5.2 The 4.3kb SV is reducing the efficacy of LLINs through genotyping of cone assays samples.
To validate the ability of the 4.3 kb SV marker to predict the impact of resistance on the efficacy of LLINs, we genotyped F1Gounougou samples obtained from cone assay with pyrethroid-only and PBO-based nets. A mortality of 100% was obtained when the Kisumu susceptible strain was exposed to these nets. Genotyping of dead and alive mosquitoes from Gounougou 2018 showed a differential survival for mosquitoes with the 4.3kb SV depending on the bed net used. Mosquitoes were phenotypically resistant to almost all the nets tested with mortality rates below 80%, 9±3.8% for Olyset, 53±11.5% for Olyset plus, 15±3.0% for PermaNet 2.0. PermaNet 3.0 was the most effective, with mortality rates of 100% when a piece of the top was used while the side gave 33 ±5.6%. A significant difference in the distribution of genotypes of the 4.3kb SV was observed for PermaNet 2.0 between dead and alive mosquitoes (χ2= 112.7; P< 0.00001) (Figure 3C; Table 1). No mortality was observed for mosquitoes with either 1 (SV+/SV-) or 2 (SV+/SV+) alleles for the structural variant. The alive PermaNet 2.0 samples were composed of 74.1% (17/27) SV+/SV+, 22.2% (6/27) SV+/SV- and 14.8% (4/27) SV-/SV- while all the dead were SV-/SV- (100%) (Figure 3C; Table 1). Comparing the proportion of each SV genotype between alive and dead mosquitoes revealed that SV+/SV+ homozygote mosquitoes were significantly more likely to survive exposure to PermaNet 2.0 than those completely lacking the 4.3kb SV (SV-/SV-) (OR: 823.45; CI: 148.4-14005.3; P<0.0001). Heterozygote (SV+/SV-) mosquitoes also exhibited a greater ability to survive exposure to PermaNet 2.0 than homozygotes susceptible wild genotype (SV-/SV-) (OR: 291.8; CI: 16.8-5059.6; P=0.0001). SV+/SV+ homozygote mosquitoes also significantly survived better than heterozygotes (SV+/SV-) (OR: 25.4; CI: 1.31-490.7; P=0.032) showing an additive effect of the 4.3kb SV on the resistance phenotype. When comparing at the allelic level, it was revealed that possessing a single 4.3kb SV+ allele confers a significantly higher ability to withstand exposure to PermaNet 2.0 compared to the SV- allele (OR: 565.08; CI: 33.9-9422.4; P<0.0001) (Fig. S3A; Table 1).
Genotyping of 43 samples (with 40 alive and 3 dead) exposed to Olyset, impregnated with permethrin, was inconclusive as only 3 mosquitoes were dead. Analysis of the Olyset plus exposed mosquitoes revealed a significant difference in the survival ability of 4.3kb SV genotypes (χ2= 56.4; P< 0.00001). The distribution of the 4.3kb SV in the alive samples was 64.3% (9/14) for the SV+/SV+, 0% for the SV+/SV- and 35.7% (5/14) for the SV-/SV- (Fig. S3B). Among the dead samples, we had 45.5% (10/22) SV+/SV+, 9.1% (2/22) SV+/SV- and 45.5% (10/22) SV-/SV-. Hence there were no significant differences in surviving exposure to Olyset Plus between the SV+/SV+ and the SV-/SV- genotype (OR: 1.8; CI: 1.1 to 3.2; P = 0.04). A similar result was obtained at the allelic level, with 64.3%of the alive having the SV+ allele and 35.7% having the SV- allele, while for the dead, both alleles were equally represented at 50% each (Fig.S3C; Table 1). No significant survival likelihood was observed when comparing SV+ and SV- (OR: 1.78; CI: 1.01 to 3.13; P = 0.05).