DSP toxin production de novo in cultures of Dinophysis acuminata (Dinophyceae) from North America

For decades, many aspects of Dinophysis biology have remained intractable due to our inability to maintain these organisms in laboratory cultures. Recent breakthroughs in culture methods have opened the door for detailed investigations of these important algae. Here, for the first time, we demonstrate toxin production in cultures of North American Dinophysis acuminata, isolated from Woods Hole, MA. These findings show that, despite the rarity of Dinophysis-related DSP events in North America, D. acuminata from this area has the ability to produce DSP toxins just as it does in other parts of the world where this species is a major cause of DSP toxicity. In our cultures, D. acuminata cells were observed feeding on Myrionecta rubra using a peduncle. Culture extracts were analyzed using LC–MS/MS, providing unequivocal evidence for the toxin DTX1 in the Dinophysis cultures. In addition, a significant amount of an okadaic acid diol ester, OA-D8, was detected. These results suggest that this Dinophysis isolate stores much of its OA as a diol ester. Also, toxin PTX-2 and a hydroxylated PTX-2 with identical fragmentation mass spectrum to that of PTX-11, but with a different retention time, were detected in this D. acuminata culture. This demonstration of toxin production in cultured North American Dinophysis sets the stage for more detailed studies investigating the causes of geographic differences in toxicity. It is now clear that North American Dinophysis have the ability to produce DSP toxins even though they only rarely cause toxic DSP events in nature. This may reflect environmental conditions that might induce or repress toxin production, genetic differences that cause modifications in toxin gene expression, or physiological and biochemical differences in prey species.     

Production and excretion of okadaic acid,pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light,food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.     

Cell cycle regulation of the mixotrophic dinoflagellate Dinophysis acuminata: Growth,photosynthetic efficiency and toxin production

The mixotrophic dinoflagellate Dinophysis acuminata is a widely distributed diarrhetic shellfish poisoning (DSP) producer. Toxin variability of Dinophysis spp. has been well studied, but little is known of the manner in which toxin production is regulated throughout the cell cycle in these species, in part due to their mixotrophic characteristics. Therefore, an experiment was conducted to investigate cell cycle regulation of growth, photosynthetic efficiency, and toxin production in D. acuminata. First, a three-step synchronization approach, termed “starvation-feeding-dark”, was used to achieve a high degree of synchrony of Dinophysis cells by starving the cells for 2 weeks, feeding them once, and then placing them in darkness for 58 h. The synchronized cells started DNA synthesis (S phase) 10 h after being released into the light, initiated G2 growth stage eight hours later, and completed mitosis (M phase) 2 h before lights were turned on. The toxin content of three dominant toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-2 (PTX2), followed a common pattern of increasing in G1 phase, decreasing on entry into the S phase, then increasing again in S phase and decreasing in M phase during the diel cell cycle. Specific toxin production rates were positive throughout the G1 and S phases, but negative during the transition from G1 to S phase and late in M phase, the latter reflecting cell division. All toxins were initially induced by the light and positively correlated with the percentage of cells in S phase, indicating that biosynthesis of Dinophysis toxins might be under circadian regulation and be most active during DNA synthesis.     

Characterization of Dinophysis spp. (Dinophyceae,Dinophysiales) from the mid-Atlantic region of the United States1

