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Article

A New Ribo-Type of Wangodinium sinense from Germination of Resting Cysts Isolated from Ballast Tank Sediments of Incoming Ships to China

by
Zhe Tao
1,3,
Caixia Yue
1,3,4,
Yuyang Liu
6,
Shuo Shi
1,3,
Ruoxi Li
1,3,
Zhaoyang Chai
1,2,
Yunyan Deng
1,2,
Lixia Shang
1,2,
Zhangxi Hu
5,
Haifeng Gu
7,
Fengting Li
1,2,* and
Yingzhong Tang
1,2,*
1
CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
2
Laboratory for Marine Ecology and Environmental Science, Qingdao Marine Science and Technology Center, Qingdao 266237, China
3
University of Chinese Academy of Sciences, Beijing 100049, China
4
Animal, Plant and Food Inspection Center of Nanjing Customs, Nanjing 210000, China
5
Department of Aquaculture, College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China
6
College of Safety and Environmental Engineering, Shandong University of Science and Technology, Qingdao 266590, China
7
Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
*
Authors to whom correspondence should be addressed.
J. Mar. Sci. Eng. 2025, 13(5), 942; https://doi.org/10.3390/jmse13050942
Submission received: 17 April 2025 / Revised: 8 May 2025 / Accepted: 9 May 2025 / Published: 12 May 2025
(This article belongs to the Section Marine Ecology)
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Versions Notes

Abstract

In recent decades, ships’ ballast water and associated sediments have been recognized globally as significant vectors for the dissemination of non-indigenous species, which has attracted extensive attention due to its ecological and economic impacts. The characteristics of production of resting cysts in the dinoflagellate life cycle further increases the risk of biological invasions through ballast tank sediments. Despite extensive research which has characterized the species diversity of dinoflagellate cysts within ballast tank sediments, the possibility and importance of invasions caused by different ribosomal types of the same species have been paid little attention. In this study, two cultures of dinoflagellates were established through cyst germination from the ballast tank sediments collected from two ships (“THETIS” and “WARIYANAREE”) arriving at the Jiangyin Port (China) and identified as Wangodinium sinense Z. Luo, Zhangxi Hu, Yingzhong Tang and H.F. Gu by comprehensive phylogenetic analysis of rDNA sequences (including LSU, SSU, and ITS1-5.8S-ITS2). Despite the rDNA sequences of the isolates showing a generally high similarity to reference sequences, the LSU D1-D6 sequences contained up to 11 stable single nucleotide polymorphisms (SNPs), while SSU and ITS1-5.8S-ITS2 sequences exhibited up to five and two divergence sites, respectively. Moreover, phylogenetic analyses based on partial LSU and SSU rDNA sequences further indicated that strains germinated from ships’ ballast tank sediments formed a strongly supported sister clade to the strains previously isolated from Chinese and Korean waters, representing a novel ribo-type distinct from Chinese and Korean strains. Detailed morphological observations using light microscopy (LM) and scanning electron microscopy (SEM) did not find differences between our isolates and the holotype of the species in key diagnostic characteristics including the position and shape of the nucleus and chloroplasts, as well as the ASC structure, which suggested that no significant morphological divergence has occurred among these ribo-types. Acute toxicity exposure assays indicated that this ribo-type of W. sinense posed no lethal effect on rotifers at concentrations ≤ 104 cells/mL, yet it remains necessary to maintain vigilance regarding the potential risk of algal blooms resulting from higher cell density or environmental changes in the invaded ecosystems. This study reports the first successful germination of W. sinense cysts from ballast tank sediments, indicating that its cysts may be widely transferred through ballast tank sediments, and presents a potential risk of bio-invasions of new genotypes of species to a region where other genotypes of the same species have been present as indigenous species.

