Kepchia D et al. (2017), Inhibition of insect olfactory behavior by an a...

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PLoS One 2017 Jan 01;125:e0177454. doi: 10.1371/journal.pone.0177454.

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Inhibition of insect olfactory behavior by an airborne antagonist of the insect odorant receptor co-receptor subunit.

Kepchia D , Moliver S , Chohan K , Phillips C , Luetje CW .

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Response to volatile environmental chemosensory cues is essential for insect survival. The odorant receptor (OR) family is an important class of receptors that detects volatile molecules; guiding insects towards food, mates, and oviposition sites. ORs are odorant-gated ion channels, consisting of a variable odorant specificity subunit and a conserved odorant receptor co-receptor (Orco) subunit, in an unknown stoichiometry. The Orco subunit possesses an allosteric site to which modulators can bind and noncompetitively inhibit odorant activation of ORs. In this study, we characterized several halogen-substituted versions of a phenylthiophenecarboxamide Orco antagonist structure. Orco antagonist activity was assessed on ORs from Drosophila melanogaster flies and Culex quinquefasciatus mosquitoes, expressed in Xenopus laevis oocytes and assayed by two-electrode voltage clamp electrophysiology. One compound, OX1w, was also shown to inhibit odorant activation of a panel of Anopheles gambiae mosquito ORs activated by diverse odorants. Next, we asked whether Orco antagonist OX1w could affect insect olfactory behavior. A Drosophila melanogaster larval chemotaxis assay was utilized to address this question. Larvae were robustly attracted to highly diluted ethyl acetate in a closed experimental chamber. Attraction to ethyl acetate was Orco dependent and also required the odorant specificity subunit Or42b. The addition of the airborne Orco antagonist OX1w to the experimental chamber abolished larval chemotaxis towards ethyl acetate. The Orco antagonist was not a general inhibitor of sensory behavior, as behavioral repulsion from a light source was unaffected. This is the first demonstration that an airborne Orco antagonist can alter olfactory behavior in an insect. These results suggest a new approach to insect control and emphasize the need to develop more potent Orco antagonists.

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Species referenced: Xenopus laevis
Genes referenced: ace hhex

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	Fig 1. Orco antagonist activity of phenylthiophenecarboxamide compounds OX1t-OX1w.Compounds were initially screened at 100 μM against ORs from Drosophila melanogaster (Dmel\Orco+Dmel\OR35a activated by 10 μM OLC12) and Culex quinquefasciatus (Cqui\Orco+Cqui\OR21 activated by 3 μM OLC12). Concentration-inhibition curves were constructed for compounds that displayed favorable antagonist activity at both receptors. OX1a (a previously identified Orco antagonist [49]) served as a reference compound. Data are presented as mean ± SEM (n = 3–11). nd, not determined.
	Fig 2. Orco antagonist OX1w inhibits odorant activation of a diverse array of ORs.(A) OX1w inhibits odorant activation of ORs from D. melanogaster and A. gambiae. Top trace, an oocyte expressing Dmel\Orco+Dmel\Or35a was challenged with a 30 sec application of 1 μM hexanol (Hex). After a 20 min wash period, 100 μM OX1w was applied for 90 sec before a second application of Hex and coapplied during the Hex application. Bottom trace, an oocyte expressing Agam\Orco+Agam\Or28 was challenged with a 30 sec application of 40 μM acetophenone (Ace). After a 20 min wash period, 100 μM OX1w was applied for 90 sec before a second application of Ace and coapplied during the Ace application. (B) Oocytes expressing a variety of ORs were activated by the appropriate odorant and tested for inhibition by OX1w as in panel A. Dmel\Orco+Dmel\Or35a was activated by 1 μM hexanol, Agam\Orco+Agam\Or15 was activated by 18 μM acetophenone, Agam\Orco+Agam\Or27 was activated by 3 μM L-fenchone, Agam\Orco+Agam\Or28 was activated by 40 μM acetophenone, Agam\Orco+Agam\Or31 was activated by 70 μM geranyl acetate, Agam\Orco+Agam\Or39 was activated by 10 μM 6-methyl-5-hepten-2-one, and Agam\Orco+Agam\Or65 was activated by 100 nM eugenol. Inhibition values were normalized to the value obtained when the assay was run in the absence of OX1w (sham). The structure of each odorant is shown. Data are presented as mean ± SEM (n = 3–8).
	Fig 3. D. melanogaster larvae are attracted to ethyl acetate (EA).(A) Diagram of larval plate assay. A starting circle was drawn at the center of a 100x15 mm polystyrene Petri dish (VWR) containing 20 ml of 1.1% agarose. A line divides the plate in half, with a small filter disk on each side. A Response Index (RI) is calculated as RI = (S − C)/(S + C), where S = number of larvae on the stimulus (EA) side and C = number of larvae on the control (vehicle) side. RI = 1 would indicate complete attraction, RI = 0 would indicate no preference, and RI = -1 would indicate complete repulsion. (B) Left panel, at the start of an experiment, larvae are in the starting circle flanked on either side by small filter disks. 10 μL of 1:1000 diluted EA was placed on the left filter disk and 10 μL of mineral oil (vehicle) was placed on the right filter disk. Right panel, the same plate, following a 5 min migration period. The majority of larvae have moved towards the filter disk containing EA. A large group of larvae is indicated by the arrow. (C) Larval chemotaxis towards EA is assayed at a series of dilutions. Data are presented as mean ± SEM (n = 4–7).
	Fig 4. Ethyl acetate attraction is inhibited by an airborne Orco antagonist.(A) Cross-section diagram of the larval plate assay with the addition of a large filter paper on the inner side of the lid. (B) Results of the larval chemotaxis assay. EA, ethyl acetate; oil, mineral oil (vehicle); D, DMSO (vehicle); OX1w, Orco antagonist; Ø, nothing added; light, fiber optic light source. Data are presented as mean ± SEM (n = 4–9). Results were compared by one-way ANOVA and Bonferroni’s multiple comparison test: for comparison to oil vs. oil control (sixth bar from top), , p<0.01; *, p<0.001. Light repulsion (bottom 2 bars) with DMSO or OX1w in the lid filter was compared by two-tailed, unpaired t-test. (C) A representative OX1w inhibition experiment. In both panels, larvae were placed in the starting circle, flanked on the left by EA and on the right by mineral oil (vehicle). In the left panel, DMSO (vehicle) was applied to the lid filter paper, while in the right panel, OX1w was applied to the lid filter paper. A large group of larvae is indicated by the arrow. (D) Cross-section diagram of the larval plate assay with addition of a fiber optic light source.

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