England, M. | bioRxiv Science

bioRxiv2025

Substantial cold tolerance in all life stages of the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae)

In temperate regions, vector-borne disease risk is mediated by cold winter conditions, however, the cold tolerance of key vector taxa remains poorly understood. Culicoides biting midges are the primary vectors of several pathogens of medical and veterinary importance including bluetongue virus, where seasonal cold weather in temperate regions limits midge activity and pathogen transmission. Here, we provide the first comprehensive assessment of cold tolerance across all developmental stages of Culicoides nubeculosus, a widely used laboratory species that is endemic to northern Europe. Eggs, first-instar larvae, fourth-instar larvae, pupae, and adults were exposed to acute (1 h) and extended (6 and 24 h) cold treatments spanning -1 to -18 {degrees}C, with survival, development, emergence, and adult wing size quantified. Culicoides nubeculosus showed substantial but stage-specific cold tolerance, with survival limits of [≤] -18 {degrees}C for eggs, -14 {degrees}C for pupae, -10 {degrees}C for L1 larvae and adults, and -7 {degrees}C for L4 larvae. While the effect of cold exposure duration varied across temperatures and life stages, extended exposure generally reduced survival at lower temperatures. Cold stress caused sublethal effects, including reduced adult emergence when eggs or larvae were exposed and reductions in adult wing size of up to [~]10%, depending on the life stage. These results reveal substantial cold tolerance across the full life history of C. nubeculosus, suggesting that factors beyond temperature influence population phenology. Our findings provide new insights into Culicoides ecology, with implications for seasonal vector population dynamics and arbovirus transmission risk in temperate regions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/692099v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@199de25org.highwire.dtl.DTLVardef@c75202org.highwire.dtl.DTLVardef@1d91e6dorg.highwire.dtl.DTLVardef@15c9984_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2025

Design and optimisation of rapid real-time PCR assays for the detection of key Culicoides species

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.

molecular biology↗