Toxic Dinoflagellate Prorocentrum Research Articles

Deciphering interactions between the marine dinoflagellate Prorocentrum lima and the fungus Aspergillus pseudoglaucus.

The comprehension of microbial interactions is one of the key challenges in marine microbial ecology. This study focused on exploring chemical interactions between the toxic dinoflagellate Prorocentrum lima and a filamentous fungal species, Aspergillus pseudoglaucus, which has been isolated from the microalgal culture. Such interspecies interactions are expected to occur even though they were rarely studied. Here, a co-culture system was designed in a dedicated microscale marine-like condition. This system allowed to explore microalgal-fungal physical and metabolic interactions in presence and absence of the bacterial consortium. Microscopic observation showed an unusual physical contact between the fungal mycelium and dinoflagellate cells. To delineate specialized metabolome alterations during microalgal-fungal co-culture metabolomes were monitored by high-performance liquid chromatography coupled to high-resolution mass spectrometry. In-depth multivariate statistical analysis using dedicated approaches highlighted (1) the metabolic alterations associated with microalgal-fungal co-culture, and (2) the impact of associated bacteria in microalgal metabolome response to fungal interaction. Unfortunately, only a very low number of highlighted features were fully characterized. However, an up-regulation of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed during co-culture in supernatants. Such results highlight the importance to consider microalgal-fungal interactions in the study of parameters regulating toxin production.

Effects of the toxic dinoflagellates Prorocentrum lima and Ostreopsis cf. ovata on immune responses of cultured oysters Crassostrea gasar.

Oyster production in Brazil has been highlighted as an important economic activity and is directly impacted by the quality of the environment, which is largely the result of human interference and climate change. Harmful algal blooms occur in aquatic ecosystems worldwide, including coastal marine environments which have been increasing over the last decades as a result of global change and anthropogenic activities. In this study, the native oysters Crassostrea gasar from Northeast of Brazil were exposed to two toxic benthic dinoflagellate species, Prorocentrum lima and Ostreopsis cf. ovata. Their respective effects on C. gasar physiology and defense mechanisms were investigated. Oyster hemocytes were first exposed in vitro to different concentrations of both dinoflagellate species to assess their effects on hemocyte functions, such as phagocytosis, production of reactive oxygen species, as well as mortality. Results highlighted an alteration of hemocyte phagocytosis and viability in presence of O. cf. ovata, whereas P. lima did not affect the measured hemocyte functions. In a second experiment, oysters were exposed for 4 days in vivo to toxic culture of O. cf. ovata to assess its effects on hemocyte parameters, tissues damages and pathogenic Perkinsus spp. infection. An increase in hemocyte mortality was also observed in vivo, associated with a decrease of ROS production. Histopathological analyses demonstrated a thinning of the epithelium of the digestive tubules of the digestive gland, inflammatory reaction and a significant increase in the level of infection by Perkinsus spp. in oysters exposed to O. cf. ovata. These results indicate that oysters C. gasar seem to be pretty resilient to an exposure to P. lima and may be more susceptible to O. cf. ovata. Furthermore, the latter clearly impaired oyster physiology and defense mechanisms, thus highlighting that harmful algal blooms of O. cf. ovata could potentially lead to increased susceptibility of C. gasar oysters to parasite infections.

Invasive clams (Ruditapes philippinarum) are better equipped to deal with harmful algal blooms toxins than native species (R. decussatus): evidence of species-specific toxicokinetics and DNA vulnerability

