Description: The marine dinoflagellate Lingulodinium polyedra is a toxigenic species capable of forming high magnitude and occasionally harmful algal blooms (HABs), particularly in temperate coastal waters throughout the world. Three cultured isolates of L. polyedra from a fjord system on the Skagerrak coast of Sweden were analyzed for their growth characteristics and to determine the effects of a strong salinity gradient on toxin cell quotas and composition. The cell quota of yessotoxin (YTX) analogs, as determined by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS), ranged widely among strains. For two strains, the total toxin content remained constant over time in culture, but for the third strain, the YTX cell quota significantly decreased (by 32%) during stationary growth phase. The toxin profiles of the three strains differed markedly and none produced YTX. The analog 41a-homo-YTX (m/z 1155), its putative methylated derivative 9-Me-41a-homo-YTX (m/z 1169) and an unspecified keto-YTX (m/z 1047) were detected in strain LP29-10H, whereas strain LP30-7B contained nor-YTX (m/z 1101), and two unspecified YTX analogs at m/z 1159 and m/z 1061. The toxin profile of strain LP30-8D comprised two unspecified YTX analogs at m/z 1061 and m/z 991 and carboxy-YTX (m/z 1173). Strain LP30-7B cultured at multiple salinities (10, 16, 22, 28 and 34) did not tolerate the lowest salinity (10), but there was a statistically significant decrease (by 21%) in toxin cell quota between growth at the highest versus lower permissible salinities. The toxin profile for strain LP30-7B remained constant over time for a given salinity. At lower salinities, however, the proportion of the unspecified YTX analog (m/z 1061) was significantly higher, especially with respect to nor-YTX (m/z 1101). This study shows high intra-specific variability in yessotoxin composition among strains from the same geographical region and inconsistency in toxin cell quota under different environmental regimes and growth stages in culture. This variation has important implications for the kinetics of YTX production and food web transfer in natural bloom populations from diverse geographical regions.

Description: The marine diatom genus Pseudo-nitzschia, the major known producer of the neurotoxin domoic acid (DA) responsible for the amnesic shellfish poisoning (ASP) syndrome in humans and marine mammals, is globally distributed. The genus presents high species richness in the Argentine Sea and DA has been frequently detected in the last few years in plankton and shellfish samples, but the species identity of the producers remains unclear. In the present work, the distribution and abundance of Pseudo-nitzschia species and DA were determined from samples collected on two oceanographic cruises carried out through the Argentine Sea (∼39–47°S) during summer and spring 2013. Phytoplankton composition was analysed by light and electron microscopy while DA was determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The genus Pseudo-nitzschia was recorded in 71 and 86% of samples collected in summer and spring, respectively, whereas DA was detected in only 42 and 21% of samples, respectively. Microscopic analyses revealed at least five potentially toxic species (P. australis, P. brasiliana, P. fraudulenta, P. pungens, P. turgidula), plus putatively non-toxigenic P. dolorosa, P. lineola, P. turgiduloides and unidentified specimens of the P. pseudodelicatissima complex. The species P. australis showed the highest correlation with DA occurrence (r = 0.55; p 〈 0.05), suggesting its importance as a major DA producer in the Argentine Sea. In the northern area and during summer, DA was associated with the presence of P. brasiliana, a species recorded for the first time in the Argentine Sea. By contrast, high concentrations of P. fraudulenta, P. pungens and P. turgidula did not correspond with DA occurrence. This study represents the first successful attempt to link toxigenicity with Pseudo-nitzschia diversity and cell abundance in field plankton populations in the south-western Atlantic.

Description: 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.

Description: The marine diatom genus Pseudo-nitzschia, the major known producer of the neurotoxin domoic acid (DA) responsible for the amnesic shellfish poisoning (ASP) syndrome in humans and marine mammals, is globally distributed. The genus presents high species richness in the Argentine Sea and DA has been frequently detected in the last few years in plankton and shellfish samples, but the species identity of the producers remains unclear. In the present work, the distribution and abundance of Pseudo-nitzschia species and DA were determined from samples collected on two oceanographic cruises carried out through the Argentine Sea (∼39–47°S) during summer and spring 2013. Phytoplankton composition was analysed by light and electron microscopy while DA was determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The genus Pseudo-nitzschia was recorded in 71 and 86% of samples collected in summer and spring, respectively, whereas DA was detected in only 42 and 21% of samples, respectively. Microscopic analyses revealed at least five potentially toxic species (P. australis, P. brasiliana, P. fraudulenta, P. pungens, P. turgidula), plus putatively non-toxigenic P. dolorosa, P. lineola, P. turgiduloides and unidentified specimens of the P. pseudodelicatissima complex. The species P. australis showed the highest correlation with DA occurrence (r = 0.55; p 〈 0.05), suggesting its importance as a major DA producer in the Argentine Sea. In the northern area and during summer, DA was associated with the presence of P. brasiliana, a species recorded for the first time in the Argentine Sea. By contrast, high concentrations of P. fraudulenta, P. pungens and P. turgidula did not correspond with DA occurrence. This study represents the first successful attempt to link toxigenicity with Pseudo-nitzschia diversity and cell abundance in field plankton populations in the south-western Atlantic.

