Metabolite exchange within the microbiome produces compounds that influence Drosophila behavior

Animals host multi-species microbial communities (microbiomes) whose properties may result from inter-species interactions; however current understanding of host-microbiome interactions is derived mostly from studies in which is it is difficult to elucidate microbe-microbe interactions. In exploring how Drosophila melanogaster acquires its microbiome, we found that a microbial community influences Drosophila olfactory and egg-laying behaviors differently than individual members. Drosophila prefers a Saccharomyces-Acetobacter co-culture to the same microorganisms grown individually and then mixed, a response mainly due to the conserved olfactory receptor, Or42b. Acetobacter metabolism of Saccharomyces-derived ethanol was necessary, and acetate and its metabolic derivatives were sufficient, for co-culture preference. Preference correlated with three emergent co-culture properties: ethanol catabolism, a distinct volatile emission profile, and yeast population decline. We describe a molecular mechanism by which a microbial community affects animal behavior. Our results support a model whereby emergent metabolites signal Drosophila to acquire its preferred multispecies microbiome.

individually and then mixed, a response mainly due to the conserved olfactory receptor, 23 Or42b. Acetobacter metabolism of Saccharomyces-derived ethanol was necessary, and 24 acetate and its metabolic derivatives were sufficient, for co-culture preference. 25 Preference correlated with three emergent co-culture properties: ethanol catabolism, a 26 distinct volatile emission profile, and yeast population decline. We describe a molecular 27 mechanism by which a microbial community affects animal behavior. Our results 28 support a model whereby emergent metabolites signal Drosophila to acquire its 29 preferred multispecies microbiome. 30 metabolic process. We therefore hypothesized that ethanol catabolism was the 154 emergent metabolic process. 155 We next measured ethanol and acetic acid levels over time (24-156 h) and 156 compared the chemical dynamics to Drosophila preference. Consistent with a acetaldehyde metabolic derivative (2,4,5-trimethyl-1,3-dioxolane), and two unknown 198 metabolites were more abundant in the co-culture relative to the separate-culture 199 were in the range of detected in the co-culture. The defined 9-metabolite mixture was 242 more attractive than all other conditions (Figure 6-figure supplement 5). In sum, 243 acetaldehyde metabolic derivatives and esters are potent Drosophila attractants whose 244 detection may signal the presence of actively metabolizing, multispecies microbial 245 communities. 246 247

