ECOLOGY OF CULICOIDES BITING MIDGES AND THEIR ROLE AS VECTORS OF EPIZOOTIC HEMORRHAGIC DISEASE VIRUS ON FLORIDA BIG GAME PRESERVES By BETHANY L MCGREGOR 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 2019 © 2019 Bethany L McGregor To my family, friends, and mentors who have always supported and encouraged me ACKNOWLEDGEMENTS I thank my family for their encouragement, support, and patience throughout my graduate education. I thank my friends for offering humor and companionship during the most stressful of times. I thank my lab mates for serving as a source of inspiration and a sounding board for ideas. I thank the FMEL lab technicians for their unending help towards completion of this research. Finally, I thank my committee and mentors for their guidance and advice throughout the PhD process. 4 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ............................................................................................... 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 12 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 14 2 HOST USE PATTERNS OF CULICOIDES SPP. BITING MIDGES AT A BIG GAME PRESERVE IN FLORIDA, USA .............................................................................. 25 Methods .................................................................................................................. 29 Field Methods .................................................................................................... 29 Laboratory Methods .......................................................................................... 31 Data Analysis .................................................................................................... 33 Results .................................................................................................................... 34 Forage Ratios .................................................................................................... 34 Habitat Associated Host Use ............................................................................. 36 White-tailed Deer Aspiration .............................................................................. 37 Discussion .............................................................................................................. 38 3 VERTICAL STRATIFICATION OF CULICOIDES BITING MIDGES AT A FLORIDA BIG GAME PRESERVE ......................................................................................... 52 Methods .................................................................................................................. 54 Field Collections ................................................................................................ 54 Laboratory Methods .......................................................................................... 56 Data Analysis .................................................................................................... 58 Results .................................................................................................................... 58 Total Collections ................................................................................................ 58 Ground versus Canopy Use by Abundant Species ........................................... 59 Funnel Suction Traps ........................................................................................ 60 Habitat Associations of Abundant Species ........................................................ 60 Physiological Status .......................................................................................... 61 Blood Meal Analysis .......................................................................................... 61 Discussion .............................................................................................................. 63 5 4 IMPLICATING CULICOIDES STELLIFER AND CULICOIDES VENUSTUS (DIPTERA: CERATOPOGONIDAE) AS VECTORS OF EPIZOOTIC HEMORRHAGIC DISEASE VIRUS ........................................................................ 79 Methods .................................................................................................................. 82 Culicoides Sampling and Virus Detection During EHDV Epizootic in Northern Florida ........................................................................................................... 82 White-tailed Deer Sampling and Virus Detection during the 2017 Epizootic ..... 84 Statistical Analysis of Virus Data ....................................................................... 85 Live Animal Aspiration ....................................................................................... 86 Blood Meal Analysis .......................................................................................... 86 Seasonal abundance of C. stellifer and C. venustus ......................................... 87 Results .................................................................................................................... 88 Virus Detection in Field Collected Midges during an EHDV Epizootic ............... 88 Animal Mortality due to EHDV ........................................................................... 89 Live Animal Aspiration ....................................................................................... 91 Blood Meal Analysis .......................................................................................... 91 Seasonal Abundance of C. stellifer and C. venustus ........................................ 92 Discussion .............................................................................................................. 92 5 VECTOR COMPETENCE OF CULICOIDES SONORENSIS FOR EPIZOOTIC HEMORRHAGIC DISEASE VIRUS SEROTYPE 2 STRAINS FROM CANADA AND FLORIDA .............................................................................................................. 106 Methods ................................................................................................................ 108 Culicoides sonorensis colony .......................................................................... 108 Viral Strains ..................................................................................................... 109 Bloodfeeding ................................................................................................... 110 Sample Collection ........................................................................................... 110 Honey Cards ................................................................................................... 111 Sample processing .......................................................................................... 112 Statistical analysis ........................................................................................... 113 Results .................................................................................................................. 114 Infection, Dissemination, and Transmission Rates .......................................... 114 Viral Titer Analysis .......................................................................................... 115 Honey Card Versus Capillary Assay Comparison ........................................... 116 Discussion ............................................................................................................ 117 6 VECTOR COMPETENCE OF CULICOIDES INSIGNIS (DIPTERA: CERATOPOGONIDAE) FOR EPIZOOTIC HEMORRHAGIC DISEASE VIRUS SEROTYPE 2 ....................................................................................................... 127 Methods ................................................................................................................ 130 Blood Feeding Trial Specimen Collection........................................................ 130 Blood Feeding Apparatus Design .................................................................... 130 6 Blood Feeding Trial Variables ......................................................................... 131 Infection Trial Culicoides insignis Collections .................................................. 131 Viral Blood Feeding ......................................................................................... 132 Sample Processing ......................................................................................... 132 RNA Extraction and Virus Detection ............................................................... 133 Statistical Analyses ......................................................................................... 134 Intrathoracic Inoculation .................................................................................. 135 Virus Screening of Field-Collected C. insignis ................................................. 135 Results .................................................................................................................. 136 Blood