黄鳍棘鲷家系亲缘关系鉴定

为了建立黄鳍棘鲷微卫星亲子鉴定技术,利用荧光引物和自动测序技术检测了自主开发的12对微卫星分子标记在505尾黄鳍棘鲷个体中的遗传多态性,并构建了亲子鉴定技术。结果显示,该研究中筛选的12个微卫星标记共检测到119个等位基因,平均等位基因数(N_a)为9.91,平均观测杂合度(H_o)为0.651,平均期望杂合度(H_e)为0.661,平均多态性信息含量(PIC)为0.621,具有丰富的多态性。此外,运用Cervus 3.0软件对已知系谱信息的112尾黄鳍棘鲷亲本和393尾子代个体进行模拟分析,结果显示,当双亲未知且置信度为95%时,12个标记的累积排除概率达99.58%;当微卫星标记数量为8时,累积排除概率达到99.1%。因此确定AL49、AL37、AL01、AL20、AL14、AL18、AL15和AL51共8个多态性较高的微卫星标记为黄鳍棘鲷微卫星亲子鉴定的核心体系。在双亲性别未知的情况下,其双亲的累积排除率为99.1%。根据黄鳍棘鲷子代的实际基因分型数据,实际鉴定率为89.31%。该研究构建的微卫星标记组合能为黄鳍棘鲷不同...     

基于微卫星标记建立翘嘴鳜亲子鉴定技术

利用9个多态性较高的微卫星标记对翘嘴鳜(Siniperca chuatsi)进行亲子鉴定分析。结果显示,22个家系翘嘴鳜个体中共观测到95个等位基因,其观测杂合度和期望杂合度的平均值(范围)分别为0.578(0.219~0.800)和0.607(0.200~0.806),多态信息含量(范围)为0.552(0.187~0.787)。通过软件Cervus统计分析得到9个微卫星座位累计排除率为99.9658%,在27个可能的父母对条件下,混养养殖家系后代个体鉴定准确率为91%,研究结果表明筛选的这些多态微卫星分子标记可以用于翘嘴鳜优势家系的鉴定和分离,通过验证的微卫星标记建立的亲子鉴定技术为基础选育翘嘴鳜生长速度快的优势家系,为培育出生长速度快、抗病力强的翘嘴鳜新品种奠定基础。     

微卫星分子标记技术在大黄鱼增殖放流效果评估中的应用

为研究微卫星分子标记在大黄鱼(Larimichthys crocea)增殖放流效果评估中的适用性, 本研究利用 8 个多态性丰富的微卫星位点, 对 100 尾放流大黄鱼亲鱼以及官井洋海域回捕的 295 尾大黄鱼样本进行亲子鉴定, 并提出了一种基于微卫星分子标记的大黄鱼回捕效果评估方法。8 个微卫星位点共检测到 78 个等位基因, 其观测杂合度为 0.511~0.939 (平均值 0.764), 期望杂合度为 0.643~0.897 (平均值 0.781), 多态性信息含量为 0.587~0.883 (平均值为 0.751); 通过 Cervus 3.0.7 软件模拟分析得到, 在置信度大于 95%的情况下, 8 个微卫星位点的个体累积排除率达到 99.99%以上水平; 2019 年 7 月从官井洋海域回捕到的 190 尾大黄鱼中, 检测出 5 尾与放流亲本存在亲子关系的个体, 占回捕样本总数的 2.63%。推算本次富发放流大黄鱼在回捕样本中所占比例为 60.53%左右, 进一步推算出 7 月富发公司放流大黄鱼的回捕率为 0.0038%左右。8 月、9 月的 105 尾回捕样品中未能检出标记个体。本研究构建的大黄鱼亲子鉴定技术体系不仅可为三沙湾大黄鱼增殖放流效果评估工作提供可靠的技术支撑, 亦可为三沙湾大黄鱼保护区的科学管理提供参考, 此外本研究对于我国其他海水鱼类的增殖放流评估工作的开展也将有重要的参考价值。     

