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.