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.

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.

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.

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.

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.

Grouper Atg14 promotes Singapore grouper iridovirus (SGIV) replication by inhibiting the host innate immune response.

As one of the important members of the autophagy-related protein family, Atg14 plays a key role in the formation and maturation of autophagosomes. However, little is known about the potential roles of fish Atg14 and its roles in virus infection. In the present study, the homolog of Atg14 (EcAtg14) from the orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The open reading frame (ORF) of EcAtg14 consists of 1530 nucleotides, encoding 509 amino acids, with a predicted molecular weight of 56.9 kDa. EcAtg14 was distributed in all tested tissues, with higher expression in liver, blood and spleen. The expression of EcAtg14 was increased in grouper spleen (GS) cells after Singapore grouper iridovirus (SGIV) infection. EcAtg14 was distributed in the cytoplasm of GS cells. Overexpression of EcAtg14 promoted SGIV replication in GS cells and inhibited IFN3, ISRE and NF-κB promoter activities. Co-immunoprecipitation results showed that there was an interaction between EcAtg14 and EcBeclin. EcAtg14 also promoted the synthesis of LC3-II in GS cells. These findings provide a basis for understanding the innate immune mechanism of grouper against viral infection.

Vibrio harveyi co-infected with Cryptocaryon irritans to orange-spotted groupers Epinephelus coioides.

The orange-spotted grouper (Epinephelus coioides) is a high economic value aquacultural fish in China, however, it often suffers from the outbreak of parasitic ciliate Cryptocaryon irritans as well as bacterium Vibrio harveyi which bring great loss in grouper farming. In the present study, we established a high dose C. irritans local-infected model which caused the mortality of groupers which showed low vitality and histopathological analysis demonstrated inflammatory response and degeneration in infected skin, gill and liver. In addition, gene expression of inflammatory cytokines was detected to assist the estimate of inflammatory response. Furthermore, we also found that the activity of Na+/K+ ATPase in gill was decreased in groupers infected C. irritans and the concentration of Na+/Cl- in blood were varied. Base on the morbidity symptom occurring in noninfected organs, we hypothesized that the result of morbidity and mortality were due to secondary bacterial infection post parasitism of C. irritans. Moreover, four strains of bacteria were isolated from the infected site skin and liver of local-infected groupers which were identified as V. harveyi in accordance of phenotypic traits, biochemical characterization and molecular analysis of 16S rDNA genes, housekeeping genes (gyrB and cpn60) and species-specific gene Vhhp2. Regression tests of injecting the isolated strain V. harveyi has showed high pathogenicity to groupers. In conclusion, these findings provide the evidence of coinfections with C. irritans and V. harveyi in orange-spotted grouper.

Oral immunizations with Bacillus subtilis spores displaying VP19 protein provide protection against Singapore grouper iridovirus (SGIV) infection in grouper.

Disease caused by Singapore grouper iridovirus (SGIV) results in major economic losses in the global grouper aquaculture industry. Vaccination is considered to be the most effective way to protect grouper from SGIV. In this study, the spores of Bacillus subtilis (B.subtilis) WB600 were utilized as the vehicle that the VP19 protein was displayed on the spores surface. To further investigate the effect of oral vaccination, the grouper were orally immunized with B.s-CotC-19 spores. After challenged, the survival rate of grouper orally vaccinated with B.s-CotC-19 spores was 34.5% and the relative percent survival (RPS) was 28.7% compared to the PBS group. Moreover, the viral load in the tissues of the B.s-CotC-19 group was significantly lower than that of the PBS group. The histopathological sections of head kidney and liver tissue from the B.s-CotC-19 group showed significantly less histopathology compared to the PBS group. In addition, the specific IgM levels in serum in the B.s-CotC-19 group was higher than those in the PBS group. In the hindgut tissue, the immune-related gene expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the B.s-CotC-19 group, suggesting that the innate and adaptive immune responses were activated. These results indicated that the oral administration of recombinant B.subtilis spores was effective for preventing SGIV infection. This study provided a feasible strategy for the controlling of fish virus diseases.

Grouper annexin A2 affects RGNNV by regulating the host immune response.

Annexin A2 (AnxA2) is ubiquitous in vertebrates and has been identified as a multifunctional protein participating in a series of biological processes, such as endocytosis, exocytosis, signal transduction, transcription regulation, and immune responses. However, the function of AnxA2 in fish during virus infection still remains unknown. In this study, we identified and characterized AnxA2 (EcAnxA2) in Epinephelus coioides. EcAnxA2 encoded a 338 amino acids protein with four identical annexin superfamily conserved domains, which shared high identity with other AnxA2 of different species. EcAnxA2 was widely expressed in different tissues of healthy groupers, and its expression was significantly increased in grouper spleen cells infected with red-spotted grouper nervous necrosis virus (RGNNV). Subcellular locatio n analyses showed that EcAnxA2 diffusely distributed in the cytoplasm. After RGNNV infection, the spatial distribution of EcAnxA2 was unaltered, and a few EcAnxA2 co-localized with RGNNV during the late stage of infection. Furthermore, overexpression of EcAnxA2 significantly increased RGNNV infection, and knockdown of EcAnxA2 reduced RGNNV infection. In addition, overexpressed EcAnxA2 reduced the transcription of interferon (IFN)-related and inflammatory factors, including IFN regulatory factor 7 (IRF7), IFN stimulating gene 15 (ISG15), melanoma differentiation related gene 5 (MDA5), MAX interactor 1 (Mxi1) laboratory of genetics and physiology 2 (LGP2), IFN induced 35 kDa protein (IFP35), tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin 6 (IL-6). The transcription of these genes was up-regulated when EcAnxA2 was inhibited by siRNA. Taken together, our results showed that EcAnxA2 affected RGNNV infection by down-regulating the host immune response in groupers, which provided new insights into the roles of AnxA2 in fish during virus infection.

