Mapping the distribution of phlebotomine sand fly species with emphasis on Leishmania vectors in Nepal and exploring the potential of DNA barcoding for their identification.

Nepal is committed to eliminating visceral leishmaniasis as a public health problem by 2030. The scattered distribution of VL cases across wide geo-ecological regions, including areas previously considered unsuitable for the survival of vectors and the transmission of the pathogen, poses a major threat to Nepal's national VL elimination programme. Regular monitoring and accurate identification of sand fly species are essential for implementing tailored vector control interventions. Hence, this study aimed to update the distribution of sand fly species with a focus on Leishmania vectors and evaluate DNA barcoding as a complementary tool for their identification. Sand flies were collected from 43 districts with active VL cases across the country between 2017 and 2022. The mitochondrial COI gene was amplified for DNA barcoding analysis. The primary vector, Phlebotomus argentipes, was present in all except three districts. Potential vectors, Ph. (Adlerius) spp. and Ph. major, were found common in high-altitude regions. The species identification success rate of generated COI barcode sequences based on the "Best Close Match" was 97%, indicating high accuracy in delineating sand flies to the species level. The information on the distribution of phlebotomine sand flies and the potential use of DNA barcoding for their identification could be milestones for sand fly research and help to guide the vector control interventions in support of VL elimination in Nepal.