Mapping the resting cysts of dinof lagellate Alexandrium catenella along the coast of Qinhuangdao, China*

Wenjiao TANG , Huixia GENG , Yanjuan XI , Qingchun ZHANG ,**,Xuexi TANG , Rencheng YU ,

1 College of Marine Life Science, Ocean University of China, Qingdao 266003, China

2 CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences,Qingdao 266071, China

3 Key Laboratory for Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, China

4 University of Chinese Academy of Sciences, Beijing 100049, China

5 Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China

6 Hebei Academy of Ocean and Fishery Science, Qinhuangdao 066200, China

Abstract Dinof lagellate Alexandrium catenella is a cosmopolitan bloom-forming species with complex life cycle, the formation and germination of resting cysts are critical for its bloom dynamics. In the coastal waters of Qinhuangdao, A. catenella has been identif ied as the major causative agent for paralytic shellf ish poisoning, but there is little knowledge concerning its resting cysts in this region. In this study, three surveys were carried out along the coast of Qinhuangdao from 2020 to 2021 to map the distribution of A. catenella resting cysts, using a quantitative PCR (qPCR) assay specif ic for A. catenella. The resting cysts were detected in surface sediments during all the three surveys, and their distribution patterns were similar. High abundance of resting cysts (maximum 1 300 cysts/g sediment (wet weight)) were found in a region (119.62°E- 119.99°E,39.67°N- 39.98°N) northeast to the coastal waters of Qinhuangdao, where surface sediments were mainly composed of clay and silt (percentage above 50%). Prior to the formation of the A. catenella bloom in March 2021, the abundance of A. catenella vegetative cells in seawater had extremely signif icant positive correlation with the abundance of resting cysts in surface sediments, ref lecting the important role of resting cysts in the initiation of A. catenella blooms. As far as we know, this is the f irst report on the distribution of A. catenella cysts along the coast of Qinhuangdao. The results will off er a sound basis for the future monitoring and mitigation of toxic A. catenella blooms and paralytic shellf ish poisoning events in this region.

Keyword: harmful algal bloom (HAB); Alexandrium catenella; resting cyst; paralytic shellf ish toxin (PST);Qinhuangdao

1 INTRODUCTION

Paralytic shellf ish toxins (PSTs), a group of potent neurotoxins, are widely distributed around the world(Asakawa et al., 1995; Lassus et al., 2016). PSTs are mainly produced by marine dinof lagellates, such asAlexandriumspp.,Gymnodiniumcatenatum,andPyrodiniumbehamensevar.compressum), and freshwater cyanobacteria in the generaAnabaena,Aphanizomenon,Cylindrospermopsis,Lyngbya,Planktothrix, andScytonema(Anderson et al.,2012a; Christensen and Khan, 2020). In marine environments, PSTs produced by toxic dinof lagellates can accumulate in f ish and shellf ish at the high trophic levels via marine food web. Consumption of contaminated animals poses a serious threat to human health, and sometimes leads to mortality of victims. About 1 600 paralytic shellf ish poisoning(PSP) episodes were recorded prior to the 1970s,mainly in Europe, East Asia, and North America. The distribution of PSP events has expanded gradually around the world since then, and more than 900 poisoning episodes were recorded globally from 1970 to 1984 (U. S. National Offi ce for Harmful Algal Blooms, https://hab.whoi.edu/). It is estimated that about 2 000 PSP incidents occur worldwide every year (Berdalet et al., 2016; Wang et al., 2016; Brown et al., 2020; Yu et al., 2020).

In China, more than 600 suspected PSP cases have been reported so far, and PSTs were detected in many shellf ish samples (FAO, 2004; Liu et al., 2016; Liang et al., 2019). In Daya Bay, for example, PST toxicity of the scallops’ digestive gland screened by mouse bioassay reached 227 340 μg STX eq./kg in 1999(Jiang et al., 2000). In another study in the northern Yellow Sea, the maximum PST toxicity of scallops reached 65 140 μg STX eq./kg in 2007 (Xia et al.,2010). Recently, PSP episodes were reported in Fujian province and Hebei province. Toxic dinof lagellateGymnodiniumcatenatumhas been identif ied as the causative agent for PSP incidents in Fujian province(Chen et al., 2018). In Hebei province, several PSP episodes were reported in Qinhuangdao, a coastal city located near the Bohai Sea (Ding et al., 2017). The maximum level of PST content in a sample of musselMytilusgalloprovincialisreached 40 561 μg/kg in the spring of 2016 (Liang et al., 2019). PSTs detected in shellf ish samples were mainly composed of carbamate toxins, andA.catenellaproducing only carbamate toxins was proposed recently as the major causative species of the PSP events in this region (Yu et al., 2021). The follow-up studies found that PST contamination in shellf ish collected from Qinhuangdao occurred shortly after the bloom ofA.catenellain April (Tang et al., 2022).

