Esterase, Glutathione S-Transferase and NADPH-Cytochrome P450 Reductase Activity Evaluation in Cacopsylla pyri L. (Hemiptera: Psyllidae) Individual Adults

SIMPLE SUMMARY: The resistance of pest insects to insecticides is a growing problem in all types of crops. Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in Spain, and the large number of insecticide treatments necessary for its control may contribute to the emergence of resistance. Laboratory insecticide toxicity (mortality assays) and biochemical assays (to know the enzymatic mechanisms of the insects in acquiring resistance) are necessary to confirm the existence of this resistance. All the previous methodologies developed to evaluate enzyme activity in C. pyri to date have been performed using “pools” of adults. In this study, we determined the optimal working conditions for the evaluation of the enzymatic activities in individual insects. Determining the frequency of resistant individuals within a population could be used as an indicator for the evolution of insecticide resistance over time. Two laboratory strains, one of them selected with cypermethrin, and two field populations were analyzed for this purpose. The data obtained revealed that one of the three resistance mechanisms studied had a high level of activity and was present in a high proportion of insects in the population selected with the insecticide as well as and in the field populations. These results validated the applied methodology. ABSTRACT: Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in Spain. The large number of insecticide treatments necessary for control may be an important contributor to the emergence of resistance. Laboratory toxicity and biochemical assays are necessary to validate the existence of insecticide resistance and establish the underlying mechanisms. All the methodologies developed to evaluate enzyme activity in C. pyri to date have incorporated “pools” of adults to detect minimum activity ranges. In this study, we determined the optimal working conditions for evaluation of the activities of esterase, glutathione S-transferase and NADPH-cytochrome P450 reductase in individual insects via colorimetric methods using a microplate reader. The main factors affecting enzymatic analysis activity, such as enzyme source and substrate concentration, filter wavelength, buffer pH, reaction time and additives, were evaluated for optimization. Determining the frequency of resistant individuals within a population could be used as an indicator for the evolution of insecticide resistance over time. Two laboratory strains, one of them selected with cypermethrin, and two field populations were analyzed for this purpose. The data obtained revealed high values and great variation in the activity ranges of esterase (EST) in the insecticide-selected population as well as in the field populations validating the applied methodology.