Synergist-Insecticide Bottle Bioassay

In a separate experiment (unpublished) in 2018, at the 143rd generation, a synergist assay with piperonyl butoxide (PBO) was undertaken to assess the role of elevated mixedfunction oxidases in resistance. One hundred, 2–5-day-old female An. gambiae Muleba-Kis mosquitoes were tested for metabolic resistance using Piperonyl butoxide (PBO) in four replicates (25 mosquitoes per bottle) at a concentration of 100 μg/mL for one hour preexposure and then followed by 30 min exposure to permethrin (21.5 μg/mL), in accordance to the CDC guidelines [52], with the exception that mortality was considered at 24 h post-exposure. In brief, mosquitoes were pre-exposed to either acetone-coated bottles or PBO for 1 h at a temperature and humidity of 27 ± 2 ◦C and 70 ± 10% RH, respectively, during and after exposure. After pre-exposure, mosquitoes were transferred to holding cages for 60 min before being exposed for 30 min to bottles coated with either 21.5 μg/mL of permethrin or acetone as a control. After exposure, the mosquitos were transferred into holding cups and provided with 10% glucose-soaked cotton pads. After 60 min, post-exposure knockdown was recorded, and mortality was recorded 24 h post-exposure.

#### 2.6.2. Genotypic Basis of Resistance

#### Detection of *kdr* and *Ace*-1 Mutations

The frequency of leucine to serine mutations (L1014S), termed *kdr* east (*kdr*-e) and *ace*-1, were assessed and frequently monitored using the method described by Bass et al. [47,53] to monitor progress in resistance development after successive insecticide selection events. For *Ace*-1 and/or *kdr*-e alleles, a total of 84–88 samples were analyzed by PCR per each test.

#### 2.6.3. Species Identification and Biometric Measures for Fitness
