Potential roles of hydroxybenzoate paralytic shellfish toxins in modulating toxin biokinetics in scallops.

Paralytic shellfish toxins (PSTs) produced by some marine dinoflagellates can cause severe human intoxication via vectors like bivalves. Toxic dinoflagellate Gymnodinium catenatum produce a novel group of hydroxybenzoate PSTs named GC toxins, but their biokinetics in bivalves haven't been well examined. In this experiment, we analyzed PSTs in bay scallops Argopecten irradians exposed to G. catenatum (strain MEL11) to determine their accumulation, elimination, anatomical distribution, and biotransformation. To our surprise, up to 30% of the PSTs were accumulated in the adductor muscle of scallops at the end of the experiment, and the toxicity of adductor muscle exceeded the regulatory limit of 800 µg STXeq/kg in only 6 days. High concentration of toxins in the adductor muscle are likely linked to the rapid transfer of GC toxins from viscera to other tissues. Moreover, most GC toxins in scallops were found rapidly transformed to decarbamoyl toxins through enzyme-mediated hydrolysis, which was further supported by the in vitro incubation experiments. Our study demonstrates that GC toxins actively participate in toxin distribution and transformation in scallops, which may increase the risks of food poisoning associated with the consumption of scallop adductor muscle. ENVIRONMENTAL IMPLICATION: The negative impacts of harmful algal blooms (HABs) have become a global environmental concern under the joint effects of cultural eutrophication and climate change. Our study, targeted on the biokinetics of paralytic shellfish toxins in scallops exposed to Gymnodinium catenatum producing unique GC toxins, aims to elucidate potential risks of seafood poisoning associated with GC toxins. The findings of this study will help us to understand the roles of GC toxins in seafood poisoning, and to develop effective management strategies against toxic algal blooms and phycotoxins.

Ultrasensitive fluorescence detection of gonyautoxins in seawater using a novel molecularly imprinted nanoprobe.

Gonyautoxins (GTXs), a group of potent neurotoxins belonging to paralytic shellfish toxins (PSTs), are often associated with harmful algal blooms of toxic dinoflagellates in the sea and represent serious health and ecological concerns worldwide. In the study, a highly selective and sensitive fluorescence nanoprobe was constructed based on photoinduced electron transfer recognition mechanism to rapidly detect GTXs in seawater, using specific entrapment of molecularly imprinted polymers (MIPs) combined with fluorescence analyses. The green emissive fluorescein isothiocyanate was grafted in a silicate matrix as a signal transducer and fluorescence intensity of the nanoprobe with a core-shell structure exhibited a strong enhancement due to efficient analyte blockage in a short response time. Under optimal conditions, the developed MIPs nanoprobe presented an excellent analytical performance for spiked seawater samples including a recovery from 94.44 % to 98.23 %, a linear range between 0.018 nmol L-1 and 0.36 nmol L-1, as well as good accuracy. Furthermore, the method had extremely high sensitivity, with limit of detection obtained as 0.005 nmol L-1 for GTXs and GTX2/3. Finally, the nanoprobe was applied for the determination of GTXs in seven natural seawater samples with GTXs mixture (0.035-0.058 nmol L-1) or single GTX2/3 (0.033-0.050 nmol L-1), and the results agreed well with those of a UPLC-MS/MS method. The findings of our study suggest that the constructed MIPs-based fluorescence enhancement nanoprobe was suitable for rapid, selective and ultrasensitive detection of GTXs, particular GTX2/3, in natural seawater samples.

A dinoflagellate bloom caused by multiple species of Kareniaceae in the coastal waters of Fujian in June 2022 and its adverse impacts on Brachionus plicatilis and Artemia salina.

Recently, dinoflagellate blooms have frequently occurred in the coastal waters of Fujian, East China Sea. In June 2022, a fish-killing bloom of Kareniaceae species occurred in this region. In this study, four species of Kareniaceae, namely, Karenia longicanalis, K. papilionacea, Karlodinium veneficum, and Karl. digitatum were identified from this bloom event based on the results of single-cell PCR and clone libraries, and intraspecies genetic diversity was found in the Karl. veneficum population. The results of acute toxicity assays of the bloom water to two zooplankton species (Brachionus plicatilis and Artemia salina) demonstrated this bloom event strongly inhibited their swimming capacities and survival. The results of this study suggested that the bloom events caused by multiple species of Kareniaceae in the Fujian coastal waters had adverse impacts on the local fishery resources and zooplankton community.

