SmC2P1, a C2 domain protein from Scophthalmus maximus that binds bacterial pathogen-infected lymphocytes and reduces bacterial survival

C2 domains are protein structural modules found in many eukaryotic proteins involved in signal transduction, membrane trafficking, and immune defense. Most of the studied C2 domain-containing proteins are multi-domained in structure, in which the C2 domain is an independently folded motif and plays an essential role in calcium-dependent membrane-targeting. Although C2 domains isolated from intact proteins have been studied for biological functions, no study on natural proteins containing C2 domain only has been documented. In this study, we identified a Scophthalmus maximus protein SmC2P1 that is comprised of a single C2 domain and lacks any other apparent domain structures. The deduced amino acid sequence of SmC2P1 contains 129 residues and shares 36-38% identities with the C2 domains of the perforins of several fish species. Like typical C2 domains, SmC2P1 is predicted to organize into eight beta-strands with a Ca2+-binding site located in inter-strand loops. SmC2P1 expression was detected, in deceasing order, in liver, spleen, blood, brain, muscle, kidney, gill, and heart. Experimental challenge of turbot with a bacterial pathogen significantly upregulated SmC2P1 expression in kidney in a time-dependent manner. Recombinant SmC2P1 purified from yeast exhibits no hemolytic activity but binds to pathogen-infected kidney lymphocytes in the presence of calcium. Furthermore, interaction of recombinant SmC2P1 with bacterium-infected lymphocytes reduced bacterial survival. These results indicate that SmC2P1 is a functional protein that is involved in host immune defense against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651 bp in length, having a 5' untranslated region (UTR) of 96 bp, a 3' UTR of 575 bp, and an open reading frame (ORF) of 1980 bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80 kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro, analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and,C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8 h and lasted to 16 h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Physiological and immune responses of zhikong scallop Chlamys farreri to the acute viral necrobiotic virus infection

The Zhikong Scallop, Chlamys farreri, is one of the most Important bivalve mollusks cultured in northern China However, mass mortality of the cultured C farreri has posed a serious threat to the maricultural Industry in recent years. Acute Viral Necrobiotic Virus (AVNV) is believed as an important etiological agent causing the scallop mass mortalities To understand the mechanism behind the AVNV associated scallop disease and mortality, we assessed the physiological and immune responses of C farreri to the virus infection using oxygen consumption rate, ammonium-nitrogen excretion rate, hemocyte copper, zinc superoxide dismutase gene expression, and plasma superoxide dismutase activity and alkaline phosphatase activity as indicators Scallops challenged by AVNV at 25 C developed typical disease signs 2 days after virus injection Before the disease manifested, scallop oxygen consumption and NH4+-N excretion rates rose and then fell back. Real-time PCR revealed that the hemocyte cytosol Cu, Zn SOD gene expression was upregulated followed by recovery The plasma SOD activity, however, augmented consistently following virus injection Moreover, plasma AKP activity first lowered and then elevated gradually to the highest level at 24 h post virus injection Scallops challenged by AVNV at 17 degrees C neither developed notable disease nor showed obvious responses that could be associated with the virus infection. While the results suggested a correlation between the elevated seawater temperature and the AVNV infection associated C farreri mortalities, they also indicated that the viral infection provoked multiple physiological and immune responses in the host scallops (C) 2010 Elsevier Ltd All rights reserved

The first molluscan TCTP in Venerupis philippinarum: Molecular cloning and expression analysis