1. Introduction

Since the 1970s, ballast water has been widely used in ocean-going vessels, pumped in from ports or coastal waters to ensure stability when the vessels are empty or lightly loaded [1]. However, the extensive transport and discharge of ballast water can have significant negative impacts on the marine ecosystems of the discharge areas, potentially leading to severe ecological disasters [1,2]. During the intake of ballast water, solid particles are simultaneously pumped into the tanks. The interaction of ballast system byproducts with both inorganic and organic constituents results in sediment deposition and accumulation at the bottom of ballast tanks [3,4]. Various studies have shown that phytoplankton diversity in ballast water decrease rapidly over time (and distance traveled) due to trophic interaction (zooplankton predation) and environmental stressors (darkness, hypoxia, and temperature) [5,6,7]. However, dinoflagellates demonstrate an adaptive strategy through the formation of resting cysts under adverse conditions, which settle into ballast sediments for long-term survival [8,9]. Therefore, comparative studies reveal that dinoflagellate abundance (per unit volume or area) in ballast sediments is more likely to exceed that in ballast water [10,11,12]. For example, estimates show that during toxic dinoflagellate blooms in Muroran, Japan, and Eden, Australia, the sediments in a single ballast tank could contain over 300 million viable Alexandrium cysts [13].
Over the past several decades, harmful algal blooms (HABs) have increased globally in terms of their frequency, scale, duration, and geographic range [14,15,16,17,18], with some species showing a tendency to spread across national and regional boundaries [19,20]. Dinoflagellates account for approximately 75% of all HABs caused by microalgae and have been widely reported as non-indigenous species introduced by ships across numerous global regions [11,21,22,23]. The formation of resting cysts serves as a critical biological mechanism driving the recurrence and geographic expansion of dinoflagellate HABs. Resting cysts, which are reproductive cells formed by dinoflagellates in response to nutrient limitation or environmental stress, remain dormant in sediments and germinate under favorable conditions to resume growth in the water column [16,24,25,26]. The tolerance of cysts to extreme environments (such as darkness, hypoxia, and low temperatures) and their survival capabilities make them more likely to spread geographically through human or natural processes, such as the transplantation of aquaculture shellfish and the transfer of ballast water and its sediments [12,27,28,29]. Additionally, resting cysts of dinoflagellate formed through sexual reproduction maintain high genetic diversity through the exchange of genetic material, enhancing their adaptability to environmental changes [25]. Consequently, the inadvertent introduction of dinoflagellate cysts through ballast sediments may pose significant threats to the biodiversity of coastal and estuarine ecosystems, human health, and the global economy [11,28,30].
Extensive research has identified various dinoflagellate cysts in ships’ ballast tank sediments, particularly a number of toxic and harmful species. For example, Hamer et al. [31] identified 48 dinoflagellate species from ballast tank sediments of ships arriving in English and Welsh ports, with toxic Alexandrium species recorded in 25% of the samples. Casas-Monroy et al. [11] identified seven harmful taxa or bloom-forming species in ballast tank sediments from ships arriving at Canada’s east and west coasts and the Great Lakes, of which six were considered non-indigenous. Shang et al. [12] applied next-generation sequencing and single-cell PCR sequencing to detect 73 dinoflagellate cysts in ballast tank sediments collected from ships entering the Great Lakes or Chesapeake Bay, including 55 reported from ballast tank sediments for the first time, 19 toxic species, and 36 HAB-forming species. Additionally, through metabarcoding, single-cyst PCR sequencing, cyst germination, and phylogenetic analysis, Shang et al. [27] identified 43 dinoflagellate resting cysts in ballast tank sediments from ships arriving in China, including 12 non-indigenous species and 23 toxic or bloom-forming species. However, current research primarily focuses on non-indigenous species, especially HAB species, while overlooking the possibility and importance of invasions caused by different ribo-types of the same species. Different ribo-types of the same species can exhibit significant ecological differences [32], such as toxicity or non-toxicity [33,34,35]. Furthermore, hybridization events, whether between native and introduced species or among distinct introduced populations, may yield progeny with increased ecological competitiveness relative to native species [29].
To assess the viability of dinoflagellate cysts in ballast tank sediments and their potential bio-invasion risk, particularly the new genotypes of species, we conducted experiments involving cyst germination and the establishment of cultures for species identification. In this study, two dinoflagellate strains were germinated from ballast tank sediments collected from the ships “THETIS” and “WARIYA NAREE” upon arrival in Chinese port, with clonal cultures subsequently established for both strains. Comprehensive rDNA sequencing and phylogenetic analyses confirmed the identity of both strains as Wangodinium sinense. Notably, multiple nucleotide polymorphisms were detected within the rDNA sequences of these W. sinense strains compared to those of strains isolated from Chinese and Korean waters, suggesting the possibility of a novel ribosomal type distinct from previously identified populations. Subsequently, detailed morphological examinations, utilizing LM and SEM, were conducted to characterize critical morphological features of the strains. Additionally, experimental evaluations were performed to assess their potential toxicity or harmful activity toward aquatic organisms.

