*Article* **Detection of the Benthic Dinoflagellates,** *Ostreopsis* **cf.** *ovata* **and** *Amphidinium massartii* **(Dinophyceae), Using Loop-Mediated Isothermal Amplification**

**Eun Sun Lee, Jinik Hwang, Jun-Ho Hyung and Jaeyeon Park \***

Environment and Resource Convergence Center, Advanced Institute of Convergence Technology, Suwon 16229, Korea; eunsun742@snu.ac.kr (E.S.L.); jinike12@snu.ac.kr (J.H.); hjh1120@snu.ac.kr (J.-H.H.) **\*** Correspondence: bada0@snu.ac.kr; Tel.: +82-31-888-9042

**Abstract:** For the in situ and sensitive detection of benthic dinoflagellates, we have established an integrated loop-mediated isothermal amplification (LAMP) assay based on *Ostreopsis* cf. *ovata* and *Amphidinium massartii*. To detect the two species, a set of species-specific primers was constructed between the ITS gene and D1–D6 LSU gene, and the reaction temperature, time, and buffer composition were optimized to establish this method. In addition, the specificity of the LAMP primers was verified both in strains established in the laboratory and in field samples collected from the Jeju coastal waters, Korea. With the LAMP assay, the analysing time was within 45 to 60 min, which may be shorter than that with the conventional PCR. The detection sensitivity of the LAMP assay for *O*. cf. *ovata* or *A. massartii* was comparable to other molecular assays (PCR and quantitative PCR (qPCR)) and microscopy examination. The detection limit of LAMP was 0.1 cell of *O*. cf. *ovata* and 1 cell of *A. massartii*. The optimized LAMP assay was successfully applied to detect *O.* cf. *ovata* and *A. massartii* in field samples. Thus, this study provides an effective method for detecting target benthic dinoflagellate species, and could be further implemented to monitor phytoplankton in field surveys as an altenative.

**Keywords:** harmful algae; molecular detection; monitoring; Jeju coastal waters

#### **1. Introduction**

The genus *Ostreopsis* Johannes Schmidt and most of the species of *Amphidinium* Claparède & J. Lachmann are benthic dinoflagellates that grow on macrophytes or are attached to sand or coral rubble [1–3]. Their occurrence has been generally reported in tropical and subtropical seas [4,5]. The global occurrence of some species in these genera has significantly increased over the last decade and is expected to expand to temperate regions. The main harmful effects of some species of benthic dinoflagellates are related to the fact that they not only affect marine life and the aquaculture industry, but also pose a threat to human health [6–10]. *Ostreopsis* species have a particularly rich mucilaginous matrix, and some of them have thus far been reported to produce several toxins [1,11–15]. Moreover, *Ostreopsis* species are potentially toxic and can affect marine organisms and humans through the food web [16]. Their toxins can cause severe irritation to human skin and respiratory problems through aerosolization. They can also cause vomiting, kidney problems, and even death in severe cases [17,18].

Over the past several decades, blooms of benthic dinoflagellates have been observed in temperate to tropical coastal waters, in both the southern and northern hemispheres, whereas the proliferation of *Ostreopsis* cf. *ovata* was found in temperate regions during the summer [5]. The expansion of toxin-producing *Ostreopsis* spp. to temperate regions can potentially occur due to ballast water discharge by cargo ships, and, mainly due to the marginal dispersal associated with global warming, can induce bloom formations [19].

The cosmopolitan dinoflagellate genus *Amphidinium* has been found in pelagic and mainly in benthic environments with frequent occurrence [20–23]. Some *Amphidinium*

**Citation:** Lee, E.S.; Hwang, J.; Hyung, J.-H.; Park, J. Detection of the Benthic Dinoflagellates, *Ostreopsis* cf. *ovata* and *Amphidinium massartii* (Dinophyceae), Using Loop-Mediated Isothermal Amplification. *J. Mar. Sci. Eng.* **2021**, *9*, 885. https://doi.org/ 10.3390/jmse9080885

Academic Editor: Feng Zhou

Received: 12 June 2021 Accepted: 13 August 2021 Published: 17 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

species, particularly *A. caterae*, have been known to produce a number of bioactive compounds with cytotoxic or hemolytic activity to marine organisms [24–28]. Moreover, the cytotoxicity of *A.* cf. *massartii* affecting *Artemia salina* mortality has been revealed [29].

Several benthic dinoflagellate species, including *Ostreopsis*, have been reported as potential causative agents of toxic poisoning in Korean coastal waters [30–35]. Recently, a rapid increase in the *O*. cf. *ovata* biomass in Jeju coastal waters has been reported [36]. Most of these reports on the occurrence of benthic dinoflagellates in Korean coastal waters were based on conventional microscopic analysis methods [37–40]. Because blooms in coastal and oceanic waters with negative impacts on environmental health have the possibility of occurrence, several toxigenic dinoflagellates belonging to the genera *Amphidinium*, *Coolia*, *Gambierdiscus*, and *Ostreopsis* (including *O*. cf. *ovata* and *A.* cf. *massartii*) have been seriously considered as harmful organism candidates in Korea [41].

Following the development of molecular techniques, a number of modifications of polymerase chain reaction (PCR) methods have been established [42,43]. However, whereas these PCR-based assays have provided a reliable, sensitive, and specific tool to detect potentially harmful dinoflagellates, their economic limitations such as dependence on an expensive apparatus, practical limitations such as low amplification efficiencies, and long reaction times ultimately restrict their widespread application. Therefore, the development of rapid, simple, and cost-effective detection methods is still necessary to effectively detect harmful dinoflagellates.

Since Loop-mediated Isothermal Amplification (LAMP) was first introduced by Notomi et al. in 2000 [44], more than 100 LAMP detection methods have been developed for animal pathogens, including humans, and are ideally used due to the advantages of low cost and high sensitivity [45–47]. Because LAMP is performed under isothermal conditions, this method facilitates the amplification of only a few copies of initial DNA to obtain approximately 10<sup>9</sup> copies in less than 1 h, which can be visualized after the reaction using SYBR Green dye. The development of a LAMP assay, therefore, enhances the detection of various dinoflagellate species [48,49].

