Molecular species delimitation analysis of Leptotrombidium spp. and other chigger species parasitizing birds in Malaysia.

Trombiculid mites (Acariformes) are unique among arthropods of medical importance in that only the larval instar (chigger) is parasitic, which can result in the transmission of zoonotic scrub typhus. The use of molecular approaches for chigger species discrimination has been very limited until recently, especially for those parasitizing bird hosts, where data remain scarce. Here, we aimed to generate DNA barcodes of chiggers parasitizing birds in Malaysia based on the mitochondrial cytochrome c oxidase subunit I (COI) gene following DNA extraction, PCR and sequencing. Fifty-four COI sequences from 8 bird-associated chigger species in Malaysia were combined with 50 GenBank sequences comprising 7 genera from various countries for DNA barcode and phylogenetic analysis. The correct identification rates for the 95 COI barcodes were 96.84% (Best Match) and 86.31% (Best-Close Match). DNA barcode analyses effectively clustered the 8 nominal species from this study into their respective genera. Genetic divergence of less than 3% was observed within Ascoschoengastia lorius, Neoschoengastia gallinarum, Parascoschoengastia heynemani, Leptotrombidium imphalum, and Blankaartia acuscutellaris, all of which formed a monophyletic clade, confirming their conspecific nature. Conversely, intraspecific divergences of 17.64%, 15.49%, and 11.63% were obtained for Toritrombicula densipiliata, Odontacarus audyi, and Leptotrombidium deliense. These divergences, supported by evidence of distinct entities through delimitation analyses, indicate potential cryptic diversity within these populations. In conclusion, this study represents the first molecular genetic analysis of bird chiggers in Malaysia, revealing varying levels of genetic divergence. Our findings highlight the utility of DNA barcoding for understanding chigger diversity and aiding in accurate identification.