B. purificata (body mass: 1.85 ± 0.56 g; shell height: 20.0 ± 2.11 mm; shell width: 14.82 ± 1.35 mm) and P. canaliculata (body mass: 6.55 ± 1.72 g; shell height: 32.68 ± 3.14 mm; shell width: 26.79 ± 3.06 mm) were co-cultured at the rice-snail demonstration base in Ligao Town, Liujiang District, Liuzhou City, Guangxi Province (23.37°N, 111.29°E). The net cages (1 m × 1 m × 1 m) were fully enclosed in structure, with an openable zipper on the top and a base that could be inserted into the sediment for fixation. They were arranged in the paddy field in a layout of 3 columns × 6 rows, totaling 18 cages, with a spacing of 1 meter between both columns and rows.

Six experimental groups were established: B. purificata monoculture (BI-30/60), P. canaliculata monoculture (PI-30/60), and co-culture with B. purificata (BM-30/60) and P. canaliculata (PM-30/60) at a 5:1 biomass ratio (BM: PM). Each group was set up with three biological replicates. The monoculture replicates contained 30 individuals of either B. purificata or P. canaliculata, whereas each replicate in the co-culture group consisted of 25 B. purificata and 5 P. canaliculata (Liu M. et al., 2025).

Sampling occurred at 30 and 60 days post-co-culture initiation following 24-h starvation to minimize intestinal food residues. To minimize bias caused by individual variation, this experiment employed a pooled sample strategy. Five pooled sequencing samples were prepared for each group. Each pooled sample was created by randomly selecting 10 individuals and combining equal amounts of their DNA samples.

Under aseptic conditions, shells were disinfected with 75% ethanol before extracting soft tissues. Tissues were rinsed thrice with ice-cold sterile 1 × PBS, and intestinal dissection was performed on sterile Petri dishes maintained on ice using sterile instruments. Whole intestinal segments were washed three times with sterile PBS, with luminal contents separately scraped using sterile surgical blades into 4% formaldehyde for dietary analysis. Residual intestinal tissues were homogenized in sterile centrifuge tubes using a Tissuelyser-LT (QIAGEN, Shanghai, China). Microbial DNA was extracted from homogenate using HiPure Stool DNA Kits (Magen, Guangzhou, China) according to manufacturer protocols. All extracts were stored at −80 °C prior to library construction.

The V3-V4 region of the 16S rRNA gene was amplified by PCR using universal primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTATCTAAT-3′) (Guo et al., 2017). Amplifications were performed in a 50 μL reaction volume containing 10 μL of 5 × Q5® Reaction Buffer, 10 μL of 5 × Q5® High GC Enhancer, 1.5 μL of 2.5 mM dNTPs, 1.5 μL of each primer (10 μM), 0.2 μL of Q5® High-Fidelity DNA Polymerase, and 50 ng of template DNA. All PCR reagents were obtained from New England Biolabs, United States. The thermal cycling conditions consisted of an initial denaturation at 95 °C for 5 min; followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min, and extension at 72 °C for 1 min; with a final extension at 72 °C for 7 min. Amplified products were pooled, purified, and quantified using a QuantiFluor™ fluorometer (Promega, Beijing, China). Purified amplicons were sequenced on the Illumina HiSeq 2500 platform using a paired-end strategy (PE250) according to the manufacturer’s standard protocols. Raw sequencing reads have been deposited into the NCBI Sequence Read Archive database under accession number PRJNA778015.

To obtain high-quality clean reads, raw reads containing over 10% unknown nucleotides (N) or with fewer than 50% of bases having a Phred quality score (Q-score) > 20 were removed using FASTP (Chen et al., 2018a). Paired-end clean reads were merged into raw tags using FLASH (v1.2.11) with a minimum overlap of 10 bp and a maximum mismatch rate of 2% (Salzberg, 2011). Noisy sequences within raw tags were filtered under specific conditions using the QIIME (v1.9.1) pipeline to generate high-quality clean tags (Caporaso et al., 2010). Briefly, raw tags were truncated starting from the position of the first low-quality base (default quality score ≤3) where the length of a continuous low-quality segment reached the threshold (default length: 3). Tags where the longest contiguous stretch of high-quality bases was shorter than 75% of the total tag length were discarded. Clean tags were then compared against the reference database¹ to perform reference-based chimera detection using the UCHIME algorithm (Knight, 2011). All chimeric tags were removed to obtain effective tags for downstream analysis.

