Toxin production of dinoflagellate Gymnodinium catenatum isolated from the East China Sea.

Dinoflagellate Gymnodinium catenatum is an important producer of paralytic shellfish toxins (PSTs), including a novel group of hydroxybenzoate derivatives named GC toxins. In the East China Sea, G. catenatum has been considered as the causative agent for several paralytic shellfish poisoning (PSP) episodes, yet the knowledge on their toxin production was still quite limited. In this study, toxins produced by a strain of G. catenatum (MEL11) isolated from the East China Sea were determined, using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Changes of toxin profile in the stain MEL11 in response to nutrient and temperature variations were also examined. A total of 11 PST components dominated by hydroxybenzoate analogs and N-sulfocarbamoyl toxins were detected, which was different from other G. catenatum strains previously established in the East China Sea in the presence of GC5 and the lack of dcGTX2&3. Cellular toxin composition and content of the strain had no apparent change within a range of temperature from 20°C to 26°C. In contrast, nutrient limitation and nitrogen source had notable impacts on toxin production. The molar percentage of GC toxins decreased remarkably at the stationary growth phase under nutrient-deprived conditions of both nitrogen (N) and phosphorus (P). The replacement of nitrate with ammonium as the source of N significantly promoted PST production by G. catenatum. The study revealed the potential diversity of toxin profiles of G. catenatum in the East China Sea, and highlighted the effects of nutrients on production of GC toxins by G. catenatum.

Distribution of Aureococcus anophagefferens in relation to environmental factors and implications for brown tide seed sources in Qinhuangdao coastal waters, China.

Brown tides caused by Aureococcus anophagefferens have occurred along the Qinhuangdao coastline in the Bohai Sea (BS) in recent years. Little is known about the spatio-temporal distribution of A. anophagefferens, particularly its profile distribution and the effects of environmental controls. In this study, four surveys were conducted in Qinhuangdao coastal waters during the brown tide from June to July 2013; another survey was conducted to cover a larger region in the BS in May 2016. Temperature, salinity, nutrients, and chlorophyll a were analyzed; and the density of A. anophagefferens was detected by a sensitive qPCR method. The intensive brown tide only occurred in Qinhuangdao inshore waters at temperatures ranging from 21.5 to 23.2 °C and relatively high salinity (> 29). Redundancy analysis indicated that the low dissolved inorganic nitrogen limited the growth of other pico- and nano-algal species; high dissolved organic nitrogen and low inorganic nutrients were suitable for the development of brown tides in Qinhuangdao coastal waters, which also contained a thermocline during the brown tide. At the early stage of the brown tide, a high abundance of A. anophagefferens appeared at the bottom of offshore waters characterized by low temperature and high salinity. The A. anophagefferens cells were speculated to originate from water mass located in a depression between the central ridge and the Qinhuangdao coastal area. In brief, this study reported the spatio-temporal variation of brown tides based on the abundance of A. anophagefferens and environmental forces and implied that A. anophagefferens could be transported from the bottom of offshore waters to promote brown tides in inshore waters of Qinhuangdao.

The dinoflagellate Alexandrium catenella producing only carbamate toxins may account for the seafood poisonings in Qinhuangdao, China.

