MORPHOLOGY, TAXONOMY AND LIFE CYCLES OF SOME SAURIAN HAEMATOZOA by Keith Robert Wallbanks B.Sc. (Lond.) A.R.C.S. 1982 A thesis submitted for the Degree of Doctor of Philosophy of the University of London Department of Pure and Applied Biology Imperial College Silwood Park Ascot Berkshire ii TO MY MOTHER AND FATHER WITH GRATITUDE AND LOVE iii Abstract The trypanosomes and Leishmania parasites of lizards are reviewed. The development of Trypanosoma platydactyli in two sandfly species, Sergentomyia minuta and Phlehotomus papatasi and in in vitro culture was followed. In sandflies the blood trypomastigotes passed through amastigote, epimastigote and promastigote phases in the midgut of the fly before developing into short, slender, non-dividing trypomastigotes in the mid- and hind-gut. These short trypomastigotes are presumed to be the infective metatrypomastigotes. In axenic culture T. platydactyli passed through amastigote and epimastigote phases into a promastigote phase. The promastigote phase was very stable and attempts to stimulate -the differentiation of promastigotes to epi- or trypo-mastigotes, by changing culture media, pH values and temperature failed. The trypanosome origin of the promastigotes was proved by the growth of promastigotes in cultures from a cloned blood trypomastigote. The resultant promastigote cultures were identical in general morphology, ultrastructure and the electrophoretic mobility of 8 enzymes to those previously considered to be Leishmania tarentolae. T. platydactyli and L. tarentolae are synonymised and the present status of saurian Leishmania parasites is discussed. Promastigote cultures of T. platydactyli formed intracellular amastigotes. in mouse macrophages, lizard monocytes and lizard kidney cells in vitro. The parasites were rapidly destroyed by mouse macrophages jlii vivo and in vitro at 37°C. Intestinal infections of trypanosomatine parasites were followed for up to 6 months in 3 Chamaeleo dilepis. The flagellates were not infective to Musca domestica or Tenebrio molitor and failed to grow in in vitro culture, iv probably because of fungal contamination of the cultures. The classification of trypanosomatine parasites of lizard intestine is discussed. The transfer of these parasites from the genus Leishmania to the genera Leptomonas, Herpetomonas and Crithidia and the necessary redefinition of these genera to include a wider host range is suggested. An attempt to discover the vector of Plasmodium agamae in the Gambia is described. Sandflies fed on lizards infected with P. agamae showed no signs of parasite gametogenesis or sporogenesis when dissected. Possible reasons for this failure including gametocyte immaturity or senility and other insects as the natural vectors, are discussed. V Acknowledgement s I am most grateful to the many people who made this work possible, both known and unknown. My supervisor, Professor E.U. Canning, and adviser Dr. R.R. Killick-Kendrick, provided advice and encouragement throughout the project. Drs. R.S. Bray, R.E. Sinden, R.J. Barker, D.P. Turner and J. Alexander provided help and advice when asked. Mrs. A. Mendis and Mr. A.J. Leaney taught me aTl I know about sandfly colonisation. L. Malone, D. Pinero, 0. and R. Rossell and especially N. Anez gave me ideas and companionship when I needed them most. Technical assistance came from Messrs. J.P. Nicholas, A.M. Page, R. Hartley andA. Righton. Mr. T.J. Wilkes helped with the entomological side of the project and also with useful introductions in the Gambia. Mrs. J. Pugh was an efficient source of stores and amusement and Mrs. M. Wilmot managed to track down many obscure references. Professor P.C.C. Garnham offered suggestions, interest, and new names for the author. I am indebted to Professor J.A. Rioux, Drs. G. Lannotte and R. Maazoun, Madame M. Bailly and their colleagues for their generosity, hospitality, patience and assistance during my visits to Southern France. My stay in the Gambia was made enjoyable by the many people who came to my aid and who were so friendly and generous in terms of their time. Dr. and Mrs. B.M. Greenwood helped to make the necessary arrangements for my visit prior to and after my arrival. Dr. and Mrs. H.A. Wilkins, Dr. and Mrs. P. Hagan, Dr. J.H. Bryan and especially Mr. P.J. Moore, went to great lengths to make me feel welcome and helped me whenever they could. I am grateful to Father H. Fagan and the Peace Corps volunteers, especially Mr. and Mrs. B. Trimble who offered hospitality at Basse and helped broadcast my need for lizards. Unpublished results, advice and information were generously given by vi Professor