Chiang Mai Journal of Science

Environmental Interactions Shaping the Intestinal Microbiota of Cultured Sandfish (Holothuria scabra)

1. INTRODUCTION

     Holothuria scabra is a species of considerable economic importance in aquaculture and traditional medicine and as a source of bioactive chemicals such as fucosylated chondroitin sulfates, peptides, and sphingolipids[1]. Declines in wild populations caused by habitat degradation and overexploitation have increased interest in aquaculture-based production of this species [2-3]. Consequently, several strategies for cultivating H. scabra have been devised to solve the problems posed by declining wild populations. Sea ranching and pond-based culture systems have been implemented across Southeast Asia; however, large-scale and consistent cultivation of H. scabra remains challenging in Thailand and other ASEAN countries due to environmental variability and system-specific limitations [4].
     The gut microbiota plays a crucial role in the biology of H. scabra, contributing to host digestion, nutrient assimilation, metabolic processes, and immune function. In deposit-feeding marine invertebrates, intestinal microbial communities also facilitate the degradation of complex organic matter and may support host adaptation to different environmental conditions [5-6]. Moreover, gut microbial composition has been proposed as a biological indicator reflecting surrounding environmental conditions [7]. Despite the acknowledged significance of host–microbiome interactions, fundamental data regarding the composition and functional potential of gut microbiota in H. scabra across different aquaculture environments is still limited.
     Previous studies have primarily focused on growth performance and survival of H. scabra cultured in different systems. For example, Indriana et al. (2017) reported that outdoors concrete tanks provided optimal circumstances for juvenile H. scabra, leading to enhanced survival and growth. Pond-based culture systems offer semi-natural conditions with greater interaction between sediment, detritus, and microbial communities, which may influence both host performance and microbiota structure [8]. Firdaus and Indriana (2019) demonstrated that tidal earthen ponds were suitable for nursery culture of juvenile sandfish [9]. In contrast, sea pen systems expose sandfish to open marine environments, potentially enhancing microbial diversity through natural ecological interactions while also introducing greater environmental variability. Sabilu et al. (2022) reported improved growth performance of H. scabra reared in sea pens, although such systems may be less consistent due to fluctuating environmental conditions [10]. Recent work on H. scabra juveniles reared in ocean nursery systems has further demonstrated that early life stages exhibit strong microbial sensitivity to environmental conditions, reinforcing the importance of characterizing microbiota across aquaculture settings [11].
     Although early research on the gut microbiota of holothurians, such as H. scabra, has provided insights into the gut microbial structure and its connections to growth, health, and disease resistance, this field is still in the early stages of development. The available information about the effects of different aquaculture systems on the diversity and composition of the gut microbiota in H. scabra is limited. The lack of information represents a knowledge gap that could prove to be critical to commercial operations since cultivation methods may significantly influence gut microbial communities and, consequently, the physiology and health of the species. The importance of filling this gap in the knowledge was highlighted by research into Holothuria glaberrima [12], which revealed substantial differences in gut microbiota composition that were driven by the variations in diet and sediment exposure between wild and cultivated habitats. However, it remains unclear how these findings apply to H. scabra and its gut microbiota across different aquaculture systems.
     The present study aims to address this gap by characterizing the gut microbiota of adult H. scabra reared in three representative aquaculture environments: concrete tanks, pond systems, and sea pens. This work was conducted as a pilot-scale study using high-throughput 16S rRNA gene sequencing to establish baseline information on environment-associated variation in gut microbiota, while acknowledging practical limitations in sample size and environmental metadata. The findings provide baseline information to support future, larger-scale and replicated investigations into host–microbiome interactions and microbiome-informed strategies for sustainable H. scabra aquaculture.

2. MATERIALS AND METHODS

2.1 Ethics
     All procedures in this work were approved by the Animal Care and Use Committee of Prince of Songkla University (Protocol Code:2022-SCI30-055).