Algae and zooplankton within Dietary Composition were identified and quantified via microscopic examination. Given that this study utilizes pooled samples to evaluate average inter-group effects and focuses primarily on community-level responses at higher taxonomic ranks, we employed the OTU-based clustering approach. Specifically, high-quality sequencing reads were clustered into operational taxonomic units (OTUs) at 97% sequence similarity using the UPARSE pipeline (v9.2.64) for downstream intestinal microbiota analyses. The most abundant sequence within each OTU was designated as its representative sequence. Venn diagrams (VennDiagram v1.6.16) and UpSet plots (UpSetR v1.3.3) were employed to visualize unique and shared OTUs across experimental groups. Representative sequences were classified against the SILVA database using the RDP Classifier (v2.2) with a naive Bayesian model at a confidence threshold of 80%. Taxonomic abundance was visualized with Krona (v2.6) (Ondov et al., 2011). Community composition was illustrated through stacked bar plots (ggplot2 v2.2.1) and ternary diagrams (ggtern v3.1.0). Alpha diversity indices (including Sobs, Chao1, ACE, Shannon and Simpson) were calculated in QIIME, with rarefaction curves and rank-abundance plots generated using ggplot2. Inter-group alpha diversity comparisons utilized Welch’s t-tests (vegan v2.5–3), while multi-group differences were assessed by Kruskal-Wallis tests with Tukey’s HSD post-hoc analysis. Beta diversity was evaluated through Principal Coordinates Analysis (PCoA) of Bray-Curtis distances (vegan v2.5-3), with statistical significance determined by PERMANOVA (adonis function). Differential abundance testing for indicator species identification was performed using Welch’s t-tests (vegan), while biomarker screening implemented the labdsv package (v2.0-1). Functional potential was predicted via KEGG pathway analysis of OTUs using PICRUSt2 (v2.1.4).

At 30 days of cultivation, the average body weights of B. purificata in the monoculture and co-culture groups were 2.85 g and 2.05 g, respectively, with average shell heights of 26.32 mm and 19.02 mm and survival rates of 96.7 and 78.9%. For P. canaliculata, the average body weights in the monoculture and co-culture groups were 10.48 g and 9.17 g, respectively, with average shell heights of 29.57 mm and 28.47 mm and survival rates of 97.8 and 93.3%.

At 60 days of cultivation, the average body weights of B. purificata in the monoculture and co-culture groups were 3.84 g and 2.45 g, respectively, with average shell heights of 27.82 mm and 23.11 mm and survival rates of 91.1 and 68%. For P. canaliculata, the corresponding values were14.41 g and 11.79 g for average body weight, 32.35 mm and 30.15 mm for average shell height, and 92.2 and 81.1% for survival rate.

These results indicate that, at both 30 and 60 days of cultivation, the body weight, shell height, and survival rate of both B. purificata and P. canaliculata in the co-culture group were significantly lower than those in their respective monoculture groups (Table 1).

The feeding habits of aquatic animals are influenced by foraging modes, growth stages, physiological activities, and environmental factors. Understanding these trophic dynamics is essential for elucidating ecological relationships between organisms and their environment, particularly regarding their nutritional niches within ecosystems (Han et al., 2013).

In all experimental groups, Chlorella, Scenedesmus, and Oscillatoria constituted predominant components of dietary composition, indicating consistent consumption of these algae by both snail species. This suggests potential interspecific competition for algal resources (Yang et al., 2024). Compared to monoculture groups, B. purificata co-culture groups exhibited an increased proportion of Eudorina but decreased Euglena in their dietary composition. For P. canaliculata co-culture groups, elevated proportions of Pinnularia, Scenedesmus, Gyrosigma, and Chlorella were observed alongside reduced proportions of Phacus, Ulothrix, and Oscillatoria. These shifts likely reflect food competition or selective feeding under co-culture conditions (Svanbäck and Bolnick, 2007). Temporal variations revealed distinct foraging preferences: after 60 days compared to 30 days, B. purificata showed increased consumption of Kirchneriella and Eudorina but decreased Melosira, while P. canaliculata exhibited reduced Eudorina intake. These ontogenetic dietary shifts align with established patterns of feeding plasticity during growth (Bryan et al., 2021; Hüne et al., 2023).

