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Insights Into Coral Bleaching Under Heat Stress from Analysis of Gene Expression in a Sea Anemone Model System

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Insights Into Coral Bleaching Under Heat Stress from Analysis of Gene Expression in a Sea Anemone Model System Insights into coral bleaching under heat stress from analysis of gene expression in a sea anemone model system Phillip A. Clevesa,1,2, Cory J. Kredieta,b,1, Erik M. Lehnerta, Masayuki Onishia,3, and John R. Pringlea,4 aDepartment of Genetics, Stanford University School of Medicine, Stanford, CA 94305; and bDepartment of Marine Science, Eckerd College, St. Petersburg, FL 33711 Contributed by John R. Pringle, September 9, 2020 (sent for review July 27, 2020; reviewed by Andrew C. Baker and Katie L. Barott) Loss of endosymbiotic algae (“bleaching”) under heat stress has symbiotic cnidarians, like most other organisms (16, 17), rapidly become a major problem for reef-building corals worldwide. To (within a few hours) up-regulate the genes encoding the identify genes that might be involved in triggering or executing heat-shock-protein (HSP) molecular chaperones (18–22). As two bleaching, or in protecting corals from it, we used RNAseq to an- of these studies (18, 19) used aposymbiotic larvae, it appears that alyze gene-expression changes during heat stress in a coral rela- the up-regulation of HSP genes does not depend on the presence tive, the sea anemone Aiptasia. We identified >500 genes that of algal symbionts but is rather an intrinsic part of the animal’s showed rapid and extensive up-regulation upon temperature in- stress response (as expected from studies in other species). crease. These genes fell into two clusters. In both clusters, most Moreover, the same two studies provided evidence that the up- genes showed similar expression patterns in symbiotic and apo- regulation is transient (again as expected from studies in other symbiotic anemones, suggesting that this early stress response is organisms), although interpretation of the data is complicated largely independent of the symbiosis. Cluster I was highly enriched both by the transcriptional changes associated with the concomi- for genes involved in innate immunity and apoptosis, and most tant larval development in these studies and by the reliance of transcript levels returned to baseline many hours before bleaching each study on a single later time point. Thus, the detailed dy- was first detected, raising doubts about their possible roles in this namics of the HSP transcriptional response have remained ob- process. Cluster II was highly enriched for genes involved in pro- scure. Nonetheless, the conclusion that HSP mRNA levels are GENETICS tein folding, and most transcript levels returned more slowly to rapidly but transiently up-regulated during heat stress in cnidar- baseline, so that roles in either promoting or preventing bleaching ians is consistent with earlier studies showing a rapid but transient seem plausible. Many of the genes in clusters I and II appear to be up-regulation of Hsp70 protein levels (23, 24). targets of the transcription factors NFκB and HSF1, respectively. Although many genes in addition to the HSPs have been We also examined the behavior of 337 genes whose much higher reported to be up- or down-regulated during heat stress, there levels of expression in symbiotic than aposymbiotic anemones in are as yet few cases in which the available data are convincing, the absence of stress suggest that they are important for the sym- consistent across multiple studies, and strongly suggestive of biosis. Unexpectedly, in many cases, these expression levels de- clined precipitously long before bleaching itself was evident, Significance suggesting that loss of expression of symbiosis-supporting genes may be involved in triggering bleaching. Coral reefs are biodiversity hotspots of great ecological, eco- nomic, and aesthetic importance. Their global decline under heat-shock proteins | innate immunity | symbiosis | nutrient transport | climate change and other stresses makes it urgent to under- reactive oxygen species stand the molecular bases of their responses to stress, includ- ing “bleaching,” in which the corals’ photosynthetic algal hallow-water coral reefs are biodiversity hotspots in the trop- symbionts are lost, thus depriving the host animals of a crucial Sical and subtropical oceans. These ecologically, economically, source of energy and metabolic building blocks. We sought and aesthetically important ecosystems are underpinned by reef- clues to the mechanisms that cause (or protect against) building corals, whose ecological and evolutionary success is due in bleaching by analyzing the patterns of gene expression in a sea large part to their mutualistic relationship with dinoflagellates in anemone relative of corals during exposure to a heat stress the family Symbiodiniaceae (1). These algal