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Microplastics have become ubiquitous in the global marine environment, posing substantial influences on marine organism health, food web function and marine ecosystem structure. Protozoan grazers are known for their ability to improve the biochemical constituents of poor-quality algae for subsequent use by higher trophic levels. However, the effects of microplastics on the trophic upgrading of protozoan grazers remain unknown. To address this knowledge gap, the ciliate Euplotes vannus and the heterotrophic dinoflagellate Oxyrrhis marina were exposed to microplastic particles (5 μm) for four days with various concentrations (1-20 mg/L). Both O. marina and E. vannus ingested microplastics. At the exposure level of 20 mg/L, the ingestion rate, growth rate, biovolume, and carbon biomass of E. vannus were significantly decreased by 28.18 %, 32.01 %, 30.46 %, and 82.27 %, respectively, while such effects were not observed for O. marina. The contents of highly unsaturated fatty acids in O. marina and E. vannus on a mixed diet of microplastic particles and green algae significantly reduced by 8.66 % and 41.49 % relative to feeding only on green algae, respectively. Besides, we also observed an increase in the composition of C18:3 (ω-3) and C20:3 (ω-3) concurrence with a significant decrease in C16:0 and C18:0 in E. vannus after 96 h exposure at 20 mg/L. These results indicate that microplastics can weaken trophic upgrading of the nutritional quality by protozoan grazers, which may consequently alter the function of food webs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study examined effects of mangrove plants Kandelia obovata and Aegiceras corniculatum on harmful algal species. While A. corniculatum leaf extract had no inhibitory effect, K. obovata leaf extract significantly inhibited the growth of two harmful algal species Alexandrium tamarense and Karenia mikimotoi. The inhibitory effect was concentration-dependent, with over 90 % inhibition at the highest concentration. Morphological changes and cell size reduction were observed in both microalgae. Excessive production of reactive oxygen species and damage to algal photosynthetic system were found. The allelopathic effect of K. obovata on K. mikimotoi with low-concentration repeated exposure was more effective than high-concentration single exposure. The EC 50 of K. obovata (0.33 g L -1 ) was lower than reported values on other coastal plants. Higher inhibitory effects of K. obovata were found on naked algal species than the armoured ones. These findings suggest potential applications of K. obovata leaf extract in controlling harmful algal blooms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Microalgae are of great interest due to their ability to produce valuable compounds, such as pigments, omega-3 fatty acids, antioxidants, and antimicrobials. The dinoflagellate genus Amphidinium is particularly notable for its amphidinol-like compounds, which exhibit antibacterial and antifungal properties. This study utilized a two-stage cultivation method to grow Amphidinium carterae CCAP 1102/8 under varying conditions, such as blue LED light, increased salinity, and the addition of sodium carbonate or hydrogen peroxide. After cultivation, the biomass was extracted and fractionated using solid-phase extraction, yielding six fractions per treatment. These fractions were analyzed using Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS/MS) to identify their chemical components. Key amphidinol compounds (AM-B, AM-C, AM-22, and AM-A) were identified, with AM-B being the most abundant in Fraction 4, followed by AM-C. Fraction 5 also contained a significant amount of AM-C along with an unknown compound. Fraction 4 returned the highest antimicrobial activity against the pathogens Staphylococcus aureus, Enterococcus faecalis, and Candida albicans, with Minimal Biocidal Concentrations (MBCs) ranging from 1 to 512 µg/mL. Results indicate that the modulation of both amphidinol profile and fraction bioactivity can be induced by adjusting the cultivation parameters used to grow two-stage batch cultures of A. carterae., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Dinoflagellates Prorocentrum donghaiense and Karlodinium veneficum are the dominant species of harmful algal blooms in the East China Sea. The role of their allelopathy on the succession of marine phytoplankton populations is a subject of ongoing debate, particularly concerning the formation of blooms. To explore the allelopathy of K. veneficum on P. donghaiense, an investigation was conducted into photosynthetic performance (including PSII functional activities, photosynthetic electron transport chain, energy flux, photosynthetic different genes and photosynthetic performance) and photosynthetic damage-induced oxidative stress (MDA, SOD, and CAT activity). The growth of P. donghaiense was strongly restrained during the initial four days (1-6 folds, C K /C P ), but the cells gradually resumed activity at low filtrate concentrations from the eighth day. On the fourth day of the strongest inhibition, allelochemicals reduced representative photosynthetic performance parameters PI and Φ PSII , disrupted related processes of photosynthesis, and elevated the levels of MDA content in P. donghaiense. Simultaneously, P. donghaiense repairs these impairments by up-regulating the expression of 13 photosynthetic genes, modifying photosynthetic processes, and activating antioxidant enzyme activities from the eighth day onward. Overall, this study provides an in-depth overview of allelopathic photosynthetic damage, the relationship between genes and photosynthesis, and the causes of oxidative damage induced by photosynthesis. ENVIRONMENTAL IMPLICATIONS: As a typical HAB species, Karlodinium veneficum is associated with numerous fish poisoning events, which have negative impacts on aquatic ecosystems and human health. Allelochemicals produced by K. veneficum can provide a competitive advantage by interfering with the survival, reproduction and growth of competing species. This study primarily investigated the effects of K. veneficum allelochemicals on the photosynthesis and photosynthetic genes of Prorocentrum donghaiense. Grasping the mechanism of allelochemicals inhibiting microalgae is helpful to better understand the succession process of algal blooms and provide a new scientific basis for effective prevention and control of harmful algal blooms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Microalgae are unicellular, photosynthetic organisms in aquatic environments and are sensitive to water quality and contaminants. While green algae and diatoms are widely used for toxicity assessments, there is a relatively limited amount of toxicity data available for freshwater dinoflagellates. Here, we evaluated the sub-lethal effects of the metals Cu, Cr, Ni, and Zn and the herbicides atrazine and S-metolachlor on the freshwater dinoflagellate Palatinus apiculatus. Based on the 72-h median effective concentration (EC 50 ), P. apiculatus showed sensitive responses to metals in the order of Cu (0.052 mg L -1 ), Cr (0.085 mg L -1 ), Zn (0.098 mg L -1 ), and Ni (0.13 mg L -1 ). Among the tested herbicides, P. apiculatus was more sensitive to atrazine (0.0048 mg L -1 ) than S-metolachlor (0.062 mg L -1 ). In addition, we observed morphological alterations and significant increases in reactive oxygen species (ROS) production in cells exposed to 0.05 mg L -1 of Cu and 0.005 mg L -1 of atrazine. These indicated that metals and pesticides induced oxidative stress in cellular metabolic processes and consequently caused severe physiological damage to the cells. Our results provide baseline data on the toxic effects of typical environmental contaminants on freshwater dinoflagellate, suggesting that P. apiculatus could be used as a bioindicator in freshwater toxicity assessments. PRACTITIONER POINTS: The sub-lethal effects of metals and pesticides on the freshwater dinoflagellate Palatinus apiculatus were evaluated. Palatinus sensitively responded to metals and pesticides; of test chemicals, atrazine (0.0048 mg L -1 of EC50) was the most sensitive. Metals and pesticides induced oxidative stress and consequently caused severe physiological damage to the Palatinus cells. The freshwater dinoflagellate Palatinus can be used as a bioindicator in freshwater toxicity assessments., (© 2024 Water Environment Federation.)

