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 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 declined 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, including “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 First published November 9, 2020.

28906–28917 | PNAS | November 17, 2020 | vol. 117 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.2015737117 Downloaded by guest on September 30, 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 of genes whose products may be protective against the knockdown and gene-knockout methods that are becoming effects of oxidative stress (14, 19, 26, 29–33), consistent with the available for symbiotic cnidarians (52–54). hypothesis that the production of reactive oxygen species (ROS) by heat-stressed algal chloroplasts plays a central role in trig- Results gering bleaching (25, 34). However, in all of the studies of which Strategy for Analysis of Gene Expression during Heat Stress. To ex- we are aware, the response detected has involved only one or a amine the transcriptional responses of Aiptasia to heat stress, we few genes (rather than the whole suite that might be expected), performed RNAseq time courses on both symbiotic and apo- the fold changes observed have been modest, and/or the par- symbiotic anemones after a shift to 34 °C (Fig. 1 A and B). For GENETICS ticular genes reported as up-regulated have been different from comparison, we also determined bleaching rates in the symbiotic those reported in the other studies. Moreover, some studies that anemones (Fig. 1 C and D and SI Appendix, Table S1). The seemingly could have seen such changes did not (18, 35–37). Thus, sampling times for RNAseq were chosen to capture changes that it has remained unclear whether there is a concerted transcrip- occurred before, during, and after bleaching. tional response to oxidative stress during heat stress and, if so, whether it depends on the presence of the algal symbionts. Rapid Up-Regulation of Many Immune-Response, Protein-Folding, and Finally, as the expression of many genes is markedly different Other Genes in both Symbiotic and Aposymbiotic Anemones. To between symbiotic and aposymbiotic animals in the absence of explore the early transcriptional responses to heat stress, we used stress (7–10, 27, 38–45), it is of interest to ask how the expression DESeq2 to identify genes that were significantly and highly up- of such “symbiosis genes” changes under conditions that will regulated (P < 0.05, fold change >4) in symbiotic anemones eventually lead to symbiosis breakdown: do their expression levels during the first 3 h after the initiation of heat stress; this revealed simply track with the numbers of remaining algae, or do they an- a set of 524 genes (Dataset S1). After normalizing the expression ticipate or lag behind the bleaching curve? To the best of our of each gene to its value at time = 0, we used k-means clustering knowledge, no studies published to date have addressed this to group the genes into two clusters (320 and 204 genes, re- question through comparative time courses of gene expression in spectively) based on the similarity of their expression patterns symbiotic and aposymbiotic animals. across the time course; we then sorted each cluster according to In summary, despite a considerable effort, we do not yet have the fold change from 0 to 3 h (Fig. 2 A, Left). The genes in the a clear picture of the transcriptional responses to heat stress in two clusters showed similarly dramatic levels of early up- symbiotic cnidarians or of which of these responses may be in- regulation (e.g., 28 and 17 genes with up-regulation by 3 h of volved in causing—or in protecting from—bleaching. The rea- more than eightfold in clusters I and II, respectively) but differed sons include at least the following: (i) The studies to date have in that expression of most genes in cluster I returned to near used a variety of cnidarian and algal species, often a variety of baseline levels by 12 h, whereas expression of most genes in genotypes and prior thermal histories for the individuals within a cluster II returned to baseline levels more slowly (Fig. 2 A, Left given species, and a variety of temperature and light regimens for and Fig. 2B). the stress experiments themselves, all of which complicate at- To ask if these early responses to heat stress are affected by tempts to compare the results of different studies (46). (ii) Such the presence of the symbiotic algae, we examined the expression comparisons are further complicated by the changes during the of the same 524 genes over an identical time course at 34 °C in past dozen years in the methods used for such studies, which aposymbiotic anemones. Strikingly, most (but not all) genes be- have progressed from an early reliance on microarrays (with haved similarly (Fig. 2 A, Right, Fig. 2B, and Dataset S1; and see limited numbers of features) and quantitative RT-PCR (of small below), suggesting that the majority of these gene-expression numbers of preselected genes) to the more comprehensive and changes are part of the animal’s core response to heat stress quantitatively reliable RNAseq. (iii) Although some studies have independent of its symbiotic state. analyzed samples collected soon after the imposition of heat To explore the functions of the genes showing an early re- stress (and thus well before bleaching is observed), and others sponse to heat stress, we performed Gene Ontology (GO)-term have analyzed samples collected considerably later (and thus analyses on clusters I and II (Dataset S2). For cluster I, the most concomitant with, or even after, bleaching), only one study of enriched GO terms were related to innate immunity and apo- which we are aware has analyzed samples of both types, and that ptosis (Fig. 2 C, Top; see also Fig. 2B, plots 1, 2, 4, and 5 for study used only aposymbiotic larvae (19). Thus, we have had no examples; Dataset S1). For cluster II, the most enriched GO real picture of the dynamics of the transcriptional response to terms were related to protein folding and the endoplasmic

Cleves et al. PNAS | November 17, 2020 | vol. 117 | no. 46 | 28907 Downloaded by guest on September 30, 2021 reticulum (ER)-stress response (Fig. 2 C, Bottom; see also Time course of temperature increase A Fig. 2B, plots 9, 10, and 12 for examples; Dataset S1). Inspection of Fig. 2A suggested that some genes did not follow 34 the general rule of rapid up-regulation in aposymbiotic animals C) o paralleling that in symbiotic animals. This set of genes was 32 revealed more clearly when we resorted the genes in each cluster by their fold change between 0 and 3 h in the aposymbiotic an- 30 imals (Fig. 2 D and E and SI Appendix, Fig. S1). Some of these genes (particularly in cluster I) seemed not to be up-regulated at all in the aposymbiotic animals during the heat stress, whereas

Temperature ( 28 others (particularly in cluster II) seemed to be up-regulated, but only after a delay relative to what was observed in the symbiotic 0 12345 animals. In total, this analysis identified 41 genes in cluster I and Time since initiation of heat stress (h) 18 genes in cluster II that were up-regulated less than twofold in aposymbiotic animals (versus their more than fourfold up- B Times of sampling for RNAseq regulation in symbiotic animals). Surprisingly, inspection of the annotations of these genes (SI Appendix, Table S2 and Dataset 043123 12 24 896S3) did not reveal any obvious common themes. In particular, none of these genes had annotations relating directly to oxidative-stress response, a surprising result given the Bleaching (Long term) common expectation that symbiotic anemones would have C higher levels of ROS due to their increased production by heat- stressed algal chloroplasts (25, 34, 55, 56). To explore this issue

nietorp gμ rep sllec sllec rep gμ nietorp further, we asked if any of 99 Aiptasia genes with putative roles 100 in the oxidative-stress response (the 97 genes manually curated

