Isotopic and biochemical trophic markers reveal the complexity of interactions at the base of pelagic food webs (Mediterranean sea).

To gain insight into the impact of bottom-up changes in the plankton community on planktivorous fish in the context of the decline of small pelagic fisheries in the Northwestern Mediterranean Sea, we have conducted an extensive year-long study. The investigation combined biochemical analyses (proteins, carbohydrates, and lipids) with C and N stable isotope analyses (SIA) to simultaneously study phytoplankton, zooplankton, and eight planktivorous fish species (Engraulis encrasicolus, Sardina pilchardus, Sardinella aurita, Sprattus sprattus, Cepola macrophthalma, Chromis chromis, Boops boops, and Spicara maena). This study is the first to analyze both stable isotope and biochemical compositions in coastal particulate organic matter (POM) size classes (0.7-2.7 µm, 2.7-20 µm, and 20-200 µm), zooplankton size classes (200-300 µm, 300-500 µm, 500-1000 µm, 1000-2000 µm, and >2000 µm), and taxonomic groups. We demonstrated that: (1) POM stable isotope compositions varied based on its spatial origin, the taxonomic composition of its biota, and its biochemical content; (2) δ15N values increased with zooplankton size classes and groups, indicating different trophic levels; (3) Phytoplankton exhibited a lipid-rich composition (∼55%), while zooplankton and fish muscles were protein-rich (∼61% and ∼66%, respectively). Bayesian stable isotope mixing models revealed that, on average: (1) POM from oceanic waters contributed the most to the POM in the bay (>51%), with a dominance of pico-POM (∼43%); (2) The 200-1000 µm zooplankton primarily consumed nano-POM, the 1000-2000 µm zooplankton mostly consumed micro-POM (∼64%), and the >2000 µm zooplankton also mostly consumed micro-POM; (3) Mesozooplankton (200-2000 µm) constituted the main portion (∼42%) of the diet for planktivorous fish species, while macrozooplankton organisms (>2000 µm) were the primary food resource (∼43%) for both B. boops and S. sprattus. Our study underscores the complexity of the pelagic food web and highlights the bottom-up transfer of organic matter from the smallest phytoplankton size fractions to planktivorous fish.

Assessing the bioavailability of black carbon-derived dissolved organic matter for marine heterotrophic prokaryotes.

Here we investigated the bioavailability of black carbon (BC)-derived dissolved organic matter (DOM) for a natural mixed community of marine heterotrophic prokaryotes. We ran an in vitro biodegradation experiment that took place over 3 months and exposed a community of organisms collected in the northwestern Mediterranean Sea (Bay of Marseille, France) to three different soluble fractions of BC prepared in the laboratory from various fossil fuel combustion particulates: standard diesel (DREF), oxidized diesel (DREF-OX), and natural samples of ship soot (DSHIP). Over the course of the three months, we observed significant decreases in the concentrations of dissolved organic carbon (DOC; from 9 to 21 %), dissolved BC (DBC; from 22 to 38 %) and dissolved polycyclic aromatic hydrocarbons (d-PAH; from 24 to 64 %) along with variability in the growth dynamics and activity of the heterotrophic prokaryotic community. The heterotrophic prokaryotic community exposed to DREF-OX treatment showed the highest values of respiration and production and the highest cell abundance, associated with the highest decrease in DOC (21 %) and d-PAH (64 %) concentrations. In the DREF and DSHIP treatments, prokaryotic activity was oriented towards anabolism. DREF treatment led to the highest decrease in DBC concentration (38 %). DSHIP treatment, which presented a substantially different d-PAH and dissolved metals content to the other two treatments, showed the lowest decreases in DOC, DBC and d-PAH concentrations, as well as the lowest prokaryotic activity and biomasses. Our results indicate that BC-derived DOM, including the most condensed fraction of this material, is partly bioavailable and therefore likely to be assimilated by marine prokaryotes. The origin of BC/soot deposited at the ocean surface turns out to be a key parameter that dictates the efficiency of biodegradation of its dissolved fraction by heterotrophic prokaryotes.

Impact of sterilization methods on dissolved trace metals concentrations in complex natural samples: Optimization of UV irradiation.

