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Ulva is a green macroalgal genus with rich species diversity and worldwide distribution. While current knowledge on Ulva diversity focuses on temperate regions, genetic and morphological data in tropical and subtropical areas are scarce and the species richness is not clearly defined. The genus is known for its bloom-forming ability that can induce green tides leading to severe environmental and economic damage. In the last two decades, several important blooms of Ulva spp. have occurred in New Caledonia, requiring further investigations to identify the species involved. As knowledge of New Caledonian Ulva diversity is limited, an update to the Ulva spp. inventory in the area is essential. Based on Ulva specimens collected throughout New Caledonia (Grande Terre, Isle of Pines and Loyalty Islands), we (1) reassessed species diversity using species delimitation methods, (2) analysed morpho-anatomical characters to identify species and/or enrich their diagnosis, and (3) reconstructed a multilocus phylogeny (ITS, rbcL, tufA) of the genus. We found 21 secondary species hypotheses (SSHs) among our dataset, from which five were successfully assigned to U. lactuca, U. ohnoi, U. tepida, U. meridionalis and U. taeniata. Ten SSHs were defined as new species for which we provided taxonomic description, and six other SSHs were singletons that will need to be data-enriched for better interpretation. Our concatenated multilocus matrix included 61 Ulva species. Of these, 15 species were found in New Caledonia and were moderately to strongly supported. Among the Ulva species found in New Caledonia, seven are known to be bloom-forming which highlights the need for strict regulation and regular monitoring of water quality, particularly in areas exposed to strong nutrient input where these species can form green tides. Highlights Ulva diversity in New Caledonia was reassessed with 15 species highlighted. Ten new species have been discovered in New Caledonia. Indigenous species caused recent green tides in New Caledonia.

One of the main challenges in coral reef conservation and restoration is the identification of coral populations resilient under global warming. Seascape genomics is a powerful tool to uncover genetic markers potentially involved in heat tolerance among large populations without prior information on phenotypes. Here, we aimed to confirm the role of several candidate heat associated loci identified using this method in driving the phenotypic response of Acropora millepora from New Caledonia to thermal stress. We subjected 7 colonies to a long-term ex-situ heat stress assay (4°C above the Maximum Monthly Mean) and investigated their physiological response along with their Symbiodiniaceae communities and genotypes. Despite having similar thermal histories and associated symbionts, these conspecific individuals differed greatly in their tolerance to heat stress. More importantly, the clustering of individuals based on their alleles at the candidate loci was able to resolve most of the phenotypic variation in heat tolerance. Colonies harboring a higher proportion of beneficial alleles sustained on average lower mortality, higher Symbiodiniaceae/chlorophyll concentrations and photosynthetic efficiency under prolonged heat stress. Together these results support the relevance of seascape genomics to reveal candidate loci for heat adaptation in corals and develop predictive models of heat tolerance.

A Tetraselmis sp. was selected for its antioxidant activity owing to its high lipid peroxidation inhibition capacity. With the aim to monitor culture conditions to improve antioxidant activity, effects of CO2-induced acidification on Tetraselmis growth, elemental composition, photosynthetic parameters and antioxidant activity were determined. Two pH values were tested (6.5 and 8.5) in batch and continuous cultures in photobioreactors. Acidification enhanced cell growth under both culture methods. However, the microalgae physiological state was healthier at pH 8.5 than at pH 6.5. Indeed, photosynthetic parameters measured with pulse amplitude modulated (PAM) fluorometry showed a decrease in the photosystem II (PSII) efficiency at pH 6.5 in batch culture. Yet, with the exception of the PSII recovering capacity, photosynthetic parameters were similar in continuous culture at both pH. These results suggest that lowering pH through CO2-induced acidification may induce a lower conversion of light to chemical energy especially when coupled with N-limitation and/or under un-balanced culture conditions. The highest antioxidant activity was measured in continuous culture at pH 6.5 with an IC50 of 3.44 ± 0.6 μg mL−1, which is close to the IC50 of reference compounds (trolox and α-tocopherol). In addition, the principal component analysis revealed a strong link between the antioxidant activity and the culture method, the photophysiological state and the nitrogen cell quota and C:N ratio of Tetraselmis sp.. These results highlight Tetraselmis sp. as a species of interest for natural antioxidant production and the potential of PAM fluorometry to monitor culture for production of biomass with a high antioxidant activity.

