Rock Bream Research Articles

Characterization and expression analysis of a goose-type lysozyme from the rock bream Oplegnathus fasciatus, and antimicrobial activity of its recombinant protein

Lysozyme (muramidase) represents an important defense molecule of the fish innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of β-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of a g-type lysozyme (RBgLyz) was identified in the Oplegnathus fasciatus rock bream fish genome by means of multi-tissue normalized cDNA pyrosequencing using Roche 454 GS-FLX™ technology. RBgLyz is composed of 669 bp, with a 567 bp open reading frame that encodes 188 amino acids. Protein motif searches indicated that RBgLyz contains the soluble lytic transglycosylase domain involved in maintaining cell wall integrity. Furthermore, RBgLyz shares significant identity (81.4%) with Chinese perch Siniperca chuatsi. Quantitative real-time RT-PCR analysis results showed that RBgLyz transcripts are constitutively expressed in various tissues from healthy rock breams. In order to determine RBgLyz function in immunity, its expression was analyzed in head kidney following exposure to known immune stimulants or pathogens. RBgLyz transcripts were significantly up-regulated in response to challenge with lipopolysaccharide (LPS) and Edwardsiella tarda, as compared to non-injected control fish. Polyinosinic:polycytidylic acid (poly I:C) dsRNA stimulated a moderate expression of RBgLyz, as did Streptococcus iniae but to a lesser extent. There were no specific time-dependent effects on RBgLyz mRNA expression observed in response to rock bream iridovirus (RBIV) infection. Taken together, the gene expression results indicated that g-type lysozyme plays a role in the innate immune response to LPS, poly I:C, E. tarda and S. iniae in rock bream. Thus, we generated recombinant RBgLyz in an Escherichia coli expression system and characterized its antimicrobial activity. Our results indicated that recombinant RBgLyz had lytic activity against Gram-negative Vibrio salmonicida, Gram-positive Listeria monocytogenes, S. iniae and Micrococcus lysodeikticus. In addition, observations by scanning electron microscope (SEM) confirmed that the cell morphology of M. lysodeikticus was altered in the presence of recombinant RBgLyz.

Characterization and expression analysis of the myeloid differentiation factor 88 (MyD88) in rock bream Oplegnathus fasciatus

Myeloid differentiation factor 88 (MyD88) is a universal adaptor protein able to activate nuclear factor-kappa B (NF-κB) through interactions with interleukin-1 receptor (IL-1R) and the Toll-like receptors (TLRs), with the exception of TLR3. Here, we describe the identification of MyD88 from the rock bream fish Oplegnathus fasciatus and its characterization based on GS-FLX™ sequencing. The cDNA of rock bream MyD88 was found to be composed of 1626bp, with an 867bp open reading frame that encodes 288 amino acids. The deduced amino acid sequence of MyD88 possessed both a conserved death domain at the amino terminus and a typical Toll-IL-1 receptor (TIR) domain at the carboxyl terminus, similar to that found in other fishes, amphibians, avians, mammals and invertebrates. The mRNA expression pattern of MyD88 in healthy and bacterially challenged rock bream were examined using quantitative real-time polymerase chain reaction (qRT-PCR). MyD88 transcripts were found to be strongly expressed in blood, gill, liver, spleen, head kidney and kidney, moderately expressed in skin, brain and intestine, and weakly expressed in muscle. Expression levels of MyD88 in blood, spleen and head kidney were dramatically up-regulated upon exposure to LPS and the Gram-negative bacteria Edwardsiella tarda, suggesting that MyD88 plays an important role in rock bream defenses against bacterial infection.

RETRACTED: Molecular characterization and expression analysis of lily type lectin-1 (OfLTL-1) in rock bream ( Oplegnathus fasciatus)

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Galectin-1 (OfGLec-1) from rock bream ( Oplegnathus fasciatus): Identification, characterization, gene expression and functional properties

Lectins may function as pattern-recognition receptors (PRRs) and play important roles in immune responses. In this study, a novel galectin-1 (designated OfGLec-1) gene was identified from rock bream, Oplegnathus fasciatus, cDNA library. The complete cDNA sequence of the OfGLec-1 gene is 993 base pairs in length, and encodes 135 amino acids. OfGLec-1 shares a highly conserved carbohydrate recognition domain with galectin-1 from other fishes. The mRNA expressions of OfGLec-1 in healthy and bacterial and viral challenged rock breams were examined using quantitative real time polymerase chain reaction (qRT-PCR). OfGLec-1 is highly expressed in head kidney and other tissues of the rock bream. The expression is strongly up-regulated after bacterial and viral challenge. To understand its biological activity, the recombinant OfGLec-1 gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant OfGLec-1 specifically agglutinated including E. coli, Vibrio alginolyticus and Edwardsiella tarda at a concentration of 50 μg/mL in a calcium independent manner. Both the gene expression analysis and the recombinant protein activity assay suggest that OfGLec-1 is an important immune gene involved in the recognition and elimination of pathogens in rock breams.