Translationally controlled tumor protein (TCTP) is one of the abundant and ubiquitously expressed proteins in metazoans In the present study, the first molluscan TCTP (denoted as VpTCTP) was identified from Venerupis philippinarum haemocytes by EST and RACE approaches The full-length cDNA of VpTCTP consisted of 1148 nucleotides with an open-reading frame of 555 bp encoding 184 amino acids The deduced amino acid sequence of VpTCTP shared high similarity with TCTPs from other species, indicating that VpTCTP should be a new member of TCTP family Several highly conserved motifs, including 5'terminal ologopyrimidine (5'TOP) starting sequence and rich AU and AUUT elements in 3'UTR, were also identified in VpTCTP The tissue and temporal expression of VpTCTP after Vi boo anguillarum challenge was recorded by quantitative real-time RT-PCR. VpTCTP transcript could be detected in all examined tissues with the highest expression level in haemocytes and the lowest in hepatopancreas Concerning the time-course expression in haemocytes, the relative expression of VpTCTP mRNA was down-regulated sharply from 6 h to 12 h post-infection. Then, the expression level was obviously up-regulated and reached 3.4-fold to that in the control group at 48 h post challenge As time progressed, the expression of VpTCTP recovered to the original level at 96 h. All these results indicated that VpTCTP was an acute-phase protein involved in the Immune response of V philippinarum (C) 2010 Elsevier Ltd. All rights reserved.

Cflec-5, a pattern recognition receptor in scallop Chlamys farreri agglutinating yeast Pichia pastoris

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively But its expression level did not change significantly during peptidoglycan (PGN) stimulation The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3) The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way The agglutinating activity could be inhibited by D-mannose. LPS and glucan, but not by D-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate Immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs (C) 2010 Elsevier Ltd All rights reserved

Identification and molecular analysis of a novel C-type lectin from Scophthalmus maximus

C-type lectins are calcium-dependent carbohydrate-binding proteins that play Important roles in innate immunity In this study, a C-type lectin homologue (SmLec1) was identified from turbot (Scophthalmus maximus) and analyzed at expression and functional levels. The open reading frame of SmLec1 is 504 bp, with a 5'-untranslated region (UTR) of 101 bp and a 3'-UTR of 164 bp The deduced amino acid sequence of SmLec1 shares 34%-38% overall identities with the C-type lectins of several fish species In silico analysis identified in SmLec1 conserved C-type lectin features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, and the mannose-type carbohydrate-binding motif In addition, SmLec1 possesses a putative signal peptide sequence and is predicted to be localized in the extracellular. Expression of SmLec1 was highest in liver and responded positively to experimental challenges with fish pathogens Recombinant SmLec1 (rSmLec1) purified from yeast was able to agglutinate the Gram-negative fish pathogen Listonella anguillarum but not the Gram-positive pathogen Streptococcus uncle The agglutinating ability of rSmLec1 was abolished in the presence of mannose and ethylenediaminetetraacetic acid and by elevated temperature (65 degrees C) Further analysis showed that rSmLec1 could stimulate kidney lymphocyte proliferation and enhance the killing of bacterial pathogen by macrophages Taken together, these results suggest that SmLec1 is a unique mannose-binding C-type lectin that possesses apparent immunomodulating property and is likely to be involved in host defense against bacterial infection (C) 2010 Elsevier Ltd. All rights reserved

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

PRELIMINARY STUDIES ON CRYOPRESERVATION OF SYDNEY ROCK OYSTER (SACCOSTREA GLOMERATA) LARVAE

In this study several parameters critical to the success of cryopreserving Sydney rock oyster (Saccostrea glomerata) larvae were investigated. They were: (1) cryoprotectants (10% dimethyl sulfoxide and 10% propylene glycol). (2) freezing protocols (with or without the seeding step). (3) larval concentrations (1,000, 3,000, 5,000, 10,000, 30,000 individuals mL(-1)). and (4) larval ages (6, 12, 24, 48 and 96 h old). The survival rates were determined as percentages of postthaw larvae performing active movements for the 6 and 12 h larvae or active cilia movement for the 24, 48 and 96 h larvae. Analyses showed that the difference in survival rates between different age classses was significant in all the experiments conducted, with the maximum survival rate being achieved in the 24-h-old larvae the postthaw survival rates of larvae cryopreserved with 10% dimethyl sulfoxide (93.1 +/- 0.2%) were significantly higher (P < 0.001) that those with 10% propylene glycol (81.5 +/- 0.4%). Differences in postthaw survival rates between different concentrations (1,000 30,000 individuals mL(-1)) were not significant within each of the three larval age classes (6-, 12-, and 24-h-old ) used.