)gniniamer %( )gniniamer in ref. 33 plus two genes [AIPGENEs 21344 and 21459] anno- 75 tated as catalases) were up-regulated early during heat stress. Strikingly, only one of these genes was in either cluster I or cluster II, and this gene was up-regulated to similar extents in 50 symbiotic and aposymbiotic anemones (Dataset S1 and SI Ap- pendix, Tables S3A and S3B: AIPGENE 8449). Thus, we found

laglA 25 no evidence of an early transcriptional response to ROS that was specific to the presence of the algal symbiont. Because our initial analysis had focused specifically on genes 0 that were strongly up-regulated during the first 3 h of heat stress, 0424 896 and it seemed possible that a transcriptional response to ROS Time since initiation of heat stress (h) would be manifested only at later times, we performed two additional analyses. First, we asked if any of the 99 genes were < > D Bleaching (Short term) strongly up-regulated (P 0.05, fold change 4) in symbiotic animals at times later than 3 h. We found only two such genes, both annotated as glutathione S-transferases (Dataset S4 and SI 100 Appendix, Table S3A: AIPGENEs 20480 and 6351). Both were ni modestly up-regulated throughout the time course and met the

e t more than fourfold cutoff only at 12 h (AIPGENE 20480) or 48

)gnin and 96 h (AIPGENE 6351). Moreover, both genes were also up-

gμ gμ regulated in aposymbiotic anemones (SI Appendix, Table S3B),

iamer %( iamer

r while neither was up-regulated in symbiotic relative to aposym-

p biotic anemones (SI Appendix, Table S3C). Second, we asked if

sllec l sllec 50 any of the 99 genes were strongly up-regulated (P < 0.05, fold difference >4) in symbiotic relative to aposymbiotic animals at any time point. We found only three such genes (SI Appendix,

aglA 25 Table S3C: AIPGENEs 8889, 22290, and 27830), and, strikingly, none of them was also up-regulated during the heat-stress time 0 course in symbiotic anemones (SI Appendix, Table S3A). Even relaxing the fold-change cutoff from fourfold to twofold revealed 0 612 24 48 no very suggestive patterns of expression (SI Appendix, Table S3 Time since initiation of heat stress (h) and Dataset S4), and, strikingly, neither of the putative catalase genes met even this relaxed criterion. Taken together, these data Fig. 1. Experimental design. (A) Gradual increase in temperature after indicate that at least for this Aiptasia strain and stress conditions, shifting a tank containing anemones in 1 L of ASW from an incubator at 27 there is no concerted and strong transcriptional response to °C to one at 34 °C. (B and C) Time courses of sampling symbiotic (CC7-SSB01) and aposymbiotic (CC7-Apo) anemones for RNAseq analyses (B) and the symbiotic anemones for assessment of bleaching (C). The tanks were shifted from an incubator at 27 °C to one at 34 °C at time = 0. In C, algal counts were experiment variability at 24 h probably reflects the fact that bleaching is just normalized to total protein in the homogenates (Materials and Methods) beginning at around this time), but the levels of bleaching at 48 h were similar. and then expressed as percentages of the value at time = 0. (D) In a separate In C and D, data are shown as means ± SEMs of the percentage values; the experiment, bleaching was assessed over a shorter time course. Less bleaching actual numbers of algae per microgram (μg) of protein are shown in SI Ap- was observed at 24 h than in the experiment of B and C (experiment-to- pendix,TableS1.

28908 | www.pnas.org/cgi/doi/10.1073/pnas.2015737117 Cleves et al. Downloaded by guest on September 30, 2021 A Symbiotic Aposymbiotic B 4 Caspase-7 NFKB1 Heat shock factor protein 1 2 AIPGENE3163 AIPGENE8848 AIPGENE11173 10000 8000 0 1 2 3 7500 4000 6000 -2 5000 4000 2000 -4 2500 2000 0 TNF receptor-associated Hypoxia-inducible factor Interferon regulatory factor 8 factor 3 1-alpha inhibitor I AIPGENE1568 AIPGENE14887 AIPGENE25750 I 300 4 5 6 4000 9000 200 3000 6000 2000 100 1000 3000 0

TNF receptor Heat shock cognate Calumenin-B superfamily member 27 71 kDa protein AIPGENE9938 1600 AIPGENE27609 120000 AIPGENE21738 6000 7 8 9 1200 90000 4000 800 60000

Expression (Normalized read count) 2000 400 30000

0 0

DnaJ homolog subfamily C Protein disulfide-isomerase A3 Protein ERGIC-53 member 22 II AIPGENE12999 AIPGENE3723 AIPGENE5858 16000 II 1000 10 11 12 12000 2000 750

8000 500 1000 4000 250

0 3 12 24 9648 0 3 12 964824 0 3 482412 96 0 3 482412 96 03 482412 96 C GENETICS GO.ID Term Annotated Significant Fisher GO:0042981 regulation of apoptotic process 843 22 5 E-06 I GO:0002224 toll-like receptor signaling pathway 59 6 3 E-05 GO:0002218 activation of innate immune response 89 11 7 E-05 GO:0006457 protein folding 138 16 1 E-15 GO:0044267 cellular protein metabolic process 4003 31 5 E-12 II GO:0034976 response to endoplasmic reticulum stress 106 8 9 E-05 GO:0006986 response to unfolded protein 80 7 9 E-05

Histamine H2 receptor Fibroblast growth factor 7 Krueppel-like factor 5 D E AIPGENE15499 AIPGENE6180 AIPGENE26044 Symbiotic Aposymbiotic 600 400 3000

400 300 II2000 II I I 200 200 1000 100

II 0 0 0 3 12 964824 0 3 12 24 9648 30 482412 96 0 3 482412 96 0 3 482412 96 F Symbiotic Aposymbiotic Symbiotic Aposymbiotic G All genes Cluster I Cluster II I

II Density

0 3 12 24 9648 0 3 12 24 9648 0.0 0.2 0.4 0.6 0.8 1.0 0 3 12 24 9648 0 3 12 24 9648 −2 −1 0 1 2 Log2 Fold-difference Sym/Apo at t = 0

