Journal of the American Mosquito Control Association, 29(2):102–107, 2013 Copyright E 2013 by The American Mosquito Control Association, Inc. EVALUATION OF METHODS FOR COLLECTING BLOOD-ENGORGED MOSQUITOES FROM HABITATS WITHIN A WILDLIFE REFUGE KRISTINA M. FRIESEN 1,2 AND GREGORY D. JOHNSON1 ABSTRACT. Mortality of American white pelican (Pelecanus erythrorhynchos) chicks attributed to West Nile virus (WNV) prompted field studies on the bionomics of mosquitoes on a wildlife refuge in northern Montana. One component of these studies was to identify blood meal sources for Culex tarsalis, the primary vector of WNV in the region, and the potential bridge vectors Aedes vexans and Culiseta inornata. To accomplish this, 3 methods were evaluated to collect bloodfed mosquitoes: a gasoline powered aspirator, CO2-baited light traps, and fiber pots in shelterbelts consisting of stands of deciduous trees and shrubs and marshes along the lake edge. Fiber pots were also deployed in open fields of prairie grasses. Overall, fiber pots were the most efficient method for collecting engorged Cx. tarsalis and Cs. inornata, largely due to shorter sampling and processing times. Aedes vexans was not collected in fiber pots but was more abundant in aspiration samples than the other 2 species. The optimal location for collecting Cx. tarsalis was dependent on trapping method. Aspirations and fiber pot placements collected more Cx. tarsalis in shelterbelts, while CO2-baited light traps collected more Cx. tarsalis in the marsh habitat. Sixteen avian and 4 mammalian hosts were identified from bloodfed Cx. tarsalis with 46 blood meals derived from birds and 49 from mammals. Aedes vexans and Cs. inornata fed predominantly on white-tailed deer (Odocoileus virginianus) and cattle (Bos taurus), respectively. Humans were identified as hosts in 33% of engorged Cx. tarsalis,4% of engorged Ae. vexans,and18% of engorged Cs. inornata. KEY WORDS Resting fiber pots, aspirator, light trap, American white pelican, West Nile virus, blood meal identification INTRODUCTION tarsalis does not frequent beach shorelines where waterfowl and shorebirds such as pelicans roost An investigation of West Nile virus (WNV) and nest (Lothrop and Reisen 2001). As a result, transmission was initiated at Medicine Lake waterfowl and shorebirds have not been described National Wildlife Refuge (MLNWR) in north- as important components of the transmission east Montana following an outbreak of WNV in cycle of WNV. a colony of American white pelicans, Pelecanus The objectives of this study were 1) to compare 3 erythrorhynchos Gmelin, in 2003. Medicine Lake methods in different habitats for collecting en- NWR provides breeding and resting habitat for gorged specimens of the primary WNV vector Cx. approximately 125 species of resident and migra- tarsalis and bridge or secondary vectors, Aedes tory birds, including the 5th largest breeding vexans (Meigen) and Culiseta inornata (Williston) colony of American white pelicans in North and 2) to identify hosts of these species. America (King and Anderson 2005, Madden and Restani 2005). Following the introduction of WNV into the area in 2002, juvenile mortality within the colony increased from an average of MATERIALS AND METHODS 4% to 32% with an estimated 1,000 deaths in Study area 2003. High mortality associated with WNV was sustained in 2004, and approximately 44% of the Located in the Prairie Pothole Region of the juveniles succumbed. northern Great Plains, Medicine Lake National Regional mosquito surveillance and investiga- Wildlife Refuge (48u279N, 104u239W) is a tions of vector competency (Goddard et al. 2002, 12,829 ha wetland formed by glacial recession. Turell et al. 2005) and WNV prevalence (Bell et In addition to a diverse avian population, the al. 2005) implicated Culex tarsalis Coquillett as refuge supports numerous small and large mam- the primary WNV vector at MLNWR. A critical mals including jackrabbits (Lepus townsendii element when characterizing arthropod-borne Bachman), ground squirrels (Spermophilus spp.), infectious diseases is the identification of hosts deer (Odocoileus virginianus Zimmerman), cattle and host-feeding patterns. Interestingly, land- (Bos taurus L.), horses (Equus ferus Boddaert), scape bionomic studies have indicated that Cx. coyotes (Canis latrans Say), badgers (Taxidea taxus Schreber), and bats (Lasiurus and Myotis 1 Department of Animal and Range Sciences, Mon- spp.). The refuge consists of 2 lakes, totaling tana State University, Bozeman, MT 59717. 3,850 ha of open water; a marsh ecotone 2 Present address: US Department of Agriculture, bordering the lake perimeter; and approximately Agricultural Research Service, Agroecosystem Manage- 7,500 ha of native grasslands and perennial grass ment Research Unit, Lincoln, NE 68583. plantings. 