5.3 Genetic crosses further reveal an association between the 4.3kb SV and pyrethroid resistance
Another approach was to use samples generated from crossing Elende field mosquitoes (SV+/SV+) and FANG lab susceptible colony (SV-/SV-), allowing them to interbreed and later conduct bioassays.
Results from deltamethrin (0.05%, type II) bioassay at F3 exposed for 60 minutes revealed 80.1 ± 5% mortality rates showing that the crossing was still resistant. Genotyping of the 4.3kb SV revealed a strong association between the presence of this structural variant and the ability to survive exposure to deltamethrin. SV+ is significantly associated with survival to deltamethrin exposure (OR: 19.4; CI: 9.5 to 39.6; P< 0.0001) (Figure 3D; Table 2). Homozygote SV+/SV+ mosquitoes had significantly higher likelihood of surviving than the heterozygotes SV+/SV- (OR: 37; CI: 2-689.9; P=0.016) and the homozygote lacking the SV SV-/SV- (OR: 1517; CI: 28.6-80383.1; P = 0.0003) genotypes. The heterozygotes also had higher chances of surviving than homozygotes SV-/SV- (OR: 41; CI: 2.2 to 761.8; P = 0.01).
Another crossing generated with the field Mibellon males (fixed for the SV+) crossed with FANG females (without this SV) at F3revealed a similar association with permethrin (0.75%, type I) and alpha-cypermethrin (0.05%, type II). This SV was found to be linked with the ability to survive permethrin exposure (OR: 5.6; CI: 3.1 to 10.4; P< 0.0001) (Figure 3F; Table 2). At the genotypic level, the SV+/SV+ genotype had increased chances of being alive after exposure to permethrin compared to the SV+/SV- (OR: 6.4; CI: 2.0-20.8; P=0.0018) and the SV-/SV- (OR: 17.7; CI: 4.3-72.3; P = 0.0001) genotypes. Heterozygotes still had higher odds of surviving than the SV-/SV- (OR: 2.8; CI: 0.7 to 10.4; P = 0.0135) (Figure 3G; Table 2).
Furthermore, examining the frequency of the 4.3kb SV in the alpha-cypermethrin 60 minutes alive and 10 minutes dead. Among the alive, 59.09% had the SV+ while 40.91% lacked the SV- (Figure 3H; Table 2), showing that this SV is also associated with survival to alpha-cypermethrin exposure (OR: 116.4; CI: 5.5 to 24.5; P< 0.0001). Calculating the odds of having the SV and surviving revealed a positive association between both. The SV+/SV+ genotype is associated with survival to alpha-cypermethrin more than the SV+/SV- (OR: 3.05; CI: 0.8-11.2; P=0.009) and the SV-/SV- (OR: 72.7; CI: 18.9-278.8; P < 0.0001) genotypes. The SV+/SV- genotype also had more chances of surviving compared to the SV-/SV- (OR: 24; CI: 10.5 to 54.2; P < 0.0001) (Figure 3I).