Brown tides linked to the unique nutrient profile in coastal waters of Qinhuangdao, China.

Brown tides caused by the pelagophyte Aureococcus anophagefferens have frequently occurred in the Bohai Sea since 2009 and have led to a dramatic collapse of the local scallop culture. To determine why brown tides occurred in the Bohai Sea rather than in other eutrophic coastal waters of China, phytoplankton communities and nutrients were evaluated and nutrient addition experiments were conducted in the Qinhuangdao coastal area. The concentration of dissolved organic nitrogen (DON) was nearly five times higher than that of dissolved inorganic nitrogen (DIN) during brown tides. High levels of phytoplankton biomass and nutrients were observed in the inshore waters, and the patterns of different nutrients were heterogeneous, which could be due to the uneven distribution of pelagophytes and non-brown tide phytoplankton populations (NBTP). The nutrient enrichment results indicated that the growth of the phytoplankton community was nitrogen-limited. Enrichment of DON, especially urea, could promote the growth of pelagophytes during the development stages of the brown tide. In brief, the results of this study imply that the unique nutrient profile (rich in DON but deficient in DIN) could support the outbreak of brown tides in the inshore waters of Qinhuangdao.

3,000 km and 1,500-year presence of Aureococcus anophagefferens reveals indigenous origin of brown tides in China.

The nonmotile, spherical, picoplanktonic (2-µm-sized) pelagophyte Aureococcus anophagefferens has caused numerous harmful blooms ("brown tides") across global marine ecosystems. Blooms have developed along the east coast of the USA since 1985, a limited number of times in South Africa around 1997, and frequently in China since 2009. As a consequence, the harmful blooms have caused massive losses in aquaculture and coastal ecosystems, particularly mortalities in cultured shellfish. Therefore, whether A. anophagefferens was recently introduced to China via natural/artificial transport of resting stage cells or has been an indigenous species has become a question of profound ecological significance and broad interest, which motivated our extensive investigation on the geographic and historical presence of this species in the seas of China. We applied a combined approach of extensive PCR-based detection and sequencing, germination experiments and monoclonal antibody staining of germlings to samples of surface sediment and sediment core (dated via combined isotopic measurements) collected from all four seas of China, and searched the supplementary data set of a recent Science publication. We discovered that A. anophagefferens does have a resting stage in the sediment, but it also has a wide geographic distribution both in China (covering a range of ~30° in latitude, ~15.7° in longitude and 2.5-3,456 m in water depth; temperate to tropical and coastal to open oceans) and in almost all oceans of the world and a historical presence of >1,500 years in the Bohai Sea, China. The work revealed that A. anophagefferens is not a recently introduced, but an indigenous species in China and has in fact a globally cosmopolitan distribution.

Comparative Genomics of Chrysochromulina Ericina Virus and Other Microalga-Infecting Large DNA Viruses Highlights Their Intricate Evolutionary Relationship with the Established Mimiviridae Family.

