Beta-N-Methylamino-L-Alanine: LC-MS/MS optimization, screening of cyanobacterial strains and occurrence in shellfish from Thau, a French Mediterannean lagoon

International audienceβ-N-methylamino-L-alanine (BMAA) is a neurotoxic non-protein amino acid suggested to be involved in neurodegenerative diseases. It was reported to be produced by cyanobacteria, but also found in edible aquatic organisms, thus raising concern of a widespread human exposure. However, the chemical analysis of BMAA and its isomers are controversial, mainly due to the lack of selectivity of the analytical methods. Using factorial design, we have optimized the chromatographic separation of underivatized analogues by a hydrophilic interaction chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) method. A combination of an effective solid phase extraction (SPE) clean-up, appropriate chromatographic resolution and the use of specific mass spectral transitions allowed for the development of a highly selective and sensitive analytical procedure to identify and quantify BMAA and its isomers (in both free and total form) in cyanobacteria and mollusk matrices (LOQ of 0.225 and 0.15 μg/g dry weight, respectively). Ten species of cyanobacteria (six are reported to be BMAA producers) were screened with this method, and neither free nor bound BMAA could be found, while both free and bound DAB were present in almost all samples. Mussels and oysters collected in 2009 in the Thau Lagoon, France, were also screened, and bound BMAA and its two isomers, DAB and AEG, were observed in all samples (from 0.6 to 14.4 μg/g DW), while only several samples contained quantifiable free BMAA

Impact of granular filtration on ultrafiltration membrane performance as pre-treatment to seawater desalination in presence of algal blooms

To mitigate fouling of the ultrafiltration (UF) membrane and improve permeate quality, we coupled granular filters (GF) with UF membrane as a pre-treatment for reconstituted seawater in the presence of algal bloom. Mono and bilayer granular filtrations were led at a mean velocity of 10 m h−1 over a 7-hour period. Both GF gave the same algal cell retention rate (∼63%) after 7 hours of filtration. Turbidity reduction rate was 50% for the monolayer filter and 75% for the bilayer filter. Resulting organic matter removal rate was 10% for the monolayer filter and 35% for the bilayer filter. Dissolved organic carbon removal was low (20%) with the bilayer filter and non-existent with the monolayer filter. GF-coupled UF reduced humic acids in the permeate (20%) compared with UF alone. Peak pressure of 3 bars was reached at the end of 30 minutes of UF in both direct UF or UF after monolayer GF. The filtrate from the bilayer GF enables UF over a longer period (7 hours)

Effect of environmental and nutritional factors on growth and azaspiracid production of the dinoflagellate Azadinium spinosum

Azadinium spinosum, a small dinoflagellate isolated from the North Sea, is a producer of azaspiracids (AZAs), a group of biotoxins associated with human illness following ingestion of contaminated shellfish. Using batch and continuous cultures of A. spinosum, the present study investigated the effects of different environmental and nutritional factors (salinity, temperature, photon flux density, aeration, culture media, nitrogen sources, phosphate source, and N/P ratios) on growth, maximum cell concentration, and AZA cell quota. Azadinium spinosum grew in a wide range of conditions; from 10˚ C to 26˚ C and salinities from 30 to 40, under irradiances ranging from 50 mmol m�2 s�1 to 250 mmol m�2 s�1, with or without aeration. Growth and maximum cell concentration were highest at a salinity of 35, at temperatures between 18˚ C and 22˚ C, and with aeration. Concerning AZA cell quota, the most significant effect was observed at low temperature; the AZA cell quota was more than 20 times higher at 10˚ C (220 fg cell�1) than at temperatures between 18˚ C and 26˚ C. A. spinosum grew on all media tested with only slight differences in growth rate and AZA cell quota. In continuous culture, lowering the concentration of nutrients (0.5 strength of a modified K-medium) in the inflow improved AZA cell quota whereas higher concentration (doubling the normal strength of K-medium) improved maximal cell concentration. A. spinosum grew on different sources of nitrogen tested (nitrate, urea, ammonium) with almost no effect on toxin cell quota and growth, except that adding ammonium caused a decrease in growth. These first experiments on Azadinium spinosum increased our knowledge on factors affecting its growth and toxin production; furthermore, these results allowed and improved particularly A. spinosum production in pilot scale photobioreactors for AZA isolation

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 × 105 and 1 × 104 cell mL-1. Subsequently, we dissected and analysed digestive glands, gills and remaining flesh. Mussels (whole flesh) accumulated AZAs 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 a short-term salinity stress on the growth, biovolume, toxins, osmolytes and metabolite profiles on three strains of the Dinophysis acuminata-complex (Dinophysis cf. sacculus)

International audienceDinophysis is the main dinoflagellate genus responsible for diarrheic shellfish poisoning (DSP) in human consumers of filter feeding bivalves contaminated with lipophilic diarrheic toxins. Species of this genus have a worldwide distribution driven by environmental conditions (temperature, irradiance, salinity, nutrients etc.), and these factors are sensitive to climate change. The D. acuminata-complex may contain several species, including D. sacculus. The latter has been found in estuaries and semi-enclosed areas, water bodies subjected to quick salinity variations and its natural repartition suggests some tolerance to salinity changes. However, the response of strains of D. acuminata-complex (D. cf. sacculus) subjected to salinity stress and the underlying mechanisms have never been studied in the laboratory. Here, a 24 h hypoosmotic (25) and hyperosmotic (42) stress was performed in vitro in a metabolomic study carried out with three cultivated strains of D. cf. sacculus isolated from the French Atlantic and Mediterranean coasts. Growth rate, biovolume and osmolyte (proline, glycine betaine and dimethylsulfoniopropionate (DMSP)) and toxin contents were measured. Osmolyte contents were higher at the highest salinity, but only a significant increase in glycine betaine was observed between the control (35) and the hyperosmotic treatment. Metabolomics revealed significant and strain-dependent differences in metabolite profiles for different salinities. These results, as well as the absence of effects on growth rate, biovolume, okadaic acid (OA) and pectenotoxin (PTXs) cellular contents, suggest that the D. cf. sacculus strains studied are highly tolerant to salinity variations

