Dinoflagellate Dinophysis Acuta Research Articles

From lipophilic to hydrophilic toxin producers: Phytoplankton succession driven by an atmospheric river in western Patagonia

Phytoplankton succession is related to hydroclimatic conditions. In this study we provide the first description of a toxic phytoplankton succession in the Patagonian Fjord System. The shift was modulated by atmospheric-oceanographic forcing and consisted of the replacement of the marine dinoflagellate Dinophysis acuta in a highly stratified water column during austral summer by the diatom Pseudo-nitzschia calliantha in a mixed water column during late summer and early autumn. This transition, accompanied by a change in the biotoxin profiles (from lipophilic dinophysis toxins to hydrophilic domoic acid), was induced by the arrival of an intense atmospheric river. The winds in Magdalena Sound may have been further amplified, due to its west-east orientation and its location within a tall, narrow mountain canyon. This work also documents the first known appearance of toxic P. calliantha in Northern Patagonian. The potential impacts of the biotoxins of this species on higher trophic levels are discussed.

Effects of the DSP-toxic dinoflagellate Dinophysis acuta on clearance and respiration rate of the blue mussel, Mytilus edulis.

Diarrheic Shellfish Poisoning toxins (DST) are a severe health risk to shellfish consumers and can be a major problem for the shellfish industry. Bivalve molluscs can accumulate DST via ingestion of toxic dinoflagellates like Dinophysis spp., which are the most prominent producers of DST. The effects of DST-containing dinoflagellate Dinophysis acuta on bivalve clearance and respiration rate were investigated in the blue mussel (Mytilus edulis) exposed to different algal densities in a controlled laboratory study. Results showed that M. edulis exposed to D. acuta displayed a reduced clearance rate compared to M. edulis exposed to equivalent bio-volumes of the non-toxic cryptophyte Rhodomonas salina. Furthermore, M. edulis ceased to feed on D. acuta after 1 to 4 h, depending on D. acuta densities. The quickest response was observed at the highest densities of D. acuta. The estimated total amount of DST accumulated in the M. edulis exceeded the regulatory limit for human consumption and furthermore, intoxication of the M. edulis seemed to occur faster at high cell toxicity rather than at high cell density. However, respiration rates were, similar, irrespective of whether M. edulis were fed single diets of R. salina, D. acuta or a mixed diet of both algal species. In conclusion, the DST-containing D. acuta had a severe negative effect on the clearance of M. edulis, which can affect the conditions of the M. edulis negatively. Hence, DST may cause low quality M. edulis, due to reduced feeding when exposed to DST-containing D. acuta.

Inter-species variability of okadaic acid group toxicity in relation to the content of fatty acids detected in different marine vectors

ABSTRACTOkadaic acid group (OA-group) is a set of lipophilic toxins which are characterised by being produced by species associated with the genera Dinophysis and Prorocentrum. OA-group has been regularly detected in endemic shellfish species from the southern zone of Chile only through the mouse bioassay. The purpose of this work was to determine the variability of OA-group toxins in endemic aquatic organisms (bivalves, crabs, gastropods and fish) and to establish the relationship with the concentration of fatty acids (FAs) detected in the evaluated species. The toxicity of OA-group and the FA profiles were determined using LC-MS/MS and gas chromatography with flame-ionisation detection, respectively. In the study area, the dinoflagellate Dinophysis acuta was detected in densities ≈2000 cells ml−1 with a toxicity ≈18.3 pg OA equiv cel−1. The analysis identified OA and dinophysistoxin-1 in shellfish in a range of ≈90 to ≈225 μg OA eq kg−1, where no toxins in fish were detected. A positive relationship between the FA level and the concentration of OA-group toxins in the digestive glands of bivalves and gastropods was established, noted for high levels of saturated FAs (C14:0 and C16:0). The toxic variability of OA-group toxins determined in the different species allowed us to establish that the consumption of these vectors, regulated by non-analytical methods, can be harmful when consumed by humans, thus suggesting that the sanitary regulations for the control of OA-group in Chile should be updated.