This study aims to assess and compare the kinetics (accumulation/elimination) of the marine biotoxins okadaic acid (OA) and dinophysistoxin-1 (DTX1), between native (Ruditapes decussatus) and invasive (Ruditapes philippinarum) clam species, and their genotoxic effects and DNA recover capacity after, exposure to toxic dinoflagellates Prorocentrum lima. Clams were fed with P. lima for 5 days and then to non-toxic algae (post-exposure) during other 5 days. Toxin concentrations determined in clams by LC-MS/MS were related with DNA damage and repair assessment through the comet and base excision repair (BER) assays, respectively. Differential accumulation patterns were observed between the invasive and native species. The invasive species consistently and progressively accumulated the toxins during the first 24 h of exposure, while the native clams showed drastic variations in the toxin accumulation. Nevertheless, at the end of a 5 days of exposure period, the native clams presented higher toxin concentrations, nearly reaching the legal regulatory limit for human consumption. In addition, native clams were vastly affected by OA and DTX1, presenting an increment in the DNA damage since the first day, with a correspondent increase in the repair activity. On the other hand, invasive clams were not affected by the dinoflagellate toxins, exhibiting only some signs of the challenge, namely an increase in the DNA repair mechanisms in the post-exposure period. Invasive clams R. philippinarum are better adapted to cope with harmful algal blooms and OA-group toxins than native species. These results may increase farming interest and may lead to new introductions of the invasive clams. In sympatry sites, exposure to OA-group toxins may unbalance clams species biomass and distribution as exposure to toxic dinoflagellates affects the native clams from cellular to a population level, representing a significant threat to development and maintenance of R. decussatus populations.

The interplay of two invaders: can blooms of the potentially toxic dinoflagellates Prorocentrum cordatum be downregulated by the neritic copepods Acartia tonsa?

The interplay of two invaders: can blooms of the potentially toxic dinoflagellates Prorocentrum cordatum be downregulated by the neritic copepods Acartia tonsa?

Plankton community of trafficked ports as a baseline reference for Non Indigenous Species arrivals. Case study of the Port of Bar (South Adriatic Sea)

Plankton (ichthyo, zoo and phyto) communities were studied in the temperate shallow waters of the Port of Bar, one of the main cargo ports on the south-eastern Adriatic coast. Sampling was undertaken in February, April, June and October 2015 at 12 stations using the BALMAS Port Baseline Survey protocol. The research was conducted to determine the presence of invasive and potentially toxic plankton species in the port.The most dominant species of ichthyoplankton were eggs and larvae of families Engraulidae, Bothidae and Sparidae with a dominance of Engraulis encrasicolus, Arnoglossus laterna and Diplodus annularis. In addition to ichthyoplankton, sampling of phyto and zooplankton was performed to estimate the abundance and diversity of species.The most numerous zooplankton species throughout investigated period were Penilia avirostris, Euterpina acutifrons, Oithona nana, Acartia clausi, Centropages kroyeri, Paracalanus parvus, Oncaeidae and larvae of Bivalvia. One very unusual occurrence was the spawning of parrotfish Sparisoma cretense (Linnaeus, 1758), a species with Atlantic origin and tropical affinities, whose presence throughout the Mediterranean has shown an increasing trend over the last decade. The most dominant species of phytoplankton were the diatoms Chaetoceros affinis and Chaetoceros spp., Asterionellopsis glacialis, Pseudo-nitzschia spp., Thalassionema nitzschioides, and dinoflagellates Gymnodinium spp. and Prorocentrum triestinum. The potentially toxic species from genus Pseudo- nitzschia reached an abundance of 104 cells L-1. Toxic dinoflagellates Prorocentrum cordatum and P. micans reached values of 103 cells L-1.Although there were no HAOP species found during the survey, presence of several potentially toxic and toxic phytoplankton species whose impact is not sufficiently known indicates the necessity of introduction of regular monitoring activities and definition of preventive protection measures.

The tellurite-reducing bacterium Alteromonas macleodii from a culture of the toxic dinoflagellate Prorocentrum foraminosum

The Alteromonas macleodii strain 2328 was isolated from a clonal culture of the toxic dinoflagellate Prorocentrum foraminosum. The strain exhibits a resistance to high K2TeO3 concentrations (2500 μg/mL). A study of the growth dynamics of the strain exposed to K2TeO3 has shown a longer lag phase and a reduced stationary phase compared to those during cultivation with no toxicant. The fatty acids profile is dominated by 16:1 (n-7), 16:0, 17:1, 15:0, 18:1 (n-7), and 17:0. The 2328 strain belongs to the Gammaproteobacteria and is related to the genus Alteromonas with 99–100% sequence similarity to some intra-genome allele variants (paralogues) of 16S rRNA from A. macleodii. A phylogenetic reconstruction (ML and NJ), based on HyHK amino acid sequences, has revealed that the analyzed 2328 strain forms a common cluster with A. macleodii strains. In the presented work, the ability of A. macleodii to reduce potassium tellurite to elemental tellurium has been recorded for the first time. Bacteria reduce potassium tellurite to Te (0), nanoparticles of which become distributed diffusely and in the form of electron-dense globules in cytoplasm. Large polymorphous metalloid crystals are formed in the extracellular space. Such feature of the A. macleodii strain 2328 makes it quite attractive for biotechnological application as an organism concentrating the rare metalloid.

