Morphological, molecular, and toxin analysis of field populations of Alexandrium genus from the Argentine Sea.

In the Argentine Sea, blooms of toxigenic dinoflagellates of the Alexandrium tamarense species complex have led to fish and bird mortalities and human deaths as a consequence of paralytic shellfish poisoning (PSP). Yet little is known about the occurrence of other toxigenic species of the genus Alexandrium, or of their toxin composition beyond coastal waters. The distribution of Alexandrium species and related toxins in the Argentine Sea was determined by sampling surface waters on an oceanographic expedition during austral spring from ~39°S to 48°S. Light microscope and SEM analysis for species identification and enumeration was supplemented by confirmatory PCR analysis from field samples. The most frequent Alexandrium taxon identified by microscopy corresponded to the classical description of A. tamarense. Only weak signals of Group I from the A. tamarense species complex were detected by PCR of bulk field samples, but phylogenetic reconstruction of rDNA sequences from single cells from one station assigned them to ribotype Group I (Alexandrium catenella). PCR probes for Alexandrium minutum and Alexandrium ostenfeldii yielded a positive signal, although A. minutum morphology did not completely match the classical description. Analysis of PSP toxin composition of plankton samples revealed toxin profiles dominated by gonyautoxins (GTX1/4). The main toxic cyclic imine detected was 13-desMe-spirolide C and this supported the association with A. ostenfeldii in the field. This study represents the first integrated molecular, morphological and toxinological analysis of field populations of the genus Alexandrium in the Argentine Sea.

Guanidinium Toxins and Their Interactions with Voltage-Gated Sodium Ion Channels.

Guanidinium toxins, such as saxitoxin (STX), tetrodotoxin (TTX) and their analogs, are naturally occurring alkaloids with divergent evolutionary origins and biogeographical distribution, but which share the common chemical feature of guanidinium moieties. These guanidinium groups confer high biological activity with high affinity and ion flux blockage capacity for voltage-gated sodium channels (NaV). Members of the STX group, known collectively as paralytic shellfish toxins (PSTs), are produced among three genera of marine dinoflagellates and about a dozen genera of primarily freshwater or brackish water cyanobacteria. In contrast, toxins of the TTX group occur mainly in macrozoa, particularly among puffer fish, several species of marine invertebrates and a few terrestrial amphibians. In the case of TTX and analogs, most evidence suggests that symbiotic bacteria are the origin of the toxins, although endogenous biosynthesis independent from bacteria has not been excluded. The evolutionary origin of the biosynthetic genes for STX and analogs in dinoflagellates and cyanobacteria remains elusive. These highly potent molecules have been the subject of intensive research since the latter half of the past century; first to study the mode of action of their toxigenicity, and later as tools to characterize the role and structure of NaV channels, and finally as therapeutics. Their pharmacological activities have provided encouragement for their use as therapeutants for ion channel-related pathologies, such as pain control. The functional role in aquatic and terrestrial ecosystems for both groups of toxins is unproven, although plausible mechanisms of ion channel regulation and chemical defense are often invoked. Molecular approaches and the development of improved detection methods will yield deeper understanding of their physiological and ecological roles. This knowledge will facilitate their further biotechnological exploitation and point the way towards development of pharmaceuticals and therapeutic applications.

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean).

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.

Transcriptomic characterisation and genomic glimps into the toxigenic dinoflagellate Azadinium spinosum, with emphasis on polykeitde synthase genes.

