Lactiplantibacillus plantarum O126 postbiotics boost growth, antioxidant levels, disease resistance, and gut health in sea cucumber (Apostichopus japonicus)

Lactiplantibacillus plantarum O126 postbiotics boost growth, antioxidant levels, disease resistance, and gut health in sea cucumber (Apostichopus japonicus) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Lactiplantibacillus plantarum O126 postbiotics boost growth, antioxidant levels, disease resistance, and gut health in sea cucumber (Apostichopus japonicus) Feng-Li Chen, Hui-Ting Zhao, Wei Gu, Zong-Xiu Wu, Jing-Wen Xiang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7545463/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Feb, 2026 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted 12 You are reading this latest preprint version Abstract Lactiplantibacillus plantarum ( L. plantarum ) postbiotics promote animal health, but their application in Apostichopus japonicus is currently limited. This study added L. plantarum O126 postbiotics to the diet at concentrations of 1‰, 2‰, and 4‰ (w/w) for 42 days to assess their impacts on A. japonicus. The results showed that, in terms of growth performance, the medium-dose (2‰, LPP-M) and high-dose (4‰, LPP-H) groups significantly increased the final body weight and specific growth rate (SGR), and reduced the feed conversion ratio (FCR). Regarding digestion and gut morphology, the LPP-M and LPP-H groups significantly enhanced gut trypsin and lipase activities and increased the height and width of midgut villi. With respect to antioxidant capacity, L. plantarum O126 postbiotics increased T-AOC, CAT, and SOD levels and upregulated the expression of antioxidant-related genes PRDX5 and PRDX6 . In terms of immunoregulation, L. plantarum O126 postbiotics increased ACP and AKP activities and upregulated the expression of immune-related genes Rel , P50 , and C3-2 . Regarding disease resistance, after the Vibrio splendidus challenge, all experimental groups exhibited reduced evisceration rates, with the LPP-M group showing the lowest rate (40%). In terms of gut microbiota, the α-diversity of the experimental groups was significantly increased, with higher levels of beneficial bacteria ( Bacillus , Exiguobacterium , Vagococcus ) observed in the LPP-M and LPP-H groups. Overall, L. plantarum O126 postbiotics improved the growth and disease resistance of A. japonicus by enhancing digestion, antioxidant capacity, immune function, as well as by regulating gut microbiota, with a medium dose (2‰, w/w) showing the best effect. Lactiplantibacillus plantarum postbiotics Apostichopus japonicus disease resistance gut microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Sea cucumber ( Apostichopus japonicus ), long regarded as a premium tonic food in Asia, particularly in China, has gained widespread popularity among consumers due to its unique nutritional and medicinal properties. The sustained growth in market demand has driven the rapid development of A. japonicus aquaculture. A. japonicus aquaculture stands as a key pillar in China's marine aquaculture industry [ 1 ]. However, there are numerous challenges, including persistently high feed costs, deterioration of aquaculture water environments, frequent disease outbreaks, and the overuse of pesticides and antibiotics. These issues not only reduce the economic benefits of A. japonicus aquaculture but also lead to secondary problems such as environmental pollution. To address these issues, probiotics have been introduced as feed additives in A. japonicus aquaculture, demonstrating beneficial effects on promoting growth, improving gut health, and modulating microbiota balance [ 2 ]. Given their potential as alternatives to antibiotics, probiotic applications could potentially reduce the risk of antimicrobial resistance dissemination and mitigate public health concerns related to antibiotic residues in food products [ 3 – 5 ]. However, live probiotics may destabilize aquaculture ecosystems and exhibit poor processing and storage stability. According to the International Scientific Association for Probiotics and Prebiotics (ISAPP ) , postbiotics denote inactivated microbial entities and/or constitutive elements that provide health benefits to the host, thus representing a safer and more promising alternative [ 6 , 7 ]. Lactiplantibacillus plantarum ( L. plantarum ), a typical gut-commensal lactic acid bacterium, has been shown to possess multiple probiotic properties [ 8 , 9 ]. Research has demonstrated significant beneficial effects conferred by L. plantarum postbiotics on marine organisms. Nguyen et al. found that heat-killed L. plantarum L-137 significantly improved immune capacity in Nile tilapia [ 10 ]. Similarly, Zheng et al. verified that cell-free extracts of L. plantarum could effectively regulate gut microbiota in Pacific white shrimp [ 11 ]. Additionally, Rawling et al. found that heat-inactivated L. plantarum HA-122 postbiotics could enhance gut immune function in zebrafish by reinforcing the gut mucosal barrier [ 12 ]. The above research outcomes robustly support the use of L. plantarum -derived postbiotics in aquaculture, though their specific functions in A. japonicus aquaculture remain insufficiently studied. This study systematically evaluated the impacts of postbiotics derived from L. plantarum O126 on the growth, antioxidant and immune capacity, disease resistance, and gut health in A. japonicus . This supports their application as a sustainable strategy for improving the productivity and health management of A. japonicus aquaculture. Materials and Methods Research animals and rearing conditions A. japonicus individuals were sourced from an A. japonicus aquaculture facility in Weihai, Shandong, China. After a 7-day acclimation period, 480 healthy individuals with an average body weight of 17.35 ± 1.76 g were divided into 12 aquariums (100 × 80 × 60 cm³). There were three aquariums in each group, with 40 individuals in each aquarium. Each animal received a single daily feeding at 18:00, with the amount allotted equal to 3% of its total body weight. The water conditions were consistently maintained with the temperature at 15 ± 1°C, dissolved oxygen > 8 mg/L, salinity of 30‰, and a pH of 8.0 ± 0.05. Diet composition Purchased from Guangdong Haid Group Co., Limited (Guangzhou, China), the composition of the basal feed is shown in Table S1 . L. plantarum O126 postbiotics were provided by Shandong Baolai-Leelai Bioengineering Co., Ltd. (Yantai, China). These postbiotics contained inactivated microbial biomass exceeding 1 × 10 11 cells per gram, with lactic acid content > 10% and crude polysaccharide content > 5%. L. plantarum O126 postbiotics were supplemented to the basal diet of A. japonicus at distinct doses: 1‰ (w/w) (designated as the LPP-L group), 2‰ (w/w) (designated as the LPP-M group), and 4‰ (w/w) (designated as the LPP-H group). The control group received solely a standard basal diet. Sample collection At weeks 0, 2, 4, and 6, we dissected six A. japonicus individuals per group under sterile conditions to obtain body wall, muscle, respiratory tree, foregut, midgut, and hindgut tissues. The coelomic fluid was aspirated using sterile syringes and centrifuged at 4°C for 15 min at 800 ×g , with the resulting supernatant aliquots subjected to enzymatic assays [ 13 ]. At week 6, the midgut tissues of A. japonicus (three individuals per group) were fixed in tissue fixative (10% neutral-buffered formalin) for histological examination. Meanwhile, gut contents were collected from A. japonicus (six individuals per group) for 16S rRNA amplicon sequencing. Growth indicators In determining the growth indicators for A. japonicus , the following formulas were applied [ 14 ]: Specific growth rate (SGR, %/d) = 100 × [(lnW 42 - lnW 0 ) /t] Feed conversion ratio (FCR) = [F /(W 42 - W 0 )] In the above formulas, W 42 designates the final body weight of A. japonicus at the termination of the experiment, whereas W 0 signifies the weight at its initiation; t represents the duration of the trial; F signifies the intake of desiccated feed. Enzyme activity assays of the gut and coelomic fluid To examine the digestive enzyme activities (trypsin, lipase, and amylase), the entire gut samples were homogenized in a 0.9% NaCl solution and centrifuged at 10000 ×g for 15 min under refrigerated conditions (4°C) [ 15 ]. The resulting supernatant was then used for analysis. The coelomic fluid was utilized for assays of antioxidant capacity and immune parameters. The antioxidant parameters measured included the total antioxidant capacity (T-AOC), catalase (CAT), and superoxide dismutase (SOD). The immune parameters included acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM). Each experiment adhered meticulously to the protocols outlined in the assay-specific kits provided by the Jiancheng Institute of Bioengineering. RT-qPCR analysis Real-time quantitative PCR (RT-qPCR) was used to examine the expression levels of antioxidant genes (Peroxiredoxin 5, PRDX5 ; Peroxiredoxin 6, PRDX6 ) and non-specific immune genes (Nuclear Factor Kappa B Transcription Factor p65, Rel ; Nuclear Factor Kappa B Subunit 1, P50 ; Complement Component 3 − 2, C3-2 ) in various tissues of A. japonicus , including the body wall, muscle, respiratory tree, foregut, midgut, and hindgut. Total RNA was extracted from each tissue using the RNA extraction kit (Bioflux-Bioer, Hangzhou, China). Reverse transcription was performed based on the guidelines of the PrimeScript™ RT reagent kit (Takara, Tokyo, Japan). The RT-qPCR reactions were conducted in a final volume of 20 µL, containing 10 µL TransStart® Top Green qPCR SuperMix (2×), 1 µL forward primer (10 µM), 1 µL reverse primer (10 µM), 1 µL cDNA template (1000 ng/µL), and 7 µL ddH 2 O. Internal references were β-actin and β-tubulin . The 2 −ΔΔCT technique was applied to determine the levels of gene expression [ 16 ]. A list of all primers is provided in Table S2. Morphology of gut structure The midgut samples preserved in the tissue fixative underwent dehydration using ethanol, were then cleared with xylene, and were subsequently embedded in paraffin. Serial sections (approximately 5 µm thick) were cut, followed by hematoxylin and eosin (H&E) staining [ 17 ]. The SlideViewer software (3DHistotech Ltd., Budapest, Hungary) was utilized for digital imaging and morphometric analysis. Challenge test After 6 weeks of feeding, we selected 75 A. japonicus individuals from each group and evenly distributed them into three different replicate tanks for an immersion challenge with Vibrio splendidus (1 × 10 8 CFU/mL). The evisceration rate of each group was recorded for 14 consecutive days to assess the possible defensive effects of postbiotics derived from L. plantarum O126 against infections caused by V. splendidus . (Evisceration rate, %) = (n e /n o ) × 100 In the formula, n e denotes the number of cumulative eviscerated individuals, and n o denotes the initial number of A. japonicus in the experiment. Gut microbiota analysis Genomic DNA was extracted from intestinal content samples using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany). Subsequently, 