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\begin{document}

\title{Preconditioning improves bleaching susceptibility in the reef-building
coral Pocillopora acuta through modulations in autophagy pathway}



\author[1]{Eva Majerova}%
\author[1]{Fiona Carey}%
\author[1]{Crawford Drury}%
\author[1,2]{Ruth Gates}%
\affil[1]{University of Hawai'i at Mānoa Hawaiʻi Institute of Marine Biology}%
\affil[2]{University of Hawaii at Manoa}%


\vspace{-1em}



  
  \date{May 08, 2024}


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\selectlanguage{english}
\begin{abstract}
Stony corals heavily rely on their intracellular algal symbionts for
energetical supply. Increasing extreme weather driven by climate change
often leads to disruption of the symbiosis and to coral death,
threatening the sole existence of coral reefs, the key underwater
ecosystems. As climate change mitigation outcomes are uncertain, it is
important to search for ways to increase coral resilience towards future
climate conditions, thermal extremes in particular. It has been shown
that corals can withstand stress conditions better after previous
exposure, but the mechanism remains unclear. Here we show that after
three-day thermal preconditioning, stony coral Pocillopora acuta becomes
more resilient to acute heat stress through modulations in cell
signaling. In preconditioned corals, the expression of pro-survival gene
pBcl-2 increases relatively to pro-death genes pBak and pBax during
thermal stress, and the coral bleaching rate significantly decreases.
After pBcl-2 activity inhibition, preconditioned corals lose the
acquired beneficial phenotype and bleach at the same rate as
non-preconditioned corals, which confirms the crucial role of programmed
cell death in coral bleaching and acclimatization. The detailed analysis
points to the involvement of autophagy/symbiophagy rather than apoptosis
in the process. A similar shift in gene expression also occurs in
thermally stressed corals that have previously acclimatized to summer
temperatures in Kaneohe Bay, Hawai'i, suggesting that corals can
naturally increase their resilience to warming events during high-risk
periods through alterations in cell signaling. An in-depth understanding
of molecular mechanisms underlying coral acclimatization and resilience
could open the way for restoration practices such as human-assisted
evolution.%
\end{abstract}%



\sloppy


\section*{Keywords}

{\label{keywords}}

Stony corals, coral bleaching, autophagy, programmed cell death,
assisted evolution, acclimatization

\textbf{Introduction}

Reef building stony corals live in a mutually beneficial partnership
with photosynthetic algae that provide them with essential nutrients and
oxygen. Stressful conditions such as high temperature or oceanic
acidification lead to the disruption of symbiosis known as coral
bleaching, and eventual coral death. Climate-change related thermal mass
bleaching events have become increasingly common, affecting about 75\%
of coral reefs in Hawaii and 93\% of surveyed corals in Great Barrier
Reef, with over 50\% mortality in some regions (Couch et al., 2017;
Hoegh-Guldberg et al., 2015). Current climate-change mitigation measures
(e.g. the Paris agreement), aiming to limit global warming to 2, seem
not being aggressive enough to effectively moderate impacts on coral
reefs, leaving these ecosystems under an imminent threat of collapsing
and disappearing by 2050 (Ainsworth et al., 2016; Frieler et al., 2013).

Coral reefs are of particular importance for life on Earth. They produce
atmospheric oxygen -- while absorbing carbon dioxide -- through their
photosynthesizing symbiotic algae, they also offer necessary shelter for
marine life, serve as nurseries for fishery species, protect coastal
regions from wave exposure and provide food for more than 3 billion
people (Vercelloni et al., 2018). The World Wildlife Fund for Nature
estimates that the range of products and services that coastal and
marine environments provide can be valued conservatively at \$2.5
trillion each (Hoegh-Guldberg et al., 2015). So, not surprisingly,
searching for ways to better protect coral reefs became the kernel of
coral research all over the world.

Animals and plants are known to acclimatize to stress conditions and
several recent studies show that heat acclimatization might help corals
better withstand thermal stress and reduce the severity of coral
bleaching and mortality (Bay \& Palumbi, 2015; Bellantuono,
Granados-Cifuentes, Miller, Hoegh-Guldberg, \& Rodriguez-Lanetty, 2012;
Palumbi, Barshis, Traylor-Knowles, \& Bay, 2014). Unfortunately, this
natural protection mechanism could be lost under future climate-change
scenarios (Ainsworth et al., 2016). One proposed solution for helping
corals withstand future climate conditions is human-assisted evolution
(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; Oppen et al., 2017). Whether it is selective breeding, managed
relocation (assisted gene flow and migration), or genetic manipulations,
the main setback is the fundamental lack of knowledge underlying
mechanisms of coral-algal symbiosis maintenance and disruption. If we
don't understand the mechanism of bleaching and natural
acclimatization/adaptation of corals to heat stress, we can't
responsibly define the attributes of resilience. Reversely, with a
proper understanding of coral resilient traits and their trade-offs,
current restoration projects could be scaled up to dramatically increase
impact on coral reefs worldwide.