Digital holographic microscopy is an imaging process that encodes the 3D information of a sample into a single 2D hologram. The holographic reconstruction that decodes the hologram is conventionally based on the diffraction formula and involves various iterative steps in order to recover the lost phase information of the hologram. In the past few years, the deep-learning-based model has shown great potential to perform holographic reconstruction directly on a single hologram. However, preparing a large and high-quality dataset to train the models remains a challenge, especially when the holographic reconstruction images that serve as ground truth are difficult to obtain and can have a deteriorated quality due to various interferences of the imaging device. A cycle generative adversarial network is first trained with unpaired brightfield microscope images to restore the visual quality of the holographic reconstructions. The enhanced holographic reconstructions then serve as ground truth for the supervised learning of a U-Net that performs the holographic reconstruction on a single hologram. The proposed method was evaluated on plankton images and could also be applied to achieve super-resolution or colorization of the holographic reconstructions.

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Foraminifera are ubiquitously distributed in marine habitats, playing a major role in marine sediment carbon sequestration and the nitrogen cycle. They exhibit a wide diversity of feeding and behavioural strategies (heterotrophy, autotrophy and mixotrophy), including species with the ability of sequestering intact functional chloroplasts from their microalgal food source (kleptoplastidy), resulting in a mixotrophic lifestyle. The mechanisms by which kleptoplasts are integrated and kept functional inside foraminiferal cytosol are poorly known. In our study, we investigated relationships between feeding strategies, kleptoplast spatial distribution and photosynthetic functionality in two shallow-water benthic foraminifera (Haynesina germanica and Elphidium williamsoni), both species feeding on benthic diatoms. We used a combination of observations of foraminiferal feeding behaviour, test morphology, cytological TEM-based observations and HPLC pigment analysis, with non-destructive, single-cell level imaging of kleptoplast spatial distribution and PSII quantum efficiency. The two species showed different feeding strategies, with H. germanica removing diatom content at the foraminifer’s apertural region and E. williamsoni on the dorsal site. All E. williamsoni parameters showed that this species has higher autotrophic capacity albeit both feeding on benthic diatoms. This might represent two different stages in the evolutionary process of establishing a permanent symbiotic relationship, or may reflect different trophic strategies.

To assess the photophysiological capacity of the recently described Ulva batuffolosa to form blooms in coral reefs, we monitored its biomass and photophysiological capacity with pulse amplitude modulated (PAM) fluorometry over a year period on a coastal barrier reef in New Caledonia, along with temperature and light. Effective and maximum quantum efficiencies of the photosystem II measured on this Ulva species indicated that the algae was in a “good health” all over the year with high quantum efficiencies under either light (Fq’/Fm’) or dark incubated conditions and (Fv/Fm). Photo-acclimation and -regulation status used by this Ulva sp. were driven by seasons (i.e., light and temperature) in the lagoon. Although photo-inhibition was an evidence during the warm period, U. batuffolosa was overall well adapted to tolerate the range of irradiance and temperature that characterized the lagoon over the year, which suggests that photosynthesis is not an impediment to green tides by this species.

Nitrogen availability often limits biological productivity in marine systems, where inorganic nitrogen, such as ammonium is assimilated into the food web by bacteria and photoautotrophic eukaryotes. Recently, ammonium assimilation was observed in kleptoplast-containing protists of the phylum foraminifera, possibly via the glutamine synthetase/glutamate synthase (GS/GOGAT) assimilation pathway imported with the kleptoplasts. However, it is not known if the ubiquitous and diverse heterotrophic protists have an innate ability for ammonium assimilation. Using stable isotope incubations (15N-ammonium and 13C-bicarbonate) and combining transmission electron microscopy (TEM) with quantitative nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, we investigated the uptake and assimilation of dissolved inorganic ammonium by two heterotrophic foraminifera; a non-kleptoplastic benthic species, Ammonia sp., and a planktonic species, Globigerina bulloides. These species are heterotrophic and not capable of photosynthesis. Accordingly, they did not assimilate 13C-bicarbonate. However, both species assimilated dissolved 15N-ammonium and incorporated it into organelles of direct importance for ontogenetic growth and development of the cell. These observations demonstrate that at least some heterotrophic protists have an innate cellular mechanism for inorganic ammonium assimilation, highlighting a newly discovered pathway for dissolved inorganic nitrogen (DIN) assimilation within the marine microbial loop.

