盐度胁迫下点带石斑鱼(Epinephelus malabaricus)胚胎及卵黄囊仔鱼的形态变化

将点带石斑鱼的受精卵置于不同盐度的水体中,观察受盐度胁迫下点带石斑鱼胚胎的沉浮情况、卵径、油球径、孵化率、畸形率,以及卵黄囊仔鱼的形态变化和营养物质的消耗状况。结果表明,点带石斑鱼受精卵在盐度25(含25)以下的水体中为沉性,在盐度35(含35)以上的海水中全部漂浮于水表层,在盐度30和对照组31的水体中大部分胚胎浮在水上层。受精卵卵径随孵化盐度降低而减小。在水温26—28℃的条件下,点带石斑鱼受精卵经21—26h可孵化成仔鱼,盐度对孵化时间影响不显著(P>0.05)。盐度在15—50范围内都能孵出仔鱼,但孵化率和畸形率因盐度不同而有显著差异。综合分析表明点带石斑鱼受精卵孵化的适合盐度范围为26.7—41.1。最适孵化盐度范围在30.5—32.5之间。盐度对卵黄囊仔鱼可量性状的影响主要表现在卵黄囊容积和全长的变化,高盐度(≥45)下胚后仔鱼卵黄囊容积、全长和体高显著减小(P<0.05),油球径不因盐度不同而改变。

Identification of Epinephelus malabaricus and Epinephelus coioides using DNA markers

Using multi-molecular marker technologies and based on morphological criteria, the genetic relationship between Epinepheelus malabaricus and E, coioides was examined in the hope of resolving the long-standing issue of identifying these two species. Results showed that： （ 1 ） E. coioides and E. malabaricus should be identified as two species, the consistency of mitochondrial DNA cytochrome b gene sequence between E. malabaricus and E. coioides is 94.4%, and the genetic similarity by AFLP was 0.753 9 ; （2） Hybridization exists between E. malabaricus and E. coioides, the specific RAPD and AFLP fragments are found to be useful in the identification of these two species, and the genetic properties （both with exterior and inheritance） of hybrid is significantly biased to the male parents ; and （3） AFLP was a potentially powerful tool in constructing the genetic linkage map for these two groupers.

Assessment of Genetic Variability and Inter-specific Relationships among Four Species of Groupers (Genus Epinephelus) from South China Sea Using Microsatellite Markers

[Objective] The study aimed to evaluate the genetic variability and interspecific relationships among four species of groupers from South China Sea, including E. fario, E. merra, E. malabaricus and E. coioides. [Method] Twenty one mircosatellite loci of groupers were selected from GenBank and eight high polymorphic loci were used to further genetic analysis. [Result] The mean number of alleles per locus （A）, effective number of alleles （Ne）, mean polymorphism information content （PIC）, observed heterozygosity （Ho） and expected heterozygosity （He） were 4.38±1.60, 3.69±0.86, 0.69±0.08, 0.67±0.08, 0.72±0.06 in E. malabaricus; 3.88±1.13, 3.55±1.04, 0.66±0.10, 0.68±0.21, 0.70±0.08 in E.coioides; 6.00±1.07, 4.68±0.65, 0.78±0.03, 0.73±0.25, 0.79±0.03 in E. fario; 5.50±1.07, 4.58±0.80, 0.76±0.05, 0.75±0.18, 0.78±0.04 in E. merra, respectively. [Conclusion] We compared the values above, the order of the genetic variability among these grouper species was E. fario E. merra E. malabaricus E. coioides. We found that the level of genetic variability of these groupers species was relatively higher than that of other marine fish, so their genetic status was good. In addition, the analyisis of genetic relationship showed that E. malabaricus and E. coioides was the closest and it was the farthest between E. merra and E. coioides.

Diagnosis of nervous necrosis virus in orange-spotted grouper,Epinephelus coioides, by a rapid and convenient RT-PCR method

Viral nervous necrosis （VNN） causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction （RT-PCR） was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necro- sis virus （NNV）, in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus （IPNV）, infectious hematopoietic necrosis virus （IHNV）, spring viraemia of carp virus （SVCV）, epizootic haematopoietic necrosis virus （EHNV）, and large yellow croaker iridovirus （LYC1V）. With this method, the orange-spotted grouper （Epinephelus coioides） fry from hatcheries with or without incidence of the VN- N epidemic in Fujian Province were detected. The results showed that all or 93% of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40% or 25% of fry from the t- wo hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.

