Comparative Transcriptome Analysis of the Phototrophic Dinoflagellate Biecheleriopsis adriatica Grown Under Optimal Temperature and Cold and Heat Stress

Dinoflagellates are a major component of marine ecosystems, and very cold and hot water may affect their survival. Global warming has amplified the magnitude of water temperature fluctuations. To investigate the molecular responses of dinoflagellates to very cold and hot water, we compared the differentially expressed genes of the phototrophic dinoflagellate Biecheleriopsis adriatica grown under optimal temperature and cold and heat stress. The number of genes upregulated or downregulated between optimal temperature and cold stress was twice than that between optimal temperature and heat stress. Moreover, the number of upregulated genes was greater than that of the downregulated genes under cold stress, whereas the number of upregulated genes was less than that of the downregulated genes under heat stress. Furthermore, among the differentially expressed genes, the number of genes upregulated under cold stress and with unchanged expression under heat stress was the highest, while the number of the genes downregulated under cold stress, but not under heat stress, was the second-highest. Facilitated trehalose transporter Tret1 and DnaJ-like subfamily B member 6-A were upregulated and downregulated, respectively, under cold stress; however, their expression remained unchanged under heat stress. In contrast, Apolipoprotein d lipocalin and Troponin C in skeletal muscle were upregulated and downregulated, respectively, under both cold and heat stress. This study provides insight into the genetic responses of dinoflagellates to climate change-driven large water temperature fluctuations.

Temperature is a major factor affecting dinoflagellate survival, growth rate, and abundance (Hinder et al., 2012;Kohli et al., 2014;Ok et al., 2019;Jang and Jeong, 2020;You et al., 2020). In particular, many dinoflagellates cannot survive in very cold or hot waters (Matsubara et al., 2007;Kibler et al., 2012;Xu et al., 2016;Lim et al., 2019;Kang et al., 2020a). Global warming, which is accelerating presently, has increased air and sea surface temperatures (Hansen et al., 2006;Smith et al., 2015). Furthermore, it amplifies the magnitude of water temperature fluctuations (Han and Lee, 2020). Thus, very cold periods in the winter and very hot periods in the summer are likely to affect the survival of dinoflagellates. An abnormal change in the ambient water temperature in coral reefs breaks the symbiotic relationship between the host and symbiotic dinoflagellates and subsequently causes coral bleaching (McClanahan et al., 2007;Veron et al., 2009;Graham and Nash, 2013). Thus, many studies on the effects of temperature on coral bleaching have focused on the changes in the eco-physiology and molecular responses of the host and symbiotic dinoflagellates (Jones et al., 2000;Saxby et al., 2003;Franklin et al., 2004;Leggat et al., 2011;Rosic et al., 2011;Bayer et al., 2012;Krueger et al., 2014;Levin et al., 2016;Gierz et al., 2017;Davies et al., 2018). However, very few studies have focused on understanding the effects of temperature on the molecular responses in dinoflagellates.
The temperature range for the survival of dinoflagellates is important for cell cycle, photosynthesis, distribution, and bloom dynamics (Iglesias-Prieto et al., 1992;Pospelova et al., 2008;Jeong et al., 2015;Jang and Jeong, 2020;Kang et al., 2020a). Dinoflagellates have experienced fluctuation in seawater temperature for a long time, and some species have evolved to adapt and survive in these environments (Brinkhuis et al., 1998;Crouch et al., 2001;Sluijs et al., 2005). Each dinoflagellate species has an optimal temperature for the maximum growth rate and the lowest and highest temperature limits for survival, which are different for each dinoflagellate species (Matsubara et al., 2007;Xu et al., 2010;Kibler et al., 2012;Jeong et al., 2018;Lim et al., 2019;Ok et al., 2019;Kang et al., 2020a;You et al., 2020). Some dinoflagellate species can survive at 4 • C, while some can survive at 34 • C (Morton et al., 1992;Band-Schmidt et al., 2003;Tong et al., 2010;Zheng et al., 2012;Xu et al., 2016). The survival of a dinoflagellate at a certain temperature may be affected by the presence and expression of temperature stress-regulating genes (Levin et al., 2016;Gierz et al., 2017;Davies et al., 2018).
To understand the responses of a dinoflagellate to a certain temperature that causes mortality, transcriptomes of the dinoflagellate grown under optimal temperature and cold and heat stress should be compared simultaneously. There have been several studies on comparative transcriptome analysis of dinoflagellates grown under optimal temperature and heat stress (Barshis et al., 2014;Fridey, 2015;Levin et al., 2016;Gierz et al., 2017;Davies et al., 2018;Bellantuono et al., 2019;Lin et al., 2019). However, only a few studies have compared the expression of a few target genes of dinoflagellates grown under optimal temperature and cold stress or optimal temperature and cold and heat stress (Deng et al., 2019;Kim et al., 2021;Wang et al., 2021).
