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

Abstract

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 introduce a method 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 allows us to uncouple the study of the microbiota in larvae and adults. In adults, we detect no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, data from our transcriptome analysis and diet supplementation experiments following decolonisation suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development.

Mosquito-bacteria interactions during larval development trigger metabolic changes with carry-over effects on adult fitness

“…In addition to the expected differences between life stages, we found that most of the transcriptional changes were related to metabolic processes. This is consistent with other studies suggesting that bacteria play a nutritional role during larval development in mosquitoes [28,32] and fruit flies [33,34]. This is also in line with the results from a transcriptional comparison of axenic and gnotobiotic Ae.…”

“…It has been proposed that bacteria in the larval gut provide the essential micronutrient riboflavin (vitamin B2), whose lack results in gut hypoxia and developmental arrest [29][30][31]. Other studies suggested that the role of bacteria during larval development is primarily nutritional [28], possibly by contributing to folate biosynthesis and/or by enhancing energy storage [32]. This is consistent with work in Drosophila showing that larval gut bacteria cooperate to establish an integrated nutritional network supporting host growth [33,34].…”

The Axenic and Gnotobiotic Mosquito: Emerging Models for Microbiome Host Interactions

“…aegypti larvae through a process of “transient colonization.” Briefly, axenic larvae are colonized by a genetically modified strain of E. coli that can be removed during larval development, referred to as decolonization. The decolonized larvae go on to produce axenic adult mosquitoes ( Romoli et al, 2021 ).…”

“…Yet, several lines of evidence suggest that the hypoxic model may be incorrect. First, there was no difference in oxygen concentration between colonized larvae and decolonized axenic larvae ( Romoli et al, 2021 ). Here, we show that axenic Ae.…”

“…For instance, larvae reared on a high-density diet of liver and yeast extract have to be reared in the dark to protect a light-sensitive component in the food, presumably a B vitamin ( Hyde et al, 2019a ). Transcriptional data from decolonized axenic larvae showed that folate biosynthesis, folate transport, and thiamine metabolism were all upregulated in comparison with their microbially colonized cohorts, additionally pointing to a B vitamin deficiency in axenic larvae ( Romoli et al, 2021 ). Furthermore, supplementing the diet of axenic larvae with folate increased larval survival 4-fold ( Romoli et al, 2021 ).…”