Owing to the recent increase in the abundance of benthic dinoflagellates in Korean coastal waters, their rapid detection and extensive monitoring are required. In this study, we developed a highly specific LAMP assay for the sensitive detection of two species of benthic dinoflagellates, *O.* cf. *ovata* and *A. massartii*. Moreover, we established the proper LAMP conditions for each species and applied them to the field samples to verify the sensitivity.

#### **2. Materials and Methods**

#### *2.1. Sampling Site and Establishment of Dinoflagellate Strains*

Macrophytes samples were collected by scuba divers in May 2018 within a water depth of 10 m at Seongsan, Jeju Island (33◦27.35- N, 126◦56.01- E). The seawater temperature and salinity were recorded as 17.4 ◦C and 33.2, respectively, at that time. The collected macrophytes were transferred into a 1 L bottle, which was filled with filtered seawater, and then shaken vigorously to detach the attached dinoflagellates. The samples were filtered by a 100 μm mesh to separate the macrophyte from seawater, and then transferred to the laboratory.

To establish a single cell strain, 5 mL of the sample was placed in a six-well plate, and single-cell isolation was performed under a dissecting microscope (SZX10, Olympus, Tokyo, Japan). After the clonal cultures of *O.* cf. *ovata* and *A. massartii* were established, the strains were transferred into 30 mL flasks containing fresh f/2-Si medium. The cultures were placed under white fluorescent lights at 22 ◦C with a continuous illumination of <sup>20</sup> <sup>μ</sup>E·m−2·s<sup>−</sup>1.

#### *2.2. DNA Extraction and Species Identification*

The DNA sequences of these cells were analysed when the concentration of each strain was more than 103 cells mL−1. The dense culture (10 mL) was centrifuged at 10,000× *<sup>g</sup>* for 3 min at room temperature and the pellet was used for DNA extraction. Genomic DNA was extracted using an AccuPrep Genomic DNA Extraction Kit (BIONEER, Daejeon, Korea). The quality and purity of the DNA were assessed using agarose gel electrophoresis and spectrophotometry. For species identification, the SSU, ITS1, 5.8S, ITS2, and LSU rDNA sequences were amplified using universal eukaryotic primers [50,51]; the obtained DNA sequences were confirmed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, NCBI (accessed on 3 September 2018). The rDNA sequences of the two species were identical to those of the Korean strain (*Ostreopsis* cf. *ovata* [52]; *Amphidinium massartii* [53]).

#### *2.3. Construction of LAMP Primers*

Primers for LAMP assay were designed based on the internal transcribed spacer region (ITS gene) of *O.* cf. *ovata* (HE793379.2) and the D1–D6 region of large subunit rRNA (LSU gene) of *A. massartii* (AY455670.1) using Primer Explorer V5 (http://primerexplorer. jp/lampv5e/index.html (accessed on 17 September 2018) software. A total of six distinct sequences (B1, B2, B3, F1, F2, and F3) in the target DNA were designed for LAMP assay (B3, F3, backward inner primer (BIP), and forward inner primer (FIP)). Primer details are listed in Table 1. The primer sequences and their respective binding sites are shown in Figure 1. Although LAMP reaction could be accelated using additional primers (termed loop primers), no suitable loop primer was found within the target gene. The LAMP reactions of the designed primer sets were confirmed as typical ladder-like patterns by gel electrophoresis as well as direct color change by SYBR Green dye [44].

**Table 1.** Oligonucleotide primers developed for detecting *Ostreopsis* cf. *ovata* and *Amphidinium massartii* using loop-mediated isothermal amplification (LAMP), PCR, and qPCR.


**Figure 1.** Nucleotide sequence used to design the primers for loop-mediated isothermal amplification (LAMP). The square boxes indicate the recognition sequences of the primers. The right arrow indicates that a sense sequence was used for the primers. The left arrow indicates that a complementary sequence was used for the primer. (**a**) Primer sequences for *Ostreopsis* cf. *ovata.* (**b**) Primer sequences for *Amphidinium massartii*. (**c**) Schematic representation of the primers used in this study. Construction of two outer (F3 and B3) and two inner (forward inner primer (FIP) and backward inner primer (BIP)) primers for loop-mediated isothermal amplification (LAMP).

#### *2.4. Optimization of LAMP Reaction Conditions*

To determine the optimal conditions of LAMP assay for detecting *O*. cf. *ovata* and *A. massartii*, experiments were performed in which the different variables known to affect this assay, such as reaction times, temperatures, and buffer composition concentrations (dNTP, *Bst* polymerase, and MgSO4), were altered (Table 2). The reaction conditions and buffer compositions were varied to include ranges of 0–16 units of *Bst* DNA polymerase, 0–18 mM of MgSO4, 0–4.5 mM of dNTP each, 54–64 ◦C of temperatures, and 15–90 min of reaction time. After that, the LAMP reaction mixture contained 20 mM tris-HCl, 50 mM KCl, 10 mM (NH4)2SO4, 1.0 μM of each FIP and BIP, 0.4 μM of each of the outer F3 and B3

primers, and 4 μL of genomic DNA extracted from 100 cells using syringe filter set, which resulted in a final reaction volume of 20 μL.

**Table 2.** Conditions and tested ranges of each factor for optimizing LAMP.


The detectable ranges for the species (*O*. cf. *ovata* or *A. massartii*) included the concentraions of compositon in the commercial LAMP mix (2× LAMP Maeter mix, NEB E1700L): 8 U *Bst* polymerase, 1.4 mM dNTPs, and 8 mM MgSO4. Therefore, the following tests (sensitivity, specific-specificity, and field samples application) were performed with the commercial LAMP mix, at 62 ◦C in *O*. cf. *ovata* and 60 ◦C *A. massartii* for 60 min. The reaction temperature was determined to the median between the minimum reaction temperature and the manufacturer's recommended temperature.

