Insights into how characteristics of dissolved algal organic matter affect the efficiency of modified clay in controlling harmful algal blooms.

Dissolved algal organic matter (dAOM) originating from harmful algal blooms (HABs) can deteriorate the quality of municipal water supplies, threaten the health of aquatic environments, and interfere with modified clay (MC)-based HABs control measures. In this study, we explored the composition of dAOM from Prorocentrum donghaiense, a typical HAB organism, and assessed the influence of dAOM on MC flocculation. Our results suggested that dAOM composition was complex and had a wide molecular weight (MW) distribution. MW and electrical properties were important dAOM characteristics affecting flocculation and algal removal efficiency of MC. Negatively charged high-MW components (>50 kDa) critically affected algal removal efficiency, reducing the zeta potential of MC particles and leading to small and weak flocs. However, the effect of dAOM depended on its concentration. When the cell density of P. donghaiense reached HAB levels, the high-MW dAOM strongly decreased the algal removal efficiency of MC.

Uncovering nutrient regeneration, transformation pattern, and its contribution to harmful algal blooms in mariculture waters.

The prevalence of harmful algal blooms (HABs), especially in mariculture waters, has become a concern for environmental and human health worldwide. Notably, the frequent occurrence of HABs relies upon a substantial supply of available nutrients, which are influenced by nutrient recycling. However, nutrient regeneration, transformation pattern, and their contribution to HABs in mariculture waters remain largely unknown. In this study, by combining field investigation and incubation experiments from June to September 2020, the temporal variations in nutrients and algal composition were revealed. In addition, the nutrient regeneration and assimilation rates in the water column during two continuous algal blooms were measured. The results indicated that organic nutrients, which were the dominant components, strongly stimulated nutrient regeneration. High regeneration rates were observed, with dissolved inorganic nitrogen (DIN) and phosphorous (DIP) regeneration rates ranging from 0.25 to 2.64 µmol/L·h and 0.01 to 0.09 µmol/L·h, respectively. Compared to the direct uptake of organic nutrients, the rapid regeneration of inorganic nutrients played a vital role in sustaining continuous algal blooms, as regenerated DIN contributed 100 % while regenerated DIP contributed 72-100 % of the algal assimilation demand. Furthermore, the redundancy analysis and inverse solution equations indicated that different N transformation patterns and utilization strategies occurred during Heterosigma and Nannochloris blooms. The shorter N recycling pathway and faster NH4+ supply rates provided favorable conditions for the dominance of Nannochloris over Heterosigma, which had a preference for the uptake of NO3-. In conclusion, we propose that nutrient regeneration is a key maintenance mechanism underlying the maintenance of continuous algal blooms, and different N transformation patterns and utilization strategies regulate algal communities in mariculture waters.

Novel insights into toxin changes associated with the growth of Alexandrium pacificum: Revealing active toxin-secretion ability and toxin cell quota variation.

Paralytic shellfish toxins (PSTs) are widely distributed globally and are produced by Alexandrium pacificum in marine system. However, the characteristics of toxins producing and secreting associated with growth phases are still unclear, especially whether A. pacificum has the ability to actively secrete PSTs is controversial. In this study, variation characteristics of intracellular and extracellular PSTs contents associated with A. pacificum growth phases were investigated thoroughly. The results showed that intracellular and extracellular PSTs contents increased sharply during the exponential phase. But during the stationary phase, the intracellular PSTs content increased by only 26 %, and the extracellular PSTs content did not increase significantly. Since the increase in extracellular PSTs content mainly occurred at the exponential phase, when most cells were living, we speculated that active PSTs secretion of living cells might be an important production pathway of extracellular toxins besides leakage from dead cells. Furthermore, toxin cell quota variation associated with the growth phase was analysed. In the exponential phase, the toxin cell quota first increased and then decreased, with a maximum of 19.02 ± 1.80 fmol/cell at 6 d. However, after entering the stationary phase, this value slowly increased again, suggesting that vigilance should be raised for the plateau of Alexandrium blooms. In addition, cells in the exponential phase mainly produced O-sulfated components such as GTX1&4, cells in the stationary phase mainly produced O-sulfate-free components such as GTX5. In this study, the toxigenic rules of A. pacificum were comprehensively uncovered, which provided theoretical guidance for the prevention and mitigation of A. pacificum blooms.

Uncovering the regulation effect of modified clay on toxin production in Alexandrium pacificum: From physiological insights.

