Molecular survey of the haemosporidians Haemoproteus and Leucocytozoon in Culicoides (Diptera: Ceratopogonidae) from the Brazilian Amazon

Haemosporidians belonging to the subgenus Haemoproteus (Parahaemoproteus) and the genus Leucocytozoon rely on dipteran vectors for transmission, with biting midges (Culicoides spp.) and black flies (Simuliidae), respectively, playing essential roles in their life cycles. However, little is known about the potential role of Culicoides species as vectors for haemosporidians outside Europe and Asia. This study aimed to investigate the presence of Haemoproteus spp. and Leucocytozoon spp. DNA in Culicoides spp. from the Brazilian Amazon. A total of 345 midges (95 Culicoides foxi, 218 Culicoides hylas, and 32 Culicoides leopoldoi) were collected between February 2022 and February 2023 in the Amazon National Park, Pará. Specimens were morphologically identified, and DNA was extracted using the TRIzol technique. PCR targeting the endogenous cox-1 gene confirmed successful DNA extraction in 86.7% (299/345) of samples. Nested PCR assays targeting the cytB gene of Haemoproteus spp. and Leucocytozoon spp. did not detect DNA in any sample. The absence of detection suggests that the sampled Culicoides species are unlikely vectors, possibly due to feeding preferences or ecological limitations. Future studies targeting both DNA and sporozoites in salivary glands of engorged female Culicoides spp. are needed to clarify the vectors involved in the transmission of haemosporidians.