We address the targeted destruction of Karenia brevis using the algaecide calcium peroxide, in tandem with the flocculation and sinking of the species. The specific aspect of the approach is the incorporation of the algaecide within the floc to rapidly kill K. brevis, thus minimizing escape of cells from the floc and reentry to the water column. CaO₂ gradually produces H₂O₂, which diffuses through cell membranes and induces oxidative stress, leading to cell death via excessive reactive oxygen species (ROS) formation. The effect of varying doses of calcium peroxide on K. brevis cells was measured with pulse amplitude modulated fluorometry and indicated that doses as low as 30 mg/L when integrated into flocs are effective in suppressing photosynthesis. Cell viability assays also indicate that such low levels are sufficient to cause cell death in a 3-6 hour time period. Thus, the proposed technology involving the incorporation of calcium peroxide in a cationic flocculating agent (polyaluminum chloride, PAC) leads to an inexpensive and scalable technology to mitigate harmful algal blooms of K. brevis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Red tides not only destroy marine ecosystems but also pose a great threat to human health. The traditional anti-red tide materials are difficult to degrade effectively in the natural environment and there may be risks of environmental leakage and secondary pollution. Furthermore, they cannot reduce the toxicity of toxins released by algae. It is very important to prepare degradable materials that can effectively control red tide and reduce their toxins in the future. Herein, degradable CDs (De-CDs) with biocompatibility and non-toxicity is successfully prepared using the one-step electrolytic method. De-CDs can effectively inhibit P. globosa (algae associated with red tide) growth. More importantly, the De-CDs not only can attenuate the toxicity of toxins released by P. globosa, but also can be degraded under visible-light irradiation in the seawater and avoids environmental leakage. The successful preparation of De-CDs provides a new idea for degradable materials with anti-red tide algae in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The present study compares the CMFDA/FDA + motility- and the Most Probable Number (MPN) Dilution Culture + Motility methods for testing the viability of ≥10-<50 μm organisms in chlorine treated ballast water. The results of both methods were within the regulatory compliance criterion <10 organisms/mL, but the MPN-method revealed that growth-outs did occur. While the CMFDA/FDA method showed <0.5 organisms/mL, the MPN-method gave approx. 6 organisms/mL. This demonstrated that false negatives, i.e. living but not stained organisms, may occur when using the CMFDA/FDA-method for compliance testing of chemical treated ballast water. Organisms surviving the treatment were primarily the dinoflagellate Scrippsiella sp. and various coccoid chlorophytes present in a brackish- and freshwater test, respectively. It is suggested that their resilience to the chemical treatment is the ability to transform into a temporary cyst (Scrippsiella sp.) or the presence of a chemical resistant cell wall (certain chlorophytes)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Due to the increase in nanoplastics (NPs) abundance in aquatic environments, their effects on phytoplankton have aroused large research attention. In this study, 100 nm sized polystyrene NPs were chosen to investigate their effecting performance and mechanisms on a typical dinoflagellates Alexandrium tamarense . The results indicated the population growth and photosynthetic efficiencies of A. tamarense were significantly inhibited by NPs exposure, as well as the increase in cellular total carotenoids and paralytic shellfish toxins (PSTs). Meanwhile, the cellar ROS levels increased, corresponding to the increased activities or contents of multiple antioxidant components, including SOD, CAT, GPX, GR, GSH and GSSG. The transcriptional results support the physiological-biochemical results and further revealed the down-regulation of genes encoding the light reaction centers (PSI and PSII) and up-regulation of genes encoding the antioxidant components. Up-regulation of genes encoding key enzymes of the Calvin cycle and glycolytic pathway together with the TCA cycle could accelerate organic carbon and ATP production for A. tamarense cells resistant to NPs stress. Finally, more Glu and acetyl-CoA produced by the enhanced GSH cycle and the glycolytic pathway, respectively, accompanied by the up-regulation of Glu and Arg biosynthesis genes supported the increase in the PST contents under NPs exposure. This study established a data set involving physiological-biochemical changes and gene information about marine dinoflagellates responding to NPs, providing a data basis for further evaluating the ecological risk of NPs in marine environments.