Environmental control of harmful dinoflagellates and diatoms in a fjordic system

Fjordic coastlines provide an ideal protected environment for both finfish and shellfish aquaculture operations. This study reports the results of a cruise to the Scottish Clyde Sea, and associated fjordic sea lochs, that coincided with blooms of the diarrhetic shellfish toxin producing dinoflagellate Dinophysis acuta and the diatom genus Chaetoceros, that can generate finfish mortalities. Unusually, D. acuta reached one order of magnitude higher cell abundance in the water column (2840cellsL−1) than the more common Dinophysis acuminata (200cellsL−1) and was linked with elevated shellfish toxicity (maximum 601±237μg OA eq/kg shellfish flesh) which caused shellfish harvesting closures in the region. Significant correlations between D. acuta abundance and that of Mesodinium rubrum were also observed across the cruise transect potentially supporting bloom formation of the mixotrophic D. acuta. Significant spatial variability in phytoplankton that was related to physical characteristics of the water column was observed, with a temperature-driven frontal region at the mouth of Loch Fyne being important in the development of the D. acuta, but not the Chaetoceros bloom. The front also provided important protection to the aquaculture located within the loch, with neither of the blooms encroaching within it. Analysis based on a particle-tracking model confirms the importance of the front to cell transport and shows significant inter-annual differences in advection within the region, that are important to the harmful algal bloom risk therein.

The use of a mucus trap by Dinophysis acuta for the capture of Mesodinium rubrum prey under culture conditions

A capture mechanism observed in a culture of the dinoflagellate Dinophysis acuta when preying on the ciliate Mesodinium rubrum (also sometimes referred to as Myrionecta rubra) is described. The dinoflagellate released cohesive clumps of mucilage into the culture media. When M. rubrum cells came into contact with this mucilage, they were immediately immobilized, but remained alive for a short period of time. Observations of D. acuta cells ‘visiting and probing’ trapped M. rubrum cells were made and at a critical point D. acuta cells removed individual M. rubrum cells from the mucus to swim away with them. The removal of M. rubrum from the mucus coincided with the cells losing all their cilia and becoming swollen, presumably signifying the death of the cell. These changes may enable the D. acuta peduncle to penetrate the ciliate cell cortex. It is hypothesized that toxins produced by D. acuta play a role in the immobilization process within the mucilage trap.

Photoregulation in a Kleptochloroplastidic Dinoflagellate, Dinophysis acuta.

Some phagotrophic organisms can retain chloroplasts of their photosynthetic prey as so-called kleptochloroplasts and maintain their function for shorter or longer periods of time. Here we show for the first time that the dinoflagellate Dinophysis acuta takes control over “third-hand” chloroplasts obtained from its ciliate prey Mesodinium spp. that originally ingested the cryptophyte chloroplasts. With its kleptochloroplasts, D. acuta can synthesize photosynthetic as well as photoprotective pigments under long-term starvation in the light. Variable chlorophyll fluorescence measurements showed that the kleptochloroplasts were fully functional during 1 month of prey starvation, while the chlorophyll a-specific inorganic carbon uptake decreased within days of prey starvation under an irradiance of 100 μmol photons m-2 s-1. While D. acuta cells can regulate their pigmentation and function of kleptochloroplasts they apparently lose the ability to maintain high inorganic carbon fixation rates.

Accumulation, transformation and breakdown of DSP toxins from the toxic dinoflagellate Dinophysis acuta in blue mussels, Mytilus edulis

Okadaic acid (OA), dinophysistoxins (DTX) and pectenotoxins (PTX) produced by the dinoflagellates Dinophysis spp. can accumulate in shellfish and cause diarrhetic shellfish poisoning upon human consumption. Shellfish toxicity is a result of algal abundance and toxicity as well as accumulation and depuration kinetics in mussels. We mass-cultured Dinophysis acuta containing OA, DTX-1b and PTX-2 and fed it to the blue mussel, Mytilus edulis under controlled laboratory conditions for a week to study toxin accumulation and transformation. Contents of OA and DTX-1b in mussels increased linearly with incubation time, and the net toxin accumulation was 66% and 71% for OA and DTX-1b, respectively. Large proportions (≈50%) of both these toxins were transformed to fatty acid esters. Most PTX-2 was transformed to PTX-2 seco-acid and net accumulation was initially high, but decreased progressively throughout the experiment, likely due to esterification and loss of detectability. We also quantified depuration during the subsequent four days and found half-life times of 5–6 days for OA and DTX-1b. Measurements of dissolved toxins revealed that depuration was achieved through excreting rather than metabolizing toxins. This is the first study to construct a full mass balance of DSP toxins during both accumulation and depuration, and we demonstrate rapid toxin accumulation in mussels at realistic in situ levels of Dinophysis. Applying the observed accumulation and depuration kinetics, we model mussel toxicity, and demonstrate that a concentration of only 75 Dinophysis cells l−1 is enough to make 60 mm long mussels exceed the regulatory threshold for OA equivalents.

