豹纹鳃棘鲈差异流速下肝脏转录组分析

水流速度是影响鱼类生长的重要环境因子之一.为探究豹纹鳃棘鲈(Plectropomus leopardus)在不同流速条件下相关基因的功能和表达情况,利用RNA-Seq技术对差异流速下的豹纹鳃棘鲈肝脏组织进行了转录组分析.挑选相同繁育批次中规格一致的豹纹鳃棘鲈幼苗,分别在正常流速(0.1 m·s?1,Low flow v...     

豹纹鳃棘鲈消化道粘液细胞的类型与分布

应用AB-PAS染色法对工厂化养殖条件下豹纹鳃棘鲈(Plectropomus leopardus)消化道粘液细胞的类型与分布进行了观察研究。结果显示:豹纹鳃棘鲈粘液细胞在消化道各部位均有分布,不同部位的粘液细胞密度和类型均有差异,豹纹鳃棘鲈消化道粘液细胞可分为Ⅰ型、Ⅱ型、Ⅲ型、Ⅳ型。其中,食道粘液细胞以Ⅲ型为主,胃以Ⅰ型为主,幽门盲囊和肠以Ⅲ型为主,直肠以Ⅲ型和Ⅳ型为主;胃部粘液细胞分布密度最大,达(111.37±21.74)cell·mm~(-2),极显著高于消化道其它部分,直肠、幽门盲囊、肠的粘液细胞分布密度分别为(35.09±8.74)、(30.23±5.37)、(28.00±4.67)cell·mm~(-2),相互之间差异不显著,食道的粘液细胞分布密度最低,为(10.83±1.94)cell·mm~(-2),与消化道其它部分相比差异极显著。另外,消化道各段单位面积内粘液细胞的分泌能力顺序为食道直肠肠幽门盲囊胃。通过对消化道各部位粘液细胞的分类对比可以发现,粘液细胞的类型和分布与其所在部位的功能有密切关系。     

工厂化养殖条件下豹纹鳃棘鲈消化系统组织学的观察

采用解剖和组织切片技术,对工厂化养殖条件下全程投喂人工配合饲料的豹纹鳃棘鲈(Plectropomus leopardus)消化系统形态学和组织学进行研究。豹纹鳃棘鲈的消化道由食道、胃、幽门盲囊和肠道构成,消化道管壁由粘膜层、粘膜下层、肌层和浆膜层组成。食道短而粗,粘膜层向内腔突起形成纵行褶皱,上皮表层排列有杯状细胞。胃部膨大,为"Y"形,未见有杯状细胞,食道与胃的肌层均发达。幽门盲囊有3条,组织学结构与小肠相似。肠道在腹腔内呈"Z"形排列,比肠长为0.72±0.04,肠粘膜层向腔内突起形成发达的褶皱,多数形成二级褶皱,肠道各段均分布有杯状细胞。消化腺由肝胰脏和胆囊组成,肝脏分三叶,胰腺在肝脏内呈弥散性分布,胆囊呈长条状。本研究讨论了工厂化养殖条件下豹纹鳃棘鲈消化系统组织学特征与消化、吸收的关系,认为豹纹鳃棘鲈消化系统具备典型肉食性鱼类消化系统特征。     

豹纹鳃棘鲈抗哈维氏弧菌遗传参数分析

哈维氏弧菌(Vibrio harveyi)是引起豹纹鳃棘鲈(Plectropomus leopardus)患“烂身病”的主要致病菌,每年6―8月发病率极高,严重影响了该品种养殖业的可持续发展。因此,培育抗病良种是豹纹鳃棘鲈养殖业的迫切需求。为评估豹纹鳃棘鲈抗哈维氏弧菌遗传参数,本研究基于高密度单核苷酸多态性位点构建的基因组亲缘关系矩阵,使用4种模型(BLM、BTM、LLM和LTM)拟合了2种抗病表型(测试日性状,TDS;二元死亡存活性状,TS),并用约束最大似然法(REML)估算方差组分。经分析,豹纹鳃棘鲈抗哈维氏弧菌遗传力为0.182~0.486,属中高遗传力性状,加性遗传方差为0.071~0.262。其中,利用线性模型(BLM和LLM)估算的遗传力分别为0.382和0.476,利用阈值模型(BTM和LTM)估算的遗传力分别为0.182和0.207。表明可以通过遗传选育提高豹纹鳃棘鲈抗弧菌能力。对不同模型估算的基因组估算育种值(GEBV)进行相关性分析,不同模型拟合同种抗病表型时,GEBV之间相关系数> 0.9,属于高强度正相关关系,表明使用同种表型定义时,阈值或线性模型对GEBV排名影响很小。对不同模型估算的GEBV与不同表型进行相关性分析的结果显示,纵向模型(LLM和LTM)估算的GEBV与表型TS之间的相关系数高于横截面模型(BLM和BTM),说明表型TDS可能比表型TS更适合作为抗病表型。此外,在线性模型中,使用表型TDS和表型TS估算的GEBV之间的相关系数< 0.85,说明采用2种表型定义下估计的豹纹鳃棘鲈抗哈维弧菌GEBV排名不一致。但基于表型TDS估算的GEBV与表型TS之间的相关系数较强(0.824),表明使用表型TDS和纵向模型(LLM)估算豹纹鳃棘鲈抗哈维氏弧菌遗传参数更有优势。本研究补充了豹纹鳃棘鲈抗哈维氏弧菌遗传参数研究,为豹纹鳃棘鲈抗哈维氏弧菌良种选育提供了参考。     

