Introduction

The solar, tidal, and lunar cycles have significant effects on the behavior, metabolism and gene transcription of organisms in marine ecosystems1. These diel rhythms lead to the development of circadian clocks, which help regulate various biological processes and improve the fitness and survival of different kingdoms of life, including cyanobacteria2, fungi3, plant4 and animal5,6.

Coral reefs, intricate ecosystems providing habitants for abundant species, are primarily constructed by reef-building corals7. The success of coral reefs in nutrient-poor seawater is attributed to the mutually beneficial symbiotic relationship between coral and photosynthetic Symbiodiniaceae8. This partnership offers an avenue to explore the influence of diel rhythm on the coordination of the symbiosis between coral and its hosted Symbiodiniaceae9. Some studies have observed rhythmic and coordinated behavior between coral and Symbiodiniaceae10,11,12,13. For example, one research on sea anemone Aiptasia showed that symbiotic Symbiodiniaceae can influence the transcriptional rhythm of host14. Similarly, study on the mesophotic coral Euphyllia paradivisa has shown that this species has an internal clock, which is influenced by its symbiotic Symbiodiniaceae15. Rhythmic processes were also observed in symbiotic Symbiodiniaceae, proposed the presence of endogenous clocks in both coral and its hosted Symbiodiniaceae10,11,12,13.

It is well known that elevated temperatures significantly impact coral and Symbiodiniaceae growth and survival16,17. Coral bleaching, a major threat to reef ecosystems, is primarily driven by elevated temperature18. It is well established that elevated temperature has a great effect on the diel rhythms of gene transcription in plants (such as soybean)19, animals (such as Drosophila)20, and bacteria (such as Klebsiella aerogenes)21,22. However, the influence of elevated temperatures on the rhythms of gene transcription that possibley regulate symbiosis during coral bleaching is poorly understood.

To address this gap, our study examines changes in physiology, biochemistry, cellular processes, and transcriptome over a 48–72 h period under three conditions: constant darkness (DD), light/dark cycle (LD), and LD with elevated temperature (HLD). Using Acropora tenuis-Symbiodiniaceae (Cladocopium sp. genus) as a model holobiont, we aims to investigate how elevated temperatures affect the diel rhythms of coral holobiont. The availability of sequenced genomes for both A. tenuis and Cladocopium makes this holobiont an ideal model for studying symbiosis23,24. Through rhythmic analysis, we identified the 24-h periods of gene transcription patterns in both A. tenuis and its symbiotic Symbiodiniaceae, revealing distinct diel gene transcription patterns under LD and HLD conditions.

Results

Physiology and metabolism rhythms of coral-Symbiodiniaceae holobiont

Colonies of A. tenuis were subjected to three conditions: constant darkness at 27 °C (DD), light/dark cycle at 27 °C (LD), and LD at 32 °C (HLD) (Fig. 1a). Visual observations indicated thermal bleaching in A. tenuis colonies (Fig. 1a), with an 86% reduction in algal symbiont density after 72 h of HLD cultivation (Fig. 1b). The thermal bleaching of A. tenuis was further linked to significant diel rhythmic oscillations (24-h) of photosynthetic efficiency (Fv/Fm, Fig. 1c), reactive oxygen species (ROS, Fig. 1d) leakage, and lipid peroxidation (Fig. 1e) in the A. tenuis holobiont (Cosinor algorithm, p-values < 0.05, Table 1, Supplementary Data 1). ROS leakage and host lipid peroxidation peaked at dusk (18:00), while Fv/Fm peaked at midday (12:00). Elevated temperature amplified oscillation amplitude of ROS leakage, lipid peroxidation, and Fv/Fm. Compared to LD, HLD resulted in a decrease in Fv/Fm (7–30%) and increases in ROS (>43%) and lipid peroxidation (48–63%).

