The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Animal-associated microorganisms (microbiota) dramatically influence the nutritional and physiological traits of their hosts. To expand our understanding of such influences, we predicted bacterial genes that influence a quantitative animal trait by a comparative genomic approach, and we extended these predictions via mutant analysis. We focused on starvation resistance (SR). We first confirmed that SR responds to the microbiota by demonstrating that bacterium-free flies have greater SR than flies bearing a standard 5-species microbial community, and we extended this analysis by revealing the species-specific influences of 38 genome-sequenced bacterial species on SR. A subsequent metagenome-wide association analysis predicted bacterial genes with potential influence on SR, among which were significant enrichments in bacterial genes for the metabolism of sulfur-containing amino acids and B vitamins. Dietary supplementation experiments established that the addition of methionine, but not B vitamins, to the diets significantly lowered SR in a way that was additive, but not interactive, with the microbiota. A direct role for bacterial methionine metabolism genes in SR was subsequently confirmed by analysis of flies that were reared individually with distinct methionine cycle mutants. The correlated responses of SR to bacterial methionine metabolism mutants and dietary modification are consistent with the established finding that bacteria can influence fly phenotypes through dietary modification, although we do not provide explicit evidence of this conclusion. Taken together, this work reveals that SR is a microbiota-responsive trait, and specific bacterial genes underlie these influences. Extending descriptive studies of animal-associated microorganisms (microbiota) to define causal mechanistic bases for their influence on animal traits is an emerging imperative. In this study, we reveal that starvation resistance (SR), a model quantitative trait in animal genetics, responds to the presence and identity of the microbiota. Using a predictive analysis, we reveal that the amino acid methionine has a key influence on SR and show that bacterial methionine metabolism mutants alter normal patterns of SR in flies bearing the bacteria. Our data further suggest that these effects are additive, and we propose the untested hypothesis that, similar to bacterial effects on fruit fly triacylglyceride deposition, the bacterial influence may be through dietary modification. Together, these findings expand our understanding of the bacterial genetic basis for influence on a nutritionally relevant trait of a model animal host.

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