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

Abstract 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 °C, with survival, development, emergence, and adult wing size quantified. Culicoides nubeculosus showed substantial but stage-specific cold tolerance, with survival limits of ≤ −18 °C for eggs, −14 °C for pupae, −10 °C for L1 larvae and adults, and −7 °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. Competing Interest Statement The authors have declared no competing interest.