Transcriptome analysis reveals immune-related genes in tissues of Vibrio anguillarum-infected turbot Scophthalmus maximus

Turbot Scophthalmus maximus is an important mariculture fish species with high economic value.However,the bacterial diseases caused by Vibrio anguillarum infection bring huge economic losses to the turbot aquaculture industry.To understand the immune response of the turbot against V.anguillarum infection and to explore novel immune-related genes,the transcriptome analysis of turbot spleen and gills were conducted after V.anguillarum infection.Differentially expressed genes(DEGs)were identified in spleen and gill of the turbot amounted to 17261 and 16436,respectively.A large number of immunerelated DEGs were enriched in cytokine-cytokine receptor interaction signaling pathway,and the others by the kyoto encyclopedia of genes and genomes(KEGG)enrichment.The gene ontology(GO)classification analysis revealed that V.anguillarum infection had the greatest effect on biological processes and cellular components.Twelve immune-related DEGs were identified in the spleen(cstl.1,egfl6,lamb21,v2rx4,calcr,and gpr78a)and gills(ghra,sh3gl2a,cst12,inhbaa,cxcl8,and il-1b)by heat map.The proteinprotein interaction(PPI)networks were constructed to analyze the immune mechanism.The results demonstrate that the maturation and antigen processing of major histocompatibility complex(MHC)class II molecule,and calcitonin-or adrenomedullin-regulated physiological activity were important events in the immunity of turbot against V.anguillarum infection.In the gills,the protein interactions in TGF-βsignaling pathway,production of inflammatory factors,and endocytosis regulation were most significant.Our research laid a foundation for discovering novel immune-related genes and enriching the knowledge of immune mechanisms of turbot against V.anguillarum infection.

The ontogeny of acidic digestive tract in larval turbot Scophthalmus maximus based on H+/K+-ATPase and pepsinogen

Acidic digestion is an important digestive process of marine fish.In fish stomach,two enzymes are involved in the secretion of hydrochloric acid(HCl)and proteomic digestion:H^(+)/K^(+)-ATPase and pepsinogen.However,the starting of digestive function in fish is still unclear.To reveal the details of acidic digestion of turbot Scophthalmus maximus in early development,a 40 day of turbot larvae culture was conducted.The H^(+)/K^(+)-ATPase gene from the turbot S.maximus(smH^(+)/K^(+)-ATPase)was identified and characterized.Based on our previous discription on pepsinogen of turbot S.maximus,we combined pepsinogen and H^(+)/K^(+)-ATPase and analyzed the mechanism of acidic digestion in turbot.Results show that the spatial and temporal expression profiles of H^(+)/K^(+)-ATPase agreed with pepsinogen A and C in turbot,indicating a synergetic action between H^(+)/K^(+)-ATPase and pepsinogen during the acidic digestion process.In addition,the turbot juveniles showed a faster growth after the expressions of H^(+)/K^(+)-ATPase gene and pepsinogen gene,demonstrating that pepsin had a higher digestive efficiency,for which a compound diet should be provided to the fish from Day 22 onward.This study provided a reference for biology research and aquaculture of turbot and other marine fishes.

Establishment,Characterization and Expression Pattern of a Spleen Cell Line,SMSP,Derived from Turbot(Scophthalmus maximus)

A turbot(Scophthalmus maximus)cell line named SMSP was obtained from the spleen.The origin of the cells was identified by morphology,chromosome number and COI gene.The optimal basic medium,serum concentration and growth temperature of the cells were detected.SMSP cell line is mainly composed of fibroblast-like cells.Most of the SMSP cells contained 44 chromosomes,and the sequence of COI gene confirmed that the cells were originated from turbot.The optimal culture conditions were 24℃,DMEM+10%FBS.The cell line had high transfection efficiency for siRNA and plasmid.After stimulation with lipopolysaccharide(LPS)or poly(I:C),the expressions of immune-related genes such as TNF-β,IL-12s,IL-10 and IL-1βwere up-regulated significantly in the early stage(P<0.05).This study will provide a model for exploring immune mechanism of turbot against pathogen in vitro.

Isolation and characterization of 45 polymorphic 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.

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.

