The composition of both yeast and bacteria in the food microbiota shifts as fermentation progresses, independently of the presence of larvae (A-F) Sampling designs and results for the analyses of microbial compositions in natural food sources of Drosophila (A and B), effects of larval presence on the microbial composition shift (C and D), or microbial compositions in adult flies, larvae, and their foods (E and F). (A, C, and E) Designs of the samplings for microbial analyses. Traps with bananas were set up outdoors near human habitations in order to collect food samples on which wild Drosophila spp. lay eggs. Fermented food samples were collected from the traps at two time points: the early stage (just after trap collection), and the late stage (when larvae in the foods developed into late 3rd instars). Collected samples are indicated in bold letters. (C) In this sampling, the collected early-stage food was crushed in PBS, and the liquid portion (microbial suspension) was added to fresh bananas with or without germ-free embryos (Cg-Gal4, UAS-mCD8:GFP). After incubation, late-stage food and surface-sterilized larvae were collected. (E) In addition to the food samples, adult flies in the traps and larvae from the late-stage foods were collected. This sampling was conducted independently from that shown in (C) and (D), although the sampling places were in common (see Table S1A). Both adults and larvae were surface-sterilized. Note that all of the adults in the traps at Place 7 and Place 8 were collected for the microbial analysis, while 20 out of 37 were collected at Place 9. (B, D, and F) The relative abundances of fungi or bacteria in the fermented banana or fly samples. The compositions were analyzed using primer sets amplifying the fungal ITS region or bacterial 16S rDNA region. Operational taxonomic units (OTUs) accounting for >1% in any of the samples were grouped by families, as shown. The ratio of those accounting for <1% was summed and is indicated as “Others.” The genera belonging to uncertain families (incertae sedis) are indicated by their genus name in parentheses. (B) The result corresponding to the sampling depicted in (A). The numbers on the horizontal axis indicate each numbered sampling location. “No-fly” indicates the food samples from sampling place 6, where no fly or larva was found in the trap or the foods, respectively. (D and F) The result corresponding to the sampling depicted in (C) and (E), respectively. E, early-stage food; L, late-stage food; Susp, microbial suspension; w/o L, late-stage food without larvae; w/ L, late-stage food with larvae; Lar, larvae.

Prominent acceleration of larval development occurred with early-stage-dominant yeast alone, as well as in combination with late-stage-dominant acetic acid bacteria and other microbes (A-F) The percentage and timing of pupariation of larvae feeding on microbes detected in the early-stage food (A and B), or five (C and D) or three (E and F) microbes detected in the late-stage food from Sampling Place 1 in Figure 1B. (A, C, and E) Each data point represents the average percentage of individuals per tube that pupariated by each day. n=3-4. Colored lines are for mixed conditions, while gray lines are for mono-associated or germ-free conditions. The codes for individual species are provided in the chart at the bottom of (B), (D), and (E), respectively. Error bars represent SEM. (C) The color coding for the respective foods is as follows: includes yeasts only (orange); includes yeast(s) and LAB but no AAB (red); includes LAB and AAB (blue). (B, D, and F) Percentage of the larvae pupariated (pupariation ratio; upper) and the timing at which 50% of the pupariation ratio was achieved (lower). Boxes represent upper and lower quartiles, while the central lines indicate the median. Whiskers extend to the most extreme data points, which are no more than 1.5 times the interquartile range. Unique letters indicate significant differences between groups (Tukey-Kramer test, p < 0.05). Pi. kluyveri, Pichia kluyveri; Pa. agglomerans, Pantoea agglomerans; St. bacillaris, Starmerella bacillaris; Le. mesenteroides, Leuconostoc mesenteroides; Entero., Enterobacterales; GF, germ-free; NA, not applicable; days AEL, days after egg laying.

List of microbial species used in the feeding experiments.

Percentages indicate the relative abundance of each species in the foods shown in Figure 1B.