An outbreak of paralytic shellfish poisoning, recorded in April 2016 in Qinhuangdao China, was suspected to be caused by a toxic species in genus Alexandrium. Shortly after the poisoning outbreak, shellfish and net-concentrated phytoplankton samples were collected from the Bohai Sea, and analysed using high performance liquid chromatography coupled with fluorescence detection. Paralytic shellfish toxins (PSTs) were detected in both phytoplankton and shellfish samples, with similar toxin profiles dominated by carbamate toxins. High throughput sequencing data for phytoplankton samples collected previously in the coastal waters of Qinhuangdao were then analysed, and 8 operational taxonomic units (OTUs) were assigned to Alexandrium affine, A. andersonii/A. ostenfeldii, A. catenella, A. fraterculus, A. hiranoi/A. pseudogonyaulax, A. margalefii, A. pacificum and A. pohangense, among which A. catenella, A. pacificum and A. ostenfeldii could be potential producers of PSTs. During a cruise in 2019, three isolates of Alexandrium were established by cyst germination, and identified as A. catenella based on the sequences of the 28S ribosomal RNA gene (28S rDNA) D1-D2 region. Interestingly, all the three strains had the same toxin profile consisting of gonyautoxins 1, 3, 4 (GTX1, 3, 4) and neosaxitoxin (NEO). The toxin profile is similar to those of phytoplankton samples collected previously in the coastal waters of Qinhuangdao, but remarkably different from the general toxin profile of A. catenella dominated by N-sulfocarbamoyl toxins C1-2 in the Bohai Sea and the Yellow Sea. The results suggest that A. catenella is most likely to be the causative species of the poisoning outbreak in Qinhuangdao. As far as we know, this is the first report of A. catenella in the Bohai Sea producing PSTs dominated by high potent gonyautoxins GTX1-4. Occurrence of the highly toxic A. catenella will increase the risk of paralytic shellfish poisoning, which necessitates in-depth mechanism studies and increasing monitoring efforts.

Resting cysts of Alexandrium catenella and A. pacificum (Dinophyceae) in the Bohai and Yellow Seas, China: Abundance, distribution and implications for toxic algal blooms.

The Alexandrium tamarense species complex consists of 5 closely related species that are important bloom-forming dinoflagellates with a complex life cycle. The formation of resting cyst is a key strategy to resist harsh environmental conditions. In this study, the resting cysts of two major bloom-forming species of the A. tamarense species complex in China, A. catenella (Whedon & Kof.) Balech (previously A. fundyense, or A. tamarense species complex Group I) and A. pacificum Litaker (A. tamarense species complex Group IV), were studied in surface sediment collected from the Bohai Sea (BS) and Yellow Sea (YS) during two cruises conducted in 2012 and 2015. Cyst abundance of the A. tamarense species complex was first quantified by the primuline-staining method, and cysts of the two species were subsequently determined using two real-time quantitative PCR (qPCR) assays. Results showed that resting cysts of the A. tamarense species complex were more abundant in the YS than the BS (mean of 480 and 33 cysts g dry weight, DW-1 of sediment, respectively). Cysts were mainly found in the central portion of the northern YS, the area SE (southeast) of the Shandong peninsula, and the area near the Subei Shoal in the southern YS, where surface sediment had a high percentage of clay and silt (particle size < 63 µm) content. The maximum cyst abundance recorded was 3090 cysts g DW-1 of sediment in 2012 and 3448 cysts g DW-1 in 2015, respectively. Cysts were mainly composed of A. catenella in the YS and the BS, while those of A. pacificum were only detected occasionally at some sampling sites in the YS. Highly abundant resting cysts in surface sediment of the YS may serve as "seed banks" for recurrent toxic blooms of A. catenella and the associated shellfish contamination by paralytic shellfish toxins in the YS.

Spatiotemporal variation of paralytic shellfish toxins in the sea area adjacent to the Changjiang River estuary.

The Changjiang (Yangtze River) River estuary (CRE) and its adjacent coastal waters is a notable region for nutrient pollution, which results in severe problems of coastal eutrophication and harmful algal blooms (HABs). The occurrence of HABs, particularly those of dinoflagellate Alexandrium spp. capable of producing paralytic shellfish toxins (PSTs), has an increasing risk of contaminating seafood and poisoning human-beings. The investigation of PSTs, however, is often hampered by the relatively low abundance of Alexandrium spp. present in seawater. In this study, a monitoring strategy of PSTs using net-concentrated phytoplankton from a large volume of seawater was employed to examine spatiotemporal variations of PSTs in the CRE and its adjacent waters every month from February to September in 2015. Toxins in concentrated phytoplankton samples were analyzed using high-performance liquid chromatography coupled with a fluorescence detector (HPLC-FLD). The results showed that PSTs could be detected in phytoplankton samples during the sampling stage in the CRE and its adjacent waters. Toxin content increased gradually from February to May, reached the peak in June, and then decreased rapidly from July to September. The maximum value of PST content was 215 nmol m-3 in June. Low-potency toxins N-sulfocarbamoyl toxins 1/2 (C1/2) were the most dominant components of PST in phytoplankton samples from February to June in 2015, while high-potency gonyautoxin 4 (GTX4) became the dominant component from July to September. Toxins were mainly detected from three regions, the sea area north to the CRE, the sea area east to the CRE, and sea area near Zhoushan Island south to the CRE. Based on the results of this study, it can be inferred that the three regions around the CRE in May and June is of high risk for PST contamination and seafood poisoning.