Oxygen availability impacts the marine nitrogen cycle at a range of spatial and temporal scales. Invasive organisms have shown to sustainably affect sediment geochemistry and benthic ecology. Nonionella sp. T1 was recently described as an invasive benthic foraminifer in the North Sea region. Here, we demonstrate the impact of this denitrifying species on the foraminifera fauna and the nitrogen cycle of the Gullmar Fjord (Sweden). The foraminifera contribution to benthic denitrification was estimated by coupling living foraminifera micro-distribution, denitrification rate measurement and sedimentary nitrate 2D distribution. Nonionella sp. T1 dominated the foraminifera fauna and could denitrify up to 50–100 % of nitrate porewater in oxygenated bottom waters of the fjord. Contrastingly, at the deepest hypoxic low-nitrate station, denitrifying foraminifera species were scarce and did not contribute to nitrogen removal (~ 5 %). Our study showed that benthic foraminifera can be a major contributor of nitrogen mitigation in oxic coastal ecosystems and should be included in ecological and diagenetic models aiming at understanding biogeochemical cycles coupled to nitrogen.

A Tetraselmis sp. was selected for its antioxidant activity owing to its high lipid peroxidation inhibition capacity. With the aim to monitor culture conditions to improve antioxidant activity, effects of CO

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 13C-bicarbonate, 15N-ammonium and 34S-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 13C 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 15N-ammonium and 34S-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. (Less)

The demand for natural products isolated from microalgae has increased over the last decade and has drawn the attention from the food, cosmetic and nutraceutical industries. Among these natural products, the demand for natural antioxidants as an alternative to synthetic antioxidants has increased. In addition, microalgae combine several advantages for the development of biotechnological applications: high biodiversity, photosynthetic yield, growth, productivity and a metabolic plasticity that can be orientated using culture conditions. Regarding the wide diversity of antioxidant compounds and mode of action combined with the diversity of reactive oxygen species (ROS), this review covers a brief presentation of antioxidant molecules with their role and mode of action, to summarize and evaluate common and recent assays used to assess antioxidant activity of microalgae. The aim is to improve our ability to choose the right assay to assess microalgae antioxidant activity regarding the antioxidant molecules studied.

Haynesina germanica is a coastal benthic foraminifer known to sequester chloroplasts from benthic pennate diatoms. This study investigates its cellular organization, as well as the oxygen consumption and production rates under dark and light conditions. The implications of the sequestered chloroplasts are subsequently discussed at an individual and ecological level. Living specimens collected in Arcachon Basin showed well-preserved chloroplasts in the cytoplasm in observations using transmission electron microscopy. Microelectrodes were used to measure oxygen consumption and production rates in incubated specimens in the laboratory. In the dark, oxygen consumption rates ranged from 662 ± 12 to 1082 ± 59 pmol O 2 ind -1 d -1 . Under illuminated conditions (i.e., about 300 μmol photon -1 m -2 s -1 ), we observed and quantified substantial photosynthetic activity, up to 199 pmol O 2 ind -1 d -1 . It appears that H. germanica combines autotrophic and heterotrophic nutritional modes, thereby reflecting a mixotrophic strategy. In Arcachon Basin, H. germanica potentially uses chloroplasts acquired from microphytobenthos to carry out photosynthesis and produce oxygen and organic matter. Stored chloroplasts could constitute an additional food source during impoverished periods, providing substantial competitive advantage to this species. This putative behavior could also affect the biogeochemistry of the sediment at a microscale by creating oxygenated microniches around individual foraminifera.

