海洋生态与环境科学重点实验室知识库

    随着人类活动的不断增强以及富营养化在全球沿岸海域的愈演愈烈，海洋低氧区的分布范围和严重程度都呈现出快速上升的态势。在全球变化的大背景下，海洋低氧与赤潮、酸化等其他海洋灾害的关系愈发紧密，构成了海洋变化的重要方面。尽管一些游泳能力较强的鱼类可以通过运动逃离低氧区，但移动能力较差的底栖生物却经常在严重低氧事件中发生大规模死亡，从而使现有海洋生态系统的面貌发生极大改变，引发诸如水母暴发等严重的海洋生态灾害。而在养殖区，由高密度养殖和富营养化加剧等因素导致的低氧现象使养殖生物面临的生存风险日益增加，从而给渔业经济的发展带来了潜在的威胁。尽管关于海洋生物对低氧耐受性的研究自上世纪六十年代就已经展开，但由于实验方法各异、实验条件不尽相同，我们难以对不同海洋生物的耐受性进行准确的比较、难以建立海洋生物耐受性与溶解氧的准确关系。此外，关于海洋生物在低氧压力下的响应，仍有某些行为学现象、关键呼吸酶活变化以及细胞损伤等重要问题亟待解决。

    菲律宾蛤仔作为一种广温性与广盐性的重要经济贝类，广泛分布于西太平洋与欧洲温带陆架区等众多沿岸海区。由于生长速度快、繁殖能力强，其在很多海域建立了优势地位，并构成了食物网与生物地化循环中的关键一环。栉孔扇贝，一种广泛分布于我国北方海域营附着生活的底栖贝类，其养殖规模在过去几十年呈现了快速的增长，并与菲律宾蛤仔一起构成了我国主要的养殖贝类之一。但是，沿岸与河口区低氧的不断恶化对菲律宾蛤仔和栉孔扇贝的生存以及养殖业的安全提出了日益严峻的挑战。在我国，作为菲律宾蛤仔与栉孔扇贝的重要栖息地与养殖区域，山东半岛的夏季低氧事件愈发严重——莱州湾和乳山湾夏季溶氧的最低值均已降低到2.0 mg/L以下；小清河口溶氧的最低值则只有0.5 mg/L；胶州湾扇贝养殖区的溶氧一度降低到2.0 mg/L；桑沟湾富营养化和低氧的风险也由于养殖活动的增加而显著上升。在日本，由低氧导致的菲律宾蛤仔大规模死亡事件时有发生，给水产养殖业造成了重创。然而，在此背景下，关于菲律宾蛤仔和栉孔扇贝对低氧的耐受性研究却仍然非常缺乏。

    本研究中，我们设计了一种精准度与稳定性较高的新型海洋低氧环境模拟系统，利用一套标准和统一的实验方法，考察了菲律宾蛤仔和栉孔扇贝的存活率以及行为学、生理学和关键呼吸酶活的响应。此外，还调查了菲律宾蛤仔在长期低氧环境下的细胞损伤。

    由结果可知，菲律宾蛤仔对低氧具有强大的耐受力——20天半致死浓度为0.57 mg/L，在0.5 mg/L的极端低氧环境下半致死时间为422h，且在实验最初的7天内得以全部存活。相比而言，栉孔扇贝对低氧更加敏感——20天半致死浓度为1.8 mg/L，1.5 mg/L溶氧下的半致死时间为432h，且当溶氧降低时，其存活率会立即下降。此外，菲律宾蛤仔在低氧环境下的存活率受底质影响较大，而栉孔扇贝在二次低氧事件中的耐受力会出现显著的下降。

    在行为学响应上，菲律宾蛤仔主要通过由底内向底表迁移来获取更多的溶解氧，而栉孔扇贝则表现出了对低氧可能的规避反应。

    在生理学响应上，菲律宾蛤仔首先通过对呼吸活动的调节来保证氧气的供应。此后，通过对呼吸活动进行抑制来降低新陈代谢速率。与此同时，菲律宾蛤仔在低氧环境下倾向于以碳水化合物和脂肪取代蛋白质作为供能物质。与菲律宾蛤仔不同的是，栉孔扇贝的耗氧率随溶氧的下降而立即下降，但在2.0-3.0 mg/L的溶氧区间内能基本维持稳定。此外，栉孔扇贝的心率在1.5 mg/L的低氧环境下会出现显著的抑制现象。

