Impact of captivity and natural habitats on gut microbiome in Epinephelus akaara across seasons.

BACKGROUND: The gut microbiota significantly influences the health and growth of red-spotted grouper (Epinephelus akaara), a well-known commercial marine fish from Fujian Province in southern China. However, variations in survival strategies and seasons can impact the stability of gut microbiota data, rendering it inaccurate in reflecting the state of gut microbiota. Which impedes the effective enhancement of aquaculture health through a nuanced understanding of gut microbiota. Inspired by this, we conducted a comprehensive analysis of the gut microbiota of wild and captive E. akaara in four seasons. RESULTS: Seventy-two E. akaara samples were collected from wild and captive populations in Dongshan city, during four different seasons. Four sections of the gut were collected to obtain comprehensive information on the gut microbial composition and sequenced using 16S rRNA next-generation Illumina MiSeq. We observed the highest gut microbial diversity in both captive and wild E. akaara during the winter season, and identified strong correlations with water temperature using Mantel analysis. Compared to wild E. akaara, we found a more complex microbial network in captive E. akaara, as evidenced by increased abundance of Bacillaceae, Moraxellaceae and Enterobacteriaceae. In contrast, Vibrionaceae, Clostridiaceae, Flavobacteriaceae and Rhodobacteraceae were found to be more active in wild E. akaara. However, some core microorganisms, such as Firmicutes and Photobacterium, showed similar distribution patterns in both wild and captive groups. Moreover, we found the common community composition and distribution characteristics of top 10 core microbes from foregut to hindgut in E. akaara. CONCLUSIONS: Collectively, the study provides relatively more comprehensive description of the gut microbiota in E. akaara, taking into account survival strategies and temporal dimensions, which yields valuable insights into the gut microbiota of E. akaara and provides a valuable reference to its aquaculture.

Function analysis of fish PACT gene in response to virus infection.

PACT (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a cellular protein which can activate PKR in dsRNA-independent manner. However, the role of PACT in fish virus infection remains largely unknown. In this study, a PACT homologue from grouper (Epinephelus coioides)(EcPACT) was cloned and characterized. The open reading frame of EcPACT has a full length of 924 bp and encodes a protein of 307 amino acids with a predicted molecular weight of 33.29 kDa. Similar to mammals, EcPACT contains three dsRBD domains. EcPACT shares 99.67 % homology with E. lanceolatus. Real-time fluorescence quantitative PCR results showed that EcPACT mRNA was widely expressed in all tissues and abundantly expressed in brain, blood, head kidney and kidney. In addition, SGIV and RGNNV infection significantly upregulated the transcript levels of EcPACT. Subcellular localization analysis showed that EcPACT was mainly distributed in the nucleus. Overexpression of EcPACT inhibited the replication of SGIV and RGNNV in vitro and positively regulated the expression of interferon (IFN) and pro-inflammatory factors. The results provide a better understanding of the relationship between PACT and viral infection in fish.

Dual-specificity phosphatase 1 inhibits Singapore grouper iridovirus replication via regulating apoptosis in Epinephelus coioides.

The dual-specificity phosphatase (DUSP) family plays key roles in the maintenance of cellular homeostasis and apoptosis etc. In this study, the DUSP member DUSP1 of Epinephelus coioides was characterized: the length was 2371 bp including 281 bp 5' UTR, 911 bp 3' UTR, and a 1125 bp open reading frame encoding 374 amino acids. E. coioides DUSP1 has two conserved domains, a ROHD and DSPc along with a p38 MAPK phosphorylation site, localized at Ser308. E. coioides DUSP1 mRNA can be detected in all of the tissues examined, and the subcellular localization showed that DUSP1 was mainly distributed in the nucleus. Singapore grouper iridovirus (SGIV) infection could induce the differential expression of E. coioides DUSP1. Overexpression of DUSP1 could inhibit SGIV-induced cytopathic effect (CPE), the expressions of SGIV key genes, and the viral titers. Overexpression of DUSP1 could also regulate SGIV-induced apoptosis, and the expression of apoptosis-related factor caspase 3. The results would be helpful to further study the role of DUSP1 in viral infection.

Orange-spotted grouper IFNh response to NNV or MSRV and its potential antiviral activities.