Dinof lagellateA.catenellahas a complex life cycle and diverse modes of reproduction (Anderson et al.,2012a), and the formation ofA.catenellabloom is closely associated with its resting cysts (Ribeiro et al.,2011; Lundholm et al., 2011; Miyazono et al., 2012;Dai et al., 2020). Vegetative cells ofA.catenellain seawater mainly reproduce asexually by binary f ission.When exposed to sudden environmental changes or other stimuli, the vegetative cells ofA.catenellamay lose their f lagella and form temporary cysts(Anderson et al., 2012b). The temporary cysts can return to the stage of vegetative cells quickly under optimal environmental conditions. More importantly,the vegetative cells ofA.catenellacan undergo sexual reproduction to form thick-walled, non-motile resting cysts, which signif icantly improves its adaptability to environmental changes and the capability of population dispersal (Erdner et al., 2010; Genovesi et al., 2015). The resting cysts ofA.catenellaformed in the water column deposit to the bottom and reserve in sediments. Therefore, the distribution of resting cysts in sediments is generally consistent with the distribution of vegetative cells in seawater (Yamaguchi et al., 1995; Shimada and Miyazono, 2005), and the variation of resting cyst abundance in a sediment core can ref lect the long-term changes ofA.catenellapopulation in a specif ic region (He et al., 2008;Anderson et al., 2014; Tang et al., 2016). Moreover,the germination ofA.catenellaresting cysts releases vegetative cells into seawater, which could proliferate rapidly under optimal environmental conditions to form a bloom (Bravo et al., 2006; Kim et al., 2020).Based on the data of cyst abundance and distribution in surface sediments, numerical models have been successfully developed to predict the blooms ofA.catenella(previously named asA.fundyense) in the Gulf of Maine, America (Anderson et al., 2005,2014; He et al., 2008).

Several strains ofA.catenellahave been established using the resting cysts isolated from the coastal region of Qinhuangdao (Gu et al., 2013; Yu et al., 2021), but the knowledge on the distribution ofA.catenellaresting cysts is still quite limited in this region. In previous studies, a sensitive quantitative PCR (qPCR) assay specif ic forA.catenellahas been established and applied successfully to detect vegetative cells and resting cysts ofA.catenellain the Bohai Sea and Yellow Sea (Gao et al., 2015; Dai et al., 2020). In this study, the qPCR assay was used to detectA.catenellaresting cysts in surface sediments,as well as its vegetative cells in seawater, during the three surveys in 2020 and 2021. The study aims to map the distribution ofA.catenellaresting cysts along the coast of Qinhuangdao and to explore their relationship withA.catenellablooms. The results would be helpful to understand the mechanisms ofA.catenellablooms and to develop monitoring strategies accordingly.

2 MATERIAL AND METHOD

2.1 Sample collection

Fig.1 The sampling sites along the coast of Qinhuangdao in the Bohai Sea

Three cruises were carried out in June and October 2020, and March 2021. Each cruise covered about 40 sampling sites along 8 sections (Fig.1).Environmental parameters including temperature and salinity were measured using a self-contained STD prof iling system (RBR, Linkocean Technologies Ltd., Canada). Surface sediment samples were collected using a grab sampler (DDC1-2, GuoKe).Approximately 200-g sediment was stored in a freezer at -20 °C to detectA.catenellaresting cysts and to measure sediment grain size. A total of 111 sediment samples (32, 40, and 39 samples in June and October 2020, and March 2021, respectively)were collected during the three cruises. In June 2020 and March 2021, phytoplankton samples were also collected to determine the abundance and distribution ofA.catenellavegetative cells. Surface seawater was collected using a submersible pump at each sampling site, and 1-L seawater was passed through a sieve (mesh size 200 μm) to remove zooplankton.The f iltrate was then f iltered through a 0.4-μm polycarbonate f iber membrane (HTTP, Millipore,Ireland) to collect phytoplankton. The membranes were stored at -80 ℃ for qPCR assay. A total of 32 phytoplankton samples were collected in June 2020,and 40 samples were collected in March 2021.

