A biosensor monitoring approach for toxic algae: Construction of calibration curves to infer cell numbers in field material.

A variety of shellfish toxin-producing Harmful Algal Blooms (HABs) occur every year in coastal temperate waters worldwide. These toxic HABs may cause lengthy (months) harvesting bans of mussels and other suspension feeding bivalves exposed to their blooms. To safeguard public health and the shellfish industry, European Union regulations request periodic monitoring of potentially toxic microalgae in seawater and phycotoxins in live bivalve molluscs from shellfish production areas. Monitoring of other toxic microalgae, e.g., fish killers, is based solely on cell counts. Morphological identification and quantification of microalgal cells with light microscopy is time-consuming, requires a good expertise, and accurate identification to species level (e.g., Pseudo-nitzschia species) may require electron microscopy. Toxicity varies among morphologically similar species; there are toxic and non-toxic strains of the same species. Molecular techniques using ribosomal DNA sequences offer a possibility to identify and detect precisely the potentially toxic genus/species. In an earlier project (MIDTAL), specific probes against rRNA sequences of all HAB taxa, known at the time of the project, affecting shellfish areas worldwide were designed, and those affecting Europe were tested and calibrated against rRNA extracts of clonal cultures and field samples. Microarray technology was adopted to relate to cell numbers the fluorescence signal from the reaction of all target species probes spotted in the microarray slides with those present in a single sample extract. The EMERTOX project aimed to develop a more automatic "Lab on a chip" (LOC) technology, including a non- (cell) disruptive water concentration system and biosensors for HAB cells detection. Here, calibration curves are presented against toxic microalgae (cultures and field samples) causing endemic and emerging toxicity events in Galicia (NW Spain) and Portugal. Results here relating cell numbers to electrochemical signals will be used in an early warning biosensor for toxic algae.

An Unprecedented Bloom of Oceanic Dinoflagellates (Karenia spp.) Inside a Fjord within a Highly Dynamic Multifrontal Ecosystem in Chilean Patagonia.

At the end of summer 2020, a moderate (~105 cells L-1) bloom of potential fish-killing Karenia spp. was detected in samples from a 24 h study focused on Dinophysis spp. in the outer reaches of the Pitipalena-Añihue Marine Protected Area. Previous Karenia events with devastating effects on caged salmon and the wild fauna of Chilean Patagonia had been restricted to offshore waters, eventually reaching the southern coasts of Chiloé Island through the channel connecting the Chiloé Inland Sea to the Pacific Ocean. This event occurred at the onset of the COVID-19 lockdown when monitoring activities were slackened. A few salmon mortalities were related to other fish-killing species (e.g., Margalefidinium polykrikoides). As in the major Karenia event in 1999, the austral summer of 2020 was characterised by negative anomalies in rainfall and river outflow and a severe drought in March. Karenia spp. appeared to have been advected in a warm (14-15 °C) surface layer of estuarine saline water (S > 21). A lack of daily vertical migration patterns and cells dispersed through the whole water column suggested a declining population. Satellite images confirmed the decline, but gave evidence of dynamic multifrontal patterns of temperature and chl a distribution. A conceptual circulation model is proposed to explain the hypothetical retention of the Karenia bloom by a coastally generated eddy coupled with the semidiurnal tides at the mouth of Pitipalena Fjord. Thermal fronts generated by (topographically induced) upwelling around the Tic Toc Seamount are proposed as hot spots for the accumulation of swimming dinoflagellates in summer in the southern Chiloé Inland Sea. The results here provide helpful information on the environmental conditions and water column structure favouring Karenia occurrence. Thermohaline properties in the surface layer in summer can be used to develop a risk index (positive if the EFW layer is thin or absent).

The niche of a stress-tolerant specialist, Dinophysis acuminata, in a coastal upwelling system.

