Development of an inactivated iridovirus vaccine against turbot viral reddish body syndrome

Turbot(Scophthalmus maximus L.) reddish body iridovirus(TRBIV) was propagated in turbot fin cells(TF cells) and inactivated as the TRBIV vaccine with its protection efficiency evaluated in this study.TF cells were cultured in 10% bovine calf serum(BCS)-containing MEM medium(pH7.0) at 22℃,in which TRBIV propagated to a titer as high as 105.6 TCID50 mL-1.The TRBIV was inactivated with 0.1% formalin and formulated with 0.5% aluminum hydroxide.The inactivated vaccine caused neither cytopathogenic effect(CPE) on TF cells nor pathogenic effect on turbots.After being administered with the vaccine twice via muscle injection,the turbot developed high-tittered TRBIV neutralizing antibodies in a dose-dependent manner.The vaccine protected the turbot from dying with an immunoprotection rate of 83.3% as was determined via subcutaneous vaccination in the laboratory and 90.5% via bath vaccination in turbot farms,respectively.The inactivated vaccine was very immunogenic,efficiently preventing tur-bot from death.It holds the potential of being applied in aquaculture.     

Cloning and sequence analysis of a full-length cDNA of <Emphasis Type="Italic">SmPP1cb</Emphasis> encoding turbot protein phosphatase 1 beta catalytic subunit

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 （PP1） is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase l（PPlcb）, was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPPlcb, by the rapid amplification of cDNA ends （RACE） technique. The cDNA sequence of SmPPlcb we obtained contains a 984 bp open reading frame （ORF）, flanked by a complete 39 bp 5＇ untranslated region and 462 bp 3＇ untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B （PP2B）. And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPPlcb is extremely conserved in both amino acid and nucleotide acid levels compared with the PPlcb of other vertebrates and invertebrates, and its Kozak motif contained in the 5＇UTR around ATG start codon is GXXAXXGXXATGG, which is different from mammalian in two positions A6 and G3, indicating the possibility of different initiation of translation in turbot, and also the 3＇UTR of SmPPlcb is highly diverse in the sequence similarity and length compared with other animals, especially zebraf＇lsh. The cloning and sequencing of SmPPlcb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.     

Effect of stocking density on performances of juvenile turbot (Scophthalmus maximus) in recirculating aquaculture systems

Limited information has been available about the influence of loading density on the performances of Scophthalmus maximus, especially in recirculating aquaculture systems (RAS). In this study, turbot (13.84±2.74 g; average weight±SD) were reared at four different initial densities (low 0.66, medium 1.26, sub-high 2.56, high 4.00 kg/m²) for 10 weeks in RAS at 23±1°C. Final densities were 4.67, 7.25, 14.16, and 17.47 kg/m², respectively, which translate to 82, 108, 214, and 282 percent coverage of the tank bottom. Density had both negative and independent impacts on growth. The final mean weight, specific growth rate (SGR), and voluntary feed intake significantly decreased and the coefficient of variation (CV) of final body weight increased with increase in stocking density. The medium and sub-high density groups did not differ significantly in SGR, mean weight, CV, food conversion rate (FCR), feed intake, blood parameters, and digestive enzymes. The protease activities of the digestive tract at pH 7, 8.5, 9, and 10 were significantly higher for the highest density group, but tended to be lower (not significantly) at pH 4 and 8.5 for the lowest density group. The intensity of protease activity was inversely related to feed intake at the different densities. Catalase activity was higher (but not significantly) at the highest density, perhaps because high density started to induce an oxidative effect in turbot. In conclusion, turbot can be cultured in RAS at a density of less than 17.47 kg/m². With good water quality and no feed limitation, initial density between 1.26 and 2.56 kg/m² (final: 7.25 and 14.16 kg/m²) would not negatively affect the turbot cultured in RAS. For culture at higher density, multi-level feeding devices are suggested to ease feeding competition.     

Variation in the immunoglobulin levels in turbot （Scophthalmus maximus） after vaccination with Streptococcus iniae

A pathogenic bacterium(S636),identified as Streptococcus iniae,was isolated from turbot(Scophthalmus maximus) in 2005.We immunized turbot with formalin-killed S.iniae four times(on days 1,14,21,and 28) by intraperitoneal inoculation.After each vaccination,we obtained serum samples and isolated the lymphocytes from the peripheral blood,spleen,pronephros,and mesonephros.We measured surface Ig-positive(sIg+) lymphocytes and serum antibody levels from these organs using flow cytometry and enzyme-linked immunoso...     