The phototrophic dinoflagellate Biecheleriopsis adriatica, which belongs to the family Suessiaceae, was first described in 2009 (Moestrup et al., 2009). This dinoflagellate has been reported in the waters of many countries, including Korea, China, Greece, Japan, Palau Island, Philippines, and Singapore, as well as in the Adriatic Sea (Moestrup et al., 2009;Takahashi et al., 2014;Jang et al., 2015;Luo et al., 2015;Benico et al., 2019;Kang et al., 2019b;Tsipas, 2020). Furthermore, B. adriatica was reported to form a bloom with the phototrophic dinoflagellate Takayama sp., resulting in mass mortality of farmed milkfish in 2016 in Bolinao, Philippines (Benico et al., 2019). It is known that B. adriatica is consumed by many common heterotrophic dinoflagellates such as Aduncodinium glandula, Oxyrrhis marina, Gyrodinium dominans, Gyrodinium moestrupii, Luciella masanensis, Pfiesteria piscicida, and Oblea rotunda and the common naked ciliates Strombidinopsis sp. and Pelagostrobilidium sp. (Kang et al., 2019a). Thus, B. adriatica likely plays important ecological roles in marine ecosystems. Biecheleriopsis adriatica is positioned at the base of the clade consisting of the phototrophic dinoflagellate Ansanella granifera and species belonging to the family Symbiodiniaceae (LaJeunesse et al., 2018). Thus, B. adriatica is an important species for understanding the evolution of the order Suessiales. Cells of B. adriatica have been found in oceans worldwide at water temperatures of 17.7-32.0 • C. The winter temperature of Korean waters is often below 5 • C, whereas the summer temperature is above 30 • C (Korea Hydrographic and Oceanographic Agency, 2021) 1 . Thus, in Korean waters, B. adriatica may produce coldresistant and heat-resistant enzymes under cold and heat stress, respectively. Therefore, temperature-regulation-related genes may be differentially expressed under optimal temperature and cold and heat stress conditions. This study explored differential gene expression of B. adriatica grown under optimal temperature and cold and heat stress. Furthermore, the differential expression pattern (upregulated, unchanged, and downregulated) of genes under cold stress, optimal temperature, and heat stress was investigated. The differential expression of genes under cold stress and optimal temperature and that under optimal temperature and heat stress yielded nine patterns. The results of this study provide a basis for understanding the molecular signs of responses of dinoflagellates to cold and heat stress and their adaption to global climate changes.

Preparation of Experimental Organism and Incubation at the Target Temperatures
Biecheleriopsis adriatica was isolated from plankton samples collected from surface waters off the coast of Tongyoung, southern Korea, using plankton samplers in August 2006, when the water temperature and salinity were 28.0 • C and 31.0, respectively (Jang et al., 2015). The collected samples were filtered using a 154-µm Nitex mesh, and a clonal culture of B. adriatica BATY06 was established using two consecutive single-cell isolations (Jang et al., 2015). When B. adriatica concentration increased sufficiently, it was transferred to 32-, 270-, and 500-mL polycarbonate bottles containing f/2-Si medium (Guillard and Ryther, 1962). The bottles were placed on a shelf at 20 • C for the cold stress experiment and 25 • C for the optimal temperature and heat stress experiments under a 14 h:10 h light:dark cycle and illuminated with 50 µmol photons m −2 s −1 using cool white fluorescent light.
To determine the optimal, lowest, and highest water temperatures for survival, the growth rates of B. adriatica were measured at 14-34 • C. The growth rates were almost zero at 14 and 32 • C; thus, these two temperatures were used for cold and heat stress. The optimal temperature for maximum growth rate was 25 • C.
For cold stress, a dense B. adriatica culture (approximately 100,000 cells mL −1 ) grown autotrophically at 20 • C was distributed to three 800-mL tissue culture flasks (Falcon; Heidelberg, Germany) containing fresh f/2-Si medium. For heat stress and optimal temperature, a dense B. adriatica culture (approximately 100,000 cells mL −1 ) grown autotrophically at 25 • C was distributed to six 800-mL tissue culture flasks containing fresh f/2-Si medium.
For RNA sequencing, the triplicate flasks for each target temperature were incubated in three temperature-controlled chambers set up at 14, 25, and 32 • C for 11 days after gradual acclimation ( Figure 1A). The temperature in each chamber was monitored using HOBO data loggers (HOBO MX2202 Pendant MX Temperature/Light Data Logger; Onset; Bourne, MA, United States).
To monitor B. adriatica cell density at each target temperature, a 5-mL aliquot was collected from each flask and fixed with 5% acidic Lugol's solution daily. Aliquots were collected from the fixed samples, and ≥ 200 B. adriatica cells were enumerated using a Sedgewick-Rafter counting chamber to determine B. adriatica density. After incubating for 6 days at 25 • C, B. adriatica reached the stationary phase and were transferred into new 800-mL flasks containing fresh f/2-Si medium.
To confirm that nutrient concentration did not affect B. adriatica growth, a 10-mL aliquot was collected from each flask on days 0, 6, and 11, gently filtered through GF/F filters (Whatman Inc., Floreham Park, NJ, United States), and stored at -20 • C until nutrient concentration measurement. The concentrations of nitrate plus nitrite (hereafter NO 3 ), phosphate (PO 4 ), and silicate (SiO 2 ) were measured using a nutrient autoanalyzer system (QuAAtro; Seal Analytical GmbH; Nordestedt, Germany). The samples diluted to 1/10 were used for the measurement to consider the limit concentration of the autoanalyzer system, and it was considered when the final nutrient concentration was calculated after the measurement.
Harvesting, RNA Extraction, and cDNA Library Construction To harvest the similar cell number of B. adriatica (1 × 10 7 cells) after incubating for 11 days, 600, 200, and 300 mL aliquots were collected from each flask at 14, 25, and 32 • C, respectively. The cells in the aliquots were collected by centrifugation at 2,808× g for 5 min (Labogene 1696R; Gyrozen Co., Gimpo, Korea), immediately frozen in liquid nitrogen, and stored at -80 • C until RNA extraction.