BACKGROUND: Unicellular dinoflagellates are an important group of primary producers within the marine plankton community. Many of these species are capable of forming harmful algae blooms (HABs) and of producing potent phycotoxins, thereby causing deleterious impacts on their environment and posing a threat to human health. The recently discovered toxigenic dinoflagellate Azadinium spinosum is known to produce azaspiracid toxins. These toxins are most likely produced by polyketide synthases (PKS). Recently, PKS I-like transcripts have been identified in a number of dinoflagellate species. Despite the global distribution of A. spinosum, little is known about molecular features. In this study, we investigate the genomic and transcriptomic features of A. spinosum with a focus on polyketide synthesis and PKS evolution. RESULTS: We identify orphan and homologous genes by comparing the transcriptome data of A. spinosum with a diverse set of 18 other dinoflagellates, five further species out of the Rhizaria Alveolate Stramelopile (RAS)-group, and one representative from the Plantae. The number of orphan genes in the analysed dinoflagellate species averaged 27%. In contrast, within the A. spinosum transcriptome, we discovered 12,661 orphan transcripts (18%). The dinoflagellates toxins known as azaspiracids (AZAs) are structurally polyethers; we therefore analyse the transcriptome of A. spinosum with respect to polyketide synthases (PKSs), the primary biosynthetic enzymes in polyketide synthesis. We find all the genes thought to be potentially essential for polyketide toxin synthesis to be expressed in A. spinosum, whose PKS transcripts fall into the dinoflagellate sub-clade in PKS evolution. CONCLUSIONS: Overall, we demonstrate that the number of orphan genes in the A. spinosum genome is relatively small compared to other dinoflagellate species. In addition, all PKS domains needed to produce the azaspiracid carbon backbone are present in A. spinosum. Our study underscores the extraordinary evolution of such gene clusters and, in particular, supports the proposed structural and functional paradigm for PKS Type I genes in dinoflagellates.

Functional diversity of bacterial microbiota associated with the toxigenic benthic dinoflagellate Prorocentrum.

Interactions between bacterial microbiota and epibenthic species of the dinoflagellate Prorocentrum may define the onset and persistence of benthic harmful algal blooms (bHABs). Chemical ecological interactions within the dinoflagellate phycosphere potentially involve a complex variety of organic molecules, metabolites, and toxins, including undefined bioactive compounds. In this study, the bacterial diversity and core members of the dinoflagellate-associated microbiota were defined from 11 strains of three epibenthic Prorocentrum species, representing three geographically disjunct locations within Mexican coastal waters. Microbiota profiles in stable monoclonal Prorocentrum cultures were obtained by sequencing amplicons of the V3-V4 region of the 16S rRNA gene. Thirteen classes of bacteria were identified among dinoflagellate clones, where Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia were consistently dominant. The bacterial community structure exhibited significantly different grouping by the location of origin of dinoflagellate clones. No significant diversity difference was found among free-living or unattached bacteria in the dinoflagellate culture medium (M) compared with those in closer association with the dinoflagellate host cells (H). Twelve taxa were defined as core members of the bacterial assemblage, representing the genera Algiphilus, Cohaesibacter, Labrenzia, Mameliella, Marinobacter, Marivita, Massilia, Muricauda, Roseitalea, and an unclassified member of the Rhodobacteraceae. The core members are inferred to significantly contribute to primary and secondary metabolic functions, but no direct correlation with dinoflagellate toxigenicity was apparent. Overall the bacterial profile and implied gene functionality indicated a suite of positive interactions, suggesting either mutualism or commensalism with the dinoflagellate. The further characterization and interpretation of specific gene functions and interactions between bacteria and dinoflagellates, such as epibenthic members of genus Prorocentrum, are key to understanding their role in toxigenesis and bHAB development.

Emerging phylogeographic perspective on the toxigenic diatom genus Pseudo-nitzschia in coastal northern European waters and gateways to eastern Arctic seas: Causes, ecological consequences and socio-economic impacts.

