大菱鲆趋化因子受体CCR3和CCR9基因的克隆及组织表达

本实验克隆了大菱鲆趋化因子受体基因CCR3和CCR9的全长cDNA, 并进行了鉴定。其中, 全长cDNA的克隆采用了5′和 3′-RACE。CCR3 cDNA全长1 451 bp, 包括92 bp的5′非编码区, 276 bp的3′非编码区以及编码360个氨基酸的1 083 bp的开放阅读框。CCR9 cDNA全长1 441 bp, 包括59 bp 的5′非编码区, 278 bp 的3′非编码区, 以及编码367个氨基酸的1 104 bp的开放阅读框。两种受体均具有7次跨膜结构, 符合G蛋白偶联受体的序列特征。系统进化分析表明, 大菱鲆CCR3与其他鱼类CCR3聚为一支, 大菱鲆CCR9也与其他鱼类的CCR9聚为一支; 系统进化树显示的亲缘关系符合各物种的进化地位。实时定量 PCR (qRT-PCR)表明这两个基因在大菱鲆正常组织中均有一定量的表达, 尤其是它们在头肾、脾及心脏中均有高水平的表达。在脂多糖(Lipopolysaccharide, LPS)诱导之后, CCR3在脾和肝中的表达水平与对照组有显著性差异(P<0.05), 而CCR9的表达水平仅在肝中与对照组有显著性差异(P<0.05)。两个基因在免疫相关组织中表达水平较高, 并且能够对LPS产生免疫应答, 说明它们在大菱鲆免疫系统中发挥了重要功能。

     

光照强度对青色系与红色系仿刺参生长与能量分配的影响

实验所用仿刺参(Apostichopus japonicus)红色系和青色系体质量分别为 (6.28±0.02) g和 (6.34 ± 0.04) g。实验设定5个光强处理组(50、300、1 000、2 000、3 500 lx) 和对照组(0 lx)。光照强度通过调节灯泡功率、数量以及灯源距离获得, 光照周期为12L︰12D,  养殖水温16~17℃。结果表明, 两种色系仿刺参生长速度与光强呈负相关, 弱光照时(50 lx)时生长速度最快。黑暗(0 lx)时两种色系仿刺参生长较弱光时(50 lx)略慢(P>0.05)。过强光照   (3 500 lx)抑制仿刺参的摄食率(P<0.05), 降低其食物转化效率(P<0.05)。两种色系仿刺参的耗氧率随光照强度增强而逐渐增大。各处理组两种色系间仿刺参生长速率不存在显著差异(P>0.05)。仿刺参生长能(G/C)约7%, 粪便能(F/C)和呼吸能所占比重超过90%。强光(3 500 lx)下仿刺参排便能损失的比例增至59%, 而生长能降至2%。从提高能量分配和饵料利用效率看, 两种色系仿刺参生长速度较适宜光强为弱光(50 lx)。本研究旨在通过分析光照强度对生长、摄食、耗氧率和能量分配影响, 并以青色系仿刺参作为对比, 为红色系仿刺参在中国近海养殖提供基础参数。

     

牙鲆连续三代抗鳗弧菌病家系的筛选与分析

为了培育牙鲆(Paralichthys olivaceus)抗鳗弧菌(Vibrio anguillarum)病的品系或品种, 2009和2012年利用中国牙鲆抗病群体、从日本和韩国引进的牙鲆群体以及2007年和2009年从大量牙鲆家系中选留的优良家系为亲本, 通过巢式杂交、三元杂交及雌核发育等方法, 分别于2009年和2012年建成牙鲆家系43个和65个, 选取2009年的33个家系和2012年的43个家系进行鳗弧菌感染实验, 共筛选出13个抗病家系, 其中3个家系的存活率极显著高于对照组(P<0.01), 另外10个家系的存活率显著高于对照组(P<0.05), 这13个家系包含F3家系、雌核发育一代和二代家系各1个, F2家系3个, 在以上6个家系中,除了1个F2家系, 其他家系的亲本均来自于抗病家系且鳗弧菌感染存活率的变异系数都低于10%。对连续三代抗病家系进行分析, 发现在上述13个抗病家系和2007年筛选出的3个抗病家系共16个抗病家系中有13个家系来自于中国牙鲆抗病群体相关, 结果表明, 部分F1、F2、F3和雌核发育家系较好地遗传了其亲本的抗病性能, 抗病性能稳定, 为培育抗鳗弧菌病的牙鲆品系或品种奠定了基础。

     

