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University of Florida Thesis Or Dissertation Formatting Template BIOLOGY AND CONTROL OF TWO SPECIES OF HUMAN PARASITIC BED BUGS: CIMEX LECTULARIUS LINNAEUS AND CIMEX HEMIPTERUS FABRICIUS By BRITTANY ELISE DELONG CAMPBELL 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 2018 © 2018 Brittany Elise Delong Campbell To my parents for their endless love and support ACKNOWLEDGMENTS I am extremely grateful for all the time and knowledge my committee members have dedicated to me over the previous few years. I am grateful to my committee chair, Dr. Rebecca Baldwin, for her kind advice, support, and an ability to bring happiness at moments that I felt at a loss. Secondly, I am beyond grateful for my co-committee chair, Dr. Phil Koehler. He believed in me and provided the support necessary to complete this degree with an unyielding optimistic attitude and quirky sense of humor. I thank my committee member, Dr. Salvador Gezan for his heaps of patience and guidance with statistical analyses. I am also very grateful for my committee member, Dr. Emma Weeks. Emma is an academic saint and helped me tremendously with designing projects and science, but more importantly, with navigating PhD life and struggles. Many more people were influential and helpful throughout this process. Thank you, members of the urban lab, Liz Pereira, Roberto Pereira, Casey Parker, Heather Erskine, Kristen Stevens, Mark Mitola, Ben Hottel, Richard Murphy, Tanner Felbinger, WinDI Sanchez, Tiny Willis, and Lettie Cronin. Heather and Casey, thank you for a wonderful friendship outside of work and your companionship; you are both one of my best friends. Many thanks to Mark Mitola for his help with experimental design and top- notch pranks. Liz, thank you for being a second mom in the lab and for your political views. Thank you Tiny, for your Louisiana tales and the millions of store errands for supplies. There are too many people to thank outside of the urban laboratory, but a special thanks to all the friendships that I made with fellow graduate students at UF. I could not imagine doing this degree without the support, encouragement, and moaning fests that were beyond necessary for peace of mind. Just to name a few people that I owe my 4 sanity to, Mike Bentley, Oliver Keller, Haleigh Ray, Mike Vickers, and Chase Kimmel. Thank you. You made graduate school so much more than earning a degree. Many thanks to Amin Emanifar and Oliver Keller for their help with taxonomic and phylogenetic analyses. A tremendous thanks to Al Estep and Neil Sanscrainte at USDA ARS in Gainesville for their help with Nano injection. I’ve met very few people who are as nice and witty as those two; they brought tremendous joy and enthusiasm daily for science. Lastly, I thank my family for all of their support. I have been in school for most of my life now and I greatly appreciate the constant encouragement. Thanks to my husband, Josh Campbell, for dealing with all the stresses that this degree has brought and for always being a sense of calm in the storm. Thanks to my pup, BoJack. He sacrificed lots of walking and swimming because I had to be in the lab. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 2 LITERATURE REVIEW .......................................................................................... 16 Bed Bug Biology ..................................................................................................... 16 Bed Bug Origins ...................................................................................................... 19 Cimex hemipterus and Cimex lectularius Distribution ............................................. 20 Medical Importance of Bed Bugs ............................................................................ 21 Bed Bug Control Methods ....................................................................................... 22 Pyrethroid Resistance ............................................................................................. 25 Juvenile Hormone Analogues and Chitin Synthesis Inhibitors ................................ 26 3 RECENT DOCUMENTATION OF THE TROPICAL BED BUG IN FLORIDA SINCE THE COMMON BED BUG RESURGENCE (HEMIPTERA: CIMICIDAE) ... 29 Introduction ............................................................................................................. 29 Materials and Methods............................................................................................ 30 Results and Discussion........................................................................................... 31 4 MORPHOLOGICAL AND MITOCHONDRIAL DNA VARIATION BETWEEN COMMON BED BUGS (CIMEX LECTULARIUS) AND TROPICAL BED BUGS (CIMEX HEMIPTERUS) .......................................................................................... 35 Materials and Methods............................................................................................ 38 Bed Bug Strains ............................................................................................... 38 Morphological Analyses.................................................................................... 39 Statistical Analyses of Morphological Characteristics ....................................... 39 DNA Extraction, PCR Amplification, and Sequencing ...................................... 40 Phylogenetic Analyses ..................................................................................... 41 Results .................................................................................................................... 42 Morphological Differentiation ............................................................................ 42 Genetic Diversity .............................................................................................. 43 Phylogenetic Analyses and Haplotype Network ............................................... 44 Discussion .............................................................................................................. 44 6 5 LOCOMOTION INHIBITON OF CIMEX LECTULARIUS L., FOLLOWING TOPICAL, SUBLETHAL DOSE APPLICATION OF THE CHITIN SYNTHESIS INHIBITOR LUFENURON ...................................................................................... 75 Materials and Methods............................................................................................ 78 Insects .............................................................................................................. 78 Insecticide Dilutions .......................................................................................... 78 Insect Growth Regulator Topical Application Bioassay .................................... 79 Pulling Force Assay .......................................................................................... 80 Statistical Analysis ............................................................................................ 82 Results .................................................................................................................... 82 Discussion .............................................................................................................. 83 6 LUFENURON EFFECTS ON BED BUG FECUNDITY AND EGG DEVELOPMENT ..................................................................................................... 94 Material and Methods ............................................................................................. 95 Insects .............................................................................................................. 95 Topically Treated Egg Dose Bioassay .............................................................. 96 Topically Treated Egg Age Bioassay ................................................................ 97 Topically Treated Adult Males and Females with Lufenuron ............................ 97 Statistical Analysis ............................................................................................ 98 Results .................................................................................................................... 99 Discussion ............................................................................................................ 100 7 EXTERNAL AND INTERNAL APPLICATION OF LUFENURON AGAINST TROPICAL BEDBUGS VIA TOPICAL APPLICATION, FEEDING, AND INJECTION BIOASSAYS ..................................................................................... 106 Materials and Methods.......................................................................................... 108 Insects ............................................................................................................ 108 Insect Growth Regulator Topical Application Bioassay .................................. 108 Insect Growth Regulator Feeding Bioassay ................................................... 109 Insect Growth Regulator Injection Bioassay
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