Sand samples were collected using a 5 cm sampling tube during low tide at sandy beach of Geoje Island, Korea (34°58′15.59″ N, 128°42′00.74″ E) on Jan 29, 2024, and transported directly to the laboratory. The sand samples were placed into a Petri dish and stirred with filtered seawater to separate dinoflagellates and light particles from heavier grains of sand. The resulting suspension was collected and examined with an inverted microscope (Eclipse 50i; Nikon, Tokyo, Japan). This process was repeated several times. Cells were isolated using a capillary pipette and inoculated into individual wells of 48-well culture plates (Eppendorf, Hamburg, Germany) filled with f/2-Si culture medium (Marine Water Enrichment Solution; Sigma-Aldrich, St. Louis, MO, USA) and cultured at a temperature of 15°C and ca. 80 μmol photons m⁻² s⁻¹ cool-white illumination under a 12 L: 12 D photo-cycle. The cultured cells were transferred to individual wells of 6-well culture plates and after sufficient growth, the cells were transferred to a culture flask (70025; SPL Life Science, Pocheon, Korea) containing 35 mL of sterile f/2-Si culture medium. The monoclonal culture of Pseudadenoides kofoidii was successfully established and deposited as strain KMCC-4011 (MABIK PD00002585) in the Korean Microalgae Culture Collection at Pukyong National University and then has been maintained at a temperature of 20°C and ca. 100 μmol photons m⁻² s⁻¹ cool-white illumination under a 12 L: 12 D photo-cycle.

Living cells of the strains were photographed at ×1,000 magnification using an ultra-high resolution digital camera (DS-Ri2; Nikon) on an upright microscope (ECLIPSE Ni; Nikon). Cell size was measured based on light microscopy images (NIS-Elements D 4.30; Nikon). For fluorescence microscopy, approximately 1 mL of culture was transferred to a 1.7-mL microcentrifuge tube, and SYTOX Green Nucleic Acid Stain (Molecular Probes, Eugene, OR, USA) was added at a final concentration of 1.0 μM. The cells were incubated in the dark at room temperature for 10 min. The nuclear fluorescence in the cell was observed using a fluorescence LED illumination system (D-LEDI; Nikon) with an excitation wavelength of 475 nm and photographed using a Digital Sight 10 camera on an Eclipse Ci upright microscope (Nikon).

For scanning electron microscopy (SEM), 2 mL of mid-exponential batch cultures of the Korean isolate of Pseudadenoides kofoidii were fixed by Lugol’s iodine solution (0.1% final concentration) for 1 h at room temperature, then rinsed with deionized water. After rinsing, the samples were dehydrated in a graded ethanol series (10–99% in seven steps) for 15 min at each step and then critical point dried using an Autosamdri-815 critical-point dryer (Tousimis Co., Rockville, MD, USA) with liquid CO₂. Finally, the samples were coated with platinum and examined under a JEOL JSM 7600F field emission scanning electron microscope (SEM, JEOL Ltd., Tokyo, Japan).

Genomic DNA was extracted from 2 mL of exponentially growing cultures of Pseudadenoides kofoidii using the DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA, USA) following the manufacturer’s instructions. The SSU rRNA gene sequence was amplified using the primer pairs SR1 and SR12b (Takano and Horiguchi 2006), and the partial LSU rRNA gene sequence was amplified using the primer pairs 25F1 and R2 (Yamaguchi and Horiguchi 2005, Takano and Horiguchi 2006). Polymerase chain reaction (PCR) was performed on a total volume of 25 μL; 12.5 μL of GoTaq G2 Colorless Master Mix (Promega, Madison, WI, USA), 1 μL of forward primer, 1 μL of reverse primer, 4 μL of template DNA, and 6.5 μL of distilled water. PCR was conducted using a Thermal Cycler (Mastercycler nexus; Eppendorf) at 98°C for 5 min, followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 58°C for 15 s, and extension at 68°C for 2 min. The reaction was completed with a final elongation at 68°C for 5 min. The PCR products were confirmed by 1% agarose gel electrophoresis. The PCR products were purified using the Labopass PCR Purification Kit (Cosmogenetech, Seoul, Korea) according to the manufacturer’s instructions. The cycle sequencing reaction was performed using the ABI PRISM BigDyeTM Terminator ver. 3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA).

