Orange-spotted Grouper Epinephelus Coioides Research Articles

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.

An Integrated in silico and in vivo study of nucleic acid vaccine against Nocardia seriolae infection in orange-spotted grouper Epinephelus coioides

Nocardiosis in aquatic animals caused by Nocardia seriolae is a frequently occurring serious infection that has recently spread to many countries. In this study, DNA vaccines containing potential bacterial antigens predicted using the reverse vaccinology approach were developed and evaluated in orange-spotted groupers. In silico analysis indicated that proteins including cholesterol oxidase, ld-transpeptidase, and glycosyl hydroxylase have high immunogenicity and are potential vaccine candidates. In vitro assays revealed the mature and biological configurations of these proteins. Importantly, when compared to a control PBS injection, N. seriolae DNA-based vaccines showed significantly higher expression of IL1β, IL17, and IFNγ at 1 or 2 days, in line with higher serum antibody production and expression of other cellular immune-related genes, such as MHCI, CD4, and CD8, at 7 days post-immunization. Remarkably, enhanced immune responses and strong protective efficacy against a highly virulent strain of N. seriolae were recorded in DNA vaccine-cholesterol oxidase (pcD::Cho) injected fish, with a relative survival rate of 73.3%. Our results demonstrate that the reverse vaccinology approach is a valid strategy for screening vaccine candidates and pcD::Cho is a promising candidate that can boost both innate and adaptive immune responses and confer considerable protection against N. seriolae infection.

Pathogenicity characterization of nocardiosis in orange-spotted grouper (Epinephelus coioides) via exposure to nine distinct isolates of Nocardia seriolae and four administrative routes.

Nocardia seriolae causes chronic nocardiosis in various marine and freshwater aquatic animals; however, grouper species have rarely been investigated. This study evaluated the pathogenicity of nocardiosis following N. seriolae infection in the orange-spotted grouper Epinephelus coioides. Nine identified genetic isolates of N. seriolae were tested in vivo using the intraperitoneal method and observed daily for 35 days. The most virulent isolate was then used to evaluate transmission through different routes (intraperitoneal IP, intramuscular IM, oral OR, and immersion IS) in the same fish model and was observed daily for 42 days. The results showed mild variation in virulence among N. seriolae isolates. AOD107132-2 K and OT103003-N11 strains displayed the highest and lowest risk virulence, respectively, based on the accumulation and kinetics of mortality. IM and IP administrations showed an early phase response with early mortality by 5 dpc (30%-100%), while slower kinetics of nocardiosis occurred in the OR and IS routes with slow mortality at 35 dpc (4%-8%). Histopathology revealed typical granulomas, confirming the progression of nocardiosis in the diseased fish. These results provide the basis for further studies on the virulence profile of N. seriolae in Taiwan and a well-suited route of administration in orange-spotted groupers for further prevention development.

Antiviral immunity of grouper MAP kinase phosphatase 1 to Singapore grouper iridovirus infection

Singapore grouper iridovirus (SGIV), with various mechanisms for evading and modulating host, has inflicted heavy economic losses in the grouper aquaculture. MAP kinase phosphatase 1 (MKP-1) regulates mitogen-activated protein kinases (MAPKs) to mediate the innate immune response. Here, we cloned EcMKP-1, an MKP-1 homolog from the orange-spotted grouper Epinephelus coioides, and investigated its role in the infection of SGIV. In juvenile grouper, EcMKP-1 was highly upregulated and peaked at different times after injection with lipopolysaccharide, polyriboinosinic polyribocytidylic acid and SGIV. EcMKP-1 expression in heterologous fathead minnow cells was able to suppress SGIV infection and replication. Furthermore, EcMKP-1 was a negative regulator of c-Jun N-terminal kinase (JNK) phosphorylation early in SGIV infection. EcMKP-1 decreased the apoptotic percentage and caspase-3 activity during the late stage of SGIV replication. Our results demonstrate critical functions of EcMKP-1 in antiviral immunity, JNK dephosphorylation and anti-apoptosis during SGIV infection.