豹纹鳃棘鲈室外大型水泥池的人工育苗技术

为了探索利用室外大型水泥池进行豹纹鳃棘鲈(Plectropomus leopardus)人工育苗的技术模式,通过在室外大型水泥池顶部加盖遮光太阳板半封闭顶棚,对育苗水体进行控光、控温和防雨,进行该鱼人工育苗过程中水处理技术、饵料培育技术和育苗方法的研究。2010年3～7月,累计放卵5批次,共培育出全长2.5～3.0 cm的鱼苗21.9×104ind,受精卵平均孵化率达81.4%,育苗平均成活率达5.59%;初孵仔鱼平均全长1.6mm,在早期发育阶段每经历一次饵料转变,都会带来仔稚幼鱼的快速增长;该鱼摄食时间主要集中在白天,随着个体生长发育,饵料出现顺序依次为原生动物、轮虫、桡足类、蒙古裸腹蚤。研究表明,豹纹鳃棘鲈室外大型水泥池人工育苗模式克服了池塘育苗受自然环境影响大和室内水泥池育苗水体小、开口饵料匮乏的缺点,育苗成功率和出苗率比较稳定。     

4月龄豹纹鳃棘鲈形态性状对体质量的影响分析

摸清豹纹鳃棘鲈(Plectropomus leopardus)形态性状与体质量的关系对其选育工作具有重要意义。以同一批次159尾4月龄豹纹鳃棘鲈为试验对象,测量并探寻其形态性状对体质量的影响效应。结果表明,豹纹鳃棘鲈吻长与体质量的相关性为显著水平(P<0.05),其余形态性状与体质量的相关性均为极显著水平(P<0.01);通径分析结果发现,体长、体高、体宽、头高和吻长是影响4月龄豹纹鳃棘鲈体质量的主要形态性状,其中体高主要是通过直接作用影响体质量,而体长、体宽和头高则主要通过体高间接影响体质量;体长、体高、体宽、头高和吻长性状对体质量的共同决定系数之和大于0.85,说明4月龄豹纹鳃棘鲈体质量主要受这5个性状影响。通过回归分析建立多元回归方程,剔除与体质量相关性不显著的形态性状,所得方程保留的形态指标与通径分析结果一致。     

养殖密度对豹纹鳃棘鲈生长和血液生化指标的影响

采用实验生态学方法研究了饥饿对豹纹鳃棘鲈(Plectropomus leopardus)仔鱼形态发育、生长和成活的影响,并测定了饥饿仔鱼的初次摄食率和不可逆点(PNR)。结果显示,水温23~24 ℃下豹纹鳃棘鲈仔鱼2.5 DPH(孵化后天数)开口摄食,3.0 DPH卵黄囊和油球消失,混合营养期仅0.5 d,容易遭受饥饿胁迫。饥饿仔鱼最高初次摄食率为65%,出现在3.5 DPH; PNR发生在4.5~5.0 DPH,仔鱼具有初次摄食能力的时间仅2.0~2.5 d。对照组和饥饿组仔鱼在3.5 DPH前全长无明显差异(P>0.05),之后饥饿组仔鱼出现生理性萎缩,全长开始与对照组差异显著(P < 0.05)。饥饿组仔鱼在内源性营养消耗完毕后出现了一系列因饥饿所导致的形态发育特征,并于6.5 DPH全部死亡,因此豹纹鳃棘鲈仔鱼最佳投喂时间为2.5~3.5 DPH。

