ECOLOGY OF CULEX (MELANOCONION) CEDECEI, VECTOR OF EVERGLADES VIRUS IN FLORIDA By ISAIAH J. HOYER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017 © 2017 Isaiah J. Hoyer To my father, Kevin R. Hoyer and mother Ava L. Hoyer. You have both supported me from Alaska to Florida, and everywhere in between. ACKNOWLEDGMENTS I would like to thank Dr. Nathan Burkett-Cadena for his guidance and council. Much of the work presented is attributed to Dr. Burkett-Cadena’s ingenuity and shared interest in sylvatic cycles of mosquito-host interactions. I thank Dr. Jonathan Day and Dr. Phil Lounibos for their insightful feedback and support. I extend my gratitude to Dr. Erik Blosser for his occasional visits to the field, shared knowledge, and support. I thank Lary Reeves for acquiring the Everglades National Park permit, his company in the Everglades, and his contagious broad exuberant enthusiasm for the natural world. I further extend my thanks to the FMEL technicians who performed a large part of bloodmeal extractions and PCR assays, Carolina Acevedo, Tanise Stenn, Anna Thompson, and Jordan Vann; additionally, thanking Glauber Rocha Pereira for his assistance identifying CO2-baited CDC light-trap mosquito samples. Lastly, I express warm regards to my friends and family for their unwavering encouragement. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................ 4 LIST OF TABLES ...................................................................................................... 6 LIST OF FIGURES .................................................................................................... 7 ABSTRACT ............................................................................................................... 9 CHAPTER 1 INTRODUCTION .............................................................................................. 11 2 INVESTIGATING SEASONAL AND REGIONAL PATTERNS OF CULEX (MELANOCONION) CEDECEI HOST UTILIZATION ....................................... 16 Materials and Methods...................................................................................... 16 Data Analysis .................................................................................................... 25 Results .............................................................................................................. 28 3 UNDERSTANDING PATTERNS OF MOSQUITO HOST UTILIZATION THROUGH PASSIVE AND ACTIVE CAPTURE TECHNIQUES ...................... 46 Materials and Methods...................................................................................... 46 Data Analysis .................................................................................................... 52 Results .............................................................................................................. 54 4 DISCUSSION ................................................................................................... 64 IRC Resting Shelter Sampling .......................................................................... 64 ENP Mosquito Collections ................................................................................ 65 Bloodmeal Analysis of Blood-Engorged Culex (Melanoconion) cedecei ........... 69 Mosquito Drift Fence, Wildlife Cameras, and Modified no. 17 Trinidad Trap .... 73 Implications for Everglades Virus Transmission in Florida ................................ 78 LIST OF REFERENCES ......................................................................................... 82 BIOGRAPHICAL SKETCH ...................................................................................... 86 5 LIST OF TABLES Table page 2-1 Adult mosquitoes sampled from resting shelters in Indian River County ...... 33 2-2 Adult mosquitoes sampled by resting shelters from Everglades National Park .............................................................................................................. 34 2-3 Adult mosquitoes sampled by CO2-baited CDC miniature light-traps from Everglades National Park ............................................................................. 35 2-4 Hosts of Culex (Melanoconion) cedecei from Everglades National Park and Indian River County ............................................................................... 36 2-5 Total Culex cedecei bloodmeals from brown rat/roof rat, hispid cotton rat, and cotton mouse in Everglades National Park ............................................ 37 3-1 Adult mosquitoes sampled from mosquito drift fence ................................... 57 6 LIST OF FIGURES Figure page 2-1 Wireframe resting shelter, sensu Burkett-Cadena (2011a) ........................... 38 2-2 PVC resting shelter ....................................................................................... 39 2-3 Pop-up resting shelter .................................................................................. 40 2-4 Demonstration of sampling mosquitoes from pop-up resting shelter in Everglades National Park ............................................................................. 41 2-5 Hand-held aspirator fitted collection cup and removable funnel ................... 42 2-6 Monthly wireframe resting shelter collections of Culex cedecei from Indian River County ...................................................................................... 43 2-7 Comparison of PVC and wireframe resting shelter sampling methods in Indian River County ...................................................................................... 43 2-8 Seasonal patterns of Culex cedecei abundance from sampling sites in Everglades National Park ............................................................................. 44 2-9 Comparison of PVC resting shelter and natural aspiration sampling method in Everglades National Park ............................................................ 44 2-10 Seasonal patterns of host use by Culex cedecei from Indian River County ...................................................................................... 45 2-11 Seasonal patterns of host use by Culex cedecei from Everglades National Park ............................................................................. 45 3-1 Mosquito drift fence ...................................................................................... 58 3-2 Close-up of a “sticky-clock” suction device and mosquito drift fence ............ 58 3-3 Wildlife camera (Stealth Cam G42NG), used for capturing animal activity patterns ........................................................................................................ 59 3-4 Modified no. 17 Trinidad trap ........................................................................ 60 3-5 Circadian activity of Culex cedecei ............................................................... 61 3-6 Day and night activity patterns of mammal (10 species) and mourning dove in Indian River County .......................................................... 61 3-7 Attraction of Culex cedecei to modified no. 17 Trinidad trap baited with animal, compared to unbaited traps ............................................................. 62 7 3-8 Circadian activity of Culex cedecei and mammalian host animals................ 62 3-9 Relationship between nocturnal activity and host use by Culex cedecei for 10 mammal species ................................................................................ 63 8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ECOLOGY OF CULEX (MELANOCONION) CEDECEI, VECTOR OF EVERGLADES VIRUS IN FLORIDA By Isaiah J. Hoyer May 2017 Chair: Nathan Burkett-Cadena Major: Entomology and Nematology Culex (Melanoconion) cedecei is the principal vector of Everglades virus in Florida, a subtype of Venezuelan equine encephalitis virus complex, circulating among wild rodents and mosquitoes. Field studies were conducted in Florida to elucidate Culex cedecei and vertebrate host interactions in Indian River County and Everglades National Park, Florida, USA. Blood-engorged female Culex cedecei were sampled using primarily resting shelters. Host associations were determined using PCR bloodmeal analysis techniques targeting 16s and cytochrome b mitochondrial DNA of host animals. Host and vector activity patterns were quantified using wildlife camera and mosquito drift fence trapping, respectively. Results of these studies revealed that Culex cedecei primarily feeds upon mammals, particularly rodents, the reservoir hosts of Everglades virus. In Everglades National park, nearly 98% of bloodmeals were identified as rodents, in contrast to previous work from the late 1970’s in which rodents accounted for approximately half of Culex cedecei hosts. This shift in host use is likely related to changes in the mammal community in the Everglades, with decreased availability of medium and large mammals. Host circadian activity patterns were generally similar to 9 that of Culex cedecei, however some mammals whose activity patterns overlapped with Culex cedecei were underrepresented in the bloodmeal identifications. These results may indicate that animal circadian activity does not necessarily conform with vector-host use. In conclusion, these findings suggest an expected increase in prevalence of Everglades virus in