Seasonal Changes of Host Use by Culiseta Melanura (Diptera: Culicidae) in Central Florida

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Journal of Medical Entomology, 57(5), 2020, 1627–1634 doi: 10.1093/jme/tjaa067 Advance Access Publication Date: 13 April 2020 Vector/Pathogen/Host Interaction, Transmission Research

Seasonal Changes of Host Use by Culiseta melanura (Diptera: Culicidae) in Central Florida

Richard G. West,1 Derrick K. Mathias,1 Jonathan F. Day,1 Carolina Acevedo,1

Thomas R. Unnasch,2 and Nathan D. Burkett-Cadena1,3, Downloaded from https://academic.oup.com/jme/article/57/5/1627/5819317 by Rutgers University user on 03 June 2021

1Florida Medical Entomology Laboratory, University of Florida IFAS, 200 9th Street SE, Vero Beach, FL 32962, 2Global Health Infectious Disease Research Program, University of South Florida, 3720 Spectrum Boulevard, Suite 304, Tampa, FL 33612, and 3Corresponding XX author, e-mail: [email protected] Subject Editor: Theodore Andreadis

XXXX Received 4 January 2020; Editorial decision 10 March 2020

XXXX Abstract The mosquito Culiseta melanura (Coquillett) is the primary enzootic vector of eastern equine encephalitis virus (EEEV), a zoonotic Alphavirus endemic to eastern North America. In its northern range, Cs. melanura is considered a strict avian biter, transmitting EEEV among susceptible birds in a cycle of enzootic amplification. In its southern range, however, Cs. melanura is more general in host use, feeding heavily upon birds but also reptiles and mammals. The goal of this study was to better understand how host use of Cs. melanura changes throughout the year in Florida, where year-round EEEV transmission is observed. Mosquitoes were sampled in 2018 from nine sites across three central Florida counties. In total, 213 Cs. melanura bloodmeals were identified by PCR consisting of 39 species of birds, reptiles, and mammals. Avian bloodmeals were prominent throughout the year (range = 30–85%), and songbirds were a large portion of identified bloodmeals (37.1%). Reptiles surpassed birds only in spring (April–June), and brown anole (Anolis sagrei Duméril and Bibron, 1837 [Reptilia: Dactyloidae]) was the most commonly detected single host species (22.1% overall). Mammalian bloodmeals were mainly observed in summer, with humans being the most fed on mammal (12.7% overall). This study reveals that in southern foci of EEEV transmission, Cs. melanura host use varies throughout the year with reptiles providing the majority of blood meals in spring (51.3%), and birds are fed on more than other host groups during all other seasons (50.6–70.1%). In addition, feeding on mammals increases during summer months, which may implicate Cs. melanura in epizootic transmission in Florida.

Key words: Culiseta, arbovirus, seasonal, host use, Anolis

The mosquito Culiseta melanura (Coquillett) is the primary enzootic location. In the northeastern United States, few nonavian bloodmeals vector of eastern equine encephalitis virus (EEEV; family Togaviridae, have been observed for Cs. melanura, with studies reporting multiple genus Alphavirus), a highly pathogenic zoonotic agent endemic different avian species fed on in large numbers. In Virginia, 43% to eastern North America (Bigler et al. 1976). Enzootic transmis- of Cs. melanura bloodmeals were from American robin (Turdus sion of EEEV is typically described as involving Cs. melanura and migratorius), followed by 16% from northern cardinal (Cardinalis passeriform birds (songbirds) in freshwater swamps, which represent cardinalis) and 11% from European starling (Sturnus vulgaris; foci of amplification. Numerous studies investigating host use by Cs. Molaei et al. 2015b). In New York, 24% of bloodmeals were from melanura have demonstrated that it primarily feeds on songbirds wood thrush (Hylocichla mustelina) and 9% from American robin (Cupp et al. 2003, Bingham et al. 2014, Molaei et al. 2015a), with up (Molaei et al. 2006). In Connecticut, 23% were from American to 95% of bloodmeals taken from avian hosts (Molaei et al. 2015b). robin and 13% from wood thrush (Molaei and Andreadis 2006). Circulation of EEEV outside of the avian enzootic cycle takes place In Vermont, green heron (Butorides virescens) and American robin primarily when the avian amplifying hosts are fed on by multiple composed 17 and 12% of bloodmeals from Cs. melanura, respec- mosquito vectors with transmission spilling over into mammalian or tively (Molaei et al. 2015a). In the southeastern United States other dead-end hosts (Molaei et al. 2015b). (Alabama, Florida, North Carolina), Cs. melanura also feeds sub- Because the distribution of Cs. melanura is wide (southern stantially upon mammals and/or reptiles (Nasci and Edman 1981, Canada through southern Florida), the host species encountered and Burkett-Cadena et al. 2008, Blosser et al. 2017), in addition to birds. fed on by this mosquito are numerous and differ from location to In these southern states, important hosts include various wading

© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. 1627 All rights reserved. For permissions, please e-mail: [email protected]. 1628 Journal of Medical Entomology, 2020, Vol. 57, No. 5 birds (Bingham et al. 2014), northern cardinal (Estep et al. 2011), Materials and Methods and the brown anole lizard (Anolis sagrei; Blosser et al. 2017), among others. Field Locations Host use by Cs. melanura is also reported to change season- Sampling was conducted in the central region of Florida with nine ally, in the northern, central, and southern portions of its range. In sites in Orange (3), Polk (3), and Volusia (3) counties (Fig. 1). Polk Vermont and Virginia, Molaei et al. (2015a,b) found that host pref- County marks the southern edge of the region with high EEEV erence shifted between several species of bird, including songbirds transmission in the state of Florida as recorded over the past 10 yr and wading birds. In North Carolina, Cs. melanura was found to (Florida Department of Health 2019). In these counties, there is also feed upon birds throughout most of the year but took a substan- historic evidence of consistent EEEV circulation, with 21, 30, and 11 tial proportion of bloodmeals from mammals in summer months virus isolations from vertebrate and invertebrate made in Orange, (Nasci and Edman 1981). In Florida, Blosser et al. (2017) found that Polk, and Volusia counties, respectively, from 1955 to 1974 (Bigler

Cs. melanura fed mostly on birds in winter and early spring but fed et al. 1976). Many Cs. melanura have been collected at these sites Downloaded from https://academic.oup.com/jme/article/57/5/1627/5819317 by Rutgers University user on 03 June 2021 primarily on reptiles in late spring, with the shift being driven by previously with conventional light traps, and five sites have sentinel warmer temperatures and longer days in late spring. Sampling for chicken coops present (data not shown). Blosser et al. was terminated in May, so seasonal patterns of host use of Cs. melanura during peak transmission season (June–August) in Mosquito Sampling Florida is not currently known. Weekly mosquito collections were made at these nine sites from Seasonal patterns of host use are important to quantify for vector October 2017 to January 2019 using six artificial resting shelters species because seasonal shifts can influence amplification and drive at each site for a total of 54 resting shelter points. Resting shelters spillover of vector-borne pathogens (Kilpatrick et al. 2006). To date, serve as dark, protected refuges that mosquitoes seeking a place to seasonal patterns of host use for Cs. melanura are not known in rest. Resting shelters and resting boxes have been used in studies to Florida, where EEEV is transmitted year-round (Bigler et al. 1976). sample various species of Culex (Hoyer et al. 2017) and have been In addition, Florida may serve as a geographic reservoir for EEEV found to be an effective method to collect bloodfed Cs. melanura fe- strains that circulate in northern foci (Heberlein-Larson et al. 2019). males (Edman et al. 1968, Bingham et al. 2014). The resting shelters The goal of this study was to quantify seasonal host use by Cs. used in the current study were made by placing a fitted black trash melanura in central Florida using PCR-based bloodmeal analysis of compactor bag over a cylindrical frame made with three 86-cm-long females collected from three counties. ½ inch PVC pipes, six PVC tees, ½ inch PEX pipe shaped into two