Determination of biological parameters of 4 marine fish species (sea bass, cobia, orange-spotted grouper, and short yellowfin pompano) for assessment of the holding capacity of a location

A biochemical experiment on four marine fish species commonly farmed in Vietnam, sea bass Lates calcarifer(Bloch, 1790), cobia Rachycentron canadum (Linnaeus, 1766), orange-spotted grouper Epinephelus coioides(Hamilton, 1822), and short yellowfin pompano Trachinotus ovatus (Linnaeus, 1758), was carried out at Cat Ba island, Hai Phong province. The mean weight of fish used in the experiment were 2, 5, 2, and 0.6 kg.ind-1, respectively. The experiment aimed to determine the biological parameters of the farmed fish (Ap, Af, Ac) and related nutritional parameters (Pp, Pf, Fp, Ff, Fc), for assessment of the holding capacity of a location. The results showed that the apparent digestibility coefficient of protein and fat in trash fish for sea bass were 92.90%, 93.84%, respectively; cobia 91.17%, 95.53%; orange-spotted grouper 95.20%, 96.40%. The apparent digestibility coefficient of protein, fat and carbohydrate in pellet feed for short yellowfin pompano were 85.83, 76.47, and 13.68%, respectively. The protein content and fat content in the muscle of sea bass were 19.28%, 6.27%; cobia 19.13%, 13.85%; orange-spotted grouper 17.37%, 7.32%; and short yellowfin pompano 18.31%, 15.08%, respectively. The protein content and fat content of trash fish were 16.99%, 7.19%, respectively. The proportion of protein, fat and carbohydrate of pellet feed were 42.01, 9.93, and 27.82%, respectively. There are no carbohydrates in the muscle of farmed fish as well as trash fish.

Iron loss and hypoxia are involved in lethal Pseudomonas plecoglossicida infections in the orange-spotted grouper Epinephelus coioides

Pseudomonas plecoglossicida is a causative agent of disease in aquaculture, leading to massive mortality in a variety of economically important cultured fish and resulting in great economic losses. Here we revealed that P. plecoglossicida infection caused a 62% probability of visceral white spot disease in orange-spotted grouper (Epinephelus coioides). After P. plecoglossicida challenge, the spleen mass increased significantly and the liver mass decreased significantly. Spleens had the highest bacterial load, at 1.38 × 107 colony forming units per gram. In addition, the number of blood red blood cells decreased. Moreover, diseased fish suffered from anemia and hypoxia. In addition, ferritin in the spleen was reduced, and there was a loss of iron in the fish. Furthermore, histological examination of the spleen showed that abundant red blood cells had infiltrated and that collagen fibers had been dissolved. In contrast, the survivors' spleens were rich in ferritin, having few red blood cell infiltration and collagen fibers that were intact. In summary, spleen collagen fibrinolysis, red blood cell infiltration, and ferritin loss were demonstrated to cause hypoxia in the fish, which may be the reasons of death caused by P. plecoglossicida in orange-spotted grouper.

Allopodocotyle palmi sp. nov. and Prosorhynchus maternus Bray & Justine, 2006 (Digenea: Opecoelidae & Bucephalidae) from the Orange-Spotted Grouper Epinephelus coioides (Hamilton, 1822) off Bali, Indonesia, Described Using Modern Techniques