Fourier Transform Infrared Spectrometry Detection of Phaeodactylum tricornutum Biomacromolecules in Response to Environmental Changes.

Environmental factors play an important role in the lipid, protein, and carbohydrate compositions of microalgae, wherein temperature and light are key influencing factors. Fourier transform infrared (FTIR) spectrometry was used in this study to detect biomacromolecules in Phaeodactylum tricornutum cells under different temperatures (10, 15, 20, and 25 °C) and different illumination conditions (1000, 2000, 3000, and 4000 lx) to study the corresponding changes in lipid, protein, and carbohydrate contents. Results indicate that the biomacromolecule content at different temperatures has different patterns. Specifically, the patterns at 15 and 25 °C are similar to each other and the contents accumulate with extended culture time. However, the pattern at 20 °C is different. The carbohydrate and protein contents peaked during the early stage of the exponential phase, whereas lipid accumulation lagged behind the former two, peaking during the middle of the culture stage and then decreasing. Lipid content was analyzed by transmission electron microscopy (TEM), which revealed that the highest lipid content was observed at 15 °C. Results also show that all of the lipid, protein, and carbohydrate contents in cells were the highest when the illumination was at 2000 lx and that the contents decreased with increasing illumination. By using FTIR, less samples were needed as compared to the traditional chemical quantitative detection methods. Moreover, the relative content changes of various biomacromolecules during the growth of P. tricornutum could be accurately determined by a single detection, thereby providing a new technique for the further study of metabolic mechanisms.

Marked shifts of harmful algal blooms in the Bohai Sea linked with combined impacts of environmental changes.

The Bohai Sea, a semi-enclosed inland sea in China and an important mariculture region, has experienced extensive harmful algal blooms (HABs) and their negative impacts for several decades. To investigate the changes of HABs and their potential drivers over time and space, a dataset of 230 HAB events (1952-2017), along with corresponding environmental data (1990-2017) was compiled. The frequency of HAB events in the Bohai Sea has increased over time but plateaued in the last decade, and our analysis showed that history of HABs in the Bohai Sea could be categorized into three periods based on their frequency, scale, and HAB-forming species. The seasonal window of HAB events has started earlier and lasted longer, and the main hotspot has moved from Bohai Bay to coastal waters of Qinhuangdao over time. There were marked shifts in the representative HAB-forming microalgae, from dinoflagellates in the first period (before 2000) to haptophytes in the second period (2000-2009), and pelagophytes in the third period (2009 onwards). These community changes are accompanied by a trend toward diversification of HAB-forming microalgae, decrease in cell-size, and increase in negative impacts. Statistical analyses indicate that long-term changes in HABs in the Bohai Sea are linked with the combined effects of climate change, eutrophication and mariculture development. The results of the present study require to refine future monitoring programs, develop adaptive management strategies and predictive models for HABs in the Bohai Sea.

An investigation on bloom dynamics of Alexandrium catenella and A. pacificum and toxin accumulation in shellfish along the coast of Qinhuangdao, China.

The dinoflagellate genus Alexandrium comprises most of the toxic bloom-forming species producing paralytic shellfish toxins (PSTs) in the sea. Recently, repeated paralytic shellfish poisoning episodes have been recorded in Qinhuangdao located at the west coast of the Bohai Sea. To elucidate the relationship between toxic Alexandrium blooms and the poisoning episodes, a year-round investigation was carried out in this region from July 2020 to July 2021. Two qPCR assays were used to detect A. catenella and A. pacificum, and LC-MS/MS was applied to analyze PSTs in phytoplankton and shellfish samples. The blooms of A. catenella and A. pacificum were found in April and July, respectively, and PST content in three bivalves exhibited notable increase following the bloom of A. catenella. The results revealed bloom dynamics of the two toxic Alexandrium species in the Bohai Sea for the first time, and further confirmed A. catenella as the causative agent of poisoning episodes.

Evolution of harmful algal blooms in the East China Sea under eutrophication and warming scenarios.

