海洋生态与环境科学重点实验室知识库

近几十年来，受全球变化和人类活动的影响，海洋生物正面临着升温、低氧、酸化和重金属污染等多重环境压力的威胁。本研究以生活习性和生境迥异的重要经济物种半滑舌鳎（Cynoglossus semilaevis Günther, 1873）和菲律宾蛤仔（Ruditapes philippinarum Adams et Reeve, 1850）作为实验对象，通过开展受控毒理实验对比研究海水酸化和近海常见污染物镉的二元环境胁迫下两种生物早期生活阶段的抗氧化防御的生理响应和策略。将半滑舌鳎稚鱼和菲律宾蛤仔幼贝分别置于不同组合的pH水平（pH 8.10，7.70和7.30；对应于IPCC预测的2100年和2300年海洋酸化情景）和镉浓度（稚鱼：0，5，10和50 μg L^-1；幼贝：0，10，50和100 μg L^-1；对应近海背景相关镉浓度至高污染水平）的海水中连续暴露21天。实验结束后，测定生物抗氧化剂的含量或活性，包括还原型谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GPx）、谷胱甘肽S-转移酶（GST）、谷胱甘肽还原酶（GR）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT），解析稚鱼和幼贝对两种胁迫的抗氧化防御响应；通过分析生物体内丙二醛（MDA）的含量变化研究其脂质过氧化水平（LPO）即氧化损伤程度；利用整合生物标志响应指数（IBR）和主成分分析（PCA）评估生物对共同胁迫的整体响应，筛选潜在的敏感生物标志物。在此基础上，对比分析早期生活阶段鱼类和贝类的抗氧化防御对两种环境胁迫的生理响应及防御策略的差异。研究结果有助于科学认识二者共同胁迫对海洋鱼类和贝类抗氧化防御交互作用的毒理学机制，为揭示海洋生物在未来海洋酸化背景下对重金属胁迫的响应机理提供科学参考。

1. 揭示了早期生活阶段半滑舌鳎稚鱼和菲律宾蛤仔幼贝对海水酸化和镉胁迫的抗氧化防御响应。单一镉效应显著影响稚鱼体内GSH相关抗氧化剂（GPx、GST和GR），但各抗氧化剂对镉胁迫的响应方式存在差异；海水酸化效应显著抑制GSH、GPx和GR但诱导SOD和CAT。两种环境应激的显著交互作用仅出现在GPx和GST中。随着胁迫水平的增加，稚鱼的抗氧化防御通过不同策略对氧化应激做出适应性响应：GSH相关防御系统呈现抗氧化剂的不同活性变化以响应升高的金属浓度，但海水酸化条件下相关抗氧化剂对镉效应的响应以受抑制为主。SOD和CAT的抗氧化防线总体上对镉胁迫的敏感程度较低，但受海水酸化水平的显著被诱导，其活性的同步响应突出两种抗氧化剂在应对海水酸化诱导的氧化应激中的重要作用。整体而言，在本实验环境胁迫水平下，稚鱼的抗氧化防御系统对海水酸化胁迫比对镉胁迫的响应更敏感。

镉胁迫显著影响幼贝中除CAT外的其他抗氧化剂；海水酸化胁迫和二者交互作用显著影响所有抗氧化剂。GPx受单一镉效应显著被抑制，而GSH和GST以相似的变化趋势应对金属浓度升高的胁迫。海水酸化效应总体上抑制GSH相关抗氧化剂，出现幼贝的总可用GSH资源库被过度消耗而减少的现象。在此情况下，镉效应对幼贝的GSH防御系统的影响因海水酸化效应的叠加而发生改变，二者表现出一定的拮抗效应。幼贝SOD和CAT抗氧化防线随各胁迫水平的升高呈现相反的酶活变化趋势，但两种抗氧化剂对单一胁迫的响应模式相似，涉及类似的特异性抗氧化防御策略。

2. 阐释了海水酸化和镉胁迫下稚鱼和幼贝的整体氧化损伤程度。二种胁迫的交互作用导致稚鱼体内脂质过氧化（LPO）水平显著升高，机体氧化应激加剧。在所有镉浓度下，海水酸化效应均显著提高稚鱼的LPO水平，且高于单一镉效应诱导的LPO水平。海水酸化和镉的相互作用会诱导氧化应激，从而加剧鱼体内的脂质过氧化损伤。而在高度海水酸化水平下，海水酸化效应会掩盖镉胁迫对氧化损伤的负面效应，弱化了镉效应在诱导LPO水平中的作用。

