Seasonal variability of lipophilic toxins during a Dinophysis acuta bloom in Western Iberia: Differences between picked cells and plankton concentrates

This work describes and compares the seasonal variability of toxin profiles and content, estimated by LC–MS analyses, in picked cell of Dinophysis acuta Ehrenberg, in plankton concentrates rich in this species, and in extracellular lipophilic toxins collected by adsorbent resins during weekly sampling in a Galician ría (Western Iberia) from October 2005 to January 2006. Picked cells of D. acuta—which exhibited a fairly stable OA:DTX2 ratio, close to 3:2, but a variable okadaates:PTX2 ratio—showed a 9-fold variation in cell toxin quota, which was partly related to cellular volume, with maximum values (19 pg cell⁻¹) observed during the exponential decline of the population. Large differences in toxin profiles and content were observed between picked cells and plankton concentrates (up to 73 pg cell⁻¹ in the latter), that were most conspicuous after the bloom decline. The toxin profile of picked cells was more similar to that observed in the adsorbent resins than to the profiles of plankton concentrates. Their continued detection several weeks after the disappearance of Dinophysis spp. indicates that these toxins may take a long time to be degraded. It is concluded that analyses of picked-cells are essential to determine the contribution of each species of Dinophysis to a toxic outbreak. Estimates of cellular toxin content from plankton concentrates can lead to considerable overestimates after Dinophysis blooms decay due to extracellular toxins that persist in the water column, possibly bound to organic aggregates and detritus, and are retained (>0.22 μm) in the filters.     

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.     

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.     

Regulation of spiroimine neurotoxins and hemolytic activity in laboratory cultures of the dinoflagellate Alexandrium peruvianum (Balech & Mendiola) Balech & Tangen

Defined experimental regimes were used to determine the effects of nutrient limitation on the toxicity of Alexandrium peruvianum in batch culture. Subsamples for cell counts and spiroimine analysis at six day intervals were used to investigate the concentrations and composition of these compounds throughout growth. An erythrocyte lysis assay for hemolytic activity was performed on cell pellets and supernatants also collected every six days over the entire growth period from all treatments. From the data, growth rates, cellular spiroimine quotas and effective concentration-fifty (EC₅₀s) for cellular and supernatant associated hemolytic activity were calculated. Phosphate limitation was identified as a key regulator of toxicity in this species, yielding maximum values of 54.1 pg cell⁻¹ for 13-desmethyl spirolide C, 96.4 pg cell⁻¹ for 12-methylgymnodimine and a potent hemolytic EC₅₀ value of 7.1 × 10³ cells. The concentrations of spiroimines detected in A. peruvianum among various treatments, in addition to a unique profile of paralytic shellfish poisoning toxins, is unique in the body of microalgal literature. Because of the multiple toxin arsenal produced by this organism, the evaluation of a single toxin clearly would have underestimated the potential virulence and significance of this clone. This study provides the first evidence that growth and toxin production of A. peruvianum are influenced by altered nutrient ratios.     

Alexandrium pacificum Litaker sp. nov (Group IV): Resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia,Southern Mediterranean Sea)

A high spatial resolution sampling of Alexandrium pacificum cysts, along with sediment characteristics (% H₂O, % organic matter (OM), granulometry), vegetative cell abundance and environmental factors were investigated at 123 study stations in Bizerte Lagoon (Tunisia). Morphological examination and ribotyping of cells obtained from a culture called ABZ1 obtained from a cyst isolated in lagoon sediment confirmed that the species was A. pacificum. The toxin profile from the ABZ1 culture harvested during exponential growth phase was simple and composed of the N-sulfocarbamoyl toxins C1 (9.82 pg toxin cell⁻¹), the GTX6 (3.26 pg toxin cell⁻¹) and the carbamoyl toxin Neo-STX (0.38 pg toxin cell⁻¹). The latter represented only 2.8% of the total toxins in this strain.High abundance of A. pacificum cysts correlated with enhanced percentages of water and organic matter in the sediment. In addition, sediment fractions of less than 63 μm were examined as a favorable potential seedbed for initiation of future blooms and outbreaks of A. pacificum in the lagoon. A significant difference in the cyst distribution pattern was recorded among the lagoon's different zones, with the higher cyst abundance occurring in the inner waters. Also, no correlation due to the specific hydrodynamics of the lagoon was observed in the spatial distribution of A. pacificum cysts and vegetative cells.     

