Pomacea canaliculata snails were cultured in cement ponds located at the Ecological Teaching and Research Farm (23° 16′N, 113° 36′E) of South China Agricultural University (SCAU) in Guangzhou, China. The region experiences a humid subtropical monsoon climate. Yearly average temperature is 21.5°C, with January and July marking the coolest and warmest periods, respectively. Annual rainfall fluctuates between 1,612 and 1909 mm, predominantly occurring from April to September, which accounts for over 80% of the total precipitation. Prior to the experiment, these snails were transported to the laboratory for sex and size selection. After screening, female snails with a shell height of 30–35 mm were selected for in situ hibernation experiments.

The in-situ hibernation experiment (Figure 1a) was conducted in the paddy field of the Ecological Teaching and Research Farm at SCAU from December 2022 to April 2023 (120 days in total). Specifically, a plastic mesh basket (31.5 cm × 23 cm × 10 cm) was filled with 5 cm of in situ soil which was taken from a depth of 5–10 cm, and then 14 female snails were evenly placed on the soil surface. Next, the basket was continued filling with soil from 0 to 5 cm depth until it was nearly full. Once the snails and soil were properly arranged, the basket was covered with a mesh bag to prevent snail escape. Finally, the entire basket was placed into pre-dug trenches approximately 10 cm deep, covering the surface with a small amount of soil to make it level with the ground.

A total of 25 baskets were set up for the in situ experiment. At 15th, 30th, 60th, 90th, and 120th days of the experiment, five randomly selected baskets were retrieved from the field and transported back to the laboratory for snail sampling. The buried female snails were collected by carefully removing the soil. The number of dead snails was recorded. From each basket, one snail was randomly selected and dissected on a sterile workbench. The dissected intestines (from the pylorus to the hindgut) were placed in 2 mL sterile cryovials (Bikeman Biotechnology Co., Ltd., Hunan, China). These cryovials containing the intestinal samples were immediately frozen in liquid nitrogen. Once the sampling process was completed, the cryovials were stored at −80°C for preservation.

Microbial DNA was extracted from 30 intestinal samples (each sample represents the intestine of a single snail from a different basket) using the FastDNA® Kit (MP Biomedicals, CA, USA) according to the manufacturer’s protocol, respectively. The quality and quantity of DNA were evaluated by 1% agarose gel electrophoresis. A NanoDrop2000 spectrophotometer (Thermo Scientific, Wilmington, USA) was employed to determine DNA purity and concentration.

The V3-V4 region of the bacterial 16S rRNA genes was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The amplified products were purified using the AxyPrepDNA kit (AXYGEN, USA) and quantified using the QuantiFluorTM-ST (Promega, USA). High-throughput sequencing of the PCR products was performed on an Illumina MiSeq PE300 platform at Majorbio BioPharm Technology Co., Ltd. (Shanghai, China).

Raw data was subjected to bioinformatics analysis using QIIME 1.9.1 software. The raw fastq files were processed for demultiplexing and denoising using FLASH 1.2.11 and Trimmomatic, respectively. The sequence data were assigned to operational taxonomic units (OTUs) using USEARCH 7.1 software with a 97% identity threshold (Edgar et al., 2011). RDP Classifier 2.13 was utilized to assign taxonomy to each 16S rRNA gene sequence by comparing it against the Silva 138 (rRNA database) (Quast et al., 2012; Bokulich et al., 2018). The taxonomic identity of the unranked OTUs at the genus level was determined by querying them against the NCBI database using BLAST. Alpha-diversity indices (Chao1, Shannon, Simpson (not Gini-Simpsion), PD, and Pielou evenness) of the microbial community were calculated using QIIME. Phenotypic properties (Gram Negative, Gram Positive, Pathogenic, Mobile Element Containing, Oxygen Utilizing, Biofilm Forming, and Oxidative Stress Tolerant) were performed using Bugbase software (Zhang et al., 2019).

