海洋生态与环境科学重点实验室知识库

    海洋酸化和污染是备受人们关注的海洋环境问题。海洋酸化不仅直接影响海洋生物的繁殖发育和生长存活等生命过程，而且影响重金属等污染物的生物毒性效应。因此，二者复合胁迫对海洋生物早期生命过程的作用是海洋生态学研究的一个前沿科学问题。本论文通过开展系列受控实验，研究褐牙鲆不同早期发育阶段（胚胎、仔鱼和幼鱼）重要生理生命过程（孵化发育、生长存活、抗氧化防御系统、免疫功能和生物矿化等）对海水酸化和镉（Cd）暴露复合胁迫的响应，构建综合生物标志物响应指数（IBR）评估二者复合胁迫对褐牙鲆早期发育阶段的影响。开展本研究有助于科学评估海洋酸化和重金属复合胁迫对渔业种群资源补充过程和数量变动的毒理效应，同时为深入认识未来海洋酸化情景下鱼类早期生活阶段的生物响应机理提供科学参考。主要研究结果如下：

        1、海水酸化（pH 7.70和7.30）和镉暴露（0.01，0.15和0.50 mg L^-1 Cd²⁺）会抑制胚胎孵化和卵黄囊吸收、加剧仔鱼形态发育畸形，导致其生长存活能力降低。镉暴露会造成胚胎活动的减弱和蛋白酶水解，造成与孵化相关的生物酶活性和功能异常，阻碍初期仔鱼破膜过程，影响其孵化能力。海水酸化会通过影响这些生理过程，延迟胚胎孵化时间或加剧其死亡，降低孵化成功率。仔鱼最常见的形态发育畸形为骨骼（脊柱和尾部骨骼等）畸形。这可能与海水酸化和镉暴露抑制仔鱼的骨钙素（oc）基因的表达、影响成骨细胞的分化和活化、抑制与生物矿化相关的碳酸酐酶（CA）和Ca-ATP酶的活性有关。这些作用会阻碍仔鱼对Ca2+的吸收，导致其骨骼系统发育所必需的肌球蛋白和肌节减少，对骨骼的形成和发育造成影响，加剧仔鱼的骨骼发育畸形。

        2、镉在褐牙鲆仔鱼生物体或幼鱼肝脏、鳃和肌肉等生物组织内的蓄积量与镉暴露浓度呈正剂量关系，且具有组织特异性，依次为肝脏＞鳃＞肌肉。但海水酸化未显著影响镉在仔鱼或幼鱼各组织内的蓄积。肝脏是幼鱼的主要解毒和代谢器官，从皮肤、鳃或肠道中吸收的Cd2+随血液或淋巴循环最终被转运到肝脏或肾脏组织进行解毒。在这一过程中，Cd2+与肝脏中富含巯基（-SH）的还原型谷胱甘肽（GSH）和金属硫蛋白（MT）结合形成毒性较小的络合物并长期滞留于组织内，加剧镉在肝脏中的蓄积。鳃是幼鱼呼吸和渗透调节以及酸碱平衡调节的主要器官，不断过滤海水保证体内溶氧充足和维持渗透平衡和酸碱平衡，因而容易直接从海水中富集镉并蓄积于鳃组织内。肌肉内的镉主要来源于血液和淋巴循环等生理代谢过程的转入，浓度相对低，且肌肉组织质量相对较大，具有质量稀释效应，降低了肌肉内镉蓄积水平。镉蓄积的组织特异性会影响到仔鱼体内或幼鱼生物组织内的抗氧化防御、免疫功能和生物矿化等对二者复合胁迫的响应。

        3、海水酸化（pH 7.70和7.30）和镉暴露（0.01和0.15 mg L^-1 Cd²⁺）均显著影响褐牙鲆仔鱼的抗氧化防御系统、免疫功能和生物矿化作用。总体而言，镉暴露下，超氧化物歧化酶（SOD）和谷胱甘肽-S转移酶（GST）的活性以及还原型谷胱甘肽（GSH）的含量被诱导以应对镉胁迫造成的氧化应激损伤，而过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GPx）的活性则被过量的活性氧自由基（ROS）所抑制。另一方面，海水酸化对仔鱼的抗氧化防御系统的影响因生物标志物种类和镉暴露浓度而异，表明海水酸化会影响抗氧化防御系统对镉暴露的响应。二者复合胁迫提高了仔鱼的脂质过氧化水平，加剧了其氧化损伤程度。