1% agarose gel electrophoresis was employed to determine the purity and concentration of the DNA. Amplify the V3-V4 region of the 16S rRNA gene using the specific primers 341F and 806R [ 18 ] (Table S2) . PCR products were purified with the Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany), and sequencing was facilitated by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The QIIME2 software was used for primer removal, quality filtering, denoising via DADA2, and generation of unique Amplicon Sequence Variants (ASVs). For each representative sequence, taxonomic annotation was performed using the Silva database ( http://www.arb-silva.de/ ). For the analysis of sequencing data, the Chao1 and Shannon indices enable the calculation of α-diversity. Meanwhile, β-diversity can be assessed through Principal Coordinates Analysis (PCoA) utilizing the weighted UniFrac distance and Non-metric Multidimensional Scaling (NMDS) employing the Jaccard distance to visualize sample dissimilarities in complex multidimensional datasets. To identify the representative bacterial taxa in each treatment, Linear Discriminant Analysis Effect Size (LEfSe) was performed using the Galaxy module ( http://huttenhower.sph.harvard.edu/galaxy ), with a Linear Discriminant Analysis (LDA) score threshold set at 4.0 [ 19 ]. Statistical analysis SPSS v26.0 (IBM, NY, USA) was used to conduct statistical evaluations, and data visualization was achieved through GraphPad Prism v9.0 (GraphPad Software, CA, USA) to ensure both analytical rigor and intuitive presentation. The heatmap of Spearman correlation analysis for various indicators was created with Origin v10.0 (OriginLab, MA, USA). A one-way analysis of variance (ANOVA) was utilized to assess differences among groups, while Duncan's test was used to facilitate multiple comparisons. Statistical significance was confirmed at P < 0.05. The statistics are presented as mean ± standard deviation (SD). Results Effects of L. plantarum O126 postbiotics on the growth of A. japonicus In Table 1 , we outline the effects of introducing different dosages of L. plantarum O126 postbiotics into the diet of A. japonicus . The LPP-M and LPP-H groups experienced a substantial increase in final body weight and SGR ( P < 0.05), along with a notable decrease in FCR ( P 0.05). The LPP-L group, on the other hand, displayed no noticeable changes in body weight, SGR, or FCR ( P > 0.05). The results indicate that the addition of medium and high doses of L. plantarum O126 postbiotics to the diet significantly enhances the growth performance of A. japonicus . Table 1 Effects of L. plantarum O126 postbiotics on growth performance of A. japonicus . Diet groups W 0 (g) W 42 (g) SGR (%/d) FCR Control 17.66 ± 1.01 27.56 ± 1.71 a 1.06 ± 0.15 a 2.21 ± 0.09 b LPP-L 17.81 ± 2.33 29.28 ± 1.27 ab 1.18 ± 0.10 a 2.16 ± 0.07 b LPP-M 16.96 ± 2.19 31.11 ± 2.17 b 1.44 ± 0.17 b 1.90 ± 0.08 a LPP-H 16.98 ± 1.50 30.10 ± 1.17 b 1.36 ± 0.09 b 1.89 ± 0.05 a Note: Different letters within the same column indicate significant differences among groups ( P < 0.05). Effects of L. plantarum O126 postbiotics on gut digestive enzymes and morphology in A. japonicus Results illustrating the influence of L. plantarum O126 postbiotics addition at different doses on digestive enzyme activities and the morphology of the midgut in A. japonicus are presented in Fig. 1 . At weeks 4 and 6 after the addition of L. plantarum O126, the trypsin activity in the LPP-M and LPP-H groups was significantly higher than that in the control group ( P 0.05) ( Fig. 1 a ) . Lipase activity showed a significant increase at weeks 2 and 4 ( P < 0.05), with all experimental groups exhibiting an initial rise, peaking at week 4, followed by a gradual decline ( Fig. 1 b ) . In contrast, supplementation with L. plantarum O126 postbiotics had no noticeable effect on amylase activity ( P > 0.05) ( Fig. 1 c ) . Results of gut morphological analysis are presented in Fig. 1 d-g. Compared with the control and LPP-L groups, villus height and width increased in the LPP-M and LPP-H groups ( P 0.05) ( Fig. 1 h ) . Effects of L. plantarum O126 postbiotics on the antioxidant capacity of A. japonicus The impact of varying doses of L. plantarum O126 postbiotics on the antioxidant levels of A. japonicus is depicted in Fig. 2 . Following supplementation with L. plantarum O126 postbiotics, the T-AOC levels of all experimental groups were higher than those of the control group at all time points, with the LPP-M group showing higher levels compared to both the LPP-L and LPP-H groups ( P < 0.05) ( Fig. 2 A1) . However, the L. plantarum O126 postbiotics did not exhibit a significant effect on CAT activity in A. japonicus , with the CAT levels in the LPP-M group increasing only at week 2 ( P 0.05) ( Fig. 2 A2) . Compared with the control group, the elevation of SOD activity in the LPP-M group showed consistent significance at all sampling times ( P < 0.05). In the LPP-H group, SOD activity showed enhancement only at week 4 ( P < 0.05), while the LPP-L group showed no significant changes at any time point ( Fig. 2 A3) . Dietary supplementation with L. plantarum O126 postbiotics significantly altered the expression patterns of antioxidant-related genes PRDX5 and PRDX6 in most of the examined tissues of A. japonicus , as shown in Fig. 2 B and C . The postbiotics markedly enhanced PRDX5 and PRDX6 expression in the body wall, respiratory tree, and gut tissues ( P < 0.05). Notably, both genes exhibited a consistent expression pattern in these tissues (body wall, respiratory tree, and gut), characterized by an initial increase peaking at weeks 2–4, followed by a gradual decline. In contrast, the supplementation generally showed no significant effects on PRDX5 and PRDX6 expression in muscle tissue. Effects of L. plantarum O126 postbiotics on the immune modulation in A. japonicus The impacts of immune parameters observed following dietary supplementation with varying levels of L. plantarum O126 postbiotics in A. japonicus are shown in Fig. 3 . All experimental groups exhibited an initial increase in immune enzyme activities, followed by a gradual decline after week 4. Compared with the control and LPP-L groups, the ACP activity was elevated in both LPP-M and LPP-H groups at all time points ( P 0.05) ( Fig. 3 A1) . The AKP levels of all experimental groups were notably elevated at weeks 4 and 6, showing dose-dependent effects ( Fig. 3 A2) . However, L. plantarum O126 postbiotics supplementation had limited effects on LZM activity, with only the LPP-M group showing a significant effect at week 4 ( P 0.05) ( Fig. 3 A3) . The relative expression levels of immune-related genes ( Rel , P50 , and C3-2 ) in various tissues of A. japonicus are shown in Fig. 3 B-D. L. plantarum O126 postbiotics significantly influenced the expression of the Rel gene in the body wall, muscle, respiratory tree, and midgut of A. japonicus , showing a consistent pattern of initial increase, peaking at week 4, followed by a gradual decline. In contrast, the effects on the expression of the Rel gene in the foregut and hindgut were minimal, showing no statistical differences among all groups at most time points ( P > 0.05). Interestingly, the influence on tissues of both the respiratory tree and gut appeared to be temporary: by week 6, there were no statistical differences among all groups ( P > 0.05) ( Fig. 3 B ) . Similar expression patterns were observed for the P50 gene in the body wall, respiratory tree, and gut tissues, exhibiting an increase until week 4, followed by a gradual decline. However, P50 upregulation in muscle tissue remained limited, with only the LPP-M group showing significantly elevated levels at week 4 ( P 0.05) (Fig. 3 C ) . The expression of the C3-2 gene in the body wall, respiratory tree, and gut tissues was significantly affected by L. plantarum O126 postbiotics, displaying a characteristic rise-and-fall pattern, with peak expression occurring at week 4. Interestingly, in muscle tissue, the C3-2 expression showed delayed enhancement, with upregulation only appearing at week 6 ( P < 0.05) (Fig. 3 D ) . Notably, in the aforementioned results, the LPP-M group demonstrated immune enzyme activities and related gene expression levels that were similar to, or even higher than, those observed in the LPP-H group. Furthermore, in gut tissues, the expression levels of immune genes varied across different gut segments. Overall, their expression was higher in the midgut and hindgut compared to the foregut, demonstrating a certain concentration-dependent pattern. Effects of L. plantarum O126 postbiotics on the enhancement of disease resistance in A. japonicus The evisceration rates of A. japonicus following V. splendidus infection are shown in Fig. 4 . After the challenge, both experimental and control groups exhibited gradually increasing evisceration rates that stabilized after 9 days. The final evisceration rates were 64% (control), 52% (LPP-L), 40% (LPP-M), and 44% (LPP-H) ( Fig. 4 e ) . In contrast to the control group, the evisceration rates in the experimental groups were significantly reduced ( P < 0.05), with the most significant reduction observed in the LPP-M group. Effects of L. plantarum O126 postbiotics on the gut microbiota community of A. japonicus The impacts of dietary L. plantarum O126 postbiotics on the gut microbiota community of A. japonicus are shown in Fig. 5 . The Venn diagram revealed 696 shared ASVs among the four groups, while the control, LPP-L, LPP-M, and LPP-H groups contained 1203, 1742, 2419, and 2407 unique ASVs, respectively ( Fig. 5 a ) . Chao1 and Shannon indices revealed that, compared with the control group, α-diversity was elevated in experimental groups, with the LPP-M and LPP-H groups showing more prominent enhancement in α-diversity ( P < 0.05) ( Fig. 5 b and c) . β-diversity analysis using PCoA and NMDS revealed considerable variations in gut microbiota structure under different diets ( Fig. 5 d and e) . In addition, at the phylum and genus levels, differences in the composition of the gut microbiota among different groups are also shown in Fig. 5 f and g . At the phylum level ( top 10 phyla, Fig. 5 f ) , compared with the control group, all experimental groups showed significantly reduced relative abundance of Proteobacteria but increased relative abundance of Firmicutes and Actinobacteria. Figure 5 g shows the distribution of relative abundance for the top 10 dominant bacterial genera at the genus level in the gut microbiota. To further investigate the effects of different doses of L. plantarum O126 postbiotics on the gut microbiota composition of A. japonicus , the Linear Discriminant Analysis (LDA) method was used in LEfSe analysis to identify differential microbial taxa among groups. It was determined that the control group was rich in genus-level differential microbial taxa, including Sulfitobacter , Brevundimonas and Vibrio ; the LPP-L group was rich in Haloferula , Pseudoruegeria , Pseudopelagicola , Pseudomonas , and Marimicrobium ; the LPP-M group was rich in Bacillus and Exiguobacterium ; and the LPP-H group was rich in Vagococcus (Fig. 6 a and b) . The significance