Effects of different slaughtering methods on the energy metabolism, apoptosis process and quality of grouper (Epinephelus fuscoguttatus) during cold storage at 4 °C.

BACKGROUND: The aquatic processing industry is increasingly aware of the need to ensure that slaughtering is carried out under high welfare standards, so there is a need to explore the impact of slaughter methods on fish fillets. This study aimed to investigate the effects of different slaughtering methods (M1, lethality by hammering; M2, gas mixture causing death; M3, lethality by clove oil anesthesia + ice slurry; M4, lethality by ice slurry; M5, lethality by gradient cooling) on the energy metabolism, apoptosis and flesh mass in grouper (Epinephelus fuscoguttatus). RESULTS: Therefore, 120 fish (24 per treatment) were slaughtered by the five methods. The results showed that the succinate dehydrogenase (SDH) enzyme activity of M5 sample was higher. The serum glucose level of M2 samples and DAPI staining fluorescence of M2 samples were the highest, indicating that the stress response of M2 was strong. In addition, the texture, pH, total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA) and K value results showed M5 samples had better flesh quality. CONCLUSION: Gradient cooling lethality had the least effect on oxidative damage and apoptosis in grouper during cold storage as the gradient cooling lethality had the least effect on antioxidant enzyme activities. © 2024 Society of Chemical Industry.

The toxic effect of lead exposure on the physiological homeostasis of grouper: Insight from gut-liver axis.

The heavy metal lead (Pb) pollution in marine environment has been widely concerned. The liver and intestine are important for the health of fish. In this study, the grouper were exposed to 1 µg/L Pb for 14 days, and the physiological homeostasis changes were explored via gut-liver axis. The results showed that Pb stress caused liver morphological changes, oxidative stress, and the accumulation and peroxidation of the lipids. The liver metabolism were disturbed, especially amino acid metabolism and the synthesis and degradation of ketone bodies. Pb stress also caused intestinal mucosal ablation, tight junction dysfunction and inflammatory response. Additionally, intestinal microbial diversity was decreased, and the community composition was altered especially several bacteria genera (Ruminococcus UCG-005, Ruminococcus UCG-014, Oscillibacter, and Streptococcus) were significantly correlated with the physiological indexes and metabolites of the liver. These results reveal that Pb stress negatively affect the physiological homeostasis of the grouper via gut-liver axis.

Establishment, characterization, and sensory characteristics (taste and flavor) of an immortalized muscle cell line from the seven-band grouper Epinephelus septemfasciatus: implications for cultured seafood applications.

Grouper muscle satellite cells (GMSCs) from the seven-band grouper (Epinephelus septemfasciatus) were isolated, and their growth conditions were optimized (10% fetal bovine serum, 24°C, 10 ng/mL bFGF). The cells were immortalized at passage 14 and designated as grouper immortalized muscle satellite cells (GIMSCs). DNA barcoding confirmed the grouper origin of both GMSC and GIMSC lines. GIMSCs exhibited enhanced proliferation, accelerated differentiation, and robust myotube formation compared to pre-crisis GMSCs. Western blot analysis showed upregulation of key myogenic factors (Pax7, MyoD, MyoG) and structural proteins (Desmin) in GIMSC, indicating the differentiation potential. The immortalized GIMSC line maintained consistent morphology, growth rates, and viability across multiple passages. Biocompatibility studies showed GIMSCs were compatible with bio-inks (sodium alginate, gelatin, κ-carrageenan) at 250 to 10,000 µg/mL, retaining ~ 80% viability at the highest concentration. Taste sensory analysis revealed GMSCs had the highest umami and lowest saltiness and sourness, contrasting with the muscle of the seven-band grouper, which had higher saltiness and sourness. Flavor analysis identified pronounced fishy, hot fat, and ethereal flavors in the cells at higher level than in the muscle. These findings suggest GMSCs and GIMSCs are promising for producing cultured meat with enhanced umami taste and flavors, advancing cellular agriculture and sustainable food production.

DNA Methylation and Subgenome Dominance Reveal the Role of Lipid Metabolism in Jinhu Grouper Heterosis.

