Generation of axenic Aedes aegypti demonstrate live bacteria are not required for mosquito development

Abstract

The mosquito gut microbiome plays an important role in mosquito development and fitness, providing a promising avenue for novel mosquito control strategies. Here we present a method for rearing axenic (bacteria free) Aedes aegypti mosquitoes, consisting of feeding sterilized larvae on agar plugs containing a high concentration of liver and yeast extract. This approach allows for the complete development to adulthood while maintaining sterility; however, axenic mosquito’s exhibit delayed development time and stunted growth in comparison to their bacterially colonized cohorts. These data challenge the notion that live microorganisms are required for mosquito development, and suggest that the microbiota’s main role is nutritional. Furthermore, we colonize axenic mosquitoes with simplified microbial communities ranging from a single bacterial species to a three-member community, demonstrating the ability to control the composition of the microbiota. This axenic system will allow the systematic manipulation of the mosquito microbiome for a deeper understanding of microbiota-host interactions.

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

“…Transient colonisation with auxotrophic bacteria allows the production of germ-free adult mosquitoes After microbiological sterilisation of mosquito eggs, hatchlings provided with autoclaved usual rearing food are able to survive for weeks but remain first-instar larvae; their development can be rescued by bacterial colonisation, for instance with Escherichia coli (Coon et al, 2014;Correa et al, 2018). Therefore, we hypothesised that transient bacterial colonisation would be an efficient way to support larval development and produce germ-free adults.…”

“…The current study allowed the development of a new approach to produce fully germ-free adult mosquitoes and sheds light on the metabolic contribution of the microbiota to larval development. The production of a relevant negative control has long been a technical issue for the study of the microbiota in adult mosquitoes, which was either solved by allowing normal development and treating emerging mosquitoes with antibiotics or by producing stunted germ-free mosquitoes (Correa et al, 2018;de Gaio et al, 2011). However, antibiotic treatments are known to reduce bacterial load and modify microbiota composition rather than produce germfree mosquitoes, and off-target effects cannot be ruled out.…”

“…Therefore, most studies investigating the mosquito microbiota were based on conventional rearing and use of antibiotics at adult emergence, which may have off-target effects and does not fully sterilize mosquitoes. In the past, several studies proposed rearing protocols of germfree larvae, which could not be reproduced in recent experiments (Correa et al, 2018;Golberg et al, 1945;Lang et al, 1972). Lately, mosquito larvae have been shown to develop in the absence of bacteria when provided an enriched diet delivered as bits rather than mashed food, but development time is tripled and germ-free mosquitoes are smaller than their conventional controls (Correa et al, 2018).…”

“…In the past, several studies proposed rearing protocols of germfree larvae, which could not be reproduced in recent experiments (Correa et al, 2018;Golberg et al, 1945;Lang et al, 1972). Lately, mosquito larvae have been shown to develop in the absence of bacteria when provided an enriched diet delivered as bits rather than mashed food, but development time is tripled and germ-free mosquitoes are smaller than their conventional controls (Correa et al, 2018). The exact contribution of the microbiota to larval development is still a conundrum.…”

“…It was proposed that the bacteria growing in the mosquito gut consume oxygen and that the resulting hypoxia is a developmental signal for moulting from one stage to the next . However, successful larval development in fully germ-free conditions challenged this model and metabolic contribution of bacteria to larval development was alternatively proposed (Correa et al, 2018). We developed a transient bacterial colonisation method to support larval development while producing germ-free adults.…”