Description: The overall objective of the NORCOHAB cruise was to study the coastal oceanographic processes and mechanisms underlying the dynamics of key toxic bloom species and the biogeographical distribution of their toxins in the water column of different North Sea coastal waters. The cruise was endorsed by SCOR-IOC Programme GEOHAB (Global Ecology and Oceanography of Harmful Algal Blooms), under the auspices of the Core Research Project on HABS in Fjords and Coastal Embayments. Following the prescribed format of GEOHAB research the studies were international, multidisciplinary and comparative with the ultimate aim of modeling dynamics and behavior.We assessed and compared key genotypic and phenotypic properties of HAB species in relation to grazing and toxic and allelochemical interactions in Scottish and Norwegian/Danish (Skagerrak) coastal areas. For the first time, on board measurements of known phycotoxins collected directly from the plankton were subjected to analysis by high resolution tandem mass-spectrometry coupled with liquid chromatography (LC-MS/MS). These measurements included domoic acid and a large variety of lipophilic toxins associated with marine plankton. We also performed grazing experiments with protists and copepods on selected key toxin algal species in on-board incubations. The following main goals of the cruise programme were successfully addressed: 1) the phenotypic and genotypic characteristics of populations of selected HAB species were compared, including that of a new dinoflagellate identified as the proximal source of azaspiracid poisoning (AZP) in the North Sea; 2) the toxin profile and content of HAB species and toxin transfer and metabolism to the next trophic level were assessed and compared; and 3) insights into the relative importance of grazing by both metazoa and protists as a potential “top down” regulatory mechanism for population dynamics of HAB species was achieved.

Description: Bacterial diversity was explored among field samples and cultured isolates from coral reefs within the Veracruz Reef System. Bacterioplankton and bacteriobenthos were characterized by pyrosequencing 16S rRNA genes. Identified sequences belonged to the kingdom Bacteria and classified into 33 phyla. Proteobacteria (likely SAR11 clade) dominated in collective field samples, whereas Firmicutes were the most abundant taxa among cultured isolates. Bioinformatic sorting of sequences to family level revealed 223 bacterial families. Pseudomonadaceae, Exiguobacteraceae and Bacillaceae were dominant among cultured isolates. Vibrionaceae, Alteromonadaceae, and Flavobacteriaceae dominated in reef-associated sediments, whereas Rickettsiaceae and Synechoccaceae were more highly represented in the water column. Bacterial communities from sediments were more diverse than from the water column. This study reveals cryptic bacterial diversity among microenvironmental components of marine microbial reef communities subject to differential influence of anthropogenic stressors. Such investigations are critical for constructing scenarios of environmentally induced shifts in bacterial biodiversity and species composition.

Description: Benthic dinoflagellates produce a wide array of bioactive compounds, primarily polyketides, that cause toxic effects on human consumers of seafood and perhaps mediate species interactions in the benthic microenvironment. This study assesses toxic and other bioactive effects of the benthic dinoflagellate Amphidinium operculatum (strain AA60) in two targeted bioassays. The brine shrimp (Artemia salina) bioassay revealed lethal effects of direct exposure to live dinoflagellate cells (Treatment A) and even higher potency with ethanolic extracts of lysed cells (Treatment D). There were no inimical bioactive effects of components released to the aqueous growth medium (Treatment B) or from aqueous cell lysates (Treatment C). The hypothesis that released bioactive compounds provide a chemical defense against metazoan grazers is therefore not supported by these results. The cytotoxic effect of ethanolic crude extracts of this dinoflagellate exhibited mild to high growth reduction effects on six human cancer cell lines. In particular, crude cell-free extracts proved highly growth-inhibitory activity towards breast and lung cancer cell lines MCF-7 and SKLU-1, respectively. Preliminary anti-cancer results indicate that natural bioactive compounds from Amphidinium are worthy of structural characterization and further toxicological investigation as potential therapeutants.

Description: Climate change alters environmental conditions that are expected to have a profound effect on the biodiversity, community composition, and metabolic processes of microeukaryotic plankton in Arctic and Subarctic coastal waters. The molecular biodiversity [large subunit (LSU) rRNA gene] of three plankton size-fractions (micro-, nano-, and picoplankton) from coastal waters of ice-influenced west Greenland was compared with fractions from ice-free northwest Iceland within their summer environmental context. Putative metabolic functions were determined by differentially expressed mRNA (metatranscriptomics) of the microplankton. Temperature and salinity variations were more closely correlated than inorganic macronutrients with metabolic functions and community composition. Temperature explained much of the community variance, approximately 20% among micro- and nanoplankton, whereas other environmental factors accounted for rather low fractional variance (〈7%). Species of smaller cell-size were more evenly distributed (Pielou’s evenness index J) across regions, with a higher diversity and total abundance, and thereby indicating high plasticity. The metatranscriptomic profiles in these respective microeukaryotic communities revealed that diatoms were more plastic in their gene expression than dinoflagellates, but dinoflagellates had a more diverse, albeit homogeneously expressed, gene pool. This could be interpreted as expression of alternative lifestyle strategies, whereby the functionally more conservative diatoms fill their niches primarily through variable resource use, whereas dinoflagellates apparently differentiate their niches through more diverse lifestyles. Patterns of microeukaryotic diversity are thus primarily associated with differences in metabolic function and activity of diatom- versus dinoflagellate-dominated communities in Arctic and Subarctic waters during summer.

