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. Documents Reimbursable with CSREES. Log 38112. Formerly 5352-22000-017-35R (5/10);5352-22000-019-13R (1/11). Formerly 5352-22000-021-02R (2/11).
3. Progress Report:
The work summarized in this progress report relates to objective number 2 in the Project Plan for 001-00D: 2. Identify genes, receptor proteins, and respective ligands that are critical to codling moth (CM) development and reproduction. A key first step in understanding semiochemical-induced behavioral response is the interaction of the chemical ligand with its membrane bound receptor. Identification of chemosensory receptors in insects has been mainly limited to those species with sequenced genomes. Unfortunately, the genomes of many of the major crop pests in the U.S. have not been sequenced and identification of their chemosensory receptors has been elusive. Molecular and bioinformatic techniques were developed to address this problem. We identified chemosensory receptors expressed in the CM using two different approaches: 1) Polymrase Chain Reaction (PCR) amplification of codling moth receptors using degenerate primers designed against conserved regions of “pheromone” sub-family receptors already identified in insects with sequenced genomes; and 2) direct sequencing of cDNA prepared from RNA transcripts extracted from CM chemosensory organs. Each approach was used successfully to identify putative “pheromone” receptors in CM, but both approaches were necessary to identify a more complete complement of CM odorant receptors. Significant Findings from identification of chemosensory receptors: 1) PCR amplification of CM receptors using degenerate primers. Odorant receptors, especially those of the “pheromone” receptor subfamily are critical to nearly all aspects of pest insect reproduction, from mate-finding and courtship, to host-finding and oviposition, as well as locating and recognizing food sources. Research to identify and characterize “pheromone” receptors expressed by lepidopteran crop pests has increased in hopes of achieving a more complete understanding of the moth chemosensory system. In this project, a cost effective, targeted method was developed to identify gene transcripts expressed in CM antennae that encode for “pheromone” receptors. First, a highly conserved amino acid sequence was identified from “pheromone” sub-family members of Bombyx mori and Heliothis virescens. By reverse translation, degenerate oligonucleotide primers specific for “pheromone” sub-family receptors were designed and then used in PCR reactions to amplify gene transcripts encoding for these receptors. Five putative “pheromone” receptors were identified among gene transcripts expressed in CM antennae. To demonstrate the universality of this method, transcripts encoding for “pheromone” receptors have been cloned from cDNAs from 18 lepidopteran species. Furthermore, this technique was used in collaborations with researchers at Montana State University and the University of California Davis, to quickly identify pheromone receptors expressed in the European corn borer and the navel orangeworm. Additionally, this method has been used to identify more than 75 “pheromone” receptors from 29 species of Lepidoptera (unpublished results) as further demonstration of its efficacy. 2) Identification of chemosensory proteins and other receptors expressed in CM The degenerate primer approach for identification of “pheromone” receptors in CM and other lepidopteran species is a very targeted approach. To identify a fuller complement of receptors, as well as, other proteins involved in the senses of smell and taste, we generated a transcriptome by direct sequencing of cDNA prepared from RNA transcripts extracted from CM chemosensory organs. The CM transcriptomes have yielded over 190,000 contigs assembled from over 80 million bases of sequenced RNA (as cDNA). Annotations identified transcripts encoding for over 30 odorant binding proteins, including three pheromone binding proteins and three general odorant binding proteins1. Through manual annotations, gene transcripts encoding for chemosensory system proteins includes over 50 odorant receptors, 20 which have not been previously reported, 12 gustatory receptors, two sensory neuron membrane proteins, 47 ionotropic receptors, 17 chemosensory binding proteins and 30 putative odorant degrading enzymes. Because transcriptomes do not necessarily provide full-length transcript information, we are currently in the process of completing the cloning of all transcripts potentially involved in chemosensation to verify their authenticity and for use in future projects. Our second objective was to develop a high-throughput cell-based assay that can be used to test ligands against chemosensory receptors. Once putative “pheromone” receptors are identified, a functional assay is needed to determine their ligands. Traditionally, assays used have either been to clone receptors into Drosophila null neurons and then to perform electroantennagrams to detect ligand activation or to express receptors in frog oocytes and determine activation using a patch-clamp technique. We proposed to develop a cell-based assay in which we would generate stably transfected cell lines expressing the “pheromone” receptor of interest, along with the ubiquitous co-receptor OR2 and olfactory-specific G proteins. This proved technically challenging as our microplate reader was not designed to monitor intracellular calcium. To overcome this difficulty, a chimeric G-protein which converts calcium signaling into cyclic AMP production was cloned into our cell lines. Using cell lines stably expressing a receptor of interest, the co-receptor and the chimeric G-protein have proved successful in identification of ligand receptor pairs. Using the cell based assay and monitoring for cAMP production, a receptor that interacts with codlemone has been identified. However, a shortcoming of this technique is that the assay is not sensitive enough to give a pharmacological profile. We have recently gained access to a microplate reader capable of measuring intracellular calcium and are currently finishing up receptor ligand pharmacology studies.