Despite global ecological relevance, surprisingly little is known about
the cellular and molecular mechanisms of bleaching. In general, the
observations point to two prevalent strategies involved in bleaching --
apoptosis and autophagy/symbiophagy (Downs et al., 2009; Dunn,
Schnitzler, \& Weis, 2007; Kvitt, Rosenfeld, \& Tchernov, 2016; Pernice
et al., 2011; Tchernov et al., 2011). These two evolutionary conserved
programmed cell death (PCD) pathways may occur independently but often
simultaneously interplay or compensate for each other (Denton \& Kumar,
2019; Dunn et al., 2007). For example, key pro-survival
genes\emph{Bcl-2} (B-cell lymphoma 2) and \emph{BI-1} (Bax inhibitor 1)
inhibit both apoptosis and autophagy, and pro-death genes BAX (Bcl2
Associated X) and BAK (Bcl2 Antagonist/Killer) activate them (Castillo
et al., 2011; Karch et al., 2017; Xu et al., 2013).

Molecular analyses from previous bleaching experiments showed
correlation of -- among others - HSP70 (heat shock protein 70), and PCD
genes Bcl-2, BI-1, BAK, and BAX expressions with bleaching, and proved
the involvement of caspases -- apoptotic proteases - in the process
(Kvitt et al., 2016; Pernice et al., 2011; Tchernov et al., 2011).
Transcriptomic studies in preconditioned/acclimatized corals focused
mostly on the longer-term changes in gene expression profiles and found
differences in the expressions of e.g. heat shock family proteins, genes
involved in oxidative stress and various pleiotropic cell signaling and
transcription factors such as TNFR (tumor necrosis factor receptor) or
NFkB inhibitor (NFkBI)(Bay \& Palumbi, 2015; Bellantuono et al., 2012;
Palumbi et al., 2014; Thomas et al., 2018). The early phase acute heat
stress response and the role of PCD in preconditioning and acquired
thermal resilience in corals is therefore not well understood. Moreover,
the functional analyses of particular genes in the process are missing.

In our study, we used the heat susceptible stony coral \emph{Pocillopora
acuta} and we show that three-day exposure to sublethal temperature
makes it more resilient to subsequent acute thermal stress via
modulations in cell signaling. In preconditioned corals, the expression
of pro-survival gene \emph{pBcl-2} increases relatively to pro-death
genes \emph{pBak} and \emph{pBax} during early phase of the thermal
stress. After pBcl-2 activity inhibition, preconditioned corals lose the
acquired phenotype and bleach at the same rate as non-preconditioned
corals, which determines the crucial role of pBcl-2 and programmed cell
death in coral bleaching and acclimatization. Detailed analysis points
to the involvement of autophagy/symbiophagy rather than apoptosis in the
process. Corals exposed to natural oceanic summer temperatures show
stress-induced gene expression profile similar to the experimentally
preconditioned corals which suggests that during periods with high
probability of extreme temperature events, corals can naturally increase
their resilience through alterations in cell signaling\textbf{.}

\section*{Results}

{\label{results}}

\textbf{Thermal preconditioning slows down the bleaching rate in P.
acuta}

We split colonies collected in Kaneohe Bay into three parts for
preconditioned (PC), non-preconditioned (NPC) and control treatments.
After 3 weeks of acclimatization to indoor tanks environment at winter
ambient temperature (26\selectlanguage{ngerman}ºC), PC part of each colony was exposed to the
usual summer maximal temperature (29ºC) for 3 days and then rested at
ambient 26ºC for two weeks (Fig.1A). To assess thermal tolerance, we
measured bleaching rate in each coral during a sudden heat stress (32ºC)
using fluorescence confocal microscopy (Fig.1B, C). Each fragment of PC,
NPC and control coral was live imaged at the beginning of the experiment
and after 3 and 6 days. Red (algae) to green (host) signal ratio was
calculated to quantify the density of \emph{Symbiodinium} cells in coral
tissue. Our results show that corals previously exposed to sublethal
temperatures are more tolerant to acute heat stress and bleach at slower
rate than non-preconditioned corals (two-way ANOVA, signal\_ratio
\textasciitilde{} time * conditioning, p = 0.000) (Fig. 1C). To
eliminate that the shift in red to green signal ratio is caused by
increased level of pGFP in coral tissue as response to heat stress, we
analyzed \emph{pGFP} expression via qPCR and observed no differences
(data not shown).