Harmful algal blooms (HABs) worldwide are experiencing obvious changes under the combined impacts of global warming, eutrophication, and other driving forces. In the East China Sea (ECS), large-scale blooms caused by dinoflagellates occurred since 2000 and there has been an apparent shift of bloom-causative microalgae from diatoms to dinoflagellates. To predict the future evolution of HABs in this region, a model was developed based on the competition between diatoms and dinoflagellates, which would serve to reproduce the seasonal succession of microalgal blooms driven by multiple environmental factors. The evolution features of HABs were then projected under different scenarios of eutrophication and global warming. Under the 'business as usual' scenario, dinoflagellate blooms are expected to become more frequent with higher peak biomass concentrations over the next 30 years. Changes in nutrient composition of the Changjiang riverine discharge may largely give rise to this phenomenon, and accelerated warming associated with climate change may result in earlier occurrence of dinoflagellate blooms. To prevent further intensification of dinoflagellate blooms, efforts could be made to reduce nitrogen inputs and maintain or even increase silicate inputs from the Changjiang river.

Intense blooms of Phaeocystis globosa in the South China Sea are caused by a unique "giant-colony" ecotype.

The haptophyte Phaeocystis globosa, an important causative agent of harmful algal blooms globally, exhibits varying morphological and physiological features and high genetic diversity, yet the relationship among these has never been elucidated. In this study, colony sizes and pigment profiles of 19 P. globosa isolates from the Pacific and Atlantic Oceans were determined. Genetic divergence of these strains was analyzed using the chloroplast rbcS-rpl27 intergenic spacer, a novel high-resolution molecular marker. Strains could be divided into four genetic clades based on these sequences, or two groups based on colony size and the identity of diagnostic pigments (19'-hexanoyloxyfucoxanthin, hex-fuco, and 19'-butanoyloxyfucoxanthin, but-fuco). Three strains from the South China Sea (SCS), all belonging to the same genetic clade, have unique biological features in forming giant colonies and possessing but-fuco as their diagnostic pigment. Based on these findings, we propose that these SCS strains should be a unique "giant-colony" ecotype of P. globosa. During the period 2016-2021, more than 1000 rbcS-rpl27 sequences were obtained from 16 P. globosa colony samples and 18 phytoplankton samples containing solitary P. globosa cells in the SCS. Phylogenetic analysis indicated that >95% of the sequences from P. globosa colonies in the SCS were comprised of the "giant-colony" ecotype, whereas the genetic diversity of solitary cells was much higher. Results demonstrated that intense blooms of P. globosa featuring giant colonies in the SCS were mainly caused by this giant-colony P. globosa ecotype.

Rising CO2 will increase toxicity of marine dinoflagellate Alexandrium minutum.

Ocean acidification caused by increasing emission of carbon dioxide (CO2) is expected to have profound impacts on marine ecological processes, including the formation and evolution of harmful algal blooms (HABs). We designed a set of experiments in the laboratory to examine the effects of increasing CO2 on the growth and toxicity of a toxic dinoflagellate Alexandrium minutum producing paralytic shellfish toxins (PSTs). It was found that high levels of CO2 (800 and 1200 ppm) significantly promoted the growth of A. minutum compared to the group (400 ppm) representing the current CO2 level. The total yields of PSTs by A. minutum, including both intracellular and extracellular toxins, were significantly enhanced, probably due to the induction of core enzyme activity and key amino acids synthesis for PST production. More interestingly, high level of CO2 promoted the transformation from gonyautoxin2&3 to gonyautoxin1&4 and depressed the release of PSTs from inside to outside of the cells. All these processes collectively led to an apparent increase of A. minutum toxicity. Our study demonstrated that rising CO2 would increase the risk of toxic A. minutum based on the comprehensive analyses of different processes including algal growth and toxin synthesis, transformation and release.

First record of a Takayama bloom in Haizhou Bay in response to dissolved organic nitrogen and phosphorus.