Chrysochromulina ericina virus CeV-01B (CeV) was isolated from Norwegian coastal waters in 1998. Its icosahedral particle is 160 nm in diameter and encloses a 474-kb double-stranded DNA (dsDNA) genome. This virus, although infecting a microalga (the haptophyceae Haptolina ericina, formerly Chrysochromulina ericina), is phylogenetically related to members of the Mimiviridae family, initially established with the acanthamoeba-infecting mimivirus and megavirus as prototypes. This family was later split into two genera (Mimivirus and Cafeteriavirus) following the characterization of a virus infecting the heterotrophic stramenopile Cafeteria roenbergensis (CroV). CeV, as well as two of its close relatives, which infect the unicellular photosynthetic eukaryotes Phaeocystis globosa (Phaeocystis globosa virus [PgV]) and Aureococcus anophagefferens (Aureococcus anophagefferens virus [AaV]), are currently unclassified by the International Committee on Viral Taxonomy (ICTV). The detailed comparative analysis of the CeV genome presented here confirms the phylogenetic affinity of this emerging group of microalga-infecting viruses with the Mimiviridae but argues in favor of their classification inside a distinct clade within the family. Although CeV, PgV, and AaV share more common features among them than with the larger Mimiviridae, they also exhibit a large complement of unique genes, attesting to their complex evolutionary history. We identified several gene fusion events and cases of convergent evolution involving independent lateral gene acquisitions. Finally, CeV possesses an unusual number of inteins, some of which are closely related despite being inserted in nonhomologous genes. This appears to contradict the paradigm of allele-specific inteins and suggests that the Mimiviridae are especially efficient in spreading inteins while enlarging their repertoire of homing genes.IMPORTANCE Although it infects the microalga Chrysochromulina ericina, CeV is more closely related to acanthamoeba-infecting viruses of the Mimiviridae family than to any member of the Phycodnaviridae, the ICTV-approved family historically including all alga-infecting large dsDNA viruses. CeV, as well as its relatives that infect the microalgae Phaeocystic globosa (PgV) and Aureococcus anophagefferens (AaV), remains officially unclassified and a source of confusion in the literature. Our comparative analysis of the CeV genome in the context of this emerging group of alga-infecting viruses suggests that they belong to a distinct clade within the established Mimiviridae family. The presence of a large number of unique genes as well as specific gene fusion events, evolutionary convergences, and inteins integrated at unusual locations document the complex evolutionary history of the CeV lineage.

Proteomic profile in the mussel Perna viridis after short-term exposure to the brown tide alga Aureococcus anophagefferens.

Blooms of Aureococcus anophagefferens, referred to as brown tides are responsible for massive mortalities and recruitment failure of some bivalves. However, the molecular mechanisms underlying the toxicity remain elusive despite its biological significance, and the information currently available on the molecular effects is still insufficient. In this study, to evaluate the toxicity and associated mechanism of A. anophagefferens on bivalves, we analyzed the protein expression profiles in digestive glands of the A. anophagefferens-exposed Perna viridis by using iTRAQ. A total of 3138 proteins were identified in the digestive glands of A. anophagefferens-exposed P. viridis based on iTRAQ. Amongst, a repertoire of 236 proteins involved in cell, cell part, catalytic activity, metabolic process, biological regulation, immune system process, and response to stimulus were found to be differentially expressed. Functional analysis of the differentially expressed proteins demonstrated that innate immune system of P. viridis was activated, and some proteins associated with stress response and lipid metabolism were induced after exposure to A. anophagefferens. Additionally, MDA content, SOD activity and GSH-Px activity was increased significantly in the digestive gland of A. anophagefferens-exposed P. viridis. Taken together, our results indicated that the A. anophagefferens could induce oxidative stress, activate complement system and alter fat acid metabolism of P. viridis.

Relationship between phytoplankton community succession and environmental parameters in Qinhuangdao coastal areas, China: A region with recurrent brown tide outbreaks.

The picoplanktonic pelagophyte Aureococcus anophagefferens could trigger harmful algal blooms (HABs) to discolor water in brown, known as brown tide. Since 2009, large-scale brown tides, caused by A. anophagefferens, had been occurred in early summer for three consecutive years in the coastal waters of Qinhuangdao, China and resulted considerable deleterious effects on the scallop mariculture industry. The causes for the occurrence of brown tides were not fully understood. Therefore, we conducted a one-year survey from June 2013 to May 2014 to study the seasonal succession of the phytoplankton community, including A. anophagefferens and its relationship with environmental variables in the area. The results revealed that the population dynamics of the phytoplankton community were significant variation with seasonal succession, in which A. anophagefferens played an important role during the entire year. The trend of the whole diversity index indicated that the community structure became more stable in winter. The results of principle component analysis (PCA) applied to the environmental factors indicated four major seasonal groups in the environmental variables. The water temperature, silicate and total nitrogen were contributed to the environment in summer, autumn and spring, respectively. In addition, a few another environmental factors commonly contributed to the winter waterbody, indicated that the aquatic environment is more complex in the cold season. The result revealed that the phytoplankton community structure and its variation were mainly affected by the hydrological factors, by using the redundancy analysis (RDA) for the relationship between dominant species and the environment. Furthermore, we inferred Chaetoceros decipiens as a potential species for the breakout of harmful algae blooms (HABs) by RDA ordination. We concluded that the key factor for the seasonal variations in the dynamics of phytoplankton community could be the hydrological parameters in Qinghuangdao coastal area. This research may provide more insight into the occurrence mechanism of brown tide.