Heparin cofactor II (RbHCII) from rock bream ( Oplegnathus fasciatus): Molecular characterization, cloning and expression analysis

Heparin cofactor (HCII) is a serine protease inhibitor (SPI), and plays important physiological roles in various biological events including hemostasis. The gene encoding the HCII was isolated from GS-FLX™ genomic data of rock bream ( Oplegnathus fasciatus), designated as RbHCII. The RbHCII (1950 bp) consists of a 1512 bp open reading frame (ORF) encoding 504 amino acids (aa), with a signal peptide of 19 aa residues. The predicted molecular mass and the estimated isoelectric point of RbHCII were 58 kDa and 5.9, respectively. The deduced aa sequence of RbHCII displayed a characteristic serpin domain and a serpin signature motif (FTVDQPFLFLI). RbHCII demonstrated homology with vertebrate HCIIs and the greatest degree of similarity (90.1%) was observed with Gasterosteus aculeatus HCII. Various functional domains including the reactive center loop (RCL), glycosaminoglycan (GAG) and thrombin binding sites and acidic repeats of human and RbHCII were found to be orthologs through the molecular modeling studies. Phylogenetic analysis revealed that RbHCII belongs to the clade D serpins, and is closely related to the clade A members. Constitutive expression of RbHCII mRNA was detected at different levels in various tissues in a tissue-specific manner. Interestingly, RbHCII transcription was significantly downregulated ( p < 0.05) in liver after challenge with lipopolysaccharide (LPS), Edwardsiella tarda and rock bream iridovirus (RBIV). However, after the immune challenges, RbHCII showed a significant downregulation in blood tissue only at the late-phase of investigation. The recombinant RbHCII (rRbHCII) was overexpressed in Rosetta-gami (DE3) cells and purified using the pMAL™ system. The rRbHCII inhibited thrombin and chymotrypsin in a dose-dependent manner. Remarkably, heparin was found to be an enhancer of RbHCII’s thrombin-inhibitory activity. Correlating the heparin-dependent thrombin-inhibition activity of RbHCII with its temporal downregulation against immune stimulants, it could be suggested that it is not only involved in the blood coagulation cascade, but also plays an incognito role in immune modulation.

WITHDRAWN: A mannose binding lily type lectin-3 (OfLTL-3) from rock bream (Oplegnathus fasciatus): Characterization, cloning and gene expression.

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Complete genome sequence of a Megalocytivirus (family Iridoviridae) associated with turbot mortality in China

BackgroundTurbot reddish body iridovirus (TRBIV) causes serious systemic diseases with high mortality in the cultured turbot, Scophthalmus maximus. We here sequenced and analyzed the complete genome of TRBIV, which was identified in Shandong province, China.ResultsThe genome of TRBIV is a linear double-stranded DNA of 110,104 base pairs, comprising 55% G + C. Total 115 open reading frames were identified, encoding polypeptides ranging from 40 to 1168 amino acids. Amino acid sequences analysis revealed that 39 of the 115 potential gene products of TRBIV show significant homology to other iridovirus proteins. Phylogenetic analysis of conserved genes indicated that TRBIV is closely related to infectious spleen and kidney necrosis virus (ISKNV), rock bream iridovirus (RBIV), orange-spotted grouper iridovirus (OSGIV), and large yellow croaker iridovirus (LYCIV). The results indicated that TRBIV belongs to the genus Megalocytivirus (family Iridoviridae).ConclusionsThe determination of the genome of TRBIV will provide useful information for comparative study of Megalocytivirus and developing strategies to control outbreaks of TRBIV-induced disease.

Identification of the VP92R gene from infectious spleen and kidney necrosis virus

Megalocytiviruses, which belong to the family Iridoviridae, are among the most harmful viruses to cultured fishes. Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus. The genome of ISKNV was sequenced and annotated by He et al. (Virology 291:126-139, 2001) in 2001 and re-annotated by Eaton et al. (Virol. J. 4:11, 2007) in 2007. The ORF092R of ISKNV was identified in the annotations of He et al. but was not reported by Eaton et al. In this study, by using Northern-blot and RACE assays, we identified the ORF092R transcript, indicating that ORF092R was indeed transcribed in the ISKNV-infected cells. Immunofluorescence and Western-blot showed that VP92R protein was expressed in the ISKNV-infected cells, and the molecular mass of VP92R is consistent with the theoretical one. Sequence comparison demonstrated that the VP92R orthologous protein is present in large yellow croaker iridovirus (LYCIV), but not in rock bream iridovirus (RBIV) or orange-spotted grouper iridovirus (OSGIV). Therefore, VP92R may have specific functions during ISKNV pathogenesis and could be a subject for studying the differences between megalocytiviruses.

