000 citations from the biomedical literature for the years 1967 through 1972. It will be impossible to record them all, and I must be very selective about what I report here. I have chosen individuaI case reports only to emphasize some clinical problems, and have concentrated on long-term clinical studies, the work on the life cycle and development of the parasite, epidemiology, and immunology. When the previous review in “Advances in Parasitology” was prepared, the finding of Hutchison (1965) of an infective stage in cat feces after feeding Toxoplasma cysts to cats, was the principal issue of importance. According to Hutchison (1965, 1967), the nematode Toxocara cati, was identified as the means by which Toxoplasma remained infective in the feces of cats, possibly protected within the egg of the helminth. My review in “Advances” cited instances in which Marjorie Melton and I obtained transmission of T . gondii from cat feces which did not contain Toxocara catieggs. I was reluctant at that 631

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time to discount the importance of Toxocara cati in the transmission of Toxoplasma because experiments on filtering aqueous suspensions of ZnS04 floats of cat feces through filters capable of holding back nematode eggs had given negative results in Hutchison’s experiments (1967). Frenkel et al. (1969) have since then provided an explanation of this phenomenon, in that the infective forms of Toxoplasma in feces tend to become aggregated on other particles such as nematode eggs and thus may not come through filters large enough to accommodate their passage. My report of the transmission of toxoplasmosis by cat feces without the presence of nematode eggs stimulated additional investigation by other workers on the contaminative route of infection of Toxoplasma gondii. The elucidation of the complete life history of this parasite is the result of work by a number of very able workers who became involved in this enterprise, in a very competitive way. Because of this, priority can be judged not only on the date of publication of a report, but also on the completeness of the information given in it. Hutchison et al. (1968) reported on the transmission of Toxoplasma by the feces of one cat, in the absence of nematode eggs. This work was done after Hutchison had observed that transmission of Toxoplasma occurred when floats of cat feces containing only partially developed T. cati eggs-incapable of hatching-were fed to mice. At that time, he had carried out experiments on 32 cats, 17 of which were positive for T. cati and 14 of which produced infective feces after they were fed Toxoplasma cysts; on the other hand he had been unsuccessful in producing toxoplasmosis in mice with fecal material obtained from 15 nematode-negative cats which were similarly fed tissues containing Toxoplasma cysts. Hutchison et al. stated that they were also able to repeat the transmission of Toxoplasma with the feces of another Toxocara-negative cat fed on Toxoplasma cysts. However, in view of the micro-isolation experiments reported by Dubey (1967), they felt that the question of the nematode transmission of toxoplasmosis was still open. They were also convinced that there exist unknown forms of T.gondii which are passed independently in feces and capable of surviving for at least three months under moist conditions. Dubey (1968), in the discussion of a paper entitled “Feline toxo-plasmosis and its nematode transmission”, mentioned an observation in his thesis, produced two years earlier, that he had isolated Toxoplasma from the feces of a helminth-free cat, 22 and 25 days after feeding it Toxoplasma cysts. However, he did not discuss the possible explanation or significance of the observation. Subsequent contributions on this subject were made by Sheffield and Melton (1969) who removed Toxocara cati eggs from the uteri of worms recovered from 7 cats orally infected with T. gondii 6-13 days previously. No transmissions occurred in mice fed the eggs immediately or at intervals up to 6 months. On the other hand, flotations of feces collected from these cats between 4 and 10 days after infection and incubated for from 5 to over 100 days produced toxoplasmosis. Subsequently they treated Toxocara-infected cats to remove the worms and then fed the animals Toxoplasma-infected tissues. The results were again positive with floats of the cat feces. They concluded that a new resistant form of T. gondii develops in the cat, and indicated that further studies would be needed to identify this form. In the same issue of the same

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journal, Frenkel et al. (1969) reported confirmatory results. They separated T. cati eggs from T.gondii “fecal forms” by filtration and produced taxoplasmosis in mice with the “fecal form” suspensions. They also obtained infective forms from the feces of a cat which was Toxocara-free. Work and Hutchison (1969a) in a preliminary report and later (1969b) in a more complete description of their work identified a different cyst form of Toxoplasma in cats previously fed the parasite cysts. They correlated the dose of these cysts to mice, by titration, with the occurrence of Toxoplasma infection in the mice. They also produced the infection in mice with single fecal “cysts” which they micro-isolated from washed floats of cat feces. They did not, in these first two papers, attempt to identify the fecal “cyst” even though the second paper contains an excellent photomicrograph of an oocyst of a coccidian, and they observed the maturation of this structure and the formation of two sporocysts within it. In a later paper, however, Siim et al. (1969) reported on work in which they centrifuged and sectioned suspensions of these structures, described them as oocysts and sporocysts, and mentioned their similarity to Isospora bigemina. In an addendum to this paper, they explain that they have applied coccidial terminology to these Toxoplasma forms because of having observed typical coccidial schizogony and gamogony in the intestine of a specific-pathogen-free cat which was studied after being fed Toxoplasma tissue cysts. Kiihn and Weiland (1969) also stated that the feces of Toxoplasma-infected cats can be a potential source of infection, even if free of ascarids, although they did not describe in that paper the observations to support it. These authors also noted the presence, in the infective cat feces, of small organisms similar to coccidian oocysts, but stated that they could not establish that these forms were related to Toxoplasma. Thus, over a period of four years, the original clue of Hutchison (1965) that orally fed cats excreted infective forms of Toxoplasma associated with Toxocara cati led to the independent discovery in various laboratories that the nematode egg was not necessary in the life cycle of the protozoan. A number of investigators added to the knowledge of the coccidial nature of Toxoplasma. Garnham (1971), in a review article, recounts that in December 1969 Hutchison telephoned him “from his laboratory in Scotland to say that the discovery had just been made. In the previous few weeks, the Scottish, Danish, and North American workers had recognized that the cysts were in fact oocysts, whose contents were seen to divide into two bodies (sporocysts) after some days, and the sporocysts were eventually found to contain four elongated bodies (sporozoites). The inevitable, however incredible, conclusion was reached that the oocysts were coccidial in nature. . .” Hutchison called to tell Garnham that he had now found profuse developmental stages of the parasite in the intestinal epithelium of a cat. “Within 10 days,” Garnham wrote, “a letter had appeared in the British Medical Journal and within a month, the full paper (Hutchison et al., 1970).” Garnham’s date agrees with the date of the addendum in the paper by Siim et al. (1 969). Twenty days after the appearance of this paper, two papers were published in Science. Sheffield and Melton (1970) were able to identify the oocysts seen in infectivecat feces as forms of Toxoplasma.These oocysts were seen only after feeding cats infective meals, and only during a period corresponding to that of

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the infectivity of the feces for mice. Moreover, these workers artificially released sporozoites from the oocysts by fracturing the oocyst wall and incubating the sporocysts in a tissue culture medium with bile and trypsin to cause them to excyst. They then washed the sporozoites and introduced them into tissue cultures of primary monkey kidney cells, from which they later produced toxoplasmosis in mice. Thus, they removed all doubt as to the relationship of the oocyst and Toxoplasma, already suggested by Work and Hutchison (1969) and Weiland and Kiihn (1970). In a paper immediately following this report Frenkel et al. (1970) identified the oocysts as Toxoplasma by the close quantitative and biological correlation, in tests using 9 kittens, between the appearance of oocysts and the infectivity of feces. They also examined sections of cat intestine for developing forms, and identified schizogonic stages, macrogametocytes, and microgametocytes typical for coccidia. They reinforced the identity of these forms as Toxoplasma by means of indirect fluorescent antibody tests on sections and smears containing these developmental stages and by the obliteration of fluorescence when the fluorescein-tagged antibody was absorbed with T. gondii proliferative forms from mouse peritoneal exudate. Their photomicrographs can be compared with the two drawings of Hutchison et al. (1970). Overdulve (1970) also identified the infective forms of Toxoplasma as coccidia and considered that they were Isospora. Weiland and Kiihn (1970), following very closely on the other contributions, published photomicrographs showing schizogonic and gametogonic stages in the intestinal epithelium of cats killed on various days after the infective meal. Thus, we see what is always true when scientific opportunity presents itself to vigorous investigators. There has been a convergence on the problem, and the utilization of a variety of methodologies to solve it (Frenkel, 1970). The dates of acceptance of the individual papers indicate that all the investigators approached the solution at the same time, and as I have said above, the reports must also be judged on their completeness. Hutchison has the distinction of identifying the cat as the source of fecal forms. All of the other workers will share in the history of research on Toxoplasma. I doubt that it pays to sort out the priorities. They were all 011 the right track, and the accolades should fall on all of them. Since these initial contributions, a number of papers have appeared, further characterizing the morphology of the intestinal schizogonic and gametogonic stages of Toxoplasma gondii (Dubey et al., 1970; Hutchison et al., 1971; Piekarski and Witte, 1971; Dubey and Frenkel, 1972a). It seems clear that Toxoplasmagondii is a coccidian which may be identical with Isospora bigemina. Dubey et al. (1970) have discussed the taxonomy of the organism and recommend retention of the name Toxoplasma. Hutchison et al. (1971) have given a very studious analysis of the taxonomic questions related to the new observations on Toxoplasma. They state, and I concur, that at this point they are entirely opposed to any alteration in the nomenclature of T. gondii. They predict the enormous confusion which would occur, if consistent with the change from Toxoplasma to Isospora, the clinical disease became similarly changed in name. They cite Article 23 a (ii) of the International Code of Zoological Nomenclature which makes provision for the retention of well known

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names which have been in general use for a considerable period of time, irrespective of priority. Quite correctly, they point out that taxonomic and systematic changes would be influenced by the elucidation of the life cycles of Besnoitia, Sarcocystis, and Frenkelia. Moreover, they, as did Dubey et al., point out that endodyogeny has not, thus far, been observed in any species of Isospora, nor has tissue cyst formation. Zaman (1970) has reported on the measurements of the oocysts and sporocysts of T. gondii in comparison with those of other coccidia. He gives the following ranges (in micrometres): Isosporu felis Isospora rivoltu Isospora bigemina Toxoplasma gondii Sporocysts-Isospora felis Isospora rivolta Isospora bigemina Toxoplasma gondii

Oocysts-

38-48~25-38 20-26 x 15-20 18-23 (diameter) 10-13 x 9-11 21-26 x 17-22 14-19x 11-13 10-13x9-11 6- 7 x 4 5

Dubey and Frenkel(1972b) addressed themselves to the problem of whether or not, in (usually) intestinal coccidiosis of cats, the parasite could be found in extra-intestinal tissues. Using both Isospora rivoltu and I. felis, they were able to obtain infections by feeding kittens less than one day old with ground-up liver and spleen combined, mesenteric lymph nodes, brain, and lung from previously infected kittens. The extra-intestinal stages apparently persisted beyond the time when oocysts of I. felis were no longer present in the feces, three months after the cat was infected. This suggests that these stages may be biologically important. Dubey and Frenkel also cite the work of Box (1970) who found schizonts of “Atoxoplasma” in the liver, spleen, and lungs of canaries infected with I. lacazei; and Long (1970) who found extra-intestinal schizonts in sheep and goats and in chickens. Coulston (1942) made a similar observation in sparrows, identifying extra-intestinal coccidial merozoites as what had been called “avian Toxoplasma”. However, there is no evidence from any of these studies that they were dealing with different stages of the parasite than were to be found in the intestinal epithelium. This is the crucial point, which as yet distinguishes Toxoplasma. Toxoplasma has highly successful tissue-invasive forms which are capable of proliferating in many hosts other than that in which its sexual stages are capable of developing. The only indication, thus far, that extra-intestinal stages of coccidia can remain in the tissues of animals other than their normal hosts is the report of Frenkel and Dubey (1972). These authors reported that mice, rats, and hamsters, which they infected with oocysts of Isospora felis and I. rivolta retained infective stages of these coccidia in lung, liver, or spleen, which were infectious for kittens for as long as 67 days after the rodents were infected. The specificity of the sexual stages of Toxoplasma gondii is indeed notable. In my earlier review, I cited a number of reports which recorded failure in attempts to transmit toxoplasmosis with fecal forms derived from a variety of other animals which had received meals of Toxoplasma cyst-infected tissues.

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To these can be added the report of Nakayama et al. (1969) that feeding floats of the feces of pigs (containing Ascaris eggs) did not produce toxoplasmosis in recipient mice. Rommel et al. (1968) made a similar report of studies on swine, sheep, dogs, cats, mice, and chickens. Jewell et al. (1972) tested members of the raccoon family, Procyonidae, which failed to produce oocysts although they became infected. Thus it appears that the lack of host-specificity of the extraintestinal stages known prior to 1969, which always perplexed workers in respect to the relationship of T. gondii to the Sporozoa, is not similarly characteristic of its schizogonic, gametogonic, and sporogonic forms. These forms occur only in members of the Felidae. In addition to the domestic cat, Jewell et al. (1972) did isolate oocysts from the feces of one jaguarundi (Felis yagouaround) and two ocelots (Felis pardalis) which initially lacked antibody to Toxoplasma.Milleretal. (1972) reported that bobcats(Lynxrufus),Asianleopard cats (Felis bengalensis) and a mountain lion (Felis concolor) also shed oocysts after oral infection. Janitschke and Werner (1972) also reported oocyst development in Felis bengalensis after feeding of Toxoplasma cysts; they obtained negative results with related animals of the family Viverridae. A number of other Felidae were also tested by Jewell et al., such as margay cats, jaguars, pumas, and a tiger, but except for the margay cats, these animals had dye test antibodies and could have been immune. Immunity to the intestinal stages in cats is apparently not absolute. Piekarski and Witte (1971) report that when the antibody titer related to a first infection had fallen, cats could be successfully reinfected, with renewed oocyst-production. Kiihn and Weiland (1969) also reported renewed oocyst-shedding after a second infection in one cat of 5 ; the second exposure was 34 days after the first. On the other hand, Dubey et al. (1970) make the observation that generally there is immunity to the intestinal infection and failure of oocyst-production in cats for 1-5 months after an initial infective meal. They consider it likely that after several months cats develop a less solid immunity to the intestinal forms. We will consider other aspects of immunity in toxoplasmosis later in this review. Hutchison et al. (1971) have addressed themselves to the question of the terminology of the stages of Toxoplasma. They point out that while the new stages, such as schizonts and gametocytes which have been described in the intestinal epithelium fall into already well-defined categories firmly established for coccidia, terms such as “trophozoite” and “zoite”, for the rapidly multiplying and cyst forms in extra-intestinal sites probably cannot be retained. Hoare (1972) agrees and suggests the names “endozoite” for the rapidly multiplying extra-intestinal forms, and “cystozoite” for those which develop within cysts. “Trophozoite” must be considered for the pre-schizogonic intestinal stage, and the term “zoite”itse1f is too undefined in relation to “merozoite”. Frenkel has proposed two terms which may be useful. He suggests “tachyzoite” for the rapidly multiplying extra-intestinal forms of the acute infection, which reproduce by endodyogeny and eventually rupture their host cells; and “bradyzoite” for the more slowly multiplying (by endodyogeny) encysted forms, characteristic of the chronic infection. He suggests that “trophozoite” no longer be used, because it refers to feeding forms and could

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apply to many stages. On the other hand, I believe that, as Hutchison et al. and Hoare have pointed out, this term should be retained for the pre-schizont in the intestinal epithelium. Frenkel also suggests that the term “pseudocyst” is confusing, in that it has been used for both aggregations of tachyzoites and for true cysts; he suggests “group” instead. I would prefer, however, to retain “pseudocyst” for the aggregations of rapidly dividing tissue forms, or “tachyzoites”, within a host cell, because it has been used for a longtime, and its misuse can be avoided by redefinition now. It is useful also in regard to other protozoa such as Leishmania. Thus, in summary, I would suggest the following terms: Trophozoite : Schizont : Merozoite : Gametocyte : Gamete : Zygote : Oocyst : Sporont : Sporoblast :

Intestinal Epithelium Stages The form of parasite which, within cells of the intestinal epithelium, grows, and prepares for schizogony. The form which produces merozoites by multiple formation of cytoplasmic components and nuclear replication. Individual product of schizogony. Precursor to male or female gamete, to be designated male or female on the basis of presence or absence of nuclear divisions. (Male or female) product of gametogony. Product of fertilization of female gamete by male gamete. Zygote with a heavy protective wall. Zygote undergoing division into sporoblasts. Derivative of sporont undergoing further division into sporozoites.

Tissue Stages Rapidly multiplying forms reproducing by endodyogeny, Tachyzoite or Endozoite: filling and distending the vacuole of the host cell until it is destroyed. Host cell containing numerous tachyzoites (endozoites). Pseudocyst : Slowly (by endodyogeny) reproducing parasites withm a Bradyzoite or Cystozoite: cyst wall. A collection of bradyzoites (cystozoites) within a dense cyst: wall-not the residuum of the host cell wall but a separate structure laid down on the internal membrane of the host cell vacuole containing the parasites, and extending among the individual parasites as well. Originally intracellular, the cyst may eventually become extracellular because of distention and rupture of the host cell (Van der Zypen, 1966).

A number of electron microscopic studies of tachyzoites and bradyzoites have been made (Rondanelli el al., 1968, 1969; Van der Zypen and Piekarski, 1967a, b; Piekarski et al., 1971; Sheffield and Melton, 1968). The papers by Rondanelli et al. are concerned with the occurrence of cell lysis after proliferation of the parasites within them. They found electron-dense particles in the 23

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nuclei of parasitized cells, which they regard as indicators of nucleic acid activity stimulated by intracellular parasites rather than a specific effect of Toxopfasma.They also observed that, while the parasites usually distend the cell and cause it to burst, sometimes cells occupied by small numbers of parasites rupture, because of different mechanisms. Van der Zypen and Piekarski describe a quite different electron-dense body in the parasite nucleus, rather than in the host cell, which seems to have a special role as the initiating factor in endodyogeny. They identify this “E body” as DNA, which protrudes itself from the maternal nucleus, divides, and forms the matrix for the development of the daughter cell nuclei. The maternal nucleus, they state, apparently is not fully divided between the daughter cells. Dubey and Frenkel (1972a) made an intensive light microscope study of the schizogonic and gametogonic stages of T.gondii cyst-produced infections in the cat. Piekarski et af. (1971) and Sheffield (1970) also did electron microscopy on the intestinal stages found in the cat following oral infection with cyst forms. Piekarski et al. describe schizogony as occurring in a manner different from that in other coccidia. While in the latter forms, organelles form in “buds” on the periphery of the schizont, and nuclei are later produced by multiple divisions of the schizont nucleus, in Toxopfasmathe schizont undergoes two nuclear divisions before these organelles form, and these organelles are not peripherally distributed. Werner and Janitschke (1970) state that they prefer the term “agamogony” to “schizogony” because they are not convinced of the latter. What they have seen is a type of binary fission but different from endodyogeny. Sheffield (1970), in describing the same events, noted also that one or more divisions of the nucleus preceded the formation of merozoites, which is characterized by the formation of anterior organelles within a coneshaped membrane near the nuclei. It is not clear from the study of Piekarski el al. that formation of merozoites occurs only in a two-fold fashion, from each previously formed nucleus; indeed, their description states that 32 merozoites are formed from a schizont and there are only 4 nuclei at the start of the process, which makes their term “endopolygeny” highly unlikely. Colley and Zaman (1970) state that they observed merozoites forming adjacent to the nucleus of the schizont, following which the outer membrane of the schizont folds inward to separate the individual merozoites. The number of merozoites they found within schizonts ranged from 8 to 14. I am inclined to reiterate my earlier statement that endodyogeny is a special form of schizogony, rather than coming full circle to the conclusion that schizogony is a special form of endodyogeny. Apparently schizogony occurs in a variety of ways among the coccidia (Scholtyseck, 1965; Sheffield and Hammond, 1967), and there is no reason to expect that the process should be uniform throughout the group. At some stage,whether the nucleus is single or already divided once or twice, messages to the cytoplasmic RNA result in the formation of multiple new orgacelles or new organizational arrangements of the endoplasmic reticulum, within which nuclear replicates become organized as individuals. Apparently there are merely differences in the timing of the information transfer, and these do not represent completely different phenomena. Pelster and Piekarski (197 1) have also reported on an electron microscope

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study ofthe development of microgametes of T.gondii. The formation of gametes begins with the multiplication of the nucleus, and then the formation of many protuberances on the surface into each of which a single nucleus migrates. Within each protuberance, two flagella develop from each basal body. The protuberance grows and buds off to form the gamete, which then lies free in the vacuole surrounding the parasite. The description of the surface protuberances is similar to schizogony in some coccidia in the sense that merozoites form peripherally. Their observations of groups of microgametes being shed sequentially by the gametocyte is unusual, however, and undoubtedly will prompt additional studies. It is clear from a number of studies, including the routine use of the mouse in revealing the infectivity of fecal forms in cats, that the oocysts of Toxoplasma are infective to other animals by the parenteral as well as the oral route. Also, animals infected by the oral route with oocysts develop generalized toxoplasmosis with parasites (tachyzoites in pseudocysts and bradyzoites in cysts) demonstrable in their tissues at various times after infection (Kiihn et al., 1972; Dubey and Frenkel, 1972; Miller et al., 1972). Cats have been demonstrated to develop the intestinal stages after feeding of cysts or oocysts or after parenteral inoculation of cysts, tachyzoites or oocysts. There seem to be some differences in the times at which feces become infective after infection of the cat by different routes. Dubey et al. (1970) reported that it requires 20-24 days for cats fed oocysts to produce a new generation of oocysts, as compared to 3-5 days or 7-9 days following the feeding of cysts or trophozoites. On the other hand, one cat infected by the oral route with oocysts, by Hutchison et al. (1971),produced oocysts on the 9th day. Wallace (1973) observed prepatent periods of 21-49 days in cats fed oocysts, but he did not examine the feces of these animals prior to 19 days; 3 cats examined between 8 and 30 days did not produce oocysts. These differences may be due to the use of a variety of Toxoplasma strains, or they may represent a temporal difference in the relationship between a generalized systemic infection induced by some forms prior to schizogony and gametogony in the intestinal epithelium. Janitschke (1971) reports that T. gondii was found in the epithelium of the large intestine as early as one hour after feeding cysts to cats. He concluded that the parasite first enters the wall of the small intestine and, via the lymphatics and blood stream, rapidly colonizes other organs, as well as the large intestine. Similar studies on cats fed oocysts and examined at various times thereafter would be useful to reveal the sequence of stages. 11. EPIDEMIOLOGY The relation between the life cycle of Toxoplasma gondii, as revealed by our new knowledge of the parasite’s stages, and the epidemiology of the infection, is now of considerable importance. In my earlier review (Jacobs, 1967),I reported the occurrence of Toxoplasma infectivity in 2 of 53 stray cats which also had Toxocara cati infections and Toxoplasma antibodies. Wallace (1971, 1973) isolated Toxoplasma from the feces of naturally infected cats, in the latter survey from 12 of 1604 animals

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examined, and Janitschke and Kuhn (1972) identified microscopically 7 (14 %) of 502 cats as having Toxoplasma oocysts in their feces; by inoculation of mice, 5 (1 .O%) of these samples produced toxoplasmosis. Wallace had earlier (1969) broached the idea that the occurrence of human toxoplasmosis on three Pacific atolls was related to the presence or absence of cats and rats on these diminutive bits of land. The acquisition of toxoplasmosis by herbivorous animals can now be explained in a variety of ways. Certainly, direct contamination of the feed of domestic animals, either on pasture or in feed lots, can occur if cats are allowed to defecate on the premises. Also, it appears that filth flies and cockroaches (Wallace, 1971, 1972) can disseminate the parasite. Wallace suggests (1973) that terrestrial molluscs can serve as transport hosts for grazing herbivores. Ruiz et al. (1972) have reported the isolation of Toxoplasma oocysts from naturally infected soil. It is likely that earthworms are involved in moving ToxopIasma oocysts from lower levels of soil, in which cats bury their feces, but it is also probable that the specific gravity of the oocyst is such that, when the soil is saturated with rainwater, the oocysts tend to rise to the surface, as occurs with nematode eggs which have the same specific gravity. The relation of these characteristics of T. gondii oocysts to the epidemiology of toxoplasmosis in man and animals will soon be elucidated. Frenkel and Dubey (1972) have provided valuable information on the resistance of the oocysts to experimental and field environments. The oocysts are able to survive forvery long periods,up to one year, in soil that remains moist and shaded.They are highly resistant to chemical disinfectants, but they can be killed by heating to about 65°C under soil conditions. Low humidity and heat are especially effective in rendering oocysts non-infective. Hot water at 75°C was effective in killing oocysts, when poured on moist cat feces. Heat may be the most useful method for household decontamination. My suggestion is that people who wish to retain cats as pets should use an electrically timed and heated hot tray, such as those which are sold commercially for keeping food warm, for heating the litter in cat boxes, at convenient times, to destroy oocysts. It would be important, with such a device, to assure heating of the entire litter to over 60°C for about one hour. It seems clear, from the present data, that the cat is a necessary link in the maintenance of T. gondii infection as a zoonosis. However, the question still remains as to the relative importance of other routes of infection to man. Additional reports of the occurrence of toxoplasmosis in food animals indicate that in many parts of the world, raw or rare meat can serve as the source of human toxoplasmosis. Catar et al. (1969) reported the isolation of T. gondii from 11 of 15 (73.3 %) pools of diaphragms from 75 pigs, and from diaphragm and brain of 13 of 30 (43.3 %) swine tested individually, 6 from brain alone, 7 from both diaphragm and brain, but not from diaphragm alone. Similar isolation trials performed on 85 cattle yielded 8 positives (9.4%); 4 T. gondii strains were isolated from brain, 2 from diaphragm, and 2 from both. These isolations were correlated with complement fixation test titers of 1 : 4 or higher; no isolations were obtained from sero-negative cattle. Janitschke el al. (1 967) isolated T. gondii from 6 of 50 sero-positive sheep in Germany, but not from 74

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healthy calves, 34 % of which showed low dye test titers. Work (1967) isolated T. gondii from the diaphragm of 7 of 31 sheep, and 10 of 29 swine, but not from 30 cattle. Berengo er al. (1969) obtained isolates from 18 of 60 (30 %) of pigs from Siena, Italy. Amaral and Macruz (1969) isolated the parasite from 8 of 25 hog diaphragms obtained from slaughter houses of Sgo Paulo, Brazil. Jamra et al. (1969) reported finding T. gondii in 6.8 % of 73 specimens of pork, but in none of 73 beef samples in S%o Paulo. These reports add to earlier data on the widespread occurrence of T. gondii in the flesh of meat animals. Berengo et al. state that pork diaphragms are commonly used in the preparation of sausages in the Siena region, and that these sausages are eaten raw. Other peoples of Europe are accustomed to raw sausages, and this custom has extended to the United States where raw sausage has been implicated in many outbreaks of trichinosis. (In the light of our new knowledge of toxoplasmosis, the clinical picture of trichinosis should be re-examined to explain the occurrence of central nervous system disease attributed to a muscle-invading nematode.) Kean et al. (1969) have incriminated rare hamburger as the cause of a small outbreak of acute toxoplasmosis in five medical students. While no meat used in the hamburger was available for examination, the epidemiological evidence is ample to justify this conclusion. The meat, while supposedly beef, may have been contaminated with pork or mutton. Desmonts et al. (1965) have associated the rapid rate of seroconversion from dye test negative to positive in a group of hospitalized tubercular children with the fact that these patients were fed under-cooked meat. They believe that the high incidence of toxoplasmosis in France is connected with the usual practice of eating meat which is undercooked. Other workers have noted the occurrence of toxoplasmosis in patients whose only exposure was rare meat. Although these observations are merely suggestive,enough evidence is accruing to indicate that meat is a very likely source of the infection, and can explain the prevalence of toxoplasmosis in areas where cats are rare, and among individual groups in the population who are not exposed to cats or to contaminated soil. Baruzzi et al. (1970) found that Indians of the upper Xingu river of Brazil had the same prevalence of Toxoplasmaantibodies as other populations, despite the fact that they had no horses, donkeys, oxen, pigs, goats, rabbits, or cats. It would be most interesting to discover the source of their infections. Of considerable importance, in relation to meat, is the effect of freezing on Toxoplasma cysts in the flesh of meat animals. Our own observations (Jacobs er al., 1960) led me to believe that Tuxupfasmacysts were readily killed on freezing. However, while Frenkel and Dubey (1972, personal communication) confirmed this in general, in that freezing at - 6°C killed cysts in mouse brain after 1 day and at - 21°C killed cysts in brain and muscle in 1 day, they nevertheless state a discrepant observation. The skeletal muscle of a monkey frozen at - 20°C for 16 days was used as an inoculum for mice and a strain of Toxoplasma was isolated thereby. Nakabayashi et al. (1967) found that a temperature of - 14°C for as short a time as 3 h killed T. gondii cysts in meat. Because of the considerable public health and economic importance of this aspect of the epidemiology of toxoplasmosis, it is important that further studies be done to determine the exact parameters of survival and death of T. gondii in various

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tissues preserved frozen. My own guess is that, despite the occasional positive finding, T. gondii oocysts will be found to be vulnerable to freezing for even brief periods of time at most sub-freezing temperatures.

111. ANIMAL TOXOPLASMOSIS There are some notable points to be made about T. gondii infection in meat animals. I have pointed out, in the earlier review, my own failure to isolate the parasite from beef. Remington (1968) also failed to find T. gondii in 50 beef samples. Rommel et al. (1966) in experimental infections of calves, found the parasite in the tissues only during the acute stage of the infection, and then most frequently in the lung, liver, and lymphnodes. Boch et al. (1965a) obtained only negative results in 500 slaughter cattle examined for the presence of Toxoplasma, in addition to their results in calves reported above (Rommel et al., 1966). Munday (1971) and Work (1967) failed to isolate T. gondiifrom the retina of 25 cattle or from the diaphragm of 30 cattle. On the other hand, Mayer and de Boehringer (1967) isolated the parasite from the retina of 18 % of 597 cattle. It thus appears that it is rare that Toxoplasmahas been isolated from beef muscle; Catar’s findings are the only ones from skeletal muscle. Piper et al. (1970) studied ocular and orbital tissues, including extraocular muscles, from the eyes of a number of domestic animals, either experimentally or naturally infected. Of three cattle with natural infections, presumably identified by serological tests, and seven with experimental infections, Toxoplasmawas isolated from the retina of one; although various lesions were described in the iris, ciliary body, choroid and other tissues, no organisms were seen in them. It may be that, when Toxoplasma does occur in cattle, it becomes restricted to the brain and eye. More work should be done to obtain adequate data on the occurrence of T. gondii cysts in beef. A number of studies of experimental or naturally occurring toxoplasmosis r al. in domestic animals have been reported. Work et al. (1970) and M ~ l l e et (1970) reported on clinicalillness in experimentally infected swine which received the RH strain of T. gondii or a strain of porcine origin. Both strains caused disease, with fever and anorexia, and with dyspnoea in RH infections. Death occurred in 1 of 4 pigs infected with the R H strain, but in none of those given the porcine strain. Parasitemia with both strains was demonstrable during the time of fever. However, when the surviving RH-infected animals were killed at 6 weeks post-inoculation, it was not possible to isolate the parasite from them while it was easily recovered from the pigs inoculated with the porcine strain, when they were killed 6 5 7 weeks after infection. It appears that the RH strain, which has been passaged in the laboratory for about 30 years, has lost some of its ability to produce chronic infection, at least in some animals. When pregnant sows were inoculated during the 3rd month of gestation, fatal infections resulted in 50-100 % of the litters of RH-infected animals and in 30 % of those infected with the porcine strain. When surviving piglets were killed, 3-4 weeks after birth, T . gondii was recovered from those infected with the porcine strain but not from RH-infected animals. The same was true of the sows killed 25-79 days post-inoculation. Again, the RH strain had apparently

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disappeared, or its numbers were so small in the tissues that it could not be recovered on inoculation of a small sample, even though parasitemia and congenital transmission was demonstrated earlier. It is apparent that congenital infection can occur readily in sows that acquire the infection during pregnancy. This may occur in nature, as has been reported by Sanger and Cole (1955), but it apparently does not usually occur in the same epidemic form that is found in sheep. Nobuto et al. (1969) found strikingly different rates of infection in various pig-breeding farms, and observed, on contaminated farms, a rise in positive serological tests in piglets with increasing age, which they interpreted as postnatally acquired infection. Boch et al. (1965b) have shown that congenital toxoplasmosis does not occur in swine that acquired infection prior to pregnancy. It is likely that in the contaminated Japanese farms, the pigs’ infections occur before pregnancy. It would be interesting to observe whether or not congenital toxoplasmosis would appear in epidemic form if negative sows were introduced into contaminated farms during their breeding seasons. Congenital toxoplasmosis in sheep does occur with high frequency in New Zealand, Australia and England (Hartley and Moyle, 1968;Watson and Beverley, 1971a). For example, Munday (1971) estimated that toxoplasmosis was the cause of 46% of outbreaks of abortion and neonatal deaths in sheep in Tasmania during the period 1962-1968. The occurrence of the infection in sheep may be the most important agricultural problem with this parasite. I reported in 1963 that experimental congenital transmission of ovine toxoplasmosis occurred only in ewes infected during pregnancy, not prior to pregnancy. In seronegative ewes infected at about 30 days of pregnancy, congenital infection resulted frequently in early death or mummification of the foetus. In ewes infected at 90 days of pregnancy, congenital transmission occurred frequently, but only a small percentage of these ewes aborted. In many cases they gave birth to live lambs of which about 20% died. Hartley (I 964) found, similarly, considerable immunity in ewes which had naturally acquired Toxoplasma antibodies prior to experimental challenge, but he did find congenital transmission and some abortions among the ewes with dye test antibody. Watson and Beverley (1971b) observed experimentally infected ewes through a second experimental infection in the next breeding season. Of 16 ewes challenged with T. gondii when about 90 days pregnant, 14 had live uninfected lambs and healthy placentas, one was barren, and one had a live but infected lamb. In another test (Beverley and Watson, 1971), they found that experimental infection of ewes prior to mating will generally prevent abortion due to toxoplasmosis after challenge in mid-pregnancy. Subsequently, Beverley et al. (1971) reported a trial of a killed vaccine in the prevention of ovine abortion due to experimental toxoplasmosis. Only ewes which developed relatively high fluorescent antibody titers (greater than 1 : 128) following vaccination with lysed Toxoplasma manifested any protection against foetal death following a challenge inoculum. This was only partial; the pregnancy was more often normal, but the foetus and placenta were infected. There have been other reports of toxoplasmosis in dogs, cats, pigs, and other domestic animals. In general, infections induced with cysts and tachyzoites

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show the picture of subclinical infection or mild illness in mature animals. There is greater susceptibility in the young, with more extensive tissue invasion and sometimes death, when large inocula are used. Dubey and Frenkel(1972a) reported diarrhea in young kittens fed cysts, and later hepatitis, myocarditis, myositis, pneumonia, and encephalitis. Older kittens showed no disease. After oocyst infection, the animals retain cysts in their tissues in the same type of distribution as is found following inoculation with other stages of the parasite. When infections are produced with oocysts, there may be diarrhea and vomiting as well as fever and lymphadenopathy, as was demonstrated by Kuhn et al. (1972) in dogs. Vainisi and Campbell (1969) have described ocular toxoplasmosis in cats.

IV. HUMAN TOXOPLASMOSIS While toxoplasmosis usually occurs in individual cases, the small outbreak, traced to hamburger, described by Kean et at. (1969) is indicative of its capability of occurring in epidemic form. A much larger epidemic has been described by Magaldi et al. (1969a) in the state of Silo Paulo. These authors claim that within a 3-month period at a university, 110people including 96 students and 14 non-students, were diagnosed clinically and/or serologically as having acute toxoplasmosis. The symptoms were principally lymphadenopathy and fever. Over half of the students had dye test titers over 1 : 4000, ranging up to 1 : 256 OOO, and similar titers were found with the indirect fluorescent antibody test. While they were unsuccessful in some attempts at isolating Toxoplasrna from lymph nodes, they did observe some increasing titers, and the histopathology of biopsied nodes was considered compatible with toxoplasmosis. While this report does not provide definitive proof, it is likely that these investigators were dealing with an outbreak of the infection, even though some of the cases they identified should best be excluded because antibody levels were low and stable. The authors state that most of the subjects claimed to have eaten undercooked meat,but in a follow-up paper (Magaldi etal., 1969b)they reported that they could not identify the source of infection in this situation. In another report from Brazil, Amato Net0 et al. (1967) describe a household situation in which there seemed to be a number of infections recently acquired. This household was overcrowded, and there was very poor hygiene both inside and outside the structure. Eight families, in total 31 persons, lived in 12 rooms, an average of one family per room. There were also numerous cats, rats, and pigeons in and around the house. One girl, aged 12, was the first patient diagnosed. She had glandular toxoplasmosis and a high dye test titer. Twenty-seven inhabitants of the household were then tested; 13 were negative in the dye test, but the rest had titers of 1 : 64 (2 persons) to as high as 1 : 128000. Five were children under 10 years of age, all of whom had titers of 1 : 1024 or higher. In 2 cases, a second test revealed a higher titer, suggesting that the subclinical infection was very recently acquired. The coincidence of infection in this group of people may have been due to contamination with cysts from cat feces, since they lived in a highly unsanitary situation. The descriptions of the Brazilian outbreaks point up the difficulty of establish-

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ing a definitive diagnosis of acute toxoplasmosis. Feldman (1968), Remington and Gentry (1970), and Remington (1970) have paid special attention to this problem in review articles. These authors have pointed out that the persistence of dormant Toxoplasma infections, and of antibodies, makes it difficult to relate positive serological and parasitological findings definitively to a current disease. Remington et al. (1968a) developed an indirect fluorescent antibody test for identification of specificIgM antibodies in acute toxoplasmosis. Theoretically,the demonstration of IgM antibodies would be a very significant indication of recent infection, because IgM antibodies appear early in the infection. Also, in the newborn, IgM antibodies would represent, in the absence of a placental leak, manufacture of antibodies by the infant, because these larger globulin molecules do not traverse the placental barrier. In actual practice, however, the test for IgM antibodies in acquired toxoplasmosis is not always conclusive (Remjngton, 1969), in that IgM antibodies have persisted in some cases for some years (Remington et al., 1968b).However, false negatives have not been seen, fortunately; so the absence of IgM is probably indicative of past infection. Vitali et al. (1969) report that, in 2 cases of acquired toxoplasmosis, confirmed both serologically and parasitologically, IgM antibodies had disappeared 3 and 4 months after onset of disease. Thiermann and Stagna (1972) followed 27 cases of acute toxoplasmosis and 33 cases they considered chronic infections for various periods of time. The titer curves they obtained indicate that IgM antibodies appear early in acute infection and reach their peak earlier than IgG, then disappear during the 3rd to 5th months. They may reappear later and persist for a year. In chronic cases, IgM may appear sporadically and persist a short time, or on occasion remain for several months. It is interesting to conjecture that the reappearance of IgM antibodies represents a localized flare-up of the infection which is not usually discernible clinically. In the newborn, occasional “false positive” IgM titers have been observed in the absence of evidence of maternal macroglobulins in the infant’s serum and, also, enough “false negatives” occur to make this test uncertain (Remington, 1970; Desmonts, 1971). Possibly, quantitation of IgM may eventually result in the IgM-IFA test becoming useful as a screen for detecting congenital infection, as demonstrated by the reports of Alford et al. (1969a, b). These workers detected 6 cases of congenital toxoplasmosis in 29 16 consecutive live births, by quantitative tests for IgM and subsequent determinations of the etiological agent by other serological techniques. Various problems of acute and chronic toxoplasmosis still require resolution, as is indicated by the reports appearing concerning them. Involvement of the myocardium has been reported in severe acute toxoplasmosis on a number of occasions. However, it is rare to observe myocarditis in the absence of other signs or symptoms representative of multiple organ involvement in addition to lymphadenopathy. Mullan et al. (1968) report such a case, in which the diagnosis of acute toxoplasmosis was established serologicallyand by isolation of the parasite from a biopsied lymph node. The patient was a 21-year-old woman, whose principal complaints were low-grade fever, cervical adenitis, and frequent ventricular ectopic beats; no pneumonitis was evident clinically, nor were any other signs of widespread tissue involvement. Her course was

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relatively prolonged, from an onset in June until early December, despite chemotherapy with pyrimethamine and sulfadiazine administered in late September and early October. Some 21 months after the onset of her illness, she still had some ventricular ectopic heart beats. Discussions of myocarditis and pericarditis possibly associated with toxoplasmosis are contained in a few recent papers (Durge et al., 1967; Buhler, 1971 ; Theologides and Kennedy, 1969). However, the diagnostic criteria for attributing myocarditis to Toxoplasma infection decrease in certainty as one proceeds from the acute generalized disease in man to less severe infection involving many organs, and to lymphadenopathic disease without other manifestations than irregularities of conduction. The diagnosis of chronic toxoplasmic myocarditis is the least certain. Henry and Beverley (1969) gives a good discussion of these diagnostic problems in a paper on experimental toxoplasmic myocarditis and myositis in mice, in which they found Toxoplasma lesions in a high percentage of animals infected with virulent and avirulent strains. Chandar et a/. (1968) have reported a case of polymyositis in a 9-year-old boy, coincident with toxoplasmic lymphadenopathy. The boy had such muscle weakness in his neck, torso, and limbs, that he was unable to lift his head, or to sit up or walk. Histological examinations of biopsied muscle revealed T. gondii. He was treated with pyrimethamine, sulfa, and folic acid, and apparently responded. Coincidentally, again, the boy’s sister was found to have a dye test titer of 1 : 32 000. It was recalled that she had had cervical node enlargement two weeks before her brother’s illness began. This coincidenceprobably represents another instance of infection from the same source. A similar case of polymyositis was reported by McNicholl and Underhill (1970). This was in a boy, 9 years of age, with increasing muscular pain and weakness associated with lymphadenopathy and a dye test titer of 1 : 16000. The questions of the definitive diagnosis of ocular toxoplasmosis and of the evaluation of therapy of the ocular disease, which are of course inter-related, are still with us. Desmonts (1966) has utilized the comparison of titers in aqueous humor and serum, in relation to the concentration of the globulins in these fluids, as a means of arriving at a definitive serological method of diagnosis. A coefficient, the aqueous humor antibody coefficient, is derived by multiplying the ratio of antibody titer in the aqueous to that in serum by the ratio of the concentration of globulins in serum to that in aqueous. As the result of the study of 596 patients, Desmonts concludes that if the coefficient is 2-7, the diagnosis of ocular toxoplasmosis is suggested; if it is 8 or higher, the diagnosis is probable. Analyzing his cases, he found that 33.9 o/, of 309 patients with posterior uveitis had a coefficient of 2 or higher, and 24.2 % had a coefficientof 8 or higher. On the other hand, of 215 patients with anterior uveitis, only 4.6 % had a coefficient of 2 or higher, and only 1.8 % had a coefficient of 8 or higher. These data are strongly confirmatory of earlier conclusions by Woods et al. (1953) that ocular toxoplasmosis is principally manifested as a posterior uveitis. Remky (1968), in a study of 1000 cases of ocular disease, has confirmed Desmonts’ findings.