Rapid Identification and Quantification of Aureococcus anophagefferens by qPCR Method (Taqman) in the Qinhuangdao Coastal Area: A Region for Recurrent Brown Tide Breakout in China.

Since 2009, Aureococcus anophagefferens has caused brown tide to occur recurrently in Qinhuangdao coastal area, China. Because the algal cells of A. anophagefferens are so tiny (~3 µm) that it is very hard to identify exactly under a microscope for natural water samples, it is very urgent to develop a method for efficient and continuous monitoring. Here specific primers and Taqman probe are designed to develop a real-time quantitative PCR (qPCR) method for identification and quantification continually. The algal community and cell abundance of A. anophagefferens in the study area (E 119°20'-119°50' and N 39°30'-39°50') from April to October in 2013 are detected by pyrosequencing, and are used to validate the specification and precision of qPCR method for natural samples. Both pyrosequencing and qPCR shows that the targeted cells are present only in May, June and July, and the cell abundance are July > June > May. Although there are various algal species including dinoflagellata, diatom, Cryptomonadales, Chrysophyceae and Chlorophyta living in the natural seawater simultaneously, no disturbance happens to qPCR method. This qPCR method could detect as few as 10 targeted cells, indicating it is able to detect the algal cells at pre-bloom levels. Therefore, qPCR with Taqman probe provides a powerful and sensitive method to monitor the brown tide continually in Qinhuangdao coastal area, China. The results provide a necessary technology support for forecasting the brown tide initiation, in China.

Prediction of Aureococcus anophageffens using machine learning and deep learning.

The recurrent brown tide phenomenon, attributed to Aureococcus anophagefferens (A. anophagefferens), constitutes a significant threat to the Qinhuangdao sea area in China, leading to pronounced ecological degradation and substantial economic losses. This study utilized machine learning and deep learning techniques to predict A. anophagefferens population density, aiming to elucidate the occurrence mechanism and influencing factors of brown tide. Specifically, Random Forest (RF) algorithm was utilized to impute missing water quality data, facilitating its direct application in subsequent algal population prediction models. The results revealed that all four models-RF, Support Vector Regression (SVR), Multilayer Perceptron (MLP), and Convolutional Neural Network (CNN)-exhibited high accuracy in predicting A. anophagefferens population densities, with R2 values exceeding 0.75. RF, in particular, showed exceptional accuracy and reliability, with an R2 value surpassing 0.8. Additionally, the study ascertained five critical factors influencing A. anophagefferens population density: ammonia nitrogen, pH, total nitrogen, temperature, and silicate.

Sublethal effects on the hard clam Mercenaria mercenaria after exposure to Aureococcus anophagefferens Chinese strain isolates.