Previous experiments have already shown the ability of corals to
increase their tolerance to heat stress after exposure to sublethal
temperatures (Ainsworth et al., 2016; Bay \& Palumbi, 2015; Bellantuono
et al., 2012). It seems that the length of sublethal temperature
exposure along with the length and temperature of the resting period are
more important for the acquired thermal resilience than the
preconditioning temperature. Our experiment is unique because it
combines relatively short preconditioning period with relatively long
resting time.

\textbf{pHSP70 expression increases after preconditioning}

To unveil the molecular mechanisms underlying acquired higher thermal
resilience in preconditioned corals, we first analyzed the early
hyperthermal stress response of PC and NPC corals by RT-qPCR. We
expected lively cell signaling reflected in rapid changes in gene
expression to occur in corals during the first 24h of acute heat stress.
To increase the chance of capturing these changes, we sampled PC and NPC
corals (n = 10 per conditioning) exposed to 32ºC at 0, 1, 3, 6, 12, 24,
48, and 72 hours. To exclude the influence of natural circadian gene
expression oscillation, control samples (NPC corals at ambient
temperature) were sampled as well. We expressed gene expression level
relatively to control at time 0. Considering the qPCR calculation method
and the fact that gene expression changes are very dynamic and can
happen in only a very short time window with no regards to previous or
future levels, we analyzed the results independently for every timepoint
using one-way ANOVA.

Heat shock proteins (HSP) are considered first-line stress responders,
protecting other proteins from misassembling during extreme conditions.
In our system, the expression of \emph{pHSP70} was very heterogeneous
and colony-specific (Fig. 2A). We observed a small increase
in\emph{HSP70} gene expression in NPC corals compared to control after
heat stress. This result supports previous experiments that showed high
variability in basal expression of HSP proteins depending on colony
depth (Poli, Fabbri, Goffredo, Airi, \& Franzellitti, 2017). Corals
living in shallow waters (up to 3m) have high basal
\emph{pHSP70}expression and do not increase it significantly during heat
shock, suggesting that their molecular guard mechanisms stay on alert
all the time. \emph{Pocillopora} corals for our experiment were
collected from shallow waters (1-4m), which potentially clarifies why we
did not detect changes in p\emph{HSP70} expression in NPC corals.
Nevertheless, after preconditioning, p\emph{HSP70} gene expression
increased at two timepoints - 3h (p =0.053) and 24h (p = 0.070) compared
to NPC corals. The function of HSP in preconditioning and
acclimatization remains disputed. Palumbi et al. observed alterations in
HSP family gene expression after acclimatization (Palumbi et al., 2014),
but others detected only limited (Bay \& Palumbi, 2015) or no
(Bellantuono et al., 2012) differences. Considering the dynamics of
HSP70 expression, detection of its alterations is largely dependent on
the experimental design and different observations might be attributed
to different sampling times.

Transcription of HSP is regulated by HSF1 transcription factor
(Pirkkala, Nykänen, \& Sistonen, 2001). In our experiment, pHSF1 basal
expression level was higher in non-stressed PC corals (p = 0.000) but
after the beginning of heat stress, the levels of PC and NPC corals
aligned until day 3 when we observed significant decrease of HSF1
expression in NPC corals (p = 0.001) (Figure S1). The expression of
pHSF1 does not correlate with the expression of HSP70, which is not much
surprising since changes in gene expression may be caused by many other
factors such as transcription factor localization, promoter
accessibility etc.

\textbf{Thermal preconditioning shifts cell signaling in favor of
pro-life genes pBcl-2 and pBI-1}

