Microplate adaptation of Gomori's assay for quantitative determination of general esterase activity in single insects

Esterase activity is monitored in mosquitoes and other arthropod species because high levels of these enzymes can be associated with pesticide resistance. In the 1950s, G. Gomori devised a colorimetric method to detect esterase activity based on their capacity to hydrolyze aryl-esters. We modified this method for use in microtiter plates. Mosquito homogenates (Culex quinquefasciatus Say and C. pipiens L.) from strains susceptible and resistant to insecticides were allowed to hydrolyze alpha-naphthyl acetate in the presence of Triton X-100 and a specific acetylcholinesterase inhibitor. The alpha-naphthol product was detected colorimetrically by a diazo-coupling reaction with Fast Garnet GBC salt. Triton X-100 improved the extraction of esterases and maintained the azo compound in solution. The linear range of the method was 2-20 nmoles of alpha-naphthol; this high sensitivity permitted accurate determinations in 1/30 portions of single adult mosquitoes from the strain with the lowest esterase activity. To avoid variations due to changes in temperature and duration of assay, results were normalized to equivalent enzyme activity units obtained in a spectrophotometer at 25 degrees C. Depending on the number of homogenate dilutions required, performance of the assay in microplates allowed the simultaneous analysis of 20-80 samples. Female mosquitoes showed higher enzyme activity than males when expressed in nmoles/min per mosquito, but differences were reduced when results were expressed as specific activity (nmoles/min per mg protein). A mosquito strain resistant to organophosphates due to the presence of high levels of esterases showed about 200 times more esterase activity than a susceptible strain or a strain resistant due to insensitive acetylcholinesterase.