Fig. 2. Rapid up-regulation of many genes in response to heat stress in both symbiotic and aposymbiotic anemones. (A) Heatmaps of genes significantly and highly up-regulated in symbiotic anemones in response to a shift from 27 °C to 34 °C (Left; see text for details) and of the same genes in aposymbiotic anemones (Right). Genes were grouped into two clusters by k-means clustering based on their expression patterns in the symbiotic animals and are sorted by their extents of up-regulation from 0 to 3 h in those animals. Expression values for each gene in the aposymbiotic animals (Right column) were also normalized to the value for that gene in the symbiotic animals at time = 0, and the genes are shown in the same order as in the Left column. Colors in the

heatmap are based on a log2 scale (as shown). (B) Expression patterns for example genes in clusters I (plots 1 through 6) and II (plots 7 through 12). Data are shown as raw read counts normalized to library size (Materials and Methods). Red, data for symbiotic animals; blue, data for aposymbiotic animals. (C) Results from GO-term-enrichment analyses of each cluster. The terms shown are those significant in each cluster (Fisher’s P value <1e-4) and represented by ≥6 genes; the full outputs of these analyses are provided in Dataset S2.(D) Heatmaps (color scale as in A) showing genes from clusters I and II after normalizing each gene’s expression level to its mean value in aposymbiotic animals at time = 0 and then sorting by the degree of up-regulation from 0 to 3 h in aposymbiotic animals. Shown here are only the genes in which up-regulation in the aposymbiotic animals was less than twofold at 3 h; the complete heatmaps are shown in SI Appendix, Fig. S1.(E) Examples of genes showing differential regulation between symbiotic and aposymbiotic anemones. The cluster number for each gene is indicated. Red, data for symbiotic animals; blue, data for aposymbiotic animals. (F) Heatmaps (color scale as in A) of genes as in A but after resorting based on the fold difference between symbiotic and aposymbiotic animals at time = 0. All genes are shown in which this difference was statistically significant (adjusted P value <0.05), independent of the magnitude of the expression difference or of whether expression was higher at 0 h in symbiotic or aposymbiotic animals. The dashed line separates the two groups, and the complete heatmaps are shown in SI Appendix, Fig. S2.(G) Smoothened-density histograms

(Materials and Methods)oflog2 fold differences between symbiotic and aposymbiotic animals at 0 h.

Cleves et al. PNAS | November 17, 2020 | vol. 117 | no. 46 | 28909 Downloaded by guest on September 30, 2021 A 500 bp Promoter Heat-Responsive Gene Top 100 upregulated genes (3 h:0 h)

B 2 C Motif 1 Motif 2 2 P ~ 4 x 10-29 1 P -18 1 bits ~ 2 x 10 bits Sites Found: 29 GT C TT A T T T C A CC Sites Found: 30 AG T C G GA CA GA G G A G A A T TCGT CC C C A T T TC T AC C GACCATC T G GG C GT GT A T A GAA CT AT A 0 AA GG TT CCC 0 A T G

2 2 Top hit: NFкB Top hit: HSF1 1 1 bits q ~ 8 x 10-4 bits A q ~ 2 x 10-3 T TA A C C G TT GAG CG CT G C A TAG T T TC A A GAA T A 0 GGGGA TCCCC 0 TC G D Putative E Putative 0.6 NFкB Binding 0.6 HSF1 Binding Sites Sites All Genes 0.4 All Genes 0.4 N ≥ 2 N ≥ 2 N ≥ 3 N ≥ 3 Density Density N ≥ 4 0.2 N ≥ 4 0.2 N ≥ 5 N ≥ 5 N ≥ 6 0.0 0.0 50−5 10 50−5 10 2015

Log2 Fold-change (3 h:0 h) Log2 Fold-change (3 h:0 h)

Log Expression F 2 G

Putative NFкB targets −2−3−1 0 1 2 3 Putative HSF1 targets

7 RCHY1 (E3 ubiquitin−protein ligase) 7 HSP90 (Heat shock protein) MAFG (Transcription factor) HSP70 (Heat shock protein) 6 BCL3 6 DTX3L (E3 ubiquitin−protein ligase) No Annotation 5 HSPA8 (Heat shock protein) ADRA1A (Adrenergic receptor α−1A) GBP5 (Guanylate−binding protein 5)

5 No Annotation 4 No Annotation HSP20 (Heat shock protein) TNF receptor-assiociated factor 3 HSPA8 (Heat shock protein) Cyclic GMP−AMP synthase No Annotation DZIP3 (E3 ubiquitin−protein ligase) IFIH1 l(2)efl (HSP20 Family) 4 IFIH1 PXMP2 (Peroxisomal membrane protein) 3 Number of binding sites Number of binding sites No Annotation BAG domain-containing protein 3 0 3 12 24 48 96 0 3 12 24 48 96 Time (h) In Cluster I In Cluster II Time (h)

Fig. 3. Enrichment of potential NFκB and HSF1 binding sites in the promoter regions of genes that are highly up-regulated during heat stress. (A) Strategy used to search for transcription-factor binding sites involved in the response to heat stress; see text for details. (B and C) Logo plots showing the top two hits returned by the MEME search (Upper plots) and their correspondence to canonical binding sites (Lower plots) for transcription factors NFκB(B) and HSF1 (C); see text for details. (D–G) Correlations between numbers of putative binding sites for NFκB(D and F) and HSF1 (E and G) and degrees of up-regulation of the

associated genes. (D and E) Smoothened-density histograms for the indicated numbers of binding sites plotted as a function of the log2 fold changes in transcript levels between 0 and 3 h after the shift to 34 °C for all genes in the genome. (F and G) Heatmaps showing changes in transcript levels (log2 scale; see key) as a function of time since the initiation of heat stress for all of the genes with four or more putative NFκB(F) or HSF1 (G) binding sites plus some of the genes with three such sites in each case. Annotations were obtained from ref. 7 and by manual BLASTP searches against nonredundent databases on NCBI for genes with no annotation (99); red font shows genes that are known to be regulated by the respective transcription factor in at least one other organism.

oxidative stress during heat stress despite the occurrence of heat stress were also expressed at substantially higher levels in nearly complete bleaching during the time course (Fig. 1). symbiotic than in aposymbiotic animals in the absence of stress. Inspection of Fig. 2A also suggested that some of the genes Such genes were revealed more clearly when we resorted the that underwent dramatic up-regulation during the first 3 h of genes in each cluster according to the magnitude of the difference

