Acetobacter pomorum in the Drosophila gut microbiota buffers against host metabolic impacts of dietary preservative formula and batch variation in dietary yeast

Abstract

Gut microbiota are fundamentally important for healthy function in animal hosts. Drosophila melanogaster is a powerful system for understanding host-microbiota interactions, with modulation of the microbiota inducing phenotypic changes that are conserved across animal taxa. Qualitative differences in diet, such as preservatives and dietary yeast batch variation, may affect fly health indirectly via microbiota, and may potentially have hitherto uncharacterized effects directly on the fly. These factors are rarely considered, controlled, and are not standardized among laboratories. Here, we show that the microbiota's impact on fly triacylglyceride (TAG) levels-a commonly-measured metabolic index-depends on both preservatives and yeast, and combinatorial interactions among the three variables. 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 caused more variance in host TAG than microbiota status, and certain yeast-preservative combinations even reversed effects of microbiota on TAG. Preservatives had major effects in axenic flies, suggesting either direct effects on the fly or indirect effects via media. However, Acetobacter pomorum buffers the fly against this effect, despite the preservatives inhibiting growth, indicating that this bacterium benefits the host in the face of mutual environmental toxicity. Our results suggest that antimicrobial preservatives have major impacts on host TAG, and that microbiota modulates host TAG dependent on the combination of the dietary factors of preservative formula and yeast batch. IMPORTANCE Drosophila melanogaster is a premier model for microbiome science, which has greatly enhanced our understanding of the basic biology of host-microbe interactions. However, often overlooked factors such as dietary composition, including yeast batch variability and preservative formula, may confound data interpretation of experiments within the same lab and lead to different findings when comparing between labs. Our study supports this notion; we find that the microbiota does not alter host TAG levels independently. Rather, TAG is modulated by combinatorial effects of microbiota, yeast batch, and preservative formula. Specific preservatives increase TAG even in germ-free flies, showing that a commonplace procedure in fly husbandry alters metabolic physiology. This work serves as a cautionary tale that fly rearing methodology can mask or drive microbiota-dependent metabolic changes and also cause microbiota-independent changes.

Keywords: Acetobacter pomorum; Drosophila melanogaster; Levilactobacillus brevis; diet; microbiota.

Fig 1

Metabolic impact of microbiota depends on the combination of yeast batch and preservative formula. (A) and (C) show relative TAG levels in two different experiments, separated by preservative conditions (columns, shown at top), and yeast batch (rows, shown at side). In both experiments, relative TAG was calculated by normalizing TAG density (µg per mg fly wet weight) to the mean of axenic flies without preservatives on yeast A. (B) and (D) show effect size calculations for main effects and interaction terms in the two experiments, color-coded by statistical significance. (A) Comparisons between axenic (Ax) and conventional (Cv) flies show that, on yeasts used in this experiment, relative TAG is reduced only in conventional flies when no preservatives are present. On Yeast A, adding preservative set 2 reversed the sign of the effect of eliminating the microbiota. (B) In the experiment shown in panel A, comparing axenic and conventional flies, preservatives are the biggest source of variance in relative TAG, with both a statistically significant effect (P < 0.05), and the biggest-sized effect. The bacteria-by-preservative interaction is the next biggest-sized effect, suggesting that impacts of eliminating the microbiota are contingent on preservatives. (C) Comparisons of relative TAG between axenic (Ax), Levilactobacillus brevis DmCS003 (Lb), and Acetobacter pomorum DmCS004 (Ap) associated flies show that A. pomorum reduces TAG levels relative to axenic flies in most conditions. Preservative set 2 elevated TAG on both yeasts (noting that it only did so on Yeast B in the first experiment), but A. pomorum abrogated this effect. (D) In the experiment shown in panel C, comparing axenic to monoassociated flies, bacteria and preservatives are equally major contributors to the variance in TAG observed, with their interaction being another significant contributor: again this indicates that the impact of variation in microbiota is contingent on preservatives.