Since 1990s, harmful algal blooms (HABs) of Kareniaceae, primarily caused by species of Karenia and Karlodinium and rarely by Takayama species, have been substantially increasing in frequency and duration in the coastal waters of China. In this study, we recorded a bloom of high abundance of T. acrotrocha in the Haizhou Bay, the Yellow Sea in September 2020, which is the first record of a Takayama bloom in the temperate coastal waters of China. We found that high concentrations of DON and DOP accelerated the proliferation of T. acrotrocha in the Haizhou Bay. Intensive mariculture, and terrestrial nitrogen and phosphorus input may be responsible for the eutrophication in the Haizhou Bay featuring high concentrations of DON and DOP, and high DIN/DIP ratios. The results suggested that, under ocean warming, the HABs of Kareniaceae are becoming increasingly dominant in eutrophic temperate coasts with intensive mariculture activities.

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.

Green Tides in the Yellow Sea Promoted the Proliferation of Pelagophyte Aureococcus anophagefferens.

Harmful algal blooms formed by fast-growing, ephemeral macroalgae have expanded worldwide, yet there is limited knowledge of their potential ecological consequences. Here, we select intense green tides formed by Ulva prolifera in the Yellow Sea, China, to examine the ecological consequences of these blooms. Using 28-isofucosterol in the surface sediment as a biomarker of green algae, we identified the settlement region of massive floating green algae in the area southeast of the Shandong Peninsula in the southern Yellow Sea. The responses of the phytoplankton assemblage from the deep chlorophyll-a maximum layer were then resolved using high-throughput sequencing. We found striking changes in the phytoplankton community in the settlement region after an intensive green tide in 2016, characterized by a remarkable increase in the abundance of the pelagophyte Aureococcus anophagefferens, the causative species of ecosystem disruptive brown tides. Our study strongly suggests that the occurrence of massive macroalgal blooms may promote blooms of specific groups of microalgae through alteration of the marine environment.

Toxic effects, mechanisms, and ecological impacts of harmful algal blooms in China.

Over the last 30 years, harmful algal blooms (HABs) have occurred frequently in the coastal waters of China, resulting in financial losses of over 5.9 billion yuan (about 0.87 billion US dollars) due to massive fish and shellfish mortalities and negative impacts on tourism. To better understand HABs in China, herein we summarized bloom events with massive fish/shellfish mortalities and/or economic losses. Our results suggest that the diversity of HAB species has increased over the last 30 years, with the main causative species shifting from the raphidophyte Chattonella marina and dinoflagellates Gymnodinium spp. to various other species, including the dinoflagellates Karenia mikimotoi and Prorocentrum donghaiense, the haptophyte Phaeocystis globosa, and the pelagophyte Aureococcus anophagefferens. In addition, new types of HABs, such as macroalgal blooms, emerged with severe ecological impacts. We also reviewed the toxic effects, mechanisms, and ecological impacts of common HAB causative species in China. Analysis of the toxic effects of three types of harmful algae (toxin-producing, fish killing, and ecosystem disruptive algae) on marine organisms commonly found in China at different trophic levels revealed that HABs often had toxic effects on multiple organisms in addition to fish or shellfish, with species-specific impacts. Common mechanisms of intoxication include shifting environmental parameters, shellfish poisoning, reactive oxygen species, and haemolytic/cytotoxic toxins. The main mechanism appears to vary with the type of HAB species, and for some notorious algae such as K. mikimotoi and C. marina, further investigations are needed to identify their intoxication mechanism.

Pigment Characterization of the Giant-Colony-Forming Haptophyte Phaeocystis globosa in the Beibu Gulf Reveals Blooms of Different Origins.