Due to the increasing prevalence of Dinophysis spp. and their toxins on every US coast in recent years, the need to identify and monitor for problematic Dinophysis populations has become apparent. Here, we present morphological analyses, using light and scanning electron microscopy, and rDNA sequence analysis, using a ~2-kb sequence of ribosomal ITS1, 5.8S, ITS2, and LSU DNA, of Dinophysis collected in mid-Atlantic estuarine and coastal waters from Virginia to New Jersey to better characterize local populations. In addition, we analyzed for diarrhetic shellfish poisoning (DSP) toxins in water and shellfish samples collected during blooms using liquid-chromatography tandem mass spectrometry and an in vitro protein phosphatase inhibition assay and compared this data to a toxin profile generated from a mid-Atlantic Dinophysis culture. Three distinct morphospecies were documented in mid-Atlantic surface waters: D. acuminata, D. norvegica, and a “small Dinophysis sp.” that was morphologically distinct based on multivariate analysis of morphometric data but was genetically consistent with D. acuminata. While mid-Atlantic D. acuminata could not be distinguished from the other species in the D. acuminata-complex (D. ovum from the Gulf of Mexico and D. sacculus from the western Mediterranean Sea) using the molecular markers chosen, it could be distinguished based on morphometrics. Okadaic acid, dinophysistoxin 1, and pectenotoxin 2 were found in filtered water and shellfish samples during Dinophysis blooms in the mid-Atlantic region, as well as in a locally isolated D. acuminata culture. However, DSP toxins exceeded regulatory guidance concentrations only a few times during the study period and only in noncommercial shellfish samples.     

Diarrheic toxins in field-sampled and cultivated Dinophysis spp. cells from southern Brazil

In southern Brazil, mixotrophic dinoflagellates belonging to the Dinophysis acuminata complex have recently been involved in diarrheic shellfish poisoning episodes through the production of lipophilic toxins such as okadaic acid (OA) and dinophysistoxin-1 (DTX-1). The present investigation used a combination of laboratory cultures and field surveys at three large estuarine systems in that region to examine toxin retention in Dinophysis spp. cells under optimum or growth-limiting conditions. This study represents the first successful culture of a Dinophysis isolate from the Atlantic South America region. Starved D. acuminata complex cells reached 5.6-fold higher cellular OA quotas (up to 18 pg cell^?1) than Mesodinium rubrum-fed cultures 20 days after inoculation in the laboratory. Moreover, in field samples, light-limited cells at the bottom of a stratified water column were less abundant, yet 6.6- to 11-fold more toxic (up to 26.4 pg OA and 1.7 pg DTX-1 cell^?1) than those located at the illuminated surface. This phenomenon of toxin retention by slow-dividing cells may partially explain the enormous variation in cell toxin quota found within Dinophysis spp. populations from a single location, and it may have serious implications for cell count-based monitoring program in bivalve aquaculture areas. In fact, only low to moderate OA levels were detected in the digestive glands of oysters Crassostrea spp. (up to 17.8 ng g^?1) and the guts and livers of filter-feeding fish (44.7 ng g^?1) during the present study, despite the relatively high Dinophysis cell densities (up to 19,500 cells L^?1) found in the field.     

Are the mitochondrial cox1 and cob genes suitable markers for species of Dinophysis Ehrenberg?

The identification of Dinophysis species with similar morphology but different toxic (Diarrhetic Shellfish Poisoning, DSP) potential is a crucial task in harmful algae monitoring programmes. The taxonomic assignment of Dinophysis species using molecular markers is a difficult task due to extremely low interspecific variability within their nuclear ribosomal genes and intergenic regions. Mitochondrial cox1 gene has been proposed as a better specific marker for Dinophysis species based on its higher resolution for two morphologically related species (Dinophysis acuminata and Dinophysis ovum) of the “Dinophysis acuminata complex”. In this study, the potential of two mitochondrial genes (mt cox1 and cob) to discriminate among six Dinophysis species (field isolates and cultures) associated with DSP events was explored. Neither mt cox1 nor cob genes provided enough resolution for all species of Dinophysis. The cob gene showed very poor resolution and grouped all Dinophysis spp. in a common clade. In contrast, the cox1 phylogeny distinguished 5 clades in the Dinophysiales – the “acuminata complex”, the “caudata group”, “acuta + norvegica” and Phalacromaspp. However, within the “D. acuminata complex” mtcox1 is so far the unique marker that differentiates D. acuminata from other species: isolates of D. ovum and Dinophysis sacculus had almost identical sequences (only four mismatches), but they were well separated from D. acuminata. D. acuminata and Dinophysis skagii (considered a life cycle stage of the former) showed identical cox1 sequences. Probes towards this gene can be useful in Mediterranean and Western Iberia sites where the co-occurrence of close morphotypes of D. acuminata and D. sacculus pose a problem for monitoring analyses. This is the first report on cultures of D. sacculus and its phylogenetic relation with other species of the D. acuminata complex.     