Expression of pro-survival genes p\emph{BI-1} and p\emph{Bcl-2} and
pro-death genes p\emph{BAK} and p\emph{BAX} increased during first six
hours of the stress (Fig. 2A). After preconditioning, the increase of
p\emph{BI-1} , \emph{pBcl-2} and p\emph{BAX} expression was even more
pronounced (p\textsubscript{(BI-1,3h)} = 0.006, p\textsubscript{(Bcl-2,
0h)} = 0.076, p\textsubscript{(Bcl-2, 3h)} = 0.002,
p\textsubscript{(BAX, 1h)} = 0.027, p\textsubscript{(BAX, 3h)} = 0.014),
but the expression of pro-death \emph{BAK} gene in PC corals dropped to
control levels at 6 hours, differing from the NPC corals (p = 0.084).
Upon proper signaling, BAK and BAX heterodimerize and permeabilize
mitochondrial membrane, releasing cytochrome c and considerably
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 an apoptosis inhibitor
but the exact mechanism is still not fully understood. PCD signaling
consists of a complex protein networking where single gene expression
profile does not reflect the entirety of the process. For this reason,
we calculated the expression ratio of pro-survival vs. pro-death genes
to better express the prevalent signalization in the cell. After
preconditioning, the pBcl/pBAK, pBcl/pBAX, and pBI/pBAX ratios shift
towards the pro-survival partner during the early heat-stress response
(Fig. 2B). p\emph{BI-1} expression level is higher than the
p\emph{BAX}expression level in PC vs. NPC corals at the first hour after
stress (p = 0.058). p\emph{Bcl-2} is expressed considerably more than
the p\emph{BAK} gene at 3 hours (p = 0.002), while compared to
p\emph{BAX} , it's the basal expression of p\emph{Bcl-2} in non-stressed
PC corals and the expression around 1h of the stress that is higher
(p\textsubscript{(0h)} = 0.005, p\textsubscript{(1h)} = 0.084). All
these results suggest that during thermal preconditioning, coral PCD
signaling is manipulated to favor the pro-survival genes.

\textbf{Stress response in naturally acclimatized corals correspond to
experimentally preconditioned corals}

\emph{In vitro} experiments may not always reflect the natural behavior
of the organism. To analyze the stress response of naturally
acclimatized corals, we collected six \emph{Pocillopora acuta} colonies
living in the outdoor flow-through tanks (WT) at the beginning of
September 2019, when water temperatures peaked 29ºC in Kaneohe bay (Fig.
S2). We let them acclimate to winter ambient 26ºC to simulate our
preconditioning experiment and then exposed them to acute heat stress
(32ºC). The correlation of gene expression between WT and PC corals
during acute heat stress was surprisingly high (Fig 3, S2) showing that
corals react to peaking temperatures in a similar way as to natural
temperature profile. It also suggests that during summer, when the risk
of extreme weather causing warming events and mass coral bleaching is
the highest, corals naturally acclimatize to warm temperatures through
modulations in the PCD pathways, which increase their survival chances
by slowing down the bleaching rate.

\textbf{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 1\selectlanguage{greek}μ\selectlanguage{english}M 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\textasciitilde{} time
* conditioning * treatment; \$conditioning : treatment -
p\textsubscript{(ven-)} = 0.000, p\textsubscript{(ven+)} = 0.464). Our
data show for the first time the direct functional involvement of PCD
genes in coral thermal tolerance and preconditioning.

\textbf{AMPK gene expression and caspase-3 activity points to
autophagy/symbiophagy as the main bleaching pathway in P. acuta}

Bcl-2 gene is involved in both apoptosis and autophagy pathways (Xu et
al., 2013). In order to distinguish between these two programmed cell
death pathways, we analyzed activity of caspase-3, one the key apoptotic
effector proteases. We performed caspase-3 activity test in thermally
stressed PC and NPC corals at times 0, 3 days and 5 days, and in PC and
NPC corals treated with venetoclax to exclude the switch in PCD pathways
upon treatment. Both PC and NPC corals showed slightly increased
caspase-3 activity under thermal stress, but significantly lower
compared to colchicine-induced apoptosis (Fig. 5A).

Moreover, we analyzed expression profile of \emph{pAMPK} (adenosine
monophosphate-activated protein kinase), a major signal inducer of
autophagy pathway, triggered by ROS(Villanueva-Paz et al.,
2016).\emph{pAMPK} was significantly upregulated at 6 hours post-stress
in NPC vs PC (p = 0.010) and after 24 hours we observed a gradual
increase in AMPK levels in NPC, peaking at 72 hours with almost three
times higher mean expression than in PC corals (p = 0.077) (Fig. 5B).

