Simulium ochraceum is recognized as the primary vector of human onchocerciasis in southern Mexico. It can also transmit other pathogens, including Mansonella ozzardii and vesicular stomatitis virus; however, its role in the transmission of bacteria remains unknown. Metagenomic approaches have enabled significant advances in the study of vector bacteriomes, with considerable impact on public health. These studies have identified symbiotic bacteria associated with reduced vector competence, as well as others with potential applications in biological control. However, the bacteriome of S.ochraceum remains uncharacterized.

Therefore, this study examines the bacteriome of S.ochraceum from former onchocerciasis-endemic areas in Mexico, using a 16S rRNA metagenomics analysis. Adult female black flies were collected via humanlanding catches in November 2024 and morphologically identified using taxonomic keys. A pool of 50 individuals were used for genomic DNA extraction with the DNeasy Blood and Tissue Kit. Amplicons of the V4-V4 region were sequenced using the Illumina MiSeq platform. Bioinformatic analysis was conducted using EzBioCloud and CZ ID, which included the estimation and analysis of diversity, and estimation of bacteria associated with human and animal pathogens. The bacteriome was dominated by Pseudomonadota, with abundances of Bacillota, Bateroidota, and Actinomycetota. The identification of diverse bacterial taxa revealed the presence of potential human and animal pathogens, including members of the genera Escherichia, Streptococcus, Acinetobacter, and Rickettsia. Additionally, unclassified bacteria were detected, as well as microorganisms associated with environmental, plant-related, and insect-symbiotic niches. To our knowledge, this is the first report on the bacteriome of S. ochraceum on a global scale. The microbial diversity suggests a complex ecosystem with potential implications for public health, vector ecology, and environmental microbial dynamics.