Molecular cloning,characterization and expression analysis of a C-type lectin (Ec-CTL) in orange-spotted grouper,Epinephelus coioides

C-type lectins play crucial roles in pathogen recognition, innate immunity, and cell–cell interactions. In this study, a new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-CTL was composed of 840 bp with a 651 bp open reading frame (ORF) that encodes a 216-residue protein. The deduced amino acid sequence of Ec-CTL possessed all conserved features crucial for the fundamental structure, such as the four cysteine residues (Cys⁷¹, Cys¹⁵², Cys¹⁶⁷, Cys¹⁷⁵) involved in the formation of disulphide bridges and the potential Ca²⁺/carbohydrate-binding sites. Ec-CTL contains a signal peptide and a single carbohydrate recognition domain (CRD). The genomic DNA of the gene consists of three exons and two introns. Ec-CTL showed high similarity of 54% to the C-type lectin of killifish Fundulus heteroclitus. Ec-CTL mRNA is predominately expressed in liver and skin, and lower expressed in kidney, intestine, heart, brain and spleen. The expression of Ec-CTL was differentially up-regulated in orange-spotted grouper challenged with Saccharomyces cerevisiae, Vibrio vulnificus, Staphyloccocus aureus and Singapore grouper iridovirus (SGIV). Recombinant mature Ec-CTL (rEc-CTL) was expressed in E. coli BL21, purified and characterized as a typical Ca²⁺-dependent carbohydrate-binding protein possessing hemagglutinating activity. It bound to all examined bacterial and yeast strains, and aggregated with S. cerevisiae, V. vulnificus and S. aureus in a Ca²⁺-dependent manner.     

Cloning and immune characterization of the c-type lysozyme gene in red-spotted grouper,Epinephelus akaara

Lysozyme is an important component of the innate immune response against pathogen infection. The gene coding for c-type lysozyme in red-spotted grouper Epinephelus akaara was cloned and designated EaClys. The complete cDNA contains a 432 bp open reading frame encoding a protein of 144 amino acids displaying 65–91% similarity with the amino acid sequences of human, mouse, chicken, and fish counterparts. Recombinant EaClys (rEaClys) was expressed in Escherichia coli, displayed antibacterial activity against Gram-positive and Gram-negative bacteria, and possessed bactericidal activity against Vibrio alginolyticus. EaClys mRNA was constitutively expressed in all tested E. akaara tissues, and its expression increased after pathogen challenge. Most notably, challenges with LPS, SGIV or V. alginolyticus upregulated EaClys mRNA expression in the head, kidney, and blood. Its expression peaked between 16 and 24 h after challenge before dropping back to the baseline level. By using recombinant cytokines as signaling pathway mimetics and blocking antibodies and chemical inhibitors as pathway inhibitors, we show that LPS-induced lysozyme release from macrophages is promoted by cytokines TNF-α and IL-1β, and dependent on NF-κB pathway activation. These data suggest that EaClys is a constitutive and inducible acute-phase protein that is involved in the innate immune defense of E. akaara, and provide new clues about the molecular mechanisms that regulate innate immune responses in fish.     

Identification and characterization of interferon regulatory factor-1 from orange-spotted grouper (<Emphasis Type="Italic">Epinephelus coioides</Emphasis>)

Interferon-regulatory factor 1 (IRF-1) is the first member of IRF family, which is involved in many biological processes such as immune response, antiviral defense, cell growth regulation, and apoptosis. In this study, an IRF-1 gene, EcIRF-1, was isolated and characterized from orange-spotted grouper (Epinephelus coioides). The full-length cDNA of EcIRF-1 is 1,730 bp, including an open reading frame of 906 bp, a 5′-terminal untranslated region (5′-UTR) of 153 bp, and a 3′-UTR of 671 bp. The EcIRF-1 gene consists of 10 exons and 9 introns, spanning over approximate 4.3 kb of genomic sequence. The 5′-UTR sequence contains an exon and an intron, and the 3′-UTR sequence is included in the last exon. Expression analysis by real-time PCR reveals that the EcIRF-1 gene is ubiquitously expressed in various healthy fish tissues, whereas its expression is upregulated in vivo in response to polyinosinic–polycytidylic acid or lipopolysaccharide stimulation. Subcellular localization analysis shows the EcIRF-1 is an intranuclearly localized and immobile protein in the cultured fish cells. Data presented in this paper provide an important base to further understand EcIRF-1 gene function and its regulation associated with interferon immune system in orange-spotted grouper.     

Cloning of an orange-spotted grouper Epinephelus coioides heat shock protein 90AB (HSP90AB) and characterization of its expression in response to nodavirus

The heat shock proteins (HSPs) family which consists of HSP90, HSP70, and low molecular mass HSPs are involved in chaperone activity. Here, we report the cloning and characterization of HSP90AB gene from orange-spotted grouper, Epinephelus coioides. The full-length of grouper HSP90AB was 727 amino acids and possessed an ATPase domain as well as an evolutionarily conserved molecular chaperone. The HSP90AB-green fluorescent protein fusion protein was evenly distributed in the cytoplasm. Immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) analyses indicated that the expression of grouper HSP90AB was marginally increased following nodavirus infection. Grouper E. coioides that received HSP90 inhibitor geldanamycin (GA) showed an increase in HSP90AB expression and growth of nodavirus supporting nodavirus replication.     

