Functional Characteristics of the Flying Squirrel's Cecal Microbiota under a Leaf-Based Diet, Based on Multiple Meta-Omic Profiling

Abstract

Mammalian herbivores rely on microbial activities in an expanded gut chamber to convert plant biomass into absorbable nutrients. Distinct from ruminants, small herbivores typically have a simple stomach but an enlarged cecum to harbor symbiotic microbes; however, knowledge of this specialized gut structure and characteristics of its microbial contents is limited. Here, we used leaf-eating flying squirrels as a model to explore functional characteristics of the cecal microbiota adapted to a high-fiber, toxin-rich diet. Specifically, environmental conditions across gut regions were evaluated by measuring mass, pH, feed particle size, and metabolomes. Then, parallel metagenomes and metatranscriptomes were used to detect microbial functions corresponding to the cecal environment. Based on metabolomic profiles, >600 phytochemical compounds were detected, although many were present only in the foregut and probably degraded or transformed by gut microbes in the hindgut. Based on metagenomic (DNA) and metatranscriptomic (RNA) profiles, taxonomic compositions of the cecal microbiota were dominated by bacteria of the Firmicutes taxa; they contained major gene functions related to degradation and fermentation of leaf-derived compounds. Based on functional compositions, genes related to multidrug exporters were rich in microbial genomes, whereas genes involved in nutrient importers were rich in microbial transcriptomes. In addition, genes encoding chemotaxis-associated components and glycoside hydrolases specific for plant beta-glycosidic linkages were abundant in both DNA and RNA. This exploratory study provides findings which may help to form molecular-based hypotheses regarding functional contributions of symbiotic gut microbiota in small herbivores with folivorous dietary habits.

Keywords: animal-microbe interaction; ecological adaptation; gut microbiota; metabolomics; metagenomics; metatranscriptomics.

Figure 1

Digestive strategies of the leaf-eating flying squirrel. (A) White-faced flying squirrel (Petaurista alborufus lena) occupies a unique feeding niche in the treetop. (B) The skull structure (mouth with a broad gap) enables temporarily holding a large volume of tree leaves before chewing them. (C) Molars with multiple narrow folds on the crown enable tree leaves to be well chewed. (D) Anatomical/physiological characteristics (mean ± SD) of the four main gut compartments; note the extremely enlarged cecum which contained the majority of feed contents for microbial fermentation. (E–G) The cecum harbored large numbers of microorganisms that act on plant debris. Photo credits: (A) Hsueh-Chen Chen; (B,C) Ji-Fan Hsieh; and (E–G) Han-Chen Ho.

Figure 2

PCoA plot of metabolomic compositions revealing distinct chemical environments across gut compartments of the flying squirrel (N = 3). For each individual, 2, 5, 4, and 4 metabolomic samples were collected from the stomach, small intestine, cecum, and large intestine, respectively (see Figure S1 for details).

Figure 3

Numbers of phytochemical compounds determined across gut compartments of the flying squirrel (N = 3), for (A) entire group of phytochemicals or (B–F) each subgroup. ^a–cColumns without a common letter differed (P < 0.05); error bars are standard error of the mean (SEM) for number of compounds detected within each gut region.

Figure 4

Genus-level taxonomic compositions of metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Top 30 abundant genera that constituted >0.5% in either library are shown, with their phyla in parentheses.

Figure 5

Abundance distributions of COGs in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant COGs that constituted >0.5% in either library are shown.

Figure 6

Abundance distributions of KOs in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant KOs that constituted >0.5% in either library are shown.

Figure 7

Glycoside hydrolases (GHs) detected in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant GHs that constituted >0.05% in either library are shown.

Figure 8

NMDS plot based on COG compositions, revealing functional similarity / dissimilarity among mammalian gut metagenomes. In addition to two datasets from the flying squirrel (FS) cecum of this study, publicly available gut metagenomes, including four datasets from the cow's rumen and 39 datasets from the fecal samples of zoo mammals (categorized by diets: carnivore, omnivore, and herbivore), were included for comparison.

Figure 9

Diagrammatic illustration of potentially crucial microbial functions in the flying squirrel's cecum, based on genes and compounds detected in the metagenome, metatranscriptome, and metabolome. From top to bottom: simple sugars were transported into microbial cells through various ABC importers, and subsequently fermented into short-chain fatty acids. Meanwhile, substrate-binding components of ABC importers were also involved in chemotaxis signaling, enabling bacteria to move toward higher concentrations of sugars by flagellar motility. Left: ABC exporters involved in defense mechanisms may enable gut microbes to export toxic phytochemical derivatives.