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Rottini et al. (1971) describe the use of the indirect immunofluorescense test and quantitative assays, to measure IgM, IgG, and IgA, in the sera of 10 cases of active or reactivated ocular toxoplasmosis. IgM antibodies were constantly present, and there was also a high titer of IgA as well as IgG. The IgA titers were reported to be higher than those of IgG or IgM in some cases. In most of the cases, the titers of three globulin classesshowed parallel increasing and decreasing curves. These findings may be illustrative of the reappearance of IgM related to foci of reactivation of infection, as I conjectured above. More work should be done along these lines. Friedman and Knox (1969), in an attempt to define a natural history for recurrent retinochoroiditis, have pointed out the difficulties of obtaining such knowledge on the basis of clinical history, serological and skin tests, and subsequent observation. They stress, also, that in order to evaluate therapy, it will be necessary to perform double-blind studies on patients who are adequatelyclassified on clinical criteria and serological test results. It is to be hoped that such studies will be performed, utilizing the new methods available in the clinical laboratory, and with adequate adherence to rigidly defined clinical criteria. Friedmann and Knox point out that in their series of cases, first symptoms appeared between the ages of 7 and 57 years of age, with 75 % of the cases first experiencing visual difficulties between 10 and 35 years. It is easy to believe that a considerable percentage of toxoplasmic retinochoroiditis seen between early childhood and early adult life is due to congenital toxoplasmosis. However, this does not exclude the possibility that some cases of acquired toxoplasmosis eventually result in ocular infection. Several cases of ocular manifestations, coincident with or closely following systemic toxoplasmosis, have been reported in earlier literature. Ramsdell and Gamero (1967) have reported an additional one, which is acceptable although the proof is not conclusive. There is no reason to deny the possibility that in acquired toxoplasmosis, as in the congenital infection, ocular manifestations may be temporarily far removed from the other signs of toxoplasmosis. When one considers the bleak prognosis for progressive retinochoroiditis, as reported by Friedmann and Knox, it is clear that more work on ocular toxoplasmosis and its chemotherapy is merited, even though, statistically, it is a much smaller threat to vision than diseases such as glaucoma. The man going blind from toxoplasmic retinochoroiditis is not comforted by the fact that his condition occurs less frequently than other causes of loss of vision. In view of the difficulties in defining the natural history of toxoplasmic uveitis and evaluating its therapy, and in view of the pharmacokinetics of antiToxoplasma drugs (Kaufman and Caldwell, 1959), I hesitate to place much credence in the report of Linton (1969) on the use of low doses of pyrimethamine in the prevention of recurrent uveitis due to toxoplasmosis. Nevertheless, his enterprise should be noted. Analogizingfrom the use of drugs in the suppression of malaria, he placed 15 patients, whom he had seen during the previous 10 years, on pyrimethamine, 25 mg once a week, after he had treated them effectively for acute episodes of toxoplasmic uveitis according to the regimen suggested by Kaufman and Caldwell. Aside from one case, in which a young schoolboy had forgotten to take his pill, he observed no recurrences among

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these patients until oral pyrimethamine was temporarily taken off the market. During the next few months, 8 of his patients experienced acute exacerbation of the ocular lesions, and he had to find new supplies of the drug in order to treat them. Since these patients had presented themselves to him at various times in the past decade, it seems unlikely that the exacerbations were all coincidental. While Linton’s observations are equivocal, the idea of suppressive treatment might well be considered in any further studies of the natural history and clinical management of toxoplasmic retinochoroiditis. O’Connor (1970) has given a scholarly paper on the mechanisms of pathogenesis of ocular toxoplasmosis in relation to treatment and in relation to his own experimental studies on these mechanisms in animals. Remington (1970 and 1972)has discussed toxoplasmosis in the compromised host, due either to the basic underlying disease such as hematologic malignancy, or to the therapy such patients receive, such as high doses of corticosteroids and cytotoxic agents. Increasing numbers of cases of this type are being reported (Gelderman et al., 1968; Cohen, 1970). Vietzke et al. (1968) described clinical serological and pathological findings in 6 cases of toxoplasmosis observed in the National Cancer Institute wards of the Clinical Center, National Institutes of Health. Encephalitis, myocarditis, and pneumonitis due to Toxoplasma were found. Dubin et al. (1971) have similarly observed acute toxoplasmosis in a case of lupus erythematosus treated with immunosuppressive agents. Since active infection is treatable, it is important to consider toxoplasmosis in the differential diagnosis of widespread systemic and central nervous system disease occurring in patients undergoing therapy for malignancies. Vogel and Lunde (1969) and Lunde et al. (1970) have discussed the serological diagnosis in such cases. Observations on the effects of immunosuppressive agents on chronic Toxoplasma infections in animals will be discussed below in relation to new knowledge of the mechanisms of immunity in toxoplasmosis. The occurrence of parasitemia during chronic Toxoplasma infections in animals and man was mentioned in my earlier review (Jacobs, 1967). Miller et al. (1969) reported instances of such late parasitemias in two patients with toxoplasmosis. One of these was studied and reported earlier, and another case was more recently observed. In the first case, isolation of T. gondii from peripheral blood was accomplished 14 months after the woman delivered a congenitally infected infant. She had never experienced illness. In the second case, the isolation from bIood was made two months after an attack of lymphadenopathic toxoplasmosis, at a time when the patient was asymptomatic. The significance of these observations relates to the possibility of the transmission of toxoplasmosis by blood transfusion. Siege1 et al. (1971) report on four cases of toxoplasmosis acquired as a result of transfusion of leucocytes from two donors who were patients with chronic myelogenous leukemia who showed elevated Toxoplasma antibody titers. Roth et al. (1971) reported on the course of the infection in one of these cases. Although the initial Toxoplasma infection was controlled with anti-Toxoplasma drugs, pyrimethamine and sulfadinzine, a recurrent episode about one month after treatment was fulminating, probably because she was also being treated with cyclophosphamide

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and prednisone. Here again, we must refer to later remarks on immunity, but these cases do signal the importance of awareness of toxoplasmosis in patients receiving whole blood or platelets or leucocytes by transfusion. The question of congenital transmission of toxoplasmosis during the chronic or latent stage in animals and human beings is still a subject of discussion in various clinical situations. I believe, however, that it is being resolved. Some definitive studies in the last few years have narrowed the arguments concerning congenital transmission and the circumstances of its occurrence. Janitschke and Jorren (1970) report on the examination of 270 foetuses from 39 rabbits which were experimentally infected with Toxoplasmagondii between the 267th day and the 7th day before mating. Those rabbits which were infected earliest, up to 35 days before mating, produced normal offspring with no evidence of toxoplasmosis. When the experimental infection of the dams took place no earlier than 32 days before mating, a small number (3 in 25) of the foetuses were infected. In contrast, when experimental infection was done early after conception, a very high rate of congenital transmission occurred, with 50 of 60 foetuses invoIved. Intra-uterine transmission was less usual when the dams were experimentally infected towards the end of pregnancy, in the last 10 days, when only 19 of 55 foetuses showed evidence of toxoplasmosis. These results in rabbits are similar to those obtained in prospective clinical studies in man, as I will describe further on in this section. Werner and Egger (1969) have described additional experiments on murine congenital toxoplasmosis. When a strain of T. gondii which had “only little affinity” to the reproductive organs was used, 85 of 120 mice infected up to 30 weeks prior to conception delivered normal young. The uterine tissues of 6 of these female mice contained Toxoplasma, but the parasite was not demonstrable in the foetuses. The remaining 35 female mice showed infection of the foetus with varying frequency; the uteri of these dams were infected, but the foetuses, although infected, had no developmental anomalies. Werner and Egger conclude that the occurrence of congenital toxoplasmosis is dependent on the virulence of the strain of the parasite, its toxicity for embryonic tissue, and its affinity for the reproductive organs, because their results with the Weiss strains are different from results with other strains they had used earlier. Nakayama (1968) used a porcine strain (S-273) of Toxoplusma, of low virulence for mice, in a study of the effects of challenge of “immune” animals on the outcome of pregnancy. Congenital transmission of R H strain Toxoplasma was observed only rarely in mice, challenged with the RH strain I to 6 months after the initial infection with the S-273 strain; only 1 foetus of 310 examined was found infected. Of 104 foetuses of 44mice in this study, 104 were examined microscopically to detect cysts of T.gondii, and all were negative. The remaining 205 progeny were challenged with the RH strain, from the peritoneal exudate of mice, and only 3 survived; these 3 are presumed to have been previously infected. The controls of Nakayama are even more interesting. His results indicate that strain S-273 was rarely transmitted congenitally. He attributes this to the fact that strain S-273 does not proliferate in mice to the same extent as the Beverley

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strain, with which many of the earlier studies of congenital transmission in mice were conducted. For instance, he found that T. gondii, S-273, could be isolated from 55 % of the uteri of chronically infected mice, while 86 % of mice chronically infected with the Beverley strain were demonstrated to harbor the parasite. Possibly more worthy of notice are the results Nakayama obtained with pregnant mice infected with the R H strain as controls for the experiments described above. Here he found that practically all of the placentas of these mice were infected; only 7 of 464 escaped, and these were examined very early. However, only 52 of the 464 foetuses involved were shown to have toxoplasmosis. These experiments involved pregnant mice which were killed and examined very early, 1-6 days after inoculation with the RH strain of T. gondii In other experiments, mice were inoculated with the S-273 strain and with RH trophozoites about 2-23 months later. Then they were mated and allowed to suckle their young. None of the young examined up to two weeks after birth were found infected. Some of the young examined at 3, 5, or 7 weeks after birth were infected with the S-273 strain. These experiments indicate a certain degree of immunity to congenital infection even in mice, which have been the model on which the question of congenital transmission in humans has been posed. So far as experience on congenital transmission of human toxoplasmosis is concerned, the latest papers of Couvreur (1971) and Desmonts (1971), who have had an extensive collaborative study at the St Vincent de Paul Hospital in Paris for a number of years (Desmonts and Couvreur, 1967), are of special note. Couvreur reports that, in the prospective study of more than 25 000 pregnant women, no case of congenital toxoplasmosis was found whenever maternal infection occurred prior to pregnancy. There were 118 cases of maternal infection closely related, in time, to pregnancy. The outcome of these 118 pregnancies were : abortion or neonatal death, without confirmation by examination of the foetus, in 9 cases (7.6 %), congenital toxoplasmosis in 39 cases (3373, including 2 perinatal deaths, and subclinical infection in 28 cases. Seventy (59.3 %) children born of mothers who, on the basis of serology, were infected during pregnancy, proved to be free of infection. “According to the present series, there is strong evidence that the time of maternal infection during pregnancy is an important factor in foetal risk. There was no proven case of foetopathy whenever maternal infection was close to conception. Infection in the first trimester was less frequently associated with foetal infection but carried a higher risk of severe foetopathy. Maternal infection in the third trimester resulted in more frequent foetal infection which was always subclinical. The results were intermediate between these two extremes for maternal infection during the second trimester (Couvreur, 1971).” Desmonts (1971), whose serological and parasitological studies are the basis of the clinical observations reported by Couvreur, has described in considerable detail the difficulties attending the diagnosis of toxoplasmosis in an infant born of a mother with a high antibody titer. Hopefully, newer methods of quantitating IgM and IgA as discussed above will be useful in revealing true cases.

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Of the neonatal cases who were found infected during pregnancy, follow-up studies will probably reveal some instances of retinochoroiditis. However, these long-range studies of Desmonts and Couvreur have established, with very little equivocation, that maternal infection prior to pregnancy does not usually result in foetal damage. Janssen et al. (1970) examined, by inoculation of mice, 218 maternal or foetal tissues obtained from 172 cases of abortion and from 10 cases of curettage of non-pregnant women who had had abortions. Seventy per cent of these women had a positive dye test, and 29% had a positive complement fixation test for toxoplasmosis. In only 1 case, which was definitely a latent infection, was it possible to isolate T. gondii from curettage material taken after a second abortion. Products obtained from an abortion that this woman experienced 5 months earlier had been negative, and at that time her dye test titer was 1 : 256 and her complement fixation titer 1 : 5. A third abortion, 7 months later, was very thoroughly studied. Practically all of the material obtained, placenta, fetus, and products of curettage, were inoculated into mice. All the mice remained negative. Janssen et al. concluded that it cannot be stated with certainty that T. gondii was responsible for the abortion even in this positive case. I agree; since T. gondii can encyst in uterine tissues, its demonstration in material obtained by curettage after abortion may have been coincidental. These authors then give a good discussion of the technical problems associated with the demonstration of Toxoplasma and with the reports of other workers who have claimed that there is a relation between latent T. gondii infection and habitual abortion. Their conclusion is that such a relation has not been demonstrated. Kimball et al. (1971) examined, serologically,4048 women during pregnancy, in a prospective study of the occurrence of congenital toxoplasmosis. Of these, 2765 were negative and therefore at risk. Six of these converted to positive, and two of them transmitted the infection to their infants. Of the 1283women who were originally positive, 17 showed a substantial rise in titer on repeat tests, and one of these transmitted T. gondii to her infant. This woman was first tested 8 weeks prior to delivery, when her dye test was positive but the complement fixation test was negative. At term the dye test titer was elevated and the complement fixation test was positive, suggesting that infection had occurred during pregnancy.

V. SEROLOGYAND IMMUNOLOGY I have already mentioned the possibilities of the usefulness of immunofluorescence techniques for the identification of specific immunoglobulins. Possibly the most notable development in the serology of toxoplasmosis is the practically universal acceptance of the indirect immunofluorescence technique (IFA) in the diagnosis of human and animal infection with T . gondii (Remington, 1970).In addition to the references cited by Remington, there are additional evaluations of the IFA by Fairchild et al. (1967), Stagna et al. (1970), Kramer et al. (1970), Muller (1971), Ambroise-Thomas e f al. (1971), and Archer et al. (1971). IFA reveals antibodies as early as the dye test and yields titers compar-

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able to those of the dye test. From a practical standpoint, it is easier to perform and also much less hazardous, because there is no need to handle living organisms. Therefore, it can have much wider use in laboratories that have fluorescence microscopy equipment. The dye test should be maintained as the reference in those specialized laboratories that have developed the competence to perform it (Ambroise-Thomas et al., 1971). Araujo et al. (1971) have pointed out that false-positive anti-Toxoplasma IFA reactions can occur in the sera of patients with antinuclear antibodies, and there may be other circumstances in which the IFA should be checked against the dye test or the hemagglutination test. It appears logical to assume that both IFA and the dye test detect antibodies that act on surface antigens of T. gondii. On the other hand, Lunde and Jacobs (1967) showed that fractions of T.gondii eluates, separated by chromatography, could be divided into those which stimulated the production of only hemagglutinating antibodies, when inoculated into rabbits, and those which stimulated both HA and dye test antibodies. When “ghosts” of lysed T. gondii were separated from the soluble components of the organisms by high-speed centrifugation and repeated washings, and inoculated into rabbits, only low transient levels of HA antibody were produced, but high dye test titers. When the supernate of lysed T. gondii preparations were separated from the “ghosts” by high speed centrifugation and inoculated into rabbits, predominantly HA antibodies were produced. These results suggest that the sequence of antibody production in acute toxoplasmosis, with dye test antibodies appearing a week before HA antibodies are detectable, is the result of an initial stimulus by surface antigens of the parasite and to a later stimulus by internal components when some of the parasites are destroyed. A few studies on the accessory factor required in the dye test should be mentioned. Kobayashi et al. (1968) studied the effects of anticoagulants on the dye test, and concluded that the use of citrate made Toxoplasma antibodynegative sera more suitablefor accessoryfactor, inhibiting non-specificreactions caused by heat-stable anti-Toxoplasma factors. They conjecture that the effect is probably due to chelation of Mg and Ca cations, more of which may be necessary for the non-specific than for the specific reaction. The amount of citrate used was insufficient to effect complete chelation of the cations; when this was accomplished by high doses of citrate or ethylene diamine tetra-acetate, the dye test reaction with positive sera was obliterated. The use of citrate may be justified in situations where it is difficult to obtain adequate accessory factor sera, but this certainly requires that there be ample controls to assure that the sensitivity of the test is maintained. Wallace (1969) also found citrate useful. Kobayashi et al. (1969) studied the usefulness of guinea pig sera as accessory factor. When Alsever solution was added, 20% of the serum specimens lost the non-specific anti-Toxoplasma factor. Also, 60 % of these sera, used in dye tests with known positive sera, produced titers higher by one dilution tube, or 4-fold, than were obtained with a standard human accessory factor serum. The latter observation suggests that many negative control sera should be used in such tests, to be sure that false positive reactions are not enhanced. Strannegard (1967a) showed that the heat-stable anti-Toxoplasma factor

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(THF) in normal animal sera is dependent on activator factors present in normal serum components. He suggested that THF may be a fast-moving immunoglobulin,possibly of theIgAtype. In anotherpaper( 1967b),he reports that when rabbits ingest Toxoplasma antigen, IgA antibodies occur in their serum. Much more must be investigated about the Toxoplasma-hostile factor and its occurrence in other animals, especially ruminants, in which it is so common. This is especially important in determining the specificity of anti-Toxoplasrna antibodies in beef cattle, despite the general failure to detect T. gondii in beef. It is conceivable that, as in rabbits, ruminants can ingest Toxoplasma or its antigens, and respond with IgA antibodies, without experiencing a generalized infection with the live organism. The subject of humoral immunity to Toxoplasma has been extensively explored. I will not cite earlier papers on this subject, but will confine my review to those published in 1966 or thereafter. Gunnel Huldt (1966a) found that high humoral antibody levels in rabbits induced by vaccination with dead T.gondii did not confer any immunity to challenge with T. gondii. Only animals with high serological test titers following previous infection with T. gondii exhibited real protection against a challenge. Foster and McCullough (1968) also failed to find anything but a slight increase in survival time of animals challenged with T. gondii after being passively immunized with sera from convalescent animals. They also could not demonstrate complete protection from challenge in animals which had recovered from an initial infection. Nakayama (1969) found that one-third to one-half of mice repeatedly vaccinated with heat-killed T. gondii, and showing HA antibodies, could survive challenge with highly virulent strains 2-6 weeks after the last immunizing injection. All survivors became infected. On the other hand, if challenge was postponed to 8-14 weeks, very few animals survived, even though they still had high antibody titers. Krahenbuhl et al. (1972), using a low challenge dose of a strain of lower virulence, were able to demonstrate protection of some mice vaccinated with various preparations of formalinized killed T. gondii and also of soluble or “particulate” T. gondii antigens, administered alone or with Freund’s incomplete or complete adjuvant. They also showed some protection of mice by passive transfer of immune sera from chronically infected mice. Undoubtedly their ability to discern protective effects was enhanced by the use of a challenge strain of low virulence, and a relatively small challenge dose. Krahenbuhl et al. found that administration of Freund’s complete adjuvant alone also conferred some protection. A protective effect of Toxoplasma antibodies is demonstrable in the dye test. This has also been shown in tissue culture (Yanagawa and Hirato, 1963). Nevertheless, it is clear that humoral antibodies alone do not play the principal role in immunity in toxoplasmosis. Acquired celIuIar resistance must play a major role. Huldt (1966b)pointed out that no significantdifferences in antibody level existed in two groups of rabbits immunized by vaccination with killed organisms or by past infection, but the former group had no less susceptibility to challenge with the RH strain than non-immunized while the latter had no illness even though they showed some parasitemia. In sub-groups of these

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animals that were treated with cortisone for the entire time of the experiment, the cortisone appeared to enhance the infection in non-immunized animals and in those which had received dead vaccine, but not in those previously immunized by a past infection. She suggests that the enhancement was due to suppression of cellular immunity, through, possibly, the ability of corticosteroids to increase the stability of lysosomes. Using similar groups of rabbits, Huldt (1967) studied the transformation to blastoid cells in lymphocyte cultures derived from their peripheral blood, lymph nodes, and spleen. She observed that, on stimulation with toxoplasmin or with live T. gondii, a significant increase in blastogenesis occurred in all cultures derived from rabbits that exhibited delayed hypersensitivity in the skin test and had positive serology after a previous T.gondiiinfection. Lymphocytes from rabbits that were seropositive but lacked delayed hypersensitivity following vaccination with killed organisms exhibited a significant increase in blastogenesis only in cultures derived from spleen. She did not find, in cultures from seronegative and toxoplasmin-negative rabbits, a significant increase in blastogenesis. She suggested that these findings may indicate an immune protective property of lymphocytes. She also observed that, after transformation, blastoid cells were more easily infected and that the parasites multiplied more rapidly within them. Thus, whatever the function of the blastoid cells in immunity, they also are not involved alone. Frenkel (1967) demonstrated adoptive immunity to Toxoplasma and Besnoitia infections in hamsters by transfer of spleen and lymph node cells derived from immune animals. Antiserum from the immune animals had a slight additive protective effect. Both clinical immunity and transferable cellular immunity appeared in the recipients after three weeks; delayed hypersensitivity and antibody appeared much earlier, by the 5th day of infection. This is the first definite demonstration of cells as the principal agents of specificacquired immunity in these protozoan infections. The specific cell type or types capable of transferring immunity is (are) still to be defined. Tremonti and Walton (1970) used histological methods to study blast transformation in cultures of peripheral lymphocytes derived from patients with serological (IFA) evidence of past infection and from three cases of acute infection confirmed parasitologically. They observed no difference between seronegative controls and individuals with past infections, in that Toxoplasma antigens stimulated blast transformation in both. However, the cultures derived from the acute cases showed no blastogenesis. They also observed, in preparations from guinea pigs which had been experimentally infected with T.gondii and protected from death with sulfadiazine, the occurrence of macrophage inhibition when Toxoplasmaantigens were added. These guinea pigs were reactive to toxoplasmin in the skin test. The patients were not skin-tested. Krahenbuhl et al. (1972) used the incorporation of tritiated thymidine into lymphocytes in culture as the measure of blast transformation. They found that cells from individuals with serological evidence of past toxoplasmosis of at least one year’s duration transformed more frequently on stimulation with Toxoplasma antigen than lymphocytes from negative people. Here again, cells from patients with suspected acute toxoplasmosis had less transformation. No

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skin tests were done, but it is intriguing to think that blastogenesis is related to delayed hypersensitivity; the skin test becomes positive late after the acute infection. In a subsequent paper, Gaines et al. (1972) reported inhibition of macrophage migration with cells derived from the same group of seropositive subjects studied by Krahenbuhl et al., and no inhibition of cells from the seronegative individuals. In other experiments designed to demonstrate the importance of cellular elements in immunity to toxoplasmosis, Nakayama and Aoki (1970a) studied the effect of antilymphocyte sera (ALS). When they treated the animals with ALS and infected them with endozoites of the Beverley strain, they obtained only 9 % survival, as compared to 85 % survival in animals infected but not given ALS. Similar results were obtained with another relatively avirulent strain of T. gondii. Mice which had humoral antibodies against Toxoplasma were given ALS and then challenged with the RH strain. The mortality rate among them was substantially greater than in similar mice not given ALS. They also noted that the number of Toxoplasmain the peritonealexudateof Beverley strain-infected mice was 40-fold higher when the animals were given ALS. Strannegard and Lycke (1972) had similar findings with antithymocyte serum (ATS) which reduced the survival time of mice infected with strains of high virulence. ATS also prolonged the illness and increased deaths among mice infected with avirulent strains. In a series of studies in Remington’s laboratory, non-specific resistance has been shown to develop following infection with a number of intracellular parasites, including Toxoplasma and Besnoitia. Long-term resistance against challenge with ordinarily lethal doses of Listeria monocytogenes, Brucella melitensis, and Salmonella typhimurium, and Mengo virus was conferred by prior Toxoplasma infection (Ruskin and Remington, 1968a, b ; Remington and Merigan, 1969). The converse was also true; Listeria infection conferred resistance against T. gondii. Also, challenge with Listeria of cultures of macrophages derived from mice chronically infected with Toxoplasma or Besnoitia showed the macrophages to be much more resistant to necrotization than macrophages from non-immune mice; and again the converse was true (Ruskin and Remington, 1968b; Ruskin et al., 1969). Krahenbuhl and Remington (1971) were able to confer in vitro non-specific resistance to Listeria when cultured macrophages from guinea pigs with chronic infections of T. gondii were sensitized by blastoid cells stimulated by Toxoplasma antigens or by the supernatants from such lymphocyte cultures. These results and those of Frenkel and Huldt suggest a major role for cellular mechanisms in resistance to intracellular parasites. Ruskin and Remington (1971) have also shown that vaccination of mice with killed antigen, in this case Toxoplasmain Freund’s incomplete antigen, conferred protection against Listeria challenge. They showed, in this work, that peritoneal macrophages from mice given this vaccine were activated and thereby became resistant to necrotization by Listeria. Under some conditions the protection afforded by vaccination appears to be mediated by nonspecific cellular mechanisms, which also seem to develop from persistent infection with an intracellular organism. The activated macrophage appears to be a major cell involved.

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Gentry and Remington (1971) also found increased resistance to Cryptococcus in mice chronically infected with Besnoitia or Toxoplasma, as revealed by fewer deaths and prolonged survival time, and by resistance of macrophages in vitro. Hibbs et al. (1971) noted increased resistance to both spontaneous mammary carcinoma and transplanted leukemia and sarcoma in mice chronically infected with these protozoa. Their results are similar to those obtained with BCG. Activated macrophages from Toxoplasma- or Besnoitia-infected mice appear to be able to recognize and destroy tumor cells but not normal cells (Hibbs et al., 1972a, b). Another type of non-specific resistance induced by T.gondii is the production of interferon. Rytel and Jones (1966) and Freshman et al. (1966) both reported that interferon was demonstrable in serum and peritoneal exudate of mice infected with the RH strain, as evidenced by increased survival and prolongation of life of mice challenged with vesicular stomatitis or Mengo virus. Remington and Merigan (1968) also demonstrated that interferon induced by T.gondii is active in vitro. Remington’s group, however, checked for the presence of interferon in the studies of cross-resistance cited above, and did not find evidence of it. The timing was considerably different. The term “resistance” is apt when applied to these demonstrations of nonspecific protection. The resistance was measured in fewer deaths and prolonged survival time. The type of immunity seen in animals that have survived a previous infection with the same parasite was not observed. Some new information on the cellular basis for specific immunity has recently been forthcoming. Jones and Hirsch (1972) have observed that, in cultured macrophages infected with live T. gondii, lysosomes (phagosomes) do not fuse with the vacuoles containing the parasite. They also noted that there were two populations of T. gondii in such cells, in about equal numbers. Vacuoles containing what were apparently dead or dying parasites did fuse with lysosomal vacuoles. This was somewhat surprising, since viability tests of the T. gondii suspensions used indicated that at least 95% of the organisms were alive at the time of inoculation. These observations suggest some activity on the part of the live parasite in preventing the fusion of the vacuoles, and possibly a lesser capacity for this in some parasites than in others. Alternatively, the macrophages may have some latent capacity to destroy some of the invading parasites. The experiments suggest additional work to see if macrophages from immune animals, activated by transformed lymphocytes, have the ability to overcome the resistance of T. gondii to vacuolar fusion. Remington (personal communication) has very recently been successful in activating macrophages against T. gondii. I do not have the details of his experiments, but they will be published shortly in Infection and Immunity.

VI. BIOLOGY Lycke et al. (1965) described the entry of T. gondii into cells as an active process of penetration, which was enhanced by hyaluronidase or lysozyme. Norrby et al. (1968) demonstrated by histochemical methods that T.gondii contains acid phosphatase-positive granules near the anterior pole, and that

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these granules appear to be diminished after penetration of the cell. Hanson and Sourander (1968) demonstrated lysosomes in T. gondii by electron microscope studies and stated that they have a high activity of both aryl sulfatase and acid phosphatase. They discussed these findings in relation to penetration. Lycke et al. (1968) and Norrby (1970, 1971) described a penetration factor associated with active entry by the parasite. Exposure to host cells stimulated synthesis of this factor. In addition to these observations, Bommer et al. (1968) described microcinematographic studies which showed that T. gondii apparently makes a hole in the host cell membrane, the borders of which are rigid, and the parasite squeezes its way through this hole. On the other hand, Jones et al. (1972), in a carefully conducted electron microscope study, designed to capture the early stages of entrance of T. gondii into cells, found that the process is one of phagocytosis. They identified micropseudopods of the cell around the parasite, which was not oriented so that its anterior pole was usually against the host cell wall. They suggest that whatever penetration factor exists in T. gondii is stimulatory of phagocytosis by cells which ordinarily do not exhibit this activity. The findings cited above are not incompatible with this suggestion. Sheffield and Melton (1968) and Jones et al. (1972) have observed fine microtubules extending from the wall of parasite-containing vacuoles into the cytoplasm of the host cell. This is probably indicative of some process involved in the nutrition of T.gondii. The search for a biochemical deficiency in T. gondii, which makes it dependent on an intracellular situation, has thus far not been revealing. Lund et al. (1966) could not find any lack of glycolytic or oxidative activity. Dumas (1970) also studied respiration of T. gondii and found that normal rabbit and guinea pig sera aided respiration. Sera of immune animals caused only slight inhibition. In addition to energy-producing mechanisms, T. gondii seems to have considerable capacity for synthesis. Remington et al. (1970) found that tritiated uridine is incorporated into the RNA of the parasite even when it is maintained outside of cells, indicating that the free organism can synthesize RNA. Perrotto et al. (1971) demonstrated that T.gondii can also synthesizeDNA independently of the host cell. While pyrimidines and their precursors are incorporated into the DNA, only preformed purines are utilized. This is suggestive of the question: is the inability of T. gondii to use purine precursors important in relation to its obligate intracellular nature ? One final note on the biology of T. gondii: Ben Rachid (1970) reported on infecting Ctenodactylus gundi with oocysts derived from cat feces. The gundis died 6 to 7 days later, with numerous parasites demonstrable. There was a cat in the laboratory of the Pasteur Institute in Tunis when the gundis were brought in from the field and died.

VII. CHEMOTHERAPY This subject will be mentioned only briefly. Ohshima et al. (1967) tested a derivative of diaminodiphenyl sulfone (SDDS) in mice with encouraging results. These authors followed this study in 1971 by a test of prophylactic

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effect of SDDS in swine which were given repeated oral doses of T. gondii cysts. The animals were continuously medicated with the drug at doses of 2-5-10 mg/kg/day starting 7 days before infection. There were no clinical symptoms of parasitemia in the treated pigs, in contrast to controls, and when the pigs were slaughtered 6 weeks after infection no parasites could be found. These results are encouraging because there are few compounds in our armamentarium against Toxoplasma. Spiramycin and acetylspiramycin, which are derivatives of erythromycin, have been tested by a number of workers and have been used in France in the clinic. The results with acetylspiramycin have not been encouraging in experimental trials (Nakayama and Aoki, 1970b; Gill and Prakash, 1970). Nevertheless, I expect that it will receive further evaluation, and it may be found to have usefulness when combined with other drugs (Aoki, 1969). Couvreur (1971) indicated that spiramycin may have had some effect in the prevention of congenital toxoplasmosis in humans, in that there were fewer instances of overt infection and death in the offspring of treated women, and also less subclinical congenital infection. These results will require statistical analysis; they show, at this time, that such treatment is not completely effective. Kraubig (1966) treated women in the 2nd and 3rd trimesters of pregnancy with pyrimethamine and sulfa drugs. He found that the frequency of Toxoplasma infection in the children born of treated women was only one-third of that in the untreated control group. A recent study by McMaster et al. (1973) reports that two chlorinated lincomycin analogues are effective against RH strain T. gondii infection in mice. The drugs are clindamycin and N-dimethyl-4'-pentyI clindamycin. Their activity is similar to that of pyrimethamine and pyrimethamine-sulfadiazine combinations, in that they produce radical cure of the infection when administered on the day of infection or day 1 and continued for 2 weeks. Clindamycin is already commercially available; so it is to be hoped that there will be additional evaluations of its efficacy stimulated by this report. VIII. NEWKNOWLEDGE OF SARCOCYSTIS

Despite the deficiencies in reporting on Toxoplasma, I would be remiss if I did not mention recent findings on Sarcocystis which were stimulated by the elucidation of the life cycle of T. gondii. In a series of 3 papers, Rommel el al. (1972), Heydorn and Rommel(1972), and Rommel and Heydorn (1972) have reported on the results of feeding Sarcocystis tenella of sheep to cats and dogs, S.fusiformis of cattle to cats, dogs, and humans, and S. miescheriana of swine to cats and humans. In these experiments, they were able to demonstrate the production of sporulated sporocysts, sometimes paired and enveloped in a thin oocyst wall and frequently single. The occurrence of oocysts rather than sporocysts in infections with S. fusiformis seemed to depend on the host, being more common in the cat and man than in the dog. S. fusiformis produced the sporocysts or oocysts in all of these hosts. S. tenella produced sporocysts in the cat but not the dog. S. miescheriana produced sporocysts in man but not the cat, The evidence seems to indicate that Sarcocystis from sheep and cattle

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produce forms in the feces of cats that resemble isosporu bigemina var. cuti and that Sarcocystis from cattle and swine produce forms in man that resemble I. hominis. There is relatively little immunity. Dogs and cats can be reinfected, and probably man as well, and shedding of the fecal forms occurs for 30-40 or more days. We are inclined to accept these findings already because of the rigorous proofs which were required for the life history of T. gondii. However, these studies must obviously be followed by attempts to produce the muscle stages of Sarcocystis in animals by the feeding of the sporocysts or oocysts. The host-specificity of the intestinal stages needs careful study. More work along these lines will undoubtedly follow, to reveal the life cycle of similar parasites such as Besnoitiu aud Frenkelia.

IX. CONCLUSION It has obviously been impossible, in the space allotted, to give adequate attention to all of the contributions of investigators studying the various facets of toxoplasmosis. I may have concentrated too much on some areas at the expense of others. It seemed to me best, however, to give as complete a story as possible on the developments concerning the life cycle of the parasite and their implications in regard to epidemiology. In all of the other areas, the story is incomplete, but I have attempted to highlight problems and identify some advances towards their solution. Certainly it can be expected that, in a further review, say 5 years hence, the balance will shift, probably to more understanding of the immunology and the physiology and biochemistry of the parasite. Hopefully, it will be possible, also, to report some successes in the diagnosis of chronic disease and in chemotherapy. REFERENCES

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The Biology of the Acanthocephala

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W L NICHOLAS

Department of Zoology. Australian National University. Canberra. Australia I . Introduction .................................................................................... I1. Morphology. Functional Anatomy and Histology .................................... A . Proboscis ................................................................................. B. Trunk ....................................................................................... C Uterine Bell .............................................................................. D. Acanthor ................................................................................. E. Nuclei and Nucleoli..................................................................... 111. Development ................................................................................. A . In the Intermediate Host ............................................................... B. In the Definitive Host .................................................................. N . Fine Structure ................................................................................. A . Adult Tegument ........................................................................ B. Acanthor Tegument..................................................................... C. Spermiogenesis and Spermatozoon Ultrastructure .............................. D. Egg Envelopes ........................................................................... V . Physiology ....................................................................................... A . Osmotic Regulation ..................................................................... B. Hatching of the Acanthor ............................................................ VI . Biochemistry .................................................................................... A . Composition .............................................................................. B. Assimilation .............................................................................. C. Intermediary Metabolism ............................................................ VIL. Host-Parasite Relationship ............................................................... A . With the Arthropod Intermediate Host .......................................... B. Pathology in the Definitive Host ................................................... C. Medical and Veterinary Importance ................................................ D. Ecological Factors ..................................................................... VIIL. Summary and Conclusions .................................................................. Acknowledgements ........................................................................... References.......................................................................................

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I. INTRODUCTION This review is concerned with work published since September 1966. when the author’s previous review of acanthocephalan biology went to the editor (Nicholas. 1967). Earlier work will be referred to only where necessary to introduce a topic. Journals published after the beginning of 1972 were not received in time to be included.Included within its terms of reference are studies of morphology. functional anatomy. histology. cytology. ultrastructure. 671

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development, biochemistry, host-parasite relationships, epidemiology, and medical and veterinary helminthology. It will not cover taxonomic or zoogeographic studies or records of new hosts, or data on incidence of infection, and infection rates. An exception is made, because of its importance to students of the Acanthocephala, in noting a revision of the superfamily Echinorhynchoidea by Golvan (1969). This publication, it is hoped, will be the first part of a major taxonomic review of the Acanthocephala. Interest in the Acanthocephala has increased and diversified, during the last 5 years. Ultrastructural and biochemical studies have been in the forefront, while studies of development and host-parasite relationships, at the whole animal level, have perhaps become less popular. However, some problems in the functional significance of organs peculiar to the Acanthocephala have been looked at again in the light of modern techniques. A book devoted to the Acanthocephala has been published by Crompton (1970), entitled “An Ecological Approach to Acanthocephalan Physiology”. The book brings together a great deal of information on infection, growth, and reproduction, much of it in tabular or graphical form. The author’s analyses of the physical, chemical and biotic properties of the arthropod haemocoel and the vertebrate intestine, as environments in which parasites develop, will prove particularly interesting to many parasitologists.

FUNCTIONAL ANATOMY A N D HISTOLOGY 11. MORPHOLOGY, A. THE PROBOSCIS

1. Attachment to the host The function of the acanthocephalan proboscis has been re-examined by Hammond in a series of papers on Acunthocephalus runue, a parasite of toads. The proboscis (Fig. l), which is armed with rows of hooks, serves primarily as an attachment organ to the vertebrate intestinal wall, and in some species becomes firmly embedded in a nodule of host origin, though in others, such as A . runue, it is less firmly attached. The effects on the host will be dealt with in a later section (Section VII). Hammond (1966a, b; 1967b) studied the in vitro behaviour of A . runue and the mechanics of proboscis evagination and invagination by isolated worms as well as the means of attachment of the worm to excised portions of host intestine. The acanthocephalan body contains two separate hydraulic systems which can act independently of one another, the first comprising the fluid-filled cavity of the proboscis and proboscis receptacle, and the second the body cavity of the trunk. When unattached, in vitro, A . runue periodically shows spontaneous cycles of proboscis withdrawal and eversion (Fig. 2). In vivo, the same action probably serves two purposes, first to catch the proboscis hooks on the intestinal epithelium, and, second, forcibly to draw the proboscis into the intestinal wall. Contraction of neck retractors will then tighten the grip of the proboscis by forcing fluid out of the spongy lemnisci into the lacuna canal system of the proboscis. Hammond concluded that the lemnisci do not aid in the evagination ofthe proboscis, as has sometimes been suggested. The neck retractor muscles

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-G -RE

I

0.4 mm.

I

FIG.1. Diagram of the anterior part of Acanthocephalus ranae. G , ganglion; H, hook; L, lemniscus;LC, lacunar channel;LS, ligament sac; N, neck; NR, neck retractors;P, partition; PR, proboscisretractor; PW, probosciswall; R, receptacle;RE, retinaculum;RR,receptacle retractor;T, testis; TW,trunk wall. Reproduced from Hammond (1966b). J. exp. Biol. 45, 203-21 3.

might perhaps be better termed lemniscal compressors, though neither term is entirely appropriate. The lemnisci may play a part in the exchange of lipid with the host by way of the proboscis (see Section VI). Independence of movements of trunk. Because the actions of the two hydraulic systems are independent, movements of the trunk within the intestines of its host do not loosen the hold of the proboscis. The longitudinal and circular muscles of the body wall contract as units, acting primarily through changes in pressure, though some local movements are possible. Although the fibrous body wall is extensile, up to 40-50%, in A. ranae, it is much less so than in many other pseudocoelomates, and wrinkles on shortening.

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FIG.2. Operation of the proboscis. (a) Proboscis invaginated, proboscis apparatus fully withdrawn; (b) trunk maximally extended; (c) trunk extended, proboscis evaginated ; (d) proboscis evaginated, neck retractor muscles contracted. L, lemniscus; LS, ligament sac; NR,neckretractors; P, proboscis; PR, proboscis retractor; R, receptacle; RE, retinaculum; RR, receptacle retractor. Reproduced from Hammond (1966b). J. exp. Biol. 45,203-213.

2. Proboscis armature Miller and Dunagan (1971) made the interesting discovery, when studying the proboscis of Macracanthorhynchushirudinaceus with the scanning electron microscope, that a groove runs along the outer curvature ofthe hooks from what appears to be a pore. They speculated that secretions synthesized within the hook may be released from the pore to reach the host tissues through the groove, and that the secretions may be responsible for the marked inflammatory reaction characteristic of this species, in contrast to two other species, Neoechinorhynchus sp. and Echinorhynchus thecarus, in which no evidence could be found of either groove or pore.

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B. TRUNK

Holloway and Nicol (1970) have described the morphology of the trunk of Corynosoma hamanni. I feel that, though useful, work of this kind cannot be effectively summarized. C. THE UTERINE BELL

The function of another unique acanthocephalan organ, the uterine bell, has been investigated by Whitfield. Fertilization and embryonic development are internal in the Acanthocephala, so that the female gonoduct, comprising vagina, uterus and uterine bell, must permit entry of spermatozoa to the body cavity and the exit of embryonated eggs. In Whitfield’s (1970) opinion, Kaiser’s conclusion (in 1893) that the uterine bell organ sorts fully developed acanthors from immature stages for passage to the exterior, was later contested on insufficient grounds. There can now be little doubt from Whitfield’s work that in Polymorphus minutus, and probably in other Acanthocephala, the uterine bell does sort out the mature acanthors from the immature forms. Whitfield (1968) has described the complex histology of the bell apparatus and associated structures, and studied its action in in vitro preparations (Whitfield, 1970). The entry of acanthors from the body cavity, and their passage through the bell apparatus to the uterus, depends on periodic peristaltic waves which pass down the bell to the uterus. Two possible routes may be followed by eggs, one leading back to the body cavity, the other to the uterus. Sorting depends on length and fully developed acanthors are slightly longer than earlier stages, and cannot pass back into the body cavity by way of two slits, as immature acanthors can. Instead, they are pushed into the uterine duct and transported by a peristaltic contraction to the uterus. In P. minutus, the efficiency of sorting is very high, and very few immature acanthors are passed to the uterus. D. THE ACANTHOR

The acanthor (enclosed within a complex shell) is the only free-living stage in the acanthocephalan life-cycle and becomes infective when ingested by the appropriate arthropod intermediate host. Whitfield (1971a) has described the structure, and analysed the locomotion, of the hatched acanthor of Moniliformis dubius. According to Whitfield, Grabda-Kazubska (1964) had previously described the armature of the acanthor of M. hirudinaceus, which closely resembles that of M. dubius. Whitfield used a polarizing microscope to view the internal musculature and cinephotomicrography to analyse movement. 1. Blades and spines The surface of the acanthor is covered with minute backwardly directed spines, set in about 20 anteriorlposterior helical rows of long-pitch. The spines become larger anteriorly, and, together with two pairs of much larger hooked blades, constitute the rostellar apparatus or “aclid organ” (so called to distinguish the acanthor apparatus from the adult proboscis, sometimes referred

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to as t i rostelluni). Each pair of blades is united by a hinge, located at the anterior tip of the acanthor. The body, posterior to the aclid, is encircled by 40-50 skeletal hoops, visible by phase contrast microscopy.

2. Muscles About 10 anterior/posterior muscles lie next to the surface of the body. They arise at the level of the aclid and take a helical course to insert at the posterior extremity of the acanthor. Two oblique aclid retractor muscles arise laterally, about half way along the body, to insert at the anterior tip, beneath the hing of the median blades. Analyses of movement suggest that, in addition, a circular band of muscle may surround the acanthor at the posterior margin of the aclid, but this muscle could not be seen with the polarizing microscope. 3. Locomotion In locomotion, the aclid is periodically retracted, bringing the tips of the blades to the anterior end of the acanthor, then everted so as to sweep the blades forward and outward. Reciprocal contraction and relaxation of the aclid retractors and longitudinal muscles, acting on the body fluid, brings about the movements of the aclid organ as well as locomotion. The surface spines are important in preventing backward slip as longitudinal muscles contract, and skeletal hoops encompassing the hind body ensure that, as internal hydrostatic pressure rises, the aclid everts. The blades sweep out a cavity ahead of the advancing acanthor as it moves forward (normally within the gut or gut wall of its host). The pattern of locomotion appears highly stereotyped. E. NUCLEI AND NUCLEOLI

1. Giant nuclei of tegument Barabashova (1968) has re-examined the remarkable giant tegumentary nuclei, using the Feulgen reaction for DNA, and Brachet’s methyl green pyronin reaction for RNA. In M. hirudinaceus DNA is unevenly dispersed throughout the large dendritic nuclei, which contain a number of pyroninpositive vacuolated nucleoli. Cytoplasmic RNA was evident in the radial fibre layer. In Polymorphus magnus, A . ranae and A . lucii the nuclei fragment amitotocally. In the former two species DNA occurs in irregular aggregates of particulate material and 1-3 nucleoli per nucleus were located peripherally. In A . lucii DNA staining was very faint and nucleoli were not found. Marshall et al. (1973) studied the DNA content of embryonic and larval nuclei of M. dubius by Feulgen microspectrophotometry. They found polar body nuclei were haploid, n, oogonia nuclei equivalent to 4n, while cleavage nuclei varied from 2n to 4n, indicative of nuclei in GI, S and GZphases of the cell cycle. The DNA content of muscle nuclei was close to 4n in the early acanthella, suggesting these nuclei were in G2, but may have risen to 8n in the cystacanth. In the cortical and lemniscal nuclei, DNA increased progressively with development, and values between loon and 350n were measured in the cystacanth. Probably tegumentary nuclei have higher values in the adult.

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2. Numbers of nuclei: symmetry Cable and Dill (1967) have drawn attention to the tendency for acanthocephalan nuclei to occur in threes, or multiples of three, in various organs, for example three in the proboscis wall and three in the lemnisci of Puulisentis fractus, upsetting bilateral symmetry. The tendency is particularly well seen in neoechinorhynchids, such as P. fructus, because of the small total number of cells.

111. DEVELOPMENT A.

IN THE INTERMEDIATE HOST

1, Morphogenesis

The reviewer has found no recent studies of embryonic development, but further studies of larval development have been reported (Table I). Awachie (1966), King and Robinson (1967), Cable and Dill (1967), Uglem and Larson (1969), Robinson and Jones (1971) and Olson and Pratt (1971) all give detailed accounts of organogenesis in the intermediate host. Only minor variations from the now well established pattern of larval development are reported. The mode of formation of proboscis hooks might repay more critical observation. So far as is known, Paulisentisfractus is unusual amongst neoechinorhynchids in utilizing a copepod and not an ostracod as an intermediate host, and in the rapidity with which it develops, becoming infective in 13 days. Cable and Dill (1967) point out that the term cystacanth is a misnomer so far as neoechinorhynchids are concerned, since they do not become enclosed by a membrane in the intermediate host, and they prefer the earlier term, juvenile, for the fully developed acanthocephalan in its intermediate host. King and Robinson (1967), prompted by the increasing use of Moniliformis dubius for experimental work, have redescribed its larval development. They point out that the cystacanth of this species is flattened laterally, one surface being convex and the other flat or slightly concave, and is not flattened dorsoventrally as has sometimes been assumed. The two bands of undifferentiated cells constituting the trunk muscle primordia are therefore lateral. These do not differentiate until the parasite is inside the definitive host. Sensitivity to X-irradiation and to abnormally high temperatures can be correlated with morphogenetic activity (Robinson and Jones, 1971). The acanthors of M . dubius are relativelyinsensitive to X-irradiation when compared with the acanthella stage. X-irradiation of the acanthella produces characteristic malformations of the proboscis and the genitalia, failure to invaginate the proboscis, and deletion of nuclei from the muscle bands. It is probable that tissues involved in morphogenetic movements are as sensitiveto damage as those engaged in mitotic activity. High temperatures (the cockroach is more tolerant than the parasite) produce similar malformations. Butterworth's ( I 969) work on Polymorphus minufirs is centred primarily on ultrastructure and will be referred to in Section IV.

TABLE I Recent studies of post-embryonic development in the intermediate and definitive host Species

Echinorhynchus truttae P Moniliformis dubius (A) Moniliformis dubius (A) Paulisentis fractus (E)

Intermediate host

Gammarus pulex G. lacustris (Amphipoda) Freshwater Periplaneta americana Panesthia sp. (Dictyoptera) Terrestrial Periplaneta americana Panesthia sp. (Dictyoptera) Terrestrial Tropocylopsprasinus (Copepoda) Freshwater Gammarus pulex

Definitive host

Author Awachie, 1966

Salmo trutta S. gairdneri (Pisces) Laboratory rat

King and Robinson, 1967

Laboratory rat

Robinson and Jones, 1971

Semotilus atromaculatus (Pisces) (Waterfow1)a

Cable and Dill, 1967

Polymorphus minutus (P) Polymorphus minutus ( P ) Neoechinorhynchus saginatus ( E )

(Gammarus pulex)a

Domestic Duck

Cypridopsis vidua (Ostracoda) Freshwater

Semotilus atromaculatus

Echinorhynchus lageniformis (P)

Corophium spinicorne (Amphipoda) Marine

(Platichthys ste1latus)a (Pisces)

Butterworth, 1969 Crompton and Whitfield, 1968a Uglem and Larsen, 1969

(Pisces)

~~

(P) Palaeacanthocephala. (A) Archiacanthocephala. (E) Eoacanthocephala. a Development in this host not studied in paper cited.

Olson and Pratt, 1971

r

?cn

T H E BIOLOGY OF THE ACANTHOCEPHALA

679

2. Hatching and infection of the intermediate host Lackie (1972) has studied the statistics of acanthor hatching and cockroach infection with M. dubius. Acanthors from the pseudocoel of the female worm were experimentally hatched in 0.3 M NaHC03 (Edmonds, 1966). Storage for 1-2 days, or exposure to 60 sucrose (possibly simulating natural dehydration in the faeces of rats), slightly increased the proportion of acanthors hatching in vitro. Acanthors were successfully stored for 3 months at 4°C in 60 % sucrose. Lackie also studied the factors that may contribute to the highly variable infection rate in the cockroach. OnIy 20-30 % of the eggs capable of hatching in vitro gave rise to cystacanths when fed to cockroaches. The wastage occurred very early in the infection process, and the success rate was not improved by injecting artificially hatched acanthors into the haemocoel. The proportion of acanthors completing development fell markedly, and variability in recovery increased, as the infective dose was increased. Adult male cockroaches gave slightly better recoveries than adult females, a statistically significant difference which could not be demonstrated when hatched acanthors were injected directly into the haemocoel. The growth of the larval stages in terms of increases in weight and N/weight ratio were studied. No evidence couId be found of significant reduction in amino acids in the haemocoel as a result of infection,

B. IN THE DEFINITIVE HOST

Crompton and Whitfield (1968a) have examined the statistics of attachment, development, and egg production by P. minutus in its definitivehost, the domestic duck. Crompton and Walters (1972) confirm that females of M . dubius survive longer than males in the rat and found that worms of both sexes persist longer in male rats than in females. A biochemical evaluation of the develop ment of M . dubius in the rat (Crompton, 1972) will be discussed in Section VI. P. minutus shows some posterior movement along the host’s intestines during the course of infection, while Echinorhynchus truttae shows much greater movement in the trout, from the upper intestine to the rectum (Awachie, 1966).M. dubius, in contrast, moves its point of attachment forward as it grows in the intestines, though the mid point of the body retains the same relative position in the gut. Crompton and Whitfield (1968b) suggested that the growth of M. dubius in the rat is sufficient to make the parasite relatively long in relation to the host’s gut, and forward movement keeps the body surface in the optimum region for assimilation. A similar situation applies to the cestode Hymenolepis diminuta in the rat where, as with M. dubius, the body surface is the assimilatory organ. Crompton (1970) has brought together and collated in his monograph on the Acanthocephala much information on the statistics of growth, development, reproduction, and egg viability, both from his own work and from the literature, some of it in the form of tables and graphs which cannot usefully be summarized further here.

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Iv. A.

