As the global population continues to rise and the demand for sustainable aquaculture increases, the industry is challenged to enhance production while conserving energy and reducing emissions, hence the intensive culture methods are rapidly developing (El-Saadony et al., 2022). Although this farming model has temporarily boosted aquaculture productivity and economic benefits, it has also led to environmental pressures such as water quality deterioration and spatial limitations that weaken the immune function of aquatic animals, promote disease spread, and lead to frequent outbreaks of diseases in aquatic animals (Assefa and Abunna, 2018; Legrand et al., 2020). However, the disease outbreaks under environmental pressures are often closely associated with alterations and imbalances in the host’s intestinal microbial community (Xiong et al., 2015). The intestinal microbiota in aquatic animals, existing in a state of dynamic equilibrium, is crucial for maintaining host health, particularly through the facilitation of immune system development, immune response regulation, and pathogen colonization inhibition, while, disturbances from environmental stress in aquaculture can induce microbiota dysbiosis, leading to pathogen invasion and subsequent disease outbreaks (Huang et al., 2018; Rowland et al., 2018). Especially, this disturbance in the intestinal microbiota (microbiota dysbiosis) will further increase the host’s susceptibility to diseases (Petersen and Round, 2014). Therefore, due to the close relationship between the instability of the intestinal microbial community and host health, efforts have been made to monitor the health of aquatic animals by identifying the signatures of intestinal microbiota dysbiosis in response to diverse environmental stresses, with the aim of timely Bacteroidetes microbiota homeostasis to reduce host morbidity (Xiong et al., 2019).

Currently, the signatures of intestinal microbiota dysbiosis in aquatic animals under various aquaculture environmental pressures have been extensively explored. Specifically, alterations in water quality parameters in aquaculture (e.g., temperature, salinity, ammonium, and nitrite) have been shown to impact the diversity and composition of intestinal microbial community in aquatic organisms (Huang et al., 2018; Wang et al., 2021). A previous study has reported that the species evenness (Shannon index) of the hind-intestine microbial community in sea cucumbers (Apostichopus japonicus) significantly decreases under high temperature (20°C), high concentrations (50 mg/L) of ammonium or nitrite stresses (Zhang Z. et al., 2019). Under elevated salinity conditions (30 practical salinity units), there was an observed increase in the abundance of potential pathogens such as Photobacterium and Pseudoalteromonas, coupled with a reduction in the abundance of beneficial bacteria including the Bacteroidetes in the intestines of Litopenaeus vannamei (Zhang et al., 2016). Furthermore, the environmental stresses (e.g., inadequate culturing space and food) resulting from inappropriate culturing practices has been shown to impact the composition and function of the intestinal microbial community (Dai et al., 2018; Zheng et al., 2023). For instance, the Mycoplasma, a potential pathogen, was notably enriched in the intestines of juvenile Micropterus salmoides under high culturing density stress (160 fish/m³) (Zheng et al., 2023). Under starvation stress, a shift from Firmicutes to Proteobacteria was observed in the dominant phylum in the intestines of leopard coralgrouper (Plectropomus leopardus), and the functional pathways related to nutrient metabolism of the intestinal microbial community in starved L. vannamei were enhanced (Dai et al., 2018; Mekuchi et al., 2018). These findings illustrate the distinct response patterns of intestinal microbiota in aquatic animals under various environmental stresses, which is crucial for enabling early detection and intervention prior to the host progressing to an irreversible disease state by identifying the prevalent indicators of intestinal microbiota dysbiosis (Xavier et al., 2023).

