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      A natural polymorphism alters odour and DEET sensitivity in an insect odorant receptor

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          Abstract

          Blood-feeding insects such as mosquitoes are efficient vectors of human infectious diseases because they are strongly attracted by body heat, carbon dioxide, and odours produced by their vertebrate hosts. Insect repellents containing DEET (N,N-diethyl-meta-toluamide) are highly effective, but the mechanism by which this chemical wards off biting insects remains controversial despite decades of investigation 1- 11 . DEET appears to act both at close range as a contact chemorepellent by acting on insect gustatory receptors 12 and at long range by acting on the olfactory system 1- 11 . Two opposing mechanisms for the observed behavioural effects of DEET in the gas phase have been proposed: that DEET interferes with the olfactory system to block host odour recognition 1- 7 or that DEET actively repels insects by activating olfactory neurons that elicit avoidance behaviour 8- 11 . Here we show that the insect repellent DEET functions as a modulator of the odour-gated ion channel formed by the insect odorant receptor (OR) complex 13, 14 . The functional insect OR complex consists of a common co-receptor, Orco (ref. 15 , formerly called Or83b, ref 16 ), and one or more variable OR subunits that confer odour-selectivity 17 . DEET acts on this complex to potentiate or inhibit odour-evoked activity or to inhibit odour-evoked suppression of spontaneous activity. This modulation depends on the specific OR and the concentration and identity of the odour ligand. We identify a single amino acid polymorphism in the second transmembrane domain of Or59b in a Drosophila melanogaster strain from Brazil that renders this receptor insensitive to inhibition by the odour ligand and modulation by DEET. These data provide the first evidence that natural variation can modify the sensitivity of an odour-specific insect OR to odour ligands and DEET. Our data support the hypothesis that DEET acts as a molecular “confusant” that scrambles the insect odour code and provide a compelling explanation for the broad-spectrum efficacy of DEET against multiple insect species.

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          An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.

          Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a phiC31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the phiC31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the phiC31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, high-throughput screening of large cDNA sets and regulatory elements.
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            Insect olfactory receptors are heteromeric ligand-gated ion channels.

            In insects, each olfactory sensory neuron expresses between one and three ligand-binding members of the olfactory receptor (OR) gene family, along with the highly conserved and broadly expressed Or83b co-receptor. The functional insect OR consists of a heteromeric complex of unknown stoichiometry but comprising at least one variable odorant-binding subunit and one constant Or83b family subunit. Insect ORs lack homology to G-protein-coupled chemosensory receptors in vertebrates and possess a distinct seven-transmembrane topology with the amino terminus located intracellularly. Here we provide evidence that heteromeric insect ORs comprise a new class of ligand-activated non-selective cation channels. Heterologous cells expressing silkmoth, fruitfly or mosquito heteromeric OR complexes showed extracellular Ca2+ influx and cation-non-selective ion conductance on stimulation with odorant. Odour-evoked OR currents are independent of known G-protein-coupled second messenger pathways. The fast response kinetics and OR-subunit-dependent K+ ion selectivity of the insect OR complex support the hypothesis that the complex between OR and Or83b itself confers channel activity. Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The ligand-gated ion channel formed by an insect OR complex seems to be the basis for a unique strategy that insects have acquired to respond to the olfactory environment.
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              The PredictProtein server.

              PredictProtein (http://www.predictprotein.org) is an Internet service for sequence analysis and the prediction of protein structure and function. Users submit protein sequences or alignments; PredictProtein returns multiple sequence alignments, PROSITE sequence motifs, low-complexity regions (SEG), nuclear localization signals, regions lacking regular structure (NORS) and predictions of secondary structure, solvent accessibility, globular regions, transmembrane helices, coiled-coil regions, structural switch regions, disulfide-bonds, sub-cellular localization and functional annotations. Upon request fold recognition by prediction-based threading, CHOP domain assignments, predictions of transmembrane strands and inter-residue contacts are also available. For all services, users can submit their query either by electronic mail or interactively via the World Wide Web.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                0028-0836
                1476-4687
                15 August 2011
                21 September 2011
                27 April 2012
                : 478
                : 7370
                : 511-514
                Affiliations
                [1 ]Laboratory of Neurogenetics and Behaviour, The Rockefeller University, 1230 York Avenue, Box 63, New York, NY 10065 USA
                [2 ]Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 63, New York, NY 10065 USA
                [3 ]Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology Hans Knöll Str. 8, 07745 Jena, Germany
                Author notes
                Correspondence and requests for materials should be addressed to L.B.V. ( Leslie.Vosshall@ 123456rockefeller.edu ).
                [†]

                Present addresses: Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA 94720 USA (M.P.); Integrated Ph.D. Program in Cellular, Molecular and Biomedical Studies, Columbia University, New York, NY 10032 USA (N.S.).

                Article
                nihpa317226
                10.1038/nature10438
                3203342
                21937991
                693f341c-d3b0-40ab-99ad-2475462b632c

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Institute on Deafness and Other Communication Disorders : NIDCD
                Award ID: R01 DC008600-03 || DC
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