STUDIES ON LUNG FLUKES OF RANA SPP. By SURIAYA MOGHAL M. Sc A thesis submitted for the Degree of Master of Philosophy of the University of London and for the Diploma of Membership of the Imperial College. October, 1979 Department of Zoology and Applied Entomology, Imperial College of Science and Technology, South Kensington, London, SW 7. 2 ABSTRACT Miss Suriaya Moghal. "Studies on lung flukes of Rana spp" Studies were made on (1) the life cycle of Haematoloechus similis and H. variegatus from Rana ridibunda (2) Egg hatching in vitro of a, Haplometra cylindracea (from Rana temporaria) b, Haema- toloechus spp from Rana pipiens and R. ridibunda (3) In vitro culture of lung of Rana temporaria and R. pipiens. The development of Haematoloechus similis and H. variegatus in their intermediate hosts was investigated. Planorbis planorbis was found to serve as the first intermediate host for both these species. The second intermediate host Coenagrion puella was exposed to cercariae of Haematoloechus similis and 1 - 2 unencysted metacercariae were found free in the haemocoele on the following day. Coenagrion puella was also exposed to the cercariae of H. variegatus but no penetration was observed, again 1 - 2 metacercarial cysts were found. Out of 10 nymphs unexposed to cercariae which were used as controls, one was positive. Hence an experimental infection was not unequivocally demonstrated. The complete life cycle of Haematoloechus similis and H. variegatus in the U.K. thus remains undetermined. Attempts were made to obtain experimental infections of Haplometra cylindracea in snails of various species of laboratory bred snails such as Physa sp, Planorbis planorbis, Lymnaea peregra, L. stagnalis, L. palustris and L. truncatula. All these attempts were unsuccessful. It was, therefore, decided to attempt to hatch the eggs in vitro. The effects of different factors on the eggs of Haplometra cylindracea in vitro, such as osmotic pressure, hydrogen ion concentration, digestive enzymes, temperature, gas - 3 - phase, reducing agents, bile salts, NaOCland various other factors were examined. All attempts to hatch the eggs outside the host were unsuccessful but hatching took place in NaOC1, but only non-motile miracidia were observed since NaOClis toxic to miracidia. Eggs of Haematoloechus spp. hatched when placed in "snail conditioned water" (using Planorbis Planorbis) suggesting that some factor or factors which induce hatching, such as an enzyme, are released from the snail. Attempts were made to culture the lung tissue of Rana temporaria and R. pipiens in vitro in the hope that this might make possible culture of trematodes within cultured lung tissue. The maximum time of survival of this tissue culture was 114 days. 4 ACKNOWLEDGEMENT It is my first duty to express my sincerest thanks to my supervisor, Professor J. D. Smyth, for introducing me into such an interesting field, for his encouragement, continuous help and patience which have been priceless throughout my work and the preparation of my thesis. My sincere thanks to Dr D. P. McManus and Dr C. J. Davies for helpful advice and encouragement throughout the course of my work. Thanks are also due to Dr Heather Paterson and Mrs Nicola Barrett for their advice and suggestions. Special thanks to Mrs G. Adams for her advice and encouragement during my stay. I would like to thank Mrs A. Meredith for her care in typing the manuscript. I am very grateful to Mr C. L. Meredith, Mr I. J. French, Mr J. Harmer, Miss S.R. Harper, Mrs A. Merry, Mr R. Krafft, Mrs I. Gadd, Mrs B. Stronach, Mr S. V. Stammers and others for help in various ways. I am grateful to all my colleagues and friends. Thanks are due to Dr Paul Sterry, Dr S. Cole, Mrs J. Yeadon and other librarian staff. The work was supported by a central training overseas scheme of the Government of Pakistan. I am grateful to the Government of Pakistan for awarding me the scholarship for this re search. Finally, my gratitude is due to my parents, M.A. Moghal and Dr Sanjida Moghal for their help and moral support. 5 CONTENTS Page No. Title Page ... ... ... ... .. .. ... ... .. ... ... 1 Abstract ... ... ... ... ... ... ... ... ... ... ... 2 Acknowledgement ... ... ... ... ... ... ... ... ... 4 Contents ... ....... ... ... ... ... ... ... 5 List of Figures ... ... ... ... ... ... ... ... ... ... 8 List of Plates . , . • . ... ... ... ... ... ... ... ... 10 List of Tables ... ... ... ... ... ... ... ... ... ... 12 General Introduction • ••• •• • ••• ••• •.. 16 Part I The life cycle of Haematoloechus similis and H. variegatus ••• ••• ••• ••• ••• ••• ••• 18 Introduction ••• ••• ••• ••• ••• ••• ••• ••• ,.. 19 Materials and Methods ••• ••• •• • • .. ••• ••• 36 Results of the examination of Planorbis planorbis exposed to infection of Haematoloechus egg. ... •• 63 Discussion ... ... ... ... ... ... ... 