As a common dinoflagellate, Alexandrium pacificum can produce paralytic shellfish toxins (PSTs). It can be removed from water by Polyaluminium chloride modified clay (PAC-MC), but it is unclear whether PAC-MC can prevent PSTs content and toxicity from increasing and whether PAC-MC can stimulate PSTs biosynthesis by A. pacificum. Effect of PAC-MC on PSTs and the physiological mechanism were analysed here. The results showed total PSTs content and toxicity decreased respectively by 34.10 % and 48.59 % in 0.2 g/L PAC-MC group at 12 days compared with control group. And the restriction of total PSTs by PAC-MC was mainly achieved via inhibition of algal cell proliferation, by affecting A. pacificum physiological processes and changing phycosphere microbial community. Meanwhile, single-cell PSTs toxicity did not increase significantly throughout the experiment. Moreover, A. pacificum treated with PAC-MC tended to synthesize sulfated PSTs such as C1&2. Mechanistic analysis showed that PAC-MC induced upregulation of sulfotransferase sxtN (related to PSTs sulfation), and functional prediction of bacterial community also showed significant enrichment of "sulfur relay system" after PAC-MC treatment, which might also promote PSTs sulfation. The results will provide theoretical guidance for the application of PAC-MC to field control of toxic Alexandrium blooms.

Mechanisms of Phaeocystis globosa blooms in the Beibu Gulf revealed by metatranscriptome analysis.

The haptophyceae Phaeocystis globosa is a species responsible for harmful algal blooms in the global ocean, forming blooms in the Beibu Gulf annually since 2011. This species can alternate between solitary free-living cells and colonies. Colonies are the dominant morphotype during blooms. To date, the underlying mechanism of P. globosa blooms in the Beibu Gulf is poorly understood. After combining results of ecological surveys, laboratory studies, and metatranscriptome and bioinformatics analyses, it was found that low temperatures, high nitrate, and low organic phosphorus induced P. globosa blooms in the Beibu Gulf. Additionally, the unique genetic and physiological characteristics that allow P. globosa to stand out as a dominant species in such an environment include (1) several genes encoding high-affinity nitrate transport proteins that could be highly expressed under sufficient nitrate conditions; (2) energy metabolism genes involved in photosynthesis and oxidative phosphorylation that were actively expressed at low temperatures to carry out carbon and energy reversion and produce sufficient ATP for various life activities, individually; (3) abundant glycan synthesis genes that were highly expressed at low temperatures, thus synthesizing large quantities of proteoglycans to construct the mucilaginous envelope forming the colony; (4) cells in colonies exhibited active gene expression in DNA replication contributing to a faster growth rate, which could help P. globosa occupy niches quickly; and (5) the energy and material expenditure was redistributed in colonial cells accompanied with chitin filaments and flagella degraded, more expenditure was used for the synthesis of the mucilaginous envelope and the rapid proliferation.

Metabarcoding analysis identifies high diversity of harmful algal bloom species in the coastal waters of the Beibu Gulf.

Harmful algal blooms (HABs) have occurred more frequently in recent years. In this study, to investigate their potential impact in the Beibu Gulf, short-read and long-read metabarcoding analyses were combined for annual marine phytoplankton community and HAB species identification. Short-read metabarcoding showed a high level of phytoplankton biodiversity in this area, with Dinophyceae dominating, especially Gymnodiniales. Multiple small phytoplankton, including Prymnesiophyceae and Prasinophyceae, were also identified, which complements the previous lack of identifying small phytoplankton and those unstable after fixation. Of the top 20 phytoplankton genera identified, 15 were HAB-forming genera, which accounted for 47.3%-71.5% of the relative abundance of phytoplankton. Based on long-read metabarcoding, a total of 147 OTUs (PID > 97%) belonging to phytoplankton were identified at the species level, including 118 species. Among them, 37 species belonged to HAB-forming species, and 98 species were reported for the first time in the Beibu Gulf. Contrasting the two metabarcoding approaches at the class level, they both showed a predominance of Dinophyceae, and both included high abundances of Bacillariophyceae, Prasinophyceae, and Prymnesiophyceae, but the relative contents of the classes varied. Notably, the results of the two metabarcoding approaches were quite different below the genus level. The high abundance and diversity of HAB species were probably due to their special life history and multiple nutritional modes. Annual HAB species variation revealed in this study provided a basis for evaluating their potential impact on aquaculture and even nuclear power plant safety in the Beibu Gulf.

