qRT-PCR Analysis of COEs in B. tabaci

The specific primers for COEs of B. tabaci MED were designed using Primer Premier 5.0 and used to verify the expression levels of COEs by qRT-PCR (Table S2). qRT-PCR was performed using an ABI 7500 system (Applied Biosystems) with the 25-μL reaction containing 0.5 μL of each specific primer, 0.5 μL of 50 × ROX reference dye (TIANGEN, Beijing, China), 1 μL of cDNA template, 10 μL of ddH₂O, and 12.5 μL of 2 × SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China). The qRT-PCR programme was as follows: 95 °C for 10 min (initial denaturation), followed by 40 cycles of 95 °C for 5 s (denaturation), 60 °C for 15 s (annealing), and 72 °C for 35 s (elongation). Only the qRT-PCR primers with 90–110% amplification efficiencies were used for the subsequent data analysis.
Relative expression levels were quantified using the 2^−ΔΔCt method [66 (link)]. The geometric mean of the reference genes 60S ribosomal protein L29 (RPL29) (GenBank accession no. EE596314) and elongation factor 1 alpha (EF1-α) (GenBank accession no. EE600682) was used to normalize the expression of target genes [67 (link),68 (link)]. Three biological replicates and four technical replicates were performed for each sample. One-way analysis of variance (ANOVA) (SPSS 23) was used to detect significant differences between samples.