Subcellular effects and lipid metabolism alterations in the gilthead seabream Sparus aurata fed on ovatoxins-contaminated mussels.

The marine microalgae Ostreopsis cf. ovata are a well-known producer of palytoxin (PlTXs) analogues, i.e. ovatoxins (OVTXs) among others, which arouse concern for animal and human health. Both in field and laboratory studies, presence of OVTXs, detected in species directly feeding on O. cf. ovata, was frequently correlated with impairment on organisms' physiology, development and behaviour, while similar knowledge is still lacking for animals feeding on contaminated preys. In this study, transfer and toxicity of OVTXs were evaluated in an exposure experiment, in which gilthead seabream Sparus aurata was fed with bivalve mussel Mytilus galloprovincialis, contaminated by a toxic strain of O. cf. ovata. Mussels exposed to O. cf. ovata for 21 days accumulated meanly 188 ± 13 µg/kg OVTXs in the whole tissues. Seabreams fed with OVTX-contaminated mussels started to reject the food after 6 days of contaminated diet. Although no detectable levels of OVTXs were measured in muscle, liver, gills and gastro-intestinal tracts, the OVTX-enriched diet induced alterations of lipid metabolism in seabreams livers, displaying a decreased content of total lipid and fatty acid, together with overexpression of fatty acid biosynthetic genes, downregulation of ß-oxidation genes and modulation of several genes related to lipid transport and regulation. Results from this study would suggest the hypothesis that OVTXs produced by O. cf. ovata may not be subject to bioaccumulation in fish fed on contaminated preys, being however responsible of significant biological effects, with important implications for human consumption of seafood products.

Marine phycotoxin levels in shellfish-14 years of data gathered along the Italian coast.

Along the Italian coasts, toxins of algal origin in wild and cultivated shellfish have been reported since the 1970s. In this study, we used data gathered by the Veterinary Public Health Institutes (IZS) and the Italian Environmental Health Protection Agencies (ARPA) from 2006 to 2019 to investigate toxicity events along the Italian coasts and relate them to the distribution of potentially toxic species. Among the detected toxins (OA and analogs, YTXs, PTXs, STXs, DAs, AZAs), OA and YTX were those most frequently reported. Levels exceeding regulatory limits in the case of OA (≤2,448 µg equivalent kg-1) were associated with high abundances of Dinophysis spp., and in the case of YTXs (≤22 mg equivalent kg-1) with blooms of Gonyaulax spinifera, Lingulodinium polyedra, and Protoceratium reticulatum. Seasonal blooms of Pseudo-nitzschia spp. occur all along the Italian coast, but DA has only occasionally been detected in shellfish at concentrations always below the regulatory limit (≤18 mg kg-1). Alexandrium spp. were recorded in several areas, although STXs (≤13,782 µg equivalent kg-1) rarely and only in few sites exceeded the regulatory limit in shellfish. Azadinium spp. have been sporadically recorded, and AZAs have been sometimes detected but always in low concentrations (≤7 µg equivalent kg-1). Among the emerging toxins, PLTX-like toxins (≤971 µg kg-1 OVTX-a) have often been detected mainly in wild mussels and sea urchins from rocky shores due to the presence of Ostreopsis cf. ovata. Overall, Italian coastal waters harbour a high number of potentially toxic species, with a few HAB hotspots mainly related to DSP toxins. Nevertheless, rare cases of intoxications have occurred so far, reflecting the whole Mediterranean Sea conditions.

A Hotspot of TTX Contamination in the Adriatic Sea: Study on the Origin and Causative Factors.

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels (Mytilus galloprovincialis) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin's origin and the way through which it accumulates in mussels. A two-year study (spring-summer 2020-2021) confirmed the recurrent presence of TTX (11-68 µg kg-1) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus. Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9-296 µg kg-1) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a "potential vector" should therefore be further investigated.

Tetrodotoxins (TTXs) and Vibrio alginolyticus in Mussels from Central Adriatic Sea (Italy): Are They Closely Related?

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg-1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018-2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8-26 µg kg-1) and 3 (2%) contained levels above the EFSA safety threshold (61-76 µg kg-1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.

Survey of the allelopathic potential of Mediterranean macroalgae: production of long-chain polyunsaturated aldehydes (PUAs).

