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
This project is an extension of research on the management of insect pests of temperate tree fruits and addresses objective 2 of the related in-house project. The chemosensory system is a target of codling moth control by using sex pheromones in mating disruption programs, and pheromones or kairomones for monitoring field populations or for the development of attract and kill technologies. In alignment with National Program 304 2A, Protection of Agricultural and Horticultural Crops, Biology and Ecology of Pests and Natural Enemies, the goal of this project is to identify critical protein receptors within the codling moth chemosensory system that regulate recognition and behavioral response to pheromones and kairomones. We have generated 12 mammalian cell lines stably expressing gene transcripts encoding proteins involved in codling moth perception of pheromones and kairomones. We have developed cell-based assays to monitor cyclic AMP and calcium (two intracellular signaling molecules activated in response to pheromone interaction with its receptor) to determine receptor function. Using the cell-based assay systems, we have identified a codling moth pheromone receptor that is responsive to codlemone, the codling moth sex pheromone component that is attractive to males and used in the orchard for mating disruption. We are currently refining our procedures to adapt the mammalian cell lines for use in high-throughput assays to identify novel and more potent attractants or disruptants for use in codling moth control programs. Monitoring of activities and progress on this project was accomplished by direct supervision of on-site employees, and use of site visits, email and telephone to communicate with off-site collaborators.