Electropherograms of forward and reverse reads were examined, and de novo assembly and consensus sequence generation were performed using the DNA Baser sequence assembly software (https://www.dnabaser.com/). The sequence of a Korean isolate of Pseudadenoides kofoidii was aligned with those of related species using the MUSCLE algorithms implemented in Molecular Evolutionary Genetics Analysis 5 (MEGA5) software, followed by manual refinement (Tamura et al. 2011, Gu et al. 2018).

Phylogenetic analyses were conducted separately for the SSU and LSU rRNA gene datasets. The SSU rRNA gene dataset included 64 taxa and comprised 1,397 base pairs, while the LSU rRNA gene dataset included 54 taxa and comprised 1,092 base pairs. For the phylogenetic analysis based on SSU rRNA gene sequences, Cryptosporidium parvum (Conoidasida), Perkinsus marinus (Perkinsea), Theileria parva (Aconoidasida), and Toxoplasma gondii (Conoidasida) were selected as the outgroup taxa (Hoppenrath et al. 2017). For the phylogenetic analysis based on LSU rRNA gene sequences, the outgroup included Acavomonas peruviana (Acavomonadea), Atoxoplasma sp. (Conoidasida), Babesia bigemina (Aconoidasida), Colponema vietnamica (Colponemea), Eimeria anguillae (Conoidasida), Perkinsus andrewsi (Perkinsea), Plasmodium falciparum (Aconoidasida), and Sarcocystis corvusi (Conoidasida) (Hoppenrath et al. 2017). Phylogenetic trees were inferred using both maximum likelihood (ML) analyses (RAxML version 8.2.10) (Stamatakis 2014) and Bayesian inference (BI) (MrBayes version 3.7) (Ronquist et al. 2012). ML bootstrap values were calculated using 1,000 pseudo-replicates with the substitution model GTR + Γ. The best-fitting model for the dataset was selected using the Bayesian information criterion in jModelTest2 (Posada and Crandall 1998) with the selected GTR + I + Γ model. Each analysis was performed using a Metropolis-coupled Markov chain Monte Carlo (MC³) approach with 10,000,000 generations, sampling one tree every 1,000 generations. The first 30% of sampled trees were discarded as burn-in after confirming stationarity by visually inspecting trace plots of log-likelihood and model parameters in Tracer v1.7.2 (Rambaut et al. 2018). The remaining 7,001 trees were used to calculate posterior probabilities of each clade. Convergence of the Markov chain Monte Carlo runs was assessed by examining the average standard deviation of split frequencies (<0.01; final value = 0.003). The trees were visualized using FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

To estimate the growth responses of Pseudadenoides kofoidii culture exposed to drastic changes of temperature, experiments were performed under four different water temperatures (10, 15, 20, and 25°C) without acclimation of the culture. Cultures maintained at 20°C were directly transferred to 25 and 15°C, and cultures maintained at 15°C were transferred to 10°C. The target temperatures were established in temperature-controlled incubators. The culture experiments to investigate the effect of temperature were performed at a salinity of 35 psu. For the culture experiments, aliquots of P. kofoidii with concentration of ca. 200 cells mL⁻¹ were inoculated into 2-L culture bottles (SPL Life Science) filled with f/2-Si culture medium using seawater from Busan, Korea (35°09′14.73″ N, 129°07′45.41″ E), and incubated at the target temperature under ca. 100 μmol photons m⁻² s⁻¹ cool-white illumination and a 12 L: 12 D photo-cycle. All experiments on growth were conducted in triplicate.