    在免疫应答方面，二者复合胁迫导致仔鱼体内溶菌酶（LZM）的活性显著下降，即先天性免疫功能受到抑制；而免疫球蛋白M（IgM）的含量升高，即获得性免疫功能表现出免疫刺激。镉暴露会诱导仔鱼体内MT和热休克蛋白（HSP70）的产生，但海水酸化仅诱导HSP70产生，未影响MT产生。

    海水酸化和镉暴露均显著诱导三种生物矿化酶（碳酸酐酶CA、钙-ATP酶Ca-ATPase和钠/钾-ATP酶Na/K-ATPase）的活性，以应对二者胁迫造成的酸碱平衡和渗透平衡的变化。

        4、镉暴露（0.01，0.30和3.0 mg L^-1 Cd²⁺）显著影响褐牙鲆幼鱼肝脏、鳃和肌肉组织的抗氧化防御系统和免疫功能，它们对镉暴露的响应具有组织差异性。总体上，肝脏组织对二者复合胁迫的响应比鳃和肌肉组织更为显著。这种对二者复合胁迫生理响应的组织差异性可能与镉蓄积量的组织特异性相关。但海水酸化对褐牙鲆幼鱼三个生物组织内的抗氧化防御系统和免疫功能相关的各生物标志物无显著影响。

    具体而言，在最高镉暴露（3.0 mg L^-1）下，肝脏内抗氧化防御系统（SOD、CAT、GSH、GST和GPx）和免疫功能（LZM、ACP、AKP和IgM）相关的生物标志物的酶活性或含量被抑制；鳃组织内的CAT、GSH、GST和GPx等抗氧化物以及LZM和ACP等免疫功能酶的活性或含量被抑制，而SOD、AKP和IgM的活性或含量被诱导；肌肉组织内的SOD、GSH、GST、GPx、ACP和AKP的活性或含量被诱导，但CAT、LZM和IgM的活性或含量无显著变化。在三个生物组织内，高浓度镉暴露诱导MT含量，并造成组织的脂质过氧化损伤。一般而言，当组织内镉蓄积量过高时，会破坏蛋白质结构，酶活性受到抑制，影响各生理、生化过程。例如，肝脏组织内镉蓄积量高，超出自身的清除能力，造成肝脏内抗氧化防御系统和免疫功能相关酶的活性被抑制；而肌肉组织内镉蓄积量远低于肝脏中的蓄积量，抗氧化防御系统和免疫功能相关酶被诱导以应对镉和海水酸化胁迫造成的损伤。

    海水酸化（pH 7.70和7.30）仅对幼鱼肝脏和鳃组织中的CAT活性和肌肉中ACP的活性产生影响，未显著影响三个生物组织内其他抗氧化防御响应和免疫应答相关生物标志物。这可能与幼鱼已经形成了完善的酸碱调节系统有关。海水酸化下生物体内多余的CO₂和H⁺在酸碱调节酶（CA）、缓冲离子和跨膜蛋白（Na/K-ATP酶和H⁺-ATP酶）作用下被及时清除，从而保护幼鱼各组织免受海水酸化的影响。但是，海水酸化在一定程度上会加剧褐牙鲆幼鱼对镉暴露的抗氧化防御响应和免疫应答。

        5、构建了综合生物标志物响应指数（IBR）以评估海水酸化和镉复合胁迫对褐牙鲆仔鱼或幼鱼的抗氧化防御系统和免疫应答-生物矿化的毒性效应。仔鱼或幼鱼各组织的抗氧化防御和免疫功能-生物矿化对复合胁迫比对单一胁迫的响应更加敏感，即海水酸化会加剧镉暴露对褐牙鲆仔鱼和幼鱼的毒性效应。此外，仔鱼对复合污染的相关生理响应比幼鱼更敏感和显著。相较于单一的生物标志物评价法，IBR法能够更加客观地评估复合胁迫对受试生物的综合生物毒性效应。