102 JUNE 2013 HOST IDENTIFICATION 103 Mosquito collection traps were hung at a height of 1.5 m and programmed to turn on an incandescent light Mosquito sampling was conducted from July bulb and fan at dusk and to release CO at a rate through September 2005 in 3 habitats around the 2 of 500 ml/min. Light trap containers with adult refuge: the marsh ecotone, grasslands, and mosquitoes were retrieved the following morning shelterbelts. The marsh vegetation included dense and placed in coolers with blue ice while being stands of cattail (Typha spp.), common reed transported to a 220uC freezer at the refuge (Phragmites australis (Cavanilles)), reed canary- headquarters. Each collection was processed grass (Phalarus arundinacea L.), and hardstem separately on a chill table. Mosquitoes were bulrush (Schoenoplectus acutus (Muhlenberg ex ´ counted and identified to species (Darsie and Bigelow) A.Lo¨ve and D. Lo¨ve var. acutus). The Ward 2005). Females were classified as bloodfed grassland vegetation included a mixture of short, if the abdomen was swollen red and gravid if medium, and tall native and exotic grasses (needle swollen white. As part of a larger program in- and thread (Hesperostipa comata (Trinius and vestigating the transmission of WNV, CO2-baited Ruprecht) Barkworth), blue grama (Bouteloua CDC light traps were operated at additional sites gracilis (Willd ex Kunth) Lag ex Griffiths), on the refuge from which engorged females were smooth brome (Bromus inermis Leyss), crested also submitted for host identification. Although wheatgrass (Agropyron cristatum (L.) Gaertner), light traps were operated at 8 locations, only prairie cordgrass (Spartina pectinata Bosc ex collections from a total of 3 traps placed at the 2 Link), and prairie sandreed (Calamovilfa long- shelterbelts and marsh were used for evaluating ifolia (Hooker) Scribner)). The shelterbelts con- trap and habitat types. However, all bloodfed Cx. tained dense stands of Russian olive (Elaeagnus tarsalis, Ae. vexans,andCs. inornata specimens angustifolia L.) and caragana (Caragana arbor- collected from the 8 light traps were included for escens (Lamarck)). host identification. Three methods were evaluated for collecting The average number of unengorged and bloodfed mosquitoes from the different habitats: engorged Cx. tarsalis collected were ln (y + 1) fiber pots (Western Pulp Products Co., Corvallis, transformed and compared with an ANOVA OR), a gasoline powered Agricultural Backpack using habitat type, method, and habitat 3 2-Cycle Aspirator (John W. Hock Co., Gaines- method interaction as main effects. A 1-way ville, FL), and CO2-baited light traps (John W. ANOVA analyzed unengorged and engorged Cx. Hock Co.). Fiber pots, molded from recycled tarsalis collection with respect to fiber pot wood pulp and in the shape of truncated placement (marsh, trees, grass). All statistics were pyramids (height, 28 cm; open end, 28 3 28 cm; performed with RE software (version 2.12, R closed end, 15 3 15 cm) were spray painted flat Development Core Team 2011). black (Komar et al. 1995). Ten pots were randomly placed in the marsh ecotone, 10 pots in each of 2 shelterbelts, and 5 pots in grasslands. Host identification At each location, fiber pots were oriented so that Blood-engorged females collected from the as- the opening faced west or north (i.e., away from pirator, fiber pots, and all light traps were sorted, the sun). Pots were deployed before sunset for 2 identified, and placed individually in microcen- consecutive nights each week. Mosquitoes were trifuge tubes for later assay. Bloodfed specimens collected between 0700–1000 h the next day with were sent to US Department of Agriculture— a mouth aspirator or battery-operated aspirator Agricultural Research Service (USDA ARS) and placed in a cooler with blue ice while being Arthropod-Borne Animal Diseases Research transported to a 220uC freezer at the refuge Unit, Laramie, WY (currently located in Man- headquarters. hattan, KS) and the University of Alabama in Mosquitoes were collected once per week with Birmingham. Species level identification of hosts a gasoline powered Agricultural Backpack 2- was made using polymerase chain reaction– Cycle Aspirator from the marsh ecotone and heteroduplex analysis (Lee et al. 2002). Briefly, shelterbelts. Grasslands were excluded because bloodfed mosquitoes were homogenated in the lack of naturally occurring shaded sites DNAzol-BD solution. The DNA pellets were rendered this habitat an unlikely location for formed after the addition of 80 ml of isopropol to postfeeding resting sites of Cx. tarsalis. Three the solution followed by centrifugation. The aspirations approximately 15–25 min each in DNA pellets were washed with 95% ethanol, duration were