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.