pBcl-2 inhibition leads to the loss of higher thermal resilience in preconditioned corals
Previous experiments focused on the correlation of gene expression differences with preconditioning or acclimatization (Bay & Palumbi, 2015; Bellantuono et al., 2012; Palumbi et al., 2014), but deeper insight into mechanisms of such processes is missing. Here, we investigated whether higher pro-survival gene expression prevents coral from bleaching, by inhibiting the effect of pBcl-2 in PC corals. If the acquired higher thermotolerance is caused by the pro-survival shift in PCD pathway, inhibition of pBcl-2 function should result in the loss of the beneficial phenotype. Venetoclax is a BH3 mimetic small molecule that binds to Bcl-2 protein binding domain, blocking its molecular function (Souers et al., 2013). Six PC and NPC corals were exposed to acute heat stress (32ºC) and simultaneously treated with 1μM venetoclax or with DMSO (for control). Fragments were also treated with venetoclax at ambient temperature to exclude the impact of venetoclax itself to coral bleaching. The bleaching ratio was assessed via confocal microscopy at the beginning of the experiment and after 5 days of heat stress. Control samples showed no effect of venetoclax to bleaching rate at ambient temperature, but we observed significant differences in the phenotype of corals treated and not treated with venetoclax under heat stress (Fig. 4, S3). After the application of venetoclax, the beneficial phenotype was completely lost, and PC corals bleached at the same rate as NPC corals (multi-way ANOVA microscopy_signal~ time * conditioning * treatment; $conditioning : treatment - p(ven-) = 0.000, p(ven+) = 0.464). Our data show for the first time the direct functional involvement of PCD genes in coral thermal tolerance and preconditioning.