test analysis revealed that the relative abundance of Sulfitobacter and Brevundimonas was significantly downregulated in all experimental groups, while Pseudomonas was downregulated in the LPP-M and LPP-H groups compared with the control group ( P < 0.05) ( Fig. 6 c-e ) . Pseudoruegeria was significantly upregulated in the LPP-L and LPP-M groups compared with the control group ( P < 0.05) ( Fig. 6 f ) . Interestingly, the LPP-M and LPP-H groups showed consistency in genus composition, with Bacillus , Exiguobacterium , and Vagococcus showing increased abundance ( P < 0.05) ( Fig. 6 g-i ) . Correlation analysis Correlation analysis revealed that growth indicators and evisceration rate were significantly correlated with gut morphology and immune-antioxidant indicators ( Fig. 7 ) . Specifically, the final body weight and SGR exhibited a positive correlation with VH and VW ( P < 0.01). FCR exhibited negative correlations with VH and VW ( P < 0.01). Additionally, the evisceration rate was negatively correlated with final body weight, SGR, VH, VW, ACP, and P50 ( P < 0.01). The correlation heatmap analysis further indicated potential associations between the gut microbiota and other health indicators. The genera Haloferula and Pseudomonas also showed negative correlations with final body weight, SGR, VH, and VW ( P < 0.01). Furthermore, the genus Sulfitobacter demonstrated negative correlations with final body weight, SGR, VH, VW, SOD, PRDX5 , ACP, Rel , P50 , and C3-2 ( P < 0.01). Moreover, the genus Brevundimonas demonstrated negative correlations with final body weight, SGR, VH, VW, ACP, P50 , and C3-2 ( P < 0.01). Conversely, Pseudoruegeria showed significant positive correlations with final body weight, MT, and P50 ( P < 0.05). In addition, the genera Bacillus , Exiguobacterium , and Vagococcus showed positive correlations with final body weight, SGR, VH, VW, PRDX5 , ACP, and P50 ( P < 0.01), while exhibiting negative correlations with the evisceration rate ( P < 0.01). Discussion Probiotics, widely used as bioregulators in aquaculture, can enhance production efficiency and animal health status [ 20 ]. However, as live microbial products, they are highly susceptible to environmental conditions during production, storage, and application [ 6 ]. In contrast, postbiotics, which consist of inactivated microbial cells and/or their extracellular metabolites, can possess superior stability and safety advantages. These properties facilitate large-scale production and storage. They have been shown to enhance the health of aquatic organisms such as Nile tilapia, Pacific white shrimp, and black sea bream, among others. However, research on their effects on A. japonicus remains limited. Consequently, this investigation focused on the impacts of L. plantarum O126 postbiotics on growth, antioxidant capacity, disease resistance, and gut health in A. japonicus . A considerable body of evidence supports the notion that postbiotic additives can promote growth in aquatic organisms [ 21 , 22 ]. In this study, the notable increases in body weight and specific growth rate among the LPP-M and LPP-H groups indicate that L. plantarum O126 postbiotics significantly improved the growth of A. japonicus . This positive effect is likely linked to increased activity of digestive enzymes, as documented in earlier studies [ 23 , 24 ]. Supporting this notion, our findings revealed a marked boost in trypsin and lipase activities within the gut. However, amylase activity remained unaffected, contrasting with previous reports by Yang et al. [ 25 ], potentially due to differences in probiotic strains, dosage regimens, or intervention durations. Notably, this study also observed significant increases in both villus height and width in the midgut. Such morphological modifications can enhance nutrient absorption efficiency by expanding the surface area of gut villi [ 26 ]. Correlation analysis also indicates that they are crucial factors contributing to the observed growth promotion in the subjects of this study. Furthermore, the improved growth performance may also be associated with enhanced antioxidant capacity, immune status, and modulation of gut microbiota [ 27 ]. Antioxidant capacity serves as a critical indicator of animal health status. T-AOC is a vital measure for assessing the overall antioxidant levels in various organisms [ 28 , 29 ]. SOD facilitates the conversion of superoxide anion radicals (O₂⁻) into hydrogen peroxide (H₂O₂), and CAT further converts H₂O₂ into water and oxygen, thus safeguarding the organism against damage from free radicals [ 30 ]. This study revealed that the inclusion of L. plantarum O126 postbiotics in the diet enhanced the activities of T-AOC, SOD, and CAT to varying degrees in the coelomic fluid of A. japonicus , indicating a bolstered antioxidant capacity in this species. Peroxiredoxin (Prx), as a core component of the organism's antioxidant system, can catalyze the reduction of H₂O₂ to water. The proteins encoded by the PRDX5 and PRDX6 genes belong to the Prx superfamily [ 31 – 33 ]. This research found that adding L. plantarum O126 postbiotics to the diet notably boosted the levels of PRDX5 and PRDX6 gene expression across various tissues, including the body wall, respiratory tree, and gut in A. japonicus , with the results reaching statistical significance ( P 0.05), demonstrating distinct tissue-specific expression patterns for these antioxidant genes. The antioxidant regulatory effects of L. plantarum postbiotics may originate from their inherent bioactive compounds, including γ-aminobutyric acid (GABA), exopolysaccharides, and lipoteichoic acid (LTA) [ 34 , 35 ], which can directly scavenge free radicals and enhance antioxidant capacity. Furthermore, our results demonstrated that these postbiotics significantly upregulated antioxidant enzyme activities in A. japonicus . This regulatory pattern has been similarly validated in black sea bream [ 36 ] and Nile tilapia [ 37 ], indicating an additional indirect antioxidant protective mechanism through activation of the host's endogenous antioxidant enzyme system. A. japonicus lacks an adaptive immune system, relying on non-specific immunity as its main form of immune defense [ 38 ]. The non-specific immune enzymes secreted by coelomocytes constitute an indispensable component of non-specific immunity, playing a critical role in immune defense and pathogen clearance [ 39 , 40 ]. Our findings demonstrated that L. plantarum O126 postbiotics can enhance non-specific immune enzymes of A. japonicus . Furthermore, numerous studies have reported that postbiotics can enhance innate immune responses by regulating immune-related genes [ 12 ]. Thus, we further examined the expression of essential immune-related genes ( Rel , P50 , and C3-2 ) in A. japonicus . Although tissue-specific differential expression of these genes was observed, dietary supplementation with L. plantarum O126 postbiotics significantly upregulated their expression across all examined tissues, suggesting that these postbiotics effectively enhance cellular immune responses in A. japonicus . Notably, the midgut exhibited higher immune gene expression levels than the foregut and hindgut, likely because of its more diverse microbial community [ 41 ], which produces immune-modulating enzymes and bioactive compounds [ 42 , 43 ]. Furthermore, the challenge test revealed that A. japonicus fed with L. plantarum O126 postbiotics exhibited significantly lower evisceration rates, indicating that they have potential applications for enhancing resistance against V. splendidus infection. The immune-boosting and infection-fighting properties of L. plantarum O126 postbiotics are likely due to various bioactive molecules, such as antimicrobial peptides, exopolysaccharides, and short-chain fatty acids (SCFAs). By engaging with pattern recognition receptors (PRRs) on immune cells, these molecules activate signaling pathways like NF-κB or MAPK that modulate the immune response [ 44 ]. The gut microbiota plays a crucial role in maintaining the health of aquatic animals [ 45 , 46 ]. Numerous studies have reported that postbiotics can enhance microbial diversity and sustain gut microecological homeostasis in animals [ 47 ]. Consistent with these findings, the LPP-M and LPP-H groups exhibited notably higher α-diversity indices of gut microbiota, indicating that L. plantarum O126 postbiotics improved species richness and evenness in the gut microbiota of A. japonicus . Furthermore, β-diversity analysis revealed distinct alterations in microbial composition among postbiotics-supplemented groups and control group, demonstrating that L. plantarum O126 postbiotics serve as key modulators of the gut microbiota in A. japonicus . Phylum-level analysis showed a notable decrease in Proteobacteria proportions within the experimental groups. As a phylum ubiquitously distributed in marine environments, Proteobacteria encompasses various pathogenic genera, including Pseudomonas , Aeromonas , and Vibrio , among others. Elevated proportions of Proteobacteria in the gut microbiota typically indicate microbial community instability, which may trigger inflammatory responses and disease onset [ 48 ]. Conversely, L. plantarum O126 postbiotics increased the relative abundances of Firmicutes and Actinobacteria. Notably, Firmicutes are capable of producing various active enzymes that aid hosts in food decomposition and digestion, followed by fermentative production of SCFAs [ 49 ]. Actinobacteria can maintain gut microbiota homeostasis, as well as modulate both immune system function and metabolic processes [ 49 ]. The increased relative abundances of these phyla suggest that L. plantarum O126 postbiotics may promote both growth and immunity in A. japonicus . At the genus level, LEfSe analysis revealed that different dosage additions of L. plantarum O126 postbiotics could lead to distinct colonization patterns of gut microbiota in A. japonicus . Sulfitobacter exhibited decreased relative abundance across all experimental groups, and correlation analysis indicated a negative relationship between this genus and key physiological health indicators, including growth and immunity. Although studies on Sulfitobacter in animal gut microbiota remain limited, our findings suggest that it may play a harmful role in the gut microbiota of aquatic animals. Additionally, Brevundimonas was significantly downregulated in all experimental groups, while Pseudomo nas was significantly downregulated in the LPP-M and LPP-H groups. Both Brevundimonas and Pseudomonas are frequently reported as common pathogenic bacteria in aquatic animals [ 50 , 51 ]. Conversely, Pseudoruegeria was significantly upregulated in the LPP-L and LPP-M groups. Currently, there are limited reports on the genus Pseudoruegeria . Ko et al. [ 50 ] identified a species within this genus that possesses metabolic pathways for all seven B vitamins, while Cho et al. [ 52 ] discovered that certain species within the same genus could eliminate harmful algae. This study also found a positive correlation between Pseudoruegeria and the growth and immune status of A. japonicus , suggesting its potential role