The algal culture used in this study was isolated from tropical coral reefs of the South China Sea (E109.476, N18.211°) according to our previous methods (Gong et al., 2018, 2019). The unialgal strain of symbiotic Symbiodiniaceae was obtained according to Xiang’s method (Xiang et al., 2013). The 18 S rRNA sequence of the culture was amplified using the primer pair ss5 and ss3z (Rowan and Powers, 1991) and was used as the query for a BLAST search against the National Center for Biotechnology Information (NCBI) database¹ with the default settings. The results showed that the 18 S rRNA gene sequence (Supplementary File 1) of the culture was highly similar to E. voratum SMFL (HE653238.1), with 98.6% identity and 100% query coverage. Therefore, the culture was named E. voratum SCS01 (Supplementary Figure 1).

Stock cultures of E. voratum SCS01 were maintained in 250-mL flasks with 100 mL of f/2 medium (Guillard and Ryther, 1962) at an in situ temperature of 26°C under a light intensity of 90 μmol photon m^–2 s^–1 and a light:dark cycle of 12 h:12 h.

The growth of E. voratum SCS01 at different temperatures was determined by inoculating the exponential-phase cells in 500-mL flasks with 250 mL of f/2 medium, followed by incubation at 20, 23, 26, 29, or 32°C for 10 days under the same light intensity and light:dark cycle indicated above.

To monitor the photobleaching and recovery of E. voratum SCS01, its cells were initially cultured at the optimal growth temperature (26°C) for 4 days. Exponential-phase cells were gently centrifuged (5,000 rpm, 5 min) and transferred to 3 500-mL flasks with 250 mL of f/2 medium. The flasks were cultured at 32°C. After 6 days of incubation, the cells cultured at 32°C were harvested and transferred to fresh medium as described above and cultured again at 26°C.

At 18:00 every day, 1-mL aliquots of the cultures in each flask under different temperature treatments were collected and fixed with glutaraldehyde at a final concentration of 2.5%, and cell numbers were counted under a microscope (Leica DMRXA, Germany). The exponential growth phase was determined, and the maximal specific growth rate was calculated following Mou’s method (Mou et al., 2017). The morphology of E. voratum SCS01 cells was recorded using a fluorescence microscope (Leica DMRXA, Germany) under bright-field conditions and/or fluorescent light. The subcellular structures of E. voratum SCS01 cells were imaged via transmission electron microscopy according to our previously reported method (Gong et al., 2018).

The maximum photochemical efficiency of photosystem II (PS II) of E. voratum SCS01 was measured using a pulse-amplitude modulated (PAM) fluorometer (Water-PAM WALZ, Germany) after dark adaptation for 20 min in a custom-made acrylic black box at room temperature. Briefly, we measured the maximal chlorophyll fluorescence (F_m) of the dark-adapted cells under a saturating blue light pulse (3,000 μmol photons m^–2 s^–1, 1 s), and we measured their minimal fluorescence (F_O) in the presence of weak, modulated measurement light. We then calculated the maximum PSII photochemical quantum yield (Fv/Fm) (van Kooten and Snel, 1990) as follows:

The photosynthetic and respiration rates of E. voratum SCS01 cells were measured using the oxygen electrode method. Culture was collected from each flask, centrifuged (5,000 × g, 5 min) (5804 R Eppendorf, Germany), resuspended to 5 mL in remaining media, and dark adapted for 5 min in a chamber with temperature control to the same as growth temperature. The incident irradiance in the chamber was supplied by LED flexible rope lights and measured using a microspherical quantum sensor (QSL2100, Biospherical Instruments Inc., United States). The decline in oxygen concentration under dark conditions and the increase in oxygen concentration under illumination (90 μmol photon m^–2 s^–1) were tracked using a liquid oxygen electrode (Chlorolab 2, Hansatech Instrument Ltd., United Kingdom). The photosynthetic and respiration rates of E. voratum SCS01 were calculated according to the oxygen increase and decline rates (μmol O₂ S^–1 per 10⁶ cells), respectively.