The Impact of Temperature Stress on DNA and RNA Synthesis in Potentially Toxic Dinoflagellates Prorocentrum minimum

Biomarkers of temperature stress were studied as major characteristics crucial for the understanding complex processes that underlie the response of marine planktonic microorganisms to environmental factors and their sublethal effects. Using the potentially toxic dinoflagellates Prorocentrum minimum as a model object, the impact of temperature stress on viability, cell cycle, RNA synthesis and DNA replication in these protists was evaluated. It was shown by flow cytometry that stress evoked by a temperature increase from 25°C (control) to 37 or 42°C during 15 to 60 min did not cause any considerable alterations in the cell cycle, while cell death rate increased from ≤ 1% (control) to 2–12% at 37°C and 4–22% at 42°C. Along with a relatively low cell death rate, following a temperature increase to 37 and/or 42°C, P. minimum displayed the ability to boost the synthesis of DNA (1.7–1.9 and 1.2–1.6 times, respectively) and especially RNA (2.5–3.1 and 1.7–2.8 times, respectively) during the first 15–30 min after stress. At certain stages of the life cycle, this effect can be critical for maintaining the viability and normal development of the P. minimum population. The obtained results demonstrate that a significantly elevated synthesis of nucleic acids can serve as an indicator (biomarker) of sublethal environmental stress.

The toxic benthic dinoflagellate Prorocentrum maculosum Faust is a synonym of Prorocentrum hoffmannianum Faust

Three strains of the toxic benthic dinoflagellate Prorocentrum hoffmannianum were isolated in the Canary Islands (north-east Atlantic Ocean, Spain). The identity of the strains was determined by phylogenetic analyses of partial LSU rDNA (D1-D2 regions) but their morphology based on SEM images corresponded to P. maculosum. Their toxin profiles were analyzed by liquid chromatography and high resolution mass spectrometry analysis (LC–HRMS) on cell extracts and culture media. Okadaic acid and three analogs were detected in all strains. Rather, in culture media the detected compounds were variable among strains, two of them being okadaic acid analogs not found on cell extracts. As a result, the taxonomy of the species was revised and P. maculosum is proposed as a junior synonym of P. hoffmannianum whose description is emended.

Salinity stress response of the invasive dinoflagellate Prorocentrum minimum

Estuarine and coastal environments are vulnerable to alien species invasions. Many nonindigenous organisms demonstrate high ecological plasticity, which is particularly well expressed in unicellular eukaryotes. In this study, responses of the potentially toxic invasive dinoflagellate Prorocentrum minimum, initially acclimated to salinity 17 (control), to short-term stress by salinities 4, 8 and 35 were investigated experimentally by determination of mortality, alterations in cell cycle and chromosome fine structure, RNA synthesis and DNA replication. The percentage of dead cells within the P. minimum population after stress by critical salinity 8 was lower compared to the other tested conditions. Salinity stress caused only moderate impact on the cell cycle pattern; changes of chromosome fine structure at salinities 4 and 8 were evident though reversible. We disclosed the elevated RNA synthesis and DNA replication which can play a ‘compensational’ role by speeding up the metabolic (synthetic) activity of these protists at the critical salinity conditions. The increased RNA synthesis in phase G1 and additional DNA replication in phase S of the cell cycle likely enhance salinity tolerance and cause relatively low mortality of these micro-eukaryotes at salinity 8. We infer that the enhanced synthesis of DNA and RNA can be one of the advanced ecological strategies of P. minimum which along with the other cellular and physiological characteristics (small body size, planktonic mode of life, fast reproduction, high evolution rates, mixotrophy, ability to form cysts, etc.) helps these dinoflagellates to invade successfully and populate permanently coastal ecosystems, remaining highly competitive in the unstable brackishwater environments.

