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Article

Resting Cysts of the Toxic Dinoflagellate Gymnodinium catenatum (Dinophyceae) Ubiquitously Distribute along the Entire Coast of China, with Higher Abundance in Bloom-Prone Areas

by
Xiaohan Liu
1,4,
Zhe Tao
1,5,
Yuyang Liu
1,3,
Zhangxi Hu
1,6,
Yunyan Deng
1,2,3,
Lixia Shang
1,2,3,
Po-Teen Lim
7,
Zhaoyang Chai
1,2,3,* and
Ying-Zhong Tang
1,2,3,*
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 266071, China
3
Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
4
National Marine Environmental Monitoring Center, Dalian 116023, China
5
University of Chinese Academy of Sciences, Beijing 100049, China
6
Department of Aquaculture, College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China
7
Institute of Ocean and Earth Sciences, University of Malaya, Bachok 16310, Kelantan, Malaysia
*
Authors to whom correspondence should be addressed.
J. Mar. Sci. Eng. 2024, 12(9), 1651; https://doi.org/10.3390/jmse12091651
Submission received: 14 August 2024 / Revised: 11 September 2024 / Accepted: 12 September 2024 / Published: 14 September 2024
(This article belongs to the Special Issue Studies on Marine Microbial Ecology (2nd Edition))
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Versions Notes

Abstract

:
Blooms of Gymnodinium catenatum have occurred occasionally in different areas of China and caused tremendous economic loss and even threatened human health. Not only is G. catenatum an important harmful-algal-bloom (HAB)-causing species, but also the only gymnodinioid dinoflagellate known to produce paralytic shellfish poisoning toxins (PSTs). Due to the germination of resting cysts, which often initiates blooms, the distribution and abundance of cysts in sediments and particularly the confirmation of cyst beds are important information for understanding and predicting dinoflagellate blooms. In this research, 199 sediment samples were collected from China’s coastal seas, ranging from the Beidaihe in the Bohai Sea (BS) to the southernmost sample from the Nansha Islands of the South China Sea (SCS). TaqMan quantitative PCR (qPCR) assays with species-specific primers and probes were developed to specifically detect the distribution and abundance of cysts in the 199 samples. The detection revealed that G. catenatum cysts were widely present in the sediments (126 of the 199 samples), with 93.55%, 74.65%, 42.37%, and 50% of the samples detected positively from the BS, YS, ECS and SCS, respectively, and covering the vast sea area from Nansha Islands to the Beidaihe area. The single-cyst morpho-molecular identification in the samples from Beidaihe confirmed the existence of G. catenatum cysts in the BS, and the positive detections of G. catenatum cysts using the qPCR methods. While G. catenatum cysts were widely distributed in all four seas of China, the average abundance was relatively low (1.0 cyst per gram of wet sediment). Three samples from the East China Sea (ECS), however, contained G. catenatum cysts at a relatively higher level (23 cysts g−1 wet sediment) than other sea areas, suggesting a pertinence of cyst abundance to the frequent occurrences of G. catenatum blooms in the area during recent years. Collectively, for G. catenatum being such an important toxic and HAB-causing species globally, the ubiquitous distribution of its cysts along the coastal waters of China and higher abundance in the bloom-prone areas warns us of a risk that cyst beds, although currently low in abundance, may seed HABs in any and many sea areas of China at any forthcoming year, and particularly those areas with records of frequent HABs outbreaks in the past.