Resolving phytoplankton taxa based on high-throughput sequencing during brown tides in the Bohai Sea, China.

Large-scale blooms formed by pico-sized phytoplankton, which strongly inhibited feeding activity and growth of cultured scallops, have been recorded along the coast of Qinhuangdao in the Bohai Sea since 2009. Based on pigment profiles and clone library analysis of phytoplankton samples during the blooms, the major bloom-forming species was primarily identified as pelagophyte Aureococcus anophagefferens Hargraves et Sieburth, the causative species of intensive brown tides in the United States and South Africa. Due to the indistinct morphological features of the bloom-forming microalgae and limited knowledge on the composition of phytoplankton communities, there were still disputes concerning the causative species of the blooms. In this study, the method of high-throughput sequencing targeted 18S rDNA V4 region was used to study the composition of pico- and nano-sized phytoplankton communities in 2013 and 2014. A total of 18 groups of eukaryotic microalgae at the class level and more than 2000 operational taxonomic units (OTUs) were identified in phytoplankton samples collected from the brown-tide zone in the Qinhuangdao coastal waters. For both years, A. anophagefferens was the most dominant species during the bloom period and its maximum relative abundance exceeded 60 percent. Along with other evidence, the results further confirm that A. anophagefferens is the major causative species of the pico-sized phytoplankton blooms in the Bohai Sea. The outbreak of brown tide exhibited a strong inter-annual variation between 2013 and 2014, and an increasing dominance of dinoflagellates could be observed in the Qinhuangdao coastal waters.

Contamination status of lipophilic marine toxins in shellfish samples from the Bohai Sea, China.

Lipophilic marine toxins in shellfish pose significant threats to the health of seafood consumers. To assess the contamination status of shellfish by lipophilic marine toxins in the Bohai Sea, nine species of shellfish periodically collected from five representative aquaculture zones throughout a year were analyzed with a method of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), homo-yessotoxin (homo-YTX), azaspiracids (AZA2 and AZA3), gymnodimine (GYM), and 13-desmethyl spirolide C (13-DesMe-C), were detected in more than 95 percent of the shellfish samples. Toxins PTX2, YTX, 13-DesMe-C and GYM were predominant components detected in shellfish samples. Scallops, clams and mussels accumulated much higher level of lipophilic marine toxins compared to oysters. Toxin content in shellfish samples collected from different sampling locations showed site-specific seasonal variation patterns. High level of toxins was found during the stages from December to February and June to July in Hangu, while from March to April and August to September in Laishan. Some toxic algae, including Dinophysis acuminata, D. fortii, Prorocentrum lima, Gonyaulax spinifera and Lingulodinium polyedrum, were identified as potential origins of lipophilic marine toxins in the Bohai Sea. The results will offer a sound basis for monitoring marine toxins and protecting the health of seafood consumers.

Genetic evidence in tracking the origin of Ulva prolifera blooms in the Yellow Sea, China.