2.2 Sample preparation

Extraction of the genomic DNA of resting cysts was performed according to the protocol described by Kamikawa et al. (2007) and Dai et al. (2020). Brief ly,the frozen sediment samples were thawed at room temperature, and about 5-g sediment was weighed and put into a beaker. The sediment was mixed with seawater f iltered through cellulose-ester membranes(pore diameter 0.4 μm), and further treated in an ultrasonic cleaner (Branson, 2510, USA). The mixture was then f iltered successively through 120-μm and 20-μm sieves, and the fraction between 20 and 120 μm was collected and centrifuged at 3 000×gfor 10 min using a high-speed refrigerated centrifuge (Sigma,3-16 K, Germany). The precipitate was carefully weighed to extracted DNA using the PowerSoil DNA Isolation Kit (Qiagen, USA), following the instruction manual. The DNA recovered from the silica gel f ilter membrane was used for qPCR assay.

The genomic DNA of phytoplankton samples was extracted using a cetyltrimethyl ammonium bromide(CTAB) method described by Winnepenninckx et al.(1993). The DNA was dissolved in 30-μL TE buff er and stored at -20 °C for qPCR assay.

2.3 qPCR assay for Alexandrium catenella

A qPCR assay developed by Gao et al. (2013,2015) was used to determine the abundance ofA.catenellavegetative cells in seawater and resting cysts in sediments. A Bio-Rad CFX96 TouchTMreal-time PCR detection system was used to perform the analysis. The primers were AtI-F(5ʹ-GCTTGGTGGGAGTGTTGCAC-3ʹ) and AtI-R(5ʹ-TAAGTCCAAGGAAGGAAGCATC-3ʹ), and the TaqMan probe was AtI-P (5ʹ-AGAGCTTTGGGCTGTGGGTGTA-3ʹ). The volume of qPCR reaction mixture was 10 μL, containing 5-μL 2× qPCR buff er(TaKaRa, China), 0.4 μL of 10-nmol/L forward primer, 0.4 μL of 10-nmol/L reverse primer, 0.4 μL of 10-nmol/L probes labeled with Texas Red and BHQ2 at the 5ʹ and 3ʹ ends, 2.8 μL of ddH2O, and 1 μL of DNA template. The qPCR reaction started from an initial denaturing at 95 °C for 30 s, followed by 40 cycles of denaturing at 95°C for 5 s and annealingelongating at 60 °C for 30 s.

Fig.2 Distribution and abundance of Alexandrium catenella resting cysts in the coastal waters of Qinhuangdao in June (a),October (b) 2020, and March 2021 (c)

A strain ofA.catenella(MEL91) isolated from the coastal waters of Qinhuangdao was cultured in laboratory (Yu et al., 2021), and the resting cysts were induced by nutrient starve. The cysts were collected using the sodium polytungstate gradient centrifugation method (Bolch, 1997) and picked out under a microscope. The cysts were used to optimize qPCR conditions and to establish a standard calibration curve for the qPCR assay. Vegetative cells of the strain MEL91 were also used to establish the calibration curve to determineA.catenellacells in seawater.

2.4 Sediment analysis

Sediment samples collected in March 2021 were thawed at room temperature, and approximately 0.2-g sediment were placed into a beaker and mixed with the seawater f iltered through cellulose-ester membranes(pore diameter 0.4 μm). After ultrasonic treatment for 1 min in an ultrasonic cleaner, the samples were analyzed using a laser particle size analyzer (1190,Cilas, France). The measurement range of particle size was from 0.04 to 2 500 μm, the accuracy was less than 3%, and the repeatability was less than 1%.Following the protocol of Folk-Walker classif ication(Folk et al., 1970), the sediment particles were divided into three parts based on the measured grain sizes, namely clay (<4 μm), silt (4- 63 μm), and sand(>63 μm). Among them, the clay and silt were named collectively as f ine-grained particles (<63 μm).

