High performance liquid chromatography analysis of diarrhetic toxins inDinophysis spp. from the French coast

Diarrhetic shellfish poisoning has been a recurrent problem along the Brittany coast (France) since 1983. Okadaic acid (O.A.), the main toxin detected in mussels, is generally associated with the presence ofDinophysis cells in sea water. We report here the results of okadaic acid analyses by high performance liquid chromatography, on planktonic samples collected during the summer of 1991.D. sacculus, the majorDinophysis species, throughout this period, showed low okadaic acid content in raw extract, whereas toxic levels were 3- to 12-fold higher in sorted samples than in raw ones. A maximum O.A. concentration of 29.6 pg cell^-1 was found inD. sacculus/D. cf. acuminata extract. Similarly, higher O.A. levels were noted in raw samples when these two species were associated. Generally speaking, variations in raw samples were similar to those in sorted samples. Nevertheless, whenD. rotundata reached a concentration equal to that ofD. sacculus in sorted samples, the O.A. level was lower.     

Okadaic acid accumulation in macrofilter feeders subjected to natural blooms of Dinophysis acuminata

Thermaikos Gulf is a eutrophic area located in the Northwestern part of the Aegean Sea in the Eastern Mediterranean. Interspecific differences among various filter feeders in their ability to accumulate okadaic acid, were observed during natural blooms of Dinophysis acuminata in the gulf. Okadaic acid analyses by high performance liquid chromatography (HPLC) were performed on benthic specimens and D. acuminata cell densities and cell toxin content were estimated in water samples. Seven filter feeding species were collected in the gulf during two DSP outbreaks in May 2003 and March 2004. The various species showed a different potential to accumulate okadaic acid in their tissues. The highest concentrations were found in the mussel populations (Mytilus galloprovincialis and Modiolus barbatus), while among the non-bivalve filter feeders, ascidians were the main accumulators of okadaic acid. The rest of shellfish populations (Flexopecten proteus, Chlamys varia and Venus verrucosa) were found to contain toxins only during 2004, when D. acuminata densities were found above 10000 cells l⁻¹. M. galloprovincialis was proved to be the most appropriate indicator for a safe warning of okadaic acid contamination in Thermaikos Gulf.     

DSP toxin contents inDinophysis fortii and scallops collected at Mutsu Bay,Japan

Cell densities of toxic phytoplankton species responsible for diarrhetic shellfish poisoning (DSP) were monitored at a sampling site in Mutsu Bay, Japan, in 1995.Dinophysis fortii almost completely dominated the toxic phytoplankton community. Okadaic acid (OA) and dinophysistoxin-1 (DTX1) contents in bothD. fortii cells and midgut glands of scallops collected at the same sampling site were determined by HPLC — fluorometry. DTX1 was detected fromD. fortii and scallops. The contents of DTX1 inD. fortii changed markedly during the experimental periods (5–252 pg cell^–1). The highest concentration of DTX1 in the midgut glands of scallops coincided with the period of relatively high cell densities ofD. fortii with the highest content of DTX1 (252 pg cell^–1). The results demonstrate that toxin content in the cells is an important factor affecting the toxicity of shellfish.     

Determination of diarrhetic shellfish toxins in various dinoflagellate species

Sixteen species of unialgal samples of dinoflagellate, either wild or cultured, were tested for production of diarrhetic shellfish toxins such as okadaic acid (OA), dinophysistoxin-1 (DTX1), and pectenotoxins (PTXs). Determination of micro-quantities of the toxins was facilitated by fluorometry and UV HPLC. Seven Dinophysis species were confirmed to produce either OA or DTX1, or both. Toxin content and composition varied regionally and seasonally. Intraspecies variation was also observed among cultured strains of Prorocentrum lima. PTX2 was the only toxin detected among PTX family, and D. fortii was the only species to contain this toxin. author for correspondence     

Occurrence of Prorocentrum lima,a DSP toxin-producing species from the Atlantic coast of Canada

A species of Prorocentrum (Dinophyta, Prorocentrales), isolated from a phytoplankton net sample from the Atlantic coast of Nova Scotia, has been brought into unialgal culture. The sample was collected at an aquaculture site immediately following an incident of diarrhetic shellfish poisoning (DSP) due to the consumption of contaminated mussels. This clonal isolate has been identified as P. lima, based on its morphological characteristics. Analysis of the culture extract, using high performance liquid chromatography (HPLC) with fluorescence detection, indicated the presence of the DSP toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX-1).     