By adding 1 μL of fluorescent dye SYBR Green I (×1000, Thermo Fisher Scientific, Waltham, MA, USA) after completing the LAMP reaction, the positive reactions could be confirmed with the naked eye. The colour of positive reactions turned orange to yellow-green, while negative reactions remained orange. Moreover, by performing the gel electrophoresis, the LAMP products could be double-checked. The positive sample of LAMP showed a band in the ladder pattern, while the negative sample had no band in the gel. The photographs of the agarose gel were taken using an Azure c200 gel imaging workstation (Azure Biosystems, Dublin, CA, USA).

#### *2.5. Sensitivity of LAMP*

#### 2.5.1. Sensitivity Test from Extracted DNA

The sensitivity tests were evaluated using DNA templates extracted from the clonal cultures of *<sup>O</sup>*. cf. *ovata* or *A. massartii* each (3 × 103 cells of *<sup>O</sup>*. cf. *ovata* and 10<sup>4</sup> cells of *A. massartii*). The cells were concentrated with GF/C filters (Whatman® glass microfiber filters, Little Chalfont, UK) using a syringe filter, and then the genomic DNAs were extracted using an AccuPrep Genomic DNA Extraction Kit (BIONEER, Daejeon, Korea). The extracted DNA was diluted serially.

#### 2.5.2. Sensitivity Test from 10 Cells Directly

To evaluate the detection limit more precisely, we used the cells isolated from the clonal cultures as the DNA templates directly without the column-based DNA extraction processing. Ten cells were isolated each from the culture of *O*. cf. *ovata* or *A. massartii* using a sterile micropipette under a dissecting microscope (SZX10, Olympus, Tokyo, Japan). Individual cells were transferred to sterile seawater to remove any contaminants and then suspended in approximately 1 μL of TE buffer. The ten cells were lysed by freeze-thawing process and diluted ten-fold serially, by adding sterile distilled water (Invitrogen, Carlsbad, CA, USA), from 1 to 10<sup>−</sup>4. All samples were frozen at −20 °C in preparation for the test.

#### 2.5.3. Comparison of the Detection Sensitivity with Other Molecular Assays

We carried out the conventional PCR and qPCR (quantitative PCR) for comparison with LAMP. The PCR was performed on a Mastercycler Nexus (Eppendorf, Hamburg, Germany) in a 20 μL PCR mixture containing 4 μL of HiPi PCR premix, 1 μL of 10 μM each forward/reverse primers, 10 μL of sterile distilled water, and 4 μL of DNA template. PCR conditions of *O.* cf. *ovata* and *A. massartii* were as follows: an initial denaturation step of 95 ◦C for 15 min, followed by 35 cycles of denaturation at 94 ◦C for 30 s, and then

an annealing step at 55 ◦C for 30 s, and elongation at 72 ◦C for 50 s, followed by a final extension step 72 ◦C for 7 min. The PCR products were analysed on a 1.5% agarose gel using gel electrophoresis.

Quantitative PCR (qPCR) was performed in duplicates with a PCR max Eco 48 real-time PCR system (PCR max, Stone, UK) using qPCRBIO probe Mix No-ROX (PCR biosystms, London, England) following the manufacturer's guidelines: 10 μL of 2× qPCRBIO Probe Mix, 1 μL of 10 μM each of forward/reverse primers, 0.5 μL of 10 μM probe labelled at the 5 and 3 ends with the fluorescent dye 6-FAM and BHQ-1, 4.5 μL of distilled water, and 3 μL of DNA template. The primers and TaqMan probes (coupled to for qPCR were also designed to amplify the ITS gene (Table 3). Thermal cycling was performed under the following conditions: 95 ◦C for 3 min of initial denaturation, then 40 cycles of amplification at 95 ◦C for 10 s and 60 ◦C for 30 s. The standard templates (3 × 103 cells of *<sup>O</sup>*. cf. *ovata*) were concentrated with GF/C filters using a syringe filter. Following DNA extraction and qPCR methods were as described above. The eluted genomic DNA was diluted to the equivalent of 1000, 300, 100, 30, and 10 cells. In the application of the field samples, the relatively quantified cell concentrations were calculated according to Park et al. [36].

**Table 3.** List of dinoflagellate species used to confirm the species specificity of the two sets of LAMP primers (*O*. cf. *ovata* and *A. massartii*).


#### *2.6. Confirmation of Species-Specific LAMP Primers*

To confirm species specificity and exclude the possibility of false positives about the two sets of LAMP primers (*O*. cf. *ovata* and *A. massartii*), DNAs of eleven dinoflagellate species (*Alexandrium tamarense* (Lebor) Balech; *Amphidinium carterae* Hulbert; *Coolia malayensis* Leaw, P.-T. Lim & Usup; *Gambierdiscus jejuensis* S. H. Jang & H. J. Jeong; *Gymnodinium aureolum* (Hulburt) G. Hansen; *Heterocapsa steinii* Tillmann, Gottschling, Hoppenrath, Kusber & Elbrächter; *Ostreopsis lenticularis* Y. Fukuyo; *Prorocentrum minimum* (Pavillard) Schiller; *Prorocentrum koreanum* M.-S. Han, S. Y. Cho & P. Wang; *Scrippsiella acuminata* (Ehrenberg) Kretschmann, Elbrächter, Zinssmeister, S. Soehner, Kirsch, Kusber & Gottschling); and *Symbiodinium voratum* Jeong, Lee, Kang, LaJeunesse) were extracted and used as a template for the LAMP reaction (Table 3). All strains, mentioned above, have been established and maintained in our cell culture laboratory (at 22 ◦C under continuos illumination of <sup>20</sup> <sup>μ</sup>E·m−2·s<sup>−</sup>1) of the Advanced Institute of Convergence Technology (AICT, Suwon, Korea), excluding *Amphidinium carterae* strain (CCMP1314). Two thousand cells from each species were isolated from the dense culture media of the dinoflagellate strains and mixed in a tube. The mixture was concentrated with GF/C filters using a syringe filter. Then, the genomic DNA was extracted using an AccuPrep Genomic DNA extraction kit (Bioneer, Daejeon, Korea). The positive mixture including either *O*. cf. *ovata* or *A. massartii* and the negative mixture without *O*. cf. *ovata* and *A. massartii* were used as the controls. The LAMP reaction was conducted at 62 ◦C in *O*. cf. *ovata* and 60 ◦C in *A. massartii* for 60 min.

