The recent development of biological sensors has extended marine plankton studies from conducting laboratory bench work to in vivo and real-time observations. Flow cytometry (FCM) has shed new light on marine microorganisms since the 1980s through its single-cell approach and robust detection of the smallest cells. FCM records valuable optical properties of light scattering and fluorescence from cells passing in a single file in front of a narrow-collimated light source, recording tens of thousands of cells within a few minutes. Depending on the instrument settings, the sampling strategy, and the automation level, it resolves the spatial and temporal distribution of microbial marine prokaryotes and eukaryotes. Cells are usually classified and grouped on cytograms by experts and are still lacking standards, reducing data sharing capacities. Therefore, the need to make FCM data sets FAIR (Findability, Accessibility, Interoperability, and Reusability of digital assets) is becoming critical. In this paper, we present a consensus vocabulary for the 13 most common marine microbial groups observed with FCM using blue and red-light excitation. The authors designed a common layout on two-dimensional log-transformed cytograms reinforced by a decision tree that facilitates the characterization of groups. The proposed vocabulary aims at standardising data analysis and definitions, to promote harmonisation and comparison of data between users and instruments. This represents a much-needed step towards FAIRification of flow cytometric data collected in various marine environments.

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 C-13 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.

The PHYTOBS dataset includes long-term time series on marine microphytoplankton, since 1987, along the whole French metropolitan coast. Microphytoplankton data cover microscopic taxonomic identifications and counts. The whole dataset is available, it includes 25 sampling locations. PHYTOBS network studies microphytoplankton diversity in the hydrological context along French coasts under gradients of anthropogenic pressures. PHYTOBS network allows to analyse the responses of phytoplankton communities to environmental changes, to assess the quality of the coastal environment through indicators, to define ecological niches, to detect variations in bloom phenology, and to support any scientific question by providing data. The PHYTOBS network provides the scientific community and stakeholders with validated and qualified data, in order to improve knowledge regarding biomass, abundance and composition of marine microphytoplankton in coastal and lagoon waters in their hydrological context. PHYTOBS originates of two networks. The historical REPHY (French Observation and Monitoring program for Phytoplankton and Hydrology in coastal waters) supported by Ifremer since 1984 and the SOMLIT (Service d'observation en milieu littoral) supported by INSU-CNRS since 1995. The monitoring has started in 1987 on some sites and later in others. Hydrological data are provided by REPHY or SOMLIT network as a function of site locations.

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.

In low-nutrient low-chlorophyll areas, such as the Mediterranean Sea, atmospheric fluxes represent a considerable external source of nutrients likely supporting primary production, especially during periods of stratification. These areas are expected to expand in the future due to lower nutrient supply from sub-surface waters caused by climate-driven enhanced stratification, likely further increasing the role of atmospheric deposition as a source of new nutrients to surface waters. Whether plankton communities will react differently to dust deposition in a warmer and acidified environment remains; however, an open question. The potential impact of dust deposition both in present and future climate conditions was investigated in three perturbation experiments in the open Mediterranean Sea. Climate reactors (300 L) were filled with surface water collected in the Tyrrhenian Sea, Ionian Sea and in the Algerian basin during a cruise conducted in the frame of the PEACETIME project in May–June 2017. The experiments comprised two unmodified control tanks, two tanks enriched with a Saharan dust analogue and two tanks enriched with the dust analogue and maintained under warmer (+3 ∘C) and acidified (−0.3 pH unit) conditions. Samples for the analysis of an extensive number of biogeochemical parameters and processes were taken over the duration (3–4 d) of the experiments. Dust addition led to a rapid release of nitrate and phosphate, however, nitrate inputs were much higher than phosphate. Our results showed that the impacts of Saharan dust deposition in three different basins of the open northwestern Mediterranean Sea are at least as strong as those observed previously, all performed in coastal waters. The effects of dust deposition on biological stocks were different for the three investigated stations and could not be attributed to differences in their degree of oligotrophy but rather to the initial metabolic state of the community. Ocean acidification and warming did not drastically modify the composition of the autotrophic assemblage, with all groups positively impacted by warming and acidification. Although autotrophic biomass was more positively impacted than heterotrophic biomass under future environmental conditions, a stronger impact of warming and acidification on mineralization processes suggests a decreased capacity of Mediterranean surface plankton communities to sequester atmospheric CO2 following the deposition of atmospheric particles.

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.

