Diptera: Culicidae) in RELATION to EPIZOOTIC TRANSMISSION of EASTERN EQUINE ENCEPHALITIS VIRUS in CENTRAL FLORIDA

SEASONAL CHANGES IN HOST USE AND VECTORIAL CAPACITY OF Culiseta melanura (Diptera: Culicidae) IN RELATION TO EPIZOOTIC TRANSMISSION OF EASTERN EQUINE ENCEPHALITIS VIRUS IN CENTRAL FLORIDA

RICHARD G. WEST

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

2019

ACKNOWLEDGMENTS

I would like to thank my advisor Nathan Burkett-Cadena for his invaluable guidance and instruction and Derrick Mathias and Jonathan Day for serving on my committee and sharing their expertise and helpful input.

I would like to thank the following for their assistance with mosquito sampling:

Carl Boohene, Jackson Mosley, Hugo Ortiz Saavedra, and Roger Johnson at Polk

County Mosquito Control District; Kelly Deutsch, Rafael Melendez, and others at

Orange County Mosquito Control District; and Sue Bartlett, Miranda Tressler, Hong

Chen, Drake Falcon, Tia Vasconcellos, and Brandi Anderson at Volusia County

Mosquito Control District. This study could not have been done without their cooperation and hard work.

I would also like to thank Carolina Acevedo for help with bloodmeal analysis, Erik

Blosser for help with mosquito identifications, Diana Rojas and Annsley West for helping with field collections, and to all the faculty, staff, and students at FMEL for their support and encouragement. Finally, I thank my wife Annsley for her faithful encouragement and love and for my Lord Jesus and family for their support.

This research is supported by the CDC Southeast Gateway Center of Excellence and the University of Florida.

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... 3

LIST OF TABLES ...... 6

LIST OF FIGURES ...... 7

LIST OF DEFINITIONS AND ABBREVIATIONS ...... 8

ABSTRACT ...... 9

CHAPTER

1 INTRODUCTION ...... 11

Eastern Equine Encephalitis Virus and Culiseta melanura ...... 11 Seasonality of EEEV ...... 13

2 SEASONAL HOST USE OF Culiseta melanura ...... 15

Materials and Methods...... 17 Field Locations ...... 17 Mosquito Sampling ...... 17 Bloodmeal Analysis ...... 18 Results ...... 21 Culiseta melanura Bloodmeals ...... 21 Seasonal Host Use ...... 22 Avian Host Diversity and Residency ...... 23 Mammalian Bloodmeals ...... 24 Discussion ...... 25

3 SEASONAL CHANGES IN VECTORIAL CAPACITY OF Culiseta melanura FOR EEEV IN CENTRAL FLORIDA ...... 41

Seasonality of EEEV in Florida ...... 41 Vectorial Capacity ...... 41 Materials and Methods...... 46 Parity Determinations ...... 46 Vectorial Capacity Calculations ...... 46 Results ...... 49 Mosquito Abundance and Host Feeding ...... 49 Parity Rate, Gonotrophic Cycle Length, and Extrinsic Incubation Length ...... 49 EEEV Transmission in Central Florida ...... 50 Vectorial Capacity ...... 51 Discussion ...... 52

Vectorial Capacity ...... 52 Abundance ...... 52 Host use ...... 54 Mosquito Survival ...... 55 Conclusion ...... 55

LIST OF REFERENCES ...... 64

BIOGRAPHICAL SKETCH ...... 72

LIST OF TABLES

Table page

2-1 Collection sites of Cs. melanura in central Florida...... 30

2-2 Forward and reverse primers used for bloodmeal host identification...... 31

2-3 Vertebrate hosts of Cs. melanura by county ...... 32

2-4 Vertebrate hosts of Cs. melanura by month...... 34

3-1 Monthly abundance of Cs. melanura and other mosquito species ...... 57

3-2 Vectorial capacity variables of Cs. melanura for EEEV ...... 58

LIST OF FIGURES

Figure page

2-1 Map of collection sites in central Florida, USA...... 36

2-2 Mosquito resting shelter ...... 37

2-3 Aspirator used in mosquito collection from resting shelters ...... 38

2-4 Seasonal host use by Cs. melanura in central Florida ...... 39

2-5 Monthly resident and nonresident bird host detection ...... 40

3-1 Ovaries from Cs. melanura at 100X magnification ...... 59

3-2 Mean density of Cs. melanura from Orange and Polk County, FL in 2018 ...... 60

3-3 Parity of Cs. melanura from Orange and Polk County, FL in 2018 ...... 61

3-4 Vectorial capacity (C) of Cs. melanura for EEEV against EEEV equine cases from 2018 and past decade ...... 62

3-5 Relationship of central Florida EEEV equine cases and vectorial capacity of Cs. melanura ...... 63

LIST OF DEFINITIONS AND ABBREVIATIONS

Bridge vector A species of arthropod that acquires an infectious agent from an infected wild animal and then transmits the agent to a non- amplifying host.

C Vectorial capacity; the average number of new vertebrate infections per day resulting from an initial index case.

Dilution host A species that has low host competence for an infectious agent which reduces transmission by decreasing contact between competent vectors and competent hosts.

EEEV Eastern equine encephalitis virus.

Encephalitis Inflammation of the brain.

Endemic Regularly found among particular group or in a certain area.

Enzootic In relation to a disease regularly affecting nonhuman animals.

Epizootic An outbreak of disease in nonhuman animals; in relation to an epizootic disease in animals.

Extrinsic incubation The interval between the acquisition of an infectious agent by a period vector and the point at which the vector is able to transmit the agent to other susceptible vertebrate hosts.

Host competence The physiological ability of a host organism to acquire, maintain and transmit an infectious agent.

Parity The reproductive state of an organism; whether a female has completed a reproductive cycle.

Vector competence The physiological ability of a vector organism to acquire, maintain and transmit an infectious agent.

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

SEASONAL CHANGES IN HOST USE AND VECTORIAL CAPACITY OF Culiseta melanura (Diptera: Culicidae) IN RELATION TO EPIZOOTIC TRANSMISSION OF EASTERN EQUINE ENCEPHALITIS VIRUS IN CENTRAL FLORIDA

Richard G. West

August 2019

Chair: Nathan D. Burkett-Cadena Major: Entomology and Nematology

The mosquito Culiseta melanura (Coquillett) is the primary enzootic vector of eastern equine encephalitis virus (EEEV), which circulates in the eastern United States in avian hosts and causes disease primarily in equids but also in humans. Our goal was to expand the understanding of how host use and vectorial capacity of Cs. melanura for

EEEV changes seasonally and its relationship with EEEV risk. This was done by quantifying seasonal host use and vectorial capacity (C) as a function of season for Cs. melanura in central Florida in 2018. Mosquitoes were collected at nine sites in central

Florida. Cs. melanura females were dissected for parity state, and bloodmeals were identified by PCR to quantify host usage. PCR-based bloodmeal analysis identified 277 bloodmeals from Cs. melanura that consisted of 39 species of birds, reptiles, and mammals. Most common species identified were brown anole (Anolis sagrei, 22.1%), human (Homo sapiens, 12.7%), northern cardinal (Cardinalis cardinalis, 12.2%), and

Carolina anole (Anolis carolinensis, 11.7%). Avian bloodmeals were prominent throughout the year and were primarily from northern cardinal, red-eyed vireo (Vireo olivaceus), mourning dove (Zenaida macroura), pine warbler (Setophaga pinus), and

Carolina wren (Thryothorus ludovicianus). Songbirds were a large portion of all bloodmeals identified (37.1%). Of the avian hosts, 12 species were nonresidents of

Florida and were detected primarily in winter, but no significant shift to resident birds was found. Reptiles and mammals contributed to 50.7% of total meals. Reptile feeding peaked in April and a significant shift in host feeding occurred in July from reptiles to mammals. Calculation of C of Cs. melanura utilized the density of the vector, proportion of avian hosts fed upon, parity state of the vector, and mean temperature of the study area. The C value calculations were highest in the summer but also had a peak in

December. Linear regression revealed a significant positive correlation between C values and Florida EEEV equine cases in 2018 as well as cases reported during the last decade. This study reveals that in southern foci of EEEV transmission, Cs. melanura can feed heavily on reptiles, which may amplify or suppress transmission. In addition to birds and reptiles, Cs. melanura can also feed heavily on mammals, which may result in epizootic cases transmitted by this mosquito vector. The relationship between virus infections in equids and C values give support to the large effect enzootic transmission by Cs. melanura has on epizootic outbreaks.

CHAPTER 1 INTRODUCTION

Eastern Equine Encephalitis Virus and Culiseta melanura

Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) is a vector-borne pathogen found in the eastern United States. The primary region of

EEEV distribution stretches from New Hampshire to Florida and along the Gulf of

Mexico to Texas (Scott and Weaver 1989). Previously, EEEV was grouped with three related virus lineages found in Central and South America that are now called

Madariaga virus (MADV) (Arrigo et al. 2010).

Circulation of EEEV takes place primarily in avian amplifying hosts by multiple mosquito vectors with transmission spilling over into mammalian or other dead-end hosts (Molaei et al. 2015b). The virus was first reported as the cause of disease in horses in 1933, with reports primarily from coastal areas in the northeastern United

States in the states of Virginia, Delaware, New Jersey, and Maryland (Tenbroeck et al.

