title: Facile functional analysis of insect odorant receptors expressed in the fruit fly: validation with receptors from taxonomically distant and closely related species.
abstract: With the advent of genomic sequences and next-generation sequencing technologies (RNA-Seq), multiple repertoires of olfactory proteins in various insect species are being unraveled. However, functional analyses are lagging behind due in part to the lack of simple and reliable methods for heterologous expression of odorant receptors (ORs). While the Xenopus oocyte recording system fulfills some of this lacuna, this system is devoid of other olfactory proteins, thus testing only the "naked" ORs. Recently, a moth OR was expressed in the majority of neurons in the antennae of the fruit fly using Orco-GAL4 to drive expression of the moth OR. Electroantennogram (EAG) was used to de-orphanize the moth OR, but generic application of this approach was brought to question. Here, we describe that this system works with ORs not only from taxonomically distant insect species (moth), but also closely related species (mosquito), even when the fruit fly has highly sensitive innate ORs for the odorant being tested. We demonstrate that Orco-GAL4 flies expressing the silkworm pheromone receptor, BmorOR1, showed significantly higher responses to the sex pheromone bombykol than the control lines used to drive expression. Additionally, we show that flies expressing an OR from the Southern house mosquito, CquiOR2, gave significantly stronger responses to the cognate odorants indole and 2-methylphenol than the "background noise" recorder from control lines. In summary, we validate the use of Orco-GAL4 driven UAS-OR lines along with EAG analysis as a simple alternative for de-orphanization and functional studies of insect ORs in an intact olfactory system.
authors.affiliation: Department of Molecular and Cellular Biology, University of California, 1 Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Carlos
authors.initials: C
authors.lastname: Ueira-Vieira
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Kimbrell
authors.forename: Washington J
authors.initials: WJ
authors.lastname: de Carvalho
authors.forename: Walter S
authors.initials: WS
authors.lastname: Leal
mesh_terms: D000818:Animals; D057506:Arthropod Antennae / Q000502:physiology; D012831:Bombyx / Q000235:genetics / Q000502:physiology; D003465:Culex / Q000235:genetics / Q000502:physiology; D004330:Drosophila / Q000235:genetics* / Q000502:physiology*; D005260:Female; D015870:Gene Expression; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D008297:Male; D010675:Pheromones / Q000378:metabolism; D018035:Receptors, Odorant / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics / Q000502:physiology; D019076:Transgenes
chemical_list: D019476:Insect Proteins; D010675:Pheromones; D018035:Receptors, Odorant