【作者】 王国红；

【导师】 李镇宇； Hans Smid；

【作者基本信息】 北京林业大学 ， 森林保护学， 2008， 博士

【摘要】 动物学习和记忆是当前生态学和行为学研究的热点。由于近缘物种寄生蜂间学习表现和记忆特性的比较研究,能很好地阐明进化和生态学因素对学习行为和记忆的影响,以此为模型开展对学习和记忆过程的分子和细胞水平分析,已经成为了现代行为生态学研究的前沿。本博士论文以粉蝶盘绒茧蜂(Cotesia glomerata)和微红盘绒茧蜂(Cotesiarubecula)两个寄生蜂近缘种为材料,在分析它们取食行为差异与记忆的获得方式关系的基础上,通过设置经典的条件反射实验,使用抑制蛋白质合成的转录抑制剂放线菌酮D(actinomycin D,ACD)和翻译抑制剂茴香霉素(anisomycin,ANI)阻断长时记忆以及冷休克阻断短时记忆,结合寄生蜂不同的产卵学习方式,研究了这两种近缘种寄生蜂学习和记忆特征,取得了如下的重要研究成果:(1)粉蝶盘绒茧蜂和微红盘绒茧蜂长时记忆形成中的特异性粉蝶盘绒茧蜂一次产卵学习后,其24小时记忆保持力能被蛋白质合成的转录抑制剂放线菌酮D(ACD)完全抑制,表现出经处理的粉蝶盘绒茧蜂与未经历产卵的寄生蜂趋向旱金莲无差异,表明粉蝶绒茧蜂一次产卵经历后就形成了依赖于蛋白质合成的长时记忆。微红盘绒茧蜂一次产卵学习后,形成了对旱金莲趋向的记忆,但记忆持续时间短于粉蝶盘绒茧蜂。两种寄生蜂3次连续产卵学习的记忆与一次产卵学习记忆结果相似。同未经历产卵的寄生蜂相比,粉蝶盘绒茧蜂3次连续产卵在24小时同样产生强的记忆保持力,这种记忆保持力同样能被蛋白合成的转录抑制剂放线菌酮D强烈抑制;但微红盘绒茧蜂3次连续产卵后在24小时的记忆和未经历产卵的寄生蜂是相似的,不存在显著的差异。进一步研究发现,与未经历产卵的寄生蜂相比,两种寄生蜂经过3次间隔产卵学习记忆均可产生持续5天的记忆保持力,但它们之间长时记忆巩固的时间动态有差异。粉蝶盘绒茧蜂在4小时就形成了依赖于蛋白质合成的记忆,放线菌酮D明显抑制粉蝶盘绒茧蜂的4小时及以后的记忆保持力。放线菌酮D和茴香霉素对微红盘绒茧蜂24小时的记忆保持力部分抑制,在其24小时的记忆中,存在依赖于蛋白质合成和不依赖于蛋白质合成的两种方式。但在72小时,其记忆则完全依赖于蛋白质合成,表现出茴香霉素明显抑制微红盘绒茧蜂72小时的记忆保持力。因此,粉蝶盘绒茧蜂长时记忆产生早,巩固快;微红盘绒茧蜂形成长时记忆较晚,72小时后才形成。72小时后,茴香霉素处理的记忆保持力在两种寄生蜂之间没有差异。(2)粉蝶盘绒茧蜂和微红盘绒茧蜂的短时记忆粉蝶盘绒茧蜂1小时的记忆保持力受到冷休克干扰,4小时后记忆保持力部分受影响,8小时后记忆不受冷休克影响。影响长时记忆的抑制剂的抑制效果显示,对粉蝶盘绒茧蜂一次产卵后1小时的记忆的抑制作用没有影响,4小时部分受到抑制,8小时记忆显著抑制。以上结果均表明粉蝶盘绒茧蜂的麻醉敏感记忆(AnesthesiaSensitive Memory,ASM)能持续几小时,一次产卵后8小时完全形成长时记忆。而微红盘绒茧蜂ASM衰退速度比粉蝶盘绒茧蜂要快,一次产卵后20分钟处理,对冷休克干扰敏感,1小时后明显减弱。对两种寄生蜂一次产卵后间隔20分钟、1小时、2小时和3小时进行冷休克处理结果显示:随着时间间隔的延长,冷休克处理对寄生蜂记忆的干扰作用逐渐减弱。间隔20分钟内冷休克处理的效果明显,但间隔1小时后冷休克处理的效果变得不明显。(3)粉蝶盘绒茧蜂和微红盘绒茧蜂的记忆结构本研究表明,在粉蝶盘绒茧蜂中,麻醉敏感记忆ASM的衰退和长时记忆(Long-term memory,LTM的巩固阶段发生在一个相似的时间段,这表明ASM直接巩固为LTM,不需要抗麻醉记忆(Anesthesia-resistant memory,ARM)作为过渡。粉蝶盘绒茧蜂中LTM的形成阻断了ARM的形成,两者不能共存。但在微红盘绒茧蜂中,麻醉敏感记忆(ASM)的衰退比长时记忆LTM时相巩固要开始提前几个小时,它的记忆首先巩固为抗麻醉记忆阶段,随后与长时记忆(LTM)记忆共存,然后在48小时后衰退。以上结果表明,这两种寄生蜂间记忆巩固形成的ARM和长时记忆(LTM)的巩固存在明显的差异。(4)creb基因在粉蝶盘绒茧蜂脑部的表达creb基因在长时记忆形成中起重要作用。为了揭示creb基因与记忆的关系,本论文通过原位杂交,检测了creb基因在寄生蜂脑中的表达特征,发现粉蝶盘绒茧蜂记忆形成过程中,脑部蕈体的Kenyon细胞有creb基因优势表达;而且在视叶的神经节层和外髓等部位也检测到creb基因的表达。本博士论文通过对两种近缘种寄生蜂的学习行为的研究,发现粉蝶盘绒茧蜂和微红盘绒茧蜂记忆形成和巩固的种的特异性不同,学习方式和学习次数影响昆虫长时程记忆形成和巩固是适应于取食策略的不同的进化结果;首次表明两个近缘种间在形成LTM时,必须的学习事件中在数量和质量上存在明显的自然差异。从而阐明了进化和生态环境对昆虫学习行为和记忆的作用,揭示了LTM形成和巩固在不同近缘种之间差异产生的机制,丰富和发展了昆虫行为学的理论。更多还原

【Abstract】 The ecology and behaviour of animal learning and memory have been studied extensively Differences in learning between closely related species create excellent opportunities for studying species-specific adaptations of learning. Parasitic wasps are good model systems for exploring learning and memory formation and their underlying mechanisms. We investigated learning and memory formation of two closely related parasitic wasp species, Cotesia. glomerata and Cotesia. rubecula, to reveal natural differences in memory acquisition. We wanted to establish the type of memory that is different between these wasp species. Using a strictly controlled classical conditioning set-up, we defined differences in specific forms