**5. Conclusions**

This study demonstrates that different species within a species complex, collected from the same geographical area (including two originating from the same larval collections) and hence presumably under similar selection pressures, can evolve multiple, different resistance mechanisms. This may be indicative of the exceptionally strong selection pressure exerted on Anopheles mosquitoes in this major agricultural region in Burkina Faso but it presents a major challenge for existing and new insecticide based control tools. As the strains have been maintained under selection pressure in the laboratory, the fitness costs of alternative mechanisms, and hence their stability under natural settings, are unknown but nevertheless the strains represent a valuable biological resource for the screening of new insecticides for potential resistance liabilities. From an evolutionary perspective, genomic sequencing of these strains, coupled with further sampling of sympatric members of the species complex in the region, provides an opportunity to investigate the role of introgression versus de novo mutation, in the evolution of resistance, and in assessing the response to the introduction of ITNs with new classes of insecticides.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/insects13030247/s1, Supplementary Table S1. Complete mortality data for standard PBO bioassays plus PBO: perm synergism ratios for five resistant strains. Mean mortality rates (%) across three reps 24 h after exposure are given, minimum sample size 75. Supplementary Table S2. Complete mortality data for simultaneous and sequential exposures of PBO with either permethrin and deltamethrin plus PBO synergism rations for five resistant strains. Mean mortality rates (%) across three reps 24 h after exposure are given, minimum sample size 75. Supplementary Table S3. The total number of genes differentially expressed across resistant compared to susceptible strains. Supplementary Table S4. qPCR data showing expression levels of the panel of detoxification genes in the five resistant strains. Mean fold changes from 3 biological replicates and 3 technical replicates, relative to susceptible strain(s), SD = standard deviation. Data have been normalised against expression in the susceptible strain as described in the methods. Supplementary Figure S1: PBO synergism results for three resistant anopheline strains with simultaneous and sequential exposures to PBO and permethrin (Perm) or deltamethrin (Delta). Mortality rates % (24) hours after exposure. Minimal sample size n = 80. Error bars represent 95% binomial confidence intervals. Statistical differences between insecticide only and PBO + insecticide are indicated as \* *p* < 0.05, or ns- not significant. Supplementary Figure S2: RNA Correlation matrix for five resistant strains and three susceptible strains. Red represents a strong correlation and blue represents a disassociation. VK7 = VK7 2014, Bak = Bakaridjan, T = Tiefora, K = Kisumu, B = Banfora M, NG = N'Gousso, M = Moz, G= Gaoua-ara. Supplementary Figure S3: GO terms enrichment up regulation for five resistant strains. Supplementary Figure S4: GO terms enrichment down regulation for five resistant strains. Supplementary Figure S5: Expression of genes in the cuticular hydrocarbon production pathway in five resistant strains. Blue represents genes down regulated and green represents genes upregulated. Reduct = reductase, Elong = elongase, Desat = desaturase, FAS = fatty acid synthase. Supplementary Figure S6: qPCR P450 expression in five resistant strains. Error bars represent standard deviations, statistically significant differences in expression level relative to susceptible strains are indicated as \**p* < 0.05, \*\**p* < 0.01, \*\*\* *p* ≤ 0.001 ANOVA test followed by Dunnett's or Dunn test.

**Author Contributions:** Conceptualization, H.R.; Data curation, V.A.I.; Formal analysis, J.W. and V.A.I.; Funding acquisition, V.A.I. and H.R.; Investigation, J.W., V.A.I. and M.M.; Resources, K.H.T., A.S.H., J.C.M., R.K.D., W.M.G. and N.S.; Supervision, R.K.D., W.M.G., N.S. and H.R.; Visualization, J.W. and V.A.I.; Writing—original draft, J.W., V.A.I. and H.R.; Writing—review and editing, K.H.T., W.M.G. and N.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a PhD studentship to J.W from IVCC, an MRC Skills Development Fellowship to V.I (MR/R024839/1). We also acknowledge support from Wellcome Trust, Grant/Award Number: 200222/Z/15/Z and the Medical Research Council of the UK (grant number MR/P027873/1) through the Global Challenges Research Fund.

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

**Data Availability Statement:** All RNAseq data is freely available on SRA under accessions PR-JNA780362 and PRJNA750256. Custom code for enrichment analysis is available on GitHub at https://github.com/VictoriaIngham/BurkinsStrains.

**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 assistance with field collections of mosquitoes; 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 assistance with rearing mosquitoes and conducting the routine profiling of colonies.

**Conflicts of Interest:** The authors declare no conflict of interests.
