低盐胁迫下LXRα激动剂添加和RNA干扰对大菱鲆(Scophthalmus maximus)幼鱼脂质代谢的影响

大菱鲆是我国重要的养殖经济鱼类,随着养殖水域逐渐向滩涂和内陆拓展,亟待研究其低盐适应性机制,进而为培育耐低盐良种提供参考。通过腹腔注射干扰剂dsRNA以及激动剂T091317,以进一步检测下游脂质代谢相关基因的表达以及血清脂代谢产物的变化。研究设置两个盐度梯度(5和30),每个盐度设置4个处理组(干扰组、激动组、生理盐水组和空白对照组),实验周期72 h。结果显示:在dsRNA注射后,肝脏中LXRα基因的相对表达量呈现出显著的抑制作用,尤其是在盐度30下;盐度5的肝脏组以及不同盐度的肠道组中,干扰效果不明显。激动剂注射后LXRα基因的相对表达量在不同盐度组中均呈现了激动效应,但维持时间较短。低盐可以抑制SREBP-1、CYP7A1和ApoA-IV基因的表达,当LXRα基因受到抑制或激动后,三者尽管表达趋势不同,但整体上均呈现出相应的干扰和激动效应。血清生化结果显示,低盐显著抑制了TG和HDL的含量,对T-CHO以及LDL的含量无显著影响。30盐度下,当LXRα基因受到抑制或激动后,TG的含量呈现出相应的干扰和激动效应。以上表明在腹腔注射干扰剂dsRNA以及激动剂后,都能够相应的导致LXRα基因表达量降低或者升高。LXRα基因受到抑制后,可以引起下游转录因子SREBP-1基因表达的降低,进而抑制了TG的合成,因此血清中甘油三酯的含量可以进一步作为大菱鲆耐低盐选育的表型。

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.

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.

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.

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 rnaxirnus, and then isolated and characterized 45 microsateIIite 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 0.9254 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 rnoseri, 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.

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.

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 ll-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）.

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.

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.

Dietary Protein Requirement of Juvenile Turbot(Scophthalmus maximus Linnaeus)

The dietary protein requirement of juvenile turbot(initial average weight, 38.2 g ± 0.1 g) reared indoor in aerated aquaria was determined in this study. Five energy equal experimental diets were formulated with fish meal as protein source, which contained different concentrations of protein(47.2%, 51.0%, 54.6%, 59.3% and 63.6% of dry diet). Three groups of fish with 18 individuals in each, were cultured in 300-L tanks and fed twice a day for 8 weeks. During culture, temperature was controlled between 15.0 and 18.0℃, salinity was controlled between 28.5 and 32.0, acidity was controlled between p H7.8 and p H8.5, and ammonia nitrogen was maintained below 0.03 mg L-1 and dissolved oxygen was maintained about 7 mg L-1. Results showed that the growth of fish was significantly affected by dietary protein content(P < 0.05). Specific growth rate(SGR) of turbot increased when dietary protein content varied between 47.2% and 51.0%(P < 0.05), and then kept stable when dietary protein content was higher than 51.0%. Fish which were fed the diet containing 63.6% protein showed the highest SGR while those fed the diet containing 59.3% protein showed the highest feed efficiency rate. No significant difference of feed intake and protein efficiency ratio was found among experimental diets(P > 0.05). Broken-line regression analysis of SGR showed that the optimal dietary protein requirement of turbot was about 57.0%.

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%.

Identification and characterization of pathogen to bacterial septicaemia in cultured turbot, Scophthalmus maximus

Bacteria-infected turbots Scophthalmus maximus with septicaemia were examined between 2001 and 2004 in aspects of the conditions of disease occurrence, clinical syndromes and pathological changes. The phenotypic information of pathogenic bacteria was studied, including morphology, physiological and biochemical characteristics, and the mol% G+C of the DNA. In addition, representative strains (S010623-1, LH031120-1) were selected for molecular identification by partial 16S rRNA gene sequencing. The results show that the isolates (LH031120-1 to LH031120-6, HT040308-1 to HT040308-6, HT040620-1 to HT040620-6) from three farms were identified as Edwardsiella tarda. The isolates (S010610-1 to S010610-10, S010623-1 to S010623-20) from one farm were identified as Listonella anguillarum. We conducted studies on the pathogenicity of isolates by artificial infection, and revealed all infected groups in morbidity and mortality. The septicaemia infected turbot showed a syndrome similar to that of the naturally infected fish. Antibiotic sensitivity showed that of 37 antimicrobial agents, E. tarda was sensitive to 27 agents, and L. anguillarum was sensitive to 21 agents.

