双壳贝类幼虫变态诱导及其机理研究

IOCAS-IR > 海洋环流与波动重点实验室

	双壳贝类幼虫变态诱导及其机理研究
	张涛
学位类型	博士
	2000
学位授予单位	中国科学院海洋研究所
学位授予地点	中国科学院海洋研究所
学位专业	海洋生物学
关键词	双壳贝类变态诱导机理
摘要	变态过程是双壳贝类由幼虫向成体转变的一个必不缺少的发育阶段。研究双壳贝类幼虫的变态过程及其机理，对于阐明它们的种群数量变动，促进重要经济双壳贝类增养殖的发展有重要的理论和实践意义。本论文除了用化学物质对几种双壳贝类(海湾扇贝、墨西哥湾扇贝和硬壳蛤)幼虫的变态进行诱导外，主要以激素和神经递质的作用方式为基础，通过直接测定双壳贝类(以海湾扇贝为代表)幼虫体内激素和神经递质、第二信使cAMP等生化物质含量的变化来研究双壳贝类幼虫变态过程中的信息传递途径，从分子生物学和神经生物学角度阐明双壳贝类幼虫变态机理。主要结果如下：1．通过参考国内外大量文献的基础上，较为系统地评述了近二十年来海洋无脊椎动物幼虫附着变态研究的一些进展情况，主要包括诱导因子、附着变态机理模型、人工诱导物的应用和延迟变态四个方面。到目前为止，人们已经发现了许多海洋无脊椎动物幼虫附着变态的诱导物质，主要分为天然诱导物和人工诱导物两大类，一些人工诱导物如GABA、肾上腺素和去甲肾上腺素已经在经济贝类苗种生产中得到应用。幼虫附着变态机理模型主要有长牡蛎(Crassostrea gigas)幼虫附着变态的双调控模型、红鲍(Haliotis rufescens)幼虫附着变态的上行调节模型以及多毛类Phragmatopoma california幼虫附着变态的脂肪酸调控模型。本论文还评述了海洋无脊椎动物幼虫发生延迟变态的原因以及延迟变态对海洋无脊椎动物造成的影响，并提出了解决的方法和今后研究的重点问题。2．在室内用氯化乙酰胆碱、ATP和CaCl_2 3种化学物质对海湾扇贝幼虫的变态进行了诱导实验。结果表明，虽然在个别浓度和处理时间氯化乙酰胆碱和ATP有诱导作用，但总体诱导效果不显著。而10×10~(-3)～40×10~(-3M的CaCl_2在处理12～24h后诱导效果较显著，其诱导效果对处理时间的依赖性较显著，在浓度为40×10~(-3)M和处理时间为24h时诱导效果最好，与对照组相比，变态率提高23.18％。3种诱导物对幼虫死亡率均有显著影响，并且死亡率对浓度和处理时间均有显著的依赖性，浓度越高，处理时间越长，死亡率越高。3．用KCl、肾上腺素、去甲肾上腺素和氯化胆碱进行了墨西哥湾扇贝(Argopectenirradians concentricus Say)幼虫变态的诱导作用实验。结果表明，KCl、肾上腺素、去甲肾上腺素和氯化胆碱对墨西哥湾扇贝幼虫变态均有显著诱导作用。KCl在处理时间为12h～48h范围内均有诱导作用；13.42×10~(-3)M和20.13×10~(-3)M的KCl诱导效果较好，变态率平均提高10％以上。1．O×10~(-6)M～50×10~(-6)M的肾上腺素在处理时间为lh～12h较适宜，此时变态率均提高10％以上。1.0×10~(-6)M～50×10~(-6)M的去甲肾上腺素在处理时间为1h～24h都较适宜，变态率平均均提高10％以上，最高可提高31．07％。0.01×10~(-4)M～1.O×10~(-4)M的氯化胆碱在处理时间为12h～48h时诱导效果均较好，它们之间的平均变态提高率并没有显著差别，均在12％～13％之间。10×10~(-4)M的氯化胆碱在处理时间为12h时诱导效果较明显，变态率可以提高19.14％，超过12h，变态率明显下降，100×10~(-4)M的氯化胆碱明显产生毒害作用，幼虫变态率均为零，而幼虫的死亡率均为100％。4．用KCl、肾上腺素、去甲肾上腺素、L-DOPA、5-羟色胺(5-hydroxytryptamine，Serotonin，5-HT)和GABA(γ-氨基丁酸)进行了不同浓度不同处理时间对硬壳蛤(Mercenaria mercenaria L.)幼虫变态诱导实验。结果表明，KCl、肾上腺素、去甲肾上腺素、L-DOPA和5-羟色胺对硬壳蛤幼虫的变态均有诱导作用，而GABA的诱导作用不显著。KCl的最佳诱导浓度随处理时间不同而有所不同。当处理时间为1～24h时，KCl的最佳诱导浓度为33.56×10~(-3)M，此时幼虫变态率均提高24％以上，当处理时间为48h时，KCl的最佳诱导浓度为20.13～26.85×10~(-3)M，处理时间为72h时，最佳诱导浓度为13.42×10~(-3)M。肾上腺素和去甲肾上腺素的诱导作用与浓度和处理时间均有关。肾上腺素的最佳处理浓度为100×10~(-6)M，最佳处理时间均为8h，此时幼虫变态率提高最大，为36.97％。当去甲肾上腺素的诱导浓度为100×10~(-6)M，处理时间为8h～16h时，幼虫变态提高率较高，均大于18％，死亡提高率均低于30％，当去甲肾上腺索诱导浓度为500×10~(-6)M时，虽然在8h～16h的处理时间范围内，幼虫变态提高率也较高，均大于18％，但当处理时间超过8h，在16～48h范围内，幼虫死亡提高率明显升高，均大于50％。L-DOPA的适宜诱导浓度为10×10~(-6)M～50×10~(-6)M，适宜处理时间为8～24h，此时幼虫变态率均提高30％以上，最高可提高79.43％。5-羟色胺的诱导作用较强，其适宜诱导浓度为100×10~(-6)M—1000×10~(-6)M，适宜处理时间为0.5～24h，此时幼虫变态率提高均在30％以上，当处理时间为8h时，最佳诱导浓度为1000×10~(-6)M，此时幼虫变态率提高57.5％，当处理时间为24h时，最佳诱导浓度为100×10~(-6)M，此时幼虫变态率提高69.29％。GABA的诱导作用较弱，最佳诱导浓度随处理时间的不同而有所不同。处理时间为24h和48h时，最佳诱导浓度为0.1×10~(-6)M；处理时间为0.5～16h时，最佳诱导浓度为100×10~(-6)M。5．KCl、肾上腺素、去甲肾上腺索、L-DOPA、5-羟色胺、GABA、茶碱和咖啡因8种诱导物对不同发育阶段海湾扇贝幼虫变态的诱导作用是不同的。13．42×10~(-3)M和20.13×10~(-3)M的KCl对第12天幼虫的变态有抑制作用，变态提高率为负值；之后当幼虫发育至第13和14天时，两浓度的KCl能够明显诱导幼虫变态，变态提高率均高于20％，而对于第16天的幼虫诱导作用有所减弱，变态提高率有所降低；26.85×10~(-3)M的KCl对第12和13天幼虫的变态均有抑制作用，变态提高率为负值，对第14和16天幼虫的变态却有明显的持续的诱导作用，变态提高率分别为22.98％和37.5％。