Chemical interactions between macroalgae and other organisms play an important role in determining species compositions and dominance patterns, and can explain the widespread success of some species in establishing their predominant populations in a specific coastal area. Allelopathy could act as a self-regulatory strategy of the algal community, being not only a succession regulator but also an active mechanism maintaining the species diversity especially in a delimited environment, such as the benthic ecosystem. Polyunsaturated aldehydes (PUAs) are among the most studied allelopathic compounds and are commonly released into the aquatic environment by different phytoplankton species in response to environmental stressors (e.g. wounding, grazing, or competition for nutrients). Diatom-released PUAs were observed to affect phytoplankton community dynamics and structure, and showed inhibitory effects on the reproduction and development of marine invertebrates. As for macroalgae, there are only a few reports that attest to the production of PUAs, and mostly refer to Ulva spp. In this study, the production of PUAs by several Mediterranean macroalgae was investigated at different sampling times, aiming at providing the first evidence of potential allelochemical activity. Results highlighted the potential production by macroalgae of a variety of aldehydes, among which some have never reported so far. Some species (i.e. D. polypodioides and U. cf. rigida) were found to produce higher PUAs amounts than others, and even a wider variety of structures (e.g. length of the carbon chain); these species might exert strong effects on epiphytic species or other organisms of the benthic community, especially considering the differential sensitivities of the various taxa. A high dPUA concentration (order of µM) potentially due to the release of PUAs by algal species was found, and might affect the population dynamics of the epiphytic organisms (e.g. microalgae, meiofauna), of grazers, as well as of the microbial community.

Monitoring of cyanobacterial blooms and assessing polymer-enhanced microfiltration and ultrafiltration for microcystin removal in an Italian drinking water treatment plant.

The water intake of a drinking water treatment plant (DWTP) in Central Italy was monitored over six bloom seasons for cyanotoxin severity, which supplies drinking water from an oligo-mesotrophic lake with microcystin levels up to 10.3 µg/L. The historical data showed that the water temperature did not show extreme/large seasonal variation and it was not correlated either with cyanobacterial growth or microcystin concentration. Among all parameters, the cyanobacteria growth was negatively correlated with humidity and manganese and positively correlated with atmospheric temperature. No significant correlation was found between microcystin concentration and the climatic parameters. Polymer(chitosan)-enhanced microfiltration (PEMF) and ultrafiltration (PEUF) were further tested as an alternative microcystin removal approach from dense cyanobacteria-rich flows. The dominant cyanobacteria in the water intake, Planktothrix rubescens, was isolated and enriched to simulate cyanobacterial blooms in the lake. The PEMF and PEUF were separately applied to enriched P. rubescens culture (PC) (microcystin = 1.236 µg/L) as well as to the sand filter backwash water (SFBW) of the DWTP where microcystin concentration was higher than 12 µg/L. The overall microcystin removal rates from the final effluent of PC (always <0.15 µg/L) were between 90.1-94.7% and 89.5-95.4% using 4 and 20 mg chitosan/L, respectively. Meanwhile, after the PEMF and PEUF of SFBW, the final effluent contained only 0.099 and 0.057 µg microcystin/L with an overall removal >99%. The presented results are the first from the application of chitosan to remove P. rubescens as well as the implementation of PEMF and PEUF on SFBW to remove cyanobacterial cells and associated toxins.

Morphological Variability of Pseudo-nitzschia pungens Clade I (Bacillariophyceae) in the Northwestern Adriatic Sea.

Pseudo-nitzschia pungens is a common component of the phytoplankton community of the northern Adriatic Sea. In this study, an in-depth morphological analysis of P. pungens was carried out in both cultured strains isolated in different periods and field samples, revealing a surprisingly wide variability in a number of details, with both the gross morphology and ultrastructural levels deviating from the nominal P. pungens. Colonies showed an overlap (from one-third to one-sixth) and a transapical axis (rarely reaching 3 µm), strongly differing from the original description of the species. Moreover, valves may be either symmetrical or slightly asymmetrical, with striae almost always biseriate but sometimes uniseriate or triseriate. Poroids' morphology in cingular bands was characterized by a wide variability (square, circular, or rectangular poroids without or with up to two hymen sectors), with several combination of them, even within the same cingular band. Phylogenetic analyses based on ITS rDNA showed that the P. pungens of the northern Adriatic Sea belonged to clade I. Domoic acid was not detected.

Distribution and potential toxicity of benthic harmful dinoflagellates in waters of Florida Bay and the Florida Keys.