BackgroundThe most convincing species of Allopodocotyle Pritchard, 1966 (Digenea: Opecoelidae) are known overwhelmingly from groupers (Serranidae: Epinephelinae). Six species of Allopodocotyle have been reported, collectively, from species of Cromileptes Swainson, 1839, Epinephelus Bloch, 1793 and Plectropomus Oken, 1817. These are A. epinepheli (Yamaguti, 1942), A. heronensis Downie & Cribb, 2011, A. manteri (Saoud & Ramadan, 1984), A. mecopera (Manter, 1940), A. plectropomi (Manter, 1963) and A. serrani (Yamaguti, 1952). In addition, a not yet fully described and unnamed seventh species, morphologically and phylogenetically close to A. epinepheli, was isolated from the orange-spotted grouper Epinephelus coioides (Hamilton, 1822) off Bali, Indonesia in 2016. An eighth species, again from E. coioides off Bali is described herein.MethodsMorphological and phylogenetic analyses justify the recognition of A. palmi sp. nov., which is also genetically different from the as yet unnamed congener from the same host and locality. For the first time, 3D confocal laser scanning microscopy was applied to study and distinguish Digenea taxonomically. We introduce the ‘Palm pattern’, a new simplified way to visualise morphometric differences of related digenean taxa.ResultsAllopodocotyle palmi sp. nov. is distinguished from its congeners that infect groupers by its elongate body with a size > 2.7 mm and diagonal testes. The ovary is located mainly, and the anterior testis completely, in the posterior half of the body; the uterine coils are in the fourth eighth of the body. The cirrus-sac is 0.75–1.4 (1.1) mm long, its posterior extremity is well separated from the anterior extent of the vitelline fields, just reaching the anterior border of uterine coils. In addition, Prosorhynchus maternus Bray & Justine, 2006 (Bucephalidae) was isolated from E. coioides, representing the first record in Indonesia and the third record for this fish species.ConclusionThe biodiversity research in Indonesia is enhanced with a new species description based on modern and newly applied techniques.

First Record of Orange Spotted Grouper Epinephelus coioides (Hamilton, 1822) from the Iskenderun Bay, the northeastern Mediterranean

A single specimen of orange spotted grouper Epinephelus coioides (Hamilton, 1822) was captured by a fisherman with a fishing line from the Iskenderun Bay (Konacık, Turkey) at 52 m depth. The morphological and color descriptions of E. coioides agree with previous descriptions. This record indicates that the eastward migration of this species in the Mediterranean coast of Turkey and fills the gap in the distribution range of the serranid species.

Digestibility of dehulled lupine meal, corn distiller’s dried grain with solubles and de-fatted soybean meal for orange-spotted grouper Epinephelus coioides

ABSTRACT Our study investigated the apparent digestibility coefficients (ADCs) of corn distiller’s dried grains with solubles (DDGS), dehulled lupine meal (DHLM) and de-fatted soybean meal (DSBM) for orange-spotted grouper Epinephelus coioides. The ADCcp of DHLM (97%) was the highest while the ADCcp of DDGS (90%) was similar to that of DSBM (89%). Digestible protein (DP) of DHLM (523 g kg−1) was significantly higher than that of DSBM (495 g kg−1) while DP of DDGS (457 g kg−1) was the lowest. We found no difference in digestible energy (DE) between DSBM (17.53 MJ kg−1) and DHLM (16.99 MJ kg−1) while DE of DDGS (15.77 MJ kg−1) was also the lowest. The relationship between fiber class and ADCs suggested that reducing fiber class in DHLM and DDGS should be helpful for the orange-spotted grouper’s digestibility.

Two Foxo1 homologues in the orange-spotted grouper Epinephelus coioides: sequences, expression, and possible involvement in the activation of cyp19a1a expression in the ovary.

Foxo1, a member of Foxo transcription factor family, is involved in a number of physiological processes including metabolism, cell cycle progression, aging, and apoptosis. In the ovarian granulosa cell of mouse, Foxo1 is implicated to inhibit the expression of Cyp19a1, a gene encoding the aromatase that converts androgens into estrogens. Currently, the information about the expression and physiological relevance of Foxo1 homologues in the ovary of teleosts is scarce. In the present study, cDNAs encoding two forms of Foxo1, Foxo1a and Foxo1b, were isolated from the orange-spotted grouper. Phylogenetic analysis indicated that the orange-spotted groupers Foxo1a and Foxo1b were closely related to the counterparts of the ricefield eel. RT-PCR analysis showed that the orange-spotted groupers foxo1a and foxo1b were expressed in a wide range of tissues, with high levels detected in the brain regions, liver, and intestine. Quantitative real-time PCR analysis showed similar expression profiles for cyp19a1a, foxo1a, and foxo1b in the ovary during development from the primary growth to mature stages, with peak values detected at the vitellogenic stage. In situ hybridization detected mRNA of foxo1a, foxo1b, and cyp19a1a in granulosa cells surrounding vitellogenic oocytes. In vitro transfection showed that both Foxo1a and Foxo1b upregulated the orange-spotted grouper cyp19a1a promoter activities, possibly through the conserved Foxo binding site. Collectively, these results suggest that both Foxo1a and Foxo1b may be involved in the regulation of the ovarian functions in the orange-spotted grouper and the physiological roles of Foxo1 homologues in the ovary may be diversified in vertebrates.

