天津沿海菲律宾蛤仔人工育苗技术的初步研究

利用自然海水和天然饵料,采用无泥砂采苗方法,在天津沿海首次成功开展了菲律宾蛤仔工厂化育苗。幼虫初始壳长93.8μm,壳长平均日增长8.3μm,成活率41.0%;第15-23 d为变态期,壳长平均日增长12.9μm,变态率47.8%;变态后至第55 d,壳长平均日增长33.2μm,成活率76.0%。本次育苗试验充分利用天津沿海水质条件与环境特点,改进育苗方法,降低生产成本,建立了适于天津海区的菲律宾蛤仔人工育苗工艺,共得到双水管稚贝约1 800万枚。     

4种壳色蛤仔TYR基因的表达特性研究

为了解色素控制基因与壳色的关系,本实验用荧光定量聚合酶链式反应(Polymerase Chain Reaction,PCR)技术分析了酪氨酸酶基因(Tyrosinase,TYR)在菲律宾蛤仔(Ruditapes philippinarum)4种壳色蛤仔和7种组织中的表达特性。结果表明,酪氨酸酶基因在鳃、外套膜、闭壳肌、性腺、内脏团、水管和唇瓣中均有表达,其中外套膜中表达量较高,其次为鳃,在闭壳肌中表达量最少。不同的酪氨酸酶基因在4种壳色的表达量不同,酪氨酸酶基因TYR2、TYR3、TYR6和TYR9在白斑马蛤中表达量居高,且与斑马蛤和白蛤差异显著(P0.05)。TYR2、TYR6和TYR10在橙蛤中表达量最高,推测与橙色形成有关。TYR11在斑马蛤中表达量最高,且与白蛤和白斑马蛤差异显著(P0.05),推测与背景色形成有关。系统发育分析结果表明,TYR3与马氏珠母贝(Pinctada martensii)同源性最高为64%。TYR10与加州双斑蛸(Octopus bimaculoides)同源性最高,为53%。与长牡蛎进化关系最近。     

蛤仔回交家系建立及早期生长发育比较

2009年9月,作者以蛤仔(Ruditapes philippinarum)杂交家系 HD(♀H×♂D)及其亲本家系 H、D 为材料,采取单对交配策略建立了 B1F1(♀HD×♂H)、B2F1(♀D×♂HD)、B3F1(♀HD×♂D)和 B4F1(♀H×♂HD)4个回交家系,并对各家系蛤仔早期生长发育进行比较.结果表明:家系间蛤仔的卵径、受精率、孵化率和初孵 D 形幼虫壳长差异不显著(P>0.05);浮游幼虫期, B1F1和 B3F1家系幼虫生长速度显著快于B2F1和 B4F1(P<0.05),9日龄时 B2F1和 B4F1幼虫存活率显著低于 B1F1和 B3F1(P<0.05);变态期, B2F1和B4F1家系幼虫表现出明显的变态时间延长、变态规格小型化和变态率低等近交衰退现象;稚贝期, B1F1和 B3F1家系稚贝生长优势开始显现,而 B2F1和 B4F1家系稚贝生长和存活劣势依旧明显.综上可见以杂交 F1(♀)为母本的 B1F1和 B3F1家系早期生长和存活性状显著优于以杂交 F1(♂)为父本的 B2F1和 B4F1家系,因此在蛤仔回交育种中以杂交 F1(♀)为母本更有利于选择.     

辽宁沿海菲律宾蛤仔不同地理群体形态差异研究

利用多变量形态度量学方法,对辽宁沿海菲律宾蛤仔(Ruditapes philippinarum)长兴岛(CXD)、旅顺(LS)、营口(YK)、盘锦(PJ)、锦州(JZH)、海洋岛(HYD)、黑石礁(HSJ)以及金州(JZ)8个野生群体进行形态学比较分析。结果表明:主成分分析得到了3个主成分,贡献率分别为40.21%,19.98%和11.57%,累积贡献率为71.75%。聚类分析结果显示,旅顺群体和锦州群体形态最为接近,而与黑石礁群体差异较大。营口群体的趋异程度最大,长兴岛群体、金州群体和海洋岛群体的趋异程度居中。长兴岛群体壳型最扁,贝壳较厚且重;营口群体软体部最肥;金州群体壳型"凸"型最明显,且质量最大,而黑石礁群体贝壳最薄。这些明显特征揭示了不同群体的形态差异。研究结果为菲律宾蛤仔的地理群体识别、种质资源保护与恢复提供了重要依据。     