    在关键呼吸酶活的响应上，菲律宾蛤仔在0.5 mg/L的极端低氧环境下通过提高磷酸果糖激酶和丙酮酸激酶的活性来加快糖酵解速率，进而满足氧供应不足时闭壳肌较高的能量需求。而栉孔扇贝则主要通过激活磷酸烯醇式丙酮酸羧化酶和延胡索酸还原酶来实现糖酵解过程的异化，从而为机体提供更多的ATP。但相比于菲律宾蛤仔，栉孔扇贝在低氧环境下乳酸脱氢酶的活性普遍较高，这可能导致了其细胞内乳酸的过量积累，并进一步引发了内稳态的打破和最终的死亡。在低氧压力下，菲律宾蛤仔的细胞出现了明显的损伤特征——线粒体嵴崩塌、线粒体和细胞空泡化以及肌丝溶解等，而这将导致菲律宾蛤仔在面对其它环境压力时抗性下降。

With the intensified human activities and the worsen eutrophication in coastal regions worldwide, both the coverage and the strength of hypoxia have risen sharply. In the background of global change, the relationships between hypoxia and other marine ecological disasters such as algal bloom and ocean acidification have become closer. Though some fishes with strong mobility can flee the hypoxic zones, most marine benthos with poor mobility often suffer mass mortalities, and as a result, the current marine ecosystems would change greatly with some serious ecological disasters such as jellyfish bloom frequently breaking out. In the culture area, the hypoxia caused by high-density mariculture and eutrophication challenge the survival of the cultured animals, bring potential threat to the industry. The studies on the tolerance of marine organisms to hypoxia have begun since 1960s. However, due to the difference in experimental method and condition, it is hard for us to compare the tolerance of different marine organisms precisely. It is also difficult to establish an accurate relationship between the tolerance of these annimals and the dissolved oxygen. Additionally, for the research on the responses of marine organism to hypoxia, some key parts are still missing. For example, some behavioral phenomenon, the key respiratory enzyme activity changes and the cellular damage remain unknown.

The commercially important Manila clam Ruditapes philippinarum has strong adaptability to a wide range of salinities and water temperatures; the species has a widespread natural distribution in coastal areas of the western Pacific and has become established on the Atlantic coast of Europe. As a common and dominant species in many coastal regions, it functions as a key part of food webs and the biogeochemical cycle in these marine and brackish ecosystems. Zhikong scallop, a sessile and filter-feeding benthic animal, is widely distributed along the coast of northern China. Both the culture area and the density of Zhikong scallop have soared in the past 30 years. In particular, Zhikong scallop and Manila clam constitute the major cultured shellfish in China. However, worsening hypoxia in shallow coastal waters and estuaries increasingly challenges the survival of Manila clam and Zhikong scallop as well as the mariculture industry development. In China, the severity of summer hypoxia has been rising in coastal areas of Shandong Peninsula — an important natural habitat and mariculture region for Manila clam. Dissolved oxygen (DO) in Laizhou Bay and Rushan Bay dropped below 2.0 mg/L, and the situation was worse in the Xiaoqing River estuary, with DO concentrations of less than 0.5 mg/L being recorded. In Jiaozhou Bay, the culture of Zhikong scallops and bay scallops (Argopecten irradians) resulted in a decrease of DO levels to around 2.0 mg/L. In Sanggou Bay, another important culture area for Zhikong scallops, the risk of eutrophication and hypoxia have risen sharply due to aquaculture activities. In Japan, mass mortalities of Manila clam caused by hypoxia have occurred many times during summer, bringing tremendous economic loss to the fishery. However, studies on the effects of hypoxia on both mollusks are still limited.

In the present study, we designed a novel hypoxia simulation device which featured high accuracy and good stability. We established a standard and uniform method to investigate the effects of hypoxia on survival, behavioral, physiological and key enzyme activities involved in respiration. Besides, we observed the cellular damage of Manila clam caused by hypoxia.

As the results showed, Manila clam is tolerant to hypoxia as its 20-day LC₅₀ for DO was 0.57 mg/L and its LT₅₀ at 0.5 mg/L DO was 422 hours. All the clams survived the hypoxic challenge after 7 days of the experiment. In contrast, Zhikong scallop is sensitive to hypoxia as its 20-day LC₅₀ was estimated to be 1.8 mg/L and its LT₅₀ at 1.5 mg/L DO was 432 hours. When DO started to drop, the survival rate of Zhikong scallop decreased immediately. Additionally, the survival of Manila clam was significantly affected by the sediment type, and the survival rate of Zhikong scallop would reduce largely in a second hypoxia event.

For behavioral response, Manila clam mainly emerged from the sediment to get more oxygen, while Zhikong scallop showed possible escape responses.

For physiological response, Manila clam would firstly ensure the oxygen supply and then depress its respiration to reduce its metabolic rate; Manila clam preferred the use of carbohydrate and fats instead of protein when oxygen is insufficient. Unlike Manila clam, Zhikong scallop would first depress its respiration and then keep it stable when DO dropped to 2.0-3.0 mg/L. At the DO concentration of 1.5 mg/L, Zhikong scallop would depress its heart rate (HR) at the same time.