滤食性贝类养殖活动对海域生态系统的影响及生态容量评估

滤食性贝类以水体中的浮游植物和有机碎屑为主要食物，养殖海域的初级生产力水平、水动力学特性等生态环境因子的差异，不仅直接影响养殖贝类的产量，而且也与贝类养殖活动对生态环境的压力密切相关。由于养殖的种类、密度、方式及养殖海域的特性不同，关于贝类对生态环境的影响往往有不同的结果。本文以我国北方大连獐子岛扇贝底播海区和荣成桑沟湾贝类筏式养殖区为研究对象，采用现场调查、室内受控实验及生态数值模型模拟方法，分析研究了滤食性贝类对海域生态系统的影响，对这两个海域贝类养殖的生态容量进行了初步的评价。 主要结果： 1. 獐子岛海域底播贝类养殖活动对该海域生态系统的影响较小。非参数统计—符号检验的结果显示，养殖区与非养殖区之间的溶解性无机氮、磷酸盐浓度、氮磷摩尔比及浮游植物群落结构没有统计学上的差异（p>0.05）。但是从变化的趋势上来看，贝类养殖活动对水域环境的某些参数有一定程度的影响。例如，獐子岛底播贝类养殖海域的溶解性无机氮以氨氮为主，可能与贝类的代泄活动有关；不论是叶绿素浓度，还是网采浮游植物的生物量都是贝类高密度养殖区<贝类低密度养殖区<非养殖区（7月份除外），这种趋势可能与贝类的摄食压力有关。 桑沟湾各环境指标表现出明显的区域性。除春季外，非养殖区的DIN浓度高于各养殖区。在春季和冬季，贝类区的磷酸盐浓度显著降低；而硅酸盐浓度在夏季和秋季显著增大。综合分析DIN、PO4-P及SiO3-Si三个参数的四季变化，海带区、贝藻区及贝类区发生显著性变异的概率分别为25%，42%和50%，贝类区的变异较大。浮游植物、小型浮游动物的生物多样性指数都是以非养殖区为最高，贝类区的多样性指数最低。尤其是浮游动物的丰度，贝类区显著低于非养殖区。 2. 利用挪威的MOM (Modelling-Ongrowing fish farms-Monitoring)评价系统，评价了桑沟湾长期大规模的贝藻筏式养殖活动对底质环境的压力。在桑沟湾设10个取样站位，共获得66个底泥样品。比较了MOM－B评价系统的3组参数的季节变化特性。结果显示，底质条件属于1级，说明桑沟湾贝藻长期大规模的养殖活动对底质环境的压力较低。结合桑沟湾的环境及养殖特点，分析了压力较低的原因。 3. 经计算，2006年中国海水养殖的贝类和藻类使用浅海生态系统的碳可达396万吨，并通过收获从海中移出至少136.9万吨的碳。从1995年至2006年，养殖大型藻类和贝类累计移出的碳分别约为365万吨和893万吨，总计达1258万吨。证明了浅海的贝类和藻类养殖活动直接或间接地使用了大量的海洋碳，提高了浅海生态系统吸收大气CO2的能力。 4. 采用模拟现场生物沉积法测定了虾夷扇贝的滤水率、摄食率等生理指标及其与贝类个体大小、水温的关系。虾夷扇贝单位个体的滤水率与组织干重的关系符合幂函数方程CR=a×DWb，b值在0.45～0.65范围内；水温对虾夷扇贝滤水率的影响极其显著(p<0.01)，温度（T）与滤水率（CR）呈抛物线的关系：CR=-0.0009T2+0.0188T-0.0306，水温为10℃时，虾夷扇贝的滤水率、摄食较大。 5. 采用模拟现场流水法测定了3种滤食性贝类的食物选择性。紫贻贝、长牡蛎及栉孔扇贝分别对直径4m, 6m 和 8m颗粒的保留效率达到最大值；对小颗粒（直径2m）的保留效率分别为17%, 19% 和 8％。栉孔扇贝对食物数量和质量浓度的变化相对敏感，随着数量浓度的增加，栉孔扇贝倾向于摄食较大的颗粒；随着颗粒食物质量浓度的增加，倾向于摄食较小的颗粒。 6. 獐子岛海域四个航次的调查结果显示，叶绿素浓度在1.23～2.85mg.m-3范围内，均值为1.78±0.57 mg.m-3；初级生产力的变化范围为30.4～117.0 mg C. m-2.d-1，平均值为76.6±41.9 mg C. m-2.d-1。通过虾夷扇贝生物量断面调查，获得了虾夷扇贝的壳高频率分布情况，7月份的众数值出现在100 mm，10月份壳高的众数值为80 mm。利用以上测定的虾夷扇贝的滤水率等基本生物学特性，结合虾夷扇贝的年产量、海域面积和有关的水文状况等数据，计算了滤水效率、摄食压力、调节比率3个食物限制性指标参数，全年的均值分别为0.048, 0.31和 0.16，都小于1，说明目前该海域虾夷扇贝的养殖量未达到养殖容量。 7. 利用STELLA软件，建立的桑沟湾贝藻养殖的数值模型，模拟了叶绿素a浓度及氮磷营养盐的周年变化情况，及其对贝类养殖生物量变化的响应。以叶绿素a浓度为指标，初步探讨了桑沟湾贝类的生态容量。

