Magda Vila

Development of a Real-Time PCR Assay for Rapid Detection and Quantification of Alexandrium minutum (a Dinoflagellate)

ABSTRACT 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.

CHARACTERIZATION OF OSTREOPSIS AND COOLIA (DINOPHYCEAE) ISOLATES IN THE WESTERN MEDITERRANEAN SEA BASED ON MORPHOLOGY, TOXICITY AND INTERNAL TRANSCRIBED SPACER 5.8S rDNA SEQUENCES†

Several isolates of epiphytic dinoflagellates belonging to the genera Ostreopsis Schmidt and Coolia Meunier from the western Mediterranean Sea were examined by LM and EM, toxicity assays, and internal transcribed spacer (ITS) regions of nuclear rDNA, and 5.8S rDNA were sequenced. Morphological comparisons based on the analyses of cell shape, size, thecal plates, and surface ornamentation revealed two distinct species in the western Mediterranean: O. cf. siamensis Schmidt from the Catalan, Andalusian, and Sicilian coasts and O. ovata Fukuyo from the Ligurian coast, southern Tyrrhenian Sea, and Balearic Islands. Both Ostreopsis species were toxic; however, no differences in toxicity were detected between the two Ostreopsis species. Coolia monotis Meunier was nontoxic. The morphological studies were supported by phylogenetic analyses; all western Mediterranean isolates of O. cf. siamensis showed ITS and 5.8S rDNA sequences identical to each other and so did those of O. ovata, whereas high genetic diversity was detected between the western Mediterranean and Asian isolates of O. ovata. The nucleotide sequence analyses of the C. monotis strains showed that all C. monotis isolates from Europe formed a homogeneous clade. Further, the genetic diversity was high between the European and Asian C. monotis isolates. In this study, genetic markers combined with morphology and toxicity analyses was useful in the taxonomic and phylogenetic studies of the Ostreopsidaceae in a temperate area.

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.

Phylogenetic relationships among the Mediterranean Alexandrium (Dinophyceae) species based on sequences of 5.8S gene and Internal Transcript Spacers of the rRNA operon

A phylogenetic analysis of the genus Alexandrium, including both the most common and rare species from coastal areas of the Mediterranean Sea was carried out. Nucleotide sequences of 5.8 S gene and Internal Transcribed Spacer regions of the rRNA operon were examined and analysed together with isolates of Alexandrium spp. from elsewhere in the world. These rDNA ribosomal markers were useful in delineating the phylogenetic position of species in the genus, as well as in determining relationships among isolates within each species collected from different localities. Results of phylogeographical analyses within the ‘Alexandrium tamarense’ species complex identified three lineages in the Mediterranean Sea: the Mediterranean (ME), Western European (WE) and Temperate Asian (TA) clades. The phylogenetic grouping of the isolates is consistent with the ribotype clades, but not with the morpho-species that constitute the complex. Additional non-toxic isolates were included in the ME clade. The NA (North Atlantic) clade is the fourth group within the ‘Alexandrium tamarense’ species complex identified by phylogenetic analyses. Based on its higher genetic diversity and phylogeographical relationships, it can be hypothesized that the NA clade represents the ancestral group of the ‘Alexandrium tamarense’ species complex. Alexandrium minutum isolates of the NW Mediterranean clustered with strains from Brittany and Australia. Alexandrium minutum constituted a sister clade of A. tamutum, which is another species strongly associated with the Mediterranean area. Another typical Mediterranean species, A. taylori, was placed as a sister clade of A. pseudogoniaulax by the phylogenetic analysis. Finally, the phylogenetic relationships of some Alexandrium morpho-species that were infrequently observed in the Mediterranean Sea have been resolved.

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.

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.

Short-term variations in development of a recurrent toxic Alexandrium minutum–dominated dinoflagellate bloom induced by meteorological conditions1

Development of an Alexandrium minutum Halim bloom affecting a Mediterranean harbor was monitored in detail using a multidisciplinary approach. A. minutum was by far the most abundant species at and near the bloom maximum, but always coexisted with members of three additional dinoflagellate genera and prasinophytes. Bloom initiation (early February) occurred during prolonged influences of sunny weather conditions, when day length exceeded 10.5 h and water temperatures reached 10.2°C. Subsequent development toward its maximum (end of March) also relied on good weather conditions, with specific wind directions favoring accumulation of cells. Arrival of rainy weather, associated with frontal boundaries of large‐scale low‐atmospheric‐pressure systems and characterized by reduced solar irradiance (heavy cloud coverage), opposite wind directions, and enhanced wind speeds, always caused temporal declines of the bloom. These declines were attributed to dispersal or displacement of algae, but a vertical migration of A. minutum cells toward the sediment was not excluded. Delayed inflows of excess terrestrial rainwater along the inner harbor wall strongly reduced salinity and prolonged a temporal decline far beyond influences of bad weather. The associated nutrient supply favored development of the phytoplankton population but reduced the toxin production of A. minutum cells. The HPLC‐determined Gonyautoxin (GTX) 1 + 4/GTX 2 + 3 ratio strongly increased toward the bloom maximum. This ratio was influenced by nutrient status and cell density and has a potential value for monitoring developmental stages of blooms. Prolonged bad weather conditions eventually hindered continuation of bloom development, and subsequent declines of algal biomass were attributed to grazing.

Genetic diversity of the genus Ostreopsis Schmidt: Phylogeographical considerations and molecular methodology applications for field detection in the Mediterranean Sea

Abstract This study reports some recent phylogeographical considerations on the genus Ostreopsis distribution worldwide, with particular attention to the Mediterranean Sea, and new recent advances on the quali-quantitiative detection of Ostreopsis species along coastal areas of the Mediterranean Sea based on the PCR and quantitative real time PCR (qrt-PCR) assays. It was found that O. cf. ovata is widely dispersed throughout tropical and warm temperate coastal areas. In the Atlantic/Mediterranean region it represents a panmictic population that is highly divergent from Indo-Pacific populations. Furthermore, we demonstrated that the developed qrt-PCR assay is specific, robust and high sample throughput for the quantification of the toxic O. cf. ovata in the environmental samples. This molecular approach may be considered alternative to traditional methods of microscopy and applied for the survey of benthic toxic microalgal species in marine ecosystems.

Management of Ostreopsis Blooms in Recreational waters along the Catalan Coast (NW Mediterranean Sea): Cooperation between a Research Project and a Monitoring Program

Abstract As shown in this report, the integration of a research project with a monitoring program improves the detection and management of Ostreopsis blooms in Catalonia. The research project benefits from information previously obtained from several localities by the monitoring program, which in turn profits from the specific findings and conclusions contributed by the research project.

The genus Ostreopsis along the Algerian coastal waters (SW Mediterranean Sea) associated with a human respiratory intoxication episode

Abstract This work describes the first toxic event (human intoxications and sea urchin mortality) related to Ostreopsis bloom in Algerian coastal waters. The distribution of Ostreopsis is presented in three rocky beaches of Algiers Wilaya, where several cases of human intoxications were registered during July 2009. The maximum abundances attained by Ostreopsis in other stations of the network established by the Agency for the Protection and Promotion of Algiers Wilaya coasts (APPL) for monitoring HABs species are also presented. We discuss some of the factors that could explain this toxic episode.