Daily variations of Ostreopsis cf. ovata abundances in NW Mediterranean Sea.

Ostreopsis cf. ovata is a benthic dinoflagellate very common in tropical and temperate coastal areas, particularly in the Mediterranean Sea. This species is also found in the plankton, i.e. swimming in the water column or in aggregates floating at the sea surface. The potential links between the planktonic and benthic populations influencing their relative distribution in the water column and attached to the benthic substrate are poorly understood. To shed light on this question, a high-frequency temporal monitoring was conducted in the Villefranche bay (France) to determine the abundance of (1) epibenthic cells attached to macroalgae, (2) planktonic cells in the water column and (3) cells in aggregates floating at the sea water surface (hereafter, referred to sea surface cells) . This monitoring was realized over 3 consecutive years (2018, 2019 and 2020) and at different phases of the bloom (exponential phase - 2020, peak - 2019 and decline phase - 2018). Strong variations in benthic and planktonic O. cf. ovata abundances were observed over the 24 h sampling cycles conducted in three consecutive years. The three populations, planktonic, benthic and sea surface cells, exhibited the highest numbers during the day (light) hours and lowest values at night in 2018 and 2019. In 2020, however, benthic abundances did not differ significantly between light and dark periods. Moreover, epibenthic cells abundances peaked in the morning, followed by the peak of the cells in the plankton and in the surface aggregates during the afternoon. Monitoring of O. cf. ovata is often based on a single sampling per day without precise indications of sampling time and shows great variability in O. cf. ovata abundances. Our observations of daily variations in cell abundances along the water column clearly indicate that time and water column depth of sampling constitute a great source of variability and have to be considered when designing new monitoring strategies to reduce variability and to harmonize data acquisition and international comparisons.

Subcellular architecture and metabolic connection in the planktonic photosymbiosis between Collodaria (radiolarians) and their microalgae.

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium). We showed that this symbiosis is very dynamic whereby symbionts interact with different host cells via extracellular vesicles within the colony. 3D electron microscopy revealed that the photosynthetic apparatus of the microalgae was more voluminous in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were stored in the symbiotic microalga in starch granules and purine crystals respectively. Nitrogen was also allocated to the algal nucleolus. In the host, low 13 C transfer was detected in the Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (c. 40 ppm) and twofold higher in the host, whereas copper concentration increased in symbionts and was detected in the host cell and extracellular vesicles. Sulfur concentration was around two times higher in symbionts (chromatin and pyrenoid) than their host. This study improves our understanding on the functioning of this oceanic photosymbiosis and paves the way for more studies to further assess its biogeochemical significance.

Atypical Membrane-Anchored Cytokine MIF in a Marine Dinoflagellate.

Macrophage Migration Inhibitory Factors (MIF) are pivotal cytokines/chemokines for vertebrate immune systems. MIFs are typically soluble single-domain proteins that are conserved across plant, fungal, protist, and metazoan kingdoms, but their functions have not been determined in most phylogenetic groups. Here, we describe an atypical multidomain MIF protein. The marine dinoflagellate Lingulodinium polyedra produces a transmembrane protein with an extra-cytoplasmic MIF domain, which localizes to cell-wall-associated membranes and vesicular bodies. This protein is also present in the membranes of extracellular vesicles accumulating at the secretory pores of the cells. Upon exposure to biotic stress, L. polyedra exhibits reduced expression of the MIF gene and reduced abundance of the surface-associated protein. The presence of LpMIF in the membranes of secreted extracellular vesicles evokes the fascinating possibility that LpMIF may participate in intercellular communication and/or interactions between free-living organisms in multispecies planktonic communities.

Efficient, fast and inexpensive bioassay to monitor benthic microalgae toxicity: Application to Ostreopsis species.

Even though HPLC-MS is commonly used to quantify the toxin content of Ostreopsis spp. cells, there is a need to develop easy-to-use toxicological tests to set thresholds during Ostreopsis spp. blooms. The crustacean Artemia has been widely used to evaluate the presence and toxicity of chemicals and biological contaminants and we anticipated that it could also be useful to test Ostreopsis spp. toxicity. Its relevance was first assessed by investigating the variability of the toxic effects among Ostreopsis spp. strains and throughout the dinoflagellate life cycle in combination with chemical analyses of the toxinic content by UHPLC-HRMS. After testing the toxicity of fractions prepared from Ostreopsis spp. cells, the known ova- and paly-toxins were not the only toxic metabolites to Artemia franciscana, indicating that other toxic compounds synthesized by Ostreopsis spp. still remain to be identified. To extend the bioassay to in situ monitoring, the toxicity of the benthic microalgal consortium was tested during a natural bloom of Ostreopsis cf. ovata in the NW Mediterranean Sea. The results highlight the accuracy and sensitivity of the ecotoxicological assay with Artemia franciscana to assess the toxicity of Ostreopsis spp. blooms.

