Multiplex PCR for the differentiation of Simulium asakoae and molecular detection of pathogens in the S. asakoae species group in Thailand.

A human-biting black fly, Simulium (Gomphostilbia) asakoae Takaoka & Davies, 1995, together with certain other members of the S. asakoae species group, is of medical and veterinary importance as a vector of various pathogens. However, accurate species identification remains challenging due to the high morphological similarity among related members and the limitations of cox1 barcoding. To address this challenge, this study developed a multiplex PCR assay based on the cox1 gene to differentiate S. asakoae. In addition, diverse pathogens circulating in the S. asakoae species group collected from Chiang Mai Province, northern Thailand, were molecularly detected. The multiplex PCR assay was developed using two allele-specific forward primers combined with universal cox1 primers as an internal control. This assay demonstrated 100% sensitivity and specificity, successfully differentiating S. asakoae from other Thai-related species in the same species group. Pathogen screening of 100 individual specimens and 20 pooled samples of the S. asakoae species group revealed the highest infection rate for Leucocytozoon (14% in individuals and 8.5% minimum infection rate (MIR) in pools), followed by trypanosomatids (10% in individuals and 4% MIR in pools) and filarial nematodes (5% in individuals and 2.5% MIR in pools). Molecular analysis further confirmed the first detection of Leucocytozoon sabrazesi, Trypanosomatidae spp. (including Leptomonas spiculata), Onchocerca sp. Type I, and a possible new Onchocerca sp. in the S. asakoae species group in Thailand. To the best of our knowledge, this study provides the first multiplex PCR assay for the rapid and correct identification of S. asakoae and confirms the circulation of diverse pathogens in the S. asakoae species group, thereby supporting enhanced surveillance and vector control programmes in Thailand.