IN Y1YQ STUDIES OF SUSPECTED MECHANISMS OF DDT-RESISTANCE IN BLATTELLA GERM.ANICA (L.) by George Lawrence Rolof son Thesis submitted to the Graduate Faculty of the Virginia Polytechnic Institute in partial fulfillment for the degree of DOCTOR OF PHILOSOPHY in Entomology APPROVED: Donald G. Cochran James McD. Grayson Mary H. Ross Ryland E. Webb David A. West Blacksburg, Virginia May 1968 TABLE OF CONTENTS Page I. INTRODUCTION , . 1 II. LITERATURE REVIEW . 3 Early Insecticide Resistance •• . 3 Development of DDT • . 4 Development of DDT Resistance in Houseflies . 6 DDT-Resistance in Cockroaches and Other Insects 13 Inheritance of DDT Resistance . 18 Mode of Action of DDT . ~ . 23 DDT Synergism by Sesamex • • • • . .. 35 III. METIIODS AND MATERIALS . 42 Cockroach Strains • • • • • • 42 Treatment Procedure . 43 Sample Extraction and Cleanup it • • • • • • • • 44 Quantitation of DDT and Metabolites . 47 Thin Layer Chromatography . 48 IV. RESULTS AND DISCUSSION • • • . 50 Toxicological Date • . • • • if • • • 50 DDT Recovery , • • • • • • • • . 52 Penetration . 52 Detoxication • . • • 66 Excretion • • • • • • • • • • • • • • • • • • • 102 Combined Effects • • • ~ • • ~ j • • • • • ~ • ~ • • • • 122 ii iii Page v. STJMMARY 133 VI. REFERENCES CITED . .. 135 VII. VITA. 154 ACKNOWLEDGEMENTS The writer wishes to express his appreciation to Dr. Donald G. Cochran for his helpful criticisms and suggestions throughout the duration of this program. Appreciation is also extended to Dr. Jack L. Bishop for his helpful suggestions in the early part of this work and to Dr. James McD. Grayson for his continuous thoughtful encouragement. The writer is grateful to Drs. Cochran, Graysoni Ross, Webb and West for their critical reading of this manuscript and to Professor Rodney Young for the use of his laboratory and equipment. Appreciation is also extended to Mrs. Jean Dickinson, Mr. J. E. Dunwoody, and Mr. Ty Ku who provided valuable consultation throughout this study. Finally, the writer would like to acknowledge his laboratory assistants, Mrs. Frank Marshall and Mrs. Herbert Thomas, for their faithful work in this project. Recognition is due to Mrs. John Proco for typing this manuscript. iv LIST OF ABBREVIATIONS 1. DDT - 2,2-bis-(p-chlorophenyl)-1,1,1-trichloroethane. 2. DDE ... 2,2-bis-(p-chlorophenyl)-1,1-dichloroethylene. 3. ODA - di(p-chlorophenyl) acetic acid. 4. DBP ... 4,4·' ·dichlorobenzophenone. 5. DDD (TDE) - 2,2-bis-(p-chlorophenyl)-1,1-dichloroethane. 6. dicofol - 2,2-bis-(p-chlorophenyl)-1,1,1-trichloroethanol. 7. sesamex (sesoxane) - acetaldehyde 2·(2-ethoxy-ethoxy) ethyl 3:4 methylene-dioxyphenyl acetal. 8. F-DMC - bis-(p-chlorophenyl)-trifluoromethyl-carbinol. 9. NAD - oxidized nicotinamide adenine dinucleotide. 10. NADPH - reduced nicotinamide adenine dinucleotide phosphate. 11. DMC • bis•(p-chlorophenyl)•methyl-carbinol. v LIST OF FIGURES Figure Page 1. Penetration of 8 ug. of DDT in male and female susceptible- strain German cockroaches . • 56 2. Penetration of 8 ug. of DDT in resistant and susceptible male German cockroaches • . • • 58 3. Penetration of ·30 ug. of DDT in resistant and susceptible German male cockroaches • t • I • e f I e f + • • t t f • • • 60 4. Penetration of 30 ug. of DDT in VPI•DDT-strain male and female German cockroaches . • • • 62 5. Penetration of 30 ug. of DDT in male and female Landsthul- strain German cockroaches . • • 64 6. Penetration of three DDT treatment levels in male VPI- DDT-strain German cockroaches • • • • • • • • • • • • • • • • 67 7. Penetration of two DDT treatment levels in male Landsthul• strain German cockroaches . - . • • • • 69 8. Penetration of 8 ug. of DDT in susceptible-strain male cockroaches pretreated with two different levels of sesamex • • • • • • • • • • • t • • • • • • • • • - • • • • • 71 9. Penetration of 30 ug. of DDT in VPI-DDT•strain male German cockroaches following pretreatment with three levels of sesamex • • • • • • • • • • • • • • • • • • • • •.• 73 10. Penetration of 30 ug. of DDT in Landsthul•strain male German cockroaches following pretreatment with three levels of sesamex • • • • • • • • • • • • • • • • • • • • • • • 75 vi vii Figure Page 11. Conversion of 8 ug. of DDT to dicofol by susceptible•strain male and female German cockroaches . 79 12. Conversion of 8 ug. of DDT to dicofol by male VPI•DDT- and susceptible~strain German cockroaches • . • • • • • • . 81 13. Conversion of 30 ug. of DDT to dicofol in males of two resistant and one susceptible strain of German cockroaches 83 14. Conversion of 30 ug. of DDT to dicofol by male and female VPI•DDT•strain German cockroaches . • • • • • • • • • • • • • 87 15. Conversion of 30 ug. of DDT to dicofol by male and female Landsthul-strain German cockroaches • • • • • • • • • • • • • 89 16. Conversion of the penetra~ed dose from three different DDT treatments to dicofol by VPI-DDT•strain male German cockroaches • • • • • • . .. 