Hibernation alters the diversity and composition of mucosa‐associated bacteria while enhancing antimicrobial defence in the gut of 13‐lined ground squirrels

Abstract

The gut microbiota plays important roles in animal nutrition and health. This relationship is particularly dynamic in hibernating mammals where fasting drives the gut community to rely on host-derived nutrients instead of exogenous substrates. We used 16S rRNA pyrosequencing and caecal tissue protein analysis to investigate the effects of hibernation on the mucosa-associated bacterial microbiota and host responses in 13-lined ground squirrels. The mucosal microbiota was less diverse in winter hibernators than in actively feeding spring and summer squirrels. UniFrac analysis revealed distinct summer and late winter microbiota clusters, while spring and early winter clusters overlapped slightly, consistent with their transitional structures. Communities in all seasons were dominated by Firmicutes and Bacteroidetes, with lesser contributions from Proteobacteria, Verrucomicrobia, Tenericutes and Actinobacteria. Hibernators had lower relative abundances of Firmicutes, which include genera that prefer plant polysaccharides, and higher abundances of Bacteroidetes and Verrucomicrobia, some of which can survive solely on host-derived mucins. A core mucosal assemblage of nine operational taxonomic units shared among all individuals was identified with an average total sequence abundance of 60.2%. This core community, together with moderate shifts in specific taxa, indicates that the mucosal microbiota remains relatively stable over the annual cycle yet responds to substrate changes while potentially serving as a pool for 'seeding' the microbiota once exogenous substrates return in spring. Relative to summer, hibernation reduced caecal crypt length and increased MUC2 expression in early winter and spring. Hibernation also decreased caecal TLR4 and increased TLR5 expression, suggesting a protective response that minimizes inflammation.

Integrative Physiology of Fasting

“…This community, which is closely associated with the mucus layer, is thought to be more resistant to perturbations such as those induced by fasting and refeeding (483). Like the luminal community, the mucosal microbiota undergoes similar seasonal shifts over the hibernation cycle, although differences in relative abundance of specific taxa are less pronounced (144). The relative persistence of bacterial taxa within the squirrel mucosal microbiota across the annual cycle, along with a core assemblage of operational taxonomic units (analogous to species) that makes up 36% to 76% of total sequences in all animals regardless of season suggest that the relative stability of this community is made possible because of continued host production of their main energy source, mucin glycans (144).…”

“…In ground squirrels, crypt lengths in the cecum fall progressively with time in hibernation, from a high in summer through 1 and 4 months of winter fasting. Two weeks after refeeding in spring, crypt length is greater than in hibernation but is still reduced compared with summer values (144).…”

“…Elevated permeability can increase movement of bacterial products, such as lipopolysaccharide (LPS), across the epithelium and induce inflammation in the intestine and other organs (573). In support, hibernation is associated with increased epithelial expression of Toll-like receptor TLR5, and reduced expression of TLR4 (144). Binding of TLR4 by LPS activates the transcription factor NF-κB which can induce inflammatory responses, whereas TLR5 activation by its ligand, flagellin, is associated with protective, anti-inflammatory responses and maintenance of intestinal barrier function.…”

“…The relative persistence of bacterial taxa within the squirrel mucosal microbiota across the annual cycle, along with a core assemblage of operational taxonomic units (analogous to species) that makes up 36% to 76% of total sequences in all animals regardless of season suggest that the relative stability of this community is made possible because of continued host production of their main energy source, mucin glycans (144). Relative to summer squirrels, protein expression of the main intestinal mucin, MUC2, is greater in squirrels after 1 month of hibernation and in spring after two weeks of refeeding, and expression levels were unchanged even after 4 to 5 months of hibernation (144).…”

A place for host–microbe symbiosis in the comparative physiologist's toolbox

“…For example, the circannual rhythms in food intake and metabolism that are hallmarks of mammalian hibernation alter microbiota structure and function Dill-McFarland et al, 2014;Stevenson et al, 2014). Although more research is needed to determine whether seasonal remodeling of the gut microbiota contributes to the hibernation phenotype, a recent study in brown bears suggests that gut microbes may provide physiological benefits to their hosts.…”