Recovery and fate of three species of marine dinoflagellates after yellow clay flocculation

The recovery and fate of three species of dinoflagellates, Alexandrium tamarense, Cochlodinium polykrikoides and Scrippsiella trochoidea, after having been sedimented by yellow clay, were investigated in the laboratory. The effect of burying period in yellow clay pellet and mixing on the recovery of settled algal cells were studied. The morphological changes of algal cells in yellow clay pellet were also tracked. Results showed that there was almost no recovery for A. tamarense and C. polykrikoides, and the cells decomposed after 2-3 days after visible changes in morphology and chloroplasts. There was some recovery for S. trochoidea. Moreover, S. trochoidea cysts were formed in clay pellet during the period of about 14 days, with the highest abundance of 87 000 cysts g(-1) clay and the incidence of cyst formation of 6.5%, which was considered as a potential threat for the further occurrence of algal blooms. S. trochoidea cysts were isolated from yellow clay and incubated to test their viability, and a germination ratio of more than 30% was obtained after incubation for 1 month. These results showed the species specificity of the mitigation effect of yellow clay. It is suggested that cautions be taken for some harmful species and thorough risk assessments be conducted before using this mitigation strategy in the field.

Modelling the Stoichiometric Regulation of C-Rich Toxins in Marine Dinoflagellates

Toxin production in marine microalgae was previously shown to be tightly coupled with cellular stoichiometry. The highest values of cellular toxin are in fact mainly associated with a high carbon to nutrient cellular ratio. In particular, the cellular accumulation of C-rich toxins (i.e., with C:N > 6.6) can be stimulated by both N and P deficiency. Dinoflagellates are the main producers of C-rich toxins and may represent a serious threat for human health and the marine ecosystem. As such, the development of a numerical model able to predict how toxin production is stimulated by nutrient supply/deficiency is of primary utility for both scientific and management purposes. In this work we have developed a mechanistic model describing the stoichiometric regulation of C-rich toxins in marine dinoflagellates. To this purpose, a new formulation describing toxin production and fate was embedded in the European Regional Seas Ecosystem Model (ERSEM), here simplified to describe a monospecific batch culture. Toxin production was assumed to be composed by two distinct additive terms; the first is a constant fraction of algal production and is assumed to take place at any physiological conditions. The second term is assumed to be dependent on algal biomass and to be stimulated by internal nutrient deficiency. By using these assumptions, the model reproduced the concentrations and temporal evolution of toxins observed in cultures of Ostreopsis cf. ovata, a benthic/epiphytic dinoflagellate producing C-rich toxins named ovatoxins. The analysis of simulations and their comparison with experimental data provided a conceptual model linking toxin production and nutritional status in this species. The model was also qualitatively validated by using independent literature data, and the results indicate that our formulation can be also used to simulate toxin dynamics in other dinoflagellates. Our model represents an important step towards the simulation and prediction of marine algal toxicity.

A molecular basis for polyketide biosynthesis in marine dinoflagellates with emphasis on Karenia brevis

Polyketides derived from dinoflagellates are among the most complex and unique structures identified to date. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). No studies of the biosynthesis of dinoflagellate derived polyketides at the genomic level have been reported to date. Nine strains representing seven different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKS) by PCR and RT-PCR. Seven of the nine strains yielded products that were homologous with known and putative type I polyketide synthases. In each case, the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S rRNA gene. However, residual phylogenetic signals, resistance to methylation sensitive restriction enzymes and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes. ^ A more detailed analysis of Karenia brevis, a toxic marine dinoflagellate endemic to the Gulf of Mexico, also supports the hypothesis that dinoflagellates have polyketide synthase genes. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. PKS encoding genes amplified from K. brevis culture were found to be similar to PKS genes from the closely related protist, Cryptosporidium parvum. This suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. This dissertation reports the localization of these PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate. ^

Combined effects of ocean acidification and light or nitrogen availabilities on 13C fractionation in marine dinoflagellates