Soil temperature was monitored in real-time during the in-situ experiment using a temperature intelligent monitoring device (developed by CIMC Intelligent Cold Chain Technology, Beijing, China), which uploaded temperature data every hour. The temperature probe was placed at a 5 cm depth in the soil of the experimental site. During each sampling event, soil samples from the 0–10 cm depth were collected and brought back to the laboratory. Soil water content was determined by drying samples at 105°C.

At each sampling time point (15th, 30th, 60th, 90th, and 120th days), five baskets were collected and transported to the laboratory. The female snails were delicately separated from the soil, and dead individuals were recorded. The survival of each snail was determined by assessing the presence of odor indicating decay or gently testing whether the operculum would retract upon light touch. Finally, the survival rate of the snails was calculated.

Beta diversity was analyzed using non-metric multidimensional scaling (NMDS). The vegan package was used for permutational multivariate analysis of variance (PERMANOVA), with Bray–Curtis dissimilarity as the distance measure. Intestinal microbial composition differences between hibernation periods were analyzed using Linear Discriminant Analysis (LDA) Effect Size (LEfSe), considering only those features with an absolute LDA score greater than 4 (p < 0.05) (Li et al., 2022b). The relationship between hibernation duration and specific phyla, genera, alpha diversity indices, and phenotypic properties was established using generalized additive models (GAMs). The ecological process of community assembly was estimated using the iCAMP R package’s phylogenetic bin-based null model (Ning et al., 2020). The normalized stochasticity ratio (NST) was used to quantify the relative importance of stochastic and deterministic processes in gut microbiota assembly, with a threshold of 50% set to determine the dominance of either deterministic or stochastic processes (Ning et al., 2019).

During the 120-day hibernation period, the average soil temperature was 18.66°C. The average soil temperatures for the periods of day 0–30, day 30–60, day 60–90, and day 90–120 were 15.5°C, 16.7°C, 20.3°C, and 22°C, respectively. The lowest soil temperature occurred on the 55th day, reaching 8.5°C (Figure 1c). The highest soil temperature was recorded on the 93rd and 95th days, reaching 34°C (Figure 1c). Soil moisture content was at its lowest on the 30th day, at 16.3%, and reached its peak on the 60th day, at 24.1% (Figure 1c). After 120 days of hibernation, the survival rate of female snails was 85.7% (Figure 1b).

A total of 30 snail gut samples, collected from 6 periods (0, 15th, 30th, 60th, 90th, and 120th days), underwent 16S rRNA high-throughput sequencing analysis. The analysis yielded 2,443,935 valid sequences, identifying 2,821 OTUs at a 97% similarity threshold. The species accumulation curve of all samples (Supplementary Figure S1) demonstrated that the observed species richness approached saturation, indicating the reliability of sequencing data and its suitability for subsequent analysis.

The relationship between microbial diversity indices and hibernation duration was analyzed using the generalized additive model (Figure 2). Hibernation duration had no significant effect on the Sobs (observed species), phylogenetic diversity (PD), or Chao1 indices (Figures 2a,b,d). In contrast, the Pielou evenness, Shannon, and Simpson indices exhibited significant correlations with hibernation duration (Figures 2c,e,f). Specifically, both the Pielou evenness and Shannon indices increased over time (Figures 2c,e), indicating enhanced community evenness and diversity. Conversely, the Simpson index decreased with hibernation duration (Figure 2f).

Before hibernation initiation (day 0), Firmicutes was the dominant phylum in the gut microbiota (Figure 3a). The dominant phylum gradually shifted from Firmicutes to Bacteroidota (Bacteroidetes) with increasing hibernation duration (Figure 3a). At the genus level, before hibernation, Lactococcus was the dominant genus (Figure 3b). The dominant genus gradually shifted from Lactococcus to Bacteroides with increasing hibernation duration (Figure 3b).