Fig. 1. Map of sampling locations in central Florida. Journal of Medical Entomology, 2020, Vol. 57, No. 5 1629 rings with a diameter of 46 cm, two PEX couplers, and rubber bands and absence of bands. PCR with no band were discarded. Unpurified and binder clips to hold the tees and bag in place (Hoyer et al. 2017, PCR products from reactions with visible bands were sent for Sanger Burkett-Cadena et al. 2019). Mosquitoes were collected from shel- sequencing, which was done in forward direction only (Eurofins ters by aspiration with a modified vacuum (BDH1800S Ni-Cd 18V MWG Operon, Huntsville, AL). Dustbuster, Black & Decker, MD) and collection cup (BioQuip Sequence files were annotated of undetermined nucleotides and Products, Rancho Dominguez, CA; Blosser et al. 2017). The vacuum were compared with sequence database using the BLASTn in the was modified with a wide dust mop brim attachment that fit snugly NCBI database GenBank. Species identification, percent match, inside of the shelters, thus preventing mosquitoes from escaping query coverage, query length, and match length were recorded for around the edge of the bag during aspiration (Burkett-Cadena et al. each sequence. Only samples that were categorized as 1- or 2-d post- 2019). blood feeding were considered identified and were analyzed and Collected female mosquitoes were identified to the species level presented in the results (Reeves et al. 2016). Identified bloodmeals by examining morphological traits using a dissecting microscope were those which had a ≥95% match of the sequence and a >3% Downloaded from https://academic.oup.com/jme/article/57/5/1627/5819317 by Rutgers University user on 03 June 2021 and dichotomous keys updated in 2018 (Darsie and Morris 2003, mismatch from the next closest species. Suspected mixed bloodmeals Darsie and Ward 2005, Burkett-Cadena 2013). The presence of a from more than one host from a low sequence match or DNA chro- tuft of setae on ventral basal surface of subcostal wing vein and matogram with many double peaks were tested with all primers. prespiracular setae separated Cs. melanura from other species, including Culex spp. Once identified, Cs. melanura females were Data Handling and Statistical Analysis stored in Thermo Scientific Microcentrifuge tubes at −20°C for fu- Positive bloodmeals were categorized by host, class, and order. ture analysis. Resident status for avian hosts was determined to better understand the host use of Cs. melanura throughout the year. Birds not typi- Bloodmeal Analysis cally found in Florida year-round were categorized as either summer The hosts fed on by Cs. melanura were determined by bloodmeal resident or winter resident based on their peak season of residency. analysis consisting of DNA extraction, PCR, and sequencing. This was determined by using Florida bird frequency data from Extraction was performed using InstaGene by homogenizing each the Weekly Bar Chart feature on Cornell University’s eBird (2019) individual mosquito in 150 µl of 0.9% NaCl solution with 2-mm website. Chi-square test of independence was used to test whether glass beads using the Bullet Blender Storm 24 Homogenizer on speed the distribution of bloodmeals derived from vertebrate classes (rep- setting 8 for 5 min. Tubes were briefly centrifuged at 6,000 rpm. Then tile, bird, mammal) differed between counties. Fisher exact test was 200 µl of InstaGene was added to tubes following the manufacturer’s used to test for seasonal shifts in host use between vertebrate classes protocol. Tubes were incubated in a 98°C water bath for 10 min and and between resident, summer resident, and winter-resident birds. then centrifuged at 6,000 rpm for 5 min. The supernatant containing Seasons were categorized based on 3-mo periods as follows: winter the DNA was transferred to a new, sterile tube and stored at −20°C (January–March), spring (April–June), summer (July–September), until subsequent PCR analyses. and fall (October–December). Five primer pairs were used to detect vertebrate host bloodmeals in a series of PCR reactions. DNA from PCR with negative results were run with the subsequent primer pair in a hierarchal approach. Results The 16L1/H3056 (Lzrd) primer pair, which targets the 16S rRNA gene, producing 450 bp amplicon, was used to detect reptilian Culiseta melanura Bloodmeals blood with the positive control from a yellow rat snake (Hass et al. During the 2018 sampling year, 7,125 female mosquitoes, comprised 1993, Vidal et al. 2000). To amplify avian bloodmeals, the L0/H1 of 24 different species, were collected. The most abundant mosquito primer pair, which targets a cytochrome b gene, producing 220 bp species were Cx. erraticus (n = 3,288) and Cs. melanura. (n = 1,241). amplicon, was used with a positive control from the common yel- Of the female Cs. melanura collected, 277 (22.3%) were bloodfed. lowthroat (Lee et al. 2008). The H2714/L2513 primer pair was used Culiseta melanura fed on a diverse set of hosts with variation in to amplify mammalian and amphibian blood and targets the cyto- hosts by site and month sampled. Bloodmeals that were considered chrome b gene, producing 300 bp amplicon (Kitano et al. 2007). three or more days post-blood feeding by visual estimates of egg de- Black rat and Cuban treefrog DNA were positive controls for the velopment and were not considered identifiable (n = 33). Bloodmeals mammalian and amphibian primer pair. Last, the VertCO1_7194_F/ 3 d and older have often had poor sequence results (Reeves et al. ModREPCO1_R and ModREPCO1_F/VertCO1_7216_R primer 2016). Of the 33 bloodmeals 3 d or older not included in the pre- pairs, targeting cytochrome oxidase I gene, were used to amplify sent study, 14 (45%) had poor sequence results. Of the 244 females remaining unidentified vertebrate bloodmeals (Reeves et al. 2018). less than 3 d post-bloodfed, 213 (87.3%) were identified. The hosts Each sample was composed of 2.5 µl of bloodmeal DNA and 22.5 µl identified included 29 bird species, six mammal species, and four of master mix. The master mix was composed of 12.5 µl of Platinum reptile species (Table 1) distributed among 15 orders and 31 families Green 2X Master Mix, 9.0 µl molecular grade water, and 0.5 µl of of vertebrates. Combined, birds contributed the greatest number of 20 µM forward primer and 0.5 µl of 20 µM reverse primer. A neg- bloodmeals (n = 105; 49.3%), followed by reptiles (n = 74; 34.7%), ative control of 2.5 µl of molecular grade water was added. PCR and mammals (n = 34; 16.0%). Amphibian bloodmeals were not were run using published thermocycling conditions for the specific observed. Overall, the largest number of bloodmeals from any primers used (Blosser et al. 2017). single vertebrate species was derived from the brown anole (Anolis PCR products were electrophoresed in a 1% agarose gel and sagrei, n = 47; 22.1%), followed by human (Homo sapiens, n = 27; viewed with a blue light with the aid of SYBR Safe gel stain. A ladder 12.7%), northern cardinal (n = 26; 12.2%), and Carolina anole (5 µl; O’GeneRuler 100 bp DNA Ladder, Thermo Scientific, MA) (Anolis carolinensis, n = 25; 11.7%; Table 1). Dominant host species was placed in the first well followed by 10 µl of PCR product in each varied by county (Table 1), although and no significant difference other well. Each gel was electrophoresed at 100 V for 30–45 min between host class was observed between counties (χ 2 = 4.7; df = 4; and then was transferred to a blue light for recording the presence P = 0.318). 1630 Journal of Medical Entomology, 2020, Vol. 57, No. 5

