Phenoloxidase Activity

Phenoloxidase (PO) activity was evaluated using the methodology proposed by Cruz et al. (2014). For each treatment, three repetitions with up to eight insects each were conducted. From each of the insects, 15 μl of hemolymph was removed, as in previous procedures, and diluted in 1× PBS at proportions 1:1. A 10 μl aliquot of this hemolymph was placed in a well with 35 μl of 1× PBS from a cell culture plate. After filling all the wells, 15 μl of 3,4-dihydroxy-l-phenylalanine (l-DOPA; Sigma–Aldrich) saturated in 1× PBS (4 mg/ml) was added and then the plate was placed in an absorbance microplate reader with a 492 nm filter and the absorbance (the radiation absorbed by the sample) was recorded after 60 min.

Also, for each reading, four wells were filled with only 45 μl of 1× PBS and 15 μl of l-DOPA, with no hemolymph added (blank space). Absorbance values were subtracted from the mean of blank spaces at each reading to obtain corrected absorbance. The mean corrected absorbance values of each of the treatments were compared by ANOVA and the means compared by Tukey’s test (α = 0.05), both using the statistical program IBM SPSS version 23 (IBM 2015).