First Report of the Dinoflagellate Genus Effrenium in the East Sea of Korea: Morphological, Genetic, and Fatty Acid Characteristics

: Most species in the family Symbiodiniaceae are symbiotic partners to invertebrate and protist hosts, but a few live freely in water columns. Here, a free-living dinoflagellate was isolated from the waters off the Dokdo Islands in the East Sea of Korea. Morphological and molecular analyses show this isolate belongs to Effrenium voratum. Prior to the present study, E . voratum had been reported to live in the waters in the temperate latitudes in the western North Pacific, the southwest Western Pacific, the eastern North Pacific, the eastern Atlantic, and the Mediterranean Sea. To our knowledge, this is the highest latitude in the western North Pacific, where E . voratum has been reported. This report extends the known range of this dinoflagellate to the temperate waters of the western North Pacific Ocean. The sequence of the D1/D2 region of the large subunit ribosomal DNA (LSU rDNA) was identical to E . voratum found in Jeju Island, Korea, Tsushima Island, Japan, and Cook Strait, New Zealand, suggesting this species is cosmopolitan. However, it was different by 1 bp from those found in Blanes, Spain and Santa Barbara, USA. In the phylogenetic tree built on the basis of the LSU (D1-D2) rDNA region sequences, this dinoflagellate was clustered within a clade, including all the other E . voratum strains. Morphological characteristics were like those of strains found in the waters of Jeju Island. This is the first report conducted on the fatty acid profile of fully characterized E . voratum . Importantly, the isolate possessed a high ratio of long-chain omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to total lipid. This dinoflagellate could be a candidate for commercial applications, such as aquaculture feed and essential omega-3 PUFA productions.


Introduction
Dinoflagellates are ubiquitous protists that can be found in almost every conceivable marine environment [1]. Recently, they have drawn increasing attention from both the scientific and public communities because of the following reasons: Some dinoflagellates form red tides and/or harmful algal blooms, which often cause large-scale mortality of fish [2], they play indispensable ecological roles in marine plankton communities, serving as prey for a variety of predators and as a predator on diverse microorganisms [3], some dinoflagellates are used as various biological resources and they are also treated as one of the most promising bio-resources for new high-value products [4][5][6]. In particular, dinoflagellates are known to be rich in long-chain omega-3 PUFAs, such as EPA (C20:5 n-3) and DHA (C22:6 n-3) [7][8][9]. Thus, the amount and composition of omega-3 PUFAs from dinoflagellates in the phytoplankton community are strongly intertwined with higher trophic levels, eventually affecting the nutritional values of higher-order consumers [7,10,11]. Therefore, to better understand the role of dinoflagellates in the marine ecosystems and their potential commercial applications, the establishment of a clonal culture is a critical first step for accurate identification of an isolate of interest.
The family Symbiodiniaceae (Order Suessiales) comprises symbiotic dinoflagellates, most of which are symbiotic with invertebrate and protist hosts [12][13][14][15][16][17], even though some species exist as free-living forms [18]. These dinoflagellates are vital components of the coral reef ecosystems, and they are promising resources in the production of valuable pigments [19] and toxin compounds, such as zooxanthella toxins [20]. Despite their ecological and economic importance in the marine ecosystems and biotechnology, little information is available on their taxonomy. This is due to, in part, the difficulties in culturing and making morphological observations, which must be done by scanning electron and transmission electron microscopy (SEM and TEM). Therefore, their roles as essential components of the marine ecosystems and their potential for biotechnology are often overlooked. Accurate identification and establishment of a clonal culture of these dinoflagellates are essential for further research and commercial application.
The systematics of Symbiodiniaceae was revised, and distinct clades within the family were reassigned into seven genera in 2018 [21]. Symbiodinium voratum was first described and named by Jeong et al. [18], it was renamed as Effrenium voratum by LaJeunesse et al. [21]. This is the only taxonomically accepted species in the genus Effrenium [21]. E. voratum is found in the Pacific and Atlantic Oceans, and it grows at sub-tropical and temperate latitudes [18,[21][22][23][24]. Jeong et al. [18] reported the Korean E. voratum strains that had been isolated from waters off Jeju Island. However, no additional documentation of E. voratum colonies in Korean water have been reported.
In this study, we isolated and identified a unicellular dinoflagellate E. voratum from seawater off the Dokdo Islands, Dokdo-ri, Ulleung-eup, Ulleung-gun, Gyeongsangbuk-do, Korea. The present report provides information about the morphological, molecular, and chemotaxonomic features on the first record of this species living in the East Sea of Korea.