Fig. 1: Physiology and metabolism rhythms of A. tenuis-Cladocopium sp. holoboint.
figure 1

The experimental design involved cultivating reef-building A. tenuis samples under three light conditions: DD, LD and HLD (a). The horizontal boxes represent the light and dark phases during sampling, with blue boxes indicating light and gray boxes indicating dark. The arrows (pointing upwards) mark the sampling time points. Sampling commenced at midnight (time 0 = 24:00) on November 13, 2021, and continued at 6-h intervals for 48–72 h. The oscillation plots depict variations in Symbiodiniaceae cell densities (b), photochemical efficiency of Photosystem II (Fv/Fm) (c), relative release of reactive oxygen species (ROS) by symbiotic Symbiodiniaceae (d), host lipid peroxidation levels (e). Error bars represent mean values ± standard deviation (SD), with n = 9.

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Table 1 Rhythmicity test of algal density, photosynthetic efficiency, reactive oxygen species (ROS) leakage, and lipid peroxidation in the A. tenuis holobiont (Cosinor algorithm) under DD, LD and HLD conditions
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Transcriptome rhythmic analysis of coral-Symbiodiniaceae holobiont

To reveal the molecular causes behind the observed phenotype, physiology and metabolism, we further examined the rhythmic genes and their oscillation patterns in the A. tenuis-Symbiodiniaceae holobiont at 6-h intervals under DD, LD and HLD conditions.

Firstly, a rigorous analysis was conducted to identify rhythmic genes (JTK + Cosinor, q-values < 0.05), resulting in 0, 34 and 0 genes identified in the coral host under DD, LD, and HLD conditions, respectively (Supplementary Data 2). For the symbiont, we found 0, 1 and 0 rhythmic genes under the same conditions (Supplementary Data 3). Considering the difficulty to identify rhythmic genes with q-values < 0.05, we then opted for p-values < 0.02 to identify rhythmic genes, acknowledging the risk for false-positive results. Under DD, LD, and HLD conditions, 100, 522, and 72 rhythmic genes were identified in the host, respectively (JTK + Cosinor, p-values < 0.02, Fig. 2a, Supplementary Data 2). In contrast, 75, 64, and 93 rhythmic genes were detected in the symbiont (JTK + Cosinor, p-values < 0.02, Fig. 2c, Supplementary Data 3). Among the identified rhythmic genes, 25 exhibited oscillatory transcription in the host under both DD and LD conditions. In the symbiont, we also detected several rhythmic genes, which exhibiting similar oscillations under both DD and LD conditions. These genes were designated as candidate clock or clock-controlled genes (Supplementary Data 4).

Fig. 2: The overall rhythmic transcription of genes in the A. tenuis-Cladocopium sp. holoboint.
figure 2

Based on Cosinor and JTK algorithms (p-values < 0.02), numerous rhythmic genes were identified in A. tenuis (a1) and its hosted symbiont (b1) under DD, LD and HLD conditions. Venn diagrams illustrate the overlap of rhythmic genes in A. tenuis (a2) and its symbiont (b2). Heatmaps depict the median gene expression values of rhythmic genes identified from DD, LD and HLD conditions (JTK + Cosinor, p-values < 0.02, after deduplication) over time points (n = 3 biological replicates), where each row represents a rhythmic gene in the host (c) or symbiont (d).

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Analysis of the identified rhythmic genes from DD, LD and HLD conditions (JTK + Cosinor, p-values < 0.02, after deduplication) and their heatmaps (Fig. 2b, d) revealed that the coral host displayed more robust diel gene transcription than its symbiont under LD. However, HLD resulted in a loss of rhythmicity in the majority of host genes.

Furthermore, we observed that the overall oscillation patterns of rhythmic genes identified under LD (JTK + Cosinor, p-values < 0.02) were disrupted in the coral (Fig. 3a) and shifted in the symbiont under HLD (Fig. 3b). These disordered oscillation patterns were further supported by Gaussian mixture model (GMM) (Fig. 3c, d) clustering of rhythmic genes identified under LD and HLD (JTK + Cosinor, p-values < 0.02), respectively. It was observed that most rhythmic genes (>90%) had high transcriptional abundance (peak) at dawn (6:00) or midday (12:00) in both the coral host and symbiont under LD. Conversely, under HLD, the patterns were altered, with 84% of rhythmic genes in the symbiont peaking at dusk (18:00).

