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Palytoxin is one of the most toxic marine toxins known. Distributed worldwide, it poses a potential human health risk linked to the consumption of contaminated seafood. Despite its high parenteral toxicity, the lethal oral dose of palytoxin is several times higher than the intraperitoneal lethal dose. In the present study, we investigated the passage of palytoxin through the human intestinal barrier by employing a well-characterized and accepted in vitro model of intestinal permeability that uses differentiated Caco-2 cell monolayers. Trans-epithelial electric resistance measurements showed that palytoxin disrupts the integrity of Caco-2 monolayers at concentrations > 0.135 n m. However, confocal microscopy imaging showed that the tight-junction protein occludin was not affected by palytoxin in the nanomolar range. This finding was supported by transmission electron microscopy imaging, where tight-junctions appeared to be unaffected by palytoxin treatment. In addition, the nuclear envelope does not appear to be altered by high concentrations of palytoxin. However, palytoxin-treated cells showed electron-dense and damaged mitochondria. Toxin exposure also induced the disappearance of the differentiated Caco-2 microvilli and organelles, as well as chromatin de-condensation. Permeability assays showed that palytoxin could not significantly pass the Caco-2 monolayer, despite the lack of epithelium integrity, suggesting that palytoxins would be poorly transported to blood, which may explain its lower oral toxicity. These data can help to achieve a better understanding of palytoxin poisoning. However, more studies regarding its repeated administration and chronic effects are needed. [ABSTRACT FROM AUTHOR]

Highlights: [•] 15 species of edible marine organisms sampled on 4 sites on the French Mediterranean coast were analysed for PlTXs. [•] Herbivorous and omnivorous species were more contaminated than carnivorous ones. [•] The major components of the toxin profile were OVTX-a (90%) and PlTX. [•] Occasionally the toxin content in the whole flesh was higher than 30μg PlTX+OVTX-a/kg meat. [•] Toxins were sequestered in the digestive tube and not in the remaining tissue or in the roe. [Copyright &y& Elsevier]

Since 1953, a total of 27 human poisoning cases caused by the consumption of blue humphead parrotfish,Scarus ovifrons, have been reported in Japan. Characteristic symptoms are severe muscle pain associated with rhabdomyolysis. Although it is believed that palytoxin, which is one of the most potent non-protein marine biotoxins, is the most likely causative toxin in blue humphead parrotfish poisoning, palytoxin has not been proven conclusively as the causative toxin because of lack of a reliable and sensitive analytical method for palytoxin. In 2011, human poisoning cases caused by the consumption of blue humphead parrotfish occurred in Miyazaki and Tokyo. In our present study, an LC-MS/MS method for palytoxin and its analogues in the blue humphead parrotfish samples causing the human poisoning cases in 2011 was developed and the samples were analysed by using the newly developed LC-MS/MS method. Palytoxin and its analogues were not detected in the samples from the food poisoning cases. The LC-MS/MS findings therefore do not support the recently accepted hypothesis that palytoxin is the causative agent in blue humphead parrotfish poisoning in Japan. [ABSTRACT FROM PUBLISHER]

We report the development and validation of a qPCR based method for estimation of the toxic benthic dinoflagellate Ostreopsis cf. ovata in the complex matrix of marine aerosol at Sant Andreu de Llavaneres beach (northwestern Mediterranean Sea). Toxic events in humans after inhalation or cutaneous contact have been reported during O. cf. ovata blooms and were attributed to palytoxin (PLTX)-like compounds produced by this microalga. Similar PCR efficiencies of plasmid and cellular environmental standard curves (98 and 100%, respectively) allowed obtaining the rDNA copy number per cell. The analytical sensitivity was set at 2 × 100 rDNA copy number and 8 × 10-4 cell per reaction. Based on spiking experiments, we evaluated the aerosol filter inhibitory activity and recovery rate of cells from filters, then normalized the abundance data of toxic O. cf. ovata. The abundance in marine aerosol during the bloom varied in the range of 1-102 cells per filter. Analytical determinations were also applied to detect palytoxin in field samples. No palytoxin was detected in the aerosol filters, and the estimation of PLTX like-compound concentrations in microepiphytic assemblages varied between 0.1 and 1.2 pg/cell. [ABSTRACT FROM AUTHOR]

Abstract: In recent decades, the North West Mediterranean Sea has been seriously affected by the development of the toxic benthic dinoflagellate Ostreopsis cf. ovata, which is associated with harmful effects on human health and the environment. The present work aims to provide a large overview of the multiple environmental factors assumed to regulate or influence the growth of Ostreopsis. An intensive sampling campaign over a full annual cycle was performed along the French and Italian coasts (in six sites from Cassis to Genoa), to determine patterns of temporal and spatial distributions of both O. cf. ovata epiphytic and planktonic cells. Results highlighted substantial seasonal variations in the abundance of Ostreopsis. These variations correlated to seawater temperature, with an optimum growth temperature ranging from 23°C to 27.5°C. Phosphate concentration, rather than nitrogen or silicate, was also positively associated with Ostreopsis. Decreases in oxygen and increases in chlorophyll a concentrations were recorded during the summer blooming period. The maximal Ostreopsis epiphytic abundance was generally higher on Dictyota spp. than on the other two sampled macroalgae (up to 8.54×106 cellsg−1 FW), even though statistical analysis did not support a clear substrate preference. Epiphytic abundances were significantly higher at a very shallow depth (0.5m), than at 1 and/or 3m depths. High anthropogenic pressure (related to population density) seems to have promoted the occurrence of blooms in urbanized areas, which could partly explain the strong demarcation in Ostreopsis development between Western and Eastern sampling sites. The ecological niche of Ostreopsis cf. ovata needs precise definition, which will require further in situ and in vitro experimental studies, to determine the relative importance of distinct environmental parameters. [Copyright &y& Elsevier]

Objectives: Palytoxin (PTX), a marine toxin isolated from the Cnidaria (zooanthid) Palythoa caribaeorum is one of the most potent non-protein substances known. It is a very complex molecule that presents both lipophilic and hydrophilic areas. The effect of PTX was investigated in a series of experiments conducted in head and neck squamous cell carcinoma (HNSCC) cell lines and xenografts. Materials and methods: Cell viability, and gene expression of the sodium/potassium-transporting ATPase subumit alpha1 (ATP1AL1) and GAPDH were analyzed in HNSCC cells and normal epithelial cells after treatment with PTX using cytotoxicity-, clonogenic-, and enzyme inhibitor assays as well as RT-PCR and Northern Blotting. For xenograft experiments severe combined immunodeficient (SCID) mice were used to analyze tumor regression. The data were statistically analyzed using One-Way Annova (SPSS vs20). Results: Significant toxic effects were observed in tumor cells treated with PTX (LD50 of 1.5 to 3.5 ng/ml) in contrast to normal cells. In tumor cells PTX affected both the release of LDH and the expression of the sodium/ potassium-transporting ATPase subunit alpha1 gene suggesting loss of cellular integrity, primarily of the plasma membrane. Furthermore, strong repression of the c-Jun N-terminal kinase 3 (JNK3) mRNA expression was found in carcinoma cells which correlated with enhanced toxicity of PTX suggesting an essential role of the mitogen activated protein kinase (MAPK)/JNK signalling cascades pathway in the mechanisms of HNSCC cell resistance to PTX. In mice inoculated with carcinoma cells, injections of PTX into the xenografted tumors resulted within 24 days in extensive tumor destruction in 75% of the treated animals (LD50 of 68 ng/kg to 83 ng/kg) while no tumor regression occurred in control animals. Conclusions: These results clearly provide evidence that PTX possesses preferential toxicity for head and neck carcinoma cells and therefore it is worth further studying its impact which may extend our knowledge of the biology of head and neck cancer. [ABSTRACT FROM AUTHOR]

