Growth dynamics in relation to the production of the main cellular components in the toxic dinoflagellate Ostreopsis cf. ovata

In the last decade Ostreopsis cf. ovata blooms have been among the most intense along the entire Mediterranean coast, leading to ecological and human health problems, that are associated with the toxins (palytoxin-like compounds) produced by these algal cells. These compounds are secondary metabolites, whose rates of synthesis depend on the metabolism of their precursors. In general, growth dynamics and toxicity of dinoflagellates reflect the physiological status of the organism. The aim of the present study was to investigate the cellular production of the main biochemical compounds likely involved in the growth and toxicity dynamics of O. cf. ovata during exponential to the late stationary phase in batch cultures of an Adriatic strain. Removal of major nutrients from the medium was monitored along with concentration, biovolume and production of the main cellular components (e.g. polysaccharides, proteins, lipids and toxins). Nutrient uptake, as well as toxin production rates were calculated in the different growth periods. Nutrients (N and P) were completely depleted when cells entered stationary phase and the greatest net toxin production rate (R_TOX) occurred during the first days of growth. The various palytoxins reported a relative abundance quite stable during the different growth phases, while the total toxin cellular amount increased along the growth curve. Total and extracellular released polysaccharides, as well as the lipid content increased greatly during the stationary phase, while proteins were mainly produced by cells during the exponential phase. The continuous release of polysaccharides could facilitate cell aggregation and the formation of the benthic community during algal blooms. The trend of production of the main cellular compounds in O. cf. ovata and the growth dynamics of this species lead us to hypothesize that the fast growth of this dinoflagellate, associated with the rapid use of environmental resources (nutrients, and phosphates in particular), may be an ecological/adaptive strategy which could favor this organism in competition with other species.     

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.     

Microelectrode array (MEA) platform as a sensitive tool to detect and evaluate Ostreopsis cf. ovata toxicity

In the last decade, the occurrence of harmful dinoflagellate blooms of the genus Ostreopsis has increased both in frequency and in geographic distribution with adverse impacts on public health and the economy. Ostreopsis species are producers of palytoxin-like toxins (putative palytoxin and ovatoxins) which are among the most potent natural non-protein compounds known to date, exhibiting extreme toxicity in mammals, including humans. Most existing toxicological data are derived from in vivo mouse assay and are related to acute effects of pure palytoxin, without considering that the toxicity mechanism of dinoflagellates can be dependent on the varying composition of complex biotoxins mixture and on the presence of cellular components.In this study, in vitro neuronal networks coupled to microelectrode array (MEA)-based system are proposed, for the first time, as sensitive biosensors for the evaluation of marine alga toxicity on mammalian cells. Toxic effect was investigated by testing three different treatments of laboratory cultured Ostreopsis cf. ovata cells: filtered and re-suspended algal cells; filtered, re-suspended and sonicated algal cells; conditioned growth medium devoid of algal cells. The great sensitivity of this system revealed the mixture of PTLX-complex analogues naturally released in the growth medium and the different potency of the three treatments to inhibit the neuronal network spontaneous electrical activity. Moreover, by means of the multiparametric analysis of neuronal network activity, the approach revealed a different toxicity mechanism of the cellular component compared to the algal conditioned growth medium, highlighting the potential active role of the first treatment.     

Bacterial communities and toxin profiles of Ostreopsis (Dinophyceae) from the Pacific island of Okinawa,Japan

Variations in toxicity of the benthic dinoflagellate Ostreopsis Schmidt 1901 have been attributed to specific molecular clades, biogeography of isolated strains, and the associated bacterial community. Here, we attempted to better understand the biodiversity and the basic biology influencing toxin production of Ostreopsis. Nine clonal cultures were established from Okinawa, Japan, and identified using phylogenetic analysis of the ITS-5.8S rRNA and 28S rRNA genes. Morphological analysis suggests that the apical pore complex L/W ratio could be a feature for differentiating Ostreopsis sp. 2 from the O. ovata species complex. We analyzed the toxicity and bacterial communities using liquid chromatography-mass spectrometry, and PCR-free metagenomic sequencing. Ovatoxin was detected in three of the seven strains of O. cf. ovata extracts, highlighting intraspecies variation in toxin production. Additionally, two new potential analogs of ovatoxin-a and ostreocin-A were identified. Commonly associated bacteria clades of Ostreopsis were identified from the established cultures. While some of these bacteria groups may be common to Ostreopsis (Rhodobacterales, Flavobacteria-Sphingobacteria, and Enterobacterales), it was not clear from our analysis if any one or more of these plays a role in toxin biosynthesis. Further examination of biosynthetic pathways in metagenomic data and additional experiments isolating specific bacteria from Ostreopsis would aid these efforts.     

