Metabolite exchange between microbiome members produces compounds that influence Drosophila behavior
- Yale University, United States
- University of Georgia, United States
- University of Connecticut, United States
Figures
Figure 1 with 1 supplement
Drosophila detection of microbe-microbe metabolite exchange.
(A) T-maze setup and quantification. (B) Drosophila behavior toward yeasts (blue), acetic acid bacteria (red), and lactic acid bacteria (brown) (Supplementary file 2). Mean ± SEM of 12–36 replicates …
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Figure 1—source data 1
Raw Drosophila preference data for Figure 1B,C.
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Figure 1—source data 2
Raw Drosophila preference data for Figure 1D.
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Figure 1—source data 3
Raw Drosophila preference data for Figure 1E.
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Figure 1—figure supplement 1
Drosophila melanogaster olfactory behavior toward different culture volumes of Saccharomyces cerevisiae and Acetobacter malorum.
The top three experimental groups are controls: Mock (empty tube versus empty tube) recapitulates alternating of test and control arms, as in all experimental groups; apple cider vinegar (ACV [25% …
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Figure 1—figure supplement 1—source data 1
Raw Drosophila preference data for Figure 1—figuresupplement 1.
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Figure 2 with 1 supplement
Drosophila temporal preference for metabolite exchange.
(A) S. cerevisiae and A. malorum viable populations. Mean ± SEM of 2–3 experiments with one pooled replicate (2–3 cultures from the same colony) per experiment. Limit of detection is 20 CFU/mL. A …
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Figure 2—source data 1
Raw Drosophila preference data for Figure 2B & Figure 2—figure supplement 1C.
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Figure 2—source data 2
Raw Drosophila preference data, microbial population data, and pH data for Figure 2A,C,D & Figure 2—figure supplement 1A,B.
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Figure 2—figure supplement 1
Properties of the co-culture and its relationship to Drosophila preference.
(A) pH of experimental groups as a function of microbial growth time. Mean pH ± SEM of three experiments with one pooled replicate per experiment. (B) Relationship between A. malorum populations and …
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Figure 2—figure supplement 1—source data 1
Raw Drosophila preference data and microbial population data for Figure 2—figure supplement 1D,E.
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Figure 3 with 1 supplement
Role of olfactory receptor mutants in Drosophila detection of inter-species microbial interactions.
(A) The mean rank of the response index of the various Drosophila mutants toward the co-culture was compared with the mean rank of wild-type fly behavior toward the co-culture using the …
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Figure 3—source data 1
Raw Drosophila preference data and microbial population data for Figure 3A and Figure 3—figure supplement 1.
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Figure 3—source data 2
Raw Drosophila preference data for Figure 3B.
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Figure 3—figure supplement 1
Effect of co-culture age on Drosophila attraction and microbial density.
(A) Attraction of wild-type Drosophila to different aged co-cultures (grown 67–163 hr, S. cerevisiae and A. malorum). Mean ± SEM of 12–24 replicates per group (n = 2–4 experiments). A one-way ANOVA …
Figure 4 with 1 supplement
Drosophila behavior and ethanol catabolism.
(A) Dynamics of ethanol, acetic acid, and Drosophila co-culture preference. Acetic acid was only detected in the co-culture. The abundance was derived from a linear regression calculated from …
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Figure 4—source data 1
Raw spectral abundance data associated with metabolites graphed in Figure 4A.
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Figure 4—source data 2
Raw Drosophila preference data for Figure 4B.
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Figure 4—source data 3
Raw Drosophila preference data for Figure 4C.
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Figure 4—figure supplement 1
Drosophila behavior toward the co-culture using A. malorum or A. pomorum.
Drosophila behavior toward co-cultures grown for 96 hr using A. malorum or A. pomorum versus a media control (AJM = apple juice medium). Result of two experiments with six replicates each. Data …
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Figure 4—figure supplement 1—source data 1
Raw Drosophila preference data for Figure 2—figure supplement 1.
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Figure 5 with 1 supplement
Acetaldehyde metabolic derivatives as attractive microbial community generated metabolites.
(A) Representative chromatogram of m/z 88.05 in the tri-culture (S. cerevisiae-A. malorum-L. plantarum) compared to the co-culture (S. cerevisiae and A. malorum). (B) Estimated quantification is …
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Figure 5—source data 1
Extracted ion current for m/z 88.05 in Figure 5A.
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Figure 5—source data 2
Peak areas associated with acetoin for Figure 5B.
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Figure 5—source data 3
Raw Drosophila preference data for Figure 5C.
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Figure 5—figure supplement 1
Acetoin linear regression.
The curve is based on maximum m/z values (88.05) of three concentrations of acetoin. One replicate per concentration (n = 1 experiment). The linear regression was used to estimate acetoin …
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Figure 5—figure supplement 1—source data 1
Extracted ion current for Figure 5—figure supplement 1.
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Figure 6 with 2 supplements
Drosophila behavior toward 21 metabolite mixtures .
