Dose responses of phenoloxidase
To determine the presence of phenoloxidase (PO) activity within a scleractinian coral, a dose response curve was created using pooled sample extracts from colonies (n = 5) of a massive Poritessp.. Within 96-well microtiter plates, six wells each of 5, 10, 15, 20 and 25 μl of the pooled sample extract were set up and the corresponding 55 to 35 μl of phosphate buffer (50 mmol.l-1 pH 7.5) added to make a total well volume of 60 μl. Next, 25 μl of double distilled water (ddH2O) was added to each well and plates were left to incubate at room temperature for 20 min. Then 30 μl of 10 mmol.l-1 solutions of one of the following colourless substrates was added to two of the wells of each extract volume (0 to 20 μl): Tyramine (Fluka 93810) for mono-phenoloxidase activity, dopamine hydrochloride (Sigma-Aldrich H8502) for o-diphenoloxidase activity and hydroquinone (Sigma H9003) for p-diphenoloxidase (laccase) activity (Jordan & Deaton 2005; Table 2.1). In addition, wells controlling for auto-oxidation of the substrates were included, where the sample extract was replaced with ddH2O, sample effects were tested (and not detected) in the absence of substrate and no colour change was detected using boiled sampled extracts (data not shown). The absorbance at 410 nm was recorded every 5 min for 45 min using a Spectramax M2 (Molecµlar Devices) spectrophotometer, and the change in absorbance for the linear portion of the reaction curve was plotted as PO activity.
Data were tested for normality and homoscedasticity (Shapiro-Wilks and Levene’s tests, respectively) prior to performing statistical analyses. Data sets that did not meet the criteria were log transformed to meet the parametric criteria of normality and equal variances. To determine if enzyme activities were correlated with sample extract volumes, analyses of variance of regression (May & Bigelow 2005) were conducted using Sigma Plot (10.0). Enzyme activity in the presence and absence of inhibitors was compared using a Mann-Whitney U test, as parametric assumptions were not met in the latter case.
Dose-dependent responses were significant for all three types of phenoloxidase (PO) activity investigated (Figure S1A). Activity was positively correlated with sample extract volume for each PO, with the regression explaining 90% of activity variation in mono-phenoloxidase (R2 = 0.90, F (1, 4) = 29.89, P = 0.01), 93% in o-diphenoloxidase (R2 = 0.93, F (1, 4) = 38.62, P = 0.01) and 84% in p-diphenoloxidase (R2 = 0.84, F (1, 4) = 16.38, P = 0.03). At 20 µl, mono-phenoloxidase had approximately 10-fold lower activity than either p-diphenoloxidase or o-diphenoloxidase. Additionally, although the variation was very high for p-diphenoloxidase activity with greater sample extract, activity appeared to plateau from 20µl, indicating there may be asubstrate limitation with greater extract volumes. The presence of PO activity within corals was further confirmed via the significant (~85%) inhibition of o-diphenoloxidase with sodium azide (Figure S1B; n = 18; Mann-Whitney U = 3.0, P < 0.001).
Figure S1: Phenoloxidase (PO) activity of pooled Porites massive sp. samples showing; a) dose-response curves of each PO, which demonstrated an increase in activity with sample volume (mono-phenoloxidase: R2 = 0.90, F (1, 4) = 29.89, P = 0.01; o-diphenoloxidase: R2 = 0.93, F (1, 4) = 38.62, P = 0.01; and p-diphenoloxidase: R2 = 0.84, F (1, 4) = 16.38, P = 0.03) and b) a comparison of mean o-diphenoloxidase activity (± SE) between assays with (inhibited) and without (normal) the addition of sodium azide (n = 18 samples; Mann-Whitney U = 3.0, P < 0.001).
Jordan, P. J. & Deaton, L. E. 2005 Characterization of phenoloxidase from Crassostrea virginica hemocytes and the effect of Perkinsus marinus on phenoloxidase activity in the hemolymph of Crassostrea virginica and Geukensia demissa. Journal of Shellfish Research24, 477-482.
May, S. & Bigelow, C. 2005 Modeling nonlinear dose-response relationships in epidemiologic studies: statistical approaches and practical challenges. Dose Response3, 474-490.
Influence of pH on phenoloxidase activity
As enzyme activity may vary under different pH conditions, to establish the influence of pH on the hard coral PO assays, 20 µl were combined with 25 µl of ddH2O and 40 µl of phosphate buffer at either pH 6.4, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 or 8.2, for three samples of A. millepora, in a 96-well plate. To activate the assay, 30 µl of 10 mM dopamine hydrochloride was added to each sample well. Additionally, sample blank and dopamine hydrochloride blank wells were included for each pH to measure any substrate auto-oxidation or sample extract effects. The change in absorbance over 15 min (within the linear portion of the reaction curve) at 410 nm was recorded using a Spectramax M2 (Molecular Devices) spectrophotometer.
There was no significant difference in mean PO activity (o-diphenoloxidase) using phosphate buffers of pH 6.4 to 8.2 (F(9, 29) = 0.408, P = 0.916), although the highest activities were between pH7.4 and 7.8 (Fig. S2) and a pH of 7.5 was subsequently used throughout the study. Additionally, there was no auto-oxidation of dopamine hydrochloride under any tested pH.
Figure S2: Phenoloxidase activity (± SE) using phosphate buffer of different pH.