At the phylum level, the relative abundance of Firmicutes showed a significant relationship with hibernation duration (Figure 3c). Its relative abundance decreased from 92.3% before hibernation (day 0) to 19.7% after 120 days of hibernation. In contrast, the relative abundances of Bacteroidota and Proteobacteria significantly increased with the duration of hibernation (Figure 3c). The relative abundance of Bacteroidota and Proteobacteria increased from 0.4% and 2.1% before hibernation to 61.7% and 10.6% after 120 days of hibernation, respectively.

At the genus level, Lactococcus showed a pronounced decline in relative abundance with increasing hibernation duration (Figure 3d), dropping from 47.2% before hibernation to just 0.2% after 120 days. In contrast, the genera Acetobacteroides, Macellibacteroides, Cetobacterium, and Mycoplasma exhibited significant increases. Notably, Acetobacteroides increased from a negligible 0.0008% at day 0 to 17.4% after 120 days of hibernation.

The NMDS plot and PERMANOVA analysis revealed that hibernation duration significantly influenced gut microbiota composition (Figure 4a). The intestinal bacterial community structure of snails at the 60th, 90th, and 120th days of hibernation showed significant differences compared with that before hibernation (Figures 4d–f). The intestinal bacterial community structure changed after 60 days of hibernation.

During the 120-day hibernation period, a total of 208 OTUs were shared across all six time periods (Figure 5a). Additionally, there were 390, 126, 128, 231, 254, and 386 unique OTUs specifically presenting in the snail gut microbiota at the 0th, 15th, 30th, 60th, 90th, and 120th days of hibernation, respectively (Figure 5a).

LEfSe was used to compare the impact of different hibernation periods on the gut microbiota (Figures 5b,c). The histogram of LDA scores revealed clear differences in microbial abundance among time points. The phylum Firmicutes, class Bacilli, order Lactobacillales, family Streptococcaceae, and genus Lactococcus were much enriched at the pre-hibernation period (Figures 5b,c). The order Flavobacteriales, family Weeksellaceae, and genus Cloacibacterium significantly enriched at 15th day of hibernation (Figures 5b,c). Similarly, the families Bacteroidaceae and Shewanellaceae, and genus Bacteroides enriched much at 30th day of hibernation (Figures 5b,c). The phylum Desulfobacterota, class Desulfovibrionia, order Desulfovibrionales, family Desulfovibrionaceae, and genus Desulfovibrio were significantly enriched at 60th day of hibernation (Figures 5b,c). The phylum Proteobacteria, class Gammaproteobacteria, order Burkholderiales, families Williamwhitmaniaceae and Rhodocyclaceae, and genus Acetobacteroides enriched much at 90th day of hibernation (Figures 5b,c). The phylum Bacteroidota, class Bacteroidia, order Bacteroidales, family Tannerellaceae, and genus Macellibacteroides significantly enriched at 120th day of hibernation (Figures 5b,c).

The normalized stochasticity ratio (NST) was used to quantify the relative contributions of deterministic (niche-based) and stochastic (neutral) processes in shaping gut microbiota during hibernation (Supplementary Figure S2). Prior to hibernation, NST values averaged 32.5%, remaining below the 50% threshold, indicating that deterministic processes predominated in gut microbial community assembly. In contrast, during hibernation (15th to 90th days), NST values exceeded 50%, suggesting a shift toward stochastic dominance in community assembly. Gut bacterial community assembly in snails involved five ecological processes, with three being predominant: homogeneous selection, dispersal limitation, and drift (Figure 6). Notably, the contribution of drift significantly increased during hibernation (15th to 120th days) compared with pre-hibernation levels (Figure 6h). In contrast, dispersal limitation showed a significant decline at several time points during hibernation (15th, 30th, and 90th days) relative to pre-hibernation (Figure 6g).