All DNA templates tested in this work were from fresh samples only that had not been stored for more than two days. The amplification results were obtained following electrophoresis, and gels were stained to verify specificity.

#### *2.7. Testing of Field Samples*

To test the *O*. cf. *ovata* LAMP primers using field samples, four different macroalgal species were randomly collected from two sampling sites on Jeju Island (Sasu and Seongsan) in 2019 (January, March, and June) (Figure 2). Macroalgae living within 10 m of water depth were collected by scuba divers and pooled in a 1 L bottle with ambient seawater. The bottle was shaken vigorously to detach the benthic dinoflagellates. After that, macroalgae were frozen on dry ice immediately and the water sample was filtered using a 100 μm mesh to remove macroalgal particles and zooplankton. Each water sample (50 mL) was concentrated with a GF/C filter using a syringe filter. The GF/C filter was placed in a 1.5 mL tube and kept frozen immediately until DNA extraction. Genomic DNAs were extracted with a Bioneer AccuPrep® Genomic DNA Extraction kit, and a LAMP assay was performed. The LAMP reaction was conducted at 62 ◦C for 60 min.

**Figure 2.** Map of Korea and Jeju Island indicating the sampling sites.

To compare microscopic detection, 300 mL of water sample was fixed with formalin (final concentration, 1%). The fixed samples were concentrated for microscopic observation. Fixed samples were left overnight to allow the cells to sink, and the supernatant was removed to adjust the final volume to 50 mL. A solution of Calcofluor (Sigma-Aldrich, St. Louis, MO, USA) was added at a final concentration of 10 μg mL−<sup>1</sup> for 1–2 min in the dark before observation. Then, 1 mL samples were observed in a Sedgewick-Rafter chamber (SPI Supplies, West Chester, PA, USA) at 100× magnification using an epifluorescence microscope (BX 53, Olympus, Tokyo, Japan) to confirm the presence of cells. For the same reason, dense plankton samples were collected at each sampling sites by towing a 20 μm mesh sized plankton net along the water column for 1 min. Collected samples were fixed with formalin and observed with light and epifluorescence microscopes (BX 53, Olympus, Tokyo, Japan).

#### **3. Results**

#### *3.1. Optimization of LAMP Conditions*

LAMP reaction in *O*. cf. *ovata* required at least 45 min and occurred at a temperature of 56 ◦C or higher. We confirmed that LAMP reactions occurred even at two units of *Bst* polymerase per reaction tube. To find an optimal dNTP concentration, each 0–4.5 mM of dNTPs were tested in 20 μL of the reaction mixture. LAMP reaction occurred in 1.0–2.5 mM dNTPs, but it did not occur in 0.5 and 4.5 mM dNTPs. In the concentration test of MgSO4 per reaction tube, LAMP reaction occurred from 6 to 16 mM of MgSO4 (Figure 3a). LAMP reaction in *A. massartii* showed that the reaction was initiated at least after 30 min and at all temperatures from 54 to 64 ◦C. The required concentration of dNTP per reaction tube was from 1.0 (very weak) to 2.5 mM. Compared to what was confirmed in *O*. cf. *ovata* LAMP reactions, LAMP reactions in *A. massartii* were in a narrow range of MgSO4 concentration, from 8 to 14 uM per reaction tube (Figure 3b). Too low or too high dNTP and MgSO4 concentrations resulted in false-negative results in both *O*. cf. *ovata* and *A. massartii*. These composition (*Bst* polymerase, dNTPs, MgSO4) concentration tests were performed at 62 ◦C in *O*. cf. *ovata* and 60 ◦C *A. massartii* for 60 min.

#### *3.2. Comparison of the Sensitivity of LAMP and Other Molecular Assays* 3.2.1. *Ostreopsis*. cf. *ovata*

When tested with extracted DNA from 3 × 103 cells concentrated with GF/C filter, the detection limit of LAMP was 10 cells (Figure 4a). Meanwhile, when we tested using lysed DNA from 10 cells isolated directly, sensitivity was increased as 0.1 cell of *O*. cf. *ovata* (Figure 4b). The sensitivity was higher when performing LAMP with isolated cells than with extracted DNA. The sensitivity of qPCR was similar to the LAMP result while the PCR was ten-fold more sensitive than the LAMP assay (Figure 4a). The qPCR was only performed for *O*. cf. *ovata* as suitable primers and probes, but could not be designed for *A. massarti*.


**Figure 4.** Comparison of the detection sensitivity with loop-mediated isothermal amplification (LAMP) and conventional PCR. (**a**) Samples in each line were serially diluted (from 3 <sup>×</sup> <sup>10</sup><sup>3</sup> cells in *<sup>O</sup>*. cf. *ovata* and 104 cells in *A. massartii*) from genomic DNA which was extracted from the sample concentrated with GF/C filter. (**b**) Samples in each line were serially diluted from directly isolated ten cells without the column-based DNA extraction processing. cq = quantification cycle.

#### 3.2.2. Amphidinium Massartii

The detecton limit of *A. massartii* was 1 cell in both DNA extracted from 104 cells concentrated with GF/C filter (Figure 4a) and DNA lysed from 10 cells isolated directly (Figure 4b). The PCR detection limit of *A. massartii* was similar to that of the LAMP results.

#### *3.3. Confirmation of Species-Specific LAMP Primers*

By performing LAMP reactions on the DNA mixture containing eleven strains of dinoflagellates (*A. tamarense*, *A. carterae*, *C. malayensis*, *G. jejuensis*, *G. aureolum*, *H. steinii*, *O. lenticularis*, *P. minimum*, *P. koreanum*, *S. acuminata*, and *S. voratum*), we confirmed that the positive LAMP reactions were only observed for target species and positive control mixtures containing either *O*. cf. *ovata* or *A. massartii*, while no products were amplified in both negative control and negative control mixture (Figure 5). Therefore, the results showed that the designed LAMP primers for the two species react specifically.

