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.     

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.     

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.     

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.     

Seasonal dynamics and spatial distribution of epiphytic dinoflagellates in Peter the Great Bay (Sea of Japan) with special emphasis on Ostreopsis species

Studies of epiphytic dinoflagellates in Peter the Great Bay, Sea of Japan in 2008–2011 revealed the presence of 13 species. Five of the species are known as potentially toxic: Amphidinium carterae, A. operculatum, Ostreopsis cf. ovata, O. cf. siamensis and Prorocentrum lima. The maximum species richness and abundance of epiphytic dinoflagellates were observed in autumn (from September to October). Ostreopsis spp. were most widely distributed and predominated, amounting to 99% of the total density of dinoflagellates. Multi-year seasonal dynamics of Ostreopsis spp. in Peter the Great Bay showed that these cells appear as epiphyton in August after maximum warming of surface waters (22–24 °С) and disappear in early November, when the water temperature decreases below 7 °С. Ostreopsis spp. proliferation occurred in September, when the water temperature was 17.2–21.0 °C. The highest densities of Ostreopsis spp. were recorded on September 9, 2010 on the rhodophyte Neorhodomela aculeata – 230 × 10³ cells g⁻¹ DW or 52 × 10³ cells g⁻¹ FW. The spatial distribution of epiphytic dinoflagellates was investigated in the near-shore areas of Peter the Great Bay during the second half of September 2010 to evaluate the role of hydrodynamic conditions. Epiphytic dinoflagellates were not found in sheltered sites having weak mixing hydrodynamics. However, the abundances of Ostreopsis spp. were significantly higher at sites having moderate turbulence compared to biotopes experiencing strong wave action. Densities of Ostreopsis spp. were not significantly different on macrophytes with branched thallus of all taxonomic divisions. However, the average cell densities of Ostreopsis spp. on green algae with branched thallus were significantly higher than on green algae having laminar thallus.     

Uptake of dissolved inorganic and organic nitrogen by the benthic toxic dinoflagellate Ostreopsis cf. ovata

Environmental factors that shape dynamics of benthic toxic blooms are largely unknown. In particular, for the toxic dinoflagellate Ostreopsis cf. ovata, the importance of the availability of nutrients and the contribution of the inorganic and organic pools to growth need to be quantified in marine coastal environments. The present study aimed at characterizing N-uptake of dissolved inorganic and organic sources by O. cf. ovata cells, using the ¹⁵N-labelling technique. Experiments were conducted taking into account potential interactions between nutrient uptake systems as well as variations with the diel cycle. Uptake abilities of O. cf. ovata were parameterized for ammonium (NH₄⁺), nitrate (NO₃⁻) and N-urea, from the estimation of kinetic and inhibition parameters. In the range of 0 to 10 μmol N L⁻¹, kinetic curves showed a clear preference pattern following the ranking NH₄⁺ > NO₃⁻ > N-urea, where the preferential uptake of NH₄⁺ relative to NO₃⁻ was accentuated by an inhibitory effect of NH₄⁺ concentration on NO₃⁻ uptake capabilities. Conversely, under high nutrient concentrations, the preference for NH₄⁺ relative to NO₃⁻ was largely reduced, probably because of the existence of a low-affinity high capacity inducible NO₃⁻ uptake system. Ability to take up nutrients in darkness could not be defined as a competitive advantage for O. cf. ovata. Species competitiveness can also be defined from nutrient uptake kinetic parameters. A strong affinity for NH₄⁺ was observed for O. cf. ovata cells that may partly explain the success of this toxic species during the summer season in the Bay of Villefranche-sur-mer (France).     

Ostreol A: A new cytotoxic compound isolated from the epiphytic dinoflagellate Ostreopsis cf. ovata from the coastal waters of Jeju Island,Korea

Ostreol A was isolated from cultures of the epiphytic dinoflagellate Ostreopsis cf. ovata from the coastal waters of Jeju Island, Korea. The compound, a non-palytoxin derivative, has a polyhydroxy chain ending with the primary amino group and contains an amide bond, along with two tetrahydropyran rings in the chain. Its chemical structure was elucidated by nuclear magnetic resonance (NMR) spectroscopy methods and confirmed by mass analysis. The compound exhibited significant cytotoxicity in the brine shrimp lethality test at a concentration of 0.9 μg/mL.     

Physiological responses of Ostreopsis ovata to changes in N and P availability and temperature increase

Ostreopsis ovata is a benthic dinoflagellate that produces palytoxin and ovatoxins. Blooms of O. ovata causing human health problems and mortality of benthic fauna have been reported from many tropical and temperate marine waters. In the present study we examined the combined effects of temperature and different nutrient conditions on the biochemical composition, growth, toxicity and carbohydrate production of an O. ovata strain originating from the Tyrrhenian Sea. O. ovata cultures with N:P ratios of 1.6, 16 and 160 (N deficient, NP sufficient and P deficient, respectively) were grown at 20 °C and 30 °C. Biomass accumulation, growth rates, cell volumes, biochemical composition, cell toxicity and carbohydrate production in each treatment were studied. Results indicated that under nutrient sufficiency O. ovata biomass accumulation increased significantly compared to N and P deficiency and also that N limitation severely affected growth. The highest growth rates were recorded at 30 °C. Cellular contents and the atomic ratios of C, N and P were higher in the cells grown at 20 °C than in those grown at 30 °C. O. ovata cell volumes increased at 20 °C. N deficiency significantly increased cell toxicity. Toxicity per cell was higher at 20 °C, but per carbon was highest at 30 °C. The highest carbohydrate production was found in conditions of N deficiency and at the lower temperature.Our study suggests that temperature increases due to global warming and nutrient enrichment of coastal waters stimulate the proliferation of O. ovata, particularly for the strains that have become adapted to warm temperate waters.     