Type I interferon (IFN) plays a crucial role in the antiviral immune response. Nervous necrosis virus (NNV) and Micropterus salmoides rhabdovirus (MSRV) are the most important viruses in cultured larvae and juveniles, causing great economic losses to fish farming. To better understand the antiviral activities and immunoregulatory role of IFN from orange-spotted grouper (Epinephelus coioides), EcIFNh was cloned from NNV infected sample. EcIFNh has an open reading frame (ORF) of 552 bp and encodes a polypeptide of 183 amino acids. Phylogenetic tree analysis showed that EcIFNh was clustered into the IFNh branch. The tissue distribution analysis revealed that EcIFNh was highly expressed in the liver and brain of healthy orange-spotted grouper. The mRNA levels of EcIFNh were significantly upregulated after poly (I:C) stimulation and NNV or MSRV infection. Furthermore, the promoter of EcIFNh was characterized and significantly activated by EcMDA5, EcMAVS, EcSTING, EcIRF3, and EcIRF7 in the luciferase activity assays. We found that EcIFNh overexpression resisted the replication of NNV and MSRV, while EcIFNh silencing facilitated NNV replication in GB cells. In addition, EcIFNh recombinant protein (rEcIFNh) enhanced the immune response by inducing the expression of ISGs in vivo and in vitro, suggesting the potential application of rEcIFNh for anti-NNV and anti-MSRV. Taken together, our research may offer the foundation for virus-IFN system interaction in orange-spotted grouper.

Functional properties of ATPIF1 in the orange-spotted grouper (Epinephelus coioides) in response to viral infection.

ATP synthase inhibitory factor 1 (ATPIF1) can activate mitochondrial autophagic pathway and mediates immune response by regulating ATP synthase activity. However, the role of fish ATPIF1 on viral infection is still unknown. In this study, we identified an ATPIF1 homolog (Ec-ATPIF1) from orange-spotted grouper (Epinephelus coioides). Ec-ATPIF1 is mainly expressed in the kidney and liver. The expression of Ec-ATPIF1 was significantly up-regulated after RGNNV stimulation in vitro. Further experiments showed that overexpression of Ec-ATPIF1 inhibited the expression of viral genes (CP and RdRp) and intracellular ATP synthesis. Ec-ATPIF1 overexpression also promoted the expression of mitophagy related genes (PINK1, Parkin, BNIP3, NIX, FUNDC1, LC3), inflammation-related factors (IL-1ß, IL-6, IL-8, IL-10, TNF-α, TLR2) and interferon pathway factors (IRF1, IRF3, IRF7, MX1, ISG15, ISG56, MDA5, TRIF). While the knockdown of Ec-ATPIF1 exhibited the opposite effects on the expression of viral genes and immune-related factors above. These data suggest that Ec-ATPIF1 can impact viral infection by regulating mitophagy, ATP synthesis, the expression of inflammatory factors and interferon pathway factors. These findings will be beneficial to better explore the immune regulatory mechanisms of fish respond to viral infection.

Characterization and functional analysis of SOCS9 from orange-spotted grouper (Epinephelus coioides) during virus infection.

The suppressor of cytokine signaling (SOCS) proteins family have twelve members including eight known mammalian SOCS members (CISH, SOCS1-7) and four new discovery members (SOCS3b, SOCS5b, SOCS8 and SOCS9) that is regarded as a classic feedback inhibitor of cytokine signaling. Although the function of the mammalian SOCS proteins have been well studied, little is known about the roles of SOCS in fish during viral infection. In this study, the molecular characteristics of SOCS9 from orange-spotted grouper (Epinephelus coioides, EcSOCS9) is investigated. The EcSOCS9 protein encoded 543 amino acids with typical SH2 (389-475aa) and SOCS_box (491-527aa), sharing high identities with reported fish SOCS9. EcSOCS9 was expressed in all detected tissues and highly expressed in kidney. After red-spotted grouper nervous necrosis virus (RGNNV) infection, the expression of EcSOCS9 was significantly induced in vitro. Furthermore, EcSOCS9 overexpression enhanced RGNNV replication, promoted virus-induced mitophagy that evidenced by the increased level of LC3-Ⅱ, BCL2, PGAM5 and decreased level of BNIP3 and FUNDC1. Besides, EcSOCS9 overexpression suppressed the expression levels of ATP6, CYB, ND4, ATP level and induced ROS level. The expression levels of interferon (IFN) related factors (IRF1, IRF3, IRF7, P53), inflammatory factors (IL1-ß, IL8, TLR2, TNF-α) and IFN-3, ISRE, NF-κB, AP1 activities were also reduced by overexpressing EcSOCS9. These date suggests that EcSOCS9 impacts RGNNV infection through modulating mitophagy, regulating the expression levels of IFN- related and inflammatory factors, which will expand our understanding of fish immune responses during viral infection.