Dinophysis acuminata, the main cause of shellfish harvesting bans in Europe, blooms in the Galician Rías (NW Spain) throughout the upwelling season (ca. March to September). Here we illustrate rapid changes in vertical and across ría-shelf distributions of diatoms and dinoflagellates (including D. acuminata vegetative and small cells) in Ría de Pontevedra (RP) and Ría de Vigo (RV) during transitions from spin-down to spin-up phases of upwelling cycles. A subniche approach based on a Within Outlying Mean Index (WitOMI) showed that under the transient environmental conditions met during the cruise, both vegetative and small cells of D. acuminata colonized the Ria and Mid-shelf subniches, exhibiting good tolerance and extremely high marginality, in particular the small cells. Bottom-up (abiotic) control overwhelmed biological constraints, and shelf waters became a more favourable environment than the Rías. Contrasting higher biotic constraints inside the Rías were found for the small cells, with a subniche possibly controlled by unsuitable physiological status (notwithstanding the higher density) of the vegetative cell population. Results here on behaviour (vertical positioning) and physiological traits (high tolerance but very specialized niche) of D. acuminata give new insights into the ability of this species to remain in the upwelling circulation system. Higher shelf-ría exchanges in the Ría (RP) with more dense and persistent D. acuminata blooms reveal the relevance of transient event-scales and species- and site-specific characteristics to the fate of these blooms. Earlier statements about simple linear relationships between average upwelling intensities and the recurrence of Harmful algae bloom (HAB) events in the Galician Rías Baixas are questioned.

Dinophysis acuminata or Dinophysis acuta: What Makes the Difference in Highly Stratified Fjords?

Dinophysis acuminata and D. acuta, which follows it seasonally, are the main producers of lipophilic toxins in temperate coastal waters, including Southern Chile. Strains of the two species differ in their toxin profiles and impacts on shellfish resources. D. acuta is considered the major cause of diarrhetic shellfish poisoning (DSP) outbreaks in Southern Chile, but there is uncertainty about the toxicity of D. acuminata, and little information on microscale oceanographic conditions promoting their blooms. During the austral summer of 2020, intensive sampling was carried out in two northern Patagonian fjords, Puyuhuapi (PUY) and Pitipalena (PIT), sharing D. acuminata dominance and D. acuta near detection levels. Dinophysistoxin 1 (DTX 1) and pectenotoxin 2 (PTX 2) were present in all net tow samples but OA was not detected. Although differing in hydrodynamics and sampling dates, D. acuminata shared behavioural traits in the two fjords: cell maxima (>103 cells L-1) in the interface (S ~ 21) between the estuarine freshwater (EFW)) and saline water (ESW) layers; and phased-cell division (µ = 0.3-0.4 d-1) peaking after dawn, and abundance of ciliate prey. Niche analysis (Outlying Mean Index, OMI) of D. acuta with a high marginality and much lower tolerance than D. acuminata indicated an unfavourable physical environment for D. acuta (bloom failure). Comparison of toxin profiles and Dinophysis niches in three contrasting years in PUY-2020 (D. acuminata bloom), 2018 (exceptional bloom of D. acuta), and 2019 (bloom co-occurrence of the two species)-shed light on the vertical gradients which promote each species. The presence of FW (S < 11) and thermal inversion may be used to provide short-term forecasts of no risk of D. acuta blooms and OA occurrence, but D. acuminata associated with DTX 1 pose a risk of DSP events in North Patagonian fjords.

Interannual variability in mesoscale distribution of Dinophysis acuminata and D. acuta in Northwestern Patagonian fjords.

