After sampling the branches, the maximum quantum efficiency
of Photosystem II (Fv/Fm) was measured using a junior PAM (Walz,Germany) after 30 min of dark adaptation. Coral tissues were
treated according to Higuchi et al. (2009). Tissue homogenates
were prepared using the air-jet method, in which 100 mM phosphate
buffer was sprayed to strip the tissue from the coral skeleton.
Then, 15 ml of the non-fixed sample was separated into host coral
tissue and endosymbiont zooxanthellae (Zoox) by centrifugation
(time and speed as in Higuchi et al., 2009). The zooxanthellae fraction
was used for counting zooxanthellae density. Zooxanthellae
were lysed by sonication in phosphate buffer with 0.025% Triton
X-100. SOD activity, which reflects changes in superoxide anion
concentrations, was assayed spectrophotometrically as described
by Elstner and Heupel (1976) and Oyanagui (1984). Standards for
activity were prepared using bovine erythrocytic SOD (Sigma) for
each set of samples. CAT activity, which is responsible for inactivating
hydrogen peroxide (H2O2), was measured by determining
depletion of H2O2 at 240 nm (Beers and Sizer, 1952). All assays
were conducted at 25 °C immediately after sampling, and enzyme
activity is expressed as units (U) per mg protein. Protein content
was determined by the Bradford assay (Bradford, 1976). Statistical
analyses were performed using three-way analysis of variance
(ANOVA) and Tukey–Kramer honestly significant difference (HSD)
tests (JMP 8.0, SAS).