中红侧沟茧蜂嗅觉受体介导的寄主挥发物识别机制

The host volatile signals play an important role in the host location of parasitic wasps，as well as the olfactory receptors recognizing these volatiles. It is critical to understanding the significance of host-related volatile signals guiding parasitoids in host location process and the olfactory communication mechanisms, which can provide theoretical basis for parasitoid behavior regulation and utilization, so as to improve the biological control efficacy of parasitoids. This research was planed to illustrate the important effect of host volatiles in the host location of Microplitis mediator, and the functional characteristics of olfactory receptors in detecting the host-related volatile signals. Specific objectives are: (1) Identify the electrophysiological and behavioral active compounds to parasitoids from host body and frass by using method of SPME, GC-MS, GC-GC-TOF，GC-EAD and insect olfactometer; (2) Compare the olfactory receptors transcript level of naive wasps and experienced wasps to identify candidate olfactory receptors by using RNA-seq and qRT-PCR methods;（3）Investigate the expression profiles of the candidate olfactory receptor in the olfactory receptor neurons by in situ hybridization and immunohistochemistry; (4) Explore the relationships between olfactory receptors and volatiles compounds by two-electrode voltage-clamp recording (TEVC) and RNAi technologies.

寄主挥发物在寄生蜂寄主定位中发挥重要作用，嗅觉受体蛋白是寄生蜂识别寄主挥发性物质的关键因子。深入解析寄生蜂寄主定位过程中寄主挥发物的功能及嗅觉识别机制，可以为寄生蜂行为调控和保护利用提供新思路和新方法。本申请以中红侧沟茧蜂及其寄主棉铃虫幼虫为研究对象，采用固相微萃取、GC-MS、GC-GC-TOF、GC-EAD及昆虫嗅觉仪等鉴定棉铃虫幼虫虫体及虫粪中对中红侧沟茧蜂具有电生理及行为活性的挥发物组分。利用转录组测序等技术鉴定寄生蜂嗅觉受体基因，通过qPCR检测气味诱导及寄生经历引起的受体基因表达变化，利用原位杂交和免疫组化手段对候选嗅觉受体进行组织定位。通过卵母细胞表达结合双电极电压钳系统解析嗅觉受体与寄主气味分子的对应识别关系，采用RNAi沉默目标嗅觉受体并研究基因缺失对寄生蜂识别寄主挥发物行为的影响。最终阐明中红侧沟茧蜂嗅觉受体介导的寄主挥发物识别机制。

通过基因组结合转录组测序，在中红侧沟茧蜂触角中总计鉴定169个气味受体MmedORｓ。其中，25个MmedOR基因（OR15，OR16，OR18，OR20，OR21，OR22，OR23，OR24，OR25，OR29，OR30，OR31，OR33，OR34，OR35，OR36，OR37，OR41，OR43，OR45，OR47，OR48，OR49，OR50和OR52）在雌触角表达水平高于雄触角，表明这些气味受体在雌蜂寻找寄主过程中发挥重要作用。两个OR基因（OR19和OR26）在雄触角的表达高于雌触角，暗示这两个基因可能参与雄蜂对雌蜂性信息素的识别。在中红侧沟茧触角转录组中共鉴定17个的新离子型受体IRs。非洲爪蟾卵母细胞表达结合双电极电压钳系统测定发现，中红侧沟茧蜂气味受体MmedOR25特异识别辛醛，MmedOR19能识别壬醛和辛醛，而MmedOR48识别谱较广，能够感受正辛醛、壬醛和癸醛等醛类化合物，推测这些气味受体参与寄生蜂识别寄主生境化学信号。离子型受体MmedIR64a1-MmedIR8a可广泛的识别6-8个碳原子的酸、醛、酯及醇类，这些物质多为栖境信号，即高挥发性的、远距离大量释放的植物挥发物，可指示潜在的寄主存在的区域；而MmedIR64a2-MmedIR8a相对特异的识别9-13个碳原子的酸和醛，包括低挥发性的植物挥发物、寄主体表挥发物，还包括一种鳞翅目性信息素的主要组分Z9:14-Ald，这些低挥发性信号有助于寄生蜂在近距离范围内更精确地定位寄主。此外，MmedIR64a1-MmedIR8a和MmedIR64a2-MmedIR8a的多数主要配体化合物对寄生蜂具有电生理和行为活性，我们推测，MmedIR64a1和MmedIR64a2复合体可以协作识别不同空间范围内的栖境信号和寄主信号，以帮助中红侧沟茧蜂更高效节能的定位寄主。. 在Plant Biotechnology Journal，Pest Management Science，Insect Biochemistry and Molecular Biology等学术期刊发表SCI论文13篇，中文核心期刊论文2篇，授权专利1项。