Effects of temperature,salinity and their interaction on growth of benthic dinoflagellates Ostreopsis spp. from Thailand

Benthic dinoflagellates Ostreopsis spp. are known as producers of palytoxin and its analogs, resulting occasionally in human health problems worldwide. Although distribution of Ostreopsis spp. along the Thai coasts has been reported, little is known about their growth characteristics. To discuss the bloom dynamics of Ostreopsis spp. in Thailand, first we tested four kinds of media to optimize growth conditions and then clarified the effects of temperature, salinity and temperature–salinity interaction on the growth of strains of the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade, Ostreopsis sp. 6 and Ostreopsis sp. 7. We showed that the f/2 medium was a suitable medium which gave the highest cell yields for all the strains tested. The strains of the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade and Ostreopsis sp. 6 grew in the temperature range 20–32.5 °C, whereas the strain of Ostreopsis sp. 7 grew in 20–30 °C. The semi-optimal temperature ranges (≧80% of the maximal growth rate) for the former three strains were 22.7–27.4 °C, 27.9–30.8 °C and 23.5–26.4 °C, respectively, whereas that of the latter strain was 23–27.2 °C. The optimal temperature for the O. cf. ovata South China Sea subclade was 30 °C, whereas for the others it was 25 °C. All the Ostreopsis strains tested could grow in a salinity range of 20–40. The semi-optimal salinities for the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade Ostreopsis sp. 6 and Ostreopsis sp. 7 were 28.7–35, 23.8–30.8, 29.8–36 and 28–36, respectively. The optimal salinities for the O. cf. ovata Thailand subclade and O. cf. ovata South China Sea subclade were 30 and 25, respectively, whereas for Ostreopsis sp. 6 and Ostreopsis sp. 7 it was 35. In this study, our results suggested that the optimal and tolerable temperature–salinity conditions differ among the Thai Ostreopsis species/clades/subclades. Tolerances of the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade and Ostreopsis sp. 6 to the high temperature of 32.5 °C may allow these organisms to be distributed in the tropical areas, where the water temperature often reaches >30 °C.     

Investigation of toxin profile of Mediterranean and Atlantic strains of Ostreopsis cf. siamensis (Dinophyceae) by liquid chromatography–high resolution mass spectrometry

Blooms of Ostreopsis spp. once confined to tropical and subtropical areas have recently spread to more temperate regions such as the Mediterranean and the Southern-Atlantic coasts of Europe. However, while O. confronta (cf.) ovata has caused several toxic outbreaks, the presence of O. cf. siamensis has been reported rather occasionally and in very few regions; as a consequence, O. cf. ovata toxin profile has been in-depth studied while poor information exists on toxicity of the Mediterranean and Atlantic O. cf. siamensis. In the present study toxin profile of Mediterranean and Atlantic O. cf. siamensis isolates also phylogenetically related has been studied through liquid chromatography–high resolution mass spectrometry (LC–HRMS) versus a palytoxin standard, a crude extract of O. cf. ovata containing all the ovatoxins so far known (ovatoxin-a to -f), and a Japanese O. siamensis extract which contained ostreocin-d and ostreocin-b. The Mediterranean and Atlantic O. cf. siamensis strains were shown not to produce either ostreocins, which are produced by the Japanese O. siamensis strain, or ovatoxins, which are produced by the Mediterranean O. cf. ovata. Only sub-fg levels of palytoxin on a per cell basis were detected in the Mediterranean strain. This study demonstrates that the Mediterranean and the Atlantic O. cf. siamensis strains are devoid of any appreciable toxicity. Thus, at least in the European area, O. cf. siamensis seems to present a much lower risk to human health than O. cf. ovata.     

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.     

Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand

The toxic benthic dinoflagellate genus Ostreopsis has been connected to the production of palytoxin and its analogs in many tropical and temperate areas. Although the type species, O. siamensis, was originally described from the Gulf of Thailand in 1901, little is known about the species composition and distribution of the genus Ostreopsis in Thailand. In this study, a total of 64 Ostreopsis strains isolated from the Andaman Sea as well as the Gulf of Thailand were investigated by analyzing the nucleotide sequences of the LSU rDNA D1/D2, D8/D10 and ITS-5.8S rDNA regions. Phylogenetic analyses (BI and ML) resulted in some of the strains being assigned to previously described clades, O. cf. ovata and Ostreopsis sp. 6, and revealed the existence of a novel clade named Ostreopsis sp. 7, which exhibited large genetic distances from the other clades. Among O. cf. ovata, several strains from Thailand were formed into a new subclade, the Thailand subclade, whereas a few strains belonged to the South China Sea subclade. Morphometric characteristics such as the cell sizes of the two O. cf. ovata subclades and those of Ostreopsis sp. 7 were not significantly different from each other (p > 0.05). Their characteristics were similar but slightly different from those of O. ovata and were significantly different from those of Ostreopsis sp. 6 (p < 0.05). Toxicities of Ostreopsis from Thailand were evaluated using mouse bioassay. Strains of Ostreopsis sp. 6 and Ostreopsis sp. 7 tested were highly toxic, while the two subclades of O. cf. ovata strains seemed to be nontoxic. This study suggests that toxic Ostreopsis sp. 7 is distributed in the Andaman Sea, whereas the two subclades of O. cf. ovata and toxic Ostreopsis sp. 6 are distributed in the Gulf of Thailand.     

Phylogenetic structure of bacterial assemblages co-occurring with Ostreopsis cf. ovata bloom

Extensive blooms of the toxic epiphytic/benthic dinoflagellate Ostreopsis cf. ovata are being reported with increasing frequency and spatial distribution in temperate coastal regions including the Mediterranean. These blooms are of human and environmental health concern due to the production of isobaric palytoxin and a wide range of ovatoxins by Ostreopsis cf. ovata. Bacterial-microalgal interactions are important regulators in algal bloom dynamics and potentially toxin dynamics. This study investigated the bacterial assemblages co-occurring with O. cf. ovata (OA) and from ambient seawaters (SW) during the early and peak phases of bloom development in NW Adriatic Sea. Fractions of the bacterial assemblages co-occurring with O. cf. ovata (OA) and more closely associated to the mucilage layer (LA) embedding O. cf. ovata cells were also reported.In total, 14 bacterial phyla were detected by targeted 454 pyrosequencing of the 16S rRNA gene. The dominant bacterial phyla in the OA assemblages were Proteobacteria and Bacteroidetes; while at the class level, Alphaproteobacteria were the most abundant (83 and 66%, relative abundance, early and peak bloom phases), followed by Flavobacteria (7 and 19%, early and peak phases). Actinobacteria and Cyanobacteria were of minor importance (<5% of the relative bacterial abundance each). Gammaproteobacteria showed a notably presence in OA assemblage only at the early phase of the bloom (genus Haliea, 13%). The Alphaproteobacteria were predominately composed by the genera Ruegeria, Jannaschia and Erythrobacter which represented about half of the total phylotypes’ contribution of OA at both early and peak phases of the O. cf. ovata bloom, suggesting interactions between this consortium and the microalga. Moreover, the highest contribution of Ruegeria (30% of the total phylotypes) was observed at the early phase of the bloom in LA assemblage.Microbial assemblages associated with the ambient seawaters while being also dominated by Alphaproteobacteria and Flavobacteria were partially distinct from those associated with O. cf. ovata due to the presence of genera almost not retrieved in the latter assemblages.     

Ostreopsis cf. siamensis and Ostreopsis cf. ovata from the Atlantic Iberian Peninsula: Morphological and phylogenetic characterization

Individuals of Ostreopsis, a genus containing potentially toxic species which affects human health, were collected during summer-autumn 2010 and 2011 from 17 sites located along the Atlantic coast of the Iberian Peninsula, a temperate area which during summer presents contrasting seawater temperatures. Ostreopsis cells were obtained by shaking macroalgae collected from rocky-shore areas bordering accessible beaches. Isolated strains and field samples were analyzed for morphological and phylogenetic characterization where sequences of the ITS1-5.8S-ITS2 region of the rDNA delineated two different species fitting Ostreopsis cf. ovata and Ostreopsis cf. siamensis. By means of calcofluor staining and scanning electron microscopy, it was observed that field samples of both species exhibited a wide and overlapping range of dorsoventral as well as width values. Those cells presented 11–18 pores/100 μm² and were also similar concerning plates shape and size. The main differential feature between the two species was the presence of two sizes of thecal pores (0.07–0.13 μm and 0.15–0.39 μm) in Ostreopsis cf. siamensis and one size (0.24–0.56 μm) in Ostreopsis cf. ovata. A comparison of field vs. cultured cells indicated that field isolates presented larger cells than in culture.     