Fig. 3: Overall oscillation of rhythmic genes in the A. tenuis-Cladocopium sp. holobiont.
figure 3

The overall oscillation of rhythmic genes identified under LD (JTK + Cosinor, p-values < 0.02) in the host (a1-LD, a2-HLD) and symbiont (b1-LD, b2-HLD) under different conditions. Lines are based on sinusoidal function. the lines are fitted using a sinusoidal function. Pie charts present the distribution (percentages) of rhythmic genes identified under LD and HLD conditions (JTK + Cosinor, p-values < 0.02) within different clusters (determined by Gaussian mixture model) for both the host (c) or symbiont (d).

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The oscillation of candidate clock or clock-controlled genes in coral holobiont under elevated temperature

In the coral host, rhythmic genes essential to the circadian clock (possibly clock genes-C) were identified (Fig. 4, Supplementary Data 4), including CIPC (CLOCK-interacting pacemaker-like), Cry1 (cryptochrome 1), phrB (Deoxyribodipyrimidine photolyase). The CLOCK(Circadian locomoter output cycles kaput protein) and BMAL1(Aryl hydrocarbon receptor nuclear translocator) genes were actively transcribed but did not exhibit significant rhythmicity. Cry1 peaked at midday, while CIPC and phrB peaked at dawn.

Fig. 4: Diel oscillation of candidate clock genes in the A. tenuis-Cladocopium sp. holoboint.
figure 4

Oscillation plots for candidate circadian clock genes cycling with a 24-h period in host under DD, LD and HLD conditions. The x-axis of the plots represents the experimental time course (with time 0 set at 24:00), while the y-axis displays normalized expression levels. Note that the scales differ across plots. Data points are presented as mean values (n = 3 biological replicates).

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Additional rhythmic genes (possibly clock-controlled genes-CCG) (Fig. 5, Supplementary Fig. S1, Supplementary Data 4) included ANP1 (Atrial natriuretic peptide receptor 1), Ang1 (Angiopoietin-1 receptor), DHRS12 (Dehydrogenases with different specificities) TEF (Thyrotroph embryonic factor-like), HEY (Hairy and enhancer of split), UTP18 (U3 small nucleolar RNA-associated protein 18), CBM21 (Carbohydrate/starch-binding module (family 21), PNPO (Pyridoxamine-phosphate oxidase), SOUL (SOUL heme-binding protein), LX3 (Legumain-like isoform X3) and and others, exhibiting significant rhythmic oscillations under both DD and LD conditions. These genes involved in diverse processes such as signaling, transcription regulation, translation, cofactor and lipid metabolism, and transportation (Fig. 5).

Fig. 5: The gene function of candidate clock or clock-controlled genes in the A. tenuis-Cladocopium sp. holoboint.
figure 5

Heatmap visualizes the median gene expression values across time points (n = 3 biological replicates). Each row represents a rhythmic gene in the host or symbiont under DD, LD and HLD conditions. A list of genes and their potential functions is provided to the right of the heatmap. Up and down arrows represent increased and decreased gene transcription levels under elevated temperature. The capital letters of C and CCG represent candidate clock gene, and clock-controlled gene, respectively.

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Notably, the CBM21 and SOUL genes showed the highest transcription abundance, implicated in polysaccharide utilization and cellular oxidation-reduction equilibrium, respectively. Intriguingly, these genes exhibited comparable rhythmic patterns in LD and HLD conditions (Fig. 5, Supplementary Fig. S1). However, elevated temperature reduced the transcription abundance of most core rhythmic genes at their peaks.

In the symbiont, several rhythmic genes (possibly clock-controlled genes-CCG) were observed, including APA1 (Aspartic proteinase A1), BMY1 (Beta-amylase), P4H (Prolyl 4-hydroxylase), ARSB (N-acetylgalactosamine 4-sulfatase), SLC (Solute carrier family) (Fig. 5, Supplementary Fig. S1, Supplementary Data 4). These genes may be involved in carbohydrate metabolism and transport (Fig. 5). Notably, the BMY1 gene, involved in polysaccharide metabolism, showed significant rhythmic oscillations under all conditions. However, its transcription abundance at peaks was diminished by elevated temperature.

