Environment and diet shape the geography-specific Drosophila melanogaster microbiota composition

Abstract

Geographic and environmental variation in the animal microbiota can be directly linked to the evolution and wild fitness of their hosts, but it can be difficult to explain the underlying patterns. Here, we sought to better understand wild variation in the microbiota composition of Drosophila melanogaster. First, environmental temperature predicted geographic variation in fly microbial communities more consistently than latitude did. The microbiota also differed between wild flies and their diets, supporting previous conclusions that the fly microbiota is not merely a reflection of diet. Flies feeding on different diets varied significantly in their microbiota composition, and flies sampled from individual apples were exceptionally depauperate for the lactic acid bacteria (LAB), a major bacterial group in wild and laboratory flies. However, flies bore significantly more LAB when sampled from other fruits or compost piles. Follow-up analyses revealed that LAB abundance in the flies uniquely responds to fruit decomposition, whereas other microbiota members better indicate temporal seasonal progression. Finally, we show that diet-dependent variation in the fly microbiota is associated with phenotypic differentiation of fly lines collected in a single orchard. These last findings link covariation between the flies' dietary history, microbiota composition, and genetic variation across relatively small (single-orchard) landscapes, reinforcing the critical role that environment-dependent variation in microbiota composition can play in local adaptation and genomic differentiation of a model animal host.IMPORTANCEThe microbial communities of animals influence their hosts' evolution and wild fitness, but it is hard to predict and explain how the microbiota varies in wild animals. Here, we describe that the microbiota composition of wild Drosophila melanogaster can be determined by temperature, humidity, geographic distance, diet decomposition, and diet type. We show how these determinants of microbiota variation can help explain lactic acid bacteria (LAB) abundance in the flies, including the rarity of LAB in some previous studies. Finally, we show that wild fly phenotypes segregate with the flies' diet and microbiota composition, illuminating links between the microbiota and host evolution. Together, these findings help explain how variation in microbiota compositions can shape an animal's life history.

Keywords: lactic acid bacteria; latitude; life history; local adaptation; neutral theory; photoperiod; rapid adaptation; temperature.

Fig 1

Latitude- and temperature-dependent variation in microbiota composition of D. melanogaster from the eastern USA in multiple years and samplings. D. melanogaster from (A) the same general area in the eastern USA was sampled and sequenced across portions of the 16S rRNA V4 (A and E) or V1–V2 (C and D) regions by two laboratories in (B) 2009 (apples and peaches, N = 15), (C) 2018 (grapes, N = 71), (D) 2018 (apples, N = 79), and (E) 2021 (apples, N = 60). Taxon plots are an average of samples, which are pools of flies (B, rarefaction threshold [rt] = 475) or individual flies (C–E, rt = 500). Mantel tests reporting the relationship of the Bray-Curtis and environmental distances between each sample were also performed, with environmental distance calculated from distance matrices based on (F–I) latitude or (J–M) latitude plus maximum temperature and minimum relative humidity (RH) on the day of sampling. Letters over F–M report the Mantel test correlation coefficient ρ and the P-value results when the results are (blue) or are not (black) significant. (N–U) A similar analysis as for eastern USA flies was conducted using sequences extracted from whole-genome sequencing of flies collected with the DEST data set. (N and O) Maps, (P and Q) taxon plots, and (R–U) Mantel test results for DEST flies from (N, P, R, and S) North America and (O, Q, T, and U) Europe are reported. Samplings from Guadeloupe, a department of France in the Caribbean, are not shown on the map. (V and W) Common garden populations of individual isofemale lines from each location in Fig. S6A were reared under six-species gnotobiotic conditions in the laboratory until adult flies were 3 days old, transferred to test conditions for 3 days, then the microbiota composition of pools of two surface-sterilized adults was analyzed by homogenization and dilution plating (two pools of two females and two males per vial, three vials per common garden population in each experiment, and three separate experiments in time). The relative (V) and absolute (W) abundances of AAB (red) and LAB (blue) CFUs in flies reared at varying temperatures. Relative abundances are shown as the mean of AAB counts divided by the mean of LAB counts, with the fraction of LAB shown as a white point and the overlayed violin plot showing the distribution of fractional LAB abundance. Significant differences in relative abundances of LAB were determined by PERMANOVA of CFU counts rarefied to 10,000 CFUs per sample (Table S2). Absolute CFU abundances are shown as the mean and SEM of all replicates. Significant between-group differences in absolute AAB and LAB abundances are shown as determined by a Kruskal-Wallis test with a post-hoc Dunn test, and different letters over (AAB) or under (LAB) the bars report significant differences in their abundance.