Cyclic imine toxins survey in coastal european shellfish samples: Bioaccumulation and mode of action of 28-O-palmitoyl ester of pinnatoxin-G. first report of portimine-A bioaccumulation

International audienceCyclic imine toxins exhibit fast acting neurotoxicity and lethality by respiratory arrest in mice explained by their potent antagonistic activity against muscular nicotinic acetylcholine receptors. We performed a survey of gymnodimine-A, 13-desmethyl spirolide-C, 13,19-didesmethyl spirolide-C, 20-methyl spirolide-G, pinnatoxin-A, pinnatoxin-G, portimine-A and 28-O-palmitoyl ester of pinnatoxin-G in 36 shellfish samples collected in coastal areas of 8 European countries using a microplate receptor binding assay and UPLC-MS/MS for toxin identification and quantification. The major toxins found in these samples were pinnatoxin-G, 20-methyl spirolide-G, 13-desmethyl spirolide-C, gymnodimine-A and portimine-A. Traces of 13,19-didesmethyl spirolide-C, pinnatoxin-A and 28-O-palmitoyl ester of pinnatoxin-G were also detected. The rapid death of mice was correlated with higher pinnatoxin-G concentrations in mussel digestive gland extracts injected intraperitoneally. Our survey included nontoxic control samples that were found to contain moderate to trace amounts of several cyclic imine toxins. Shellfish may bioaccumulate not only cyclic imine toxins but also a large number of acyl derivatives as a product of metabolic transformation of these neurotoxins. This is the first report in which portimine-A and 28-O-palmitoyl ester of pinnatoxin-G were detected in shellfish extracts from digestive glands of mussels collected in Ingril lagoon. The bioaccumulation of portimine-A is particularly of concern because it is cytotoxic and is able to induce apotosis. The mode of action of 28-O-palmitoyl ester of pinnatoxin-G was studied by receptor binding-assay and by two-electrode voltage clamp electrophysiology. The antagonistic behavior of the acylated pinnatoxin-G towards nicotinic acetylcholine receptor of muscle type is shown here for the first time. Since cyclic imine toxins are not regulated further monitoring of these emerging toxins is needed to improve evidence gathering of their occurrence in shellfish commercialized for human consumption in Europe given their potent antagonism against muscle and neuronal nicotinic acetylcholine receptors

Gut passage times in two bivalve molluscs fed toxic microalgae:

The occurrence of new phytoplankton species in a coastal area may be explained by the import of shellfish containing whole live algal cells in their digestive tracts. Indeed, shellfish containing toxic algal cells can induce both primary contaminations in safe areas (initially free from toxic microalgae), and secondary contaminations of other shellfish growing in the same area via the faeces of the imported animals. To mitigate this problem, shellfish need to be placed in a separate holding tank and their intestinal content purged. For a deeper understanding of the risks associated with transferring contaminated shellfish, oysters (Crassostrea gigas) and mussels (Mytilus edulis) were purposely fed either Alexandrium minutum or A. catenella (Dinophyceae) or Pseudo-nitzschia calliantha (Bacillariophyceae) toxic algae for 2 h. They were then transferred into individual tanks where they were continuously fed with a non-toxic alga, Tetraselmis suecica. Biodeposit production, faeces composition, and filtration rates were monitored for the shellfish over a 6-h period. The effect of temperature differences and different initial toxic algae concentrations were compared. This study revealed a relationship between temperature and cell lysis in the oyster digestive tract. It also indicated that toxic algae concentration did not seem to influence gut passage time in oysters, while a significant effect was observed in mussels, and confirmed the existence of a difference between oyster and mussel feeding patterns

DataSheet_1_Advection and Composition of Dinophysis spp. Populations Along the European Atlantic Shelf.pdf

The main objective was to study relationships between the regional biogeography of Dinophysis species and water masses circulation along the European Atlantic coast. Hydrodynamic connectivities were estimated with a Lagrangian approach. Available and validated physical hindcasts from regional hydrodynamical models, with different resolutions were used. The target area is the Bay of Biscay (NE Atlantic) and connectivity was evaluated between a set of spatially distributed stations and during temporally specified periods. Different indexes related to connectivity properties such as mean, median, most frequent transit times were calculated. To illustrate the dispersion pattern, a molecular approach was jointly set-up to describe the species composition of this genus. At the seasonal scale, a high connectivity within the Bay of Biscay was observed with a slight northward connectivity from Galicia coastal waters to the Shelf of the Bay of Biscay. By comparison to the connectivity between shelf waters of French Brittany and English Channel waters, a higher connectivity between shelf waters of French Brittany and the Celtic Sea shelf was observed. The species mixing in the Bay of Biscay from Galicia waters to the Celtic Sea was confirmed by the genetic analyses despite the absence of Dinophysis sacculus in natural samples. The molecular methodology developed for this work, permitting at least the description of the species composition, also highlights, at the European scale, an unexpected low genetic variability which echoes the complex taxonomic classification inside the genus and the difficulties encountered by national monitoring programs to reach a taxonomic resolution at species level. It is now necessary to start some monitoring at the species level before realizing mid- or long-term forecasts.</p