Effects of temperature change on the innate cellular and humoral immune responses of orange-spotted grouper Epinephelus coioides and its susceptibility to Vibrio alginolyticus

Orange-spotted grouper Epinephelus coioides held at 27 °C were then further cultured at 19, 27 (control), and 35 °C, and were examined for innate cellular and humoral responses after 3–96 h. The total leucocyte count, respiratory burst, and phagocytic activity significantly decreased 3, 48, and 96 h after fish were transferred to 19 and 35 °C. Both the alternative complement pathway (ACH₅₀) and the lysozyme activity significantly decreased at 3–96 h after fish were transferred to 19 and 35 °C. In another experiment, groupers reared at 27 °C at 34‰ salinity were injected with Vibrio alginolyticus grown in tryptic soy broth (TSB) at a dose of 2.3 × 10⁹ colony-forming units (cfu) fish^?1, and then further reared in water temperatures of 19, 27 (control), and 35 °C. The cumulative mortalities of V. alginolyticus-injected fish held in 19 and 35 °C were significantly higher than that of injected fish held in 27 °C. Resistance had decreased after 12 h for the challenged grouper held at 35 °C. All injected fish held in 19 °C had died after 72 h. It was concluded that at 12 h after transfer of grouper from 27 to 19 and 35 °C, immunity was suppressed and resistance against V. alginolyticus had decreased.     

Myostatin gene organization and nodavirus-influenced expression in orange-spotted grouper (Epinephelus coioides)

The relationship(s) between nodavirus infection and myostatin expression in the skeletal muscle tissue of grouper is unclear. To investigate, the grouper (Epinephelus coioides) myostatin gene was cloned and cDNA was utilized to examine the expression of the gene in skeletal muscle and serum of healthy (uninfected) grouper and fish naturally infected with nodavirus. The myostatin gene comprises three exons and two introns and is transcribed as a 2778-bp mRNA length that encodes a 376-aa precursor protein. All exon–intron boundaries conformed to the consensus sequences. Alignment of the upstream sequences indicated that the grouper myostatin promoter has been highly conserved during evolution. Sequence analyses of 1936 bp of the upstream region revealed ten E-box motifs. The protein was consistent with the predicted molecular weight (approximately 42 kDa) of the unprocessed monomeric precursor protein and the processed myostatin form of the protein secreted into the plasma. Transient transfection studies revealed that the activity of the myostatin promoter decreased in a subset of viral titers. Grouper naturally infected with nodavirus displayed downregulation of the myostatin protein.     

Expressed sequence tags (ESTs) based identification of genes and expression analysis of leukocyte cell-derived chemotaxin-2 (LECT2) from Epinephelus bruneus

The kelp grouper, Epinephelus bruneus, is an economically important intensively cultured species in Southeast Asia. Despite the insatiable demand its large-scale production has been hindered by problems associated with water quality, nutrition, and diseases especially due to increased rearing density. It is generally accepted that in fish both innate and adaptive immune system provide protection from diseases. In the present study a cDNA library of Streptococcus iniae-challenged kelp grouper was constructed to identify the genes that reveal molecular mechanism, physiological functions, and gene expression in different tissues using expressed sequence tags (ESTs) and RT-PCR strategy. Of a total of 2170 ESTs examined 279 (12.9%) were identified as contig and 860 (39.6%) as singletons. A total of 190 important immune and enzyme related genes (16.7%) were identified in both contig and singletons. The key immune molecules identified comprise complement factors, chemotaxin, chemokine, Fas ligand, ferritins, hepcidin, lysozyme c, MHC, and TLR which are involved in the innate or adaptive immune system. Among the genes a full-length cDNA of leukocyte cell-derived chemotaxin-2 (EbLECT2) with 540 base pair (bp) was identified; it consists of a 5′-untranslated region (UTR) of 17 bp, a 3′-UTR of 76 bp, and a stop codon TAA in 3′-UTR. The EbLECT2 is an important molecule in the innate immunity. It is a multifunctional protein involved in cell growth, differentiation, and autoimmunity. The open reading frame (ORF) of the EbLECT2 encodes with 155 amino acid (aa) residues with a predicted molecular weight and isoelectric point (pI) of 17 kDa and 9, respectively. The close phylogenetic relationship of EbLECT2 shares the highest similarity with the already reported LECT2 from Epinephelus coioides (96%) and Epinephelus akaara (94%). EbLECT2 mRNA was expressed predominantly in liver, spleen, and kidney while the expression was moderate in gills, heart, and muscle in E. bruneus after being challenged with LPS from Escherichia coli and pathogenic bacterium Vibrio anguillarum both of which involve the immune defense system. Further, the recombinant mature EbLECT2 (rEbLECT2) was successfully expressed in E. coli BL21 (DE3), and the antiserum against EbLECT2 was obtained for further investigations. The significant number of ESTs genome results obtained constitutes a powerful resource for further investigation to establish the gene discovery, functional genomic research, molecular mechanisms, and development of microarrays for the gene expression studies in kelp grouper.     