The diatom Pseudo-nitzschia H. Peragallo is perhaps the most intensively researched genus of marine pennate diatoms, with respect to species diversity, life history strategies, toxigenicity, and biogeographical distribution. The global magnitude and consequences of harmful algal blooms (HABs) of Pseudo-nitzschia are particularly significant because of the high socioeconomic impacts and environmental and human health risks associated with the production of the neurotoxin domoic acid (DA) among populations of many (although not all) species. This has led to enhanced monitoring and mitigation strategies for toxigenic Pseudo-nitzschia blooms and their toxins in recent years. Nevertheless, human adaptive actions based on future scenarios of bloom dynamics and proposed shifts in biogeographical distribution under climate-change regimes have not been implemented on a regional scale. In the CoCliME (Co-development of climate services for adaptation to changing marine ecosystems) program these issues were addressed with respect to past, current and anticipated future status of key HAB genera such as Pseudo-nitzschia and expected benefits of enhanced monitoring. Data on the distribution and frequency of Pseudo-nitzschia blooms in relation to DA occurrence and associated amnesic shellfish toxin (AST) events were evaluated in a contemporary and historical context over the past several decades from key northern CoCliME Case Study areas. The regional studies comprised the greater North Sea and adjacent Kattegat-Skagerrak and Norwegian Sea, eastern North Atlantic marginal seas and Arctic gateways, and the Baltic Sea. The first evidence of possible biogeographical expansion of Pseudo-nitzschia taxa into frontier eastern Arctic gateways was provided from DNA barcoding signatures. Key climate change indicators, such as salinity, temperature, and water-column stratification were identified as drivers of upwelling and advection related to the distribution of regional Pseudo-nitzschia blooms. The possible influence of changing variables on bloom dynamics, magnitude, frequency and spatial and temporal distribution were interpreted in the context of regional ocean climate models. These climate change indicators may play key roles in selecting for the occurrence and diversity of Pseudo-nitzschia species within the broader microeukaryote communities. Shifts to higher temperature and lower salinity regimes predicted for the southern North Sea indicate the potential for high-magnitude Pseudo-nitzschia blooms, currently absent from this area. Ecological and socioeconomic impacts of Pseudo-nitzschia blooms are evaluated with reference to effects on fisheries and mariculture resources and coastal ecosystem function. Where feasible, effective adaptation strategies are proposed herein as emerging climate services for the northern CoCLiME region.

The globally distributed genus Alexandrium: multifaceted roles in marine ecosystems and impacts on human health.

The dinoflagellate genus Alexandrium is one of the major harmful algal bloom (HAB) genera with respect to the diversity, magnitude and consequences of blooms. The ability of Alexandrium to colonize multiple habitats and to persist over large regions through time is testimony to the adaptability and resilience of this group of species. Three different families of toxins, as well as an as yet incompletely characterized suite of allelochemicals are produced among Alexandrium species. Nutritional strategies are equally diverse, including the ability to utilize a range of inorganic and organic nutrient sources, and feeding by ingestion of other organisms. Many Alexandrium species have complex life histories that include sexuality and often, but not always, cyst formation, which is characteristic of a meroplanktonic life strategy and offers considerable ecological advantages. Due to the public health and ecosystem impacts of Alexandrium blooms, the genus has been extensively studied, and there exists a broad knowledge base that ranges from taxonomy and phylogeny through genomics and toxin biosynthesis to bloom dynamics and modeling. Here we present a review of the genus Alexandrium, focusing on the major toxic and otherwise harmful species.

Temporal and spatial distribution of epibenthic dinoflagellates in the Kattegat-Skagerrak, NE Atlantic-Focus on Prorocentrum lima and Coolia monotis.

Epibenthic dinoflagellates occur globally and include many toxin-producing species of concern to human health and benthic ecosystem function. Such benthic harmful algal blooms (BHABs) have been well described from tropical and sub-tropical coastal environments, but assessments from north temperate waters, e.g., northern Europe, and polar regions are scarce. The present study addressed the biodiversity and distribution of potentially toxic epibenthic dinoflagellate populations along the west coast of Sweden (Kattegat-Skagerrak) by morphological and molecular criteria. Morphological analysis conducted by light- and electron-microscopy was then linked by DNA barcoding of the V4 region of 18S rRNA gene sequences to interpret taxonomic and phylogenetic relationships. The presence of two potentially toxigenic epibenthic dinoflagellates, Prorocentrum lima (Ehrenberg) F.Stein and Coolia monotis Meunier was confirmed, along with a description of their spatial and temporal distribution. For P. lima, one third of the cell abundance values exceeded official alarm thresholds for potentially toxic BHAB events (>1000 cells gr-1 of macroalgae fresh weight). The same species were recorded consecutively for two summers, but without significant temporal variation in cell densities. SEM analyses confirmed the presence of other benthic Prorocentrum species: P. fukuyoi complex, P. cf. foraminosum and P. cf. hoffmannianum. Analyses of the V4 region of the 18S rRNA gene also indicated the presence P. compressum, P. hoffmannianum, P. foraminosum, P. fukuyoi, and P. nanum. These findings provide the first biogeographical evidence of toxigenic benthic dinoflagellates along the west coast of Sweden, in the absence of ongoing monitoring to include epibenthic dinoflagellates. Harmful events due to the presence of Coolia at shellfish aquaculture sites along the Kattegat-Skagerrak are likely to be rather marginal because C. monotis is not known to be toxigenic. In any case, as a preliminary assessment, the results highlight the risk of diarrhetic shellfish poisoning (DSP) events caused by P. lima, which may affect the development and sustainability of shellfish aquaculture in the region.

