Hydrogeochemistry of formation water in relation to overpressures and fluid flow in the Qikou Depression of the Bohai bay basin, China

The Qikou Depression is the largest hydrocarbon bearing depression in the western part of the Bohai bay basin, dominated by fan delta and lacustrine strata with volcanic and volcaniclastic rocks. In this study, the formation pressures and hydrochemistry of the formation water in the Qikou depression are investigated. It is found that a significant overpressure occurs in the Dongying (Ed) Formation and the first member (Est), the second member (Es2), the third member (Es3) of the Shahejie Formation. The pressure coefficients commonly range from 1.2 to 1.6 with the highest pressure coefficient being 1.7. The analysis of hydrochemistry data shows that the whole depression is dominated by NaHCO3 water type. The concentration of total dissolved solid (TDS) ranges from 2.13 to 53.16 g/L and shows a distinct vertical variation of salinity and ion ratios. High salinity water (TDS> 10 g/L) occurs below a depth of 2500 m, which coincides with the presence of the overpressured system. However, the increasing trend of TDS is diminished below 3500 m because the generation of organic acids in Qikou Depression is inhibited in the presence of overpressure. The analysis of the relationship among different ions indicates that the present-day characteristics of the formation water result from the albitization of feldspar and the dissolution of sodium-rich silicate minerals and halite in the different hydrochemical and pressure systems. (C) 2009 Elsevier B.V. All rights reserved.

黄海纤毛虫的丰度和生物量

海洋浮游纤毛虫是在海洋中浮游生活的一类单细胞原生动物，主要是指寡毛类纤毛虫(Oligotrich ciliates)，隶属原生动物界(Protozoa)、纤毛门(Ciliophora)、寡毛纲(Oligotrichea)，分属于Oligotrichina 和Tintinnina两个亚纲。它们个体微小，粒径在5-200 µm之间，是微型浮游动物和海洋微食物环(Marine Microbial Food Web)的重要组成部分。 2006年4月至2007年12月，在黄海(包括胶州湾)采样分析海洋浮游纤毛虫的种类组成(砂壳纤毛虫)、丰度和生物量，分析纤毛虫在这一海区的季节变化和空间变化。 纤毛虫丰度和生物量的研究方法为：Rosette采水器(胶州湾用Niskin采水器)采集水样，取1 L水样，加Lugol’s试剂固定(终浓度1%)，Utermöhl方法100倍镜检。测量虫体的体长、体宽，按最接近的几何形状(圆柱体、球体和圆锥体)计算体积。生物量由体积乘转换系数(0.19 pgC/µm3)得到，砂壳纤毛虫的肉体体积按照壳体积的1/3近似。 本文的结果表明，胶州湾各站纤毛虫平均丰度于6月达到全年最高值6065 ind./L，12月为全年丰度最低值843 ind./L。平均生物量8月达全年最高值(18.5 µg C/L)，6月为全年最低值(0.6 µg C/L)。砂壳纤毛虫种丰富度于8月达到最高值，共发现25种砂壳纤毛虫，1月种类最少(6种)。湾内站位的纤毛虫平均丰度比湾外的高(6月和8月除外)。砂壳纤毛虫在纤毛虫总丰度中的比例较小，平均为25%，范围为8-57%，分别于1月和8月达到最低和最高值。 两次冷水团大面调查结果表明，4月表层纤毛虫平均丰度(1490 ind./L)要高于10月(972 ind./L)。10月表层纤毛虫生物量0.14-5.33 µg C/L，14194站、15694站和15894站生物量较高，为4.08-5.33 µg C/L。无壳纤毛虫优势种Laboea strobila在两个航次中均呈现斑块分布，4月航次丰度0-10000 ind./L，10月航次丰度11-350 ind./L；砂壳纤毛虫优势种Ptychocylis obtusa仅在4月航次发现，最大丰度2895 ind./L，10月航次未发现。4月航次砂壳纤毛虫有百乐拟铃虫(Tintinnopsis beroidea)，丰度为0-1920 ind./L；卡拉直克拟铃虫(Tintinnopsis karajacensis)，丰度很小(10-93 ind./L)。10月航次砂壳纤毛虫优势种Tintinnidium primitivum，丰度为35-700 ind./L；也出现了尖底类瓮虫(Amphorellopsis acuta)和网纹虫(Favella spp.)，但丰度不大(0-210 ind./L)；运动类铃虫(Codonellopsis mobilis)、筒状拟铃虫(Tintinnopsis tubulosoides)和Eutintinnus sp.丰度也较低(35-105 ind./L)；Craterella torulata丰度为0-120 ind./L，主要分布于15694站。10月航次已经出现了温跃层，位于30 m左右水层，纤毛虫主要分布于温跃层之上。 六次黄海断面航次表明：温跃层在5月已经出现，到12月消失。在有温跃层的5月、6月、8月、9月，纤毛虫主要分布于温跃层(30 m左右)之上。其中8月份航次纤毛虫丰度最高，表层平均丰度3103 ind./L。12月份纤毛虫丰度最低，表层平均丰度406 ind./L。纤毛虫生物量春夏季为0.02-5.5 µg C/L，冬季为0.04-1.99 µg C/L。小型无壳纤毛虫占优势，砂壳纤毛虫东方拟铃虫(Tintinnopsis orientalis)、筒状拟铃虫、运动类铃虫、Craterella torulata和Tintinnidium primitivum几乎在各个航次均有分布。