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.     

Harmful effects of Dinophysis to the ciliate Mesodinium rubrum: Implications for prey capture

Toxigenic Dinophysis spp. are obligate mixotrophic dinoflagellates that require a constant supply of prey—Mesodinium rubrum—to achieve long-term growth by means of kleptoplasty. Mesodinium rubrum is, however, a fast moving, jumping ciliate exhibiting an effective escape response from suspensivorous predators. In the present study, a series of laboratory experiments evaluating the motility and survival of M. rubrum in the presence of Dinophysis cells and/or substances contained in their culture medium was designed, in order to assess the mechanisms involved in prey capture by Dinophysis spp. Cell abundance of M. rubrum decreased in the presence of Dinophysis cf. ovum cells producing okadaic acid (OA; up to 7.94 ± 2.67 pg cell⁻¹) and smaller amounts of dinophysistoxin-1 (DTX-1) and pectenotoxin-2 (PTX-2). Prey capture was often observed after the ciliate had been attached to adhesive “mucus traps”, which only appeared in the presence of Dinophysis cells. Before being attached to the mucus traps, M. rubrum cells reduced significantly their swimming frequency (from ∼41 to 19 ± 3 jumps min⁻¹) after only 4 h of initial contact with D. cf. ovum cells. M. rubrum survival was not affected in contact with purified OA, DTX-1 and PTX-2 solutions, but decreased significantly when the ciliate was exposed to cell-free or filtered culture medium from both D. cf. ovum and D. caudata, the latter containing moderate concentrations of free eicosapentaenoic acid and docosahexaenoic acid. The results thus indicate that Dinophysis combines the release of toxic compounds other than shellfish toxins, possibly free PUFAs, and a “mucus trap” to enhance its prey capture success by immobilizing and subsequently arresting M. rubrum cells.     

Morphology and molecular phylogeny of Nitzschia bizertensis sp. nov.—A new domoic acid-producer

A new toxin-producing marine diatom, Nitzschia bizertensis sp. nov., isolated from the Bizerte Lagoon (Tunisia, Southwest Mediterranean Sea) is, based on studies on eight different strains, characterized morphologically by light microscopy, transmission and scanning electron microscopy, and phylogenetically using the nuclear rDNA regions: SSU, ITS1, 5.8S, ITS2 and D1–D3 of the LSU. The species belongs to the sections Lanceolatae or Lineares as defined by Cleve and Grunow (1880). These sections are characterized by species having linear-lanceolate valves with an eccentric raphe where the fibulae does not extend into the valve, and are otherwise famous for the lack of characters useful for delineation of species. Nitzschia bizertensis differs from most other species in these sections by having a high density of interstriae. The morphological and phylogenetic studies and comparisons with previously described Nitzschia species showed Nitzschia bizertensis sp. nov. to be a new species. Batch culture experiments were conducted for estimations of maximum growth rate and production of domoic acid (DA). Maximum cellular DA content of the examined strains ranged from 2 × 10⁻⁴ to 3.6 × 10⁻² pg cells⁻¹. The total DA concentration (pg mL⁻¹) was high already in exponential growth phase maybe due to reinoculation of “old” stationary phase cells, and increased into stationary growth phase where it reached a stationary level varying among the strains from ca. 4500 to 9500 pg mL⁻¹. Nitzschia bizertensis represents a new domoic acid-producing diatom and is the second toxin producing Nitzschia species. The resolution of Nitzschia bizertensis and Nitzschia navis-varingica in different parts of the LSU phylogenetic tree, and the recovery of the Pseudo-nitzschia species phylogenetically distant from those two species suggests that the ability to produce DA either evolved multiple times independently or was lost multiple times.     

