Elucidation of Insect Olfaction in the Pea Aphid, Acyrthosiphon pisum

蚜虫(同翅目：蚜科)是西欧和北欧地区农田和园艺作物的主要害虫，通过取食或通过传播植物病原体如大麦黄矮病毒(BYDV)间接造成危害。豌豆蚜(Acyrthosiphon Pisum)的性信息素成分是由两种环烯醚类化合物(环戊烷类化合物)、(4As，7S，7AR)-内酯和(1R，4AS，7S，7AR)-内酯组成的混合物，由雌性在其生活史的有性阶段释放。雄性蚜虫会被性信息素吸引，并以物种特有的方式对信息素组分的比例做出反应。它们的对映体(4AR，7R，7AS)-内酯和(1S，4AR，7R，7AS)-内酯不是天然存在的，不具有生物活性，但它们与性信息素成分具有相同的物理化学性质，只是与镜像的三维结构不同。由于(I)已经确定的气味物质，特别是上述NAT中描述的性信息素成分，因此，由于(I)已经确定的气味物质，因此，A.pisum可以作为一个容易处理的模型系统来研究动物对气味物质的分子识别机制。戳。代表2013，30：1277-1283(Ii)豌豆蚜虫基因组注释的最新进展，特别是能够识别和区分不同气味的嗅觉蛋白(PLoS Biol 2010，8：e1000313；昆虫分子)。比奥尔。2010、19：113-122和(Iii)容易接触到的嗅觉器官，即蚜虫触角。尽管嗅觉在蚜虫生物学中起着至关重要的作用，但目前还不可能明确地阐明和区分支持外围水平嗅觉感知的分子识别机制的两个假说。(I)气味直接激活气味受体(ORs)(假设A)，以及(Ii)气味结合蛋白(OBP)-气味复合体激活ORs(假设B)。该项目寻求使用包括分子生物学、电生理学和分析/合成有机化学在内的多学科方法来测试两个假说，表明OBP在确定气味的特定分子识别方面是否具有重要作用。利用实时RT-qPCR技术，将鉴定和定量检测豌豆根结线虫基因组中的雄性触角特异的OR和OBP基因。雄性特有的OR基因将在非洲爪哇卵母细胞中进行功能性表达，用于使用双电极电压钳技术(PLoS One 2013，8：E62098)进行功能鉴定。将合成性信息素成分的非天然对映体，并用于探索在OR水平上的对映体识别。在有和没有OBPs的情况下，将测量雄虫对蚜性信息素成分及其对映体的专一性和反应动力学。将使用建立的高通量ESI-MS分析来测量男性天线特异性OBP区分对映体的能力，该分析将由一个合作实验室(Ales Svatos博士，Max-Planck化学生态研究所，耶拿)完成。

Aphids (Homoptera: Aphididae) are the major pests of arable and horticultural crops in Western and Northern Europe, causing direct damage through their feeding or indirectly through the transmission of plant pathogens eg Barley Yellow Dwarf Virus (BYDV). The sex pheromone components of the pea aphid, Acyrthosiphon pisum, comprise of a mixture of two iridoids (cyclopentanoids), (4aS,7S,7aR)-nepetalactone and (1R,4aS,7S,7aR)-nepetalactol that are emitted by females during the sexual phase of their life cycle. Male aphids are attracted to the sex pheromone and respond to the ratio of the pheromone components in a species-specific manner. Their enantiomers, (4aR,7R,7aS)-nepetalactone and (1S,4aR,7R,7aS)-nepetalactol are not naturally-occurring and do not possess biological activity although they have the same physical and chemical properties as the sex pheromone components and only differ in their three-dimensional structure as mirror images. A. pisum can serve as a tractable model system for investigation of the mechanisms underlying molecular recognition of odorants in animals because of (i) already identified odorants, in particular sex pheromone components described above Nat. Prod. Rep. 2013, 30:1277-1283 (ii) recent advances in the annotation of the pea aphid genome, specifically olfactory proteins that can recognise and discriminate between different odorants (PLoS Biol 2010, 8: e1000313; Insect Mol. Biol. 2010, 19:113-122 and (iii) readily accessible olfactory organs, i.e., aphid antennae. Despite the crucial role that olfaction plays in aphid biology, it has not been possible to elucidate and discriminate unequivocally between two hypotheses underlying the molecular recognition mechanisms underpinning olfactory perception at the peripheral level, ie. (i) odorants directly activate odorant receptors (ORs) (Hypothesis A), and (ii) ORs are activated by odorant-binding protein (OBP)-odorant complexes (Hypothesis B). This project seeks to use a multi-disciplinary approach including molecular biology, electrophysiology and analytical/synthetic organic chemistry to test the two hypotheses showing whether or not there is an essential role for OBPs in determining specific molecular recognition of odorants. Male-antenna specific OR and OBP genes in the A. pisum genome will be identified and quantified using real time RT-qPCR. Male-specific OR genes will be functionally expressed in Xenopus oocytes for functional characterisation with a two electrode voltage clamp technique (PLoS One 2013, 8:e62098). The non-natural enantiomers of the sex pheromone components will be synthesized and used to explore enantiomer discrimination at the OR level. The specificity and response kinetics of male ORs to aphid sex pheromone components and their enantiomers will be measured with and without OBPs. The ability of male-antenna specific OBPs to discriminate enantiomers will be measured using an established high-throughput ESI-MS assay to be done by a collaborating lab (Dr Ales Svatos, Max-Planck Institute for Chemical Ecology, Jena.