Impact of elevated pCO2 on paralytic shellfish poisoning toxin content and composition in Alexandrium tamarense

Ocean acidification is considered a major threat to marine ecosystems and may particularly affect primary producers. Here we investigated the impact of elevated pCO2 on paralytic shellfish poisoning toxin (PST) content and composition in two strains of Alexandrium tamarense, Alex5 and Alex2. Experiments were carried out as dilute batch to keep carbonate chemistry unaltered over time. We observed only minor changes with respect to growth and elemental composition in response to elevated pCO2. For both strains, the cellular PST content, and in particular the associated cellular toxicity, was lower in the high CO2 treatments. In addition, Alex5 showed a shift in its PST composition from a nonsulfated analogue towards less toxic sulfated analogues with increasing pCO2. Transcriptomic analyses suggest that the ability of A. tamarense to maintain cellular homeostasis is predominantly regulated on the post-translational level rather than on the transcriptomic level. Furthermore, genes associated to secondary metabolite and amino acid metabolism in Alex5 were down-regulated in the high CO2 treatment, which may explain the lower PST content. Elevated pCO2 also induced up-regulation of a putative sulfotransferase sxtN homologue and a substantial down-regulation of several sulfatases. Such changes in sulfur metabolism may explain the shift in PST composition towards more sulfated analogues. All in all, our results indicate that elevated pCO2 will have minor consequences for growth and elemental composition, but may potentially reduce the cellular toxicity of A. tamarense.

Identification and localization of polyketide synthase genes from cultures of diarrheic shellfish poisoning toxin producing dinoflagellates of the genus Prorocentrum

Aquatic toxins are responsible for a number of acute and chronic diseases in humans. Okadaic acid (OA) and other dinoflagellate derived polyketide toxins pose serious health risks on a global scale. Ingestion of OA contaminated shellfish causes diarrheic shellfish poisoning (DSP). Some evidence also suggests tumor promotion in the liver by OA. Microcystin-LR (MC-LR) is produced by cyanobacteria and is believed to be the most common freshwater toxin in the US. Humans may be exposed to this acute hepatotoxin through drinking or recreational use of contaminated waters. ^ OA producing dinoflagellates have not been cultured axenically. The presence of associated bacteria raises questions about the ultimate source of OA. Identification of the toxin-producing organism(s) is the first step in identifying the biosynthetic pathways involved in toxin production. Polyketide synthase (PKS) genes of toxic and non-toxic species were surveyed by construction of clonal libraries from PCR amplicons of various toxic and non-toxic species of Prorocentrum in an effort to identify genes, which may be part of the biosynthetic pathway of OA. Analysis of the PKS sequences revealed that toxic species shared identical PKS genes not present in non-toxic species. Interestingly, the same PKS genes were identified in a library constructed from associated bacteria. ^ Subsequent bacterial small subunit RNA (16S) clonal libraries identified several common bacterial species. The most frequent 16S sequences found were identified as species of the genus Roseobacter which has previously been implicated in the production of OA. Attempts to culture commonly occurring bacteria resulted in the isolation of Oceanicaulis alexandrii , a novel marine bacterium previously isolated from the dinoflagellate Alexandrium tamarense, from both P. lima, and P. hoffmanianum. ^ Metabolic studies of microcystin-LR, were conducted to probe the activity of the major human liver cytochromes (CYP) towards the toxin. CYPs may provide alternate routes of detoxification of toxins when the usual routes have been inhibited. For example, some research indicates that cyanobacterial xenobiotics, in particular, lipopolysaccharides may inhibit glutathione S-transferases allowing the toxin to persist long enough to be acted upon by other enzymes. These studies found that at least one human liver CYP was capable of metabolizing the toxin. ^

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Pectenotoxins - an issue for public health - A review of their comparative toxicology and metabolism

