**1. Introduction**

Pyrethroid insecticides have played a key role in interrupting malaria transmission. All insecticide treated nets (ITNs) in use contain pyrethroids; they are the major active ingredient in insecticidal household aerosol sprays and coils and, prior to the advent of widespread resistance, they were the preferred chemistry for use in indoor residual spraying programmes [1]. Malaria vectors will also likely encounter pyrethroids in their aquatic habitats as this insecticide class is still widely used in agriculture, and mosquito breeding sites in rural areas frequently contain detectable levels of insecticides utilised to spray nearby crops [2,3].

Resistance to pyrethroids was first detected in African malaria vectors in the 1970s [4] and is now widespread [5], prompting the search for new chemistries for use in vector control tools. Whether re-purposing chemistries used to control other pest species, or searching for new insecticide classes, the identification of suitable chemistries requires a robust screening pipeline that includes screening potential compounds against a range of mosquito populations resistant to current chemistries [6,7]. Whilst ultimately testing against natural wild populations will be required, the availability of stable laboratory colonies of the predominant vector species, containing the major resistance mechanisms circulating in the field can greatly accelerate the insecticide screening pipeline by identifying resistance liabilities at an early stage [8].

We have previously described the properties of several colonies of *Anopheles* mosquitoes that have been widely used in insecticide discovery programmes; these contain well characterised target site mutations and metabolic resistance conferred by elevated levels of specific pyrethroid metabolising cytochrome P450s [9]. However, recent studies on *Anopheles gambiae* s.l. mosquito populations from West Africa have identified additional, potent pyrethroid resistance mechanisms such as reduced penetration caused by cuticular thickening [10,11], insecticide sequestration by pyrethroid binding proteins in the mosquito appendages and novel resistance associated haplotypes of the pyrethroid target site, the voltage gated sodium channel (VGSC) [12–14]. Several of these resistance mechanisms could potentially cause cross resistance to existing or new classes of insecticides; thus, we sought to establish new colonies of pyrethroid resistant *An. gambiae* s.l. from Burkina Faso, stabilise and quantify their pyrethroid resistance phenotypes and determine the underpinning mechanisms responsible for resistance.

*An. gambiae* is a species complex of at least nine morphologically identical species. Three of these (*An. gambiae* s.s, *Anopeheles coluzzii* and *Anopheles arabiensis*) are amongs<sup>t</sup> the most important malaria vectors and are found in Burkina Faso [15]. Introgression of genes under selection pressure is not uncommon between members of the complex with several well documented cases of exchange of haplotypes containing point mutations in insecticide target sites [16,17]. The Southwest region of Burkina Faso is an important agricultural region of the country and also an area of stubbornly persistent malaria transmission, perhaps partially linked to the exceptionally high levels of pyrethroid resistance in the malaria vectors from this region [18,19]. We established three new colonies from larval collections in the Cascades and Southwest regions of Burkina Faso between 2015 and 2018, encompassing each of the three members of the *An. gambiae* complex found in the country. Phenotyping and molecular characterisation of these new colonies, the previously established Banfora

M colony (Cascades region) and the VK72014 colony (neighbouring Hauts Basin region), revealed high levels of pyrethroid resistance with four colonies meeting the WHO definition of high intensity resistance and the fifth with moderate intensity. Genotyping and RNAseq identified resistance mechanisms in common between strains but also key differences that may have implications for susceptibility to alternative insecticide classes.

#### **2. Materials and Methods**

#### *2.1. Establishment of Strains*

Details of the strains used in this study are provided in Table 1. The origins of the susceptible strains Kisumu and Moz and the pyrethroid resistant Burkina Faso populations VK7 2014 and Banfora M have been described previously [9]. Larval collections from multiple villages in the Comoé Province, Cascades region of Burkina Faso in 2015 led to the establishment of two strains: Bakaridjan and Banfora. Briefly, larvae were reared to adults, allowed to mate and then females transferred to Eppendorf tubes to oviposit individually as described previously [20]. Females were killed by freezing after oviposition. Dried females, and egg papers were transported to the Liverpool School of Tropical Medicine. Species ID on the F0 female was performed [21] and egg batches from *An. gambiae* (s.s.) or *An. coluzzii* females were pooled to establish two separate colonies. The *An. coluzzii* colony was named 'Banfora' after the Banfora district as the colony was established from collections from several villages within this district (Tiefora, Pont Maurice, Sikane and Djomale; Figure 1). The *An. gambiae* s.s. strain was named 'Bakaridjan' as the majority of egg batches used to establish this strain were collected from this village. The *An. coluzzii* Tiefora strain and the *An. arabiensis* Gaoua-ara strains were established as above from larval collections performed in Tiefora Village Comoé Province, Banfora District and Gaoua District, Poni Province in 2018. The insecticide-susceptible colony N'Gousso originated from Cameroon [22].

## *2.2. Mosquito Rearing*

Insectaries were maintained under standard conditions at 26 ± 2 ◦C and 70% relative humidity ±10% under L12:D12 h light:dark photoperiod. All stages of larvae were fed on ground fish food (TetraMin® tropical flakes, Tetra®, Blacksburg, VA, USA) and adults were provided with 10% sucrose solution ad libitum.

#### *2.3. Selection and Resistance Profiling*

The five insecticide resistant strains were routinely selected every 3rd to 5th generation with 0.05% deltamethrin to preserve their resistant phenotype. Insecticide papers were purchased from the WHO facility at the University Sains Malaysia (USM), Penang, Malaysia and used a maximum of 6 times following the WHO procedure [24]. Selection was undertaken at the adult stage (2–5 days old) using the WHO susceptibility bioassay [24]. Exposure times varied between strains to ensure at least 50% survival (VK7 2014 2 h, Banfora M and Bakaridjan 2–3 h, Gaoua-ara 2–4 h and Tiefora 4–5 h). All adults from the generation to be selected were exposed, with results scored from at least 100 individuals. Following exposure, the mosquitoes were transferred to holding tubes and supplied with 10% sucrose solution and the initial knockdown effect was scored immediately post exposure. At 24 h post exposure, mortality rates were recorded. Bioassays and 24 h holding periods were conducted at 26 ± 2 ◦C and 80 ± 10% RH.


**Table 1.** Summary of the *Anopheles gambiae* s.l. mosquito strains used in the study.

Each strain was profiled annually against eight insecticides (except VK7 2014 which was profiled against six insecticides) representing the different insecticide classes currently used for mosquito control, to monitor the stability of their resistance phenotype; as described in [9] insecticides used were permethrin, deltamethrin, alpha-cypermethrin, DDT, dieldrin, bendiocarb, propoxur and fenitrothion. Results for VK7 2014 and Banfora M have been reported previously [9], but are included here for comparative purposes.

The intensity of resistance was evaluated in the different strains using papers treated with 5× and 10× the diagnostic dose of permethrin following the WHO procedure [24].

**Figure 1.** Map of Burkina Faso showing mosquito collection sites.
