ISSN 0-70 4 -3716- Canadian Translation of Fisheries and Aquatic Sciences No. 5079 Biology of a microsporidian: Glugea atherini n.sp., parasite of the atherine Atherina boyeri Risso, 1810, (Pisces - Teleost) occurring in coastal lagoons P. Berrebi Original title: Biologie d'une microsporidie: Glugea atherini n.sp. Parasite de l'atherine: Atherina boyeri Risso, 1810, (Poisson - Teleosteen) des etangs cotiers In: Thèse pour obtenir le grade de Docteur de 3ème cycle, Université des Sciences et Techniques du Languedoc, (France), 1978 Original language: French ' Available from: Canada Institute for Scientific and Technical Information National Research Council Ottawa, Ontario, Canada R1A 0S2 1984 238 typescript pages Academy of montpellier CTF/ S 5-e-fej Languedoc University of Science and Technology THESIS presented at the Languedoc University of Science and Technology for the Doctorate Parasitology, Pathology, Ecophysiological Relationships Contribution to the Biological Study of Brackish Areas of the French Mediterranean Coast. BIOLOGY OF A MICROSPORIDIAN: Glugea atherini n. sp. PARASITE OF THE ATHERINE Atherina boyeri Risso, 1810, (PISCES - TELEOST) OCCURRING IN COASTAL LAGOONS.' • by Patrick Berrebi Defended in June 1978 before the Examining Board Mr. L. Euzet, Chairman Mr. C. Vago, Member of the Institute . Mr, G. Bouix . Assessors Mr. J.P. Trilles • FORWARD I take this opportunity to thank Professor G. Bouix, my thesis director, who devoted a great deal of his time to supervising my research and the subseouent writing of this thesis. I greatly appreciated the atmosphere of directness and informality in which our discussions were held and am very grateful that through Professor Bouix's open-minded attitude, I was able to adopt a biochemical approach that is seldom used in the Laboratory to carry out my research. I should also like to thank: Professor L. Euzet, Chairman of the Board, who always wel- comed me cordially in his department where Mr. Gabrion and Mr. Maillard helped me in the field of helminthology. Professor C. Vago, a member of the Institute, who accepted to iudge this work. Professor J.P. Trilles, who, through our discussions, helped me considerably in the complex field of physiology. It is not possible to mention all the other investigators who provided invaluable assistance. I thank them all, in particular: Professor A. Raibaut and Professor J.P. guignard in the field of parasitic copepods, ichthyology and lagoonal environments. Mr. Loubès who aided me a great deal in the electron micro- scopic and histochemical study of microsporidians. Janice, my devoted friend; Mrs. Nicole Pasteur as well as Professor Thaler's entire laboratory team for their practical and theoretical assistance in the field of enzyme electrophoresis. All the laboratory staff, in particular, Mr. J. Barrai, Mrs. 5. nustau and Mrs. j. Boyer who displayed considerable patience in typing this text. The many fishermen, who are vital props in this kind of work, • especially Mr. Scopel, who . was always ready to help and be of service, Mr. Laplace and the fishermen of the Rhâne Mort. Last but certainly not least, my parents and Ghislaine, to them I wish to express my affection. • TABLE OF CONTENTS Page CHAPTER ONE. Introduction 1 II, A - Historical Background and General Comments 2 B - The Microsporidia of Fish 7 C - The Microsporidian Infesting Atherines 17 CHAPTER TWO. The Microsporidian and Its Host 18 A - Environment 20 B - Study of the Host 33 C - Study of the Parasite 56 1/ Procedure 56 2/ Study of Parasite Implantation 57 3/ Ultrastructural Study of the Parasitic Cycle 88 4/ Histochemistry 132 5/ Taxonomic Position 141 CHAPTER THREE. Geographic Distribution 145 A - Range of Microsporidiosis 146 B - Explanations for the Geographic Range 151 CHAPTER FOUR. Development of Microsporidiosis in Atherines 166 A - Contamination of the Host 167 B - Internal Phase 181 C - Free-Living Phase 200 CHAPTER FIVE. Development of Parasitism Over Time: The Seasonal Cycle 203 1/ Digestive Tube Xenomas 205 2/ Body Cavity Xenomas 206 GENERAL CONCLUSIONS 211 SUMMARY 216 BIBLIOGRAPHY 217 • CHAPTER ONE INTRODUCTION • • 2. I - INTRODUCTION A/ Microsporidia: Historical Background and General Comments For a long time, these organisms were considered pluricellular and classified as a subphylum of Cnidosporida. It was only with the development of electron microscopy that they were found to be unicellular with highly specific characteristics, warranting re- classification among the MICROSPORA (Sprague, 1977). Arthropods are the most common hosts of microsporidians which attack both larval and adult forms of insects and crusta- ceans alike, producing cysts and resulting in diffuse invasion of the tissues and even total invasion of the host. Nosemia bombycis Naegeli, 1857, was one of the first spe- cies described because of its economic significance. This parti- cular parasite attacks silkworms in many parts of the world, i.e., France, Italy, Germany, Australia, Hungary, Japan, India, etc.... It has a rapid cycle, weakening the larva which becomes less active, loses its appetite and finally dies. Spots cover the larva in a disease called "pébrine" which causes particular ravages in breed- ing farms because it can be transmitted via the ovary. Fish seem to be the most common victims among vertebrates although other groups are also subject to microsporidian infestation: a) Batrachians: Pleistophora myotrophica Canning, Elkan and Trigg, 1964, attacks muscle fibers in the toad, Bufo bufo. The result is a number of white, thread-like lesions which gradually destroy the motor muscles (Canning et al., 1964). Death eventually ensues. One species of microsporidians is also found in newt larvae where it forms white cysts. 