Microbiota-induced changes in drosophila melanogaster host gene expression and gut morphology

Abstract

To elucidate mechanisms underlying the complex relationships between a host and its microbiota, we used the genetically tractable model Drosophila melanogaster. Consistent with previous studies, the microbiota was simple in composition and diversity. However, analysis of single flies revealed high interfly variability that correlated with differences in feeding. To understand the effects of this simple and variable consortium, we compared the transcriptome of guts from conventionally reared flies to that for their axenically reared counterparts. Our analysis of two wild-type fly lines identified 121 up- and 31 downregulated genes. The majority of these genes were associated with immune responses, tissue homeostasis, gut physiology, and metabolism. By comparing the transcriptomes of young and old flies, we identified temporally responsive genes and showed that the overall impact of microbiota was greater in older flies. In addition, comparison of wild-type gene expression with that of an immune-deficient line revealed that 53% of upregulated genes exerted their effects through the immune deficiency (Imd) pathway. The genes included not only classic immune response genes but also those involved in signaling, gene expression, and metabolism, unveiling new and unexpected connections between immunity and other systems. Given these findings, we further characterized the effects of gut-associated microbes on gut morphology and epithelial architecture. The results showed that the microbiota affected gut morphology through their impacts on epithelial renewal rate, cellular spacing, and the composition of different cell types in the epithelium. Thus, while bacteria in the gut are highly variable, the influence of the microbiota at large has far-reaching effects on host physiology.

Importance: The guts of animals are in constant association with microbes, and these interactions are understood to have important roles in animal development and physiology. Yet we know little about the mechanisms underlying the establishment and function of these associations. Here, we used the fruit fly to understand how the microbiota affects host function. Importantly, we found that the microbiota has far-reaching effects on host physiology, ranging from immunity to gut structure. Our results validate the notion that important insights on complex host-microbe relationships can be obtained from the use of a well-established and genetically tractable invertebrate model.

FIG 1

Composition and characteristics of D. melanogaster gut-associated bacteria. (A) Relative frequency of bacterial 16S rRNA clones from libraries constructed from two laboratory-derived wild-type populations (Oregon^R and Canton^S). (B) Variability in CFU isolated from dissected guts of individual or pooled (n = 5 guts; n = 7 replicates) 4- to 7-day-old flies. (C) Counts of culturable bacteria (CFU) isolated from the guts of young (4 to 7 days [d] old), mid-age (20 to 25 days old), and old (40 to 43 days old) flies. Total culturable bacteria from individual guts are shown. The means ± SEM for three biological replicates (n = 10 flies each) are shown. Mean counts for the guts of old flies are significantly higher (P = 0.0002) than counts in guts of young and mid-aged flies. Data shown are from analysis of Oregon^R flies; similar results were obtained with Canton^S. (D) Frequency distributions of bacterial cells (CFUs) (top; n = 52), food intake (middle; n = 143), and expression of the antimicrobial peptide gene diptericin reporter (Dpt-lacZ, bottom; n = 105) in the guts of individual flies. Bin numbers for each panel represent percentage deciles calculated based on the maximum measured parameter. For example, bin 1 represent 0 to 10%, Bin 2 represents 11 to 20%, and so on. The top-panel bin percentages were calculated from a maximal CFU of 48,000 per gut. Middle and bottom panel bin percentages were calculated from maximal OD values (see Materials and Methods for more details).

FIG 2

Impact of microbiota on host gene expression in the gut. (A) Venn diagram depicting the core set of genes whose expression was altered in the gut of wild-type flies by microbiota. Two laboratory-derived wild-type populations were compared (Oregon^R and Canton^S). The number of altered genes specific to each strain is indicated, and highlighted pathways and genes of the core response are listed. (B) Proportion of upregulated (top) and downregulated (bottom) genes in each of six gene ontology categories or unknowns. The total gene number in each category is indicated in its respective section. (C) Highlight of core genes altered in the gut of young flies by microbiota. Across the different gene ontology categories, our microarray showed that microbiota were important basal inducers of immune and homeostatic pathways in the gut. Fold changes in expression for each wild-type condition tested (Oregon^R young, Canton^S young, and Canton^S old) are shown. In addition, genes whose expression was impacted in young immune-deficient (Relish^E20) flies (“*” denotes Imd-regulated genes; “$” indicates Imd-altered genes) or displayed abnormal regulation with aging (#) are indicated. CR, conventionally reared; AX, axenically reared. (D) Distribution of the core set of upregulated (blue) and downregulated (red) genes in different regions of the gut. Classification for the gut regions was made based on their enrichment per the classification given elsewhere (21). The number of genes enriched in the crop, regions 1 to 5 of the midgut (R1 to R5), or the hindgut (HG) are shown (the scheme of gut regions is depicted below). (E) The proportion of microbiota-induced genes in each gene ontology category per region (see panel D) is shown. (F) Distribution of the core set of microbiota-induced genes based on their fold change in Oregon^R (black) and Canton^S (gray) flies.

FIG 3

Impact of microbiota on host gene expression increases as flies age and is mainly local. Venn diagram and GO category proportions depicting the impact of microbiota on host gene expression as flies age. Genes that were up- or downregulated in the guts of young (4 to 7 days old) and old (35 to 40 days old) Canton^S flies were compared to identify genes specific to young or old flies, as well as genes whose expression was affected by microbiota at both ages.