Few studies have been carried out on benthic dinoflagellates along the Florida Keys, and little is known about their distribution or toxicity in Florida Bay. Here, the distribution and abundance of benthic dinoflagellates was explored in northern and eastern Florida Bay and along the bay and ocean sides of the Florida Keys. Isolates were brought into culture and their toxicity was tested with oyster larvae bioassays. Seven genera were detected, including Prorocentrum, Coolia, Ostreopsis, Amphidinium, Gambierdiscus, Fukuyoa (all included potentially toxic species) and Sinophysis. In general, distribution increased with water temperature and nutrient availability, especially that of phosphate. This study documented the first record of Coolia santacroce in the Florida Keys. Potential toxic effects of Gambierdiscus caribaeus, the abundance of which exceeded 1000 cells g-1 fw at some sites, were established using oyster larvae as a bioassay organism. These findings suggest a potential risk of ciguatera fish poisoning in this area.

Biological Effects of the Azaspiracid-Producing Dinoflagellate Azadinium dexteroporum in Mytilus galloprovincialis from the Mediterranean Sea.

Azaspiracids (AZAs) are marine biotoxins including a variety of analogues. Recently, novel AZAs produced by the Mediterranean dinoflagellate Azadinium dexteroporum were discovered (AZA-54, AZA-55, 3-epi-AZA-7, AZA-56, AZA-57 and AZA-58) and their biological effects have not been investigated yet. This study aimed to identify the biological responses (biomarkers) induced in mussels Mytilus galloprovincialis after the bioaccumulation of AZAs from A. dexteroporum. Organisms were fed with A. dexteroporum for 21 days and subsequently subjected to a recovery period (normal diet) of 21 days. Exposed organisms accumulated AZA-54, 3-epi-AZA-7 and AZA-55, predominantly in the digestive gland. Mussels' haemocytes showed inhibition of phagocytosis activity, modulation of the composition of haemocytic subpopulation and damage to lysosomal membranes; the digestive tissue displayed thinned tubule walls, consumption of storage lipids and accumulation of lipofuscin. Slight genotoxic damage was also observed. No clear occurrence of oxidative stress and alteration of nervous activity was detected in AZA-accumulating mussels. Most of the altered parameters returned to control levels after the recovery phase. The toxic effects detected in M. galloprovincialis demonstrate a clear biological impact of the AZAs produced by A. dexteroporum, and could be used as early indicators of contamination associated with the ingestion of seafood.

Role of temperature and nutrients on the growth and toxin production of Prorocentrum hoffmannianum (Dinophyceae) from the Florida Keys.

The benthic dinoflagellate Prorocentrum hoffmannianum M.A. Faust is typical of tropical warm waters and produces biotoxins responsible for diarrhetic shellfish poisoning (DSP). In this study, the effect of temperature and nutrient limitation on growth and toxin production of P. hoffmannianum isolated from field samples collected in the Florida Keys was investigated. Batch culture experiments were ran at two temperatures (i.e. 21 ± 0.1 and 27 ± 0.1 °C) and under nitrogen-limited (14.7 µmol L-1 N-NO3- and 18.1 µmol L-1 P-PO43-) and phosphorus-limited (441 µmol L-1 N-NO3- and 0.6 µmol L-1 P-PO43-) levels with respect to control nutrient conditions (441 µmol L-1 N-NO3-and 18.1 µmol L-1 P-PO43-). Both temperature and nutrient conditions significantly affected growth rates and maximum yield of P. hoffmannianum with the maximum values being recorded at the higher temperature and in the replete medium. Production of okadaic acid was induced under all conditions (from 13.5 to 859.8 pg cell-1), with values up to one order of magnitude higher than those observed in other DSP toxin producing species. Toxin production was enhanced under P limitation at 27 °C, corroborating the theory that toxin production is modulated by cell physiological conditions, which are in turn affected by a wide spectrum of factors, including environmental stressors such as nutrient availability. Toxin fraction released in the growth medium was negligible. No okadaic acid esters were detected in this strain of P. hoffmannianum.

Plankton dynamics across the freshwater, transitional and marine research sites of the LTER-Italy Network. Patterns, fluctuations, drivers.

