Seasonal variability of Protoceratium reticulatum and yessotoxins in Japanese scallop Patinopecten yessoensis in northern Yellow Sea of China

Toxicon 139 (2017) 31e40

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Seasonal variability of Protoceratium reticulatum and yessotoxins in Japanese scallop Patinopecten yessoensis in northern Yellow Sea of China Lei Liu a, b, Ning Wei c, Yuxiao Gou c, Dongmei Li b, Yubo Liang b, *, Daoyan Xu b, Renyan Liu b, Shifeng Sui b, Tianjiu Jiang a, ** a b c

College of Life Science and Technology, Jinan University, Guangzhou, 510632, China National Marine Environmental Monitoring Center, SOA, Dalian, 116023, China Dalian Ocean University, Dalian, 116023, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 May 2017 Received in revised form 26 September 2017 Accepted 28 September 2017 Available online 29 September 2017

This paper reports a toxic strain of Protoceratium reticulatum, its morphology, phylogeny, yessotoxins (YTXs) production and abundance in northern Yellow Sea of China from 2011 to 2015 was investigated. YTXs in hepatopancreas and edible parts of bottom sowing cultured Japanese scallop Patinopecten yessoensis in this sea area were determined weekly for 5 years. Other potential producers of YTXs, Gonyaulax spinifera and Lingulodinium polyedrum, were also investigated. Results revealed that Protoceratium reticulatum strain from the northern Yellow Sea belongs to a geographically widely distributed species. Motile cells of Protoceratium reticulatum contribute to YTXs in Japanese scallop, and G. spinifera may also be a potential contributor. Resting cysts of Protoceratium reticulatum, G. spinifera, and L. polyedrum in sediments were possibly important origins of YTXs in scallop cultured at sea bottom. YTXs in scallop decreased from 2011 to 2015, most toxins were concentrated in hepatopancreas, while a small portion in edible parts which was safe for consumption the whole year around. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Protoceratium reticulatum Yessotoxin Patinopecten yessoensis Northern Yellow Sea

1. Introduction Accumulation of phytotoxins in bivalve shellfish is harmful to human health and aquaculture industry. Yessotoxins (YTXs) are polyether toxins and were first isolated from digestive glands of the Japanese scallop Patinopecten yessoensis (Murata et al., 1987). Dinoflagellates Protoceratium reticulatum (syn.: Gonyaulax grindleyi) (Satake et al., 1997), Lingulodinium polyedrum (syn.: Gonyaulax polyedra Stein) (Paz et al., 2004), and Gonyaulax spinifera (Rhodes et al., 2006; Riccardi et al., 2009) may produce YTXs, which can damage cardiac muscles, liver, pancreas and neuronal tissues in
rez et al., 2016). YTXs are considered as potential risk mice (Sala-Pe for human health with a lethal dose between 80 and 750 mg/kg (Paz et al., 2008). European Union established 1 mg kg
1 (EC 853/ 2004) as maximum level permitted in shellfish, this value was

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (T. Jiang).

(Y.

https://doi.org/10.1016/j.toxicon.2017.09.015 0041-0101/© 2017 Elsevier Ltd. All rights reserved.

Liang),

[email protected]

amended to 3.75 mg kg
1 recently (EC 786/2013). YTXs were also monitored in shellfishes from Japan, Norway, Chile, New Zealand, Italy, and Spain (Paz et al., 2013). The toxic dinoflagellate Protoceratium reticulatum is widely distributed in coastal waters around the world and was reported in Argentina, Arctic, Brazil, Canada, Chile, Japan, Italy, Mexico, New € der Zealand, Norway, Russia, South Africa, Spain, UK, and USA (Ro
rez et al., et al., 2012; Paz et al., 2013; Akselman et al., 2015; Sala-Pe 2016). Protoceratium reticulatum generates more than 100 YTX analogues, and its toxin profiles differ among origin of strains from New Zealand, Japan, Norway, Italy, UK, Canada, Spain, and USA (Paz et al., 2013). YTX is the major toxin in Protoceratium reticulatum, and only homoYTX was found to be the main toxin in two strains from Japan and Spain (Konishi et al., 2004; Paz et al., 2007). YTXs produced in cultured Protoceratium reticulatum measured 0.9e79 pg YTX cell
1, 1.5 pg YTX cell
1 in L. polyedrum, and 200 pg YTX cell
1 in G. spinifera € der et al., 2012). Quantity of YTXs secreted by Protoceratium (Ro reticulatum were reported to be influenced by temperature, light, growth phase, and nutritional conditions (Guerrini et al., 2007; Mitrovic et al., 2005; Paz et al., 2006, 2013).

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L. Liu et al. / Toxicon 139 (2017) 31e40

YTXs were detected in bivalves of Patinopecten yessoensis, Chlamys ferreri, Argopectens irrardias, Crassostrea gigas, and Mytilus edulis in coastal waters of Yellow Sea in China (Gao et al., 2010, 2012; Chen et al., 2014). This study aimed to isolate Protoceratium reticulatum strain from northern Yellow Sea in China and describe its taxonomical and toxicological features. By counting dinoflagellates of Protoceratium reticulatum, L. polyedrum, and G. spinifera motile cells in water column and resting cysts in sediment and detecting YTXs in scallop Patinopecten yessoensis cultured at sea bottom every month all year, this study attempted to determine the relationship between toxic dinoflagellate biomass and toxin contents in scallops.

