**5. Conclusions**

While this study uses only one insecticide and one mosquito species, the insecticide chosen is a heavily used insecticide for profiling with a well-understood mechanism of action and effect on mosquito populations. The strains tested are also well-established and well-characterized laboratory strains, one of which is wholly susceptible and another of which is highly resistant. With variable results seen for this combination of insecticide and mosquito, it is possible that even more variable results would be seen with a more moderately resistant strain and with novel or less well-understood mode of action with changes to the investigated parameters.

As a result, we make the following recommendations for this bioassay method:

	- a. Holding/exposure temperature
	- b. Holding/exposure humidity
	- c. Source of insecticide-treated papers
	- d. Expiry date or batch number
	- e. Reporting of negative control data
	- f. Reporting of total N per treatment
	- g. Reporting of number of mosquitoes per test unit.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/insects13060544/s1, Supplementary File S1: Table detailing all peer reviewed publications included in the review of the literature and all raw data, and reproducing Table 2.

**Author Contributions:** Conceptualization, G.P. and R.S.L.; methodology, G.P., A.S. and R.S.L.; formal analysis, G.P., D.P.M. and R.S.L.; investigation, G.P.; data curation, G.P.; writing–original draft preparation, G.P.; writing–review and editing, G.P., D.P.M., A.S. and R.S.L.; visualization, G.P. and D.P.M.; project administration, A.S. and R.S.L.; funding acquisition, A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Bill and Melinda Gates Foundation, gran<sup>t</sup> number INV004350, through Innovation to Impact (I2I) at the Liverpool School of Tropical Medicine.

**Institutional Review Board Statement:** Not applicable. **Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article or supplementary material.

**Acknowledgments:** The authors would like to thank the members of the Liverpool Insect Testing Establishment (LITE) in the Liverpool School of Tropical Medicine (LSTM) team for rearing all mosquitoes, Rajpal Singh Yadav for generously sharing WHO documentation to support the review of the literature, and Vincent Corbel for providing feedback on manuscript drafts.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **Appendix A. Odds Ratio Plots**

**Figure A1.** Odds ratio plot for the number of mosquitoes per test unit. The dataset was screened using a binomial GLM, binomial GLMM with random effect for biological replicate, and binomial GLMM with a random effect for biological replicate, and a nested random effect for technical replicate using the glmmTMB package in R. For this analysis, the variable was the age as a factor with 25 mosquitoes used as reference. P-value significance indicated by '\*' if ≤0.05, '\*\*' if ≤0.01 and '\*\*\*' if ≤0.001.

**Figure A2.** Odds ratio plot for age. The dataset was screened using a binomial GLM, binomial GLMM with random effect for biological replicate, binomial GLMM with a random effect for biological replicate and a nested random effect for technical replicate using the glmmTMB package in R. For this analysis, the variable was the age as a factor with 5 days used as a reference. Age groups are represented by the oldest possible age within that group. P-value significance indicated by '\*\*' if ≤0.01 and '\*\*\*' if ≤0.001.
