Extracellular microbes are required for mosquito development even in the presence of Wolbachia.

Wolbachia, an endosymbiotic bacterium infecting a wide array of invertebrates, has gained attention for its potential in vector control. Its capacity to colonise host populations primarily relies on vertical transmission and reproductive manipulation in arthropods. This endosymbiont is additionally mutualistic in some hosts, across several Wolbachia supergroups; notably, in nematodes and, as recently demonstrated, in planthoppers and bedbugs, it functions as an essential nutritional symbiont by providing vitamins to its host. Since mosquito larvae require microbe-derived nutrients for development, we investigated whether Wolbachia alone can support larval development in Culex quinquefasciatus mosquitoes. Our findings reveal that Wolbachia alone is insufficient to support larval development. Using transient colonisation with Escherichia coli, we developed a protocol to produce adult Culex quinquefasciatus mosquitoes harbouring Wolbachia only (germ-freeWol+). These results suggest that E. coli can support larval development in this species, which typically thrives in murky water; they also underscore the importance of extracellular microbes in larval growth. Furthermore, when Wolbachia infection was suppressed in germ-freeWol+ larvae using tetracycline treatment, we observed enhanced larval development, suggesting that Wolbachia acts as a metabolic parasite. In summary, this study opens the way for gnotobiology research in Culex quinquefasciatus and highlights the intricate interactions between Wolbachia and other members, which collectively influence mosquito development.