Water environment samples (WE-30/WE-60) demonstrated stable algal composition, with Chlorella, Scenedesmus, Euglena, and Phacus dominating both periods. Notably, these algae that were abundant in the water environmental samples consistently comprised a high proportion across all samples. Although Cyclotella accounted for 4% of the composition in the WE-30 water sample, it was only detected in the BI-30 culture group. This pattern highlights both the similarities and distinctions between the environmental availability of resources and the dietary composition. Therefore, it can be concluded that both the culture method (monoculture or co-culture) and the duration of culture (30 d or 60 d) had significant effects on the gut dietary composition of B. purificata and P. canaliculata at the genus level. The shifts in the relative abundance of various genera reflect their feeding preferences and ecological adaptability under different environmental conditions. Furthermore, a close trophic association was observed between the aquatic animals and their surrounding water environment.

Based on the α- and β-diversity analyses of the intestinal dietary composition in B. purificata and P. canaliculata, this study revealed patterns of food resource utilization by these two snail species under different cultivation modes.

At 30 days of cultivation, the monoculture groups of both snail species exhibited significantly higher α-diversity indices compared to their co-culture groups. A significant difference in intestinal food community structure was observed between the monoculture and co-culture groups of B. purificata, whereas no such significant difference was found for P. canaliculata. Therefore, it is hypothesized that interspecific competition may have led to a compression of their dietary niches (Schulze et al., 2012). P. canaliculata, potentially due to its larger body size, may hold a competitive advantage in resource acquisition, or its broader dietary range may contribute to the stability of its gut food community structure (Wang Y. et al., 2025). In contrast, B. purificata may have undergone adjustments in its feeding behavior in response to the pressure from interspecific competition (Charette et al., 2024).

At 30 days of cultivation, the monoculture groups of both snail species exhibited notably higher α-diversity indices compared to their co-culture groups. A significant difference in intestinal food community structure was observed between the monoculture and co-culture groups of B. purificata, whereas no such marked difference was found for P. canaliculata. Therefore, it is hypothesized that interspecific competition may have led to a compression of their dietary niches. P. canaliculata, potentially due to its larger body size, may hold a competitive advantage in resource acquisition, or its broader dietary range may contribute to the stability of its gut food community structure. In contrast, B. purificata may have undergone adjustments in its feeding behavior in response to the pressure from interspecific competition.

At 60 days of cultivation, no substantial differences were detected in either α-diversity indices or intestinal food community structure between the co-culture and monoculture groups for either snail species. This may be attributed to the fact that, in response to the prolonged stress from P. canaliculata, B. purificata may have enhanced its utilization of dominant food resources. This behavioral adjustment could have facilitated the avoidance of direct competition and a reduction in niche overlap, ultimately leading to a gut food composition in the co-culture group that approximated its state under monoculture conditions (Duan, 2013).

Under monoculture conditions, B. purificata exhibited a trend of decreasing dietary diversity over time, whereas the opposite trend was observed under co-culture conditions. This likely reflects adjustments in its feeding behavior in response to interspecific competitive pressure. In contrast, P. canaliculata showed no significant temporal changes in diversity under either monoculture or co-culture conditions, demonstrating greater stability in biotic interaction environments.

The lack of significant differences in the α-diversity of the aquaculture water and in Chao1 and ACE indices among all groups indicates that the observed changes in food composition are more likely attributable to active feeding selection and behavioral adaptation by the snails, rather than to alterations in the available food resource pool itself.

Collectively, these results suggest that B. purificata is initially more sensitive to competitive pressure but gradually exhibits dietary adaptation over time, whereas P. canaliculata maintains a relatively stable pattern.

Dominant microbial phyla in aquatic organisms predominantly include Proteobacteria, Bacteroidota, and Firmicutes (Shi et al., 2023). Proteobacteria encompasses diverse bacteria with functional roles in nutrient metabolism (Feng et al., 2008), immunomodulation (Sugita et al., 1996), and environmental adaptation (Xiang et al., 2022), though many species within this phylum are pathogenic. While Enterobacteriaceae can convert non-essential amino acids into essential amino acids or proteins (Ben-Yosef et al., 2010), pathogenic members like Escherichia and Salmonella may cause diarrhea and enteritis (Janda and Abbott, 2021). Similarly, Aeromonadaceae species produce extracellular enzymes enhancing host digestion (Zeng and Yuang, 1999; Koca et al., 2015), yet Aeromonas veronii induces intestinal inflammation in fish (Yin, 2022), and A. hydrophila causes bacterial septicemia (Sun et al., 2023). Firmicutes contribute crucially to host nutritional metabolism and immunoregulation, facilitating dietary energy harvest through carbohydrate fermentation while producing anti-inflammatory compounds that maintain intestinal homeostasis (Säemann et al., 2000; Magne et al., 2020). For instance, Lactococcus can modulate lysozyme activity, inhibit pathogen growth, promote mucus secretion, and strengthen the physical barrier function (Paritova et al., 2024), and Bacillus species induce antimicrobial peptide production, enhancing mucosal defense (Safarchi et al., 2025).

