Stable Host Gene Expression in the Gut of Adult Drosophila melanogaster with Different Bacterial Mono-Associations

Abstract

There is growing evidence that the microbes found in the digestive tracts of animals influence host biology, but we still do not understand how they accomplish this. Here, we evaluated how different microbial species commonly associated with laboratory-reared Drosophila melanogaster impact host biology at the level of gene expression in the dissected adult gut and in the entire adult organism. We observed that guts from animals associated from the embryonic stage with either zero, one or three bacterial species demonstrated indistinguishable transcriptional profiles. Additionally, we found that the gut transcriptional profiles of animals reared in the presence of the yeast Saccharomyces cerevisiae alone or in combination with bacteria could recapitulate those of conventionally-reared animals. In contrast, we found whole body transcriptional profiles of conventionally-reared animals were distinct from all of the treatments tested. Our data suggest that adult flies are insensitive to the ingestion of the bacteria found in their gut, but that prior to adulthood, different microbes impact the host in ways that lead to global transcriptional differences observable across the whole adult body.

Fig 1. Limited variation in gut gene expression with bacterial mono-association.

Expression data from guts dissected from five-day post-eclosion, Wolbachia-free, mated female CantonS D. melanogaster individuals mono-associated with one of three bacteria (Ap = A. pasteurianus, Lbrev = L. brevis, Lp = L. plantarum) were clustered by gene (average linkage, uncentered correlation) after first filtering out genes that lacked three instances of FPKM greater than two (Gene Cluster 3.0). FPKM values for each gene were normalized to range from -1 to 1 before plotting. A) Samples arranged by bacterial treatment. B) Samples arranged by date of experiment. Scale bars for each heatmap are given to the right of the plot.

Fig 2. Genes showing greatest difference in expression values from dissected adult guts as determined by one-way ANOVA.

A) Scatterplot of log10-transformed FPKM values for each bacteria mono-associated gut replicate (Ap = A. pasteurianus, Lbrev = L. brevis, Lp = L. plantarum). Genes are ordered from lowest ANOVA p-value (top) to highest (bottom). P-values have undergone a Bonferroni correction for multiple testing. B) Data from A presented as a heatmap. FPKM values for each gene are linearly normalized to range from -1 to 1 before plotting.

Fig 3. Yeast drives genome-wide difference in gut gene expression.

A) Average linkage hierarchical clustering was performed in Gene Cluster 3.0 across all genes that are expressed at least at two FPKM in at least two out of 11 samples. Bacteria mono-association data has been averaged across each treatment to collapse down into a single column. FPKM values for each gene are normalized to range from -1 to 1 before plotting. Abbreviations: Ap avg = average for A. pasteurianus-mono-associated samples; Lbrev avg = average for L. brevis-mono-associated samples, Lp avg = average for L. plantarum-mono-associated samples, 3bac = poly-associated (without yeast), Ax = axenic, Conv = conventional, Yeast = S. cerevisiae-mono-associated, 4mic = poly-associated (with yeast). Scale bar is shown at bottom right. B) Top) heatmap of 579 genes that are overexpressed in axenic, bacteria-mono-associated and poly-associated (without yeast) guts compared to other gut samples (Bonferroni p-value>0.05, ANOVA). Bottom) Results from Panther GO-SLIM biological function enrichment test [26] for gene set above compared to reference set of all genes observed across all gut datasets (556 were identified by Panther and used for analysis out of 579) C) Top) Heatmap of 1728 genes that are overexpressed in conventional, yeast mono-associated and poly-associated (with yeast) compared to other gut samples (Bonferroni p-value>0.05, ANOVA). Results from Panther GO-Slim biological processes enrichment test with 1728 (1663 identified) genes compared to reference set of all genes observed across all gut datasets. Note for B) and C): all individual sample values were used for ANOVA analysis, not the average value as plotted in A).

Fig 4. Analysis of gene expression trends from gnotobiotic whole flies.

A) Transcriptome-wide heatmap from axenic, conventional, yeast-mono-associated, bacteria-mono-associated and poly-associated whole flies clustered by gene expression. Average linkage hierarchical clustering using an uncentered correlation similarity metric was performed in Gene Cluster 3.0 across all genes that are expressed at least at two FPKM across two out of eleven samples. Abbreviations: Ap = A. pasteurianus-mono-associated; Lbrev = L. brevis-mono-associated, Lp = L. plantarum-mono-associated, 3bac = poly-associated without yeast, Ax = axenic, Conv = conventional, Yeast = S. cerevisiae-mono-associated, 4mic = poly-associated with yeast. Scale bar is shown at bottom right. B) Top) heatmap of 1159 of 1385 genes that are overexpressed in conventional whole flies compared to other whole fly samples (Bonferroni p-value>0.05, ANOVA). Genes absent in heatmap did not pass filtering criteria. Bottom) Results from Panther GO-SLIM biological function enrichment test [26] for gene set above (1278 genes were identified of 1385) compared to reference set of all genes observed across all whole fly datasets. C) Top) Heatmap 351 that are overexpressed in all non-conventional whole-fly samples compared to conventional whole flies (Bonferroni p-value>0.05, ANOVA). Results from Panther GO-Slim biological processes enrichment test with gene set above (348 genes were identified out of 351) compared to reference set of all genes observed across all whole fly datasets.

Fig 5. Microbial load of female D. melanogaster individuals.

A) Log10 transformed average number of colony forming units (CFU) from plating individual gnotobiotic and conventional, lab-reared CantonS, Wolbachia-free, mated, 5-day post-eclosion females on two separate plates. B) Log10 transformed average number of CFU and estimated microbial cells (yeast and bacteria combined) by qPCR for individual, female, wild D. melanogaster raised from embryos (ranging from three to ten days post-eclosion) or caught as adults (of unknown age). The mean for each group is plotted as a horizontal line.