DEVELOPMENT IN VITRO OF MICROSPORIDIA FROM INSECT HOSTS by XIE WEI-DONG, M.Sc. (Zhongshan University, Guangzhou) Thesis submitted for the Degree of Doctor of Philosophy University of London and for the Diploma of Imperial College Imperial College of Science & Technology Department of Pure & Applied Biology Silwood Park Ascot Berkshire November 1985 2 ABSTRACT Five species of microsporidia derived from insect hosts, Nosema sp. from Spodoptera litura, N. heliothidis, N. locustae, Vairimorpha necatrix and Pleistophora opero- phterae, were successfully established in £5. frugiperda cell cultures by addition of haemolymph from larvae infected with the microsporidia. By using these systems, the growth and development of the 5 species of microsporidia and spread of infection by microsporidia from cell to cell were investi­ gated in vitro. Four of the species of microsporidia did grow at 30°C, but P. operophterae did not. The most favourable temperatures for growth of microsporidia were 20°C and 25°C. Extracellular vegetative stages of micro­ sporidia were capable of initiating infections in new cells, but their ability was almost completely lost within 30 min by the Nosema sp., 60 min by N. heliothidis and V. necatrix, 120 min by P. operophterae and 180 min by N. locustae. Addition of ATP to the medium resulted in more cells becoming infected with Nosema sp. and N. heliothidis and further confirmed that extracellular infective stages are responsible for infection of new cells. Meronts were observed by light and electron microscopy attached to the surface of host cells. A dose dependent inhibition of uptake of both parasites and red blood cells into Spodoptera cells by Cytochalasin B, provided evidence for entry of parasites into cells by phagocytosis. Inhibition of 3 infection by lectins and carbohydrates suggested that glycoproteins and/or glycolipids on parasites and host cell surfaces interact during parasites entry into cells. Phagosome-lysosome fusion was prevented in cells containing the vegetative stages of P. operophterae but not in cells containing only spores. Nosema locustae, a microsporidia infecting locusts was shown to be dimorphic at low temperatures vivo producing Nosema type free spores and membrane bound octospores. In an ultrastructural study of N. locustae, it was revealed that synaptonemal complexes occur in early stages of the octosporoblastic sequence, implying that meiosis occurs at the beginning of this sporogony, so that 8 haploid spores are produced. This type of development is characteristic of the genus Vairimorpha. Neither N. locustae nor V. necatrix produced octospores jjn vitro and attempts to induce dimorphism in vitro by applying juvenile hormone or p-ecdysone to the medium failed. Spores hatched when subjected to a shift from alkaline conditions (pH 11-13 for Nosema sp., and N. heliothidis or pH 9.5 for Orthosoma operophterae, Glugea anomala and Amblyospora sp., to acid on neutral conditions (pH 6-7) in 1M KC1 buffer solution. 4 ACKNOWLEDGEMENTS I wish to express my gratitude to Professor E.U. Canning for her supervision and encouragement throughout the course of my study and for many helpful and stimulating discussions. I am grateful to Dr. D.C. Kelly and his colleagues, of Institute of Virology, NERC, Oxford, for supplying Spodoptera frugiperda cell line and Heliothis zea and Marnestra brassicae eggs and to Dr. R. Gordon for Heliothis virescens eggs,, originally from the Bioenvironmental Insect Laboratory, Mississippi, U.S.A. I am indebted to the following people for the supply of samples of microsporidian spores: Professor E.U. Canning for Pleistophora operophterae and Orthosoma operophterae spores; Dr. J.V. Maddox of the Illinois Natural History Survey, Urbana, Illinois U.S.A., for Vairimorpha necatrix spores; Dr. J.E. Henry of the Rangeland Insect Laboratory, Montana State University, Bozeman, Montana, U.S.A., for Nosema locustae spores; Dr. R. Ishihara, of the Faculty of Agriculture and Veterinary Medicine, Nihon University, Kanagana, Japan and Mr. Z. Wang, of the Department of Sericulture, South China Agricultural University, China, for Nosema bombycis spores; Dr. J. Weiser, of the Institute of Entomology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia, for Pleistophora schuberqi spores; Dr. W.M. Brooks of the North Carolina University, Raleigh, North Carolina, U.S.A., for Nosema heliothidis spores. 