Distribution of Dinophysis species and their association with lipophilic phycotoxins in plankton from the Argentine Sea

Dinophysis is a cosmopolitan genus of marine dinoflagellates, considered as the major proximal source of diarrheic shellfish toxins and the only producer of pectenotoxins (PTX). From three oceanographic expeditions carried out during autumn, spring and late summer along the Argentine Sea (∼38–56°S), lipophilic phycotoxins were determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in size-fractionated plankton samples. Lipophilic toxin profiles were associated with species composition by microscopic analyses of toxigenic phytoplankton. Pectenotoxin-2 and PTX-11 were frequently found together with the presence of Dinophysis acuminata and Dinophysis tripos. By contrast, okadaic acid was rarely detected and only in trace concentrations, and dinophysistoxins were not found. The clear predominance of PTX over other lipophilic toxins in Dinophysis species from the Argentine Sea is in accordance with previous results obtained from north Patagonian Gulfs of the Argentine Sea, and from coastal waters of New Zealand, Chile, Denmark and United States. Dinophysis caudata was rarely found and it was confined to the north of the sampling area. Because of low cell densities, neither D. caudata nor Dinophysis norvegica could be biogeographically related to lipophilic toxins in this study. Nevertheless, the current identification of D. norvegica in the southern Argentine Sea is the first record for the southwestern Atlantic Ocean. Given the typical toxigenicity of this species on a global scale, this represents an important finding for future surveillance of plankton-toxin associations.     

Co-occurrence of Dinophysis tripos and pectenotoxins in Argentinean shelf waters

The species Dinophysis tripos is a widely distributed marine dinoflagellate associated with diarrheic shellfish poisoning (DSP) events, which has been recently identified as a pectenotoxin (PTX) producer. In two sampling expeditions carried out during austral autumns 2012 and 2013 along the Argentine Sea (≈38–56° S), lipophilic phycotoxins were measured by tandem mass spectrometry coupled to liquid chromatography (LC–MS/MS) in size-fractionated plankton samples together with microscopic analyses of potentially toxic phytoplankton. PTX-2, PTX-11 and PTX-2sa were recurrently detected in the 50–200 μm fractions, in association to D. tripos. PTX-2 was also widely distributed among the 20–50 μm fractions, mostly related to Dinophysis acuminata. Okadaic acid or its analogs were not detected in any sample. This is the first report of D. tripos related to PTX in the Argentine Sea and the first record of PTX-11 and PTX-2sa for this area. The morphological variability of D. tripos, including the presence of intermediate, small and dimorphic cells, is described. Also, the micro- and mesoplanktonic potential grazers of Dinophysis spp. were explored.     

The emergence of Dinophysis acuminata blooms and DSP toxins in shellfish in New York waters

