**11. Conclusions**

Evidence suggests that in areas where pyrethroid resistance exists, different results in insecticide susceptibility assays with specific pyrethroids currently in common use (deltamethrin, permethrin, α-cypermethrin, and <sup>λ</sup>-cyhalothrin) are not necessarily indicative of an operationally relevant difference in potential performance. Consequently, it is not advisable to use rotation between these pyrethroids as an insecticide resistance managemen<sup>t</sup> strategy. Less commonly used pyrethroids (bifenthrin and etofenprox) may have sufficiently different modes of action, though further work would be needed to examine how this may apply to insecticide resistance management.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2075-4 450/12/9/826/s1, Figure S1: Dose response curves for deltamethrin (A–M) and permethrin (N–Y) using original raw data from the 1998 WHO multicentre study [8]; Table S1: Probit analysis of deltamethrin and permethrin using original raw data from the 1998 WHO multicentre study [8]. Analysis was conducted using PoloJR program within PoloSuite (Ver 2.1). The discriminating dose is twice the LD99. Abbreviations: LD = Lethal dose, DD = Discriminating dose; Figure S2: Calculated discriminating doses (%) for (A) deltamethrin and (B) permethrin. Points show individual sites/strain combinations, and data pooled by species or overall, by insecticide. Site/strain testing <3 concentrations of an insecticide and datasets not robust enough to calculate lethal dose matrixes are excluded. Discriminating doses are set at 2 x the calculated lethal dose at which 99% (LD99) of test mosquitoes were killed. LSHTM *An. stephensi* Beech data included; Figure S3: Mosquito mortality (%) following exposure to deltamethrin in WHO tube bioassays of site/strain combinations for (A) *An. albimanus*, (B) *An. gambiae*, and (C) *An. stephensi*. Numbers above bars show number of exposed mosquitoes; Figure S4: Mosquito mortality (%) following exposure to permethrin in WHO tube bioassays of site/strain combinations for (A) *An. albimanus*, (B) *An. gambiae*, and (C) *An. stephensi*. Numbers above bars show number of exposed mosquitoes. Table S2: Summary statistics of variability in mosquito mortality following exposure to pyrethroids in standard WHO tube or CDC bottle bioassays. Mosquitoes were exposed to insecticides following the recommended methods for each test and mortality in each replicate tube or bottle was recorded 24-h post-exposure. The strains detailed here are those maintained by the Ranson Group or LITE at LSTM, UK. Abbreviations: R = Insecticide resistant mosquito strain, S = Insecticide susceptible mosquito strain, IKR = interquartile range. Table S3: *p*-values (Welch's *t*-test) comparing mean mosquito mortality following exposure to α-cypermethrin 0.05%, deltamethrin 0.05%, or permethrin 0.75% in a standard WHO tube bioassay. Values significant at the 5% level (*p* = < 0.05) are highlighted in bold. Abbreviations; Delta = deltamethrin, Perm = permethrin, Alpha = α-cypermethrin; Figure S5: Mosquito mortality over time following exposure to pyrethroids in a standard WHO tube bioassay. Ranson group (A–J) and LITE (K–O) mosquito strains were exposed to deltamethrin 0.05%, permethrin 0.75% and α-cypermethrin 0.05% in a standard 1-h WHO tube bioassay, and their 24-h mortality was recorded. Coloured circles indicate each individual replicate tube; Figure S6: Box plot summarising mosquito mortality following exposure to deltamethrin 0.05% (top), permethrin 0.75% (middle), or α-cypermethrin 0.05% (bottom) in a standard WHO tube bioassay in Ranson group strains. Each box represents a different mosquito strain. Coloured circles and n-numbers indicate each individual tube replicate; Figure S7: Box plot summarising mosquito mortality following exposure to deltamethrin 0.05% (top), permethrin 0.75% (middle), or α-cypermethrin 0.05% (bottom) in a standard WHO tube bioassay in LITE strains. Each box represents a different mosquito strain. Coloured circles and n-numbers indicate each individual tube replicate; Figure S8: Distribution of larval relatedness of (A) An. gambiae and (B) An. Coluzzii within breeding sites sampled across multiple locations in 2016 from southern Ghana. Individuals (median N = 7 per site) were genotyped by reduced coverage whole genome sequencing and pairwise relatedness categories estimated from chromosome 3 data (2229 SNP markers) as siblings or unrelated using ML-Relate [41]. The percentage of sibling relationships is shown. Sample site numbers are nominal and are not equivalent between plots.

**Author Contributions:** Conceptualization, R.S.L.; data curation, N.L., M.D.K., J.E., H.M.I., T.S.C., B.L., C.L.M., G.P. and D.W.; formal analysis, N.L., M.D.K., J.E., H.M.I., T.S.C., B.L., C.L.M., G.P. and D.W.; funding acquisition, R.S.L.; investigation, N.L., M.D.K., J.E., H.M.I., T.S.C., B.L., A.L., P.J.M., C.L.M., G.P., D.W. and R.S.L.; methodology, N.L., M.D.K., H.M.I., T.S.C., B.L., C.L.M., D.W. and R.S.L.; project administration, N.L. and R.S.L.; supervision, H.M.I., T.S.C., M.J.I.P., D.W. and R.S.L.; visualization, N.L., M.D.K., H.M.I., T.S.C., B.L. and C.L.M.; writing—original draft, N.L. and R.S.L.; writing—review and editing, N.L., M.D.K., H.M.I., T.S.C., B.L., A.L., P.J.M., C.L.M., M.J.I.P., G.P., D.W. and R.S.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a Global Fund to Fight AIDS, Tuberculosis, and Malaria contract awarded to R.S.L. M.D.K. is funded by the Medical Research Council Doctoral Training Partnership (MRC-DTP). T.S.C., and B.L. acknowledge funding from the MRC Centre for Global Infectious Disease Analysis (reference MR/R015600/1), jointly funded by the UK Medical Research Council (MRC) and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement, which is also part of the EDCTP2 programme supported by the European Union. J.E. was funded by a Wellcome Trust Training Fellowship.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data generated or analyzed during this study are included in this published article.

**Acknowledgments:** The authors would like to thank the teams at Centre National de Recherche et de Formation sur le Paludisme (CNRFP) and Institut de Recherche en Sciences de la Santé (IRSS) for establishing the mosquito colonies used in Section 4; Helen Williams, the members of Liverpool Insect Testing Establishment (LITE) team, and the member of the Vector Biology Insectary team at the Liverpool School of Tropical Medicine (LSTM) for rearing mosquitoes and conducting the routine profiling of colonies described in Section 4; Kyle Walker and Sade Oladepo for searching the literature and laboratory data for quantitative toxicity of pyrethroids against malaria and dengue vectors (Section 3); Rachel Davies for her work on PBO bottle bioassays (Section 4); Hilary Ranson for contributions to the final report; and Martin Donnelly, Bashir Adam Ismail, and Hmooda Toto Kafy for provision of the Sudan data for Figure 8.

**Conflicts of Interest:** The authors declare no conflict of interest. Kate Kolaczinski (The Global Fund to Fight AIDS, Tuberculosis, and Malaria) contributed to meetings in which the results were discussed. However, the funders had no role in the design of the study; in the collection or analyses of data; in the writing of the manuscript; or in the decision to publish the results. The findings and conclusions in this paper are those of the authors, and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