Growth was monitored at 2-d intervals. For cell enumeration, subsamples were fixed with Lugol’s solution (final concentration 1%). Because of the relatively large cell size, counts were performed using a Sedgewick–Rafter counting chamber. A 100 μL aliquot was examined under an upright microscope (ECLIPSE Ni; Nikon), and cell densities were calculated and expressed as cells mL⁻¹. Growth rates during the exponential growth phase (μ, d⁻¹) were calculated using the equation of Guillard (1973).

To estimate the fatty acid contents in cultures of the isolate of Pseudadenoides kofoidii, triplicate samples were harvested during the exponential growth phase (day 32). The samples were centrifuged by a Combi R515 centrifuge (Hanil, Gimpo, Korea) at 3,515 ×g for 10 min. The supernatant was gently removed, and the cell pellets were resuspended with distilled water and centrifuged to remove residual salts. This step was repeated three times. The cell pellets were frozen at a −20°C, and the frozen cell pellets were lyophilized using a freeze-dryer (FRD-16; DAIHAN Scientific, Wonju, Korea) at −80°C under vacuum for 1 d and weighed. Biomass production was determined by measuring the dry cell weight (mg L⁻¹).

Total fatty acid methyl esters (FAMEs) were analyzed by gas chromatography (GC2400; PerkinElmer, Waltham, MA, USA). For FAMEs extraction, freeze-dried samples of approximately 10 mg and glass micro-beads were combined in a 2 mL screw cap tube to which 0.9 mL of a 5: 100 v/v acetyl chloride: methanol (MeOH) solution and 0.1 mL of methyl heptadecanoate (3 mg mL⁻¹) dissolved in hexane were added. The use of methanol (MeOH) and acetyl chloride as esterification agents is common, because it efficiently converts free fatty acids into their methyl esters, which are easier to analyze by gas chromatography (GC). Methyl heptadecanoate (C17:0) was used as an internal standard to normalize variability during sample preparation and GC analysis, ensuring accurate fatty acid quantification. Cell disruption was performed using a Mini-beadbeater 24 (Biospec Products, Bartlesville, AZ, USA) for 2 min. The samples were incubated at 80°C, shaken at 300 rpm for 1 h using a thermomixer (Eppendorf), and then cooled for 1 min on ice, after which 1 mL of n-hexane was added and mixed for 1 min with a vortex mixer. The supernatant was separated, and 1 μL of the extract was injected into a DB-23 column (60 m × 0.25 mm internal diameter, 0.15 μm film thickness). The split ratio was 1/10 and N₂ was used as the carrier gas. Column temperature programs used the following procedure: 50°C for 1 min, increased to 175°C min⁻¹ at 25°C min⁻¹, and then increased to 230°C for 5 min at 2°C min⁻¹. The injector and detector were set at 250°C and 280°C, respectively. FAME peaks were determined by comparing the retention times between the reference standard (Supelco 37-component FAME mix; Sigma-Aldrich) and the samples and quantified as the percentage area of each component of FAME. The signal data at each retention time were compared with those of the internal standard for a quantitative analysis.

Data were presented as the mean ± standard deviation of triplicate samples. The assumptions of normality and homogeneity of variance were tested by Shapiro-Wilk’s W and Levene’s test, respectively. A linear mixed-effects model (LMM) was fitted with temperature and day as fixed effects and triplicate as a random intercept to account for repeated measurements over time. In addition, one-way analysis of variance (ANOVA) followed by Tukey’s test was performed for multiple comparisons of differences between treatments. All statistical analyses were conducted using R (R Foundation for Statistical Computing, Vienna, Austria). Differences were considered significant when p < 0.05.

Cells were light yellowish, asymmetrical, round to squarish and flattened laterally, and were 26.3–41.5 μm long (34.8 ± 3.6 μm) and 19.8–40.8 μm deep (28.9 ± 4.8 μm) (n = 50) (Fig. 1). A flagellar pore and two pyrenoids were visible in the ventral view (Fig. 1A), and in the dorsal view, two pores were clearly present in the antapical area of the cell (Fig. 1B). The cells contained small ellipsoidal granules, and one or two pyrenoids surrounded by a starch sheath was observed in both right and left lateral views (Fig. 1C & D). In the apical view, the transverse flagellum was clearly visible (Fig. 1E). A large, round to oval nucleus in the left lateral view was centrally located in the dorsal part of the cell (Fig. 1F).

