Rapid detection of SNPs in candidate genes regulating the growth of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), using semiconductor sequencing

Orange‐spotted grouper (Epinephelus coioides) is one of the most important marine food species in Southeast Asia and China and has been cultured for decades. In this study, we fully utilized the limited capacity of semiconductor sequencing, the high efficiency of long‐range PCR for target enrichment and a non‐indexed pooling strategy to screen single‐nucleotide polymorphisms (SNPs) in a breeding population of orange‐spotted grouper. Forty‐one genomic DNA fragments, with a total length of approximately 180 kb, including 22 candidate genes that control growth, and from a DNA pool of 20 heaviest and 22 lightest individuals of the sampled population were successfully sequenced using an Ion Torrent Personal Genome Machine. 3 503 466 clear reads were produced with a length of 192 ± 56 bp, 86.8% of which were mapped to the reference with an average coverage depth of 2567‐fold and physical coverage of 98.8%. Finally, 1623 high‐quality SNPs were adopted. Compared with Sanger sequencing of three random common regions, the sensitivity and specificity of our approach were 39.4% and 100.0% respectively. A mutation located at the third position of the previously labelled start codon of growth hormone receptor type 1 invalidated the start codon. Furthermore, comparison of the frequencies of genotypes and alleles of this site between the two extreme groups, prediction of signal peptide and identification of conservative mRNA sequences suggested that the functional start codon is likely located at the position of another downstream in‐frame ATG in the mutant. These detected SNP markers will provide important tools for the selective breeding of orange‐spotted grouper.     

Effects of dietary protein and lipid levels with different protein‐to‐energy ratios on growth performance,feed utilization and body composition of juvenile red‐spotted grouper,Epinephelus akaara

An 8‐week feeding trial was conducted to assess dietary protein and lipid levels on growth performance, feed utilization and body composition of juvenile red‐spotted grouper (7.85 ± 0.03 g fish^?1). Nine semi‐purified diets were formulated containing varying protein levels (440–520 g kg^?1, dry matter) and lipid levels (60–120 g kg^?1, dry matter). The weight gain of juvenile Epinephelus akaara was affected by dietary protein (p = .005) and its interaction with dietary lipid (p = .020). Viscerosomatic index, intraperitoneal fat ratio and whole‐body lipid level increased with increasing dietary lipid level (p < .001). Nitrogen retention was not affected by dietary protein and lipid, while lipid retention decreased with increasing dietary lipid level (p < .001). The plasma blood urea nitrogen increased with increasing dietary protein level (p = .003). This study showed that diet with 520 g kg^?1 protein and 60 g kg^?1 lipid with 30.58 mg kJ^?1 P:E provided a maximal growth for this species. Moreover, an increase in dietary lipid levels (from 60 to 90 g kg^?1) could reduce the protein requirement (from 520 to 480 g kg^?1) without affecting the growth performance, while higher fat deposition was observed in fish fed high‐lipid diets.     

荧光定量PCR法检测点带石斑鱼神经坏死病毒研究

根据己公布的神经坏死病毒全基因序列,应用引物设计软件依据Gene Bank所收录的各种石斑鱼神经坏死病毒RNA2基因组全序列选择保守区域,根据Taqman探针引物设计原则,设计Taq Man探针及其引物。采用Taq Man探针技术,并且将假病毒技术构建的含有神经坏死病毒RNA2基因组的MS2噬菌体假病毒作为阳性质控品,建立Taq Man探针检测点带石斑鱼神经坏死病毒的荧光定量PCR方法,拟合标准曲线,能够在病毒检测中进行绝对定量,能检测到10个病毒拷贝模板数,在临床检测中得到很好运用。本研究建立实时荧光定量RT-PCR,有助于日常检验工作,为鱼类神经坏死病毒的筛选和监测提供基础,有助于控制神经坏死病的疫情传播。     