Long Double-stranded RNA Induces Sequence-specific RNA Interference and Type I Interferon Responses in Rock Bream (Oplegnathus fasciatus)

To determine whether long double-stranded RNA (dsRNA) induces RNA interference and type I interferon (IFN) responses in fish, long dsRNAs encoding enhanced green fluorescent protein (EGFP), GFPuv, and polyinosinic-polycytidylic acid sequences were co-injected with an EGFP expressing plasmid, into rock bream (Oplegnathus fasciatus). We investigated the EGFP mRNA and protein levels, and the transcriptional responses of dsRNA-dependent protein kinase and Mx1 genes. Long dsRNAs were strong inducers of a type I IFN response in rock bream, resulting in nonspecific suppression of exogenous gene expression. Furthermore, sequence-specific knockdown of exogenous gene expression at the mRNA level was detected at an early phase (24 h). These results suggested that long dsRNA may inhibit exogenous gene expression through an early mRNA interference response and a later type I IFN response in fish.

시중 유통 자연산 및 양식산 활어의 항생제 잔류

Wild and cultured fish including olive flounder, sea bass, rock bream, yellowtail, gray mullet, gizzard shad, black rockfish, red seabream and squid were collected from a fish market located on the coast of Korea, and the antibiotic content of their muscle was investigated. Tetracycline group antibiotics were not detected in the 108 individuals of 9 species of wild fish. However, oxytetracycline (OTC) and tetracycline(TC) were detected in some samples of the 111 individuals in 7 cultured live fish species. The detected ranges of OTC and TC were ND~ 0.06 and ND~ 0.03, respectively. Five different fluoroquinolone antibiotics were also tested for, but were not detected in the wild fish species. Only small amount of criprofloxacin(ND~0.029 mg/kg) were detected in a few cultured fish samples. Oxolinic acid was not detected in either wild and cultured fish samples. Results showed that even very low levels of antibiotics could be detected by the testing methods used. Antibiotics were identified in a few fish samples but levels were far below the maximum allowable limits of the Korean Food Code, and the safety of fish being sold in markets, with regard to antibiotic levels, was confirmed.

Genetic Phylogeny among Three Species Red Seabream, Black Seabream and Rock Bream Based on Mitochondrial DNA Sequences

The Perciformes include approximately 40% of all bony fishes and are the largest order of vertebrates. This order includes some of the most economically relevant marine fishes, particularly the red seabream, black seabream and rock bream. A 409 bp fragment of the cytochrome b (cyt b) gene and 403 bp and 518 bp fragments of ribosomal RNA (12S and 16S rRNA, respectively) were sequenced from five populations of natural and cultured red seabreams, natural black seabream, and natural and cultured rock breams. The mitochondrial DNA sequences were utilized for the genetic identification and population structural analyses of these three species. Phylogenetic relationships of intra- and inter-species were elucidated using three types of molecular genetic markers from three species of the order Perciformes in Korea. We noted no significant differences in the intra-specific variation of the cyt b and rRNA genes in each population however, inter-specific divergences were greater than intra-specific variation. Inter-specific variation was induced more by transition than transversion type in the cyt b and rRNA genes. The cyt b gene and rRNA genes make it possible to determine the inter-species divergence. The rRNA genes have more conserved sequences than the cyt b gene. Therefore, these genes are expected to prove useful among species belonging to the different genera or families.

Molecular cloning and expression analysis of double-stranded RNA-dependent protein kinase (PKR) in rock bream ( Oplegnathus fasciatus)

We have cloned and characterized rock bream double-stranded RNA-dependent protein kinase (PKR), which is a key component of type I IFN inducible innate immune system. Full-length rock bream PKR cDNA consists of 2115 bp ORF encoding 704 amino acids, 124 bp 5′ UTR, and 529 bp 3′ UTR. Conserved domain analysis revealed that rock bream PKR contained two tandem dsRBM and kinase domain consisted of 11 kinase sub-motifs, which are characteristics found in other PKRs. Rock bream PKR was constitutively expressed in the spleen of rock bream and, upon injection of poly I:C, up-regulated not later than 12 h post injection and returned to baseline level at 48 h post injection. Although the eIF2α kinase activity of rock bream PKR was not examined in the present study, dsRNA inducible nature of rock bream PKR implies the possible important role of this gene in innate immune system of rock bream as suggested in zebrafish and flounder.