of memory between the two species., by measuring the dynamics of memory formation. For this purpose, we applied (1) retrograde amnesia by cooling directly after learning to inhibit anesthesia sensitive memory (ASM) or (2) inhibition of translation or transcription with anisomycin (ANI) and actinomycin D (AcD), respectively, to block long-term memory (LTM). The two species were then tested for memory retention after different conditioning trials. Following these treatments, we studied the memory dynamics in two closely related species. The main results were as follow listed below:C. glomerata memory retention was completely inhibited by actinomycin D (ACD) after one conditioning trial for 24 hrs. When ACD-fed wasps were given a single conditioning trial, 24hrs memory was reduced to a level that was not different from naive ACD-fed wasps, showing that 24h memory was composed entirely of a transcription-dependent memory component. In C. rubecula a single conditioning does not induce 24 hr memory. Memory for nasturtium is formed after a single trial, but lasts only some hours. A massed learning protocol (3 subsequent oviposition experiences on nasturtium without a rest interval) yielded for both species displayed similar result for 24h memory with single trial learning. For C. glomerata, massed learning gave a strong memory compared to naive wasps and a strong reduction of this memory by ACD treatment to a level not different from naive, ACD-fed, but in C. rubecula, 24 hour memory retention was again similar to naive wasps.A spaced learning protocol (3 oviposition experiences on nasturtium with a 10 min interval) yielded a stable memory lasting 5 days in contrast with naive wasps for both species. There was, however, a difference in the temporal dynamics of LTM. In C. glomerata, maximum inhibition of memory performance in ANI- fed and ACD-fed wasps was reached and maintained stable after 4 h a showing that consolidation of a protein synthesis-dependent memory was complete after 4h. In C. rubecula 24h memory was only partially inhibited by ACD and anisomycin (ANI). This shows that a protein synthesis-independent memory coexists with a protein synthesis-dependent memory in C. rubecula at 24 h, while memory is entirely protein synthesis dependent at 72hrs. At 72hrs, the memory retention was completely inhibited by ANI. LTM consolidation took 72h in C. rubecula.In C. glomerata, wasps treated by a cold shock directly after a single trial had a similar memory performance at 1 h after a single trial as naive wasps, showing that memory was completely inhibited by cold shock. 