(0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.9) (0.9) (0.9) (0.9) (0.9) (1.4) (1.9) (0.5) (1.9) (0.5) (1.9) (0.5) (2.8) (0.5) (2.8) (1.4) (3.3) (4.7) (5.2) (0.5) (0.5) (22.1) (34.7) (12.7) (16.0) (12.2) (49.3) (11.7) Total (%) Total 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 3 4 1 4 1 4 1 6 1 6 3 7 1 1 47 74 27 10 34 11 26 25 213 105

(2.7) (2.7) (2.7) (2.7) (2.7) (2.7) (2.7) (2.7) (5.4) (2.7) (8.1) (10.8) (27.0) (18.9) (18.9) (21.6) (51.4) (13.5) Volusia Downloaded from https://academic.oup.com/jme/article/57/5/1627/5819317 by Rutgers University user on 03 June 2021 4

1 1 2 7 7 8

1 3

5 10 37 19

(1.0) (1.0) (1.0) (1.0) (1.0) (1.0) (1.0) (1.0) (1.0) (1.9) (1.9) (1.0) (1.0) (2.9) (2.9) (1.0) (2.9) (3.8) (1.0) (3.8) (1.0) (2.9) (2.9) (7.6) (5.7) (20.0) (32.4) (15.2) (18.1) (49.5) (12.4) Polk 1 1

1 1

1 1 1

2 2 1 1 3

3 1 3

4 1 4 1 3 3 8

13 21 34 16 19 52 105

(9.9) (1.4) (1.4) (1.4) (1.4) (1.4) (1.4) (1.4) (2.8) (1.4) (1.4) (1.4) (1.4) (1.4) (1.4) (2.8) (5.6) (9.9) (4.2) (1.4) (31.0) (42.3) (23.9) (47.9) Orange 7

1 1

1 2

1 1

1 1 2 4

7 3

1 22 30 71 17 34

Resident Resident Resident Resident Resident Winter Winter Winter Resident Winter Winter Resident Winter Resident Winter Resident Winter Winter Winter Resident

