PO was estimated spectrophotometrically by recording the formation of dopachrome using L-3,4 dihydroxyphenylalanine (L-DOPA; Sigma, USA) as substrate according to Hernández-López et al. (1996).
Briefly, 50 μl haemolymph supernatant was incubated with 50 μl trypsin (0.1% in CAC buffer: 0.45 M sodium chloride, 0.10M trisodium citrate, 0.01 M sodium cacodylate, pH 7.0) in 96 wells micro plate at 25 °C for 10 min, and then 50 μl L-DOPA (0.3% in CAC buffer) was added