1935). Disease caused by EEEV was confirmed in humans in 1938 after the original report of a horse outbreak in Massachusetts in 1831 (Fothergill et al. 1938, Scott and

Weaver 1989). EEEV is the deadliest vector-borne pathogen in the United States. Over

30% of symptomatic cases in humans are fatal and a high proportion of infected individuals that survive have residual neurological sequelae (Bigler et al. 1976, Villari et al. 1995). Occasional outbreaks with high fatality rates occur, such as the original report in Massachusetts, where 74% of the 34 infected people died (Fothergill et al. 1938). In the United States an average of six human cases are reported for the disease each year (Day and Shaman 2011). The largest equine outbreak that has occurred was a widespread epizootic in Texas and Louisiana in 1947 when 14,334 horses were

11 reported to be infected with EEEV and 11,727 of the infections were fatal (Scott and

Weaver 1989). Ten human cases of EEEV were associated with the 1947 epizootic, 7 of which were fatal (Beadle 1952).

Within avian hosts, the virus causes low but varied levels of mortality depending on the bird species (Komar et al. 1999). The virus also causes high mortality in emu, game birds, and in swine younger than two months old (Scott and Weaver 1989, Tully

Jr et al. 1992, Elvinger et al. 1994). While an EEEV vaccine is available for horses, no practical vaccine or effective treatment is available for humans (Armstrong and

Andreadis 2013). An EEEV vaccine for humans has been developed but is only used by at-risk laboratory workers and requires a booster shot 28 days after the initial dose to be effective (Bartelloni et al. 1970).

The mosquito Culiseta melanura (Coquillett) (Diptera: Culicidae) is the primary enzootic vector of EEEV throughout eastern North America. Its distribution matches that of EEEV, occurring along the eastern coast of the United States and stretching west to

Kansas (Darsie and Ward 2005). Virus infection in this species has been recorded from transmission foci in states including Maine (Lubelczyk et al. 2013), Vermont (Molaei et al. 2015a), Maryland (Saugstad et al. 1972), Virginia (Molaei et al. 2015b), Alabama

(Cupp et al. 2003) and Florida (Bingham et al. 2014). Because Cs. melanura has the highest competence for EEEV of any mosquito species and has a short extrinsic incubation period of three days, it is an efficient vector of the virus (Scott and Burrage

1984, Vaidyanathan et al. 1997). Birds which are fed on by Cs. melanura serve as amplifying hosts for the virus (Bingham et al. 2014, Blosser et al. 2017).

Other mosquito species which serve as bridge vectors of EEEV to mammalian, reptilian, amphibian, or non-amplifying avian hosts include Aedes vexans (Meigen),

Aedes sollicitans (Walker), Coquillettidia perturbans (Walker), Culex erraticus (Dyar and

Knab), Culex nigripalpus Theobald, Culex territans Walker, Culex peccator Dyar and

Knab, and Uranotaenia sapphirina (Osten Sacken) (Crans et al. 1986, Crans and

Schulze 1986, Cupp et al. 2003, Cupp et al. 2004, Cohen et al. 2009). In some studies,

Cx. erraticus is implicated as the primary EEEV vector because of its high abundance and presence of the virus (Cupp et al. 2003, Mukherjee et al. 2012, Bingham et al.

2015). Analyses of abundance and feeding preferences of these additional species were not addressed in this study but should be incorporated into disease models when considering the entire EEEV disease system. The focus on Cs. melanura in this study is based on it being the primary enzootic vector of the disease (Armstrong and Andreadis

2010).

Seasonality of EEEV

Transmission of EEEV in the Unites States is seasonal. In most endemic regions, epizootic transmission occurs primarily in summer, peaks in late summer, and declines in fall (Tenbroeck et al. 1935,). In temperate regions, the first heavy frost in November or December ends virus transmission in mosquitoes (Scott and Weaver 1989). Because of the warmer climate in the southern US, transmission can also occur in winter (Bigler et al. 1976, Burkett-Cadena et al. 2015). In Florida, reported equine cases are sporadic and occur in isolated foci (Day and Shaman 2011). Sixty-six percent of EEEV equine cases in Florida from 2009 to 2018 were reported in summer (Florida Department of

Health 2019). Although yearly number of cases may be low in some years and regions of Florida, horse cases are the most reliable indicator of virus activity in the southeast 13

(Bigler et al. 1976). The seroconversions of sentinel chicken flocks that are used for detection of arboviruses have a similar trend of summer (May-September) transmission in Florida (Heberlein-Larson et al. 2019). The summer peak in transmission also coincides with increased mosquito abundance and EEEV antibody seroprevalence in young birds (Tenbroeck et al. 1935, Elias et al. 2017). One cause of seasonality and severity of transmission is seasonal weather patterns. A study by Skaff et al. (2017) found that rainfall in the fall and winter of the previous year and rainfall in the current transmission season were associated with increased EEEV transmission and Cs. melanura abundance. An additional factor that increases transmission of arboviruses in

Florida is drought conditions during spring, which causes avian hosts to encounter more mosquitoes (Day and Shaman 2011, Shaman et al. 2005).

As demonstrated by Cs. melanura and EEEV, the disease cycles in which vectors operate are complex and need to be better understood to adequately predict and control epizootic outbreaks. The goals of this study were to (1) explore seasonal host use by Cs. melanura and (2) quantify vectorial capacity (C) as a function of season for EEEV and its vector Cs. melanura in central Florida. This is reported in the following two chapters and was achieved by determining mosquito abundance, extent of seasonal variation in host feeding, and parity of Cs. melanura throughout the year.

These variables were incorporated into the C equation using temperature data and previous data on extrinsic incubation period (n) and gonotrophic cycle (gc) of Cs. melanura.

CHAPTER 2 SEASONAL HOST USE OF Culiseta melanura

The mosquito Culiseta melanura is the primary enzootic vector of EEEV throughout eastern North America and feeds primarily on avian hosts, with occasional feeding on mammals and reptiles. Numerous studies investigating host use by this vector have demonstrated that it primarily feeds on songbirds in swamps (Cupp et al.

2003, Bingham et al. 2014, Molaei et al. 2015a), with up to 95% taken from avian hosts

(Molaei et al. 2015b). Because the distribution of Cs. melanura is wide, the host species this mosquito feeds upon are numerous and differ from location to location. In the northern United States, avian species such as green heron (Butorides virescens),

American robin (Turdus migratorius), and common yellowthroat (Geothlypis trichas) were identified as the main source of blood for Cs. melanura (Molaei and Andreadis

2006, Molaei et al. 2006, Molaei et al. 2015b, Molaei et al. 2015a). In the southern

United States, wading birds (Bingham et al. 2014) and northern cardinal (Cardinalis cardinalis) (Estep et al. 2011, Blosser et al. 2017) have been determined to be the main hosts. In the study by Molaei et al. (2015a) in Vermont, host preference changed seasonally with the main host being green heron in June and July, American robin in

August, and common yellowthroat in September. Additionally, in Virginia the main hosts changed from American robin in May, to Carolina wren (Thryothorus ludovicianus) in

June, to American robin in July, to northern cardinal in August and September, and back to American robin in October (Molaei et al. 2015b). However, a study in Florida on temporal shift in host use by Blosser et al. (2017) found that Cs. melanura fed mostly on northern cardinal and other birds in winter and early spring but fed primarily on reptiles in late spring.

The role of reptiles in the EEEV transmission cycle have been researched by many over the years (Karstad 1961, Hayes et al. 1964, White et al. 2011, Graham et al.

2012, Blosser et al. 2017). Many reptile and amphibian species have been found positive for EEEV virus or antibody (Craighead et al. 1962, Bingham et al. 2012,

Graham et al. 2012). In a study on seroprevalence of EEEV in reptiles and amphibians in Alabama, eight of nine species of snake tested were seropositive for EEEV (Graham et al. 2012). The individual snakes from this study had 35% seropositive conversions.

Successful inoculation and high viremia in snake and lizard species have also been documented (Karstad 1961, Craighead et al. 1962, White et al. 2011). Whether the reptiles that Cs. melanura may feed on are competent hosts would influence their role in virus transmission.

The goal of this chapter was to explore host use by Cs. melanura in central

Florida, which may influence the transmission of EEEV in its southern region. This was achieved by determining the extent of seasonal variation in host feeding of Cs. melanura throughout the year using mosquito sampling and bloodmeal identification.

Consistent feeding shifts by Cs. melanura are reported where reptiles were fed on more in the summer than were birds. Feeding shifts between migratory and resident birds are also reported.

Materials and Methods

Field Locations

Sampling was conducted in the central region of Florida with nine sites in Orange

(3), Polk (3), and Volusia (3) counties (Table 2-1, Figure 2-1). Polk County marks the southern edge of the region with high EEEV transmission in the state of Florida in the past 10 years (Florida Department of Health 2019). In these counties there is also historic evidence of consistent EEEV circulation, with 21, 30 and 11 vertebrate and invertebrate isolations made in Orange, Polk, and Volusia counties, respectively, from

1955 to 1974 (Bigler et al. 1976). Many Cs. melanura have been collected previously at these sites with conventional light traps and five sites have sentinel chicken coops present. Sites are surrounded by a mixture of forest, wetland, and residential areas

(Table 2-1).

Mosquito Sampling

Weekly mosquito collections were made at these nine sites from October 2017 to

January 2019 using six artificial resting shelters at each site for a total of 54 resting sites. Resting shelters serve as dark, protected refuges that attract blood-engorged females seeking a place to rest and digest their meals. They have been used in studies to sample various species of Culex (Hoyer et al. 2017) and have been found to be the most effective method to collect bloodfed Cs. melanura females (Bingham et al. 2014).

