Cecilia Battocchi

Unique Toxin Profile of a Mediterranean Ostreopsis cf. ovata Strain: HR LC-MSn Characterization of Ovatoxin-f, a New Palytoxin Congener

Currently, the benthic dinoflagellate Ostreopsis cf. ovata represents a serious concern to human health in the whole Mediterranean basin due to the production of palytoxin congeners, a putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d/-e), listed among the most potent marine toxins. High resolution liquid chromatography-mass spectrometry (HR LC-MS) based investigation of a North Western Adriatic strain of Ostreopsis cf. ovata collected at Portonovo (Italy) in 2008 is reported herein. Toxin profile was different from those previously reported for other O. cf. ovata, both qualitatively and quantitatively. For the first time, ovatoxin-a did not dominate the toxin profile, and a new palytoxin congener, here named ovatoxin-f, was detected. Ovatoxin-f and its elemental formula present C(2)H(4) more than ovatoxin-a. HR CID MS(n) experiments allowed us to restrict structural differences between ovatoxin-a and -f to the region between C-95 and C-102, a region not previously been described to be modified in other palytoxins. Ovatoxin-f represents the major component of the toxin profile of the analyzed strain accounting for 50% of the total toxin content, while ovatoxin-a, the dominant toxin in most of the Mediterranean O. cf. ovata strains we have analyzed so far, is the second major component of the toxin profile (23%). Thus, the presence of ovatoxin-f should be taken into account when monitoring programs for palytoxin-like compounds in microalgae and/or seawater are carried out.

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.

First finding of Ostreopsis cf. ovata toxins in marine aerosols.

Since the late 1990s, a respiratory syndrome has been repetitively observed in humans concomitant with Ostreopsis spp. blooms (mainly O. cf. ovata) in the Mediterranean area. Previous studies have demonstrated that O. cf. ovata produces analogues of palytoxin (ovatoxins and a putative palytoxin), one of the most potent marine toxins. On the basis of the observed association between O. cf. ovata blooms, respiratory illness in people, and detection of palytoxin complex in algal samples, toxic aerosols, containing Ostreopsis cells and/or the toxins they produce, were postulated to be the cause of human illness. A small scale monitoring study of marine aerosol carried out along the Tuscan coasts (Italy) in 2009 and 2010 is reported. Aerosols were collected concomitantly with O. cf. ovata blooms, and they were analyzed by both PCR assays and LC-HRMS. The results, besides confirming the presence of O. cf. ovata cells, demonstrated for the first time the occurrence of ovatoxins in the aerosol at levels of 2.4 pg of ovatoxins per liter of air. Given the lack of toxicological data on palytoxins by inhalation exposure, our results are only a first step toward a more comprehensive understanding of the Ostreopsis-related respiratory syndrome.

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.

New Insights on Cytological and Metabolic Features of Ostreopsis cf. ovata Fukuyo (Dinophyceae): A Multidisciplinary Approach

The harmful dinoflagellate Ostreopsis cf. ovata has been causing toxic events along the Mediterranean coasts and other temperate and tropical areas, with increasing frequency during the last decade. Despite many studies, important biological features of this species are still poorly known. An integrated study, using different microscopy and molecular techniques, Raman microspectroscopy and high resolution liquid chromatography-mass spectrometry (HR LC-MS), was undertaken to elucidate cytological aspects, and identify main metabolites including toxins. The species was genetically identified as O. cf. ovata, Atlantic-Mediterranean clade. The ultrastructural results show unique features of the mucilage network abundantly produced by this species to colonize benthic substrates, with a new role of trichocysts, never described before. The amorphous polysaccharidic component of mucilage appears to derive from pusule fibrous material and mucocysts. In all stages of growth, the cells show an abundant production of lipids. Different developmental stages of chloroplasts are found in the peripheral cytoplasm and in the centre of cell. In vivo Raman microspectroscopy confirms the presence of the carotenoid peridinin in O. cf. ovata, and detects in several specimen the abundant presence of unsaturated lipids structurally related to docosahexaenoic acid. The HR LC-MS analysis reveals that ovatoxin-a is the predominant toxin, together with decreasing amounts of ovatoxin-b, -d/e, -c and putative palytoxin. Toxins concentration on a per cell basis increases from exponential to senescent phase. The results suggest that benthic blooms of this species are probably related to features such as the ability to create a unique mucilaginous sheath covering the sea bottom, associated with the production of potent toxins as palytoxin-like compounds. In this way, O. cf. ovata may be able to rapidly colonize benthic substrates outcompeting other species.