\textbf{The expression of pNFkB in PC and NPC corals supports the
involvement of autophagy/symbiophagy in \emph{P. acuta} bleaching}

pBcl-2 and other involved proteins are the effectors in the PCD
signaling, not the triggers. That's why we analyzed the expression rate
of NFkB, a pleiotropic transcription factor (TF) involved in many
crucial cellular processes. In response to various stressors, NFkB is
translocated to nucleus where it usually triggers transcription of
pro-survival genes such as Bcl-2 (Luo, Kamata, \& Karin, 2005).
Increased expression levels of \emph{NFkB} as a result of hyperthermal
shock have been observed in coral \emph{Acropora palmata} (DeSalvo,
Sunagawa, Voolstra, \& Medina, 2010). Moreover, Bellantuono et al.
observed increased levels of NFkB inhibitor (\emph{NFKBI} ) in thermally
PC corals (Bellantuono et al., 2012). Our results also show
increased\emph{NFKBI} expression levels in PC vs NPC corals
(p\textsubscript{(0h)} = 0.003, p\textsubscript{(1h)} = 0.013) (Fig.
5B). It suggests the role of NFkB in corals is to promote, not to
prevent cell death. It is known that NFkB is involved in innate immune
system across species (Gilmore \& Wolenski, 2012). It promotes
organismal immune reaction to get rid of the pathogen while it enhances
the capability of the host cells to survive. One of the algal-coral
symbiosis theories suggests that coral symbiont forms an intracellular
organelle called the symbiosome, which is a late endosome in the state
of arrested phagocytosis (Davy, Allemand, \& Weis, 2012). Under stress
conditions, the host innate immune response is reactivated leading to a
specific type of autophagy called symbiophagy. Autophagy and NFkB
pathway share common upstream signals and can control each other
(Trocoli \& Djavaheri-Mergny, 2011). During signaling, NFkB represses
autophagy through activation of mTOR, but under specific conditions such
as presence of reactive oxygen species (ROS) or after heat shock, NFkB
directly activates autophagy (Trocoli \& Djavaheri-Mergny, 2011). It has
been well documented that symbionts release increased ROS during heat
stress due to overexcitation of their photosynthetic systems, and
symbiophagy is one of the main observed mechanisms of algal loss in
hyperthermia (Downs et al., 2009; Dunn et al., 2007). NFkB has the
potential to link these two processes.

\section*{Discussion}

{\label{discussion}}

Our data show that heat-susceptible stony coral \emph{Pocillopora
acuta}can become more heat stress tolerant after previous exposure to
sublethal temperatures through modulations in cell signaling. In
preconditioned corals, the expression of pro-survival gene pBcl-2
increases relatively to pro-death genes pBak and pBax during acute
thermal stress. After pBcl-2 activity inhibition, this beneficial
phenotype is lost and preconditioned corals bleach at the same rate as
non-preconditioned ones. It implies direct involvement of programmed
cell death pathways in coral bleaching, but due to the complexity and
interconnection of these pathways, it is difficult to identify it
precisely (Castillo et al., 2011; Denton \& Kumar, 2019; Dunn et al.,
2007; Karch et al., 2017; Xu et al., 2013). Yet, our results indicate
that autophagy, not apoptosis, underlies bleaching in \emph{P.
acuta}during acute heat stress. Corals activate autophagy as a part of
their immune system to eliminate intracellular bacteria (Fuess, Pinz\selectlanguage{ngerman}ón
C, Weil, Grinshpon, \& Mydlarz, 2017). From this point of view,
autophagy is mostly regarded as a cell rescue pathway that allows
organism to get rid of pathogens and reuse autophaged cell components as
a new energetic source. In the light of existing literature and our
results, we hypothesize that upon acute heat stress, coral induces
symbiophagy through AMPK signaling to eliminate algae producing high
levels of ROS and other toxins. At the same time, it recycles algal
cellular components to meet its energetic demands. This strategy of
feeding on its symbionts would solve two problems at once and would
allow coral to survive for prolongated time even without its symbiont.
Our hypothesis is partially backed up by the recent findings showing
that corals in Kaneohe Bay did not increase heterotrophic nutrition
during a 2014 mass bleaching event but significantly lowered their
biomass, suggesting that they could have digest symbionts and their own
tissues to survive stress conditions (Wall, Ritson\selectlanguage{english}-Williams, Popp, \&
Gates, 2019). After preconditioning, cell signalization changes, and
corals slow down the bleaching rate thus extending the period of
autotrophic feeding and decreasing the chances of death by starvation.

Analysis of heat stress-induced gene expression in corals naturally
acclimatized to summer temperatures showed striking correlation to gene
expression in experimentally preconditioned corals but not in
non-preconditioned corals acclimatized to winter temperature. It
suggests that such preconditioning is a natural phenomenon that serves
to temporarily increase heat resilience in corals during summer, when
the risk of extreme weather event increases, but is lost towards winter
months.

What exactly triggers this process and what the downstream consequences
of keeping the symbionts throughout hostile thermal conditions are needs
to be addressed and elucidated in future experiments.

