Low temperature stress on the hematological parameters and <Emphasis Type="Italic">HSP</Emphasis> gene expression in the turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis>

To study the effect of low temperature stress on hematological parameters and HSP gene expression in the turbot (Scophthalmus maximus), water temperature was lowered rapidly from 18 to 1°C. During the cooling process, three individuals were removed from culture tanks at 18, 13, 8, 5, 3, and 1°C. Blood samples and tissues were taken from each individual, hematological indices and HSP gene expression in tissues were measured. The red blood cell count, white blood cell count, and hemoglobin concentration decreased significantly (P < 0.05) as temperature decreased. Enzyme activities of plasma alanine transaminase and creatine kinase increased as temperature decreased, whereas aspartic transaminase and γ-glutamyl transpeptidase activities displayed no obvious changes above 1°C and lactate dehydrogenase activity increased first and then decreased. Blood urea nitrogen and uric acid levels were highest at 8°C, and creatinine concentration was highest at 3°C. The concentrations of plasma cortisol, cholesterol, and triglyceride all increased significantly (P < 0.05) as temperature decreased. The serum glucose concentration increased first and then decreased to the initial level. The HSP70 mRNA expression showed various patterns in different tissues, whereas HSP90 mRNA expression showed the same tendency in all tissues. Overall, these results indicate that temperature decreases in the range of 8 to 5°C may induce a stress response in S. maximus and that temperature should be kept above 8°C in the aquaculture setting to avoid damage to the fish.     

Estimation of genetic parameters for upper thermal tolerances and growth-related traits in turbot Scophthalmus maximus

An abnormally high temperature produces a stress response in turbot causing large economic losses in the turbot aquaculture industry of China.A genetic improvement of the upper thermal tolerance(UTT)of turbot could allow cultured fish to adapt.A genetic evaluation of UTT is required for determining the practicability of including this trait into a breeding program.In this study,data were recorded from a temperature tolerance test conducted on 3 200 individual turbots from 32 full-sib groups.A cross-sectional linear model and a cross-sectional threshold probit model were used to analyze the test-period survival and a cross-sectional threshold logit model was used to analyze the test-day survival.In addition,phenotypic and genetic correlations between body weight and survival data were estimated.The estimated heritability values obtained from the cross-sectional linear model(CSL),the cross-sectional threshold(probit)model(THRp),and the cross-sectional threshold(logit)model(THR1)were 0.247 9±0.108 3,0.288 3±0.161 2,and 0.106 9±0.045 2,respectively.The correlation coefficients among the full-sib family estimated breeding values(EBVs)obtained from the three models were greater than 0.998 6 and all models produced an almost identical family ranking.The accuracies of selection obtained with the CSL,THRp,and THR1 model were0.773 8,0.775 4,and 0.784 4,respectively,the greatest from the THR1 model.The genetic correlations between body weight and survival data EBVs from the CSL,THRp,and THR1 models were 0.020 1,-6.201 1 ×10-4,and-3.115 4×10-4,respectively,and the phenotypic correlations between the two traits were-0.837 1 and-0.667 1,respectively.The findings of this study provide background information to determine the best strategy of selection for the genetic improvement of UTT in turbot.     

Interaction of temperature and salinity on the expression of immunity factors in different tissues of juvenile turbot Scophthalmus maximus based on response surface methodology

Central Composite Design(CCD) and response surface methodology were used in the experiment to examine the combined effect of temperature(16-28℃) and salinity(18-42) on Hsp70 and IgM genes expression levels in turbot(Scophthalmus maximus) liver and kidney.The results showed that the coefficients of determination(R~2=0.965 2 for liver Hsp70,0.972 9 for kidney Hsp70,0.921 for liver IgM and 0.962 1 for kidney IgM) and probability values(P0.01) were significant for the regression model.The interactive effect between temperature and salinity on liver Hsp70,kidney Hsp70 and liver IgM were not significant(P0.05),while the interactive effect between temperature and salinity on kidney IgM was significant(P0.01).The model equation could be used in practice for forecasting Hsp70 and IgM genes expression levels in the liver and kidney of juvenile turbot via applying statistical optimization of the response of interest,at which the maximum liver Hsp70,kidney Hsp70,liver IgM and kidney IgM of1.48,1.49,2.48,and 1.38,respectively,were reached.The present model may be valuable in assessing the feasibility of turbot farming at different geographic locations and,furthermore,could be a useful reference for scientists studying the immunity of turbot.     