Epiphytic abundance and toxicity of Prorocentrum lima populations in the Fleet Lagoon, UK

Planktonic Dinophysis spp. and epiphytic Prorocentrum lima (Ehrenberg) Dodge are known dinoflagellate producers of okadaic acid (OA) and dinophysistoxins (DTX), causative phycotoxins of diarrhetic shellfish poisoning (DSP). Underestimation of toxic dinoflagellates associated with a toxic event may be due to the lack of sampling of species with epiphytic and epibenthic strategies, such as P. lima. As Dinophysis spp. is not found in the Fleet Lagoon, Dorset, but previous DSP events have closed the Crassostrea gigas oyster farm, P. lima is the most likely causative organism. A field assay for separating microalgal epiphytes and concentrating wild cells on to filters was successfully applied to sub-samples of a variety of macroalgae and macrophytes (seagrass) collected from the Fleet during summer 2002. P. lima was present in increasing cell densities on most substratum species, over the sampling period, from 10² to 10³ cells g⁻¹ fresh weight (FW) plant biomass. LC–MS analysis detected OA and DTX-1 in extracts of wild P. lima cells, in ratios characteristic of P. lima strains previously isolated from the Fleet. No toxins, however, were detected in oyster flesh.     

Parasitism as a biological control agent of dinoflagellate blooms in the California Current System

Amoebophrya is a marine parasite recently found to infect and kill bloom-forming dinoflagellates in the California Current System (CCS). However, it is unknown whether parasitism by Amoebophrya can control dinoflagellate blooms in major eastern boundary upwelling systems, such as the CCS. We quantified the abundance of a common bloom-forming species Akashiwo sanguinea and prevalence of its parasite (i.e., % infected cells) in surface water samples collected weekly from August 2005 to December 2008 at the Santa Cruz Wharf (SCW), Monterey Bay, CA. Additionally, we measured physical and chemical properties at the SCW and examined regional patterns of wind forcing and sea surface temperature. Relative abundance of the net phytoplankton species was also analyzed to discern whether or not parasitism influences net phytoplankton community composition. Epidemic infection outbreaks (>20% parasite prevalence in the host species) may have contributed to the end or prevented the occurrence of A. sanguinea blooms, whereas low parasite prevalence was associated with short-term (≤2 weeks) A. sanguinea blooms. The complete absence of parasitism in 2007 was associated with an extreme A. sanguinea bloom. Anomalously strong upwelling conditions were detected in 2007, suggesting that A. sanguinea was able to outgrow Amoebophrya and ‘escape’ parasitism. We conclude that parasitism can strongly influence dinoflagellate bloom dynamics in upwelling systems. Moreover, Amoebophrya may indirectly influence net phytoplankton species composition, as species that dominated the net phytoplankton and developed algal blooms never appeared to be infected.     

A procedure to estimate okadaic acid in whole dinoflagellate cells using immunological techniques

A single procedure to detect and estimate okadaic acid in isolated whole cells was developed based on immunofluorescence and microscope photometry. This procedure allows the study of variations in okadaic acid concentration per cell although it is no substitute for HPLC procedures. Cells from mid-log exponential and stationary phase from two different clonal cultures of the okadaic-acid-producing dinoflagellate Prorocentrum lima (PI 5V and PI 7V) were analyzed. The results showed that: (1) cells from saturated phase cultures contain more okadaic acid than those from exponentially-growing mid-log phase; (2) genetic differences exist in okadaic acid production between the clones used; (3) okadaic acid is synthesized continuously during the whole cell cycle.     

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.     