The molecular mechanisms and environmental effects of modified clay control algal blooms in aquacultural water.

Modified clay (MC) technology is an effective method for controlling harmful algal blooms (HABs). Based on field experience, a bloom does not continue after treatment with MC, even though the residual HAB biomass accounts for 20-30% of the initial biomass. Laboratory studies using unialgal cultures have found that MC could inhibit the growth of the residual algal cells to prevent HABs. Nevertheless, the phytoplankton in field waters is diverse. Therefore, unclassified complex mechanisms may exist. To illustrate the molecular mechanisms through which MC controls HABs in the field and verify the previous laboratory findings, a series of experiments and bioinformatics analyses were conducted using bloom waters from aquacultural ponds. The results showed that a 72.29% removal efficiency of algal biomass could effectively control blooms. The metatranscriptomic results revealed that the number of downregulated genes (131,546) was greater than that of upregulated genes (24,318) at 3 h after MC addition. Among these genes, several genes related to DNA replication were downregulated; however, genes involved in DNA repair were upregulated. Metabolism-related pathways were the most significantly upregulated (q < 0.05), including photosynthesis and oxidative phosphorylation. The results also showed that MC reduced most of the biomass of the dominant phytoplankton species, likely by removing apical dominance, which increased the diversity and stability of the phytoplankton community. In addition to reducing the pathogenic bacterial density, MC reduced the concentrations of PO43- (96.22%) and SiO32- (66.77%), thus improving the aquaculture water quality, altering the phytoplankton community structure (the proportion of Diatomea decreased, and that of Chlorophyta increased), and inhibiting phytoplankton growth. These effects hindered the rapid development of large phytoplankton biomasses and allowed the community structure to remain stable, reducing HAB threats. This study illustrates the molecular mechanisms through which MC controls HABs in the field and provides a scientific method for removing HABs in aquacultural waters.

The synthesis of an acrylamide copolymer and its synergistic effects on clay flocculation of red tide organisms.

The modified clay (MC) method is a common emergency treatment technology for red tides, and the selection of surface modifiers is the key to the MC technology. A cationic polymeric modifier, the copolymer of dimethyl diallyl ammonium chloride and acrylamide (P (DMDAAC-co-AM), PDA) was optimized via a visible-light-induced polymerization technique. The PDA-modified clay (PDAMC) was prepared with strong salt tolerance and achieved efficiencies of 86% at the concentration of 50 mg L-1, and the dose was 90% lower than that of aluminum polychloride-modified clay (PACMC). While polyacrylamide and commercial PDA can achieve efficiencies of only 25 and 67%, respectively, but high doses were required. This is because PDA changed the surface charges of clay particles from negative to positive, which promotes the formation of the polymer-chains bridging network to overcome the difficulties of curling in seawater. According to the analysis of flocculation parameters and spatial conformation of PDAMC, the high salinity tolerance of the PDAMC was attributed to the synergistic processes of charge neutralization and the three-dimensional network bridging. Therefore, this study has developed a highly effective flocculant material used in seawater and provided an important reference for the management of red tide organisms.

Degradation of paralytic shellfish toxins during flocculation of Alexandrium pacificum by an oxidized modified clay: A laboratory experiment.

Paralytic shellfish toxins (PSTs), produced by Alexandrium pacificum in the marine environment, are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells. During the toxigenic A. pacificum blooms outbreaks, PSTs can be accumulated through the food chain and finally enter the human body, posing a significant threat to human health and safety. This study experimented with a novel type of oxidized modified clay, potassium peroxymonosulfate modified clay (PMPS-MC), which could remove A. pacificum cells as well as reduce intracellular and extracellular PSTs toxicity rapidly. For the extracellular PSTs, its content decreased to below the detection limit rapidly through oxidative degradation within 15 min of 10 mg/L PMPS-MC treatment. Whereafter, although the residual cells in water column and some viable cells in flocculated sediment continued to secrete toxins, the extracellular PSTs content and toxicity in the PMPS-MC treatment groups remained significantly lower than those in the control group. For the intracellular PSTs, PMPS-MC might induce the transformation of more toxic GTX1&4 to less toxic GTX2&3 and C1&2, resulting in intracellular PSTs toxicity reduced within 15 min. In addition, intracellular PSTs content and toxicity in the PMPS-MC treatment groups were consistently lower than the control group within 48 h, possibly by inhibiting the A. pacificum cells growth. These results will provide a scientific basis for the field application of modified clay to control A. pacificum blooms.