28910 | www.pnas.org/cgi/doi/10.1073/pnas.2015737117 Cleves et al. Downloaded by guest on September 30, 2021 in expression between symbiotic and aposymbiotic animals at break down under heat stress, we first identified the genes that time = 0(Fig.2F and SI Appendix,Fig.S2); they were present in showed strong differential expression (P < 0.05; fold differ- both clusters (based on P < 0.05; fold difference > 2) but more ence >4or<0.25) in our RNAseq dataset at time = 0, finding numerous in cluster I (57 genes, ∼18%) than in cluster II (22 337 that were up-regulated and 150 that were down-regulated in genes, ∼11%) (SI Appendix,TableS4and Dataset S5). Such genes symbiotic relative to aposymbiotic anemones. We then followed were also enriched relative to their frequency among all genes, these differences in expression during the period of heat stress particularly in cluster I (Fig. 2G; P < 2e-16 for cluster I and P < (Fig. 4A). Not surprisingly, for many genes, the expression levels 0.02 for cluster II by two-sample Kolmogorov–Smirnov tests). in the symbiotic and aposymbiotic anemone populations gradu- Many of these genes have annotations related to immune response ally converged as the symbiotic animals lost algae through bleaching or apoptosis (particularly in cluster I: SI Appendix, Table S4A)and (and thus approached the aposymbiotic state). Unexpectedly, how- protein folding (particularly in cluster II: SI Appendix, Table S4B). ever, for some genes the expression levels had changed dramatically These data suggest that there may be stress arising from the by 12 or even 3 h (Fig. 4A), although no bleaching was detected until presence of the algae even at a nominally nonstressful temperature 24 h or later (Fig. 1 C and D). (Discussion). To investigate these genes further, we first ranked the 337 genes that were initially expressed at higher levels in symbiotic Apparent Involvement of the NFκB and HSF1 Transcription Factors in anemones (green dots in Fig. 4A) by their fold decrease in ex- the Early Up-Regulation of Stress-Responsive Genes. The seemingly pression in symbiotic anemones between 0 and 12 h (SI Appen- coordinated up-regulation of many genes during the early hours dix, Table S5A and Dataset S6). Strikingly, ∼25% (84 of 337) of of heat stress suggested that there might be common control by these genes fell 3-fold or more during the first 12 h of heat ex- specific transcription factors. To explore this possibility, we first posure, with ∼5% (18 of 337) dropping 10-fold or more. Inter- identified the 100 genes with the greatest up-regulation over the estingly, among the genes whose expression dropped most rapidly first 3 h of heat stress in symbiotic animals; 58 of these genes were ones encoding ammonium-, glucose-, and sterol-transport were in cluster I of Fig. 2A, and 42 were in cluster II. In each case proteins (Fig. 4 B–D and SI Appendix, Table S5A), all of which in which the genome assembly contains 500 bp immediately are likely to be involved in metabolite exchanges critical for sup- upstream of the putative transcription start site (Fig. 3A;96of port of the symbiosis. Other genes with dramatic early decreases the 100 genes), we searched these 500 bp for significantly in expression (Fig. 4 E–G and SI Appendix, Table S5A) include enriched DNA motifs (Materials and Methods). Remarkably, ones encoding extracellular-matrix/cell-adhesion, innate-immunity/ these searches identified two DNA motifs with high confidence stress-response, and potential signaling proteins, as well as proteins (Fig. 3 B and C, Upper) that corresponded (again with high involved in lipid (including arachidonic acid) metabolism, all of GENETICS confidence) to canonical binding sites for the transcription fac- which may also be important to support the symbiosis. Thus, the tors NFκB and HSF1 (Fig. 3 B and C, Lower). In the 96 upstream host begins to reduce the expression of many symbiosis-supporting regions, we found 29 putative NFκB binding sites (20 in 10 genes genes long before bleaching is detected (Discussion). of cluster I; 9 in 9 genes of cluster II) and 30 putative HSF1 We performed similar analyses of the 150 genes that were binding sites (9 in 8 genes of cluster I; 21 in 10 genes of cluster initially expressed at lower levels in symbiotic anemones (black II). Consistent with the hypothesis that NFκB and HSF1 are dots in Fig. 4A), keeping in mind the ambiguity as to whether important in driving the early burst of gene up-regulation upon these genes should be viewed as down-regulated in unstressed heat stress, the genes encoding these two transcription factors symbiotic anemones, up-regulated in unstressed aposymbiotic were themselves highly up-regulated early in the course of heat anemones, or both. We first ranked the genes in this group by stress (Fig. 2B, plots 2 and 3). their fold increases in expression between 0 and 12 h in symbiotic To explore further whether the identified motifs, and their anemones. However, this analysis was uninformative: only 10 associated transcription factors, are indeed involved in the up- genes had fold changes more than twofold, and their associated regulation of genes in response to heat stress, we performed two annotations did not fit any evident pattern (SI Appendix, Table additional bioinformatic analyses. First, for all 22,872 annotated S5B and Dataset S7). We then ranked the same genes according Aiptasia genes with 500 bp of immediately upstream sequence to their fold decreases in expression between 0 and 12 h in present in the genome assembly, we searched these 500-bp seg- aposymbiotic anemones, obtaining potentially more informative ments for the presence of each motif and then correlated the results. Again, just 10 genes had fold changes <0.5-fold, but numbers of motifs found with the degrees of up-regulation found several of these did have annotations suggestive of possible roles during the first 3 h of heat stress (Materials and Methods). The in symbiont uptake or the digestion of food (both processes that smoothened-density plots showed a strong correlation between might well be enhanced in aposymbiotic animals) (SI Appendix, the numbers of such motifs and the degrees of up-regulation Table S5C and Dataset S8). However, why heat stress would (Fig. 3 D and E). Second, we examined the specific genes with drive down the expression of these genes remains mysterious. three or more putative NFκB or HSF1 binding sites in their upstream regions. Strikingly, not only did many of these genes Discussion show strong up-regulation during the first 3 h of heat stress, but It is important to understand both the mechanisms that cause many of them are the Aiptasia homologs of genes known to be coral bleaching under heat stress and those that may help protect targets of these same transcription factors in other organisms against it. To this end, we used RNAseq to examine gene ex- (Fig. 3 F and G). Moreover, the putative NFκB and HSF1 target pression in the sea anemone Aiptasia through a time course that genes identified in this way had expression patterns similar to began soon after the start of heat stress and continued until those typical of cluster I (rapid return to baseline) and cluster II bleaching was essentially complete. By using closely controlled (slower return to baseline), respectively, suggesting the existence laboratory conditions and clonal populations of both the animal of distinct patterns of regulation in the response to heat stress host and the algal symbiont, we eliminated some of the variables that are driven at least in part by NFκB and HSF1. that have complicated interpretation of many earlier studies (46). By focusing on genes with relatively large changes in mRNA Surprisingly Rapid Changes in Expression of Many Genes with levels, we tried to identify cases in which there would actually be Putative Roles in Symbiosis Maintenance. Previous studies have significant changes at the protein level [for which mRNA levels identified many genes that are differentially expressed between are only a rather crude proxy (33, 57)]. And by subjecting both symbiotic and aposymbiotic cnidarians (in the Introduction). To symbiotic and aposymbiotic anemones to the full time course investigate the behavior of such genes as symbiosis begins to in parallel, we could ask which aspects of the animal’sresponse

