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).