Description: The diversity and biogeography of populations of the toxigenic marine dinoflagellate genus Alexandrium, a major global cause of paralytic shellfish poisoning (PSP), are represented by only a few studies based upon a low number of cultured isolates and remain poorly described in Arctic and sub-Arctic waters. Multiple clonal isolates (n = 22) of the Alexandrium tamarense species complex, and a single isolate of A. tamutum, were collected from the water column while on board an oceanographic expedition to the west coast of Greenland. After culturing of these isolates under controlled conditions, their phylogenetic affinities within the genus Alexandrium were characterized by sequence analysis of nuclear large subunit (LSU) rDNA. Based upon morphological and molecular genetic criteria, all isolates of the A. tamarense species complex were consistent with membership in the Group I ribotype (previously known as the North American ribotype). Phenotypic signatures were also analyzed based upon their respective profiles of paralytic shellfish toxins (PST) and allelochemical interactions against a target cryptophyte Rhodomonas, as determined by lytic potency. All isolates conforming to the A. tamarense Group I produced PST, but no toxins were detected in A. tamutum P2E2. Unusually, only carbamoyl toxins were produced among the A. tamarense Group I isolates from Greenland; sulfocarbamoyl derivatives, generally present in A. tamarense population from other locations, including the Arctic, North Pacific and North Atlantic, were absent from all isolates. Allelochemical activity, causing cell lysis of Rhodomonas, but generally being unrelated to cellular PST, was expressed by all A. tamarense isolates and also by A. tamutum, but varied widely in potency. Comparison of the genotypic (rDNA) and phenotypic (PST profile, allelochemical activity) characteristics of Greenland isolates with those of other Arctic populations reveals a complex pattern of intra-specific diversity. Estimation of diversity relationships is problematic because of the distinct patterns of divergence and lack of evidence of linkage among the alternative biomarkers and morphology. Nevertheless, such studies are necessary as the basis for constructing hindcasting scenarios and predicting changes in Alexandrium species distribution in the Arctic from the regional to the global scale.

Description: Detection of paralytic shellfish poisoning (PSP) toxins in scallops from the west coast of Greenland exceeding the 800 μg toxin/kg shellfish limit led to an investigation with the aim of finding the responsible organism(s). Three strains of Alexandrium Halim were established from single cell isolations. Morphological identification of the strains and determination of their position within the genus by LSU rDNA sequences was carried out. Light microscopy revealed that the three strains was of the Alexandrium tamarense morphotype, and bayesian and neighbor-joining analyses of the LSU rDNA sequences placed them within Group I of the A. tamarense species complex. The toxicity and toxin profiles of the strains were measured by liquid chromatography fluorescence detection (LC-FD) and their identity was confirmed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The three strains all turned out to be toxic and all produced large proportions (>60% total mol) of gonyautoxins 1 and 4 (GTX1/GTX4). This is the first record of saxitoxin producers from western Greenland. The toxin profiles were atypical for A. tamarense in their absence of N-sulfocarbanoyl C1/C2 or B1/B2 toxins. Rather the high molar percentage of GTX1/GTX4, the lesser amounts of only carbamoyl toxins and the absence of decarbamoyl derivatives are more characteristic features of A. minutum strains. This may indicate that the genetically determined toxin profiles in Alexandrium species are more complex than previously appreciated.