坛紫菜“申福2号”的SSR分子标记

利用微卫星(SSR)分子标记技术, 对坛紫菜(Porphyra haitanensis)优良品系“申福2号”的丝状体和叶状体分别进行了特异性分子标记鉴定, 结果发现: 用9^#引物(序列为F: TCACAATGGGTGATATGGC; R: CCACAT TTAAGTCCGACTCTG) 对“申福2号”丝状体的DNA进行扩增, 出现了能区别于其他14个品系(6个优良品系, 3个杂交品系, 5个野生品系)的特异性条带。用该引物对室内培养的“申福2号”叶状体的DNA进行扩增, 也均获得了与丝状体相同的特异性条带。此外, 用该引物分别对栽培在不同海区和不同时期采收的“申福2号”叶状体进行验证, 结果均出现了与丝状体相同的特异性条带。该特异性条带的DNA测序结果证实, 9^#引物产生的SSR标记反映了微卫星DNA重复序列的变化。通过SSR Hunter软件搜索到了设计引物时的核心序列, 所得产物大小在预期长度范围内, 是特异性扩增。通过BLAST比对得知, 该序列在核酸数据库中没有同源序列, 是一个新序列。通过DNAMAN软件分析得知, “申福2号”、“申福1号”之间序列差异较小, 与坛紫菜霞浦野生种差异较大。这证实该标记反映的是种内品系间的差异, 可以用于种内品系鉴定。上述结果表明, 由9^#引物扩增出的这一特异性条带可以认为是“申福2号”丝状体和叶状体的特异性标记, 可用于该品系的种质鉴定。

     

海湾扇贝南部亚种闭壳肌质量的双性状选择效应

采用双性状选择方法在经群体继代大壳高选育的海湾扇贝(Argopecten irradians concentricus Say)南部亚种F₃养殖群体中进行闭壳肌定向选育, 以评价闭壳肌的选择效果和现实遗传力, 为海湾扇贝南部亚种育种提供理论依据。结果表明, 在以壳高为第一选择性状、体质量为第二选择性状、壳高选择压力为10%、体质量选择压力为1%情况下, 正、负向选择组、对照组在产卵量、受精率、孵化率、幼虫期壳长增长率、中培期存活率、成体各形态参数等方面的差异不显著(P>0.05); 在稚贝育成率、养成期存活率、成体各质量性状等方面均存在显著差异(P<0.05), 各指标值均呈现正向选择组>对照组>负向选择组; 正、负向选择组中, 体质量与闭壳肌质量均出现较高的现实遗传力, 且正向选择组高于负向选择组; 双性状选择时, 体质量与闭壳肌质量的当代遗传获得明显高于单性状选择结果。壳高和体质量对闭壳肌质量均具有密切的遗传相关, 任何一个性状的选择均可对闭壳肌质量产生显著影响, 双性状同时选择可对闭壳肌质量产生叠加效应, 使闭壳肌质量性状的遗传进展达到最大。

     

浒苔对刺参幼参生长影响的初步研究

对浒苔(Entero­morpha prolifera)的一般营养成分、氨基酸及脂肪酸含量进行测定, 并以浒苔为原料投喂体质量(5.26±0.14) g的刺参(Apostichopus japonicas Selenka)幼参, 与幼参常用饵料如鼠尾藻(Sargassum thunbergii)、马尾藻(Sargassum muticum)、海带(Laminaria japonica)进行效果对比。营养成分检测结果显示, 浒苔蛋白质含量为15.7%, 其中含量较高的氨基酸有天门冬氨酸、谷氨酸、丙氨酸、亮氨酸等; 脂肪含量低(1%), 多不饱和脂肪酸比例较高(0.24%); 浒苔中各重金属(无机砷、镉、铅、甲基汞)含量均低于国家相关限量标准。以4种海藻为饵料投喂幼参70 d后, 各组刺参幼参生长良好, 成活率无明显差异(P>0.05), 幼参体质量均显著增长(P<0.05); 各组刺参的特定生长率(SGR)由高到低依次为鼠尾藻组、浒苔组、马尾藻组、海带组; 摄食率(IR)由高到低依次为海带、马尾藻、浒苔、鼠尾藻, 饵料转化率(FE)由高到低依次为鼠尾藻组、浒苔组、马尾藻组、海带组。浒苔、鼠尾藻、马尾藻3个投喂组刺参幼参特定生长(SGR)无显著差异(P>0.05), 但显著高于海带组(P<0.05)。本研究说明, 浒苔蛋白含量高、脂肪含量低, 可全部或部分替代鼠尾藻添加到幼参饵料。

     

大眼鳜与翘嘴鳜正反交及其正交子代自交的胚胎发育观察

对大眼鳜(Siniperca kneri)(♀)×翘嘴鳜(Siniperca chuatsi)(♂)正交F1(DQ)、翘嘴鳜(♀)×大眼鳜(♂)反交F1(QD)、大眼鳜(♀)×翘嘴鳜(♂)正交F1的自交F2(F2)的胚胎发育进行观察, 详细记录了受精卵、分裂期、囊胚期、原肠胚期、胚体形成期、破膜期6个胚胎发育时期的卵径、孵化破膜时间、初孵仔鱼大小以及胚胎发育特征。大眼鳜受精卵卵径为(1.231±0.057) mm, 翘嘴鳜卵径为(1.197±0.052) mm, 显著大于正交F1雌鱼的卵径(1.723±0.0519) mm(P<0.05)。DQ受精卵在水温25.5~27.7℃经过37 h孵化破膜, QD受精卵在水温27.5~28.7℃经过30 h孵化破膜, F2受精卵在21.6~24.1℃经过40 h 57 min孵化破膜。DQ、QD、F2初孵仔鱼大小分别为(4.1±0.4) mm、(4.0±0.2) mm、(3.5±0.2) mm。鳜属鱼类中翘嘴鳜与大眼鳜胚胎发育各时期特征基本一致, 3种杂交鳜胚胎发育特征与其父母本也基本一致。经过比较发现, 其色素的形成与运动有鳜属的特异性: 在胚孔封闭后, 黑色素开始形成并逐渐扩散覆盖整个卵黄囊, 中期黑色素呈现星芒状, 并在油球处有集中现象, 后期色素则逐渐出现在眼和头部。水温21.6~28.7℃时3种杂交鳜的胚胎发育时间都偏向与翘嘴鳜的胚胎发育时间一致, 在水温23~26.5℃时DQ、F2与大眼鳜的胚胎发育时间有较大差别。

