Impact of atmospheric deposition on the dynamics of ultraphytoplanktonic populations in the Gulf of Gabès during an intense dust event (MERITE-HIPPOCAMPE campaign).

The ultraphytoplankton composition and dynamics were assessed during a Saharan dust event occurring off the southern Tunisian coasts during the MERITE-HIPPOCAMPE Trans-Mediterranean oceanographic cruise. The composition of atmospheric dust was characterized in terms of nutriments and trace metals. Data-assimilative hydrodynamic model revealed no differences in the hydrological features along the sampling track and almost no water transport occurred during the period of atmospheric deposition. Dust deposition increased the growth rates and the productivity of the major phytoplanktonic cytometric groups, resulting in the highest surface biomass along the Mediterranean transect. One group, distinguished by low fluorescence and nanoplanktonic size, reacted to dust deposition within hours, exhibiting the highest growth rate and net productivity. The dust composition showed a substantial enrichment with organic phosphorous representing (56 % of Total phosphorus) and trace metals mainly Fe, Mn and V.

Ultraphytoplankton community structure in subsurface waters along a North-South Mediterranean transect.

Here we assessed the subsurface ultraphytoplanktonic (< 10 µm) community along a North-South round-trip Mediterranean transect as part of a MERITE-HIPPOCAMPE cruise campaign in April-May 2019. Temperature, salinity, and nutrient concentrations in subsurface waters (2-5 m depth) were also measured along the transect. The subsurface ultraphytoplankton community structure was resolved with a spatial resolution of few kilometers and temporal resolution of 30-min intervals using automated pulse shape recording flow cytometry. The subsurface waters were clustered into seven areas based on temperature and salinity characteristics. Synechococcus were by far the most abundant group in all prospected zones, and nanoeukaryotes were the main biomass component, representing up to 51 % of ultraphytoplanktonic carbon biomass. Apparent net primary productivity (NPP) followed a decreasing gradient along the transect from north to south and was mostly sustained by Synechococcus in all zones. These findings are likely to have implications in terms of the trophic transfer of contaminants in planktonic food webs, as they highlight the potential role of nanoplankton in contaminants bioaccumulation processes and the potential role of Synechococcus in a likely transfer via grazing activities.

Mercury stable isotopes constrain atmospheric sources to the ocean.

Human exposure to toxic mercury (Hg) is dominated by the consumption of seafood1,2. Earth system models suggest that Hg in marine ecosystems is supplied by atmospheric wet and dry Hg(II) deposition, with a three times smaller contribution from gaseous Hg(0) uptake3,4. Observations of marine Hg(II) deposition and Hg(0) gas exchange are sparse, however5, leaving the suggested importance of Hg(II) deposition6 ill-constrained. Here we present the first Hg stable isotope measurements of total Hg (tHg) in surface and deep Atlantic and Mediterranean seawater and use them to quantify atmospheric Hg deposition pathways. We observe overall similar tHg isotope compositions, with median Δ200Hg signatures of 0.02‰, lying in between atmospheric Hg(0) and Hg(II) deposition end-members. We use a Δ200Hg isotope mass balance to estimate that seawater tHg can be explained by the mixing of 42% (median; interquartile range, 24-50%) atmospheric Hg(II) gross deposition and 58% (50-76%) Hg(0) gross uptake. We measure and compile additional, global marine Hg isotope data including particulate Hg, sediments and biota and observe a latitudinal Δ200Hg gradient that indicates larger ocean Hg(0) uptake at high latitudes. Our findings suggest that global atmospheric Hg(0) uptake by the oceans is equal to Hg(II) deposition, which has implications for our understanding of atmospheric Hg dispersal and marine ecosystem recovery.

Surface ocean microbiota determine cloud precursors.

One pathway by which the oceans influence climate is via the emission of sea spray that may subsequently influence cloud properties. Sea spray emissions are known to be dependent on atmospheric and oceanic physicochemical parameters, but the potential role of ocean biology on sea spray fluxes remains poorly characterized. Here we show a consistent significant relationship between seawater nanophytoplankton cell abundances and sea-spray derived Cloud Condensation Nuclei (CCN) number fluxes, generated using water from three different oceanic regions. This sensitivity of CCN number fluxes to ocean biology is currently unaccounted for in climate models yet our measurements indicate that it influences fluxes by more than one order of magnitude over the range of phytoplankton investigated.

Nanoplanktonic diatoms are globally overlooked but play a role in spring blooms and carbon export.

Diatoms are one of the major primary producers in the ocean, responsible annually for ~20% of photosynthetically fixed CO2 on Earth. In oceanic models, they are typically represented as large (>20 µm) microphytoplankton. However, many diatoms belong to the nanophytoplankton (2-20 µm) and a few species even overlap with the picoplanktonic size-class (<2 µm). Due to their minute size and difficulty of detection they are poorly characterized. Here we describe a massive spring bloom of the smallest known diatom (Minidiscus) in the northwestern Mediterranean Sea. Analysis of Tara Oceans data, together with literature review, reveal a general oversight of the significance of these small diatoms at the global scale. We further evidence that they can reach the seafloor at high sinking rates, implying the need to revise our classical binary vision of pico- and nanoplanktonic cells fueling the microbial loop, while only microphytoplankton sustain secondary trophic levels and carbon export.