Heritability and genetic correlation of survival in turbot(Scophthalmus maximus)

We analyzed the survival data of the offspring from 21 sires and 42 dams of turbot.The results show that the cumulative survival rates for turbot from 2 to 18 months range from 17.5% to 28.5%;main mortality occurred during months 2-5;and the highest survival rates of families were 97.9%,98.8%,99.4%,99.7% during months 2-5,5-6,6-8,8-11,and 11-18,respectively,and 99.5%,being 53.5%,23.8%,19.5%,14.9%,and 13.2% higher,respectively,than the mean values in each period.In all periods,the estimated heritabilities for survival were very low without significant difference from zero(P0.05)(values ranged from 0.06 to 0.12),indicating low additive genetic effects.The genetic correlations of survival among families in different periods were all positive,but low in magnitude(values range from 0.03 to 0.31).Genetic correlations between long-term survival and other periods' survival had negative values(-0.06 and-0.15) and three positive values(0.16,0.12 and 0.14).Genetic correlations between survival and weight were all positive,except for survival at months 2-5 and weight at 18 months,which was not significantly negative(-0.18).     

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.     

Heritability and genetic correlation of survival in turbot (Scophthalmus maximus)

We analyzed the survival data of the offspring from 21 sires and 42 dams of turbot. The results show that the cumulative survival rates for turbot from 2 to 18 months range from 17.5% to 28.5%; main mortality occurred during months 2–5; and the highest survival rates of families were 97.9%, 98.8%, 99.4%, 99.7% during months 2–5, 5–6, 6–8, 8–11, and 11–18, respectively, and 99.5%, being 53.5%, 23.8%, 19.5%, 14.9%, and 13.2% higher, respectively, than the mean values in each period. In all periods, the estimated heritabilities for survival were very low without significant difference from zero (P≫0.05) (values ranged from 0.06 to 0.12), indicating low additive genetic effects. The genetic correlations of survival among families in different periods were all positive, but low in magnitude (values range from 0.03 to 0.31). Genetic correlations between long-term survival and other periods’ survival had negative values (−0.06 and −0.15) and three positive values (0.16, 0.12 and 0.14). Genetic correlations between survival and weight were all positive, except for survival at months 2–5 and weight at 18 months, which was not significantly negative (−0.18).     

Pharmacokinetics and acetylation of sulfamethoxazole in turbot Scophthalmus maximus after intravascular administration

The pharmacokinetic profi les and sulfamethoxazole(SMX) acetylation process in turbot reared at 18°C were investigated. Either SMX(parent drug) or its acetylized metabolite, N4-acetylsulfamethoxazole(Ac SMX), was administered intravascularly to turbot at a dosage of 50 mg/kg BW. Serum concentrations of the parent drug and its metabolite were both measured by HPLC, and the changes in concentration over time were analyzed in two- and non-compartment models because SMX treatment produced multiple peaks. The results demonstrated that the elimination half-life of the parent drugs, SMX and Ac SMX, were 159.2 and 5.9 h, respectively. The apparent volume of distribution was 0.2 and 0.8 L/kg, and the clearance was 0.038 and 0.222 L/(h·kg), for SMX and Ac SMX, respectively. SMX acetylation in turbot was 2.8%, and the deacetylation of Ac SMX was 0.2%. These fi ndings may be useful in optimizing SMX dosage regimens in turbot aquaculture.     