Abstract: Blooms of Ostreopsis spp. once confined to tropical and subtropical areas have recently spread to more temperate regions such as the Mediterranean and the Southern-Atlantic coasts of Europe. However, while O. confronta (cf.) ovata has caused several toxic outbreaks, the presence of O. cf. siamensis has been reported rather occasionally and in very few regions; as a consequence, O. cf. ovata toxin profile has been in-depth studied while poor information exists on toxicity of the Mediterranean and Atlantic O. cf. siamensis. In the present study toxin profile of Mediterranean and Atlantic O. cf. siamensis isolates also phylogenetically related has been studied through liquid chromatography–high resolution mass spectrometry (LC–HRMS) versus a palytoxin standard, a crude extract of O. cf. ovata containing all the ovatoxins so far known (ovatoxin-a to -f), and a Japanese O. siamensis extract which contained ostreocin-d and ostreocin-b. The Mediterranean and Atlantic O. cf. siamensis strains were shown not to produce either ostreocins, which are produced by the Japanese O. siamensis strain, or ovatoxins, which are produced by the Mediterranean O. cf. ovata. Only sub-fg levels of palytoxin on a per cell basis were detected in the Mediterranean strain. This study demonstrates that the Mediterranean and the Atlantic O. cf. siamensis strains are devoid of any appreciable toxicity. Thus, at least in the European area, O. cf. siamensis seems to present a much lower risk to human health than O. cf. ovata. [Copyright &y& Elsevier]

Some species belonging to Ostreopsis, a benthic dinoflagellate genus, are known to produce palytoxin analogues. Around the coastal regions of Japan, the toxic Ostreopsis sp. 1 and Ostreopsis sp. 6 which are genetically divergent from other species of Ostreopsis are present from the southern to northern regions and in the southern region, respectively. The present study examined the growth responses of these strains to seven temperatures (15-35 °C) in combination with five salinities (20-40) and discusses the effects of temperature and salinity on their distribution and bloom dynamics in Japan. Tolerable temperatures and salinities ranged 15-30 °C and 25-40 for Ostreopsis sp. 1, and 17.5-30 °C and 20-40 for Ostreopsis sp. 6. The optimal temperature ranges which gave growth rates of >90 % of maximal growth rate of each strain were 22-25 °C for Ostreopsis sp. 1 and 24-30 °C for Ostreopsis sp. 6. Therefore, Ostreopsis sp. 1 is putatively tolerant to lower temperatures and thus possesses adaptability to colder waters of relatively higher latitude regions of Japan, whereas Ostreopsis sp. 6 presumably possesses adaptability to warmer waters of the southern region. We conclude that growth responses of Japanese toxic Ostreopsis sp. 1 and Ostreopsis sp. 6 to temperature-salinity affect their distribution and bloom dynamics in Japan. [ABSTRACT FROM AUTHOR]

Ciguatera fish poisoning (CFP), a disease caused by consuming fish that have accumulated ciguatoxins (CTXs) in their tissue, is regarded as the most prevalent form of intoxication in French Polynesia. Recently, the Australes, one of the least affected archipelago until the early 1980s, has shown a dramatic increase in its incidence rates in 2009 with unusual CFP cases. In the present work, potential health hazards associated with the proliferation of various marine phytoplankton species and the consumption of fish and marine invertebrates highly popular among local population were assessed in three Australes islands: Raivavae, Rurutu and Rapa. Extracts from the marine dinoflagellatesGambierdiscus,Ostreospisand mat-forming cyanobacteria as well as fish, giant clams and sea urchin samples were examined for the presence of CTXs and palytoxin (PLTX) by using the neuroblastoma cell-based assay (CBA-N2a). Cytotoxic responses observed with both standards (Pacific CTX-3C and PLTX) and targeted marine products indicate that CBA-N2a is a robust screening tool, with high sensitivity and good repeatability and reproducibility. In Rurutu and Raivavae islands, our main findings concern the presence of CTX-like compounds in giant clams and sea urchins, suggesting a second bio-accumulation route for CFP toxins in the ciguatera food chain. In Rapa, the potential CFP risk fromGambierdiscusbloom and fish was confirmed for the first time, with levels of CTXs found above the consumer advisory level of 0.01 ng Pacific CTX-1B g−1of flesh in three fish samples. However, despite the presence of trace level of PLTX inOstreopsisnatural assemblages of Rapa, no sign of PLTX accumulation is yet observed in tested fish samples. Because this multi-toxinic context is likely to emerge in most French Polynesian islands, CBA-N2a shows great potential for future applications in the algal- and toxin-based field monitoring programmes currently on hand locally. [ABSTRACT FROM PUBLISHER]

Palytoxin (PLTX) is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+ /K+-ATPase. Indeed, ouabain (OUA), a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na+/K+ -ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a Kd of 3 x 10-10 M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3-1.0 x 10-7 M) and possibly as a non-competitive antagonist against low PLTX concentrations (0.1-3.0 x 10-9 M). Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 x 10-5 M). However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites. [ABSTRACT FROM AUTHOR]

Palytoxins are potent marine biotoxins that have recently become endemic to the Mediterranean Sea, and are becoming more frequently associated with seafood. Due to their high toxicity, suitable methods to quantify palytoxins are needed. Thus, we developed an indirect sandwich ELISA for palytoxin and 42-hydroxy-palytoxin. An intralaboratory study demonstrated sensitivity (limit of detection, LOD = 1.1 ng/mL; limit of quantitation, LOQ = 2.2 ng/mL), accuracy (bias of 2.1%), repeatability (RSDr = 6% and 9% for intra- and interassay variability, respectively) and specificity: other common marine toxins (okadaic acid, domoic acid, saxitoxin, brevetoxin-3, and yessotoxin) do not cross-react in this assay. It performed well in three different matrices: observed LOQs were 11.0, 9.6, and 2.4 ng/mL for mussel extracts, algal net samples and seawater, respectively, with good accuracy and precision. The LOQ in seafood is 11 μg palytoxin/kg mussel meat, lower than that of the most common detection technique, LC-MS/MS. [ABSTRACT FROM AUTHOR]

ABSTRACT Palytoxin (PTX) is a potent marine toxin that causies serious damage to various tissues and organs. It has been reported to affect the transport of cations across the plasma membranes, which is commonly recognized as being the principal mechanism of its highly toxic action on mammals, including humans. However, although some marine toxins have been shown to cause toxic effects on the nervous system by interfering with the transmission of nerve impulses, the effect of PTX on neuronal cells has not yet been fully elucidated. Therefore, the toxic action of PTX on PC12 cells was examined as an in vitro model experiment to elucidate the neurotoxic properties of this toxin, and PTX was shown to reduce the viability of PC12 cells in a concentration-dependent manner. The cytotoxic action of PTX was not significantly altered by the presence of the antioxidant N-acetylcysteine and reduced-form glutathione in the cultures. Fluorescence staining of the cells and the electrophoretic analysis of genomic DNA showed that PTX failed to cause chromatin condensation and DNA fragmentation within the cells. On the other hand, the exposure to PTX caused positive staining of the cytoplasmic space of the cells with propidium iodide and the release of lactate dehydrogenase into the culture medium. Based on these observations, PTX is considered to cause cell death as a consequence of disrupting the plasma membranes, thus causing nonoxidative necrotic damage to PC12 cells. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Ostreopsis ovata is a benthic dinoflagellate that produces palytoxin and ovatoxins. Blooms of O. ovata causing human health problems and mortality of benthic fauna have been reported from many tropical and temperate marine waters. In the present study we examined the combined effects of temperature and different nutrient conditions on the biochemical composition, growth, toxicity and carbohydrate production of an O. ovata strain originating from the Tyrrhenian Sea. O. ovata cultures with N:P ratios of 1.6, 16 and 160 (N deficient, NP sufficient and P deficient, respectively) were grown at 20°C and 30°C. Biomass accumulation, growth rates, cell volumes, biochemical composition, cell toxicity and carbohydrate production in each treatment were studied. Results indicated that under nutrient sufficiency O. ovata biomass accumulation increased significantly compared to N and P deficiency and also that N limitation severely affected growth. The highest growth rates were recorded at 30°C. Cellular contents and the atomic ratios of C, N and P were higher in the cells grown at 20°C than in those grown at 30°C. O. ovata cell volumes increased at 20°C. N deficiency significantly increased cell toxicity. Toxicity per cell was higher at 20°C, but per carbon was highest at 30°C. The highest carbohydrate production was found in conditions of N deficiency and at the lower temperature. Our study suggests that temperature increases due to global warming and nutrient enrichment of coastal waters stimulate the proliferation of O. ovata, particularly for the strains that have become adapted to warm temperate waters. [Copyright &y& Elsevier]

