2.8. Acetylcholinesterase (AChE) Activity Assay

For each replication, 20 mg of mixed-sex insects were homogenized in Sorensen’s buffer (0.05 M; pH 7.4) in a 1:10 ratio. Thereafter, the homogenate was centrifuged (10,000 RPM, 10 min, 4 °C). Blind tests were prepared using buffers instead of homogenates. All measurements were performed with the Tecan M200 spectrophotometer in Corning^® 96-well UV-Transparent microplates. In the samples, the protein content was determined using the Bradford method, and then the enzyme activity was converted into Δ/min/mg of protein [11].

The AChE activity was determined by the colorimetric method of Ellman et al. [12], based on the changes in the absorbance of 412 nm light by DTNB (Ellman’s reagent) over time in the presence of AChE. The reaction mixture consisted of 150 μL DTNB (0.01 M), 20 μL AChTI (0.075 M), 10 μL probe. The eight consecutive measurements were performed every 30 s.