Discussion 
Our data show that the heat susceptible coral Pocillopora acuta can become more heat tolerant after previous exposure to sublethal temperatures through modulations in programmed cell death pathways. These data provide insight into the mechanism underlying the ability of corals to increase their tolerance to heat stress after pre-exposure.  
Our first aim was to analyze the expression rate of the pa-HSP70, one of the most studied heat shock proteins in corals. While we expected to see a clear overexpression in all heat-stressed corals, we detected large heterogeneity in the expression among colonies and only a mild, non-significant increase in non-preconditioned (NPC) corals during heat stress (Fig. 2A). In their work, Poli, Fabbri, Goffredo, Airi, & Franzellitti (2017) also observed high variability in basal expression of HSP proteins depending on colony depth. Pocillopora damicornis living in shallow waters (<3m) had high basal HSP70 expression and did not increase it significantly during heat shock, suggesting that molecular stress response mechanisms are always alert. Corals in our experiment were also collected from shallow waters (1-4m), which may be why we did not detect large changes in pa-HSP70 expression in NPC corals. However, the overexpression of pa-HSP70 in preconditioned (PC) corals at 3h (Fig. 2A) suggests that after preconditioning, corals may strengthen the first-line stress protection and employ more heat shock proteins. 
The function of HSP in preconditioning and acclimatization remains disputed. Palumbi et al. (2014) observed alterations in HSP family gene expression after acclimatization, but others detected only limited (Bay & Palumbi, 2015) or no (Bellantuono et al., 2012) differences. Considering the dynamics of HSP70 expression, detection of change is largely dependent on the experimental design and different observations might be attributed to different sampling times.
When analyzing the expression profile of pa-HSP70’s most plausible transcription factor – pa-HSF1, we did not find any relationship with the pa-HSP70 expression (Fig. S2). HSF1 and Hsp70 regulate each other’s expression through a negative feedback loop where trimerized HSF1 triggers transcription of HSP70 which in turn dissolves the HSF1 trimers and represses its action (Kmiecik, Le Breton, & Mayer, 2020; Krakowiak et al., 2018). These results suggest that transcriptional regulation of pa-HSP70 is not solely dependent on the transcription level of pa-HSF1 but rather relies on its multiple posttranslational modifications or cellular localizations.  
Thermal preconditioning of P. acuta significantly modulated important components of PCD pathways – pa-BI-1, pa-Bcl-2, pa-BAK, and pa-BAX. Upon proper signaling, BAK and BAX heterodimerize and permeabilize mitochondrial membrane, releasing cytochrome c and progressing towards cell death (Galluzzi et al., 2018; Karch et al., 2017). Bcl-2 can antagonize this process via direct protein-protein interaction with both BAK and BAX. BI-1 acts as a PCD inhibitor but the exact mechanism is still not fully understood. Their gene expression is generally studied separately or as a ratio of corresponding pro-life to pro-death partners which captures the PCD pathway state more accurately. This approach is typical of  Bcl2 to BAK in the study of apoptosis in human disease and cancer (Mackey, Borkowski, Amin, Jacobs, & Kyprianou, 1998; Salakou et al., 2007). In corals, the ratios of Bcl-2 to BAK and BAX were shown to increase during heat-stressed Acropora millepora (Pernice et al., 2011). We observed upregulation of pro-life genes pa-BI-1 and pa-Bcl2and downregulation of pro-death pa-BAK in PC corals compared to NPC (Fig. 2A). The pro-death pa-BAX expression profile was substantially different through our timeseries, where PC corals peaked rapidly around 3h, but NPC corals gradually increased until 6h before decreasing. When analyzed as ratios, we saw a clear shift towards the pro-life partner in all studied interactions (Fig. 2B), suggesting that after preconditioning, the PCD pathway is modulated to promote cell survival. 
We presumed that the observed increase in pa-Bcl-2 expression is reflected in higher pa-Bcl-2 protein level in PC corals, which then protects cells from PCD pathway activation and consecutive loss of symbionts (bleaching). To examine this hypothesis, we inhibited pa-Bcl2 activity in heat-stressed PC corals with venetoclax (ABT-199) and analyzed the bleaching rate. Venetoclax is not common in coral research, so we examined potential side effects of the drug treatment that could impact our experimental setup and conclusions. These results showed that venetoclax is not toxic for corals and does not cause symbiont or tissue loss (Fig. 4A, S6). 
Venetoclax acts at the protein level without impacting Bcl-2 gene expression, translation, or protein turnover rate (Souers et al., 2013), so we relied on a series of indirect indices to analyze Bcl-2/venetoclax interactions. 
We hypothesized that if preconditioning-based improved bleaching rate in PC corals is caused independently of pa-Bcl-2, venetoclax will either have no effect (in case pa-Bcl-2 does not control bleaching mechanism at all) or will negatively impact the bleaching rate in all venetoclax-treated heat-stressed corals, PC and NPC because it will trigger a novel pathway, not involved in preconditioning. This pathway would – in theory – have additional or synergistic effect on an already active bleaching pathway in corals. Conversely, if pa-Bcl-2 gene upregulation is the key modulation that happens in corals upon preconditioning, then venetoclax treatment will impair only bleaching rate in PC corals as it will have a complementary effect in NPC corals where the bleaching pathway controlled by the pa-Bcl-2 function has already been triggered. 