Total RNA was extracted individually from each sample using the RNeasy Plant Mini Kit (Cat. No. 74903; Qiagen; Hilden, Germany) and treated with RNase-Free DNase set (Cat. No. 79254;Qiagen) to eliminate residual genomic DNA contaminants. The extracted RNA samples were purified using a RNeasy Kit (Cat. No. 74104;Qiagen), and the total RNA amount was assessed using Quant-IT RiboGreen (Cat. No. R11490; Invitrogen; Carlsbad, CA, United States). The integrity of the total RNA was evaluated using the TapeStation RNA Screen Tape (Cat. No. 5067-5576; Agilent Technologies; Santa Clara, CA, United States), and only RNA preparations with RNA integrity number (RIN) > 7.0 were used for RNA library construction.
The nine cDNA libraries were separately constructed using an Illumina TruSeq Stranded mRNA LT Sample Prep Kit (San Diego, CA, United States), and paired-end (2 × 100 bp) RNA of the pooled libraries was sequenced on an Illumina NovaSeq 6000 at Macrogen (Seoul, Korea). In the first step of this process, only eukaryotic polyadenylated mRNA was filtered to eliminate potential bacterial contaminants from the total RNA of B. adriatica.
The quality, total bases, total reads, GC content (%), and basic statistics of the sequenced raw reads of the nine libraries were calculated, and the raw reads of low-quality, adapter, contaminant DNA, or PCR duplicates were removed using Trimmomatic program (Bolger et al., 2014). The quality of the trimmed reads (clean reads) was checked using FastQC v0.11.7 2 before assembly.

Transcriptome Analyses
The clean reads for the nine libraries were assembled de novo using Trinity with the "-SS_lib_type FR" option, which is typically used for de novo transcriptome reconstruction (Grabherr et al., 2011). It combines the read sequences of a certain length of overlap to form long fragments without N gaps, called contigs. Only contigs longer than 200 bp were selected. Average concentration (µM) of nitrate plus nitrite, silicate, and phosphate in the triplicate flasks at days 0, 6, and 11 under each temperature condition. After incubating for 11 days under each condition, B. adriatica cells were harvested for RNA sequencing. Purple triangles, black circles, and red rectangles indicate CS, OT, and HS data points, respectively. Vertical symbols represent treatment means ± 1 SE.
The longest assembled contigs were filtered, and redundant contigs were removed (i.e., unigenes) using CD-HIT-EST, with a clustering identity of 0.98 (Li and Godzik, 2006).
For functional annotation, the unigenes were searched against NCBI non-redundant (NCBI nr) and Gene Ontology (GO) using BLASTx of DIAMOND software with an E-value default cutoff of 1.0E-5 (Buchfink et al., 2015).
Unigene expression levels were estimated using RSEM v1.3.1 (Li and Dewey, 2011). The clean reads from each sample were mapped back onto the assembled unigenes, and the read count value of each unigene was obtained from the mapping results. The unigene expression level (log 2 fold change) between the samples under cold stress and optimal temperature or under heat stress and optimal temperature was calculated as fragments per kilobase of unigene per million mapped reads (FPKM). If more than one FPKM value was 0, it was excluded from the analysis to avoid reducing the power of the statistical hypothesis testing. After the addition of + 1 to each of the FPKM values from filtered unigenes, they were transformed to log 2 scale and subjected to relative log expression (RLE) normalization. Statistically significant results of differential gene expression were obtained with |log 2 fold change| ≥ 1 and p < 0.05, using the nbinomWaldTest of DESeq2 R library (Anders and Huber, 2010). Hierarchical clustering analysis of differentially expressed genes (DEGs) was performed using the Euclidean method and complete linkage, representing the similarity of expression patterns between unigenes and samples.
Based on the GO annotation results, functional unigene classification and enrichment were analyzed using Web Gene Ontology Annotation Plot (WEGO) 3 . The Pearson's chi-square test was automatically carried out using WEGO to test whether the GO categories were significantly differentiated between the upregulated and downregulated gene datasets (Ye et al., 2006). Differences in GO categories between the two datasets were considered significant at a p-value < 0.05. In addition, the number of DEGs obtained with |log 2 fold change| ≥ 1 and adjusted p-value (padj) < 0.05 was also determined (Benjamini and Hochberg, 1995).

