刺参（Apostichopus japonicus Selenka）对典型环境胁迫的生理生态学响应及其机制研究
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摘要
刺参（Apostichopus japonicus Selenka）属于棘皮动物门（Echinodermata）、海参纲（Holothroidea），是我国重要的海水养殖经济种类。近年来，由于其较高的经济价值，刺参养殖业在我国发展很快，目前已成为海水养殖的支柱产业之一。本论文以刺参为研究对象，设计了典型的环境胁迫因子，包括温度、氨氮、饥饿和夏眠、运输、排脏等，对比研究了胁迫种类、胁迫强度、胁迫作用方式等对刺参生理和生态学的不同影响。通过本研究的进行，不仅可以揭示环境胁迫对刺参影响的生理和生态学机制，丰富刺参生物学研究内容，还有助于找到消除或缓解环境胁迫对刺参负面影响的方法，从而为改进刺参养殖技术、促进刺参养殖业的健康发展提供必要的理论依据，因此具有重要的理论和实际意义。研究结果总结如下：
1.采用实验生态学的方法，研究了不同变温模式(变温回复、变温维持)、变温强度(16±4℃，±8℃，±12℃)及变温方向(升温、降温)对刺参(Apostichopusjaponicus Selenka)幼参(体重3.37-4.61g)热休克蛋白70(Hsp70)表达的影响。主要结果如下：(1)升温组在不同变温模式下刺参热休克蛋白70表达量在实验开始后均迅速升高，说明温度胁迫可以迅速诱导热休克蛋白70的表达(P <0.05)。(2)在升温回复实验中，温度可以诱导热休克蛋白70的迅速表达(P<0.05)，当放回正常温度时，热休克蛋白70水平逐渐下降，下降趋势一直保持到72h，其中R24组在第72h与对照组无显著差异(P>0.05)，R20组在第48h与对照组无显著差异(P>0.05)，这说明热应激后热休克蛋白70表达迅速但维持时间较短。(3)升温维持组刺参热休克蛋白70表达在实验开始后迅速升高，之后缓慢降低但始终维持在一个较高的水平上，说明持续的热应激可以诱导热休克蛋白70持续表达。(4)刺参热休克蛋白70的诱导强度不仅与变温持续时间有关，并且也与变温强度相关联。在本实验中，升温组变温幅度越大，刺参热休克蛋白70的表达越迅速，表达强度越高，维持时间越长。(5)本实验研究发现，升温组刺参热休克蛋白表达在应激开始后2h迅速升高，但是降温组刺参热休克蛋白表达在应激开始后4h内保持相对稳定，然后迅速升高至第8h达到最高。刺参热休克蛋白70的表达对不同变温方向的相应不同，相比降温波动，刺参对升温波动更加敏感。综上，更多的研究应当以此为基础，关注于更复杂的、贴近自然状态的热应激条件下刺参热休克蛋白表达的规律和分子机制。
2.以初始体重为(50.50±1.67)g左右的刺参(Apostichopus japonicus Selenka)为研究对象，研究了不同氨氮浓度对刺参体壁和体腔液中酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性及Hsp70表达的影响。20天慢性毒性实验研究结果表明：(1)刺参体壁和体腔液ACP、AKP活性在0.51和2.04mg/L氨氮浓度下表现出先上升后降低的趋势，而在5.09、10.19和50.93mg/L下，则表现出降低的趋势；(2)不同浓度氨氮胁迫下，刺参体壁CAT活性均呈现降低趋势，且与氨氮浓度呈现显著的负相关性(P<0.05)，而刺参体腔液CAT活性在0.51、5.09、10.19和50.93mg/L浓度下则均随取样时间而降低；(3)在氨氮浓度0.51~5.09mg/L时，刺参体壁和体腔液SOD活性均表现出不同程度的先增高后降低的趋势，而在高浓度下则与氮氮浓度显著负相关；(4)在氨氮胁迫下，刺参体壁、呼吸树、肠道和体腔液中Hsp70表达量迅速升高，氨氮胁迫第5天时检测到达到最高；随后各组织Hsp70的表达量缓慢降低，但在氨氮胁迫第20天时，各组织Hsp70的表达量仍显著高于氨氮胁迫前的水平(P<0.05)。各组织中Hsp70最高表达量与氨氮浓度呈现明显的正相关性。综合本研究结果可以看出，随着氨氮胁迫浓度的增加和胁迫时间的延长，刺参免疫酶活性趋于降低，同时机体清除自由基的能力下降，机体非特异性免疫防御系统遭到损伤。而Hsp70表达量维持在较高水平，对于增强机体的抗逆性具有重要的作用。
3.采用实验生态学方法，研究了刺参(Apostichopus japonicus Selenka)在夏眠、实验性夏眠、饥饿状态下生长、代谢及免疫应答的变化。通过测定刺参体重、耗氧率(OCR)、体壁和体腔液中免疫酶活性包括酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性以及热休克蛋白70(Hsp70)表达，以评估刺参在不同处理下的生理反应。主要结果如下：(1)在所有三个处理中刺参体重显著下降(P <0.05)。(2)饥饿组、实验性夏眠组刺参OCR下降，但自然夏眠组刺参COR逐渐升高。(3)三个处理组体壁和体腔液中ACP、AKP活性逐渐降低，饥饿组和实验性夏眠组刺参体壁和体腔液中SOD、CAT活性及Hsp70表达逐渐降低，但是自然夏眠组刺参体壁和体腔液中SOD、CAT活性及Hsp70表达逐渐升高。结果显示，刺参在饥饿条件下，体重、代谢速率及免疫应答由于停止摄食而降低。当水温保持在24℃时，刺参进入夏眠状态，但是实验性夏眠与自然夏眠状态在耗氧率、免疫应答及Hsp70表达方面不尽相同。这揭示了夏眠的机制是复杂的，不能仅仅归咎于环境因子的变化，比如温度。