Possible importance of algal toxins in the Salton Sea, California

In response to wildlife mortality including unexplained eared grebe (Podiceps nigricollis) die-off events in 1992 and 1994 and other mortality events including large fish kills, a survey was conducted for the presence of algal toxins in the Salton Sea. Goals of this survey were to determine if and when algal toxins are present in the Salton Sea and to describe the phytoplankton composition during those times. A total of 29 samples was collected for toxicity analysis from both nearshore and midlake sites visited biweekly from January to December 1999. Dinoflagellates and diatoms dominated most samples, but some were dominated by a prymnesiophyte (Pleurochrysis pseudoroscoffensis) or a raphidophyte (Chattonella marina). Several types of blooms were observed and sampled. The dinoflagellate Gyrodinium uncatenum formed an extensive, dense (up to 310000 cells ml^–1) and long-lasting bloom during the winter in 1999. A coccolithophorid, Pleurochrysis pseudoroscoffensis, occurred at high densities in surface films and nearshore areas during the spring and summer of 1999. These surface films also contained high densities of one or two other species (an unidentified scrippsielloid, Heterocapsa niei, Chattonella marina). Localized blooms were also observed in the Salton Sea. An unknown small dinoflagellate reached high densities (110000 cells ml^–1) inside Varner Harbor, and an unidentified species of Gymnodinium formed a dense (270000 cells ml^–1) band along part of the southern shoreline during the summer. Three species known to produce toxins in other systems were found. Protoceratium reticulatum (=Gonyaulax grindleyi) and Chattonella marina were found in several samples taken during summer months, and Prorocentrum minimum was found in low densities in several samples. Extracts of most samples, including those containing known toxic species, showed a low level (<10% mortality across all concentrations) of activity in the brine shrimp lethality assay and were not considered toxic. All sample extracts tested in the mouse bioassay showed no activity. One sample extract taken from the bloom of the small dinoflagellate was highly active (100% mortality across all concentrations) in the brine shrimp lethality assay, but the active material could not be isolated. While dense algal blooms are common at the Salton Sea, no evidence gathered in this study suggests that algal toxins are present within phytoplankton cells; however, toxins actively excreted by cells may have been missed. Blooms of phytoplankton likely contribute to wildlife mortality at the Salton Sea. Possible mechanisms including intoxication due to ingestion of feathers in grebes and waterlogging caused by changes in surface tension are discussed.     

Sulfated diesters of okadaic acid and DTX-1: Self-protective precursors of diarrhetic shellfish poisoning (DSP) toxins

Many toxic secondary metabolites used for defense are also toxic to the producing organism. One important way to circumvent toxicity is to store the toxin as an inactive precursor. Several sulfated diesters of the diarrhetic shellfish poisoning (DSP) toxin okadaic acid have been reported from cultures of various dinoflagellate species belonging to the genus Prorocentrum. It has been proposed that these sulfated diesters are a means of toxin storage within the dinoflagellate cell, and that a putative enzyme mediated two-step hydrolysis of sulfated diesters such as DTX-4 and DTX-5 initially leads to the formation of diol esters and ultimately to the release of free okadaic acid. However, only one diol ester and no sulfated diesters of DTX-1, a closely related DSP toxin, have been isolated leading some to speculate that this toxin is not stored as a sulfated diester and is processed by some other means. DSP components in organic extracts of two large scale Prorocentrum lima laboratory cultures have been investigated. In addition to the usual suite of okadaic acid esters, as well as the free acids okadaic acid and DTX-1, a group of corresponding diol- and sulfated diesters of both okadaic acid and DTX-1 have now been isolated and structurally characterized, confirming that both okadaic acid and DTX-1 are initially formed in the dinoflagellate cell as the non-toxic sulfated diesters.     

