Seasonal dynamics, Leishmania diversity, and nanopore-based metabarcoding of blood meal origins in Culicoides spp. in the newly emerging focus of leishmaniasis in Northern Thailand

Abstract Background Clinical cases of leishmaniasis caused by Leishmania ( Mundinia ) parasites have been increasingly reported in Southeast Asia, particularly Thailand. Recent evidence has shown that Leishmania ( Mundinia ) parasites successfully developed into infective metacyclic promastigotes in Culicoides biting midges, strongly supporting their putative role in disease transmission. However, Culicoides diversity, host preference, and Leishmania prevalence in endemic areas remain largely unknown. Methods We investigated the seasonal dynamics, infection prevalence, and blood meal identification of Culicoides collected from the emerging focus of visceral leishmaniasis in Lampang Province, Northern Thailand, during 2021–2023. Midge samples were molecularly screened for Leishmania using SSU rRNA -qPCR and ITS1 -PCR, followed by Sanger plasmid sequencing, and parasite haplotype diversity was analyzed. Host blood meal origins were comparatively identified using host-specific Cytb -PCRs and a nanopore-based metabarcoding approach. Results A total of 501 parous and gravid females and 46 blood-engorged ones belonging to at least 17 species of five subgenera ( Remmia , Trithecoides , Avaritia , Hoffmania , and Meijerehelea ) and two species groups ( Shortti and Calvipalpis ) were collected with temporal differences in abundance. Leishmania was detected by SSU rRNA -qPCR in 31 samples of at least 11 midge species, consisting of Culicoides oxystoma , C. guttifer , C. orientalis , C. mahasarakhamense , C ( Trithecoides ) spp., C. innoxius , C. shortti , C. arakawae , C. sumatrae, C. actoni , and C. fulvus , with the overall infection prevalence of 5.7%. The latter six species represent the new records as putative leishmaniasis vectors in Northern Thailand. The ITS1 -PCR and plasmid sequencing revealed that Leishmania martiniquensis was predominantly identified in all qPCR-positive species, whereas L. orientalis was identified only in three C. oxystoma samples. The most dominant haplotype of L. martiniquensis in Thailand was genetically intermixed with those from other geographical regions, confirming its globalization. Neutrality test statistics were also significantly negative on regional and country-wide scales, suggesting rapid population expansion or selective sweeps. Nanopore-based blood meal analysis revealed that most Culicoides species are mammalophilic, with peridomestic and wild mammals (cow, pig, deer, and goat-like species) and humans as hosts, while C. guttifer and C. mahasarakhamense fed preferentially on chickens. Conclusions This study revealed seasonal dynamics and sympatric circulation of L. martiniquensis and L. orientalis in different species of Culicoides . Evidence of human blood feeding was also demonstrated, implicating Culicoides as putative vectors of human leishmaniasis in endemic areas. Further research is therefore urgently needed to develop vector control strategies and assess the infection status of their reservoir hosts to effectively minimize disease transmission. Graphical Abstract