Heterosis of growth traits in economic fish has benefited the production of aquaculture for many years, yet its genetic and molecular basis has remained obscure. Nowadays, a new germplasm of hybrid Jinhu grouper (Epinephelus fuscoguttatus ♀ × E. tukula ♂), abbreviated as EFT, exhibiting paternal-biased growth heterosis, has provided an excellent model for investigating the potential regulatory mechanisms of heterosis. We integrated transcriptome and methylome to unravel the changes of gene expression, epigenetic modification, and subgenome dominance in EFT compared with maternal E. fuscoguttatus. Integration analyses showed that the heterotic hybrids showed lower genomic DNA methylation levels than the purebred parent, and the up-regulated genes were mostly DNA hypomethylation. Furthermore, allele-specific expression (ASE) detected paternal subgenome dominance-regulated paternal-biased heterosis, and paternal bias differentially expressed genes (DEGs) were wholly up-regulated in the muscle. Multi-omics results highlighted the role of lipid metabolism, particularly "Fatty acid synthesis", "EPA biosynthesis", and "Signaling lipids", in EFT heterosis formation. Coherently, our studies have proved that the eicosapentaenoic acid (EPA) of EFT was greater than that of maternal E. fuscoguttatus (8.46% vs. 7.46%). Finally, we constructed a potential regulatory network for control of the heterosis formation in EFT. Among them, fasn, pparg, dgat1, igf1, pomca, fgf8a, and fgfr4 were identified as key genes. Our results provide new and valuable clues for understanding paternal-biased growth heterosis in EFT, taking a significant step towards the molecular basis of heterosis.

Epinephelus coioides NLRP3 inhibits SGIV infection by upregulating Capspase-1 activity.

NLRP3 has an important role in the immune response and viral infection as an essential inflammasome component. However, it is unclear whether the grouper immune system is regulated by NLRP3 inflammasome. In this study, we cloned the NLRP3 gene from Epinephelus coioides. Ec-NLRP3 encodes 893 amino acids and contains two major structural domains, the NACHT domain (69-234aa) and the LRR domain (477-893aa). Tissue distribution analysis showed that Ec-NLRP3 was expressed in all tissues tested, with the spleen exhibiting the highest expression. Additionally, after being infected with SGIV, the expression of the Ec-NLRP3 gene was significantly increased. The results of subcellular localization revealed that Ec-NLRP3 was distributed throughout GS cells. In addition, Ec-NLRP3 co-localized with Ec-ASC and was observed as a cytosolic speck. Ec-NLRP3 overexpression significantly inhibited SGIV infection, which was further inhibited by co-overexpression of Ec-NLRP3 and Ec-ASC. Further studies revealed that overexpression of Ec-NLRP3 significantly upregulated caspase-1 activity, and co-overexpression of Ec-NLRP3 and Ec-ASC further upregulated caspase-1 activity. In addition, inhibition of Caspase-1 activity with VX-765 significantly increased the infection of SGIV. Furthermore, the NLRP3 inflammasome activator Nigericin was able to inhibit the infection of SGIV significantly. The above findings suggest that Ec-NLRP3 inhibits SGIV infection by upregulating caspase-1 activity.

ß-glucan improves intestinal health of pearl gentian grouper via activation of the p38 mitogen-activated protein kinase signaling pathway.

