Probiotics provide an efficient and relatively safe method for preventing disease and increasing production in aquaculture. During the screening of beneficial bacteria in the economically important mud crab (Scylla paramamosain), an indigenous gut strain, Cetobacterium somerae R9, was isolated for the first time as a butyrate producer. This study aimed to evaluate the probiotic potential of C. somerae R9 both in vitro and in vivo. In vitro assays revealed that C. somerae R9 exhibited grow at pH 7-9, NaCl concentrations of 0.5-2.5%, and bile salt concentrations of 0.4-1.0%, and displayed susceptibility to most of the antibiotics tested. In the in vivo study, dietary supplementation with C. somerae R9, either alone or in combination with prebiotics (resistant starch and galactooligosaccharides), enhanced growth, improved antioxidative status (evidenced by elevated SOD and CAT activity and reduced MDA content), reduced hepatopancreatic damage (reduced AST activity), and maintained intestinal integrity. Supplementation also selectively enriched beneficial gut microbiota (e.g., members of Fusobacteriota). Transcriptome analysis showed that C. somerae R9 appeared to activate the PI3K-Akt signaling pathway, focal adhesion, and ECM-receptor interaction, while the combination of C. somerae R9 and prebiotics activates complement and coagulation cascades, amino sugar and nucleotide sugar metabolism, and protein digestion and absorption. Furthermore, mud crabs fed diets supplemented with C. somerae R9 exhibited significantly higher survival after challenge with Vibrio parahaemolyticus, with the synbiotic providing greater benefits than the probiotic alone. These findings collectively suggest that C. somerae R9 is a promising probiotic candidate (used either alone or in combination with prebiotics) for mud crab aquaculture.

The mud crab (Scylla paramamosain) is an important economically farmed crustacean species in Southeast Asia, with continually increasing annual yields and high economic value. However, in recent years, the mud crab aquaculture industry has been afflicted by major disease outbreaks, among which Mud Crab Reovirus (MCRV) is the principal pathogen causing mass mortality of the species and posing a severe threat to industry development. In this study, transcriptomic analysis of mud crab hemocytes following MCRV infection was conducted to elucidate the molecular mechanisms underlying antiviral responses. The results revealed that MCRV infection significantly enriches apoptosis-related pathways, with widespread upregulation of genes, such as p53, caspase-3, bax, and bag-1. Functional experiments showed that promoting apoptosis effectively reduces viral loads, while inhibiting apoptosis accelerates viral replication. In addition, screening of immune signaling pathways showed that the transcription factor SpFoxO in the AMPK-FoxO pathway is markedly upregulated, and SpFoxO regulates key genes of the RNAi pathway (including dicer-2, ago2, and draper) to enhance antiviral capacity. Silencing the SpFoxO gene led to increased viral load and decreased survival rate in mud crabs. In contrast, classical immune pathways such as TLR-Dorsal, IMD-Relish, and JAK-Stat showed weaker or reduced responses. This study provided evidence that mud crabs primarily rely on apoptosis and FoxO-mediated antiviral responses to combat MCRV infection, offering a theoretical basis for future antiviral intervention and disease management in aquaculture.

The aquaculture sector is vulnerable to disease outbreaks, environmental stressors, and antibiotic overuse due to high stocking densities and intensive production practices, necessitating the adoption of sustainable health management strategies. Dietary functional additives such as probiotics, prebiotics, and synbiotics have emerged as promising alternatives to antibiotics in fish farming. Probiotics are live, non-pathogenic microorganisms that confer health benefits to the host, whereas prebiotics are indigestible substrates that selectively stimulate beneficial gut microbiota; synbiotics combine both components to enhance their functional complementarity. The individual effects of probiotics and prebiotics are well-documented. Nevertheless, there is limited integrative knowledge on the optimal dosage and combination strategies. This review critically synthesizes synbiotic supplementation (prebiotics and probiotics) in fish farming, highlighting the underlying mechanisms and the synbiotic dosages that maximize synergistic effects on fish growth, immune response, and disease resistance. By consolidating experimental evidence and identifying knowledge gaps, it offers a practical and research-oriented framework to guide the effective application of synbiotics in sustainable aquaculture.