(Eclipse E100, Nikon, Japan). Then, cells were transferred to individual wells of tissue culture plates (Costar 96 well, Corning, USA) containing 200 mL of sterile native seawater at a salinity of 33 from the collected site and cultured in an illumination incubator (HPG 4000G, Ruihua Instrument & Equipment Co., Ltd., Wuhan, China) at 15  C under artificial light under an irradiance of 100e125 mmol photons m
2$s
1 on a 12:12 h light/dark regimen. Parafilm was used to prevent evaporation in culture medium. After four weeks, uni-algal isolates were transferred to 24-well tissue culture plates (Costar 24 well, Corning, USA), L1-Si medium (Guillard and Ryther, 1962) was used. Two months later, only one strain, named ZZD01, survived, and cultures were maintained and grown in 100 mL Erlenmeyer flasks under the same conditions as described above.

2. Materials and methods 2.3. Morphological identification of Protoceratium reticulatum 2.1. Study area Zhangzi Island (122.73310 E, 39.04078 N) is located in northern Yellow Sea of China on the path of Yellow Sea Warm Current (Fig. 1). This island comprises an area measuring 2200 km2 and with a maximum depth of 45 m. In this area, the Yellow Sea Warm Current is enhanced in winter and weakened in summer. Annual cycle is characterized by cold mixing period in winter, followed by stratification period during spring and summer (Bao et al., 2009). Seawater temperature measures 22.5  C at the surface and 12.8  C at the bottom in July, which is the largest thermocline for an entire year. The maximum mean column temperature (19.6 ± 3.1  C) occurs in August, whereas the lowest (1.3 ± 1.3  C) can be observed in February (Zhang et al., 2016). Japanese scallop Patinopecten yessoensis cultivation began in 1982 and became a dominant maricultural species since 1998. Sampling stations for Japanese scallop are located in cultural areas around this island. 2.2. Isolation of Protoceratium reticulatum Seawater samples for Protoceratium reticulatum isolation were collected from coastal waters of Station No. 15 on April 2010 and placed in an 800 mL plastic bottle. Single motile cells of Protoceratium reticulatum were isolated with capillary pipette and washed four times with filtered seawater under a microscope

2.3.1. Light microscopy Motile cells Protoceratium reticulatum were examined under a microscope (Eclipse 80i, Nikon, Japan) equipped with microphoto system (DS-Ri2, Nikon, Japan), and light micrographs were obtained. For identifying thecal plates, samples were stained by 1% fluorescent brightener 28 (Sigma-Aldrich, USA) to discern plates in motile cells following the method of Fritz and Triemer (1985). 2.3.2. Scanning electron microscopy (SEM) To clarify classification suggested by light microscopy, thecal plate analysis was combined with SEM for definitive identification. To remove thick membrane and impurities on surface of algae cells, 5% Trition X-100 (Sigma-Aldrich, USA) was added and maintained in ultrasonic water bath for 10 min. Then, cells were primarily fixed by 4% glutaraldehyde (EM grade, SPI-CHEM, USA) in 0.1 M sodium cacodylate buffer (pH ¼ 7.5) to one volume of culture for 1 h at 4  C. After pelleting and washing in filtered seawater for five times and as described above, samples were fixed again by 1% osmic acid (enzyme reagent, Beijing Zhongjingkeyi Technology Co., Ltd., China) for 40 min at 4  C. Cells were dehydrated in gradient ethanol/water solutions of 20%, 30%, 50%, 70%, 80%, 90%, and 100% twice for 10 min each and washed thrice with 90% and 100% ethanol/water dehydration solution. Then, cells were dried under a critical point dryer (HCPD-15-500, Jeol, China), collected on a cover

Fig. 1. Geography of northern Yellow Sea area and the investigated station (C) in the study, where the clonal strain of Protoceratium reticulatum was isolated at station 15.

