Identification and growth of Prorocentrum cf. cassubicum (Dinoflagellata: Prorocentraceae) from Bahía de La Paz: first record for the Gulf of California

2.1 Sampling site

Both strains were isolated in August 2018 from the beach in Ensenada de La Paz, a shallow coastal lagoon connected with Bahía de La Paz through an inlet of 1.2 km width (Sandoval and Gomes-Valdes 1997) in Baja California Sur, Mexico (Figure 1). This region has a dry or semi-desertic climate, with an average annual temperature of 24 °C (minimum = 5 °C, maximum = 41.5 °C) and an annual rainfall that fluctuates between 180 and 250 mm (Chávez 1985) with the maximum in September and the minimum from March to June (Carmona et al. 2002). It has a mixed tidal regime with a predominance of semi-diurnal tides (Álvarez-Arellano et al. 1997). The Ensenada de La Paz lagoon is anti-estuarine in character, as salinity increases towards the interior of the lagoon due to the shallowness, high rate of evaporation, low precipitation and absence of freshwater runoff (Espinoza 1977). In particular, the sampling site is between 1 and 2 m deep, with water temperatures that fluctuate from 22 to 28.4 °C, dissolved organic matter from 423.1 to 549.40 μg l⁻¹, pH from 7 to 8.3, and salinity from 27.2 to 40.3 (Cruz-Escalona, V.H. pers comm).

Figure 1:

Isolation site of the strains PNCETMAR-1 and PNCETMAR-2 used in this study.

2.2 Sampling method and isolation

Samples were collected with phytoplankton nets of 10- and 20-μm mesh size, using shallow horizontal tows during 10 min at an approximate depth of 0.5 m. This depth was chosen considering that the sampling sites have maximum depths of 2 m. In the laboratory, cells were isolated under a Zeiss AXIO Vert A1 inverted microscope with fine tip capillaries. Subsequently, cells were transferred to 96-well plates with culture medium and were progressively scaled-up to 20–25 ml of media in 50-ml culture tubes.

2.3 Strains and growth conditions

Strains used in this study were provided by the IPN-CICIMAR (Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas) dinoflagellate collection (Table 1). The PNCETMAR-1 strain was maintained in modified GSe medium (Blackburn et al. 2001; Bustillos-Guzmán et al. 2015) and the PNCETMAR-2 strain in modified K medium (Keller et al. 1987), both were modified with the addition of vermicompost extract. Strains were maintained at a salinity of 34, at 24 °C ± 1 °C, and 120–150 μmol m⁻² s⁻¹ in a 12 h light: 12 h dark cycle. Strains were maintained in 50-ml culture tubes with 20 ml of medium and 1 ml of stock culture (inoculum) with monthly reseeding.

2.4 Light microscopy

For morphological identification, in general, cell shape and size, symmetry/asymmetry of the lateral thecal plates, periflagellar platelets, pore types and their distribution pattern on the thecal plates were considered. Light microscopy (bright field) photographs were obtained from live cells (Axio Vert.A1 inverted microscope, Carl Zeiss, Germany). An epifluorescence microscopy was used (Axio Scope.A1 compound microscope, Carl Zeiss, with EC-Neofluar Plan 40×/0.75 and 63×/0.95 objectives, and 6-megapixel Axiocam 506 color camera), applying 0.2 % Calcofluor White M2R stain (Fritz and Triemer 1985) for the theca and DAPI (Sigma; 4′,6-diamidino-2-phenylindole; Kapuscinski 1995) to visualize the position and size of the nucleus.

2.5 Scanning electron microscopy

Two techniques were used for scanning electron microscope (SEM) analysis, from which micrographs were obtained. Samples of 2 ml of strain PNCETMAR-1 were fixed with 4 % formaldehyde and processed as follows: a preliminary wash in distilled water was done to remove the fixative and salts (five times), followed by dehydration in a series of ethanol solutions of increasing concentration (30, 50, 70, 90 and 100 %) and two washes with 100 % ethanol (30 min per step in 1.5-ml Eppendorf tubes, without centrifugation). The samples were mounted directly on a 12.7-mm aluminum specimen stubs with a micropipette, dried at room temperature, and coated by high-resolution sputtering (Polaron SC7640, Quorum Technologies, Newhaven, SXE, UK) with Au-Pd. Observations were made and micrographs were taken with a JEOL JSM-7600F SEM at 5 kV, at a working distance of 15.9–16.7 mm, in the Laboratorio Nacional para el Estudio de Nano y Biomateriales of the Departamento de Física Aplicada del Centro de Investigación y Estudios Avanzados of the Instituto Politécnico Nacional (CINVESTAV-IPN), Unidad Mérida, Yucatán, Mexico.

