Gut yeasts accelerate chill coma recovery in <i>Drosophila melanogaster</i>

The role of microbial symbionts in host stress tolerance remains underexplored. Gut microbiome studies in Drosophila melanogaster have largely focused on bacteria, whereas yeasts have been assumed to provide nutrition rather than engage in true symbiosis. We explored the effect of gut yeasts on chill coma recovery time (CCRT, a proxy for cold tolerance) and its yeast species-specificity and dependence on live yeast cells. We generated flies with distinct gut microbiota conditions: axenic (microbe-free), with their native microbiota (derived from the microbes associated with flies conventionally reared in our colony), or gnotobiotic flies mono-associated with either live or heat-killed yeasts (Saccharomyces cerevisiae - not normally associated with Drosophila guts, and three species previously isolated from wild flies - Lachancea kluyveri, Pichia kluyveri, or P. nakasei). We quantified yeast abundance, sex differences in yeast ingestion, and measured CCRT after exposure to 0 °C for 8 hours. Female axenic flies recovered 42% more slowly than those with their native microbiota, but this delay was fully rescued by live L. kluyveri, P. kluyveri, or P. nakasei, not by S. cerevisiae or dead yeasts. The effect was rapid (occurs within 48 h), sex-specific (restricted to females), and appeared to be dose-dependent. We also confirmed that yeasts in the gut are alive, facilitating a true (albeit transient) symbiotic interaction. Our findings show that yeast symbionts may contribute to natural variation in thermal tolerance and may broadly impact host phenotypes. Excluding yeasts or assuming a solely nutritional role risks overlooking key symbiotic interactions that have profound functional consequences.