Sample Collection and Isolation
Plankton samples from the Dokdo Islands in the East Sea of Korea (37.240486 N, 131.870853 E), were collected using a water sampler during September 2016, when the water conditions were 24 °C and 35 practical salinity unit (PSU), respectively (Table 1, Figure 1). The samples were gently filtered through a 154 μm Nitex mesh and placed in six-well tissue culture plates. A clonal culture of E. voratum was established by using two serial single-cell isolations. Polycarbonate (PC) bottles containing f/2 medium (AusAqua, Wallaroo, SA, Australia) and isolated E. voratum cells were filled with filtered seawater, capped, and incubated at 20 °C, under illumination by cool white fluorescent lights at approximately 20 μmol photons m −2 s −1 in a 14:10 h light-dark cycle. As the concentration of E. voratum increased, the cells were transferred to 50, 125, and 500 mL PC bottles containing fresh f/2 media. Once dense cultures of E. voratum were obtained, they were transferred approximately every four weeks to new 500 mL PC bottles filled with fresh f/2 media. When sufficient volumes of E. voratum culture were available, genomic DNA was extracted, and the DNA sequence of the cultured cells was analyzed. After genetic identification, the morphology and cellular fatty acid composition of the dinoflagellate were examined. For morphological and fatty acid analyses, cells were collected in the exponential growth phase.

Morphological Identification
An inverted microscope was used to examine the morphology of photosynthetically grown living cells. The length and width of the live cells were measured with the aid of a digital camera (Zeiss AxioCam MRc5; Carl Zeiss, Göttingen, Germany). For SEM, 10 mL aliquots of cultures at ~1000 cells mL −1 were fixed for 10 min in osmium tetroxide (OsO4; Electron Microscopy Sciences, EMS hereafter, Hatfield, PA, USA) at a final concentration of 2% (v/v) in seawater. Fixed cells were collected on a 3 μm pore-sized PC membrane filter without additional pressure and rinsed three times with distilled water to remove residual salts. Cells were dehydrated in an ethanol series (Merck, Darmstadt, Germany) and dried using a critical point dryer (CPD 300, Bal-Tec, Balzers, Liechtenstein). The dried filters were mounted on a stub and coated with gold-palladium in a sputter coater (SCD 005; Bal-Tec). Cells were viewed with an FE-SEM (S-4800; Hitachi, Hitachinaka, Japan). For TEM, cells from a dense culture were transferred to a 10 mL tube and fixed in 2.5% (v/v) glutaraldehyde (final concentration, EMS) in a culture medium. After 1.5-2 h, the contents of the tube were placed in a 10 mL centrifuge tube and concentrated at 1610× g for 10 min in a Vision Centrifuge VS-5500 (Vision Scientific, Bucheon, Korea). The pellet was transferred to a 1.5 mL tube and rinsed in 0.2 M sodium cacodylate buffer (EMS) at pH 7.4. After several rinses in the buffer, cells were postfixed for 1.5 h in 1% (w/v) OsO4 in deionized water. The pellet was embedded in agar (EMS) and dehydrated in a graded ethanol series (50%, 60%, 70%, 80%, 90%, and 100% ethanol, followed by two changes in 100% ethanol). The material was embedded in Spurr's low-viscosity resin (Low Viscosity Embedding Media Spurr's Kit; EMS). Sections were prepared on an RMC MT-XL ultramicrotome (Boeckeler Instruments, Tucson, AZ, USA) and stained using 3% (w/v) aqueous uranyl acetate (EMS) followed by lead citrate (EMS). The sections were viewed using a JEOL-1010 TEM (Tokyo, Japan).