浅海贝藻养殖水域沉积物再悬浮的发生机制及生态效应

沉积物再悬浮作为一个比较普遍的物理现象，对浅海生态系统污染物的生物地球化学循环具有强烈的干扰作用。本研究以我国北方重要养殖海湾——桑沟湾为研究对象，从物理、化学、生物三个角度出发，研究了沉积物再悬浮的发生过程以及再悬浮介质-沉积物的源汇转换角色及其与养殖藻类的关系，构建了波流耦合模型和再悬浮颗粒物浓度预测数学模型。主要研究结果如下： 1）桑沟湾的海湾动力比约为1.54，沉积物具有发生再悬浮的潜在动力条件；推导出波流耦合切应力的计算公式。 2）悬浮颗粒物浓度（SSC）与浊度（NTU）之间符合线性方程SSC=15.908×ln（NTU）+7.0888（n＝33，R2＝0.7209）；碎屑有机碳库是桑沟湾养殖生态系统中最大的有机碳库，占总POC库储量的81.87%。 3）沉积物再悬浮的临界切应力在0.059 N/m2左右，耦合切应力与悬浮颗粒物浓度符合方程= 238.06 SSC + 25.215（n=25，R2 = 0.7298）；最大剪切深度可达8.81 cm；桑沟湾沉积物再悬浮通量的数量级在10-5~10-6 kg·m-2·s-1之间，再悬浮临界风速约为5.51 m/s，全年约有171天沉积物处于再悬浮状态；构建了沉积物再悬浮颗粒物浓度预测数学模型。 4）桑沟湾表层沉积物总氮的含量范围313.09~1094.44µg/g，有机氮是总氮的主要形态，平均占总氮的60.86%；交换态氮是无机氮的主要形式，平均占无机氮的71.40%，交换态氮中NO3--N的含量最大；桑沟湾表层沉积物的TOC/TN比值为9.38，表明沉积物中有机质具有混合来源的特征；无机磷是桑沟湾表层沉积物中磷的主要形态，平均占总磷的73.33%，钙结合磷是无机磷的主要赋存形态；表层沉积物中潜在生物有效性磷的含量占总磷的86.54%，具有很强的释磷潜力。桑沟湾重金属的潜在生态危害指数RI约为36.17，表明重金属的潜在生态危害轻微。 5）再悬浮过程中沉积物春季表现为氮磷源，释放溶解无机氮和磷酸盐；夏、秋季表现为氮汇磷源，释放磷酸盐而吸附溶解无机氮；冬季表现为氮磷汇，吸附磷酸盐和溶解无机氮。

