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ZEEB ROAD, ANN ARBOR, Ml 48106 18 BEDFORD ROW. LONDON WC1R 4EJ, ENGLAND 8022354 St r e e t t , D o u g l a s A l l e n ELECTROPHORETIC SEPARATION OF SPORE POLYPEPTIDES FOR THE IDENTIFICATION AND CLASSIFICATION OF MICROSPORIDIA The Ohio State University Ph.D. 1980 University Microfilms International 300 N. Zeeb Road, Ann Arbor, M I 48106 18 Bedford Row, London WC1R 4EJ, England Copyright 1980 by Streett, Douglas Allen All Rights Reserved PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark . 1. Glossy photographs v 2. Colored illu stra tio n s _________ 3. Photographs with dark background I ' 4. Illu stra tio n s are poor copy _ _ _ _ _ _ 5. Drin t shows through as there is text on both sides of page __________ 6. In d istin ct, broken or small p rin t on several pages _________ throughout 7. Tightly bound copy with p rin t lost in spine ________ 8. Computer printout pages with in d istin ct p rin t ________ 9. Page(s) ^ lacking when material received, and not available 10o Page(s) _________ seem to be missing in numbering only as text follows _______ 11. Poor carbon copy _________ 12. Not original copy, several pages with blurred type _______ 13. Appendix pages are poor copy _______ 14. Original copy with lig h t type _________ 15. Curling and wrinkled pages ________ 16. Other University M icrd fiim s Internarional 200 \ C = = = 3 0 43 30(5 Ml - 8 ’■ 06 ' 312: 761-4700 ELECTROPHORETIC SEPARATION OF SPORE POLYPEPTIDES FOR THE IDENTIFICATION AND CLASSIFICATION OF MICROSPORIDIA DISSERTATION Presented in Partial Fulfillm ent of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Douglas Allen Streett, B.S., M.S. "k •k i t k k The Ohio State University 1980 Reading Committee: proved* By. Dr. John L. Crites Dr. David H. Ives dviser Dr. Emanuel D. Rudolph t of Entomolo ACKNOWLEDGEMENTS , I would lik e to thank my advisor, Dr. John D. Briggs who was always present when I needed advice and friendship, and for his care­ ful consideration of my research. I would also lik e to thank my parents for th e ir moral support, and Dr. Victor Sprague who introduced me to microsporidia and continues to advise me in my research. In addition, I want to thank Dr. Robert M. P fister, one of my favorite faculty members, and Sheila Milligan whom I w ill always consider a close friend. Finally, I'd lik e to thank Adrienne M. Van Zwoll, without whose support and assistance I would never have completed the doctoral program at The Ohio State University. VITA April 19, 1952 ................................ Born - Baltimore, Maryland 1974 .................................................... B.S., St. Mary's College of Maryland, St. Mary's City, Maryland. 1974-1977 ............................................ Graduate Research Assistant, The Ohio State University, Columbus, Ohio. 1977 .................................................... M.S., The Ohio State University, Columbus, Ohio. 1977-1980 ............................................ Graduate Research Assistant, The Ohio State University, Columbus, Ohio PUBLICATIONS Streett, D, A. and V. Sprague. 1974. A Mew Species of Pleistophora (Microsporida: Pleistophoridae) Parasitic in the Shrimp Paleomonetes pugio, J. Invertebr. Pathol., 23:153-156. Streett, D. A., V. Sprague and D. M. Harmon. 1975. Brief Study of Microsporidian Pathogens in the White Pine Weevil Pissodes s tro b i. Ches. Sci., 16:32-38. Service, M. W. and D. A. Streett, 1976. A Pathogenic Mosquito Iridescent Virus in Aedes cantans. Trans. Royal Soc. Trop. Med. Hyg., 70:18. Streett, D. A. 1976. Analysis of Microsporidian Spore Proteins by Electrophoresis on SDS Polyacrylamide Gels: Taxonomic Consider­ ations. Proc. 1st Int. Colloq. on Invertebr. Pathol., Kingston, Canada: 361^362. Streett, D, A. 1977. AmblyOspora sp. from the blackfly Simulium vittatum . Roy. Soc. Trop. Med. Hyg. Symp: Medical Entomology Centenary. i i i Streett, D.A. and W.F. Hink. 1978. Oxygen consumption of Trichiplusia ni (TN-368) Insect Cell Line Infected with Autographa californica Nuclear Polyhedrosis Virus. J. Invertebr. P a th o l.,321112-113. Streett, D.A. and J.Y. Bradfield IV. 1978. Juvenile Hormone / Activity in Microsporidian Spores. Proc. Ivth International Colloq.Invertebr. Pathol., Prague, Czechoslovakia. 199-200. Streett, D.A., D. Ralph and W.F. Hink. 1980. Replication of Nosema algerae in Three Insect Cell Lines. J. Protozool. 27:113-1177 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS........................................................................................... i i VITA ............................................................................................................... i i i LIST OF TABLES........................................................... v ii LIST OF FIGURES.......................................................................................v iii INTRODUCTION................................................................................................... 1 Classification of the Microsporidia ..................................... 2 Life Cycle of the Microsporidia ............................................. 3 Taxonomy of the Microsporidia ................................................. 4 Electrophoretic Analysis of Microsporidian Parasites . 8 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. 10 MATERIALS AND METHODS ............................................................................... 13 Microsporidia ........................................................... 13 Infection of Insects ..... ................................................... 15 Isolation of Microsporidian Spores ............................................ 16 Preparation of Spore Proteins for Electrophoresis .......... 23 Polyacrylamide Gel Electrophoresis (PAGE) of Micro­ sporidian Spore Polypeptides ......................................... 28 Data Analyses of Electrophoretic P rofile s.......................... 33 Investigation of Spore Surface Contaminants and Proteinaceous Exospore Layer ......................................... 35 Detection of Protein Contamination in Spore Storage Buffer. 36 Protein Determination ................................................................. 36 Effect of Different Host Rearing Temperatures on the Electrophoretic Profile ....................................................... 38 Effect of Different Host Species on the Electrophoretic P r o f ile ................................................................................. 38 Effect of Spore Mixtures on the Electrophoretic Profile. 39 RESULTS........................................................................................................... 41 DISCUSSION....................................................................................................... 82 V CONCLUSIONS....................................................................................................... APPENDIXES A. Taxonomic Classification of the Microsporidia ................ B. Glossary fo r the Microsporidia ................................................ C. Preparation of Semisynthetic Insect Diet ........................... D. Instructions for Preparation of Spore Sample for SDS Polyacrylamide Gel Electrophoresis ........................................ E. Table for Preparing SDS Acrylamide Gel Concentrations . F. Stock Solutions for SDS Electrophoresis ............................. G. Instructions for Preparation of SDS Polyacrylamide Gel. H. Separation of Spore Polypeptides with