BIOLOGICAL STUDIES ON THE GUT SYMBIONT BURKHOLDERIA ASSOCIATED WITH BLISSUS INSULARIS (HEMIPTERA: BLISSIDAE) By YAO XU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2015 1 © 2015 Yao Xu 2 ACKNOWLEDGMENTS I am fortunate to have been mentored by Dr. Drion Boucias during my doctoral program. His constructive criticism, guidance, and generosity of time and resources allowed me to achieve both breadth and depth in research. Without his inspirational ideas and timely feedback, this dissertation would never have been accomplished on time. I owe my deepest gratitude to my co- advisor, Dr. Eileen Buss, for her encouragement, support, and advice on my academic and personal development. I thank her for admitting me, guiding me to enter the world of Southern chinch bugs, and trusting me. I also would like to thank my other committee members, Drs. Frederick Fishel (Department of Agronomy, UF), Kevin Kenworthy (Department of Agronomy, UF), and Cindy McKenzie (United States Department of Agriculture-Agricultural Research Service). I appreciate their time, comments, and encouragement on my research and this dissertation. Many scientists and colleagues have been helpful to me during my doctoral program. First, I thank Dr. Michael Scharf (Department of Entomology, Purdue University) for his valuable comments on the detoxification enzyme work, and especially for hosting me in his laboratory in March 2014. Second, I thank Dr. Paul Linser (Whitney Laboratory for Marine Bioscience, UF) for his guidance on the confocal microscopy and allowing me to use the microscopes in his laboratory in April 2015. Third, I thank Drs. Maurice Marshall, Yavuz Yagiz (Food and Environmental Toxicology Laboratory, UF) and Wlodzimierz Borejsza-Wysocki (IR- 4 Southern Region Laboratory) for their training on the use of gas chromatography and high- performance liquid chromatography. Thanks to Chelsea Verhoeven, Jenna McDaniel, Dr. Navneet Kaur, Dr. Shweta Sharma, Qinwen Xia, Mengyi Gu, Dr. Chao Chen, Tabitha Henson, and Sarah Rachel for their technical assistance. I am grateful to Drs. Ron Cherry and Bryan Unruh (UF), Daniel Dye (Florida Pest 3 Control and Chemical Company, FL), Roi Levin (Environmental Pest and Lawn Services, FL), and John McDaniel (SummerGlen Golf Club Community, FL) for providing some of the Southern chinch bugs and sampling sites used in this research. Special thanks are given to Dr. Aurelien Tartar (Nova Southeastern University) for providing helpful insight on phylogenetic analyses. I thank Dr. Savita Shanker and her crew at the ICBR Sequencing Core (UF) for their technique supports on sequencing. Karen Kelly and Rudy Alvarado at the Electron Microscopy Laboratory (UF) took all the transmission electron microscopy images in this research. I thank Lyle Buss for taking some of the great images and training me on photography. I also thank Judy Boucias for devoting her time and expertise to edit this dissertation. My thanks are given to all faculty members, staff, and graduate students in the Department of Entomology and Nematology for their support and friendship. I am grateful to the Insecticide Resistance Action Committee, Valent USA, Arysta LifeScience, Syngenta, and FMC Corp. for providing the funding and/or the insecticides needed to conduct this research. To my loving parents, Shengjie Xu and Min Zhu: thank you for your selfless love and emotional support. To my husband and my soul mate, Dr. Xuxuan Ma: thank you for believing in me and standing by my side when times go hard. I would never have made it here without all of you. 4 TABLE OF CONTENTS Page ACKNOWLEDGMENTS ...............................................................................................................3 LIST OF TABLES ...........................................................................................................................9 LIST OF FIGURES .......................................................................................................................11 ABSTRACT ...................................................................................................................................15 CHAPTER 1 LITERATURE REVIEW .......................................................................................................17 The Southern Chinch Bug, Blissus insularis ..........................................................................17 Life Cycle and General Biology ......................................................................................17 Damage and Chemical Control .......................................................................................20 Mechanisms of Xenobiotic Detoxification and Resistance ....................................................21 Metabolic Detoxification Mechanism .............................................................................21 Target-site Insensitivity Mechanism ...............................................................................24 Physiological, Behavioral, and Symbiont-mediated Mechanisms ..................................25 Bacterial Symbionts ................................................................................................................26 Bacterial Endosymbionts in Hemiptera ...........................................................................27 Endocellular symbionts ............................................................................................27 Exocellular symbionts ..............................................................................................29 Biological Functions of Bacterial Endosymbionts in Insects ..........................................31 Nutrient provision ....................................................................................................31 Protection against environmental stresses ................................................................34 Members of the Genus Burkholderia .....................................................................................35 Background ......................................................................................................................35 Catabolism of Organic Compounds ................................................................................37 Symbiotic Burkholderia ..................................................................................................38 Research Hypotheses and Objectives .....................................................................................42 2 SELECTION OF BIFENTHRIN RESISTANCE AND EXAMINATION OF METABOLIC DETOXIFICATION ENZYME ACTIVITIES IN BLISSUS INSULARIS ....50 Introduction .............................................................................................................................50 Materials and Methods ...........................................................................................................51 Insects and Plants ............................................................................................................51 Bifenthrin Screening and Assays .....................................................................................51 Clothianidin Assays .........................................................................................................52 Selection for Insecticide Resistance ................................................................................53 Enzyme Assays of B. insularis ........................................................................................54 Results.....................................................................................................................................57 Susceptibilities of Field-collected Populations to Bifenthrin and Clothianidin ..............57 5 Insecticide Resistance Development ...............................................................................58 Enzyme Activities ...........................................................................................................60 Discussion ...............................................................................................................................62 3 BACTERIAL SYMBIONTS ASSOCIATED WITH BLISSUS INSULARIS ........................76 Introduction .............................................................................................................................76 Materials and Methods ...........................................................................................................77 Insect Dissection and Genomic DNA Extraction ............................................................77 Mitochondrial DNA Sequences of Host B. insularis ......................................................77 PCR Amplification and Sequencing of 16S rRNA Genes ..............................................78 PCR Amplification and Sequencing of Burkholderia 16S rRNA Gene ..........................79 Sequence Assembly and Phylogenetic Analyses ............................................................79 Fluorescence in situ Hybridization (FISH) ......................................................................81 Quantitative Real-time PCR (qPCR) ...............................................................................82 Nucleotide Sequence Accession Numbers ......................................................................84 Results.....................................................................................................................................84