Samples (1 ml) of the PNCETMAR-2 strain were prefixed with 0.5 ml of 4 % glutaraldehyde and left in darkness for 90 min. Subsequently, post fixation was performed with 2 % osmium tetroxide (OsO₄); the samples were left in darkness for 2 h until cells were blackened (indicating complete fixation). Prefixation and postfixation was done at room temperature (∼24 °C). After fixation, several centrifugation washes (Thermo Scientific™ Sorvall™ Legend™ XTR, USA) were performed with cold sterile distilled water to remove fixative residues. Gradual dehydration was performed with ethanol (EtOH) at 10, 20, 30, 40, 40, 50, 60, 70, 80, 90 % and twice at 100 %. At each dehydration step, cells were gently resuspended for 1 min and centrifuged at 800g at 4 °C for 5 min. To the decanted samples, 100–200 μl of hexamethyldisilazane (HMDS) were added, and samples were then placed in 0.30-mm metal sample holders (1 cm² surface area), and left to dry for 12 h at room temperature. Samples were kept in a desiccator with silica gel until they were sent to the Servicio Académico de Microscopía Electrónica de Barrido (SAMEB) of the Instituto de Ciencias del Mar y Limnología (ICMyL), Universidad Nacional Autónoma de México (UNAM) in Mexico City. The samples were coated with Au in an ionizer (Ion Sputter JEOL-JFC-1100, Japan) for 5 min and examined in the JEOL JMS-6360-LV type SEM at a voltage of 10–20 kV and a working distance of 20 mm. The micrographs were edited with Photoshop CS6 Portable^® (v. 13.1.2, 2013). We followed unified terminology for morphological characters of the Prorocentrum species (Hoppenrath et al. 2013; Tillmann et al. 2019).

2.6 DNA extraction, amplification, and sequencing

DNA extraction was performed using the Quick-DNA™ Miniprep plus universal (Zymo Research, Irvine, CA, USA). A mixture of 4 µl of PCR Master mix 5X (Gene and Cell Technologies, USA), 14.8 µl of H₂O milli-Q, 2 µl of primers and 2 µl of DNA was used for amplification. For DNA amplification, 1:100 and 1:500-µl dilutions (DNA: H₂O milli-Q) were used. Published primers for the 28S and ITS regions of rDNA were used (Table 2). All PCR reactions were performed using an iCycler PCR System (Bio-Rad Laboratories, California, USA). The amplification program for the 28S region (gen D1–D2) of the rDNA consisted of a denaturation step at 95 °C for 5 min, followed by 35 cycles at 95 °C for 60 s, alignment temperature of 52 °C for 60 s, 72 °C for 120 s, and finally an extension at 72 °C for 7 min. The program for the ITS region consisted of a denaturation at 94 °C for 230 s, followed by 35 cycles at 94 °C for 50 s, alignment temperature of 47 °C for 60 s and 72 °C for 80 s, ending with an extension at 72 °C for 10 min. PCR products were purified using Quick-DNA^TM Miniprep kit (Zymo Research) and sent for sequencing to MCLAB (San Francisco, CA, USA).

2.7 Sequence alignment and phylogenetic analyses

Sequences were edited with Sequencher v. 4.1.4. BLAST analyses of the consensus sequences of this study were performed with sequences from the GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences from the corresponding regions (28S and ITS) were selected for the reconstruction of the phylogenetic trees, and sequences from Karenia brevis (C.C. Davis) Gert Hansen et Moestrup were chosen as an outgroup. Sequence alignment was performed with the program MEGA11 v. 10.0.5 (Tamura et al. 2021) using the multiple alignment algorithm. With JModel test v. 2.1.10 (Darriba et al. 2012), the models that best described the nucleotide substitution rates of the sequences were selected. Mega v. 10.0.5 was used for the maximum likelihood (ML) and maximum parsimony (MP) algorithms, and Mr. Bayes v. 3.2.7a (Huelsenbeck and Ronquist 2001) for Bayesian inference (BI). The construction of the trees was performed with a bootstrap of 1000 replicates for ML and MP, and 3 × 10⁶ generations for BI. The trees were edited with FigTree v. 1.4.4-2006.