For key respiratory enzyme activity response, Manila clam exhibited significantly high activities of phosphofructokinase (PFK) and pyruvate kinase (PK) under hypoxia, which indicate the accelerated glycolysis. For Zhikong scallop, the phosphoenolpyruvate carboxylase (PEPC) and fumaric reductase (FR) were significantly activated, suggesting the dismutation of glycolysis. However, the activated lactate dehydrogenase (LDH) may be responsible for the accumulation of excessive lactate and the final death of Zhikong scallop. Under hypoxic stress, the cellular damage of Manila clam was obvious with collapsed cristae, shriveled membranes and induced cell inclusion, which would possibly reduce their resistance to other environmental stressors.

第一章 引言.... 1

1.1 海洋低氧的发展态势... 1

1.2 海洋低氧的生态后果... 3

1.3 海洋低氧与海洋生物对低氧耐受性的研究现状... 4

1.4 本研究的目的与意义... 8

第二章 一种海洋低氧环境模拟系统.... 11

2.1 前言... 11

2.2 系统的构造... 13

2.3 系统的使用与运行... 17

2.4 系统的性能... 18

2.5 讨论... 21

2.6 小结... 23

第三章 菲律宾蛤仔与栉孔扇贝在低氧环境下的存活.... 25

3.1 前言... 25

3.2 材料与方法... 27

3.2.1 实验动物与底质... 27

3.2.2 菲律宾蛤的存活实验... 29

3.2.3 栉孔扇贝的存活实验... 33

3.2.4 统计分析... 34

3.3 结果... 34

3.3.1 菲律宾蛤仔的存活... 34

3.3.2 栉孔扇贝的存活... 39

3.4 讨论... 41

3.4.1 菲律宾蛤仔对低氧的耐受... 41

3.4.2 栉孔扇贝对低氧的耐受... 43

3.5 小结... 46

第四章 菲律宾蛤仔与栉孔扇贝在低氧环境下的行为学响应.... 47

4.1 前言... 47

4.2 材料与方法... 49

4.2.1 菲律宾蛤仔的行为学响应实验... 49

4.2.2 栉孔扇贝的行为学响应实验... 50

4.2.3 统计分析... 51

4.3 结果... 52

4.4 讨论... 59

4.4.1 菲律宾蛤仔的行为学响应... 59

4.4.2 栉孔扇贝的行为学响应... 60

4.5 小结... 62

第五章 菲律宾蛤仔与栉孔扇贝在低氧环境下的生理学响应.... 63

5.1 前言... 63

5.2 材料与方法... 64

5.2.1 菲律宾蛤仔耗氧率、排氨率以及氧氮比的响应实验... 64

5.2.2 栉孔扇贝耗氧率、排氨率、氧氮比以及心率（HR）的响应实验... 65

5.2.3 栉孔扇贝耗氧率的连续观测实验... 66

5.2.4 统计分析... 67

5.3 结果... 68

5.3.1 菲律宾蛤仔的耗氧率、排氨率以及氧氮比... 68

5.3.2 栉孔扇贝的耗氧率、排氨率、氧氮比以及心率... 70

5.3.3 栉孔扇贝耗氧率的连续变化... 72

5.4 讨论... 76

5.4.1 菲律宾蛤仔的生理学响应... 76

5.4.2 栉孔扇贝的生理学响应... 77

5.5 小结... 79

第六章 菲律宾蛤仔与栉孔扇贝在低氧环境下的关键呼吸酶活响应与细胞损伤响应 81

6.1 前言... 81

6.2 材料与方法... 82

6.2.1 菲律宾蛤仔关键呼吸酶活的测量... 82

6.2.2 菲律宾蛤仔细胞变化的观察... 83

6.2.3 栉孔扇贝关键呼吸酶活的测量... 84

6.2.4 统计分析... 84

6.3 结果... 84

6.3.1 菲律宾蛤仔的关键呼吸酶活响应... 84

6.3.2 菲律宾蛤仔的细胞损伤... 87

6.3.3 栉孔扇贝的关键呼吸酶活响应... 89

6.4 讨论... 91

6.4.1 菲律宾蛤仔与栉孔扇贝关键呼吸酶活的响应... 91

6.4.2 菲律宾蛤仔的细胞损伤... 94

6.5 小结... 95

第七章 结论与展望.... 97

7.1 主要结论... 97

7.2 创新点... 99

7.3 展望... 99

参考文献.... 101

致 谢.... 111

作者简历及攻读学位期间发表的学术论文与研究成果.... 113