Estimation of gene expression of key P450s in triple mutant haplotype
To determine whether the presence of the triple mutant haplotype type was associated with differential expression of genes, we examined individual females from the BusiaUg colony (Triple mutant Freq. 0.297; 95%CI 0.233-0.370). Two legs were removed from individual mosquitoes for DNA analysis with the remaining mosquito kept for RNA analysis. DNA was extracted from legs by boiling in STE buffer at 95°C for 90 minutes and individuals were genotyped using the LNA qPCR assays. RNA was then extracted individually from 8 mosquitoes in each genotypic group - homozygotes for the triple mutant haplotype, wild-type homozygotes, and heterozygotes, using the Arcturus Picopure RNA isolation kit (Thermofisher). We then performed SYBR green based qPCR to measure the expression of Cyp6aa1 and Cyp6p4 together with the known resistance-linked variant Cyp6p3 using the housekeeping genes 40s ribosomal protein S7 (AGAP010592) and elongation factor Tu (AGAP005128) for normalisation. The ΔΔCT values were tested for normality and homogeneity of variances using the Shapiro-Wilks test, and the Bartlett test, respectively. A significant difference in gene expression between the genotypic groups was determined by a two-tailed two-sample Student’s t-test on ΔΔCT values, with a threshold of P=0.05.