鳙鱼微囊藻毒素去毒酶基因cDNA全序列的克隆与序列分析

淡水鱼类可溶性谷胱甘肽S-转移酶(soluble glutathione S-transferase,sGST)在微囊藻毒素去毒代谢过程中起着关键作用,又被称为微囊藻毒素去毒酶.通过简并引物克隆鳙鱼肝脏sGST基因cDNA核心序列,应用5'-RACE和3'-RACE技术分别扩增该序列的5'末端和3'末端序列,最后通过序列拼接获得鳙鱼肝脏sGST基因cDNA全序列.序列分析结果表明,鳙鱼肝脏sGST基因cDNA全长934 bp,其中5'-UTR长104bp,3'-UTR长158 bp,编码区672 bp,编码223个氨基酸.鳙鱼与海水鱼类、鸟类、哺乳类等不同类群sGST的N-末端功能域氨基酸同源性较高,达75%左右,而C-末端功能域氨基酸同源性较低,仅为43.6%-55.9%,这与脊椎动物sGST的N-末端功能域均存在相同的GSH结合位点,而C-末端功能域存在不同的底物结合位点相一致.鳙鱼sGST的C-末端功能域的低同源性可能与其承担微囊藻毒素去毒代谢之特殊功能有关.     

三种裸腹溞的16S rDNA序列分析与系统分类探讨

为了检验已记录的3种裸腹溞(发头裸腹溞(Moina irrasa)、短型裸腹溞(Moina brachiata)、微型裸腹溞(Moina micrura))的系统分类,用试剂盒法分别提取3种裸腹溞的基因组DNA.利用特异性引物,通过PCR扩增了3种裸腹溞的16S r DNA部分序列,并与来自Gen Bank中每个种类相似度较高的裸腹溞属序列进行分析.结果表明,3种裸腹溞的平均种间相似度为88.7%,碱基中A+T含量均明显高于G+C含量.本研究的发头裸腹溞的16S r DNA序列与Gen Bank所下载的多刺裸腹溞(Moina macrocopa)的16S r DNA序列相似度为99%,遗传距离(K2P双参考模型)为0.5%,属种内范围;两个地区的短型裸腹溞测得的16S r DNA序列与Gen Bank下载的欧洲短型裸腹溞的16S r DNA序列序列相似度相对较低(88%~90%),遗传距离较大(13.2%~13.5%左右),已达到属内种间分化水平.基于16S r DNA构建的NJ树和贝叶斯树也支持以上结论.结果表明,本研究的发头裸腹溞可能为多刺裸腹溞,本研究用的短型裸腹溞与Gen Bank下载的欧洲短型裸腹溞已经达到种间分化的标准.由于缺乏物种形态资料和其他分子标记的对比,3种裸腹溞的分类地位还需进行更深入的探讨.     

基于COI基因序列的太湖新银鱼遗传多样性

利用线粒体细胞色素C氧化酶I(COI)分子标记分析长江中下游太湖新银鱼(Neosalanx taihuensis)8个地理种群132个样本的遗传多样性.该基因630 bp片段的碱基序列共检出8个核苷酸变异位点(变异率1.27%),其中局域性单倍型居多(75%),群体单倍型多样性较高(h=0.576±0.036),而核苷酸多样性较低(π=0.00112±0.00204).不同地理种群遗传多样性差异显著:有人工移植历史种群遗传多样性较高、隔离度较高的种群遗传多样性较低,但大部分的遗传变异来自于种群内(54.83%),反映出地理隔离和人为干扰对太湖新银鱼遗传格局影响显著.研究表明COI基因适于银鱼科鱼类物种鉴别和系统发育研究,同时可为同种种群间遗传关系分析提供一定的信息.     