2.5 Statistical analysis

Statistical analyses were performed using IBM SPSS 26.0 (IBM, Armonk, NY, USA), and the correlation between two factors was analyzed using the Pearson’s chi-squared test. The correlation was considered signif icant whenP<0.05 (2-sided signif icance testing). The diff erence analysis between two data was analyzed usingt-test, and the diff erence was considered signif icant whenP<0.05(2-sided signif icance testing). The distribution map ofA.catenellaresting cysts, vegetative cells, and environmental parameters in the study region were prepared using Surfer 13.0 (Golden Software, Inc.,USA).

3 RESULT

3.1 Distribution and abundance of Alexandrium catenella resting cysts in surface sediments

The abundance ofA.catenellaresting cysts in surface sediment samples collected in June and October 2020 and March 2021 are illustrated in Fig.2. In June 2020, the cyst abundance in 32 samples was in a range of ND (not detected) to 1 300 cysts/g sediment (wet weight, (WW)), with the average abundance of 162 cysts/g sediment (WW). In October 2020, the cyst abundance in 40 samples ranged from ND to 424 cysts/g sediment (WW), and with the average abundance of 39 cysts/g sediment (WW). In March 2021, the cyst abundance of 39 samples was in a range of ND to 459 cysts/g sediment (WW) with the average abundance of 59 cysts/g sediment (WW).

Distribution patterns ofA.catenellaresting cysts during the three surveys were very similar. Cyst abundance in the northern part of the study region(sections 1- 4) was signif icantly higher than the southern part (sections 5 - 8) (P<0.05). In June 2020,the abundance of resting cysts on average in the northern and southern parts of the study region was 238 and 33 cysts/g sediment (WW), respectively. In October 2020, the values decreased to 68 and 4 cysts/g sediment (WW), respectively. The abundance of resting cysts in the two parts was 109 and 0 cysts/g sediment (WW), respectively, in March 2021.

Fig.3 Distribution and abundance of Alexandrium catenella vegetative cells in the coastal waters of Qinhuangdao in June 2020 (a) and March 2021 (b)

3.2 Distribution and abundance of Alexandrium catenella vegetative cells in seawater

The abundance ofA.catenellavegetative cells in seawater collected in June 2020 and March 2021 was determined by qPCR assay. The abundance was extremely low in the whole survey region in June 2020, andA.catenellacells were only detected at the site 1-1 (7 cells/L) and the site 3-0 (4 cells/L) (Fig.3a).In March 2021,A.catenellacells were widely detected in the study region, and the abundance ranged from 1 to 206 cells/L, averaged 45 cells/L. The maximum abundance ofA.catenellawas recorded at the site 2-0 (Fig.3b). The average abundance ofA.catenellacells in the northern part (57 cells/L, sections 1- 4)was higher than that in the southern part (31 cells/L,sections 5- 8).

3.3 Spatial variation of environmental parameters

Seawater temperature and salinity in the coastal waters of Qinhuangdao were measured in June 2020 and March 2021 (Fig.4). Seawater temperature was in a range of 18.7- 24.2 °C in June 2020, and the salinity was in a range of 31.9- 32.3. In the southern part of the study region, there was an apparent onshore intrusion of warm seawater with relatively high salinity along the sections 7 & 8. In the central part of the study region, there was a similar coastward intrusion of high salinity tongue along the sections 3 & 4, but the temperature of seawater was much lower. In March 2021, seawater temperature was in a range of 2.4- 7.4 °C, and salinity was in a range of 31.4- 32.3. Seawater with relatively high salinity and low temperature occupied the northern part of the study region.

In the study region, approximately one third (32.5%)of sediment samples were composed of silt and clay(grain size≤63 μm), and the others were mainly composed of sand (grain size>63 μm) (Fig.5). Silt and clay were primarily distributed in the northern part of the study region (sections 1- 4), and the percentage of f ine-grained particles in the northern part was higher than 50%. In contrast, grain size of sediments in the sections 5- 8 was much coarser, and the percentage of f ine-grained particles was generally less than 40%,except for the sampling sites 5-4 and 8-2.