FINE. STKUCTURE ADULT TEGUMENT

The acanthocephalan body wall or tegument is an interesting structure which, in the absence of an alimentary canal and, in most species, specific excretory organs, must serve assimilatory and osmoregulatory functions as well as structural and protective roles. It has attracted the attention of a number of electron microscopists since the first detailed descriptions were given by Crompton and Lee (1965) of Polymorphus minutus and by Nicholas and Mercer (1965) of Moniliformis dubius. The fine structure has been described for Pumphor~~ynch~s laevis by Stranach et al. (I 966), Acantjacephalus ranae by Hammond (1967a) and ~ c h i n o ~ gadi ~ ~ by ~ Lange c j ~ ~(1970). Butterworth (1969) has described the development of the tegument in the larval stages of P. minutus. Later work has added substantially to our knowledge, but has revealed a basically similar structure in all the species studied. Terminology has created difficulty because the organ is complex and names were originally given to the different regions seen by light microscopy, which only partially resolves their structures. Though basically similar in all the species studied, the degree of development of the different regions varies and, therefore, so does their resolution by light microscopy. 1. Epicuticle

The surface of the tegument is formed by an extracellular fibrous layer. Since it lies exterior to the outer plasma membrane, Nicholas and Mercer (1965) called this a cuticle, a term previously applied by light microscopists to an underlying layer which the electron microscope shows to lie within the plasma membrane. Others have termed it an epicuticle (Crompton and Lee, 1965; Wright and Lumsden, 1968), a term which may prove to be the most generally acceptable. Wright and Lumsden (1968) described the fine structure of the epicuticle in M . dubius and investigated its composition by light and electron microscopy. It is composed of a felt-work of fine fibres, 2.5 nm in diameter, extending out from the plasma membrane for about 0.5 pm in the adult worm. It is fully developed in the cystacanth, forming a somewhat thicker zone. Histochemical studies suggest the presence of weakly acidic mucopolysaccharides (by Alcian blue, colloidal iron and thorotrast binding at controlled pH) and neutral polysaccharides or glycoproteins (PAS and PA silver methenamine tests). On this basis the epicuticle resembles the glycocalyx of other cells, and, in particular, the surface coat of intestinal villi. The host's intestine may contribute to it as Lee (1966) suggested, but it cannot originally be formed in this way because it is evident in the cystacanth, when taken directly from the arthropod haemocoel. Wright and Lumsden (1968) speculated on a number of possible functions for the epicuticle, based on a stable polyanionic layer. Their suggestions are the inactivation of the host's digestive enzymes by charge effects, concentration of nutrients facilitating assimilation by pinocytosis, ion accumulation, and the facilitation of ion transport through the tegument.

THE BIOLOGY OF THE ACANTHOCEPHALA

68 1

2. Pore canals Large numbers of closely packed pores cover the surface of the tegument (20 nm in diameter in M. dubius). The outer plasma membrane of the syncytial tegument is tucked in at these pores to line closely packed, more or less parallel, structures which have been variously termed pore canals, radial canals, channels, and caveolae. Evidently, this great increase in surface area must facilitate assimilation by the tegument, but whether the apparent vesicular structures lying close to the base of the canals can correctly be interpreted as evidence of pinocytosis (Nicholas and Mercer, 1965; Lange, 1970) is open to question. Stranach ef al. (1966) suggested that in P. laevis the canals may connect with the smooth endoplasmic reticulum. Wright and Lumsden (1969) found no evidence of direct continuity with deeper organelles in M . dubius but concluded, from studies of the penetration of colloidal lanthanum nitrate, that the lumen of the canal was in contact with the external medium. The pore canals, which branch and anastomose, are about 4 pm deep in M . dubius. Butterworth (1969) suggested, from studies of the development of the cystacanth of P. minutus, that pore canals may increase in number by sub-division during growth. Rothman(I967) reported phosphatases in the pore canals of M . dubius.

3 . Plasma and sub-plasma membrane The plasma membrane is thinner where it lines a canal (9 nm) than between the pores (12 nm), which in Wright and Lumsden’s view indicates a relatively permanent structure and not pinocytosis. Lipids were found in the lumen of the canals in A . ranae (see Hammond, 1967a). A narrow electron dense layer lies beneath the plasma membrane but is not drawn into the pore canals; it was called the sub-plasma membrane by Nicholas and Mercer (1965) and it has been likened by Wright and Lumsden (1969), who disliked the term “membrane”, to the terminal web of other types of cells, where a supportive role has been suggested.

4.“Cuticle” andJibres Beneath these surface structures lies an electron dense region, honeycombed by the pore canals, referred to as the “cuticle” by earlier microscopists. It merges into the “striped” layer as it becomes attenuated to radial supporting bars between the deeper regions of the pore canals. A complex of electron dense fibres permeates the bulk of the tegument, forming a felt-work beneath the radial bars of the striped layer, with which it connects, but taking a predominantly radial orientation in the deeper and thickest region of the tegument. The details of this system of supporting fibres vary in different species.

5 . Organelles The tegument contains an extensive system of smooth endoplasmic reticulum (ER), Golgi clusters, mitochondria, lipid and glycogen deposits, vesicles of various kinds, and many free ribosomes. In some species a definite band of vesicles lies between the base of the pore canals and the felt layer. Stranach et al. (1966) found a close association between the fibres and both the smooth ER

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and small dense granules, suggesting the involvement of the ER in their synthesis. Butterworth (I969), believed the fragmentation of dendritic cortical nuclei in P . minutus was associated with fibre formation. Lange (1970) reported crystalline inclusions in E. gadi. Stranach et al. (1966) also described the tegumentary nuclei of P. Zaevis. They observed finely granular nucleoplasm and electron dense aggregates, identified as nucleoli, as well as scattered dense granules. They noted the unusually narrow gap (15nm) separating the two nuclear membranes and a paucity of nuclear pores. The lacunar canal system, which has been such a marked feature of light microscope studies of the tegument, has been poorly resolved by electron microscopy. Stranach et al. (1966) and Hammond (1967a) observed large membrane-bounded lacunar spaces. Stranach et al. observed a close association with elements of the smooth ER and Hammond the presence of droplets, probably lipid, within them. Butterworth (1969), however, describing the development of the cortex of the larval stages of P. minutus, suggested that the lacunar canals are spaces between the tegumentary fibres, normally packed with lipid, which is readily lost during preparation of tissues for microscopy. The innermost region of the tegument contains many small vacuoles and is demarcated from the underlying connective tissues by a deeply infolded plasma membrane. A basement lamina underlies the plasma membrane. The infolded plasma membrane recalls the structures of the cells lining the proximal convoluted tubule of the mammalian nephron (Stranach et al., 1966) and probably indicates a region of active ion transport. 6. Presoma tegument

Hammond (1967a) has compared the structure of the tegument of the presoma (the proboscis, neck and lemnisci) with that of the metasoma, or trunk, in A . ranae. Though generally similar to that of the metasoma, the tegument of the presoma is much thinner. The pore canals are shorter and less numerous and the inter-canal electron dense matrix or “cuticle” constitutes a shallower superficial zone, giving place first to lamellae and then to a layer of fibres running parallel to the surface. There is a single open lacework of fibres occupying most of the tegument, in place of distinctive felt and radial layers. Hammond suggested that these differences render the presoma more flexible than the metasome. The surface of the tegument is coated with a thick layer of dense material, believed to be lipid, which obscures the epicuticle and extends down into the pore canals. 7. Lemnisci The lemnisci in A . ranae resemble the inner region of the presoma (see Hammond, 1967a). They are ensheathed by the neck retractor muscles, from which they are separated by connective tissue, and a basement lamina. Wright (1970) has drawn attention to their similarity to the deeper layers of the tegument in M. dubius. The pseudocoelomic surface showed a 10nm plasma membrane, half-desmosome-like structures, extensive infolding of the membrane,

THEBIOLOGYOFTHEACANTHOCEPHALA

683

glycogen, lipoidal material and cytoplasmic inclusions. Lange (1970) also described the lemnisci of E. gadi. B. ACANTHOR TEGUMENT

Wright and Lumsden (1970) have described the tegument of the acanthor of M. dubius immediately after being “hatched” in vitro. No fibrous epicuticle was found, but a thin layer of amorphous material coated the outer plasma membrane (like that found beneath the epicuticle and plasma membrane of the cystacanth), which bound acidic preparations of thorotrast or colloidal iron. Two felt layers lie beneath the plasma membrane (6nm), one adjacent to it (20nm thick), with an inner dense margin, the second (about 1.1 pm thick), lying against the hypodermal muscles. The two layers fuse at the base of the cuticular spines. Pores open from the surface of the tegument to an extensive labyrinthine intrahypodermal cavity. The walls give rise to microvilli and the lumen, as shown by means of colloidal La(N03)2, opens to the exterior. Hypodermal muscles, with thin and thick filaments and dense bodies are illustrated. C. SPERMIOGENESIS AND SPERMATOZOON ULTRASTRUCTURE

The structure and development of the spermatozoa of P. minutus has been described by Whitfield (1971b) (Fig. 3). Clusters of spermatidsdevelop synchronously as part of a syncytial morula. The early spermatid shows typical cellular organization at the ultrastructural level. An active golgi body of unusual form develops and numerous membrane bound dense bodies appear in the cytoplasm. A simple centriole is either transformed into or replaced by one of unusual structure, initiating the development of the flagellum within a cytoplasmic cleft, which indents the spermatid nucleus. A great elongation of the flagellum, the cytoplasm and nucleus follows, so that the flagellum comes to lie within a shallow cellular groove, from which it projects terminally. The fully developed spermatozoon is about 60 pm in length, 0.3-0.5pm in diameter, and capable of sinuous movement. The flagellum, inserted close to one end, and adjacent to the unusual centriole, shows a typical 9+2 pattern of tubules, in which sub-fibres A and B and their associated arms are recognizable. Glycogen deposits, and two lateral rows of dense bodies (about 150 in each) are located in the cytoplasm, which lacks mitochondria, although these were present in the spermatid. The nucleolus and nuclear envelope of the spermatid breaks down, though a pentalaminate membrane, derived from condensation of the nuclear envelope adjacent to the flagella cleft, persists, parallel to the flagellar groove. An arc-shaped reticulum of condensed chromatin forms along the length of the cell between the evenly spaced dense bodies and along the abflagellar surface. D. EGG ENVELOPES

Theacanthor, when passed bythefemaleworm,isenclosedby four membranes or envelopes. Their fine structure and histochemistry have been described in

684

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THE BIOLOGY OF T H E A C A N T H O C E P H A L A

685

M. dubius (Table 11) by Wright (1971). Stranach (1972) has described their development in P.luevis. Stranach recognized only three major layers, although these became complex and sub-divided into sub-layers as development proceeded within the female worm. His thin outer layer (la) and thicker layer (1 b) probably correspond to the outer and fibrillar envelopes, respectively, of other workers. These envelopes are the first to form, following fertilization, probably from globules present at the periphery of the ovum, corresponding to the true fertilization membrane. The remaining layers develop later, and thick fibres may become evident in 1b. V. PHYSIOLOGY A. OSMOTIC REGULATION

Adult Acanthocephala show little evidence of osmotic regulation in vitro, swelling or shrinking according to the tonicity of the medium. A flaccid condition is probably the normal state in the host's intestine. Some Acanthocephala possess a protonephrial excretory organ, others do not. The primordium of an excretory organ has been found in the larva of M . dubius, but this fails to develop in the adult. Branch (1970b) has studied in vitro the osmotic behaviour in A4. dubius. The adult worm swells and soon dies in distilled water, but shrinks in hyperosmotic sucrose, like an imperfect osmometer. At 37"C, the adults show considerable swelling in hypertonic solutions of NaCl or KCI, but not at 4"C, suggesting the active uptake of Na+ and K+ against a concentration gradient. Similar swelling occurs in more dilute solutions of these salts, also suggesting that uptake is not purely dependent on diffusion. Whether swelling occurs depends on the accompanying anion, suggesting the presence of pores in the plasma membrane of the same order of magnitude as the hydrated anion. The worm behaves as though its body surface were permeable to C1-, HC03and C(h2-, and marginally permeable to Nos-, but not to sod2-.It is apparently impermeable to Ca2f and Mgz+, Lif can replace Na+ or K+, i.e. the worm swells in hypertonic LiCl; while in the case of NHdCI, the result depends on the sex of the worm, some uptake occurring in male worms. Hanks BSS provides a good isotonic maintenance medium, with little change in the worm's FIG.3. Spermatozoa and spermatid of Poiymorphrrs minutus.A. Diagram of the ultrastructural organization of the late preflagellar spermatid of P. minutus. c., Cytophoral stalk; h., homogeneous nucleoplasm; d., dense inclusion; g., Golgi body; m., mitochondrion; n., nuclear envelope with pores; p., polysomal ribosomes; f., flagellar centriole. B. Simplified cut-away stereogram of the mature spermatozoon of P. minutus. The drawing is based on thin-section electron micrographs and omits large portions of the cell in the cut regions. a., Anterior portion of the spermatozoan body with no dense inclusions; d., dense inclusion; f., flagellum; fc, flagellar centriole; s., spermatozoan body. C. A diagram to show the general ultrastructural features of an acanthocephalan spermatozoon. The diagram is based n the spermatozoon of Polyniorphus minut/rs and represents a transverse section through the gamete. c., Chromatin of nuclear origin; d., membrane-bound dense inclusion; f., 9 2 flagellum; g., glycogen granules; p., pentalaminate remnant of nuclear envelope. A and B reproduced from Whitfield (1971b). Parasitology 62,415-430. C reproduced from Whitfield (1971~).Parasitology 63,49-58.

+

TABLE 11 The egg envelopes of Moniliformis dubius, adapted from Wright (1971)

Thickness (pm)

Designation Outer Shell I Shell TI

Live -

2.0 7.5

Shell 111 Third

0.5

Inner Acanthor surface

1.0

2.0

-

Histochemistry (+ = positive test, - =negative)

Thick epon sections

Protein

0.05 1.9

+ +

4.5

0-5 2.0 1.0 -

-

+ + + +

bonds

Carbohydrate, diastase stable

+

-

-s-s-

+

-

-

+ -

+

+

-

+

+

*Weak reaction, probably from acid mucopolysaccharide.

Chitin

-a

Acid mucopolysaccharide -

-

-

Granular and fibrillar components Sheet-like filaments, lucent Granular Striated 25 nm periodicity Layered

+

-

-

-

-

+ -

Electronmicroscopy

-

+

E r 2:

w

c.

z

0

p

>

m

687

THEBIOLOGYOFTHEACANTHOCEPHALA

water balance, despite a lower osmotic pressure than that of solutions referred to above. Comparisons between the freezing point of the worm's tissue fluids and rat intestinal juice (Table 111) (Branch, 1970a) have shown the worm to be hyperosmotic to intestinal juice, and measurements of ionic composition (Branch, 1970a) have shown that the worm maintains a higher K+ and lower Na+ concentration than in the host's intestine (Table IV, Section VI). The magnitude of these differences varies with the sex of the worm. Branch (1970a) found, by using 24Na and 42K, that worms incubated in Hanks BSS at 37", under an atmosphere of 8 % C02 and 92 % Nz, rapidly exchanged Na and K with the external medium. It appears that, when transferred from the gut to Hanks BSS, a new equilibrium is reached, in which Naf loss and K+ accumulation take place against a concentration gradient. TABLE 111 The mean freezing point depression estimates (+ 1 standard deviation) of the concentration (mM NaCl) of Moniliformis dubius componentfluidsand the rat intestinalfluids together with the real osmotic pressures these solutions would exert (mm Hg)

estimates

Mean equivaIent concn (m)

Mean osmotic pressure (mm Hg)

4 4 22 24 16 4

168+ 14 245 35 261 k 34 364k41 312k69 563+ 3

6020 8690 9250 12820 11020 19590

No. of

Component Rat s e m Rat intestinal fluids Female P/coela Female B.W.b Male P/coeP Male B.W.b ~~

~

P/coel = pseudocoel. b B.W. = body wall. Reproduced from Branch (1970). ExpI Parasit. 2 7 , 3 3 4 3 .

a

Histochemical studies (Branch, 1970a) show accumulations of Kf beneath the pore canal zone, and of Na adjacent to the basal plasma membrane of the tegument. Chloride was concentrated at both zones. The fine structure of both regions is compatible with sites of active ion transport, with the basal region of the tegument resembling the basal region of the kidney proximal convoluted tubule, a region of active Na+ transport. Crompton and Edmonds (1969) studied the osmotic environment of P. minutus in the duck, using canulae to remove samples of intestinal fluid from living unanaesthetized ducks, as well as taking samples from freshly killed ducks. The osmotic pressure in the region occupied by the worm, expressed as the concentration of a cryoscopically equivalent solution of NaCI, was about 1 7 4 m ~NaCl, increasing slightly from the anterior to posterior points of attachment of the worms, and fluctuating appreciably about the mean value throughout the day. This is close to the osmotic pressure of pseudocoel liquid taken from living worms, immediately after removal from the duck, and to that

688

W. L . NICHOLAS

o f wornis which had bccn experitnentally suspended by canulae in the host’s intestinal lumen for short periods of time. B.

HATCHING OF THE ACANTHOR

The conditions under which the acanthors of different species of Acanthocephalae hatch are probably rather different (Crompton, 1970). The stimuli which have been shown to induce hatching in M . dubius (Edmonds, 1966) reflect the condition met in the mid-gut of its host, the cockroach. Even here, acanthors hatch within 2-6 h in vitro, whereas they may take 2 4 4 8 h to hatch in the cockroach. Edmonds (1966) found optimal hatching in vitro at 2-3.5 M, a pH above 7, the presence of HC03- ions, and a temperature above 15°C. A pH of 8 gave a better percentage hatch than 7, but hatching could be stimulated at a pH as low as 6 by bubbling COa through the suspension. Reducing substance produced no discernible effect. In practice a solution of 0.25 M NaHC03 gives good results. The acanthor, when hatched in vitro, liberates a chitinase which may facilitate active penetration of the acanthor shell by the acanthor (Whitfield, 1971a; Wright, 1971), perhaps as well as of the host’s peritrophic membrane.

VI. BIOCHEMISTRY A.

COMPOSITION

1. Inorganic Measurements have been made of major cations in various tissues ofMoniliformis dubius and Macracanthorhynchus hirudinaceus by Branch (197Oa), by atomic absorption spectrophotometry (Table IV). Calculations (assuming that each cation is associated with the chloride ion, and that ions are free in solution) show the worms to be appreciably hyperosmotic to the rat’s intestinal juice. As already noted (Section V, Table HI), Branch came to the same conclusion from freezing point measurements. 2. Proteins and nucleic acids Crompton (1972) doubts the value of wet weight as a basis for experimental comparisons between worms. He studied the changes in protein N, RNA and DNA associated with the growth of M . dubius in the male rat. The starting point for chemical determinations was a pellet obtained by centrifugation of worm tissue minced in 0.2 N cold perchloric acid. DNA and RNA were estimated spectrophotometricaIly, using the diphenylamine and orcinol reagents, respectively, in a hot perchloric acid extract of the residual pellet. Nitrogen was determined spectrophotometrically from an alkaline hydrolysate of the residual pellet. Nitrogen, DNA and RNA increase at similar rates in both sexes up to the third week of infection (the time of copulation), and diverge thereafter. At 6 weeks, the mean N per female is 2.8 mg and eventually reaches a mean weight of 7 mg, while the male contains 0.6 mg at 6 weeks and rises to 1.2 mg. In the female, from 6 weeks on, the eggs account for about 28 ”/, of N, 80 % of DNA

THE DIOLOGY OF T H E A C A N T H O C E P H A L A

689

and 38 7; of KNA in the whole worm, but considerable individual variation occurs. Cronipton’s (1972) paper tabulates the means of N, RNA and DNA per worm, and per mg N, for both sexes on a weekly basis (for up to 18 weeks in females), and their partition between eggs and body for gravid females. 3 . Collagen Collagen forms a major constituent of acanthocephalan connective tissues. Collagens from different animals and tissues differ in two characteristic transition temperatures, (a) T,, the bulk melting temperature at which they shrink in dilute neutral saline, and (b) Tt, the temperature of shrinkage in 0.1 N HCl, equal to TO, the molecular melting temperature (Rigby, 1968a, b). It has been suggested that these two transition temperatures are determined by the value ofproline plus hydroxyprolineper 1000amino acid residues. However, Rigby did not find a good correlation between the content of these amino acids and the transition temperatures for M . hirudinaceus, Ascaris and several nonparasitic earthworms, though there was a correlation with their maximum environmental temperatures. M . hirudinaceus collagen gave a high angle X-ray diffraction repeat of 60-70 nm and the following values:

Ts= 62°C (in neutral 0.9 % saline) Tt (=TO)=42°C (in 0.1 N HCl) Proline + hydroxyproline/ 1000 amino acid residues = 167. 4. Sterols Sterols are believed to be essential dietary nutrients for Arthropods, Platyhelminthes, Nematodes, Molluscs, Echinoderms and terrestrial Annelids. It seems, from studies of sterol metabolism in M. dubius and M . hirudinaceus by Barrett et al. (1970), that the Acanthocephala can probably be added to the list. Studies of their non-saponifiable lipids, by thin layer Chromatography and gas-liquid chromatography, identified cholesterol as the major sterol present, but a number of phytosterols, reflecting the diets of their hosts, were also present. No evidence was found after incubation of M . dubius in l4Cacetate, or the injection of 14C-mevalonate into M . hirudinaceus, that either could synthesize sterols. Nor did injection of M . hirudinaceus with labelled /3-sitosterol show any capacity to derive cholesterol from this plant sterol by dealkylation of the side chain, such as occurs in some phytophagous animals. It would appear that cholesterol must either be derived from the host’s secretions or from its diet. P . minutus, in common with some other Acanthocephala from crustacean hosts, is a bright orange colour. Barrett and Butterworth (1968) found that esterified astaxanthin, concentrated in the radial layer of the tegument and the lemnisci, was selectively absorbed from its host, which contains a number of other carotenoids in addition to astaxanthin. Adults of P. minutus, which are normally tinged orange when recovered from ducks, also contain esterified astaxanthin, but become very pale in ducks fed on diets deficient in carotenoids, though this does not affect egg-production by the worm. Probably astaxanthin comes from p-carotene in the diet of the shrimp.

TABLE IV l?te mean Nu, K, Ca, and Mg content (f1 standard deviation) of Moniliformis dubius components and of the rat intestinal fluids( g element per g water x 10-3) Component Female P/coeP (ant.) Female P/coela (post.) Female B.W.b (ant.) Female B.W.b (post.) Whole female (ant.) Whole female (post.) Whole male Rat intestinal fluids

No. of estimates

Na

K

1-76f 0.37 1-64k 0.50 0.65 f0 -12 0.63 0.18 0-88 & 0.16 0.81 f0.13 1 -48f0.37 2.56 f0.29

1-43fO.46 1*98f0.54 6 * 18k 0-96 6.23 f0.69 3.86f0.46 4.43 + 0 - 8 5 8 -87If:2.89 1 *08f0.27

+

P/coel= pseudoccel. B.W.= body wall. Reproduced from Branch (1970). ExpZ Parusit. 2 7 , 3 3 4 3 .

a

b

No. of estimates 5 5 5

5 6 5

Ca 0.62 k 0-38 0.76 k 0 -32 0.42 f0.58 0-35+0-21 0.38+0*17 0.67 f0.43

Mg 0.53f0-18 0*59k0*14 0.53+0*08 0*53+0.09 0.39fO-17 0*38+0-04

$

2 c)

Z

$

THE BIOLOGY OF THE ACANTHOCEPHALA

69 1

According to Crompton (1970), Butterworth found that cystacanths of P. rninutus contained 28 % lipid, on a dry weight basis, 95 % of which was neutral lipid, mostly located in the radial layer of the tegument. B. ASSIMILATION

1. Lipids

The presoma, buried in the host’s tissues, is in a different environment from the metasoma, which is exposed to the intestinal juices, and the two regions may differ in their assimilatory powers. Hammond (1968), following up earlier observations of Pflugfelder (1949), investigated lipid assimilation by A . ranae. He observed the accumulation of fat-soluble dyes, when these were fed to infected toads in olive oil, and their loss when the parasite was subsequently transferred to a new host. Hammond (1968) concluded that the dyes were probably exclusively adsorbed through the metasoma, and only later became concentrated in droplets within the lemnisci and presomal wall. It seemed possible that the dyes were eventually excreted through the surface of the presoma. Hammond (1968) studied the assimilation of 3H-glyceryl trioleate by A . ranae, in vitro, using light and electron microscope autoradiography. The results indicated that this fat was exclusively assimilated by the metasoma, possibly by way of the pore canals, occurring in droplets in the felt and radial fibre layers of the tegument, some being metabolized in the latter region.

2. Amino acids, nucleotides, sugars The small size ofPauZisentisfractus, a fish parasite, favours autoradiographic studies of assimilation. It reaches 8 mm in length when fully grown. Hibbard and Cable (1968) studied the uptake of 3H-glucose, 3H-tyrosineYand 3Hthymidine, by parasites in short-term incubations in saline under aerobic conditions. Each of the tritiated substrates was rapidly absorbed by the metasoma though not by the presoma. Glucose was rapidly converted to glycogen (identified histochemically), concentrations becoming evident within 1 h in deeper tissues, e.g. the musculature. Tyrosine was incorporated into protein (i.e., incorporation inhibited by cycloheximide), particularly in the male bursa. Thymidine was incorporated in DNA (from sensitivity to enzyme digestion), located in the peri-lacunar regions in the tegument. Only in ovarian masses and the testes did nuclei become labelled by 3H-thymidineYwhich is consistent with the restriction of nuclear division in the adult. The authors suggest that tegumental concentrations may represent mitochondria1 DNA, the growth of which may have been stimulated by aerobic conditions. Tritium, from labelled glucose, rapidly appeared in excreted lipids. The medium, following 3H-glucose assimilation, was extracted with isooctane, and tritium estimated with a scintillation counter. Studies of the kinetics of uptake of 14C-labelled nutrients by helminths in vitro have been used as evidence for specific active transport mechanisms at the ~ was unable to confirm the surface of the body. Branch ( 1 9 7 0 ~ )however, earlier conclusion of Rothman and Fisher (1 964) that the assimilation of neutral amino acids by M. dubius followed Michaelis-Mentenfirst-order kinetics.

692

W. L. NICHOLAS

Crompton and Lockwood (1968) studied ~-U-14C-glucoseabsorption in vifroby P . minutus. Their results were consistent with the operation of a carrier system. The carrier was half-saturated by an external glucose concentration of 0.25 mg ml-1, and approached saturation at 2-0 mg ml-1. They estimated that P . minutus requires 4 pg glucose per mg wet weight of worm per hour in vivo, with external glucose concentrations of not less than 2-0 mg ml-1. After 4 h incubation, radiocarbon was found in glycogen, glucose, sugar phosphates, maltotriose, UDPG, (uridine diphosphate glucose), amino acids, amino sugars and organic acids. Surprisingly, trehalose was not found in P . minutus, and after confirming previous reportsof its presence in M . dubius,the authors speculated that its absence from P. minutus may be linked phylogenetically to glucose as the main blood sugar of its host, whereas trehalose fills that role in the host of M . dubius. Butterworth (cited by Crompton, 1970) measured the rate of absorption of radioglucose by different development stages of P . minutus, and gaseous exchange in vitro. Radioglucose was most rapidly assimilated by the early acanthella, but decreased sharply later in development. Beitinger and Hammen (1971) found glucose was readily assimilated by a fish parasite, Echinorhynclzus gadi, in vitro. Crompton (1970) discussed at length in his monograph the physiological environment in which acanthocephalan assimilation occurs drawing from his own studies on the physiological and chemical environment in the intestine of birds. It is appropriate to introduce at this point the work of Dunagan and Yau (1968) on the oligosaccharidases of Macracanthorhynclzus hirudinaceus because their presence may be related to the capacity of the parasite to utilize different dietary carbohydrates (though their findings are based on the use of cell-free homogenates). Yeast cultures, capable of utilizing specific hexoses were combined with a manometric determination of COZto show the presence of hydrolytic enzymes in the cell-free homogenate. More conventional spectrophotometric enzyme assays were also used. Dunagan and Yau found evidence of oligosaccharidases acting on maltose, dextrin, trehalose, turanose, and possibly on melezitose and lactose in that order of activity. The hydrolysis of indin, sucrose, raffinose, melibiose, a-D-methylglucopyranoside and a - ~ methyl mannoside were not detected. A single isozyme of maltose was found by starch gel electrophoresis. Data on the optimum conditions for maltose and trehalose are reported. McAlister and Fisher (1972) suggest trehalose synthesis from glucose in the body wall of M . dubius may accelerate the inward diffusion of glucose from the host’s intestines, until synthesis is subject to endproduct inhibition (see next section). C.

INTERMEDIARY METABOLISM

1. Utilization of lipid and carboliydrate reserves

Korting and Fairbairn (1972) found no evidence for the depletion of the substantial lipid content of adult M . dubius of either sex when incubated anaerobically (95 Na; 5 COZ)in saline. However, carbohydrate reserves

THE B I O L O G Y O F T H E A C A N T H O C E P H A L A

693

were used under these conditions, but t o a greater extent by female than by male worms. Assays for enzymes involved in lipid metabolism showed that one enzyme from the Boxidation pathway, enoyl-CoA hydratase, was absent from the adult worm, and that a different enzyme, ,!?-hydroxyacyl-CoAdehydrogenase, was lacking in the cystacanth. Horvath (1971) has shown that glycogen metabolism is stimulated in the cystacanth of M . dubius when activated physiologically by bile salts, following the technique of Graff and Kitzman (1965). The inactivated cystacanth did not use endogenous glycogen in vitro but, following activation, consumed about 35 % in 4 h incubation. Pulse labelling with 14C-glucose showed that the rate of glucose incorporation into glycogen rose as glycogen levelsfell. Glycogen was depleted more rapidly in 95 % N2 + 5 % C02 than in air, air + 5 % C02, or nitrogen alone. Horvath suggests that the anaerobic formation of succinate requires C02 fixation in a 1 : 1 ratio. 2. Glycolysis The Embden-Meyerhof sequence of glycolytic enzymes are active in Acanthocephala, as in other intestinal helminths, Dunagan and Scheifinger (1966b) measured the specific activity of several enzymes from the sequence in M. lzirudinaceus. They studied the hexokinase and found high glucokinase and fructokinase activity in homogenates. Glucosamine kinase and galactokinase activity were low, glucosamine phosphorylation probably being dependent on glucokinase, while mannose was not phosphorylated. The presence of glutamic dehydrogenase and high levels of glucose-6-phosphate dehydrogenase were reported, but alcohol dehydrogenase was not found. Korting and Fairbairn (1972) have reported specific activities of glycolytic enzymes in M. dubius. 3. Tricarboxylic acid cycle An incomplete TCA cycle has been reported from Acanthocephala, as in several other intestinal helminths. Recent studies by Dunagan and Scheifinger (1 966a) on mitochondria1 preparations from M. liirudinaceus (and supernatants) failed to show aconitase, or isocitrate dehydrogenase, though both fumarate hydratase (very low) and malate dehydrogenase (high) were present. Similarly, Korting and Fairbairn (1972) reported the absence of citrate synthase, aconitate hydratase, NADP-dependent isocitrate dehydrogenase and fumarate hydratase from adult M . dubius, though citrate synthase, aconitate hydratase, NADP-dependent isocitrate dehydrogenase and fumarate hydratase were detected in the cystacanth. In contrast, experiments on Echinorhynchus gadi, a parasite of the flounder (Pseudopleuronectesamericanus), by Beitinger and Hammen (1 971) suggested a complete TCA cycle. 14C from labelled glucose or bicarbonate became incorportated into succinate, a-ketoglutarate, malate, citrate, fumarate and oxaloacetate in vitro. It may be that parasites of poikilotherms, such as E. gadi, differ in this respect from the better known parasites of homoiotherms. E. gadi respires aerobically for many days in vitro at YC, with an initial R.Q. of 0.78

694

W . L. NICHOLAS

rising to 1.0 over several days. However, the addition of glucose enhanced anaerobic COZproduction (in Nz) 40 %. Acanthocephala from homoiotherms also use oxygen in vitro, though Ward and Crompton (1969) argued that respiration by the adult worm is predominantly anaerobic in vivo. 4. COzjixation The coupling of glycolysis to a reversed dicarboxylic acid sequence of the TCA cycle, and the associated fixation of COz into the cycle, has been investigated by several workers. Korting and Fairbairn (1972) found high levels of phosphoenolpyruvate carboxykinase in the adults and cystacanths of M. dubius, suggesting that this enzyme provides the major link between the glycolytic pathway and the TCA cycle. Possible alternative enzymes involved in COZ fixation into the TCA cycle, phosphoenolpyruvate carboxylase and phosphoenolpyruvate transphosphorylase were not found, while only a trace of malate dehydrogenase (decarboxylating) was detected but only in the cystacanth. They found phosphoenolpyruvate carboxykinase was most active in the presence of MnZ+ and IDP. Horvath and Fisher (1971) confirmed that in both larval and adult M. dubius phosphoenolpyruvate carboxykinase is the principle enzyme involved in COz fixation, and that activity is optimal in the presence of Mnz+, and with ITP in place of ATP. They, too, found malic enzyme activity low.

5 . End products of respiration Crompton and Ward (1967) found lactate and succinate to be the main products of 14C-glucose utilization in vitro by P. minutus under conditions simulating those found in the host’s intestines. Worms remained active for many days in vitro and were successfully re-introduced into the host’s gut. Hanks BSS and low levels of 0 2 were used for the incubation medium. Surprisingly, M. dubius excreted large quantities of ethanol when incubated in Tyrodes BSS, whether in air or nitrogen (Ward and Crompton, 1969). After 2+ h incubation in nitrogen, 95 % of 14C-glucose supplied was utilized, 60 % being recovered in the incubation medium or as COz. Most of the radiocarbon was excreted as ethanol or C02, with small amounts in lactate and traces in succinate, acetate or butyrate. Ethanol plus lactate accounted for 97 % of the activity in the solution, in a ratio of 6 molecules to 1, and ethanol for twice as much as COZ. Korting and Fairbairn (1972) confirmed the high levels of ethanol excreted by M . dubius anaerobically, and reported the presence of the appropriate enzymes in both adult and cystacanth, i.e. pyruvate kinase, pyruvate decarboxylase, lactate dehydrogenase, and alcohol oxidoreductase. The latter was notable for NADP- rather than NAD-dependence. Smaller amounts of lactate were excreted. It seems, therefore, that despite the presence of high levels of phosphoenolpyruvate carboxykinase in this species, pyruvate kinase may be more active in the intact animal, so that more ethanol and lactate are excreted than succinate. Beitinger and Hammen (1971) reported that E. gadi, when incubated anaerobically with 4C-glucose,excreted nearly equal amounts of radiocarbon

THE BIOLOGY OF T H E A C A N T H O C E P H A L A

695

as lactate and succinate. In air, most radiocarbon was excreted as lactate with small amounts as succinate.

6. Nitrogenous excretion Janssens and Bryant (1969) were unable to find evidence of an active ornithine-urea cycle in adult M. dubius, or in a number of other parasitic helminths, in a comparative study of urea synthesis. It is unlikely that urea forms an important nitrogenous excretory product in adult M. dubius. The failure of tissue homogenates of M. dubius to incorporate radiocarbon from NaH14C03 into citrulline suggested the lack of carbamyl phosphate synthetase, a key enzyme in the urea-ornithine cycle. The presence of ornithine transcarbamylase, the only enzyme of the cycle found in M. dubius, could be linked to carbamyl phosphate synthesis from citrulline to supply a pyrimidine precursor. The apparent absence of arginase, present in the other helminths investigated by Janssens and Bryant, where it was suggested it may be an important step in collagen synthesis, was surprising. The incorporation of radiocarbon from carbamyl-labelled citrulline and from 14C-labelled aspartate into malate, fumarate and succinate, and, with citrulline, into aspartate as well, is consistent with the pathway leading to succinate which Scheibel and Saz (1966) have suggested may be a common feature of parasitic helminths. 7. Trehalose metabolism McAlister and Fisher (1972) studied trehalose synthesis in M. dubius. Considerable quantities of trehalose were present in the body wall, body fluids and acanthors. A level of about 26 pmol g-1 wet weight, or about 1.9 % of the wet weight, was found in the intact worm representing perhaps 3 % of tissue colloids. Trehalose synthesis could be demonstrated in the body wall but not in the body fluids or acanthors. McAlister and Fisher (1972) suggested that, as in other organisms, two enzymes are involved, trehalose phosphate synthetase and trehalose phosphate phosphatase, the latter requiring divalent cations. Probably, in M. dubius, glucose-6-phosphate and UDPG are cosubstrates. They found Mg2+, Fe2-b and to a lesser extent Zn2+ supported synthesis(Ca2+,Cu2+wereinhibitory), at an optimum pH of 8.0 and an optimum temperature of 50°C. Data were also given on the kinetics and activation energy of the reaction. Synthesis was inhibited by trehalose and high levels of ATP. End-product inhibition may stabilize the concentration of trehalose in tissues which may be important in osmotic regulation and the assimilation of glucose. 8. Glucose-6-phosphate dehydrogenase As already noted, Dunagan and Scheifinger (1966b) found high levels of this enzyme in M. hirudinaceus. Stein (1971) has partially purified the enzyme (E.C.1.1.1.49) from this species. He found activity was optimal at 40°C, pH 7.6, in the presence of 0.033 M MgC12. The enzyme was more concentrated in the posterior part of the body and much more so in the body wall than in the pseudocoel liquids. Korting and Fairbairn (1972) reported high levels of both

696

W . L. N I C H O L A S

glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from adults and cystacanths of M . dubius. 9. Isozymes Dunagan and Luque (1966) have found isozymes of lactate dehydrogenase and malate dehydrogenase in M . liirudinaceus by starch gel electrophoresis. Dunagan and Yau (1968) reported only a single isozyme of maltose in the same species.

VII. HOST-PARASITE RELATIONSHIP A.

WITH THE ARTHROPOD INTERMEDIATE HOST

Arthropods generally encapsulate foreign objects in their tissues. The capsule is usually formed initially by the aggregation of haemocytes, which become transformed into a multilayered capsule and within which, in insects and some other arthropods, a melanin-like pigment is deposited. Potential parasites invading the tissues of unsuitable hosts become treated as a foreign object. In the appropriate host they may remain non-encapsulated, or a non-melanized capsule of a different kind may form around them. There are exceptions to these generalizations, and the role of humoral, as distinct from cellular responses, in killing or inhibiting potential parasites, and in capsule formation, requires clarification. Salt (I 970) amongst others, has recently reviewed the field of arthropod reactions to parasites at length. The larval stages of some neoechinorhynchid Acanthocephalans remain unencapsulated within the haemocoel of the arthropod host, e.g. Paulisenfis fractus (Cable and Dill, 1967). Other Acanthocephala become encapsulated within their hosts by a thin envelope unlike that formed around foreign objects. Crompton ( 1970) has discussed earlier descriptive studies of this capsule by various authors. Capsules of this kind, or envelopes as they are more correctly termed, are formed in both crustacean and insect hosts and, as Bowen (1967) has shown, in millipedes. Crompton (1967) experimented with inter- and intraspecific transfers of the acanthellae of Polymorphus minutus from one Gammarus (Amphipoda; Crustacea), to another. The interpretation of these interesting experiments is complicated by physiological differences between species of Gammarus, interspecific differences in the parasite, and the effects of major surgery on the host. Nonetheless, the results show that acanthellae can complete development when transferred from one host to another of the same species, but not when transferred to another species, where they were treated as foreign objects and “melanized”. Apparently, the living parasite within its envelope is treated like healthy host tissue, following both intra- and interspecific transfers. Removal of the envelope before transplantation was always lethal to the parasite, but its death preceded the typical foreign body reaction. In general, the reactions of these amphipods to foreign bodies closely resembled those of insects, and were not successfully suppressed by X-irradiation. Mercer and Nicholas (I 967) described the fine structure of the envelope

THE BIOLOGY OF THE ACANTHOCEPHALA

697

surrounding the acanthella and cystacanth of Moniliformis dubius in the cockroach, Periplaneta americana. In the acanthella it is formed from a thick zone of adherent vesicles. In the cystacanth, this zone of vesicles becomes separated from the surface of the parasite by the accumulation of fluid between the envelope and the parasite, and amorphous material accumulates between the less closely packed vesicles. Butterworth (1969) showed that the envelope surrounding P. minutus in the amphipod host was similar in fine structure. Rotherham and Crompton (1972), have shown that the vesicular layer is not primarily, as suggested by Mercer and Nicholas (1967), a product of the host’s haemocytes, although they may possibly contribute to the envelope. The vesicles, which are its principle constituent as seen with the electron microscope, arise instead from the plasma membrane of the parasite. The vesicles are bound by a trilaminar membrane resembling the plasma membrane. Rotherham and Crompton (1972) described the envelope in its earliest phase as formed from microvilli extending from the parasite’s body surface. Haemocytes aggregate around the acanthor, as soon as it emerges on to the outer surface of the gut, but later largely disperse (Robinson and Strickland, 1969; Rotherham and Crompton, 1972). Sometimes, in contrast, a typical foreign body reaction occurs, and a melanized cellular capsule forms around part or the whole of the parasite, which does not complete development (Robinson and Strickland, 1969). Macracanthorhynchus hirudinaceus, when fed to P. americana, an unsuitable host, successfully hatches and penetrates the gut; it sometimes reaches the haemocoel, but provokes an intense cellular reaction within the gut and haemocoel and becomes heavily melanized. Tissue damage is not an essential precursor of the foreign body reaction, because when artificially hatched acanthors of M. hirudinaceus are injected directly into the haemocoel of the cockroach they are similarly melanized, whereas those of M . dubius often complete normal development. The removal of the envelope prevents the development of M. dubius acanthellae experimentally transferred to a new host, and leads to a melanized capsule. Lackie’s (1972) experiments, referred to in Section 111, show that unknown factors operate to restrict the numbers of M . dubius developing in primary infections of P. americana. The proportion of acanthors completing development in experimental infections varies greatly in different cockroaches, but declines rapidly with increasing dosage. However, some Acanthocephala may cause significant mortality in their normal hosts under natural conditions, e.g. P. minutus in G . pulex (see Crofton, 1971). 8.

PATHOLOGY IN THE DEFINITIVE HOST

Chaicharn and Bullock (1967) compared histopathology in two species of catastomid fish (Catastomus cammersoni and Erymyzon oblongus) arising from infection by four different Acanthocephala. The histopathology can be directly related to thedepthofpenetration of the host’s intestines. With Pomphorhynchus bulbocholli, the proboscis and bulbous top of the neck penetrated through the intestinal wall inducing the formation by the host of a fibroblastic, multi-

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layered collagenous capsule. The filiform sub-terminal neck passed through the entire intestinal wall in a tunnel produced by the complete elimination of the host’s intestinal tissues. The proboscis of the female of Octospinifer macilentus reached, but did not penetrate the muscle layer of the intestines, inducing fibroblast infiltration and leading to the formation of a nodule within the intestinal wall. The male penetrated less deeply. With Neoechinorhynchus cristatus the lamina propria was penetrated and the tissues became infiltrated by granular cells. N . prolixoides only disrupted the epithelium, producing minimal host reaction. Miller and Dunagan (1971) have suggested that a further factor may be the secretion by some Acanthocephala of a toxin from pores on the proboscis hooks (see Section II), causing an intense inflammatory response in the host. Such pores were found in M. hirudinaceus but not in NeoechinorJzynchus sp. or Echinorhynchus thecatus, which do not produce such an intense host reaction. C. MEDICAL A N D VETERINARY IMPORTANCE

1. Human infections Human infections by M . hirudinaceus and M . dubius occur sporadically in many parts of the world, but cannot be considered a significant medical problem, although it has been reported that M . hirudinaceus may once have been a common human parasite in Russia. Sahba et al. (1970) have reported another human case of M . dubius in Iran. Three more species have been reported recently from modern man and one from prehistoric man. Juveniles of Corynosoma strumosum, which normally mature only in the seal, are found in a variety of fish-eating mammals. A juvenile recovered at Chevak (Alaska) from the stool of an Eskimo who had been treated with atabrine, was identified by Schmidt (1971). Eskimos may be infected commonly by juveniles as a result of eating raw fish. Schmidt (1971) reports that Golvan and Houin identified as Acanthocephalus rauschi a specimen recovered from the peritoneum of an Alaskan Eskimo. Schmidt (1971) also identified several male A . bugonis in material from a routine autopsy in Djakarta, Indonesia. A . buffonis is a common parasite of amphibia in S.E. Asia. Moore et al. (1969) found acanthors, possibly of Moniliformis clarki, in human coprolites from an archaeological excavation at Danger Cave, Utah, dated at 1869f 160 years B.C. and 20 k 240 A.D. A . clarki is common today in animals of the vicinity.

2. Veterinary medicine Corynosoma has been implicated in serious outbreaks of disease amongst farmed mink (Mustela vision) in Finland (Nuorteva, 1966). The symptoms were bloody diarrhoea and anaemia and many animals died. C. semerne was found in many surviving mink, and higher infection rates were associated with clinical anaemia. C . strumosum was found in two dead animals examined and possibly is more pathogenic than C . semerne. Both species are parasites of seals, Phoca hispida in the Baltic, Lake Ladogoda (as glacial relics), and other fresh water

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lakes in Finland (Nuorteva, 1965). The mink undoubtedly became infected when fish, several species of which can serve as transport hosts, were used for food. D.

