Figures legends
Figure 1: Identification and genotyping of a 4.3kb structural variant. A)Transposon insertion position detected by visually inspecting the intergenic region between CYP6P5 andCYP6P9b in IGV. B) PCR amplification of the CYP6P9b andCYP6P5 intergenic region. Lane 1 and 19 are the 1-kilobasepair DNA size marker;2 to 16 are field samples from Cameroon-Mibelong (2016); 17 is the negative control and 18 is a positive control. C) Schematic representation of CYP6P9b and CYP6P5 intergenic region with the structural variant and without the structural variant and Coverage summary of pooled WGS data from Uganda aligned to CYP6P5, CYP6P9b and the 5.5 kb intergenic region. The plot shows that the coverage depth is approximately 40x across the genes and part of the intergenic region, but increases to >100x across the middle portion, indicating that it is a multi-copy transposon. D) Schematic representation of the 4.3kb SV diagnostic assay, consisting of 2 primers flanking the insertion site and 1 in the 4.3kb SV and electropherogram showing the different genotypes. +/+= SV+/SV+, +/- =SV+/SV- and -/- =SV-/SV-.
Figure 2: Spatial and temporal distribution of the 4.3kb SV across Africa. A) Geographical distribution of the 4.3kb SV inAn. funestus population collected across Africa showing elevated frequencies in Cameroon and Uganda and absence in Ghana and Mozambique. Allelic (B) and genotypic (C) frequencies of 4.3kb SV in Tibati showing a decrease in SV- allele and increase in SV+ over the time. Allelic (D) and genotypic (E) frequencies of 4.3kb SV in Mibellon showing a decrease in SV- allele and increase in SV+ over the time. Genotypic frequency of the 4.3kb SV in Gounougou from 2014 to 2021 (F).
Figure 3: Association of 4.3kb SV with pyrethroid resistance. A & B Genotyping results of the 4.3kb SV among the Gounougou 2018 alive and dead deltamethrin post exposure reveal a strong association between the 4.3kb SV and ability to survive. A) shows the genotype distribution and B) allelic distribution. C) Genotype frequencies of the 4.3kb SV in alive and dead mosquitoes exposed to PermaNet2 .0 bed nets showing a positive association between the 4.3kb SV and resistance. D & E illustrate the strong association between 4.3kb SV and the ability to survive exposure to deltamethrin by looking at its genotypic and allelic distribution among dead and alive F3 Elende-Fang crossing mosquitoes. F & G illustrate the strong association between 4.3kb SV and the ability to survive exposure to Permethrin by looking at its genotypic and allelic distribution among dead and alive F3 mibellon-Fang crossing mosquitoes. H & I illustrate the strong association between 4.3kb SV and the ability to survive exposure to α-cypermethrin by looking at its genotypic and allelic distribution among dead and alive F3 mibellon-Fang crossing mosquitoes.
Figure 4: Impact of 4.3kb structural variant on expression of nearby genes and Plasmodium infection. A) Differential qRT-PCR expression for different structural variant genotypes of three cytochrome P450 genes in the immediate vicinity of the 4.3kb SV. Error bars represent standard deviation (n = 3). ns= not statistically significant; *= significantly different at p < 0.05. 4.3kb SV genotypes (B) and alele (C) distribution among Plasmodiuminfected and non-infected samples collected from Obout-Cameroon 2016 showing that samples with the 4.3kb SV are less infected than those without the 4.3kb SV.
Table 1: Association between insecticide susceptibility as determined by WHO tube bioassay or WHO cone bioassay and 4.3kb SV genotype in wild-caught, female Anopheles funestus from Gounougou Cameroon in 2018.