Growth and metabolic responses of orange spotted grouper Epinephelus coioides (Hamilton, 1822) fingerlings at different salinity regimes

A feeding trial was conducted for 60 days to delineate the effect of different salinities on growth and metabolic responses in fingerlings of orange spotted grouper Epinephelus coioides (Hamilton, 1822). The grouper fingerlings (N=1050; Av. wt. 2.0±0.01 g) were distributed equally into seven treatments of varying salinities viz., 5±1‰ (T1), 10±1‰ (T2), 15±1‰ (T3), 20±1‰ (T4), 25±1‰ (T5), 30±1‰ (T6) and 35±1‰ (T7) in triplicates. Fingerlings were fed with commercial diet containing 45% crude protein and 10% lipid. Results from the experiment concluded that growth performance of fingerlings of T4 group was better with minimum metabolic enzyme activity. The liver glycogen was significantly lower (p<0.05) in T4 group. Oxygen consumption rate (OCR) was recorded maximum at 15±1‰ and was significantly reduced for the fingerlings maintained at high salinity (p<0.05) from 20±1 to 35±1‰. Orange spotted grouper fingerlings maintained at salinity of 20±1‰ confirmed that, the animal spent less metabolic energy for maintaining the ionic balance at this optimum salinity compared to other salinities and it was reflected in better growth performance with minimum OCR and metabolic enzyme activities.

Jumonji family histone demethylases in Epinephelus coioides gonad

The Jumonji family (Jmj family) is a large family of proteins that contain the JmjC domain. Various members of this family are lysine (K)-specific demethylases, which remove methylation from the lysines of histones, and thus, are known as Jmj-KDMs. In this study, all Jmj-KDMs were identified in the orange-spotted grouper Epinephelus coioides and compared with those from Homo sapiens, Danio rerio, Takifugu rubripes, Oreochromis niloticus, and Xenopus tropicalis. The comparison revealed the teleost-specific loss of jmjd1a and the teleost-specific expansion of kdm5b, jmjd3, jmjd2a, and kdm6a in E. coioides. Analysis of the temporal expression pattern of Jmj-KDM genes in E. coioides enabled their division into different types according to their sex-related expression patterns. The kdm6 subfamily genes kdm6aa and jmjd3 (kdm6bb), which are also teleost specific, were selected for further analysis. Quantitative PCR analysis revealed the significantly higher expression of kdm6bb in bisexual-phase gonads and testes compared with ovary. Histological expression pattern analysis based on in situ hybridization (ISH) showed the high expression of kdm6aa in primary-growth stage oocytes, cortical-alveolus stage oocytes and granulosa cells, whereas kdm6aa and jmjd3 were highly expressed in Sertoli cells and spermatogonium. These results suggest that Jmj-KDMs, especially the kdm6 subfamily genes kdm6aa and jmjd3, are involved in sex-related processes in E. coioides.

Physical interactions facilitate sex change in the protogynous orange-spotted grouper, Epinephelus coioides.

Sex change in teleost fishes is commonly regulated by social factors. In species that exhibit protogynous sex change, such as the orange-spotted grouper Epinephelus coioides, when the dominant males are removed from the social group, the most dominant female initiates sex change. The aim of this study was to determine the regulatory mechanisms of socially controlled sex change in E. coioides. We investigated the seasonal variation in social behaviours and sex change throughout the reproductive cycle of E. coioides, and defined the behaviour pattern of this fish during the establishment of a dominance hierarchy. The social behaviours and sex change in this fish were affected by season, and only occurred during the prebreeding season and breeding season. Therefore, a series of sensory isolation experiments was conducted during the breeding season to determine the role of physical, visual and olfactory cues in mediating socially controlled sex change. The results demonstrated that physical interactions between individuals in the social groups were crucial for the initiation and completion of sex change, whereas visual and olfactory cues alone were insufficient in stimulating sex change in dominant females. In addition, we propose that the steroid hormones 11-ketotestosterone and cortisol are involved in regulating the initiation of socially controlled sex change.

