Elucidation of Insect Olfaction in the Pea Aphid, Acyrthosiphon pisum

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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.