Classification of Unigenes by the Trend in Expression Among the Conditions
The unigenes were classified into nine types based on the trends in expression under cold and heat stress against that under optimal temperature (Figure 2). Type 1 was composed of DEGs upregulated under both cold and heat stress; Type 2 consisted of unigenes upregulated under cold stress, but not significantly differentially expressed between optimal temperature and heat stress; Type 3 was composed of DEGs upregulated under cold stress but downregulated under heat stress; Type 4 was composed of unigenes not significantly differentially expressed between cold stress and optimal temperature but upregulated under heat stress; Type 5 consisted of unigenes not significantly differentially expressed between cold stress and optimal temperature and also between heat stress and optimal temperature; Type 6 consisted of unigenes not significantly differentially expressed between cold stress and optimal temperature but downregulated under heat stress; Type 7 was composed of DEGs downregulated under cold stress but upregulated under heat stress; Type 8 was composed of unigenes downregulated under cold stress but not significantly differentially expressed between optimal temperature and heat stress; Type 9 consisted of DEGs downregulated under both cold and heat stress. The number of unigenes in each type and the proportion of each type to all unigenes or DEGs was determined. To investigate the functions of representative DEGs belonging to each type, the unigenes annotated against eukaryotic organisms in the NCBI nr database with E-value cutoff E-30 were ranked by their expression level, and the functions of the high-ranking unigenes were examined. The hypothetical or predicted proteins were excluded from the identification and further screened using an NCBI conserved domain search. Unigenes with functions revealed previously were obtained. The rank of the unigenes was determined as follows: In the first step, the fold change values of the unigene expression under cold stress were arranged in the descending or ascending orders if they were upregulated or downregulated, respectively. In the second step, the unigenes were ranked. In the third step, the first and second steps were repeated for the fold change values of the same unigenes under heat stress. In the fourth step, the two rank numbers assigned to each unigene under cold and heat stress were added, and the unigenes were arranged according to the ascending order of the added number. If there was no differential expression of unigenes under cold or heat stress depending on the type, they were not calculated. Therefore, Type 5 was excluded from this calculation and examination of the function of the unigenes.
The average NO 3 , PO 4 , and SiO 2 concentrations on days 0, 6, and 11 under optimal temperature and heat stress were similar to those under cold stress (Figures 1C,D).

General Transcriptome Features of B. adriatica
Illumina sequencing of B. adriatica generated 130-202 million paired-end 100-bp clean reads per library (nine libraries = three triplicates at each temperature condition), with a trimming process for removing adapter sequences and poor-quality bases ( Table 1). The GC content of the clean reads was between 54.30 and 54.92% and the ratio of bases with Phred quality scores ≥ 20 and 30 (Q20 and Q30) of the clean reads exceeded 99.14 and 96.70%, respectively (Table 1). A total of 198,703 unigenes were generated by a combined de novo assembly with an average 850 bp length and a 1,562 bp N50 (Table 1). Among the 198,703 unigenes,35,370 (17.8%) and 65,580 (33.0%) were functionally annotated against the GO and NCBI nr databases, respectively, with an E-value default cutoff of 1.0E -5.
The number of DEGs in B. adriatica under cold and heat stress were 24,916 and 13,366, respectively, at the expression level of |log 2 fold change| ≥ 1 and p < 0.05 (Figure 3). Under cold stress, 13,159 and 11,757 unigenes were upregulated and downregulated, respectively. In comparison, 6,345 upregulated and 7,021 downregulated unigenes were identified under heat stress (Figure 3). At the expression level of |log 2 fold change| ≥ 1 and padj < 0.05, the number of DEGs in B. adriatica under cold and heat stress were 23,965 and 12,185, respectively (Supplementary Figure 1), and 12,812 and 11,153 genes were upregulated and downregulated, respectively, whereas 5,892 upregulated and 6,293 downregulated genes were identified under heat stress.
The heatmap for the DEGs depicting the result of hierarchical clustering analysis showed more similar patterns of DEGs between heat stress and optimal temperature than those between cold stress and optimal temperature (Figure 4).

Functional Classification of B. adriatica Differentially Expressed Genes Under Cold Stress
The biological function of the identified DEGs of B. adriatica under cold stress was investigated using GO analysis. The results of GO functional classification and enrichment were visualized and compared using WEGO (Figure 5). Under cold stress, 4,370 upregulated and 2,866 downregulated DEGs were annotated to the GO database (29.0% DEGs under cold stress), which were classified into 21, 18, and 37 GO subcategories in cellular component (CC), molecular function (MF), and biological process (BP) categories, respectively, in GO level 2 (Figure 5 and Supplementary Table 1).
In the MF category, most DEGs belonged to "catalytic activity" and "binding" subcategories ( Figure 5B and Supplementary Table 1). Four of the 18 subcategories had significantly different percentages of upregulated and downregulated DEGs (Pearson's chi-squared test; p < 0.05; Figure 5B, Table 2, and Supplementary Table 1). The percentage of upregulated DEGs was higher than that of downregulated DEGs in the "structural molecule activity" subcategory (Pearson chi-squared test; p = 0.001), whereas the percentage of downregulated DEGs was higher than that of upregulated DEGs in "transporter activity, " "signal transducer activity, " and "binding" subcategories (Pearson's chi-squared test; p < 0.001, p < 0.001, p < 0.001; Figure 5B, Table 2 and Supplementary Table 1).
In the BP category, "cellular process" subcategory had the highest percentage of DEGs, followed by "metabolic process" and "biological regulation" subcategories ( Figure 5C and Supplementary Table 1). Eleven of the 37 subcategories had significantly different percentages of upregulated and downregulated DEGs (Pearson's chi-squared test; p < 0.05; Figure 5C, Table 2, and Supplementary Table 1). Among them, the percentage of upregulated DEGs in "metabolic process" subcategory was higher than that of downregulated DEGs (Pearson's chi-squared test; p = 0.001; Figure 5C, Table 2, and  Supplementary Table 1).
At the expression level of |log 2 fold change| ≥ 1 and padj < 0.05, there was no change in the trend of the results of GO analysis at level 2 under cold stress (Supplementary Figure 2).
