Apoptosis and autophagy as mechanisms of dinoflagellate symbiont release during cnidarian bleaching: every which way you lose

Abstract

Cnidarian bleaching results from the breakdown in the symbiosis between the host cnidarian and its dinoflagellate symbiont. Coral bleaching in recent years has increasingly caused degradation and mortality of coral reefs on a global scale. Although much is understood about the environmental causes of bleaching, the underlying cellular mechanisms of symbiont release that drive the process are just beginning to be described. In this study, we investigated the roles of two cellular pathways, host cell apoptosis and autophagy, in the bleaching process of the symbiotic anemone Aiptasia pallida. Host cell apoptosis was experimentally manipulated using gene knockdown of an anemone caspase by RNA interference, chemical inhibition of caspase using ZVAD-fmk and an apoptosis-inducer wortmannin. Autophagy was manipulated by chemical inhibition using wortmannin or induction using rapamycin. The applications of multiple single treatments resulted in some increased bleaching in anemones under control conditions but no significant drop in bleaching in individuals subjected to a hyperthermic stress. These results indicated that no single pathway is responsible for symbiont release during bleaching. However, when multiple inhibitors were applied simultaneously to block both apoptosis and autophagy, there was a significant reduction in bleaching in heat-stressed anemones. Our results allow us to formulate a model for cellular processes involved in the control of cnidarian bleaching where apoptosis and autophagy act together in a see-saw mechanism such that if one is inhibited the other is induced. Similar interconnectivity between apoptosis and autophagy has previously been shown in vertebrates including involvement in an innate immune response to pathogens and parasites. This suggests that the bleaching response could be a modified immune response that recognizes and removes dysfunctional symbionts.

Figure 1

Expulsion of symbiotic dinoflagellates (error bars, s.d.) in response to separate manipulation of apoptosis or autophagy. Results of multiple independent tests (one-way ANOVA and post hoc Tukey pairwise comparison): between CT (white bars) and HTS (grey bars; filled circles, p≤0.01); between control untreated and treated: open triangle, p≤0.05 and filled triangle, p≤0.01; and between HTS untreated and treated, p≥0.05. N=4.

Figure 2

Expulsion of symbiotic dinoflagellates (+s.d.) in response to simultaneous manipulation of apoptosis and autophagy. Inhibitors applied were as follows: 10 μM 3MA, 500 nM wortmannin and 12.5 μM ZVAD-fmk in 0.2% DMSO. White bars, CT; grey bars, HTS. (Intra-treatment test: Kruskal–Wallis, adjusted to Bonferroni significance level, open triangle, p>0.05, N=5.)