Discussion: 248
Here, we have demonstrated how emergent properties of a microbial 249 community-volatile profile, population dynamics, and pH-influence Drosophila 250 attraction, survival, and egg-laying behaviors. Our study is the first to identify the 251 consequences of microbe-microbe metabolic exchange on animal behavior and 252 discovers additional microbial interactions for further mechanistic study ( Figure 1D). 253 Microbe-microbe metabolic exchange generates unique and quantitatively 254 different volatiles from those resulting from individual microbial metabolism (Tables 1 &  255 2, Figure 7). Acetobacter-generated acetate coupled to Saccharomyces-derived 256 alcohols spawn diverse acetate esters (Table 1 & Table 2). We hypothesize that more 257 complex and diverse communities, comprising alcohol-producing yeasts, acetate-258 producing Acetobacter, and lactate-producing Lactobacillus, will generate a wider array 259 of attractive esters (Figure 7). The community of S. cerevisiae, A. malorum, and L. 260 plantarum emitted higher levels of acetoin and attracted Drosophila more strongly than 261 the co-culture of S. cerevisiae and A. malorum ( Figure 1D, Figure 6). Acetoin and 2,3-262 butanedione are formed by an α-acetolactate intermediate in bacteria and directly from 263 acetaldehyde in yeast (52). We therefore hypothesize that communities of yeasts and 264 bacteria may emit high levels of attractive acetaldehyde metabolic derivatives ( Figure  265   7). 266 Drosophila behavioral studies have mostly focused on yeasts, even though 267 Drosophila evolved in a bacterial-rich environment. Our results suggest that non-yeast 268 microorganisms, especially when grown in microbial communities, affect Drosophila 269 behaviors. We reason that additional investigations that couple chemical microbial 270 ecology with Drosophila behavior will herald the discovery of microbe-influenced 271 behaviors and microbial community-generated metabolites. 272 This study demonstrates the coordination of ethanol synthesis and catabolism by 273 S. cerevisiae and Acetobacter, respectively, and the role of ethanol in Drosophila 274 behavior and survival. Non-Saccharomyces Drosophila microbiome members also 275 make ethanol (53) and diverse acetic acid bacteria catabolize ethanol, generalizing our 276 findings to other microbial community combinations. Ethanol can have deleterious or 277 beneficial fitness consequences for Drosophila depending on concentration (54,55) and 278 ecological context (56). Our results show that Drosophila uses products of inter-species 279 microbiome metabolism to detect a community that titrates ethanol concentration 280 optimally for the host. Work that further dissects the consequences of acetic acid and 281 ethanol concentrations on Drosophila biology and investigates other community-level 282 metabolic profiles will be of interest to enrich the chemical and ecological portrait of the Our work raises questions about the consequences of the observed behavior on 285 microbiome assembly and stability in the Drosophila intestine. Drosophila possesses 286 specific and regionalized gut immune responses to the microbiome (57-60) implying a 287 tolerant environment in which privileged microbiome members are maintained and 288 reproduce in the Drosophila intestine. Other work suggests that Drosophila acquires its 289 adult microbiome from exogenous sources, that adult microbiome abundance drops 290 without continuous ingestion of exogenous microorganisms, and that the microbiome 291 can be shaped by diet (19,25,26). As such, a combination of internal mechanisms, 292 exogenous factors, and host behavior likely sculpt the microbiome; determining the 293 relative contribution of each will be important moving forward. Complicating our 294 understanding of the contribution of these factors is the opaque distinction between 295 'microbiome' and 'food', since both are ingested from the environment (61). To dissect 296 the formation and stability of the Drosophila microbiome, the fate of ingested 297 microorganisms needs to be monitored and microbial intestinal reproduction needs to 298 be surveyed as a function of Drosophila behavior, age, immune status, microbiome 299 membership, and nutritional state (e.g. using synthetic diets without yeast; (34, 62)). 300 In sum, our results support a model in which the Drosophila olfactory system is 301 tuned to fruity (e.g., esters) and buttery (several acetaldehyde metabolic derivatives, 302 such as 2,3-butanedione) smelling metabolites promoted by microbe-microbe 303 interactions. We anticipate that accounting for microbial interactions in diverse host-304 microbe studies will lead to new insights into diverse aspects of microbial-animal 305 symbioses.  (Table S1). Mean ± SEM of 12-36 replicates (n=2-6 experiments). Each T-maze replicate uses a technical replicate of a microbial culture and one cohort of Drosophila maintained in separate vials for 3-5 days. Mock (two empty tubes), ACV (25% apple cider vinegar versus water), and benzaldehyde (1% versus paraffin oil (PO)). A one-sample t-test assessed deviance from 0. Symbols: NS p >0.05; * p ≤0.05; ** p ≤0.01; *** p Differences between groups were assessed by ANOVA. A Tukey's multiple comparison test was used to assess significant differences between groups (p≤0.05), as indicated by unique letters.

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Culture were grown at and after peak Drosophila attraction. Differences were assessed by a one-way 396 ANOVA within each culture age. A Tukey's multiple comparison test was used to assess significant

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Drosophila co-culture preference is the mean value of the preference shown in Figure 2B.

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Groupings were based on concentrations of metabolites estimated from pre-ethanol catabolism ( (Table 2) Table 4 contains the concentrations of all mixtures (in 50% AJM). The co-culture (S. cerevisiae and A. malorum, purple bar) was grown for 96 h. The pink bar is metabolite mixture #21 and most closely resembled the co-culture.

Fly maintenance
Fly stocks, genotypes, and sources are listed in Methods The percent composition (v/v) of the metabolite mixtures used in fig. S1 the life sciences. PNAS 110:3229-3236.