In GO level 3, 77 subcategories had significant differences between the percentages of the upregulated and downregulated genes under cold stress (Pearson's chi-squared test; p < 0.05; Supplementary Table 2). Among them, 23, 13, and 41 subcategories belonged to the CC, MF, and BP categories, respectively (Supplementary Table 2). Furthermore, the percentage of upregulated DEGs of 17 subcategories was higher than that of downregulated DEGs, whereas the remaining 60 subcategories had a higher percentage of downregulated DEGs than that of upregulated DEGs (Supplementary Table 2). The top 5 subcategories in GO level 3 with the largest differences between the percentage of the upregulated and downregulated genes significantly in B. adriatica under cold stress, based on the p-value, were "methylation, " "ion binding, " "cell communication, " "transmembrane transporter activity, " and "catalytic activity, acting on RNA" in sequence (Table 3).

Functional Classification of B. adriatica Differentially Expressed Genes Under Heat Stress
When the biological function of the identified B. adriatica DEGs under heat stress was investigated, 1,451 upregulated and 1,504 downregulated genes were annotated to the GO database (22.1% DEGs under heat stress), which were classified into 21, 12, and 33 GO subcategories in the CC, MF, and BP categories, respectively, at GO level 2 (Figure 6).
In the CC category, "cell, " "cell part, " "organelle, " and "membrane" subcategories were dominant (Figure 6A and Supplementary Table 3). There were no subcategories The numbers in parentheses in the higher percentage column indicate the number of the categories under cold and heat stress, respectively. The subcategories in each category were sorted based on the p-values from Pearson's chi-squared test. U, upregulated genes; D, downregulated genes. with significant differences between the percentages of upregulated and downregulated genes ( Figure 6A, Table 2,  and Supplementary Table 3).
In the MF category, "catalytic activity" subcategory had the highest percentage of DEGs, followed by "binding" subcategory ( Figure 6B and Supplementary Table 3). The "catalytic activity" subcategory had a higher percentage of downregulated genes than that of upregulated genes (Pearson's chi-squared test; p < 0.001), but the percentage of downregulated genes in other subcategories was not significantly different from that of the upregulated genes ( Figure 6B, Table 2, and Supplementary Table 3).
In the BP category, most DEGs were involved in the "cellular process" and "metabolic process" (Figure 6C and Supplementary Table 3). Four of the 33 subcategories had significantly different percentages of upregulated and downregulated DEGs (Pearson's chi-squared test; p < 0.05; Figure 6C, Table 2, and Supplementary Table 3). The percentage of the downregulated genes in "nitrogen utilization, " "response to stimulus, " "metabolic process, " and "cellular process" subcategories were significantly higher than that of the upregulated genes (Pearson's chi-squared test; p = 0.005, p = 0.006, p = 0.012, p = 0.016), whereas there were no significant differences between the percentage of the upregulated and downregulated genes in other subcategories ( Figure 6C, Table 2, and Supplementary Table 3).
At the expression level of |log 2 fold change| ≥ 1 and padj < 0.05, there was no change in the trend of the results of GO analysis under heat stress (Supplementary Figure 3).
In GO level 3, 29 subcategories had significant differences between the percentages of the upregulated and downregulated genes (Pearson's chi-squared test; p < 0.05) under heat stress, with 9, 7, and 13 subcategories belonging to the CC, MF, and BP categories, respectively (Supplementary Table 4). Most subcategories had a higher percentage of downregulated genes than that of the upregulated genes (24 subcategories; Supplementary Table 4). The top 5 subcategories in GO level 3 with the largest differences between the percentage of the significantly upregulated and downregulated genes in B. adriatica under heat stress, based on the p-value, were "regulation of biological quality, " "small molecule metabolic process, " "response to abiotic stimulus, " "carbohydrate derivative binding, " and "ion binding" in sequence ( Table 3).

DISCUSSION
Dinoflagellates have a very large genome size ranging from 1.5 to 112 Gbp (Murray et al., 2016). In general, studies focus on the gene expression of dinoflagellates because it is difficult to determine their whole genome sequence (Murray et al., 2016). With advances in sequencing technology, complex transcriptional responses of dinoflagellates to diverse conditions have been reported, revealing a large number of unigenes (Murray et al., 2016;Caron et al., 2017). There have been many studies of de novo transcriptomics of dinoflagellates under different conditions of diverse environmental factors such as water temperature, nutrients, salinity, and light (e.g., Jaeckisch et al., 2011;Lowe et al., 2011;Morey et al., 2011;Bayer et al., 2012;Levin et al., 2016;Shi et al., 2017). The results of these studies provide a basis for better understanding of the responses of dinoflagellates to changes in environmental factors with regard to ecology and molecular biology.