单一海水酸化或镉胁迫均未显著加剧幼贝体内的脂质过氧化损伤，二者胁迫的显著交互作用也未出现在LPO水平中。虽然海水酸化效应未直接提高幼贝的LPO水平，但是，只有在海水酸化胁迫条件下才出现高浓度镉胁迫诱导了LPO水平显著增加的现象。这些结果表明抗氧化防御系统未能充分保护幼贝免受高浓度镉和海水酸化共同胁迫所诱导的体内脂质过氧化损伤。

3. 评估了稚鱼和幼贝对海水酸化和镉共同胁迫的整体抗氧化防御响应，筛选了响应的潜在敏感生物标志物。整合生物标志响应指数（IBR）分析表明，海水酸化和镉胁迫均显著增加稚鱼的整合抗氧化响应。与镉胁迫相比，海水酸化对稚鱼的整合抗氧化响应有更明显的加剧效应，导致稚鱼处于较高的环境压力中。高度海水酸化水平下，抗氧化剂整体表现出对镉胁迫响应的减弱，海水酸化效应可能超过并掩盖了稚鱼抗氧化生物标志物对镉胁迫的响应。主成分析（PCA）结果表明，与GPx和GST相关的主成分可以有效地区分镉浓度处理组，其酶活变化是机体应对镉胁迫的重要抗氧化防御途径；稚鱼的抗氧化防御对海水酸化胁迫响应的潜在敏感生物标志物包括GSH、GR、SOD和CAT，对镉胁迫响应的主要是GPx和GST。

单一的海水酸化或镉效应均导致幼贝处于更高的环境压力下，显著增强了整合生物标志物响应。但在高镉浓度或海水酸化水平胁迫下，幼贝长期处于抑制状态，IBR表现相对稳定。总体上，镉胁迫趋向于诱导幼贝的整合生物标志物响应，而海水酸化抑制其整合生物标志物响应，且抑制程度随金属浓度升高而增加。PCA能有效区分未海水酸化下的镉浓度组，但无法有效区分海水酸化下的镉浓度组。海水酸化造成的生物标志物的被抑制状态导致抗氧化剂对金属浓度升高的响应减弱，不利于抗氧化系统的有效运转。GSH和SOD是幼贝对海水酸化抗氧化响应的潜在敏感生物标志物，而MDA和GST适用于评估镉胁迫下其抗氧化防御响应。

综合上述研究结果发现，1）稚鱼和幼贝的GSH防御系统总体上均受单一镉胁迫显著被诱导，而海水酸化条件下相关抗氧化剂对镉胁迫的响应以被抑制为主。但两种生物中同一抗氧化剂对各环境胁迫的响应方式及敏感程度各不相同，说明其抗氧化防御途径或策略存在差异。2）稚鱼的SOD和CAT抗氧化防线受镉胁迫响应较弱，但积极应对海水酸化胁迫。而幼贝的该抗氧化防线采取相似的响应模式应对两种环境胁迫。3）在设定的镉浓度下，单一镉胁迫未显著增加幼贝体内的LPO水平，且相对于幼贝，海水酸化胁迫和二者的交互作用更明显地加剧稚鱼体内的脂质过氧化损伤。4）稚鱼和幼贝的抗氧化防御系统均对高度海水酸化胁迫响应敏感，且在高度海水酸化水平下呈现出对镉胁迫响应总体减弱的趋势。

In the last few decades, marine organisms have been facing threats from various environmental factors such as elevated seawater temperature, hypoxia, acidification, and heavy metal pollution, due to global changes and human activities. For this study, the experimental animals chosen were tongue sole (Cynoglossus semilaevis Günther, 1873) and Manila clam (Ruditapes philippinarum Adams et Reeve, 1850), which are commercial species with different living habits and habitats. Two toxicological experiments were carried out to explore the antioxidant defense responses of these organisms during early life stages (ELSs) when exposed to combined stressors of seawater acidification (SA) and cadmium (Cd), a prevalent coastal pollutant. The juvenile tongue sole and clams were exposed to varying combinations of pH levels (pH 8.10, 7.70 and 7.30; reflecting ocean acidification scenarios projected for 2100 and 2300 by IPCC) and Cd concentrations (juvenile fish: 0, 5, 10 and 50 μg L^-1; juvenile clams: 0, 10, 50 and 100 μg L^-1; representing background-related Cd concentrations from coastal waters to highly polluted levels) for a duration of 21 days. Subsequently, the levels of antioxidants and activities, including reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT), were measured to assess the antioxidant defense responses of juvenile fish and clams to the two environmental stressors. The malondialdehyde (MDA) content of the organisms was also analyzed to evaluate the lipid peroxidation (LPO), which indicated oxidative damage levels. Integrated biomarker response (IBR) and principal component analysis (PCA) methods were employed to comprehensively analyze the overall antioxidant responses of the organisms to the combined stressors and identify potentially sensitive biomarkers. Based on this experimental framework, the study conducted a comparative analysis of the physiological responses and defense mechanisms of fish and clams at ELSs to the two environmental stressors. The results of this study are anticipated to contribute scientific insights into the toxicological mechanisms underlying interactions among multiple environmental stressors on the antioxidant defenses of marine fish and clams, thereby providing valuable references for understanding the response mechanisms of marine organisms exposed to heavy metals under future ocean acidification scenarios.

