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.