Bleaching rate assessment
Fragments of PC and NPC corals were live imaged with Zeiss LSM-710
confocal microscope at the beginning of the experiment and at days 3 and
5 of the heat stress to assess the rate of symbiont loss, or only at day
5 for venetoclax experiment. EC Plan-Neofluar 2.5X/0.075 objective was
used for the imaging resulting in total magnification 25X. Images were
acquired with Zen Imaging Software (Black edition) by Zeiss. Host cell
fluorescence was excited with the 405nm laser and collected in emission
range 454-621nm with master gain set at 650, digital gain 1.0, laser
power 28% and pinhole 35.5. Symbiodinium fluorescence was
excited with the 405nm laser and collected in emission range 655-718nm
with master gain set at 575, digital gain 1.0, laser power 28% and
pinhole 35.5. Fragments were kept in saltwater of respective temperature
(26°C and 32°C) during all the microscoping. Three snap images of each
fragment were taken using two fragments per treatment (control, ven+ at
26°C, ven- at 32°C, and ven+ at 32°C) per colony, with the total of 6
colonies. Images were analyzed with ZEN Imaging Software. The ratio of
green to red signal intensity (in fluorescence units) was analyzed in
ten circular areas along the coral tissue (avoiding mouth and tentacles
area) in each image. Two-way ANOVA (microscopy_signal ~
time * conditioning) with Tukey’s post hoc test was used to compare
bleaching rate between PC and NPC corals. Multiway ANOVA analysis with
Tukey’s post hoc test (microscopy_signal ~ time *
conditioning * treatment) was used to test the similarity of bleaching
rate between PC and NPC corals treated with venetoclax or with DMSO.
Controls (corals treated with venetoclax or with DMSO at ambient
temperature) were analyzed as an independent experiment.