Diverse methanogens, bacteria and tannase genes in the feces of the endangered volcano rabbit (Romerolagus diazi)

Abstract

Background: The volcano rabbit is the smallest lagomorph in Mexico, it is monotypic and endemic to the Trans-Mexican Volcanic Belt. It is classified as endangered by Mexican legislation and as critically endangered by the IUCN, in the Red List. Romerolagus diazi consumes large amounts of grasses, seedlings, shrubs, and trees. Pines and oaks contain tannins that can be toxic to the organisms which consume them. The volcano rabbit microbiota may be rich in bacteria capable of degrading fiber and phenolic compounds.

Methods: We obtained the fecal microbiome of three adults and one young rabbit collected in Coajomulco, Morelos, Mexico. Taxonomic assignments and gene annotation revealed the possible roles of different bacteria in the rabbit gut. We searched for sequences encoding tannase enzymes and enzymes associated with digestion of plant fibers such as cellulose and hemicellulose.

Results: The most representative phyla within the Bacteria domain were: Proteobacteria, Firmicutes and Actinobacteria for the young rabbit sample (S1) and adult rabbit sample (S2), which was the only sample not confirmed by sequencing to correspond to the volcano rabbit. Firmicutes, Actinobacteria and Cyanobacteria were found in adult rabbit samples S3 and S4. The most abundant phylum within the Archaea domain was Euryarchaeota. The most abundant genera of the Bacteria domain were Lachnoclostridium (Firmicutes) and Acinetobacter (Proteobacteria), while Methanosarcina predominated from the Archaea. In addition, the potential functions of metagenomic sequences were identified, which include carbohydrate and amino acid metabolism. We obtained genes encoding enzymes for plant fiber degradation such as endo 1,4 β-xylanases, arabinofuranosidases, endoglucanases and β-glucosidases. We also found 18 bacterial tannase sequences.

Keywords: Fecal metagenome; Herbivorous animals; Microbiome; Tannase enzymes; Volcano rabbit.

Figure 1. Phyla of the bacteria and archaea domain found in the fecal microbiome of the volcano rabbit.

Relative abundance was obtained by dividing the number of contigs assigned per phylum by the total number of contigs for the Bacteria and Archaea domains, respectively. The figures were constructed considering only phyla with a relative abundance equal to or greater than 0.5%, which means that rare bacteria and archaea were not considered. (A) Percentage of abundance of the main phyla from the Bacteria domain found in feces of the volcano rabbit. (B) Percentage of abundance of the main phyla from the Archaea domain found in feces of the volcano rabbit.

Figure 2. Genera of the bacteria and archaea domain found in the fecal microbiome of the volcano rabbit.

Relative abundance was obtained by dividing the number of contigs assigned per genus by the total number of contigs for the Bacteria and Archaea domains, respectively. The figures were constructed considering only genera with a relative abundance equal to or greater than 0.5%, which means that rare bacteria and archaea were not considered. (A) Percentage of abundance of the most representative genera per sample within Bacteria domain. (B) Percentage of abundance of the most representative genera per sample within Archaea domain.

Figure 3. Sample coverage (SC) per sample, taxonomic level, and diversity (q0).

SC ranged from 0.0 to 1.0. (A) Sample coverage for the phyla of the Bacteria domain. (B) Sample coverage for the phyla of Archaea domain. (C) Sample coverage for the genera of the Bacteria domain. (D) Sample coverage for the genera of the Archaea domain.

Figure 4. Species Richness (q0) per sample and taxonomic level.

(A) Species richness for the phyla of the Bacteria domain. (B) Species richness for the phyla of Archaea domain. (C) Species richness for the genera of the Bacteria domain. (D) Species richness for the genera of the Archaea domain.

Figure 5. Enzymes annotated in the fecal metagenome of the volcano rabbit.

Comparison of enzymes according to their catalytic action: Transferases, Hydrolases, Oxidoreductases, Ligases, Lyases and Isomerases per sample and by number of contigs (COUNTS) using PROKKA.

Figure 6. Functional annotation categories in the fecal metagenome of the volcano rabbit.

Comparison of categories of functional annotation by sample and by number of contigs (COUNTS) using GhostKOALA.

Figure 7. Enzymes (class 3) associated in the degradation of cellulose and hemicellulose of plants.

Comparison of the enzymes associated with plant fiber degradation by sample and by number of contigs (COUNTS).