The dynamics of Dinophysis acuminata and its associated diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX1) as well as pectenotoxins (PTXs), were investigated within plankton and shellfish in Northport Bay, NY, USA, over a four year period (2008–2011). Over the course of the study, Dinophysis bloom densities ranged from ~10⁴ to 10⁶ cells L⁻¹ and exceeded 10⁶ L⁻¹ in 2011 when levels of total OA, total DTX1, and PTX in the water column were 188, 86, and 2900 pg mL⁻¹, respectively, with the majority of the DSP toxins present as esters. These cell densities exceed – by two orders of magnitude – those previously reported within thousands of samples collected from NY waters from 1971 to 1986. The bloom species was positively identified as D. acuminata via scanning electron microscopy and genetic sequencing (cox1 gene). The cox1 gene sequence from the D. acuminata populations in Northport Bay was 100% identical to D. acuminata from Narragansett Bay, RI, USA and formed a strongly supported phylogenetic cluster (posterior probability = 1) that included D. acuminata and Dinophysis ovum from systems along the North Atlantic Ocean. Shellfish collected from Northport Bay during the 2011 bloom had DSP toxin levels (1245 ng g⁻¹ total OA congeners) far exceeding the USFDA action level (160 ng g⁻¹ total OA of shellfish tissue) representing the first such occurrence on the East Coast of the U.S. D. acuminata blooms co-occurred with paralytic shellfish poisoning (PSP) causing blooms of Alexandrium fundyense during late spring each year of the study. D. acuminata cell abundances were significantly correlated with levels of total phytoplankton biomass and Mesodinium spp., suggesting food web interactions may influence the dynamics of these blooms. Given that little is known regarding the combined effects of DSP and PSP toxins on human health and the concurrent accumulation and depuration of these toxins in shellfish, these blooms represent a novel managerial challenge.     

A survey of Dinophysis spp. and their potential to cause diarrhetic shellfish poisoning in coastal waters of the United States

Multiple species of the genus Dinophysis produce diarrhetic shellfish toxins (okadaic acid and Dinophysis toxins, OA/DTXs analogs) and/or pectenotoxins (PTXs). Only since 2008 have DSP events (illnesses and/or shellfish harvesting closures) become recognized as a threat to human health in the United States. This study characterized 20 strains representing five species of Dinophysis spp. isolated from three US coastal regions that have experienced DSP events: the Northeast/Mid-Atlantic, the Gulf of Mexico, and the Pacific Northwest. Using a combination of morphometric and DNA-based evidence, seven Northeast/Mid-Atlantic isolates and four Pacific Northwest isolates were classified as D. acuminata, a total of four isolates from two coasts were classified as D. norvegica, two isolates from the Pacific Northwest coast were identified as D. fortii, and three isolates from the Gulf of Mexico were identified as D. ovum and D. caudata. Toxin profiles of D. acuminata and D. norvegica varied by their geographical origin within the United States. Cross-regional comparison of toxin profiles was not possible with the other three species; however, within each region, distinct species-conserved profiles for isolates of D. fortii, D. ovum, and D. caudata were observed. Historical and recent data from various State and Tribal monitoring programs were compiled and compared, including maximum recorded cell abundances of Dinophysis spp., maximum concentrations of OA/DTXs recorded in commercial shellfish species, and durations of harvesting closures, to provide perspective regarding potential for DSP impacts to regional public health and shellfish industry.     

Nitrogenous Nutrients Promote the Growth and Toxicity of Dinophysis acuminata during Estuarine Bloom Events

Diarrhetic Shellfish Poisoning (DSP) is a globally significant human health syndrome most commonly caused by dinoflagellates within the genus Dinophysis. While blooms of harmful algae have frequently been linked to excessive nutrient loading, Dinophysis is a mixotrophic alga whose growth is typically associated with prey availability. Consequently, field studies of Dinophysis and nutrients have been rare. Here, the temporal dynamics of Dinophysis acuminata blooms, DSP toxins, and nutrients (nitrate, ammonium, phosphate, silicate, organic compounds) were examined over four years within two New York estuaries (Meetinghouse Creek and Northport Bay). Further, changes in the abundance and toxicity of D. acuminata were assessed during a series of nutrient amendment experiments performed over a three year period. During the study, Dinophysis acuminata blooms exceeding one million cells L-1 were observed in both estuaries. Highly significant (p<0.001) forward stepwise multivariate regression models of ecosystem observations demonstrated that D. acuminata abundances were positively dependent on multiple environmental parameters including ammonium (p = 0.007) while cellular toxin content was positively dependent on ammonium (p = 0.002) but negatively dependent on nitrate (p<0.001). Nitrogen- (N) and phosphorus- (P) containing inorganic and organic nutrients significantly enhanced D. acuminata densities in nearly all (13 of 14) experiments performed. Ammonium significantly increased cell densities in 10 of 11 experiments, while glutamine significantly enhanced cellular DSP content in 4 of 5 experiments examining this compound. Nutrients may have directly or indirectly enhanced D. acuminata abundances as densities of this mixotroph during experiments were significantly correlated with multiple members of the planktonic community (phytoflagellates and Mesodinium). Collectively, this study demonstrates that nutrient loading and more specifically N-loading promotes the growth and toxicity of D. acuminata populations in coastal zones.     