Discussion

Oscillation of candidate clock genes in coral-Symbiodiniaceae holobiont under elevated temperature

In animal models, a CLOCK-BMAL1 heterodimer activates the transcription of its negative regulators, such as Cry, Per, Tim genes25,26. This study identifies active transcription of clock genes (CLOCK, BMAL1, Cry) in A. tenuis as well. Notably, this study reports the first identification of the CIPC gene in A. tenuis. CIPC encodes the CLOCK-interacting pacemaker-like protein, a newly discovered protein that functions as a circadian feedback loop negative regulator in mammals27. In A. tenuis, CIPC exhibits the highest transcriptional abundance among clock genes and displays a robust rhythmic oscillation, peaking at dawn under both DD and LD conditions, supporting its role as a core clock gene in coral.

The Cry and phrB genes encode evolutionarily related flavoproteins, cryptochrome and deoxyribodipyrimidine photolyase, respectively. Cryptochromes regulate growth, development, and the circadian clock in plants and animals28,29, while photolyases repair UV-induced DNA damage in a light-dependent manner30. In the A. tenuis, both Cry (Cry1) and phrB genes show rhythmic oscillations under DD and LD conditions. Cry (Cry1) peaks at midday, while phrB peaks at dawn. While diel rhythmic oscillations of Cry genes have been reported previously in corals9,13,14, this study is the first to report the diel rhythmic oscillation of the phrB gene. Interestingly, CIPC, Cry1, and phrB genes exhibit similar rhythmic oscillation patterns in both LD and HLD conditions. However, their peak transcription levels were changed by elevated temperature, suggesting that coral host circadian clock gene transcription is buffered against temperature changes. Our results indicated that the negative feedback in the coral circadian clock is probably divided into distinct pathways, and that the addition of the new clock genes, such as CIPC and phrB, has contributed to the complexity of coral clocks.

In photosynthetic organisms, including algae, the circadian clock comprises a large number of transcription factors organized in multiple feedback loops, including clock genes of MYB (MYB-related transcription factors), LHY (LATE ELONGATED HYPOCOTYL), CCA1 (CIRCADIAN CLOCK ASSOCIATED 1), TOC1 (TIMING OF CAB2 EXPRESSION1) families4,31,32. However, we does not detect rhythmic genes in coral-hosted Symbiodiniaceae that have been established as clock genes in photosynthetic organisms. This could be due to weak diel rhythmicity in symbiotic Symbiodiniaceae or host-mediated effects on the diel rhythmicity of symbiotic Symbiodiniaceae, requiring further investigation.

Diel rhythmic oscillation of genes related to coral bleaching

Coral bleaching is primarily driven by elevated temperature, but the behind mechanisms remain elusive due to the complexity of the coral-Symbiodiniaceae holobiont33. In this study, significant rhythmic oscillations in photosynthetic efficiency (Fv/Fm), reactive oxygen species (ROS) leakage, and lipid peroxidation were observed in the A. tenuis, particularly during coral bleaching under elevated temperature. Previous studies has highlighted the importance of diel rhythmicity in coral bleaching. Baird et al. demonstrated that daytime bleaching is more pronounced than nighttime bleaching, supporting the role of diel cycles in coral bleaching responses34. Wooldridge and Levy observed that antioxidant defenses in corals exhibit diel rhythms, which may influence their resilience to oxidative damage during bleaching35,36. Our findings corroborate with their observations and provide further evidence for the role of diel rhythmicity in photosynthetic inhibition, oxidative damage of coral holobiont. We observed that the Fv/Fm exhibited its lowest oscillation value at midday, while ROS leakage from isolated algal symbionts and lipid peroxidation levels in the host peaked at dusk under elevated temperature. This suggests that increased oxidative damage to the coral host resulting from ROS leakage lags behind photosynthetic inhibition in symbiont.