Two cytokine receptor family B (CRFB) members in orange-spotted grouper Epinephelus coioides, EcCRFB3 and EcCRFB4, negatively regulate interferon immune responses to assist nervous necrosis virus replication.

Receptors of type I interferon (IFNR) play a vital role in the antiviral immune response. However, little is known about the negative regulatory role of the IFNR. Nervous necrosis virus (NNV) is one of the most significant viruses in cultured fish, resulting in great economic losses for the aquaculture industry. In this study, two orange-spotted grouper (Epinephelus coioides) cytokine receptor family B (CRFB) members, EcCRFB3 and EcCRFB4 were cloned and characterized from NNV infected grouper brain (GB) cells. The open reading frame (ORF) of EcCRFB3 consists of 852 bp encoding 283 amino acids, while EcCRFB4 has an ORF of 990 bp encoding 329 amino acids. The mRNA levels of EcCRFB3 or EcCRFB4 were significantly upregulated after NNV infection and the stimulation of poly (I:C) or NNV-encoded Protein A. In addition, EcCRFB3 or EcCRFB4 overexpression facilitated NNV replication, whereas EcCRFB3 or EcCRFB4 silencing resisted NNV replication. Overexpressed EcCRFB3 or EcCRFB4 inhibited the expression of IFN-I-induced ISGs. Taken together, our research provides the first evidence in fish demonstrating the role of IFNRs to regulate the IFN signaling pathway negatively. Our findings enrich the understanding of the functions of IFNRs and reveal a novel escape mechanism of NNV.

Epinephelus coioides Sec3 promotes Singapore grouper iridovirus infection by negatively regulates immune response.

Exocyst, a protein complex, plays a crucial role in various cellular functions, including cell polarization, migration, invasion, cytokinesis, and autophagy. Sec3, known as Exoc1, is a key subunit of the Exocyst complex and can be involved in cell survival and apoptosis. In this study, two subtypes of Sec3 were isolated from Epinephelus coioides, an important marine fish in China. The role of E. coioides Sec3 was explored during Singapore grouper iridovirus (SGIV) infection, an important pathogen of marine fish which could induce 90 % mortality. E. coioides Sec3 sequences showed a high similarity with that from other species, indicating the presence of a conserved Sec3 superfamily domain. E. coioides Sec3 mRNA could be detected in all examined tissues, albeit at varying expression levels. SGIV infection could upregulate E. coioides Sec3 mRNA. Upregulated Sec3 significantly promoted SGIV-induced CPE, and the expressions of viral key genes. E. coioides Sec3 could inhibit the activation of NF-κB and AP-1, as well as SGIV-induced cell apoptosis. The results illustrated that E. coioides Sec3 promotes SGIV infection by regulating the innate immune response.

Grouper TIM-1 promotes nodavirus infection by inhibiting immune and inflammation response.

T-cell/transmembrane immunoglobulin and mucin domain-containing (TIM) protein family has attracted particular attention because of their broad immune functions and the response to viral infections. TIM-1, a member of the TIM family, has been demonstrated to play an important role in viral infections. However, its roles during fish nodavirus infection still remained largely unknown. In this study, a homolog of TIM-1 from orange-spotted grouper (Epinephelus coioides) (EcTIM-1) was identified, and characterized. EcTIM-1 encoded a 217-amino acids protein, containing one Immunoglobulin domain. Homology analysis showed that EcTIM-1 shared 98.62 % and 42.99 % identity to giant grouper (E. lanceolatus) and human (Homo sapiens). Quantitative Real-time PCR analyses indicated that EcTIM-1 was expressed in all examined tissues, with higher expression in liver, spleen, skin, and heart, and was significantly up-regulated in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTIM-1 was distributed in the cytoplasm, and partly co-localized with Golgi apparatus and lysosomes in vitro. The ectopic expression of EcTIM-1 promoted RGNNV replication by increasing the level of viral genes transcription and protein synthesis. Besides, overexpression of EcTIM-1 decreased the luciferase activity of type I interferon (IFN1), interferon stimulated response elements (ISRE) and nuclear factor kappa-B (NF-κB) promoters, as well as the transcription of pro-inflammatory factors and interferon related genes. EcTIM-1 significantly suppressed the luciferase activity of IFN1, ISRE and NF-κB promoters evoked by Epinephelus coioides melanoma differentiation-associated gene 5 (EcMDA5), mitochondrial antiviral signaling protein (EcMAVS), stimulator of IFN genes (EcSTING) or TANK-binding kinase 1 (EcTBK1). Collectively, EcTIM-1 negatively regulated interferon and inflammatory response to promote RGNNV infection. These results provide a basis for a better understanding of the innate immune response of TIM-1 in fish.