Brown tides caused by Aureococcus anophagefferens occur frequently worldwide and have contributed to the collapse of Mercenaria mercenaria farming in the United States. This economically valuable hard clam has been used in China for more than 20 years. To date, it has remained unknown whether A. anophagefferens Chinese strain has an impact on hard clam cultivation in the coastal areas of China or other sea areas worldwide if it enters through ship ballast water and other ways. In this study, a Chinese strain of A. anophagefferens isolated from the brown tide waters of Bohai Bay, China, was selected to explore its influence on the feedback of hard clams. After being fed with A. anophagefferens, hard clams showed characteristics similar to starvation. The reduced feeding efficiency of hard clams leads to reduced energy intake. However, the immune response and oxidative stress, result in increased energy consumption. An imbalance in the energy budget may be an important reason for hard clam starvation. This study has described the response characteristics of the A. anophagefferens Chinese strain to M. mercenaria, explored the reasons for the negative impact of A. anophagefferens on hard clams, and provides ideas for reducing shellfish aquaculture caused by brown tides.

Covariance of Marine Nucleocytoplasmic Large DNA Viruses with Eukaryotic Plankton Communities in the Sub-Arctic Kongsfjorden Ecosystem: A Metagenomic Analysis of Marine Microbial Ecosystems.

Nucleocytoplasmic large DNA viruses (NCLDVs) infect various marine eukaryotes. However, little is known about NCLDV diversity and their relationships with eukaryotic hosts in marine environments, the elucidation of which will advance the current understanding of marine ecosystems. This study characterizes the interplay between NCLDVs and the eukaryotic plankton community (EPC) in the sub-Arctic area using metagenomics and metabarcoding to investigate NCLDVs and EPC, respectively, in the Kongsfjorden ecosystem of Svalbard (Norway) in April and June 2018. Gyrodinium helveticum (Dinophyceae) is the most prevalent eukaryotic taxon in the EPC in April, during which time Mimiviridae (31.8%), Poxviridae (25.1%), Phycodnaviridae (14.7%) and Pandoraviridae (13.1%) predominate. However, in June, the predominant taxon is Aureococcus anophagefferens (Pelagophyceae), and the NCLDVs, Poxviridae (32.9%), Mimiviridae (29.1%), and Phycodnaviridae (18.5%) appear in higher proportions with an increase in Pelagophyceae, Bacillariophyceae, and Chlorophyta groups. Thus, differences in NCLDVs may be caused by changes in EPC composition in response to environmental changes, such as increases in water temperature and light intensity. Taken together, these findings are particularly relevant considering the anticipated impact of NCLDV-induced EPC control mechanisms on polar regions and, therefore, improve the understanding of the Sub-Arctic Kongsfjorden ecosystem.

Kratosvirus quantuckense: the history and novelty of an algal bloom disrupting virus and a model for giant virus research.

Since the discovery of the first "giant virus," particular attention has been paid toward isolating and culturing these large DNA viruses through Acanthamoeba spp. bait systems. While this method has allowed for the discovery of plenty novel viruses in the Nucleocytoviricota, environmental -omics-based analyses have shown that there is a wealth of diversity among this phylum, particularly in marine datasets. The prevalence of these viruses in metatranscriptomes points toward their ecological importance in nutrient turnover in our oceans and as such, in depth study into non-amoebal Nucleocytoviricota should be considered a focal point in viral ecology. In this review, we report on Kratosvirus quantuckense (née Aureococcus anophagefferens Virus), an algae-infecting virus of the Imitervirales. Current systems for study in the Nucleocytoviricota differ significantly from this virus and its relatives, and a litany of trade-offs within physiology, coding potential, and ecology compared to these other viruses reveal the importance of K. quantuckense. Herein, we review the research that has been performed on this virus as well as its potential as a model system for algal-virus interactions.

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.

Mechanism by Which MC Controls Harmful Algal Blooms Revealed by Cell Morphology of Aureococcus anophagefferens.