     

豹纹鳃棘鲈类结节症病原的分离鉴定

为查明养殖豹纹鳃棘鲈类结节症的病因,自患病豹纹鳃棘鲈内脏中分离到1株优势菌株061101,人工感染试验证实,该菌具有强毒力,能够引起被感染鱼内脏出现白色结节并死亡,为引起此次豹纹鳃棘鲈类结节症的病原菌。采用形态学、生化特性测定对菌株061101进行鉴定,同时采用细菌16S rRNA通用引物进行PCR扩增得到该菌的16S rRNA基因序列,测序后比对,构建系统发育树。试验结果表明,菌株061101与发光杆菌属中的美人鱼发光杆菌杀鱼亚种基本特征相符;基于16S rRNA基因序列构建的系统发育树将菌株061101与美人鱼发光杆菌杀鱼亚种聚为一支,置信度达100%。据此确定,此次豹纹鳃棘鲈类结节症的病原为美人鱼发光杆菌杀鱼亚种。药敏结果显示,该菌株仅对链霉素(300μg/片)、庆大霉素(120μg/片)敏感,对多黏菌素B、阿洛西林等中度敏感,对氟苯尼考、恩诺沙星、强力霉素等均耐药。     

不同流速对豹纹鳃棘鲈摆尾频率和血液生理的影响

水流是影响鱼类生理和生长的重要生态因子之一,探究豹纹鳃棘鲈(Plectropomus leopardus)受流速胁迫呈现的应激水平变化,掌握其耐受的最大流速,可为网箱养殖选址、集约化流水养殖和深远海工船养殖提供理论基础。以体长(11.38±1.48) cm、体质量(34.71±11.57) g的豹纹鳃棘鲈为研究对象,通过自制鱼类游泳实验装置,设计静水对照组[0 cm·s^-1或0 BL·s^-1 (体长·秒-1)]和3组实验组(11.4、22.8和34.2 cm·s^-1对应1、2和3 BL·s^-1),探究40 min水流刺激对豹纹鳃棘鲈摆尾频率变化、血糖、血液中乳酸和皮质醇含量的影响。结果显示,摆尾频率与水流速度之间呈线性正相关关系;血糖和血液中皮质醇含量随摆尾次数增多呈线性增长趋势,乳酸随摆尾次数增多呈非线性增长趋势。超过2 BL·s^-1流速会导致豹纹鳃棘鲈血液中皮质醇含量显著升高(P<0.05)。当流速达3 BL·s^-1时,血液中乳酸和血糖含量显著升...     

海南椰林湾工厂化养殖豹纹鳃棘鲈的病害及其防控

为了解海南琼海椰林湾地区豹纹鳃棘鲈在工厂化养殖期间的病害情况,于2011~2012年进行了调查。结果:豹纹鳃棘鲈工厂化养殖期间的寄生虫病主要由纤毛类原生动物,如淀粉卵鞭虫、刺激隐核虫、车轮虫等病原引起,偶尔会寄生鱼蛭、本尼登虫等大型寄生虫,纤毛类原生动物寄生虫病一般在每年的4、5、6和9、10、11月份发病率较高,而其它类寄生虫病发病率较低;由弧菌引起的烂身病主要在每年的12、1、2月份水温低于23℃时暴发,养殖用水未经紫外线消毒时,发病率可高达30%以上,而养殖用水经紫外线消毒后,发病率低于5%。针对上述发病规律,定期对鱼和水体进行药物预防,可起到很好的防控效果。     

Increasing survival and growth in larval leopard coral grouper (Plectropomus leopardus) using intensively cultured Parvocalanus crassirostris nauplii