2.8 Growth curve

The growth curve of the PNCETMAR-1 strain was determined in GSe (Blackburn et al. 2001) and f/2 media (Guillard and Ryther 1962) modified with the addition of vermicompost extract, pre-adapting the cells to each culture medium for at least three generations. For this, monocultures were grown in 250-ml Erlenmeyer flasks with an initial concentration of 500 cells ml⁻¹ in triplicate. Growth curves were determined by cell counts carried out every second day; for this, 2 ml of each replicate was taken and fixed with acid Lugol (Throndsen 1979). Counts were performed in a Sedgwick-Rafter chamber with a 1-ml capacity in an Axio Vert.A1 inverted microscope in brightfield. The identification of the growth phases (lag, exponential, stationary, and decay phases) were carried out following the criteria of Vonshak and Maske (1982). The growth rate (K) in the exponential phase was determined according to Guillard (1973), where C1 is the cell density (cells ml⁻¹) at the initial time (T1) of the exponential phase, and C2 is the cell density (cells ml⁻¹) at the final time (T2) of the exponential phase.

3.1 Morphological observations

Single cells, growing in clusters on the culture tube wall (Figure 2A and B), had one to three pusules comprising a quarter of the cells (Figure 2C–G). The cells are oval to oblong (Figures 2C–H, 4A and B and 5A–E). Generally, a circular pyrenoid is in the center of the cells (diameter of 3.28 ± 0.33 µm, n = 10); in a few cases, two pyrenoids per cell could be observed (Figures 2H–K and 4A–B). Chloroplasts were generally peripheral, with a coloration from green to yellow (Figure 4A–E). Cells with round green bodies, possibly oil droplets, were observed, although in some cases the latter showed autofluorescence, so they could be pigment accumulation bodies (Figures 2L–N and 4C–J). The nucleus is located posteriorly in the cells (Figure 4K–Q). The right thecal plate is convex (slight indentation in the center), while the left thecal plate is more flattened (Figures 4R and S and 5A–F). In the right lateral view, they show an asymmetry with respect to their sagittal axis (Figures 2C and D, 4R and S and 5A–E). Cells of strain PNCETMAR-1 measured 18.79 ± 1.25 µm long and 12.15 ± 1.01 µm deep (n = 30), while strain PNCETMAR-2 measured 18.47 ± 1.53 µm long and 11.83 ± 1.05 µm deep (n = 38). Cells did not show a developed apical spine (Figure 5A–F). The intercalary band (IB) that forms the junction of the lateral thecal plates is thin in non-dividing cells, while in cells showing possible division, the IB is broad, with a smooth, spaced horizontal and transverse striation (Figure 5G and H). In the mid-apical region, a tubular-shaped protuberance may be present, which is an extreme intercalary band along the periflagellar area with a smooth striation on its surface (Figure 5I and J). The periflagellar area has a “V” shaped form consisting of seven visible platelets (Figure 5I and J). The thecal plates do not show a regular pattern of pore distribution, and only one type of pore, although variable in size (Figure 5K–L) with a center mostly devoid of pores (Figure 5C–E), was observed, and a total of 108 fine pores (diameter of 0.10 ± 0.02 µm) were counted on the right thecal plate (Figure 5C–E).

Figure 2:

Cells of strain PNCETMAR-2 under a light microscope. (A and B) Cell clusters. (C–H, J, K) General view, showing periflagellar area, flagella, pyrenoid, pusules, chloroplasts, nucleus and concave margin (white arrow with margin). (I, L) Granulated cells in the process of division (red arrowhead). (L–N) Irregular planozygotes. Py, pyrenoid; Pa, periflagellar area; Pu, pusule; N, nucleus. Scale bars: 10 µm in (C–G, I and L–N); 5 µm in (H, J, K).

Figure 3:

Cells of strain PNCETMAR-2 under a light microscope. (A–D) Planozygotes initiating cell division; the red arrowhead indicates deformation at cell contact or junction, division planes are indicated by asterisks (*) and a concave margin (white arrow). (E–J) Gametes fused in the central valve zone of the lateral thecal platelets, finishing division in the periflagellar area. (K–L) Three- and four-cell stages (arrows indicate the number of planozygotes). LF, longitudinal flagellum; ab, accumulation bodies; Py, pyrenoid; Pu, pusule; tr, translucent rounded bodies; N, nucleus; Chl, chloroplasts. Scale bars: 10 µm in (A, D, E, F, J and L); 5 µm in (C, B, G, H, I and K).