神经递质肾上腺素、去甲肾上腺素、L-DOPA、5-羟色胺和GABA的诱导作用规律基本相似，即对第13天海湾扇贝幼虫的变态有明显的抑制作用，变态提高率均为负值，而对第14天幼虫的诱导作用较显著。茶碱和咖啡因作为影响细胞内cAMP的物质，它们的诱导作用规律与神经递质有所不同。它们对第13天海湾扇贝幼虫变态的诱导效果最好。6．测定了不同发育阶段及人工诱导后海湾扇贝幼虫体内去甲肾上腺素、多巴胺和5-羟色胺含量的变化规律。结果表明，海湾扇贝幼虫体内去甲肾上腺索含量在变态前和变态后没有明显变化，变态前为2352(pg／mg湿重)，变态后为2770(pg／mg湿重)。多巴胺和5-羟色胺含量在变态前随幼虫的发育而增加，变态前(第13天)急剧增加，第13天的幼虫比第12天的幼虫分别增加了2.8倍和5.7倍，变态后急剧下降，变态后幼苗比第13天的幼虫分别降低了25.1倍和16.4倍。海湾扇贝幼虫体内DA：NE比和5-HT：NE比在变态前和变态后变化比较剧烈。DA：NE比和5-HT：NE比在变态前(第13天)急剧增加，第13天的幼虫比第12天的幼虫增加了3.O倍(DA：NE比)和5.0倍(5-HT:NE比)；变态后急剧降低，变态后幼苗比第13天的幼虫降低了29.8倍(DA：NE比)和19.5倍(5-HT：NE比)。海湾扇贝幼虫经KCl和氯化钙诱导24h后，体内去甲肾上腺素、多巴胺和5-羟色胺以及DA：NE比和5-HT：NE比均有所降低。本实验的结果表明，多巴胺和5-羟色胺可能启动了海湾扇贝幼虫的变态过程。7．茶碱和咖啡因对墨西哥湾扇贝幼虫的变态均有明显诱导作用。它们的诱导作用均对浓度的依赖性较强，对处理时间的依赖性较弱。10×10~(-4)M的茶碱诱导效果最好，平均变态提高率达33％，其次为1.0×10~(-4)M和100×10~(-4)M的茶碱，平均变态提高率分别为23.15％和21.97％。处理时间对茶碱诱导效果影响不显著，在1～24h范围内，平均变态提高率在19.07～26.1％之间变动。10×10~(-4)M的咖啡因诱导效果最佳，4个处理时间的平均变态提高率为36.01％，其次为100×10~(-4)M，平均变态提高率为26.43％。处理时间对茶碱的诱导效果影响不大，在1～24h范围内，平均变态提高率在19.65～22.02％之间变动。8．采用直接测定cAMP的方法来研究cAMP是否参与了海湾扇贝幼虫的变态过程。结果表明，cAMP参与了海湾扇贝幼虫的变态过程。海湾扇贝幼虫体内cAMP含量随着发育阶段的不同而有所变化。在D形幼虫期最低，为73 pmol／(mg蛋白质)；当到达壳顶期幼虫时cAMP含量明显增加，比D形幼虫期提高了12.7倍。从壳顶期幼虫到眼点幼虫(100％，第13天)cAMP含量增加速度较慢，各发育阶段分别比前一发育阶段增加了0.4倍、0.3倍和0.2倍。但当幼虫变态后，体内cAMP含量又急剧增加，幼苗体内cAMP含量比眼点幼虫(100％，第13天)增加了6.1倍。当用KCl、肾上腺索和L-DOPA诱导后，幼虫体内cAMP含量明显增加，分别比对照组提高了7.8倍、1.5倍和10.7倍，说明cAMP参与了这3种诱导物诱导海湾扇贝幼虫变态的过程。9．在前面实验结果和参考有关文献的基础上，初步提出了以海湾扇贝为代表的双壳贝类幼虫变态机理模型：幼虫变态分为两个过程：启动过程和后续过程。当幼虫发育到一定阶段，在外界刺激因子的作用下，体内分泌多巴胺和5-羟色胺，多巴胺和5-羟色胺通过某种信号转导途径(如以DG和IP_3为第二信使)启动变态过程，变态过程启动后，又激活以cAMP为第二信使的信号转导途径(暂时称为后续过程)，两者共同完成了幼虫的变态过程。
其他摘要	Metamorphosis is an important stage from larva to juvenile in bivalvia. Studying themetamorphosis process and mechanism of bivalvia is important for us to determine their population variation and increase the economic efficiency of bivalvia cultivation industries. In this paper, apart from the induction of metamorphosis by chemical matter in bivalvia (Argopecten irradians, Argopecten irradians concentricus and Mercenaria mercenaria), we study the metamorphosis mechanism of bivalvial larvae at molecular biology and neurobiology levels, which through determining the hormone and neurotransmitter content and the second messenger cAMP content in bay scallop Argopecten irradians larvae. The main results are as follows. 1. The advancement in researching on settlement and metamorphosis of marine invertebrate larvae are reviewed in detail according to a lot of references. There are many factors that affecting these processes, including biotic and abiotic factors. Biotic factors involve larval behavior, abiotic factors involve physical and chemical factors. Last decade many achievements about mechanism of settlement and metamorphosis of marine invertebrate larvae have been achieved, at neurology and