91 17. Actual dicofol produced by VPI•DDT•strain male German cockroaches following treatment wi~h three different dosage levels of DDT • • • • • • • • • • • • • • • • • • • · • 93 18. Conversion of 8 ug. of DDT to dicofol by susceptible• strain male German ~ockroaches in the presence of two levels of sesamex • • • • • . Ill • • • • • • • • • 96 19. Conversion of 30 ug. of DDT to dicofol by VPI•DDT•strain male German cockro•ches following pretreatment with three different levels o~ sesamex • • • • • • • • • , • • • • • • • 98 viii Figure 20. Conversion of 30 ug. of DDT to dicofol by Landsthul~strain male German cockroaches following pretreatment with three different levels of sesamex • • • • • • • • • • • • • • • • • 100 21. Excretion of DDT by male and female susceptible-strain German cockroaches following treatment with 8 ug. of DDT per insect • • • • • • • • • • • • • • • • • • • • • • • • • • 104 22. Excretion of DDT by male and female VPI•DDT•strain German cockroaches following a treatment of 30 ug. of DDT per insect • • • • • • • • • • • • • • • • • • • • • • • • • • • • 106 23. Excretion of .DDT by male and female Landsthul-strain German. cockroaches following a 30 ug. treatment of DDT per insect • • 108 24. Excretion of DDT by VPI-DDT- and susceptible-strain male German cockroaches following treatment with 8 ug. of DDT per insect • • • • • • • • • • • • • • • • • • • • • • • • • • 111 25. Excretion of DDT following a 30 ug. DDT treatment by two resistant• and one susceptible-strain of male German cockroaches • • • • • • • • • • • • • • • • • • • • • • • •• 113 26. Excretion of DDT by VPI•DDT-strain male German cockroaches after treatment with three different dosage levels of DDT . ·• • • • • • • • • • • • • • • • • • • • • • • • .. • • 115 27. Actual ug. of DDT excreted by male VPI .. DDT•strain German cockroaches following DDT treatments of three different dosage levels • • • • • • • • • • • • • • • • • • • • • • • • • • • 118 ix Figure 28. Excretion of DDT by susceptible-strain male German cock- roaches following treatment with 8 ug. of DDT in combination with two levels of sesamex • • • • • • • • • • • • • • • • • 120 29. Excretion of DDT by VPI-DDT-strain male German cockroaches following treatment with 30 ug. of DDT in three different combinations with sesamex • • • • • • • • • • • • • • • • • 123 30. Excretion of DDT by Landsthul-strain male German cockroaches following treatment with 30 ug. of DDT in three different combinations with sesamex • • • • • • • • • • • • • • • • • 125 31. Internal DDT concentrations in VPI•DDT•strain male German cockroaches following three different treatments of DDT • • 128 LIST OF TABLES Table Page I. Six-day mortality counts in susceptible-strain male German cockroaches following treatment with 8 ug. of DDT alone and in combination with several levels of sesamex (Ses.) ••• • • 51 II. A typical thin-layer chromatographic confirmatory analysis for DDT and dicofol in internal and excreta sample fractions • 53 III. Penetration of several dosage levels of DDT in resistant and susceptible German cockroaches in presence and absence of sesamex (Ses.) • • • • • • • • • • • • • • • • • • • • • • • • . 54 rv. Detoxication of several dosage levels of DDT in resistant and susceptible German cockroaches in presence and absence of sesamex {Ses.) • • • • • . • • • • • • • • • • • • • • • • • • 78 V. Excretion of DDT by resistant and susceptible German cock• roaches following treatment with several dosage levels of DDT in presence and absence of sesamex (Ses.) • • • • • • • • 103 VI. Internal concentrations of DDT in resistant and susceptible German cockroaches following three different treatment levels and 127 in presence absence of sesamex (Ses.) • • • • • • • • • • • LIST OF TABLES IN APPENDIX. A Table Page I. DDT and dicofol recovered from resistant and susceptible German cockroaches after treatment with 8 ug. of DDT per insect . • • • • • • • • • • • • • • • • • • • • • • 156 II. DDT and dicofol recovered from susceptible German cock· roaches after treatment with sesamex and 8 ug. of DDT per insect • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1S7 III. DDT and dicofol recovered from resistant and susceptible German cockroaches after treatment with 30 ug. of DDT • • • • 158 rv. DDT and dicofol recovered from resistant VPI-DDT-strain German cockroaches after treatment with sesamex and 30 ug. of DDT per insect • • • • • • • • •. • • • • • • • • • • • • • 159 v. DDT and dicofol recovered from resistant Landsthul-strain German cockroaches after treatment with 30 ug. of DDT per insect • • • • • • • • • • • • • • • • • • • • ........ •• 160 VI. DDT and