Figure 4:

Micrographs of Prorocentrum cf. cassubicum: (A–C, E–G, I, K–Ñ) strain PNCETMAR-1; (D, H, J, O–S) strain PNCETMAR-2. (A–B, I, J, M and P) Cells under phase-contrast microscope; concave margin [broad white arrow in (B)]. (D–H, K, L, N–O and Q–S) Cells under epifluorescence microscope, stained with DAPI, showing the shape and position of the nucleus. (D–H) Epifluorescence images of chloroplasts. (F–H) Accumulation bodies with autofluorescence are shown. (R and S) Cells stained with Calcofluor White M2R; the thecal surface is shown. Pa, periflagellar area; LF, longitudinal flagellum; ab, accumulation bodies; Py, pyrenoid; Pu, pusule; N, nucleus; Chl, chloroplast. Scale bars: 10 µm in (A, M, N, F, P and Q); 5 µm in (C, D, G, H–L, Ñ, O, P–S).

Figure 5:

Prorocentrum cf. cassubicum cells in a scanning electron microscope. (A–B, F, G and L) strain PNCETMAR-2. (C–E and H–K) strain PNCETMAR-1. (G) Antapical view of a cell with a broad intercalary band (BI). (H) Cells of different sizes; black arrows indicate horizontal striation; yellow arrows show transverse striation. (I–J) Rounded cell with prolongation of the periflagellar area. (K–L) Detail of thecal surface. IB, intercalary band; RTP, right thecal plate; LTP, left thecal plate; as, posterior tip of the periflagellar area; SS, sagittal suture. Scale bars: 4 µm in (A–H); 1 µm in (I–L).

3.2 Reproductive stages

Different stages of cell division were documented in the PNCETMAR-2 strain. Deformed motile cells have a widened periflagellar zone; granular cells with colorless circular bodies generally marked the beginning of the division process (Figure 3A–D). The possible fusion of gametes starting from the intercalary band zone was observed, showing changes in morphology in the contact zone (Figure 3A). Other reproductive stages were documented in cells with preserved longitudinal flagella showing the division lines (Figure 3B–D). Possible asexual reproductive stages were documented in daughter cells, which separated from the posterior zone ending in the periflagellar area (Figure 3E–J). Dyads, triads and tetrads were also observed (Figure 3J–L). The pusule (one to three per cell) and the pyrenoid were observed in both motile and reproducing cells (generally immotile), the latter being larger.

3.3 Phylogenetic position of Prorocentrum cf. cassubicum

Phylogenetic analysis of the 28S rDNA region, using the Kimura 2-parameter model, grouped the sequence of strain of PNCETMAR-2 into a well-supported clade (bootstrap = 100) with the sequence strain of PNCETMAR-1 (Figure 6). These two sequences showed a degree of genetic divergence of 0.02. Both sequences in this study diverged from the five sequences identified as P. cf. norrisianum from the study of Chomérat et al. (2019), with a genetic distance of 0.22 with respect to the sequences in this study, that were also separated from the P. lima sequences with a genetic distance of 0.29. In contrast, employing the Kimura 2-parameter model, the PNCETMAR-2 (PQ182182) strain sequence of the ITS region clustered with those of P. cassubicum (EU927557) and Prorocentrum sp. (FJ160593) with a 100 % bootstrap support, and genetic distances of 0.030 and 0.032, respectively (Figure 7).

Figure 6:

Phylogenetic tree of Prorocentrum from the 28S rDNA region. The phylogenetic tree was obtained using the maximum parsimony (MP), maximum likelihood (MV) and Bayesian inference (BI) methods. The nodes show the bootstrap percentage and posterior probability (MP/MV/BI). The analysis included 19 sequences; dataset based on 660 bp.

Figure 7:

Phylogenetic tree of Prorocentrum from the ITS region of rDNA. The phylogenetic tree was developed using the maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) methods. The percentage of bootstrap and posterior probability (MP/ML/BI) are shown at the nodes. The analysis included 19 sequences; dataset based on 524 bp. ND, no data.