原生动物单细胞PCR应用初探——以天蓝喇叭虫为例

以天蓝喇叭虫(Stentor coeruleus)为研究对象,探索了单细胞单基因PCR扩增及单细胞全基因组PCR扩增技术在原生动物中的应用.经过不断探索和优化条件后,试验取得了理想的结果.在40例单细胞单基因(SSU rDNA基因全序列)PCR的一次性扩增中,新鲜细胞和经过中性红染色的细胞都获得了100%的成功率,室温下酒精(95%)保存一周的细胞获得了82.5%的成功率.在单细胞全基因组PCR扩增中,采用高效高保真的phi29 DNA聚合酶结合随机引物(Random Primer)进行扩增,获得了丰富且质量较高的PCR产物.以全基因组扩增产物(稀释10倍)对四个常用的基因位点(TEF1、SSU rRNA、18S-ITS1-5.8S、a-tubulin)进行扩增,均成功获得相应的基因片断.     

基于分子标记的藻类鉴定研究进展

藻类鉴定被广泛应用于藻类遗传学、生理学、生态学和应用藻类学,尤其是藻类调查和评估.然而,基于形态学的鉴定往往因为分类特征未出现或不典型、设备限制和人员经验欠缺等原因带来较大误差.随着测序技术的不断发展,分子标记已成为藻类鉴定的一个通用工具.由于藻类类群众多且差异很大,分子标记的选择成为藻类鉴定的关键.本文综述了蓝藻、硅藻、绿藻、甲藻、裸藻、隐藻、金藻、黄藻、红藻和褐藻等主要门类分子标记的选择及应用进展,包括分子标记选择原则、常用标记和相应序列数据库,以及各个分子标记在不同类群应用中的优缺点等.藻类分子鉴定源于编码核糖体RNA的基因（rDNA）,发展于细胞核、线粒体、叶绿体DNA等.然而,当前藻类分子鉴定逐渐细化和完善,单一的核糖体DNA、内转录间隔区（ITS）和保守蛋白编码基因等短序列分子标记已经很难满足藻类鉴定的需求,多标记组合成为一种必然选择.同时,线粒体基因组、叶绿体基因组、核基因组、转录组和宏基因组等提供了更多遗传进化信息,弥补了短序列分子标记在系统分类应用中的不足.对于藻类鉴定,单纯依赖分子标记或形态学都不足以保证鉴定的准确性,采用将分子生物学、形态学、生理生化学等结合的多相学方法,才能准确地完成鉴定工作.此外,藻类分子数据库的建立和完善是未来分子鉴定的重要工作,快速鉴定方法也必将在未来获得广泛的应用和发展.     

广东省水库枝角类组成特征的初步研究

于2000年丰水期和枯水期调查了广东省19座典型大中型水库枝角类,随后对其中3座代表性水库进行了4年的枝角类种类定性调查.在鉴定出的枝角类16属24种中有13个广温种、7个嗜暖种、3个嗜寒种和1个特有种;每个水库的平均种类数5-6种.从个体大小来看,水库枝角类以小个体种类为主,有14种的体长小、于1 mm,它们分布广且数量多,其中优势种主要为长额象鼻溞、秀体溞、微型裸腹溞、颈沟基合溞和角突网纹溞等.透明溞和透明薄皮溞是主要的大个体种类,出现在4座贫、中营养水库的水样中.水库枝角类的丰度较低,在丰水期,平均丰度小于10 ind./L和10-20 ind./L的水库分别为14座和5座,在枯水期,为12座和5座,而高州水库和河溪水库中的平均丰度达67ind./L.主要优势种在不同营养类型水库中分布差别不大.枝角类的丰度受水力滞留时间影响,直流型水库的枝角类种群丰度的分布明显受到水流速度的影响,湖泊区枝角类个体及其食物均随水库放水输出而减少.由于采样点主要位于大坝附近,枝角类以敞水区的种类为主.从种类组成和数量来看,广东省水库枝角类具有明显的热带特点,枝角类的生物量小,以个体较小的种类优势种,大型种类的个体数量极低且身体透明.     

五个湖泊中华刺鳅(Mastacembelus sinensis)种群线粒体细胞色素b遗传多样性分析

采用特异性引物对中华刺鳅基因组DNA进行扩增和测序,获得了江苏省5个湖泊的中华刺鳅种群线粒体cyt b基因767 bp的序列.分析表明,cyt b基因中A+T含量略高于G+C含量,144个中华刺鳅共获得cyt b基因单倍型28个,变异位点38个,其中单倍型Hap1分布最为广泛,可能为祖先型单倍型.平均单倍型多样性和平均核苷酸多样性指数分别为0.58751和0.00214,遗传多样性较低.群体间遗传分化系数为0.0356,绝大多数的遗传变异都来自群体内部,而群体间的遗传分化极小.构建的系统进化树未出现明显的以单个湖泊群体的聚簇,5个湖泊的种群间基因交流极其强烈.研究结果表明,当前5个湖泊的中华刺鳅遗传多样性水平比较低,亟待加强保护.     