The thecal plate pattern of cells based on the SEM observations is shown in Fig. 2, and schematic drawings based on the observations are shown in Fig. 3. The cells displayed a plate formula of apical pore complex (APC), 4′, 6C, 4S, 5‴, 5p and 1‴′ (Figs 2 & 3). There were no precingular plates. Thecal pore were randomly distributed, with an average diameter of 0.2 μm (n = 30) (Fig. 2). The APC contained angular apical pore plate (Po) with a round central cover plate (Fig. 2F & G). The Po was surrounded by four apical plates (Fig. 2F & G), and a few marginal thecal pores (7 or 8 pores) were observed on it (Fig. 2F & G). The apical plates 1′ and 4′ were in contact with the anterior sulcal plate (Sa) (Fig. 2F, G & I). Plate 4′ was the largest among the apical plate series and plate 2′, which touched cingulum plates C2 and C3, was narrow (Fig. 2F & G). The shallow cingulum consisted of six plates, and there were four sulcal plates surrounding the flagellar pore (Fig. 2F, G & I). The right sulcal plate (Sd) was the smallest among the sulcal plates, and other plates were similar in size (Fig. 2F, G & I). There were eleven plates in the hypotheca, including five post cingular plates, five posterior intercalary plates, and one antapical plate (Fig. 2B–E & H). The first (1‴) and second (2‴) postcingular plates were located on the left lateral side of the cell (Fig. 2A & D), and the fourth (4‴) and fifth (5‴) postcingular plates were on the right lateral side of the cell (Fig. 2A, C, E & F). The third (3‴) postcingular plates was visible in dorsal view (Fig. 2C). Among these postcingular plates, plate 5‴, which was in contact with the sulcus, was the smallest, and plate 4‴ was the largest (Fig. 2A, E & F). Five posterior intercalary plates are quite large and covered most of the hypotheca (Fig. 2A–E). In ventral view, the first (1p) and fifth (5p) posterior intercalary plates were in contact with the left sulcal (Ss) and posterior sulcal (Sp) plates, and plate 5‴ (Fig. 2A). The plates 1p and 5p were in contact with each other (Fig. 2A). The second (2p) posterior intercalary plate touched the posterior margins of plates 1‴ and 2‴ in the left lateral view (Fig. 2B & D). In dorsal view, the third (3p) and fourth (4p) posterior intercalary plates were in contact with the posterior margin of plate 3‴ (Fig. 2C & F). Two large pores (0.59 ± 0.08 μm), which were visible in light microscopy, were present at the posterior margins of plates 3p and 4p (Fig. 2B). Two pores on the 3p plate were occasionally present (30%, n = 20) (Fig. 2C & H). Large pores with an internal sieve were observed in plates 3p and 4p (Fig. 2C, inset). One antapical plate (1‴′) was located at the posterior end of the cell (Fig. 2H).