温度变化对七带石斑鱼早期发育及开口摄食的影响

观察比较了9个温度梯度(13、15、17、19、21、23、25、27、29℃)对七带石斑鱼(Epinephelus septemfasciatus)受精卵孵化时间、孵化率和畸形率的影响。用在(21±0.5)℃条件下孵出的健康仔鱼进行耐饥饿和摄食实验,实验温度处理设两种方式:一、处理温度始终保持不变;二、将温度在处理48 h后统一调节至21℃。实验期间,每天统计仔鱼死亡数,测定不投饵条件下的存活指数(SAI),并观察仔鱼投饵后的开口摄食情况及其形态发育状况。结果表明:1)受精卵孵化的最适温度范围为17~23℃,当温度低于13℃或高于27℃时,受精卵不能孵化。21℃时,受精卵孵化率最高,为(93.67±1.52)%;而畸形率最低,为(1.06±1.06)%。当温度低于或高于21℃时其孵化率降低,而畸形率升高;2)在13~28℃范围内,初孵仔鱼的SAI值随着温度的上升先升高后逐渐降低,21℃时的SAI值最高,为(20.26±0.44)%。各组温度保持不变时,13℃和27℃的SAI值分别为(2.18±0.01)%、(8.47±0.28)%;调整一致后分别为(6.90±0.44)%、(13.30±0.31)%。温度调节组与不调节组相比,其SAI值明显提高。温度调节后,每个梯度组的畸形率也出现了不同程度的降低;3)不同温度处理的仔鱼开口摄食存在差异。21℃时,仔鱼的初始摄食率和饱食率分别可达80.0%和40.0%。随着温度偏离21℃,摄食率和饱食率均逐渐降低。温度调节恢复组的仔鱼摄食情况改善明显,其中17℃不变和恢复组对比变化最为明显:当保持17℃不变时仔鱼的摄食率和饱食率分别为20.0%、13.3%,而处理48 h后恢复到21℃后,两者分别提高到66.7%和33.3%,分别提高了2.3和1.5倍。     

鞍带石斑鱼仔稚（幼）鱼的发育和生长研究

对鞍带石斑鱼的仔稚幼鱼形态发育的各个阶段进行了观察与研究,详细描述从初孵仔鱼到幼鱼各个发育时期的形态特征和发育时间。根据卵黄囊的变化,长鳍棘的长出与收缩,鳞片和体色斑纹的出现,鞍带石斑鱼胚后发育可以划分为仔鱼期、稚鱼期、幼鱼期。仔鱼期又可分为卵黄囊期仔鱼和后期仔鱼。水温27～30℃,盐度27～31,pH值8.0～8.4的海水中培育,初孵仔鱼至孵化后2日龄为卵黄囊期仔鱼。2日龄仔鱼开口,3日龄至20日龄为后期仔鱼,22日龄至30日龄为稚鱼期,31日龄进入幼鱼期。鞍带石斑鱼胚后发育过程中最明显的变化是背鳍棘和腹鳍棘的生长和收缩,也是生产育苗当中比较关键的仔稚幼鱼变态过程。     

Changes in Na+,K+-ATPase activity and gill chloride cell morphology in the grouper Epinephelus coioides larvae and juveniles in response to salinity and temperature

The activity of the enzyme Na⁺,K⁺-ATPase and morphological changes of gill chloride cells in grouper, Epinephelus coioides larvae and juveniles were determined 6–48 h after abrupt transfer from ambient rearing conditions (30–32 ppt, 26.5–30 °C) to different salinity (8, 18, 32, 40 ppt) and temperature (25, 30 °C) combinations. Na⁺,K⁺-ATPase activity in day 20 larvae did not change at salinities 8–32 ppt. Activity decreased significantly (P <0.01) after exposure to 40 ppt at 25–30 °C, which was accompanied by an increase (P <0.05) in density and fractional area of chloride cells. Enzyme activity in 40 ppt did not reach a stable level and larvae failed to recover from an osmotic imbalance that produced a low survival at 25 °C and death of all larvae at 30 °C. Enzyme activity and chloride cell morphology in day 40 groupers did not change in 8–40 ppt at 25 °C and 8–32 ppt at 30 °C. A significant decrease and a subsequent increase in Na⁺,K⁺-ATPase activity in 40 ppt at 30 °C was associated with the increase in chloride cell density resulting in an increased fractional area but a decreased cell size. Enzyme activity and chloride cells of day 60 grouper were unaffected by abrupt transfer to test salinities and temperatures. These results demonstrate that grouper larvae and juveniles are efficient osmoregulators over a wide range of salinities. Salinity adaptation showed an ontogenetic shift as the larvae grew and reached the juvenile stage. This development of tolerance limits may reflect their response to actual conditions existing in the natural environment.     

