INFORMATION TO USERS This material was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1.The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete. 4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced. 5. PLEASE NOTE: Some pages may have indistinct print. Filmed as received. Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 77-24,697 SCHREIBER, Frederick Erwin, 1943- INDUCED ANTIBACTERIAL ACTIVITY AGAINST PSEUDOMONAS AERUGINOSA (SCHROETER) MIGULA IN THE L arvae of th e tobacco hornworm, MANDUCA SEXTA (L.) The Ohio State University, Ph.D., 1977 Entomology Xerox University Microfilms,Ann Arbor, Michigan 48106 PLEASE NOTE: Some print throughout this text is broken and light. This is the best available copy. Filmed as received. University Microfilms INDUCED ANTIBACTERIAL ACTIVITY AGAINST PSEUDOMONAS AERUGINOSA (SCHROETER) MIGULA IN THE LARVAE OF THE TOBACCO HORNWORM, MANDUCA SEXTA (L.) DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Frederick Erwin Schreiber, B.S., M.S The Ohio State University 1977 Reading Committee: Approved by W. Fred Hink John D. Briggs William J. Collins Adviser Frank W. Fisk Department of Entomology Ruth, Robert and Mark ACKNOWLEDGMENTS I thank my adviser Dr. W. Fred Hink for his * 0 assistance and encouragement#during the course of this study. I also greatly appreciate his help, some of it above and beyond the call of duty, during the preparation and completion of this dissertation. The rearing of Manduea required a considerable amount of space and equipment. The Department of Entomology and the individual faculty members involved were more than generous in making available to me what I needed. A fellow graduate student, Frank Eischen, suggested the use of Manduea sexta as an experimental animal. Having just completed his thesis on Manduca, he also taught me how to rear the animal and gave me the needed equipment. At the time this research was done, A1 Pye was studying the role of phenoloxidase in wax moth immunity. He shared literature, ideas, and equipment, all of which proved very helpful. Dave Chesemore provided valuable assistance in choosing and interpreting statistical tests. Ruth, my wife, typed the first drafts of this dissertation. That was probably the least of her sacrifices. Though the gesture is inadequate, I thank her. iii VITA April 27, 1943 ......... Born - Wetaskiwin, Alberta, Canada 1967 .................... B.S., Northern Illinois University, DeKalb, Illinois 1967-1969 ............. Teaching Assistant, Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 1969 .................... M.S., Northern Illinois University, DeKalb, Illinois 1969-1973 ............. Teaching Associate, Department of Entomology, The Ohio State University, Columbus, Ohio 1973-1977.. ............. Assistant Professor, Department of Biology, California State University, Fresno, Fresno, California FIELDS OF STUDY Studies in Insect Pathology Associate Professor W. Fred Hink Studies in Insect Toxicology Associate Professor William J. Collins Studies in Insect Physiology Professor Frank W. Fisk Studies in Insect Morphology Professor Donald J'. Borror Studies in Biochemistry Professor James 0. Alben Professor Gerald P. Brierly Professor Harold Broquist iv VITA (Continued) Associate Professor E. L. Gross Professor D. H. Ives Professor Keith E. Richardson Professor R. A. Scott Professor George Serif Professor Howard W. Sprecher Professor Thomas E. Webb Studies in Biophysics Associate Professor Joseph Y. Cassim Studies in Cell Biology Associate Professor Thomas J. Byers Associate Professor Lynn B. Graves, Jr Professor Knut J. Norstog v TABLE OF CONTENTS Page DEDICATION .................................... ii ACKNOWLEDGMENTS ......................................... iii VITA .................................................... iv LIST OF TABLES ......................... viii LIST OF FIGURES ......................................... x INTRODUCTION AND LITERATURE REVIEW .................. 1 METHODS AND MATERIALS .................................. 31 R e a r i n g ............................................. 31 Bacterial Strains Used ........................... 34 Preparation of Bacterin ........................... 34 Immunization and Challenge ...................... 35 Agar Well Diffusion A s s a y ......................... 37 Preparation of Hemolymph ......................... 37 In Vitro Techniques ................................ 39 Lysozyme Assay .................................... 41 RESULTS AND DISCUSSION ................................ 44 Response of M. sexta to P. aeruginosa and S. marcescens .................................... 44 The LD50 of P. aeruginosa and S. marcescens for M. s e x t a .................................. 44 The pathology of infected larvae .............. 51 In vitro growth of P. aeruginosa and S. marcescens in normal serum ................ 55 Sensitivity of P. aeruginosa and £3. marcescens to dietary antimicrobial c o m p o u n d s ...................................... 60 Page In Vivo and In Vitro Immunity and Melanization ..................................... 62 Integumental melanization occurring with immunization .............................. 62 Toxicity of bacterin ........................... 65 In vivo immunity ................................ 70 In vitro antibacterial activity and melanization .................................. 78 In Vitro Antibacterial Activity, Its Stability and Relationship to Melanization ..................................... 87 Classification of immunized larvae ............. 87 Antibacterial activity of the three classes of hemolymph ......................... 89 Stability of antibacterial activity ...... 94 Studies on the relationship between in vitro melanization and antibacterial a c t i v i t y ....................................... 104 Hemolymph Lysozyme Activity ....................... Ill GENERAL DISCUSSION AND CONCLUSIONS .................. 118 SUMMARY ................................................... 125 LITERATURE C I T E D .............. 127 I LIST OF TABLES Table Page 1. Artificial Diet for Tobacco Homworm L a r v a e ...................................... 33 2. L D 5 q °f P- aeruginosa in Various Insects . 48 3. ^ 5 0 marcescens in Various Insects . 50 4. Inhibition of Bacterial Growth by M. sexta Dietary Antimicrobial Compounds in the Agar Well Diffusion A s s a y ........................................ 61 5. Weight Gain of Immunized M. sexta Larvae . 68 6 . Survival of Immunized M. sexta Larvae After Challenge with P. aeruginosa .... 71 7. Survival of M. sexta Larvae Immunized with 1x10? S." marcescens to Homologous Challenge ...................... 75 8 . In Vitro Antibacterial Activity and Melanization of Three Immune Serum C l a s s e s ...................................... 91 9. Stability of Antibacterial Activity of Class II Hemolymph to Heating in a Boiling Water Bath for Five Minutes .... 95 10. Stability of Serum Bactericidal Activity During Storage at 4 ° C ...................... 98 11. Stability of Class II Serum Antibacterial Activity to Treatment with Equal Volumes of Acid or B a s e .................... 102 12. Effect of Adding Bacterin to Depression on IS Vitro Melanization and Bactericidal Activity ...................... 106 • • • Vlll Table Page 13. Effect of Heating on Melanization and Antibacterial Activity of Serum from Saline Injected and Class I Larvae.... 109 14. The Effect of Adding 0.001 M Tyrosine on In Vitro Melanization and Antibacterial Activity .................. 110 15. Units of Lysozyme Activity of Hemolymph from Saline Injected Larvae and Class I, II, and III Immunized Larvae . 114 16. Bactericidal Activity of Serum in Relation to Melanization ................. 121 \ ix LIST OF FIGURES Figure Page 1. Typical arrangement of sera and bacteria