Abstract: Ostreopsis ovata Fukuyo is a benthic dinoflagellate widespread from tropical to subtropical and warm temperate coastal areas world-wide. Since the species produces palytoxin-like substances that can accumulate in seafood, the apparent expansion of its range in recent years represents an increasing risk for human health. This leads to the necessity of monitoring protocols that enable the rapid detection of the presence of this microalga in environmental samples and sea-food. We developed an identification protocol based on real-time PCR (qPCR) to detect O. cf. ovata presence in different matrices. The protocol was proved to be able to reveal microalgal traces in both soft tissues and intervalvar liquid of mussels exposed to O. cf. ovata in natural and experimental conditions as well as in seawater samples. O. cf. ovata could also be detected in mussel tissues after the end of the bloom, when it was no longer detectable in sea water. We were able to detect O. cf. ovata in copepods fed on unialgal cultures as well. Cell density estimates based on standard curves resulted to be comparable to direct microscopical counts. The method is therefore suitable to ascertain the origin of palytoxin-like substances in toxic seafood. In addition, our results confirm that mussels and other predators can actually ingest O. cf. ovata cells and act as a vector for toxin transfer through both benthic and planktonic food webs. [Copyright &y& Elsevier]

Abstract: In the last decades, massive blooms of palytoxin (PLTX)-producing Ostreopsis cf. ovata have been observed along Mediterranean coasts, usually associated to human respiratory and cutaneous problems. At the molecular level, PLTX induces a massive intracellular Na+ influx due to the transformation of Na+/K+ ATPase in a cationic channel. Recently, we have demonstrated that Na+ overload is the crucial step in mediating overproduction of reactive oxygen species (ROS) and cell death in human HaCaT keratinocytes, tentatively explaining PLTX-induced skin irritant effects. In the present study the molecular mechanisms of ROS production induced by PLTX-mediated Na+ intracellular overload have been investigated. In HaCaT cells, PLTX exposure caused accumulation of superoxide anion, but not of nitric oxide or peroxynitrite/hydroxyl radicals. Even if RT-PCR and western blot analysis revealed an early NOX-2 and iNOS gene and protein over-expressions, their active involvement seemed to be only partial since selective inhibitors did not completely reduce O2 − production. A significant role of other enzymes (COX-1, COX-2, XO) was not evidenced. Nigericin, that counteracts Na+-mediated H+-imbalance, dissipating ΔpH across mitochondrial inner membrane, and the uncouplers DNP significantly reduced O2 − production. These inhibitions were synergistic when co-exposed with complex-I inhibitor rotenone. These results suggest a novel mechanism of O2 − production induced by PLTX-mediated ionic imbalance. Indeed, the H+ intracellular overload that follows PLTX-induced intracellular Na+ accumulation, could enhance ΔpH across mitochondrial inner membrane, that seems to be the driving force for O2 − production by reversing mitochondrial electron transport. [Copyright &y& Elsevier]

Among marine biotoxins, palytoxins (PlTXs) and cyclic imines (CIs), including spirolides, pinnatoxins, pteriatoxins, and gymnodimines, are not managed in many countries, such as the USA, European nations, and South Korea, because there are not enough poisoning cases or data for the limits on these biotoxins. In this article, we review unregulated marine biotoxins (e.g., PlTXs and CIs), their toxicity, causative phytoplankton species, and toxin extraction and detection protocols. Due to global warming, the habitat of the causative phytoplankton has expanded to the Asia-Pacific region. When ingested by humans, shellfish that accumulated toxins can cause various symptoms (muscle pain or diarrhea) and even death. There are no systematic reports on the occurrence of these toxins; however, it is important to continuously monitor causative phytoplankton and poisoning of accumulating shellfish by PlTXs and CI toxins because of the high risk of toxicity in human consumers.

Abstract: The dinoflagellate genus Ostreopsis is known as a potential producer of palytoxin analogues. Recently a surprisingly diverse and widespread distribution of Ostreopsis along coastal waters in Japan was reported in our previous paper. In the present study, detailed toxin profiles of palytoxin analogues in several Japanese Ostreopsis strains were clarified for the first time by liquid chromatography–mass spectrometry (LC–MS). An Italian Ostreopsis strain (KAC 85) was also analyzed by LC–MS as a reference material of Ostreopsis and the presence of ovatoxin-a, -b, -c, -d, and -e was re-confirmed. The most dominant palytoxin analogue in Japanese Ostreopsis was a novel palytoxin analogue, which was identified as an isomer of ovatoxin-a. Novel isomers of ovatoxin-b, -d, and -e were also found in Japanese Ostreopsis. The isomers of ovatoxin-a, -b, -d, and -e detected in Japanese Ostreopsis were tentatively named ovatoxin-a AC, -b AC, -d AC and -e AC. LC–MS analysis suggests that ovatoxin-a AC is dideoxy-42-hydroxy-44-deoxy-palytoxin. The unidentified deoxy-positions can be assigned both between C16 and C20 and between C53 and C73. Toxin profiles of Ostreopsis collected in the middle to southern coastal area in Japan were similar irrespective of clades classified by the phylogenetic analysis reported in our previous study. The toxin contents of some strains collected in Japan were comparable to that obtained in a strain (KAC 85) collected in the Italian coast where human health problems occurred, suggesting a potential risk of Japanese Ostreopsis to human health. [Copyright &y& Elsevier]

Abstract: Massive blooms of the harmful benthic dinoflagellate Ostreopsis cf. ovata are of growing environmental concern in the Mediterranean, having recently caused adverse effects on benthic invertebrates and also some intoxication episodes to humans. The toxicological potential of produced palytoxin-like compounds was investigated in the present study on a typical marine sentinel species, the mussel Mytilus galloprovincialis. Organisms were sampled during various phases of a O. cf. ovata bloom, in two differently impacted sites. The presence of the algal toxins was indirectly assessed in mussels tissues (mouse test and hemolysis neutralization assay), while biological and toxicological effects were evaluated through the measurement of osmoregulatory and neurotoxic alterations (Na+/K+-ATPase and acetylcholinesterase activities), oxidative stress responses (antioxidant defences and total oxyradical scavenging capacity), lipid peroxidation processes (level of malondialdehyde), peroxisomal proliferation, organelle dysfunctions (lysosomal membrane stability, accumulation of lipofuscin and neutral lipids), immunological impairment (granulocytes percentage). Obtained results demonstrated a significant accumulation of algal toxins in mussels exposed to O. cf. ovata. These organisms exhibited a marked inhibition of the Na+/K+-ATPase activity and alterations of immunological, lysosomal and neurotoxic responses. Markers of oxidative stress showed more limited variations suggesting that toxicity of the O. cf. ovata toxins is not primarily mediated by an over production of reactive oxygen species. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess biological alterations and toxicological events associated to blooms of O. cf. ovata in marine organisms. [Copyright &y& Elsevier]

