RT-PCR

Gene expression analysis was carried on D. suzukii adults from a population collected in Trento Province (Italy) and maintained in our laboratory on a standard Drosophila semiartificial diet (Drosophila species stock center, https://stockcenter.ucsd.edu/info/food_cornmeal.php) at a temperature of 23–25°, relative humidity of 65 ± 5%, and 16L:8D photoperiod. We dissected flies from both sexes using forceps, and we pooled males and females to obtain four different samples: heads (n = 20), thoraxes (including wings and legs; n = 10), abdomens (n = 10), and antennae (n = 300 pairs). A fifth sample was composed of third instar larvae (n = 10) that were processed as whole body samples. Additionally, two more samples consisting of female forelegs and male forelegs (n = 100 legs each) were prepared. All samples were placed immediately in cold RNAlater (LifeSciences) and stored at −80° until crushed in Trizol (LifeSciences) using Tissue Lyser II (QIAGEN). Total RNA was extracted followed the Trizol manufacturer’s instructions. Extracted RNA was treated with RNAse-free DNAse (LifeSciences) and then used for first strand cDNA synthesis using Superscript RT III (LifeSciences). One µl of cDNA diluted 1:5 was amplified by PCR with GreenTaq (Promega) according to the manufacturer’s instructions using 32 amplification cycles. To control for genomic DNA contamination, each batch of total RNA underwent a parallel mock reverse transcription step in which the reverse transcriptase was omitted. The cDNA quality was checked by tubulin amplification. PCR primers (listed in Table S4) were first checked against genomic DNA. Two biological replicates were done for each sample, and each amplification was repeated at least twice.