Introduction
The control of vector-borne diseases, which accounts for more than 17%
of all infectious diseases reported in 2020, still greatly relies on
using insecticides despite a recently recommended vaccine (WHO, 2021).
Insecticides have effectively reduced the number of cases and deaths due
to these diseases, with over 80% of the reduction in malaria cases
between 2000 and 2015 attributed to their use (Bhatt et al., 2015). All
LLINs currently used, including the new generation nets, contain a
pyrethroid insecticide due to their high potency and low toxicity in
humans (Mosha et al., 2022). However, malaria vectors have developed
resistance to pyrethroids which has spread widely in field populations
(Riveron et al., 2019), severely affecting our ability to controlAnopheles with evidence that it could be impacting malaria
transmission (Mosha et al., 2022; Protopopoff et al., 2018).
Insecticide toxicity can act as intense selective pressure, leading to
the rapid evolution of resistance through the overexpression or modified
activity of detoxification enzymes such as cytochrome P450s (Weedall et
al., 2019;), alteration of the target site (Martinez-Torres et al.,
1998; Weill et al., 2004), thickening of the cuticle (Balabanidou et
al., 2016) and behavioural changes (Kreppel et al., 2020). The
widespread insecticide resistance is a major global challenge
threatening the efficacy of current and future vector control tools
(Challenger et al., 2023).
Most studies on the molecular bases of insecticide resistance have
focused on single nucleotide polymorphisms and small indels because they
can be readily identified with short reads (Duneau, 2018; Weedall et
al., 2020; Weetman, 2018). However, growing evidence shows that
structural variants (SVs) contribute to adaptive mechanisms, including
insecticide resistance (Lucas et al., 2019).
SVs represent an essential source of genetic variation and are defined
as large DNA sequence variations, including duplications, deletions,
insertions, inversions, mobile-element transpositions, and
translocations throughout the genome (Alkan, Coe, & Eichler, 2011).
Structural variants are abundant across chromosomes. They are frequently
found near genes where they are often associated with expression and
likely contribute to phenotypic variations (Alonge et al., 2020) and are
predominantly shaped by transposons (Brookfield, 2004). Decades of
research have shown that the alteration of cis-regulatory regions by SVs
can lead to perturbation of gene expression and phenotype (Alonge et
al., 2020; Weischenfeldt et al., 2013). In Anopheles mosquitoes,
gene copy number variations have been identified and correlated with
increased expression of insecticide resistance-associated genes (Lucas
et al., 2019; Weedall et al., 2020). A 6.5kb structural variant inAn. funestus was recently shown to be associated with the
increased expression of two cytochrome P450 genes whose overexpression
confers high resistance to pyrethroids ( Mugenzi et al., 2020) in
Southern Africa and is absent elsewhere (West, Central and East Africa)
suggesting a restriction to gene flow (Weedall et al., 2020). Population
studies have shown a clear demarcation between Southern and
Central/Western/Eastern populations with the clustering of Ghanaian,
Cameroonian and Ugandan populations (Weedall et al., 2020). This points
to the possibility of resistance-associated mutations not only emerging
independently within a population but also spreading from another
resistant population as seen for the cytochrome P450s CYP6P9a andb in southern Africa (Barnes et al., 2017).
A previous study on the promoter region of an insecticide resistance
gene CYP6P9b in An. funestus showed that this gene’s 1kb
upstream region failed to amplify in certain countries (Uganda and
Cameroon) while it was successfully amplified in most regions (Mugenzi
et al., 2019). Therefore, it was hypothesised that a structural variant
in this region could prevent PCR amplification.
Following this observation, we identified a 4.3 kb insertion in the
p450s loci rp1 previously identified by QTL mapping, where two
insecticide resistance genes, CYP6P9a and CYP6P9b are
found (Wondji et al., 2009). This 4.3kb SV is shown to be present in
Central, East and West Africa while absent in southern regions. Temporal
monitoring of this structural variant in An. funestus populations
of Cameroon revealed that it was under strong selection, showing that it
might be an adaptive variation or be linked with a nearby adaptive
mutation. Genetic crosses showed a strong association between this
structural variant and resistance to pyrethroids, and an association was
found between the presence of this structural variant and the
overexpression of nearby genes.