Molecular Identification
For molecular analysis, approximately 10 mL of a dense E. voratum culture was concentrated by centrifugation at 2190× g for 15 min at room temperature, and the pellet was used for genomic DNA extraction. The DNA extraction, including the amplification of the LSU rDNA, PCR reaction, sequencing, and alignment of DNA sequences, were performed as previously reported by Kang et al. [25]. The primers that were used to amplify the LSU region of rDNA are reported in Table 2. Reverse primer LSUB LSU rDNA ACG AAC GAT TTG CAC GTC AG [27] Alignments, phylogenetic, and molecular evolutionary analyses of the D1-D2 LSU rDNA sequences were performed using MEGA v.4 [28] and Clustal X2 [29], with diverse assemblages using available data in the National Center for Biotechnology Information (NCBI) GenBank database, for other species. Bayesian analyses were performed using MrBayes v.3.1 [30,31] with the default GTR + G + I model, to determine the best model for the data in each region. For all sequence regions, four independent Markov Chain Monte Carlo (MCMC) runs were performed, as described by Kang et al. [32]. Maximum likelihood (ML) analyses were conducted using the RAxML 7.0.3 program [33]. We allowed for 200 independent free inferences and used the -# option to identify the best tree. Bootstrap values were calculated using 1000 replicates under the same substitution model.

Fatty acid Composition Analysis
Lipid extraction was carried out using the modified Bligh-Dyer method developed by Breuer et al. [34]. Fatty acid methyl ester (FAME) composition was analyzed using a 7890A gas chromatograph, equipped with a 5975C mass selective detector (Agilent, Santa Clara, CA, USA), based on our previous publication [35]. Compound identification was completed by matching the mass spectra with those in the Wiley/NBS registry of mass spectral data, searches with a match value higher than 90% were valid.

Results
Mastigote, coccoid, and doublet cells occurred in all cultures examined under light microscopy, and they appeared to be like one another (Figure 2A-C). Motile cells of E. voratum were mushroomedshaped with the hemispherical episome slightly larger than the hemispherical hyposome ( Figure 2A). The nucleus was in the episome ( Figure 2A). The ranges (mean ± standard error, n = 30) of the living cell length and width were 9.42-15.6 μm (12 ± 0.2) and 7.08-11.9 μm (9.7 ± 0.2), respectively ( Table 3). The ratios of the cell length to the width (mean ± standard error, n = 30) ranged from 1.12 to 1.4 (1.2 ± 0.01). When observed under SEM, fixed cells were slightly smaller than unfixed cells. The cell length under SEM had a range of 7.3-12.7 μm (9.6 ± 0.2) and the width had a range of 5.52-11.4 μm (7.7 ± 0.2), respectively ( Table 3). The ratio of the length to the width under SEM had a range of 0.83-1.4 μm (1.2 ± 0.02) ( Table 3).  Mean values are shown in parentheses. AP = anteroposterior; EAV = elongated amphiesmal vesicle, PE = peduncle.