5 I am grateful to Mr. J.P. Nicholas and Mr. R. Hartley for their valuable technical assistance. I would also like to thank Mr. Z. Liu, Institute of Entomology, Zhongshan University, Guangzhou, China, for drawing the diagram of fine structure of microsporidian spore, and Mr. Ian Fosbrooke for generating three-dimensional graphs. My thanks also go to all my friends and colleagues at Imperial College, Silwood Park, for their help and advice. I should like to thank Zhongshan University, Guangzhou, China, for financial support during this period of my study in U.K. I would like to express my sincere thanks to Professor Pu Zhelong, Institute of Entomology, Zhongshan University, Guangzhou, China, whose careful and stimulating supervision developed my first interest in microsporidiology. Finally, my love and thanks to my parents, my sisters, particularly my wife, for their constant encouragement and support over these years. 6 TABLE OF CONTENTS Page ABSTRACT 2 ACKNOWLEDGEMENTS 4 TABLE OF CONTENTS 6 LIST OF FIGURES. 12 LIST OF TABLES 20 1 INTRODUCTION 21 2 LITERATURE REVIEW 22 2.1 Germination of spores jji vitro 22 2.2 Microsporidia in tissue cultures 26 2.2.1 Techniques for the establishment 26 of microsporidia in tissue cultures 2.2.2 Factors affecting microsporidian 31 infectivity and multiplication in vitro 2.3 Current taxonomic position of three 33 genera of microsporidia and their developmental cycles 2.3.1 The genus Nosemaand its related 33 genera 2.3.2 The genus Vairimorpha and other 36 dimorphic genera 2.3.3 The genus Pleistophora and 39 related genera 7 Page 2.4 Interaction of parasitic protozoa with 42 their host cells 2.4.1 Invasion into the host cells 43 2.4.1.1. Microsporidia 43 2.4.1.2. Other protozoan parasites 51 2.4.2 Fate of parasitic protozoa in host 55 cells. 3. MATERIALS AND METHODS 59 3.1 Microsporidian species 59 3.2 Infection of insects 59 3.3. Collection and purification of spores 61 from infected insects. 3.4 Induction of spore hatching 62 3.5 Maintenance of insect cell line 64 3.6 Infection of cell cultures 65 3.6.1 Inoculation of cell cultures with 65 spores 3.6.2 Inoculation of cell cultures with 65 the haemolymph from infected insects 3.7 Maintenance of raicrosporidia in cultures 66 of £5. frugiperda cells 3.8 The effect of temperature on development 67 and multiplication of microsporidia in cultures 3.9 The effect of antibiotics and ATP on the 68 growth of Nosema sp. and Nosema heliothidis 8 Page 3.10 Isolation of pre-spore stages (meronts 68 and sporonts). 3.11 Determination of viability of 70 extracellular parasites 3.12 Determination of the phagocytic ability 70 of 55. frugiperda cells 3.13 Determination of the effect of 71 antiphagocytic reagent on infection of £5. frugiperda cells by N. heliothidis 3.14 Demonstration of possible receptor 72 involvement in microsporidian invasion of cells 3.15 Preparation of electron microscopy 73 3.15.1 General method 73 3.15.2 Detection of acid phophastase by 73 electron microscopy in 55. frugiperda cells infected with P. operophterae and N. heliothidis 3.15.3 Detection of secondary lysosomes 74 in 55. frugiperda cells infected with P. operophterae and N. heliothidis 3.16 Attempts to induce dimorphism ir\ vitro by 74 applying hormones 9 Page 4 RESULTS 77 4.1 Germination of spores jin vitro 77 4.1.1 pH optima for pretreatment and 77 treatment 4.1.2 Time for completion of spore 79 germination 4.2 Establishment of microsporidia in the 79 Spodoptera frugiperda cell line 4.2.1 Attempt to infect cell cultures 79 with spores 4.2.2 Infection of cell cultures with 81 infected haemolymph 4.3 The effect of temperature on multi- 81 plication of microsporidia in £>. frugiperda cells 4.3.1 Nosema heliothidis 83 4.3.2 Nosema sp. 86 4.3.3 Nosema locustae 89 4.3.4 Vairimorpha necatrix 89 4.3.5 Pleistophora operophterae 94 4.4 The effect of antibiotics on infectivity 97 to J3. frugiperda of Nosema sp. and N. heliothidis 4.4.1 The effect of antibiotics on 97 N . heliothidis 4.4.2 The effect of antibiotics on 97 Nosema sp. 4.5 Viability of extracellular stages of 99 microsporidia in TC-100 medium 10 Page 4.6 The effect of adenosine triphosphate 105 (ATP) on invasion of cells by Nosema sp. and N. heliothidis 4.7 The effect of Cytochalasin B on entry of 107 N. heliothidis into £3. frugiperda cells 4.8 The effect of lectins and carbohydrates 110 on invasions of S. fruqiperda cells by different species of microsporidia 4.9 Attempts to induce dimorphism in vitro 114 4.10 The morphology and life cycle of micro- 114 sporidia studied by light and electron microscopy 4.10.1 N. heliothidis in vitro 114 4.10.2 Nosema sp. from Spodoptera litura 118 in vitro 4.10.3 Pleistophora operophterae in vitro 119 4.10.4 Life cycle of N. locustae in vivo 121 4.10.5 Attempt to induce dimoirphism in 125 Nosema bombycis 4.10.6 Nosema locustae in vitro 125 4.10.7 Light microscope observation on 125 Vairimorpha necatrix in vitro 4.10.8 Phase contrast light microscope 126 and electron microscope observations of infective stages of N . heliothidis 4.11 Fate of P. operophterae inside the 127 £>. frugiperda cells. 11 Page 4.11.1 Labelling of secondary lysosomes 127 in £3.