Dinoflagellates experience both cold winter and hot summer in temperate regions (Selina et al., 2014;Kang et al., 2019b;Jang and Jeong, 2020). In temperate regions such as Korea, Japan, and China, sea waters become cold due to cold waves caused by the weakness of the stratospheric polar vortex by Arctic seaice loss or hot due to heat waves caused by the heat domes due to strengthening of the power of the North Pacific and Tibetan high pressures (Kim et al., 2014;Han and Lee, 2020;Lee et al., 2020;Inoue et al., 2021). Therefore, differences in growth and differential gene expression among very cold or hot and optimal temperatures should be understood to explore the survival of dinoflagellates in terms of eco-physiology and gene expression with respect to molecular biology. However, only three studies have explored the differential expression of two target genes of dinoflagellates grown under optimal temperature and cold stress or under optimal temperature and cold and heat stress (Deng et al., 2019;Kim et al., 2021;Wang et al., 2021). The expression of two cold shock domain protein genes in the mixotrophic dinoflagellate Prorocentrum cordatum exposed to low temperatures (4, 12, or 16 • C) increased compared to that exposed to 20 • C; the expression of saxitoxin biosynthesis gene sxtA4 increased in the toxic dinoflagellate Alexandrium catenella by cold shock (20→16 • C), while that of sxtG increased by cold shock (20→16 • C) and heat stress (12→20 • C); heat shock protein 60 and 10 expressions were upregulated in the mixotrophic dinoflagellate Scrippsiella acuminata after exposure to lower (15, 10, and 5 • C) and higher (25 and 30 • C) temperatures compared to optimal temperature (20 • C). This study showed that 5,233 genes were differentially expressed under both cold and heat stress. Furthermore, 19,683 genes were differentially expressed only under cold stress, while 8,133 genes were differentially expressed only under heat stress. Therefore, comparative transcriptome analysis of dinoflagellates grown under optimal temperature and cold and heat stress should be conducted simultaneously to understand the pattern of all expressed genes, and the present study provides a milestone for this topic.
Positive growth during the first 3 days under heat stress was observed. This likely reflected residual growth during preincubation. However, during the last 8 days, the abundance of B. adriatica continuously decreased, indicating that the cells were affected by heat stress when they were harvested.
The cultures used in this study were not completely axenic, although we attempted to minimize the effect of bacteria on the mRNA dataset of B. adriatica while preparing the experimental organisms and during RNA sequencing. It is generally very difficult for dinoflagellates to grow without bacteria; studies shown that many dinoflagellates have endosymbiotic relationships with bacteria or require bacteria for their survival (Alavi et al., 2001;Croft et al., 2005;Bolch et al., 2011;Cruz-López and Maske, 2016). Nutrient concentrations during incubation under the three different temperature conditions were sufficiently high to enable dinoflagellate growth. Nutrient concentration is one of the major factors affecting dinoflagellate growth (Hwang and Lu, 2000;Nagasoe et al., 2010). Furthermore, nutrient limitations in ambient waters are known to affect gene expression in dinoflagellates (Morey et al., 2011;Lauritano et al., 2017).

General Transcriptome Features of B. adriatica
Some studies determined the number of DEGs of dinoflagellates at the expression level of p < 0.05 (e.g., Guo et al., 2016;Jang et al., 2019), whereas others determined that at the expression level of padj < 0.05 (e.g., Barshis et al., 2014;Bellantuono et al., 2019;Lin et al., 2019). The number of DEGs in B. adriatica at the expression level of p < 0.05 and padj < 0.05, were 33,049 and 30,877, respectively, indicating a 6.6% difference. The results of GO analysis and expression type at the expression level of p < 0.05, and padj < 0.05, did not change significantly. Furthermore, there was no change in the trends of any of the results.
At the expression level of p < 0.05, the larger number of upregulated DEGs than downregulated DEGs under cold stress indicates that B. adriatica expresses more genes in response to cold stress. In contrast, the higher number of downregulated DEGs than upregulated DEGs under heat stress suggests that B. adriatica restricts gene expression to save energy or that genes are damaged by heat shock. At the geological scale, the sea surface temperature during the mid-and late-Pleistocene (∼1.2 Ma) showed a cooling trend, although a glacial-interglacial cycle with large amplitudes of air temperature change was repeated (McClymont et al., 2013). The period of global warming in the last century was significantly shorter than that in the ice age. Thus, dinoflagellates may have acclimated to cold stress better than to heat stress. Furthermore, dinoflagellates conduct diurnal vertical migration, and some dinoflagellates pass through the thermocline and remain in cold water, although the surface water temperature increases significantly . However, vertical migration may not provide a refuge when the surface water temperature decreases significantly. Thus, dinoflagellates may upregulate or downregulate gene expression under cold stress than under heat stress.

Functional Classification of B. adriatica Differentially Expressed Genes Under Cold and Heat Stress
The "structural molecule activity, " "membrane-enclosed lumen, " and "organelle part" subcategories in GO level 2 are related to cell structure and organelles. Cold stress may damage the protist cell structure. The morphology of the green alga Chlamydomonas reinhardtii changed significantly and its organelles were progressively disorganized and disrupted after decreasing the water temperature from 25 to 5 • C (Valledor et al., 2013). After 24 h of water temperature decrease, the nucleolus of C. reinhardtii could not be distinguished; vacuoles and starch sheaths increased. Furthermore, the chloroplast changed its shape and flagella were shortened and disappeared after 120 h (Valledor et al., 2013). Similarly, under cold stress, B. adriatica might upregulate genes related to maintaining cell integrity instead of genes with other functions.
Under stress, microalgae tend to reserve extra metabolites by promoting metabolic processes or save energy by reducing metabolic processes (Levin et al., 2016;Sun et al., 2018). In this study, the "metabolic process" subcategory had a significantly higher percentage of upregulated DEGs than that of downregulated DEGs under cold stress. Therefore, B. adriatica may respond to cold stress by enhancing metabolic processes to reserve extra metabolites.
Under cold stress, the "signaling" subcategory had the greatest difference between the percentages of upregulated and downregulated DEGs, that is, the smallest p-value. This was followed by "response to stimulus" and "biological regulation." The basic activities of B. adriatica, which perceive environmental change, transmit signals, and respond to changes by modulating biological processes or functions, seemed to be disturbed by cold stress.