1. Introduction

Harmful algal blooms (HABs) have been recorded more frequently during the past few decades, posing serious threats to fishery, tourism, human health, and marine ecosystems [1,2,3,4,5,6]. About 75% of HAB events are caused by dinoflagellates [7], and dinoflagellates account for 44% of the HAB-causing species [8]. Dinoflagellates have remarkable adaptive strategies, making them strong competitors in the formation of HABs [3,9,10,11,12]. For many dinoflagellates, one of these strategies is forming resting cysts in their lifecycle [13]. Resting cysts play an important role in the biology and ecology of dinoflagellates, associated with resisting adverse environments [11], geographical expansion [14], recurrence and termination of algal blooms [15,16], and seeding HABs [17].
The dinoflagellate Gymnodinium catenatum, an important HABs-causing species, is the only reported athecate dinophyte that produces paralytic shellfish toxins [18]. As a cosmopolitan species, it has been observed and reported from coastal waters in areas including North and South America [19,20,21], Europe [22,23], Africa [24], Asia [25,26,27], and Oceania [28]. As one of the common causative species of HABs in China, G. catenatum have bloomed in all four seas. At the Chinese coast, the species was first reported in 1998 in the Pearl River estuary [29], followed by Haizhou Bay [30], Zhimao Bay [31], and the coastal waters of Fujian province [32]. In particular, toxic blooms of G. catenatum occurred in Zhangzhou and Quanzhou in 2017, and led to human poisoning in many places in the Fujian Province due to the consumption of contaminated mussels [32].
While the formation of cysts is considered to be a general survival strategy for dinoflagellates to cope with adverse environments, it has been proven that cysts also play vital roles in the formation of blooms, as cyst germination functions as an inoculation and initiation process of HABs and the abundance of cysts in sediments may act as an important predictor of HABs [10,15,33]. Therefore, it is of great significance to investigate the distribution and abundance of G. catenatum cysts in marine sediments, particularly from the HABs-prone areas where more abundant cysts may be preserved in the sediments.
The presence of G. catenatum cysts has been reported in multiple coastal areas of China, mostly based on light microscopic (LM) identification. Qi et al. (1996) observed G. catenatum cysts with LM for the first time in Dapeng Bay, Guangdong province [34]. Also based on LM, Wang et al. (2003) reported G. catenatum cysts from 10 sampling stations of the Yellow Sea (YS), East China Sea (ECS), and South China Sea (SCS), with the highest abundance of 71.1 cysts g−1 DW in a site located in Daya Bay, Guangdong province [35]. Using LM identification, Liu et al. (2020) noticed an elevated abundance of G. catenatum cysts in the sediment of the Taiwan Strait nine months after a bloom, reaching a maximum density up to 689 cysts cm−3 [36]. A similar but more recent investigation found that G. catenatum cysts were produced during a bloom that occurred in Fujian province in 2018, with a maximum cyst abundance reached at the bloom termination [37]. Generally, investigations into the presence and distribution of G. catenatum cysts in China has been “patchy” and mostly used LM identification, leaving the Bohai Sea (BS) as an unexplored sea. While these surveys laid important stepping stones for the cyst ecology of G. catenatum, those published in the early years used LM as the sole identification method, which possibly contained mis-identification, as the cysts of G. catenatum, G. microreticulatum, and G. nolleri have similar sizes and microreticulate morphology [38]. In addition, the area coverage of these surveys was highly “patchy”, with the Bohai Sea, in particular, left unexplored. Therefore, taking into consideration the importance of this highly toxic species and the history of its HAB events at discrete locations, we believe it is worthwhile to accomplish a comprehensive investigation into the distribution of G. catenatum cysts along the entire coasts of all four seas of China by means of more intensive sampling and a more affirmative quantification technique.
In this study, G. catenatum cysts in 199 surface sediment samples that were collected along the coastal seas of China were quantified with quantitative PCR (qPCR) using specially designed species-specific primers and probes targeting the D1–D2 region of 28S rDNA of G. catenatum. Meanwhile, we used an approach called single-cyst morpho-molecular identification to identify dinoflagellate cysts both morphologically and molecularly from sediment samples, which led us to findings of G. catenatum cysts from the sediments of the Beidaihe, BS, and thus provided confirmatory evidence for the existence of G. catenatum cysts in the BS as well as a partial validation for the qPCR results.

2. Materials and Methods

2.1. Marine Sediment Sample Collection

A total of 199 sediment samples were collected from four seas of China (BS, YS, ECS, and SCS) during research cruises from the years 2014 to 2018 using a box corer (geographical coordinates, water depth, and sampling dates were shown in Table S1), with the longitude covering from 108.33° E to 124° E and the latitude from 10.01° N to 39.85° N (Figure 1, Table S1). The upper 2 cm of each surface sediment sample was transferred to sterile plastic bags or cryotubes and stored in the refrigerator (4 °C in darkness) until further treatment.