4.采用实验生态学的方法，研究了带水运输法不同运输温度(10℃、15℃、20℃)、密度和运输时间(<24h)对刺参(Apostichopus japonicus Selenka)幼参行为、排脏及存活的影响，并在此基础上确定不同温度下刺参幼参运输的临界密度以研究不同温度、密度和运输时间对刺参机体生理指标的影响以及机体对运输过程中胁迫因子的响应机制。通过研究发现，运输作为一种多因子协同作用的急性胁迫过程，运输密度、运输温度及运输时间对刺参的行为、存活及生理指标均具有显著地影响。采用塑料袋加水充氧的方法以较高的密度运输刺参稚参是可行的。保持较低温度(10℃)可以提高刺参的运输密度(140头/2kg水)，并可以延长运输时间(24h)，但较低的温度、较高的密度及较长的运输时间会延长刺参休克后恢复的时间。因而，在10℃下保持140头/2kg水的密度运输24h是可行的，但我们建议刺参的运输采用更加保守的范围，降低胁迫强度以利于释放后更快的恢复。
5.于2009年5月17日到2009年6月30日，采用实验生态学的方法，研究了人工诱导排脏对刺参(Apostichopus japonicus Selenka)生长、代谢及免疫应答的影响。通过测定体重、特定生长率(SGR)、耗氧率(OCR)及体壁、肠道、呼吸树中免疫酶活力，包括酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)、微量丙二醛(MDA)以评估刺参排脏及内脏再生过程中的生理反应。结果显示，排脏后刺参体重显著下降然后随着消化功能的恢复逐渐上升，然而排脏45天后末体重始终极显著低于对照组(P <0.01)。实验第10天至第20天再生组SGR上升而在实验结束时与对照组无显著差异(P>0.05)。刺参排脏后OCR迅速降低随后逐渐升高并在第45天与对照组无显著差异(P>0.05)。刺参再生期间的免疫应答具有明显的组织特异性。排脏后体壁内ACP和AKP活力升高，于第10天达到峰值然后逐渐降低并于第45天降至正常水平(P>0.05)。20天后再生组织中ACP和AKP活力存在不同的上升趋势，在实验结束时呼吸树中ACP活力、肠道和呼吸树AKP活力恢复正常水平(P>0.05)，而肠道ACP活力仍然显著高于对照组(P <0.05)。体壁SOD活力受排脏影响不大而再生组织中SOD活力显著升高并于实验结束时极显著高于对照组(P<0.01)。体壁CAT活力升高并于20天达到峰值且在实验过程中始终显著高于对照组(P <0.05)，呼吸树中CAT活力持续升高并在第45天与对照组无显著差异(P>0.05)，而肠道中CAT活力受排脏影响不大(P>0.05)。排脏后三种组织中T-AOC和MDA均显著升高(P <0.05)，但在实验结束时均恢复正常水平(P>0.05)。本研究说明排脏对刺参生长、代谢以及免疫应答均具有显著的影响。尽管再生组体重与对照组具有显著差异，但SGR、OCR和主要非特异性免疫因子均在排脏后45天之内恢复正常水平，从生理学上说明刺参已从排脏中恢复。
1. Sea cucumber (Apostichopus japonicus Selenka) is an important marine culturespecies in northern China. Using experimental ecology methods, the influence ofdifferent temperature fluctuation modes and intensities and directions to heat shockprotein70(Hsp70) were studied. The results are as follows:(1). Hsp70contentsincreased rapidly initially under different temperature fluctuation modes intemperature increase treatment.(2). Hsp70contents decreased as temperature turnsback to normal level, the R24and R20group got no significant difference with thecontrol group(P>0.05) at72h and48h, respectively.(3) Hsp70contents increased\apidly and maintained at a high level in consecutive heating-up treatment, whichillustrate that Hsp70contents can be continuous expressed with consecutivetemperature stimulus.