Under cold stress, "methylation" had the highest difference between upregulated and downregulated genes in GO level 3. In organisms, methylation plays diverse roles, such as heavy metal modification, gene expression and protein function regulation, and RNA processing (Lee et al., 2010;Li et al., 2018). DNA methylation, which adds a methyl group to the DNA, can affect the gene expression of dinoflagellates, and temperature change can regulate DNA methylation levels (Bayer et al., 2012;Naydenov et al., 2015;Li et al., 2018). Therefore, cold stress may affect DNA methylation and gene expression in B. adriatica.
The number of subcategories showing a significant difference between the percentages of upregulated and downregulated DEGs under heat stress was lower than that under cold stress. The catalytic activity generally increases with temperature, but it decreases sharply due to enzyme denaturation if the temperature exceeds a certain threshold (Daniel and Danson, 2013). Therefore, enzyme denaturation in B. adriatica cells may occur at 32 • C. Furthermore, nitrogen utilization efficiency decreases at non-optimal temperatures owing to the changes in cytoplasmic viscosity. Heat stress may also affect the cytoplasmic viscosity and nitrogen utilization efficiency in B. adriatica (Raven and Geider, 1988). Levin et al. (2016) reported that the downregulated DEGs of the thermo-tolerant dinoflagellate Cladocopium sp. under heat stress were distributed in more GO categories related to the metabolic and biosynthetic processes than those of thermo-sensitive Cladocopium sp. In some organisms, reduced metabolic and biosynthetic activity under stress is correlated with the increased survival time of organisms while saving energy (Hand and Hardewig, 1996). Therefore, B. adriatica may try to acclimate to high water temperatures with reduced energy consumption.
Under heat stress, only "regulation of biological quality" had a higher percentage of the upregulated DEGs than that of the downregulated DEGs among the top 5 subcategories. It includes many processes that modulate a qualitative or quantitative trait with a measurable attribute in an organism or part of an organism, such as size, mass, shape, and color (Gene Ontology). It was found that increasing the water temperature by 1 • C caused an approximately 2.5% intra-specific decrease in protist cell volume (Atkinson et al., 2003). Furthermore, changes in cell morphology, such as cell shape, size, and color, due to heat stress have been observed in microalgae (Neustupa et al., 2008;Chokshi et al., 2015). The "responses to abiotic stimulus" subcategory had a higher percentage of downregulated genes than that of upregulated genes, suggesting that high temperature may not cause significant stress to B. adriatica or it adapts to high temperature rapidly.

Classification of Unigenes by the Expression Trend Among the Temperature Conditions
The number of unigenes differentially expressed under only cold or heat stress from that under optimal temperature (Types 2, 4, 6, and 8) was significantly more than that detected under both cold and heat stress (Types 1, 3, 7, and 9). This suggests that the mechanism of molecular responses of B. adriatica to cold stress is different from that to heat stress. Interestingly, the number of unigenes belonging to Types 1 (upregulated under both cold and heat stress) and 9 (downregulated under both cold and heat stress), Types 2 (upregulated under cold stress and unchanged under heat stress) and 8 (downregulated under cold stress and unchanged under heat stress), Types 3 (upregulated under cold stress and downregulated under heat stress) and 7 (downregulated under cold stress and upregulated under heat stress), and Types 4 (unchanged under cold stress and upregulated under heat stress) and 6 (unchanged under cold stress and downregulated under heat stress) were similar to a mirror image. Thus, B. adriatica may balance upregulation and downregulation of gene expression in response to temperature stress.
The "apolipoprotein d lipocalin (ApoD)" identified in Type 1 is an evolutionarily conserved anti-stress protein that enhances tolerance to freezing and oxidative stress in Arabidopsis thaliana. It was upregulated in the branching stony coral Acropora millepora under cool and warm treatments and the phototrophic dinoflagellate Prorocentrum donghaiense under phosphorusdepleted conditions (Charron et al., 2008;Ganfornina et al., 2008;Muffat and Walker, 2010;Wuitchik, 2018;Zhang et al., 2018). Furthermore, the unigene encoding "alphaprotein kinase vwkA" belonged to Type 1. The alpha-kinase family is involved in diverse cellular processes such as protein translation, Mg 2+ homeostasis, intracellular transport, cell migration, adhesion, and proliferation (Middelbeek et al., 2010). Betapudi et al. (2005) reported vwkA involvement in regulating myosin II abundance and assembly behavior in the amoeba Dictyostelium discoideum. Myosin II is an actindependent molecular motor protein important for cell motility and cytokinesis (Maciver, 1996;Foth et al., 2006). Therefore, temperature stress may affect B. adriatica cell movement and division processes.
The unigenes encoding "facilitated trehalose transporter Tret1" and "glutathione S-transferase class-mu 28 kDa isozyme" belonged to Type 2. Trehalose improves insect and plant tolerance to cold stress (Kikawada et al., 2007;Iordachescu and Imai, 2008;Benoit et al., 2009). Therefore, trehalose may play the same role in B. adriatica as in insects and plants. Furthermore, glutathione S-transferase (GST) is an antioxidant; thus, it is considered to be a potential oxidative stress biomarker (Guo et al., 2014). In the dinoflagellate Fugacium sp., most of the 10 GST transcripts detected were upregulated under heat stress (Gierz et al., 2017). However, in this study, the B. adriatica GST gene was highly expressed under cold stress, but not heat stress. Therefore, this gene may be expressed by temperature stress, but whether the temperature stress is cold or heat stress is dependent on dinoflagellate species.