Dinophysis acuminata and D. acuta, which produce diarrheogenic toxins and pectenotoxins in southern Chile, display site-specific differences in interannual variability (2006 - 2018) in Reloncaví, Pitipalena and Puyuhuapi fjords (41 - 46 °S), Chilean Patagonia. Linear Models show decreasing trends in rainfall and river discharge. Latitudinal decreasing gradients in SST temperature and vertical salinity gradients were observed. A brackish water layer (FW salinity <11 psu), permanently present in Reloncaví, decreased in thickness with time in Pitipalena and was usually absent in Puyuhuapi, the only fjord where D. acuta reached bloom (>103 cells L‒1) densities every season. Dinophysis acuminata, associated with toxin profiles in shellfish that include only pectenotoxins, bloomed everywhere with a poleward increasing gradient. Absence of the FW layer provides a possible index of risk for D. acuta blooms. An apparent poleward shift of D. acuta populations, responsible for DSP outbreaks in Reloncaví in the 1970s, and the recent EU deregulation of pectenotoxins will have a positive impact on the mussel industry in Los Lagos Region. Changes to ongoing monitoring protocols to improve risk assessment capabilities are suggested.

Lipophilic Toxins in Chile: History, Producers and Impacts.

A variety of microalgal species produce lipophilic toxins (LT) that are accumulated by filter-feeding bivalves. Their negative impacts on human health and shellfish exploitation are determined by toxic potential of the local strains and toxin biotransformations by exploited bivalve species. Chile has become, in a decade, the world's major exporter of mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) and has implemented toxin testing according to importing countries' demands. Species of the Dinophysis acuminata complex and Protoceratium reticulatum are the most widespread and abundant LT producers in Chile. Dominant D. acuminata strains, notwithstanding, unlike most strains in Europe rich in okadaic acid (OA), produce only pectenotoxins, with no impact on human health. Dinophysis acuta, suspected to be the main cause of diarrhetic shellfish poisoning outbreaks, is found in the two southernmost regions of Chile, and has apparently shifted poleward. Mouse bioassay (MBA) is the official method to control shellfish safety for the national market. Positive results from mouse tests to mixtures of toxins and other compounds only toxic by intraperitoneal injection, including already deregulated toxins (PTXs), force unnecessary harvesting bans, and hinder progress in the identification of emerging toxins. Here, 50 years of LST events in Chile, and current knowledge of their sources, accumulation and effects, are reviewed. Improvements of monitoring practices are suggested, and strategies to face new challenges and answer the main questions are proposed.

Identification of 24-O-ß-d-Glycosides and 7-Deoxy-Analogues of Okadaic Acid and Dinophysistoxin-1 and -2 in Extracts from Dinophysis Blooms, Dinophysis and Prorocentrum Cultures, and Shellfish in Europe, North America and Australasia.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-ß-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1-5, the 24-O-ß-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4-6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

Niche differentiation of Dinophysis acuta and D. acuminata in a stratified fjord.

Dinophysis acuta and D. acuminata are associated with lipophilic toxins in Southern Chile. Blooms of the two species coincided during summer 2019 in a highly stratified fjord system (Puyuhuapi, Chilean Patagonia). High vertical resolution measurements of physical parameters were carried out during 48 h sampling to i) explore physiological status (e.g., division rates, toxin content) and ii) illustrate the fine scale distribution of D. acuta and D. acuminata populations with a focus on water column structure and co-occurring plastid-bearing ciliates. The species-specific resources and regulators defining the realized niches (sensu Hutchinson) of the two species were identified. Differences in vertical distribution, daily vertical migration and in situ division rates (with record values, 0.76 d-1, in D. acuta), in response to the environmental conditions and potential prey availability, revealed their niche differences. The Outlying Mean Index (OMI) analysis showed that the realized niche of D. acuta (cell maximum 7 × 103 cells L-1 within the pycnocline) was characterized by sub-surface estuarine waters (salinity 23 - 25), lower values of turbulence and PAR, and a narrow niche breath. In contrast, the realized niche of D. acuminata (cell maximum 6.8 × 103 cells L-1 just above the pycnocline) was characterized by fresher (salinity 17 - 20) outflowing surface waters, with higher turbulence and light intensity and a wider niche breadth. Results from OMI and PERMANOVA analyses of co-occurring microplanktonic ciliates were compatible with the hypothesis of species such as those from genera Pseudotontonia and Strombidium constituting an alternative ciliate prey to Mesodinium. The D. acuta cell maximum was associated with DSP (OA and DTX-1) toxins and pectenotoxins; that of D. acuminata only with pectenotoxins. Results presented here contribute to a better understanding of the environmental drivers of species-specific blooms of Dinophysis and management of their distinct effects in Southern Chile.