Cleves et al. PNAS | November 17, 2020 | vol. 117 | no. 46 | 28911 Downloaded by guest on September 30, 2021 A 337 Genes 10

0 fold-difference between 2 −5 Log 150 Genes symbiotic and aposymbiotic anemones 0 3 12 24 48 96 Time since initiation of heat stress (h)

B Nitrogen Transport C Glucose Transport D Lipid Metabolism

Ammonium 120 15 30 Glut8 (18406) NPC2 (22473) transporter Rh type B 1000 100 (18105) NPC2 (22527) 90 Putative ammonium 750 Aldehyde dehydrogenase 75 transporter 1 10 20 family 3 member B1 (17420) (21619) 60 500 50 5 10 30 250 25 Relative expression

0 0 0 0 0 Relative expression (solid line) Relative expression (solid lines) Relative expression (dotted line) 0 3 12 24 48 96 Relative expression (dotted line) 0 3 12 24 48 96 0 3 12 24 48 96

EFG Extracellular matrix Immunity and Stress Arachidonic Acid Metabolism

25 Sulfotransferase 1C2A Phospholipase A2 (11619) Aggrecan core protein 30 8 (20198) (11251) Arachidonate 12-lipoxygenase 10 Nidogen-2 (11248) 20 Scavenger receptor class 6 (21885) B member 1 (25617) Arachidonate 5-lipoxygenase 6 Vitrin (6809) 20 15 (21881) TNFR27 (27690) 4 4 5 10 TGFa (894) 10 2 2 5 Relative expression

0 0 0 0 0 Relative expression (solid line) Relative expression (solid lines) Relative expression (dotted lines) 30 482412 96 30 482412 96 30 2412 48 96 Relative expression (dotted lines) Time since initiation of heat stress (h)

Fig. 4. Rapid changes in expression of many symbiosis-related genes after onset of heat stress. In the experiment of Fig. 1 A–C, genes with strong differential expression (≥4-fold, P < 0.05) between symbiotic and aposymbiotic anemones prior to heat stress were identified and analyzed. (A) Dot plots of the ratios of

expression levels between symbiotic and aposymbiotic anemones for individual genes (log2 scale) as a function of time after the temperature shift. Both genes expressed at higher levels at time = 0 in symbiotic anemones (green dots) and those expressed at higher levels in aposymbiotic anemones (black dots) are shown. (B–G) Examples of genes whose expression is rapidly down-regulated in symbiotic anemones after the temperature shift. The mean expression values for each gene at each time in symbiotic anemones are shown relative to the mean expression of that gene in aposymbiotic anemones at time = 0. See SI Appendix, Table S5A and the text for more details.

