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.