Along with increasing oceanic CO2 concentrations, enhanced stratification constrains phytoplankton to shallower upper mixed layers with altered light regimes and nutrient concentrations. Here, we investigate the effects of elevated pCO2 in combination with light or nitrogen-limitation on 13C fractionation (epsilon p) in four dinoflagellate species. We cultured Gonyaulax spinifera and Protoceratium reticulatum in dilute batches under low-light (LL) and high-light (HL) conditions, and grew Alexandrium fundyense and Scrippsiella trochoidea in nitrogen-limited continuous cultures (LN) and nitrogen-replete batches (HN). The observed CO2-dependency of epsilon p remained unaffected by the availability of light for both G. spinifera and P. reticulatum, though at HL epsilon p was consistently lower by about 2.7 per mil over the tested CO2 range for P. reticulatum. This may reflect increased uptake of (13C-enriched) bicarbonate fueled by increased ATP production under HL conditions. The observed CO2-dependency of epsilon p disappeared under LN conditions in both A. fundyense and S. trochoidea. The generally higher epsilon p under LN may be associated with lower organic carbon production rates and/or higher ATP:NADPH ratios. CO2-dependent epsilon p under non-limiting conditions has been observed in several dinoflagellate species, showing potential for a new CO2-proxy. Our results however demonstrate that light- and nitrogen-limitation also affect epsilon p, thereby illustrating the need to carefully consider prevailing environmental conditions.

Development and validation of an ultrasensitive fluorescence planar waveguide biosensor for the detection of paralytic shellfish toxins in marine algae

Marine dinoflagellates of the genera Alexandrium are well known producers of the potent neurotoxic paralytic shellfish toxins that can enter the food web and ultimately present a serious risk to public health in addition to causing huge economic losses. Direct coastal monitoring of Alexandrium spp. can provide early warning of potential shellfish contamination and risks to consumers and so a rapid, sensitive, portable and easy-to-use assay has been developed for this purpose using an innovative planar waveguide device. The disposable planar waveguide is comprised of a transparent substrate onto which an array of toxin-protein conjugates is deposited, assembled in a cartridge allowing the introduction of sample, and detection reagents. The competitive assay format uses a high affinity antibody to paralytic shellfish toxins with a detection signal generated via a fluorescently labelled secondary antibody. The waveguide cartridge is analysed by a simple reader device and results are displayed on a laptop computer. Assay speed has been optimised to enable measurement within 15 min. A rapid, portable sample preparation technique was developed for Alexandrium spp. in seawater to ensure analysis was completed within a short period of time. The assay was validated and the LOD and CCß were determined as 12 pg/mL and 20 pg/mL respectively with an intra-assay CV of 11.3% at the CCß and an average recovery of 106%. The highly innovative assay was proven to accurately detect toxin presence in algae sampled from the US and European waters at an unprecedented cell density of 10 cells/L. © 2012 Elsevier B.V. All rights reserved.

Novel hydrolysis-probe based qPCR assay to detect saxitoxin transcripts of dinoflagellates in environmental samples

Paralytic Shellfish Poisoning (PSP) is a serious human illness caused by ingestion of seafood enriched with paralytic shellfish toxins (PSTs). PSTs are neurotoxic compounds produced by marine dinoflagellates, specifically by Alexandrium spp., Gymnodinium catenatum and Pyrodinium bahamense. Every year, massive monitoring of PSTs and their producers is undertaken worldwide to avoid PSP incidences. Here we developed a sensitive, hydrolysis probe-based quantitative PCR (qPCR) assay to detect a gene essential for PST synthesis across different dinoflagellate species and genera and tested it on cDNA generated from environmental samples spiked with Alexandrium minutum or Alexandrium fundyense cells. The assay was then applied to two environmental sample series from Norway and Spain and the results were complemented with cell counts, LSU-based microarray data and toxin measurements (enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor method). The overall agreement between the results of the qPCR assay and the complementary data was good. The assay reliably detected sxtA transcripts from Alexandrium spp. and G. catenatum, even though Alexandrium spp. cell concentrations were mostly so low that they could not be quantified microscopically. Agreement between the novel assay and toxin measurements or cell counts was generally good; the few inconsistencies observed were most likely due to disparate residence times of sxtA transcripts and PSTs in seawater, or, in the case of cell counts, to dissimilar sxtA4 transcript numbers per cell in different dinoflagellate strains or species. © 2013 Elsevier B.V.