Proteobacteria and Firmicutes constituted the predominant phyla in the gut microbiota of B. purificata and P. canaliculata, representing over 99 and 85% of the total abundance, respectively. At the genus level, Lactococcus, Enterobacter, and Aeromonas collectively accounted for over 85 and 73% in all groups of the two species, respectively. These taxa represent the core microbiota at the phylum and genus levels in both snails, indicating fundamental roles in host metabolic and immune processes.

The gut tract is one of the most closely connected interfaces between the host and the external environment. Previous studies have shown that the invasion of P. canaliculata can occupy the living areas of native species, reduce food abundance, and secrete harmful substances that alter the aquatic environment (such as pH and transparency), leading to disease or death among local snails (Liu M. et al., 2025).

In this study, at 30 days of cultivation, the gut microbial abundance showed no significant difference between co-culture and monoculture groups of B. purificata, while the microbial diversity was appreciably higher in co-culture groups. By 60 days, neither microbial abundance nor diversity differed decidedly between co-culture and monoculture groups of B. purificata. These results indicate that co-culture did not significantly affect microbial abundance in either snail species, but it transiently increased gut microbial diversity in the short term. However, this effect diminished over time. Studies on yellow catfish (Zhu et al., 2021), opsariichthys (Cui et al., 2025), grass carp (Xiao et al., 2023) and kelp grouper (Li et al., 2015) have generally reported that polyculture can enhance the diversity of intestinal microbiota in aquatic animals. In contrast, our study observed higher microbial diversity in monoculture groups at 60 days, which may be associated with changes in environmental biochemical factors, distinct growth stages, feeding habits, and immune-metabolic adaptations (Li et al., 2015; An, 2024).

Both monoculture and co-culture groups showed a decreasing trend in gut microbial abundance and diversity over time. However, aside from a significant reduction in microbial diversity observed in the co-culture groups at 60 days compared to 30 days, no other differences were statistically significant. This pattern may be attributed to the prolonged consumption of resources in the water environment, which led to a decline in microbial availability in the aquaculture system, thereby reducing gut microbial diversity (Wang et al., 2025).

Notably, no significant difference was observed in the microbial community structure between the monoculture and co-culture groups of B. purificata at 30 days, whereas a statistically significant difference emerged by 60 days. This indicates that the stress exerted by P. canaliculata on B. purificata exhibits a time-dependent effect. The underlying reason may be that prolonged invasion by P. canaliculata may alter physicochemical factors of water environment and impose competitive pressure on B. purificata in terms of food resources and habitat space (Zhang et al., 2017). Such environmental changes could induce a stress response in B. purificata, thereby altering its gut microbial structure (Wei et al., 2024). Alternatively, B. purificata may adjust its feeding habits to alleviate interspecific competition for limited food resources (Li J. et al., 2025; Tao et al., 2023), resulting in structural and functional adaptations of the gut microbiota to dietary shifts, which help maintain nutrient absorption and intestinal homeostasis (Zhao et al., 2025). It is thus inferred that the significant shift in the microbial community structure of B. purificata may represent a responsive adaptation to the influence of P. canaliculata invasion (Givens et al., 2015).

Furthermore, the significant differences in gut microbial community structure observed between 30-day and 60-day monoculture groups may reflect functional changes in the gut microbiota associated with snail growth and developmental stages (Liu et al., 2022). This finding is consistent with numerous studies reporting that the intestinal microbiota of aquatic animals undergoes dynamic successional changes throughout development (Yan et al., 2012).