endosymbionts enable sufficient to induce bleaching. The results challenge some corals to thrive in nutrient-poor waters by providing them with current ideas about bleaching while also suggesting hypothe- photosynthetically derived energy (via the transfer of glucose and ses and identifying genes that are prime targets for future perhaps other compounds) and metabolic building blocks, and in genetic analyses. return the coral hosts provide the algae with shelter and inorganic nutrients, including ammonium (2–11). However, corals are en- Author contributions: P.A.C., C.J.K., E.M.L., M.O., and J.R.P. designed research; P.A.C., dangered globally by a variety of anthropogenic stressors, including C.J.K., and E.M.L. performed research; P.A.C., C.J.K., E.M.L., M.O., and J.R.P. analyzed data; the rising sea-surface temperatures associated with climate change. and P.A.C., C.J.K., and J.R.P. wrote the paper. These stresses have increased the frequency of coral bleaching, in Reviewers: A.C.B., University of Miami; and K.L.B., University of Pennsylvania. which the algal symbionts are lost from the coral tissue; when The authors declare no competing interest. prolonged, bleaching leads to coral death (12, 13). Published under the PNAS license. Because of the critical threat posed by heat-induced bleaching, 1P.A.C. and C.J.K. contributed equally to this work. a major focus of recent research has been to investigate the mo- 2Present address: Department of Embryology, Carnegie Institution for Science, Baltimore, lecular bases of the coral response to heat stress and the cellular MD 21218. mechanisms underlying heat-induced bleaching. Among other 3Present address: Department of Biology, Duke University, Durham, NC 27708. approaches, numerous transcriptomic studies over the past ∼15 y 4To whom correspondence may be addressed. Email: [email protected]. have examined the gene-expression responses to heat stress in This article contains supporting information online at https://www.pnas.org/lookup/suppl/ corals and related symbiotic cnidarians (recently reviewed in refs. doi:10.1073/pnas.2015737117/-/DCSupplemental. 14, 15). From these studies, it seems clear that heat-stressed www.pnas.org/cgi/doi/10.1073/pnas.2015737117 PNAS Latest Articles | 1of12 Downloaded by guest on October 2, 2021 relevant biological mechanisms. For example, one study has heat stress as it relates to bleaching itself. (iv) Although some reported a large increase in mRNA for the transcription factor studies have used aposymbiotic larvae, only two studies of which NFκB (a major regulator of innate immunity) during the first we are aware have attempted systematic comparisons of gene few hours of heat stress (22), consistent with the hypothesis (25) expression under heat stress in symbiotic and aposymbiotic ani- that activation of innate-immunity and apoptotic pathways plays mals (27, 39), and each of those studies involved a single time of a central role in bleaching. However, that study only examined sampling. Thus, there has been little information available on gene expression during the first few hours of heat stress, so that how the presence of the symbiotic algae might influence the the full dynamics of the NFκB response remained unclear. dynamics of the heat-stress response, a point of particular in- Moreover, another study that examined early time points did not terest given the prominent hypothesis suggesting that bleaching note changes in NFκB mRNA levels (19), whereas of two studies is triggered by the release of ROS by heat-stressed algae (25, 34). that examined a later time point (∼24 h of heat stress), one In this study, we attempted to achieve a clearer picture of gene reported elevated levels of NFκB mRNA (26) while the other did expression during heat stress by using RNAseq to analyze sam- not (27). A sustained elevation of NFκB mRNA during heat- ples obtained over a full time course that began soon after the induced bleaching would be consistent with the evidence that imposition of heat stress and continued until bleaching was es- NFκB protein is present at lower levels in symbiotic than in sentially complete. We used the small sea anemone Aiptasia aposymbiotic animals, which may be necessary to avoid innate- (sensu Exaiptasia pallida), a model system with great advantages immune rejection of the foreign cells (28). In summary, the for study of many aspects of cnidarian-dinoflagellate symbiosis available data, although intriguing, have not yet provided a clear (47–49), including the long-term viability of fully aposymbiotic picture of the expression behavior of NFκB (and thus, presum- animals (43, 50, 51). The results obtained suggest hypotheses ably, of its targets) during heat stress. about the molecular and cellular mechanisms of heat-stress re- Similarly, several studies have reported up-regulation during sponse and bleaching that should be testable using the gene- heat stress
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