*2.3. Topical Application*

Cohorts of 10 2–5 day old female mosquitoes at a time were knocked down using CO2 for 20 s before being transferred to a petri dish with filter paper dampened with purified water. While knocked down, the mosquitoes were positioned ventrally so that their dorsi were easily accessible. 0.2 μL aliquots of insecticide in acetone solution were applied to the dorsal side of each mosquito thorax using a 10 μL Hamilton syringe (Scientific Laboratory Supplies Ltd. (SLS), Nottingham, UK). Mosquitoes were then transferred back into holding cups and knock down or mortality was scored at 30 min, 60 min, and 24 h post-exposure. As well as an acetone-only negative control and a positive control of Permethrin at a concentration of 0.1%, six doses of dinotefuran were applied to Kisumu (0.0002%, 0.0005%, 0.0001%, 0.0025%, 0.004%, and 0.005% *w*/*v*). These six and a further four concentrations

were applied to VK7 2014 (0.01%, 0.02%, 0.04%, and 0.1% *w*/*v*). For the *Anopheles funestus* strains, the range was reduced to three concentrations in addition to the positive and negative controls: 0.0004%, 0.004%, and 0.02% *<sup>w</sup>*/*<sup>v</sup>*. Three replicates were performed, each using different generations of each strain such that 60 Kisumu individuals were treated with each concentration of insecticide and 50 for each control. Similarly, at least three replicates totaling 60 VK7 2014 individuals were tested at each concentration. However, only 20 VK7 2014 individuals were tested at 0.1% as a part of range finding where mortality had already reached 100% in lower concentrations. For both Fang and FUMOZ-R strains, at least 90 mosquitoes were tested at each concentration over three replicates. Data sets from 24 h post exposure were used to generate values for lethal doses (LD). All raw bioassay data is available in Supplementary Materials.

#### *2.4. Establishing Dose Response Curves*

A dose response dataset was established for dinotefuran applied by topical application, as well as by sugar feeding assay in a susceptible strain of *Anopheles gambiae* (Kisumu, [9]) by applying a range of concentrations which gave mortality ranging from 0 to 100%. Topical application gives doses in nanograms per mosquito, converted to nanograms per milligram of mosquito by taking the averages of sample weights of 20 mosquitoes. For the ingestion assay, doses in nanograms per milligram of mosquito were found by estimating the average meal sizes of 10% sugar solution and 0.8% Uranine using fluorimetry (see Appendix A— Quantifying the Average Size of a Sugar Meal Using Fluorescein Sodium Salt (Uranine)). Dose was then inferred through the estimated average meal size of 0.4 μL per feed against the average mosquito mass of 20 individuals per sample.

#### *2.5. Calculating LD Values and Resistance Ratios*

LD50 and LD95 values with associated 95% confidence intervals were obtained for each strain by fitting the data to a dose response model ('drc' package [11] in R Studio [12].

Susceptibility to dinotefuran was compared between strains by calculating a resistance ratio by dividing the LD50 of the pyrethroid resistant strain in each species pair by the LD50 of the susceptible strain.