FIG 4

Impact of the Imd pathway on gut microbiota composition, density, and localization. (A) Relative frequency of bacterial 16S rRNA clones from libraries constructed from Imd-deficient flies (Relish^E20) compared to laboratory-derived wild-type flies (Oregon^R). (B) Density of culturable bacteria in the guts of individual wild-type (Oregon^R) and Imd-deficient (Relish^E20) flies. (C) Density of bacteria in the guts of Oregon^R (top) and Relish^E20 flies (middle), compared to immune pathway activity in different regions of the gut of wild-type (Oregon^R) flies as measured by RT-qPCR. The ratio of total 16S rRNA is shown relative to a host housekeeping gene (Rpl32) in each gut region. The antimicrobial peptide diptericin gene (Dpt) was used as a readout of immune pathway activity (bottom) in the same regions of the guts of wild-type flies (Oregon^R). (D) Representative images of the localization of bacteria in the guts from wild-type (Oregon^R) and immune-deficient (Rel^E20) flies. Dyes from the Live/Dead BacLight bacterial viability kit (Syto 9 [green] and propidium iodide [red]) were fed to adult female flies to visualize the location of bacteria in the gut. Guts were dissected after 2 h of feeding, fixed, and stained with DAPI to mark the gut prior to imaging. Composite gut images were stitched together from overlapping single 10× three-channel (DAPI, GFP, and red fluorescent protein [RFP]) image tiles of the gut using the Axioplot imager and MosaiX program (Zeiss). The stitched composite image was then processed to correct for shading differences across the individual tiles.

FIG 5

Impact of microbiota on cell identity of the gut. (A) The mitotic activity of the guts of conventionally reared flies is significantly higher than that of axenically reared flies, as measured by immunostaining with anti-Ph3 antibody. The impacts of microbiota on mitotic activity are more pronounced in the posterior region of the gut. Mean values from four experiments (n = 10 guts each) ± SE are shown; anterior, P = 0.04; middle, P = 0.39; posterior, P = 0.0019. Results from Oregon^R are shown; similar results were obtained with Canton^S. (B) There is no significant difference in the number of midgut enterocytes in axenic and conventionally reared flies (mean values from 10 guts each ± SE; P = 0.4995). (C to F) Composition of cell types (eb, enteroblasts; ee, enteroendocrine cells) is altered in the guts of axenic versus conventionally reared flies. Quantitative measurements (C) of the ratio of cell types per region are shown. The expression of cell identity markers (D to F) in the guts of axenic or conventionally reared flies was compared. Expression of green fluorescent protein (GFP) under the control of progenitor cell (D) (stem cell and enteroblast; esg-Gal4^TS; UAS-nlsGFP) or enteroblast (E) [Su(H)GBE-Gal4; UAS-mcd8GFP] reporter genes was monitored. (C and F) Enteroendocrine cells were quantified using anti-Prospero antibody. Representative images of GFP or anti-Prospero antibody signal from the guts of 4- to 7-day-old flies are shown. Guts were stained with DAPI and examined by fluorescence microscopy at magnification ×20, and images were taken of the anterior (R2) and posterior (R5) regions of the gut. Quantitative measurements were made by counting the number of GFP⁺ or anti-Prospero cells and total number of DAPI-positive cells per field of 10 individual guts and replicated at least three times (for panel D, anterior, P < 0.0001; posterior, P = 0.0003; for panel E, anterior, P = 0.106; posterior, P = 0.005; for panel F, anterior, P = 0.0002; posterior, P = 0.0017).

FIG 6

Microbiota alter gut morphology. (A) Nuclear staining (DAPI) of guts from axenically reared (Ax) flies reveals a decrease in cell density compared to that of the guts of conventionally reared (CR) flies. Representative images of the posterior midgut (R5) were taken at magnification ×20. Scale bars = 50 µm. (B) Quantification of the mean distance between the nearest adjacent nuclei (distance, internuclei [DIN]) in guts from AX and CR flies. The mean DIN for each condition was calculated from the average measure for individual guts (20 random measures per gut using DAPI-stained images as for panel B), and then the mean ± SE for 70 guts per condition were compared; P < 0.0001. (C) Mean length ± SE of the midgut and hindgut of Ax and CR flies. Lengths were measured from images of DAPI-stained guts from the midline of the proventriculous to the pyloric valve (midgut) and then from the pyloric valve to the beginning of the rectal ampulla (hindgut). Values are from four experiments (n = 6 to 10 guts each). For midgut, P < 0.0001; for hindgut, no significant difference. (D) The impact of the microbiota on the mean length for different midgut regions was assessed by measuring the length of the anterior (R1-R2), middle (R3), and posterior (R4-R5) midguts of both Ax and CR flies from images of DAPI-stained guts. Mean values for each region from three experiments (n = 5 guts each) ± SE are shown; anterior, P = 0.0018; middle, P = 0.09; posterior, P = 0.002. For simplification, data obtained from measures of guts of axenic and conventionally reared Oregon^R flies are shown; however, similar values were obtained from the guts of Canton^S, esg-Gal4>UAS-mCD8-GFP, Su(H)GBE-Gal4>UAS-mCD8-GFP, and prospero-Gal4>UAS-mCD8-GFP flies.