Nitrate dynamics and source apportionment on the East China Sea shelf revealed by nitrate stable isotopes and a Bayesian mixing model.

The excess input of nitrate is one of the primary factors triggering nearshore eutrophication. To estimate the source apportionment of nitrate on the East China Sea (ECS) shelf, the nitrogen and oxygen stable isotopes in nitrate (δ15N-NO3- and δ18O-NO3-) collected in winter and late spring 2016 were analyzed alongside essential physical, chemical and biological parameters. The temporal and spatial distributions and characteristic values of nitrate-bearing water masses were presented. Accordingly, the end-member mixing model and Rayleigh model were applied to systematically analyze biogeochemical processes. The biogeochemical processes of nitrate were weak in winter, except in the southern ECS, where assimilation and nitrification probably occurred. In contrast, the biogeochemical processes were intensive in spring. The stable isotopic fractionations of N and O were unified in the whole area, and the ratio between δ18O-NO3- and δ15N-NO3- was 1.81 ± 0.04, which indicated significant assimilation accompanying nitrification in spring. Furthermore, a Bayesian stable isotope mixing model was used to reveal the source contributions of nitrate on the ECS shelf for the first time, demonstrating that the Changjiang Diluted Water and Kuroshio Subsurface Water were always sustained and provided steady nitrate sources for the whole ECS. The nitrate inputs from the Yellow Sea to the northern ECS increased from approximately 30 % in spring to nearly 70 % in winter, while that from the Taiwan Strait Warm Water to the southern ECS decreased from approximately 40 % in spring to zero in winter. Moreover, although the nitrate contributions from nitrification were significantly weak in the middle and northern ECS during winter, they were important over the entire ECS during spring. This study qualitatively and quantitatively improves the understanding of seasonal nitrate control from various sources, and these findings are important for nutrient management and policy making to mitigate nearshore eutrophication.

A New Perspective: Revealing the Algicidal Properties of Bacillus subtilis to Alexandrium pacificum from Bacterial Communities and Toxins.

Algicidal bacteria are important in the control of toxic dinoflagellate blooms, but studies on the environmental behavior of related algal toxins are still lacking. In this study, Bacillus subtilis S3 (S3) showed the highest algicidal activity against Alexandrium pacificum (Group IV) out of six Bacillus strains. When treated with 0.5% (v/v) S3 bacterial culture and sterile supernatant, the algicidal rates were 69.74% and 70.22% at 12 h, respectively, and algicidal substances secreted by S3 were considered the mechanism of algicidal effect. During the algicidal process, the rapid proliferation of Alteromonas sp. in the phycosphere of A. pacificum may have accelerated the algal death. Moreover, the algicidal development of S3 released large amounts of intracellular paralytic shellfish toxins (PSTs) into the water, as the extracellular PSTs increased by 187.88% and 231.47% at 12 h, compared with the treatment of bacterial culture and sterile supernatant at 0 h, respectively. Although the total amount of PSTs increased slightly, the total toxicity of the algal sample decreased as GTX1/4 was transformed by S3 into GTX2/3 and GTX5. These results more comprehensively reveal the complex relationship between algicidal bacteria and microalgae, providing a potential source of biological control for harmful algal blooms and toxins.

Establishment and preliminary study of electrophysiological techniques in a typical red tide species.

Electrophysiology studies the electrical properties of cells and tissues including bioelectrical signals and membrane ion channel activities. As an important means to reveal ion channel related physiological functions and the underlying mechanisms, electrophysiological techniques have been widely used in studies of animals, higher plants and algae that are closely related to higher plants. However, few electrophysiological studies have been carried out in red tide organisms, especially in dinoflagellates, which is mainly due to the complex surface structure of dinoflagellate amphiesma. In this study, the surface amphiesma of Alexandrium pacificum, a typical red tide species, was removed by centrifugation, low-temperature treatment and enzymatic treatment. In all three treatments, low-temperature treatment with 4 °C for 2 h had high ecdysis rate and high fixation rate, and the treated cells were easy to puncture, so low-temperature treatment was used as a preprocessing treatment for subsequent current recording. Acquired protoplasts of A. pacificum were identified by calcofluor fluorescence and immobilized by poly-lysine. A modified "puncture" single-electrode voltage-clamp recording was first applied to dinoflagellates, and voltage-gated currents, which had the characteristics of outward K+ current and inward Cl- current, were recorded and confirmed by ion replacement, indicating the voltage-gated currents were mixed. This method can be used as a technical basis for the electrophysiological study of dinoflagellates and provides a new perspective for the study of stress tolerance, red tide succession, and the regulation of physiological function of dinoflagellates.

