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Marine diatoms of the genus Pseudo-nitzschia are widespread and occur in various environments. About half of the species described today have been shown to be toxic, producing the potent neurotoxin, domoic acid. The Pseudo-nitzschia species are a common component of the Adriatic phytoplankton community and are occasionally predominant. In the present study, the composition of the Pseudo-nitzschia species was studied in four distinct aquaculture areas from February 2022 to February 2023. These are the first results of a comprehensive study of Pseudo-nitzschia diversity combining morphological and molecular analyses. The integrated approach resulted in the confirmation of Pseudo-nitzschia brasiliana, Pseudo-nitzschia hasleana, Pseudo-nitzschia galaxiae, and Pseudo-nitzschia linea. The species, P. galaxiae, was the most frequently occurring, P. linea was found in all investigated areas, while P. hasleana and P. brasiliana were rare, and their distribution was limited mainly to Kaštela Bay. The findings of P. brasiliana and P. hasleana represent the first confirmation for the Adriatic Sea, while P. galaxiae and P. linea have not been previously reported along the eastern Adriatic coast.

This study provides the first report of the presence of Coolia malayensis in the Mediterranean Sea, co-occurring with C. monotis. Isolated strains from the Gulf of Gabès, Tunisia (South-eastern Mediterranean) were identified by morphological characterization and phylogenetic analysis. Examination by light and scanning electron microscopy revealed no significant morphological differences between the Tunisian isolates and other geographically distant strains of C. monotis and C. malayensis. Phylogenetic trees based on ITS1-5.8S-ITS2 and D1‒D3/28S rDNA sequences showed that C. monotis strains clustered with others from the Mediterranean and Atlantic whereas the C. malayensis isolate branched with isolates from the Pacific and the Atlantic, therefore revealing no geographical trend among C. monotis and C. malayensis populations. Ultrastructural analyses by transmission electron microscopy revealed the presence of numerous vesicles containing spirally coiled fibers in both C. malayensis and C. monotis cells, which we speculate to be involved in mucus production.

Plastic pollution is a global issue posing a threat to marine biota with ecological implications on ecosystem functioning. Micro and nanoplastic impact on phytoplankton autotrophic species (e.g., cell growth inhibition, decrease in chlorophyll a and photosynthetic efficiency and hetero-aggregates formation) have been largely documented. However, the heterogeneity of data makes rather difficult a comparison based on size (i.e. micro vs nano). In addition, knowledge gaps on the ecological impact on phytoplankton assemblage structure and functioning are evident. A new virtual meta-analysis on cause-effect relationships of micro and nanoplastics on phytoplankton species revealed the significant effect posed by polymer type on reducing cell density for tested PVC, PS and PE plastics. Linked with autotrophic phytoplankton role in atmospheric CO2 fixation, a potential impact of plastics on marine carbon pump is discussed. The understanding of the effects of microplastics and nanoplastics on the phytoplankton functioning is fundamental to raise awareness on the overall impact on the first level of marine food web. Interactions between micro and nanoplastics and phytoplankton assemblages have been quite documented by in vitro examinations; but, further studies considering natural plankton assemblages and/or large mesocosm experiments should be performed to evaluate and try predicting ecological impacts on primary producers.

Cyst abundance and identity are essential for understanding and predicting blooms, and for assessing the dispersal of toxic target dinoflagellate species by natural or human mediated ways, as with ballast waters. The aim of this study was to apply rapid, specific and sensitive qPCR assays to enumerate toxic dinoflagellate cysts in sediment samples collected from Adriatic harbours. The molecular standard curves of various target species allowed obtaining the rDNA copy number per cyst. The analytical sensitivity for specific standard curves was determined to be 2 or 10 rDNA copies per reaction. The abundance varied in the range of 1–747 dinoflagellate cysts g−1 dry weight. The assays showed greater sensitivity as compared to counts by light microscopy. This qPCR method revealed a powerful tool for the quantification of cysts from toxic dinoflagellate resting stages in sediment samples from Adriatic ports.

