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.

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.

First identification of a C9-diol-ester of okadaic acid in Dinophysis acuta from Galician Rías Baixas (NW Spain).

Dinoflagellates of the genus Dinophysis produce okadaic acid (OA) and analogues. Cultures of a strain of Dinophysis acuta isolated from the Galician Rías contained high levels of pectenotoxins but little OA. Isomeric forms of the latter were suspected as the concentration of OA increased after alkaline hydrolysis. High resolution mass spectra of candidate compounds suggest the presence of an OA-C9-diol ester, reported for the first time in Dinophysis acuta.

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.

Morphological and molecular study of the cyanobiont-bearing dinoflagellate Sinophysis canaliculata from the Canary Islands (eastern central Atlantic).

The presence of the benthic dinophysoid dinoflagellate Sinophysis canaliculata has been reported in the Canary Islands (eastern central Atlantic) in live field observations and on fixed macroalgal samples from intertidal ponds (26 sampling sites from El Hierro, Tenerife, Gran Canaria, Fuerteventura and Lanzarote islands). In vivo Sinophysis cells were typically pale pink colored. Light microscopy and scanning electron microscopy micrographs showed a small characteristic narrow hypothecal cut that matched the original description of S. canaliculata. SSU rRNA gene (rDNA) nuclear phylogeny showed that S. canaliculata is closely related to S. microcephalus. Sinophysis specimens displayed cyanobacterial endosymbionts with orange autofluorescence from phycoerythrins. SSU rDNA analyses of the cyanobionts nearly matched a former sequence obtained from S. canaliculata in the Pacific Ocean (Japan). S. canaliculata survived up to 5 months in the original seawater samples. During that period cyanobionts were always present and maintained their orange autofluorescence, although the pink color gradually vanished (<1 month) in most individuals. Molecular similarity of Sinophysis cyanobionts from the Canary Islands and Japanese waters suggest a deterministic relationship, likely a temporary maintenance inside their host via some specific grazing system.

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.