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.

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.

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.

Development and immune evaluation of LAMP1 chimeric DNA vaccine against Singapore grouper iridovirus in orange-spotted grouper, Epinephelus coioides.

Grouper is one of the most important and valuable mariculture fish in China, with a high economic value. As the production of grouper has increased, massive outbreaks of epidemic diseases have limited the development of the industry. Singapore grouper iridovirus (SGIV) is one of the most serious infectious viral pathogens and has caused huge economic losses to grouper farming worldwide due to its rapid spread and high lethality. To find new strategies for the effective prevention and control of SGIV, we constructed two chimeric DNA vaccines using Lysosome-associated membrane protein 1 (LAMP1) fused with major capsid proteins (MCP) against SGIV. In addition, we evaluated the immune protective effects of vaccines including pcDNA3.1-3HA, pcDNA3.1-MCP, pcDNA3.1-LAMP1, chimeric DNA vaccine pcDNA3.1-MLAMP and pcDNA3.1-LAMCP by intramuscular injection. Our results showed that compared with groups injected with PBS, pcDNA3.1-3HA, pcDNA3.1-LAMP1 or pcDNA3.1-MCP, the antibody titer significantly increased in the chimeric vaccine groups. Moreover, the mRNA levels of immune-related factors in groupers, including IRF3, MHC-I, TNF-α, and CD8, showed the same trend. However, MHC-II and CD4 were significantly increased only in the chimeric vaccine groups. After 28 days of vaccination, groupers were challenged with SGIV, and mortality was documented for each group within 14 days. The data showed that two chimeric DNA vaccines provided 87 % and 91 % immune protection for groupers which were significantly higher than the 52 % protection rate of pcDNA3.1-MCP group, indicating that both forms of LAMP1 chimeric vaccines possessed higher immune protection against SGIV, providing the theoretical foundation for the creation of novel DNA vaccines for fish.

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.

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.

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.

Sea perch (Lateolabrax japonicus) UBC9 augments RGNNV infection by hindering RLRs-interferon response.

Small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various biological processes in eukaryotes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2-conjugating enzyme responsible for SUMOylation and plays an important role in essential cellular functions. Here, we cloned the UBC9 gene from sea perch (Lateolabrax japonicus) (LjUBC9) and investigated its role in regulating the IFN response during red-spotted grouper nervous necrosis virus (RGNNV) infection. The LjUBC9 gene consisted of 477 base pairs and encoded a polypeptide of 158 amino acids with an active site cysteine residue and a UBCc domain. Phylogenetic analysis showed that LjUBC9 shared the closest evolutionary relationship with UBC9 from Paralichthys olivaceus. Tissue expression profile analysis demonstrated that LjUBC9 was significantly increased in multiple tissues of sea perch following RGNNV infection. Further experiments showed that overexpression of LjUBC9 significantly increased the mRNA and protein levels of RGNNV capsid protein in LJB cells infected with RGNNV, nevertheless knockdown of LjUBC9 had the opposite effect, suggesting that LjUBC9 exerted a pro-viral effect during RGNNV infection. More importantly, we found that the 93rd cysteine is crucial for its pro-viral effect. Additionally, dual luciferase assays revealed that LjUBC9 prominently attenuated the promoter activities of sea perch type Ⅰ interferon (IFN) in RGNNV-infected cells, and overexpression of LjUBC9 markedly suppressed the transcription of key genes associated with RLRs-IFN pathway. In summary, these findings elucidate that LjUBC9 impairs the RLRs-IFN response, resulting in enhanced RGNNV infection.

Involvement of tumor necrosis factor receptor-associated factor 6 (TRAF6) in NF-κB activation and antiviral immunity: Molecular and functional characterization of TRAF6 in red-spotted grouper (Epinephelus akaara).