东海沿岸贝类体内石油烃和有机氯农药的分布特征及评价

本研究分别于2006、2007年5月在东海沿岸海域22个站位采集牡蛎、贻贝、四角蛤蜊、文蛤、花蛤5种贝类样品，采用荧光分光光度法和气相色谱法检测贝类体内的石油烃（TPHs）和有机氯农药(HCHs和DDTs) 含量，分析东海沿岸海域贝类体内的TPHs、HCHs和DDTs的时空分布特征和HCHs、DDTs的组分特征，比较不同贝类体内污染物含量的差异，探讨HCHs和DDTs的来源，并对贝类进行初步海洋生物质量评价和人体健康风险评价。研究结果表明： （1）东海沿岸海域贝类体内TPHs含量范围为0.40 74.40 mg kg-1，平均含量为9.42 mg kg-1；HCHs含量为ND～5.42×10-3mg kg-1，平均含量为1.47×10-3 mg kg-1；DDTs含量为2.09×10-3～197.66×10-3mg kg-1，平均含量为38.05×10-3 mg kg-1。其中，TPHs含量在长江口及邻近海域较高， HCHs和DDTs含量在江苏省的如东、海门和浙江省的各海域含量较高。与2006年相比，2007年贝类体内TPHs含量与2006年基本持平，HCHs含量有稍微降低，但是DDTs含量有明显升高趋势。 （2）HCHs和DDTs组分分析表明，α-HCH和β-HCH占HCHs总量的绝大多数，γ-HCH和δ-HCH仅有极少量检出，大多数区域的α-HCH/γ-HCH比值在4-7之间，说明研究区域内有新的HCHs污染源； DDTs 中O,P’-DDT/P,P'-DDT的比值远高于工业DDTs中O,P’-DDT/P,P'-DDT的比值，分析认为其与周围区域使用三氯杀螨醇密切有关。 （3）贝类体内的TPHs、HCHs和DDTs与水体、沉积物中的相应有机物存在正相关性。相对于TPHs和HCHs，DDTs更容易在贝类体内富集；牡蛎对TPHs和HCHs、DDTs的累积能力明显高于四角蛤蜊和贻贝。 （4）质量评价显示TPHs在局部海域超标，HCHs未超标，DDTs含量超标严重。人体健康风险评估结果显示，东海沿岸海域贝类体内HCHs和DDTs的致癌风险和暴露风险均在可接受的范围之内，消费者因食用监测海域的贝类而受到HCHs和DDTs危害的可能性不大。 （5）与世界其他海域比较表明，东海海域贝类TPHs和HCHs含量处于中等水平， DDTs含量处于比较高的水平。