Butyrate-producing bacteria (BPB) are widely used as additives in aquaculture because of the beneficial effects of the butyrate they produce. However, most available BPB currently are of terrestrial origin, which are quite different from aquatic animals, resulting in profoundly unstable probiotic performance. This study isolated a novel, indigenous BPB, Clostridium moniliforme G18, from the gut of mud crabs, Scylla paramamosain. The strain demonstrated robust butyric acid production, tolerance to gastrointestinal conditions (pH 4–9, 0–3

Mitochondria, as central hubs of cellular metabolism and signaling, play a pivotal role in mediating the physiological response of aquatic animals to environmental stressors, largely through their involvement in oxidative stress pathway and quality control mechanism. Understanding these molecular pathways is crucial for addressing key challenges in both aquaculture and environmental toxicology. Rather than focusing on the established fact of stress induced mitochondrial damage, this review synthesizes current knowledge to highlighting the emerging role of the mitochondrial quality control (MQC) system as a decisive determinant of stress resilience. A key research evolution is documented, showing a shift from describing oxidative stress towards exploiting MQC for adaptation and performance optimization. Furthermore, a novel theoretical framework is proposed, explaining how aquatic animals perceive and respond to environmental stress through a multi-stage process: ‘stress perception-metabolic reprogramming-quality control’. This framework not only integrates a wide range of existing research but also pinpoints key intervention points for enhancing stress resilience. Collectively, these findings provide a significant theoretical foundation and practical guidance for stress tolerance breeding in aquaculture, precise environmental management, and sustainable development.

Optimizing metabolic health in farmed fish is increasingly important as aquaculture production intensifies. Gracilaria lemaneiformis-derived polysaccharide (GLP), a natural sulfated polysaccharide, has emerged as a promising candidate for mitigating lipid metabolic disorders. To elucidate the mechanisms through which GLP modulates hepatic lipid deposition, we established complementary in vivo feeding trial in rabbitfish (Siganus canaliculatus) under standard culture conditions and in vitro palmitic acid-induced steatosis model. A total of 450 fish (initial body weight: 3.11 ± 0.01 g) were randomly distributed into three experimental groups with three replicate cages per group (50 fish per cage). Fish were fed a basal diet (control) or basal diet supplemented with 1.0 g GLP/kg diet (GLP1) or 1.5 g GLP/kg diet (GLP2) for 60 days. Results showed that the GLP supplementation promoted growth performance, with the GLP1 achieving the greatest final body mass, weight gain, and thermal growth coefficient (P < 0.05), whereas GLP2 yielded superior economic conversion efficiency and improved population uniformity (P < 0.05). In parallel, GLP reduced circulating total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels (P < 0.05). Histological assessment further indicated that GLP effectively attenuated hepatic injury and lipid droplet accumulation. Transcriptomic profiling demonstrated that differentially expressed genes were primarily enriched in pathways related to lipid metabolism, amino acid metabolism, and signal transduction. Mostly, GLP enhanced lipid catabolism and β-oxidation by upregulating genes such as acbd4 and pnpla2, while concurrently suppressing lipogenic genes including sqle and lss. Consistent with the in vivo findings, in vitro assays showed that GLP mitigated hepatocyte steatosis in parallel with downregulation of sqle and its downstream lipogenic regulators (acc, fas, and ppar-γ). While pharmacological inhibition of SQLE with terbinafine recapitulated the lipid lowering effects of GLP, identifying SQLE as a transcriptional correlate of GLP action rather than establishing direct mechanistic targeting. Collectively, these results establish GLP as an effective lipid-lowering feed additive that simultaneously promotes lipid breakdown and inhibits lipid synthesis, providing a practical nutritional strategy to enhance metabolic health and production efficiency in aquaculture.