Benthic dinoflagellates of the genus Ostreopsis produce palytoxin ( PTX)-like compounds. The worldwide distributed Ostreopsis ovata/ O. cf. ovata is potentially responsible for outbreaks of human health problems around the coasts of tropical, subtropical, and temperate regions. The present study examined growth responses of an O. cf. ovata strain s0662 collected from coastal waters of Japan with 35 different combinations of temperature (15-35° C) and salinity (20-40) and discusses the bloom dynamics of the organism in Japanese coastal environments. The O. cf. ovata strain s0662 tolerated a wide range of temperature (17.5-35° C) and salinity (25-40). Results of a two-way ANOVA showed significant effects of temperature-salinity interaction on growth rates and biomass yields of the O. cf. ovata strain ( F(24,70) > 127, P < 0.001). The strain showed a maximal growth rate (1.03 divisions day−1) and biomass yield (240 relative fluorescence) at temperature 25° C and salinity 30. The high growth rates of over 1.0 division day−1 were obtained in conditions of temperature 25-30° C and salinity 30-35, which indicates that strain s0662 prefers high temperature and salinity conditions. The growth rates of O. cf. ovata under the optimal conditions were higher than those of other benthic toxic-dinoflagellates, Coolia monotis, Gambierdiscus toxicus, and Prorocentrum lima ( Dinophyceae) previously reported. Taken together, we suggest that O. cf. ovata is able to grow faster than the other benthic dinoflagellates in waters of high temperature and salinity. The physiological feature probably confers an ecological advantage on O. cf. ovata in the bloom development during warmer seasons in Japan and may be responsible for outbreaks of PTX-like poisoning in the region especially during the warmer seasons. [ABSTRACT FROM AUTHOR]

Abstract: Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 μg kg−1 for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 μg kg−1) from shellfish and fish tissues were 114–119% (as amine aldehyde) and 90–115% (as amide aldehyde) with RSDs for both of ≤18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL−1 and the LOQ 4 ng mL−1, which corresponds to 10 μg kg−1 in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required. [Copyright &y& Elsevier]

No abstract available Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Palytoxin (PLTX) is one of the most toxic seafood contaminants ever isolated. Reports of human food-borne poisoning ascribed to PLTX suggest skeletal muscle as a primary target site. Primary cultures of mouse skeletal muscle cells were used to study the relationship between Ca2+ response triggered by PLTX and the development of myotoxic insult. Ca2+ imaging experiments revealed that PLTX causes a transitory intracellular Ca2+ response (transient phase) followed by a slower and more sustained Ca2+ increase (long-lasting phase). The transient phase is due to Ca2+ release from intracellular stores and entry through voltage-dependent channels and the Na+/Ca2+ exchanger (reverse mode). The long-lasting phase is due to a massive and prolonged Ca2+ influx from the extracellular compartment. Sulforhodamine B assay revealed that the long-lasting phase is the one responsible for the toxicity in skeletal muscle cells. Our data analyzed, for the first time, pathways of PLTX-induced Ca2+ entry and their correlation with PLTX-induced toxicity in skeletal muscle cells. The cellular morphology changes induced by PLTX and the sensitivity to gadolinium suggest a role for stretch-activated channels. [ABSTRACT FROM AUTHOR]

Abstract: Blooms of the toxic benthic dinoflagellate Ostreopsis cf. ovata are associated with noxious effects on human health and mortality of benthic organisms, due to the production of palytoxin-like compounds. We investigated the O. cf. ovata bloom along the Conero Riviera (northern Adriatic Sea) during summer 2009 and 2010, in order to analyse morphometric characteristics in relation to bloom phases and environmental conditions. Up to 1000 cells were measured in each study year in natural samples. The morphometric analysis showed a marked variability in cell size (DV: 18.75–75μm; W: 12.5–60μm), and a relationship between cell dimensions and bloom phases was shown: cells in the final phase had a significantly longer DV than those in both the initial and in the proliferation phases. O. cf. ovata showed significantly lower DV in sheltered sites compared with exposed ones, suggesting that the former represent favourable sites for intense O. cf. ovata proliferation and that turbulence can affect O. cf. ovata growth rate and consequently its cell size. The dimensions of Ostreopsis cells in the water column were significantly higher than those of epiphytic ones, suggesting that resuspended cells in the water column probably derived from a mature benthic population. O. cf. ovata developed from August to November, with the highest abundances on 9th September in 2009 (1.3×106 cellsg−1 fw, corresponding to 1.6×107 cellsg−1 dw and 63.8×103 cellscm−2) and on 19th August in 2010 (1.2×106 cellsg−1 fw corresponding to 1.6×107 cellsg−1 dw and 47.8×103 cellscm−2). The abundances of O. cf. ovata were significantly higher in sheltered than in exposed sites, but such differences were significant only during the proliferation phase, suggesting that hydrodynamics affect abundances mainly during the proliferation bloom phase when the highest abundances are reached. [Copyright &y& Elsevier]

Blooms of toxic dinoflagellates of the genus Ostreopsis, which is known as a producer of palytoxin (PTX) analogs, may pose a threat to human health in tropical, subtropical, and temperate regions around the world. In the present study, we established a suitable culture method for Ostreopsis spp. isolated from Japanese coastal waters and characterized their growth potential using the method to discuss their bloom dynamics. Each clonal strain of Ostreopsis cf. ovata, Ostreopsis sp. 1, Ostreopsis sp. 5, and Ostreopsis sp. 6 was incubated in 25 × 150 mm test tubes with a flat bottom containing various kinds of medium. Since Ostreopsis spp. strains grew well in IMK and/or f/2 media, we selected these media for cultivation of all the Ostreopsis spp. isolates. Growth rates of O. cf. ovata (0.834 divisions/day), Ostreopsis sp. 1 (0.619 divisions/day), and Ostreopsis sp. 6 (1.04 divisions/day) that produce PTX analogs significantly differed ( p < 0.05) respectively and are clearly higher than those of other reported epiphytic dinoflagellate Gambierdiscus toxicus, Prorocentrum lima, and Coolia monotis cultures, which suggest that these species have ecological advantages to predominate through the algal succession in Japanese coastal waters, resulting in a potential risk to human health in this region. [ABSTRACT FROM AUTHOR]

Currently, the benthic dinoflagellate Ostreopsiscf. ovatarepresents a serious concern to humanhealth in the whole Mediterranean basin due to the production of palytoxincongeners, a putative palytoxin and ovatoxins (ovatoxin-a, -b, -c,-d/-e), listed among the most potent marine toxins. High resolutionliquid chromatography–mass spectrometry (HR LC-MS) based investigationof a North Western Adriatic strain of Ostreopsiscf. ovatacollected at Portonovo (Italy) in 2008 is reportedherein. Toxin profile was different from those previously reportedfor other O.cf. ovata, both qualitativelyand quantitatively. For the first time, ovatoxin-a did not dominatethe toxin profile, and a new palytoxin congener, here named ovatoxin-f,was detected. Ovatoxin-f and its elemental formula present C2H4more than ovatoxin-a. HR CID MSnexperimentsallowed us to restrict structural differences between ovatoxin-a and-f to the region between C-95 and C-102, a region not previously beendescribed to be modified in other palytoxins. Ovatoxin-f representsthe major component of the toxin profile of the analyzed strain accountingfor 50% of the total toxin content, while ovatoxin-a, the dominanttoxin in most of the Mediterranean O.cf. ovatastrains we have analyzed so far, is the second majorcomponent of the toxin profile (23%). Thus, the presence of ovatoxin-fshould be taken into account when monitoring programs for palytoxin-likecompounds in microalgae and/or seawater are carried out. [ABSTRACT FROM AUTHOR]