Based on phenotypic trait predictions from BugBase, the relative abundances of mobile element-containing and Gram-positive bacteria significantly declined with increasing hibernation duration (Figures 7b,d). In contrast, the relative abundances of anaerobic, Gram-negative, and potentially pathogenic bacteria significantly increased over time (Figures 7a,c,e). No significant correlation was observed between the relative abundance of oxidative stress-tolerant bacteria and hibernation duration (Figure 7f).

Our findings indicated that the richness (Sobs and Chao1 indices) of gut microbiota remained unchanged after the snail entered hibernation (Figures 2a,d), while alpha diversity (Shannon index) increased with hibernation duration (Figure 2e). Moreover, the Pielou evenness of gut microbiota also increased over time (Figure 2c). These results suggest that during the hibernation of P. canaliculata, while the number of species in its gut microbiota remained relatively stable, the relative abundance distribution of species changed significantly with hibernation duration, leading to an increase in alpha diversity (Figure 8). Consequently, the observed increase in the Shannon index with hibernation duration may indicate an enhanced capacity of gut microbiota in P. canaliculata to adapt to external disturbances during hibernation (Stoffel et al., 2020). Zhou J. et al. (2022) revealed that gut microbiota alpha diversity (Shannon index) in the vertebrate Siberian chipmunk (Tamias sibiricus) was significantly higher after hibernation (from December to March) than pre-hibernation (November), which aligns with the results of this experiment. Similarly, Bosmans et al. (2018) also observed that gut microbiota alpha diversity (Shannon index) in the invertebrate Bombus terrestris was significantly higher after 16 weeks of artificial hibernation at 3°C than that of the non-hibernating group. Conversely, most previous studies supported that the alpha diversity (Shannon index) of gut microbiota in hibernating animals was typically lower during hibernation compared with active periods. Examples include mammals like Urocitellus parryii (Stevenson et al., 2014), Ictidomys tridecemlineatus (Carey et al., 2013; Dill-McFarland et al., 2014), bear Ursus arctos (Sommer et al., 2016), and Rhinolophus ferrumequinum (Xiao et al., 2019), as well as amphibians such as Rana dybowskii (Tong et al., 2019), Polypedates megacephalus (Weng et al., 2016), and Strauchbufo raddei (Cao et al., 2023). This study showed that during hibernation, the gut microbiota of snails exhibited an increase in alpha diversity due to a rise in evenness, which differed from the traditional pattern of decreased alpha diversity observed in hibernating animals. This observed difference could potentially stem from the remarkable hibernation adaptability and resilience exhibited by the invasive snail P. canaliculata.

Short-term hibernation (15–30 days) had a limited effect on the gut microbiota community structure of snails (Figure 4). However, long-term hibernation (60–120 days) significantly altered the community structure (Figure 8). Similarly, in some animals, the beta diversity of gut microbiota during hibernation significantly differed from pre-hibernation or non-hibernation periods, indicating pronounced alterations in gut microbiota structure during post-hibernation. For instance, in mammals, examples include U. parryii (Stevenson et al., 2014), I. tridecemlineatus (Dill-McFarland et al., 2014), U. arctos (Sommer et al., 2016), R. ferrumequinum (Xiao et al., 2019), and Tamias sibiricus (Zhou J. et al., 2022); in amphibians such as R. dybowskii (Tong et al., 2020) and P. megacephalus (Weng et al., 2016); and in invertebrates like Bombus terrestris (Bosmans et al., 2018).

Before hibernation, Firmicutes dominated the snail gut microbiota, accounting for 92.3% of the relative abundance (Figure 3a). During the 15–60 days of hibernation, Bacteroidota also became dominant, reaching approximately equal relative abundance to that of Firmicutes (Figure 3a). As hibernation progressed to 90–120 days, the dominant phylum shifted from Firmicutes to Bacteroidota, with the latter reaching a relative abundance of approximately 60% (Figures 3a, 8). Previous studies have consistently found that Bacteroidota, Firmicutes, and Proteobacteria are among the three most dominant phyla (not in any particular order) in the gut microbiota of hibernating animals during their hibernation period (Sonoyama et al., 2009; Wiebler et al., 2018; Tong et al., 2020). Notably, Bacteroidota often emerges as the most dominant phylum during hibernation (Dill-McFarland et al., 2014; Sommer et al., 2016; Weng et al., 2016; Tong et al., 2019).