This study probed the impacts of dietary octacosanol supplementation at 0 (control), 6, 12, and 18 mg/kg diets on the growth parameters, intestinal enzyme activities, biochemical, antioxidant status, gene expression pattern, and hypoxia tolerance of whiteleg shrimp (Litopenaeus vannamei, initial weight 1.49 ± 0.01 g, n = 3, total shrimp = 360) during a 60-day feeding trial. To assess growth efficiency, final weight, weight gain, and specific growth rate were recorded, which were significantly (p < 0.05) improved with octacosanol supplementation, with optimal responses observed at 12 mg/kg. Feed conversion ratio decreased quadratically, reaching optimal efficiency at approximately 10.83 mg/kg. Intestinal enzyme activities (amylase, lipase, and protease) and antioxidant indicators (SOD, CAT, GPx, and TAC) were markedly enhanced at 12 mg/kg, while lipid peroxidation (MDA) was minimized. Hemolymph biochemical indices, including total protein, albumin, globulin, HDL-c, and lysozyme, increased significantly (p < 0.05), whereas AST and ALT decreased. Furthermore, transcriptional expression of immune- and antioxidant-related genes (Mn-SOD, CAT, GSH-Px, LYZ, and ProPO) was upregulated, coinciding with elevated antioxidant capacity and immune responses. Shrimp fed octacosanol diets exhibited enhanced hypoxia tolerance, with LT50 extended by approximately 30–40 min compared to the control. Collectively, the results demonstrate that dietary octacosanol at 12 mg/kg optimizes growth, physiological health, and stress resilience in L. vannamei, supporting its potential as a natural dietary additive for sustainable shrimp farming. Future studies are needed to explore octacosanol’s long-term effects on shrimp under various stress conditions, its molecular mechanisms, and its impact on other species, as well as its synergy with other feed additives.

Outer membrane vesicles (OMVs) are extracellular nanostructures secreted by bacteria, which carry various virulence factors and play a pivotal role in the pathogenesis of bacterial infections. In this study, outer membrane vesicles derived from Vibrio parahaemolyticus (Vp-OMVs) were isolated to examine their effects on innate immunity in mud crabs (Scylla paramamosain). Characterization of the Vp-OMVs confirmed the presence of vesicles exhibiting typical OMV morphology and size. Vp-OMVs were internalized by host cells through clathrin-mediated endocytosis, leading to cytotoxicity, increased mortality, tissue vacuolation, and reduced cell viability. Furthermore, Vp-OMVs were found to contain lipopolysaccharide (LPS), which activates the Toll2 receptor, thereby initiating the Toll2 signaling pathway and promoting the expression of antimicrobial peptides. Additionally, internalized Vp-OMVs triggered inflammasome activation and mitochondrial dysfunction, resulting in inflammation and apoptosis. These findings highlight the critical role of Vp-OMVs in the pathogenesis of V. parahaemolyticus infections in mud crabs and offer valuable insights into potential therapeutic strategies for managing these infections.

Exosomes play an important role in the innate immune system. Many types of cells can secrete exosomes in both normal and abnormal status. However, the mechanism of exosome secretion in invertebrates has not been reported. In this study, the mechanism of the secretion of exosomes regulated by the Ca2+ signaling pathway in the hemocytes of mud crabs (Scylla paramamosain) under infection with Vibrio parahaemolyticus was investigated. The results showed that the increase of exosome secretion in mud crabs infected with V. parahaemolyticus was caused by the increase in intracellular Ca2+ level. In addition, transcriptome sequencing and differential expression analysis revealed that SpVAMP2, a vesicle-associated membrane protein 2, was found to be involved in the exosome secretion regulated by Ca2+ signaling. Further research revealed that SpVAMP2 can inhibit MVB from entering the lysosome for degradation and promote the release of exosomes. Additionally, the secretion of exosomes upregulated the expression of anti-lipopolysaccharide factors and enhanced the survival rate of mud crabs after V. parahaemolyticus infection. This study clarified the exosome secretion regulated by the Ca2+-SpVAMP2 pathway, which, in turn, increased the innate immune response in mud crabs to resist the invasion of V. parahaemolyticus.