Multiscale physical background to an exceptional harmful algal bloom of Dinophysis acuta in a fjord system.

Dinophysis acuta produces diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins (PTX). It blooms in thermally-stratified shelf waters in late summer in temperate to cold temperate latitudes. Despite its major contribution to shellfish harvesting bans, little effort has been devoted to study its population dynamics in Chilean Patagonia. In 2017-2018, mesoscale distribution of harmful algal species (75 monitoring stations) revealed the initiation (late spring) and seasonal growth of a dense D. acuta population in the Aysén region, with maximal values at Puyuhuapi Fjord (PF). Vertical phytoplankton distribution and fine-resolution measurements of physical parameters along a 25-km transect in February 16th identified a 15-km (horizontal extension) subsurface thin layer of D. acuta from 4 to 8 m depth. This layer, disrupted at the confluence of PF with the Magdalena Sound, peaked at the top of the pycnocline (6 m, 15.9 °C, 23.4 psu) where static stability was maximal. By February 22nd, it deepened (8 m, 15.5 °C; 23.62 psu) following the excursions of the pycnocline and reached the highest density ever recorded (664 × 103 cells L-1) for this species. Dinophysis acuta was the dominant Dinophysis species in all microplankton net-tows/bottle samples; they all contained DSP toxins (OA, DTX-1) and PTX-2. Modeled flushing rates showed that Puyuhuapi, the only fjord in the area with 2 connections with the open sea, had the highest water residence time. Long term climate variability in the Southern hemisphere showed the effects of a Southern Annular Mode (SAM) in positive mode (+1.1 hPa) overwhelming a moderate La Niña. These effects included positive spring precipitation anomalies with enhanced salinity gradients and summer drought with positive anomalies in air (+1 °C) and sea surface (+2 °C) temperature. Locally, persistent thermal stratification in PF seemed to provide an optimal physical habitat for initiation and bloom development of D. acuta. Thus, in summer 2018, a favourable combination of meteorological and hydrographic processes of multiple scales created conditions that promoted the development of a widespread bloom of D. acuta with its epicentre at the head of Puyuhuapi fjord.

Structural variation of potentially toxic epiphytic dinoflagellates on Thalassia testudinum from two coastal systems of Colombian Caribbean.

Some benthic dinoflagellates produce toxins that can affect other organisms including humans, and their proliferation seems to be related to the environmental variability. For this reason, the present study aims to compare the structural variation of potentially toxic dinoflagellates associated with the seagrass Thalassia testudinum from two nearby systems, with different environmental characteristics in Colombian Caribbean, corresponding to a brackish water coastal lagoon and an adjacent bay. Between January 2014 and December 2015, leaves of T. testudinum were collected monthly to obtain the dinoflagellates. Salinity, temperature, dissolved oxygen, pH, nutrients and total suspended solids (TSS) were measured, and precipitation data and the Oceanic Niño Index (ONI) were obtained. Dinoflagellates were detached from the leaves, morphologically identified by analyzing their thecal plates arrangements, and quantified using a Sedgewick-Rafter chamber. The information was analyzed using standard statistics and regression models. Fourteen species of potentially toxic epiphytic dinoflagellate belonging to four genera were recorded, being Prorocentrum the most representative in number of species. The maximum density, dominated by P. lima, were found in Bahía Chengue during the rainy season of 2014 (18452 and 20109 cells g-1 w.w.), with salinity of 35.50, high temperatures (>29.60 °C), dissolved oxygen >6 mg L-1, pH close to 8 and TSS >85 mg L-1. Densities at the Lagoon were lower than 80 cells g-1 w.w. with the highest values of Prorocentrum sp.1 under different environmental conditions. With the statistical relationships between the most abundant species and the main environmental variables, fundamental niche models were proposed in which cells could proliferate. The degree of risk to human health due to the presence of these potentially toxic epiphytic dinoflagellates will not be resolved until their toxicity discarded.