Pectenotoxins (PTXs) are a group of toxins associated with diarrhetic shellfish poisoning (DSP) and isolated from DSP toxin-producing dinoflagellate algae. Consumption of shellfish contaminated with PTXs has been associated with incidences of severe diarrhetic illness resulting in hospitalisation. Concern has been raised for public health following the discovery that these toxins are not only hepatotoxic and can cause diarrhetic effects in mammals, but that they are potently cytotoxic to human cancer cell lines and have been found to be tumour promoters in animals. With advances in knowledge and technology, more PTXs are being identified, but little is known of their toxicology and the potential impact these toxins may have on public health in the long term. Without such information, adequate health-risk assessments for the consumption of shellfish contaminated with PTXs cannot be performed. This review gives a brief introduction to diarrhetic shellfish toxins, details the known toxicology and metabolism of PTXs in animals, and discusses known incidences of PTX poisoning in humans. (C) 2001 Elsevier Science Ltd. All rights reserved.

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freezethaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A2 were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 +/- 34.7% of control twitch height, which were blocked with either 100 mu m of phentolamine or 1m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-NG-Nitroarginine methyl ester (0.5 m m) or 1m m of lidocaine. PHY-N (1, 3 and 10 mu g ml(-1)) induced an increase in tonus of the biventercervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 mu m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release. Copyright (C) 2011 John Wiley & Sons, Ltd.

Expression and cellular localization of Copper transporter 2 (Ctr2) in Mus musculus

The Ctr family is an essential part of the copper homeostasis machinery and its members share sequence homology and structural and functional features. Higher eukaryotes express two members of this family Ctr1 and Ctr2. Numerous structural and functional studies are available for Ctr1, the only high affinity Cu(I) transporter thus far identified. Ctr1 holigotrimers mediate cellular copper uptake and this protein was demonstrated to be essential for embryonic development and to play a crucial role in dietary copper acquisition. Instead very little is known about Ctr2, it bears structural homology to the yeast vacuolar copper transporter, which mediates mobilization of vacuolar copper stores. Recent studies using over-expressed epitope-tagged forms of human Ctr2 suggested a function as a low affinity copper transporter that can mediate either copper uptake from the extracellular environment or mobilization of lysosomal copper stores. Using an antibody that recognizes endogenous mouse Ctr2, we studied the expression and localization of endogenous mouse Ctr2 in cell culture and in mouse models to understand its regulation and function in copper homeostasis. By immunoblot we observed a regulation of mCtr2 protein levels in a copper and Ctr1 dependent way. Our observations in cells and transgenic mice suggest that lack of Ctr1 induces a strong downregulation of Ctr2 probably by a post-translational mechanism. By indirect immunofluorescence we observed an exclusive intracellular localization in a perinuclear compartment and no co-localization with lysosomal markers. Immunofluorescence experiments in Ctr1 null cells, supported by sequence analysis, suggest that lysosomes may play a role in mCtr2 biology not as resident compartment, but as a degradation site. In appendix a LC-mass method for analysis of algal biotoxins belonging to the family of PsP (paralytic shellfish poisoning) is described.

Entwicklung eines immunologischen Testsystems zur Detektion des marinen Biotoxins Domoinsäure