(4) The induction of Hsp70is in connection with not only thecontinuity of temperature changing but also the intensities of temperature fluctuation.The more the temperature changed, the quicker and the more Hsp70was induced.(5)In this experiment, Hsp70contents increased rapidly after2h in temperature-uptreatment while increased slowly in temperature-drop treatment and didn’t reach thepeak value until8h. Therefore we estimated that Hsp70was more sensitive to thetemperature-up treatment than to the temperature-drop treatment. In conclusion, moreresearch based on this experiment should be done to fully understand the laws ofexpress for heat shock protein under more complicated condition which is similarwith the natural environment.
2. The influence of chronic stress of ammonical nitrogen on the activities of immunoenzymes (acidic phosphatase (ACP), alkaline phosphatase (AKP), catalase(CAT) and superoxide dismutase (SOD) and heat shock protein70(Hsp70) contentwere investigated in Sea Cucumber, Apostichopus japonicus Selenka. Body wall andcoelomic fluid samples of sea cucumbers in5ammonical nitrogen concentration(ANC) groups (0.51、2.04、5.09、10.19and50.93mg/L) were collected anddetermined every5days till the20thday. ACP and AKP activities in both tissuesincreased at first and decreased afterwards in0.51and2.04mg/L groups whiledecreased in5.09、10.19and50.93mg/L groups. CAT activities in body walldecreased in all5groups and were reversely correlated with ANC, while those incoelomic fluid decreased in0.51、5.09、10.19and50.93mg/L groups. SOD activitiesin both tissues increased firstly and then decreased in0.51、2.04、5.09mg/L groupswhile those were reversely correlated with ANC in high concentrations. Hsp70content in body wall, intestine, respiratory tree and coelomic fluid rose rapidly underammonical nitrogen stress and peaked on the5thday. The Hsp70content were stillmarked higher than the control on the20thday (P<0.05). The maximum Hsp70content in4tissues weas positively correlated with ANC. All the results indicated theactivities of immunoenzymes tended to decrease and the non-specific immune systemof sea cucumbers were damaged as ANC increased and time went by. However, Hsp70concentration maintained high level, which was important to promote the stressresistance of sea cucumbers.