Leopard coral grouper, Plectropomus leopardus are a heavily exploited, high-value fish commonly found in the Asian live reef food fish trade. In past decades, many attempts at the mass culture of various grouper species have been undertaken; however, their small mouth gape at first feed has resulted in very low survival when using traditional live feeds such as rotifers. The use of wild caught or extensively cultured copepods has yielded potentially promising increases in survival and growth, but overall survival to the juvenile stage remains low, making mass culture currently impractical. The current study sought to build on past developments in grouper culture and recent advancements in copepod culture technology by observing how growth and survival were influenced by the addition of intensively cultured copepods to the early diet of P. leopardus larvae. Six tanks of larvae, three replicates per treatment, were fed either eggs and nauplii of the calanoid copepod Parvocalanus crassirostris, at a starting density of 5 mL⁻¹, and the rotifer Brachionus rotundiformis, at a starting density of 10 mL⁻¹, or were fed only B. rotundiformis, at a density of 15 mL⁻¹, starting on the evening of 2 days post-hatch (dph) and continuing until 9 dph. After this initial period, all larvae were fed the same diet of rotifers, Artemia, and dry feed until the cessation of the trial at 21 dph. Larvae fed P. crassirostris in addition to rotifers had a significantly higher survival, 9.9 versus 0.5%, than those fed only rotifers. Growth was also significantly enhanced in larvae offered copepods. Larvae only fed rotifers were, on average, 1.5 mm shorter at 21 dph than those that had been fed copepods. More rapid development and the earlier onset of flexion were also noted in the larvae that were offered copepods. The use of intensively cultured copepods, in this study, increased survival tenfold over previous studies, with P. leopardus larvae fed wild-caught copepods. The application of intensively cultured copepods to the early diet of P. leopardus, along with future research to evaluate late-stage mortality issues, may facilitate commercial production of this species.     

罗非鱼HSP70(tHSP70)蛋白的生物信息学分析、真核表达及质谱鉴定

运用生物信息学技术分析罗非鱼热休克蛋白70(tilapia heat shock protein,tHSP70)的理化特性、糖基化位点、跨膜区域、蛋白的细胞定位及信号肽与虹鳟等其他动物HSP70的相似性,以人工合成cDNA为模板,通过聚合酶链式反应扩增其完整CDS,并将此DNA片段与真核表达载体pGAPZa-A连接,构建pGAPZa-HSP70表达质粒,在毕赤酵母GS115中表达tHSP70蛋白,对表达上清液进行SDS-PAGE及Western-blotting分析后,采用Ni²⁺IDA层析脱盐纯化目的蛋白,并对所表达的蛋白进行糖基化PAS染色鉴定。对表达的蛋白进行SDS-PAGE后,将所获得的非预定大小(约100 ku)条带进行电离飞行时间质谱鉴定,并确定其蛋白种类。结果表明:tHSP70所含氨基酸数量为640,分子量为70 274.5 u,等电点为5.49。在哺乳动物网织红细胞(体外)的半衰期为30 h,在酵母内半衰期大于20 h,而在大肠杆菌内的半衰期也大于10 h,不稳定指数为36.64,脂肪族指数为85.58。可能分别含有6个N-和O-糖基化位点,所克隆tHSP70基因与目的基因完整编码区序列完全一致,所构建的pGAPZa-HSP70质粒能在GS115中成功表达,诱导表达上清液经SDS-PAGE及Western-blotting分析后发现,除70 ku目的蛋白外,还出现一条100 ku条带。经糖基化PAS染色证明,此100 ku蛋白可能是HSP70糖基化的结果,且能被人的兔抗HSP70抗体识别,质谱鉴定证明该条带即是tHSP70蛋白。本研究所表达tHSP70蛋白为后续罗非鱼细胞学及抗原提呈等免疫学研究奠定了基础。     

Morphological development and growth patterns of the leopard grouper Mycteroperca rosacea during larval development

At 25 °C, metamorphosis in leopard grouper Mycteroperca rosacea larvae took 60 days after hatching. The total length at day 1 was 1.95±0.22 mm and juveniles reached a length of 30.64±0.23 mm at day 60; the increase was approximately linear. We describe eight stages of development during this period. Larvae with the yolk sac attached occur from days 1 to 4 (Stages 1 and 2). The preflexion occurs on days 5–20 (Stages 3 and 4). Bending notochord occurred at day 25 (Stage 5). The other morphological changes that precede the juvenile phase occurred progressively until day 60 (Stages 6–8). Allometric growth in the height and length of the head, trunk length, height and length of the tail and the diameter of the eye compared with the total length showed two distinct stages of growth. Inflexion point, where growth is positive, occurred when larvae reached between 18.75 and 21.59 mm, which corresponds to larvae at days 35–40.     