ECOLOGICAL FACTORS

The development of parasites in poikilothermichosts is strongly influenced by seasonal changes, especially temperature. Walkey (1967) concluded, from studies of natural infections of the stickleback, Gasterosteus aculeatus, by Neoechinorhynchus rutili, in a pond in northern England, that an annual cycle was initiated by the rapid maturation of immature worms in spring and early summer. Infected fishes passed acanthors, and the intermediate hosts probably became infected, throughout much of the year, with the spent female worms being lost late in the year. Fishes were infected at all times of the year, the larger fish carrying heavier parasite burdens as a consequence of their feeding habits. Two species of ostracods, Cypria ophthalmica and Candona candida served as intermediate hosts. Both were experimentally infected in the laboratory, though a number of other possible intermediate hosts, including Sialis (previously reported as a host), could not be infected. Tedla and Fernand (1970) found that Echinorhynchus salmonis completed one generation per year in Lake Ontario in its host, Perca,fluviatilis.Acanthors were passed in early summer, the parasite then died, and new hosts became infected in autumn and winter. Olson and Pratt (1971) found that Echinorhynchus lagenformis survived for about 1 year in its host-the marine flounder, Platichthys stellatus. Development in Corophium spinicorne, the intermediate host, probably occurred in spring and summer. Fishes between 1 and 2 years old became infected because of their feeding preferences. Denny (1969) gave some data on the effect of temperature on the development of three species of Polymorphus; P . marilis, P . contortus and P. paradoxus, in Gammaruslacustris (Amphipoda) from Alberta, Canada, in apaper primarily concered with taxonomy and host records. Crofton (197 1)suggestedthat the ecology of parasitism should be approached quantitatively. To illustrate his views he analysed again the data of Hynes and Nicholas (1963) on natural infections of G. pulex by P . minutus. They reported the infection rate and parasites per host at collection sites spaced out at various distances along a stream from the source of infection, a duck farm. Crofton (1971) found the number of parasites per host at each site followed a truncated negative binomial distribution which improved the fit where infection rates were highest, probably reflecting increased mortality in the host with higher levels of infection. VIII. SUMMARY AND CONCLUSIONS When the writer’s review of this subject was published in “Advances in Parasitology” (Nicholas, 1967), studies of the fine structure of Acanthocephala had just begun. Now, 5 years later, many of the organ systems have been described in several species and we may soon have a fairly complete description

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of acantlioccplialai~ fine structure, although the neuroniuscular system has received little attention so far. However, the functional significance of some of the details peculiar to the group are not well understood; e.g. the relationship between assimilatory mechanisms and ion pumps in the tegument and fine structure are unresolved. The way in which the tegument grows and develops presents interesting unsolved problems, the more so because of its syncytial nature, and the presence of a complex intracytoplasmic framework of supporting bars and fibres. A combination of experimental physiology and histology has given a clearer understanding of how the uterine bell apparatus and proboscis work, in comparison to work based on earlier purely histological studies. The movement of the acanthor has also been elucidated. Similarities between cestode and acanthocephalan morphology and life cycles have frequently been commented on by helminthologists in the past, notably by Van Cleave. Such comparisons can now profitably be extended to fine structure and biochemistry. In both groups, the tegument is an assimilatory organ, with its surface greatly expanded, by microvilli in cestodes and by pore canals in Acanthocephala. The surface is coated by a fibrous layer of mucopolysaccharide, which in cestodes has been the subject of a number of recent studies, particularly by Lumsden. Wright and Lumsden’s comments on its properties and possible function in Acanthocephala have been discussed in Section IV A, 1 (Epicuticle). A similar overall pattern of predominantly anaerobic respiration characterizes a number of the larger parasitic helminths. Glycolysis, by the EmbdenMeyerhof pathway, is often coupled by phosphoenolpyruvate carboxykinase to an incomplete TCA cycle. The dicarboxylic acid sequence of the TCA cycle operates in reverse, culminating in the production of succinate, or a derivative of succinate. It has been postulated that in some helminths, for example the cestode Hymenolepis diminuta (Scheibel and Saz, 1966; Scheibel et al., 1968), this modification of orthodox glycolysis, culminating in the production of lactate, may be advantageous to helminths by providing an additional phosphorylation step associated with the reduction of fumarate in succinate. Studies of Acanthocephala have shown that there may be important differences between the different groups and even between genera, in the operation of this pathway. Despite the high levels of phosphoenolpyruvate carboxykinase, Moniliformis dubius excretes C02, ethanol and lactate rather than succinate under anaerobic conditions. Polymorphus minutus and Echinorhynchus gadi excrete significant amounts of both lactate and succinate anaerobically. E. gadi may possess a complete TCA cycle, and part of its respiration may be aerobic in the gut of its host, a fish. It is possible that parasites of poikilotherms differ from homoiotherms in this respect. It will be interesting in the future to compare the respiratory metabolism of acanthellae from the haemocoel of arthropods with that of the adult forms from the intestines of vertebrates. Some studies have been made of the cystacanth, but as this is a resting, transitional stage, the significance of the comparison between it and the adult is difficult to evaluate. New avenues of research into the properties of collagens, sterol metabolism

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and isozyiiies of Acanthocephala liavc been explored. Acanthocephala must now be included with other major invertebrate groups which require a sterol in the diet. Of the two commonly expressed views on the phylogenetic affinities of the Acanthocephala, neither of which is supposedly close, that of an ill-defined relationship with the Aschelminthes is gaining some support at the expense of a distant affinity with the Platyhelminthes. Whitfield has described the fine structure of acanthocephalan spermatozoa (Section IV) and in a later paper (Whitfield, 1971b) compared acanthocephalan with platyhelminth spermatozoa. Spermatozoon structure seems to be rather a consistent character within the major phyla, and acanthocephalan spermatozoa resemble those of Aschelminthes (though not those of Priapulida, the group usually considered closest) rather than those of Platyhelminthes. There are also some suggested similarities between the tegument and muscles of Acanthocephala and Rotifers (Aschelminthes). Nicholas (1971) has argued that some of the peculiarities of acanthocephalan embryonic and larval development can be given a phylogenetic explanation. It is suggested that the ancestors of Acanthocephala may have been members of the marine interstitial fauna of sands and muds, a habitat which leads to certain well marked adaptations, among them miniaturization. The restriction of cell division to an early developmental stage may have arisen in this way as a concomitant of small size. The subsequent adoption of parasitism would have permitted a great increase in size, as it has in other invertebrate groups, e.g. nematodes, and would superimpose a new set of adaptations. The consequences could have been the development of larger syncytial organs, such as the tegument, characterized by a few very large nuclei. If the many similarities between Acanthocephala and Cestoda have arisen by convergent evolution from very different free-living ancestral forms, an understanding of their adaptive significance should add to our understanding of the origins and nature of parasitism in helminths.

ACKNOWLEDGEMENTS I am grateful to Dr R. A. Hammond, Department of Zoology, University College, Cardiff;and Dr P. J. Whitfield, Department of Zoology, King’s College, London, for permission to reproduce figures from their papers.

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Crompton, D. W. T. and Whitfield, P. J. (1968b). A hypothesis to account for the anterior migrations of adult Hymenolepis diminuta (Cestoda) and Moniliformis dubius (Acanthocephala) in the intestine of rats. Parasitology 58,227-229. Denny, M. (1969). Life cycles of helminth parasites using Gammarus lacustris as an intermediate host in a Canadian Lake. Parasitology 59,795-827. Dunagan, T. T. and Luque, 0. de (1966). Isozyme patterns for lactic and malic dehydrogenases in Macracanthorhynchus hirudinaceus (Acanthocephala). J. Parasit. 52,727-129. Dunagan, T. T. and Scheifinger,C. C. (1966a). Studies on the TCA cycle of Macracanthorhynchus hirudinaceus (Acanthocephala). Comp. Biochem. Physiol. 18, 663-667. Dunagan, T. T. and Scheifinger, C. C. (1966b). Studies on glycolytic enzymes from Macracanthorhynchushirudinaceus (Acanthocephala).J. Parasit. 52,730-734. Dunagan, T . T. and Yau, T. M. (1968). Oligosaccharidasesfrom Macracanthorhynchus hirudinaceus (Acanthocephala) from swine. Comp. Biochem. Physiol. 26, 28 1-289. Edmonds, S. J. (1966). Hatching of the eggs of Moniliformis dubius. Expl Parasit. 19, 216-226. Golvan, Y . J. (1969). Syst6matique des Acanthodphales (Acanthocephala Rudolphe 1801). Premibre partie: L'ordre des Palaeacanthocephala Meyer 1931, premier fasicule: la super-famille des Echinorhynchoidea (Cobbold, 1876) Golvan et Houin 1963. MJm. Mus. natn. Hisr. nut., Paris (Serie A, Zool.) 57,l-373. Grabda-Kazubska, B. (1964). Observations on the armature of embryos of acanthocephalans. Actaparasit.polon. 12,215-231 [fromcitation in Whitfield, 1971al. Graff, D. J. and Kitzman, W. B. (1965). Factors influencingthe activation of acanthocephalan cystacanths. J. Parasit. 51,424429. Hammond, R. A. (1966a). Changes of internal hydrostatic pressure and body shape in Acanthocephalus ranae. J. exp. Biol. 45, 197-202. Hammond, R. A. (1966b). The proboscis mechanism of Acanthocephalus ranae. J. exp. Biol. 45,203-213. Hammond, R. A. (1967a). The fine structure of the trunk and praesoma wall of Acanthocephalus ranae (Schrank, 1788) Liihe 1911. Parasitology 57,475-486. Hammond, R. A. (1967b). Themode of attachment within the host of Acanthocephalus ranae (Schrank, 1788) Liihe 1911. J. Helminth. 41,321-328. Hammond, R. A. (1968). Some observations on the role of the body wall of Acanthocephalus ranae in lipid uptake. J. exp. Biol. 48,217-225. Hibbard, K. M. and Cable, R. M. (1968). The uptake and metabolism of tritiated glucose, tryosine, and thymidine by adult Pauksentis fractus Van Cleave and Bangham, 1949 (Acanthocephala: Neoechinorhynchidae).J. Parasit. 54,517-523. Holloway, H. L., Jr. and Nicol, B. B. (1970). Morphology of the trunk of Corynosoma hamanni (Acanthocephala: Polymorphidae). J. Morph. 130,151-162. Horvath, K. (1971). Glycogen metabolism in larval Moniliformis dubius (Acanthocephala). J. Parasit. 57, 132-136. Horvath, K. and Fisher, F. M., Jr. (1971). Enzymes of CO, fixation in larval and adult Moniliformis dubius (Acanthocephala). J. Parasit. 57,440-442. Hynes, H. B. N. and Nicholas, W. L. (1963). The importance of the acanthocephalan Polymorphus minutus as a parasite of domestic ducks in the United Kingdom. J. Helminth. 37, 185-198. Janssens, P. A. and Bryant, C . (1969). The ornithine-urea cycle in some parasitic helminths. Comp. Biochem.Physiol. 30,261-272. King, D. and Robinson, E. S. (1967). Aspects of the development of Moniliformis tlrr6itis. J . Pnmsit. 53, 142-149.

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Wright, R. D. (1971). The egg envelopes of Moniliformis dubiiis. J. Purasit. 57, 122131. Wright, R. D. and Lumsden, R. D. (1968). Ultrastructural and histochemical properties of the acanthocephalan epicuticle. J. Parasit. 54, 1111-1123. Wright, R. D. and Lumsden, R. D. (1969). Ultrastructure of the tegumentary porecanal system of the acanthocephalan Moniliformis dubiia. J. Parusit. 55, 9931103. Wright, R. D. and Lumsden, R. D. (1970). The acanthor tegument of Moniliformis dubius. J. Parasit. 56,721-735.

The Post-Embryonic Developmental Stages of Cestodes MARIETTA VOGE

Department of Medical Microbiology and Immunology, U.C.L.A.School of Medicine, Los Angeles, California, U.S.A. 1. Introduction .................................................................................... 11. Life Cycles and Larval Growth ............................................................ A. Tetraphyllidea ...........................................................................

111.

IV.

V. VI. VIT.

B. Pseudophyllidea.. ........................ ........................................... C. Cyclophyllidea ........................................................................... Histology, Histochemistry and Fine Structure .......................................... A. Lecanicephalidea ........................................................................ B. Pseudophyllidea. .......................................................................... C. Cyclophyllidea .......................... .......................................... Host-Parasite Relationships.. ................................................................ A. Invertebrate Hosts.. ...................................................................... B. Vertebrate Intermediate Hosts.. ....................................................... C. Vertebrate Hosts: Immunity ......................................................... Metabolism....................................................................................... Growth in vitro ................................................................................. Conclusions .................................................................................... References .......................................................................................

707 708 708 708 709 71 1 71 1 712 713 714 714 715 717 718 720 722 723

I. INTRODUCTION Since the previous review on the biology of post-embryonic developmental stages of cestodes (Voge, 1967) the trends of research have shifted more perceptibly toward investigations on fine structure, immunology, metabolism, and other areas now popular in the biology of free-living organisms. This does not mean that life histories and purely morphological aspects of cestodes have been neglected. Indeed, the total number of publications on all aspects of cestode larval stages is so great that citation of all pertinent references is not feasible within the scope of this review. The availability of recent books and reviews on growth in vitro (Taylor and Baker, 1968; Silverman and Hansen, 1971) and on immunology (Weinmann, 1970) makes complete coverage of these fields unnecessary. Echinococcus biology is omitted because it will be covered separately elsewhere. Assignment of some papers to the subdivisions used here is of necessity somewhat arbitrary because many studies contain information pertinent to more than one area of 707

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research. It is hoped that the following pages will be useful in bringing illto sharper focus at least some of the recent accomplishments and that the author will be forgiven if the choice of subjects discussed appears unduly limited.

11. LIFECYCLES AND LARVAL GROWTH The importance of the structure and biology of pre-adult developmental stages in cestode systematics was discussed by Voge (1969) in relation to several cestode groups and in particular with regard to the tenuous position of mesocestoidid tapeworms within the Cyclophyllidea. The need to re-evaluate our ideas about cestode evolution and the possible relationship among different cestode groups is exemplified by the studies of Freeman (1970) and Jarecka (1970a) who attempted to modify some of the existing ideas by an analysis of the different types of larval stages. One of the distinctive differences in larval developmental patterns according to Freeman (1970), is the presence or absence of a primitive lacuna during early post-oncospheral development. The possible significance of the cercomer, and the tissues surrounding the scolex in the different types of larvae, are also discussed. Evolutionary trends with hypothetical sequences of structural types are discussed and shown diagramatically by Jarecka (1970a), who also presents different life cycle schemes with the host environments in which the different larval stages of a cycle occur. While our knowledge of cestode life cycles is still inadequate the progress made during the last few years permits some generalizations based on the more firmly established developmental patterns. A.

TETRAPHYLLIDEA

The occurrence of larval Echeneibothrium in two species of clams in California was reported by Katkansky et al. (1969) who found the larvae encysted in the foot and also free in the intestinal lumen. The finding of stages intermediate between the ciliated embryo and the plerocercoid suggests that clams may serve as suitable intermediate hosts. Warner and Katkansky (1969) also found Echeneibotlzrium larvae encysted in a third species of clam in California. Fried1 and Simon (I 970) found tetraphyllidean larvae, presumably onchobothriids, in marine prosobranch gastropods in Florida. B. PSEUDOPHYLLIDEA

While many miscellaneous facts have been reported by various workers on diphyllobothriid life histories, no unusual new information has been published within the last few years. An exception is the experimentally demonstrated growth of tail fragments of spirometrid plerocercoids (Mueller, 1972a), studied in four different strains of Spirometra. Tail fragments of all strains showed the ability to grow when injected into mice but the extent of growth varied with the age of the worm and with the region of the “tail” from which fragments were obtained for injection.

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C . CYCLOPHYLLIDEA

1 . Mesocestoididae It is with some hesitation that I am discussing this group within the order Cyclophyllidea since I do not believe that the mesocestoidids belong here (Voge, 1969). A definitive decision regarding the position and the relationships of mesocestoidids to other tapeworms will be easier when more is known about their life history. Recent findings (Hart, 1968)that fragments of tetrathyridia of Mesocestoides corti can regenerate the whole organism in vivo as well as in vitro further broadens the versatility of this unusual organism. In addition, Eckert et al. (1969) showed that tetrathyridia, when fed to dogs or skunks, continue asexual multiplication in the intestines of the definitive host, thereby increasing their number significantly before transforming into adult worms. Thus, a dog fed about 1500 tetrathyridia harbored more than 45 000 worms several weeks later. Tetrathyridia are also able to multiply in the peritoneal cavity of dogs and to migrate from the peritoneal cavity to the intestinal lumen. Whether or not tetrathyridia of other mesocestoidid species have similar abilities remains to be determined. Additional information on the sequence of events during asexual multiplication and growth of Mesocestoides in different strains of laboratory mice is given by Novak (1972). Novak and Lubinsky (1972) demonstrated enhanced multiplication of tetrathyridiain hosts treated with agents shown to be cytostatic for other helminths. 2. Hymenolepididae Several interesting findings have been reported for species from mammalian hosts. The occurrence of cysticercoids of Hymenolepis nana in extra-intestinal sites was reported by Solonenko (1969,1970)who found the larvaein mesenteric lymph nodes of white rats, and by Astafev (1970) who described and illustrated the cysticercoids from lymphatics of white mice; some of the cysts have two scoleces, others have various structural abnormalities. DiConza (1970) showed that oncospheres of H. nana injected subcutaneously into mice developed to cysticercoids in that site. Cockroaches (Blatella germanica) were reported as intermediate hosts of H. nana (see Furukawa, 1970). The apparently related species, H. microstoma, has been found to develop in mice from eggs to adult worms, especially if eggs were kept for 24 h at 4°C before they were fed to mice (Actor et al., 1968).The site and mode of cysticercoid development in the mouse was not studied and should be investigated. The life cycle of Hymenolepis nagatyi from shrews was experimentally demonstrated by Hunkeler (1969) who obtained infective cysticercoids in the beetle Tenebrio molitor. Cysticercoids of another parasite of shrews, Vigisolepis spinulosa, were found in natural infections in Collembola (ProkopiE, 1968). Quentin et al. (1971) reported the occurrence of apparently fully developed cysticercoids of Hymenolepis brusatae in two species of Phleboromus. This finding extends the intermediate host range of hymenolepidids of insectivores. Reports on life histories of avian hymenolepidids are much more numerous. Pike (I 968) found cysticercoids of Dicranoraenia coronula and Microsoma-

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canthus compressa in the stomach contents of lymnaeid snails. Whether these are accidental findings or whether they are of significance in the development of these cysticercoids must be determined experimentally. The same author also found cysticercoids of Haploparaksis cirrosa and Kowalewskiusparvula in leeches. Hymenolepidids with copepod intermediate hosts, as demonstrated experimentally, are Tschertkovilepis krabbei (see Czaplinski and Jarecka, 1967), and Gastrotaenia paracygni (see Wisniewski, 1971). Wisniewski (1971) also showed that ostracods are intermediate hosts of Nematoparataenia southwelli. Other species having ostracod intermediate hosts are Monosaccanthes streperae as reported by Czaplinski and Wilanowicz (I 969), and Hamatolepis teresoides (see Wisniewski, 1970). The life cycle of Hymenolepis hopkinsi was shown to require amphipods for cysticercoid development (McLaughlin and Burt, 1970). Detailed descriptions of the cysticercoids and their developmental stages are included in all of the studies mentioned above.

3. Dilepididae Several recent studies on life histories of dilepidids with larval stages in fish hosts raise questions about the affinity of these species to other cestode groups and on the present conglomeration of species placed into this family, Jarecka (1970b) studied the life cycle of Vulipora campylancristrota in which a cysticercoid-like larva develops in copepods. Jarecka proposed the name cercoscolex for this larva which bears a very small tail appendage. However, the precise differences between this larval type and a cysticeroid are not clear, except that this larva is transferred from the copepod to a fish host. Whether the fish serves only as a paratenic host, transferring the larvae to birds where the adults develop, or whether any essential growth occurs in the fish is not yet known. Similar life cycles and larval stages were described by Jarecka (1970~)for Paradilepis scolecina and Neogryporhynchus cheilancristrotus where the first intermediate hosts are copepods, whence apparently fully developed larvae are transferred to fishes. Stages of these species found in fishes are described by Kozicka (1971). A careful histological and developmental comparison of these larvae with cysticercoids would be desirable. The life cycles of two species of Choanotaenia from shrews were studied in experimental infections of the mollusc Arion lusitanicus by Jourdane (1972). No development occurred at temperatures below 10°C.At the optimal temperature of 15"C, C. crassiscolex requires 40 days to complete development, while C. estavarensis requires 50 days at the same temperature. 4. Davaineidae

The life history of Cotugnia srivastavai n. sp. is described by Malviya and Dutt (1970) who found the fully developed cysticercoids in naturally infected ants. Larvae removed from ants were infective to pigeons. Developmental stages were not studied. 5. Taeniidae

Of interest is the report by Opuni (1970) on the occurrence of asexual reproduction in Tuenia pisiformis, either by transverse fission of the bladder or

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71 1

through budding at the neck region to produce two scoleces. As stated by the author, additional studies are needed to determine whether or not this is a normal process for the species. Growth rates of two strains of Taenia crassiceps are compared by Dorais and Esch (1969) who found the greater larval proliferation in one of the strains associated with hook abnormalities. Authors believe that these characteristics are the results of mutation. X-irradiation of both strains resulted in significant stimulation of reproduction in the ORF strain but had no effect on KBS larvae (Beavers et al., 1971). Subsequently, Smith et al. (1972) showed that the ORF strain contained only 14 instead of 16 chromosomes, and that the abnormalities are due to a missing homologous pair, the result of aneuploidy. As this strain (ORF) has been maintained in the laboratory for nearly 20 years, changes in overall behaviour of the organism are to be expected. However, we know very little about the kind or frequency of mutations in organisms we have been working with for many years. Hopefully, studies similar to those described above will increase in number in the future. Evagination of Taenia hydatigena cysticerci in vivo and in vitro was studied by Featherston (1 97 1) who found that a large portion of the larval tissue was shed in the dog's small intestine. Experiments with different solutions showed that maximal evagination in vitro occurred in the presence of dog's bile, but that larval tissues never were shed in vitro. Data on relative densities of taeniid larvae in their hosts were given by Filippov and Kosminkov (1970) who showed that cysticerci of Taenia saginata in experimentally infected calves were most numerous in the masseter muscles. Gemmell (1970a) showed that distribution of Taenia ovis larvae in sheep depended somewhat on intensity of infection, the fore- and hind-legs containing the highest percentage of Iarvae when infections were heavy. In moderate or light infections, however, the pattern of distribution was variable. The migration of oncospheres of three taeniids was studied by Heath (1971) who presented information on the path of migration in the vertebrate hosts and also on the numbers of oncospheres or eggs administered by different routes and the number of cysticerci resulting from the infection. The loss of embryos occurring even in normal hosts and normal modes of infection (e.g. T.pisiformis in rabbits) is impressive and reiterates that the parasitic way of life can be a biological extravagance.

111. HISTOLOGY, HISTOCHEMISTRY AND FINE STRUCTURE A.

LECANICEPHALIDEA

Study of the larvae of Tylocephalum by Rifkin et al. (1970) revealed an unusual type of microvillus arising from the tegument, each terminating in a prominent spherical vesicle. The authors suggest that the microvillar layer may serve to prevent intimate encapsulation by fibrous elements from the host and that the terminal vesicles may function in secretion. It seems reasonable to assume that the ubiquitous microvillar layer may serve in both an absorptive

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as well as a secretory capacity, the latter perhaps also functioning as a defensive mechanism for the tissue-dwelling stages. At least, this is an interesting hypothesis which should be tested if possible. B. PSEUDOPHYLLIDEA

Considerable progress has been made in our knowledge of cellular organization and structure of this group of cestodes. Braten (1968a) studied the fine structure of the procercoid of Diphyllobothrium latum, in particular the tegument and microvilli covering the surface of the organism. Charles (1970) studied the fine structure of the developing pseudophyllid tegument in larvae of Schistocephaliis and Ligula, identifying developing microtriches in procercoids as early as 2-4 days after penetration of oncospheres into the copepod hemocoel. He also compared the procercoid and plerocercoid teguments as to structure and number of microtriches, Golgi activity, and other parameters. Halvorsen (1970), in a detailed, comparative study of plerocercoid structure, included many excellent illustrations of sections showing cell density and distribution in different areas of plerocercoids and measurements of the thickness of tissues at different stages of development. The cellular composition of Diphyllobothrium dendriticum plerocercoids was studied by von Bonsdorff et al. (1971) who presented in cross section a diagrammatic distribution of the different cell types and their relative abundance ;he identified germinal, muscle and parenchyma cells, as well as three different types of nerve cells, in addition to the sub-integumental cells and the flame cells. A discussion of each cell type and of observations made follows the description. The primary “anlage” formation in D. dendriticum was described by Wikgren et al. (1971) by the use of autoradiographic techniques. The authors suggested that genital primordia are formed by the migration of germinal cells to the inner parenchyma, and stated that the process resembles blastema formation during regeneration in planarians. The scolex of the plerocercoid of Spirometra erinacei was studied by Kwa (1972a) in an attempt to determine the presence or absence of histolytic glands. However, no glands were found, even although biochemical assays revealed the presence of proteolytic enzyme in the tegument (Kwa, 1972b). Kwa (1972~) also studied the fine structure of the tegument of the scolex, and described two new organelles in the tegument. He suggested that the granules found within them might represent enzymic proteins transported to the outside during tissue penetration. Other observations of the structure of the tegument were similar to those made by Yamane (1968). Comparison of the tegument of plerocercoids and of adults of Diphyllobotlzrium latum (see Braten, 1968b)revealed several striking changes in the adult worm; an increase in the size and number of mitochondria, an increase in the length and number of microvilli, and the disappearance of the “lamellated bodies” present in the procercoid and plerocercoid. Growth of subtegumental tissue apparently occurs from migration of germinative cells (Wikgren and Knuts, 1970). Wikgren and Gustafsson (1971) described the germinal cells in plerocercoids

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of D. dendriticum,basophilic cells constituting33 of the whole cell population. Distribution of cells in mitosis and DNA-synthesizing cells closely corresponded to the distribution of germinative cells which, according to the authors, form a pool from which the various specialized cells are drawn. According to the cell maps included in this study, germinative cells are present throughout the plerocercoid body. C. CYCLOPHYLLIDEA

1. Hymenolepididae The first demonstration of microvilli on the surface of larval hymenolepidids was made by Collin (1969, 1970) in early developmental stages of Hymenolepis citelli from Tribolium confusum. The whole surface of the “hollow ball” stage was covered with slender projections from the surface, 1-4 pm in length. Structurally, these microvilli were unlike those in adult worms. Collin also described the cell types found in early developmental stages and their position within the organism. Ubelaker et al. (1970) described microvilli on the capsule of the fully developed cysticercoid of Hymenolepis diminuta. Some of these villi are shown to have a bulbous, extended tip. The cells beneath the surface of the cysticercoid form a syncytium. Morphogenesis of the cysticercoid of Hymenolepis microstoma was studied by Goodchild and Stullken (1970) in histological sections and by light microscopy. In general, development of this species conformed to that known for other hymenolepidids. The presence of two phosphatases, a cholinesterase, lactic, succinic, and malic dehydrogenases, and other enzymes, was demonstrated histochemically in cysticercoids of H. microstoma and H. diminuta by Bogitsh (1967). The fine structural localization of acid phosphatase and aryl sulfatase was described by Bogitsh (1969) in the intermediate cell layer of H. diminuta.

2. Davaineidae The histology, histochemistry and fine structure of the fully developed cysticercoid of Raillietina cesticillus were described by Baron (1971). Study of the layer beneath the hyaline coat showed that it consisted of many globules and that its surface was raised in many microthrix-like projections. The sequence of layers comprising the cyst envelope is described and illustrated, and comparison is made between the cyst wall and the scolex. Fine structure of the latter resembles other cyclophyllid scoleces. Baron suggested that the globules may be a secretory substance, possibly connected with an antigenantibody reaction between host and parasite. While the secretory function is a very reasonable hypothesis, the antigen-antibody reaction is not, as no one has yet been able to demonstrate this type of reaction in insects. However, the secretions, if present, may function to minimize the effect of the host cellular response (see section on host-parasite relationships). 3. Taeniidae In studies on the fine structures of Taenia taeniaefurmis larvae, Nieland and Weinbach (1968) include comparative assays for glucose in bladder fluid and

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in host plasma. Results showed that glucose levels in bladder fluid were in most instances higher than those in host plasma. As to fine structure, the network of ducts and their cytoplasmic connections are very well illustrated. A discussion on the bladder fluid in relation to nutrition of the larva is included in this study. The composition of larval hooks of T. taeniaeformis was studied by Dvorak (1969a, b) who found a single N-terminal amino acid, cystine, in the hook protein molecules. Perhaps because of its convenience as a “laboratory animal”, Taenia crassiceps has been studied extensively during the past few years. Histogenesis of the rostellum was described by Bilqees and Freeman (1969) who stated that hooks are not produced from fused microtriches but arise de novo. Mount (1970), on the basis of fine structure, stated that the hooks originate through enlargement of specializedmicrotriches on which hook protein is then deposited. The visual evidence for this claim by Mount is not wholly convincing but the problem merits further study. Baron (1968) stated that the rostellar hooks appear first as small cones to which a base is then added. Chemical tests by Baronshowed that mature aswell as immature hooks probably contain keratin. An interesting study by Bilqees (1970) on the sequence of events in bud formation of T. crassiceps showed that the subtegumental cells are very important in the differentiation of buds. The changes during bud formation are: a thickening of the tegument; an increase in the number of subtegumental cells by division with subsequent folding of the tegument; and an apparent breakage of subtegumental muscle fibers. Diagrams showed these and other steps in bud formationin histological section. Theexistence of a budding gradient was also noted by Bilqees (1968a) who showed that more buds develop at the posterior than the anterior end of the larva. She also illustrated some primitive cell types (Bilqees, 1969) and mitotic figures in somatic cells (Bilqees, 1968b). Numerous studies on the structure of cysticerci of Taenia solium and T. saginata culminated in a detailed presentation of morphology and pathology of these organisms by h i s (1970a). The book contains numerous plates, several in color, showing the histologic structure of cysticerci in their different growth forms. Some of the conclusions reached are that it is possible to differentiate T. solium from T. saginata cysticerci on sections from the bladder wall alone, and that larvae in skeletal muscle actually develop in the lymphatic system. Some aspects of the fine structure of the cysticerci were also presented by Slais (1970b). IV. HOST-PARASITE RELATIONSHIPS A. INVERTEBRATE HOSTS

The effects of infection upon invertebrate animals and the responses of invertebrates to the presence of foreign substances have been of increasing interest in recent years. Invertebrate pathology and invertebrate “immunity” have become major fields of investigation. While our knowledge of invertebrate host responses to infection with larval tapeworms is scanty, a few recent publications might illustrate investigative trends in this field. Rifkin and Cheng (1968)

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studied encapsulation of Tylocephalum larvae by the oyster Crassotrea virginica and described the origin and composition of the host-cystic tissue. Cheng and Rifkin (1968) showed that many of the larvae become resorbed owing to massive host cell responses to the infection. The fine structure of the encapsulating host tissue was described by Rifkin el al. (1969) who found that cystic tissue consisted of both fibers and cells, the fibers being non-collagenous. Authors speculate on the possible nature of the intimate contact between the cestode microvilli and the fibers of the host capsule, suggesting that the fibers might represent a source of food, or that this adherence has a role in the larval defense mechanisms. Cellular responses of insects to infection with cestode larvae were described by Collin (1970) who presented pictorial evidence that the host cells from Tribolium confusum which come to surround the early developmental stages of Hymenolepis citelli eventually disintegrate, perhaps in response to some substance released by the microvillar projections at the parasite’s surface. Comparison of the response by Tribolium confusum infected with Hymenolepis diminuta, H. microstoma or H . citelli showed that only H. citelli larvae become temporarily encapsulated while larvae of the other species do not (Heyneman and Voge, 1971). Also, no evidencewas found that challenge infections with the same or a different species of Hymenolepis in any way differed from those of previously uninfected controls. Melanization or resorption of the parasites did not occur in Tribolium. However, Cavier and LBger (1965) showed that in unusual hosts (cockroaches) cysticercoids of H. nana were destroyed and melanization also occurred. Other host-parasite effects, especially those involving host susceptibility, were studied by Dunkley and Mettrick (1971) who showed that temperature, age of the host, and the period of host starvation all influenced the parasite burden in Tribolium exposed to H. diminuta. Soltice et al. (1971) showed that infection of Tribolium with H. diminuta was not influenced by the sex of the beetles. They also showed that the respiratory rate of the insects increased upon infection. Retardation of growth and development of Tribolium larvae infected with H. microstoma was reported by Tan and Jones (1969) who showed that this delay was longer when the number of parasites was greater. The effect of crowding on Sobolevieanthus gracilis in the ostracod intermediate host was studied by Misiura (1971) who found that the final length of rostellar hooks of cysticercoids varied with the intensity of infection in individual hosts, larger hooks developing in hosts with fewer parasites. B.

VERTEBRATE INTERMEDIATE HOSTS

One of the important aspects of host-parasite relationships, pathogenesis and pathology caused by cestode larvae, will be mentioned only briefly because of the extensive treatment of this subject by Smyth and Heath (1970). Also, Slais (1970) should be consulted for the pathology of cysticercosis, and Specht and Widmer (1972) for mouse liver pathology caused by the invasion of tetrathyridia of Mesocestnides. Among the several studies on the problem of host susceptibility, the relation-

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ship between host size and intensity of infection with Schistocephulirs was studied by Pennycuick( 1971)in sticklebacks. She found nodifference in intensity of infection between male and female fish. Distinct differencesin susceptibility of male and female mice infected with comparable numbers of Mesocestoides tetrathyridia were reported by Novak (1972). In her experiments with different strains of laboratory mice, tetrathyridia always multiplied more rapidly in males than in females. Similarly, an analysis of rabbits infected with cysticerci of Tueniu pisiformis (Berg and Beck, 1968) showed a much higher incidence of infection in males than in females. Differences in growth and in the extent of liver invasion by tetrathyridia were noted by Novak (1 972) in birds, cotton rats, ground squirrels and muskrats, all of which were susceptible to the infection. Eckert (1 970) gave the Chinese hamster as an additional host for the tetrathyridia, and reported subcutaneous infection in mice and transplacental infection in rats. Regarding age susceptibility, Vegors and Lucker (1 971) showed that 3-4 year old cattle were as susceptible to infection with Taeniu saginutu as 9 month old animals, while the 7 year old ones appeared much more resistant. One of the most striking and still obscure phenomena involving hormonelike substances and helminths is that of the effect of Spirometru mansonoides spargana on the vertebrate host. Recent studies by Harlow et a/. (1967) and Mueller (1968) demonstrated the insulin-like activity of extracts from S. mansonoides and the growth-stimulating effect of spargana implanted into thyroidectomized or hypophysectomized rats. Comparison of the effect of different strains of Spirometra (see Mueller, 1970a) showed that the effect of S. mansonoides was much greater than that of three other strains tested. Mueller (1970b) also showed the relation between numbers of implanted spargana and the growth response in hypophysectomized rats. Steelman et ul. (1970) confirmed the growth hormone-like action of spargana by comparing growth stimulating activity of growth hormone with that of plasma from rats implanted with spargana. Other properties and metabolic actions of spargana were investigated by Steelman et ul. (1971). The number of implanted spargana in relation to host weight gain was further investigated by Mueller (1972~)who found an increasing host response up to 40 worms per rat. No weight gain was noted beyond that number. Of interest is the technique of tracing parasitic infections by an analysis of host responses. As shown by Bundesen and Janssens (1971), the path of Tueniu pisgormes through the liver coincided with enhanced activity of glutamate pyruvate transaminase and glutamate dehydrogenase in the rabbit host. The authors also showed that it was possible to assess parasite burdens by determining plasma enzyme activity levels. One of the largely unsolved problems concerns the reasons for site specificity or predilection of tapeworm larvae within the vertebrate host. Kearney (1970) stated that cysticerci of Taeniu saginutu tend to settle in the deeper muscles of the body, which have a rich supply of myoglobin and a higher respiratory activity. This may be the reason why most larvae of Tueniu ovis are found in muscle (Geminell, 1970). $lais (1970) showed that T. soliurn cysticerci were actually located in the lymph spaces which should provide a rich nutritive

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environment for the parasites. In this connection, the report by Jolly and Pallis (1971) on muscular pseudohypertrophy in human cysticercosis raises questions on the mechanism underlying the symmetrical enlargement of muscles and the increase of muscle bulk in some infected persons. While all cestode larvae probably have “preferred” sites for growth, environmental tolerance ranges differ depending on the cestode species. Recent observations on Hymenolepis nana have demonstrated that cysticercoids may develop in various extra-intestinal sites, that eggs injected subcutaneously into mice will hatch and the oncospheres develop into cysticercoids 4 to 5 days later (DiConza, 1968). Thus, site tolerance within a host species may be much broader than expected. Since we know almost nothing about the attributes of different environments such as lungs, peritoneal cavity, liver, etc., it is understandable that the precise basis for “preferred” locations is unknown. Similarly, lack of broad host tolerance as exemplified by Taenia crassiceps which does not develop in rabbits, hooded rats or guinea-pig (Lubinsky and Baron, 1970) cannot yet be explained since both the essential environmental requirements of the parasite as well as the differences in environments within various hosts remain largely unknown. C. VERTEBRATE HOSTS: IMMUNITY

Different aspects of immunity to infection with cestodes were reviewed by Weinmann (1970). For this reason, and because there is relatively little additional information, this subject will be treated very briefly. A report by Kowalski and Thorson (I 972) presents information on protective immunity achieved against tetrathyridia of Mesocestoides by passive transfer of “immune” serum to mice. The extent of protection achieved by the development of antibodies against Mesocestoides is by no means defined. It is puzzling that multiplication of tetrathyridia in mice generally continues for the lifetime of the host, indicating that protective antibody, although present, is not of great consequence in arresting the infection. Obviously, additional work is needed to clarify this situation. DiConza (1969) achieved protective immunity in mice by transfer of serum from animals infected with Hymenolepis nana. The anti-parasitic activity of the serum was found to be associated with the IgG (7s)immunoglobulin fraction. Mouse tissue responses to subcutaneously infected H. nana were also studied by DiConza (I 970) and compared in normal and immune mice, while the effect of various types of stress upon acquired immunity to H. nana was studied by Weinmann and Rothman (1967). These authors found that severe nutritional stress made immune mice susceptible to re-infection. Weinmann (1968) also studied the effect of splenectomy and thymectomy on acquired immunity to H. nana and found little difference between experimental mice and controls. Assays for histamine in the intestines of normal rats and rats harboring cysticercoids of H. nana showed that infected rats contained twice as much histamine as uninfected ones (Katiyar and Sen, 1970). Considerable work has been done with immunity to larval taeniids, primarily for the purpose of laboratory diagnosis of infections and vaccination of domes-

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tic animals. Only a few papers relevant to these subjects will be mentioned. A study by Morris et al. (1968) showed that the great complexity of cysticercus antigens used in serological tests was the reason for relative lack of specificity. Lamina and Hein (1970) reported on their results with complement fixation tests on calves infected with Taenia saginata, and rabbits infected with T. pisiformis. They demonstrated that this test could be used for a period of 2-3 months only and was, therefore, not useful as a diagnostic procedure for the detection of cysticercosis in cattle. Blundell et al. (1967) injected activated Taenia oncospheres into sheep, and showed production of antibody which provided some protection against challenge infections upon transfer of this serum to non-immune animals. Gemmell et al. (1968) reported on the optimal time intervals between artificial immunization and “challenge” infection of sheep with Taenia hydatigena, and Gemmell (1 970) gave additional results with hyperimmunized animals. Wikerhauser et al. (1970) discussed immunization of calves against bovine cysticercosis by injection of activated oncospheres and reported considerable protection against challenge infection of these animals. The effect of immune serum upon larvae of Taenia taeniaeformis was studied by Murrell(l970) who found that such serum altered the in vitro absorption of glucose of the larva. Analysis of Taenia saginata, Cysticercus tenuicollis and C. longicollis showed that a number of antigens are shared by the three species (Enyenihi, 1970). Purification of the antigens resulted in fewer cross reactions with other helminths. Study of two different strains of Taenia crassiceps by Fox et al. (1971) showed that the KBS strain has two unique antigens, thus differing from the ORF strain which had mutated and showed altered growth patterns (see section on life cycles). This represents an interesting method for the demonstration of genetic alterations in organisms kept for long periods in the laboratory.

V. METABOLISM Studies on various aspects of metabolism reflect the “domestication” or easy availability of certain cestode species or groups of species for scientific work. Among the pseudophyllids, Diphyllobothrium dendriticum was chosen by Reuter (1967a, byc, 1971a, b) for studies on dry/fresh weight ratios of plerocercoids under various environmental conditions, the effect of different gas phases and osmotic concentrations on metabolism, and the influence of different temperatures on rates of lactic acid and succinic acid production. Reuter found that the lack of carbon dioxide in the gas phase greatly reduced the production of succinic acid and the utilization of glycogen. However, in the presence of carbon dioxide with or without oxygen there was no difference in succinic acid production. He also found that differences in ionic concentration of the incubation media did not affect the rate of glycogen utilization but that hypertonic solutions impeded the rate of oxygen consumption and succinic acid production, and stimulated the production of lactic acid. Reuter also discussed the possibility of a biochemical lesion produced by the hyper-

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tonic medium. Further studies by Reuter (1971 a, b) were conducted on plerocercoids maintained in a nutrient medium rather than Hanks’ solution. Aerobic “cultivation” of plerocercoids at 20°C or 29°C resulted in no loss of glycogen content, while maintenance under anaerobic conditions resulted in glycogen loss. Reuter interpreted these results as being related to a stimulating effect of oxygen upon glycogen synthesis. As might be expected, the rate of glycogen depletion changed in response to environmental temperature. Temperature coefficients of the different processes tested were normal between 20°C and 38”C, but were greatly elevated between 38°C and 41°C. The rise in lactic acid production at high temperatures is interpreted as a sign of damage. As to temperature tolerance, Mueller (1972b) reports on the remarkable tolerance of Mesocestoides tetrathyridia to cold. Fifty per cent of the larvae survived 3-5°C for 4 months in a tube with culture medium which was not changed during this period. Some of the larvae survived for 74 months. Comparative observations on Spirometra mansonoides spargana showed that this species was unable to survive such conditions for more than 3 months. Studies on the respiratory metabolism in developing embryos and coracidia of Diphyllobothrium latum and Triaenophorus nodulosus by Michajlow et al. (1971) showed that oxygen consumption changes during development and that absorption is highest during organogenesis. Coracidia of D . latum, however, apparently do not take up oxygen, although they live in an aerobic environment. Respiration of Mesocestoides tetrathyridia was studied by Weinbach and Eckert (1969) who found that respiration was significantly diminished at an 0 2 tension of 2 %. Glycerol had a pronounced stimulatory effect on the respiratory rate while cyanide strongly inhibited respiration. Malonate apparently had no effect whatsoever. Purine and pyrimidine biosynthesis in Mesocestoides was studied by Heath (1970) and by Heath and Hart (1970). When tetrathyridia were maintained in medium containing preformed purines and pyrimidines, de novo synthesis did not occur (Heath, 1970). However, when in a medium lacking these compounds, tetrathyridia incorporated labelled orotic acid into pyrimidines, indicating that pyrimidine biosynethesis is under metabolic control by the end products of the pathway, either by feedback inhibition or gene repression. The aerobic and anaerobic metabolism of Taenia taeniaeformis was studied by von Brand et al. (1968), von Brand and Stites (1970), and Weinbach and von Brand (1970). Glycogen synthesis in vitro occurred both in the presence and in the absence of carbon dioxide. Glucose, galactose and glycerol, singly or in combination, served as substrates for glycogen synthesis (von Brand and Stites, 1970). Extensive studies on the aerobic metabolism of mitchondria (Weinbach and von Brand, 1970) provided information on oxidative capacity in the presence of various substrates and on inhibitors of oxidation. Flavoprotein dehydrogenases, non-heme iron, pyridine nucleotide and a branched cytochrome system are reported to be components of the T.taeniaeformisrespiratory chain. Studies on larvae of Taeniacrassiceps by Murrell(l968) indicated that glucose absorption was a mediated process and that aerobic and anaerobic conditions did not significantly affect total glucose consumption. Studies on amino acid

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absorption of T. crassiceps by Haynes and Taylor (1968) showed that L-valine and L-methionine were absorbed by a process of active transport; Haynes (1 970) demonstrated active transport in the uptake of lysine, tyrosine and phenylalanine. Esch and Kuhn (1971) reported on the uptake of 14C-Chlorella protein by T. crassiceps, and showed differences in the amounts taken up by the ORF and the KBS strains. Authors provide convincing arguments that the protein was taken up by the larvae without prior hydrolysis. Esch (1969) also studied carbohydrate metabolism in T. crassiceps in vitro and reported that insulin was stimulatory under certain environmental conditions. Esch (1968) gave evidence that glucose levels in cyst fluid or glycogen levels in the cyst wall of Taenia multiceps were not altered in hyperglycemic hosts. Comparative studies on DNA, RNA and protein synthesis by Kuhn and Esch (1970) showed a much greater rate of DNA synthesis in the ORF strain of T. crassiceps than in rhe KBS strain. IN VI. GROWTH

VITRO

A review of achievements in growing tapeworm larval stages makes it possible to assign the different studies roughly to three categories: (a) success in (somewhat haphazardly) growing certain organisms by trying a variety of environmental conditions, without determining limiting or essential growth factors; (b) successful determination of single or multiple factors essential for the development of a given stage; and (c) the use of in vitro methods to study activities or characteristics of the organism in a relatively controlled environment. While the ability to grow any tapeworm is commendable as an initial task, failure to elicit some definite information about the over-all biology, or particularly the biochemical versatility and limitations of the organism grown in vitro, reduces the initial achievement to a somewhat meaningless “tour de force”, especially when the media or experimental conditions are so complex that definition of essential factors or substances is virtually impossible. With this in mind, most achievements in the past 5 years are still fairly pedestrian, even though some of the information obtained is useful for further work. Monoxenic cultivation of cysticercoids of Hymenolepis diminuta (Graham and Berntzen, 1970) was successful with rat fibroblast cells in mammalian tissue culture media. The same media without cells did not support growth. The critical factors in this environment are obviously supplied directly or indirectly by the mammalian cells and are so far unknown. Since in vivo development of H. diminuta cysticercoids is limited to the insect body cavity, the relation between the artificial and the usual host environment is obscure. It would seem that many tapeworm larvae, regardless of their host occurrence, are much more tolerant to a variety of environmental conditions (see also section on life histories and host relations) than hitherto supposed. Hymenolepis nana, known to develop to the cysticercoid in either the insect or the mammalian host, was grown axenically to the cysticercoid in a “mammalian” medium (Berntzen, 1970). Details on some procedures were not included in the rCsumC. Partial development of Hymenolepis citelli cysticercoids (Voge, 1972) was

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obtained axenically in a medium designed for the growth of cockroach cells. Results with medium designed for growth of mammalian cells were negative as were attempts with a variety of monoxenic cultures. Aside from many qualitative differences between mammalian and the cockroach tissue culture media, there is a pronounced difference in osmolarity (more than 100 OMSM/l), a seemingly very important and much neglected environmental factor in the growth of larval stages adapted to invertebrate hosts. Medium for H. citelli was supplemented with foetal calf serum (several other sera were unsuitable) and the serum concentration in the medium was critical in that too much was at least as deleterious as too little. It is unfortunate that so little is known about the constituents of different animal sera routinely used in growth media. Until more is known about the composition of animal sera, interpretation of our results will remain at a very primitive level. The importance of using appropriate sera was clearly demonstrated by Heath and Smyth (1970) who showed that taeniid larvae developed only in media containing serum from the natural intermediate host. They also reported that fresh serum from young animals was better than commercially obtained serum. They showed comparative developmental rates in vitro of Taeniapisiformis, T. Iiydatigena, T. serialis, T. ovis and Echinococcus granulosus. Best results with T. pisiformis were obtained if the rabbit serum was used without prior inactivation. It might be important to keep this in mind for future in vitro studies, since routine inactivation at 56°C probably destroys various serum constituents which may be growth-promoting. Studies by Heath and Elsdon-Dew (1972) on the importance of serum in culture media indicated that taeniid oncospheres become coated with serum constituents which might protect the oncosphere from “recognition” by its host. The authors also presented ample experimental evidence of the variation in growth-promoting factors contained in sera from different hosts. Dependence of continued development upon a single additional substance in the culture medium was demonstrated by Voge and Seidel (1968) with Mesocestoides tetrathyridia which attained complete development only in the presence of hemoglobin (or, as shown subsequently, hemin). In this study, the complete larval cycle, beginning with the hatched oncosphere and terminating with asexually multiplying tetrathyridia, was carried in vitro and showed that progression of larval development in this species entails distinct morphological as well as metabolic changes which then result in a change of environmental tolerances, particularly of temperature. The usefulness of in vitro methods in the study of cestode larvae was demonstrated by Hart (1 968) who achieved regeneration of Mesocestoides tetrathyridia and obtained regrowth of a complete organism from fragments containing one or more suckers. It is strange that asexual multiplication does not occur in a medium which does promote regeneration after injury (NCTC 135 with horse serum). One might speculate that an organism under stress has a different biochemical potential, or that the requirements for asexual reproduction are more stringent than those for regeneration. Heath and Hart (1970) also demonstrated that tetrathyridia were able t o synthesize pyrimidines de novo when placed in a synthetic medium deficient

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in all purine and pyrimidine derivatives. However, this ability of de iiovo synthesis is masked when organisms are grown in medium containing pyrimidine derivatives. Thus, the biochemical abilities of organisms cannot be determined accurately from observation of only one growth environment. Because of the “masking effect” and the repression of certain pathways depending on the environment presented to the organism, all statements about “genetic losses” of parasites should be evaluated critically and cautiously. Heath and Hart also showed that two-sucker fragments of tetrathyridia regenerated whole organisms in “deficient” medium (=deficient NCTC 135 without serum) but that similar fragments, when placed in NCTC 135 with purine and pyrimidine precursors did not regenerate unless horse serum was added to the medium. VII. CONCLUSIONS From the many contributions reviewed in the preceding pages several generalizations can be made. First, the structural plan of the cestode surface is comparable in all the different species studied. There are differencesat various stages of larval growth, and between surfaces of pre-adult forms of the same species, but the basic organization is apparently similar in all. Microvilli form early during post-embryonic development and then persist (or reform?) throughout the life of the individual. Regarding host sex susceptibility to infection, it seems that females are more resistant than males and that asexually multiplying species reproduce more rapidly in male than in female hosts. Of interest also is the apparently unequivocal proof of strain differences in Tueniu crussiceps and the usefulness of the methods used for the detection of mutations. In vitro studies on growth and development reaffirm the contention that different species of cestodes may have very different requirements in axenic environments, and also that these requirements may change at different stages of growth of one and the same species. Some of the areas requiring much additional study are the following: (a) lifehistories of lesser known cestode groups, with careful description of pre-adult stages so that this information could form the “backbone” of a more rational systematic grouping; (b) studies on the biochemical behaviour of apparently related species, and comparison of the behaviour of any one species in different in vitro environments; (c) additional data on environmental tolerances of a species at different stages of development; (d) frequencies of mutations in our laboratory strains, and experimental work on the induction of mutations by means other than X-irradiation; (e) the response of invertebrate hosts to infection and the nature of parasite defense mechanisms (if present); (f) the possibility that cestodes release proteolytic enzymes through the body surface to the outside for the partial breakdown and utilization of host proteins, and that this process is more or less continuous in vivo as well as in vitro. One of the most important problems, however, concerns our ignorance of the different internal environments presented to the parasite by the host. We know virtually nothing about the differences of a given environment (peritoneal cavity, for instance) in different host species, or of the difference in various

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areas of the body in the same host individual. We are equally ignorant about the composition of host body fluids (blood, serum) which we use daily in the growth of cells or of whole organisms. Accumulation of data on serum constituents of different animal donors and comparative studies on animal sera would be effort well spent and could make our accomplishments o r our failures more intelligible and meaningful. Hopefully, studies in the near future will provide answers t o some of these questions. REFERENCES Actor, P., Kunkle, D. and Pagano, J. F. (1968). Infection of the mouse with Hymenolepis microstoma ova. 43rd Annual Meeting, Amer. SOC.Parasit., Abstract No. 150. Astafev, B. A. (1970). [Germination of tissue larvae of Hymenolepis nana] Medskaya Parazit. 39, 299-302. Baron, P. J. (1968). On the histology and ultrastructure of Cysticercus longicollis, the cysticercus of Taenia crassiceps Zeder, 1800 (Cestoda: Cyclophyllidea). Parasitology 58, 497-513. Baron, P. J. (1971). On the histology, histochemistry and ultrastructure of the cysticercoid of Raillietina cesticillus (Molin, 1858) Fuhrrnann, 1920 (Cestoda: Cyclophyllidea). Parasitology 62, 233-245. Beavers, P. E., Esch, G. W. and Kuhn, R. E. (1971). Some effects of sublethal Xirradiation on reproduction and development in two strains of larval Taenia crassiceps. Int. J. Parasit. 1, 235-239. Berg, E. and Beck, R. D. (1968). Possible role of a sex factor in rabbit hosts naturally infected with Taeniupisiformiscysticerci.J. Parasit. 54,1252-1253. Berntzen, A. K. (1970). Continuous axenic culture of the successive stages of the life cycle of Hymenolepis nana. 2nd Intl Congress Parasit. Washington, D.C., RBsumB No. 714, J. Parasit. 56, Sect. 11, Part 2. Bilqees, F. M. (1968a). Observations on budding gradient in Taenia crassiceps(Zeder, 1800) Rud, 1810. Riv. Parusit. 29,261-264. Bilqees, F. M. (1968b). Somatic cell division in Taenia crassiceps (Zeder, 1800) (Cestoda). Zool. Anz. 181,450-454. Bilqees, F. M. (1969). Early development of Taenia crassiceps in the intermediate host with a note on primitive cell types. Aust. J . 2001.17,487493. Bilqees, F. M. (1970). Histological study of external budding in Taenia crassiceps. Aust. J. 2001.18, 1-7. Bilqees, F. M. and Freeman, R. S. (1969). Histogenesis of the rostellum of Taenia crassiceps (Zeder, 1800) (Cestoda), with special reference to hook development. Can. J. 2001. 47,251-261. Blundell, S. K., Gemmell, M. A. and Macnamara, F. M. (1968). Immunological responses of the mammalian host against tapeworm infections. VI. Demonstration of humoral immunity in sheep induced by the activated embryos of Taenia hydatigena and T. ovis. Expl. Parasit. 23, 79-82. Bogitsh, B. J. (1967). Histochemicallocalization of some enzymes in cysticercoids of two species of Hymenolepis. Expl Pnrusif. 21,373-379. Bogitsh, B. J. (1969). Fine structural localization of acid phosphatase and aryl sulfatase activities in the intermediate layer of Hymenolepis diminuta cysticercoids. Trans. Am. microsc. SOC.88,411-419. Bonsdorff, C. H. von, Forssten, T., Gustafsson, M. K. S. and Wikgren, B. J. (1971). Cellularcomposition of plerocercoids of Diphyllobotiiriurncfendriticum(Ces toda). Acta zool. fenn. 132, 1-25.