3. Growth, metabolism and physiological responses of sea cucumber, Apostichopusjaponicus Selenka, during periods of inactivity, were investigated in this study. Thebody weight, oxygen consumption rate (OCR), activities of immunoenzymesincluding acidic phosphatase (ACP), alkaline phosphatase (AKP), catalase (CAT) andsuperoxide dismutase (SOD), and Heat shock protein70(Hsp70) contents in bodywall and coelomic fluid were measured to evaluate the growth, metabolism andphysiological responses of A. japonicus during starvation, experimental aestivationand aestivation. The results showed that body weight of sea cucumbers in all threetreatments dropped significantly (P <0.05). OCR reduced in starvation, experimental aestivation treatments but increased gradually in aestivation group. Both ACP andAKP activities in all treatments decreased gradually, and SOD and CAT activities andHsp70contents in starvation and experimental aestivation treatments decreased,however, SOD and CAT activities and Hsp70contents increased in aestivationtreatment. Results of this study indicated that growth, metabolism and physiologicalresponses declined in A. japonicus due to the stress of fasting. The sea cucumber A.japonicus enters a state of aestivation when the temperature is maintained at24°C.However, the experimental aestivation is different from aestivation to some extent inmetabolism and physiological responses. This suggest that the mechanism ofaestivation in A. japonicus is complex and may not be attributed only toenvironmental changes, such as temperature, as shown in previous studies.
4. Sea cucumber (Apostichopus japonicus Selenka) is an important marine culturespecies in northern China. Using experimental ecology methods, the influence ofdifferent transportation densities and temperatures (10℃、15℃、20℃) and duration(<24h) were to behaviors and evisceration and survival of A. japonicus juveniles werestudied, and the critical densities under different temperatures were determined toinvestigate the influence of different transportation densities and temperatures (10℃、15℃、20℃) and duration to physiological parameters and response mechanism of A.japonicus juveniles. The results showed that as an acute environmental stress undermulti-factor synergy, transportation densities and temperatures and durationsignificantly influence the behaviors and survival and physiological parameters of A.japonicus. It’s feasible to transport A. japonicus juveniles using plastic sea water bagswith oxygen under high densities. Low temperature (10℃) is benefit to increasetransportation density (140juveniles per bag) and prolong duration (24h), however,shock duration will be prolonged and recovery will be delayed under this conditions.Our results showed that transportation at densities of140juveniles per bag (2L water,4L oxygen) for24h at10℃is feasible, but we recommend transportation for A.japonicus juveniles at more conservative densities and durations where possible tominimize transportation stress. 5. The growth, metabolism and immune responses of sea cucumber, Apostichopusjaponicus Selenka, following evisceration induced artificially, were investigated inthis study. The body weight, specific growth rate (SGR), oxygen consumption rate(OCR), activities of immunoenzymes including acidic phosphatase (ACP), alkalinephosphatase (AKP), catalase (CAT) and superoxide dismutase (SOD), totalantioxidant capacity (T-AOC) and malonyl-dialdehyde (MDA) in muscle, intestineand respiratory tree were measured to evaluate the physiological responses of A.japonicus to evisceration and the regeneration of viscera. The results showed that thebody weight of sea cucumber significantly dropped following evisceration and thenincreased gradually with digestive function resumed. Accelerated growth rates wereobserved in the regeneration group from the10thday to20thday, but there was nosignificant difference between the regeneration group and control group at the end ofthe experiment. As for OCR, it reduced rapidly after evisceration, then increasedgradually afterwards. Compared to the control, no significant difference in OCR wasfound on the45thday (P>0.05). The immune responses of A. japonicus were highlytissue-specific during the regeneration of the viscera. Both ACP and AKP activities inmuscle enhanced and peaked on the10thday, then decreased gradually to normal levelon the45thday (P>0.05). ACP and AKP activities in the regenerated tissuesexhibited a differential rising trend from the20thday after evisceration. SOD activityin muscle was not significantly influenced by evisceration with respected to thecontrol; however, those in the regenerated tissues significantly enhanced and weresignificantly higher than those in the control on the45thday (P <0.01). CAT activityin muscle of the regeneration group was significantly higher than that in the controlduring the whole period of the experiment (P <0.05). CAT activity in the regeneratedrespiration tree enhanced invariably after evisceration, while that in the intestine werenot significantly affected by evisceration (P>0.05). Significant increase was found inboth T-AOC and MDA in all three tissues after evisceration (P <0.05), however, theyall returned to normal at the end of the experiment. The results of this study indicatedthat the growth, metabolism and immune responses in sea cucumber weresignificantly influenced by evisceration. Although the regeneration group did not catch up with the control in body weight, SGR, OCR and major non-specific immuneparameters have resumed to normal level within45days after evisceration, whichmight mean that A. japonicus has recovered in physiology from evisceration.