A first synoptic and trans-domain overview of plankton dynamics was conducted across the aquatic sites belonging to the Italian Long-Term Ecological Research Network (LTER-Italy). Based on published studies, checked and complemented with unpublished information, we investigated phytoplankton and zooplankton annual dynamics and long-term changes across domains: from the large subalpine lakes to mountain lakes and artificial lakes, from lagoons to marine coastal ecosystems. This study permitted identifying common and unique environmental drivers and ecological functional processes controlling seasonal and long-term temporal course. The most relevant patterns of plankton seasonal succession were revealed, showing that the driving factors were nutrient availability, stratification regime, and freshwater inflow. Phytoplankton and mesozooplankton displayed a wide interannual variability at most sites. Unidirectional or linear long-term trends were rarely detected but all sites were impacted across the years by at least one, but in many case several major stressor(s): nutrient inputs, meteo-climatic variability at the local and regional scale, and direct human activities at specific sites. Different climatic and anthropic forcings frequently co-occurred, whereby the responses of plankton communities were the result of this environmental complexity. Overall, the LTER investigations are providing an unparalleled framework of knowledge to evaluate changes in the aquatic pelagic systems and management options.

Allelopathic effects of diatom filtrates on the toxic benthic dinoflagellate Ostreopsis cf. ovata.

Ostreopsis blooms regularly occur in many Mediterranean coastal areas in late summer-autumn. In the northern Adriatic Sea, Ostreopsis blooms affect diatom-dominated microphytobenthic communities. In this study, the effects of the filtrates of some diatom species, both benthic (Tabularia affinis, Proschkinia complanatoides and Navicula sp.) and planktonic (Thalassiosira sp. and Skeletonema marinoi) on cell morphology, cytological features and growth of O. cf. ovata were investigated. Our results showed a marked decrease of O. cf. ovata growth when cells were exposed to all diatom filtrates tested. The highest inhibitions were observed for exposures to P. complanatoides and Navicula sp. filtrates (92.5% and 80%, respectively) and increased with the age of diatom culture. Moreover, a clear DNA degradation and abnormal forms of O. cf. ovata cells (83.8% of the total) were found at the highest concentrations using Navicula sp. filtrate after 10 days of the inoculum.

Influence of environmental factors on the toxin production of Ostreopsis cf. ovata during bloom events.

Intense blooms of the toxic dinoflagellate Ostreopsis have been a recurrent phenomenon along several Mediterranean coasts. Blooms have been associated with noxious effects on human health and mortality of marine organisms, due to the production of palytoxin-like compounds. We analyzed the toxin concentrations throughout an O. cf. ovata bloom to highlight their relationships with environmental parameters in the Conero Riviera, northern Adriatic Sea. High temperature and balanced nutrient conditions were the optimal environmental conditions to start and sustain blooms as well as to maximize toxin production. Ostreopsis showed a gradual decrease of toxin content throughout the bloom ascribed to the occurring of the same non-optimal conditions that led to the bloom decline. Moreover, our results suggest that toxin fraction released during bloom could be higher than that released in batch culture. Results from this study pointed out that the first bloom phase is potentially the most dangerous to human health.

Phosphatase activities of a microepiphytic community during a bloom of Ostreopsis cf. ovata in the northern Adriatic Sea.

It is becoming more apparent that increased organic nutrient loads deriving from anthropogenic activities and natural processes frequently cause the eutrophication of coastal waters. Concurrently, an increasing number of phototrophs have been shown to make use of organic nutrients, mainly through indirect studies of surface enzyme activities or through direct studies of growth in media containing organic-only nutrients. The potential utilization of dissolved organic phosphorus (DOP) by microepiphytic-mats associated with frequently problematic, toxic dinoflagellate Ostreopsis cf. ovata, was investigated throughout a full cycle of a bloom that occurs annually (over the last few decades) during summer along several stretches of the Mediterranean coast. Measurements of phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities of the epiphytic mats (including cells and exopolymeric substances) and a range of chemico-physical parameters were made from late summer to early autumn at a eutrophic site. Analyses of ambient nutrient fractions revealed very high aquatic N:P values (TN:TP = 178 ± 50), very low filterable reactive P (FRP) concentrations (13 out 19 under the limits of detection) and DOP concentrations that were on average 85% of the total dissolved P. We recorded a rapid increase in PMEase and PDEase activities in the epiphytic microalgal community that coincided with the onset of a proliferation of the Ostreopsis population. Chromogenic staining of samples showed that activity was closely associated with the Ostreopsis cells, located both extracellularly (cell surface and within the EPS) and intracellularly (ventral cytoplasm). The increase in both phosphatases indicates that Ostreopsis can utilise a wide range of DOP types. The intense activity in the EPS was suggested to aid in the efficient entrapment and processing of high concentration nutrient pulses, for extracellular processing of larger organic materials and to prevent loss of substrates and products to the surrounding water. Based on the present findings, Ostreopsis seems to have adaptations that allow it to thrive in P-limited environments where organic P is the main source of P.