We add new pieces to the understanding of coral plasticity in responding
to heat stress and their capability to acclimatize to changing
conditions via preconditioning. Nevertheless, 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 et al., 2019; Oppen et al., 2017), but without proper
understanding of resilient traits and their cellular and molecular
background, they have only a limited chance to succeed.

\section*{Methods and Material}

{\label{methods-and-material}}

\textbf{Coral growth, preconditioning and treatments}

Colonies of \emph{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 26degC. 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 29degC temperature
for 72 hours and then returned to ambient 26degC (0.5degC 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 26degC to 32degC in \textasciitilde{}2 hours. At
time points 1h, 3h, 6h, 12h, 24h, and 72h, one fragment of each
treatment (PC and NPC at 32degC and control at 26degC) was cut to 0.5 - 2cm
long pieces, aliquoted, washed with filtered sea water (FSW) and stored
dry at -80degC.

For Bcl-2 inhibition, PC and NPC fragments were treated with 1\selectlanguage{greek}μ\selectlanguage{english}M
venetoclax for the first 6 hours of the acute heat stress and then
placed to venetoclax-free sea water (32\selectlanguage{ngerman}°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.

\textbf{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. \emph{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 \textasciitilde{}
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 \textasciitilde{} 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.

\textbf{Gene expression analysis}

Total RNA was extracted from dry frozen coral samples (size
\textasciitilde{}0.5 - 1.5cm) with RiboZol\textsuperscript{TM} RNA
Extraction Reagent (VWR Life Science) followed by DNase treatment
(DNaseI, Zymo Research) and phenol-chloroform extraction. We performed
the DNase treatment in 50 \selectlanguage{greek}μ\selectlanguage{english}l with 1U of DNase I at 37\selectlanguage{ngerman}°C for 15 min and
-- to increase the efficiency -- with additional 1U for 25 min. 1\selectlanguage{greek}μ\selectlanguage{english}g of
RNA was reversely transcribed with High-Capacity cDNA reverse
transcription kit (ThermoFisher Scientific) with RiboLock RNase
inhibitor (ThermoFisher Scientific). Reverse transcription quantitative
PCR (RT-qPCR) reactions were ran in 12 \selectlanguage{greek}μ\selectlanguage{english}l with
PowerUp\textsuperscript{TM} SYBR\textsuperscript{TM} Green Master Mix
(Applied Biosystems) for 40 cycles. Four gene expression profiles
--\emph{pActin, pEF-1a (elongation factor 1), pCalm} (calmodulin)
and\emph{pAHC} (AdenosylHomoCysteinase) - were compared in five
different treatments and pEF-1a showed the highest expression stability
upon heat stress and thus chosen as the reference gene. All primer
sequences are listed in Table S1.

Expression of target genes was calculated relatively to the control
treatment at time 0 with \selectlanguage{greek}ΔΔ\selectlanguage{english}Ct method.

\begin{equation}
\selectlanguage{greek}\text{ΔΔ}\selectlanguage{english}\text{Ct}=2^{-\left[\left(\text{Ct}_{target,\ treated}-\text{Ct}_{ref,\ treated}\right)-(\text{Ct}_{target,\ control}-\text{Ct}_{ref,\ control}\right]}\nonumber \\
 \end{equation}

\selectlanguage{greek}ΔΔ\selectlanguage{english}Ct calculation method returns gene expression relative to control
coral at time 0 without reflecting RNA levels at other timepoints.
Moreover, we analyzed mostly genes involved in cell signaling whose gene
expression can change very promptly after the signaling occurs and the
change can last for only a very short period of time. Thus, the gene
expression levels are similar between treatments for most of the time,
with only a few differing timepoints. For these reasons, we excluded
time as a variable from our statistical analysis and analyzed expression
rates individually for each timepoint using One-way ANOVA with Tukey's
post hoc test. We sampled corals at different times only to increase the
chance of capturing gene expression changes, not to compare the entire
gene expression patterns between different treatments. Including time as
a variable decreases the probability that a gene expression difference
will be detected.

To compare gene expression profiles between experimentally
preconditioned (PC) and naturally acclimatized (WT) corals during heat
stress, linear regression model with Pearson correlation was calculated
for mean single gene expression and mean gene expression ratios between
PC and WT.