According to the results of the experimental study, the main regularities of changes in morphological, structural-functional and fluorescent indices of P. cordatum were established when zinc oxide nanoparticles ZnO NPs (0.3-6.4 mg L -1 ) and Zn in form of salt (0.09-0.4 mg L -1 ) were added to the medium. The studied pollutants have cytotoxic (growth inhibition, development of oxidative stress, destruction of cytoplasmic organelles, disorganization of mitochondria) and genotoxic (changes in the morphology of nuclei, chromatin condensation) effects on microalgae, affecting almost all aspects of cell functioning. Despite the similar mechanism of action of zinc sulfate and ZnO NPs on P. cordatum cells, the negative effect of ZnO NPs is also due to the inhibition of photosynthetic activity of cells (significant decrease in the maximum quantum yield of photosynthesis and electron transport rate), reduction of chlorophyll concentration from 3.5 to 1.8 pg cell -1 , as well as mechanical effect on cells: deformation and damage of cell membranes, aggregation of NPs on the cell surface. Apoptosis-like signs of cell death upon exposure to zinc sulfate and ZnO NPs were identified by flow cytometry and laser scanning confocal microscopy methods: changes in cell morphology, cytoplasm retraction, development of oxidative stress, deformation of nuclei, and disorganization of mitochondria. It was shown that the first signs of cell apoptosis appear at 0.02 mg L -1 Zn and 0.6 mg L -1 ZnO NPs after 72 h of exposure. At higher concentrations of pollutants, a dose-dependent decrease in algal enzymatic activity (up to 5 times relative to control) and mitochondrial membrane potential (up to 4 times relative to control), and an increase in the production of reactive oxygen species (up to 4-5 times relative to control) were observed. The results of the presented study contribute to the disclosure of fundamental mechanisms of toxic effects of pollutants and prediction of ways of phototrophic microorganisms reaction to this impact., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The presence in marine shellfish of toxins and pollutants like rare earth elements (REEs) poses a major threat to human well-being, coastal ecosystems, and marine life. Among the REEs, neodymium (Nd) stands out as a widely utilized element and is projected to be among the top five critical elements by 2025. Gymnodinum catenatum is a phytoplankton species commonly associated with the contamination of bivalves with paralytic shellfish toxins. This study evaluated the biological effects of Nd on the mussel species Mytilus galloprovincialis when exposed to G. catenatum cells for fourteen days, followed by a recovery period in uncontaminated seawater for another fourteen days. After co-exposure, mussels showed similar toxin accumulation in the Nd and G. catenatum treatment in comparison with the G. catenatum treatment alone. Increased metabolism and enzymatic defenses were observed in organisms exposed to G. catenatum cells, while Nd inhibited enzyme activity and caused cellular damage. Overall, this study revealed that the combined presence of G. catenatum cells and Nd, produced positive synergistic effects on M. galloprovincialis biochemical responses compared to G. catenatum alone, indicating that organisms' performance may be significantly modulated by the presence of multiple co-occurring stressors, such those related to chemical pollution and harmful algal blooms. ENVIRONMENTAL IMPLICATIONS: Neodymium (Nd) is widely used in green technologies like wind turbines, and this element's potential threats to aquatic environments are almost unknown, especially when co-occurring with other environmental factors such as blooms of toxic algae. This study revealed the cellular impacts induced by Nd in the bioindicator species Mytilus galloprovincialis but further demonstrated that the combination of both stressors can generate a positive defense response in mussels. The present findings also demonstrated that the impacts caused by Nd lasted even after a recovery period while a previous exposure to the toxins generated a faster biochemical improvement by the mussels., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The bloom-forming dinoflagellate Alexandrium tamarense is one of the most important producers of paralytic shellfish poisoning toxins. Annually recurrent blooms of this dinoflagellate species is associated with the incremental nitrogen influx, especially excessive nitrate input. However, limited studies have been conducted on the toxin production and underlying molecular regulation mechanisms of A. tamarense under various nitrate (N) conditions. Therefore, toxin production and transcriptomic responses of this species were investigated. The toxin profile of A. tamarense was consistently dominated by the C2-toxins, and the cellular toxicity increased with N concentrations peaking at 9.23 ± 0.03 fmol/cell in the 883 μM N-added group. Under lower N conditions, expressions of two STX-core genes, sxtA and sxtG, were significantly down-regulated, suggesting that N regulated sxt expression and triggered responses related to toxin biosynthesis. Results of this study provided valuable insights into the ecophysiology of A. tamarense, enhancing our understanding of the occurrence of toxification events in natural environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Endosymbionts (Symbiodiniaceae) play a vital role in the health of corals. Seawater pollution can harm these endosymbionts and dispersants used during oil spill cleanup can be extremely toxic to these organisms. Here, we examined the impact of oil and a specific dispersant, Corexit-9500, on two representative endosymbionts - Symbiodinium and Cladocopium - from the Southwestern endemic coral Mussismilia braziliensis. The survival and photosynthetic potential of the endosymbionts decreased dramatically after exposure to the dispersant and oil by ~25 % after 2 h and ~50 % after 7 days. Low concentrations of dispersant (0.005 ml/l) and dispersed oil (Polycyclic Aromatic Hydrocarbons, 1132 μg/l; Total Petroleum Hydrocarbons, 595 μg/l) proved highly toxic to both Symbiodinium and Cladocopium. These levels triggered a reduction in growth rate, cell size, and cell wall thickness. After a few hours of exposure, cellular organelles were damaged or destroyed. These acute toxic effects underline the fragile nature of coral endosymbionts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Microplastics are well known as contaminants in marine environments. With the development of biofilms, most microplastics will eventually sink and deposit in benthic environment. However, little research has been done on benthic toxic dinoflagellates, and the effects of microplastics on benthic dinoflagellates are unknown. Prorocentrum lima is a cosmopolitan toxic benthic dinoflagellate, which can produce a range of polyether metabolites, such as diarrhetic shellfish poisoning (DSP) toxins. In order to explore the impact of microplastics on marine benthic dinoflagellates, in this paper, we studied the effects of polystyrene (PS) on the growth and toxin production of P. lima. The molecular response of P. lima to microplastic stress was analyzed by transcriptomics. We selected 100 nm, 10 μm and 100 μm PS, and set three concentrations of 1 mg L -1 , 10 mg L -1 and 100 mg L -1 . The results showed that PS exposure had limited effects on cell growth, but increased the OA and extracellular polysaccharide content at high concentrations. After exposure to PS MPs, genes associated with DSP toxins synthesis, carbohydrate synthesis and energy metabolism, such as glycolysis, TCA cycle and pyruvate metabolism, were significantly up-regulated. We speculated that after exposure to microplastics, P. lima may increase the synthesis of DSP toxins and extracellular polysaccharides, improve the level of energy metabolism and gene expression of ABC transporter, thereby protecting algal cells from damage. Our findings provide new insights into the effects of microplastics on toxic benthic dinoflagellates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The dinoflagellate Karenia brevis is a causative agent of red tides in the Gulf of Mexico and generates a potent family of structurally related brevetoxins that act via the voltage-sensitive Na + channel. This project was undertaken to better understand the neurotoxicology and kdr cross-resistance to brevetoxins in house flies by comparing the susceptible aabys strain to ALkdr (kdr) and JPskdr (super-kdr). When injected directly into the hemocoel, larvae exhibited rigid, non-convulsive paralysis consistent with prolongation of sodium channel currents, the known mechanism of action of brevetoxins. In neurophysiological studies, the firing frequency of susceptible larval house fly central nervous system preparations showed a > 200% increase 10 min after treatment with 1 nM brevetoxin-3. This neuroexcitation is consistent with the spastic paralytic response seen after hemocoel injections. Target site mutations in the voltage-sensitive sodium channel of house flies, known to confer knockdown resistance (kdr and super-kdr) against pyrethroids, attenuated the effect of brevetoxin-3 in baseline firing frequency and toxicity assays. The rank order of sensitivity to brevetoxin-3 in both assays was aabys > ALkdr > JPskdr. At the LD 50 level, resistance ratios for the knockdown resistance strains were 6.9 for the double mutant (super-kdr) and 2.3 for the single mutant (kdr). The data suggest that knockdown resistance mutations may be one mechanism by which flies survive brevetoxin-3 exposure during red tide events., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Modified clay compounds are used globally as a method of controlling harmful algal blooms, and their use is currently under consideration to control Karenia brevis blooms in Florida, USA. In 1400 L mesocosm tanks, chemical dynamics and lethal and sublethal impacts of MC II, a polyaluminum chloride (PAC)-modified kaolinite clay, were evaluated over 72 h on a benthic community representative of Sarasota Bay, which included blue crab (Callinectes sapidus), sea urchin (Lytechinus variegatus), and hard clam (Mercenaria campechiensis). In this experiment, MC II was dosed at 0.2 g L - 1 to treat bloom-level densities of K. brevis at 1 × 10 6 cells L - 1 . Cell removal in MC II-treated tanks was 57% after 8 h and 95% after 48 h. In the water column, brevetoxin analogs BTx-1 and BTx-2 were found to be significantly higher in untreated tanks at 24 and 48 h, while in MC II-treated tanks, BTx-3 was found to be higher at 48 h and BTx-B5 was found to be higher at 24 and 48 h. In MC II floc, we found no significant differences in BTx-1 or BTx-2 between treatments for any time point, while BTx-3 was found to be significantly higher in the MC II-treated tanks at 48 and 72 h, and BTx-B5 was higher in MC II-treated tanks at 24 and 72 h. Among various chemical dynamics observed, it was notable that dissolved phosphorus was consistently significantly lower in MC II tanks after 2 h, and that turbidity in MC II tanks returned to control levels 48 h after treatment. Dissolved inorganic carbon and total seawater alkalinity were significantly reduced in MC II tanks, and partial pressure of CO 2 (pCO 2 ) was significantly higher in the MC II-only treatment after 2 h. In MC II floc, particulate phosphorus was found to be significantly higher in MC II tanks after 24 h. In animals, lethal and sublethal responses to MC II-treated K. brevis did not differ from untreated K. brevis for either of our three species at any time point, suggesting MC II treatment at this dosage has negligible impacts to these species within 72 h of exposure. These results appear promising in terms of the environmental safety of MC II as a potential bloom control option, and we recommend scaling up MC II experiments to field trials in order to gain deeper understanding of MC II performance and dynamics in natural waters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Dinoflagellates are among the most diverse group of microalgae. Many dinoflagellate species have been isolated and cultured, and these are used for scientific, industrial, pharmaceutical, and agricultural applications. Maintaining cultures is time-consuming, expensive, and there is a risk of contamination or genetic drift. Cryopreservation offers an efficient means for their long-term preservation. Cryopreservation of larger dinoflagellate species is challenging and to date there has been only limited success. In this study, we explored the effect of cryoprotectant agents (CPAs) and freezing methods on three species: Vulcanodinium rugosum, Alexandrium pacificum and Breviolum sp. A total of 12 CPAs were assessed at concentrations between 5 and 15%, as well as in combination with dimethyl sulfoxide (DMSO) and other non-penetrating CPAs. Two freezing techniques were employed: rapid freezing and controlled-rate freezing. Breviolum sp. was successfully cryopreserved using 15% DMSO. Despite exploring different CPAs and optimizing the freezing techniques, we were unable to successfully cryopreserve V. rugosum and A. pacificum. For Breviolum sp. there was higher cell viability (45.4 ± 2.2%) when using the controlled-rate freezing compared to the rapid freezing technique (10.0 ± 2.8%). This optimized cryopreservation protocol will be of benefit for the cryopreservation of other species from the family Symbiodiniaceae., (© 2022. The Author(s).)

Dinoflagellates are main primary producers in the oceans, the cause of algal blooms and endosymbionts of marine invertebrates. Much remains to be understood about their biology, including their peculiar crystalline chromosomes. We assembled 94 chromosome-scale scaffolds of the genome of the coral endosymbiont Symbiodinium microadriaticum and analyzed their organization. Genes are enriched towards the ends of chromosomes and are arranged in alternating unidirectional blocks. Some chromosomes are enriched for genes involved in specific biological processes. The chromosomes fold as linear rods and each is composed of a series of structural domains separated by boundaries. Domain boundaries are positioned at sites where transcription of two gene blocks converges and disappear when cells are treated with chemicals that block transcription, indicating correlations between gene orientation, transcription and chromosome folding. The description of the genetic and spatial organization of the S. microadriaticum genome provides a foundation for deeper exploration of the extraordinary biology of dinoflagellates and their chromosomes.

Microplastics (MP) widely distributed in aquatic environments have adverse effects on aquatic organisms. Currently, the impact of MP on toxigenic red tide microalgae is poorly understood. In this study, the strain of Alexandrium pacificum ATHK, typically producing paralytic shellfish toxins (PST), was selected as the target. Effects of 1 and 0.1 μm polystyrene MP with three concentration gradients (5 mg L -1 , 25 mg L -1 and 100 mg L -1 ) on the growth, chlorophyll a (Chl a ), photosynthetic activity (F v /F m ) and PST production of ATHK were explored. Results showed that the high concentration (100 mg L -1 ) of 1 μm and 0.1 μm MP significantly inhibited the growth of ATHK, and the inhibition depended on the size and concentration of MP. Contents of Chl a showed an increase with various degrees after MP exposure in all cases. The photosynthesis indicator F v /F m of ATHK was significantly inhibited in the first 11 days, then gradually returned to the level of control group at day 13, and finally was gradually inhibited in the 1 μm MP treatments, and promotion or inhibition to some degree also occurred at different periods after exposure to 0.1 μm MP. Overall, both particle sizes of MP at 5 and 25 mg L -1 had no significant effect on cell toxin quota, and the high concentration 100 mg L -1 significantly promoted the PST biosynthesis on the day 7, 11 and 15. No significant difference occurred in the cell toxin quota and the total toxin content in all treatments at the end of the experiment (day 21). All MP treatments did not change the toxin profiles of ATHK, nor did the relative molar percentage of main PST components. The growth of ATHK, Chl a content, F v /F m and toxin production were not affected by MP shading. This is the first report on the effects of MP on the PST-producing microalgae, which will improve the understanding of the adverse impact of MP on the growth and toxin production of A. pacificum .