Preventive and reparative potentials of heat-inactivated and viable commensal Bacillus pumilus SE5 in ameliorating the adverse impacts of high soybean meal in grouper (Epinephelus coioides).

Probiotic Bacillus pumilus SE5, heat-inactivated (HSE5) or active (ASE5), were supplemented to high soybean meal (HSM) (36 %) diet at whole term (0-56 days) and middle term (29-56 days) to investigate the preventing and repairing effects of B. pumilus SE5 in ameliorating the adverse effects of HSM in Epinephelus coioides. The results suggested that the HSM significantly decreased the weight gain rate (WGR), specific growth rate (SGR), and increased the feed conversion rate (FCR) at day 56 (P < 0.05), while HSE5 and ASE5 promoted the growth performance. The HSE5 and ASE5 showed preventive and reparative functions on the antioxidant capacity and serum immunity, with significantly increased the total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-PX) activities, and reduced malondialdehyde (MDA) level, and increased acid phosphatase (ACP), alkaline phosphatase (AKP), immunoglobulin M (IgM) and complement 3 (C3). The HSM impaired the intestinal health (destroyed the intestinal structure, significantly increased the contents of serum D-lactic acid and diamine oxidase, and reduced the expressions of claudin-3 and occludin), while HSE5 and ASE5 improved them at whole term and middle term. The HSM impaired the intestinal microbiota and reduced its diversity, and the HSE5 or ASE5 improved the intestinal microbiota (especially at whole term). HSE5 and ASE5 improved the intestinal mRNA expressions of anti-inflammatory genes (il-10 and tgf-ß1) and reduced the expressions of pro-inflammatory genes (il-1ß, il-8, il-12), and promoted the expressions of humoral immune factor-related genes (cd4, igm, mhcII-α) and antimicrobial peptide genes (ß-defensin, epinecidin-1 and hepcidin-1), and decreased the expressions of NF-κB/MAPK signaling pathway-related genes (ikk-α, nf-κb, erk-1), and improved the expressions of MAPK signaling pathway-related gene p38-α (P < 0.05). In conclusion, the heat-inactivated and active B. pumilus SE5 effectively prevented and repaired the suppressive effects of soybean meal in E. coioides, which underscored the potential of B. pumilus SE5 as a nutritional intervention agent in HSM diet in aquaculture.

Molecular features, antiviral activity, and immunological expression assessment of interferon-related developmental regulator 1 (IFRD1) in red-spotted grouper (Epinephelus akaara).

Interferon-related developmental regulator 1 (IFRD1) is a viral responsive gene associated with interferon-gamma. Herein, we identified the IFRD1 gene (EaIFRD1) from red-spotted grouper (Epinephelus akaara), evaluated its transcriptional responses, and investigated its functional features using various biological assays. EaIFRD1 encodes a protein comprising 428 amino acids with a molecular mass of 48.22 kDa. It features a substantial domain belonging to the interferon-related developmental regulator superfamily. Spatial mRNA expression of EaIFRD1 demonstrated the highest expression levels in the brain and the lowest in the skin. Furthermore, EaIFRD1 mRNA expression in grouper tissues exhibited significant modulation in response to immune stimulants, including poly (I:C), LPS, and nervous necrosis virus (NNV) infection. We analyzed downstream gene regulation by examining type Ⅰ interferon pathway genes following EaIFRD1 overexpression. The results demonstrated a significant upregulation in cells overexpressing EaIFRD1 compared to the control after infection with viral hemorrhagic septicemia virus (VHSV). A subcellular localization assay confirmed the nuclear location of the EaIFRD1 protein, consistent with its role as a transcriptional coactivator. Cells overexpressing EaIFRD1 exhibited increased migratory activity, enhancing wound-healing capabilities compared to the control. Additionally, under H2O2 exposure, EaIFRD1 overexpression protected cells against oxidative stress. Overexpression of EaIFRD1 also reduced poly (I:C)-mediated NO production in RAW267.4 macrophage cells. In FHM cells, EaIFRD1 overexpression significantly reduced VHSV virion replication. Collectively, these findings suggest that EaIFRD1 plays a crucial role in the antiviral immune response and immunological regulation in E. akaara.

