军曹鱼的胚胎发育及仔稚鱼形态观察

为了探明军曹鱼早期发育阶段的特征和规律,本实验通过连续显微观察,记录分析其胚胎及仔稚鱼发育各时期的形态特征和发育特点。结果显示,军曹鱼受精卵呈圆球形,为浮性卵,卵径(1.245±0.065) mm,油球1个,油球直径(0.325±0.027) mm。在水温(27.0±0.5)℃、盐度29、pH值8.3的条件下,军曹鱼卵从受精至孵化出膜约需26 h 30 min,经历合子、卵裂、囊胚、原肠胚、神经胚、器官形成和孵化出膜7个阶段,共24个时期。初孵仔鱼平均全长(3.254±0.096) mm,仔鱼在孵化后第3天开口摄食,眼囊变黑,卵黄囊被消耗约80%,第5天卵黄囊消失,第7天油球耗尽,转化为外源性营养。孵化后第14天全长(19.933±1.118) mm,各鳍鳍条形成,进入稚鱼期;22日龄稚鱼全长(41.140±3.779) mm,尾柄部开始出现鳞片,46日龄的后期稚鱼全长达(116.667±5.916) mm,体表完全覆盖鳞片,除尾鳍形状外,体形与成鱼相似。     

病原诱导水产动物细胞凋亡途径研究进展

军曹鱼 (Rachycentron canadum) 鳃组织对水温变化敏感。为探究低温胁迫对军曹鱼幼鱼鳃组织的影响,揭示该鱼应对低温胁迫的响应机制,实验设置2个低温胁迫组 (18 ℃、21 ℃) 和1个对照组 (28 ℃),比较分析鳃组织在胁迫后第0、第4和第7天的氧化应激状态、细胞凋亡和组织结构情况。结果显示,低温胁迫下鳃组织超氧化物歧化酶 (SOD)、过氧化氢酶 (CAT) 和谷胱甘肽过氧化物酶 (GSH-Px) 活性显著低于对照组 (P<0.05),丙二醛 (MDA) 浓度显著高于对照组 (P<0.05);低温胁迫组鳃组织凋亡相关基因bax、caspase-9、caspase-3、p53和mdm2表达量在第4和第7天时相较于对照组显著升高 (P<0.05),bcl-2基因表达量显著降低 (P<0.05);TUNEL检测显示低温组鳃组织细胞凋亡率升高;组织学分析表明低温胁迫下鳃组织出现了不同程度的鳃小片排列紊乱、基部增生、融合,上皮细胞和泌氯细胞空泡化等现象。研究表明,低温胁迫抑制了军曹鱼幼鱼的鳃组织抗氧化酶活性,造成氧化损伤,进一步诱导细胞凋亡,破坏鳃组织结构完整性。     

Amending reduced fish‐meal feeds with marine lecithin,but not soy lecithin,improves the growth of juvenile cobia and may attenuate heightened responses to stress challenge

Sparing of marine resources in aquafeeds can be environmentally and economically advantageous; however, fish meal (FM) replacement can affect the production performance and physiological competence. Phospholipids are increasingly understood to be involved in maintaining growth and vigour in fish and may be deficient in reduced FM formulations. Accordingly, we evaluated the growth and stress tolerance of juvenile cobia fed typical (50% FM) or reduced FM feeds (12% FM) with or without phospholipid amendment [1% marine lecithin (12% FM + Marine PL) or soy lecithin (12% FM + Soy PL)] for 6 weeks in triplicate tanks (N = 3) in a recirculation aquaculture system. The 50% FM feed yielded significantly superior growth and growth efficiency in comparison with the 12% FM and 12% FM+ Soy PL feeds, but the 12% FM+ Marine PL feed yielded comparable results to 50% FM feed. A low‐water stress challenge induced elevated plasma glucose, cortisol and lactate levels in all treatments. However, a significant interaction (diet × stress) effect suggested a lesser cortisol response among fish fed the 12% FM+ Marine PL and 50% FM diets. These findings demonstrate that growth performance and, perhaps, resilience of cobia raised on reduced FM feeds may be improved by the addition of marine‐origin phospholipid to the diet.     