28912 | www.pnas.org/cgi/doi/10.1073/pnas.2015737117 Cleves et al. Downloaded by guest on September 30, 2021 relate directly to the presence of algal symbionts. The results have slower return of mRNA levels to baseline during continued heat provided a clearer picture of the heat-stress response than has stress (Fig. 2, cluster II). Many of these genes encode proteins been available previously and suggest numerous hypotheses for expected to function in protein folding and the response to ER which critical experimental tests should now be possible (58). stress. Early up-regulation of some of these genes in heat- stressed corals had been reported previously (18–22) and is Highly Transient Up-Regulation of a Large Set of Genes Including consistent with the very wide conservation of these heat-stress Many Implicated in Innate-Immunity Function. Our analyses responses in animals and other eukaryotes (16, 17). Many of the revealed a set of >300 genes that were rapidly (within <3 h) and up-regulated genes appear to be targets of the transcription extensively (more than fourfold) up-regulated upon exposure of factor HSF1, as expected from extensive studies in other or- symbiotic anemones to heat stress but whose transcript levels ganisms (62), and the HSF1 gene itself was rapidly and exten- also returned rapidly (by ≤12 h) to near-baseline levels even sively up-regulated in our experiment (Fig. 2B, plot 3). during continued heat stress (Fig. 2, cluster I). Many of these Like the cluster I genes (see above), most of the cluster II genes encode proteins with putative functions in innate immunity genes showed up-regulation in aposymbiotic anemones that was or apoptosis, and indeed many of them appear to be targets of very similar to that in symbiotic anemones. However, as argued the innate-immunity transcription factor NFκB. The finding of above, the products of these genes might still play roles either in conserved NFκB binding sites in the Aiptasia genome was not in promoting bleaching or in protecting the animals from it. Indeed, itself surprising (59), but their concentration in the regions up- given the well established role of the heat-shock proteins in stream of genes that are highly up-regulated during heat stress protecting various types of cells from protein damage during was striking. The NFκB gene itself is in cluster I (Fig. 2B, plot 2), heat stress, it seems likely that at least some of the cluster II gene as are the genes encoding several proteins likely to be upstream products will prove to be protective (up to a point) against the or downstream of NFκB in a TNF-like signaling pathway stresses that lead to bleaching. In this regard, we have recently (Fig. 2B, plot 4 and Dataset S1), while several other functionally shown that a CRISPR-induced knockout of the gene encoding related genes were in a second group (cluster II) of rapidly up- HSF1 produces increased sensitivity to heat-stress-induced death regulated genes (Fig. 2B, plot 8 and Dataset S1; see also below). in aposymbiotic larvae of the coral Acropora millepora (54). As Rapid up-regulation of some of these genes under heat stress Acropora larvae can be infected with Symbiodiniaceae strains had been reported previously [e.g., NFκB (22); TRAF3 (26, 32, and then subjected to bleaching tests (63, 64), it should now be 37)] but neither the full extent of this response nor its striking straightforward to ask whether a knockout of HSF1 (or of any of transience had been clear. its targets) also produces an increased sensitivity to heat-induced NFκB-regulated activation of innate-immunity and apoptotic bleaching. GENETICS pathways has been proposed to play a major role in bleaching (25, 60), a model consistent with recent evidence indicating that Apparent Absence of a Gene-Expression Response to Oxidative Stress NFκB protein and activity levels are reduced in symbiotic rela- during Heat Exposure. About 10% of the genes in clusters I and II tive to aposymbiotic Aiptasia (28). However, our results pose differed from the majority in showing a delayed and/or much significant challenges for this model. First, the mRNA levels for weaker up-regulation under heat stress in aposymbiotic than in most of these genes have returned to near-baseline levels well symbiotic anemones (Fig. 2 D and E). Examination of the an- before bleaching is even detectable, much less complete. These notations of these genes did not reveal any obvious common observations are not necessarily incompatible with the model, themes, and, in particular, none of them encodes a protein known because it is possible that the levels of the relevant protein to be directly involved in response to oxidative stress. More ex- products stay elevated long after the mRNA levels have declined tensive analyses of our data also failed to find any substantial and/or that the early activation of these genes triggers a later signature of up-regulation of oxidative-stress-response genes dur- wave(s) of activation of other genes whose products are more ing heat exposure (Results), even though bleaching was essentially directly involved in bleaching. However, it should be noted that complete by the end of the time course. the brief elevation of caspase 7 mRNA levels (Fig. 2B, plot 1) is These results were initially surprising, given the prominence of paralleled by a similarly brief elevation of caspase enzyme- models in which photosynthetically produced ROS play a central activity levels during heat stress (61), one of several lines of ev- role in triggering bleaching (25, 34, 55, 56). However, examina- idence suggesting that apoptosis does not play a major role in tion of the literature revealed that previous gene-expression bleaching under these conditions (61). Nonetheless, as NFκB has studies have also found either the up-regulation of only one or many roles, it may be involved in bleaching in some way other than a few oxidative-stress-response genes during heat exposure, and its promotion of apoptosis through caspases. Second, most of the generally with only modest levels of up-regulation (14, 19, 26, genes in this group had very similar expression profiles in apo- 29–33) or no such up-regulation at all (18, 35–37). Thus, to date, symbiotic anemones, indicating that this aspect of the response to gene-expression studies in a variety of symbiotic cnidarians, using heat stress is inherent to the animal and not specific to the presence a variety of light- and heat-stress conditions, have failed to of the symbiotic algae. However, these observations are also not produce appreciable support for the ROS-induced-bleaching necessarily incompatible with the model, because it is possible that model. an ancestral animal response to heat stress was co-opted during However, these results do not necessarily invalidate the model. evolution to provide a mechanism for ridding the animal cells of First, it is possible that increased levels of ROS during heat stress algal partners that have become unwanted under stress conditions. result in changes in transcript levels that are biologically mean- Resolution of these questions is likely to come only when the ingful but less than the twofold cutoff used in our analyses, that rates of bleaching can be evaluated in animals in which the genes the relevant genes are not known to be involved in response to encoding NFκB and/or others of this early up-regulated set have oxidative stress, or both. Second, it is possible that there is rel- been inactivated. Fortunately, such experiments should soon be evant regulation, but all at the posttranscriptional level. This might possible using the recently developed methods for morpholino- explain the apparent discrepancy between results at the mRNA based gene knockdown (52) and CRISPR-based gene knockout level and those reported at the protein and enzyme-activity levels (53, 54) in corals. (34, 65–72). Third, our experiment used rather low light levels and a spectrum with little UV, and it is possible that experiments with Longer Lasting Up-Regulation of a Large Set of Genes Including Many more intense light or a full-sunlight spectrum would have yielded Implicated in Protein Homeostasis. A second group of >200 genes different results. However, it must be noted both that the condi- showed equally striking early up-regulation but, in most cases, a tions in our experiment did lead to full bleaching within ∼4dand