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Brand, T. Von, Churchwell, F. and Eckert, J. (1968). Aerobic and anaerobic metabolism of larval and adult Taenia taeniaeformis: V. Glycogen synthesis, metabolic endproducts, and carbon balances of glucose and glycerol utilization. Expl Parasit. 23, 309-318. Brand, T. Von and Stites, E. (1970). Aerobic and anaerobic metabolism of larval and adult Taenia taeniaeformis: VI. Glycogen synthesis from single substrates and substrate mixtures. Expl Parasit. 27, 444-453. Brbten, T. (1968a). An electron microscope study of the tegument and associated structures of the procercoid of Diphyllobothrium latum (L.). 2. ParasitKde 30, 95-103. BrZiten, T. (1968b). The fine structure of the tegument of Diphyllobothrium latum (L.). A comparison of the plerocercoid and adult stages. 2. ParasitKde 30, 104-112. Bundesen, P. G . and Janssens, P. A. (1971). Biochemical tracing of parasitic infections. 11. Taenia pisiformis in rabbits-a quantitative study. Int. J. Parasit. 1, 15-20. Cavier, R. and Lkger, N. (1965). A propos de I’kvolution d’Hymenolepis nana var. ,fraterna chez des hbtes intermediaires inhabituels. Annls Parasit. hum. comp. 40, 651-658. Charles, G. H. (1970). The ultrastructure of the developing pseudophyllid tegument (epidermis) with reference to the larval stages of Schistocephalus solidus and Ligula intestinalis. 2nd Intl Congress Parasit. Washington, D.C., RCsumC No. 929, J. Parasit. 57, Sect. 11, Part I. Cheng, T. C. and Rifkin, E. (1968). The occurrence and resorption of Tylocephulum metacestodes in the clam Tapessemidecussata. J Invert. Path. 10,65-69. Collin, W. K. (1969). Morphology of post-embryonic stages of the tapeworm Hymenolepis cifelli. 44th Annual Meeting, Amer. SOC.Parasit. Abstract No. 134. Collin, W. K. (1970). Electron microscopy of postembryonic stages of the tapeworm Hymenolepis citelli. J. Parasit. 56, 1159-1 170. Czaplinski, B. and Jarecka, L. (1967). Morphologie et cycle Cvolutif de Tschertkovilepis krabbei (Kowalewski, 1895) comb. n.-Syn. Drepanidotaenia przewalskii (Skrjabin, 1914), nec Taenia tenuirostris Rud., 1819(Cestoda, Hymenolepididae). Acfaparasit. pol. 15, 289-304. Czaplinski, B. and Wilanowicz, H. (1969). Anatomy and development in the intermediate host of Monosaccanthes streperae sp. n. (Cestoda, Hymenolepididae) from the ceca of Anas strepera L. Actaparasit. pol. 17,103-108. DiConza, J. J. (1968). Hatching requirements of dwarf tapeworm eggs (Hymenolepis nana) in relation to extraintestinal development of larval stages in mice. 2.ParnsitKde 31, 276-281. DiConza, J. J. (1970). Hymenolepis nana: mouse subcutaneous tissue responses to larval stages. Expl Parasit. 28, 482-492. Dorais, F. J. and Esch, G . W. (1969). Growth rate of two Taenia crassiceps strains. Expl Parasit. 25,395-398. Dunkley, L. C. and Mettrick, D. F. (1971). Factors affecting the susceptibility of the beetle ’Ikibofiumcottjiusum to infection by Hymenolepis diminuta. JI N . Y .ent. SOC. 79, 133-138. Dvorak, J. A. (1969a). Hydatigera taeniaeformis: Strobilocerci hooks. TI. Solubility and structural homogeneity. Expl Parasit. 26, 122-127. Dvorak, J. A. (1969b). Hydatigera taeniaeforrnis.Strobilocerci hooks. III. Molecular weight and N-terminal amino acid determinations. Expl Purnsit. 26. 128-1 33. Eckert, J., Brand,T.VonandVoge, M. (1 969). Asexual multiplicationof Mesorestoides corti(Cestoda) in the intestine of dogs and skunks. J. Parasit. 55,241-249.

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Eckert, J . (1971)). Biologie uiid Pathologie der hlcsuccJtoides Infektion von Hund und Maus. 2. ParasitKdc 34, 26-27. Enyenihi, U. K. (1970). Analysis, purification, and aerologic evaluation of antigens for diagnosis of bovine cysticercosis and human taeniasis. 2nd Intl Congress Parasit. Washington, D.C., RBsumk No. 165: J. Parasit. 56, Sect. 11, Part 4. Esch, G. W. (1968). Studies on carbohydrates in tissues and coenurus fluid of larval Taenia multiceps from normal and alloxanized mice. Parasitology 58,619-623. Esch, G. W. (1969). Taenia crassiceps: Insulin and carbohydrate metabolism in larval forms. Expl Parasit. 25,210-216. Esch, G. W. and Kuhn, R. E. (1971). The uptake of W-Chlorella protein by larval Taenia crassiceps (Cestoda). Parasitology 62, 21-29. Featherston, D. W. (1971). Taenia hydatigena. 11. Evagination of cysticerci and establishment in dogs. Expl Parasit. 29, 242-249. Filippov, V. V. and Kosminkov, N. E. (1970). D a t a on occurrence of infection in different groups of skeletal muscles of cattle with C. bouis.] Medskaya Parazit. 39, 306-310. Fischer, H. (1 968). The life cycle of Proteocephalusfluviatilis Bangham (Cestoda) from small mouth bass, Micropterm dolomieni LacCede. Can. J. Zool. 46,569-579. Fox, L. L., Kuhn, R. E. and Esch, G. W. (1971). Taenia crassiceps: Antigenic comparison of two larval strains. Expl Parasit. 29, 194-196. Freeman, R. S. (1970). Terminology of cestode development. 2nd Intl Congress Parasit. Washing, D.C., RCsumC No. 192;J. Parasit. 56, Sect. 11, Part 1. Friedl, F. E. and Simon, J. L. (1970). A tetraphyllidean tapeworm larva from the marine snail Fasciolaria tulipa in Florida. J. Parasit. 56,400-401, Furukawa, T. (1970) German cockroaches (Blatella germanica) as an intermediate host of Hymenolepis nana. Jap. J. Parasit. 19,482-486. Gemmell, M. A. (1970a). Hydatidosis and cysticercosis.2. Distribution of Cysticercus ovis in sheep. Aust. vet. J. 46,22-24. Gemmell, M. A. (1970b). Hydatidosis and cysticercosis. 3. Induced resistance to the larval phase. Aust. Vet. J. 46, 366-369. Gemmell, M. A., Blundell, S. K. and Macnamara, F. N. (1968). Immunological responses of mammalian host against tapeworm infections. VII. The effect of the time interval between artificial immunication and the ingestion of egg on the development of immunity by sheep to Taenia hydatigena. Expl Parasit. 23, 83-87. Goodchild, C. G. and Stullken, R. E. (1970). Hymenolepis microstoma: cysticercoid morphogenesis. Trans. Am. microsc. SOC.89,224-229. Graham, J. J. and Berntzen, A. K. (1970). The monoxenic cultivation of Hymenolepis diminuta cysticercoids with rat fibroblasts. J. Parasit. 56, 1184-1 188. Halvorsen, 0.(1970). Studies on the helminth fauna of Norway. XV. On the taxonomy and biology of plerocercoids of Diphyllobothrium Cobbold, 1858 (Cestoda, Pseudophyllidea) from North-Western Europe. Nytt. Mag. Zool. 18, 113174. Harlow, D. R., Mertz, W. and Mueller, J. F. (1967). Insulin-like activity from the sparganum of Spirometra mansonoides. J. Parasit. 53, 449-454. Hart, J. L. (1968). Regeneration of tetrathyridia of Mesocestoides (Cestoda: Cyclophyllidea) in vivo and in vitro. J. Parasit. 56, 950-956. Haynes, W. D. G. (1970). Taenia crassiceps: uptake of basic and aromatic amino acids and imino acids by larvae. Expl Parasit. 27,256-264. Haynes, W. D. G. and Taylor, A. E. R. (1968). Studies on the absorption of amino acids by larval tapeworms (Cyclophyllidea: Taenia crassiceps). Parasitology 58, 47-59.

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Heath, D. D. (1971). ’I’he niigralion of oncospheres of 7henicl pisijormis. 7 . scricllis and Echinococcus granulosiis within the intermediate host. Znt. J . Parasit. 1, 145-152. Heath, D. D. and Elsdon-Dew, R. (1972). The in vitro culture of Taenia saginata and Taenia taeniaeformis larvae from the oncosphere, with observations on the role of serum for in vitro culture of larval cestodes. Znr. J. Parasit. 2, 119-130. Heath, R. L. (1970). Biosynthesisde novo of purines and pyrimidines inMesocestoides (Cestoda). 1. J. Parasit. 56,98-102. Heath, R. L. and Hart, J. L. (1970). Biosynthesis de novo of purines and pyrimidines in Mesocestoides (Cestoda). 11. J. Parasit. 56,340-345. Heath, D. D. and Smyth, J. D. (1970). In vitro cultivation of Echinococcirs granulosus, Taenia hydatigena, T. ovis, T.pisifbrmis and T. seriafis from oncosphere to cystic larva. Parasitology 61, 329-343. Heyneman, D. and Voge, M. (1969). Host response of the flour beetle Tribolium confusum, to single and challenge infection with Hymenolepis dimincita and H . microstoma. 44th Annual Meeting, Amer. SOC.Parasit., Abstract No. 93. Heyneman, D. and Voge, M. (1971). Host response of the flour beetle Tribolilrm confusum to infections with Hymenolepis diminuta, H . microstoma and H . citelli (Cestoda: Hymenolepididae).J. Parasit. 57, 881-886. Hunkeler, P. (1969). La larve d’Hymenolepis nagatyi Hilmy, 1936 (Cestoda, Cyclophyllidea). Z. ParasitKde 32, 176-180. Jarecka, L. (1970a). Zagadnienia ewolucji i filogenezy Cestoda w swietle ich rozwoju ontogenetycznego. Kosmos Az. 1 (102), pp. 1-27. Jarecka, L. (1970b). Life cycle of Valipora campylancristrota (Wedl, 1855) Baer and Bona, 1958-1960 (Cestoda-Dilepididae) and the description of cercoscolexa new type of cestode larva. Bull. Acad. pol. Sci. Cl. ZZSPr. Sci. biol. 18,99-102. Jarecka, L. (1970~).On the life cycles of Paradilepis scolecina (Rud, 181 9) Hsu, 1935, and Neogryporhynchus cheilancristrotus (Wedl, 1855) Baer and Bona, 19581960(Cestoda-Dilepididae). Bull. Acad.po1. Sci. Cl. ZZSir. Sci. biol. 18,159-163. Jolly, S . S. and Pallis, C. (1971). Muscular pseudohypertrophy due to cysticercosis. J. neurol. Sci. 12, 155-162. Jourdane, J. (1972). &ude experimentale du cycle biologique de deux espkces de Choanotueniaintestinaux des Soricidae.Z. ParasitKde 38, 333-343. Katiyar, J. C. and Sen, A. B. (1970). Occurrence of histamine in the intestines of rats harbouring cysticercoids of Hymenolepis nana. Indian J. exp. Biol. 9, 191-193. Katkansky, S. C., Warner, R. W. and Poole, R. L. (1969). On the occurrence of larval cestodes in the Washington clam, Saxidomus nuttalli and the gaper clam, Tresus nuttalli from Drakes Estero, California. California Fish and Game 55, 317-322. Kearney, A. (1970). Cysticercus bouis-some factors which niay influence cyst distribution. 2nd Intl Congress Parasit. Washington, D.C., RtsumC No. 332; J. Parasit. 56, Sect. 11, Part 1. Kowalski, J. C. and Thorson, R. E. (1972). Protective immunity against tetrathyridia of Mescocestoides corti by passive transfer of serum in mice. J. Parasit. 58, 244246. Kozicka, J. (1971). Cestode larvae of the family Dilepididae Fuhrmann, 1907 parasitizing fresh-water fish in Poland. Actaparasit. pol. 19, 81-93. Kuhn, R. E. and Esch, G . W. (1970). Comparative studies on protein RNA and DNA synthesis in normal and neoplastic strains of larval Taenia crassiceps (Cestoda). 2nd Intl Congress Parasit. Washington, D.C., RBsum6 No. 353; J. Parasit. 56, Sect. II, Part 1. Kwa, B. H. (1972a). Studies on the sparganum of Spirometra erinacei. I. The histology and histochemistry of the scolex. Znt. J. Parasit. 2,23-28.

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Kwa, B. H. (1972b). Studies on the sparganum of Spirometra erinacei. 11. Proteolytic enzyme(s) in the scolex. Znt. J. Parasit. 2,29-33. Kwa, B. H. (1972~).Studies on the sparganum of Spirometra erinacei. 111. The fine structure of the tegument in the scolex. Znt. J . Parasit. 2,3543. Lamina, J. and Hein, B. (1970). Untersuchungen zur Frage des immunologischen Nachweises einer Zystizerkose am lebenden Tier. 11. Mitteilung: Die Komplementbindungsreaktion. Dt. tierarztl. Wschr. 77,273-278. LCger, N. and Cavier, R. (1970). Reaction de divers insectes A I’ infestation par Hymenolepis nana var. fraterna. 2nd Intl Congress Parasit. Washington, D.C., RCsumC No. 372; J. Parasit. 56, Sect. II, Part 1. Lubinsky, G, and Baron, R. (1970). Taenia crassiceps (Zeder, 1800) in metropolitan Winnipeg, Manitoba. Can. J. 2001.48, 1144-1 145. Mackiewicz, J. S. (1972). Caryophyllidea (Cestoidea): A Review. Parasitology 31, 417-512. Malviya, H. C. and Dutt, S. C . (1970). Morphology and life history of Cotugnia srivasfuuai: n. sp. (Cestoda: Davaineidae) from the domestic pigeon. H. D. Srivastava Commem. Vol., Indian Veterinary Research Inst., Izatnagar, pp. 103-108. Michajlow, W., Guttowa, A. and Grabiec, S. (1971). On the respiratory metabolism in the embryos and coracidia of some Pseudophyllidea(Cestoda). Actaparasit.po1. 18, 275-283. Misiura, M. (1971). Morphological variation in Sobolevicanthus gracilis (Zeder, 1803) (Cestoda, Hymenolepididae). I. Variability of length of the rostellar hooks and its cause. Actaparasit. pol. 19,6%80. Morris, N., Proctor, E. M. and Elsdon-Dew, R. (1968). A physicochemical approach to the serological diagnosis of cysticercosis. J. S.Afr. vet. med. Ass. 39,41-43,44. Mount, P. M. (1970). Histogenesis of the rostellar hooks of Taenia crassiceps (Zeder, 1800) (Cestoda). J. Parasit. 56, 947-961. Mueller, J. F. (1968). Growth stimulating effect of experimental sparganosis in thyroidectomized and hypophysectomized rats, and comparative activity of different species of Spirometra. J. Parasit. 54,795-801. Mueller, J. F. (1970a). Comparison of the growth-promoting effect of Spirometra mansonoides vs. three oriental forms in intact mice and hypophysectomized rats. J. Parusit. 56, 842-844. Mueller, J. F. (1970b). Quantitative relationship between stimulus and response in the growth-promotifig effect of Spirometra mansonoides spargana on the hypophysectomized rat. J. Parasit. 56,840-842. Mueller, J. F. (1972a). Occurrence of growth zones in the “tail” region of spirometrid plerocercoids. J . Parasit. 58, 407409. Mueller, J. F. (1972b). Survival and longevity of Mesocestoides tetrathyridia under adverse conditions. J. Parasit. 58, 228. Mueller, J. F. (1972~).Further studies on sparganum growth factor in the hypophysectomized rat: response to large numbers of spargana. J. Parasit. 58,438-443. Murrell, K. D. (1968). Respiration studies and glucose absorption kinetics of Taenia crassiceps larvae. J. Parasit.. 54, 1147-1 150. Murrell, K. D. (1970). Some physiologic effects of antibodies on Taenia taeniaeformis lawae. 2nd jlntl Congress Parasit. Washington, D.C., R6sum6 No. 818; J. Parasit. 56, Sect. II, Part 2. McLaughlin, J. D. and Burt, M. D. B. (1970). Observations on the morphology and life cycle of Hymenolepi,s hopkinsi Schiller 1951 (Ccstoda: Cyclophyllidea) a parasite of black ducks (AM/.(.ruhripes Brewster). Carl. .I. Z d .48, 1043-1046.

728

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Nieland, M.L. and Weinbach, E. C. (1968). The bladder of Cysticercus fasciolaris: Electron microscopy and carbohydrate content. Parasitology 58,489496. Novak, M. (1972). Quantitative studies on the growth and multiplication of tetrathyridia of Mesocestoides corti Hoeppli, 1925 (Cestoda: Cyclophyllidea) in rodents. Can. J. 2001. 50, 1189-1196. Novak, M. and Lubinsky, G . (1973). Acceleration of the growth of populations and of the multiplication of tetrathyridia of Mesocestoides corti Hoeppli, 1925 (Cestoda: Cyclophyllidea) by some cytostatic agents. Can. J. 2001. 51, 83-90. Opuni, E. K. (1970). Asexual multiplication in Cysticercus pisiformis (Cestoda). J. Helminth. 44, 321-322. Pennycuick, L. (1971). Differences in the parasite infections in three-spined sticklebacks (Gasterosteus aculeatus L.) of different sex, age and size. Parasitology 63, 407-418. Pike, A. W. (1968). Notes on some cysticercoids from pulmonate molluscs and leeches in British fresh waters. J. Helminth. 42, 131-138. ProkopiE, J. (1968). A description of the cysticercoid of the cestode Vigisolepis spinulosa (Cholodkowsky, 1906) found in Collembola. Folia parasit., Praha 15, 266. Quentin, J. C., Jourdane, J., Rioux, J. A., LCger, N., Houin, R. andCroset, H. (1971). PrCsence du cysticercoide &Hymenolepis brusatae Vaucher, 1971 chez Phlebotomus perniciosus Newstead, 191 1 et Phlebotomus mascittii Grassi, 1908. Annls Parasit. hum. comp. 46, 589-594. Reuter, J. (1967a). Studies on plerocercoids of Diphyllobothrirrm dendriticum. I. The dry weight/fresh weight ratio of the tissues under various physiological conditions. Acta Acad. Aboensis Ser. B 27, 1-15. Reuter, J. (1967b). Studies on plerocercoids of Diphyllobothrium dendriticum. 11. The dependence of lactic and succinic acid excretion on the gas phase. Acta Acad. Aboensis, Ser. B 27, 1-7. Reuter, J. (1967~).Studies on plerocercoids of Diphyllobothrium dendriticum. 111. The oxygen uptake and the excretion of lactic and succinic acid in media with different osmotic concentrations. Acta Acad. Aboensis, Ser. B 27, 1-10. Reuter, J. (1971 a). Studies on plerocercoids of Diphyllobothrium dendriticum. IV. The influence of oxygen and of a tissue homogenate on the glycogen contents and weight changes of the larvae. Acta Acad. Aboensis, Ser. B 31, 1-6. Reuter, J. (1971b). Studies on plerocercoids of Diphyllobothrium dendriticum. V. Rates of acid production, change in weight and depletion of glycogen during in vitro cultivation at different temperatures. Acta Acad. Aboensis, Ser. B 3 1 , 1 4 . Rifkin, E. and Cheng, T. C. (1968). The origin, structure, and histochemical characterization of encapsulating cysts in the oyster Crassotrea virginica parasitized by the cestode Tylocephalumsp. J. invert. Path. 1 0 , 5 4 6 4 . Rifkin, E., Cheng, T. C. and Hohl, H. R. (1969). An electron-microscope study of the constituents of encapsulating cysts in the American oyster, Crassotrea virginica, formed in response to Tylocephahm inetacestodes. J . invert. Path. 14, 21 1-226. 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. Mofph. 130,ll-24. Silverman, P. H. and Hansen, E. L. (1971). In iitro cultivation procedures for parasitic helminths: Recent advances. In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 9, pp. 227-258. Academic Press, London and New York. Slais, J. (1970a). The electron microscopical characteristics of the bladder wall of the cysticercus. 2nd Intl Congress Parasit. Washington, D.C., Resume No. 590; J . Pnrasit. 56, Sect. TT, Part 1.

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Slais, J. (1970b). “The Morphology and Pathogenicity of the Bladdcr Worm”. Dr W. Junk N.V. Publishers, The Hague, Netherlands. Smith, J. K., Esch, G. W. and Kuhn, R. E. (1972). Growth and development of larval Taenia crassiceps (Cestoda). I. Aneuploidy in the anomalous ORF strain. Int. J. Parasit. 2, 261-263. Smyth, J. D. and Heath, D. D. (1970). Pathogenesis of larval cestodes in mammals. Helminth. Abstracts Series A 39, 1-24. Solonenko, I. G. (1969). Occurrence of Hymenolepis nana cysticercoids in the mesenteric lymph nodes of white rats. Parazitologiya 3,74-75. Solonenko, I. G. (1970). Migration and development of Hymenolepis nuna larvae in mesenteric lymph nodes of rats. Parazitologiya 4 476479. Soltice, G. W., Arai, H. P. and Scheinberg, E. (1971). Host-parasite interactions of Tribolium confusurn and T. castaneum with Hymenolepis diminuta. Can. J. Zool. 49, 265-275. Specht, D. and Widmer, E. A. (1972). Response of mouse liver to infection with tetrathyridia of Mesocestoides (Cestoda). J. Parasit. 58,431437. Steelman, S. L., Morgan, E. R., Cuccaro, A. J. and Glitzer, M. S. (1970). Growth hormone-like activity in hypophysectomized rats implanted with Spirometra mansonoides spargana. Proc. SOC.exp. Biol. Med. 133,269-273. Steelman, S. L., Glitzer, M. S., Ostlind, D. A. and Mueller, J. F. (1971). Biological properties of the growth hormone-like factor from the plerocercoid of Spirometra mansonoides. Recent Progress in Hormone Research 27,97-120, Tan, B. D. and Jones, A. W. (1969). Hymenolepis microstoma: retardation of growth and development of larval and pupal stages of Tribolium confusum. Expl Parasit. 26, 393-397. Taylor, A. E. R. and Baker, J. R. (1968). “The Cultivation of Parasites in vitro”. Blackwell Scientific Publications, Oxford and Edinburgh. Ubelaker, J. E., Cooper, N. D. and Allison, V. F. (1970). The fine structure of the cysticercoid of Hymenolepis diminuta. I. The outer wall of the capsule. Z . ParasitKde 34, 258-270. Vegors, H. H. and Lucker, J. T. (1971). Age and susceptibility of cattle to initial infection with Cysticercus bovis. Proc. helminth. SOC.Wash. 38, 122-127. Voge, M. (1967).The post-embryonic developmental stages of cestodes. In “Advances in Parasitology” (Ed. Ben Dawes), Vol. 5, pp. 247-297. Academic Press, London and New York. Voge, M. (1969). Systematics of cestodes-present and future. Zn “Problems in the Systematics of Parasites” (Ed. G . D. Schmidt), pp. 49-73. University Park Press, Baltimore. Voge, M. (1972). Axenic development of post-embryonic stages of Hymenolepis citelli (Cestoda). 47th Annual Meeting, Amer. SOC.Parasit., Abstract No. 203. Voge, M. and Seidel, J. S. (1968). Continuous growth in vitro of Mesocestoides (Cestoda) from oncosphere to fully developed tetrathyridium. J. Parasit. 54, 269-271. Warner, R. W. and Katkansky, S. C. (1969). Infestation of the clam, Protothncu staminea by two species of tetraphyllidean cestodes (Echeneibothriumspp.). J. Invert. Path. 13, 129-133. Weinbach,E. C. and Brand, T.Von (1970). The biochemistry of cestode mitochondria. I. Aerobic metabolism of mitchondria from Taenia taeniaeformis. Znt. J. Biochem. 1, 39-56. Weinbach, E. C. and Eckert, J. (1969). Respiration of the larvae (Tetrathyridia) of Mcsocesto ides cor t i. Expl Pww‘t. 24, 54-62.

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Weinmann, C. J. (1968). Effects of splenectomy and neonatal thymectomy on acquired immunity to the dwarf tapeworm, Hymenolepis nana. Expl Parasit. 22,68-72. Weinmann, C. J. (1970). Cestodes and acanthocephala. In “Immunity to Parasitic Animals” (Eds G . L. Jackson et al.), Vol. 2, pp. 1021-1059. Appleton-CenturyCrofts, New York. Weinmann, C .J. and Rothman, A. H. (1967). Effectsof stress upon acquired immunity to the dwarf tapeworm, Hymenolepis nana. Expl Parusit. 21,61-67. Wikerhauser, T., hkoviC, M. and Dzakula, N. (1970). Vaccination against bovine cysticercosis. 2nd Intl Congress Parasit., Washington, D.C., R h m B No. 678 ; J . Parusit. 56, Sect. II, Part 1 . Wikgren, B. P. and Gustafsson, M. K. S. (1971). Cell proliferation and histogenesis in diphyllobothrid tapeworms (Cestoda). Acta Acud. Aboensis, Ser. B 31, 1-10. Wikgren, B. P. and Knuts, G. M. (1970). Growth of subtegumental tissue in cestodes by cell migration. Actu Acud. Aboensis, Ser. B 30, 1-6. Wikgren, B. P., Gustafsson, M. K. S. and Knuts, G. M. (1971). Primary anlage formation in diphyllobothrid tapeworms. 2.PurasitKde 3b, 131-1 39. Wisniewski, R. J. (1970). Experimental development of Hamutolepis tevesoides (Fuhrmann, 1906) Spassky, 1962 (Cestoda, Hymenolepididae) in intermediate hosts. Actu purasit. pol. 18, 315-322. Wisniewski, R. J. (1971). Studies on the development of Nematopurutaenia southwelli Fuhrmann, 1934 and Gastrotueniuparacygni Czaplinski et Ryzikov, 1966 (Cestoda, Hymenolepididae) in the intermediate hosts. Actupurusit.poZ. 19,49-61. Yamane, Y . (1968). On the fine structure of Diphyllobofhvium erinucei with special reference to the tegument. Yonago Acta Medicu 12,169-181.

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

A Abdou, A. H., 502, 516, 521,548 Abede, A. van den, 25,61 Abuladze, K. I., 415, 428,466, 481, 486, 490, 504, 507, 508,548 Acanfora, G., 5, 9,47, 58 Actor, I?., 709, 723 Adam, J. P., 608, 610, 629 Adams, T. S., 131, 137,188 Adelson, E., 31, 71 Agapova, A. I., 431,433,466,470 Aikawa, M., 621,622,623,624,626,629 Akin, G. C., 376,384 Alfaro, M., 39, 69 Alford, C. A., 645, 659 Alicata, J. E., 517, 529, 551, 562, 564, 596 Aliev, A. A., 512, 588 Aligon, C., 421, 466 Allee, W. C., 361, 362, 384 Allen, A. V. K., 94, I12 Allen, R. W., 522, 528,548 AIlison,V. F., 447,448,467,478,713,729 Almeida, F. B., 614, 626 Alsop, N., 143, 145, I89 Altman, R. M., 159, I86 Alyarado, S., 5, 33, 37, 46, 54, 55, 66 Alworth, W. L., 213,214,230,437,438, 473 Amaral, V. do, 641, 659 Amato Neto, V., 644, 659 Ambroise-Thomas, P., 605, 626, 651, 652,659 Amin, M. A., 322,390 Anantaraman, S., 411,415,467 Anderson, F. M., 534, 548 Anderson, J. R., 6, 37, 52, 58 Anderson, M. G., 534, 548 Anderson, P. J. S., 573, 586, 588, 595, 598 Andrk, R. G., 609, 627 Andrewartha, H. G., 316, 317, 384 Angulo, J. J., 463, 467

Angus, M. G. W., 617,626 &Antonio, L. E., 618, 627 Anwar, M., 627 Aoki, I., 655,665 Aoki, T., 636, 658, 659, 665 Arai, H. P., 715, 729 Araoz, J., de, 88, 112 Araujo, F. G., 652, 655, 660, 661 Archer, D. M., 492,493, 505, 548 Archer, J. F., 643, 651, 660 Archibald, R. D., 248,297 Arfaa, F., 698, 705 Arme, C., 207, 215, 229, 437, 438, 439, 448, 467, 473 Arnold, J. D., 617, 626 Aronsen, W. J., 648, 664 Artyukh, E. S., 481, 548 Ascher, K. R. S., 140, 183 Ashenbrucker, H., 29, 31, 58 Assel, S., van, 20, 70 Astafev, B. A,, 709, 723 Astaphiev, B. A., 462, 480 Athens, J. W., 29, 31, 58 Athias, M., 2, 5, 11, 22, 45, 47, 62 Atlavinyte, O., 561, 597 Audy, J. R., 321, 389 Augustine, D. L., 318, 385 Avkrinzev, S., 45, 51, 58 Awachie, J. B. E., 677, 678, 679, 701 Ax, P., 196, 198, 217, 228 Axmann, M. C., 268, 271,297 Ayad, N., 312,384 Ayala, S. C., 6, 10, 12, 18, 19, 37,40,45, 52,58 Azzaretti, G., 637, 666 B Babudieri, B., 47, 58 Baer, J. G., 428,467,481,482,485,487, 492,493, 498, 501, 502, 503, 504, 505, 506, 507, 508, 518, 524, 528, 529, 531, 533, 538,539, 540, 544,548,551, 513, 578,592

731

732

AUTHOR lNDEX

Bafort, .I,, 608, 610, 626 Bafort, J. M., 608, 610, 626 Bahr, Ci. F., 604, 623, 626, 629 Bailenger, J., 129, 183 Bailey, H. H., 201, 210, 228 Bailey, J., 96, I13 Bailey, J. K., 6, 37, 58 Baily, J. L., Jr, 360, 384 Baird, C. R., 154, 183 Baker, D. W., 575, 576,593 Baker, J. A. F., 167, 168, 169, 183 Baker, J. R., 9, 10, 39, 58, 70, 614, 623, 625, 626, 628, 707, 729 Baker, K. P., 173, 174, 175, 183 Baker, K. F., 242, 297 Bd, A. K., 240, 295, 301 Baldwin, J., 438, 471 Baldwin, K. F., 188 Balfour, A., 50, 58 Ball, G. H., 618, 626 Ball, 1. R., 351, 373, 384 Bancroft, T. L., 51, 58 Bang, Y. H., 124,191 Baqai, M. U., 646, 661 Barabashova, V. N., 676, 701 Barber, V. C., 220, 228 Barbosa, F. S., 318, 384, 390 Barbosa de Almeida, 614, 626 Bardsley, J. E., 2, 13, 21, 24, 25, 26, 27, 28, 29, 34, 35, 36, 37, 57, 58 Barker, M., 293, 294,301 Barnett, E. V., 652, 660 Barnett, S. F., 167, 183 Baron, P. J., 440,441,446,467,713,714, 723 Baron, R., 717, 727 Barr, A. R., 133, 192 Barr, M., 160, 183 Barratt, M. E. J., 569, 588 Barrett, J., 689, 701, 702 Barrow, G. I., 96, 113 Barrow, J. H., 7, 10, 17, 21, 22, 23, 24, 31, 36, 37, 58 Barrow, L. J., 167,187 Bartels, E., 441, 451, 467 Barton, M., 460, 477 Barua, D., 86, 88, 112, 113 Baruzzi, R. G., 641, 660 Basch, P. F., 321, 389 Bass, C. C., 618, 626 Bauman, P. M., 291,302 Baumhover, A. H., 183

Bay, E. C., 130, 183 Bayne, K. A., 372,384 Beale, A. J., 109, 112 Beaudoin, R. L., 623, 626 Beavers, P. E., 711, 723 Bechelli, G., 641, 660 Becht, H., 568, 588 Beck, A. J., 321,389 Beck, R. D., 716, 723 Becker, R. P., 622, 628 Beesley, W. N., 122, 146, 147, 148, 149, 154, 183 Befus, A. D., 490, 494, 497, 498, 500, 502, 509, 510, 527,548 BCguin, F., 450, 467 Behbehani, G., 190 Beitinger, T. L., 692, 693, 694, 702 Belkin, G. Y., 565, 588 Bellier, L., 608, 628 Belobrad, G., 567, 589 Belton, C. M., 235,237, 244, 247, 297 Belton, J. C., 235, 237, 244, 297 Beneson, A. S., 291, 302 Benenson, A. S., 110, 112 Benex, J., 322, 386, 436, 460, 464, 467 Bennett, G., 214, 228 Bennett, H. S., 238, 242, 297 Ben Rachid, M. S., 657, 660 Benton, J. W., Jr, 645, 659 Berard, C. W., 648, 666 Berecky, I., 584, 598 Berenberg-Gossler, H. V. von, 614 Berengo, A., 641, 660 Beresford-Jones, W. P., 575, 580, 584, 589 Berestnev, N. M., 27, 49, 59 Berg, C. O., 314, 318, 384 Berg, E., 716, 723 Bergendahl, E., 372, 392 Bergendi, L., 640, 660 Berger, A. E., 617, 626 Berger, J., 216, 228, 437, 467 Bernard, J., 433, 462, 467 Berndt, W. L., 139, 186 Berner, R. A., 373, 384 Berntzen, A. K., 412,438,439,463,471, 478, 492, 493, 548, 552, 720, 723, 725 Berrie, A. D., 310, 311, 312, 320, 361, 374,384 Berry, E. G., 245, 304, 312, 394 Best, J. B., 198, 228, 364, 376, 384

AUTHOR I N D E X

Beverley, J. K. A., 643, 645, 646, 651, 660, 662,664, 668 Bibby, M. C., 250, 255, 294, 297 Bier, J. W.,235, 298 Bierman, H. R., 31, 59 Biguet, J., 458, 469 Billet, A., 14, 16, 35, 47, 56, 59 Bilqees, F. M., 494, 514, 519, 548, 714, 723 Bilquees, F. M., 423, 443, 467 Bils, R. F., 206, 216, 228, 234, 235, 237, 245,294,295,297 Birch, L. C., 316, 317, 384 Bird, R. G., 131, 183, 293,294,301, 623, 625, 626,628 Bischoff, R., 210, 229 Bishopp, F. C., 152, 183 Bittner, B. A., 183 Bjorkman, N., 268, 284,297, 304 Blackman, C. G., 147, 190 Blair, D. M., 312, 386 Blankenship, W. J., 645, 659 Blazkovec, A. A., 653, 663 Bloomfield, M. M., 657, 666 Blundell, S. K., 718, 723, 725 Boag, B., 571, 598 Boch, J., 642, 643, 660 Boehringer, I. K., de, 642, 664 Boertje, S. B., 490,493, 522, 548 Boev, S. N., 574, 575, 576,589,596 Bogitsch, B. J., 446, 456, 467 Bogitsh, B. J., 212, 214, 228, 232, 264, 269, 270, 271, 277, 279, 280, 283, 294, 297,301,713, 723 Boll, C. D., 361, 387 Bolla, R. I., 444,468 Bollinger, R. R., 24, 25, 27, 29, 53, 59 Bommer, W., 657, 660 Bondareva, V. I., 424,427,459,460,468 Bonk, G., 14, 39,40,59 Bonk, G. J., 19, 20, 70 Bonner, J. T., 380,385 Bonner, T. P., 217, 229 BonsdorfF, C. H. v, 410, 468, 492, 548, 712, 723 Boray, J. C., 368, 385 Borchert, A., 572, 589 Borkovec, A. B., 132,190 Borodina, V. A., 572, 589 Bortoletti, G., 421, 450, 468 Boschman, T. A., 29,60 Bott, A., 460, 468 26

733

Bouchite, B., 125, 190 Bouet, G., 18, 24, 38, 47, 49, 59 Boulard, V., 629 Boulard, Y., 608, 610, 629 Bourgeois, J. G., 283, 298 Bourtcart, N., 15, 16, 17, 19, 21, 22, 27, 33, 35, 56, 60, 62 Bovet, J., 219, 229 Bowen, R.C., 696, 702 Bowers, E. A., 234, 237, 295, 301 Bowman, M. C., 149,184 Box, E. D., 635, 660 Boycott, A. E., 370,385 Brack, C., 12,59 Bradford, J., 52, 60 Bragdon, J. H., 326, 385 Branch, S. I., 685,687,688,690,691, 702 Brand, T., von, 35, 59, 438, 439, 463, 468, 469, 474, 709, 719, 724, 729 Brandt, B. B., 24, 28, 38, 53, 57,59 Brandt, P. W., 242, 297 BrAten, T., 408, 409, 439, 468, 712, 724 Braun, M., 499, 509, 516, 520,548 Bray, R. S., 104, 112 Bres, P., 92, 112 Bresciani, J., 194, 196, 207, 208, 210, 214, 217, 220, 224, 229 Breuning, J., 636, 666 Breza, M., 567, 589 Brock, T. D., 373,385 Broden, A., 11, 50,59 Brooker, B., 219, 220, 229 Brooker, B. E., 290,291, 297 Brooks, G. D., 125, 191 Brown, A. W. A., 119, 124, 125, 126, 127, 131, 137, 138, 140, 141, 145, 147, 158, 159, 160, 162, 167, 169,183,187 Brown, B. W., 438,439,471 Brown, I. N., 604, 626 Brown, J. A., 648, 667 Brown, K. N., 604,626 Brown, L. R., 307,385 Brown, P. R. M., 147, 148, 184,192 Brownlee, I., 645, 666 Brownlee, I. E., 656, 661 Brownlie, W. M., 173, 184 Bruce, J. I., 250, 251, 261, 277, 280, 297 Bruce-Chwatt, L. J., 76,83,98, 100,101, 103,112,113, 116,184 Brumpt, E., 5, 10, 15, 17, 27, 32, 33, 34, 35,36,40,45,47,48, 51,54,55,56,59, 60

734

AUTHOR INDEX

Brundrett, H. N., 152, 183 Bry, R. E., 149, 184 Bryan, J. H., 134, 184 Bryant, C., 695, 703 Brzheskiy, V. V., 435, 436, 468 Bucci, A., 605, 627 Buch, B., 257, 304 Buchanan, R. D., 621, 627 Buchwald, K. W., 26,29, 60 Buchwalder, R., 560, 568,593,595 Buck, G., 609, 627 Bueding, E., 269, 272, 283, 284, 297, 298, 305, 700, 705 Biihler, F., 646, 660 Bullock, W. L., 697, 702 Bullough, W. S., 320, 378, 385 Bundesen, P. G., 716, 724 Burden, C. S., 159, I84 Burgers, A. C. J., 29, 60 Burges, H. D., 117, 184 Burgess, L., 37, 60 Burikova, Y. N., 593 Burnett, G. F., 144, 145, 184 Burrows, W., 88,112 Burt, M. D. B., 196, 229, 484, 490, 525, 544,554, 710, 727 Burton, P. R., 233, 244, 245, 262, 266, 267,298 Bushland, R. C., 183 Butcher, G. A., 605, 618, 627 Butler, C. G., 320, 385 Butt, K. M., 162, 184 Butterworth, P. E., 217, 229, 677, 678, 680, 681, 682, 689, 697, 701, 702 Buttner, A., 15, 16, 17, 19,21, 22,23, 27, 33, 34, 35, 56, 60, 62 Bychowsky, B. E., 532, 548 Byram, J., 111, 400, 453, 472 Byron, R. L., 31, 59 C Cable, R. M., 291, 300, 677, 678, 691, 696, 702, 703 Cadigan, F. C., 619, 620, 621, 627 Cadigan, F. L., 609, 627 Cahill, Kevin M., 76, 95, 113 Caid, G. D., 689, 702 Cail, R. S.,149, 184 Caillon, L., 40,47, 64 Caldwell, L. A., 647, 663 Calentine, R. L., 412, 468

Call, R. N., 676, 704 Cameron, T. W. M., 460,462,479, 531, 533, 534,549, 578,589 Cammell, M. E., 320, 380, 393 Campbell, J. W., 373, 392 Campbell, L. H., 644, 668 Campbell, R. A., 53, 60 Campbell, W. C., 456,458, 468 Campesi, G . , 455, 468 Cankovic, M., 585, 596 Canning, E., 623, 624, 627 Cannon, D. A., 112,113 Cardarelli, N. F., 314, 385 Cardell, R. R., 208, 232 Carini, A., 14, 15, 27, 45, 54, 60 Carlisle, D. B., 320, 385 Carneiro, E. W. B., 627 Carney, D. M., 321, 390 Carosi, G., 637, 666 Carpenter, C. C. J., Jr, 88, 112 Carson, P. E., 622, 628 Carson, R., 116, 146, 184 Carter, G. S., 28, 29, 60 Carter, R., 608, 627, 630 Cartwright, G. E., 29, 31, 58 Casarosa, L., 565, 589 Cash, R. A., 88, 112 Casida, J. E., 117, 176, 187 Cassady, G., 645, 659 Castagnari, L., 436, 460, 474 Catar, G., 640, 660 Catts, E. P., 156, 184 Caughey, W., 144, 185 Cavallini, F., 641, 660 Cavallini-Sampiere, L., 641, 660 Cavallini-Sampieri,C., 645, 668 Cavier, R., 715, 724, 727 Center for Disease Control, 90, 93, 98, 112, 113 Centurier, H., 639, 644, 664 Cerdas, L., 640, 666 Chabaud, A., 610, 629 Chabaud, A. G., 560,589 Chaicharn, A., 697, 702 Chaicumpa, V., 620, 627 Chalupsky, J., 651, 663 Chamberlain, W. F., 160, 184 Chambers, C. V., 122, 188 Chandar, K., 646, 660 Chao, C. S.,17, 37, 61 Chao, J., 618, 626 Chapman, H. D., 250, 298