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…     

有效群体大小对蛤仔F1生长和存活影响

对不同有效群体大小(Ne=2、4、8、16、32、200)的蛤仔F1代生长和存活性状进行了比较。结果表明:浮游期,各试验组幼虫壳长均未表现出显著性差异(P0.05),实验组Ne=200的存活率低于其他各实验组,并随着日龄的增长,差异越来越显著;稚贝期,各实验组的壳长和存活率的变化规律和浮游期基本一致。对壳长变异情况分析表明,浮游期,随着群体有效含量的增大,子代开始出现小型个体和大型个体,壳长分布变异逐渐明显;稚贝期,各试验组组内壳长变异进一步加剧,除Ne=2试验组稚贝壳长介于400μm和600μm之间外,其他各试验组均出现明显的小型和大型个体,表现为有效群体越大,组内稚贝壳长变异越为明显。     

恩诺沙星在菲律宾蛤仔体内的消除规律研究

以5mg/L恩诺沙星浸浴菲律宾蛤仔(Ruditapes philippinarum)24h,取血液、鳃、内脏团、足、闭壳肌5种组织,反相高效液相色谱法测定其中的药物浓度。结果表明,恩诺沙星按一级动力学过程从体内消除,在5种组织中消除速率快慢不一,其在菲律宾蛤仔血液、鳃、内脏团、足、闭壳肌中的消除曲线方程分别为c=6.995e-0.0853t,c=2.7957e-0.1177t,c=1.539e-0.1013t,c=1.7015e-0.1535t,c=2.010e-0.1414t,消除半衰期T1/2分别为6.284,5.889,6.483,4.516,3.679h。恩诺沙星在水产品中的最大残留限量为50μg/kg,在本实验条件下,建议临床休药期不小于2d。     

柴油水溶性成分胁迫下菲律宾蛤仔溶酶体生物标志物的应答

溶酶体生物标志物是环境污染引起生物效应的早期预警信号。石油类污染物对菲律宾蛤仔(Ruditapes philippinarum)溶酶体完整性的影响尚未研究。本研究以0号柴油的水溶性成分(WSF)为污染物,以养殖的菲律宾蛤仔为受试生物,测试3种溶酶体生物标志物对柴油WSF胁迫应答的剂量-效应关系和时间-效应关系。采用室内模拟暴露实验,将菲律宾蛤仔置于添加不同浓度(0.1~1.6 mg/L)柴油WSF的海水中5 d,结果显示:不同浓度的暴露组血细胞溶酶体中性红保留时间(NRRT)值比对照组低23.1%~48.5%(p<0.05或p<0.01)。而各浓度暴露组的鳃和内脏团酸性磷酸酶(ACP)活性均与对照组无显著差异。选取柴油WSF为0.8 mg/L进行污染的时间-效应实验,于第0、1、3、7、11天取样测定,结果显示:暴露组NRRT值始终低于对照组,且在第1、3天差异显著(p<0.05)。暴露组鳃和内脏团ACP活性都是先逐步升高到第7天达最大值后迅速下降,鳃ACP活性从第3天到第11天与对照组差异显著(p<0.01),内脏团ACP活性从第7天到第11天与对照组差异显著(p<0.05)。本研究未能获得基于N-乙酰-β-氨基己糖苷酶的组织化学染色反应上的溶酶体膜稳定性(LMS)应答。研究表明,菲律宾蛤仔血细胞NRRT和鳃ACP活性可作为海洋污染生物效应监测的潜在生物标志物。     

Risk assessment of TBT in the Japanese short-neck clam (Ruditapes philippinarum) of Tokyo Bay using a chemical fate model

A risk assessment of Tributyltin (TBT) in Tokyo Bay was conducted using the Margin of Exposure (MOE) method at the species level using the Japanese short-neck clam, Ruditapes philippinarum. The assessment endpoint was defined to protect R. philippinarum in Tokyo Bay from TBT (growth effects). A No Observed Effect Concentration (NOEC) for this species with respect to growth reduction induced by TBT was estimated from experimental results published in the scientific literature. Sources of TBT in this study were assumed to be commercial vessels in harbors and navigation routes. Concentrations of TBT in Tokyo Bay were estimated using a three-dimensional hydrodynamic model, an ecosystem model and a chemical fate model. MOEs for this species were estimated for the years 1990, 2000, and 2007. Estimated MOEs for R. philippinarum for 1990, 2000, and 2007 were approximately 1–3, 10, and 100, respectively, indicating a declining temporal trend in the probability of adverse growth effects.A simplified software package called RAMTB was developed by incorporating the chemical fate model and the databases of seasonal flow fields and distributions of organic substances (phytoplankton and detritus) in Tokyo Bay, simulated by the hydrodynamic and ecological model, respectively.     