Uptake of Inorganic and Organic Nitrogen Sources by Dinophysis acuminata and D. acuta.

Dinoflagellate species of Dinophysis are obligate mixotrophs that require light, nutrients, and prey for sustained growth. Information about their nitrogenous nutrient preferences and their uptake kinetics are scarce. This study aimed to determine the preferred nitrogen sources in cultures of D. acuminata and D. acuta strains from the Galician Rías Baixas (NW Spain) and to compare their uptake kinetics. Well-fed versus starved cultures of D. acuminata and D. acuta were supplied with N15 labeled inorganic (nitrate, ammonium) and organic (urea) nutrients. Both species showed a preference for ammonium and urea whereas uptake of nitrate was negligible. Uptake rates by well-fed cells of D. acuminata and D. acuta were 200% and 50% higher, respectively, than by starved cells. Uptake of urea by D. acuminata was significantly higher than that of ammonium in both nutritional conditions. In contrast, similar uptake rates of both compounds were observed in D. acuta. The apparent inability of Dinophysis to take up nitrate suggests the existence of incomplete nitrate-reducing and assimilatory pathways, in line with the paucity of nitrate transporter homologs in the D. acuminata reference transcriptome. Results derived from this study will contribute to understand Harmful Algal Blooms succession and differences in the spatio-temporal distribution of the two Dinophysis species when they co-occur in stratified scenarios.

Effects of small-scale turbulence on two species of Dinophysis.

Dinoflagellate species of Dinophysis, in particular D. acuminata and D. acuta, produce lipophilic toxins that pose a threat to human health when concentrated in shellfish and jeopardize shellfish exploitations in western Europe. In northwestern Iberia, D. acuminata has a long growing season, from spring to early autumn, and populations develop as soon as shallow stratification forms when the upwelling season begins. In contrast, D. acuta blooms in late summer, when the depth of the pycnocline is maximal and upwelling pulses are moderate. In situ observations on the hydrodynamic regimes during the two windows of opportunity for Dinophysis species led us to hypothesize that D. acuta should be more sensitive to turbulence than D. acuminata. To test this hypothesis, we studied the response of D. acuminata and D. acuta to three realistic turbulence levels low (LT), ε ≈ 10-6 m2 s-3; medium (MT), ε ≈ 10-5 m2 s-3 and high (HT), ε ≈ 10-4 m2 s-3generated by Turbogen, a highly reproducible, computer-controlled system. Cells of both species exposed to LT and MT grew at rates similar to the controls. Marked differences were found in the response to HT: D. acuminata grew slowly after an initial lag phase, whereas D. acuta cell numbers declined. Results from this study support the hypothesis that turbulence may play a role in shaping the spatio-temporal distribution of individual species of Dinophysis. We also hypothesize that, in addition to cell disturbance affecting division, sustained high shear generated by microturbulence may cause a decline in Dinophysis numbers due to decreased densities of ciliate prey.

Cell Cycle, Division Rate, and Feeding of the Heterotroph Phalacroma rotundatum in a Chilean Fjord.