Recurrent green tides have been recorded in the Yellow Sea for 11 consecutive years. The origin of floating green algae in the Yellow Sea, however, remains a subject of debate. Previous studies suggest that the major bloom-forming green alga Ulva prolifera represent a unique ecotype different from other attached populations of U. prolifera in China. In this study, 97 green algal samples collected during the 2012 green-tide event and from other locations along the coastline of China were analyzed. Based on the sequences of nuclear ribosomal RNA gene (rDNA) internal transcribed spacer (ITS) region and 5S rDNA spacer region, the green alga U. prolifera in the samples were identified. The intraspecific genetic diversity within U. prolifera was then examined using sequences of 5S rDNA spacer and a marker of sequence characterized amplified region (SCAR) highly specific for bloom-forming U. prolifera in the Yellow Sea. The screening results for SCAR marker demonstrated that U. prolifera attached to aquaculture rafts in Subei Shoal belong to the same ecotype of the bloom-forming U. prolifera in the Yellow Sea. These findings offer genetic evidence that aquaculture rafts in Subei Shoal are a major source of floating green algae in the Yellow Sea.

Analysis of sterols in selected bloom-forming algae in China.

Sterols, a group of stable lipid compounds, are often used as biomarkers in marine biogeochemical studies to indicate sources of organic matter. In this study, sterols in 13 species of major bloom-forming algae in China, which belong to Dinophyceae, Bacillariophyceae, Ulvophyceae, and Pelagophyceae, were analyzed with gas chromatography-mass spectrometry (GC-MS) to test their feasibility in representing different types of harmful algal blooms (HABs). It was found that (24Z)-stigmasta-5,24-dien-3ß-ol (28-isofucosterol) was a major sterol component in green-tide forming macroalga Ulva prolifera. In bloom-forming dinoflagellates Alexandrium spp., Prorocentrum micans and Scrippsiella trochoidea, (22E)-4α,23-dimethyl-5α-ergost-22-en-3ß-ol (dinosterol) was detected in addition to cholest-5-en-3ß-ol (cholesterol), (22E)-ergosta-5,22-dien-3ß-ol, (22E)-stigmasta-5,22-dien-3ß-ol and other minor sterol components. In brown-tide forming pelagophyte Aureococcus anophagefferens, (24E)-24-propylcholesta-5,24-dien-3ß-ol ((24E)-24-propylidenecholesterol) and (24Z)-24-propylcholesta-5,24-dien-3ß-ol ((24Z)-24-propylidenecholesterol) were detected together with cholesterol, (22E)-stigmasta-5,22-dien-3ß-ol, stigmast-5-en-3ß-ol and campest-5-en-3ß-ol. Among the selected bloom-forming diatoms, Chaetoceros sp. and Pseudo-nitzschia spp. only produced cholesterol, while Cylindrotheca closterium produced solely (22E)-ergosta-5,22-dien-3ß-ol. Sterol content in four bloom-forming algal species correlates well with their biomass or abundance. It's proposed that 28-isofucosterol could serve as a promising biomarker for green algae in green-tide studies. Dinosterol and (24Z)-24-propylidenecholesterol can be used as potential biomarkers to represent bloom-forming dinoflagellates and pelagophytes, while (22E)-ergosta-5,22-dien-3ß-ol is not a good indicator for diatoms.

Lipophilic marine toxins discovered in the Bohai Sea using high performance liquid chromatography coupled with tandem mass spectrometry.

Some dinoflagellates can produce lipophilic marine toxins, which pose potent threats to seafood consumers. In the Bohai Sea, an important semi-closed inland sea with intensive mariculture industry in China, there is little knowledge concerning lipophilic marine toxins and their potential threats. In this study, net-concentrated phytoplankton samples were periodically collected from 5 typical mariculture zones around the Bohai Sea, including Laishan (LS), Laizhou (LZ), Hangu (HG), Qinhuangdao (QHD) and Huludao (HLD) in 2013 and 2014, and a method using high performance liquid chromatography (HPLC) coupled with a Q-Trap mass spectrometer was applied to analyze seven representative lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), azaspiracid-1 (AZA1), gymnodimine (GYM), and 13-desmethyl spirolide C (desMeC). The method had high sensitivity and repeatability, and exhibited satisfactory recoveries for most of the lipophilic marine toxins (92.1-108%) except for AZA1 (65.8-68.9%). Nearly all the lipophilic marine toxins could be detected in phytoplankton samples from the Bohai Sea. OA, DTX1 and PTX2 were predominant components and present in most of the phytoplankton samples. The maximum content of lipophilic marine toxin in phytoplankton samples concentrated from seawater (OA 464 pg L-1; DTX1 783 pg L-1; YTX 86.6 pg L-1; desMeC 15.6 pg L-1; PTX2 1.11 × 103 pg L-1) appeared in June 2014. Based on toxins present in phytoplankton samples, it is implied that seafood in the Bohai Sea is more likely to be contaminated by OA group and PTX group toxins, and spring is the high-risk season for toxin contamination.