Plastic debris carry fouling a variety of class-size organisms, among them harmful microorganisms that potentially play a role in the dispersal of allochthonous species and toxic compounds with ecological impacts on the marine environment and human health. We analyzed samples of marine plastics floating at the sea surface using a molecular qPCR assay to quantify the attached microalgal taxa, in particular, harmful species. Diatoms were the most abundant group of plastic colonizers with maximum abundance of 8.2 x 10(4) cells cm(-2) of plastics, the maximum abundance of dinoflagellates amounted to 1.1 x 10(3) cells cm(-2) of plastics. The most abundant harmful microalgal taxon was the diatom Pseudo-nitzschia spp., including at least 12 toxic species, and the dinoflagellate Ostreopsis cf. ovata with 6606 and 259 cells cm(-2), respectively. The abundance of other harmful microalgal species including the toxic allochthonous dinoflagellate Alexandrium pacificum ranged from 1 to 73 cells cm(-2). In the present study, a direct relationship between the abundance of harmful algal species colonizing the plastic substrates and their toxin production was found. The levels of potential toxins on plastic samples ranged from 101 to 10(2) ng cm(-2), considering the various toxin families produced by the colonized harmful microalgal species. We also measured the rate of adhesion by several target microalgal species. It ranged from 1.8 to 0.3 day(-1) demonstrating the capacity of plastic substrate colonizing rapidly by microalgae. The present study reports the first estimates of molecular quantification of microorganisms including toxin producing species that can colonize plastics. Such findings provide important insights for improving the monitoring practice of plastics and illustrate how the epi-plastic community can exacerbate the harmful effects of plastics by dispersal, acting as an alien and toxic species carrier and potentially being ingested through the marine trophic web. (C) 2018 Elsevier Ltd. All rights reserved.

The aim of the present study isto characterise the diatom Pseudo-nitzschia community during a bloom period together with environmental conditions. High proliferation of Pseudo-nitzschia spp. was observed in September 2017 at the shellfish breeding area in the Krka River estuary (Central eastern Adriatic Sea). The peak of abundance (1.8 x 10 6 cells L -1 ) was recorded at 7 m depth, and the increased abundance persisted for four weeks.Morphological analyses of field samples based on scanning electron microscopy (SEM) revealed that Pseudo-nitzschia cf. arenysensis was prevailing (94%) in the Pseudo-nitzschia assemblage. Several strains were successfully isolated from net samples in order to better define morphological features and phylogenetic characterisation. The isolated Pseudo-nitzschia strains corresponded morphologically to the P. cf. arenysensis from the field samples, based on our SEM observations. Phylogenetic analysis demonstrated that the Croatian strains grouped with P. arenysensis using the ITS and LSU rDNA sequences. Spearman rank correlation showed that salinity was an important environmental factor affecting the vertical distribution of Pseudo-nitzschia spp. in this highly variable area. Availability of increased concentration of orthophosphates and ammonium and low Si: TIN ratio may have promoted the bloom of P. cf. arenysensis in the estuary.

Phytoplankton species can produce resting stages that persist in the sediment over long periods and accomplish important ecological functions. With the aim of investigating the phytoplankton assemblage structure in relation to environmental drivers and human pressures, we analyzed resting stages of diatoms and dinoflagellates, including harmful algal bloom taxa, in the surface sediments of 3 Mediterranean regional areas. Abundance of resting stages was determined by molecular quantitative polymerase chain reaction assay. Multivariate data analysis confirmed that the abundance of resting-stage assemblages seemed related to depth. Regional differences in the composition of the resting-stage assemblages were evident and environmental drivers were correlated with those regional differences. Three main groups of samples were defined according to SST and depth thresholds. Samples from the northern and central Adriatic Sea (average SST < 18°C) formed the richest assemblages, both in terms of abundance and species richness, while deep samples from all other basins (depth > 368 m) were poorer and less diverse than those from shallower sites (depth ≤ 368 m). Resting-stage taxa contributed differently to the 3 groups. Diatom spores and dinoflagellate cysts were the most abundant taxa, but Alexandrium minutum cysts and Ditylum brightwellii spores also accounted for a large share of the overall inter-group compositional distance. The structure of resting-stage assemblages can be regarded as a time- and space-integrated response of a subset of phytoplankton species to environmental conditions, including the physical oceanographic dynamics that favor or prevent sedimentation of resting stages.

