Species Identification

To ensure colony species purity, at 43, 99, 131, 150, 162, 168, 178, 188, 190, 198, and 204th generations, the PCR for species identification was conducted using single nucleotide polymorphism genotyping [47]. At each generation, a total of 84-88 mosquito samples were tested.

Biometric Measurements

The size of individual adult females was estimated by the average length of left wings, while weight was measured by weighing the whole mosquito. To measure the wing length, a total of one hundred Muleba-Kisumu females were randomly sampled from five selected mosquito-rearing cages quarterly, covering the 99th to 204th mosquito generations. The wings were cut and placed on a stage micrometer (10 mm long with 100 × 0.1 mm (100 μm) divisions). Wing length was measured as the distance from the alula to the end of the wing where vein three ends [54–56] using an ocular micrometer at 2X objective magnification on a Nikon stereomicroscope, Model; SMZ 645 [Nikon Instruments, 1300 Walt Whitman Road, Melville, NY 11747-3064, U.S.A.], see Figure 3 below.

**Figure 3.** Image of wing aligned on a micrometer indicating the ocular gradations which correspond to the distance on the stage micrometer. Number 1–5 indicates the wing veins, where vein 3 is used for measuring the distance from the alula to the wing fringe (wing length). This photo was copied from PAMVERC Test Facility SOP with permission, originally taken and donated by MK (co-author).

Wing length and weight were continuously monitored in succeeding years regardless of whether selection with insecticide selection was done or not.

## *2.7. Statistical Analysis*

The WHO criteria were used to classify the resistance or susceptibility status of the tested mosquito populations [51]. Descriptive analysis was performed to check for normality on wing and weight measures from the samples. Wing length and mosquito weight measures were all normally distributed. Using Stata [57], two sample T-tests were performed to compare wing length or mosquito weight across the years for the *An. gambiae* Muleba-Kisumu, and differences in mortality between *An. gambiae* Kisumu and *An. gambiae* Muleba-Kisumu across different concentrations in the CDC bottle bioassay.