L. Liu et al. / Toxicon 139 (2017) 31e40

glass (18 mm  18 mm), platinum-palladium sputter-coated (JFC1600, JOEL, Japan), and examined with SEM (JSM-7800F, JOEL, Japan). 2.4. Molecular phylogenetic analysis About 1  104 cells were collected by low-speed centrifugation from 100 mL culture of strains ZZD01 and 2776 (from USA) of motile Protoceratium reticulatum. DNA was extracted using a DNA extraction kit (Sangon, Shanghai, China). Partial D1-D2 of LSU rDNA and ITS regions were amplified using primers D1R and D2C (Scholin et al., 1994) and primers ITSA and ITSB (Adachi et al., 1996). PCR products were isolated and sent to Takara Biotechnology (Dalian) Co., Ltd. for sequencing. ITS and 18S rDNA sequence of ZZD01 and 2776 strains obtained in the study were aligned with those available in GenBank. 2.5. YTXs profile and quantification of intracellular and extracellular in ZZD01 To identify differences in YTX profiles and quantity in intracellular and extracellular ZZD01, 50 mL of ZZD01 strain cultured in stationary phase was centrifuged at 3000 rpm for 10 min at 4  C using a refrigerated centrifuge (3e18 K, Sigma, Germany). Pellet was extracted twice with 4 mL methanol under ultrasonication (VCX130, Sonics, USA) for 10 min, and unbroken cells were not observed by microscopy. Supernatants were combined, volume was adjusted to 10 mL by 100% methanol, and filtration step was performed before analysis. Another solid-phase extraction (SPE) method (Gerssen et al., 2009) was performed using cell-free supernatant to calculate extracellular YTXs in cultured medium. A Strata™-X cartridge (Phenomenex, USA) was activated with 1 mL methanol and equilibrated using 1 mL 30% (v/v) methanol. A total of 28 mL supernatant was diluted with 12 mL methanol and loaded onto the cartridge after washing with 1 mL 20% (v/v) methanol to remove salts and polar compounds. Toxins were eluted by 1.2 mL methanol containing 0.3% (v/v) ammonium hydroxide. Eluent was filtered through a methanol compatible 0.22 mm syringe filter (Jinteng, Tianjin, China) and transferred to vials before analysis. 2.6. Sample collection and processing of phytoplankton, sediments and scallop Phytoplankton water samples, which were used for counting € hl toxic and potential toxic species, were collected by the Utermo (1958) method at Stations 01e16 and 18e30 (Fig. 1) every midmonth from January 2011 to December 2015. Samples were preserved in dark plastic sample bottles with 4% neutralized formalin to ensure long-term preservation. Phytoplankton composition and abundance were quantified with an inverted microscope at 400 magnification (Eeclipse 80i, Nikon, Japan). Sediment sampling was carried out at Stations 02, 04, 21, 23, 25, and 29 from January to December 2013. Samples were collected on a bottom area of 0.04 m2 with undisturbed sediment cores by a van Veen grab, stored in the dark, and kept at a low temperature. After transport to the laboratory, sediment cores were sectioned at 2 cm interval and stored in Ziploc bags at 4  C in the dark until analysis. For dinocyst analysis, the top fraction (0e2 cm) representing recent sediments was palynologically processed (Matsuoka and Fukuyo, 2000). Sediment samples were desalted, acid-treated, sonicated and sieved through 100 and 10 mm mesh sizes. Residues retained on the 10 mm mesh were transferred to a vial and suspended in 10 mL of distilled water. Aliquots of 0.1 mL processed sample were added to a phytoplankton count plate (20  20 mm) and scanned under an inverted microscope (Eeclipse 80i, Nikon, Japan) at 100 to 400

33

times of magnification. Cyst abundance was expressed as number of cysts per gram of wet sediment. For the Japanese scallop Patinopecten yessoensis, at least 60 individuals on two stations in investigated sea area (Fig. 1) were collected weekly by trawling from 2011 to 2015. Samples were immediately transported to laboratory at low temperature, cleaned with freshwater, and tissues were drained in a sieve to dislodge saltwater after removing shells. Twenty scallops were dissected for their digestive glands and edible parts for YTXs analysis. To determine distribution of YTXs in different shellfish tissues, another 20 individuals were further dissected into five parts, including adductor, digestive glands, mantel, gill, and gonad from October 2010 to September 2011. Each part was well mixed and homogenized in a blender. Samples were extracted following standard operating procedures (EURLMB, 2011). A 2.00 ± 0.05 g tissue homogenate was extracted twice by 9.0 mL 100% methanol after centrifugation at 3000 rpm for 10 min. Two supernatants were transferred to a 20 mL volumetric flask and used to prepare 20 mL extract with 100% methanol. To remove matrix effects in liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) analysis, a Strata™-X cartridge (Phenomenex, USA) was selected to perform an SPE clean-up protocol (Gerssen et al., 2009), and the cartridge was activated with 1 mL methanol and equilibrated using 1 mL 30% (v/v) methanol. A 1.2 mL crude extract was diluted with 2.8 mL deionized water and loaded onto the cartridge after washing with 1 mL 20% (v/v) methanol to remove polar compounds. Toxins were eluted by 1.2 mL methanol containing 0.3% (v/v) ammonium hydroxide. Purified extracts were filtered through a methanol compatible 0.22 mm syringe filter (Jinteng, Tianjin, China) and transferred to vials for analysis.