Toxigenic phytoplankton and concomitant toxicity in the mussel Choromytilus meridionalis off the west coast of South Africa

The variability of toxigenic phytoplankton and the consequent uptake and loss of toxins by the mussel Choromytilus meridionalis was investigated in the southern Benguela at the event scale (3–10 days) in response to the upwelling–downwelling cycle. Phytoplankton and mussel samples were collected daily (20 March–11 April 2007) from a mooring station (32.04°S; 18.26°E) located 3.5 km offshore of Lambert's Bay, within the St Helena Bay region. Rapid changes in phytoplankton assemblages incorporated three groups of toxigenic phytoplankton: (1) the dinoflagellate Alexandrium catenella; (2) several species of Dinophysis, including Dinophysis acuminata, Dinophysis fortii, Dinophysis hastata and Dinophysis rotundata; and (3) members of the diatom genus Pseudo-nitzschia. Analysis of phytoplankton concentrates by LC–MS/MS or LC-FD provided information on the toxin composition and calculated toxicity of each group. Several additional in vitro assays were used for the analysis of toxins in mussels (ELISA, RBA, MBA for PSP toxins; and ELISA for DSP toxins). Good correspondence was observed between methods except for the MBA, which provided significantly lower (approximately 2-fold) estimates of PSP toxins. PSP and DSP toxins both exceeded the regulatory limits in Choromytilis meridionalis, but ASP toxins were undetected. Differences were observed in the composition of both PSP and DSP toxins in C. meridionalis from that of the ingested dinoflagellates (PSP toxins showed an increase in STX, C1,2, and traces of dcSTX and GTX1,4 and a decrease in NEO; DSP toxins showed an increased in DTX1, and traces of PTX2sa, and a decrease in OA). The rate of loss of PSP toxins following dispersal of the A. catenella boom was 0.12 d⁻¹. Variation in the loss rates of different PSP toxins contributed to the change in toxin profile in C. meridionalis. Prediction of net toxicity in shellfish of the nearshore environment in the southern Benguela is limited due to rapid phytoplankton community changes, high variability in cellular toxicity, and the selective uptake and loss of toxins, and/or transformation of toxins.     

Growth and domoic acid content of Pseudo-nitzschia australis isolated from northwestern Baja California,Mexico, cultured under batch conditions at different temperatures and two Si:NO3 ratios

Domoic acid (DA) poisoning in the southern part of the California Current System has been associated typically with blooms of Pseudo-nitzschia australis. The environmental variables that promote growth and DA production in the Mexican part of this system have not been identified. The present study investigated the effect of temperature and two nutrient ratios on the growth characteristics and DA content of two (BTS-1, BTS-2) P. australis strains isolated from the Pacific coast of northern Baja California peninsula, México. Of the different temperatures assayed (10, 12, 14, 15, 18 and 20 °C), the maximum cell abundance was detected at 12 °C for BTS-2 and 14 °C for BTS-1. The highest maximum specific growth rate (1.69 day⁻¹) was measured at 15 °C for BTS-2. With the exception of cells maintained at 15 °C, growth characteristics were similar in P. australis cultured in a high Si:NO₃ (2.5) or low Si:NO₃ (0.5) ratio at each temperature. Dissolved (dDA) and cellular (cDA) DA content measured at the stationary phase of growth was similar in cells cultivated at the different temperatures. No difference in cDA (between 0.11 and 1.87 pg DA cell⁻¹) was observed in cells cultivated at the two nutrient ratios. To evaluate if P. australis accumulates DA (cDA + dDA) at different stages of the culture and not only during the stationary phase of growth, the BTS-1 strain was cultivated at 14 °C and the content of this toxin was measured during culture development. The cultures were maintained at high (HL; 200 μmol quanta m⁻² s⁻¹) and low light (LL; 30 μmol quanta m⁻² s⁻¹) and in the two nutrient ratios to evaluate the effect of these variables on DA content. The photosynthetic performance and pigment concentration were measured as indicators of the physiological condition of the cells. cDA was detected in all culture conditions and during the different stages of growth. The highest DA content was measured during the lag phase of growth and it was present mainly in the medium (dDA = 70.83 pg DA cell⁻¹). Cells cultivated at HL produced more DA than LL cultured cells. P. australis cultured in HL presented lower photosynthetic rates than LL cells and had similar concentrations of photoprotective pigments and the highest maximum photosynthetic rates were detected during the lag phase of growth in all culture conditions. The results demonstrate that P. australis from northern Baja California peninsula presents a narrow temperature range for optimal growth under batch culture conditions. P. australis produce DA at different stages of growth, and DA content was related to the light intensity at which the cells were cultivated.     