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a member of the TRAF family of adaptor proteins involved in the signal transduction pathways of both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. In this study, red-spotted grouper (Epinephelus akaara) TRAF6 (EaTraf6) was identified and characterized. The open reading frame of EaTraf6, 1713 bp in length, encodes a putative protein of 570 amino acids and has a predicted molecular weight and theoretical isoelectric point of 64.11 kDa and 6.07, respectively. EaTraf6 protein contains an N-terminal RING-type zinc finger domain, two TRAF-type zinc finger domains, a coiled-coil region (zf-TRAF), and a conserved C-terminal meprin and TRAF homology (MATH) domain. EaTraf6 shared the highest amino acid sequence identity with its ortholog from Epinephelus coioides, and phylogenetic analysis showed all fish TRAF6s clustered together and apart from other species. qRT-PCR results revealed that EaTraf6 was ubiquitously expressed in all examined tissues, with the highest level detected in the blood. In the immune challenge, EaTraf6 exhibited modulated mRNA expression levels in the blood and spleen. The subcellular localization analysis revealed that the EaTraf6 protein was predominantly present in the cytoplasm; however, it could translocate into the nucleus following poly (I:C) stimulation. The antiviral function of EaTraf6 was confirmed by analyzing the expression of host antiviral genes and viral genomic RNA during viral hemorrhagic septicemia virus infection. Additionally, luciferase reporter assay results indicated that EaTraf6 is involved in the activation of the NF-κB signaling pathway upon poly (I:C) stimulation. Finally, the effect of EaTraf6 on cytokine gene expression and its role in regulating macrophage M1 polarization were demonstrated. Collectively, these findings suggest that EaTraf6 is a crucial immune-related gene that significantly contributes to antiviral functions and regulation of NF-κB activity in the red-spotted grouper.

Identification of candidate SNPs and genes associated with resistance to nervous necrosis virus in leopard coral grouper (Plectropomus leopardus) using GWAS.

The leopard coral grouper (Plectropomus leopardus), which has become increasingly popular in consumption due to its bright body color and great nutritional, holds a high economic and breeding potential. However, in recent years, the P.leopardus aquaculture industry has been impeded by the nervous necrosis virus (NNV) outbreak, leading to widespread mortality among fry and juvenile grouper. However, the genetic basis of resistance to NNV in P. leopardus remains to be investigated. In the present study, we conducted a genome-wide association analysis (GWAS) on 100 resistant and 100 susceptible samples to discover variants and potential genes linked with NNV resistance. For this study, 157,926 high-quality single nucleotide polymorphisms (SNPs) based on whole genome resequencing were discovered, and eighteen SNPs loci linked to disease resistance were discovered. We annotated six relevant candidate genes, including sik2, herc2, pip5k1c, npr1, mybpc3, and arhgap9, which showed important roles in lipid metabolism, oxidative stress, and neuronal survival. In the brain tissues of resistant and susceptible groups, candidate genes against NNV infection showed significant differential expression. The results indicate that regulating neuronal survival or pathways involved in lipid metabolism may result in increased resistance to NNV. Understanding the molecular mechanisms that lead to NNV resistance will be beneficial for the growth of the P. leopardus breeding sector. Additionally, the identified SNPs could be employed as biomarkers of disease resistance in P. leopardus, which will facilitate the selective breeding of grouper.

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.

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.

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.

Comparative proteome analysis revealed potential biomarkers and the underlying immune mechanisms in Vibrio-resistant hybrid grouper, Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂.

Vibrio alginolyticus is the causative agent of vibriosis, a common bacterial infection in grouper aquaculture that is associated with the development of haemorrhagic and non-haemorrhagic ulcerations on the fish. In the present study, comparative proteome analysis was performed on serum samples from Vibrio-resistant and Vibrio-susceptible grouper. Samples were analysed using high-throughput LC-MS/MS and identified 2770 unique peptides that corresponded to 344 proteins. Subsequent analysis identified 21 proteins that were significantly up-regulated in the resistant group compared to the control and the susceptible groups. Those proteins are associated with immunostimulatory effects, signalling and binding cascade, metabolism, and maintaining tissue integrity and physiological condition. Besides, potential protein biomarkers related to the immune system were identified, which could be associated with the disease-resistant phenotype. These data provide insights into the underlying immune mechanism of hybrid groupers upon Vibrio sp. infection.

Effects of vitamin C combined with sodium alginate on serum biochemistry, oxidative stress, gill tissue morphology, and muscle quality of pearl gentian grouper during waterless transport.

