Trypanosomatid detection in Culicoides biting midges (Diptera: Ceratopogonidae) from leishmaniasis-endemic Songkhla Province, Southern Thailand: Microscopy and nanopore metabarcoding reveal parasite diversity

Leishmaniasis caused by Leishmania ( Mundinia ) spp. has recently emerged in Southeast Asia, including Thailand. Although molecular studies have detected Leishmania DNA in Culicoides midges, complete parasite development, which proves vector competence, remains undemonstrated in field specimens. We collected 215 parous/gravid ( n = 195) and blood-engorged ( n = 20) female Culicoides representing at least 10 species from two leishmaniasis foci in Songkhla Province, Southern Thailand. Specimens underwent microscopic dissection, culturing in Schneider's medium, ITS1/SSU rRNA-PCR for Leishmania /trypanosomatids, cox 1-PCR for blood meals, and MinION® nanopore metabarcoding. Microscopy revealed live metacyclic trypomastigotes in one C. guttifer , which was confirmed as avian Trypanosoma bennetti -related by both culture and phylogenetics. No Leishmania promastigotes were observed, yet L. martiniquensis and L. orientalis were molecularly detected in C. guttifer , C. huffi , C. ( Trithecoides ) spp., and C. orientalis with an overall prevalence of 16.3%. Trypanosoma bennetti -related sequences and monoxenous trypanosomatids ( Herpetomonas and Critidia ) were also identified (7.0% combined). Chickens were primary hosts for C. guttifer , C. huffi , and C. mahasarakhamense , with opportunistic human feeding in C. guttifer . These findings reveal that nanopore metabarcoding enables the detection of low-abundance, multi-parasite communities, the resolution of haplotype diversity, and the identification of blood meal sources. However, microscopy remains essential for differentiating viable trypanosomatid infections from non-viable, residual DNA that persists across gonotrophic cycles. While Leishmania DNA detection suggests that the midges feed on infected hosts, the absence of microscopically observable promastigotes prevents confirmation of vector competence. Conversely, natural T. bennetti -related trypomastigotes confirm C. guttifer as a competent vector for avian trypanosomes. These findings establish an integrated microscopy-metabarcoding approach for trypanosomatid surveillance in Thai Culicoides , providing baseline data on parasite detection and host feeding patterns in emerging leishmaniasis foci. • First natural Trypanosoma bennetti -related trypomastigotes were identified in field-collected C. guttifer , confirming vector competence for this avian parasite. • Leishmania ( Mundinia ) spp. DNA was detected in 16.3% of Culicoides from two Songkhla leishmaniasis foci using nanopore metabarcoding. • T. bennetti -related trypanosomes and monoxenous trypanosomatids ( Herpetomonas and Crithidia ) were identified (combined prevalence of 7%). • Metabarcoding reveals opportunistic human blood-feeding in primarily ornithophilic C. guttifer . • Microscopy remains essential to distinguish viable trypanosomatid infections from residual DNA, confirming vector competence.