Integrative molecular identification and mitochondrial genetic diversity of cryptic Anopheles vectors in malaria hotspots of northwestern Thailand

The Anopheles barbirostris complex comprises several sibling species capable of transmitting malaria and lymphatic filariasis and is increasingly recognised as a potential contributor to residual malaria transmission in the Greater Mekong Subregion (GMS). However, the morphological similarity among sibling species complicates accurate species identification and limits understanding of their epidemiological importance. Understanding the species composition and genetic diversity of Anopheles mosquitoes is necessary to improve vector surveillance and support effective malaria control strategies. Therefore, this study assessed the species composition and genetic diversity of Anopheles species complexes in malaria hotspot villages in Tha Song Yang District, Tak Province, northwestern Thailand. Adult Anopheles mosquitoes were collected in two villages in 2024 using CDC light traps and human landing catches. Specimens were identified morphologically using standard taxonomic keys and then characterized using multiplex PCR. Species assignments were validated by ITS2 sequencing and phylogenetic analysis. Mitochondrial cox1 and cox2 sequences were generated for genetic diversity analyses. A total of 385 adult Anopheles mosquitoes representing six species were identified. Anopheles peditaeniatus was the predominant species, accounting for 70.6% (272/385) of the total collection, followed by An. sinensis at 14.0% (54/385). The remaining species were identified as An. dissidens (12.5%, 48/385), An. annularis (1.0%, 4/385), An. wejchoochotei (0.8%, 3/385), and An. minimus (0.5%, 2/385), all of which were detected at low frequencies. ITS2 phylogenetic analysis showed clear clustering of study sequences with reference taxa, supporting species-level identification. In An. dissidens , An. peditaeniatus , and An. sinensis , mitochondrial analyses indicated moderate-to-high haplotype diversity with low nucleotide diversity (Hd = 0.593–0.917; π = 0.0017–0.0076). Tajima’s D values were generally negative, with a significantly negative value for cox2 in An. peditaeniatus ( p < 0.01), consistent with recent population expansion and/or purifying selection. Morphological identification alone was insufficient to reliably distinguish cryptic Anopheles taxa in northwestern Thailand. The integration of ITS2 and mitochondrial markers improved species discrimination and genetic characterization, highlighting the value of molecular approaches for strengthening vector surveillance and supporting targeted malaria control in border endemic areas.