Effects of acute hyperglycaemia stress on indices of glucose metabolism and glucose transporter genes expression in cobia (Rachycentron canadum)

The objective of the present study is to preliminarily clarify the mechanism of carbohydrates metabolism in cobia (Rachycentron canadum) (85 ± 3 g) receiving injection of glucose solution. We examined plasma glucose (GLU), total protein (TP), triglyceride (TG), cholesterol (CHOL), insulin, liver glycogen and muscle glycogen, activities of hepatic hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), as well as relative expressions of glucose transporter 1 (GLUT1), GLUT2, GLUT3, GLUT4, GLUT5 and GLUT9 in hemocyte, liver and muscle of R. canadum when fish were injected with 200 μl of glucose solution (255 mg/ml) after 0, 1, 2, 4, 8, 12, 24 and 48 hr. Fish received injection of 0.68% saline served as control. Results indicated that the plasma GLU, TG and CHOL increased and reached peak at 1, 8 and 48 hr postinjection (hpi) respectively. The hepatic glycogen increased from 1 hpi, and reached peak at 8 hpi, plasma insulin increased at 1 hpi, and reached peak at 2 hpi, and activity of hepatic PK peaked at 8 hpi. Furthermore, the relative expressions of GLUT1, GLUT2, GLUT3, GLUT4 and GLUT5 in hemocytes reached peak at 1,4, 8, 4 and 8 hpi, respectively, relative expressions of GLUT2, GLUT3, GLUT5 and GLUT9 in liver reached peak at 24, 24, 12 and 24 hpi, respectively, and relative expressions of GLUT1 and GLUT3 in muscle were significantly higher at 2 and 2–4 hpi, respectively compared with those in controls. In conclusion, low ability of utilizing glucose in R. canadum may be attributed to insufficient insulin secretion, low activities of key glycolytic enzymes (HK, PFK and PK) regulated by glucose injection and slow increase of GLUTs.     

军曹鱼MHC-Ⅰα基因全长cDNA的克隆及其组织表达分析

采用同源克隆和末端快速扩增（RACE）方法,得到1330bp的军曹鱼（Rachycentroncanadum）MHC-Ⅰα全长cDNA片段。该序列包括76bp的5’末端非编码区（UTR）,189bp的3’UTR及1065bp的开放阅读框（ORF）,编码354个氨基酸,预测其蛋白质分子量约40．10kDa,等电点5．70。构建MHC-Ⅰα氨基酸序列的系统进化树并进行氨基酸相似性比对,结果表明,军曹鱼和已知鱼类及人类（Homosapiens）MHC-Ⅰα氨基酸的同源性在27．9％～67．1％之间。所推测的蛋白序列具有一些重要特征,包括前导肽、α1、α2、α3区、CP／TM／CYT区和保守的半胱氨酸等。Real—timePCR检测结果显示,MHC-Ⅰα基因在各个正常军曹鱼组织中均表达,但表达量各有不同,其中较强的表达于头肾;中等程度表达于鳃、脾和肠;在心、脑和肌肉中表达较弱。     

军曹鱼Ig κ轻链cDNA克隆及组织表达分析

应用同源克隆和RACE技术克隆了军曹鱼(Rachycentron canadium Linnaeus)Igκ轻链的cDNA全序列,并分析其在组织中的表达.军曹鱼Igκ的cDNA全长969 bp,3'端的非编码区域(UTR)为188 bp,5'UTR为52 bp,开放阅读框为729 bp,编码242个氨基酸,分子量约为26.255 kD,理论等电点7.52.推测的军曹鱼Igκ恒定区氨基酸序列同鰤(Seriola quinqueradiata)、大西洋鲑(Salmo salar)的Ig轻链同源性最高,达77%以上,可变区氨基酸序列与鰤的相应序列同源性最高,达87%.通过构建系统进化树可以看出,军曹鱼Igκ恒定区氨基酸序列同鰤、大西洋鲑的L3、斑点叉尾鲴(Ictalurus punctatus)的G等鱼的轻链均为κ型的聚为一支,同大西洋鲑的L2、斑马鱼(Danio rerios)的L2、鲤(Cyprinus carpio)的L2等均为λ型的明显不同,由此可推断此序列属于Igκ型.利用半定量PCR技术,发现在健康鱼体中κ链基因在肝脏和鳃中的表达量较高,在肠和脑组织中几乎没有表达.经鲨鱼弧菌(Vibrio carchariae)JT2刺激192 h后,κ链基因在采样的各个组织中均有表达,尤其在头肾、肠、鳃和脾中的表达量较正常水平明显升高,而肝的表达量有所下降,脑组织有κ链基因的少量表达.说明经刺激后头肾、脾脏、肠、鳃是免疫球蛋白的主要表达场所,在抵御病原感染过程中发挥着重要作用.     

