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A unialgal culture of aPseudo-nitzschiaspecies dominant in the plankton of Algoa Bay in the spring of 2012 was established by isolation of clonal chains of cells. Identification of the species asPseudo-nitzschia multiserieswas based on frustule morphometrics provided by light and scanning electron microscopy, and confirmed by phylogenetic analysis of the LSU rDNA gene. Cultures were shown to produce domoic acid (DA) as measured by ELISA and LC/MS-MS methods, and levels of cellular DA were ∼0.1 pg cell–1. Although it is recognised as a cosmopolitan species, these observations provide the first account of this toxic diatom in the coastal waters of South Africa. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Martínez-Mercado et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

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.

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., (Copyright © 2021. Published by Elsevier B.V.)

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., Competing Interests: The authors declare no conflict of interest.

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 (Na V ). 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 Na V 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., Competing Interests: The authors declare no conflict of interest.

Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na⁺ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

The public health, tourism, fisheries, and ecosystem impacts from harmful algal blooms (HABs) have all increased over the past few decades. This has led to heightened scientific and regulatory attention, and the development of many new technologies and approaches for research and management. This, in turn, is leading to significant paradigm shifts with regard to, e.g., our interpretation of the phytoplankton species concept (strain variation), the dogma of their apparent cosmopolitanism, the role of bacteria and zooplankton grazing in HABs, and our approaches to investigating the ecological and genetic basis for the production of toxins and allelochemicals. Increasingly, eutrophication and climate change are viewed and managed as multifactorial environmental stressors that will further challenge managers of coastal resources and those responsible for protecting human health. Here we review HAB science with an eye toward new concepts and approaches, emphasizing, where possible, the unexpected yet promising new directions that research has taken in this diverse 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.

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.

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.

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.

Biosynthetic origins of the cyclic imine toxin 13-desmethyl spirolide C were determined by supplementing cultures of the toxigenic dinoflagellate Alexandrium ostenfeldii with stable isotope-labeled precursors [1,2-13C2]acetate, [1-13C]acetate, [2-13CD3]acetate, and [1,2-13C2,15N]glycine and measuring the incorporation patterns by 13C NMR spectroscopy. Despite partial scrambling of the acetate labels, the results show that most carbons of the macrocycle are polyketide-derived and that glycine is incorporated as an intact unit into the cyclic imine moiety. This work represents the first conclusive evidence that such cyclic imine toxins are polyketides and provides support for biosynthetic pathways previously defined for other polyether dinoflagellate toxins.

Using LC/MS methodology, spirolides were detected in two clonal isolates of Alexandrium ostenfeldii isolated from Limfjorden, Denmark. Examination of the LC/MS profiles of extracts from these Danish cultures revealed the presence of two dominant peaks representing two previously unidentified spirolide components and one minor peak identified as the previously reported desmethyl spirolide C (1). Culturing of these clonal strains, LF 37 and LF 38, of A. ostenfeldii resulted in the accumulation of sufficient cell biomass to allow for the isolation and structure elucidation of two new spirolides, 13,19-didesmethylspirolide C (2) and spirolide G (3). While 2 was found to differ from 1 only in that it contained one less methyl group, 3 was the first spirolide to be isolated that contained a 5:6:6-trispiroketal ring system. The effect of this new feature on the toxicity of 3 relative to other spirolides is presently being pursued.

Three additional marine toxins, spirolides A (1), C (3), and 13-desmethyl-C (7), were isolated from contaminated scallops and phytoplankton collections obtained from a Nova Scotian aquaculture site, as well as from batch cultures of the dinoflagellate Alexandrium ostenfeldii obtained as a single-cell isolate from these phytoplankton assemblages. The structures of these new spirolide derivatives, characterized by mass spectrometry and NMR, indicate a close relationship with spirolides B (2) and D (4) isolated previously from contaminated shellfish in the same area. All of these compounds display "fast-acting" toxicity in the traditional bioassay used for monitoring shellfish, and this is related to the presence of a cyclic imine function in all these compounds. Those spirolides containing a vicinal dimethyl group in the seven-membered ring are resistant to oxalic acid hydrolysis, whereas those that do not are readily hydrolyzed. These observations suggest that the extra methyl group on the seven-membered imine ring of 3, 4, and 7 appears to block the process of imine hydrolysis perhaps by stereochemical interference.

A novel micro-extraction technique was applied to the extraction of biologically active macrocyclic imines known as spirolides from pooled individual cells isolated from spirolide-rich plankton material. For comparison, this method was also applied to pooled individual cells isolated from a unialgal culture of the marine dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech & Tangen, a species known to produce spirolides. Both athecate cells and motile forms of gonyaulacoid dinoflagellates derived from size-fractionated plankton material from Nova Scotia, Canada were sorted and pooled by the glass micropipette isolation technique and by flow cytometry. The development of a highly sensitive analytical method for spirolides (detection limit 2 ng ml(-1) for spirolide B) using liquid chromatography-mass spectrometry (LC-MS) and application to micro-extracted samples allowed the accurate determination of spirolide composition in as few as 50 cells. Total spirolide concentrations (fmol cell(-1)) calculated from pooled micropipette isolated cells were very consistent with those based upon bulk- or micro-extractions of A. ostenfeldii cells from unialgal batch cultures in exponential growth phase. The results of the pooled cell selection from field material from two sites in Nova Scotia confirmed the association of spirolides with vegetative cells of A. ostenfeldii and related athecate forms. Combining these techniques represents a highly sensitive method for the analysis of marine toxins within complex plankton matrices, even when the toxigenic species is in low abundance, by enrichment of the target organism.