The marine bacterium, Bacillus sp. SY-1, produced algicidal compounds that are notably active against the bloom-forming alga Cochlodinium polykrikoides. We isolated three algicidal compounds and identified these as mycosubtilins with molecular weights of 1056, 1070, and 1084 (designated MS 1056, 1070, and 1084, respectively), based on amino acid analyses and 1 H, 13 C, and two-dimensional nuclear magnetic resonance spectroscopy, including 1 H- 15 N heteronuclear multiple bond correlation analysis. MS 1056 contains a β-amino acid residue with an alkyl side chain of C 15 , which has not previously been seen in known mycosubtilin families. MS 1056, 1070, and 1084 showed algicidal activities against C. polykrikoides with 6-h LC 50 values of 2.3 ± 0.4, 0.8 ± 0.2, and 0.6 ± 0.1 μg/ml, respectively. These compounds also showed significant algicidal activities against other harmful algal bloom species. In contrast, MS 1084 showed no significant growth inhibitory effects against other organisms, including bacteria and microalgae, although does inhibit the growth of some fungi and yeasts. These observations imply that the algicidal bacterium Bacillus sp. SY-1 and its algicidal compounds could play an important role in regulating the onset and development of harmful algal blooms in natural environments.

Calmodulin (CaM) is one of the major Ca 2+ -binding proteins in the cells, and it plays multiple roles in several Ca 2+ signaling pathways and regulating the activities of other proteins. In the present study, we characterized CaM genes from the marine dinoflagellates Amphidinium carterae, Cochlodinium polykrikoides, Prorocentrum micans, and P. minimum, and examined their expression patterns upon the addition and chelation of calcium. Their cDNAs had same ORF length (450 bp) and encoded the same protein, but with few nucleotide differences in the ORF and different 3'- and 5' untranslated regions (UTRs). The four CaM proteins consist of four EF-hand Ca 2+ -binding motifs, two N-terminal domains and two C-terminal domains, and they were highly conserved within eukaryotes. The CaM gene expressions in the tested species increased by calcium treatments; however, they were significantly down-regulated by the calcium-chelator EGTA. The CaM genes of the test species were inducible and regulated by different calcium doses, suggesting their major role in calcium regulation in dinoflagellates., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Harmful algal blooms (HABs) affect both freshwater and marine systems. Laboratory experiments suggest an exudate produced by the bacterium Shewanella sp. IRI-160 could be used to prevent or mitigate dinoflagellate blooms; however, effects on non-target organisms are unknown. The algicide (IRI-160AA) was tested on various ontogenetic stages of the copepod Acartia tonsa (nauplii and adult copepodites), the blue crab Callinectes sapidus (zoea larvae and megalopa postlarvae), and the eastern oyster Crassostrea virginica (pediveliger larvae and adults). Mortality experiments with A. tonsa revealed that the 24-h LC50 was 13.4% v/v algicide for adult females and 5.96% for early-stage nauplii. For C. sapidus, the 24-h LC50 for first-stage zoeae was 16.8%; results were not significant for megalopae or oysters. Respiration rates for copepod nauplii increased in the 11% concentration, and in the 11% and 17% concentrations for crab zoeae; rates of later stages and oysters were unaffected. Activity level was affected for crab zoeae in the 1%, 11%, and 17% treatments, and for oyster pediveliger larvae at the 17% level. Activity of later stages and of adult copepods was unaffected. Smaller, non-target biota with higher surface to volume could be negatively impacted from IRI-160AA dosing, but overall the taxa and stages assayed were tolerant to the algicide at concentrations required for dinoflagellate mortality (EC50 =  ~ 1%).

Academic research on dinoflagellate, the primary causative agent of harmful algal blooms (HABs), is often hindered by the coexistence with bacteria in laboratory cultures. The development of axenic dinoflagellate cultures is challenging and no universally accepted method suit for different algal species. In this study, we demonstrated a promising approach combined density gradient centrifugation, antibiotic treatment, and serial dilution to generate axenic cultures of Karenia mikimotoi (KMHK). Density gradient centrifugation and antibiotic treatments reduced the bacterial population from 5.79 ± 0.22 log 10 CFU/mL to 1.13 ± 0.07 log 10 CFU/mL. The treated KMHK cells were rendered axenic through serial dilution, and algal cells in different dilutions with the absence of unculturable bacteria were isolated. Axenicity was verified through bacterial (16S) and fungal internal transcribed spacer (ITS) sequencing and DAPI epifluorescence microscopy. Axenic KMHK culture regrew from 1000 to 9408 cells/mL in 7 days, comparable with a normal culture. The established methodology was validated with other dinoflagellate, Alexandrium tamarense (AT6) and successfully obtained the axenic culture. The axenic status of both cultures was maintained more than 30 generations without antibiotics. This efficient, straightforward and inexpensive approach suits for both armored and unarmored dinoflagellate species.

Ciguatera poisoning is a foodborne disease caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the genera Gambierdiscus and Fukuyoa . Ciguatera outbreaks are expected to increase worldwide with global change, in particular as a function of its main drivers, including changes in sea surface temperature, acidification, and coastal eutrophication. In French Polynesia, G. polynesiensis is regarded as the dominant source of CTXs entering the food web. The effects of pH (8.4, 8.2, and 7.9), Nitrogen:Phosphorus ratios (24N:1P vs. 48N:1P), and nitrogen source (nitrates vs. urea) on growth rate, biomass, CTX levels, and profiles were examined in four clones of G. polynesiensis at different culture age (D10, D21, and D30). Results highlight a decrease in growth rate and cellular biomass at low pH when urea is used as a N source. No significant effect of pH, N:P ratio, and N source on the overall CTX content was observed. Up to ten distinct analogs of Pacific ciguatoxins (P-CTXs) could be detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in clone NHA4 grown in urea, at D21. Amounts of more oxidized P-CTX analogs also increased under the lowest pH condition. These data provide interesting leads for the custom production of CTX standards.

The effects of allelopathy and the potential harm of several isolated allelochemicals have been studied in detail. Microorganisms in the phycosphere play an important role in algal growth, decay and nutrient cycling. However, it is unknown and often neglected whether allelochemicals affect the phycosphere. The present study selected a phenolic acid protocatechuic acid (PA) - previously shown to be an allelochemical. We studied PA at a half maximal effective concentration of 0.20 mM (30 mg L -1 ) against Scrippsiella trochoidea to assess the effect of PA on its phycosphere in an acute time period (48 h). The results showed that: 1) OTUs (operational taxonomic units) in the treatment groups (31.4 ± 0.55) exceeded those of the control groups (28.2 ± 1.30) and the Shannon and Simpson indices were lower than the control groups (3.31 ± 0.08 and 0.84 ± 0.02, 3.45 ± 0.09 and 0.88 ± 0.01); 2) Gammaproteobacteria predominated in the treatment groups (44.71 ± 2.13 %) while Alphaproteobacteria dominated in the controls (67.17 ± 3.87 %); 3) Gammaproteobacteria and Alphaproteobacteria were important biomarkers in the treatment and control groups respectively (LDA > 4.0). PA improved the relative abundance of Alteromonas significantly and decreased the one of Rhodobacteraceae. PICRUSt analysis showed that the decrease of Rhodobacterceae was closely related with the decline of most functional genes in metabolism such as amino acid, carbohydrate, xenobiotics, cofactors and vitamins metabolism after PA-treated., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mussel-inspired catechol-based strategy has been widely used in the development of underwater adhesives. Nonetheless, the properties of the adhesives were still severely limited under harsh environments. A facile approach was proposed herein to prepare a double network hydrogel adhesive with low swelling rate and high strength in seawater, where the first network was polyacrylamide (PAM) and the second network was alginate (Alg). Meanwhile, polydopamine (PDA) nanoparticles, which were formed through self-polymerization as adhesion anchoring sites, distributed evenly throughout the double network hydrogel and effectively enhanced the adhesion capability of the hydrogel. The properties of the resulting hydrogel have been fully characterized. The optimal adhesion strength of the hydrogel adhesive in seawater was as high as 146.84 ± 7.78 kPa. Furthermore, the hydrogel also has excellent ability to promote the growth of zooxanthellae. Our studies provide useful insights into the rational design of underwater adhesives with high performances even beyond nature.

