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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