3.2.1. WHO Susceptibility

The mortality observed in adult Muleba Kis exposed to permethrin (0.75%) test papers in the WHO susceptibility test was high (91% mortality) in the 17th generation (G17) and decreased to less than 20% at G25, then increased at G35. This follows a similar trend to the larvae mortality during selection procedures. The larvae selection with permethrin (pyrethroid types I) was not associated with a reduction in adult susceptibility when exposed to alpha-cypermethrin and deltamethrin (pyrethroid type II insecticides) using the WHO susceptibility bioassay at G35. Resistance to permethrin was the highest compared to the two other pyrethroids (α-cypermethrin and δ-methrin) from the 35th to 125th generations. In parallel with permethrin papers, WHO susceptibility bioassays conducted against bendiocarb papers (0.1%) and pirimiphos methyl (0.25%) resulted in 100% mortality, indicating that Muleba-Kis is fully susceptible to these insecticides. Mortality to DDT was consistently below 89% (Figure 5).

**Figure 5.** WHO susceptibility profiling of adult An. gambiae Muleba-Kisumu across generations. Mortality less than 90% indicates resistance, WHO (51). G = Generation.

On average, the Muleba-Kisumu strain's mortality was below the cutoff point (90%) when tested against permethrin (type I pyrethroid) and DDT papers. Only results with control mortality that were less than 20% were considered for analysis; tests when control mortality was higher than 20% were rejected. When tested against alpha-cypermethrin and deltamethrin (type II pyrethroids), the resistance level was low and above the cutoff value, which is suggestive of susceptibility to this pyrethroid class. However, although several mosquito mortalities were above 90%, during the 35th, 89th, 97th, and 125th generations mortalities scored below 98%, which could imply existence of resistance.

#### 3.2.2. Synergist-Insecticide Bottle Bioassay

CDC bottle bioassays were conducted with permethrin (PRM) and piperonyl butoxide (PBO) against a susceptible strain and a resistant strain. The susceptible strain showed >98% knockdown and mortality after exposure to permethrin, both with and without pre-exposure to PBO. Muleba-Kis showed resistance to PRM (73% mortality), which was restored to susceptible levels (94% mortality) after pre-exposure to PBO, indicating likely involvement of metabolic resistance mechanism in the *An. gambiae* Muleba-Kis strain; see Figure 6 below.

**Figure 6.** The knockdown and mortality rates of An. gambiae Kisumu and An. gambiae Muleba-Kis with and without PBO pre-exposure. Error bars are equivalent to 95% confidence intervals. PRM = Permethrin, PBO = Piperonyl butoxide.

#### *3.3. Polymerase Chain Reaction (PCR) for Species Identification and Resistance Status*

The *kdr* L1014S allele reached fixation in *An. gambiae* s.s. Muleba-Kis populations, coincident with the insecticide selection (Table 1).


**Table 1.** Molecular assays for Muleba-Kis strain over generations.

Note: Ar = *An. arabiensis*, Ga = *An. gambiae* ss, Ace-1 = insensitive acetylcholinesterase, RRe = homozygous mutant, RSe = heterozygous mutant, SSe = homozygous susceptible. When an assay was not done it is coded as N.

#### *3.4. Resistance Strength of the Selected Colony: CDC Bottle Bioassay*

Results from the CDC Bottle bioassay indicate that *An. gambiae* Muleba-Kisumu mosquitoes have lower mortality than *An. gambiae* Kisumu (Figure 7), which is suggestive of a higher level of pyrethroid resistance in the strain.

**Figure 7.** Mortality percentage of An. gambiae Kisumu and An. gambiae Muleba-Kis to varying concentrations of α-cypermethrin in CDC bottle bioassay.

At one and two times the diagnostic concentration of alphacypermethrin—12.5 μg/bottle and 25 μg/bottle, respectively—the Muleba-Kis strain showed significantly higher mortality than the Kisumu strain (two-sample *t*-test, *p* < 0.001). At four times the diagnostic concentration of the same insecticide—52.5 μg/bottle—there was no significant difference in mortality between the two strains.

Exposure of the Kisumu strain against alphacypermethrin in CDC bottles resulted in high mortality, indicating susceptibility against all doses, starting with a low dosage of 1.466 μg/bottle to the highest at 52.5 μg/bottle. On the other hand, exposure to the Muleba-Kis strain showed a dose-response, with mortality as low as 37% against the lowest dose and increasing to 98% mortality at four times the diagnostic dose.

#### *3.5. Biometric Measures for Fitness*

A total of 450 mosquitoes were analyzed: 50 in 2016, 150 in 2017, 150 in 2019, and 100 in 2020.

Data for female mosquito weight and wing length were normally distributed, hence we used the two-sample T-test to compare results between consecutive years. These results indicated that mosquito mean weight in 2017 was significantly higher than all other years, while the other years were similar to each other (Table 2).


**Table 2.** Dynamics in mosquito wing length across years 2016, 2017, 2019, and 2020.

\* Two-sample T-test.

On the other hand, mean wing length was only significantly higher in 2019 compared to the other years (Table 3).


**Table 3.** Dynamics in mosquito weight across years 2016, 2017, 2019, and 2020.

\* Two-sample T-test.