Most tintinnid species have a shortest linear dimension < 50 μm. Hence, a priori, nets of mesh sizes ≥ 50 μm will likely under‐sample most tintinnid species. However, studies often appear (23 since 2015) using sampling with nets of meshes sizes ≥ 50 μm, reporting both tintinnid concentrations, and community composition. How biased are results from using coarse mesh nets? We provide a comparison of whole water vs. net sampling based on fortuitous, that is, unplanned, parallel sampling. Pairs of samples from a standard monitoring station in the Bay of Villefranche (N.W. Mediterranean Sea) taken on 44 dates from 2013 to 2018 were compared. Tintinnids were enumerated in settled material from a water column sample, an integration of six discrete depth samples between 5 and 70 m, prepared for analysis of phytoplankton composition and in material from a plankton net (52 μm mesh) tow from 70 to 0 m, taken the same day. Despite the large confidence limits due to low raw cell counts from whole water samples, cell concentration estimates were about an order of magnitude higher than those from plankton net samples and frequently biomass estimates as well. Community composition also differed. The most common species in whole water samples were small (diameter ≤ 20 μm), and some common forms were absent, or nearly, from the net samples. We show that, while valuable for collecting larger and rarer species, coarse net samples do not yield robust estimations of overall concentrations, nor allow identification of the dominant tintinnid species.

Phycotoxins synthesized by benthic dinoflagellates are known to bioaccumulate in macrofauna and hence represent a risk for human health. However, the presence of toxins synthesized by benthic dinoflagellates in smaller marine organisms than macrofauna has not been considered despite the fact that such small organisms have an important ecological role in the benthic food web. This present study quantified, for the first time, the trophic relationship between benthic dinoflagellates and meiofauna by using stable isotope enriched dinoflagellates during ingestion experiments. Results showed that harpacticoid copepods were not able to discriminate, during ingestion, between the potentially toxic cells of Ostreopsis cf. ovata and the non-toxic cells of Amphidinium cf. carterae, even when another food resource, such as diatoms (e.g. Odontella sp.), was provided (Kruskal Wallis test, p > 0.05).

The effect of flagellate grazing on bacterioplankton community composition, e.g. cell size and diversity, has been well studied in fresh waters, but much less is known for marine systems. We conducted experiments with communities from an oligotrophic bay in the NW Mediterranean Sea; size fractionation was used to alter grazing by flagellates, and incubation in dialysis bags was used to retain otherwise <i>in situ</i> conditions. Grazing reduced bacterial abundance and growth. In contrast to a current model of the effects of grazing, cell size distribution was unimodal and not bimodal. Significant shifts towards larger cell size classes were observed. This is in accordance with the idea that increasing the cell length is a bacterial strategy to reduce susceptibility to grazing by small flagellates. Only weak evidence was found for the complementary strategy of reducing cell size. In all experiments, bacterial community structure as assessed by PCR-based 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) showed significant differences associated with the presence of grazers. Sequence analyses of DGGE bands were used to select probes for catalyzed reporter deposition-fluorescence <i>in situ</i> hybridization analysis for specific taxa. In our experiments, <i>Polaribacter</i> showed patterns of defense against grazing, while <i>Roseobacter</i> was highly susceptible to grazing. Although the effect of grazing may be stronger in freshwater than in oligotrophic marine systems, our data support the hypotheses that (1) increasing the cell size is a bacterial defense strategy against grazing by small flagellates typical in marine systems and (2) grazing influences bacterial community composition.

For the first time, distribution and abundances (cells per gram of fresh macrophyte weight) of potentially toxic benthic dinoflagellates were studied around Guadeloupe (20 sites) and Martinique (six sites) islands (Lesser Antilles, Caribbean Sea). Benthic dinoflagellates were identified at the genus level and cell counts were undertaken on different host species of macroalgae and seagrasses. Abundance values of potentially toxic benthic dinoflagellates were one order of magnitude higher in Guadeloupe than in Martinique. The highest abundances of benthic dinoflagellates were found in the northern part of Guadeloupe Island, while their distribution was more homogeneous in Martinique. Ostreopsis was the dominant genus in Guadeloupe and Martinique. Regarding biotic substrate preferences, Phaeophyceae hosted the highest total abundances of benthic dinoflagellates on both islands, while the lowest total abundances were observed on Ulvophyceae in Guadeloupe and Florideophyceae in Martinique. The genus Gambierdiscus, known as the causal agent of the ciguatera fish poisoning (CFP), developed on all macrophyte groups on both islands without showing any preferences towards biotic substrates. The presence of this potentially harmful dinoflagellate genus in both islands could explain the existence of local cases of CFP in Guadeloupe and Martinique islands.

Is there a mesopelagic protist fauna composed of species different from that of the overlying surface community? Does the mesopelagic community show seasonal changes in abundances and species composition? We addressed these questions by considering three distinct groups in which species identification is relatively unambiguous: tintinnid ciliates, phaeodarian radiolarians, and amphisolenid dinoflagellates. We sampled weekly at 250 m and 30 m depth from January to June a deep-water coastal site characterized by seasonal changes in water column structure; notably, in winter the mixed layer extends down into mesopelagic depths. We found a deep-water community of tintinnid ciliates comprised of forms apparently restricted to deep waters and species also found in the surface layer. This latter group was dominant during the winter mixis period when tintinnid concentrations were highest and subsequently declined with water column stratification. Phaeodarian radiolarians and the amphisolenid dinoflagellates were regularly found in deep samples but were largely absent from surface water samples and showed distinct patterns in the mesopelagic. Phaeodarian radiolarians declined with water column mixing and then increased in concentration with water column stratification whilst amphisolenid dinoflagellates concentrations showed no pattern but species composition varied. We conclude that for all three protists groups there appear to be both distinct mesopelagic forms and seasonal patterns.