Plastics are the most abundant marine debris globally dispersed in the oceans and its production is rising with documented negative impacts in marine ecosystems. However, the chemical-physical and biological interactions occurring between plastic and planktonic communities of different types of microorganisms are poorly understood. In these respects, it is of paramount importance to understand, on a molecular level on the surface, what happens to plastic fragments when dispersed in the ocean and directly interacting with phytoplankton assemblages. This study presents a computer-aided analysis of electron paramagnetic resonance (EPR) spectra of selected spin probes able to enter the phyoplanktonic cell interface and interact with the plastic surface. Two different marine phytoplankton species were analyzed, such as the diatom Skeletonema marinoi and dinoflagellate Lingulodinium polyedrum, in absence and presence of polyethylene terephthalate (PET) fragments in synthetic seawater (ASPM), in order to in-situ characterize the interactions occurring between the microalgal cells and plastic surfaces. The analysis was performed at increasing incubation times. The cellular growth and adhesion rates of microalgae in batch culture medium and on the plastic fragments were also evaluated. The data agreed with the EPR results, which showed a significant difference in terms of surface properties between the diatom and dinoflagellate species. Low-polar interactions of lipid aggregates with the plastic surface sites were mainly responsible for the cell-plastic adhesion by S. marinoi, which is exponentially growing on the plastic surface over the incubation time.

Long-term data series (1971&ndash, 2015) of physical and biogeochemical parameters were analyzed in order to assess trends and variability of oceanographic conditions in the northern Adriatic Sea (NAS), a mid-latitude shallow continental shelf strongly impacted by river discharges, human activities and climate changes. Interpolation maps and statistical models were applied to investigate seasonal and spatial variability, as well as decadal trends of temperature, salinity, chlorophyll-a and nutrients. This analysis shows that sea surface temperature increased by +0.36% year&minus, 1 over four decades. Annual mean flow of the Po River markedly changed due to the occurrence of periods of persistent drought, whereas the frequency of flow rates higher than 3000 m3 s&minus, 1 decreased between 2006 and 2015. Moreover, we observed a long-term decrease in surface phosphate concentrations in Po River water (&minus, 1.34% year&minus, 1) and in seawater (in summer &minus, 2.56% year&minus, 1) coupled, however, to a significant increase in nitrate concentration in seawater (+3.80% year&minus, 1) in almost all seasons. These changes indicate that the nutrient concentrations in the NAS have been largely modulated, in the last forty years, by the evolution of environmental management practices and of the runoff. This implies that further alteration of the marine environment must be expected as a consequence of the climate changes.

In the Adriatic Sea, massive rainfall events are causing flooding of rivers and streams, with severe consequences on the environment. The consequent bacterial contamination of bathing water poses public health risks besides damaging tourism and the economy. This study was conducted in the framework of WATERCARE, an EU Interreg Italy-Croatia Project, which aims at reducing the impact of microbial contamination on Adriatic bathing water due to heavy rainfall events drained in the local sewage network and; enhancing the quality of local waters; and providing support for the decision-making processes regarding the management of bathing water in line with EU regulations. The study involved the development of an innovative water quality integrated system that helps meet these objectives. It consists of four components: a real time hydro-meteorological monitoring system; an autosampler to collect freshwater samples during and after significant rainfall events; a forecast system to simulate the dispersion of pollutants in seawater; and a real-time alert system that can predict the potential ecological risk from the microbial contamination of seawater. The system was developed and tested at a pilot site (Fano, Italy). These preliminary results will be used to develop guidelines for urban wastewater and coastal system quality assessments to contribute to develop policy actions and final governance decisions.

During the past decade, next generation sequencing (NGS) technologies have provided new insights into the diversity, dynamics, and metabolic pathways of natural microbial communities. But, these new techniques face challenges related to the genome size and level of genome complexity of the species under investigation. Moreover, the coverage depth and the short-read length achieved by NGS based approaches also represent a major challenge for assembly. These factors could limit the use of these high-throughput sequencing methods for species lacking a reference genome and characterized by a high level of complexity. In the present work, the evolutionary history, mainly consisting of gene transfer events from bacteria and unicellular eukaryotes to microalgae, including harmful species, is discussed and reviewed as it relates to NGS application in microbial communities, with a particular focus on harmful algal bloom species and dinoflagellates. In the context of genetic population studies, genotyping-by-sequencing (GBS), an NGS based approach, could be used for the discovery and analysis of single nucleotide polymorphisms (SNPs). The NGS technologies are still relatively new and require further improvement. Specifically, there is a need to develop and standardize tools and approaches to handle large data sets, which have to be used for the majority of HAB species characterized by evolutionary highly dynamic genomes.