2.7. LC-MS/MS analyses of yessotoxins YTXs in samples were analyzed by a validated LC-MS/MS method. The instrument consisted of an API4000 (AB SCIEX, MA, USA) and a high performance liquid chromatography system which included an UltiMate 3000 binary pump system, an autosampler, and an Ultimate 3000 column compartment oven (Dionex, CA, USA). The analytical column was Waters X-Bridge C18 (150  3.0 mm, 3.5 mm), coupled with a security guard column (Phenomenex, Torrance, CA, USA). Mobile phase A consisted of 0.05% (v/v) ammonia in water; mobile phase B consisted of 0.05% (v/v) ammonia in 90% acetonitrile with a flow rate set at 0.40 mL min
1. Injection volume was set at 10 mL. Analyses were separated by gradient elution. Initial composition was 10% mobile phase B for 1.0 min, then increased to 90% in 9 min, maintained at 90% for 3 min, followed by a change to the initial condition in 2 min and re-equilibration at 10% mobile phase B for 4 min; total run time was 19 min. Temperature of column was set at 40  C. Mass spectra were acquired by the API 4000 MS/MS system equipped with electrospray ionization interface with turbo spray ion source. Ion spray voltage was set at 4500 V, and temperature was maintained at 600  C. Nebulizing gas was high-purity nitrogen, and gasses 1 and 2 were set at 60 and 50 L min
1, respectively. Curtain and collision gasses were 13 and 5, respectively. Quantification was performed in multiple reactions monitoring (MRM) mode with a dwell time of 125 ms for each transition. Selected reaction monitoring in negative ion mode was used for YTX (1141.5 > 1061.7/855.5) and homoYTX (1155.5 > 1075.5/869.5). Sample concentrations were calculated by external standard method using Analyst software (version 1.5.1, AB SCIEX, Framingham, MA, USA).

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L. Liu et al. / Toxicon 139 (2017) 31e40

2.8. Statistical analysis All data acquired on yessotoxins in phytoplankton and scallop samples were processed with Microsoft Excel 2013. The results represent the mean ± standard deviation (SD). Further graphing and data analysis were processed using origin software 2016 (OriginLab Corp, Northampton, MA). 3. Results 3.1. Confirmation of Protoceratium reticulatum Motile cells of Protoceratium reticulatum from Zhangzi Island were golden brown in general with a long flagellum (Fig. 2A). Cells cultured at 15  C measured 31.27 ± 4.14 mm (n ¼ 50) in length and 28.22 ± 3.03 mm (n ¼ 50) in width, indicating slightly longer length than width. Protoceratium reticulatum cell consists of conical epithecal and hemispherical hypothecal parts surrounded by a slightly left-handed cingulum upon which small and open pores are uniformly distributed (Fig. 2J and K). Each thecal plate was covered by several reticulations with 0e2 small pores within each reticulation (Fig. 2IeL). Ventral view shows a narrow, elongated apical pore directly in contact with an ear-shaped 10 plate within which a ventral pore (Vp) exists at the left margin (Fig. 2E). At the edge of

each thecal plate were raised little girdles, which were nearly vertical to those of plates (Fig. 2IeL). Apical pore complex was broad, slit-shaped, and featured a comma-shaped pore encircled by several visible pores (Fig. 2J). A highlighted ventral pore was present at the margin of right concave side of 10 plate attached to 30 plate (Fig. 2J). Epithecal tabulation was Po, 30 , and 1a, 600 (Fig. 2G and H), and hypothecal tabulation was 6000 , 1p, and 10000 (Fig. 2H and J). Thus, plate formula includes Po, 30 , 1a, 600 , 6000 , 1p, and 10000. Cysts (Matsuoka, 1985) produced by ZZD01 strain were spherical, transparent, and with an intracellular bubble and membrane with closed capitate extremities (Fig. 2B). Our results showed that the ZZD01 strain from Zhangzi Island and 2776 strain from the USA were identical to most other sequences of Protoceratium reticulatum included in multiple sequence alignment by maximum likelihood analysis of ITS sequence data. ZZD01 strain of Protoceratium reticulatum genes were closest to species of Japanese (AB727654) and Swedish strains (AB727655) (Mertens et al., 2012) and presented a short distance with the 2776 strain (Fig. 3). Phylogenetic analyses of 18S rDNA region showed that ZZD01 and 2776 strain of Protoceratium reticulatum contained short genetic distances from L. polyedrum and G. spinifera, as described in
rez et al., 2016) (Fig. 3). literature (Akselman et al., 2015; Sala-Pe

Fig. 2. (A, E-H) Light microscopy of Protoceratium reticulatum strain ZZD01 motile cells. (A) showing a flagellum(F) and chloroplast. (B) P. reticulatum living cyst generated from the strain ZZD01. (C) Gonyaulax spinifera living cyst collected from the Zhangzi Island sediment. (D) Lingulodinium polyedrum living cyst collected from the Zhangzi Island's sediment. (EH) showing 20 , 30 , 1a, Po plate and ventral pore (Vp), cingular plate (CI), sulcal plate (SU). (IeL) Scanning electron microscopy of Protoceratium. reticulatum strain ZZD01 motile cells showing Po, 30 , 600 , 6000 , 1p, 10000, SU, CI and Vp. Scale bars ¼ 10 mm.

L. Liu et al. / Toxicon 139 (2017) 31e40

35

Fig. 3. Phylogeny of Protoceratium reticulatum and Lingulodinium polyedrum, Gonyaulax spinifera inferred partial sequence of the ITS and 18S rDNA based on maximum likedhood. Sequences of the strain ZZD01 isolated from northern Yellow Sea and the strain 2776 came from USA fall in the same Clade A.