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.

Harmful algal blooms are a source of increasing concern within the health, economic and ecological sectors. In the Mediterranean Sea, severe blooms of the benthic dinoflagellate Ostreopsis cf. ovata have been occurring since the beginning of the century, causing human intoxications by inhalation of bio-aerosols or direct contact with cells. The toxicity of this dinoflagellate is attributed to the presence of palytoxin and several of its analogs called ovatoxins, palytoxin being one of the most potent marine toxins. While mass mortalities of marine invertebrates have already been reported in relation with O. cf. ovata blooms, the toxic effects of this dinoflagellate on benthic organisms is still poorly documented. In the present study, laboratory experiments were performed on a meiobenthic copepod (Sarsamphiascus cf. propinquus), which naturally lives on macrophytes in close contact to O. cf. ovata, in order to assess its potential toxic effects on mortality, fecal pellet production (as a proxy of feeding), as well as fecundity and fertility ratios. Both, O. cf. ovata as well as a non-toxic competitive diatom (Licmophora paradoxa), were used as food in the experiments. Regarding acute toxicity evaluation, this copepod proved to be the most tolerant organism to O. cf. ovata reported to date. Nevertheless, its fecundity and fertility ratios were lower when fed with the toxic dinoflagellate, indicating a possible reprotoxic effect. Moreover, although fecal pellet production decreased significantly when the copepod was fed with a mono-diet of O. cf. ovata, epifluorescence microscopy observations revealed the presence of the toxic cells inside the digestive track, hence suggesting that these primary grazers could be a vector of toxins through the marine food web.

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.

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 bio-mass 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%). Picoeu-karyote 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 Pro-chlorococcus 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, Prochloro-coccus 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.

The effect of ocean acidification and changing water conditions on primary (and secondary) marine aerosol emissions is not well understood on a regional or a global scale. To investigate this effect as well as the indirect effect on aerosol that changing biogeochemical parameters can have, ~ 52 m3 pelagic mesocosms were deployed for several weeks in the Mediterranean Sea during both winter pre-bloom and summer oligotrophic conditions and were subjected to various levels of CO2 to simulate the conditions foreseen in this region for the coming decades. After seawater sampling, primary bubble-bursting aerosol experiments were performed using a plunging water jet system to test both chemical and physical aerosol parameters (10–400 nm). Comparing results obtained during pre-bloom and oligotrophic conditions, we find the same four log-normal modal diameters (18.5 ± 0.6, 37.5 ± 1.4, 91.5 ± 2.0, 260 ± 3.2 nm) describing the aerosol size distribution during both campaigns, yet pre-bloom conditions significantly increased the number fraction of the second (Aitken) mode, with an amplitude correlated to virus-like particles, heterotrophic prokaryotes, TEPs (transparent exopolymeric particles), chlorophyll a and other pigments. Organic fractions determined from kappa closure calculations for the diameter, Dp ~ 50 nm, were much larger during the pre-bloom period (64 %) than during the oligotrophic period (38 %), and the organic fraction decreased as the particle size increased. Combining data from both campaigns together, strong positive correlations were found between the organic fraction of the aerosol and chlorophyll a concentrations, heterotrophic and autotrophic bacteria abundance, and dissolved organic carbon (DOC) concentrations. As a consequence of the changes in the organic fraction and the size distributions between pre-bloom and oligotrophic periods, we find that the ratio of cloud condensation nuclei (CCN) to condensation nuclei (CN) slightly decreased during the pre-bloom period. The enrichment of the seawater samples with microlayer samples did not have any effect on the size distribution, organic content or the CCN activity of the generated primary aerosol. Partial pressure of CO2, pCO2, perturbations had little effect on the physical or chemical parameters of the aerosol emissions, with larger effects observed due to the differences between a pre-bloom and oligotrophic environment.

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 cm3. 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.

Identification of dinoflagellates to specific level is sometimes arduous due to high diversity and frequent morphological variability. Focusing on Neoceratium genus, often used as ecological indicator, our collaborative web site provides to scientists an accessible and detailed taxonomic tool, allowing accurate identification of its numerous species and varieties. This original web site offers the possibility to visualize several taxa to avoid confusion between infraspecific taxa of the same species and between morphologically close taxa, belonging to different species.

The main goal of the multidisciplinary MediOs project was to acquire and analyze pertinent scientific knowledge in fields as diverse as ecology, biology, chemistry, epidemiology or socio-economics concerning the occurrence of species belonging to genus Ostreopsis (toxic benthic dinoflagellates) in the Mediterranean. The economic impact of Ostreopsis was estimated, based on the intensity and frequency of blooms. Results obtained allowed science-based recommendations concerning the management of risk, with suggestions to optimize environmental surveys, to initiate a food risk survey, and to structure actions at the national level.