Sea cucumber has emerged as an important pivotal species in mariculture in Asian countries like China, Japan, and South Korea, owing to its considerable nutritional and economic value (Han et al., 2016). As a stenohaline temperate marine species, sea cucumbers have stringent requirements for water quality conditions, including temperature and salinity, and are highly susceptible to disturbances from toxic factors (e.g., ammonium and nitrite) and other aquaculture environmental stresses (e.g., crowding and starvation stress) (Han et al., 2016; Tian et al., 2014; Zhang et al., 2018; Zhang Z. et al., 2019). Accordingly, it is imperative to identify the signatures of intestinal microbiota dysbiosis in sea cucumbers under various aquaculture stresses to dynamically monitor the host’s health status and prevent disease outbreaks. However, the majority of current studies predominantly focus on examining the characteristics of intestinal microbiota dysbiosis in sea cucumbers under different levels of single environmental stress (Pei et al., 2024; Zhao et al., 2019; Zhao Y. et al., 2023; Zhao Z. L. et al., 2023), while lacking investigations into common features of intestinal microbiota dysbiosis under diverse perturbations (Zhang Z. et al., 2019).

Thus, the growth performance, oxidative stress levels, and immune defenses of sea cucumber under various common stresses in aquaculture environments (thermal, hypoosmotic, ammonium, nitrite, crowding, and starvation stress) will be detected to identify the changes in growth and physiological responses of sea cucumber under each aquaculture stress in this study. Moreover, the signatures of intestinal microbiota dysbiosis, along with the crosstalk effect between alterations in the intestinal microbiota and nonspecific defenses in sea cucumbers under different aquaculture pressures will be further explored based on the 16S rRNA gene amplicon sequencing analysis. The findings of this study will advance the comprehension of interplay between intestinal microbiota homeostasis and the health of aquatic organisms, providing reliable indicators for the dynamic surveillance of intestinal microbiota homeostasis in sea cucumber.

Induced by global climate change and unsustainable aquaculture practices, common aquacultural environmental stresses (such as fluctuations in water temperature and salinity, accumulation of toxic compounds like ammonium and nitrite, and limitations in farming space and food resources) can disrupt the equilibrium of intestinal microbiota in aquatic species, leading to an ecological imbalance that increases the host’s susceptibility to pathogenic bacteria (Petersen and Round, 2014). Consequently, researchers have extensively compared gut microbiome-host interactions under normal and various stressful conditions to identify common microbial characteristics linked to intestinal dysbiosis in aquaculture animals (Infante-Villamil et al., 2021; Xavier et al., 2023). However, there remains a paucity of such comprehensive studies on the economically significant species like the sea cucumber. Therefore, this study investigated the effects of thermal, hypoosmotic, ammonium, nitrite, crowding, and starvation stress on the growth, antioxidant, and immune defenses of the sea cucumber, and further attempts to summarize the common and differential microbial characteristics of intestinal microbial dysbiosis in sea cucumber under different stresses.

Physiological variations such as growth and digestive performance, in aquatic species can reflect their adaptive capacity to specific environments (Duan et al., 2017; Limbu et al., 2018; Xie et al., 2023). In this study, a significant negative growth in the body weight of sea cucumber was observed after a 60-day fasting period. Moreover, when under diverse stressful conditions that included a temperature of 23°C, salinity of 22, 0.5 mg/L NH₄⁺-N, 0.25 mg/L NO₂⁻-N, or a stocking density of 6 kg/m³ after 30 or 60 culturing days, there was a notable impairment in the growth performance (as indicated by the WGR and SGR) and digestive capability (as represented by the ADR and FER) of sea cucumber across all experimental groups. These findings suggest the significant inhibition in the physiological process of sea cucumbers under these six stressful conditions.

As a vital component of the sea cucumber’s nonspecific immune system, coelomocytes produce and release large amounts of reactive oxygen species (ROS) and bactericidal humoral factors to enhance immune defense against pathogens and environmental stimuli (Wu et al., 2020). However, prolonged exposure to stress can trigger an oxidative stress response in aquatic species, disrupting the balance between ROS production and removal by the antioxidant system (comprised of various cytokines and antioxidant enzymes), thus resulting in oxidative damage (Li et al., 2023; Zhao Y. et al., 2023). Hence, within the 30-day exposure groups, the substantial increase in the activity of the antioxidant enzyme CAT under hypoosmotic stress, and the augmentation of antioxidant enzyme GSH-PX activity under thermal, ammonium, and nitrite stress, indicating excessive ROS production in coelomocytes under these conditions (Huo et al., 2019a). The marked increase in MDA concentration further reflects lipid peroxidation due to ROS accumulation, leading to peroxidative damage to coelomocytes (Lushchak, 2011). In contrast, persistent fasting stress significantly decreased CAT and GSH-PX activities, likely due to the adverse effects of sustained fasting on the physiological condition of sea cucumbers, thereby weakening their oxidative defense capabilities (Llesuy et al., 2001). Additionally, the minor changes in antioxidant indices observed in sea cucumbers under high-density stress may be attributed to a relatively weaker stress effect that are insufficient to elicit a strong oxidative stress response (Wang et al., 2019).