    Ocean acidification (OA) and marine pollution are two globally marine environmental problems that have increasingly come to the foreground and public notice in past years. Ocean acidification not only directly affects the reproduction, development, growth and survival of marine organisms, but also influences the biotoxicity of pollutants, such as heavy metals. Therefore, the issues how OA and heavy metal exposure affect marine organisms at early life stages (ELSs) have become hot research topics in marine ecotoxicology studies. In this paper, a series of laboratory experiments were conducted to investigate how physiological life processes (e.g., hatching, development, growth, survival, antioxidative defense, immune function and biomineralization) of the flounder Paralichthys olivaceus at ELSs (embryos, larvae and juveniles) responded to OA and cadmium (Cd) exposure. Subsequently, the integrated biomarker response (IBR) was established and used to evaluate the intergrative effects of the two stressors on the physiological processes of the flounder larvae and juveniles. This study could help improve the assessment of the toxic effects of the two stressors on the recruitments and population dynamics of fishery species. Additionally, the results are expected to provide knowledge for better understanding how marine fishes at ELSs respond to OA scenarios in the future.

    (1) Seawater acidification (pH 7.70 and 7.30) and Cd exposure (0.01, 0.15 and 0.50 mg L^-1 Cd²⁺) could inhibit the hatchability and yolk absorption and increase the morphological abnormalities of the larvae, leading to decreases in larval growth and survival. Cd exposure would reduce the activity of embryos and induce the protease hydrolysis, resulting in the abnormal functions of hatching-related enzymes and thus decreasing hatching success. Seawater acidification could affect the above-mentioned physiological and biochemical processes induced by Cd exposure. This could further cause a delay in hatching or aggravate the embryonic mortality, leading to low hatchability. The morphological deformity of the larvae mainly occurred in the skeleton (e.g., spine and tail). The deformity might be related to the down-regulated oc (osteocalcin) gene and reduced CA (carbonic anhydrase) and Ca-ATPase activities under seawater acidification and Cd exposure, which would hinder the absorption of calcium (Ca) and lead to a decrease of myosin and sarcomere and aggravate the developmental malformation in the skeleton of the larvae.

    (2) The Cd accumulation in the larvae or in the tissues (liver, gill and muscle) of the juveniles was postively dose-depndent on Cd and tissue-specific. The Cd accumulation increased following the order of liver>gill>muscle. However, seawater acidification did not affect the Cd accumulation either in the larvae or in the tissues of the juveniles. The liver is the main organ for detoxification and metabolism of the metals. The Cd that was absorbed by skin, gill and intestine was eventually transported into the liver and kidney for detoxification through the blood and lymph circulations. The Cd²⁺ could bind to the -SH in the reduced glutathione (GSH) and metallothionein (MT), and is synthesized to low toxic compounds. The compounds could remain in the tissues for a long time and increase the Cd accumulation in the livers. The gill is the main organ for respiration, osmotic regulation and acid-base balance regulation of the juveniles. Seawater was continuously filtered to provide sufficient dissolved oxygen or maintained the osmotic balance and acid-base balance, increasing the Cd accumulation in the gills. The Cd in the muscles mainly comes from metal transfer via physiological metabolic processes such as blood and lymph circulations, which is commonly at relatively low concentration. Moreover, muslces have mass dilution effects due to their relatively large mass, potentially reducing the Cd accumulation level in muscles. Meanwhile, the tissue-specific Cd accumulation could affect the antioxidative and immune responses of the larvae and juveniles to the interactive stresses of the two stressors.