3.4 Growth curve

The growth curves of strain PNCETMAR-1 showed no significant differences when grown in GSe and f/2 media. In both culture media the adaptation phase lasted 10 days (Figure 8). The exponential phase in f/2 medium ended on day 34 and in GSe medium on day 36, reaching a maximum biomass of 74,312 ± 2,008 cells ml⁻¹ and 69,766 ± 2,154 cells ml⁻¹, respectively (Figure 8). The growth rate in f/2 medium was 0.428 ± 0.002 divisions per day and with GSe medium was 0.464 ± 0.006 divisions per day. This strain is characterized by individual free-swimming, highly motile vegetative cells during the adaptive and exponential phase; they decrease in motility and form clumps during the late exponential phase.

Figure 8:

Growth curve of the PNCETMAR-1 strain in GSe () and f/2 () medium at 24 °C, salinity of 34 and a 12 h light: 12 h dark cycle. Error bars indicate standard deviations (n = 3).

4.1 Morphology

Prorocentrum norrisianum is from 14 to 25 µm long and from 10 to 16 µm wide (Faust 1997; Hoppenrath et al. 2023; Mohammad-Noor et al. 2007). Strains identified as Prorocentrum cf. norrisianum have larger cells, ranging from 20 to 37 µm long and 20–26 µm wide (Chomérat et al. 2019). One of the characteristics previously considered to differentiate P. norrisianum from P. cassubicum is its size; the cells of the former are smaller (Table 3). However, there is a discrepancy between the reported sizes because there is an overlap in the cell size ranges reported to date (Table 3). Another species often confused with P. norrisianum and P. cassubicum is P. lima; however, the latter is larger, with a length from 30 to 57 µm and depth from 21 to 46 µm (Hoppenrath et al. 2023; Nava-Ruiz and Valadez-Cruz 2012).

Another trait that has also been proposed to distinguish between these two species is the shape of the periflagellar platelets: P. cassubicum has curved platelets, while the apical platelets of P. norrisianum are rectangular, vertical or curved (Faust 1997). In summary, due to phenotypic plasticity, the descriptions used for P. norrisianum and P. cassubicum could apply to both species. For example, P. norrisianum is described as an oval species with a length between 20 and 25 µm, with a central pyrenoid (Al-Has and Mohammad-Noor 2011); both features are also reported for P. cassubicum (Dodge and Bibby 1973). Also, cells of P. norrisianum are described as compressed, with the lateral thecal plates ornamented with fine pores and a triangular or V-shaped periflagellar area (Al-Has and Mohammad-Noor 2011); these characteristics are also described in P. cassubicum (Nava-Ruiz and Valadez-Cruz 2012). Therefore, it is recommended to perform an exhaustive analysis in strains that are identified both morphologically and molecularly, in which there are non-contradictory morphometric differences that will help to establish characters to differentiate these two species in future studies.

In Bahía de La Paz, Villa-Arce (2021) reported P. cassubicum with a description of the morphological characteristics and a sequence of the 28S rDNA region of strain PNCETMAR-1. The only difference between the strain of this study (PNCETMAR-2) and strain PNCETMAR-1 was its size (Table 3). Therefore, the biological material used by Villa-Arce (2021) was requested for review, and micrographs were taken to measure the cells, as well as epifluorescence micrographs to observe other characteristics, such as the position of the nucleus and the pigment distribution. This strain presented smaller sizes than those originally reported by the author. Therefore, this study complements the information on both strains to determine if they are distinct species or, given the limited information, to report them as P. cf. cassubicum until more precise, non-contradictory and detailed information on both species is available.