Furthermore, the imbalance of intracellular redox homeostasis induced by stress can lead to the disruption of the intestinal barrier function in sea cucumbers, and further triggering inflammatory responses and other immune defense processes mediated by the nuclear factor-κB (NF-κB) signaling pathway (Wang et al., 2013; Zhao et al., 2019). Consistently, within the 30-day exposure groups, an overall upregulation trend in the expression of Aj-rel and Aj-p50, genes involved in the NF-κB signaling pathway, was observed in the mid-intestine of sea cucumber. In contrast, Aj-p105 expression showed no significant difference or a decrease compared to the control group, suggesting that stress stimulation may have induced the degradation of Aj-p105 protein and the nuclear translocation of Aj-rel and Aj-p50 proteins (Wang et al., 2013). Additionally, the heat shock protein coding gene (Aj-hsp90) exhibited an upregulation trend in the aforementioned stress groups to protect proteins from oxidative damage and maintain normal cellular physiological functions. These results indicate that the NF-κB signaling cascade and HSPs-related genes are critical for inflammatory and immune responses under different stressful conditions (Huo et al., 2019b). The significant changes in NF-κB pathway gene expression under starvation and crowding stress further highlight its pivotal role in the sea cucumber’s adaptive response to diverse stress. Additionally, due to the flexible nature of lysozyme-induced immune defense in sea cucumbers under stress, variability was observed in the expression of the lysozyme-encoding gene (Aj-lsz) among different stress groups (Huo et al., 2019b).

Previous research has demonstrated that the crosstalk between the innate immune system of aquatic animals and their intestinal microbiota is beneficial for enhancing the host’s immune responses and maintaining the stability of the intestinal microbial ecosystem (Cao et al., 2021). The significant correlation between the expression levels of lectin-encoding genes in the respiratory tree of sea cucumber and the relative abundance of Flavirhabdus was revealed by Song et al. (2019). Similarly, research by Yang et al. (2015) demonstrated that the enrichment of Rhodobacteraceae enhanced the expression of the NF-κB signaling pathway, thereby strengthening the immune defense of the sea cucumber. Consistently, a significant correlation between the relative abundance of most differential species (identified by LDA analysis) between stressed and healthy sea cucumbers and the expression levels of intestinal immune-related genes was revealed in this study. Moreover, the function of intestinal microbiota involved in host immunity in five stress groups (excluding the High density group) was all weakened, which further highlights the close interrelationship between intestinal microbiota dysbiosis under environmental stress and the host’s immune defense functions. Therefore, it is critical to identify the characteristics of microbial imbalances in sea cucumbers under different stress and determine if these responses follow a consistent pattern, allowing for early intervention before the host reaches an irreversible disease state.