Cleves et al. PNAS | November 17, 2020 | vol. 117 | no. 46 | 28913 Downloaded by guest on September 30, 2021 that some of the other studies cited above used more intense light, likely to require experiments in which the relevant genes have full-spectrum sunlight, or both, so that this possibility does not been ectopically expressed from constitutive promoters, knocked actually seem likely. Moreover, in a previous study, we observed down or knocked out, or both; such experiments should now be that bleaching under heat stress could occur as rapidly in the dark feasible (52–54, 80). as in the light both in Aiptasia and in several species of corals (73), Of perhaps greater interest, however, is to ask how the rapid results clearly incompatible with the hypothesis that photosyn- down-regulation of symbiosis-related genes relates to the break- thetically derived ROS are necessary to trigger bleaching. Other down of the symbiosis that follows later. Possible models include studies have also reported results that are difficult to reconcile at least the following: with the ROS-induced bleaching model (41, 44, 71, 74–76). We (i) Breakdown of the symbiosis at the cellular level (ejection of suggest that the possible role, if any, of photosynthetically pro- algae from host gastrodermal cells) actually occurs much duced ROS in bleaching will not be settled until bleaching rates earlier than is evident from whole-animal color or algal have been measured in animals in which genes encoding proteins counts on whole-animal homogenates. This possibility involved in controlling oxidative stress have been knocked down or should be testable by real-time fluorescence video micros- knocked out, experiments that should now be feasible (52–54). copy that takes advantage of the intrinsic chlorophyll fluorescence of the algae. Evidence That the Presence of Algae May Be Stressful Even in the (ii) Very soon after the onset of heat stress, the algae cease to Absence of Heat. Among the genes up-regulated early in response to heat stress were a substantial number that were already release something that they normally provide to the host [e.g., glucose (6) or sterols (8, 10)], in response to which the up-regulated in symbiotic relative to aposymbiotic anemones host turns off genes necessary for support of the algae [e.g., under nominally nonstressful conditions (Fig. 2F). Such genes the symbiosome-membrane NH transporter(s)], resulting were much more frequent than expected by chance, particularly 3 in algal loss. This model is attractive in part because it fits in cluster I (Fig. 2G), and annotations relating to immunity, well with an increasing body of evidence for the importance apoptosis, and protein folding appeared to be particularly fre- of nutritional interactions (and, especially, the regulation of quent among them (SI Appendix, Table S4), even given the high nitrogen supply) in the control of algal numbers in symbi- overall frequencies of such annotations in the full clusters I and otic corals and anemones (4, 5, 9, 11, 81–88). Evaluation of II. Taken together, these results suggest that even at nonstressful this model should probably begin by establishing [by gene temperatures, symbiotic anemones experience some stress simply knockdown or knockout (52–54)] which of the metabolite- due to the presence of the algal symbionts. Although it is not yet transporter genes [e.g., GLUT8 (43), the two NPC2s (8, 10), clear what that stress might be, it may represent a physiological and the two NH -transporter genes (9, 43) (Fig. 4B)] are cost to the host that partially offsets the benefits of the symbiosis, 3 actually essential for symbiosis maintenance. Evaluating as has been observed in many other mutualisms (77–79). heat-induced bleaching in animals expressing the appropri- Possible Roles in Bleaching of a Rapid Down-Regulation of Many ate NH3 transporter(s) from a constitutive promoter [which Symbiosis-Related Genes. As observed also by others (7–10, 27, should soon be possible (54, 80)] might ultimately provide 38–45), we found that many genes were highly up-regulated in an incisive test of the model. symbiotic relative to aposymbiotic anemones prior to stress (Fig. (iii) Very soon after the onset of heat stress, the algae begin to 4A, 0 h). The differential expression of these genes and putative release something (e.g., ROS; see, however, above) that is functions of their products suggest that they have direct and toxic to the host, in response to which the host turns off important roles in supporting the symbiosis. Thus, we expected genes necessary for support of the algae, resulting in bleach- that in most cases, their levels of up-regulation would decline in ing. This model might also be tested by establishing consti- parallel with the numbers of algal cells during bleaching, and tutive expression of (for example) an NH3 transporter, such a gradual decline was indeed observed for many genes. although the consequences of thus potentially forcing reten- Unexpectedly, however, we also found that in addition to some tion of a source of toxic molecules are difficult to predict. genes in this group that were actually up-regulated in response to (iv) Very soon after the onset of heat stress, the host initiates a heat stress (see preceding section), ∼25% were very rapidly response in which it turns off genes necessary for support of down-regulated (to essentially aposymbiotic levels) by 12 or even the algae, resulting in bleaching. It is difficult to see why the 3 h (Fig. 4), and thus long before any bleaching was detected. host would do this, except in the context of model 2 or 3, The products of the genes showing this behavior include trans- but experimental tests could proceed along lines similar to porters likely to be involved in metabolite exchange between the those discussed above. host and alga, extracellular-matrix proteins that might be involved in surface contacts between host and algal cells, enzymes Rapidly Responding Genes as Possible Early Biomarkers for Coral of lipid metabolism, and other proteins that might be involved in Bleaching. The identification of many genes that undergo large signaling between the symbiotic partners. Thus, it appears that changes in expression (up or down) many hours in advance of the host transcriptional support for important aspects of the symbi- actual loss of algae from the host raises the possibility of using osis shuts down well before any actual loss of algae occurs. An these changes in expression as early biomarkers of incipient coral important remaining question is whether the levels of the cor- bleaching. This idea presupposes only that the kinds of changes responding gene products also decline precipitously during the seen here in Aiptasia will be observed also in corals of ecological early hours of heat stress; this question should be answerable by interest, which should be straightforward to test and indeed is experiments using appropriate antibodies or proteomic methods. already supported by considerable published data (18–22, 37). It will be of interest to determine what causes the rapid down- Such tests may be easier to deploy in the field if they can be done regulation of transcript levels. In this regard, it may be relevant using protein rather than mRNA levels, but the degree to which that at least 20 putative transcription factors are in the sets of protein levels track mRNA levels in this system is an issue that very rapidly up-regulated and very rapidly down-regulated genes needs to be explored in any case, as already noted above. (Datasets S1 and S6). Regulation by miRNAs might also be a factor (45). Further insight might be gained by examining the Concluding Remarks. It is important to note several limitations of relative timing of the various transcriptional changes in an this study. First, we examined a single combination of host and RNAseq time course with multiple time points during the first algal strain and subjected it to a moderate heat stress under low few hours of heat stress. However, a deeper understanding is light, such that bleaching occurred to near completion over ∼4d.

28914 | www.pnas.org/cgi/doi/10.1073/pnas.2015737117 Cleves et al. Downloaded by guest on September 30, 2021 Examination of a more (or less) sensitive holobiont or the use of manufacturer’s instructions to produce indexed libraries. The resulting li- more extreme conditions (e.g., higher light) might yield at least braries were pooled based on their indices (as described in the kit instruc- some different results. Second, except for the potential oxidative- tions), and sequencing of 36-bp single-end reads was performed by the stress-response genes, we focused here entirely on genes whose Stanford Center for Genomics and Personalized Medicine using an Illumina HiSeq 2000 sequencer. expression changed dramatically in the first few hours of heat stress. It is possible that an analysis focused on genes whose Analysis of Differential Gene Expression and GO-Term Analyses. Reads were expression is altered strongly when bleaching is in full swing (e.g., aligned to the Aiptasia genome (version 1.0) (7) using STAR (version 2.5.1b) ∼48 h in this study) would lead to additional interesting findings. (95) under default alignment parameters. Read counts for each gene were Third, our analysis was done with mRNAs extracted from whole then generated using the Aiptasia gene models (version 1.0) using HTSeq animals, so that we might have missed changes in expression that (version 0.6.1) under default parameters (96). To generate library- were strong but occurred only in specific cell types. Fortunately, normalized expression counts for each gene, raw read counts were nor- methods for doing such cell-type-specific analyses have emerged malized to the total numbers of reads from the corresponding libraries using and are improving rapidly (89, 90). Nonetheless, despite its limi- the function counts with the parameter normalize = TRUE in DESeq2 (97). To tations, this study has suggested a variety of specific hypotheses identify differentially expressed genes, we used DESeq2 with the default parameters to generate fold-change (log2) expression ratios and adjusted P that should be susceptible to rigorous experimental tests using values (97). These expression ratios were used for all analyses except the methods that are already available or rapidly emerging, as ranking of genes for the motif analysis (see below) and for the analysis of indicated above. symbiosis-associated genes (SI Appendix, Table S5). In these cases, shrunken

