Molecular cloning,characterization and expression analysis of glucose transporters from Rachycentron canadum

It is very necessary to clarify the mechanism of carbohydrate metabolism in fish due to their poor ability to utilize carbohydrates. Glucose transporters (GLUTs) are important carriers involved in glucose transport across the plasma membrane, which is the first rate‐limiting step of carbohydrate metabolism. In this study, five glucose transporters (RcGLUT1, RcGLUT2, RcGLUT3, RcGLUT5 and RcGLUT9) were obtained from Rachycentron canadum. The complete cDNAs open reading frames (ORF) of RcGLUT1, RcGLUT2, RcGLUT3, RcGLUT5 and RcGLUT9 were 1,476 bp, 1,530 bp, 1,551 bp, 1,539 bp and 1,578 bp (GenBank accession no. MK560257 , MK560258 , MH184460 , MH184461 and MH184462 ), encoding 491, 509, 516, 512 and 525 amino acid (aa) residues respectively. All five RcGLUTs contained a Sugar_tr domain, which is an important feature of the GLUT gene family. The multiple sequence alignment and phylogenetic relationship analyses showed that RcGLUTs were highly conserved and homologous from fish to mammals. Examination of tissue distribution showed that RcGLUTs were expressed constitutively in R. canadum. RcGLUT1‐5 and RcGLUT9 had the highest expression in the foregut, kidneys, haemocytes, muscles, liver and heart respectively. All six RcGLUTs first increased to peak levels of expression and then reduced both in the liver and muscle after fasting. The exception is that the expression levels of RcGLUT4 and RcGLUT5 in muscle were significantly lower than the control. In response to hypoxia, only RcGLUT2 in the liver and muscle and RcGLUT9 in muscle were expressed at a significantly lower level relative to the control. In sum, all RcGLUTs in the liver and muscles showed significant changes in response to fasting and hypoxia, suggesting that GLUT genes may play a role in the response to common physiological stresses.     

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.     

Dietary methionine affects growth and the expression of key genes involved in hepatic lipogenesis and glucose metabolism in cobia (Rachycentron canadum)

Juvenile cobia (Rachycentron canadum), a carnivorous fish, was chosen to examine the effects of methionine (Met) supplementation of low fishmeal diets on growth and the expression of key genes related to hepatic lipogenesis and glucose metabolism. Seven isonitrogenous and isolipidic diets were formulated to contain 7.2, 9.0, 10.0, 12.4, 14.1, 16.3 and 18.6 g/kg dietary Met levels. Healthy cobia (initial average weight ± SE = 9.79 ± 0.04 g) were randomly assigned into seven groups in triplicate with 40 individuals per sea cage for 8 weeks. The results showed that the weight gain rate (WGR) of fish increased as the dietary Met was increased up to 12.4 g/kg and then decreased. The expression of insulin‐like growth factor‐I (IGF‐I) and target of rapamycin (TOR) genes was inhibited by the Met‐deficient diets. The mRNA levels of fatty acid synthase (FAS) and sterol regulatory element‐binding protein‐1 (SREBP1) genes increased with increasing dietary Met until 12.4 g/kg after which the former remained constant, whereas the latter decreased from 12.4 to 18.6 g/kg Met. The mRNA levels of pyruvate kinase (PK) increased as the dietary Met levels increased, reaching a peak at 12.4 g/kg, and then decreased with further increases in Met. However, the mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) decreased as the dietary Met was increased up to 12.4 g/kg and then remained constant from 12.4 to 18.6 g/kg Met. In conclusion, based on the straight broken‐line analysis of WGR against dietary Met levels, the optimal dietary Met requirement for juvenile cobia was estimated to be 12.4 g/kg (26.9 g/kg dietary protein). A diet with the optimal level of Met induces fish growth and is associated with increased IGF‐I and TOR.     

Acute responses of juvenile cobia Rachycentron canadum (Linnaeus 1766) to acid stress

Fish are potentially submitted to water acidification when reared in recirculating aquaculture systems. This study evaluated the responses of juvenile cobia Rachycentron canadum after acute exposure to acid water. Juvenile cobia (12.6 ± 0.5 g; 14.2 ± 0.2 cm) were acutely exposed to four pH levels (7.9 (control), 6.5, 6.0, and 5.5). After 24 h of exposure to different pH values, fish were sampled for physiological and histopathological evaluation. Acid water affected physiological parameters and induced morphological histopathologies on gill and skin of juvenile cobia, and these effects were more conspicuous with decreasing pH values. Acid stress induced blood acidosis in juvenile cobia, coupled to a decrease in bicarbonate (HCO₃^?) and saturated O₂ (sO₂) in fish blood. On the other hand, haematocrit, haemoglobin and glucose concentration increased their values (P < 0.01) comparing to control level. Hyperplasia with completely fusion of secondary lamella was observed in all pH treatments (6.5. 6.0 and 5.5), while telangiectasia and proliferation of chloride cells were present for fish exposed to pH 6.0 and 5.5. In skin hyperplasia and hypertrophy of mucous cells, necrosis of these cells for fish exposed to pH 6.0 and 5.5 was observed. The results of this study demonstrate that acute acid water exposition affected physiology and histopathology in juvenile cobia, especially at pH values below 6.5. Accordingly, particular attention must be given to pH during cobia reared in recirculating aquaculture.     

