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

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.

Ovicidal and repellent activities of several plant essential oils against Periplaneta americana L. and enhanced activities from their combined formulation

“…Strong potency of trans-anethole against mosquitoes and houseflies was reported by Aungtikun et al 25 and Pavela 55 . Its mechanism of action was reported in Jankowska et al 54 and Bosch-Serra et al 56 : namely, trans-anethole inhibited the development of embryo by blocking cytochrome P450 detoxification enzymes, retarding cell growth and juvenile hormone production and weakening the immune system, leading to eventual embryo death.…”

Age matters: Life-stage, tissue, and sex-specific gene expression dynamics in Ips typographus (Coleoptera: Curculionidae: Scolytinae)

“…To consider differences in the enzyme activity levels between the ESBB developmental stages, biochemical assays for Glutathione S-transferase (GST) and Cytochrome P450 Reductase (CYP450) were performed as described previously (Habig and Jakoby, 1981;Ortego et al, 1999;Bosch-Serra et al, 2021). The developmental stages and gut samples of ESBB were collected and homogenized in sodium phosphate buffer (50 mM) solution at pH 7.0.…”

“…The activity of glutathione S-transferase (GST) was measured using a spectrophotometric method with 5 mM reduced glutathione (GSH) and 1.5 mM 1-chloro-2, 4-dinitrobenzene (CDNB) as substrates (Habig and Jakoby, 1981;Bosch-Serra et al, 2021). One unit of glutathione S-transferase activity is the amount of enzyme-catalyzing 1 µmol of CDNB -GSH conjugate per minute at 30 • C. For the GST activity, the reaction solution contained 20 µl of protein extract (Supplementary Table 2), 170 µl of 5 mM GSH in sodium phosphate buffer (pH 7.0), and 10 µL1.5 mM CDNB to make a 200 µl total reaction volume in a transparent micro-plate (96-well Thermo Scientific TM Sterilin TM Clear Microtiter TM Plates).…”

“…Cytochrome P450 Reductase (CYP450) activity was measured according to Ortego et al (1999) and Bosch-Serra et al (2021). The 200 µl reaction was prepared in a clear micro-plate (96well Thermo Scientific TM Sterilin TM Clear Microtiter TM Plates).…”

Comparative gut proteomics study revealing adaptive physiology of Eurasian spruce bark beetle, Ips typographus (Coleoptera: Scolytinae)

“…Biochemical techniques, including colourimetry, successfully identify and quantify detoxification enzymes such as esterase, glutathione S-transferase, cytochrome, etc. (Bosch-Serra et al, 2021). Transcriptomic approaches reflect organism biology because they allow the investigation of genes that are expressed in a tissue or organism under certain conditions.…”