2.2. Quantitative PCR (qPCR) Assay

2.2.1. Sample Processing and DNA Extraction

For the samples with the red dots shown in Figure 1, 32 g (wet weight) sediment samples from each site (red open circles and red closed circles in Figure 1) were concentrated through a density gradient centrifugation with sodium polytungstate (SPT) [39]. Subsequently, 0.25 g quartz sands and 800 μL buffer GP1 (Tiangen, Beijing, China) were added to the concentrated cyst, and lysed thoroughly with the FastPerp-24 classic grinder (MP Biomedicals, Santa Ana, CA, USA). The total genomic DNA was extracted according to the protocol of the plant genomic DNA kit (Tiangen, Beijing, China). As for the samples with the black dots in Figure 1, the amount of sample was not enough to perform the SPT concentration protocol, and therefore 1.0 g each of these sediment samples was transferred to Lysing Matrix E tubes (MP Biomedicals) for direct DNA extraction. Then, the DNA of the lysis was extracted using the Fast DNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, USA), according to the manufacturer’s protocol. The quality and quantity of all extracted DNA were measured with an ND-2000 NanoDrop spectrophotometer (Thermo Fisher Scientific, Somerset, NJ, USA). All genomic DNA was stored at −80 °C prior to the cyst quantification below.

2.2.2. Primers and TaqMan Probes for the Detection of G. catenatum Cysts

The partial LSU rDNA sequences of G. catenatum, including the D1–D2 region were obtained from NCBI (https://www.ncbi.nlm.nih.gov, accessed on 13 August 2024), and then assembled into a contig using the DNAstar software (version 7.1.0; https://www.dnastar.com, accessed on 3 July 2022). After alignment with other related dinoflagellates (Table S2) through ClustalX (version 2.0; [40]), the primers and probe for G. catenatum were designed. The primer pairs were GCF, 5′-AGATTGTCGCACGCAGCA-3′; GCR, 5′-TGTCCCCACCTCCAAACTGA-3′, and the TaqMan probe was GC-P CGCTTTCGCTGGAATAGAAGGTGAT. Their specificity was tested by BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 18 April 2023), and the probe was labeled with FAM at the 5′ end and BHQ1 at the 3′ end (Tsingke company, Beijing, China). A 120 bp LSU rDNA fragment was targeted.

2.2.3. Detection and Quantification of G. catenatum Cysts in Surface Sediment

A Taqman-qPCR assay was used for the detection of G. catenatum cysts that targeted the D1–D2 region of LSU rDNA; the specific primers and probe are described above. Cyst abundance in surface sediment samples was quantified based on LSU rDNA copy numbers [41]. The 20 μL PCR reaction system contained 10 μL Premix Ex Taq II (2×), 0.8 μL of each PCR primer (10 μM), 0.8 μL probe (10 μM), 2 μL of the DNA template, and 5.6 μL nucleotide-free water. PCR reactions were conducted with the following parameters: pre-heating at 94 °C for 10 min, 40 cycles at 94 °C for 20 s, and then 60 °C for 45 s. Triplicate reactions, as well as the negative controls (i.e., template control), were performed for each DNA template. Genomic DNA of G. catenatum vegetative cell used for the construction of plasmid DNA was extracted using the plant genomic DNA kit, as described above. Calibration curves were constructed using 10-fold serial dilutions of plasmid DNA (approximately 102–108 copies of recombinant plasmid). The amplification efficiency of the real-time PCR was 98% with R2 = 0.9995 (Figure S1), showing a sound linear relationship between the gene copy number and Ct value. One third of the qPCR products amplified from all sediment samples was cloned and sequenced to confirm the specificity of assays. The qPCR products were separated by electrophoresis in 1% agarose gels and verified visually under UV light, purified using a DNA gel extraction kit (Generay, Shanghai, China), ligated into the PMD19-T at 16 °C overnight, and then imported into Trans1-T Escherichia coli (Trans, Beijing, China). After colony PCR, the positive transformants with correct inserts were chosen for Sanger sequencing according to TSINGKE, China. All sequences retrieved were identified using BLASTn. The G. catenatum cyst abundance in marine sediments was quantified on the basis of the assumption that the rDNA copy number in a resting cyst (diploid) is twice that in a vegetative cell (haploid), and the formula to calculate the cell density by copy numbers was the same as in our previously published study [42]. The following adjustments were made to the quantification results for G. catenatum cyst in sediment samples: By comparing the number of G. catenatum vegetative cells through two methods—microscopic count and qPCR—the extraction efficiency of genomic DNA was estimated. The LSU rRNA gene copies in each sediment sample were then multiplied by this coefficient.