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

Juvenile turbot （Scophthalmus maximus） the initial stock densities at 0.28, 0.87, 1.12, 1.16, 2.75 were reared in five different experiment groups in kg/m^2 respectively for the study of the density effect on growth, pigmentation and feed coefficient rate. The experiment lasted for 60 days with final stock densities at 1.91, 6.31, 8.86, 11.97, 17.67 kg/m^2 respectively. Result showed that, in the same experiment condition, the stocking density has a positive effect on growth in low density and negative in high density. The SGR （special growth rate） was 3.189, 3.304, 3.447, 3.341, and 3.087 respectively. The uniformity of weight distributing among experiment groups decreased with increasing density. The stocking density had positive effect on feed coefficient rate. Group 1 had the least feed coefficient rate 0.96, and highest at 1.25 in Group 5, the highest density group. High stocking density inhibited the growth and increased the feed coefficient rate. The stocking density had negative relationship to pigmentation improvement for whitened fish.

大菱鲆(Scophthalmus maximus L.)胚胎及仔稚幼鱼发育研究

采用体视镜对大菱鲆胚胎期发育全过程进行连续观察 ,研究其胚胎正常发育特征 ,并继续对胚后期的仔、稚、幼鱼发育阶段采用测量、手绘、照相等方法进行形态学与生态学研究。结果表明 ,大菱鲆胚胎发育与其他硬骨鱼浮性卵鱼类基本相似 ,属端黄卵 ,盘状分裂。在水温 1 3℃条件下 ,约经 1 1 6h完成孵化。胚后仔、稚、幼鱼阶段 ,体背部色素逐步增深 ,鱼体逐渐由透明变为不透明 ,由两侧对称变为不对称。 3日龄仔鱼开口 ;1— 9日龄仔鱼 ,体色呈红色 ,称之为“红苗” ;1 0— 2 4日龄 ,黑色素增多 ,称之为“黑苗” ;2 5日龄以上 ,体披大量花状色素 ,称之为“花苗” ,由此鱼体变宽 ,右眼开始上升 ;30日龄苗 ,右眼已上移至头顶背部 ;35— 38日龄苗 ,右眼已完全转移至左侧 ,变态完成而达鲆鲽类所特有的形态、生态和生理状态 ;60—90日龄苗 ,背部正常体色为沙色 (sandcolor) ,全长已达 5 0— 60mm ,可以作为商品苗出售。

进口大菱鲆Scophthalmus maximus L.苗种的遗传结构分析

利用随机扩增多态性DNA(RAPD)和微卫星两种分子标记技术分析了从法国 (F)、英国 (E)、西班牙 (S)引进我国的三个大菱鲆群体的遗传结构。 2 0个RAPD随机引物对每个群体各 2 0尾大菱鲆的基因组DNA进行扩增 ,共获得 1 2 5个重复性好且谱带清晰的RAPD标记 ,片段大小在 2 0 0— 2 5 0 0bp之间 ,三群体的多态位点比例在 1 2 80 %— 2 0 0 0 %之间 ,Nei基因多样性指数在 0 0 1 42— 0 0 35 2之间 ,Shannon多样性指数在 0 0 4 2 3— 0 0 72 0之间。微卫星引物的分析结果与RAPD一致。总体上 ,三个群体的遗传多样性均较低 ,其中西班牙群体遗传多样性水平最高 ,英国群体次之 ,而法国群体最低。利用Shannon多样性指数和Nei多样性指数估算群体遗传变异的来源 ,两者得出一致的结论 :群体间遗传分化很弱。AMOVA分析结果进一步证实了Shannon多样性指数和Nei基因多样性指数的分析结果 :群体的遗传变异有 97%以上是由群体内个体间的遗传变异引起的 ,遗传变异主要来自于群体内个体间 ,显著性检验表明群体间的变异对总变异的影响不显著。

大菱鲆(Scophthalmus maximus)四个不同地理群体数量形态特征比较

采用聚类分析、单因子方差分析和主成分分析方法,对4个不同地理群体大菱鲆进行了12项形态性状的测定,比较了4个群体的外部形态特征。聚类分析结果表明,4种大菱鲆群体中,丹麦和挪威之间及英国和法国之间的差异较小,丹麦、挪威和英国、法国之间的差异较大。方差分析多重比较结果表明,4个群体在部分形态特征上表现出明显差异,其中,英国和法国群体、挪威和丹麦群体之间形态特征差异显著指标较少,丹麦群体和英国群体、丹麦群体和法国群体、挪成群体和英国群体、挪威群体和法国群体之间差异显著指标较多。主成分分析构建了3个反映形态特征信息的综合性指标——主成分1、主成分2和主成分3,三者的贡献率分别为38.663%、14.117%和8.976%,三个主成分的累积贡献率为61.756%.分析结果显示,英国和法国群体、挪威和丹麦群体之间形态差异不显著,丹麦群体和英国群体、丹麦群体和法国群体、挪威群体和英国群体、挪威群体和法国群体之间形态差异显著,且差异主要来源于主成分1,而主成分1主要由经方差分析多重比较差异显著的指标组成,显然,主成分分析与聚类分析、方差分析的结论基本上是类似的,它们从不同的角度反映了群体间的形态学差异.

