Release of hydrogen peroxide and antioxidants by the coral Stylophora pistillatato its external milieu

Hydrogen peroxide (H2O2), a common reactive oxygen species, plays multiple roles in coral health and disease. Elevated H 2O2 production by the symbiotic algae during stress may result in symbiosis breakdown and bleaching of the coral. We have recently reported that various Red Sea corals release H 2O2 and antioxidants to their external milieu, and can influence the H 2O2 dynamics in the reef. Here, we present a laboratory characterization of H 2O2 and antioxidant activity release kinetics by intact, non-stressed Stylophora pistillata. Experimenting with bleached and nonbleached corals and different stirring speeds, we explored the sources and modes of H 2O2 and antioxidant release. Since H2O2 is produced and degraded simultaneously, we developed a methodology for resolving the actual H 2O2 concentrations released by the corals. H2O2 and antioxidant activity steadily increased in the water surrounding the coral over short periods of 1–2 h. Over longer periods of 5–7 h, the antioxidant activity kept increasing with time, while H2O2 concentrations were stabilized at ∼ 1 μM by 1–3 h, and then gradually declined. Solving for H2O2 release, corals were found to release H 2O2 at increasing rates over 2–4 h, and then to slow down and stop by 5–7 h. Stirring was shown to induce the release of H 2O2, possibly since the flow reduces the thickness of the diffusive boundary layer of the coral, and thus increases H 2O2 mass flux. Antioxidant activity was released at similar rates by bleached and non-bleached corals, suggesting that the antioxidants did not originate from the symbiotic algae. H 2O2, however, was not released from bleached corals, implying that the symbiotic algae are the source of the released H 2O2. The observed flow-induced H2O2 release may aid corals in removing some of the internal H2O2 produced by their symbiotic algae, and may possibly assist in preventing coral bleaching under conditions of elevated temperature and irradiance.


Supplement S1. Changes with time in k antiox and its calculation for missing time points
In all experiments k antiox values increased with time. The release kinetics could be fitted with a linear or an exponential function. To determine the more appropriate function we fitted both of these functions to data from 15 long incubations (100-240 min) and compared the resulting R 2 values of the fits (Table S1). We found that R 2 values of linear correlations (0.84±0.15) are significantly higher (p=0.019, paired T-Test) then those of exponential correlations (0.77±0.13; see Table S1). Table S1. Equation fitting through the antioxidant activity data. Curve-fitting for the increase in k antiox over time for 15 long incubations (100-240min). R 2 values of linear and exponential correlations were calculated and compared. R 2 values of linear correlations are significantly higher (p=0.019, paired T-Test) then those of exponential correlations.
The change with time in the antioxidant activity and its curve fitting is important for our understanding of this phenomenon and for practical reasons of calculating missing data points. The later issue of missing data points is of significance for the calculation of H 2 O 2 release rates according to equation 5. This calculation is conducted for small time Exp Normalization of the measured rates to coral related parameters A clear benefit to our newly described phenomenon can stem from normalization of the rates of H 2 O 2 and antioxidant activity release to some parameters of the coral that generate them. However, at current, the "appropriate" parameter of normalization is not yet known since the source of H 2 O 2 and antioxidants are not yet fully resolved. Several parameters can be considered such as coral size (surface area, volume, and weight), protein content, zooxanthellae density, tissue or mucus thickness etc. The data set described in the paper was conducted using similar sized coral fragments and similar water volumes, in an attempt generate comparable experiments. It is however possible that some of the variability observed between experiments reflect coral parameters that were not measured.
Release of antioxidant activity by corals with different sizes A preliminary attempt towards obtaining a normalization factor was done by incubating six coral fragments of different sizes in containers with different water volumes. Following 1 h of incubation the coral water was assayed for antioxidant activity. These results were normalized to a constant water volume (to avoid dilution effect) and were plotted against coral size expressed in volume (Fig. S2). The coral volume was estimated by a simple technique of water displacement. In this technique corals are introduced to a beaker completely filled with seawater and the overflowing water are collected and weighted. This simple technique is rather accurate (given sufficient repetitions) and imposes minimal stress on the corals. In our case, we allowed the corals to recover for one weak between this measurement and the incubation experiment. The experiment show a general trend of increase in antioxidant activity with the coral (Fig. S2). However we found a rather weak linear correlation (R 2 = 0.44). Considering these results, we think that that normalization of the observed rates to coral size could introduce large noise to the data. Figure S2. The effect of coral size on antioxidant activity release. Following 1 h of incubation that antioxidant activity in the incubation water was determined for six coral fragments of varying sizes. The coral volumes were examined by the water displacement technique and the antioxidant activities were normalized to a constant water volume.
Recommended optimal coral size for our experiments Our experiments were conducted using 20cm 2 surface area coral fragments and water volume of 100ml. In order to achieve reliable measurements we recommend keeping this ratio of coral size to water volume. Note that H 2 O 2 concentration in the sample is influenced by the coral H 2 O 2 releases rates, the antioxidant activity that degrade H 2 O 2 simultaneously and the stirring speed in the beaker. In addition, the quality of the measurements is also influenced by the assay accuracy and sensitivity as well as reliable blanks and trustful calibration curve. If one can develop sensitive and accurate H 2 O 2 assays, then a smaller ratio of coral size to water volume can be used. We did manage to measured H 2 O 2 and antioxidants from much smaller corals (~2 cm 2 ) with a similar water volume, but this required experienced personal. For larger corals, larger water volumes are required and rates are expected to be kept on the same order of magnitude. To enable readers to repeat our experiments we included few photos of the experimental setup for measuring H 2 O 2 and antioxidant activity release rates.