3.2. YTXs produced by ZZD01 strain Protoceratium reticulatum YTX in ZZD01 strain Protoceratium reticulatum dominated 94% of all YTXs in stationary phase cells. YTX presented a concentration of

21.67 ± 2.28 pg cell
1 and included low-level homoYTX measuring 0.62 ± 0.27 pg YTXeq cell
1 in the motile cell at 15  C. Trace levels of 45-OH YTX, 45-OH homoYTX, 45,46,47-trinorYTX, 45,46,47-trinor homoYTX, 23-YTX, 35-YTX, 38-YTX, 41-YTX, 49-YTX, and 52-YTX

36

L. Liu et al. / Toxicon 139 (2017) 31e40

were detected in motile cells; very low levels of YTX, 45-OH YTX, 45-OH homoYTX, 45,46,47-trinorYTX, 45,46,47-trinor homoYTX, and 22-YTX were observed in cultured medium (Table 1, Fig. 4). Cysts produced by motile cells of ZZD01 strain Protoceratium reticulatum in laboratory, and YTX content in resting cysts included 89.00 ± 0.78 pg cyst
1 and reached four times that of motile cells. However, homoYTX content in resting cyst totalled 0.60 ± 0.98 pg YTXeq cyst
1 at the same level of motile cell. 3.3. Variability of YTXs in scallop Patinopecten yessoensis YTX toxin was detected in hepatopancreas of scallops every week during 2011e2015, and the highest amount of 5667.5 ± 421.3 mg kg
1 was observed in August 2011. Average YTX value of 2066.8 ± 1813.7 mg kg
1 in 2011 was also the highest, and it decreased to 165.6 ± 83.2 mg kg
1 in 2015 (Fig. 5). YTXs in edible parts, including gill, mantle, gonad, and adductor measured 170.6 ± 82.8 mg kg
1 in 2011e2013 and 2015, but were not detected in most months of 2014 (Fig. 6). The 45-OH YTX toxin in hepatopancreas of scallops was detected weekly in 2012e2015. Highest amounts reached 148.65 ± 37.99 mg YTXeq kg
1 in August 2012, whereas 10.07 ± 0.15 mg YTXeq kg
1 observed in October 2015 was the least. The 45-OH YTX value in hepatopancreas was relatively constant at 13% of YTXs in the same month (Fig. 7), this YTX was not detected in edible parts. YTXs in scallop tissues were monitored every week from October 2010 to September 2011. In hepatopancreas, amount of YTX that ranged from 3.9 ± 0.1 mg kg
1 to 212.5 ± 94.3 mg kg
1 every month were the highest among all tissues and beyond the 3.75 mg kg
1 limit (EC, 2013) in August and September 2011 (Fig. 8). About 98% of YTX was distributed in hepatopancreas of whole scallops. In gills, YTX measured 118.5 ± 30.0 mg kg
1, and approximately 1% was distributed in gills of whole scallops. In mantles, YTX ranged between 32.9 ± 2.6 and 10.4 ± 0.8 mg kg
1 each month, and average value reached 0.8% in that of whole scallops. In gonads, YTXs were detected in October to December 2010 and June, July, and September 2011. YTXs were not detected in adductors muscle in indicated observation periods (Fig. 8).

Fig. 4. Multiple reaction monitoring (MRM) chromatogram showing the presence of numerous yessotoxins in the strain ZZD01 Protoceratium reticulatum cells.

Fig. 5. Month variability of YTX content in hepatopancreas of scallop Patinopecten yessoensis during 2011e2015. Mean ± SD (n ¼ 4).

3.4. Variability of dinoflagellates produced YTXs In the investigated area of northern Yellow Sea, dinoflagellates producing YTXs species, Protoceratium reticulatum, and potential toxic algae, L. polyedrum and G. spinifera, in seawater column were counted monthly from January 2011 to November 2015.

Motile cells of Protoceratium reticulatum in some samples at surface, middle, and bottom seawater at all stations were frequently observed from March to June when water temperature ranged from 5  C to 20  C and occasionally from December to

Table 1 Profiles of yessotoxins produced by the ZZD01 strain Protoceratium reticulatum under laboratory culture revealed by LC-MS/MS. Entry

Q1

Q3

Retention time min

Intracellular YTXeq pg cell
1

Extracellular YTXeq fg cell
1

Compounds name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1141.5 1155.5 1157.5 1157.5 1171.0 1101.0 1115.0 985.0 985.0 1061.0 1119.0 1143.0 1201.0 1221.0 955.0

1061.5, 855.5 1075.0, 869.5 1077.5, 871.5 1077.5, 871.5 1091.5, 869.5 1021.0 1035.0 905.0; 815.0 905.0; 815.0 924.0; 855.0 1039.0 1063.0 1121.0 1141.0 875.0

7.98 8.06 7.50 8.05 8.05 7.51 7.51 7.16 7.85 7.92 7.38 7.91 9.15 7.52 5.19

21.67 0.62 0.12 0.079 0.013 0.25 0.32 0.004 0.008 0.013 0.011 0.025 0.006 0.004 ND

138.89 ± 114.59 ND Trace level ND ND 88.70 ± 22.64 3.27 ± 2.28 ND ND ND ND ND ND ND Trace level

YTX homoYTX 45OHYTX 45OHYTX 45OHhomoYTX 45,46,47-trinorYTX 45,46,47-trinorhomoYTX 23YTX 23YTX 35YTX 38YTX 41YTX 49YTX 52YTX 22YTX

L. Liu et al. / Toxicon 139 (2017) 31e40

Fig. 6. Month variability of YTX content in edible part of scallop Patinopecten yessoensis during 2011e2015. Mean ± SD (n ¼ 4).