as a probiotic in the gut microbiota. However, further investigation is needed to elucidate the underlying mechanisms. Interestingly, both the LPP-M and LPP-H groups consistently showed significantly increased abundances of three genera: Bacillus , Exiguobacterium , and Vagococcus . The genus Bacillus has been widely utilized as a probiotic in aquaculture and has demonstrated immunomodulatory activities [ 53 – 55 ]. Exiguobacterium , a prospective probiotic for aquatic animals, synthesizes glutathione (GSH) to boost the host's antioxidant defenses [ 56 , 57 ]. Some strains of the genus Vagococcus have been proven to help aquatic animals resist Vibrio infection [ 58 , 59 ]. Correlation analysis suggests that microbial population shifts may contribute to enhancing growth velocities, amplified antioxidant protection, and strengthened immune responses in A. japonicus . Conclusion Taken together, our results suggest that L. plantarum O126 postbiotics effectively enhance growth performance, improve gut morphology, promote digestion, boost immunity, enhance antioxidant capacity, and increase gut microbial diversity in A. japonicus , while concurrently increasing resistance against V. splendidus . These results indicate that L. plantarum O126 postbiotics have potential for application in sustainable A. japonicus aquaculture. Declarations Author Contribution Feng-Li Chen: Investigation, Formal analysis, Software, Writing-original draft; Hui-Ting Zhao: Investigation, Formal analysis; Wei Gu: Project administration, Resources; Zong-Xiu Wu: Investigation, Formal analysis; Jing-Wen Xiang: Investigation; Qing Yang: Investigation; Yu-Lin Yang: Investigation; Lei Tan: Investigation; Meng-Xia Sun: Investigation; Wei Cong: Writing-review & editing; Shu Li: Writing-review & editing; Bin-Tong Yang: Project administration, Funding acquisition, Writing-review & editing; Yuan-Huan Kang: Project administration, Funding acquisition, Writing-review & editing. Funding This work was supported by the National Natural Science Foundation of China (Youth Science Foundation Program, Grant No. 32301410), Tai'an City Science and Technology Innovation ''Double Ten Engineering'' (Major Technological Breakthrough) Project (23JSGG02) and Key R&D Program (Rural Revitalization Technological Innovation Boosting Action Plan) of Shandong Province, China (Program No. 2024TZXD006). Data availability The underlying data for this study are available from the corresponding author upon request. Ethics approval All animal experiments carried out in this study strictly adhered to the guidelines set forth by Shandong University's Regulations for Animal Experimentation, as well as the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No.1438023). Conflict of interest The authors declare no conflicts of interest. 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16:07:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5699836,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7545463/v1/87cbe400-9206-49a6-9575-2aecb1ebc6ee.pdf"},{"id":97682154,"identity":"c5f5d8c6-0c9c-4e77-8ec5-19b75d2ed09b","added_by":"auto","created_at":"2025-12-08 10:02:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21933,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7545463/v1/7619b230c852da338aa7e533.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lactiplantibacillus plantarum O126 postbiotics boost growth, antioxidant levels, disease resistance, and gut health in sea cucumber (Apostichopus japonicus)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSea cucumber (\u003cem\u003eApostichopus japonicus\u003c/em\u003e), long regarded as a premium tonic food in Asia, particularly in China, has gained widespread popularity among consumers due to its unique nutritional and medicinal properties. The sustained growth in market demand has driven the rapid development of \u003cem\u003eA. japonicus\u003c/em\u003e aquaculture. \u003cem\u003eA. japonicus\u003c/em\u003e aquaculture stands as a key pillar in China's marine aquaculture industry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, there are numerous challenges, including persistently high feed costs, deterioration of aquaculture water environments, frequent disease outbreaks, and the overuse of pesticides and antibiotics. These issues not only reduce the economic benefits of \u003cem\u003eA. japonicus\u003c/em\u003e aquaculture but also lead to secondary problems such as environmental pollution.\u003c/p\u003e\u003cp\u003eTo address these issues, probiotics have been introduced as feed additives in \u003cem\u003eA. japonicus\u003c/em\u003e aquaculture, demonstrating beneficial effects on promoting growth, improving gut health, and modulating microbiota balance [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Given their potential as alternatives to antibiotics, probiotic applications could potentially reduce the risk of antimicrobial resistance dissemination and mitigate public health concerns related to antibiotic residues in food products [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, live probiotics may destabilize aquaculture ecosystems and exhibit poor processing and storage stability. According to the International Scientific Association for Probiotics and Prebiotics (ISAPP\u003cem\u003e)\u003c/em\u003e, postbiotics denote inactivated microbial entities and/or constitutive elements that provide health benefits to the host, thus representing a safer and more promising alternative [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e (\u003cem\u003eL. plantarum\u003c/em\u003e), a typical gut-commensal lactic acid bacterium, has been shown to possess multiple probiotic properties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Research has demonstrated significant beneficial effects conferred by \u003cem\u003eL. plantarum\u003c/em\u003e postbiotics on marine organisms. Nguyen et al. found that heat-killed \u003cem\u003eL. plantarum\u003c/em\u003e L-137 significantly improved immune capacity in Nile tilapia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Similarly, Zheng et al. verified that cell-free extracts of \u003cem\u003eL. plantarum\u003c/em\u003e could effectively regulate gut microbiota in Pacific white shrimp [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, Rawling et al. found that heat-inactivated \u003cem\u003eL. plantarum\u003c/em\u003e HA-122 postbiotics could enhance gut immune function in zebrafish by reinforcing the gut mucosal barrier [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The above research outcomes robustly support the use of \u003cem\u003eL. plantarum\u003c/em\u003e-derived postbiotics in aquaculture, though their specific functions in \u003cem\u003eA. japonicus\u003c/em\u003e aquaculture remain insufficiently studied.\u003c/p\u003e\u003cp\u003eThis study systematically evaluated the impacts of postbiotics derived from \u003cem\u003eL. plantarum\u003c/em\u003e O126 on the growth, antioxidant and immune capacity, disease resistance, and gut health in \u003cem\u003eA. japonicus\u003c/em\u003e. This supports their application as a sustainable strategy for improving the productivity and health management of \u003cem\u003eA. japonicus\u003c/em\u003e aquaculture.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eResearch animals and rearing conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. japonicus\u003c/em\u003e individuals were sourced from an \u003cem\u003eA. japonicus\u003c/em\u003e aquaculture facility in Weihai, Shandong, China. After a 7-day acclimation period, 480 healthy individuals with an average body weight of 17.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76 g were divided into 12 aquariums (100 \u0026times; 80 \u0026times; 60 cm\u0026sup3;). There were three aquariums in each group, with 40 individuals in each aquarium. Each animal received a single daily feeding at 18:00, with the amount allotted equal to 3% of its total body weight. The water conditions were consistently maintained with the temperature at 15\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, dissolved oxygen\u0026thinsp;\u0026gt;\u0026thinsp;8 mg/L, salinity of 30\u0026permil;, and a pH of 8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDiet composition\u003c/h2\u003e\u003cp\u003ePurchased from Guangdong Haid Group Co., Limited (Guangzhou, China), the composition of the basal feed is shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics were provided by Shandong Baolai-Leelai Bioengineering Co., Ltd. (Yantai, China). These postbiotics contained inactivated microbial biomass exceeding 1 \u003cem\u003e\u0026times;\u003c/em\u003e 10\u003csup\u003e11\u003c/sup\u003e cells per gram, with lactic acid content\u0026thinsp;\u0026gt;\u0026thinsp;10% and crude polysaccharide content\u0026thinsp;\u0026gt;\u0026thinsp;5%. \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics were supplemented to the basal diet of \u003cem\u003eA. japonicus\u003c/em\u003e at distinct doses: 1\u0026permil; (w/w) (designated as the LPP-L group), 2\u0026permil; (w/w) (designated as the LPP-M group), and 4\u0026permil; (w/w) (designated as the LPP-H group). The control group received solely a standard basal diet.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eAt weeks 0, 2, 4, and 6, we dissected six \u003cem\u003eA. japonicus\u003c/em\u003e individuals per group under sterile conditions to obtain body wall, muscle, respiratory tree, foregut, midgut, and hindgut tissues. The coelomic fluid was aspirated using sterile syringes and centrifuged at 4\u0026deg;C for 15 min at 800\u003cem\u003e\u0026times;g\u003c/em\u003e, with the resulting supernatant aliquots subjected to enzymatic assays [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. At week 6, the midgut tissues of \u003cem\u003eA. japonicus\u003c/em\u003e (three individuals per group) were fixed in tissue fixative (10% neutral-buffered formalin) for histological examination. Meanwhile, gut contents were collected from \u003cem\u003eA. japonicus\u003c/em\u003e (six individuals per group) for 16S rRNA amplicon sequencing.\u003c/p\u003e\n\u003ch3\u003eGrowth indicators\u003c/h3\u003e\n\u003cp\u003eIn determining the growth indicators for \u003cem\u003eA. japonicus\u003c/em\u003e, the following formulas were applied [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]:\u003c/p\u003e\u003cp\u003eSpecific growth rate (SGR, %/d)\u0026thinsp;=\u0026thinsp;100 \u0026times; [(lnW\u003csub\u003e42\u003c/sub\u003e - lnW\u003csub\u003e0\u003c/sub\u003e) /t]\u003c/p\u003e\u003cp\u003eFeed conversion ratio (FCR) = [F /(W\u003csub\u003e42\u003c/sub\u003e - W\u003csub\u003e0\u003c/sub\u003e)]\u003c/p\u003e\u003cp\u003eIn the above formulas, W\u003csub\u003e42\u003c/sub\u003e designates the final body weight of \u003cem\u003eA. japonicus\u003c/em\u003e at the termination of the experiment, whereas W\u003csub\u003e0\u003c/sub\u003e signifies the weight at its initiation; t represents the duration of the trial; F signifies the intake of desiccated feed.