1.采用实验生态学的方法，研究了不同变温模式(变温回复、变温维持)、变温强度(16±4℃，±8℃，±12℃)及变温方向(升温、降温)对刺参(Apostichopusjaponicus Selenka)幼参(体重3.37-4.61g)热休克蛋白70(Hsp70)表达的影响。主要结果如下：(1)升温组在不同变温模式下刺参热休克蛋白70表达量在实验开始后均迅速升高，说明温度胁迫可以迅速诱导热休克蛋白70的表达(P <0.05)。(2)在升温回复实验中，温度可以诱导热休克蛋白70的迅速表达(P<0.05)，当放回正常温度时，热休克蛋白70水平逐渐下降，下降趋势一直保持到72h，其中R24组在第72h与对照组无显著差异(P>0.05)，R20组在第48h与对照组无显著差异(P>0.05)，这说明热应激后热休克蛋白70表达迅速但维持时间较短。(3)升温维持组刺参热休克蛋白70表达在实验开始后迅速升高，之后缓慢降低但始终维持在一个较高的水平上，说明持续的热应激可以诱导热休克蛋白70持续表达。(4)刺参热休克蛋白70的诱导强度不仅与变温持续时间有关，并且也与变温强度相关联。在本实验中，升温组变温幅度越大，刺参热休克蛋白70的表达越迅速，表达强度越高，维持时间越长。(5)本实验研究发现，升温组刺参热休克蛋白表达在应激开始后2h迅速升高，但是降温组刺参热休克蛋白表达在应激开始后4h内保持相对稳定，然后迅速升高至第8h达到最高。刺参热休克蛋白70的表达对不同变温方向的相应不同，相比降温波动，刺参对升温波动更加敏感。综上，更多的研究应当以此为基础，关注于更复杂的、贴近自然状态的热应激条件下刺参热休克蛋白表达的规律和分子机制。
2.以初始体重为(50.50±1.67)g左右的刺参(Apostichopus japonicus Selenka)为研究对象，研究了不同氨氮浓度对刺参体壁和体腔液中酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性及Hsp70表达的影响。20天慢性毒性实验研究结果表明：(1)刺参体壁和体腔液ACP、AKP活性在0.51和2.04mg/L氨氮浓度下表现出先上升后降低的趋势，而在5.09、10.19和50.93mg/L下，则表现出降低的趋势；(2)不同浓度氨氮胁迫下，刺参体壁CAT活性均呈现降低趋势，且与氨氮浓度呈现显著的负相关性(P<0.05)，而刺参体腔液CAT活性在0.51、5.09、10.19和50.93mg/L浓度下则均随取样时间而降低；(3)在氨氮浓度0.51~5.09mg/L时，刺参体壁和体腔液SOD活性均表现出不同程度的先增高后降低的趋势，而在高浓度下则与氮氮浓度显著负相关；(4)在氨氮胁迫下，刺参体壁、呼吸树、肠道和体腔液中Hsp70表达量迅速升高，氨氮胁迫第5天时检测到达到最高；随后各组织Hsp70的表达量缓慢降低，但在氨氮胁迫第20天时，各组织Hsp70的表达量仍显著高于氨氮胁迫前的水平(P<0.05)。各组织中Hsp70最高表达量与氨氮浓度呈现明显的正相关性。综合本研究结果可以看出，随着氨氮胁迫浓度的增加和胁迫时间的延长，刺参免疫酶活性趋于降低，同时机体清除自由基的能力下降，机体非特异性免疫防御系统遭到损伤。而Hsp70表达量维持在较高水平，对于增强机体的抗逆性具有重要的作用。
3.采用实验生态学方法，研究了刺参(Apostichopus japonicus Selenka)在夏眠、实验性夏眠、饥饿状态下生长、代谢及免疫应答的变化。通过测定刺参体重、耗氧率(OCR)、体壁和体腔液中免疫酶活性包括酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性以及热休克蛋白70(Hsp70)表达，以评估刺参在不同处理下的生理反应。主要结果如下：(1)在所有三个处理中刺参体重显著下降(P <0.05)。(2)饥饿组、实验性夏眠组刺参OCR下降，但自然夏眠组刺参COR逐渐升高。(3)三个处理组体壁和体腔液中ACP、AKP活性逐渐降低，饥饿组和实验性夏眠组刺参体壁和体腔液中SOD、CAT活性及Hsp70表达逐渐降低，但是自然夏眠组刺参体壁和体腔液中SOD、CAT活性及Hsp70表达逐渐升高。结果显示，刺参在饥饿条件下，体重、代谢速率及免疫应答由于停止摄食而降低。当水温保持在24℃时，刺参进入夏眠状态，但是实验性夏眠与自然夏眠状态在耗氧率、免疫应答及Hsp70表达方面不尽相同。这揭示了夏眠的机制是复杂的，不能仅仅归咎于环境因子的变化，比如温度。
4.采用实验生态学的方法，研究了带水运输法不同运输温度(10℃、15℃、20℃)、密度和运输时间(<24h)对刺参(Apostichopus japonicus Selenka)幼参行为、排脏及存活的影响，并在此基础上确定不同温度下刺参幼参运输的临界密度以研究不同温度、密度和运输时间对刺参机体生理指标的影响以及机体对运输过程中胁迫因子的响应机制。通过研究发现，运输作为一种多因子协同作用的急性胁迫过程，运输密度、运输温度及运输时间对刺参的行为、存活及生理指标均具有显著地影响。采用塑料袋加水充氧的方法以较高的密度运输刺参稚参是可行的。保持较低温度(10℃)可以提高刺参的运输密度(140头/2kg水)，并可以延长运输时间(24h)，但较低的温度、较高的密度及较长的运输时间会延长刺参休克后恢复的时间。因而，在10℃下保持140头/2kg水的密度运输24h是可行的，但我们建议刺参的运输采用更加保守的范围，降低胁迫强度以利于释放后更快的恢复。