牡蛎染色体的分子生物学分析

本研究应用显带技术和荧光原位杂交(Fluorescence in situ hybridization，FISH)技术，鉴定了牡蛎的染色体；应用FISH方法定位了一系列的重复序列和大分子的P1克隆DNA；制备了染色体特异性探针。应用FISH特异性探针成功地鉴定了长牡蛎的三体10。结果如下：1．分析了G带和C带在美洲牡蛎染色体上的分布。G带在每一条染色体上的带型不同，某些染色体间(如第1对和第4对染色体，第7对和第9对染色体)的带型差别不是很明显。G带型容易受染色体收缩程度的影响。C带型重复性较好，染色体带型较清楚，分布在染色体的端粒区域和着丝粒区域。G带和C带带型能够用来鉴定牡蛎的染色体，但是重复性低和带型差异不显著，并不适合常规的染色体鉴定。2．早期胚胎和担轮幼虫制备的染色体适合于FISH分析。染色体制备方法重复性好，可适用于其它贝类的染色体制备。3．研究了重复序列基因--rDNA的定位：1)18S-5.8S rDNA在研究的五种巨蛎属Crassostrea牡蛎均只有一个位 点。太平洋种(C.gigas，C. ariakensis和C. plicatula)中，杂交信号位于最短的染色体一第10对染色体长臂的端粒区域，在大西洋种(C. virginica和C. rhizophorae)中，同一序列定位在第2对染色体短臂的端粒区域。2)18S-28S rDNA在两种蛤中有两个位点。rDNA探针定位在侏儒蛤(Mulinis Lateralis)的第15对和第19对染色体的端粒区域，同一序列定位在硬壳蛤(Mercenaria mercenaria)的第10对染色体的长臂和第12对染色体短臂的端粒区域。信号强度在两对染色体之间有差异。 3)5s rDNA位于美洲牡蛎的第5对染色体的短臂上靠近着丝粒区域和第6 对染色体的短臂的中间区域。信号强度在两对染色体之间没有显著差异。5S rDNA探针可以作为鉴定和识别第5对和第6对染色体的特异性探针。4．研究了一些重复序列的定位1)两个短的重复序列1G8，1P2均产生很强的荧光信号分布在美洲牡蛎所有的染色体上。在低严谨条件下，这些序列均产生很强的信号散布在所有的染色体上。在高严谨条件下，信号强度大大减弱，但是信号仍散布在所有的染色体上。这些重复序列散布在美洲牡蛎的整个基因组中。2)高度重复序列Cgl70产生的信号分布在长牡蛎的7对染色体的着丝粒区域，没有发现间区信号。在第1对，第2对，第4对和第7对染色体上的荧光信号强且稳定。在第5对，第8对和第10对染色体上的信号相对弱且不稳定。在剩余的染色体上(第3对，第6对和第9对染色体)没有检测到荧光信号。结果表明此卫星序列是一个着丝粒卫星序列。在美洲牡蛎的染色体上没有检测到荧光信号，表明了这个着丝粒卫星序列在这两种牡蛎中的分布存在着显著的差异。3)脊椎动物端粒序列(TTAGGG)n的FISH信号局限在四种双壳贝类(美洲牡蛎，the mangrove oyster，硬壳蛤，侏儒蛤)所有染色体的端粒区域，没有发现间区信号的存在。研究结果与已报道的研究结果表明脊椎动物端粒序列或许存在于所有双壳贝类的染色体末端。双壳贝类是目前研究过的唯一含有脊椎动物端粒序列DNA的无脊椎动物。4)研究了RAPD探针在美洲牡蛎染色体上的定位。大多数RAPD探针产生了多个信号散布在间期细胞核和所有的染色体上。引物OPX-03，OPX-04，OPX—06，OPG-02，OPM—04，OPM-11，0PS-02制备的探针在适宜的条件下产生特异性荧光 信号，分布在牡蛎的特定的染色体上。PCR特异性带产生的探针OPX—06—310和0PG-02—300产生了特异性的荧光信号：OPX—06—310产生的信号位于第5对染色体的短臂的近端粒区域，0PG—02—300探针定位到第3对染色体的短臂上。这两个探针是鉴定美洲牡蛎单条染色体的特异性探针。5．研究了大分子Pl克隆DNA(插入片断为80～100 kb)在美洲牡蛎染色体上的定位。Pl克隆DNA通过切口平移方法标记digoxigenin—11-dUTP用作FISH的探针。Cot-1 DNA作为竞争剂有效地抑制了Pl克隆序列中的重复序列产生的信号。杂交信号用fluorescein标记的anti—digoxigenin抗体来检测，用两层抗体rabbit-anti-sheep抗体和FITC anti—rabbit抗体来扩增信号。9个P1探针成功地定位在特定的染色体上。46—1探针杂交到第1对染色体的长臂靠近着丝粒区域；47-10探针定位到第2对染色体的长臂近端粒区域；Cvpl和48-13两探针定位到第3对染色体上：Cvpl位于短臂的端粒区域，48-13探针位于长臂的近着丝粒区域；48—10探针杂交到第4对染色体的长臂上；48-1探针杂交到第5对染色体长臂的近着丝粒区域；49-11探针位于第7对染色体长臂上；探针49-10和44-11位于第8对染色体长臂上。同时我们成功地将2个P1探针杂交到同一染色体分裂相中，进一步确定了Pl探针在美洲牡蛎染色体 上的定位。6．应用18S-28S rDNA探针成功地鉴定出长牡蛎非整倍体中的三体10。经鉴定AF-35，AF-39和AF-3三体家系属于三体10家系。rDNA探针分布在三条染色体上，即多出的一条染色体为染色体10。相应地在间期细胞核上有三个信号出现。AF-34和AF-36家系不属于三体10家系。rDNA探针分布在两条染色体上，相应地在间期细胞核上有两个信号出现。FISH和染色体特异性探针为非整倍体的鉴定提供了一个快速准确可靠的方法和途径。

