1 Introduction
1.3 Seasonal alterations on gut microbiota in hibernators
1.3.1 Compositional changes of gut microbiota during hibernation
Hibernators, such as amphibians, few mammalians and birds, have evolved the physiological features to survive unfavourable winter environment. Hibernation is a biological adaptation for animals that helps animals to conserve energy during foodbody temperature to only a few degrees above ambient temperatures to reduce metabolic activity and energy expenditure (Book 1974). Torpor bouts are interrupted by reactivating animal metabolic activities when body temperature is restored.
Seasonal patterns in physiology of hibernators are accompanied by marked changes.
For example, gastrointestinal tract of hibernators reduces in crypt depth, villus length, and total mucosal surface area. Epithelial permeability, a major component of the intestinal barrier that restricts passage of luminal molecules such as bacterial lipopolysaccharide, increases during hibernation (Carey and Physiology 1990).
Hibernation also reduces digestive enzyme activities (Balslev-Clausen, McCarthy et al. 2003) and affects the function of the innate and the adaptive systems (Bouma, Carey et al. 2010).
1.3.2 Seasonal shifts of dominant bacterial populations in hibernators identified by conventional methods
Microbial diversity in the intestine of most hibernating animal varies temporally in response to seasonal changes due to the food availability across seasons. Food availability determines the temporal variation of dietary composition for these hibernators and their gut microbiota. As a result, phylogenetic compositions of gut microbiota in hibernators can be altered dramatically across seasons due to the variability of diets and patterns of nutrition intake (Goldizen, Terborgh et al. 1988, Overdorff, Strait et al. 1997, Sommer, Ståhlman et al. 2016). The nutritional shifts and the changes in morphology and physiology of intestine across seasons suggest strong impacts on gut microbiota in microbial composition, diversity, and metabolic
functions in hibernators.
There are varieties of conventional approach on exploring the impacts of hibernation on gut microbiota in amphibians. For example, studies on artificial hibernation of northern leopard frogs (Rana pipiens) and bullfrogs demonstrated a drop in bacterial counts and a change in the composition of gut microbiota (Carr, Amborski et al. 1976,
Gossling, Loesche et al. 1982). In addition, studies on hibernating amphibians also characterized a higher risk of pathogenic infections by identifying the increase of pathogenic populations in the intestine. Hibernating northern leopard frogs (Van der Waaij, Cohen et al. 1974) and chilled southern bullfrogs (Rana catesbiana) (Carr, Amborski et al. 1976) harbored potentially pathogenic facultative bacteria in the intestine. Facultative (preferentially aerobic but facultatively anaerobic) bacteria from the intestines of frogs have been investigated as a source of septicemia, often
associated with chilling and hibernation (Gibbs, Gibbs et al. 1966, Carr, Amborski et al. 1976), which occasionally kills large numbers of frogs in the laboratory and in the wild (Gibbs, Gibbs et al. 1966, Hawksworth 1974). Carr et al. (Carr, Amborski et al.
1976) and Gibbs et al. (Gibbs, Gibbs et al. 1966) also found that hibernation can alter the relative concentrations and proportions of facultative versus anaerobic bacteria, leading to disease. These studies suggest that during hibernation, gut microbiota of amphibian reduces the amounts of bacterial populations, and it may increase the risk of pathogenic infections due to the increase of pathogenic populations.
1.3.3 Seasonal restructure of gut bacterial assembly in hibernators characterized by advanced sequencing technology
Hibernating ground squirrel has been studied extensively with regard to the gut microbiota. Research on ground squirrels extend the comparisons of gut microbiota between hibernating and non-hibernating stage into the seasonal changes of gut microbiota. Based on cultivated approach, Barnes and Burton reported that the total number of viable bacteria in the intestine decreases during hibernation (Barnes and Burton 1970). Later on, Carey et al. took advantage of the improvement of
sequencing throughputs to extend the research on gut microbiota of ground squirrel into seasonal-wise scale, and explored the effect of the annual hibernation cycle across different seasons on microbial diversity and composition using deep
sequencing of 16S rRNA genes from fecal contents (Carey, Walters et al. 2013). In
their work, they found that the phylogenetic diversity was the lowest in late winter, and the highest in spring after two weeks period of re-feeding, suggesting hibernation causes the reduction of microbial complexity, and the complexity is able to return back to the original status. This study also demonstrated the compositional changes of gut microbiota across the seasons. The most dominant phyla in ground squirrel are Bacteroidetes, Firmicutes, and Verrucomicrobia. The relative abundance of
Bacteroidetes and Verrucomicrobia that contain species, e.g., Akkermansia
muciniphila, capable of surviving on host-derived substrates such as mucins increases
in hibernating ground squirrel. However, Firmicutes reduces relative abundance in hibernating ground squirrel. It may be due to the large amounts of species inFirmicutes prefer dietary polysaccharides and thus reflects the reductions in relative abundance of Firmicutes in hibernating ground squirrels. These results suggest that the gut microbiota of ground squirrel is restructured across seasons that reflects differences in microbial preferences for host-derived substrates or dietary intake across seasons.
Another approach using high-throughput sequencing technology on exploring the changes of gut microbiota across seasons is brown bear (Sommer, Ståhlman et al.
2016). Compared with the active seasons, gut microbiota in hibernating season has reduced in microbial diversity, levels of Firmicutes and Actinobacteria, and increased levels of Bacteroidetes, which are also found in ground squirrels. In order to
understand the connections between the changes of gut microbiota and host
physiologies, they found that several metabolites involved in lipid metabolism were also affected by gut microbiota in hibernation shown in their study. Transplantation of the gut microbiota of brown bear from winter to germ-free mice also demonstrate some seasonal metabolic features observed in brown bear such as physiological energy absorption, suggesting that seasonal variation in the microbiota may contribute
to host energy metabolism in the hibernating brown bear. These results concluded that gut microbiota of brown bear changes in composition and metabolic functions across the seasons, and manipulation of microbial composition in the intestine can modulate host physiology.
The population of P. megacephalus have widely distributed in Asia. However, the knowledge of gut microbiota of this invasive species is still lacking, including composition and functions. Although evidences consistently indicated that gut
microbiota of varies of hibernators restructure in microbial composition and metabolic functions, however, none of the studies had addressed gut microbiome in this invasive species. We have limited knowledge of how microbial composition could alter
physiology of P. megacephalus. Furthermore, the impacts of seasonal changes on gut microbiome of P. megacephalus is also unclear. It is crucial to have a deep
understanding of gut microbiota to reach a global view to understand this invasive species.