molecular biology levels. So far there are three models for explaining the settlement and metamorphosis mechanism of marine invertebrate larvae: the two control pathyways model for Pacific oyster (Crassostrea gigas) larvae, the up-regulation model for red abalone (Haliotis rufescens) larvae and the free fatty acid control model for polychaete Phragmatopoma california larvae. Some inductive matters such as GABA, epinephrine, norepinephrine have been successfully applied to economical molluscan seed-production. The delayed metamorphosis of marine invertebrate larvae is also reviewed, including the cause occurring delayed metamorphosis and effects of delayed metamorphosis on metamorphosis, growth and mortality. 2. Bay scallop A. irradians larvae are treated with Ach Cl, ATP and CaCl_2 to test the ability to induce metamorphosis during 3～8 April, 2000. Ach Cl and ATP are found ineffective exception at special concentrations and exposure time tested. CaCl_2 has been identified as active inducer of metamorphosis. Exposure to 10 * 10~(-3)～40 * 10~(-3)M CaCl_2 for 12～24h is sufficient to promote >10% metamorphosis. The inductive effect is dose-dependent with a maxima under 40 × 10~(-3)M and 24h, at which the percentage metamorphosis increment is 23.18%. All the three chemical matters affect the morality of bay scallop larvae. The effect is dose- and time-dependent, the higher the concentration and the longer the exposure time, the higher the mortality. 3. The induction of metamorphosis of larvae of the southern bay scallop Argopecten irradians concentricus Say by KC1, epinephrine (EPI), norepinephrine (NE) and choline chloride have studied. The results suggest that KCl, EPI, NE and choline chloride are all the active inducers of metamorphosis. KCl can increase metamorphosis at all concentrations (6.71 * 10~(-3)～33.56 * 10~(-3)M) and exposure time (12～48h). When exposure to 13.42 * 10~(-)3M and 20.13 * 10~(-3)M KC1 for 12～48h, the average percentage metamorphosis increment are all > 10%. The better inductive effect of EPI and NE are all archived at 1.0 * 10~(-6)M～50 * 10~(-6)M when exposure to lh～12h, the average percentage metamorphosis increment is >10%. The inductive effect of choline chloride is dose-dependent, but not time-dependent. The average percentage metamorphosis increment do not vary significantly with the exposure times 12h～48h at 0.01 * 10~(-4)M～l.0 * 10~(-4)M (12%～13%). Exposure to 10 * 10~(-4)M choline chloride for 12h is sufficient to increase 19.14% metamorphosis, and the metamorphosis decrease significantly excess 12h. Choline chloride is toxic at 100 * 10~(-4)M, the metamorphosis is zero while the mortality is 100%. 