Supplement S4. H 2 O 2 release by S. pistillata fragments under complete darkness, low light intensity, and high light intensity.
In general, our experiments were conducted under fluorescence laboratory light of ~10 µmole quanta m -2 s -1 . This low illumination is sufficient for only minimal photosynthesis (or none at all), as it is far below the compensation light intensity of these corals. Several experiments were conducted in complete darkness to allow comparison with our standard low light conditions. In these experiments, the initial H 2 O 2 accumulation rates calculated in nmol per min were similar between dark and low light conditions (Fig. S4a).
These results suggest that the H 2 O 2 released during our standard experiments was not produced via photosynthesis. The effect of light on H 2 O 2 release by the coral is intriguing since the symbiotic algae, identified as the H 2 O 2 source, are strongly influenced by light. To conduct light experiments we had to change the experimental setup and use temperature controlled chambers (metabolic cells). These chambers had a volume of 0.5L and hence larger coral colonies were used (~ 100 cm 2 ). Corals were allowed to acclimate in the dark for 1-2 hrs in the chamber. Before each experimental stage (i.e. light or dark) the incubation water was replaced with a peristaltic pump to wash out the accumulated antioxidants. These washes seem to influence the coral and result in lower H 2 O 2 release with time (Fig. S4b). Due to this effect it is more appropriate to compare separately each set of dark and light treatments (i.e. prior to 130 min and after 140 min). While H 2 O 2 levels off after a short while, the initial rate of its accumulation in each of these sets is higher in the light compared to that in the dark. Further research is required to test the effect of light on H 2 O 2 release in a more optimized system. Figure S4b. H 2 O 2 release by S. pistillata colony in the dark and in the light. The experiment was preformed in a sealed metabolic cell with stirring and oxygen concentrations were determined. Prior to each stage the water in the chamber was replaced by a peristaltic pump to wash out antioxidants. Light was supplied by halogen lamps at intensity of 300 µmole quanta m -2 s -1 .

Supplement S5. H 2 O 2 release from S. pistillata under high natural irradiance.
The experiments reported in the manuscript are restricted to low light conditions, where photosynthesis is negligible. Since the symbiotic algae are the source of the released H 2 O 2 (Fig. 4), it is highly feasible that upon illumination and the commencement of photosynthesis corals will release more H 2 O 2. We are currently investigating these issues and have been establishing a different experimental setup to examine the effect of light on H 2 O 2 release dynamics. This setup involves a flow-through system in a water table where constant water exchange enables long experiments under natural irradiance and constant seawater temperature. Analytically it is rather challenging to obtain an appropriate water exchange rate that does not wash out the coral produced H 2 O 2 nor allow too much antioxidants to accumulate. Such an experiment was run with a large coral fragment of ~ 100 cm 2 , in a 600 mL glass beaker, with water flow rate of   Fig. S5). This experiment indeed shows higher H 2 O 2 concentrations and release rates at noon, when high solar irradiation was measured. We have not measured the coral photosynthesis rate in this experiment, and can relate at current only light and H 2 O 2 release. However, this is a promising first step in studying the link between H 2 O 2 release dynamics and photosynthesis.

Supplement S6. A complimentary experiment with decreased stirring speed
An experiment with reversed stirring regime (compared to those in Fig. 5) was conducted to test the effect of reducing flow speed on H 2 O 2 and antioxidants release. At the beginning of the experiment, fast flow speed was applied and after 60 min the flow was reduced dramatically to slow speed. H 2 O 2 concentrations increased initially and then dropped immediately when the flow was reduced (Fig, S6a). Antioxidant activity on the other hand continued accumulating throughout the experiment (Fig. S6a). From these parameters we calculated the amount of H 2 O 2 released during each time interval. We then present the cumulative H 2 O 2 released (Fig. A6b). It is apparent from the slopes, that the rate of change, which is the rate of H 2 O 2 accumulation, is faster when stirring speed is rapid.  Figure S7. H 2 O 2 and antioxidant activity release kinetics over long incubation experiments of four individual coral fragments (a-d) showing comparable patterns of antioxidant activity accumulation (red diamonds) and changing H 2 O 2 accumulation (blue squares) and release rate (green triangle) as showed also in Fig. 3. The total H 2 O 2 amount been released by the corals (indicated in the title) were summed using the frequent H 2 O 2 release rates calculations.