2.4. Sublethal dose determination

Three replicates of 20 unfed, 2–3‐day‐old male and female Ae. aegypti from the S‐Cairns strain were exposed to a dilution series of permethrin (Merck, analytical standard) using CDC bottle bioassay protocols. 28 The number of mosquitoes that were scored as ‘knocked down’ (unable to fly or right themselves when the bottle is gently rotated), appearing as dead or remaining active was recorded after 30 min of exposure (diagnostic time point). After exposure, mosquitoes were transferred to untreated containers with continuous access to 10% sucrose. Mortality was scored 24 h post‐exposure. The LD₂₅ for the susceptible S‐Cairns strain was determined using nonlinear regression (GraphPad Prism version 7.00) and established as 1 μg/bottle. In response to 30 min of exposure to that dose, male and female R‐BC and R‐TL mosquitoes exhibited no knockdown at the diagnostic time point and no mortality 24 h after exposure. Only surviving mosquitoes from this exposure protocol were used in the following comparisons. Mosquitoes used as controls in the following comparisons were treated following the CDC bottle bioassay protocol 28 using Wheaton bottles coated with acetone in the absence of insecticide.