**Figure 5.** Species-specificity of loop-mediated isothermal amplification (LAMP) primers. LAMP reactions were performed by visual inspection with diluted SYBR Green I and electrophoresis with EtBr. (**a**) *Ostreopsis* cf. *ovata.* (**b**) *Amphidinium massartii*.

#### *3.4. Application to Field Samples*

The LAMP assay for *O*. cf. *ovata* was applied using the field samples collected from Jeju, Korea. PCR and qPCR were also performed to confirm the accuracy and the reliability of LAMP results. The result of LAMP showed that *O.* cf. *ovata* was detected in both Sasu and Seongsan in January and April, but detected only in Seongsan in June. Specifically, the positive LAMP results of *O*. cf. *ovata* were obtained only in samples estimated to be more than 10 cells mL−<sup>1</sup> in qPCR assay, while PCR results showed that they were amplified even at less than 10 cells mL<sup>−</sup>1. In the microscopy examination results, *O*. cf. *ovata* was not observed in some of the samples (Seongsan no.4 in Jan. and no.1, 2 in Jun.) in which it was confirmed by all molecular assays, including LAMP (Figure 6).


**Figure 6.** Field sample detection using loop-mediated isothermal amplification (LAMP) primers. Detection of each species was confirmed using a microscope, LAMP, PCR, and qPCR in seawater samples from Sasu and Seongsan of Jeju Island, 2019. The numbers (1 to 4) in the table indicate the randomly collected macrophytes (unidentified).

#### **4. Discussion**

Traditional PCR and qPCR assays using *Taq* polymerase have already been used to detect or quantitatively evaluate many dinoflagellates with high sensitivity and specificity. However, these assays require special and expensive devices, such as a thermal cycler. LAMP assay using *Bst* polymerase is a simple and fast molecular diagnostic tool that does not require an expensive thermocycler because *Bst* polymerase amplifies nucleic acids under isothermal conditions [54]. Until now, attempts have been made to apply LAMP to some dinoflagellates related to harmful algal blooms, but no studies have been conducted on the early detection of benthic dinoflagellates. Novel and highly specific LAMP assays for the sensitive detection of benthic dinoflagellates *O.* cf. *ovata* and *A. massartii* were established in this study. Moreover, another purpose of this work was to evaluate the detection capabilities of LAMP compared to other molecular assays. The LAMP assay was sensitive enough to detect for *O.* cf. *ovata* and *A. massartii*, similar to both PCR and qPCR results.

The LAMP assay provides several advantages over a conventional PCR assay in that it can amplify target DNA sequence under isothermal conditions faster (≤1 h) and does not require the use of sophisticated or expensive equipment. Thus, in laboratories or isolated areas where equipment is minimal, the entire reaction process can still be performed using only a heat block or temperature-controlled water tank. However, the most substantial feature is the ability to visually detect amplification through the addition of fluorescent dyes such as SYBR Green I [55], making it suitable for implementation in rapid field trials.

Recently, LAMP has been applied in the detection of marine dinoflagellates such as *Alexandrium* sp., *Karenia mikimotoi*, and *Prorocentrum* sp., and many studies have been conducted for the rapid detection of harmful dinoflagellates [56–58]. To date, most LAMP methods for detecting dinoflagellates have been limited to planktonic dinoflagellates. In the present study, we successfully detected benthic dinoflagellates and confirmed that the detection threshold of LAMP was similar to that of PCR and qPCR assays. When the LAMP method was used, we completed the analysis and obtained the results in situ with high accuracy within 60 min to detect the occurrence of *O.* cf. *ovata* and *A. massartii*. Generally, LAMP positive products could be confirmed in the ladder pattern band through the gel electrophoresis. However, we found that the samples kept frozen for a long time (more than a month) or repeated freezing-thawing several times caused false-positive results with a smear band rather than a ladder pattern. This is why we only used the fresh samples to avoid false-positive results.

In the field samples detached from macrophytes, we observed not only *O*. cf. *ovata* and *A. massartii* but also diverse benthic dinoflagellates (such as *Ostreopsis lenticularis*, *Coolia* spp., *Gambierdiscus* spp., *Prorocentrum* spp.) with high abundances, but there was no LAMP reaction from the samples without *O*. cf. *ovata* and *A. massartii*. The LAMP sets in this study have been proven to react only with *O*. cf. *ovata* or *A. massartii*.

#### **5. Conclusions**

In this study, we developed a LAMP method for in situ and precise detection of benthic dinoflagellate species, *O.* cf. *ovata* and *A. massartii*. This provides a useful detection technique for field research, owing to the use of simple reaction conditions and inexpensive equipment and the lack of a need for an expert phycologist for algal identification by microscopy. Thus, the LAMP method can be applied to monitor the occurrence and distribution of benthic dinoflagellates in large-scale environments such as the coastal area of Korea.

**Author Contributions:** Data curation, formal analysis, writing—original draft preparation, E.S.L., J.H.; field investigation, methodology, J.-H.H.; conceptualization, supervision, project administration, writing—review and editing, J.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was a part of the project titled "Improvement of management strategies on marine disturbing and harmful organisms (No. 20190518)" funded by the Ministry of Oceans and Fisheries, Korea and supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. NRF-2021R1A2C1005943).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank E. J. Kim and S. W. Kim for providing technical support and field sampling. We thank the reviewers for their comments.

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

#### **References**


## *Article* **Numerical Study on the Massive Outbreak of the** *Ulva prolifera* **Green Tides in the Southwestern Yellow Sea in 2021**

**Bin Wang 1,2,\* and Lei Wu 1,2**


**Abstract:** The most massive outbreak on record of the *Ulva prolifera* green tides in the southwestern Yellow Sea occurred in summer of 2021. The environmental factors were investigated based on observations and simulations. The results suggested that the significantly enhanced discharge of the Changjiang River since winter 2020–2021 was crucial for the outbreak of the *Ulva prolifera* green tides in the southwestern Yellow Sea, which could significantly have contributed to the nutrient enrichment off the Subei coast. Additionally, the southerly wind stress anomaly during winter 2020–2021 favored the upwind transport of Changjiang water. Numerical experiments showed that the remaining winter freshwater coming from the Changjiang River, which persisted in the Subei coast's upper layer until spring 2021, exceeded the long-term average value by 20%. We demonstrated that these large amount of nutrient inputs, as an effective supplement, were the reason the green tides sharply emerged as an extensive outbreak in 2021. The easterly wind anomaly during spring 2021 contributed to the landing of *Ulva prolifera* off the Lunan coast.