Apparent biogeographical trends in Alexandrium blooms for northern Europe: identifying links to climate change and effective adaptive actions.

The marine dinoflagellate Alexandrium Halim represents perhaps the most significant and intensively studied genus with respect to species diversity, life history strategies, toxigenicity, biogeographical distribution, and global magnitude and consequences harmful algal blooms (HABs). The socioeconomic impacts, environmental and human health risks, and mitigation strategies for toxigenic Alexandrium blooms have also been explored in recent years. Human adaptive actions based on future scenarios of bloom dynamics and shifts in biogeographical distribution under climate-change parameters remain under development and not yet implemented on a regional scale. In the CoCliME (Co-development of climate services for adaptation to changing marine ecosystems) project these issues were addressed with respect to past, current and anticipated future status of key HAB genera and expected benefits of enhanced monitoring. Data on the distribution and frequency of Alexandrium blooms related to paralytic shellfish toxin (PST) events from key CoCliME Case Study areas, comprising the North Sea and adjacent Kattegat-Skagerrak, Norwegian Sea, and Baltic Sea, and eastern North Atlantic marginal seas, were evaluated in a contemporary and historical context over the past several decades. The first evidence of possible biogeographical expansion of Alexandrium taxa into eastern Arctic gateways was provided from DNA barcoding signatures. Various key climate change indicators, such as salinity, temperature, and water-column stratification, relevant to Alexandrium bloom initiation and development were identified. The possible influence of changing variables on bloom dynamics, magnitude, frequency and spatial and temporal distribution were interpreted in the context of regional ocean climate models. These climate change impact indicators may play key roles in selecting for the occurrence and diversity of Alexandrium species within the broader microeukaryote communities. For example, shifts to higher temperature and lower salinity regimes predicted for the southern North Sea indicate the potential for increased Alexandrium blooms, currently absent from this area. Ecological and socioeconomic impacts of Alexandrium blooms and effects on fisheries and aquaculture resources and coastal ecosystem function are evaluated, and, where feasible, effective adaptation strategies are proposed herein as emerging climate services.

Spatial and biological oceanographic insights into the massive fish-killing bloom of the haptophyte Chrysochromulina leadbeateri in northern Norway.

A bloom of the fish-killing haptophyte Chrysochromulina leadbeateri in northern Norway during May and June 2019 was the most harmful algal event ever recorded in the region, causing massive mortalities of farmed salmon. Accordingly, oceanographic and biodiversity aspects of the bloom were studied in unprecedented detail, based on metabarcoding and physico-chemical and biotic factors related with the dynamics and distribution of the bloom. Light- and electron-microscopical observations of nanoplankton samples from diverse locations confirmed that C. leadbeateri was dominant in the bloom and the primary cause of associated fish mortalities. Cell counts by light microscopy and flow cytometry were obtained throughout the regional bloom within and adjacent to five fjord systems. Metabarcoding sequences of the V4 region of the 18S rRNA gene from field material collected during the bloom and a cultured isolate from offshore of Tromsøy island confirmed the species identification. Sequences from three genetic markers (18S, 28S rRNA gene and ITS region) verified the close if not identical genetic similarity to C. leadbeateri from a previous massive fish-killing bloom in 1991 in northern Norway. The distribution and cell abundance of C. leadbeateri and related Chrysochromulina species in the recent incident were tracked by integrating observations from metabarcoding sequences of the V4 region of the 18S rRNA gene. Metabarcoding revealed at least 14 distinct Chrysochromulina variants, including putative cryptic species. C. leadbeateri was by far the most abundant of these species, but with high intraspecific genetic variability. Highest cell abundance of up to 2.7 × 107 cells L - 1 of C. leadbeateri was found in Balsfjorden; the high cell densities were associated with stratification near the pycnocline (at ca. 12 m depth) within the fjord. The cell abundance of C. leadbeateri showed positive correlations with temperature, negative correlation with salinity, and a slightly positive correlation with ambient phosphate and nitrate concentrations. The spatio-temporal succession of the C. leadbeateri bloom suggests independent initiation from existing pre-bloom populations in local zones, perhaps sustained and supplemented over time by northeastward advection of the bloom from the fjords.

