Characterization of toxin-producing strains of Dinophysis spp. (Dinophyceae) isolated from French coastal waters, with a particular focus on the D. acuminata-complex.

Dinoflagellates of the genus Dinophysis are the most prominent producers of Diarrhetic Shellfish Poisoning (DSP) toxins which have an impact on public health and on marine aquaculture worldwide. In particular, Dinophysis acuminata has been reported as the major DSP agent in Western Europe. Still, its contribution to DSP events in the regions of the English Channel and the Atlantic coast of France, and the role of the others species of the Dinophysis community in these areas are not as clear. In addition, species identification within the D. acuminata complex has proven difficult due to their highly similar morphological features. In the present study, 30 clonal strains of the dominant Dinophysis species have been isolated from French coasts including the English Channel (3 sites), the Atlantic Ocean (11 sites) and the Mediterranean Sea (6 sites). Morphologically, strains were identified as three species: D. acuta, D. caudata, D. tripos, as well as the D. acuminata-complex. Sequences of the ITS and LSU rDNA regions confirmed these identifications and revealed no genetic difference within the D. acuminata-complex. Using the mitochondrial gene cox1, two groups of strains differing by only one substitution were found in the D. acuminata-complex, but SEM analysis of various strains showed a large range of morphological variations. Based on geographical origin and morphology, strains of the subclade A were ascribed to 'D. acuminata' while those of the subclade B were ascribed to 'D. sacculus'. Nevertheless, the distinction into two separate species remains questionable and was not supported by our genetic data. The considerable variations observed in cultured strains suggest that physiological factors might influence cell contour and bias identification. Analyses of Dinophysis cultures from French coastal waters using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) revealed species-conserved toxin profiles for D. acuta (dinophysistoxin 2 (DTX2), okadaic acid (OA), pectenotoxin 2 (PTX2)), D. caudata (PTX2) and D. tripos (PTX2), irrespective of geographical origin (Atlantic Ocean or Mediterranean Sea). Within the D. acuminata-complex, two different toxin profiles were observed: the strains of 'D. acuminata' (subclade A) from the English Channel and the Atlantic Ocean contained only OA while strains of 'D. sacculus' (subclade B) from Mediterranean Sea/Atlantic Ocean contained PTX2 as the dominant toxin, with OA and C9-esters also being present, albeit in lower proportions. The same difference in toxin profiles between 'D. sacculus' and 'D. acuminata' was reported in several studies from Galicia (NW- Spain). This difference in toxin profiles has consequences in terms of public health, and consequently for monitoring programs. While toxin profile could appear as a reliable feature separating 'D. acuminata' from 'D. sacculus' on both French and Spanish coasts, this does not seem consistent with observations on a broader geographical scale for the D. acuminata complex, possibly due to the frequent lack of genetic characterization.

Nutrient ratios influence variability in Pseudo-nitzschia species diversity and particulate domoic acid production in the Bay of Seine (France).

The population dynamics of different Pseudo-nitzschia species, along with particulate domoic acid (pDA) concentrations, were studied from May 2012 to December 2013 in the Bay of Seine (English Channel, Normandy). While Pseudo-nitzschia spp. blooms occurred during the two years of study, Pseudo-nitzschia species diversity and particulate domoic acid concentrations varied greatly. In 2012, three different species were identified during the spring bloom (P. australis, P. pungens and P. fraudulenta) with high pDA concentrations (∼1400ngl-1) resulting in shellfish harvesting closures. In contrast, the 2013 spring was characterised by a P. delicatissima bloom without any toxic event. Above all, the results show that high pDA concentrations coincided with the presence of P. australis and with potential silicate limitation (Si:N<1), while nitrate concentrations were still replete. The contrasting environmental conditions between 2012 and 2013 highlight different environmental controls that might favour the development of either P. delicatissima or P. australis. This study points to the key role of Pseudo-nitzschia diversity and cellular toxicity in the control of particulate domoic acid variations and highlights the fact that diversity and toxicity are influenced by nutrients, especially nutrient ratios.

Effect of filtration rate on coal-sand dual-media filter performances for microalgae removal.

