Assessment of the expression of genes in proximity to the 4.3kb structural variant using qRT-PCR.
To determine if the 4.3kb SV affected the expression of nearby genes, qPCR was used to compare the expression of 3 genes (CYP6P5 ,CYP6P9a, and CYP6P9b ) found near it. The Elende x Fang cross was used to generate pools of mosquitoes with different genotypes: homozygous for SV (SV+/SV+), heterozygous for SV (SV+/SV-), and homozygous no SV (SV-/SV-).
After rearing Elende x Fang mosquitoes to the F3generation, adult females aged 3-5 days old were collected and kept at -80oC. DNA was extracted from the legs as described previously (Mugenzi et al., 2020) and used for the 4.3kb insertion genotyping. Bodies were kept in RNAlater (ThermoFisher scientific) and stored at -80oC until genotyping was completed. The bodies were grouped in triplicates of 8 mosquitoes, each according to their genotypes; SV+/SV+, SV+/SV-, and SV-/SV-. RNA was extracted by genotype using the Arcturus PicoPure RNA Isolation Kit (Life Technologies), and cDNA was synthesised using the Superscript III (Invitrogen) as previously described (Mugenzi et al., 2020). Expression levels of CYP6P5 , CYP6P9a, and CYP6P9b were assessed relative to the susceptible FANG strain on the Agilent MX3005. Relative expression and fold-change of each target genes were calculated according to the 2−ΔΔCT method incorporating PCR efficiency (Schmittgen & Livak, 2008) after normalisation with theAn. funestus housekeeping ribosomal protein S7 (RSP7) and actin 5C genes.