Studies on the edwardsiellosis and characterization of pathogenic bacteria from diseased flounder (Paralichthys olivaceus L.) and turbot (Scophthalmus maximus L.)

Edwardsiellosis of flounder and turbot occurring in different mariculture farms during 2001～ 2004 was examined, including the conditions of disease occurrence, clinical signs and pathological changes. The results showed that all diseased fishes expressed bacterial septicaemia. A total of 148 strains were identified using a combination of traditional physiological and biochemical tests and partial 16S rRNA gene analysis. In addition, the mole fraction G ＋ C ratio of the DNA of representative strain of isolates and serum homology were detected, and pathogenicity tests of isolates were conducted by experimental infection. The results revealed that 148 strains were identified as E. tarda of genus Edwardsiella, all the isolates are of serologic similarity, and have strong pathogenicity to flounder and turbot.

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 identifi ed it as Uronema marinum based on morphological and molecular evidence. We then infected the turbots in artifi cial 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 confi rmed the presence of the ciliate in the tissues of those laboratory-infected turbots. To our best knowledge, this is the fi rst report of scuticociliatosis caused by U . marinum in farm-raised turbot S . maximus in China.

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).

Effects of Dietary Carbohydrate-to-Lipid Ratio on the Growth Performance and Feed Utilization of Juvenile Turbot(Scophthalmus maximus)

A 9-week feeding trial was conducted to investigate the effects of dietary carbohydrate to lipid ratio(CHO:LIP) on the growth performance and feed utilization of juvenile turbot Scophthalmus maximus(initial body weight 8.75 g ± 0.04 g). Four isonitrogenous and isoenergetic low protein level(39%) diets were formulated with increasing ratios of dietary carbohydrate to lipid(2:18, 6:18, 18:12 and 28:6). A high protein level(50%) diet with the 2:12 ratio of carbohydrate to lipid was used as the control. Results showed that the survival rate, contents of moisture, crude protein and ash in muscle were not significantly affected by dietary treatments. With the dietary CHO:LIP ratio increased from 2:18 to 18:12, weight gain rate significantly increased(P < 0.05). Higher dietary CHO:LIP ratio(28:6) resulted in the significantly decreased weight gain rate(P < 0.05). Meanwhile, this treatment also resulted in the highest daily feed intake and liver glycogen content, as well as the lowest feed efficiency(P < 0.05). Muscle glycogen content in fish fed the diet with 2:12 or 2:18 CHO:LIP ratio was significantly lower than those fed with the other three diets(P < 0.05). The present results confirmed that the juvenile turbot can utilize carbohydrate. Furthermore, the appropriate ratio of dietary carbohydrate to lipid was important to the growth and feed utilization of turbot. The proper CHO:LIP ratio based on the growth performance in the present study was determined to be 18:12 when the dietary protein level was 39%.

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(P<0.01) were significant for the regression model.The interactive effect between temperature and salinity on liver Hsp70,kidney Hsp70 and liver IgM were not significant(P>0.05),while the interactive effect between temperature and salinity on kidney IgM was significant(P<0.01).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.

Developmental quantitative genetic analysis of body weights and morphological traits in the turbot, Scophthalmus maximus

In order to elucidate the genetic mechanism of growth traits in turbot during ontogeny, developmental genetic analysis of the body weights, total lengths, standard lengths and body heights of turbots was conducted by mixed genetic models with additive-dominance effects, based on complete diallel crosses with four different strains of Scophthalmus maximus from Denmark, Norway, Britain, and France. Unconditional genetic analysis revealed that the unconditional additive effects for the four traits were more significant than unconditional dominance effects, meanwhile, the alternative expressions were also observed between the additive and dominant effects for body weights, total lengths and standard lengths. Conditional analysis showed that the developmental periods with active gene expression for body weights, total lengths, standard lengths and body heights were 15–18, 15 and 21–24, 15 and 24, and 21 and 27 months of age, respectively. The proportions of unconditional/conditional variances indicated that the narrow-sense heritabilities of body weights, total lengths and standard lengths were all increased systematically. The accumulative effects of genes controlling the four quantitative traits were mainly additive effects, suggesting that the selection is more efficient for the genetic improvement of turbots. The conditional genetic procedure is a useful tool to understand the expression of genes controlling developmental quantitative traits at a specific developmental period（t-1→t） during ontogeny. It is also important to determine the appropriate developmental period（t-1→t） for trait measurement in developmental quantitative genetic analysis in fish.