Protective immunity of orange-spotted grouper(Epinephelus coioids) against a nervous necrosis virus isolated from China,and determination of the complete sequences of the virus

On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus（ OGNNV）, China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV. The major capsid protein （ MCP）gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction （RT-PCR） and ligated into the pET32a expression plasmid. The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa. Recombinant protein with a molecular mass of 57.4 kDa was expressed in E. coli BL21 （DE3）. Vaccine was prepared from the recombinant protein expressed in recombinant cells. The juvenile orange-spotted groupers （8 cm in average length） were immunized by intraperitoneal injection. Group A was challenged with infected tissue filtrates 25 d post-vaccination. The mortality in the vaccined group （ A1,30% ） was a little higher than the unvaccined group （ B2, 27.8% ）. Group B was challenged after three vaccine injections. The mortality in the vaccined group （B1, 16.7% ） was lower than the unvaccined group （132, 27.8% ）, And the relative percentage survival （RPS） value of vaccined group, compared with the unvaccined group, was 40%. The anti-recombinant protein sera with a 1 ： 100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4 ℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper. Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish （ Group C1, mortality of 18.18% ）, compared with the fish （ Group C2, mortality of 40% ） which received infected tissue filtrates treated with control serum. Results implied the potential use of the capsid protein in immunization against OGNNV.

DISEASE RESISTANCE AND HUMORAL IMMUNOMODULATORY EFFECTS OF VITAMIN C ON GROUPER, EPINEPHELUS AWOARA

The disease resistance and humoral immunomodulatory effects of vitamin C administered orally to grouper, Epinephelus awoara maintained on a frozen fish diet supplemented with vitamin C at 500, 1000, 1500 and 2000 mg/kg were investigated. After 20 weeks, the growth rates of the groups with high level of vitamin C apparently increased. The untreated fish had symptoms of vitamin C deficiency. The endogenous liver tissue vitamin C levels were found to reflect well the dietary treatments. After intraperitoneal injection or bath challenge with a virulent strain of Vibrio vulnificus , fish fed with high level vitamin C showed significantly higher survival rate compared with the normal control group. Vaccination with formalin inactivated V. vulnificus significantly enhanced the specific antibody production in fish treated with vitamin C, and completely protected from strong bacterial challenge the groups fed on fish with vitamin C 1500 and 2000 mg/kg diet.

High-throughput simple sequence repeat (SSR) markers development for the kelp grouper (<i>Epinephelus bruneus</i>) and cross-species amplifications for Epinephelinae species

The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. To provide tools for addressing both population genetics studies and gene mapping, dito pentanucleotide simple sequence repeat (SSR) markers were developed using 454 pyrosequencing. Among the 1466 SSR markers developed, 1244 primer sets produced strong PCR products, of which 905 (72.7%) were polymorphic in kelp grouper. Cross-species utility of the 905 polymorphic SSR markers was tested in four additional Epinephelinae species of Hyporthodus septemfasciatus, Plectropomus leopardus, Epinephelus lanceolatus and Epinephelus coioides. Results revealed that, respectively, 401 (44.3%), 136 (15.0%), 434 (49.0%) and 538 (59.4%) SSRs showed specific polymorphic products. Of these, 40 SSR markers (33 di-, 1 tri- and 6 tetra-nucleotides) showed polymorphism in all species tested. Additionally, three AGAT SSR motifs which accounted for 42.9% of the nondi-nucleotide markers were found in the 40 SSR markers. This indicates that the AGAT SSR motif has a high potential as a highly versatile SSR marker in grouper Epinephelinae. The SSR markers developed in this study can be employed to obtain reliable genetic variability estimates for groupers (Epinephelinae).

Advances in the Study on Nutrient Requirements of Grouper (Epinephelus sp.): a Review

The paper reviews the recent advances in studying grouper nutrition requirement for the development of cost-effective and environmentally friendly artificial diets. It consists of seven parts: protein and amino acid, lipid and essential fatty acid, carbohydrate, vitamin, mineral, alternative protein source, broodstock and larval nutrition. The review provides some basic information for further investigation of nutrient requirements of groupers.

Optimum temperature for the growth performance of juvenile orange-spotted grouper (Epinephelus coioides H.)