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.

Isolation and identification of pectenotoxins-13 and -14 from Dinophysis acuta in New Zealand

Two novel pectenotoxins (PTXs), PTX-13 and -14, were isolated from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compounds were identified as oxidized analogues of PTX-2 by NMR spectroscopic and LC-MS studies. PTX-13 (32 R-hydroxyPTX-2) corresponds to the unidentified analogue PTX-11x reported by [Suzuki et al., 2003. Liquid chromatography-mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analysis of novel pectenotoxin isomers in the toxic dinoflagellate Dinophysis acuta from New Zealand. J. Chromatogr. A 992, 141–150]. PTX-13 underwent slow deuteration at the 13β-position during NMR analysis. PTX-14 corresponds to the 32,36-dehydration product of PTX-13, and may be an artifact.

Identification of Pectenotoxin-11 as 34S-Hydroxypectenotoxin-2, a New Pectenotoxin Analogue in the Toxic Dinoflagellate Dinophysis acuta from New Zealand

A new pectenotoxin, which has been named pectenotoxin-11 (PTX11), was isolated from the dinoflagellate Dinophysis acuta collected from the west coast of New Zealand. The structure of PTX11 was determined as 34S-hydroxypectenotoxin-2 by tandem mass spectrometry and UV and NMR spectroscopy. PTX11 appears to be only the third pectenotoxin identified as a natural biosynthetic product from algae after pectenotoxin-2 and pectenotoxin-12. The LD50 of PTX11 determined by mouse intraperitoneal injection was 244 microg/kg. The LD(min) of PTX11 in these experiments was 250 microg/kg. No signs of toxicity were recorded in mice following an oral dose of PTX11 at 5000 microg/kg. No diarrhea was observed in any of the animals administered with the test substance by either route of administration. Unlike pectenotoxin-2 (PTX2), PTX11 was not readily hydrolyzed to its corresponding seco acid by enzymes from homogenized green-lipped mussel (Perna canaliculus) hepatopancreas.

Discovery of okadaic acid esters in the toxic dinoflagellate Dinophysis acuta from New Zealand using liquid chromatography/tandem mass spectrometry.

The dinoflagellate Dinophysis acuta has been associated with various incidents of diarrhetic shellfish poisoning. A sample of Dinophysis acuta collected from New Zealand waters in 2002 was previously found to contain high levels of pectenotoxins, but only a very low level of the diarrhea-inducing okadaic acid (OA). After hydrolysis under basic conditions, however, the concentration of OA increased substantially, indicating the presence of conjugated forms of OA. Using various liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) techniques, a number of OA esters were detected in the original extract. The principal compound was identified as a C8 diol-ester of OA (OA-D8), which had been identified previously in another dinoflagellate, Prorocentrum lima. The retention time, as well as positive and negative ion MS, MS/MS and UV spectra of the D. acuta compound, matched exactly those of OA-D8 isolated from P. lima. In addition to OA-D8, several other novel OA esters were detected in the D. acuta but these have not yet been identified. This is the first report identifying the presence of OA esters in Dinophysis species.

Liquid chromatography–mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analysis of novel pectenotoxin isomers in the toxic dinoflagellate Dinophysis acuta from New Zealand

The acid-catalyzed inter-conversion of spiroketal isomers of pectenotoxins PTX1, PTX6 and PTX2 were studied by liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–MS–MS). Using a C 8-silica reversed-phase column and a mobile phase of aqueous acetonitrile containing 2 m M ammonium formate and 50 m M formic acid, the known spiroketal stereoisomers of PTX1 eluted in order of PTX1, PTX4 and PTX8, while those of PTX6 eluted in the order PTX6, PTX7 and PTX9. Acid treatment of PTX2 yielded two novel spiroketal stereoisomers, which have been named PTX2b and PTX2c. LC–MS–MS spectra obtained for the [M+NH 4] + ions of PTX2, PTX2b and PTX2c were essentially identical. As an application of the LC–MS–MS methodology, a sample of the toxic dinoflagellate Dinophysis acuta collected from the coast of New Zealand was analyzed for pectenotoxins. PTX2 and a new pectenotoxin, which has been named PTX11, were detected as the most predominant compounds. Novel PTX2 and PTX11 isomers were also found in the D. acuta although the levels of these compounds were low.

