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 8.061024 cell per reaction for plasmid and gold standards, respectively; the sensitivity of the assay was of cells g21 fw or 121 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

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

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

Palytoxins: Toxicological Profile

Palytoxin (PLTX), identified in Palythoa corals, Ostreopsis dinoflagellates, and Trichodesmium cyanobacteria, is considered one of the most complicate and toxic molecules found in nature. The recent appearance of Ostreopsis spp. in the Mediterranean Sea as well as the uncontrolled diffusion of Palythoa corals, widely used as aquarium decorative elements, pose serious concerns for human health. The concomitant detection of putative PLTX and of PLTX analogues poses new problems regarding the toxicological potential of PLTXs, already known for their high toxicity since the 1970s. In general, adverse effects in humans can occur through different exposureroutes: ingestion, dermal, inhalational, or ocular exposure. Among them, oral exposure after ingestion of contaminated seafood is the most dangerous one for human health, although it seems to be limited to tropical and subtropical regions. On the contrary, in temperate areas, such as the Mediterranean Sea, human intoxications are frequently associated with inhalational exposure to aerosolized seawater during Ostreopsis blooms. In vivo toxicity studies indicate that the toxicity of PLTX by oral route is lower than that after parenteral injection, but the European Food Safety Authority suggested an admissible limit of 30 \u3bcg PLTX/kg shellfish meat. Also the potential toxicity of PLTXs after aerosol exposure needs to be urgently addressed due to increasing blooms of Ostreopsis associated with respiratory and ocular problems as well as dermotoxicity in people exposed to seawater both for recreational and working activities