大菱鲆(Scophthalmus maximus)大规模家系选育优良家系的生长发育规律

运用Logistic、Gompertz和von Bertalanffy3种生长模型分别对大菱鲆的5个优良家系E♂1×E♀2、F♂4×N♀3、F♂2×E♀4、E♂2×F♀1、F♂1×F♀4及对照组群体的生长曲线进行了拟合和比较分析。结果表明,家系E♂1×E♀2、F♂2×E♀4和E♂2×F♀1的最优拟合模型为Gompertz模型,家系F♂4×N♀3和F♂1×F♀4的最优拟合模型为vonBertalanffy模型,而对照组则更适应于Logistic模型拟合其生长曲线。不同月龄家系的瞬时生长率均高于对照组,大多数相对生长率高于对照组,拐点月龄均较对照组拖后,拐点体重均高于对照组,快速生长区间的始速点均比对照组提前,快速生长区间长度均大于对照组。瞬时生长加速率在不同月龄则存在较大变化。

大菱鲆(Scophthalmus maximus)选育家系的构建和培育技术研究

采用巢式设计方法和人工采卵授精技术,对大菱鲆大规模家系的构建与培育技术进行研究。按照1雄配2雌的原则,选取英国、法国、丹麦和挪威4个群体的大菱鲆进行定向交配,并对大菱鲆早期发育阶段苗种进行了环境标准化和一级、二级、三级数量标准化培育。结果表明,构建父系半同胞家系的初始成功率不高,1/3父系半同胞家系的初始构建成功率≤20%;由于家系成功初始构建的不同步性,拉长了家系的培育时间,对同一发育阶段家系早期培育的同步性较差,导致对处于同一发育阶段、但在不同时期培育的家系所进行的环境标准化效果不好,每次数量标准化时,存在部分全同胞家系间数量差异显著。基于早期阶段家系构建和培育存在的问题,提出了拟解决的方法和对策,为大规模建立大菱鲆家系提供参考。

蛹肽蛋白替代鱼粉对大菱鲆(Scophthalmus maximus L.)幼鱼生长、饲料利用、消化代谢酶及免疫性能的影响

选取蛹肽蛋白分别替代大菱鲆幼鱼配合饲料中0、15%、30%、45%、60%、75%的鱼粉,配制成6种等氮等能的饲料饲喂大菱鲆幼鱼(19.84±0.04 g)56 d,以研究蛹肽蛋白替代鱼粉的效果。结果显示,大菱鲆幼鱼的特定生长率、饲料效率随替代水平的升高而降低,30%及以上替代组显著低于对照组(P<0.05);鱼体粗蛋白、粗脂肪水平随替代水平的升高而降低,分别在75%和30%及以上替代组显著降低,鱼体水分和灰分含量随替代的升高而升高,分别在75%和30%及以上替代组显著升高(P<0.05)。摄食率随替代水平升高呈先上升后下降的趋势,在30%替代组呈现出最高的摄食率,且显著高于对照组(P<0.05)。饲料干物质的表观消化率在42.53%-54.36%之间,且当替代水平达到75%时显著低于对照组(P<0.05);而蛋白质表观消化率在71.67%-86.89%之间,仅45%和75%替代组显著低于对照组(P<0.05)。各替代组肝脏超氧化物歧化酶活性均高于对照组,在45%和60%替代组出现显著性差异(P<0.05)。综上所述,蛹肽蛋白可以替代大菱鲆幼鱼饲料中15%的鱼粉而不影响其生长摄食、饲料利用以及与消化、代谢、免疫相关的酶的活性。

大菱鲆(Scophthalmus maximus)耐高温品系选育及耐温性能评估

采用家系选育方法,进行了大菱鲆耐高温性状选育研究,对两代家系耐温性能进行测定,并利用bCOX回归分析了各家系的耐温性优势比。结果表明:各家系耐温性差异达到了显著水平(P<0.05),并且耐高温能力的优势比也存在显著差异,这反映出在大菱鲆耐高温选育中家系选择的高效性;经过一代选育,获得4个耐高温家系,并据此构建选育二代家系。耐温性能评估,选育二代比一代有显著提高,在27℃高温条件下,选育一代的平均死亡率为49%,选育二代的平均死亡率为10.26%;28℃条件下,选育二代耐高温子代的平均死亡率为32.83%,对照组的平均死亡率为88.9%;经过筛选后,选育二代的耐温性比选育一代提高了1—2℃左右。