**Keywords:** *Ulva prolifera*; Changjiang; southwestern Yellow Sea; outbreak mechanisms; wind anomaly

#### **1. Introduction**

The Yellow Sea, surrounded by Mainland China and the Korean Peninsula, is a characteristic continental shelf sea (Figure 1). It plays an important role in the environment of China and the Korean Peninsula. The bathymetry of the Yellow Sea is generally shallow and complex. The southwestern Yellow Sea is listed as a marginal sea with various contributions from land, rivers, and tides, which has been the focus of multi-disciplinary research in recent years.

Over the past 15 years (from 2007 to 2021), successively occurrences of *Ulva prolifera* green tides have become a striking recurrent phenomenon in the southwestern Yellow Sea. The average distribution area and the cover area were reported to be 37,000 km2 and 450 km2, respectively, in recent ten years. The general consensus is that the green tide originates from the coast of Subei in late spring. It migrates northward with the ocean current [1–3] and lands on the Lunan coast over long-distance migration every summer. The *Ulva prolifera* green tides have had detrimental effects on the local marine environment and ecosystem [4]. According to the previous studies, *Ulva prolifera* has wide adaptability to temperature and salinity [5–7]. The freshwater from the Changjiang River and local rivers provide abundant nutrients into the southwestern Yellow Sea and is also suitable for the growth of *Ulva prolifera*. Dissolved inorganic nitrogen (DIN) has been suggested to play a crucial role in the bloom of *Ulva prolifera* [8–11]. Chen et al. [12] found that the nutrient distribution and structure in the Jiangsu coast was affected by the land-based load, and the ratio of nitrogen to phosphorus could affect the bloom of *Ulva prolifera* significantly. Wang et al. [13] suggested the phosphate limitation on the initial growth of *Ulva prolifera* seedling can occur in the southern Yellow Sea. Later, Sun et al. [14] confirmed that the DIN was the most critical nutrient controlling the magnitude and time of the green tide rather

**Citation:** Wang, B.; Wu, L. Numerical Study on the Massive Outbreak of the *Ulva prolifera* Green Tides in the Southwestern Yellow Sea in 2021. *J. Mar. Sci. Eng.* **2021**, *9*, 1167. https:// doi.org/10.3390/jmse9111167

Academic Editors: Zhun Li and Bum Soo Park

Received: 28 September 2021 Accepted: 22 October 2021 Published: 24 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

than phosphorus, based on a dynamic growth model. According to the observations, the DIN concentration in the southwestern Yellow Sea is lowest in the winter and reaches its highest value in April [7,11,15], which implies that overwintering banks of nutrients might be an important population trigger of *Ulva prolifera* [16,17].

**Figure 1.** Model domain with geography and bathymetry (unit: m).

However, effective solutions for preventing the harmful algal bloom have not been found so far [17,18]. According to the local news, the worst *Ulva prolifera* green tides in last 15 years occurred in the summer of 2021. The maximal distribution area and the maximal cover area have been over 60,000 km<sup>2</sup> and 1700 km2, respectively. The amount of the *Ulva prolifera* was about 2.3 times that of 2013, which was reported to be the worst year previously.

Thus, based on observational data and numerical modelling, the hydrographical features of the southwestern Yellow Sea from the winter 2020–2021 to summer 2021 were compared with the long-term averaged status. Section 2 comprises a description of the methodology and data used in this study. Section 3 addresses the model results about the inter-annual variation of the green tides. Section 4 is a summary of the study.

#### **2. Data and Methodology**

#### *2.1. Observation Data*

The water depth in the nearshore region of Subei is quite shallow, and the observation data are relatively scarce. Supported by the Project on Coastal Investigation and Research (i.e., Project 908) of China, four cruises in four seasons were conducted during 2006 and 2007, mostly covering the Subei coast region. The measured salinity data were reported by Zhang et al. [19]. The digitalized isohaline was compared with the simulated sea surface salinity.

Merged satellite and in situ global daily sea surface temperatures (MGDSSTs) of the Japan Meteorological Agency, with a resolution of 0.25◦ × 0.25◦, were used to validate the simulated sea surface temperatures of the present model.

Furthermore, in late April of 2021, four GPS-tracked ARGOS surface floats were deployed in the southwestern Yellow Sea to measure the currents off the Subei coast. The floats were released along the edge of the Subei Bank to track the movement of the Subei coastal current. The trajectories of four surface ARGOS drifters were used to check the simulated circulation.

#### *2.2. Numerical Modeling*

Based on the Princeton Ocean Model (POM), we established a high-resolution regional circulation model over the Bohai Sea, the Yellow Sea, and a part of the East China Sea. The model, with a horizontal resolution of 1/12◦ × 1/12◦, covered (Figure 1) the domain of 27◦ N–41◦ N, 117◦ E–128◦ E. The vertical sigma coordinates were layered in the following proportions from top to bottom: 0.000, −0.003, −0.006, −0.013, −0.025, −0.050, −0.100, −0.200, −0.300, −0.400, −0.500, −0.600, −0.700, −0.800, −0.900, and −1.00. The model topography was based on the 1- × 1- Shuttle Radar Topography Mission (SRTM) database. The minimum and maximum depths in this model were set to 10 m and 4000 m, respectively.

The boundary conditions were determined by the simulated results of a 1/12◦ × 1/15◦ Eastern Asian Marginal Seas data assimilation model [20]. The monthly mean results from 2001 to 2020 were averaged as the climatological open boundary of the present study. The eight main tidal components (M2, S2, K1, O1, N2, P1, K2, and Q1) also were considered from the open boundary. The tidal harmonic constants were decided by the results of the NAO.99b model [21].