1. The antioxidant defense responses of juvenile tongue sole and clams to seawater acidification (SA) and cadmium (Cd) were investigated. Significant effects of Cd on GSH-related antioxidants (GPx, GST and GR) in juvenile fish were observed, with distinct responses of each antioxidant to Cd stress. Conversely, SA effects notably suppressed GSH, GPx and GR, while stimulating SOD and CAT. Interactions between the two stressors were mainly evident for GPx and GST. Additionally, the antioxidant defenses of juvenile fish demonstrated adaptive responses to oxidative stress under increasing environmental stressors. The GSH defense system exhibited varying activity patterns among the GSH-related antioxidants in reaction to different Cd concentrations in the absence of SA, while these antioxidants tended to be inhibited in response to Cd effects under SA conditions. Furthermore, the defense system of SOD and CAT showed lower sensitivity to Cd stress but was significantly induced by SA levels. The concomitant responses underscored the significance of these antioxidants in combating oxidative stress induced by SA. The findings suggested that the antioxidant defense system of juvenile fish was more susceptible to SA compared to Cd stress under the environmental stress conditions examined in this study.

In juvenile clams, the impacts of Cd stress on antioxidants were significant, with the exception of CAT. Both SA exposure and the combined effects of two environmental stressors had a significant influence on all antioxidants. The single effect of Cd apparently inhibited GPx, while GSH and GST exhibited a similar trend in response to increasing Cd concentrations. Under SA conditions, most GSH-related antioxidants were inhibited and the depletion of the total available GSH pool was observed as a result of excessive consumption in juvenile clams. An antagonistic relationship between the two stressors was observed, resulting in changes of the antioxidant responses of juvenile clams to Cd stress. Interestingly, the defense mechanisms of SOD and CAT in juvenile clams displayed opposite trends with increasing stress levels, while their activities demonstrated a similar response pattern to single exposure, suggesting a consistent specific antioxidant defense strategy.

2. The interactions between SA and Cd stress were investigated to assess the overall oxidative damage in juvenile fish and clams. In juvenile fish, the combined exposure to SA and Cd led to a significant increase in LPO levels, inducing oxidative stress. The effects of SA further heightened LPO levels at all Cd concentrations, surpassing the levels induced by Cd stress alone. This suggested that the interactions between SA and Cd exacerbated oxidative stress and augmented LPO damage in juvenile fish. Specifically, in high SA conditions, the effects of SA masked the impacts of Cd on oxidative damage and mitigated the influence of Cd on LPO levels. In contrast, in juvenile clams, single exposure to either SA or Cd did not significantly aggravate LPO damage and no significant interactions between the two stressors were observed in LPO levels. While SA did not directly increase LPO levels in juvenile clams, elevated LPO levels by high Cd concentrations were only evident under SA conditions. These findings indicated that the antioxidant defense system may not offer complete protection in juvenile clams against LPO damage caused by the combined exposure to SA and high Cd concentrations.

3. The overall responses of juvenile fish and clams to the combined stressors of SA and Cd were evaluated and the potential sensitive biomarkers for oxidative stress were identified. The Integrated biomarker response (IBR) revealed that both SA and Cd stress significantly increased the integrated biomarker response of juvenile fish, with SA exposure having a more pronounced effect on promoting these responses, which caused juvenile fish under severe environmental stress. Furthermore, under high SA levels, the overall responses of antioxidants to Cd exposure were diminished, indicating that the impacts of high SA levels may overshadow the antioxidant responses to Cd exposure in juvenile fish. The principal component analysis (PCA) results showed that the principal component associated with GPx and GST effectively distinguished the Cd treatments, suggesting that the activities of these enzymes played a crucial role in an antioxidant defense mechanism against Cd stress in organisms. The identified potential sensitive biomarkers of antioxidant defense in juvenile fish in response to SA included GSH, GR, SOD and CAT, whereas GPx and GST were found to be more suitable biomarkers for Cd stress.