3.4 Relationships between abundance of Alexandrium catenella resting cysts, vegetative cells, and environmental parameters

The abundance ofA.catenellaresting cysts had a signif icant positive correlation with the percentage of f ine-grained particles in both June 2020 and March 2021 (P<0.05 andP<0.01) (Tables 1 & 2). Resting cysts ofA.catenellawere mainly detected at the sites with the percentage of f ine-grained sediment above 50%. Almost noA.catenellaresting cysts were detected in the region with the percentage of f inegrained particles lower than 50%. The abundance ofA.catenellavegetative cells was extremely low in June 2020, and it had a signif icant negative correlation with temperature (P<0.05). Prior to the formation ofA.catenellabloom in March 2021, the abundance ofA.catenellavegetative cells had little correlation with seawater temperature and salinity, but it had an extremely signif icant positive correlation with resting cyst abundance in surface sediment (P<0.01)(Table 2).

Fig.4 Temperature (a, b) and salinity (c, d) of surface seawater in the coastal waters of Qinhuangdao in June 2020 (a, c) and March 2021 (b, d)

4 DISCUSSION

4.1 Distribution of Alexandrium catenella resting cysts along the coast of Qinhuangdao

In our previous studies, three isolates ofA.catenellahave been established by germination of resting cysts collected from surface sediment along the coast of Qinhuangdao (Yu et al., 2021). The results could conf irm the presence ofA.catenellaresting cysts in this region, but there is little information available about their distribution characteristics. In this study,the distribution ofA.catenellaresting cysts along the coast of Qinhuangdao was investigated for the f irst time using a qPCR assay specif ic forA.catenella.The resting cysts ofA.catenellawere detected in surface sediments during all three cruises, suggesting that the resting cysts exist all year round in the coastal waters of Qinhuangdao. High abundance ofA.catenellaresting cysts were found in the northern part of the study region (119.62°E- 119.99°E,39.67°N- 39.98°N), which is northeast to the coastal waters of Qinhuangdao.

Fig.5 Percentage of f ine-grained particles in surface sediments in the coastal waters of Qinhuangdao in March 2021

Table 1 Correlation between abundances of Alexandrium catenella resting cysts, vegetative cells, and seawater temperature and salinity in June 2020

Table 2 Correlation between abundances of Alexandrium catenella resting cysts, vegetative cells, and seawater temperature and salinity in March 2021

Many processes, such as cyst formation and germination, sediment composition, hydrodynamic processes, and animal predation, could aff ect the distribution ofA.catenellaresting cysts (Persson,2000; Anderson et al., 2005; Richlen et al., 2016).Sediment composed of f ine-grained particles can provide a better condition for the preservation of resting cysts, while the cysts in sediment composed of sand are easily lost under the external impacts (Anderson et al., 2003). A previous study in the northern Yellow Sea found that there was a strong positive correlation between the abundance ofAlexandriumcysts and the percentage of f ine-grained particles, while a negative correlation between cyst abundance and sand percentage (Shi et al., 2011). Similarly, the investigations in the Yellow Sea and the Bohai Sea also found strong correlation between the abundance ofA.catenellaresting cysts and the percentage of f ine-grained particles in surface sediments (Dai et al.,2020). In this study, the region with high abundance ofA.catenellaresting cysts is also featured by a high percentage of f ine-grained particles. It can be implied that sediment composition is a critical factor aff ecting the distribution of resting cysts (Anderson et al., 2005;Shin et al., 2013). Besides, hydrodynamic processes also aff ect the distribution of resting cysts. The Bohai Sea is strongly aff ected by tidal currents, but there is an amphidromic point close to Qinhuangdao (Zhang and Yang, 2013; Luo and Liu, 2015). Therefore, the coastal waters of Qinhuangdao could off er a stable environment for the growth ofA.catenellavegetative cells, and for the deposition and preservation of its resting cysts formed during the bloom.