Effects of temperature, pH and NaC1 on protease activity in digestive tract of young turbot, Scophthalmus maximus

The protease activity in digestive tract of young turbot Scophthalmus maximum was studied, and the optimal pH, temperature and NaCl concentration were determined for different portions of the fish＇s internal organs. The optimal activity in the fish＇s stomach was at pH of 2.2, while that in the intestinal extracts was within the alkaline range from 9.5 to 10.0. In hepatopancreas, the optimal pH was in low alkalinity at 8.5. The optimal reaction temperature was above 40℃ in stomach, intestine and hepatopancreas. With increasing temperature, the pH value increased in stomach, while in the intestine, an opposite tendency was observed due to combined effect of pH and temperature. NaCl concentration showed inhibitory impact on protein digestion in hepatopancreas. The main protease for protein digestion in turbot seemed to be pepsin. Moreover, the maximum protease activity in different segments of intestine existed in the hindgut.     

Effects of temperature, pH and NaCl on protease activity in digestive tract of young turbot, Scophthalmus maximus

1 INTRODUCTION Digestive enzyme activity is one of important issues for learning digestive physiology of fish, and widely applied in commercial fish culture. So far, the data obtained in fish showed that the diges- tive enzymes were qualitatively similar …     

Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXXATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.     

Characterization of Turbot(Scophthalmus maximus) Skin and the Extracted Acid-Soluble Collagen

The biochemical composition of the turbot skin was investigated. The moisture level of the skin was found to be 51.4%. Based on dry matter content, there were relatively high protein(82.1%) and lipid(13.1%) concentrations in the turbot skin. Mineral element analysis revealed that the turbot skin had high Ca content(2069.0 mg kg^-1), and the concentrations of toxic heavy metals Hg and Pb were less than 0.005 mg kg^-1, which indicates that the turbot skin is a safe resource for collagen production. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis showed that acid-soluble collagen(ASC) extracted from the turbot skin was type-I collagen. The imino acid content of the ASC was 241.6 per 1000 amino acids, which suggests a relatively high denaturation temperature. The Fourier transform infrared(FTIR) spectra of ASC reflected a highly stable structure, and the measured denaturation temperature of ASC was 29.5℃, which is higher than those from many temperate fishes. ASC was the most soluble at pH 4.0, and when the pH values were below or above 4.0, the solubility decreased rapidly. The ASC exhibited a relatively high solubility when NaCl concentration was lower than 2%. These results show that turbo skin can be employed as a source for producing collagen with high quality.     

Distribution and ultrastructure of pigment cells in the skins of normal and albino adult turbot, Scophthalmus Maximus

The distribution and ultrastructure of pigment cells in skins of normal and albino adult turbots were examined with transmission electron microscopy (TEM). Three types of pigment cells of melanophore, iridophore and xanthophore have been recognized in adult turbot skins. The skin color depends mainly on the amount and distribution of melanophore and iridophore, as xanthophore is quite rare. No pigment cells can be found in the epidermis of the skins. In the pigmented ocular skin of the turbot, melanophore and iridophore are usually co-localized in the dermis. This is quite different from the distribution in larvae skin. In albino and white blind skins of adult turbots, however, only iridophore monolayer still exists, while the melanophore monolayer disappears. This cytological evidence explains why the albino adult turbot, unlike its larvae, could never resume its body color no matter what environmental and nutritional conditions were provided. Endocytosis is quite active in the cellular membrane of the iridophore. This might be related to the formation of reflective platelet and stability of the iridophore.     

The eco-nutrition requirements for dietary protein and its rhomb characteristics in juvenile turbot (Scophthalmus maximus L.)

We evaluated the dietary protein requirements of juvenile turbot (Scophthalmus maximus L.) and their effects on aquatic quality. Five experimental diets were formulated containing 450, 480, 500, 520, and 540 g/kg. Each diet was randomly assigned to triplicate groups of juvenile turbot (mean initial body weight 34.5 ± 5.5 g) for 88 d. Both the weight gain ratio and feed efficiency increased with increasing dietary protein up to 500 g/kg, but no further improvement was detected when dietary protein levels were >500 g/kg. Protein intake and digestion increased with protein levels, while fecal nitrogen and nitrogen content in seawater increased only when dietary protein exceeded 500 g/kg. Protein digestibility was highest at intermediate dietary protein levels. Chemical oxygen demand, nitrite-nitrogen (NO2--N) and phosphatic-phosphor (PO43--P) levels increased in the rearing water as dietary protein levels increased. The optimum eco-nutrition level of dietary protein for juvenile turbot was 500 g/kg under the current experimental conditions. The diets containing 540 and 500 g/kg protein had similar growth rates and feed conversion ratios, but levels of ammonia (NH4+) and nitrogen were considerably higher in the water and feces, respectively, at the higher level of dietary protein. The difference in the pattern of change between body weight gain and ammonia concentration in water with increasing dietary protein is described by rhomb characteristics.     