\u003c/p\u003e\n\u003ch3\u003eEnzyme activity assays of the gut and coelomic fluid\u003c/h3\u003e\n\u003cp\u003eTo examine the digestive enzyme activities (trypsin, lipase, and amylase), the entire gut samples were homogenized in a 0.9% NaCl solution and centrifuged at 10000\u003cem\u003e\u0026times;g\u003c/em\u003e for 15 min under refrigerated conditions (4\u0026deg;C) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The resulting supernatant was then used for analysis. The coelomic fluid was utilized for assays of antioxidant capacity and immune parameters. The antioxidant parameters measured included the total antioxidant capacity (T-AOC), catalase (CAT), and superoxide dismutase (SOD). The immune parameters included acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM). Each experiment adhered meticulously to the protocols outlined in the assay-specific kits provided by the Jiancheng Institute of Bioengineering.\u003c/p\u003e\n\u003ch3\u003eRT-qPCR analysis\u003c/h3\u003e\n\u003cp\u003eReal-time quantitative PCR (RT-qPCR) was used to examine the expression levels of antioxidant genes (Peroxiredoxin 5, \u003cem\u003ePRDX5\u003c/em\u003e; Peroxiredoxin 6, \u003cem\u003ePRDX6\u003c/em\u003e) and non-specific immune genes (Nuclear Factor Kappa B Transcription Factor p65, \u003cem\u003eRel\u003c/em\u003e; Nuclear Factor Kappa B Subunit 1, \u003cem\u003eP50\u003c/em\u003e; Complement Component 3\u0026thinsp;\u0026minus;\u0026thinsp;2, \u003cem\u003eC3-2\u003c/em\u003e) in various tissues of \u003cem\u003eA. japonicus\u003c/em\u003e, including the body wall, muscle, respiratory tree, foregut, midgut, and hindgut. Total RNA was extracted from each tissue using the RNA extraction kit (Bioflux-Bioer, Hangzhou, China). Reverse transcription was performed based on the guidelines of the PrimeScript\u0026trade; RT reagent kit (Takara, Tokyo, Japan).\u003c/p\u003e\u003cp\u003eThe RT-qPCR reactions were conducted in a final volume of 20 \u0026micro;L, containing 10 \u0026micro;L TransStart\u0026reg; Top Green qPCR SuperMix (2\u0026times;), 1 \u0026micro;L forward primer (10 \u0026micro;M), 1 \u0026micro;L reverse primer (10 \u0026micro;M), 1 \u0026micro;L cDNA template (1000 ng/\u0026micro;L), and 7 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO. Internal references were \u003cem\u003eβ-actin\u003c/em\u003e and \u003cem\u003eβ-tubulin\u003c/em\u003e. The 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e technique was applied to determine the levels of gene expression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A list of all primers is provided in \u003cb\u003eTable S2.\u003c/b\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMorphology of gut structure\u003c/h2\u003e\u003cp\u003eThe midgut samples preserved in the tissue fixative underwent dehydration using ethanol, were then cleared with xylene, and were subsequently embedded in paraffin. Serial sections (approximately 5 \u0026micro;m thick) were cut, followed by hematoxylin and eosin (H\u0026amp;E) staining [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The SlideViewer software (3DHistotech Ltd., Budapest, Hungary) was utilized for digital imaging and morphometric analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eChallenge test\u003c/h3\u003e\n\u003cp\u003eAfter 6 weeks of feeding, we selected 75 \u003cem\u003eA. japonicus\u003c/em\u003e individuals from each group and evenly distributed them into three different replicate tanks for an immersion challenge with \u003cem\u003eVibrio splendidus\u003c/em\u003e (1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL). The evisceration rate of each group was recorded for 14 consecutive days to assess the possible defensive effects of postbiotics derived from \u003cem\u003eL. plantarum\u003c/em\u003e O126 against infections caused by \u003cem\u003eV. splendidus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e(Evisceration rate, %) = (n\u003csub\u003ee\u003c/sub\u003e/n\u003csub\u003eo\u003c/sub\u003e) \u0026times; 100\u003c/p\u003e\u003cp\u003eIn the formula, n\u003csub\u003ee\u003c/sub\u003e denotes the number of cumulative eviscerated individuals, and n\u003csub\u003eo\u003c/sub\u003e denotes the initial number of \u003cem\u003eA. japonicus\u003c/em\u003e in the experiment.\u003c/p\u003e\n\u003ch3\u003eGut microbiota analysis\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from intestinal content samples using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany). Subsequently, 1% agarose gel electrophoresis was employed to determine the purity and concentration of the DNA. Amplify the V3-V4 region of the 16S rRNA gene using the specific primers 341F and 806R [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] \u003cb\u003e(Table S2)\u003c/b\u003e. PCR products were purified with the Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany), and sequencing was facilitated by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China).\u003c/p\u003e\u003cp\u003eThe QIIME2 software was used for primer removal, quality filtering, denoising via DADA2, and generation of unique Amplicon Sequence Variants (ASVs). For each representative sequence, taxonomic annotation was performed using the Silva database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.arb-silva.de/\u003c/span\u003e\u003cspan address=\"http://www.arb-silva.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor the analysis of sequencing data, the Chao1 and Shannon indices enable the calculation of α-diversity. Meanwhile, β-diversity can be assessed through Principal Coordinates Analysis (PCoA) utilizing the weighted UniFrac distance and Non-metric Multidimensional Scaling (NMDS) employing the Jaccard distance to visualize sample dissimilarities in complex multidimensional datasets. To identify the representative bacterial taxa in each treatment, Linear Discriminant Analysis Effect Size (LEfSe) was performed using the Galaxy module (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://huttenhower.sph.harvard.edu/galaxy\u003c/span\u003e\u003cspan address=\"http://huttenhower.sph.harvard.edu/galaxy\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with a Linear Discriminant Analysis (LDA) score threshold set at 4.0 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eSPSS v26.0 (IBM, NY, USA) was used to conduct statistical evaluations, and data visualization was achieved through GraphPad Prism v9.0 (GraphPad Software, CA, USA) to ensure both analytical rigor and intuitive presentation. The heatmap of Spearman correlation analysis for various indicators was created with Origin v10.0 (OriginLab, MA, USA). A one-way analysis of variance (ANOVA) was utilized to assess differences among groups, while Duncan's test was used to facilitate multiple comparisons. Statistical significance was confirmed at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The statistics are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eEffects of L. plantarum O126 postbiotics on the growth of A. japonicus\u003c/h2\u003e\u003cp\u003eIn Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, we outline the effects of introducing different dosages of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics into the diet of \u003cem\u003eA. japonicus\u003c/em\u003e. The LPP-M and LPP-H groups experienced a substantial increase in final body weight and SGR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), along with a notable decrease in FCR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no statistical differences were found between the LPP-M and LPP-H groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The LPP-L group, on the other hand, displayed no noticeable changes in body weight, SGR, or FCR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The results indicate that the addition of medium and high doses of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics to the diet significantly enhances the growth performance of \u003cem\u003eA. japonicus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics on growth performance of \u003cem\u003eA. japonicus\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDiet groups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eW\u003csub\u003e0\u003c/sub\u003e (g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eW\u003csub\u003e42\u003c/sub\u003e (g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSGR (%/d)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFCR\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e17.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLPP-L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e17.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLPP-M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e16.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLPP-H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e16.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: Different letters within the same column indicate significant differences among groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eEffects of L. plantarum O126 postbiotics on gut digestive enzymes and morphology in A. japonicus\u003c/h2\u003e\u003cp\u003eResults illustrating the influence of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics addition at different doses on digestive enzyme activities and the morphology of the midgut in \u003cem\u003eA. japonicus\u003c/em\u003e are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. At weeks 4 and 6 after the addition of \u003cem\u003eL. plantarum\u003c/em\u003e O126, the trypsin activity in the LPP-M and LPP-H groups was significantly higher than that in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, there were no statistically significant differences in trypsin activity between the LPP-L and control groups across all time points (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Lipase activity showed a significant increase at weeks 2 and 4 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with all experimental groups exhibiting an initial rise, peaking at week 4, followed by a gradual decline \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. In contrast, supplementation with \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics had no noticeable effect on amylase activity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eResults of gut morphological analysis are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-g. Compared with the control and LPP-L groups, villus height and width increased in the LPP-M and LPP-H groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the muscle layer thickness showed no significant changes across all groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eEffects of L. plantarum O126 postbiotics on the antioxidant capacity of A. japonicus\u003c/h2\u003e\u003cp\u003eThe impact of varying doses of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics on the antioxidant levels of \u003cem\u003eA. japonicus\u003c/em\u003e is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Following supplementation with \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics, the T-AOC levels of all experimental groups were higher than those of the control group at all time points, with the LPP-M group showing higher levels compared to both the LPP-L and LPP-H groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003eA1)\u003c/b\u003e. However, the \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics did not exhibit a significant effect on CAT activity in \u003cem\u003eA. japonicus\u003c/em\u003e, with the CAT levels in the LPP-M group increasing only at week 2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Beyond this specific case, no statistical differences were observed among all groups at any measured time points (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003eA2)\u003c/b\u003e. Compared with the control group, the elevation of SOD activity in the LPP-M group showed consistent significance at all sampling times (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the LPP-H group, SOD activity showed enhancement only at week 4 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the LPP-L group showed no significant changes at any time point \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003eA3)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDietary supplementation with \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics significantly altered the expression patterns of antioxidant-related genes \u003cem\u003ePRDX5\u003c/em\u003e and \u003cem\u003ePRDX6\u003c/em\u003e in most of the examined tissues of \u003cem\u003eA. japonicus\u003c/em\u003e, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB \u003cb\u003eand C\u003c/b\u003e. The postbiotics markedly enhanced \u003cem\u003ePRDX5\u003c/em\u003e and \u003cem\u003ePRDX6\u003c/em\u003e expression in the body wall, respiratory tree, and gut tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, both genes exhibited a consistent expression pattern in these tissues (body wall, respiratory tree, and gut), characterized by an initial increase peaking at weeks 2\u0026ndash;4, followed by a gradual decline. In contrast, the supplementation generally showed no significant effects on \u003cem\u003ePRDX5\u003c/em\u003e and \u003cem\u003ePRDX6\u003c/em\u003e expression in muscle tissue.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eEffects of L. plantarum O126 postbiotics on the immune modulation in A. japonicus\u003c/h2\u003e\u003cp\u003eThe impacts of immune parameters observed following dietary supplementation with varying levels of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics in \u003cem\u003eA. japonicus\u003c/em\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. All experimental groups exhibited an initial increase in immune enzyme activities, followed by a gradual decline after week 4. Compared with the control and LPP-L groups, the ACP activity was elevated in both LPP-M and LPP-H groups at all time points (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), though there were no statistical differences between them (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003eA1)\u003c/b\u003e. The AKP levels of all experimental groups were notably elevated at weeks 4 and 6, showing dose-dependent effects \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003eA2)\u003c/b\u003e. However, \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics supplementation had limited effects on LZM activity, with only the LPP-M group showing a significant effect at week 4 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant differences were observed among all groups at any time point (\u003cem\u003eP\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003eA3)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe relative expression levels of immune-related genes (\u003cem\u003eRel\u003c/em\u003e, \u003cem\u003eP50\u003c/em\u003e, and \u003cem\u003eC3-2\u003c/em\u003e) in various tissues of \u003cem\u003eA. japonicus\u003c/em\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D. \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics significantly influenced the expression of the \u003cem\u003eRel\u003c/em\u003e gene in the body wall, muscle, respiratory tree, and midgut of \u003cem\u003eA. japonicus\u003c/em\u003e, showing a consistent pattern of initial increase, peaking at week 4, followed by a gradual decline. In contrast, the effects on the expression of the \u003cem\u003eRel\u003c/em\u003e gene in the foregut and hindgut were minimal, showing no statistical differences among all groups at most time points (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Interestingly, the influence on tissues of both the respiratory tree and gut appeared to be temporary: by week 6, there were no statistical differences among all groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Similar expression patterns were observed for the \u003cem\u003eP50\u003c/em\u003e gene in the body wall, respiratory tree, and gut tissues, exhibiting an increase until week 4, followed by a gradual decline. However, \u003cem\u003eP50\u003c/em\u003e upregulation in muscle tissue remained limited, with only the LPP-M group showing significantly elevated levels at week 4 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); moreover, no intergroup differences were observed at any other time points (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. The expression of the \u003cem\u003eC3-2\u003c/em\u003e gene in the body wall, respiratory tree, and gut tissues was significantly affected by \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics, displaying a characteristic rise-and-fall pattern, with peak expression occurring at week 4. Interestingly, in muscle tissue, the \u003cem\u003eC3-2\u003c/em\u003e expression showed delayed enhancement, with upregulation only appearing at week 6 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eNotably, in the aforementioned results, the LPP-M group demonstrated immune enzyme activities and related gene expression levels that were similar to, or even higher than, those observed in the LPP-H group. Furthermore, in gut tissues, the expression levels of immune genes varied across different gut segments. Overall, their expression was higher in the midgut and hindgut compared to the foregut, demonstrating a certain concentration-dependent pattern.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eEffects of L. plantarum O126 postbiotics on the enhancement of disease resistance in A. japonicus\u003c/h2\u003e\u003cp\u003eThe evisceration rates of \u003cem\u003eA. japonicus\u003c/em\u003e following \u003cem\u003eV. splendidus\u003c/em\u003e infection are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. After the challenge, both experimental and control groups exhibited gradually increasing evisceration rates that stabilized after 9 days. The final evisceration rates were 64% (control), 52% (LPP-L), 40% (LPP-M), and 44% (LPP-H) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. In contrast to the control group, the evisceration rates in the experimental groups were significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the most significant reduction observed in the LPP-M group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEffects of L. plantarum O126 postbiotics on the gut microbiota community of A. japonicus\u003c/h2\u003e\u003cp\u003eThe impacts of dietary \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics on the gut microbiota community of \u003cem\u003eA. japonicus\u003c/em\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The Venn diagram revealed 696 shared ASVs among the four groups, while the control, LPP-L, LPP-M, and LPP-H groups contained 1203, 1742, 2419, and 2407 unique ASVs, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Chao1 and Shannon indices revealed that, compared with the control group, α-diversity was elevated in experimental groups, with the LPP-M and LPP-H groups showing more prominent enhancement in α-diversity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb \u003cb\u003eand c)\u003c/b\u003e. β-diversity analysis using PCoA and NMDS revealed considerable variations in gut microbiota structure under different diets \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed \u003cb\u003eand e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition, at the phylum and genus levels, differences in the composition of the gut microbiota among different groups are also shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef \u003cb\u003eand g\u003c/b\u003e. At the phylum level \u003cb\u003e(\u003c/b\u003etop 10 phyla, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e, compared with the control group, all experimental groups showed significantly reduced relative abundance of Proteobacteria but increased relative abundance of Firmicutes and Actinobacteria. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg shows the distribution of relative abundance for the top 10 dominant bacterial genera at the genus level in the gut microbiota.\u003c/p\u003e\u003cp\u003eTo further investigate the effects of different doses of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics on the gut microbiota composition of \u003cem\u003eA. japonicus\u003c/em\u003e, the Linear Discriminant Analysis (LDA) method was used in LEfSe analysis to identify differential microbial taxa among groups. It was determined that the control group was rich in genus-level differential microbial taxa, including \u003cem\u003eSulfitobacter\u003c/em\u003e, \u003cem\u003eBrevundimonas\u003c/em\u003e and \u003cem\u003eVibrio\u003c/em\u003e; the LPP-L group was rich in \u003cem\u003eHaloferula\u003c/em\u003e, \u003cem\u003ePseudoruegeria\u003c/em\u003e, \u003cem\u003ePseudopelagicola\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eMarimicrobium\u003c/em\u003e; the LPP-M group was rich in \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eExiguobacterium\u003c/em\u003e; and the LPP-H group was rich in \u003cem\u003eVagococcus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea \u003cb\u003eand b)\u003c/b\u003e. The significance test analysis revealed that the relative abundance of \u003cem\u003eSulfitobacter\u003c/em\u003e and \u003cem\u003eBrevundimonas\u003c/em\u003e was significantly downregulated in all experimental groups, while \u003cem\u003ePseudomonas\u003c/em\u003e was downregulated in the LPP-M and LPP-H groups compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-e\u003cb\u003e)\u003c/b\u003e. \u003cem\u003ePseudoruegeria\u003c/em\u003e was significantly upregulated in the LPP-L and LPP-M groups compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e. Interestingly, the LPP-M and LPP-H groups showed consistency in genus composition, with \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eExiguobacterium\u003c/em\u003e, and \u003cem\u003eVagococcus\u003c/em\u003e showing increased abundance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-i\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eCorrelation analysis\u003c/h2\u003e\u003cp\u003eCorrelation analysis revealed that growth indicators and evisceration rate were significantly correlated with gut morphology and immune-antioxidant indicators \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Specifically, the final body weight and SGR exhibited a positive correlation with VH and VW (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). FCR