Our previous study has demonstrated that supplementation of yeast ß-glucan improves intestinal health in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀), accompanied by the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we investigated the effects of perturbing p38 MAPK activity using an inhibitor on the intestinal health of ß-glucan-injected pearl gentian grouper to elucidate the potential molecular mechanism underlying the protective effects of ß-glucan on the fish gut. The pearl gentian grouper was categorized into four groups: PBS injected (CD group), ß-glucan injected at a dose of 80 mg/kg (ßG group), p38 MAPK inhibitor SB203580 injected at a dose of 1 mg/kg (SB203580 group), and a combination of ß-glucan (80 mg/kg) and SB203580 (1 mg/kg) injected together (ßG + SB203580 group). The results revealed that the introduction of SB203580 significantly suppressed the ß-glucan-induced increase in p38α and p38ß mRNA expression, as well as the phosphorylation of p38 MAPK. Both the ßG group and SB203580 group exhibited reduced plica height and muscularis thickness. The ßG + SB203580 group displayed a significant reduction in mucin cell level; interleukin 1ß (il1ß) mRNA expression; induced nitric oxide synthase, tumor necrosis factor α, and IL1ß concentration; catalase and total antioxidant capacity activities. Additionally, there was a significant increase in the levels of intestinal malondialdehyde in the ßG + SB203580 group compared to the ßG group. The inhibition of the p38 MAPK signaling halted the trend of apoptosis-related caspase molecular expression induced by ß-glucan. In conclusion, ß-glucan injection resulted in elevated levels of mucous cells, nonspecific immunity, antioxidant capacity, and anti-apoptosis in grouper by modulating the p38 MAPK pathway. This study offers insights into the potential molecular mechanism underlying the protective effects of ß-glucan on intestinal health in pearl gentian grouper.

Molecular characterization and gene expression of pattern recognition receptors in brown-marbled grouper (Epinephelus fuscoguttatus) fingerlings responding to vibriosis infection.

The pathogen recognition system involves receptors and genes that play a crucial role in activating innate immune response in brown-marbled grouper (Epinephelus fuscoguttatus) as a control agent against various infections including vibriosis. Here, we report the molecular cloning of partial open reading frames, sequences characterization, and expression profiles of Pattern Recognition Receptors (PRRs) in brown-marbled grouper. The PRRs, namely pglyrp5, tlr5, ctlD, and ctlE in brown-marbled grouper, possess conserved domains and showed shared evolutionary relationships with other fishes, humans, mammals, birds, reptilians, amphibians, and insects. In infection experiments, up to 50% mortality was found in brown-marbled grouper fingerlings infected with Vibrio alginolyticus compared to 27% mortality infected Vibrio parahaemolyticus and 100% survival of control groups. It is also demonstrated that all four PRRs had higher expression in samples infected with V. alginolyticus compared to V. parahaemolyticus. This PRRs gene expression analysis revealed that all four PRRs expressed rapidly at 4-h post-inoculation even though the Vibrio count was only detected earliest at 12-h post-inoculation in samples. The highest expression recorded was from V. alginolyticus inoculated fish spleen with up to 73-fold change for pglyrp5 gene, followed by 14 to 38-fold expression for the same treatment in spleen, head kidney, and blood samples for other PRRs, namely tlr5, ctlD, and ctlE genes. Meanwhile less than a 10% increase in expression of all four genes was detected in spleen, head kidney, and blood samples inoculated with V. parahaemolyticus. These findings indicated that pglyrp5, tlr5, ctlD, and ctlE play important roles in the early immune response to vibriosis infected, brown-marbled grouper fingerlings.

Nectin1 is a pivotal host factor involved in attachment and entry of red-spotted grouper nervous necrosis virus in the early stages of the viral life cycle.

Nervous necrosis virus (NNV) is a highly neurotropic virus that poses a persistent threat to the survival of multiple fish species. However, its inimitable neuropathogenesis remains largely elusive. To rummage potential partners germane to the nervous system, we investigated the interaction between red-spotted grouper NNV (RGNNV) and grouper brain by immunoprecipitation coupled with mass spectrometry and discerned Nectin1 as a novel host factor subtly involved in viral early invasion events. Nectin1 was abundant in neural tissues and implicated in the inception of tunnel nanotubes triggered by RGNNV. Its overexpression not only dramatically potentiated the replication dynamics of RGNNV in susceptible cells, but also empowered non-sensitive cells to expeditiously capture free virions within 2 min. This potency was impervious to low temperatures but was dose-dependently suppressed by soluble protein or specific antibody of Nectin1 ectodomain, indicating Nectin1 as an attachment receptor for RGNNV. Mechanistically, efficient hijacking of virions by Nectin1 strictly depended on intricate linkages to different modules of viral capsid protein, especially the direct binding between the IgC1 loop and P-domain. More strikingly, despite abortive proliferation in Nectin1-reconstructed CHSE-214 cells, a non-sensitive cell, RGNNV could gain access to the intracellular compartment by capitalizing on Nectin1, thereby inducing canonical cytoplasmic vacuolation. Altogether, our findings delineate a candidate entrance for RGNNV infiltration into the nervous system, which may shed unprecedented insights into the exploration and elucidation of RGNNV pathogenesis.IMPORTANCENervous necrosis virus (NNV) is one of the most virulent pathogens in the aquaculture industry, which inflicts catastrophic damage to ecology, environment, and economy annually around the world. Nevertheless, its idiosyncratic invasion and latency mechanisms pose enormous hardships to epidemic prevention and control. In this study, deploying grouper brain as a natural screening library, a single-transmembrane glycoprotein, Nectin1, was first identified as an emergent functional receptor for red-spotted grouper NNV (RGNNV) that widely allocated in nervous tissues and directly interacted with viral capsid protein through distinct Ig-like loops to bridge virus-host crosstalk, apprehend free virions, and concomitantly propel viral entry. Our findings illuminate the critical role of Nectin1 in RGNNV attachment and entry and provide a potential target for future clinical intervention strategies in the therapeutic race against RGNNV.