The cross-reactivity of the 6/50 monoclonal anti-okadaic acid antibody (mAb) to the recently discovered diarrhetic shellfish poisoning (DSP) metabolites dinophysistoxin-4 (DTX-4), dinophysistoxin-5 (DTX-5), and an okadaic acid (OA) diol ester was determined using a competitive indirect enzyme-linked immunosorbent assay (ELISA). The reactivity of the antibody to these molecules was compared to that with OA; the 6/50 mAb recognized all of these DSP compounds with equal sensitivity within the working range of the antibody (10-100 nM for OA). This confirms the ability of the antibody to detect all DSP compounds when used in analyses including ELISA and immunocytochemistry.

Production of domoic acid (DA) by the pennate diatom Pseudo-nitzschia multiseries is associated with physiological stress caused by silicate (Si) and/or phosphate (P) limitation. Such limitation may promote DA synthesis by (1) reducing primary metabolic activity, thus making available necessary precursors, high energy compounds, and cofactors, and (2) favoring the expression of genes involved in the biosynthesis of this toxin. In the case of Si and P-limitation, DNA synthesis and the progression through the cell division cycle are slowed, perhaps prolonging or arresting the cells in the stage of the division cycle which is most conducive to DA production. However, N-limitation results in an insufficient pool of cellular free N, which restricts synthesis of this nitrogenous toxin. A continuous supply of photophosphorylated high-energy intermediates (e.g., ATP and NADPH) is necessary for DA synthesis. In order to better understand the mechanism(s) of DA production, more studies are needed to elucidate: (1) the details of the biosynthetic pathway, (2) the regulation of enzymes involved in the pathway, (3) the relation between DA synthesis and the cell division cycle, (4) the cellular compartmentalization of DA biosynthesis, and (5) other environmental factors that may trigger DA production. Finally, these studies should be extended to include toxigenic Pseudo-nitzschia species other than P. multiseries, to confirm the commonality of these mechanisms.

Accumulation of paralytic shellfish poisoning (PSP) toxins by surfclams, Spisula solidissima, was studied over a period of two years at two inshore locations in southern Maine and at six stations on Georges Bank in the Gulf of Maine. Whole animals as well as individual tissues (siphon, mantle, digestive gland, foot, adductor muscle, gill) were analyzed for PSP-toxicity levels using the standard AOAC mouse bioassay. Analyses of gut contents were carried out on surfclams from both inshore and offshore locations to identify the type of particles ingested. Surfclams feed primarily on phytoplankton and detrital material characteristic of the overlying seawater and surface sediment. No evidence was found for any selection based on particle size or type. Elevated levels of PSP toxins were noted in surfclams from Georges Bank more than two years after initial toxification. Toxins were not evenly distributed among the various tissues of surfclams. Initially, maximum toxicity among surfclam tissues was found in digestive glands; however, subsequent analyses of samples collected later in the year indicated that toxicity in gill and mantle tissues had increased relative to initial values. No toxicity was detected in adductor muscles. Surfclams are characterized by a high variation in total toxin load among individual animals, with a tendency for decreasing variation as toxin levels increase. Archived data from the Main Department of Marine Resources revealed annual and seasonal patterns of toxin accumulation by surfclams, i.e., toxin accumulation is an annual event, with initial increases in toxicity usually occurring in early spring.

This comprehensive literature review of the phosphorus nutrition and metabolism of eukaryotic microalgae deals sequentially with (1) extracellular P-compounds available for algal utilization and growth; (2) orthophosphate uptake mechanisms, kinetics, and influence from environmental variables; (3) phosphatase-mediated utilization of organic phosphates involving multiple enzymes, induction and cellular location of repressible and irrepressible phosphatases, and their role in growth physiological processes; (4) intracellular phosphate metabolism covering diversity of phosphometabolites. ATP-linked energy regulation, polyphosphate pools and storage roles, phospholipids and phospholipases; (5) steady-state and transient-state models relating phosphate utilization to growth; (6) ecological aspects covering manifestations of phosphorus limitation, interspecific competition for phosphonutrients among microorganisms, and current views on phosphorus cycling and turnover in aquatic ecosystems. Although concentrating on the microalgae, the review often points out sounder conclusions drawn from bacteria and fungi, and includes specific macroalgae in considering certain subtopics where such algae were better investigated and provided a good basis for comparison with the microalgae.