4h memory retention after cold shock was inhibited partially compared to wasps after a single trial, while after 8h the cold shock treated wasps progressively approached the memory performance of untreated wasps after a single trial. When ACD-fed or ANI-fed wasps were given 1 single conditioning trial ,1h memory retention are not affected and 4h memory retention inhibited partially while 8h memory retention was completely inhibited by inhibitor .Thus, the ASM component after a single trial learning in C. glomerata contributes 100% to the observed memory trace at 1h and progressively decays within 8 h. In C. rubecula, the decay rate occurs at a faster rate than in C. glomerata, as cold shock treated wasps had a similar memory performance 20 min. after a single trial as naive wasps, whereas after 1h memory was significantly less affected by cold shock treatment. After 4h the cold shock treated wasps had similar memory retention levels as untreated wasps after a single trial. Thus, the ASM component after single trial learning in C. rubecula is 100% at 20 min and progressively decays within 4 h. Wasps subjected to cold shock 20min, 1h, 2h or 3h after training and then 4h retention were tested in each case. 4h retention was found to be severely disrupted when cold anesthesia was delivered 20min after training, but became increasingly resistant to cold anesthesia, as such treatment was delivered 1h after training.In C. glomerata, the ASM component decays within a similar time window as consolidation phase of LTM, suggesting that ASM consolidates directly into LTM, without an intermediate or coexisting anesthesia-resistant memory (ARM) trace. In C. rubecula, ASM decays several hours before the start of the consolidation phase of LTM, and its memory consolidates first into an ARM trace that later on coexists with an LTM trace and then decays after 48h. A model showed the differences in ARM and LTM consolidation between the two wasp species.cAMP responsive element binding protein (CREB), a transcription factor plays a key role in the LTM formation. To elucidate the role of the CREB in wasp memory formation, we analyzed creb expression in wasp brain by FISH. First results show a positive signal in the kenyon cells around the mushroom bodies, suggesting that the gene is predominantly expressed in kenyon cells around the mushroom bodies, a structure that is involved in learning and memory processes. However, expression can also be found around the lamina and medulla of the optic lobesThe species-specific difference in LTM acquisition and consolidation is adaptive given the extreme differences in the effects of learning on two parasitoid species evolved distinctly different foraging strategies. This is the first demonstration of natural differences in quantity and quality of learning events required for LTM formation between closely related species. The present studies indicate that the development of learning and memory of insects is closely associated with evolution and environment. Our results suggested that different mechanism between the closely related species underlines the formation and maintenance of LTM.更多还原