Harmful dinoflagellate Ostreopsis cf. ovata Fukuyo: detection of ovatoxins in field samples and cell immunolocalization using antipalytoxin antibodies.

Ostreopsis cf. ovata, a benthic dinoflagellate often blooming along the Mediterranean coasts, has been associated with toxic events ranging from dyspnea to mild dermatitis. In late September 2009, an Ostreopsis cf. ovata bloom occurred in the Gulf of Trieste (Northern Adriatic Sea; Italy), causing pruritus and mild dermatitis in beachgoers. An integrated study was initiated to characterize Ostreopsis cells by light and confocal microscopy, PCR techniques, immunocytochemistry, and high resolution liquid chromatography-mass spectrometry (HR LC-MS). The presence of Ostreopsis cf. ovata of the Atlantic/Mediterranean clade was unambiguously established by morphological and genetic analyses in field samples. Several palytoxin-like compounds (ovatoxin-a,-b,-c,-d,-e) were identified by HR LC-MS, ovatoxin-a being the most abundant (45-64 pg/cell). Surprisingly, no palytoxin was detected. For the first time, monoclonal and polyclonal antipalytoxin antibodies revealed the intracellular cytoplasmic localization of ovatoxins, suggesting their cross-reactivity with these antibodies. Since harmful dinoflagellates do not always produce toxins, the immunocytochemical localization of ovatoxins, although qualitative, can provide an early warning for toxic Ostreopsis cells before their massive diffusion and/or concentration in seafood.

Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-κB

Background Palytoxin and, likely, its analogues produced by the dinoflagellate genus Ostreopsis, represent a class of non-proteinaceous compounds displaying high toxicity in animals. Owing to the wide distribution and the poisonous effects of these toxins in humans, their chemistry and mechanism of action have generated a growing scientific interest. Depending on the exposure route, palytoxin and its Ostreopsis analogues may cause several adverse effects on human health, including acute inflammatory reactions which seem more typical of cutaneous and inhalation contact. These observations have led us to hypothesize that these toxins may activate pro-inflammatory signalling cascades. Methodology and Principal Findings Here we demonstrate that palytoxin and a semi-purified Ostreopsis cf. ovata toxin extract obtained from a cultured strain isolated in the NW Adriatic Sea and containing a putative palytoxin and all the ovatoxins so far known – including the recently identified ovatoxin-f – significantly increase the levels of mRNAs encoding inflammation-related proteins in immune cells, i.e. monocyte-derived human macrophages, as assessed by Real-Time PCR analysis. Western immunoblot and electrophoretic mobility shift assays revealed that nuclear transcription factor -κB (NF-κB) is activated in cells exposed to toxins in coincidence with reduced levels of the inhibitory protein IκB-α. Moreover, Mitogen-Activated Protein Kinases (MAPK) were phosphorylated in response to palytoxin, as also reported by others, and to the Ostreopsis toxin extract, as shown here for the first time. By using specific chemical inhibitors, the involvement of NF-κB and p38 MAPK in the toxin-induced transcription and accumulation of Cycloxigenase-2, Tumor Necrosis Factor-α, and Interleukin-8 transcripts has been demonstrated. Conclusions and Significance The identification of specific molecular targets of palytoxin and its Ostreopsis analogues, besides contributing to expand the still limited knowledge of the intracellular signalling cascades affected by these toxins, may have important implications in setting up focused pharmacological interventions, replacing currently used symptomatic treatments.

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.

Ecology of Ostreopsis cf. ovata Blooms in the Northwestern Adriatic Sea

Abstract The ecology of Ostreopsis cf. ovata blooms was investigated to evaluate the role of environmental factors (temperature, hydrodynamism, nutrient concentrations, depth and substratum) on the bloom dynamics. This paper reports the present knowledge on O. cf. ovata blooms along the Conero Riviera (NW Adriatic Sea), on the basis of samplings carried out from 2006 to 2010. The annual maximum of benthic cell abundance was always observed in late-summer, reaching the order of magnitude of 106 cells g-1 fw (corresponding to 107 cells g-1 dw and 104 cells cm-2) on macrophyte samples. Comparing the mean abundances settled on seaweeds with those growing on hard substrata, significantly higher abundances were observed on the latter. Hydrodynamism plays a major role in Ostreopsis blooms, as significantly higher abundances were observed in sheltered sites compared with exposed ones. The abundances of O. cf. ovata showed a marked decrease with depth. Temperature and nutrients do not seem to play an important effect on the O. cf. ovata blooms. High levels of ovatoxins were recorded in natural samples; episodes of death of both benthic invertebrates (limpets, sea urchins and mussels) and macroalgae were commonly observed during O. cf. ovata blooms.