exhibited negative correlations with VH and VW (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, the evisceration rate was negatively correlated with final body weight, SGR, VH, VW, ACP, and \u003cem\u003eP50\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe correlation heatmap analysis further indicated potential associations between the gut microbiota and other health indicators. The genera \u003cem\u003eHaloferula\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e also showed negative correlations with final body weight, SGR, VH, and VW (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, the genus \u003cem\u003eSulfitobacter\u003c/em\u003e demonstrated negative correlations with final body weight, SGR, VH, VW, SOD, \u003cem\u003ePRDX5\u003c/em\u003e, ACP, \u003cem\u003eRel\u003c/em\u003e, \u003cem\u003eP50\u003c/em\u003e, and \u003cem\u003eC3-2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Moreover, the genus \u003cem\u003eBrevundimonas\u003c/em\u003e demonstrated negative correlations with final body weight, SGR, VH, VW, ACP, \u003cem\u003eP50\u003c/em\u003e, and \u003cem\u003eC3-2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003eConversely, \u003cem\u003ePseudoruegeria\u003c/em\u003e showed significant positive correlations with final body weight, MT, and \u003cem\u003eP50\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the genera \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eExiguobacterium\u003c/em\u003e, and \u003cem\u003eVagococcus\u003c/em\u003e showed positive correlations with final body weight, SGR, VH, VW, \u003cem\u003ePRDX5\u003c/em\u003e, ACP, and \u003cem\u003eP50\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while exhibiting negative correlations with the evisceration rate (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eProbiotics, widely used as bioregulators in aquaculture, can enhance production efficiency and animal health status [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, as live microbial products, they are highly susceptible to environmental conditions during production, storage, and application [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In contrast, postbiotics, which consist of inactivated microbial cells and/or their extracellular metabolites, can possess superior stability and safety advantages. These properties facilitate large-scale production and storage. They have been shown to enhance the health of aquatic organisms such as Nile tilapia, Pacific white shrimp, and black sea bream, among others. However, research on their effects on \u003cem\u003eA. japonicus\u003c/em\u003e remains limited. Consequently, this investigation focused on the impacts of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics on growth, antioxidant capacity, disease resistance, and gut health in \u003cem\u003eA. japonicus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eA considerable body of evidence supports the notion that postbiotic additives can promote growth in aquatic organisms [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this study, the notable increases in body weight and specific growth rate among the LPP-M and LPP-H groups indicate that \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics significantly improved the growth of \u003cem\u003eA. japonicus\u003c/em\u003e. This positive effect is likely linked to increased activity of digestive enzymes, as documented in earlier studies [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Supporting this notion, our findings revealed a marked boost in trypsin and lipase activities within the gut. However, amylase activity remained unaffected, contrasting with previous reports by Yang et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], potentially due to differences in probiotic strains, dosage regimens, or intervention durations. Notably, this study also observed significant increases in both villus height and width in the midgut. Such morphological modifications can enhance nutrient absorption efficiency by expanding the surface area of gut villi [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Correlation analysis also indicates that they are crucial factors contributing to the observed growth promotion in the subjects of this study. Furthermore, the improved growth performance may also be associated with enhanced antioxidant capacity, immune status, and modulation of gut microbiota [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAntioxidant capacity serves as a critical indicator of animal health status. T-AOC is a vital measure for assessing the overall antioxidant levels in various organisms [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. SOD facilitates the conversion of superoxide anion radicals (O₂⁻) into hydrogen peroxide (H₂O₂), and CAT further converts H₂O₂ into water and oxygen, thus safeguarding the organism against damage from free radicals [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This study revealed that the inclusion of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics in the diet enhanced the activities of T-AOC, SOD, and CAT to varying degrees in the coelomic fluid of \u003cem\u003eA. japonicus\u003c/em\u003e, indicating a bolstered antioxidant capacity in this species. Peroxiredoxin (Prx), as a core component of the organism's antioxidant system, can catalyze the reduction of H₂O₂ to water. The proteins encoded by the \u003cem\u003ePRDX5\u003c/em\u003e and \u003cem\u003ePRDX6\u003c/em\u003e genes belong to the Prx superfamily [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This research found that adding \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics to the diet notably boosted the levels of \u003cem\u003ePRDX5\u003c/em\u003e and \u003cem\u003ePRDX6\u003c/em\u003e gene expression across various tissues, including the body wall, respiratory tree, and gut in \u003cem\u003eA. japonicus\u003c/em\u003e, with the results reaching statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the muscle tissue showed no noticeable changes in gene expression following supplementation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), demonstrating distinct tissue-specific expression patterns for these antioxidant genes. The antioxidant regulatory effects of \u003cem\u003eL. plantarum\u003c/em\u003e postbiotics may originate from their inherent bioactive compounds, including γ-aminobutyric acid (GABA), exopolysaccharides, and lipoteichoic acid (LTA) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], which can directly scavenge free radicals and enhance antioxidant capacity. Furthermore, our results demonstrated that these postbiotics significantly upregulated antioxidant enzyme activities in \u003cem\u003eA. japonicus\u003c/em\u003e. This regulatory pattern has been similarly validated in black sea bream [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and Nile tilapia [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], indicating an additional indirect antioxidant protective mechanism through activation of the host's endogenous antioxidant enzyme system.\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. japonicus\u003c/em\u003e lacks an adaptive immune system, relying on non-specific immunity as its main form of immune defense [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The non-specific immune enzymes secreted by coelomocytes constitute an indispensable component of non-specific immunity, playing a critical role in immune defense and pathogen clearance [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our findings demonstrated that \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics can enhance non-specific immune enzymes of \u003cem\u003eA. japonicus\u003c/em\u003e. Furthermore, numerous studies have reported that postbiotics can enhance innate immune responses by regulating immune-related genes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, we further examined the expression of essential immune-related genes (\u003cem\u003eRel\u003c/em\u003e, \u003cem\u003eP50\u003c/em\u003e, and \u003cem\u003eC3-2\u003c/em\u003e) in \u003cem\u003eA. japonicus\u003c/em\u003e. Although tissue-specific differential expression of these genes was observed, dietary supplementation with \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics significantly upregulated their expression across all examined tissues, suggesting that these postbiotics effectively enhance cellular immune responses in \u003cem\u003eA. japonicus\u003c/em\u003e. Notably, the midgut exhibited higher immune gene expression levels than the foregut and hindgut, likely because of its more diverse microbial community [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], which produces immune-modulating enzymes and bioactive compounds [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, the challenge test revealed that \u003cem\u003eA. japonicus\u003c/em\u003e fed with \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics exhibited significantly lower evisceration rates, indicating that they have potential applications for enhancing resistance against \u003cem\u003eV. splendidus\u003c/em\u003e infection. The immune-boosting and infection-fighting properties of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics are likely due to various bioactive molecules, such as antimicrobial peptides, exopolysaccharides, and short-chain fatty acids (SCFAs). By engaging with pattern recognition receptors (PRRs) on immune cells, these molecules activate signaling pathways like NF-κB or MAPK that modulate the immune response [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe gut microbiota plays a crucial role in maintaining the health of aquatic animals [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Numerous studies have reported that postbiotics can enhance microbial diversity and sustain gut microecological homeostasis in animals [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Consistent with these findings, the LPP-M and LPP-H groups exhibited notably higher α-diversity indices of gut microbiota, indicating that \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics improved species richness and evenness in the gut microbiota of \u003cem\u003eA. japonicus\u003c/em\u003e. Furthermore, β-diversity analysis revealed distinct alterations in microbial composition among postbiotics-supplemented groups and control group, demonstrating that \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics serve as key modulators of the gut microbiota in \u003cem\u003eA. japonicus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003ePhylum-level analysis showed a notable decrease in Proteobacteria proportions within the experimental groups. As a phylum ubiquitously distributed in marine environments, Proteobacteria encompasses various pathogenic genera, including \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eAeromonas\u003c/em\u003e, and \u003cem\u003eVibrio\u003c/em\u003e, among others. Elevated proportions of Proteobacteria in the gut microbiota typically indicate microbial community instability, which may trigger inflammatory responses and disease