Abstract: The asexual and sexual reproduction of Ostreopsis cf. ovata was studied in the field and in cultures isolated from two locations in the Mediterranean Sea. Asexual division took place in the motile stage by the sharing of theca (desmoschisis). High cell-size variability and differences in division capability were detected in the cultures. Thecal analyses and nuclear division patterns allowed characterization of the different phases of dividing cells obtained during an in situ cell-cycle sampling performed off Llavaneres beach (Northeast Spain). During the 45-h cycle, binucleated cells accounted for 2.6% of the population. Division was initiated with the onset of dusk and reached a maximum 3–4h before dawn. No dividing cells were detected after 09:00 AM. Sexuality occurred both in cultures and in natural populations of O. cf. ovata. Mating gamete pairs were the only sexual stages that could be distinguished from vegetative stages. The differences between these pairs and dividing cells are described herein. None of the individually isolated gamete pairs underwent fusion nor encystment, instead, in most of them one member of the gamete pair divided. Rather than plasmogamy, there was evidence of nuclear migration from one gamete to the other. Pellicle cysts, thin-walled cysts, and thecate cysts were also identified and studied. These cysts, embedded in abundant mucilage in a bloom-derived incubated sample, were able to germinate for as long as 6 months after their formation. Our results suggest that they constitute the overwintering population that causes recurrent blooms of O. cf. ovata in some areas of the Mediterranean Sea. [Copyright &y& Elsevier]

Abstract: Recently, in the Mediterranean Sea and in other temperate regions, mass mortalities of marine organisms and human diseases have been caused by blooms of the dinoflagellate genus Ostreopsis, palytoxin (PLTX) producing species. Such blooms are cause of health, economical and environmental concern, particularly when affecting edible species of commercial relevance that, additionally, play key ecological roles, with cascading effects through the whole ecosystem. The present research aims at the evaluation of the effects produced by Ostreopsis ovata blooms on the early stages of Paracentrotus lividus (Lamarck, 1816). This sea urchin species is considered one of the key controllers of the structure and dynamics of Mediterranean algal communities. Further, P. lividus is an edible species and its gonads (roe) are considered a culinary delicacy: in many areas the harvesting of this species has lead to overexploitation. Through three eco-toxicological assays we tested mortality effects produced by four O. ovata concentrations (4, 40, 400 and 4000cellml−1) on P. lividus competent larvae, using O. ovata cultured at 20°C and 25°C, and on juveniles, using O. ovata cultured at 20°C. Both O. ovata culture and growth medium devoid of algal cells were tested. Juveniles turned out to be more sensitive than larvae to O. ovata presence, the latter showing significant mortality only at extremely high O. ovata concentrations. Furthermore, temperature at which the algal cultures were grown played an additional role. The results of the present research are particularly relevant given the commercial and ecological relevance of P. lividus. [Copyright &y& Elsevier]

Toxic substances that occur in nature have various structures and functions. In fact, the very novelty of their structures and functions sometimes extends far beyond the realm of human imagination, and the capabilities of these compounds are still largely untapped despite the major advances of modern science. In this report we focus on the most recent developments in this field, with a particular emphasis on natural venoms, marine sunscreen, and marine huge molecules. [ABSTRACT FROM AUTHOR]

Dinoflagellates of the genus Ostreopsis are known to cause (often fatal) food poisoning in tropical coastal areas following the accumulation of palytoxin (PLTX) and/or its analogues (PLTX group) in crabs, sea urchins or fish. Ostreopsis spp. occurrence is presently increasing in the northern to north western Mediterranean Sea (Italy, Spain, Greece and France), probably in response to climate change. In France, Ostreopsis. cf. ovata has been associated with toxic events during summer 2006, at Morgiret, off the coast of Marseille, and a specific monitoring has been designed and implemented since 2007. Results from 2008 and 2009 showed that there is a real danger of human poisoning, as these demonstrated bioaccumulation of the PLTX group (PLTX and ovatoxin-a) in both filter-feeding bivalve molluscs (mussels) and herbivorous echinoderms (sea urchins). The total content accumulated in urchins reached 450 µg PLTX eq/kg total flesh (summer 2008). In mussels, the maximum was 230 µg eq PLTX/kg (summer 2009) compared with a maximum of 360 µg found in sea urchins during the same period at the same site. This publication brings together scientific knowledge obtained about the summer development of Ostreopsis spp. in France during 2007, 2008 and 2009. [ABSTRACT FROM AUTHOR]

Mass appearances of the toxic dinoflagellate genus Ostreopsis are known to cause dangerous respiratory symptoms in humans exposed to aerosols. The outbreaks can appear in shallow marine waters of temperate regions around the globe. We followed a massive bloom event on a public beach on the northern Adriatic coast near Rovinj, Croatia. We identified the responsible species and the produced toxins as well as the dynamics of the event with respect to environmental conditions. Ostreopsis cf. ovata appeared in masses from September through October 2010 on a public beach near Rovinj, Croatia but stayed undetected by public health organizations. Respiratory symptoms were observed whenever humans were exposed to substrate samples containing large numbers of Ostreopsis cells. During the mass abundance of O. cf. ovata also exposure to the aerosols on the beach evoked respiratory symptoms in humans. Our measurements showed high cell abundances and high toxin contents with a stable relative contribution of putative Palytoxin and Ovatoxins a-e. Artificial beach structures proved to dramatically reduce settling of the observed Ostreopsis biofilm. Blooms like those reported herein have a high potential to happen undetected with a high potential of affecting the health of coastal human populations. Increased monitoring efforts are therefore required to understand the ecology and toxicology of those bloom events and reduce their negative impact on coastal populations. [ABSTRACT FROM AUTHOR]

Abstract: The genus Ostreopsis includes several species capable of producing various palytoxin-like compounds which have harmful effects on humans and marine fauna. Species in this genus are regular members of the epiphytic community in tropical seas but their geographic range have shown an apparent expansion towards temperate regions in recent years. This is the case of the Mediterranean Sea, where Ostreopsis cf. ovata is responsible for intense blooms in summer and autumn. With the aim of describing the growth performances of O. cf. ovata, we carried out a multifactorial experiment in which strains isolated from 3 locations along the Italian coast were grown at different combinations of temperature, daylength and irradiance encompassing different seasonal conditions. Temperature constrained growth of O. cf. ovata within a window of 18–30°C, while best performances were recorded at 22 and 26°C. Growth was maximum at 12h daylength, whereas it was limited by photon flux density at short daylength (9h) and often showed photosaturation at the longest daylength (15h). Cells with anomalous shape were found during the exponential phase at the lowest and highest temperatures, while cells with a reduced size were produced at all conditions except 18°C. Total toxin concentration varied between the different environmental conditions and between exponential and post-exponential growth phases without a clear pattern, whereas toxin composition was less variable. Our results suggest that O. cf. ovata is adapted to intermediate temperatures and daylength conditions such as those recorded in the natural environment at the beginning of summer and/or at the beginning of autumn, when this dinoflagellate builds up its biomass along the coast of the Mediterranean Sea. Photosaturation instead occurs at the highest temperature and irradiance conditions, thus supporting the observations of generally lower cell abundances in late July–August. [Copyright &y& Elsevier]

When it comes to streamline organic synthesis on the way from the 20th to the 21st century, it is paramount to find ways to reduce the number of individual synthetic steps. The conceptual basis for doing this has been reviewed. [ABSTRACT FROM AUTHOR]

In order to investigate the identity, abundance and toxicity of a distinctive Ostreopsis species present in the southern-central coast of Cuba, Ostreopsis field populations were examined during January-September of 2010. The morphological and partial LSU phylogenetic data suggested the presence of O. lenticularis, although a very close species like O. labens could not be discarded. Results indicated the presence of palytoxin-like molecules in a natural extract at concentration of 0.12 pg/cell. Ostreopsis populations were very stable through the course of the study, and a maximum of 7.9 X 104 cells/g of macroalgae fresh weight was recorded on June. [ABSTRACT FROM AUTHOR]