In autumn 2020, cultured sea cucumbers (Apostichopus japonicus) in the Xiapu Sea area (Southeast China) exhibited ulcerative skin lesions. To identify the causative pathogen, the Gram-negative strain 10MKXP20 was isolated from the muscle tissue of diseased animals and identified as Photobacterium leiognathi through phenotypic and genetic analysis. The bacterium was sensitive to 14 antibiotics, including tetracycline and enrofloxacin, but resistant to gentamicin, neomycin, kanamycin, rifampicin, amikacin, and amoxicillin. Minimum inhibitory concentration (MIC) assays revealed varying efficacy among the tested antibiotics, with enrofloxacin, doxycycline, and florfenicol showing particularly low MICs, indicating strong inhibitory activity. Challenge experiments via immersion and intramuscular injection confirmed the pathogenicity of P. leiognathi 10MKXP20, with mortality rates being dose-dependent. This study is the first to identify P. leiognathi as the causative agent of ulcerative skin disease in sea cucumbers, providing crucial insights for disease diagnosis and antibiotic treatment.

Spermatogonial stem cells (SSCs) can differentiate into sperm and are important for studying on genetic information transmission of animals. However, the establishment of the SSC line in crustaceans is still in its infancy. This study aimed to establish a method for the isolation, culture, and identification of SSCs derived from the gonad of a marine crustacean (mud crab, Scylla paramamosain), and evaluate their differentiation ability and potential application in immunological research, in vitro. SSCs showed robust growth, proliferation, and passaging ability (up to 35 passages) in germ cell culture medium. Proteomic analysis showed that the protein expression profile of SSC was closely related to the gonadal tissue. SSCs were found to be able to express male-specific and pluripotent markers, such as CD9, PIWI, DDX4, DAZL, NANOG, SOX2, and EPHA1. Furthermore, SSCs were differentiated into osteoblasts and adipocytes under in vitro induction. Green fluorescent protein (GFP), packaged by lentivirus, was able to be overexpressed in SSCs after infection. In addition, the infection of white spot syndrome virus (WSSV) simulated the expression of inflammation-associated factors, including TRAF6, TNF-α, MyD88, Dorsal, and Relish, and apoptosis-related genes (BAX and Bcl2) in SSCs. Thus, SSCs were initially isolated and characterized from mud crabs for the first time. Our results proved that SSCs can be used in reproduction technology, germplasm conservation, and immunological studies in crustaceans.

Aquaculture, encompassing the cultivation of diverse saltwater and freshwater fish and shellfish species, is rapidly expanding, driven by the pressing need for sustainable food production. However, intensified farming practices often lead to significant economic losses due to fish mortality from infectious diseases and stress-related conditions. Consequently, enhancing the immune responses of farmed species has become vital for sustainable management. This has encouraged a growing interest in natural and eco-friendly bioactive compounds as alternatives to synthetic chemicals for disease prevention. Among these, polysaccharides from various natural sources have emerged as promising candidates, administered through dietary inclusion, intraperitoneal injection, or immersion. As prebiotics, these polysaccharides enhance the immunity and overall health of aquaculture species by modulating both innate and adaptive immune responses, including lysozyme activity, phagocytosis, and cytokine production. This review critically examines the latest advances in polysaccharide-induced immunoregulation, highlighting their influence on key signaling pathways, which play pivotal roles in immune modulation. Additionally, the potential of polysaccharides to mitigate stress caused by environmental factors is discussed, demonstrating their ability to enhance growth performance and disease resistance. The collective findings underscore the value of polysaccharides as sustainable alternatives to antibiotics, aligning with the increasing consumer demand for antibiotic-free aquaculture products and supporting the advancement of global aquaculture sustainability.