5.于2009年5月17日到2009年6月30日，采用实验生态学的方法，研究了人工诱导排脏对刺参(Apostichopus japonicus Selenka)生长、代谢及免疫应答的影响。通过测定体重、特定生长率(SGR)、耗氧率(OCR)及体壁、肠道、呼吸树中免疫酶活力，包括酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)、微量丙二醛(MDA)以评估刺参排脏及内脏再生过程中的生理反应。结果显示，排脏后刺参体重显著下降然后随着消化功能的恢复逐渐上升，然而排脏45天后末体重始终极显著低于对照组(P <0.01)。实验第10天至第20天再生组SGR上升而在实验结束时与对照组无显著差异(P>0.05)。刺参排脏后OCR迅速降低随后逐渐升高并在第45天与对照组无显著差异(P>0.05)。刺参再生期间的免疫应答具有明显的组织特异性。排脏后体壁内ACP和AKP活力升高，于第10天达到峰值然后逐渐降低并于第45天降至正常水平(P>0.05)。20天后再生组织中ACP和AKP活力存在不同的上升趋势，在实验结束时呼吸树中ACP活力、肠道和呼吸树AKP活力恢复正常水平(P>0.05)，而肠道ACP活力仍然显著高于对照组(P <0.05)。体壁SOD活力受排脏影响不大而再生组织中SOD活力显著升高并于实验结束时极显著高于对照组(P<0.01)。体壁CAT活力升高并于20天达到峰值且在实验过程中始终显著高于对照组(P <0.05)，呼吸树中CAT活力持续升高并在第45天与对照组无显著差异(P>0.05)，而肠道中CAT活力受排脏影响不大(P>0.05)。排脏后三种组织中T-AOC和MDA均显著升高(P <0.05)，但在实验结束时均恢复正常水平(P>0.05)。本研究说明排脏对刺参生长、代谢以及免疫应答均具有显著的影响。尽管再生组体重与对照组具有显著差异，但SGR、OCR和主要非特异性免疫因子均在排脏后45天之内恢复正常水平，从生理学上说明刺参已从排脏中恢复。
1. Sea cucumber (Apostichopus japonicus Selenka) is an important marine culturespecies in northern China. Using experimental ecology methods, the influence ofdifferent temperature fluctuation modes and intensities and directions to heat shockprotein70(Hsp70) were studied. The results are as follows:(1). Hsp70contentsincreased rapidly initially under different temperature fluctuation modes intemperature increase treatment.(2). Hsp70contents decreased as temperature turnsback to normal level, the R24and R20group got no significant difference with thecontrol group(P>0.05) at72h and48h, respectively.(3) Hsp70contents increased\apidly and maintained at a high level in consecutive heating-up treatment, whichillustrate that Hsp70contents can be continuous expressed with consecutivetemperature stimulus.(4) The induction of Hsp70is in connection with not only thecontinuity of temperature changing but also the intensities of temperature fluctuation.The more the temperature changed, the quicker and the more Hsp70was induced.(5)In this experiment, Hsp70contents increased rapidly after2h in temperature-uptreatment while increased slowly in temperature-drop treatment and didn’t reach thepeak value until8h. Therefore we estimated that Hsp70was more sensitive to thetemperature-up treatment than to the temperature-drop treatment. In conclusion, moreresearch based on this experiment should be done to fully understand the laws ofexpress for heat shock protein under more complicated condition which is similarwith the natural environment.
2. The influence of chronic stress of ammonical nitrogen on the activities of immunoenzymes (acidic phosphatase (ACP), alkaline phosphatase (AKP), catalase(CAT) and superoxide dismutase (SOD) and heat shock protein70(Hsp70) contentwere investigated in Sea Cucumber, Apostichopus japonicus Selenka. Body wall andcoelomic fluid samples of sea cucumbers in5ammonical nitrogen concentration(ANC) groups (0.51、2.04、5.09、10.19and50.93mg/L) were collected anddetermined every5days till the20thday. ACP and AKP activities in both