Marine diatoms have been identified among the most abundant taxa of microorganisms associated with plastic waste collected at sea. However, the impact of nano-sized plastic fragments (nanoplastics) at single cell and population level is almost unknown. We exposed the marine diatom Skeletonema marinoi to model polystyrene nanoparticles with carboxylic acid groups (PS–COOH NPs, 90 nm) for 15 days (1, 10, 50 μg/mL). Growth, reactive oxygen species (ROS) production, and nano-bio-interactions were investigated. No effect on diatom growth was observed, however Dynamic light scattering (DLS) demonstrated the formation of large PS aggregates which were localized at the diatoms’ fultoportula process (FPP), as shown by TEM images. Increase production of ROS and reduction in chain length were also observed upon PS NPs exposure (p < 0.005). The observed PS-diatom interaction could have serious consequences on diatoms ecological role on the biogeochemical cycle of carbon, by impairing the formation of fast-sinking aggregates responsible for atmospheric carbon fixation and sequestration in the ocean sea floor. S. marinoi exposure to PS NPs caused an increase of intracellular and extracellular oxidative stress, the reduction of diatom's chain length and the adhesion of PS NPs onto the algal surface.

Paralytic shellfish toxins (PST) and tetrodotoxin (TTX) are naturally-occurring toxins that may contaminate the food chain, inducing similar neurological symptoms in humans. They are co-extracted under the same conditions and thus their combined detection is desirable. Whilst PST are regulated and officially monitored in Europe, more data on TTX occurrence in bivalves and gastropods are needed before meaningful regulations can be established. In this study, we used three separate analytical methods - pre-column oxidation with liquid chromatography and fluorescence detection, ultrahigh performance hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (MS/MS) and HILIC high resolution (HR) MS/MS - to investigate the presence of PST and TTX in seawater and shellfish (mussels, clams) collected in spring summer 2015 to 2017 in the Mediterranean Sea. Samples were collected at 10 sites in the Syracuse Bay (Sicily, Italy) in concomitance with a mixed bloom of Alexandrium minutum and A. pacificum. A very high PST contamination in mussels emerged, unprecedentedly found in Italy, with maximum total concentration of 10851 mu g saxitoxin equivalents per kg of shellfish tissue measured in 2016. In addition, for the first time TTX was detected in Italy in most of the analysed samples in the range 0.8-6.4 mu g TTX eq/kg. The recurring blooms of PST-producing species over the 3-year period, the high PST levels and the first finding of TTX in mussels from the Syracuse bay, suggest that monitoring programmes of PST and TTX in seafood should be activated in this geographical area. (C) 2018 Elsevier Ltd. All rights reserved.

The aim of this study was to monitor for the first time the oligosaccharide pattern in the northwestern Adriatic Sea. Oligosaccharides are components of the low-molecular-weight fraction of dissolved organic matter (DOM). They perform an important ecological function in the microbial loop prompting substrates for bacterial degradation. In particular, our investigation focused on maltooligosaccharides (glucose α 1–4 linked). The analyses were carried out using a dialysis-charcoal column sorption pre-concentration method, followed by liquid chromatography-tandem mass spectrometry analyses with a triple quadrupole mass spectrometer operating in multiple reaction monitoring mode. During the study period, the concentration of maltooligosaccharides with a low degree of polymerisation (ranging from maltose to maltotetraose) was higher than other maltooligosaccharides (maltopentaose and maltoeptaose) and all together they reached their highest concentration in June. This concentration was temporally shift...

Vassalli, M. ... et al.-- 9 pages, 4 figures, 1 table, This paper describes the adoption and validation of two innovative methods for the automated count of Ostreopsis spp. concentration in sea water: a molecular assay based on RT-qPCR and an opto-electronic device implementing automatic recognition algorithms. The proposed approaches were tested on samples coming from different locations along the Mediterranean Sea and compared with the standard counting method based on microscopy observation by a taxonomy expert. The results demonstrate the effectiveness of both automatic approaches which provide a valuable tool, mostly cost and time effective, for the establishment of wide pan-Mediterranean monitoring strategies of Ostreopsis spp. blooms. Moreover, the two automatic methods demonstrated the ability to discriminate for the presence of a different but similar species, O. fattorussoi, for which new species-specific qPCR primers were developed, This publication has been produced with the financial assistance of the European Union under the ENPI CBC Mediterranean Sea Basin Programme. [...]. Magda Vila also acknowledges the support of the Spanish Government (MINECO) throught the project OstreoRisk (CTM2014-53818-R)