\textbf{Caspase activity assay}

Coral fragments (size \textasciitilde{}0.5 -- 1.5cm) were dry-frozen and
kept at -80\selectlanguage{ngerman}°C. Prior the assay, host and algal cells were separated as
follows. Frozen fragments were placed on ice in 1.5ml tubes with 250\selectlanguage{greek}μ\selectlanguage{english}l
of ice cold EB buffer (100mM Tris, 20mM EDTA, pH 8.0). Glass beads
(Sigma Aldrich, G1152) were added to the corals, each fragment was
vortexed in short pulses for 20s and then placed immediately back on
ice. Samples were centrifugated at 500g for 5min at 4\selectlanguage{ngerman}°C. 50\selectlanguage{greek}μ\selectlanguage{english}l of host
cell containing supernatant was transferred to a new tube and lysed with
Cell Lysis Buffer according to instructions in
EnzChek\textsuperscript{TM} Caspase-3 Assay Kit \#1, Z-DEVD-AMC
substrate (ThermoFisher Scientific). Protein concentration was measured
with Qubit Protein Assay Kit (Life Technologies). 50\selectlanguage{greek}μ\selectlanguage{english}g of protein in
volume of 50\selectlanguage{greek}μ\selectlanguage{english}l or lower was used to measure caspase 3 activity following
the kit instructions. Fluorescence was measured with SpectraMax2
(Molecular devices) using SoftMax Pro v4.8 software. Caspase activity
was analyzed in 6 colonies for heat stress experiment, and in 4 colonies
for the venetoclax and colchicine experiments.

\section*{Acknowledgments}

{\label{acknowledgments}}

We thank to the members and interns of the Gates Coral Lab, especially
to Vojt\selectlanguage{polish}ě\selectlanguage{english}ch Prok\selectlanguage{polish}ů\selectlanguage{english}pek, for assistance with fieldwork, coral fragmenting
and sampling, as well as to Nicole Cernohorsky, Ariana S. Huffmyer and
Crawford Drury for their valuable comments on the statistical analysis
and RStudio programming. We also thank Crawford Drury for his comments
on the manuscript. The research reported in this publication was
supported by the Paul G. Allen Foundation.

\section*{Author contributions}

{\label{author-contributions}}

E.M. and R.D.G conceived and coordinated the project, E.M. and F.C.C.
collected samples from the wild, ran the preconditioning experiment, and
assessed the bleaching rate in corals. E.M extracted RNA and analyzed
gene expression, performed Caspase-3 activity experiments and
statistically analyzed the data. E.M. wrote the manuscript. E.M. and
F.C.C. read and approved the manuscript.

\section*{Data Accessibility
Statement}

{\label{data-accessibility-statement}}

The data that supports the findings of this study are available in the
supplementary material of this article

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\section*{Figures}

{\label{figures}}

Figure 1\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\selectlanguage{ngerman}\textbf{Figure 1 Corals exposed to sublethal temperature (29°C) for 3
days become more resilient to following acute heat stress (32°C} ). A)
Graphical depiction of preconditioning experiment layout. C -- control
corals, NPC -- non-preconditioned corals, PC -- preconditioned corals.
B) Bleaching rate of PC and NPC corals upon acute heat stress expressed
as symbiont to host cell signal ratio based on confocal fluorescence
microscopy. Graph shows that PC corals are more resilient to thermal
stress and bleach with significantly slower pace than NPC corals. NPC
corals are depicted with grey dashed line, PC with black solid line.
Graph shows mean values with standard error bars. Two-way ANOVA
(microscopy\_signal \textasciitilde{} time * conditioning, p = 0.000)
was used to test the influence of conditioning to the bleaching rate. *
marks significantly different values in Tukey's post hoc test ( p \selectlanguage{english}[?]
0.001), n = 6. C) PC and NPC coral fragments after 5-day long heat
stress exposure compared to control coral. D) Confocal fluorescence
microscopy image of control (left) and bleached (right) coral. Host
cells (GFP) are depicted in green, algal cells (chlorophyll) in red.

Figure 2\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2 Expression of genes related to bleaching and programmed
cell death pathways,} relative to control at time 0h. Control samples
are depicted with dark grey dotted line, non-preconditioned (NPC) with
light grey dashed line and preconditioned (PC) with black solid line.
\selectlanguage{greek}ΔΔ\selectlanguage{english}Ct method was used to calculate the relative expression and the
ratios. Graph shows mean values with standard error bars. One-way ANOVA
with Tukey's post hoc test for each timepoint was used to test the
difference of the values. * marks significant (p [?] 0.05) difference
between PC and NPC corals, n = 10.

