The Effecting Mechanisms of 100 nm Sized Polystyrene Nanoplastics on the Typical Coastal Alexandrium tamarense.

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.

Allelopathy of extracellular chemicals released by Karlodinium veneficum on photosynthesis of Prorocentrum donghaiense.

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, CK/CP), 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 ΦPSII, 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.

Viability assessed with the most probable number dilution culture method after chemical treatment of ballast water reveals the presence of false negatives from an approved vital stain method.

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).

Environmentally degradable carbon dots for inhibiting P. globosa growth and reducing hemolytic toxin.

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.

Physiological and genetic responses of the benthic dinoflagellate Prorocentrum lima to polystyrene microplastics.

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.

Mesocosm study of PAC-modified clay effects on Karenia brevis cells and toxins, chemical dynamics, and benthic invertebrate physiology.

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 × 106 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 CO2 (pCO2) 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.

The dispersant Corexit 9500 and (dispersed) oil are lethal to coral endosymbionts.

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.

Toxin production and transcriptomic response to nitrate concentrations in the toxic dinoflagellate Alexandrium tamarense.

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.

Activation of stress reactions in the dinophyte microalga Prorocentrum cordatum as a consequence of the toxic effect of ZnO nanoparticles and zinc sulfate.

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.

Cross resistance to brevetoxin-3 by kdr and super-kdr mutations in house flies.

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 LD50 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.

Can exposure to Gymnodinium catenatum toxic blooms influence the impacts induced by Neodymium in Mytilus galloprovincialis mussels? What doesn't kill can make them stronger?

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.

Cryoprotectant treatment tests on three morphologically diverse marine dinoflagellates and the cryopreservation of Breviolum sp. (Symbiodiniaceae).

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.

Effects of Polystyrene Microplastics on Growth and Toxin Production of Alexandrium pacificum.

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 (Fv/Fm) 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 Fv/Fm 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, Fv/Fm 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.

Genetic and spatial organization of the unusual chromosomes of the dinoflagellate Symbiodinium microadriaticum.

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.

Effects of mycosubtilin homolog algicides from a marine bacterium, Bacillus sp. SY-1, against the harmful algal bloom species Cochlodinium polykrikoides.

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 1H, 13C, and two-dimensional nuclear magnetic resonance spectroscopy, including 1H-15N heteronuclear multiple bond correlation analysis. MS 1056 contains a ß-amino acid residue with an alkyl side chain of C15, which has not previously been seen in known mycosubtilin families. MS 1056, 1070, and 1084 showed algicidal activities against C. polykrikoides with 6-h LC50 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.

Characterization and Ca2+-induced expression of calmodulin (CaM) in marine dinoflagellates.

Calmodulin (CaM) is one of the major Ca2+-binding proteins in the cells, and it plays multiple roles in several Ca2+ 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 Ca2+-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.

Effects of a bacteria-produced algicide on non-target marine invertebrate species.

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%).

Establish axenic cultures of armored and unarmored marine dinoflagellate species using density separation, antibacterial treatments and stepwise dilution selection.

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 log10 CFU/mL to 1.13 ± 0.07 log10 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.

Effects of pH and Nutrients (Nitrogen) on Growth and Toxin Profile of the Ciguatera-Causing Dinoflagellate Gambierdiscus polynesiensis (Dinophyceae).

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 effect of protocatechuic acid on the phycosphere in harmful algal bloom species Scrippsiella trochoidea.

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.