The silicon transport and use inside cells are key processes for understanding how diatoms metabolize this element in the silica biogenic cycle in the ocean. A spin-probe electron paramagnetic resonance (EPR) study over time helped to investigate the interacting properties and the internalization mechanisms of silicic acid from different silicon sources into the cells. Diatom cells were grown in media containing biogenic amorphous substrates, such as diatomaceous earth and sponge spicules, and crystalline sodium metasilicate. It was found that the amorphous biogenic silicon slowed down the internalization process probably due to formation of colloidal particles at the cell surface after silicic acid condensation. Weaker interactions occurred with sponge spicules silicon source if compared to the other sources. The EPR results were explained by analyzing transcript level changes of silicon transporters (SITs) and silaffins (SILs) in synchronized Thalassiosira pseudonana cultures over time. The results indicated that the transport role of SITs is minor for silicic acid from both biogenic and crystalline substrates, and the role of SIT3 is linked to the transport of silicon inside the cells, mainly in the presence of sponge spicules. SIL3 transcripts were expressed in the presence of all silicon sources, while SIL1 transcripts only with sponge spicules. The data suggest that the transport of silicic acid from various silicon sources in diatoms is based on different physico-chemical interactions with the cell surface.

Special issue on International Cooperation in Harmful Algal Bloom Science.-- 10 pages, 4 figures, Shallow, well-illuminated coastal waters from tropical to temperate latitudes are attractive environments for humans. Beaches and coral reefs have provided lodging and food to coastal communities for centuries. Unfortunately, tropical regions traditionally have been threatened by outbreaks of the toxic benthic dinoflagellate Gambierdiscus, which is associated with ciguatera fish poisoning. The ciguatoxins produced by Gambierdiscus bioaccumulate in reef fishes and are responsible for the most common algal toxin-related illnesses, globally affecting the greatest number of victims and often with significant long-term health effects. Recently, Gambierdiscus has been documented in subtropical and temperate latitudes. Blooms of another benthic and toxic dinoflagellate, Ostreopsis, have become more frequent and intense, especially in temperate waters. Ostreopsis produces palytoxins and analogues, and some outbreaks have been associated with massive benthic faunal damage and respiratory irritations in humans exposed to aerosols. The increased frequency of harmful events and the biogeographic extension of benthic microalgae incentivized the launch of the Global Ecology and Oceanography of Harmful Algal Blooms (GEOHAB) Core Research Project on “Benthic Harmful Algal Blooms” in 2010. This article summarizes the main scientific advances and gaps in related knowledge as well as advances the project has made toward managing and mitigating the impacts of benthic HABs on human illnesses and marine resource losses, The authors, on behalf of the international community working on benthic HABs, thank the Intergovernmental Oceanographic Commission (IOC) of UNESCO and the Scientific Committee on Oceanic Research (SCOR) for their financial and logistic support for the launch and implementation of the GEOHAB CRP Harmful Algal Blooms in Benthic Systems program. The authors received further support from the National Oceanic and Atmospheric Administration (NOAA), the Contrat de Projets État-Pays of French Polynesia and the National French Project ANR OCEAN 2015, the International Atomic Energy Agency (IAEA), the Spanish funded projects OstreoRisk (CTM2014-53818-R) and CICAN (CGL2013-40671-R), the M3-HABs Project (ENPI CBC MED Programme, European Commission), the YEOSU project funded by the government of the Republic of Korea, grant DIP-2012-023 from the Universiti Kebangsaan Malaysia, and the “Ostreopsis Project” granted by the Assessorato alla Sanità of the Campania Region (Italy)

Studying taxonomic and ecological diversity of phytoplankton assemblages is often difficult because morphological analysis cannot provide a complete description of their composition. Therefore, more robust and feasible approaches have to be chosen to elucidate the interactions between environmental and human pressures and phytoplankton assemblages. The Ocean Sampling Day (OSD) allowed collecting seawater samples from a wide range of oceanic regions including the Mediterranean Sea. In this study, a total of 754,167 V4-18S ribosomal DNA (rDNA) metabarcodes derived from 20 plankton samples collected at 19 sampling sites across the coastal areas of the Mediterranean Sea were analyzed to explore the relationships between phytoplankton assemblages' composition, sub-regional environmental features and human pressures. We reduced the whole set of autotroph plankton (1398 OTUs) to a smaller number of ecologically relevant entities (205 taxa) and used the latter for analysing the structure of phytoplankton assemblages. Chaetoceros was the only genus occurring in all the samples, while the number of taxa was maximum in the W Mediterranean. Based on the assigned OTUs, the structure of E Mediterranean phytoplankton was the most homogeneous. Further, phytoplankton assemblages from the three Mediterranean sub-regions (Western, Adriatic and Eastern) were significantly different (R=0.25, p=0.0136) based on Jaccard similarity. We also observed that phytoplankton diversity and human impact on marine ecosystems were not significantly related to each other based on Mantel's test.