AUTHOR INDEX

Chapman, R. F., 366, 385 Charles, G. H., 712, 724 Chatterjee, A. B., 377, 389 Chaussat, J. B., 50, 60 Chaykin, S., 326, 385 Cheng, T. C., 235, 298, 411, 447, 475, 483,486, 507, 524, 549, 711, 714, 715, 724, 728 Cheong, W. H., 619, 630 Chernin, E., 312, 318, 321, 360, 361, 365, 367, 370, 372,385 Chien, S., 618, 622, 629 Chitty, D., 320, 379, 385 Chizhova, T. P., 408, 468 Christensen, H. A., 10, 60 Christenson, W. N., 641, 644, 663 Christian, J. J., 320, 374, 379, 385 Chu, K. Y.,318, 319,386 Chung, H. L., 6, 10, 37, 47, 61 Churchwell, F., 719, 724 Churchwell, F. K., 438, 468 Claflin, J. L., 40, 65 Clark, G. W., 52, 60 Clark, K. M., 198, 207, 208, 216, 217, 229 Clark, L. R., 314, 320, 386 Clarke, V. de V., 312,386 Claus, C., 537,549 Clegg, J. A., 213,215,229,250, 251,254, 255,269,298 Cleland, J. B., 51, 52, 60 Clements, A. N., 485, 549 Cleveland, L. R., 39, 60 Cloudsley-Thompson, J. L., 122, 188 Coates, A., 448, 467 Coatney, G. R., 604, 607, 609, 611, 614, 615, 616, 619, 621, 627, 630 Coatney, J. R., 619, 627 Coelho, M. V., 318, 390 Cohen, S.,605, 618, 627 Cohen, S. N., 648, 660 Coil, H. W., 399, 404, 468 Coil, W. H., 399, 404,469, 493,494,549 Cole, C. R., 642, 643, 665, 667 Cole, M. M., 159, 184 Colglazier, M. L., 173, 184, 569, 589 Colley, F. C., 621, 627, 638, 669 Collier, J., 39, 60 Collin, W. K., 400, 401, 402, 403, 416, 447, 469,493, 494, 496, 549, 713, 715, 724 Collins, H. M., 573, 586, 595, 598

735

Collins, R. C., 184 Collins, W. E., 604, 607, 609, 611, 612, 614, 615, 616, 627 Coluzzi, M., 134, 184 Contacos, P., 626, 629 Contacos, P. C., 615, 628 Contacos, P. G., 604,607,609,611, 612, 614, 615, 616, 627, 628, 629 Conway, E. J., 326, 386 Cook, G. M. W., 268, 298 Cook, J., 116, 146, 184 Cook, M. K., 646, 668 Cooman, E. P. De, 469 Cooper, N. B., 447, 448, 467, 478 Cooper, N. D., 713, 729 Cordes, F., 31, 59 Cordes, F. L., 31, 59 Cori, C. F., 26, 29, 60 Cornet, A., 651, 652, 659 Cornet, M., 92, 113 Corradetti, A., 605, 627 Cornea, L. R., 321, 390 CoscinA, A. L., 644, 664 Cosgrove, G. E., 412, 473 Coudurier, J., 609, 627 Coulston, F., 635, 660 Courmes, E., 322, 386 Courter, M. H., 648, 661 Cousineau, G. H., 240, 295,301 Coutelen, F., 458, 460, 469 Coutinho, A. B., 311, 318,386 Coutinho, F. A, B., 311, 318, 386 Couvreur, J., 641, 650, 658, 660, 661 Cox, F. E. G., 605, 627 Craig, G. B., 131, 187 Crampton, H. E., 361,387 Crans, W. J., 72, 73 Creemers, J., 13, 32, 33, 36, 39, 61 Crofton, H. D., 697, 699, 702 Crompton, D. W. T., 268,298,672,678, 679, 680, 687, 688, 689, 691, 692, 694, 696, 697, 702, 703, 705 Crosby, W. H., 31, 71 Croset, H., 420, 474, 709, 728 Cross, J. H., 609, 629 Crosskey, M. E., 136,184 Crosskey, R. W., 136, 184 Crumley, F. G., 149,184 Cruse, G. T., 31, 64 Crusz, H., 421, 442, 462, 469 Crystal, M. M., 150, 184 Cuccaro, A. J., 716, 729

736

A U T H O R INDEX

Cuperlovic, K., 583, 592, 594, 597 Currih, G. A., 268,298 Cvjetanovic, B., 86, 88, 112, 113 Czaplinski, B., 710, 724

D Dagert, C., 5, 6, 21, 24, 37, 55, 57, 69 Dailey, M. D., 495, 498, 502, 503, 507, 510, 512, 513, 524, 534, 539, 540, 550, 553 DaIchow, W., 636, 666 Danilewsky, B., 15, 22, 27, 45, 48, 61 Dannelley, C. E., 154, 188 Dardmg, R. L., 366, 388 Darrow, D. I., 153, 169, 184,185 Daubney, R., 571, 572,589 Davey, T.H., 234,249, 300 Davidson, G., 134,184 Davies, C. W., 158,185 Davies, E. E., 624, 625, 628 Davies, J. B., 136, 184 Davis, B. O., Jr, 513, 516, 518, 550 Davis, D. E., 320,373, 374,379,386 Davis, G. M., 361, 393 Davis, L. V., 320, 376, 386 Davtian, E. A., 574, 575, 576, 577, 584, 589 Davudov, D. M., 572,590,593 Dawes, B., 284, 292, 298 Dean, A. C. R.,361,386 De Andrade, R. W., 318,386 Deane, L. M., 612, 613, 614,626,627 Deane, M. P., 641, 662 Deblock, St., 458, 469 Decker, H. A,, 651,661 Dedek, W.,154,184 Delain, E., 14, 68 De Lalla, F., 641, 660 Delic, S., 425, 475, 585, 596 Della Croce, G., 582, 585, 590 De Maria, M., de, 322, 391 Demilo, A. B., 150, 184 Dempster, J. P., 314, 317, 386 Denev, J., 582, 595 Denev, Y., 568, 569, 582,590 Denison, J., 205, 216, 232, 292, 305 Dennison, W. L., 242,301 Denny, M., 493, 522,529,549, 699, 703 Department of Health and Social Security U.K., 95, 112, 113

De Queiroz, J. C., 644,664 Dermott, E., 207, 229, 248, 300 Deschiens, R., 322,386 Deslandes, N., 360, 392 Desmonts, G., 641, 645, 646, 650, 660, 661 Desowitz, R. S., 621, 627 Despeignes, M. J., 651, 652, 659 Desser, S. S., 72, 73, 625, 626, 627, 628 Deutschman, Z., 77, I13 Devauchelle, G., 608, 623, 630 DIM, F., 421,469,499, 514, 519,549 Dewhirst, L. W., 184 De Witt, R. M., 361, 362, 386 Diamond, L. S., 5, 6, 7, 8, 15, 16, 17, 19, 22, 23, 27, 32, 35, 36, 39, 40, 45, 52, 53, 54, 57, 61 Dick, J. R., 569, 590 Di Conza, J. J., 709, 717, 724 Dierickx, K., 25, 61 Dike, S. C., 214,230,277,279,280,298, 438,469 Dill, W. T., 677, 678, 696, 702 Dissanaike, A. S., 426, 469, 609, 627 Dixon, C. W., 88,113 Dixon, J. B., 568, 590 Dixon, K. E., 206, 229, 235, 237, 250, 252,295,299,302 Dixon, M. A., 569,590 Dobell, C. C., 49, 61 Dobrikov, D. M., 593 Dobrovolny, C. G., 312,394 Doby, J. M., 458,469 Docurnenta Geigy Scientific Tables, 370, 386 Doenhoff, A. E., von, 618, 627 Doflein, F., 14, 18, 32, 33, 34,39,40,41, 61 Dollfus, R. Ph.,431, 469, 499, 510, 524, 533, 540,549 Dollfus, R. P. F., 48, 61 Dorais, F. J., 711, 724 Dorey, A. E., 194, 196, 202, 229 Dorney, R. S., 569,590 Dorolle, P., 76, 79, 81, 90,93, 113 Dorrington, J. E., 585, 590 Dorsey, C. H., 247, 248, 299 Downs, W., 92, I13 Draper,C. C.,98,100,103,113,131,183 Drummond, R. O., 150, 153, 155, 156, 157,184,185 D'Souza, B. A., 569, 597

737

A U T H O R INDEX

Dubey, J. P., 632, 633, 634, 635, 636, 638, 639, 640, 641, 644, 661, 664 Dubin, H. V., 648, 661 Dubois, G., 234, 299 Ducceschi, V., 38, 61 Dudley, F. H., 183 Duerr, F. G., 372,386 Duflo, T., 137, 186 Duke, B. 0. L., 106, 113 Dumas, N., 657, 661 Dunachie, J. F., 632, 633, 634, 636, 639, 662 Dunagan, T. T., 674,692,693,695,696, 698, 703, 704 Dunavan, C. A., 312,385 Dunkley, L. C., 715, 724 Dunn, D. R., 560, 564,590 Dunn, F. L., 621, 627 Dunn, M. A,, 610,627 Durge, N. G., 646,661 Durkee, T., 626, 629 Dusanic, D. G., 234, 271, 294, 299 Du Toit, R., 144, 185 Dutt, S. C., 710, 727 Dutton, J. E., 5, 11, 14,45, 50, 51, 61 Dvorak, J. A., 403,442,469, 714, 724 Dyce, A. L., 137, 190 Dyer, W. G., 483, 552 Dzakula, N., 718, 730

E Eagle, H., 366, 386 Ebrahimzadeh, A., 245, 247, 248, 299 Eckert, J., 463, 469, 709, 716, 719, 724, 725, 729 Edeson, J. F. B., 106, 113 Edmonds, S. J., 679, 687, 688, 702, 703 Edwards, T. W., 319, 391 Egger, I., 649, 668 Egorov, Y.G., 575, 576, 585, 590 Ehrlich, I., 272, 302 Eisenberg, R. M., 316, 317, 323, 361, 368, 386 EIder, J. E., 214, 232 Elizian, M., 585, 586, 597, 598 Elkan, E., 33, 68 Elkis, H., 644, 664 Ellis, D. S., 131, I83 Ellis, P. E., 320, 385 El Nahal, H. M. S., 625, 626,628

Elsdon-Dew, R., 421,471,718,721, 726, 727 Emerson, A. E., 361, 362, 384 Englemann, F., 362, 376, 377,386 Englert, E., Jr, 698, 704 Enyenihi, U. K., 718, 725 Enzie, F. D., 173, 184, 569, 589 Erasmus, D. A., 238,247, 248, 249, 260, 262, 267, 299,303 Erhardova, B., 572, 590 Eriksen, L., 642, 664, 669 Esch, G. W., 425, 437, 441, 443, 449, 454,462,472, 474,476, 477, 523,549, 71 1, 718, 720, 723, 724, 725, 726, 728 Eslami, A. H., 585, 597 Esozed, S., 123, 186 Essex, H. E., 495, 500, 549 Etges, F. J., 318, 386, 387 Euzet, L., 483, 488, 489, 499, 533, 539, 544,549 Evans, A. S., 242,299 Ewers, W. H., 51, 61, 319, 387 Ewing, S. A., 560, 565, 569, 590 F

Faich, G. A., 87, 113 Fain, A., 433, 470 Fairbairn, D., 689, 692, 693, 694, 695, 702, 704 Fairchild, G., 651, 661 Falconer, E. H., 31, 66 Falyushin, V. S., 585, 594 Fantham, H. B., 15, 27, 36, 37, 45, 49, 50, 51, 52, 53, 61 Farbman, A. I., 282, 299 Farner, D. S., 220, 231 Farooq, M., 312, 387 Farr, A. L., 326,389 Fauran, P., 322,386 Faust, G. E., 248,299 Favati, V., 571, 582, 585, 590 Favento, R., 646, 666 Fawcett, A. R., 643, 660 Fawcett, D. W., 264, 299 Featherston, D. W., 437, 444,458, 470, 711, 725 Feeley, J. C., 88, 112 Feldman, H. A., 645, 661 Feng, L. C., 6, 10, 17, 37, 47, 61 Fennestad, K. L., 642, 664, 669 Ferguson, D. A. M., 147, 148,184,192

738

AUTHOR INDEX

Fremount, H. N.,622,629 Ferguson, D. L.,570,590 Ferguson, F. F., 312,313,317,318,387, Frenkel, J. K., 632, 633, 634, 635, 636,

388

638,639,640,641,644,654,661,663,

664,666 Fernand, C. H., 699,705 Freshman, M. M., 656, 661 Ferreira, N. J. A., 627 Freze, V. I., 481,495,498,502,504,506, Ferretti, G., 421,450,468 507, 520,526,527, 534, 537,542, 545, Ferreira, J. M.,644,664 546,550 Fiedler, 0.G. H., 144, 157,185 Fried, B.,360,365,387 Fife, E.H., 618,627 Friedl, F. E.,372,384,708, 725 Filippov, V. V.,711, 725 Friedman, C.T.,647,661 Finerty, J. F., 658,664 Fripp, P.J., 269,270,271, 272, 282, 299 Finkelstein, N.I., 27,47, 61 Fritz, R.F.,121, 127, 192 Finnegan, C.V.,409,439,473 Fromentin, H.,39,40,62 Firket, H.,14,70 Fischer, H.,411,470,485,488,490,494, Fry, G. F., 698, 704 496,498,504, 506,509, 510, 526,549, Fuchs, F., 651,663 Fuhrmann, O.,407,413, 470, 499, 507, 725 510, 513, 515, 517, 521, 524, 529, 536, Fisher, F. M., Jr, 691,692,694,695, 703, 704, 705

539, 542,550

Fisher, R. G., 129,189 Flesher, A. R.,617,626 Fletcher, K.A.,617,624,626,629 Floch, H.,560,590 Fluch, M.,28, 29,61 Foft, J. W., 645,659 Fong, Y.L.,608,609,619,627 Foor, W.E.,237, 247,302 Forbes, G.S.,361, 387 Ford, H.R.,133, 189 Fors, M.B., 245,304 Forssten, I., 410,468 Forssten, T.,492,548,712,723 Forsyth, B. A,, 586,590 Foster, B. G., 653,661 Fournis, M.A., 651,652,659 Fox, L.L.,718, 725 FranGa, C.,2,5, 6, 11, 14,15, 16,18,19,

Fujihara, H., 580,591 Fukuto, T.R.,129,187 Fullard, J., 624,629 Furman, D.P.,149, 185 Furukawa, T.,709,725

424,428,433,443,460,467,470,482, 485, 486,488,490,492,494,496,497, 498,499,501,502,503, 504,506,508, 509, 510, 513, 514,515, 516, 517, 518, 519, 526, 527, 528,529,530,548,549, 550,708,714, 723, 725 Freese, P., 455,470 Fregeau, W.A.,250,304

606,607,609,612,614,615,616,617, 618,619,620,621,622,623,625,626, 627,628,629,630,633,661 Gaud, J., 573, 575,576,592 Gaule, J., 14,62 Gayral, P.,125,190 Gazzinelli, G., 361, 375,387 Gear, H.S.,77,113

G Gaber, S., 83, I13 Gaddy, N.K.,122,188 Gaines, J. D.,653,654,655,661,663 Galaugher, W., 462,472 Gallagher, S. S. E., 247, 273,299, 305 Gallati, W.W., 415,470 Galley, R. A. E., 185 Galliard, H.,16, 19, 21, 27, 33, 34, 39,

40, 56,62

Gallut, J. C., 88, 112 22,23,24,33,45,47,49,50,56,61,62 Gam, A.A.,24, 25, 27, 29, 53, 59 Gamero, B. A.,647,666 Franchi, P.,637,666 Gammage, K.,604,616,617,638 Frank, P.W.,361,387 Gangarosa, E.J., 87,113 Franke, I., 5, 33, 37,46, 54, 55, 66 Garcia, R.,156, 184 Frazer, A.C.,116, 146, 185 Garin, J. P.,605,627,651,652,659 Fredericks, H.K.,619,630 Freeman, R.S.,406,411,414,418,423, Garnham, P. C. C., 158, 185, 604,605,

A U T H O R INDEX

Gear, J. H. S., 260, 268, 272, 274,303 Geckler, R. P., 318, 387 Geier, P. W., 314, 320, 386 Geiman, Q. M., 619, 620, 628 Gelderman, A,, 648, 662 Gelderman, A. H., 657, 664, 665, 667, 668 Gelfand, H. M., 81, I13 GeII, L. S., 615, 627 Gemmell, M. A., 711, 716, 718, 723, 725 Gentle, M. A., 560, 590 Gentry, L. O., 645, 652, 656, 660, 662, 666 George, R. W., 585, 597 Georghiou, G. P., 126, 141, 185 Gerichter, Ch. B., 575, 576, 577, 578, 590,591 Gerna, G., 637, 666 Gething, M. A., 175, 185 Gevondian, S. A,, 574,591 Giacobbe, O., 5, 23, 27, 28, 55, 71 Gibbs, H. C., 586,591 Gibson, T. E., 152, 185, 586, 591 Gilbert, B., 361, 375, 387 Gill, H. S., 658, 662 Gillett, J. D., 130, 185 Gilula, N. B., 205, 229 Gisry, O., 39, 41, 63 Gladney, W. J., 153, 185 Gledhill, J. A., 143, 144, 145, 185, 189 Gligorijevik, J., 583, 592 Glitzer, M. S., 716, 729 Glover, P. E. 143, 185 Gluge, G., 1, 62 Gluschenko, V. V., 47, 62 Goddard, P. A,, 604, 616, 628 Goffman, W., 308, 309, 31 1, 312, 387 Gofman-Kadoshnikov, P. B., 408, 468 Goldberg, A., 573, 579, 591 Goldner, M. R., 29, 64 Goldschmidt, R., 426, 470 Goldsmid, J. M., 463, 470 Golubev, N. F., 572, 575, 576, 591 Golvan, Y.J., 672, 703 Gonder, B. 614 Gonnert, R., 233, 257, 283, 299,300 Gonzales, G., 438, 472 Gonzalez, C., 5, 33, 37, 46, 54, 55, 66 Goodchild, C. G., 360, 365, 387, 419, 456, 470, 490, 502, 513, 516, 518, 550, 713, 725 Gooding, R. H., 156, 191

739

Goodman, A. B., 364, 376,384 Goodman, N. M., 77,113 Goodwin, T. W., 39, 41, 71 Gordon, C. H., 140,188 Gordon, R. M., 234, 249,300 Goto, M., 580,591 Gouck, H. K., 129,189,190 Gourvitsch, V., 27, 62 Grabda-Kazubska, B., 675, 703 Grabiec, S., 719, 727 Gradwell, G. R., 316, 393 Graff, D. J., 693, 703 Graham, A. J., 183 Graham, D. H., 155, 156,185 Graham, H. J., 492, 526,550 Graham, J. J., 720, 725 Graham, 0. H., 153, 157,184 Grftsse, P.-P., 9, 63 Grassi, B., 5 , 27, 45, 48, 63 Graw, R. G., Jr, 648, 667 Green, P.E., 168, 189 Greenaway, P., 369, 387 Greenwald, P., 651, 661 Greenwood, D., 149,185 Gregson, J. D., 155, 185 Grembergen, G., van, 458, 475 Gresso, W., 318, 387 Gresty, R. H. C., 163, 165, 166,185 Gretillat, S., 560, 589 Greve, J. H., 560, 593 Griener, H., 28, 29, 61 Griffin, R. L., 680, 681, 682, 705 Griffiths, R. B., 240, 300 Grimley, P. M., 648, 668 Grimley, Ph. M., 648, 662 Groschaft, J., 447, 474 Grover, K. K., 132,133, I85 Gruber, F., 658, 666 Gruby, D., 1, 3, 5 , 24, 25, 26, 27, 33.45, 63 Grundmann, W. A., 37, 52, 67 Guberlet, J. E., 571, 572, 591 Gruys, P., 380, 387 Gubler, D. J., 133, 185 Gude, D. W., 412, 473 Guerrant, G. O., 125, 191 Guilhon, J., 586, 591 Guimaraes, E. C . , 641, 662 Guindy, E., 83, 113 Guinn,E. G., 611, 614, 615, 627 Gupta, V. P., 489, 542, 543, 550 Gurri, J., 463, 477

740

AUTHOR INDEX

Gustaf'sson, M. K. S., 409, 410, 439, 468, 479, 492,548,556, 712, 723, 730 Guttman, H. N., 20, 39, 63 Guttowa, A., 719, 727 Gvozdev, E. V., 431,470 Gvozdev, Ye. V., 406,476

H Haab, 0. P., 29, 31, 58 Hadaway, A. B., 144, 145, 185 Haight, A. S., 29, 72 Hairston, N. G., 308, 309, 310, 31 1, 312, 314,387,391 Halbastaedter, L., 607, 628 Hales, H., 13, 72 Halevy, S.,39, 41, 63 Halhead, W. A., 587,591 Hall, C. A., 149, 185 Halterman, R. A., 648, 667 Halton, D. W., 201, 207, 208, 210, 212, 213, 214, 215, 220, 224, 229, 231, 248, 271, 272, 281, 300 Halvorsen, O., 712, 725 Hamilton, A. G., 459, 471 Hamilton, J., 160, 183 Hamilton, W. P., 378, 379,387 Hammen, C. S., 365,388, 692, 693, 694, 702 Hammond, D. M., 638, 667 Hammond, G. H., 37, 60 Hammond, R. A., 672, 673, 674, 680, 681, 682, 691, 703 Hamon, J., 92, 113 Hanaki, T., 636, 643, 665 Hansen, E., 293, 300 Hansen, E. L., 707, 728 Hanson, H. A., 657,662 Hanson, R. P., 567,592 Hansson, H.-A., 657,664 Harbone, J. B., 320,388 Harley, K. L. S., 170, 185 Harlow, D. R., 716, 725 Harmsen, R., 2, 13, 25, 26, 29, 37, 58 Harrell, F. R., 648, 661 Harris, P. J., 244, 247, 297 Harrison, A. D., 370, 388 Harrison, D. L., 456,470,490,502,518, 550

Harrison, I. R., 147, 148, 149, 173, 184, 185, 186 Hart, J. L., 463, 471,709, 719, 721, 725,

726

Hartley, W. J., 643, 662 Harvey, S. C., 35, 63 Hasan, S., 131, 186 Hashimoto, B. Y.,5, 15, 24, 25, 46, 68 Hassell, M. P., 318, 388 Haufe, W. O., 159, 160,190 Haust, M. D., 274,300 Hawking, F., 604, 616, 617, 628 Hawkins, W. W., 148, 187 Hawley, M. K., 126, 141, 185 Haworth, J., 121, 127, 192 Haydock, K. P., 171, 186 Hayes, D. E., 605, 630 Hayes, S. L., 648, 664 Haynes, W. D. G., 454, 471, 720, 725 Heath, A. C. G., 160, 186 Heath, D. D., 404, 405, 406, 421, 460, 462,471, 486, 556, 711, 715, 721, 726, 729 Heath, R., 174, 186 Heath, R. L., 719, 721, 726 Hebden, S. P., 148, 149, I88 Hegner, R. W., 5, 23, 63 Heide, D., 585, 591 Hein, B., 718, 727 Heinz, M. L., 510,513, 540,550 Heisch, R. B., 158, I85 Held, J. R., 611, 612, 614, 627, 628, 629 Hemingway, E. E., 35, 67 Henry, H. M., 433,473 Henry, L., 645, 646, 662, 664 Henshall, T. C., 435,477 Herbert, C. N., 141, 151, I91 Herbert, I. V., 560, 568, 569, 588, 592 Herde, K. E., 501, 502, 508,550 Herman, C. M., 39, 61 Herman, R., 32, 63 Heunert, H. E., 657,660 Hewitt, M., 175, 186 Heydorn, A. O., 658,662,666 Heyneman, D., 318, 321, 322, 388, 389, 418,419,446,448,456, 471,479,498, 499, 500, 501, 513, 515, 516, 517, 519, 520, 521, 528, 529, 534, 537,550, 556, 715, 726 Hibbard, K. M., 691, 703 Hibbs, J. B., 656, 662 Hibbs, J. B., Jr, 656, 662 Hickman, J. L., 489, 490, 492, 494, 496, 497, 501, 502. 514, 516, 519, 520, 523, 529,550

74 I

AUTHOR INDEX

Hicks, R. M., 268, 300 Hiepe, T., 560, 568, 585, 591, 593, 595 Hinde, R., 342, 388 Hinshelwood, C., 361,386 Hirato, K., 653, 669 Hirsch, J. G., 656, 657, 663 Hirst, J. M., 129, 190 Hinve, A. S., 118, 176,188 Hoare, C. A., 4, 9, 12, 51, 63, 605, 628, 636, 662 Hobbs, B. C., 96, I13 Hobmaier, A., 561, 562, 564, 573, 574, 575, 576,591 Hobmaier, M., 561, 562, 564, 573, 574, 575, 576,591,592 Hockley, D. J., 206, 213, 214, 216, 217, 229,234,235,236,237,238,240,241, 242,243,244,245,246,247,248, 249, 250, 251, 252, 254, 255, 257, 260, 262, 264,266,267, 268,269, 271,275, 279, 283, 284, 285, 289, 300, 304 Hodges, T. K., 366,388 Hodjat, S. H., 361, 388 Hoffman, R. A., 161,186 Hoffmann Mendizabel, A., 37,63 Hofling, K. H., 657, 660 Hofsten, B. V., 365, 388 Hogan, B. F., 161, I86 Hogg, J., 361, 388 Hohl, H. R., 411, 447, 475, 711, 715, 728 Holm, G., 118,186 Holberton, D. V., 51, 63 Holkova, R., 640, 660 Hollo, F., 564, 565, 568, 585, 592 Holloway, H. L., Jr, 675, 703 Holtzer, H., 210, 229 Holzapfel, R., 29, 63 Honin, R., 629 Hoo, C. C., 610,628 Hoogstraal, H., 83, 113, 609, 628 Hooper, A. S. C., 370,388 Hopkins, C. A., 439, 473,492,493, 505, 548 Hopkins, D. E., 160, 183, 184 Horsfall, M. W., 521, 528, 550 Horvath, K., 693, 694, 703 Houin, R., 420, 474, 709, 728 Houssay, B. A., 28,29, 63 Howells, R. E., 452, 454, 471, 624, 625, 628,629 Howkins, A. B., 460, 477

Hoy, J. B., 130, 186 Hrab6, S.,448,471 Hubert, H. E., 38, 63 Hubner, J., 651,663 Hudson, J. E., 123, 186 Huff, C. G., 609,628 Huffaker, C. B., 318,388 Hughes, R. D., 314, 320,386 Huldt, G., 653, 654, 662 Hunkeler, P., 444,471, 709, 726 Hunter, D., 651, 660 Hunter, G. W., 111, 504, 550 Hunter, W., 49, 63 Hurlbut, H. S., 159, 186 Hussey, N. W., 117, 184 Hutchinson, G. E., 3, 63 Hutchison, W. M., 442, 471, 493, 505, 517, 529,550, 631,632,633, 634, 636, 639, 662, 667,669 Hutner, S. H., 34, 63 Hyman, L. H., 362, 388, 484, 493, 535, 536,550 Hynes, H. B. N., 699, 703

I Ikeshoji, T., 320, 376, 377, 388 Inamasu, S., 129, 188 Inami, Y., 657, 665 Inatomi, S., 237,243, 260, 300 Inglis, W. G., 534, 550 Inoue, S., 295, 301 International Panel Workshop Experimental Malaria, 605, 628 Ishikawa, H., 210, 229 Isserhoff, H., 291, 300 Itano, K., 237, 243, 260, 300 Ito, S., 237, 238, 240, 300 IvaniE, M., 14, 15, 32, 47, 63 Ivanov, A. V., 207,230

on

J Jackson, G. J., 32, 63 Jacobs, L., 639, 641, 646, 648, 652, 662, 664,668 Jacono, I., 9, 47, 63, 64 Jacox, R. F., 243, 300 Jadin, J. B., 610, 628 Jadin, J. M., 13, 32, 33, 36, 39, 61, 610, 628 Jaggers, S. E., 560, 568, 592

742

AUTHOR INDEX

James, B. L., 234, 237, 295, 300, 301 James, C., 361, 368,393 James, H. G., 462,471 Jamieson, J. L., 492,551 Jamnback, H. A., 137, 186 Jamra, L. F., 641, 662 Jamuar, M. P., 290, 291, 301 Janicki, C., von, 487, 507, 532, 551 Janitschke, K., 636, 638, 639, 640, 642, 643, 649, 660, 663, 666, 668 Janssen, P., 65 1, 663 Janssens, P. A., 695, 703, 716, 724 Jarecka, L., 482,486,487,488,489,494, 495, 499, 502, 503, 504, 506, 507, 509, 510, 513, 517, 525, 527, 529, 534, 534, 537, 542, 551, 708, 710, 724, 726 Jauregui, R. F., 29, 64 Jayasuriya, J. M. R., 629 Jeter, M. H., 615, 627 Jewell, M. L., 636, 663 Jha, R. K., 196, 213, 230, 437, 443, 444, 471 Jirovec, O., 12, 64 Jobin, W. R., 312, 313, 316, 317, 318, 323, 360, 361, 363, 314, 387, 388 Johns, F. M., 618, 626 Johnson, C. M., 619, 630 Johnson, F. H., 242, 301 Johnson, K. M., 636, 663 Johnson, R. E., 25, 66 Johnson, W. B., 10, 27, 37, 51, 65 Johnston, L. A. Y., 171,186 Johnston, M. R. L., 136, 184 Johnston, T. H., 5 , 45, 51, 52, 60, 64 Jolly, S. S., 717, 726 Jones, A. W., 677, 678, 704, 715, 729 Jones, M. F., 517,521,528,529,550,551 Jones, T. C., 656, 657,663, 667 Jones-Davies, W. J., 167, 186 Jordan, A. J., 29, 72 Jordan, H. E., 584, 592 Jordan, P., 105, 113, 307, 308, 309, 310, 311, 314, 316, 317, 318, 323,388 Jorg, M. E., 14, 27, 55, 64 Jorren, H. R., 649, 663 Jourdane, J., 420, 471, 474, 709, 710, 726, 728 JovanoviC, M., 583, 592, 597 Joyeux, C., 481,482,492,493,498, 501, 502, 503, 504, 505, 506, 507, 508, 518, 524, 528, 529, 531, 533, 538, 539, 540, 544,551, 573, 575, 576, 578,592

Jumper, J. R., 611, 612, 614, 627, 628, 629 K Kaiser, M. N., 83, 113 Kammer, H., 567,592 Kapandze, K. I., 586,592,593 Kaplan, H. M., 31, 64 Kapoor, I. P., 118, 176,188 Karin, D. S., 444, 472 Karmanova, E. M., 420,471 Kaschef, A. H., 129, 186 Kass, L., 622, 628 Kassai, T., 571, 575, 576, 578, 580, 581, 585,592 Katansky, S. C., 483, 551 Kates, K. C., 561, 592 Katiyar, J. C., 717, 726 Katkansky, S. C., 708, 726, 729 Katsuda, Y., 129, 176, 188 Kauffman, E. E., 130, 186 Kaufrnan, H. E., 647,663 Kausa, M., 451, 477 Kauzal, G., 572, 573, 577, 581, 593 Kawamoto, N. Y., 373,388 Kean, B. H., 641, 644,651, 663 Kearn, G. C., 202, 205, 206, 207, 219, 220, 230 Kearney, A., 716, 726 Kegley, L. M., 439, 471 Kegley, M. L., 438, 471 Keiding, J., 186 Keister, D. B., 657, 665 Kelley, G. W., 560, 567, 569, 593, 594, 596 Kelly, K. H., 31, 59 Kelly, R. W., 361, 387 Kemp, W. M., 238, 241, 242,301 Kempf, A. H., 243,302 Kendall, S. B., 174, 186 Kennedy, B. J., 646,667 Kennedy, C. R., 503,512, 525, 533,5552 Kennet, C. E., 318, 388 Kent, P. W., 242, 301 Kepinov, L., 25, 29,64 Kerandel, J., 50, 51,64 Kessler, H., 139, 186 Ketterer, P. J., 162, 189 Keymer, I. F., 610, 628 Khan, M. A., 153, 155, 156, 160, 186, 189

743

AUTHOR INDEX

Kien Truong, T., 651, 652, 659 Kilejian, A., 455, 471 Killick-Kendrick, R., 607, 608, 615, 618, 623, 625, 626,628 Kilpatrick, J. W., 133, 188 Kimball, A. C., 641, 644, 651, 663 King, D., 677, 678, 703 King, J. W., 438, 472 Kinoti, G. K., 290, 293, 294, 301 Kirkwood, A. C., 141, 151, 174, 175, 176,186, 191 Kirschstein, H., 220, 231 Kisielewska, K., 462, 472, 517, 529, 552 Kitaoka, M.,159, 186 Kitzman, W. B., 693, 703 Kitzmiller, J. B., 131, 187 Klomp, H., 316, 380, 382, 388, 389 Kluge, E. G., 144,185 Klyavinsh, Y.R., 488, 489, 552 Knapp, F. W., 157, 187 Knipling, E. F., 116, 131, 149,187 Knox, D. L., 647, 661 Knuts, G. M., 410, 439, 479, 712, 730 Kobayashi, A., 652, 657, 663, 665 Kobayashi, H., 220, 231 Koidzumi, M., 47, 64 Kraie, M., 194, 196, 217, 229, 240, 244, 245, 246, 247, 294, 295, 301 Koizumi, T., 643, 665 Kondrat’ev, V. P., 586, 593 Koniiiski, K., 24, 26, 27, 48, 64 Kopirin, A. V., 593 Korchagin, A. I., 586, 593 Korting, W., 692, 693, 694, 695, 704 Kosminkov, N. E., 71 1, 725 Kotecki, N. R., 417, 472, 489, 552 Kotelnichov, G. A., 593 Kowalski, J. C., 717, 726 Kozicka, J., 710, 726 Kozo Abe, 129,188 Kraft, M., 247, 248, 299 Krahenbuhl, J. L., 653, 654, 655, 661, 663

Kramar, J., 651, 663 Kramer, van der J. C., 29, 60 Kraiibig, H., 658, 664 Krause, H., 560,593 Krebs, C. J., 379, 389 Kronman, G., 610,630 Kruidenier, F. J., 237,238,240,241,301 Krupa, P. L., 240, 269, 270, 271, 294, 295, 297,301

Kruse, W., 11, 14, 32, 48, 64 Kuczkowski, St., 497, 552 Kudo, R., 5, 6, 14, 27,45, 53, 54, 57, 64 Kuhl, G.. 131. 186 Kuhlow,.F., 491, 499, 502, 504, 506, 507.552 Kiihn; D., 633, 634, 636, 639, 640, 644, 663, 664, 668 Kuhn, R. E., 443, 462, 476, 477, 711, 718, 720, 723, 725, 726, 728 Kumada, M., 652, 657, 663, 665 Kunkle, D., 709, 723 Kunsemiiller, F., 425, 472 Kuperman, B. I., 397,408,409,440,478 Kurelec, B., 272, 302 Kuttler, K. L., 609, 629 Kwa, B. H., 409, 472, 712, 726, 727 Kwo, E. H., 319, 321, 322, 389 L Laake, E. W., 152,183 La Brecque, G. C., 132, 141, 187, 188, 190 Lack, D., 316, 317, 389 Lackie, J. M., 679, 697, 704 Ladda, R. L., 622, 623, 629 Lagrange, E., 368, 389 Laird, M., 53, 64 Lal, M. B., 268,302 Lambert, L. H., Jr, 656, 662 Lamborn, W. A., 9, 70 Lamina, J., 718, 727 Lancaster, J. L., 155, 191 Land, M. F., 220,230 Landau, I., 608, 610, 629 Lang, J. H., 149,184 Lange, H., 680, 681, 682, 683, 704 Languillon, J., 615, 629 Langmuir, A. D., 81, 113 Lankester, E. R., 5 , 6, 22, 45, 47, 64 Lapeyssonnie, L., 87, 113 Larsh, J. E., 247, 257, 303, 425, 472 Larsh, J. E., Jr, 437, 441, 449, 454, 474 Larson, 0. R., 677, 678, 705 La Rue, G. R., 482, 552 Lauder, I. M., 174, 189 Laurent, M., 14, 64, 70 Laurinaitis, B., 561, 597 Lauter, F. H., 24, 28, 33, 53, 54, 64 Laven, H., 131, 133, 187 Laveran, A., 5, 11, 12, 26, 46, 50, 64

744

AUTHOR INDEX

Lavier, G., 10, 64 Laws, G. F., 404, 472 Lebailly, C., 40, 47, 64 Lebedeff, W., 14, 15, 18, 24, 32, 33, 36, 38, 48, 56, 64 Leboeuf, A., 50, 51, 66 Lebredo, M. G., 27, 54,65 Ledet, A. E., 560,593 Lee, C. W., 143, 145,189 Lee, D. L., 193, 194, 201, 208, 218, 230, 234, 268,302,436,438, 472, 680, 702, 704 Lee, H., 28, 29, 65 Lees, A. D., 317, 389 Gger, M., 5, 6, 11, 46, 49, 66 Leger, N., 420, 474, 709, 715, 724, 727, 728 Lehmann, D. L., 5,15,19,35,39,40,41, 42, 46, 52, 54, 65 Leiby, P. D., 483, 552 Leinert, E., 268, 297 Leland, S. E., 570, 593 Lelong, F., 641, 661 Lembright, H. W., 155, 189 Lemma, A., 320,389 Leonard, M. E., 31, 66 Lesinsh, K. P., 488, 489, 552 Lesser, E. J., 28, 65 Leuckart, K. G. F. R., 485, 486, 493 497, 518, 552 Levine, A. S., 648, 666, 667 Lewert, R. M., 290, 291, 301 Lewis, J., 24, 38, 65 Lichtenberg, F., von, 234, 238, 241,243, 244, 246, 254, 260,262, 264, 266, 267, 271, 272, 273, 275, 283, 304 Lie, K. J., 319, 321, 322, 389 Lieberkiihn, N., 5, 46, 65 Lien, J. C., 609, 629 Lilac, R., 583, 594 Lilly, D. M., 374, 381,389 Lim, B. L., 608, 627 Lim, H.-K., 318, 322,389 Lindquist, W. D., 570, 593 Linholm, L., 656, 665 Linton, R. G., 647, 664 Lippmann, R., 585, 591 Littlejohn, A., 173, 187 Littman, A., 31, 59 Llewellyn, J., 194, 196, 207, 218, 230, 487, 532, 533, 535, 537,552 Lloyd, J. A., 320, 374, 379, 385

Lloyd, J. E., 153, 187 Lloyd, L., 10, 27, 37, 51, 65 Lockwood, A. P. M., 692, 702 Loewi, O., 28, 29, 61 Lofgren, C. S., 133, 144, 187, 189 Logachev, E. G., 452,472 Lola, J. E., 361, 393 Long, P. L., 635, 664 Long, W. D., 25,66 Loosli, B. G., 242, 297 Loser, E., 493, 495, 552 Lowe, R. E., 188 Lowry, 0. H., 326,389 Lubinsky, G., 460, 462, 472, 709, 717, 727, 728 Lucia, J. P., 31, 66 Lucker, J. T., 565, 567,596, 716, 729 Ludwig, P. D., 149, 155, 185, 189 Liihe, M., 501, 515, 517, 519, 522, 529, 552 Lukashenko, N. P., 427, 472 Lukovich, R., 172, 189 Lumsden, R.D., 194,213,214,215,216, 230,231,237,242,247,302,305,400, 437,438,440,444,453,472,473,474, 680, 681, 683, 706 Lumsden, W. H. R., 13, 72, 104, 113 Lund, E., 657,664 Lunde, M. L., 652,664 Lunde, M. N., 648, 658, 662, 664, 667, 668 Luque, O., de, 696, 703 Luse, S.A., 622, 629 Lycke, E., 655, 656,657,664,665,667 Lyness, R. A. W., 201, 210, 229 Lyons, K. M., 194, 196, 201, 202, 205, 206, 207, 208, 210, 212, 213, 214, 215, 217,218,219,220,222,224,225,226 230,231 Lysenko, M. G., 653, 663 M McAlister, R. O., 692, 695, 704 Macan, T. T., 370, 389 McCarty, J. E., 250, 251, 261, 277, 280, 297 McClelland, W. F. J., 318, 389 McColl, E. L., 491, 556 McCraig, M. L. O., 439, 473 McCray, E. M., 133,188 McCulloch, R. N., 167, 187

A U T H O R INDEX

McCullough, W. E. 653,661 McDaniel, J. S.,295, 302 Macdonald, G., 308, 309, 310, 311, 313, 389 McFadyean, J., 580,593 MacFie, J. W. S.,14, 50, 51, 66 McGregor, I. A., 604, 629 Machado, A., 11, 13, 14, 27, 29, 55, 66 McIntosh, J., 655,667 McJunkin, F. E., 316, 318,389 McKeever, S.,433,473 Mackenzie, A., 560, 564, 565, 566, 567, 593 Mackenzie, M., 242, 299 Mackenzie, R. B., 84, 109, 113 Mackerras, I. M., 52, 66 Mackerras, M. J., 52, 66 Mackiewicz, J., 483, 484, 490, 493, 494, 495, 511, 512, 525, 541, 544,552 Mackiewicz, J. S.,412, 473 Mackinnon, J. A., 269, 298 McLaren, D. J., 243,244,245,250,251, 252, 254,255, 262, 267,300 McLaughlin, J. D., 710, 727 MacLennan, K. J. R., 143, 144,187 McLeod, J. A., 481, 483, 484, 485, 486, 490,491,492,493, 494, 495,496,498, 499, 503, 505, 507, 508, 510, 511, 512, 517, 520, 521, 524, 526, 528, 529, 533, 536, 538, 539, 540, 541, 542, 546, 556 McMahon, J. P., 134, 135, 187 McMaster, P. R. B., 658,664 Macnamara, F. M., 718, 723 Macnamara, F. N., 718, 725 McNicholl, B., 646, 664 MacRae, E. K., 198,231 Macruz, R., 641,659 McWilliams, J. G., 129, 190 Macy, R. W., 493,552 Maddrell, S. H. P., 117, 176, 187 Maegraith, B. G., 76, 84, 109, 110, 113, 617, 626, 629 Maetz, J., 313, 389 Magaldi, C., 644,664 Magat, A., 187 Mahalanabis, D., 88, I12 Mahnert, V., 433, 474 Mair, H. J., 646, 660 Mair, N. S.,646, 660 Makhano, E. V., 139, I87 Malek, E. A., 318, 389

745

Malheiros, O., Jr, 644, 659 Malmberg, G., 485, 496, 497, 532, 535, 536,552 Malviya, H. C., 710, 727 Mangoury, M. A., el, 361, 365, 381, 391 Mankau, S. K., 421,473 Manktelow, B. W., 460,477 Mann, K. H., 35,66 Manson-Bahr, P. E. C., 105, 109,114 Mansour, N. S., 48,66,67 Mapes, C. R., 575, 576,593 Marchoux, E., $46, 54,66 Marcial-Rojas, R. A., 489, 553 Markotic, R., 585,596 Markowski, S.,492, 552 Marquardt, W. C.. 148, 187 Marr, D., 137, I86 Marsden, P. D., 104,113 Marshall, A., 248, 297 Marshall, A. G., 492, 503, 515, 519, 520, 521, 528,552 Marshall, J., 676, 704 Martin, D. F., 377, 389 Martin, G., 50, 51, 66 Martin, G. N., 438,468 Martin, J. H., 247, 257, 303, 437, 441, 449, 454,474 Martin, W. E., 206, 216, 228, 234, 235, 237, 245,294, 295,297 Mason, G., 25, 21, 29, 66, 69 Massoud, J., 318, 319,386 Matekin, P. V., 574, 593 Mathevossian, E. M., 481,499,514,518, 519, 523, 530, 552 Mathews, S. A., 29, 66 Mathis, C., 5, 6, 11, 38, 46, 49, 66 Matricon-Gondran, M., 206, 231, 250, 291, 294, 295,302 Mattes, O., 292, 302 Matthysse, J. G., 152, 161, 175, 187 Mattos, R. 0. De, 560, 593 Mauer, A. M., 29, 31, 58 Mauro, F., 646,666 May, I. R., 376, 380, 390 Mayer, A. F. I. C., 3, 5, 6, 46, 66 Mayer, H. F., 642,664 Maynard Smith, J., 379,389 MaZgon, z., 569, 584,598 Mazza, S., 5, 33, 37, 46, 54, 55, 66 Mazzocco, P., 28,66 Mead, A. R., 314,390

746

AUTHOR INDEX

Mead, R. W., 488, 504, 506, 527,552 Meagher, M. J., 619, 628 Medina, F., 645, 668 Mehlhorn, H., 623, 629 Mehlman, B., 368, 393 Meifert, D. W., 141, 187 Meleney, H. E., 321, 390 Mellanby, K., 116, 146, 187 Melton, M. L., 632, 633, 637, 641, 657, 662, 667 Melvin, D. M., 418, 473 Mendeeff-Goldberg, P., 23, 32, 36, 39, 66

Menschel, E., 579, 580, 596 Menzies, C. M., 116, 187 Mercer, E. H., 235, 237, 252, 299, 680, 681, 696, 697, 704 Mercer, E. M., 250, 299 Merigan, T. C., 655, 656, 661, 666 Mertz, W., 716, 725 Mesnil, F., 11, 12, 26, 64 Meszoely, C. A., 604, 629 Metcalf, R. L., 118, 129, 176, 187, 188 Mettrick, D. F., 216, 228, 437, 467, 715, 724 Meyer, F. R., 501, 502, 504, 506, 525, 552 Meyer, M. C., 492, 552 Michajiow, W., 719, 727 Michel, J. C., 608, 610, 629 Michel, J. F., 579, 581, 582, 593, 594 Michelson, E. H., 316, 317, 318, 322, 323, 360, 361, 363, 365, 367, 370, 374, 385,388,390 Micks, D. W., 122, 188 Mikel, U., 623, 626 Miles, J. W., 125, 191 Millar, E. S., 160, 186 Millemann, R. E., 415, 473, 502, 523, 553 Miller, D. M., 674, 698, 704 Miller, L. H., 618, 621, 622, 627, 629 Miller, M. J., 645, 648, 664, 666 Miller, M. R., 26, 28, 29, 66, 72 Miller, N. L., 632, 633, 634, 636, 639, 661, 664 Miller, R. W., 140, 188 Mills, R. R., 438, 472 Milne, A., 317, 390 Ministry of Agriculture, Fisheries and Food, 148, 172,188 Minnick, D. R., 250, 304

Minning, W., 242, 305 Misiura, M., 715, 727 Mizell, S., 28, 66 Miadenovic, Z., 583, 594 Mohammed, A. H. H., 48,66,67 Mohi-ud-din, G., 585, 594 Moller, T., 642, 664, 669 Mondal, A., 88, 112 MonnC, L., 268,302 Monod, L., 605, 627 Montesionos, C. M., 5, 15, 24, 25,46, 68 Moore, D. V., 247, 257,303, 321,390 Moore, G. A., 624,629 Moore, J. G., 698, 704 Moore, J. P., 35, 67 Morgan, D. O., 574, 594 Morgan, E. R., 716, 729 Morgan, P. B., 141,188 Morgan, S.,608, 630 Morita, M., 198, 228 Morlan, H. B., 133,188 Morozov, I. G., 585,590 Morris, G. P., 207, 208, 210, 212, 213, 214, 215, 220, 224, 229, 231, 238, 242, 243, 244, 245, 246, 247, 248, 249, 260, 262,264,266,268,271,272,273, 275, 277, 279, 280, 283, 302, 439,440, 441, 473 Morris, N., 718, 727 Morris, P. G., 409, 439, 473 Morris, R. F., 314, 317, 320, 386, 390 Morrison, H., 10, 27, 37, 51, 65 Morton, H., 174,186 Morseth, D. J., 398, 437, 455, 473 Mosley, W. H., 88, 112 Moss, R., 380, 393 Most, H., 610, 619, 630 Motomura, I., 641, 665 Mount, G. A., 188 Mount, P. M., 399, 427, 443, 473, 714, 727 Movsesijan, M., 583, 592, 594, 597 Moyle, G., 643, 662 Mrazek, A., 447, 473 Mrazek, Al, 498, 502, 553 Mudry, D. R., 495, 498, 502, 503, 507, 510, 512, 524, 534, 539, 540,553 Mueller, J. F., 407, 411, 462, 473, 483, 488,492, 503, 510,526, 535,548, 553, 708, 716, 719, 725, 727, 729 Mui, P. T., 129,189 Mujata, A., 641, 665