At the phylum level, no significant differences were observed in the relative abundance of gut microbiota between monoculture and co-culture groups of B. purificata at 30 days. In contrast, at 60 days, the co-culture group showed significantly higher relative abundances of Bacteroidota, Actinobacteriota, and Planctomycetota compared to the monoculture group (p < 0.05). Bacteroidota and Actinobacteriota are considered beneficial bacterial phyla in the intestinal tracts of aquatic animals. Bacteroidota contributes to host growth by efficiently utilizing low-carbon compounds (Xu et al., 2024), while Actinobacteriota produces antibacterial substances and nutritional factors that suppress pathogenic bacteria and promote growth in aquaculture species (James et al., 2023; Liu M. Q. et al., 2025). Planctomycetota, commonly found in the intestinal tracts of aquatic animals, water environments, and sediments, plays an important role in nitrogen cycling (Huang et al., 2024).

At the genus level, after 30 days of cultivation, the co-culture group of B. purificata showed a considerably higher relative abundance of Acinetobacter but dramatically lower relative abundances of Pseudomonas, Shewanella, Edwardsiella, Candidatus_Bacilloplasma, and Candidatus_Hepatoplasma compared to the monoculture group. Most species of the genus Acinetobacter have been characterized as pathogens. Their increased presence in the gut has been linked to morphological changes in endocrine cells and a state of nutrient insensitivity, potentially even contributing to metabolic diseases (Wang et al., 2023). Pseudomonas, Shewanella, Edwardsiella, and Acinetobacter are commonly present in the intestines of healthy aquatic animals (Wang, 2021; Liu et al., 2024; Chen, 2024). Many species within Pseudomonas and Shewanella have been identified as potential probiotics and are used in aquaculture (Wu et al., 2012; Jiang, 2020). For example, supplementing grass carp feed with Pseudomonas monteilii JK-1 has been shown to increase body weight, improve specific growth rate and survival, reduce pathogen load, and enhance the expression of immune-related genes and the activity of antioxidant enzymes (Qi et al., 2024). Similarly, adding Rhodopseudomonas palustris to the diet of Penaeus japonicus greatly enhances immune and antioxidant levels, alleviating stress induced by pesticide residues (Li, 2024). Supplementing Pacific white shrimp feed with Shewanella notably improves weight gain and specific growth rate, induces the expression of antioxidant- and immune-related genes, enriches beneficial gut bacteria, and reduces the abundance of potential pathogens (Wei et al., 2021). The functional roles of Candidatus_Bacilloplasma and Candidatus_Hepatoplasma remain poorly understood. Some studies suggest that the relative abundance of Candidatus_Bacilloplasma is significantly higher in fast-growing shrimp populations compared to slow-growing ones (Imaizumi et al., 2022); while sarvation has been observed to reduce its abundance in river crabs, which recovers upon refeeding (Wang et al., 2019), implying a potential role in growth and metabolism. Few studies have focused on the influence of Candidatus_Hepatoplasma in aquatic animals, though its abundance in crab hepatopancreas is highly correlated with hepatopancreatic necrosis disease (Shen et al., 2017). In terrestrial isopods, Candidatus_Hepatoplasma is generally thought to enhance host survival under low-nutrient conditions (Leclercq et al., 2014).

After 60 days of cultivation, the co-culture group of B. purificata exhibited decidedly higher relative abundances of Aeromonas, Cloacibacterium, Kurthia, Planktothrix_NIVA-CYA_15, Gluconobacter, Microseira_Carmichael-Alabama, and Methyloparacoccus, while the relative abundances of Enterobacter, Enterococcus, Candidatus_Hepatoplasma, LactoBacillus, and Akkermansia were markedly lower compared to the monoculture group. Aeromonas and Enterobacter showed the most notable differences between monoculture and co-culture groups. Both are core microbiota in the gut tract of B. purificata, and many species within these genera have been identified as pathogen (Kang et al., 2018; Shi et al., 2021; Cui et al., 2022). In contrast, the other mentioned bacterial groups have not been reported to cause diseases in aquatic animals and are largely considered beneficial. Cloacibacterium, a core microbe in freshwater gastropods, can be vertically transmitted among hosts, promoting adaptive evolution and supporting host growth and health (Lin et al., 2023). Gluconobacter participates in the oxidation of various sugars and polyols, facilitating energy cycling within the gut microbial community. Enterococcus contributes to enhanced carbohydrate and protein metabolism, non-specific immune enzyme activity, and disease resistance in the host (Satish et al., 2011; Ju et al., 2018). LactoBacillus inhibits the growth of pathogenic microorganisms and strengthens digestive enzyme and non-specific immune enzyme activities (Zhang et al., 2017; Shui et al., 2021; Zhang et al., 2024). Akkermansia promotes intestinal mucus secretion, modulates mucosal barrier function, helps regulate blood glucose levels, and maintains glucose homeostasis (Liu Y. J. et al., 2025). Additionally, Planktothrix_NIVA-CYA_15 and Microseira_Carmichael-Alabama are common Cyanobacteria. The former is often abundant in eutrophic water bodies, and the latter proliferates in phosphorus-enriched sediments (Pancrace et al., 2017; Anderson et al., 2019). While the OTU clustering method employed in this study is suitable for detecting community changes at higher taxonomic levels, its resolution for distinguishing between strains within highly diverse genera is limited. Therefore, the observed changes in relative abundance at the genus level should be interpreted as indicators of broader microbial community restructuring under invasion pressure, rather than as definitive evidence for the increase or decrease of specific functional groups.