Bacteria associated to benthic dinoflagellate Ostreopsis cf. ovata cultures were removed to assess their effects on algal growth and toxins' production. Bacteria were removed using an antibiotic cocktail (streptomycin, ciprofloxacin, gentamicin and penicillin G). The actual axenic status of antibiotic treated cultures and bacterial growth in control cultures were assessed by epifluorescence microscopy using SYBR gold dye. The removal of bacteria unaffected algal growth, except conferring a higher cell number at mid stationary phase. Toxin profile and quantification of each toxin (PLTX, OVTX-a, -b, -c, -d, -e) were performed by HR LC-MS on both cell pellet and growth medium extracts. No changes in toxins' profile nor in cell and extra-cellular toxins' concentrations were found between bacteria-free and control cultures at the early stationary phase. Whereas, in late stationary phase axenic cultures showed significant lower cell toxins' concentrations and higher extra-cellular toxins' values, though not significantly (total cell toxins' concentrations: 39.3 and 24.9 pg cell-1; total extra-cellular toxins' concentrations: 23.8 and 28.3 &mgr;gL-1for control and bacteria-free cultures, respectively). [ABSTRACT FROM AUTHOR]

The presence of dinoflagellates of the genus Ostreopsis along Mediterranean coasts was first observed in 1972, in the bay of Villefranche-sur-Mer. However, over the past ten years, harmful events related to this benthic dinoflagellate have been reported in Italian, Spanish, Greek, French, Tunisian and Algerian coastal areas. In France, during a hot period in August 2006, cases of dermatitis and respiratory problems were registered in Marseille area. At that time, a link to the proliferation of Ostreopsis was highhghted for the first time in that area. A specific monitoring was designed and implemented in the summer 2007. Two strains of Ostreopsis cf. ovata, collected in 2008 from Villefranche-sur-Mer and Morgiret coastal waters and grown in culture, were identified by molecular analysis and studied to characterise their growth and toxin profile. Liquid chromatography-mass spectrometry (LC-MS/MS) indicated that both strains produced ovatoxin-a (OVTX-a) as the major component (ca. 90%), and traces of palytoxin (PLTX). Toxin content was determined at the end of the exponential growth phase with highest concentration of 55 pg.cell-1 of OVTX-a and 2.5 pg.cell-1 of PLTX. [ABSTRACT FROM AUTHOR]

The complexity of effects exerted by palytoxin (PITX) in biological systems is apparent from responses observed in different models. We have approached this complexity by proteomic analyses, to study responses induced by PITX at a system level. The analysis of a sub-proteome of a human cell line has shown that PITX causes changes in phosphorylation states of Hsp27 and the oxidation of DJ-1, supporting the conclusion that cell stress responses, including oxidative damage, participate to the cytotoxic effect of PITX. A descriptive meta-analysis of proteomic data about toxic responses induced by other classes of microalgal toxins, such as okadaic acid and microcystins, reveals that several protein components participating to cell stress responses, particularly oxidative stress, are affected when the systems are exposed to effective doses of those toxins. Detected changes comprise either the total levels of individual proteins, or the relative proportions of isoforms distinguishable according to their post-translational modifications. A common set of protein effectors emerges from our analysis, suggesting that cytotoxic effects exerted by some biotoxins could share molecular processes executing cell death responses. [ABSTRACT FROM AUTHOR]

Information about several papers discussed at the roundtable of International Conference on Ostreopsis Development held in April 2011 sponsored by the University of Genoa based on blooming of species is presented. Topics include the secondary metabolites and toxicological aspects of Ostreopsis species and cryptogamie. The conference featured several scientists and lecturers including Gian Paolo Rossini, Aurelia Tubaro and Takeshi Yasumoto.

Abstract: Palytoxin (PLT) is a highly toxic nonpeptidic marine natural product, with a complex chemical structure. Its mechanism of action targets Na,K-ATPase. Fluorescence polarization (FP) is a spectroscopic technique that can be used to determine molecular interactions. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, FP was used to develop a detection method based on the interaction between the Na,K-ATPase and the PLT. The Na,K-ATPase was labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of protein–dye conjugate was measured when the amount of PLT in the medium was modified. The assay protocol was first developed using ouabain as a binding molecule. The final result was a straight line that correlates FP units and PLT concentration. Within this line the PLT equivalents in a natural sample can be quantified. A selective cleaning procedure to mussel samples and dinoflagellates cultures was also developed to avoid the matrix effect. The LOQ (limit of quantification) of the method is 10nM and the LOD (limit of detection) is 2nM. This new PLT detection method is easier, faster, and more reliable than the other methods described to date. [Copyright &y& Elsevier]

Palytoxin is a potent marine toxin and one of the most complex natural compounds ever described. A number of compounds identified as palytoxin congeners (e.g., ovatoxins, mascarenotoxins, ostreocins, etc.) have not been yet structurally elucidated due to lack of pure material in quantities sufficient to an NMR-based structural investigation. In this study, the complex fragmentation pattern of palytoxin in its positive high resolution liquid chromatography tandem mass spectra (HR LC-MS) was interpreted. Under the used conditions, the molecule underwent fragmentation at many sites of its backbone, and a large number of diagnostic fragment ions were identified. The natural product itself was used with no need for derivatization. Interestingly, most of the fragments contained calcium in their elemental formula. Evidence for palytoxin tendency to form adduct ions with calcium and other divalent cations in its mass spectra was obtained. Fragmentation pattern of palytoxin was used as template to gain detailed structural information on ovatoxin-a, the main toxin produced by Ostreopsis ovata, (observe correct font) a benthic dinoflagellate that currently represents the major harmful algal bloom threat in the Mediterranean area. Either the regions or the specific sites where ovatoxin-a and palytoxin structurally differ have been identified. [ABSTRACT FROM AUTHOR]

Abstract: Harmful benthic microalgae blooms are an emerging phenomenon causing health and economic concern, especially in tourist areas. This is the case of the Mediterranean Sea, where Ostreopsis ovata blooms occur in summer, with increasing regularity. Ostreopsis species produce palytoxin (PTX) and analogues, and a number of deaths directly associated with the ingestion of PTX contaminated seafood have been reported. PTX is considered one of the most toxic molecules occurring in nature and can provoke severe and sometimes lethal intoxications in humans. So far in temperate areas, O. ovata blooms were reported to cause intoxications of humans by inhalation and irritations by contact. In addition, invertebrate mass mortalities have been reported, possibly linked to O. ovata blooms, although other causes cannot be ruled out, such as oxygen depletion or high seawater temperature. In order to improve our knowledge about the direct toxicity of this species on invertebrate and vertebrate marine organisms, we performed an ecotoxicological screening to investigate the toxic effects of different concentrations of O. ovata (cultured in the laboratory and sampled in the field during blooms) on crustaceans and fish as model organisms. Artemia salina, Tigriopus fulvus, and Amphibalanus amphitrite larvae and juveniles of the sea bass Dicentrarchus labrax were used as model species. Toxic effects associated with cultured O. ovata cells were investigated using a crossed design: testing two different temperatures (20 and 25°C), four different cell concentrations, and four treatments (untreated O. ovata culture, filtered and resuspended algal cells, growth medium devoid of algal cells, and sonicated algal cells). The results indicate that the toxicity of cultured O. ovata is related to the presence of living O. ovata cells, and that this effect is amplified by temperature. Furthermore, both tests with laboratory cultured algae and field sampled cells pointed out that A. salina is the most sensitive species even at concentrations below the Environmental Alarm Threshold set by the Italian Ministry of Health. Some possible explanations of such sensitivity are discussed, taking into account evidence of O. ovata cells ingestion and the activity of its toxins on the Na+/K+-ATPase. [Copyright &y& Elsevier]