747

AUTHOR INDEX

Mujib, B. F., 517, 553 Mukai, T., 129, 188 Mukerjee, S., 88, 112 MulIa, M. S., 320, 376, 377,388 Mullan, D. P., 645, 664 Muller, C. H., 318, 390 Muller, I., 642, 666 Muller, W. A., 651, 665 Munday, B. L., 642, 643, 665 Murdie, G., 361, 390 Murray, M. D., 137, 188 Murrell, K. D., 454, 473, 718, 719, 727 Muscatine, L., 342, 393 Muul, I., 608, 627 N Nabarro, D., 5,46, 49, 50, 67 Nachtrieb, H. F., 35, 67 Naidu, K. S., 342, 390 Nakabayashi, T., 641,665 Nakajima, M., 320, 394 Nakamura, M., 39,67 Nakanishi, M., 129, 188 Nakayama, I., 636, 649, 653, 655, 658, 665 Nakhai, R., 190 Nalin, D. R., 88,112 Nawrot, R., 619, 630 Nayak, D. P., 567, 594 Nduku, W., 370,388 Neiland, Y. A,, 561, 594 Nelson, D. R., 131, 137, 188 Nelson, G. S., 95, 106, 114, 135, 188, 322,390 Nelson, P., 629 Nelson, W. A., 151, 152, 188 Neradovk-Valkounovi, J., 417,447,473 Nesbitt, P. E., 614, 628 Neto, F. J. A., 612, 627 Nevenic, V., 560, 583,592,594, S97 New, W. D., 183 Newsom, L. D., 314, 390 Newton, L. G., 167,188 Newton, W. L., 321, 390 Nibley, C., 159, 186 Nicholas, W.L., 671, 676, 680, 681, 696, 697, 699,701, 703, 704 Nicol, B. B., 675, 703 Nicholson, A. J., 316, 380,390 Nickel, E. A., 581, 594 Nickel, S.,574, 594

Nicoli, R. M., 9, 67 Nieland, M. L., 398, 399, 439,449, 450, 454,474, 713, 727 Nigrelli, R. F., 5,7, 15,24,25,35,38,46, 52, 53, 54, 57, 67 Nilsson, O., 586, 594 Nimmo-Smith, R. H., 269, 271, 302 Noble, E. R., 486, 553 Noble, G. A., 486, 553 Nobuto, K., 636, 643, 665 Noda, H., 641, 665 Noel, J., 198, 228 Noguchi, H., 41, 67 Nollen, P. M., 269, 271, 272, 302 Noller, W., 6, 14, 15, 17, 18, 23, 32, 36, 37, 39, 48, 56, 67 Nolte, D.J., 376, 380, 390 Norrby, R., 656, 657, 664, 665 Norris, K. R., 120, 141, 178, 188, 191 Norris, M. J., 380, 381, 382, 390 Novak, M., 510,553,709,716, 727, 728 Novikoff, A. B., 13, 70 Nungester, W. J., 243, 302 Nunns, V. J., 571,598 Nuorteva, P., 698, 699, 704 Nussbaum, R., 52,60 Nussenzweig, R. S., 605, 619, 621, 624, 629,630 Nuttman, C. J., 249, 302 Nylen, M. U., 438, 468

0 Oaks, J. A., 213, 214, 215, 216,230, 231, 437,438,473, 474 Obrayashi, M., 436, 460,479 O’Brien, A. G., 117, 130, 186 O’Brien, R. D., 154, I88 OConnor, G. R., 648,665 Odening, K., 28, 67 O’Donoghue, J. G. O., 585,594 Odum, E. P., 317,379,390,488,541,553 Ogarni, H., 129, 176, 188 Ogawa, M., 11,49,67 Ogita, Z., 140, 188 Ogren, R. E., 400, 401, 402, 405, 406, 474, 484, 485, 486, 493, 494, 495, 496, 498,553 Ohman, C., 260, 299 Ohman-James, C., 439, 474 Ohshima, S., 657, 665 O’Kelly, J. C., 163, 188

748

AUTHOR INDEX

Park, T., 361, 362,384 Parker, W. H., 585, 595 Parkinson, K. D., 162, 189 Parrish, M., 443, 462, 477 Parry, J. E., 37, 52, 67 Patterson, R. S., 133, 189 Pattoli, D., 644, 664 Patton, W. S., 5, 46, 49, 56, 67 Pauluzzi, S., 436, 460, 474 Pavlov, P., 574, 575, 576, 582, 595 Pearse, A. J., 24, 67 Pearson, J. C., 534, 537, 553 Peaston, H., 234, 249, 300 Pedersen, K. J., 194, 231 Peffers, A. S. R., 96, 113 Pellegrino, J., 322, 361, 375, 387, 391 Pelster, B., 637, 638, 665 Pence, D. B., 397, 398, 399, 402, 474, 493,494,496,553 Pen-Lin Li, 580,595 Pennacchio, A. E., 605, 627 Pennoit-de-Cooman, E., 460, 474 Pennycuick, L., 716, 728 Pereira, O., 360, 391 Perez, H., 285, 302 Perez-Mendez, G., 293, 300 Perez-Reyes, R., 5, 13, 15,21, 22,23,24, 25,27,34,37, 39,40,41,46, 51,52,53, 54, 67, 68 Perlmutter, A., 320, 376, 394 Perlstein, J. M., 321, 385 P Permpanich, B., 621, 627 Perrotto, J., 657, 665 Packchanian, A., 39, 67 Pesigan, T. P., 312, 391 Pagano, J. F., 709, 723 Page, K. W., 147, 148, 152, 171, 172, Pessoa, S. B., 37, 42, 68 Peter, B., 155, 189 184,189,190, 192 Pal, R., 119, 126, 137, 138, 140, 141, 145, Peters, W., 98, 100, 102, 103, 113, 114, 149,183, 607, 615, 618, 624, 628, 629 147, 158, 159, 160, 162, 167, 169, 183, Peterson, G. D., 131, 189 187 Petit, J., 591 Pallis, C., 717, 726 Palmer, J. R.,312,313,317,318,387,388 Petitprez, A., 608, 623, 630 Pezzlo, F., 250, 251, 261, 277, 280, 297 Pan, C., 318,319,390 Pfadt, R. E., 157,189 Panasiuk, D. I., 579, 595 Pfeiffer, E. F., 28, 68 Panosian, M. A., 584,589 Pflugfelder, O., 691, 704 Pantelouris, E. M., 272, 302 Paperna, I., 308, 312, 390 Phillips, W. L., 196, 229 Pichon, G., 92, 113 Pappas, G. D., 242,297 Piekarski, G., 634, 636, 637, 638, 651, Paraense, W. L., 321,390 663, 665, 668 Parent, G., 39, 59 Pierce, N. F., 88, 112 Parfitt, J. W., 586, 591 Pierce, N. W., 188 Park, O., 361, 362, 384 Pierroti, P., 585, 595 Park, P. O., 143, 145,189

Oksche, A., 220,231 Oliver-Gonzalez, J., 291, 302 Olivier, L., 318, 390 Olsen, L. S., 569,596 Olsen, 0. W., 415, 479, 482, 488, 504, 506, 527, 529,552,553,556 Olson, R. E., 677, 678, 699, 704 Olteanu, G., 571, 572, 585,594 Omer, S. M., 122, 188 Onabanjo, A. O., 617,629 Onar, E., 586,598 O'Neilli, D. K., 148, 149, 188 O'Nuallain, T., 189 Oppermann, W. H., 639,644, 664 Opuni, E. K., 710, 728 Organization for Economic Cooperation and Development, 79, 113 Orihara, M., 421,436,442,460,474,479 Orihel, T. C., 615, 627 Osborne, D. J., 320, 385 Oschman, J. L., 194, 231 Osler, W., 54, 67 Ostlind, D. A,, 716, 729 O'Sullivan, P. J., 148, 168, 189 Otsuru, M., 611,629 Overdulve, J. P., 634, 665 Owyang, C. K., 319, 321, 322,389 Ozerskaia, V. N., 584, 585, 594

749

A U T H O R INDEX

Piez, K. A., 366, 386 Pigon, A., 364, 376, 384 Pike, A. W., 709, 728 Pillai, J. S., 131, 189 Pillai, M. K. K., 132, 133, 185 Pimentel, D., 319, 320, 391 Pinto, C., 57, 68 Piper, R. C., 642, 665 Pitchford, R. J., 319, 391 Pittaluga, G., 5, 27, 46, 47, 68 Plapp, F. W., 118, 189 Pleger, D., 560, 593 Plimmer, H. G., 5, 55, 68 Polhemus, J. A., 31, 59 Polt, S . S.,645, 659 Polzenhagen, M., 568, 595 Pomonis, J. G., 131, 137, 188 Ponselle, A., 19, 39, 68 Poole, J. B., 489, 553 Poole, R. L., 483, 551, 708, 726 Poorbaugh, J. H., 156,184 Popova, K. A., 585,594 Porchet-Hennerk, E., 608, 623, 630 Porter, A., 15, 27, 36, 37, 45, 49, 50, 51, 52, 53, 61 Porter, D. A,, 562, 563, 596 Porter, J. A., 619, 629 Potts, T. W., 365, 391 Powell, J. W., 320, 391 Powers, K., 604, 630 Powers, K. G., 658, 664 Powers, R., 616, 628 Prakash, O., 658, 662 Prashad, B., 513, 555 Pratt, I., 677, 678, 699, 704 Premier, G., 608, 623, 630 Presidente, P. J. A., 586, 595 Pretorius, J. L., 586, 595 Price, C. E., 532, 553 Probert, A. J., 562, 563, 595 Proctor, E. M., 718, 727 ProkopiE, J., 420, 433, 447, 474, 475, 709, 728 Prowazek, S., von, 607, 628 Pujatti, D., 35, 36, 49, 56, 68 Pullen, E. W., 198, 231 Pullin, J. W., 586, 591 Pullin, R., 205, 216, 232, 292, 305

Q Quentin, J. C., 420, 474, 709, 728 21

Quesnel, J. J., 609, 627 Quilici, M., 9, 67 Quinn, T. C., 610,627 Quintana, R. P., 129, 189

R Raab, S. O., 29, 31, 58 Rabbege, J., 623, 626 Rabson, A, S., 648, 662 Race, G. J., 247, 257,303, 425,437,441, 449, 454, 472, 474 Rajapaksa, N., 607, 618, 628 Rakai, I., 131, 189 Ramachandran, S.,579, 580, 595 Ramalho-Pinto, F. J., 361, 375, 387 Rambourg, A., 214,231 Ramsdell, T. G., 647, 666 Randall, R. J., 326, 389 Rappoport, S.,31, 59 RaSka, K., 81,88,112, 114 Rastegar, M., 698, 705 Rattig, A., 34, 68 Rausch, R., 421, 428, 431, 433, 475 Rausch, R. L.,492, 523, 530,553 Rawson, D., 420, 440, 475, 516, 521, 522, 554 Read, C. P., 271,272,303,305,438,450, 469,477 Readshaw, J. L., 317, 391 Reed, V., 636, 663 Rees, G., 244, 246, 247, 250, 255, 294, 295, 297, 303, 441, 451, 475, 502, 517, 523,554 Reeve, 318 Reeves, W. C., 92, 107, 108,114 Refuerzo, P. G., 563,595 Reichenbach-Klinke, H., 33, 68 Reichenbach-Klinke, H. H., 524, 540, 554 Reid, J. F. S., 174, 189 Reid, W. M., 494, 554 Reinecke, R.K., 586, 595 Reissig, M., 262,268,269, 271,273, 274, 275, 303 Remington, J. S.,641,642,645,648,651, 652, 653, 654, 655, 656, 657, 660, 661, 662, 663, 664, 666, 667 Remky, H., 646,666 Reuter, J., 718, 719, 728 Reyes, P. V., 563,595

750

A U T H O R INDEX

Reynolds, E. S., 234,238, 241, 243,244, 246, 254, 260, 262, 264, 266, 267, 271, 272, 273, 275, 283,304 Reynoldson, T. B., 316, 317, 391 Rhenova, M., 575,595 Rhesetnikov, A. G., 585, 595 Rich, G. B., 152, 155, 189 Richards, C. S., 321,391 Richards, J. G., 295, 301 Richards, M. J., 361, 365, 381, 391 Richards, 0. W., 320,391 Richardson, G., 147,189 Richardson, L. R., 15,27, 36, 37,45,49, 50, 51, 52, 53, 61 Riches, J. H., 148, 189 Richter, S., 569, 584, 598 Riddiford, L. M., 320, 391 Ridley, D. S., 94, 112 Ridley, R. K., 570, 593 Rieckmann, K. H., 622, 628 Riehl, L. H., 155, 189 Rifkin, E., 234, 235, 237, 238, 250, 251, 293, 303, 411, 447,475, 711, 714, 715, 728 Rigby, B. J., 689, 704 Rigby, J. E., 420,440,475, 516,521,522, 554 Riley, P. A., 31, 71 Riou, G., 14, 68 Rioux, J. A., 420, 474, 709, 728 Riser, N. W., 482, 496, 503, 510, 533, 535, 537, 539, 540,554 Ritchie, L. S., 319, 391 Rivetti, F. S., 644, 659 Rivosecchi, L., 137, 189 Roberts, L. S., 444, 468 Robertson, D., 560, 595 Robertson, M., 19, 68 Robertson, R. H., 156, 191 Robinson, D., 326, 391 Robinson, D. L. H., 271, 272,303 Robinson, E. S., 677,678, 697, 703, 704, 705 Robinson, M., 586, 595 Robinson, R. G., 460,477 Robinson, R. M., 609,629 Robinson, W. S., 657, 666 Robson, R. T., 238, 247, 248, 249,303 Roche, J., 269, 303 Rodel, H., 639, 644,664 Rodhain, J., 50, 51, 69 Rodin, J., 619, 622, 629

Rodriguez, J. G., 142,192 Rogers, W. P., 609, 629 Rohde, K., 200, 210, 231, 232 Romboli, B., 579, 595 Romer, A. S., 538,554 Rommel, M., 636, 640, 642, 643, 658, 660, 662, 663, 666 Rondanelli, E. G., 637, 666 Roque, A. L., 463,467 Rosa, W. A. J., 172,189 Rose, C. R., 319,387 Rose, F. C., 360, 376,391 Rose, J. H., 561,562, 563,570, 571, 572, 573, 574, 575, 576, 578, 580, 582, 584, 595,596 Rose, S. M., 360, 376, 391 Rosebrough, N. J., 326,389 Rosen, F., 485, 487, 490, 507, 551,554 Rosen, H., 326, 391 Ross, D. B., 586,596 Rossan, R. N., 604, 630 Ross Institute of Tropical Hygiene, 112, 114 Rossler, R., 449,453,475 Roth, J. A., 648, 666 Rotherham, S., 697, 705 Rothman, A. H., 214,232,438,475,691, 705, 717, 730 Rottini, G., 646, 666 Roubaud, E., 50, 51, 66 Rouge, M., 129,183 Roulston, W. J., 168, 190 Rousselot, R., 51, 69 Rowan, W. B., 322,392 Rubin, H., 364, 392 Rudzinska, M. A., 622, 624, 629 Ruiz, A., 39, 69, 636, 640, 663, 666 Rukavina, J., 425,475, 585, 596 Ruskin, J., 653, 654, 655, 663, 667 Russel, E., Jr, 657, 666 Russell, P. F., 248, 299 Rybak, V. F., 575,576,596 Rybicka, K., 206, 217, 232, 396, 398, 399,404,416,417,475,483,493,494, 495,554 Rycke, P. H., de, 442,444,455,458,460, 469, 474, 475 Ryenaer, M., 25, 61 Ryley, J. F., 39, 41, 71, 148, 149, 190 Ryiavy, B., 420, 475, 488, 489, 502, 554

Rytel, M. W., 656, 667

AUTHOR INDEX

75 1

Scholtyseck, E., 623, 629, 638, 667 Schramlova, J., 449, 450, 451, 452, 453, 476 Saavedra, P., 651,667 Schreck, C., 129,189 Sabbaghian, H., 318, 319,386 Schrevel, J., 608, 623, 630 Sack, R. B., 88,112 Schuckmann, W., von, 562,596 Sahba, G. H., 698, 705 Schulz, H.-P., 636, 666 Saint Girons, M. Ch., 431, 469 Sakamoto, T., 421, 426, 436, 450, 455, Schultz, R. S., 584, 589 Schulz, R. S., 574, 575, 576, 584, 596 460,476, 479 Schwabe, C. W., 451,455,471,477 Sakazaki, R., 88,112 Schwartz, B., 562, 563, 564, 565, 567, Sakuma, E., 200,232 596 Sakumoto, D., 237, 243,260,300 Schwarz, H., 154, 184 Salimbeni, A., 5, 46, 54, 66 Schwetz, J., 50, 51, 69 Saliou, P., 605, 626 Scorza, J. V., 5 , 6, 21, 24, 37, 55, 57, 69 Salt, G., 696, 705 Scott, H. H., 69 Samaan, S. A., 312,387 Scott, J. S., 443, 476, 497, 499, 514, 518, Sanabria, Y., 619, 630 519, 523, 530,554 Sanchez, F. F., 118,190 Sandeman, I. M., 484,490,525,544,554 Seamen, E., 648, 664 Seddon, H. R., 172,190 Sandosham, A., 610,628 Sedlmeier, H., 579, 580, 596 Sanger, V. L., 643,667 Seebeck, R. M., 163,188 Saoud, M. F. A., 322,390 Seed, J. R., 14,24, 25, 27, 29, 53,59,69 Sasaki, N., 569,596 Seidel, J., 412, 479 Satir, P., 205, 229 Seidel, J. S., 721, 729 Sauer, K., 455,471 Seifert, G. W., 171, 190 Savage, K. E., 188 Sekikawa, H., 611, 629 Sawada, I., 515, 521,554 Self, J. T., 523, 549 Sawyer, R. T., 35,69 Self, L. S., 130, 183 Saz, H. J., 695, 700, 705 Semenov, P. V., 156,190 Schanzel, H., 571, 572,596 Semper, C., 361, 392 Scheibel, L. W., 695,700, 705 Sen, A. B., 717, 726 Scheidegger, S., 428,467 Sen, H. G., 560, 567, 569, 596 Scheifinger, C. C., 693, 695, 703 Seneca, H., 372, 392 Schein, E., 636, 666 Senft, A. W., 233,270,272,280,282,303 Scheinberg, E., 715, 729 Senft, D. G., 270,303 Schiefer, B., 579, 580, 596 Schiller, E. L., 283, 298, 416, 419, 476, Senn, J., 5, 32, 69 Senterfit, L. B., 291, 303 515, 516, 519,554 Serbus, C., 449, 450, 451, 452, 453, 476 Schlechte, F. R., 317, 392 Sergent, Ed., 5,22,24, 33,46,47, 48, 69 Schletzer, A. M., 565,588 Sergent, Et., 5, 22, 24, 33, 46, 47, 48, 69 Schlossmann, A., 29,69 Schmidt, G., 481, 482, 498, 508, 510, Serra, P., 436,460, 474 Service, M. E., 138, 290 540,554 Shadduck, J. A., 642,665 Schmidt, G. D., 698, 705 Shahlapour, A. A., 585,597 Schmidt, H. D., 52, 69 Shakhmatova, V. I., 428, 476 Schmidt, K. P., 361, 362, 384 Shalashnikov, A. P., 24, 27, 46, 48, 69 Schneider, I., 618, 619, 629 Shalaeva, N. M., 574, 593 Schneider, J., 76, 114 Shanahan, G. J., 147,190 Schnell, J. V., 620, 628 Shannon, R. C., 37,69 Schnitzer, B., 626, 629 Shannon, W. A,, 214,232,264,277,279, Schnitzerling, H. J., 168, 190 280,283,297,303 Schoenfeld, C., 610,630 S

752

AUTHOR INDEX

Shaw, J. N., 560, 564, 565, 597 Shaw, R. D., 147, 167, 168, 169, 170, 183,190 Sheahan, B. J., 173, I90 Sheffield,H., 638, 667 Sheffield, H. G., 632, 633, 637, 638, 657, 667 Shemanchuk, J. A., 159, 160, 190 Sherman, M., 118, 190 Shiff, C. J., 312, 316, 361, 386, 392 Shiroishi, T., 604, 630 Shockman, G. D., 373,393 Shope, R. E., 567,596, 597 Shrivastava, S. C., 268, 302 Shults, R. S., 406, 476 Sibalic, S., 560, 594 Siddiqui, W. A., 619, 620, 628 Siebold, C. T. T. von, 11, 69 Siegel, S. E., 648, 666, 667 Siim, J. C., 633, 634, 636, 639, 642, 662, 664,667,669 Sikes, R. K., 106, 114 Silk, M. H., 257,260, 261, 264, 268,272, 274, 275, 276, 277, 279, 280, 303, 304 Silva, I. I., 9, 69 Silverman, P. H., 494, 554, 707, 728 Silverstein, R. M., 320, 394 Simon, J. L., 708, 725 Simpson, D. I. H., 92, 107, 108, 114 Simpson, H. R., 145, 192 Simpson, L., 14, 69 Sinclair, I. J. B., 582, 594 Sinden, R. E., 622, 623, 624, 627, 629 Singer, I., 32, 63 Sivers, P., 145, 190 Sivickis, P., 561, 597 Skaer, R. J., 198, 200, 202, 206, 232 Skerman, K. D., 585,597 Skinner, J. C., 614, 627 Slais, J., 268, 304, 398, 413, 422, 423, 424, 427, 428, 435, 437, 440, 442, 449, 450, 451,452, 453, 454, 455, 456, 457, 458,463,464,476,485,554,714, 715, 716, 728 Slen, S. B., 151, 188 Sloan, W., 361, 362, 386 Smith, C. E. G., 76, 84, 114 Smith, C. L., 26, 28, 29, 69 Smith, C. N., 129, 131, 132, 187, 190 Smith, C. S.,614, 629

Smith, J. H., 234,238,241,243,244,246, 254, 260, 262, 264, 266, 267, 271, 272, 273, 275, 283,304 Smith, J. K., 443,462,476,477, 711, 728 Smith, L. S., 611, 612, 614, 628 Smith, M. S., 147, 148, 184, 192 Smith, M. W., 170, 190 Smithers, S. R., 251, 254, 269, 284, 285, 289,298, 300, 304, 309, 392 Smyth, J. D., 196, 213, 232, 322, 392, 404, 405, 406, 421, 435,436, 437,443, 444,445, 456, 460,462,463, 471, 477, 486, 487, 494, 495, 497, 499, 514, 518, 519, 523, 530,555, 715, 721, 729 Snyder, N., 364,392 Soans, A. B., 319, 320, 391 Sodeman, T. M., 614, 626, 629 Sodeman, W. A., 245, 304 SofrenoviC, D., 583, 592, 597 Sogoyan, I. S., 578, 581, 597 Sokol, J., 584, 585, 586, 597, 598 Sokolic, A., 583, 592, 594, 597 Solomon, M. E., 316, 392 Solonenko, I. G., 709, 729 Soltice, G. W., 715, 729 Sommer, R., 642,666 Sorelius, L., 586, 594 Sorice, F., 436, 460, 474 Sorsoli, W. A., 19, 41, 65 Soulsby, E. J. L., 582, 597 Sourander, P., 657, 662, 664 Southgate, V. R., 205, 216, 232, 235, 237, 245, 248, 249, 252, 257, 292, 293, 304 Southward, A. J., 194, 232 Southward, E. C., 194,232 Southwell, T., 513, 539, 555 Southwood, T. R. E., 320, 391 Southworth, G. C., 25, 27, 69 Spaskii, A. A., 481, 482, 547, 555 Specht, D., 462,477, 510, 555, 715, 729 Speeg, K. V., Jr, 373,392 Spence, I. M., 257, 260, 261, 264, 268, 272, 274, 275, 276, 277, 279, 280, 303, 304 Spielberger, U., 145, 190 Spitalny, G. L., 605, 629 Spitzer, J. M., 372, 392 Spotte, S. H., 373, 375, 392 Springell, P. H., 163, 188 Sprintz, H., 622, 623, 629 Stagna, S., 645, 651, 667

A U T H O R INDEX

Standen, C. P., 269, 271, 302 Standen, 0. D., 284,304 Standfast, H. A., 137, 190 Stark, G. T. C., 512,555 Stebbins, J. H., 15, 46, 70 Steelman, S. L., 716, 729 Stein, R. L., 604, 629 Stein, T., 695, 705 Steinert, G., 14, 20, 70 Steinert, M., 13, 14, 19, 20, 39, 40,41, 59, 64, 70 Stephens, G. C., 365,392 Sterling, C. R., 621, 623, 624, 626, 629 Stevenson, A. C., 13, 51, 70 Stirewalt, M. A., 237,238,240,241,242, 247, 248,250,299, 301,304 Stites, E., 438, 468, 719, 724 Stockem, W., 268, 304 Stoker, M., 320, 361, 378, 392 Stranach, F. R., 680, 681, 682, 685, 705 Strannegard, O., 652, 653, 655,667 Stratton, V. S., 149, 185 Streber, F., 34,41, 68 Streeter, J., 317, 392 Strickland, B. C., 697, 705 Strome, C. P. A,, 623, 626 Strumwasser, F., 317, 392 Stullken, R. E., 419, 470, 513, 550, 713, 725 Stunkard, H. W., 482,484,485,486,494, 498, 531, 533, 534, 536, 539, 543, 555 Sturrock, B. M., 318, 361,392 Sturrock, R. F., 361,392 Subra, R., 125, 190 Subrahmanyan, D. V., 88,112 Subramaniam, T., 569,597 Sugimura, M., 421, 426,450,455, 476 Suguri, S.,237,243, 260, 300 Sullivan, J. F., 560, 564, 565, 597 Sullivant, N. D., 168, 190 Sutherst, R. W., 168, 190 Suzuki, J., 28, 70 Suzuki, M., 652,663 Svarc, R., 576, 577, 599 Sweatman, G. K., 435,460,477 Swiderski, Z., 397, 398,477

T Tabibzadeh, I., 190 Taliaferro, L. G., 612, 620, 629 Taliaferro, W. H., 612, 620, 629

753

Talice, R. V., 463,477 Tamaski, K., 565,597 Tan, B. D., 715, 729 Tanabe, M., 15,23, 27,48, 70 Tanaka, H., 657,665 Taniguchi, O., 569,598 Tarry, D. W., 141, 151,191 Taubr, J. H., 319,391 Taylor, A. E. R., 39, 70, 707, 720, 725, 729 Taylor, E. R., 454,471 Taylor, M. A., 83, 113 Taylor, R. T., 125, 191 Taylor, W. C., 150, 191 Tazieva, Z. Kh., 435, 477 Tedla, S.,699, 705 Teesdale, C., 318, 392 Teesdale, G., 322, 390 Telford, S. R., 10, 60 Terry, R. J., 254,269,284,285,289,302, 304, 309,392 Terwedow, H. A., 610,627 Terzakis, J. A., 251, 304 Teskey, M. J., 155, 191 Thakur, A. S.,451,477 Theakston, R. D. G., 624,629 Theologides, A., 646, 667 Thiermann, E., 645, 651, 667 Thillet, C. J., 321, 390 Thomas, B. M., 376, 380,390 Thomas, J. G., 380,393 Thomas, L. J., 504, 506, 520,555 Thomas, P. L., 148, 191 Thomas, R. J., 571, 585,597,598 Thompson, B. W., 145,191 Thompson, C. 0. M., 159, 160,190 Thompson, G. E., 167, 168, 169, 183 Thompson, W. R., 316,320,393 Thomson, J. G., 9, 70 Thorburn, J. A., 167, 168, 169,183 Thornberry, H., 173, 191 Thorpe, E., 268, 304 Thorsell, W., 268, 284, 297, 304 Thorson, R. E., 717, 726 Threadgold, L. T., 233, 247, 260, 262, 264, 266, 267, 268, 271, 272, 273, 275, 277, 279, 283, 299, 302, 305, 437,438, 450, 473, 477 Thurston, J. P., 495, 525, 555 Tilney, L. G., 208, 232 Timm, W., 572,589 Timms, A. R., 272,305

154

AUTHOR INDEX

Valencia, L. C., 5, 15, 24, 25, 46, 68 Valsamis, M. P.,648,668 Vanderberg, J., 619,622,629 Vanderberg, J. P.,619,630 Van der Borght, O., 370,393 Van der Schalie, H., 361,393 Van der Zypen, E., 637,668 Van Puymbroeck, S.,370,393 Varenika, D.,425,475 Varley, G.C.,316,393 Vegors, H.H., 716, 729 Vejlens, G.,31, 70 Venard, C.E.,492,498, 515, 519, 522, 528,555 Verolini, F., 605,627 Verster, A,, 396, 478, 573, 588, 598 Verwey, W. F., 88,112 Vickerman, K.,14,24, 70,624,629 Victor, D.A.,569,597 Vieira, E. C.,368,393 Vietzke, W.M.,648,668 Vik, R.,495,503,504,505,506,510,525, 526, 537, 542,555 Vilagiova, I., 141,191 Viles, J. M.,438,472 Villalobos, T. J., 31, 71 Villot, F. C.A., 485,555 U Vinckier, D., 608,623,630 Ubelaker, J. E., 416,447,448, 467, 478, Visser, P. S.,319,391 713,729 Visser, S. A.,320, 374,384 Uegaki, J., 49,67 Vitali, D.,645,668 Ueno, H., 569,598 Vivier, E.,608,623,630 Uglem, G.L.,677, 678, 705 Vodrazka, J., 584,598 Uilenberg, G.,605,609,628 Voge, M.,406,412,413, 416,418,419, Ulmer, M.J., 462, 471, 490, 493, 522, 420,436,445,446,447,448,456,459, 548 462,463,469,471,477,478,479,482, Umathevy, T., 321,388 483,493,497,498,499,500, 501,502, Umov, A. A., 585,598 503,505,507,510,513, 515, 516,517, Underdahl, N.R.,567,594,596 519, 520,521, 522,527,528,529,555, Underhill, D.,646,664 556,707,708, 709, 715,720,721, 724, Upmanis, R. S., 610,630 726, 729 Usami, S.,618,629 Vogel, C. L., 648,664,668 Ustinov, D.,562,563,598 Vogel, H.,242, 304, 407, 479, 502, Utech, K.B. W., 160,191 556 Uvarov, B., 320,361,393 Voller, A., 604, 630 Von Brand, T., 368,393 Voors, A. W.,311, 393 V VraZiC, O.,569, 584,598 Vucetich, M.,5, 23,27,28, 55, 71 Vago, C., 131,186 Vural, A., 586,598 Vainisis, S. J., 644,668

T i m , G.L.,158,185 Timofeev, B. A., 408,409,440,478 Timofeev, V. A.,397,409,440,477,478 Tindal, J. S.,29, 70 Tiunov, I., 563, 565,568,598 Tobey, E.N.,15, 24,27,38,45,46,50, 61, 70 Todd, A. C., 560,565,569,590 Todd, J. L..5, 11, 15, 45,50,51,61 Toennies, G.,373,393 Tofler, Alvin, 75, 114 Tompkins, S. J., 201, 210,228 Tongu, Y.,237,243,260,300 Tonn, R.J., 124,191 Trager, W.,622,629 Treeby, P.J., 148, 160,161,191 Tremonti, L.,654,667 Trench, M.E.,342,393 Trench, R.K.,342,393 Tribouley, J., 129,183 Truong, T. K.,605,627 Tsuda, A,, 129,188 Tsunematsu, Y.,652,663 Tsuong, T. K.,605,626 Tsvetaeva, N.P.,579,598 Turlygina, E. S.,574,593

AUTHOR INDEX

W Wadji, N., 292, 305, 319, 393 Wagner, A., 249, 305 Wagner, O., 424, 425,426, 479 Waitz, J. A., 442, 479 Walker, T. F., 118,191 Walkey, M., 699, 705 Wallace, F. G., 9, 11, 12, 39, 63, 71 Wallace, G. D., 639, 640, 652, 668 Walley, J. K., 569, 584, 585, 586, 598 Walliker, D., 608, 630 Walters, D. E., 679, 702 Walton, A. C., 32, 35, 37, 38,46,48,49, 50, 54, 55, 71 Walton, B. C., 654, 667 Walton, G. S., 175, 185, I86 Walton, R. L., 155, 191 Wandera, J. G., 579,598 Ward, P. F. V., 694, 702, 705 Ward, R., 646,661 Ward, R. A., 605, 620,627,630 Wardle, R. A., 481, 483, 484, 485, 486, 490, 491,492,493,494, 495,496,498, 499, 503, 505, 507, 508, 510, 511, 512, 517,520, 521, 524, 526, 528, 529, 533, 536, 538, 539, 540, 541, 542, 546, 556 Wiireborn, I., 370,393 Warner, R. W., 483, 551, 708, 726, 729 Warren, B., 139,192 Warren, K. S.,308, 309, 311, 312, 387 Warren, M., 604,607,609,611,614,615, 616, 619,627, 630 Wasielewski, T. von, 27, 71 Watanabe, K., 237, 240, 310 Watanabe, S., 569, 598 Watanabe, Y., 88,112 Waterhouse, M., 175, 186 Watson, A., 380, 393 Watson, W. A,, 643, 651,660, 668 Way, M. J., 320, 380, 393 Webbe, C., 105,113 Webbe, G., 308,309,310,311,312,314, 316, 317, 318, 319, 323, 361, 368, 388, 393,404,479 Webster, G. A., 460,462, 479 Wedderburn, N., 605,630 Wedl, C., 27, 71 Weidhass, D. E., 133, 189 Weidner, T., 366,388

755

Weiland, G., 633, 634, 636, 640, 663, 664,668 Weinbach, E. C.,438,449,450,454,468, 474, 713, 719, 727, 729 Weinmann, C. J., 448, 479, 488, 503, 556, 707, 717, 729, 730 Weinstein, P. P., 217, 229 Weintraub, J., 156, 191 Weitz, B., 191 Weller, I., 626, 627 Wells, R. W., 152, 183 Wenyon, C. M., 49, 71 Werner, H., 636, 638, 649, 663, 668 Why, M., 610, 630 Wetzel, R., 489, 515, 519, 528, 556, 572, 598 Whalley, W. B., 320, 391 Wharton, D. R. A., 361, 393 Wharton, M. L., 361,393 Wharton, R. H., 160, 167, 168,190,191 Whetstone, T. M., 169, 184 Whitcomb, R., 489, 556 White, E. G., 560, 590 White, G. B., 191 White, L. P., 31, 59 Whitehead, C. C., 175, I91 Whitfield, P. F., 678, 679, 702 Whitfield, P. J., 675, 679, 683, 685, 688, 701, 703, 705 Whitten, L. K., 586, 598 Whitten, M. J., 120, 150, 151, 178, 191 Whittlestone, P., 560, 565, 598 Wicht, M. C., 142,192 Widmer, E. A., 415, 479, 529, 556, 715, 729 Wigglesworth, V. B., 192 Wikerhauser, T., 569,584,598,718, 730 Wikgren, B. J., 410, 468, 492, 548, 712, 723, 730 Wikgren, B.-J. P., 408, 409, 410, 439, 479, 492, 556 Wilanowicz, H., 710, 724 Wilcocks, C., 105, 109,114 Wilkens, E. H., 173,184 Wilkinson, C. F., 118, I92 Wilkinson, P. R., 163,170,171,185,192 Wille, 29 Willerson, D., 622, 628 Williams, B. L., 39,41, 71 Williams, C. M., 320, 394 Williams, D. C., 378, 394 Williams, D. W., 574, 575, 576, 598

156

A U T H O R INDEX

Williams, G. C., 378, 394 Williams, H. H., 483, 533, 537, 539, 540, 556 Williams, H. U., 24, 38, 65 Williamson, M., 316, 317, 394 Wilson, A., 116,192 Wilson, B. H., 140,192 Wilson, E. O., 320, 361, 364, 365, 377, 378,394 Wilson, F., 316, 394 Wilson, G. I., 582, 583, 598, 599 Wilson, M. E., 616, 628 Wilson, R. A., 205, 216, 219, 232, 250, 292,298,305, 317, 371,394 Wintrobe, M. M.,29, 31, 58 WiSniewski, L. W., 511, 512, 525,556 Wihiewski, R. J., 482,556, 710, 730 Wisseman, C. L., Jr, 516, 521,557 Witte, H. M., 634, 636, 637, 638, 665 Woke, P. A., 37, 71 Wolf, Z. V., 576,589 Woo, P. T. K., 5, 9, 46, 52, 53, 54,!57, 72 Wood, D. L., 320,394 Wood, J. C., 147, 148,184,192 Wood, R.M., 646,668 Wooderson, L. A., 160,191 Woodhouse, M. A., 680,681,682, 705 Woodruff, A. W., 95, 106,114 Woods, A. C., 646,668 Work, K., 632, 633, 634, 636, 639, 641, 642,662,664,667,668, 669 World Health Organization, 78, 81, 83, 84,86,88,89,94,97,98,102,103,106,

110, 114, 116, 117, 119, 121, 122, 124, 125, 126, 130, 131, 142, 144, 156, 158, 162, 163, 176, 192, 307, 314, 394, 605, 630 Worley, D. E., 586, 595 Worms, J. J., 617, 628 Worms, M. J., 604, 616,628 Worte, W., 651, 663 Wright, C. A., 205, 232, 290, 292, 305, 308, 319, 320, 361, 374, 378,394 Wright, D. E., 220,228 Wright, F. J., 110, 114 Wright, J. W., 121, 127 192

Wright, K. A., 13, 72 Wright, P.A., 29, 72 Wright, R. D., 242, 305, 680, 681, 682, 683, 685, 686, 688, 706 Wright, W. H., 312,394 Wurster, D. D., 26, 29, 72 Wurster, D. H., 28, 66 Wynne-Edwards, V. C., 320, 361, 362, 374, 379,394 Y Yajima, Y., 250, 251, 261, 277, 280, 297 Yamaguti, S., 412, 479, 481, 482, 508, 509, 511, 520, 525, 538, 539, 540, 542, 557 Yamane, Y., 712, 730 Yamashita, J., 436, 460, 479 Yamomoto, I., 188 Yanagawa, R.,653,669 Yasutomi, K., 186 Yau, T. M., 692, 696, 703 Yazawa, M., 657, 665 Yeh, S.,657, 663 Yen, J. H., 133, 192 Yeo, D., 145, I92 Yeoman, G. H., 139,192 Yoeli, M., 610, 619, 622, 626, 627, 629, 630 Yonemochi, K., 643,665 Yoshida, Y., 129, 176, 188 Young, G. A., 567,596 Young, M. D., 619,630 Young, R. T., 421,449,480 Young, W. A., 10,27, 37, 51,65 Yu, M. L., 320, 376,394

Z Zaentz, S. D., 29,66 Zaman, V., 621, 627, 635, 638,669 Zdarska, Z., 268,304,454,480 Zinichenko, I. I., 585, 594 Zmornay, I., 576, 577,599 Zuckerman, A., 604,630 ZukoviC, M., 569, 584, 598, 718, 730 Zunker, M., 562,596

Subject Index Page numbers in italics indicate illustrations

A albopictus, cross-mating with vector Abida frumentum, 576, 517 species, 133-4 Acanthatrium oregonense, cercarial tegupolynesiensis ment structure, 244, 247 elimination of vector, 133-4 Acanthobothriwn, procercoid, 507 scutellaria coronatum, metacestode, 524 sterile mating, 133 olsensi, metacestode, 524 simpsoni, control, 125 Acanthocephala, development in intertaeniorhynchus,control, 122 mediate and definitivehosts, 678 “Agamospecies”, 3, 4, 16, 21 Acanthocephalus lucii, fine structure, Agriolimax, 576, 578 nuclei, 676 agrestis, 575, 576 ranae, reticulatis, 575 assimilation, 691 Airports as rat habitats, 94 nuclei, 676 Air travel, cause of increase in spread proboscis, 672-4 of disease, 79-84 tegument structure, 682 &lee effect, 365 Acanthucotyle, epidermis structure, 218 in blowfly larvae, 366 secretory inclusions, 212 in molluscs, 359-61, 362, 383 sense organs, 223, 224 Alveococcus multilocularis elegans, epidermis structure, 207, 208 cysticercus, 421 sense organs, scoleces, 462 .224 lobianchi, sense organs, 220,222, 224, Amblyomma, disease vector, 163 225 americanum, resistance to acaricide, Acanthoparyphium spinulosum, cyst, 216 169 redia, 295 Amia calva, 532 Acanthor, 675-6, 683, 688 Amoebiasis, 85, 95 Acaricides, 180-2 Amoebic dysentery, 95 tick control, 163-7 Amoebotaenia, hosts and sites, 514 Acarina, 37, 38 life cycle, 519 Acoela, 194, 196 metacestode, 513, 518 Acris gryllus, 7, 38, 72 sphenoides, Aedes aegypti metacestode, sequence and names, control 530 breeding sites, 123-4 Amphibdella, epidermis, 212, 213 measures, 122, 125, 128, 132 jlavolineata, epidermis, 207, 216 resistance to insecticides, 120 microvilli, 208 culture, 618 receptor, 220 vector of disease surface coat, 215 anuran trypanosomes, 37 Amphicotylidae, development, 401 danger of importation, 83 Amphiscolops langerhansi, epidermis, dengue, 108 194, 196 yellow fever, 92, 93, 123 Ancylostoma duodenale, 1 06 757

758

SUBJECT INDEX

Anomalurus derbianus, 608 peli, infection with malaria parasites, 608 Anomotaenia, life cycle, 515, 519 Anonchotaenia, oncosphere, 496 Anopheles adult behaviour, 122 breeding sites, 122 control, 127 resistance, 125-7 albimanus, resistance, 127, 129 (Arribalzagia) mediopunctatus, 612 atroparvus, 616 balbacensis, 122, 607, 614, 615 elegans, 612 jreeborni, 614, 617 control by fish, 130 funestus, 126 gambiae, control probfems aestivation, 122 cross matings, 134 introduction in Brazil, 82 in Philippines, 83 melas, breeding sites, 122 hyrcanus simensis, resistance, 126 (Kerteszia) crwi, 612 neivai, 612 labrmchia atroparvus, 122, 124 letifer, 610 leucosphyrus, 611 maculatus, 615 nili, 126 (Nyssorhynchus)oswaldoi, 612 pharoensis, I22 pseudopunctipennis, 126 punctipennis, 122 punctulatus, 83 rufipes, 126 sacharoyi, 122, 125 stephemi, 83, 122, 129, 614, 616, 619 sunahicus, 122 umbrosus, 609,610 vagus, 122 walkeri, 122 Anoplocephala, larval stages, 414 Anoplocephalidae,cysticercoid,418,521 Anuran trypanosomes ancestry, 10 distribution, 47-55 hosts, 45-6 vectors, arthropod, 37-8 himdinid, 56-7

Aotus trivirgatus, infection with malaria human, 619 simian, 620 Aphanostoma diversicola, epidermis, 194 Apholate, 132, 180 Apis mellifera, inhibitory chemicals, 377 Aploparaksis cercomer, 517, 521 life cycle, 515 hosts and names, 519 metacestode, sequence and names, 529 filum, cysticercoid, 420 furcigera, cysticercoid, 420 Aplysia californica, circadian rhythms, 317 Apodemus flavicollis, 431 Aporidea, relationships, 482 Archigefes, adult, 511 in invertebrate host, 484, 531 iowensis, procercoid, 412, 525 limnodrili life cycle, 490, 503 procercoid, 525 arprocarb, tsetse control, 144 Arthropods, haematophagous, as vectors, 37-8 Ascaris Iumbrlcoides, 106 Aspidogaster conchicola, epidermis, 201 Aspidogastrea, epidermis, 210, 218 Aswan dam, bridge for mosquito vector, 82 Atriotaenia procyonis, larval stages, 41 5 Aziridine chemosterilants, 132

B Bacciger bacciger, sporocyst, 294, 295 Bacillus thuringensis, spores in insecticides, 117 Balmtidium coli, 95 Bafracobdellapicfa, 36 Besnoitia, 635, 659 Benzene hexachloride (BHC), 122, 123 residue in butter, 116 resistance in human lice, 159 sheep dip, 172 tsetse control, 144 Benzene sulphonamide, 122 Biacetabulum inji-equens,procercoid, 412 macrocephalum, procercoid, 412 Bilharziasis, 105 see also Schistosomiasis

SUBJECT I N D E X

Biomphalaria angulosa, crowding effect, 361 glabrata, 308 Allee effect, 360 effects on growth and reproductive rates of age, 382 ammonia, 350-2, 372-3 381 biomass, 359 calcium, 3434, 3524, 355, 369, 370 cellulase, 346, 364, 366-7 conditioning of media by snails, 341-8 conductivity in medium, 343,344, 369 crowding, 361 grazing of snails, 363 inhibiting factors, 357, 361, 364, 365, 373, 374, 375-82 oxygen consumption, 368 PH, 342-3, 370-1 pheromones, 383 plant substances, 363 promoting factors' 3279 356-9y 363-4, 365, 382 sing1e and paired 337-40 survival rate, 319 temperature, 316 weight, 330, 334-41 sudanica tanganyiensis lethal factor, 374-5 Blowfly, biological control, 147 Boophilus, control, 164 &coloratw, resistance to acaricides, 167 microplus,eradication, 167 pest of cattle, 159, 163 resistance to acaricides, 167 Borrelia recurrentis. 157 Bothrimonus sturionis, metacestode, 525 Bothriocephalus hosts and names, 504, and sites, 506 claviceps, metacestode, sequence and names, 525 Brachyurus calms, infection with malaria parasites, 614 Bromocyclen, tsetse control, 144 Bromophos, control of sheep nasal fly, 157 Bruce effect, 377

759

Brucella melitensis, 655 Brugia malayi, distribution, 105 Bufo, 7 boreas, 6, 7, 37 bufo, 6, 7 fowleri, 38 melanostictus, 6 regularis, 6, 39 Bulinus africanus ovoideus, crowding effect, 361 B. forskali, 361, 374 B. globosus, 361 temperature and fecundity, 316 obtusispira, 361 Bupalus bipinnarius, dispersionary behaviour, 380 Butacarb, 117, 148

C Calliobothrium, metacestode, 524 Calliphoridae, U.V. light attraction, 151 Callitroga (cochliomyia) hominivorax eradication, 149-50 Candona candida, 699 Carbamate insecticides, 177, 118, 122, 146, 178 Carbawl, dose for spraying, 123 Caryophyllaeus, postplerocercoid (procercoid), 511, 512, 525 Caryophyllaeidea, 482 evolution, 539, 544, 545 exclusively freshwater, 541 life cycles, 412, 511, 512, 525 Catenotaenia. ontogeny, 505, 507, 513, 533 pusilla egg capsule, 397 embryophore, 398 metacestode, sequence and names, 528 ontogeny, 482 Cepaea vindobonensis, 577, 578 Cephalochlamysnamaguensis, metacestode, sequence and names, 525 Cercaria buccini sporocyst microvilli, 294, 295 bucephalopsis haimeana) tegument, 295 dichotoma, sporocyst, 295

