AbstractCulicoides biting midges (Diptera: Ceratopogonidae) and #mosquitoes (Diptera: Culicidae) are important vectors of pathogens affecting ruminants. On deer farms, Culicoides species transmit bluetongue virus and epizootic hemorrhagic disease virus, while #mosquitoes can cause chronic stress and even exsanguination. We evaluated the effectiveness of the insecticide ­InsectGuard (0.5% permethrin), applied as a barrier treatment and pour-on, to reduce mosquito and biting midge landings on deer in Martin County, Florida. Deer simulators baited with carbon dioxide and fitted with sticky cards were used to trap landing insects. A polyethylene fence barrier treated with InsectGuard (1.34 fl oz/m2) and an untreated fence were tested against a no-barrier control. Separately, InsectGuard pour-on (1 fl oz per deer) was compared to an untreated control. The ­InsectGuard-treated barrier and pour-on applications reduced landings of three Culicoides species, including Culicoides insignis Lutz, a key bluetongue virus vector, by 175-fold and 7-fold, respectively. The InsectGuard-treated barrier also reduced mosquito landings: #Culex spp. (15-fold), and Psorophora spp. (6-fold). While the InsectGuard pour-on caused a dramatic reduction of C. insignis, it had no measurable effect on #Culex spp., Psorophora spp., or Culicoides floridensis Beck and appeared to attract Culicoides pusillus Lutz (8-fold increase). Our findings demonstrate that permethrin-treated barriers and pour-on applications can reduce the landing and, therefore, potential infectious bites of #mosquitoes and biting midges. These interventions can be incorporated as part of an integrated vector management program for deer farms to enhance control outcomes, in combination with other strategies such as adulticide sprays, habitat management, and vaccination.

In extensive surveillance programmes of Culicoides biting midges (Diptera: Ceratopogonidae), morphological identification can be time-consuming and difficult, while DNA barcoding, although highly accurate, may not be cost-effective or suitable for rapid analysis, as it requires individual specimen processing. To address these limitations, we developed a rapid screening method using real-time PCR assays with either SYBR Green or hydrolysis probe-based detection chemistries. Species-specific primers and, where necessary, hydrolysis probes were designed based on the updated sequences of the ITS2 region of seven Culicoides species. The specificity and efficiency of these assays were validated both in silico and through real-time PCR testing on target and non-target Culicoides species, tested individually and as mixed-species samples. The new real-time PCR assays detect vector species including C. obsoletus, C. scoticus, C. chiopterus, C. dewulfi, C. pulicaris, C. punctatus, and C. impunctatus in pools of individual specimens, with single-specimen sensitivity. The molecular techniques developed in this study provide a valuable tool for accurate and high-throughput Culicoides surveillance, which can be used for year-round monitoring of adult midges in traps and larvae in environmental samples, potentially revealing novel insights into the spatial and temporal turnover of Culicoides species. These methods can be applied to large-scale vector screening programmes involving pooled samples, addressing the limitations of previously described methods used in midge surveillance.