Domoinsäure ist ein von mehreren Arten mariner Kieselalgen der Gattung Pseudonitzschia produziertes Toxin, welches während einer Algenblüte in Molluscen wie z.B. der Miesmuschel Mytilus sp. akkumuliert werden kann. Beim Verzehr solch kontaminierter Muscheln können sowohl beim Menschen als auch bei Tieren erhebliche Vergiftungserscheinungen auftreten, die von Übelkeit, Kopfschmerzen und Orientierungsstörungen bis hin zum Verlust des Kurzzeitgedächtnisses (daher auch als amnesic shellfish poisoning bekannt) reichen und in einigen Fällen tödlich enden. rnDie heute gängigen Methoden zur Detektion von Domoinsäure in Muschelgewebe wie Flüssigkeitschromatographie und Maus-Bioassay sind zeit- und kostenintensiv bzw. in Anbetracht einer Verbesserung des Tierschutzes aus ethischer Sicht nicht zu vertreten. Immunologische Testsysteme stellen eine erstrebenswerte Alternative dar, da sie sich durch eine vergleichsweise einfache Handhabung, hohe Selektivität und Reproduzierbarkeit auszeichnen.rnDas Ziel der vorliegenden Arbeit war es, ein solches immunologisches Testsystem zur Detektion von Domoinsäure zu entwickeln. Hierfür wurden zunächst Antikörper gegen Domoinsäure gewonnen, wofür das Toxin wiederum als erstes über die Carbodiimid-Methode an das Trägerprotein keyhole limpet hemocyanin (KLH) gekoppelt wurde, um eine Immunantwort auslösen zu können. Kaninchen und Mäuse wurden mit KLH-DO-Konjugaten nach vorgegebenen Immunisierungsschemata immunisiert. Nach vier Blutabnahmen zeigte das polyklonale Kaninchenantiserum eine ausreichend hohe Sensitivität zum Antigen; das nachfolgende Detektionssystem wurde mit Hilfe dieses polyklonalen Antikörpers aufgebaut. Zwar ist es gegen Ende der Arbeit auch gelungen, einen spezifischen monoklonalen Antikörper aus der Maus zu gewinnen, jedoch konnte dieser aus zeitlichen Gründen nicht mehr im Detektionssystem etabliert werden, was durchaus wünschenswert gewesen wäre. rnWeiterhin wurde Domoinsäure im Zuge der Entwicklung eines neuartigen Testsystems an die Trägerproteine Ovalbumin, Trypsininhibitor und Casein sowie an Biotin konjugiert. Die Kopplungserfolge wurden im ELISA, Western Blot bzw. Dot Blot nachgewiesen. Die Ovalbumin-gekoppelte sowie die biotinylierte Domoinsäure dienten im Folgenden als die zu messenden Größen in den Detektionsassays- die in einer zu untersuchenden Probe vorhandende, kompetitierende Domoinsäure wurde somit indirekt nachgewiesen. rnDer zulässige Höchstwert für Domoinsäure liegt bei 20 µg/g Muschelgewebe. Sowohl mit Biotin-DO als auch mit OVA-DO als den zu messenden Größen waren Domoinsäurekonzentrationen unterhalb dieses Grenzwertes nachweisbar; allerdings erwies sich der Aufbau mit Biotin-DO um das ca. 20-fache empfindlicher als jener mit OVA-DO. rnDie in dieser Arbeit präsentierten Ergebnisse könnten als Grundlage zur Etablierung eines kommerzialisierbaren immunologischen Testsystems zur Detektion von Domoinsäure und anderen Biotoxinen dienen. Nach erfolgreicher Validierung wäre ein solches Testsystem in seiner Handhabung einfacher als die gängige Flüssigkeitschromatographie und besser reproduzierbar als der Maus-Bioassay.rn

(Table S2) Cell concentrations of individual and combined Alexandrium spp. and phylogenetic groups as obtained by qPCR assay and Utermöhl counts, as well as particulate PSP toxin concentrations from discrete water depths