太湖与广东汤溪水库微囊藻gyrB基因序列分析

测定了江苏太湖和广东汤溪水库7株铜绿微囊藻(M.aeruginosa)和1株水华微囊藻(M.flos-aquae)gyrB基因部分序列(974bp),发现40个变异位点,17个简约信息位点,平均(G+C)%为47.8%,序列间相似性≥97.10%.在NJ分子系统树上,不同种类的微囊藻混杂分布,表明基因型聚类与表型无直接关系;不同地理来源的铜绿微囊藻间遗传变异小,没有明显的地理聚群,反映出地理差异并不影响遗传上的相似性;支持暂将不同藻种归为铜绿微囊藻复合种的分类处理.gyrB基因对微囊藻遗传差异的解析效果优于16S rRNA,与16S-23S ITS和cpcBA-IGS等效果相当,表明gyrB基因可能成为研究微囊藻分类和遗传变异新的良好分子标记.     

5个湖泊河川沙塘鳢(Odontobutis potamophila)种群线粒体细胞色素b基因的遗传变异分析

通过对5个湖泊的河川沙塘鳢种群的线粒体DNA细胞色素b基因进行PCR扩增、测序,获得1141 bp的序列全长.序列分析显示,cyt b基因序列中A+T含量(55.8%)略高于G+C含量(44.2%),共检测到806个多态位点,115个样本得到87个单倍型,平均单倍型多样性为0.969±0.012,核苷酸多样性为0.20081±0.00742,遗传多样性表现高度多样性.太湖种群与大纵湖种群间的遗传距离最近,为0.137,巢湖种群和大纵湖种群之间遗传距离最远,为0.424.分子方差分析表明,群体间遗传分化系数Fst为0.531,变异来自群体内及群体间.cyt b基因序列构建的UPGMA系统进化树显示,5个种群分化成不同的分支系谱,种群间存在的基因交流较少.     

南京玄武湖浮游甲壳动物的水平分布及其与环境的关系

于2010年7月对玄武湖三个区域(荷花区、湖岸区、敞水区)的浮游甲壳动物水平分布进行了分析,并探讨了浮游甲壳动物种类、丰度分布与环境因子的关系.结果显示:荷花区、湖岸区、敞水区的氮磷营养盐浓度无显著差异,三个区域pH逐渐变高.相比其他区域,荷花区叶绿素a浓度最高而总悬浮质浓度最低.荷花区浮游甲壳动物丰度显著高于敞水区和湖岸区.全湖枝角类优势种为模糊秀体溞、微型裸腹溞、尖额溞以及壳纹船卵溞.桡足类的优势种为北碚中剑水蚤和台湾温剑水蚤.与其他湖区相比,荷花区浮游甲壳动物种类最多,且检出缺刺新秀体溞等稀有枝角类.冗余分析(RDA)表明与玄武湖浮游甲壳动物优势种分布相关的重要环境因子为总悬浮质、叶绿素a和总溶解磷浓度.结合浮游甲壳动物种类、丰度分布及环境因子分析得出,模糊秀体溞更倾向于分布在敞水区,而荷花区为其余大部分浮游甲壳动物种类提供了合适的生存条件.     