The mud crab (Scylla paramamosain) is a major aquaculture species in southeastern China. However, intensifying farming has led to disease outbreaks, with Vibrio parahaemolyticus (Vp) infections causing high mortality and economic losses. Although gut microbiota is important for host health, its response to artificial Vp challenge in mud crabs remains poorly understood. In this study, we investigated gut microbiota and physiological responses to Vp infection resistant (VPS) and susceptible (VPD) mud crab groups. 16S RNA sequencing presented significant changes in microbial diversity, composition, and functional structure after Vp infection. Firmicutes significantly increased in VPS, but decreased in VPD, while Bacteroidetes significantly reduced in VPD. Compared to VPS, the structure of the intestine and hepatopancreas in the VPD group exhibited severe intestinal and hepatopancreatic damage, and reduced short chain fatty acids (including propionic and butyric acids) and antioxidant and immune enzyme activity (including superoxide dismutase, catalase, alkaline phosphatase, acid phosphatase, and polyphenol oxidase). In addition, RT-qPCR results indicated a significant upregulation of immune-related genes (including GLT, tKtA, α-2M, Lamp, proPO, Rab5, Toll1, Toll2, CTL-B, ALF5, NF-κB, and TNF-α) in the VPD. Thus, we found that during Vp infection, mud crabs in the VPS could regulate gut microbiota and restore physiological functions to normal levels, protecting them from infestation. The results of this study enhance our understanding of Vp-induced gut microbiota dysbiosis and immune activation in mud crabs, providing actionable targets for developing disease-resistant aquaculture strategies based on microbial-immune synergy.

C-type lectin (CTL) is a widespread pattern recognition receptor that recognizes and binds to pathogen-associated molecular patterns on the surface of pathogens and mediates a series of immune responses in animals. In this study, the mud crab SpCTL cDNA was cloned and functionally characterized. Phylogenetic analysis showed that SpCTL was closely related to Eriocheir sinensis with 81 % homology. The SpCTL mRNA was expressed in hemocytes and hepatopancreas under infection with Vibrio parahaemolyticus (Vp), lipopolysaccharide (LPS), white spot syndrome virus (WSSV), or Poly I: C (PIC). Furthermore, the recombinant SpCTL (rSpCTL) protein was purified, and the antimicrobial activity of rSpCTL was analyzed. The results showed that the rSpCTL had an agglutination activity on Staphylococcus aureus, Streptococcus b, Escherichia coli, Vp, Aeromonas hydrophila, Vibrio alginolyticus, and Saccharomyces cerevisiae in the presence of calcium ions. rSpCTL showed strong adhesion to Streptococcus b, E. coli and V. alginolyticus. Also, rSpCTL revealed good bacteriostatic activity on S. aureus, Streptococcus b, E. coli, Vp, A. hydrophila, and V. alginolyticus, with a dose-dependent manner, but not on S. cerevisiae. Besides, the mass spectrometry results showed that the expression of Sp(3-integrin and Spflotillin-2 (SpFLT-2) was significantly down-regulated at 6, 24, and 48 h when the expression of SpCTL was knocked down by RNAi, whereas the change of FLT-2 was not significant (first up-regulated and then down-regulated) at 6 and 24 h. Our results found that under stimulation of Vp, SpCTL recognizes the bacterium and binds to Sp(3-integrin, which transmits signals into hemocyte and subsequently regulates the expression of antimicrobial peptides (SpALF1, SpALF3-5) and protects mud crabs from infection. Collectively, the results indicate that SpCTL has agglutination, bacteriostatic, and adhesion activities. SpCTL play an important role in the innate immunity of mud crabs.

This study evaluated the synergistic effects of dietary butyrate-producing bacteria (Clostridium senegalense I5 (I5) and Paraclostridium benzoelyticum G5 (G5) and Gracilaria lemaneiformis-derived polysaccharide (GLP) in rabbitfish (Siganus canaliculatus). Both bacterial strains demonstrated high susceptibility to most antibiotics, salt tolerance up to 6.5 %, pH tolerance ranging from 2 to 10, and strong auto-aggregation abilities. In a 60-day feeding trial, rabbitfish were fed either commercial pelleted feed (CPF) as a control, or CPF supplemented with 0.10 % GLP and 10(7) cfu of G5 (GPb), or 0.10 % GLP and 10(7) cfu of I5 (GCs). GCs significantly improved growth and feed utilization compared to other diets (P < 0.05), while both GPb and GCs improved intestinal health, and digestive enzyme activity (amylase and lipase). Additionally, both GPb and GCs increased the activity of immune-related enzymes and total antioxidant capacity, while reducing malondialdehyde levels (P < 0.05). Transcriptomic analysis of liver tissue revealed differential gene expression in immune-related pathways for GCs and GPb. Fish fed GCs and GPb diets exhibited enhanced resistance against Vibrio parahaemolyticus compared to controls (P < 0.05). These findings suggest the potential of synbiotics involving I5 or G5 and GLP to improve growth, immune response, intestinal health, and disease resistance in rabbitfish, providing valuable insights for rabbitfish aquaculture.

