2.6.1. Phenotypic Resistance

#### WHO Susceptibility Test and CDC Bioassay

Insecticide susceptibility bioassays were done from the 17th to 196th generation, in accordance with WHO guidelines [51]. Bioassays were carried out using six insecticides, namely permethrin (0.75%), alphacypermethrin (0.05%), deltamethrin (0.05%), DDT (4%), bendiocarb (0.1%), and pirimiphos-methyl (0.25%), and tests were conducted at 25 ± 2 ◦C and 80 ± 10% relative humidity. Each type of insecticide bioassay was performed in 5 replicates, including one as a control. Twenty to 25, two-to-five-day-old female, blood unfed mosquitoes were tested, constituting a sample size of 100 to 125 mosquitoes for each insecticide. Tested mosquitoes were monitored for knockdown at 60 min and mortality at 24 h post exposure. In parallel with permethrin papers, limited WHO susceptibility bioassays were also conducted against bendiocarb papers (0.1%) and pirimiphos-methyl (0.25%). The insecticide resistance of the selected colony at the 190th generation was compared to the susceptible Kisumu strain using α-cypermethrin in CDC bottle bioassay [52] at concentrations of 52.5, 25.7, 12.5, 6.1, 3, 1.5, and 0 μg/bottle, where 12.5 μg/bottle acted as a discriminating concentration for *Anopheles* [51].