37

Fig. 9. Month variability of Protoceratium reticulatum cell density in sea water column during 2011e2015. Mean ± SD (n ¼ 29).

Fig. 7. Month variability of 45-OHYTX content in hepatopancreas of scallop Patinopecten yessoensis during 2012e2015. Mean ± SD (n ¼ 4).

Fig. 10. Month variability of Protoceratium reticulatum, Gonyaulax spinifera, Lingulodinium polyedrum cyst density in the sediment in 2013. Mean ± SD (n ¼ 6).

Fig. 8. Month variability of YTX content in different tissue compartments of scallop Patinopecten yessoensis from Oct. 2010 to Sep. 2011. Mean ± SD (n ¼ 4).

February of the following year. The maximum density of 46 ± 63 cells L
1 was observed in April 2014 (Fig. 9). Protoceratium reticulatum cysts were easily identified by characteristic capitate

extremities (Fig. 2B) and were detected except in October and December with peak value of 640 ± 496 cysts g
1w.w. in March 2013 (Fig. 10). Motile cells of G. spinifera at some samples from surface, middle, and bottom seawater at all stations were detected during March to November. Peak value of G. spinifera reached 449 ± 214 cells L
1 in March 2011 (Fig. 11). Cysts of G. spinifera were easily identified by complete trabecula network (Fig. 2C) and were detected in February, March, May, July, August, and September. The maximum density measured 176 ± 109 cysts g
1w.w. in July 2013 (Fig. 10). Motile cells of L. polyedrum were not detected in seawater at all stations. L. polyedrum cysts were identified by characteristic capitate extremities (Fig. 2D), but were detected in January, February, March, May, July, and August. The maximum density measured 195 ± 106 cysts g
1w.w. in August 2013 (Fig. 10). 4. Discussion Several studies reported the presence of YTXs in bivalves in coastal waters of China (Gao et al., 2010, 2012; Chen et al., 2014),

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L. Liu et al. / Toxicon 139 (2017) 31e40

of 94% in all compositions (Table 1). YTX is the major toxin profile in the ZZD01 strain, including those isolated from Japan (Suzuki et al., 2007), Norway (Samdal et al., 2004), Italy (Ciminiello et al., 2003), and News land (Finch et al., 2005). HomoYTX measured 0.62 pg cell
1, which is similar to levels of Japan strains from Matsu Bay and Okirai (Suzuki et al., 2007). HomoYTX predominated some strains, such as strains OM6-NP31 and VGO764 from Japan and Spain (Paz et al., 2007; Suzuki et al., 2007). Toxin profile difference in Protoceratium reticulatum strains were discovered to be dependent on geographical locations and may be affected by genetic variations and inhabited environments. 4.2. YTXs variability in scallops Patinopecten yessoensis

Fig. 11. Month variability of Gonyaulax spinifera cell density in sea water column during 2011e2015. Mean ± SD (n ¼ 29).

but details of their variability in Protoceratium reticulatum and toxin producing algae, such as G. spinifera, and L. polyedrum were not reported. 4.1. Morphology, phylogeny and YTXs of Protoceratium reticulatum Plate formula of ZZD01 strain Protoceratium reticulatum from the northern Yellow Sea agreed with those of previous studies on Protoceratium reticulatum (Dodge, 1989; Hansen et al., 1996/1997). Cell size is a variable that corresponds to ranges reported in liter€ der et al., 2012; ature (Dodge, 1989; Hansen et al., 1996/97; Ro
rez et al., 2016). Nearly half of ProAkselman et al., 2015; Sala-Pe toceratium reticulatum specimens from Danish field samples showed contact between 1 and pore plate (Hansen et al., 1996/ 1997). In Protoceratium reticulatum samples from South Patagonia of Argentina, the cell with plate configuration of 40 and 1a was not identified (Akselman et al., 2015). Some variability exists in the position of intercalary plate 1a as exemplified by PR3 isolate from
rez et al., 2016). The shape of intercalary plate 1a the Arctic (Sala-Pe in strain ZZD01 varied to a certain degree (Fig. 2GeH) compared with the common feature of Protoceratium reticulatum described in
rez literature (Hansen et al., 1996/97; Akselman et al., 2015; Sala-Pe et al., 2016). Strains of Protoceratium reticulatum were part of clades A or B based on phylogenetic analyses of reported DNA sequences
rez et al., 2016). The ZZD01 strain (Akselman et al., 2015; Sala-Pe from waters of northern Yellow Sea belongs to Clade A. Clade A is a geographically widely distributed sub-clade, which includes coasts of the Atlantic, the Mediterranean, and the Pacific and arctic waters. However, genetic homogeneity between ZZD01 with AB727654 (Japan) strains is the closest based on short geographical distance of Yellow and Japanese Seas but with the same affinity of European isolates (EU927654, EU 927569, EU 927572, and AB727655) in the long range (Fig. 3). Phylogenetic patterns of Protoceratium reticulatum did not reflect good correspondence with its morphological characteristics. In this study, the presence of YTX and homoYTX was confirmed through detection of their peaks at the same retention time for standards of strain ZZD01. Other related analogues containing homoYTX, 45-OH YTX, 45-OH homoYTX, 45,46,47-trinorYTX, 45,46,47-trinor homoYTX, and 41-YTX were calculated by YTX standard curve on the assumption that they yield the same molar responses as YTX. YTX measured 22 pg cell
1 and reached a quota