Phytoplankton composition of the Kandalaksha Gulf, Russian White Sea: Dinophysis and lipophilic toxins in the blue mussel (Mytilus edulis)

Dinophysis acuminata and D. norvegica were observed in plankton net samples during the summer of 2002 from the Kandalaksha Gulf in the White Sea (North European Russia). Prorocentrum lima was found as an epiphyte on subtidal macroalgae in August, but not observed in plankton net samples. Protein phosphatase 2A (PP2A) inhibition measured 127.8 ng OA-equivalent/g of mussel (Mytilus edulis) hepatopancreas from samples collected a few days after when Dinophysis was recorded at a density of 1550 cells L⁻¹. Liquid chromatography–mass spectrometry confirmed presence of several classes of lipophilic shellfish toxins associated with Dinophysis spp. in the mussels including okadaic acid, dinophysistoxin-1, pectenotoxins and yessotoxins. No azaspiracid was detected. This represents the first identification of phycotoxicity in the White Sea.     

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.     

Occurrence ofDinophysis spp. and toxic shellfish in the Northern Adriatic

The dynamics of the toxicity of the musselMytilus galloprovincialis was compared between two different shellfish farms, 5 km apart, but using the same cultivation technique. The main differences concerned the freshwater influx and the open aspect to the Gulf of Trieste. It is suggested that a deep closed bay and abundant fresh water inflow are the two main conditions for the low toxicity levels in mussels and for shorter periods of danger. A detailed study of the phytoplankton samples revealed the presence of eight species ofDinophysis in the area of both shellfish farms. During the period of the DSP outbreak in Slovenia (autumn and winter 1989).D. fortii andD. acuminata were the most frequentDinophysis species. There was a high positive correlation between the onset of mussel toxicity and the appearance ofDinophysis spp.     

Toxic and noxious phytoplankton in Big Glory Bay,Stewart Island,New Zealand

Diurnal vertical profile sampling of the water column, during a fish killing bloom of the raphidophycean alga Heterosigma akashiwo, revealed a phytoplankton population otherwise composed almost entirely of a variety of dinoflagellates. Of these Glenodinium danicum, Dinophysis acuta, Polykrikos schwartzii, Ceratium furca and Gyrodinium spirale were predominant. The distribution of the major species within the phytoplankton were documented and evidence of synchronous vertical migration of H. akashiwo, G. danicum and P. schwartzii was observed. Extracts of shellfish obtained during the bloom and tested by mouse bioassay showed no PSP toxicity but a marginal degree of DSP toxicity. During a subsequent one year phytoplankton monitoring programme another potentially noxious species (Chaetoceros convolutus) appeared and the seasonal reoccurrence of species present during the bloom (e.g. H. akashiwo) was observed. Important year to year differences in the summer phytoplankton (diatom versus flagellate dominated populations) were apparent and analysis of climate data showed that these differences related to different weather conditions prevailing during the two summer periods sampled. The data suggest the fish killing bloom was giving a chance to develop by a prolonged period of warm, calm weather (during which several heavy rainfall events occurred) leading to stable hydrographic conditions (i.e. stratification) and an increase in the retention time of water within the bay.     

Contribution to toxicity assessment of <Emphasis Type="BoldItalic">Dinophysis acuminata</Emphasis> (Dinophyceae)

Blooms of Dinophysis in French coastal waters are implicated in most bans on marketing commercial bivalves. However, the relation between Dinophysis cell density and shellfish toxicity is not always consistent. Discrepancies may be due to the simple fact that it is nearly impossible to compare an integral over a few days (shellfish toxin content) and water samples. Furthermore, it seems that cells may have a variable specific toxicity. This work focuses on the variability in cell toxicity taking into account recent findings and using liquid chromatography coupled to mass spectrometry with an ion trap and electrospray interface. Esterified analogues of okadaic acid (DTX-4 and diol-esters) have been identified in cultures of Prorocentrum lima, another okadaic acid producer. These analogues are inactive on some protein phosphatases, contrary to okadaic acid, and seem to protect the cell from harmful effects by the toxin and to be enzymatically hydrolyzed during cell lysis. In order to document specific toxicity and to validate the presence of these analogues, D. acuminata concentrates were subjected to two separate heating and freeze/thaw procedures, respectively inhibiting or promoting hydrolysis. This paper reports on the high variability of D. acuminata specific toxicity and the presence of esters found in half of the samples only.     

