AFLP analysis revealed differences in genetic diversity of four natural populations of Manila clam(Ruditapes philippinarum) in China

1 IntroductionManila clam (Ruditapes philippinarum) is aworldwide distributed species. In China, it is foundfrom the northern to southern seashores (Wang etal., 1993; Zhuang, 2001). This species is eura-line with the salinity tolerance ranging from 20 to4…     

New polymorphic microsatellite markers derived from hemocyte cDNA library of Manila clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> challenged by the protozoan parasite <Emphasis Type="Italic">Perkinsus olseni</Emphasis>

Manila clam Ruditapes philippinarum is one of the most important benthic animals in the coastal north Pacific region, where clam populations have been mixed genetically through trade and aquaculture activities. Accordingly, identification of the genetically different clam populations has become one of the most important issues to manage interbreeding of the local and introduced clam populations. To identify genetically different populations of clam populations, we developed 11 expressed sequence tag (EST)-microsatellite loci (i.e., simple sequence repeat, SSR) from 1,128 clam hemocyte cDNA clones challenged by the protozoan parasite Perkinsus olseni. Genotype analysis using the markers developed in this study demonstrated that clams from a tidal flat on the west coast contained 6 to 19 alleles per locus, and a population from Jeju Island had 4 to 20 alleles per locus. The expected heterozygosity of the 2 clam populations ranged from 0.472 to 0.919 for clams from the west coast, and 0.494 to 0.919 for clams from Jeju Island, respectively. Among the 11 loci discovered in this study, 7 loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction. The 5 loci developed in this study also successfully amplified the SSRs of R. variegatus, a clam species taxonomically very close to R. philippinarum, from Hong Kong and Jeju Island. We believe that the 11 novel polymorphic SSR developed in this study can be utilized successfully in Manila clam genetic diversity analysis, as well as in genetic discrimination of different clam populations.     

菲律宾蛤仔5个地理群体形态差异分析

应用单因素方差分析、聚类分析、主成分分析和判别分析方法,对海州(HZ)、南浦(NP)、莱州(LZ)、莆田(PT)和汕尾(SW)等不同海域的5个菲律宾蛤仔(Ruditapes philippinarum)野生群体形态数据进行比较分析。单因素方差分析表明HZ群体在多个形态参数上与其它组差异显著;聚类分析显示HZ群体与其它组距离较大;主成分分析共提取2个主成分,贡献率为28.28%和20.77%,累计贡献率为49.05%,散布图表明HZ群体与其他组区分明显;建立了5个群体的判别函数,判别准确率P1和P2均为36.0%~80.0%,综合判别率为53.6%,HZ群体的判别率最高。以上结果说明HZ群体与其它4组形态差异显著。     

Metabolic responses in gills of Manila clam Ruditapes philippinarum exposed to copper using NMR-based metabolomics

Copper is an important heavy metal contaminant with high ecological risk in the Bohai Sea. In this study, the metabolic responses in the bioindicator, Manila clam (Ruditapes philippinarum), to the environmentally relevant copper exposures were characterized using NMR-based metabolomics. The significant metabolic changes corresponding to copper exposures were related to osmolytes, intermediates of the Krebs cycle and amino acids, such as the increase in homarine, branched chain amino acids and decrease in succinate, alanine and dimethylamine in the copper-exposed clam gills during 96 h exposure period. Overall, Cu may lead to the disturbances in osmotic regulation and energy metabolism in clams during 96 h experimental period. These results demonstrate that NMR-based metabolomics is applicable for the discovery of metabolic biomarkers which could be used to elucidate the toxicological mechanisms of marine heavy metal contaminants.     

