Functional genetic validation of key genes conferring insecticide resistance in the major African malaria vector, Anopheles gambiae

Abstract

This PDF file includes: 640 Supplementary text: Cyp6 expression analysis using the ΔΔCt method 641 Figs. S1 to S4 642 Tables S1 and S2 643 29 Supplementary Text 644 Cyp6 expression analysis using the ΔΔCt method 645 Threshold cycle (Ct) values were corrected for primer efficiency applying the formula Corrected 646 Ct = Ct (Logx / Log2), where x = 2 if efficiency is >100% and x = 1+Efficiency if efficiency is 647 <100%. 648 Ct values obtained for target genes were normalised against those of the housekeeping genes 649 RPS7 and Ubiquitin using the formula ΔCt = Ct target -Ct housekeeping. 650Mean ΔCt values of GAL4/+ mosquitoes were then compared to those of GAL4/UAS mosquitoes 651 using the formula ΔΔCt = ΔCt GAL4/+ -ΔCt GAL4/UAS. Fold change (FC, x) expression 652 between the two populations was calculated with the formula FC = (2ᶺ GAL4/UAS ΔΔCt) / (2ᶺ 653 GAL4/+ ΔΔCt). 654Statistical differences in fold change expression between GAL4/UAS and GAL4/+ were 655 determined using t test (unpaired, two-tails, assuming equal variance). 656

Insecticide Resistance Profiling of Anopheles coluzzii and Anopheles gambiae Populations in the Southern Senegal: Role of Target Sites and Metabolic Resistance Mechanisms

“…Likewise, high GSTs activity was reported to be associated with insect resistance to DDT and pyrethroids [59,69]. The following candidate genes used in this study (CYP6M2, CYP6P3 [70][71][72], CYP6Z2 [73], CYP4G16, CYP4G17 [19,74,75] and CYP9K1) have been reported to be involved in pyrethroid resistance in An. gambiae in Africa [76].…”

Genome variation and population structure among 1142 mosquitoes of the African malaria vector species Anopheles gambiae and Anopheles coluzzii

“…Our data resource includes CNVs at four Cyp gene loci (Cyp6p/aa, Cyp6m, Cyp6z, and Cyp9k), which could serve as molecular markers of CYP-mediated metabolic resistance. Glutathione Stransferase enzymes are also associated with metabolic resistance to pyrethroids (Ochieng'Opondo et al 2016;Adolfi et al 2019) B A Figure 5. Nucleotide diversity within the female-specific exon 5 of the doublesex gene (dsx; AGAP004050), a key component of the sex determination pathway and a gene targeted for Cas9-based homing endonuclease gene drive (Kyrou et al 2018).…”

“…In both plots, the location of exon 5 within the femalespecific isoform (AGAP004050-RB; AgamP4.12 gene set) is shown above (black indicates coding sequence; gray, untranslated region), with additional annotations above to show the location of Cas9 target sequences containing at most one SNP, and the putative exon splice enhancing sequences ("RE") reported by Scali et al (2005). as well as other insecticide classes (Mitchell et al 2014;Riveron et al 2014;Pavlidi et al 2018;Adolfi et al 2019), and we found CNVs at the Gste locus that could serve as molecular markers of this alternative resistance mechanism, which is not inhibited by PBO. Gste CNVs were less prevalent in our data set than Cyp CNVs, and the geographical distribution also differed, suggesting they may be driven by different selection pressures (Supplemental Fig.…”

“…Metabolic resistance to pyrethroids is mediated at least in part by increased expression of CYP enzymes (Kwiatkowska et al 2013;Edi et al 2014;Ngufor et al 2015;Vontas et al 2018), and we found CNV hot-spots at two loci containing Cyp genes (Lucas et al 2019a). One of these loci occurs on chromosome arm 2R and overlaps a cluster of 10 Cyp genes, including Cyp6p3 previously shown to metabolize pyrethroids (Müller et al 2008) and recently shown to confer pyrethroid resistance when expression is experimentally increased in A. gambiae (Adolfi et al 2019). The second locus occurs on the X Chromosome and spans a single Cyp gene, Cyp9k1, which has also been shown to metabolize pyrethroids (Vontas et al 2018).…”

“…Cyp6m2 (Stevenson et al 2011), although there was only a single CNV allele at each locus. Overexpression of Cyp6m2 has been shown to confer resistance to pyrethroids but increased susceptibility to the organophosphate malathion (Adolfi et al 2019), and so, the selection pressures at this locus may be more complex. The precise phenotype of these CNVs remains to be characterized, but given the multiple lines of evidence showing that increased expression of genes at these loci confers pyrethroid resistance, it seems reasonable to assume that CNVs at these loci can serve as a molecular marker of CYP-mediated pyrethroid resistance.…”