Characterization of the Regulatory Process of Pyrethroid Resistance in the House Fly, Musca domestica by Ming Li A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama December 13, 2014 Keywords: Insecticide resistance, Regulation, Transcriptome, Cytochrome P450, Carboxylesterase, Musca domestica Copyright 2014 by Ming Li Approved by Nannan Liu, Chair, Professor of Entomology and Plant Pathology Joseph W. Kloepper, Professor of Entomology and Plant Pathology Scott R. Santos, Associate Professor of Biological Sciences Lee Zhang, Research Fellow of Entomology and Plant Pathology Abstract The house fly, Musca domestica, is a major domestic, medical and veterinary pest that causes more than 100 human and animal intestinal diseases. The major barrier in the house fly control is their remarkable ability to develop not only resistance to the insecticide used against them, but also cross-resistance to unrelated classes of insecticides. The house fly has demonstrated to be a useful model to study and predict resistance in not only themselves but also other insect species. The current study generated the first reference transcriptome from the adult house fly and a whole transcriptome analysis was conducted for the multiple insecticide resistant strain ALHF (wild-type) and two insecticide susceptible strains: aabys (with morphological recessive markers) and CS (wild type) to gain valuable insights into the gene interaction and complex regulation in insecticide resistance of house flies. A total of 1316 genes were identified as being co-up- regulated in ALHF in comparison to both aabys and CS. The majority of these up-regulated genes fell within the three key detailed function categories: redox detailed function category in metabolism, signal transduction and kinases/phosphatases in regulation, and proteases in intra- cellular processes. Genetic linkage analysis with house fly lines comparing different autosomal combinations from ALHF revealed that the up-regulation of gene expression occurred mainly through the co-regulation of factors among multiple autosomes, especially between autosomes 2 and 5, suggesting that signaling transduction cascades controlled by GPCRs, protein ii kinase/phosphates and proteases may be involved in the regulation of P450 and carboxylesterase gene expression. To characterize the cytochrome P450 and carboxylesterase genes that play important roles in the pyrethroid resistance of house flies, 86 P450 and 26 carboxylesterase genes were selected based on our whole transcriptome analysis of the house fly to conduct the expression profile analysis in different house fly strains with different levels of permethrin resistance and autosome combinations. Our study showed that multiple P450 and carboxylesterase genes were co-up- regulated in insecticide-resistant house flies compared to -susceptible house flies, and the expression of these genes was regulated by cis or trans regulatory factors/genes, which were mainly on autosomes 1, 2 and 5. Transgenic expression analysis of selected P450 and carboxylesterase genes in Drosophila melanogaster demonstrated that elevated expression of these genes confers different levels of resistance to permethrin in the transgenic Drosophila. Homology modeling and permethrin docking analysis further suggested potential ability of these genes to metabolize permethrin. Taken together, the study provides a global picture of P450 and carboxylesterase gene expression, regulation, autosomal interaction, and function in insecticide resistance of house flies, indicating multiple genes are co-responsible for detoxification of insecticides, and multiple mechanisms co-work on the development of insecticide resistance in house flies. iii Acknowledgments I am highly grateful to my major professor, Dr. Nannan Liu, for her guidance, encouragement, support, timely advice and numerous opportunities that she provided over these four years. Without her patience and help, this dissertation would never be accomplished. I feel very honored and fortunate to join Dr. Liu’s program and have such a great advisor in my academic life. Her attitudes towards life, perspectives of science, and ways of doing research will influence me forever. I also would like to thank my committee members, Dr. Scott R. Santos, Dr. Joseph W. Kloepper, Dr. Lee Zhang and my dissertation outside reader, Dr. Yucheng Feng, for their invaluable encouragement, advice and kind help. I also would like to give my many thanks to my laboratory colleagues, Dr. Chuanwang Cao, Dr. Lin He, Ms. Ting Li, Mr. Li Tian, Mr. William R. Reid, Mr. Feng Liu, Ms. Youhui Gong, Ms. Xuechun Feng, Mr, Zi Ye, Ms Fang Tang, Mr Zhou Chen and Ms Caixing Xiong for their friendly help, suggestions and kindness during the period of my Ph.D study. The support and help from other faculty members, staff and students in the Department of Entomology and Plant Pathology are also greatly appreciated. Finally, the deepest gratitude goes to my parents, wife and sister for their endless love and support. I am forever grateful for their consistent understanding and encouragement. iv Table of Contents Abstract ....................................................................................................................................... ii Acknowledgments.......................................................................................................................... iv List of Tables .................................................................................................................................. xi List of Figures ............................................................................................................................... xii List of Abbreviations..................................................................................................................... xv Chapter 1: Literature Review .......................................................................................................... 1 1.1 Insecticide resistance ................................................................................................................ 1 1.2 How insecticide resistance develops ......................................................................................... 2 1.3 Cross resistance and multiple resistances ................................................................................. 2 1.3.1 Cross resistance ...................................................................................................................... 2 1.3.2 Multiple resistance ................................................................................................................. 3 1.4 Insecticide resistance stability ................................................................................................... 3 1.5 Insecticide resistance mechanisms ............................................................................................ 4 1.5.1 Behavioral resistance ............................................................................................................. 4 1.5.2 Physiological resistance ......................................................................................................... 5 1.5.2.1 Reduced cuticle penetration rate ......................................................................................... 5 1.5.2.2 Increased sequestration storage and accelerated excretion of insecticides ......................... 6 1.5.3 Increased activities of detoxification enzymes ...................................................................... 7 v 1.5.3.1 Cytochrome P450 monooxygenases ................................................................................... 8 1.5.3.2 Esterases and hydrolases ................................................................................................... 12 1.5.3.2.1 Quantitative changes ...................................................................................................... 12 1.5.3.2.2 Qualitative changes ........................................................................................................ 13 1.5.3.3 Glutathione S-transferases ................................................................................................ 13 1.5.4 Reduced sensitivity of target sites ........................................................................................ 14 1.5.4.1 Insensitivity of acetylcholinesterase ................................................................................. 14 1.5.4.2 The GABA receptor mutation ........................................................................................... 15 1.5.4.3 Mutations in the voltage-gated sodium channel ............................................................... 16 1.6 Regulatory gene and gene expression regulation.................................................................... 18 1.6.1 Regulatory gene ................................................................................................................... 18 1.6.2 Regulation of gene expression ............................................................................................. 19 1.6.2.1 Regulation of gene expression by extracellular signals .................................................... 20 1.6.2.2 Regulation of gene expression by the structure of chromatin ........................................... 20 1.6.2.3 Regulation of gene expression by transcription