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 …     

Comparison of the morphometric dynamics of fast-growing and slow-growing strains of turbot Scophthalmus maximus

The dynamics of changes in body shape of fast-growing and slow-growing strains of turbot Scophthalmus maximus, and of the differences in body shape between the two strains, were evaluated from 3 to 27 months of age. The ratios of total length/body length, body width/body length and total length/body width were used as morphometric indices. The two strains exhibited different temporal trends in total length/body length but similar trends in body width/body length and total length/body width. Generally, body width/body length of the two strains increased with time and total length/body width decreased. Thus, the bodies of both fast-growing and slow-growing strains of turbot changed from a narrow to a more rounded shape. However, the ratio total length/body length was generally lower, body width/body length was mostly higher and total length/body width was consistently lower in the fast-growing strain than in the slowgrowing strain. Correlation analysis of the three shape ratios with body weight showed that total length/body length and total length/body width were unsuitable, and that width/body length was suitable, for use as a phenotypic marker for selective breeding of turbot for growth in weight.     

Impact assessment of modified clay on embryo-larval stages of turbot Scophthalmus maximus L.

Using modified clay is one of the most promising methods for the mitigation of harmful algal blooms(HABs). However, the environmental impact of modified clay has become a subject of global concern. In this study, turbot(Scophthalmus maximus L.) embryos were used as a model to assess the effect of modified clay on this sensitive stage of fish development. Results show that the 24 and 48 h LC_(50)(median lethal concentrations) of the modified clay were 1.70 and 1.65 g/L, and the safe concentration was 0.47 g/L, which is much higher than the concentration used to treat HAB. The modified clay did not affect significantly the hatchability of turbot embryos but when the concentration exceeded 0.50 g/L, the deformity rate of newly hatched larvae increased significantly. The total length, specific growth rate(SGR)and yolk sac absorption rate of larvae reached their peaks at 0.50 g/L and then gradually decreased as the concentration of modified clay increased. Therefore, a moderate amount of modified clay does not harm the survival and hatching of turbot embryos, or the growth and development of newly hatched larvae.     

Ciliate Uronema marinum is the causative agent of scuticociliatosis in farm raised turbot Scophthalmus maximus

To identify the pathogen that causes scuticociliatosis in farmed-raised turbot Scophthalmus maximus,we isolated a ciliate from the brain tissue of an infected turbot and identified it as Uronema marinum based on morphological and molecular evidence.We then infected the turbots in artificial laboratory settings with pure cultured U.marinum.The infected turbots showed syndromes similar to those observed in naturally infected ones.Furthermore,microscopic examination and PCR detection confirmed the presence of the ciliate in the tissues of those laboratory-infected turbots.To our best knowledge,this is the first report of scuticociliatosis caused by U.marinum in farm-raised turbot S.maximus in China.     

Morphological comparison between a selected fast-growing strain and the common cultured strain of turbot Scophthalmus maximus

To evaluate the effect of genetic improvement of the turbot Scophthalmus maximus, we analyzed morphological differences between a fast-growing strain obtained by family selection and the common cultured strain, by principal component analysis, stepwise discriminant analysis, and t-tests. Although they clearly differed morphologically, plots of the principal components of the two strains partially overlapped. However, the difference between the strains was supported with very high precision by discriminant analysis. The t-tests revealed that 4 of the 13 morphological traits analyzed were highly significantly different (P<0.01), 4 traits also differed significantly (P<0.05), and the remainder did not differ significantly. The coefficients of difference of the 13 traits were all lower than the threshold value between subspecies (1.28). Together, the results indicate that a trend for segregation of characters from the common cultured strain have already appeared in the selected fast-growing strain but the degree of segregation have not risen to subspecies level.     

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.     

