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Multi-Gene Resistance to Neonicotinoids in the Colorado Potato Beetle, Leptinotarsa Decemlineata

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Multi-Gene Resistance to Neonicotinoids in the Colorado Potato Beetle, Leptinotarsa Decemlineata Western University Scholarship@Western Electronic Thesis and Dissertation Repository 8-22-2016 12:00 AM Multi-gene resistance to neonicotinoids in the Colorado potato beetle, Leptinotarsa decemlineata Emine Kaplanoglu The University of Western Ontario Supervisor Dr. Cam Donly The University of Western Ontario Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Emine Kaplanoglu 2016 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Agriculture Commons, Entomology Commons, Molecular Biology Commons, and the Molecular Genetics Commons Recommended Citation Kaplanoglu, Emine, "Multi-gene resistance to neonicotinoids in the Colorado potato beetle, Leptinotarsa decemlineata" (2016). Electronic Thesis and Dissertation Repository. 3953. https://ir.lib.uwo.ca/etd/3953 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract The Colorado potato beetle, Leptinotarsa decemlineata, is a significant pest of potato, and its impact on agriculture is measured on a global scale. The beetle is mainly controlled by neonicotinoid insecticides, however, resistance development is a growing concern. Resistance to neonicotinoids is thought to involve elevated activity of detoxifying enzymes and xenobiotic transporters that break-down and excrete insecticide molecules. Here, using mRNA sequencing, I identified multiple detoxifying enzyme and xenobiotic transporter genes transcriptionally up-regulated in a neonicotinoid resistant strain of beetles. I then used RNA interference to knock down the transcript levels of the ten most promising genes in resistant beetles to test their possible roles in resistance. The silencing of two detoxifying enzyme genes, a cytochrome P450 (CYP4Q3) and a uridine 5'-diphospho-glycosyltransferase (UGT 2B5), significantly increased susceptibility of resistant beetles to the neonicotinoid insecticide imidacloprid. My results indicate that over-expression of these two genes contributes to neonicotinoid resistance. Keywords Colorado potato beetle, metabolic resistance, neonicotinoid resistance, cytochrome P450, esterase, uridine 5'-diphospho-glycosyltransferases, glutathione S-transferase, ATP- binding cassette transporter, mRNA sequencing, RNA interference Acknowledgments First of all, I would like to thank my supervisor, Dr. Cam Donly, for providing me with the opportunity and privilege to be a part of his research team at Agriculture and Agri- Food Canada (AAFC). I truly appreciate his kindness, patience, and guidance over the past two years. Throughout my studies, he has been always encouraging and understanding, and his support has been invaluable. I would like to also thank my co- supervisor, Dr. Graham Thompson, and advisors, Dr. Brent Sinclair and Dr. Ian Scott. Their guidance and encouragement have been instrumental in my progress as a graduate student. I feel very fortunate to have them in my team, and I really appreciate their great inputs to this project. Special thanks also go to Patrick Chapman for his assistance with RNA-seq analysis and to Alex Molnar for his help with the preparation of figures and posters. I am thankful to all current and past members of the Donly Lab, Lou Ann Verellen, Grant Favell, Elizabeth Chen, and Angela Kipp for creating a wonderful work environment in the lab. I would also like to extend my thanks to my colleagues Chelsea Ishmael, Arun Kumaran Anguraj Vadivel, Hanh Tran, Ren Na, and all other friends at AAFC for their friendship and all the great memories I will cherish for the rest of my life. Finally, I would like to thank my family for their constant support and encouragement. Most importantly, I want to thank my loving husband, Tayip Kaplanoglu, for being my source of strength and motivation throughout my school years. I could never have completed this degree without his endless and unconditional support. ii Table of Contents Abstract ................................................................................................................................ i Acknowledgments............................................................................................................... ii Table of Contents ............................................................................................................... iii List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii List of Appendices ............................................................................................................. ix List of Abbreviations .......................................................................................................... x Chapter 1. Introduction ....................................................................................................... 1 1.1 Insecticides and insecticide resistance ................................................................... 1 1.2 Insecticide resistance mechanisms ........................................................................ 2 1.3 Metabolic resistance .............................................................................................. 3 1.3.1 Phase I metabolism: Cytochrome P450s......................................................... 5 1.3.2 Phase I metabolism: Esterases ........................................................................ 6 1.3.3 Phase II metabolism: Glutathione S-transferases ........................................... 7 1.3.4 Phase II metabolism: Uridine 5'-diphospho-glycosyltransferases .................. 7 1.3.5 Phase III metabolism: ATP-binding cassette (ABC) transporters .................. 8 1.4 Constitutive and induced metabolic resistance to insecticides .............................. 9 1.5 Sites of metabolic detoxification in insects ......................................................... 10 1.6 Identification of detoxifying enzymes and ABC transporters involved in insecticide resistance ................................................................................................. 11 1.6.1 RNA sequencing (RNA-seq) to identify differentially expressed genes ...... 11 1.7 RNA interference ................................................................................................. 12 1.7.1 RNAi as a tool to study gene function .......................................................... 14 1.7.2 dsRNA delivery methods in insects .............................................................. 14 1.7.3 dsRNA uptake by cells.................................................................................. 15 1.8 The Colorado potato beetle: an agricultural pest ................................................. 16 1.8.1 Control strategies .......................................................................................... 18 1.8.1.1 Neonicotinoid insecticides ...................................................................... 19 1.8.2 Insecticide resistance in the Colorado potato beetle ..................................... 19 iii 1.8.3 Insecticide resistance mechanisms in the Colorado potato beetle ................ 20 1.9 Rationale and objectives of the study .................................................................. 21 Chapter 2. Materials and Methods .................................................................................... 22 2.1 Beetle strains and rearing conditions ................................................................... 22 2.2 Topical bioassays to determine LD50 of imidacloprid ......................................... 22 2.3 Next-generation sequencing of mRNA (mRNA-seq) ......................................... 23 2.3.1 Beetle treatment and dissections ................................................................... 23 2.3.2 Total RNA extraction and mRNA-seq .......................................................... 23 2.3.3 mRNA-seq data processing and differentially expressed sequence (DESeq) analysis ................................................................................................................... 24 2.4 Validation of DESeq results using quantitative PCR (qPCR) ............................. 25 2.4.1 Primer design ................................................................................................ 25 2.4.2 cDNA synthesis and qPCR analysis ............................................................. 30 2.5 Silencing of target genes using RNAi ................................................................. 31 2.5.1 Bacterial strain and growth conditions ......................................................... 31 2.5.2 RNAi vector L4440....................................................................................... 31 2.5.3 Cloning of gene fragments into L4440 ......................................................... 32 2.5.4 Induction of E. coli HT115 to produce dsRNA ............................................ 38 2.5.5 Confirmation of dsRNA in E. coli HT115 .................................................... 38 2.5.6 dsRNA feeding assays to silence expression of target genes in the RS beetles ...............................................................................................................................
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