2.3. Single-Cyst Morpho-Molecular Identification in the Sediment Samples from Beidaihe

Sediment samples of Beidaihe (BDH) were analyzed via single-cyst LM-PCR identification. Cysts were concentrated through a density gradient centrifugation with SPT [39], and then washed three times with sterilized distilled water. The G. catenatum-like cysts were picked out, and then observed and photographed using an inverted microscope (IX73, Olympus, Tokyo, Japan) and a light microscope (BX53, Olympus, Japan) equipped with a digital camera (DP80, Olympus, Japan). After that, individual cysts were micropipetted and transferred to a sterile slide, and then broken by another coverslip. The crushed cyst, together with the coverslip pieces, was transferred into a 250 μL centrifuge tube, and became the template to amplify about 1400 bp of the LSU rDNA (D1–D6 domains) using the primer sets D1R (F: 5′-ACCCGCTGAATTTAAGCATA-3′) [43] and 28-1483R (R: 5′-GCTACTACCACCAAGATCTGC-3′) [44]. The 30 μL PCR reaction solution included 15 μL HiFi PCR SuperMix (Transgen, Beijing, China), 1 μL of each PCR primer, 2 μL of DNA template, and 11 μL nucleotide-free water. The reaction processes were performed with the following program: 94 °C for 5 min; followed by 35 cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 2 min; final extension of 72 °C for 10 min. The PCR products were separated by electrophoresis in 1% agarose gel, and then purified using a DNA gel extraction kit (GENEray, Shanghai, China). Purified DNA fragments were processed and sequenced using the above-mentioned methods. The retrieved sequences were blasted in GenBank for their identities.

3. Results

3.1. Cyst Distribution of G. catenatum in the Sediments along the Coastal Seas of China

The distribution of G. catenatum cysts in the sediments collected from all four seas of China is shown in Figure 2. The presence of G. catenatum cysts was confirmed in 126 samples (63.32%) of the 199 sediment samples (Figure 2). The number of samples that were positively detected from the BS, YS, ECS, and SCS were 93.55% (N = 31), 74.65% (N = 71), 42.37% (N = 59), and 50% (N = 38), respectively. The highest detection rate was found in the Bohai Sea. The results showed that G. catenatum cysts were widely distributed in the sediments of the coastal seas of China, ranging from 10.01° N to 39.85° N (ca 3000 km) and from 108.33° E to 124° E, covering the vast sea area from Nansha Islands in the SCS to Beidaihe in the BS. The qPCR products from 42 positive samples that covered all four China seas were further sequenced for their LSU rDNA, while the obtained sequences were confirmed to be those of G. catenatum (coverage = 100%, identity > 99% to the reference in the NCBI database, MK215823.1, which was reported for Republic of Korea).

3.2. Abundance of G. catenatum Cysts in the Sediments along the Coastal Seas of China

The abundance of G. catenatum cysts in the sediments collected from all four seas of China is shown in Figure 3. In all sediment samples, the LSU rDNA copies ranged from 0 to 764,405 copies g−1 sediment (wet weight). Comparatively, the calculated abundance of G. catenatum cysts varied from 0 to 23 cysts g−1 sediment (wet weight). The average abundance of all the samples was 1.0 cyst per gram sediment (wet weight), with the highest abundance in the BS being 8 cysts g−1, that in the YS being 12 cysts g−1, that in the ECS being 23 cysts g−1 sediment, and that in the SCS being 6 cysts g−1; the stations with high abundances were in the ECS (ECS08, ECS09, ECS14). Comparatively, although the detection rate of G. catenatum cysts in the BS was the highest, the average abundance was low, while the detection rate in the ECS was the lowest, but the abundance was the highest among the four seas.

