Four strains of laboratory-reared mosquitoes were used in the experiments: the fully pyrethroid-susceptible Anopheles gambiae sensu stricto (s.s.) Ifakara strain; the pyrethroid-resistant (knock-down resistance; KDR) Anopheles gambiae s.s. Kisumu strain; the pyrethroid-resistant (metabolic resistance) Anopheles funestus FUMOZ strain; and the pyrethroid-susceptible Aedes aegypti Bagamoyo strain (Table 1). Colonies of these strains are maintained according to MR4 guidelines [24 ]. The larvae are fed on TetraMin fish flakes (Tetra, UK), and adults on 10% sugar ad libitum; females are membrane-fed cow’s blood for egg production. The colonies are maintained under approximately 12-h:12-h light:dark (natural light) at 27 ± 5 °C and 70 ± 30% relative humidity (RH).

Results of the World Health Organization susceptibility test for the laboratory-reared mosquitoes used in this experiment

Mosquito species (strain)24-h mortality
Permethrin (0.75%)Deltamethrin (0.05%)\documentclass[12pt]{minimal}
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\begin{document}$$\alpha$$\end{document}α-cypermethrin (0.05%)		\documentclass[12pt]{minimal}
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\begin{document}$$\lambda$$\end{document}λ-cyhalothrin (0.05%)		Bendiocarb (0.1%)Pirimiphos methyl (0.25%)
Anopheles gambiae (Ifakara)94%100%100%100%100%100%
Anopheles gambiae (Kisumu)a88%96%72%66%94%100%
Anopheles funestus (FUMOZ)40%38%13%100%96%100%
Aedes aegypti (Bagamoyo)100%100%100%100%96%100%

aKnock-down resistant (KDR)

Nulliparous 3–8 day-old mosquitoes were used for the experiments. Mosquitoes were selected by placing a hand near to their cage, and those that attempted to aggressively bite were aspirated into paper cups. When two mosquito strains of similar morphology were released simultaneously, red fluorescent pigment (Swada, Cheshire, UK) was used to mark the individuals of one of the strains so that the strains could be distinguished between. Mosquitoes were marked by dusting the mesh lid of the cup with a brush to create a cloud of pigment that was deposited onto the mosquitoes. After marking, the mosquitoes were aspirated into 10 × 10 × 10-cm release cages. The mosquitoes were transferred from the insectary to the SFS in a black cloth bag to prevent them from being damaged by the wind. Aedes mosquitoes were sugar starved for 12 h and Anopheles mosquitoes for 6 h prior to commencement of the experiments, to maximise their avidity without inducing excess mortality. Before each experiment, the mosquitoes were acclimatized for 45 min in the corridor of the SFS, which is separated from the experimental space by polyurethane sheeting to prevent the mosquitoes from coming into contact with the tested insecticides.
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