(A) Supplementary file 5 contains the concentrations of all mixtures (in 50% AJM). The co-culture was grown for 96 hr. Mean ± SEM of 4–6 replicates per experimental group. Groups were tested over …
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Figure 6—source data 1
Concentrations of mixtures and raw Drosophila preference data for Figure 6.
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Figure 6—figure supplement 1
Acetaldehyde metabolic derivatives can complement the co-culture containing A. pomorum adhA, although their physiological concentrations are unknown.
(A) Dose response of acetaldehyde was given to the co-culture containing A. pomorum adhA (along with a constant dose of 3.0% acetic acid). Metabolite additions were added to the culture in the noted …
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Figure 6—figure supplement 1—source data 1
Raw Drosophila preference data for Figure 6—figure supplement 1.
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Figure 6—figure supplement 2
Drosophila behavior toward water amended with nine metabolites (9-metabolite mixture) versus three different apple cider vinegars (ACV), a co-culture (Sc-Am = S. cerevisiae and A. malorum), or tri-culture (Sc-Am-Lp = S. cerevisiae, A. malorum, L. plantarum cs).
Cultures were grown for 72 hr and mixed 1:1 with water, as in all other experiments. Data points represent the Mean ± SEM of two experiments with twelve total replicates. A one-sample t-test …
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Figure 6—figure supplement 2—source data 1
Raw Drosophila preference data for Figure 6—figure supplement 2.
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Figure 7 with 1 supplement
Drosophila egg-laying preference, nutrition, and pathogen protection.
(A) Drosophila was given a choice to lay eggs in a co-culture containing S. cerevisiae and A. pomorum wild-type (WT) or S. cerevisiae and A. pomorum adhA. The co-cultures were grown for 96 hr and …
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Figure 7—source data 1
Raw Drosophila egg-laying preference data for Figure 7A.
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Figure 7—source data 2
Raw developmental data for Figure 7B,C,E,F.
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Figure 7—figure supplement 1
Impact of co-culture metabolites on adult survival and yeast populations.
(A) Drosophila survival in the presence of acetic acid (AA), ethanol (EtOH) or the combination of the two in water. Groupings were based on concentrations of metabolites estimated from pre-ethanol …
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Figure 7—figure supplement 1—source data 1
Raw survival proportions for Figure 7-figuresupplement1A.
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Figure 8
Model of microbe-microbe metabolite exchange.
Bolded are metabolites increased due to microbe-microbe interactions.
Tables
Table 1
Summary of volatiles detected using GC-MS. Relative abundance of volatiles in the co-culture (S. cerevisiae and A. malorum grown together) compared to the separate-culture mixture (S. cerevisiae and …
Identity | Standard confirmation | Relative quantification (co-culture: separate-culture mixture) |
|---|---|---|
Ethanol | Y | 5.0–12.6-fold reduced |
Isobutanol | Y | 7.3–24.7-fold reduced |
Isoamyl acetate | Y | unique to co-culture |
Isoamyl alcohol | Y | 3.6–6.4-fold reduced |
Acetic acid | Y | unique to co-culture |
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Table 1—source data 1
Extracted ion chromatograms of five metabolites detected by gas chromatography-mass spectrometry (GC-MS) in Table 1.
Extracted ion chromatograms of the five metabolites detected by gas chromatography- mass spectrometry (GC-MS). (A) Schematic depicting the experimental setup (B-F) Representative extracted ion chromatograms from one replicate (out of three total) of one experiment (out of 3–4 total) of m/z values corresponding to major metabolites identified in the experimental conditions along with appropriate standards. Acetic acid (B), isoamyl alcohol (C), isoamyl acetate (D) isobutanol (E), and ethanol (F) were identified as the five major metabolites in the co-culture (S. cerevisiae and A. malorum). Isoamyl alcohol (C), ethanol (E), and isobutanol (F) were identified as the major metabolites in S. cerevisiae grown alone. Extracted ion chromatograms were constructed using the m/z value in the title of each graph. For acetic acid and isobutanol, the m/z value used corresponds to the molecular weight of the molecule. For ethanol, the m/z used corresponds to the molecular weight minus one (hydrogen). For isoamyl alcohol, the m/z used corresponds to the loss of the hydroxyl group (depicted), which may have picked up hydrogen and been lost as water. For isoamyl acetate, the m/z value corresponds to the molecule shown within the graph. In all cases, figures showing the complete mass spectra between the metabolite and standard are found in Table 1—source data 2. Microorganisms were grown 72–96 hr.
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Table 1—source data 2
Representative spectra of metabolites in Table 1.
Representative spectra of acetic acid (A-B), isoamyl alcohol (C-E), isoamyl acetate (F-G), ethanol (H-J) and isobutanol (K-L) in standard and experimental samples. Standard concentrations are denoted on individual graphs. All mass spectra are one replicate (out of 3–4 experiments with three replicates per experiment).
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Table 1—source data 3
Linear regression of metabolites using GC-MS in Table 1.