Also, preconditioning probably acts on multiple levels of coral physiology, cell signaling, and metabolism (as previously shown in Bay & Palumbi, 2015; Bellantuono et al., 2012; Palumbi et al., 2014; Thomas et al., 2018). If PCD pathways are the major drivers of slower bleaching rate in PC corals, the inhibition of pa-Bcl-2 in PC corals should negate the beneficial phenotype and PC corals treated with venetoclax should bleach at the same rate as NPC corals when exposed to the acute heat stress. Conversely, if the modulations in PCD signal transduction are one of the multiple complementary effector mechanisms contributing to the different bleaching rate in PC and NPC corals (for example apoptosis and exocytosis would act in parallel), venetoclax treatment will cause only partial restoration of the original phenotype, if any, because the other mechanism will still be in play and could compensate. 
The similar rate of bleaching in NPC corals with (ven+) and without (ven-) venetoclax treatment with differences in bleaching rate in ven+ and ven- PC corals suggest that overexpression of pa-Bcl-2 as an effect of preconditioning mediates the acquisition of the improved bleaching susceptibility. Additionally, the similar bleaching rate of PC and NPC corals upon venetoclax treatment suggests that the Bcl-2-controlled molecular pathway is the major effector bleaching pathway involved in preconditioning-induced improved bleaching susceptibility in P. acuta. The observed differences in the Bcl-2 inhibition experiment (no detrimental effect of venetoclax treatment in non-heat-stressed corals, bleaching induction in heat-stressed PC corals, and no effect in heat-stressed NPC corals) suggest that the pa-Bcl-2 activity was successfully selectively inhibited with venetoclax. 
Our results imply direct involvement of PCD pathways in coral bleaching, but due to the complexity and interconnection of these pathways, it is difficult to identify specifics. Apoptosis and autophagy/symbiophagy were previously detected as the mechanisms in action in thermally induced bleaching of P. acuta from Kāne‘ohe Bay, Hawai‘i (Downs et al., 2009; Tchernov et al., 2011). In an effort to distinguish the pathway involved in P. acuta with acquired bleaching tolerance, we analyzed the effector enzymes characteristic for each. Sequential activation of caspase-3 plays a central role in the execution phase of cell apoptosis (Galluzzi et al., 2018). While evidence in many model organisms suggest that executioner caspases precipitate intrinsic apoptosis, caspase-independent apoptotic pathways have been observed as well (Galluzzi et al., 2015; Galluzzi et al., 2018). However, increased caspase activity is observed in thermally stressed A. millepora and P. damicornis corals where it precedes the loss of the symbionts (Ros et al., 2016). Caspase activity also correlates with the increase in TUNEL positive cells (i.e. presumably apoptotic cells) in bleaching Acropora millepora (Pernice et al., 2011), and the inhibition of caspases via VAD inhibitor during heat stress prevents bleaching in P. damicornis and Montipora capitata (Tchernov et al., 2011). Based on these results, we assume that thermally induced apoptosis in Pocillopora corals is typically accompanied by an increase in caspase activity. The lack of caspase response in bleaching P. acuta in our experiment thus excludes the intrinsic apoptosis pathway as an underlying mechanism (Fig. 5A). 
Corals form an intracellular organelle called the symbiosome, which is a late endosome in the state of arrested phagocytosis that houses symbiont cells (Davy et al., 2012; Mohamed et al., 2016). The symbiophagy hypothesis proposes that under stress conditions when the symbiosis is no longer beneficial, the host innate immune response is reactivated leading to the degradation of the symbiont cell. In general, corals activate autophagy as a part of their immune system, including eliminating intracellular bacteria (Fuess, Pinzón C, Weil, Grinshpon, & Mydlarz, 2017). Acid phosphatases (APs) are activated as enzymes promoting autophagosome-lysosomal fusion during the execution of this process (Fitt & Trench, 1983; Hohman et al., 1982; O’Brien, 1982). Downs et al. ( 2009) observed increased levels of lysosomal acid phosphatase (LAS) along with an increase in the prenylated form of Rab7 (that indicates phagosome maturation) during thermally induced bleaching in P. acuta corals. The correlation between APs activity (Fig. 5A) and bleaching rate (Fig. 1B, 4A) in corals from our experiments indicates that autophagy/symbiophagy is the molecular mechanism underlying coral bleaching and preconditioning in these experimental conditions. The results from the venetoclax experiment, inhibiting the activity of Bcl-2, support this conclusion (Fig 5A).