2. Materials and Methods

2.1. Sampling and Cultures Establishment

Sediment samples were collected from the ballast tanks of the vessels “THETIS” and “WARIYA NAREE” upon their arrival at the Jiangyin port in China. The sampling dates and the routes taken by the vessels are shown in Table 1. The sediment samples were collected with sterile trowels and placed into sterile sealed bags. They were then stored in a dark environment at a low temperature (4 °C) in the laboratory until further processing. Resting cysts in the ballast tank sediments were enriched using the sodium polytungstate (SPT) through density gradient centrifugation method [36]. The collected cysts were cultivated in f/2-Si medium at a temperature of 20 ± 1 °C, with a light intensity of 90 μmol photons m–2 s–1 following a 12:12 h light-dark cycle. Germinated cells were carefully transferred using a micropipette to establish clonal cultures, which were then kept under the same conditions as mentioned above. A single strain of W. sinense was successfully established from the ballast tank sediments of each of the two aforementioned vessels.

2.2. Light Microscopy Observation

Live cells and resting cysts of W. sinense were observed with an inverted microscope (IX73, Olympus, Tokyo, Japan) and upright microscope (BX53, Olympus, Tokyo, Japan) equipped with a digital camera (DP80, Olympus, Tokyo, Japan). The shape of the chloroplast was identified by autofluorescence, excited at 530–550 nm and emitted at wavelengths greater than 575 nm. The nucleus of the fixed cells, stained with SYBR Green (Solarbio, Beijing, China), was imaged using the same upright microscope, with excitation at 450–480 nm and emission at 515–525 nm. Cell sizes of W. sinense for 50 vegetative cells at the mid-exponential growth phase were measured using a DP80 digital camera (Olympus, Tokyo, Japan) at 200× magnification.

2.3. Scanning Electron Microscopy (SEM) Observation

For SEM, cells and cysts of W. sinense at the exponential growth phase were fixed with OsO4 (2% final concentration) for 50 min at room temperature. The fixed cells were gently filtered onto a Millipore nylon membrane with a pore size of 5 μm, dehydrated in an acetone series (10%, 30%, 50%, 70%, 90%, and 3 times in 100%, 15 min for each step). Following this, the samples were dried using a critical point dryer (EM CPD300, Leica, Vienna, Austria) with liquid CO2, sputter coated with gold (Sputter/Carbon Thread, EM ACE200, Leica, Austria). Finally, the cells and cysts were observed under 5-kV accelerating voltages with an S-3400N scanning electron microscope (Hitachi, Hitachinaka, Japan).

2.4. DNA Extraction, PCR Amplification, and Sequencing

The total genomic DNA of W. sinense was extracted using the Plant Genomic DNA Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. The concentration and quality of the extracted DNA were assessed using an ND-2000 NanoDrop spectrophotometer (Thermo Fisher Scientific, Somerset, NJ, USA). Three pairs of primers targeting dinoflagellate rDNA sequences were selected for DNA amplification: for the 28S region, primers D1R [37] (F: 5′-ACCCGCTGAATTTAAGCATA-3′) and 28-1483R [38] (R: 5′-GCTACTACCACCAAGATCTGC-3′); for the 18S region, primers sA [39] (F: 5′-ACCTGGTTGATCCTGCCAGT-3′) and 329R [40] (R: 5′-TGATCCTTCYGCAGGTTCAC-3′); and for the ITS1–5.8S–ITS2 region, primers ITS1 (F: 5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 [41] (R: 5′-TCCTCCGCTTATTGATATGC-3′). The PCR reaction mixture was composed of 2 μL DNA template, 12.5 μL 2× TransTaq HiFi PCR Super Mix I, 1 μL of each forward and reverse primer, and 8.5 μL sterile ultrapure water. The PCR protocol was as follows: an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C (28S), 52 °C (18S), or 55 °C (ITS1–5.8S–ITS2) for 30 s, extension at 72 °C for 1 min and 40 s (28S), 2 min (18S) or 45 s (ITS1–5.8S–ITS2), and a final extension at 72 °C for 10 min. The PCR products were then separated by electrophoresis in a 1% agarose gel, purified using a DNA gel extraction kit (GENEray, Shanghai, China). The purified DNA fragments were ligated into the PMD-19T vector (TaKaRa, Tokyo, Japan) and transformed into Escherichia coli (DH5α) competent cells (Biomed, Beijing, China). Positive transformants were confirmed by colony PCR and selected for Sanger sequencing by the TSINGKE company (Beijing, China). The DNA sequences obtained in this study have been deposited in Genbank with accession numbers PV412747-PV412766 (LSU), PV412768-PV412785 (SSU) and PV423430-PV423448 (ITS1–5.8S–ITS2).