The giant-colony-forming haptophyte Phaeocystis globosa has caused several large-scale blooms in the Beibu Gulf since 2011, but the distribution and dynamics of the blooms remained largely unknown. In this study, colonies of P. globosa, as well as membrane-concentrated phytoplankton samples, were collected during eight cruises in the Beibu Gulf from September 2016 to August 2017. Pigments were analyzed by high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). The pigment 19'-hexanoyloxyfucoxanthin (hex-fuco), generally considered a diagnostic pigment for Phaeocystis, was not detected in P. globosa colonies in the Beibu Gulf, whereas 19'-butanoyloxyfucoxanthin (but-fuco) was found in all colony samples. Moreover, but-fuco in membrane-concentrated phytoplankton samples exhibited a similar distribution pattern to that of P. globosa colonies, suggesting that but-fuco provided the diagnostic pigment for bloom-forming P. globosa in the Beibu Gulf. Based on the distribution of but-fuco in different water masses in the region prior to the formation of intensive blooms, it is suggested that P. globosa blooms in the Beibu Gulf could originate from two different sources. IMPORTANCE Phaeocystis globosa has formed intensive blooms in the South China Sea and even around the world, causing huge social economic losses and environmental damage. However, little is known about the formation mechanism and dynamics of P. globosa blooms. 19'-Hexanoyloxyfucoxanthin (hex-fuco) is often used as the pigment proxy to estimate Phaeocystis biomass, while this is challenged by the giant-colony-forming P. globosa in the Beibu Gulf, which contains only 19'-butanoyloxyfucoxanthin (but-fuco) but not hex-fuco. Using but-fuco as a diagnostic pigment, we traced two different origins of P. globosa blooms in the Beibu Gulf. This study clarifies the development process of P. globosa blooms in the Beibu Gulf, which provides a basis for the early monitoring and prevention of the blooms.

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.

Increasing dominance of dinoflagellate red tides in the coastal waters of Yellow Sea, China.

The Yellow Sea (YS) has been subjected to harmful algal blooms (HABs) for several decades. In this study, we compiled and analyzed a dataset of 165 red tides from 1972 to 2017 and a dataset of green tides from 2008 to 2017 in the YS. The most notable feature of red tides in the YS is the increasing dominance of dinoflagellate red tides in terms of frequency, scale, seasonal distribution, spatial coverage, and red tide causative species. The increasing dominance of dinoflagellate red tides is closely related to eutrophication and the development of the mariculture industry in the YS. However, the dinoflagellate red tides in the northern Yellow Sea (NYS) and the southern Yellow Sea (SYS) have different features. The apparent changes in red tides in the SYS in terms of frequency and seasonal patterns might have been caused by recurrent large-scale green tides in the last decade.

Biokinetics and biotransformation of paralytic shellfish toxins in different tissues of Yesso scallops, Patinopecten yessoensis.

Paralytic shellfish toxins (PSTs) are a group of natural toxic substances often found in marine bivalves. Accumulation, anatomical distribution, biotransformation and depuration of PSTs in different tissues of bivalves, however, are still not very well understood. In this study, we investigated biokinetics and biotransformation of PSTs in six different tissues, namely gill, mantle, gonad, adductor muscle, kidney, and digestive gland, in Yesso scallops Patinopecten yessoensis exposed to a toxic strain of dinoflagellate Alexandrium pacificum. High daily accumulation rate (DAR) was recorded at the beginning stage of the experiment. Most of the PSTs in toxic algae ingested by scallops were retained and the toxicity level of PSTs in scallops exceeded the regulatory limit within 5 days. At the late stage of the experiment, however, DAR decreased obviously due to the removal of PSTs. Fitting results of the biokinetics model indicated that the amount of PSTs transferred from digestive gland to mantle, adductor muscle, gonad, kidney, and gill in a decreasing order, and adductor muscle, kidney, and gonad had higher removal rate than gill and mantle. Toxin profile in digestive gland was dominated by N-sulfocarbamoyl toxins 1/2 (C1/2), closely resembled that of the toxic algae. In contrast, toxin components in kidney were dominated by high-potency neosaxitoxin (NEO) and saxitoxin (STX), suggesting that the kidney be a major organ for transformation of PSTs.

Distribution of Alexandrium pacificum cysts in the area adjacent to the Changjiang River estuary, China.

The coastal waters adjacent to the Changjiang River estuary (CRE) are characterized by nutrient pollution and recurrent harmful algal blooms. In this study, resting cysts of Alexandrium pacificum Litaker and A. catenella (Whedon & Kof.) Balech, two major species within the A. tamarense species complex in Chinese coastal waters, were studied using sediment samples collected from the area adjacent to the CRE in May 2014 and December 2015. Cysts were detected with two real-time quantitative PCR assays, as well as the primuline-staining method. Only cysts of A. pacificum were found in the study area, which mainly distributed in the mud depositional zone near the CRE. A low-abundance region of the cysts present in spring is in accordance with the intrusive pathway of the Nearshore Kuroshio Branch Current (NKBC), suggesting that A. pacificum blooms could be regulated by seasonal intrusion of NKBC.

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.