This research examined the effects of sodium alginate (SA) and vitamin C (Vc) soaking of pearl gentian grouper before waterless transportation from the perspectives of serum parameters, oxidative stress, muscle quality, and gill tissue morphology. After the fish reached semi-dormancy with a cooling rate of 3 °C/h, fish (420 ± 25 g) were distributed to 4 treatments as follows: S1 group (50 mg/L Vc and 0.1% SA were added), S2 group (50 mg/L Vc and 0.3% SA were added), S3 group (50 mg/L Vc and 0.5% SA were added), and control group (without soaking in protective fluid). After oxygenated packaging, samples were taken at 0, 8, and 16 h of waterless transportation and 12 h after rehydration, respectively. It was found that after 16 h of waterless transport, compared with the control group, cortisol, glucose, blood urea nitrogen (BUN), uric acid (UA), creatinine (CREA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels were significantly decreased (p < 0.05), while albumin, lysozyme (LZM), muscle pH, and total free amino acid (TFAA) contents were significantly elevated (p < 0.05) in the S3 group. Moreover, by gill tissue microscopy, it was found that the protective solution of group S3 did not cause serious deleterious morphological changes to the gill epithelium. The results showed that the grouper was soaked by protective fluid before waterless could maintain surface moisture, reduce gill and kidney function and oxidative stress damage, and maintain the stability of muscle quality. This study provides a novel transportation method for waterless preservation, which helps to reduce transportation costs and improve transportation efficiency.

Protected fish spawning aggregations as self-replenishing reservoirs for regional recovery.

Dispersal of eggs and larvae from spawning sites is critical to the population dynamics and conservation of marine fishes. For overfished species like critically endangered Nassau grouper (Epinephelus striatus), recovery depends on the fate of eggs spawned at the few remaining aggregation sites. Biophysical models can predict larval dispersal, yet these rely on assumed values of key parameters, such as diffusion and mortality rates, which have historically been difficult or impossible to estimate. We used in situ imaging to record three-dimensional positions of individual eggs and larvae in proximity to oceanographic drifters released into egg plumes from the largest known Nassau grouper spawning aggregation. We then estimated a diffusion-mortality model and applied it to previous years' drifter tracks to evaluate the possibility of retention versus export to nearby sites within 5 days of spawning. Results indicate that larvae were retained locally in 2011 and 2017, with 2011 recruitment being a substantial driver of population recovery on Little Cayman. Export to a nearby island with a depleted population occurred in 2016. After two decades of protection, the population appears to be self-replenishing but also capable of seeding recruitment in the region, supporting calls to incorporate spawning aggregation protections into fisheries management.

Screening of host gut-derived probiotics and effects of feeding probiotics on growth, immunity, and antioxidant enzyme activity of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂).

In recent years, the widespread use of antibiotics in intensive grouper mariculture has resulted in the ineffectiveness of antibiotic treatment, leading to an increasing incidence of diseases caused by bacteria, viruses, and parasites, causing serious economic losses. Hence, it is crucial to develop alternative strategies to antibiotics for healthy and sustainable development of the mariculture industry. Here, we aimed to screen host gut-derived probiotics and evaluate its effects on growth and immunity of grouper. In this study, 43 bacterial strains were isolated from the intestine of the hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂), and a potential probiotic strain G1-26, which can efficiently secrete amylase, protease, and lipase, was obtained using different screening mediums. Based on 16S rDNA sequencing, the potential probiotic strain G1-26 was identified as Vibrio fluvialis. The results of a biological characteristic evaluation showed that V. fluvialis G1-26 could grow at 25-45 °C, pH 5.5-7.5, salinity 10-40, and bile salt concentration 0-0.030%, and produce amylase, lipase, and protease under different culture conditions. Additionally, V. fluvialis G1-26 is sensitive to many antibiotics and does not exhibit aquatic biotoxicity. Subsequently, hybrid groupers were fed diets containing V. fluvialis G1-26 at different concentrations (0, 106, 108, and 1010 CFU/g) for 60 d. The results showed that V. fluvialis G1-26 at 108 CFU/g did not significantly affect the growth performance of the hybrid grouper (P > 0.05). V. fluvialis G1-26 supplementation at 108 and 1010 CFU/g significantly promoted the relative expression of immune-related genes in hybrid groupers (TLR3, TLR5, IL-1ß, IL-8, IL-10, CTL, LysC, TNF-2, and MHC-2) and improved the activities of alkaline phosphatase, acid phosphatase, total superoxide dismutase, and total protein in the liver. In conclusion, V. fluvialis G1-26, a potential probiotic strain isolated from the intestine of the hybrid grouper, can be used as an effective immunopotentiator at an optimal dose of 108 CFU/g diet. Our results provide a scientific basis for the development and utilization of probiotics in the grouper mariculture industry.

