03252nas a2200301   4500000000100000008004100001260001200042653002200054653001500076653001500091653001500106653002700121653002000148100001900168700001300187700002000200700001800220700001500238700001200253700001900265700002100284245021400305856015300519300000900672490000800681520224700689022001402936       2026                            d  c02/202610aAvian trypanosome10aCulicoides10aLeishmania10aMicroscopy10aNanopore metabarcoding10aTrypanosomatids1 aTepboonrueng P1 aLerona P1 aSrichareonsuk C1 aPromrangsee C1 aBoonserm R1 aAmpol R1 aSiriyasatien P1 aPreativatanyou K00aTrypanosomatid detection in Culicoides biting midges (Diptera: Ceratopogonidae) from leishmaniasis-endemic Songkhla Province, Southern Thailand: Microscopy and nanopore metabarcoding reveal parasite diversity.  uhttps://www.sciencedirect.com/science/article/pii/S1567134826000365/pdfft?md5=89a0225415864dd9378c6be2f8bd3705&pid=1-s2.0-S1567134826000365-main.pdf  a1-130 v1393 a<p>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&nbsp;=&nbsp;195) and blood-engorged (n&nbsp;=&nbsp;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, cox1-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.</p>
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