Ostreopsis fattorussoi sp. nov. (Dinophyceae), a new benthic toxic Ostreopsis species from the eastern Mediterranean Sea.

The new benthic toxic dinoflagellate, Ostreopsis fattorussoi sp. nov., is described from the Eastern Mediterranean Sea, Lebanon and Cyprus coasts, and is supported by morphological and molecular data. The plate formula, Po, 3', 7″, 6c, 7s, 5‴, 2'''', is typical for the Ostreopsis genus. It differs from all other Ostreopsis species in that (i) the curved suture between plates 1' and 3' makes them approximately hexagonal, (ii) the 1' plate lies in the left half of the epitheca and is obliquely orientated leading to a characteristic shape of plate 6″. The round thecal pores are bigger than the other two Mediterranean species (O. cf. ovata and O. cf. siamensis). O. fattorussoi is among the smallest species of the genus (DV: 60.07 ± 5.63 µm, AP: 25.66 ± 2.97 µm, W: 39.81 ± 5.05 µm) along with O. ovata. Phylogenetic analyses based on the LSU and internal transcribed spacer rDNA shows that O. fattorussoi belongs to the Atlantic/Mediterranean Ostreopsis spp. clade separated from the other Ostreopsis species. Ostreopsis fattorussoi produces OVTX-a and structural isomers OVTX-d and -e, O. cf. ovata is the only other species of this genus known to produce these toxins. The Lebanese O. fattorussoi did not produce the new palytoxin-like compounds (ovatoxin-i, ovatoxin-j1 , ovatoxin-j2 , and ovatoxin-k) that were previously found in O. fattorussoi from Cyprus. The toxin content was in the range of 0.28-0.94 pg · cell-1 . On the Lebanon coast, O. fattorussoi was recorded throughout the year 2015 (temperature range 18°C-31.5°C), with peaks in June and August.

Effects of the bloom of harmful benthic dinoflagellate Ostreopsis cf. ovata on the microphytobenthos community in the northern Adriatic Sea.

Composition and temporal variation of the microphytobenthos communities of the Conero Riviera (northern Adriatic Sea) were investigated in the course of an annual cycle, focusing on their relationships with blooms of the benthic dinoflagellate Ostreopsis cf. ovata. Sampling was carried out from March 2009 to March 2010 on undisturbed benthic substrata (macroalgae and pebbles). Samples for the study of microphytobenthos were collected with a monthly frequency, while those for the study of Ostreopsis bloom weekly. Benthic diatoms dominated the microphytobenthos communities for most of the annual cycle (except the summer), both in terms of abundance and biomass. In summer, cyanobacteria were dominant (54.04±9.18 and 24.29±11.11% of total abundance and biomass, respectively), while benthic dinoflagellates were an important component of the community in terms of biomass only at the peak of the Ostreopsis bloom in late summer (up to 91% of the total biomass). Among diatoms, the most abundant forms throughout the year were motile species (77.5±3.71% of the population), while erect diatoms formed the majority of the biomass in winter and spring (48.66±16.66 and 48.05±5.56% of total population, respectively). Diatoms were mainly affected by DIN availability, while the patterns of biomass of O. cf. ovata and cyanobacteria were related to salinity and temperature. The biomass of Ostreopsis was also affected by the availability of phosphorus. The results of this study suggest that the proliferation of Ostreopsis affected the structure of the benthic diatom community: motile diatoms were significantly more abundant during the Ostreopsis bloom peak than during the rest of summer, probably because they benefited from the abundant mucilaginous mat covering the benthic substrata. In the course of the O. cf. ovata bloom the diversity of the microphytobenthos was significantly lower than during the rest of the year, suggesting an influence of both the shading produced by the mucous mat and allelopathic compounds possibly produced by O. cf. ovata.

Ostreopsis cf. ovata bloom in the northern Adriatic Sea during summer 2009: ecology, molecular characterization and toxin profile.