Exotic

Resident

Winter

Resident

Summer

Resident

Residency

Passeriformes Passeriformes Passeriformes Cuculiformes Gruiformes Passeriformes Charadriiformes Coraciiformes Strigiformes Passeriformes Passeriformes Passeriformes Pelecaniformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes

Galliformes

Passeriformes

Strigiformes

Passeriformes

Columbiformes

Passeriformes

Order Anolis carolinensis Anolis sagrei

Quiscalus quiscula Poecile carolinensis Poecile Toxostoma rufum Toxostoma Coccyzus americanus Coccyzus Porphyrio martinicus Porphyrio Setophaga coronata Setophaga Sterna forsteri alcyon Megaceryle Megascops asio Megascops Spinus tristis Sayornis phoebe Sayornis Cyanocitta cristata Cyanocitta Botaurus lentiginosus Setophaga americana Setophaga Spizella passerina Geothlypis trichas Geothlypis Turdus migratorius Turdus Vireo solitarius Vireo Regulus calendula Polioptila caerulea Polioptila

Gallus gallus Procyon lotor Procyon Baeolophus bicolor Bos taurus Troglodytes aedon Troglodytes Odocoileus virginianus Setophaga pinus Setophaga Rattus spp. Strix varia Didelphis virginiana Thryothorus ludovicianus Thryothorus Homo sapiens Zenaida macroura

Vireo olivaceus Vireo Basiliscus vittatus Cardinalis cardinalis Coluber constrictor priapus

Scientific name Vertebrate hosts of Culiseta melanura by county Vertebrate

Hosts were identified by bloodmeal analysis on mosquitoes from Orange, Polk, and Volusia County, FL, in 2018. Percentages of column total in parentheses. in 2018. FL, Volusia County, and Polk, Hosts were identified by bloodmeal analysis on mosquitoes from Orange, Carolina anole Brown anole Common grackle Carolina chickadee Brown thrasher Forster’s tern Forster’s Belted kingfisher Eastern screech-owl Eastern phoebe Blue jay Northern parula Chipping sparrow Common yellowthroat Blue-headed vireo Ruby-crowned kinglet Blue-gray gnatcatcher House wren Pine warbler Black/brown rat Barred owl Carolina wren Mourning dove Red-eyed vireo Brown basilisk Northern cardinal Southern black racer Yellow-billed cuckoo Yellow-billed American purple gallinule Yellow-rumped warbler Yellow-rumped American goldfinch American bittern American robin Tufted titmouse Tufted White-tail deer Virginia opossum Virginia

Reptilia

Total Chicken Raccoon

Cattle

Human

Mammalia

Aves Common name Table 1. Table Journal of Medical Entomology, 2020, Vol. 57, No. 5 1631

Seasonality of Host Use Feeding on resident and nonresident songbirds was dynamic Birds were the most commonly bitten host class in every season ex- throughout the year. Significant shifts between resident and nonresi- cept spring, when reptiles were observed to be the most commonly dent birds were detected between winter and spring (P < 0.001) and bitten class of hosts (Fig. 2a). Significant shifts in the distributions summer and fall (P < 0.001), but not spring and summer (P = 0.194) of host class bloodmeals were observed between winter and spring or fall and winter (P = 0.741). Resident bird bloodmeals constituted (P = 0.008), and spring and summer (P < 0.001), attributed to shifts the majority of avian bloodmeals in spring (81.3%) and summer between birds and reptiles. Significant shifts in class-level host use (85.4%), but less than half of total avian meals in fall (40.0%) and were not observed between summer and fall (P = 0.838) or fall winter (41.7%). Bloodmeals from the winter-resident hosts were and winter (P = 0.764). The greatest numbers and proportions of low in spring (6.25%) and not detected in summer (0/41 avian mammal-derived bloodmeals were observed in summer months bloodmeals). The most commonly bitten nonresident birds (n = 2 or (Fig. 2a) of July (n = 9, 26.5%) and August (n = 11, 25.0%). more) were red-eyed vireo (a summer resident), house wren, blue-