四种海洋生物的化学成分及其生物活性研究

从海洋动植物中寻找和筛选具有药用价值的活性先导化合物是海洋药物研发极为重要的理论基础研究。基于中国东南热带和亚热带沿海海域丰富的生物资源，本工作以化学分析和生物活性筛选为导向，以黄海海域习见的绿藻束生刚毛藻、南海三亚附近海域的圆裂短足软珊瑚、海绵Pseudoceratina sp.及广西沿海滩涂的盐生植物海刀豆四种海洋生物为样品，应用现代分析和检测手段对其次生代谢产物及其生物活性进行较系统的研究。 综合利用现代色谱手段和波谱技术，从上述目标生物样品中共分离并结构鉴定化合物74个，其中新化合物10个，新天然产物1个，这一新发现原则性结果，属国内外首次报道。1.对黄海海域束生刚毛藻C. fascicularis的化学成分及其生物活性进行了首次研究，从中得到叶绿素类新化合物Porphyrinolactone 1和新天然产物 20-Chlor-(132-S)-hydroxyphaeophytin a 2，在避光条件下对叶绿素类化合物进行体外NF-κB活性抑制筛选实验, 结果表明化合物1-6对NF-κB都有显著的抑制活性；对叶绿素类化合物首次进行体外蛋白酶体ChT-L的抑制活性实验筛选,化合物2和4对蛋白酶体ChT-L表现出显著的抑制活性，在浓度为50μM时，抑制率分别为70%和73.5%，并对其立体构型和生物活性的构效关系进行探讨；首次从该属得到环阿尔廷三萜类化合物7—12；对所得化合物7—15在浓度为1μg/mL时对肿瘤细胞部分靶蛋白酶的抑制活性测定结果表明，这些化合物对不同的靶蛋白酶表现出较强的抑制活性。2. 从南海圆裂短足软珊瑚中发现孕甾及其糖苷类新化合物22—27。3. 南海海绵Pseudoceratina sp.中发现溴代酪氨酸生物碱类新化合物41—42，组胺酸生物碱类新化合物43，并对其生源关系进行探讨。4. 首次对海刀豆C. maritime的化学成分进行研究，发现紫檀素类化合物61—63，异黄酮4个64—67；对该类化合物61—67首次进行NF-κB和蛋白酶体ChT-L的抑制活性筛选，其中化合物63表现出较强的NF-κB的抑制活性。

利用两种大型海藻制备生物能源的探索研究

生物质燃料乙醇和沼气都属于发展潜力巨大的生物质能源，大力发展生物质燃料乙醇和沼气对解决当今能源危机、环境污染问题和促进我国经济发展具有重要意义。 本文就海带化工废弃物——海带渣糖化技术、浒苔糖化技术及初步酒精发酵技术、海带沼气发酵技术、浒苔沼气发酵技术进行了可行性研究。 1、海带渣总糖含量为52.6%，海带渣总糖中葡萄糖含量占90.9%，另外还有少量的半乳糖、甘露糖和木糖，这说明海带渣是非常优良的能源生物质。 2、海带渣糖化工艺采取稀硫酸预处理后纤维素酶酶解产糖工艺。海带渣最佳稀硫酸预处理条件为预处理温度121℃、硫酸浓度0.6%、预处理时间60 min，此时海带渣纤维素酶解产糖可达187.8 mg/g干藻。 3、优化了预处理海带渣纤维素酶酶解产糖工艺。各因素对海带渣酶解产糖的影响依次为：pH>温度>时间>酶用量，海带渣纤维素酶解产糖的最佳条件为温度45℃、pH5.2、时间48小时、酶用量16 mg/g干藻，此时糖产量为238.9 mg/g干藻。 4、浒苔总糖含量为67.2%，浒苔总糖主要有葡萄糖、木糖、葡萄糖醛酸和鼠李糖组成，其中葡萄糖和木糖可以做为酒精发酵的原料，这两种糖占总糖含量的51%。 5、以鲜浒苔为原料研究了浒苔稀硫酸水解工艺和纤维素酶酶解工艺。发现浒苔酸水解产糖效果明显优于纤维素酶酶解。 6、以干浒苔为原料研究了浒苔酸解工艺。硫酸水解干浒苔产糖能力优于盐酸、磷酸和马来酸，水解时间为60 min、硫酸浓度为1.8%的时候可用于酒精发酵的糖（葡萄糖和木糖）总产量达到最大值为230.5 mg/g干浒苔，占此条件下总还原糖产量的48.6%。同时发现干浒苔比鲜浒苔更易被硫酸水解产糖。 7、初步研究了浒苔酒精发酵工艺。初步工艺中酒精产量较低、测得酒精在发酵液中浓度为0.23%（v/v），后续工作中需要对酒精发酵工艺进行优化。 8、海带与牛粪比例为4:1（w/w）是海带与牛粪联合厌氧消化的最佳比例，此时产气时间最长，达到37天，总产气量最高，达到13600 mL。 9、在海带与牛粪联合厌氧消化中，增加接种量到15 g（干重）时发酵周期为39天，总产气量为14630 mL，TS产气量为152.4 mL/gTS，接种量为21 g（干重）时发酵周期为36天，总产气量为14090 mL，TS产气量为138.1 mL/gTS，可见适当增加接种量可促进产气量的增加。 10、浒苔与牛粪比例为4:2（w/w）是浒苔与牛粪联合厌氧消化的最佳比例，此时产气时间为33天，总产气量最高达到6785 mL。 11、在浒苔与牛粪联合厌氧消化中，增加接种量到7.5 g（干重）时发酵周期为38天，总产气量为7470 mL，TS产气量为155.6 mL/gTS，接种量为10.5 g（干重）时发酵周期也为38天，总产气量为7020 mL，TS产气量为137.6 mL/gTS，可见增加接种量可促进产气量的增加。