onset [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Conversely, \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics increased the relative abundances of Firmicutes and Actinobacteria. Notably, Firmicutes are capable of producing various active enzymes that aid hosts in food decomposition and digestion, followed by fermentative production of SCFAs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Actinobacteria can maintain gut microbiota homeostasis, as well as modulate both immune system function and metabolic processes [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The increased relative abundances of these phyla suggest that \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics may promote both growth and immunity in \u003cem\u003eA. japonicus\u003c/em\u003e. At the genus level, LEfSe analysis revealed that different dosage additions of \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics could lead to distinct colonization patterns of gut microbiota in \u003cem\u003eA. japonicus\u003c/em\u003e. \u003cem\u003eSulfitobacter\u003c/em\u003e exhibited decreased relative abundance across all experimental groups, and correlation analysis indicated a negative relationship between this genus and key physiological health indicators, including growth and immunity. Although studies on \u003cem\u003eSulfitobacter\u003c/em\u003e in animal gut microbiota remain limited, our findings suggest that it may play a harmful role in the gut microbiota of aquatic animals. Additionally, \u003cem\u003eBrevundimonas\u003c/em\u003e was significantly downregulated in all experimental groups, while \u003cem\u003ePseudomo\u003c/em\u003enas was significantly downregulated in the LPP-M and LPP-H groups. Both \u003cem\u003eBrevundimonas\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e are frequently reported as common pathogenic bacteria in aquatic animals [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConversely, \u003cem\u003ePseudoruegeria\u003c/em\u003e was significantly upregulated in the LPP-L and LPP-M groups. Currently, there are limited reports on the genus \u003cem\u003ePseudoruegeria\u003c/em\u003e. Ko et al. [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] identified a species within this genus that possesses metabolic pathways for all seven B vitamins, while Cho et al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] discovered that certain species within the same genus could eliminate harmful algae. This study also found a positive correlation between \u003cem\u003ePseudoruegeria\u003c/em\u003e and the growth and immune status of \u003cem\u003eA. japonicus\u003c/em\u003e, suggesting its potential role as a probiotic in the gut microbiota. However, further investigation is needed to elucidate the underlying mechanisms.\u003c/p\u003e\u003cp\u003eInterestingly, both the LPP-M and LPP-H groups consistently showed significantly increased abundances of three genera: \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eExiguobacterium\u003c/em\u003e, and \u003cem\u003eVagococcus\u003c/em\u003e. The genus \u003cem\u003eBacillus\u003c/em\u003e has been widely utilized as a probiotic in aquaculture and has demonstrated immunomodulatory activities [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. \u003cem\u003eExiguobacterium\u003c/em\u003e, a prospective probiotic for aquatic animals, synthesizes glutathione (GSH) to boost the host's antioxidant defenses [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Some strains of the genus \u003cem\u003eVagococcus\u003c/em\u003e have been proven to help aquatic animals resist \u003cem\u003eVibrio\u003c/em\u003e infection [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Correlation analysis suggests that microbial population shifts may contribute to enhancing growth velocities, amplified antioxidant protection, and strengthened immune responses in \u003cem\u003eA. japonicus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTaken together, our results suggest that \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics effectively enhance growth performance, improve gut morphology, promote digestion, boost immunity, enhance antioxidant capacity, and increase gut microbial diversity in \u003cem\u003eA. japonicus\u003c/em\u003e, while concurrently increasing resistance against \u003cem\u003eV. splendidus\u003c/em\u003e. These results indicate that \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics have potential for application in sustainable \u003cem\u003eA. japonicus\u003c/em\u003e aquaculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFeng-Li Chen: Investigation, Formal analysis, Software, Writing-original draft; Hui-Ting Zhao: Investigation, Formal analysis; Wei Gu: Project administration, Resources; Zong-Xiu Wu: Investigation, Formal analysis; Jing-Wen Xiang: Investigation; Qing Yang: Investigation; Yu-Lin Yang: Investigation; Lei Tan: Investigation; Meng-Xia Sun: Investigation; Wei Cong: Writing-review \u0026amp; editing; Shu Li: Writing-review \u0026amp; editing; Bin-Tong Yang: Project administration, Funding acquisition, Writing-review \u0026amp; editing; Yuan-Huan Kang: Project administration, Funding acquisition, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Youth Science Foundation Program, Grant No. 32301410), Tai\u0026apos;an City Science and Technology Innovation \u0026apos;\u0026apos;Double Ten Engineering\u0026apos;\u0026apos; (Major Technological Breakthrough) Project (23JSGG02) and Key R\u0026amp;D Program (Rural Revitalization Technological Innovation Boosting Action Plan) of Shandong Province, China (Program No. 2024TZXD006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe underlying data for this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll animal experiments carried out in this study strictly adhered to the guidelines set forth by Shandong University\u0026apos;s Regulations for Animal Experimentation, as well as the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No.1438023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRu XS, Zhang LB, Li XN, Liu SL, Yang HS (2019) Development strategies for the sea cucumber industry in China. 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Vet Microbiol 155(2\u0026ndash;4):369\u0026ndash;373. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vetmic.2011.09.013\u003c/span\u003e\u003cspan address=\"10.1016/j.vetmic.2011.09.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lactiplantibacillus plantarum, postbiotics, Apostichopus japonicus, disease resistance, gut microbiota","lastPublishedDoi":"10.21203/rs.3.rs-7545463/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7545463/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e (\u003cem\u003eL. plantarum\u003c/em\u003e) postbiotics promote animal health, but their application in \u003cem\u003eApostichopus japonicus\u003c/em\u003e is currently limited. This study added \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics to the diet at concentrations of 1\u0026permil;, 2\u0026permil;, and 4\u0026permil; (w/w) for 42 days to assess their impacts on \u003cem\u003eA. japonicus.\u003c/em\u003e The results showed that, in terms of growth performance, the medium-dose (2\u0026permil;, LPP-M) and high-dose (4\u0026permil;, LPP-H) groups significantly increased the final body weight and specific growth rate (SGR), and reduced the feed conversion ratio (FCR). Regarding digestion and gut morphology, the LPP-M and LPP-H groups significantly enhanced gut trypsin and lipase activities and increased the height and width of midgut villi. With respect to antioxidant capacity, \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics increased T-AOC, CAT, and SOD levels and upregulated the expression of antioxidant-related genes \u003cem\u003ePRDX5\u003c/em\u003e and \u003cem\u003ePRDX6\u003c/em\u003e. In terms of immunoregulation, \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics increased ACP and AKP activities and upregulated the expression of immune-related genes \u003cem\u003eRel\u003c/em\u003e, \u003cem\u003eP50\u003c/em\u003e, and \u003cem\u003eC3-2\u003c/em\u003e. Regarding disease resistance, after the \u003cem\u003eVibrio splendidus\u003c/em\u003e challenge, all experimental groups exhibited reduced evisceration rates, with the LPP-M group showing the lowest rate (40%). In terms of gut microbiota, the α-diversity of the experimental groups was significantly increased, with higher levels of beneficial bacteria (\u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eExiguobacterium\u003c/em\u003e, \u003cem\u003eVagococcus\u003c/em\u003e) observed in the LPP-M and LPP-H groups. Overall, \u003cem\u003eL. plantarum\u003c/em\u003e O126 postbiotics improved the growth and disease resistance of \u003cem\u003eA. japonicus\u003c/em\u003e by enhancing digestion, antioxidant capacity, immune function, as well as by regulating gut microbiota, with a medium dose (2\u0026permil;, w/w) showing the best effect.\u003c/p\u003e","manuscriptTitle":"Lactiplantibacillus plantarum O126 postbiotics boost growth, antioxidant levels, disease resistance, and gut health in sea cucumber (Apostichopus japonicus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 09:48:49","doi":"10.21203/rs.3.rs-7545463/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-18T05:42:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-17T18:09:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-11T08:20:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115337414748741902197996415271189207833","date":"2025-12-07T09:11:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73819261090308164155029425673444687735","date":"2025-12-07T08:15:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302603317083356913067143648838463328388","date":"2025-12-07T07:44:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109977874848334700612269425707464235119","date":"2025-12-06T05:33:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312722141741085175798053498554381957207","date":"2025-12-05T12:13:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-05T06:09:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-08T09:12:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-08T09:08:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Probiotics and Antimicrobial Proteins","date":"2025-09-05T14:59:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d449dc39-1686-4313-b1f5-8ff7cedf2401","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:04:01+00:00","versionOfRecord":{"articleIdentity":"rs-7545463","link":"https://doi.org/10.1007/s12602-026-10929-8","journal":{"identity":"probiotics-and-antimicrobial-proteins","isVorOnly":false,"title":"Probiotics and Antimicrobial Proteins"},"publishedOn":"2026-02-26 15:57:39","publishedOnDateReadable":"February 26th, 2026"},"versionCreatedAt":"2025-12-08 09:48:49","video":"","vorDoi":"10.1007/s12602-026-10929-8","vorDoiUrl":"https://doi.org/10.1007/s12602-026-10929-8","workflowStages":[]},"version":"v1","identity":"rs-7545463","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7545463","identity":"rs-7545463","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}