Liu H , Chen ZS , Zhang DJ , Lu YY .

	FIGURE 1. Statistical analysis of the differential expression of genes in male antennae of Bactrocera dorsalis flies from the MO (mineral oil) and ME (methyl eugenol) treatment groups. (A) Classification for the differential abundance of genes. (B) Volcano plots of differentially expressed genes (DEGs) between the ME and MO treatments. Y axis represents -log10 significance. X axis represents logFC (fold change). Red points indicate up-regulated expression of DEGs; green points indicate down-regulated expression of DEGs; and black points show non-differentially expressed genes. Genes with a P-value < 0.05 and fold change ≥1.0 were considered as DEGs in this study. (C) Hierarchical clustering graph of differential gene expression profiles in the ME and MO treatments. Clustering was done using RNA-Seq data derived from the six samples based on log10FPKM values. The column and row indicate the sample and gene, respectively. CK and T represent MO- and ME-treatment, respectively. Red and blue bands indicate, respectively, those genes that were significantly up-regulated and down-regulated in the ME-treated males.
	FIGURE 2. Heatmap representing the expression patterns of DEGs related to olfactory transport in male antennae from the ME (methyl eugenol) and MO (mineral oil) treatment groups. Clustering was done using RNA-Seq data derived from the six samples based on log10FPKM values. The column and row indicate the sample and gene, respectively. CK and T represent MO- and ME-treatment, respectively. Blue, white, and red indicate low, medium, and high levels of gene expression, respectively. Three biological replicates were conducted for each treatment.
	FIGURE 3. Validation of antennal DEGs (differentially expressed genes) in Bactrocera dorsalis male flies using quantitative real-time PCR (qRT-PCR). The α-tubulin gene was used as an internal control and three biological replicates were performed. The results were evaluated using a 2-ΔΔCT method, and the 2-ΔΔCT value of calibrant equals to 1.0. Bars represent mean ± SE values. ns, not significant, ∗P < 0.05, ∗∗P < 0.01 (t-tests).
	FIGURE 4. Maximum likelihood dendrogram based on protein sequences of candidate odorant receptors (ORs) in Bactrocera dorsalis and other insects. Evolutionary history was inferred using the maximum likelihood method. Bootstrap values greater than 90% (n = 1,000 replications) are displayed. ORs from Bactrocera dorsalis (Bdor), Ceratitis capitata (Ccap), and Drosophila melanogaster (Dmel) were included.
	FIGURE 5. The attractiveness effect of ME (methyl eugenol) to Bactrocera dorsalis male flies and the expression pattern of BdorOR63a-1 and BdorOR88a in the males’ antennae. (A) Responses of immature and mature male flies to ME. (B,C) BdorOR63a-1 and BdorOR88a expressed at higher levels in mature males than immature males antennae. (D) Diurnal pattern of mature male responsiveness to ME. (E,F) Diurnal pattern of BdorOR63a-1 and BdorOR88a expression levels in the mature males antennae. All the experiments were performed in triplicate. Transcription levels of the BdorOR63a-1 and BdorOR88a gene were normalized by α-tubulin gene. The results were evaluated using a 2-ΔΔCT method, and the 2-ΔΔCT value of calibrant equals to 1.0. Bars represent mean ± SE values. ns, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (t-tests).
	FIGURE 6. Responses of Xenopus oocytes with co-expressed BdorOR88a/BdorOrco or BdorOR63a-1/BdorOrco to stimulation with ME and MO compounds. (A) H2O-injected Xenopus oocytes failed to respond to the ME (methyl eugenol) and MO (mineral oil) odorants. (B) BdorOR63a-1/BdorOrco only slightly responded to the 10-3 M solution of ME. (C) BdorOR88a/BdorOrco Xenopus oocytes were stimulated with a range of ME concentrations. (D) Dose-dependent curve of BdorOR88a to ME using doses of 1.0 × 10-8 to 1.0 × 10-3 M. EC50 = 2.83 × 10-5. Symbols show the electrical current responses from the BdorOR88a/BdorOrco complex presented as the mean ± SE (n = 8). The dose-response curve shown was fitted with a sigmoidal model that had a variable slope (in Origin v9.0 software).
	FIGURE 7. Effects of RNA interference on the mortality of Bactrocera dorsalis male flies, BdorOR63a-1 and BdorOR88a expression in their antennae, and fly responsiveness to ME. (A,B) Mortality of male flies in the dsBdorOR63a-1 and dsBdorOR88a treatments in the 24 h and 48 h bioassays were calculated. Negative control males were injected with the same amount of dsGFP. Blank control groups (CK) were normally reared. (C,D) The RNAi efficiency was measured at 24 and 48 h by qRT-PCR after the dsRNA injection. α-tubulin gene was used as an internal reference gene. The results were evaluated using a 2-ΔΔCT method, and the 2-ΔΔCT value of calibrant equals to 1.0. (E,F) The attractiveness effect of ME (methyl eugenol) to males after BdorOR63a-1 and BdorOR88a genes were silenced by RNAi. Different letters among the columns within a time point indicate significant differences (ANOVA, P < 0.05). Five biological replicates were used. Bars represent mean ± SE values.
	FIGURE 8. Schematic view of the ME (methyl eugenol) odorant detection process in the antennae of mature Bactrocera dorsalis male flies. Once ME odorants penetrate pore tubules of the sensillum, they are bound and solubilized by BdorOBP83a-2 and BdorOBP2, transported through the sensillum lymph and finally reach sensory dendrites, where they activate membrane-bound BdorOR88a. Signal transduction evokes a response behavior to the detected ME. ME odorants are rapidly degraded and inactivated by odorant-degrading enzymes.

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