Japanese scallop Patinopecten yessoensis is an important seafood in China, Japan, Korea, Russia, and Canada. YTXs in Japanese scallop were first reported in Japan by Murata et al. (1987) and in China by Gao et al. (2010, 2012). This study serve as the first detailed explanation of YTXs in monthly and yearly variability of scallops and their distribution in tissues in northern Yellow Sea. YTXs in scallop hepatopancreas was safely limited under 3.75 mg kg
1, this value was only observed in August and September 2011 and never reached over that level in four years (Fig. 5). A large proportion of YTXs assembled in scallop hepatopancreas, some in edible parts without hepatopancreas, and none was detected in adductor muscle (Fig. 8). Therefore, it would be best to remove their hepatopancreas before eating the scallops from northern Yellow Sea of China. In mussel Mytilus galloprovincialis from Adriatic Sea of Italy, using the analysis method of LC-MS/MS, the major analogues of YTXs included carboxy homoYTX and homoYTX, followed by carboxyYTX, 45-OH homoYTX, 45-OH YTX, YTX, and stacked up 4 mg YTXeq kg
1 (Pierina et al., 2013). In cultivated mussels M. galloprovincialis from Baja California in Mexico, homoYTX was the most abundant analogue followed by YTX, 45-OH homoYTX, and 45-OH YTX, and with the highest concentration reaching 1.081 mg kg
1 (Ernesto et al., 2014). In the present study, the major analogues of YTXs in Japanese scallop Patinopecten yessoensis were YTX followed by 45-OH YTX, other components were not detected (Figs. 5 and 7), and YTX profile differed from that of mussels M. galloprovincialis from Italy and Mexico. These observations possibly resulted from diverse patterns of scallop and mussel in terms of YTX uptake, metabolism, and depuration, as described by John et al. (2005), and perhaps influenced by toxic algae from different geographic locations (Meredith et al., 2008). 4.3. Relationship between YTXs in scallops and toxic dinoflagellate abundance Japanese scallops Patinopecten yessoensis are cultivated at sea bottom in northern Yellow Sea. These organisms can filter plankton in seawater and resting cysts resuspended on surface of sediments. In this study, YTXs in scallop were detected every month, and peak values were not significant in August to September and April to May during 2011e2015 (Figs. 5 and 6). Toxic motile cells of Protoceratium reticulatum were identified in seawater from April to June in the investigating period (Fig. 9), and cysts in sediments were detected every month except October and December (Fig. 10). Potential toxic motile cells of G. spinifera were observed from March to November (Fig. 11), and its cysts were detected in February, March, May, July, August, and September (Fig. 10). Potential toxic cysts L. polyedrum were identified in January, February, March, May, July, and August (Fig. 10). The above results indicate that YTX level in scallops was relevant to abundance of motile cells and cysts of Protoceratium reticulatum, G. spinifera, and L. polyedrum, but this relevance was