3.3. Detection of Gymnodinium catenatum Cysts in the Sediments of Bohai Sea According to Single-Cyst PCR

The LM micrographs and ~1500 bp LSU rDNA sequences of three G. catenatum cysts in the samples collected from Beidaihe (BDH) were obtained by single-cyst LM-PCR sequencing (Figure 4). All G. catenatum cysts were spherical, dark brown, and had an accumulation body inside, with a diameter ranging from 45 to 55 µm, and they were consistent with the initial and other authors’ observations of resting cysts of the species [18,36,48]. There were no apparent ornaments around the cyst surface. Newly obtained sequences from these cysts were deposited in the GenBank with accession numbers from OQ727112 to OQ727114. All the LSU rDNA sequences were annotated as G. catenatum (coverage = 100%, identity > 99% to the reference in the NCBI database, ON392354.1, which was reported from YS, China).

4. Discussion

4.1. Application of the qPCR Method in Determining the Abundance of G. catenatum Cysts

The identification and quantification of G. catenatum cysts has mainly used light microscopic counting after a pre-treatment of the sediment by applying either a palynological method (e.g., strong acid digestion and careening) or a concentration step through sodium polytungstate (SPT) density gradient centrifugation [35,36,49]. However, the applicability of LM identification and quantification is limited by the following: (1) The similarities in general morphology, cell size, and numerous microreticulations on cyst surface among G. catenatum, G. microreticulatum, and G. nolleri may easily lead to misidentification [38]. (2) LM identification and counting are time-consuming and labor-intensive, and thus only suitable for a small number of samples. (3) LM identification needs personnel knowledgeable enough in the fine differences in the morphological features of dinoflagellate cysts. Therefore, the LM approach has possibly led to mis-identification and thus over- or under-quantification of G. catenatum cysts from marine sediments in the past and is surely inadequate for specific identification and quantification from a large number of sediment samples.
As a highly specific and efficient molecular biological detection method, quantitative PCR (qPCR) assay has been applied to detect the distribution and abundance of dinoflagellate cysts in sediments. For example, cysts of five HABs-causing dinoflagellates were detected in Adriatic harbor sediments [50]; Dai et al. (2020) investigated the distribution and abundance of Alexandrium catenella and A. pacificum cysts in sediments from the YS and BS using qPCR assay [51]; and Liu et al. (2021) quantified K. mikimotoi cysts’ abundance in sediments along the coast of China using the qPCR method, as well [52]. Also based on qPCR, Ben Amor et al. (2022) detected G. catenatum in environmental samples from Tunisia for the first time [53]. qPCR is advantageous over the LM method both in its accurate identification and high throughput, and is thus highly suitable for the cyst mapping of particular species of interest. In addition, qPCR is relatively more economical and also efficient in detecting those species with low cell abundance, as compared to LM and the metabarcoding high-throughput sequencing. Given that most investigations into the presence and distribution of G. catenatum cysts in China have used LM identification, the qPCR method used in the present study is believed to provide more reliable detection for the geographic distribution of G. catenatum cysts along the entire coast of China, including some open ocean areas. For the detected signal of G. catenatum in the SPT-concentrated samples, some may be concerned that the possible presence of “temporary cysts” might also be detected as resting cysts. This possibility could be largely excluded due to the two reasons following: (1) the absence or presence of a thick wall differentiates temporary cysts (pellicle cysts) from resting cysts, as the thin wall (singular layer) of temporary cysts prevents them from long survival buried in sediment and the resting cysts of the species have a characteristic thick and reticulated wall; (2) although the pellicle cysts of some species (e.g., Cochlodinium polykrikoides, Scripsiella hangoei, and Ostreopsis cf. ovata), although not necessarily temporary cysts, have been shown to remain viable for extended periods, their survival time is generally limited to a few months [13].

