Comparison of the gut microbiome and resistome in captive African and Asian elephants on the same diet

Abstract

Elephants are endangered species and threatened with extinction. They are monogastric herbivorous, hindgut fermenters and their digestive strategy requires them to consume large amounts of low quality forage. The gut microbiome is important to their metabolism, immune regulation, and ecological adaptation. Our study investigated the structure and function of the gut microbiota as well as the antibiotic resistance genes (ARGs) in captive African and Asian elephants on the same diet. Results showed that captive African and Asian elephants had distinct gut bacterial composition. MetaStats analysis showed that the relative abundance of Spirochaetes (FDR = 0.00) and Verrucomicrobia (FDR = 0.01) at the phylum level as well as Spirochaetaceae (FDR = 0.01) and Akkermansiaceae (FDR = 0.02) at the family level varied between captive African and Asian elephants. Among the top ten functional subcategories at level 2 (57 seed pathway) of Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the relative gene abundance of cellular community-prokaryotes, membrane transport, and carbohydrate metabolism in African elephants were significantly lower than those in Asian elephants (0.98 vs. 1.03%, FDR = 0.04; 1.25 vs. 1.43%, FDR = 0.03; 3.39 vs. 3.63%; FDR = 0.02). Among the top ten functional subcategories at level 2 (CAZy family) of CAZy database, MetaStats analysis showed that African elephants had higher relative gene abundance of Glycoside Hydrolases family 28 (GH 28) compared to Asian elephants (0.10 vs. 0.08%, FDR = 0.03). Regarding the antibiotic resistance genes carried by gut microbes, MetaStats analysis showed that African elephants had significantly higher relative abundance of vanO (FDR = 0.00), tetQ (FDR = 0.04), and efrA (FDR = 0.04) than Asian elephants encoding resistance for glycopeptide, tetracycline, and macrolide/rifamycin/fluoroquinolone antibiotic, respectively. In conclusion, captive African and Asian elephants on the same diet have distinct gut microbial communities. Our findings established the ground work for future research on improving gut health of captive elephants.

Keywords: captivity; diet; elephant; microbiome; resistome.

Figure 1

(A) Venn diagram of gene numbers between African and Asian elephants; (B) Correlation heatmap of gene abundance between samples from African and Asian elephants. The color legend on the right shows the level of correlation coefficient. The ellipse with the darker color means the higher absolute value of the correlation coefficient between samples. The right deviation of the ellipse indicates that the correlation coefficient is positive, and the left deviation is negative. The flatter the ellipse, the greater the absolute value of the correlation coefficient. FZ, FJ, and FH are African elephants. YM, YZ, YL, and YW are Asian elephants.

Figure 2

(A) Stacked bar plot depicting relative abundance of top ten bacteria phyla; (B) Stacked bar plot depicting relative abundance of top ten bacteria families in African (n = 3) and Asian (n = 4) elephants.

Figure 3

(A) Principal Co-ordinates Analysis (PCoA) of microbiota from African and Asian elephants at the phylum level (Bray-Curtis distance); (B) ANOSIM analysis of microbial composition at the phylum level. The y-axis of the plot represents the ranked Bray-Curtis distance. Between is the combined microbial information at the phylum level from both groups. A higher median line of between than the median line of the other two groups means that the variance between groups was higher than variance within groups; (C) Cluster tree based on Bray-Curtis distance at the phylum level. The left side is the Bray-Curtis distance clustering tree structure; the right side is the relative abundance distribution map of each sample at the phylum level.

Figure 4

LEfSe Analysis of Differential taxa between Groups is shown in the LDA value distribution histogram. The presented are biomarkers taxa enriched within each group (LDA > 4). The length of the histogram (LDA score) represents the effect size of each abundant taxa.

Figure 5

(A) Relative gene abundance bar plot of functional annotations at level1in KEGG database; (B) Relative gene abundance histogram of top ten functional annotations at level 2 in KEGG database in African (n = 3) and Asian (n = 4) elephants. (C) PCoA analysis of functional gene abundance at level 1 in KEGG database based on Bray-Curtis distance (D) Clustering tree based on Bray-Curtis distance (the left side is the clustering tree structure; the right side is the functional relative abundance distribution of each sample at the first level of KEGG).

Figure 6

(A) Relative gene abundance bar plot of functional annotations at level 1 in CAZy database; (B) Relative gene abundance histogram of top ten functional annotations at level 2 in CAZy database in African (n = 3) and Asian (n = 4) elephants. (C) PCoA analysis of functional gene abundance at level 1 in CAZy database based on Bray-Curtis distance. (D) Clustering tree based on Bray-Curtis distance (the left side is the clustering tree structure; the right side is the functional relative abundance distribution of each sample at the first level of CAZy).

Figure 7

(A) Venn diagrams of antibiotic resistant genes shared by African and Asian elephants and specific genes belong to each host species; (B) Absolute gene abundance of different ARGs in each sample (× 10⁶); (C) Relative abundance of top 20 ARGs.

Figure 8

Circos diagram summarizing the relationship between the resistance mechanism and the microbial taxa (combined data from both African and Asian elephants). The circos diagram is divided into two parts, with phylum-level taxa information on the right and resistance mechanism information on the left. For the outer circle, the left side is the relative proportion of the resistance genes coding for corresponding resistance mechanism within each taxa. The right side is the relative proportion of the resistance genes coding each resistance mechanism within the taxa. For the inner circle, different colors represent different taxa and resistance mechanisms, and the scale is the number of genes. The left side is the sum of the number of resistance genes coding corresponding resistance mechanism in different taxa. The right side is the sum of the number of resistance genes coding different resistance mechanisms within each taxa.