Valcarcel Olmeda, A. | bioRxiv Science

bioRxiv2026

Molecular identification of vertebrate blood meals in Culicoides biting midges reveals host-vector associations relevant to arbovirus transmission in Ireland

Understanding the vertebrate hosts exploited by Culicoides biting midges is fundamental to assessing the transmission risk of livestock arboviruses. Blood meal analysis provides direct evidence of host-vector interactions under natural conditions; however, molecular data on Culicoides host feeding are lacking in Ireland. This study aimed to identify the vertebrate blood meal hosts of Culicoides species of veterinary importance and establish host-vector associations relevant to arbovirus transmission. A total of 115 morphologically blood-fed Culicoides females collected from 13 sites across Ireland were analysed using a sequential molecular workflow. Species-specific real-time PCR assays were used to screen for vertebrate hosts, with positive detections confirmed by mitochondrial DNA sequencing. Blood-fed Culicoides were subsequently identified to species level using DNA barcoding, enabling direct linkage between vector species and their vertebrate hosts. Vertebrate DNA was detected in 50 specimens, with cattle and horses accounting for the majority of identified blood meals. Culicoides obsoletus accounted for most cattle (n = 12) and horse (n = 11) blood meals. Culicoides scoticus fed on cattle (n = 6), horses (n = 4) and sheep (n = 1), while C. dewulfi and C. chiopterus fed predominantly on cattle, with occasional horse blood meals. Four cattle blood meals were identified from C. punctatus. No cervid-derived blood meals were detected. This study provides the first molecular characterisation of Culicoides blood meal hosts in Ireland, confirming host-vector associations between major vector species and livestock hosts of veterinary importance. Although the number of successfully characterised blood meals was modest, reflecting the inherent difficulty of recovering recently blood-fed Culicoides from field collections, the findings establish an important baseline for Ireland. The sequential molecular workflow provides a practical framework for future blood meal investigations and will support improved understanding of host-vector interactions relevant to arbovirus surveillance and risk assessment.

molecular biology↗

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↗