2.3. AR measurement and tissue extraction
Each coral fragment was placed in a glass bottle (inner volume:
134mL), which contained 100 mL of filtered seawater. The filtered seawater
was originally collected from experimental tanks containing the
same nutrient condition as that in which the coral was cultured (LN or
HN) andwas filtered withmembrane filters (pore size: 0.45 μm).Microscopic
examination confirmed that there were no algae in the filtered
seawater. Triplicate control bottles, which contained only filtered seawater
without corals, were also prepared for each nutrient condition.
After the glass bottles were closed, they were placed in experimental
tanks and were incubated for 4 h (1100 to 1500 h) in the same light
and temperature conditions as those under which the corals had been
cultured. After 4 h, the corals were taken out of the bottles, and 5 mL
of 20% formalin was added to each bottle (final conc. 1% v/v) to fix
microbial activity in the seawater. The bottles were stored under dark
in a refrigerator until analysis of the abundance of zooxanthellae
released from corals.
The corals incubated for AR measurement were then separated into
organic soft tissue and the carbonate skeleton by awater-pikmethod. At
least 50 mL of tissue solution was obtained from each coral fragment
and 5 mL of the solution was fixed with 20% formalin (final conc. 2%
v/v) and stored in a refrigerator until analysis of the abundance of
symbiotic zooxanthellae. The remaining tissue solutionwas centrifuged
at 900 ×g for 5 min to separate zooxanthellae (pellet) from animal tissue
(supernatant). After the supernatant was removed, 5 mL of methanol
was added to the pellet to extract Chl a from zooxanthellae. The
methanol solution was stored at −30 °C in the dark until analysis.