2.2 Sample Collection (Experimental Animals)
     Adult sea cucumbers, Holothuria scabra, were collected from concrete tanks (CT), ponds (PO), and sea pens (SP) in the Southern Thailand region of Krabi Province in April and June 2024. The study site is not directly exposed to any major river outflow or urban wastewater inputs, thus reducing direct anthropogenic influence on the source water (Table 1).
     This study was performed as a pilot-scale investigation, focusing on comparative microbiota profiling across three representative aquaculture systems for H. scabra. Nine individuals (n= 3 in each environment) were selected to represent the key environmental conditions used in sandfish aquaculture. Despite the restricted sample size, each specimen was selected based on defined criteria to enhance biological significance and reduce confounding environmental factors.
     All collected specimens were healthy adults of similar size, weighing approximately 500-600 g. to minimize host-related variation in microbiota structure. Exact age and parentage of the specimens were not available, which is a common limitation in field-based sea cucumber collections in Thailand. During culture, individuals were not fed externally; thus, the source of nutrient intake was primarily from sediment substrate. The seawater salinity ranged from 32 to 35.5 ppt, and pH ranged from 7.32-7.60.
     All specimens were promptly transported alive to the laboratory for the experiment. To collect intestinal microbiome populations, specimens were anesthetized in ice for 15 min, then sacrificed by aseptic dissection. The ventral surface was incised with a sterile scalpel to expose the gut within the body cavity. The intestinal contents of each individual were separately placed in sterile 1.5 mL microcentrifuge tubes for further analysis and stored at −80°C.

2.3 DNA Extraction and 16S rRNA Gene Sequencing
     Genomic DNA was extracted from intestinal content samples using the QIAamp DNA Mini Kit (QIAGEN, Germany) following the manufacturer’s protocol. Genomic DNA quality was verified by 1% agarose gel electrophoresis. DNA concentration and purity were determined using the Qubit dsDNA HS Assay kit (Invitrogen) and the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Total genomics were submitted to the U2Bio sequencing service (Illumina, South Korea) for amplicon sequencing of the V3-V4 hypervariable regions of the bacterial 16s rRNA gene using the universal primer sets 341F (3’ CCTACGGGNGGCWGCAG 5’) and 805R (3’ GACTACHVGGGTATCTAATCC 5’). Primary processing was done by removing barcodes, adaptor sequences, and indices.

2.4 Data Processing and Statistical Analysis
     To analyse microbial community data, raw sequence reads were processed using the QIIME2 platform version 2023.9 [13]. High-quality reads were generated through denoising and paired-end read merging using DADA2 via q2-dada2 [14], producing clean 16S rRNA gene amplicon sequence variants (ASVs). Taxonomic assignments were carried out using a Naïve Bayes classifier trained on the SILVA database v.138.1 [15], allowing for accurate assignment of microbial taxa. Relative microbial abundance was calculated, highlighting dominant taxa in each sample. Microbial diversity was assessed using normalized data to provide insight into species richness and evenness with the alpha diversity metrics Evenness, Chao1, Shannon, and Simpson indices. Beta diversity was analyse using Bray-Curtis dissimilarity matrices, with patterns visualized via Principal Coordinates Analysis (PCoA). Differences in diversity and community structure were evaluated using statistical methods. Species variation across groups was calculated with the Kruskal-Wallis test. PERMANOVA was employed to assess beta diversity differences between groups.

2.5 Functional Prediction
     Functional predictions of the microbial communities in the different culturing environments were obtained using PICRUSt2 [16], based on ASV sequences to predict the relative abundance of functional genes, expressed as KEGG orthologs (KOs). The MetaCyc database was then utilized as the reference of metabolic pathways and enzymes [17]. Functional profiles and their distributions were visualized using STAMP software (V.2.1.3) [18] to identify key differences among samples.

