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Background   Wolbachia  are intracellular bacterial endosymbionts found in most insect lineages. In mosquitoes, the influence of these endosymbionts on host reproduction and arboviral transmission has spurred numerous studies aimed at using  Wolbachia  infection as a vector control technique. However, there are several knowledge gaps in the literature and little is known about natural  Wolbachia  infection across species, their transmission modes, or associations between various  Wolbachia  lineages and their hosts. This study aims to address these gaps by exploring mosquito- Wolbachia  associations and their evolutionary implications.    Methods  We conducted tissue-specific polymerase chain reaction screening for  Wolbachia  infection in the leg, gut and reproductive tissues of wild mosquitoes from Singapore using the  Wolbachia  surface protein gene ( wsp)  molecular marker. Mosquito- Wolbachia  associations were explored using three methods—tanglegram, distance-based, and event-based methods—and by inferred instances of vertical transmission and host shifts.    Results  Adult mosquitoes (271 specimens) representing 14 genera and 40 species were screened for  Wolbachia . Overall, 21 species (51.2%) were found positive for  Wolbachia , including five in the genus  Aedes  and five in the genus  Culex . To our knowledge,  Wolbachia  infections have not been previously reported in seven of these 21 species:  Aedes  nr.  fumidus ,  Aedes annandalei ,  Uranotaenia obscura ,  Uranotaenia trilineata ,  Verrallina butleri ,  Verrallina  sp. and  Zeugnomyia gracilis .  Wolbachia  were predominantly detected in the reproductive tissues, which is an indication of vertical transmission. However,  Wolbachia  infection rates varied widely within a mosquito host species. There was no clear signal of cophylogeny between the mosquito hosts and the 12 putative  Wolbachia  strains observed in this study. Host shift events were also observed.    Conclusions  Our results suggest that the mosquito- Wolbachia  relationship is complex and that combinations of transmission modes and multiple evolutionary events likely explain the observed distribution of  Wolbachia  diversity across mosquito hosts. These findings have implications for a better understanding of the diversity and ecology of  Wolbachia  and for their utility as biocontrol agents.

Wolbachia infection in wild mosquitoes (Diptera: Culicidae): implications for transmission modes and host-endosymbiont associations in Singapore