First evidence of the induction of domoic acid production in Pseudo-nitzschia australis by the copepod Temora longicornis from the French coast.

Diatoms of the genus Pseudo-nitzschia are widespread in marine waters. Some of them can produce the toxin domoic acid (DA) which can be responsible for amnesic shellfish poisoning (ASP) when transferred into the food web. These ASP events are of major concern, due to their ecological and socio-economic repercussions, particularly on the shellfish industry. Many studies have focused on the influence of abiotic factors on DA induction, less on the role of biotic interactions. Recently, the presence of predators has been shown to increase DA production in several Pseudo-nitzschia species, in particular in Arctic areas. In order to investigate the relationship between Pseudo-nitzschia species and grazers from the French coast, exposures between one strain of three species (P. australis, P. pungens, P. fraudulenta) and the copepod Temora longicornis were conducted for 5 days. Cellular and dissolved DA content were enhanced by 1,203 % and 1,556 % respectively after the 5-days exposure of P.australis whereas no DA induction was observed in P. pungens and P. fraudulenta. T. longicornis consumed all three Pseudo-nitzschia species. The copepod survival was not related to DA content. This study is an essential first step to better understanding the interactions between planktonic species from the French coast and highlights the potential key role of copepods in the Pseudo-nitzschia bloom events in the temperate ecosystems.

Interactions between Filter-Feeding Bivalves and Toxic Diatoms: Influence on the Feeding Behavior of Crassostrea gigas and Pecten maximus and on Toxin Production by Pseudo-nitzschia.

Among Pseudo-nitzschia species, some produce the neurotoxin domoic acid (DA), a source of serious health problems for marine organisms. Filter-feeding organisms-e.g., bivalves feeding on toxigenic Pseudo-nitzschia spp.-are the main vector of DA in humans. However, little is known about the interactions between bivalves and Pseudo-nitzschia. In this study, we examined the interactions between two juvenile bivalve species-oyster (Crassostrea gigas) and scallop (Pecten maximus)-and two toxic Pseudo-nitzschia species-P. australis and P. fraudulenta. We characterized the influence of (1) diet composition and the Pseudo-nitzschia DA content on the feeding rates of oysters and scallops, and (2) the presence of bivalves on Pseudo-nitzschia toxin production. Both bivalve species fed on P. australis and P. fraudulenta. However, they preferentially filtered the non-toxic Isochrysis galbana compared to Pseudo-nitzschia. The presence of the most toxic P. australis species resulted in a decreased clearance rate in C. gigas. The two bivalve species accumulated DA in their tissues (up to 0.35 × 10-3 and 5.1 × 10-3 µg g-1 for C. gigas and P. maximus, respectively). Most importantly, the presence of bivalves induced an increase in the cellular DA contents of both Pseudo-nitzschia species (up to 58-fold in P. fraudulenta in the presence of C. gigas). This is the first evidence of DA production by Pseudo-nitzschia species stimulated in the presence of filter-feeding bivalves. The results of this study highlight complex interactions that can influence toxin production by Pseudo-nitzschia and accumulation in bivalves. These results will help to better understand the biotic factors that drive DA production by Pseudo-nitzschia and bivalve contamination during Pseudo-nitzschia blooms.

Differential Influence of Life Cycle on Growth and Toxin Production of three Pseudo-nitzschia Species (Bacillariophyceae).

We used a multistrain approach to study the intra- and interspecific variability of the growth rates of three Pseudo-nitzschia species - P. australis, P. fraudulenta, and P. pungens - and of their domoic acid (DA) production. We carried out mating and batch experiments to investigate the respective effects of strain age and cell size, and thus the influence of their life cycle on the physiology of these species. The cell size - life cycle relationship was characteristic of each species. The influence of age and cell size on the intraspecific variability of growth rates suggests that these characteristics should be considered cautiously for the strains used in physiological studies on Pseudo-nitzschia species. The results from all three species do not support the hypothesis of a decrease in DA production with time since isolation from natural populations. In P. australis, the cellular DA content was rather a function of cell size. More particularly, cells at the gametangia stage of their life cycle contained up to six times more DA than smaller or larger cells incapable of sexual reproduction. These findings reveal a link between P. australis life cycle and cell toxicity. This suggest that life cycle dynamics in Pseudo-nitzschia natural populations may influence bloom toxicity.

Inter- and Intra-Specific Transcriptional and Phenotypic Responses of Pseudo-nitzschia under Different Nutrient Conditions.