Phalacroma rotundatum is a rare cosmopolitan heterotrophic dinoflagellate. This species, included in the IOC-UNESCO Taxonomic Reference List of Harmful Microalgae, may be a diarrhetic shellfish poisoning (DSP) toxin vector, but little is known about its ecophysiology and behavior. A vertical net haul collected during the austral summer of 2018 in Reloncaví Sound (Chilean Patagonia) revealed an unusually abundant population of P. rotundatum and prompted intensive 24 h sampling on 16-17 January to study the cell cycle and feeding behavior of this species. Hydrographic measurements from a buoy revealed the local characteristic estuarine circulation, with a brackish surface layer (salinity 26-28) separated from saltier, colder bottom waters by a pycnocline at a depth modulated by the tidal regime. A high proportion of P. rotundatum cells were packed with digestive vacuoles (peak of 70% at 14:00), and phased cell division (µ = 0.46 d-1) occurred 3 h after sunrise. The division time (TD) was 2 h. This is the first cell cycle study of P. rotundatum. The results here disagree with those of previous field studies that considered asynchronous division in some Dinophysis species to be related to heterotrophic feeding. They also question the very specific prey requirements, Tiarina fusus, reported for P. rotundatum in northern Europe.

Dinophysis Toxins: Distribution, Fate in Shellfish and Impacts.

Several planktonic dinoflagellate species of the genus Dinophysis produce one or two groups of lipophilic toxins: (i) okadaic acid (OA) and its derivatives, the dinophysistoxins (DTXs), and (ii) pectenotoxins (PTXs) [...].

Mesoscale Dynamics and Niche Segregation of Two Dinophysis Species in Galician-Portuguese Coastal Waters.

Blooms of Dinophysis acuminata occur every year in Galicia (northwest Spain), between spring and autumn. These blooms contaminate shellfish with lipophilic toxins and cause lengthy harvesting bans. They are often followed by short-lived blooms of Dinophysis acuta, associated with northward longshore transport, at the end of the upwelling season. During the summers of 1989 and 1990, dense blooms of D. acuta developed in situ, initially co-occurring with D.acuminata and later with the paralytic shellfish toxin-producer Gymnodiniumcatenatum. Unexplored data from three cruises carried out before, during, and following autumn blooms (13⁻14, 27⁻28 September and 11⁻12 October) in 1990 showed D. acuta distribution in shelf waters within the 50 m and 130 m isobaths, delimited by the upwelling front. A joint review of monitoring data from Galicia and Portugal provided a mesoscale view of anomalies in SST and other hydroclimatic factors associated with a northward displacement of the center of gravity of D. acuta populations. At the microscale, re-examination of the vertical segregation of cell maxima in the light of current knowledge, improved our understanding of niche differentiation between the two species of Dinophysis. Results here improve local transport models and forecast of Dinophysis events, the main cause of shellfish harvesting bans in the most important mussel production area in Europe.

Notes on the Cultivation of Two Mixotrophic Dinophysis Species and Their Ciliate Prey Mesodinium rubrum.

Kleptoplastic mixotrophic species of the genus Dinophysis are cultured by feeding with the ciliate Mesodinium rubrum, itself a kleptoplastic mixotroph, that in turn feeds on cryptophytes of the Teleaulax/Plagioselmis/Geminigera (TPG) clade. Optimal culture media for phototrophic growth of D. acuminata and D. acuta from the Galician Rías (northwest Spain) and culture media and cryptophyte prey for M.rubrum from Huelva (southwest Spain) used to feed Dinophysis, were investigated. Phototrophic growth rates and yields were maximal when D. acuminata and D. acuta were grown in ammonia-containing K(-Si) medium versus f/2(-Si) or L1(-Si) media. Dinophysis acuminata cultures were scaled up to 18 L in a photobioreactor. Large differences in cell toxin quota were observed in the same Dinophysis strains under different experimental conditions. Yields and duration of exponential growth were maximal for M. rubrum from Huelva when fed Teleaulax amphioxeia from the same region, versus T. amphioxeia from the Galician Rías or T. minuta and Plagioselmis prolonga. Limitations for mass cultivation of northern Dinophysis strains with southern M. rubrum were overcome using more favorable (1:20) Dinophysis: Mesodinium ratios. These subtleties highlight the ciliate strain-specific response to prey and its importance to mass production of M. rubrum and Dinophysis cultures.

Comparative ecophysiology of Dinophysis acuminata and D. acuta (DINOPHYCEAE, DINOPHYSIALES): effect of light intensity and quality on growth, cellular toxin content, and photosynthesis.