2.5. Phylogenetic Analyses

The newly acquired rDNA sequences, along with sequences of the closest relatives of the W. sinense obtained from the NCBI database were analyzed in the phylogenetic and genetic diversity. All sequences were aligned using ClustalW v2.0.10 [42], and ambiguous sequences at both ends of the sequence fragments were excluded from the alignments. Maximum likelihood analysis was conducted using the Tamura-Nei model on MEGA v10.2.6, with node support evaluated through 1000 bootstrap values [43].

2.6. Bioassays for Possible Effects on Aquatic Organisms

To test the toxic effects of W. sinense on aquatic animals, exposure experiments were conducted using live cell cultures of W. sinense on widely distributed marine zooplankton (rotifer, Brachionus plicatilis). Resting eggs of rotifer B. plicatilis were purchased from Ningbo Futian Biotechnology Co., Ltd. (Ningbo, China), and two-day-old nauplii were used in the study. The exposure experiments were conducted in 12-well culture plates, with each well containing one vigorous rotifer and 5 mL culture at the stationary growth phase (n = 6). Simultaneously, 1 mL culture was fixed with Lugol’s solution at a final concentration of 2%, diluted, and counted. The f/2-Si medium was used as blank control. The activity of the rotifers was observed at 4, 8, 12, 24, 48, 72, and 96 h, with death recorded when no movement was observed.

3. Results

3.1. Phylogenetic Analysis

The 1525 bp long partial LSU rDNA sequences of Wangodinium sinense (accession no s. PV412747-PV412766) isolated from ballast tank sediments of the ships “THETIS” and “WARIYANAREE” were 97.63~98.47% and 98.19~98.33% identical, respectively, to the holotype sequence of W. sinense (accession No. MH732680) isolated from Chinese waters, despite only having 47% coverage. Additionally, they were 98.40–98.70% and 98.20~98.70% identical, respectively, to the sequence of W. sinense (accession No. OL699923) isolated from Korean waters. Additionally, the sequence of the strain isolated from “THETIS” showed 15 and 16 nucleotide polymorphisms, respectively, compared to the reference sequences from the Chinese and Korean waters, with 7 and 11 stable nucleotide polymorphisms, respectively (Table 2, Figure S1). The sequence of the strain isolated from “WARIYANAREE” showed 12 and 13 nucleotide polymorphisms, respectively, compared to the reference sequences from the Chinese and Korean waters, with 7 and 11 stable nucleotide polymorphisms, respectively (Table 2, Figure S1). However, the minimum similarity between the LSU rDNA sequences of the two isolates germinated from ballast tank sediments was 99.80%, with only three unstable nucleotide polymorphisms (Table 2, Figure S1), indicating a likely common origin. Phylogenetic analysis using maximum likelihood was consistent with the aforementioned results. The two strains germinated from ballast tank sediments and W. sinense (OL699923 from Korea; MH732679, MH732680, and MH732681 from China) formed a coherent clade with strong support (100 BS). A separate analysis of the W. sinense clade showed that strains isolated from Chinese and Korean waters formed a relatively highly supported sister group (64 BS) with those germinated from ships’ ballast tank sediments (Figure 1).
The 1799 bp long SSU rDNA sequences of Wangodinium sinense (accession Nos. PV412768-PV412785) isolated from ballast tank sediments of the ships “THETIS” and “WARIYANAREE” were 99.65~99.88% and 99.42~99.65% identical, respectively, to the holotype sequence of W. sinense (accession No. MH732687 and MH732688) isolated from Chinese waters. However, there were five nucleotide polymorphisms between the two isolates and Chinese strains. In the holotype sequence of W. sinense, four degenerate bases (Y = C/T and R = A/G) were present, indicating that they might be 50% identical to the corresponding positions in our sequences (Table 2, Figure S2). Phylogenetic analysis based on SSU sequences using maximum likelihood established a tree similar to that derived from LSU sequences. The two strains germinated from ballast tank sediments formed a coherent clade with W. sinense (MH732687 and MH732688, from China) with high support (85 BS). A separate analysis of the W. sinense clade showed that strains isolated from Chinese waters formed a sister group with those germinated from ship ballast tank sediments (Figure 2).
The 660 bp long ITS1-5.8S-ITS2 rDNA sequences of Wangodinium sinense (accession Nos. PV423430-PV423448) isolated from ballast tank sediments of the ships “THETIS” and “WARIYANAREE” were 99.82~100% and 99.64~99.82% identical, respectively, to the holotype sequence of W. sinense (accession No. MH732671 and MH732672) isolated from Chinese waters. And the sequences of the isolates from “THETIS” and “WARIYANAREE” had only one and two unstable nucleotide polymorphisms, respectively, compared to the reference sequences isolated from Chinese waters. Phylogenetic analysis based on ITS1-5.8S-ITS2 sequences using maximum likelihood generated a tree showing that the two strains germinated from ballast tank sediments formed a coherent clade with W. sinense (MH732671 and MH732672, from China), with high support (100 BS) (Figure 3).