During the late stage, acetic acid bacteria play a crucial role in supporting larval development through interspecies interactions among the microbes (A) Heat map of gene expression values for first instar larvae fed H. uvarum or bacteria. Freshly hatched germ-free larvae were placed on banana agar inoculated with each microbe and collected after 15 h feeding to examine gene expression of the whole body. The data of the genes that were differentially expressed between “LAB”-fed conditions and the respective “LAB + AAB” conditions are shown. (B and C) Plots showing the result of GO term (B) or KEGG Pathway (C) enrichment analysis of the RNA-seq data. 10 terms/pathways that showed the smallest FDR in each analysis are shown. (D) Heat maps showing the similarity between our RNA-seq data and microarray data in a previous study (Zinke et al., 2002). Zinke et al. used larvae at 47-49 h AEL and fed them yeast paste or starved them for 12h before comparing the gene expression profiles. The data from Zinke et al., 2002. are labeled as “Fed/Starved” in the righthand column of each heat map, which show the fold change value of each gene. Only the genes exhibiting significant differences in their analysis are shown. For our data, log2(fold change) were calculated using TPM of each gene in the larvae on the supportive conditions versus those on the non-supportive conditions. (E and F) The percentage and timing of pupariation of the larvae feeding on AAB on banana agar. Graphs are presented as in Figure 2. n=3-4. (G and H) AAB load of foods inoculated with AAB, with or without other microbial species. Boxplots are depicted as in Figure 2 (Dunnett’s test, *p < 0.05, **p < 0.01, ***p < 0.001). La. pla, Lactiplantibacillus plantarum; Le. mes, Leuconostoc mesenteroides; A. ori, Acetobacter orientalis; H. uva, Hanseniaspora uvarum; St. bac, Starmerella bacillaris; Pi. klu, Pichia kluyveri; GF, germ-free; NA, not applicable; days AEL, days after egg laying.

Isolated yeast species promote larval development to varying degrees, but all support larval development upon heat killing (A and B) The percentage and timing of pupariation of larvae feeding on live yeasts on banana agar. Graphs are presented as in Figure 2. n= 3-4. (C) Heat map of gene expression values for first instar larvae fed on each yeast species. Freshly hatched germ-free larvae were placed on banana agar inoculated with each microbe and collected after 15 h feeding to examine gene expression of the whole body. The data of the genes that were differentially expressed between the H. uvarum- fed larvae and the Starmerella bacillaris-fed larvae are shown. (D and E) Plots showing the result of GO term (D) or KEGG Pathway (E) enrichment analysis of the RNA-seq data. Genes that showed significantly higher expression on each of the supportive species than on both the non- supportive species were analyzed. 10 terms/pathways that showed the smallest FDR in each analysis are shown. (F) Heat maps showing the similarity between our RNA-seq data and microarray data in Zinke et al., 2002, shown as described in Figure 3. (G-J) The percentage and timing of pupariation of the larvae feeding on heat-killed yeasts on banana agar (G and H) or live yeasts on a nutritionally rich medium (I and J). Graphs are depicted as in Figure 2. n=3-4. H. uva, Hanseniaspora uvarum; K. hum, Kazachstania humilis; M. asi, Martiniozyma asiatica; Sa. cra, Saccharomycopsis crataegensis; Pi. klu, Pichia kluyveri; St. bac, Starmerella bacillaris; BY4741, Saccharomyces cerevisiae BY4741 strain; GF, germ-free; NA, not applicable; days AEL, days after egg laying.

Metabolomic analysis of yeast cells, yeast-conditioned banana-agar plates, or cell suspension supernatants (A-F) Heat maps displaying all detected metabolites (A, C, and E) or only the metabolites included in a chemically defined synthetic (holidic) medium for Drosophila melanogaster (Piper et al., 2014) (B, D, and F) detected from the metabolomic analysis of banana-agar plates (A and B), yeast cells (C and D), or yeast-cell suspension supernatants (E and F). Row Z-scores of normalized peak areas are shown. (G and H) Normalized peak areas of leucine (G) and isoleucine (H) in yeast-cell suspension supernatants. Boxplots are presented as in Figure 2. Unique letters indicate significant differences between groups (Steel-Dwass test, p < 0.05).

Supportive yeasts facilitate larval growth by providing nutrients, including branched-chain amino acids, by releasing them from their cells (A and B) Growth of larvae feeding on yeasts on banana agar supplemented with leucine and isoleucine. (A) The mean percentage of the live/dead individuals in each developmental stage. n=4. (B) The percentage of larvae that developed into second instar or later stages. The “Not found” population in Figure 6A was omitted from the calculation. Each data point represents data from a single tube. Unique letters indicate significant differences between groups (Tukey-Kramer test, p < 0.05). (C) The biosynthetic pathways for leucine and isoleucine with Saccharomyces cerevisiae gene names are shown. The colored dots indicate enzymes that are conserved in the six isolated species, while the white dots indicate those that are not conserved. Abbreviations of genera are given in the key in the upper right corner. LEU2 is deleted in BY4741. (D-G) Representative image of Phloxine B-stained yeasts. The right-side images are expanded images of the boxed areas. The scale bar represents 50 µm. (H) Summary of this study. H. uvarum is predominant in the early- stage food and provides Leu, Ile, and other nutrients that are required for larval growth. In the late- stage food, AAB directly provides nutrients, while LAB and yeasts indirectly contribute to larval growth by enabling the stable larva-AAB association. The host larva responds to the nutritional environment by dramatically altering gene expression profiles, which leads to growth and pupariation. H. uva, Hanseniaspora uvarum; K. hum, Kazachstania humilis; Pi. klu, Pichia kluyveri; St. bac, Starmerella bacillaris; GF, germ-free.

