Gene knockdown and quantitative reverse transcription PCR (qRT-PCR)

The cDNA clones of target genes were obtained using gene-specific primers (Table S4). PCR amplicons of GFP, V-ATpase_H_N3 and caudal tailed with a T7 promoter (TAA TAC GAC TCA CTA TAG GGA GA) were used to synthesize dsRNAs using a MEGAscript T7 High Yield Transcription kit (Thermo Fisher, China). Four- to six-day-old females received a total 69 nL dsRNAs (4 μg/μL) through intra-thoracic microinjection using a Nanoject II microinjector (Drummond, USA). Injected mosquitoes were allowed to recover for four days prior to infection (Blandin et al., 2002). Silencing efficiency was verified by qPCR four days post dsRNA treatment using the primers listed in Table S4. The cDNA was prepared from total RNA using the 5XAll-in-One MasterMix (with AccuRT Genomic DNA Removal Kit) (ABM, China). Levels of target genes were determined by a Roche LightCycler 96 Real Time PCR Detection System with SYBR Green qPCR Master Mix (Biomake, China). The data were processed and analyzed with LightCycler 96 software. Ribosomal gene S7 of An. stephensi and 18S rRNA of P. berghei were used as the internal references (Baptista et al., 2010; Dimopoulos et al., 1996). Relative quantitation results were normalized with reference genes and analyzed by the 2^−ΔΔCt method (Livak and Schmittgen, 2001). Gene expression of the dsRNA treated group was normalized to that of dsGFP controls.