The relevance of marine chemical ecology to plankton and ecosystem function: an emerging field.

Marine chemical ecology comprises the study of the production and interaction of bioactive molecules affecting organism behavior and function. Here we focus on bioactive compounds and interactions associated with phytoplankton, particularly bloom-forming diatoms, prymnesiophytes and dinoflagellates. Planktonic bioactive metabolites are structurally and functionally diverse and some may have multiple simultaneous functions including roles in chemical defense (antipredator, allelopathic and antibacterial compounds), and/or cell-to-cell signaling (e.g., polyunsaturated aldehydes (PUAs) of diatoms). Among inducible chemical defenses in response to grazing, there is high species-specific variability in the effects on grazers, ranging from severe physical incapacitation and/or death to no apparent physiological response, depending on predator susceptibility and detoxification capability. Most bioactive compounds are present in very low concentrations, in both the producing organism and the surrounding aqueous medium. Furthermore, bioactivity may be subject to synergistic interactions with other natural and anthropogenic environmental toxicants. Most, if not all phycotoxins are classic secondary metabolites, but many other bioactive metabolites are simple molecules derived from primary metabolism (e.g., PUAs in diatoms, dimethylsulfoniopropionate (DMSP) in prymnesiophytes). Producing cells do not seem to suffer physiological impact due to their synthesis. Functional genome sequence data and gene expression analysis will provide insights into regulatory and metabolic pathways in producer organisms, as well as identification of mechanisms of action in target organisms. Understanding chemical ecological responses to environmental triggers and chemically-mediated species interactions will help define crucial chemical and molecular processes that help maintain biodiversity and ecosystem functionality.

Toxicity Bioassay and Cytotoxic Effects of the Benthic Marine Dinoflagellate Amphidinium operculatum.

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.

Diversity of Bacterioplankton and Bacteriobenthos from the Veracruz Reef System, Southwestern Gulf of Mexico.

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.

Harmful algal blooms and their effects in coastal seas of Northern Europe.