Dinoflagellates of the genus Dinophysis are the most persistent producers of lipophilic shellfish toxins in Western Europe. Their mixotrophic nutrition requires a food chain of cryptophytes and plastid-bearing ciliates for sustained growth and photosynthesis. In this study, cultures of D. acuminata and D. acuta, their ciliate prey Mesodinium rubrum and the cryptophyte, Teleaulax amphioxeia, were subject to three experimental settings to study their physiological response to different combinations of light intensity and quality. Growth rates, pigment analyses (HPLC), photosynthetic parameters (PAM-fluorometry), and cellular toxin content (LC-MS) were determined. Specific differences in photosynthetic parameters were observed in Dinophysis exposed to different photon fluxes (10-650 µmol photons · m-2  · s-1 ), light quality (white, blue and green), and shifts in light regime. Dinophysis acuta was more susceptible to photodamage under high light intensities (370-650 µmol photons · m-2  · s-1 ) than D. acuminata but survived better with low light (10 µmol photons · m-2  · s-1 ) and to a prolonged period (28 d) of darkness. Mesodinium rubrum and T. amphioxeia showed their maximal growth rate and yield under white and high light whereas Dinophysis seemed better adapted to grow under green and blue light. Toxin analyses in Dinophysis showed maximal toxin per cell under high light after prey depletion at the late exponential-plateau phase. Changes observed in photosynthetic light curves of D. acuminata cultures after shifting light conditions from low intensity-blue light to high intensity-white light seemed compatible with photoacclimation in this species. Results obtained here are discussed in relation to different spatiotemporal distributions observed in field populations of D. acuminata and D. acuta in northwestern Iberia.

Metabolomic Profiles of Dinophysis acuminata and Dinophysis acuta Using Non-Targeted High-Resolution Mass Spectrometry: Effect of Nutritional Status and Prey.

Photosynthetic species of the genus Dinophysis are obligate mixotrophs with temporary plastids (kleptoplastids) that are acquired from the ciliate Mesodinium rubrum, which feeds on cryptophytes of the Teleaulax-Plagioselmis-Geminigera clade. A metabolomic study of the three-species food chain Dinophysis-Mesodinium-Teleaulax was carried out using mass spectrometric analysis of extracts of batch-cultured cells of each level of that food chain. The main goal was to compare the metabolomic expression of Galician strains of Dinophysis acuminata and D. acuta that were subjected to different feeding regimes (well-fed and prey-limited) and feeding on two Mesodinium (Spanish and Danish) strains. Both Dinophysis species were able to grow while feeding on both Mesodinium strains, although differences in growth rates were observed. Toxin and metabolomic profiles of the two Dinophysis species were significantly different, and also varied between different feeding regimes and different prey organisms. Furthermore, significantly different metabolomes were expressed by a strain of D. acuminata that was feeding on different strains of the ciliate Mesodinium rubrum. Both species-specific metabolites and those common to D. acuminata and D. acuta were tentatively identified by screening of METLIN and Marine Natural Products Dictionary databases. This first metabolomic study applied to Dinophysis acuminata and D.acuta in culture establishes a basis for the chemical inventory of these species.

Forecasting the risk of harmful algal blooms.

The "Applied Simulations and Integrated Modelling for the Understanding of Harmful Algal Blooms" (Asimuth) project sought to develop a harmful algal bloom (HAB) alert system for Atlantic Europe. This was approached by combining, at a national or regional level, regulatory monitoring phytoplankton and biotoxin data with satellite remote sensing and other information on current marine conditions, coupled with regional scale models that included a representation of HAB transport. Synthesis of these products was achieved by expert interpretation within HAB risk alert bulletins that were prepared on a regular basis (typically weekly) for use by the aquaculture industry. In this preface to the Asimuth Special Issue we outline the main HAB species of concern in the region and the strengths and limitations of different methodologies to provide early warning of their blooms.