菲律宾蛤仔血淋巴溶菌酶活性的研究

通过不同的反应条件对菲律宾蛤仔(Ruditapes philippinarum)血淋巴溶菌酶活力的影响实验,发现各反应条件与菲律宾蛤仔血淋巴溶菌活力密切相关:随着血淋巴添加量的增加,菲律宾蛤仔血淋巴溶菌活力逐渐升高,添加量在150~250μL之间时,两者呈现直线关系;溶菌活力随着温度和pH的升高呈峰值变化,分别在反应温度为50℃和pH为6.4时达到最大值,但统计分析显示pH在5.2~6.4之间时,溶菌活力差异不显著(P>0.05);溶菌活力随着反应时间的延长逐渐升高,当反应时间大于100 min后趋于稳定。这为探讨菲律宾蛤仔免疫力的不同变化提供基础性的数据。     

菲律宾蛤仔不同地理群体生化遗传结构与变异的研究

采用聚丙稀酰胺凝胶电泳技术对福建平潭岛、浙江象山港、江苏海州湾和辽宁辽东湾4个海区菲律宾蛤仔(Ruditapes philippinarum)野生群体的生化遗传特征进行了分析.结果表明,菲律宾蛤仔4个群体的多态位点比例为62.5%～75.0%,平均杂合度观测值在0.274 3～0.338 7之间,与平均杂合度期望值相近;平均有效等位基因数在1.489 1～1.632 5之间,遗传变异水平较高;杂合子缺失不显著.比较群体间的遗传分化指数和群体每代迁移数发现,平潭岛、象山港和海州湾3个群体间的遗传分化不显著,而辽东湾群体与3个群体间出现遗传差异.聚类分析同样表明,平潭岛群体首先与象山港群体聚类,再与海州湾群体相聚,最后与辽东湾群体相汇.讨论了杂合子缺失不显著原因和群体间遗传分化机制.     

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.     

硬壳蛤的核型研究

基于硬壳蛤（Mercenaria mercenaria（Linnaeus,1758））是一种生长速度快、经济效益高的埋栖型双壳类,为给生产提供更多的遗传学理论依据,以硬壳蛤胚胎（4-8细胞期）为材料,通过压片法和空气干燥法进行染色体制片和核型分析。结果表明：硬壳蛤二倍体染色体数目为2n=38,其中中部着丝粒染色体（m）有14对,亚中部着丝粒染色体（sm）有4对,亚端部着丝粒染色体（sr）有1对,无端部着丝粒染色体（r）。核型公式为28m＋8sm＋2st,染色体臂数=74。未发现性染色体和随体。     

LPS胁迫下五个群体菲律宾蛤仔TYR基因在鳃和肝胰腺中表达特性研究

为了解TYR基因与蛤仔免疫的关系,本实验利用荧光定量PCR技术研究了菲律宾蛤仔五个群体(白蛤、白斑马蛤、斑马蛤、养殖和野生群体)的鳃组织和肝胰腺组织在LPS胁迫下TYR基因在不同时间(0 h、3 h、12 h、24 h、48 h)的表达特性。结果表明,在LPS注射后鳃组织中TYR6基因表达水平在白蛤和白斑马蛤3 h、6 h、12 h,野生蛤仔3 h,斑马蛤3 h、6 h,养殖群体6 h、12 h显著上调(P<0.05),3h野生蛤仔和斑马蛤达到峰值, 6 h白蛤、白斑马蛤、养殖群体达到峰值(P<0.05);在肝胰腺中,养殖群体和白斑马蛤6 h,白蛤6 h、24 h,野生群体24 h,斑马蛤3 h显著上调, 3 h野生蛤仔和斑马蛤达到峰值,6h养殖群体、白蛤、白斑马达到峰值(P<0.05);鳃组织中TYR10基因表达水平在白蛤、野生群体和白斑马蛤3 h,养殖群体3 h、6 h,斑马蛤3 h、12 h显著上调(P<0.05),肝胰腺组织中TYR10基因表达水平在白蛤3 h,野生群体3 h、6 h、12 h,斑马蛤、养殖群体6 h显著上调(P<0.05),推测T...     

养殖迁移对菲律宾蛤仔挥发性有机物质组成变化的影响

环境条件变化是造成贝类挥发性有机物质(VOCs)组成变化的主要原因。菲律宾蛤仔养殖生产中,常进行从开放式自然滩涂迁移至人工围塘养殖的活动。本文研究了这一过程前后菲律宾蛤仔(Ruditapes philippinarum)肌肉组织的VOCs组成特征、变化及其与环境因子相关性。结果显示:基于固相微萃取和气质联用技术,共鉴定出52种VOCs,分别属于醛、醇、酯、烃、呋喃、烷烃、烯、芳香烃、含硫和含氮化合物等。迁移前样本中,检出41种化合物,迁移至人工围塘养殖20 d后,检出32种。迁移造成了化合物种类数量降低、种类发生变化。醛、醇类的相对含量最高、迁移前后的变化尤为显著,呈现由醛高醇低的组成逆转为醇高醛低的特点。溶氧是影响VOCs组成变化及其醇、醛类差别的重要因子。饵料生物量、相对丰度和多样性与VOCs的组成具有显著相关性:自然滩涂微型真核生物多样性高但生物量和丰度相对低,人工围塘多样性低但生物量和丰度高,是VOCs组成差异的关键影响因子。本研究建立了贝类VOCs与关键环境因子的直接联系,为进一步解析贝类品质的环境关系、优化和调控养殖环境等研究提供了参考。     