E. voratum SCS01 cells in 100-mL aliquots of the cultures under different temperature treatments were collected via centrifugation (5,000 rpm, 5 min) and dried in a vacuum freeze dryer (SCIENTZ-18N, China). Freeze-dried cells were used to assess the biochemical composition (i.e., pigment, total carbohydrates and total lipids) under each treatment. To measure pigment contents, freeze-dried cells were extracted in 7 mL of 100% acetone for 24 h, and chlorophyll a and carotenoid contents were determined following Dere’s method (Dere et al., 1998). Total carbohydrates were extracted from freeze-dried E. voratum SCS01 cells using 1 N H₂SO₄ at 80°C. The total carbohydrate content was determined via the phenol-sulfuric acid method proposed by DuBois et al. (1956) using glucose as a standard. The extraction of total lipids and the determination of their contents were performed using a modified version of the Khozin-Goldberg’s method (Khozingoldberg et al., 2005). Pigment, total carbohydrate and total lipid contents were calculated as nanograms per cell.

E. voratum SCS01 cells for the photobleaching and recovery experiments were collected via centrifugation (5,000 rpm, 5 min) on day 4 of incubation at 26°C, on day 2 and day 6 after transfer to 32°C, and on day 4 after being returned to 26°C. The cell pellets were immediately frozen in liquid nitrogen then stored at −80°C for further RNA extraction. Total RNA was extracted from E. voratum SC01 cells using the TRIzol method (Invitrogen, Australia) (Rosic and Hoegh-Guldberg, 2010). RNA quantity and integrity were analyzed using a NanoDrop ND-1000 spectrometer (Wilmington, DE, United States) and an Agilent 2100 Bioanalyzer (Santa Clara, CA, United States). RNA samples with high purity (OD260/280 between 1.8 and 2.2) and high integrity (RNA integrity number (RIN) > 7.5) were used for further cDNA library construction. cDNA library construction and the sequencing and quality control of raw sequences were performed according to our previous study (Gong et al., 2020). The raw sequence data sets (a total of 12 sequencing libraries, the statistical information of RNA-seq data sets were supplied in Supplementary Table 1) produced in this study were deposited in the Sequence Read Archive (PRJNA699996) of the NCBI (see text footnote 1).

The obtained clean reads were assembled de novo with Trinity v2.1.1 software using the default parameters (Haas et al., 2013), and the statistical information of de novo assembled unigenes is supplied in Supplementary Table 2 and Supplementary File 2. The expression levels of the unigenes (in fragments per kilobase of exon model per million mapped fragments, FPKM) were calculated using RSEM provided within the Trinity package (Li and Dewey, 2011). All unigenes were annotated using the Basic Local Alignment Search Tool (BLAST) algorithm with a cutoff E-value of 10^–5 based on the NCBI non-redundant protein (Nr) database. The sequences of the identified differentially expressed genes (DEGs) were also subjected to BLAST searches against entries in the Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Eukaryotic Orthologous Groups of proteins (KOG) and Gene Ontology (GO) databases (Supplementary File 2). The annotation files are supplied in Supplementary File 3. The analysis of DEGs in the samples was performed using the DEGseq2 method, with a threshold q-value of < 0.001. Genes with a log2 fold change (LogFC) ≥ 1, false discovery rate (FDR) < 0.05 and FPKM ≥ 3 (in at least one group) were considered significant DEGs.

All of the data are recorded as the means ± standard deviation values from at least 3 independent biological replicates. One-way analysis of variance (ANOVA) with Tukey’s test was used to analyze the experimental data, and the differences were considered significant at P < 0.05. All analyses and visualizations of the results were performed using the vegan and pheatmap packages in R software (R 3.1.2) and/or Origin 8.5 software.

