Methods in Anopheles Research - MR4

Chapter 5 : Insecticide Resistance Monitoring5.2 Microplate Enzyme Activity AssaysPage 2 of 4(possibly corresponding to resistance) causing the distribution to be skewed to the right. Most fieldpopulations will contain both resistant and susceptible individuals. When this method is performed inconjunction with bioassays, the levels of resistance observed in the bioassay should correspond to theenzyme activity. If a higher frequency of resistance is observed in the bioassay than what is suggested bythe enzyme assay, a different resistance mechanism may be responsible.10090susceptibleresistantnumber mosqs80706050403020100susceptiblefield0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6absorbance 570 nmFigure 5.2.2. The absorbance of individuals of two different populations. Note that the referencesusceptible population contains no individuals having an absorbance > 0.9. In contrast, the fieldpopulation contains not only individuals with absorbance levels similar to those of the susceptibles butalso many with much higher absorbance levels. One may conclude that the latter individuals representinsecticide-resistant mosquitoes if similar proportions are confirmed by bioassays.It is sometimes useful to directly compare the absorbances obtained from two different enzyme assays oftwo populations when different mechanisms may be responsible for resistance observed in a bioassay.This also allows one to identify the presence of resistant individuals using a smaller sample. Such arepresentation is shown in Figure 5.2.3.Materials:• 1.5 ml tubes (e.g. Kontes)• Disposable or reusable pestles for above tubes• Pipettors and tips• Multichannel pipettors• Forceps• Freezer (used to kill or anesthetize samples)• Timer (digital counter capable of counting seconds)• Microplates• Microplate-reading spectrophotometer• Analytical balance• Dibasic potassium phosphate• Monobasic potassium phosphate