International audience; Ammonia tepida is a dominant benthic foraminifer colonizing intertidal mudflat sediments. Horizontal locomotion speeds were monitored using time-lapse image analysis over 6 and 24h. Experimental conditions were based on foraminifera exposed to dry sediment re-suspended in artificial sea water (ASW) without any nutrient addition (condition DS), to combusted sediment re-suspended in in ASW also without any nutrient addition (condition CS), or to combusted sediment re-suspended in ASW enriched with either: nitrate, urea, glucose, soil extract (SE), extracellular polymeric substances (EPS), benthic diatoms (Entomoneis paludosa) or natural microphytobenthic assemblages (MPB). Significant differences were already measured after 6h between A. tepida mean locomotion speeds at the different experimental conditions. However, differences were clearer after 24h where the slowest A. tepida mean locomotion speed was measured in specimens placed in CS (1.00±0.30mmh−1) and the highest mean locomotion speed in DS (2.99±0.22mmh−1). Three different groups were defined according to their locomotion speed, (1) foraminifera exposed to DS had a locomotion speed significantly higher than all other conditions, (2) foraminifera placed in conditions enriched in SE, Glucose, Urea and EPS had intermediary locomotion speeds (1.8–2.5mmh−1), and (3) conditions with foraminifera showing the lowest locomotion speeds (1–1.6mmh−1) were CS, nitrate, MPB and E. paludosa. Thus, foraminifera exposed to organic matter (DS, SE, Glucose and Urea) showed faster locomotion speeds than foraminifera exposed to inorganic matter (CS, nitrate) or live preys (E. paludosa, MPB).Dissolved organic matter enrichment enhanced foraminifera locomotion speed, which might be a behavioural response to satisfy their carbon and/or nitrogen requirements, and the lowest locomotion speed observed when feeding on live preys might be a consequence of longer time required for live prey phagocytosis.

The aim of this work is to document the complex nutritional strategy developed by kleptoplastic intertidal foraminifera. We study the mixotrophic ability of a common intertidal foraminifer, Elphidium williamsoni, by (i) investigating the phylogenetic identity of the foraminiferal kleptoplasts, (ii) following their oxygenic photosynthetic capacity and (iii) observing the modification in cellular ultrastructural features in response to photoautotrophic conditions. This was achieved by coupling molecular phylogenetic analyses and TEM observations with non-destructive measurements of kleptoplast O2 production over a 15-day experimental study. Results show that the studied E. williamsoni actively selected kleptoplasts mainly from pennate diatoms and had the ability to produce oxygen, up to 13.4 nmol O2 cell-1 d-1, from low to relatively high irradiance over at least 15 days. Ultrastructural features and photophysiological data showed significant differences over time, the number of lipid droplets, residual bodies and the dark respiration increased; whereas, the number of kleptoplasts decreased accompanied by a minor decrease of the photosynthetic rate. These observations suggest that in E. williamsoni kleptoplasts might provide extra carbon storage through lipid droplets synthesis and highlight the complexity of E. williamsoni feeding strategy and the necessity of further dedicated studies regarding mechanisms developed by kleptoplastidic foraminifera for carbon partitioning and storage.

This study presents a new gel based technique to describe the pore water ammonium distribution through the sediment-water interface in two dimensions at a millimeter scale. The technique is an adaptation of the classical colorimetric method based on the Berthelot's reaction. After the thin film of the gel probe was equilibrated by diffusion either in standard solutions or in pore waters, a colorimetric reagent gel was set on the gel probe, allowing development of the characteristic green color. A flatbed scanner and subsequent densitometry image analysis allowed to determine the concentration distribution of ammonium. The gel probe was tested in the laboratory for two media, deionized water and seawater, within the range 0–3000 μM in NH4+. Detection limit is about 20 μM and accuracy about ±25 μM. The field validation was realized in a tidal mudflat of the French Atlantic coast by comparison with conventional pore water extraction and colorimetric analysis. The large range of concentrations and its applicability in continental and marine sediments suggest a wide range of applications of the technique for a reasonable cost., Graphical abstract Image 1, Highlights • Fast and inexpensive ammonium 2D imaging through the sediment water interface. • Millimetric spatial resolution. • Applicable in marine and fresh aquatic systems.