Algae-bacteria interaction is one of the main factors underlying the formation of harmful algal blooms (HABs). The aim of this study was to develop a genome-wide high-throughput screening method to identify HAB-influenced specific interactive bacterial metabolites using a comprehensive collection of gene-disrupted E. coli K-12 mutants (Keio collection). The screening revealed that a total of 80 gene knockout mutants in E. coli K-12 resulted in an approximately 1.5-fold increase in algal growth relative to that in wild-type E. coli. Five bacterial genes (lpxL, lpxM, kdsC, kdsD, gmhB) involved in the lipopolysaccharide (LPS) (or lipooligosaccharide, LOS) biosynthesis were identified from the screen. Relatively lower levels of LPS were detected in these bacteria compared to that in the wild-type. Moreover, the concentration-dependent decrease in microalgal growth after synthetic LPS supplementation indicated that LPS inhibits algal growth. LPS supplementation increased the 2,7-dichlorodihydrofluorescein diacetate fluorescence, as well as the levels of lipid peroxidation-mediated malondialdehyde formation, in a concentration-dependent manner, indicating that oxidative stress can result from LPS supplementation. Furthermore, supplementation with LPS also remarkably reduced the growth of diverse bloom-forming dinoflagellates and green algae. Our findings indicate that the Keio collection-based high-throughput in vitro screening is an effective approach for the identification of interactive bacterial metabolites and related genes.

Even though HPLC-MS is commonly used to quantify the toxin content of Ostreopsis spp. cells, there is a need to develop easy-to-use toxicological tests to set thresholds during Ostreopsis spp. blooms. The crustacean Artemia has been widely used to evaluate the presence and toxicity of chemicals and biological contaminants and we anticipated that it could also be useful to test Ostreopsis spp. toxicity. Its relevance was first assessed by investigating the variability of the toxic effects among Ostreopsis spp. strains and throughout the dinoflagellate life cycle in combination with chemical analyses of the toxinic content by UHPLC-HRMS. After testing the toxicity of fractions prepared from Ostreopsis spp. cells, the known ova- and paly-toxins were not the only toxic metabolites to Artemia franciscana, indicating that other toxic compounds synthesized by Ostreopsis spp. still remain to be identified. To extend the bioassay to in situ monitoring, the toxicity of the benthic microalgal consortium was tested during a natural bloom of Ostreopsis cf. ovata in the NW Mediterranean Sea. The results highlight the accuracy and sensitivity of the ecotoxicological assay with Artemia franciscana to assess the toxicity of Ostreopsis spp. blooms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recent oil spill responses such as the Deepwater Horizon event have underscored the need for crude oil ecotoxicological threshold data for shallow water corals to assist in natural resource damage assessments. We determined the toxicity of a mechanically agitated oil-seawater mixture (high-energy water-accommodated fraction, HEWAF) of a sweet crude oil on a branched stony coral, Pocillopora damicornis. We report the results of two experiments: a 96 h static renewal exposure experiment and a "pulse-chase" experiment of three short-term exposure durations followed by a recovery period in artificial seawater. Five endpoints were used to determine ecotoxicological values: 1) algal symbiont chlorophyll fluorescence, 2) a tissue regeneration assay and a visual health metric with three endpoints: 3) tissue integrity, 4) tissue color, and 5) polyp behavior. The sum of 50 entrained polycyclic aromatic hydrocarbons (tPAH50) was used as a proxy for oil exposure. For the 96 h exposure dose response experiment, dark-adapted maximum quantum yield (Fv/Fm) of the dinoflagellate symbionts was least affected by crude oil (EC 50 = 913 μg/L tPAH50); light-adapted effective quantum yield (EQY) was more sensitive (EC 50  =  428 μg/L tPAH50). In the health assessment, polyp behavior (EC 50 = 27 μg/L tPAH50) was more sensitive than tissue integrity (EC 50 = 806 μg/L tPAH50) or tissue color (EC 50 = 926 μg/L tPAH50). Tissue regeneration proved to be a particularly sensitive measurement for toxicity effects (EC 50  = 10 μg/L tPAH50). Short duration (6-24 h) exposures using 503 μg/L tPAH50 (average concentration) resulted in negative impacts to P. damicornis and its symbionts. Recovery of chlorophyll a fluorescence levels for 6-24 h oil exposures was observed in a few hours (Fv/Fm) to several days (EQY) following recovery in fresh seawater. The coral health assessments for tissue integrity and tissue color were not affected following short-term oil exposure durations, but the 96 h treatment duration resulted in significant decreases for both. A reduction in polyp behavior (extension) was observed for all treatment durations, with recovery observed for the short-term (6-24 h) exposures within 1-2 days following placement in fresh seawater. Wounded and intact fragments exposed to oil treatments were particularly sensitive, with significant delays observed in tissue regeneration. Estimating ecotoxicological values for P. damicornis exposed to crude oil HEWAFs provides a basis for natural resource damage assessments for oil spills in reef ecosystems. These data, when combined with ecotoxicological values for other coral reef species, will contribute to the development of species sensitivity models., Competing Interests: Declarations of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This paper studied the allelopathic effect of Cylindrotheca closterium on the growth of Prorocentrum donghaiense, the model of harmful algal blooms in aquatic environment, by the co-culture tests and bioassay-guided fraction methods. The growth of P. donghaiense in co-cultures was observably suppressed by C. closterium, and P. donghaiense biomass in C/P = 3: 1 group increased slowly with a low growth rate of 0.18 d -1 after 4 days. Petroleum ether (PE) extract derived from C. closterium filtrates was isolated by C18 column and the allelopathy of all isolated fractions for P. donghaiense was investigated. After 96 h cultivation, the inhibition ratio of PE-Ⅲ and PE-Ⅷ fractions on P. donghaiense could reach up to 70.2% and 64.3% at the concentration of 10-fold when compared to control, while the other fractions displayed relatively low inhibitory effects on P. donghaiense. PE-Ⅲ and PE-Ⅷ fractions also decreased the chlorophyll content and maximum quantum yield of photosystem II (Fv/Fm) of P. donghaiense cells. The activities of superoxide dismutase (SOD), one of antioxidant enzymes, reduced around 8.3% and 13.7% following exposure to 2-fold PE-Ⅲ and PE-Ⅷ, and was significantly decreased following higher exposure concentrations. After 96 h of 10-fold PE-Ⅲ and PE-Ⅷ treatments, Catalase (CAT) activity reduced to 44.86% and 46.42% of that observed in the control group. At the same time, a significant increase in malondialdehyde (MDA) contents was observed. These findings suggested that PE-Ⅲ and PE-Ⅷ fractions contained main allelochemicals and possibly acted as promising algistatic agents for emergency handling of P. donghaiense blooms., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Labile dissolved organic carbon (DOC) is a major pollutant in coastal marine environments affected by anthropogenic impacts, and may significantly contribute to coral bleaching and subsequent mortality on coastal reefs. DOC can cause bleaching indirectly through the rapid proliferation of copiotrophic and pathogenic bacteria. Here we demonstrate that labile DOC compounds can also impair the coral-dinoflagellate symbiosis by directly affecting coral physiology on both the host and algal symbiont level. In a controlled aquarium experiment, we monitored over several weeks key physiological parameters of the tropical coral Stylophora pistillata exposed to ambient and elevated labile DOC levels (0.1 and 1.0 mM) in combination with low and high nitrogen (i.e. ammonium) conditions (0.2 and 4.0 μM). At the symbiont level, DOC exposure under low ammonium availability decreased the photosynthetic efficiency accompanied by ∼75 % Chl a and ∼50 % symbiont cell reduction. The photosynthetic functioning of the symbionts recovered once the DOC enrichment ceased indicating a reversible shift between autotrophic and heterotrophic metabolism. At the host level, the assimilation of exogenous DOC sustained the tissue carbon reserves, but induced a depletion of the nitrogen reserves, indicated by ∼35 % decreased protein levels. This suggests an imbalanced exogenous carbon to nitrogen supply with nitrogen potentially limiting host metabolism on the long-term. We also demonstrate that increased ammonium availability delayed DOC-induced bleaching likely by keeping symbionts in a photosynthetically competent state, which is crucial for symbiosis maintenance and coral survival. Overall, the present study provides further insights into how coastal pollution can de-stabilize the coral-algal symbiosis and cause coral bleaching. Therefore, reducing coastal pollution and sustaining ecological integrity are critical to strengthen the resilience of coral reefs facing climate change., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Symbiosis with zooxanthellae is essential for survival of corals. Using a bioassay, we report the H-type lectin SLL-2 purified from the octocoral Sinularia lochmodes to restrict zooxanthellae form to spherical cells. However, the factor for initiating or maintaining a symbiotic relationship between a host and zooxanthellae has not been found in many corals. This bioassay is useful for evaluating the role of a lectin as a symbiosis-related factor.