On the basis of field experience, a bloom does not continue after treatment with modified clay (MC), even though the residual harmful algal bloom (HAB) biomass accounts for 20-30% of the initial cells. This interesting phenomenon indicates that, in addition to causing flocculation, MC can inhibit the growth of residual cells. Here, from a cell morphology perspective, Aureococcus anophagefferens was used as a model organism to explore this scientific issue and clarify the mechanism by which MC mitigates harmful algal blooms (HABs). The results showed that, at an ~70% removal efficiency, neutral clay (NC) could not effectively inhibit the growth of residual cells, although it caused various forms of damage to residual cells, such as cell deformation, cell breakage, decreased extracellular polysaccharides (EPS), increased cell membrane permeability, and increased cytoplasmic granularity, due to physical collisions. After modification, some physical and chemical properties of the clay particle surface were changed; for example, the surface electrical properties changed from negative to positive, lamellar spacing increased, hardness decreased, adhesion chains increased, adhesion improved, and the number of absorption sites increased, enhancing the occurrence of chemical and electrochemical effects and physical collisions with residual cells, leading to severe cell deformation and chemical cell breakage. Thus, MC effectively inhibited the growth of residual cells and controlled HABs.

Harmful algal blooms and associated fisheries damage in East Asia: Current status and trends in China, Japan, Korea and Russia.

Occurrences of harmful algal blooms (HABs) and associated fisheries damage have been continuously monitored since the 1970s along the coasts of East Asia. Fisheries damage comprises mass mortalities of fish and shellfish mainly by harmful dinoflagellates and raphidophytes (e.g., Chattonella antiqua/marina, Cochlodinium polykrikoides and Karenia mikimotoi), and contamination of algal toxins in shellfish in particular Diarrhetic Shellfish Toxins by Dinophysis spp. and Paralytic Shellfish Toxins by Alexandrium spp. Shellfish mass mortalities due to Heterocapsa circularisquama in Hong Kong and western Japan, and fish kills by Karlodinium digitatum are unique incidents for this region, whereas C. antiqua/marina, C. polykrikoides and K. mikimotoi are common also in other regions. Time series data showed that the highest bloom numbers were recorded in 1980 (Japan), in 1998 (Korea) and in 2003 (China), followed by decreasing trends in these countries. These data suggest a shift in microalgal species composition, from dominance by diatoms to dinoflagellates after 1980s in Korea, and from diatoms to small haptophytes and cyanobacteria after 2013 in eastern Russia. HAB species composition and the changes were compared among countries, for better understanding on current status and trend of HAB species in East Asia.

[Characteristics of seawater nutrients during the occurrence of brown tide in the coastal area of Qinhuangdao, China]. / ç§¦çš‡å²›æµ·åŸŸè¤æ½®ç”Ÿæ¶ˆè¿‡ç¨‹ä¸­è¥å »ç›ç‰¹å¾.

Brown tide that occurred in Qinhuangdao coastal waters from 2009 to 2015 caused huge losses of local marine aquaculture and coastal tourism, with devastating effects on marine ecosystems. Nutrients are important biogenic elements for algal growth. It is of great significance to examine the fluctuation characteristics of nutrients in the process of brown tide to understand the nutritional mechanism of brown tide. Based on the survey data of 30 stations located in Qinhuangdao coastal area from April to June 2014, we analyzed nutrient characteristics during the occurrence of brown tide and its relationship with the population dynamics of Aureococcus anophagefferens. The results showed that the concentration of dissolved nitrogen (DN) in April, May and June 2014 was 265.65, 355.36 and 323.71 µg·L-1 respectively, and the concentration of dissolved organic nitrogen (DON) was 196.98, 242.88 and 177.69 µg·L-1, accounting for 74.2%, 68.3% and 54.9% of DN, respectively. The concentration of dissolved phosphorus (DP) in April, May and June was 15.95, 11.39 and 11.14 µg·L-1 respectively. In April and May, PO43--P accounted for a large proportion of the DP, 74.8% and 80.9% respectively. In June, the proportion of PO43--P in DP fell to 33.8%, and the proportion of dissolved organic phosphorus (DOP) in DP rose to 66.2%. The concentration of SiO32--Si in April, May and June was 70.95, 181.13 and 120.68 µg·L-1, respectively. Except for dissolved inorganic nitrogen (DIN) in May and DON, the distribution of other nutrients had clear characteristics that it decreased gradually from inshore to the offshore, with the relatively high concentrations in river mouth. Through R-factor analysis and nutrient structure analysis, it was found that in April, brown tide was at the development stage, and DOP might be the main driving factor for the growth of A. anophagefferens. In May, brown tide was at the maintenance stage, and water temperature became the main controlling factor. When water temperature was higher than 12 ℃, brown tide could occur. In June, brown tide began to decay, and PO43--P had greater effects on the community structure of phytoplankton. DON was the important factor causing the outbreak of brown tide, with a concentration threshold of 150 µg·L-1 and the ratio DON/DIN being greater than 1.