Harmful algal blooms (HAB) are recurrent phenomena in northern Europe along the coasts of the Baltic Sea, Kattegat-Skagerrak, eastern North Sea, Norwegian Sea and the Barents Sea. These HABs have caused occasional massive losses for the aquaculture industry and have chronically affected socioeconomic interests in several ways. This status review gives an overview of historical HAB events and summarises reports to the Harmful Algae Event Database from 1986 to the end of year 2019 and observations made in long term monitoring programmes of potentially harmful phytoplankton and of phycotoxins in bivalve shellfish. Major HAB taxa causing fish mortalities in the region include blooms of the prymnesiophyte Chrysochromulina leadbeateri in northern Norway in 1991 and 2019, resulting in huge economic losses for fish farmers. A bloom of the prymesiophyte Prymnesium polylepis (syn. Chrysochromulina polylepis) in the Kattegat-Skagerrak in 1988 was ecosystem disruptive. Blooms of the prymnesiophyte Phaeocystis spp. have caused accumulations of foam on beaches in the southwestern North Sea and Wadden Sea coasts and shellfish mortality has been linked to their occurrence. Mortality of shellfish linked to HAB events has been observed in estuarine waters associated with influx of water from the southern North Sea. The first bloom of the dictyochophyte genus Pseudochattonella was observed in 1998, and since then such blooms have been observed in high cell densities in spring causing fish mortalities some years. Dinoflagellates, primarily Dinophysis spp., intermittently yield concentrations of Diarrhetic Shellfish Toxins (DST) in blue mussels, Mytilus edulis, above regulatory limits along the coasts of Norway, Denmark and the Swedish west coast. On average, DST levels in shellfish have decreased along the Swedish and Norwegian Skagerrak coasts since approximately 2006, coinciding with a decrease in the cell abundance of D. acuta. Among dinoflagellates, Alexandrium species are the major source of Paralytic Shellfish Toxins (PST) in the region. PST concentrations above regulatory levels were rare in the Skagerrak-Kattegat during the three decadal review period, but frequent and often abundant findings of Alexandrium resting cysts in surface sediments indicate a high potential risk for blooms. PST levels often above regulatory limits along the west coast of Norway are associated with A. catenella (ribotype Group 1) as the main toxin producer. Other Alexandrium species, such as A. ostenfeldii and A. minutum, are capable of producing PST among some populations but are usually not associated with PSP events in the region. The cell abundance of A. pseudogonyaulax, a producer of the ichthyotoxin goniodomin (GD), has increased in the Skagerrak-Kattegat since 2010, and may constitute an emerging threat. The dinoflagellate Azadinium spp. have been unequivocally linked to the presence of azaspiracid toxins (AZT) responsible for Azaspiracid Shellfish Poisoning (AZP) in northern Europe. These toxins were detected in bivalve shellfish at concentrations above regulatory limits for the first time in Norway in blue mussels in 2005 and in Sweden in blue mussels and oysters (Ostrea edulis and Crassostrea gigas) in 2018. Certain members of the diatom genus Pseudo-nitzschia produce the neurotoxin domoic acid and analogs known as Amnesic Shellfish Toxins (AST). Blooms of Pseudo-nitzschia were common in the North Sea and the Skagerrak-Kattegat, but levels of AST in bivalve shellfish were rarely above regulatory limits during the review period. Summer cyanobacteria blooms in the Baltic Sea are a concern mainly for tourism by causing massive fouling of bathing water and beaches. Some of the cyanobacteria produce toxins, e.g. Nodularia spumigena, producer of nodularin, which may be a human health problem and cause occasional dog mortalities. Coastal and shelf sea regions in northern Europe provide a key supply of seafood, socioeconomic well-being and ecosystem services. Increasing anthropogenic influence and climate change create environmental stressors causing shifts in the biogeography and intensity of HABs. Continued monitoring of HAB and phycotoxins and the operation of historical databases such as HAEDAT provide not only an ongoing status report but also provide a way to interpret causes and mechanisms of HABs.

Diversity and regional distribution of harmful algal events along the Atlantic margin of Europe.

The IOC-ICES-PICES Harmful Algal Event Database (HAEDAT) was used to describe the diversity and spatiotemporal distribution of harmful algal events along the Atlantic margin of Europe from 1987 - 2018. The majority of events recorded are caused by Diarrhetic Shellfish Toxins (DSTs). These events are recorded annually over a wide geographic area from southern Spain to northern Scotland and Iceland, and are responsible for annual closures of many shellfish harvesting areas. The dominant causative dinoflagellates, members of the morphospecies 'Dinophysis acuminata complex' and D. acuta, are common in the waters of the majority of countries affected. There are regional differences in the causative species associated with PST events; the coasts of Spain and Portugal with the dinoflagellates Alexandrium minutum and Gymnodinium catenatum, north west France/south west England/south Ireland with A. minutum, and Scotland/Faroe Islands/Iceland with A. catenella. This can influence the duration and spatial scale of PST events as well as the toxicity of shellfish. The diatom Pseudo-nitzschia australis is the most widespread Domoic Acid (DA) producer, with records coming from Spain, Portugal, France, Ireland and the UK. Amnesic Shellfish Toxins (ASTs) have caused prolonged closures for the scallop fishing industry due to the slow depuration rate of DA. Amendments to EU shellfish hygiene regulations introduced between 2002 and 2005 facilitated end-product testing and sale of adductor muscle. This reduced the impact of ASTs on the scallop fishing industry and thus the number of recorded HAEDAT events. Azaspiracids (AZAs) are the most recent toxin group responsible for events to be characterised in the ICES area. Events associated with AZAs have a discrete distribution with the majority recorded along the west coast of Ireland. Ciguatera Poisoning (CP) has been an emerging issue in the Canary Islands and Madeira since 2004. The majority of aquaculture and wild fish mortality events are associated with blooms of the dinoflagellate Karenia mikimotoi and raphidophyte Heterosigma akashiwo. Such fish killing events occur infrequently yet can cause significant mortalities. Interannual variability was observed in the annual number of HAEDAT areas with events associated with individual shellfish toxin groups. HABs represent a continued risk for the aquaculture industry along the Atlantic margin of Europe and should be accounted for when considering expansion of the industry or operational shifts to offshore areas.