4.2. Distribution of G. catenatum Cysts in the Surface Sediments of Chinese Seas

Gymnodinium catenatum is an important HAB-causing species in China, with high risks of blooms and poisoning. As described in the introduction, it is not only a cosmopolitan species, but also has bloom records in the four seas of China. In particular, in the adjacent areas of Quanzhou and Zhangzhou in the ECS, a G. catenatum bloom broke out for two consecutive years in 2017 and 2018, which not only brought huge economic losses to the local aquaculture industry, but also caused harm to human health [37,54]. A comprehensive understanding of G. catenatum cysts’ distribution along the coastal marine sediments of China will help us to learn more about the ecological characteristics of this species, and provide important information for the prediction, warning, and risk assessment of G. catenatum blooms.
The results indicate that G. catenatum cysts are widely present in the sediments of the coastal seas of China, covering the vast sea area from Nansha Islands in the SCS to the Beidaihe area of the BS. Records indicate that G. catenatum blooms occurred in the spring and November of 1998 in the Pearl River Estuary [29,55], and six times in the Lianyungang between 2005 and 2017 [56,57]. In this study, cysts were present in samples taken from Guishan Island, located in the Pearl River Estuary, and Lianyungang. Additionally, while G. catenatum blooms were recorded in the ECS in 2017 and 2018, a relatively higher abundance of G. catenatum cysts was detected in ECS samples that were collected in 2018. As for the distribution of G. catenatum cysts in the BS, although the blooms were only recorded in Zhimao Bay (western Liaoning province, northern BS) in August 2007 and Tianjin, Bohai Bay (southwest BS), in August 2016 (Figure 2), this study found that G. catenatum cysts were widely distributed in BS, with 93.55% of the samples detected to be positive, indicating that G. catenatum is widely distributed in BS and there is a risk of G. catenatum blooms in this region in the future.
In this research, G. catenatum cysts in Beidaihe, BS, were also detected via a single-cyst PCR method, in which both the morphology and partial 28S rDNA sequences of G. catenatum cysts were obtained for individual cysts through a combination of morphological observation and single-cyst sequencing. This part of our results indicates that, at least partly, our qPCR detections mapped the distribution of G. catenatum cysts instead of the fragmental vegetative cells or DNA relics.

4.3. Association of G. catenatum Cysts with HABs

Results of qPCR assays show that G. catenatum cysts are widely distributed in all samples, but the abundance is not high in most samples. Wang et al. (2003) also found that G. catenatum cysts were widely distributed in a total of 10 stations from the YS, ECS, and SCS through LM identification, and the abundance was not high, ranging from 0 to 71.1 cysts g−1 [35]. Three samples from the East China Sea (ECS) contained G. catenatum cysts at relatively higher concentrations than other sea areas, suggesting a pertinence of cyst abundance to the frequent occurrences of G. catenatum blooms in the area during recent years. Two of the three stations were located in the area of the Taiwan Strait near Fuzhou, Fujian, and the third was located in an area of Zhejiang Province, some distance north of the Taiwan Strait, and all of them were sampled in April 2018. Notably, in June 2017, a G. catenatum bloom broke out in the areas of Zhangzhou and Quanzhou in Fujian Province, and the highest cell density reached 6.0 × 105 cell L−1 [32]. We therefore speculated that this bloom led to the more abundant cysts in the three abovementioned stations. In addition, the cyst abundance of the three stations ranged within 18–23 cysts g−1 (wet weight), which, however, was not as high as those counted for this from other regions (49.7 cysts g−1 in Sishili Bay, YS, [35]) and other well-known HAB-causing dinoflagellates (e.g., 0–1464 cysts g−1 of Alexandrium catenella cysts in the YS and BS [51]). This could be due to the gradual decomposition, current-induced dissipation, and shellfish predation of G. catenatum cysts over time [37]. Furthermore, cysts production rates and their subsequent accumulation in the sediment are also influenced by numerous factors in the waterbody where the cysts are formed, including temperature and its changing patterns, nutrients, currents, and even the presence of prey of the cyst-forming dinoflagellate [55,56,57,58]. It is noteworthy that Li et al. (2020) also found that G. catenatum cysts were the dominant species in the whole year of 2018 in the sediment investigation of Meizhou Bay, but the annual average of cyst abundance was 20.8 cysts g−1 (dry weight) [59].
It is still highly possible that Gymnodinium catenatum cysts with low abundance are the seeds that initiate blooms via germination. A recent study has proved that Alexandrium catenella blooms in the Chilean fjord system originated from low densities of cysts dispersed throughout the area [60]. Liu et al. (2020) pointed out that the germination time of G. catenatum cysts was shortened as the temperature increased; it reduced from 3 days to 1 day when the temperature increased from 20 °C to 23 °C [36]. This means that the cysts can germinate into vegetative cells in a short time and enter the water column when the temperature conditions are suitable, providing “seeds” for the initiation of blooms. The division rate of G. catenatum from the Taiwan Strait is the highest at 23 °C [36], the growth rate of G. catenatum from the Fujian coastal area is highest at 0.28 d−1, and the maximum cell density can reach 12,000 cells mL−1 [61]. After germination, G. catenatum cells can grow rapidly and reproduce under favorable environmental conditions, leading to the formation of G. catenatum bloom in the ECS. Assuming an abundance of 1 cyst g−1 wet sediment (average abundance detected in this study), a germination rate of 100%, a growth rate of 0.2 d−1, a cell mortality rate of 0, and a wet weight sediment density of 2 g cm−3 [62], it can be postulated that G. catenatum will reach the bloom benchmark density of 5 × 105 cells L−1 in 40 days. Therefore, with favorable environmental condition and enough time for vegetative growth prior to the blooming season, cysts with low abundance may seed HABs in any and many sea areas of China in any forthcoming year, and especially in areas where HABs used to occurred frequently.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jmse12091651/s1, Figure S1: Standard curve with 10-fold serial dilutions of plasmid DNA of Gymnodinium catenatum for qPCR; Table S1: Sampling information and Gymnodinium catenatum cyst abundance in the sediments along the coastal seas of China; Table S2: Representative species and Genbank accession numbers included in the alignment.