Untangling the functional basis of divergence between closely related species is a step toward understanding species dynamics within communities at both the evolutionary and ecological scales. We investigated cellular (i.e., growth, domoic acid production, and nutrient consumption) and molecular (transcriptomic analyses) responses to varying nutrient concentrations across several strains belonging to three species of the toxic diatom genus Pseudo-nitzschia. Three main results were obtained. First, strains from the same species displayed similar transcriptomic, but not necessarily cellular, responses to the experimental conditions. It showed the importance of considering intraspecific diversity to investigate functional divergence between species. Second, a major exception to the first finding was a strain recently isolated from the natural environment and displaying contrasting gene expression patterns related to cell motility and domoic acid production. This result illustrated the profound modifications that may occur when transferring a cell from the natural to the in vitro environment and asks for future studies to better understand the influence of culture duration and life cycle on expression patterns. Third, transcriptomic responses were more similar between the two species displaying similar ecology in situ, irrespective of the genetic distance. This was especially true for molecular responses related to TCA cycle, photosynthesis, and nitrogen metabolism. However, transcripts related to phosphate uptake were variable between species. It highlighted the importance of considering both overall genetic distance and ecological divergence to explain functional divergence between species.

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.

Inter and intra-specific growth and domoic acid production in relation to nutrient ratios and concentrations in Pseudo-nitzschia: phosphate an important factor.

The factors responsible for inducing the synthesis of toxins and responses from toxic phytoplankton blooms remain unclear. In this study we compare the influence of genotypic (at both the intra and interspecific levels) and environmental factors (nutrient concentration and ratio) on growth (in terms of cell densities) and domoic acid (DA) production in three Pseudo-nitzschia species: P. australis, P.pungens and P.fradulenta. A strong phosphate effect was detected. More precisely, a low initial concentration in phosphate, even at high initial nitrogen and silicate concentrations, induced the highest DA concentrations and the lowest cell densities in all strains/species studied. In contrast, a low initial concentration of nitrogen and silicate combined, with a higher phosphate concentration resulted in low cell densities, but without high DA production. Inter-species effects were also observed in DA production, where P. australis represented the most toxigenic species of all. Intra-specific variations were only moderate, except for a recently isolated P. australis strain, suggesting the influence of time since isolation on the physiology and DA production of Pseudo-nitzschia species. Overall, the lack of strong interaction between environmental and genotypic factors showed that the various genotypes investigated did not extensively diverge in their ability to respond (in terms of DA production and cell densities) to contrasting nutrient supply.

INFLUENCE OF UV-B RADIATION ON NITROGEN UTILIZATION BY A NATURAL ASSEMBLAGE OF PHYTOPLANKTON.

A 7-day mesocosm experiment was conducted in July 1996 to investigate the effects of ambient UV-B radiation (UVBR) exclusion and two UVBR enhancements above ambient levels on NO3- , NH4+ and urea utilization in a natural plankton community (<240 µm) from the Lower St. Lawrence Estuary. The phytoplankton community was dominated by diatoms during the first 3 days and, afterward, by flagellates and dinoflagellates. The results of 4-h incubations just below the water surface show that, compared with ambient UVBR conditions, UVBR exclusion generally increased NO3- , NH4+ , and urea uptakes. During the last 4 days of the experiment, the percent increase in the specific uptake rate of urea under excluded UVBR conditions varied between 17% and 130% and was a linear function of the ambient UVBR dose removed. During the first 3 days, the phytoplankton community dominated by diatoms was able to withstand UVBR enhancements without any perceptible effect on nitrogen uptake. However, during the post-diatom bloom period, UVBR enhancements resulted in decreases in NO3- , NH4+ , and urea uptake compared with ambient UVBR conditions. The reduction of urea uptake under UVBR enhancements during the last 3 days varied between 23% and 64% and was linearly related to the enhanced UVBR dose. However, the different UVBR treatments did not affect the internal organic nitrogen composition (internal urea, free amino acids, and proteins) of the phytoplankton community experiencing vertical mixing in the mesocosms. The discrepancy between short-term uptake measurements at the surface and long-term effects in the mesocosms emphasizes the importance of vertical mixing on UVBR effects in natural ecosystems. This suggests that an increase in ambient UVBR would have a minimal effect on nitrogen utilization by natural phytoplankton assemblages if these are vertically mixed.