Supplementation of heat‐inactivated Bacillus clausii DE5 in diets for grouper,Epinephelus coioides,improves feed utilization,intestinal and systemic immune responses and not growth performance

In recent years, more and more attentions have been paid to the development and application of probiotics in aquaculture, and viable probiotics have been extensively studied, while rare information was available about inactivated probiotics in aquaculture. Therefore, in this study, a feeding trial was designed to investigate the effect of dietary supplementation of heat‐inactivated probiotic Bacillus clausii DE5 on growth performance, immune response and key immune genes expression in head kidney and intestine in grouper Epinephelus coioides. Fish were fed for 60 days with control diet (C) and two experimental diets containing 1.0 × 10⁸ CFU/g live (T1) and heat‐inactivated (T2) B. clausii DE5, respectively. The probiotic treatments did not affect the final weight, weight gain (WG) and specific growth rate (SGR) of E. coioides at days 30 and 60 (p > .05), while both heat‐inactivated and live B. clausii DE5 significantly decreased the feed intake and feed conversion ratio (FCR) at day 60 (p < .05). Serum lysozyme activity and complement C3 level in the two probiotic treatments were significantly higher than those in the control (p < .05). The lysozyme activity and complement C3 level at day 60 were significantly higher than those at day 30 (p < .05), while no significant interaction effect between diet and administration date was observed. Moreover, the heat‐inactivated B. clausii DE5 significantly improved the expression of TLR5, pro‐inflammatory cytokines (IL‐8 and IL‐1β) and TGF‐β1 in head kidney and intestine (p < .05), while the live probiotic did not show any significant effect on the expression of these key immune‐related genes in head kidney and intestine. These results indicate that dietary supplementation of heat‐inactivated B. clausii DE5 effectively improved feed utilization and both the local and systemic immune responses of E. coioides.     

Effects of rhythmic temperature change on the growth,body composition and energy budget of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀)

Effects of rhythmic temperature change on the growth, body composition and energy budget of hybrid grouper (Epinephelus lanceolatus ♂ × E. fuscoguttatus ♀) were investigated. Nine groups of fish received repeating cycles of low‐temperature manipulation (22°C) for 1, 2 and 4 days followed by recovery temperature (28°C) for (3, 7 and 11 days), (6, 14 and 22 days) and (12, 28 and 44 days) respectively, designated as L1R3, L1R7, L1R11, L2R6, L2R14, L2R22, L4R12, L4R28 and L4R44 respectively. In the control group (C), fish were reared at 28°C throughout the whole experiment. After 96‐day feeding trail, the final weight, relative weight gain rate, specific growth rate, food conversion efficiency and apparent digestibility coefficient of fish in L4R28 were significantly higher than those of control (p < .05). The crude protein contents of fish in L2R14, L2R22, L4R12, L4R28 and L4R44 were significantly higher compared to the control (p < .05). According to energy budget, fish in L4R28 exhibited significantly higher proportion of food energy assimilated into growth and lower proportion consumed for excretion than the control fish (p < .05). These results indicated the occurrence of complete or over growth compensation in hybrid grouper, and the use of repeated cycles of low temperature (22°C) for 4 days followed by recovery temperature (28°C) for 28 days could obtain enhanced growth, that is overcompensation, with improved apparent digestibility coefficient, food conversion efficiency and energetic efficiency into growth. This technique would be more practically applicable in indoor culture for this species.     