Relative contribution of alternative proteins to the growth of Juvenile Cobia,Rachycentron canadum (Linnaeus)

Five isonitrogenous and isocaloric diets were fed to juvenile cobia, to assess the relative contribution of different proteins (fish meal, soybean meal, corn gluten and beer yeast) to the growth of cobia. The dietary effects on nitrogen and carbon turnover and on the isotopic diet‐consumer discrimination factors (Δ¹⁵N and Δ¹³C) were also assessed. Growth results showed that the final body weight, growth rate, feed conversion ratio and protein efficiency ratio of cobia fed diets with alternative protein were significantly lower (P < 0.05) than cobia fed diet formulated with 100% fish meal. The estimated half‐lives of nitrogen and carbon ranged between 9–11 days and 6–8 days, respectively, with significant differences among treatments (P < 0.05). Δ¹⁵N ranged between 0.0–1.2‰ and ?0.1–1.6‰ in whole fish and muscle and Δ¹³C ranged between 3.8–5.1‰ and 4.0–5.1‰ in whole fish and muscle respectively. Diets were formulated with low levels of dietary nitrogen (10%) supplied by alternative protein sources substituting fish meal. The relative contributions of the dietary nitrogen supplied from these sources to the growth of whole fish and muscle tissue ranged between 4.9–5.2% and 5.9–7.7% respectively. Results indicated that growth accounted for the majority of observed isotopic change in animals under all treatments. In whole animals and muscle tissue, isotopic change due to metabolism occurred faster for carbon stable isotopes than for nitrogen stable isotopes. Cobia fed diets formulated with alternative proteins showed reduced nitrogen turnover rate and increased Δ¹⁵N.     

我国军曹鱼研究现状

军曹鱼具有生长速度快、病害少、营养价值高等优点,是国内近几年来养殖规模迅速发展的重要海水经济鱼类,已成为海水网箱养殖的重要品种。根据国内军曹鱼的研究情况,对军曹鱼的基础生物学、人工繁养殖、营养需求、病害、遗传多样性等方面进行了综述和展望,以期为军曹鱼的进一步开发利用提供参考依据。     

微胶囊蛋氨酸或晶体蛋氨酸对军曹鱼幼鱼相关酶活性的影响

比较研究了低鱼粉饲料中添加微胶囊蛋氨酸或晶体蛋氨酸对军曹鱼(Rachycentron canadum),消化酶和蛋白质代谢酶活性的影响.将鱼粉组和减少鱼粉后蛋氨酸缺乏组分别设为正、负对照组,试验组饲料在负对照组的基础上分别添加晶体蛋氨酸(MET)、羟基蛋氨酸(MHA)、邻苯二甲酸醋酸纤维素微胶囊蛋氨酸(CAP)、丙烯酸树脂微胶囊蛋氨酸(RES)和棕榈酸甘油酯微胶囊蛋氨酸(TPA),共7组等氮等脂饲料.每个处理设3个重复,每个重复放养20尾鱼,初始体质量(5.40±0.07)g,流水养殖8周,水温29～31℃.结果显示微胶囊蛋氨酸组的军曹鱼肠道胰蛋白酶活性显著高于正对照组和MET组(P＜0.05),MET和TPA组肝脏谷草转氨酶(GOT)活性显著高于正负对照组(P＜0.05);MET组军曹鱼幼鱼摄食后0.5 h肠道Na+、K+-ATP酶活性显著高于其他各组(P＜0.05),摄食3 h后正对照组和RES组Na+、K+-ATP酶活性上升,显著高于CAP和MHA组(P＜0.05),摄食8 h后正对照组和微胶囊蛋氨酸组显著高于MHA和MET组(P＜0.05).以上结果表明,军曹鱼低鱼粉饲料中添加晶体蛋氨酸即可增进氨基酸的代谢和蛋白质的合成,微胶囊有助于蛋氨酸在肠道中的缓慢释放,提高肠道内胰蛋白酶活性.     

