Methods in Anopheles Research - MR4

Chapter 5 : Insecticide Resistance5.1 Insecticide Resistance Bioassays5.1.3 Guidelines for Evaluating Insecticide Resistance in Vectors using the CDC Bottle BioassayPage 18 of 24Use of synergistsCommonly used synergists in conjunction with the CDC bottle bioassay: Piperonyl butoxide (PBO), which inhibits oxidase activity; S.S.S-tributlyphosphorotrithioate (DEF), which inhibits esterase activity; Ethacrynic acid (EA), diethyl maleate (DM), and chlorfenethol (CF), which inhibitglutathione transferase activity.It is also possible to use a combination of the above synergists. Testing mosquitoes with synergists is atwo-step procedure. Mosquitoes are first exposed to the synergist(s) for 1 hour and then tested forinsecticide resistance using the CDC bottle bioassay. A schematic representation of performing abioassay with synergist(s) is shown in Figure 5.1.3.11.Preparation of bottles for synergist bioassaysTo use the bioassay with synergists:1. Prepare the synergist stock solution by diluting the appropriate amount of synergist in acetone ortechnical grade ethanol to be able to coat the bottles with the concentrations shown in Table 3.To make these stock solutions, use the same procedure used for making insecticide stocksolutions. In brief, dilute the appropriate amount of synergist in acetone or technical gradeethanol. To get a concentration of 400 µg/bottle of piperonyl butoxide, dissolve 400 mg inenough acetone or absolute ethanol to make 1 liter of solution. Each 1 ml of this solution willcontain 400 µg of piperonyl butoxide.2. Mark one bottle and its cap as the synergist-control bottle (without synergist);3. Mark a second bottle and its cap to be the synergist-exposure bottle;4. Add 1 ml of acetone or ethanol to the synergist-control bottle and put the cap back on tightly;5. Add 1 ml of the synergist stock solution to the synergist-exposure bottle and put the cap on backtightly;6. Coat the bottles, remove the caps, and let the bottles dry (Figures 5.1.3.3 – 5.1.3.6)7. Prepare two test sets of bottles to run the CDC bottle bioassay.Table 5.1.3.3: Synergist concentrations used in the CDC bottle bioassay.SynergistSynergist concentration(µg/bottle)Chlorfenethol 80Diethyl maleate 80Ethacrynic acid 80Piperonyl butoxide 400S.S.S-tributlyphosphorotrithioate 125