The relative abundance of phylum Firmicutes in gut microbiota of snails significantly decreased with hibernation duration (Figures 3c, 8). In contrast, phyla Bacteroidota and Proteobacteria relative abundances significantly increased with hibernation duration (Figures 3c, 8). A large body of prior research supports the trend of decreased relative abundance of Firmicutes in snail gut microbiota during hibernation compared with that at pre-hibernation or non-hibernation periods, consistent with the findings of this study. Examples include U. parryii (Stevenson et al., 2014), I. tridecemlineatus (Dill-McFarland et al., 2014), P. megacephalus (Weng et al., 2016), R. dybowskii (Tong et al., 2019), among others (Sommer et al., 2016; Xiao et al., 2019). Moreover, these studies also observed a general increase in the relative abundances of Bacteroidota and Proteobacteria during hibernation. The rising relative abundance of the phylum Bacteroidota during hibernation might be attributed to the ability of Bacteroidota-related bacteria to metabolize host polysaccharides, enabling them to thrive in the absence of food in the gut during hibernation (Sonnenburg et al., 2005). Additionally, Bacteroidota-related bacteria may also participate in metabolizing proteins and fats provided by the gut epithelium (Wu et al., 2011). The decline in the relative abundance of Firmicutes might be linked to the lack of relevant food sources during hibernation. As food was digested, the proportion of bacteria from the Firmicutes phylum involved in the metabolism of plant polysaccharides (Boutard et al., 2014) and carbohydrates (Garbacz, 2022), such as Clostridia class and Lactobacillales order, may decrease.

The relative abundance of genus Lactococcus decreased significantly with hibernation duration (Figures 3d, 8). Before hibernation, its relative abundance was 47.2%, but after 120 days of hibernation, it dropped to less than 1%. This observation aligns with the significant decrease in Streptococcaceae during hibernation in some animals, as Lactococcus belongs to this family (Sommer et al., 2016; Tong et al., 2019). Lactococcus is primarily involved in carbohydrate metabolism, and the fasting state during hibernation may lead to a dramatic decrease in the proportion of this genus (Buron-Moles et al., 2019). The decrease in the relative abundance of Firmicutes after hibernation may be related to the sharp decline in the relative abundance of Lactococcus within this phylum. In contrast, the relative abundance of family Lachnospiraceae increased slightly over the course of hibernation. This finding is contrary to some previous studies (Dill-McFarland et al., 2014; Stevenson et al., 2014), which reported decreases in Lachnospiraceae during hibernation.

SCFAs are major metabolites of the intestinal microbiota, which not only provide energy for intestinal epithelial cells, but also stimulate the proliferation of intestinal mucosal cells to maintain gut health (Peng et al., 2009; Donohoe et al., 2011; Fukuda et al., 2012; Morrison and Preston, 2016). The relative abundances of genera Acetobacteroides, Macellibacteroides, and Cetobacterium exhibited a significant increase during snail hibernation (Figure 3d). These organisms play a crucial role in metabolizing host polysaccharides to produce SCFAs such as acetate and propionate (Tsuchiya et al., 2008; Jabari et al., 2012; Zhang et al., 2015; Liu et al., 2023). These SCFAs serve as a vital energy source for the host during hibernation while also stimulating the secretion of intestinal mucin. Similarly, the relative abundances of the genus Bacteroides substantially rose during the hibernation period from 15th to 120th day (Figure 3a). Like the aforementioned genera, Bacteroides also actively participates in the metabolism of host polysaccharides to generate SCFAs (Sonnenburg et al., 2005; Rios-Covian et al., 2017). The collective surge in the abundance of these four genera, all belonging to the phylum Bacteroidota, stands as the primary driver behind the notable increase in the relative abundance of this phylum throughout the hibernation period. In addition, the relative abundance of genus Mycoplasma increased slightly during hibernation (Figures 3d, 8), which may be related to the decreased immune function of the snails in the dormant state.