Seasonal distribution of species of the toxic dinoflagellate genus Dinophysis in Maizuru Bay (Japan), with comments on their autofluorescence and attachment of picophytoplankton

The seasonal distribution of the dinoflagellate genus, Dinophysis, in Maizuru Bay, Japan, was investigated from May 1997 to December 1999. Seven species of Dinophysis were detected, including the toxic species of Dinophysis acuminata and D. fortii. The most dominant species wasD. acuminata, detected year-around and more abundantly during periods when water temperatures were between 15 and 18 °C. No relationship was found between cell abundance of Dinophysis spp. and concentrations of dissolved inorganic nutrients. Phycoerythrin containing nano- and picophytoplankton (cryptophytes and cyanobacteria), suspected to be prey of mixotrophic Dinophysis, were enumerated simultaneously. A clear relationship was not found among the cell abundances of Dinophysis spp. and nano- and picophytoplankton. Autofluorescence of Dinophysis spp. (mainly D. acuminata and D. fortii) under blue-light excitation was usually of a yellow-orange color. Occasionally, Dinophysis spp. had red autofluorescencing and yellow-orange autofluorescencing particles. The proportion of cells possessing red autofluorescence tended to be higher in the warm season. Numerous coccoid cells of picophytoplankton (ca. 1–2 μm in diameter) were found attached to the cell surface of D. acuminata, D. fortii, etc. and food vacuole-like structures also observed. These observations suggest there is a close relationship between mixotrophic Dinophysis spp. and certain picophytoplankton. Based on our observations, the possibility that the picophytoplankton found to be attached onto Dinophysis cell surfaces are a food source for Dinophysis, and a source of DSP toxins, is discussed.     

Oxidative metabolism by Thalassiosira weissflogii (Bacillariophyceae) of a diol-ester of okadaic acid, the diarrhetic shellfish poisoning

Previous investigations into the comparative toxicity of the diarrhetic shellfish poisoning (DSP) toxins to Thalassiosira weissflogii (Grun.) Fryxell et Hasle found that this diatom oxidatively metabolized okadaic acid diol‐ester (OA diol‐ester) to a more water‐soluble product. This oxidative transformation of OA diol‐ester by the diatom is significant for two reasons. First, it is known that dinophysistoxin‐4 (DTX‐4), the primary DSP toxin produced by the dinoflagellate Exuviaella lima (Ehr.) Butschli, will be hydrolyzed to the diol‐ester following cell rupture (e.g. ingestion by a predator). Second, it implies that the ester, an uncharged, lipophilic intermediate, can easily enter cells and therefore may play an important role in the uptake and transfer of DSP toxins through the food web. It has been suggested that the water soluble DTX‐4 may also be the form in which DSP toxins are excreted from the producing cell. Therefore, the stability of DTX‐4 was examined when incubated either in fresh seawater medium into which washed cells of E. lima were introduced or in seawater medium conditioned by E. lima cells. Rapid hydrolysis of DTX‐4 to the diol‐ester took place in both cases. Thus, regardless of the route by which DTX‐4 is liberated from the cell, either by cell disruption or excretion, the diol‐ester will be the dominant form of the toxin to challenge associated organisms. To examine the metabolism of OA diol‐ester by T. weissflogii in more detail, serial cultures of the diatom were challenged with OA diol‐ester at a concentration of 2.0 μg·mL^?1. The metabolism and fate of the diol‐ester in both cellular and medium fractions were monitored over 3 days using liquid chromatography with either ultraviolet (LC‐UV) or mass spectrometric (LC‐MS) detection. During the course of the experiment, all of the diol‐ester was metabolized. LC‐MS analysis revealed the presence of multiple oxidative products of OA diol‐ester in the medium fraction, including a carboxylic acid derivative. The major metabolites were isolated in sufficient quantity to permit structural elucidation by NMR and MS. All the metabolites identified resulted from oxidation of the diol‐ester side chain with the primary sites of attack at the terminal, subterminal, and unsaturated carbons. OA was found in both cellular and medium fractions, and its production was directly correlated with the metabolism of the diol‐ester. The relative partitioning of both OA diol‐ester and its oxidation products between cells and medium supports the contention that OA diol‐ester can readily enter cells, be metabolized, and then excreted in more water‐soluble forms.     