Effects of water temperature （17, 21, 25, 30 and 35℃） and body size （14.75-281.41 g initial body weight） on food consumption, growth, feed conversion, and dry matter content in orange-spotted grouper fed to satiation were investigated. The combined effect of temperature （T, ℃） and body weight （W, g） on maximum food consumption （Cmax, g/day） was described as： InCmax=-7.411＋0.828 InW＋0.317T4）.004 7T2, and the optimum feeding temperature was 33.9℃. The combined effect of temperature and body weight on growth （G） was described as： lnG=-4.461-0.2081nW＋0.394T-0.006 3T^2. The optimum growth temperature was 31.4℃, whereas overall growth rates were high at 25, 30 and 35 ℃. Feed conversion efficiencies （FCE, %）, increasing first and then decreasing with increasing temperature, averaged from 1.8 to 2.1 in terms of dry weight of food fish. The optimum temperature for FCE tended to be lower than that for growth or feeding. Dry matter content increased with both increasing water temperature （17, 25, 30 and 35℃） and body weight, and the combined effect of temperature and body weight on dry matter content （DM, %） was described as： lnDM =3.232＋0.01 4 lnW-0.004 4T＋0.001 2TInW.

Soybean meal as a source of protein in formulated diets for tiger grouper, <i>Epinephelus fuscoguttatus</i>juvenile. Part I: Effects on growth, survival, feed utilization and body compositions

The effects of fish meal (FM) replacement with graded level of soybean meal (SBM) on growth performance, feed utilization, survival rate and body composition were investigated in juvenile tiger grouper, Epinephelus fuscoguttatus (initial body weight 13.9 ±0.65 g). Six experimental diets were formulated to contain 50% crude protein, 16% crude lipid and 365.8 kcal/100g feed with SBM replacing FM protein at 0% (SM 0), 10% (SM 0), 20% (SM 20), 30% (SM 30), 40% (SM 40) and 20% with phytase (SM 20P) replacement levels. At the end of the ten-week feeding trial, there were no significant differences detected in terms of growth performances (weight gain and specific growth rate), feed conversion ratio (FCR) and survival rates of fish fed with the control diet (SM 0), SM 20, SM 30 and SM 20P. Net protein utilization of fish fed SM 20P was higher than those fed with other diets suggesting an improved utilization of nutrients with phytase addition in the diet. Replacements of FM protein with SBM at 10% and 40% have resulted in significantly lower growth and poorer FCR than other replacement levels. Survival rates remain high (≥90%) throughout the trial. Whole-body proximate composition of the fish was significantly affected by the inclusion of SBM in the diets. It can be concluded that 20%-30% of FM protein replacement with SBM is recommended and addition of phytase in the SBM-based diet should be considered to improve nutrient utilization of tiger grouper juvenile.

The mechanism of retinomotor movements in the retina of banded grouper (Epinephelus awoara)

In order to clarify the mechanism of retinomotor movements in banded grouper, this paper utilizes inhibition factors of microfilaments and microtubules cytochalasin-B and colchicine to study the roles of actin microfilaments and microtubules in the movements of the REP pigment granles and cones. It provides evidences to explain the mechanism of retinomotor movements in fish.

饲料中锌含量对斜带石斑鱼(Epinephelus coioides)生长、饲料效率、矿物质含量和体组成的影响

以ZnSO4·7H2O为锌源,用锌添加量为0、20、40、60、80和100mg/kg的实验饲料投喂斜带石斑鱼12周,研究其对斜带石斑鱼生长、饲料效率、矿物质含量和体组成的影响。结果表明,各实验组的增重率,饲料效率,存活率和全鱼、脊椎骨、鳞片的锌含量无显著差异。全鱼、脊椎骨和鳞片的锰含量均随着饲料中锌的添加而降低,锰和锌存在明显的拮抗关系。全鱼的铁含量随着饲料中锌的添加而降低。全鱼、脊椎骨和鳞片的钙、磷、镁含量均不受饲料中锌含量的影响。饲料中添加锌对全鱼水分、灰分,肌肉水分、脂肪,形态学指标,红细胞比容和血红蛋白含量没有显著影响。饲料中不添加锌时,全鱼的蛋白、脂肪和肌肉的蛋白、灰分含量较低。实验结果表明基础饲料中33mg/kg的锌已经满足斜带石斑鱼生长的需求,能使鱼体锌达到饱和。

七带石斑鱼(Epinephelus septemfasciatus)两个野生群体形态差异分析

对2种不同地理种群七带石斑鱼进行14项形态性状的测定,采用2种多元统计分析方法和t-检验方法,比较了2个群体的外部形态特征。t-检验结果表明,2群体在14个性状中有12个性状表现出极其显著差异(P<0.01),1个性状表现出差异显著(P<0.05)。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29.873%、25.091%和16.875%,三个主成分的累积贡献率为71.804%,2群体之间存在明显的偏离态势,形成两个不同的类群,2群体在主成分1轴上的差异较在主成分2轴上的差异更为明显,主成分1主要由经t-检验差异指标数量多和差异程度大的性状组成。判别分析表明,2个七带石斑鱼群体的形态差异显著(P<0.0001),建立了2个群体的判别函数,其判别准确率及综合判别率均为100%,可以认为逐步判别法对七带石斑鱼不同群体的初步鉴定是可行的。差异系数(C.D)计算的结果表明,尽管2群体在形态上存在一定的差异,但尚未达到亚种水平。主成分分析、t-检验和判别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。