log2 fold-change values (which are more accurate for gene ranking) were Materials and Methods used after generation using the function lfcshrink in DEseq2 (97). For hier- Aiptasia Strains and Husbandry. All animals were from the clonal population archical clustering, we used the function rlog in DESeq2 to generate nor- CC7, which naturally contains algal symbionts of the Symbiodiniaceae clade A malized, log2-transformed read counts and perform k-means clustering species Symbiodinium linucheae (61, 91). CC7 animals were rendered apo- using the pheatmap function and specifying the number of clusters with the symbiotic as described previously (92, 93), generating strain CC7-Apo. CC7- parameter kmeans_k. Smoothened density histograms were generated in R Apo animals were then exposed to algae of the clonal, axenic strain SSB01 using the density function with default parameters. The statistical signifi- [Symbiodiniaceae clade B species Breviolum minutum (7, 93)] and grown cance of differences between these histogram distributions was assessed under standard conditions (see below) until the algal population was in steady using a two-sample Kolmogorov–Smirnov test (function ks.test in R). state. The resulting strain CC7-SSB01 was checked periodically by sequencing To conduct the GO-term analyses, we used the R function topGO using PCR-amplified fragments of cps23S (chloroplast rDNA), 18S (nuclear rDNA), default parameters except for specifying the program to report the top 20 and/or ITS2 (nuclear rDNA), as described previously (93), to ensure that the terms and the gene-ontology level as biological process (parameters:

animals had not been repopulated by S. linuchae or another algal type. topNodes = 20 and ontology = BP). We used the GO terms for each gene GENETICS Except where noted, animal stocks were maintained at 27 °C in artificial from the Aiptasia genome version 1.0 as our database (7). Enrichment seawater (ASW) prepared using Coral Pro Salt (Red Sea) at 33.5 ppt in analysis was done for each query GO-term list against this database using a ’ = deionized water (dH2O) under a 12 h:12 h light:dark cycle at an irradiance of Fisher s exact test (parameter: statistic Fisher). All analyses in R were per- − − ∼25 μmol photons m 2 s 1 (Phillips Alto II 25 W white fluorescent bulbs). formed using RStudio version 1.1.414 with the R version 3.5.1. Anemones were fed every 2 to 3 d with freshly hatched Artemia nauplii, and the water was changed on the days after feeding. Analysis of Putative Transcription-Factor Binding Motifs. We used the function lfcShrink in DESeq2 to generate log2 fold-change ratios for 3-h read counts Design of Heat-Stress Experiments and Quantification of Bleaching. To inves- vs. 0-h read counts for all genes in symbiotic anemones, and we used these tigate the transcriptional response of Aiptasia to thermal stress, CC7-SSB01 ratios to rank the genes (97). For each of the top 100 most-up-regulated and CC7-Apo anemones were first acclimated at 27 °C for ∼2mowitha genes, we used a custom Python script (Python version 2.7.13) to extract the normal feeding regimen. For the experiment, polycarbonate tanks con- putative promoter sequence, defined as the 500 bp immediately upstream taining 1 L of ASW were used. Each of three tanks contained ∼35 CC7-SSB01 of the predicted transcription start site in the gene model. Four of the 100 anemones, while each of three other tanks contained ∼25 CC7-Apo anem- genes did not have a full 500 bp upstream of their transcription start sites in ones. Time = 0 samples were taken at 3 h into the light phase, after which the genome assembly and so were not included in the further analysis. We the tanks were shifted (within ∼20 min) to an incubator at 34 °C. The ASW then used the 96 500-bp sequences to identify enriched motifs (possible gradually warmed to 34 °C over several hours (Fig. 1A), and anemones were binding sites for transcription factors) using the program MEME with the not fed after the temperature shift. Anemones of each strain were sampled following parameters: max width = 25, motif site distribution = “any num- at 6.5 h into the light phase (thus, slightly more than 3 h after the tanks were ber of sites per sequence”, and maximum number of motifs = 2. The shifted to the 34 °C incubator: time = 3-h samples), 9 h later (time = 12-h resulting enriched motifs were then searched against a database of known samples), and 3.5 h into the light phase on each of several subsequent days transcription factor binding sites with the TOMTOM program using default (time = 24-h, 48-h, and 96-h samples). At each time of sampling, three parameters. Next, we used the program FIMO to quantify the numbers of anemones were removed from each tank, pooled (thus, six samples con- these motifs within the putative promoters of all 22,872 genes for which the taining three anemones apiece), and preserved in RNAlater (Ambion) at −20 gene models contain a full 500 bp immediately upstream of the putative °C until RNA extraction for RNAseq analysis. In addition, at times 0, 24, 48, transcription start site. To increase the specificity of the motif search, the and 96 h, eight individual CC7-SSB01 animals were removed at random from sequence of the enriched HSF1 motif (Fig. 2 C, Top) was trimmed to the three tanks (typically three from each of two tanks and two from the “GAANNTTCTAGAA” to match the size of the canonical HSF1 binding site third), transferred into separate wells of a 96-well plate containing 500 μLof (Fig. 2 C, Bottom). The enriched Aiptasia NFKB motif (Fig. 2 B, Top) was used

0.01% SDS detergent (Sigma-Aldrich) in dH2O, and frozen at −20 °C for without alteration. The MEME, TOMTOM, and FIMO programs are parts of subsequent quantification of algal numbers by flow cytometry using a the MEME suite web server (98). Guava cytometer (Millipore) as described previously (94). Each algal count was normalized to the total protein of the same anemone homogenate Data Availability. All data for this paper are provided in the main text or SI using the Thermo Scientific Pierce BCA assay (94). Appendix. Raw sequences and metadata have been deposited in the NCBI The short-term bleaching experiment was performed similarly except that BioProject database (accession no. PRJNA662400). a single tank was used; eight anemones per time point were analyzed in- dividually as just described. ACKNOWLEDGMENTS. We thank Amanda Tinoco for assistance with bleaching experiments and members of our own and the Arthur Grossman RNA Isolation and Sequencing. Total RNA was extracted from each pool of and Stephen Palumbi laboratories for many helpful discussions. Funding for three anemones using the RNAqueous-4PCR Kit (Ambion AM1914) following this study was provided by grants from the Gordon and Betty Moore Foundation (Grant 2629.01), the Simons Foundation (LIFE#336932), and the the manufacturer’s instructions. The RNA-integrity number of each sample National Science Foundation (IOS EDGE Award 1645164). This work used the was determined using an Agilent 2100 Bioanalyzer; all samples had val- Genome Sequencing Service Center of the Stanford Center for Genomics ues ≥8 and were therefore used for sequencing. For each sample, ∼1 μgof and Personalized Medicine, supported by grant awards NIH S10OD025212 total RNA was processed (including the poly-A+ selection step) using the and NIH/National Institute of Diabetes and Digestive and Kidney Diseases TruSeq RNA Sample Prep Kit (Illumina FC-122-1001) and following the P30DK116074.

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