The Korean isolate of unidentified species displayed similar morphological characteristics with the specimens of Pseudadenoides kofoidii described by Hoppenrath et al. (2003) and Gómez et al. (2015). These are characterized by a nucleus located in the dorsal half of the cell, pyrenoid surrounded by a starch sheath, the same plate formula, scattered thecal pores, a single flagellar pore, a shallow anterior cingulum, and a large hypotheca. However, there is a difference in the number of large pores on plates 3p and 4p. In P. kofoidii (=Adenoides eludens) described by Hoppenrath et al. (2003), two pores are present on both plates 3p and 4p, whereas the specimen described by Gómez et al. (2015) shows no such pores; they might not have recognized the pores. In the Korean isolate, one or two pores are present on the plate 3p, and one pore is consistently observed on plate 4p (Fig. 2B, C & H). According to Hoppenrath et al. (2017), the difference in the number of large pores on the hypotheca can be one of the distinguishing features to differentiate P. kofoidii from P. polypyrenoides. However, as our findings indicate that the number of pores on plate 3p can vary, the differences in the number of large pores may not be a reliable distinguishing feature between species. Instead, the present study proposes that the presence or absence of the large pore on plate 1‴′ provides a more reliable characteristic. P. polypyrenoides can have large pores on plates 3p, 4p, and plate 1‴′ (Hoppenrath et al. 2017), whereas the Korean isolate of P. kofoidii consistently lacks a pore on plate 1‴′ (Fig. 2H). Similarly, in Adenoides species such as A. eludens and A. sinensis, which are phylogenetically close to Pseudadenoides species, the pores on plates 3p, 4p and plate 1‴′ are also present (Gu et al. 2018). However, Adenoides species form small areas of dense pores on each plate (see figs 21 & 31–40 in Gu et al. 2018), allowing for distinguishing Adenoides species from Pseudadenoides species. In addition, according to Gu et al. (2018), Adenoides species have a ventral pore and at least 10 pores at the margins of the pore plate (Po), whereas Pseudadenoides species lack the ventral pore and fewer pores are observed on the Po (5 or 7 pores in Gómez et al. 2015, Hoppenrath et al. 2017, 7 or 8 pores in the present study). As a consequence, the location, number, and absence or presence of distinct pores on thecal plates of Adenoides and Pseudadenoides species can be significant distinguishing feature at generic and species levels.

The phylogenetic trees inferred from SSU and LSU rRNA gene sequences are shown in Figs 4 and 5. ML and BI analyses generated similar phylogenetic trees. The phylogenetic trees inferred from SSU and LSU rRNA gene sequences revealed that Pseudadenoides kofoidii, P. polypyrenoides, Adenoides sinensis, and A. eludens are distinct from each other (Figs 4 & 5). In a phylogenetic tree based on the SSU rRNA gene sequences, the genus Pseudadenoides was a monophyletic group comprising P. kofoidii and P. polypyrenoides. Korean isolate formed a clade with two Canadian strains (accession Nos. KX000289 and EF492484), Japanese (KX000291), and German (KX000290) strains of P. kofoidii (ML bootstrap support 72, BI posterior probability 0.98) (Fig. 4). This clade was closely related to P. polypyrenoides (ML bootstrap support 93, BI posterior probability 1.00). Pseudadenoides species was a sister lineage to a clade comprising A. sinensis, and the clade of A. sinensis formed a sister clade of A. eludens. The group consisting of Adenoides and Pseudadenoides species was related to Prorocentrum species.

The phylogenetic tree based on the LSU rRNA gene sequences was similar to that of SSU rRNA gene sequences (Fig. 5). In a phylogenetic tree based on the LSU rRNA gene sequences, Korean isolate of P. kofoidii was grouped with Canadian (KX000293), French (LC002848), Japanese (KX000295), and German (KX000294) strains, and this group was a sister to P. polypyrenoides. The sister clade to Pseudadenoides species was a clade comprising A. sinensis and A. eludens. The group consisting of Adenoides and Pseudadenoides species in the phylogenetic tree based on the LSU rRNA gene sequences was also closely related to Prorocentrum species.

Together with morphological characterizations, the molecular phylogeny demonstrated that the Korean isolate can be identified as P. kofoidii, and that this species may have a much broader geographical distribution than previously recognized. In addition, Pseudadenoides and Adenoides species were found to be closely related to Prorocentrum species. According to Gómez et al. (2015), the phylogenetic affinity between Adenoides and Prorocentrum species may reflect shared evolutionary adaptations associated with benthic lifestyles, since these species are characterized by the loss of cingular groove that can be interpreted as an adaptive traits for benthic habitats. Interestingly, P. kofoidii and P. polypyrenoides also lack the precingular plates. This reduction in thecal plate may likewise represent an adaptive modification associated with benthic environments (Gómez et al. 2015), or alternatively, it may indicate a lineage-specific evolutionary trajectory within the genus Pseudadenoides. However, as Adenoides and Pseudadenoides species also share notable morphological similarity (see Hoppenrath et al. 2017), further detailed morphological and molecular investigations are required to verify and clarify this relationship.