We assume that mosquitoes collected from resting shelters represent the actual population and that the resting shelters are not attracting one kind of mosquito more than another. However, these shelters do not attract species such as Aedes spp. or Cx. nigripalpus (Burkett-Cadena et al. 2019), which are also vectors of EEEV. The resting shelters used in the current study were made by placing a fitted black trash compactor

17 bag over a cylindrical frame made with three 86cm long ½ inch PVC pipes, six PVC tees, ½ inch PEX pipe shaped into two rings with a diameter of 46cm, two PEX couplers, and rubber bands and binder clips to hold the tees and bag in place (Figure 2-

2). Mosquitoes were collected from shelters by aspiration with a modified vacuum

(BDH1800S Ni-Cd 18V Dustbuster, Black & Decker, MD) and collection cup (BioQuip

Products, Rancho Dominguez, CA) (Blosser et al. 2017). The vacuum was modified with a wide brim attachment that fit the inside of the shelters, thus preventing mosquitoes from escaping around the edge of the bag during aspiration (Figure 2-3).

Collected female mosquitoes were identified to species by examining morphological traits using a dissecting microscope and dichotomous keys updated in

2018 (Darsie and Morris 2003, Darsie and Ward 2005, Burkett-Cadena 2013). Once identified, Cs. melanura females were stored in Thermo Scientific Microcentrifuge tubes at -20°C for future analysis.

Bloodmeal Analysis

The hosts fed on by Cs. melanura were determined by blood meal analysis consisting of DNA extraction, PCR, and sequencing. Extraction was performed using

InstaGene by homogenizing each individual mosquito in 150 µL of 0.9% NaCl solution with 2 mm glass beads using the Bullet Blender Storm 24 Homogenizer. Tubes were briefly centrifuged at 6,000 rpm to remove drops from inside the lid. Then 200 µL of

InstaGene were added to tubes following the label’s protocol. Tubes were incubated at

100°C for 10 minutes and then centrifuged at 6,000 rpm for 5 minutes. The supernatant with the DNA was transferred to a new tube and stored at -20°C until subsequent PCR analyses.

Five primer pairs were used to detect vertebrate host blood meals in a series of

PCR reactions as previously described (Table 2-2). DNA from PCR reactions with negative results were run with the subsequent primer pair in a hierarchal approach. The

16L1/H3056 (Lzrd) primer was used to detect reptilian blood with the positive control from a yellow rat snake. To amplify avian bloodmeals, the L0/H1 primer was used with a positive control from the common yellowthroat. The H2714/L2513 primer was used to amplify mammalian and amphibian blood with black rat and Cuban treefrog as positive controls. Lastly, the VertCO1_7194_F/ModREPCO1_R and

ModREPCO1_F/VertCO1_7216_R primer pairs were used to amplify remaining unidentified vertebrate bloodmeals. Each sample was composed of 2.5 µL of bloodmeal

DNA and 22.5 µL of master mix. The master mix was composed of 12.5 µL of Platinum

Green 2X Master Mix, 9.0 µL molecular grade water, 0.5 µL of 20 µM forward primer and 0.5 µL of 20 µM reverse primer. A negative control of 2.5 µL of molecular grade water was added. PCR reactions were run using published PCR conditions for the specific primers used (Blosser et al. 2017).

To visualize PCR results, PCR products were placed in a 1% agarose gel, electrophoresed, and viewed with a UV light. A ladder (5 µL; O’GeneRuler 100bp DNA

Ladder) was placed in the first well followed by 10 µL of PCR product in each well. Each gel was electrophoresed at 100 V for 30 to 45 minutes then was transferred to a UV light for recording presence and absence of bands. PCR reactions with no band were discarded. All positive reactions with visible bands were sent for Sanger sequencing

(Eurofins MWG Operon, Huntsville, AL).

Sequence files were trimmed of undetermined nucleotides and were identified using the BLASTn function in the NCBI database GenBank. Species identification, percent match, query cover, query length, and match length were recorded for each sequence. Only samples which were one- or two-days post blood feeding were considered identified and analyzed and presented in the results (Reeves et al. 2016).

Identified bloodmeals were those which had a ≥ 95% match of the sequence and a >3% match difference from the next closest species. The identified hosts were classified as competent or non-competent hosts from the literature for the calculation of vectorial capacity (Chapter 3). Suspected mixed bloodmeals from more than one host from a low sequence match or DNA chromatogram with many double peaks were tested with all primers.

Positive bloodmeals were categorized by host, class, and order. Resident status for avian hosts was determined to better understand the host use of Cs. melanura throughout the year (eBird 2019). Fisher’s exact test was used to determine monthly changes in host feeding.

Results

Culiseta melanura Bloodmeals

Culiseta melanura fed on a diverse set of hosts with variation in hosts by site and month sampled. From the 2018 collections, 1,241 Cs. melanura females were collected,

277 of which were bloodfed. Bloodmeals that were three or more days post blood feeding were not considered identified (n=33) (Reeves et al. 2016). Of the 244 females less than three days post bloodfed, 213 were identified. The hosts identified included 29 bird species, 6 mammal species, and 4 reptile species (Table 2-3). These 39 species belong to 15 orders and 31 families. Of the avian hosts identified, 16 were residents, 12 were nonresidents, and one was a domestic chicken. The largest number of bloodmeals came from the brown anole (Anolis sagrei, n=47), human (Homo sapiens, n=27), northern cardinal (n=26), and Carolina anole (Anolis carolinensis, n=25) (Table 2-3).

In Orange County, brown anoles were the most commonly bitten host (31.0%), followed by northern cardinal (23.9%), Carolina anole (9.9%) and human (5.6%). In Polk

County, the most commonly bitten hosts included brown anole (20.0%), human (15.2%),

Carolina anole (12.4%), and red-eyed vireo (Vireo olivaceus, 7.6%). In Volusia County, the most commonly bitten hosts were human (18.9%), mourning dove (Zenaida macroura, 18.9%), Carolina anole (13.5%), and brown anole (10.8) (Table 2-3).

Brown anole bloodmeals dominated the reptile hosts (63.5%), followed by

Carolina anole (33.8%). Two additional bloodmeals from a southern black racer

(Coluber constrictor priapus) and a brown basilisk (Basiliscus vittatus) were identified from Kelly Park (Orange Co.) and Fairgrounds (Volusia Co.) sites, respectively. The brown basilisk bloodmeal was identified from Volusia County, FL, at the Fairgrounds on

June 28, 2018. This represents the northern most location recorded for this invasive species (Krysko et al. 2006, iNaturalist 2019).

Of the 277 bloodfed Cs. melanura collected from Orange, Polk, and Volusia counties in 2018, 213 bloodmeals were successfully identified (76.9%). Few bloodmeals were observed in January (n=3), September (n=3), and in Orange and Volusia counties in November and December (n=4, Figure 2-4), despite extensive sampling. The Lzrd primer amplified many avian bloodmeals, but often a sequence would come back as a match to multiple birds including northern cardinal, American robin, and common grackle (Quiscalus quiscula). Many of these sequences were identified with the Vert

COI primer afterwards, eight of which were identified as northern cardinal.

Seasonal Host Use

Culiseta melanura collections from 2018 resulted in 105 avian (49.3%), 74 reptilian (34.7%), 34 mammalian (16.0%), and zero amphibian bloodmeals (Table 2-4).

In February and March, avian meals were most common (71%-86%, Figure 2-4). In

April, reptilian host use peaked at 62% and replaced birds as the most commonly fed upon host class. The monthly shift from avian to reptilian hosts from March to April was not significant (P=0.170) but the shift from late winter (Feb. & March) to spring (April &

May) was significant (P=0.030). Reptilian bloodmeals remained abundant in May and

June until a significant change from reptilian to mammalian bloodmeals was observed from June to July (P=0.007). Reptilian and mammalian bloodmeals had no other significant change for the remainder of the year but varied in proportion. From July to

December, bloodmeals from birds were most common (47-67%).

Birds were the most commonly bitten host every month except April, May, and

June. In September, a bloodmeal was identified from two birds and one reptile (n=3). No 22 significant change of host feeding was detected to or from birds. A small sample size from January to March may have prevented finding any significant monthly change in host use during the spring, although an increase in reptile bloodmeals was observed.

Reptile bloodmeals contributed 14% to 62% of meals throughout the year with the most reptile meals occurring in May (n=18, Figure 2-4). Reptilian host use varied monthly yet was similar for each site. There was decreased feeding on reptiles in

February (14%) and December (17%). In January there were three vertebrate bloodmeals, with one from a brown anole. Reptile use was high in April (62%), May

(49%) and June (50%).

Avian Host Diversity and Residency

Bird species identified from bloodmeals consisted of 9 orders and 22 families of birds (Table 2-3). The avian hosts most fed on were northern cardinal, red-eyed vireo, mourning dove, Carolina wren, pine warbler (Setophaga pinus), barred owl (Strix varia), and house wren (Troglodytes aedon). The largest number of bloodmeals came from songbirds in the order Passeriformes (n=79, 75% of avian hosts, 37% of total). In the winter months, 56% of bloodmeals were from songbirds.