Description: The dinoflagellate genus Prorocentrum is globally represented by a wide variety of species found upon benthic and/or epiphytic substrates. Many epibenthic Prorocentrum species produce lipophilic polyether toxins, some of which act as potent protein phosphatase inhibitors and tumor-promoters associated with Diarrheic Shellfish Poisoning (DSP). Most members of the Prorocentrum lima species complex (PLSC) commonly found in the tropics and sub-tropics are toxigenic. Epiphytic and planktonic bacteria co-occur with toxigenic Prorocentrum but reciprocal allelochemical interactions are under-investigated. The aim of the present study was to identify the culturable bacteria collected together with isolates of the PLSC from seagrass (Thalassia testudinum) and macroalgae along tropical Atlantic coasts of Mexico, and to explore potential species interactions with selected isolates. Twenty-one bacterial genera belonging to Proteobacteria, Actinobacteria, and Bacteroidetes were identified by amplification of the 16S rRNA gene marker from nine clonal Prorocentrum cultures, with g-proteobacteria comprising the dominant class. A positive correlation was found between the bacterial genera associated with two Prorocentrum clones and the esterified toxin analog DTX1a-D8, but there was no apparent correlation between the other PLSC clones and their associated bacteria with the other five DSP toxins detected. No bacteriostatic or allelochemical response was found for cell- and culture medium extracts of five Prorocentrum isolates assayed for bioactivity against Staphylococcus sp. DMBS2 and Vibrio sp. HEL66. Bulk cell-washing of Prorocentrum PA1, followed by growth with antibiotics, was only effective in reducing bacterial load in the initial growth stages, but did not yield axenic cultures or lower bacterial cell densities throughout the culture cycle. Antibiotic treatment did not impair growth or survival of the dinoflagellate, or apparently affect DSP toxin production. There was no significant correlation between Prorocentrum cell volume, growth rate, bacterial cell counts, or cellular toxin concentration over the entire time-series culture cycle. Benthic Prorocentrum and associated bacterial communities comprise highly diverse and characteristic microbiomes upon substrates, and among compartments in culture, but this study provides little evidence that allelochemical interactions among Prorocentrum cells and associated bacteria originating from epibenthic substrates play a definable role in growth and toxigenicity.

Description: Molluscan safety monitoring programs search for control systems able to give an estimation of the concentration levels of marine toxins present in water, which could represent a risk for the health of consumers of shellfish products. In this study a deployment of passive sampling devices consisting of resin filled sachets was carried out weekly during two years in Alfacs Bay, a coastal embayment of the Ebro Delta in the NW Mediterranean, Spain. Lipophilic marine toxin concentration profiles were obtained analyzing the content of the polymeric resins after extraction and analysis by liquid chromatography coupled to a quadrupole ion trap mass spectrometer.

Description: Coupling Physiological Responses of the Toxic Haptophyte Prymnesium parvum to Patterns in Gene ExpressionMichael F. Freitag1, Uwe John1 & AD Cembella11Alfred Wegener Institute for Polar and Marine research, 27576 Bremerhaven, Germany 2Sára Beszteri, Alfred Wegener Institute, Bremerhaven GermanyA normalized cDNA library2 from the toxic haptophyte Prymnesium parvum was used to design an oligonucleotide-based microarray platform. Allelopathic interactions were investigated between P. parvum strain RL10 and the heterotrophic dinoflagellate Oxyhrris marina. Cultures of Prymnesium were incubated together with both whole cells and cell-free filtrate from Oxyhrris cultures. During incubation with Prymnesium, the presence of Oxyhrris cells, compared to cell-free culture medium, showed no differential effect on the gene expression profile of the haptophyte. A bioassay measuring the toxicity of Prymnesium against the sensitive cryptophyte Rhodomonas baltica was additionally performed. In this case, Rhodomonas viabilities following incubation with Prymnesium and Prymnesium previously incubated with O. marina medium were virtually identical. This suggests no correlation between the presence of O. marina medium, and induced toxicity in P. parvum. Similar experiments, involving the dinoflagellate Heterocapsa rotunda and an unclassified toxic crooccal cyanobacteria strain: NIVA CYA 331, are currently underway. All tentative unique genes (TUGs) identified from the microarray hybridization will be further classified through the use of a Prymnesium fosmid library prepared from axenic cultures.

Description: Marine dinoflagellates of the genus Alexandrium are well known for their ability to produce the potent neurotoxin saxitoxin and other analogues responsible for paralytic shellfish poisoning (PSP). Dinoflagellates are the only known eukaryotes confirmed to produce these neurotoxins, but they are also synthesized by certain brackish and freshwater cyanobacteria giving rise to speculations about their prokaryotic origin. Thus far, no distinct enzymes or genes involved in the actual synthesis of the toxins have been unequivocally identified in either dinoflagellates or cyanobacteria. However, detailed proposals for such a pathway exist, and the requirements for biosynthesis have been examined in vitro. In order to contribute to the search for genes involved in PSP toxin production, we developed a DNA microarray based upon an expressed sequence tag (EST) library from A. minutum. This species tends to produce a limited sub-set of PSP toxins, typically dominated by the sulfated carbamate derivatives known as gonyautoxins. The array was used in gene expression studies involving both toxic and non-toxic A. minutum strains, as well as different physiological conditions within one strain. We identified genes associated with the ability to produce PSP toxins in an inter-strain comparison. Here we compare these data to gene expression correlated with differences in toxicity under different physiological conditions.