Polyp bail-out induction and RNA sampling for transcriptomic analysis (Experiment I)
To examine the transcriptomic response during initiation of polyp bail-out, we collected RNA samples at 12-hour intervals during induction of the bail-out response in P. acuta , using a hyperosmotic treatment modified from Shapiro et al. (2016). One day prior to the experiments, six coral fragments from a mother colony were clipped off and randomly placed in two 5-L indoor experimental tanks. Artificial seawater (Kaisuimaren, Japan) at 35‰ salinity was prepared in these tanks (three in the treatment group and three in the control group). Light intensity was provided at 300 μmol photons m-2s-1 with a 12-hour day-night cycle. Tank temperature was allowed to fluctuate daily in the range of 24 – 26°C. For the treatment group, high-salinity artificial seawater (48‰) was pumped into the experimental tank at a constant rate of 4 mL/min to create hyperosmotic conditions, while salinity remained unchanged for the control group. Since partial polyp detachment (an indicator of onset of polyp bail-out) was observed in the treatment group at 24 h, we collected both undetached polyps and detached polyps (10 – 15 polyps/sample) separately. The same experiment was repeated three times on different P. acuta colonies, with at least one month between experiments (N = 3).