The unigenes encoding "fatty acid desaturase" and "fructosebisphosphate aldolase (FBA)" belonged to Type 3. Temperature change affects the membrane fluidity of poikilothermic organisms such as bacteria, fungi, protozoa, plants, and animals (Murata and Los, 1997). Membrane fluidity perceives changes in environmental conditions and transduces signals to acclimate to these conditions (Murata and Los, 1997;Los and Murata, 2004). The cell membrane becomes rigid when exposed to low temperatures and becomes fluid at high temperatures (Murata and Los, 1997). The reduced membrane fluidity at low temperatures can be improved via membrane lipid desaturation by fatty acid desaturases (Sakamoto and Murata, 2002). Conversely, the increased membrane fluidity at high temperatures can be ameliorated by integrating de novo synthesized saturated fatty acids into membrane lipids and the presence of membrane-stabilizing proteins (Los et al., 2013). Therefore, the upregulation and downregulation of this gene under cold and heat stress, respectively, in this study corresponded with previous results. FBA is a key metabolic enzyme involved in glycolysis, gluconeogenesis, and the Calvin cycle (Flechner et al., 1999;Gross et al., 1999). Furthermore, it plays an important role in stress signaling in plants (Lu et al., 2012). Therefore, B. adriatica may be affected more by cold stress than that by heat stress.
The "FAD-binding domain-containing protein" belonging to Type 4 includes several genes playing key roles in many metabolic pathways, including the citric acid cycle, fatty acid beta-oxidation, and amino acid catabolism (Mansoorabadi et al., 2007;Kim and Winge, 2013). Moreover, "malate l-lactate dehydrogenase" belonging to Type 4 also functions in many metabolic pathways, including the tricarboxylic acid cycle, glyoxylate bypass, amino acid synthesis, and gluconeogenesis (Musrati et al., 1998;Minarik et al., 2002). They were upregulated only under heat stress, thus indicating that mechanisms for acclimation to temperature stress act differentially under cold and heat stress in B. adriatica metabolic pathways.
As "proline/betaine transporter" belonging to Type 6 is an amino acid transporter, its downregulation under heat stress suggests that the capacity to export amino acids in B. adriatica cell may reduce under heat stress (Li et al., 2021).
The unigenes encoding "chloroplast light harvesting complex (LHC) protein, partial" and "intraflagellar transport (IFT) protein 46-like" belonged to Type 7. LHC proteins function in light capture and photoprotection (Boldt et al., 2012). Three upregulated LHC genes were detected in Fugacium sp. under heat stress (Gierz et al., 2016). In contrast, under cold stress, LHC activity was reduced in C. reinhardtii and cereal grain Hordeum vulgare (Atienza et al., 2004;Valledor et al., 2013). Therefore, the results of this study are consistent with those of previous studies. The IFT system assembles and maintains eukaryotic cilia and flagella (Cole, 2003). The cilia and flagella are best known to play important roles as motile organelles, but they also function as sensory perception organelles that monitor the surrounding environment, such as chemicals, light, and temperature (Silflow and Lefebvre, 2001;Follit et al., 2006). Although B. adriatica speed was not measured during incubation, when observed under a microscope, B. adriatica cells swam more slowly under cold stress than under heat stress or optimal temperature.
The unigene encoding "DnaJ-like subfamily B member 6-A" was identified to belong to Type 8. Heat shock proteins (HSPs) are important chaperones that maintain cellular homeostasis under both favorable and unfavorable conditions (Wang et al., 2004). They are considered to be produced more under unfavorable conditions, such as temperature and light stress (Rajan and D'Silva, 2009). Unlike B. adriatica, which downregulates the unigene encoding DnaJ, DnaJ expression in other dinoflagellates was upregulated. The relative DnaJ transcription level in S. acuminata increased under both low and high temperatures than that under a moderate temperature; moreover, increased DnaJ expression in heat stress-exposed Cladocopium sp. and Fugacium kawagutii have been reported (Levin et al., 2016;Deng et al., 2019;Lin et al., 2019). In contrast, Gierz et al. (2017) reported that several transcripts encoding HSPs, including DnaJ, were identified in Fugacium sp. under heat stress, and the downregulated transcripts were more dominant than upregulated transcripts. Therefore, whether the expression of this gene is upregulated or downregulated is dependent on the dinoflagellate species.
The unigene encoding "troponin C, skeletal muscle" belonged to Type 9. Troponin C is a Ca 2+ homeostasis protein of the calmodulin family (DeSalvo et al., 2010;Abassi et al., 2021). In the elkhorn coral Acropora palmata, this gene was downregulated under heat stress (DeSalvo et al., 2010). The downregulation of this gene under cold and heat stress in this study showed that Ca 2+ homeostasis in B. adriatica may be affected by cold and heat stress. To elucidate this, more studies on the effect of temperature on the expression of this gene in dinoflagellates are required.
To better understand the response of dinoflagellate communities to diverse environmental changes, including cold waves, heat waves, ocean acidification, and local pollution, both eco-physiological and molecular characteristic changes should be explored together.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI (accession: PRJNA756051).