三株赤潮硅藻5.8SrDNA及转录间隔区(ITS)的克隆及序列分析

对引发赤潮的3株硅藻——1株尖刺拟菱形藻(Pseudo-nitzshia pungens)和2株中肋骨条藻(Skeletonema costatum)的5.8SrDNA和ITS(internal transcribed spacers)序列进行了PCR扩增、克隆和序列测定,并分析了硅藻门10株赤潮藻(7株从GenBank获得)的系统进化关系.研究结果表明,尖刺拟菱形藻的ITS和5.8SrDNA的长度为693bp,SK-1(分离自东海赤潮暴发区)测序得到715bp,除ITS和5.8SrDNA外,还包含部分18SrDNA和28SrDNA;SK-2(分离自青岛养殖场)的ITS和5.8SrDNA的长度为331bp,尖刺拟菱形藻与从GenBank中获得的2株尖刺拟菱形藻相似程度最高,为100%,与该属的多列拟菱形藻相似程度稍低,为82.9%.SK-2的ITS序列与SK-1的相似程度很低,只有51%,但与拟中肋骨条藻的ITS序列相似程度高,为95.5%.SK-1的ITS序列与拟中肋骨条藻的相似程度也低,为56.7%.系统进化树反映的结果与相似性反映的结果一致.研究的该株尖刺拟菱形藻从根据ITS序列研究的结果与形态鉴定的结果看是一致的;SK-2可能属于拟中肋骨条藻;SK-1比较特殊,有待于用其他的方法进一步研究确定其分类地位.     

Comparative study on in vitro transformation of paralytic shellfish poisoning (PSP) toxins in different shellfish tissues

Dissected tissues of three shellfish species,the Chinese scallop,Chlamys farreri,Manila clam,Ruditapes philippinarum,and Razor shell,Solen strictu were evaluated for in vitro transformation of paralytic shellfish poisoning(PSP) toxins. Tissue homogenates were incubated with extraction from toxic algae Alexandrium minutum to determine toxin conversion. The effects of heating and addition of a natural reductant(glutathione) on toxin conversion were also assessed. The toxin profile was investigated through high performance liquid chromatography with fluorescence detection(HPLC-FLD) . The evident variations in the toxin content were observed only in Chinese scallop viscera homogenates. The concentration of GTX4 was reduced by 45%(approximately 0.8 μmol/dm 3 ) and 25%(approximately 1 μmol/dm 3 ) for GTX1,while GTX2 and GTX3 increased by six times(approximately 1 μmol/dm 3 ) and 3 times(approximately 0.3 μmol/dm 3 ) respectively. Simultaneously,the total toxicity decreased by 38% during the 48 h incubation period,the final toxicity was 20.4 nmol STXeq/g. Furthermore,heated Chinese scallop viscera homogenates samples were compared with non-heated samples. The concentration of the GTX4 and GTX1 was clearly 28%(approximately 0.53 μmol/dm 3 ) and 17%(approximately 0.69 μmol/dm 3 ) higher in heated samples,GTX2 and GTX3 were four times(0.66 μmol/dm 3 ) and two times(0.187 μmol/dm 3 ) lower respectively. GSH(+) Chinese scallop viscera homogenates samples were compared with GSH(-) samples,the concentration in the GTX4 and GTX1 was 9%(approximately 0.12 μmol/dm 3 ) and 11%(approximately 0.36 μmol/dm 3 ) lower respectively,GTX2 and GTX3 was 17%(approximately 0.14 μmol/dm 3 ) and 19%(approximately 0.006 μmol/dm 3 ) higher respectively. In contrast,there was a little change in the concentration of PSP toxins of Manila clam and Razor shell tissue homogenates. These observations on three shellfish tissues confirmed that there were species-specific differences in PSP toxins transformation. PSP toxins transformation was more pronounced in viscera tissue than in muscle tissue. PSP toxins was possibly interfered by some carbamoylase enzyme,and the activity in Chinese scallop viscera tissue is more remarkable than in the other two species.     

