**3. Results**

#### *3.1. Discriminating Dose Assays*

Bakaridjan, Gaoua-ara, Banfora M, Tiefora and VK7 2014 are all resistant to pyrethroids and DDT according to WHO definitions [24] (Figure 2). Gaoua-ara and Tiefora are also resistant to the organochlorine dieldrin. Bakaridjan, Gaoua-ara and Tiefora are resistant to the carbamates propoxur and bendiocarb with Banfora M resistant to bendiocarb only. None of the five strains are resistant to the organophosphate fenitrothion. Kisumu and Moz are susceptible to all the insecticides tested and results have been reported previously [9]. N'Gousso showed less than 90 % mortality after exposure to propoxur (87%), DDT (61%) and dieldrin (39%) but was susceptible to other insecticides tested.

The results of profiling the five resistant strains against 5 and 10× diagnostic dose (DD) of permethrin are shown in Figure 3. All 5 strains survived exposure to 5× DD (mortality ranged from 14% to 71%). Four of the strains also showed less than 90% mortality after exposure to 10 × permethrin papers (and would be described by WHO as having high intensity resistance) whereas Gaoua-ara with 55% mortality with 5× papers, 98% mortality with 10× is defined by WHO as moderate to high intensity resistance.

#### *3.2. Impact of PBO on Pyrethroid Mortality*

All strains showed significant synergism when pre-exposed to PBO followed by a 4-h exposure to permethrin but synergism was not consistently observed with shorter pyrethroid exposures and PBO pre-exposure did not fully restore susceptibility to permethrin in any of the strains (Figure 4; full mortality results and synergism ratios are available in Supplementary Table S1). The highest synergist ratios were seen for Banfora where significant synergism was observed at all permethrin exposures greater than 2 h and PBO:permethrin synergism ratios ranged from 7:1 (2 h) to 54:1 (3 h). Negative controls (both control papers only and PBO followed by control papers) gave <4% mortality in all assays.

In a separate set of experiments the effect of sequential versus simultaneous exposure to PBO and pyrethroids was compared (Supplementary Figure S1) with pyrethroid exposure duration constant at one hour. PBO did not synergise permethrin in these experiments but the efficacy of deltamethrin was significantly improved in all three strains with both PBO exposure methods (*p* < 0.0001 in all cases). Simultaneous exposure to PBO and pyrethroids results in increased mortalities compared to PBO pre-exposure for all three strains but this was only significant (*p* < 0.05) in Bakaridjan for both insecticides and in Banfora with deltamethrin. Full mortality results and synergism ratios are available in additional Supplementary Table S2.

**Figure 2.** Mosquito mortality following exposure to insecticide papers in discriminating dose assays Mortality rates (%) 24 h after exposure for 5 strains of Anopheles mosquito (results shown from assays performed in 2019). Minimum sample size n = 80. Error bars represent 95% confidence intervals. Dotted line represents the WHO 90 % mortality resistance threshold.

**Figure 3.** Mosquito mortality following exposure to permethrin papers in WHO resistance intensity assays. Mortality rates (%) 24 h after exposure for 5 strains of Anopheles mosquito. Minimum sample size n = 80. Error bars represent 95% confidence intervals. Dotted line represents the WHO 90% mortality resistance threshold. DD: Diagnostic dose.

**Figure 4.** Mortality following exposure to permethrin with or without the synergist PBO. Mortality rates % 24 h after exposure. Minimum sample size n = 80. Error bars represent 95% confidence intervals. Statistical differences between permethrin only and PBO + permethrin for each paired combination indicated as \* *p* <0.05, \*\* *p* < 0.001, \*\*\* *p* < 0.001 Fisher's Exact test.
