Gastrointestinal Biogeography of Luminal Microbiota and Short-Chain Fatty Acids in Sika Deer (Cervus nippon)

Abstract

The gut microbiota of sika deer has been widely investigated, but the spatial distribution of symbiotic microbes among physical niches in the gastrointestinal tract remains to be established. While feces are the most commonly used biological samples in these studies, the accuracy of fecal matter as a proxy of the microbiome at other gastrointestinal sites is as yet unknown. In the present study, luminal contents obtained along the longitudinal axis of deer gastrointestinal tract (rumen, reticulum, omasum, abomasum, small intestine, cecum, colon, and rectum) were subjected to 16S rRNA gene sequencing for profiling of the microbial composition, and samples from the rumen, small intestine, and cecum were subjected to metabolomic analysis to evaluate short-chain fatty acid (SCFA) profiles. Prevotella bacteria were the dominant gastric core microbes, while Christensenellaceae_R-7_group was predominantly observed in the intestine. While the eight gastrointestinal sites displayed variations in microbial diversity, abundance, and function, they could be clustered into stomach, small intestine, and large intestine segments, and the results further highlighted a specific microbial niche of the small intestine. SCFA levels in the rumen, small intestine, and cecum were significantly different, with Bacteroidetes and Spirochaetes were shown to play a critical role in SCFA production. Finally, the rectal microbial composition was significantly correlated with colonic and cecum communities but not those of the small intestine and four gastric sites. Quantification of the compositions and biogeographic relationships between gut microbes and SCFAs in sika deer should provide valuable insights into the interactions contributing to microbial functions and metabolites. IMPORTANCE Feces or specific segments of the gastrointestinal tract (in particular, the rumen) were sampled to explore the gut microbiome. The gastrointestinal biogeography of the luminal microbiota in ruminants, which is critical to guide accurate sampling for different purposes, is poorly understood at present. The microbial community of the rectal sample (as a proxy of fecal sample) showed higher correlation with those of other large intestinal sites relative to the small intestine or stomach, suggesting that the microbial composition is specifically shaped by the unique physiological characteristics of different gastrointestinal niches. In addition, significant differences in microbiomes and SCFAs were observed among the different gastrointestinal sites.

Keywords: biogeography; deer; gastrointestinal tract; gut microbiome; ruminants; short-chain fatty acids.

FIG 1

Comparison of Shannon indices (A and C) and Simpson indices (B and D) among the gastrointestinal tract sites in sika deer. Panels A and B exhibit the differences in Shannon and Simpson index among the rumen, reticulum, omasum, abomasum, small intestine, cecum, colon, and rectum. After the data from rumen, reticulum, and omasum were pooled into forestomach and data from the cecum, colon, and rectum were pooled into large intestine, the comparison was performed again; results are shown in panels C and D. *, significant difference.

FIG 2

Core microbes at the genus level in the microbial communities of the rumen (A), reticulum (B), omasum (C), abomasum (D), small intestine (E), cecum (F), colon (G), and rectum (H) in sika deer. Numbers following the genus name are the shared OTU numbers, and numbers in the pie charts are the proportion of each genus in total shared OTUs. The genera that occurred at low relative abundance are included in “Others.”

FIG 3

Relative abundances of dominant phyla (A) and genera (B) in the microbial communities of the rumen, reticulum, omasum, abomasum, small intestine, cecum, colon, and rectum in sika deer. The taxa that occurred at low relative abundance are included in “Others.”

FIG 4

Comparison of dominant genera among the rumen, reticulum, omasum, abomasum, small intestine, cecum, colon, and rectum in sika deer. (A) Phylogenetic tree of the top 100 genera and comparison of the relative abundances of these genera. The colors of the genera indicate the phyla to which they belong. (B) Histogram of the main genera which showed significant differences among eight gastrointestinal sites.

FIG 5

(A) Linear discriminant analysis (LDA) effect size (LEfSe) analysis at the LDA threshold of 4, indicating the differently abundant taxa among the rumen, reticulum, omasum, abomasum, small intestine, cecum, colon, and rectum of sika deer. (B) Heat map analysis based on the relative abundance of genera. Different colors in the heat map represent different relative abundances; the color trend to red means higher abundance.

FIG 6

Principal-coordinate analysis (PCoA) based on the weighted UniFrac distance (A) and the nonmetric multidimensional scaling (NMDS) based on the Bray-Curtis similarities (B). The rumen, reticulum, omasum, abomasum, small intestine, cecum, colon, and rectum of sika deer are indicated by different shapes, and different colors are used to distinguish the merged data.

FIG 7

(A) LEfSe analysis at the LDA threshold of 2, which indicating the different abundances of level 2 KEGG functions among the rumen, reticulum, omasum, abomasum, small intestine, cecum, colon, and rectum of sika deer. (B) PCA based on the weighted UniFrac distance of KEGG functions. The rumen, reticulum, omasum, abomasum, small intestine, cecum, colon, and rectum are indicated by different shapes, and cycles with different colors are used to distinguish the merged data.

FIG 8

Correlation analysis of microbial community between the rectum and cecum (A) and colon (B) based on the relative abundance of OTUs in sika deer. Each point in the plots represents one OTU, and numbers on the x axis and y axis indicate the relative abundance of OTUs (percent).

FIG 9

Heat map analysis (A) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) (B to D) based on the concentrations of seven types of SCFAs in the rumen, small intestine, and cecum of sika deer. Each point in the OPLS-DA plots represents one sample. “R” refers to the samples from the rumen, “C” refers to the samples from the cecum, and “IT” refers to the samples from the small intestine. T scores on the x axis are for the dissimilarity between two groups, and T scores on the y axis are for the dissimilarity within each group.

FIG 10

(A) Canonical correlation analysis (CCA) of relationships between seven types of SCFAs and dominant genera in sika deer. Points with three different colors represents samples from the rumen, small intestine and cecum. Blue lines represent genera, and red lines represent SCFAs. The smaller the angle between lines, or the longer the lines toward the points, the stronger the positive correlation. (B) Correlation heat map analysis of relationships between seven types of SCFAs and the top 30 genera in sika deer. Trending red color means a positive correlation, and trending blue color means a negative correlation. *; significant correlation; **, extremely significant correlation.