Fungal and bacterial species compositions in natural food sources of Drosophila. (A and B) Results of microbial composition analysis corresponding to Figure 1B. The relative abundances of fungal (A) or bacterial (B) species in the fermented banana are shown. The compositions were analyzed using primer sets amplifying the fungal ITS region or bacterial 16S rDNA region. Operational taxonomic units (OTUs) accounting for >1% in any of the samples were grouped by species, as shown. The ratios of those accounting for <1% or those of unidentified species were summed and indicated as “Others.” The genera belonging to uncertain families (incertae sedis) are indicated by their genus name in parentheses. The numbers on the horizontal axis represent each numbered sampling location, corresponding to those indicated in Figure 1B. “No-fly” highlights the food samples from sampling place 6, where no fly or larva was found in the trap or the foods, respectively. E, early-stage food; L, late-stage food.

Copy numbers of microbial rDNA in fermented banana samples. (A-F) Graphs showing the copy numbers of the fungal ITS region (A, C, and E) or bacterial 16S rRNA region (B, D, and F) in the fermented banana samples shown in Figures 1A and 1B (A and B), Figures 1C and 1D (C and D), and Figures 1E and 1F (E and F). Numbers below each pair of foods indicate the sampling places, which correspond to those indicated in Figure 1. (C and D) Yeast and bacteria were also detected in Blank banana samples, in which sterile PBS instead of microbial suspensions was added. They might have invaded the fruit by passing through wounds on the peel (Oyewole, 2012). E: early-stage food; L: late-stage food.

Larvae feeding on mixtures of isolated microbial species detected in the food samples from Sampling Place 2 in Figure 1B. (A-F) The percentage and timing of pupariation of larvae feeding on (A and B) microbes derived from the early-stage food, or (C and D) five, or (E and F) three microbes derived from the late- stage food. (A, C, and E) The codes for individual species are provided in the chart at the bottom of (B), (D), and (F), respectively. (B, D, and F) Graphs are presented as in Figure 2. n=3-4. Pi. kluyveri, Pichia kluyveri; Pa. agglomerans, Pantoea agglomerans; La. plantarum, Lactiplantibacillus plantarum; Entero., Enterobacterales; GF, germ-free; NA, not applicable; days AEL, days after egg laying.

The percentage of pupariation and eclosion of the larvae feeding on microbes. (A-D) Graphs showing the percentage of larvae that pupariated in the experiments shown in Figures 2A and 2B (A), Figures 2C and 2D (B), Figures S3A and S3B (C), or Figures S3C and S3D (D). The mean percentages of the pupariated and eclosed (dark gray) or pupariated and not eclosed (light gray) individuals are shown. n=3-4. Pi. kluyveri, Pichia kluyveri; Pa. agglomerans, Pantoea agglomerans; St. bacillaris, Starmerella bacillaris; Le. mesenteroides, Leuconostoc mesenteroides; La. plantarum, Lactiplantibacillus plantarum; Entero., Enterobacterales; GF, germ-free; NA, not applicable.

Enrichment analyses of the genes upregulated in non-supportive conditions and morphology of yeast colonies grown on a nutrient-rich diet. (A-D) Plots showing the result of GO term (A and C) or KEGG Pathway (B and D) enrichment analysis of the RNA-seq data shown in Figure 3A (A and B) and Figure 4C (C and D). Plots were generated as described in Figure 3. (E) Images of single yeast colonies grown on banana agar or the nutrient-rich diet. The images were taken after a 2-day incubation at 25°C. La. pla, Lactiplantibacillus plantarum; Le. mes, Leuconostoc mesenteroides; A. ori, Acetobacter orientalis.

Sample preparations for the metabolomic analysis. Sample preparation process for the metabolomic analysis. We suspected that the supportive live yeast cells may release critical nutrients for larval growth, whereas the non-supportive yeasts may not. To test this possibility, we made three distinct sample preparations of individual yeast strains (yeast cells, yeast-conditioned banana-agar plates, and cell suspension supernatants). Yeast cells were for the analysis of intracellular metabolites, whereas yeast-conditioned banana-agar plates and cell suspension supernatants were for that of extracellular metabolites. The samples were prepared as the following procedures. Yeasts were grown on banana-agar plates for 2 days at 25°C, and then scraped from the plates to obtain “yeast cells.” Next, the remaining yeasts on the resultant plates were thoroughly removed, and a portion from each plate was cut out (“yeast-conditioned banana agar”). In addition, we suspended yeast cells from the agar plates into sterile PBS, followed by centrifugation and filtration to eliminate the yeast cells, to prepare “cell suspension supernatants.”