Transcriptome profiles of genes related to growth and virulence potential in Vibrio alginolyticus treated with modified clay.

Vibrio alginolyticus is a globally distributed opportunistic pathogen that causes different degrees of disease in various marine organisms, such as fish, shrimp and shellfish. At present, vibriosis caused by V. alginolyticus has a wide epidemic range and causes frequent outbreaks, resulting in substantial losses in aquaculture. According to previous studies, modified clay (MC) could effectively flocculate and reduce the density of Vibrio in water, but it is still unknown whether MC inhibits growth and how it affects virulence in bottom flocs. Here, we studied the response mechanism of V. alginolyticus in flocs treated with MC at the transcriptome level and verified the transcriptomic data combined with relevant physiological experiments and reverse transcription quantitative real-time PCR (RT-qPCR) for the first time. It was found that the morphology of Vibrio in the MC flocs changed, the membrane function was damaged, the antioxidant system was activated, and the material and energy metabolism also changed. In addition, MC could inhibit the expression of virulence factors of V. alginolyticus; for example, flagella, pilus, siderophores, quorum sensing, and other related genes were significantly downregulated. In general, MC effectively inhibited the growth of Vibrio and reduced its virulence potential in flocs, which could provide theoretical support for a new model of healthy aquaculture.

Effects of modified clay on the formation of Phaeocystis globosa colony revealed by physiological and transcriptomic analyses.

The harmful algal bloom (HAB) species Phaeocystis globosa is commonly observed in global temperate and tropical oceans, and colonies of P. globosa exhibit a dominant morphotype during blooms. The use of polyaluminium chloride modified clay (PAC-MC) is an effective mitigation strategy for P. globosa blooms. Although previous studies have found that PAC-MC can stimulate P. globosa colony formation at low concentrations and inhibit it at higher concentrations, the underlying mechanisms of these effects are poorly understood. Here, we comprehensively compared the physiochemical indices and transcriptomic response of residual P. globosa cells after treatment with two concentrations of PAC-MC. The results showed that PAC-MC induced oxidative stress, photosynthetic inhibition, and DNA damage in residual cells. Moreover, it could activate antioxidant responses and enhance the repair of photosynthetic structure and DNA damage in cells. The biosynthesis of polysaccharides was enhanced and genes associated with cell motility were down-regulated after treatment with PAC-MC, resulting in the accumulation of colonial matrixes. After treatment with a low concentration of PAC-MC (0.1 g/L), the residual cells were slightly stressed, including physical damage, oxidative stress and other damage, and polysaccharide synthesis was enhanced to promote colony formation to alleviate environmental stress. Moreover, the damage to residual cells was slight; thus, normal cell function provided abundant energy and matter for colony formation. After treatment with a high concentration of PAC-MC (0.5 g/L), the residual cells suffered severe damage, which disrupted normal physiological processes and inhibited cell proliferation and colony formation. The present study elucidated the concentration-dependent mechanism of PAC-MC affecting the formation of P. globosa colonies and provided a reference for the application of PAC-MC to control P. globosa blooms.

Assessing the Effect of Modified Clay on the Toxicity of Karenia mikimotoi Using Marine Medaka (Oryzias melastigma) as a Model Organism.

Blooms of the toxic dinoflagellate Karenia mikimotoi could threaten the survival of marine life, and modified clay (MC) is considered a promising method for the control of harmful algal blooms. Here, using marine medaka as the model organism, the toxicity of K. mikimotoi before and after MC disposal was investigated. The results showed that only a certain density of intact K. mikimotoi cells could cause obvious damage to fish gills and lead to rapid death. A systematic analysis of morphology, physiology, and molecular biology parameters revealed that the fish gills exhibited structural damage, oxidative damage, osmotic regulation impairment, immune response activation, and signal transduction enhancement. MC can flocculate K. mikimotoi rapidly in water and reduce its toxicity by reducing the density of intact algae cells and hemolytic toxicity. The results indicate that MC is an effective and safe method for controlling K. mikimotoi blooms.