Host Gut-Derived Probiotic, Exiguobacterium acetylicum G1-33, Improves Growth, Immunity, and Resistance to Vibrio harveyi in Hybrid Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

Several exogenous probiotics are applicable in fish culture; however, challenges in isolation and verification have hindered the full utilization of numerous host probiotics. Therefore, this study aimed to apply the host probiotic Exiguobacterium acetylicum G1-33 to hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) cultures and explore its mechanism of action. In total, 360 hybrid grouper were divided into four groups, which were fed the following for 60 days: three received commercial feed with varying concentrations of E. acetylicum G1-33 (106, 108, and 1010 CFU/g), while a control group received commercial feed. The results showed that supplementation with 106 and 108 CFU/g of E. acetylicum G1-33 enhanced gut morphology, upregulated growth-related genes (ghr1, igf-2, s6k1, tor), and promoted growth, with supplementation with 108 CFU/g resulting in the most notable enhancement. However, supplementation with 1010 CFU/g inhibited growth, possibly because of changes in intestinal morphology. Additionally, supplementation with E. acetylicum G1-33 upregulated the expression of immune-related genes (c3, myd88, Cu/Zn-sod, tlr3, and tnf2) in the liver and head kidney but led to an increase in malondialdehyde content, as well as a decrease in alkaline phosphatase and acid phosphatase activities, in the liver and serum, indicating increased oxidative stress. Moreover, supplementation with 106 and 108 CFU/g E. acetylicum G1-33 enhanced the widespread expression of immune-related genes in the head kidney and liver, respectively, and improved resistance to Vibrio harveyi, whereas supplementation with 1010 CFU/g weakened this resistance. In conclusion, E. acetylicum G1-33, particularly at 108 CFU/g, emerged as an effective probiotic, optimizing growth performance and immunity in hybrid grouper. This research is pioneering in its application of E. acetylicum in mariculture, potentially broadening the range of probiotic strategies in aquaculture.

Exploring the correlation of microbial community diversity and succession with protein degradation and impact on the production of volatile compounds during cold storage of grouper (Epinephelus coioides).

Microorganisms, proteins, and lipids play crucial and intricate roles in the aroma generation of aquatic products. To explore the impact of the interaction between microorganisms and proteins on the volatile compounds (VOCs) in grouper, this study employed whey protein isolate (WPI) to inhibit lipid oxidation and reduce mutual interference. Changes in bacterial profiles, metabolites, and VOCs were detected. Eighteen key VOCs associated with the overall flavor of grouper were identified, and the potential relationships among microorganisms, proteins, and VOCs were explored using a correlation network. Five microorganisms (Vibrio, Vagococcus, Pseudomonas, Psychrobacter, and Shewanella) closely related to characteristic flavor compounds were identified. Additionally, 30 differential metabolites related to proteins and six metabolic pathways were screened. Therefore, this study unveils the potential interaction between microorganisms and proteins in flavor formation and provides new insights into the relationships among microorganisms, proteins, and VOCs.