Responses of cytochrome P450, GST and MXR in the mussel Perna viridis to the exposure of Aureococcus anophagefferens.

The brown tide formed by a microscopic alga called Aureococcus anophagefferens has a devastating effect on filter-feeding bivalves, however, the related toxic principle remains an open question. In this study, we found that A. anophagefferens cells could motivate detoxification associated genes including CYP450, GST, P-gp and MVP, and induce SOD activity in the mussel Perna viridis. D1-like and D2-like receptors were expressed at high level in the gills of P. viridis, however, D2-like receptor transcript was too low to detect in digestive gland. The exposure of A. anophagefferens did not lead to any significant alterations in the expression of D1-like and D2-like receptors in both gills and digestive gland. These findings suggested that A. anophagefferens exhibited cytotoxicity toward bivalves, but did not obviously disrupt the dopamine system at transcriptional level in the acute exposure. Further studies are warranted to explore the nature of toxic compounds in A. anophagefferens affected bivalves.

Hemolytic and cytotoxic activity from cultures of Aureococcus anophagefferens-a causative species of brown tides in the north-western Bohai Sea, China.

Brown tides were first observed in 2009 in the north-western Bohai Sea (Qinhuangdao sea area), China, and blooms have occurred at different scales in late spring every year since then. Although the detrimental effects on marine organisms of the causative phytoplankton species Aureococcus anophagefferens have been extensively studied, the mechanism remains poorly understood. We used erythrocytes and adrenal gland chromaffin tumor cells (PC12) to explore the hemolytic activity and cytotoxicity, respectively, of chloroform and methanol extracts of cultured A. anophagefferens isolated from the north-western Bohai Sea area. The methanol extracts showed no hemolytic or cytotoxic activity. Chloroform extracts had a potent hemolytic effect on rabbit erythrocytes; thin layer chromatography (TLC) indicated that the hemolysin was a kind of glycolipid compound. Erythrocyte lysis assay showed that erythrocytes of sea bream were sensitive to the hemolysin, whereas those of human and chicken erythrocytes were insensitive. The hemolytic effects were elevated as temperatures rose from 4 °C to 37 °C. Hemolytic blocking experiments showed that sphingomyelin and d-xylose can inhibit hemolysis significantly, while osmotic protectants with different hydrated molecular diameters had no inhibition, and the hemolysins had no obvious phospholipase activity. The chloroform extracts of A. anophagefferens had significant inhibitory effects on the viability of PC12 cells, and can induce efflux of lactic dehydrogenase (LDH) of PC12 cells and lead to their necrosis.

Transcriptional Shifts Highlight the Role of Nutrients in Harmful Brown Tide Dynamics.

Harmful algal blooms (HABs) threaten ecosystems and human health worldwide. Controlling nitrogen inputs to coastal waters is a common HAB management strategy, as nutrient concentrations often suggest coastal blooms are nitrogen-limited. However, defining best nutrient management practices is a long-standing challenge: in part, because of difficulties in directly tracking the nutritional physiology of harmful species in mixed communities. Using metatranscriptome sequencing and incubation experiments, we addressed this challenge by assaying the in situ physiological ecology of the ecosystem destructive alga, Aureococcus anophagefferens. Here we show that gene markers of phosphorus deficiency were expressed in situ, and modulated by the enrichment of phosphorus, which was consistent with the observed growth rate responses. These data demonstrate the importance of phosphorus in controlling brown-tide dynamics, suggesting that phosphorus, in addition to nitrogen, should be evaluated in the management and mitigation of these blooms. Given that nutrient concentrations alone were suggestive of a nitrogen-limited ecosystem, this study underscores the value of directly assaying harmful algae in situ for the development of management strategies.