Butyrate-producing bacteria (BPB) benefit the health of aquatic animals. This current study aimed to isolate BPB from the intestines of Nibea coibor and assess their probiotic potential. The results showed that nine isolates were obtained in vitro from the gut of N. coibor, including six Clostridium butyricum, two Proteocatella sphenisci, and one Fusobacterium varium. The representative bacteria, C. butyricum CG-3 and P. sphenisci DG-1, which produce high butyrate levels, were further studied for short-chain fatty acid (SCFA) production and antibiotic susceptibility. The effects of BPB singly (CB: basal diet + CG-3 and PS: basal diet + DG-1, at 107 CFU/g) or in combination with galactooligosaccharides (GOS) (0.5%) and inulin (0.5%) (CBIG) or D-sorbitol (0.5%) (PSGS) on the growth and health status of N. coibor were investigated. Results showed an increase in growth parameters in the CB, CBIG, and PSGS groups, except for the PS group. Alterations in intestinal microbiota (including diversity, abundance, and function) were observed in four experimental groups (CB, CBIG, PS, and PSGS groups). SCFA contents increased in treated groups; butyrate production was positively related to bacterial abundance. Compared to control, levels of complement C3, complement C4, immunoglobulin M (IgM), transforming growth factor-β (TGF-β), interleukin (IL)-10, IL-1β, and lysozyme (LZM) increased, while malondialdehyde (MDA) decreased in treated groups. Contents of IL-6 (PS and PSGS groups), tumor necrosis factor-alpha (TNF-α) (CB, PS, and PSGS groups), total antioxidant capacity (T-AOC) (CB and PS groups), total superoxide dismutase (T-SOD) (PS group), catalase (CAT) (CB and PSGS groups), and activities of amylase (PS and PSGS groups), trypsin (CB group), and lipase (CBIG group) were increased. Our results suggested the potential use of C. butyricum CG-1 or P. sphenisci DG-1 singly or in combination with prebiotics improved growth and health conditions in N. coibor.

The Toll pathway is crucial for innate immune responses in organisms (including Drosophila and mammals). The Spätzle protein outside of cells acts as a ligand for Toll receptors, enabling the transfer of signals from outside the cell to the inside. However, the function of Spätzle in the immune system of mud crab (Scylla paramamosain) remains unclear. This research discovered a novel Spätzle gene (Sp-Spz) in mud crab, which showed extensive expression in all the tissues that were examined. The RNA interference exhibited the correlation between Sp-Spz and the anti-lipopolysaccharide factors (ALFs). Knockdown of Sp-Spz decreased the expression of Sp-Toll2 but not Sp-Toll1. In Drosophila Schneider 2 cells, Sp-Spz was found interacted with Sp-Toll2. Moreover, the depletion of Sp-Spz caused the separation of hepatic lobules from the basement membrane, resulting in the disruption of the structural coherence of hepatopancreatic cells. Additionally, the knockdown of Sp-Spz resulted in changes to the composition of the hemolymph microbiota, specifically affecting the proportions of different phylum and family levels. The findings indicated that Sp-Spz may promote the synthesis of ALFs via Sp-Toll2, thereby influencing the homeostasis of microbiota in the hemolymph. In this study, novel insights into mud crab immunity are provided.