Acetylthiocholine (ATC)--cleaving cholinesterase (ChE) activity as a potential biomarker of pesticide exposure in the Manila clam, Ruditapes philippinarum, of Korea

The acetylthiocholine (ATC) - cleaving cholinesterase (ChE) activity in Manila clam, which is widely distributed throughout the coastal environment of Korea, was assayed as a potentially useful biomarker of organophosphorous pesticides (OPs). A clear dose-response relationship was determined between inhibited ChE in adductor muscle of clams and four OPs (methidathion, chlorpyrifos, diazinon, IBP) which are heavily used OPs in Korea. The measured EC(50)-24?h values of methidathion, chlorpyrifos, diazinon, and IBP for Ruditapes philippinarum were 7.16?μg?l(-1), 0.34?mg?l(-1), 3.01?mg?l(-1), and 3.41?mg?l(-1), respectively. In field studies, ChE activity in Manila clams collected from 23 stations in the mid-western coastal region demonstrated spatial variation with statistical differences. These results suggest that ChE activity in R. philippinarum is a potential biomarker for assessing organophosphorous pesticide contamination in coastal environments.     

Pathologic survey on the Manila clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> (Adams and Reeve 1850) from Haeju off the western coastal Yellow Sea

Pathologic condition of Manila clam, Ruditapes philippinarum collected from Haeju off the west coast of North Korea in February and March 2007 was investigated in this study. The diagnostics included a protozoan parasite, Perkinsus olseni, the brown ring disease (BRD) caused by bacteria Vibrio tapetis and metazoan parasites Trematod and Cestod. P. olseni infection was examined using histology and Ray’s fluid thioglycollate medium (RFTM) assay along with Choi’s NaOH digestion technique. Trematode and cestode infection was also examined from the histology. A total of 140 clams with 35–45 mm in shell length (2–3 years old) were analyzed. Condition factor (CF), a ratio of the wet tissue weight to the shell dry weight, ranged from 0.445±0.074 (February) to 0.544±0.132 (March). Total body burden of P. olseni (i.e. infection intensity) ranged from 18,644±37,755 (February) to 26,933±80,611 cells/g wet tissue (March) with the prevalence of 76.7–43.3% (March) respectively. Prevalence of trematode and cestode infection in February and March varied 22.5–25.0 and 15.0–22.5% respectively. Infection intensity of V. tapetis ranged 1–2 and the prevalence was 1.4 (February) and 5.7% (March). Prevalence and infection intensity of P. olseni in clams from Haeju was comparatively lower than those of clams reported on the west coast of Korea. BRD and the other metazoan parasite infection were also relatively lower or similar to the condition of the clams reported previously. CF of Haeju clams was also relatively higher than the clams in Gyeonggi Bay and Taean, suggesting that Haeju clams had relatively better pathologic and health condition.     

黄、东海地理环境与环流系统分析——长江口及济州岛邻近海域综合调查研究报告(第二章)

简要介绍了黄海和东海的地理环境概况,着重分析调查海域的环流系统。有如下一些初步看法与结论。 台湾暖流的前缘混合水,可从长江冲淡水底层穿越而影响到苏北沿岸,直到32°N以北的浅水区域。对马暖流西侧的水体是东海混合水,而其东侧为黑潮分支。黄海暖流的流向在不同季节具有规律的摆动。黄海底层冷水团属于季节性水团,其强盛及消衰与温跃层的形成及消亡紧密相关。黄海底层冷水团与中部底层冷水并非每年彼此独立,它们的共同特征甚至比其差异更明显。夏季东海冷水不能借助爬升侵入黄海底层冷水团内部。在济州岛南部区域,中层的逆温、逆盐现象,是由黄海密度环流的扩散效应与东海冷水沿黄海底层冷水团边界的爬升这两个原因而形成的。     

TAGGING EXPERIMENTS OF THE PENAEID SHRIMP IN THE BOHAI SEA AND HUANGHAI SEA IN AUTUMN SEASON

There exists obvious difference in recapture rate of tagged shrimp in various sea areas and years in autumn season, but the majority of them (98%) are recaptured in the releasing year. Although the main portion of each shrimp group in the Bohai Sea has separate distribution area, they intermingle in autumn season to search for food, thus forming a principal fishing ground located in the central Bohai Sea.Those shrimps wintering in south-central portion of the Huanghai Sea are generally divided into two populations. One is called the Korean population, inhabiting along the western coast of Koera, which is less in abundance and smaller in shrimp size; and the other is called the Chinese population, inhibiting along the Bohai Sea coast and the Huanghai Sea coast of China, which is richer in abundance and larger in shrimp size. The spawning grounds of the latter extend throughout the Bohai Sea and off the Huanghai Sea coast of China, from the Yalujiang Estuary area in the north to the coastal waters of Haiz     