Comparison of biosensor platforms for surface plasmon resonance based detection of paralytic shellfish toxins

Paralytic shellfish poisoning (PSP) toxins are produced by certain marine dinoflagellates and may accumulate in bivalve molluscs through filter feeding. The Mouse Bioassay (MBA) is the internationally recognised reference method of analysis, but it is prone to technical difficulties and regarded with increasing disapproval due to ethical reasons. As such, alternative methods are required. A rapid surface plasmon resonance (SPR) biosensor inhibition assay was developed to detect PSP toxins in shellfish by employing a saxitoxin polyclonal antibody (R895). Using an assay developed for and validated on the Biacore Q biosensor system, this project focused on transferring the assay to a high-throughput, Biacore T100 biosensor in another laboratory. This was achieved using a prototype PSP toxin kit and recommended assay parameters based on the Biacore Q method. A monoclonal antibody (GT13A) was also assessed. Even though these two instruments are based on SPR principles, they vary widely in their mode of operation including differences in the integrated mu-fluidic cartridges, autosampler system, and sensor chip compatibilities. Shellfish samples (n = 60), extracted using a simple, rapid procedure, were analysed using each platform, and results were compared to AOAC high performance liquid chromatography (HPLC) and MBA methods. The overall agreement, based on statistical 2 x 2 comparison tables, between each method ranged from 85% to 94.4% using R895 and 77.8% to 100% using GT13A. The results demonstrated that the antibody based assays with high sensitivity and broad specificity to PSP toxins can be applied to different biosensor platforms. (C) 2011 Elsevier B.V. All rights reserved.

First report of the use of a saxitoxin-protein conjugate to develop a DNA aptamer to a small molecule toxin

Saxitoxin (STX) is a low molecular weight neurotoxin mainly produced by certain marine dinoflagellates that, along with its family of similarly related paralytic shellfish toxins, may cause the potentially fatal intoxication known as paralytic shellfish poisoning. Illness and fatality rates are low due to the effective monitoring programs that determine when toxins exceed the established regulatory action level and effectuate shellfish harvesting closures accordingly. Such monitoring programs rely on the ability to rapidly screen large volumes of samples. Many of the screening assays currently available employ antibodies or live animals. This research focused on developing an analytical recognition element that would eliminate the challenges associated with the limited availability of antibodies and the use of animals. Here we report the discovery of a DNA aptamer that targets STX. Concentration-dependent and selective binding of the aptamer to STX was determined using a surface plasmon resonance sensor. Not only does this work represent the first reported aptamer to STX, but also the first aptamer to any marine biotoxin. A novel strategy of using a toxin-protein conjugate for DNA aptamer selection was successfully implemented to overcome the challenges associated with aptamer selection to small molecules. Taking advantage of such an approach could lead to increased diversity and accessibility of aptamers to low molecular weight toxins, which could then be incorporated as analytical recognition elements in diagnostic assays for foodborne toxin detection. The selected STX aptamer sequence is provided here, making it available to any investigator for use in assay development for the detection of STX.

Experimental growth characteristics, cell size, yessotoxin values and bioassay data of Protoceratium reticulatum (Dinophyceae)

Harmful algal blooms are mainly caused by marine dinoflagellates and are known to produce potent toxins that may affect the ecosystem, human activities and health. Such events have increased in frequency and intensity worldwide in the past decades. Numerous processes involved in Global Change are amplified in the Arctic, but little is known about species specific responses of arctic dinoflagellates. The aim of this work was to perform an exhaustive morphological, phylogenetical and toxinological characterization of Greenland Protoceratium reticulatum and, in addition, to test the effect of temperature on growth and production of bioactive secondary metabolites. Seven clonal isolates, the first isolates of P. reticulatum available from arctic waters, were phylogenetically characterized by analysis of the LSU rDNA. Six isolates were further characterized morphologically and were shown to produce both yessotoxins (YTX) and lytic compounds, representing the first report of allelochemical activity in P. reticulatum. As shown for one of the isolates, growth was strongly affected by temperature with a maximum growth rate at 15 °C, a significant but slow growth at 1 °C, and cell death at 25 °C, suggesting an adaptation of P. reticulatum to temperate waters. Temperature had no major effect on total YTX cell quota or lytic activity but both were affected by the growth phase with a significant increase at stationary phase. A comparison of six isolates at a fixed temperature of 10 °C showed high intraspecific variability for all three physiological parameters tested. Growth rate varied from 0.06 to 0.19 per day, and total YTX concentration ranged from 0.3 to 15.0 pg YTX/cell and from 0.5 to 31.0 pg YTX/cell at exponential and stationary phase, respectively. All six isolates performed lytic activity; however, for two isolates lytic activity was only detectable at higher cell densities in stationary phase.