Role of the Pseudomonas plecoglossicida fliL gene in immune response of infected hybrid groupers (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

Pseudomonas plecoglossicida, a gram-negative bacterium, is the main pathogen of visceral white-point disease in marine fish, responsible for substantial economic losses in the aquaculture industry. The FliL protein, involved in torque production of the bacterial flagella motor, is essential for the pathogenicity of a variety of bacteria. In the current study, the fliL gene deletion strain (ΔfliL), fliL gene complement strain (C-ΔfliL), and wild-type strain (NZBD9) were compared to explore the influence of the fliL gene on P. plecoglossicida pathogenicity and its role in host immune response. Results showed that fliL gene deletion increased the survival rate (50%) and reduced white spot disease progression in the hybrid groupers. Moreover, compared to the NZBD9 strain, the ΔfliL strain was consistently associated with lower bacterial loads in the grouper spleen, head kidney, liver, and intestine, coupled with reduced tissue damage. Transcriptomic analysis identified 2 238 differentially expressed genes (DEGs) in the spleens of fish infected with the ΔfliL strain compared to the NZBD9 strain. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the DEGs were significantly enriched in seven immune system-associated pathways and three signaling molecule and interaction pathways. Upon infection with the ΔfliL strain, the toll-like receptor (TLR) signaling pathway was activated in the hybrid groupers, leading to the activation of transcription factors (NF-κB and AP1) and cytokines. The expression levels of proinflammatory cytokine-related genes IL-1ß, IL-12B, and IL-6 and chemokine-related genes CXCL9, CXCL10, and CCL4 were significantly up-regulated. In conclusion, the fliL gene markedly influenced the pathogenicity of P. plecoglossicida infection in the hybrid groupers. Notably, deletion of fliL gene in P. plecoglossicida induced a robust immune response in the groupers, promoting defense against and elimination of pathogens via an inflammatory response involving multiple cytokines.

In vitro antiviral activity of eugenol on Singapore grouper iridovirus.

The high mortality rate of Singapore grouper iridovirus (SGIV) posing a serious threat to the grouper aquaculture industry and causing significant economic losses. Therefore, finding effective drugs against SGIV is of great significance. Eugenol (C10H12O2) is a phenolic aromatic compound, has been widely studied for its anti-inflammatory, antioxidant and antiviral capacity. In this study, we explored the effect of eugenol on SGIV infection and its possible mechanisms using grouper spleen cells (GS) as an in vitro model. We found that treatment of GS cells with 100 µM eugenol for 4 h exhibited the optimal inhibitory effect on SGIV. Eugenol was able to reduce the expression level of inflammatory factors by inhibiting the activation of MAPK pathway and also inhibited the activity of NF-κB and AP-1 promoter. On the other hand, eugenol attenuated cellular oxidative stress by reducing intracellular ROS and promoted the expression of interferon-related genes. Therefore, we conclude that eugenol inhibits SGIV infection by enhancing cellular immunity through its anti-inflammatory and antioxidant functions.

Comparative Transcriptome Analysis of the Hypothalamic-Pituitary-Gonadal Axis of Jinhu Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus tukula ♂) and Tiger Grouper (Epinephelus fuscoguttatus).

Jinhu groupers, the hybrid offspring of tiger groupers (Epinephelus fuscoguttatus) and potato groupers (Epinephelus tukula), have excellent heterosis in fast growth and strong stress resistance. However, compared with the maternal tiger grouper, Jinhu groupers show delayed gonadal development. To explore the interspecific difference in gonadal development, we compared the transcriptomes of brain, pituitary, and gonadal tissues between Jinhu groupers and tiger groupers at 24-months old. In total, 3034 differentially expressed genes (DEGs) were obtained. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses showed that the osteoclast differentiation, oocyte meiosis, and ovarian steroidogenesis may be involved in the difference in gonadal development. Trend analysis showed that the DEGs were mainly related to signal transduction and cell growth and death. Additionally, differences in expression levels of nr4a1, pgr, dmrta2, tbx19, and cyp19a1 may be related to gonadal retardation in Jinhu groupers. A weighted gene co-expression network analysis revealed three modules (i.e., saddlebrown, paleturquoise, and greenyellow) that were significantly related to gonadal development in the brain, pituitary, and gonadal tissues, respectively, of Jinhu groupers and tiger groupers. Network diagrams of the target modules were constructed and the respective hub genes were determined (i.e., cdh6, col18a1, and hat1). This study provides additional insight into the molecular mechanism underlying ovarian stunting in grouper hybrids.

