A Multiplex Pcr Based On Mitochondrialcoisequences For Identification Of Members Of Theanopheles Barbirostriscomplex (Diptera: Culicidae) In Thailand And Other Countries In The Region

A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand:Anopheles barbirostriss.s.,An. dissidens,An. saeungae,An. wejchoochoteiandAn. barbirostrisspecies A3.Anopheles campestriswas not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucleotide segments ofCOIsequences of each species. Mismatch method and addition of long GC tail were applied for some primers. The assay provided products of 706 bp forAn. barbirostriss.s., 238 bp forAn. dissidens, 611 bp forAn. saeungae, 502 bp forAn. wejchoochoteiand 365 bp forAn. barbirostrisA3. The assay was tested using 111 wild-caught female mosquitoes from Bhutan, Cambodia, Indonesia (Sulawesi) and Thailand. The results of the multiplex PCR were in complete agreement withCOIsequencing; however, one of three specimens from Bhutan and all 11 specimens from Indonesia were not amplifiable by the assay due to their distinctCOIsequences. This, together with the distinct rDNA sequences of these specimens, suggests the presence of at least two additional new species in the Barbirostris Complex.