中国沿海典型区域织纹螺毒性及毒素成分研究

织纹螺（Nassarius spp.）味道鲜美，是中国及其它一些亚洲国家沿海地区居民习惯食用的一种水产品。但是，近几十年来，中国沿海频繁发生食用织纹螺中毒事件，严重威胁着人们的身体健康和生命安全。加之人们对织纹螺体内的毒素成分、来源及其毒性变化规律还没有清晰的认识，因此难以有效预防和控制食用织纹螺引起的中毒事件。本文根据文献报道，在中国沿海食用织纹螺中毒事件多发的典型区域，包括江苏省的连云港市和盐城市、浙江省的舟山市和宁波市、福建省的宁德市、厦门市和莆田市设立了监测点，于2006年和2007年间进行了连续采样，应用小鼠生物测试法调查了织纹螺毒性的消长情况，并利用高效液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry，LC-MS）和高效液相色谱技术（High Performance Liquid Chromatography，HPLC）对织纹螺体内的毒素成分进行了分析。 实验结果表明，2006年于江苏省盐城市射阳海域采集的织纹螺样品中，阳性样品检出率为56%，毒性在2-5 MU/g组织（湿重）之间变化，在2007年于同地采集的8个样品中，除一个样品毒性为3.14 MU/g组织（湿重）以外，其余样品均表现为阴性；而2007年采集自连云港市赣榆海域的织纹螺样品，在采样期间则呈现出极高的毒性，最高达到846.52 MU/g 组织（湿重），毒性在监测期间呈“M”状波动，在5月和7月下旬出现两个毒性高峰。2006年于浙江省宁波市象山港采集的织纹螺样品中，阳性样品检出率为25%，毒性均在2.5 MU/g组织（湿重）左右；而同年采集自舟山市定海的织纹螺样品中，阳性样品检出率为100%，最高毒性达18.40 MU/g组织（湿重），毒性在监测期间也呈“M”状波动，高峰期出现在6月初和7月底。2006年3-9月采集自福建省宁德霞浦、厦门同安和莆田涵江采集的织纹螺样品中，阳性样品检出率分别为20%、43%和14%，除7月中旬采集自宁德霞浦的一个样品毒性达到16.19 MU/g组织（湿重）之外，其余样品毒性均在2-5 MU/g组织（湿重）间波动。从阳性样品的时间分布规律来看，3月份和6、7月份是阳性样品集中出现的时期。根据以上调查结果可以看出，织纹螺的毒性消长呈现出较明显的地域性和季节性特征，不同地区的织纹螺毒性存在差异，而同一区域织纹螺毒性的消长则表现出明显的季节性集中趋势。除了2007年采集自连云港赣榆的织纹螺样品毒性与其平均个体组织重量有相似的变化趋势以外，其余地区的织纹螺样品毒性和个体大小无明显相关性。 利用LC-MS和HPLC技术对织纹螺样品中的毒素成分进行了分析，确定河豚毒素（tetrodotoxin, TTX）及其同系物（trideoxyTTX，4-epi-TTX，anhydroTTX，oxoTTX）是所采集织纹螺中的主要致毒成分，样品中没有检测到麻痹性贝毒毒素（Paralytic Shellfish Poison, PSP）。自不同地区采集的织纹螺中毒素成分基本一致，但组成存在一定差异。其中，采自江苏省连云港赣榆和浙江省舟山定海的织纹螺样品中，trideoxyTTX是主要的成分，其次是TTX；而从其它采样地点采集的织纹螺中，TTX都是主要的毒素成分，其次才是trideoxyTTX及其它同系物。对采集自江苏省连云港赣榆和浙江舟山定海的织纹螺体内毒素的解剖学分布进行了分析，结果表明肌肉、消化腺和剩余部分中的毒素组成基本一致，其中trideoxyTTX是主要的毒素成分，其次为TTX，但采自浙江舟山的织纹螺剩余部分中的TTX是主要的毒素成分。在监测期间，各组织中的毒素组成没有明显变化，但毒素含量随季节变化表现出了一定的差异。 综上所述，在中国沿海典型区域开展的织纹螺毒性调查结果表明其毒性消长具有一定的地域性和季节性特征。分析结果显示织纹螺体内的毒素成分是河豚毒素及其同系物，采自不同区域的织纹螺体内毒素成分基本一致，但毒素组成稍有差异。对织纹螺中毒素的解剖学分布研究显示，各组织中的毒素含量随季节变化而表现出一定差异，但毒素组成没有明显的季节性变化。这些结果显示中国沿海的织纹螺应具有相似的毒素来源，研究结果将为相关部门有效监测、预防和控制食用织纹螺中毒事件提供有力的科学依据。