Plasma and Tissue Depletion of Oxolinic Acid after Administration to Orange-Spotted Grouper (Epinephelus coioides), Snubnose Pompano (Trachinotus blochii) and Giant Seaperch (Lates calcarifer)

Pharmacokinetics and residues of oxolinic acid (OXO) in serum, muscle, liver, and kidney were measured in orange-spotted grouper Epinephelus coioides, snubnose pompano Trachinotus blochii and giant seaperch Lates calcarifer. Fish were treated with OXO after a single-dose trial (60 mg/kg) by oral gavages and repeat-doses of 60 mg/kg of body weight once daily in feed for five consecutive days. Analysis of OXO kinetic profiles, when determined by HPLC provided elimination half-life in muscle were 131, 26, and 85 h for 26-27 °C orange-spotted grouper, snubnose pompano, and giant seaperch, respectively. Depletion studies were conducted the time for OXO concentrations to fall below 0.1 µg/g (the current tolerance set by the European Medicines Agency (EMA) among fish species establishing an adequate depletion period). The mean tissue OXO concentration in the edible portion was below maximum residue level (MRL) of snubnose pompano at 6 d postdosing, for orange-spotted grouper and giant seaperch, at 10 d postdosing, respectively. When possible, depletion data were fitted to a one-compartment pharmacokinetic model. For all test species, the longest withdrawal period of 9 days in snubnose pompano and 15 days in orange-spotted grouper and giant seaperch were calculated because of a safety span (corresponds to 50% of the time point when at which residues fall below the MRL added to the depletion time). These results suggest withdrawal times with reference to human consumption of treated fish, to establish policy guidelines and basic principles regarding the use of OXO for fish-farming, and to fish-farmers for the proper handling to ensure safe fish that the consumer will not be at risk.

Identification of a novel splice variant isoform of interferon regulatory factor 10, IRF10, in orange spotted grouper Epinephelus coioides

Interferon regulatory factors (IRFs) are a family of transcription factors essential to the control of antiviral immune response, cell growth, differentiation and apoptosis. IRF10 was originally found in chicken, which was induced by the v-Rel oncoprotein in lymphoid cell lines and involved in the upregulation of major histocompatibility complex (MHC) class I and guanylate-binding protein. In fish, IRF10 plays negative roles in regulation of the interferon (IFN) response. Here, we identified a splice variant of IRF10, named as EcIRF10-SF in orange spotted grouper, which shares the first three exons with the long form (EcIRF10-LF) and retains part of intron 3, creating a premature termination codon. Furthermore, we observed that the EcIRF10-SF exhibits similar expression pattern compared to its native counterparts. Functional studies demonstrate that the two EcIRF10 isoforms repress DrIFNϕ1 and DrIFNϕ3 promoter activity and negatively regulate fish antiviral gene expression. Subcellular localization analysis shows that the amino acids from 57 to 86 within DBD are required for IRF10 nuclear import. Overall, our description of transcript diversification of IRF10 in the grouper provides a coherent framework to further dissect its roles in immune response.

Changing sex: it's a fishy thing.

