Haemoproteus spp. and Culicoides spp.: insights into their relationships and challenges of research, with the confirmation of Culicoides minutissimus as a competent vector of a new Haemoproteus sp. lineage.

Haemoproteus parasites, transmitted by Culicoides biting midges, can cause severe or lethal infections in birds, particularly in immunologically naïve individuals. Although blood-stage studies of haemosporidians are extensive, vector research remains limited, often relying on molecular methods that cannot confirm parasite development, whereas experimental infections are rare. The goal of this research was to investigate the presence of Haemoproteus parasites in wild-caught Culicoides, determining parasite lineages infecting these vectors and monthly prevalence of infections. In total, 1252 parous Culicoides females belonging to 13 species collected at six locations in Lithuania were dissected and screened using PCR. Individuals positive by PCR were examined microscopically to determine parasite developmental stage. The most abundant species was Culicoides kibunensis (31.8%). Parasite DNA was found in 6.9% of investigated Culicoides and 46.5% of PCR-positive individuals had sporozoite stage confirmed in salivary glands by microscopy. Overall, 19 genetic lineages of Haemoproteus (six new) and one of Plasmodium were identified. Sporozoites in salivary glands were confirmed for 12 genetic lineages; eight were found for the first time, seven in C. kibunensis and one in Culicoides minutissimus (first report as a competent vector of Haemoproteus parasites). June had the lowest infection prevalence (4.7% PCR-positive; 1.2% microscopy positive), July - the highest rate of PCR-positive individuals (9.5% PCR positive; 4.0% microscopy positive), and August - the highest sporozoite detection rate (8.6% PCR-positive; 5.8% microscopy positive). By combining PCR-based screening with microscopy, this study reveals active and seasonally variable transmission of multiple Haemoproteus lineages by Culicoides under natural conditions.