Trizol lysis reagent

Total RNA was isolated from cells or fish liver using Trizol lysis reagent (Sigma Aldrich, St. Louis, MO, USA) followed by isopropanol precipitation. The RNA pellet was washed with 70% (v/v) ethanol. RNA was quantitated using a nanodrop spectrophotometer and cDNA synthesis was performed using MMLV Reverse Transcriptase (MMLV RT [H-], Promega, Madison, WI, USA) and oligo-dT primer. For the detection of cholesterol biosynthesis pathway genes, primers listed in Supplementary Table S7 were used. PCR was performed using 2X GoTaq^® Green Master Mix (Promega).

Considering the importance of carboxypeptidase-1 enzymes of An. gambiae and An. stephensi in the sexual development of Plasmodium falciparum [70 (link), 75 (link)], the cpbAs1 gene of An. stephensi was selected as the target gene for designing and producing dsRNA molecules to evaluate their effects on the target gene expression in the future experiments. This gene has three exons and two introns in An. stephensi and 450-bp from the end of the first exon was selected as the target region for the design of dsRNA. A 150-bp linker in length was used to connect two antiparallel sequences and BamHI and XhoI restriction sites were added to the 5ʹ- and 3ʹ-ends of the designed sequence respectively. This construct was synthesized by ShineGene (Shanghai, China) in pUC57 vector and then sub-cloned into the pET-28a expression vector by digestion with BamHI and XhoI restriction enzymes. At the end, dsRNA expression was performed in E. coli BL21 (DE3) strain by 0.4 mM of IPTG induction for 4 h; the produced dsRNA molecules were purified by TRIZOL Lysis Reagent (Sigma-Aldrich, Missouri, Germany) [76 (link)–78 (link)], recovered from the agarose gel and their quality and quantity were evaluated by agarose gel electrophoresis and Colibri Microvolume Spectrophotometer (Titertek Berthold, Bad Wildbad, Germany), respectively.