Algal Remodeling in a Ubiquitous Planktonic Photosymbiosis.

Photosymbiosis between single-celled hosts and microalgae is common in oceanic plankton, especially in oligotrophic surface waters. However, the functioning of this ecologically important cell-cell interaction and the subcellular mechanisms allowing the host to accommodate and benefit from its microalgae remain enigmatic. Here, using a combination of quantitative single-cell structural and chemical imaging techniques (FIB-SEM, nanoSIMS, Synchrotron X-ray fluorescence), we show that the structural organization, physiology, and trophic status of the algal symbionts (the haptophyte Phaeocystis) significantly change within their acantharian hosts compared to their free-living phase in culture. In symbiosis, algal cell division is blocked, photosynthesis is enhanced, and cell volume is increased by up to 10-fold with a higher number of plastids (from 2 to up to 30) and thylakoid membranes. The multiplication of plastids can lead to a 38-fold increase of the total plastid volume in a cell. Subcellular mapping of nutrients (nitrogen and phosphorous) and their stoichiometric ratios shows that symbiotic algae are impoverished in phosphorous and suggests a higher investment in energy-acquisition machinery rather than in growth. Nanoscale imaging also showed that the host supplies a substantial amount of trace metals (e.g., iron and cobalt), which are stored in algal vacuoles at high concentrations (up to 660 ppm). Sulfur mapping reveals a high concentration in algal vacuoles that may be a source of antioxidant molecules. Overall, this study unveils an unprecedented morphological and metabolic transformation of microalgae following their integration into a host, and it suggests that this widespread symbiosis is a farming strategy wherein the host engulfs and exploits microalgae.

Allelopathic interactions between the benthic toxic dinoflagellate Ostreopsis cf. ovata and a co-occurring diatom.

For decades the microphytobenthos assemblage in the coastal Mediterranean Sea has been regularly colonized by the toxic benthic dinoflagellate Ostreopsis cf. ovata. This harmful algal species is a toxin producer and occupies the same ecological niche as various diatoms. Surprisingly, there are only few insights reported on the physiological responses of diatoms to blooms of O. cf. ovata The chemical interactions of O. cf. ovata with the co-occurring diatom Licmophora paradoxa was studied using a bioassay (measuring impact of cell-free culture filtrate) and a co-culture approach (separate by a membrane) to investigate the effects of the exometabolome and its mode of action. Bioassays highlighted a toxic effect of the exometabolome of O. cf. ovata on the diatom photosynthetic activity. However, the co-cultures revealed that these toxic effects do not occur through remote allelopathy. Contact or close interactions between cells of the two species is most likely needed to impair the diatom growth. Ovatoxins are suspected to be the toxic metabolites secreted by O. cf. ovata although the current set of data did not give confirmation of this assumption. Interestingly, the exometabolome of L. paradoxa impaired the growth and the photochemistry of O. cf. ovata in both bioassays and co-cultures. Some biomarkers possibly involved for the effect were identified using a metabolomic approach and may correspond to oxylipins, however a bacterial source of the bioactive metabolites is also considered.

Variability of ultraplankton composition and distribution in an oligotrophic coastal ecosystem of the NW Mediterranean Sea derived from a two-year survey at the single cell level.