基于微卫星标记对长江江苏段鳙增殖放流效果评估

为评估长江江苏段鳙(Aristichthys nobilis)增殖放流效果,本研究基于微卫星标记的亲子鉴定技术,选用 10对扩增效果稳定的微卫星荧光标记引物,结合毛细管电泳对江苏省内良种场 563 尾鳙亲鱼和长江江苏段 687 尾回捕鳙进行基因分型,并对位点多态性及亲子鉴定结果进行了分析。结果表明,各位点等位基因介于 19~55 之间,其观测杂合度为 0.530~0.909(平均值为 0.748),期望杂合度为 0.818~0.929 (平均值为 0.882),多态性信息含量为0.797~0.924 (平均值为 0.871);Cervus3.0 模拟结果显示,当双亲未知且置信度为 95%时, 10 个微卫星位点的累积非亲权排除率为 99.996%。亲子鉴定结果发现,所有回捕鳙中有 42 尾与亲本之间存在亲子关系,被确认来源于鳙增殖放流群体,并由此推算此次评估亲本的放流子代对长江江苏段野生群体的贡献率为 6.11%。结果显示该研究进行的鳙亲子鉴定技术可为长江鳙增殖放流效果评估工作提供可靠的数据支撑,为长江渔业资源管理提供参考资料。     

大黄鱼微卫星多重PCR体系的建立及其应用

利用本实验室开发的微卫星标记,通过优化退火温度、引物浓度、循环次数等条件,建立了3组大黄鱼微卫星多重PCR体系,每组包含3个微卫星位点,用3组微卫星多重PCR分析了一个大黄鱼选育群体JD-01的遗传多样性。结果显示,该群体的平均等位基因为12.111,平均有效等位基因为7.408,平均多态信息含量为0.846,平均观测杂合度和期望杂合度分别为0.825、0.868,香农多样性指数为2.165。运用Cervus 3.0软件,对已知系谱关系的9个大黄鱼家系及对应亲本进行了亲子鉴定分析,以验证3组微卫星多重PCR在亲子鉴定中的准确性。结果显示,使用该3组微卫星多重PCR体系进行亲子鉴定准确率为100%。大黄鱼微卫星多重PCR体系的建立为分析群体的遗传多样性、亲子鉴定和辅助家系管理提供了一种高效的技术手段。     

基于微卫星多重PCR的黄鳝亲子鉴定技术

为助力黄鳝(Monopterus albus)良种选育中的亲子鉴定和系谱管理问题,利用黄鳝全基因组预测并筛选获得的16个多态性较高的微卫星标记,建立了2组微卫星多重PCR体系,并成功用于11个家系的亲子鉴定。通过Cervus 3.0软件对132尾黄鳝进行遗传多样性分析,结果显示,16个微卫星标记的平均等位基因数(Na)为5.562,平均观测杂合度(Ho)和平均期望杂合度(He)分别为0.627和0.619,平均多态信息含量(PIC)为0.564。亲子鉴定结果表明,双亲基因型未知时单亲本的累积排除概率(CE-1P)为0.999 999 99,单亲基因型已知时另一亲本的累积排除概率(CE-2P)为0.999 999 91,双亲基因型未知时双亲的累积排除概率(CE-PP)为0.999 964 76。黄鳝11个家系的模拟鉴定率为99.96%,实际鉴定率为95%。模拟分析不同亲本数的结果显示,实验的16个微卫星标记在双亲性别已知的200对亲本和双亲性别未知的150对亲本的情况下,均可达到95%以上的鉴定率。利用NTSYS软件对11个家系的110尾子代个体进行聚类分析,结果显示除2尾子代外其余10...     

刺参微卫星标记与生长性状体重、体长的相关分析

运用单标记分析法分析了10个微卫星位点与控制体重、体长数量性状的QTL的连锁关系。10对微卫星引物在8月龄具有生长性状差异的60个刺参个体基因组DNA中扩增得到50个等位基因,每个微卫星位点等位基因数3~7个,平均有效等位基因个数5.0。10个微卫星位点的多态信息含量为0.268 0~0.825 6不等,观测杂合度为0.083 3~0.666 7。采用SPSS软件对刺参的体重、体长生长性状和微卫星位点相关性进行分析,结果显示,微卫星位点AjSSR02基因型AB、AD、CD与体重和体长相关,AjSSR04标记中等位基因A对生长性状有正面影响,位点AjSSR06基因型FF个体仅平均体重与其他基因型个体显著差异,AjSSR09标记的A和B等位基因分别对生长性状具有正面和负面不同影响。     

团头鲂的微卫星亲子鉴定

本试验选用8个高质量多态性的微卫星标记对团头鲂(Megalobrama amblycephala)选育世代F1、F2、F3、F4和团头鲂"浦江1号"的357个子代进行了亲子鉴定,主要方法是通过Cervus 3.0.7计算机模拟软件依次进行等位基因频率分析、模拟分析和亲子分析,根据LOD值大小判定亲子关系。结果表明在团头鲂混养家系中,微卫星位点的平均等位基因数为35,平均观测杂合度为0.867,平均期望杂合度为0.94,平均多态信息含量为0.935,最终所有子代准确地找出对应的父母本,亲子鉴定准确率为100%,证实微卫星标记在团头鲂亲子鉴定中的应用价值。     