Alachlor is one of the most widely used herbicides and can remain in agricultural soils and wastewater. The toxicity of alachlor to marine life has been rarely studied; therefore, we evaluated the physiological and transcriptional responses in the marine dinoflagellate Prorocentrum minimum . The herbicide led to considerable decreases in P. minimum cell numbers and pigment contents. The EC 50 was determined to be 0.373 mg/L. Photosynthesis efficiency and chlorophyll autofluorescence dramatically decreased with increasing alachlor dose and exposure time. Real-time PCR analysis showed that the photosynthesis-related genes PmpsbA, PmatpB , and PmrbcL were induced the most by alachlor; the transcriptional level of each gene varied with time. PmrbcL expression increased after 30 min of alachlor treatment, whereas PmatpB and PmpsbA increased after 24 h. The PmpsbA expression level was highest (5.0 times compared to control) after 6 h of alachlor treatment. There was no significant change in PmpsaA expression with varying treatment time or concentration. Additionally, there was no notable change in the expression of antioxidant genes PmGST and PmKatG , or in ROS accumulation. These suggest that alachlor may affect microalgal photosystem function, with little oxidative stress, causing severe physiological damage to the cells, and even cell death.

Antibiotics have been widely detected in the ocean and have various impacts on the environment, while knowledge of their chronic influence on phytoplankton, especially red tide algae, is still limited. Dinoflagellates and green algae are common phytoplankton in marine ecosystems. The former is the main red tide algae, and the latter is an important primary producer. We investigated the long-term responses of two representative algae, Prorocentrum lima and Chlorella sp., to two common antibiotics (sulfamethoxazole (SMX) and norfloxacin (NFX)) at environmentally relevant levels (10 and 100 ng/L) during simulated natural conditions. The cell density and activities of three antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)) were analyzed. The results showed that the influence of each antibiotic on Chlorella sp. was not significant (p > 0.05) during the first 10 days, but the influence of the antibiotics later began to show significant inhibition (p < 0.05) compared with the control group, especially during mixed exposure. P. lima was not inhibited, but its cell density increased. SMX had a superior stimulation effect on P. lima. The three enzymes activities of P. lima increased, and the antioxidant mechanism was not seriously impacted. However, for Chlorella sp., the activity of SOD increased while the activities of CAT and POD decreased, suggesting that this algae's antioxidant system was unbalanced due to oxidative stress. Based on our results, the growth of P. lima was different from green algae Chlorella sp. as well as other inhibited marine algae (such as diatom, golden algae) studied in previous studies. Therefore, as a typical pollutant in the ocean, antibiotics may play a positive role in the bloom of dinoflagellate red tides., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study investigates the effect of metals (cadmium, lead, mercury, and tellurium) and organic pollutants (benzene, diesel, lindane, and xylene) on a dinoflagellate-Prorocentrum sigmoides Böhm-and its associated culturable bacteria. Two bacterial cultures (Bacillus subtilis strain PD005 and B. xiamensis strain PD006) were isolated from P. sigmoides and characterized by scanning electron microscopy, 16S ribosomal RNA sequencing, biochemical analyses, and growth curve studies. This study points to a mutualistic relationship between P. sigmoides and its associated Bacillus isolates. P. sigmoides enhanced the growth of its associated Bacillus spp., through the secretion of extracellular exudates. In return, both Bacillus isolates contributed to the resistance of P. sigmoides to metals and organic pollutants. P. sigmoides and both Bacillus isolates exhibited concentration-dependent responses to metals and organic pollutants. An intriguing feature was the similar response of P. sigmoides and its associated Bacillus isolates to mercury and cadmium, indicating a co-selection of mercury and cadmium resistance. This provides support to the "dinoflagellate host-phycosphere bacteria" behaving as a single functional unit. However, the sensitivity profiles of P. sigmoides and its associated Bacillus isolates are different with respect to metals versus organic pollutants. These aspects need to be addressed in future studies to unravel the effect of metal and organic pollutants on dinoflagellates, an important component of the phytoplankton community, and to discern the influence of associated "phycosphere" bacteria on the response of dinoflagellates to pollutants., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the Northern Patagonian gulfs of Argentina (Golfo Nuevo and Golfo San José), blooms of toxigenic microalgae and the detection of their associated phycotoxins are recurrent phenomena. The present study evaluated the transfer of phycotoxins from toxigenic microalgae to mesozooplankton in Golfo Nuevo and Golfo San José throughout an annual cycle (December 2014-2015 and January 2015-2016, respectively). In addition, solid-phase adsorption toxin tracking (SPATT) samplers were deployed for the first time in these gulfs, to estimate the occurrence of phycotoxins in the seawater between the phytoplankton samplings. Domoic acid was present throughout the annual cycle in SPATT samplers, whereas no paralytic shellfish poisoning toxins were detected. Ten toxigenic species were identified: Alexandrium catenella, Dinophysis acuminata, Dinophysis acuta, Dinophysis tripos, Dinophysis caudata, Prorocentrum lima, Pseudo-nitzschia australis, Pseudo-nitzschia calliantha, Pseudo-nitzschia fraudulenta, and Pseudo-nitzschia pungens. Lipophilic and hydrophilic toxins were detected in phytoplankton and mesozooplankton from both gulfs. Pseudo-nitzschia spp. were the toxigenic species most frequent in these gulfs. Consequently, domoic acid was the phycotoxin most abundantly detected and transferred to upper trophic levels. Spirolides were detected in phytoplankton and mesozooplankton for the first time in the study area. Likewise, dinophysistoxins were found in mesozooplankton from both gulfs, and this is the first report of the presence of these phycotoxins in zooplankton from the Argentine Sea. The dominance of calanoid copepods indicates that they were the primary vector of phycotoxins in the pelagic trophic web. Environ Toxicol Chem 2019;38:2209-2223. © 2019 SETAC., (© 2019 SETAC.)

Dinoflagellates from the Symbiodiniaceae family and corals have an ecologically important endosymbiotic relationship. Scleractinian corals cannot survive for long periods without their symbionts. These algae, also known as zooxanthellae, on the other hand, thrives outside the coral cells. The free-living populations of zooxanthellae are essential for the resilience of the coral to environmental stressors such as temperature anomalies and ocean acidification. Yet, little is known about how ocean acidification may affect the free-living zooxanthellae. In this study we aimed to test morphological, physiological and biochemical responses of zooxanthellae from the Symbiodinium genus isolated from the coral Mussismilia braziliensis, endemic to the Brazilian coast, to acidification led by increased atmospheric CO2. We tested whether photosynthetic yield, cell ultrastructure, cell density and lipid profile would change after up to 16 days of exposure to pH 7.5 in an atmospheric pCO2 of 1633 μatm. Photosynthetic yield and cell density were negatively affected and chloroplasts showed vesiculated thylakoids, indicating morphological damage. Moreover, Symbiodinium fatty acid profile drastically changed in acidified condition, showing lower polyunsaturated fatty acids and higher saturated fatty acids contents, when compared to the control, non-acidified condition. These results show that seawater acidification as an only stressor causes significant changes in the physiology, biochemistry and ultrastructure of free-living Symbiodinium., Competing Interests: The authors have declared that no competing interests exist.