Occurrence of okadaic acid,a major diarrheic shellfish toxin,in natural populations ofDinophysis spp. from the eastern coast of North America

Okadaic acid, one of the principal toxin components implicated in cases of diarrheic shellfish poisoning (DSP), was identified for the first time in natural phytoplankton assemblages from North American waters. During periods in late summer when significant quantities of okadaic acid were detected in net haul samples in the lower estuary and Gulf of St Lawrence in eastern Canada, the phytoplankton community consistently contained species of the dinoflagellate genusDinophysis Ehrenberg. The presence of okadaic acid was detected by screening dinoflagellate extracts with an enzyme-linked immunological assay (ELISA); positive results were confirmed by reverse-phase high-performance liquid chromatography (HPLC) separation, followed by fluorescence detection. Okadaic acid was only found in phytoplankton samples in which the photosynthetic dinophysoid speciesD. norvegica andD. acuminata were prominent; blooms of the related heterotrophic speciesD. rotundata exhibited no trace of okadaic acid, nor other suspected DSP components.     

Production of dinophysistoxin-1 and pectenotoxin-2 by a culture of Dinophysis acuminata (Dinophyceae)

The production of diarrhetic shellfish poisoning toxins (okadaic acid analogues and other lipophilic toxins) by a culture of Dinophysis acuminata, fed with the autotrophic ciliate Myrionecta rubra, was confirmed by LC–MS analysis, and the toxin profile compared with that in the field assemblage of the same species. The growth response of D. acuminata to the density of the food organism was also examined in laboratory experiments. In semi-continuous culture experiments, the growth rates of D. acuminata increased with increasing density of M. rubra and a maximum growth rate of 0.67 per day was calculated. In batch culture experiments; the cellular content of PTX2 and DTX1 were 14.7–14.8 and 2.5–4.8 pg cell^?1, respectively. Okadaic acid, dinophysistoxin-3, pectenotoxin-1, pectenotoxin-6, yessotoxin (YTX) and 45-OHYTX were not detected. PTX2 was detected (cellular toxin content: 22 pg cell^?1), but DTX1 was not detected, in an extract of D. acuminata collected from natural seawater at the same location where the cultured D. acuminata specimens were isolated. These results strongly suggest that D. acuminata produces these toxins during cell growth and that environmental factors influence variations in the toxin composition and specific cellular toxicity.     