Bloodmeals identified from Cs. melanura in 2018 included 72 resident birds, 29 nonresident birds, and four domestic birds (chickens). Nonresident host species consisted of 11 winter residents and one summer resident. The non-songbird species fed on were mostly mourning dove, barred owl, and chicken (Table 2-3). The number of bloodmeals from resident and nonresident bird hosts varied monthly but variation was not significant (Figure 2-5). From March to April there was a decline in nonresident bloodmeals from four (80%) to zero. While more resident bird meals were detected during summer, bloodmeals from the winter resident hosts remained low in May and 23

June, were not found in July through September, and reappeared in October. Summer resident bloodmeals were found in June through December. No significant difference between months was found for Florida residency of bird hosts in our study (Fisher’s exact test). The most commonly bitten nonresident hosts (n=2 or more) were red-eyed vireo (a summer resident), house wren, blue-headed vireo (Vireo solitarius), ruby- crowned kinglet (Regulus calendula), American robin and chipping sparrow (Spizella passerina) (Table 2-4). The red-eyed vireo is a mainly summer resident, but removal from nonresident data did not cause significant changes.

Mammalian Bloodmeals

In addition to avian and reptilian hosts, many bloodmeals were from mammals

(Table 2-3). Species identified were human (12.7%), Virginia opossum (Didelphis virginiana, 1.4%), rats (Rattus spp., 0.5%), white-tail deer (Odocoileus virginianus,

0.5%), cattle (Bos taurus, 0.5%), and raccoon (Procyon lotor, 0.5%). Human bloodmeals were one of the four most common sources of blood in each of the three counties (5.6-18.9%) and were detected in every month of the year except February,

March, September, and December (Table 2-4). Seasonally, human bloodmeals constituted the majority of bloodmeals of any individual species in July and August

(n=15, 19.2%, Table 2-4). Polk County had the most occurrences (n=16) and Volusia

County had the highest percentage of human bloodmeals (n=7, 18.9%). In addition to the mammal species positively identified from Cs. melanura, one bloodmeal from the

Green Pond Road site was a close match (97-99%) to multiple species of exotic deer.

Discussion

Bloodmeals from Cs. melanura were collected from central Florida throughout

2018 and were identified as coming from birds, reptiles, and mammals but not amphibians. Host diversity was high, with Cs. melanura feeding on 29 species of birds as well as lizards, a snake, humans, opossums, deer, and other mammals. Results from this study support the traditional idea that Cs. melanura feeds primarily on Passerine songbirds but expands on this because of the detection of numerous bloodmeals from reptiles and mammals. While the total number of avian bloodmeals was greater than those from reptiles or mammals, combined reptile and mammal bloodmeals contributed to 50.7% of total meals and brown anole lizards and humans were the most commonly fed upon species. This study represents the first year-round seasonal record of host use by Cs. melanura in Florida.

This study found that songbirds (order Passeriformes) represented a large portion of all identified bloodmeals (37.1%). Avian bloodmeals were primarily from northern cardinal, red-eyed vireo, mourning dove, and Carolina wren. Songbirds are considered the primary amplification host for EEEV in winter (Burkett-Cadena et al.

2015), which is supported by this study. The northern cardinal was the third most common host of Cs. melanura and contributed 12.2% of total bloodmeals. Culiseta melanura has been shown to feed heavily upon northern cardinals (Estep et al. 2011,

Blosser et al. 2017) and host competence of northern cardinals for EEEV has been studied. The study by Komar et al. (1999) on competence of eight songbirds and two non-songbirds (Columbiformes) found competence of songbirds varied from high to low, with the non-songbirds having a lower level viremia. Songbirds have also had high seroconversion rates for EEEV (Elias et al. 2017). Because most birds detected in this 25 study were songbirds, it could be assumed that most birds fed on by Cs. melanura are moderately competent hosts of EEEV. There were fewer (1 of 213) bloodmeals from wading birds than in some previous studies (Bingham et al. 2014, Molaei et al. 2015a).

In the study by Bingham et al. (2014), 39.6% of Cs. melanura bloodmeals were from wading birds in winter months. Two of the collection sites of their study were adjacent to a river, whereas in the current study only Tibet-Butler Park was near a large body of water. This difference in habitat may explain the lack of wading birds from our data. In the current study there were also not many American robin bloodmeals, in contrast to studies from Virginia (Molaei et al. 2015b), Connecticut (Molaei and Andreadis 2006) and New York (Molaei et al. 2006). Weekly mosquito collection from this study was likely not able to detect the patchy spatial and temporal winter distribution of American robin in Florida. As expected, nonresident birds were detected from Cs. melanura mainly in winter. Although we sampled three counties weekly, the number of bloodmeals were likely too few to detect a significant seasonal change in host residency.

Because a non-significant change of host feeding was detected from birds to reptiles in spring, we cannot determine whether Cs. melanura feeding on reptiles influences EEEV transmission seasonally. The temporary shift in this mosquito feeding on mammals in July suggests that Cs. melanura may contribute to epizootic transmission more during summer. Even though our study only detected a single significant monthly change in host bloodmeals, other changes may exist in nature.

Because birds are the primary reservoir host for EEEV, deviations from bird feeding by

Cs. melanura may change when and how the virus is spread.

This study detected extensive feeding on reptiles. The species with the most total bloodmeals was the brown anole. Together, brown anoles and Carolina anoles made up

33.8% of all bloodmeals identified. Reptile feeding peaked in late spring and early summer. While previous research has recorded Cs. melanura feeding on reptiles, our results confirm that Cs. melanura may feed on reptiles throughout the year in its southern range. Previous research showed lower host use on reptiles of 17.2% from

November to May (Blosser et al. 2017). The average reptile host use from this study was 40.2% during the same months. The higher reptile host use observed may have been due to a greater abundance of reptiles at the study sites. The only significant shift in host use by class occurred in July when fewer reptile bloodmeals were detected than mammal bloodmeals. Brown and Carolina anoles are not thought to amplify EEEV

(White et al. 2011) and if wild lizards in Florida are not susceptible to the virus, these species may act as dilution hosts. However, snakes and turtles can be highly susceptible to EEEV infection. Six species that have been studied had a viremia lasting up to two weeks and it has been found that the garter snake (Thamnophis sirtalis) can maintain a low viremia over winter (Hayes et al. 1964, White et al. 2011). Infection of a northern black racer, a subspecies of the black racer, resulted in a viremia which lasted

10 to 14 days (Hayes et al. 1964). Our detection of Cs. melanura feeding on a southern black racer could indicate that they may serve as an amplifying or overwintering host if they are heavily fed on elsewhere.

Overall, humans were the second most fed on host species with 27 positive bloodmeal identifications (12.7%). These bloodmeals came from Cs. melanura on 20 different collection dates throughout eight months of 2018. Human bloodmeals were not

27 detected in February, March, September, or December. July and August, when human bloodmeals were most abundant (n=15, 19.2%), corresponds to the peak occurrence of

EEEV in horses and humans in 2018. The human bloodmeals were detected at seven of nine study sites, with no human host bloodmeals detected from Kelly Park in Orange

County or Deep Creek in Volusia County. Of the 27 bloodmeals, 48.1% were from sites at state parks which have active walking or biking trails. Sites within residential areas resulted in 38.9% of human bloodmeals. Because of its nocturnal biting habit, it is suspected that Cs. melanura fed upon humans which were outside for recreation during evening or morning hours at parks or place of residence. Host use on humans may occur more frequently in residential areas and parks that border hardwood swamps where Cs. melanura reproduce. Previously, mammalian hosts have composed 1-25% of

Cs. melanura bloodmeals (Nasci and Edman 1981, Apperson et al. 2004, Molaei and

Andreadis 2006, Molaei et al. 2006, Molaei et al. 2015a). The study by Nasci and

Edman (1981) detected large proportions of non-avian hosts from Cs. melanura in summer months but the proportion of human bloodmeals from their study is unknown.

The transmission of EEEV is complex because of the diversity and heterogeneity of host feeding that occurs by Cs. melanura. Although bloodmeals from birds were the most abundant, the data from this study reveal the large amount of feeding on reptiles and mammals that can take place by Cs. melanura in the southern range of EEEV transmission. A non-significant shift from bird to reptile bloodmeals occurred in early spring and reptile and mammal were present throughout most of the year. Many human bloodmeals as well as a total of seven bloodmeals from five other mammal species were observed. Even though horses were present near some sites, no horse

28 bloodmeals were found during the study period. If a large population of Cs. melanura occurs in hardwood swamps bordering residential areas or recreational parks, Cs. melanura may spread EEEV to epizootic hosts or humans.

