Location: Livestock Arthropod Pest Research Unit
Project Number: 3094-32000-041-010-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2020
End Date: Mar 31, 2025
Objective:
The objective of this research is to clone and functionally express the differentially expressed odorant receptors from screwworm towards identifying and developing sex specific baits.
Approach:
Using the wide collection of odors collected from fly cuticular hydrocarbons and rotting beef liver, we will test the olfaction receptors (ORs) as biological sensors to isolate and identify constituent odorants that will be used to develop robust and specific baits. We will clone and functionally characterize Cochliomyia homivorox ORs by employing an empty neuron system in Drosophila and the Xenopus oocyte expression system. In the Drosophila empty neuron system, full-length coding sequences of differentially expressed screwworm ORs will be inserted into a plasmid for expression in the empty neuron of Drosophila. Purified plasmid constructs will be injected into Drosophila embryos. UAS-ChomORx transgene will be inserted into 'halo genetic background by crossing. Resulting flies (w; 'halo; UAS-ChomORx) will be further crossed to w; 'halo; OR22a-Gal4 flies. Response of specific ORs in ab3-A neuron of test flies (w; 'halo; UAS-ChomORx/OR22a-Gal4) will be measured by single sensillum recordings. In the Xenopus oocyte system, ORs coding sequences will be inserted into a plasmid vector. Capped cRNAs will be synthesized in vitro from linearized plasmids encoding C. homivorax receptors by using RNA syntesis kits. Stage V oocytes will be injected with cRNA in 25 nl of water. Oocytes will be incubated in Barth’s saline for 3–7 days before electrophysiological recording. Our expectation is that this extensive screening of odorants will not only identify odor baits, but will shed new light on the genomics of ecological specialization and speciation, and will greatly improve our understanding of coevolution and interplay of ORs and ligands.
