【作者】 王清；

【导师】 杨红生；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生态学， 2010， 博士

【摘要】 本论文根据我国沿海重金属和有机物污染加剧以及滩涂贝类资源衰退的现状,开展了污染物对文蛤生活史各关键阶段特别是早期发育阶段的生态毒理效应,以及幼虫对重金属污染响应机制的研究,同时探讨了污染物对文蛤繁殖和血细胞免疫功能的影响。本研究探讨了重金属和有机污染物对双壳贝类生物资源补充的影响,为建立污染条件下资源生物的种群数量变化模型提供数据支撑,并为开展海洋生物保护和生境修复等提供科学依据。主要研究结果如下:1.研究了Pb2+,Cd2+和Hg2+三种重金属对文蛤胚胎发育,生长、存活和附着变态的影响。Pb2+,Cd2+和Hg2+对文蛤胚胎的半效应浓度(EC50)分别为297μg/L,1014μg/L和5.4μg/L。而Pb2+,Cd2+和Hg2+对文蛤D形幼虫96小时半致死浓度(LC50)分别为353μg/L,68μg/L和14.0μg/L。197μg/L Pb2+、104μg/L Cd2+和18.5μg/L Hg2+能显著地抑制D形幼虫的生长。上述三种重金属对文蛤幼虫附着变态的半效应浓度值与其对D形幼虫的48小时半致死浓度值类似,高于96小时半致死浓度值。三种重金属对文蛤幼虫的毒性由大到小为:Hg2+ > Cd2+ > Pb2+。2.探讨了苯并芘(Bap)和Aroclor1254对文蛤胚胎和幼虫发育的影响。结果表明596μg/L Bap和984μg/L Aroclor1254仅能够导致胚胎发育成功率的小幅降低。Bap和Aroclor1254对文蛤D形幼虫96 LC50分别为156μg/L和132μg/L。本研究中最为敏感的毒性测定终点是幼虫的附着变态率,Bap和Aroclor1254对附着变态率的半有效剂量浓度分别为20μg/L和35μg/L。3.从文蛤体内克隆了金属硫蛋白(命名为MmMT)基因。MmMT序列包含一个390bp的开放阅读框,编码129个氨基酸,其中有23个Cys,其C末端具有一个Cys-x-Cys-x(3)-Cys-Tyr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys的保守结构。MmMT的氨基酸序列与贻贝和牡蛎MT氨基酸序列具有57%~84%的相似性。MmMT基因和其他双壳贝类的MT基因具有很近的系统发生关系。Cd2+(25μg/L)胁迫会增加文蛤幼虫体内MmMT mRNA的表达,MmMT mRNA表达量在D形幼虫和壳顶幼虫期分别为对照组的5.04和3.99倍。免疫组织化学结果表明MmMT在Cd胁迫的担轮幼虫和稚贝几乎整个软体部都有分布,然而在受胁迫的D形幼虫和壳顶幼虫体内,MmMT只在面盘和表皮分布。在Cd胁迫下D形幼虫和壳顶幼虫体内抗氧化酶包括超氧化物歧化酶(SOD),过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPX)的活力会显著增加。4.5μg/L和50μg/L苯并芘暴露30天对文蛤性腺发育具有明显的延缓作用,其对雌性文蛤的延缓作用更加明显。100μg/L Cd2+和50μg/L Hg2+暴露15天后可能会加快雌性文蛤性腺的退化过程,但对雄性文蛤性腺发育影响相对较小。50μg/L Bap胁迫会明显破坏文蛤卵母细胞的膜结构,会阻碍卵黄蛋白原的合成,造成卵黄颗粒变形。50μg/L Bap和50μg/L Hg2+都会导致文蛤性腺谷胱甘肽(GSH)含量显著下降。所有浓度的Bap和100μg/L Cd2+会显著增加文蛤性腺GST的活力,而50μg/L Hg则会降低文蛤性腺GPx的活力。5.Bap对文蛤血细胞超微结构的损伤主要表现在导致血细胞伪足异常伸展,线粒体肿胀,细胞器数量减少,损伤更为严重的血细胞出现空泡化,细胞边缘裂解等现象。随着Bap暴露时间的增加,文蛤血细胞溶酶体中性红保持时间(NRRT)逐渐下降。5μg/L和50μg/L Bap处理组在胁迫第6天后开始,文蛤血细胞中性红保持时间开始显著低于对照组。血细胞微核(MN)率和总畸形核(TNA)率随胁迫时间的增加也逐渐增多。从暴露第6天开始,50μg/L处理组血细胞MN和TNA产生率均显著高于对照组。6.通过流式细胞仪技术,研究了Cd2+和苯并芘胁迫7天对文蛤血细胞功能的影响。结果表明,5μg/L和50μg/L的Bap胁迫会显著降低血细胞的吞噬活力,增加文蛤血细胞死亡率,而50μg/L Bap会显著增加血细胞活性氧产物。10μg/L和100μg/L Cd2+胁迫也会显著降低血细胞的吞噬活力,增加血细胞活性氧产物和死亡率。更多还原

