Synergism Study for Investigating Possible Mechanisms of Insecticide Resistance in Mosquitoes

Metabolic detoxification, in which insecticides are metabolized by enzymes, including cytochrome P450s, hydrolases, and glutathione- S -transferases (GSTs), to become more polar and less toxic, is one of the major mechanisms involved in the development of insecticide resistance. Piperonyl butoxide (PBO), S , S , S ,-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) are inhibitors of P450s, hydrolases, and GSTs, respectively, and are frequently used as insecticide synergists in assessing the metabolic mechanisms that may be involved in the detoxification of insecticides and in the development of insecticide resistance. Synergistic assays can be used to identify the detoxification enzyme that leads to resistance to a specific insecticide. Here, we describe the procedures used in synergist studies of insecticides in both mosquito larvae and adults. The synergist is applied at a maximum sublethal concentration, which is the highest concentration that produces no apparent mortality in the experimental population, above which mortality appears. Insecticide synergistic experiments measure (1) the synergism ratio (SR), which is the difference in the levels of toxicity of a specific insecticide to a strain with and without the presence of the synergists; and (2) the synergism resistance ratio (SRR), which compares SR in the resistant strain with SR in a susceptible strain. In effect, SR indicates the levels of specific enzymes involved in the detoxification of insecticide and SRR identifies the detoxification enzymes/mechanisms that may be involved in the insecticide resistance of insects.