The aim of this study was to develop and validate a high resolution melting (HRM) method for the rapid, accurate identification of the various harmful diatom Pseudo-nitzschia species in marine environments. Pseudo-nitzschia has a worldwide distribution and some species are toxic, producing the potent domoic acid toxin, which poses a threat to both human and animal health. Hence, it is important to identify toxic Pseudo-nitzschia species. A pair of primers targeting the LSU rDNA of the genus Pseudo-nitzschia was designed for the development of the assay and its specificity was validated using 22 control DNAs of the P. calliantha, P. delicatissima/P. arenysensis complex and P. pungens. The post-PCR HRM assay was applied to numerous unidentified Pseudo-nitzschia strains isolated from the northwestern Adriatic Sea (Mediterranean Sea), and it was able to detect and discriminate three distinct Pseudo-nitzschia taxa from unidentified samples. Moreover, the species-specific identification of Pseudo-nitzschia isolates by the HRM assay was consistent with phylogenetic analyses. The HRM assay was specific, robust and rapid when applied to high numbers of cultured samples in order to taxonomically identify Pseudo-nitzschia isolates recovered from environmental samples.

In the last few decades, the frequency of the toxic benthic dinoflagellate Ostreopsis cf. ovata proliferation has increased in the Mediterranean Sea. These blooms are associated with harmful effects on human health and the environment. The present work provides the first long term study on the spatio-temporal distribution of O . cf. ovata in relation to physical parameters in the Gulf of Gabes coastal waters (south-eastern Mediterranean Sea), as well as its morphological, molecular and physiological features. The strains of O. cf. ovata were identified morphologically by light and epifluorescence microscopy. The morphology and the size range of cultured strains were similar to those described regarding O . cf. ovata isolated from the Mediterranean Sea. The ultrastructural analysis of O . cf. ovata cells using the transmission electron microscopy showed the presence of numerous vesicles (VE) containing spirally coiled fibers (SCFs) connected to the mucus canal (CH). The phylogenetic tree based on the internal transcribed spacer region containing the 5.8S rDNA (ITS-5.8S rDNA) revealed that O . cf. ovata strains were placed into the Mediterranean/Atlantic clade. In addition, O . cf. ovata toxicity was evaluated by the mouse bioassay and a dose level ≥ 4 × 10 4 cells was found to be lethal to mice. The examination of the O. cf. ovata occurrence in the Gulf of Gabes at a large temporal scale (1997–2012) revealed a clear seasonal pattern with dominance from midsummer (July) to late autumn (November). Furthermore, a positive correlation was found between the abundance of O. cf. ovata and salinity, whereas no correlation was found as regards temperature. The occurrence of O. cf. ovata was only detected at salinity above 35 and the highest concentrations were observed at 45. Laboratory experiments confirmed such a result and showed that isolated O . cf. ovata strains had optimal growth at salinity ranging between 35 and 45, with its peak at 40.

Fifty-five strains of Ostreopsis were collected in the Mediterranean Sea and analyzed to characterize their toxin profiles. All the strains were grown in culture under the same experimental conditions and identified by molecular PCR assay based on the ITS-5.8S rDNA. A liquid chromatography-high resolution multiple stage mass spectrometry (LC-HRMSn) approach was used to analyze toxin profiles and to structurally characterize the detected toxins. Despite morphological and molecular characterization being consistent within the species O. cf. ovata, a certain degree of toxin variability was observed. All the strains produced ovatoxins (OVTXs), with the exception of only one strain. Toxin profiles were quite different from both qualitative and quantitative standpoints: 67% of the strains contained OVTX-a to -e, OVTX-g, and isobaric PLTX, in 25% of them only OVTX-a, -d, -e and isobaric PLTX were present, while 4% produced only OVTX-b and -c. None of the strains showed a previously identified profile, featuring OVTX-f as dominant toxin, whereas OVTX-f was a minor component of very few strains. Toxin content was mostly in the range 4-70 pg/cell with higher levels (up to 238 pg/cell) being found in strains from the Ligurian and South Adriatic Sea. Structural insights into OVTX-b, -c, -d, and -e were gained, and the new OVTX-l was detected in 36 strains.

