Detection of novel Wolbachia strains in Aedes aegypti populations from a recent arbovirus outbreak region in Pune District, Maharashtra, India (2024)

<i>Aedes aegypti</i> mosquitoes are principal vectors of arboviruses such as dengue, chikungunya, and Zika. The intracellular symbiont <i>Wolbachia pipientis</i> is known to inhibit viral replication and induce cytoplasmic incompatibility, making it a promising candidate for biological vector control. While <i>Wolbachia</i> is commonly found in <i>Ae. albopictus</i>, its natural presence in <i>Ae. aegypti</i> remains under debate, particularly in India. This study investigated the presence and diversity of <i>Wolbachia</i> in <i>Ae. aegypti</i> 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 <i>Ae. aegypti</i> and pooled (<i>n</i> = 93) for <i>Wolbachia</i> screening using 16S rRNA and <i>wsp</i> 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. <i>Wolbachia</i> was detected in 11.8% of <i>Aedes aegypti</i> pools. Phylogenetic and similarity analyses identified three distinct clusters: supergroup A (<i>n</i> = 2), B (<i>n</i> = 5), and a divergent F-type strain (<i>n</i> = 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 <i>wAlbA</i> and <i>wAlbB</i> 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 <i>Wolbachia</i>, although individual co-infection could not be confirmed. This study provides the first evidence of naturally occurring <i>Wolbachia</i> supergroups A, B, and a potentially novel F-type in <i>Ae. aegypti</i> from an arbovirus-endemic region of western India. These findings highlight the evolutionary diversity of <i>Wolbachia</i> in local vector populations and underscore the importance of integrating <i>Wolbachia</i> surveillance into vector control strategies.