COI DNA barcoding of phlebotomine sand flies from the Pamplonita River basin, Norte de Santander, Colombia.

Leishmaniasis remains a major neglected tropical disease in the Americas, where transmission involves diverse parasites, reservoirs, and phlebotomine sand fly vectors. Accurate sand fly identification is essential for surveillance, but morphology-based diagnosis may be difficult in closely related taxa and isomorphic females. This study evaluated COI DNA barcoding for identifying sand flies from the Pamplonita River basin, Colombia. Adult sand flies were collected in three ecological settings using Centers for Disease Control and Prevention (CDC) light traps, Shannon traps, and active searching. Specimens were morphologically examined and processed for DNA extraction, cytochrome c oxidase subunit I (COI) amplification, and bidirectional sequencing. Sequences were edited, aligned, screened for stop codons and indels, and analyzed using diversity estimates, Kimura two-parameter (K2P) distances, neighbor-joining (NJ), and PhyML maximum-likelihood (ML) clustering under the General Time Reversible model with invariant sites and gamma-distributed rates (GTR+I+G) with 1,000 bootstrap replicates. Sixty-two study sequences representing seven species were analyzed with three external sequences. The curated alignment contained 65 sequences of 660 bp, with no gaps or ambiguous bases. The study dataset included 24 haplotypes and 185 polymorphic sites. Mean interspecific K2P distances ranged from 3.55% between Pintomyia (Pi.) longiflocosa and Pi. torvida to 19.82% between Psathyromyia (Pa.) shannoni and Pi. evansi. Neighbor-joining and maximum-likelihood analyses recovered species-level clusters consistent with morphological assignments. COI barcoding supported the molecular discrimination of medically relevant sand flies from northeastern Colombia and provides a regional baseline for vector surveillance in an area of epidemiological interest.