3.2. Morphology of Resting Cyst and Vegetative Cell of Wangodinium sinense

The two strains of Wangodinium sinense germinated from the ballast tank sediments of the vessels “THETIS” and “WARIYA NAREE” exhibited high morphological similarity and were generally consistent with holotype descriptions [44]. The vegetative cells of W. sinense were predominantly ovoid or elliptical, ranging from 8.5 to 11.8 µm in length (n = 50) and 6.2 to 9.3 µm in width (n = 50) (Figure 4 and Figure 5). The cells were divided into the epicone and the hypocone by the cingulum; both conical parts were of similar size and spherical in shape (Figure 5A,B), with the hypocone further divided into two lobes by the deeply incised sulcus, the right lobe being more prominent (Figure 5D). The periphery of the cells contained a green to golden reticulated chloroplast. The nucleus was spherical, centrally located in the epicone, and occupied most of this region (Figure 4G–I). The cingulum was deeply incised with sharp edges and displaced approximately two cingulum widths downward (Figure 5A,D). The sulcus was deeply excavated, extending downward to the base and gradually narrowing upward into a furrow that connected to the apical structure complex (ASC) (Figure 5D). The cell surface displayed horizontally arranged amphiesmal vesicles (AVs), which were irregularly polygonal (Figure 5A,B). There were five to six rows of AVs on both the epicone and the hypocone (Figure 5A,B). The ASC, the most distinguishing feature of this species, was ring-shaped and encircled the apex on three sides, excluding where it connected to the sulcus (Figure 5E). The ASC comprised two rows of narrow AVs (central ridge, CR), though the exact number of vesicles was challenging to determine due to cellular secretions (Figure 5E). Seven irregular AVs were located in the apex and were surrounded by the ASC (Figure 5E). The cysts formed in laboratory culture were spherical, 8.3 to 11.4 µm in diameter (n = 50), with a smooth surface and no apparent adornments, filled with white granules (Figure 4E,F and Figure 5F).

3.3. Toxicity of Wangodinium sinense to Rotifer

The live cell cultures of Wangodinium sinense showed no toxicity to rotifers. The cell density of W. sinense germinated from the ballast tank sediments of the ships “THETIS” and “WARIYANAREE” was approximately 4.1 × 104 cells·mL−1 and 2.9 × 104 cells·mL−1, respectively. In the bioassays, no mortality was observed in rotifers exposed to these vegetative cell densities within 96 h (Table 3), and several rotifers were observed in some wells after 72 h, indicating asexual reproduction occurred during this period.

