Coral growth, preconditioning and treatments
Colonies of P. acuta were collected in Kaneohe Bay, Hawaii in depths ranging between 1 to 4m and placed in temperature and light controlled flow-through indoor tanks for acclimation. The ambient temperature was set to 26°C. Light controlling system (Radion XR30w G3 Pro LED Light, EcoTech Marine) template for shallow reef at 2 m depth with maximum light intensity of 35% was set to imitate natural reef light conditions. After one-month acclimation, eight coral colonies were cut to three parts for preconditioned (PC), non-preconditioned (NPC) and control treatments. Control and NCP corals were left at ambient temperature while PC corals were exposed to sublethal 29°C temperature for 72 hours and then returned to ambient 26°C (0.5°C per 4 hours decrease rate) to rest. After two days, all corals were sub-fragmented, and fragments were put in respective tanks for ambient or hyperthermal treatment. Twelve days later – two weeks after the preconditioning – PC and NPC coral fragments were exposed to an acute thermal stress when temperature ramped from 26°C to 32°C in ~2 hours. At time points 1h, 3h, 6h, 12h, 24h, and 72h, one fragment of each treatment (PC and NPC at 32°C and control at 26°C) was cut to 0.5 - 2cm long pieces, aliquoted, washed with filtered sea water (FSW) and stored dry at -80°C.
For Bcl-2 inhibition, PC and NPC fragments were treated with 1μM venetoclax for the first 6 hours of the acute heat stress and then placed to venetoclax-free sea water (32°C) for the rest of the experiment. Fragments were microscoped prior to the treatment and then at day 5 of the heat stress. The total of 7 colonies were used for the venetoclax experiment but for statistical purposes, the data of only 5 colonies were used because NPC fragments and PC fragments with venetoclax of two colonies did not survive to day 5 of heat stress.
To compare heat stress response of experimentally preconditioned corals with naturally acclimatized corals, we collected 6 colonies (WT) from our outdoor flow-through tanks at the beginning of September 2018, when water temperatures peaked 29ºC in Kaneohe bay. We let them acclimate to winter ambient 26ºC to simulate our preconditioning experiment, sub-fragmented them as described above, and then exposed them to acute heat stress (32ºC). We sampled and stored them in the same way as described above.