L. Liu et al. / Toxicon 139 (2017) 31e40

non-significant. Protoceratium reticulatum cell density at Flødevigen of Norway reached a maximum on 16 May 2001, and YTX levels in blue mussels M. edulis at the same site increased sharply by 14 May and peaked on 28 May (John et al., 2005). YTX in mussels M. californicus from Monterey Bay of California presents a measurable YTX level of up to 60 mg kg
1 after blooming of L. polyedrum (Meredith et al., 2008). In 2004, unusually high amounts of homoYTX were detected in mussels M. galloprovincialis. In this period, Protoceratium reticulatum and L. polyedrum were nearly absent in seawater, whereas dinoflagellate G. spinifera was present at high densities in New Zealand (Manuela et al., 2009). Resting cysts of dinoflagellate Alexandrium fundyense can be consumed and cause toxicity in bivalve Crassostrea virginica. Direct consumption of resting cysts may explain shellfish toxicity in areas without known blooms but with toxic resting cysts in sediments (Agneta et al., 2006). A. fundyense cysts in sediments can be resuspended and reach a concentration of at least 104 cysts m
3 in water column in the Gulf of Maine (Bradford et al., 2014). Therefore, Protoceratium reticulatum motile cells in water and cysts in sediments are responsible for YTXs in scallops. G. spinifera motile cells and its cysts as well as L. polyedrum cysts were possible contributors of YTXs because of its constant toxicity time. These results require further studies in the northern Yellow Sea. 4.4. Distribution of YTX in scallop Patinopecten yessoensis tissues In this study, most of yessotoxins concentrated in the scallop tissue of hepatopancreas, a small portion was in gills, mantle and gonad, undetected in adductor muscle (Fig. 8). The situation was consistent to the lipophilic toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), and yessotoxin (YTX) content in the hepatopancreas was the highest followed by gonad, mantle, and adductor muscle in turn (Chen et al., 2014). But 39% of hydrophilic paralytic shellfish poisoning (PSP) were accumulated in the adductor muscle, 33% in hepatopancreas, 18% in gill and mantle, 10% in gonad of the Patinopecten yessoensis (Wong et al., 2009). Therefore, the concentration of lipophilic and hydrophilic shellfish toxins in the tissues, especially in adductor muscle of Patinopecten yessoensis, was obviously discrepant. YTXs in scallops was the highest in 2011, and then decreased gradually until 2015 (Figs. 5 and 6). Meanwhile, the motile cells which can produce yessotoxin of Protoceratium reticulatum (Fig. 9) and Gonyaulax spinifera (Fig. 11) in the water also decreased year by year. The correlation between amount of yessotoxin in the scallops and the abundance of toxic algae in the water in northern Yellow Sea also verified that the yessotoxins were generated by the toxic algae Protoceratium reticulatum and Gonyaulax spinifera. 5. Conclusions The studied sea area is an important district of bottom sowing cultured Japanese scallop Patinopecten yessoensis in northern Yellow Sea of China. Motile cells and cysts of Protoceratium reticulatum were confirmed as contributors of YTXs in Japanese scallops, and G. spinifera was a potential contributor. Motile cells of L. polyedrum were not detected in seawater, but its cysts were detected in sediments, these cysts were possibly the origin of YTXs in Japanese scallops. YTXs in Japanese scallop were relevant to abundance of motile cells and cysts of Protoceratium reticulatum, G. spinifera, and L. polyedrum. YTXs in scallop decreased from 2011 to 2015, the major toxins concentrated in hepatopancreas and the little in edible parts. Therefore, the Japanese scallop edible parts from northern Yellow Sea were safe for consumption within a year.

39

Ethics statement The study proposal was approved by National Marine Environmental Monitoring Center, and didn't involved any animal testing. Acknowledgments This work was supported by the National Key Research and Development Program of China (2016YFF0201104, 2017YFC1404303), the National Nature Science Foundation of China (41576120, 41276099), and the National Marine Public Welfare Research Project of China (No. 201305010-2). We particularly thank several anonymous referees for their professional comments on this manuscript. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.toxicon.2017.09.015. References Agneta, Persson, Smith, Barry C., Wikfors, Gary H., Quilliam, Michael, 2006. Grazing on toxic Alexandrium fundyense resting cysts and vegetative cells by the eastern oyster (Crassostrea virginica). Harmful Algae 5, 678e684. Akselman, R., Krock, B., Alpermann, T.J., Tillmann, U., Borel, M., Almandoz, G.O., Ferrario, M.E., 2015. Protoceratium reticulatum (Dinophyceae) in the austral Southwestern Atlantic and the first report on YTX-production in shelf waters of Argentina. Harmful Algae 45, 40e52. Adachi, M., Sako, Y., Ishida, Y., 1996. Analysis of Alexandrium (Dinophyceae) species using sequence of the 5.8S ribosomal DNA and internal transcribed spacer regions. J. Phycol. 32, 424e432. Bao, X.W., Li, N., Yao, Z.G., Wu, D.X., 2009. Seasonal variation characteristics of temperature and salinity of the north Yellow Sea. Period. Ocean Univ. China 39 (4), ,553e562 (in Chinese, with English abstract). Bradford, Butman, Aretxabaleta, Alfredo L., Dickhudt, Patrick J., Dalyander, P. Soupy, Sherwood, Christopher R., Anderson, Donald M., Keafer, Bruce A., Signell, Richard P., 2014. Investigating the importance of sediment resuspension in Alexandrium fundyense cyst population dynamics in the Gulf of Maine. DeepSea Res. II 103, 79e95. Chen, J.H., Yu, R.C., Kong, F.Z., Gao, Y., Luo, X., Wang, Y.F., Zhou, M.J., 2014. Detection of lipophilic phycotoxin in Patinopecten yessoensis in the northern Yellow Sea. Oceanol. Limnol. Sin. 45 (4), 855e863 (in Chinese, with English abstract). Ciminiello, P., Dell-Aversano, C., Fattorusso, E., Forino, M., Magno, S., Guerrini, F., Pistocchi, R., Boni, L., 2003. Complex yessotoxins profile in Protoceratium reticulatum from north-western Adriatic sea revealed by LC-MS analysis. Toxicon 42, 7e14. Dodge, J.D., 1989. Some revisions of the family Gonyaulacaceae (Dinophyceae) based on a scanning electron microscopy study. Bot. Mar. 32, 275e298. EC No 853/2004 of the European Parliament and of the Cou g down specific hygiene rules for food of animal origin. Off. J. Eur. Commun. L 226/61. EC No 786/2013 of the European Parliament and of the Council of of 16 August 2013 amending Annex III to Regulation (EC) No 853/2004 of the European Parliament and of the Council as regards the permitted limits of yessotoxins in live bivalve molluscs. Off. J. Eur. Commun. L220/14. Ernesto, García-Mendoza, S
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