Author Contributions

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

Funding

This research was funded by the National Key Research and Development Program of China (grant No. 2022YFC3105201), the Science and Technology Innovation Project of Laoshan Laboratory (grant No. LSKJ202203700), the Key Research Infrastructures in the CAS Field Stations of the Chinese Academy of Science (grant No. KFJ-SW-YW047), and the Science and Technology Basic Resources Investigation Program of China (2018FY100200).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are contained within the article and Supplementary Material.

Acknowledgments

We would like to express our gratitude to the two anonymous reviewers for their constructive suggestions and comments. We acknowledge financial support from the National Key Research and Development Program of China, grant number 2022YFC3105201, the Science and Technology Innovation Project of Laoshan Laboratory, grant number LSKJ202203700, the Key Research Infrastructures in the CAS Field Stations of the Chinese Academy of Science, grant number KFJ-SW-YW047, and the Science and Technology Basic Resources Investigation Program of China, grant number 2018FY100200.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Jmse 12 01651 g001
Figure 1. Location of the sediments collected from all four seas of China. Notes: red dots indicate that the samples were concentrated with SPT, while the black dots indicate that the sediment samples were subjected to DNA extraction without a prior SPT concentration. Refer to Table S1 for the coordinates and other environmental conditions of all sampling sites.
Figure 1. Location of the sediments collected from all four seas of China. Notes: red dots indicate that the samples were concentrated with SPT, while the black dots indicate that the sediment samples were subjected to DNA extraction without a prior SPT concentration. Refer to Table S1 for the coordinates and other environmental conditions of all sampling sites.
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Figure 2. Distribution of Gymnodinium catenatum cysts in the sediments collected from all four seas of China. Notes: All closed circles (red and black) represent the samples detected to be positive in qPCR detections for Gymnodinium catenatum, while open circles (red and black) represent the samples detected to be negative in qPCR detection for G. catenatum. Red dots indicate that the samples were SPT-concentrated for cysts prior to DNA extraction, while the black dots indicate that the samples were subjected to DNA extraction directly. Yellow triangles represent the “hot spots” where blooms of G. catenatum have been reported. The location, date frequency, and references of these blooms are as follows: (1) Zhimao Bay, once in August 2007 [31]; (2) Tianjin Harbor Economic Zone, once in August 2016 [45]; (3) Lianyungang, five times in Haizhou Bay from 2005 to 2010 (twice in October 2005, once in January 2006, once in October 2006, and another one in July 2010) [45,46], Paidan estuary to Liezi estuary, once in May 2017 [45]; (4) Fujian Province sea area, Quanzhou and Zhangzhou, once in June 2017 [32], Quanzhou Bay, once in June 2018 [37]; and (5) Pearl River estuary, once in spring and another one in November 1998 [29,47].