烟台海域暴发浒苔ITS及5.8S rDNA的克隆及序列分析

以烟台海域引发"绿潮"的浒苔为研究对象,采用PCR技术扩增出浒苔的ITS-1、5.8SrDNA及ITS-2片段,将扩增出的片段纯化后克隆至pGEM-TEasy载体,筛选阳性克隆进行序列测定。结果表明,浒苔的ITS-1序列长度为195bp,5.8S序列为155bp,ITS-2序列为181bp,该序列与浒苔属的多种物种ITS序列具有很高的同源性,在ITS-1区、5.8SrDNA区和ITS-2区仅存在4个转换/颠换位点。结合GenBank注册序列和本研究的结果发现,单纯依靠ITS序列并不能对浒苔属种类进行有效的分类鉴定。     

胶州湾双壳类壳体中的Ca,Mg,Mn,Sr元素组成及影响因素

探讨影响双壳类元素组成的可能因素,从胶州湾沿岸不同取样点采集黄海近海常见的菲律宾蛤仔(Ruditapesphilippinarum)、褶牡蛎(Oystrea denselamellosa)和紫贻贝(Mytilus galloprovincialis),分别测量了3种壳体的矿物物相类型和壳体中的Ca,Mg,Mn,Sr元素组成。研究表明:3种壳体分别属于文石质、方解石质和混合质壳;Ca,Mn元素含量在菲律宾蛤仔、褶牡蛎和紫贻贝壳体中没有差别,而Mg元素含量在菲律宾蛤仔壳体中含量较低,在褶牡蛎壳体和紫贻贝壳体中含量较高,Sr元素恰好与Mg元素相反。双壳类壳体中元素组成主要受壳体矿物物相类型的制约,而与水体环境要素之间的关系弱。     

Genetic responses to metal contamination in two clams: Ruditapes decussatus and Ruditapes philippinarum

Coastal ecosystems are subjected to a wide variety of disturbances, including those due to xenobiotics of agricultural and industrial origin. These pollutants as heavy metals can modify the genetic diversity of populations by favouring or counter-selecting certain alleles or genotypes by differential mortality. In the present study, two genetic markers (phosphoglucomutase and glucosephosphate isomerase) and a protein marker (metallothionein) were monitored in order to determine the impact of heavy metals in different clam populations. Analysis of the genetic structure of the clam populations examined reveals that those inhabiting environments contaminated by heavy metals exhibit a higher allelic diversity and possess alleles at PGM loci that could be selected by the presence of heavy metals. The evaluation of metallothionein levels using a specific polyclonal antibody developed in the Pacific oyster (Crassostrea gigas) demonstrated the existence of a relationship between metallothionein concentrations and the level of metal pollution for clam populations sampled from different sites. An inter-specific difference was also detected between Ruditapes decussatus and Ruditapes philippinarum living in sympatry at the same site, suggesting a differential response of these two species upon exposure to an identical heavy metal concentration.     

Vertical zonation and aggregated distribution of the Manila clam on subtidal sand flats in a coastal brackish lagoon along the Sea of Japan