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.     

Growth and bioactive secondary metabolites of arctic 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 d⁻¹, and total YTX concentration ranged from 0.3 to 15.0 pg  YTX cell⁻¹ and from 0.5 to 31.0 pg YTX cell⁻¹ 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.     

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.     

The combined effect of salinity and temperature on the growth and toxin content of four Chilean strains of Alexandrium catenella (Whedon and Kofoid) Balech 1985 (Dinophyceae) isolated from an outbreak occurring in southern Chile in 2009

The toxic dinoflagellate Alexandrium catenella has been detected in the southern Chile since 1972, causing severe negative impacts on public health and aquaculture activities. Several environmental factors have been determined to affect growth and toxin production in Alexandrium strains. The aim of this study was to determine the effect of four combined conditions of two temperatures (10 and 15 °C) and two salinities (15 and 35 psu) on the growth and the Paralytic Shellfish Poisoning (PSP) toxin content and composition in four Chilean strains of A. catenella (PFB41, PFB42, PFB37 and PFB38), isolated during a summer outbreak occurred in southern Chile in 2009. The growth curves showed a higher effect of the salinity in strains PFB41 and PFB42 than in strains PFB37 and PFB38. The values of growth rates and maximum cell densities ranged from 0.25 to 0.73 div day⁻¹ and 1.1 × 10⁴ to 5.2 × 10⁴ cells mL⁻¹, respectively. All of the strains showed the highest values for both growth parameters at 15 °C and 35 psu. In general, growth parameters were higher at 35 psu independently of the temperature. On the other hand, the total PSP toxin content ranged widely from 3.99 to 239 fmol cell⁻¹. The highest values of PSP toxin content were attained at 10 °C and 35 psu for all of the strains, at both stages of growth. All of the strains displayed different toxin compositions, with neoSTX, GTX4-1, GTX3-2 and GTX5 being the main toxins detected. The results showed significant differences in the absolute values of growth and toxin production parameters among the strains grown under the same culture conditions, and for each strain grown under different combined conditions of temperature and salinity. These findings demonstrate that abiotic factors can differentially affect the population dynamics of the A. catenella toxic genotypes, thus making it extremely difficult to predict the ecological behavior of this species in the field in terms of the intensity of a potential outbreak.     

Uptake of paralytic shellfish poisoning and spirolide toxins by paddle crabs (Ovalipes catharus) via a bivalve vector

The uptake of paralytic shellfish poisoning (PSP) toxins and spirolides by the paddle crab (Ovalipes catharus) was investigated in two laboratory feeding trials using Greenshell? mussels (Perna canaliculus), which had been fed toxic strains of either Alexandrium catenella or A. ostenfeldii, as a vector. Toxin uptake by crabs occurred in both feeding trials and was limited to the visceral tissue; no toxins were detected in the body meat or the gills. The first trial utilized a strain of A. catenella that had high total PSP toxin content, 442.3 ± 91.6 fmol/cell, that was dominated by low toxicity N-sulfocarbamoyl toxins resulting in a low cellular toxicity, 5.5 ± 1.6 pg STXequiv./cell. In this trial, toxin accumulation in the crabs was highly variable and ranged from 3.8 to 221.5 μg STXequiv./100 g, with 3/4 of the crabs exceeding the regulatory limit of 80 μg STXequiv./100 g. Eight days after feeding on toxic mussels the crabs still retained high levels of toxin suggesting that depuration rates in this species may be slow. In the second feeding trial, the A. ostenfeldii strain fed to mussels produced low levels of both PSP toxins (52.0 ± 19.5 fmol/cell; 1.4 ± 0.3 pg STXequiv./cell) and spirolides (1.8 pg/cell) and, as a result, the concentration transferred to crabs via the mussels was very low-PSP toxins ranged from 2.5 to 6.8 μg STXequiv./100 g and spirolides from 6 to 7 μg/kg. The results of our study demonstrate that paddle crabs are capable of acquiring both PSP toxins and spirolides and suggest that this may occur in the wild during a toxic shellfish event. It also highlights the need to remove the viscera before consumption.     