Sterilization is essential for discriminating biotic responses from abiotic reactions in laboratory experiments investigating biogeochemical processes of complex natural samples. However, the conventional methods used to effectively sterilize materials or culture media do not allow sterilizing complex natural samples while maintaining biogeochemical balances. The aim of this study was to develop a low-cost and easy-to-use method to obtain geochemically unmodified and sterilized samples from complex lacustrine or coastal marine ecosystems. In preliminary assays, the impact of several sterilization methods (autoclaving, chemical poisoning, microwave, UV irradiation) on the trace metals balances was studied using borosilicate glass (BG), fluorinated ethylene-propylene (FEP) or polyethylene terephthalate (PET) bottles. Unlike other methods, UV sterilization had minor effects on the distribution of dissolved trace metals. Additional tests using complex lacustrine and coastal marine samples under 10 g/L sediments were performed using a homemade UV sterilization chamber designed to simultaneously irradiate a large number samples. Results showed: •very reproducible UV tests in BG and FEP bottles•faster sterilization using FEP bottles than using BG bottles•low variations of dissolved trace metals concentrations, except for Al, Cu, Fe and Zn.

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.

Spatio-Temporal Variations of Marine Biofilm Communities Colonizing Artificial Substrata Including Antifouling Coatings in Contrasted French Coastal Environments.

Surface colonization in seawater first corresponds to the selection of specific microbial biofilm communities. By coupling flow cytometry, microscopy and high throughput sequencing (HTS, 454 pyrosequencing) with artificial surfaces and environmental analyses, we intend to identify the contribution of biofilm community drivers at two contrasted French sites, one temperate and eutrophic (Lorient, Atlantic coast) and the other at a mesotrophic but highly contaminated bay (Toulon, North-Western Mediterranean Sea). Microbial communities were shaped by high temperatures, salinity and lead at Toulon by but nutrients and DOC at Lorient. Coatings including pyrithione exhibited a significant decrease of their microbial densities except for nanoeukaryotes. Clustering of communities was mainly based on the surface type and secondly the site, whereas seasons appeared of less importance. The in-depth HTS revealed that γ- and α-proteobacteria, but also Bacteroidetes, dominated highly diversified bacterial communities with a relative low ß-diversity. Sensitivity to biocides released by the tested antifouling coatings could be noticed at different taxonomic levels: the percentage of Bacteroidetes overall decreased with the presence of pyrithione, whereas the α/γ-proteobacteria ratio decreased at Toulon when increased at Lorient. Small diatom cells (Amphora and Navicula spp.) dominated on all surfaces, whereas site-specific sub-dominant taxa appeared clearly more sensitive to biocides. This overall approach exhibited the critical significance of surface characteristics in biofilm community shaping.

Assessing ultraphytoplankton and heterotrophic prokaryote composition by flow cytometry in a Mediterranean lagoon.

In the eutrophic Ghar El Melh Lagoon (GML, Tunisia), the distribution of heterotrophic prokaryotes, pico- and nanophytoplankton was studied at five stations in November 2012 at the single cell level, along with environmental factors. Flow cytometry analysis of ultraplankton (<10 µm) resolved (i) two heterotrophic prokaryote groups, low and high nucleic acid contents (LNA and HNA, respectively), and (ii) eight to nine ultraphytoplankton groups (cryptophyte-like cells, two nanoeukaryote subgroups, two picoeukaryote subgroups and three Synechococcus-like cells subgroups). Prochlorococcus was not detected. According to redundancy analysis (RDA), a significant difference was found in the distribution of the ultraplankton between stations (F = 2.61, p < 0.05); maximum proliferations of heterotrophic prokaryotes were observed in the inner parts of the lagoon at stations 3, 4 and 5 affected by urban, agricultural and industrial discharges. Ultraphytoplankton concentrations were the highest near the outlet of the lagoon at stations 1 and 2 influenced by freshwater outflow and oligotrophic Mediterranean water inflow, respectively. At station 1, the large ultraphytoplankton concentration derives from the high abundance of cryptophyte-like cells favoured by the freshwater outflow whereas at station 2, the input of oligotrophic Mediterranean water enhanced the abundance of Synechococcus and picoeukaryotes at the expense of nanoeukaryotes. Two trophic regimes were thus differentiated in GML.

Microbial composition and ecological features of phototrophic biofilms proliferating in the Moidons Caves (France): investigation at the single-cell level.

The authors investigated the microbial composition of phototrophic biofilms proliferating in a show cave using flow cytometry for the first time in such a context. Results are based on several biofilms sampled in the Moidons Caves (France) and concern both heterotrophic prokaryotes and autotrophic microorganisms. Heterotrophic microorganisms with low nucleic acid content were dominant in biofilms, as can be expected from the oligotrophic conditions prevailing within the cave. Analysis of the biofilm autotrophic components revealed the presence of several taxa, particularly the unicellular green algae Chlorella minutissima, specifically well adapted to this cave. Relationships between flow cytometry results and environmental variables determined in the cave were established and discussed so as to better understand biofilm proliferation processes in caves.