3. RESULTS AND DISCUSSION

3.1 The Most Abundant Bacterial Phyla and Dominant Microbial Composition in the Gut of H. scabra
     This preliminary study examined the effects of different aquaculture environments on the gut microbiota of H. scabra, a species of both ecological and commercial significance in Southeast Asia. Sequencing of the 16S rRNA gene generated 446,900 clean reads from nine gut content samples of H. scabra. Rarefaction curves based on ASVs indicated that all samples reached a saturation plateau, confirming that a sequencing depth of 33,205 reads was sufficient to capture the bacterial diversity in H. scabra intestines. Using the SILVA database, the sequences were classified into 845 ASVs, comprising 18 phyla, 39 classes, 93 orders, 142 families, and 186 genera. Across all environments, the gut microbial composition was shown in terms of relative abundance. At the phylum level, Proteobacteria (45.87%) was the predominant phylum, followed by Firmicutes (22.05%) and Actinobacteria (13.58%). Other phyla, including Bacteroidota (4.02%), Cyanobacteria (3.01%), and Chloroflexi (2.81%), were present at lower relative abundances (Figure 1a). This phylum distribution appears broadly similar to patterns reported in other marine invertebrates in several aquatic habitats [19-21]. Proteobacteria constituted the dominant phylum in all environments, a pattern commonly associated with versatility in nutrient acquisition and stress tolerance in marine systems[22] . Similarly, Firmicutes and Actinobacteria may play important roles in metabolism, nutrient assimilation, and host immunity [20]. Their supplementation has been shown to improve growth and immune function, indicating their importance in maintaining sea cucumbers [23]. Additionally, Firmicutes have been identified as a key phylum linked to aestivating sea cucumbers, where their increased abundance during aestivation indicates a possible involvement in metabolic adaptation required for survival during this period [24]. Actinobacteria contribute to gut stability and diversity and associate functional pathways related to amino acid metabolism [23]. Studies that report the high prevalence of Actinobacteria consistent with the antibiotic biosynthesis pathway have also discussed their ability to produce antibiotics [25].
     Comparisons among culture environments revealed differences in the relative abundance of several bacterial phyla. Sandfish reared in concrete tanks and sea pens exhibited broadly similar phylum-level compositions, dominated by Proteobacteria (CT= 50.89% and SP= 48.39%), Firmicutes (CT= 19.92% and SP= 23.47%), and Actinobacteria (CT= 11.39% and SP= 18.29%). In contrast, pond-reared individuals showed higher relative abundances of Actinobacteria (20.10%), Cyanobacteria (5.99%), and Chloroflexi (5.46%). The increased representation of Cyanobacteria and Chloroflexi in pond samples may reflect differences in environmental exposure under semi-natural pond conditions, such as sediment characteristics and light exposure, although these parameters were not directly measured in this study. Chloroflexi is widespread in anoxic environments, including deep subseafloor sediment [26]. Although the roles of Cyanobacteria in sea cucumbers remain unclear, their photosynthetic capabilities as a primary producer suggest a potential involvement in gut nutrient cycling and ecological balance [27].