P. Ranque, Drs. E.N. Arnold, J.R. Baker, P. Desjeux, D.A. Evans, U. Joger, D.M. Minter, W.F. Snow and S.R. Telford and Mr. D.J. Ball. Dr. Telford deserves a special mention for his long letters on saurian haemoflagellates, his permission to use data and the donation of slides of several parasite infections. Drs. M.L. Chance, S.L. Croft and C.J. Young are also to be thanked for donating parasite cultures. Mrs. Joan Shepherd typed my thesis with speed and efficiency and a quiet tolerance of my handwriting. The generous financial support of the Medical Research Council, London, and the Department of Pure and Applied Biology, Imperial College, London is gratefully acknowledged. Finally I must thank my family, particularly my parents and my brother Michael, for their constant support and encouragement over the years. vii CONTENTS PART A x Page LIZARD HAEMOFLAGELLATES 1 General Introduction 2 The Morphology and taxonomy of the kinetoplastid parasites of lizard blood 2.1 Lizard trypanosomes 6 2.2 List of lizard trypanosomes 6 2.3 Lizard Leishmania parasites 24 2.4 List of lizard Leishmania parasites 24 2.5 A note on reports of Leishmania parasites from some Kenyan lizards 32 An examination of the haemoflagellates of Tarentola mauritanica (L. 1758) 33 3.1 Introduction 33 3.2 Materials and methods 34 3.2.1. Collection of lizards and their maintenance 34 3.2.2. Examination of blood and tissues 34 3.2.3. Culture of parasites in axenic culture 35 3.2.3.1. Routine culture and culture "en masse" 35 3.2.3.2. Culture in a variety of media 35 3.2.3.3. Culture at different temperatures and pH values 37 3.2.3.4. Culture in medium with stimulants of differentiation 37 3.2.3.5. The use of antibiotics and routine examination of cultures 37 3.2.4. Interaction of parasites with vertebrate cells 38 3.2.4.1. Lizard blood cells (i) in vitro 38 (ii) in vivo 38 3.2.4.2. Lizard tissue cells 39 3.2.4.3. Murine macrophage cells in vitro 39 3.2.4.4. Murine macrophage cells in vivo 40 3.2.5. Cloning of blood trypomastigotes 41 3.2.6. Comparison of stock morphology and biochemistry 42 3.2.6.1. Morphological comparison (i) by light microscopy 43 (ii) by electron microscopy 43 3.2.6.2. Biochemical comparison by isoenzyme electrophoresis 44 3.2.7. Origin, collection and maintenance of sandflies 44 3.2.7.1. Phlebotomus papatasi 44 3.2.7.2. Sergentomyia minuta minuta 46 3.2.8. Infection and dissection of sandflies 47 3.2.8.1. Infection of sandflies from a gecko 47 3.2.8.2. The observation of flagellate inrections by light and electron microscopy 47 3.2.8.3. Attempt to infect sandflies by membrane feeding 48 .3. Results 49 3.3.1. Collection of lizards 49 3.3.2. Examination of blood 49 3.3.2.1. Description of parasites 49 3.3.3. Culture of parasites in axenic culture 56 3.3.4. Interaction of parasites with vertebrate cells 60 3.3.4.1. Lizard blood cells (i) in vitro 60 (ii) jLn vivo 60 3.3.4.2. Lizard tissue cells 63 viii Page 3.3.4.3. Murine macrophage cells in vitro 63 3.3.4.4. Murine cells in vivo 65 3.3.5. Cloning of blood trypomastigotes 68 3.3.6. Comparison of strain morphology and biochemistry 68 3.3.6.1. Comparison of strain morphology 68 3.3.6.2. " " " biochemistry 71 3.3.7. Origin collection and maintenance of sandflies 71 3.3.7.1. S .m.minuta 71 3.3.8. Infection of sandflies and dissection 73 3.3.8.1. Development of T. platydactyli in P. papatasi 73 3.3.8.2. " " " S. minuta 75 3.3.8.3. Electron microscopic examination of the sandfly infections 78 3.4. Discussion 87 3.4.1. The gecko haemoflagellates in vivo 87 3.4.1.1. The trypomastigotes 87 3.4.1.2. The amastigotes 87 3.4.1.3. The role of the intracellular amastigotes 90 3.4.2. The relationship between the promastigotes and trypomastigotes from the gecko 93 3.4.3. The morphological and biochemical comparison of stocks and strains of gecko haemoflagellates in culture 94 3.4.4. The development of T. platydactyli in sandflies 96 3.4.4.1. Previous observations of sandfly infections 96 3.4.4.2. Recent observations of sandfly infections 98 3.4.5. Comparison of S. minuta and P. papatasi as invertebrate hosts of T. platydactyli 100 3.4.6. The ultrastructure of T. platydactyli 100 3.4.6.1. Parasites in the sandfly 100 3.4.6.2. Parasites in in vitro culture 103 3.4.7. The interaction of promastigote cultures with vertebrate cells in vivo and iji vitro 104 3.4.7.1. The interaction of parasites with mouse cells 104 3.4.7.2. " " " " " lizard " 106 3.4.8. Promastigotes in the genus Trypanosoma and trypanosomatid phylogeny 108 3.4.9. The status of saurian leishmaniasis 109 ix PART B TRYPANOSOMATINE INFECTIONS OF LIZARD INTESTINE Introduction A study of intestinal trypanosomatine parasite infections of some Zambian Chamaeleo dilepis ,1.