The Manila clam, Ruditapes philippinarum, has maintained small‐sized populations in a semi‐enclosed brackish lake along the Sea of Japan, the Honjo area of Lake Nakaumi, although the environment and biota of this area have changed dramatically due to a large‐scale reclamation project. There should be underlying processes that enable the restoration of this species from small‐sized populations, such as the existence of source (i.e. reproductive) populations in other areas and depth zones of the lake. However, there has been no robust, properly designed evaluation of the distribution of the Manila clam in the subtidal sand flats. In order to elucidate the possible mechanisms that allow for the persistence of populations of the Manila clam, we examined the spatiotemporal and vertical variation in distributions of 0‐age clams in the subtidal zone of sand flats. Seasonal effects on population variations showed erratic changes among depth zones without a decreasing trend along the depth gradient. Further, many local populations became extinct even in the shallower zones due to seasonal (summer) hypoxia at deeper zones and hypoxia by the accumulation of key benthic species (Asian mussel and decaying macroalgae) in mats at shallower zones. A few surviving local populations were stable with a spatial‐fragmental (patchy) distribution, associated with fragmented accumulations of Asian mussels and macroalgae. Efforts to maintain stable populations and to restore this species in the subtidal area may depend on these small, restricted, patchy local populations. These findings suggest that high fertility and productivity of the Manila clam as well as patchy distribution of small populations may contribute to the maintenance of the population and the avoidance of extinction (by spatially diffusing the risk of extinction) in harsh environments resulting from the reclamation project.     

菲律宾蛤仔(Ruditapes philippinarum)金属硫蛋白基因克隆及Cd~(2+)胁迫下的表达分析

金属硫蛋白(Metallothionein,MT)是一类富含半胱氨酸的小分子蛋白质,参与机体重金属解毒和金属元素代谢等生理过程。本研究采用RACE技术,克隆获得了菲律宾蛤仔(Ruditapes philippinarum)金属硫蛋白(RpMT)的全长cDNA序列。RpMT的cDNA全长为570bp,编码75个氨基酸,包含15个MT所特有的Cys-Xn-Cys结构。采用实时荧光定量PCR技术,分析了两种壳色菲律宾蛤仔(白蛤和斑马蛤)RpMT基因在Cd2+暴露后的表达变化。结果发现:Cd2+急性和亚慢性暴露均可导致两种壳色蛤仔消化腺和鳃组织RpMT基因表达量的显著上调;暴露后两种壳色蛤仔鳃组织RpMT基因表达量的增加幅度均高于消化腺组织,且以白蛤鳃组织基因表达水平的上调幅度较高。上述结果表明,RpMT可能在菲律宾蛤仔抵御Cd2+胁迫过程中发挥了重要作用。     

龙须菜5.8S rRNA和ITS区的克隆与系统学分析

研究青岛产龙须菜居群内的遗传多样性和系统学分类,通过对龙须菜居群内不同个体的5.8S rRNA-ITS区(包括ITS1-5.8S rRNA-ITS2)进行PCR扩增和序列分析,得到扩增DNA片段长度均为1 066 bp,包含完整的ITS1(250 bp)、5.8S(159 bp)和ITS2(657 bp);利用GenBank数据库对Cracilaria(江蓠属)和Gracilariopsis属共20个物种的rRNA-ITS相应序列进行了比较和系统进化分析.结果表明,龙须莱和江蓠科19个物种中rRNA的5.8S区的长度和序列非常保守,而ITS区的长度和序列则变异较大,20种江蓠科物种的遗传距离在0.013～0.596之间,龙须菜在5.8S rRNA-ITS区特性上相比江蓠属物种与Gracilariopsis属物种更为相似;采用UPGMA法构建了这20种江蓠科物种的系统发育树;分析结果表明青岛产龙须菜的居群内不存在遗传差异,且应属于Gracilariopsis属,并且在江蓠科中较早分化,而龙须菜处于Gracilariopsis属中比较古老的位置.     