    (3) The antioxidative defense, immune responses and biomineralization of the flounder larvae were significantly affected by seawater acidification (pH 7.70 and 7.30) and Cd exposure (0.01 and 0.15 mg L^-1 Cd²⁺). Specifically, increasing Cd²⁺ concentration significantly induced the reduced glutathione (GSH) contents, the activities of superoxide dismutase (SOD) and glutathione S-transferase (GST), but inhibited the activities of catalase (CAT) and glutathione peroxidase (GPx). On the other hand, the antioxidants responded differently to seawater acidification, depending on biomarker or Cd²⁺ concentration. Seawater acidification would affect the responses of antioxidative defense to the Cd exposure. Additionally, both seawater acidification and Cd exposure induced lipid peroxidation (LPO) and aggravated the oxidative damages to the flounder larvae.

    When it comes to immune responses, lysozyme (LZM) activities were reduced and immunoglobulin M (IgM) contents were induced under seawater acidification and Cd exposure. These findings indicated that the two stressors resulted in an immunosuppression in the innate immunity and an immunostimulation in the acquired immunity in the larvae. Heat shock protein 70 (HSP70) contents were induced by Cd exposure and seawater acidification, whereas MT contents were induced by Cd exposure but were not affected by seawater acidification.

    As for biomineralization and acid-base regulation, the acitivties of the three investigated enzymes that are related to biomineralization (CA, Na/K-ATPase and Ca-ATPase) in the larvae were all significantly induced under seawater acidification and Cd exposure.

    (4) The antioxidative defense and immune responses in the liver, gill and muscle of the flounder juveniles were all significantly affected by Cd exposure (0.01, 0.30 and 3.0 mg L^-1 Cd²⁺), which were apparently tissue-specific. Liver commonly responded more sensitively to the intergrative stress of the two stressors than gill and muscle, potentially being related to the tissue-specific Cd accumulation. However, seawater acidification did not show significant effects on the antixodative and immune responses in the liver, gill and muscle of juveniles.

    Specifically, in the liver, all the investigated antioxidants (SOD, CAT, GSH, GST, GPx) and immune biomarkers (LZM, ACP, AKP and IgM) were inhibited at the highest Cd²⁺ concentration (3.0 mg L^-1). In the gill, the activities of CAT, GST, GPx, LZM and ACP and GSH contents were all inhibited, but the activities of SOD and AKP and IgM contents were induced at the highest Cd²⁺ concentration. In the muscle, the activities of SOD, GST, GPx, ACP and AKP and GSH contents were all induced at the highest Cd²⁺ concentration, but either the activities of CAT and LZM or the IgM contents were not affected by Cd exposure. Additionally, both MT content and LPO level were significantly increased at the highest Cd²⁺ concentration in all tissues. The inhibition of the antioxidative or immune biomarkers in the liver and gill might result from the high Cd accumulation in the two tissues, which could destroy the protein structures, inhibit the emzyme activities and consequently affect the physiological and biochemical processes. The Cd accumulation in the muscle was low compared to that in the liver and did not exceed the level at which the antioxidative and immune systems could cope with. Under such circumstances, the activities or contents of the antioxidative and immune biomarkers were induced so that the cells could be protected from oxidative damages.

    Overall, seawater acidification significantly affected the activities of CAT in the liver and gill and ACP in the muscle, but did not show significant effects on the other antioxidative or immune responses in any tissue of the juveniles. This could be possibly related to the fact that juveniles had developed relatively perfect mechanism of acid-base regulations. The impacts of excessive CO₂ and H⁺ in acidified seawater on the juveniles could be timely defended by the functioning of CA, buffer ions and transmembrane proteins of the fish. Nonetheless, seawater acidification increased the antioxidative and immune responses of the juveniles to Cd exposure.

    (5) IBR index was established and used to assess how antioxidative defense or immune system and biomineralization of the flounder larvae and juveniles responded to the intergrative stress of seawater acidification and Cd exposure. The related biomarkers of the larvae and juveniles obviously responded more sensitively to the intergrative stress than that by a single stressor. In other words, seawater acidification could increase the toxic effects of Cd exposure on the flounder larvae and juveniles. Furthermore, the larvae responded more sensitively to the intergrative stress than the juveniles. IBR could more reliably reflect the comprehensive biotoxic effects of muti-stressors on tested organisms than a single-biomarker assessment.