Exosomes have been considered anti-microbial immune factors in animals (including aquatic animals). However, the relationship between exosomal proteins and immune responses during bacterial infection has not been addressed. Flotillin-1 has previously been shown to enrich plasma membrane which implicates in cellular processes, including signal transduction, membrane trafficking, and molecular sorting. In this study, SpFlotillin-1 was cloned and characterized from mud crab (Scylla paramamosain). SpFlotillin-1 was found to be up-regulated in the hemocytes of mud crabs after infecting with V. parahaemolyticus. RNAi knockdown of SpFlotillin-1 showed capacity in suppressing phagocytosis and reducing the expression of antimicrobial peptides (AMPs) but increasing the ROS production in hemocytes. Furthermore, SpFlotillin-1 densely packaged in the exosomes can regulate the phagocytosis, expression of AMPs through MAPK and NF-κB pathway, and production of ROS, eventually activating the immune response to bacterial infection in mud crabs. Taken together, the results of this study provide a new finding on the mechanism that exosomal SpFlotillin-1 may participate in the V. parahaemolyticus infection through the regulation of phagocytosis and activation of AMP synthesis in mud crabs.

As an enduring hot topic in the field of innate immunity, apoptosis is widely considered as an effective approach to eliminate pathogenic microbes, and possesses crucial role during host-pathogen interactions. Recently, researchers have found that virus-containing host cells could transmit apoptotic signals to surrounding uninfected cells during infection, but the mechanism remains unclear. Here, we found that exosomes secreted by WSSV-infected mud crab hemocytes contain viral nucleic acid wsv277, which could be transported to the recipient cells and further expressed viral protein with phosphokinase activity. Besides, by using transcriptome, proteome, ChIP-seq and coIP techniques, the results revealed that wsv277 could activate the transcription and translation of apoptotic genes via interacting with CBF and EF-1α, so as to suppress the spread of virus infection by inducing apoptosis of the surrounding cells. Therefore, for the first time, our study proved that the components of DNA virus could be encapsulated into exosomes, and elucidated the mechanism of apoptotic signal transduction between cells from the perspective of exosome. Moreover, comparing those of invertebrates, we hypothesized that the innate immune system of vertebrate was weakened or degenerated, allowing exosomes to be employed by virus as the vehicle for immune escape. Significance statement Our study revealed that the components of DNA virus could be packaged and transmitted through the exosomes of lower invertebrates, which strongly demonstrated the diversity of exosome-mediated viral immunity and its universality in animals. Furthermore, we elucidated the mechanism of apoptotic signal transduction between cells from the perspective of exosome, and revealed a novel strategy for the host to cope with viral infection. Since exosome is an important innate immune regulation approach, we hypothesize that the innate immune system of vertebrates is weakened or degenerated, allowing exosomes to be employed by virus as the vehicle for immune escape.

Pacific white shrimp (Penaeus vannamei) is one of the most productive and economically important species globally. However, the development and continuous expansion of the farming scale led to an increase in the risk of disease occurrence in shrimp farming. The application of probiotics as an effective method for controlling diseases in aquaculture has been widely considered. In shrimp farming, several probiotics have been used and shown benefits to the health of the host. To diverse the sources of bacterial species as probiotics in shrimp farming, in this study, we aimed to elucidate the effects of dietary probiotics (Clostridium butyricum I9 (I9), Clostridium butyricum G15 (G15), or Paraclostridium bifermentans X13) on the growth, immune response and intestinal microbiome of white shrimp. Shrimps were fed with diets containing either phosphate-buffered saline (PBS), I9 (107 CFU/g feed), G15 (107 CFU/g feed), or X13 (107 CFU/g feed) for 30 days and followed by the challenge with Vibrio parahaemolyticus (Vp). The results showed that the survival rate, body weight gain, and special growth rate of shrimps in the I9, X13, and G15 groups significantly increased, compared to the PBS. The supplementation of probiotics increased the content of short-chain fatty acids and effectively maintained the normal morphology and structure of the intestinal tract and hepatopancreas. The I9, X13, or G15 groups showed a positive change in the diversity and abundance of gut bacteria. There was a significant up-regulation of CTL, SOD, proPO, Crustin, PEN2-4, and ALF1-3 genes in shrimps in the I9, X13, and G15. Additionally, dietary probiotics significantly increased the survival rate, maintained the intestinal structure, promoted the activities of SOD, AKP, ACP, and T-AOC enzymes, and reduced the level of MDA in shrimps after Vp infection. In conclusion, dietary supplementation of I9, G15, or X13 improved the growth, immunity, and disease resistance of Pacific white shrimp, providing a scientific basis for shrimp farming.