Both single SA and Cd exposure caused juvenile clams under higher environmental stress and heightened the IBR of juvenile clams under elevated environmental stress levels. Under high Cd concentrations and SA conditions, juvenile clams experienced long-term inhibition and exhibited a relatively stable IBR. Generally, Cd exposure led to an increase in IBR, whereas SA conditions tended to inhibit these responses, with the degree of inhibition escalating with higher Cd concentrations. PCA effectively differentiated the Cd treatments in the absence of SA but not in its presence. The inhibitory effects of SA resulted in a compromised antioxidant response to elevated metal concentrations, thereby hindering the efficient operation of the antioxidant system. GSH and SOD emerged as potential sensitive biomarkers for the antioxidant defense of juvenile clams under SA exposure, whereas MDA and GST were identified as appropriate markers for assessing Cd stress.

In summary, 1) The antioxidant responses related to the GSH defense system of both juvenile fish and clams were induced overall in response to single Cd stress, while these antioxidants were predominantly inhibited in response to Cd stress under SA conditions. However, the response patterns and sensitivity of these antioxidants to SA or Cd stress differed between the two organisms. 2) The antioxidant defense mechanisms of SOD and CAT in juvenile fish showed less susceptibility to Cd stress but exhibited positive responses to SA exposure. In contrast, the antioxidant defense in juvenile clams displayed a similar response pattern to both environmental stressors. 3) Under the specified Cd concentrations, single Cd stress did not significantly induce LPO damage in juvenile clams. And SA exposure and the interactions of the two stressors notably exacerbated LPO levels in juvenile fish. 4) The antioxidant defense systems of juvenile fish and clams exhibited sensitivity to high SA esposure, resulting in a weakening of the overall responses to Cd stress under these SA conditions.

第1章 绪论

1.1 海洋酸化

1.1.1 海洋酸化现象

1.1.2 海洋酸化对海洋生物的影响

1.2 镉及其毒性效应

1.2.1 海洋环境中镉的来源及污染状况

1.2.2 镉的生物毒性效应

1.3 海洋酸化背景下镉的生物毒性效应及其对抗氧化防御的影响

1.4 研究意义、研究内容和技术路线

1.4.1 研究目的和意义

1.4.2 研究内容

1.4.3 技术路线

第2章 海水酸化和镉胁迫下半滑舌鳎稚鱼抗氧化防御响应

2.1 材料和方法

2.1.1 实验用鱼的驯化与饲育管理

2.1.2 实验设计

2.1.3 水体取样及理化参数测定

2.1.4 鱼类取样和生化分析

2.1.5 数据分析

2.2 结果

2.2.1 实验海水的理化参数

2.2.2 抗氧化生物标志物对海洋酸化和镉胁迫的响应

2.2.3 抗氧化生物标志物对海水酸化和镉共同胁迫的整合响应

2.3 讨论

2.3.1 海水未酸化下稚鱼抗氧化防御对镉胁迫的响应

2.3.2 海水酸化下稚鱼抗氧化防御对镉胁迫的响应

2.3.3 稚鱼抗氧化防御对海水酸化和镉胁迫的整合响应

第3章 海水酸化和镉胁迫下菲律宾蛤仔幼贝抗氧化防御响应

3.1 材料和方法

3.1.1 实验用贝的采集与驯化

3.1.2 实验设计

3.1.3 水体取样及理化参数测定

3.1.4 贝类取样和生化分析

3.1.5 数据分析

3.2 结果

3.2.1 实验海水的理化参数

3.2.2 抗氧化生物标志物对海洋酸化和镉胁迫的响应

3.2.3 幼贝抗氧化防御对海水酸化和镉共同胁迫的整合响应

3.3 讨论

3.3.1 海水未酸化下幼贝抗氧化防御对镉胁迫的响应

3.3.2 海水酸化下幼贝抗氧化防御对镉胁迫的响应

3.3.3 幼贝抗氧化防御对海水酸化和镉胁迫的整合响应

3.3.4 幼贝和稚鱼抗氧化防御对海水酸化和镉胁迫响应的比较分析

第4章 结论与展望

4.1 结论

4.2 展望

参考文献

致谢

作者简历及攻读学位期间发表的学术论文与其他相关学术成果