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。     

盐度变化对军曹鱼稚鱼相关免疫因子及其生长的影响

研究了盐度(5、10、20、30及对照37)对军曹鱼(Rachycentron canadum)稚鱼生长及其血清溶菌酶、碱性磷酸酶(ALP)、旁路途径补体(ACP)溶血活性(ACH50)和总免疫球蛋白(T-Ig)含量等免疫因子的影响。实验周期为14 d。结果表明,在逐渐达到设定盐度后养殖14 d,盐度30组稚鱼特定生长率(SGR)最高(5.77%/d),而盐度5和10组的SGR(分别为4.24%/d和4.38%/d)显著低于其他组(P<0.05)。在低盐度环境中血清溶菌酶活性在第7天各组都有不同程度升高,其中以盐度20组升高最为显著,其次为盐度10和30组,但第14天各组溶菌酶活性又都回落至对照组水平;而稚鱼在低盐度环境中血清ALP活性受到明显抑制,且活性与盐度呈一定正相关;各组间ACP活性在第7天各盐度组间无明显差异,但实验结束时盐度5和10两组出现显著升高;而T-Ig含量在盐度10组中始终明显高于其他组。研究显示,军曹鱼稚鱼在盐度20~37范围内都可正常生长,尽管稚鱼有较强的低盐度耐受能力,但过低盐度明显影响其生长率并导致体色变黑、蛀鳍和应激增强等异常表现。此外,盐度变化还影响稚鱼多种免疫相关因子。[中国水产科学,2007,14(1):120-125]     

军曹鱼全基因组微卫星特征分析与多态性标记的筛选及应用

为更好地开发军曹鱼(Rachycentron canadum)高多态性微卫星分子标记,本研究基于军曹鱼全基因组测序结果,利用MISA1.0软件对其简单重复序列(simple sequence repeat, SSR)的位点信息进行检索及分析。结果显示,基因组中共筛选出1~6个核苷酸为重复单元的SSR位点344 820个,其中,以单核苷酸重复序列最多(174 146个),占SSR总数的50.50%;其次为二核苷酸重复和三核苷酸重复序列,分别占SSR总数的30.23%和14.02%。在SSR包含的重复单元中,单核苷酸重复以A/T类型为主,AC/GT是二核苷酸的优势重复单元类型。军曹鱼基因组SSR核心序列重复次数在4~275次范围内波动,单核苷酸SSR重复数为10的最多,二核苷酸SSR重复次数为6的最多。本研究设置长度≥12 bp为筛选高多态性SSR位点的标准,共获得361 684个位点。在上述位点中随机选取100个候选位点进行基因分型检测,利用从中筛选到多态性较高的10个SSR标记分别对北海、陵水、硇洲、徐闻和三亚5个养殖群体进行遗传多样性分析,145尾个体中共检测到69个等位基因,观测杂合度(Ho)、期望杂合度(He)和多态信息含量(PIC)平均值分别为0.628、0.706和0.653。相关研究表明,军曹鱼基因组中SSR位点类型较为丰富、多态性潜能较高,从中筛选获得的多态性SSR标记可为军曹鱼分子标记辅助育种、群体遗传多样性评价等研究提供有力支持。     

Partial replacement of fishmeal by soybean meal in diets for juvenile cobia (Rachycentron canadum)

An 8‐week feeding experiment was conducted in floating cages (1.5 × 1.0 × 2.0 m) to determine the potential use of defatted soybean meal (roasted and solvent‐extracted) as a partial replacement of fishmeal in the isonitrogenous (approximately 450 g kg^?1 CP [crude protein]) diet for juvenile cobia with an initial average weight of about 8.3 g. Diets were formulated to include 0, 100, 200, 300, 400, 500 and 600 g kg^?1 (diets D0, D10, D20, D30, D40, D50 and D60, respectively) of fishmeal protein being substituted by defatted soybean meal without methionine supplementation. The results showed that weight gain rate decreased significantly when the replacement level of fishmeal protein was increased from 400 g kg^?1 to 500 g kg^?1, and the D60 diet was the lowest in all groups. These results indicate that up to 400 g kg^?1 of fishmeal protein can be replaced by defatted soybean meal without causing significant reduction in growth. Feed conversion ratio (FCR) and protein efficiency ratio (PER) were significantly affected by the replacement level of fishmeal protein being substituted by defatted soybean meal, when the replacement level of fishmeal protein was 200 g kg^?1 (diet, D20), FCR was the lowest and PER was the highest. There were no significant differences in the moisture, lipid, crude protein and ash content in whole body and muscle, while lipid content in liver increased as the dietary soybean meal replacement levels increased. There were significant differences in haemoglobin, haematocrit, red blood cell, plasma glucose and triglyceride concentration in fish fed diets with different soybean meal replacement levels. Results of this trial indicated that the optimum level of fishmeal protein replacement with defatted soybean meal, determined by quadratic regression analysis was 189.2 g kg^?1, on the basis of maximum weight gain.     

