Thierry Jauffrais

Production and isolation of azaspiracid-1 and -2 from Azadinium spinosum culture in pilot scale photobioreactors.

Azaspiracid (AZA) poisoning has been reported following consumption of contaminated shellfish, and is of human health concern. Hence, it is important to have sustainable amounts of the causative toxins available for toxicological studies and for instrument calibration in monitoring programs, without having to rely on natural toxin events. Continuous pilot scale culturing was carried out to evaluate the feasibility of AZA production using Azadinium spinosum cultures. Algae were harvested using tangential flow filtration or continuous centrifugation. AZAs were extracted using solid phase extraction (SPE) procedures, and subsequently purified. When coupling two stirred photobioreactors in series, cell concentrations reached 190,000 and 210,000 cell·mL−1 at steady state in bioreactors 1 and 2, respectively. The AZA cell quota decreased as the dilution rate increased from 0.15 to 0.3 day−1, with optimum toxin production at 0.25 day−1. After optimization, SPE procedures allowed for the recovery of 79 ± 9% of AZAs. The preparative isolation procedure previously developed for shellfish was optimized for algal extracts, such that only four steps were necessary to obtain purified AZA1 and -2. A purification efficiency of more than 70% was achieved, and isolation from 1200 L of culture yielded 9.3 mg of AZA1 and 2.2 mg of AZA2 of >95% purity. This work demonstrated the feasibility of sustainably producing AZA1 and -2 from A. spinosum cultures.

Isolation, Structure Elucidation, Relative LC-MS Response, and in Vitro Toxicity of Azaspiracids from the Dinoflagellate Azadinium spinosum

We identified three new azaspiracids (AZAs) with molecular weights of 715, 815, and 829 (AZA33 (3), AZA34 (4), and AZA35, respectively) in mussels, seawater, and Azadinium spinosum culture. Approximately 700 μg of 3 and 250 μg of 4 were isolated from a bulk culture of A. spinosum, and their structures determined by MS and NMR spectroscopy. These compounds differ significantly at the carboxyl end of the molecule from known AZA analogues and therefore provide valuable information on structure-activity relationships. Initial toxicological assessment was performed using an in vitro model system based on Jurkat T lymphocyte cytotoxicity, and the potencies of 3 and 4 were found to be 0.22- and 5.5-fold that of AZA1 (1), respectively. Thus, major changes in the carboxyl end of 1 resulted in significant changes in toxicity. In mussel extracts, 3 was detected at low levels, whereas 4 and AZA35 were detected only at extremely low levels or not at all. The structures of 3 and 4 are consistent with AZAs being biosynthetically assembled from the amino end.

Dissolved azaspiracids are absorbed and metabolized by blue mussels (Mytilus edulis)

The relationship between azaspiracid shellfish poisoning and a small dinoflagellate, Azadinium spinosum, has been shown recently. The organism produces AZA1 and -2, while AZA3 and other analogues are metabolic products formed in shellfish. We evaluated whether mussels were capable of accumulating dissolved AZA1 and -2, and compared the toxin profiles of these mussels at 24 h with profiles of those exposed to live or lysed A. spinosum. We also assessed the possibility of preparative production of AZA metabolites by exposing mussels to semi-purified AZA1. We exposed mussels to similar concentration of AZAs: dissolved AZA1 + 2 (crude extract) at 7.5 and 0.75 μg L(-1), dissolved AZA1+2 (7.5 μg L(-1)) in combination with Isochrysis affinis galbana, and lysed and live A. spinosum cells at 1 × 10(5) and 1 × 10(4) cell mL(-1) (containing equivalent amounts of AZA1 + 2). Subsequently, we dissected and analysed digestive glands, gills and remaining flesh. Mussels (whole flesh) accumulated AZAs to levels above the regulatory limit, except at the lower levels of dissolved AZAs. The toxin profile of the mussels varied significantly with treatment. The gills contained 42-46% and the digestive glands 23-24% of the total toxin load using dissolved AZAs, compared to 3-12% and 75-90%, respectively, in mussels exposed to live A. spinosum. Exposure of mussels to semi-purified AZA1 produced the metabolites AZA17 (16.5%) and AZA3 (1.7%) after 4 days of exposure, but the conversion efficiency was too low to justify using this procedure for preparative isolation.