     

不同牙鲆群体遗传力和育种值分析

以牙鲆(Paralichthys olivaceus)抗病群体(RS)、RS(♂)与从日本引进的日本群体(♀)交配建立的群体(RJ)、日本群体(♂)与RS(♀)交配建立的群体(JR)、以及韩国群体(KS)为基础群体, 通过随机交配建立牙鲆家系, 研究了4个群体作为亲本的育种性能。待所建立的家系生长至19月龄左右时, 测量家系生长性状, 包括全长和体质量, 测量所得数据用SPSS及DMU软件中的REML算法和BLUP方法进行分析。结果显示, 从表型参数可以看出KS(♂)×RJ(♀)杂交后代表现出明显的生长优势。19月龄牙鲆全长和体质量的遗传力分别为0.301、0.295, 都属于中等遗传力。因此, 牙鲆群体具有较好的遗传改良潜力。全长、体质量育种值与其表型值的相关系数分别为0.838和0.827, 且呈极显著相关(P<0.01), 表明个体育种值的预测结果具有较高的准确性。对父母本分别进行育种值比较可知, 父本中KS的育种值最高, 母本中RJ的育种值最高, 因此选用KS(♂)和RJ(♀)杂交可培育出生长迅速的牙鲆新品种。本研究通过比较4个资源群体牙鲆生长性状的育种性能, 并以此作为筛选优良亲本群体的重要依据, 旨在为牙鲆新品种的成功选育奠定理论基础, 同时为牙鲆的进一步遗传改良提供重要的科学依据。

     

b-葡聚糖对暗纹东方鲀幼鱼非特异性免疫及生长性能的影响

选取健康暗纹东方鲀(Takifugu obscures)幼鱼, 体质量约50 g。根据设计项目分别进行取样分组, 各组设3个重复。分别采用注射、投喂不同剂量的β-葡聚糖和注射相应浓度的b-葡聚糖后于不同时间进行嗜水气单胞菌(Aeromonas hydrophila)的感染试验的方法, 探讨β-葡聚糖对暗纹东方鲀幼鱼非特异性免疫及生长性能的影响。结果显示: 腹腔注射β-葡聚糖试验组与对照组相比, 除C3补体外, 试验组血清溶菌酶(LSZ)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、血清总蛋白(STP)的水平出现不同程度提高(P<0.05); 另在腹腔注射不同剂量β-葡聚糖之后, 进行嗜水气单胞菌的感染试验, 试验组累计存活率显著提高(P<0.05)且注射葡聚糖5 d后再进行感染不能提高存活率; 投喂试验中, β-葡聚糖也可以增强暗纹东方鲀的非特异免疫功能(P<0.05), 但对增重率、饵料系数、特定生长率没有影响; 其中, 连续投喂和间断投喂所引起的免疫增强效果没有差异。考虑到免疫抑制以及降低生产成本, 建议生产采取间断投喂的方式。

     

基于多源数据的东海小黄鱼资源评估与管理

为建立稳定环境和波动环境机制下预防性渔业管理生物参考点, 整合调查设计和渔捞日志等多源资源指标构建混合矩阵, 利用logistic和Fox剩余产量模型的两步分析技术, 对东海区小黄鱼(Larimichthys polyactis)渔业资源动态进行评估。模型估算参数和管理参考点显示, Fox模型对渔获量和CPUE拟合的方差贡献率高于logistic模型, 两者分别为68%和57%, 环境承载力和内禀增长率相差较大。logistic模型估算了相对较低的承载力和较高的内秉增长率、初始开发率以及MSY。稳定环境下资源状况评判结果表明: 1999―2008年间多数年份的捕捞强度超过捕捞水平限制参考点, 渔业遭受过度开发, 平均资源量保持在中位水平且未达到过度捕捞状态, 但已超过目标参考点; 波动环境条件下的判别结果显示: logistic和Fox模型拟合的渔业水平均已达到过度捕捞。采用保护性捕捞参考点可增强渔业资源稳定性, 当捕捞死亡从参考点F_MSY降至预防性参考点F_opt, logistic模型估算资源量从8.1 t上升到10.1 t, 而渔获量从13.1 t下降至12.3 t; Fox模型资源量则从11 t增加到15.9 t, 相应的捕捞产量从12.8 t下降到11.6 t。Fox模型评估结果较为保守, 适合预防性渔业管理。

     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.