Pharmacology of orange-spotted grouper (Epinephelus coioides) melanocortin-5 receptor and its modulation by Mrap2.

The mammalian melanocortin-5 receptors (MC5Rs) are involved in various functions, including exocrine gland secretion, glucose uptake, adipocyte lipolysis, and immunity. However, the physiological role of fish Mc5r is rarely studied. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates pharmacological properties of melanocortin receptors. Herein, to lay the foundation for future physiological studies, we cloned the orange-spotted grouper (Epinephelus coioides) mc5r, with a 1008 bp open reading frame and a predicted protein of 334 amino acids. Grouper mc5r had abundant expression in the brain, skin, and kidney. Four ligands could bind to grouper Mc5r and dose-dependently increase intracellular cAMP levels. Grouper Mrap2 did not affect binding affinity or potency of Mc5r; however, grouper Mrap2 decreased cell surface expression and maximal binding of Mc5r. Mrap2 also significantly decreased the maximal response to a superpotent agonist but not the endogenous agonist. This study provided new data on fish Mc5r pharmacology and its regulation by Mrap2.

Treatments of orange-spotted grouper (Epinephelus coioides) against Cryptocaryon irritans with •OH, ClO2 or HCHO: Survival, physiological and histological response.

Cryptocaryon irritans causes one of the most serious diseases in various wild and cultured marine fish, leading to mass mortality and economic loss. In this study, hydroxyl radical (•OH) solution produced by strong ionization discharge combined with water jet cavitation effect was injected into orange-spotted grouper (Epinephelus coioides) aquaculture tanks for C. irritans control. The results showed that all C. irritans theronts were inactivated by •OH solution at concentrations of 0.5 mg/L within 2 min. •OH could induce alteration of shape, the absence of motility and macronucleus dispersion in theronts. A possible explanation was that the macronucleus of C. irritans might be damaged by •OH; as a result, its metabolism and life activities were disturbed. The •OH treatment increased the survival rate of E. coioides challenged with C. irritans from 64.7 ± 8.0% (mean ± SD) to 100% and reduced their infection intensity significantly. Stress response biomarkers such as malonaldehyde, glutathione, glutathione peroxidase, superoxide dismutase (SOD) and catalase levels in the gills of E. coioides at different time points were analysed. The SOD activity in the •OH group first decreased and then recovered to the initial level at the end of the experiment. The other stress response biomarkers had no significant difference from that in the uninfected control group after •OH treatment. Additionally, the gill of E. coioides in the •OH group exhibited slight and reversible transformation compared with the uninfected control group. Compared with •OH treatment, chlorine dioxide and formalin treatment reduced the survival rate, induced oxidative damage and changed the histological gill structure in E. coioides. In conclusion, •OH could be applied effectively to control C. irritans infection without affecting the normal physiological condition of E. coioides.

The Expression Pattern of Insulin-Like Growth Factor Subtype 3 (igf3) in the Orange-Spotted Grouper Epinephelus coioides and Its Function on Ovary Maturation.

A new insulin-like growth factor (Igf) subtype 3 (igf3) has recently been found in the bony fish orange-spotted grouper (Epinephelus coioides). However, the role of igf3 in the maturation of the ovary and sex differentiation in E. coioides is currently unknown. We examined the ovarian localization and receptor binding of the novel ortholog Igf3 using qRT-PCR, and Western blotting, combined with in situ hybridization and immunohistochemistry methods. Results demonstrated the presence of igf3 mRNA and protein in mature oocytes. Furthermore, Igf3 protein expression was not detected in testis, brain, kidney and liver homogenates. The calculated molecular weight of Igf3 was 22 kDa, which was consistent with the deduced amino acid sequence from the full-length open reading frame. The immunoreactivity showed that Igf3 was strongly present in the follicle staining fully-grown stage. The igf3 mRNA expression level was significantly positively correlated with ovarian follicular maturation. Meanwhile, Igf3 increased germinal-vesicle breakdown in a time- and dose-dependent manner. In vitro, treatment of primary ovarian cells with Igf3 up-regulated significantly the mRNA expression level of genes related to sex determination and reproduction such as forkhead boxl2 (foxl2), dosage-sensitive sex reversal adrenal hypoplasia critical region on chromosome x gene 1 (dax1), cytochrome P450 family 19 subfamily member 1 a (cyp19a1a), cytochrome P450 family 11 subfamily a member 1 a (cyp11a1a) and luteinizing hormone receptor 1 (lhr1). Overall, our results demonstrated that igf3 promotes the maturation of the ovary and plays an important role in sex differentiation in E. coioides.