Ciguatoxins: Cyclic polyether modulators of voltage-gated Iion channel function

Ciguatoxins are cyclic polyether toxins, derived from marine dinoflagellates, which are responsible for the symptoms of ciguatera poisoning. Ingestion of tropical and subtropical fin fish contaminated by ciguatoxins results in an illness characterised by neurological, cardiovascular and gastrointestinal disorders. The pharmacology of ciguatoxins is characterised by their ability to cause persistent activation of voltage-gated sodium channels, to increase neuronal excitability and neurotransmitter release, to impair synaptic vesicle recycling, and to cause cell swelling. It is these effects, in combination with an action to block voltage-gated potassium channels at high doses, which are believed to underlie the complex of symptoms associated with ciguatera. This review examines the sources, structures and pharmacology of ciguatoxins. In particular, attention is placed on their cellular modes of actions to modulate voltage-gated ion channels and other Na+-dependent mechanisms in numerous cell types and to current approaches for detection and treatment of ciguatera.

Diffusive and ship-mediated spread of dinoflagellates in the Baltic Sea with Prorocentrum minimum as a special case

In this thesis the role played by expansive and introduced species in the phytoplankton ecology of the Baltic Sea was investigated. The aims were threefold. First, the studies investigated the resting stages of dinoflagellates, which were transported into the Baltic Sea via shipping and were able to germinate under the ambient, nutrient-rich, brackish water conditions. The studies also estimated which factors favoured the occurrence and spread of P. minimum in the Baltic Sea and discussed the identification of this morphologically variable species. In addition, the classification of phytoplankton species recently observed in the Baltic Sea was discussed. Incubation of sediments from four Finnish ports and 10 ships ballast tanks revealed that the sediments act as sources of living dinoflagellates and other phytoplankton. Dinoflagellates germinated from all ports detected and from 90% of ballast tanks. The concentrations of cells germinating from ballast tank sediments were mostly low compared with the acceptable cell concentrations set by the International Maritime Organization s (IMO s) International Convention for the Control and Management of Ships Ballast Water and Sediments. However, the IMO allows such high concentrations of small cells in the discharged ballast water that the total number of cells in large ballast water tanks can be very high. Prorocentrum minimum occurred in the Baltic Sea annually but with no obvious trend in the 10-year timespan from 1993 to 2002. The species occurred under wide ranges of temperatures and salinities and the abundance of the species was positively related especially to the presence of organic nitrogen and phosphorus. This indicated that the species was favoured by increased organic nutrient loading and runoff from land and rivers. The cell shape of P. minimum varied from triangular to oval-round, but morphological fine details indicated that only one morphospecies was present. P. minimum also is, according to present knowledge, the only potentially harmful phytoplankton species that has recently expanded widely into new areas of the Baltic Sea.