In the northern Adriatic Sea, Mediterranean Sea, the mucilage phenomenon is usually related to the aggregation of extracellular organic matter of phytoplanktonic origin under special seasonal and trophic conditions. Environmental factors, such as temperature, salinity, nitrogen, phosphorus and N/P ratio as well as polysaccharide and monosaccharide levels in the presence and absence of mucilage were investigated in the northern Adriatic Sea. Variations in temperature and salinity during spring in years characterised by mucilage events have been recognised having an important role in preparing a favourable physical environment where mucilage formation could take place. Since the DIN/TP ratio increased markedly during the spring months (March–April) in 2002, 2003 and 2004, preceding the early summer mucilage outbreaks, it appeared that the DIN/TP ratio may be a more robust predictor than the DIN/DIP ratio, which, although more commonly used, exhibited a discontinuous trend. The contents of total carbohydrate...

In September 2015, a massive occurrence of the Ostreopsis species was recorded in central Adriatic Ka&scaron, tela Bay. In order to taxonomically identify the Ostreopsis species responsible for this event and determine their toxin profile, cells collected in seawater and from benthic macroalgae were analyzed. Conservative taxonomic methods (light microscopy and SEM) and molecular methods (PCR-based assay) allowed the identification of the species Ostreopsis cf. ovata associated with Coolia monotis. The abundance of O. cf. ovata reached 2.9 &times, 104 cells L&minus, 1 in seawater, while on macroalgae, it was estimated to be up to 2.67 &times, 106 cells g&minus, 1 of macroalgae fresh weight and 14.4 &times, 1 of macroalgae dry weight. An indirect sandwich immunoenzymatic assay (ELISA) and liquid chromatography&ndash, high-resolution mass spectrometry (LC-HRMS) were used to determine the toxin profile. The ELISA assay revealed the presence of 5.6 pg palytoxin (PLTX) equivalents per O. cf. ovata cell. LC-HRMS was used for further characterization of the toxin profile, which showed that there were 6.3 pg of the sum of ovatoxins (OVTXs) and isobaric PLTX per O. cf. ovata cell, with a prevalence of OVTXs (6.2 pg cell&minus, 1), while the isobaric PLTX concentration was very low (0.1 pg cell&minus, 1). Among OVTXs, the highest concentration was recorded for OVTX-a (3.6 pg cell&minus, 1), followed by OVTX-b (1.3 pg cell&minus, 1), OVTX-d (1.1 pg cell&minus, 1), and OVTX-c (0.2 pg cell&minus, 1).

In the last decade, various molecular methods (e.g., fluorescent hybridization assay, sandwich hybridization assay, automatized biosensor detection, real-time PCR assay) have been developed and implemented for accurate and specific identification and estimation of marine toxic microalgal species. This review focuses on the recent quantitative real-time PCR (qrt-PCR) technology developed for the control and monitoring of the most important taxonomic phytoplankton groups producing biotoxins with relevant negative impact on human health, the marine environment, and related economic activities. The high specificity and sensitivity of the qrt-PCR methods determined by the adequate choice of the genomic target gene, nucleic acid purification protocol, quantification through the standard curve, and type of chemical detection method make them highly efficient and therefore applicable to harmful algal bloom phenomena. Recent development of qrt-PCR-based assays using the target gene of toxins, such as saxitoxin compounds, has allowed more precise quantification of toxigenic species (i.e., Alexandrium catenella) abundance. These studies focus only on toxin-producing species in the marine environment. Therefore, qrt-PCR technology seems to offer the advantages of understanding the ecology of harmful algal bloom species and facilitating the management of their outbreaks.

The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions.