Daphnia magna as a Model to Assess Heavy Metal Toxicity: Comparative Assessment with Mouse System

The acute static bioassays with heavy metals at 13 ± 1.5 °C in hard water (total hardness = 240 ppm as CaCO₃) were conducted in the laboratory with Daphnia magna. The 48 hr LC values and their 95 per dent confidence limits in ppm of metal were: 0.0038 (0.0027… 0.0053) for Hg; 0.015 (0.009… 0.026) for Ag; 0.1 (0.063… 0.16) for Cu; 0.69 (0.48… 1.0) for Zn; 1.52 (1.01… 2.28) for Co; 1.8 (1.16… 2.70) for Cr; 1.5 (1.07… 2.1) for Cd; 2,63 (1.7… 4.08) for Pb; 7.3 (5.49… 9.71) for Ni and 19.5 (13.45… 28.3) for Sn. At the close of 24 h of exposure Zn and Cd solutions showed milky white precipitation. The order of toxicity of heavy metals in this model was: Hg>Ag>Cu>Zn>Cd>Co>Cr>Pb>Ni>Sn. Comparison of D. magna and mouse LC₅₀ data showed that Daphnia were sensitive at 1/2710, 1/83, 1/123 and 1/5.5, the LC₅₀ values of Hg, Cu and Zn, respectively. Toxicity test with D. magna is simple, requiring less space, time, facilities, acclimatization time and equipment. Furthermore, Daphnia is more functional, reliable and gives reproducible results. Based on this data we believe the Daphnia magna model can be used as a model to study heavy metal toxicity.     

Growth of Daphnia magna males and females fed with the cyanobacterium Microcystis aeruginosa and the green alga Scenedesmus obliquus in different proportions

Laboratory experiments were used to study the sensitivity of both male and female Daphnia magna to a toxic cyanobacterium, Microcystis aeruginosa. Male and female D. magna were fed with M. aeruginosa and a green alga (Scenedesmus obliquus) in different mixtures that included 0%, 25%, 50%, 75% and 100% Microcystis. Growth of both males and females declined with increasing proportion of Microcystis in the diet. Males were slightly less sensitive to Microcystis than females with EC₅₀ for growth inhibition at 72% and 63% Microcystis in the diet, respectively. On monospecific Microcystis diet, mortality rates for males (0.16 d^–1) and females (0.17 d^–1) were similar. The reproductive rate of females was the most sensitive endpoint with an EC₅₀ of 42% Microcystis in the diet, whereas population growth rates were the least sensitive (EC₅₀ of 74% Microcystis). Microcystis in the diet severely depressed growth and reproduction in Daphnia and poses a severe threat to its survival in which only a switch in reproductive strategy might provide Daphnia a refuge to a Microcystis environment that gradually becomes uninhabitable.     

Biodegradability and Toxicity of Anionic Surfactants

Alkyl sulphates of the general formula CH_{2n + 1}-O-SO₃Na and alkyl sulphonates of the general formula C_nH_{2n + 1}-SO₃Na, where n = 8, 10, 12, 14 in each series have been tested for biodegradability with the modified OECD screening test No. 301 E, and for toxicity with Daphnia magna and with Photobacterium phosphoreum (the Microtox^TM test). The results show that both alkyl sulphates and alkyl sulphonates are biodegraded with the alkyl sulphates degrading more rapidly under the test conditions. The alkyl sulphates also have higher toxicity to both test species and for both alkyl sulphates and sulphonates the toxicity increases linearly with increasing molecular weight of the surfactant.     

On the Toxicity and Biodegradation of Cationic Surfactants Über die Toxizität und den biologischen Abbau kationischer Tenside

Three ammonium halide type surfactants were tested under standard laboratory condition for biodegradability and acute toxicity to Daphnia magna and in the Microtox test with the luminescent bacterium Photobacterium phosphoreum. The tested compounds are dimethyldistearylammonium chloride (DSDMAC), cetyltrimethylammonium bromide (HTMAB) and cetylbenzyldimethylammonium chloride (HBDMAC). The results indicate DSDMAC is less toxic than the other two surfactants and that it biodegrades rapidly at both lower (2.5 mg/L) and higher (10 mg/L) concentrations.     

