A large number of microorganisms colonize the intestines of animals. The gut microbiota plays an important role in nutrient metabolism and affects a number of physiological mechanisms in the host. Studies have shown that seasonal changes occur in the intestinal microbes of mammals that hibernate seasonally. However, these studies only focused on ground squirrels and bears. It remains unclear how hibernation might affect the intestinal microbes of bats. In this study, we measured microbial diversity and composition in the gut of Rhinolophus ferrumequinum in different periods (early spring, early summer, late summer, torpor, and interbout arousal) using 16S ribosomal RNA gene amplicon sequencing and PICRUSt to predict functional profiles. We found seasonal changes in the diversity and composition of the gut microbes in R. ferrumequinum. The diversity of gut microbiota was highest in the late summer and lowest in the early summer. The relative abundance of Proteobacteria was highest in the early summer and significantly lower in other periods. The relative abundance of Firmicutes was lowest in the early summer and significantly increased in the late summer, followed by a significant decrease in the early winter and early spring. The relative abundance of Tenericutes was significantly higher in the early spring compared with other periods. The results of functional prediction by PICRUSt showed seasonal variations in the relative abundance of metabolism-related pathways, including lipid metabolism, carbohydrate metabolism, and energy metabolism. Functional categories for carbohydrate metabolism had significantly lower relative abundance in early winter-torpor compared with late summer, while those associated with lipid metabolism had significantly higher relative abundance in the early winter compared with late summer. Overall, our results show that seasonal physiological changes associated with hibernation alter the gut microbial community of R. ferrumequinum. Hibernation may also alter the metabolic function of intestinal microbes, possibly by converting the gut microflora from carbohydrate-related to lipid-related functional categories. This study deepens our understanding of the symbiosis between hibernating mammals and gut microbes.

Background: Diet plays a crucial role in sculpting microbial communities. Similar diets appear to drive convergence of gut microbial communities between host species. Captivity usually provides an identical diet and environment to different animal species that normally have similar diets. Whether different species’ microbial gut communities can be homogenized by a uniform diet in captivity remains unclear. Methods: In this study, we compared gut microbial communities of three insectivorous bat species (Rhinolophus ferrumequinum, Vespertilio sinensis, and Hipposideros armiger) in captivity and in the wild using 16S rDNA sequencing. In captivity, R. ferrumequinum and V. sinensis were fed yellow mealworms, while H. armiger was fed giant mealworms to rule out the impact of an identical environment on the species’ gut microbial communities. Results: We found that the microbial communities of the bat species we studied clustered by species in the wild, while the microbial communities of R. ferrumequinum and V. sinensis in captivity clustered together. All microbial functions found in captive V. sinensis were shared by R. ferrumequinum. Moreover, the relative abundances of all metabolism related KEGG pathways did not significantly differ between captive R. ferrumequinum and V. sinensis; however, the relative abundance of “Glycan Biosynthesis and Metabolism” differed significantly between wild R. ferrumequinum and V. sinensis. Conclusion: Our results suggest that consuming identical diets while in captivity tends to homogenize the gut microbial communities among bat species. This study further highlights the importance of diet in shaping animal gut microbiotas.

Rhinolophus ferrumequinum nippon and Rhinolophus ferrumequinum tragatus are two subspecies of Rhinolophus ferrumequinum currently recognized in China. In this study, their mitochondrial genomes were completely sequenced and annotated. Phylogenetic analyses indicated that R. f. nippon has a close relationship with two subspecies of R. ferrumequinum from Korea with 0.1% divergence, which indicated they are synonyms.

Arylalkylamine N-acetyltransferase (AANAT) is a crucial rate-limiting enzyme in the synthesis of melatonin. AANAT has been confirmed to be independently duplicated and inactivated in different animal taxa in order to adapt to the environment. However, the evolutionary forces associated with having a single copy of AANAT remain unclear. The greater horseshoe bat has a single copy of AANAT but exhibits different hibernation rhythms in various populations. We analyzed the adaptive evolution at the gene and protein levels of AANAT from three distinct genetic lineages in China: northeast (NE), central east (CE), and southwest (SW). The results revealed greater genetic diversity in the AANAT loci of the NE and CE lineage populations that have longer hibernation times, and there were two positive selection loci. The catalytic capacity of AANAT in the Liaoning population that underwent positive selection was significantly higher than that of the Yunnan population (p < 0.05). This difference may be related to the lower proportion of α helix and the variation in two interface residues. The adaptive evolution of AANAT was significantly correlated with climate and environment (p < 0.05). After controlling for geographical factors (latitude and altitude), the evolution of AANAT by the negative temperature factor was represented by the monthly mean temperature (r = −0.6, p < 0.05). The results identified the gene level variation, functional adaptation, and evolutionary driving factors of AANAT, provide an important foundation for further understanding the adaptive evolution of the single copy of AANAT in pteropods, and may offer evidence for adaptive hibernation rhythms in bats.

The greater horseshoe bat (Rhinolophus ferrumequinum), which is widely distributed across the temperate regions of China, primarily consists of two major evolutionary lineages: a northeastern (NE) lineage with a hibernation period of 6–8 months and a central–eastern (CE) lineage with a hibernation period of 4–5 months. This study conducted a comparative analysis of liver transcriptomes from these two lineages during the active, torpor, and arousal phases. The results indicated that the CE lineage exhibited a significantly greater number of differentially expressed genes (DEGs) compared to the NE lineage. During the torpor phase, both lineages transitioned from carbohydrate metabolism to lipid metabolism, substantially downregulating genes and pathways associated with amino acid metabolism, and upregulating immune-related genes to maintain essential defense functions. In the arousal phase, both lineages only moderately activated several genes associated with immunity and metabolic regulation to facilitate a rapid return to torpor. Notably, the number of DEGs co-regulated between the two lineages was very limited, and a large number of lineage-specific regulatory genes related to energy and metabolism were identified. This may reflect the adaptability of different bat lineages to the local environment, highlighting the importance of habitat conditions in lineage differentiation. Therefore, hibernation induces substantial transcriptomic reorganization in the liver of R. ferrumequinum, particularly affecting metabolic and immune processes. Distinct geographic lineages exhibit unique hibernation adaptation strategies through the regulation of specific genes and pathways. This study enhances the understanding of the molecular mechanisms underlying hibernation adaptation across different evolutionary lineages of the same species at the transcriptomic level, providing insights into the evolutionary adaptations of animals to environmental changes.