【Abstract】 At present, heavy metal and organic pollution have become serious problems in marine environment of China, which results in the deterioration of bivalve resources. Toxic effects of these pollutants on different life stages of Meretrix meretrix, especially the early development stages, were assessed, and the responding mechanism of the larvae to the heavy metal-stress was investigated. In addition, the impacts of these pollutants on the reproduction and immune function of hemocyte were also examined. The results discussed the effects of heavy metals and organic pollutants on recruitment amount of bivalve resources, and could provide data for establishing population-dynamic model. Moreover, these results also provide basis for marine organism protection and marine environment restoration. The details are as follows:1. The impacts of Hg2+, Cd2+ and Pb2+ on embryogenesis, survival, growth and metamorphosis of larvae were investigated. The EC50 for embryogenesis was 5.4μg/L for Hg2+, 1014μg/L for Cd2+ and 297μg/L for Pb2+, respectively. The 96 h LC50 for D-shaped larvae was 14.0μg/L for Hg2+, 68μg/L for Cd2+ and 353μg/L for Pb2+, respectively. Growth was significantly retarded at 18.5μg/L for Hg2+, 104μg/L for Cd2+ and 197μg/L for Pb2+ respectively. The EC50 for metamorphosis, similar to 48 h LC50, was higher than 96 h LC50. The heavy metals were consistently ranked in the following order from highest to lowest toxicity: Hg2+ >Pb2+> Cd2+.2. The impacts of benzo[a]pyrene (Bap) and Aroclor1254 on embryogenesis and larval development were investigated using static laboratory toxicity tests at nominal concentrations of 6.25–1600μg/L. Even 596μg/L Bap and 984μg/L Aroclor1254 only caused minor reductions in embryo development rates. The 96 h LC50 values for D-shaped larvae were 156μg/L for Bap and 132μg/L for Aroclor1254, respectively. The most sensitive toxicity endpoint in this study was metamorphosis, with an EC50 value of 20μg/L for Bap and 35μg/L for Aroclor1254.3. A new MT (designated MmMT) gene was identified and cloned from M. meretrix. The full length cDNA of MmMT consisted of an open reading frame (ORF) of 390 bp encoding a protein of 129 amino acids, with 23 cysteine residues and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys at the C-terminus. The deduced amino acid sequence of MmMT showed about 57%~84% identity with previously published MT sequences of mussels and oysters. Real-time PCR was conducted to analyze the expression level of MT mRNA in M. meretrix larvae under Cd2+ (25μg/L) exposure. Results showed that Cd2+ could induce the mRNA expression of MmMT in the larvae, and the expression level increased 5.04-fold and 3.99-fold in D-shaped larvae and pediveligers respectively. Immunolocalization of MmMT in the stressed larvae revealed that MmMT was synthesized in almost all the soft parts of the stressed larvae at trochophore and postlarva stage, while it was only synthesized in the velum and epidermis of the stressed larvae at D-shaped larva and pediveliger stages. Additionally, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were also measured in M. meretrix larvae. The results showed the increase of enzymatic activities mainly in D-shaped larvae and pediveligers under cadmium stress.4. The gonad development of M. meretrix was retarded after exposure to 5μg/L and 50μg/L Bap for 30 days. 100μg/L Cd2+ and 50μg/L Hg2+ accelerated gonad degeneration of female clams respectively after exposure for 15 days, while they had little effect on male gonad development. 50μg/L Bap not only had obvious damage to membrane structure of organelles, but also hindered vitellogenin synthesis resulting in yolk granule deformation. 50μg/L Bap and 50μg/L Hg2+ induced a decrease of the glutathione (GSH) content in gonad. Bap (all concentration) and 100μg/L Cd2+ exposure increased GST activity in gonad, while 50μg/L Hg2+ inhibited GPx activity.5. The ultrastructure damages of heamocyte exposed to Bap were described as follows: the pseudopod stretching singularly, mitochondrion becoming swollen, and the number of organelles decreasing. There were also vacuolization and lysis on the edge of some heamocytes. The neutral red retention time (NRRT) of lysosome in heamocytes decreased as exposure time prolonged. After exposure to 5μg/L and 50μg/L Bap for 6 days, NRRTs of these groups were significantly lower than those of control group. Micronucleus (MN) frequency and total nucleus abnormality (TNA) frequency in heamocytes also enhanced as exposure time increased. The MN frequency and TNA frequency of 50μg/L group increased significantly after 6-day exposure.6. The effects of cadmium and Bap on immune function of heamocytes were examined by a flow cytometer. The results indicated that 5μg/L and 50μg/L Bap inhibited phagocytosis and increased the mortality of heamocytes, while 50μg/L Bap could enhance reactive oxygen species (ROS). 10μg/L and 100μg/L Cd2+ significantly suppressed phagocytosis of heamocytes and increased mortality of heamocytes, while 100μg/L Cd2+ stimulated ROS in hemocytes.更多还原