Microbial communities of the Southeast Asian black flies (Diptera: Simuliidae) based on multiple hypervariable regions of 16S rRNA

Abstract Black flies (Diptera: Simuliidae) are important vectors of disease-causing agents, but little is known about their microbiome in Southeast Asia, highlighting the need for further investigation. In Malaysia, Simulium cheongi, Simulium jeffreyi, and Simulium vanluni are among the most abundant black fly species and are of potential medical importance, making them suitable representatives for microbiome studies. In this study, their bacterial communities were characterized using next-generation sequencing (NGS) targeting seven hypervariable regions (V2, V3, V4, V6 to V7, V8, and V9) of the 16S rRNA gene to enable comprehensive community profiling. The alpha diversity of the bacterial community showed the highest values of Shannon and Simpson indices in S. jeffreyi and increased values of observed species and Chao1 indices in S. cheongi. Males showed greater microbial diversity than females in the alpha diversity analysis, with all alpha rarefaction plots reaching a plateau. Moreover, the beta diversity of the microbial communities measured by Bray-Curtis distance indices revealed three PCs coordinates with 63.75% of the total variance. However, no significant differences in alpha and beta diversity indexes were found among the three species. The bacterial composition included six phyla, 15 classes, 37 orders, 78 families, 143 genera, and 216 bacterial species, with the V3 region having the highest taxonomic identification. The V9 region had the least detection at all taxonomic levels, emphasizing the importance of selecting appropriate hypervariable regions to accurately assess the diversity of black fly bacterial communities.