**3. Results**

#### *3.1. Establishing Dose Response Curves by Ingestion Assay*

An ingestion assay was used to plot dose response curves for orally ingested dinotefuran in a sugar solution. The ratio of LD50 values in each pair of strains, Kisumu vs. VK7 2014 and Fang vs. FUMOZ-R, were similar, so only Kisumu and VK7 2014 were selected to establish further dose response curves by the ingestion assay for comparison between the two methods.

The feeding rate between tests ranged from 70 to 98% and 80 to 97% in Kisumu and VK7 2014 cohorts, respectively, and there was no visible trend with dinotefuran concentration (see Supplementary Material). The LD50 for Kisumu was 0.08 (0.06–0.11) ng of dinotefuran per mg of mosquito and the value for VK7 2014 was 0.17 (0.12–0.23) ng of dinotefuran per mg of mosquito (Figure 3), resulting in a resistance ratio of 2.13. Lethal doses (LD50 and LD95) by ingestion are shown in Table 1.

**Table 1.** Lethal doses and lethal concentrations of dinotefuran ingested in a sugar solution in two strains of *Anopheles* mosquitoes. 95% CI given in parentheses. Kisumu is a lab strain of *Anopheles gambiae*, VK7 2014 is *An. coluzzii*.


**Figure 3.** Mortality comparison between Kisumu and VK7 strains when fed on sugar solution spiked with dinotefuran at known concentrations, resulting in doses of dinotefuran in nanograms per milligram of mosquito. Central lines of each curve represent the dose response of each species. Black lines indicate LD50 values; red refers to the mortality curve of Kisumu; blue refers to the mortality curve of VK7 2014. The shaded areas of each curve represent 95% CI values, generated by R software using the ggplot2 package [13].

#### *3.2. Establishing Dose Response Curves by Topical Application*

Because of the practical challenges in performing ingestion assays, particularly in field settings and at high throughput, dose response curves were also plotted using topically applied dinotefuran in two pairs of *Anopheles* strains as a comparator to the ingestion assay. Topical bioassays (Figure 4A,B) for Kisumu generated an LD50 value of 0.75 (0.55–1.03) ng of dinotefuran per mg of mosquito and VK7 2014 assays generated an LD50 value of 5.34 (3.97–7.19) ng of dinotefuran per mg of mosquito; together this gives a resistance ratio of 7.12. LD50 values for Fang and FUMOZ-R were 2.31 (1.63–3.27) and 7.47 (5.98–9.32) ng of dinotefuran per mg of mosquito, respectively, resulting in a resistance ratio of 3.23. Lethal doses (LD50 and LD95) by topical application are shown in Table 2.

(**A**)

**Figure 4.** *Cont.*

**Figure 4.** Mortality comparisons between (**A**) Kisumu and VK7 2014 strains and (**B**) Fang and FUMOZ-R strains in a topical application bioassay. Central lines of each curve represent the dose response of each species. Black lines indicate LD50 values on both graphs; red lines show mortality curves of the insecticide susceptible strains of each pair of strains (Kisumu and Fang); blue lines show data for insecticide-resistant strains (VK7 2014 and FUMOZ-R). Shaded areas of each curve represent 95% CI values, generated by R software using the ggplot2 package [13]. (**A**) omits data for VK7 2014 treated with 136 ng per mg of mosquito, this gave 100% mortality with no variance as did the highest represented range (54 ng per mg of mosquito, refer to Supplementary Material) and so was removed for clarity.


**Table 2.** Lethal doses and lethal concentrations in four strains of *Anopheles* mosquitoes by topical application of dinotefuran. 95% CI given in parentheses. Kisumu is a lab strain of *Anopheles gambiae*, VK7 2014 is *An. coluzzii*, and Fang and FUMOZ-R are *An. funestus*.