菲律宾蛤仔不同品系斑马蛤的选择反应及现实遗传力

对菲律宾蛤仔中的斑马蛤、黑斑马、白斑马三品系进行混合选择,测量了各实验组的壳长,计算了不同品系斑马蛤的选择反应和现实遗传力。结果表明:3个品系的上选组壳长均大于对照组,说明单向上选是行之有效的遗传改良方法。在不同的生长发育阶段,斑马蛤品系的选择反应(R)和现实遗传力(2Rh)出现阶段性变化,从平均水平上看,R在幼虫期为0.688±0.184,稚贝期为0.605±0.214,养成期为0.670±0.120;2Rh在幼虫期为0.392±0.081,稚贝期为0.344±088,养成期为0.380±0.058。从一周年的生长情况来看,三个品系总的R为0.654±0.179,斑马蛤、黑斑马蛤和白斑马蛤品系的R分别为0.655±0.155、0.525±0.116、0.782±0.170;三个品系总的2Rh为0.373±0.070,斑马蛤、黑斑马蛤和白斑马蛤品系的2Rh分别为0.372±0.042、0.300±0.028、0.445±0.031。不同品系间R和2Rh次序为白斑马斑马蛤黑斑马,说明各斑马蛤品系的选择效果是不同的,品系间存在着一定程度的遗传差异。     

胶州湾贝类体内29种元素含量的分析研究

从胶州湾不同污染程度的几个地点采集贻贝,蛤仔和牡蛎,通过用中子活化分析技术和原子吸收法,测得它们体内的２９种元素的组成,通过比较元素组成的差异性,初步分析了样品所在地的环境现状,该项研究是中国环境标本库海洋方面的重要部分。     

菲律宾蛤仔(Ruditapes philippinarum)“斑马蛤2号”筏式和底播养殖模式比较研究

为探究菲律宾蛤仔（Ruditapes philippinarum）筏式养殖模式可行性，在我国黄海北部首次开展了菲律宾蛤仔"斑马蛤2号"新品种的筏式与底播养殖模式对比试验。采用平均壳长为2.37 cm，平均全湿重为2.99 g规格的"斑马蛤2号"苗种为试验材料，对两种养殖方式的"斑马蛤2号"生长、存活、营养成分进行比较分析，结果表明，筏式养殖"斑马蛤2号"夏季（4-7月）壳长、壳宽、壳高、全湿重、软体重的相对生长速率分别比底播养殖"斑马蛤2号"快149.54%、100.61%、116.30%、152.25%、132.58%（P<0.05）。筏式养殖"斑马蛤2号"存活率为90%以上，显著高于底播养殖（P<0.05）。两种养殖方式的"斑马蛤2号"水分、灰分、粗蛋白、糖原、脂肪酸等营养成分季节变化规律基本一致。筏式养殖"斑马蛤2号"冬季糖原、单不饱和脂肪酸含量明显高于底播养殖"斑马蛤2号"（P<0.05）。研究表明，当年10月采用多层聚乙烯网笼按密度为150-200粒/层放养菲律宾蛤仔"斑马蛤2号"大规格苗种（平均壳长为2.37 cm，平均全湿重为2.99 g），可于翌年的4月、7月、10月收获平均壳长为3.03、3.36、3.51 cm的市场规格产品。筏式养殖模式具有存活率高的优点。"斑马蛤2号"筏式养殖模式试验的成功为蛤仔健康养殖提供了新途径。     

基于GLM模型Logistic回归的菲律宾蛤仔地理群体判别

菲律宾蛤仔(Ruditapesphilippinarum)是重要的海洋经济贝类和海水养殖品种,具有较强的表型可塑性特点,不同地理群体之间存在表型差异。本研究根据实测数据建立了胶州湾与天鹅湖菲律宾蛤仔壳长(SL)与壳高(SH)、壳宽(SW)和活体重(W)的回归方程,定量分析了两个地理群体的形态学差异。由于其近似圆形的形态使得所能测量的外部指标较少,限制了传统形态测量学在其地理群体判别中的应用。本研究提出了将传统形态测量学和广义线性模型(GLM)相结合的方法,建立了判别菲律宾蛤仔地理群体的逻辑斯蒂(Logistic)回归方程。结果表明:菲律宾蛤仔的天鹅湖群体比胶州湾群体具有更高的SH/SL和SW/SL比值,壳形椭圆较圆且隆起程度高,"凸"形明显;基于GLM模型的Logistic回归能够快速准确地判别菲律宾蛤仔的地理群体归属,判别正确率高达94.23%。本研究为双壳类物种地理群体判别提供了新的分析方法和技术手段。