Identification and characterization of four Bacillus species from the intestine of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂), their antagonistic role on common pathogenic bacteria, and effects on intestinal health.

As an alternative to the criticized antibiotics, probiotics have been adopted for their eco-friendly nature and ability to enhance host growth and immunity. Nevertheless, reports suggest ineffectiveness in commercially available probiotics since most are from non-fish sources; thus, this study was envisaged to isolate and characterize new Bacillus spp. from the gut of hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) which could serve as potential probiotics. The isolation and characterization were performed based on their morphological and biochemical properties, and 16S rRNA sequencing homology analysis. A subsequent 30-day in vivo biosafety feeding trial was conducted to ascertain isolates' non-pathogenicity, as well as their effects on fish growth, and intestinal mucosal microvilli via scanning electron microscopy (SEM) analysis. Four Bacillus spp. strains, namely, B. velezensis strain PGSAK01 (accession number OQ726606), B. stercoris strain PGSAK05 (accession number OQ726607), B. velezensis strain PGSAK17 (accession number OQ726601), and B. subtilis strain PGSAK19 (accession number OQ726605), were identified and characterized in the current study. The strains showed promising probiotic properties such higher adhesion capability, higher thermotolerance, displaying higher survivability to 0.5 % bile, lower pH tolerance, γ-haemolytic activity, and multispecies characteristics. Among the 24 antibiotics tested, while all isolates showed susceptibility to 21, the PGSAK01 strain showed resistance to furazolidone antibiotics. None of the isolates showed possession of i) virulence factor genes encoding enterotoxigenic (hblA, hblC, hblD, nheA, nheB, and entFM) and emetic (cereulide synthetase gene, ces) genes, and ii) streptomycin resistance gene (vat c), ampicillin-resistant genes (mecA and bla), and vancomycin-resistant gene (van B). Nevertheless, the PGSAK01 and PGSAK17 strains showed possession of tek K, cat, and ant(4')-Ia (adenylyltransferase) (except the PGSAK01) resistant genes. All isolates displayed better antimicrobial effects against pathogenic bacteria Streptococcus agalactiae, S. iniae, Vibrio harveyi, and V. alginolyticus. The in vivo biosafety trial involved hybrid grouper fish being grouped into five (average weight 32 ± 0.94 g), namely, the group fed the basal diet void of isolate's supplementation (control), and the remaining four groups fed the basal diet with 1 × 108 CFU/g diet of individual strain PGSAK01, PGSAK05, PGSAK17, and PGSAK19 supplementation. At the end of the study, a significantly higher WGR, K (except the PGSAK01 group), VSI; lysozyme (except PGSAK01 group), total antioxidant activity, alkaline phosphatase, superoxide dismutase enzyme activities; highly dense intestinal mucosal villi (based on the scanning electron microscopy analysis); and significantly lower malondialdehyde levels were witnessed in the isolated treated groups compared to the control, supporting the results obtained in the auto-aggregation and cell-surface hydrophobicity test. This work's results have provided thought-provoking targets; thus, studies involving extensive genome sequencing and functional annotation analysis will be explored to offer unfathomable insights into their mechanisms of action and potential health benefits, further establishing the four Bacillus strains' (PGSAK01, PGSAK05, PGSAK17, and PGSAK19) potential role in probiotic fields and functional foods.

Grouper OTUB1 and OTUB2 promote red-spotted grouper nervous necrosis virus (RGNNV) replication by inhibiting the host innate immune response.