Zooplankton community structure and ecology of the tropical-highland Lake Hayq, Ethiopia

Lake Hayq, a highland lake in Ethiopia, was stocked with Tilapia fish (Oreochromis niloticus) in late 1970s, offering an opportunity to study the effect of fish predation in a natural lake. Since 1930s, some limnological surveys have been done sporadically documenting a change in zooplankton composition including the disappearance of cladocerans, hypothesizing the stocked planktivorous fish could be a cause. Nevertheless, no detailed research was conducted to identify potential effects of fish stocking predominantly due to its remote location. The article presents data about zooplankton composition, abundance and biomass done between October 2007 and January 2009 on short-time intervals including the underlying limnological variables. The zooplankton community was depauperate comprising two copepods, three cladocerans, and six rotifers taxa, as typical for tropical lakes. Total mean standing biomass of all crustacean zooplankton was 237 mg dry mass m⁻³, which gave Lake Hayq an intermediate position when compared with other tropical lakes. Of copepods, Thermocyclops ethiopiensis was almost an exclusive species, and its temporal variation was influenced by food supply and water temperature. We refute the hypothesis that Tilapia was the cause for the seasonal disappearance of cladocerans, and attribute it to the adverse effect of episodic mixing. Nevertheless, the planktivorous fish probably plays a key role in structuring the cladocerans in particular the large-sized Daphnia magna. In January 2008, we observed a massive planktivorous fish mortality that triggered high algal biomass, which was later grazed by large-sized D. magna demonstrating the trophic cascade hypothesis in a natural ecosystem.     

A comparative study of toxicity identification using Daphnia magna and Tigriopus japonicus: Implications of establishing effluent discharge limits in Korea

In Korea, the new permission criteria for industrial effluents based on Daphnia magna acute toxicity tests will be gradually implemented starting from 2011. Thus, in this study, toxicity assessment and identification using a marine species (Tigriopus japonicus) and the freshwater species (D. magna) was comparatively investigated. Effluent from an acid mine drainage treatment plant showed acute toxicity toward both organisms due to low pH, which was removed by neutralization of the effluent. Additionally, evaluation of the effluent of an electronics company revealed that Cu was attributable to the observed toxicity, and the effluent was more toxic toward T. japonicus than D. magna. Moreover, effluents from a metal plating factory were acutely toxic toward D. magna (6.50 TU), while they were not toxic against T. japonicus. Toxicity identification revealed that the high level of Cl⁻ (12,841 mg L⁻¹) was the cause of toxicity. Thus, the effluents had no effect on the marine species, T. japonicus. These findings suggest that a marine species rather than a freshwater species is more desirable for toxicity assessment of industrial effluent discharged into the saltwater, and thus should be considered in the legislation of toxicity-based discharge limits in Korea.     

淡水鱼类环境DNA宏条形码引物的筛选及其在千岛湖的应用

基于环境DNA(eDNA)技术的鱼类多样性监测方法因具有灵敏度高、对目标生物无伤害以及成本低等特点,近年来在国内外得到了广泛应用。eDNA方法的有效性往往取决于宏条形码引物的选择,尽管目前已经有一些鱼类环境DNA宏条形码引物,但较少有研究综合评估这些引物的检出效果。本研究评估了来自COI、Cytb、12S rRNA和16S rRNA基因的29对鱼类eDNA宏条形码引物(包括本研究设计的2对和从国内外文献中引用的27对引物),首先基于计算机模拟PCR(in silico PCR)进行了初步分析,随后通过高通量测序对其中效果较好的17对引物开展了更进一步的验证,结果显示:计算机模拟PCR结果良好的引物在进行高通量测序时并非全部表现良好,表明引物的筛选不能仅仅依靠计算机模拟PCR;具有更长扩增片段长度的宏条形码引物并没有获得理想中更好的扩增效果,表现效果好的引物大多是扩增片段长度处于200~300 bp之间的引物;相对于COI和Cytb而言,12S rRNA引物与16S rRNA引物均具有良好的扩增效果,适宜作为鱼类环境DNA宏条形码而用于鱼类多样性研究;同时,鉴于当前的鱼类DNA条形码数据库尚不完备以及不同引物的特性不同,使用多对引物将大大增加物种的检出概率和eDNA研究的可信性。本研究展示了eDNA在评估生物多样性方面的潜力,有助于将来的鱼类eDNA研究,从而为鱼类多样性的保护提供参考。     