Antifouling coatings influence both abundance and community structure of colonizing biofilms: a case study in the Northwestern Mediterranean Sea.

When immersed in seawater, substrates are rapidly colonized by both micro- and macroorganisms. This process is responsible for important economic and ecological prejudices, particularly when related to ship hulls or aquaculture nets. Commercial antifouling coatings are supposed to reduce biofouling, i.e., micro- and macrofoulers. In this study, biofilms that primarily settled on seven different coatings (polyvinyl chloride [PVC], a fouling release coating [FRC], and five self-polishing copolymer coatings [SPC], including four commercial ones) were quantitatively studied, after 1 month of immersion in summer in the Toulon Bay (Northwestern Mediterranean Sea, France), by using flow cytometry (FCM), microscopy, and denaturing gradient gel electrophoresis. FCM was used after a pretreatment to separate cells from the biofilm matrix, in order to determine densities of heterotrophic bacteria, picocyanobacteria, and pico- and nanoeukaryotes on these coatings. Among diatoms, the only microphytobenthic class identified by microscopy, Licmophora, Navicula, and Nitzschia were determined to be the dominant taxa. Overall, biocide-free coatings showed higher densities than all other coatings, except for one biocidal coating, whatever the group of microorganisms. Heterotrophic bacteria always showed the highest densities, and diatoms showed the lowest, but the relative abundances of these groups varied depending on the coating. In particular, the copper-free SPC failed to prevent diatom settlement, whereas the pyrithione-free SPC exhibited high picocyanobacterial density. These results highlight the interest in FCM for antifouling coating assessment as well as specific selection among microbial communities by antifouling coatings.

Seasonal distribution of ultraphytoplankton and heterotrophic prokaryotes in relation to abiotic variables on the north coast of Sfax after restoration.

The Taparura project was set up to restore the north Sfax coast (Tunisia) by shutting down the northern phosphate plant responsible for chronic pollution and uncontrolled phosphogypsum dumping. The restoration effect on coastal ultraphytoplankton (<10 µm) and heterotrophic prokaryotes was investigated using conventional flow cytometry over four successive seasons during 2009-2010. Cell concentrations were generally higher than values reported for the open sea, both in the western and eastern Mediterranean basins. One striking point was that chl a concentration on the north Sfax coast was unchanged after restoration but was still one order of magnitude higher than in the Gulf of Gabès. Restoration of pH, following the shutdown of the phosphate processing plants on the north coast, appeared to reach normal levels for seawater during the study, whereas seawater acidification persisted on the south coast where plants are still in operation. The largest ultraphytoplankton biomass was from an unknown cell group, whose identity and role needs to be established.

Do human activities affect the picoplankton structure of the Ahe atoll lagoon (Tuamotu Archipelago, French Polynesia)?

The spatial variations of the picoplankton (photoautotrophic and heterotrophic microorganisms) in the Ahe atoll lagoon were studied in May and October 2008 to assess whether they were affected by human activities along the atoll. Spatial patterns were studied using 10 sampling stations chosen according to the location of the anthropogenic activities (pearl farming, harbor). Experiments were also carried out to determine whether bacterial growth, with or without predators, was limited by inorganic (N and P) substrates. The results showed that heterotrophic bacterioplankton abundance was superior to the photoautotrophic organisms, especially in May. Significant increases in bacterial abundance were observed in May after 24 h incubation with +P and +N (but not in October). All samples complied with the quality levels for fecal indicator bacteria (FIB) defined by the European Union and there was no evidence that human sewage had any impact on picoplankton over the whole atoll.

Abnormalities of satellite cells function in amyotrophic lateral sclerosis.

Abstract Amyotrophic lateral sclerosis (ALS) is characterized by progressive denervation leading to muscle atrophy prevented, during the early phase, by compensatory reinnervation. Little is known about muscle fibre regeneration capacity in ALS. We have carried out in vivo and in vitro investigation of skeletal muscle in ALS. Seven ALS patients underwent a deltoid muscle biopsy. Immunohistochemical analysis revealed various degrees of denervation- and reinnervation-related changes in the ALS muscle biopsies including satellite cells (SCs) activation and regenerating fibres. Only 3/7 primary cultures of ALS muscle cells were successfully established and had sufficient myogenicity, as assessed by desmin positivity, to be used without further purification. This was in contrast with the cultures derived from control muscles, predominantly desmin-positive cells. Although capable to proliferate in vitro, ALS-derived SCs presented an abnormal senescent-like morphology. Markers of senescence, including senescent-associated (SA)-ßGal activity and p16 expression, were increased. Furthermore, ALS-derived SCs were also unable to fully differentiate in vitro as shown by abnormal myotubes morphology and reduced MHC isoform expression, compared to control myotubes. Our study suggests that SC function is altered in ALS. This could limit the efficacy of compensatory processes and therefore could contribute to the progression of muscle atrophy and weakness.