Growth curves and biomass production of the Korean isolate of Pseudadenoides kofoidii exposed to abrupt temperature changes are shown in Fig. 6. At 25°C, P. kofoidii did not grow, and cell density decreased below the initial inoculum level after inoculation. Cell growth at 15°C was similar to 20°C (LMM, p > 0.05): cells exhibited continuous growth during the cultivation period (32 d), and the highest cell density was observed at the end of cultivation (5,502 ± 699 cells mL⁻¹ at 15°C and 4,992 ± 420 cells mL⁻¹ at 20°C) (Fig. 6A). The growth with relatively low cell density was observed in P. kofoidii cultivated at 10°C (<1,952 ± 161 cells mL⁻¹) (Fig. 6A). At 15 and 20°C, the growth rate in the exponential phase was similar (0.10 ± 0.01 d⁻¹ at 15°C and 0.11 ± 0.01 d⁻¹ at 20°C). The highest biomass, expressed as the dry cell weight, was observed at 20°C (18.1 mg L⁻¹), followed by 15°C (11.2 mg L⁻¹) and 10°C (5.7 mg L⁻¹) (Fig. 6B). Relatively high cell densities were accompanied by high biomass.

Previous studies suggested that temperature can be a major factor influencing benthic dinoflagellate communities (Huang et al. 2020, Reñé et al. 2021), and Shah et al. (2014) reported that higher abundances of sand-dwelling and epiphytic dinoflagellates along the coast of Jeju Island, Korea were observed at low to moderate temperatures (12–20°C). The Korean isolate of P. kofoidii also appears to prefer moderate temperature (15 and 20°C) rather than higher temperature (25°C). In addition, under the present experimental conditions, growth was sustained after transfer from 20°C to 10°C and 15°C, although long-term acclimation to these temperatures was not specifically tested. These results suggest that P. kofoidii may have limited tolerance to higher temperatures. Benthic habitats generally provide a thermally buffered microenvironment compared with seawater and air (Cho et al. 2005), and peak temperatures in coastal sandy habitats are often lower than those in the water column (e.g., Koyama and Inui 2024). Indeed, many planktonic dinoflagellates inhabiting the water column exhibit their highest growth rates at relatively higher temperatures, reflecting adaptation to warmer surface-water conditions (e.g., Kim et al. 2004, Kwon and Oh 2014). Therefore, the inability of P. kofoidii to grow at 25°C may reflect an ecological adaptation to relatively moderate benthic thermal conditions.

The fatty acid profile of Pseudadenoides kofoidii remained qualitatively similar across temperature treatments (Table 1, Fig. 7), indicating that temperature did not substantially alter the overall fatty acid composition. The fatty acid composition of P. kofoidii at all tested temperatures consistently included the saturated fatty acids C12:0 (lauric acid), C14:0 (myristic acid), C16:0 (palmitic acid), C18:0 (stearic acid), and C20:0 (arachidic acid), the monounsaturated fatty acids C15:1 (pentadecenoic acid), C16:1 (palmitoleic acid) and C18:1 (oleic acid), the PUFAs C20:5 (eicosapentaenoic acid; EPA) and C22:6 (docosahexaenoic acid; DHA) (Table 1, Fig. 7). The fatty acid compositions of planktonic dinoflagellates have been reported in many previous studies (e.g., Harrington et al. 1970, Fuentes-Grünewald et al. 2009, Jang et al. 2017, Xu et al. 2020, Calderini et al. 2023), whereas the investigations on benthic-epiphytic dinoflagellates remain relatively scarce. In some benthic-epiphytic dinoflagellates, such as Amphidinium carterae and Ostreopsis spp., EPA and DHA have been reported as the predominant PUFA under various experimental conditions (e.g., salinity, light quality [LED], and bacteria association) (Carballeira et al. 1998, Molina-Miras et al. 2018, Mendoza-Flores et al. 2025), but the proportions were considerably lower than those observed in P. kofoidii in this study.

