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.