Basin scale variability of Ostreopsis spp. blooms provides evidence of effectiveness of an integrated sampling approach.

Ostreopsis spp. blooms have been occurring in the last two decades in the Mediterranean Sea in association with a variety of biotic and abiotic substrata (macroalgae, seagrasses, benthic invertebrates, sand, pebbles and rocks). Cells proliferate attached to the surfaces through mucilaginous trichocysts, which lump together microalgal cells, and can also be found in the plankton and on floating aggregates: such tychoplanktonic behavior makes the quantitative assessment of blooms more difficult than planktonic or benthic ones. Different techniques have been so far applied for quantifying cell abundances of benthic microalgae for research, monitoring and risk assessment purposes. In this context, the Benthic Dinoflagellates Integrator (BEDI), a non-destructive quantification method for benthic dinoflagellate abundances, was developed and tested within the EU ENPI-CBCMED project M3-HABs. This device allows mechanical detachment of cells without collecting the benthic substrate, providing an integrated assessment of both epiphytic and planktonic cells, i.e. of the number of cells potentially made available in the water volume from "resuspension" which could have harmful effects on other organisms (including humans). The present study confirms the effectiveness of the BEDI sampling device across different environments across the Mediterranean Sea and constitutes the first large-scale study of Ostreopsis spp. blooms magnitude in function of different macro- and meso­habitat features across the basin.

Quantification of the toxic dinoflagellate Ostreopsis spp. by qPCR assay in marine aerosol.

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 × 10(0) 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.

Environmental, human health and socioeconomic impacts of Ostreopsis spp. Blooms in the NW Mediterranean.

This paper summarizes the research conducted by the partners of the EU co-funded CoCliME project to ascertain the ecological, human health and economic impacts of Ostreopsis (mainly O. cf. ovata) blooms in the NW Mediterranean coasts of France, Monaco and Spain. This knowledge is necessary to design strategies to prevent, mitigate and, if necessary, adapt to the impacts of these events in the future and in other regions. Ostreopsis proliferations in the Mediterranean have been related to massive mortalities of benthic organisms and to symptoms of respiratory and cutaneous irritation in humans. A six-year epidemiologic study in a Ostreopsis hot spot in Catalonia and the accumulated experience of the French Mediterranean National Ostreopsis Surveillance Network confirm the main effects of these blooms on human health in the NW Mediterranean. The impacts are associated to direct exposure to seawater with high Ostreopsis cell concentrations and to inhalation of aerosols containing unknown irritative chemicals produced under certain circumstances during the blooms. A series of mild acute symptoms, affecting the entire body as well as the ophthalmic, digestive, respiratory and dermatologic systems have been identified. A main remaining challenge is to ascertain the effects of the chronic exposure to toxic Ostreopsis blooms. Still, the mechanisms involved in the deletereous effects of Ostreopsis blooms are poorly understood. Characterizing the chemical nature of the harmful compounds synthesized by Ostreopsis as well as the role of the mucus by which cells attach to benthic surfaces, requires new technical approaches (e.g., metabolomics) and realistic and standardized ecotoxicology tests. It is also necessary to investigate how palytoxin analogues produced by O. cf. ovata could be transferred through the marine food webs, and to evaluate the real risk of seafood poisonings in the area. On the other hand, the implementation of beach monitoring and surveillance systems in the summer constitutes an effective strategy to prevent the impacts of Ostreopsis on human health. In spite of the confirmed noxious effects, a survey of tourists and residents in Nice and Monaco to ascertain the socioeconomic costs of Ostreopsis blooms indicated that the occurrence of these events and their impacts are poorly known by the general public. In relationship with a plausible near future increase of Ostreopsis blooms in the NW Mediterranean coast, this survey showed that a substantial part of the population might continue to go to the beaches during Ostreopsis proliferations and thus could be exposed to health risks. In contrast, some people would not visit the affected areas, with the potential subsequent negative impacts on coastal recreational and touristic activities. However, at this stage, it is too early to accurately assess all the economic impacts that a potentially increasing frequency and biogeographic expansion of the events might cause in the future.

Effects of the harmful dinoflagellate Ostreopsis cf. ovata on different life cycle stages of the common moon jellyfish Aurelia sp.

The frequency and geographic extension of microalgae and gelatinous zooplankton blooms seem to have been increasing worldwide over recent decades. In particular, the harmful dinoflagellate Ostreopsis cf. ovata and the Schyphozoan jellyfish Aurelia sp. are two of the most frequent and long lasting species forming blooms in the Mediterranean Sea. A kind of interaction among any of their life cycle stages (i.e. planula-polyp-ephyrae vs Ostreopsis cells) can likely occur, although in this area there are no data available on the co-occurrence of these species. The aim of this study was to investigate, for the first time, the potential noxious effect of O. cf. ovata on different life stages of Aurelia sp. (polyps and ephyrae), testing several concentrations of whole algal culture. Rsults of toxicity bioassay highlighted that ephyrae, but not polyps, are affected by this harmful dinoflagellate and comparisons among other model organisms show that Aurelia sp. ephyrae are the most sensitive model organism tested so far (EC50-24h=10.5cells/mL). These findings suggest an interesting scenario on the interaction of these two bloom forming species in the natural marine environment.

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.

SHORT-TERM TEMPORAL VARIABILITY OF AMMONIUM AND UREA UPTAKE BY ALEXANDRIUM CATENELLA (DINOPHYTA) IN CULTURES(1).

In batch cultures of four Mediterranean strains (from France, Italy, and Spain) of Alexandrium catenella (Whedon et Kof.) Balech growing on a daily light cycle, ammonium and urea uptake were estimated by the (15) N tracer technique. Ammonium uptake could be described by Michaelis-Menten kinetics along a substrate gradient of 0.1-10 µgat N · L(-1) for the four strains, while two different patterns were observed for urea uptake with Michaelis-Menten kinetics for one strain and linear kinetics for the others. In all cases, an increase in uptake rates with time was noted over the daylight period. This trend led to a net increase in the maximum uptake rate (Vmax ; for saturable kinetics) and in the initial slope α. For ammonium, Vmax increased by a factor of 2-10 depending on the strain, and, for urea, the maximal uptake rates measured increased by a factor of 2-18. Temporal variations of half-saturation constants (Ks ) for both nutrients did not show a clear trend. Increases in Vmax and α showed an acclimation of the cells' uptake system over time to a N pulse, which may be explained by the light periodicity. For two strains, extensive ammonium release was observed during urea assimilation. This mechanism removes urea from the medium, so it is no longer available to other potential competitors, but supplies N back to the medium in the form of ammonium. From a methodological point of view, the phenomenon leads to considerable underestimates of the contribution of urea to phytoplankton growth.

Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a Dinoflagellate).

The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.