At 30 days of cultivation, stress from P. canaliculata led to a significant increase in Acinetobacter within the gut microbiota of B. purificata, which may contribute to metabolic disorders in this species. Concurrently, the relative abundances of several probiotic bacteria decreased, indicating a notable negative impact of co-culture with P. canaliculata on B. purificata at this stage. By 60 days of cultivation, the relative abundances of both pathogenic and beneficial bacteria in the gut microbiota of B. purificata showed simultaneous increases and decreases. This pattern of change may be attributed to multiple factors including the environment, diet, and host growth. The results indicate that the stress from P. canaliculata still exerted some influence on B. purificata, though its adverse effects were less pronounced. Some of the observed shifts further suggest that B. purificata may have developed a degree of adaptation to the stress imposed by P. canaliculata. For instance, the increased relative abundance of Cloacibacterium may have promoted adaptive evolution in B. purificata (Lin et al., 2023). Shifts in dietary composition, such as increased proportions of Chroococcus, Stauroneis, and Pediastrum, and decreased proportions of Cymbella and Oscillatoria—suggest altered feeding strategies in response to food competition with P. canaliculata. Furthermore, the increased abundance of Methyloparacoccus, which uses methane as its sole carbon source (Deng et al., 2015), may reflect metabolic adaptations to energy resource competition.

At both the 30-day and 60-day culture periods, the relative abundance of Euglena in the intestine of B. purificata in the co-culture group was higher than that in the monoculture group. Although Euglena is considered a beneficial alga that can activate the immune system, promote beneficial bacteria, and inhibit pathogens (Rodríguez-Zavala et al., 2010; Suzuki et al., 2018), its relative abundance in this study showed a significant positive correlation with the relative abundance of the conditional pathogen Aeromonas. Existing studies have confirmed that the efficacy of prebiotics is closely related to the physiological state of the host (Hashemitabar and Hosseinian, 2024; Zhang et al., 2024). For instance, probiotics that enhance the immunity of rainbow trout under 20 °C heat stress become ineffective under the more severe stress of 25 °C (Nakhei et al., 2023). It is therefore hypothesized that stress induced by P. canaliculata on B. purificata alters the intestinal microenvironment, leading to an anomalous relationship between the abundances of Euglena and Aeromonas.

PICRUSt2 analysis revealed that metabolism-related pathways accounted for the highest proportion of enriched functional genes in the intestinal microbiota of both P. canaliculata and B. purificata, which is consistent with the findings reported by Li Han in different geographic populations of Quasipaa spinosa (Li et al., 2025b). This suggests that the gut microbiota may play an important role in the digestion and metabolism of complex biochemical substances within the intestine.

At 30 days of cultivation, the co-culture group of B. purificata exhibited appreciably higher enrichment in pathways associated with neurodegenerative diseases, and significantly lower enrichment in immune diseases and signaling molecules and interaction pathways compared to the monoculture group. This implies that short-term stress from P. canaliculata might negatively affect might negatively affect the neural and immune functions in B. purificata. In contrast, the co-culture group of B. purificata at 60 days showed considerably higher enrichment in pathways related to cell motility, cellular community – prokaryotes, and the digestive system compared to the 30-day group. These observations suggest that prolonged stress from P. canaliculata could be associated with enhances the motility and digestive intensity of the intestinal microbiota in B. purificata, potentially representing an adaptive strategy to improve competitive advantage during development.

Overall, these predictive functional results indicate short-term stress from P. canaliculata may have adverse effects on B. purificata, while long-term stress appears to be linked to regulatory and adaptive responses in its intestinal microbiota. It should be noted that these interpretations are derived from in silico predictions, and further experimental validation is needed to confirm the associated phenotypic and physiological implications.