Sex is a much more fluid affair for fishes than for terrestrial vertebrates. Many species (around 2%) show sequential hermaphroditism, switching from male to female (protandry), from female to male (protogyny) or - more rarely - from one to the other then back again. Such sexual plasticity likely confers an adaptive advantage during changes in the social milieu. Protogynous fishes, such as groupers and wrasses, were previously thought to remain as males after having made the switch once. However, Jiaxing Chen and colleagues from Sun Yet-Sen University (Guangzhou, China) show in the current issue of Journal of Fish Biology that, in the orange-spotted grouper Epinephelus coioides, some males will make the reverse switch back to females when placed in male-only groups. Interestingly, it was the smaller males, likely lower in the pecking order, that tended to make this switch. As might be expected, plasma levels of 11-keto testosterone (11-KT; the main androgen in teleosts) fell significantly in those males that reversed sex, whilst there was a peak of 17β-estradiol (the major estrogen) at six weeks in the same fish. Expression of genes coding for other sex hormones, namely gonadotropin-releasing hormone (GnRH) in the hypothalamus, plus follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary glands, of sex-reversed fish were similar to that of females, as were those of some other genes thought to be involved in gonadal sex-change (cyp191a1a, the gene coding for aromatase, for example). Intriguingly, however, this last example only became elevated after the sex-reversal was complete, raising doubts as to its involvement in the initiation of the process. The authors suggest that, in this species at least, FSH may be more important than LH in the regulation of sex-reversal, the latter more important in maintaining male gametes. Whilst the involvement of the hypothalamus-pituitary-gonadal (HPG) axis in sex-change is beyond doubt, the precise details may be species or, as the current study suggests, context-dependent. As Mark Lokman (University of Otago, New Zealand), who works on sex-change in wrasse (but was not involved in the current study) comments, “The ability to change sexual phenotype is not uncommon in fishes..”. However, since the work of Chen et al. in groupers, and Kuwamura et al. (2007) in wrasse (also published in Journal of Fish Biology),, he continues, “…it is possible that sexual phenotype in fish is more plastic than generally accepted or appreciated.”

Socially controlled male-to-female sex reversal in the protogynous orange-spotted grouper, Epinephelus coioides.

Socially controlled sex change in teleosts is a dramatic example of adaptive reproductive plasticity. In many cases, the occurrence of sex change is triggered by a change in the social context, such as the disappearance of the dominant individual. The orange-spotted grouper Epinephelus coioides is a typical protogynous hermaphrodite fish that changes sex from female to male and remains male throughout its life span. In this study, male-to-female sex reversal in male Epinephelus coioides was successfully induced by social isolation. The body length and mass, gonadal change, serum sex steroid hormone levels and sex-related gene expression patterns during the process of socially controlled male-to-female sex reversal in E. coioides were systematically examined. This report investigates the physiological mechanisms of the socially controlled male-to-female sex reversal process in a protogynous hermaphrodite grouper species. The results enable us to study the physiological control of sex change, not only from female to male, but also from male to female.

Effects of fish oil with difference oxidation degree on growth performance and expression abundance of antioxidant and fat metabolism genes in orange spotted grouper,Epinephelus coioides

The present study was designed to assess the effects of fish oil with different oxidation degree on growth performance, serum biochemistry parameters and expressive abundance of oxidative stress and fat metabolism genes of orange spotted grouper Epinephelus coioides. The oxidized fish oil was conducted as follows: storage temperature: 4°C, ambient temperature (AT, [31.5 ± 3.5]°C); storage time: 45, 90, 135 days; antioxidant contents: 30 mg/kg (ethoxyquin [EQ]), 300 mg/kg Higher EQ (HEQ). According to the different treated conditions, 14 kinds of fish oil with different oxidation degree were obtained: TF+EQ [positive control (fresh oil + EQ)], TF (negative control [fresh oil]), T4°C+45d+EQ, T4°C+45d+HEQ, T4°C+90d+EQ, T4°C+90d+HEQ, T4°C+135d+EQ, T4°C+135d+HEQ, TAT+45d+EQ, TAT+45d+HEQ, TAT+90d+EQ, TAT+90d+HEQ, TAT+135d+EQ, TAT+135d+HEQ. Groupers were fed isonitrogenous and isolipidic diets containing 14 kinds of fish oil for 8 weeks, respectively. The results showed that survival, weight gain rate and thermal growth coefficient decreased as oxidation degree of dietary fish oil increased (p < 0.05). Higher serum total protein, triglyceride and glucose were observed with ascending oxidation degree of fish oil (p < 0.05). The genes expression levels of catalase, superoxide dismutase and glutathione peroxidase were up-regulated with dietary oxidized level increasing (p < 0.05). In addition, the similar status also appeared in expression of peroxisome proliferator-activated receptor gamma (PPARγ), hormone-sensitive lipase (HSL) and fatty acid synthase (FAS) genes. In conclusion, the fish oil would show negative influence on the fish health until peroxide value and p-anisidine value in oil exceed 12.96 meq/kg and 20.89. The best storage condition for fish oil is 4°C, 45 days and 30 mg/kg EQ which could keep fish oil available property to grouper.