Some benthic foraminifera have the ability to incorporate functional chloroplasts from diatoms (kleptoplasty). Our objective was to investigate chloroplast functionality of two benthic foraminifera (Haynesina germanica and Ammonia tepida) exposed to different irradiance levels (0, 25, 70 µmol photon m−2 s−1) using spectral reflectance, epifluorescence observations, oxygen evolution and pulse amplitude modulated (PAM) fluorometry (maximum photosystem II quantum efficiency (Fv/Fm) and rapid light curves (RLC)). Our results clearly showed that H. germanica was capable of using its kleptoplasts for more than 1 week while A. tepida showed very limited kleptoplastic ability with maximum photosystem II quantum efficiency (Fv/Fm = 0.4), much lower than H. germanica and decreasing to zero in only 1 day. Only H. germanica showed net oxygen production with a compensation point at 24 µmol photon m−2 s−1 and a production up to 1000 pmol O2 cell−1 day−1 at 300 µmol photon m−2 s−1. Haynesina germanica Fv/Fm slowly decreased from 0.65 to 0.55 in 7 days when kept in darkness; however, it quickly decreased to 0.2 under high light. Kleptoplast functional time was thus estimated between 11 and 21 days in darkness and between 7 and 8 days at high light. These results emphasize that studies about foraminifera kleptoplasty must take into account light history. Additionally, this study showed that the kleptoplasts are unlikely to be completely functional, thus requiring continuous chloroplast resupply from foraminifera food source. The advantages of keeping functional chloroplasts are discussed but more information is needed to better understand foraminifera feeding strategies.

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

International audience; Assimilation, sequestration and maintenance of foreign chloroplasts inside an organism is termed “chloroplast sequestration” or “kleptoplasty”. This phenomenon is known in certain benthic foraminifera, in which such kleptoplasts can be found both intact and functional, but with different retention times depending on foraminiferal species. In the present study, seven species of benthic foraminifera (Haynesina germanica, Elphidium williamsoni, E. selseyense, E. oceanense, E. aff. E. crispum, Planoglabratella opercularis and Ammonia sp.) were collected from shallow-water benthic habitats and examined with the transmission electron microscope (TEM) for cellular ultrastructure to ascertain attributes of kleptoplasts. Results indicate that all these foraminiferal taxa actively obtain kleptoplasts but organized them differently within their endoplasm. In some species, the kleptoplasts were evenly distributed throughout the endoplasm (e.g., H. germanica, E. oceanense, Ammonia sp.), whereas other species consistently had plastids distributed close to the external cell membrane (e.g., Elphidium williamsoni, E. selseyense, P. opercularis). Chloroplast degradation also seemed to differ between species, as many degraded plastids were found in Ammonia sp. and E. oceanense compared to other investigated species. Digestion ability, along with different feeding and sequestration strategies may explain the differences in retention time between taxa. Additionally, the organization of the sequestered plastids within the endoplasm may also suggest behavioral strategies to expose and/or protect the sequestered plastids to/from light and/or to favor gas and/or nutrient exchange with their surrounding habitats.

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.

Benthic diatoms are the dominant microalgae in intertidal mudflats and are in constant interaction with their surrounding bacteria. This study was designed to investigate the effect of bacteria on growth, biomass, elemental (C & N) and biochemical composition, and extracellular polymeric substances (EPS) excretion by two marine benthic diatoms, Halamphora coffeaeformis and Entomoneis paludosa . The experiments were conducted on diatom cultures previously exposed or not to antibiotics. The treatment with antibiotics caused a decrease of bacterial abundance from 24 to fewer than 1 bacteria per algal cell. In non-treated cultures of E . paludosa and H . coffeaeformis , the bacteria phylogenetic affiliation was equally distributed between Bacteroidetes (Flavobacteriia) and Proteobacteria (alpha- and gammaproteobacteria). After treatment with antibiotics, the residual bacterial community was ~ 37% Flavobacteriia ( Winogragskyella genus), 34% for the alphaproteobacteria (mainly Roseibacterium sp and Antarctobacter sp.) and 29% for the gammaproteobacteria (mainly Methylophaga sp. and Stenotrophomonas sp.). Growth of H . coffeaeformis and E . paludosa in non-treated cultures was enhanced by the abundance of the associated bacteria, with mean growth rate of 1 day − 1 compared to 0.7 for antibiotic treated cultures. In E . paludosa , maximal cell abundance was higher in the presence of bacteria while the final carbon biomass did not vary, but in H . coffeaeformis maximal cell abundance did not vary significantly while final carbon biomass was higher in the presence of bacteria. By contrast, for both diatoms, cellular content of protein and lipids decrease significantly, as did extracellular carbon (EPS fraction) in the presence of bacteria. However, only a minor effect was observed on cellular carbohydrates, C/N ratio, and pigments (Chl a ). Diatoms carbon fluxes towards the main biochemical components were also modified, with the protein carbon fraction significantly lower relative to other carbon compounds in the presence of high bacterial biomass. These results showed the complex interactions between diatoms and their associated bacteria. Promotion of diatoms growth by the presence of bacteria appears linked to change in microalgae biochemical composition that will modify the biofilm. Our results might help understanding the regulation of benthic biota in mudflat ecosystems.