DIFFERENCES IN THE PRODUCTION AND EXCRETION KINETICS OF OKADAIC ACID,DINOPHYSISTOXIN‐1, AND PECTENOTOXIN‐2 BETWEEN CULTURES OF DINOPHYSIS ACUMINATA AND DINOPHYSIS FORTII ISOLATED FROM WESTERN JAPAN1

We established clonal cultures of Dinophysis acuminata Clap. et Lachm. and D. fortii Pavill. isolated from western Japan and examined toxin production in them, focusing on intracellular production and extracellular excretion. At the end of incubations, the total amounts of pectenotoxin‐2 (PTX‐2), dinophysistoxin‐1 (DTX‐1), and okadaic acid (OA) in the D. acuminata cultures reached up to 672.7 ± 14.7 (mean ± SD), 88.1 ± 2.8, and 539.3 ± 39.7 ng · mL^?1, respectively, and the excreted extracellular amounts were equivalent to 5.1, 79.5, and 79.5% of the total amounts, respectively. Similarly, at the end of incubations, the total amounts of PTX‐2, DTX‐1, and OA in the D. fortii cultures reached up to 526.6 ± 52.6 (mean ±SD), 4.4 ± 0.4, and 135.9 ± 3.9 ng · mL^?1, respectively, and the excreted extracellular amounts were equivalent to 1.8, 80.1, and 86.6% of the total amounts, respectively. Further, we tested the availability of cell debris and dissolved organic substances that originated from the ciliate prey Myrionecta rubra for growth and toxin production in D. acuminata. Although no significant growth was observed in D. acuminata in the medium containing the cell debris and organic substances originated from M. rubra, the toxicity was significantly greater than that in the control (P < 0.05–0.001); this finding suggested the availability of organic substances for toxin production. However, toxin productivity was remarkably lower than that of Dinophysis species feeding on living M. rubra.     

Toxin production,retention, and extracellular release by Dinophysis acuminata during extended stationary phase and culture decline

Dinophysis spp. produce diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins. The extent to which the dinoflagellate cells retain their toxicity in stationary phase, a period when cells are most toxic, and their transition into cell death is not known. Here we present results on the production, recycling, retention, and release of toxins from a monoculture of Dinophysis acuminata during these two important stages. Once stationary phase was reached, cultures were divided between light and dark treatments to identify if light influenced toxin dynamics. Light was required for long-term cell maintenance (>2 months) of D. acuminata in the absence of prey, however, in the dark, cells in stationary phase survived on reserves alone for four weeks before beginning to decline. Cells maintained relatively constant levels of intracellular OA (0.39 ± 0.03 pg/cell, 0.44 ± 0.05 pg/cell), DTX1 (0.45 ± 0.09 pg/cell, 0.64 ± 0.10 pg/cell) and PTX2 (10.4 ± 1.4 pg/cell, 11.0 ± 1.9 pg/cell) in the dark and light treatments, respectively, throughout stationary phase and into culture decline. Toxin production was only apparent during late exponential and early stationary growth when cells were actively dividing. In general, the concentration of dissolved (extracellular) toxin in the medium significantly increased upon culture aging and decline; cells did not appear to be actively or passively releasing toxin during stationary phase, but rather extracellular release was likely a result of cell death. Light availability did not have an apparent effect on toxin production, quotas, or intracellular vs. extracellular distribution. Together these results suggest that a bloom of D. acuminata would retain its cellular toxicity or potency as long as the population is viable, and that cells under conditions of low light (e.g., at the boundary or below euphotic zone) and/or minimal prey could maintain toxicity for extended periods.     

Effects of small-scale turbulence on two species of Dinophysis

Dinoflagellate species of Dinophysis, in particular D. acuminata and D. acuta, produce lipophilic toxins that pose a threat to human health when concentrated in shellfish and jeopardize shellfish exploitations in western Europe. In northwestern Iberia, D. acuminata has a long growing season, from spring to early autumn, and populations develop as soon as shallow stratification forms when the upwelling season begins. In contrast, D. acuta blooms in late summer, when the depth of the pycnocline is maximal and upwelling pulses are moderate. In situ observations on the hydrodynamic regimes during the two windows of opportunity for Dinophysis species led us to hypothesize that D. acuta should be more sensitive to turbulence than D. acuminata.To test this hypothesis, we studied the response of D. acuminata and D. acuta to three realistic turbulence levels low (LT), ε ≈ 10⁻⁶ m² s^-3; medium (MT), ε ≈ 10^-5 m² s^-3 and high (HT), ε ≈ 10^-4 m² s^-3 generated by Turbogen, a highly reproducible, computer-controlled system. Cells of both species exposed to LT and MT grew at rates similar to the controls. Marked differences were found in the response to HT: D. acuminata grew slowly after an initial lag phase, whereas D. acuta cell numbers declined. Results from this study support the hypothesis that turbulence may play a role in shaping the spatio-temporal distribution of individual species of Dinophysis. We also hypothesize that, in addition to cell disturbance affecting division, sustained high shear generated by microturbulence may cause a decline in Dinophysis numbers due to decreased densities of ciliate prey.     