Table 2-1. Collection sites of Cs. melanura in central Florida. Site name County Coordinates Arbovirus monitoring Habitat Deen Still Road Polk 28.2568°, -81.6850° Light trapping Forest Fussell Road Polk 28.2163°, -81.7720° Light trapping Forest, pasture, and residential Green Pond Road Polk 28.3693°, -81.8185° Light trapping Pasture, forest, and wetland New Independence Orange 28.4663°, -81.5982° Former sentinel flock Forest, wetland, and residential Kelly Park Orange 28.7583°, -81.5022° Sentinel flock Forest, wetland Tibet-Butler Park Orange 28.4437°, -81.5432° Sentinel flock Forest, wetland, and residential Deep Creek Volusia 28.9496°, -81.1028° Sentinel flock Forest, pasture, residential Fairgrounds Volusia 29.0100°, -81.2218° Sentinel flock Forest, pasture, wetland, Needles Volusia 29.2137°, -81.2303° Sentinel flock Forest, residential, wetland

Table 2-2. Forward and reverse primers used for bloodmeal host identification. Primer pair Sequence Amplicon Host type Citations size (bp) VertCOI_7194_F 5’-CGMATRAAYAAYATRAGCTTCTGAY -3’ 395 Vertebrate Reeves et al. ModREPCOI_R 5’-TTCDGGRTGNCCRAARAATCA -3’ 2018 ModRepCOI_F, 5’-TNTTYTCMACYAACCACAAAGA -3’ 244 Vertebrate Reeves et al. VertCOI_7216_R 5’-CARAAGCTYATGTTRTTYATDCG -3’ 2018 L2513 5′-GCCTGTTTACCAAAAACATCAC-3′ 300 Mammalian and Kitano et al. H2714 5′-CTCCATAGGGTCTTCTCGTCTT-3′ amphibian 2007 L0 5′-GGACAAATATCATTCTGAGG-3′ 220 Avian Lee et al. 2008 H1 5′-GGGTGGAATGGGATTTTGTC-3′ 16L1 5′-CTGACCGTGCAAAGGTAGCGTAATCACT-3′ 450 Reptilian Hass et al. 1993, H3056 5′-CTCCGGTCTGAACTCAGATCACGTAGG-3′ Vidal et al. 2000

Table 2-3. Vertebrate hosts of Cs. melanura by county. Hosts were identified by bloodmeal analysis on mosquitoes from Orange, Polk, and Volusia County, FL, in 2018. Percentages of column total in parentheses. Common name Scientific name Order Residency Orange Polk Volusia Total (%) Aves 34 (47.9) 52 (49.5) 19 (51.4) 105 (49.3) Northern cardinal Cardinalis cardinalis Passeriformes Resident 17 (23.9) 6 (5.7) 3 (8.1) 26 (12.2) Red-eyed vireo Vireo olivaceus Passeriformes Summer 3 (4.2) 8 (7.6) 11 (5.2) Mourning dove Zenaida macroura Columbiformes Resident 3 (2.9) 7 (18.9) 10 (4.7) Carolina wren Thryothorus ludovicianus Passeriformes Resident 2 (2.8) 3 (2.9) 2 (5.4) 7 (3.3) Barred owl Strix varia Strigiformes Resident 1 (1.4) 4 (3.8) 1 (2.7) 6 (2.8) Pine warbler Setophaga pinus Passeriformes Resident 1 (1.4) 4 (3.8) 1 (2.7) 6 (2.8) House wren Troglodytes aedon Passeriformes Winter 3 (2.9) 1 (2.7) 4 (1.9) Tufted titmouse Baeolophus bicolor Passeriformes Resident 1 (1.4) 3 (2.9) 4 (1.9) Chicken Gallus gallus Galliformes Exotic 3 (2.9) 1 (2.7) 4 (1.9) Blue-gray gnatcatcher Polioptila caerulea Passeriformes Resident 2 (2.8) 1 (1.0) 3 (1.4) Ruby-crowned kinglet Regulus calendula Passeriformes Winter 1 (1.4) 1 (1.0) 2 (0.9) Blue-headed vireo Vireo solitarius Passeriformes Winter 2 (1.9) 2 (0.9) American robin Turdus migratorius Passeriformes Winter 2 (1.9) 2 (0.9) Common yellowthroat Geothlypis trichas Passeriformes Resident 1 (1.4) 1 (2.7) 2 (0.9) Chipping sparrow Spizella passerina Passeriformes Winter 1 (1.4) 1 (1.0) 2 (0.9) Northern parula Setophaga americana Passeriformes Resident 1 (1.0) 1 (0.5) American bittern Botaurus lentiginosus Pelecaniformes Winter 1 (1.0) 1 (0.5) Blue jay Cyanocitta cristata Passeriformes Resident 1 (1.4) 1 (0.5) Eastern phoebe Sayornis phoebe Passeriformes Winter 1 (1.0) 1 (0.5) American goldfinch Spinus tristis Passeriformes Winter 1 (1.0) 1 (0.5) Eastern screech-owl Megascops asio Strigiformes Resident 1 (2.7) 1 (0.5) Belted kingfisher Megaceryle alcyon Coraciiformes Winter 1 (1.4) 1 (0.5) Forster's tern Sterna forsteri Charadriiformes Winter 1 (2.7) 1 (0.5) Yellow-rumped warbler Setophaga coronata Passeriformes Winter 1 (1.0) 1 (0.5) American purple gallinule Porphyrio martinicus Gruiformes Resident 1 (1.0) 1 (0.5)

Table 2-3 Continued. Common name Scientific name Order Residency Orange Polk Volusia Total (%) Yellow-billed cuckoo Coccyzus americanus Cuculiformes Resident 1 (1.4) 1 (0.5) Brown thrasher Toxostoma rufum Passeriformes Resident 1 (1.4) 1 (0.5) Carolina chickadee Poecile carolinensis Passeriformes Resident 1 (1.0) 1 (0.5) Common grackle Quiscalus quiscula Passeriformes Resident 1 (1.0) 1 (0.5) Reptilia 30 (42.3) 34 (32.4) 10 (27.0) 74 (34.7) Brown anole Anolis sagrei 22 (31.0) 21 (20.0) 4 (10.8) 47 (22.1) Carolina anole Anolis carolinensis 7 (9.9) 13 (12.4) 5 (13.5) 25 (11.7) Southern black racer Coluber constrictor priapus 1 (1.4) 1 (0.5) Brown basilisk Basiliscus vittatus 1 (2.7) 1 (0.5) Mammalia 7 (9.9) 19 (18.1) 8 (21.6) 34 (16.0) Human Homo sapiens 4 (5.6) 16 (15.2) 7 (18.9) 27 (12.7) Virginia opossum Didelphis virginiana 1 (1.4) 1 (1.0) 1 (2.7) 3 (1.4) Black/brown rat Rattus spp. 1 (1.0) 1 (0.5) White-tail deer Odocoileus virginianus 1 (1.4) 1 (0.5) Cattle Bos taurus 1 (1.0) 1 (0.5) Raccoon Procyon lotor 1 (1.4) 1 (0.5) Total 71 105 37 213

Table 2-4. Vertebrate hosts of Cs. melanura by month. Hosts were identified by bloodmeal analysis of mosquitoes from Orange, Polk, and Volusia County, FL, in 2018. Common name Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total Aves 1 6 5 4 15 13 17 22 2 8 8 4 105 Northern cardinal 1 3 7 7 4 1 3 26 Red-eyed vireo 1 2 2 1 2 1 2 11 Mourning dove 1 2 7 10 Carolina wren 3 2 1 1 7 Pine warbler 1 2 1 2 6 Barred owl 1 1 2 1 1 6 Chicken 1 2 1 4 Tufted titmouse 2 1 1 4 House wren 1 1 1 1 4 Blue-gray gnatcatcher 1 1 1 3 Blue-headed vireo 2 2 Ruby-crowned kinglet 1 1 2 American robin 2 2 Chipping sparrow 1 1 2 Common yellowthroat 1 1 2 Blue jay 1 1 American purple gallinule 1 1 American bittern 1 1 Yellow-billed cuckoo 1 1 Northern parula 1 1 Forster's tern 1 1 Belted kingfisher 1 1 Carolina chickadee 1 1 Yellow-rumped warbler 1 1 Brown thrasher 1 1 American goldfinch 1 1 Eastern screech-owl 1 1 Eastern phoebe 1 1

Table 2-4 Continued. Common name Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total Common grackle 1 1 Reptilia 1 1 2 8 18 14 8 11 1 5 4 1 74 Brown anole 1 1 4 10 10 5 8 1 4 2 1 47 Carolina anole 1 1 4 8 3 3 2 1 2 25 Southern black racer 1 1 Brown basilisk 1 1 Mammalia 1 1 4 1 9 11 4 2 1 34 Human 1 1 4 1 7 8 4 1 27 Virginia opossum 1 1 1 3 Raccoon 1 1 Cattle 1 1 White-tail deer 1 1 Black/brown rat 1 1 Total 3 7 7 13 37 28 34 44 3 17 14 6 213

Figure 2-1. Map of collection sites in Orange, Polk, and Volusia County, Florida, USA.

Figure 2-2. Mosquito resting shelter. A) Shelter placement in shaded area. B) Resting shelter with view of frame. Photos courtesy of author.

Figure 2-3. Aspirator used in mosquito collection from resting shelters. A) Aspirator with attachment. B) Collection cup secured in aspirator. Photos courtesy of author.

1.00 Aves 0.90 Reptilia 0.80 Mammalia 0.70 0.60 0.50

Proprotion 0.40 0.30 0.20 0.10 0.00 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec (3) (7) (7) (13) (37) (28) (34) (44) (3) (17) (14) (6)

Figure 2-4. Seasonal host use by Cs. melanura in central Florida. Points represent proportion of host class fed on each month in Orange, Polk, and Volusia County, FL (January - December 2018). Sample sizes in parentheses.

Summer resident Winter resident Resident 25

Count 10

0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Figure 2-5. Monthly summer resident, winter resident, and resident avian host use by Cs. melanura. Bird species detected by bloodmeal analysis of Cs. melanura in Orange, Polk, and Volusia County, FL in 2018. N=101.