     At the family level, several dominant taxa were shared across all environments (Figure 1b), including Rhodobacteraceae (CT= 23.73%, PO= 8.93%, SP= 10.38%), Lactobacillaceae (CT= 16.33%, PO= 12.30%, SP= 8.74%), Oxalobacteraceae (CT= 5.21%, PO= 6.30%, SP= 4.65%), Enterobacteriaceae (CT= 5.03%, PO= 16.36%, SP= 3.09%), Sphingomonadaceae (CT= 3.43%, PO= 3.92%, SP= 1.81%), Propionibacteriaceae (CT= 2.11%, PO= 3.50%, SP= 1.18%), Saccharimonadales (CT= 1.90%, PO= 2.42%, SP= 0.54%), Staphylococcaceae (CT= 1.71%, PO= 7.07%, SP= 1.45%), Cyanobiaceae (CT= 1.54%, PO= 0.21%, SP= 3.91%), and Moraxellaceae (CT= 1.38%, PO= 1.25%, SP= 3.89%). The composition varied across environments, with Rhodobacteraceae most prevalent in the concrete tank, while Enterobacteriaceae showed the highest abundance in the sea pen. The prevalence of Rhodobacteraceae, Lactobacillaceae, and Enterobacteriaceae at the family level in all environments may indicate potential core taxa associated with the gut community of H. scabra. Rhodobacteraceae (Proteobacteria) and Lactobacillaceae (Firmicutes) were most abundant in the concrete tank environment. Rhodobacteraceae, representing the most diverse aquatic bacterial groups, comprise aerobic photoheterotrophs and chemoheterotrophs [28]. Meanwhile, Enterobacteriaceae (Proteobacteria) were significantly enriched in sea pens and are a diverse family linked to pathogenesis and virulence [29].
     At the genus level (Figure 1c), Pediococcus was dominant in the gut microbiota of H. scabra (12.16%) with the highest abundance in concrete tanks (15.6%), followed by sea pens (12.17%) and ponds (8.67%). Escherichia-Shigella was the most abundant genus in sea pens with 16.36%, while being less abundant in concrete tanks (4.5%) and ponds (3.09%). Ruegeria exhibited the opposite pattern by reaching its predominance in concrete tanks (12.81%) and lower abundances in ponds (2.7%) and sea pens (4.68%). Staphylococcus was detected only in sea pen samples within this dataset, showing a relatively higher abundance (7.07%), though broader sampling is needed to verify this pattern. Functionally, Pediococcus (Firmicutes) was dominant in concrete tanks, contributes to the lactic acid fermentation process, and has probiotic properties, including pathogen suppression and enhancement of growth performance and enzyme activities in juvenile sea cucumbers [30]. In contrast, most abundances of Escherichia-Shigella in sea pens raise concerns, as it is a member of Enterobacteriaceae (Proteobacteria), which is commonly found in fish and recognized as a potential pathogenic species [21]. Its presence may reflect interactions with environmental bacteria or potential pathogens ingested with particulate material affected by bioturbation [31]. The predominant genus, Ruegeria, a member of Rhodobacteraceae (Proteobacteria) in concrete tanks widely distributed in marine ecosystems, plays roles in nutrient recycling and symbiont interactions [32-33] and is linked to macromolecule polysaccharide degradation, which contributes to digestion and improves disease resistance [34]. A genus-level Venn diagram revealed 48 taxa were shared in three environments, while each environment exhibited a unique microbiota consisting of 48 taxa in concrete tanks, 38 taxa in sea pens, and 81 taxa in the pond environment (Figure 1d). The higher number of unique taxa observed in pond-reared individuals implies greater environmental heterogeneity and microbial exposure in semi-natural pond systems.
     Despite the limited number of biological replicates, similar taxonomic patterns were observed among individuals within each aquaculture environment, suggesting preliminary trends in gut microbial composition. These results provide a descriptive overview of dominant bacterial taxa associated with H. scabra across different culture systems and form a basis for subsequent analyses of diversity patterns and environment-associated variation.

3.2 Bacterial Community Comparison Among Various Aquaculture Environments
3.2.1 Alpha and beta diversity of microbial communities
     Alpha diversity of the gut microbiota in H. scabra was assessed using Evenness, Chao1, Shannon, and Simpson indices. The Evenness and Chao1 indices indicated that pond-reared individuals tended to exhibit higher microbial richness than those from concrete tanks and sea pens. However, Kruskal–Wallis tests showed no significant differences among environments for any alpha diversity metric (P > 0.05; Table 2). Similar trends were observed for the Shannon and Simpson indices, with higher mean diversity values in pond samples but no statistically significant differences. These results suggest that overall microbial richness was largely comparable across culture systems, although pond environments may support slightly greater diversity.
   Beta diversity analysis based on Bray–Curtis dissimilarity suggested that rearing environment contributed to variation in gut microbial community composition. PERMANOVA analysis (adonis2, 999 permutations) indicated a significant overall effect of rearing environment (pseudo-F = 1.286, R² = 0.300, P = 0.042; Supplementary Table S1). Consistent with the PCoA ordination (Figure 2), pond-associated samples tended to cluster separately, whereas concrete tank and sea pen samples showed partial overlap. Pairwise comparisons between individual culture systems were not statistically significant the after false discovery rate correction (FDR) (Supplementary Table S2), likely reflecting limited replication and within-group variability.
     To further assess phylogeny-informed community differences, PERMANOVA was also performed using unweighted and weighted UniFrac distance matrices. In contrast to the Bray–Curtis results, PERMANOVA based on unweighted UniFrac distances (Supplementary Table S3–S4) and weighted UniFrac distances (Supplementary Table S5–S6) did not detect statistically significant differences in gut microbial community structure among rearing environments (P > 0.05). Similarly, no significant pairwise differences were observed after FDR correction for either UniFrac metric.
     Taken together, the contrasting results between Bray–Curtis and UniFrac-based PERMANOVA suggest that variation in gut microbiota among rearing environments was primarily driven by shifts in the relative abundance of shared taxa rather than by changes in phylogenetic community membership. This pattern is consistent with subtle restructuring of dominant microbial groups across aquaculture systems and should be interpreted cautiously due to the pilot-scale sampling and limited replication.

