赤眼蜂对寄主受精卵和未受精卵嗅觉识别机制研究

The olfactory identification mechanism of parasitic wasps to their host is one of the key problems in pest biological control. Numerous studies have shown that parasitic wasps can recognize different hosts by the volatile substances from the host surface. In our previous research, we found that the Trichogramma can identify fertilized host eggs and the unfertilized host eggs by different volatile substances released from host egg surface. However, the identification mechanisms are still unknown. In the present project, we plan to use the Trichogramma dendrolimi as an model to research the olfactory identification mechanism. 1) Identify the key component of volatile substances of fertilized-unfertilized ricemoth egg by GC-MS and direct observation. 2) Identify the different expression of olfactory genes between male and female Trichogramma dendrolimi by qRT-PCR, aiming to get the specific or highly expressed olfactory genes based on the work of olfactory gene screening. 3) By the methods of fluorescence competitive binding experiment and the icro-electrode voltage-clamp technique in Xenopus laevis oocytes to screen the key odour receptor genes of host selection of Trichogramma dendrolimi. Hence, this project can reveal olfactory identification mechanism of Trichogramma to fertilized eggs and the unfertilized eggs. The results can help to reveal the relationship between the olfactory system and their life activities of parasitic wasps and provide new ideas for the development of sustained release agents for Trichogramma attraction, which could promote the development of pest biological control technology in future.

寄生蜂对寄主的嗅觉识别机制是害虫生物防治研究中的一个焦点问题。大量研究表明，寄生蜂可通过寄主自身释放的挥发性物质识别不同寄主。本课题组研究发现，赤眼蜂能够利用卵表挥发性气味辨别寄主受精卵和未受精卵，并偏好寄生受精卵。但是，这种识别机制还尚不清楚。本项目以松毛虫赤眼蜂为研究对象，米蛾受精卵与未受精卵为寄主，1）拟通过GC-MS和行为观测手段，明确导致赤眼蜂分辨两种米蛾卵的关键差异气味组分；2）在嗅觉基因筛选工作上，利用qRT-PCR等方法分析雌雄赤眼蜂嗅觉基因的表达差异，获取特异或高表达的嗅觉基因。3）以上述研究为基础，利用荧光竞争结合实验、非洲爪蟾母细胞双电极钳记录技术，筛选调控赤眼蜂寄主选择差异化的关键气味受体基因，最终阐明赤眼蜂对寄主受精卵和未受精卵的嗅觉识别机制。研究结果有助于揭示寄生蜂嗅觉系统与其生命活动之间的联系，为研发对赤眼蜂有吸引作用的缓释剂、制定害虫防治策略提供新思路。

寄生蜂可通过寄主自身释放的挥发性物质识别不同寄主，而嗅觉相关蛋白对于昆虫与外界进行信息交流具有重要意义。嗅觉蛋白可以识别环境中的特异性化学气味分子,并依此引导昆虫觅食、寻偶、定位寄主植物、庇护、选择产卵场所等行为。本课题组研究发现，赤眼蜂能够利用卵表挥发性气味辨别寄主受精卵和未受精卵，并偏好寄生受精卵。本项目通过GC-MS明确两种米蛾卵卵表存在12种（邻苯二甲酸二丁酯，丙烯酸十二烷酯，对苯二甲酸，2,4-二甲基-1-庚烯，氯丙嗪、甲酚，酞酸二丁酯，甲基己基甲酮，异戊烯醛，2,4-二甲基-1-庚烯，正己醇，1,2-苯二羧酸双(2-甲基丙基)酯，多聚异戊二烯醇，安眠酮）差异性的挥发物质。并通过行为观测手段，明确丙烯酸十二烷酯、2,4-二甲基-1-庚烯、甲基己基甲酮、苯二甲酸、安眠酮和而甲酚能够导致赤眼蜂存在差异性的选择行为。通过对不同寄主（受精卵S、未受精卵N、无寄主CK）不同时间（0h、2h、4h、6h、12h、24h）诱导的松毛虫赤眼蜂转录组差异基因变化，共鉴定65个气味受体（TdenORs）、6个离子型受体（TdenIRs）和 2个味觉受体（TdenGRs）。利用RACE-PCR技术获得40个ORs、6个IRs和2个GRs的全长序列。RT-PCR结果显示TdenOR24，TdenOR26，TdenOR28，TdenOR29，TdenOR31，TdenOR32，TdenOR34，TdenOR36-OR43，TdenOR45，TdenOR46，TdenOR48，TdenOR49，TdenOR50，TdenOR52和TdenOR53在雌虫和雄虫中特异性表达，表明这些基因在寄生蜂触角嗅觉识别中发挥重要作用。6个IR基因和2个GR基因几乎在所有检测组织中均有分布。通过体外表达和荧光结合实验初步明确了ORs能够结合差异性的挥发性化合物。其中ORs17有很强的结合能力，对甲基己基甲酮和苯二甲酸产生电位反应,，暗示松毛虫赤眼蜂ORs17参与米蛾识别受精卵未受精卵识别过程。研究结果有助于揭示寄生蜂嗅觉系统与其生命活动之间的联系，为研发对赤眼蜂有吸引作用的缓释剂、制定害虫防治策略提供新思路。