Production of germ-free mosquitoes via transient colonisation allows stage-specific investigation of host-microbiota interactions

The mosquito microbiota impacts the physiology of its host and is essential for normal larval development, thereby influencing transmission of vector-borne pathogens. Germ-free mosquitoes generated with current methods show larval stunting and developmental deficits. Therefore, functional studies of the mosquito microbiota have so far mostly been limited to antibiotic treatments of emerging adults. In this study, we developed a novel approach to produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with bacteria genetically modified to allow complete decolonisation at any developmental stage. We show that, unlike germ-free mosquitoes previously produced using sterile diets, reversibly colonised mosquitoes show no developmental retardation and reach the same size as control adults. This allowed us to uncouple the study of the microbiota in larvae and adults. In adults, we detected no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, we performed a transcriptome analysis and diet supplementation experiments following decolonisation during the third larval instar. Our data suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a novel tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development.