Discussion
The key for differentiating families in the order Suessiales [36] is the morphology of the apical furrow: Species in the family Tovelliaceae have an apical line of narrow plates (ALP), species in the family Borghiellaceae have a pair of elongated anterior vesicles (PEV), and species in the family Symbiodinaceae have a single EAV [37]. All Symbiodinaceae contain a type E eyespot next to the sulcus groove of the motile cell. The motile stage (mastigote) has only seven latitudinal series of amphiesmal vesicles [21]. Strain MABIKLP88 had the EAV, type E eyespot, and seven latitudinal series of amphiesmal vesicles, these morphologies fit the criteria for the family Symbiodinaceae.
The morphological features of this isolate resembled those of the strain SvFL 1 and CCMP421 because it had a type E eyespot, a peduncle, two-stalked pyrenoid, peripheral chloroplasts, and a Kofoidian series of the small plate (x), the EAV, 5′, 5a, 8′′, 9s, 17-20c, 6′′′, and 2′′′′ ( Table 3). The length and the width of living E. voratum MABIKLP88 cells were 9.42-15.6 μm and 7.08-11.9 μm, respectively. The sequence of the D1/D2 region of LSU rDNA was also identical to those of strains SvFL 1 and CCMP421 (Table 4). Although these E. voratum strains were isolated from distinct sites (the Dokdo Islands in Korea, Jeju Island in Korea, and Cook Strait in New Zealand), they all share the typical morphologies of E. voratum and identical LSU rDNA sequences. Thus, this newly isolated Korean strain could serve as a good example of a cosmopolite species found in different oceans. The length and width of living cells of the E. voratum MABIKLP88 (9.42-15.6 μm and 7.08-11.9 μm, respectively) were comparable to those of other strains of E. voratum reported at 10.1-17.1 μm and 7.8-14.4 μm, respectively (Table 3). In SEM micrographs of cells, the width and EAV length of the E. voratum MABIKLP88 (5.52-11.4 μm and 1.71-2.72 μm, respectively) were also similar to those of other strains of E. voratum (5.8-10.9 μm and 1.75-3.19 μm, respectively) ( Table 3). The ratio of cell length to the width of the E. voratum MABIKLP88 (0.83-1.4) was comparable to those of other strains of E. voratum (1.2-1.4) ( Table 3). The ratio of cingulum displacement to cell length of the E. voratum MABIKLP88 (0.11-0.26) was similar to those of other strains of E. voratum (0.1-0.21) (Table 3). Therefore, our report provides new information on the extended ranges of cell length, cell width, EAV length, the ratio of cell length to cell width, and the ratio of cingulum displacement to cell length in E. voratum. These results showed more variability on the measured morphological traits of the species than previously reported E. voratum strains.
The sequence of the D1/D2 region of LSU rDNA was identical to the of the E. voratum strains located in the waters off the Jeju Island of Korea, Tsushima Island of Japan, and Cook Strait of New Zealand. Our phylogenetic analysis confirmed that the strain MABIKLP88 belonged to E. voratum (Table 4, Figure 6). Molecular phylogeny results from the LSU rDNA sequences supported the morphological identification by forming a well-supported clade, including the sequences from the original description.
Prior to the present study, E. voratum had been reported to live in waters in the temperate latitudes in the western North Pacific, the southwest Western Pacific, the eastern North Pacific, the eastern Atlantic, and the Mediterranean Sea [18,[21][22][23][24][38][39][40]. E. voratum appears to be distributed across high sub-tropical and low temperate latitudes. E. voratum is cold-water-adapted and may tolerate temperatures as low as 10-12 °C. This is supported by physiological measurements taken on culture rt-383, which showed that this strain grew optimally at 15-20 °C, but also grew at 12 °C and 28 °C [41]. This wide temperature range tolerance may enable E. voratum to survive in a wide variety of oceanic environments around the world (Figure 7). Additional physiological tests on rt-383 and other E. voratum strains are needed to elucidate their thermal tolerance. In addition, the maximal growth of strain MABIKLP88 was obtained at 22-28 °C and 300-700 μmol photons m −2 s −1 (unpublished data). Moreover, E. voratum has an ability to feed on bacteria and other macroalgae, proving a possible survival strategy for Effrenium to persist in nutrient-poor conditions [39].
It seems that this wide temperature range tolerance of the species, coupled with tolerance to high light intensities and mixotrophic ability, attributed to E. voratum's survival and presence in a wide variety of oceanic environments around the world. Thus, this newly isolated Korean strain could serve as a good example of a candidate for cosmopolite species in high sub-tropical and low temperate latitudes. Exploration of this dinoflagellate's distribution in greater detail would be worthwhile.