蒽、菲、芘、■混合液对菲律宾蛤仔抗氧化酶活性的影响

以菲律宾蛤仔（Ruditapes philippinarum）为研究对象，在实验生态条件下采用不同浓度的蒽（Fluoranthene）、菲（Phenaphthene）、芘（Pyrene）、[艹屈]（chrysene）混合液进行染毒实验，研究菲律宾蛤仔体内超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）和谷胱苷肽过氧化物酶（glutathione peroxidase，GPx）的剂量-效应关系和时间-效应关系。实验的3种混合液浓度为5μg／L（低浓度组），50μg/L（中浓度组），500μg／L（高浓度组）。结果表明，在低浓度混合液下（5μg／L），CAT比SOD和GPx更快受到诱导；4种多环芳烃混合污染物对SOD、CAT和GPx活性的影响均为先诱导后抑制的变化过程。     

一株光合细菌菌株RPD-1的生物学特性及其在菲律宾蛤仔育苗中的应用

RPD-1菌株分离自青岛栈桥近岸海水,根据其生理生化特征鉴定为紫色非硫菌科红假单胞菌属(Rhodopseudomonas)。菌株RPD-1适盐范围较广,在NaCl含量为0～8%的培养基中均可生长,在NaCl为3%的培养基中生长最快;在初始pH为6.0～9.0的培养基中均可生长,pH为7.5时生长最快;不同光源的光照条件下,生长速度不同,白炽灯光照下,生长速度快,日光灯照下,生长速度慢;RPD-1菌株蛋白质含量为58.5%,含各种必需氨基酸。菲律宾蛤仔(Ruditapes philippinarum)育苗实验结果表明,RPD-1菌株能提高菲律宾蛤仔幼虫的存活率和变态率;水体氨态氮测定结果表明,RPD-1菌株能显著降低养殖水体中的氨氮。     

Tapes philippinarum seed exposure to metals in polluted areas of the Venice lagoon

The concentration changes of 12 metals (As, Hg, Cd, Cr, Co, Cu, Pb, Mn, Ni, V, Zn, Fe, and Hg) in the soft tissues of Tapes philippinarum during growth were investigated. Clams were seeded in two contaminated areas of the Venice lagoon (San Giuliano and Fusina) and in an area of the Marano lagoon (Lignano Sabbiadoro) close to the clam-farm where the seed was produced. Metal trends were very different according to the considered element, the study site and the growth period.Arsenic was always higher in clam tissues than in surface sediments and suspended particles in all the three stations. Mercury, Cd and Cu were higher in the clams from Marano and Fusina but not in those from San Giuliano. Zinc and Co in clams exceeded the concentrations in the sediment and suspended particles only at Marano. The other elements (Cr, Pb, Mn, Ni, and Fe) were always higher in SPM and sediments. In general metal concentrations in clams were more highly correlated to concentrations in the suspended particles rather than in the surface sediments and in suspended clams rather than in bottom clams, nevertheless significant differences between stations and contaminants were found. Metal concentrations in clams were always lower than the European regulatory limits.     

Response of antioxidant systems to copper in the gills of the clam Ruditapes decussatus

Copper (Cu) is an essential element for biological systems, however, when present in excess, is toxic. Metallothioneins can play an important role in Cu homeostasis and detoxification. Moreover, Cu can catalyse the production of toxic hydroxyl radicals that cause lipid peroxidation but defence systems in the cells can limit the oxidative damage. The present study was performed to investigate the effect of three Cu concentrations (0.5, 2.5 and 25 μg l⁻¹) on the response of antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT), selenium-dependent glutathion peroxidase and total glutathion peroxidase), total proteins, metallothioneins (MT), malondialdehyde (MDA) concentrations in the gills of the clam, Ruditapes decussatus. The activity of antioxidant enzymes and total proteins, MT and MDA concentrations were measured in the gills of the clams after 1, 3, 7, 14, 21 and 28 days of Cu exposure. Results indicate that Cu only induces an imbalance in the oxygen metabolism during the first week of Cu exposure due to a decrease in mitochondrial SOD and CAT, selenium-dependent and total glutathion peroxidase activities. Cu also causes lipid peroxidation, measured by the MDA formation, that was Cu dependent. In the gills of clams exposed to 25 μg Cu/l, the excess of Cu triggers the induction of MT synthesis after 3 days of exposure.