Effect of packing density on selected tissue biochemical parameters of hatchery produced fingerlings of orange spotted grouper Epinephelus coioides (Hamilton, 1822) during transportaion

&lt;p&gt;Effect of different packing densities on water quality parameters, survival and selected tissue biochemical parameters during transportation of hatchery produced fingerlings of orange spotted grouper &lt;em&gt;Epinephelus coioides&lt;/em&gt; (Hamilton, 1822) was investigated. Fingerlings (weight 3.0±0.2 g and length 6.0±0.2 cm) were packed in sealed double layered oxygen packed polythene bags (water and oxygen ratio 1:3) at different packing densities of 20, 30, 40 and 50 no. l-1. The packed fishes were transported for 6 h. After transportation, water samples and tissue samples from fishes were collected for further analyses. Levels of tissue glucose and selected metabolic enzymes (lactate dehydrogenase, LDH; aspartate amino transferase, AST and alanine amino transferase, ALT) significantly (p&amp;lt;0.05) increased with increased packing density. Water quality parameters &lt;em&gt;viz.,&lt;/em&gt; pH, dissolved oxygen, CO2, alkalinity, total ammonia nitrogen (TAN) and nitrite nitrogen (NO2-N) were also significantly different at higher packing densities (p&amp;lt;0.05). However, levels of all the tissue biochemical parameters tested were in tolerable range and no mortality of fingerlings was recorded at any of the packing densities. Though the tissue enzyme levels were significantly higher and water quality was significantly deteriorated at the highest packing density of 50 no. l-1, it did not lead to mortality of fish. Therefore, it is inferred that this density can be used for short distance transportation of fingerlings of orange spotted grouper.&lt;/p&gt;

Grouper STAT1a is involved in antiviral immune response against iridovirus and nodavirus infection

Signal Transducer and Activator of Transcription 1 (STAT1) has been demonstrated to function as a critical mediator in multiple cell processes, such as cell proliferation, cell death, and innate immune response. Interestingly, two orthologues of human STAT1, including STAT1a and STAT1b genes have been identified in different fish. However, the detailed roles of fish STAT1a in virus replication still remained largely uncertain. Here, we cloned a STAT1a from orange-spotted grouper Epinephelus coioides (EcSTAT1a) and characterized its roles during fish virus infection. EcSTAT1a encoded a 751-aa peptide which shared 97% and 93% identity to STAT1 from mandarin fish (Siniperca chuatsi) and Malabar grouper (Epinephelus malabaricus), respectively. Amino acid alignment analysis showed that EcSTAT1a contained a STAT-int domain, a STAT-alpha domain, a STAT-bind domain (DNA binding domain), a SH2 domain and a STAT1-TAZ2 bind domain. In examined tissues from healthy grouper, the expression of EcSTAT1a was predominant in intestine, gill and liver. In grouper cells, the relative expression levels of EcSTAT1a was significantly increased during red-spotted grouper nervous necrosis virus (RGNNV) or Singapore grouper iridovirus (SGIV) infection. Under fluorescence microscopy, we found that EcSTAT1a mainly localized in the cytoplasm. The ectopic expression of EcSTAT1a in vitro significantly delayed the cytopathic effect (CPE) progression evoked by RGNNV and SGIV. Further studies showed that the expression levels of viral genes, including SGIV major capsid protein (MCP), VP19, ICP-18, LITAF and RGNNV coat protein (CP), RNA-dependent RNA polymerase (RdRp) were all significantly reduced in EcSTAT1a overexpressing cells compared to the control vector transfected cells, suggested that EcSTAT1a exerted antiviral activity against iridovirus and nodavirus. Furthermore, overexpression of EcSTAT1a significantly increased the expression of interferon related cytokines or effectors and pro-inflammatory factors. Together, our results elucidated that EcSTAT1a might function as a critical antiviral factor by regulating the host interferon immune and inflammation response.