The maximal specific growth rate (μ_max) of E. voratum SCS01 increased from 0.55 to 0.69 d^–1 when the temperature increased from 20 to 26°C and then decreased to 0.15 d^–1 with a further increase in temperature to 32°C (Figure 1A and Supplementary Figure 2). The Fv/Fm ratio of E. voratum SCS01 gradually decreased from 0.64 to 0.61 as the temperature increased from 20 to 29°C and then dramatically declined to 0.47 at 32°C (Figure 1A). Visible plastid photobleaching was observed in E. voratum SCS01 coccoid and mastigote cells at 32°C (Figure 1B and Supplementary Figure 3), which was supported by a significant decline in pigment content (chlorophyll and carotenoid contents) (Figure 1C) and Fv/Fm values (Figure 1A).

The physiological and morphological changes observed in E. voratum SCS01 during the bleaching and recovery processes are shown in Figure 2. Cell counts increased from 2.3 × 10⁵ cells mL^–1 to 2.5 × 10⁶ cells mL^–1 from days 0 to 4, when E. voratum SCS01 was incubated at 26°. After transfer to 32 °C, cell counts decreased dramatically to 4.6 × 10⁵ cells mL^–1 on day 10. After 4 days of recovery at 26°C, the cell count increased again to 1.5 × 10⁶ cells mL^–1 (Figure 2A). The Fv/Fm value exhibited a similar variation trend. Morphological measurements clearly showed pigment loss in mastigote and coccoid cells on day 6 and day 10 due to thermal stress (32°C), but the cells recovered a brownish color after being returned to 26°C for 4 days (day 14) (Figure 2B and Supplementary Figure 4). In accordance with the observed morphological changes, cellular chlorophyll and carotenoid contents decreased with thermal stress then increased after the stress was removed (Figure 2C). The net photosynthetic rate was lower, and the respiration rate was higher at 32°C than at 26°C in the initial and recovery cultures (Figure 2D).

Two distinct life forms of E. voratum SCS01 were identified during the bleaching and recovery periods, including coccoid cells (vegetative) and mastigote cells (flagellated) (Figure 3A and Supplementary Figure 5). Mastigote cells dominated the initial cultures (day 4) and constituted over 85% of the total cells. At this stage, coccoid cells accounted for approximately 10% of the total cells (Figure 3B). During the bleaching period (days 4 and 10), the proportion of coccoid cells increased to over 50%. The proportion of mastigote cells simultaneously declined to 13% (Figure 3B). When the photobleached E. voratum SCS01 cells were returned to 26°C, the proportions of coccoid and mastigote cells recovered to the levels observed in the initial period (day 4).

The transcriptomes of E. voratum SCS01 were analyzed in different stages of bleaching and recovery processes (Figure 3C and Supplementary File 4). Three genes related to cell motility mediated by the flagellum were downregulated during the bleaching period (Figure 3C). These genes encoded sperm motility kinase 2B (2.1-fold downregulation in day 10 bleaching samples compared to the initial day 4 samples, q-value < 0.001), sperm acrosomal protein FSA-ACR.1 (2.5-fold downregulation in day 10 bleaching samples compared to the initial day 4 samples, q-value < 0.001), and cation channel sperm-associated protein 1 (2.0-fold downregulation in day 10 bleaching samples compared to the initial day 4 samples, q-value < 0.001). These results were consistent with the decrease in mastigote cells during the bleaching period. Genes encoding MEI2 protein family members (ML1, ML2, ML3, ML4, and ML5, over 2.1-fold downregulation in day 10 bleaching samples compared to the initial day 4 samples, q-value < 0.001), the meiotically up-regulated gene 158 protein (2.7-fold downregulation in day 10 bleaching samples compared to the initial day 4 samples, q-value < 0.001) and meiotic recombination protein DMC1-like subunits (2.7-fold downregulation in day 10 bleaching samples compared to the day 4 initial samples, q-value < 0.001), which may be involved in meiosis, showed decreased expression with increasing temperature (Figure 3C).