The surface wind stress was determined by the monthly forcing of the ERA5 dataset with a high horizontal resolution of 0.25◦ × 0.25◦. The long-term averaged values were calculated during the same 20-year period described in the previous paragraph as the climatological forcings. The surface heat flux was calculated using a bulk formula [22]. The net heat flux was expressed as the sum of shortwave radiation, longwave radiation, sensible heat flux, and latent heat flux, and all of these components followed the empirical formulas of Hirose et al. [20]. The penetration of the shortwave radiation was also considered, and the water quality was type II [23].

The freshwater flux was estimated with the precipitation (P) and evaporation (E) as well as the river runoffs (R), asP+R − E. Precipitation (P) data were also obtained from the long-term averaged monthly ERA5 dataset. The corresponding evaporation (E) was obtained when calculating the latent heat flux as mentioned above. The amounts of the freshwater from Changjiang River were retrieved from the climatological monthly transports at Datong station, which are reported by the Chinese River Sediment Bulletin. The multi-year mean discharges from Subei local rivers were retrieved from the Jiangsu Province Water Resources Bulletin, which are represented by the Guanhe River and the Sheyang River in this study [24]. The discharges of the Guanhe River and Sheyang River and their seasonal variations were also allotted based on the work of Yang [24]. There were no other relaxations for temperature and salinity in the present model.

The model was first integrated with the climatological fields for 4 years. An additional two-year calculation was conducted due to the overwinter effects, using the above last month's status for the restart conditions. The experiment that used the climatological meteorological forcings and boundary conditions was named Exp.C (referring to climatological meteorological forcings). To investigate the environmental features in the winter 2020–2012, a comparative experiment was designed (Table 1). The realistic monthly meteorological forcings and boundary conditions were employed and named Exp.R (referring to realistic forcings). Exp.R was established under the daily meteorological forcings from January 2020 to June 2021, and the Changjiang River discharges were also changed to the realistic monthly discharges. The values and their comparisons to meteorological values during the interested period are listed in Table 2.


**Table 1.** List of experiments.



#### *2.3. Passive Tracer*

Salinity distribution can present the pathway of the Changjiang River qualitatively. To quantitatively discuss the roles of the Changjiang River discharges, the passive tracer was continuously released in the numerical experiments from December to the following June at the Changjiang River estuary. The initial value of the passive tracer was 0, which means the nutrient content from the Changjiang River was 0 at the beginning. The tracer concentration at the estuary was set to 1, which was dimensionless and represented the nutrient concentrations inputted from the Changjiang River. According to Zhang et al. [11], the area south of 35◦ N and west of 122◦ E is one in which the *Ulva prolifera* was rapidly developed. Therefore, the persisted tracer content in the upper layer (within 5 m depth) of the region 32–35◦ N, 119–122◦ E was also calculated, following Formula (1):

$$T\_{\mathfrak{c}} = \int \mathfrak{c} \, dv \,\tag{1}$$

where *Tc* is the content of tracer in the upper layer, and *c* is the tracer's concentration.

#### **3. Results**

#### *3.1. Model Validations*

The simulated sea surface temperatures (SSTs) in Exp.C were compared with MGDSSTs. The basic features of the SST in summer and winter were well-represented (Figure 2). Our model successfully reproduced the surface cold patch off the Subei coast in the summer and the intrusion of the high temperature water in the winter. There were slightly larger differences in the nearshore region (shallower than 10 m) between the simulated SST and MGDSSTs. This may have been due to the lower horizontal resolution of MGDSSTs. Furthermore, shown in Figure 3, the root-mean-square errors between simulated SST and MGDSSTs were 0.99 ◦C in the summer and 1.15 ◦C in the winter after interpolating the MGDSSTS to the model grids.

The digitalized isohalines in the southwestern Yellow Sea in each seasons were overlaid on the simulated results of Exp.C (Figure 4). It should be noted that the digitalized isohalines were based on observations during a particular period. The simulated salinities represent the climatological distributions. Therefore, more attention should be paid to the pattern of simulated isohalines instead of the reproduction of isolated low-salinity water patches. The low-salinity water (<30 PSU) of Changjiang diluted water occupies most of the southwest Yellow and East China Seas in the summer (Figure 4c). Though with a significantly reduced range, it retreats to the coast with large horizontal gradients in other seasons. The 30 and 31 PSU isohalines in individual seasons were also found to be in good agreement with observations. All the characteristics, including the seasonal evolutions

of the isohalines (Figure 4a–d), were well-simulated by Exp.C, implying that the present model is able to capture the essential mass transportations in this area.

**Figure 2.** Simulated summer sea surface temperature of (**a**) Exp.C and (**b**) MGDSSTs; simulated winter sea surface temperature of (**c**) Exp.C and (**d**) MGDSSTs (unit: ◦C).

**Figure 3.** Validations of simulated surface temperature with MGDSSTs. Black and blue dots represent summer and winter values, respectively (unit: ◦C).

Four ARGOS surface drifts (Figure 5a) were deployed off the Subei coast since late April of 2021. For comparison, in the realistic case of Exp.R, eleven modeled drifters (Figure 5b) were released at the sea surface. The initial locations of the simulated drifters followed those of the ARGOS drifts. Simulated circulation was represented by the pathway of the modeled surface drifters. The trajectories of the modeled drifters, which were deemed to be Lagrangian particles, were calculated by the fourthorder Runge–Kutta scheme [25]. The positions of the drifters were calculated every

3 computational hours until end of June 2021. Figure 5 suggests that the pattern of the modeled drifter trajectories successfully reproduced the observed ones of ARGOS surface drifters. The characteristics of the ARGOS trajectories were well-simulated by this experiment, once again showing that the present model can capture the realistic features of regional circulation.

**Figure 4.** Simulated climatological salinity at 10 m of Exp.C in (**a**) winter, (**b**) spring, (**c**) summer, and (**d**) autumn (unit: PSU). The solid line is the simulated isohaline, and the dashed line is the observed isohaline digitalized from the work of Zhang et al. [19]. The blue and yellow lines represent the 30 and 31 PSU, respectively.

**Figure 5.** Observed (**a**) and simulated (**b**) trajectories of surface ARGOS drifters released in late April 2021 off the Subei coast. Star markers represent the released locations.