海滨锦葵种子油脂提取及制备生物柴油研究

生物柴油是绿色清洁可再生能源，大力发展生物柴油对解决影响我国经济可持续发展的能源危机和环境危机具有重要意义。 本文就海洋滩涂能源油料植物海滨锦葵油脂的提取和制备生物柴油技术进行了研究。 1、利用超临界CO2流体萃取技术提取海滨锦葵籽油。结果表明超临界CO2流体萃取技术提取海滨锦葵籽油的最佳工艺参数为：萃取压力25MPa，萃取温度45℃，CO2流量18kg.h-1，萃取时间为120min，在该工艺条件下萃取三次，海滨锦葵籽油萃取率达到19.35%。 2、以海滨锦葵籽仁为原料，利用水酶法提取海滨锦葵籽仁油。水酶法提取海滨锦葵籽油的最佳工艺参数为：酶用量0.024ml.g-1，提取温度63℃，固液比1/6，提取时间为230min，在该工艺条件下海滨锦葵籽油提取率达到24.28%。 3、海滨锦葵油制备生物柴油的最佳工艺参数为：搅拌强度为1800r.min-1，催化剂KOH用量为海滨锦葵油质量的1%，醇油摩尔比6/1，反应时间50min，反应温度65℃，在该工艺条件下，酯交换反应三次，酯交换率达到97.8%。 4、利用固定化脂肪酶Novo435催化海滨锦葵油酯交换制备生物柴油。结果表明海滨锦葵油固定化脂肪酶催化法制备制备生物柴油的最优工艺参数为反应温度47℃，反应时间31h，催化剂用量18%，搅拌强度900r.min-1，醇油摩尔比3.2/1。在该工艺条件下酯交换率达到92.68%。 5、利用超临界法制备生物柴油，结果表明海滨锦葵油超临界法制备生物柴油的最佳工艺条件为：反应温度为300℃，反应压力为12MPa，反应时间为9min，搅拌强度为300r.min-1，醇油摩尔比为30/1。在此条件下，酯交换反应三次，酯交换率可达97.62%。 6、利用超声波辅助法制备生物柴油。结果表明海滨锦葵油超声波辅助法制备生物柴油的最佳工艺参数为：超声波功率为180W，催化剂KOH用量为海滨锦葵油质量的0.6%，反应温度65℃，醇油摩尔比7/1，在该工艺条件下酯交换反应三次，酯交换率达到99.85%。

