Molecular Basis of Alarm Pheromone Detection in Aphids Ruibin Zhang, Bing Wang, Gerarda Grossi, Patrizia Falabella, Yang Liu, Shanchun Yan, Jian Lu, Jinghui Xi, Guirong Wang  Current Biology  Volume 27, Issue 1, Pages 55-61 (January 2017) DOI: 10.1016/j.cub.2016.10.013 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2017 27, 55-61DOI: (10.1016/j.cub.2016.10.013) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 SSR of LP and SP Sensilla on the Fifth and Sixth Antennal Segments (A) Spontaneous activity (500 ms) of a placoid sensillum. Individual action potentials (spikes) are labeled A, B, or C according to their amplitude and shape. (B) Single-sensillum recordings of neurons in LP (large placoid) or SP (small placoid) sensilla on the sixth antennal segment and LP sensilla on the fifth antennal segment of A. pisum in response to paraffin oil (solvent), EBF, and allyl isothiocyanate (10−3 g loaded onto filter paper). (C) Electrophysiological responses of LP neurons on the fifth antennal segment (LP 5) and LP or SP neurons on the sixth antennal segment (LP 6 and SP 6) to EBF (10−3 g loaded onto filter paper) (mean ± SEM, n = 7–17; GLM followed by Duncan’s multiple range test, F = 26.03, p < 0.01). Bars labeled with different lowercase letters are significantly different. (D) Dose-response curves for LP neurons on the sixth antennal segment to EBF. A stimulus air pulse was added for 300 ms. All error bars indicate the SEM (n = 6; EC50 = 1.00 × 10−4 g). (E) Electrophysiological responses of LP neurons on the fifth antennal segment and LP or SP neurons on the sixth antennal segment to allyl isothiocyanate (mean ± SEM, n = 6–8; GLM followed by Duncan’s multiple range test, F = 39.05, p < 0.01). Bars labeled with different lowercase letters are significantly different. See also Figure S1. Current Biology 2017 27, 55-61DOI: (10.1016/j.cub.2016.10.013) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Functional Characterization of ApisOR5 (A) Recordings of different odorant-induced currents in Xenopus oocytes co-expressing ApisOR5 and ApisOrco. (B) Responses of ApisOR5. Responses are measured as induced inward currents. The error bars indicate the SEM (n = 9). (C) Dose-response curves of ApisOR5 and AgosOR5 to EBF when expressed in Xenopus oocytes. (D) Dose-response curves of ApisOR5 and AgosOR5 when expressed in Xenopus oocytes. Each point represents the mean current value (mean ± SEM, n = 5–8). (E) Action potentials recorded from at1 neurons in transgenic flies in response to solvent and EBF at a concentration of 10−3 g. (F) Mean responses of at1 neurons in transgenic flies from the Or67dGAL4 line, UAS-ApisOR5 line, and UAS-ApisOR5; Or67dGAL4 homozygous line in response to EBF. Significant responses to EBF are only found in the UAS-ApisOR5; Or67dGAL4 fly line (mean ± SEM, n = 6–8; GLM followed by Duncan’s multiple range test, F = 54.90, p < 0.01). Bars labeled with different letters are significantly different. (G) EBF-induced dose-response curves from at1 sensillum neurons expressing ApisOR5 (UAS-ApisOR5; Or67dGAL4 line; mean ± SEM, n = 8) and without ApisOR5 expression (UAS-ApisOR5 line; mean ± SEM, n = 6). The concentrations range from 10−8 to 10−3 g. The EC50 value for EBF is 1.30 × 10−5 g. A stimulus air pulse was added for 300 ms. See also Figures S3 and S4 and Table S1. Current Biology 2017 27, 55-61DOI: (10.1016/j.cub.2016.10.013) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Behavioral and Electrophysiological Responses of Pea Aphids to EBF after ApisOR5, ApisOBP3, and ApisOBP7 Knockdown by RNAi (A) Behavioral responses in a Y tube olfactometer. The pea aphids were tested 72 hr after dsGFP (control) or dsApisOR5 injection. Bars represent the overall percentages of aphids choosing either of the odor sources; the number in the bar indicates the total number of aphids choosing the arm. The white bar represents hexane treatment (control) and the black bar represents EBF treatment (ns, no significant difference, p > 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; two-sided binominal test). (B) EAG responses of pea aphids 72 hr after dsGFP (control) or dsApisOR5 injection to EBF, (Z)-3-hexen-1-ol, and (E)-2-hexenyl acetate (mean ± SEM, n = 19–24; GLM followed by Duncan’s multiple range test). Bars labeled with different letters are significantly different. (C) Behavioral responses in a Y tube olfactometer. Pea aphids were tested 72 hr after dsGFP (control) or dsApisOBP3 and/or dsApisOBP7 injection. Bars represent the overall percentages of aphids choosing either of the odor sources; the number in the bar indicates the total number of aphids choosing the arm. The white bar represents hexane treatment (control) and the black bar represents EBF treatment (ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; two-sided binominal test). (D) EAG tests of RNAi-treated aphids. EAG responses of pea aphids 72 hr after dsGFP (control) or dsApisOBP3 and/or dsApisOBP7 injection to EBF, (Z)-3-hexen-1-ol, and (E)-2-hexenyl acetate (mean ± SEM, n = 13–34; GLM followed by Duncan’s multiple range test). Bars labeled with different letters are significantly different. See also Figure S2 and Movie S1. Current Biology 2017 27, 55-61DOI: (10.1016/j.cub.2016.10.013) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Screening and Characterization of ApisOR5 Ligands In Vitro and In Vivo (A) ApisOR5 is narrowly tuned to EBF and its analog geranyl acetate when expressed in Xenopus oocytes with ApisOrco. Sixty-one chemicals were tested in this study. (B) Comparison of the induced currents by EBF and its analog geranyl acetate in ApisOR5/Orco-expressed Xenopus oocytes (mean ± SEM, n = 9; Student’s t test, ∗p < 0.05). (C) SSR traces showing control and geranyl-acetate-evoked activity of neurons in LP sensilla on the sixth antennal segment at a concentration of 10−3 g. (D) SSR traces showing geranyl acetate-evoked activity of at1 neurons from UAS-ApisOR5 and Or67dGAL4; UAS-ApisOR5 homozygous lines at concentration of 10−3 g. (E) Mean SSR responses to geranyl acetate in LP sensilla on the fifth antennal segment and in LP or SP sensilla on the sixth antennal segment, 10−3 g loaded on a filter paper (mean ± SEM, n= 7–17, GLM followed by Duncan multiple range test, F = 72.13, p < 0.01). Bars labeled with different letters are significantly different. (F) Mean SSR responses to geranyl acetate of at1 neurons from different fly lines (mean ± SEM, n = 6–17; Student’s t test, ∗∗p < 0.01). (G) Geranyl-acetate-induced dose-response curves from at1 neurons expressing ApisOR5 (UAS-ApisOR5; Or67dGAL4). A stimulus air pulse was added for 300 ms. All error bars indicate the SEM (n = 10). The EC50 value for geranyl acetate is 1.24 × 10−4 g. (H) The repellent behavior of pea aphids elicited by EBF or geranyl acetate. Bars represent the overall percentages of aphids choosing either of the odor sources; the number in the bar indicates the total number of aphids choosing the arm. The white bar represents hexane treatment (control) and the black bar represents EBF or geranyl acetate treatment (two-sided binominal test, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Table S2. Current Biology 2017 27, 55-61DOI: (10.1016/j.cub.2016.10.013) Copyright © 2017 Elsevier Ltd Terms and Conditions