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.

Physiological response dynamics of the brown tide organism Aureococcus anophagefferens treated with modified clay.

On the basis of experiences in mitigating harmful algal blooms (HABs) with modified clay (MC), a bloom does not continue after the dispersal of the MC, even though the density of the residual cells in the water remains as high as 20-30% of the initial cell density. This interesting phenomenon indicates that in addition to flocculation, MC has additional mechanisms of HAB control. Here, Aureococcus anophagefferens was selected as a model organism to study the physiological response dynamics of residual cells treated with MC, and RT-qPCR was used to measure the differential expression of 40 genes involved in anti-oxidation, photosynthesis, phospholipid synthesis, programmed cell death and cell proliferation at five time points. The results showed that every functional gene category exhibited a "V" shaped pattern with a turning point. It was reflected that there were two processes for MC inhibiting the growth of residual cells. One is the oxidative stress process (OSP) caused by ineffective collision with MC, whose effect weakened gradually; another is the programmed cell death process (PCDP) caused by the lysis of damaged residual cells, whose effect enhanced two days after MC treatment. In addition, the scanning electron micrographs verified that some of the residual cells were deformed or even lysed. Combined with the effects of OSP and PCDP in dynamics, the growth of residual cells was inhibited and was followed by gradual bloom disappearance. This study further elucidates the mechanism of MC controlling HABs at the molecular level and enable a more comprehensive understanding of HAB mitigation using MC.

Spatial-temporal variation of Aureococcus anophagefferens blooms in relation to environmental factors in the coastal waters of Qinhuangdao, China.

The brown tides occurring in the coastal scallop cultivation area of Qinhuangdao, China, in recent years are caused by Aureococcus anophagefferens and significantly impact the scallop industry and the marine ecosystem in this region. Long-term investigations of phytoplankton and hydrological variables in the Qinhuangdao sea area were conducted in this study to understand the spatial-temporal variations of A. anophagefferens in relation to environmental factors. Samples were collected during twelve cruises from July 2011 to December 2013 and were analyzed for the temperature, salinity, dissolved oxygen (DO), nutrients and phytoplankton pigments. All diagnostic pigments of A. anophagefferens, such as chlorophyll c3 (Chl c3), Chl c2, 19'-butanoyloxyfucoxanthin (But-fuco), fucoxanthin (Fuco), and diadinoxanthin (Diad), were detected in the surface water by using high-performance liquid chromatography (HPLC). The highest concentrations of But-fuco (5.64 µg L-1), Fuco (37.94 µg L-1) and chlorophyll a (Chl a, 17.25 µg L-1) occurred in different seasons and sampling sites. The A. anophagefferens bloom (as indicated by But-fuco) usually expanded from the south to the north of the Qinhuangdao sea area, close to scallop-culturing regions. The bloom unusually starts in May, reaches its peak in June and almost disappears in August, with the temperature ranging from ca. 19 °C to 23 °C. The redundancy analysis (RDA) indicated that relatively high salinity (>29) and low inorganic nutrients were suitable for the development of the A. anophagefferens bloom. The ratios of diagnostic pigments to Chl a were not constant during different cruises and generally obeyed two different linear relationships, thus indicating the co-occurrence of the blooms of A. anophagefferens and other species, such as Minutocellus polymorphus. In summary, our work reports the long-term variation of A. anophagefferens blooms based on diagnostic pigments and environmental controls, which may provide more insights into the formation mechanisms of the brown tide in this region.