Characterization of heterotrophic prokaryote subgroups in the Sfax coastal solar salterns by combining flow cytometry cell sorting and phylogenetic analysis.

Here, we combined flow cytometry (FCM) and phylogenetic analyses after cell sorting to characterize the dominant groups of the prokaryotic assemblages inhabiting two ponds of increasing salinity: a crystallizer pond (TS) with a salinity of 390 g/L, and the non-crystallizer pond (M1) with a salinity of 200 g/L retrieved from the solar saltern of Sfax in Tunisia. As expected, FCM analysis enabled the resolution of high nucleic acid content (HNA) and low nucleic acid content (LNA) prokaryotes. Next, we performed a taxonomic analysis of the bacterial and archaeal communities comprising the two most populated clusters by phylogenetic analyses of 16S rRNA gene clone library. We show for the first time that the presence of HNA and LNA content cells could also be extended to the archaeal populations. Archaea were detected in all M1 and TS samples, whereas representatives of Bacteria were detected only in LNA for M1 and HNA for TS. Although most of the archaeal sequences remained undetermined, other clones were most frequently affiliated to Haloquadratum and Halorubrum. In contrast, most bacterial clones belonged to the Alphaproteobacteria class (Phyllobacterium genus) in M1 samples and to the Bacteroidetes phylum (Sphingobacteria and Salinibacter genus) in TS samples.

Age-related changes in the mitotic and metabolic characteristics of muscle-derived cells.

Age-related sarcopenia could partly result from cumulative repeated episodes of incomplete repair and regeneration. We hypothesized that mitotic and metabolic events associated with satellite cell activation and proliferation could be altered with aging. Muscle-derived cells (mdc) were isolated from gastrocnemius and quadriceps muscles of young (3 wk old), adult (9 mo old), and old (24 mo old) Sprague-Dawley male rats (n = 10/group). The mdc from young growing rats started to proliferate earlier compared with adult and old animals. Cell cycle duration was significantly reduced with aging from 36.5 +/- 3.2 to 28.0 +/- 2.2 h. However, the proportion of noncycling (G0 phase) and cycling (G1 + S + G2 + M phases) cultured mdc was statistically unchanged among the three age groups. Significantly lower increase in c-met and proliferating cell nuclear antigen expression were observed in cultured mdc of old rats upon serum stimulation. Major changes in the expression of citrate synthase, lactate dehydrogenase, proteasome, caspase 3, plasminogen activators (PAs), and matrix metalloproteinase 2-9 (MMP2-9) were observed upon serum stimulation, but no age-related difference was noted. However, when measured on crushed muscle extracts, PAs and MMP2-9 enzyme activities were significantly decreased with aging. Our results show that cellular and biochemical events associated with the control of mdc activation and proliferation occur with aging. These alterations may participate in the accumulation of repeated episodes of incomplete repair and regeneration throughout the life span, thus contributing to the loss of skeletal muscle mass and function with aging.

Mitotic activity of rat muscle satellite cells in response to serum stimulation: relation with cellular metabolism.

Cellular and molecular adaptations of satellite cells isolated from rat hindlimb muscles (n = 10) were investigated in response to serum stimulation. Flow cytometry analysis of the amounts of DNA and RNA indicated that 97.7 +/- 0.7% of satellite cells were in G0 at the end of the isolation procedure, whereas 93.2 +/- 2.0% of cells were cycling after serum exposure. The length of cell division was 34.0 +/- 2.8 h. Myoblast proliferation was asynchronous, suggesting the existence of heterogeneous cell populations in skeletal muscle. Myoblast proliferation was also accompanied by a significant increase in c-met expression, and major adaptations of energetic and proteolytic metabolisms, including an increase in the relative contribution of glycolytic metabolism for energy production, an increase in proteasome and matrix metalloproteinases 2 and 9 activities, and a decrease in plasminogen activator activities. Our data suggest that, along with molecular adaptations leading to cell cycle activation itself, adaptations in energetic and proteolytic metabolisms are crucial events involved in satellite cell activation and myoblast proliferation.