Ultraplankton [heterotrophic prokaryotes and ultraphytoplankton (<10 µm)] were monitored weekly over two years (2009 & 2010) in a coastal area of the NW Mediterranean Sea. Six clusters were differentiated by flow cytometry on the basis of their optical properties, two heterotrophic prokaryote (HP) subgroups labelled LNA and HNA (low and high nucleic acid content respectively), Prochlorococcus, Synechococcus, autotrophic picoeukaryotes and nanoeukaryotes. HP represented an important component of the microbial assemblage over the survey with relatively small abundance variation through seasons. The carbon biomass ratio HP/ultraphytoplankton averaged 0.45, however this ratio exceeded 1 during spring. Ultraphytoplankton biomass made about 50% of the total autotrophic carbon estimates but this contribution increased up to 97% and 67% during the 2009 and 2010 spring periods respectively. Within ultraphytoplankton, nanoeukaryote represent the most important ultraphytoplankton group in terms of autotrophic carbon biomass (up to 70%). Picoeukaryote maximum abundance occurred in winter. Synechococcus was the most abundant population (maximum 1.2 x 10 5 cells cm-3) particularly in spring where it represented up to 54% of ultraphytoplankton carbon biomass. The warmer winter-spring temperatures and the lengthening of the stratification period created a favorable situation for the earlier appearance of Synechococcus and its persistence throughout summer, paralleling Prochlorococcus development. Prochlorococcus was dominant over summer and autumn with concentrations up to 1.0 × 10 5 cells cm-3. While the abundance of Synechococcus throughout survey was of the same order as that reported in western Mediterranean Sea, Prochlorococcus was more abundant and similar to the more typical oligotrophic and warm waters. The abundance variation of the ultraplankton components through the survey was relatable to variations in the hydrological and nutrient conditions.

Onset of the spring bloom in the northwestern Mediterranean Sea: influence of environmental pulse events on the in situ hourly-scale dynamics of the phytoplankton community structure.

Most of phytoplankton influence is barely understood at the sub meso scale and daily scale because of the lack of means to simultaneously assess phytoplankton functionality, dynamics and community structure. For a few years now, it has been possible to address this objective with an automated in situ high frequency sampling strategy. In order to study the influence of environmental short-term events (nutrients, wind speed, precipitation, solar radiation, temperature, and salinity) on the onset of the phytoplankton bloom in the oligotrophic Bay of Villefranche-sur-Mer (NW Mediterranean Sea), a fully remotely controlled automated flow cytometer (CytoSense) was deployed on a solar-powered platform (EOL buoy, CNRS-Mobilis). The CytoSense carried out single-cell analyses on particles (1-800 µm in width, up to several mm in length), recording optical pulse shapes when analyzing several cm(3). Samples were taken every 2 h in the surface waters during 2 months. Up to 6 phytoplankton clusters were resolved based on their optical properties (PicoFLO, Picoeukaryotes, Nanophytoplankton, Microphytoplankton, HighSWS, HighFLO). Three main abundance pulses involving the 6 phytoplankton groups monitored indicated that the spring bloom not only depends on light and water column stability, but also on short-term events such as wind events and precipitation followed by nutrient pulses. Wind and precipitation were also determinant in the collapse of the clusters' abundances. These events occurred within a couple of days, and phytoplankton abundance reacted within days. The third abundance pulse could be considered as the spring bloom commonly observed in the area. The high frequency data-set made it possible to study the phytoplankton cell cycle based on daily cycles of forward scatter and abundance. The combination of daily cell cycle, abundance trends and environmental pulses will open the way to the study of phytoplankton short-term reactivity to environmental conditions.

Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1.

The TGF-beta family member Nodal is essential for specification of the dorsal-ventral axis of the sea urchin embryo, but the molecular factors regulating its expression are not known. Analysis of the nodal promoter is an excellent entry point to identify these factors and to dissect the regulatory logic driving dorsal-ventral axis specification. Using phylogenetic footprinting, we delineated two regulatory regions located in the 5' region of the nodal promoter and in the intron that are required for correct spatial expression and for autoregulation. The 5' regulatory region contains essential binding sites for homeodomain, bZIP, Oct, Tcf/Lef, Sox and Smad transcription factors, and a binding site for an unidentified spatial repressor possibly related to Myb. Soon after its initiation, nodal expression critically requires autoregulation by Nodal and signaling by the maternal TGF-beta Univin. We show that Univin is related to Vg1, that both Nodal and Univin signal through Alk4/5/7, and that zygotic expression of univin, like that of nodal, is dependent on SoxB1 function and Tcf/beta-catenin signaling. This work shows that Tcf, SoxB1 and Univin play essential roles in the regulation of nodal expression in the sea urchin and suggests that some of the regulatory interactions controlling nodal expression predate the chordates. The data are consistent with a model of nodal regulation in which a maternal TGF-beta acts in synergy with maternal transcription factors and with spatial repressors to establish the dorsal-ventral axis of the sea urchin embryo.