饲料中高水平铜对斜带石斑鱼(Epinephelus coioides)生长和铜、铁、锰、锌含量的影响

采用铜适量(4.47mg/kg)和铜过量(1127.51mg/kg)的两种饲料投喂初始体重为(12.9±0.2)g的斜带石斑鱼(Epinephelus coioides)8周,研究饲料中过量铜对斜带石斑鱼生长性能、形态学指标、体组成和鱼体微量元素含量的影响。结果表明,当饲料铜含量由4.47mg/kg升高到1127.51mg/kg时,斜带石斑鱼增重率由228%下降到63%,饲料效率由1.12下降到0.51,肠脂比由2.90%下降到1.69%。全鱼和肝脏中的铜含量均随饲料铜水平的增加而显著增加,斜带石斑鱼全鱼铜含量由2.98mg/kg上升到34.98mg/kg,肝脏铜含量由36.6mg/kg上升到1364.0mg/kg。但饲料中过量铜对斜带石斑鱼的存活率、鱼体肥满度、脏体比、肝体比和全鱼体组成没有显著性影响。摄食高铜饲料的斜带石斑鱼肝脏锌含量显著升高,锌可能在铜的解毒过程中起着重要的作用。

斜带石斑鱼(Epinephelus coioides)饲料中铜的适宜添加量研究

以初始体重为(13.0±0.3)g的斜带石斑鱼(Epinephelus coioides)为实验对象,用铜添加量为0、1、3、5、10和20mg/kg的实验饲料投喂斜带石斑鱼8周,研究斜带石斑鱼的铜需求量。结果表明,饲料中添加0—20mg/kg铜对石斑鱼生长、饲料效率、存活率、形态学指标和红细胞比容没有显著性影响。随饲料铜水平的上升,斜带石斑鱼全鱼和肝脏的铜含量呈先上升后平稳的趋势,当饲料铜添加量达到或超过3mg/kg时,石斑鱼全鱼和肝脏的铜含量趋于平稳。折线模型表明,饲料中铜总含量为5—5.5mg/kg时,石斑鱼全鱼和肝脏的铜含量能够达到饱和。当饲料中铜总含量≥6.00mg/kg时,全鱼的铁、锰、锌含量显著下降。因此,根据全鱼和肝脏铜含量的折线模型分析,考虑到饲料铜对全鱼铁、锰、锌含量的影响,作者认为当基础饲料铜含量为1.50mg/kg时,饲料中的最适铜添加量为3mg/kg,饲料中铜总量为5—5.5mg/kg左右。

维生素C对点带石斑鱼(Epinephelus coioides)生长及组织中维生素C积累量的影响

采用单因素实验设计方法 ,进行了饲料中添加维生素C对点带石斑鱼生长和组织中维生素C积累量影响的研究 ,设计了 6个不同维生素C水平 ( 0、35、70、1 40、2 80、70 0mg/kg)的等氮等能饲料 ,对石斑鱼幼鱼进行为期 8周的生长实验 ,每个水平三个重复 ,每个养殖单元放养初始体重为 1 0g左右的点带石斑鱼幼鱼 2 0尾。饲养实验在海水网箱 ( 1 .5m× 1m× 2 .0m)中进行。结果表明 ,饲料中未添加维生素C组的增重率、特定生长率以及存活率显著低于维生素C添加组 ,饲料中添加维生素C的浓度为 70mg/kg组的增重率和特定生长率最高。对鱼体生物学指标的分析表明 ,饲料中添加维生素C并不影响鱼体的肥满度和内脏比指数 ,却显著影响肝体比指数。鱼体水分、蛋白质和灰分含量不受饲料中维生素C添加水平的影响 ,然而对鱼体脂肪含量的影响显著。随着饲料中维生素C添加水平的提高 ,石斑鱼肌肉和肝脏中维生素C的积累量显著升高。以增重率和特定生长率为指标 ,点带石斑鱼维生素C的适宜需要量为70mg/kg。

