Extracellular microbes are required for mosquito development even in the presence ofWolbachia

Wolbachia is an endosymbiotic bacterium infecting a wide array of invertebrates that gained attention for its potential to curb the transmission of vector-borne diseases. Its capacity to colonize arthropod populations is generally driven by vertical transmission and reproductive manipulation. In some insect species, Wolbachia additionally became an essential nutritional symbiont, providing vitamins to its host. As mosquito larvae require microbe-derived vitamins for development, we studied whether such a support of Wolbachia would exist in mosquitoes but be masked by the presence of other microbes. We chose Culex quinquefasciatus species to address this question, as it is highly colonized with Wolbachia . We developed a method to produce Culex quinquefasciatus devoid of extracellular microbiota and demonstrated that Wolbachia alone is insufficient to support larval development. Using transient colonization with Escherichia coli , we managed to produce adult Culex quinquefasciatus harboring Wolbachia only. When curbing Wolbachia infection of these E. coli -colonized larvae via tetracycline treatment, we obtained a higher larval development. Together, our data indicate that Wolbachia does not support development but rather acts here as a metabolic burden, and that E. coli is sufficient for development success even in a species that grows in “dirty” water. This opens the way towards gnotobiology studies in Culex quinquefasciatus and highlights the complex relationships between Wolbachia and its mosquito host. Author summary Wolbachia is a bacterium infecting many invertebrates that gained attention for its potential to curb the transmission of vector-borne diseases, such as dengue. In some insect species, Wolbachia provides vitamins to its host. As mosquito larvae require microbe-derived vitamins for development, we studied whether such a support of Wolbachia would exist in mosquitoes but be masked by the presence of other microbes. We chose Culex quinquefasciatus species to address this question, as it is highly colonized with Wolbachia . We developed a method to produce Culex quinquefasciatus devoid of extracellular microbiota and demonstrated that Wolbachia alone is insufficient to support larval development. We transiently colonized these larvae with Escherichia coli to rescue larval development and to produce adults harboring Wolbachia only. When curbing Wolbachia infection of these E. coli -colonized larvae via tetracycline treatment, we obtained a higher larval development. Together, our data indicate that Wolbachia does not support development but rather acts here as a metabolic burden, and that E. coli is sufficient for development success even in a mosquito species that grows in “dirty” water.