Abstract
Aedes aegypti mosquitoes are principal vectors of arboviruses such as dengue, chikungunya, and Zika. The intracellular symbiont Wolbachia pipientis is known to inhibit viral replication and induce cytoplasmic incompatibility, making it a promising candidate for biological vector control. While Wolbachia is commonly found in Ae. albopictus, its natural presence in Ae. aegypti remains under debate, particularly in India. This study investigated the presence and diversity of Wolbachia in Ae. aegypti mosquitoes collected from 21 locations across Pune district, Maharashtra, during a 2024 arbovirus outbreak. A total of 1,020 adult mosquitoes and 1,000 larvae and pupae were morphologically and molecularly confirmed as Ae. aegypti and pooled (n = 93) for Wolbachia screening using 16S rRNA and wsp gene-specific PCRs. Positive samples were sequenced and subjected to phylogenetic and intergenomic similarity analyses. Simultaneously, dengue, chikungunya, and Zika virus screening was conducted via RT-qPCR. Entomological indices were calculated to assess vector density. Wolbachia was detected in 11.8% of Aedes aegypti pools. Phylogenetic and similarity analyses identified three distinct clusters: supergroup A (n = 2), B (n = 5), and a divergent F-type strain (n = 2). Nucleotide gene sequence similarity analysis corroborated the phylogenetic structure, showing high intra-supergroup similarity and low inter-supergroup similarity, consistent with deep evolutionary divergence among supergroups. Supergroup A and B sequences exhibited close affinity to known wAlbA and wAlbB lineages, respectively, whereas the F-type sequence formed a distinct cluster with low intergenomic similarity to A and B members, indicating a divergent lineage. Dengue virus RNA was detected in two pools, one co-occurring with Wolbachia, although individual co-infection could not be confirmed. This study provides the first evidence of naturally occurring Wolbachia supergroups A, B, and a potentially novel F-type in Ae. aegypti from an arbovirus-endemic region of western India. These findings highlight the evolutionary diversity of Wolbachia in local vector populations and underscore the importance of integrating Wolbachia surveillance into vector control strategies.