Effect of Azadinium spinosum on the feeding behaviour and azaspiracid accumulation of Mytilus edulis

Azadinium spinosum, a small toxic dinoflagellate, was recently isolated and identified as a primary producer of azaspiracid toxins (AZAs). Previous experiments related to AZA accumulation in blue mussels upon direct feeding with A. spinosum revealed increased mussel mortality and had negative effects on the thickness of the digestive gland tubules. Therefore we conducted follow up experiments in order to study effects of A. spinosum on mussel feeding behaviour. Individual assessment of mussel feeding time activity (FTA), clearance rate (CR), filtration rate (TFR), absorption rate (AR), faeces and pseudofaeces production were carried out on mussel fed either toxic (A. spinosum) or non-toxic (Isochrisis aff. galbana (T-Iso)) diets. Furthermore, AZA accumulation and biotransformation in mussels were followed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A. spinosum had a significant effect on mussel feeding behaviour compared to T-Iso: CR was lower by a factor of 6, FTA by a factor of 5, TFR by a factor of 3 and AR even decreased to negative values for the last day of exposure. Even so, a rapid AZA accumulation was observed during the first hours of the trial; less than 6h of feeding were required to reach AZA concentration in mussel above regulatory level. In consistence with physiological observations, AZA concentration of about 200 μg kg(-1) did not increase further until the end of the study. AZA bioconversion was also found to be a fast process: after 3h of exposure AZA17, -19 and AZA7-10 were already found, with a proportion of AZA17 equal to AZA2. These results show a negative effect of A. spinosum on blue mussel feeding activity and indicate a possible regulation of AZA uptake by decreasing filtration and increasing pseudofaeces production.

Effects of Organic and Inorganic Nitrogen on the Growth and Production of Domoic Acid by Pseudo-nitzschia multiseries and P. australis (Bacillariophyceae) in Culture

Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in estuarine and coastal waters reinforces the need to understand better the environmental control of its growth and domoic acid (DA) production. Here, we document Pseudo-nitzschia spp. growth and toxicity on a large set of inorganic and organic nitrogen (nitrate, ammonium, urea, glutamate, glutamine, arginine and taurine). Our study focused on two species isolated from European coastal waters: P. multiseries CCL70 and P. australis PNC1. The nitrogen sources induced broad differences between the two species with respect to growth rate, biomass and cellular DA, but no specific variation could be attributed to any of the inorganic or organic nitrogen substrates. Enrichment with ammonium resulted in an enhanced growth rate and cell yield, whereas glutamate did not support the growth of P. multiseries. Arginine, glutamine and taurine enabled good growth of P. australis, but without toxin production. The highest DA content was produced when P. multiseries grew with urea and P. australis grew with glutamate. For both species, growth rate was not correlated with DA content but more toxin was produced when the nitrogen source could not sustain a high biomass. A significant negative correlation was found between cell biomass and DA content in P. australis. This study shows that Pseudo-nitzschia can readily utilize organic nitrogen in the form of amino acids, and confirms that both inorganic and organic nitrogen affect growth and DA production. Our results contribute to our understanding of the ecophysiology of Pseudo-nitzschia spp. and may help to predict toxic events in the natural environment.

Ostreopsis cf. ovata in the French Mediterranean Coast: Molecular Characterisation and Toxin Profile

Abstract The presence of dinoflagellates of the genus Ostreopsis along Mediterranean coasts was first observed in 1972, in the bay of Villefranche-sur-Mer. However, over the past ten years, harmful events related to this benthic dinoflagellate have been reported in Italian, Spanish, Greek, French, Tunisian and Algerian coastal areas. In France, during a hot period in August 2006, cases of dermatitis and respiratory problems were registered in Marseille area. At that time, a link to the proliferation of Ostreopsis was highlighted for the first time in that area. A specific monitoring was designed and implemented in the summer 2007. Two strains of Ostreopsis cf. ovata, collected in 2008 from Villefranche-sur-Mer and Morgiret coastal waters and grown in culture, were identified by molecular analysis and studied to characterise their growth and toxin profile. Liquid chromatography-mass spectrometry (LC-MS/MS) indicated that both strains produced ovatoxin-a (OVTX-a) as the major component (ca. 90%), and traces of palytoxin (PLTX). Toxin content was determined at the end of the exponential growth phase with highest concentration of 55 pg.cell-1 of OVTX-a and 2.5 pg.cell-1 of PLTX.

Functional xanthophyll cycle and pigment content of a kleptoplastic benthic foraminifer: Haynesina germanica

Some shallow water benthic foraminifera are able to retain functional chloroplasts (kleptoplasts) from their food source, i.e. diatoms. Here we assessed the functionality of the kleptoplast xanthophyll cycle (XC, i.e. the main diatom short-term photo-regulation mechanism) and we surveyed Haynesina germanica kleptoplast pigment composition over time and at different light regimes. Six common diatom lipophilic pigments were detected, two chlorophylls (Chl a, Chl c) and four carotenoids (fucoxanthin and by-products, diadinoxanthin, diatoxanthin and β-carotene), the same pigment profile as the diatom species frequently isolated at the sampling site. The xanthophyll cycle (XC) was functional with kleptoplast diatoxanthin (DT) content increase with concomitant diadinoxanthin (DD) decrease after short term light exposure. DT/(DT+DD) and DT/DD ratios increased significantly in specimens exposed to low light and high light in comparison to specimens maintained in the dark. Specimens placed in very low light after the light treatments reverted to values close to the initial ones, suggesting that H. germanica XC is functional. A functional XC is an indication of H. germanica kleptoplasts capacity for short-term photo-protection from photo-oxidative damages caused by excess of light. Furthermore, the pigment survey suggests that H. germanica preserved some chloroplasts over a longer time than others and that pigment content is influenced by previous light history. Finally, the current study highlighted seasonal differences, with higher pigment contents in winter specimens (27.35 ± 1.30 ng cell-1) and lower in summer specimens (6.08 ± 1.21 ng cell-1), a quantitative and qualitative composition suggesting light acclimation to low or high light availability, according to the season.

