First molecular detection of avian haemosporidian parasites in Leptoconops (Diptera: Ceratopogonidae) biting midges

Avian malaria is a widespread vector-borne disease caused by haemosporidian parasites that can negatively affect avian survival, reproduction, and population dynamics. Mosquitoes, Culicoides biting midges, louse flies, and black flies are recognised vectors of avian haemosporidians. Here, we molecularly investigated the presence of these parasites in Leptoconops (Diptera: Ceratopogonidae), a genus of haematophagous biting midges closely related to Culicoides . A total of 5,417 female Leptoconops collected in Andalusia (southern Spain) during 2024 were morphologically identified, grouped into 203 pools, and screened using nested PCR targeting the avian haemosporidian barcode region of the cytochrome b gene. Positive amplicons were sequenced to estimate parasite prevalence and determine lineage identity. Haemosporidian DNA was detected in eight of the Leptoconops pools analysed. Positive samples corresponded to two of the Leptoconops species examined, including Leptoconops communis and Leptoconops nigrithorax. We identified different lineages corresponding to the three haemosporidian genera analysed: Haemoproteus noctuae (CIRCUM01), Haemoproteus sp. (COLPAL01), Plasmodium matutinum (LINN1), Leucocytozoon sp. COLIV04 and AEMO02. Additionally, two novel lineages were detected and designated as Plasmodium P_LEPTO01and Haemoproteus H_LEPTO01. The known host range associations of these parasite lineages provide insights into the potential avian blood-meal sources of Leptoconops. To our knowledge, this study provides the first molecular detection of avian haemosporidians in Leptoconops biting midges worldwide. Although vector competence has not been demonstrated, these findings suggest interactions between Leptoconops midges and infected avian hosts . The potential role of Leptoconops in the transmission of avian haemosporidian parasites is unknown and merits further investigation.