Evaluation of the effects of light intensity on growth of the benthic dinoflagellate Ostreopsis sp. 1 using a newly developed photoirradiation-culture system and a novel regression analytical method

Benthic dinoflagellates of the genus Ostreopsis are found all over the world in temperate, subtropical, and tropical coastal regions. Our recent studies revealed that a putative “cryptic” species of Ostreopsis ovata is present widely along Japanese coasts. This organism, Ostreopsis sp. 1, possesses palytoxin analogs and thus its toxic blooms may be responsible for potential toxification of marine organisms. To evaluate the bloom dynamics of Ostreopsis sp. 1, the present study examined the growth responses of Ostreopsis sp. 1 strain s0716 to various light intensities (photon flux densities: μmol photons m⁻² s⁻¹) using a newly devised photoirradiation-culture system. This novel system has white light-emitting diodes (LEDs) capable of more closely simulating the wavelength spectrum of light entering the oceanic water column than do fluorescent tubes and halogen lamps. In this system, the light intensity of the white LEDs was reduced through two polarizing filters by varying the rotation angles of the filters. Thereby, the new system was capable of culturing microalgae under well-controlled light intensity conditions. Ostreopsis sp. 1 grew proportionally when light intensity was increased from 49.5 to 199 μmol photons m⁻² s⁻¹, but its growth appeared to be inhibited slightly at ≥263 μmol photons m⁻² s⁻¹. The relationship between observed growth rates and light intensity was calculated at R > 0.99 (P < 0.01) using a regression analysis with a modified equation of the photosynthesis-light intensity (P-L) model. The equation determined the critical light intensities for growth of Ostreopsis sp. 1 and the organism's growth potential as follows: (1) the threshold light intensity for growth: 29.8 μmol photons m⁻² s⁻¹; (2) the optimum light intensity (L_m) giving the maximum growth rate (μ_max = 0.659 divisions day⁻¹): 196 μmol photons m⁻² s⁻¹; (3) the optimum light intensity range (L_opt) giving ≥95% μ_max: 130–330 μmol photons m⁻² s⁻¹; (4) the semi-optimum range (L_sopt) giving ≥80% μ_max: 90 to over 460 μmol photons m⁻² s⁻¹. The L_sopt represents 4.5–23% ambient light intensity present in surface waters off of a temperate region of the Japanese coast, Tosa Bay; putatively, this semi-optimum range of light intensity appears at depth of 12.9–27.8 m. Considering these issues, our data indicate that Ostreopsis sp. 1 in coastal environments may form blooms at ca. ∼28 m depth in regions along Japanese coasts.     

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.     

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.     

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.     

CHARACTERIZATION OF OSTREOPSIS AND COOLIA (DINOPHYCEAE) ISOLATES IN THE WESTERN MEDITERRANEAN SEA BASED ON MORPHOLOGY,TOXICITY AND INTERNAL TRANSCRIBED SPACER 5.8S rDNA SEQUENCES1

Several isolates of epiphytic dinoflagellates belonging to the genera Ostreopsis Schmidt and Coolia Meunier from the western Mediterranean Sea were examined by LM and EM, toxicity assays, and internal transcribed spacer (ITS) regions of nuclear rDNA, and 5.8S rDNA were sequenced. Morphological comparisons based on the analyses of cell shape, size, thecal plates, and surface ornamentation revealed two distinct species in the western Mediterranean: O. cf. siamensis Schmidt from the Catalan, Andalusian, and Sicilian coasts and O. ovata Fukuyo from the Ligurian coast, southern Tyrrhenian Sea, and Balearic Islands. Both Ostreopsis species were toxic; however, no differences in toxicity were detected between the two Ostreopsis species. Coolia monotis Meunier was nontoxic. The morphological studies were supported by phylogenetic analyses; all western Mediterranean isolates of O. cf. siamensis showed ITS and 5.8S rDNA sequences identical to each other and so did those of O. ovata, whereas high genetic diversity was detected between the western Mediterranean and Asian isolates of O. ovata. The nucleotide sequence analyses of the C. monotis strains showed that all C. monotis isolates from Europe formed a homogeneous clade. Further, the genetic diversity was high between the European and Asian C. monotis isolates. In this study, genetic markers combined with morphology and toxicity analyses was useful in the taxonomic and phylogenetic studies of the Ostreopsidaceae in a temperate area.     