Development of siRNA expression vector utilizing rock bream β-actin promoter: a potential therapeutic tool against viral infection in fish

In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream beta-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream beta-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream beta-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.

Molecular cloning of rock bream ( Oplegnathus fasciatus) tumor necrosis factor-α and its effect on the respiratory burst activity of phagocytes

Rock bream ( Oplegnathus fasciatus) tumor necrosis factor-α (rbTNF-α) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-α on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-α was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-α gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-α, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-α was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-α might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.

Influence of temperature shifts on the onset and development of red sea bream iridoviral disease in rock bream Oplegnathus fasciatus

The effects of various water temperature treatments on the development of red sea bream iridovirus disease (RSIVD) in rock bream Oplegnathus fasciatus challenged with iridovirus Sachun (IVS-1) were determined by measuring the mortality and the viral concentration in the spleen of infected fish. Experimental infections of rock bream with IVS-1 at water temperatures of 18, 21, and 25 degrees C resulted in a cumulative mortality of 100%, but infections at 13 degrees C resulted in 0% mortality, even after 45 d. The disease progressed more rapidly at higher water temperatures; at 25, 21, and 18 degrees C, the mean numbers of days until death were 17, 20, and 30 d, respectively. When the water temperature for fish infected with iridovirus by intramuscular injection was shifted from 13 to 25 degrees C, the cumulative mortality reached 100%, with rapid onset of the disease, independent of the time at which the temperature was shifted, i.e. 7, 14, or 30 d after injection at 13 degrees C. Real-time PCR data revealed that the viral genome copy number in the spleen of rock bream maintained at 13 degrees C increased with time, suggesting the occurrence of viral replication even at 13 degrees C. In the reverse experiment, when the water temperature for fish that were infected at a higher temperature was shifted to 13 degrees C, 3 or 7 d after injection at 25 degrees C, the fish showed 100% cumulative mortality, although the mean number of days until death was higher than that observed for fish maintained at a constant temperature of 25 degrees C. The viral DNA concentration in the spleen of rock bream that had been shifted down to 13 degrees C, 3 or 7 d after injection at 25 degrees C, was not suppressed, but increased and eventually reached levels sufficient to induce mortality at 13 degrees C. However, the level of viral genome copy numbers in the spleen of dead fish at 25 degrees C, regardless of whether those fish were held at a constant temperature of 25 degrees C or shifted up from 13 degrees C, appeared to be greater than the level found in the dead fish shifted down to 13 degrees C after inoculation at 25 degrees C.

Effect of Starvation on Kidney Melano-macrophage Centre in Sub-adult Rock Bream, Oplegnathus fasciatus (Temminck and Schlegel)

We conducted a histological analysis to investigate the influence of nutritional changes on melano-macrophages (MMs) accumulation in the kidney of sub-adult rock bream (Oplegnathus fasciatus). Four experimental groups were established (initial control, control, fed and starved), and fed commercial feed amounting to 1-3% of their body weight for 2 weeks prior to initiation of experiments. Kidney MMs with dark brown pigment were randomly observed in the kidneys of starved fish, increasing rapidly after 4 weeks, while deposition levels remained low throughout the experiment in the control and fed groups. These results suggest that catabolic tissue breakdown is a major factor contributing to the formation of pigments within MMs. Results also suggest that the degree of MMs deposition in the kidney can be used as alternative indicators in identifying starvation in wild and cultured rock bream.

Efficacy and physiological responses of rock bream, Oplegnathus fasciatus to anesthetization with clove oil

We tested the efficacy (e.g., induction time, recovery time) of clove oil as an anesthetic for rock bream Oplegnathus fasciatus. In addition, we also evaluated the physiological response of fish to the anesthetic by measuring plasma cortisol and glucose. In general, fish exposed to higher anesthetic doses were rapidly induced but took longer to recover, while lower water temperatures resulted in longer induction and recovery times. Optimal anesthetic dose and water temperature were estimated to be 150 mgL − 1 at 20 °C, 100 mgL − 1 to 125 mgL − 1 at 24 °C, and 50 mgL − 1 to 75 mgL − 1 at 28 °C. Following the administration of 100 mgL − 1 of clove oil at 24 °C, the plasma cortisol level was highest (1.70 ± 0.148 μg/dL) after 1 h while the plasma glucose level was highest (80.0 ± 1.41 mg/dL) after 2 h. It took 2 days for the plasma cortisol and plasma glucose concentrations to return to pre-exposure levels.