Deterministic and stochastic processes are thought to play simultaneous roles in the assembly of microbial communities (Chase, 2010; Ning et al., 2020). In this study, deterministic processes dominated the assembly of the gut microbiota in snails before hibernation (Supplementary Figure S2), a pattern consistent with observations in shrimp (Xiong et al., 2017). In contrast, for sea cucumbers, Drosophila simulans (fly), and Dicranocephalus wallichii bowringi (beetle), gut microbiota assembly was mainly controlled by stochastic processes (Zhao et al., 2022; Zhu et al., 2022). These findings reflect that the community assembly of gut microbiota may be influenced by host species. However, some recent studies indicated that microbial community assembly is also related to geographic location, pH, temperature, and other environmental factors (Martinson et al., 2017; Jiao and Lu, 2019). Our results demonstrated that hibernation altered the assembly of the snail gut microbiota (Figure 6; Supplementary Figure S2). During hibernation, stochastic processes (e.g., ecological drift) dominated the assembly of gut microbiota, resulting in divergent temporal succession of microbial communities. In this study, the relative importance of dispersal limitation in the gut microbiota of snails during hibernation decreased, indicating that the restrictions on the transmission or migration of gut microbes within the snails reduced (Zhou and Ning, 2017). The increased diversity of the snail gut microbiota during hibernation may be associated with the decline in dispersal limitation.

We found that the abundance of anaerobic bacteria in the gut of hibernating snails increased significantly with hibernation duration (Figures 7a, 8). During hibernation, the slowed metabolism and reduced gut motility of snails resulted in an anaerobic environment in the gut, leading to a substantial increase of anaerobic bacteria. Some anaerobes can ferment to produce acids, providing energy for the hibernating host and protecting the intestinal mucosa (Sonoyama et al., 2009; Sommer et al., 2016). Interestingly, before hibernation, the snail gut enriched with abundant mobile elements (Figure 7b). However, studies have shown that these mobile elements are significantly positively correlated with intrinsic antibiotic resistance genes (Su et al., 2020). A gut metagenomic study of P. canaliculata showed that its gut microbiota had resistance against environmental pollution stresses like heavy metals and pesticides (Liu et al., 2018). This is analogous to the stress resistance of snails before hibernation in this experiment. But after hibernation, bacteria carrying mobile elements gradually decreased over time. Notably, the potential pathogenicity of the gut microbiota significantly increased with the duration of hibernation (Figure 7e). During hibernation, the metabolic rate of snails decreases to conserve energy, which may compromise their immune system (e.g., through reduced hemolymph circulation and suppressed immune cell activity), thereby diminishing pathogen defense. In addition, the reduced dispersal limitation in the assembly of the gut microbiota may facilitate the invasion and enrichment of pathogens. However, in this study, even after prolonged hibernation (up to 120 days), the snails exhibited a high survival rate of 85%. This suggests that the invasive snail P. canaliculata exhibits unique adaptive strategies during hibernation, enabling it to tolerate the enrichment of intestinal pathogens.

Overall, after the invasive snail P. canaliculata enters hibernation, significant changes occur in the diversity, composition, phenotypic characteristics, and community assembly of its gut microbiota. Post-hibernation, the snails are in a fasting state, and nutrients such as carbohydrates in the intestinal contents are gradually depleted, leading to substantial alterations in the gut environment. This may drive adaptive changes in the gut microbiota to cope with the harsh conditions during hibernation.