Population genetic diversity and natural Wolbachia infection in Aedes aegypti from Pakistan

Aedes aegypti, the principal vector of dengue and other arboviruses, is widely distributed in Pakistan, yet its population genetics and endosymbiont status remain poorly characterized. This study aimed to investigate the genetic structure, haplotype diversity, and phylogeographic patterns of Ae. aegypti in dengue-endemic regions of Pakistan, and to screen for natural Wolbachia infections to provide baseline data for surveillance and vector control.

Ovitrap collections were conducted in 2021 across the provinces of Punjab (Bakkar) and Khyber Pakhtunkhwa (Charsadda, DI Khan, Kohat, and two sites within Peshawar: Hayat Abad and Tarnab). Following the morphological identification of adult Ae. aegypti, we extracted genomic DNA from confirmed specimens to amplify and sequence a 658-bp fragment of the mitochondrial cytochrome c oxidase I (COI) gene. Phylogenetic analyses, haplotype network construction, and population differentiation statistics were performed. Additionally, 300 field-caught adult mosquitoes were screened for Wolbachia using validated conventional and quantitative PCR assays targeting the Wolbachia surface protein (wsp) gene.

Phylogenetic analysis of 166 COI sequences (92 from Pakistan) revealed a monophyletic Ae. aegypti clade with 99.65-100% sequence identity, with Pakistani isolates clustering with those from Saudi Arabia, Iran, and India. In total, 13 global haplotypes were identified, with Hap_3 dominating (53%) and shared across regions. Within Pakistan, eight haplotypes were detected, including region-specific variants, yielding high overall diversity (Hd 0.69; π = 0.007). District-level analysis showed that DI Khan and Bakkar had the highest haplotype diversity (Hd 0.73 and 0.71) but low nucleotide diversity (π = 0.005-0.006), whereas Kohat exhibited no haplotype diversity. Population structure was higher in Pakistan (FST 0.26; Nm 0.7) than globally (FST 0.17; Nm 1.19), consistent with low gene flow among Pakistani populations. No natural Wolbachia infections were detected in Ae. aegypti.

Aedes aegypti in Pakistan belong to a globally monophyletic lineage and show moderate mitochondrial diversity with higher population structure than the global population. The lack of detected Wolbachia infections suggests that natural strains are either absent or occur at very low prevalence. These findings provide a baseline for surveillance and support integrating Wolbachia-based biocontrol alongside conventional interventions in Pakistan.