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.

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.

Genetic parameter estimation for juvenile growth and upper thermal tolerance in turbot(Scophthalmus maximus Linnaeus)

Twenty-six half-sib groups （53 full-sib families） of turbot,Scophthalmus maximus Linnaeus, were obtained by artificial insemination. We measured growth in the offspring （40-50 individuals/family） and subjected them to a thermal tolerance challenge over a period of 34 d. There was no significant difference in daily mor-tality （range： 0.580%-1.391%） between Days 1-13 during the thermal tolerance challenge. However, daily cumulative mortality increased rapidly between Days 14 and 29, especially on Days 15 and 16 （20.232% and 34.377%, respectively）. Mortality was highest on Day 16 （14.145%）. We estimated the genetic parameters using the average information restricted maximum likelihood method. We used a likelihood ratio test to evaluate the significance of effects in models with and without identity as an effect, and compared the final log-likelihoods （maximum log L）. Lastly, we estimated phenotypic and genetic correlation between the up-per thermal tolerance limit （UTT） and body weight （BW）. In this study, the positive phenotypic correlation was low between UTT and BW （0.093±0.029）. The genetic correlation between UTT and BW was negative （-0.044±0.239）. The heritability for upper thermal tolerance was low （0.087±0.032）, which is of approximate-ly moderate heritability. The heritability for body weight was high （0.303±0.074）. Our results suggest there is significant potential for improvement in the culture of turbot by selective breeding.

Genetic parameters and response to selection for body weight in turbot(Scophthalmus maximus, Linnaeus)

Genetic parameters and response to selection were estimated for harvest body weight in turbot. The data consisted of 10 952 individuals of 508 full-sib families from three generations（G0, G1, and G2）. The heritability estimates for G0, G1, and G2 were 0.11±0.08, 0.18±0.09, and 0.17±0.07, respectively. Over three generations, the heritability estimate was 0.19±0.04. Maternal and common environmental effects were 0.10±0.04, 0.14±0.04, and0.13±0.03 within each generation and 0.12±0.01 across generations. The selection differential in growth was 18.24 g in G0 and 21.19 g in G1 corresponding to an average of 19.72 g per generation. The genetic gains were also calculated, they were 22.06 g in G1 and 11.93 g in G2, corresponding to 6.36% and 3.52% body weight. The total genetic gain after two generations was 10.10% body weight, which indicated that the selective breeding program for the body weight trait in turbot was successful.

Administration of Yeast Glucan on Immunity of Offspring in Turbot Scophthalmus maximus: A Trans-Generational Immune-Enhancing Effect

β-glucan has been shown to increase immunity and survival in various juvenile and adult fish both in freshwater and marine aquaculture species as well as in marine fish larvae. However, information about the trans-generational immune-enhancing effects of β-glucan remains rather limited. Here we clearly show that dietary intake of β-glucan enhanced the levels of C3, Bf and lysozyme in the serum of turbot as well as in their eggs released. We also show that yeast glucan induced a significant increase in lysozyme activity in both the serum and eggs. Moreover, the embryos derived from yeast glucan-treated turbot were more resistant to bacterial challenge than control embryos. By contrast, the administration of yeast glucan on female turbots had little influence on the egg development and embryonic development. Collectively, these data indicate that yeast glucan can be safely used to promote the non-specific trans-generational immunity in offspring of turbot.

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 fi sh 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 (THRl) were 0.247 9±0.108 3, 0.288 3±0.161 2, and 0.106 9±0.045 2, respectively. The correlation coeffi cients 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 THRl model were 0.773 8, 0.775 4, and 0.784 4, respectively, the greatest from the THRl model. The genetic correlations between body weight and survival data EBVs from the CSL, THRp, and THRl 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.

Rearing in intermediate salinity enhances immunity and disease-resistance of turbot (Scophthalmus maximus L.)