Identification and functional characterization of interleukin-22 (IL-22) in orange-spotted grouper (Epinephelus coioides).

In mammals, IL-22 is considered as a critical cytokine regulating of immunity and homeostasis at barrier surfaces. Although IL-22 have been functional characterization in different species of fish, the studies about distinct responses of IL-22 in different organs/tissues/cell types is rather limited. Here, we identified and cloned IL-22 gene (named as Ec-IL-22) from grouper (Epinephelus coioides). Ec-IL-22 gene was detected in all orangs/tissues examined, and was induced in intestine, gill, spleen, head kidney, and primary head kidney/intestine leukocytes following the stimulation of LPS and poly (I:C), as well as Vibrio harveyi and Singapore grouper iridovirus infection (SGIV). In addition, the stimulation of DSS could induce the expression of Ec-IL-22 in intestine and primary leukocytes from intestine. Importantly, the treatment of recombinant Ec-IL-22 induced the mRNA level of proinflammatory cytokines in primary intestine/head kidney leukocytes. The present results improve the understanding of expression patterns and functional characteristics of fish IL-22 in different organs/tissues/cell types.

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.

Coexistence, resource partitioning, and fisheries management: A tale of two mesopredators in equatorial waters.

Rock hind (Epinephelus adscensionis) and spotted moray (Gymnothorax moringa) are ubiquitous mesopredators that co-occur in the nearshore waters of Ascension Island in the South Atlantic Ocean, where they have significant cultural and subsistence value, but management of their non-commercial take is limited. This isolated volcanic system is home to high biomass and low species diversity, which poses two key questions: How can two mesopredators that perform similar ecological roles coexist? And if these two species are so ecologically similar, can they be managed using the same approach? Here, we combined acoustic telemetry, stomach content analysis, and stable isotope analysis to (i) explore space use and diet choices within and between these two species and (ii) to assess appropriate species-specific management options. Although rock hind had high residency and small calculated home ranges (0.0001-0.3114 km2), spotted moray exhibited shorter periods of residency (<3 months) before exiting the array. Vertical space use differed significantly across the 20-month tracking period, with individual differences in vertical space observed for both species. A hierarchical generalized additive model using 12-h averaged depth data identified that rock hind occurred lower in the water column than spotted moray, with both species occupying moderately deeper depths at night versus day (+1.6% relative depth). Spotted moray depth was also significantly predicted by lunar illumination. Aggregating samples by species and tissue type, Bayesian ecological niche modeling identified a 53.14%-54.15% and 78.02%-97.08% probability of niche overlap from fin clip and white muscle, respectively, whereas limited stomach content data indicated a preference for piscivorous prey. Variability in niche breadth between years suggests these species may exploit a range of prey items over time. These findings indicate that although these two species perform a similar ecological role by feeding on prey occupying the same trophic levels, subtle differences in movement behaviors between them suggest a one-rule-fits-all management approach is not likely the most effective option.

Effect of temperature fluctuation on the physiological stress response of hybrid pearl gentian grouper during waterless keeping alive.