Transcriptional and biochemical analysis of antioxidant enzymes in the mussel Mytilus galloprovincialis during experimental exposures to the toxic dinoflagellate Prorocentrum lima

The genotoxic and cytotoxic effects of Diarrhetic Shellfish Poisoning (DSP) toxins have been widely investigated in bivalve molluscs, representing the main vectors of these compounds in the Atlantic coast of Europe. DSP toxins are produced by Harmful Algal Blooms (HABs) of Dinophysis and Prorocentrum dinoflagellates, being subsequently accumulated by marine organisms and biomagnified throughout trophic webs. Yet, bivalves display increased resistance to the harmful effects of these toxins during HAB episodes. While previous reports have suggested that such resilience might be the result of an increased activity in the bivalve antioxidant system, very little is still known about the specific mechanism underlying the protective effect observed in these organisms. The present work aims to fill this gap by studying transcriptional expression levels and biochemical activities of antioxidant enzymes in different tissues the mussel Mytilus galloprovincialis during experimental exposures to DSP toxins produced by the dinoflagellate Prorocentrum lima. Results are consistent with the presence of a compensatory mechanism involving a down-regulation in the expression of specific genes encoding antioxidant enzymes [i.e., SuperOxide Dismutase (SOD) and CATalase (CAT)] which is counterbalanced by the up-regulation of other antioxidant genes such as Glutathione S-Transferase pi-1 (GST-pi) and Selenium-dependent Glutathione PeroXidase (Se-GPx), respectively. Enzymatic activity analyses mirror gene expression results, revealing high antioxidant activity levels (consistent with a protective role for the antioxidant system) along with reduced lipid peroxidation (increasing the defense against oxidative stress).

The morphology and seasonal dynamics of the potentially toxic microalga Prorocentrum foraminosum Faust 1993 (Dinophyta) in Peter the Great Bay, the Sea of Japan

The morphology of the potentially toxic dinoflagellate Prorocentrum foraminosum was examined in detail using light and scanning electron microscopy for the first time for the seas of Russia. New morphological features of this species were found and documented. Long-term observations showed that P. foraminosum occurred regularly from April to November in the epiphytic assemblages of Peter the Great Bay. The highest cell abundance was recorded in June (1.5 × 103 cells/g macrophyte dry weight). This dinoflagellate was also found rarely at a low cell densities in coastal sands.

A dual-species co-cultivation system to study the interactions between Roseobacters and dinoflagellates.

Some microalgae in nature live in symbiosis with microorganisms that can enhance or inhibit growth, thus influencing the dynamics of phytoplankton blooms. In spite of the great ecological importance of these interactions, very few defined laboratory systems are available to study them in detail. Here we present a co-cultivation system consisting of the toxic phototrophic dinoflagellate Prorocentrum minimum and the photoheterotrophic alphaproteobacterium Dinoroseobacter shibae. In a mineral medium lacking a carbon source, vitamins for the bacterium and the essential vitamin B12 for the dinoflagellate, growth dynamics reproducibly went from a mutualistic phase, where both algae and bacteria grow, to a pathogenic phase, where the algae are killed by the bacteria. The data show a “Jekyll and Hyde” lifestyle that had been proposed but not previously demonstrated. We used RNAseq and microarray analysis to determine which genes of D. shibae are transcribed and differentially expressed in a light dependent way at an early time-point of the co-culture when the bacterium grows very slowly. Enrichment of bacterial mRNA for transcriptome analysis was optimized, but none of the available methods proved capable of removing dinoflagellate ribosomal RNA completely. RNAseq showed that a phasin encoding gene (phaP1) which is part of the polyhydroxyalkanoate (PHA) metabolism operon represented approximately 10% of all transcripts. Five genes for aerobic anoxygenic photosynthesis were down-regulated in the light, indicating that the photosynthesis apparatus was functional. A betaine-choline-carnitine-transporter (BCCT) that may be used for dimethylsulfoniopropionate (DMSP) uptake was the highest up-regulated gene in the light. The data suggest that at this early mutualistic phase of the symbiosis, PHA degradation might be the main carbon and energy source of D. shibae, supplemented in the light by degradation of DMSP and aerobic anoxygenic photosynthesis.