Abstract: Ostreopsis cf. ovata is an epiphytic/benthic dinoflagellate that produces palytoxin-like compounds (putative palytoxin, ovatoxin-a, -b, -c, d and -e). Here we report on effects of nitrogen (N) and phosphorous (P) limited conditions on cell growth, cell size, biovolume, and toxin production of an O. cf. ovata strain isolated from the Adriatic Sea (Italy). Experiments were carried out in batch cultures using nitrate (NO3 −) and phosphate (PO4 3−) as nutrient sources, and testing N:P ratios of 16, 5, 92 (control, N-limited and P-limited conditions, respectively). Residual N and P in the medium, cell yield, toxin concentrations, and toxin composition were analyzed throughout the growth. Two distinct cell size classes were identified and named Class 1 (small cells) and Class 2 (large cells), whose relative contribution under control condition was about 30 and 70%, respectively. N-limitation affected cell size, with significantly higher abundance (16%) of small cells being recorded under N stress than under control and P stress conditions. Conversely, P-limitation induced an increase of cell volume all over the growth cycle. Nutrient limitations affected growth rates and reduced final cell yields of 2.2-fold and 1.8-fold for N- and P-limited treatments vs control, respectively. Under all tested conditions O. cf. ovata showed the same qualitative profile, leading to a slight different contribution of each toxin to the total toxin content. On overall, toxins showed increasing concentrations from early to late stationary growth phase; particularly under control condition total toxin content increased from 13 to 24pgcell−1. Nutrient limitations affected toxin production, which resulted significantly lower than control in late stationary phase, especially under N-deficiency: a 53% and 40% decrease in toxin cell content was observed under N- and P-limited conditions, respectively. [Copyright &y& Elsevier]

Abstract: Palytoxin (PlTX) is a marine toxin originally isolated from the zoantharians of the genus Palythoa. It is considered to be one of the most lethal marine toxins that block the Na+/K+-ATPase. This study was designed to investigate the acute effects of PlTX and ouabain, also an Na+/K+-ATPase blocker, on the mammalian peripheral nervous system using an ex vivo electrophysiological preparation: the isolated mouse sciatic nerve. Amplitude of the evoked nerve compound action potential (nCAP) was used to measure the proper functioning of the sciatic nerve fibres. The half-vitality time of the nerve fibres (the time required to inhibit the nCAP to 50% of its initial value: IT50) incubated in normal saline was 24.5 ± 0.40 h (n = 5). Nerves incubated continuously in 50.0, 10.0, 1.0, 0.5, 0.250 and 0.125 nM of PlTX had an IT50 of 0.06 ± 0.00, 0.51 ± 0.00, 2.1 ± 0.10, 8.9 ± 0.30, 15.1 ± 0.30 h, and 19.5 ± 0.20 h, respectively (n = 5, 3, 4, 4, 10). PlTX was extremely toxic to the sciatic nerve fibres, with a minimum effective concentration (mEC) of 0.125 nM (n = 5) and inhibitory concentration to 50% (IC50) of 0.32 ± 0.08 nM (incubation time 24 h). Ouabain was far less toxic, with a mEC of 250.0 μM (n = 5) and IC50 of 370.0 ± 18.00 μM (incubation 24.5 h). Finally, when the two compounds were combined – e.g. pre-incubation of the nerve fibre in 250.0 μM ouabain for 1 h and then exposure to 1.0 nM PlTX – ouabain offered minor a neuroprotection of 9.1–17.6% against PlTX-induced neurotoxicity. Higher concentrations of ouabain (500.0 μM) offered no protection. The mouse sciatic nerve preparation is a simple and low-cost bioassay that can be used to assess and quantify the neurotoxic effects of standard PlTX or PlTX-like compounds, since it appears to have the same sensitivity as the haemolysis of erythrocytes assay – the standard ex vivo test for PlTX toxicity. [Copyright &y& Elsevier]

Abstract: Of the known benthic, toxigenic microalgae, species from the genera, Gambierdiscus and Ostreopsis, present the biggest threats to human and environmental health. Gambierdiscus spp. produce gambiertoxins which are precursors of ciguatoxins, the compounds that cause ciguatera, the most prevalent, phycotoxin-related form of seafood poisoning across the globe, affecting 25,000 to 500,000 people annually. Recent Ostreopsis spp. blooms in the Mediterranean Sea and coastal waters of New Zealand have led to concerns for seafood safety due to the palytoxin analogs produced by these dinoflagellates. Recent advances in molecular genetics have demonstrated that the taxonomy of both genera were in dire need of revision. Subsequently, Gambierdiscus was revised, and efforts to do the same are ongoing for Ostreopsis. The purpose of this review, therefore, is to update the state of knowledge of these genera, with particular emphasis on the validation or dismissal of earlier findings in light of these revisions. The review is divided into four sections (taxonomy, geography, ecophysiology, and toxicology) in an effort to cover all major aspects of study of these genera. A concluding section closes the review with a synthesis of the state of knowledge on these dinoflagellates and identifies gaps as suggested areas for future research efforts. [Copyright &y& Elsevier]

Abstract: Palytoxin (PLTX), found in Palythoa zoanthids and Ostreopsis dinoflagellates, has also been detected in crabs and fish, through which it can enter into the food chain. Indeed, PLTX is considered the causative agent of several cases of human seafood poisoning resulting in systemic symptoms. Available epidemiological data on PLTX human toxicity suggest that the intestinal tract may be one of its in vivo targets and its potential site of access into the bloodstream. Hence, the purpose of this study was to investigate the suitability of the human intestinal Caco-2 cell line for evaluating PLTX oral absorption. A detailed analysis of PLTX cytotoxicity revealed a high sensitivity of Caco-2 cells: 4h toxin exposure reduced mitochondrial activity (MTT assay, EC50 of 8.9±3.7×10−12 M), cell density (SRB assay, EC50 of 2.0±0.6×10−11 M) and membrane integrity (LDH release, EC50 of 4.5±1.4×10−9 M and PI uptake, EC50 of 1.0±0.8×10−8 M). After low PLTX concentration (1.0×10−11 M) exposure for 1–8h, followed by 24h recovery time in toxin-free medium, cell density reduction was only partially reversible. These results indicate that, due to the high susceptibility to PLTX cytotoxic effects, Caco-2 cells do not represent an appropriate and reliable model for investigating intestinal barrier permeation by this toxin. [Copyright &y& Elsevier]

Abstract: Since 1998, blooms of the epi-benthic dinoflagellate, Ostreopsis cf. ovata Fukuyo have been reported in the Arraial do Cabo area, Rio de Janeiro, Brazil. This dinoflagellate can produce one of the most lethal marine toxins, palytoxin (PLTX) and its analogues. In the study reported herein, the growth rate, cellular dimensions and toxin profile of two O. cf. ovata strains isolated from Armação dos Búzios, Rio de Janeiro were investigated. Molecular analysis (5.8S rDNA gene and ITS regions) of one strain was also performed. Respectively, strains LCA-E7 and LCA-B7 showed growth rates of 0.15 and 0.10divday−1, and cell dimensions of the strains and field population were similar to those found for O. ovata from the Mediterranean Sea. Ribosomal DNA genetic sequences obtained from the nuclear region (ITSs and 5.8S) of strain LCA-E7 confirmed that this is O. cf. ovata and grouped this isolate in a robust clade with isolates from Brazil and the Mediterranean. The production of putative (p)PLTX and ovatoxin (OVTX) was assessed using ouabain inhibited hemolytic assays. Extracts of the two strains caused hemolysis and was positively related to O. cf. ovata cell numbers. Analysis by liquid chromatography tandem mass spectrometry identified OVTX-a, -b, -c, -d and -e. Estimated concentrations varied between 20 and 171pg[OVTX-a]cell−1, 23 and 205pg[OVTX-b]cell−1, 3 and 37pg[OVTX-c]cell−1 and 3 and 80pg[OVTX-d+e]cell−1. OVTX-a and -b were the major components of the toxin profile produced by strains LCA-E7 and LCA-B7 and represented between 45 and 95% of the total OVTX+pPLTX content of these cells. Concentrations of OVTXs produced by the Brazilian strains were higher than those found in an O. cf. ovata Adriatic isolate, although in the same magnitude. [Copyright &y& Elsevier]

