2.5.1 double-stranded RNA (dsRNA) synthesis and injection
dsRNAs of five target esterase genes (dsCXE1 , dsCXE5 ,dsCXE13 , dsCXE14 and dsCXE21 ) were synthesized with the MEGAscriptTM T7 high yield transcription kit (Thermo Fisher Scientific, Vilnius, Lithuania) following the manufacturer’s instructions. The cDNA of larva heads and adult antennae was used as the templates. The primers used for dsRNA synthesis are shown in Table S2. ds EGFP (GenBank number: ACY56286) was used as a control in all RNAi experiments. The concentration of dsRNAs dissolved in DEPC-treated water was determined by a NanoDrop 2000 spectrophotometer, and its integrity was analyzed on 1.0% agarose gels. Then, 5 μg dsRNA were microinjected into each individual (injection sites: the base of thorax foot of fourth instar larvae and anterior thorax of male adults) with a microINJECTOR™ System MINJ-1 (Tritech Research, Los Angeles, CA, USA).