Gene transcription analysis under LD cycles revealed diel rhythms in specific genes of both coral and Symbiodiniaceae. However, we noted that elevated temperature disrupted this diel rhythmicity of gene transcription observed under LD in both coral and Symbiodiniaceae. Notably, the transcription abundance of several clock-controlled genes (e.g., ANP1, Ang1, DHRS12, TEF, HEY, UTP18, CBM21, PNPO, SOUL, LX3 BMY1, P4H, ARSB and SLC) involved in signaling, cellular processes, transcription regulation, translation, cofactor metabolism, lipid metabolism, carbohydrate metabolism, transporter processes, and stress responses was altered by elevated temperature. The results align with the findings in model organisms9,19,37, which have consistently demonstrated that elevated temperature disrupts the diel rhythms of gene transcription in coral and Symbiodiniaceae. Moreover, the alteration of clock-controlled genes involved in various cellular processes is a common observation across these studies, suggesting a critical role for temperature in regulating circadian rhythmicity and overall physiological responses in these organisms20,38. Among the identified core rhythmic genes in A. tenuis, CBM21 and SOUL participating in polysaccharide utilization and cellular redox equilibrium39,40,41, exhibited the highest transcriptional abundance. Their reduced transcriptional abundance under elevated temperature implies that diel rhythmicity in polysaccharide utilization and cellular redox equilibrium may be negatively affected in the coral host. In the symbiont, the BMY1 gene (a possible circadian clock controlled gene), associated with polysaccharide metabolism42, also showed reduced transcriptional abundance under elevated temperature, indicating that diel rhythmic polysaccharide utilization in symbiont may be impaired as well.

In all, this study provide further evidence for the role of diel rhythmicity in coral bleaching (Fig. 6). The present findings suggest that the coral host and its symbiont maintain a synchronized rhythmicity in gene transcription under normal conditions, which supports symbiosis. However, elevated temperatures disrupt this rhythmicity, potentially contributing to thermal bleaching of coral. Importantly, the identification of rhythmic genes and their oscillation patterns under elevated temperature could inform future research on the development of strategies to mitigate the impacts of elevated temperatures on coral reefs.

Fig. 6: Conceptual summary of rhythmic gene transcription in A. tenuis and its hosted Cladocopium sp. under elevated temperature, causing coral bleaching.
figure 6

It illustrate distinct response of A. tenuis and its hosted Cladocopium sp. to elevated temperature in rhythmic patterns. The response of candidate circadian clock genes and several circadian clock-controlled genes (involved polysaccharide utilization and cellular redox equilibrium, such as CLOCK, BMAL1, CIPC, Cry1, phrB CMB21, BYM1 and SOUL was illustrated.

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Methods

Coral collection and experimental design

Twenty mature colonies of the reef-building coral A. tenuis (measuring 10–20 cm) were collected from a depth of 3- to 4-m on a tropical coral reef in Sanya (109° 29’ E, 18° 12’ N), Hainan Island, China. The colonies were fragmented into branches (measuring height ~5 cm, total of ~212 branches). The branches were then placed in a 500-L outdoor tank under natural condition for an acclimatization period of 20 days. The outdoor tank (open system) supplied with seawater collected from the Sanya reef at a depth of 3- to 4-m. After the acclimation period under natural condition, the branches were randomly placed into 9 indoor tanks (150-L) and cultivated under a simulated LD cycle (i.e., sunrise at 06:00 and sunset at 18:00 on November 6, 2021; light intensity of 20 ± 5 to 200 ± 10 μmol photons m−2 s−1 during daytime and seawater temperature of 27 ± 0.5 °C during 7 days for entrainment). Subsequently, branches were divided into three experimental subgroups: corals cultivated under LD (27 ± 0.5 °C and 12:12 light:dark cycle), DD (27 ± 0.5 °C and 0:24 light:dark cycle) and HLD conditions (32 ± 0.5 °C and 12:12 light: dark cycle). Temperature conditions reflecting the annual mean temperature (27 °C; control) or elevated summer temperature (32 °C; heat stress) in Sanya reef.

Sampling began at night (i.e., time 0 = 24:00) of 13 November 2021 and was performed at 6-h intervals over 48–72 h. The indoor tanks underwent a daily seawater renewal rate of 25% using pre-warmed in-situ seawater.