The abundance of genes involved in pigment metabolism, photosynthesis, respiration, carbohydrate metabolism and antioxidant activities at different temperatures was also compared (Figure 4 and Supplementary File 5). During the bleaching period, genes involved in carotenoid synthesis (i.e., CHLP and VDE) were significantly downregulated (over twofold downregulation in day 10 bleaching samples compared to the initial day 4 samples, q-value < 0.001), and genes related to carotenoid degradation (i.e., carotenoid 9,10 cleavage dioxygenase 1 and the pheophytinase (PPH) responsible for chlorophyll and carotenoid cleavage) were upregulated (over twofold upregulation in day 10 bleaching samples compared to the initial day 4 samples, q-value < 0.001) (Figure 4A). Thermal stress universally upregulated the transcription of genes related to photosynthesis, respiration and antioxidant activities [i.e., PsbB, PsbC, PsbD, PsbO, fucoxanthin-chlorophyll as binding protein (FCPA), light harvesting chlorophyll (LHC), PsaA, PsaB, ATPF1B, and ATPF1A, involved in chloroplast photosynthesis; cyclooxygenase 1 (COX1), COX2, COX3, and ATP5O and ATP5E of ATPase, involved in mitochondrial respiration; and HSP70, superoxide dismutase (SOD) and chloramphenicol acetyltransferase (CAT), involved in antioxidant activities] (Figures 4A,B). The genes encoding cytochrome b-c1 complex subunit Rieske-2 (UQCRDS6), NADH dehydrogenase beta subcomplex subunit 2 and ATP synthase subunit epsilon (mitochondrial, ATP5E) were upregulated over 7-fold in bleaching samples on day 10 compared to the initial samples on day 4 (q-value < 0.001). The genes encoding HSP70, SOD and CAT were upregulated over 5.5-fold in bleaching samples on day 10 compared to the initial samples on day 4 (q-value < 0.001).

For carbohydrate metabolism, we observed the downregulation of genes encoding proteins related to CO₂ fixation and starch and cellulose synthesis [i.e., ribulose bisphosphate carboxylase large chain precursor (RBCL), starch synthase 1 (SS1) and callose synthase (CALS)] and the upregulation of genes encoding proteins related to lipid synthesis (i.e., MCAT, HSD17B4, and ASAH2) under thermal stress (Figure 4A). The expression of genes during the recovery period exhibited a recovery trend when E. voratum SCS01 was cultured at the optimal temperature (26°C) again, except for several genes related to photosynthesis and carbohydrate metabolism (Figure 4A).

Increasing the temperature from 20 to 32°C altered the growth of free-living E. voratum SCS01, and notable photosynthesis inhibition occurred at 32°C (Figure 1 and Supplementary Figure 2). This phenomenon of photosynthesis inhibition under elevated temperature was also observed in Symbiodinium and Breviolum, but not in Durusdinium and Fugacium (Takahashi et al., 2013; Karim et al., 2015). These results suggest that the sensitivity of PSII (Fv/Fm) to elevated temperature differs among Symbiodiniaceae phylotypes. The researchers responsible for earlier findings revealed that photoinhibition of Symbiodiniaceae is largely an impairment of PSII due to ROS production caused by high irradiance during thermal stress with the rate of photoinactivation of the core PSII proteins exceeding the rate of repair (Rehman et al., 2016; Goyen et al., 2017; Lesser, 2019). At the gene transcriptional level, we observed that antioxidant activity-related genes (i.e., genes encoding HSP70, SOD and CAT) and photosynthesis-related genes (i.e., genes encoding PsbB, PsbC, PsbD, PsbO, FCPA, and LHC in PSII; PsaA and PsaC in PSI; ATPF1B and ATPF1A of ATPase in chloroplasts) were significantly upregulated under thermal stress (Figure 4, especially at day 10), which is uncorrelated with declines in Fv/Fm. Thus, the observed photoinhibition of E. voratum SCS01 in this study is likely due to that the repair mechanism via antioxidant-related system does not match the rate of photoinactivation of the core PSII proteins and other photosynthetic related proteins. The previous study has been inferred that initial damage to the carbon fixation (dark reactions) at bleaching-relevant temperatures resulted in an electron sink limitation and, consequently, PSII photoinhibition (Jones et al., 1998). In this study, we observed that the gene encoding the Rubisco (RBCL) for carbon fixation was significantly downregulated, but the gene encoding Ribulose bisphosphate carboxylase/oxygenase activase (RCA) was significantly upregulated under thermal stress (Figure 4, especially at day 10), suggesting Rubisco activity is heat sensitive and it might be regulated by Rubisco activase in E. voratum SCS01. Furthermore, we initially observed significant photosynthesis inhibition of E. voratum SCS01 under thermal stress (at day 6), but the degree of photosynthesis inhibition was alleviated in E. voratum SCS01 at day 10 (Figure 2). Together, these results indicated that E. voratum SCS01 cells exhibited thermal acclimation potential as well, which involved the repair of photosynthetic related proteins via antioxidant systems.