Fig 2

The total and resident D. melanogaster microbiota composition is distinct from the diets of wild flies. (A) Principal coordinates ordination of Bray-Curtis dissimilarity distances for the microbiota of individual wild D. melanogaster, their diets, and nearby soil locations from multiple locations in the eastern USA. Flies were immediately frozen after collection (“flies [total]”) or starved in empty vials for >2 h (“flies [resident]”) after transient microorganisms had passed through the fly gut with the bulk flow of diet. The OTU table was rarefied to 500 reads per sample. (B) Venn diagrams showing the average fractional abundance of ASVs ± s.e.m. that were unique to or shared between sampled fruit and the total or resident fly microbiota. (C) AIC values for neutral models calculated with the group indicated in the “reference” row were compared to itself (“Reference,” ■) or the group in the “comparison” (●) row. (D) Principal coordinates ordination of Bray-Curtis dissimilarities for flies collected from individual apples or compost piles at multiple locations in the eastern USA, rarefied to 845 reads per sample. Reads assigned to the genera (E) Leuconostoc and (F) Weissella differed significantly in abundance between locations as determined by ANCOM.

Fig 3

The microbiota composition of wild and laboratory D. melanogaster varies with diet type. We sequenced the 16S rRNA V4 region of the D. melanogaster microbiota collected from different fruits in the wild and the laboratory. (A and C) Composition of the resident microbiota in wild male D. melanogaster collected from fallen fruit at Lyman Orchards in Middlefield, CT. (A) Bars are the averages of multiple samples, each rarefied to 120 reads (N = mean 3.2 ± sem 0.25, min = 2, and max = 4 samples per bar). (C) Principal coordinate ordination of Bray-Curtis dissimilarity distances. (B and D) Composition of the total microbiota in gnotobiotic 12-sp D. melanogaster CantonS male flies. (B) Bars are the averages of multiple samples, each rarefied to 200 reads (N = mean 5.3 ± sem 0.54, min = 3, and max = 7 samples per bar). (D) Principal coordinate ordination of Bray-Curtis dissimilarity distances. Clustering confidence was set to 0.7 to draw ellipses.

Fig 4

The microbiota composition of wild D. melanogaster varies with time and diet decomposition. We sequenced the 16S rRNA V4 region of the resident microbiota in individual wild Drosophila collected from 1-bushel piles of fruit established at different times in an experimental orchard in Provo, UT. The same data are shown on two timescales, either relative to (A–G) calendar date (time = 0 when the first piles were established) or (H–N) pile establishment time (time = 0 for each pile when it was established). (A and H) Taxon plot, plus a timeline showing the establishment and sampling times (S) from each of four apple (A) and three peach (P) piles. (B and I) Mantel test showing the relationship of the weighted Unifrac and calendar date (B) or pile establishment time (I) distances between each sample, including the slope of the trendline (blue). Plots showing the abundances of specific members of the microbiota that varied in (C–E and J–L) apples and peaches, or only in peaches (F, G, M, and N) in one or both timescales. The Spearman’s rank correlation coefficient (S) and P-value (p), plus a trendline for change in abundance over time (black line), are shown for each. Samples were rarefied to 500 reads before processing.

Fig 5

D. melanogaster phenotypes segregate with the diet from which they are collected. (A) Wild D. melanogaster was sampled from a compost pile (□) or individual fallen apples directly under an apple tree (○, ∆) throughout a single orchard, shown in a digital recreation of an Apple maps screenshot taken in Summer 2024. Green circles represent apple tree positions in the sampled orchard. (B) Isofemale lines derived from the wild collections were reared in the laboratory under gnotobiotic or bacteria-free conditions, and their development time to adulthood (eclosion) was measured. Significant differences between groups were determined by a Cox mixed-effects model and are shown as compact letter displays in the legend.