3. b) Reptiles are not spared. Pleistophora danilewskyi (Pfeiffer, 1895) produces parallel, elongate lesions of the muscles in various species. c) Birds are attacked by only one species, i.e., Encephalitozoon sp. Kemps and Kluge, 1975. The spores develop in the kidneys » gall bladder, liver and intestines. Infection is fatal in some lovebirds. d) Mammals are even more subject to infection. Thelohania apo- demi Doby et al., 1963, among many other species, has been found in field mice where it causes hypertrophy of the spleen. Encephalito- zoon cuniculi Levaditi, Nicolau and Schoen, 1923, has been studied in mice and has also been found to be pathogenic in rabbits and foxes. Infection with Encephalitozoon sp. Siebold and Fussel, 1973 has been described'in the monkey, Callicebus moloch. Lastly, infection with Nosema connori Sprague, 1974, was reported in a child (Margileth et al., 1973) and with Encephalitozoon matsu- bayashii Sprague, 1977, in adults. Some microsporidians, such as Encephalitozoon cuniculi, pro- duce ascites and hypertrophy of the liver and spleen in mice (Morris et al., 1956; Nelson, 1962; Lainson et al., 1964). There is evidence of a relationship between this particular microsporidian and cancer in mammals. Petri and Schi/Sdt, 1966, showed that the parasite in- 110 hibited cancer in rats with malignant cells and to some extent immunized the animals against "transmissible" malignant tumours. 4 . Lastly, Arison et al, 1966, demonstrated that the microsporidians causing ascites in mice had an inhibiting effect on some mice tumours. The spore is the key stage in identifying microsporidians. It is during this final stage of parasitic development that micro- sporidiosis can be macroscopically detected. In the case of arthro- pods, the tissues acquire a characteristically white colour only when a very large number of spores have accumulated in the hemolymph or muscles. In fish, cysts of vary.ing sizes usually develop but they are not detected until great numbers of spores have formed. Spore- formation is the only stage that can be seen with the naked eye, if only because of the vast number of spores produced. It is for this reason that early studies focussed on this particular stage. Further- more, it is the only stage in which the microsporidian assumes a definite shape and there exists an internal structure made up of characteristic components. The typical spore consists of the followings - a very solid protective envelope containing an endospore and an exospore. Neither dessication, osmotic shock or mechanic action such as crushing, or ultrasonic vibrations seem to be able to break it (personal observations). Only some stains are capable of crossing the barrier and impregnating the contents. - sporoplasm which is reduced and limited by a cytoplasmic 5. membrane on the inner side of the envelope. The nucleus is not always visible. There are no mitochondria, but occasionally a number of ribosomes may be seen in alignment. - a posterior vacuole, which is occasionally visible although it can not always be seen with the electron microscope. - the polar filament, which constitutes one of the basic ele- ments of the spore. The posterior portion of the filament is gen- erally coiled. A cross section reveals up to twelve concentric layers. According to the most genei-ally accepted theories, the filament consists of a hollow tube which is extruded by eversion. The anterior portion is essentially straight. It thickens into a complex of structures representing over half of the spore. The polar cap undoubtedly serves to attach the filament. - the polaroplast. Regardless of whether it is lamellar or vacuolar, it is thought to swell as a result of special osmotic properties and to increase the internal pressure of the spore. The filament then pierces the weaker anterior portion of the enve- lope and is violently expelled. This structural system provides the parasite with a free-living phase (in the water, in the case of fish-infesting microspori- dians). Dissemination, like penetration of the new host's diges- tive tract, is an easy and entirely passive process. In the living environment, there is an as yet unexplained stimulus which 6 . induces sudden extrusion of the filament which may be up to 25 times as long as the spore. The sporoplasm (or at least the nucleus, according to some authors, i.e., Sprague and Vernick, 1968) is thought to be carried along and injected into the host.