3.2.2 Analysis of the differences in specific microbial taxa among the three environments
     Significant differences in microbial composition were observed among environments. These differences may reflect habitat-associated influences on gut microbial assembly, although further environmental measurements are required to support this. The Planctomycetota and Acidobacteriota phyla exhibited significantly (P < 0.05) increased abundance in the pond (Figure 3); both are commonly found in natural pond ecosystems. A similar pattern has been observed in Apotichopus japonicus, where sediment significantly influenced gut microbiota and metabolic processes [35]. The distinct microbial niche in H. scabra may be shaped by unique characteristics in the pond environment, the water condition, microbial diversity, and their interactions in organic matter sediment. The enrichment of Planctomycetota may related to their known roles in maintaining water quality and ecosystem stability through anaerobic ammonium oxidation (anammox), which reduces ammonia accumulation in water that can result from feed residue and waste excretion. Similarly, Acidobacteriota are recognized for their ability to degrade complex organic compounds and utilize nitrite as a nitrogen source, which also supports nitrogen cycling [36]. Their higher abundance in pond samples may be associated with environmental conditions characteristic of pond substrates, where nitrogen compounds are abundant due to the decomposition of detritus residues. Considering the family level, Cyanobiaceae, particularly the Synechococcus genus, was observed in exclusively high abundance in the pond compared to other environments. As a primary producer within phytoplankton communities in freshwater and tropical marine environments, Synechococcus is known for primary production in aquatic systems; its detection here may reflect ingestion of sediment-associated phytoplankton rather than active ecological functioning within the gut [37]. Regarding the gut of H. scabra, its existence could be attributed to the consumption of sediment-associated phytoplankton and a trophic interaction with other microbial communities where photosynthetically fixed organic carbon supported heterotrophic intestinal microbes [38-39]. Given that no formulated feed was applied, environmental microbial exposure is a plausible contributor to the observed variation, although this cannot be confirmed without environmental microbiome data.