CHAPTER 3 SEASONAL CHANGES IN VECTORIAL CAPACITY OF Culiseta melanura FOR EEEV IN CENTRAL FLORIDA

Seasonality of EEEV in Florida

Transmission of EEEV in the United States is seasonal, and epizootic cases primarily occur in the summer months (Bigler et al. 1976, Scott and Weaver 1989). In southern states, mosquitoes can be present year-round and are thought to maintain

EEEV transmission in the enzootic cycle even when epizootic transmission is not observed (Burkett-Cadena et al. 2015). In most endemic regions, epizootic transmission occurs primarily in summer, peaks in late summer, and declines in fall (Tenbroeck et al.

1935). Cases in equids can also occur during winter in the southern US because of its warmer climate (Bigler et al. 1976, Bingham et al. 2015, Burkett-Cadena et al. 2015).

The summer peak in transmission also coincides with increased mosquito abundance and EEEV antibody seroprevalence in young birds (Tenbroeck et al. 1935, Elias et al.

2017).

The epizootic transmission of EEEV in Florida can fluctuate greatly from year to year and varies in severity between regions within the state of Florida. The last large equine epidemic occurred in 2003 with 207 confirmed cases. Between 2004 and 2018, there have been six years with fewer than 30 cases a year and four years with more than 90 cases. In the past decade (2009-2018), an average of 41.4 cases a year was reported in Florida. Monthly cases peak in June at an average of 11.5 cases and are lowest in February with an average of 0.7 cases.

Vectorial Capacity

Many factors determine the extent of transmission of a vector-borne pathogen.

Vectorial capacity is an equation that accounts for the major factors of pathogen

transmission by mosquitoes and is defined as the average number of new vertebrate infections per day resulting from an initial index case (Garrett-Jones 1964a). Calculating vectorial capacity can help assess the possible transmission intensity by a vector population (Smith et al. 2014). The origins of this equation are rooted in efforts to better understand Plasmodium transmission and assess the effectiveness of malaria control in

Africa. Nevertheless, with modifications it can be applied to other vector-borne disease systems and is especially relevant to pathogens vectored by mosquitoes or insects with similar life cycles (Macdonald 1957, Garrett-Jones and Shidrawi 1969, Smith et al.

2014). Vectorial capacity (C) is calculated by equation 3-1, where ma represents the man-biting rate, a is the man-biting habit, p is the vector’s daily probability of survival, n is the extrinsic incubation period, and v is the vector competence (Macdonald 1957,

Garrett-Jones 1964a).

푚푎2푝푛푣 (3-1) 퐶 = −ln푝

The man-biting rate (ma) in the vectorial capacity formula for transmission cycles in which humans are the primary host (e.g., yellow fever) can be estimated by human landing catch (Garrett-Jones 1964b, Almeida et al. 2005). Because Cs. melanura feeds on birds at night, estimating mosquito density from human landing catches is not tractable and using sentinel chickens would not be effective because the phenology of sentinel chicken seroconversion more closely approximates epizootic transmission than enzootic amplification (Crans 1986). Substitution of m was done in one study with the relative population density of Cs. melanura and Cx. erraticus (Bingham et al. 2015). The estimation of vector density with non-human biting mosquitoes must be done with a trapping method.

There are multiple ways in which the man-biting habit can be measured (Garrett-

Jones 1964b, Almeida et al. 2005). In a study by Bingham et al. (2015), a of Cx. erraticus and Cs. melanura for EEEV was calculated as the proportion of avian bloodmeals. The a variable can be more accurately measured by the proportion of vectors feeding on competent hosts divided by length of the gonotrophic cycle (Garrett-

Jones and Shidrawi 1969, Rubio-Palis 1994). Estimation of the gonotrophic cycle can

푡 − 6.4 be calculated with the equation 푔 = 1/푉 where 푉 = and t is temperature in 푐 95.87

Celsius (Mahmood and Crans 1997). At higher temperatures, egg maturation within the female mosquito is accelerated and the gonotrophic cycle is shorter. Therefore, at high temperatures the period between a female feeding on hosts is shorter and the man- biting habit is higher.

Daily probability of survival (p) is calculated as the probability of daily survival of mosquitoes under certain conditions over the course of the extrinsic incubation period.

P can be estimated by rearing vectors in the lab and measuring their survival by counting the number of surviving individuals from day to day. However, Cs. melanura is difficult to colonize and laboratory survival rates of the species have not been determined. The older a population, the more likely it is that individual females have fed upon a viremic host, become infected and can transmit a pathogen to additional susceptible vertebrate hosts. Another method used to estimate p is by calculating the

푔 proportion of parous mosquitoes in the field and estimated with 푝 = 푐√푀 , where M is equal to the proportion parous and gc is the length of the gonotrophic period (Almeida et al. 2005). In some vector species such as Cq. perturbans, survival may be reduced by infection with EEEV (Moncayo et al. 2000). The extrinsic incubation period (n) of the

virus in the vector is used as the exponent of p. The extrinsic incubation period is the interval between the acquisition of an infectious agent by a vector and the vector's ability to transmit the agent to other susceptible vertebrate hosts. To transmit the virus to a host, the virus must pass through the mosquito’s midgut escape barrier, midgut dissemination barrier, and salivary gland barrier. If the vector has a short n, the value of

C will be larger from the increase in p because p is always less than one.

For a vector-borne disease to be sustained and amplified in nature, the pathogen must be transmitted from competent infected vectors to competent susceptible hosts where the virus replicates to levels sufficient to infect additional susceptible vectors.

Vector competence is an additional factor that is important to vectorial capacity and is represented by v in the numerator of the vectorial capacity equation. It is defined as a composite index that captures average mosquito susceptibility to infection with a pathogen and its innate ability to transmit the agent through saliva. This can be calculated with v =DT, the product of the proportion of mosquitoes that develop a disseminated infection (D) and the proportion of mosquitoes with disseminated infection that transmit virus by bite (T) (Sardelis et al. 2001).

Culiseta melanura feeds on hosts with varying degrees of competence which will influence EEEV transmission via the biting rate. Host competence is calculated as the product of the duration of viremia and the proportion of mosquitoes which become infected after feeding on a viremic host (Komar et al. 1999). An assumption of vectorial capacity that is not upheld in the EEEV transmission cycle is that hosts have equal competence. Competence values for birds for EEEV via subcutaneous inoculation are reported to be high in European starlings, northern cardinals, grackles, and swamp

sparrows (Komar et al. 1999). Mourning doves and brown-headed cowbirds (Molothrus ater) had low competence for EEEV and American robins had moderate competence

(Komar et al. 1999). Reptiles are potential reservoir hosts for EEEV with garter snake viremia lasting up to 14 days post infection (White et al. 2011). In addition, EEEV can overwinter in garter snakes (White et al. 2011). Green anoles have low competence for

EEEV with mean viremia of 2.44 log 10 PFU/mL, which is below the 4.0 log 10 PFU/mL infectious dose for Cs. melanura (Komar et al. 1999, White et al. 2011).

The goal of this study was to quantify vectorial capacity (C) as a function of season for EEEV and its vector Cs. melanura in central Florida. This was achieved by determining Cs. melanura abundance, using the variation in host feeding to calculate a, and determining parity of Cs. melanura throughout the year. These variables were incorporated into the C equation using temperature data and previous data on extrinsic incubation period (n) and gonotrophic cycle (gc) of Cs. melanura. Quantifying vectorial capacity of Cs. melanura will expand the knowledge of how Cs. melanura ecology affects EEEV transmission.

Materials and Methods

The methods used in this study utilized a combination of blood meal analysis and

DNA sequencing to determine host usage and parity determination to measure probability of daily survival (p) of Cs. melanura. These methods contributed to the calculation of vectorial capacity for Cs. melanura in all seasons of 2018. A linear regression analysis was done on vectorial capacity values and EEEV transmission in equids to improve the understanding of the relationship between enzootic and epizootic transmission. The mosquitoes used were those collected from resting shelters in

Orange and Polk County from January through December of 2018.

Parity Determinations

After mosquitoes were sorted and identified, unfed or freshly bloodfed Cs. melanura females were dissected to determine parity. The presence of tracheal skeins in the ovaries indicates that female mosquitoes are nulliparous, while a tracheal net indicates a parous female (Figure 3-1). To make this diagnosis, ovaries were extracted into a drop of water on a slide and left to dry, making the tracheoles visible for inspection (Detinova 1962). This method was tested on Aedes vigilax (Skuse) for accuracy and was 83.7-89.8% reliable (Hugo et al. 2014). All female Cs. melanura were dissected but individuals whose parity could not be determined were excluded from the total for the parity calculation.

Vectorial Capacity Calculations

The C values for monthly periods were calculated following Macdonald (1957) with modifications including adjustments for density and biting rate of an ornithophilic mosquito species, calculating p for a wild field population, and incorporating the monthly temperature of the study area. With the assumption that host density is equal between

sites and seasons, the variable m was calculated as the number of female Cs. melanura per resting shelter per month. Host density is likely to be higher during spring and autumn but was not measured or incorporated into the vectorial capacity calculation. To calculate a, the proportion of Cs. melanura feeding on competent (avian) hosts was divided by length of the gonotrophic cycle (gc) (Rubio-Palis 1994). The gonotrophic cycle length of lab reared Cs. melanura varies greatly from 23.2 days at 10 °C to 4.5 days at 28 °C (Mahmood and Crans 1997). Estimation of gc was done using the

푡 − 6.4 equation 푔 = 1/푉 where 푉 = and t is temperature in Celsius (Mahmood and 푐 95.87

Crans 1997). The values of t for the study period were the mean daily temperature taken from historical weather data from the National Climatic Data Center weather stations at Gilbert Airport in Winter Garden, FL for the samples from Polk County and at

Executive Airport in Orlando, FL for the samples from Orange County (National Climatic

푔 Data Center 2019). The value of p was estimated with 푝 = 푐√푀 , where M is equal to the proportion parous and gc is the length of the gonotrophic cycle (Almeida et al. 2005).