In the current study, the disruption of intestinal physiological homeostasis in sea cucumbers under various environmental stress-induced intestinal microbiota dysbiosis and altered intestinal microbial composition and structure (Duan et al., 2021). Specifically, these pressures result in disturbed redox homeostasis of intestinal environments, which could potentially lead to the accumulation of ROS and/or reactive nitrogen species (RNS), and might further lead to a surge proliferation of facultative anaerobic Proteobacteria and a decreasing abundance of obligate anaerobes, including specific taxa within Bacteroidota and Firmicutes (Winter and Bäumler, 2014). Therefore, there was a significant decline in the Bacteroidota: Proteobacteria (B/P) and Firmicutes: Proteobacteria (F/P) abundance ratios in each environmental stress group. Previous studies have shown that certain taxa within Bacteroidota (Karlsson et al., 2011) and Firmicutes (Sun et al., 2023) play a positive role in intestinal homeostasis regulation by participating in the fermentation of complex carbohydrates to modulate intestinal epithelial barrier function and cytokine production. Therefore, a decrease in B/P and F/P ratios often indicates alterations in intestinal microbiota homeostasis and may even indicate the occurrence of intestinal inflammatory responses (Xavier et al., 2023). In this study, it was also found that the healthy sea cucumbers exhibited a greater number of intestinal microbiota network nodes classified as Firmicutes. Additionally, a significant positive correlation was identified between the proportion of core network nodes classified as Bacteroidota and the proportion of negative interspecies interactions, which serve as indicators of microbiota network stability. These findings suggest that the reduced relative abundance of Bacteroidota and Firmicutes may diminish their positive roles in maintaining host intestinal health and suppressing the growth of harmful bacteria. This may induce the outbreak of opportunistic pathogens in the stressed sea cucumbers, such as Vibrionaceae (Xavier et al., 2023), Moraxellaceae (Welter et al., 2021), and Shewanellaceae (Zhang et al., 2016), and then alter the interspecies interactions within the intestinal microbiota network and affect network stability. These results underscore the critical role of Bacteroidota and Firmicutes in sustaining intestinal microbiota homeostasis (Ruzauskas et al., 2021; Xiong et al., 2017). Interestingly, alterations in the intestinal microenvironment also led to a significant enrichment of beneficial microorganisms, such as Achromobacter and Rhodobacteraceae, which are known to promote host growth and immune defense, they were particularly enriched under hypoosmotic and starvation stress, respectively (Wang et al., 2024; Zhang et al., 2020). In both environmental stress groups, relatively higher stability within the intestinal microbiota network was also obtained, which may reflect an adaptive response of intestinal microbiota in sea cucumber to these specific stress. A similar observation was also found in a previous study which revealed an increased stability of the intestinal microbiota in zebrafish exposed to silver nanoparticles (Chen et al., 2021).

The significant shifts in the abundance of these dominant taxa under the distinct stress influenced the survival niches of other taxa, ultimately leading to marked changes in microbiota diversity (Invsimpson index) within each stress group and an increase in microbiota network complexity. These findings of this study are consistent with those reported by Dai et al. (2020), which identified significant alterations in gut microbiota α-diversity associated with dysbiosis. These findings of this study are consistent with those reported by Dai et al. (2020), which identified significant alterations in gut microbiota α-diversity associated with dysbiosis. However, they contrast with findings from studies that consistently observed reductions in both community diversity and complexity under dysbiosis (Barko et al., 2018; Rosenfeld, 2017). These discrepancies may reflect variations in the intestinal microbiota’s responses to different types and intensities of stressors examined across studies. Moreover, it was revealed that the High density group, which might experienced relatively mild stress, did not exhibit the significant alterations in intestinal microbiota structure observed in the other stress groups, in the present study. This finding also underscores the importance of considering both the type and intensity of stress when using significant microbiota structural changes as indicators of ecological dysbiosis in aquatic animals (Xavier et al., 2023). PICRUSt analyses further revealed that organisms adopt distinct/ similar strategies to cope with different stress, different stress drives differential/ consistent functional responses of intestinal microbiota while all consistently exhibit a diminished role in host immunity. For example, under hypoosmotic stress, the intestinal microbiota adapted to environmental changes and maintained basic cellular functions by increasing energy production, adjusting membrane lipid composition, and secreting antioxidants, which collectively led to a significant upregulation in metabolic pathways related to energy metabolism, lipid metabolism, and the metabolism of terpenoids and polyketides (Xie et al., 2023). While, under the chemical stresses of ammonium or nitrite, the intestinal bacterial community of sea cucumbers showed increased involvement in pathways related to cellular communities and signaling interactions, which suggests that the intestinal microbiota enhances interactions within the microbial community and with host cells to cope with environmental stress (Duan et al., 2024). These findings align with the significant indicator species analysis, which indicated that the ammonium and nitrite stress groups shared a significant number of indicator species, accounting for 47.22 and 34.00% of the total identified in each group, respectively. Among these shared species unique to these two stress groups, 29.63% were classified as Flavobacteriaceae, known for promoting carbohydrate utilization, and 11.11% were classified as Sphingomonadaceae, which help regulate intestinal immune responses and maintain gut homeostasis by modulating natural killer T (NKT) cell functions (Oh et al., 2021; Zhang H. et al., 2019).