As shown in Figure 7, E. voratum strains are present in many locations, and the current report further describes the occurrence of E. voratum in the western North Pacific Ocean (Table 6, Figure 7). The latitude of the Dokdo Islands, where strain MABIKLP88 was isolated, is 37.2 N. The locations where additional stains of the western North Pacific have been reported range from 22.5 N to 36.0 N ( Table 6). To date, the waters around the Dokdo Islands are the highest latitude in the western North Pacific, where E. voratum has been found. The findings in this report expand upon the understanding of the geographic distribution of E. voratum in the western North Pacific. It may also show that climate changes caused by global warming have taken place in the oceans around the Korean Peninsula since the Dokdo Islands are located at the front between the warm Kuroshio and the cold Kuril currents.  [18,[21][22][23][24][38][39][40], while the dark star indicates our MABIKLP88 strain of E. voratum. Analysis of the cellular fatty acid composition of the strain MABIKLP88 revealed that it was rich in C16:0 (22.1%), SFA and C18:4 n-3 (15.2%), C20:5 n-3 (10.9%), and C22:6 n-3 (25.4%) PUFAs. The fatty acid profile of different dinoflagellate species has been extensively studied in order to find valuable strains with high potentials for commercial applications [7][8][42][43][44][45][46]. The profile has also been used as a general chemotaxonomic guide to defining a variety of taxonomic groups since different microalgal groups show distinct fatty acid distributions [44,47,48]. In particular, the C18 fatty acids and > C20 PUFAs are generally regarded as the signature fatty acids for dinoflagellates [8,48]. In this study, E. voratum MABIKLP88 was found to be rich in the C18 fatty acid, EPA, and DHA. It has also been reported that dinoflagellates typically have higher DHA than EPA concentrations [7][8][9], and the isolate exhibited much higher DHA (25.4%) content than EPA (10.9%). Several previous studies have shown that the essential omega-3 PUFAs have a variety of beneficial health effects [49]. Importantly, EPA and DHA, which are known essential omega-3 fatty acids, have been reported to be beneficial to human health [50,51]. Most of the omega-3 PUFAs come from marine sources, such as fish oils and a variety of commercial products that are available worldwide. Using marine fish as a sustainable and safe resource of omega-3 is in question because of global climate change, overfishing issues, and the increasing levels of environmental pollutants, such as heavy metals and radioactive materials found in the ocean [52,53]. Some marine protists have high contents of EPA and/or DHA [6,[54][55][56][57]. Therefore, this marine microalga may have the potential to be a clean and sustainable omega-3 source alternative to fish-based oil. On the other hand, an excessive intake of omega-6 causes negative health outcomes [58,59], even though both omega-3 and omega-6 PUFAs are essential, and they are required for many biological processes. Due to the ever-growing demands for food, the fish farming industry has rapidly expanded over the last few decades and aquaculture now accounts for over 50% of the fish consumed worldwide [60]. This has caused further shortage of wild fish as feed at fish farms, and thus, fish oil in the aquaculture feed has been increasingly replaced by terrestrial vegetable oils that normally lack EPA and DHA, but often contain high levels of omega-6 PUFAs [61]. The imbalance between omega-3 and omega-6 levels has deteriorated the nutritional quality of farmed fish such as Atlantic salmon that contains less EPA and DHA and more omega-6 PUFAs than before [62,63]. Thus, strain MABIKLP88 could be used for the production of designed aquafeeds for balancing the dietary omega-6 and omega-3 ratios. Furthermore, several authors reported that the amount of omega-3 PUFAs in the phytoplankton community is reflected in the nutritional quality of predatory fish [7,10,11]. It should also be noted that the availability of omega-3 PUFAs in aquatic ecosystems is closely related to egg production and hatching success of marine copepods [64,65] and the fish larvae survival [66]. Hence, the presence and abundance of this Korean dinoflagellate may have a potentially positive impact on the sustainability of both capture fisheries and aquaculture in Korea.
In this study, we report the first record of E. voratum from the Dokdo Islands, in the East Sea of Korea. This marine dinoflagellate would serve as potential biological resources to produce aquaculture feeds and biochemicals of commercial interests. Importantly, a clonal culture was established and deposited in a national culture collection to allow for further research.

Conflicts of Interest:
The authors declare no conflict of interest.