DISCUSSION
In this study, we conducted the first metagenomic next-generation sequencing analysis of the gut microbiome of the common Asian bamboo pitviper. Results identified Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota as the most dominant phyla, contrasting with previous studies on snakes that identified Proteobacteria, Bacteroidetes, and Firmicutes (see Appendix 1), demonstrating that dominant microbes differ among different snake species. Compared to other snakes, our findings indicated that V. stejnegeri possessed a unique gut microbiota, predominantly comprised of Actinobacteria. In contrast, lizards have been shown to harbor a gut microbiota primarily composed of Firmicutes, Bacteroidetes, and Proteobacteria (Hong et al., 2011; Kohl et al., 2017; Zhang et al., 2018; Zhu et al., 2020; Baldo et al., 2023; Gao, Yang & Shi, 2023 ). Thus, while the dominant bacterial communities in snakes and lizards appear to be largely influenced by Proteobacteria and Bacteroidetes, their composition and abundance at the phylum level exhibit distinct differences.
Molecular phylogeny analyses indicated that the Guizhou and Hunan populations exhibited a closer relationship within the three populations (Guo et al., 2016 ). Based on the PCoA results, however, the bacterial diversity among the three populations was not consistent with the phylogenetic relationships (Fig. 4), suggesting that bacterial diversity is not influenced by population relatedness. Our findings demonstrated that the three populations shared similar dominant microbial phyla. At the genus level with relative abundance greater than 3%, except for Salmonella and Citrobacter , distinct dominant genera were observed, including Yokenella ,Enterobacter , and Serratia in the Guizhou population,Bacteroides in the Hunan population and Yokenella in the Anhui population.
Previous studies have indicated that reptiles, such as iguanas, turtles, crocodiles, and snakes, can act as vectors for transmitting bacteria to humans, potentially causing paratyphoid fever (Cohen et al., 1980; Mermin, Hoar & Angulo, 1997; Waterman et al., 1990; Schröter et al., 2004; Grupka et al., 2006; McLaughlin, Cochran & Dowd, 2015 ). In the current study, Salmonella was the most abundant genus in the gut microbiota of V. stejnegeri , with S. enterica , the most prevalent species within the genus (32.01% ± 23.73%), constituting 98.42% of the Salmonella detected. In addition, the conditionally pathogenic bacterium Citrobacter , a facultative anaerobe known to cause enteritis in animals (Mundy, Macdonald & Dougan, 2010 ), was also found at a relatively high abundance, second only to Salmonella . The significant presence ofSalmonella , Citrobacter , and S. enterica in the gut of V. stejnegeri underscores the need for careful and considered conservation and management of wild snake populations.
The composition and abundance of the host gut microbiota are influenced by multiple factors (Zhu et al., 2022; Zhang et al., 2018 ). The three geographic populations of V. stejnegeri were generally consistent in latitude, climate, and prey but varied in altitude and habitat. Notably, the Anhui, Hunan, and Guizhou populations are located at altitudes of 197–220 m, 318–319 m, and 941–1 232 m, respectively. Furthermore, the Anhui and Hunan population habitats are mainly characterized by shrub and bamboo forests adjacent to streams, while the Guizhou population habitat primarily features wooded areas alongside streams. Here, PCoA revealed significant differences in the intestinal microflora among the three populations (Fig. 4, R2 = 0.48,P = 0.01), suggesting that altitude and habitat likely influence the composition and abundance of gut microbes.
CAZymes play a key role in carbon source metabolism. The CAZy database categorizes enzyme families that catalyze the degradation, modification, and biosynthesis of carbohydrates into five major classes and one related module. For example, glycoside hydrolases (GHs) are involved in the hydrolysis and rearrangement of glycosidic bonds, glycosyl transferases (GTs) participate in the formation of glycosidic bonds, polysaccharide lyases (PLs) function in the non-hydrolytic cleavage of glycosidic bonds, carbohydrate esterases (CEs) are involved in the hydrolysis of carbohydrate esters and auxiliary activities (AAs) is a redox enzymes that act in conjunction with CAZymes, and carbohydrate-binding modules (CBMs) facilitate adhesion to carbohydrates (Lombard et al., 2014; Wardman et al., 2022 ). Previous Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of P. dhumnades has shown that genes related to metabolism are most prominent at the first level, encompassing six types of biometabolic pathways, with carbohydrate metabolism being the most abundant at the second level, involving 43 pathways (Li, Sun & Xu, 2021 ). Metabolic profiling has also shown that carbohydrate metabolism is highly dominant inCrotalus horridus and is the main metabolic pathway in R. subminiatus (McLaughlin, Cochran & Dowd, 2015; Tang et al., 2019 ). The gut microbiota CAZyme annotations for the three V. stejnegeri populations showed that the Anhui and Guizhou populations were mainly composed of GT2 (8.93% ± 1.68%, 8.82% ± 1.29%), GT4 (6.97% ± 2.25%, 5.04% ± 0.90%), GH23 (4.14% ± 1.32%, 4.79% ± 0.98%), and GT51 (3.31% ± 0.74%, 3.35% ± 0.40%), while the Hunan population was primarily composed of GT2 (10.73% ± 0.57%), GT4 (6.39% ± 0.28%), and GH2 (3.05% ± 0.14%) (relative abundance ≥ 3%). These findings show that the composition and abundance of dominant CAZymes differed among the three populations. The LEfSe results further highlighted marked disparities in CAZyme abundance across the three populations. Additionally, certain Proteobacteria strains (Gammaproteobacteria, Enterobacterales, Enterobacteriaceae,Salmonella ) and a higher proportion of certain Bacteroidetes strains (Bacteroidia, Bacteroidales, Bacteroidaceae, Bacteroides ) (relative abundance ≥ 40%) were noted in the Hunan population.Bacteroides species, which metabolize polysaccharides and oligosaccharides, provide nutrition and vitamins to the host and other intestinal microbial residents (Zafar & Saier, 2021 ). It was speculated that this difference ultimately led to differences in CAZymes composition and abundance among the three populations. However, the specific substrates and chemical mechanisms acted upon by these CAZymes in snake intestines remain unclear. Further research is needed to elucidate how the gut microbiota affects specific carbohydrate metabolism mechanisms.