激素诱导斜带石斑鱼(Epinephelus coioides)雄性化的研究

石斑鱼属雌雄同体雌性先成熟鱼类,低龄鱼卵巢先成熟,表现为雌性,高龄鱼才性转变为雄性。本实验通过持续跟踪取样,分析了人工养殖条件下斜带石斑鱼(Epinephelus coioides)在36月龄内的性腺发育过程,发现其性分化完成后,雌性卵巢在9月龄后开始发育。因此,采取投喂含有雄激素甲基睾酮(MT)饲料的方法,可以在石斑鱼卵巢发育之前,成功诱导8月龄的斜带石斑鱼幼鱼向雄性方向发育。结果表明,以MT 10mg/kg饲料的剂量投喂3个月能够有效诱导卵巢未发育的幼鱼往雄性方向发育;但停止投喂MT后,幼鱼精巢又开始出现退化,不会继续往雄性方向发展,而转变为雌性。以上结果表明,斜带石斑鱼性别决定和分化除了受遗传因子和内分泌调控外,还受到年龄、体型和群体、环境等因子的影响,人工投喂MT激素的作用并不能从根本上影响石斑鱼幼鱼的性腺发育的方向,只有在持续剂量的MT激素诱导下,石斑鱼幼鱼才能发育为成熟的雄鱼。

斜带石斑鱼(Epinephelus coioides)两种催乳素受体基因的cDNA克隆和mRNA表达分析

采用RT-PCR方法克隆了斜带石斑鱼两种催乳素受体(Prolactin receptor,PRLR)的cDNA序列,序列分析表明:PRLR1开放阅读框为1947bp,共编码649个氨基酸,PRLR2开放阅读框为1749bp,共编码487个氨基酸,PRLR1与PRLR2的氨基酸同源性为40.4%。用Real-time RT-PCR方法研究了PRLR1和PRLR2在各组织和早期发育不同阶段的表达情况,结果表明:PRLR1和PRLR2在所检测的12种组织中均有表达,其中以鳃、肾、肠表达量较高;PRLR1在受精期表达最高,PRLR2在视囊形成期表达最高,而且除受精卵期外PRLR2在各时期的表达量均高于PRLR1。

半胱胺对斜带石斑鱼(Epinephelus coioides)生长激素及其受体mRNA水平的影响

以海水硬骨鱼类斜带石斑鱼为研究对象,通过投喂和腹腔注射半胱胺盐酸盐(CSH),研究半胱胺对斜带石斑鱼生长激素(GH)及其受体(GHR)mRNA水平的影响,以初步揭示半胱胺促进斜带石斑鱼生长的作用机理。结果表明,长期投喂CSH可以显著提高斜带石斑鱼的相对体重增长率。投喂和腹腔注射不同剂量的CSH均能明显促进斜带石斑鱼脑垂体GHmRNA水平及肝脏2种gGHRmRNA水平,且促进效果与CSH浓度呈剂量依存关系。本研究还就CSH对斜带石斑鱼2种不同类型GHR作用的剂量效应和时间效应进行了初步探讨。通过注射不同剂量CSH发现,低剂量CSH(20μg/g体重)只可以引起gGHR2mRNA水平的显著提高。中剂量CSH(50μg/g体重)可同时显著提高gGHR1和gGHR2mRNA水平,但gGHR2水平明显高于gGHR1水平;通过单独注射50μg/g体重CSH溶液发现,注射后2h,2种gGHRmRNA水平均有显著升高,其中gGHR2水平明显高于gGHR1。注射后6h,gGHR2mRNA水平率先达到最高值。注射后12h,gGHR1mRNA水平达到最大值。由此可以推测gGHR2对于CSH所引起刺激的剂量敏感性及时间敏感性均高于gGHR1。

斜带石斑鱼(Epinephelus coioides)GnRH Ⅱ基因的克隆及启动子分析

促性腺激素释放激素(Gonadotrophin-releasing hormone,GnRH)是调控脊椎动物生殖的关键因子之一。文章克隆了经济鱼类斜带石斑鱼(Epinephelus coioides)GnRHⅡ基因的cDNA、基因组序列及其启动子,发现GnRHⅡ基因启动子中与组织特异性表达相关的区域位于距转录起始点上游2005 bp～956 bp之间。以GnRHⅡ启动子驱动绿色荧光蛋白在斑马鱼胚胎中表达的结果显示,GnRHⅡ神经元主要分布在中脑附近和眼部。文章为深入了解GnRHⅡ基因表达的调控机制及GnRHⅡ基因的功能提供了基础信息。