4. Discussion

The resting cysts, characterized by their thick walls and low metabolic activity, can endure harsh environmental conditions, allowing them to survive in marine sediments for extended periods (ranging from several months to over 100 years) and germinate under favorable environmental conditions, thus providing the “seed” for bloom formation [8,45,46,47]. This ability to form cysts facilitates their geographic dispersal through both anthropogenic and natural processes, such as ballast water/sediment and farmed shellfish introductions [13,19,28,29]. Consequently, it is inferred that dinoflagellates producing resting cysts can theoretically be dispersed via ballast tanks, posing serious threats to invaded water bodies [30]. Previous studies have successfully germinated various dinoflagellate cysts, such as Pseudocochlodinium profundisulcus [30], Scrippsiell acuminata [31], Alexandrium insuetum and Scrippsiell enormis [48]. In this study, two strains of Wangodinium sinense have been germinated firstly from ships’ ballast tank sediments, demonstrating that these cysts remain viable after enduring long-distance transoceanic voyages and prolonged harsh conditions. This finding underscores the necessity of implementing effective monitoring and treatment strategies to mitigate the risks associated with the introduction of alien species via ship ballast water and sediments.
Wangodinium sinense has been identified in coastal waters of Xiamen, Lianyungang, Beihai in China, and Myeongseon Island of Ulsan in South Korea, within the latitude range of 21°21′–35°23′ N [44,49]. The two strains of dinoflagellates germinated from ballast tank sediments showed cellular and cyst morphology consistent with W. sinense, and their rDNA sequences exhibited a generally high similarity to reference sequences (LSU sequence similarity >97%, SSU and ITS1-5.8S-ITS2 sequence similarity >99%). Based on these morphological and molecular characteristics, the two strains germinated from ballast tank sediments can be confidently annotated as W. sinense. Notably, the rDNA sequence differences between the strains germinated from ballast tank sediments and those isolated from Chinese and South Korean waters were greater than the intra-group differences (despite the limited reference sequence data), particularly in the LSU rDNA sequences (Table 2). Moreover, in the phylogenetic trees based on LSU and SSU rDNA sequences, the strains germinated from ballast tank sediments formed a relatively high-supported sister group with those from Chinese and South Korean waters (Figure 3 and Figure 4). Therefore, it is reasonable to infer that the W. sinense strains germinated from two different ships might have the same origin and could represent a new ecotype distinct from the Chinese and South Korean strains. Further analysis of the shipping routes revealed a repeated segment of the route: Bangladesh–India–Singapore. This suggests that these W. sinense strains might originate from the port waters of these three countries, thereby expanding their known distribution range. Compared to the previously known distribution, these countries have lower latitudes and higher average water temperatures, further supporting that the two strains germinated from ballast tank sediments may represent a different ecotype of W. sinense.
The characteristic of dinoflagellates to form resting cysts not only facilitates population survival under adverse environmental conditions but also ensures high genetic diversity through the exchange of genetic material during sexual reproduction, thereby enhancing their environmental adaptability [30,50,51,52]. Consequently, once dinoflagellate cysts from ballast tanks are released into invaded aquatic ecosystems and germinate under favorable conditions, they may introduce new genotypes into the invaded ecosystem [53,54]. Furthermore, genetic exchanges between the invasive and native may lead to hybridization, potentially producing offspring with superior ecological adaptability [29]. For instance, mating experiments conducted by Blackburn et al. [55] with 21 strains of Gymnodinium catenatum from four different global populations revealed significant differences in progeny in terms of gamete production, cyst production, and cyst dormancy between interpopulation crosses. It is noteworthy that intraspecific genetic diversity often corresponds to substantial trait variation, which enables species in enhancing their competitive success and adapting more rapidly to environmental changes [56]. For example, the variability among Margalefidinium polykrikoides populations allows blooms to persist through intraspecific bloom succession even after environmental conditions change [57]. Thus, although this study and previous research indicated that the currently ribo-types of W. sinense did not exhibit toxic effects on aquatic animals, it remains necessary to maintain vigilance regarding the possible emergence of HABs driven by increased cell density, shifting environmental factors, or genetic hybridization, as these phenomena may pose significant risks to invaded ecosystems.

5. Conclusions

This study successfully established two clonal cultures of Wangodinium sinense via cyst germination from the ballast tank sediments of two ships arriving in China. Through sequence alignment and phylogenetic analysis based on rDNA sequences, several stable nucleotide polymorphisms were identified, suggesting that these strains may represent a novel ribo-type, while no significant morphological divergence was observed using LM and SEM. Acute toxicity experiments indicated that this novel ribo-type exhibited no lethal effects on rotifers at the tested concentrations. However, it remains essential to closely monitor the potential risk of invasions by this new genotype, particularly in regions where indigenous genotypes of the same species are already established.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jmse13050942/s1, Figure S1: Nucleotide polymorphisms in the 28S rDNA D1-D6 sequences of Wangodinium sinense from different geographical origins; Figure S2: Nucleotide polymorphisms in the 18S rDNA sequences of W. sinense from different geographical origins; Figure S3: Nucleotide polymorphisms in the ITS1-5.8S-ITS2 sequences of W. sinense from different geographical origins.

Author Contributions

Conceptualization, Y.T. and F.L.; methodology, Z.H. and Y.L.; software, C.Y. and S.S.; validation, C.Y. and R.L.; formal analysis, Z.T.; investigation, Z.T. and Y.L.; resources, Y.T.; data curation, Z.T.; writing—original draft preparation, Z.T.; writing—review and editing, F.L., H.G. and Y.T.; visualization, Z.C.; supervision, Y.D. and L.S.; project administration, Y.T. and Z.C.; funding acquisition, Y.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Research Infrastructures in the CAS Field Stations of the Chinese Academy of Science (grant No. KFJ-SWYW047), the National Natural Science Foundation of China (grant No. 41976134), and the National Natural Science Foundation of China (grant No. 42376138).