All indoor tanks were filled with seawater collected from Sanya, with a pH of 8.15 ± 0.003, a salinity of 33.50 ± 0.015, a NH4+ concentration of 10 ± 2 μg/L, a NO3− concentration of 32 ± 2 μg/L, a PO43− concentration of 9 ± 1 μg/L. The light and temperature of the tanks were generated and maintained according to the methods presented by Gong et al.43. The methods of monitor temperature, salinity, pH and inorganic nutrients (ammonia, nitrate, nitrite and phosphate) in the cultivation systems were recorded according to Gong et al.43. The occurrence of visible coral bleaching was recorded through snapshots taken by a GoPro (HERO9 Black, USA). Spectral characteristics of coral growth sites and study site (depth of 3–4 m) were observed using Diving-PAM-II (Walz, Germany).

Symbiodiniaceae cell density and maximal PSII quantum yield (F V /F M)

At each time point, nine coral branches were used to monitor Symbiodiniaceae cell density and FV/FM. To measure Symbiodiniaceae cell density, coral tissue was removed using a Waterpik containing filtered seawater (0.45 μm). The initial volume of the resulting slurry was measured with a graduated cylinder. The slurry was homogenized by votex and subsampled into four 3-ml aliquots. Subsamples were centrifuged (6500 r/min) for 5 min. After discarding the supernatant, the pellet containing algal cells was preserved in 1 ml of 5% formaldehyde at 4 °C for further analysis. The cell density of Symbiodiniaceae was counted using a hemocytometer counts under a microscope (CX21, Olympus, Japan). Density of Symbiodiniaceae cells was normalized to coral surface area by correlation between weight and surface area of aluminum foil imprints44.

The highest photosystem II (PS II) photochemical quantum yield in coral holobiont, known as FV/FM value, was assessed using pulse-amplitude modulation fluorometry (Diving-PAM-II, Walz, Germany) according to our previous study43.

Measurement of the metabolitic features of coral holobiont

The production of ROS by Symbiodiniaceae cells in A. tenuis was qualified according to a recent study45. For this, we first obtained freshly isolated Symbiodiniaceae cells in A. tenuis according to the above method. Then, the pellet containing freshly isolated Symbiodiniaceae cells was placed in 1 mL Eppendorf tubes and incubated under different conditions (similar with each time point for sampling) for 30 min. To measure ROS production, 5 μM CellROX orange (Life Technologies) was added to cultivated cells. Subsequently, cells were removed by centrifugation and the remaining supernatant (two-hundred-microliter aliquots) was immediately transferred to a 96-well plate and incubated in the dark at 37 °C for another 30 min. The fluorescence intensity of CellROX was quantified using a SpectraMax to calculate the relative release of ROS.

To assess the level of oxidation stress in the tissues of the host, we conducted an analysis of lipid peroxidation by quantifying the total malondialdehyde content45. The malondialdehyde content was then normalized to coral surface area.

RNA extraction, sequencing and metatranscriptomic analysis

At each time point, total RNA was extract from three coral branches following the protocol provided by the Qiagen RNeasy Kit (Qiagen, Hilden, Germany). The quality and quantity of extracted RNA were assessed using Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) and NanoDrop ND-1000 spectrometer (Wilmington, DE, USA). RNA samples with OD260/280 values ranging from 1.8 to 2.1 were selected for further cDNA library construction. The raw RNA sequences were analyzed by using the SqueezeMeta software, an automatic pipeline for metagenomics/metatranscriptomics data sets46. The LCA algorithm in SqueezeMeta assigned taxonomic information to the assembled genes by identifying the last common ancestor hit through a Diamond search against the GenBank nr database (included the annotated genomes of Symbiodiniaceae and Acropora)46. The software Bowtie247 was used to align original reads to contigs resulting from assembly to estimate the abundance of assembled genes in different samples. RSEM software48 computed average coverage and normalized TMP values.

Rhythmicity analysis of physiology and metabolism and gene transcription

Rhythmicity in physiology and metabolism and gene transcription was identified using DiscoRhythm (an online tool to explore cyclical temporal data)49. For diel gene transcription analyses, genes with a p value < 0.02 were considered confident cyclers. To perform clustering of the rhythmic transcripts based on GMMs, we used the Mclust function on the R mclust package (V.5.2). The analyses were run separately for the 24-h oscillating genes from coral host or its hosted Symbiodiniaceae. Heatmaps were generated using the heatmap.2 function on the R package gplots (V.2.17.0). Venn diagrams were generated using the web tool Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.