4. The ability of KCl, epinephrine (EPI), norepinephrine (NE), L-DOPA, 5-HT(5- hydroxytryptamine, Serotonin) and GABA( γ-aminobutyric acid) to induce metamorphosis of Mercenaria mercenaria L. larvae have been studied. The results suggested that KCl, EPI, NE, L-DOPA, 5-HT are active inducers of metamorphosis, whilst GABA is less effective inducer. The optimum concentrations of KCl vary with exposure time different. The optimum concentration is 33.56 * 10~(-3)M when the exposure times are 1～24h, the percentage metamorphosis increments are all >24%. The optimum concentrations of KCl are 20.13～26.85 * 10~(-3)M and 13.42 * 10~(-3)M when the exposure time lasted 48h and 72h, respectively. The inductive effects of EPI and NE are dose- and time-dependent. The optimum concentration of EPI is 100 * 10~(-6)M at 8h exposure time, the highest percentage metamorphosis increment is archived, 36.97%. The percentage metamorphosis increment are >18% when exposure to 100 * 10~(-6)M NE for 8h～16h, while the percentage mortality increment are all <30%. Exposure to 500 * 10~(-6)M NE for 8～16h are also sufficient to increase > 18% metamorphosis, but prolonged exposure times (16～48h) cause toxic effect, the mortality increase significantly, the percentage mortality increment are all >50%. The better inductive effects of L-DOPA are archived at 10 * 10~(-6) and 50 * 10 ~(-6)M exposure to 8～24h, the percentage metamorphosis increment are >30%. 5-HT is the most active inducer of these six compounds. The better inductive effects are archived at 100 * 10~(-6)～1000 * 10~(-6)M exposure to 0.5～24h, the percentage metamorphosis increment are >30%. The optimum concentrations are 1000 * 10~(-6)M and 100 * 10~(-6)M when the exposure time lasted 8h and 24h, at which the percentage metamorphosis increment are 57.5% and 69.29%, respectively. In contrast to other compounds, GABA is a 1ess effective inducer. The optimum concentrations vary with the exposure time different. When the exposure time are 24～48h and 0.5～16h, the optimum concentrations are 0.1 * 10~(-6)M and 100 * 10~(-6)M, respectively. 5. Larval age influences the inductive effects of KCl, epinephrine (EPI), norepinephrine (NE), L-DOPA, 5-HT, GABA, theophylline and caffeine. 13.42 * 10~(-3)M and 20.13 * 10~(-3)M KCl inhibit the metamorphosis of 12-day-old larvae, but significantly increase the metamorphosis of 13- and 14-day-old larvae, the percentage metamorphosis increment are >20%. The inductive effect reduce on day 16.26.85 * 10~(-3)M KCl inhibits the 12- and 13-day-old larvae metamorphosis, but promote the 14- and 16-day-old larvae metamorphosis, on which the percentage metamorphosis increment are 22.98% and 37.5%, respectively.
页数	126
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/315
专题	海洋环流与波动重点实验室海洋生态与环境科学重点实验室
推荐引用方式 GB/T 7714	张涛. 双壳贝类幼虫变态诱导及其机理研究[D]. 中国科学院海洋研究所. 中国科学院海洋研究所,2000.

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