Based on the metagenomic profile of cecal microbiota, the giant flying squirrel underwent profound changes to adapt it to a diet of high-fiber, low-quality leaves. As reported for other small herbivores
, the prominent cecum of the giant flying squirrel is apparently an anaerobic chamber for microbial breakdown of plant materials, consistent with an important role for cecal microbiota. It is noteworthy that cecal microbiota of the flying squirrel differed from their functional counterpart (rumen microbiota) of cattle (which has been much better characterized). Furthermore, the microbiota of the flying squirrel were also different from those of the Prevost's squirrel and laboratory mice, although they are close relatives. In the case of the flying squirrel and cattle, we concluded that independent evolutionary routes lead to similar functions. However, in the case of the flying squirrel and two omnivorous rodents (the Prevost's squirrel and lab mice), the influence of diet apparently confounded the phylogeny. In particular, the present study, based on wild-caught mammals, represented gut microbial communities under natural conditions and contributed important new knowledge regarding intricate mechanisms and interactions of the mammalian "super-organism". Moreover, most studies on gut microbes have been based on fecal samples (“output” of the digestive system) that may not reflect the actual reactions and processes involved in digestion of foods (“input”). If the digestive tract is regarded as a “production line”, the present study of cecal microbiota could elucidate the true “power house” for liberation of energy from a diet that is generally resistant to digestion, and thus offer insights into processes shaped by evolution for use of novel energy sources.
Based on a comparison of gut microbiota of flying squirrels (hindgut fermenter) and cattle (foregut fermenter), these 2 animals have distinct bacterial compositions, although both rely on the microbiota for the conversion of plant materials into nutrients. They had different phylotypes within Firmicutes, Actinobacteria, Proteobacteria, and Verrucomicrobia. These differences might be driven by diet (tree leaves versus forage and legumes), gut physiology (cecum versus rumen), and co-evolution within 2 host lineages (Rodentia versus Artiodactyla). Also, the gut microbiota of the mouse and flying squirrel were compared, since both species are phylogenetic kins (Order Rodentia). On the basis of observations in mouse models
[14, 18], the relative abundance of Firmicutes and Bacteroidetes was associated with the capacity to harvest energy. Compared with lean mice, obese mice had a relatively high fermentative capability, which was associated with an increased number of Firmicutes[14, 18]. Since the cecal microbiota of the flying squirrel contained a high percentage of Firmicutes and harbored many genes involved in carbohydrate metabolism, we inferred that this system might be efficient at extracting energy from dietary polysaccharides, as reported in obese mice
In addition to the host digestive system, microbial genomes encoding proteins with metabolic functions are responsible for conversion of dietary substances into absorbable nutrients
[19, 20]. The present sequence-based study provided a comprehensive method to reconstruct the primary metabolic profile of the cecal microbiota which enables the flying squirrel to survive on a leaf-based diet. According to the metagenomic data, the 3 main aspects of this complex degradation system are: 1) Plant polysaccharides are broken down into monosaccharides and disaccharides by various microbial glycoside hydrolases, and these simple sugars are transported into bacterial cells and fermented into short-chain fatty acids (principally butyrate, acetate, propionate, and lactate), which provide energy for the gut epithelium and other tissues
[1, 21]. 2) Genes involved in protein biosynthesis were much more abundant than those in protein degradation, consistent with other herbivorous microbiomes
. Due to the low protein content of a leaf-based diet, the cecal microbiota of the flying squirrel require specialized mechanism to derive nitrogen from limited sources. In that regard, the cecal microbiome contained genes related to hydrolysis of urea (derived from the host) into ammonia for synthesis of amino acids and derivatives. 3) The cecal microbiota synthesizes several vitamins, especially B-complex vitamins, which may meet the host’s need for these compounds
Although several studies have focused on polysaccharide utilization by gut microbiota
[15, 24–27], there is a paucity of knowledge regarding gut microbial constituents and their functional interactions with the host, especially in wild animals. According to the CAZy database, multiple enzymes with the ability to catabolize dietary carbohydrates were detected in the cecal microbiome of the flying squirrel. Presumably metagenomic studies on the microbiota of wild herbivores that consume a wide range of plants will provide further insights regarding conversion of plant polysaccharides into monosaccharides. Based on the distribution of CAZy families detected in our fosmid library, we inferred that enzymes for plant oligosaccharide degradation (GH2, GH3, GH29, GH35, and GH39) may be more vital than those for degradation of crystalline cellulose (GH9) in the cecum, because the digesta has already been substantially degraded by physical and chemical digestion before it reaches the cecum. Furthermore, based on functional annotations, it appeared that Firmicutes has an important role in hydrolyzing indigestible dietary polysaccharides, such as components of plant cell walls (e.g. cellulose, xylan and pectin) and undigested starch, consistent with previous reports
In general, metagenomic samples from environments with a stable input and turnover of complex plant biomass have a higher abundance of GHs than those from other samples
. The GH homologs in our dataset accounted for approximately 1.5% of the total predicted genes, a similar to that reported in gut metagenomes from the termite, human, and mouse
. In addition, the present fosmid library contained more than 16 GH families that were highly diverse; this diversity was comparable to that in other cellulolytic bacterial genomes and metagenome datasets
[15, 31, 32]. In general, sequence-based searches are more efficient than function-based screening in prospecting for novel enzymes, since target genes can be directly discovered from metagenomic datasets using bioinformatics tools
. Although metagenomic approaches were used to quickly annotate various carbohydrate-active enzymes, functional assays will be required for confirmation, since sequence homology does not guarantee functional identity. Considerable additional studies are required to further elucidate and characterize the diverse plant biomass-degrading genes of the cecal microbiome.
High-throughput sequencing has been used to generate numerous gene candidates for biocatalysts; thereafter, their enzymatic activities have been characterized, with a substantial proportion of putative GHs having predicted enzyme activities
. However, most sequence-based metagenomic studies have limitations for downstream cloning and expression of genes, since the coverage is not enough to assemble full-length ORFs, due to the high microbial complexity of most environmental samples
. We therefore constructed a fosmid library, in which each clone contained an insert of ~40 kb of genomic sequence, long enough to reveal the cluster of genes in a genome, thereby improving characterization of the cecal microbiome. In this study, 2 fosmid inserts representing a total of 60 ORFs were identified as genomic fragments of Firmicutes, the most abundant and diverse phylum among the mammalian indigenous microbial communities
. These 2 inserts contained large gene clusters associated with plant polysaccharide utilization, including transcriptional regulators, glycoside hydrolases, sugar transporters, and downstream genes. The genomic arrangement of these 2 fragments verified that genes of associated metabolic pathways typically clustered together
. In prokaryotes, functionally related genes tend to form operons; conservation of neighboring genes suggested co-regulation and co-expression
. Based on sequence comparison, our results confirmed co-occurrence of Bgl3D and Bgl3E in several bacterial genomes, consistent with a functional interaction between this pair of GH3 enzymes.