Red-spotted grouper nervous necrosis virus (RGNNV) is a major viral pathogen of grouper and is able to antagonize interferon responses through multiple strategies, particularly evading host immune responses by inhibiting interferon responses. Ovarian tumor (OTU) family proteins are an important class of DUBs and the underlying mechanisms used to inhibit interferon pathway activation are unknown. In the present study, primers were designed based on the transcriptome data, and the ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) and OTUB2 genes of Epinephelus coioides (EcOTUB1 and EcOTUB2) were cloned and characterized. The homology alignment showed that both EcOTUB1 and EcOTUB2 were most closely related to E. lanceolatus with 98 % identity. Both EcOTUB1 and EcOTUB2 were distributed to varying degrees in grouper tissues, and the transcript levels were significantly up-regulated following RGNNV stimulation. Both EcOTUB1 and EcOTUB2 promoted replication of RGNNV in vitro, and inhibited the promoter activities of interferon stimulated response element (ISRE), nuclear transcription factors kappaB (NF-κB) and IFN3, and the expression levels of interferon related genes and proinflammatory factors. Co-immunoprecipitation experiments showed that both EcOTUB1 and EcOTUB2 could interact with TRAF3 and TRAF6, indicating that EcOTUB1 and EcOTUB2 may play important roles in interferon signaling pathway. The results will provide a theoretical reference for the development of novel disease prevention and control techniques.

The toxic effects of tetracycline exposure on the physiological homeostasis of the gut-liver axis in grouper.

Antibiotic residues, such as tetracycline (TET), in aquatic environments have become a global concern. The liver and gut are important for immunity and metabolism in aquatic organisms. In this study, juvenile groupers were subjected to 1 and 100 µg/L TET for 14 days, and the physiological changes of these fish were evaluated from the perspective of gut-liver axis. After TET exposure, the liver showed histopathology, lipid accumulation, and the elevated ALT activity. An oxidative stress response was induced in the liver and the metabolic pattern was disturbed, especially pyrimidine metabolism. Further, intestinal health was also affected, including the damaged intestinal mucosa, the decreased mRNA expression levels of tight junction proteins (ZO-1, Occludin, and Claudin-3), along with the increased gene expression levels of inflammation (IL-1ß, IL-8, TNF-α) and apoptosis (Casp-3 and p53). The diversity of intestinal microbes increased and the community composition was altered, and several beneficial bacteria (Lactobacillus, Bacteroidales S24-7 group, and Romboutsia) and harmful (Aeromonas, Flavobacterium, and Nautella) exhibited notable correlations with hepatic physiological indicators and metabolites. These results suggested that TET exposure can adversely affect the physiological homeostasis of groupers through the gut-liver axis.

Singapore grouper iridovirus VP146 modulates the cGAS-STING signaling pathway to escape the interferon immune response.

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that has caused significant economic losses to the grouper aquaculture industry. So far, the structure and function of SGIV proteins have been successively reported. In the present paper, the protein of SGIV VP146 was cloned and identified. VP146 was whole-cell distributed in GS cells. VP146 promoted SGIV replication and inhibited the transcription of interferon-related genes as well as pro-inflammatory cytokines in GS cells. In addition, VP146 was involved in the regulation of the cGAS-STING signaling pathway, and decreased cGAS-STING induced the promoter of ISRE and NF-κB. VP146 interacted with the proteins of cGAS, STING, TBK1, and IRF3 from grouper, but did not affect the binding of grouper STING to grouper TBK1 and grouper IRF3. Interestingly, grouper STING was able to affect the intracellular localization of VP146. Four segment structural domains of grouper STING were constructed, and grouper STING-CTT could affect the intracellular localization of VP146. VP146 had no effect on the self-binding of EcSITNG, nor on the binding of EcSTING to EcTBK1 and EcIRF3. Together, the results demonstrated that SGIV VP146 modulated the cGAS-STING signaling pathway to escape the interferon immune response.

Pathogenicity associated with an infestation of the marine leech parasite Pterobdella arugamensis in farmed fish.