中国近海沉积物中黑碳和多环芳烃的研究

黑碳和多环芳烃都是燃烧产生的污染物，并广泛存在于土壤及海洋特别是近海沉积物中。由于黑碳和多环芳烃对环境及生态系统具有不同的影响途径和危害，多年来一直受到环境研究者的极大重视。本文对我国近海几个海区（渤海湾、北黄海、胶州湾、南海珠江口及其邻近海区）沉积物中黑碳和多环芳烃的含量、分布及来源进行了研究，并首次对各海区表层沉积物中黑碳和多环芳烃的相关性进行了分析。 数据显示，中国近海各海区（渤海湾、北黄海、胶州湾、南海珠江口及其邻近海区）表层沉积物中黑碳的浓度范围为0.1 - 2.45 mg/gdw，占总有机碳浓度的1 - 41%，具有较大的空间差异。渤海湾表层沉积物中黑碳浓度最高（平均为2.18 mg/gdw），占沉积物中总有机碳浓度的27 - 41%。相比之下，北黄海、胶州湾和南海珠江口及其邻近海区表层沉积物中黑碳浓度较低，分别占沉积物中总有机碳浓度的6%、8%和5%。黑碳的分布主要受其来源的影响，陆地来源和河流输入是中国近岸海区表层沉积物中黑碳的重要来源。该研究显示黑碳在沉积物中的埋藏可能代表我国近海各海区碳循环中的一个重要碳汇。 中国近海各海区（渤海湾、北黄海、南海珠江口及其邻近海区）表层沉积物中多环芳烃的浓度范围为47.5 – 3673.5 ng/g（均为干重），亦具有很大的空间差异。渤海湾中多环芳烃的浓度最高（82.9 – 3673.5 ng/g），反映了该地区受人类污染严重的特征。在三个海区表层沉积物中，单组分多环芳烃的分布也具有明显不同的特征，渤海湾主要以5、6环的多环芳烃为主，而北黄海和南海珠江口及其邻近海区3、4环多环芳烃的浓度较高。基于多环芳烃的单组分分布特征和一些特殊指数，可以得到渤海湾表层沉积物中的多环芳烃以木材、煤的不完全燃烧来源为主，而北黄海海区和南海珠江口及其邻近海区的多环芳烃显示出木材、煤的燃烧和石油燃烧的混合来源。 中国近海表层沉积物中黑碳浓度与多环芳烃的浓度并不存在很好的相关性，这说明中国近海表层沉积物中的黑碳和多环芳烃具有不同的来源，且在沉积物中的埋藏受控于不同的地球化学作用。

海湾扇贝超氧化物歧化酶家族基因结构、表达和多态性分析

海湾扇贝Argopecten irradian Lamarck于1982年从美国引种到中国，由于具有较快的生长速度和很高的经济效益，海湾扇贝成为中国最主要的养殖贝类之一。近年来海湾扇贝养殖遇到了死亡率高等问题，深入开展海湾扇贝功能基因的研究，尤其是免疫相关基因及其机制研究并在此基础上寻找扇贝疾病防治的有效方法对海湾扇贝的健康养殖十分重要。 对于贝类免疫系统来说，其血细胞在先天性免疫防御中起着重要的作用。当受到外界病原侵染时，贝类血细胞的一个重要免疫反应就是吞噬作用。在吞噬病原过程中，受到病原侵染的贝类还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发与呼吸链相耦联的活性氧（ROS）的大量产生。这些活性氧具有极强的反应特性，能破坏病原微生物的结构和功能分子，实现对入侵病原的杀灭。利用活性氧对被吞噬的病原进行杀灭，这是吞噬作用消除病原抵御侵染的重要机制。但由于活性氧分子反应的非特异性，它们也会破坏宿主机体细胞内的功能蛋白分子、不饱和脂肪酸分子和核酸等，对细胞造成严重的伤害，进而导致机体生理机能的损伤和免疫系统的破坏。所以，及时消除病原感染机体内过量产生的ROS，维持相关细胞的正常代谢，对提高机体抵抗力和免疫力具有重要的作用。O2-是生物体内产生的第一种活性氧分子，其他的活性氧分子也是由它衍生而来，消除过量O2-是消除过量活性氧危害的第一步也是关键一步。生物体内，超氧化物歧化酶（SOD）是催化O2-发生歧化反应，消除O2-的关键酶。 首先，本文通过RACE方法获得了海湾扇贝SOD家族全部三种基因的cDNA全长并对其进行了序列的生物信息学分析，海湾扇贝AiCuZnSOD全长cDNA为1047个碱基，其中开放阅读框为459个碱基，编码152个氨基酸，与栉孔扇贝Chlamys farreri的CuZnSOD相似度为77.5%，与长牡蛎Crassostrea gigas的相似度为75%，与人的相似度为74.7%。AiMnSOD全长cDNA为1207个碱基，其中开放阅读框为678个碱基，编码226个氨基酸，序列比对结果发现AiMnSOD的氨基酸序列与虾夷扇贝Mizuhopecten yessoensis和皱纹盘鲍Haliotis discus hannai的相似度分别为85%和78.4%，与哺乳动物相似度也在68%~72%之间。AiECSOD全长cDNA为893个碱基，其中开放阅读框为657个碱基，编码218个氨基酸。AiECSOD与其它物种ECSOD相似度比较低。与线虫Brugia pahangi的相似度为27.9%，与疟蚊Anopheles gambiae的相似度为31.4%，与斑马鱼Danio rerio的相似度为27.8%，与人的相似度也只有28.6%，与同是贝类的长牡蛎ECSOD也只有28.1%的相似性。主要原因是AiECSOD的信号肽和肝磷脂结合区域在各物种中无同源性。 其次，采用qRT-PCR（quantitative real time PCR）方法分析三种SOD基因在不同组织中的表达情况，结果表明三种SOD基因的组织表达有所差异。AiCuZnSOD基因在鳃中表达水平最高，其次是血细胞和性腺，在外套膜、闭壳肌和肝胰脏表达水平较低。AiMnSOD基因在鳃中表达水平最高，其次是外套膜，在血细胞、性腺，而在肝胰脏和闭壳肌表达较弱。AiECSOD基因在血细胞中表达水平最高，其次是肝胰脏，在鳃、闭壳肌表达水平较低，而性腺和外套膜没有检测到。同时，采用qRT-PCR对鳗弧菌Vibrio angullarum感染后海湾扇贝血细胞中三种SOD基因mRNA表达变化进行了检测。AiCuZnSOD表达量在各个时间段没有显著差异(P > 0.05)。AiMnSOD的表达量在1.5 h时略有下降，在3 h时达到最高表达量，是空白组（0h）的3倍(P < 0.01)，从6 h到24 h表达量逐渐下降，24 h时表达量是空白组的1.6倍，24 h到48 h又稍有升高。AiECSOD的表达量在1.5 h时有所下降，是空白组的0.3倍（P < 0.05)，随后逐渐升高，在12 h时达到最高表达量，是空白组（0h）的4.5倍（P < 0.01），从24 h到48 h表达量逐渐下降并恢复到空白组的水平。在对照组，各个时间点没有显著差异（P > 0.05）。在鳗弧菌感染后，海湾扇贝三种SOD的表达并不一致，且差异比较显著。AiCuZnSOD被认为是构成性表达基因，其受外界刺激的影响最小，AiMnSOD和AiECSOD受刺激后表达上调比较明显。 第三，采用Genome-walking的方法得到了海湾扇贝三种SOD基因的基因组全长和近端启动子序列并对其进行了相关分析。AiCuZnSOD的基因组序列全长为4279bp，包含有4个外显子和3个内含子。AiMnSOD的基因组序列全长为10692bp，包含有4个外显子和3个内含子。AiECSOD的基因组序列全长为5276bp，包含有5个外显子和4个内含子。三种基因外显子和内含子的结合处序列遵循-AT/GT-原则。我们把海湾扇贝SOD家族的三个基因的近端启动子进行了比较分析。发现三种SOD在靠近起始密码子的位置都有Oct-1结合位点。三种SOD共有的转录位点有：Oct-1、C/EBPalp、Oct2.1、Sp-1和GATA-1。AiCuZnSOD和AiMnSOD共有的转录位点有：ICSBP、Ftz、TATA-box、C/EBPbeta和Antp。AiCuZnSOD和AiECSOD共有的转录位点有：AP-1和NFκB。AiMnSOD和AiECSOD共有的转录位点有：GR和ER。AiCuZnSOD独有的位点有：SRF、YY-1和NF-1。AiMnSOD独有的位点有：HNF-1、Hb、MEB、NF-muE1、Pit-1a和Eve。AiECSOD独有的位点有：CREB、RATA-alph、Kruppel-like和AP-3。 此外，通过构建原核表达载体，本研究对AiCuZnSOD和AiECSOD基因进行了体外重组表达，并对纯化的重组蛋白进行了酶活分析。酶活分析表明，重组AiCuZnSOD蛋白有较高的酶活和稳定性。 最后，我们对海湾扇贝三种SOD基因的部分区域，包括启动子、编码区，部分内含子区域进行了SNP检测，并对SOD基因部分SNP位点多态性和鳗弧菌敏感性进行了相关分析。三种SOD基因中，我们共发现了59个SNP位点，其中AiECSOD的SNP位点最多，特别是在启动子区，AiCuZnSOD和AiMnSOD多态性较低。其中AiCuZnSOD启动子区的-1739 T-C 位点的基因型和等位基因，AiECSOD启动子区的-498 A-T和-267 G-A等位基因频率，AiECSOD的第一个外显子38 Thr-Lys的多态性在敏感和抗菌群体中存在显著差异（P < 0.05）。