Effects of probiotic on microfloral structure of live feed used in larval breeding of turbot Scophthalmus maximus

The effects of an exogenous probiotic( Bacillus amyloliquefaciens) on microbial community structure of B ranchionus plicatils and A rtemia sinica were evaluated in this study during turbot( Scophthalmus maximus) larval breeding. The analysis and comparison of the microfloral composition of live feed with probiotic was conducted using the Illumina HiSeq PE250. The abundance of microbial species and diversity of microflora in live feed with B. amyloliquefaciens were higher than those in the control. The microfloral composition was similar among the three replicate experimental groups of B. plicatils compared with the control after enrichment. L actococcus, Pseudoalteromonas, and Alteromonas were always dominant. Additionally, some other bacterial species became dominant during the enrichment process. The microbial community during nutrient enrichment of A. sinica was rather similar among the three control replicates. Relative abundance of Cobetia sp., the most dominant species, was 54%–65.2%. Similarity in the microbial community was still high after adding B. amyloliquefaciens. Furthermore, P seudoalteromonas and Alteromonas replaced Cobetia as the dominant species, and the abundance of Cobetia decreased to 4.3%–25.3%. Mean common ratios at the operational taxonomic unit level were 50%–60% between the two B. plicatils and A. sinica treatments. Therefore, the microbial community structure changed after adding B. amyloliquefaciens during nutrient enrichment of B. plicatils or A. sinica and tended to stabilize. Additionally, the abundance of V ibrio in any kind of live feed was not significantly dif ferent from that in the control. These results will help improve the microflora of B. plicatils and A. sinica and can be used to understand the multiple-level transfer role of probiotic species among probiotic products, microflora of live feed, and fish larvae.     

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.     

Molecular characterization and expression analysis of Lily-type lectin (SmLTL) in turbot Scophthalmus maximus,and its response to Vibrio anguillarum

A full-length lily-type lectin(Sm LTL) was identified from turbot(Scophthalmus maximus) in this study. By searching database for protein identification and function prediction, Sm LTL were confirmed. The full-length c DNA of Sm LTL is composed of 569 bp and contains a 339 bp ORF that encodes 112 amino acid residues. The Sm LTL peptide is characterized by a specific β-prism architecture and contains three mannose binding sites in a three-fold internal repeat between amino acids 30–99; two of the repeats share the classical mannose binding domain(Qx Dx Nx Vx Y) while the third binding site was similar to other fish-specific binding motifs(Tx Tx Gx Rx V). The primary, secondary, and tertiary structures of Sm LTL were predicted and analyzed, indicating that the S m LTL protein was hydrophilic, contained 5.36% α-helices, 39.29% extended strands, 16.07% β-folds, and 39.29% random coils, and three β-folds. Quantitative realtime polymerase chain reaction(qPCR) analysis revealed that the Sm LTL mRNA was abundantly expressed in skin, gill, and intestine. Low levels of Sm LTL expression were observed in other tissues. The expression of Sm LTL in gill, skin and intestine increased at m RNA level after stimulation of V ibrio anguillarum, our results suggest that Sm LTL serve as the first line of defence against microbial infections and play a pivotal role in the innate mucosal immune system. The current study indicates that Sm LTL is a member of the lilytype lectin family and the information reported here will provide an important foundation for future research on the role of this protein.     

Protein-sparing effect of carbohydrate in diets for juvenile turbot Scophthalmus maximus reared at different salinities