Figure 3\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} \textbf{Correlation of gene expression between PC and
WT corals during heat stress.} Linear regression model with Pearson
correlation was calculated for mean single gene expression (A) and mean
gene expression ratio (B) of preconditioned (PC) and wild-type (WT)
corals during acute heat stress (32\selectlanguage{ngerman}ºC), n = 6. PC corals were exposed to
three-day sublethal temperature (29ºC) and then rested for two weeks at
ambient temperature (26ºC). WT corals were exposed to natural summer
fluctuating oceanic temperatures in Kaneohe Bay, Hawaii with peaks
around 29ºC. Pearson correlation factors show very similar response to
sudden heat stress in both groups of corals pointing to the natural
ability of corals to acclimatize to summer temperatures through
modulations in the signaling pathways.

Figure 4\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4 Preconditioned (PC) corals treated with Bcl-2 inhibitor
venetoclax lost the more thermal-resilient phenotype} and bleached at
the same rate as non-preconditioned (NPC) corals. A) Fluorescence
confocal microscopy pictures of coral fragments that underwent 5-day
long heat stress without or with venetoclax. Host cells are visualized
in green, algal cells in red. B) Bleaching rate of PC and NPC corals
based on calculating the symbiont: host cells signal ratio. A set of
control experiments was designed to rule out the effect of venetoclax
itself to coral symbiont density. Multiway ANOVA analysis with Tukey's
post hoc test (microscopy\_signal \textasciitilde{} time * conditioning
* treatment) was used to test the similarity of bleaching rate. The
analysis showed a significant difference in bleaching rate between PC
and NPC corals not treated with venetoclax (p = 0.000) but no difference
in corals treated with venetoclax (p = 0.464) pointing to the crucial
role of pBcl-2 in acclimatization and coral bleaching. Graphs show mean
values with standard error bars, n = 5.

Figure 5\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5 Analyses of programmed cell death pathway point to the
prevalent role of symbiophagy (autophagy) in \emph{P. acuta} thermally
induced bleaching.} A) Caspase-3 activity in NPC and PC corals under
different conditions. i) Caspase-3 activity in NPC and PC corals at day
3 of the heat stress. ii) Caspase-3 activity in NPC and PC corals
without (dashed lines) and with (black) venetoclax after 5-days long
thermal stress compared to control (dotted) corals. iii) Caspase-3
activity in corals treated with apoptosis activator colchicine compared
to controls. Error bars represent standard deviations (n = 4). B)
Expression of genes related to programmed cell death pathways, relative
to control at time 0h. Control samples are depicted with dark grey
dotted line, non-preconditioned (NPC) with light grey dashed line and
preconditioned (PC) with black solid line. \selectlanguage{greek}ΔΔ\selectlanguage{english}Ct method was used to
calculate the relative expression and the ratios. Graph shows mean
values with standard error bars. * marks significant (p [?] 0.05)
difference between PC and NPC calculated using one-way ANOVA with
Tukey's post hoc test for each timepoint individually, n = 10.

\section*{Supplementary Figures}

{\label{supplementary-figures}}

Figure S1\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure S1} \textbf{Expression of pHSF1, the major transcription
factor of heat shock protein family} . Expression of pHSF1 relative to
control at time 0h. Control samples are depicted with dark grey dotted
line, non-preconditioned (NPC) with light grey dashed line and
preconditioned (PC) with black solid line. \selectlanguage{greek}ΔΔ\selectlanguage{english}Ct method was used to
calculate the relative expression and the ratios. Graph shows mean
values with standard error bars. * marks significant (p [?] 0.05)
difference between PC and NPC calculated using one-way ANOVA with
Tukey's post hoc test for each timepoint individually, n = 10.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Figure S2

\textbf{Figure S2 Corals acclimatized to natural oceanic temperatures
show similar gene expression profile than corals preconditioned in
lab}A) Natural oceanic summer temperature profile around Coconut Island,
Hawaii, during summer 2018. It shows that corals were naturally exposed
to sublethal temperatures several times during the period. B) Gene
expression in naturally acclimatized corals (light grey solid line)
compared to experimentally preconditioned corals (black solid line) and
control corals (dotted dark grey line). \selectlanguage{greek}ΔΔ\selectlanguage{english}Ct method was used to
calculate the relative expression and the ratios. Graph shows mean
values with standard error bars. n = 6

Figure S3\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Figure S3 Visualized coral bleaching upon acute heat stress with
or without the addition of venetoclax, Bcl-2 inhibitor} .
Non-preconditioned (NPC) corals bleach in both conditions but
preconditioned (PC) corals show acquired higher thermal resilience that
is lost after addition of venetoclax. It points to the crucial role of
pBcl-2 in coral acclimatization and bleaching.

Table S1\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/default-figure}

\end{center}
\end{figure}

\textbf{Table S1 Primer sequences used for RT-qPCR experiments}

\selectlanguage{english}
\FloatBarrier
\end{document}

