Outcome of Drosophila microbiota manipulation depends on dietary preservative formula and batch variation in dietary yeast

Gut microbiota are fundamentally important for healthy function in their animal hosts. The fly Drosophila melanogaster is a powerful system for understanding the underlying host/microbe interactions, with modulation of the microbiota inducing phenotypic changes that are conserved across animal taxa. The context-dependence of these responses has not been explored systematically, which may confound repeatability. Here we show that the microbiotas impact on fly triacylglyceride (TAG) levels - a commonly-measured metabolic index - depends on factors in fly media that are rarely considered or controlled, and are not standardized among laboratories: media preservative formula, yeast batch, and the interactive effect of their combinatorial variation. In studies of conventional, axenic and gnotobiotic flies, we found that microbial impacts were apparent only on specific yeast-by-preservative conditions, with TAG levels determined by a tripartite interaction of the three experimental factors. When comparing axenic and conventional flies, we found that preservatives rather than microbiota status was the main driver of variance in host TAG, and certain yeast-preservative combinations reversed microbiota effects on TAG levels. Further, comparisons between TAG levels of axenic flies and those associated with Acetobacter pomorum or Levilactobacillus brevis determined that preservatives, microbiota status, and their interaction were the major drivers of TAG variation. Our results suggest that the microbiota shapes the host TAG response in a manner dependent on the combination of the dietary factors of preservative formulation and yeast batch, with implications for repeatability, interpretation, and optimal experimental standards. ImportanceDrosophila melanogaster is a premier model for microbiome science, which has greatly enhanced our understanding of the basic biology of host-microbe biology. However, often overlooked factors such as dietary composition, including yeast batch variability and preservative formulation used, may cofound data interpretation of experiments within the same lab and lead to different findings when comparing between labs. Our study supports this concept; we find that host TAG levels are not solely dependent on the presence or absence of microbiota members, but rather the combinatorial effects of microbiota members, yeast batch, and preservative formulation used, with preservatives being the largest driver. It serves as a cautionary tale that underappreciated components of fly rearing can mask or drive phenotypes that are believed to be impacted by microbiota members.