The aim of the present study was to investigate the protein-sparing effect of carbohydrate in diets for juvenile turbot(Scophthalmus maximus) reared at five salinities(12,18,24,30,and 36).The fish were fed three isocaloric and isolipidic diets for 60 days.The results show that specific growth rate(SGR)and feed conversion efficiency(FCE) were higher in fish reared at salinities of 18 and 36,but lower at 12.Fish fed with diet C25P40(25%carbohydrate and 40%protein) had lower SGR and FCE values compared with those fed with the C5P52(5%carbohydrate and 52%protein) and C15P46(15%carbohydrate and 46%protein) diets;however,there was no statistical difference between diet C5P52 and C15P46.SGR and FCE values were unaffected by diet composition in fish reared at salinity 36.Hepatic lipogenic enzyme activities were higher in fish reared at 18 and 36,but lower at 12,while glucokinase(GK) activity was higher in fish reared at 12,and lower at 18 and 36.Dietary starch enhanced GK activity while depressing lipogenic enzyme activity.However,lipogenic enzyme activity increased with increasing dietary starch in fish reared at 36.It is recommended that salinity should be maintained 12 in the farming of juvenile turbot.In addition,an increase in gelatinized starch from 5%to 15%could spare 6%dietary protein in fish reared at salinities of 18-30,while higher salinity(36) could improve dietary carbohydrate use and enhance the protein-sparing effect,which is linked with the induction of lipogenic capacities.     

Pharmacokinetics and tissue behavior of enrofloxacin and its metabolite ciprofloxacin in turbot Scophthalmus maximus at two water temperatures

Turbot Scophthalmus maximus, an important aquaculture species in China, currently suffers from epizootic diseases because of high density aquaculture. Enrofloxacin has been used to treat various systemic bacterial fish infections. However, studies concerning the pharmacokinetics of enrofloxacin in turbot are limited. In this study, the pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin, were investigated in the turbot following intravenous and oral administration at 10 mg enrofloxacin/kg body weight, at 16°C and 10°C water temperatures. The concentrations of enrofloxacin and ciprofloxacin in the main tissues (plasma, muscle, liver and kidney) were detected by HPLC. The results show that the plasma concentration-time data for enrofloxacin were best described as a two-compartment open model after intravenous and oral administration. Three pharmacokinetic equations were established between the concentrations and temperatures. The kinetic profile of enrofloxacin was temperature dependent. The absorption half-life of enrofloxacin was 1.99 h and 2.17 h after oral administration, whereas the elimination half-life of the drug was 98.63 h and 136.59 h at 16°C and 10°C, respectively. The peak concentration of enrofloxacin in plasma and tissues was higher at 16°C than that at 10°C, and the peak plasma concentration time in the liver was the shortest at both temperatures among those of other tissues. The plasma C max /MIC ratio varied between 11.08 and 5 540.00 at 16°C; and between 7.92 and 3 960.00 at 10°C. The AUC/MIC ratio was 467.82-280 690.00 at 16°C, and 359.48-215 690.00 at 10°C. These ratios indicate that it is possible to obtain therapeutic efficacy. Very low levels of ciprofloxacin were detected. The AUC ratios of ciprofloxacin and enrofloxacin in plasma suggest that plasma ciprofloxacin might play a minor role in enrofloxacin treatment for turbot.     

Isolation and characterization of 45 Polymorphie microsatellite loci of turbot (Scophthalmus maximus) and cross-species amplification

Turbot(Scophthalmus maximus) is a flatfish species commercially important for aquaculture.In this study,we generated a microsatellite-enriched genomic DNA library for Scophthalmus maximus,and then isolated and characterized 45 microsatellite loci by genotyping 30 individuals.The observed number of alleles ranged from 2 to 19 with an average of 6.24,while the effective number of alleles ranged from 1.30 to 11.11 with an average of 3.66.The expected heterozygosities varied from 0.235 to 0.925 4 and polymorphic information content ranged from 0.204 4 to 0.903 3,with an average of 0.622.Twelve loci deviated significantly from Hardy-Weinberg equilibrium,and no significant linkage disequilibrium was observed between any pair of loci after Bonferroni correction.In cross-species amplification,five flatfish species(Paralichthys lethostigma,Verasper moseri,platichthys stellatus,Hippoglossoides dubius and Cynoglossus semilaevis) showed at least one polymorphic locus.These polymorphic microsatellite loci should prove useful for population analysis of turbot and other related species.