1a. Objectives (from AD-416):
The long term goal of this project is to identify key proteins involved in the codling moth's ability to smell, taste, and regulate feeding and reproduction as targets for novel insect control compounds. In this proposed research project, I will clone and identify odorant and gustatory receptors from sensory tissues. Once the molecules that bind to the receptors are identified, a cell-based assay will be developed that can be used to identify strong agonist and antagonists that can be used in codling moth control programs. The specific objectives of this proposal include: 1) construct tissue specific cDNA pools from codling moth sensory organs; 2) sequence individual cDNA pools and perform computer searches to identify target receptors; 3) amplilfy potential receptors from cDNA pools using degenerate oligonucleotide primers; 4) clone target receptors into an expression system suitable for analysis in insect cell lines; and 5) develop and implement assays to identify receptors for pheromones and kairomones used in codling moth control programs.
1b. Approach (from AD-416):
Odorant and gustatory receptors are members of the G protein-coupled receptor (GPCR) family. These receptor proteins have a conserved 7 transmembrane domain structure and are identified by this structural feature. I will take advantage of the 7 transmembrane domain structure to identify potential dormant and gustatory receptors in the codling moth. The following approach will be used: 1) isolate mRNA transcripts from codling moth sensory organs (transcripts should be highly enriched for our target receptors). The mRNA transcripts will be converted to cDNA for use in PCR analysis and direct pyrosequence analysis; 2) cDNA pools will be sequented from each of the target tissue or organ types and the generated DNA sequencees will be analyzed by computer searches against a database for identification. Those sequences that have a predicted 7 transmembrane domain structure will be further analyzed for predicted similarities to know odorant and gustatory receptors; 3) cDNA pools from each of the target tissue or organ types will also be used to amplify potential receptors using degenerate oligonuceotide primers designed from odorant and gustatory receptors identified in other insects; and 4) potential odorant receptor clones isolated from each of the target tissue or organ types will be used in a cell based expression system to identify receptors for codling moth pheromones and kairomones. Potential gustatory receptor clones will be used in the assay system to identify potential taste receptors.
3. Progress Report:
The chemosensory system is a target of codling moth control, using sex pheromones in mating disruption programs, and pheromones or kairomones for monitoring field populations or for the development of attract and kill technologies. The goal of this project is to identify biological targets that are critical for codling moth survival, exploiting the physiology, biochemistry and genetics of chemosensory systems that regulate recognition and behavioral response to pheromones and kairomones. A transcriptome generated from codling moth chemosensory tissues was generated and characterized. From this transcriptome, gene transcripts encoding seven pheromone receptors, three general odorant binding proteins, three pheromone binding proteins, two sensory neuron membrane proteins and two odorant degrading enzymes, all of which are thought to play key roles in pheromone recognition and signaling, were identified. Further characterization of the general odorant binding proteins showed that their transcripts are expressed not only in antennae, but also in mouthparts, another chemosensory tissue, and for general odorant binding protein 2, expression was also detected in the female abdomen tip, the site of pheromone production and oviposition. Mammalian cell lines stably expressing genes involved in codling moth perception of pheromones and kairomones were generated, and experiments to determine receptor function are in progress. Results suggest that one of the receptors we have identified is responsive to codlemone, the codling moth sex pheromone component that is attractive to males, and used in the orchard for mating disruption. The work reported here addresses objectives 1 and 2 in the parent project plan.