Hypoxia formation in the East China Sea by decomposed organic matter in the Kuroshio Subsurface Water.

Although internal decomposition of organic matter (OM) in the Kuroshio Subsurface Water (KSSW) is a crucial factor for hypoxia formation in the East China Sea (ECS), the seasonal source and contributions of this OM remain debated. This study applied datasets collected in June and October 2015 to discuss these issues qualitatively and quantitatively. According to the variations in several parameters along the KSSW route, N2 fixation signals related to decomposed OM were apparent in the southern ECS during June, while terrestrial input signals were revealed in the northern ECS during June and most of the ECS during October. The terrestrial input contributed 47% of the decomposed OM near the historic hypoxic area in June, indicating that the terrestrial and marine sources contributed almost equally to the development of ECS hypoxia. These results provide vital information for understanding the mechanism of hypoxia formation driven by eutrophication and oceanic circulation.

Healthier Communities of Phytoplankton and Bacteria Achieved via the Application of Modified Clay in Shrimp Aquaculture Ponds.

The composition and stability of microbial communities in aquaculture water are crucial for the healthy growth of shrimp and present considerable risk to aquatic ecosystems. The modified clay (MC) method has been proposed as an efficient and safe solution for the mitigation of harmful algal blooms (HABs). Currently, the effects of MC on microbial communities in aquaculture water remain unknown. Here, we adopted the MC method to regulate shrimp-culture water quality and evaluated the effects of MC on the composition and stability of phytoplankton together with bacteria communities through high-throughput sequencing. On the one hand, a prominent change in the composition of microbial community was observed, with green algae becoming the most abundant genera and pathogens being infrequent in the MC-treated pond, which was more conducive to the growth of shrimp than that in the control pond. Moreover, MC could increase the diversity and stability of the microbial community structure in the water column, which had a higher anti-interference ability, as demonstrated by the analysis of the diversity and molecular ecological network. Taken together, MC could reduce the possibility for the occurrence of HABs and maintain a stable microbial community, which is beneficial for the health and high yield of shrimp.

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.

Programmed cell death induced by modified clay in controlling Prorocentrum donghaiense bloom.

Modified clay (MC), an effective material used for the emergency elimination of algal blooms, can rapidly reduce the biomass of harmful algal blooms (HABs) via flocculation. After that, MC can still control bloom population through indirect effects such as oxidative stress, which was initially proposed to be related to programmed cell death (PCD) at molecular level. To further study the MC induced cell death in residual bloom organisms, especially identifying PCD process, we studied the physiological state of the residual Prorocentrum donghaiense. The experimental results showed that flocculation changed the physiological state of the residual cells, as evidenced by growth inhibition and increased reactive oxygen species production. Moreover, this research provides biochemical and ultrastructural evidence showing that MC induces PCD in P. donghaiense. Nuclear changes were observed, and increased caspase-like activity, externalization of phosphatidylserine and DNA fragmentation were detected in MC-treated groups and quantified. And the mitochondrial apoptosis pathway was activated in both MC-treated groups. Besides, the features of MC-induced PCD in a unicellular organism were summarized and its concentration dependent manner was proved. All our preliminary results elucidate the mechanism through which MC can further control HABs by inducing PCD and suggest a promising application of PCD in bloom control.

Effects of Modified Clay on Phaeocystis globosa Growth and Colony Formation.

Phaeocystis globosa is a globally distributed harmful algal blooms (HABs) species dominated by the colonial morphotype, which presents dramatic environmental hazards and poses a threat to human health. Modified clay (MC) can effectively flocculate HAB organisms and prevent their subsequent growth, but the effects of MC on colony-dominated P. globosa blooms remain uncertain. In this paper, a series of removal and incubation experiments were conducted to investigate the growth, colony formation and colony development of P. globosa cells after treatment with MC. The results show that the density of colonies was higher at MC concentrations below 0.2 g/L compared to those in the control, indicating the role of P. globosa colonies in resistance to environmental stress. Concentrations of MC greater than 0.2 g/L could reduce the density of solitary cells and colonies, and the colony diameter and extracellular polysaccharide (EPS) content were also decreased. The adsorption of MC to dissolved inorganic phosphorus (DIP) and the cell damage caused by collision may be the main mechanisms underlying this phenomenon. These results elucidate that the treatment with an appropriate concentration of MC may provide an effective mitigation strategy for P. globosa blooms by preventing their growth and colony formation.