Contrasting key roles of Ruppia cirrhosa in a southern Mediterranean lagoon: Reservoir for both biodiversity and harmful species and indicator of lagoon health status

The distribution of Ruppia cirrhosa meadow density and its epiphytic organisms in relation with environmental factors were studied in summer 2011 at five stations in the Ghar El Melh lagoon (GML; southern Mediterranean Sea). Eleven epiphytic groups were recognised among which diatoms and dinoflagellates were the dominant groups and greatest contributors to temporal dissimilarity. An overwhelming concentrations of harmful microalgae was recorded, mainly represented by the toxic dinoflagellate Prorocentrum lima with maximal concentrations attaining 6×105cells 100g−1 of Ruppia fresh weight. The epifauna community accounted for only 1.4% of total epiphyte abundance and was comprised predominantly of nematodes (47.51%), ciliates (32.59%), fish eggs (7.2%) and larvae (4.95%). PERMANOVA analyses revealed a significant spatio-temporal variation of all epiphytic groups (p<0.01). In this study, R. cirrhosa and its epiphytes were studied as potential early warning indicators of the health status of GML waters.

Evaluation of Genotoxicity in Gills and Hemolymph of Clam Ruditapes decussatus Fed with the Toxic Dinoflagellate Prorocentrum lima

Diarrheic shellfish poisoning (DSP) is a gastrointestinal (GIT) disease that appears a few hours after ingesting okadaic acid (OA)-contaminated mollusks; okadaic acid is present in dinoflagellates of the genera Dinophysis and Prorocentrum. Toxic manifestations occur all year round at a higher or lesser intensity, and as a consequence, extractive production factories need to be closed during these periods which affects the economy of aquaculture industries. Although the concentration of harmful algae is usually found at high levels in clam digestive gland, bivalve mortality was not increased. In this study, the genotoxic effects produced by OA in clam Ruditapes decussatus were determined using the comet assay. In vitro (exposing hemocytes to different concentrations of OA) and in vivo (feeding clams with toxic dinoflagellate Prorocentrum lima) experiments were conducted in order to determine the genotoxic effects of OA on bivalve cells. Hemocytes and gill cells were analyzed by in vivo and in vitro approaches. While the in vitro study showed a rapid effect of OA on hemocytes, data obtained in the in vivo experiment reflected contradictory results dependent upon the concentration of OA and cell type evaluated. An increase in DNA damage was observed at the lower concentration and only in gill tissue. The results obtained may contribute to a better understanding of the mechanisms underlying genotoxic effects induced by OA on bivalves.

Diatom induction of reproductive failure in copepods: The effect of PUAs versus non volatile oxylipins

Several species and strains of diatoms were tested for their effects on egg production, hatching success, faecal pellet production, ingestion rate and food selection in the copepod Temora stylifera. Two of the diatom species tested were well known polyunsaturated aldehyde (PUA) producers: Skeletonema marinoi (SM) and Thalassiosira rotula (TR1) , while the other species tested, Skeletonema pseudocostatum, was a non-PUA producing species. Another T. rotula strain (TR2) was also tested since previous studies had reported contradictory results, due either to absence of PUA production or production in small amounts. Our results showed strong inhibitory effects on reproductive parameters of copepods with all diatom species tested. Although we confirm that S. pseudocostaum did not produce PUAs, this diatom produced large quantities of other oxylipins such as (5Z,8Z,11Z,13E,15S,17Z)-15-hydroxy-5,8,11,13,17-eicosapentaenoic acid (15S-HEPE) and 13,14-13R-hydroxy-14S,15S-trans-epoxyeicosa-5Z,8Z,11Z,17Z-tetraenoic acid (13,14-HEpETE) and 15-oxo-5Z,9E,11E,13E-pentadecatetraenoic acid (15-oxoacid), all of which have already been found in S. marinoi (Fontana et al., 2007a) and Pseudonitzschia delicatissimia (d'Ippolito et al., 2009). Another unidentified oxylipin was also present in high quantities in LC–MS profiles. Some of these oxygenated fatty acid derivatives were also found, together with PUAs, in TR1 and in TR2. Both PUA-producing and non-producing diatoms caused negative effects on hatching success with induction of apoptosis in newly hatched nauplii. This suggests that negative diatom effects can also depend on the production of non volatile oxylipins. Copepod feeding behaviour was found to be non-selective when each diatom species was offered together with the non toxic dinoflagellate Prorocentrum minimum suggesting that grazers are not aware of the toxicity of their food.

