Natural Occurrence and Molecular Diversity of Wolbachia Endosymbiont DNA in Wild Mosquito Populations of Central India

Background: Utilizing Wolbachia for mosquito biocontrol requires baseline understanding of natural infection dynamics, lineage diversity, and host-symbiont phylogenetic concordance across native vector populations. Methods: We collected 2,167 adult mosquitoes ( Anopheles , Culex , Aedes , and Mansonia ) across five districts in central India (Madhya Pradesh and Chhattisgarh). Specimens were screened in 263 genus-level pools via wsp PCR. Positive pools were brought to individuals level, typed across three different loci ( ftsZ , gltA , and GroEL ) of Wolbachia spp., and validated by mosquito barcoding ( cox1 , ITS2). Maximum likelihood phylogenies were constructed for all six loci both for Wolbachia (four) and mosquitoes (two). Results: Overall, 27 pools (10.26%) and 49 individuals (21.21%) were wsp -positive that includes 17 Anopheles , 18 Culex , 9 Aedes , and 5 Mansonia . Culicines exhibited robust multilocus amplification, whereas all 17 Anopheles failed to amplify at ftsZ , gltA , and GroEL . Phylogenetic trees of the mosquito-host cox1 and ITS2 resolved all four genera and five wsp -positive Anopheles species into monophyletic clades with 84–100% bootstrap support. In Wolbachia phylogenies, culicine isolates clustered in Supergroup B ( w Pip), with ftsZ and gltA trees resolving Mansonia symbionts into a well-supported, distinct lineage (95–97% bootstrap). Crucially, the wsp tree revealed that four An. annularis sequences formed a monophyletic clade (89–98% bootstrap), clustering with the bat-fly endosymbiont Penicillidia jenynsii rather than mosquito-adapted strains. Primary malaria vectors ( An. culicifacies and An. fluviatilis ) tested negative for Wolbachia . Conclusions: Phylogenetic divergence and housekeeping gene dropout indicate that wsp detection in wild Anopheles represents transient or horizontal acquisition rather than stable intracellular endosymbiosis. Multilocus phylogenetic validation is critical prior to deploying symbiont-based biocontrol.