Potential of the seaweed Gracilaria lemaneiformis for integrated multi-trophic aquaculture with scallop Chlamys farreri in North China

In this study the red alga, Gracilaria lemaneiformis, was cultivated with the scallop Chlamys farreri in an integrated multi-trophic aquaculture (IMTA) system for 3 weeks at the Marine Aquaculture Laboratory of the Institute of Oceanology, Chinese Academy of Sciences (IOCAS) in Qingdao, Shandong Province, North China. The nutrient uptake rate and nutrient reduction efficiency of ammonium and phosphorus from scallop excretion were determined. The experiment included four treatments each with three replicates, and three scallop monoculture systems served as the control. Scallop density (407.9 +/- 2.84 g m(-3)) remained the same in all treatments while seaweed density differed. The seaweed density was set at four levels (treatments 1, 2, 3, 4) with thallus wet weight of 69.3 +/- 3.21, 139.1 +/- 3.80, 263.5 +/- 6.83, and 347.6 +/- 6.30 g m(-3), respectively. There were no significant differences in the initial nitrogen and phosphorus concentration between each treatment and the control group (ANOVA, p > 0.05). The results showed that at the end of the experiment, the nitrogen concentration in the control group and treatment 1 was significantly higher than in the other treatments. There was also a significant difference in phosphorus concentration between the control group and the IMTA treatments (ANOVA, p < 0.05). Growth rate, C and N content of the thallus, and mortality of scallop was different between the IMTA treatments. The nutrient uptake rate and nutrient reduction efficiency of ammonium and phosphorus changed with different cultivation density and time. The maximum reduction efficiency of ammonium and phosphorus was 83.7% and 70.4%, respectively. The maximum uptake rate of ammonium and phosphorus was 6.3 and 3.3 A mu mol g(-1) DW h(-1). A bivalve/seaweed biomass ratio from 1:0.33 to 1:0.80 (treatments 2, 3, and 4) was preferable for efficient nutrient uptake and for maintaining lower nutrient levels. Results indicate that G. lemaneiformis can efficiently absorb the ammonium and phosphorus from scallop excretion and is a suitable candidate for IMTA.

