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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.

BackgroundWe 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/FindingsThe 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/SignificanceWe 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.

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<p>The genus <em>Ostreopsis</em> includes species largely distributed from tropical to temperate marine areas worldwide. Among the nine species of the genus, <em>O. siamensis</em>, <em>O. mascarenensis</em>, <em>O. lenticularis</em> and <em>O. </em>cf.<em> ovata</em> can produce toxins of the palytoxin group. In the last decade <em>Ostreopsis</em> cf. <em>ovata</em> and <em>O</em>. cf. <em>siamensis</em> originated intense blooms in all the rocky Mediterranean Sea coastal areas, typically during summer-late summer. The correct identification of <em>Ostreopsis</em> species in field samples is often problematic as <em>Ostreopsis</em> species are morphologically plastic and hardly discriminable under light microscopy and, therefore, molecular analyses are required. <em>Ostreopsis</em> blooms are often associated with noxious effects on health of both humans and benthic marine organisms mainly carried by aerosol and direct contact with seawater. Environmental factors have been shown to affect toxin content of <em>Ostreopsis</em> which generally produces more toxins per cell when growing under suboptimal conditions.<em> O</em>. cf. <em>ovata</em> is able to produce both temporary and resting cysts. In particular, the resting cysts are able to germinate in laboratory conditions for as long as 5 months after their formation at 25°C, but not at 21°C; the presence of a temperature threshold affecting cyst germination in the laboratory suggests that temperature represents a key factor for <em>Ostreopsis</em> cf. <em>ovata</em> bloom onset in natural environments as well. Several studies conducted to assess the role of abiotic factors (mainly hydrodynamics, water temperature and nutrients) on the bloom dynamics, revealed that the synergic effects of hydrodynamics, temperature and N:P ratios would lead the <em>Ostreopsis</em> blooms in temperate areas. <em>Ostreopsis</em> abundances showed a significant decrease with depth, likely related to light availability, although there are conflicting data about the relationship between light intensity and <em>Ostreopsis</em> growth in experimental conditions. The relationship between <em>Ostreopsis</em> blooms and salinity is not completely clear, complicated by the influence of high nutrient levels often associated to low salinity waters. Finally,<em> Ostreopsis</em> colonize a variety of substrata, although living substrata seems to allow lower concentration of epibionts than any other substrate, probably due to the production of some allelopathic compounds.</p>

The new benthic toxic dinoflagellate, Ostreopsis fattorussoi sp. nov., is described from the Eastern Mediterranean Sea, Lebanon and Cyprus coasts, and is 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 internal transcribed spacer rDNA shows that O. fattorussoi belongs to the Atlantic/Mediterranean Ostreopsis spp. clade separated from the other Ostreopsis species. Ostreopsis 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-j , ovatoxin-j , 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 . On the Lebanon coast, O. fattorussoi was recorded throughout the year 2015 (temperature range 18°C-31.5°C), with peaks in June and August.

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