Tissue Expression Pattern of RferOr6, RferOr40, and RferOr87

Real-time quantitative PCR (RT-qPCR) was used to evaluate the expression levels of RferOr6, RferOr40, and RferOr87 in different tissues of RPW. Total RNA was isolated from 7- to 14-day-old adults of antennae (A), legs (L), head (H), proboscides (P), and body (B) of both sexes, respectively. Tubulin and β-actin were used as reference genes (Antony et al., 2018). The specific primers were designed using Primer Premier 5.0 (PREMIER Biosoft International, CA, United States) and listed in Supplementary Table 1. The TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) was used for RT-qPCR. The RT-qPCR mix (20 μl) contained 1 μl of cDNA template, 0.6 μl of each primer (10 μM), 10 μl of 2 × TransStart Tip Green qPCR SuperMix, 0.4 μl of Passive Reference Dye (50×), and 7.4 μl of nuclease-free water. The RT-qPCR reactions were performed using the Applied Biosystems 7500 Fast. The real-time PCR system (ABI) was performed under the following conditions: 94°C for 30 s, 40 cycles of 94°C for 5 s, and 60°C for 34 s. The melting curve analysis was followed by the fluorescence measurement with one cycle of 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s and suggested amplification of a single product. The RT-qPCR experiments were repeated three times using three independently isolated RNA samples. The relative expression level of genes was calculated using the 2^–ΔΔSM1Ct method (Livak and Schmittgen, 2001). Statistical significance between males and females was analyzed by using Student’s t-test, and relative expression levels of antennal expression of three candidate genes were compared by using one-way ANOVA with IBM SPSS Statistics 25.0 (IBM Corp., Armonk, New York).