Data Availability Statement

The data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors state that there are no conflicts of interest. The funding sources did not influence the study design, data collection, analysis, or interpretation, nor did they contribute to the manuscript writing or the decision to publish the findings.

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Jmse 13 00942 g001
Figure 1. Maximum-likelihood (ML) phylogenetic tree showing the positions of partial large subunit (LSU) rDNA sequences of Wangodinium sinense. Akashiwo sanguinea (EF613348) was selected as outgroups. ML bootstrap support values are displayed on the branches, with only those exceeding 50% displayed. Branch lengths correspond to the number of substitutions per site.
Figure 1. Maximum-likelihood (ML) phylogenetic tree showing the positions of partial large subunit (LSU) rDNA sequences of Wangodinium sinense. Akashiwo sanguinea (EF613348) was selected as outgroups. ML bootstrap support values are displayed on the branches, with only those exceeding 50% displayed. Branch lengths correspond to the number of substitutions per site.
Jmse 13 00942 g001
Jmse 13 00942 g002
Figure 2. Maximum-likelihood (ML) phylogenetic tree showing the positions of partial small subunit (SSU) rDNA sequences of Wangodinium sinense. Akashiwo sanguinea (AY421771) was selected as outgroups. ML bootstrap support values are displayed on the branches, with only those exceeding 50% displayed. Branch lengths correspond to the number of substitutions per site.
Figure 2. Maximum-likelihood (ML) phylogenetic tree showing the positions of partial small subunit (SSU) rDNA sequences of Wangodinium sinense. Akashiwo sanguinea (AY421771) was selected as outgroups. ML bootstrap support values are displayed on the branches, with only those exceeding 50% displayed. Branch lengths correspond to the number of substitutions per site.
Jmse 13 00942 g002
Jmse 13 00942 g003
Figure 3. Maximum-likelihood (ML) phylogenetic tree showing the positions of partial ITS1, 5.8S, and ITS2 sequences of Wangodinium sinense. Akashiwo sanguinea (AY831412) was selected as outgroups. ML bootstrap support values are displayed on the branches, with only those exceeding 50% displayed. Branch lengths correspond to the number of substitutions per site.
Figure 3. Maximum-likelihood (ML) phylogenetic tree showing the positions of partial ITS1, 5.8S, and ITS2 sequences of Wangodinium sinense. Akashiwo sanguinea (AY831412) was selected as outgroups. ML bootstrap support values are displayed on the branches, with only those exceeding 50% displayed. Branch lengths correspond to the number of substitutions per site.
Jmse 13 00942 g003
Jmse 13 00942 g004
Figure 4. Micrographs (LM) of Wangodinium sinense strain “THETIS”. Ventral view (A), apical view (B), lateral view (C) and dorsal view (D) of vegetative cells. Surface view (E) and deeper focus (F) of resting cysts observed in culture; (G–I) Bright-field and epifluorescence light microscopy observation on the same vegetative cell. Scale bars = 10 μm.
Figure 4. Micrographs (LM) of Wangodinium sinense strain “THETIS”. Ventral view (A), apical view (B), lateral view (C) and dorsal view (D) of vegetative cells. Surface view (E) and deeper focus (F) of resting cysts observed in culture; (G–I) Bright-field and epifluorescence light microscopy observation on the same vegetative cell. Scale bars = 10 μm.
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Jmse 13 00942 g005
Figure 5. Scanning electron microscopy micrographs of Wangodinium sinense strain “THETIS”. (A) Lateral view; (B) Dorsal view showing cingulum and amphiesmal vesicles on cell surface; (C) Antapical view showing vesicles on the cell base; (D) Ventral view showing the longitudinal and transverse flagella positioned in the sulcus and cingulum, respectively, with sulcal intrusion linking to the apical structure complex and cingulum displacement; (E) Apical view showing apical structure complex (ASC), with seven irregular AVs (A1-A7) surrounded by the ASC; (F) SEM micrograph of a resting cyst. Scale bars for (A–D,F) = 5 µm, (E) = 1 µm.
Figure 5. Scanning electron microscopy micrographs of Wangodinium sinense strain “THETIS”. (A) Lateral view; (B) Dorsal view showing cingulum and amphiesmal vesicles on cell surface; (C) Antapical view showing vesicles on the cell base; (D) Ventral view showing the longitudinal and transverse flagella positioned in the sulcus and cingulum, respectively, with sulcal intrusion linking to the apical structure complex and cingulum displacement; (E) Apical view showing apical structure complex (ASC), with seven irregular AVs (A1-A7) surrounded by the ASC; (F) SEM micrograph of a resting cyst. Scale bars for (A–D,F) = 5 µm, (E) = 1 µm.
Jmse 13 00942 g005
Table 1. The sampling dates and shipping routes of two sampling ships.
Table 1. The sampling dates and shipping routes of two sampling ships.
Sampling ShipsSampling DateShipping Route
THETIS30 October 2020Russia-Philippines-South Korea-Canada-Bangladesh-India-Singapore-China
WARIYA NAREE10 March 2021United Arab Emirates-Bangladesh-India-Singapore-Indonesia-China
Table 2. Nucleotide differences in the partial rDNA (including LSU, SSU and ITS1-5.8S-ITS2) sequences of Wangodinium sinense from different geographic origins, with the positions of differing nucleotides referenced against the sequences obtained in this study.
Table 2. Nucleotide differences in the partial rDNA (including LSU, SSU and ITS1-5.8S-ITS2) sequences of Wangodinium sinense from different geographic origins, with the positions of differing nucleotides referenced against the sequences obtained in this study.
OriginLSU
155240532533549562567568569572573578597604661662681
CHINAACTTGTG/CC/TAG/CTTCTGTA
KOREAAATTGTCCAGCTCTGTG
WARIYANAREEAAG/TA/TTCGTCCTCTCGTA
THETISA/GAG/TA/TTCGTCCTCTCG/CC/TA
OriginSSUITS1-5.8S-ITS2
98516558797108784583
CHINAC/TAC/TA/GA/GA/TC
KOREA-------
WARIYANAREECGTAGTC/T
THETISCGTAGTC
Table 3. The bioassay for possible toxicity of two strains of Wangodinium sinense using the rotifers (Brachionus plicatilis). The f/2-Si medium was used as a blank control.
Table 3. The bioassay for possible toxicity of two strains of Wangodinium sinense using the rotifers (Brachionus plicatilis). The f/2-Si medium was used as a blank control.
GroupsCell Density (Cells/mL)Number of Test Rotifers per Well (n = 6)Viable Rotifers at Different Exposure Time (Hour)
481224487296
W. sinense (THETIS)4.1 × 10416666669
W. sinense (WARIYANAREE)2.9 × 104166666810
f/2-Si medium016666689
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Tao, Z.; Yue, C.; Liu, Y.; Shi, S.; Li, R.; Chai, Z.; Deng, Y.; Shang, L.; Hu, Z.; Gu, H.; et al. A New Ribo-Type of Wangodinium sinense from Germination of Resting Cysts Isolated from Ballast Tank Sediments of Incoming Ships to China. J. Mar. Sci. Eng. 2025, 13, 942. https://doi.org/10.3390/jmse13050942