Blooms of Ostreopsis spp. have been recently reported along the Mediterranean coasts of Spain, France, Italy, and Greece posing serious risks to human health. Occurrence of Ostreopsis spp. may result in palytoxin contamination of seafood and, in order to prevent sanitary risks, the need exists to develop efficient extraction procedures to be coupled to rapid and sensitive monitoring methods of palytoxin-like compounds in seafood. In the present study, the best conditions for both extraction of palytoxin from seafood and palytoxin quantification by using liquid chromatography tandem mass spectrometry (LC-MS/MS) were investigated. Three seafood matrices (mussels, sea-urchins, and anchovies) were selected and five different extraction systems were tested, namely: the official protocol for extraction of lipophilic toxins and various aqueous methanol or acetonitrile solutions (MeOH/HO 1:1, MeOH/HO 8:2, MeCN/HO 8:2 and MeOH 100%). Extraction with MeOH/HO 8:2 provided the best results in terms of accuracy and matrix interference on LC-MS/MS detection of palytoxin. Accuracy and intra-day reproducibility ( n = 3) were evaluated for all the selected matrices but only for mussels at three spiking concentration levels, including the provisional limit proposed by the Community Reference Laboratory for marine biotoxins (250 μg kg). Limits of quantitation of palytoxin in mussels, sea-urchins and anchovies tissues were calculated using matrix-matched standards; taking into account extraction efficiency of MeOH/HO 8:2, they resulted to be 228, 343, and 500 μg kg, respectively. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

In the last years Ostreopsis spp. blooms have been reported in several Italian coastal stretches, showing a clear trend towards their extension. In 2005, during summer, an O.ovata bloom occurring in the coastal area in front of Genoa was tentatively associated with some human effects. Symptoms like rhinorrea, cough, fever, conjunctivitis, bronchoconstriction with mild dyspnea, and wheezes were observed in people exposed to marine aerosols, possibly containing algal fragments and/or its toxin. With less severe symptoms, similar episodes have been sporadically recorded in other coastal areas. Up to these events, the institutional monitoring activities were focused only on the surveillance of planktonic microalgae whereas benthonic ones, such as Ostreopsis spp. were ignored. To fill this gap, in 2006 a group of experts was appointed by the Italian Ministry of Health with the task to elaborate specific guidelines to manage the possible riskassociated to Ostreopsis spp. blooms. After their formal endorsement, the guidelines were published in the official site of the Ministry and implemented by local structures. The guidelines identify surveillance activities to be carried out, subdividing them into the routine, alert and alarm phases. These guidelines are currently under revision in order to update the scientific background and refine the approach on the basis of the two-year application experience. [ABSTRACT FROM AUTHOR]

Background: A dinoflagellate genus Ostreopsis is known as a potential producer of Palytoxin derivatives. Palytoxin is the most potent non-proteinaceous compound reported so far. There has been a growing number of reports on palytoxin-like poisonings in southern areas of Japan; however, the distribution of Ostreopsis has not been investigated so far. Morphological plasticity of Ostreopsis makes reliable microscopic identification difficult so the employment of molecular tools was desirable. Methods/Principal Finding: In total 223 clones were examined from samples mainly collected from southern areas of Japan. The D8-D10 region of the nuclear large subunit rDNA (D8-D10) was selected as a genetic marker and phylogenetic analyses were conducted. Although most of the clones were unable to be identified, there potentially 8 putative species established during this study. Among them, Ostreopsis sp. 1-5 did not belong to any known clade, and each of them formed its own clade. The dominant species was Ostreopsis sp. 1, which accounted for more than half of the clones and which was highly toxic and only distributed along the Japanese coast. Comparisons between the D8-D10 and the Internal Transcribed Spacer (ITS) region of the nuclear rDNA, which has widely been used for phylogenetic/phylogeographic studies in Ostreopsis, revealed that the D8-D10 was less variable than the ITS, making consistent and reliable phylogenetic reconstruction possible. Conclusions/Significance: This study unveiled a surprisingly diverse and widespread distribution of Japanese Ostreopsis. Further study will be required to better understand the phylogeography of the genus. Our results posed the urgent need for the development of the early detection/warning systems for Ostreopsis, particularly for the widely distributed and strongly toxic Ostreopsis sp. 1. The D8-D10 marker will be suitable for these purposes. [ABSTRACT FROM AUTHOR]

Palytoxin (PLTX), a polyether marine toxin originally isolated from the zoanthid Palythoa toxica, is one of the most toxic non-protein substances known. Fatal poisonings have been linked to ingestion of PLTX-contaminated seafood, and effects in humans have been associated with dermal and inhalational exposure to PLTX containing organisms and waters. Additionally, PLTX co-occurrence with other well-characterized seafood toxins (e.g., ciguatoxins, saxitoxins, tetrodotoxin) has hindered direct associations of PLTX to seafood-borne illnesses. There are currently no validated methods for the quantitative detection of PLTX(s). As such, a well-characterized, robust, specific analytical technique is needed for the detection of PLTX(s) in source organisms, surrounding waters, and clinical samples. Surface plasmon resonance (SPR) biosensors are ideally suited for antibody characterization and quantitative immunoassay detection. Herein, we describe a newly developed SPR assay for PLTX. An anti-mouse substrate was used to characterize the kinetic values for a previously developed monoclonal anti-PLTX. The characterized antibody was then incorporated into a sensitive, rapid, and selective PLTX assay. Buffer type, flow rate, analyte-binding time, and regeneration conditions were optimized for the antibody-PLTX system. Cross-reactivity to potentially co-occurring seafood toxins was also evaluated. We show that this optimized assay is capable of measuring low- to sub-ng/mL PLTX levels in buffer and two seafood matrices (grouper and clam). Preliminary results indicate that this SPR biosensor assay allows for (1) rapid characterization of antibodies and (2) rapid, sensitive PLTX concentration determination in seafood matrices. Method development information contained herein may be broadly applied to future PLTX detection and/or antibody characterization efforts. [ABSTRACT FROM AUTHOR]

Palytoxin (PTX) is classified as one of the most powerful marine biotoxins (of high molecular weight and no protein origin) because it is able to interact strongly with important cellular structures influencing their function in different biological processes. This study of the effects of PTX on red blood cells (RBC) extends the knowledge about its toxicity, which concerns not only the well-known action on Na(+)/K(+)-ATPase but also band 3 protein (B3 or AE1), the role of which is essential for anion transport and for the structure, function, and metabolic integrity of the erythrocyte. The effects of PTX on RBC can be summarized as follows: it alters the anionic flux and seriously compromises not only CO(2) transport but also the metabolic modulation centered on the oxy-deoxy cycle of hemoglobin; it stabilizes the plasma membrane by preventing lipid peroxidation; and its effect does not lead to activation of caspases 3 and 8. From what is reported in steps 2 and 3, and on the basis of the results obtained on hemolysis, methemoglobin levels, and phosphatase activity, an increase of the reducing power of the erythrocytes (RBC) in the presence of PTX clearly emerges. The results have enabled us to outline some metabolic adaptations induced in the RBC by PTX. [ABSTRACT FROM AUTHOR]