Figure 1.
Morphology of coral polyps at different stages of thermal stress and recovery.
Fragments of S. pistillata were placed in an aquarium and the temperature was raised to 34°C for 24 h and then back to 32°C for 1 month (Exp. 3), then ambient temperature (24°C) was restored. Pictures were taken at characteristic stages of thermal stress: (A) 0 h, (B) 24–48 h and (C) 168 h. (D) one month after ambient temperature was restored.
Figure 2.
Caspase activities and quantitative analysis of StyBcl-2-like and StyCasp.
Fragments of S.pistillata were placed in two aquaria (control at 24°C and thermal stress at 32°C in Exp. 1 and 2, or 34°C in Exp. 3). Fragments from both aquaria were sampled over 72 h (Exp. 1, n = 4), over 216 h (Exp. 2, n = 6) or over 168 h (Exp. 3, n = 6). Results are expressed as means ± SE of independent extractions from distinct fragments incubated in control (white) or subjected to thermal stress (gray). Values were tested by One-Way ANOVA and a t-test. Asterisks indicate significant difference between control and thermal stress of the same time point (* represents P<0.05, ** represents P<0.01, and *** represents P<0.001). (A) Caspase 3-like activities were assayed by fluorometric method using Ac-DEVD-AFC, measured in relative units of fluorescence (RFU's), and expressed as RFU/µg protein−1 h−1. (B), (C) Quantitative analysis of StyBcl-2-like and StyCasp expression, respectively, normalized to adenosyl-homocysteinase (AdoHcyase).
Figure 3.
Detection and quantification of apoptosis-like activity in S. pistillata.
(A) Temperature-induced DNA fragmentation detected in situ in tissues of S. pistillata. Fragments were incubated in control (24°C) or thermal stress (34°C). Sections (7 µm-thick) were attached to slides and apoptotic cells were detected using the TUNEL technique. (1) control; (2) 6 h, (3) 24 h, (4) 48 h, (5) 72 h and (6) 168 h of thermal stress. Arrowheads indicate apoptotic nuclei stained brown, while intact nuclei are counterstained green. Scale bar, 20 µm. Ep, epithelium. Ga, Gastroderm. (B) Percentage of TUNEL positive cells in tissues of S. pistillata incubated in control (24°C, white) or subjected to thermal stress of 34°C for 24 h (gray) and 168 h (black). Sections (7 µm-thick) were attached to slides and apoptotic cells were detected using the TUNEL technique. The mean percentage of TUNEL positive cells of individual fragments (n = 3) was derived by comparing the number of TUNEL positive cells to the total number of cells in the same image field. A total of five image fields per fragment section was analysed and averaged. Means were compared using a One-Way Anova with Tukey post-hoc testing. Letters above bars denote statistical significance; two means are significantly different (P<0.05) if their letters are different.
Figure 4.
Phylogenetic relationships of metazoan Bcl-2 family members including StyBcl-2-like based on amino acid sequences.
The alignments and UPGMA tree were obtained using the CLUSTAL-W and PAUP software. Bootstrap values (%) from 1000 bootstrap replicates are shown for neighbor-joining/maximum likelihood estimation/maximum parsimony at major nodes on the tree. Nodes receiving bootstrap values of <50% are indicated by (#). The scale bar represents genetic distance marker (amino acid substitutions). Accession numbers of sequences downloaded from GenBank are listed in Materials and Methods.
Figure 5.
Phylogenetic relationships of metazoan caspases 3,7 and 8 including StyCasp based on amino acid sequences.
The alignments and neighbor-joining tree sequences (large and small subunits only, excluding the prodomain) were obtained using the CLUSTAL-W and PAUP software. Bootstrap values (%) from 1000 bootstrap replicates are shown for neighbor-joining/maximum likelihood estimation/maximum parsimony at major nodes on the tree. Nodes receiving bootstrap values of <50% are indicated by (#). The scale bar represents genetic distance marker (amino acid substitutions). Accession numbers of sequences downloaded from GenBank are listed in Materials and Methods.