Recovery and fate of three species of marine dinoflagellates after yellow clay flocculation

The recovery and fate of three species of dinoflagellates, Alexandrium tamarense, Cochlodinium polykrikoides and Scrippsiella trochoidea, after having been sedimented by yellow clay, were investigated in the laboratory. The effect of burying period in yellow clay pellet and mixing on the recovery of settled algal cells were studied. The morphological changes of algal cells in yellow clay pellet were also tracked. Results showed that there was almost no recovery for A. tamarense and C. polykrikoides, and the cells decomposed after 2-3 days after visible changes in morphology and chloroplasts. There was some recovery for S. trochoidea. Moreover, S. trochoidea cysts were formed in clay pellet during the period of about 14 days, with the highest abundance of 87 000 cysts g(-1) clay and the incidence of cyst formation of 6.5%, which was considered as a potential threat for the further occurrence of algal blooms. S. trochoidea cysts were isolated from yellow clay and incubated to test their viability, and a germination ratio of more than 30% was obtained after incubation for 1 month. These results showed the species specificity of the mitigation effect of yellow clay. It is suggested that cautions be taken for some harmful species and thorough risk assessments be conducted before using this mitigation strategy in the field.

Transfer of paralytic shellfish toxins via marine food chains: A simulated experiment

Objective To study the transfer of paralytic shellfish toxins (PST) using four simulated marine food chains: dinoflagellate Alexandrium tamarense -> Arterriia Artemia salina -> Mysid shrimp Neomysis awatschensis; A. tamarense-N. awatschensis: A. taniarense A. salina -> Perch Lateolabrax japonicus; and A. tamarense -> L. japonicus. Methods The ingestion of A. tamarense, a producer of PST, by L. japonicus, N. awatschensis, and A. salina was first confirmed by microscopic observation of A. tamarense cells in the intestine samples of the three different organisms, and by the analysis of Chl.a levels iii the samples. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly ibrough the vector of A. salina was then studied. The toxicity of samples was measured using the AOAC mouse bioassay method, and the toxin content and profile of A. tamarense were analyzed by the HPLC method. Results Both A. salina and N. awatschensis could ingest A. tamarense cells. However, the ingestion capability of A. salina exceeded that of N. awatschensis. After the exposure to the culture of A. tamarense (2 000 cells(.)mL(-1)) for 70 minutes, the content of ChLa in A. salina and N. awatschensis reached 0.87 and 0.024 mu g-mg(-1), respectively. Besides, A. tamarense cells existed in the intestines of L. japonicus, N. awatschensis and A. salina by microscopic observation. Therefore, the three organisms could ingest A. tamarense cells directly. A. salina could accumulate high content of PST, and the toxicity of A. salina in samples collected on days 1, 4, and 5 of the experiment was 2.18, 2.6, and 2.1 MU(.)g(-1), respectively. All extracts from the samples could lead to death of tested mice within 7 minutes, and the toxin content in arternia sample collected on the 1st day was estimated to be 1.65x10(-5) pg STX equa Vindividual. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly froin the vector of A. salina was also studied. The mice injected with extracts from L. japonicus and N. awatschensis samples that accumulated PST either directly or indirectly showed PST intoxication symptoms, indicating that low levels of PST existed in these samples. Conclusion Paralytic shellfish toxins can be transferred to L. japonicus, N. awatschensis, and A. salina from A. taniarense directly or indirectly via the food chains.

Seasonal variation in metabolism of cultured Pacific oyster, Crassostrea gigas, in Sanggou Bay, China

Rates of respiration and excretion of the Pacific oyster, Crassostrea gigas, were measured seasonally from June 2002 to July 2003 under ambient conditions of food, water temperature, pH, and salinity in Sanggou Bay, an important mariculture coast in north China. The aim of this study is to obtain fundamental data for further establishing an energy budget model and assessing the carrying capacity for cultivation of C. gigas in north China. Oysters were collected monthly or bimonthly from the integrated culture areas of bivalve and kelp in the bay. Oxygen consumption and ammonium and phosphorus excretion rates were measured, and ratios of O/N and NIP were calculated. One-way ANOVA was applied to determine differences among these parameters that act as a function of seasonal variation. All the physiological parameters yielded highly significant variations with season (P<0.01) The rate of respiration varied seasonally, with the highest oxygen consumption rate in July and the lowest rate in January, ranging from 0.07 to 2.13 mg O-2 h(-1) g(-1) dry tissue weight (DW). Maximum and minimum ammonium excretion rates were recorded in August and January, respectively, ranging from 0.51 to 5.40 mu mol NH4-N h(-1) g(-1) DW. Rates of phosphorus excretion varied from 0.11 (in January) to 0.64 (in July) mu mol PO4-P h(-1) g(-1) DW. The O/N and N/P ratios changed from 9.2 (in January) to 59.8 (in July) and from 4.6 (in January) to 10.9 (in August), respectively. For each season, the allometric relationship between the physiological response (e.g., rate of oxygen consumption, ammonium and phosphorus excretion) and DW of the animal was estimated using the formula: Y=a x DWb. (C) 2005 Elsevier B.V. All rights reserved.