Temperature fluctuations are inevitable and have an important impact on the survival of fish during transportation. Therefore, the effect of temperature fluctuation (15 ± 1 °C, 15 ± 2 °C, 15 ± 3 °C) on the muscle quality, physiological, and immune function of hybrid pearl gentian grouper before waterless keeping alive, during keeping alive (0 h, 3 h, 6 h, 9 h, 12 h), and after revival for 12 h was investigated. The plasma glucose concentration of grouper gradually decreased to 0.645 ± 0.007 mg/mL, 0.657 ± 0.006 mg/mL, and 0.677 ± 0.004 mg/mL after keeping alive for 12 h under different temperature fluctuations of 15 ± 1 °C, 15 ± 2 °C, and 15 ± 3 °C, respectively. The cortisol concentration and lysozyme activity of pearl gentian grouper significantly increased (P < 0.05) during the keeping alive period. The results suggested that fish bodies would produce acute stress response, strengthen immune defense ability, and quickly consume a lot of energy to adapt to the low-temperature anhydrous environment. In all treatment groups, the activities of plasma alanine transaminase (ALT) and aspartate aminotransferase (AST) and the content of creatinine gradually increased with the prolongation of the survival time. The hardness and springiness of muscle decreased from 5965.99 ± 20.15 and 0.90 ± 0.00 to 3490.69 ± 27.59 and 0.42 ± 0.01, respectively. In the meanwhile, the change of glycogen and lactic acid content was opposite, indicating that temperature fluctuation harmed the liver, kidney function, and muscle quality. In the later stage of keeping alive, the superoxide dismutase (SOD) and catalase (CAT) activities decreased, especially in the temperature fluctuation group of ±3 °C (125.99 ± 5.48 U/mgprot, 44.21 ± 0.63 U/mgprot), leading to an imbalance of fish immunity. In summary, higher temperature fluctuation would influence the physiological function and immune defense ability and decrease the quality of pearl gentian grouper.

Oleic acid enriched diet affects the metabolome composition of the hybrid grouper infected with vibriosis.

This study focuses in investigating the fatty acid contents of surviving infected hybrid grouper fed with oleic acid immunostimulant. After a 6-week feeding trial, Epinephelus fuscoguttatus × Epinephelus lanceolatus fingerlings were infected with Vibrio vulnificus. One week after bacterial challenge, fish oil was extracted from body tissue of surviving infected fingerlings using the Soxhlet extraction method. The extracted samples were then sent for GC-MS analysis. The raw GC-MS data were analyzed using software programs and databases (i.e., MetaboAnalyst, SIMCA-P, NIST Library, and KEGG). A total of 39 metabolites were putatively identified, with 18 metabolites derived from the fatty acid group. Our further analysis revealed that most metabolites were highly abundant in the oleic acid dietary samples, including oleic acid (4.56%), 5,8,11-eicosatrienoic acid (3.45%), n-hexadecenoic acid (3.34%), cis-erucic acid (2.76%), and 9-octadecenoic acid (2.5%). Worthy of note, we observed a greater abundance of α-linoleic acid (15.57%) in the control diet samples than in the oleic acid diet samples (14.59%) with no significant difference in their results. The results obtained from this study revealed that surviving infected hybrid grouper expressed more immune-related fatty acids due to the effect of oleic acid immunostimulant. Therefore, in this study, we propose oleic acid as a potential immunostimulant in enhancing fish immunity in aquaculture industry.

Dextran Sulfate Sodium Salt (DSS) induced enteritis in Orange-spotted grouper, Epinephelus coioides.

The enteritis is a common disease in fish farming, but the pathogenesis is still not fully understood. The aim of the present study was to investigate the inducement of Dextran Sulfate Sodium Salt (DSS) intestinal inflammation on Orange-spotted grouper (Epinephelus coioides). The fish were challenged with 200 µl 3% DSS via oral irrigation and feeding, an appropriate dose based on the disease activity index of inflammation. The results indicated that the inflammatory responses induced by DSS were closely associated with the expression of pro-inflammatory cytokines including interleukin 1ß (IL-1ß), IL-8, IL16, IL-10 and tumor necrosis factor α (TNF-α), as well as NF-κB and myeloperoxidase (MPO) activity. At day5 after DSS treatment, the highest levels of all parameters were observed. Also, the severe intestinal lesions (intestinal villus fusion and shedding), strong inflammatory cell infiltration and microvillus effacement were seen through histological examination and SEM (scanning electronic microscopy) analysis. During the subsequent 18 days of the experimental period, the injured intestinal villi were gradually recovery. These data is beneficial to further investigate the pathogenesis of enteritis in farmed fish, which is helpful for the control of enteritis in aquaculture.

flgC gene is involved in the virulence regulation of Pseudomonas plecoglossicida and affects the immune response of Epinephelus coioides.