Evolution of the scattering properties of phytoplankton cells from flow cytometry measurements.

After the exponential growth phase, variability in the scattering efficiency of phytoplankton cells over their complete life cycle is not well characterised. Bulk measurements are impacted by senescent cells and detritrus. Thus the analysis of the evolution of the optical properties thanks to their morphological and/or intra-cellular variations remains poorly studied. Using the Cytosense flow cytometer (CytoBuoy b.v., NL), the temporal course of the forward and sideward efficiencies of two phytoplankton species (Thalassiosira pseudonana and Chlamydomonas concordia) were analyzed during a complete life-cycle. These two species differ considerably from a morphological point of view. Over the whole experiment, the forward and sideward efficiencies of Thalassiosira pseudonana were, on average, respectively 2.2 and 1.6 times higher than the efficiencies of Chlamydomonas concordia. Large intra-species variability of the efficiencies were observed over the life cycle of the considered species. It highlights the importance of considering the optical properties of phytoplankton cells as a function of the population growth stage of the considered species. Furthermore, flow cytometry measurements were combined with radiative transfer simulations and biogeochemical and optical measurements. Results showed that the real refractive index of the chloroplast is a key parameter driving the sideward signal and that a simplistic two-layered model (cytoplasm-chloroplast) seems particularly appropriate to represent the phytoplankton cells.

Determination of backscattering cross section of individual particles from cytometric measurements: a new methodology.

A methodology is developed to derive the backscattering cross section of individual particles as measured with the CytoSense (CytoBuoy b.v., NL). This in situ flow cytometer detects light scatter in forward and sideward directions and fluorescence in various spectral bands for a wide range of particles. First, the weighting functions are determined for the forward and sideward detectors to take into account their instrumental response as a function of the scattering angle. The CytoSense values are converted into forward and sideward scattering cross sections. The CytoSense estimates of uniform polystyrene microspheres from 1 to 90 µm are compared with Mie computations. The mean absolute relative differences ΔE are around 33.7% and 23.9% for forward and sideward scattering, respectively. Then, a theoretical relationship is developed to convert sideward scattering into backscattering cross section, from a synthetic database of 495,900 simulations including homogeneous and multi-layered spheres. The relationship follows a power law with a coefficient of determination of 0.95. To test the methodology, a laboratory experiment is carried out on a suspension of silica beads to compare backscattering cross section as measured by the WET Labs ECO-BB9 and derived from CytoSense. Relative differences are between 35% and 60%. They are of the same order of magnitude as the instrumental variability. Differences can be partly explained by the fact that the two instruments do not measure exactly the same parameter: the cross section of individual particles for the CytoSense and the bulk cross section for the ECO-BB9.

Consequence of a sudden wind event on the dynamics of a coastal phytoplankton community: an insight into specific population growth rates using a single cell high frequency approach.

Phytoplankton is a key component in marine ecosystems. It is responsible for most of the marine primary production, particularly in eutrophic lagoons, where it frequently blooms. Because they are very sensitive to their environment, the dynamics of these microbial communities has to be observed over different time scales, however, assessment of short term variability is often out of reach of traditional monitoring methods. To overcome these limitations, we set up a Cytosense automated flow cytometer (Cytobuoy b.v.), designed for high frequency monitoring of phytoplankton composition, abundance, cell size, and pigment content, in one of the largest Mediterranean lagoons, the Berre lagoon (South-Eastern France). During October 2011, it recorded the cell optical properties of 12 groups of pico-, nano-, and microphytoplankton. Daily variations in the cluster optical properties were consistent with individual changes observed using microscopic imaging, during the cell cycle. We therefore used an adaptation of the size-structured matrix population model, developed by Sosik et al. (2003) to process the single cell analysis of the clusters and estimate the division rates of 2 dinoflagellate populations before, during, and after a strong wind event. The increase in the estimated in situ daily cluster growth rates suggest that physiological changes in the cells can prevail over the response of abundance.

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.

Functional analysis and classification of phytoplankton based on data from an automated flow cytometer.