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 μmol m−2 s−1 to 250 μmol 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.

Benthic diatoms are dominant primary producers in intertidal flats. This study investigated the effect of different nitrogen sources and concentrations on Entomoneis paludosa growth and physiological responses. Six nitrogen sources either inorganic (nitrate and ammonium) or organic (urea, arginine, glutamine and glycine) supplied at two concentrations (40 and 400 µM-N) induced significant effects on growth, carbon, nitrogen, pigment content and maximum PSII quantum efficiency (F v /F m). Entomoneis paludosa grew under all nitrogen sources albeit showing differences in lag phase, growth rate and cell yield. Inorganic nitrogen, urea and arginine induced higher growth, whereas glycine did not support high biomass. F v /F m showed variability dependent on nitrogen source and C/N ratio. F v /F m varied between 0.55 and 0.65 at 400 μM-N with the highest values observed in glycine, glutamine and urea, whereas nitrate, ammonium and arginine induced lower F v /F m. All cellular components decreased in the 40 µM-N treatments, with nitrogen and pigments being lower than carbon content. Light-harvesting pigment ratios Chl c/Chl a and photoprotective pigment ratios (diatoxanthin + diadinoxanthin)/Chl a increased, while fucoxanthin/Chl a ratios were unaffected by N-limitations. Entomoneis paludosa was capable of quickly adapting and using a wide variety of nitrogen sources. This adaptability may contribute to microphytobenthos diatom ecological success in mudflat ecosystems.

Some benthic foraminifera have the ability to incorporate functional chloroplasts from diatoms (kleptoplasty). Our objective was to investigate chloroplast functionality of two benthic foraminifera (Haynesina germanica and Ammonia tepida) exposed to different irradiance levels (0, 25, 70 μmol photon m-2 s-1) using spectral reflectance, epifluorescence observations, oxygen evolution and pulse amplitude modulated (PAM) fluorometry. Our results clearly showed that H. germanica was capable of using its kleptoplasts for more than one week while A. tepida showed very limited kleptoplastic ability with maximum photosystem II quantum efficiency (Fv/Fm = 0.4), much lower than H. germanica and decreasing to zero in only one day. Only H. germanica showed net oxygen production with a compensation point at 24 μmol photon m-2 s-1 and a production up to 1000 pmol O2 cell-1 day-1 at 300 μmol photon m-2 s-1. Haynesina germanica Fv/Fm slowly decreased from 0.65 to 0.55 in 7 days when kept in darkness; however, it quickly decreased to 0.2 under high light. Kleptoplast functional time was thus estimated between 11 and 21 days in darkness and between 7 and 8 days at high light. These results emphasize that studies about foraminifera kleptoplasty must take into account light history. Additionally, this study showed that the kleptoplasts are unlikely to be completely functional, thus requiring continuous chloroplast resupply from foraminifera food source. The advantages of keeping functional chloroplasts are discussed but more information is needed to better understand foraminifera feeding strategies.