99 Summary . .•• ••• 112 - 6 Page No. Part II 1. In vitro hatching of the eggs of 114 Haplometra cylindracea (R. temporaria) 2. In vitro hatching of the eggs of Haematoloechus (R. pipiens) 3. In vitro hatching of the eggs of Haematoloechus (R. ridibunda) Introduction ••• ••• ••• ••• ••• ••• ••• • •• 115 Materials and Methods ,,, ,,, 120 I In vitro hatching of the eggs of Haplometra cylindracea , , , 124 Experimental procedure and results II In vitro hatching of the eggs of Haematoloechus (R. pipiens) 148 Experimental procedure and results III In vitro hatching of the eggs of Haematoloechus (R. ridibunda) 152 Experimental procedure and results Discus sion ••• ••• ••• ••• ••• ••• ••• ••• 165 Summary •• • ••• ••• ••• ••• ••• ••• ••• 176 Part III In vitro culture of lung of Rana temporaria and R. pipiens ••• ••• ••• ••• ••• ••• ••• 177 Introduction ••• ••• ••• ••• ••• ••• ••• ••• 178 Materials and Methods ••• ••• ••• ••• 183 7 Page No. Result and Discussion ... ... ... ... ... ... 185 Summary ... ... ... ... ... ... 190 Conclusions and scope for further work 191 References... ... ... ... ... ... ... ... ... ... 192 - 8 - LIST OF FIGURES Figure Page No. 1. Mother sporocyst, 4 days old, of Haematoloechus sp. 65 2. Daughter sporocysts, 32 days old, of Haematoloechus sp. 65 3. Early cercarial stages, 32 days old, of Haematoloechus sp. 65 - 66 4. Mature daughter sporocyst containing an immature cercaria, 49 days after infection of Haematoloechus sp. 68 5. Tail of cercaria of Haematoloechus similis. 84 6. Stylet of cercaria of Haematoloechus similis. 84 7. Tail of cercaria of Haematoloechus variegatus. 88 8. Stylet of cercaria of Haematoloechus variegatus. 88 9. Metacercarial cyst removed from the nymph Coenagrion puella. 95 10. The life cycle of Haematoloechus similis. 97 9 Figure Page No. 11. (a) Cercaria of Haematoloechus similis. 106 (b) Stylet of cercaria of Haematoloechus similis. 12. Cercaria of Haematoloechus variegatus. 110 1.3. Complete anaerobic condition applied in the case of eggs of Haplometra cylindracea in vitro. 12.3 14. The digestive system of Lymnaea stagnalis . 164 - 10 - LIST OF PLATES Plate Page No. 1. Maintenance of uninfected and infected Snails, Planorbis planorbis. 39 2. Dorsal view of second intermediate host damsel fly nymph, Coenagrion puella. 41 3. Maintenance of second intermediate host damsel fly nymph, Coenagrion puella. 43 4. Maintenance of experimental infected second intermediate host damselfly nymph, Coenagrion puella. 45 5. Haematoloechus similis, a common parasite in the lungs of frog Rana ridibunda. 49 6. Adult Haematoloechus variegatus, a common lung fluke of Rana ridibunda. 55 7. Experimentally infected Snail, Planorbis planorbis. 62 8. Photomicrograph of 42 days old daughter sporocyst of Haematoloechus sp. 71 9. Photomicrograph of 42 days old immature cercaria of haematoloechus sp. 73 Plate Page No. 10. Photomicrograph of 49 days old section of infected snail, Planorbis planorbis containing daughter sporocysts . 75 11. Encysted metacercarial cyst removed from the second intermediate host, the damselfly nymph, Coenagrion puella. 93 12. Excysted metacercaria. 93 13. Eggs of lung fluke, Haplometra cylindracea of frog Rana temporaria. 143 14. Egg hatching of the lung fluke, Haplometra cylindracea of frog Rana temporaria. 143 15- 19. Photomicrographs showing hatching of the egg of the lung fluke Haematoloechus sp. 155 - 158 20. Lung of Rana pipiens (not cultured). 189 21. Section of lung of Rana pipiens after 114 days in culture. Mitotic divisions. 189 - 12 - LIST OF TABLES Table Page No. 1. The complete life cycle of the species of the genus Haematoloechus as reported by various workers. 25 - 27 2. Lists of the species of genus Coenagrion Kirby, 1890 and their distribution (After Hammond, 1977). 35 3. Dimensions of Haematoloechus similis. 50 4. Variability of Haematoloechus similis as recorded by the various authors. (After Prokopic & Krivanec, 1974). 51 - 52 5. Dimensions of Haematoloechus variegatus. 56 6. Variability of Haematoloechus variegatus as recorded by the various authors (After Prokopic & Krivanec, 1974). 57 - 58 7. Experimental infection of snails Planorbis planorbis with Haematoloechus sp. 76 - 77 8. Comparison of sporocysts sizes of the species of the genus Haematoloechus. 78 - 13 - Table Page No. 9. Comparative dimensions of cercariae of Skrjabinoeces similis = Haematoloechus similis and present material (After Ginetsinskaia & Dobrovol'skii, 1968). 80 10. Exposure of cercariae of Haematoloechus similis to the damselfly Coenagrion puella. 82 11. Comparison of measurements made in different conditions of cercariae of Haematoloechus variegatus. 86 12. Comparative dimensions of cercariae of Pneumonoeces variegatus