Intense blooms of the benthic dinoflagellate Ostreopsis cf. ovata have occurred in the northern Adriatic Sea since 2006. These blooms are associated with noxious effects on human health and with the mortality of benthic organisms because of the production of palytoxin-like compounds. The O. cf. ovata bloom and its relationships with nutrient concentrations at two stations on the Conero Riviera (northern Adriatic Sea) were investigated in the summer of 2009. O. cf. ovata developed from August to November, with the highest abundances in September (1.3×10(6) cells g(-1) fw corresponding to 63.8×10(3) cells cm(-2)). The presence of the single O. cf. ovata genotype was confirmed by a PCR assay. Bloom developed when the seawater temperature was decreasing. Nutrient concentrations did not seem to affect bloom dynamics. Toxin analysis performed by high resolution liquid chromatography-mass spectrometry revealed a high total toxin content (up to 75 pg cell(-1)), including putative palytoxin and all the ovatoxins known so far.

New approach using the real-time PCR method for estimation of the toxic marine dinoflagellate Ostreopsis cf. ovata in marine environment.

BACKGROUND: We describe the development and validation of a new quantitative real time PCR (qrt-PCR) method for the enumeration of the toxic benthic dinoflagellate Ostreopsis cf. ovata in marine environment. The benthic Ostreopsis sp. has a world-wide distribution and is associated during high biomass proliferation with the production of potent palytoxin-like compounds affecting human health and environment. Species-specific identification, which is relevant for the complex of different toxins production, by traditional methods of microscopy is difficult due to the high morphological variability, and thus different morphotypes can be easily misinterpreted. METHODOLOGY/FINDINGS: The method is based on the SYBR I Green real-time PCR technology and combines the use of a plasmid standard curve with a "gold standard" created with pooled crude extracts from environmental samples collected during a bloom event of Ostreopsis cf. ovata in the Mediterranean Sea. Based on their similar PCR efficiencies (95% and 98%, respectively), the exact rDNA copy number per cell was obtained in cultured and environmental samples. Cell lysates were used as the templates to obtain total recovery of DNA. The analytical sensitivity of the PCR was set at two rDNA copy number and 8.0×10(-4) cell per reaction for plasmid and gold standards, respectively; the sensitivity of the assay was of cells g(-1) fw or 1(-1) in macrophyte and seawater samples, respectively. The reproducibility was determined on the total linear quantification range of both curves confirming the accuracy of the technical set-up in the complete ranges of quantification over time. CONCLUSIONS/SIGNIFICANCE: We developed a qrt-PCR assay specific, robust and high sample throughput for the absolute quantification of the toxic dinoflagellate Ostreopsis cf. ovata in the environmental samples. This molecular approach may be considered alternative to traditional microscopy and applied for the monitoring of benthic toxic microalgal species in the marine ecosystems.

Trends in Ostreopsis proliferation along the Northern Mediterranean coasts.

Harmful benthic microalgae blooms represent an emergent phenomenon in temperate zones, causing health, ecological and economic concern. The main goal of this work was to compile records of Ostreopsis at large temporal and spatial scales, in order to study the relationship between cell abundances, the periodicity and intensity of the blooms and the role of sea water temperature in 14 Spanish, French, Monegasque and Italian sites located along the northern limits of the Mediterranean Sea. General trends were observed in the two considered basins: the north-western Mediterranean Sea, in which higher cell abundances were mostly recorded in mid-summer (end of July), and the northern Adriatic Sea where they occur in early fall (end of September). The sea-water temperature does not seem to be a primary driver, and the maximal abundance periods were site and year specific. Such results represent an important step in the understanding of harmful benthic microalgae blooms in temperate areas, and provide a good base for policy makers and managers in the attempt to monitor and forecast benthic harmful microalgae blooms.

Monitoring toxic microalgae Ostreopsis (dinoflagellate) species in coastal waters of the Mediterranean Sea using molecular PCR-based assay combined with light microscopy.

A molecular PCR-based assay was developed and applied to macrophyte and seawater samples containing mixed microphytobenthic and phytoplanktonic assemblages, respectively, in order to detect toxic Ostreopsis species in Mediterranean Sea. The specificity and sensitivity of the molecular PCR assay were assessed with both plasmidic and genomic DNA of the target genus or species using taxon-specific primers in the presence of background macrophyte DNA. The PCR molecular technique allowed rapid detection of the Ostreopsis cells, even at abundances undetectable within the resolution limit of the microscopy technique. Species-specific identification of Ostreopsis was determined only by PCR-based assay, due to the inherent difficulty of morphological identification in field samples. In the monitoring of the toxic Ostreopsis blooms PCR-based methods proved to be effective tools complementary to microscopy for rapid and specific detection of Ostreopsis and other toxic dinoflagellates in marine coastal environments.