Turbot Scophthalmus maximus：stocking density on growth, pigmentation and feed conversion

1 INTRODUCTION Turbot is a type of marine flatfish naturally distributing along the coastal area of European. It is highly demanded for good taste. The price therefore, is very expensive. To meet the consump- tion, large-scale turbot cultures were perform…     

Cloning, Expression and Characterization of Translationally Controlled Tumor Protein （TCTP） Gene from Flatfish Turbot （Scophthalmus maximus）

A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 451 bp and an open reading flame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5'UTR of SmTCTP started with a 5'-terminal oligopyrimidine tract (5'-TOP), a typical feature for translationaily controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known verte-brate TCTPs in a range of 58.8% to 64.1%. The length offish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch (Lateolabrax japonicus) is 170 aa in length, while that of zebrafish (Danio rer/o) and rohu (Labeo rohita) is 171 aa in length. North-ern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further inves-tigation of the biological function of TCTP in fish.     

Enhancing the culturability of bacteria from the gastrointestinal tract of farmed adult turbot Scophthalmus maximus

Eighteen agar media were tested for the culture of gut-associated bacteria from farmed adult turbot(Scophthalmus maximus),including 16 agar media with or without 1% gastrointestinal(GI)supernatant,or with 2% or 4% GI supernatant.A total of 1 711 colonies were analyzed and 24 operational taxonomic units(OTUs)were identifi ed.The greatest bacterial diversity was isolated on Zobell 2216E/Zobell 2216E+ agar media,whereas MRS/MRS+ agar media produced a low diversity of colonies.Agar media with GI supernatant(1%,2%,or 4%)showed increased diversity and yielded different profi les of OTUs from the corresponding original media,suggesting that GI supernatant provides substances that enhance the culture effi ciency of bacteria from the turbot GI tract.The large majority of the colonies(82%)were γ-Proteobacteria,whereas 15.6% and 2.4% of colonies were Firmicutes and Actinobacteria,respectively.At the genus level,49.4% of all colonies were assigned to Vibrio.Other potential pathogens,including Pseudomonas,Photobacterium,and Enterobacter,and potential probiotics,including Bacillus,Paenibacillus,and Pseudomonas,were also isolated on agar media.Most cultured bacteria belonged to species that were fi rst described in the turbot GI tract.The impact of these species on turbot physiology and health should be investigated further.     

Proline with or without Hydroxyproline Influences Collagen Concentration and Regulates Prolyl 4-Hydroxylase α(I) Gene Expression in Juvenile Turbot(Scophthalmus maximus L.)

This study was conducted to investigate the effect of dietary proline(Pro), and Pro and hydroxyproline(Hyp) in combination on the growth performance, total Hyp and collagen concentrations of tissues, and prolyl 4-hydroxylase α(I)(P4H α(I)) gene expression in juvenile turbot feeding high plant protein diets. A diet containing 50% crude protein and 12% crude lipid was formulated as the basal and control, on which other two protein and lipid contents identical experimental diets were formulated by supplementing the basal with either 0.75% Pro(Pro-0.75) or 0.75% Pro and 0.75% Hyp(Pro+Hyp). Four groups of fish in indoor seawater recirculating systems, 35 individuals each, were fed twice a day to apparent satiation for 10 weeks. The results showed that dietary Pro and Hyp supplementation had no significant effect on growth performance and feed utilization of juvenile turbot(P 0.05). Total Hyp and collagen concentrations in muscle were significantly increased when dietary Pro and Hyp increased(P 0.05), and fish fed diet Pro+Hyp showed significantly higher free Hyp content in plasma than those fed other diets(P 0.05). The expression of P4 H α(I) gene in liver and muscle was significantly up regulated in fish fed diet Pro-0.75 in comparison with control(P 0.05); however the gene was significantly down regulated in fish fed diet Pro+Hyp in muscle in comparison with fish fed diet Pro-0.75(P 0.05). It can be concluded that supplement of crystal L-Pro and L-Hyp to high plant protein diets did not show positive effects on growth performance of juvenile turbot, but enhanced total collagen concentrations in muscle.