*2.4. Synergist Bioassays*

The impact of the synergist piperonyl butoxide (PBO) on pyrethroid induced mortality in each of the resistant strains was assessed in two separate experiments. Firstly, 2–5 day old female mosquitoes were pre-exposed to PBO papers impregnated with PBO (4%) followed by 1, 2, 3 or 4 h exposures to papers impregnated with permethrin (0.75%) according to the WHO protocol [24].

In the second experiment, mortality rates following sequential PBO then pyrethroid exposure were compared with simultaneous exposure to insecticide and synergist. Adult females from three strains were exposed to either (1) a pyrethroid only 1-h exposure; (2) a 1-h PBO pre-exposure followed by a 1-h pyrethroid exposure, or (3) a 1-h combination exposure (with PBO and either pyrethroid on the same paper). These experiments were performed separately using 0.75% permethrin papers and 0.05% deltamethrin papers.

In both experiments, solvent only paper (no AI) and a PBO control, where a 1-h PBO exposure was followed by 1-h blank exposure were included. Differences in mortality with and without PBO exposure were analysed for significance using Fisher's exact test.