Effects of dietary immunostimulant combination on the growth performance,non‐specific immunity and disease resistance of cobia,Rachycentron canadum (Linnaeus)

The aim of this study was to examine the effects of the immunostimulant combination (IC) containing β‐glucan, A3α‐peptidoglycan, vitamin C and vitamin E on the growth performance, non‐specific immunity and protection against Vibrio harveyi infection in cobia (Rachycentron canadum). Fish were fed diets containing six graded levels of IC (0, 1, 2, 3, 4 and 5 g kg^?1 diet) for 8 weeks. The results showed that the survival rate ranged from 81.1 to 84.4% with no significant difference among all the groups (P > 0.05) after the feeding experiment. Dietary IC significantly increased the specific growth rate (SGR), serum lysozyme, alternative complement pathway (ACH50) activity, phagocytosis percentage (PP) and respiratory burst activity of head kidney macrophages of cobia. Moreover, feeding of supplemented diets containing 3.0 g kg^?1 IC resulted in significantly lower mortality against the pathogens, V. harveyi compared with the control group. To elevate the growth and immune resistance ability of cobia, the optimal dose of dietary IC administration, determined by second‐order polynomial regression analysis was 3.43 and 2.71 g kg^?1 diet, respectively, on the basis of the SGR and mortality after challenge with V. harveyi.     

军曹鱼仔稚鱼脊柱和附肢骨骼的发育特征

为探明军曹鱼仔稚鱼早期脊柱及附肢骨骼的发育特征,本研究采用软骨-硬骨双染色技术,分别利用阿利新兰、茜素红对软骨、硬骨以及1～33日龄军曹鱼仔稚鱼全骨骼进行染色,系统观察并描述其脊柱和附肢骨骼的发育特征。结果显示,军曹鱼脊柱开始发育的标志为7日龄仔鱼中脉弓和神经弓的出现;13日龄稚鱼椎骨、神经弓和脉弓开始骨化;背肋、腹肋分别于17、20日龄开始骨化;29日龄稚鱼脊柱骨化完成。附肢骨骼骨化起始顺序依次为胸鳍、尾鳍、腹鳍、背鳍和臀鳍。胸鳍匙骨于4日龄出现,肩胛骨孔于12日龄出现,同时上匙骨开始骨化;乌喙骨与肩胛骨于20日龄开始骨化;第1尾下骨于5日龄出现,15日龄稚鱼尾杆骨、侧尾下骨和尾鳍鳍条开始骨化,18日龄稚鱼尾下骨开始骨化;腹鳍支鳍骨于17日龄延伸至匙骨,同时腹鳍开始骨化;臀鳍和背鳍于17日龄由前向后开始骨化。研究表明,军曹鱼在13日龄进入稚鱼期,早期发育阶段的骨骼发育特征与其功能性适应密切相关。本研究结果对研究军曹鱼早期骨骼发育与功能适应、优化养殖条件有重要作用。     

饲料中添加胆汁酸对军曹鱼生长及体组成的影响

在4组等氮等能的军曹鱼（Rachycentron canadum）实用饲料中分别添加不同梯度水平的胆汁酸（0,0.015%,0.030%和0.045%）,连续投喂初始体质量为（5.4±0.12）g的军曹鱼8周后,通过测定军曹鱼增重率（WGR,%）、特定生长率（SGR,%）和饲料系数（FCR）,并进行肝脏成分分析和血清分析,比较各组军曹鱼生长和体组成的影响。结果表明,添加水平为0.03%组的增重率高于对照组和0.015%添加组,饲料系数低于上述2组;军曹鱼血清胆固醇和肝脏脂肪的质量分数均与饲料中胆汁酸质量分数呈负相关;4组的成活率、特定生长率、全鱼和肌肉灰分、水分质量分数没有显著性差异。由此可见,饲料中添加胆汁酸能够促进军曹鱼的生长,并能降低脂肪在军曹鱼体内中的沉积。     

Mass mortality associated with a Sphaerospora-like myxosporidean infestation in juvenile cobia, Rachycentron canadum (L.), marine cage cultured in Taiwan

Cultured cobia, Rachycentron canadum , of 45–80 g exhibited anaemia and ascites, and a mottled red and grey, extremely enlarged kidney with cream-coloured patches or spherical nodules. Cumulative mortality was about 90% within 1 month. Extrasporogonic or sporogonic stages of a myxosporean appeared in the blood, glomerulus, renal tubules and renal interstitium. The renal tubules were the main target tissue of the parasite and were completely occluded by sporogonic pseudoplasmodia at various degrees of maturity. Many sporogonic stages were attached to the brush border of the epithelium of the renal tubules. Mature spores were seen in the lumen of the tubules. They were elongated or spherical with numerous refractile granules in the cytoplasm. The polar filament formed 3–5 coils. No bacteria or viruses were isolated from the diseased fish. Based on the results of microbiological, histopathological and electron microscopical examinations, the cobia disease was believed to be caused by a Sphaerospora -like myxosporean. This is the first report of a myxosporean in cobia in aquaculture.