This research work was carried out to determine the effects of water contamination on the fatty acid (FA) profile of periphyton, zoobenthos, two Chinese carps and a common carp ( Hypophthalmichthys molitrix, Ctenopharygodon idella and Cyprinus carpio ), captured from highly polluted (HP), less polluted (LP), and non-polluted (NP) sites of the Indus river. We found that the concentration of heavy metals in the river water from the polluted locations exceeded the permissible limits suggested by the World Health Organization (WHO) and the US Environmental Protection Agency (EPA). Fatty acid profiles in periphyton, zoobenthos, H. molitrix , C. idella , and C. carpio in the food web of river ecosystems with different pollution levels were assessed. Lauric acid and arachidic acids were not detected in the biomass of periphyton and zoobenthos from HP and LP sites compared to NP sites. Alpha-linolenic acid (ALA), eicosadienoic acid and docosapentaenoic acid were not recorded in the biomass samples of periphyton and zoobenthos in both HP and LP sites. Caprylic acid, lauric acid, and arachidic acid were not found in H. molitrix , C. idella , and C. carpio captured from HP. In this study, 6 and 9 omega series FAs were identified in the muscle samples of H. molitrix , C. idella and C. carpio captured from HP and LP sites compared to NP sites, respectively. Less polyunsaturated fatty acids were observed in the muscle samples of H. molitrix , C. idella , and C. carpio collected from HP than from LP. The heavy metals showed significant negative correlations with the total FAs in periphyton, zoobenthos, and fish samples.

Antifoulant Irgarol 1051 (2-methythiol-4-tert-butylamino-6-cyclopropylamino-s-triazine) can be photodegraded into M1 (2-methylthio-4-tert-butylamino-6-amino-s-triazine) and M2 (3-4-tert-butylamino-6-methylthiol-s-triazin-2-ylamino]propion-aldehyde). M3 (2-methylthio-4,6-bis-tert-butylamino-s-triazine) was also detected as a side-product in Irgarol. This study aimed to investigate the combined toxicity of a mixture of these s-triazine compounds to eight marine organisms. A degraded mixture of Irgarol in artificial seawater was obtained by photolysis over 42 d and its composition was quantified by HPLC-UV analyses. Based on short-term toxicity tests on eight selected marine species, the mixture posed significant phytotoxic effects to the cyanobacteria (Chroococcus minor and Synechococcus sp.), the diatoms (Skeletonema costatum and Thalassiosira pseudonana), the macroalgae (Ulva lactuca and Caulerpa peltata) and the dinoflagellate (Prorocentrum dentatum), though the mixture was less toxic to the copepod Tigriopus japonicus. Both Independent Action and Concentration Addition models can generate reasonably satisfactory predictions on the overall mixture toxicity to the two diatoms, implying that the four compounds likely share a similar mode of action and resemble an additive effect in the mixture., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The dinoflagellate Alexandrium minutum produces toxic compounds, including paralytic shellfish toxins, but also some unknown extracellular toxins. Although copper (Cu) is an essential element, it can impair microalgal physiology and increase their toxic potency. This study investigated the effect of different concentrations of dissolved Cu (7 nM, 79 nM and 164 nM) on A. minutum allelochemical potency, here defined as negative effects of a protist on competing protists through the release of chemicals. This was studied in relation to its physiology. The effects of Cu were assessed on A. minutum growth, reactive oxygen species level, photosynthesis proxies, lipid metabolism, exudation of dissolved organic compounds, allelochemical potency and on the associate free bacterial community of A. minutum. Only the highest Cu exposure (164 nM) inhibited and delayed the growth of A. minutum, and only in this treatment did the allelochemical potency significantly increase, when the dissolved Cu concentration was still toxic. Within the first 7 days of the high Cu treatment, the physiology of A. minutum was severely impaired with decreased growth and photosynthesis, and increased stress responses and free bacterial density per algal cell. After 15 days, A. minutum partially recovered from Cu stress as highlighted by the growth rate, reactive oxygen species level and photosystem II yields. This recovery could be attributed to the apparent decrease in background dissolved Cu concentration to a non-toxic level, suggesting that the release of exudates may have partially decreased the bioavailable Cu fraction. Overall, A. minutum appeared quite tolerant to Cu, and this work suggests that the modifications in the physiology and in the exudates help the algae to cope with Cu exposure. Moreover, this study shows the complex interplay between abiotic and biotic factors that can influence the dynamic of A. minutum blooms. Modulation in allelochemical potency of A. minutum by Cu may have ecological implications with an increased competitiveness of this species in environments contaminated with Cu., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The dinoflagellate Alexandrium minutum (A. minutum) which can produce paralyticshellfish toxins (PSTs) is often used as a model to study the migration, biotransformation,accumulation, and removal of PSTs. However, the mechanism is still unclear. To provide a new toolfor related studies, we tried to label PSTs metabolically with 15N stable isotope to obtain 15 N-PSTsinstead of original 14 N, which could be treated as biomarker on PSTs metabolism. We then culturedthe A. minutum AGY-H46 which produces toxins GTX1-4 in f/2 medium of different 15N/Pconcentrations. The 15 N-PSTs' toxicity and toxin profile were detected. Meanwhile, the 15N labelingabundance and 15N atom number of 15N-PSTs were identified. The 14N of PSTs produced by A.minutum can be successfully replaced by 15 N, and the f/2 medium of standard 15 N/P concentrationwas the best choice in terms of the species' growth, PST profile, 15N labeling result and experimentcost. After many (>15) generations, the 15 N abundance in PSTs extract reached 82.36%, and the 15 Natom number introduced into GTX1-4 might be 4⁻6. This paper innovatively provided the initialevidence that 15 N isotope application of labeling PSTs in A. minutum is feasible. The 15 N-PSTs asbiomarker can be applied and provide further information on PSTs metabolism., Competing Interests: The authors declare no conflict of interest.

