Fig 1.
Four possible cellular mechanisms of cnidarian bleaching under stress.
During stable symbiosis (a), algae reside within gastrodermal cells of the cnidarian host and are surrounded by the host-derived symbiosome membrane. Under stress, algae could be lost (b) by in situ degradation (involving fusion of the symbiosome with lysosomes, autophagy, and/or a cell-death reaction of the algae themselves), (c) by expulsion of healthy and/or degraded algae, (d) by detachment of algae-containing host cells, (e) by death of algae-containing host cells through apoptosis or necrosis, or by some combination of these mechanisms.
Table 1.
Temperature and light treatments used to apply stress.
Fig 2.
Lack of correlation between the timing of apoptosis (as judged by caspase activation) and that of bleaching in Aiptasia under heat and/or light stress.
(A-F) Symbiotic anemones previously acclimated under standard culture conditions (27°C, 25 μmol photons m-2 s-1) were exposed to (A,B) heat stress alone (34°C, 25 μmol photons m-2 s-1), (C,D) heat and light stress (34°C, 150 μmol photons m-2 s-1), or (E,F) light stress alone (27°C, 500 μmol photons m-2 s-1) for the durations indicated, and samples were taken from each tub for the determination of caspase activities (A,C,E) and numbers of algae per unit protein (B,D,F) in homogenates of individual animals (see Materials and Methods). Two (A,B,E,F) or three (C,D) independent experiments (i.e., animals in separate tubs—see Materials and Methods) were performed, and caspase activities and algal counts were determined for 4–5 individual animals per time point in each experiment. The values for all individual animals are shown using different symbols for the independent experiments (as indicated). [Note that the experiments of Figs 3A and 5A (samples with no caspase inhibitor) provide additional independent replications for the most important time points in regard to heat and light stress.] For each panel, values were normalized to the mean values for all animals from all experiments before exposure to the stress conditions at 0 d (set at 1.0 for caspase or 100% for algal counts). The mean value for all animals at each time-point is indicated by a horizontal line; note that one animal in A, Experiment 1, appeared to give an artifactually high caspase activity (12.96 normalized value) and was excluded in calculating the mean activity for that time-point. Ctrl, anemones left at 27°C, 25 μmol photons m-2 s-1, for the duration of the experiment; Sta, anemones incubated overnight (27°C; 12 h of dark bracketed by ~1-h periods at 25 μmol photons m-2 s-1) with 3 μM staurosporine to induce apoptosis (this test was performed in just one of the three replicate experiments for C). (G) Aposymbiotic animals were analyzed as for symbiotic animals in C; three independent experiments were performed. In panels B, D, and F, mean values that appear significantly different (P ≤ 0.005) from the day-0 means are indicated by a ★ (grouped ANOVA) or # (unpaired t test) (see Materials and Methods).
Fig 3.
Lack of bleaching despite transient stimulation of apoptosis in Aiptasia exposed to limited-duration heat and light stress.
Experiments were identical to those of Fig 2C and 2D except that after 2 d of stress, anemones were returned to standard culture conditions for the duration of the experiment. (A) Caspase activities were measured during a single experiment of this type. However, note that for the first 2 d, the experimental conditions were identical to those of the three replicate experiments of Fig 2C. (B) Algal numbers were measured during three replicate experiments of this type, one of which was the same as that used to obtain the caspase-activity values of panel A. In each panel, horizontal lines indicate the mean values for all animals at each time-point; none of these means appeared significantly different from the day-0 mean by the grouped ANOVA test.
Fig 4.
Imperfect correlation between the timing of apoptotic activity and that of bleaching under mild or acute cold stress.
(A,B) Caspase activation and bleaching were assessed as in Fig 2 during mild cold stress (18°C, 25 μmol photons m-2 s-1); three independent experiments were performed. Note that the data for the 5-d time-point in A are from a single experiment and do not fit the overall trends; we presume that some unknown anomaly produced these outlier measurements. (C,D) Symbiotic anemones previously acclimated under standard culture conditions were shifted abruptly to 4°C in the dark, held for 4 h, and then returned to standard culture conditions for the duration of each experiment. Samples from two independent experiments were analyzed as in A,B. Note that the scale for C differs by a factor of two from that in the other panels showing caspase data. (E) Aposymbiotic animals were analyzed as in C; three independent experiments were performed. Scale as in panel C. In all panels, horizontal lines indicate the mean values for all animals at that time-point. In B and D, the results of grouped ANOVA and unpaired t tests are indicated as in Fig 2.
Fig 5.
Occurrence of bleaching under heat and light stress despite inhibition of caspases (and hence presumably of apoptosis).
