Total RNA extraction and cDNA synthesis

Total RNA samples were extracted from eggs and Malpighian tubules using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), as previously described [20]. Total RNAs were isolated from the other samples using the HiPure Total RNA Micro Kit (Magen, Shanghai, China), in accordance with the manufacturer’s instructions. Gel electrophoresis and the NanoDrop One spectrophotometer (Thermo Fisher Scientific) were used to determine the quantity of total RNA. This was then dissolved in 10–70 μl ddH₂O to obtain the following RNA concentrations (mean ± standard error of the mean): 200.7 ± 29.4 ng/μl for eggs; 284.4 ± 16.4 ng/μl for first instars; 637.8 ± 52.5 ng/μl for second instars; 563.0 ± 96.8 ng/μl for third instars; 864.3 ± 177.3 ng/μl for fourth instars; 804.9 ± 34.4 ng/μl for pupae; 831.4 ± 88.1 ng/μl for male adults; 866.6 ± 129.6 ng/μl for female adults; 279.8 ± 18.5 ng/μl for heads; 821.2 ± 140.4 ng/μl for carcasses; 359.5 ± 104.1 ng/μl for midguts; 553.2 ± 149.1 ng/μl for Malpighian tubules; 290.67 ± 28.71 ng/μl for first instars at 8°C; 240.57 ± 53.29 ng/μl for first instars at 25°C; and 277.10 ± 53.19 ng/μl for first instars at 35°C. The 260/280 nm optical density ratios were between 1.9 and 2.1 for all samples. The PrimeScript RT kit (containing gDNA Eraser, Perfect Real Time; TaKaRa, Dalian, China) was used to prepare first-strand cDNA for gene expression analysis. The cDNA was diluted tenfold prior to the following RT-qPCR investigations.