#### *3.2. Hydrography in the Summer of 2021*

The simulated tracer distributions at the surface until late April are shown in Figure 6. In Exp.C, a part of the Changjiang River water drifted northward, which could reach 33 ◦N in late April. A relative high concentration surface patch was identified off the Subei coast (Figure 6a), which was reported as the first observed region of *Ulva prolifera* in several years (Bulletin of China Marine Disaster). In the meantime, as listed in Table 2, the discharges of the Changjiang River were significantly increased since late 2020 compared with the climatological ones. Correspondingly, the simulated tracer concentration in Exp.R showed a large positive anomaly distribution along the Subei coast. In other words, the larger Changjiang River input in the winter 2020–2021 and spring of 2021 significantly contributed to the nutrient enrichment off the Subei coast, which led to the most massive outbreak of the *Ulva prolifera* green tides in the southwestern Yellow Sea the in summer of 2021.

**Figure 6.** Simulated distribution of the passive tracer (**a**) in Exp.C and (**b**) anomaly distribution in Exp.R at the sea surface in late April with the bathymetry.

The tracer content results showed that in late April, *Tc* was found to be 1.20 × 1010 m3 in Exp.C, which was almost 5% of the total Changjiang input, while in Exp.R, *Tc* approached 1.56 × 1010 m3 in late April, which was more than 6% of the total Changjiang input during this month. The northward transportation proportion of Changjiang water also was increased. Thus, we checked the wind distribution in the winter of 2020–2021 (Figure 7). In the winter of 2020–2021, the southerly anomaly led to a much weaker north–south component of wind (Figure 7b). Thus, the weakened southward wind-driven current was at a disadvantage in competition with the northward tidal residual current [26,27]. The counter-wind transport caused by the northward tidal residual current was dominant during this winter. Thus, there was more remaining winter freshwater from Changjiang at the Subei coast in the spring 2021. Compared with the climatological status, it increased by about 30%.

Until June, the remaining tracer content from the Chanjiang River was found to be significantly increased (Figure 8a) compared to that at the end of April, which provided necessary nutrient supplements for the population of *Ulva prolifera*. The passive tracer concentration in early summer of 2021 presented a stronger positive anomaly related to the long-term averaged case (Figure 8b). Without considering the biological absorption process, the passive tracer content results suggested that until late June, *Tc* was 4.79 × <sup>10</sup><sup>10</sup> m3 and 5.21 × <sup>10</sup><sup>10</sup> <sup>m</sup><sup>3</sup> for Exp.C and Exp.R, respectively.

The climatological wind during spring over the southwest Yellow Sea distributed northwestward (Figure 9a). It can be seen that there was a clear easterly wind anomaly during spring of 2021 (Figure 9b). The enhanced westward wind component contributed to the shoreward transport of *Ulva prolifera*, which led to its landing along the Lunan coast over long-distance migration.

**Figure 7.** (**a**) Climatological winter wind distribution and (**b**) wind anomaly in the winter of 2020–2021 at 10 m above the southwestern Yellow Sea.

**Figure 8.** Simulated distribution of the passive tracer (**a**) in Exp.C and (**b**) anomaly distribution in Exp.R at the sea surface in late June with the bathymetry.

**Figure 9.** (**a**) Climatological spring wind distribution and (**b**) wind anomaly in the spring of 2021 at 10 m above the southwestern Yellow Sea.

Altogether, these results suggest that the nutrient contents from the Changjiang River were increased in the winter 2020–2021 and spring 2021 due to the increase of discharges. Additionally, the southerly wind stress anomaly during winter 2020–2021 also favored the upwind transport of Changjiang water. According to the tracer content, overall, the remaining freshwater coming from the Changjiang River, which persisted in the upper layer of 32–35◦ N, 119–122◦ E region during spring 2021 (April, May, June), exceeded the climatological value by nearly 20%. At the same time, the stronger easterly wind

component during spring of 2021 was conducive to the landing of *Ulva prolifera* along the Lunan coast. It should be pointed out that these experiments did not consider the nutrient concentration from Changjiang River in the individual years. Further quantitative investigations remain to be conducted in the future.

#### **4. Concluding Remarks**

The most massive outbreak on record of the *Ulva prolifera* green tides in the southwestern Yellow Sea occurred in summer of 2021. The environmental factors were investigated based on observations and simulations.

The results suggested that the enhanced discharges of the Changjiang River and the southerly wind stress anomaly in winter 2020–2021 were crucial for the outbreak of the *Ulva prolifera* green tides in the southwestern Yellow Sea, which could remarkably contribute to the nutrient enrichment off the Subei coast. The remaining freshwater coming from the Changjiang River, which persisted on the coast of Subei in the spring 2021, exceeded the climatological value by nearly 20%. We demonstrated that these large amounts of nutrient inputs, as an effective supplement, were the reason the green tides sharply emerged as an extensive outbreak in 2021. Subsequently, the strong easterly wind anomaly during spring of 2021 led to shoreward transport, which was helpful for the large number of *Ulva prolifera* to land off the Lunan coast.

**Author Contributions:** Conceptualization, B.W.; Research approach, B.W.; Data analysis and writing, B.W. and L.W.; Figures, L.W.; Project administration, B.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Key Research and Development Project (2018YFD0900906), the Fundamental Research Funds for the Central Universities (B210203027), and National Natural Science Foundation of China (Project 41706023).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** MGDSSTs data were provided by the Japan Meteorological Agency, http://ds.data.jma.go.jp/gmd/goos/data/pub/JMA-product/ (accessed on 10 October 2021). ERA5 reanalysis meteorological forces and C3S sea level gridded data were provided by the European Centre for Medium-Range Weather Forecasts, https://www.ecmwf.int/en/forecasts/datasets (accessed on 10 October 2021). The data of Changjiang River Discharge were provided by the Chinese River Sediment Bulletin.

**Acknowledgments:** The authors thank Zhixin Zhang of the First Institute of Oceanography, Ministry of Natural Resources, for providing the measured isohaline data. The authors also thank the editors and anonymous reviewers for their work on this paper.

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

#### **References**


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