Influences of chronic copper exposure on intestinal histology, antioxidative and immune status, and transcriptomic response in freshwater grouper (Acrossocheilus fasciatus).

Copper (Cu) contamination is commonly found in both natural water environments and fish farms, and it can cause severe damage to different fish organs, but Cu-induced intestinal damage has been rarely studied. This study subjected three groups of freshwater grouper (Acrossocheilus fasciatus) (initial weight: 1.56 ± 0.10 g) to 0 mg/L, 0.01 mg/L, and 0.04 mg/L Cu2+ for 30 days, named Con, Cu0.01, and Cu0.04 groups, respectively. The histological observation indicated that the Cu0.04 group caused a significant decrease in villus length, lamina propria width, and muscular thickness compared to the Con group (P < 0.05). Additionally, the Cu0.04 group significantly increased intestinal superoxide dismutase (SOD), glutathione peroxidase (GPx), lysozyme (LZM) activities, as well as malondialdehyde (MDA) content than the Con group (P < 0.05). Meanwhile, the Cu0.01 and Cu0.04 groups showed significantly increased immunoglobulin M (IgM), complement 3 (C3), and glutathione (GSH) contents than the Con group (P < 0.05). Transcriptomic analysis revealed a total of 101 differentially expressed genes (DEGs), including 47 up-regulated and 54 down-regulated DEGs, were identified between the Cu0.04 and Con groups. Notably, the DEGs were mainly related to intestinal structure construction, immune functions, apoptosis, and resistance to DNA damage and pathogen infection. The findings suggest that chronic Cu exposure caused intestinal histological alterations, activated the antioxidative and immune systems, and induced systematic adaptation to cope with the physical barrier injury, DNA damage, and potential pathogen growth.

Antiviral role of grouper FoxO1 against RGNNV and SGIV infection.

As a key regulator of the innate immune system, FoxO1 has a variety of activities in biological organisms. In the present study, grouper FoxO1 (EcFoxO1) was cloned and the antiviral activity in red grouper neuron necrosis virus (RGNNV) and Singapore grouper iridescent virus (SGIV) was examined. The open reading frame (ORF) of EcFoxO1 contains 2,034 base pairs that encode a protein of 677 amino acids with a predicted molecular weight of 73.21 kDa. EcFoxO1 was shown to be broadly distributed in healthy grouper tissues, and was up-regulated in vitro in response to stimulation by RGNNV and SGIV. EcFoxO1 has a whole-cell distribution in grouper spleen (GS) cells. EcFoxO1 decreased the replication of RGNNV and SGIV, and activated interferon (IFN) 3, IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB) promoter activities. EcFoxO1 could interact with EcIRF3. Together, the results demonstrated that EcFoxO1 might be an important regulator of grouper innate immune response against RGNNV and SGIV infection.

An improved oral vaccine with molecular adjuvant ß-defensin protects grouper against nervous necrosis virus infection.

Nervous necrosis virus (NNV) is one of the most important fish viral pathogens infecting more than 120 fish species worldwide. Due to the mass mortality rates often seen among larvae and juveniles, few effective vaccines against NNV were developed up to now. Here, the protective effect of recombinant coat protein (CP) from red-spotted grouper nervous necrosis virus (RGNNV) fused with grouper ß-defensin (DEFB) as an oral vaccine was evaluated using Artemia as a biocarrier delivery system in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). Feeding with Artemia encapsulated with E. coli expressing control vector (control group), CP, or CP-DEFB showed no obvious side effects on the growth of groupers. ELISA and antibody neutralization assay showed that CP-DEFB oral vaccination group induced higher anti-RGNNV CP specific antibodies and exhibited higher neutralization potency than the CP and control group. Meanwhile, the expression levels of several immune and inflammatory factors in the spleen and kidney after feeding with CP-DEFB were also significantly increased compared with the CP group. Consistently, after challenge with RGNNV, groupers fed CP-DEFB and CP exhibited 100% and 88.23% relative percentage survival (RPS), respectively. Moreover, the lower transcription levels of viral genes and milder pathological changes in CP-DEFB group were detected compared with the CP and control group. Thus, we proposed that grouper ß-defensin functioned as an efficient molecular adjuvant for an improved oral vaccine against nervous necrosis virus infection.