Colorimetric DNA-based assay for the specific detection and quantification of Ostreopsis cf. ovata and Ostreopsis cf. siamensis in the marine environment

Ostreopsis is a toxic benthic dinoflagellate largely distributed worldwide in tropical and temperate areas. In the Mediterranean Sea, periodic summer blooms have been reported and have become a serious concern due to their direct impact on human health and the environment. Current microalgae identification is performed via light microscopy, which is time-consuming and is not able to differentiate among Ostreopsis species. Therefore, there is mature need for rapid, specific and easy-to-use detection tools. In this work, a colorimetric assay exploiting a combination of recombinase polymerase amplification (RPA) and a sandwich hybridisation assay was developed for O. cf. ovata and O. cf. siamensis detection and quantification. The specificity of the system was demonstrated by cross-reactivity experiments and calibration curves were successfully constructed using genomic DNA, achieving limits of detection of 10 and 14 pg/μL for O. cf. ovata and O. cf. siamensis, respectively. The assay was applied to the analysis of planktonic and benthic environmental samples from different sites of the Catalan coast. Species-specific DNA quantifications were in agreement with qPCR analysis, demonstrating the reliability of the colorimetric approach. Significant correlations were also obtained between DNA quantifications and light microscopy counts. The approach may be a valuable tool to provide timely warnings, facilitate monitoring activities or study population dynamics, and paves the way towards the development of in situ tools for the monitoring of harmful algal blooms.     

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.     

Benthic Dinoflagellate Integrator (BEDI): A new method for the quantification of Benthic Harmful Algal Blooms

Despite the potential negative human health, ecological and economic impact, the ecology of harmful benthic dinoflagellate blooms remains largely unknown. This is probably due to the complex interactions among biotic and abiotic drivers that influence blooms, but also to the difficulty in quantifying cell abundance in a comparable way over large spatial and temporal scales. One of the recognized priorities for bHABs (benthic Harmful Algal Blooms) assessment is developing and standardizing methods that can provide comparable data. In this context, the Benthic Dinoflagellates Integrator (BEDI), a new non-destructive quantification method for benthic dinoflagellate abundances, has been developed and tested within the present study. The rationale behind the BEDI standard assessment method is that mechanical resuspension of cells enables the quantification of abundances as cells per unit of seabed surface area (i.e. cells mm⁻²) or as Potentially Resuspended cells per unit of volume (PRcells ml⁻¹), by integrating both cells in the biofilm and those in the surrounding water. Estimations of Ostreopsis performed with BEDI method are independent of the substratum (i.e. macroalgal species) or the dominant ecosystem (i.e. algal forests or turfs, seagrass beds, coral reefs) and potentially allow the comparison of benthic dinoflagellate blooms over broad temporal and spatial scales. The first application of the BEDI method, presented in this study, gave encouraging results: the characterization of blooms of Ostreopsis cf. ovata at three sites in the NW Mediterranean Sea is consistent with results derived from the other commonly applied methods. Quantification of the ratio between abundances of cells in the biofilm and in the surrounding water was calculated for the first time per unit of seabed surface area, demonstrating that the highest abundances of cells (the stock), and therefore the associated risk for human health, are in the biofilm. For risk assessment purposes, conversion values for commonly used monitoring alert thresholds of Mediterranean Ostreopsis blooms are provided.     

The prevalence of benthic dinoflagellates associated with ciguatera fish poisoning in the central Red Sea

This study confirms the presence of the toxigenic benthic dinoflagellates Gambierdiscus belizeanus and Ostreopsis spp. in the central Red Sea. To our knowledge, this is also the first report of these taxa in coastal waters of Saudi Arabia, indicating the potential occurrence of ciguatera fish poisoning (CFP) in that region. During field investigations carried out in 2012 and 2013, a total of 100 Turbinaria and Halimeda macroalgae samples were collected from coral reefs off the Saudi Arabian coast and examined for the presence of Gambierdiscus and Ostreopsis, two toxigenic dinoflagellate genera commonly observed in coral reef communities around the world. Both Gambierdiscus and Ostreopsis spp. were observed at low densities (<200 cells g⁻¹ wet weight algae). Cell densities of Ostreopsis spp. were significantly higher than Gambierdiscus spp. at most of the sampling sites, and abundances of both genera were negatively correlated with seawater salinity. To assess the potential for ciguatoxicity in this region, several Gambierdiscus isolates were established in culture and examined for species identity and toxicity. All isolates were morphologically and molecularly identified as Gambierdiscus belizeanus. Toxicity analysis of two isolates using the mouse neuroblastoma cell-based assay for ciguatoxins (CTX) confirmed G. belizeanus as a CTX producer, with a maximum toxin content of 6.50 ± 1.14 × 10⁻⁵ pg P-CTX-1 eq. cell⁻¹. Compared to Gambierdiscus isolates from other locations, these were low toxicity strains. The low Gambierdiscus densities observed along with their comparatively low toxin contents may explain why CFP is unidentified and unreported in this region. Nevertheless, the presence of these potentially toxigenic dinoflagellate species at multiple sites in the central Red Sea warrants future study on their possible effects on marine food webs and human health in this region.