饲料中添加甜菜碱对军曹鱼生长、体营养成分和血清生化指标的影响

通过8周的养殖试验,评估了饲料中添加甜菜碱对军曹鱼（Rachycentron canadum）生长、体营养成分、血清生化指标和肝脏中酶活性的影响。共设计6种饲料,甜菜碱添加水平分别为0、0．05％、0．10％、0．20％、0．30％和0．40％。结果显示,添加水平为0．05％时增重率（WG）和特定生长率（SGR）最高,肝脏组织状况最好,全鱼和肌肉中蛋白质质量分数最高;添加水平超过0．20％时肥满度（cF）随甜菜碱添加量的上升而下降。各添加组的肝体比（HSI）均显著低于对照组且全鱼和肝脏中（0．10％添加组除外）的脂肪质量分数均低于对照组;血清指标中各添加组的血清甘油三酯（TG）浓度均高于对照组,但随着添加量的提高有先升后降的趋势;肝脏中谷丙转氨酶（GPT）的活性显著低于对照组。由此可见,饲料中添加适量的甜菜碱可以促进军曹鱼的生长,具有一定的降脂作用,利于脂肪代谢。     

军曹鱼养殖水体及其肠道弧菌的耐药性研究

选用常用的10种抗生素,通过纸片扩散法(Kirby-Bauer纸片扩散法,简称K-B法),参照NCCLS抗生素敏感试验操作标准,并以金黄色葡萄球菌Staphylococcus aureus ATCC25923和大肠埃希菌Esсheriсhia coliATCC25922为质控菌株,对周年监测分离自养殖军曹鱼水体及肠道的41株优势弧菌(水体菌18株,肠道菌23株)进行了药物敏感性研究。结果显示,抑制弧菌效果最好的药物是氯霉素(100%敏感)、庆大霉素(水体菌100%敏感,肠道菌敏感率超过90%),其次是诺氟沙星、复方新诺明和多粘菌素B。青霉素类抑菌效果则较差,如试验肠道弧菌对青霉素G的耐药率超过78%,对氨苄青霉素的耐药率超过60%。不同来源(即分别来自养殖水体和鱼肠道)的弧菌菌株对同种抗生素的敏感情况存在一定程度的差异。     

Ocular localization of mycobacterial lesions in tank‐reared juvenile cobia,Rachycentron canadum

Severe clinical mycobacteriosis with consistent ocular lesion localization was diagnosed in a population of 800 juvenile tank‐reared Cobia (Rachycentron canadum) which experienced a sudden increase in mortality approximately 5 months after arriving into Trinidad and Tobago from Florida, USA. Moderate daily mortality (15–20 animals per day) persisted for just over 1 month. Moribund fish displayed circling behaviour and had an open‐mouth gape upon death. Fish consistently presented with bilateral exophthalmia, corneal cloudiness and hyphema. Non‐branching acid‐fast rods were detected in aqueous humour touch preparations. Histological analysis revealed severe bilateral intra‐ocular granulomatous responses in all specimens. Mycobacterium sp. was identified using a real‐time PCR assay detecting the RNA polymerase β‐subunit (rpoB) gene in different tissue samples. Specimens did not present with characteristic granulomatous responses usually seen in viscera. To the best of our knowledge, this represents only the third documentation of piscine mycobacterial infection presenting with only localized ocular lesions, and the second documented case of mycobacteriosis in cobia. It is, however, the first documentation of an ocular presentation of mycobacteriosis in a marine species and is the first documentation of such a presentation in cobia.     

Effects of dietary starch levels on the growth,plasma metabolites and expression of genes involved in glucose transport and metabolism in Pelodiscus sinensis juveniles