Inorganic carbon and nitrogen assimilation in cellular compartments of a benthic kleptoplastic foraminifer

Haynesina germanica, an ubiquitous benthic foraminifer in intertidal mudflats, has the remarkable ability to isolate, sequester, and use chloroplasts from microalgae. The photosynthetic functionality of these kleptoplasts has been demonstrated by measuring photosystem II quantum efficiency and O2 production rates, but the precise role of the kleptoplasts in foraminiferal metabolism is poorly understood. Thus, the mechanism and dynamics of C and N assimilation and translocation from the kleptoplasts to the foraminiferal host requires study. The objective of this study was to investigate, using correlated TEM and NanoSIMS imaging, the assimilation of inorganic C and N (here ammonium, NH4+) in individuals of a kleptoplastic benthic foraminiferal species. H. germanica specimens were incubated for 20 h in artificial seawater enriched with H13CO3− and 15NH4+ during a light/dark cycle. All specimens (n = 12) incorporated 13C into their endoplasm stored primarily in the form of lipid droplets. A control incubation in darkness resulted in no 13C-uptake, strongly suggesting that photosynthesis is the process dominating inorganic C assimilation. Ammonium assimilation was observed both with and without light, with diffuse 15N-enrichment throughout the cytoplasm and distinct 15N-hotspots in fibrillar vesicles, electron-opaque bodies, tubulin paracrystals, bacterial associates, and, rarely and at moderate levels, in kleptoplasts. The latter observation might indicate that the kleptoplasts are involved in N assimilation. However, the higher N assimilation observed in the foraminiferal endoplasm incubated without light suggests that another cytoplasmic pathway is dominant, at least in darkness. This study clearly shows the advantage provided by the kleptoplasts as an additional source of carbon and provides observations of ammonium uptake by the foraminiferal cell.

Kleptoplastidic benthic foraminifera from aphotic habitats: insights into assimilation of inorganic C, N and S studied with sub-cellular resolution: N and S uptake by kleptoplastidic foraminifera

The assimilation of inorganic compounds in foraminiferal metabolism compared to predation or organic matter assimilation is unknown. Here, we investigate possible inorganic-compound assimilation in Nonionellina labradorica, a common kleptoplastidic benthic foraminifer from Arctic and North Atlantic sublittoral regions. The objectives were to identify the source of the foraminiferal kleptoplasts, assess their photosynthetic functionality in light and darkness and investigate inorganic nitrogen and sulfate assimilation. We used DNA barcoding of a ~ 830 bp fragment from the SSU rDNA to identify the kleptoplasts and correlated transmission electron microscopy and nanometre-scale secondary ion mass spectrometry (TEM-NanoSIMS) isotopic imaging to study 13 C-bicarbonate, 15 N-ammonium and 34 S-sulfate uptake. In addition, respiration rate measurements were determined to assess the response of N. labradorica to light. The DNA sequences established that over 80% of the kleptoplasts belonged to Thalassiosira (with 96%-99% identity), a cosmopolitan planktonic diatom. TEM-NanoSIMS imaging revealed degraded cytoplasm and an absence of 13 C assimilation in foraminifera exposed to light. Oxygen measurements showed higher respiration rates under light than dark conditions, and no O2 production was detected. These results indicate that the photosynthetic pathways in N. labradorica are not functional. Furthermore, N. labradorica assimilated both 15 N-ammonium and 34 S-sulfate into its cytoplasm, which suggests that foraminifera might have several ammonium or sulfate assimilation pathways, involving either the kleptoplasts or bona fide foraminiferal pathway(s) not yet identified.

Effect of Dilution Rate on Azadinium spinosum and Azaspiracid (AZA) Production in Pilot Scale Photobioreactors for the Harvest of AZA1 and -2

Azadinium spinosum, a small dinoflagellate has recently been discovered and identified as the primary producer of azaspiracid-1 (AZA) and -2. Since AZA poisoning has been reported following consumption of contaminated shellfish it is important to have these toxins available for toxicological studies, and a sustainable production of AZAs as calibrants in monitoring programs without having to rely on natural events.