The total fatty acid content of P. kofoidii at 10, 15, and 20°C was 97.8 ± 27.8, 79.9 ± 3.0, and 75.5 ± 3.3 mg g⁻¹, respectively (Supplementary Table S1). In P. kofoidii cultured at 10°C, relatively higher DHA content (29.5%, 29.2 mg g⁻¹) was detected (one-way ANOVA, p < 0.05), whereas no significant difference was observed at 15 (24.9%, 19.9 mg g⁻¹) and 20°C (26.8%, 20.2 mg g⁻¹) (one-way ANOVA, p > 0.05) (Table 1, Fig. 7, Supplementary Table S1). A similar temperature-dependent enhancement of DHA has been reported in Crypthecodinium cohnii, where a shift from 25 to 15°C resulted in a 19.9% increase in cellular DHA content and a 6.5% increase in productivity (Jiang and Chen 2000). Previous studies have also shown that environmental stress can enhance fatty acid accumulation while suppressing cell growth, as the biosynthesis and deposition of storage neutral lipids represent a protective response to stress (Ma et al. 2016, Oakley et al. 2022, Han et al. 2025, Mendoza-Flores et al. 2025). In the present study, the highest DHA proportion was observed at 10°C where growth and biomass were relatively low. This result indicates that DHA accumulation in P. kofoidii at 10°C may be closely associated with stress-related physiological responses. In contrast, EPA did not show a simple temperature-dependent response: EPA was detected at all tested temperatures, and the EPA content was significantly higher at 15°C (21.6%) than at 10°C, and even higher than at 20°C, where biomass was highest and similar growth was observed (one-way ANOVA, p < 0.05) (Fig. 7). Recently, Calderini et al. (2023) found no clear pattern in EPA content in response to temperature changes among the tested microalgal species, and Kim et al. (2025) reported that in cultures of dinoflagellate Akashiwo sanguinea, temperature effects on PUFA content were strain-dependent. In addition, Strandberg et al. (2022) reported that in mesocosm experiments, nutrient additions had a stronger influence on EPA and DHA concentrations of microalgae than temperature. These results indicate that EPA content in microalgae are likely regulated by complex interactions among multiple environmental and physiological factors, rather than by temperature alone. Further studies are required to clarify how multiple environmental and physiological factors interact to regulate EPA production in P. kofoidii.

Although Pseudadenoides kofoidii exhibited temperature-dependent variation in PUFA (DHA and EPA) production, the present study demonstrates that this sand-dwelling, epiphytic dinoflagellate nevertheless maintains remarkably high levels of both DHA and EPA in its fatty acid composition (>42% of the total fatty acid content), highlighting its potential as a promising new marine fatty acid resource. High proportions of PUFA are well documented in several dinoflagellates (Harrington et al. 1970, Mansour et al. 1999, Jang et al. 2017, Peltomaa et al. 2017, Xu et al. 2020), and DHA-rich dinoflagellates have already attracted attention for aquaculture nutrition and bioindustry applications (Jiang et al. 1999, Renaud et al. 1999). P. kofoidii is an epiphytic, substrate-attached dinoflagellate, and in the preliminary experiment this species showed no acute toxicity in a 24-h Artemia franciscana bioassay at the tested concentrations (Supplementary Table S2). Neither mortality nor abnormal behavior was observed. Although further toxicological evaluation is required, the preliminary observation suggests potential suitability for aquaculture-related applications. In aquaculture systems, benthic diatoms have long been widely used as feed for bivalve and gastropod spat because their attached growth enables direct grazing and stable delivery of particulate nutrition (Whyte et al. 1989). Similar to benthic diatoms, P. kofoidii exhibits a substrate-attached lifestyle, which may facilitate grazing and biofilm-based cultivation. These characteristics suggest that this species could be explored as a potential benthic feed resource. However, practical application would require further studies on large-scale cultivation, nutritional performance in target organisms, and production stability under aquaculture conditions.