Molecular methods provide promising tools for routine detection and quantification of toxic microalgae in plankton samples. To this end, novel TaqMan minor groove binding probes and primers targeting the small (SSU) or large (LSU) ribosomal subunit (rRNA) were developed for two species of the marine dinoflagellate genus Alexandrium (A. minutum, A. tamutum) and for three groups/ribotypes of the A. tamarense species complex: Group I/North American (NA), Group II/Mediterranean (ME) and Group III/Western European (WE). Primers and probes for real-time quantitative PCR (qPCR) were species-specific and highly efficient when tested in qPCR assays for cross-validation with pure DNA from cultured Alexandrium strains. Suitability of the qPCR assays as molecular tools for the detection and estimation of relative cell abundances of Alexandrium species and groups was evaluated from samples of natural plankton assemblages along the Scottish east coast. The results were compared with inverted microscope cell counts (Utermöhl technique) of Alexandrium spp. and associated paralytic shellfish poisoning (PSP) toxin concentrations. The qPCR assays indicated that A. tamarense (Group I) and A. tamutum were the most abundant Alexandrium taxa and both were highly positively correlated with PSP toxin content of plankton samples. Cells of A. tamarense (Group III) were present at nearly all stations but in low abundance. Alexandrium minutum and A. tamarense (Group II) cells were not detected in any of the samples, thereby arguing for their absence from the specific North Sea region, at least at the time of the survey. The sympatric occurrence of A. tamarense Group I and Group III gives further support to the hypothesis that the groups/ribotypes of the A. tamarense species complex are cryptic species rather than variants belonging to the same species.

Intraspecific facilitation by allelochemical mediated grazing protection within a toxigenic dinoflagellate population, link to supplementary material

Dinoflagellates are a major cause of harmful algal blooms, with consequences for coastal marine ecosystem functioning and services. Alexandrium tamarense is one of the most abundant and widespread toxigenic species in the temperate northern and southern hemisphere, and produces paralytic shellfish poisoning toxins as well as lytic allelochemical substances. These bioactive compounds may support the success of A. tamarense and its ability to form blooms. Here we investigate the impact of grazing on monoclonal and mixed set-ups of highly (Alex2) and moderately (Alex4) allelochemically active A. tamarense strains and on a non-allelochemically active conspecific (Alex5) by the heterotrophic dinoflagellate Polykrikos kofoidii. While Alex4 and particularly Alex5 were strongly grazed by P. kofoidii when offered alone, both strains grew well in the mixed assemblages (Alex4+Alex5 and Alex2+Alex5). Hence, the allelochemical active strains facilitated growth of the non-active strain by protecting the population as a whole against grazing. Based on our results, we argue that facilitation among clonal lineages within a species may partly explain the high genotypic and phenotypic diversity of Alexandrium populations. Populations of Alexandrium may comprise multiple cooperative traits that act in concert with intraspecific facilitation, and hence promote the success of this notorious harmful algal bloom species.

Seawater carbonate chemistry and toxicity of Pseudo-nitzschia fraudulenta in a laboratory experiment

Anthropogenic CO2 is progressively acidifying the ocean, but the responses of harmful algal bloom species that produce toxins that can bioaccumulate remain virtually unknown. The neurotoxin domoic acid is produced by the globally-distributed diatom genus Pseudo-nitzschia. This toxin is responsible for amnesic shellfish poisoning, which can result in illness or death in humans and regularly causes mass mortalities of marine mammals and birds. Domoic acid production by Pseudo-nitzschia cells is known to be regulated by nutrient availability, but potential interactions with increasing seawater CO2 concentrations are poorly understood. Here we present experiments measuring domoic acid production by acclimatized cultures of Pseudo-nitzschia fraudulenta that demonstrate a strong synergism between projected future CO2 levels (765 ppm) and silicate-limited growth, which greatly increases cellular toxicity relative to growth under modern atmospheric (360 ppm) or pre-industrial (200 ppm) CO2 conditions. Cellular Si:C ratios decrease with increasing CO2, in a trend opposite to that seen for domoic acid production. The coastal California upwelling system where this species was isolated currently exhibits rapidly increasing levels of anthropogenic acidification, as well as widespread episodic silicate limitation of diatom growth. Our results suggest that the current ecosystem and human health impacts of toxic Pseudo-nitzschia blooms could be greatly exacerbated by future ocean acidification and 'carbon fertilization' of the coastal ocean.

Toxicokinetics of domoic acid in a nonhuman primate model (Macaca fascicularis)

Thesis (Master's)--University of Washington, 2016-06

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.