tissuesincreased at first and decreased afterwards in0.51and2.04mg/L groups whiledecreased in5.09、10.19and50.93mg/L groups. CAT activities in body walldecreased in all5groups and were reversely correlated with ANC, while those incoelomic fluid decreased in0.51、5.09、10.19and50.93mg/L groups. SOD activitiesin both tissues increased firstly and then decreased in0.51、2.04、5.09mg/L groupswhile those were reversely correlated with ANC in high concentrations. Hsp70content in body wall, intestine, respiratory tree and coelomic fluid rose rapidly underammonical nitrogen stress and peaked on the5thday. The Hsp70content were stillmarked higher than the control on the20thday (P<0.05). The maximum Hsp70content in4tissues weas positively correlated with ANC. All the results indicated theactivities of immunoenzymes tended to decrease and the non-specific immune systemof sea cucumbers were damaged as ANC increased and time went by. However, Hsp70concentration maintained high level, which was important to promote the stressresistance of sea cucumbers.
3. Growth, metabolism and physiological responses of sea cucumber, Apostichopusjaponicus Selenka, during periods of inactivity, were investigated in this study. Thebody weight, oxygen consumption rate (OCR), activities of immunoenzymesincluding acidic phosphatase (ACP), alkaline phosphatase (AKP), catalase (CAT) andsuperoxide dismutase (SOD), and Heat shock protein70(Hsp70) contents in bodywall and coelomic fluid were measured to evaluate the growth, metabolism andphysiological responses of A. japonicus during starvation, experimental aestivationand aestivation. The results showed that body weight of sea cucumbers in all threetreatments dropped significantly (P <0.05). OCR reduced in starvation, experimental aestivation treatments but increased gradually in aestivation group. Both ACP andAKP activities in all treatments decreased gradually, and SOD and CAT activities andHsp70contents in starvation and experimental aestivation treatments decreased,however, SOD and CAT activities and Hsp70contents increased in aestivationtreatment. Results of this study indicated that growth, metabolism and physiologicalresponses declined in A. japonicus due to the stress of fasting. The sea cucumber A.japonicus enters a state of aestivation when the temperature is maintained at24°C.However, the experimental aestivation is different from aestivation to some extent inmetabolism and physiological responses. This suggest that the mechanism ofaestivation in A. japonicus is complex and may not be attributed only toenvironmental changes, such as temperature, as shown in previous studies.
