多次交配促进广聚萤叶甲成虫寿命与繁殖的分子调控机制

Multiple mating is ubiquitous in insects, and has a significance in insect population reproduction. Ophraella communa is an important natural enemy of Ambrosia artemisiifolia L., and it has a significant control effect on A. artemisiifolia. The results of our previous study showed that multiple mating could positively facilitate longevity and fecundity of O. communa adults. Based on this result of our study, we hypothesize that multiple mating not only can meliorate physiological conditions of O. communa adults, but also facilitate development of female’s genital system, thus female fecundity increase in response to multiple matings. To test this hypothesis, the technologies and methods of population ecology, insect reproductive biology, insect physiology, molecular biology, proteomics and transcriptomics are used in this project. Firstly, changes in intake food doses of O. communa adults are studied under different mating times. Secondly, electrophysiologic responses and nutrient metabolite contents of O. communa adults are compared under different mating times. Thirdly, ovary structure and development and ovum occurrence of female O. communa are observed under a microscope. Finally, the contents of reproduction related and life associated proteins are determined, and their functions are tested under different mating times. The results of this projects aim to reveal molecular regulation mechanisms that multiple mating facilitates population reproduction of O. communa adults. This affords theoretical basis to deeply study the reproductive biology of O. communa and well use it to manage A. artemisiifolia.

多次交配是昆虫界中常有的现象，对昆虫种群繁衍具有重要意义。广聚萤叶甲是入侵杂草豚草的重要天敌，对豚草控制效果显著。前期研究发现，多次交配可明显促进广聚萤叶甲成虫的寿命和繁殖。基于这一研究结果，我们假设：多次交配不仅使广聚萤叶甲成虫生理状况得以改善，且雄虫精液蛋白可促进雌虫生殖系统发育，从而提高繁殖力。为验证这一假设，本项目采用种群生态学、繁殖生物学、昆虫生理学、分子生物学、蛋白组和转录组等技术与方法，首先，研究不同交配次数下广聚萤叶甲成虫日取食量变化；其次，比较不同交配次数下成虫电生理反应与体内营养代谢物质含量；进而观察不同交配次数下雌虫卵巢结构与发育及卵子发生情况；最后，检测不同交配次数下雌虫繁殖与寿命相关的精液蛋白含量，并对其功能进行验证。研究结果揭示多次交配促进广聚萤叶甲种群繁衍的分子调控机制，这为了开展该叶甲繁殖生物学更深层次的研究及其更好的利用奠定了理论基础。

本项目通过相关内容的研究，顺利完成研究任务，主要研究结果如下：.随着交配次数增加，广聚萤叶甲雌雄成虫体能消耗变大，取食量显著增加，以维持自身生命活动。在不同交配次数处理下，广聚萤叶甲雌雄成虫对豚草气体挥发提取物α-蒎烯均有反应，三次交配处理雌、雄成虫触角对α-蒎烯EAG的反应值最大，显著高于未交配对照和一次交配处理。鉴于α-蒎烯属于豚草的挥发物并对广聚萤叶甲成虫有强烈的吸引作用，说明多次交配增加广聚萤叶甲雌雄成虫对寄主豚草挥发性物质的反应，这种反应是取食选择的第一步。交配后供豚草叶片给广聚萤叶甲成虫取食补充营养，测定结果发现三次交配处理雌、雄成虫体内果糖、肝糖、总糖的含量都显著高于未交配对照、一次交配和二次交配处理雌雄虫体内的含量处理。.广聚萤叶甲雌虫繁殖力随交配次数升高而增高，未交配处理组产卵量最少，终身产卵量平均为319.3粒，4次交配处理组产卵量最高，为681.8粒卵。在不同交配次数下处女雌的卵巢所布卵粒始终少于具有交配行为的卵巢。交配次数越高，卵巢管松散越明显，该现象表明卵巢产出过较多的卵粒，证明多次交配刺激了广聚萤叶甲雌虫卵巢卵子的发生。利用qPCR技术检测精液蛋白基因的时空表达动态，根据定量结果，获得10个在雄性生殖系统表现出显著偏向性表达的精液蛋白基因，分别为：分别为CPB、CA、PRCP、PLA2、HK、PGK、sucA、ENO、SDH、PLA2b基因。其中， 5个基因（CPB、CA、PRCP、PLA2、HK）在雄虫中被干扰后与之配对的雌虫产卵量显著低于对照组，15d内产卵量分别下降12.61%、13.43%、19.03%、27.08%、22.89%。交配结束后15min-3h，交配囊中的CA基因逐渐被雌虫消耗，至3h时消耗量达64.3%；CPB基因也在该时段呈现出递减规律，在3h时被消耗38.9%；解剖观察注射dsCPB和dsCA的雄虫配偶的卵巢，与对照相比，卵巢中所布卵子减少，由此说明CPB、CA基因是广聚萤叶甲成虫在多次交配中具有调控作用的精液蛋白基因。. 项目共发表论文13篇，其中SCI论文11篇（9篇为第一标注），中文核心期刊论文2篇（均为第一标注）。