Changes in salinity are known to alter the morphology of protists, and we hypothesized that these changes subsequently alter also the predatory behavior of the dinoflagellate Oxyrrhis marina. Oxyrrhis was grown in media of 33, 25, 20, and 10% of the regular salinity of f/2 medium (31-32‰). In all cases, the cells discharged trichocysts and swelled. Cell surfaces and volumes increased with decreasing salinity, such that cell surface area at least doubled at 10% and the cell volume increased approximately fourfold. After 1 h, the cells started to regain their regular shape, which was almost completed after 24 h. Oxyrrhis immediately regained its regular shape when culture medium was added 5-10 min after the osmotic stress. When incubated with Pyramimonas grossii as prey, those short-term stressed cells showed no significant different prey uptake in comparison to non-stressed cells. In contrast, 24 h after the addition of prey, short-term stressed Oxyrrhis cells had, with weak statistical significance, more Pyramimonas cells engulfed than non-stressed cells. These results indicated that (1) trichocysts were most likely not involved in prey capture and (2) salinity-stressed Oxyrrhis either enhanced its capability to capture more prey, or its digestion apparatus was hampered., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Microplastics pose a great threat to entire marine ecosystems, but little is known about their impacts on phytoplankton, especially for the harmful dinoflagellates. In this study, effects of micro polyvinyl chloride (mPVC) on the growth, chlorophyll content and photosynthetic efficiency of the dinoflagellate Karenia mikimotoi at different periods (0, 24, 48, 72 and 96 h) were assessed using gradient concentrations (0, 5, 25, 50 and 100 mg L -1 ) of mPVC with a size of 1 μm. PVC microplastics had dose-dependent adverse effects on K. mikimotoi growth, chlorophyll content and photosynthetic efficiency. The density of algal cell decreased with increasing mPVC concentrations and the highest inhibitory rate (IR) was 45.8% at 24 h under 100 mg L -1 of mPVC. The total chlorophyll content and chlorophyll content in a single algal cell decreased at 96 h and the ФPSⅡ and Fv/Fm decreased 25.3% and 17.1%, respectively. The SEM images provided an intuitive visual method to observe the behaviors and interactions between microplastics and microalgae. It was found from the SEM images that microalgae was wrapped by microplastic beads. The physical blockage and aggregation were also responsible for the cytotoxicity of K. mikimotoi. Our study clarified that PVC microplastics can reduce algal growth, chlorophyll content and photosynthetic efficiency, and it is beneficial to evaluate the possible impact of plastics on aquatic ecosystems., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Harmful algal blooms (HAB), heavily influenced by human activities, pose serious hazard to aquatic ecology and human health. In this study, we monitored the physiological responses and paralytic shellfish poisoning toxins (PSTs) of the toxin-producing HAB species Alexandrium tamarense under titanium dioxide nanoparticles ( n TiO 2 ) exposure in the concentration range of 2-320 mg L -1 over a period of 13 days. The results showed the acute inhibition of n TiO 2 on the algal growth, photosynthetic efficiency and esterase activity at all concentrations except 2 mg L -1 . Nonetheless, they recovered after 13 days n TiO 2 exposure from 20 to 80 mg L -1 . The EC 50 value increased from 85.1 mg L -1 in Day 4 to 140.9 mg L -1 in Day 13. The physiological recovery after prolonged exposure may result from the elimination of excess reactive oxygen species (ROS), a combined outcome of increased n TiO 2 aggregation and algal antioxidant defense mechanisms. This observation is supported by the immediately increased antioxidant enzyme activities, including the superoxide dismutase (SOD) and catalase (CAT) activities upon n TiO 2 exposure. Moreover, the production of PSTs in A. tamarense significantly increased by 1.41-1.76 folds after chronic n TiO 2 exposure at all tested concentrations (p < 0.05), which might also be an adaptive response for the microalgae to overcome the stresses. In particular, the proportions of highly-toxic PSTs analogues GTX2/3, STX and dcSTX were significantly increased upon n TiO 2 exposure (p < 0.05). Hence, the chronic n TiO 2 exposure might aggravate the ecological impact of HABs. Furthermore investigations on different HAB species, especially those toxin-producing ones, and detail physiological responses are obviously needed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A cysteine protease belonging to peptidase C1A superfamily from the eukaryotic, symbiotic dinoflagellate, Symbiodinium sp. strain KB8, was characterized. The protease was purified to near homogeneity (566-fold) by (NH4)2SO4 fractionation, ultrafiltration, and column chromatography using a fluorescent peptide, butyloxycarbonyl-Val-Leu-Lys-4-methylcoumaryl-7-amide (Boc-VLK-MCA), as a substrate for assay purposes. The enzyme was termed VLKP (VLK protease), and its activity was strongly inhibited by cysteine protease inhibitors and activated by reducing agents. Based on the results for the amino acid sequence determined by liquid chromatography-coupled tandem mass spectrometry, a cDNA encoding VLKP was synthesized. VLKP was classified into the peptidase C1A superfamily of cysteine proteases (C1AP). The predicted amino acid sequence of VLKP indicated a tandem array of highly conserved precursors of C1AP with a molecular mass of approximately 71 kDa. The results of gel-filtration chromatography and SDS-PAGE suggested that VLKP exists as a monomer of 31-32 kDa, indicating that the tandem array is likely divided into two mass-equivalent halves that undergo equivalent posttranslational modifications. The VLKP precursor contains an inhibitor prodomain that might become activated after acidic autoprocessing at approximately pH 4. Both purified and recombinant VLKPs had a similar substrate specificity and kinetic parameters for common C1AP substrates. Most C1APs reside in acidic organelles such as the vacuole and lysosomes, and indeed VLKP was most active at pH 4.5. Since VLKP exhibited maximum activity during the late logarithmic growth phase, these attributes suggest that, VLKP is involved in the metabolism of proteins in acidic organelles., Competing Interests: The authors have declared that no competing interests exist.

Phthalate esters (PAEs), one class of the most frequently detected endocrine-disrupting chemicals (EDCs) in marine environment, have aroused wide public concerns because of their carcinogenicity, teratogenicity, and mutagenicity. However, the environmental fate of PAEs in the occurrence of harmful algal blooms remains unclear. In this research, four PAEs with different alkyl chains, i.e., dimethyl phthalate (DMP), diethyl phthalate (DEP), diallyl phthalate (DAP), and dipropyl phtalate (DPrP) were selected as models to investigate toxicity, uptake, and degradation of PAEs in seawater grown with K. brevis, one of the common harmful red tide species. The 96-h median effective concentration (96h-EC 50 ) values followed the order of DMP (over 0.257 mmol L -1 ) > DEP (0.178 mmol L -1 ) > DAP (0.136 mmol L -1 ) > DPrP (0.095 mmol L -1 ), and the bio-concentration factors (BCFs) were positively correlated to the alkyl chain length. These results indicate that the toxicity of PAEs and their accumulation in K. brevis increased with increasing alkyl chains, due to the higher lipophicity of the longer chain PAEs. With growth of K. brevis for 96 h, the content of DMP, DEP, DAP, and DPrP decreased by 93.3%, 68.2%, 57.4% and 46.7%, respectively, mainly attributed to their biodegradation by K. brevis, accounting for 87.1%, 61%, 46%, 40% of their initial contents, respectively. It was noticed that abiotic degradation had little contribution to the total reduction of PAEs in the algal cultivation systems. Moreover, five metabolites were detected in the K. brevis when exposed to DEP including dimethyl phthalate (DMP), monoethyl phthalate (MEP), mono-methyl phthalate (MMP), phthalic acid (PA), and protocatechuic acid (PrA). While when exposed with to DPrP, one additional intermediate compound diethyl phthalate (DEP) was detected in the cells of K. brevis in addition to the five metabolites mentioned above. These results confirm that the main biodegradation pathways of DEP and DPrP by K. brevis included de-esterification, demethylation or transesterification. These findings will provide valuable evidences for predicting the environmental fate and assessing potential risk of PAEs in the occurrence of harmful algal blooms in marine environment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The problem of marine life attachment and its pollution to facilities has caused a lot of great troubles in the development and application of marine resources. The holes generated by the photocatalytic coating materials under sunlight may produce strong oxidizing species and showed a significant effect on the degradation and bactericidal performance of environmental organic matter. In this paper, a novel bismuth vanadate/indium vanadate (BiVO 4 /InVO 4 ) composite with cherimoya-like microstructure was fabricated using new vanadium source. It is found that the composite materials showed enhanced photocatalytic antifouling property. The degradation efficiency of the model pollutes (Rhodamine B, RhB) achieved 99.775% within 280 min over BiVO 4 /InVO 4 nanostructures, and the sterilization rate of E. coli, S. aureus, P. aeruginosa and A. carterae achieved 99.7148%, 99.5519%, 99.5411% and 96.00%, respectively. Moreover, the circulate photocatalytic degradation of antibacteria experiments demonstrated the outstanding stability and reusability of BiVO 4 /InVO 4 composite. According to the active free radical trapping experiments, the hydroxyl radical (OH) and superoxide radical (O 2- ) were certified to be the main reactive oxygen species in the BiVO 4 /InVO 4 system. The distinctly enhanced photocatalytic performance of BiVO 4 /InVO 4 nanomaterial primarily resulted from the narrow bandgap (about 1.86 eV). This study not only provides a new method for developing novel antibacterial materials, but also introduces a visible light-driven photocatalyst for water treatment and marine antifouling, especially for red tide control., (Copyright © 2018. Published by Elsevier Inc.)

Due to increasing evidence of key chemically mediated interactions in marine ecosystems, a real interest in the characterization of the metabolites involved in such intra and interspecific interactions has emerged over the past decade. Nevertheless, only a small number of studies have succeeded in identifying the chemical structure of compounds of interest. One reason for this low success rate is the small size and extremely polar features of many of these chemical compounds. Indeed, a major challenge in the search for active metabolites is the extraction of small polar compounds from seawater. Yet, a full characterization of those metabolites is necessary to understand the interactions they mediate. In this context, the study presented here aims to provide a methodology for the characterization of highly polar, low molecular weight compounds in a seawater matrix that could provide guidance for marine ecologists in their efforts to identify active metabolites. This methodology was applied to the investigation of the chemical structure of an algicidal compound secreted by the bacteria Shewanella sp. IRI-160 that was previously shown to induce programmed cell death in dinoflagellates. The results suggest that the algicidal effects may be attributed to synergistic effects of small amines (ammonium, 4-aminobutanal) derived from the catabolization of putrescine produced in large quantities (0.05⁻6.5 fmol/cell) by Shewanella sp. IRI- 160.