麻痹性贝毒毒素的制备和活性研究

麻痹性贝毒（Paralytic Shellfish Poisons, PSP）毒素主要是由海洋甲藻产生的一类活性物质，在赤潮毒素监测、神经与分子生物学基础研究以及新型药物开发方面都有潜在的应用价值。然而，纯化毒素的缺乏在很大程度上限制了相关工作的开展。本文通过大量培养塔玛亚历山大藻（Alexandrium tamarense）来提取PSP毒素，完善了毒素的分离纯化过程，并对纯化毒素的活性进行了研究和探讨。在实验室进行了塔玛亚历山大藻的规模培养，对其生长和培养条件进行了优化，探讨了有毒藻大量培养的采收方式，并获取了一批用于纯化毒素的有毒藻细胞。将藻细胞破碎后离心分离，提取出含PSP的粗毒素，再用大孔径凝胶过滤去除粗毒素中的大部分杂质，然后用小孔径凝胶和离子交换树脂将不同毒素逐级分离、纯化。结果表明，大孔径凝胶能有效地去除粗毒素中的杂质，小孔径凝胶过滤能将膝沟藻毒素（GTX）和C毒素分开，应用离子交换树脂能将GTX2,3和GTX1,4分开，最终得到GTX2,3和GTX1,4的纯化产品。分离纯化过程始终以小鼠法和荧光法进行毒素在线监测。以提取的毒素对小鼠进行毒性测试，小鼠呈现典型的PSP毒素中毒死亡症状；电生理分析的结果表明PSP毒素能够有效地阻断小鼠运动神经末梢钠电流，并可逆地阻断NG108-15全细胞的钠电流，与PSP毒素的作用机理一致；高效液相色谱分析结果表明，纯化毒素与标准毒素的保留时间完全一致；经质谱分析，纯化毒素与标准毒素具有相同的分子离子峰和碎片离子峰。应用运动模型研究了纯化毒素的药理活性。分别采用家兔椎管麻醉法、小白鼠热板法、小白鼠攀网法研究了GTX2,3对家兔的局麻作用、对小鼠的镇痛和肌松作用。结果表明，GTX2,3按剂量0.4μg STX equivalent/kg给予家兔椎管注射时，具有显著的局麻作用。GTX2,3按剂量5.99μg STX equivalent/kg给予小鼠肌肉注射时，具有显著的镇痛作用。GTX2,3按剂量6.49μg STX equivalent/kg给予小鼠皮下注射时，具有显著的肌松作用。

