DNA barcoding of sand flies from Chandipura-affected districts of Gujarat, India.

Phlebotomine sand flies are important vectors of various pathogens, including the Chandipura virus (CHPV), a neurotropic rhabdovirus associated with fatal pediatric encephalitis outbreaks in India. Gujarat, a CHPV-endemic region, lacks comprehensive molecular data on sand fly species. Accurate species identification is important for effective vector surveillance and control strategies. 2950 sand flies were collected from 14 districts using aspirators and traps. Morphological identification was performed by using standard taxonomic keys. DNA barcoding of the mitochondrial cytochrome c oxidase subunit I (COI) gene was performed using universal barcoding primers. Sequence alignment, phylogenetic analysis, and genetic divergence calculations were performed through MEGA and BOLD Systems databases. Seven species were identified: Sergentomyia babu, Se. punjabensis, Se. insularis, Se. bailyi, Se. shorttii, Phlebotomus argentipes, and Ph. papatasi. Phylogenetic clustering showed strong bootstrap support (>90%), confirming congruence between morphological and molecular identification. Intraspecific divergence was low (0.5 to 2.0%), while interspecific divergence was comparatively higher (5.4 to 18.9%), indicating a clear "barcode gap" that validated species boundaries. COI DNA barcoding reliably distinguished sand fly species from CHPV-endemic Gujarat. The study highlights the presence of epidemiologically relevant vectors, supports the use of molecular taxonomy in entomological surveillance, and emphasizes the need to expand DNA barcode reference libraries for Indian sand flies.