Description: Harmful Algal Bloom (HAB) surveillance is complicated by high diversity of species and associated phycotoxins. Such species-level information on taxonomic affiliations and on cell abundance and toxin content is, however, crucial for effective monitoring, especially of aquaculture and fisheries areas. The aim addressed in this study was to determine putative HAB taxa and related phycotoxins in plankton from aquaculture sites in the Ebro Delta, NW Mediterranean. The comparative geographical distribution of potentially harmful plankton taxa was established by weekly field sampling throughout the water column during late spring–early summer over two years at key stations in Alfacs and Fangar embayments within the Ebro Delta. Core results included not only confirmed identification of HAB taxa that are common for the time period and geographical area, but also provided evidence of potentially new taxa. At least 25 HAB taxa were identified to species level, and an additional six genera were confirmed, by morphological criteria under light microscopy and/or by molecular genetics approaches involving qPCR and next generation DNA pyrosequencing. In particular, new insights were gained by the inclusion of molecular techniques, which focused attention on the HAB genera Alexandrium, Karlodinium, and Pseudo-nitzschia. Noteworthy is the discovery of Azadinium sp., a potentially new HAB species for this area, and Gymnodinium catenatum or Gymnodinium impudicum by means of light microscopy. In addition, significant amounts of the neurotoxin domoic acid (DA) were found for the first time in phytoplankton samples in the Ebro Delta. While the presence of the known DA-producing diatom genus Pseudo-nitzschia was confirmed in corresponding samples, the maximal toxin concentration did not coincide with highest cell abundances of the genus and the responsible species could not be identified. Combined findings of microscopic and molecular detection approaches underline the need for a synoptic strategy for HAB monitoring, which integrates the respective advantages and compensates for limitations of individual methods.

Description: The ichthyotoxic and mixotrophic prymnesiophyte Prymnesium parvum is known to produce dense virtually monospecific blooms in marine coastal, brackish, and inshore waters. Fish-killing Pyrmnesium blooms are often associated with macronutrient imbalanced conditions based upon shifts in ambient nitrogen (N): phosphorus (P) ratios. We therefore investigated nutrient-dependent cellular acclimation mechanisms of this microalga based upon construction of a normalized expressed sequence tag (EST) library. We then profiled the transcriptome of P. parvum under nutrient- replete conditions as well as under nitrogen (N) and phosphorus (P) limitation via microarray analyses. Twenty three genes putatively involved in acclimation to low nutrient levels were identified, among them three phosphate transporters, which were highly upregulated under P-starvation. In contrast, the expression of genes involved in transport and acquisition of ammonium or nitrate/nitrite was unaltered in N-starved cells. We propose that genes upregulated under P- or N-starvation lend themselves as potential tools to monitor nutrient limitation effects at the cellular level and indirectly the potential for initiation and maintenance of toxic blooms of P. parvum.

Description: Surfclams,Spisula solidissima, pose a particular health risk for human consumption as they are characterized by accumulation of extremely high levels of toxins associated with paralytic shellfish poisoning (PSP), slow toxin elimination and an extremely high post-ingestive capacity for toxin bioconversion. Surfclam populations experience a wide range of temperatures along the NW Atlantic continental shelf, and are undergoing range contraction that has been attributed to global warming. In this study the influence of temperature (5, 12 and 21 C) on detoxification kinetics of individual PSP toxins in two tissue compartments of juvenile surfclams (∼35 mm shell length) was determined under controlled laboratory conditions, over prolonged (2.4 months) depuration. Clams were toxified with a representative regional Gulf of Maine isolate of the dinoflagellate Alexandrium fundyense of known toxin profile, allowing tracking of changes in toxin composition and calculated toxicity in surfclam tissues. The visceral mass detoxified at all temperatures, although toxin loss rate increased with increasing temperature. In contrast, total toxin content and calculated toxicities in other tissues remained constant or even increased during depuration, suggesting a physiological or biochemical toxin-retention mechanism in this tissue pool and temperature-independent detoxification. In vivo toxin compositional changes in surfclam tissues found in this study provide evidence of specific toxin conversion pathways,involving both reductive and decarbamoylation pathways. We conclude that such toxin biotransformations, especially in non-visceral tissues, may introduce a discrepancy in describing kinetics of total toxicity (in saxitoxin equivalents [STXeq]) of S. solidissima over prolonged detoxification. Nevertheless, use of total toxicity values generated by routine regulatory monitoring based upon mouse bioassays or calculated from chemical analytical determination of molar toxin concentrations is adequate for first-order modeling of toxin kinetics in this species. Furthermore, the differential detoxification response of viscera and other tissues in relation to temperature emphasizes the need for two-compartment modeling to describe the fate of PSP toxins in this species. Finally, key parameters were identified that may prove useful in hindcasting the timing of toxic blooms or new toxin input in deep offshore waters where routine monitoring of toxic phytoplankton is impractical.

Description: 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 degrees S; 18.26 degrees 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 SIX, 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.