The marine leech Pterobdella arugamensis is a hematophagous parasite, and the extent of injury to the host largely depends on the number of attached leeches. This study aimed to assess the pathogenicity of marine leeches in Asian seabass (Lates calcarifer) and tiger grouper (Epinephelus fuscoguttatus) fingerlings under laboratory conditions. Five groups of healthy Asian seabass and tiger grouper were exposed to varying numbers of marine leeches (0, 1, 10, 30, or 70 per fish) for 7 d. Infested Asian seabass and tiger grouper both showed pathological changes even with only 1 leech, manifesting as clinical signs like haemorrhages. The cumulative mortality at 7 d post-exposure (dpe) was 11 or 33% for Asian seabass infested with 1 or 10 marine leeches, respectively. Fish with 30 or 70 marine leeches showed higher rates of mortality (56%). A similar trend was seen in tiger grouper, with mortality rates reaching 78% in fish with 30 or 70 marine leeches, and 56 or 33% in fish with 10 leeches or 1 leech, respectively. Factorial analysis of mortality after 7 dpe between both species showed significant differences (2-way ANOVA p = 0.001) when exposed to varying numbers of marine leeches. The haematocrit values differed significantly between Asian seabass or tiger grouper infested with either 0 or 1 marine leech and those infested with 10, 30, or 70 marine leeches (1-way ANOVA, p = 0.0001). This suggests that marine leech infestation has a measurable impact on both species. Consequently, fish farmers should promptly address leech infestation upon discovery in their cages.

Enhanced effectiveness in preventing Nocardia seriolae infection utilizing heterologous prime-boost approach in orange-spotted grouper Epinephelus coioides.

Persistent nocardiosis has prompted exploration of the effectiveness of heterologous approaches to prevent severe infections. We have previously reported the efficacy of a nucleic acid vaccine in protecting groupers from highly virulent Nocardia seriolae infections. Ongoing research has involved the supplementation of recombinant cholesterol oxidase (rCho) proteins through immunization with a DNA vaccine to enhance the protective capacity of orange-spotted groupers. Recombinant rCho protein exhibited a maturity and biological structure comparable to that expressed in N. seriolae, as confirmed by Western blot immunodetection assays. The immune responses observed in vaccinated groupers were significantly higher than those observed in single-type homologous vaccinations, DNA or recombinant proteins alone (pcD:Cho and rCho/rCho), especially cell-mediated immune and mucosal immune responses. Moreover, the reduction in N. seriolae occurrence in internal organs, such as the head, kidney, and spleen, was consistent with the vaccine's efficacy, which increased from approximately 71.4 % to an undetermined higher percentage through heterologous vaccination strategies of 85.7 %. This study underscores the potential of Cho as a novel vaccine candidate and a heterologous approach for combating chronic infections such as nocardiosis.

Response signatures of intestinal microbiota and gene transcription of the pearl gentian grouper to Vibrio harveyi infection.

Vibrio harveyi causes high mortality and severely limits grouper culture. The gut microbiota is an important biological barrier against pathogen invasion. In this study, we investigated dynamic changes in the intestinal microbial community, gene transcription and immune responses signatures of pearl gentian grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀) at 0, 3 and 7 days (referred to as d0, d3 and d7 groups, respectively) after infection with V. harveyi. The results demonstrated that the d7 treatment reduced the gut microbial diversity and increased the proportion of Proteobacteria and Cyanobacteria. Notably, several putative pathogenic genera (Sphingomonas and Bacteroides) proliferated, while putative probiotic genera (Rhodococcus and Lactobacillus) reduced, and these changes in intestinal bacteria might be correlated to the alterations of host immune-related molecules. The d3 and d7 treatments also altered the histomorphology and gene transcription profiles mainly associated with immune function in intestine, such as 'MAPK signaling pathway', 'Apoptosis' and 'Toll-like receptor (TLR) signaling pathway'. Furthermore, d3 group induced a homeostatic dysregulation of the antioxidant system, cytokines and TLR signaling, with a tendency to gradually return to a normal state in d7 group, along with the apoptosis process. The pathogenic infection suppressed the expression of JNK pathway and enhanced the ERK pathway. In conclusion, the dysbiosis of the intestinal bacterial communities caused by the immune changes that occurred during V. harveyi infection disrupted the intestine health in the pearl gentian grouper. These results provided a comprehensive understandings of the immune defense mechanisms in fish and valuable references to develop disease control strategies in grouper aquaculture.