Pectenotoxin and okadaic acid-based toxin profiles in Dinophysis acuta and Dinophysis acuminata from New Zealand

The major pectenotoxin and okadaic acid group toxins in Dinophysis acuta and Dinophysis acuminata cell concentrates, collected from various locations around the coast of the South Island of New Zealand (NZ), were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). PTX2 and PTX11 were the major polyether toxins in all Dinophysis spp. cell concentrates. D. acuta contained PTX11 and PTX2 at concentrations of 4.7–64.6 and 32.5–107.5 pg per cell, respectively. The amounts of PTX11 and PTX2 in D. acuminata were much lower at 0.4–2.1 and 2.4–25.8 pg per cell, respectively. PTX seco acids comprised only 4% of the total PTX content of both D. acuta and D. acuminata. D. acuta contained low levels of OA (0.8–2.7 pg per cell) but specimens from the South Island west coast also contained up to 10 times higher levels of OA esters (7.0–10.2 pg per cell). Esterified forms of OA were not observed in D. acuta specimens from the Marlborough Sounds. D. acuta did not contain any DTX1 though all D. acuminata specimens contained DTX1 at levels of 0.1–2.4 pg per cell. DTX2 was not present in any New Zealand Dinophysis spp. specimens. Although the total toxin content varied spatially and temporally, the relative proportions of the various toxins in different specimens from the same location appeared to be relatively stable. The total PTX/total OA ratios in different isolates of D. acuta were very similar (mean±S.E.: 14.9±1.9), although the Marlborough Sounds D. acuminata isolates had a higher total PTX/total OA ratio (mean±S.E.: 22.7±2.4) than the Akaroa Harbour isolates (8.0). No evidence of azaspiracids were detected in these specimens. These results show that the LC–MS/MS monitoring of plankton for PTX group toxins (e.g. PTX2) and their derivatives (e.g. PTX2 seco acid) may provide a sensitive, semi-quantitative, indicator of the presence of more cryptic OA group toxins (e.g. OA esters).     

An assessment of Pseudo-nitzschia population dynamics and domoic acid production in coastal Louisiana

Over 1200 samples were collected from Louisiana estuarine and coastal shelf waters between 1989 and 2002, and analyzed to examine the population dynamics of Pseudo-nitzschia and to assess the potential threat posed by domoic acid (DA), a potent neurotoxin produced by some members within this toxigenic diatom genus. Results demonstrated that three species in this region (Pseudo-nitzschia multiseries, P. pseudodelicatissima complex, P. delicatissima) produce DA, and that particulate toxin levels were highest (up to 3.05 μg L⁻¹) during the spring bloom, while cellular concentrations were highest in the winter/early spring when P. multiseries was most abundant (up to 30 pg cell⁻¹). These particulate toxin levels are comparable to those seen in other regions (e.g., United States west coast) where DA poisoning events have occurred in the past. Pseudo-nitzschia were most abundant under dissolved inorganic nitrogen-replete conditions coupled with lower silicate and/or phosphate concentrations, and in the early spring months when temperatures were cooler. Pseudo-nitzschia were occasionally well-represented in the phytoplankton assemblage (≥10⁶ cells L⁻¹ in 14% of samples, over 50% of total phytoplankton in 5% of samples), indicating that planktivores (e.g., Gulf menhaden, Brevoortia patronus) may have little choice but to consume Pseudo-nitzschia cells, thereby providing potential vectors for DA transfer to higher trophic levels. By comparison, eastern oysters (Crassostrea virginica) present in estuarine waters may be more exposed to this toxin when Pseudo-nitzschia cells are part of a mixed assemblage, reducing selective grazing by these bivalves. C. virginica may thus represent the most effective vector for DA exposure in humans.     

