Bioassays

The conventional WHO bioassay³¹ was used to test the susceptibility of MRS to six insecticides at diagnostic concentrations: two pyrethroids, deltamethrin (0.05%) and permethrin (0.75%); one pseudo-pyrethroid, etofenprox (0.50%); one carbamate, bendiocarb (0.10%); one organophosphate, fenitrothion (1%); and one organochlorine, DDT (4%). At 2–5 days old, MRS females were exposed to impregnated papers for 1 hour according to the standard WHO bioassay³¹. Susceptible Kisumu females were used to verify the quality of the impregnated papers. The number of mosquitoes knocked down during exposure was regularly recorded to determine KdT₅₀, the time point at which 50% of the mosquitoes are knocked down, and KdT₉₅, the time point at which 95% of the mosquitoes are knocked down. After 1 hour of exposure, the mosquitoes were transferred to holding tubes without insecticide and fed with a 10% honey solution. Mortalities were recorded 24 h post-exposure. For each test, a batch of 25 MRS females were also exposed to untreated paper as a negative control group, and Abbott’s formula was used to correct the mortality in the treated group when control mortalities exceeded 10%. According to the resistance cut-offs established by the WHO, mortality ≤90% indicates a population resistant to the insecticide, >97% a susceptible population, and 90–97% suspected resistance. After 24 h, control MRS were individually stored at −80 °C for biochemical analysis. Other batches of unexposed MRS from the same population and the 24 h survivors were preserved in RNA extraction buffer (NucleoSpin, MACHEREY – NAGEL) and kept at −80 °C for transcriptome analysis.

Bioassays were also conducted after pre-exposure of mosquitoes to the synergist piperonyl butoxide (PBO), an inhibitor of cytochrome P450 monooxygenase (CYP450). A significant increase in the 24 h mortality rate after PBO exposure before insecticide would indicate the contribution of CYP450 to insecticide resistance. The decrease of resistance level when compared to a susceptible reference strain tested under the same conditions allows to estimate the relative contribution of CYP450 to the overall resistance. To this end, females from Kisumu and MRS strains were exposed to three different treatments in WHO test tubes: 1 h with PBO (4%), 1 h with deltamethrin (0.05%), and 1 h with PBO (4%) followed by 1 h with deltamethrin (0.05%). The number of mosquitoes knocked down during each treatment was recorded at 5, 10, 15 and 20 minutes, then every 10 minutes until 1 h for KdT determination.