放流三疣梭子蟹遗传多样性和贡献率初步研究

近年来我国沿海三疣梭子蟹自然资源明显下降,为恢复现有资源,辽宁省自2012年开始,连续实施了4年大规模人工放流工作。在目前的增殖放流中,为检测参与繁殖的三疣梭子蟹亲本和即将放流的子代间的遗传差异以及亲本对其子代的繁殖贡献率水平,利用10对具有丰富遗传多态性的微卫星分子标记,分别对9只三疣梭子蟹雌性亲本和179只即将放流的子代进行了遗传多样性分析及亲子鉴定。结果发现,子代的平均观测杂合度、平均期望杂合度和平均多态性信息含量数值均低于参与繁殖的雌性亲本,亲本和放流子代之间在观测杂合度、多态性信息含量参数方面并无显著差异(P0.05),但期望杂合度遗传参数存在显著差异(P0.05),子代的遗传多样性较雌性亲本呈下降的趋势。使用4个微卫星分子标记时,累积排除率≥0.998,亲子鉴定的准确率为56.98%;微卫星分子标记为6个时,累积排除率≥0.9999,准确率达到97.21%;当使用8个微卫星分子标记鉴定时,准确率达100%。同时发现,9只雌性亲本对子代均有贡献,最高为29.61%,最低为3.35%,不同亲本之间的贡献率存在显著差异(P0.05)。研究表明,微卫星可以作为有效的标记手段用于三疣梭子蟹增殖放流遗传评估中。上述试验结果将为我国三疣梭子蟹增殖放流的科学发展提供基础资料。     

中华绒螯蟹微卫星多重PCR体系的建立及其亲子鉴定应用

利用自主开发的微卫星标记对中华绒螯蟹(Eriocheir sinensis)进行微卫星多重PCR体系构建,最终成功建立4组多重体系,每组体系包含4个微卫星位点,并成功应用于3个家系的亲子鉴定中。结果显示:(1)本研究筛选的16个微卫星标记,平均观测杂合度为0.8206,平均期望杂合度为0.8164,平均多态信息含量为0.7927,具有丰富的多态性;(2)运用Cervus 3.0软件对已知系谱信息的3个中华绒螯蟹家系共95个子代个体进行亲子鉴定分析,结果显示,选用任意两组微卫星多重PCR体系时,累积实际正确鉴定率均超过94.74%;使用任意三组微卫星多重PCR体系时,累积实际正确鉴定率均大于98.95%;使用四组微卫星多重PCR体系时,累积实际正确鉴定率达到100%。并且当选用组合1、2和组合3,或者选用组合1、3和组合4时,对3个家系的累积正确鉴定率达到了100%,因此选用这些组合不但可以获得准确的系谱信息,还能减少工作量,降低成本。本研究构建的微卫星多重PCR体系能为中华绒螯蟹的种群选育和家系管理提供便捷、高效的途径。     

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).     

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.     

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.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

Status of aquatic and riparian biodiversity in artificial lake ecosystems with and without management for recreational fisheries: Implications for conservation

1. Humanity is facing a biodiversity crisis, with freshwater-associated biodiversity in a particularly dire state. Novel ecosystems created through human use of mineral resources, such as gravel pit lakes, can provide substitute habitats for the conservation of freshwater and riparian biodiversity. Many of these artificial ecosystems are subject to a high intensity of recreational use, however, which may limit their biodiversity potential.
2. The species richness of several taxa (plants, amphibians, dragonflies, damselflies, waterfowl, and songbirds) was assessed and a range of taxonomic biodiversity metrics were compared between gravel pit lakes managed for recreational fisheries (n = 16) and unmanaged reference lakes (n = 10), controlling for non-fishing-related environmental variation.
3. The average species richness of all the taxa examined was similar among lakes in both lake types and no substantial differences in species composition were found when examining the pooled species inventory. Similarly, there were no differences between lake types in the presence of rare species and in the Simpson diversity index across all of the taxa assessed.
4. Variation in species richness among lakes was correlated with woody habitat, lake morphology (surface area and steepness), and land use, but was not correlated with the presence of recreational fisheries. Thus, non-fishing-related environmental variables had stronger effects on local species presence than recreational fisheries management or the presence of recreational anglers.
5. Collectively, no evidence was found that anglers and recreational fisheries management constrain the development of aquatic and riparian biodiversity in gravel pit lakes in the study region; however, the conservation of species diversity in gravel pit lakes could benefit from an increasing reliance on habitat enhancement activities.