Singapore grouper iridovirus,a large DNA virus,induces nonapoptotic cell death by a cell type dependent fashion and evokes ERK signaling

Virus induced cell death, including apoptosis and nonapoptotic cell death, plays a critical role in the pathogenesis of viral diseases. Singapore grouper iridovirus (SGIV), a novel iridovirus of genus Ranavirus, causes high mortality and heavy economic losses in grouper aquaculture. Here, using fluorescence microscopy, electron microscopy and biochemical assays, we found that SGIV infection in host (grouper spleen, EAGS) cells evoked nonapoptotic programmed cell death (PCD), characterized by appearance of cytoplasmic vacuoles and distended endoplasmic reticulum, in the absence of DNA fragmentation, apoptotic bodies and caspase activation. In contrast, SGIV induced typical apoptosis in non-host (fathead minnow, FHM) cells, as evidenced by caspase activation and DNA fragmentation, suggesting that SGIV infection induced nonapoptotic cell death by a cell type dependent fashion. Furthermore, viral replication was essential for SGIV induced nonapoptotic cell death, but not for apoptosis. Notably, the disruption of mitochondrial transmembrane potential (ΔΨm) and externalization of phosphatidylserine (PS) were not detected in EAGS cells but in FHM cells after SGIV infection. Moreover, the extracellular signal-regulated kinase (ERK) signaling was involved in SGIV infection induced nonapoptotic cell death and viral replication. This is a first demonstration of ERK-mediated nonapoptotic cell death induced by a DNA virus. These findings contribute to understanding the mechanisms of iridovirus pathogenesis.     

Expression and functional characterization of a gene associated with retinoid-interferon-induced mortality 19 (GRIM-19) from orange-spotted grouper (Epinephelus coioides)

GRIM-19 is a nuclear encoded subunit of complex I that has been implicated in apoptosis. The protein participates in multiple functions including the innate immune response. GRIM-19 has been studied in humans and other mammals; however, fish GRIM-19 has not been well characterized. In this study, a new GRIM-19 gene, EcGRIM-19, was isolated from the orange-spotted grouper (Epinephelus coioides) cDNA library, which was constructed following LPS treatment. EcGRIM-19 is a 582-bp gene that encodes a 144-amino acid protein. The gene is a true ortholog of mammalian GRIM-19. EcGRIM-19 exhibits ubiquitous and constitutive expression in the different tissues of the orange-spotted grouper. The expression levels of EcGRIM-19 are altered in the gill, spleen, kidney and liver after induction with LPS. The subcellular localization analysis demonstrated that the EcGRIM-19 protein is localized predominantly in the mitochondria. In addition, amino acids 30–50 of the protein are responsible for the mitochondrial localization of EcGRIM-19. The caspase assay demonstrated that the overexpression of GRIM-19 enhanced the cellular sensitivity to interferon(IFN)-β- and retinoic acid (RA)-induced death in HeLa cells. The data presented in this study are important for further understanding the EcGRIM-19 gene function in fish.     

Draft Genome Sequence of the Fish Pathogen Vibrio harveyi Strain ZJ0603

Vibrio harveyi is an important pathogen that causes vibriosis in various aquatic organisms. Here, we announce the draft genome sequence of V. harveyi strain ZJ0603, which was isolated from diseased Orange-spotted grouper (Epinephelus coioides) in Guangdong, China.     

Orange-spotted grouper (Epinephelus coioides) orexin: molecular cloning, tissue expression, ontogeny, daily rhythm and regulation of NPY gene expression

Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4 h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro.     

Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5′ and 3′ rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709 bp and the coding sequence is flanked by a 67 bp 5′-UTR and a 358 bp 3′-UTR. The full-length cDNA sequence of S. aurata is 1599 bp, and the coding sequence is flanked by a 48 bp 5′-UTR and a 273 bp 3′-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.     

Establishment and characterization of a brain‐cell line from kelp grouper Epinephelus moara

A new brain‐cell line, EMB, was developed from kelp grouper Epinephelus moara, a cultured marine fish. The EMB cells were subcultured for more than 60 passages. The cells were cultured in Leibovitz's L‐15 medium (L15) supplemented with antibiotics, foetal bovine serum (FBS), 2‐mercaptoethanol (2‐ME) and basic fibroblast growth factor (bFGF). The cells could grow at 18–30° C, with the maximum growth between 24 and 30° C. The optimum FBS concentration for the cells growth ranged between 15 and 20%. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 45. After being transfected with pEGFP‐N3 plasmid, the cells could successfully express green fluorescence protein (GFP), implying that this cell line can be used for transgenic studies. A significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or red spotted grouper nervous necrosis virus (RGNNV) and the viral replication was confirmed by quantitative real‐time PCR (qrt‐PCR) assay, which suggested EMB's application potential for studies of SGIV and RGNNV.