Full text not available from this repository. The following URI links to the publishers’ website where the full text version of this article can be found: http://www.sciencedirect.com/science/article/pii/S156898831100076X, peer-reviewed, Azaspiracids (AZAs) are a group of lipophilic polyether compounds first detected in Ireland which have been implicated in shellfish poisoning incidents around Europe. These toxins regularly effect shellfish mariculture operations including protracted closures of shellfish harvesting areas for human consumption. The armoured dinoflagellate Azadinium spinosum Elbrächter et Tillmann gen. et sp. nov. (Dinophyceae) has been described as the de novo azaspiracid toxin producer; nonetheless the link between this organism and AZA toxin accumulation in shellfish has not yet been established. In August 2009, shellfish samples of blue mussel (Mytilus edulis) from the Southwest of Ireland were analysed using liquid chromatography–tandem-mass spectrometry (LC–MS/MS) and were found to be above the regulatory limit (0.16 μg g−1 AZA-equiv.) for AZAs. Water samples from this area were collected and one algal isolate was identified as A. spinosum and was shown to produce azaspiracid toxins. This is the first strain of A. spinosum isolated from Irish waters. The Irish A. spinosum is identical with the other two available A. spinosum strains from Scotland (3D9) and from Denmark (UTHE2) in its sequence of the D1–D2 regions of the LSU rDNA. A 24 h feeding trial of blue mussels (M. edulis) using an algal suspension of the Irish A. spinosum culture at different cell densities demonstrated that A. spinosum is filtered, consumed and digested directly by mussels. Also, LC–MS/MS analysis had shown that AZAs were accumulating in the shellfish hepatopancreas. The toxins AZA1 and -2 were detected in the shellfish together with the AZA analogues AZA3, AZA6, AZA17 and -19 suggesting that AZA1 and -2 are metabolised in the shellfish within the first 24 h after ingestion of the algae. The levels of AZA17 detected in the shellfish hepatopancreas (HP) were equivalent to the levels of AZA1 but in the remainder tissues the levels of AZA17 were four to five times higher than that of AZA1, only small quantities of AZA3 and -19 were present with negligible amounts of AZA6 detected after the 24 h period. This could have implications in the future monitoring of these toxins given that at present according to EU legislation only AZA1–AZA3 is regulated for. This is the first report of blue mussels’ (M. edulis) feeding on the azaspiracid producing algae A. spinosum from Irish waters.

Intertidal mudflats are important feeding areas for migrating and wintering shorebird species. Partial release of organic and inorganic nutrients through bird excretion may enrich the nutrients flow of mudflat ecosystems, especially in areas with a low flushing rate. The objective of the present work was to experimentally evaluate the effect of Dunlin droppings on the growth and biochemical composition of the microphytobenthic diatom, Entomoneis paludosa (W. Smith) Reimer. Different nutritional conditions were tested to investigate the impact on E. paludosa growth rate, biomass, elemental (CHN) and biochemical composition (lipids, proteins, carbohydrates, pigments). Various culture media were used going from plain artificial seawater to F/2 culture media containing variable nitrate concentrations (0; 50 or 882 μM-NO3) to which bird dropping extract was added or not. The faeces extracts contained inorganic nitrogen (9.1 μM-NH4), inorganic phosphorus (8.2 μM-PO4), traces of silicate (0.2 μM-Si), organic nitrogen in the form of urea (16 μM-N) and other dissolved organic nitrogen (120 μM-N). Faeces extract inartificial seawater was sufficient to sustain E. paludosa growth (up to 6.8 cell divisions in 9 days). A significant growth rate increase (+ 20%) and higher biomasses were observed when faeces extract was added to inorganic media enriched with 50 μM-NO3. Bird droppings had a significant effect in E. paludosa final biochemical composition with the addition of faeces extract to a culture medium containing 50 μM-NO3 increasing E. paludosa protein content and decreasing carbohydrate content. Pigment content per cell increased with the addition of bird dropping extract but ratios of light-harvesting and photo-protective pigments to chlorophyll a were unaffected. E. paludosa grown with faeces extract showed high cellular nitrogen and carbon contents, close to those obtained when cells were grown in F/2 medium. This study showed that shorebird droppings, through the addition of dissolved material, can significantly affect microphytobenthic diatom growth and biochemical composition.

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 mu g of 3 and 250 mu g 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.