4. Sea cucumber (Apostichopus japonicus Selenka) is an important marine culturespecies in northern China. Using experimental ecology methods, the influence ofdifferent transportation densities and temperatures (10℃、15℃、20℃) and duration(<24h) were to behaviors and evisceration and survival of A. japonicus juveniles werestudied, and the critical densities under different temperatures were determined toinvestigate the influence of different transportation densities and temperatures (10℃、15℃、20℃) and duration to physiological parameters and response mechanism of A.japonicus juveniles. The results showed that as an acute environmental stress undermulti-factor synergy, transportation densities and temperatures and durationsignificantly influence the behaviors and survival and physiological parameters of A.japonicus. It’s feasible to transport A. japonicus juveniles using plastic sea water bagswith oxygen under high densities. Low temperature (10℃) is benefit to increasetransportation density (140juveniles per bag) and prolong duration (24h), however,shock duration will be prolonged and recovery will be delayed under this conditions.Our results showed that transportation at densities of140juveniles per bag (2L water,4L oxygen) for24h at10℃is feasible, but we recommend transportation for A.japonicus juveniles at more conservative densities and durations where possible tominimize transportation stress. 5. The growth, metabolism and immune responses of sea cucumber, Apostichopusjaponicus Selenka, following evisceration induced artificially, were investigated inthis study. The body weight, specific growth rate (SGR), oxygen consumption rate(OCR), activities of immunoenzymes including acidic phosphatase (ACP), alkalinephosphatase (AKP), catalase (CAT) and superoxide dismutase (SOD), totalantioxidant capacity (T-AOC) and malonyl-dialdehyde (MDA) in muscle, intestineand respiratory tree were measured to evaluate the physiological responses of A.japonicus to evisceration and the regeneration of viscera. The results showed that thebody weight of sea cucumber significantly dropped following evisceration and thenincreased gradually with digestive function resumed. Accelerated growth rates wereobserved in the regeneration group from the10thday to20thday, but there was nosignificant difference between the regeneration group and control group at the end ofthe experiment. As for OCR, it reduced rapidly after evisceration, then increasedgradually afterwards. Compared to the control, no significant difference in OCR wasfound on the45thday (P>0.05). The immune responses of A. japonicus were highlytissue-specific during the regeneration of the viscera. Both ACP and AKP activities inmuscle enhanced and peaked on the10thday, then decreased gradually to normal levelon the45thday (P>0.05). ACP and AKP activities in the regenerated tissuesexhibited a differential rising trend from the20thday after evisceration. SOD activityin muscle was not significantly influenced by evisceration with respected to thecontrol; however, those in the regenerated tissues significantly enhanced and weresignificantly higher than those in the control on the45thday (P <0.01). CAT activityin muscle of the regeneration group was significantly higher than that in the controlduring the whole period of the experiment (P <0.05). CAT activity in the regeneratedrespiration tree enhanced invariably after evisceration, while that in the intestine werenot significantly affected by evisceration (P>0.05). Significant increase was found inboth T-AOC and MDA in all three tissues after evisceration (P <0.05), however, theyall returned to normal at the end of the experiment. The results of this study indicatedthat the growth, metabolism and immune responses in sea cucumber weresignificantly influenced by evisceration. Although the regeneration group did not catch up with the control in body weight, SGR, OCR and major non-specific immuneparameters have resumed to normal level within45days after evisceration, whichmight mean that A. japonicus has recovered in physiology from evisceration.
引文
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