Under environmental stress, significant changes in the relative abundance of certain beneficial microorganisms or potential pathogens can serve as sensitive indicators for diagnosing the host’s health status. Therefore, the differential species between stressed and non-stressed sea cucumbers were further compared, aiming to identify diagnostic biomarkers that distinguish between stressed and normal intestinal microbiota. LEfSe analysis revealed that the maintenance of intestinal microbial homeostasis in healthy sea cucumbers largely depends on key taxa belonging to Bacteroidota and Firmicutes, such as Lutibacter and Lactobacillaceae, which secrete complex polysaccharide-degrading enzymes and further produce short-chain fatty acids (Barbato et al., 2022; Sartor and Wu, 2017), as well as unclassified_f_Rhodobacteraceae, which enhances the non-specific immune system of sea cucumbers (Yang et al., 2015). Interestingly, in stressed sea cucumbers, along with the enrichment of dysbiosis biomarkers like Paracoccus (Sheu et al., 2018) and potential pathogens such as Aquibacter (Kopprio et al., 2021) and Thalassobius (Tapia-Paniagua et al., 2018), it was also observed that Rubritaleaceae, which belongs to Verrucomicrobiota, Verrucomicrobiae, and Verrucomicrobiales, was significantly enriched at all taxonomic levels (Quan et al., 2019; Zhao Z. L. et al., 2023). In the previous studies, Verrucomicrobia species have also been identified as the significant microbial feature in the gut of grass carp (Ctenopharyngodon idella) and sea cucumbers under nanoplastics stress, and zebrafish exposed to combined polyethylene and 9-Nitroanthracene stress (Li et al., 2023; Zhang et al., 2021; Zhao Z. L. et al., 2023). This might be associated with the important role of Verrucomicrobia species in alleviating chronic low-grade inflammation and enhancing the intestinal barrier in dysbiotic host guts (Lindenberg et al., 2019; Zhao et al., 2024). Consistently, a random forest analysis based on these differential species combinations, validated by a confusion matrix with 95% accuracy, indicated that significant changes in the relative abundance of Verrucomicrobiae, Verrucomicrobiota, and Verrucomicrobiales are the most predictive features for identifying stressed sea cucumbers, further underscoring the potential of Verrucomicrobia species as diagnostic biomarkers for intestinal microbiota dysbiosis in aquatic animals.

This study revealed that under six different types, intensities, and durations of environmental stress stresses (including thermal, hypoosmotic, ammonium, nitrite, starvation, and crowding stress) that inhibited the growth and digestive capacity of sea cucumbers, the organisms exhibited varying degrees of oxidative stress response, which subsequently activated the NF-κB signaling pathway, and induced both common and unique features of intestinal microbiota dysbiosis. The critical roles of Bacteroidota and Firmicutes in maintaining intestinal microbiota homeostasis were demonstrated, while the significant enrichment of Verrucomicrobia species, particularly Verrucomicrobiae, as an important predictive indicator for distinguishing stressed individuals, was identified in this study. The findings indicated that significant alterations in intestinal microbiota diversity, notable decreases in the B/P and/or F/P ratios, and the enrichment of Verrucomicrobia species were common characteristics of intestinal microbiota dysbiosis in sea cucumber under various environmental stresses, these results would help establish efficient health monitoring strategies for sea cucumbers.