Studies were conducted to investigate the non-specific immune responses and disease-resistance of juvenile turbot Scophthalmus maximus,cultured at four different salinities（8,20,32 and 40） . Three concentrations（3.75 × 10 7,3.75 × 10 8 and 3.75 × 10 9 CFU/ml） of Vibrio anguillarum suspension were employed at each salinity to determine the 4-day LD 50 . The serum lysozyme activity,the alternative complement pathway activity（ACH50） and the phagocytosis percentage of head kidney in turbot were tested at 24,48 and 72 h post-challenge of V. anguillarum（1.1 × 10 8 CFU/ml,0.1 ml） ,respectively,to evaluate the non-specific immune responses at the selected rearing salinities. Fish reared at salinity 20 had the lowest mortality,namely,the highest 4-day LD 50 value（8.88 ± 0.17） . Besides,the lysozyme activity,ACH50 and the phagocytosis of turbot were the highest at the salinity 20,but with the lowest at the salinity 40 treatment regardless of sampling time. In addition,the non-specific immune activities kept increasing within 72 h post-challenge of V. anguillarum,except that the lysozyme activity increased from 24 to 48 h,and then decreased from 48 to 72 h at 40 significantly. These results together indicate that rearing in intermediate salinity（20） was able to enhance the immunity and disease-resistance of turbot.

Estimates of heritability and correlation for growth traits of Turbot(Scophthalmus maximus L.) under low temperature conditions

The objectives of this present research were to assess the heritability of growth traits under low temperature conditions in turbot（Scophthalmus maximus L.）, and to analyze the correlation between body weight（BW） and body length（BL）. There were 536 individuals from 25 full- and half-sib families involved in this study. During the entire 90-day period, which was initiated at 233 dph（day old） and ended at 323 dph, the individuals＇ BW and BL were weighed consecutively six times every 18 days. The heritability of BW and BL and the correlation between these two traits were estimated based on an individual animal model with the derivative-free restricted maximum likelihood（DFREML） method. These results showed that the specific growth rates（SGR） of 25 families was from 0.75±0.11 to 1.05±0.14 under water temperature of 10.5–12°C. In addition, the heritability of BW and BL estimated under low-temperature were 0.32±0.04 and 0.47±0.06, respectively. The BW had a medium heritability（0.2–0.4）, and the BL had a high heritability（〉0.45）, which suggested that selection for increased weight and length was feasible. Moreover, there was potential for mass selection on growth. The genetic and phenotypic correlations between BW and BL were 0.95±0.01 and 0.91±0.01（P 〈 0.01）, respectively. A significant correlation between BW and BL showed that BL could be instead of BW for indirect selection, which could be effectively implemented in the breeding program.

Transcriptomic analysis reveals putative osmoregulation mechanisms in the kidney of euryhaline turbot Scophthalmus maximus responded to hypo-saline seawater

Turbot harbor a relatively remarkable ability to adapt to opposing osmotic challenges and are an excellent model species to study the physiological adaptations of flounder associated with osmoregulatory plasticity.The kidney transcriptome of turbot treated 24 h in water of hypo-salinity(salinity 5)and seawater(salinity 30)was sequenced and characterized.In silico analysis indicated that all unigenes had significant hits in seven databases.The functional annotation analysis of the transcriptome showed that the immune system and biological processes associated with digestion,absorption,and metabolism played an important role in the osmoregulation of turbot in response to hypo-salinity.Analysis of biological processes associated with inorganic channels and transporters indicated that mineral absorption and bile secretion contributed to iono-osmoregulation resulting in cell volume regulation and cell phenotypic plasticity.Moreover,we analyzed and predicted the mechanisms of canonical signaling transduction.Biological processes involved in renin secretion,ECM-receptor interaction,adherens junction,and focal adhesion played an important role in the plasticity phenotype in hypo-stress,while the signal transduction network composed of the MAPK signaling pathway and PI3K-Akt signaling pathway with GABAergic synapse,worked in hypoosmoregulation signal transduction in the turbot.In addition,analysis of the tissue specificity of targeted gene expression using qPCR during salinity stress was carried out.The results showed that the kidney,gill,and spleen were vital regulating organs of osmotic pressure,and the osmoregulation pattern of euryhaline fish dif fered among species.