Bcl-2 is usually considered a key apoptotic inhibitor through its interaction with BAK and BAX but it has been shown in model organisms that Bcl-2 can suppress both apoptosis and autophagy and the switch between the two pathways is largely dependent on the expression levels of Beclin 1 –direct Bcl-2 interacting partner and core autophagy initiator (Lorenzo Galluzzi et al., 2018; Marquez & Xu, 2012). Venetoclax–- a Bcl-2 inhibitor – has been shown to release Beclin1 from the complex with Bcl-2 and to induce autophagy in human cells (Alhoshani et al., 2020; Bodo et al., 2016). We analyzed the activity of caspase 3 and APs in corals treated with venetoclax to see whether it triggers autophagy/symbiophagy in venetoclax treated (ven+) PC corals and to exclude a possible switch in PCD pathways (Fig. 5A). 
We did not observe any significant changes in caspase-3 activity, ruling out the pathway switch in venetoclax treated corals. Interestingly, we saw an increase in APs activity in PC corals not treated with venetoclax (i.e., in the corals with improved thermal tolerance and slower bleaching rate). APs activity in NPC corals and venetoclax treated (ven+) PC corals were slightly but not significantly increased, which may be due to the late timepoint of the enzymatic assay. NPC and ven+ PC corals were visibly bleached at the time of sample collection so we presume the number of cells with ongoing symbiophagy could have been reduced. From the heat stress experiment, we see the highest APs activity at day 3 of the heat stress in NPC corals (similar level as in rapamycin-treated corals). On day 5 APs activity was declining, probably as the density of symbionts in tissue, and so the need for symbiophagy. PC corals, on the other hand, acquired higher heat tolerance that parallels the slower, ongoing bleaching that peaked at later timepoints. We speculate that this is why we observe high APs activity at 5 days in ven- PC corals but not in ven+ PC that have bleached at a similar rate as NPC corals and thus show similar APs activities as NPC corals. 
Modulations in molecular pathways usually depend on the cell signaling and the broad spectrum of activators, inhibitors, and transcription factors. Based on other works, we assumed the NFkB pathway may play important role in the regulation of coral bleaching pathways. Increased expression levels of NFkB as a result of hyperthermal shock have been observed in coral Acropora palmata (DeSalvo, Sunagawa, Voolstra, & Medina, 2010). Moreover, Bellantuono et al. (2012) observed increased levels of NFkB inhibitor (NFKBI) in thermally preconditioned corals. This suggests the role of NFkB in coral thermal stress response is to promote, not prevent PCD. 
Autophagy and NFkB pathways share common upstream signals and can control each other (Trocoli & Djavaheri-Mergny, 2011). NFkB is tightly bound to its inhibitor NFKBI in the cytoplasm. Upon signaling, IkB kinase phosphorylates NFKBI, resulting in its degradation and in the release and subsequent translocation of NFkB to the nucleus where it acts as a transcription factor (reviewed in (Luo et al., 2005). Usually, NFkB represses autophagy through activation of mTOR, but under specific conditions such as the presence of reactive oxygen species (ROS) or after heat shock, NFkB directly activates autophagy in an mTOR-independent way (Trocoli & Djavaheri-Mergny, 2011). It has been well documented that symbionts release increased ROS levels during heat stress due to overexcitation of their photosynthetic systems (reviewed in (Oakley & Davy, 2018). In theory, increased levels of ROS produced by the symbiont can surpass the ability of the host to scavenge it and the ROS signaling may then trigger NFkB-mediated autophagy independently of mTOR in NPC corals. Increased steady expression of pa-NFKBI observed in corals after thermal preconditioning (Fig. 5B) might help them to keep the pa-NFkB/pa-NFKBI complex more stable, and less likely to activate autophagy during heat stress. 
These data suggest a model in which preconditioned corals bleach at a slower rate during acute heat stress due to modulations in the autophagy pathway favoring symbiosome survival, mostly via the increase in pa-Bcl-2 gene expression. Future research should examine the triggers and downstream consequences of this process, including the implications of keeping symbionts throughout hostile thermal conditions.
Analysis of heat stress-induced gene expression in corals naturally acclimatized to summer temperatures showed a striking correlation to gene expression in experimentally PC corals but not in NPC corals acclimatized to winter temperatures (Fig 3, S3). This result suggests that preconditioning is a natural phenomenon that serves to temporarily increase heat resilience in corals during summer when the risk of extreme weather events increases but is lost in cooler months. 
Unfortunately, some future climate scenarios predict changes so severe that this natural mechanism could be inefficient (Ainsworth et al., 2016). Thus, it is important to search for other ways to protect coral reefs. Human-assisted evolution approach supported by technology development (e.g. CRISPR/Cas mediated genome editing made possible in corals (Cleves, Strader, Bay, Pringle, & Matz, 2018)) offers various solutions for scientists and coral restoration practitioners (Committee on Interventions to Increase the Resilience of Coral Reefs, Ocean Studies Board, Board on Life Sciences, Division on Earth and Life Studies, & National Academies of Sciences, Engineering, and Medicine, 2019; van Oppen et al., 2017), but without a proper understanding of resilient traits and their cellular and molecular background, they have only a limited chance to succeed.