不同维生素E水平饲料对云纹石斑鱼幼鱼低盐胁迫前后抗氧化和渗透压调节功能的比较

维生素E(VE)在鱼类繁殖、生长代谢、抗氧化能力和免疫机能等方面有至关重要的作用。为探究饲料VE添加量对云纹石斑鱼低盐度胁迫下抗氧化和渗透压调节功能的作用,本实验设计0、20、40、80和160 mg/kg等5个VE水平饵料添加量,依次为A、B、C、D、E组,饲喂云纹石斑鱼幼鱼56 d。然后进行6 h低盐度(12)胁迫实验,测试皮质醇(COR)、葡萄糖(GLU)、乳酸(LD)等血清生化指标,超氧化物岐化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、总抗氧化能力(T-AOC)和丙二醛(MDA)等抗氧化指标,以及鳃Na⁺/K⁺-ATP酶(NKA)、Ca²⁺-ATP酶活力等。结果显示,血清COR含量最高为盐度胁迫后A(32.74±1.53) ng/mL,最低为胁迫前E(19.06±3.88) ng/mL;GLU含量最高为盐度胁迫后A(16.46±0.99) mmol/L,最低为养殖前O(7.90±0.34) mmol/L;LD含量最高为低盐度胁迫前C(1.94±0.15) mmol/L,最低为胁迫后E组(1.30±0.06) mmol/L。说明VE的添加可以增强云纹石斑鱼抗应激的能力,且在47.524~134.566 mg/kg(B~D)的添加范围,效果较好。血清SOD、CAT、GSH-Px活力、T-AOC和MDA含量与投喂饲料中VE水平密切相关,SOD最高为胁迫前C(105.29±9.07) U/mL,最低为胁迫后A(11.23±2.30) U/mL;CAT最高为养殖前O(4.09±0.17) U/mL,最低为胁迫后A(0.35±0.10) U/mL;GSH-Px最高为胁迫前E(972.58±55.35) U,最低为胁迫后A(47.90±10.64) U;MDA最低为胁迫前D(33.48±2.34) nmol/mL,最高为胁迫后A(101.79±7.79) nmol/mL。饲料中添加91.378~178.924 mg/kg(C~E)水平VE对其抗氧化能力有增强作用。VE可增强NKA活力,当VE添加量为91.378 mg/kg(C)时,低盐度胁迫对NKA影响较小。而添加91.378~178.924 mg/kg(C~E)水平VE可增强云纹石斑鱼Ca²⁺-ATP酶活力。研究表明,饲料中添加91.378~134.566 mg/kg范围的维生素E,可以增强云纹石斑鱼的机体活力、抗应激及环境胁迫能力。     

七带石斑鱼精子的超微结构

利用透射电子显微镜技术研究了七带石斑鱼（Epinephelus septemfasciatus）精子的超微结构,结果发现：七带石斑鱼精子由头部和尾部（鞭毛）两部分组成,没有明显的中片,头部前段无顶体,主要有高电子密度的球形细胞核,核中主要是致密的染色质,还分布有一些核泡,核外由核膜包围,将核与细胞质分隔开,核膜最前端紧贴质膜,核后端有植入窝,近端中心粒和中心粒间体位于其中。头部后端为袖套结构,由质膜包围的袖套结构延伸到后部,使整个头部呈椭球形,质膜后端沿鞭毛形成一个袖套腔,袖套结构中分布有中心粒复合体、线粒体以及囊泡等。精子细胞的细胞膜不光滑,呈现小的皱折。七带石斑鱼精子的尾部细长,紧接于头部细胞核,穿过袖套腔,通过基体与核紧密结合,中心粒复合体也位于此,且近端中心粒和远端中心粒呈钝角,鞭毛的轴丝由此形成,基体与鞭毛间的过渡区轴丝无中央微管,呈“9+0”型。尾部的主要结构为轴丝,其轴丝为典型的“9+2”结构,其起始端开始于前端基体,轴丝外仅有少许细胞质。