Ostreopsis cf. ovata in the Gulf of Gabès (south-eastern Mediterranean Sea): morphological,molecular and ecological characterization

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 Gabès 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 × 10⁴ cells was found to be lethal to mice. The examination of the O. cf. ovata occurrence in the Gulf of Gabès 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.     

Ocurrence of Ostreopsis in two temperate coastal bays (SW iberia): Insights from the plankton

The benthic genus Ostreopsis contains toxic-bloom forming species and is an important cause of concern in warm-temperate and tropical waters. On the coast of Portugal, NE Atlantic, the occurrence of Ostreopsis cf. siamensis and Ostreopsis cf. ovata has been reported since 2008 and 2011, respectively. This work aims to understand the favorable conditions for high concentrations of Ostreopsis cells in the plankton at two sites, Lagos and Lisbon Bays, located in the South and West coast of Portugal, respectively. This study is based on weekly Ostreopsis abundance data in the plankton, from 2011 to 2017, daily satellite and in situ sea surface temperature (SST), and meteorological and sea state parameters, namely wind stress and significant wave height. The molecular identification of local Ostreopsis spp. is also presented. The maximum cell densities occur between late-summer and autumn. The distribution range of Ostreopsis cf. ovata is restricted to the South coast, while Ostreopsis cf. siamensis has a wider distribution range, being also present on the West coast. In the study period, there was only one occurrence of Ostreopsis spp., in Lagos Bay, with concentrations within the alert phase of monitoring. In Lagos Bay, high Ostreopsis spp. concentrations were related with positive SST anomalies. These high concentrations were often recorded after a period of almost 2-weeks to more than 4-weeks of low sea state (<0.6 m), followed by short time events of onshore wind and moderate waves (0.6–1 m). The former conditions are interpreted as favoring bloom development on the substrate and the latter as causing the re-suspension of Ostreopsis cells in the water column. In Lisbon Bay, O. cf. siamensis occurred in the plankton in few occasions and no clear relation could be established with the studied environmental variables. It is here hypothesized that the recent records of O. cf. siamensis in Lisbon Bay may correspond to an early colonization stage of an invasion process. Knowledge gained on Ostreopsis dynamics along the Portuguese coast can be used for both the improvement of benthic harmful algal blooms (BHABs) monitoring in the region and as a basis to design forecasting models.     

A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt

Aim Ostreopsis is a benthic and epiphytic dinoflagellate producing potent toxins widespread in tropical and warm temperate coastal areas world‐wide. We tested the hypothesis that as it is benthic, it would show distinct biogeographical patterns in comparison with planktonic species. Here, we analyse sequence variability in ribosomal DNA markers to provide the first phylogeographical study of this toxic benthic dinoflagellate. Location Mediterranean Sea, Atlantic Ocean, Pacific Ocean. Methods Ribosomal DNA sequence data from partial nuclear LSU (D1/D2 domains) and 5.8S genes and non‐coding internal transcribed spacer (ITS) regions were obtained from 82 isolates of Ostreopsis species, collected at 26 localities throughout the world. Molecular sequence data were analysed using maximum parsimony, maximum likelihood and Bayesian methods for phylogenetic inference. A statistical parsimony network was obtained based on concatenated LSU and 5.8S rDNA–ITS region sequences of the Mediterranean/Atlantic Ostreopsis cf. ovata isolates to infer haplotype distribution over their geographical range. Light epifluorescence microscopy analyses were performed on cultured and field Ostreopsis material for taxonomic identification, while laboratory experiments for encystment induction were carried out on selected O. cf. ovata isolates. Toxin assays of Ostreopsis species isolates were carried out using the haemolytic‐based method. Results Analyses based on single and concatenated ribosomal genes gave substantially similar results. The rDNA phylogeny revealed different clades corresponding to different species within the genus Ostreopsis. In the species O. cf. ovata, different genetic lineages were correlated with macrogeographical distribution. A network of haplotypes inferred from the Atlantic and Mediterranean isolates of O. cf. ovata revealed that these two areas might host a single panmictic population. The Atlantic/Mediterranean population of O. cf. ovata was differentiated considerably from the Indo‐Pacific populations. Other species of Ostreopsis were found, but they turned out to be restricted to just one of the two main warm‐water oceanic basins, the Mediterranean/Atlantic and the Indo‐Pacific. Main conclusions Ostreopsis cf. ovata was found to be widely dispersed throughout the coastal areas of tropical and some warm temperate seas. In the Atlantic/Mediterranean region it may constitute a panmictic population that is highly distinct from Indo‐Pacific populations. Ostreopsis cf. siamensis was found only in the Mediterranean Sea, and strains identified as Ostreopsis lenticularis and Ostreopsis labens were found only in the Indo‐Pacific region.     