Changes in digestive enzyme activities during larval development of leopard grouper (Mycteroperca rosacea)

The leopard grouper is an endemic species of the Mexican Pacific with an important commercial fishery and good aquaculture potential. In order to assess the digestive capacity of this species during the larval period and aid in the formulation of adequate weaning diets, this study aimed to characterize the ontogeny of digestive enzymes during development of the digestive system. Digestive enzymes trypsin, chymotrypsin, acid protease, leucine–alanine peptidase, alkaline phosphatase, aminopeptidase N, lipase, amylase and maltase were quantified in larvae fed live prey and weaned onto a formulated microdiet at 31 days after hatching (DAH) and compared with fasting larvae. Enzyme activity for trypsin, lipase and amylase were detected before the opening of the mouth and the onset of exogenous feeding, indicating a precocious development of the digestive system that has been described in many fish species. The intracellular enzyme activity of leucine–alanine peptidase was high during the first days of development, with a tendency to decrease as larvae developed, reaching undetectable levels at the end of the experimental period. In contrast, activities of enzymes located in the intestinal brush border (i.e., aminopeptidase and alkaline phosphatase) were low at the start of exogenous feeding but progressively increased with larval development, indicating the gradual maturation of the digestive system. Based on our results, we conclude that leopard grouper larvae possess a functional digestive system at hatching and before the onset of exogenous feeding. The significant increase in the activity of trypsin, lipase, amylase and acid protease between 30 and 40 DAH suggests that larvae of this species can be successfully weaned onto microdiets during this period.     

Influence of light intensity on feeding, growth, and early survival of leopard coral grouper (Plectropomus leopardus) larvae under mass-scale rearing conditions

This study investigated the effect of different light intensities on feeding, growth and survival of early stage leopard coral grouper Plectropomus leopardus larvae. Four different light intensities (0, 500, 1000 and 3000 lx) were used and larvae were kept under constant light conditions from 0 day after hatching (DAH) to 5 DAH. The larvae were fed a small S-type of Thai strain rotifers at a density of 20 individuals/mL from 2 DAH. The number of rotifers in larval digestive organ and total length of larvae were examined at 3 h intervals between 04:00 and 22:00 h on 3 DAH, and thereafter at 6 h intervals until the end of the experiment (5 DAH). Four experimental trials of the larval rearing were repeated using by 60 kL mass-scale rearing tanks. The results indicate that coral grouper larvae are visual feeders and their food intake increases with increasing light intensity. Food intake of larvae reared at 3000 lx was significantly higher than those reared at 0–1000 lx on 3 DAH despite being the first-feeding day (P < 0.01). On 4 DAH, total length of larvae reared at 3000 lx was significantly larger than those reared at the lower light intensities (0, 500 and 1000 lx), and thereafter light intensity significantly influenced larval feeding and growth until the end of the experiment. Survival on 5 DAH did not show a significant difference between light intensities, but survival rate at 3000 lx and 1000 lx had a tendency to be higher than those reared at the lower light intensities (0 and 500 lx). In contrast, larvae reared at 0 lx exhibited stagnant and/or negative growth. These results indicate that light intensity is significantly the factor affecting larval feeding, growth, and survival in coral grouper larvae under the rearing conditions.     

Influence of dietary protein level on growth performance,digestibility and activity of immunity‐related enzymes of leopard coral grouper,Plectropomus leopardus (Lacépède, 1802)

An 8‐week feeding trial was conducted to assess the interaction between dietary protein levels and fish growth, digestibility and activity of immunity‐related enzymes of Plectropomus leopardus. Five diets with different protein levels (400 g/kg, 450 g/kg, 500 g/kg, 550 g/kg and 600 g/kg protein) were designed. P. leopardus fed with 500 g/kg, 550 g/kg and 600 g/kg dietary protein, showed higher weight gain rates than fish fed 400 g/kg and 450 g/kg dietary protein. Ingestion rate in fish fed with 500 g/kg dietary protein was significantly higher than those with other diets. P. leopardus fed with 500 g/kg, 550 g/kg and 600 g/kg dietary protein, showed that feed coefficients were significantly lower than those fed with 400 g/kg and 450 g/kg dietary protein. Net protein utilization was significantly lower in fish fed with 400 g/kg diet than those with other diets. Fish fed with 400 g/kg and 450 g/kg dietary protein had an apparent feed digestibility coefficient for dry matter that was significantly lower than that with other diets. Protease activity was highest in fish fed on 500 g/kg dietary protein. Fish fed with 500 g/kg dietary protein, had the highest superoxide dismutase activity. Fish fed with 600 g/kg dietary protein, had the highest alkaline phosphatase activity. Thus, a diet containing 500 g/kg protein is recommended for P. leopardus aquaculture.