Estimation of volatile quantity using GC-MS. Separate experiments are graphed in panels (A-E) and (F-J). (A-E) Data points represent the value of a single replicate per concentration for each standard. The abundance of a single m/z value at a specific retention time was chosen for each standard. The values were fitted with a linear regression and the equation was used to estimate the concentration of the five metabolites in the experimental samples from the same experiment. (F-J) Data points represent the mean ± SEM of three replicates for a given concentration for each standard. The abundance of a single m/z value at a specific retention time was chosen for each standard. The values were fitted with a linear regression. The equation was used to estimate the concentration of the five metabolites in the experimental samples from the same experiment. When applicable an equation was calculated when the line was forced to go through X,Y = 0,0; these equations were used to calculate the concentrations of isoamyl alcohol, isoamyl acetate, and isobutanol.
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Table 1—source data 4
Raw spectral abundance data as a function of concentration used for linear regressions in Table 1—source data 3.
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Table 2
Estimated concentrations of key metabolites in the co-culture using SPME GC-MS. Estimated concentrations of differentially concentrated or unique metabolites in the co-culture. Linear regression …
Metabolite | Lin. Reg. eq. 1 | Lin. Reg. eq. 2 | Normalized peak area (single experiment) | Normalized peak area (Average, All experiments) | Estimated concentration (%) |
|---|---|---|---|---|---|
Isobutyl acetate | Y = 4151X − 0.1319 | Y = 3252X − 0.07251 | 0.29 | 1.16 | 0.00023 |
Isoamyl acetate | y = 8158X | Y = 7800X | 0.78 | 3.8 | 0.00026 |
2-Phenethyl acetate | Y = 5129X −0.04011 | Y = 6972X −0.2013 | 1.2 | 1.9 | 0.00028 |
2-Methylbutyl acetate acetate | Y = 8995X − 0.05042 | Y = 8087X−0.1307 | 0.56 | 3.1 | 0.00023 |
Methyl acetate | Y = 75.22X+0.004457 | NA | 0.018 | 0.040 | 0.00033 |
Ethyl acetate | NA | NA | NA | NA | ~0.02* |
Acetic acid | NA | NA | NA | NA | ~3.0* |
Acetoin | NA | NA | NA | NA | ~0.01* |
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Table 2—source data 1
Extracted ion chromatograms of differentially emitted or unique metabolites in the co-culture in Table 2.
Extracted ion chromatograms of differentially emitted or unique metabolites in the co-culture according to solid phase microextraction gas chromatography-mass spectrometry (SPME GC-MS). Specific metabolites are displayed above each panel. For each panel, the left-most plot compares the co-culture containing S. cerevisiae and A. malorum to S. cerevisiae grown alone, A. malorum grown alone, or media (AJM [apple juice medium]); the right-most plot compares the co-culture containing S. cerevisiae and A. pomorum wild-type to the co-culture containing S. cerevisiae and A. pomorum adhA, since A. pomorum adhA is required for Drosophila co-culture preference (Figure 5A). The two plots within the same panel contain the same standard. The y-axis for each plot is the ion current for a m/z value that discriminates the metabolite of interest over a specific retention time window. The following m/z values were chosen for each metabolite based on standards or, in the cases of putative and unknown metabolites (I and J) were chosen from the experimental groups: (A) m/z 74.04 (B) m/z 88.08 (C) m/z 73.03 (D) 87.05 (E) 74.02 (F) 104.04 (G) 60.05 (H) 88.05 (I) 101.06 (J) 101.06. Each panel is one representative replicate of 1 experiment (out of 3–5 total replicates in three experiments).
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Table 2—source data 2
Linear regression of metabolites in defined metabolite mixtures in Table 2.
Normalized peak areas corresponding to metabolites in a defined metabolite mixture (from SPME GC-MS). A linear regression was calculated to quantify the metabolites in the co-culture. Each concentration is from one replicate. A-E and F-I are two separate experiments. Linear regression was used to estimate the concentration of the metabolites in the co-culture containing S. cerevisiae and A. malorum (Table 2) and to complement the co-culture containing A. pomorum adhA (Figure 4C).
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Table 2—source data 3
Peak area as a function of concentration used to estimate metabolite concentrations in co-cultures in Table 2.
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Table 2—source data 4
Extracted ion chromatograms of various m/z values used in.
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Table 2—source data 5
Peak areas as a function of metabolite concentration used in linear regression in Table 2—source data 2A–E.
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Table 2—source data 6
Peak areas as a function of metabolite concentration used in Table 2—source data 2F–I.
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Additional files
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Supplementary file 1
Drosophila melanogaster stocks used in experiments.
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Supplementary file 2
Microorganisms used in experiments and their sources
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Supplementary file 3
Chemicals or solutions used in T-maze and GC-MS experiments.
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Supplementary file 4
Metabolite mixture concentrations used for identification and quantification in SPME GC-MS.
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Supplementary file 5
Composition of metabolite mixtures 1-21 used in Figure 6.
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