headed vireo (Vireo solitarius), ruby-crowned kinglet (Regulus ca- Downloaded from https://academic.oup.com/jme/article/57/5/1627/5819317 by Rutgers University user on 03 June 2021 Avian Host Diversity and Residency lendula), American robin, and chipping sparrow (Spizella passerina; Table 2). Avian bloodmeals of Cs. melanura consisted of nine orders and 22 families of birds (Table 1). Overall, the avian hosts most fed on were northern cardinal, red-eyed vireo, mourning dove, Carolina wren, Reptilian Host Use pine warbler (Setophaga pinus), barred owl (Strix varia), and house Reptile bloodmeals were detected in every month of the year and wren (Troglodytes aedon; Table 1). The largest number of avian contributed 14–62% of monthly bloodmeals (Fig. 2a). The greatest bloodmeals were from songbirds, order Passeriformes (n = 79, 75% proportions of reptile bloodmeals were observed in spring, consti- of avian hosts, 37% of total). The nonsongbird species fed on were tuting 61.5, 48.6, and 50.0% of total bloodmeals in April, May, and mostly mourning dove, barred owl, and chicken (Table 1). Sixteen of June, respectively. Brown anole bloodmeals dominated the reptile the avian host species identified were year-round residents, 12 were hosts (63.5%), followed by Carolina anole (33.8%). Additional nonresidents, and one was an exotic species. single reptile bloodmeals from southern black racer (Coluber constrictor priapus) and a brown basilisk (Basiliscus vittatus) were identified from Orange Co. and Volusia Co., respectively. The brown basilisk bloodmeal identified from the Volusia County Fairgrounds represents the northernmost location recorded for this invasive species (Krysko et al. 2006, iNaturalist 2019).

Mammalian Bloodmeals Bloodmeals from mammals (Table 1) were observed from human (12.7%), Virginia opossum (Didelphis virginiana, 1.4%), invasive rats (Rattus spp., 0.5%), white-tailed deer (Odocoileus virginianus, 0.5%), cattle (Bos taurus, 0.5%), and raccoon (Procyon lotor, 0.5%). Human bloodmeals were among the four most common individual host species fed on 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). Seasonally, human bloodmeals were greatest in July and August (n = 15, 19.2%; Table 2). 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. Of the 277 bloodfed Cs. melanura collected from Orange, Polk, and Volusia counties in 2018, 213 bloodmeals were success- fully 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; Fig. 2a), despite extensive sampling. The Lzrd primer pair 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 VertCOI forward and ModREPCOI reverse primer pair, eight of which were identified as northern cardinal.

Fig. 2. Seasonal host use by Culiseta melanura in central Florida, 2018. (a) Host use by vertebrate class, where lines represent the total bloodmeals taken from birds (blue), reptiles (green), and mammals (black). (b) Avian Discussion host use by seasonal residency, where lines represent the total bloodmeals Bloodmeals from Cs. melanura were collected from central Florida taken from bird species that are year-round residents (purple), summer residents (yellow), and winter residents (light blue). Domestic birds (chickens) throughout 2018. Identified bloodmeals were derived from birds, not shown. reptiles, and mammals, while no amphibian bloodmeals were found. 1632 Journal of Medical Entomology, 2020, Vol. 57, No. 5

Table 2. Vertebrate hosts of Culiseta melanura by month

Common name Jan. Feb. Mar. Apr. May June July Aug. Sept. 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 Downloaded from https://academic.oup.com/jme/article/57/5/1627/5819317 by Rutgers University user on 03 June 2021 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 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

Hosts were identified by bloodmeal analysis of mosquitoes from Orange, Polk, and Volusia County, FL, in 2018.

