2013 Annual Report
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:.
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Additionally, this method has been used to identify more than 75 “pheromone” receptors from 29 species of Lepidoptera (unpublished results) as further demonstration of its efficacy.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.