Prorocentrum lima (Dinophyceae) in northeastern USA coastal waters: II: Toxin load in the epibiota and in shellfish

The seasonal variation in diarrhetic shellfish poisoning (DSP)-type toxins was followed in the epibiotic community and in shellfish between 41° and 44°N in coastal waters of the northwest Atlantic during a 2-year period. Low levels of okadaic-acid equivalents were detected at all stations in the <90 μm fraction of the collected epibiota as measured by the protein phosphatase inhibition assay, but only 3.5% of the samples had values greater than 100 ng (g dry weight of epibiota)⁻¹. No seasonal pattern could be detected due to differences in intensity, duration and timing of toxin content in the epibiota between the 2 years and between stations. Nevertheless, the concentration of DSP-type toxins in the epibiota correlated weakly but significantly with the abundance of Prorocentrum lima, when data from all stations were considered. A very limited toxin uptake by shellfish was measured at only one station in October and November 2001 and in June and July 2002 at times of maximum cell concentration of P. lima in the epibiota. Toxin levels in shellfish remained well below regulatory limits that would have required quarantine or bans on harvesting. Results from our 2-year survey suggest that, at this time, the threat of DSP events appears minimal. However, the presence of a known toxin producer and its demonstrated ingestion by shellfish would argue for further studies to better understand conditions leading to DSP outbreaks generated by an epiphytic dinoflagellate.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Investigations of the temporal variation of cyanobacterial and other phytoplanktonic cells at the offtake of a large reservoir,and their survival following passage through it

The survival and subsequent growth potential of Anabaena spp. and other filamentous cyanobacteria and the cells of Aulacoseira spp. (diatom) and Ceratium hirundinella (dinoflagellate) following passage through the Multi Level Inlet Tower (MLIT) and offtake works at Chaffey Reservoir in New South Wales, Australia was investigated in late summer. The study aimed to test whether the phytoplankton cells were destroyed or otherwise rendered less viable during passage through the outlet works. The reservoir was strongly thermally stratified with a shallow surface mixed layer, which contributed to considerable temporal variability in the numbers of phytoplankton cells present immediately opposite the intake portal of the outlet works. To compensate, considerable replicate sampling was undertaken both upstream and downstream of the MLIT. Results indicate limited destruction of cyanobacteria, with fewer cells present immediately downstream compared to upstream. Greater destruction of cells was indicated at lower mean daily discharge rates compared to higher discharge rates. Filament lengths of both cyanobacteria and Aulacoseira were also reduced during passage. There was no apparent reduction in Ceratium cell number. Laboratory incubation studies on surviving cells collected downstream indicated no impairment on the viability of any taxa. Calculations of rates-of-strain likely to be experienced by the phytoplankton as they transited through the offtake revealed very high stress being applied to the filaments and cells at the valve, and within the spillway sections of the works. These were several orders of magnitude greater than published values shown to disrupt cells and filaments, and to impair viability for subsequent growth in laboratory studies. However, exposure times to the high rates-of-strain at Chaffey Reservoir were brief, which may reduce the impacts of the high turbulence. The conclusions were that unless cyanobacterial cell destruction during passage through an outlet works can be shown to be more effective at larger reservoirs, the withdrawal of warm, cyanobacterial infested waters from close to the surface is unlikely to provide an acceptable management action for the prevention of cold water pollution downstream. Handling editor: D. Ryder     

Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile

The morphology and toxicity of the four ubiquitous species belonging to the genus Pseudo-nitzschia found in mixed blooms of phytoplankton from northern Chilean waters were studied. The phytoplankton samples and cultures obtained were identified by scanning electron microscopy, revealing the presence of Pseudo-nitzschia australis, P. calliantha, P. pseudodelicatissima and P. subfraudulenta. This is the first report of P. calliantha in northern Chile. Toxin analyses using the LC–MS method confirmed the presence of domoic acid in P. australis and P. calliantha. Domoic acid was not detected in cultures of P. subfraudulenta. This study therefore confirms P. australis and P. calliantha as an unequivocal source of domoic acid in Chilean waters. P. australis is probably the most important producer of amnesic shellfish toxin in view of its domoic acid content. However, more research is needed to evaluate the potential for toxin production in P. pseudodelicatissima.