LC–MS/MS analysis of okadaic acid analogues and other lipophilic toxins in single-cell isolates of several Dinophysis species collected in Hokkaido,Japan

Quantification of diarrhetic shellfish poisoning (DSP) toxins (okadaic acid analogues), and other lipophilic toxins in single-cell isolates of the dinoflagellates Dinophysis fortii, D. acuminata, D. mitra, D. norvegica, D. tripos, D. infundibulus and D. rotundata, collected in coastal waters Hokkaido, Japan in 2005, was carried out by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Okadaic acid (OA), dinophysistoxin-1 (DTX1), 7-O-palmitoyldinophysistoxin-1 (DTX3), pectenotoxin-1 (PTX1), pectenotoxin-11 (PTX11), pectenotoxin-2 (PTX2), pectenotoxin-6 (PTX6), pectenotoxin-2 seco-acid (PTX2sa), yessotoxin (YTX) and 45-hydroxyyessotoxin (45-OHYTX) were quantified by LC–MS/MS. PTX2 was the dominant toxin in D. acuminata, D. norvegica and D. infundibulus whereas both DTX1 and PTX2 were the principal toxins in D. fortii. None of the toxins were detected in D. mitra, D. rotundata and D. tripos. These results suggest that D. fortii is the most important species responsible for DSP contamination of bivalves in Hokkaido. This is the first finding of PTX2 in D. infundibulus, and confirms the presence of PTX2 in Japanese D. acuminata and D. norvegica collected from natural seawater.     

Diversity and plastid types in Dinophysis acuminata complex (Dinophyceae) in Scottish waters

Dinophysis acuminata produces lipophilic shellfish toxins (LSTs) that have economic and ecological impact on marine invertebrates in NE Atlantic where aquaculture farming is prevalent. Identification of D. acuminata can be complex. Cells exhibit a variety of morphotypes that overlap between species making identification using routine light microscopy difficult. These cells are mixotrophic and their population size is influenced by hydrographic conditions and prey populations. Dinophysis cells are able to acquire and temporarily keep prey plastids from a variety of photosynthetic unicellular sources. The Dinophysis community in Scottish waters tend to be dominated by cells with morphologies that appear to be variants of D. acuminata/norvegica complex particularly during late spring/early summer. To determine the identity of these morphotypes, DNA barcoding was performed on 32 single cell isolates from sites around the Scottish coast using the ribosomal internal transcribed spacer 1 (ITS1) and a partial cytochrome oxidase I (COI) fragment on the same single cells. Although the cells exhibited a variety of morphotypes, most were restricted to one cluster containing D. acuminata and three grouped with Dinophysis ovum. This is the first molecular confirmation of the presence of D. ovum in Scottish waters. Two isolates showed considerable divergence – one was unidentifiable from the public databases, whilst the other matched a Dinophysis cf. acuta isolate from Canada. To investigate prey plastids, molecular analysis of these Dinophysis single cells was conducted with a partial fragment of the plastid ribosomal marker (16S). Most cells harboured plastids from the cryptophyte Teleaulax – the most commonly reported plastid type, however one cell harboured a Rhodomonas/Storeatula derived plastid. This finding increases the range and variety of cryptophyte plastids found in Dinophysis and increases the range of prey-types.