Toxic strains of the Alexandrium ostenfeldii complex in southern South America (Beagle Channel,Argentina)

During phytoplankton monitoring in the Beagle Channel (≈54°52′ S, 67°32′ W) a previously undetected Alexandrium species was observed in coincidence with mouse bioassay toxicity. Detailed thecal plates analysis using epifluorescence and scanning electron microscopy revealed the presence of the Alexandrium ostenfeldii species complex, showing a mixture of the diagnostic features usually used to discriminate between the morphospecies A. ostenfeldii and A. peruvianum. Cells of the A. ostenfeldii complex were commonly observed during spring after the main annual diatom bloom, when temperatures and salinities were respectively around 7.5–10 °C and 30–30.5 psu, and nutrients showed a seasonal decrease. Toxin analysis by liquid chromatography–mass spectrometry revealed the production of 13-desmethyl spirolide C and 20-methyl spirolide G in cell cultures. The cellular contain of spirolides during exponential phase growth was 0.5906 ± 0.0032 and 0.1577 ± 0.0023 pg cell⁻¹ for 13-desMe-C and 20-Me-G, respectively. A third unknown compound, with a structure resembling that of spirolides was also detected in culture. Moreover, an additional compound with a similar m/z (692) than that of 13-desMe-C but presenting a higher retention time (Rt = 40.5 min) was found in high proportions in mussel samples. PSP toxins were present at low concentration in mussels but were not detected in cultures. These results extend the world-wide distribution of toxic strains of the A. ostenfeldii complex to the Beagle Channel (southern South America), where toxic events have been traditionally linked to the presence of Alexandrium catenella. This is the first confirmed occurrence of spirolides in mussels and plankton from Argentina, which highlights the importance of monitoring these toxins and their producing organisms to protect public health and improve the management of shellfish resources.     

Recurrent vernal presence of the toxic Alexandrium tamarense/Alexandrium fundyense (Dinoflagellata) species complex in Narragansett Bay,USA

The vernal occurrence of toxic dinoflagellates in the Alexandrium tamarense/Alexandrium fundyense species complex in an enclosed embayment of Narragansett Bay (Wickford Cove, Rhode Island) was documented during 2005 and 2009–2012. This is the first report of regular appearance of the Alexandrium fundyense/Alexandrium tamarense species complex in Narragansett Bay. Thecal plate analysis of clonal isolates using SEM revealed cells morphologically consistent with both Alexandrium tamarense Lebour (Balech) and Alexandrium fundyense Balech. Additionally, molecular analyses confirmed that the partial sequences for 18S through the D1–D2 region of 28S were consistent with the identity of the two Alexandrium species. Toxin analyses revealed the presence of a suite of toxins (C1/2, B1 (GTX-5), STX, GTX-2/3. Neo, and GTX-1/4) in both Alexandrium tamarense (6.31 fmol cell⁻¹ STX equiv.) and Alexandrium fundyense (9.56 fmol cell⁻¹ STX equiv.) isolated from Wickford Cove; the toxicity of a Narragansett Bay Alexandrium peruvianum isolate (1.79 fmol cell⁻¹ STX equiv.) was also determined. Combined Alexandrium tamarense/Alexandrium fundyense abundance in Wickford Cove reached a peak abundance of 1280 cells L⁻¹ (May of 2010), with the combined abundance routinely exceeding levels leading to shellfishing closures in other systems. The toxic Alexandrium tamarense/Alexandrium fundyense species complex appears to be a regular component of the lower Narragansett Bay phytoplankton community, either newly emergent or previously overlooked by extant monitoring programs.