The Function And Interaction Of Pheromone Receptors And Pheromone Binding Proteins In The Diamondback Moth, Plutella Xyllostella

Insect dependent on a key physiological element: the olfactory system to prominent among otheranimals in the environment. Olfaction plays an indispensable role in mediating insect behavior such asfood selection, predators and noxious agents avoidance, and appropriate mating partners choice. Thediamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), is an important vegetable pest in theworld. Many crucifers are damaged much because of the moths’ invasion every year. Further study onolfaction accociated proteins in the diamondback moth could contribute to further research of olfactionmechanism in the moth and lay the foundation on the study of use of new drone in the pest control. Inthis paper, we primarily cloned and studied the functions of three PBPs genes and six candidate PRsgenes, and preliminarily the interplay between PBPs and PRs. The primary results are as follows:1. We cloned all the three PBPs in the diamondback moth. The expression profiles showed thesePxylPBP genes most expressed in antennae and PxylPBP1and PxylPBP3biased expressed in maleantennae than in female. Fluorescence competition binding assay showed that three PBPs can bindpheromone components of P. xyllotella with different sensitivities. PxylPBP1, PxylPBP2and PxylPBP3are most sensitive to Z11-16:OH, Z9-14:Ac and Z11-16:Ald, respectively. Fluorescence displacementbinding assay indicate that the three PBPs have strong affinities to the pheromone analogs with at leastone double-bond such as Z9-16:Ald, Z9, E12-14:Ac, while decreasing binding affinities to thepheromone analogs without any double-bond such as16:Ac and16:OH. Presence and absence ofunsaturated bonds in chemicals largely influence the binding capacities of PBPs in P. xyllostella.Although PBPs bound both pheromones and corresponding analogs in the fluorescence displacementbinding assay, the male antennae of P. xyllostella only robustly respond to all pheromone components,while very weakly respond to pheromone analogs if any in electroantennogram recording.2. In the present study, we have identified six receptors from P. xylostella which could be assigned tothe relatively conserved group of moth pheromone receptors in phylogenetic tree. The predominantexpression of six receptors in male antennae in the experiments of expression profiles further supportedthe notion that they may represent receptors for pheromones. Expect for male-biased expression of allsix receptors, PxylOR1and PxylOR6genes were faintly expressed in labial palps in male moths andPxylOR6gene was also very faintly expressed in proboscises. In the Xenopus-based functional studiesin the present study, PxylOR1is the receptor for the main compound in the P. xylostella pheromoneblend. PxylOR4was particularly interesting as it robustly responded to pheromone component, Z9-14:Ac as well as analog, Z9, E12-14: Ac. However, PxylOR4is tuned to pheromone component Z9-14: Acmore sensitively. By comparing expression patterns between PxylOR1and PxylOR4in in situhybridization experiments, the observation that the novel receptor type PxylOR4was expressed in cellsthat could be confined to long sensilla trichodea further supports PxylOR4is pheromone receptor.3. The Xenopus-based functional study were performed to assessed the impression of the three PBPs(PxylPBP1-3) to the sensitivity of identified pheromone receptor, PxylOR1and PxylOR4. For PxylOR1, PxylPBP1and PxylPBP3could increase the sensitivity of the complex expressing cells to Z11-16: Aldto about one or two order of magnitude. While for PxylOR4, PxylPBPs could increase the sensitivity ofthe complex expressing cells to pheromone component Z9-14: Ac but significant decrease theresponsiveness of the complex expressing cells to analog Z9, E12-14: Ac. According to this result, wededuce that activating pheromone receptor in olfactory receptor neuron need correct conformation ofpheromone/PBP complex. Pheromone components and pheromone binding proteins in the same speciescould form correct complex conformation and activate downstream pheromone receptors. If thechemical is not pheromone component in spite of pheromone analog with resemble structure, thecomplex would decrease the ability to activate downstream pheromone receptors.