Paralytic shellfish toxins in phytoplankton and shellfish samples collected from the Bohai Sea, China.

Phytoplankton and shellfish samples collected periodically from 5 representative mariculture zones around the Bohai Sea, Laishan (LS), Laizhou (LZ), Hangu (HG), Qinhuangdao (QHD) and Huludao (HLD), were analysed for paralytic shellfish toxins (PSTs) using an high-performance liquid chromatography (HPLC) method. Toxins were detected in 13 out of 20 phytoplankton samples, and N-sulfocarbamoyl toxins (C1/2) were predominant components of PSTs in phytoplankton samples with relatively low toxin content. However, two phytoplankton samples with high PST content collected from QHD and LS had unique toxin profiles characterized by high-potency carbamoyl toxins (GTX1/4) and decarbamoyl toxins (dcGTX2/3 and dcSTX), respectively. PSTs were commonly found in shellfish samples, and toxin content ranged from 0 to 27.6nmol/g. High level of PSTs were often found in scallops and clams. Shellfish from QHD in spring, and LZ and LS in autumn exhibited high risks of PST contamination.

Impact of CYP2C19 Variants on Clinical Efficacy of Clopidogrel and 1-Year Clinical Outcomes in Coronary Heart Patients Undergoing Percutaneous Coronary Intervention.

The impact of pharmacogenetic variants of cytochrome P450 2C19 (CYP2C19) on clopidogrel-mediated effects on platelet inhibition, inflammatory response and endothelial function, as well as risk of major adverse cardiovascular events (MACE), in coronary heart patients undergoing percutaneous coronary intervention (PCI) was investigated. To this end, we assessed the residual platelet aggregation rate (RPA), maximal aggregation rate (MAR) and plasma levels of sCD40L, sP-selectin, MMP-9, sVCAM-1 and sE-selectin after 24 h of PCI in 559 patients treated with clopidogrel and followed up for 1 year for evidence of MACE. CYP2C19*2 and *3 variants were identified using a clopidogrel-sensitive gene detection kit. Our results showed higher RPA and MAR as well as increased sE-selectin, sCD40L, sP-selectin, MMP-9, and sVCAM-1 levels in CYP2C19 intermediate metabolizer (IM, CYP2C19*1/*2, or *1/*3), poor metabolizer (PM, CYP2C19*2/*2, *2/*3, or *3/*3) and combined IM+PM groups, relative to those in extensive metabolizers (EM, CYP2C19*1/*1). In total, 519 patients completed 1 year of follow-up, among which 69 (13.3%) experienced MACE. The risk of MACE in CYP2C19 IM+PM patients was 2.664 times higher than that in CYP2C19 EM patients (OR = 2.664 (1.397-5.193), P = 0.004). The data suggest that CYP2C19*2 and *3 variants modulate the drug efficacy of clopidogrel in coronary heart patients undergoing PCI and further enhance the risk of MACE. Accordingly, CYP2C19 pharmacogenetic profiling may be beneficial for coronary heart patients undergoing PCI to predict the efficacy of treatment with clopidogrel. We propose that IM and PM patients should benefit from treatment with higher clopidogrel doses to improve efficacy and reduce the incidence of MACE.