AMA Style

Tao Z, Yue C, Liu Y, Shi S, Li R, Chai Z, Deng Y, Shang L, Hu Z, Gu H, et al. A New Ribo-Type of Wangodinium sinense from Germination of Resting Cysts Isolated from Ballast Tank Sediments of Incoming Ships to China. Journal of Marine Science and Engineering. 2025; 13(5):942. https://doi.org/10.3390/jmse13050942

Chicago/Turabian Style

Tao, Zhe, Caixia Yue, Yuyang Liu, Shuo Shi, Ruoxi Li, Zhaoyang Chai, Yunyan Deng, Lixia Shang, Zhangxi Hu, Haifeng Gu, and et al. 2025. "A New Ribo-Type of Wangodinium sinense from Germination of Resting Cysts Isolated from Ballast Tank Sediments of Incoming Ships to China" Journal of Marine Science and Engineering 13, no. 5: 942. https://doi.org/10.3390/jmse13050942

APA Style

Tao, Z., Yue, C., Liu, Y., Shi, S., Li, R., Chai, Z., Deng, Y., Shang, L., Hu, Z., Gu, H., Li, F., & Tang, Y. (2025). A New Ribo-Type of Wangodinium sinense from Germination of Resting Cysts Isolated from Ballast Tank Sediments of Incoming Ships to China. Journal of Marine Science and Engineering, 13(5), 942. https://doi.org/10.3390/jmse13050942

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