Proliferation of the toxic dinoflagellate Ostreopsis cf. ovata in relation to depth,biotic substrate and environmental factors in the North West Mediterranean Sea

In recent decades, the North West Mediterranean Sea has been seriously affected by the development of the toxic benthic dinoflagellate Ostreopsis cf. ovata, which is associated with harmful effects on human health and the environment. The present work aims to provide a large overview of the multiple environmental factors assumed to regulate or influence the growth of Ostreopsis. An intensive sampling campaign over a full annual cycle was performed along the French and Italian coasts (in six sites from Cassis to Genoa), to determine patterns of temporal and spatial distributions of both O. cf. ovata epiphytic and planktonic cells. Results highlighted substantial seasonal variations in the abundance of Ostreopsis. These variations correlated to seawater temperature, with an optimum growth temperature ranging from 23 °C to 27.5 °C. Phosphate concentration, rather than nitrogen or silicate, was also positively associated with Ostreopsis. Decreases in oxygen and increases in chlorophyll a concentrations were recorded during the summer blooming period. The maximal Ostreopsis epiphytic abundance was generally higher on Dictyota spp. than on the other two sampled macroalgae (up to 8.54 × 10⁶ cells g⁻¹ FW), even though statistical analysis did not support a clear substrate preference. Epiphytic abundances were significantly higher at a very shallow depth (0.5 m), than at 1 and/or 3 m depths. High anthropogenic pressure (related to population density) seems to have promoted the occurrence of blooms in urbanized areas, which could partly explain the strong demarcation in Ostreopsis development between Western and Eastern sampling sites. The ecological niche of Ostreopsis cf. ovata needs precise definition, which will require further in situ and in vitro experimental studies, to determine the relative importance of distinct environmental parameters.     

Life cycle stages of the benthic palytoxin-producing dinoflagellate Ostreopsis cf. ovata (Dinophyceae)

The asexual and sexual reproduction of Ostreopsis cf. ovata was studied in the field and in cultures isolated from two locations in the Mediterranean Sea. Asexual division took place in the motile stage by the sharing of theca (desmoschisis). High cell-size variability and differences in division capability were detected in the cultures. Thecal analyses and nuclear division patterns allowed characterization of the different phases of dividing cells obtained during an in situ cell-cycle sampling performed off Llavaneres beach (Northeast Spain). During the 45-h cycle, binucleated cells accounted for 2.6% of the population. Division was initiated with the onset of dusk and reached a maximum 3–4 h before dawn. No dividing cells were detected after 09:00 AM. Sexuality occurred both in cultures and in natural populations of O. cf. ovata. Mating gamete pairs were the only sexual stages that could be distinguished from vegetative stages. The differences between these pairs and dividing cells are described herein. None of the individually isolated gamete pairs underwent fusion nor encystment, instead, in most of them one member of the gamete pair divided. Rather than plasmogamy, there was evidence of nuclear migration from one gamete to the other. Pellicle cysts, thin-walled cysts, and thecate cysts were also identified and studied. These cysts, embedded in abundant mucilage in a bloom-derived incubated sample, were able to germinate for as long as 6 months after their formation. Our results suggest that they constitute the overwintering population that causes recurrent blooms of O. cf. ovata in some areas of the Mediterranean Sea.     