Minor sesquiterpenes with new carbon skeletons from the brown alga Dictyopteris divaricata

Five minor sesquiterpenes (1-5) with two novel carbon skeletons, together with a minor new oplopane sesquiterpene ( 6), have been isolated from the brown alga Dictyopteris divaricata. By means of spectroscopic data including IR, HRMS, 1D and 2D NMR, and CD, their structures including absolute configurations were assigned as (+)-(1R, 5S, 6S, 9R)3- acetyl-1-hydroxy-6-isopropyl-9-methylbicyclo[4.3.0] non-3-ene ( 1), (+)-(1R, 3S, 4S, 5R, 6S, 9R)-3-acetyl-1,4-dihydroxy-6- isopropyl-9-methylbicyclo[4.3.0] nonane (2), (+)-(1R, 3R, 4R, 5R, 6S, 9R)-3-acetyl-1,4-dihydroxy-6-isopropyl-9-methylbicyclo[ ;4.3.0] nonane ( 3), (+)-(1S, 2R, 6S, 9R)-1-hydroxy-2-(1-hydroxyethyl)-6-isopropyl-9-methylbicyclo[4.3.0] non-4-en-3-one (4), (-)-( 5S, 6R, 9S)-2-acetyl-5-hydroxy-6-isopropyl-9-methylbicyclo[4.3.0] non-1-en-3-one ( 5), and (-)-( 1S, 6S, 9R)- 4-acetyl- 1-hydroxy-6-isopropyl-9-methylbicyclo[ 4.3.0] non-4-en-3-one ( 6). Biogenetically, the carbon skeletons of 1-6 may be derived from the co-occurring cadinane skeleton by different ring contraction rearrangements. Compounds 1-6 were inactive (IC50 > 10 mu g/mL) against several human cancer cell lines.

Residues of enrofloxacin, furazolidone and their metabolites in Nile tilapia (Oreochromis niloticus)

The residues of enrofloxacin and its metabolite in Nile tilapia (Oreochromis niloticus) were studied after oral dose of 50 mg/kg for 7 days. To find the differences between Nile tilapia and Chinese shrimp (Penaeus chinensis), the residues of enrofloxacin in P chinensis were also studied under the same conditions. The results showed that enrofloxacin metabolized into ciprofloxacin in both Nile tilapia and P chinensis, the maximal concentration of enrofloxacin in muscle, liver and plasma of Nile tilapia were 3.61 mu g/g, 5.96 mu g/g, 1.25 mu g/ml respectively, and ciprofloxacin in muscle was 0.22 mu g/g. The maximal concentration of enrofloxacin and ciprofloxacin in P chinensis were 1.68 mu g/g and 0.07 mu g/g respectively. The predicted withdrawal time for Nile tilapia was 22 days, and P. chinensis was 12 days under our experiment conditions. The residues of fitrazolidone [3-(5-nitrofurfurylidenamino)-2-oxazolidinone] and its main metabolite 3-amina-2-oxazolidinone (AOZ) in Nile tilapia were first determined by HPLC/MS. Results showed that after oral dose of 30 mg/kg for 7 days, the maximum concentration of farazolidone in Nile tilapia was 413 mu g/kg after 6 h, whereas AOZ residue reached its maximum (31 mu g/kg) right after stopping treatment. In contrast to the high metabolic rate of furazolidone, AOZ was very difficult to eliminate in vivo, thus the withdrawal time of furazolidone in Nile tilapia was 22 days at least. (c) 2005 Elsevier B.V. All rights reserved.

Copper complexation by fulvic acid affects copper toxicity to the larvae of the polychaete Hydroides elegans

Copper toxicity is influenced by a variety of environmental factors including dissolved organic matter (DOM). We examined the complexation of copper by fulvic acid (FA), one of the major components of DOM, by measuring the decline in labile copper by anodic stripping voltammetrically (ASV). The data were described using a one-site ligand binding model, with a ligand concentration of 0.19 mu mol site mg(-1) C, and a logK' of 6.2. The model was used to predict labile copper concentration in a bioassay designed to quantify the extent to which Cu-FA complexation affected copper toxicity to the larvae of marine polychaete Hydroides elegans. The toxicity data, when expressed as labile copper concentration causing abnormal development, were independent of FA concentration and could be modeled as a logistic function, with a 48-h EC50 of 58.9 mu g 1(-1). However, when the data were expressed as a function of total copper concentration, the toxicity was dependent on FA concentration, with a 48-h EC50 ranging from 55.6 mu g 1(-1) in the no-FA control to 137.4 mu g 1(-1) in the 20 mg 1(-1) FA treatment. Thus, FA was protective against copper toxicity to the larvae, and such an effect was caused by the reduction in labile copper due to Cu-FA complexation. Our results demonstrate the potential of ASV as a useful tool for predicting metal toxicity to the larvae in coastal environment where DOM plays an important role in complexing metal ions. (c) 2007 Elsevier Ltd. All rights reserved.