Xu L , Jiang HB , Yu JL , Pan D , Tao Y , Lei Q , Chen Y , Liu Z , Wang JJ .

	Fig. 1. Olfactory preference and oviposition behavior in B. dorsalis induced by mango and 1-octen-3-ol.a The preference index of mango flesh for 3-day-old immature females, 15-day-old virgin females and 15-day-old mated females. b The preference index of 1-octen-3-ol for 3-day-old immature females, 15-day-old virgin females, and 15-day-old mated females. c Schematic diagram of the experimental setup for oviposition behavior assays. d–g Oviposition behavior induced by mango and/or 1-octen-3-ol. The heat map is based on fly tracks monitored using EnthoVision XT software. The color scheme represents the density of tracks and time spent of eight females, with red indicating the highest density. The images show the extent of oviposition after 24 h. The histograms show the average number of eggs laid by each female. Data are means ± SE of n ≥ 7 biological replicates. Statistical significance was determined using Student’s t-test (***p < 0.001). d Mango flesh vs MO. e 1-octen-3-ol vs MO. f Mango flesh vs 1-octen-3-ol. g Mango flesh + 1-octen-3-ol vs mango flesh + MO.
	Fig. 2. Responses of BdorOR7a-6/BdorOrco and BdorOR13a/BdorOrco to 1-octen-3-ol.a–c Responses to 1-octen-3-ol of Xenopus oocytes co-expressing BdorOR7a-6/BdorOrco or BdorOR13a/BdorOrco. a Xenopus oocytes were injected with the appropriate constructs and stimulated with 1-octen-3-ol at different concentrations. The oocytes were injected with (i) water as a control, (ii) cRNA of BdorOR7a-6/BdorOrco, (iii) cRNA of BdorOR13a/BdorOrco. b Dose-response curve of BdorOR7a-6 to 1-octen-3-ol. EC50 = 1.05 × 10−4 M. c Dose-response curve of BdorOR13a to 1-octen-3-ol. EC50 = 1.268 × 10−6 M. Symbols show the current responses from the BdorOR/BdorOrco complex presented as means ± SE (n = 8). The dose–response curves were fitted using GraphPad 8.0. d–h Responses to 1-octen-3-ol of HEK 293 cells co-expressing BdorOR7a-6/BdorOrco or BdorOR13a/BdorOrco. d Fluorescence images of HEK 293 cells incubated with Fluo4-AM and transfected with the candidate OR genes. Fluo4-AM was used to indicate the fluorescence change (ΔF) over time, and mCherry was used as the reporter to observe the successfully transfected cells. The proportional scale is 50 μm. e Change in fluorescence intensity of cells expressing BdorOR7a-6/BdorOrco following stimulation with 10−4 M of 1-octen-3-ol. f Dose–response curve of BdorOR7a-6 to 1-octen-3-ol. EC50 = 1.561 × 10−5 M. g Change of fluorescence intensity of cells expressing BdorOR13a/BdorOrco following stimulation with 10−4 M of 1-octen-3-ol. h Dose–response curve of BdorOR13a to 1-octen-3-ol. EC50 = 1.128 × 10−5 M.
	Fig. 3. CRISPR/Cas9 mutagenesis of BdorOR7a-6 and BdorOR13a.a Rates of survival and mutagenesis after microinjection. b The target region of BdorOR7a-6. (i) Gene structure of BdorOR7a-6 based on the genome sequence. Exons are shown as gray boxes and introns as lines. The gRNA target in the first exon contains a 20-nt guide sequence and the adjacent CGG highlighted in green is the protospacer adjacent motif (PAM). (ii) The genotypes of G0 mutants were determined by TA cloning and Sanger sequencing (deletions are shown as dashes highlighted in yellow). The identifier assigned to the mosaic G0 flies is shown in the front of each sequence, and the number of the deleted nucleotides is shown after each sequence. (iii) Sanger sequencing traces for WT, BdorOR7a-6−/+, and BdorOR7a-6−/− flies. The black region is the gRNA target site. (iv) Predicted protein sequences of the BdorOR7a-6 WT and mutant (−4 bp) alleles. c The target region of BdorOR13a, with (i–iv) indicating the same details as provided for above the BdorOR7a-6 mutants.
	Fig. 4. The phenotypes of BdorOR7a-6−/− and BdorOR13a−/− mutants.a EAG responses of WT, BdorOR7a-6−/− and BdorOR13a−/− mutants to different concentrations of 1-octen-3-ol and other volatiles. Data are means ± SE (n = 15–20). Statistical significance was determined using Student’ s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). b Oviposition behavior induced by 1-octen-3-ol in WT and mutant females. The heat map shows the density of tracks and time spent of eight females, with red indicating the highest density. The images show the extent of oviposition after 24 h, and the histogram shows the average number of eggs laid by each fly. Data are means ± SE (n ≥ 8 biological replicates). Statistical significance was determined using Student’s t-test (***p < 0.001).
	Fig. 5. Molecular docking of BdorOR7a-6 and BdorOR13a to 1-octen-3-ol, and site-directed mutagenesis to confirm the essential residues.a Structure of BdorOR7a-6 predicted using AlphaFold 2.0. b Residue of BdorOR7a-6 required for binding to 1-octen-3-ol. c Structure of BdorOR13a predicted using AlphaFold 2.0. d Residues of BdorOR13a required for binding to 1-octen-3-ol. e Response of BdorOR7a-6 Asn86Ala mutant to 1-octen-3-ol based on calcium imaging. f Responses of BdorOR13a Asp320Ala and Lys323Ala mutants to 1-octen-3-ol based on calcium imaging.

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