Topical application and bottle bioassays measure the responses of adult mosquitoes to specific doses of an insecticide (dose-response). Topical application bioassays are generally used to measure the dose-response of adult mosquitoes to insecticides in which the amount (dose) of insecticides the mosquitoes receive is known in the laboratory. Here, a 0.5-µL drop of the insecticide dissolved in a relatively nontoxic solvent, such as acetone, is applied to the thorax of insects, and the insects' susceptibility to the insecticide is determined in terms of either the median lethal dose (LD50) or 90% of the mortality lethal dose (LD90). Bottle bioassays measure the dose-responses in which the exact amount of insecticide in a bottle is known while the exact amount of insecticide that mosquitoes (field-collected or laboratory-susceptible) receive is not known. Bottle bioassays can be either single dose tests or multiple dose applications. The bottle bioassay described in this protocol is a modified form of the World Health Organization (WHO) and U.S. Centers for Disease Control (CDC) bottle bioassays. For the single bottle assay, a detailed protocol with the amount (dose/bottle) of each insecticide and threshold times is provided by the CDC; here we provide protocols for topical and bottle bioassay applications with multiple doses.
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