Use of long‐term data and multivariate ordination techniques to identify environmental factors governing estuarine phytoplankton species dynamics

A continuous, 13‐yr record of environmental data and phytoplankton species and assemblage structure in the mesohaline Neuse River Estuary (biweekly, April‐October; monthly, November‐March) was used to evaluate phytoplankton assemblage responses to changing environmental conditions. Ordination techniques including nonmetric multidimensional scaling, indicator species analysis, and BIO‐ENV software were used to investigate potential environmental predictors of phytoplankton assemblage patterns under chronic eutrophication. Phytoplankton assemblages were strongly related to temperature and total nitrogen : total phosphorus ratios, with expected seasonal changes in species composition. Interannual changes in river discharge influenced whether phytoplankton assemblages were dominated by diatoms and phototrophic flagellates or by mixotrophic and heterotrophic dinoflagellates. Increasing ammonium concentrations also have been an important influence on phytoplankton assemblages. Raphidophytes (including the potentially toxic species Heterosigma akashiwo), haptophytes, chlorophytes, and the bloom‐forming dinoflagellate Heterocapsa rotundata have increased in more recent years (2000–2006), concomitant with increasing ammonium concentrations. Abundance of the potentially toxic dinoflagellates Prorocentrum minimum and the grouping Pfiesteria spp., “pfiesteria‐like” dinoflagellates, and Karlodinium veneficum remained stationary over time and rarely exceeded 103 cells mL−1. Abundance of P. minimum was positively related to dissolved organic nitrogen and suspended solids concentrations, whereas the highest abundance of the grouping Pfiesteria spp., “pfiesteria‐like” dinoflagellates, and K. veneficum occurred during summer and fall, related to high total phosphorus concentrations, temperature, and salinity. Overall, this study provides new species‐level insights to advance understanding about anthropogenic influences on phytoplankton assemblages. The data suggest an increasingly important role of ammonium in controlling phytoplankton assemblage structure, including increased abundance of some harmful species, in eutrophic estuaries.

Prorocentrum minimum (Dinophyceae) in the Baltic Sea: morphology, occurrence—a review

The potentially toxic dinoflagellate Prorocentrum minimum (Pavillard) Schiller has successfully established in the Baltic Sea in the last two decades. A review of the invasion history is presented as well as new data on the spatial and inter-annual variability of this species and its relation to salinity, temperature, and nutrient concentrations. A short literature review of the morphological characters of the Baltic P. minimum is also included. From 1993 to 2002, P. minimum was a regular component of the summer and autumn plankton flora of the Baltic Sea proper and the Gulf of Finland. Its abundance varied considerably inter-annually and did not show any clear trends during the period. Abundance of P. minimum was significantly higher in the nutrient-enriched Bay of Mecklenburg (German coast) and the southern Baltic proper than in the central and northern Baltic proper and the Gulf of Finland, where its abundance was mostly sparse. In coastal waters P. minimum occasionally reached densities of several million cells per litre and dominated phytoplankton biomass (>90%). Abundance of the Baltic P. minimum was generally not related to salinity or temperature. It could be a dominant species at both high and low salinity (over 15 and 4.8 PSU), and its temperature range was broad (from 2.7 to 26.4 °C). However, dense populations usually occurred from July to October at temperatures above 10 °C. Further, there appears to be a positive correlation between the success of P. minimum in the Baltic Sea and high concentrations of total phosphorus and nitrogen. This tolerant and morphologically variable dinoflagellate seems to be a morphospecies without subtaxa, which can expand its range in the Baltic Sea, especially in nutrient-rich coastal waters.