Esterification of DSP toxins by Portuguese bivalves from the Northwest coast determined by LC-MS—a widespread phenomenon

Okadaic acid (OA) and dinophysistoxin-2 (DTX2) were confirmed by liquid chromatography with mass spectrometry detection both in extracts of digestive glands and edible parts of Portuguese shellfish. No dinophysistoxin-1 was found even in highly contaminated samples examined. However, only in blue mussel ( Mytilus edulis) were these two parent toxins commonly found in a free form. Usually they were found largely esterified in all remaining shellfish species—common cockle ( Cerastoderma edule), peppery furrow shell ( Scrobicularia plana), carpet shell ( Venerupis pullastra), oyster ( Crassostrea japonica), razor clam ( Ensisspp.), and clam ( Ruditapes decussata). Oysters were the least toxic. In mussels esterified OA did not surpass 50% of the total OA found in edible parts, while DTX2 esterification rates were usually much lower. In remaining shellfish species usually more than 95% of the total OA was found esterified, while free DTX2 was rarely found. Also ratios of total DTX2/total OA were higher in mussels than in the remaining species examined. From all these species commercially exploited at the northern coast, mussels and cockles contained the highest levels of DSP toxins, thus representing the highest theoretical health risk. The previous association of DTX2 with the dinoflagellate Dinophysis acuta was confirmed with selective MS detection; while OA was the only parent diarrhoeic toxin found associated with Dinophysis acuminata.

Conversion of pectenotoxin-2 to pectenotoxin-2 seco acid in the New Zealand scallop, Pecten novaezelandiae

SUMMARY: Comparison of pectenotoxin (PTX) profiles between the toxic dinoflagellate Dinophysis acuta and scallops Pecten novaezelandiae collected at Wedge Point, Queen Charlotte Sound, New Zealand was carried out by liquid chromatography–mass spectrometry (LC-MS) with turbo-ionspray ionization. Although the major PTX homolog in D. acuta was pectenotoxin-2 (PTX2), the scallops contained pectenotoxin-2 seco acid (PTX2SA) as the predominant toxin. Pectenotoxin-2 isolated from D. acuta was rapidly converted to PTX2SA and its epimer 7-epi-pectenotoxin-2 seco acid (7-epi-PTX2SA) in the scallop extracts. These results indicate that PTX2SA and 7-epi-PTX2SA arose from the conversion of PTX2 by scallop tissue. The results indicate that New Zealand scallops have an ability to reduce the cytotoxicity of PTX2 by conversion to PTX2SA.

Pectenotoxin-2 seco acid: a toxin converted from pectenotoxin-2 by the New Zealand Greenshell mussel, Perna canaliculus

Comparison of pectenotoxin (PTX) profiles of toxic dinoflagellate Dinophysis acuta, Greenshell mussels ( Perna canaliculus) and Blue mussels ( Mytilus galloprovincialis) collected from Wedge Point, Queen Charlotte Sound, New Zealand was carried out by liquid chromatography–mass spectrometry with turbo-ionspray ionization. Although the major PTX homologue in D. acuta was pectenotoxin-2 (PTX2), both Greenshell and Blue mussels contained pectenotoxin-2 seco acid (PTX2SA) as the predominant toxin. More than 90% of PTX2 isolated from D. acuta was rapidly converted to PTX2SA and its epimer 7- epi-pectenotoxin-2 seco acid (7- epi-PTX2SA) in the Greenshell mussel extracts. The conversion from PTX2 to PTX2SA and 7- epi-PTX2SA was not observed in phosphate buffers at various pH ranging from 4.1 to 9.1. These findings indicate that PTX2SA and 7- epi-PTX2SA are not artifact toxins resulting from hydrolysis of PTX2, but arise from the conversion of PTX2 by mussel tissues.

Structures of New Pectenotoxin Analogs, Pectenotoxin-2 Seco Acid and 7-epi-Pectenotoxin-2 Seco Acid, Isolated from a Dinoflagellate and Greenshell Mussels

Abstract Two new pectenotoxin analogs, pectenotoxin-2 seco acid (PTX2SA) and 7-epi-pectenotoxin-2 seco acid (7-epi-PTX2SA), were isolated from the dinoflagellate Dinophysis acuta and greenshell mussels, Perna canaliculus. Their structures were elucidated by NMR and negative ion FAB CID MS/MS experiments.