Comparative gene expression in toxic versus non-toxic strains of the marine dinoflagellate Alexandrium minutum.

BACKGROUND: The dinoflagellate Alexandrium minutum typically produces paralytic shellfish poisoning (PSP) toxins, which are known only from cyanobacteria and dinoflagellates. While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production in dinoflagellates remains elusive. RESULTS: We constructed and analysed an expressed sequence tag (EST) library of A. minutum, which contained 15,703 read sequences yielding a total of 4,320 unique expressed clusters. Of these clusters, 72% combined the forward-and reverse reads of at least one bacterial clone. This sequence resource was then used to construct an oligonucleotide microarray. We analysed the expression of all clusters in three different strains. While the cyanobacterial PSP toxin genes were not found among the A. minutum sequences, 192 genes were differentially expressed between toxic and non-toxic strains. CONCLUSIONS: Based on this study and on the lack of identified PSP synthesis genes in the two existent Alexandrium tamarense EST libraries, we propose that the PSP toxin genes in dinoflagellates might be more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer. As a starting point to identify possible PSP toxin-associated genes in dinoflagellates without relying on a priori sequence information, the sequences only present in mRNA pools of the toxic strain can be seen as putative candidates involved in toxin synthesis and regulation, or acclimation to intracellular PSP toxins.

Implications of life-history transitions on the population genetic structure of the toxigenic marine dinoflagellate Alexandrium tamarense.

Genotypic or phenotypic markers for characterization of natural populations of marine microalgae have typically addressed questions regarding differentiation among populations, usually with reference to a single or few clonal isolates. Based upon a large number of contemporaneous isolates from the same geographical population of the toxigenic species Alexandrium tamarense from the North Sea, we uncovered significant genetic substructure and low but significant multilocus linkage disequilibrium (LD) within the planktonic population. Between the alternative molecular genotyping approaches, only amplified fragment length polymorphism (AFLP) revealed cryptic genetic population substructure by Bayesian clustering, whereas microsatellite markers failed to yield concordant patterns. Both markers, however, gave evidence for genetic differentiation of population subgroups as defined by AFLP. A considerable portion of multilocus LD could be attributed to population subdivision. The remaining LD within population subgroups is interpreted as an indicator of frequency shifts of clonal lineages during vegetative growth of planktonic populations. Phenotypic characters such as cellular content and composition of neurotoxins associated with paralytic shellfish poisoning (PSP) and allelochemical properties may contribute to intra- or inter-annual differentiation of planktonic populations, if clonal lineages that express these characters are selectively favoured. Nevertheless, significant phenotypic differentiation for these characters among the genetically differentiated subgroups was only detected for PSP toxin content in two of the four population subgroups. By integrating the analysis of phenotypic and genotypic characteristics, we developed a conceptual population genetic model to explain the importance of life-cycle dynamics and transitions in the evolutionary ecology of these dinoflagellates.

Preliminary characterization of extracellular allelochemicals of the toxic marine dinoflagellate Alexandrium tamarense using a Rhodomonas salina bioassay.