Effects of macroalgae Ulva pertusa (Chlorophyta) and Gracilaria lemaneiformis (Rhodophyta) on growth of four species of bloom-forming dinoflagellates

The effects of fresh thalli and culture medium filtrates from two species of marine macroalgae, Ulva pertusa Kjellm (Chlorophyta) and Gracilaria lemaneiformis (Bory) Dawson (Rhodophyta), on growth of marine microalgae were investigated in co-culture under controlled laboratory conditions. A selection of microalgal species were used, all, being identified as bloom-forming dinoflagellates: Prorocentrum donghaiense Lu sp., Alexandrium tamarense (Lebour) Balech, Amphidinium carterae Hulburt and Scrippsiella trochoide (Stein) Loeblich III. Results showed that the fresh thalli of either U. pertusa or G. lemaneiformis significantly inhibited the microalgal growth, or caused mortality at the end of the experiment. However, the overall effects of the macroalgal culture filtrates on the growth of the dinoflagellates were species-specific (inhibitory, stimulatory or none) for different microalgal species. Results indicated an allelopathic effect of macroalga on the co-cultured dinoflagellate. We then took P. donghaiense as an example to further assess this hypothesis. The present study was carried out under controlled conditions, thereby excluded the fluctuation in light and temperature. Nutrient assays showed that nitrate and phosphate were almost exhausted in G. lemaneiformis co-culture. but remained at enough high levels in U pertusa co-culture, which were well above the nutrient limitation for the microalgal growth, when all cells of P. donghaiense were killed in the co-culture. Daily f/2 medium enrichment greatly alleviated the growth inhibition on P. donghaiense in G. lemaneiformis co-culture, but could not eliminate it. Other environmental factors, such as carbonate limitation, bacterial presence and the change of pH were also not necessary for the results. We thus concluded that allelopathy was the most possible reason leading to the negative effect of U. pertusa on P. donghaiense, and the combined roles of allelopathy and nutrient competition were essential for the effect of G. lemaneiformis on P. donghaiense. (c) 2006 Elsevier B.V. All rights reserved.

Interactions between the bloom-forming dinoflagellates Prorocentrum donghaiense and Alexandrium tamarense in laboratory cultures

Interactions between Prorocentrum donghaiense and Alexandrium tamarens, two bloom-forming dinoflagellates, were investigated using bi-algal cultures. All R donghaiense died, but A. tamarense was hardly affected by the end of the experiment when the initial cell density was set at 1.0 X 10(4) cells mL(-1) for P. donghaiense and 0.28 x 10(4) cells mL(-1) for A. tamarense. However, significant growth suppression occurred in either species when the initial cell density of P donghaiense increased to I. 0 X 105 Cells mL(-1) in the bi-algal culture, but no out-competement was observed. The simultaneous assay on the culture filtrates showed that P donghaiense filtrate prepared at a lower initial density (1.0 X 10(4) cells mL(-1)) stimulated growth of the co-cultured A. tanzarense (0.28 x 10(4) cells mL(-1)), but filtrate at a higher initial density (1.0 x 10(5) cells mL(-1)) depressed its growth. The filtrate of A. tamarense at a density of 0.28 x 10(4) cells mL(-1) killed all R donghaiense at a lower density (1.0 x 10(4) cells mL(-1)), but only exhibited an inhibitory effect on it at a higher density (1.0 x 10(5) cells mL(-1)). It is likely that these two species of microalgae interfere with each other mainly by releasing allelochemical substance(s) into the culture medium, and a direct cell-to-cell contact was not necessary for their mutual interaction. The allelopathic test further proved that A. tamarense could affect the growth of co-cultured P. donghaiense by producing allelochemical(s); moreover, A. tamarense culture filtrate at the stationary growth phase (SP) had a strongly inhibitory effect on P donghaiense compared to that at the exponential phase (EP). Results also demonstrated a dose-dependent relationship between the microalgal initial cell density and the degree of the allelopathic effect. The growth of R donghaiense and A. tamarense in the bi-algal cultures was simulated using a mathematical model to quantify the interaction. The estimated parameters from the model showed that the inhibition exerted by A. tamarense on P. donghaiense was about 17 and 8 times stronger than the inhibition P. donghaiense exerted on A. tamarense, when the initial cell density was set at 1.0 X 10(4) and 1.0 X 10(5) cells mL(-1) for P donghaiense, respectively. and 0.28 x 10(4) cells mL(-1) for A. tamarense in the bi-algal cultures. A. tamarense seems to have a survival strategy that is superior to that of P. donghaiense in bi-algal cultures under controlled laboratory conditions. (c) 2006 Elsevier B.V. All rights reserved.