2.3. Physiological indicators
The samples were rinsed with an equal volume of sterilized seawater, and divided into 6 portions, each containing 12 mL, and then subjected to centrifugation at 4 °C for 10 minutes at 4000 rpm. The precipitates were used to determine the density and Chl a content of the zooxanthellae, while, the supernatants were used to measure the activity of various enzymes. After drying, the surface area of the skeletons was determined using the aluminum foil technique (Marsh, 1970; Fu et al., 2022).
For the analysis of endosymbiont density and Chl a content, three portions of the precipitate were washed and suspended in 5 mL of formaldehyde. The density of the endosymbiont was determined using a microscope with a blood counting plate. The remaining three samples were resuspended in 8 mL of methanol and extracted at 4 °C for 24 hours. After centrifugation at 4 °C for 10 minutes at 4000 rpm, Chl acontent was determined using a UV-visible spectrophotometer (Ritchie, 2006).
Quantitative supernatants were collected for further analysis of GST, Caspase-3, SOD, and CAT activities using commercial dilution kits (AKPR013U, AKPR027-1, AKBL006C, AKEN001U, BOXBIO, Beijing, China). Finally, the protein concentrations were determined using a Bradford kit (AKPR015, BOXBIO, Beijing, China). The enzyme activity units were normalized to U mgprot-1. Data conversion was performed using Excel 2016, while graphing was done using GraphPad Prism 8.0.2.