We also observed dramatic loss of photosynthetic pigments and/or plastid photobleaching under thermal stress. The pigment content of free-living Symbiodiniaceae E. voratum SCS01 was dramatically reduced, and the plastids of E. voratum SCS01 were bleached at 32°C. For symbiotic Symbiodiniaceae cells isolated from corals, previous studies showed that Symbiodinium sp. CCMP827 lost 50% of its initial pigment content at 34°C (Takahashi et al., 2013), but Symbiodinium sp. (Avir) tolerated a wide range (10–35°C) of temperatures (Pierangelini et al., 2020). These results indicated that Symbiodiniaceae taxa exhibited different photobleaching threshold temperatures, which caused a dramatic loss of photosynthetic pigments and/or plastid photobleaching. The transcriptomic analysis of E. voratum SCS01 under thermal stress revealed an increase in the expression of genes involved in chlorophyll and carotenoid breakdown. These results supported the conclusion from previous studies that the degradation of pigments under thermal stress is an important reason for algal photobleaching (Asada, 2004; Andreeva et al., 2007; Takahashi et al., 2013). The present study further found that genes (i.e., genes encoding CHLP and VDE) related to the biosynthesis of carotenoids were significantly downregulated under high-temperature stress. This upregulation indicated that thermal stress caused photobleaching of E. voratum SCS01 via the degradation of pigments and the slowing of carotenoid biosynthesis.

The present study observed that bleached E. voratum SCS01 cells recovered their photosynthesis activity and brownish color quickly after the thermal stress was removed (Figure 2). Notably, we observed that the proportions of different E. voratum life forms changed during photobleaching and recovery (Figures 3, 5). The main life forms of Symbiodiniaceae include coccoid cells (vegetative) and mastigote cells (flagellated) (Fitt and Trench, 1983). The motility of mastigote cells of Symbiodiniaceae is the main characteristic of the free-living state (Fitt and Trench, 1983). Therefore, the proportions of mastigote cells accounted for approximately 85% of the total E. voratum SCS01 cells under the optimum growth temperature of 26°C in the present study. However, the proportion of coccoid cells significantly increased and mastigote cells significantly decreased when the temperature increased from 26°C (day 4) to 32°C (day 10). After removal of the thermal stress, the proportion of coccoid cells significantly decreased and the mastigote cells recovered to initial levels. These results suggested that coccoid cells are more tolerant to short-term thermal stress than mastigote cells of E. voratum. Gene transcription analysis observed that genes related to antioxidant activities (i.e., HSP70, SOD, and CAT) and respiration recovered quickly, and genes related to pigment metabolism (i.e., CCD, VDE, and CHLP), carbohydrate metabolism and photosynthesis partially recovered to their initial levels after the removal of thermal stress. The genes encoding a sperm motility kinase, sperm acrosomal protein and cation channel sperm-associated protein also exhibited recovery trends. These genes play important roles in flagellated sperm motility and development (Bissonnette et al., 2009) and may be involved in the decrease in the proportion of mastigote cells under high-temperature stress. The functions of mastigote cells appear to include migration and grazing on bacteria and other unicellular eukaryotes (Jeong et al., 2012), which suggests that short-term high temperature affects the movement ability and feeding behavior of Symbiodiniaceae, but it recovers after the removal of thermal stress.