Analytical flow cytometry (FCM) is well suited for the analysis of phytoplankton communities in fresh and sea waters. The measurement of light scatter and autofluorescence properties of particles by FCM provides optical fingerprints, which enables different phytoplankton groups to be separated. A submersible version of the CytoSense flow cytometer (the CytoSub) has been designed for in situ autonomous sampling and analysis, making it possible to monitor phytoplankton at a short temporal scale and obtain accurate information about its dynamics. For data analysis, a manual clustering is usually performed a posteriori: data are displayed on histograms and scatterplots, and group discrimination is made by drawing and combining regions (gating). The purpose of this study is to provide greater objectivity in the data analysis by applying a nonmanual and consistent method to automatically discriminate clusters of particles. In other words, we seek for partitioning methods based on the optical fingerprints of each particle. As the CytoSense is able to record the full pulse shape for each variable, it quickly generates a large and complex dataset to analyze. The shape, length, and area of each curve were chosen as descriptors for the analysis. To test the developed method, numerical experiments were performed on simulated curves. Then, the method was applied and validated on phytoplankton cultures data. Promising results have been obtained with a mixture of various species whose optical fingerprints overlapped considerably and could not be accurately separated using manual gating.

Phytoplankton distribution during two contrasted summers in a Mediterranean harbour: combining automated submersible flow cytometry with conventional techniques.

Automated in situ flow cytometry, high-pressure liquid chromatography (HPLC), optical microscopy and fluorometry were combined to monitor phytoplankton over two summer periods (2005 and 2006). In 2006, temperature was higher and nutrients lower than in 2005, generating differences in the phytoplankton assemblages (i.e., abundance and structure). Pigment-size classes based on daily HPLC analysis provided evidence for higher proportions of picoplankton and nanoplankton with higher biomass in 2005 and a dominance of microplankton with lower biomass in 2006, the latter with lower specific diversity, as evidenced by weekly microscopy analyses. Total chlorophyll a estimations from fluorometry measurements recorded every 30 min were higher in 2005 than in 2006, as for the HPLC chlorophyll a concentrations. An automated in situ flow cytometer (Thyssen et al., J Plankton Res 30(9):1027-1040, 2008a) sampled seawater every 30 min. Data analysis yielded the resolution of seven clusters based on light scatter and fluorescence. In 2006, an increase in abundance of the largest cells was observed, confirming pigment and microscopy data. The results suggest that the ecosystem was on a constant renewing process in summer 2005 due to a strong wind event and on a highly productive and recycling way in summer 2006 due to stratification of the upper water layer. Automated submersible flow cytometry confirms to be a powerful tool providing high-resolution data by monitoring phytoplankton at the single cell level. This technology gives access to the shape of the light scatter and fluorescence signals generated by each cell passing through a laser beam and that are linked to size, structure and pigment content of the target cell. When combined with conventional techniques, it further improves our understanding of phytoplankton assemblages.

Relationship among several key cell cycle events in the developmental cyanobacterium Anabaena sp. strain PCC 7120.

When grown in the absence of a source of combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 develops, within 24 h, a differentiated cell type called a heterocyst that is specifically involved in the fixation of N(2). Cell division is required for heterocyst development, suggesting that the cell cycle could control this developmental process. In this study, we investigated several key events of the cell cycle, such as cell growth, DNA synthesis, and cell division, and explored their relationships to heterocyst development. The results of analyses by flow cytometry indicated that the DNA content increased as the cell size expanded during cell growth. The DNA content of heterocysts corresponded to the subpopulation of vegetative cells that had a big cell size, presumably those at the late stages of cell growth. Consistent with these results, most proheterocysts exhibited two nucleoids, which were resolved into a single nucleoid in most mature heterocysts. The ring structure of FtsZ, a protein required for the initiation of bacterial cell division, was present predominantly in big cells and rarely in small cells. When cell division was inhibited and consequently cells became elongated, little change in DNA content was found by measurement using flow cytometry, suggesting that inhibition of cell division may block further synthesis of DNA. The overexpression of minC, which encodes an inhibitor of FtsZ polymerization, led to the inhibition of cell division, but cells expanded in spherical form to become giant cells; structures with several cells attached together in the form of a cloverleaf could be seen frequently. These results may indicate that the relative amounts of FtsZ and MinC affect not only cell division but also the placement of the cell division planes and the cell morphology. MinC overexpression blocked heterocyst differentiation, consistent with the requirement of cell division in the control of heterocyst development.

The bactericidal effect of TiO2 photocatalysis involves adsorption onto catalyst and the loss of membrane integrity.

The bactericidal effect of photocatalysis with TiO2 is well recognized, although its mode of action is still poorly characterized. It may involve oxidation, as illuminated TiO2 generates reactive oxygen species. Here we analyze the bactericidal effect of illuminated TiO2 in NaCl-KCl or sodium phosphate solutions. We found that adsorption of bacteria on the catalyst occurred immediately in NaCl-KCl solution, whereas it was delayed in the sodium phosphate solution. We also show that the rate of adsorption of cells onto TiO2 is positively correlated with its bactericidal effect. Importantly, adsorption was consistently associated with a reduction or loss of bacterial membrane integrity, as revealed by flow cytometry. Our work suggests that adsorption of cells onto aggregated TiO2, followed by loss of membrane integrity, is key to the bactericidal effect of photocatalysis.