The strains included in this study were isolated from Ensenada de La Paz, a coastal lagoon in the southwest of the Gulf of California. Samples were collected in the water column in a shallow area between 1 and 2 m. These conditions make it difficult to determine whether the isolated cells are benthic or planktonic in nature, since they could have been resuspended in the water column by winds. According to existing reports, P. cassubicum appears to be planktonic, while P. norrisianum is more associated with a benthic habitat (Table 3). Also, precedents indicate that P. cassubicum has been observed in brackish-water environments with a salinity of <10 (Nava-Ruiz and Valadez-Cruz 2012; Wołoszyńska 1928), while P. norrisianum has been recorded in areas with salinity higher than 30 (Faust 1997; Table 3). Originally, P. cassubicum was described from brackish water in Gdansk Bay, the southern Baltic Sea, on the Polish coast. Wołoszyńska (1928) did not indicate the salinity of the sampled water; however, we infer a range between 2 and 12, based on recent studies, although they referred to the period of 1950–2015 in a shallow coastal zone between Hel and Władisławowo (Świątek 2019). Therefore, the difference in the salinity in the two habitats (Gdansk Bay and the southern Gulf of California) is significant and could be considered an argument against the conspecificity of the cells inhabiting the two regions in temperate and subtropical zones, respectively. However, the available information is still limited because not all reports include information on the environment from which they are isolated, and this prevents us from establishing definitive criteria for their ecology and distribution patterns.

In culture, cells with asexual processes and colorless translucent rounded bodies were observed, indicating the initiation of cell division. Cells in pairs, in which two longitudinal flagella were observed, were probably evidence of a sexual process (indicative of gamete union); furthermore, these cells were deformed in the union zone. Deformed gametes in the contact zones have also been reported in Prorocentrum cordatum (Ostenfeld) J.D. Dodge (syn.: Prorocentrum minimum (Pavillard) J. Schiller) (Berdieva et al. 2018).

4.2 Molecular phylogeny

Phylogenetic reconstruction with sequences from the 28S rDNA region grouped the sequence of PNCETMAR-1 and PNCETMAR-2 within the same clade with 100 % bootstrap support. Phylogenetic analyses indicate that both strains belong to the same species. Based on the above, it was possible to amplify the ITS region for strain PNCETMAR-1. The phylogenetic reconstruction of this region showed a clade including this sequence with the sequences of P. cassubicum (EU927557) and Prorocentrum sp. (FJ160593) with bootstrap values of 100 %. However, it is crucial to highlight the lack of sequences of P. norrisianum from the ITS region in the GenBank database for comparison. Although there are seven sequences identified as P. cf. norrisianum for the 28S rDNA region, there are only two sequences of P. cassubicum for the same rDNA region; however, the latter sequences are very short and are not included in this analysis. It is important to mention that the seven sequences of P. cf. norrisianum analyzed in this study come from the strains with cell sizes that agree with the original description for P. cassubicum, showing a slightly larger size than P. norrisianum, and with the sizes reported in this study for P. cf. cassubicum. There are no sequences of P. norrisianum and P. cf. norrisianum for the ITS region for comparison. In addition, the sequence of P. cassubicum (EU927557), used for the phylogenetic reconstruction of the ITS region does not come from published material. The lack of verified sequences may cause a bias in the phylogenetic relationships that have been determined so far; therefore, they should be treated with caution. In addition, the lack of sequence information on P. cassubicum from the area of its first record (Baltic Sea) makes it difficult to determine whether it is one or two distinct species.

Current morphometric information is contradictory for both species, even with the few studies that have been carried out that complement morphological analysis with molecular analysis. It is likely that the limited information in the descriptions or reports of these two species is due to the fact that their precise identification cannot be achieved under light microscopy after sampling, due to their small cell size. This study reports for the first time the presence of P. cf. cassubicum in the region of the Ensenada de La Paz and the Gulf of California.

4.3 Growth curve

Prorocentrum cf. cassubicum grows slowly under the culture conditions tested, taking more than 30 days to reach its maximum biomass. No significant differences in maximum biomass (69,722–74,312 cells ml⁻¹), nor in growth rate (0.43–0.46 divisions day⁻¹) were observed when cultivating strain PCCETMAR-1 in f/2 and GSe media at 24 °C. No growth data are available for other strains of P. cassubicum and P. norrisianum. In general, epibenthic dinoflagellate species exhibit slow growth. Because of its toxicity, one of the most studied species is the P. lima complex (Grigoriyan et al. 2024). Numerous experiments have been carried out in the laboratory with various strains under different conditions where it has been determined that temperature is the main factor affecting their growth, registering growth rates between 0.30 and 0.75 divisions day⁻¹ and maximum biomasses of 600 to 70,000 cells ml⁻¹ (Grigoriyan et al. 2024). Several species of Prorocentrum meet the characteristics of being halotolerant and euryhaline (Glibert 2020). More knowledge of epibenthic species is needed to understand their physiology, life cycles, nutrition, and the environmental factors that regulate their growth.