Transmission of iridovirus from freshwater ornamental fish (pearl gourami) to marine fish (rock bream)

Freshwater pearl gourami Trichogaster leeri and seawater rock bream Oplegnathus fasciatus infected by the iridoviruses PGIV-SP and IVS-1 were carrying similar numbers of viral particles (2.52 x 10(8) and 2.46 x 10(8) viral genome copies mg(-1) spleen tissue, respectively). The viral genome copy number for both iridoviruses decreased much faster in seawater than in freshwater, reaching a concentration of less than 0.5%, versus 26 to 54% in freshwater, after 4 d of incubation at 25 degrees C. The decrease in copy number altered the infectivity of the viruses, as reflected by the decreased cumulative mortality of rock bream injected intraperitoneally with the incubated iridoviruses. Moreover, uninfected rock bream cohabitated with PGIV-SP-challenged rock bream showed 100% cumulative mortality; a similar experiment using IVS-1 had the same result, implying the potential for iridoviral transmission from freshwater ornamental fish to marine fish even in a marine environment. Of 58 outwardly healthy marine fish groups collected from various markets, 2 rock bream groups and 1 sea perch group Lateolabrax sp. tested positive for PGIV-SP by 2-step polymerase chain reaction (PCR). Thus, PGIV-SP from freshwater ornamental fish may have crossed both environmental and species barriers to infect marine fish such as rock bream.

Molecular cloning and expression analysis of three Mx isoforms of rock bream, Oplegnathus fasciatus

Complementary DNAs (cDNAs) corresponding to three isoforms of rock bream ( Oplegnathus fasciatus) Mx (RbMx1, RbMx2 and RbMx3) were cloned using RACE reactions. Analysis of deduced amino acid sequences revealed that the tripartite GTP-binding domain, the dynamine family signature and the leucine zipper repeat were present in all three rock bream Mx isoforms. Cloning of genomic DNA sequence and Southern blot analysis showed that three rock bream Mx isoforms were encoded by different genomic loci, and they were not alternative splicing variants, although some alternative splicing variants were found in RbMx1 and RbMx2. When comparing amino acid sequence identity, RbMx1 shares about 60–70% identities with other fish Mx proteins, whereas both RbMx2 and RbMx3 share slightly high identity of 70–90%. As a result of expression analysis using RT-PCR, RbMx1 was constitutively expressed in the spleen and kidney of rock bream yearling, but RbMx2 and RbMx3 were rarely detected in both organs. When injected with synthetic double-stranded RNA polyinosinic:polycytidylic acid (poly I:C), expression of all rock bream Mx isoforms was up-regulated in spleen and head kidney. RbMx1 was continuously up-regulated throughout experimental period of 72 h but RbMx2 and RbMx3 were down-regulated to almost non-detectable level at 48 h post-injection.

Functional characterization of the RNase III gene of rock bream iridovirus

All of the fully sequenced iridoviruses have an ORF resembling a putative RNase III gene. However, to the best of our knowledge, functional characterization of the iridovirus-encoded RNase III has not been done. In the present study, we have characterized the putative RNase III of rock bream iridovirus (RBIV), the major cause of mass mortality of cultured rock bream Oplegnathus fasciatus in Korea. RBIV RNase III has a single N-terminal endonuclease domain followed by a C-terminal double-stranded RNA (dsRNA) binding domain. The true presence of the predicted ORF encoding RNase III in RBIV was confirmed by temporal transcription analysis of the ORF in RBIV-infected grunt fin (GF) cells. Comparing the catalytic activity to that of previously reported RNase III proteins, including Escherichia coli RNase III, the present RBIV RNase III had different features in that: (1) the dsRNA substrate was cleaved by the RBIV RNase III at high concentrations of Mg(2+) (5-20 mM) at low salt concentration (50 mM), but the enzyme activity was completely inhibited at 200 mM NaCl (within physiological ranges) irrespective of Mg(2+) concentrations (0.5-20 mM); (2) the substrate dsRNA was cleaved at low concentrations of Mn(2+) (0.5-1 mM) at low salt concentration (50 mM) and was cleaved by increasing Mn(2+) (5-20 mM) at 200 mM salt. These features of RBIV RNase III are similar to E. coli RNase III devoid of the C-terminal dsRBD region. The exact role of the RNase III in RBIV replication is not known, and further studies are needed to elucidate whether the RNase III is involved in the suppression of host RNA interference, which attacks viral mRNAs, or in the processing of viral RNAs for effective replication.