Author Contributions
PS. Chuang: Conduction of experiments, design of research, data
analysis, manuscript writing
S. Mitarai: Design of research, manuscript writing
Figure 1. Principal component analysis classified RNA-seq
libraries by treatments and replicates. The first principal component is
concordant with the salinity gradient, while the second principal
component reflects variation between biological replicates. Analysis was
based on 17,908 functionally annotated transcripts.
Figure 2. Pocillopora acuta polyps bail out in response
to hyperosmotic stress. Morphological features of polyp bail-out, e.g.,
coenosarc degradation and polyp detachment, were observed at 24 h.
Labels of samples and seawater salinity at specific times (unit: ‰) are
indicated on the photos.
Figure 3. Quantitative PCR (qPCR) of ten stress genes showed
three distinct expression profiles. The FAS and CASP8 genes were
significantly upregulated during the first 12 h and remained stable
afterward (profile I). The FGFR2, FGF2, RHO, MMP19, and MMP24 genes
displayed a common expression profile that showed little upregulation
for the first 6 h and remarkable upregulation from 6 h to 18 h, after
which expression stabilized (profile II). For the JNK, NFKB1, and XIAP
genes, linear upregulation was observed during the time-course of the
experiment (profile III). Statistical analysis was conducted using
Welch’s ANOVA with a Games-Howell post-hoc test. Groups with significant
differences (p < 0.05) are indicated. Data were
log2-transformed and are expressed as means ± SD (standard deviation).
Figure 4. Parallel signaling pathways trigger polyp bail-out.
Molecular mechanisms leading to polyp bail-out are hypothesized based on
our transcriptomic and qPCR data. Environmental stress triggers the TNF
and FGF signaling pathways, which activate the caspase (CASP) and matrix
metalloproteinase (MMP) cascades, respectively. Activation of caspases
leads to apoptosis in the coenosarc and to its subsequent degradation,
while activation of MMPs, likely through Rho family GTPases (RHO),
results in extracellular matrix (ECM) degradation and detachment of
polyps. In polyp bodies, anti-apoptotic and cell survival signals
mediated by genes such as JNK, NFKB, and XIAP, are activated to suppress
the apoptotic response, which in turn promotes polyp survival.
Figure S1. Pocillopora acuta responded differently at
different levels of hyperosmotic stress. An acute hyperosmotic stress
was created by addition of 48‰ salinity seawater to the experimental
tank at 11.2 mL/min, while a mild stress was created by addition of 48‰
salinity seawater at 4 mL/min for 12 h with unchanging salinity
thereafter. Under acute hyperosmotic stress, tentacle retraction was
observed at 12 h (salinity: 46‰). Detachment of polyps was observed at
24 h (salinity: 48‰) without complete colony dissociation, resulting in
release of both solitary and clustered polyps (upper). Under mild
hyperosmotic stress, coenosarc degradation was observed at 48 h, but no
detached polyps were seen during the experiment (lower). Seawater
salinity is indicated on the side of each photo (unit: ‰).
Table 1. Apoptosis and several signaling pathways are activated
during polyp bail-out induction. Biological processes and signaling
pathways showing significant enrichment at both 12 h and 24 h are
indicated in bold (p<0.05 ). P -values and fold
enrichment (FE) are indicated for each GO category.