Cercarienhiillenreaktion (CHR), 239, 242, 250

760

S U B J E C T INDEX

Cestoda adult reproduction in invertebrates, 533 in gut of vertebrates, 537, 541 epidermis, 218 development embryogenesis, 396, 494,495 larvae bladder, function, 451 structure, 450, 454 genera based on metacestode, 508 hosts and names, 504, 519 and sites, 506, 514 metacestode sequenceand names, 524-30

tail function, 448 evolution adaptations to terrestrial hosts, 534, 542

origin of aquatic life cycles, 5334, 538, 541

Chagasia bonneae, 612 Chagas's disease, 103 see also trypanosomiasis Chemosterilants in insect control, 132 house flies, 141 screw worm flies, 150 tsetses, 143 Cheyletiella, mite of pets, 175 Chlorbicyclen for tsetse control, 144 Chlorphenamidine for tick control, 171 Choanotaenia crassiscolex cysticercoid, 420 primary lacuna, 5 16 scolex, 522 development, 710 inf d i b u l u m cysticercoid, 420,445, 522, 528 primary lacuna, 521 Cholera, 868, 111 distribution, 85 extension, 87 immunization, 88 regulations, 81 vaccination, 111 Chondrula tridens, 577 Christianella, life cycle, 540 Chrysomyia putoria, breeding sites, 139 Cittotaenia ctenoides metacestode sequence and names, 528 Cladotaenia

hosts and names, 504 hosts and sites, 506 metacestode, 513 circi, larval stages, 414 globifera, larval stages, 414, 528 Clindamycin treatment of toxoplasmosis, 658 Cloacitrema narrabeenensis tegument, 237, 252,295 Clonal aggregates as separate species, 8 Clostridiumperfringens, 96 Cockroaches, effective insecticides, 129 Coenurosis in man, 433,463 Coenurus larva scolex rudiments, 424, 425 Coenurus bovis, cysticercus, 440 bladder, 449-50,451,452-3 scolex, 422, 423,424,457 C. cellulosae, cysticercus, 435, 440 hooks, structure, 442 proliferation of bladder, 463 C. cras siceps, cysticercus, 427-8 microtriches, 440 scolex, 424 C. pisiformis, cysticercus evagination of scolex, 457-8, 459 C. skrjabini, cysticercus scolex, 425 Convoluta roscofensis, epidermis, 194 Coracidium, 397,483, 534, 539-40 Corallobothrium, metacestode, 500 fimbriatum metacestode sequence and names, 527

Corallotaenia minutia, metacestode, 527 plerocercoid, 509 Corynosoma hamanni, trunk, 675 semerni, infection, 698 strumosum, 698 Cotugnia srivastavai, 710 Crassostrea virginica, 196, 411, 715 Crotoxyphos, protection of sheep against head fly, 139 Crufomate, control of cattle warble fly, 153, 155

Cryptococcus resistance in mice infected with Toxoplasma, 656 Cryptocotyle lingua, epithelium, 240,295 Ctenocephalides canis, dichlorvos collar, 162

Culexfatigans, insecticide resistance, 126 p@iens, feeding habits during hibernation, 124

SUBJECT INDEX

Culex fatigans (cont.) p . fatigetis control by fish, 130 genetic, 133 hempa (chemosterilant), 132 improved hygiene, 178 larvicides, 125 infestation, 124 resistance, 126 p . pallens, effective insecticides, 129 p . quinquefasciatus eradication project, 133 resistance to DDT, 129 quinquefasciatus, 37 tarsalis, 124 control by fish, 130 resistance and genes, 127 territans, possible vector, 72 tritaeniorhynchus, 124 resistance, 126 Cyanacethydrazide treatment for pig lungworm, 569, 584 Cyathocephalus, adult, 511 oncospheres, 496, 512, 533 truncatus, 525 Cyathocotyle bushiensis, tegument, 267 Cyclophyllidea, 482, 538, 709 development egg capsule, 397, 398 oncosphere, 405,494 larvae, 415, 512, 513 evolution, 541-2 ontogeny hosts and names, 504, 519 metacestode neoteric development, 515, 520 sequence and names, 521-30 Cypria ophthalmica, 699 Cysticercoid, 406,415,440,483,520,521 Cysticercosis of cattle, 718 of man, 435,463, 717 Cysticercus, 406, 415, 422-7, 520, 523 Cysticercus arionis, excretory ducts, 516 longicollis, 718 tenuicollis, 718 Cystocaulus ocreatus, 570-1 bionomics, 574 development, 577-9 in sheep, 580-1 immune response in sheep, 584

761

D Damalinia (Bovicola) bovis, infestation in poorly fed cattle, 159 Davainea, cercomer, 521 hosts and names, 519 ontogeny, 515 proglottina, 516 metacestode sequence and names, 528 Davaineidae, 710, 713 Dengue, 108 DDT, 122 analogues, 118, 129 control of flies, 139 body lice, 158 mosquitoes, 124, 127, 134, 178 tsetse, 144 residue in butter, 116 resistance to 119, 125-6, 140, 159, 162 spray, 123, 135 Demodex, 173 andersoni, 171 Dermanyssus gallinae, 175 Derocercas reticulatus, 576, 577 Diaminodiphenyl sulfone (SDDS) in treatment Of toxop~asmosis~ 658 Diazinon resistance, 147 Dichlorvos, 125, 157, 162 dispensers, 125 spray, 123 Diclidophora, epidermis, 212, 2 13 merlangi, epidermis, 207, 208 opisthaptor, 215 receptor, 220, 224 Dictyocaulus filaria, 5 70 life cycle, 571-3 control, 587-8 immune response in sheep, 581-3 infectivity, 581 pathology, 579 treatment, 584-6 Dictyocotyle coeliaca, epithelium, 207, 208 Dieldrin, 122, 124, 139, 144 residue in kidney fat, 116 resistance, 119, 126, 146, 147 spray, 123 withdrawal, 116, 176 Diethylcarbamazine, treatment against sheep lungworms, 585 Difenphos, 117,122, 125

762

SUBJECT INDEX

Echinococcus ontogeny Digenea epidermis, 218, 234 hosts and names, 519 Dilepididae, 710 and sites, 514 Diorchis ransomi development, 416, 417 protoscolex microtriches, 441 Diphyllidium caninum, 515, 519 scolex, 425, 426 development granulosus epidermis, 213 oncosphere, 397, 398, 399 genital organs, 445 metacestode, 492, 521-2 larvae sequence and names, 528 oncosphere, 405 transmission by flea vector, 161 cysticercus bladder, 421,455 Diphyllobothriidae larvae, 408-9 abnormal growth, 464 Diphyllobothrium ontogeny metacestode sequence and names, hosts and names, 504 530 and sites, 506 protoscolex, 458 metacestode, 503 scolex, 444,518 plerocercoid, 441, 505, 542 multilocularis, 523, 530 procercoid, 507 protonephridial systems, 532 Echinorhynchoidea, 672 strobila, 491 Echinorhynchus gadi assimilation, 692 Diphyllobothrium dendriticum metabolism, 693-5, 700 germinative cells, 410, 721-3 lageniformis, ecology, 699 metacestode sequence and names, 526 hosts, 678 plerocercoid, 409, 439, 492 metabolism, 6945 erinacei europaei, plerocercoid, 492 salmonis, annual cycle, 699 latum, life cycle, 487 thecatus, proboscis, 674 oncosphere, 4 9 4 5 truttae, hosts, 678, 679 metacestode movement, 490 Echinostoma malayanum, effect on snail sequence and names, 526 hosts, 321-2 procercoid, 407, 408 Echinus esculentus, 196 tegument, 712 Electrocution fly trap, 51 plerocercoid, 409, 439, 487 Encephalitis, 84, 163 respiratory, metabolism, 719 Endosulfan, tsetse control, 144 osmeri, plerocercoid, 409 Entamoeba histolytica, 95 Diplogonoporus, 492 Enterobius vermicularis, 106 Diplophallus polymorphus Entobdella soleae epidermis, 201-6, 212, embryophore, 399 213,216 oncosphere, 404 eyes, 219, 220, 221, 222 Diplostornum phoxini metacercaria, 255 haptor, 218,225-226,227 sporocyst, 294 microvilli, 208, 214, 218 Diplozoon paradoxum, 219,228 receptor, 220, 223, 224, 225 Drepanidotaenia bisacculina Ephemeral fever, 137 cercomer, 517 Ethoxychlor, 118 metacestode sequence and names, 529 Eubothrium ontogeny Dugesia dorotocephala, epidermis, 198 hosts and sites, 506 tigrina, epidermis, 198 crassurn procercoid, 407 Dursban, 122 salvelini, hosts and names, 504 ULV spray, 123, 125 metacestode sequence and names Dysentery distribution, 85 525 tertiary segments of adult, 492 E Earthworm hosts of pig lungworms, 563 Eucestoda, 396 Euomphalia strigella, 577 Echeneibothriurn larvae in clams, 708 Euparypha piscina, 578 polycephalism, 510

S U B J E C T INDEX

F Fannia canicularis, breeding sites, 140 control by light traps, 141 insecticide resistance, 141 Fasciola hepatica cyst formation, 251 epidermis, 204-5, 216, 233, 235, 237, 273, 292 miracidium ciliary eye, 219 Felidae, infection with Toxoplasma, 636 Fenchlorphos control of warble flies, 153 Fenitrothion, 122 Fenthion, 122, 124, 144, 153, 157 spray, 123 Filariasis, 105, 106 vector control, 133-4 Fimbriariafasciolaris, cysticercoid, 4178, 447 “Food poisoning”, 96

763

Haematophagous insects, vectors of anuran trypanosomes, 72 Haemorrhagic fevers, transmission by rodents, 109 Haemosporidia, 606 Hannemania penetrans, 38 Haplobothriidae, evolution, 539 Haplobothrium strobila, 491, 492 globiformae, metacestode, 526 three-host cycle, 531-2 Haplometra cylindracea tegument elongate inclusion bodies, 267 Helicella barbesiana, 576, 577, 578 bolli, 576 ericetorum, 576 obvia, 576, 577, 578 vestali joppensis, 576 Helicigonafaustina, 577 Helisoma duryi, pheromones, 364 Foot and mouth disease of cattle, Helix pomatia, 577 man as carrier, 82 Helminthiasis, 106 Frenkelia, 635, 659 Helobdella algira, 16, 17, 35 Fruticicolafruticum, 577 Hemiclepsis marginata, 17 Hempa, 132 G Hepatitis, 112 Gambusia afinis, mosquito control, 130 Heptachlor, 122 Gasterosteus aculeatus, 699 Heterotylenchus autumnalis, 141 Gasterophilus, 157 Hirudinea, vectors of anuran trypanoGastrocotyle trachuri, 207 somes, 35, 56-7 Giardia lamblia, 95 Hog cholera virus, 567 Glossina austeni, chromosome translo- House flies, effective insecticides, 129 cation, 143 Hyalina cellaria, 575 morsitans, control, 144, 145 Hyalomma, disease vector, 163 resting sites, 143 Hydatid disease (echinococcosis), transpalpalis, distribution, 104 mission by rodents, 84 tachinoides, 10, 37, 144, 145 Hydrolagus colliei, 201 Glycocalyx, 213,214, 227 Hydrotoea irritans, 138, 139 Gonopore, 257 Hyla crepitans, 21 Gorgoderina tegument, 266 versicolor, 7, 38, 72 attenuata tegument, 269 Hylobates moloch, infection with malaria Grammomys surfater, infection with parasites, 614 malaria parasites, 608 Hymenolepididae, life cycles and hosts, Grillotia erinaceus coracidium. 540 709-10 Gyrodactylus epidermis, 206, 208, 212, oncosphere, 404 213. 217 cystieercoid, 446-8 receptor, 220 morphogenesis, 713 sensilla, 223, 224 Hymenolepis, ontogeny, 500, 515, 520 hosts and names, 519 H cantaniana, metacestode sequence and Haematobia irritans, 139, 140, 141 names, 529 Haematoloechus medioplexus, epidermis, scolex budding, 517 212, 214, 269 citelli, culture, 720-1

764

SUBJECT INDEX

Hymenolepis (cont.) epidermis, 214 larvae oncosphere, 400, 401, 403 cysticercoid, 416, 447-8, 713 response by insect to infection, 715 diminuta, absorption, 448, 679 larvae oncosphere, 397,398,400-3,402, 405 cysticercoid, 416-7, 497, 713 cyst wall, 447 encystment, 456 metacestode, 528 culture, 720 microstoma, 709, 715 development oncosphere, 403 cysticercoid cercomer, 448, 513 scolex, 419-20, 513 nagatyi, 709 scolex, 444-5 nana, 503 development in extra-intestinal sites, 717 in invertebrate host, 416 variety of hosts, 488 larvae cysticercoid, 419, 521, 709 cercomer, 517 excretory ducts, 516 metacestode sequence and names, 529 scolex evagination, 516 ffydatigera krepkorski, scoleces, 432, 433 Hypoderma, 155-6 bovis, 152 diana, 152 lineatum, 152

I Immunity in malaria, 604, 605 Znfula macrophallus, embryophore, 399 oncosphere, 404 Insecticide evaluation, 128 residues in animal products, 146 resistance, 116, 119 importance of genes, 177 in lice of cattle, 160 in Musca domestica, 140 in tsetses, 145

Insecticides biodegradable, 179 common, chemical and trade names, 180-2 composition of, 117 mode of action, 117-8 in sheep dips, 148 systemically active, 153-5 Insectivorous fish, 117 Intestinal infections, prevention, 97 Iodofenphos, 125 Ips confusus, 364 Irrigation projects and disease, 76, 82, 105, 108 Isobenzan, tsetse control, 144 Isopora bigemina, 633, 634, 635 felis, 635 lacazei, 635 rivolta, 635

J Juvenile hormone for insect control, 120

K Kaburakia excelsa, epidermis, 200 Kala-azar, 104 Kalotermes, pheromones, 377 Khawia simensis, oncosphere, 494 Kikuthrin, 117, 129 Kronborgia amphipodicola, epidermis, 194, 196,217

L Lacistorhynchus, procercoid, 507 tenuis, coracidium, 496 development of larvae, 407 metacestode sequence and names, 524 Larvicidal chemicals, 122, 124 Lateriporus development excretory ducts, 516 metacestode, 517 sequence and names, 529 strobilo-cysticercoid, 522 Lebistes reticulatus control of mosquitoes, 130 predator of cercariae, 322 Le Boot effect, 377 Lecanicephalum,metacestode, 524 Leech vectors of anuran trypanosomes, 4, 7, 36 Leishmania, 63, 84-5

SUBJECT INDEX

Leishmaniasis, 84, 85 Lemur macaca fulvus, 609 L. m. macaca, infection with malaria, 609 Lepeoptherirus longipes, 207 Leprosy, 109 Leptocotyle minor, epidermis, 207 receptor, 220 Leptodactylus bolivianus, 21 ocellatus, 29, 37 Leptospirosis, transmission by rodents, 84 Levantina escheriana, 577, 578 Ligula procercoid microtriches, 712 intestinalis, biochemistry, 439 Limax flavus, 577 Limnaea columella crowding effect, 361 elodes, 361, 368 population regulation, 323 palustris elodes, 361 stagnalis influence of ammonia, 372 calcium balance, 369-70 crowding effect, 361 uptake of sodium, 369 truncatula, contents of mucus trails, 371 Linstowiidae development oncosphere, 415-6 Liponyssus (Ornithonyssus) silviarum, 175 Listeria, 655 monocytogenes, 655 Locusta migratoria, inhibitory pheromones, 381-2 Lucilia, 148 cuprina control genetic, 120 sterilisation, 150 sericata, 149 dieldrin resistance, 147 lungworm larvae, earthworm hosts, 563 Lutzomyia (=Phlebotomus) vexatrix, 6, 18 M Macaca cyclopis, 611 fuscata, 611 Macracanthorhynchus hirudinaceus assimilation, 692 behaviour in unsuitable host, 697 biochemistry, 688, 689 metabolism, 693, 695, 696

765

proboscis, 674 Malaria (see also Plasmodium) antigenic variants, 604 control, 79 distribution, 85 eradication, 99, 116, 120-3, 126 immunity to, 604 imported, 84 incidence, 98 in Brazil, 82 in Nile Valley, 83 periodicity of gametocytes, 604 relapse bodies, 604 Malathion, 122, 124, 127 spray, 123, 125 tolerance, 126 Marburg disease, transmission by monkeys, 84 Marisa cornuarietis, 317-8 Marsipometra, metacestode, 513 hastata, metacestode hosts and names, 504 and sites, 506 sequence and names, 525 Mazocraeoidea, 206, 210 Measles, improved control, 79 Megalodiscus temperatus tegument, 214, 269 Menacanthus stramineus, 161 Menetus dilatatus buchannensis, Allee effect, 360 Mesocestoides larvae, 412, 507 asexual reproduction, 510, 709 culture, 721-2 Immunity, 717 incidence of infection, 716 metabolism, 719 three host cycle, 489 corti metacestode sequence and names, 527 litteratus tetrathyridium, 413, 414 Metacestodes, 406, 484-7, 502-3 genera based on, 508 hosts and names, 504 and sites, 506 sequence and names, 524-30 Metastrongylus elongatus, 559-60 life cycle, 561-4 pathology in pig host, 564-6 madagascariensis, 560 pudendotectus, 560, 561-4 salmi, 560, 562

766

SUBJECT INDEX

Methiochlor, 118 Methyridine, 585 Metroliasthes lucida metacestode, 528 Micropore, 622-3 Microtriches of cestodes, 216, 218, 4378, 440, 441 of bladders of cysticerci, 449-50 Microvilli, 194, 196,208,218,279 on cercariae, 245 Milax sowerbyi, 575 Mollusca intermediate hosts of sheep lungworms, 5768, Table 1, 577 photosynthesis by, 342 Molluscan hosts of schistosomes Allee effects, 362 inhibitory pheronomes, 320 see also Biomphalaria glabrata Molluscicides, disadvantages of, 313-6 plant factors as, 320 Monacha syriaca, 576, 577 Moniliformis dubius biology egg envelopes, 685, 686 acanthor, 675, 688 larval development, 677-9 biochemistry, 688-90 metabolism, 692-6, 700 osmotic behaviour, 685, 687 tegument, 214, 680-1 acanthor, 683 nuclei Monkeys of the New World susceptible to human Plasmodium, 620 Monochoides incarnata, 577 Monoecocestus ontogeny, 515, 520 hosts and names, 519 metacestode excretory ducts, 516 scolex, 516 americanus, metacestode, 528 sigmodontus, cysticercoid, 418 Monopisthocotylinea, 206, 207, 212, 219 Mosquito -borne viruses, 107 coils, 123 fish, 130 repellents, 129 species, new, in Guam, 83 Muellerius capillaris, 570 life cycle, 573-9 pathology in sheep, 580, 583-4 treatment with tetramisole, 586

Multicephalo-cysticercus, 523 Multicotyle purvisi, epidermis, 200 Musca autumnalis, 140, 141 M . domestica, breeding sites, 139 control, 141 resistance and genes, 119, 126 insecticide, 140 M. vetustissima, biological control, 141 Muscidae, U.V. light attraction, 151 rnyiasis, 152

N Necator americanus, 106 Nematodes transmitted by mosquitoes, 105 Nematodiriasis, 588 Neoechinorhynchid, 677 Neoechinorhynchus, proboscis, 674 cristatus, pathology in definitive host, 698 proiixoides, 698 rutili, seasonal cycle, 699 saginatus, hosts, 678 Neogryporhynchus cheilancristrotus metacestode sequence and names, 527 Neophasis lageniformis, redia, 295 Neoplerocercoid, 511,521,523 Neostrongylus linearis, incidence, 571 Nippostrongylus brasiliensis multinucleate hypodermis, 217 Nippotaenia chaenogobii, metacestode, 525 Nippotaeniidea evolution, 541, 544 metacestode sequence and names, 525 NosopsyIlus fasciatus, vector of plague 162 Notocotylus attenuatus, cyst formation, 252 tegument, 237 Notoedres, cause of mange in cats, 174

0 Octospinifer macilentus, pathology in definitive host, 698 Odocolius virginianus, 609 Oestrus ovia, control, 157 Onchocerca leucostoma, 141 Onchocerciasis, 106, 1 3 6 7 eradication, 135 transmission, 136 vectors, 134 Onthophagus gazella, 141

SUBJECT INDEX

Oncosphere, 396, 483, 485, 493-6, 543, 544 Oochoristica ontogeny hosts and names, 519 and sites, 514 metacestode, 513 precysticercus, 523 scolex, 516 deserti larval stages, 415 Opalina ranarum, 35 Ophiotaenia hosts and names, 504 and sites, 506 scolex, 501 0.filaroides metacestode movement from one site to another, 488 sequence and names, 527 0 . osherofi metacestode sequence and names, 527 0.perspicua, plerocercoid, 520 0. vacuolta, metacestode, 529 Opisthaptor, 206,208 Orangutan, infection with malarial parasites, 607 Organo-phosphorus insecticides, 117, 122, 146, 178 control of cattle warble fly, 153 in sheep dips, 148 potency of synthetic, 176 resistance to, 119, 126, 141 systemically active, 153, 155 Organo-tins, control of blow-fly, 149 Otodectes cynotis, 174 P Panorchis acanthus cercaria microvilli, 245-6 redia, absorption by, 295 Parabissacanthus philactes cysticercoid, 417 Parachristianella ontogeny, 540 monoegacantha,life cycle, 512-3 metacestode sequence and names, 524 Paradilepis scolecina metacestode, 527 Paranephrops neozealanicus, 198 Paricterotaenia ontogeny, 514, 519 P. paradoxa cysticercoid, rostellar hooks, 443 metacestode, 518 sequence and names, 530

767

Paruterina candelabraria metacestode sequence and names, 528 plerocercoid, 414 rauschi, plerocercoid, 414 Paulisentisfractus assimilation, 691, 692 hosts, 677, 678, 696 nuclei in threes, 677 Pediculosis, 158 Pediculus humanus corporis, 157 Perca fluviatilis, 699 Periplaneta americana, 697 Petaurista elegans, infection with malaria parasites, 608 Petinella petronella, 576 Pheromones, 119, 377-8, 380, 381, 383 Philophthalamus megalionis glucose absorption, 270-1 Phlebotomus vectors of toad trypanosomes, 38 squamirostris, 6, 17, 37 vexator occidentis, 37 Phoxin, 123, 125 Phthirius pubis, treatment, 159 Phyllomedusa bicolor, 21-2 Phytolacca dodecandra molluscicidal properties, 320 Placobdella brasiliensis, 17, 35 catenigera, 35 ceylonica, 35 phalera, 17, 36 Plague, 85 menace due to increased air travel, 79 protection regulations, 77, 81 transmission by rodents, 84 vaccine, 94 vectors, 161-2 Plasmodium incubation periods, 100 micropore, 622-3 New World monkeys susceptible to human, 620 simian species, 611; in Brazil, 613 subgenera, 605 anomaluri, 608 berghei infectivity and hormones, 617 sporozoites, 619 h e structure, 622 nucleus, 624, 625 subspecies, 608

768

SUBJECT INDEX

Plasmodium (cont.) b. berghei, merozoites, 626 b. yoelii, 610 merozoites, 626 oocyst nucleus, 623 booliati, 608 brasilianum, 607, 612, 613, 614,

620 fine structure, mitochondria, 624 ontogeny, 621 brucei, 610 cathemerium, 616 cephalothi, 610 coatneyi, 617, 621 pathogenesis, 618 schizogony, 611-2 cynomolgi, 607 culture, 619 midnight infectivity, 616 oocyst nuclear changes, 624 x-radiation on sporozoites, 604 c. bastianelli relapse variants, 605 schizonts, 626 eylesi, 615 falciparum, 616, 620,621 gametocyte, fine structure, 622 importation into Europe, 98 infection, 97, 100 in owl monkey, 619 pathogenesis, 617, 618 resistant strains, 102-3 fieldi, 612 fragile, 612 gallinaceurn, culture, 618 nuclear division, 623 schizogony, 624 girardi, infection in lemur, 609 gonderi, 607 hylobali, 614 inui, 621 i. cyclopis, 611 i. shortii, 612 jefleri, sporogonic cycle, 615 kno wlesi immunity, 618 infection in owl monkey, 620 infectivity of gametocytes, 616 pathogenesis, 617-8 relapse variants, 605 lemuris, 609 malariae, 619,120

incubation period, 100 mitochondria, 624 “zoonosis in reverse”, 621 ovale, 615, 616, 621 incubation period, 100 transmission by monkeys, 619-20 pitheci, 607, 615 quadrigemina, 609 rodhaini, 621 schwetzi, 615, 616, 621 silvaticum, 607, 615 pathogenesis, 618 simiovale schizogony, 612 simium, 612, 613, 614, 621 traguli, 609, 610 vinckei, 608 vivax, 620, 621 importation into Europe, 98 incubation period, 100 infection in owl monkey, 619 wutteni, parasite of ffying squirrel, 609 youngi, 615 Plectanocodyle, epidermis, 210, 212-3 gurnardi, epidermis, 207,208 Plerocercoid, 406, 483, 486, 504, 505-7, 509-10, 542 Plerocercus, 510-1 1 Poecilia (lebistes) reticulatus, 130 Poliomyelitis, 85, 109, 111 PoIyceIis nigra, pharynx epithelium, 200 tenuis epidermis, 200, 206 Polycladida, 200 Polymorphus minutus biochemistry, 689, 691 ecology, 699 hosts, 678 intermediate, 696, 697 definitive, 679 metabolism, excretion, 700 osmotic environment, 687-8 spermatozoa, 683, 684 uterine bell, 675 Polyopisthocotylinea, 206, 207, 212, 227 larval eyes, 219 sensilla, 224 Polystoma, epidermal permeability, 228 integerrimum epidermis, 207, 208, 210 sensilla, 224 Pomaceu haustrwn, 318 Pomphorhynchus bulbocholli pathology in definitive host, 697-8 Precysticercoid, 522

769

SUBJECT I N D E X

Precysticercus, 523 Procercoid, 406, 483, 504, 507 Proglottidation, 483, 537 Propoxychlor, 118 Proteocephalidea, 397, 482 evolution, 539, 541 larval stages, 41 1 oncosphere, 404,494 metacestode, 490 hosts and names, 504 sequence and names, 526-7 Proteocephalus plerocercoids from dead copepods, 535 ambloplitis metacestode hosts and names, 504 sites, 506 movement from one site to another, 488 sequence and names, 526 plerocercoid, 41 1, 510 firicollis metacestode, 497, 504, 506, 526 plerocercoid, 520 JIuviatilisontogeny hosts and names, 504 sites, 506 metacestode sequence and names, 526 plerocercoid, 510 parallacticus metacestode, 526 plerocercoid, 510 Prothrin, 129 Protostrongylineae, molluscan intermediate hosts of, 577 Protostrongylus rufescens, 570, 571 development, 574, 576, 578 pathology in sheep, 580 treatment, 584, 586-7 Pseudodiorchisprolifer scolex budding, 517 Pseudophyllidea, 482, 539, 708 life cycles, 488, 504 capsule, operculate, 397 coracidium, 403-4, 540 larvae, 406,407, 408, 439 metacestodes, 525-6 proglottidation, 490 Pseudophyllidean ancestor without coracidia, 541 Pseudodiorchisprolifer metacestode sequence and names, 529

Pseudopleuronectes americanus, 693 Psoroptes communis ovis, 171 Pulex irritans, plague vector, 162 Pyrethroid insecticides, 117, 176 Pyrethrum in tsetse control, 144

Quarantine, 77, 80

Q

R Rabies, 107 Raia clavata, 207 R. naevus, 207 Raillietina, 515, 519 cercomer, 521 cesticillus cercomer, 516 larval stages cysticercoid, 420, 713 cyst wall, 445-6 scolex, 440 metacestode sequence and names, 528 Rqjonchocotyle emarginata epidermis, 207,208,210,211,212 inclusions, 212, 213 surface coat, 215 Rana catesbeiana, 38 trypanosome parasitaemia, 25, 36 clamitans, 24, 72 esculenta, 21, 22, 26, 33, 35, 38 pipiens, 33, 376-7 sphenocephala, 33, 38 temporaria, 33, 207 Reduviidae, 103 Relapsing fever, 85 control of vectors, 163 Repellents, insect for “fly worry”, 139 new mosquito, 129 Residues of insecticides in animal products, 146 Resmethrin, 117 Resistance, 147, 159, 168-9 Rhipicephalus disease vector, 163 appendiculatus control, 164-6 vector of E. Coast fever, 168 Rhodnius prolixus, 37 Rhombomys opimus, 431, 432 Rickettsia prowazeki, 157

770

SUBJECT INDEX

Rodents in transmission of disease, 84, 94,109 Ropartz effect, 377-8

heptalaminate membrane, 261, 262, 263, 276,285, 286 oesophagus, 276-80 sensory organelles, 275, 276 S structure, 261, 263, 265 Saimiri sciurens, 614 uterus, 280 Salmonella, 95, 109 cercaria, 234-50 paratyphi, A, B, C , 95, 96 cytochemistry, 240, 241 typhi, 95, 96 functions, 238, 240, 242 typhimurium, 96, 655 structure, 238, 239, 243-50 Sandfly vector of anuran trypanosomes, miracidium, 205, 219, 290-3 6, 7, 10, 37 schistosomulum, 213, 250-7 Sarcocystis, 635 absorption, 257 fusifopmis, 548 adaptations to vertebrate host, miescheriana, 658 250 tenella, 658 changes, 253 Sarcoptes scabiei, 173 sporocyst, 293-6,294 Scab mite resistance, 172 mattheei miracidium, 290 Schistocephalus procercoid rodhaini tegument microtriches, 712 enzyme activity, 272 erinacei plerocercoid scolex, 409 spindale control in snail hosts, 322 pungitii plerocercoid Schistosomiasis, 84, 85, 105 microtriches, 409 molluscan hosts, 308-21 tegument changes, 439-40 parasites, 321-3 solidus plerocercoid prevention of transmission, 382 microtriches, 409 Sciomyzidae, potential predators of piscine host, 439 snails, 318 Schistocerca gregaria Sciurius vulgaris, 428 pheromone accelerating maturation, Scolex, 417, 443, 501, 502 381-2 development, 498-9 Schistosoma, 84 functions, 536 douthitti, 277, 279 Screw-worm flies, 131 haematobium, 105 genetic control, 117, 132 cytochemistryof tegument, 269 Sebastes maliger, 207 death rates, 311 Sex attractants, see Pheromones water velocity and snail hosts, 316 Sheep dip preparations, 145 japonicum, 105, 311 Sheep scab, 171-2 miracidium tegument, 290,291-2 Shigella, 88, 95 mansoni dysenteriae, 96 culture, 283 somnei, 96 death rate, 311 Simian Plasmodia, host species, 613 distribution, 105 Simulium damnosum, eradication diffieffect of drugs, 2834 culties, 135, 136 host immunity, 284-9 neavei, 135, 136 host-parasite relationship, 289 ornatum, vector of onchocerca in snail hosts and water velocity, 316 cattle, 137 tegument, 213, 214-5, 216 Smallpox, 79,85,89,90 adult, 257-89 surveillance regulations, 81 cytochemistry, 268-72 vaccination, 90-2, 110-1 1 functions, 282, 296 Sobolevicanthus gracilis gynaecophoric canal, 257,258, crowding effect in ostracod host, 715 260, 262, 275 Solea solea, 205

771

SUBJECT INDEX

Sparganum prolifer budding in pleroceraids, 462 Spathebothriidea, 511, 512, 525 evolution, 539, 541, 544 Spathebothrium, lack of scolex, 512 Spirometra plerocercoids asexual reproduction, 510 emergence from dead copepods, 535 growth of tail fragments in mice, 708 erinacei, scolex, 712 mansoides, development, 407,526 spargana temperature tolerance, 719 effect on vertebrate host, 719 Staphylocystis furcate cysticercoid, 420 Steatomys, infection with malaria parasites, 610 Stomoxys calcitrans, 138, 140, 141 Strobilo-cysticercoid, 522 Strobilo-cysticercus, 523 Strongyloides stercoralis, 106 Succinea pfeifferi, 576 putris, 576, 577

Swine vesicular disease, possible origin and transmission, 82 Syndesmis echinorum epidermis, 195, 196,198,218

T Taenia, 513, 716 brauni, scoleces of coenurus, 433 crassiceps antigens, 718 histogenesis, 714 metabolism, 719-20 mutation, 711

ontogeny cysticercus, 423, 427, 523 metacestode, 517, 519 hosts and sites, 514 sequence and names, 530 reproduction, asexual, by budding, 460-1, 517, 518

strain differences, 722 endothoracicus, 430, 431 hydatigena adult, 437,444 cysticercus, 435-6, 711 bladder, 422,451 scolex, 458, 462 martis armatetrathyridium, 428, 429 over age larval forms, 464 multiceps scolex, 425, 426, 441 mult&phalic, 517

mustelae development, 519 cysticercus, 433

metacestode hosts and sites, 514 sequence and names, scolex, 517 nodulosus, 407,408,409 ovis larvae, 711 parenchymatosa cysticercus, 436 pisiformis, incidence of infection, 716 larval stages, 405, 421, 435-6 asexual reproduction, 710 triple bladders, 462 polyacantha larval stages, 428, 429, 464,465 saginata, 106

age susceptibility in cattle, 716 antigens, 718 larval stages, oncosphere, 404-5 cysticercus, 523, 711, 714 metacestode, 529 serialis, oucosphere, 405, 425 solium, 106

larval stages, 404, 529, 714 scolex, 435 taeniaeformis embryophore, 398 larval stages, 7 1 3 4 cysticercus, 421 bladder, 433,454 nephridial system, 451-2 metacestode, 529 scolex, 434, 493 strobilocercus, 421, 422, 433, 441-2

strobilocysticercus, 522, 523 metabolism, 719 taxidiensis, cysticercus, 523 metacestode, 529 twitchelli

polycephalic bladders, 431 scoleces, 433 Taeniidae, 710, 713 larvae, culture, 721 cysticerci, 520 embryophores, 398 oncospheres, 404, 405 Taeniocotyle,epidermis, 201 Tarebia granifera, 3 18 Temnocephala epidermis, 216-7 novae zealandiae epidermis, 197, 198, I99

Tetrachlorvinphos (Gardona) larvicide, 125

772

S U B J E C T INDEX

Tetrahymena crithidia, 44 Tetramisole, 569, 586-7 Tetraphyllidea, 482, 708 evolution, 539, 541 larval stages, 41 1, 524 Tetrarhynchidea larval stages, 415, 482 Tetrathyridium, 412 4 Thammomys rutilans, 608, 610 Theba carthusiana, 577, 578 Thiabendazole, 586 Thiotepa, 132 Thymallus arcticus baicalensis plerocercoid, 410 Thysaniezia ovilla excretory ducts, 452 Thysanosoma actinoides metacestode, 528 Tick paralysis, 169, 171 Torpedo nobiliana, 207 Toxocara cati, 631-3 Toxoplasma gondii life cycle, 631-9 intestinal epithelium stages, 637 oocyst measurements, 635 effect of freezing on, 641 infectivity, 639 resistance of, 640 tissue stages, 637 Occurrence in cattle, 642, 653 Penetration factor, 657 relationship to Sporozoa, 636 resistance to Cryptococcus, 656 ' non-specific, after infection, 655-6 production of interferon, 656 transmission by cat faeces, 631 Toxoplasmosis in domestic animals, 643-4 immunity in cats, 636 food animals, 6404 pigs, 642-3 sheep, 643 man, 644-51 congenital, 649-51 ocular, 6468 transmission by blood ttansfusion, 648-9 by helminth-free cat faeces, 632-3 Toxorhynchitis brevipalpis, 131 splendens, 131 Tragulusjavanicus, 609 Trianenophoridae plerocercoids, 408 Triaenophorus nodulosus coracidium,

397-8 metabolism, 719 Triatoma sanguisuga, 37 Triatomid bugs, 103 Tribolium, 362, 415 confusum, 416, 713, 715 Trichia hispida, 576 Trichiuris trichiura, 106 Trichlorphon, control of warble fly, 153 Tricladida epidermis, 198-200 Triturus, 7 viridenceus, 21 Trombicula autumnalis, 175 Trypanorhyncha evolution, 541, 544 ontogeny, 406, 524, 540 Trypanosoma in anuran hosts, 45-6 control system and pathways, 30 distribution, 47-55 DNA synthesis, 20 hirudinid vectors, 56-7 hyperglycaemic agents, 25 localization, 27-8 peripheral parasitaemia, 24-5, 31 arcei effect of host species, 22 aurorae fission, 15 belli slender appearance, bocagei, 6, 8, 10 reproduction in sandfly, 17 borelli, 21 brucei, 24 b. gambiense, 103 b. rhodesiense, 103 bufophlebotomi culture, 40 sandfly vector, 6, 10 reproduction in, 18 speciation, 8 chattoni, 12 culture, 40 fission, 15 speciation, 6, 8 costatum, 40 cruzi, 103 diamondi, culture, 40 diemyctyli, 7, 8 morphology, 21,22, 23 transmission by leech vector, 36 elegans, 11, 22 galbae, culture, 40 glycogen reserves, 34 gambiense, 4 gaumontis, 15 grandis, 40

773

SUBJECT INDEX

Trypanosoma (con t.) grylli, 8, 38, 72 in tree frog host, 7 hippicum,inhibition of glucose uptake, 35 hylae, pathogenicity, 33 inopinatum, 11, 15, 16 culture, 19, 3 9 4 0 leech vector, 10, 16-17, 35 morphology, 21, 22 pathogenicity, 33 peripheral parasitaemia, 26 species, status, 41,42 trypanocides, effect of, 34 karyozeukton pathogenicity, 33 lavalia, 15 leptodactyli, 37 leech vector, 17,35, 38 morphs in vertebrate host, 21 multiple fission, 15 pathogenicity, 33 loricatum, fission, 15, 40 mega, 11, 13, 39, 41 cytoIogy, 13-4 DNA synthesis, 20 morphogenesis, 19-20 montezumae, 21, 34, 40 montrealis, 15 neveu-lemairei, 40 parroti, 15, 33, 40 pipientis, 6, 8, 15, 16, 17 culture, 19,40 leech vector, 36 species status, 42 ranarum, 6, 8, 11 culture medium, 39 and temperature, 40 morphs, 22 oxygen consumption, 41 rhodesiense, 4 rotatorium, 6, 7, 8, 11, 15, 72 biology, 41 culture forms, 13, 19 media, 38-39 and temperature, 40 cytology, 13, 29 infectivity in fly, 10, 37 leech, 16, 35 tadpole, 32 pathogenicity, 33, 34 species status, 41 sanguinis, 11, 33

schmidti, 40 sergenti, 40 undulans, 22, 33 Trypanosomatidea, 9, 11 Trypanosomiasis, 84,85,103,104,142 Trypanozoon subgenus, 4 , 7 Tsetse control, 143, 144, 145 Tuberculosis, improved control, 79 Tunga penetrans, 161 Tjdocephalumontogeny, 41 1, 507, 715 metacestode sequence and names, 524 microvilli, 711 Typhoid, 85, 95, 97, 111 Typhus, 79, 84, 85 vectors, 157, 161

U Udonella epidermis, 212, 216 caligorum, epidermis, 207, 208, 209 Urastoma cyprinae, epidermis, 196 Utricularia, 124 V Vaccination certificates, 90-91 validity periods, 111 Vaccine, rabies, 107 smallpox, 91 yellow fever, 93 Valipora campylancristrota ontogeny, 504, 506, 710 metacestode sequence and names, 527 Vampirolepishamanni, cysticercoid, 447 Vector control, 177, 178 in aircraft, 83 of dengue, 108 encephalitis, 124 onchocerciasis, 134, 136, 137 trypanosomiasis, 6, 103, 104, 142 Velacumanthus australis, 319 Venezuelan equine encephalitis (VEE), 84, 108 Veterinary products safety precautions scheme (VPSPS),new products, 1167 Vibrio parahaemolyticus, 96

W Warble eradication, 155-6 Wardoides nyrocae cysticercoid, 417 Whitten effect, 360-1 Wuchereriabancrofri, 105

774

SUBJECT I N D E X

X Xenopsylla brasiliensis, 162 cestia, 162 cheopsis, 162

Y Yaws, 79 Yellow fever, 77, 85 control, 79 surveillance regulations, 81 vaccination, 92-3, 11 1

Yersiniapestis, 94 Z

Zebrina detrita, 517, 518 Zonitoides arboreus, 575 Zoogonoides viviparus tegument inclusion, 245 microvilli, 246 mitochondria, 244 surface coat, 240

Cumulative List of Authors In Volumes 1-10 Numbers in bold face indicate the volume number of the series; asterisks indicate short, updated reviews. Adler, S., 2, 35 Alicata, Joseph, E., 3, 223 Arthur, Don R., 3,249,8, 275' Baker, John R., 1 0 , l Bennett, Gordon F., 10, 1 Berg, Clifford O., 2,259 Berrie, A. D., 8,43 Bertram, D. S.,4, 255 Bishop, Ann, 5,93 Boray, J. C., 7, 96 Bryant, C., 8,139 Cameron, Thomas W. M., 2, 1 Clark, Glen W., 10, 1 Dawes, Ben, 2, 97, 8, 259* Elsdon-Dew, Ronald, 6, 1 Fletcher, Alexander, 10, 31, 49 Garnham, P. C. C., 5, 139 Gibson, T. E., 2, 221, 7, 350 Hansen, Eder L., 9, 227* Heyneman, Donald, 10, 192 Hoare, Cecil A., 5,47 Horak, I. G., 9, 33 Horton-Smith, C., 1, 68, 6, 313* Huff, Clay G., 1, 1, 6, 293* Hughes, D. L., 2,97, 8, 259* Inglis, W. Grant, 9, 185 Jacobs, Leon, 5, 1 Jennings, J. B., 9, 1 Jirovec, Otto, 6, 117 Katz, Naftale, 6, 233 Kendall, S. B., 3, 59, 8, 251* Knapp, Stuart E., 8 , l Koberle, Fritz, 6, 63 Komiya, Yoshitaka, 4, 53 Laird, Marshall, 10, 1

Larsh, John E., Jnr., 1,213,6, 361* Lee, D. L., 4, 187, 10, 347' Lim, Hok-Kan, 10, 192 Llewellyn, J., 1, 287, 6, 373* Long, P. L., 1, 68,6, 313* Lumsden, W. H. R., 3, 1,8,227* Maegraith, Brian, 6, 189, 10, 31, 49 Michel, J. F., 7, 211 Millemann, Raymond E., 8, 1 Miller, Thomas A., 9, 153 Muller, Ralph, 9, 73 Neal, R. A., 4, 2 Nelson, George S., 8, 173 Nicholas, W. L., 5, 205 Ollerenshaw, C. B., 7, 283 Pawlowski, Zbigniew, 10, 269 Pearson, J. C., 10,153 Pellegrino, J., 6, 233 Petru, Miroslav, 6, 117 Poynter, D., 1, 179,4, 321, 6, 349* Rogers, W. P., 1, 109, 6, 327* Rohde, Klaus, 10,78 Rybicka, Krystyna, 4, 107 Schultz, Myron G., 10, 269 Silverman, Paul H., 3, 159, 9, 227* Sinclair, I. J., 8, 97 Smith, L. P.,7, 283 Smithers, S. R., 7, 41 Smyth, J. D., 2, 169, 7, 327 Sommerville, R. I., 1, 109, 6, 327* Terry, R. J., 7,41 Voge, Marietta, 5, 247 Webster, J. M., 7, 1 Yokogawa, Muneo, 3,99,7,374

175

Cumulative List of Chapter Titles Numbers in bold face indicate the volume of the series; asterisks indicate short, updated reviews. Advances in Veterinary Anthelmintic Medication, 7, 350* Aspidogastrea, Especially Multicotyle purvisi Dawes, 1941, 10, 78 Avian Blood Coccidians, 10, 1 Biological Aspects of Trypanosomiasis Research, 3, 1 Biological Aspects of Trypanosomiasis Research, 1965 ; a Retrospect, 1969, 8, 227* Biology and Distribution of the Rat Lungworm, Angiostrongylus cantonensis, and its Relationship to Eosinophilic Meningoencephalitis and other Neurological Disorders of Man and Animals, 3,223 Biology of the Acanthocephala, 5, 205 Biology of the Hydatid Organisms, 2, 169, 7, 327* Biology of Nunophyetus salmincolu and “Salmon Poisoning” Disease, 8, 1 Chagas’ Disease and Chagas’ Syndromes: The Pathology of American Trypanosomiasis, 6, 63 Clonorchis and Clonorchiasis, 4, 53 Coccidia and Coccidiosis in the Domestic Fowl, 6, 313* Coccidia and Coccidiosis in the Domestic Fowl and Turkey, 1, 68 Dracunculus and Dracunculiasis, 9, 73 Dynamics of Parasitic Equilibrium in Cotton Rat Filariasis, 4, 255 Electron Transport in Parasitic Helminths and Protozoa, 8, 139 Embryogenesis in Cestodes, 4, 107 Epidemiology and Control of Some Nematode Infections of Grazing Animals, 7, 211 Epidemiology of Amoebiasis, 6, 1 Evolutionary Trends in Mammalian Trypanosomes, 5, 47 Experimental Chemotherapy of Schistosomiasis mansoni, 6,233 Experimental Fascioliasis in Australia, 7, 96 Experimental Research on Avian Malaria, 1, 1 Experimental Studies on Entumoebu with Reference to Speciation, 4, 2 Experimental Trichiniasis, 1, 213, 6, 361* Fascioliasis: the Invasive Stages of Fusciola hepatica in Mammalian Hosts, 2, 97 Fascioliasis: the Invasive Stages in Mammals, 8, 259* Feeding in Ectoparasitic Acari with Special Reference to Ticks, 3, 249 Host-Parasite Relationships of Plant-Parasitic Nematodes, 7, 1 Host Specificity and the Evolution of Helminthic Parasites, 2, 1 Immunology of Schistosomiasis, 7,41 Infectious Process, and its Relation to the Development of Early Parasitic Stages of Nematodes, 6, 327* Infective Stage of Nematode Parasites and its Significance in Parasitism, 1, 109 Intramolluscan Inter-trematode Antagonism: a Review of Factors Influencing the Host-Parasite System and its Possible Role in Biological Control, 10, 192 In vitro Cultivation Procedures for Parasitic Helminths, 3, 159 776

C U M U L A T I V E INDEX

777

In vitro Cultivation Procedures for Parasitic Helminths: Recent Advances, 9, 227* Larvae and Larval DeveIopment of Monogeneans, 1, 287 Leishmania, 2, 35 Liver Involvement in Acute Mammalian Malaria with Special Reference to Plasmodium knowlesi Malaria, 6, 189 Malaria in Mammals Excluding Man, 5, 139 Metabolism of the Malaria Parasite and its Host, 10, 31 Meteorological Factors and Forecasts of Helminthic Disease, 7, 283 Onchocerciasis, 8, 173 Paragonimus and Paragonimiasis, 3, 99, 7, 375* Paramphistomiasis of Domestic Ruminants, 9, 33 Parasitic Bronchitis, 1, 179, 6, 349* Parasitism and Commensalism in the Turbellaria, 9, 1 Pathogenesis of Mammalian Malaria, 10, 49 PhyIogeny of Life-cycle Patterns of the Digenea, 10, 153 Post-embryonic DeveIopmental Stages of Cestodes, 5, 247 Problems in the Cultivation of some Parasitic Protozoa, 5, 93 Recent Advances in the Anthelmintic Treatment of the Domestic Animals, 2,221 Recent Experimental Research on Avian Malaria, 6, 293* Relationship between Circulating Antibodies and Immunity to Helminthic Infections, 8, 97 Relationships between the Species of Fasciola and their Molluscan Hosts, 3, 59, 8. 251* Snail ‘Control in Trematode Diseases: the Possible Value of Sciomyzid Larvae Snail-Killing Diptera, 2, 259 Snail Problems in African Schistosomiasis, 8, 43 Some Tissue Reactions to the Nematode Parasites of Animals, 4, 321 Speciation in Parasitic Nematodes, 9, 185 Structure and Composition of the Helminth Cuticle, 4, 187 Structure of the Helminth Cuticle, 10, 347* Taeniasis and Cysticercosis (Tuenia saginata), 10, 269 Tick Feeding and its Implications, 8, 275* Toxoplasma and Toxoplasmosis, 5, 1 Trichomonas vaginalis and Trichomoniasis, 6, 117 Vaccination Against the Canine Hookworm Diseases, 9, 153

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