Molecular identification of Ostreopsis cf. ovata in filter feeders and putative predators

Ostreopsis ovata Fukuyo is a benthic dinoflagellate widespread from tropical to subtropical and warm temperate coastal areas world-wide. Since the species produces palytoxin-like substances that can accumulate in seafood, the apparent expansion of its range in recent years represents an increasing risk for human health. This leads to the necessity of monitoring protocols that enable the rapid detection of the presence of this microalga in environmental samples and sea-food. We developed an identification protocol based on real-time PCR (qPCR) to detect O. cf. ovata presence in different matrices. The protocol was proved to be able to reveal microalgal traces in both soft tissues and intervalvar liquid of mussels exposed to O. cf. ovata in natural and experimental conditions as well as in seawater samples. O. cf. ovata could also be detected in mussel tissues after the end of the bloom, when it was no longer detectable in sea water. We were able to detect O. cf. ovata in copepods fed on unialgal cultures as well. Cell density estimates based on standard curves resulted to be comparable to direct microscopical counts. The method is therefore suitable to ascertain the origin of palytoxin-like substances in toxic seafood. In addition, our results confirm that mussels and other predators can actually ingest O. cf. ovata cells and act as a vector for toxin transfer through both benthic and planktonic food webs.     

Modelling the Stoichiometric Regulation of C-Rich Toxins in Marine Dinoflagellates

Toxin production in marine microalgae was previously shown to be tightly coupled with cellular stoichiometry. The highest values of cellular toxin are in fact mainly associated with a high carbon to nutrient cellular ratio. In particular, the cellular accumulation of C-rich toxins (i.e., with C:N > 6.6) can be stimulated by both N and P deficiency. Dinoflagellates are the main producers of C-rich toxins and may represent a serious threat for human health and the marine ecosystem. As such, the development of a numerical model able to predict how toxin production is stimulated by nutrient supply/deficiency is of primary utility for both scientific and management purposes. In this work we have developed a mechanistic model describing the stoichiometric regulation of C-rich toxins in marine dinoflagellates. To this purpose, a new formulation describing toxin production and fate was embedded in the European Regional Seas Ecosystem Model (ERSEM), here simplified to describe a monospecific batch culture. Toxin production was assumed to be composed by two distinct additive terms; the first is a constant fraction of algal production and is assumed to take place at any physiological conditions. The second term is assumed to be dependent on algal biomass and to be stimulated by internal nutrient deficiency. By using these assumptions, the model reproduced the concentrations and temporal evolution of toxins observed in cultures of Ostreopsis cf. ovata, a benthic/epiphytic dinoflagellate producing C-rich toxins named ovatoxins. The analysis of simulations and their comparison with experimental data provided a conceptual model linking toxin production and nutritional status in this species. The model was also qualitatively validated by using independent literature data, and the results indicate that our formulation can be also used to simulate toxin dynamics in other dinoflagellates. Our model represents an important step towards the simulation and prediction of marine algal toxicity.     

Genetic and toxinological characterization of North Atlantic strains of the dinoflagellate Ostreopsis and allelopathic interactions with toxic and non-toxic species from the genera Prorocentrum,Coolia and Gambierdiscus

The genus Ostreopsis includes several toxic species that can develop blooms in benthic ecosystems, with potential harmful consequences for human health and marine invertebrates. Despite of this, little is known about the allelopathic interactions between these organisms and other co-occurring microalgae that exploit similar spatial and nutrient resources in benthic ecosystems. The aim of this study was to follow these interactions in cultures of two Ostreopsis ribotypes with different toxin profiles (O. cf. ovata contained ovatoxins-a, b, c and e, while only ovatoxin-d was found in O .sp. “Lanzarote-type”), mixed with species of three benthic dinoflagellate genera (Coolia, Prorocentrum and Gambierdiscus), isolated from the same area (North East Atlantic, Canary Islands). In a first experiment, the potential allelopathic effects on growth rates were followed, in mixed cultures of Coolia monotis (a non toxic species) exposed to the clarified medium and to cells of O. sp.“Lanzarote-type” and O. cf. ovata. Growth delayed in C. monotis was observed specially in clarified medium, while the O. sp. “Lanzarote-type” strain attained much lower densities in mixed cultures. In a second experiment, we examined the potential effects of clarified media from O. sp.“Lanzarote-type” and O. cf. ovata on the adherence capacity in two toxic species (Prorocentrum hoffmannianum and Gambierdiscus excentricus). Contrasting effects were found: a significant increase of adherence capacity in P. hoffmannianum vs attachment decline in G. excentricus, that experienced also severe deleterious effects (cell lysis). Our results suggest the existence of weak to moderate allelopathic interactions between the studied organisms, although the outcome is dependent on the species involved.