The variable M is the proportion of parous mosquitoes of the females with a positive parity determination. The mosquitoes with undetermined parity were not included in the

C calculation and includes gravid females, females with a bloodmeal several days old, and females with damaged ovaries. The n of EEEV in Cs. melanura was found to be three days when kept at 25 to 30 °C but should fluctuate with temperature (Scott and

Burrage 1984). The n of EEEV at different temperatures has been measured in Aedes triseriatus (Say) (Chamberlain and Sudia 1955). It is assumed to be approximately the same for Cs. melanura in this study. We estimated n for our study with the TREND function in Excel using the number of days at which 50% of Ae. triseriatus were capable

of transmission, the experimental temperatures used, and our t values for the study period.

When these calculations were complete, the determined value of C for each period was compared to the mean monthly number of EEEV equine cases in Florida from the last decade and cases from 2018 (Florida Department of Health 2019).

Regression analyses were used to investigate the relationship of C values and epizootic

EEEV transmission.

Results

Mosquito Abundance and Host Feeding

During the 2018 sampling year 6,093 female mosquitoes, comprised of 24 different species, were collected from six sites in Orange and Polk County, Florida (Table 3-1).

The most abundant mosquito species were Cx. erraticus (n= 2,720) and Cs. melanura.

(n=1,036). Abundance of Cs. melanura showed a seasonal pattern of increased numbers in summer months, which was three times larger than winter months (Figure 3-

2). The density of Cs. melanura peaked in July when a mean of 1.29 females were captured per shelter (n=170, Table 3-2). In August the density remained high but in a large decrease in density to 0.29 occurred in September. The smallest density occurred in February with 0.18 mosquitoes per shelter. Abundance and density of Cs. melanura were significantly correlated (R2=0.868, df=13, P<0.0001).

Host use by Cs. melanura in 2018 was previously determined by bloodmeal analysis and resulted in 105 avian (49.3%), 74 reptilian (34.7%), and 34 mammalian

(16.0%) bloodmeals. Avian host use varied but remained high throughout most of the year (31-86%). Bird bloodmeals were most common every month except April, May, and June. In September, only three bloodmeals were identified. Bird species identified from bloodmeals consisted of 9 orders and 22 families of birds. The avian hosts most fed on were northern cardinal, red-eyed vireo, mourning dove, Carolina wren, barred owl, pine warbler, and house wren. The largest number of bloodmeals came from songbirds (Order Passeriformes) with 79 positive identifications (75% of avian hosts).

Parity Rate, Gonotrophic Cycle Length, and Extrinsic Incubation Length

Parity was determined for 653 Cs. melanura females of 1,036 collected (63.0%).

The proportion of parous females ranged from 0.13 in January to 0.38 in December of

2018 (Figure 3-3). From January to May, the proportion of parous females increased gradually to 0.28. The parity rate decreased slightly in July with the rate increasing in

October and a large peak occurring in December. Although variation was observed, differences in monthly parity rates were not significant. Of 257 blood engorged Cs. melanura females from Orange and Polk County, only 65 were successfully determined for parity. Of these, 7 (10.8%) were found to be parous.

Incorporating the mean monthly temperatures from the study sites enabled the calculation of n and gc, the latter of which is also used to estimate a and p. The lowest mean temperature of Orange and Polk Counties was recorded at 14.8 °C in January and the highest mean temperature of 29.4 °C was recorded in September. The monthly temperatures in Orange and Polk County were similar, with a mean difference of 0.3 °C.

At these temperatures, the gc for Cs. melanura was calculated to be 4.2 to 11.4 days in length (Table 3-2). The estimated n for Cs. melanura was 7.9 in September to 21.0 days in January (mean=12.9 days).

EEEV Transmission in Central Florida

The epizootic transmission of EEEV in Florida can fluctuate greatly from year to year and was higher than average in 2018, with a total of 55 equine cases (Figure 3-4).

Epizootic transmission from January to May of 2018 was over twice as high as the mean cases in January through May of the last decade. Monthly cases from July to

October were slightly less than the last decade of EEEV transmission and no cases occurred in November or December. In Orange and Polk County in 2018, 2 horse cases and 13 sentinel chicken seroconversions to EEEV were reported. In the last decade,

Orange and Polk county have reported 2 and 12 total EEEV horse cases, respectively.

The whole state of Florida has had an average of 41.4 cases a year. The average number of cases per month range from 11.5 in June to 0.7 in February.

Vectorial Capacity

Using data from mosquito abundance, parity, bloodmeal source, and temperature, vectorial capacity (C) values of Cs. melanura for EEEV were calculated for the months of 2018 (Table 3-2). The m variable is traditionally calculated as the number of vectors per host. Because the density variable used in this study is the number of mosquitoes per shelter, the m is likely underestimated and C values are relatively smaller than previous studies (Garrett-Jones and Shidrawi 1969). Values of C had a minimum of 0.00004 in January. The values rose in May and had a peak of 0.00305in

June and then decreased in September. The C values obtained for the 2018 study were significantly correlated with Florida EEEV equine cases in 2018 (R2=0.349, df=10,

P=0.043) and the mean monthly number of cases from the last decade (R2=0.537, df=10, P=0.007). During 2018, 55 positive EEEV cases were reported from equids as well as five emu flocks. Epizootic transmission from January to May was over twice as high in 2018 than the mean of the last decade.

Discussion

Vectorial Capacity

In this study, the C of Cs. melanura was calculated with mosquito sampling, parity determination, and bloodmeal analysis and was found to vary by season. The significant correlation of C values with epizootic cases of EEEV could mean that amplification of the virus in birds peaks in summer, resulting in the spillover to epizootic hosts which occurs in the summer months. Additionally, Cs. melanura may be contributing to the epizootic cases in mammals. The r-squared value from the regression of C values on EEEV equine cases per month from 2009-2018 indicates that

53.7% of the variation in recent equid cases is explained by vectorial capacity of Cs. melanura. Vectorial capacity of Cs. melanura has higher correspondence with EEEV activity of historical epizootic cases than current epizootic cases. A high proportion of avian host use and high proportion of parous females in December caused a peak of C in December. This month had few identified bloodmeals (n=6) and mosquitoes with parity determinations (n=16). Including December in the regression analysis reduces the correspondence between C and EEE in horses. If the C value from December is removed, vectorial capacity of Cs. melanura had a stronger correlation with 2018 cases

(R² =0.6691, df=10, P=0.002) and cases from the last decade (R² = 0.8304, df=10,

P<0.0001).

Abundance

The density of Cs. melanura in central Florida had the same trend of C and was highest in the summer, which contrasts with results from Edman et al. (1972) and

Blosser et al. (2017) where decreases of abundance in late spring were reported. The sharp increase of Cs. melanura that occurred from April to May 2018 matches the

increase in EEEV cases in horses that occurred in May 2018. Previously, high Cs. melanura abundance was found to be significantly correlated with EEEV infection in Cs. melanura (Skaff et al. 2017). A regression of the density values of Cs. melanura on

EEEV equine cases per month from 2009-2018 indicates that 63.5% of the variation in recent equid cases is explained by density of Cs. melanura alone (R2=0.6349). Wet hydrological conditions have been associated with Cs. melanura abundance and may be sign of increased transmission (Skaff et al. 2017).

Using the number of females per resting shelter is an indirect method of estimating density, in contrast to measuring incidence of bites per host per day.

Previously, measuring density indirectly resulted in three to four times lower density of

Anopheles than using direct measurements. If density of Cs. melanura was measured directly using bird landing rates, density values may be higher and more accurate than in this study.

The sharp decrease in Cs. melanura in September did not occur for other commonly collected mosquitoes such as Cx. erraticus or Cq. perturbans (Table 3-1).

September was the warmest month in 2018 with a mean temperature of 29.4 °C and a mean high temperature of 33.9 °C. Research on temperature effects on Cs. melanura displayed that an increase from 28 °C to 32 °C during larval development decreased emergence success from 59.6% to 1.0% in a lab colony from New Jersey (Mahmood and Crans 1998). The high temperature during the summer in Florida may cause the low abundance of Cs. melanura seen in early fall and restrict the distribution of the species from expanding southward. The abundance and distribution of Cs. melanura is

also dependent on availability of larval habitat in forested freshwater swamps, which may have a temperature dependent distribution (Skaff et al. 2017).

Host use

Heterogeneity in host availability and host use can occur seasonally within a region, as the 39 species that Cs. melanura fed in our study displays. One assumption of vectorial capacity is that the pathogen is transmitted by only one species of vector to one type of host (Dye 1986). The current study addresses this assumption by using the proportion of avian hosts in the vectorial capacity equation. Host availability and preference by Cs. melanura changes seasonally and is affected by host dispersal, migration, brumation, and hibernation (Hassan et al. 2003). It has been observed that heterogeneity in host feeding by Culex mosquitoes had a significant impact on West

Nile virus transmission (Kilpatrick et al. 2006). This also may be the case with EEEV if host feeding is not homogeneous in regions of EEEV transmission.