Members of the marine dinoflagellate genus Alexandrium are known to exude allelochemicals, unrelated to well-known neurotoxins (PSP-toxins, spirolides), with negative effects on other phytoplankton and marine grazers. Physico/chemical characterization of extracellular lytic compounds of A. tamarense, quantified by Rhodomonas salina bioassay, showed that the lytic activity, and hence presumably the compounds were stable over wide ranges of temperatures and pH and were refractory to bacterial degradation. Two distinct lytic fractions were collected by reversed-phase solid-phase extraction. The more hydrophilic fraction accounted for about 2% of the whole lytic activity of the A. tamarense culture supernatant, while the less hydrophilic one accounted for about 98% of activity. Although temporal stability of the compounds is high, substantial losses were evident during purification. Lytic activity was best removed from aqueous phase with chloroform-methanol (3:1). A "pseudo-loss" of lytic activity in undisturbed and low-concentrated samples and high activity of an emulsion between aqueous and n-hexane phase after liquid-liquid partition are strong evidence for the presence of amphipathic compounds. Lytic activity in the early fraction of gel permeation chromatography and lack of activity after 5 kD ultrafiltration indicate that the lytic agents form large aggregates or macromolecular complexes.

Novel insights into evolution of protistan polyketide synthases through phylogenomic analysis.

Polyketide synthase (PKS) enzymes are large multi-domain complexes that structurally and functionally resemble the fatty acid synthases involved in lipid metabolism. Polyketide biosynthesis of secondary metabolites and hence functional PKS genes are widespread among bacteria, fungi and streptophytes, but the Type I was formerly known only from bacteria and fungi. Recently Type I PKS genes were also uncovered in the genomes of some alveolate protists. Here we show that the newly sequenced genomes of representatives of other protist groups, specifically the chlorophytes Ostreococcus tauri, O. lucimarinus, and Chlamydomonas reinhardtii, and the haptophyte Emiliania huxleyi also contain putative modular Type I PKS genes. Based on the patchy phylogenetic distribution of this gene type among eukaryotic microorganisms, the question arises whether they originate from recent lateral gene transfer from bacteria. Our phylogenetic analyses do not indicate such an evolutionary history. Whether Type I PKS genes originated several times independently during eukaryotic evolution or were rather lost in many extant lineages cannot yet be answered. In any case, we show that environmental genome sequencing projects are likely to be a valuable resource when mining for genes resembling protistan PKS I genes.

Unambiguous identification of pectenotoxin-1 and distribution of pectenotoxins in plankton from the North Sea.

Lipophilic phycotoxins in size-fractionated plankton net tows (20 mum mesh-size) were measured on-board during a month-long oceanographic cruise in North Sea coastal waters. Tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS) was employed for the detection and quantification of a broad spectrum of known and putative phycotoxins. For pectenotoxins (PTXs) the following ion masses ([M + NH(4)](+)) were monitored: m/z 876 for PTX-2, m/z 892 for PTX-11 and PTX-13, and m/z 874 for PTX-12 and PTX-14. The PTX levels in net plankton were highest along the Danish north coast, but levels over 50 ng per net tow were also detected on the southern Scottish East coast and in the northern Skagerrak. Abundance of PTXs was highly correlated with the occurrence of the marine dinoflagellate Dinophysis spp. Whereas in the eastern North Sea PTX-2 was the most abundant PTX, in the western North Sea PTX-1 was the major component, but it was also present in lower proportions in the Norwegian and Danish waters than in the western North Sea. Isobaric PTX-11 was absent or only detected at trace levels throughout the entire cruise, and PTX-13 and PTX-14 were not detected at all. The identity of PTX-1 was confirmed by comparison of retention time and mass spectrum of the North Sea phytoplankton sample to PTX-1 previously isolated from shellfish. Statistical analysis showed the best correlation between the occurrence of PTX-1 and Dinophysis acuminata cell concentration. Nevertheless, we could not rule out the possibility of metabolic transformations of PTXs by organisms that have grazed upon Dinophysis. Such biotransformations could conceivably occur in heterotrophic dinoflagellates or ciliates, or even via oxidation in copepod fecal pellets. In any case, this study confirmed the presence of PTX-1 in the plankton and is the first definitive report of this toxin in the North Sea.