Metagenomic Exploration of the Bacteriome Reveals Natural Wolbachia Infections in Yellow Fever Mosquito Aedes aegypti and Asian Tiger Mosquito Aedes albopictus.

Dengue and associated complications are spreading to non-endemic regions of Pakistan. Vector control, the foremost and widely adopted strategy for managing dengue has been implemented through various measures in Pakistan. Biological control through the use of Wolbachia, a bacterium naturally present in various insect genera, including Aedes, has demonstrated promising results globally. In this study we collected Aedes species and investigated its microbiomes with a particular focus on identifying the endosymbiont Wolbachia. Mosquitoes were collected via Gravitraps in the Peshawar region of Pakhtunkhwa province in the northwest of Pakistan. The identity of the mosquitoes was initially confirmed through morphological characters followed by molecular identification using species-specific Cytochrome oxidase I (COI) primers. The DNA from female Ae. aegypti and Ae. albopictus was further subjected to 16 S rRNA sequencing. The hypervariable regions V3/V4 of 16 S rRNA were used for sequencing using the paired-end Illumina MiSeq platform. The phylogenetic analysis of the COI gene in our samples demonstrated similarity to Aedes species previously documented in Pakistan. In comparative analysis of the microbiomes, Ae. albopictus was found to harbor 921 bacterial species, while Ae. aegypti only had 239 species. The metagenomic analysis revealed single-strain Wolbachia pipientis infection in Ae. aegypti, while Ae. albopictus harbored a double-strain infection involving a supergroup A strain (referred to as Wolbachia pipientis in 16 S EzBioCloud database) and a supergroup B strain (referred to as Wolbachia bourtzisii in16S EzBioCloud database).