2.5. Phenoloxidase assaysPhenoloxid

2.5. Phenoloxidase assays
Phenoloxidase activity was assayed spectrophotometrically in 96-well flat-bottomed microtiter plates (Sarstedt, Technology Park,SA). Hydroquinone monomethyl ether (4HA; Sigma Aldrich, Castle
Hill, Australia) was used to measure monophenolase activity.
This assay was adapted from Aladaileh et al. (2007).
One hundred microliters of HL was added per well in triplicate for each sample. The HL
was then incubated with 100 μL 4HA (5 mM in FSW),
containing 1 mM 3-methyl-2-benzothiazoline hydrazone (MBTH; Sigma Aldrich, Castle Hill, Australia). The samples were held at room temperature
(in the dark) and the absorbance was measured at 490 nm at 0 and 60 min after addition of the substrate.
Phenoloxidase activity was calculated as a rate over time. Data were adjusted for the total protein content (Bio-Rad Bradford assay) of each sample and the absorbance of blanks (wells containing PO substrates without HL).
Data at each time-point are presented as fold differences relative to the mean PO activity of the 0 min controls
(no stress treatment).