桑沟湾贝藻养殖区附着生物生态效应研究

附着生物又称污损生物，是附生在海洋设施和生物体表面的动物、植物和微生物等生物的总称（Azis et al., 2001）。附生在养殖器材和生物体表面的数量巨大的附着生物，对贝类养殖和海湾生态系统内的物质和营养盐循环等多个方面产生影响。本研究以北方重要的养殖海湾----桑沟湾为研究对象，对贝藻养殖区附着生物的群落演替及其生态效应进行了研究。主要研究结果如下： ① 2007年5月至2008年5月，采用挂网的方法对桑沟湾栉孔扇贝和海带混养区的附着生物的季节变化进行了研究。结果显示挂网上的附着生物具有显著的季节变化特征，网片上的附着生物湿重与水温的变化相一致，生物量为3~1210 g•m-2。2月份附着生物的生物量最低，8月份最高。2007年9月至11月，对栉孔扇贝养殖笼上和贝壳上的附着生物种类和数量进行了研究。结果显示9月份养殖笼上附着生物的湿重约为1.94 kg，10月份降至0.99 kg，11月份又稍有增加，为1.03 kg。扇贝壳上的附着生物变化趋势与养殖笼上的相同，9~11月份壳上附着生物的数量约0.49~2.09 g。扇贝养殖笼上可鉴定的大型附着生物约23种，包括藻类、海鞘类、苔藓虫类、环节动物、腔肠动物、软体动物、甲壳动物和海绵动物等。玻璃海鞘、柄海鞘、紫贻贝和苔藓虫等是附着生物群落中的优势种。 ② 通过在栉孔扇贝和虾夷扇贝上壳上添加不同重量的“模拟附着生物”（速凝水泥）的方法，研究了贝壳上附着生物的重量对这两种扇贝生长和存活的影响。结果显示水泥重量是上壳重0.5-3倍的各组实验组扇贝的生长和存活与对照组（未添加水泥的扇贝）之间没有显著差异。说明贝壳上附着生物重量为上壳的3倍重时，也不会显著影响扇贝生长存活。9-11月份贝壳上的自然附着生物的重量约为1.47-2.09 g，为上壳重的28.16 (±38.6)%—31.29 ± (31.63)%。因此，贝壳上附着的生物重量不太可能对扇贝的生长存活造成显著的负面影响。 ③ 在桑沟湾现场测定了玻璃海鞘和柄海鞘的生物沉积速率。9月份（水温约24℃）玻璃海鞘和柄海鞘的生物沉积速率分别为32.14和90.06 mg•ind-1•d-1或（858.99 和467.76 mg•gdw-1•d-1），据此计算，养殖笼上的两种海鞘的生物沉积速率约为84.29 mg•m-2•d-1。海区的自然沉积速率为41.49 mg•m-2•d-1；玻璃海鞘和柄海鞘沉积物中有机质含量分别为14.34%和13.77%，对照组海区自然的有机质含量为14.36%；以上三者有机碳的含量分别为24.72%，23.74%和24.76%；氮的含量分别为0.27%和0.25%，自然沉积物中的氮含量为0.30%。9月份扇贝养殖笼上附着的海鞘将产生2588.16吨的沉积物，即向底部沉积363.77吨的有机物、6.99吨的氮和1.79吨的磷。 ④ 通过测定扇贝养殖笼上优势种附着生物--玻璃海鞘、柄海鞘和贻贝的摄食、呼吸和排泄，研究了这些优势种类对贝类养殖和海湾环境的影响。9月份（水温约24.5℃）玻璃海鞘和柄海鞘对颗粒有机物（POM）的摄食率分别为14.30 和17.01 mg• h-1•ind-1。根据实验结果计算这两种海鞘摄取的颗粒有机物相当于312个扇贝的摄取量，大于笼内养殖的扇贝的摄取量；玻璃海鞘和柄海鞘的耗氧率分别约为0.32和0.18 mg•h-1•ind-1，养殖笼上的这两种海鞘消耗的溶解氧约等于75个扇贝消耗的溶解氧。栉孔扇贝、玻璃海鞘、柄海鞘和贻贝的排氨率分别为33.66 ±11.34，117.90±23.46，35.91±6.22，28.08±3.41 ug NH4-N•gdw-1•h-1。以此估算，9月份玻璃海鞘、柄海鞘和贻贝每天排泄的氨氮约为654.08 kg，相当于16467吨栉孔扇贝（鲜重）排泄的氨氮。海鞘和贻贝排泄的氨氮可提供浮游植物等所需的2.75%的氮，可以提供1204吨海带的生长所需的氮。 ⑤ 一个养殖笼内的栉孔扇贝和全部附着生物（Scallop Culture Unit, SCU）在夏季（6-9月）对颗粒有机物的摄食速率约为43.13-98.94 mg/h，平均74.05 mg/h，期间桑沟湾养殖的栉孔扇贝及附着生物摄取的POM约为1279.58吨；同期，SCU对氨氮和磷（PO4-P）的排泄速率分别为125.59-1432.23 μmol•h-1和76.2-252.89μmol•h-1，期间桑沟湾养殖扇贝及附着生物排泄的氮磷分别为211.09 吨和83.79 吨。一串牡蛎及吊绳和牡蛎壳上的附着生物（Oyster Culture Unit, OCU），夏季摄食率为5-41.43μmol•h-1，耗氧率为16.54-41.76μmol•h-1，对氨氮和磷（PO4-P）的排泄速率分别为35.56-489.34μmol•h-1 和9.92-16.68μmol•h-1。以此估算，夏季OCU可摄取POM535.68吨，消耗溶解氧955.58吨，排泄氮磷分别为62.37 吨和15.50 吨。