太湖不同生态型湖区湖鲚(Coilia ectenes taihuensis)食物组成及其季节变化

利用单船表层单囊拖网于2013年2 12月分别在太湖不同生态型湖区(敞水湖区和草型湖区)采集湖鲚(Coilia ectenes taihuensis)样品,同时用刺网补充大个体样品.通过胃含物鉴定,分析湖鲚饵料生物的种类及数量季节和湖区间差异,同时测定湖鲚摄食率、肥满度和食物选择系数等.结果显示,湖鲚主要食物为浮游枝角类和桡足类,此外,水生昆虫、轮虫和小型鱼虾也是湖鲚的重要食物来源.湖鲚食物组成季节变化明显,冬季以桡足类的哲水蚤(Calanus spp.)、剑水蚤(Cyclops spp.)为主,夏、秋季则以枝角类的象鼻溞(Bosmina spp.)、裸腹溞(Moina spp.)和盘肠溞(Chydorus spp.)为主.不同湖区湖鲚食物种类差异明显,草型湖区水生昆虫、鱼虾较敞水湖区多,桡足类较敞水湖区少.不同湖区间湖鲚食物数量差异明显,敞水湖区湖鲚平均食物数量为788.98±262.4 ind./尾,而草型湖区仅为298.85±71.1 ind./尾.湖鲚主动摄食大型枝角类,如僧帽溞(Daphnia cucullata)、低额溞(Simocephalus spp.)和大型溞(Daphnia magna),选择指数(Ii)介于0.59~1.00之间.小型裸腹溞(Moina spp.)也是湖鲚主动选择的食物(0.62Ii0.95),但湖鲚对桡足类选择性较低,这可能与桡足类较强的逃避能力有关.湖鲚对食物选择性的季节变化明显,如冬季回避透明薄皮溞(Leptodora kindti),而夏季则主动选择,但不同湖区湖鲚对食物的选择差别较小.本研究结果有利于从食物网层面剖析湖鲚种群增长原因,进而通过生物操纵理论对湖鲚种群进行控制,最终为形成合理的湖泊渔业结构和湖泊环境修复提供科学依据.     

Separation of phytoplanktonic pigments by HPLC for the study of phyto-zooplankton trophic relationships

Phyto-zooplankton trophic relationships were studied using phytoplanktonic pigments (chlorophylls and carotenoids) as organic natural markers. Pigments were separated by high-pressure liquid chromatography (HPLC).Comparison of pigment profiles from monospecific cultures of various taxonomic groups (Chlorophyceae, Bacillariophyceae and Cyanobacteria) and from Cladocera crustaceans (Daphnia magna) fed with these cultures, showed that the characteristic pigment associations of the different taxa are conserved during their transfer from primary producers to secondary consumers.Chromatographic profiles of the Bacillariophyceae and Chlorophyceae type were obtained fromDaphnia respectively fed with mixtures of a Chlorophyceae and a diatom species and mixture of a Chlorophyceae and a Cyanobacterium. This showed the importance of this method in demonstrating a possible selective feeding by the herbivorous zooplankton.The observation of pigment profiles of the Dinophyceae type following feeding of a zooplankton assemblage from Lake Pavin within this natural medium (phytoplankton dominated by a Dinophyceae) and of a Chlorophyceae type profile as the same assemblage was fed in the laboratory on phytoplankton from Lake Villerest (composed of about 80% Cyanobacteria and 20% Chlorophyceae), suggested that this method could be applied to the natural environment.     

Influence of selected fluorescent dyes on small aquatic organisms

Rhodamine B and Rhodamine WT are fluorescent dyes commonly used as tracers in hydrological investigations. Since introducing intensely red substances into rivers raises understandable doubts of ecological nature, the authors aimed at examining the influence of these dyes on small water fauna using bioindication methods. Quantitative results, calculated with the use of Bliss-Weber probit statistical method, were achieved by means of standardized ecotoxicological tests containing ready-to-hatch resting forms of fairy shrimp (Thamnocephalus platyurus). Qualitative studies included observation of water flea crustacean (Daphnia magna) and horned planorbis snail (Planorbis corneus), both typically present in rivers and representative for temperate climate, as well as guppy fish (Poecilla reticulata), paramecium protozoan (Paramaecium caudatum) and the above-mentioned fairy shrimp. The investigation revealed that both dyes in concentrations used for hydrological purposes are low enough to exert almost no toxic impact on water fauna considered.