Ostreopsis spp. once confined to tropical and subtropical areas have recently spread to temperate regions such as the Mediterranean and the Southern-Atlantic coasts of Europe with relevant negative impact on human health through skin contact and inhalation of toxic aerosols. Following Ostreopsis-related outbreaks, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the analysis of algal samples. Our studies characterized O. cf. ovata as producer of palytoxin congeners, including a putative palytoxin and ovatoxins. Toxin profiles of different algal strains appear sometimes different both qualitatively and quantitatively and the presence of further new ovatoxins is emerging. Herein we report on a systematic study of over 40 Mediterranean strains of O. cf. ovata. All of them showed to produce ovatoxins although at different levels. Four types of toxin profiles were identified. Most of the strains produced ovatoxin-a as major component of the toxin profile while a significant number of strains did not produce ovatoxin-b and -c. Unique toxin profiles were found in a strain from the Adriatic coast of Italy, in which ovatoxin-f was the dominant toxin, and in a strain from the French Mediterranean coast, which produced only ovatoxin-b and c. On a per cell basis, toxin contents were in the range 5-230 pg/cell. In light of the structural complexity of ovatoxins, a LC-HRMSn based approach was employed to gain structural insights on known and unknown components of toxin profiles that could be available in too small amounts to be studied by NMR.

The new benthic toxic dinoflagellate Ostreopsis fattorussoi sp. nov. is described from eastern Mediterranean Sea Lebanon and Cyprus coasts, 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 ITS rDNA shows that O. fattorussoi belongs to the Atlantic/Mediterranean Ostreopsis spp. clade separated from the other Ostreopsis species. O. 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. In Lebanon coast, O. fattorussoi was recorded throughout the year 2015 (temperature range 18-31.5 °C), with peaks in June and August. This article is protected by copyright. All rights reserved.

Giussani, Valentina ... et al.-- 10 pages, 4 figures, 5 tables, 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 (EC50–24 h = 10.5 cells/mL). These findings suggest an interesting scenario on the interaction of these two bloom forming species in the natural marine environment, This work was supported by RITMARE (Ricerca Italiana per il MARE) Flagship Project, a National Research Programme funded by the Italian Ministry of University and Research (MIUR), the Programme ENPI (European Neighbourhood and Partnership Instrument) CBCMED (Cross Border Cooperation in the Mediterranean) M3-HABs II-B/2.1/0096, and OstreoRisk (CTM2014-53818-R)

Chattonella species in a Mediterranean lagoon (Santa Giusta Lagoon, Sardinia, Italy) were identified by applying a molecular approach to fixed natural phytoplankton samples collected over the last two decades. Like the other raphidophytes, Chattonella cells are naked and lose their shape when fixed, making species identification difficult on the basis of their morphological characteristics. Employing species-specific primers (oBTG-005-F, oBTG-027-R, oBTG-028-R) for the amplification of the ITS-5.8S rDNA region, we established the occurrence of C. subsalsa in fixed natural phytoplankton samples collected in coincidence with fish death events. Additionally, we established the presence of the recently discovered C. cf. subsalsa Adriatic genotype by analysing cellular cultures obtained from the same lagoon in 2013. This is the second worldwide record of C. cf. subsalsa Adriatic genotype. Our results revealed that the species-specific primers oBTG-005-F and oBTG-028-R distinguished this new genotype only when present singularly. This study provides valuable data that increase knowledge of C. subsalsa genotypes and of the long-term occurrence of Chattonella blooms in a transitional ecosystem through the use of samples up to 20 years old. Se identificaron especies de Chattonella mediante la aplicación de técnicas moleculares en muestras naturales de fitoplancton. Las muestras fueron recogidas y fijadas durante las últimas dos décadas en una laguna litoral mediterránea (Laguna de Santa Giusta, Cerdeña, Italia). Al igual que otras rafidoficeas, las células de Chattonella no poseen teca y, por lo tanto, pierden su forma cuando se fijan lo que dificulta la identificación basada en características morfológicas. Con el uso de cebadores específicos a nivel de especie (OBTG-005-F, OBTG-027-R, OBTG-028-R) diseñados para la amplificación de la región ITS- 5.8S rDNA, se detectó la presencia de C. subsalsa en las muestras recogidas en periodos coincidentes con eventos de muerte de peces. A través del análisis de los cultivos celulares obtenidos de la misma laguna en el año 2013, se identificó la presencia, por segunda vez a nivel mundial, del recientemente descubierto genotipo Adriático de C. cf. subsalsa. Los resultados revelaron que los cebadores oBTG-005-F y oBTG-028-R amplifican este nuevo genotipo sólo cuando está presente individualmente. En este estudio se presentan datos relevantes para el conocimiento de los genotipos de C. subsalsa y sobre la presencia recurrente de proliferaciones de especies de Chatonella en un ecosistema de transición a través de la utilización de muestras recogidas durante los últimos veinte años y analizadas hoy en día.