4.2 The relationship between Alexandrium catenella resting cysts and its blooms

Many studies demonstrate the important role of resting cysts in the initiation ofA.catenellablooms(Kim et al., 2002; Genovesi et al., 2009; Natsuike et al., 2017; Shin et al., 2021). Our study in the coastal waters of Qinhuangdao found thatA.catenellabloomed in April 2021 (Tang et al., 2022). Prior to the formation of the bloom, vegetative cells ofA.catenellawere detected in the water column during the cruise in March 2021. It was found that vegetative cells were mainly distributed in the northern part of the study region, similar to the resting cysts in surface sediment. There is a signif icant positive correlation between the abundance of vegetative cells and the abundance of resting cysts, while no signif icant correlation was found between vegetative cells and other environmental parameters. It can be deduced that vegetative cells ofA.catenellain seawater prior to the bloom formation were mainly resulted from the germination of resting cysts in surface sediment.The vegetative cells could then grow rapidly under optimal conditions to form the bloom. Therefore,resting cysts preserved in sediments could serve as a seed bank to modulate the dynamics ofA.catenellablooms in the coastal waters of Qinhuangdao.

Temperature is a critical factor for the germination of resting cysts and the formation of blooms(Anderson and Morel, 1979; Anderson et al., 1990;Kim et al., 2002; Natsuike et al., 2017).A.catenellais a cold-water species, which forms blooms generally in the temperature range of 5- 15 °C. In the Gulf of Maine, for example, recurrent blooms ofA.catenellawere found in a temperature range of 10- 13 ℃, and the germination ofA.catenellacysts mainly occurred in a range of 7- 15 ℃ (Tobin et al., 2019). In a study in the coastal waters of South Korea, it was also found that up to 73% of theA.catenellaresting cysts germinated in November. The vegetative cells ofA.catenellagerminated from resting cysts had low cell density in winter, and grew rapidly to form a bloom(about 4×104cells/L) in mid-April when seawater temperature reached 15 °C (Kim et al., 2020). The results in the coastal waters of Qinhuangdao are in accordance with the f indings in the Gulf of Maine and the coastal waters of South Korea. From winter to spring, the gradual increase of seawater temperature promotes the germination ofA.catenellaresting cysts and drives the bloom formation. The studies also demonstrate impacts ofA.catenellavegetative cells on the resting cysts in sediment. Among the three cruises, the distribution patterns of resting cysts were similar in the study region, but the abundance of resting cysts in June 2020 was much higher than March 2021 and October 2020. Our study found that the bloom ofA.catenelladeclined in May (Tang et al., 2022), probably due to the inhibition of rising temperature on the growth ofA.catenella. Water temperature along the coast of Qinhuangdao exceeds 20 °C in summer, beyond the optimal temperature range for the growth ofA.catenella(Tobin et al.,2019). The resting cysts formed at the late stage of bloom deposit to the bottom and lead to the relatively higher abundance of resting cysts in June. In the Gulf of Maine, it was also found that remaining cells after the bloom ofA.catenellaformed resting cysts, which deposited to sediment and contributed to the seed bed of resting cysts (Anderson, 1998).

5 CONCLUSION

In this study, three surveys were carried out in the coastal waters of Qinhuangdao to map the distribution ofA.catenellaresting cysts in surface sediments and to explore their relationships withA.catenellablooms. The distribution pattern ofA.catenellacysts along the coast of Qinhuangdao was revealed for the f irst time using a species-specif ic qPCR assay. A region with high abundance of resting cysts (119.62°E- 119.99°E, 39.67°N- 39.98°N)was identif ied northeast to the coastal waters of Qinhuangdao, where surface sediments were mainly composed of f ine-grained particles (percentage above 50%). Prior to the formation of theA.catenellabloom in March 2021, the abundance ofA.catenellavegetative cells in seawater had a signif icant correlation with the abundance of resting cysts in surface sediments, ref lecting the important role of resting cysts in the initiation ofA.catenellablooms.The results will off er a sound basis for the future monitoring and mitigation of toxicA.catenellablooms and PSP events in this region.

6 DATA AVAILABILITY STATEMENT

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

7 ACKNOWLEDGMENT

We acknowledge all the researchers who worked on survey cruises for the collection of f ield samples,and Professors Jiabo ZHANG and Gang WANG from Marine Geological Resources Survey Center of Hebei Province for providing seawater temperature and salinity data.