(A,B) Four independent experiments were like those of Fig 2C and 2D except that some tubs were treated with the caspase inhibitor Ac-DEVD-CHO at 2 μM (added from a stock solution in DMSO) or with the same concentration of DMSO alone (see Materials and Methods). Experiment 4 was of only short duration and was intended only to provide additional evidence of the effectiveness of the caspase inhibitor during the time when caspase activities would otherwise be elevated; no algal counts were performed. (A) Caspase activities. As in Figs 2–4, the section labeled "Ctrl" shows measurements on anemones that were held under control conditions (27°C, no additives) for the full 12 (Experiments 1 and 3) or 4 (Experiment 2) days of the experiments. (B) Numbers of algae per unit protein. In each panel, horizontal lines indicate the mean values for all animals at that time-point. In B, the results of grouped ANOVA and unpaired t tests are indicated as in Fig 2. (C,D) In a single experiment, anemones under otherwise standard culture conditions (27°C, 25 μmol photons m-2 s-1) were treated with Ac-DEVD-CHO (2 μM final concentration) and monitored for caspase activities (C) and numbers of remaining algae (D).
Fig 6.
Increased in situ degradation of algae during acute cold shock but not under other stress conditions.
In experiments like those of Figs 2A–2F and 4A–4D, symbiotic anemones were fixed after collection from populations under standard culture conditions (control experiments 1–4) or under stress conditions at times coinciding with maximal rates of bleaching (for acute cold shock, this is1 d after the return to standard culture conditions). Anemone sections were then examined by electron microscopy as described in Materials and Methods. In each experiment, every algal profile observed in multiple fields of a randomly chosen section of one animal was classified as Intact, Degrading, or Ambiguous following the examples shown in a section of an anemone from standard culture conditions (A; and see Materials and Methods). (B) Percentages of algae classified into each category in anemones exposed to the various stresses. Total numbers of algal profiles scored are indicated in parentheses. (C) A typical section from one of the acute-cold-shock experiments.
Fig 7.
Apparent predominance of algal expulsion over host-cell detachment during bleaching under various stress conditions.
In experiments like those of Figs 2A–2F and 4A–4D, samples of released material (typically small boluses) were collected from the ASW containing anemones under standard culture conditions and at times coinciding with significant rates of bleaching under stress (for acute cold shock, this is 1 d after the return to standard culture conditions). These samples were fixed, stained with DAPI, and examined by confocal microscopy as described in Materials and Methods. For each independent experiment, three separate samples of released material were examined in a z-stack of ~20 optical sections, and each nucleus was scored for whether it was inside an algal cell (an algal nucleus) or not (a host-cell nucleus), as judged by comparison to the chlorophyll fluorescence from the algal chloroplasts (which are largely concentrated in the cell cortex—see Figs 6A and 8C). The same scoring strategy was used with physical sections of intact anemones embedded as described in Materials and Methods. One of the three animals in which sections were examined had been grown under standard conditions; the other two had been subjected to heat and light stress for 6 and 48 h, respectively. (A) Diagram illustrating the scoring strategy. Expulsion of algae would yield "naked" algae in which the associated nuclei (N) would be seen to be within the algal cells (left), whereas host-cell detachment would reveal host-cell nuclei that were associated with, but not within, algal cells (right). (B and C) Single confocal optical sections of (B) a physical section of an intact anemone and (C) a sample of material released from animals subjected to acute cold shock, to illustrate the scoring diagrammed in A. The outlines of nuclei determined by image-analysis software (see Materials and Methods) were overlaid on the red-channel images of algal cells revealed by chlorophyll autofluorescence; arrowheads indicate nuclei that appear to be outside algae. (D) Percentages of nuclei scored as inside and outside algal cells in anemone sections and in the material released under different conditions; independent experiments are shown separately, and the total numbers of nuclei scored in each such experiment are indicated in parentheses.
Table 2.
Evidence against a quantitatively significant role for complete degradation in situ during bleaching of Aiptasia under heat-and-light stressa.
Fig 8.
Severe damage to algae exposed to acute cold shock either in hospite or in culture.
(A) In an experiment like that of Fig 4C and 4D, algae collected from the ASW 24 h after the end of the cold shock and return of the anemones to standard culture conditions were fixed and examined by electron microscopy; algae in hospite in unperturbed animals (Fig 6A) provide a comparison. (B) Maximum quantum yields of Photosystem II as judged by Fv/Fm for (i) algae in hospite in anemones under standard culture conditions (n = 3 animals; mean ± SD is shown); (ii) algae collected from the ASW in tubs that contained ~45 (left) or ~75 (right) anemones 24 h after an acute cold shock to the anemones [as in Fig 4C and 4D; means ± SDs of two (left) or three (right) technical replicates are shown]; (iii) cultured algae (strain SSA01) growing at 27°C, 10 μmol photons m-2 s-1 (three replicate experiments were performed using separately grown cultures; mean ± SD is shown); and (iv) cultured algae that were collected 24 h after an acute cold shock as in Fig 4C and 4D but with the incubation after the shock at 27°C, 10 μmol photons m-2 s-1 (three replicate experiments were performed using separately grown cultures; mean ± SD is shown). (C,D) Cultured algae of strain SSA01 were fixed and examined by electron microscopy (C) during growth at 27°C, 10 μmol photons m-2 s-1 or (D) 24 h after a 4-h cold shock in the dark as in Fig 4C and 4D but with the incubation after the shock at 27°C, 10 μmol photons m-2 s-1.