As a pathogen of cultured teleosts, Pseudomonas plecoglossicida has caused significant economic losses. flgC plays an important role in encoding flagellar basal-body rod proteins. Our previous studies revealed the high expression of P. plecoglossicida flgC in infected Epinephelus coioides. To explore the role of flgC in the virulence of P. plecoglossicida and the immune response of E. coioides to the infection of P. plecoglossicida, flgC gene of P. plecoglossicida was knocked down by RNA interference (RNAi). The results showed that the flgC gene in all four mutants of P. plecoglossicida was significantly knocked down, and the mutant with the best knockdown efficiency of 94.3% was selected for subsequent studies. Compared with the NZBD9 strain of P. plecoglossicida, the flgC-RNAi strain showed a significantly decrease in chemotaxis, motility, adhesion, and biofilm formation. Furthermore, compared with the E. coioides infected with the NZBD9 strain, the infection of flgC-RNAi strain resulted in the infected E. coioides a 1.5-day delay in the time of first death and an 80% increase in survival rate, far fewer white nodules upon the spleen surfaces, and lower pathogen load in the spleens. RNAi of flgC significantly influenced the metabolome and transcriptome of the spleen in infected E. coioides. KEGG enrichment analysis exhibited that the Toll-like receptor signaling pathway was the most enriched immune pathway; the most significantly enriched metabolic pathways were associated with Linoleic acid metabolism, Choline metabolism in cancer, and Glycerophospholipid metabolism. Further combined analysis of transcriptome and metabolome indicated significant correlations among pantothenate and CoA biosynthesis, beta-alanine metabolism, lysosome metabolites, and related genes. These results suggested that flgC was a pathogenic gene of P. plecoglossicida; flgC was associated with the regulation of chemotaxis, motility, biofilm formation, and adhesion; flgC influenced the immune response of E. coioides to the infection of P. plecoglossicida.

Effects of Cryptocaryon irritans infection on the histopathology, oxidative stress, immune response, and intestinal microbiota in the orange-spotted grouper Epinephelus coioides.

Cryptocaryon irritans is a parasitic ciliate of marine fish, causing serious mortality and economic loss of grouper. In this study, the orange-spotted grouper (Epinephelus coioides) were separately exposed to C. irritans infection for 72 h at a dose of 5000 or 10000 active theronts per fish, and we evaluated the changes in histopathology, oxidative stress, immune response, and intestinal microbiota composition. The results showed that C. irritans infection caused pathological alteration on the skin, gills, and liver of E. coioides. Oxidative stress responses occurred in the liver and gills, reflected in the corresponding antioxidant enzyme and gene indexes. The mRNA expression levels of inflammation-related genes (IL-1ß, IL-6, and IL-8) and the mediators of apoptosis (casp3, casp9, and cytc) were increased in the liver and gills of the fish. C. irritans infection also affected the diversity and composition of intestinal microbiota. Specifically, the relative abundance of Firmicutes was increased, whereas that of Proteobacteria was decreased. Several potentially beneficial bacteria (Pandoraea, Clostridium sensu stricto 1, Christensenellaceae R-7 group, and Weissella) were decreased, whereas pathogenic bacteria (Streptococcus and Acinetobacter) were increased. In conclusion, this study reveals that C. irritans infection caused histopathology, immune disorders, and intestinal microbial community variation in E. coioides.

Identification of circRNAs and circRNA-mRNA network of Epinephelus coioides during Singapore grouper iridovirus infection.

Circular RNA (circRNA), one of the important non-coding RNA molecules with a closed-loop structure, plays a key regulatory role in cell processing. In this study, circRNAs of Epinephelus coioides, an important marine cultured fish in China, were isolated and characterized, and the network of circRNAs and mRNA was explored during Singapore grouper iridovirus (SGIV) infection, one of the most important double stranded DNA virus pathogens of marine fish. 10 g of raw data was obtained by high-throughput sequencing, and 2599 circRNAs were classified. During SGIV infection, 123 and 37 circRNAs occurred differential expression in spleen and spleen cells, indicating that circRNAs would be involved in the viral infection. GO annotation and KEGG demonstrated that circRNAs could target E. coioides genes to regulate cell activity and the activation of immune factors. The results provide some insights into the circRNAs mediated immune regulatory network during bony fish virus infection.