Host diversity was high, with Cs. melanura feeding on 29 species mourning dove, and Carolina wren. Songbirds are considered the of birds as well as lizards, a snake, humans, opossums, deer, and primary amplification host for EEEV in winter (Burkett-Cadena other mammals. Results from this study support the widely held et al. 2015). The northern cardinal was the third most common idea that Cs. melanura feeds primarily on Passerine songbirds, con- host of Cs. melanura and contributed 12.2% of total bloodmeals. firms a previous report that this mosquito bites reptiles in the spring Culiseta melanura has been shown to feed heavily upon northern (Blosser et al. 2017), and demonstrates that mammals constitute cardinals (Estep et al. 2011, Blosser et al. 2017) and reservoir com- an important host group in summer. Although the total number of petence of northern cardinals for EEEV has been studied. The study avian bloodmeals was greater than those from reptiles or mammals, by Komar et al. (1999) found that the EEEV competence of eight combined reptile and mammal bloodmeals contributed up to 50.7% songbird species varied from high to low (2.0–0.2), with northern of total meals and brown anole lizards and humans were the most cardinals moderately competent (0.8) and two nonsongbird species commonly fed upon individual host species. This study represents (Columbiformes) were poorly competent (0.1). Songbirds have also the first year-round seasonal record of host use by Cs. melanura in had high seroconversion rates for EEEV (Elias et al. 2017). There Florida. were far fewer (1 of 213) bloodmeals from wading birds than in This study found that songbirds (order Passeriformes) repre- some previous studies (Bingham et al. 2014, Molaei et al. 2015a). sented a large portion of all identified bloodmeals (37.1%). Avian In the study by Bingham et al. (2014), 39.6% of Cs. melanura bloodmeals were primarily from northern cardinal, red-eyed vireo, bloodmeals were from wading birds in winter months. Two of the Journal of Medical Entomology, 2020, Vol. 57, No. 5 1633 collection sites of their study were adjacent to a river, whereas in the hosts. Although bloodmeals from birds dominated in summer, present study, only Tibet-Butler Park was near a large body of water. fall, and winter, this study reveals a relatively large amount This difference in habitat may explain the lack of wading birds of feeding on reptiles and mammals by Cs. melanura in the from our data. In the present study, there were also few American southern range of EEEV transmission. A significant shift from robin bloodmeals, in contrast to studies from Virginia (Molaei et al. bird to reptile bloodmeals occurred from winter to spring, and 2015b), Connecticut (Molaei and Andreadis 2006), and New York reptile and/or mammal bloodmeals were detected throughout (Molaei et al. 2006). Weekly mosquito collection from this study the year. Even though horses were present near some sites, no was likely not able to detect the patchy spatial and temporal winter horse bloodmeals were found during the study period. If a large distribution of American robin in Florida. As expected, nonresident population of Cs. melanura occurs in hardwood swamps bor- birds were detected from Cs. melanura mainly in winter, and a signif- dering residential areas or recreational parks, Cs. melanura may icant shift from winter-resident to resident birds was detected during spread EEEV to epizootic hosts or humans. this transition. Downloaded from https://academic.oup.com/jme/article/57/5/1627/5819317 by Rutgers University user on 03 June 2021 The significant change in host use from birds to reptiles in spring could have important implications on EEEV amplification, either Acknowledgments enhancing or suppressing transmission, depending on the compe- We thank the following for their assistance with mosquito sampling: Carl tency of individual reptile species for EEEV. Carolina anoles are not Boohene, Jackson Mosley, Hugo Ortiz Saavedra, and Roger Johnson at considered capable of producing EEEV viremia sufficient to infect Polk County Mosquito Control District; Kelly Deutsch, Rafael Melendez, feeding vectors (White et al. 2011). Noncompetent species such as and others at Orange County Mosquito Control District; and Sue Bartlett, anole lizards may act as dilution hosts in the system by occupying Miranda Tressler, Hong Chen, Drake Falcon, Tia Vasconcellos, and Brandi vector bloodmeals in place of competent species such as songbirds. Anderson at Volusia County Mosquito Control District. This study could However, generalizing across reptile species may not be warranted, not have been done without their cooperation and hard work. We also thank as some snake and turtle species are highly susceptible to EEEV in- Erik Blosser for help with mosquito identifications and Diana Rojas and fection. Six species that have been studied maintained detectable vi- Annsley West for help with field collections. This publication was supported remia up to 2 wk and the garter snake (Thamnophis sirtalis) can by Cooperative Agreement Number U01CK000510, funded by the Centers for Disease Control and Prevention. Its contents are solely the responsibility of maintain a low viremia over winter (Hayes et al. 1964, White et al. the authors and do not necessarily represent the official views of the Centers 2011). Infection of a northern black racer, a subspecies of the black for Disease Control and Prevention or the Department of Health and Human racer, resulted in a viremia which lasted 10–14 d (Hayes et al. 1964). Services. Our detection of a single southern black racer bloodmeal con- firms findings of Blosser et al. (2017) that snakes are bitten by Cs. melanura. If snakes are heavily fed on elsewhere, they may serve References Cited as an amplifying or overwintering host of EEEV. Additional studies Apperson, C. S., H. K. Hassan, B. A. Harrison, H. M. Savage, S. E. Aspen, are needed to assess the roles of reptile species in the transmission A. Farajollahi, W. Crans, T. J. Daniels, R. C. Falco, M. Benedict, et al. of EEEV. 2004. 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