Characterizing the microbiota in gastrointestinal tract segments of Rhabdophis subminiatus: Dynamic changes and functional predictions

Abstract

The gut microbiota helps the host to absorb nutrients and generate immune responses that can affect host behavior, development, reproduction, and overall health. However, in most of the previous studies, microbiota was sampled mainly using feces and intestinal contents from mammals but not from wild reptiles. Here, we described the bacterial profile from five different gastrointestinal tract (GIT) segments (esophagus, stomach, small intestine, large intestine, and cloaca) of three wild Rhabdophis subminiatus using 16S rRNA V4 hypervariable amplicon sequencing. Forty-seven bacterial phyla were found in the entire GIT, of which Proteobacteria, Firmicutes, and Bacteroidetes were predominant. The results showed a significant difference in microbial diversity between the upper GIT segments (esophagus and stomach) and lower GIT segments (large intestine and cloaca). An obvious dynamic distribution of Fusobacteria and Bacteroidetes was observed, which mainly existed in the lower GIT segments. Conversely, the distribution of Tenericutes was mainly observed in the upper GIT. We also predicted the microbial functions in the different GIT segments, which showed that microbiota in each segments played an important role in higher membrane transport and carbohydrate and amino acid metabolism. Microbes in the small intestine were also mainly involved in disease-related systems, while in the large intestine, they were associated with membrane transport and carbohydrate metabolism. This is the first study to investigate the distribution of the gut microbiota and to predict the microbial function in R. subminiatus. The composition of the gut microbiota certainly reflects the diet and the living environment of the host. Furthermore, these findings provide vital evidence for the diagnosis and treatment of gut diseases in snakes and offer a direction for a model of energy budget research.

Keywords: Rhabdophis subminiatus; Proteobacteria; diets; gut microbiota; microbial function.

Figure 1

Relative abundance of gut microbiota composition in different GIT segments and individual samples at the phyla level. Gut microbiota composition in different GIT segments (a) and individual samples (b). The top 16 abundant taxa are shown with a pie and bar chart

Figure 2

Relative abundance of microbial composition in different GIT segments and individual samples at the genus level. Gut microbiota composition in different GIT segments (a) and individual samples (b). The top 16 abundant taxa are shown with a bar chart (An underlined representative was classified only to the family level and the genus name was not accurately defined)

Figure 3

Heatmap of hierarchy cluster results for the abundance of the top 30 genera in different GIT segments (An underlined representative was classified only to the family level and the genus name was not accurately defined, “Other” representative when denoting classification, the program cannot judge which category should be classified according to the rules)

Figure 4

Linear discriminant analysis effect size (LEfSe) analysis of bacterial taxa was significantly different in the different GIT segments of R. subminiatus by the default parameters. A histogram of the LDA scores that were computed highlights different abundance among different GIT segments (a) (histograms of different colors represent the most significant differences in different GIT segments, abundance annotation represents phylum, class, order, family, and genus). Cladogram of bacterial taxa that were differentially abundant in different GIT segments (b)

Figure 5

Differences in bacterial community structures and relationship between five GIT segments with unweighted UniFrac distances. Principal coordinate analysis (PCoA) of bacterial community structures of the gut microbiota in the five GIT Segments (a). Each solid circle symbol represented each gut microbiota and shows distinct bacterial communities between different GIT segments. The UPGMA tree analysis of five GIT segments through evolution (b)

Figure 6

The OTU numbers of different GIT segments for the Venn diagram (The overlap regions show the common OTU numbers among different GIT segments)

Figure 7

Microbial functional differences in different GIT segments. The relative proportions of the most abundant metabolism‐related KEGG pathways (level 2) predicted by PICRUSt between similar GIT segments of the top 15 (a). The error bars are standard deviations. The star indicates (p < 0.05) using Welch's t test (There was no significant difference in the content denoted with the same letter, but there was a significant difference in content denoted with the different letter). Comparison of microbial functions significant differences in different GIT segments (b)