Figure 2. Distribution of Gymnodinium catenatum cysts in the sediments collected from all four seas of China. Notes: All closed circles (red and black) represent the samples detected to be positive in qPCR detections for Gymnodinium catenatum, while open circles (red and black) represent the samples detected to be negative in qPCR detection for G. catenatum. Red dots indicate that the samples were SPT-concentrated for cysts prior to DNA extraction, while the black dots indicate that the samples were subjected to DNA extraction directly. Yellow triangles represent the “hot spots” where blooms of G. catenatum have been reported. The location, date frequency, and references of these blooms are as follows: (1) Zhimao Bay, once in August 2007 [31]; (2) Tianjin Harbor Economic Zone, once in August 2016 [45]; (3) Lianyungang, five times in Haizhou Bay from 2005 to 2010 (twice in October 2005, once in January 2006, once in October 2006, and another one in July 2010) [45,46], Paidan estuary to Liezi estuary, once in May 2017 [45]; (4) Fujian Province sea area, Quanzhou and Zhangzhou, once in June 2017 [32], Quanzhou Bay, once in June 2018 [37]; and (5) Pearl River estuary, once in spring and another one in November 1998 [29,47].
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Figure 3. Abundance of Gymnodinium catenatum cysts in the sediments collected from all four seas of China ((A). Bohai Sea and Yellow Sea; (B). East China Sea; (C). South China Sea).
Figure 3. Abundance of Gymnodinium catenatum cysts in the sediments collected from all four seas of China ((A). Bohai Sea and Yellow Sea; (B). East China Sea; (C). South China Sea).
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Figure 4. Cysts of Gymnodinium catenatum in the sediments collected from Beidaihe, with their identity confirmed using the single-cyst-PCR and sequencing ((A). cyst 1; (B). cyst 2; (C). cyst 3). Scale bars: 20 μm.
Figure 4. Cysts of Gymnodinium catenatum in the sediments collected from Beidaihe, with their identity confirmed using the single-cyst-PCR and sequencing ((A). cyst 1; (B). cyst 2; (C). cyst 3). Scale bars: 20 μm.
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Liu, X.; Tao, Z.; Liu, Y.; Hu, Z.; Deng, Y.; Shang, L.; Lim, P.-T.; Chai, Z.; Tang, Y.-Z. Resting Cysts of the Toxic Dinoflagellate Gymnodinium catenatum (Dinophyceae) Ubiquitously Distribute along the Entire Coast of China, with Higher Abundance in Bloom-Prone Areas. J. Mar. Sci. Eng. 2024, 12, 1651. https://doi.org/10.3390/jmse12091651

AMA Style

Liu X, Tao Z, Liu Y, Hu Z, Deng Y, Shang L, Lim P-T, Chai Z, Tang Y-Z. Resting Cysts of the Toxic Dinoflagellate Gymnodinium catenatum (Dinophyceae) Ubiquitously Distribute along the Entire Coast of China, with Higher Abundance in Bloom-Prone Areas. Journal of Marine Science and Engineering. 2024; 12(9):1651. https://doi.org/10.3390/jmse12091651

Chicago/Turabian Style

Liu, Xiaohan, Zhe Tao, Yuyang Liu, Zhangxi Hu, Yunyan Deng, Lixia Shang, Po-Teen Lim, Zhaoyang Chai, and Ying-Zhong Tang. 2024. "Resting Cysts of the Toxic Dinoflagellate Gymnodinium catenatum (Dinophyceae) Ubiquitously Distribute along the Entire Coast of China, with Higher Abundance in Bloom-Prone Areas" Journal of Marine Science and Engineering 12, no. 9: 1651. https://doi.org/10.3390/jmse12091651

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