A 10‐week feeding trial was conducted to evaluate the growth performance, glucose transport and metabolism of Chinese soft‐shelled turtles (Pelodiscus sinensis) exposure to graded levels of dietary starch (0.52%, 7.43%, 14.74%, 22.99% and 31.38%). The 360 turtles (initial body weight, 12.94 ± 0.50 g) with 12 replicates were randomly assigned to five experimental diets. The highest weight gain and specific growth rate (SGR) were observed in 7.43% group and the lowest in 31.38% group. The protein efficiency ratio, whole‐body lipid contents, hepatic glycogen contents and the 4‐hr postprandial plasma glucose levels were significantly increased with the increment of starch levels (p < .05). In contrast, the daily feed intake and feed conversion ration were significantly declined (p < .05). The mRNA levels of glucose transporter 2, glucokinase, pyruvate kinase, malic enzyme and acetyl‐CoA carboxylase alpha genes in the liver significantly increased as the increase in starch levels at 4‐hr and 24‐hr post feeding (p < .05). No significant differences were observed in the expression of gluconeogenesis genes at each time point (p > .05). These results suggested that dietary addition of starch up‐regulated hepatic glycolysis, glycogenesis and lipogenesis genes expression, but the deficient response of gluconeogenesis to dietary starch might be part of the causes limited the starch utilization. Based on the secondary polynomial regression of SGR, y = ?0.0011x² + 0.028x + 1.63 (R² = 0.9292), the 12.73% inclusion level of dietary starch was recommended in juvenile turtles.     

饥饿及再投喂处理对草鱼生长、葡萄糖代谢和转运蛋白1表达的影响

设置持续投喂组(C,持续投喂8周)、饥饿再投喂组(R,饥饿4周+再投喂4周)和持续饥饿组(S,饥饿8周)3个处理组,研究3种不同饥饿处理对草鱼(Ctenopharyngodon idellus)血清生化指标、糖原和糖代谢相关酶和葡萄糖转运蛋白1(GLUT1)的影响,同时在此实验基础上研究草鱼在急性高糖负荷胁迫下的糖耐受能力、糖代谢相关酶和GLUT1的变化规律,旨在阐明草鱼在饥饿及再投喂处理条件下的糖代谢特征。选取初重为(125.35±0.54)g的草鱼,饲养8周后以30 mg/100 g体重的剂量腹腔注射葡萄糖研究其糖耐受能力。结果显示,S组肝糖原和血清的血糖、甘油三酯含量均最低。饥饿处理对草鱼糖耐受能力影响显著,S组血糖含量在各时间点上显著低于其余两组(P0.05),肝糖原在6 h达到峰值;饥饿处理对草鱼肝脏糖代谢关键酶影响显著,饥饿处理(S组)诱使磷酸烯醇式丙酮酸激酶(PEPCK)活性上升但抑制丙酮酸激酶(PK)和果糖-6-磷酸激酶(PFK)的活性(P0.05),而饥饿再投喂(R组)后PEPCK、PK和PFK酶活性恢复到持续投喂(C组)处理水平。注射葡萄糖后S组肝脏GK酶活性增幅最大,PK酶活性呈上升趋势,而R组则呈先下降后上升的趋势;饥饿处理对草鱼肝脏和肌肉GLUT1表达影响显著,注射葡萄糖后,除R组肝脏组织外,其余各组草鱼肝脏和肌肉组织GLUT1表达量均呈先上升后下降的趋势,且S组肌肉GLUT1表达量在各个时间点上均高于其余两组(P0.05)。研究结果表明,在不同饥饿处理下,草鱼可通过消耗肝糖原和甘油三酯及降低肝脏糖酵解相关酶(PK和PFK)活性和促进糖异生PEPCK酶活性来应对饥饿胁迫,而饥饿处理可诱使GK和PK酶活性上升、促进糖原合成和激活GLUT1基因的表达和转运来缓解草鱼急性高糖负荷,从而提高其糖耐受能力。     

Effects of dietary carbohydrate sources on the growth performance and hepatic carbohydrate metabolic enzyme activities of juvenile cobia (Rachycentron canadum Linnaeus.)

An 8-week feeding trial was conducted to evaluate the effect of dietary carbohydrate sources on the growth performance and hepatic carbohydrate metabolic enzyme activities of juvenile cobia. Six experimental diets were formulated to contain 20% glucose, sucrose, maltose, dextrin, corn starch and wheat starch respectively. The results indicated that fish fed the wheat starch and dextrin diets showed significantly better weight gain, specific growth rate and protein efficiency ratio compared with those fed the other diets. However, fish fed the glucose diet had a significantly lower survival and condition factor than those fed the other diets. There were significant differences in the total plasma glucose and triglyceride concentration in fish fed diets with different dietary carbohydrate sources. Haematocrit, haemoglobin, red blood cell and leucocytes were significantly affected by the dietary carbohydrate sources. The activities of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphofructokinase (PFK) and fructose-1,6-bisphosphatase (FBPase) were significantly affected by the dietary carbohydrate sources, while fish fed the glucose diet showed higher G6PD, PFK and FBPase activities than those fed the other diets. These data indicated that dextrin and wheat starch were the most optimal carbohydrate sources for juvenile cobia.