Detection of Karenia mikimotoi by spectral absorption signatures

This study investigated the performance of a spectral similarity index and a multivariate partial least-squares regression technique for detecting the presence of the gyroxanthin-diester-carrying toxic dinoflagellate Karenia mikimotoi from measurements of spectral light absorption. The methods were applied to fourth-derivative absorption spectra of K. mikimotoi mixed with the toxic dinoflagellate Prorocentrum minimum, which does not contain gyroxanthin-diester. Acclimating the cultures to different light and nutrient conditions allowed us to evaluate the sensitivity of the methods to changes in pigmentation and intracellular light absorption (pigment packaging) of the algae. Both methods were able to determine the fraction of K. mikimotoi and the gyroxanthin-diester concentration. However, whereas the partial least-squares predictions were almost insensitive to the induced variability in optical properties of the algae, predictions based on the similarity index differed significantly depending on the acclimation of the algae. Furthermore, discriminating K. mikimotoi from natural phytoplankton assemblages indicated a significant influence of cell-size composition, through pigment packaging, on the accuracy of the similarity index. Our results suggest that optical discrimination of phytoplankton species from spectral absorption signatures will improve significantly by applying the partial least-squares regression technique.

Effects of toxic extracts and purified borbotoxins from Prorocentrum borbonicum (Dinophyceae) on vertebrate neuromuscular junctions

Benthic dinoflagellates of the genus Prorocentrum are common in tropical and subtropical water and several species produce phycotoxins potentially involved in human toxic outbreaks. The toxic dinoflagellate Prorocentrum borbonicum collected at La Réunion Island (France) was cultured in laboratory. A crude extract of the organism displayed significant toxicity in mice characterized by progressive limb paralysis, severe dyspnea, and death, and the toxicity was retained, after partition, in the extract's butanol-soluble fraction (BSF). Electrophysiological experiments characterizing the fraction's effect on isolated vertebrate neuromuscular preparations revealed that it depolarizes the muscle membrane and reduces the driving force for endplate potentials (EPPs) evoked by nerve stimulation, blocking directly- and indirectly-elicited muscle twitches. The depolarization induced by P. borbonicum BSF was not due to Na + influx through voltage-dependent Na + channels, since tetrodotoxin neither prevented nor suppressed the depolarization. However, ouabain, a specific ligand of the Na/K ATPase, reduced the depolarization. These results suggest the presence of palytoxin-like compounds in the fraction. HPLC–MS and MS/MS analysis showed the presence of several toxins having identical UV absorbance, among which two new isomeric toxins, borbotoxin-A and -B, of molecular mass of 1037.6 Da were isolated. The purified borbotoxin-A, had no effect on the resting membrane potential of muscle fibers and did not affect directly-elicited muscle twitches. However, the toxin reduced nerve-evoked muscle twitches, in a dose-dependent manner, reduced EPPs' amplitudes and completely blocked miniature endplate potentials. These observations suggest that the main action of borbotoxin-A is to block post-synaptic nicotinic ACh receptors.

Toxin composition of the toxic dinoflagellate Prorocentrum lima isolated from different locations along the Galician coast (NW Spain)

The DSP toxin composition of 19 Prorocentrum lima isolates from different locations of the Galician rias (Vigo and Pontevedra) was investigated by high performance liquid chromatography coupled with fluorimetric detection. Boiling and freeze/thaw/hydrolyse methodology were applied during extraction to detect OA, DTX1, DTX2 and their esterified derivatives. OA and DTX2 were detected in both free and esterified form, the latter always in very low amounts, whilst DTX1 was always present in the free form. This indicate that the hypothesized self-protection mechanism of toxin storage in the less active esterified forms does not seem to apply to DTX1. A slight increase in the toxin concentration per cell was found during growth, although toxin composition did not vary appreciably. Toxin production and toxin profile varied significantly depending on the isolate. Four groups of P. lima were differentiated by cluster analysis according to their toxin composition. It is noteworthy that one of the clusters comprised all the strains collected from one location characterised by its geographical isolation, whereas the other clusters consisted of isolates from different locations. The differences in the toxin profile from P. lima strains and from the DSP contaminated shellfish, together with the very good correlation between Dinophysis spp occurrence and DSP toxicity in shellfish, support that these planktonic species are the main agents responsible for DSP events in Galicia.