The a for our calculation assumes that all avian host are equally competent. But avian EEEV competence has not been measured in all avian hosts found in this study.

The host competence of an avian species may differ depending on family, size, recent exposure to an exotic species, or could differ between individuals (Komar et al. 1999).

The seasonal host data from this study supports the northward spread of EEEV by competent migrating avian hosts such as the red-eyed vireo and house wren. The red- eyed vireo is a migratory species that breeds in North America and migrates to Central

America in the winter (eBird 2019). The species is present in Florida in spring and fall with a lower abundance in summer. The house wren is a winter resident in Florida and can have high EEEV antibody prevalence (Crans et al. 1994). American robin has been implicated in EEEV transmission. However, this species is present in Florida periodically

from November to March when EEEV transmission by Cs. melanura is low. Further research on the competence and migratory patterns of the various hosts of Cs. melanura may reveal how they contribute to EEEV transmission.

Mosquito Survival

The p of Cs. melanura was highest in January and December, which is expected in months with low temperatures (Chamberlain and Sudia 1955). From July until late fall a decrease in p was observed, caused by the decrease in parous mosquitoes and higher temperatures. The much higher parity rate and p seen in December is consistent with parity rates from Cx. pipiens in the northern US, which were high at the start of winter and decreased significantly over the course of winter (Andreadis et al. 2010). The parity rates of January through March and September through December were determined with sample sizes of less than 30 per month. These sample sizes are four- to nine-fold fewer than from June through August and may have influenced the high parity rate observed in December. The p variable in C is assumed to be constant over the lifespan of the mosquito, but in most cases, it is not (Clements and Paterson 1981).

In addition to environmental factors such as predation, infection with EEEV may decrease the survival rate of mosquitoes (Scott and Lorenz 1998, Moncayo et al. 2000,

Kramer and Ciota 2015).

Conclusion

In conclusion, a positive association between virus infections in equids and C values was found and gives support to the large effect that enzootic transmission by Cs. melanura has on epizootic outbreaks. The C values indicate that mean winter transmission of EEEV by Cs. melanura is lower than in summer. The largest C value occurred in June, when the proportion of reptile bloodmeals was high. Further

investigation into the role reptiles have in transmission could reveal that anole lizard in

Florida contribute to EEEV amplification or that they suppress transmission as dilution hosts. This study reveals more of the complex effects Cs. melanura has on the spread and maintenance of EEEV as its primary enzootic vector. The seasonality of hosts of

Cs. melanura supports the spread of EEEV by competent migrating avian hosts.

Knowledge of seasonal changes in C and the host use of Cs. melanura may aid the prevention, monitoring, and control of EEEV by indicating that enzootic amplification peaks in summer months but is affected by many factors.

Table 3-1. Monthly abundance of Cs. melanura and other mosquito species. Mosquitoes were collected using resting shelters in Orange and Polk County, FL in 2018. Species Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total % of total Cx. erraticus 150 94 46 47 196 343 476 452 252 185 358 121 2720 44.6 Cs. melanura 31 19 47 74 163 166 170 199 33 45 59 30 1036 17.0 An. crucians complex 38 69 67 51 96 137 95 119 63 51 95 36 917 15.1 Cx. cedecei 2 3 1 22 128 129 27 5 317 5.2 Cq. perturbans 1 9 6 9 20 111 64 22 22 3 267 4.4 Cx. territans 11 7 30 35 45 43 11 8 4 4 18 6 222 3.6 Ur. sapphirina 1 1 23 62 32 1 5 4 1 130 2.1 Cx. nigripalpus 4 8 1 2 13 18 21 34 14 7 3 2 127 2.1 Cx. pilosus 1 1 1 14 73 22 7 1 120 2.0 An. quadrimaculatus 3 5 3 12 10 9 5 6 2 3 58 1.0 Cx. quinquefasciatus 5 8 6 15 10 1 1 1 1 48 0.8 Cx. peccator 7 11 7 2 1 28 0.5 Mn. titillans 1 3 2 3 2 2 2 7 22 0.4 Ur. lowii 1 1 2 1 7 1 13 0.2 Ae. atlanticus/tormentor 1 6 7 0.1 Ae. albopictus 3 1 2 6 0.1 Unidentifiable 1 3 10 17 31 0.5 Minor species (n<5) 1 2 1 5 7 5 2 1 0 0 24 0.4 Total 236 208 219 229 595 888 1122 1057 451 337 551 200 6093

Table 3-2. Vectorial capacity variables of Cs. melanura for EEEV. Mosquitoes were collected in Orange and Polk County, FL in 2018. Month m H gc t a2 M p n C Jan 0.34 0.33 11.44 14.8 0.00 0.13 0.83 21.0 0.0000 Feb 0.18 0.86 5.95 22.5 0.02 0.14 0.72 14.1 0.0001 Mar 0.38 0.71 7.51 19.2 0.01 0.18 0.80 17.1 0.0003 Apr 0.59 0.31 5.64 23.4 0.00 0.24 0.78 13.3 0.0002 May 0.88 0.41 5.05 25.4 0.01 0.28 0.78 11.5 0.0013 Jun 1.06 0.46 4.38 28.3 0.01 0.28 0.75 8.9 0.0030 Jul 1.29 0.50 4.32 28.6 0.01 0.19 0.68 8.6 0.0017 Aug 1.28 0.50 4.25 28.9 0.01 0.20 0.68 8.3 0.0020 Sep 0.29 0.67 4.18 29.4 0.03 0.15 0.64 7.9 0.0005 Oct 0.42 0.47 4.77 26.5 0.01 0.23 0.73 10.5 0.0005 Nov 0.82 0.57 6.29 21.6 0.01 0.15 0.74 14.9 0.0003 Dec 0.38 0.67 8.32 17.9 0.01 0.38 0.89 18.2 0.0024 m, density of mosquito vector (mean no. of mosquitoes/shelter); H, proportion of avian hosts from Cs. melanura; gc, gonotrophic cycle = 1/V, V=(t-6.4)/95.87; t, mean daily temperature of Polk and Orange County in Celsius; a=H/gc, the biting rate of the vector; M, proportion of parous 푔 mosquitoes; p= 푐√푀, vector’s daily probability of survival; n, extrinsic incubation period; C, vectorial capacity.

Figure 3-1. Ovaries from Cs. melanura at 100X magnification. A) Ovaries with tracheal skeins from nulliparous Cs. melanura. B) Ovaries with tracheal nets from a Cs. melanura suspected to be parous. Photos courtesy of author.

2.5

1.5

Mean no. insects/shelterno.Mean 0.5

0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Figure 3-2. Mean density of Cs. melanura from Orange and Polk County, FL in 2018. Vertical axis represents the mean number of female mosquitoes per artificial resting shelter and standard error represented by error bars.

0.40 0.35 0.30

0.25 Cs. melanura Cs. 0.20 0.15 0.10 0.05 0.00 Proportion of parousof Proportion Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec (24) (14) (22) (46) (95) (137) (127) (111) (13) (22) (26) (16)

Figure 3-3. Parity of Cs. melanura from Orange and Polk County, FL in 2018. Vertical axis is the proportion of parous females from those with positive parity determination. Sample sizes in parentheses. No significant difference was detected between months.

EEEV Equine Cases, 2018 EEEV Equine Cases, 2009-2018 C 16 0.0035

14 0.0030

12 0.0025

10 0.0020 8 C 0.0015 6

Equine cases of EEEV of cases Equine 0.0010 4

2 0.0005

0 0.0000 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Figure 3-4. Vectorial capacity (C) of Cs. melanura for EEEV against EEEV equine cases from 2018 and prior decade in Florida. The left vertical axis is the number of reported EEEV cases in equids. The right vertical axis is the C value for Cs. melanura for each month of 2018.

16 2018 Cases 2018 EEEV Equine Cases y = 2829.8x + 1.3266 R² = 0.3491 2009-2018 EEEV Equine 14 P=0.043 Cases 2009-2018 Cases y = 2761.3x + 0.6168 12 R² = 0.537 P=0.007

6 EEEV equine casesequineEEEV

0 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 C

Figure 3-5. Relationship of Florida EEEV equine cases and vectorial capacity of Cs. melanura. Values of C were calculated using Cs. melanura collected from resting shelters in central Florida.

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BIOGRAPHICAL SKETCH

Richard West was born in Orlando, FL to Rick and Lyn West and was raised in

Webb, AL along with his four siblings. After graduating high school in 2011, he completed an Associate in Science degree at Wallace Community College in Dothan,

AL. He then transferred to Auburn University in 2013 to obtain a Bachelor of Science degree in Organismal Biology from the Department of Biological Sciences. While in

Auburn, he had the opportunity to serve as an Undergraduate Teaching Assistant, a laboratory technician in Dr. Brian Helms’ lab processing invertebrate stream samples, a volunteer for Auburn’s Museum of Natural History, and a technician for Dr. Charles Ray identifying insect pitfall traps. After taking two classes in entomology, he discovered he had an interest in medical entomology and started part-time work for Dr. Derrick

Mathias collecting and identifying mosquitoes. A year after graduating from Auburn in

May of 2016, he accepted a graduate research position at the Florida Medical

Entomology Laboratory in Vero Beach, FL. Richard received his Master of Science degree in Entomology from the Department of Entomology and Nematology at

University of Florida in August 2019.