This study tested the efficiency of granular filtration using a bilayer sand filter for microalgae removal from culture dilutions ranging from 10,000 to 17,000 cells/mL. The objective is to evaluate the removal capacity of the filter without chemical coagulation. Two filter media, sand and anthracite, with mean grain sizes of 0.395 and 1.2 mm, respectively, were used in constant-flow-rate experiments (down-flow mode) with suspensions containing Heterocapsa triquetra microalga. The conventional rapid filtration which usually operates at a constant rate of approximately 5 m3/m2 h is compared to high-rate filtration. Two filtration velocities (5 and 10 m/h) were investigated with bed depth of 1100 mm. Average microalgal cell removal rates were 90% at 5 m/h and 68% at 10 m/h. Turbidity removal was more than 71% at 5 m/h but just 57% at 10 m/h. Head losses did not increase significantly, and values measured at process end were 32 mbar at 5 m/h and 78 mbar at 10 m/h. Retention probabilities were calculated from experimental data. A theoretical model was used to evaluate the contributions of the different drivers of microalgae removal. Hypotheses are developed on the understanding of change in the mechanisms of retention as a function of filtration velocity.

Photoperiod length paces the temporal orchestration of cell cycle and carbon-nitrogen metabolism in Crocosphaera watsonii.

We analysed the effect of photoperiod length (PPL) (16:8 and 8:16 h of light-dark regime, named long and short PPL, respectively) on the temporal orchestration of the two antagonistic, carbon and nitrogen acquisitions in the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii strain WH8501 growing diazotrophically. Carbon and nitrogen metabolism were monitored at high frequency, and their patterns were compared with the cell cycle progression. The oxygen-sensitive N2 fixation process occurred mainly during the dark period, where photosynthesis cannot take place, inducing a light-dark cycle of cellular C : N ratio. Examination of circadian patterns in the cell cycle revealed that cell division occurred during the midlight period, (8 h and 4 h into the light in the long and short PPL conditions, respectively), thus timely separated from the energy-intensive diazotrophic process. Results consistently show a nearly 5 h time lag between the end of cell division and the onset of N2 fixation. Shorter PPLs affected DNA compaction of C. watsonii cells and also led to a decrease in the cell division rate. Therefore, PPL paces the growth of C. watsonii: a long PPL enhances cell division while a short PPL favours somatic growth (biomass production) with higher carbon and nitrogen cell contents.

Influence of Nutrient Stress on the Relationships between PAM Measurements and Carbon Incorporation in Four Phytoplankton Species.

Two methods of measuring primary production, modulated fluorimetry (PAM) and the traditional carbon incorporation method ((13)C), were compared in four phytoplankton species, two diatoms (Pseudo-nitzschia pungens and Asterionellopsis glacialis), and two dinoflagellates (Heterocapsa sp and Karenia mikimotoï), under N (nitrogen), P (phosphorus) and Si (silicon) limited semi-continuous culture. N and Si-limited cultures showed relatively high quantum efficiency of the PSII (Fv/Fm) values, confirming that Fv/Fm is not a good proxy for nutrient stress in balanced systems, whereas P limitation had a drastic effect on many physiological parameters. In all species, the physiological capacity of phytoplankton cells to acclimate to nutrient limitations led to changes in the cellular biochemical composition and the structure of the photosynthetic apparatus. The observed physiological responses were species and nutrient specific. The values of the chlorophyll-specific absorption cross section (a*) increased with nutrient limitation due to package effect, while the carbon/Chl a ratio was higher under N and P limitations. In diatoms, Si limitation did not affect photosynthesis confirming the uncoupling between Si and carbon metabolisms. In all four species and under all treatments, significant relationships were found between photosynthetic activities, ETR(Chl) (electron transport rate) and P(Chl) (carbon fixation rate) estimated using PAM measurements and (13)C incorporation, showing that the fluorescence technique can reliably be used to estimate carbon fixation by phytoplankton. The relationship between ETR(Chl) and P(Chl) can be described by the shape and the slope of the curve (ΦC.e). Linear relationships were found for dinoflagellates and P. pungens under all treatments. A decrease in ΦC.e was observed under N and P limitation probably due to structural damage to the photosynthetic apparatus. A. glacialis showed a logarithmic relationship in N and P limited conditions, due to the alternative electron flow which takes place to optimise photosynthetic performances under high light and/or nutrient stress.