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.