3.3 Functional Prediction of Gut Microbiota Among Three Environments
      Identifying the functional profiles of gut microbiota in H. scabra across distinct environments, using PICRUSt2, mapped KEGG Orthologs (KOs) to 400 metabolic pathways in the MetaCyc database. Metabolic pathways with q-values less than 0.05 were considered significant. Comparative analysis of microbial taxa in concrete tanks revealed 25 abundant paths different from the pond environment, with 4 significantly enriched pathways involved in fatty acid and lipid biosynthesis (fatty acid elongation-saturated), methylphosphonate degradation I, and mio-chiro-scyllo-inositol degradation (Figure 4a). Compared with sea pens, the CMP3-deoxy-D-manno-octulosonate biosynthesis pathway was significantly more abundant in the concrete tank environment, while the 4-aminobutanoate degradation V pathway was significantly enriched in the sea pen environment (Figure 4b). PICRUSt2-based predictions suggested higher relative representation of lipid biosynthesis pathways in concrete tank samples. This pattern may relate to characteristics of the closed tank environment, although organic matter levels were not measured. The CMP-3-deoxy-D-manno-octulosonate biosynthesis is another enriched pathway linked to lipopolysaccharide biosynthesis in Gram-negative bacteria [40]. The dominant phyla in the concrete tank were Proteobacteria and Bacteroidota, which are known for their association with lipid metabolism and organic compound degradation. The interactions among these taxa might facilitate metabolic networks of microbial survival in the concrete tank environments. The enriched pathways in sea pens may be associated with the degradation of 4-aminobutanoate (GABA), suggesting a microbial role in nitrogen and carbon metabolism [41], potentially driven by organic input and sediment interactions in open aquaculture environments. Additionally, the prediction pathway in the pond environment identified 7 pathways that were significantly enriched compared with sea pens. Among these 7 pathways, glycogen biosynthesis I and glycogen degradation I were highly enriched (Figure 4c), highlighting microbial processes that were adapted to the cycling of organic compounds. Within the pond environment, predicted carbohydrate metabolism and energy storage pathways were more prominent, which may reflect differences in microbial functional potential across rearing environments. In particular, the glycogen biosynthesis I implied a propensity to glycogen accumulation that might promote bacterial resistance to abiotic stresses [42-43] and enable metabolic stability during periods of nutrient scarcity. This functional shift in pond environments aligns with the observed increased abundance of Cyanobacteria and Chloroflexi, as glycogen serves as a crucial carbon sink, facilitating metabolic processes that support photosynthesis and cellular respiration [44].
     Manipulation of the microbiome has been suggested in alternative aquaculture systems, and the patterns observed in this study may help guide future investigative efforts for H. scabra. Taxa such as Pediococcus (beneficial lactic acid bacteria) and Ruegeria (associated with polysaccharide degradation and potential pathogen suppression) may represent candidate probiotic taxa. In addition, conditioning pond or tank substrates to promote beneficial microbial consortia could be explored as a strategy to enhance nutrient cycling and host-associated microbial stability in future studies.

4. CONCLUSIONS

     As a pilot-scale assessment, this study provides baseline information on the gut microbiota of Holothuria scabra across three representative aquaculture environments and highlights the importance of integrating environmental metadata and larger sample sizes in future investigations. The results suggest that gut microbial community composition and predicted functional profiles vary among concrete tank, pond, and sea pen systems. Across all environments, Firmicutes, Actinobacteria, and Proteobacteria were consistently dominant, indicating their potential relevance to host-associated microbial communities involved in nutrient processing and physiological functions. Pond-reared individuals showed a tendency toward higher microbial richness and an increased relative abundance of Cyanobacteria and Chloroflexi, which may reflect differences in environmental exposure under semi-natural culture conditions. Functional predictions based on PICRUSt2 suggested environment-associated variation in microbial metabolic potential, with lipid-related pathways more represented in concrete tanks, carbohydrate and energy storage pathways more prominent in ponds, and nitrogen-related pathways more represented in sea pen systems. These functional patterns should be interpreted cautiously, as they are based on predictive analyses and limited replication.
     Overall, this study identifies preliminary compositional and functional trends that may inform future microbiome-focused research in sandfish aquaculture. Further studies incorporating larger, replicated sampling designs, shotgun metagenomics, host–microbiome interaction analyses, and comprehensive environmental measurements will be essential to validate these patterns and to support the development of microbiome-informed strategies for sustainable H. scabra aquaculture.

AUTHOR CONTRIBUTIONS

     Warapond Wanna: Conceptualization, Methodology, Project administration, Funding acquisition, Writing - Original draft preparation, Writing - Reviewing and Editing. Nittaya Jaeram: Investigation, Formal analysis, Data curation, Visualization, Writing - Original draft preparation, Writing- Reviewing and Editing. Aekkaraj Nualla-ong: Investigation, Resources. Komwit Surachat: Formal analysis.

CONFLICT OF INTEREST STATEMENT

     The authors have no competing interests to declare.

FUNDING

     This research was financially supported by the National Science, Research and Innovation Fund (NSRF) and Prince of Songkla University (Grant No. AGR6701237a). Ms. Nittaya Jaeram was supported by the Graduate School, Prince of Songkla University: Grant No. PSU_PHD2565-004.

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